Govind Singh Saharan

Naresh Mehta
Prabhu Dayal Meena

Alternaria Diseases
of Crucifers:
Biology, Ecology
and Disease
Management
Alternaria Diseases of Crucifers:
Biology, Ecology and Disease
Management
Govind Singh Saharan • Naresh Mehta
Prabhu Dayal Meena

Alternaria Diseases
of Crucifers: Biology,
Ecology and Disease
Management
Govind Singh Saharan Naresh Mehta
Plant Pathology Plant Pathology
CCS Haryana Agricultural University CCS Haryana Agricultural University
Hisar, Haryana, India Hisar, Haryana, India

Prabhu Dayal Meena

Crop Protection Unit
ICAR
Bharatpur, Rajasthan, India

ISBN 978-981-10-0019-5 ISBN 978-981-10-0021-8 (eBook)

DOI 10.1007/978-981-10-0021-8

Library of Congress Control Number: 2015958091

Springer Singapore Heidelberg New York Dordrecht London

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Dedicated to Beloved Dr Prithwi Raj Verma

(10 January 1940–07 Feb 2015)

Foreword

Brassica species including B. campestris, B. juncea,

B. napus and B. carinata are an important group of
oilseed crops, constituting almost 13.2 % of the
world edible oil requirement. Together these occupy
about 29.39 million hectares of area with an annual
production of 53.01 million tons in the world. These
have wide adaptability and are often grown under
varied agroclimatic conditions throughout the world.
During the last two decades, the area and production
of these crops have increased substantially and the
total production has almost trebled.
Considerable potential exists for improving production and productivity
of oilseed brassicas, as well as cruciferous vegetables through breeding
improved varieties that are resistant to both biotic and abiotic stresses. Among
biotic factors limiting the productivity, the diseases like Alternaria blight,
white rust, downy mildew, Sclerotinia stem rot and powdery mildew are the
serious one. Presently, Alternaria diseases are due to four species, viz. A.
brassicae (Berk.) Sacc., A. brassicicola (Schwein.) Wiltsh., A. raphani
Groves and Skolko and A. alternata (Fr.) Kiessl, causing heavy yield losses
annually in cruciferous crops.
To have an in-depth knowledge of Alternaria diseases of crucifers and to
evolve effective management strategies, it is necessary that a comprehensive
review of literature relating to pathogen and its taxonomy, process of infec-
tion, pathogenesis, fine structure, biochemistry of host–pathogen interaction,
phytotoxins, pathogenic variability, distribution, yield losses, disease cycle,
epidemiology and forecasting, resistance, and sources; various techniques for
germ-plasm screening; in vitro studies; biotechnological approaches; and

vii
viii Foreword

disease management practices is available for the workers. Presently such a

comprehensive document is lacking. This book “Alternaria Diseases of
Crucifers: Biology, Ecology and Disease Management” is an outcome of
sincere efforts of the authors focusing on oilseed crops’ diseases. The sugges-
tions by the authors on priority areas of research will benefit the Brassica
researchers to plan their research in a better way.
I congratulate the authors for bringing out this long-awaited publication
and believe that this book will be of immense use to the scientists, teachers,
students, extension specialists and all those interested in the production of
crucifer crops.

New Delhi, India S. Ayyappan

22 April 2015
Preface

The aim of this book Alternaria Diseases of Crucifers: Biology, Ecology and
Disease Management is to present a comprehensive information available in
literature on fundamental and applied knowledge of Alternaria species
infecting Brassicaceae crops and weeds. Since the first publication of
Monograph on Alternaria Diseases of Crucifers (Verma and Saharan, 1994),
voluminous valuable research data have been generated and published that
encouraged the authors to update the information in the form of a book. The
four species of Alternaria, viz. A. brassicae (Berk.) Sacc., A. brassicicola
(Schwein.) Wiltsh., A. raphani Groves and Skolko. and A. alternata (Fr.)
Kiessl., are most widely distributed and cause severe quantitative and quali-
tative losses in crucifers where these crops are grown in the world. Brassica
crops are grown for high-quality edible (rapeseed–mustard, canola and other
rapes) and industrial (Crambe) oil, common vegetables (cabbage, cauli-
flower, radish, kohlrabi, broccoli, Brussels sprouts, kales and other Brassica
vegetables) and a few weeds. For convenience of the readers and coherence
of the text, the information has been arranged in 13 chapters with several
subsections. The arrangement includes the disease, geographical distribu-
tion, symptoms on different hosts, host range, yield losses and disease
assessment method, while detailed description on pathogen includes taxo-
nomic position, phylogeny, variability, sporulation, perpetuation and spore
germination, host–parasite interactions in the form of seed infection, disease
cycle, process of infection and pathogenesis, epidemiology, forecasting, fine
structures, biochemical changes and phytotoxins, host defence mechanism,
techniques to study host–parasite relationships, management practices
(including cultural, chemical, biological control practices) and deployment
of host resistance. The last section deals with gaps in our understanding, and
knowledge about management of these diseases, and offers suggestions for
future research priorities. The subject matter has been vividly illustrated
with photographs, graphs, figures, histogram, tables and coloured plates,
which makes it stimulating, effective and easy to comprehend by the readers.
The headings and subheadings of each chapter have been arranged in num-
bered series to make the subject matter contiguous. All important and rele-
vant references have been included for further consultations by the
researchers, teachers and students.

ix
x Preface

We believe that this book will be immensely useful to researchers, teach-

ers, extension specialists, students and all others who are interested in the
diagnosis and management of Alternaria diseases of crucifers. Suggestions
for the improvement are always welcomed.

Hisar, India Govind Singh Saharan

Naresh Mehta
Bharatpur, India Prabhu Dayal Meena
Acknowledgements

Authors are highly grateful to the following persons/scientists/publishers/

societies/journals/institutes/websites and all others whose valuable materials
such as photographs (macroscopic, microscopic, electron micrographs, scan-
ning electron micrographs), drawings, figures, histograms, graphs, tables,
flow charts, etc., have been used through reproduction in the present docu-
ment. The address of the author(s)/source(s) from where the material is
adapted can be obtained from the reference which has been cited in the refer-
ence section of the book.

A. Persons/Scientists
A. Tsuneda
B. M. Pryor
B. P. H. J. Thomma
Claudia A. Jasalavich
D. P. Lawrence
E. G. Simmons
F. M. Humpherson-Jones
J. H. C. Woudenberg
J. P. Tewari
M. Soledade C. Pedras
P. R. Verma
P. Parvatha Reddy
P. S. Bains
Philippe Simonea
R. P. Awasthi
R. Y. Parada
S. J. Kolte
S. K. Shrestha
Yangrae Cho

B. Journal
African Journal of Biotechnology
Australian Journal of Agricultural Research
Botany
Brazilian Phytopathological Society
Canadian Journal of Botany

xi
xii Acknowledgements

Canadian Journal of Plant Pathology

Crop Protection
European Foundation for Plant Pathology
Fungal Genet & Biology
Indian Phytopathology
International Journal of Agriculture, Environment & Biotechnology
Journal of Mycology and Plant Pathology
Journal of Oilseed Brassica
Journal of Phytopathology
Molecular Microbiology
Molecular Plant Microbe Interaction
Molecular Plant Pathology
Molecules
Mycologia
Mycological Progress
Mycological Research
Phytochemistry
Plant Molecular Biology
Plant Pathology
Plant Pathology
PLoS Pathogens
Proceedings of the National Academy of Sciences USA
Review of Plant Pathology
Seed Science & Technology Journal
The Canadian Journal of Plant Pathology
The Plant Pathology Journal
Transaction of British Mycological Society
Tropical Plant Pathology

Website
http://cals.arizona.edu/PLP/pryorlab/alternaria.html
http://nt.ars-grin.gov/fungaldatabases/
http://www.mycobank.org
http://www.marinespecies.org
www.elsevier.com/locate/yfgbi
www.sciencedirect.com

Publishers
CABI, UK
CSIRO Publishing
Elsevier
John Wiley & Sons, Inc.
Springer, Netherlands
Studium Press LLC, USA
Taylor & Francis Group
Acknowledgements xiii

Institutions
Canadian Phytopathological Society
Indian Phytopathological Society
Indian Society of Mycology and Plant Pathology
Korean Society of Plant Pathology
Mycological Society of America
Pryor Laboratory, University of Arizona School of Plant Sciences, USA
Society for Rapeseed-Mustard Research
The American Phytopathological Society
The Australasian Plant Pathology Society
The British Society for Plant Pathology
The Korean Society of Plant Pathology

Databases
MycoBank, International mycological Association
Systematic Mycology and Microbiology Laboratory Fungal Database, US
Department of Agriculture
Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Status of Genus Alternaria. . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Brassica Crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.3 The Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.4 The Pathogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.5 Epidemiology and Forecasting . . . . . . . . . . . . . . . . . . . . . . 7
1.6 Pathogenic Variability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.7 Fine Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.8 Biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.9 Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.10 Phytotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.11 Disease Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.12 Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2 The Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.2 Synonyms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.3 Symptomatology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.3.1 Rapeseed–Mustard . . . . . . . . . . . . . . . . . . . . . . . 18
2.3.2 Taramira (Eruca sativa) . . . . . . . . . . . . . . . . . . . 19
2.3.3 Crambe (Crambe abyssinica) . . . . . . . . . . . . . . 19
2.3.4 Garden Stock (Matthiola incana) . . . . . . . . . . . 22
2.3.5 Vegetable Crops (Cruciferous Vegetables). . . . . 22
2.3.6 Weeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.4 Geographical Distribution . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.5 Host Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.6 Yield Losses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.6.1 Rapeseed–Mustard . . . . . . . . . . . . . . . . . . . . . . . 23
2.6.2 Crambe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
2.6.3 Vegetable Crops (Cruciferous Vegetables). . . . . 38
2.7 Disease Assessment Keys/Severity Charts. . . . . . . . . . . . . 39
2.7.1 Visual Assessment Methods. . . . . . . . . . . . . . . . 39
2.7.2 Incidence–Severity Relationships . . . . . . . . . . . 43
2.7.3 Inoculum–Disease Intensity Relationships . . . . 43
2.7.4 Remote Sensing Method . . . . . . . . . . . . . . . . . . 43

xv
xvi Contents

2.7.5 Video Image Analysis . . . . . . . . . . . . . . . . . . . . 43

2.7.6 Stress Tolerance Attributes. . . . . . . . . . . . . . . . . 44
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
3 Pathogen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.2 Historical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.3 Phylogeny. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.4 Taxonomy, Nomenclature and Morphology . . . . . . . . . . . 57
3.4.1 Type species: Alternaria alternata
(Fr.) Keissl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
3.5 Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
3.6 Morphology of Alternaria species Pathogenic
on Cruciferous Crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
3.6.1 Type Species: Alternaria brassicicola
(Schw.) Wiltshire . . . . . . . . . . . . . . . . . . . . . . . . 59
3.6.2 Type Species: Alternaria brassicae
(Berk.) Sacc.. . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
3.6.3 Type Species: Alternaria raphani Groves
& Skolko . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
3.6.4 Type Species: Alternaria cheiranthi
(Lib.) Bolle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
3.7 The Infection Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3.8 Identification of Alternaria Genes . . . . . . . . . . . . . . . . . . . 64
3.9 Nuclear Ribosomal DNA Sequences . . . . . . . . . . . . . . . . . 65
3.10 Identification, Cloning and Sequencing
of Virulence Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
3.11 Identification of Pathogenicity Factors . . . . . . . . . . . . . . . 67
3.12 Growth and Sporulation . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
3.12.1 Culture Media . . . . . . . . . . . . . . . . . . . . . . . . . . 68
3.12.2 Temperature and Relative Humidity . . . . . . . . . 70
3.12.3 Hydrogen Ion Concentrations (pH) . . . . . . . . . . 70
3.12.4 Light and Darkness . . . . . . . . . . . . . . . . . . . . . . 71
3.13 Perpetuation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
3.14 Spore Germination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
3.14.1 Effect of Culture Media . . . . . . . . . . . . . . . . . . . 74
3.14.2 Effect of Temperature and Relative Humidity . . 76
3.14.3 Effect of Host Extract and Exudates . . . . . . . . . 76
3.14.4 Effect of Light Intensity . . . . . . . . . . . . . . . . . . . 77
3.15 Seed Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
3.15.1 Location of Seed-Borne Infection . . . . . . . . . . . 78
3.15.2 Disease Transmission in the Field . . . . . . . . . . . 78
3.15.3 Effect of Seed Treatment . . . . . . . . . . . . . . . . . . 82
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
4 Infection Process, Pathogenesis and Disease Cycle . . . . . . . . . . 87
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
4.2 Infection and Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . 87
4.3 Transcription Factors Associated with Pathogenesis . . . . . 89
Contents xvii

4.3.1 Melanin Biosynthesis and Virulence

in A. brassicicola . . . . . . . . . . . . . . . . . . . . . . . . 90
4.3.2 Mutation of the Amr1 Gene Unexpectedly
Causes Increased Virulence . . . . . . . . . . . . . . . . 92
4.4 The Cause of Increased Virulence in Δamr1 Mutants . . . . 92
4.5 Evolution of Virulence in A. brassicicola . . . . . . . . . . . . . 93
4.6 Identification of Pathogenicity Factors . . . . . . . . . . . . . . . 94
4.7 Disease Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
5 Epidemiology and Forecasting . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
5.2 Disease Development in Relation
to Environmental Conditions . . . . . . . . . . . . . . . . . . . . . . . . 99
5.3 Disease Development in Relation to Nutrition
and Cultural Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . 109
5.4 Disease Development in Relation to Host Resistance . . . 110
5.5 Disease Development in Relation to Flea Beetle . . . . . . . 110
5.6 Disease Development in Relation to Barrier Crops . . . . . 112
5.7 Models to Describe the Progress of the Disease . . . . . . . 112
5.8 Disease Forecasting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
6 Pathogenic Variability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
6.2 Historical Developments . . . . . . . . . . . . . . . . . . . . . . . . . . 125
6.3 Pathological Variations. . . . . . . . . . . . . . . . . . . . . . . . . . . 127
6.4 Symptomatological Variations . . . . . . . . . . . . . . . . . . . . . 133
6.5 Morphological and Cultural Variations . . . . . . . . . . . . . . 133
6.6 Genetic Variability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
6.7 Molecular Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
6.8 Proteome Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
6.9 Nutritional Variability . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
6.10 Biochemical Variability . . . . . . . . . . . . . . . . . . . . . . . . . . 145
6.11 Fungicidal and Plant Extracts Sensitivity . . . . . . . . . . . . 145
6.12 Thermal Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
6.13 Identification and Nomenclature of Pathotypes . . . . . . . . 154
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
7 Fine Structures and Electron Microscopy . . . . . . . . . . . . . . . . 163
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
7.2 Fine Structures and Electron Microscopy . . . . . . . . . . . . 163
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
8 Biochemistry of Host–Pathogen Interaction . . . . . . . . . . . . . . 167
8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
8.2 Biochemical Changes in the Host . . . . . . . . . . . . . . . . . . 167
8.3 Biochemical Changes in the Pathogen . . . . . . . . . . . . . . . 170
8.4 Glucosinolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
8.5 Metabolites Produced. . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
xviii Contents

9 Resistance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
9.2 Genetics of Host–Parasite Interaction . . . . . . . . . . . . . . . 176
9.2.1 Inheritance of Resistance . . . . . . . . . . . . . . . . . 176
9.2.2 Disease Tolerance . . . . . . . . . . . . . . . . . . . . . . 178
9.2.3 Components of Horizontal Resistance . . . . . . . 180
9.3 Morphological Resistance . . . . . . . . . . . . . . . . . . . . . . . . 180
9.4 Epicuticular Wax . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
9.5 Biochemical Basis of Resistance . . . . . . . . . . . . . . . . . . . 185
9.6 Proteome-Level Resistance . . . . . . . . . . . . . . . . . . . . . . . 185
9.7 Induced Resistance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
9.8 Identification, Cloning and Sequencing
of Resistant Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
9.9 Elicitation of Phytoalexins . . . . . . . . . . . . . . . . . . . . . . . . 191
9.10 Calcium Sequestration . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
9.11 Sources of Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
9.11.1 Sources of Resistance from Cruciferous
Relatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
9.12 Sources of Multiple Disease Resistance . . . . . . . . . . . . . 195
9.13 Relationship between Major Foliar Diseases . . . . . . . . . . 195
9.14 Development of Resistant Cultivars . . . . . . . . . . . . . . . . . 196
9.15 Strategies and Methods of Screening for Resistance . . . . 197
9.16 Bottlenecks in Resistance Breeding . . . . . . . . . . . . . . . . . 199
9.17 Biotechnological Approaches . . . . . . . . . . . . . . . . . . . . . 200
9.17.1 In Vitro Embryo Rescue . . . . . . . . . . . . . . . . . . 200
9.17.2 Somatic Hybridization . . . . . . . . . . . . . . . . . . . 201
9.17.3 Somaclonal Variations . . . . . . . . . . . . . . . . . . . 201
9.17.4 Genetic Transformation . . . . . . . . . . . . . . . . . . 201
9.17.5 Molecular Markers . . . . . . . . . . . . . . . . . . . . . . 202
9.17.6 Induction of Systemic Resistance . . . . . . . . . . 203
9.17.7 Genetic Engineering. . . . . . . . . . . . . . . . . . . . . 203
9.18 Factors Affecting Plant Disease Resistance . . . . . . . . . . . 203
9.19 Accessing and Exploiting Genetic Diversity . . . . . . . . . . 204
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
10 Phytotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
10.2 Historical Developments . . . . . . . . . . . . . . . . . . . . . . . . . 212
10.3 Metabolites from Alternaria . . . . . . . . . . . . . . . . . . . . . . 214
10.3.1 Classification and Occurrence . . . . . . . . . . . . . 214
10.4 Effect on Plants at Physiological, Biochemical
and Molecular Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
10.4.1 Physiological Level . . . . . . . . . . . . . . . . . . . . . 218
10.4.2 Biochemical Level . . . . . . . . . . . . . . . . . . . . . . 219
10.4.3 Molecular Level . . . . . . . . . . . . . . . . . . . . . . . . 220
10.5 Role of Toxins in the Infection Process . . . . . . . . . . . . . . 222
10.6 Toxin Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
Contents xix

10.7 Mode of Action of Host-Specific Toxins . . . . . . . . . . . . . 225

10.8 Role of Toxin in Host Defence against
Alternaria species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
10.9 In Silico Protein–Protein Interaction . . . . . . . . . . . . . . . . 230
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
11 Disease Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
11.2 Cultural Measures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
11.3 Seed Treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
11.3.1 Hot Water Treatment . . . . . . . . . . . . . . . . . . . . 240
11.3.2 Chemical Treatment . . . . . . . . . . . . . . . . . . . . . 240
11.3.3 Bioagent Treatment . . . . . . . . . . . . . . . . . . . . . 241
11.4 Chemical Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
11.4.1 Effect of Fungicides on Host Growth . . . . . . . 247
11.5 Biological Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
11.5.1 Plant Extracts as Fungitoxicants . . . . . . . . . . . 249
11.5.2 Antagonists for Biocontrol. . . . . . . . . . . . . . . . 250
11.5.3 Mechanisms of Biocontrol . . . . . . . . . . . . . . . . 252
11.5.4 Biological Control vs. Biochemical
Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
11.6 Host Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
11.7 Fungicidal Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
11.8 Integrated Disease Management . . . . . . . . . . . . . . . . . . . 259
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
12 Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
12.2 Stem Explant Culture Inoculation . . . . . . . . . . . . . . . . . . 273
12.3 Leaf Disc Inoculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
12.4 Detached Leaf and Pod Inoculation . . . . . . . . . . . . . . . . . 274
12.5 Detached Leaf Inoculation . . . . . . . . . . . . . . . . . . . . . . . . 274
12.6 Greenhouse Method for Testing Resistance. . . . . . . . . . . 274
12.7 Brassica Germ-Plasm Screening for Resistance
through AB Toxin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
12.8 Semi-Selective Medium for Detecting Seed-Borne
A. brassicicola . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
12.9 Radish Root Extract Agar for A. brassicae
Sporulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
12.10 Inducing Sporulation of A. brassicae. . . . . . . . . . . . . . . . 276
12.11 Brassica Callus Culture to Induce Sporulation
in Alternaria brassicae . . . . . . . . . . . . . . . . . . . . . . . . . . 276
12.12 Method of Estimating Alternaria brassicicola in Seed . . 277
12.13 Identification of Fungicide Antagonists
in Leaf Exudates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
12.14 Ovary and Ovule Culture . . . . . . . . . . . . . . . . . . . . . . . . . 277
12.15 Method for Evaluating Partial Resistance
to Alternaria brassicicola . . . . . . . . . . . . . . . . . . . . . . . . 278
12.16 Tissue Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
xx Contents

12.17 Inoculation Methods for Pathogenesis

of Alternaria brassicae . . . . . . . . . . . . . . . . . . . . . . . . . . 278
12.18 PCR-Based Assay for Detecting Alternaria
brassicae in Cruciferous Seed . . . . . . . . . . . . . . . . . . . . . 279
12.18.1 Preparation of Seed Samples . . . . . . . . . . . . . . 279
12.18.2 DNA Manipulation . . . . . . . . . . . . . . . . . . . . . 280
12.18.3 PCR-Based Assay . . . . . . . . . . . . . . . . . . . . . . 280
12.19 Quantitative Inoculation Method . . . . . . . . . . . . . . . . . . . 281
12.20 Assessment of Methods of Inoculation
for Resistance to Alternaria . . . . . . . . . . . . . . . . . . . . . . . 281
12.20.1 Method of Inoculation . . . . . . . . . . . . . . . . . . . 281
12.20.2 Detached True Leaf Inoculation. . . . . . . . . . . . 281
12.20.3 Infection Score and Disease Index . . . . . . . . . . 282
12.21 Image-Based Disease Identification . . . . . . . . . . . . . . . . . 282
12.22 ELISA Diagnostic Kits . . . . . . . . . . . . . . . . . . . . . . . . . . 282
12.23 Direct Tissue Blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
12.24 Nucleic Acid Probes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
12.24.1 Squash Blot Method. . . . . . . . . . . . . . . . . . . . . 284
12.24.2 Polymerase Chain Reaction (PCR) . . . . . . . . . 284
12.24.3 DNA Microarray Technology . . . . . . . . . . . . . 285
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286
13 Future Strategies and Priorities for the Management
of Alternaria Diseases of Crucifers . . . . . . . . . . . . . . . . . . . . . . 289
13.1 Disease Epidemiology and Forecasting . . . . . . . . . . . . . . 289
13.2 Physiological Specialization . . . . . . . . . . . . . . . . . . . . . . 289
13.3 Genetics of Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
13.4 Genetics of Virulence . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
13.5 Exploitation of Morphological, Structural
and Biochemical Basis of Resistance . . . . . . . . . . . . . . . 290
13.6 Comparative Studies on All Aspects of Host–Parasite
Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
13.7 Phytotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
13.8 Genome Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
13.9 Disease Control Strategy . . . . . . . . . . . . . . . . . . . . . . . . . 290
13.10 Integrated Management . . . . . . . . . . . . . . . . . . . . . . . . . . 291

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
About the Authors

Govind Singh Saharan Renowned Educationist and Plant Pathologist has

contributed in the diverse fields of Plant Pathology including standardization
of artificial inoculation techniques, identification of resistance sources, patho-
genic variability, genetics of host–parasite interaction, epidemiology and
management of several diseases. He has about 250 publications in peer-
reviewed journals and 10 books and supervised over 11 MSc and PhD stu-
dents. Professor Saharan is internationally recognized a visiting Professor at
the University of Alberta, Edmonton, Canada (1991 and 1994); Saskatoon
Research Station, Canada (1991, 1994, 1997); and Rothamsted Research, UK
(1994, 1997). He has been on the panel of experts of SAUs, ICAR, CSIR,
UGC and DBT in India.

Naresh Mehta Associate Dean, Professor (Plant Pathology), Gold medalist,

Educationist and Plant Pathologist, has contributed in diverse field of plant
pathology covering pathogenic variability, genetics of host–pathogen inter-
action, epidemiological studies, identification of resistant sources, biochem-
ical/genetical basis for resistance, residual analysis of fungicides and disease
management. He has published more than 96 research papers in peer-
reviewed journals, 2 books, 10 manuals, 8 review articles, 18 book chapters,
18 lead lectures in the international and national conferences and 79 research
papers presentations in the international/national conferences. He has super-
vised seven MSc and PhD students. Professor Mehta has been admitted as
fellow of prominent Plant Pathological Society of India; councillor (North
Zone) and editor-in-chief, ISMPP; and member of editorial board of
INSOOP and Indian Phytopathological Soc. (IPS), New Delhi, India.
Professor Mehta has been a visiting scientist at the University of Alberta,
Edmonton, Canada, and invited to deliver lecture in the 9th International
Congress of Plant Pathology at Torino, Italy. Professor Mehta is on the panel
of experts of SAUs.

Prabhu Dayal Meena is presently working as Principal Scientist (Plant

Pathology) at ICAR-Directorate of Rapeseed-Mustard Research, Bharatpur
(Rajasthan), India. He has made important contributions to the various aspects
including resistance, epidemiology, forecasting and biocontrol for the man-
agement of rapeseed–mustard diseases. He has published about 60 research
papers, two books and four bulletins and has supervised nine MSc and

xxi
xxii About the Authors

co-supervised a PhD student. He has been honoured with fellow of ISMPP,

PPAI, ISOR and Dr PR Kumar Outstanding Brassica Scientist Award (2011)
of SRMR and Best Scientist Award of DRMR for 2012–2013. Meena is
internationally recognized and has been working at Rothamsted Research,
UK (2007).
List of Figures

Fig. 2.1 A schematized drawings of (a) leaves and (b) siliquae

of crucifers showing Alternaria infection grades ................ 42
Fig. 3.1 Bayesian 50 % majority-rule consensus tree based
on the SSU, LSU and RPB2 sequences of 74 strains
representing the Pleosporineae. The Bayesian posterior
probabilities (PP) and RAxML bootstrap support values
(ML) are given at the nodes (PP/ML). Thickness lines
a PP of 1.0 and ML of 100. The tree was rooted
to Julella avicenniae ............................................................ 56
Fig. 3.2 Alternaria alternata ............................................................. 60
Fig. 3.3 Alternaria brassicicola ........................................................ 60
Fig. 3.4 Alternaria brassicae ............................................................ 61
Fig. 3.5 Alternaria raphani ............................................................... 61
Fig. 3.6 Alternaria cheiranthi ........................................................... 62
Fig. 3.7 The consensus tree generated by global parsimony
bootstrap analysis of alignment of the 5–8s rDNA
and flanking internal transcribed spaces (ITS1 and ITS 2)
sequences. The percentages represent the proportion
of 1000 bootstrap replications in which the taxa to the right
of the node were placed together by the programme
DNA PARS with randomization of the sequence input
order. The other numbers represent the steps. Branch length
(drawn in the horizontal dimension only) is the maximum
likelihood estimates made by the programme DNAML.
When the user tree was defined as the bootstrap consensus
tree, the length of the vertical lines has no meaning
and was adjusted arbitrarily for ease in labelling
termini. Leptosphaeria doliolum was designated
as the out-group ................................................................... 66
Fig. 3.8 The consensus tree generated by global parsimony
bootstrap analysis of alignment of the 18s rDNA.
The percentages represent the proportion of 1000 bootstrap
replications in which the taxa to the right of the node were
placed together by the programme DNA PARS with
randomization of the sequence input order. The other

xxiii
xxiv List of Figures

numbers represent the steps. Branch length (drawn in the

horizontal dimension only) is the maximum likelihood
estimates made by the programme DNAML. When the
user tree was defined as the bootstrap consensus tree,
the length of the vertical lines has no meaning and
was adjusted arbitrarily for ease in labelling termini.
Neurospora crassa was designated as the out-group ........... 66
Fig. 3.9 Influence of temperature on spore germination
of A. brassicae ..................................................................... 76
Fig. 3.10 (a) Relationship between seed infection and seedling
infection due to A. brassicae in rapeseed and mustard
in field. Correlation coefficient for both seasons was
highly significant (r = 0.0705; p < 0.001) in 1991. (b)
Figures showing the relationship for the year 1992
(r = 0.801; P < 0.001). Similarly, the correlation between
seedling infection and first true leaf infection was
also significant (r = 0.562; P < 0.01) ..................................... 81
Fig. 4.1 Expression of melanin biosynthesis-associated genes
and four hydrolytic enzyme-coding genes. (a–c) Relative
transcription abundance of each gene was determined
in comparison to actin gene transcripts in the same
tissue. Y-axes show relative abundance of the transcript
compared to the actin gene. (d) Expression ratio between
the Δamr1 and wild type during the late stage of infection.
A total of three biological replicates (N = 3) were used
for this study. Bars represent standard error. Wt=wild type,
∆a = Δamr1: Amr1pGFP, GY=glucose yeast extract
broth. SCD1=scytalone dehydratase, Brn1-13HN
reductase, Brn2-14HN reductase, Cbh7=cellobiohydrolase,
Amr1=Alternaria melanin regulation, chymo=
chymotrypsin ....................................................................... 94
Fig. 4.2 Disease cycle of Alternaria on crucifers.............................. 96
Fig. 5.1 Influence of weather variables on the development
of Alternaria blight .............................................................. 101
Fig. 5.2 Temporal progression of Alternaria leaf blight
on five varieties of rapeseed–mustard during
1988 to 1990 crop seasons (pooled data) ............................. 103
Fig. 5.3 Progress of Alternaria leaf blight severity on cultivar
Varuna of Indian mustard in different dates of sowing
at Bharatpur in 2001–2002 .................................................. 104
Fig. 5.4 The mean daily concentration of Alternaria
brassicicola spores in the air within Brassica
oleracea seed production crops. (a) 1976
and (b) 1977 study ............................................................... 105
Fig. 5.5 The hourly concentration of Alternaria brassicicola
spores in the air within a cabbage seed crop........................ 106
List of Figures xxv

Fig. 5.6 (a) Disease incidence (%) of dark leaf spot on Chinese
cabbage and the Gompertz growth curve and model;
(b) spore concentration of Alternaria brassicicola
in the air; (c, d) meteorological data during the
experimental period in summer .......................................... 107
Fig. 5.7 Spore percentage (A. brassicae) trapped over crop
canopy during different time interval of the day ................. 108
Fig. 5.8 Weekly average number of spores of Alternaria
brassicae .............................................................................. 108
Fig. 5.9 Increase in number of Alternaria lesions
on rapeseed–mustard cultivars ............................................. 110
Fig. 5.10 Increase in size of Alternaria lesions
on rapeseed–and mustard cultivars ...................................... 111
Fig. 5.11 Representation curves of eight gradient models.
The curves from the bottom to the top of the figure
at x = 1.0 were generated by the following models:
y = axb (Gregory); 1n[y/(1 − y)] vs.log (x); y = a exp
(bxn), n = 0.2 (Lambert et al.,); Y = axb exp(nx), n = −0.2
(Hoerl); −1n [−1n(y)] vs. log (x); y = a exp (−bx)
(Kiyosawa and Shiyomi); 1n[y/(1 − y)] vs. x; and −1n
[−1n(y)] vs. x. All curves began with proportion y = 0.6
at distance x = 0.1 and decreased to y = 0.01 at x =10.0.
Different curve shapes are possible with the models
of Lambert et al. and Hoerl by using other values
for the shape parameter (n). The curve shown for
the Gregory model typifies many of the steep gradients
observed for Alternaria brassicicola on cabbage in
which y = disease proportion and x = metres......................... 113
Fig. 6.1 Pathogenic reaction of different isolates of Alternaria
brassicae on a set of host differential .................................. 130
Fig. 6.2 Dendogram showing pathogenic variability among 13
A. brassicae isolates in respect of five qualitative characters, i.e.
spot colour, periphery colour, presence
or absence of concentric rings, central point and yellow
halo region, and one qualitative characters, i.e. per cent
disease severity ................................................................... 135
Fig. 6.3 Dendogram showing molecular variability among
13 A. brassicae isolates based on RAPD
fingerprints obtained from 100 RAPD primers ................... 135
Fig. 6.4 (a) Disease index. (b) Average number of spores/10 cm2.
(c) Average size of spot (cm) on leaves of Divya
inoculated by isolates grown on the PDA medium
on 5, 10 and 15 days after inoculation (DAI) ...................... 142
Fig. 6.5 Genetic divergence among ten isolates of Alternaria
brassicae based on UPGMA cluster analysis ...................... 143
Fig. 9.1 Metabolism of destruxin B by white mustard
and rapeseed......................................................................... 185
xxvi List of Figures

Fig. 9.2 Functional classification of upregulated genes

in Alternaria brassicicola, AI-ITC (a) and camalexin;
(b) treated conidia according to their putative
biological function ............................................................... 190
Fig. 9.3 Structures of camalexin (1), 6-methoxycamalexin (2)
and N-methylcamaxin (3) .................................................... 191
Fig. 9.4 Scheme to develop high-yielding disease-resistant
cultivars ................................................................................ 199
Fig. 10.1 Detoxification pathway of the phytoalexin brassinin
by the pathogen A. brassicicola ........................................... 217
Fig. 10.2 Detoxification pathway of the phytotoxin B
and homodestruxin B by the hosts Brassica napus
and Sinapis alba ................................................................... 217
Fig. 10.3 Phytotoxin are produced by microbial plant pathogens ...... 228
Fig. 10.4 Enzymatic reactions of plants to the pathogen
causing Alternaria black spot .............................................. 228
Fig. 10.5 Defence responses of plants resistant and susceptible
to Alternaria black spot ....................................................... 229
Fig. 10.6 Transformation of the phytoalexin cyclobrassinin
by different pathogens of Brassica ...................................... 229
Fig. 11.1 The effect of Streptomyces griseoviridis preparation
on (a) percentage emergence and (b) percentage
disease-free seedlings of cabbage cultivar Celtic.
O….O untreated seed; □….□ seed treated with
S. griseoviridis preparation; and ∆…….∆ seed
treated with iprodione .......................................................... 241
Fig. 11.2 The biological control of (a) seed-borne Alternaria
brassicicola and (b) A. brassicicola content
of seedlings grown from naturally infected Brassica
seed with a powdery preparation of Streptomyces sp. ......... 251
Fig. 12.1 ELISA diagnostic kit ............................................................ 283
Fig. 12.2 Direct tissue blotting............................................................. 284
Fig. 12.3 Nucleic acid probe squash blot method ................................ 285
List of Tables

Table 1.1 Yield losses in crucifer crops due to Alternaria diseases

in different countries of the world ................................... 3
Table 1.2 Genomic designations of varietal or subspecific
taxa of agriculturally important Brassicas and radish ..... 4
Table 2.1 Records of Alternaria species on Brassicaceae............... 24
Table 2.2 Host species susceptible to Alternaria brassicae,
Alternaria brassicicola, Alternaria raphani
and Alternaria alternata .................................................. 32
Table 2.3 Assessment of yield losses in rapeseed–mustard
due to Alternaria ............................................................. 35
Table 2.4 Influence of Alternaria pod infection on yield
components of raya cultivar Prakash ............................... 35
Table 2.5 Influence of Alternaria pod infection on yield
components of Brown Sarson.......................................... 36
Table 2.6 Influence of Alternaria pod infection on yield
components of Yellow Sarson ......................................... 36
Table 2.7 Effect of black spot on number of infected seeds
per pod (a) per cent seed germination (b)
and per cent oil content (s) of rapeseed–mustard ............ 37
Table 2.8 Influence of Alternaria infection on siliquae
of Crambe abyssinica ...................................................... 38
Table 2.9 Keys for the assessment of Alternaria disease
severity on crucifers ........................................................ 40
Table 2.10 Growth stage key for oilseed rape ................................... 41
Table 3.1 Identification characteristics of Alternaria species
infecting rapeseed–mustard ............................................. 54
Table 3.2 Culture media for growth and sporulation
of Alternaria species pathogenic on Brassicaceae .......... 69
Table 3.3 Cultural characters of Alternaria brassicae
on different culture media ............................................... 72
Table 3.4 Biomass and sporulation index of A. brassicae
and A. brassicicola at different pH levels........................ 73
Table 3.5 Influence of light intensity on A. brassicae infection ...... 73
Table 3.6 Survival of Alternaria brassicae in rapeseed–mustard
seed during storage at Ludhiana and Hisar in 1979 ........ 73

xxvii
xxviii List of Tables

Table 3.7 Alternaria blight incidence (%) in rapeseed–

mustard following inoculation with diseased
debris at different depth ................................................... 74
Table 3.8 Effect of leaf exudates and leaf extracts from
different cultivars of rapeseed and mustard
on spore germination of A. brassicae .............................. 76
Table 3.9 Location and sporulation of Alternaria brassicae
in different parts of Brassica seeds examined after
2, 4, 6, 8 and 10 days of infection ................................... 80
Table 3.10 Transmission of Alternaria brassicae in the field
by infected Brassica seeds in 1991 ................................. 80
Table 3.11 Transmission of Alternaria brassicae in the field
by infected Brassica seeds in 1992 ................................. 80
Table 3.12 Effect of seed treatment fungicides on seed-borne
infection of Alternaria brassicae and on subsequent
seedling infection in mustard .......................................... 81
Table 4.1 Summary of Alternaria brassicicola transcription
factor domains based on Pfam scans ............................... 92
Table 5.1 Weather parameters congenial for Alternaria brassicae
under (a) field and (b) laboratory conditions ................... 100
Table 5.2 Influence of temperature and leaf wetness
on infection of A. brassicae ............................................. 102
Table 5.3 Effect of age of radish plants on blight intensity
caused by Alternaria raphani .......................................... 109
Table 5.4 Effect of age of culture (Alternaria raphani)
on blight of radish ........................................................... 109
Table 5.5 Effect of inoculum (Alternaria raphani) load
on blight intensity in radish ............................................. 109
Table 5.6 Factors influencing resistance/susceptibility
of different cultivars of rapeseed–mustard
against Alternaria brassicae ............................................ 111
Table 5.7 Effect of barrier crop (oat) on the development
of Alternaria blight of rapeseed–mustard ....................... 112
Table 5.8 Prediction equations of two different growth models
and untransformed data and their comparable
factors at five different dates of sowing........................... 116
Table 5.9 Models to forecast different characters of Alternaria
blight in mustard crop along with coefficient
of determination and MAPE in different varieties .......... 118
Table 6.1 Physiological races of Alternaria brassicae .................... 126
Table 6.2 Reaction of different isolates of A. brassicae
on B. juncea host differentials ......................................... 128
Table 6.3 Incubation and latent period (in days)
of Alternaria brassicae isolates on host
differentials under controlled conditions ......................... 128
List of Tables xxix

Table 6.4 Differential reactions of different isolates

of A. brassicae when inoculated on a set
of host differentials .......................................................... 129
Table 6.5 Reaction of different isolates of Alternaria brassicae
on Brassica differentials .................................................. 130
Table 6.6 Pathogenic reaction of various isolates
of A. brassicae from India on selected
host differentials .............................................................. 131
Table 6.7 Pathogenic behaviour of different isolates
of Alternaria brassicae from India on a set
of host differentials .......................................................... 132
Table 6.8 Differences in morphological characters of different
isolates of Alternaria brassicae from India ..................... 136
Table 6.9 Conidial size of Alternaria brassicae from
different locations ............................................................ 138
Table 6.10 Sporulation index of Alternaria brassicae isolates
on different culture media ............................................... 140
Table 6.11 Conidial size of different geographical isolates
of A. brassicae ................................................................. 141
Table 6.12 Mycelial growth of A. brassicae under different
temperature and relative humidity conditions ................. 144
Table 6.13 Effect of various carbon sources on the radial
growth (cm) of various isolates of Alternaria brassicae
from Haryana, India ........................................................ 146
Table 6.14 Effect of various carbon sources on the sporulation
of various isolates of Alternaria brassicae from
Haryana, India ................................................................. 147
Table 6.15 Effect of various nitrogen sources on the radial
growth of different isolates of Alternaria brassicae
from Haryana, India ........................................................ 148
Table 6.16 Effect of various nitrogen sources on the sporulation
of different isolates of Alternaria brassicae
from Haryana, India ........................................................ 149
Table 6.17 Biochemical constituents (mg/g) in isolates
of Alternaria brassicae .................................................... 150
Table 6.18 Differential behaviour of various isolates
of Alternaria brassicae against fungicides ...................... 151
Table 6.19 Differential behaviour of various isolates
of Alternaria brassicae against neem products ............... 152
Table 6.20 Sensitivity of different isolates of Alternaria brassicae
collected from Haryana, India, to various plant extracts . 153
Table 6.21 Sensitivity of different isolates of Alternaria brassicae
from India to various fungicides ..................................... 155
Table 6.22 Differential behaviour of various isolates
of A. brassicae from India against different
fungicides ........................................................................ 156
Table 6.23 Variation in thermal sensitivity of different isolates
of Alternaria brassicae from Haryana, India .................. 157
xxx List of Tables

Table 6.24 Determinants of variability in Alternaria

infecting crucifers ............................................................ 158
Table 8.1 Carbohydrates, phenols and chlorophyll content
(mg/g dry weight) in leaves of Indian mustard
as influenced by Alternaria blight ................................... 168
Table 9.1 Per cent disease intensity (PDI) of Alternaria leaf
blight (A. brassicae) on different generations
of Brassica crosses .......................................................... 177
Table 9.2 Estimates of components of generation means
on three-parameter model for A. brassicae
on different crosses of oilseed Brassica .......................... 177
Table 9.3 Estimates of gene effects under six-generation
mean analysis for A. brassicae in different crosses
of oilseed Brassica .......................................................... 178
Table 9.4 Estimates of disease stress tolerance attributes
from the potential yield and yield under disease
stress environment (DSI = 0.195) in Indian mustard
(B. juncea) under normal date of sowing ........................ 179
Table 9.5 Estimates of disease stress tolerance attributes
from the potential yield and yield under disease
stress environment (DSI = 0.209) in Indian mustard
(B. juncea) under late date of sowing .............................. 179
Table 9.6 Components of Alternaria blight disease resistance
and yield of mustard (B. juncea) genotypes .................... 181
Table 9.7 Correlation coefficients (R) among different
components of Alternaria blight disease resistance
and yield of mustard (B. juncea) genotypes .................... 182
Table 9.8 Factors influencing resistance/susceptibility
of different cultivars of rapeseed–mustard against
Alternaria brassicae ........................................................ 182
Table 9.9 Specific activity of peroxidase enzyme in hypocotylar
calli of Brassica species raised on MS medium
supplemented with or without fungal culture
filtrate (FCF) of Alternaria brassicae ............................. 187
Table 9.10 Specific activity of enzyme catalase in hypocotylar
calli of Brassica species raised on MS medium
supplemented with or without fungal culture filtrate
(FCF) of Alternaria brassicae ......................................... 187
Table 9.11 Specific activity of enzyme polyphenol oxidase
in hypocotylar calli of Brassica species raised
on MS medium supplemented with or without fungal
culture filtrate (FCF) of Alternaria brassicae ................. 187
Table 9.12 Brassica germplasm holdings at different
organizations and research centres in the world .............. 192
Table 9.13 Sources of resistance to Alternaria brassicae
and Alternaria brassicicola ............................................. 193
List of Tables xxxi

Table 9.14 Classification of 38 Brassica coenospecies based

on the reaction to Alternaria brassicae under in vitro
and in vivo inoculation conditions .................................. 194
Table 9.15 Sources of multiple disease resistance
in oilseed Brassica........................................................... 195
Table 9.16 Relationship among major foliar diseases ....................... 196
Table 9.17 Sources of cruciferous genetic variability
in the world...................................................................... 198
Table 10.1 Isolated metabolites from Alternaria pathogenic
to crucifers ....................................................................... 215
Table 11.1 Chemicals tested against species of Alternaria
attacking Brassicaceae..................................................... 243
Table 11.2 Optimum time for spraying Difolatan
(2 g product/l water) for the control of Alternaria
leaf blight of raya at different locations
in 1979–1980 ................................................................... 245
Table 11.3 The efficacy and economics of fungicidal
spray on Alternaria leaf spot of raya at Hisar
in 1977–1978 ................................................................... 246
Table 11.4 Optimum growth stage of B. juncea for reducing
black spot through fungicidal sprays ............................... 246
Table 11.5 The effect of fungicide and insecticide mixture
on Alternaria leaf spot and aphid population
on Brown Sarson (BSH-1) at Hisar in 1977–1978 .......... 247
Table 11.6 The effect of Dithane M-45 and Ridomil MZ-72
on in vitro pollen germination (%), and tube
length (μm) in two cultivars of B. juncea ........................ 248
Table 11.7 Plant extracts tested against Alternaria spp.
attacking crucifers ........................................................... 249
Table 11.8 Effect of fungicidal seed treatment on plant
stand of mustard plants .................................................... 259
Table 11.9 Integrated disease management module
(seed treatment, spray schedule and fertilizer
doses for the control of DM, WR and AB) and its
significance in achieving higher yield of mustard
during 1999–2000 to 2001–2002 .................................... 260
Table 11.10 Some micronutrients as possible inducer for multiple
disease resistance in rapeseed–mustard........................... 261
Table 11.11 Effect of different chemicals, plant extracts
and bioagents on Alternaria leaf blight severity ............. 262
Table 11.12 Effect of different chemicals, plant extracts
and bioagents on Alternaria pod blight severity ............. 263
Table 11.13 Effect of different non-toxic chemicals,
plant extracts and bioagents on Alternaria
leaf blight severity ........................................................... 264
Table 11.14 The effect of different treatments on the initial
plant stand of Indian mustard .......................................... 265
xxxii List of Tables

Table 11.15 The effect of different treatments on Alternaria

blight severity (percent) in Indian mustard
90 days after sowing ........................................................ 265
Table 11.16 The effect of different treatments on Alternaria
blight severity (percent) in Indian mustard 120 days
after sowing ..................................................................... 266
List of Plates

Plate 2.1 Alternaria blight symptoms on various

rapeseed–mustard plant parts; (a) Alternaria infection
on cotyledons leaves; (b) Alternaria brown lesion
on cotyledons leaves; (c) Alternaria infection
on first leaves; (d) Alternaria spots on leaf ........................ 19
Plate 2.2 Alternaria blight symptoms on various rapeseed–mustard
plant parts; (a) Lesions increase in size and cover larger
area. (b) Concentric rings in the spot. (c) Alternaria
lesions on stem. (d) Severe Alternaria infection on the
lower portion of the plant ................................................... 20
Plate 2.3 Alternaria blight symptoms on various rapeseed–mustard
plant parts. (a) Alternaria infection on siliquae. (b) Deep
Alternaria infection on siliquae. (c) Severely infected
siliquae. (d) Alternaria-infected seeds (right) ................... 21

Plate 3.1 Light micrographs showing the development

of microsclerotium from Alternaria brassicae conidium:
(a) conidium, (b) initial stage in the formation of a
microsclerotium, (c) half-developed microsclerotium
with about 50 cells, (d) mature microsclerotium still
showing head and beak of the original conidium × 400,
(e) germination of a mature microsclerotium to form
hyphae × 400 and (f) germination of a frozen–thawed
microsclerotium showing many new conidia × 650........... 75
Plate 3.2 Scanning electron micrographs of the surfaces of
cabbage seeds inoculated with Alternaria brassicicola
and germinated on water agar for 24 h at 21 °C: (a)
germinated spores with collapsed germ tube and initials
of turgid aerial hyphae, (b) germinated spore with
collapsed germ tube and turgid aerial hyphae, (c) turgid
aerial germ tube with collapsed tip, (d) profuse develop-
ment of hyphae on damaged seed, (e) roughened hilum
area with broken vascular elements of the funiculus and
(f) spiral thickening of xylem vessels of the broken
vascular elements of the hilum shown in figure ................. 79

xxxiii
xxxiv List of Plates

Plate 4.1 Alternaria brassicae on leaves of Brassica rapa cv.

Candle. Bar = 5.0 μm: (a) light micrographs showing
germinating conidia (broad arrows) and subcuticular
hypha (SH) penetrating (asterisk) into epidermal cell (E)
(narrow arrows) of cuticle × 1200; (b) transmission
electron micrograph showing subcuticular hyphae (SH),
epicuticular wax layer (W) and electron-dense layer (ED)
of cuticle × 7000; and (c) light micrograph showing a
hypha penetrating through a stroma (narrow arrow)
and another one penetrating into a subepidermal
position (broad arrow) without becoming
subcuticular × 800 ............................................................... 90
Plate 4.2 Transmission electron micrographs of ultra-thin sections
of A. brassicae on the leaves of Brassica napus cv. Altex.
Bar = 2.0 μm: (a) a penetrating germ tube (broad arrow),
a subcuticular hypha (SH) and two other hyphae (H),
embedded in the cell wall of the host adaxial leaf
epidermal cell. Note the electron-dense material at the
point of penetration (narrow arrows), the layer of
epicuticular was (W) detached from the electron-dense
layer of the cuticle (ED) × 4200; (b) a penetrating germ
tube (broad arrow). Note the electron-dense material at
the point of penetration (narrow arrows), the detached
epicuticular wax layer (W) and electron-translucent layer
(EL) of the cuticle × 8050; (c) junctional region between
the two epidermal cells (E) showing a subcuticular
hyphae (SH) and glancing section of a hypha passing
through the cell wall (asterisk). The latter hypha is
penetrating between the two cells × 7400; (d) section
showing two subcuticular hyphae (SH), one of which is
penetrating into the cell wall of the epidermal cell
(E) × 4200; and (e) section showing subcuticular hyphae
(SH) a few other hyphae (H) in the epidermal cells and
one hypha (asterisk) below the epidermis close to the
palisade mesophyll tissue × 3000........................................ 91
Plate 6.1 Symptomatological variations of four isolates of
Alternaria brassicae on Brassica juncea. Fig. 1, RTK;
Fig. 2, BWL; Fig. 3, HSR; and Fig. 4, REW ..................... 134
Plate 6.2 Morphological variations in conidia of Alternaria
brassicae from India .......................................................... 137
Plate 6.3 Morphological variations in conidia of Alternaria
brassicae from Haryana (India) ......................................... 139
Plate 7.1 Conidiophores of Alternaria brassicicola showing
(a) uni- and binucleate cells and apical cell with pore and
annulus; (b) typical complement of nuclei in basal cells,
nucleate terminal cells (right and bottom), pore and
annulus (right) and nuclear material and cytoplasm
List of Plates xxxv

wedged in pore in terminal cell (left); (c) typical

complement of nuclei in subtending, basal and terminal
cells; (d) nucleus wedged in pore of terminal cell;
(e) developing conidia (only one shown) with
nucleus wedged in pore between terminal cell of
conidiophore and basal cell of developing conidium;
and (f, g) conidia with thick, roughened wall material
which stained intensely with Giemsa ................................. 164
Plate 7.2 Conidia of Alternaria brassicicola showing (a) small,
intensely staining nuclei of the terminal cells and the
wide pores of the basal cells; (b) septal pores, typical
distribution of nuclei and small, intensely staining
nucleus of the terminal cell; (c) Giemsa-stained material
wedged in septal pores between adjacent conidia and in a
septal pore within a conidium; (d, e) Giemsa-stained
material wedged in the septal pores between adjacent
conidia in a conidial chain; (f) Giemsa-stained material
wedged in the terminal pore of a conidium; and (g)
Giemsa-stained material wedged in basal and interstitial
pores of a conidium ............................................................ 165
Plate 9.1 Scanning electron micrograph of air-dried, osmium
vapour-fixed and gold-coated middle leaves of Brassica
rapa cv. Candle showing wax crystals. Bar = 1 µm ........... 183
Plate 9.2 Scanning electron micrograph of air-dried, osmium
vapour-fixed and gold-coated stem of Brassica rapa cv.
Tobin showing flat and erect wax crystals. Bar = 1 µm ..... 183
Plate 9.3 (a) Adaxial surface of an upper leaf of Brassica napus
cv. Altex showing platelike wax crystals (arrows); (b)
stem surface of cultivar Altex from the middle of a plant
showing rods (arrows); (c) adaxial surface of an upper
leaf of B. rapa cv. Tobin showing a fused rod (arrow);
(d) adaxial surface of an upper leaf of B. napus cv.
Westar showing fused rods (arrows) and growth rings in
wax crystals; (e) adaxial surface of an upper leaf
cv.Tobin showing filamentous wax crystals (arrows); and
(f) adaxial surface of a middle leaf cv. Tobin showing a
branched filamentous wax crystal (arrow). The plant
surfaces depicted in a-f were prepared for SEM by
air-drying method. Bar = 1 µm ........................................... 184
Plate 10.1 Symptoms caused by inoculation of Brassica napus cv.
Altex leaf with Alternaria brassicae (left leaf).
The right leaf is the control ................................................ 212
Plate 10.2 Symptoms caused by application of destruxin B on
Brassica napus cv. Altex leaf (left leaf). Compare
with Plate 10.1. The right leaf is the control ...................... 213
xxxvi List of Plates

Plate 11.1 Biocontrol of seed-borne Alternaria raphani and

A. brassicicola: (a) coiling of Trichoderma harzianum
ATCC 56678 hyphae around A. brassicicola mycelium.
Bar = 70 µm; (b) hyphae of T. harzianum 420
growing towards hyphae of A. raphani. Bar = 80 µm;
(c) parallel growth of hyphae of Fusarium sp. along
A. brassicicola hyphae. Bar = 25 µm; (d) scanning
electron micrographs of Chaetomium globosum hyphae
coiling around conidia of A. brassicicola. Bar = 35 µm;
(e) scanning electron micrographs of C. globosum
hyphae coiling around hyphae of A. brassicicola Bar = 7
µm; (f) scanning electron micrographs of C. globosum
hyphae coiling around A. raphani conidia. Bar = 25 µm;
and (g) reaction zones (arrows) of A. brassicicola
under the stimulus of C. globosum. Bar = 25 µm .............. 242
Plate 11.2 The effectiveness of antagonistic fungi against Alternaria
brassicicola; (a) Penicillium corylophilum-36 and
(b) Trichoderma harzianum-22 coiled around the conidia
on agar medium; (c) T. harzianum-22 coiled around a
conidium on a cabbage seed; and (d) P. citrinum
coiled around the conidial germ tube on a
cabbage root ....................................................................... 252
Plate 11.3 Excised non-dried rapeseed leaf inoculated with
Alternaria brassicae alone (left half of leaf) and with the
mixture of A. brassicae and Nectria inventa (right half of
leaf) at 1 week after inoculation. In the necrotic areas
(NA) caused by A. brassicae, note the presence of a
white mycelia mass of A. brassicae with conidia (AB) on
the left half and its absence on the right half of the leaf
with abundant conidia of N. inventa (NI). × 1.5 ................ 253
Plate 11.4 Phase contrast light micrographs of the conidia of
Alternaria brassicae parasitized by Nectria inventa:
(a) healthy-appearing conidium and infected mature
and juvenile conidia. Note non-infected cells (arrows)
in the heavily infected conidium. × 1,200; and
(b) profuse growth of parasitic hyphae around a host
conidium. × 1,200. Legend: P = parasite, C = host
conidiophore, MC = mature host conidium,
JC = juvenile host conidium .............................................. 254
Plate 11.5 Hyphae of Alternaria brassicae parasitized by Nectria
inventa: (a) light micrograph of parasite hyphae parallel
to a host hypha. Note the swollen appressorium-like
body of the parasite. × 4300; and (b, c) scanning
electron micrographs of the parasite coiling around
host hyphae. ×15,000. Legend: P = parasite, H = host,
AB = appressorium-like body of parasite,
HB = hyphal branch of the host.......................................... 255
List of Plates xxxvii

Plate 11.6 Scanning electron micrograph of mycoparasite Nectria

inventa hyphae growing on Alternaria brassicae;
(a) parasite hyphae occurring predominantly in the septal
area (arrows) and the basal portions of the germ tubes
(G) of a host conidium. × 1800; (b) appressorium-like
bodies (A) formed on the host conidium. Note the
presence of adhesive material under these bodies.
× 11,000; and (c) appressorium-like body (A) with
fibrous adhesive material (arrow) formed on host
hypha (H). × 25,000 ........................................................... 256
Plate 11.7 The Alternaria brassicae–Nectria inventa host–parasite
interface; (a) Mature conidium of A. brassicae penetrated
by hyphae of N. inventa. Note collapsed cell wall of
the conidium (arrow) × 1.800; (b) enlarged view of
penetration site. A large hole develops in the wall of the
host cell, and a meshwork of material appears at the
penetration site (arrow) × 27,000; (c) penetration of a
juvenile conidium (JC) of A. brassicae by N. inventa (P).
The host conidium is penetrated through the basal pore.
MC, infected cell of a mature conidium. CP, conidio-
phore produced by the mature conidium × 2500;
(d) light micrograph of a thin section of a normal
juvenile conidium of A. brassicae showing the basal pore
and septal pore (arrow) × 2800; and. (e) Scanning
electron micrograph of a basal pore in a juvenile
conidium of A. brassicae × 3500 ....................................... 257
 Introduction
1

1.1 Status of Genus Alternaria Alternaria was originally described by Nees

(1816) based on A. tenuis as the only species.
Alternaria is an ubiquitous fungal genus that Characteristics of the genus included the pro-
includes saprobic, endophytic and pathogenic duction of dark-coloured phaeodictyospores in
species. It is associated with a wide variety of chains and a beak of tapering apical cells. Von
substrates including seeds, plants, agricultural Keissler (1912) synonymized both A. tenuis and
products, animals, soil and atmosphere. Species Torula alternata (Fries 1832) with Alternaria
of Alternaria are known as serious plant patho- alternata, due to ambiguities in Nees’ descrip-
gens, causing major losses on a wide range of tion of A. tenuis. Description of two additional
crops. There are over 4000 Alternaria/host asso- genera, Stemphylium (Wallroth 1833) and
ciations recorded in the USDA Fungal Host Ulocladium (Preuss 1851) in phaeodictyosporic
Index ranking the genus 10th among nearly 2000 hyphomycetes, further complicated the taxo-
fungal genera based on the total number of host nomic resolution in this group of fungi. Several
records. While very few Alternaria species redescriptions and revised criteria of these gen-
appear to have a sexual stage (Pleospora) to their era (Saccardo 1886; Elliot 1917; Wiltshire 1933,
life cycle, the most lack sexuality altogether. 1938; Joly 1964) resulted in a growing number
Several species of Alternaria with their taxo- of new species. Results of lifetime study on
nomic and morphological characteristics, patho- Alternaria taxonomy based upon morphological
genic nature, and synonymous along with characteristics were summarized in a classical
distribution have been described extensively identification manual by Simmons (2007), in
(Elliott 1917; Neergaard 1945; Ellis 1971, 1976; which 275 Alternaria species were recognized.
Simmons 1967, 2002, 2007). One species was transferred to the genus
Several taxa are also important postharvest Prathoda and three new genera, Alternariaster,
pathogens and causative agents of phaeohypho- Chalastospora and Teretispora, were segregated
mycosis in immunocompromised patients or air- from Alternaria. Molecular studies revealed
borne allergens. Because of the significant multiple non-monophyletic genera within the
negative health effects of Alternaria on plants Alternaria complex, and Alternaria species
and their surroundings, a correct and rapid iden- clades, which do not always correlate to species
tification of Alternaria species would be of great group upon morphological characteristics
value to agriculturists, researchers, medical described (Pryor and Gilbertson 2000; Chou and
mycologists and the public alike (Woudenberg Wu 2002; de Hoog and Horre 2002; Pryor and
et al. 2013). Bigelow 2003; Hong et al. 2005; Inderbitzin

© Springer Science+Business Media Singapore 2016 1

G.S. Saharan et al., Alternaria Diseases of Crucifers: Biology, Ecology and Disease Management,
DOI 10.1007/978-981-10-0021-8_1
2 1 Introduction

et al. 2006; Pryor et al. 2009; Runa et al. 2009; 1.2 Brassica Crops
Wang et al. 2011; Lawrence et al. 2012). The A.
alternata, A. brassicicola, A. infectoria, A. porri Brassica oilseed crops occupy over 29.39-million
and A. radicina species group were strongly sup- hectares of the world’s agricultural lands with
ported by these studies; two new species groups, 53.01 mt total production yielding on an average
A. sonchi (Hong et al. 2005) and A. alternan- of 1700 kg/ha. Their ability to survive and grow
therae (Lawrence et al. 2012), and three new at low temperatures enables the oilseed Brassica
genera, Crivellia (Inderbitzin et al. 2006), to be cultivated successfully in cool agricultural
Undifilum (Pryor et al. 2009) and Sinomyces regions, at high elevations and as winter crops in
(Wang et al. 2011), were described. The latest the subtropics. The small round seeds of Brassica
molecular revision of Alternaria (Lawrence oilseed crops contain over 40 % oil on a dry
et al. 2013) introduced two new species groups, weight basis and, after the extraction of oil, pro-
A. panax and A. gypsophilae, and elevated eight vide a meal containing over 40 % high-quality
species groups to sections within Alternaria. protein. In the Western countries, the meal is uti-
The sexual phylogenetic Alternaria lineage, the lized exclusively as a feed for livestock and poul-
A. infectoria species group, did not get the status try, but in many Asian countries, it is used as an
of section, in contrast to the eight asexual phylo- organic fertilizer for field crops.
genetic lineages. The Alternaria complex cur- The Brassicaceae family, to which the genus
rently comprises the genera Alternaria, Brassica belongs, contains approximately 3500
Chalastospora (Simmons 2007), Crivellia, species in 350 distinct genera of many important
Embellisia, Nimbya, Stemphylium, Ulocladium, crop plants yielding high-quality, edible and
Undifilium and the recently described Sinomyces industrial oils, common vegetables and weeds.
together with eight sections of Alternaria and Based on the evidence that some vegetable types
the A. infectoria species group. were in common use in the Neolithic age (Chang
Woudenberg et al. (2013) conducted a study to 1968; Hyams 1971), and direct reference to oil-
delineate the phylogenetic lineages within seed rape and mustard in the ancient Indian
Alternaria and allied genera to create a robust Sanskrit writings of 2000–1500 BC (Singh 1958),
taxonomy. Phylogenetic inferences were drawn the Brassica vegetables and oilseeds may well
on sequence data of parts of the 18S nrDNA have been among the earliest plants domesticated
(SSU), 28S nrDNA (LSU), the internal tran- by man. Greek, Roman and Chinese writings of
scribed spacer regions 1 and 2 and intervening 500–200 BC also mention these crops and their
5.8S nrDNA (ITS), glyceraldehydes-3-phosphate medicinal value (Prakash and Hinata 1980).
dehydrogenase (GAPDH), RNA polymerase sec- Oilseed rape was introduced in China and Japan
ond largest subunit (RPB2) and translation elon- around the time of Christ (Hougen and Stefansson
gation factor 1-alpha (TEF1) gene regions of 1983). Although its cultivation began in the thir-
ex-type and reference strains of Alternaria spe- teenth century in Europe, its industrial use was
cies and all available allied genera. not widespread until its superior qualities as lubri-
The most common and destructive diseases of cant oil were recognized (Shahidi 1990). Its use as
Brassicaceae crops worldwide are those caused an edible vegetable oil in Western countries is
by four species of Alternaria, viz. A. brassicae very recent. Unlike most other oilseeds, rapeseed
(Berk.) Sacc., A. brassicicola (Schwein.) Wiltsh., comes from several species of genus Brassica
A. raphani Groves and Skolko and A. alternata (Shahidi 1990) including B. napus L., B. rapa L.
(Fr.) Keissl. Although attack by Alternaria at the (B. campestris L.) and B. juncea (L.) Czern. &
seedling stage can lead to death of young plants, Coss., which are known as rape, turnip rape and
infection on leaves, stems and siliquae generally leaf mustard, respectively. Common names for B.
results in heavy losses in seed yield and quality napus are rape, rapeseed, oil rape, colza, oilseed
(Table 1.1). rape, swede rape and Argentine rape; for B. rapa
1.2 Brassica Crops 3

Table 1.1 Yield losses in crucifer crops due to Alternaria diseases in different countries of the world
Crop/cultivar Country (location) Yield losses (%) References
Rapeseed Germany 75 Klemm (1938), Raabe (1939)
Rapeseed Germany 20–50 Daebeler et al. (1986)
Rapeseed England 60 Smith et al. (1988)
Rapeseed Lithuanian 37–100 Brazauskiene and Petraitiene
(2006)
Brassica napus Canada 42 Degenhardt et al. (1974)
Brassica napus India (Kangra) 17 Kumar (1997)
Brassica rapa Canada 70 Degenhardt et al. (1974)
Brassica carinata India (Kangra) 11 Kumar (1997)
Brassica juncea cv. Prakash India (Hisar) 18 Saharan (1984)
Brassica juncea cv. Varuna India (Hisar) 17 Saharan (1984)
Brassica juncea cv. Varuna India (Pantnagar) 34 Saharan (1984)
Brassica juncea cv. RLM-514 India (Ludhiana) 49 Saharan (1984)
Brassica juncea cv. Varuna India (Kanpur) 21–22 Prasad et al. (2003)
Brassica juncea cv. Varuna Nepal 32–57 Shrestha et al. (2005)
Brassica campestris var. India (Hisar) 26 Saharan (1984)
Brown Sarson cv. BSH-1
Brassica campestris var. India (Hisar) 35 Saharan (1984)
Yellow Sarson cv. YSPb-24
Brassica campestris var. India (Pantnagar) 45 Saharan (1984)
Yellow Sarson cv. YS-151
Brassica campestris var. India (Kangra) 28 Kumar (1997)
Yellow Sarson
Eruca sativa India 56 Jain (1992)
Cabbage, cauliflower and USA 50 Ramsey and Smith (1961)
broccoli
Cauliflower Germany 50 Stoll (1948)
Cabbage Germany 70–90 Domsch (1957)
Cabbage Germany 80–100 Gorshkov (1976)
Brassica oleracea England 80 Smith et al. (1988)
Radish India 18 Suhag et al. (1983)

are rapeseed, oil turnip and Polish rape; and for B. A. Braun (Abyssinian mustard, Ethiopian mustard),
juncea are brown mustard, oriental mustard, B. tournefortii Gouan (wild turnip), Eruca sativa
Indian mustard and rapeseed. In China, all three Mill. (E. vasicaria spp. sativa (Mill.) Thell.) (gar-
species are grown, but winter-grown rape consti- den rocket, taramira), Camelina sativa Crantz.
tutes the major source of rapeseed. In India, turnip (false flax, Dutch flax, gold-of-pleasure), Crambe
rape and mustard may be considered rapeseed, abyssinica Hochst. ex. O.E. Schulz and C. hispanica
and in North America and Europe, rape and turnip L. The genome designations of varietal or subspe-
rape are regarded as rapeseed. The other oilseed cific taxa of agriculturally important brassicas and
crops of the Brassicaceae include B. rapa L. var. radish are given in Table 1.2 (Anonymous 1985).
toria (turnip rape, toria), B. rapa L. var. Brown The traditional varieties of rapeseed that are
Sarson (turnip rape, Brown Sarson), B. rapa L. being grown in the Asian countries contain 22–60 %
var. Yellow Sarson (turnip rape, Yellow Sarson), erucic acid in their oil and high percentage of gluco-
B. nigra (L.) Koch (black mustard), B. hirta Moench sinolate in their defatted meal. The presence of eru-
(Sinapis alba L.) (white mustard), B. carinata cic acid compromises the nutritional value of oil,
4 1 Introduction

Table 1.2 Genomic designations of varietal or subspecific taxa of agriculturally important Brassicas and radish
(Anonymous 1985)
Brassica sp. (n) spp. or var. 2n genome descriptor Common name
nigra (8) – bb Black mustard
oleracea (9) – cc Cole crops
acephala cc. a Kales
alboglabra cc.al Chinese kale, kai-lan
botrytis cc.b Cauliflower, heading
broccoli
capitata cc.c Cabbage
costata cc.co Portuguese cabbage
gemmifera cc.g Brussels sprouts
gongylodes cc.go Kohlrabi
italic cc.i Broccoli, calabrese
medullosa cc.m Marrow stem kale
palmifolia cc.p Kale (Jersey kale)
ramosa cc.ra Thousand-head kale
sabauda cc.s Savoy cabbage
sabellica cc.sa Collards
selensia cc.se Borecole
campestris (10) syn. rapa – aa –
chinensis aa.c Pak choi
narinosa aa.na –
nipposinica aa.n –
oleifera aa.o Turnip rape, toria
parachinensis aa.pa Choy sum
pekinensis aa.p Chinese cabbage, pechay
perviridis aa.pe Tender green, komatsuna,
mustard spinach
rapifera aa.r Turnip
trilocularis aa.t Sarson
utilis aa.u –
carinata (17) – bbcc Ethiopian mustard
juncea (18) – aa.bb –
capitata aabb.c Head mustard
crispifolia aabb.cr Cut leaf mustard
faciliflora aabb.f Broccoli mustard
lapitata aabb.l Large petiole mustard
multiceps aabb.m Multishoot mustard
oleifera aabb.o Oilseed mustard, raya
rapifera aabb.r Root mustard
rugosa aabb.ru Leaf mustard
spicea aabb.sp Mustard
tsa-tsai aabb.t Big stem mustard
napus (19) – aacc Fodder rape
oleifera aacc.o Oil rape
rapifera aacc.r Swede, rutabaga
(continued)
1.3 The Disease 5

Table 1.2 (continued)

Brassica sp. (n) spp. or var. 2n genome descriptor Common name

Raphanus sativus (9) – rr Radish
radicola rr.r Radish, daikon
oleifera rr.o Oil radish
caudatus rr.c Rat tail radish

and glucosinolate reduces the feeding value of the Alternaria species cause symptoms in the seed-
meal. Since the late 1970s, both B. napus and B. ling stage on cotyledons and in the adult stage on
rapa Canadian varieties have been genetically mod- leaves, leaf petiole, stem, inflorescence, siliquae
ified to contain low erucic acid and glucosinolates and seeds. In general, symptoms are similar on
and named these ‘double-low’ cultivars as ‘canola’ all infected host species in the form of lesions.
in 1979. The term canola thus refers to a rapeseed There may be variations in shape, size, colour,
cultivar that contains less than 30 μmol/g of one or formation of concentric rings, yellow halo around
any combination of the four known aliphatic gluco- the lesions under different agro-ecological zones,
sinolates (gluconapin, progoitrin, glucobrassi- host genotypes, nutritional status of soil and
canapin and napoleiferin) in its defatted meal, and pathotypes involved. In rapeseed, effects on cell
less than 2 % of the fatty acyl content of the oil is membrane, chloroplast and mitochondria have
erucic acid. Canada recently has also developed been recorded (Verma and Saharan 1994; Tewari
canola-type B. juncea cultivars. 1991b). The disease is known to be distributed all
The main groups of cultivated Brassica vegeta- over the world wherever host pathogens interact
bles are kales (B. oleracea L. var. acephala) includ- under suitable environmental conditions. Host
ing kitchen kale, green kale, dwarf Siberian kale, range of all the four Alternaria species is very
narrow stem kale, collards and trochunda; cab- wide infecting oilseed Brassica, cruciferous veg-
bages (B. oleracea L. var. captitata, var. sabauda, etables, wild cruciferous hosts and weeds.
var. bullata) including headed cabbages, Brussels Quantitative and qualitative losses in yield of oil-
sprouts and savoy cabbage; kohlrabi (B. oleracea seeds and vegetables range from 11 to 100 %
L. var. gongylodes); inflorescence kales (B. olera- (Table 1.1) depending upon time of infection,
cea L. var. botrytis, var. italica) including cauli- prevailing environmental conditions after infec-
flower, broccoli and sprouting broccoli; branching tion and strategies used for its control (Verma and
bush kales (B. oleracea L. var. fruticosa) including Saharan 1994; Czyzewska 1969; Kadian and
cow kale, borecole, thousand-headed kale); Saharan 1983, 1984; Kolte 1985; Tewari and
Chinese kale (B. alloglabra L.) (Snogerup 1980); Conn 1993; Seidle et al. 1995; Kumar 1997;
and radishes (Raphanus sativus L.). Gupta et al. 1998; Meah et al. 2002; Prasad et al.
Crambe is a newly emerging oilseed crop with 2003; Mondal et al. 2007; Brazauskiene and
increasing commercial acreage in the USA, Petraitiene 2006; Smith et al. 1988; Gorshkov
Poland and some other countries of the world. 1976; Stoll 1948; Domsch 1957). The disease
Crambe oil is a potential raw material for rubber has been assessed using several methods includ-
and plastic industries. The prospective species ing descriptive keys (Mayee and Datar 1986;
for crambe cultivation are Crambe abyssinica, C. Saharan 1991; Horsfall and Barratt 1945; Fontem
hispanica and some other annuals. et al. 1991; Redman et al. 1967), standard area
diagrams (Conn et al. 1990; James 1974), inci-
dence–severity relationships (Seem 1984), inoc-
1.3 The Disease ulum disease intensity relationships (Saharan and
Kadian 1983b; Dueck and Degenhardt 1975),
The disease is known by more than 40 names in infection type and host reaction as resistant and
the world on the basis of symptoms produced on susceptible (Krishnia et al. 2000a) and disease
different parts of cruciferous crops. All the four stress tolerance attributes (Gupta et al. 2002).
6 1 Introduction

1.4 The Pathogen lation of all the three species is 6–8. Maximum
growth and sporulation of A. brassicae occurs
Alternaria is an ubiquitous fungal genus that with alternating light and darkness (Ansari et al.
includes saprobic, endophytic and pathogenic spe- 1989; Verma and Saharan 1994; Taber 1964).
cies. Four species of Alternaria, viz. A. alternata, The pathogen survives and perpetuates through
A. brassicae, A. brassicicola and A. raphani are infected seeds, diseased plant debris and patho-
pathogenic on cruciferous crops. Alternaria bras- gen propagules in the soil and other crucifers/
sicae is a major pathogen of oil-yielding Brassica, weed hosts in a particular agroecosystem (Chupp
while other three are more common on vegetable and Sherf 1960; Dixon 1981; Ellis 1968a, b;
crops. As early as 1836, Berkeley identified the Verma and Saharan 1994). Optimum temperature
causal fungus on Brassicaceae as Macrosporium for spore germination is 20–25 °C at 90 % or
brassicae, which was later renamed as A. brassi- more relative humidity. Continuous light
cae. Phylogeny, taxonomy, morphology, classifi- completely inhibits sporulation (Verma and
cation, infection process, identification Saharan 1994; Singh and Suhag 1983). All four
characteristics and synonyms have been well doc- species of Alternaria are seed borne in crucifers.
umented (Verma and Saharan 1994; Woudenberg The pathogen has been detected from all the parts
et al. 2013; Simmons 2007; Thomma 2003; Ellis of infected seeds. Its transmission through seed is
1968a, b, 1971; Neergaard 1945). Identification, very high under congenial temperature condi-
cloning and sequencing of virulence genes of tions (Atkinson 1950; Vannacci and Pecchia
Alternaria species infecting crucifers will resolve 1988; Sivapalan and Browning 1992; Kubota
many doubts about their relationship with cruci- et al. 2006; Shrestha et al. 2000). The disease
fer’s hosts (Cramer et al. 2006; Jasalavich et al. cycle starts from the primary infections initiated
1995; Kim et al. 2007). Pathogenicity factors and directly from infected seed, spores on crops resi-
transcription factor Amr1 have been identified in dues and on cruciferous hosts, weeds or possibly
A. brassicicola (Mamgain et al. 2013; Cho et al. from microsclerotia and chlamydospores pro-
2012). A non-ribosomal peptide synthase gene duced on infected debris. Spores are produced
(AbNPS2) is important for cell wall integrity, abundantly in wet weather and are dispersed
conidial viability and virulence of aged spores of locally by rain splash and wind. Under congenial
A. brassicicola (Kim et al. 2007). More than 100 weather conditions, lesions develop and produce
genes have been functionally analysed through windborne spores, which cause secondary infec-
various techniques like gene knockout and overex- tions during crop season. The cycle continues
pression making A. brassicicola the species of throughout the season when conditions are
choice for functional genomic research (Oide et al. favourable to infect seed and other parts of plants,
2006; Cho et al. 2006, 2007, 2009; Kim et al. which become the source of survival of the patho-
2007; Mamgain et al. 2013). gen (Kolte 1985; Saharan 1992; Verma and
Twenty-four liquid and solid media for growth Saharan 1994; Mehta et al. 2005).
and sporulation of Alternaria species pathogenic The process of infection and pathogenesis of
on crucifers have been reported (Verma and four Alternaria species on cruciferous hosts has
Saharan 1994); Alternaria brassicae, A. brassici- been very well understood. The role of external
cola and A. raphani grow well on most carbon and internal factors during host–pathogen inter-
sources (Taber et al. 1968). The optimum tem- actions, enzymes, toxins and genes governing
peratures for growth in culture of A. brassicae, A. pathogenesis has been determined. Identification
brassicicola and A. raphani are between 20 and of A. brassicicola genes, AbVF 19 and Amr1,
25 °C along with 95–100 % relative humidity for makes this pathogen as an efficient and success-
good sporulation (Ansari et al. 1989; Taber et al. ful facultative parasite of crucifers (Verma and
1968; Changsri and Weber 1960, 1963). The Saharan 1994; Cho et al. 2007, 2009, 2012; Giri
optimum pH requirement for growth and sporu- et al. 2013; Mamgain et al. 2013).
1.6 Pathogenic Variability 7

1.5 Epidemiology cidal sensitivity. Initial observations on variations

and Forecasting in cultural characteristics and pathogenesis of
different isolates of Alternaria were made by
Alternaria blight of crucifers develops in epi- Stoll (1952), in A. brassicicola–vegetables, by
demic form when temperature ranges from 15 to Van Schreven (1953) in A. brassicae–Brassica
25 °C, relative humidity is >90 % and wind veloc- and by Atkinson (1953) in A. raphani–radish–
ity is 2–5 km/h and during the presence of inter- host pathosystem. Alternaria alternata strains
mittent rains (Ansari et al. 1988; Saharan 1991; showed differences in their physiological and
Awasthi and Kolte 1989b; Verma and Saharan pathological characteristics isolated from
1994; Yadav and Brar 2003). Closer spacing crambe. Strain B is most virulent, strain A is
(30 × 15 cm), high doses of nitrogen (80 Kg N/ha) moderately virulent, and strain C is least patho-
and frequent irrigation rapidly increase severity of genic on crambe (Czyzewska 1969, 1971). On
disease in rapeseed–mustard (Saharan 1991; rapeseed–mustard group of crops, three races of
Stankova 1972; Verma and Saharan 1994). A. brassicae, viz. RM-1, RM-2 and V-3, are viru-
Various models have been developed to measure lent. Race RM-1 isolated from rapeseed–mustard
the temporal progression of disease under field is avirulent on B. oleracea var. capitata, Race
conditions when crops are sown on different dates RM-2 isolated from B. rapa is avirulent on both
under varied environmental conditions. Disease B. oleracea var. capitata and B. oleracea var.
forecasting models have also been developed botrytis, and Race V-3 is most virulent infecting
(Verma and Saharan 1994; Awasthi and Kolte all the test host differentials. This race was iso-
1994; Dang et al. 2006; Magarey et al. 2005; lated from vegetable host species including rad-
Mehta et al. 2002, 2008; Kumar et al. 2013; ish, cabbage and cauliflower (Saharan and Kadian
Mahapatra and Das 2014; Mehta 2014) after tak- 1983a). Thirteen isolates of A. brassicae tested
ing into account the leaf wetness period, mini- on selected cultivars of winter rape differed in
mum and maximum temperature, relative their virulence (Mridha 1983). Stoll (1952) char-
humidity, date of sowing, crop age and variety acterized three pathotypes of A. brassicae infect-
and species of Brassica crops under different ing siliquae of cauliflower as highly aggressive,
agro-ecological conditions. Models based on less aggressive and non-pathogenic. Atkinson
weekly weather data beginning from week of (1953) classified A. raphani isolates in to two
sowing till up to 6 weeks of crop growth can be races as ‘wild type’ and ‘variant type’. Three iso-
used for reliable forewarning of Alternaria blight; lates of A. brassicae designated as A, C and D
reliable forewarning regarding the crop age at first differ in their morphology, growth, sporulation
appearance of disease, peak severity of disease and cultural characteristics along with virulence
and maximum severity of disease in different crop on B. carinata (Kolte et al. 1989, 1991; Awasthi
varieties is possible (Kumar et al. 2013). and Kolte 1989a; Vishwanath and Kolte 1997).
Four pathotypes of A. brassicae from B. juncea
were identified and designated as Bj-4, Bj-5, Bj-6
1.6 Pathogenic Variability and Bj-7 by Gupta et al. (2004), on the basis of
host differentials and symptomatological varia-
Pathogenic variability in the form of pathotypes/ tions. Pathotypes DLK, RSR-1 and GDP of A.
races/strains/variants has been identified in four brassicae were identified by Mehta et al. (2003).
species of Alternaria causing blight and black Some researchers have mentioned only the num-
spot disease in crucifers. The pathogenic vari- ber of isolates exhibiting differential reactions
ability in Alternaria species is governed by deter- without designating pathotypes (Kumar et al.
minant attributes, viz. pathological, 2003; Mehta et al. 2003; Sangwan and Mehta
symptomatological, morphological, cultural, 2007). In the absence of host differentials, other
nutritional, biochemical, genetical, molecular parameters have been used to identify pathogenic
and proteome level and both thermo- and fungi- variability in Alternaria species infecting crucifers.
8 1 Introduction

Out of these criteria, symptomatology (Gupta intraspecific crosses of B. juncea and B. carinata
et al. 2004; Kolte et al. 1991; Goyal et al. 2013), to A. brassicae is governed by additive genes,
morphology (Kolte et al. 1989, 1991), genetics dominant genes, additive x additive-type epistatic
(Sharma et al. 2013; Priyanka et al. 2014) and genes, additive x dominance and dominance x
proteome level have been correlated with viru- dominance type of non-allelic interaction genes
lence of pathotypes. (Singh and Singh 1989; Krishnia et al. 2000b).
Inter-mating between resistance plants helps in
increasing the level of resistance against A.
1.7 Fine Structures brassicae through pyramiding of resistant genes.
High level of horizontal resistance in genotypes
Fine structures of A. brassicicola have been stud- of oilseed Brassica has been recorded (Saharan
ied through electron microscopy. The sequence and Kadian 1983b; Saharan and Krishnia 2001).
of events in the production and maturation of Brassica genotypes, Rajat, Kranti, RH-781 and
spores has been described. The changes in the RL-1359, have been identified with disease toler-
internal organelles of hyphae, conidia and conid- ance attributes (Gupta et al. 2002). Genotypes
iophore have been observed. Conidiophores have PR-8988 and PR-9024 show higher degree of
a similar structure to mature hyphae, except that partial resistance or slow blighting (Kumar and
after spore production, they have a pore in the tip Kolte 2001). Epicuticular wax (Candle, Tobin,
and an annule. There are variations in the number Altex, Midas, Tower) and low number and nar-
of nuclei in the cells of vegetative hyphae, conid- row stomatal aperture (Tower, RC-781) provide
iophores and conidia with six chromosomes in resistance to Alternaria infection in Brassica
dividing nuclei of vegetative hyphae (Campbell species (Saharan and Kadian 1983b; Conn et al.
1970a, b, 1972; Knox-Davis 1979). 1984; Tewari 1991a, b). The concentration of
phenolic compounds, activation of polyphenol
oxidase and catalase are higher in tolerant geno-
1.8 Biochemistry type of mustard (Gupta et al. 1990; Kiran et al.
2002). Chitinase-modifying proteins, Cmps, are
During Alternaria–crucifers host–pathogen secreted by fungal pathogens of crucifers, which
interactions, a number of biochemical changes interfere with fungalysin Cmp activity to improve
take place in the host as well as in the pathogen. plant resistance to multiple fungal diseases
These biochemical changes produce various (Naumann and Wicklow 2013). GLIP1 in asso-
kinds of primary and secondary metabolites, ciation with ethylene signalling may be a critical
which influence the host defence system and component in plant resistance (A. thaliana) to A.
pathogen virulence. Alternaria brassicicola pro- brassicicola (Oh et al. 2005). Brassica juncea
duces compounds like anti-tumouric depudecin, plants transformed with chitinase gene tagged
antibiotic complex brassicicolin and phytotoxic with an overexpressing promoters, 35S CaMV,
brassicicenes. The production of glucosinolates give defence response by degrading the cell walls
and phytoalexins has been correlated with host of invading fungi (Mondal et al. 2003). Increased
resistance (Verma and Saharan 1994; Atwal et al. level of PAL, PPO and peroxidase plays an
2003; Mathpal et al. 2011; Sharma et al. 2010; important role in the defence mechanisms of B.
Doughty et al. 1996; Jung et al. 2002). juncea genotypes against Alternaria pathogene-
sis (Parihar 2012). Treatment by β-aminobutyric
acid leads to proper balance of oxidant and anti-
1.9 Resistance oxidants suitable for expression of resistance in
B. carinata against A. brassicae by curtailing
Host resistance in crucifers against Alternaria pathogens ingress during early stages of coloni-
species has various components and it is multi- zation (Chavan et al. 2013). Zeatin, a cytokinin,
layered. Inheritance of resistance in inter- and up-regulates plant immunity via an elevation of
1.11 Disease Management 9

MAPK-4 and antagonizes the effects of A. bras- spite of several bottlenecks in the development of
sicae (Marmath et al. 2013a, b). Transgenic resistance cultivars, various methods/techniques
expression of hevein, the rubber tree lectin in B. including conventional as well as biotechnological
juncea cv. RLM-198, confers protection against approaches are being utilized to incorporate desired
A. brassicae (Kanrar et al. 2002); β-aminobutyric traits in cruciferous crops against Alternaria dis-
acid pretreatment of B. juncea plants induces A. ease (Saharan et al. 2003; Nowicki et al. 2012;
brassicae resistance mediated through an Aneja and Agnihotri 2013).
enhanced expression of pathogenesis-related pro-
tein genes, independent of SA and JA accumula-
tion (Kamble and Bhargava 2007). Hypersensitive 1.10 Phytotoxins
response gene (hsr 203J) like homologues of
Brassica plays important role in differential Alternaria species pathogenic to crucifers
defence response against A. brassicae (Mishra produce host-specific and non-host-specific
et al. 2010). The cDNA encoding Pm-AMP1 has toxins, which facilitate their pathogenic process
been successfully incorporated into the genome to become successful pathogen. Prior to
of B. napus, and its in planta expression confers colonization, necrotrophs must kill their host
greater protection against A. brassicae. A cells at a distance by producing both toxins and
cysteine-rich antimicrobial peptide, Pm AMP1, lytic enzymes often by triggering genetically
was isolated from Pinus monticola (Verma et al. programmed apoplastic pathways or by directly
2012). Combined expression of a barley class II causing cell damage resulting in necrosis.
chitinase and type I ribosome-inactivating pro- Alternaria brassicae and A. brassicicola
tein in transgenic B. juncea provides resistance pathogenic to crucifers produce a number of
against A. brassicae (Chhikara et al. 2012). toxins and metabolites belonging to chemical
Transcriptional responses to exposure to the bras- groups containing terpenoids, pyranones, steroids
sicaceous defence metabolites camalexin and and nitrogen. Effects of toxins on plants at
allyl isothiocyanate in A. brassicicola have been physiological, biochemical and molecular level
recorded (Sellam et al. 2007). have been investigated. The role of toxins in the
Elicitation and accumulation of phytoalexins in process of infection, their biosynthesis, mode of
crucifers after exposure to Alternaria and their role action, chemical structure, role in host defence
in disease resistance have been demonstrated and transformation into phytoalexins has been
(Verma and Saharan 1994). Calcium sequestration understood (Verma and Saharan 1994; Bains and
property of A. brassicae can be used in enhancing Tewari 1989; Buchwaldt and Green 1992; Lou
resistance to this pathogen in rapeseed by soil or et al. 2013; Chen et al. 2005; Li et al. 2008;
foliar application of calcium compounds (Tewari Rotem 1994; He et al. 1998; Pedras et al. 1998,
1991a, b). Numerous sources of resistance to 2001, 2002, 2009; Thomma 2003; Marmath et al.
Alternaria species have been identified from differ- 2013a, b).
ent Brassica species and their near and distantly
related coenospecies, but very few have been uti-
lized to develop resistant cultivars (Verma and 1.11 Disease Management
Saharan 1994; Saharan 1992, 1997; Saharan et al.
2003; Sharma et al. 2002). Sources of multiple dis- Common cultural practices like the use of clean,
ease resistance in B. juncea, B. rapa, B. carinata bold, healthy and treated seed of recommended
and B. napus have also been identified. Reports of cultivars, long crop rotation (3–4 years), sanita-
strong and positive correlation in the increased tion, weed control, shallow (2-cm depth) planting
level of resistance against different Brassica patho- at recommended time, use of balanced nutrients,
gens will facilitate improvement in accumulation proper plant density (45 × 30 cm), proper
of resistance to multiple disease resistance (Kumar drainage in the field, plant debris management,
and Saharan 2002; Mitchell-olds et al. 1995). In use of tolerant/resistant cultivars, application of
10 1 Introduction

chemicals/bioagents at a proper time with Tsuneda and Skoropad 1980; Danielsson et al.
adequate foliage coverage and education of farm- 2006; Sharma et al. 2010). The use of resistant
ers about the importance of appropriate practices cultivars is the most easy, economical, environ-
have been advocated to manage Alternaria mental friendly and safest way of plant disease
diseases of Brassica crops (Verma and Saharan control. However, worldwide efforts are being
1994; Kolte 1985; Saharan and Chand 1988; made to develop and release resistant cultivars of
Saharan 1992, 1997; Peruch and Michereff crucifers to mange Alternaria disease (Verma
2007). Seed treatment at 50 °C for 20 min is and Saharan 1994; Saharan et al. 2003).
highly effective in controlling the seed-borne Integration of all plant disease control strate-
inoculum. Numerous chemicals and bioagents gies, viz. cultural, chemical, biological, nutri-
have been recommended to control seed-borne tional, host resistance, biotechnological and
infection of Alternaria (Verma and Saharan genetic engineering including pest management,
1994; Vannacci and Harman 1987; Latif et al. is the best way to manage Alternaria disease of
2006). In vitro and in vivo testing of a large num- crucifers (Verma and Saharan 1994; Saharan and
ber of chemicals against Alternaria species Mehta 2002; Kolte 2005; Mehta 2014).
infecting crucifers has been found very effective
in controlling the disease and increasing the yield
under field conditions. The number of sprays, 1.12 Techniques
optimum doses, optimum crop growth stage,
spraying time, compatibility of different fungi- Twenty-four standardized, reproducible meth-
cides with insecticides, residual toxicity, nature ods, procedures and tools have been described,
of persistence, interval of sprays and cost–benefit which will help the researchers to refine and
ratio of the most effective fungicides have been reconfirm their findings in the future (Verma and
determined (Verma and Saharan 1994; Singh and Saharan 1994).
Singh 2005; Meena et al. 2004; Khan et al. 2007; The last chapter of the book highlights some
Mondal et al. 2007; Sultana et al. 2009; Saharan priority areas of research to be conducted by
1991, 1992; Marshall and Haris 1984; Bonin and future researchers to manage and understand
Fratczak 1987; Brazauskiene and Peteraitiene Alternaria–crucifer pathosystem in a better way.
2004; Davies et al. 1986). Indiscriminate use of
high doses of fungicides may affect pollen biol-
ogy of the crop resulting into adverse effect on References
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2000). Fungicides like iprodione, procymidone Aneja JK, Agnihotri A (2013) Alternaria blight of oilseed
brassicas: epidemiology and disease control strategies
and fludioxonil have shown resistance against
with special reference to use of biotechnological
isolates of A. brassicicola, which may affect their approaches for attaining host resistance. J Oilseed
efficacy under field conditions to control the dis- Bras 4:1–10
ease (Huang and Levy 1995; Iacomi-Vasilescu Anonymous (1985) Crucifer genetics cooperative (GrGc).
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brassicicola (Schw.) Wiltsh. on Brassica oleracea Verma PR, Saharan GS (1994) Monograph on Alternaria
seeds. Aust J Exp Agric 32:535–537 diseases of crucifers. Saskatoon Research Centre,
Smith IM, Dunez J, Phillips DH, Lelliott RA, Archer SA Technical Bulletin 1994–6E, Agriculture and Agri-
(1988) European handbook of plant diseases. Food Canada, Saskatoon, SK, Canada, 162 p
Blackwell Scientific Publications, Oxford Verma SS, Yajima WR, Rahman MH, Shah S, Liu JJ,
Snogerup S (1980) The wild forms of Brassica oleracea Ekramoddoullah AK, Kav NN (2012) A cysteine-rich
group (2n = 18) and their possible relations to the cul- antimicrobial peptide from Pinus monticola
tivated ones. In: Tsunoda S, Hinata K, Gomez-campa (PmAMP1) confers resistance to multiple fungal
C (eds) Brassica crops and wild allies, vol 7. Japan pathogens in canola (Brassica napus). Plant Mol Biol
Scientific Society, Press, Tokyo, pp 121–132 79:61–74
Stankova J (1972) Varietal variability of winter rape in its Vishwanath K, Kolte SJ (1997) Variability in Alternaria
susceptibility to dark leaf spot and the factors influenc- brassicae: response to host genotypes, toxin produc-
ing the development of the disease. Rostlinna Vyroba tion and fungicides. Indian Phytopathol 50:373–381
18:625–630 Von Keissler K (1912) Zur Kenntnis Pilzflora Krains.
Stoll K (1948) On the Alternaria blackening of Brassicas. Beihefte zum Botanischen Zentralblatt 29:434
NachrBl Dtsch PflSch Dienst Berl N F 2:174–178 Wallroth CFW (1833) Flora Cryptogamica Germaniae
Stoll K (1952) The origin, injurious effect and control of Sectio 2. J.L. Schrag, Nürnberg
Brassica blackening. NachrBl Dtsch Pflsch Dienst Wang Y, Geng Y, Ma J, Wang Q, Zhang XG (2011)
Berl N F 6:81–85 Sinomyces: a new genus of anamorphic Pleosporaceae.
Suhag LS, Singh R, Malik YS (1983) Assessment of Fungal Biol 115:188–195
losses caused by Alternaria alternata on radish seed White JG, Linfield CA, Lahdenpera ML, Uoti J (1990)
crop and its control by chemicals. Indian Phytopathol Mycostop- a novel bio-fungicide based on
36:758–760 Streptomyces griseoviridis. In: Proceedings of British
Sultana NA, Khan MAH, Islam MN, Kamran N (2009) crop protection conference, pests and diseases,
Evaluation of appropriate time for the application of Brighton, 1, pp 221–226
rovral against alternaria blight incidence and yield of Williams PH (1985) Crucifer genetics cooperative
mustard. Int J Sustain Agric 1:20–23 (GrGc). Cruciferae Newsl 10:1–2
Taber RA (1964) A study of pathogenicity and in vitro Williams J, Pink DAC (1987) Development of an in vitro
growth of Alternaria spp. isolated from Brassica spp. screen in Brussels sprout for resistance to Alternaria
with special reference to A. raphani from rape. M.Sc. brassicae. Cruciferae News Lett 12:91
thesis, University of Saskatchewan, Saskatoon, Canada Wiltshire SP (1933) The foundation species of Alternaria
Taber RA, Vanterpool TC, Williard AT (1968) A compara- and Macrosporium. Trans Br Mycol Soc 18:
tive nutritional study of Alternaria raphani, A. brassi- 135–160
cae and A. brassicicola with special reference to A. Wiltshire SP (1938) The original and modern conceptions
raphani. Phytopathology 58:609–616 of Stemphylium. Trans Br Mycol Soc 21:211–239
Tewari JP (1991a) Structural and biochemical bases of the Woudenberg JHC, Groenewald JZ, Binder M, Crous PW
black spot disease of crucifers. Adv Struct Biol (2013) Alternaria redefined. Stud Mycol 75:
1:325–349 171–212
Tewari JP (1991b) Current understanding of resistance to Yadav MS, Brar KS (2003) Relationship between meteo-
Alternaria brassicae in crucifers. In: Proceedings of rological factors and incidence of Alternaria blight
GCIRC 8th International Rapeseed Congress, 9–11 July, and white rust of Indian mustard in South–western
Saskatoon, Canada, p 84 (Abstr.), vol 2, pp 471–476 Punjab. Plant Dis Res 18:80–82
 The Disease
2

2.1 Introduction Alternaria blight, powdery mildew and white rust

can be observed on B. juncea leaves.
Alternaria blight caused by four species of
Alternaria is known by several names (synonyms)
on the basis of colour, shape, size and location of 2.2 Synonyms
lesions on different parts of cruciferous host spe-
cies. General symptoms are similar on all plant The diseases of Brassicaceae crops caused by
hosts with minor variations influenced by envi- four species of Alternaria are known by many
ronmental conditions, host species, genotypes, names: Alternaria leaf spot; Alternaria pod spot;
nutritional conditions and pathotypes. Pathotype- Alternaria leaf and pod spot; Alternaria blight;
specific symptoms can be observed on Brassica Alternaria leaf blight; Alternaria pod blight;
genotypes. The disease is widely distributed all Alternaria leaf and pod blight; Alternaria dark
over the world wherever the congenial environ- leaf spot; black spot; brown spot; black ring spot;
mental conditions and host–pathogen interaction blight pod spot; stem streak; seed rot; siliquae
occur. Its host range is very wide ranging from mould; black mould; grey leaf spot; grey leaf
oil-yielding Brassica to vegetables and a large mould of rapeseed, mustard and crucifers; rape
number of cruciferous weeds. The disease causes black; brassicaceous black; black spot of cab-
heavy yield losses in the form of quality and bage; brown rot of cauliflower; Alternaria stor-
quantity of cruciferous crops (11–100 %). The age rot of vegetables; Alternaria blight of
disease incidence and intensity can be accessed vegetables; Alternaria sooty spot; black pod
through various keys, scales and diagrams and blotch; brown rot; curd blight; curd drying; root
using modern techniques including remote sens- and foot rot; and damping-off (Anonymous
ing, video image and PCR. Host resistance and 1985a, b, c, d; Czyzewska 1969, 1970, 1971;
categorization of Brassica genotypes for breeding Eddins 1952; Ellis 1968a, b; Harter and Jones
resistance varieties can be done through interna- 1923; Kolte 1985a, b; Leppik 1973; Neergaard
tionally acceptable procedures and methods sug- 1945; Petrie 1975; Pound 1946; Pound et al.
gested in this chapter. Sometimes, under field 1951; Sherf and Macnab 1986; Subramanian
conditions, symptoms of mixed infections of 1971; Tewari 1985; Van Schreven 1953; Walker

© Springer Science+Business Media Singapore 2016 17

G.S. Saharan et al., Alternaria Diseases of Crucifers: Biology, Ecology and Disease Management,
DOI 10.1007/978-981-10-0021-8_2
18 2 The Disease

1952; Weber 1973; Weimer 1924; Wiltshire brown to black in colour than on B. juncea where
1947; Yoshii 1933). they are light coloured with distinct grey centres.
The diseased seeds just beneath the black spot are
small, shrivelled and grey to brown in colour. In
2.3 Symptomatology years of severe outbreaks, infection of the stem
may be sufficiently intense to cause the whole
2.3.1 Rapeseed–Mustard plant to wither before many of the pods have even
been matured or formed (Kolte 1985a, b; Saharan
On oil-yielding Brassica crops, all three species of and Chand 1988; Vasudeva 1958). Detailed
Alternaria (A. brassicae, A. brassicicola and A. symptomatology of Brassica species infected
raphani) are reported to cause symptoms at the with A. brassicae has been described by Conn
seedling stage on cotyledons (Plate 2.1a, b) and at et al. (1990) and Kadian and Saharan (1983).
the adult stage on leaves, leaf petiole, stem, inflores- In A. brassicae infections in rapeseed, the chlo-
cence, siliquae and seeds. There may be variations roplasts are more severely affected than the mito-
in shape, size, colour and intensity of lesions on dif- chondria. Changes in chloroplasts include swelling
ferent host tissues and species under different agro- and eventual disintegration. The mitochondria,
ecological zones. In general, lesions produced by A. although, show swelling of intra-cristal spaces, but
brassicae are grey compared to black sooty velvety without disintegration. In severely affected cells,
by A. brassicicola. All three species produce dis- vesiculated plasma membranes with electron-
tinct lesions with yellow halos around them. The dense deposits, intact grana from disintegrated
disease first appears on the cotyledons and hypocot- chloroplasts and mitochondria with swollen intra-
yls in the form of small light brown lesions which cristal spaces are recognizable (Tewari 1991a, b).
soon turn black due to the appearance of spore On Brassicaceae, the lesions on leaves due to
masses (under humid conditions) which act as a A. brassicicola are sooty black, velvety and copi-
source of infection for the other parts of the plant. ously covered with black conidiophores and
Damping-off of rapeseed seedlings due to A. bras- conidia, whereas those caused by A. brassicae
sicicola is common in Finland (Tahvonen 1979). are grey, dense and sparsely covered with brown
The initial infection by A. brassicae on the conidiophores and conidia (Changsri 1961).
lower leaves starts as minute brown to blackish The lesions caused by A. raphani are small
lesions, which multiply rapidly and later spread with raised margins and are surrounded by a yel-
to the upper leaves, stem and siliquae (Plate 2.1c– low translucent halo. Isolate specific lesions have
d). On the leaves, lesions may vary in size from 1 been recorded on B. juncea genotypes which vary
to 20 mm increases and cover more areas in size, shape, colour, number of concentric rings
(Plate 2.2a–b). In some Brassica species, forma- in each spot and presence and absence of yellow
tion of concentric rings in the lesions, and a zone halo around the spots (Gupta et al. 2004). Under
of yellow halo around the lesions, is very promi- field conditions on B. juncea leaves, symptoms of
nent (Kadian and Saharan 1983). Several lesions mixed infections of Alternaria blight, powdery
on the leaves coalesce to cause blighting and mildew and white rust can also be observed.
defoliation under humid weather (Plate 2.2a–b). Alternaria alternata causes root rot of rape (R.
The lesions on the stem are at first linear and then rapa) in Canada (Berkenkamp and Vaartonou
expand but remain usually elongated with pointed 1972) and root and foot rot of Eruca sativa in
ends (Plate 2.2c–d). In severe attacks, the upper India (Bhargava et al. 1980). The first evidence of
part of the stem and siliquae withers. Siliquae the disease in the field is poor germination of
may show sunken, dark-brown to black circular seeds. The infected cotyledons first become
lesions. Deep lesions on the siliquae cause infec- ­chlorotic and then necrotic. The necrotic lesions
tion in the seed (Plate 2.3a–b). On siliquae of B. extend on the stem up to 6–7 cm above the soil
rapa var. toria and Yellow and Brown Sarson, level. The root system of severely infected plants
lesions are more prominent, enlarged and dark is completely destroyed (Bhargava et al. 1980).
2.3 Symptomatology 19

Plate 2.1 Alternaria blight symptoms on various c­ otyledons leaves; (c) Alternaria infection on first leaves;
­rapeseed–mustard plant parts; (a) Alternaria infection on (d) Alternaria spots on leaf
cotyledons leaves; (b) Alternaria brown lesion on
­

2.3.2 Taramira (Eruca sativa) 2.3.3 Crambe (Crambe abyssinica)

The disease appears on pods as small lesions, cir- Alternaria brassicae infects mainly leaves and
cular and blackish in colour. As the disease pro- stems, A. brassicicola is a major pathogen of stem
gresses, several lesions coalesce and cover large and siliquae, A. alternata infects all above-­ground
areas, resulting in the death of the entire pod. plant parts, and A. raphani has not been recorded
Similar lesions are also noticed on stems where on this crop (Czyzewska 1971). Generally, symp-
they occur as circular yellow areas enlarging into toms caused by all three species are similar.
concentric circles of black sooty colour. Alternaria-infected seeds when sown cause
20 2 The Disease

Plate 2.2 Alternaria blight symptoms on various rape- Alternaria lesions on stem. (d) Severe Alternaria infec-
seed–mustard plant parts. (a) Lesions increase in size and tion on the lower portion of the plant
cover larger area. (b) Concentric rings in the spot. (c)

p­ re- and post-emergence damping-off of seedlings. small, dark brown, which quickly become large
In dry seasons, diseased cotyledons turn yellow, and cover all the tissues. Such cotyledons generally
shrivel and dehisce prematurely. During humid become slimy with mycelium, conidiophores and
weather, initial spots on the infected cotyledons are conidia leading to quick seedling death.
2.3 Symptomatology 21

Plate 2.3 Alternaria blight symptoms on various rapeseed–mustard plant parts. (a) Alternaria infection on siliquae.
(b) Deep Alternaria infection on siliquae. (c) Severely infected siliquae. (d) Alternaria-infected seeds (right)

Leaf lesions are round (0.5–2 mm dia.), rings appear on one spot. Larger spots are lighter
slightly depressed with a smooth surface and brown in colour and have a slight violet tint.
brown to dark brown in colour. The tissues Spots on the infected stems are dark brown to
around the spots turn lighter in colour and then black, mostly elongated in the form of streaks and
become yellow. Sometimes, the brown dead tis- somewhat recessed. In damp weather, infected
sues in the central part of the spot crumble and stems become constricted causing the seedlings to
fray at the leaf edge in the form of 0.5-mm-width topple. The infected collar region appears
brown tissue border. The spots caused by A. bras- recessed with elongated black to olive lesion of
sicae are larger (1–10 mm) and appear isolated up to 2 cm encircling the stem partially or com-
with visible infection centre. The concentric pletely. On siliquae, initial lesions are small
rings in the lesion are more sparsely arranged (0.3–3 mm), round and olive brown in colour
usually at a distance of 1–2 mm; two to three which later turn blackish brown after ­sporulation.
22 2 The Disease

Infected siliquae are small and deformed and The lesions are linear on stem, petiole and
produce small, discoloured, shrivelled seeds, ­siliquae (Chupp 1925; Chupp and Sherf 1960).
which are non-viable (Czyzewska 1969, 1971; Cauliflower and broccoli heads show a brown-
Holcomb and Newman 1970; Leppik 1973). ing, beginning at the margin of the individual
flower and flower clusters. On plants grown for
seed, dark necrotic lesions occur on the main axis,
2.3.4  arden Stock (Matthiola
G the inflorescence branches and on the siliquae.
incana) These lesions coalesce rapidly and cause prema-
ture ripening and splitting of the siliquae resulting
The initial symptoms appear on the lower leaves in high levels of seed infection. Infected seeds are
first, which gradually spread upward onto the small, shrunken, discoloured or covered with fun-
leaves, stem and flowers. The lesions first appear gal growth and have low viability. Cankers may
round and then elongate to 3–15 mm in size. form just below the nearly mature cabbage heads
Older lesions are pale or greyish green with con- resulting in stump rot and death of plants.
centric zones with brown centres. Such lesions On turnips and rutabaga, when foliage is
become dark brown to black during sporulation. infected, the roots may also become infected and
On stem, and flowers, lesions with water-soaked develop symptoms, especially during storage.
margins are also common (Davis et al. 1949). The leaf spots are nearly circular, often zonate,
and are of various shades of brown to black. Dark
spores may cover the spot if temperature is high
2.3.5  egetable Crops (Cruciferous
V during storage (Sherf and Macnab 1986).
Vegetables) Alternaria raphani is common on radish plants
kept for seed purpose. Leaf lesions are raised,
The symptoms produced by A. brassicae and A. spherical to elliptical and up to 1 cm in diameter;
brassicicola are similar and often indistinguish- black sporulation may be seen on the lesions. The
able. Seed infection by either fungus may cause centre soon dries and may drop off (Singh 1987).
severe damping-off or a stunting of young plants. The natural infection of radish seeds with A.
On infected seedlings, the pathogen produces raphani may result in a poor germination, pre- or
dark-brown necrotic areas on the cotyledons and post-emergence blight, appearance of distinctive
similar coloured streaks on the hypocotyl. On lesions on cotyledons and hypocotyls, presence
older plants, all above-ground parts are attacked of scab-like lesions on table radish and spotting
including leaves, stems, Brussels sprouts buttons, and blighting of leaves, stalks and siliquae
cauliflower curds, inflorescence and siliquae (Atkinson 1950).
including seeds in seed crop. On kohlrabi (Brassica oleracea var. gongy-
In cruciferous vegetables, on leaves, symp- lodes L.) plants, disease symptoms first appear as
toms first appear as minute dark-brown to black small dark brown, which turn black on leaves;
spots each surrounded by a halo of chlorotic tis- spots vary in size from pinpoints to 1 cm in diam-
sues. Leaf spots that vary in size from pinpoints eter. As the spots enlarge, a definite zonation, or
to 2–3 inches in diameter are common on old concentric rings, becomes evident and slightly
lower leaves. The enlargement of the spots may zonate giving a target spot appearance to the
be in concentric circles. Older lesions are circu- lesion. As spores are produced, spots in the cen-
lar, often zonate with a papery, thin centre, and tre become darker, and the dead centre might
may be covered with a mat of spores, which are later tear off, or partially drop away, giving the
yellow in the case of A. brassicae and dark olive leaf spots a shot-hole appearance. As the disease
brown in the case of A. brassicicola. The centres progresses, numerous spots and spotted leaves
of the lesions may fall out to give a shot-hole turn yellow and die prematurely. The pathogen
effect. Severe defoliation by A. brassicae has isolated and identified is A. brassicicola
been reported in some cultivars of stubble turnips. (Schwein) Wiltshire (El-Mohamedy 2007).
2.6 Yield Losses 23

2.3.6 Weeds Denmark, France, Germany, Great Britain,

Holland, Ireland and Italy (Ellis 1971).
A brassicaceous perennial, exotic, rangeland Alternaria raphani, which occurs on various
weed (Lepidium draba L.) shows tiny black spots brassicaceous hosts, has been recorded from
on leaves enlarging over time to become circular Canada, Denmark, Egypt, Greece, India, Iran,
to irregular and cream coloured around the initial Japan, the Netherlands and the USA (Ellis 1971).
black spots and sometimes with dark-brown bor- It is most common on radish, but also occurs on
ders or chlorotic holes (Caesar and Lartey 2009). other Brassica vegetables and oil-yielding crops
(Verma and Saharan 1993, 1994; Nourani et al.
2008).
2.4 Geographical Distribution Alternaria alternata has been reported from
Canada, Denmark, France, Poland, Latvian SSR,
Among the different Alternaria species reported Leningrad USSR and Maryland, USA
to infect brassicaceous hosts, A. alternata is an (Czyzewska 1969, 1970; Leppik 1973; Neergaard
extremely common saprophyte and a weak patho- 1945). The chronological records of Alternaria
gen found on many plants and other substrata. It species causing diseases of brassicaceous hosts
is cosmopolitan and reported to be widespread on reported from various countries of the world are
all kinds of brassicaceous plants including rape- given in Table 2.1. The names of various hosts in
seed–mustard, Crambe and Brassica vegetables this table are as reported in the original papers.
(Ellis 1971; Verma and Saharan 1993, 1994).
Alternaria brassicae (CMI map 353) and A.
brassicicola (CMI map 457) have been reported 2.5 Host Range
from almost every continent on Brassicaceae
hosts (Anonymous 1969). Alternaria brassicae is Alternaria species occur worldwide and attack a
considered most destructive on oil-yielding bras- large number of brassicaceous crops. Among the
sicas, and both are common on vegetable cruci- common hosts of economic importance are
fers. They are known to occur in Africa, Argentina, rapeseed–mustard, cabbage, Chinese cabbage,
Australia, Austria, Bangladesh, Brazil, Britain, cauliflower, broccoli, horse radish, turnip, radish,
Bhutan, Bulgaria, Burma, Canada, Chile, China, Brussels sprouts, collards, kohlrabi, rutabaga and
Cyprus, Czechoslovakia, Denmark, Egypt, swedes. Apart from these, the inventory of hosts
England, Ethiopia, Finland, France, Germany, infected by A. brassicae, A. brassicicola, A.
Ghana, Guinea, Holland, Hong Kong, Hungary, raphani and A. alternata is given in Table 2.2
India, Iran, Ireland, Italy, Jamaica, Japan, Kenya, (Ansari et al. 1990; Atkinson 1950; Chupp 1925;
Libya, Malawi, Malaya, Mauritius, Morocco, Czyzewska 1969; Ellis 1968a, b, 1971; Kadian
Mozambique, Nepal, Netherland, New Guinea, and Saharan 1983; Kolte 1985a, b; McDonald
New Zealand, Nicaragua, Nigeria, Norway, 1959; Putnam et al. 1972; Rai and Sinha 1963;
Pakistan, the Philippines, Poland, Rhodesia, Saharan et al. 1982; Tewari and Conn 1993;
Romania, Russia, Sabah, Saudi Arabia, Scotland, Verma and Saharan 1993, 1994; Walker 1927;
Sierra Leone, Singapore, Spain, South Africa, Sri Weber 1932).
Lanka, Sudan, Sweden, Switzerland, Taiwan,
Tanganyika, Tanzania, Trinidad, Turkey, Uganda,
the USA, USSR and Zambia (Anonymous 1980, 2.6 Yield Losses
1981, 1984, 1985a, b, c, d; Ellis 1971; Kolte
1985a; Saharan 1992b; Verma and Saharan 1993, 2.6.1 Rapeseed–Mustard
1994; Nourani et al. 2008).
Alternaria cheiranthi (Lib.) Bolle. is common Heavy infections on leaves, stems and siliquae
on wallflowers and occasionally recorded on adversely influence both quantity and quality of
Brassicaceae. It is recorded from Belgium, yield of Brassica crops (Butler 1918). In India,
24 2 The Disease

Table 2.1 Records of Alternaria species on Brassicaceae (Verma and Saharan 1993, 1994, updated 2014)
Recording
Alternaria species Host Disease Location year Reference
A. alternata Cauliflower Pox Italy 1932 Verona, (1932)
(A. tenius)
Cabbage Seed crop, black USSR 1959 Nelen, (1959)
spot
B. campestris and Black spot Canada 1963 Taber and
B. napus Vanterpool (1963)
Brassica spp. Black spot Canada 1967 Conners (1967)
C. abyssinica Black spot Poland 1969 Czyzewska (1969)
B. campestris Root rot Canada 1971 Berkenkamp and
Vaartonou (1972)
Garden rocket Black spot Saudi Arabia 1978 Sheir et al. (1981)
Brassica spp. Black spot USA 1978 Babadoost and
Gabrielson (1979)
Eruca sativa Root and foot India 1979 Bhargava et al.
rot (1980)
Radish Leaf and pod India 1980 Suhag et al. (1983)
blight
A. brassicae Cabbage Leaf spot USA 1909 Fawcett (1909)
(Macrosporium Collards Black mould USA 1913 Higgins (1917)
brassicae; A.
Cauliflower Leaf spot and USA 1918 Weimer (1924)
herculea)
brown rot
Cabbage Black leaf spot USA 1918 Harter and Jones
(1923)
Cabbage Leaf spot USA 1922 Chupp (1923)
Cabbage Leaf spot USA 1922 Milbraith (1922)
Cabbage and Leaf spot Russia 1922 Estifeyeff (1925)
radish
Cabbage Black leaf spot Trinidad 1922 Stell (1922)
Brassica spp. Leaf spot Holland 1924 Bolle (1924)
Brassica spp. Black leaf speck USA 1926 Nelson (1926)
Cabbage Leaf spot China 1926 Porter (1926)
Brassicaceous Leaf spot USA 1926 Weimer (1926)
vegetables
B. pekinensis Leaf spot USA 1927 Gardner (1929)
Brassica spp. Leaf spot Dominican 1927 Ciferri and
Republic Gonzalez (1927)
Cauliflower Brown rot USA 1927 Walker (1927)
Cauliflower Black leaf spot Italy 1928 Gardner (1929)
Cauliflower Black spot Colombia 1929 Toro (1929)
Cabbage Leaf spot and North 1930 Kikoina (1930)
storage rot Caucasus
Cabbage Seed infection Holland 1930 Voisenat (1930)
Cabbage Leaf spot Italy 1931 Pollacci (1932)
Cabbage and Leaf spot Japan 1933 Yoshii (1933)
radish
Brassicaceous Leaf spot UK 1933 Moore (1944)
vegetables
(continued)
2.6 Yield Losses 25

Table 2.1 (continued)

Recording
Alternaria species Host Disease Location year Reference
Cauliflower and Black leaf spot Canada 1934 Conners (1935)
broccoli and grey leaf
spot
Chinese cabbage Leaf spot USA 1934 Davis (1934)
Brassica spp. Seed infection Germany 1934 Juhans (1934)
Brassicaceous Leaf spot Trinidad, 1934 Fajardo and Palo
vegetables Philippines (1934)
Matthiola incana Leaf spot Great Britain 1935 Ware (1936)
Turnip Root rot USA 1935 Chupp (1935)
Cabbage Leaf spot Morocco 1937 Malencon and
Delecluse (1937)
B. oleracea Leaf spot Africa 1937 Roger and
Mallamaire (1937)
Colza and rape Leaf spot Germany 1938 Klemm (1938)
Cauliflower head Grey rot Brazil 1938 Arruda (1938)
Horse radish Leaf spot Germany 1938 Boning (1938)
Iberis umbellata Leaf spot Denmark 1938 Neergaard (1939)
Horse radish Leaf spot USA 1940 Kadow and
Anderson (1940)
Cabbage Leaf spot China 1940 Teng (1940)
Turnip Leaf spot Australia 1941 Anonymous (1941)
Cabbage Black spot USA 1941 Godfrey (1941)
Cabbage Storage rot and USA 1942 Myers (1942)
ball head
Cabbage and Leaf spot USA 1942 Snyder and Baker
cauliflower (1943, 1945)
Radish and turnip Leaf spot Argentina 1943 Marchionatto
(1947)
Turnip Leaf spot UK 1943 Moore (1948)
Colza and white Black spot Sweden 1944 Bjorling (1944)
mustard
Cabbage Seed infection USA 1944 Anonymous (1944)
Cauliflower and Head browning USA 1944 Rangel (1945)
broccoli
Cabbage and Brown rot Tanganyika 1945 Wallace and
turnip Wallace (1945)
Turnip, kohlrabi, Leaf spot Norway 1945 Jorstad (1945)
cabbage and
radish
Brassicaceous Leaf spot Denmark 1945 Neergaard (1948)
vegetable, M.
incana and I.
umbellata
Broccoli Dark leaf spot UK 1946 Moore (1948)
Radish Leaf spot UK 1946 Moore (1948)
Cabbage and Leaf spot Ceylon 1947 Bond (1947)
mustard
(continued)
26 2 The Disease

Table 2.1 (continued)

Recording
Alternaria species Host Disease Location year Reference
Rape Seedling blight Canada 1948 Vanterpool (1950)
C. maritima Grey leaf spot France 1949 Darpoux and
Faivre-Amiot
(1949)
Cabbage, mustard Leaf spot Holland 1949 Flik and Saaltink
and turnip (1950)
Turnip Leaf spot New Zealand 1949 Brien and Dingley
(1953)
Brassica spp. Leaf spot Holland 1950 Anonymous
(1951b)
Lunaria annua Leaf spot USA 1950 Baker and Davis
(1950)
Cabbage Leaf spot Nyasaland 1950 Gillman (1952)
Sugar beet Leaf spot USA 1950 McFarlane et al.
(1954)
Brassica spp. Leaf spot India 1950 Mehta et al. (1950)
B. napus Dark leaf Holland 1950 Van Schreven
(1953)
Turnip Leaf spot Kenya 1951 Anonymous
(1951a)
Cabbage Leaf spot New 1951 Bugnicourt et al.
Caledonia (1951)
Turnip Leaf spot Sudan 1951 Tarr (1954)
Chinese cabbage Leaf spot USSR 1952 Nelen (1966)
Candytuft Stem spot Tanganyika 1952 Wallace (1954)
C. abyssinica Grey leaf Latvian SSR 1954 Leppik (1973)
Brassica spp. Dark leaf spot Ireland 1956 McKay (1956)
Rape Grey leaf spot Canada 1956 McDonald (1959)
Cabbage Seedling blight USSR 1956 Tupenevich and
Shirko (1956)
Cabbage and Leaf spot Nicaragua 1957 Litzenberger and
turnip Stevenson (1957)
Colza Black spot France 1958 Louvet (1958)
Rape Siliquae and Poland 1958 Czyzewska (1958)
seed infection
Cabbage Leaf spot Greece 1959 Koleva-­
Sekutkovska
(1959)
Turnip and Leaf spot Australia 1959 Anonymous (1959)
Sisymbrium
orientale
Cabbage Leaf spot Primorskii 1959 Nelen (1959)
Krai, USSR
L. annua and L. Leaf spot Romania 1959 Negru (1959)
rediviva
Rape Grey leaf spot Canada 1961 Downey and
Bolton (1961)
(continued)
2.6 Yield Losses 27

Table 2.1 (continued)

Recording
Alternaria species Host Disease Location year Reference
Turnip Root rot and USA 1961 Ramsey and Smith
leaf spot (1961)
Radish Leaf spot Canada 1963 Taber and
Vanterpool (1963)
Cruciferous Leaf spot New Zealand 1964 Morton (1964)
Brassica Leaf spot New Zealand 1964 Morton (1964)
Thlaspi arvense Leaf spot Canada 1966 Petrie and
and Lepidium Vanterpool (1966)
spp.
Sinapis alba Seed infection Denmark 1967 Jorgensen (1967)
Radish Leaf spot Romania 1967 Barbu and Dinescu
(1969)
Taramira (Eruca Black spot India 1969 Prasada et al.
sativa) (1970)
B. alboglabra Leaf spot Singapore 1969 Seow and Lim
(1969)
C. abyssinica Grey leaf spot Poland 1970 Czyzewska (1970)
C. abyssinica Grey leaf spot USSR 1970 Leppik (1973)
Rape Black spot Western 1971 Bokor (1972)
Australia
Radish Leaf spot India 1977 Rao (1977)
Turnip rape Black spot Spain 1979 Romero and
Jimenez (1979)
Turnip Leaf spot Bulgaria 1979 Khristov (1979)
B. napus Leaf spot Mexico 1980 Ponce and
Mendoza (1983)
Rape Leaf spot Sweden 1981 Doughty et al.
(1991)
Anagallis Leaf spot India 1982 Saharan et al.
arvensis, (1982)
Convolvulus
aruensis
Rape Leaf spot UK 1982 Anonymous
(1983a, b)
Brassica spp. Dark leaf spot Ireland 1982 Anonymous
(1983a, b)
Charlock weed Leaf spot Ireland 1982 Anonymous
(1983a, b)
Cabbage, Curds spot UK 1982 Anonymous
Brussels sprouts (1983a, b)
and cauliflower
curds
Brassica spp. Leaf spot Ireland 1983 Ryan et al. (1984)
Swede rape Leaf spot Russia 1983 Vakhurusheva
(1983)
B. napus and B. Leaf spot Italy 1985 Tosi and Zazzerini
campestris (1985)
(continued)
28 2 The Disease

Table 2.1 (continued)

Recording
Alternaria species Host Disease Location year Reference
Oilseed rape Dark leaf spot UK 1985 Anonymous
(1985d)
Candytuft (Iberis Leaf spot India 1987 Gurha and Dhar
sp.) (1987)
B. napus and B. Leaf spot Ethiopia 1987 Kidane and Bekele
carinata (1987)
Rape Dark leaf spot Germany 1988 Daebeler and
Amelung (1988)
Chinese cabbage Brazil 2003 Reis and Boiteux
(2010)
Pak choi Brazil 2003 Reis and Boiteux
(2010)
Turnip Brazil 2003 Reis and Boiteux
(2010)
Chinese cabbage Brazil 2003 Reis and Boiteux
(2010)
Leaf mustard Brazil 2003 Reis and Boiteux
(2010)
Salad rocket Brazil 2004 Reis and Boiteux
(2010)
Oilseed rape Brazil 2004 Reis and Boiteux
(2010)
Wild mustard Brazil 2004 Reis and Boiteux
(2010)
Wild radish Brazil 2004 Reis and Boiteux
(2010)
Radish Brazil 2004 Reis and Boiteux
(2010)
Crambe Leaf spot Australia 2004 You et al. (2005)
abyssinica
Stem radish Brazil 2005 Reis and Boiteux
(2010)
Leaf mustard Brazil 2005 Reis and Boiteux
(2010)
Chinese cabbage Leaf spot Brazil 2005 Michereff et al.
(2012)
Lepidium draba Leaf spot America 2007 Caesar and Lartey
(2009)
Brassica napus Leaf spot Iran 2007 Nourani et al.
(2008)
A. brassicicola Cabbage Leaf spot Holland 1924 Bolle (1924)
Cabbage and Damping-off Burma 1934 Su (1934)
cauliflower and dark spot
Cauliflower Inflorescence Mauritius 1943 Coombes and
blight Julien (1949)
Chinese cabbage Leaf spot Jamaica 1944 Anonymous
(1945a, b)
(continued)
2.6 Yield Losses 29

Table 2.1 (continued)

Recording
Alternaria species Host Disease Location year Reference
Godetia hybrida Seed infection Denmark 1945 Anonymous
(1945a, b)
C. abyssinica Black spot Denmark 1945 Neergaard (1945)
Vegetable Leaf spot Denmark 1945 Neergaard (1945)
Brassica and
weeds
Cabbage, Black spot UK 1947 Green (1947)
cauliflower and
kale
Kohlrabi Leaf spot Ceylon 1947 Bond (1947)
Cabbage Leaf spot Nyasaland 1950 Gillman (1952)
Turnip, Leaf spot Sudan 1951 Tarr (1951, 1954)
cauliflower and
radish
Cabbage Leaf spot New Zealand 1952 Brien and Dingley
(1955)
Cabbage, Leaf spot and Germany 1952 Stoll (1952)
Brussels sprouts blackening
and radish
Cabbage and Damping-off Finland 1952 Linnasalmi (1952)
cauliflower
Cabbage Seed infection Turkey 1954 Gobelez (1956)
Brassica spp. Dark leaf spot Ireland 1956 McKay (1956)
Cabbage Dark spot USSR 1959 Nelen (1959)
B. rapa Leaf spot Malaya 1963 Jamil (1966)
Brassica spp. Leaf spot Brunei 1963 Johnston (1964)
Rape Leaf spot Chile 1963 Bertossi (1963)
Crucifers Leaf spot New Zealand 1964 Morton (1964)
Brassica
Cabbage Leaf spot Saudi Arabia 1967 Anonymous (1967)
Brassica spp. Leaf spot Canada 1967 Conners (1967)
C. abyssinica Black spot Poland 1969 Czyzewska (1969)
C. abyssinica Black spot USA 1969 Holcomb and
Newman (1970)
Brassica spp. Leaf spot Sarawak 1972 Kueh (1972)
White cabbage Storage rot UK 1973 Geeson (1979)
Crucifers Leaf spot India 1977 Rao (1977)
Cabbage Leaf spot Saudi Arabia 1978 Sheir et al. (1981)
Brassica spp. Leaf spot USA 1978 Babadoost and
seed crop Gabrielson (1979)
Cabbage Storage rot Finland 1981 Tahvonen (1981)
Cabbage Leaf spot Brazil 1982 Bolkan et al.
(1983)
Brassica spp. Leaf spot UK 1982 Anonymous
(1983a, b)
Brassica spp. Leaf spot Ireland 1983 Ryan et al. (1984)
Republic
(continued)
30 2 The Disease

Table 2.1 (continued)

Recording
Alternaria species Host Disease Location year Reference
E. sativa Leaf blight India 1984 Sharma (1985)
Brassica spp. Black spot Spain 1984 Galvez and
oilseeds Romero (1988)
Rape Dark leaf spot Germany 1988 Daebeler and
Amelung (1988)
Cabbage Sooty spot Japan 1998 Kubota and Abiko
(1998)
Chinese cabbage Brazil 2003 Reis and Boiteux
(2010)
Chinese cabbage Brazil 2003 Reis and Boiteux
(2010)
Kohlrabi Leaf spot Egypt 2004 El-Mohamedy
(2007)
Oilseed rape Brazil 2004 Reis and Boiteux
(2010)
Wild radish Brazil 2005 Reis and Boiteux
(2010)
Stem turnip Brazil 2005 Reis and Boiteux
(2010)
Leaf mustard Brazil 2005 Reis and Boiteux
(2010)
Cabbage Leaf spot Brazil 2005 Michereff et al.
(2012)
Cauliflower Leaf spot Brazil 2005 Michereff et al.
(2012)
Broccoli Leaf spot Brazil 2005 Michereff et al.
(2012)
Salad rocket Brazil 2006 Reis and Boiteux
(2010)
Turnip Brazil 2006 Reis and Boiteux
(2010)
Wild mustard Brazil 2006 Reis and Boiteux
(2010)
B. napus Leaf spot Iran 2007 Nourani et al.
(2008)
Radish Brazil 2008 Reis and Boiteux
(2010)
A. raphani Brassica spp. Leaf spot Denmark 1945 Neergaard (1948)
Garden stock Leaf spot California 1946 Davis et al. (1949)
Radish Seed, leaf, stem, USA 1947 Mclean (1947)
pods and root
infection
Matthiola spp. Leaf spot Canada 1953 Conners (1954)
B. oleracea var. Leaf spot Greece 1953 Critopoulos (1953)
capitata
Cabbage USSR 1959 Nelen (1959)
(continued)
2.6 Yield Losses 31

Table 2.1 (continued)

Recording
Alternaria species Host Disease Location year Reference
Brassica spp. Leaf spot Canada 1963 Taber and
Vanterpool (1963)
Radish and T. Leaf spot Canada 1966 Petrie and
arvense Vanterpool (1966)
Radish Leaf spot Saudi Arabia 1978 Sheir et al. (1981)
I. amara Leaf spot India 1981 Narian et al.
(1982)
Turnip Leaf spot USA 1982 Cotty and Alcorn
(1984)
B. campestris var. Leaf spot India 2002 Sangwan et al.
rapa (2002)
B. campestris var. Leaf spot India 2002 Sangwan et al.
toria (2002)
B. carinata Leaf spot India 2002 Sangwan et al.
(2002)
B. napus Leaf spot India 2002 Sangwan et al.
(2002)
B. campestris var. Leaf spot India 2002 Sangwan et al.
Yellow Sarson (2002)
B. campestris var. Leaf spot India 2002 Sangwan et al.
Brown Sarson (2002)
B. chinensis Leaf spot India 2002 Sangwan et al.
(2002)
B. juncea Leaf spot India 2002 Sangwan et al.
(2002)
B. tournefortii Leaf spot India 2002 Sangwan et al.
(2002)
B. pekinensis Leaf spot India 2002 Sangwan et al.
(2002)
B. alba Leaf spot India 2002 Sangwan et al.
(2002)
Eruca sativa Leaf spot India 2002 Sangwan et al.
(2002)
B. napus Leaf spot Iran 2007 Nourani et al.
(2008)

yield losses in Alternaria-infected plants increase Alternaria brassicae infection is also known to
considerably after winter rains (Dey 1948). affect chemical composition of seed including
Shrivelling of seeds and reduction in quantity of protein, total carbohydrates and ash (Degenhardt
oil content is the major effect in severe infections et al. 1974; Nijhawan and Hussain 1964).
(Chahal and Kang 1979; Chohan 1978; Kaushik In Canada, Degenhardt et al. (1974) reported
et al. 1984; Milbraith 1922; Nijhawan and that the combined effect of A. brassicae and A.
Hussain 1964; Vasudeva 1958). The seed produc- raphani infection under artificially inoculated
tion of brassicas is significantly reduced by field conditions resulted in 70 and 42 % losses in
Alternaria infections, which invade siliquae and yield of B. campestris (B. rapa) and B. napus,
penetrate the seeds besides damaging the assimi- respectively. According to their estimates, A.
latory tissues of the leaves and stems brassicae alone can cause 63 % loss in B. camp-
(Bandyopadhya et al. 1974; Nielsen 1933). estris and 42 % in B. napus; yield reductions due
32 2 The Disease

Table 2.2 Host species susceptible to Alternaria brassicae, Alternaria brassicicola, Alternaria raphani and Alternaria
alternata (Verma and Saharan 1994, updated 2014)
Alternaria species Host Common name Reference
A. brassicae Armoracia rusticana Horse radish Tewari and Conn (1993)
A. lapathifolia Bolle (1924)
Anagallis arvensis Saharan et al. (1982)
Brassica oleracea var. Kohlrabi Weiss (1960)
gongylodes
B. oleracea var. viridis Kale Weiss (1960)
B. napobrassica Rutabaga Weiss (1960)
B. oleracea var. gemmifera Brussels sprouts Weiss (1960)
B. oleracea var. botrytis Cauliflower Weiss (1960)
B. oleracea var. capitata Cabbage McDonald (1959)
B. napus ssp. oleifera Rapeseed McDonald (1959)
B. tournefortii Wild mustard Kadian and Saharan
(1983)
B. nigra Black mustard Kadian and Saharan
(1983)
B. chinensis Chinese cabbage Kadian and Saharan
(1983)
B. pekinensis Chinese cabbage Kadian and Saharan
(1983)
B. rugosa Kadian and Saharan
(1983)
B. rapa var. Brown Sarson Kadian and Saharan
(1983)
B. rapa var. Yellow Sarson Kadian and Saharan
(1983)
B. rapa var. toria Kadian and Saharan
(1983)
B. hirta White mustard Kadian and Saharan
(1983)
B. kabar White mustard Weiss (1960)
B. rapa ssp. oleifera Canola Tewari and Conn (1993)
B. rapa ssp. rapifera Turnip Tewari and Conn (1993)
B. carinata Abyssinian mustard Tewari and Conn (1993)
B. orientalis Bolle (1924)
B. japonica Banga et al. (1984)
B. rapa Rai and Sinha (1963)
B. juncea Mustard Mehta et al. (1950)
B. alboglabra Seow and Lim (1969)
Chenopodium album Tripathi and Kaushik
(1984)
Cleome ciliata Lapis and Ricaforte (1974)
Convolvulus arvensis Saharan et al. (1982)
Citrus aurantium Rao (1977)
Cyamopsis psoraloides Guar Siddiqui (1963)
Crambe abyssinica Crambe Czyzewska (1970)
C. maritima Crambe Leppik (1973)
(continued)
2.6 Yield Losses 33

Table 2.2 (continued)

Alternaria species Host Common name Reference
Cheiranthus cheiri Wall flower McDonald (1959)
Eruca sativa Taramira Kadian and Saharan
(1983)
Erysimum cheiranthoides Wormseed mustard Tewari and Conn (1993)
Iberis amara Candytuft McDonald (1959)
Iberis umbellata Neergaard (1939)
Isatis tinctoria Bolle (1924)
Lallemantia iberica Darpoux (1945)
Lepidium sativum Garden cress McDonald (1959)
L. latifolium McDonald (1959)
Lactuca sativa Lettuce Lapis and Ricaforte (1974)
Lunaria annua Honesty Baker and Davis (1950)
Matthiola incana Stock Ware (1936)
Portulaca oleracea Lapis and Ricaforte (1974)
Raphanus sativus Radish McDonald (1959)
Sinapis alba White mustard Tewari and Conn (1993)
S. arvensis Wild mustard Tewari and Conn (1993)
Sisymbrium officinale Hedge mustard Putnam et al. (1972)
S. altissimum Tumbling weed Putnam et al. (1972)
S. orientale Anonymous (1959)
Thlaspi arvense Stink weed Petrie and Vanterpool
(1966)
A. brassicicola B. oleracea var. capitata Cabbage Rai and Sinha (1963)
B. oleracea var. botrytis Cauliflower Rai and Sinha (1963)
B. oleracea var. caulorapa Rai and Sinha (1963)
B. juncea Mustard Bolkan et al. (1983)
B. pekinensis Chinese cabbage Bolkan et al. (1983)
B. rapa Turnip Jamil (1966)
B. carinata Abyssinian mustard Galvez and Romero
(1988)
C. abyssinica Crambe Czyzewska (1969)
E. sativa Taramira Sharma (1985)
Godetia hybrida Anonymous (1946)
I. umbellata Neergaard (1948)
Lunaria rediviva Negru (1959)
R. sativus Radish Rao (1977)
A. raphani B. rapa var. toria Rai and Sinha (1963)
B. juncea Indian mustard Rai and Sinha (1963)
B. oleracea var. botrytis Cauliflower Atkinson (1950)
B. oleracea var. capitata Cabbage Atkinson (1950)
B. napabrassica Swedes Atkinson (1950)
Cheiranthus cheiri Wall flower Atkinson (1950)
E. sativa Taramira Rai and Sinha (1963)
I. amara Candytuft Atkinson (1950)
I. armena Narain et al. (1982)
(continued)
34 2 The Disease

Table 2.2 (continued)

Alternaria species Host Common name Reference
Lactuca sativa Lettuce Atkinson (1950)
M. incana Stock Baker and Davis (1950)
T. arvense Stink weed Petrie and Vanterpool
(1966)
B. campestris var. rapa Sangwan et al. (2002)
B. campestris var. toria Sangwan et al. (2002)
B. carinata Sangwan et al. (2002)
B. napus Sangwan et al. (2002)
B. campestris var. Yellow Sangwan et al. (2002)
Sarson
B. campestris var. Brown Sangwan et al. (2002)
Sarson
B. chinensis Sangwan et al. (2002)
B. juncea Sangwan et al. (2002)
B. tournefortii Sangwan et al. (2002)
B. pekinensis Sangwan et al. (2002)
B. alba Sangwan et al. (2002)
Eruca sativa Sangwan et al. (2002)
A. alternata B. rapa var. Brown Sarson Singh and Suhag (1983)
B. rapa var. toria Singh and Suhag (1983)
B. juncea Indian Mustard Singh and Suhag (1983)
B. oleracea var. capitata Cabbage Singh and Suhag (1983)
B. oleracea var. botrytis Cauliflower Rai and Sinha (1963)
B. oleracea var. caulorapa Rai and Sinha (1963)
Beta vulgaris Spinach Singh and Suhag (1983)
Convolvulus arvensis Hiran Khuri Singh and Suhag (1983)
C. abyssinica Crambe Czyzewaska (1969)
E. sativa Taramira Rai and Sinha (1963)
1. amara Rai and Sinha (1963)
Lepidium sativum Rai and Sinha (1963)
Lycopersicon esculentum Tomato Singh and Suhag (1983)
R. sativus Radish Rai and Sinha (1963)
Trianthema monogyna Santhi Singh and Suhag (1983)

to A. raphani alone were 42 % and 34 %, respec- due to Alternaria infection. The losses in yield
tively. In 1955 and 1956 crop seasons in Canada, (Tables 2.3, 2.4, 2.5, 2.6 and 2.7) have been
about 20 % yield losses in rapeseed were attrib- reported from 35 to 45 % in the case of Yellow
uted to A. brassicae infection (McDonald 1959). Sarson (Table 2.3; Saharan 1984), 25–45 % in
However, Tewari and Conn (1988) estimated an Brown Sarson cv. BSH-1 (Table 2.3; Chahal and
average 30 % yield loss from central region of Kang 1979) and 17–48 % in raya (mustard)
Alberta. According to Daebeler et al. (1986), (Table 2.3; Saharan 1984). Kolte (1982) reported
Alternaria leaf spot damage in winter rapeseed 17–60 % loss in yield of Rai and Sarson. Yield
ranged from 20 to more than 50 % in the German losses ranging from 10 to 75 % have been
Democratic Republic. reported in different oil-yielding crops from India
Kanwar and Khanna (1979) reported consid- (Saharan 1992a). Yield losses are heavier in
erable deterioration in seed quality and quantity Yellow Sarson (38–45 %) followed by Brown
2.6 Yield Losses 35

Table 2.3 Assessment of yield losses in rapeseed–mustard due to Alternaria (Saharan 1984)
Disease intensity (%) Reduction
in disease Reduction Reduction in oil
Cultivar Location Sprayed* Unsprayed intensity (%) in yield (%) content (%)
Prakash Hisar 18.0 71.1 74.6 17.9 3.4
(raya)
Varuna Hisar 20.6 67.4 67.9 16.8 2.5
(raya)
BSH-1 Hisar 19.7 62.7 68.5 25.6 –
(Brown
Sarson)
YSPb-24 Hisar 19.7 69.9 71.5 35.4 3.5
(Yellow
Sarson)
RLM-514 Ludhiana 41.5 62.5 33.8 48.5 –
(raya)
YS-151 Pantnagar 11.0 14.9 26.6 45.0 –
(Yellow
Sarson)
Varuna Pantnagar 9.2 15.2 40.0 34.1 –
(raya)
Difolatan @ 2-g product/L water, Blitox @ 2.5-g product/L water and Dithane M-45 @ 2-g product/L water were
sprayed at Hisar, Ludhiana and Pantnagar, respectively.
* Significant @5%

Table 2.4 Influence of Alternaria pod infection on yield components of raya cultivar Parkash (Kadian and Saharan
1983)
Yield components
Oil
Category of Pod lengtha No. of seeds/ No. of infected 1000-seed Seed content
infection (cm) pod seeds/pod weight (g) germination (%) (%)
0. Healthy pods 7.2 19.4 0.0 2.3 93 40.5
1. Superficial 7.1 19.4 0.0 2.3 93 40.2
lesions on pod
2. One to two deep 7.1 19.4 0.9 2.3 91 37.9
lesions/pod
3. Three to five 6.4 17.4 2.4 2.2 82 37.7
deep lesions/pod
4. More than five 6.4 18.1 3.6 2.1 72 36.6
deep lesions/pod
Correlation (r) 0.92 0.97 0.89
Average of 1000 pods
a

Sarson (26 %) and mustard (17–18 %) (Saharan Bandyopadhya et al. 1974; Chahal and Kang,
1984, 1992a). There is a reduction in oil content 1979; Kadian and Saharan 1983; Nijhawan and
from 1 to 10 % in the infected seeds (Tables 2.4, Hussain 1964; Kolte et al. 1987; Saharan 1984;
2.5, 2.6 and 2.7). Deep lesions on the Brassica Singh and Bhowmik 1981; Tripathi et al. 1987).
siliquae increased the percentage of seed infec- Daebeler and Amelung (1988) and Saharan
tion and decreased pod length, seeds per pod, (1991) correlated disease intensity on foliage
1000-seed weight, percent seed germination and and/or siliquae with the components of yield
percent oil content (Tables 2.3, 2.4, 2.5 and 2.6; losses of rapeseed–mustard. The increase in
36 2 The Disease

Table 2.5 Influence of Alternaria pod infection on yield components of Brown Sarson (Kadian and Saharan 1983)
Yield components
No. of Seed
Pod lengtha No. of infected 1000-seed germination Oil content
Category of infection (cm) seeds/pod seeds/pod weight (g) (%) (%)
0. Healthy pods 5.9 18.4 0.0 2.3 97.5 43.4
1. Superficial 4.8 18.2 0.5 2.2 96.5 43.2
lesions on pod
2. One to two deep 4.8 18.1 2.8 2.1 85.5 42.5
lesions/pod
3. Three to five deep 5.0 17.9 3.9 2.0 81.0 41.3
lesions/pod
4. More than five 4.9 17.9 4.8 2.0 70.0 38.9
deep lesions/pod
Correlation (r) 0.98 0.92 0.87
a
Average of 1000 pods

Table 2.6 Influence of Alternaria pod infection on yield components of Yellow Sarson (Kadian and Saharan 1983)
Yield components
Pod Seed Oil
lengtha No. of seeds/ No. infected 1000-seed germination content
Category of infection (cm) pod seeds/pod weight (g) (%) (%)
0. Healthy pods 7.3 19.3 0.0 3.0 95.0 45.7
1. Superficial lesions 7.3 19.3 0.0 3.0 95.0 45.6
on pod
2. One to two deep 7.2 17.6 1.5 2.9 90.0 44.3
lesions/pod
3. Three to five deep 7.0 17.8 1.8 2.6 84.0 43.6
lesions/pod
4. More than five 7.6 19.6 3.5 2.2 69.5 40.3
deep lesions/pod
Correlation (r) 0.91 0.86 0.90
Average of 1000 pods
a

number of deep lesions on pods causes a steep Verma and Saharan (1994) reported yield losses
rise in the number of infected seeds per pod, and ranging from 10 to 75 % in oilseed Brassica in
sharp decline in percent seed germination, and India. In a field experiment, Barman and Bhagwati
percent oil content of rapeseed–mustard crops (1995) found that severely infected plants yield
(Saharan 1991; Table 2.7). According to Ansari fewer seeds as compared to the healthy plants.
et al. (1988), the loss in oil content of the seed Alternaria infection generally increases green
from rapeseed-diseased plants, over the seeds seed counts in B. rapa and causes seed shrivel-
from healthy plants, ranged between 14.6 and ling and substantial yield reductions in B. rapa
36.0 %. Under Nepal conditions, Alternaria and B. napus (Seidle et al. 1995). Pod infection
blight of mustard causes 32–57 % loss in yield results in increased losses compared to the infec-
along with 4.2 to 4.5 % losses in oil content tion on leaves. Deep lesions on the pods and
(Shrestha et al. 2005). increased seed infection reduce pod length, seed/
Jain (1992) reported yield loss up to 56 % in cer- pod, 1000-seed weight, seed germination and oil
tain Eruca sativa cultivars, and in Yellow Sarson, content. Losses due to pod infection have been
the loss in yield went up to the extent of 70 %. found to be heavier in B. campestris than other
2.6 Yield Losses 37

Table 2.7 Effect of black spot on number of infected seeds per pod (a) per cent seed germination (b) and per cent oil
content (s) of rapeseed–mustard (Saharan 1991)

Infection B. juncea B. campestris var. Brown Sarson B. campestris var. Yellow Sarson
category a b c a b c a b c
0 0 95.5 40.5 0 96.5 43.5 0 95.0 45.7
1 0 95.0 40.0 0.54 96.0 43.2 0.85 94.0 45.0
2 0.85 91.5 37.8 2.76 82.5 42.5 2.56 90.5 44.0
3 2.40 83.0 37.0 3.90 80.0 40.3 2.96 79.5 43.2
4 3.62 71.5 36.5 4.95 69.5 36.9 3.69 65.0 39.0
5 6.56 50.5 32.0 5.50 42.0 34.9 4.50 41.5 34.5
– −0.97 −0.89 – −0.92 −0.87 – −0.86 −0.90
0 = Healthy pods, 1 = minute superficial lesions on pods, 2 = one to two deep lesions on pods, 3 = three to five deep
lesions on pods, 4 = five to eight deep lesions on pods, 5 = more than nine deep lesions on pods

Brassica spp. Alternaria brassicae causes maxi-

mum average yield loss of 27.53 % in Yellow Y healthy seeds = 217.13 - 15.53 DS ( r 2 = 0.875 ) and
Sarson, 17.16 % in B. napus and 10.72 % in B. Y diseased seeds = 136. 57 - 7.10 DS ( r 2 = 0.844 ) .
carinata in Himachal Pradesh (Kumar 1997).
Gupta et al. (1998) in their detailed studies on
Alternaria leaf blight-induced changes in fatty However, the percent yield loss caused by 1 %
acid composition of siliquae wall and seeds of DS is 1.66 % in 1997–1998, but 7.2 and 5.2 %,
mustard (Brassica juncea L.) observed that eru- respectively, for healthy and diseased seeds in
cic acid decreases, while linoleic acid increases 1998–1999.
in the siliquae walls and seeds of A. brassicae- Prasad et al. (2003) assessed yield losses due
and A. brassicicola-infected seeds while linoleic to Alternaria blight in (1999–2001) Indian mus-
acid remained unchanged in the seeds. Disease tard genotypes PAB 9534, PAB 9511, JMM 915,
increased palmitic acid and oleic acid in the sili- RN 490 and Varuna under protected and unpro-
quae. However, it does not affect palmitic acid, tected conditions using Varuna and PAB 9511 as
but oleic acid is lower in diseased than in healthy the susceptible and resistant controls, respec-
seeds. Ram and Chauhan (1998) estimated the tively. The protected plots were sprayed with
loss in seed yield from 28.6 to 71.4 %. Seed ger- 0.25 % mancozeb starting from 40 days after
mination is adversely affected in Indian mustard, sowing and 3 subsequent sprays at 15-day inter-
and B. napus at infection levels 3 to 5, while seed vals. The disease appeared 45 days after sowing.
germination is adversely affected in Brassica The highest disease intensity was recorded at
campestris at levels 2 to 5 (Kumar 2001). flowering and pod formation stage. Treatment
Meah et al. (2002) observed quantitative rela- with mancozeb reduced disease incidence in all
tionships between yield of mustard (Brassica the genotypes. The highest disease intensity (46.1
campestris cv. BINA 2) and disease parameters and 41.95 %) was recorded in Varuna for both
of Alternaria blight in five field trials conducted years. In both years, the highest reduction (72.6
under natural conditions in Bangladesh. Yield and 59.0 %) in disease severity was recorded in
(Y) measured as seed weight in g/m2 is linearly RN 490 and lowest (17.8 and 16.1 %) in the pro-
related to disease severity (DS), assessed at tected plots compared with the unprotected plots
65-day-age growth stage (1997–1998): (39.6 and 32.5 %). The highest seed yield loss
(20.8 and 21.9 %) was observed in Varuna under
Y = 203.78 - 3.38 DS ( r 2 = 0.47 ) . unprotected conditions; however, it also gives the
highest seed yield (20.3 and 19.5 q/ha) followed
DS significantly reduced Y and this relation is by RN 490 (18.5 and 18.3 q/ha) in the protected
influenced by seed quality (1998–1999). plots. Pooled analysis of data revealed that
38 2 The Disease

Varuna produced the highest disease intensity Table 2.8 Influence of Alternaria infection on siliquae
of Crambe abyssinica (Czyzewska 1971)
(22.0 and 44.0 %) and yield performance (19.9
and 15.7 q/ha) in protected and unprotected plots, 1000-
respectively. The 1000-g seed weight of RN Seed Healthy seed
Infection germination seedlings weight
490 in protected (5.2 g) and unprotected (4.8 g) categories (%) (%) (g)
plots was similar to Varuna. In West Bengal, 1. None 96.0 67.0 7.9
Alternaria blight of rapeseed–mustard causes 2. Slight 95.0 62.5 7.7
loss in yield of about 47 % (Mondal et al. 2007). 3. Moderate 94.5 45.5 6.9
In Europe, epidemics occur on rape about two 4. Strong 82.0 45.0 3.2
years in every five, and in these years losses may 5. Very 66.2 39.5 1.7
be as high as 60 % in individual crops. In rape, Strong
and other brassicaceous seed crops, A. brassicae 1. No infection
causes yield loss due to premature ripening and 2. Slight infection: siliquae normal in size, several small
spots on the fruit pod surface, normally developed seeds
shedding of seed before harvest and by reducing
3. Moderate infection: siliquae somewhat smaller or nor-
the 1000 grain weight. In England, the disease mal in size, numerous spots on fruit hull, seeds smaller
first caused serious losses in rape in 1980–81, 4. Strong infection: siliquae smaller in size, almost the
and since then an estimated £3–4 million have whole surface of the fruit pod covered with black spots,
seeds small
been spent annually on fungicidal control (Smith
5. Very strong infection: siliquae deformed, the whole sur-
et al. 1988). In Germany, A. brassicae caused face of the fruit pod covered with black spots, seeds
75 % losses in rape (Klemm 1938; Raabe 1939). shrunken, small, sometimes dry and black, in which case
In Lithuanian, Alternaria blight damaged 37.2 to only the seed pod is left
100 % rape siliquae with disease severity of 6.66
to 7.24 % (Brazauskiene and Petraitiene 2006).
heads of cabbage, turnip and rutabaga. The disease
can be destructive in seedbeds, especially in cab-
2.6.2 Crambe bage, cauliflower and Brussels sprouts. Spotting
and browning of cauliflower, broccoli and cabbage
In Poland during 1953 and 1954, Alternaria blight heads reduce quality and market value of these
of C. abyssinica caused very poor germination, crops (Sherf and Macnab 1986). Alternaria bras-
and 80 % of the planting had to be ploughed in or sicicola is the most important pathogen of B.
sown again (Czyzewska 1969). Severely diseased oleracea seed crops. Reduction in seed yield may
plants produced small, wrinkled, deformed sili- be as high as 80 %, and the pathogen may severely
quae with small shrivelled and discoloured seeds depress germination to the extent that infected
having poor viability. Diseased seeds cause damp- seeds may be unsalable (Smith et al. 1988).
ing-off of seedlings leading to lower plant popula- Alternaria brassicicola has been recognized as an
tion in the field. The weight of 1000 siliquae can important cause of deterioration of white cabbage
drop three times depending on the degree of infec- in cold storage (Kear et al. 1977). According to
tion, and the weight of 1000 seeds can drop more Gorshkov (1976), damping-off of cabbage by A.
than four times. The decrease in germination and brassicae may result into 80–100 % losses. In
the number of healthy seedlings can be more than wet seasons in the USA, A. brassicae leaf spot on
30 % (Table 2.8) (Czyzewska 1969, 1971; cabbage, cauliflower, broccoli and other brassi-
Holcomb and Newman 1970; Leppik 1973). cas has been known to reduce the yield by more
than 50 %. The market losses are due to decay,
which develops in transit and storage (Ramsey
2.6.3  egetable Crops (Cruciferous
V and Smith 1961). In Magdeburg district of
Vegetables) Germany, Alternaria caused seed losses of up to
50 % in cauliflower seed plants (Stoll 1948) and
In vegetable crops, losses occur from damping-­off 70 to 90 % in rape and seed cabbage plantings
of seedlings, and spotting of lower leaves, and (Domsch 1957).
2.7 Disease Assessment Keys/Severity Charts 39

Alternaria alternata infection in radish keys for assessment of disease severity used by
reduces pod length, number of seeds per pod and different workers are given in Table 2.9. These
thousand-seed weight leading to reduction in keys measure the disease in a scoring scale of
seed yield as much as 18 % (Suhag et al. 1983). 0–5 or 0–9. The zero (0) score indicates no symp-
toms on any part of the host, and 1–5 or 1–9
scores indicate the presence of different degrees
2.7  isease Assessment Keys/
D of disease symptoms on various host parts. A
Severity Charts score of 1 is indicative of the least amount of dis-
ease, and scores 5 or 9 indicate the maximum
Alternaria diseases can be assessed by various disease (75 % or more) of the infected host.
methods described below: These descriptive keys are being successfully
used for estimating disease severity of plants
with differentiated disease resistance or of plants
2.7.1 Visual Assessment Methods subjected to different environmental conditions
or cultural procedures. Host resistance is mea-
There are two well-documented visual assess- sured by grouping into resistant (0–1), moder-
ment methods. ately resistant (1.1–3), moderately susceptible
(3.1–5), susceptible (5.1–7) and highly suscepti-
2.7.1.1 Descriptive Keys ble (7.1–9). Quantitative estimates of the disease
In diagrams, the plants with varying amounts and indices are calculated using numerical rating
kinds of disease symptoms are categorized with based on the severity of the disease (McKinney
an accompanying description. The descriptive 1923):

Disease Index ( % ) =
åsample frequency ´ numerical rating1) +¼sample frequency ´ numerical rating 5 or 9) ´100
Total no. samples ´ maximum numerical rating 5 or 9

Disease assessments recorded at different growth (Table 2.9). Several plants (20 or more) at ran-
stages of the crop will help produce temporal dis- dom are graded.
ease progress curves. Growth stage key of rape-
grade reading
seed is given in Table 2.10. Mean grade =
number of reading
An improved grading system for assessing
plant diseases proposed by Horsfall and Barratt A calibration curve is set up with grade num-
(1945) has been successfully used by Fontem bers on the x-axis and percentage disease on a
et al. (1991) to measure the temporal progress special semilog. Y-axis with one and one-half
and spread of dark leaf spot in cabbage. This phases from either end up to 50 %. This scheme
grading is based on the principle that according has been very useful in testing the efficacy of
to the Weber–Fechner law, the human eye distin- fungicides and varietal resistance and in surveys
guishes according to the logarithm of the light of plant disease. Fontem et al. (1991) used the
intensity. Hence, the grades should be based on conversion table of Redman et al. (1967) to con-
the ability to distinguish, rather than the extent of vert the Horsfall–Barratt rating score to disease
the disease. Below 50 %, the eye sees the amount proportions in black spot of cabbage.
of diseased tissue. Above 50 %, it sees the amount
of diseased free tissue. This scoring system 2.7.1.2 Standard Area Diagrams
(1–12) is based on 50 % as a midpoint. The Pictorial representation of the host plant with known
grades differ by a factor of two in either direction and graded amounts of disease is compared with
40 2 The Disease

Table 2.9 Keys for the assessment of Alternaria disease 7. 50–75 % 62.50 62.50
severity on crucifers (Mayee and Datar 1986; Verma and area
Saharan 1994) diseased
Scoring scale Description 8. 75–87 % 81.50 81.25
0. No symptoms on leaf area
diseased
1. Small, round, black dots
covering 1 % or less of the 9. 87–94 % 91.00 90.63
leaf area area
diseased
3. Grey, circular spots
containing concentric rings 10. 94–97 % 96.50 96.31
to cover 1–10 % area area
diseased
5. Lesions enlarge, grey
circular spots with 11. 97–100 % 98.50 97.66
concentric rings and a black area
border to cover 11–25 % of diseased
the leaf area 12. 100 % area 100 100
7. Lesions enlarge and coalesce diseased
with each other to cover a
Redman et al. (1967)
26–50 % of the leaf area
9. Lesions coalesced with Braverman (1971)
concentric rings and black 0. Zero percent leaf area
border to cover 50 % or affected
more of leaf area; defoliation 0.5 Traces to 10 % leaf area
occurs affected
Saharan, 1991 1.0 11–20 % leaf area affected
0. Healthy pods 1.5 21–30 % leaf area affected
1. Minute superficial lesions 2.0 31–40 % leaf area affected
per pod 2.5 41–50 % leaf area affected
2. One to two deep lesions per 3.0 51–60 % leaf area affected
pod
3.5 61–70 % leaf area affected
3. Three to five deep lesions
4.0 71–80 % leaf area affected
per pod
4.5 81–90 % leaf area affected
4. Five to eight deep lesions
per pod 5.0 91–100 % leaf area affected
5. More than nine deep lesions Alternaria blight leaf phase, scoring using 0–5
per pod modified scale (Saharan 1997; Krishnia et al. 2000)
Scoring scale: Alternaria blight leaf phase
Horsfall and Barratt (1945), Fontem et al. (1991)
Grade Description Disease reaction
Values to convert back to 0%
0 No Resistant
Midpoint Elanco Formula %a
symptoms
1. No 0 0 on leaf
symptoms 1 Small,
2. 0–3 % area 1.50 2.34 round, black
diseased dots
3. 3–6 % area 4.50 4.68 covering up
diseased to 1 % of
4. 6–12 % area 9.00 9.37 the leaf area
diseased 2 Grey circular
5. 12–25 % 18.50 18.75 spots
area containing
diseased concentric
rings to cover
6. 25–50 % 37.50 37.50
1.1–10 % of
area
leaf area
diseased
(continued)
(continued)
2.7 Disease Assessment Keys/Severity Charts 41

Table 2.9 (continued) Table 2.10 Growth stage key for oilseed rape (NIAB
1985)
Grade Description Disease reaction
3 Lesions Susceptible Decimal
enlarge, Growth stage code Characteristics
grey circular Germination 0.0 Dry seed
spots with Emergence 0.8 Cotyledons emerged
concentric
Leaf production 1.0 Cotyledons unfolded
rings and
black 1.01 First true leaf
boarder to emerged
cover 1.02 to 1.09 Second true leaf
10.1–25 % emerged
of the leaf Stem extension 2.00 No internodes
area (rosette)
4 Lesions 2.01 One internode
enlarge and 2.02 to 2.09 Two internodes
coalesce
Flower and bud 3.0 Leaf buds only
with each
development 3.3 Green flower buds
other to
cover visible
25.1–50 % 3.7 Yellow flower buds
leaf area visible
5 Lesions Flowering 4.1 First flower opened
coalesced, 4.2 20 % buds on
with terminal racemes
concentric flowered
rings and 4.3 to 4.9 30 % buds flowered
black
boarder to Pod 5.1 Lowest pods 2 cm
cover 50 % development long
of the leaf 5.2 20 % pods 2-cm long
area 5.3 to 5.9 30 % pods 2-cm long
The Alternaria blight infection scoring on siliquae Seed 6.1 Seeds present
using 0–5 modified disease rating scale development 6.2 Seeds green
Scoring scale: Alternaria blight siliquae phase 6.5 Seeds brown
Grade Description Disease reaction 6.7 Seeds black but soft
0 Healthy Resistant 6.9 All seeds black and
siliquae hard
1 Minute
superficial
lesions per
siliquae
2 One to two
deep lesions diseased leaves and/or siliquae to allow estimation
per siliquae of disease severity. It consists of a set of pictures
3 Three to five Susceptible giving a schematized illustration of the grades dis-
deep lesions tinguished as in Fig. 2.1 (Conn et al. 1990).
per siliquae
In contrast to descriptive keys, standard area
4 Six to nine
diagrams allow estimation of intermediate levels
deep lesions
per siliquae of disease severity by comparing a diseased plant
5 More than with diagrams showing both more and less
nine deep ­disease. To calculate the disease severity, the leaf
lesions per and/or siliquae to be assessed are matched to
siliquae
one of the diagrams of the black areas, or area
42 2 The Disease

d­ amaged, shown (representing 1, 5, 10, 20, 30 by the actual lesions. Disease severity is
and 50 %) for each leaf and/or siliquae covered ­calculated using the following formula:

Area of the plant tissue affected by disease

Disease severity ( Area ) % = - ´100
Total area

Fig. 2.1 A schematized drawings of (a) leaves and (b) siliquae of crucifers showing Alternaria infection grades (Conn
et al. 1990)
2.7 Disease Assessment Keys/Severity Charts 43

Although only a few representative percentage especially in understanding the type of disease
infections (1, 5, 10, 20, 30, 50) are given in the spread in a sampling unit in Alternaria diseases.
diagrams for assessment of severity, interpolations
for arriving at the middle-level percentage such as
2, 3, 15, 30, 40, 60 etc. can be easily practised and 2.7.3 Inoculum–Disease Intensity
recorded. The extent of interpolation will be dic- Relationships
tated by the ability of the observer to detect par-
ticular differences. Once the severity percentages An alternate method of disease assessment, espe-
are decided upon, the visual scales/keys can be cially in studies of host resistance, is based on the
used for categorization. However, once the number of spores produced per lesion. While
observer becomes familiar with the diagrams, the analysing components of horizontal resistance in
ranking numbers can be used as such for compari- rapeseed–mustard cultivars against A. brassicae,
son between the genotypes on 0–5 or 0–9 scale. It Saharan and Kadian (1983) indicated spore count
is expected that 10 % of the representative popula- per lesion as one of the most important parame-
tion be considered for assessment. The percentage ters. An optimum inoculum concentration must
severity data as such can be transformed for any be determined to obtain differences in suscepti-
subsequent epidemiological analysis by using the bility among cultivars/genotypes (Dueck and
disease index formula given earlier. The standard Degenhardt 1975).
area diagrams constructed by James (1974)
account for the logarithmic decrease in the activity
of the eye in estimating disease severities 2.7.4 Remote Sensing Method
approaching 50 % in their selection of representa-
tive keys. Estimations of disease severity interme- Aerial infrared photography using remote sens-
diate between two keys are often made by careful ing procedure is commonly utilized to detect
interpolation. As logistic transformation fits many plant diseases. The detection of diseased plant
disease progress curves, equal interval grades on tissue on false-colour infrared film is due to its
the logit scale are being often used. greater reflection of near infrared light (700–
950 nm) compared to healthy tissue. Colour
infrared photographs have been analysed with
2.7.2 Incidence–Severity micro-densitometers or other types of electronic
Relationships scanning devices to quantify the disease severity.
This technique is useful to employ in areas where
The relationship between disease incidence and the same crop is contiguous over large areas. The
disease intensity (severity) (I–S relationship) is use of this technique in assessing incidence of
an epidemiologically significant concept. Since Alternaria diseases has not yet been reported.
incidence is easier to measure than severity, any
quantifiable relationship between the two mea-
sures permits estimation of severity based upon 2.7.5 Video Image Analysis
incidence data, which are more precise and easily
acquired. Where resource limitations prevent the This method can be easily employed for assess-
collection of accurate severity measurements, ing Alternaria diseases of brassicaceous plants
estimation of severity based upon incidence data since its lesions and healthy tissue have different
will be highly beneficial in disease and yield loss colours. Recent advances in electronic and com-
assessments. The I–S relationship can be used puter technology allow video cameras to inter-
through the analysis of correlation, and regres- face directly with a microcomputer. Rapid,
sion, multiple infection methods, and measure- automated, nonsubjective estimates of disease
ment of aggregation (Seem 1984). There is severity are made possible by computer-­
enormous scope in exploring the I–S relationship controlled analysis of video images. The ­accuracy
44 2 The Disease

of estimates of disease severity, obtained using t­rials, the correlations between MP and Yp and
video analysis, is high and appears to be indepen- MP and YS would be positive. Thus, selection
dent of both the complexity of the host–pathogen based on MP generally increases the average per-
system and disease severity. formance in both disease and no disease environ-
ments. However, MP fails to distinguish the
group A and group B genotypes.
2.7.6 Stress Tolerance Attributes
Disease Tolerance ( TOL ) = Yp - Ys
Gupta et al. (2002) assessed stress tolerance attri- A larger value of TOL represents relatively
butes for the performance of cultivars. Genotypes more sensitivity to disease stress; thus, a smaller
can be categorized in the following four groups value of TOL is favoured. Selection based on
based on their performance in disease and no dis- TOL favours genotypes with low yield potential
ease stress environment: group A (genotypes under no disease stress conditions and high yield
expressing uniform superiority in both disease under disease stress conditions. Under most yield
and no disease stress environment), group B trails, the correlations between TOL and Yp
(genotypes performing favourably only in no dis- would be positive. Thus, TOL fails to distinguish
ease stress environment), group C (genotypes between group C and group A.
yielding relatively higher only in disease stress
environment) and group D (genotypes perform- 1 - Ys / Yp
ing poorly in both disease and no disease stress Diseasestresssusceptibilityindex ( DSSI ) =
DSI
environment).
The optimal selection criteria should distin-
guish group A from the other three groups. The smaller the value of DSSI, the greater is the
Let disease stress tolerance. Under most yield trials,
TOL and DSSI are positively correlated. Selection
YP = the potential yield of a given genotype in no based on DSSI favours genotypes with low yield
disease stress environment potential and high yield stress conditions. Thus,
YS = the yield of a given genotype in disease DSSI also fails to distinguish group A from group
stress environment C.
YP− = mean yield in no disease stress
Geometric mean productivity ( GMP ) = Ys ´ Yp
environment
YS− = mean yield in disease stress environment
GMP is based on the arithmetic means, and there-
The following stress tolerance attributes were fore, it has an upward biasness due to a relatively
defined from these four yield measurements: larger difference between Yp and Ys, whereas
YS- the geometric mean is less sensitive to large
DiseaseStress Intensity ( DSI ) = extreme values. Thus, GMP is a better indicator
1 - YP- than MP in separating group A from other groups.

It ranges between 0 and 1, and the larger the

value of DSI, the more severe is the stress Disease stress tolerance index ( DSTI ) =
(Y )(Y )
p s

(Y )
2
intensity.
p-
Y + YP
Mean productivity ( MP ) = S
2
DSTI is estimated based on GMP and thus the
This index favours higher yield potential and rank correlation between DSTI and GMP is equal
lower disease stress tolerance. Rosielle and to 1. The higher the value of DSTI for a geno-
Hamblin (1981) showed that under most yield type, the higher are its disease tolerance and yield
References 45

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An Foras Taluntais, pp 45–46
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3

3.1 Introduction pathogen survives through infected seed, crop

residue, cruciferous hosts, weeds, microsclerotia
Alternaria species pathogenic on cruciferous and chlamydospores and all act as source of pri-
crops are A. alternata, A. brassicae, A. brassici- mary inoculum. Secondary infection during the
cola and A. raphani. Among the four species of crop season is through conidia produced on
Alternaria, A. brassicae is more common on oil- infected host plants (under high humidity), which
yielding crops, while other three species are spread through rain splashes and wind to com-
major pathogens of vegetable crops. Phylogeny, plete the disease cycle. Major identification char-
taxonomy, morphology, classification, infection acteristics of Alternaria species infecting
process, pathogenesis, disease cycle and identifi- rapeseed–mustard crops are given in Table 3.1.
cation characteristics along with synonymous of
all four species have been well documented.
Identification, cloning and sequencing of viru- 3.2 Historical
lence genes of Alternaria infecting crucifers have
resolved some doubts about their relationship As early as 1836, Berkeley identified the causal
with cruciferous hosts. Pathogenicity factor and fungus on plants belonging to the Brassicaceae as
transcription factor Amr 1 have been identified in Macrosporium brassicae Berk., which was later
A. brassicicola. A non-ribosomal peptide syn- renamed as A. brassicae (Berk.) Sacc. by
thase gene (AbNPs2) is important for cell wall Saccardo (1886). In 1922, 1926 and 1945, respec-
integrity, conidial viability and virulence of aged tively, Milbraith, Weimer and Rangel also
spores of A. brassicicola. More than 100 genes described the same fungus. Then in 1947,
have been functionally analysed through various Wiltshire separated the small and big spore forms
techniques like gene knockout and overexpres- as A. brassicicola (Sch.) Wiltshire and A. brassi-
sions making A. brassicicola the species of cae (Berk.) Sacc., respectively.
choice for functional genomics research. For Every modern attempt to define the phaeodic-
minimum, maximum and optimum growth, and tyosporic form genera Alternaria Nees ex Fries
sporulation of Alternaria species pathogenic on and Stemphylium Wallroth has involved the prob-
crucifers, in vitro studies have been carried out. lem of treating numerous taxa which superfi-
The suitable culture media, temperature, relative cially resemble the type species of one or the
humidity, pH, nutrient sources and light and other of these genera, but which are sufficiently
darkness conditions for growth, sporulation and different as to leave a measure of doubt. Chief
spore germination have been determined. The among these anomalous species are Stemphylium

© Springer Science+Business Media Singapore 2016 53

G.S. Saharan et al., Alternaria Diseases of Crucifers: Biology, Ecology and Disease Management,
DOI 10.1007/978-981-10-0021-8_3
54 3 Pathogen

Table 3.1 Identification characteristics of Alternaria species infecting rapeseed–mustard

Fungal structures A. brassicae A. brassicicola A. raphani
Mycelium Septate, brownish grey Septate, olive grey to Cottony whitish to
greyish black greenish grey or dark grey
Conidiophore Dark, septate, arise in Olivaceous, septate, Septate, olive brown,
fascicles, 14–74 μ × 4–8 μ branched, 35–45 μ × 5–8 μ single or branched,
29–160 μ × 4–8 μ
Conidia Brownish black, obclavate, Dark cylindrical to oblong, Olive brown to dark,
muriform, produced singly muriform, produced in obclavate, muriform, more
or in chains or 2–3 chains of 8–10 spores or less pin pointed at each
end, appears singly or in
chains of up to 6 spores
Spore body (μ) 96–114 × 17–24 45–55 × 11–16 45–58 × 13–21
Spore beak length (μ) 45–65 None 1–25
Spore transverse septation 10–11 5–8 6–9
Longitudinal septation 0–6 0–4 3–6
Rate of growth and Rudimentary slow growth Black sooty colony with Cottony mycelial colony
sporulation on media distinct zonations, fast with less abundant
growing with abundant sporulation
sporulation
Infection Penetrates leaf only Penetrates leaf directly or Direct penetration
through stomata through stomata

lanuginosum Harz. and Macrosporium consor- inal sense (and in the sense of Wiltshire’s section
tiale Thiimen, both of which, at one time or Eustemphylium) and transferred to Alternaria
another, have been named Alternaria, several of the taxa similar to S. lanuginosum
Stemphylium or Pseudostemphylium (Simmons which were considered controversial by earlier
1967). students of the group. Most taxonomists recog-
Wiltshire (1933, 1938) pioneered the basic nized that Alternaria and Stemphylium were
studies of this group of Hyphomycetes and pub- inappropriate generic designations for species
lished the results of his examination of the avail- similar to S. lanuginosum. By their nomencla-
able type specimens. His descriptive literature tural proposals or, more importantly, by their
was fundamental to the prevailing concepts of invariably excellent illustrations, conidiophores
Alternaria, Macrosporium and Stemphylium. His of members of the S. lanuginosum group bear no
major conclusions were that Macrosporium resemblance whatsoever to those of S. botryo-
should be suppressed as a nomen ambiguum in sum, and the conidial morphology of the group is
favour of Alternaria, typified by A. tenuis Nees, fundamentally different from that of the type spe-
the type specimen of which Wiltshire was unable cies of Alternaria.
to locate for examination, and that the limits of There are two species of Alternaria infecting
Stemphylium should be modified to include two Brassica species which are probably the most
sections. controversial than any other species in this genus
Neergaard (1945), in his extensive treatment (Wiltshire 1947). The first is A. brassicae (Berk.)
of species of Alternaria and Stemphylium occur- Sacc. described as Macrosporium brassicae
ring in Denmark, recognized the same taxonomic Berk. in 1836, and the second is the fungus com-
problems in handling species similar to S. lanugi- monly known as A. circinans (Berk. & Curt.)
nosum. He also followed the lead of Wiltshire by Bolle. or A. oleracea Milbraith or incorrectly as
retaining the two sections proposed for A. brassicae (Berk.) Sacc. for which Wiltshire
Stemphylium. Joly (1964) in his survey of (1947) proposed the name A. brassicicola
Alternaria differentiated Stemphylium in its orig- (Schwein.).
3.3 Phylogeny 55

3.3 Phylogeny U. capsici clusters separate from sect.

Pseudoulocladium. The TEF1 phylogenies did
For redefining the taxonomy of Alternaria and not support sect. Nimbya and show relative low
allied genera, 121 strains were included in the support for sect. Cheiranthus, sect. Dianthicola,
Alternaria complex alignment by Woudenberg sect. Embellisioides, sect. Panax, sect.
et al. (2013). The alignment length and unique Phragmosporae and sect. Radicina and A. cumini
site patterns of the different genes and gene com- clusters outside sect. Eureka. In the two-region
binations were well defined. The original ITS phylogenies, U. capsici clusters outside sect.
alignment consisted of 577 characters of which Pseudoulocladium based on GAPDH and RPB2,
the first 78 were excluded as this contained a non- E. indefossa clusters outside sect Cheiranthus
alignable region. In the original TEF1 alignment, based on GAPDH and TEF1 and sect. Eureka are
375 characters were coded as major inserts, poorly supported based on RPB2 and TEF1. The
which otherwise would negatively influence the combined phylogeny based on the GAPDH,
phylogeny, resulting in a TEF1 alignment of 269 RPB2 and TEF1 sequences is displayed, as these
characters. All the phylogenies, different phylo- are the genes with the best resolution.
genetic methods and gene regions or gene combi- The final Pleosporineae alignment included
nations used on this dataset (data not shown; 74 strains, representing six families, and consisted
trees and alignment lodged in TreeBASE) show a of 2506 characters (SSU 935, LSU 796. RPB2
weak support at the deeper nodes of the tree. The 775) of which 700 were unique site patterns (SSU
only well-supported node (Bayesian posterior 111 LSU 145. RPB2 444). In the SSU alignment,
probability of 1.0, RAxML Maximum Likelihood a large insertion at position 446 in the isolates
support value of 100) in all phylogenies separates Chaetosphaeronema hispidulum CBS 216. 75,
Embellisia annulata CBS 302.84 and the Pleospora fallens CBS 161.78, Pleospora flavi-
Ploeospora/Stemphylium clade from the gena CBS 314.80 and Ophiosphaerella herpotri-
Alternaria complex. In the Alternaria clade, six chia CBS 620.86 was excluded from the
monotypic lineages and 24 internal clades occur phylogenetic analyses. A total of 43 202 trees
consistently in the individual, and combined phy- were sampled after the burn-in. The type species
logenies, although positions vary between the of Clathrospora, C. elynne, forms a well-sup-
different gene regions or combinations used. The ported clade, located basal to the Pleosporaceae
support values for the clades within Alternaria (Fig. 3.1) outside the Alternaria complex. The
(called sections) were plotted in a heat map per type species of Comoclathris, C. lanata, was not
gene and phylogenetic method used. The support available for the study, but the two Comoclathris
values for the different phylogenetic methods compressa strains cluster in a well-supported
vary, with the Bayesian posterior probabilities clade within the Pleosporaceae outside
being higher than the RAxML bootstrap support Alternaria s. str.
values. The SSU, LSU and ITS phylogenies dis- The genus Alternariaster with Alternariaster
play a low resolution, which reflects in poor to no helianthi as type and only species also clusters
support of the sections. Therefore, Woudenberg outside the Alternaria complex and even outside
et al. (2013) choose not to include them in the Pleosporaceae; it belongs to the Leptosphaeriaceae
multigene alignments, except in the all-gene instead (Fig. 3.1). Embellisia annulata is identical
alignment. In the GAPDH phylogenies, sect. to Dendryphiella salina and forms a well-sup-
Cheiranthus, sect. Nimbya and sect. ported clade in the Pleosporaceae together with
Pseudoulocladium are poorly supported, and A. Dendryphiella arenaria. As the type species of
resedae clusters separate from sect. Cheiranthus. Dendryphiella, D. vinosa, clusters outside the
In the RPB2 phylogenies, the support values for Pleosporineae (Cruz 2006; Jones et al. 2008),
sect. Alternata, sect. Embellisioidos and sect. Dendryphiella salina and D. arenaria are placed
Euroka are relatively low; A. cumini clusters in in a new genus, Paradendryphiella (Woudenberg
sect. Embellisioidos instead of sect. Eureka and et al. 2013).
56 3 Pathogen

Fig. 3.1 Bayesian 50 % majority-rule consensus tree (ML) are given at the nodes (PP/ML). Thickness lines a
based on the SSU, LSU and RPB2 sequences of 74 strains PP of 1.0 and ML of 100. The tree was rooted to Julella
representing the Pleosporineae. The Bayesian posterior avicenniae (BCC 184220 (Woudenberg et al. 2013)
probabilities (PP) and RAxML bootstrap support values
3.4 Taxonomy, Nomenclature and Morphology 57

3.4 Taxonomy, Nomenclature = Nimbya E.G. Simmons, Sydowia 41: 316,

and Morphology 1989
= Allewia E.G. Simmons, Mycotaxon 38:
Based on DNA sequence data in combination with 260, 1990
a review of literature and morphology, the species = Crivellia Shoemaker & Inderb, Canad.
within the Alternaria clade, 121 strains represent- J. Bot. 84: 1308, 2006
ing the Alternaria complex, were all recognized = Chalastospora E.G. Simmons, CBS
Alternaria by Woudenberg et al. (2013). This puts Biodiversity Ser. (Utrecht) 6: 668, 2007
the genera Allewia, Brachycladium, = Teretispora E.G. Simmons, CBS
Chalastospora, Chmelia, Crivellia, Embellisia, Biodiversity Ser. (Utrecht) 6: 674, 2007
Lewia, Nimbya, Sinomyces, Teretispora, = Undifilum B.M. Pryor, Creamer, Shoemaker,
Ulocladium, Undifilum and Ybotromyces in syn- McLaln-Romero & Hambl., Botany 87: 190, 2009
onymy with Alternaria, resulting in the proposal = Sinomyces Yong Wang bis & X.G. Zhang,
of 32 new combinations, 10 new names and the Fungal Biol. 115: 192, 2011
resurrection of 10 names. Species of Alternaria
were assigned to 24 Alternaria sections, of which Colonies effuse usually grey, dark blackish
16 are newly described and six monotypic lin- brown or black. Mycelium immersed or partly
eages. The (emended) description of the genus superficial; hyphae colourless, olivaceous brown
Alternaria, the Alternaria sections and monotypic or brown. Stroma rarely formed. Setae and hypho-
lineages with new Alternaria names and name podia absent. Conidiophores macronematous,
combinations have been treated in alphabetical mononematous, simple or irregularly and loosely
order. Finally, the description of the new genus branched, pale brown or brown, solitary or in fas-
Paradendryphiella is also provided (Woudenberg cicles. Conidiogenous cells integrated, terminal
et al. 2013). becoming intercalary, polytretic, sympodial or
sometimes monotretic, cicatrized. Conidia cate-
Alternaria Nees, Syst. Pilze (Würzburg) 72: nate or solitary, dry, ovoid, obovoid, cylindrical,
1816 [1816–1817] narrowly ellipsoid or obclavate, beaked or non-
= Elosia Pers. Mycol. Eur. (Erlanga) 1: 12, 1822 beaked, pale or medium olivaceous brown to
= Macrosporium Fr. Syst. Mycol. (Lundae) 3: brown, smooth or verrucose, with transverse, and
373, 1982 with or without oblique or longitudinal septa.
= Rhopalidium Mont. Ann. Sci. Nat. Bot. Ser. Septa can be thick, dark and rigid, and an internal
2, 6: 30: 1836 cell-like structure can be formed. Species with
= Brachycladlum Corda, Icon Fungorum meristematic growth are known. Ascomata small,
hucusque Cogn (Prague) 2, 14: 1838 solitary to clustered, erumpent to (nearly) superfi-
= Ulocladium Preuss, Linnaea 24: 111, 1851 cial at maturity, globose to ovoid, dark brown,
= Chmelia Svob. Pol., Biologia (Bratislava) smooth, apically papillate, ostiolate. Papilla short,
21: 82, 1966 blunt; peridium thin. Hamathecium of cellular
= Embellisia E.G. Simmons, Mycologla 63: pseudoparaphyses. Asci few to many per ascoma,
380, 1971 (4–6) 8-spored, basal, bitunicate, fissitunicate,
= Trichoconiella B. L. Jain, Kavaka 3: 39, cylindrical to cylindro-clavate, straight or some-
1976 [1975] what curved, with a short, furcate pedicel.
= Botryomyces De Hoog & C. Rubio, Ascospores muriform, ellipsoid to fusoid, slightly
Sabouraudia 20: 19, 1982 (nom illegit.) constricted at septa, yellow-brown, without
= Lewia M.E. Barr& E.G. Simmons. guttules, smooth, 3–7 transverse septa, 1–2 series
Mycotaxon 25: 289, 1986 of longitudinal septa through the two original
= Ybotromyces Rulamort, Bull. Soc. Bot. central segments, end cells without septa, or
Centre-Ouest, Nouv. Ser. 17: 192, 1986 with 1 longitudinal or oblique septum or with a
58 3 Pathogen

Y-shaped pair of septa (Ellis 1971; Holliday spores). These conidia are broadest near the base
1980; Woudenberg et al. 2013). and gradually taper to an elongated beak, provid-
ing a club-like appearance. Conidia are produced
in single or branched chains on short, erect conid-
3.4.1 Type species: Alternaria iophores. Alternaria forms conidia that arise as
alternata (Fr.) Keissl protrusions of the protoplast through pores in the
conidiophore cell wall. At the onset of conidial
The description of Alternaria s. str. by Woudenberg development, the apex of the conidiophore thick-
et al. (2013) is supported by (1) a well-supported ens, and a ring-shaped electron-transparent struc-
phylogenetic node in multiple analyses; (2) high ture is deposited at the apical dome. At the central
similarity of clades within Alternaria based on cavity of this electron-transparent structure, a pore
SSU, LSU and ITS data; and (3) variation in the is formed through the dissolving of the cell wall.
order of clades between the different gene phylog- Through this pore, cytoplasm only covered by the
enies, which is in congruence with low support val- plasma membrane is pushed out. The turgor pres-
ues at these deeper nodes. They have followed the sure required to push the cytoplasm through the
procedure introduced by Lawrence et al. (2013) to pore is presumably provided by the well-
assign the taxonomic status of sections of Alternaria developed, large vacuole that appear at this stage
for the different clades found, thus allowing retain- in the conidiophore cell. Subsequent to this, a
ing the former generic names but associated with a nucleus migrates into the newborn conidium, and
different taxonomic status. For end users, this later on a cell wall is deposited (Honda et al. 1987,
seems to be a more stable and understandable tax- 1990). The melanin that is present in the conidia is
onomy and nomenclature (Woudenberg et al. 2013) concentrated in the outer region of the primary cell
walls, which are derived from the original wall of
the developing spore, and in the septa, which
3.5 Classification delimit individual spore cells in the multicellular
conidium. After the cells have been delimited by
The genus Alternaria belongs to the kingdom septa, secondary cell walls are deposited, but these
Mycota, phylum Ascomycota, division remain unmelanized, suggesting a developmental
Deuteromycota, subdivision Pezizomycotina, regulation of melanin deposition during conidio-
class Dothideomycetes, order Pleosporales and genesis (Campbell 1969; Carzaniga et al. 2002;
family Pleosporaceae. It has several species as Kawamura et al. 1997). Melanin is probably
saprophytes and parasites. Some species of actively involved in conidial development, since
Alternaria are the asexual anamorph of the asco- disruption of a melanin biosynthesis gene in A.
mycetes Pleospora, while others are speculated to alternata reduced conidial size as well as septal
be anamorph of Leptosphaeria. The two major number (Kawamura et al. 1999).
features of Alternaria species are the production A classification based on conidial characteris-
of melanin especially in the spores and production tics is complicated by the existence of other fun-
of host-specific toxins in the case of pathogenic gal genera, such as Stemphylium and Ulocladium,
species. The genus Alternaria was established in which produce phaeodictyosporic conidia that
1817 with A. alternata (originally A. tenuis) as the resemble those of Alternaria. Based on the char-
type isolate. Because of the absence of an identi- acteristics defined by Simmons (1995),
fied sexual stage for the vast majority of Alternaria Stemphylium and Alternaria species are discrimi-
species, this genus was classified into the division nated by the appearance of the conidiophore apex
of mitosporic fungi or the phylum Fungi imper- and Ulocladium and Alternaria species by the
fecti. The key taxonomic feature of the genus appearance of the basal end of immature conidia.
Alternaria is the production of large, multicellular, This differentiation is largely supported by a
dark-coloured (melanized) conidia with longitudi- molecular analysis of ribosomal DNA sequences
nal as well as transverse septa (phaeodictyo- (Pryor and Gilbertson 2000).
3.6 Morphology of Alternaria species Pathogenic on Cruciferous Crops 59

Within the genus Alternaria, species are also = Torula alternata Fr., 1832, Syst. Mycol., 3: 500
primarily defined upon conidium characteristics. = A. tenuis C.G. Nees, 1816/17, Syst. Pilze
Over 100 species occurring worldwide have been Schwamme: 72
described (Simmons 1992). However, errors in
the taxonomy of Alternaria species have arisen Additional synonyms listed in Simmons
due to the variability of its morphological charac- (2007).
ters, which are not only affected by intrinsic fac- The reasons why the epithet alternata should
tors, but also by environmental conditions. As a be used instead of the more commonly accepted
result of this, it is feasible that single species have one tenuis are clearly stated by Simmons
been accidentally divided into several (Rotem (1967).
1994). This is illustrated by the observation that Colonies usually black or olivaceous black,
species of Alternaria alone is capable of attacking sometimes grey. Conidiophores arising singly or
over 100 hosts, and in addition, Groves and in small groups, simple or branched, straight or
Skolko (1944) found that this species typically flexuous, sometimes geniculate, pale to mild oli-
displays morphological variations. Obviously, vaceous or golden brown, smooth, up to 50-μ
this phenotypic variation does not justify assign- long, 3–6-μ thick with 1 or several conidial scars.
ing A. alternata like specimens to other species. Conidia formed in long, often branched chains;
However, it is this morphological variation that obclavate, obyriform, ovoid or ellipsoidal, often
has probably resulted the description of certain with a short conical or cylindrical beak; some-
Alternaria species that have never been verified times up to but not more than one-third the
by others (Rotem 1994). length of the conidium; pale to mid golden
Because of the large diversity of Alternaria brown; smooth or verruculose with up to 8 trans-
species, a division into subgeneric groups has verse and usually several longitudinal or oblique
occasionally been proposed. However, to date septa; overall length 20–63 (37) μ, × 9–18 (13)
there has not been one general classification of μ; thick in the broadest part; beak pale, 2–5 μ
Alternaria. Neergaard (1945) proposed a clas- thick (Fig. 3.2). An extremely common sapro-
sification based on catenulation (chain forma- phyte found on many kinds of plants and other
tion of conidia), while more recently an substrata including foodstuff, soil and textiles;
organization of the genus into species groups, cosmopolitan (Ellis 1971; Holliday 1980).
each typified by a representative species, was
proposed (Simmons 1992). Because of morpho-
logical similarity but pathological differences, 3.6.1 Type Species: Alternaria
strains of a certain species, especially A. alter- brassicicola (Schw.) Wiltshire
nata, A. brassicae and A. brassicicola, have
been defined as formae species or ‘pathotypes’ Alternaria brassicicola (Schw.) Wiltshire in
(Nishimura and Kohmoto 1983; Verma and Mycol. Pap. 20:8, 1947
Saharan 1994; Thomma 2003). = Helminthosporium brassicicola Schweinitz
(as Helminthosporium brassicola) in Trans.
AM. Phil. Soc. N.S., 4: 279, 1832
3.6 Morphology of Alternaria = Macrosporium cheiranthi Fr. var. circinans
species Pathogenic Berk. & Curt. in Grevilles, 3: 105, 1875
on Cruciferous Crops = Alternaria circinans (Berk. & Curt.) Bolle
in Meded. Phytopath. Lab. Willie comme-
The following species of this genus cause eco- lin Scholten, 7:26, 1924
nomically important diseases in Brassicaceae: = Alternaria oleracea Milbraith in Bot. Gaz.,
74: 320t 1922 (Full synonymy given by
Alternaria alternata (Fr.) Keissler, Beih. Bot. Wiltshire in Mycol. Pap. 20: 1947)
Zbl., 29: 434,1912
60 3 Pathogen

Fig. 3.3 Alternaria brassicicola (×650) Ellis 1971

Fig. 3.2 Alternaria alternata (×650) Ellis 1971

Additional synonyms listed in Simmons 3.6.2 Type Species: Alternaria

(2007). brassicae (Berk.) Sacc.
Colonies amphigenous, effused, dark oliva-
ceous brown to dark blackish brown, velvety. Alternaria brassicae (Berk.) Sacc. in Michelia,
Mycelium immersed; hyphae branched septate, 2:129 see also p. 172, 1880
hyaline at first, later brown or olivaceous brown, = Macrosporium brassicae Berk. in Smith’s
inter- and intracellular, smooth, 1.5–7.5-μ thick. Engl. Fl. 5, pt. 2 : 339, 1836. (Full synon-
Conidiophores arising singly or in groups of ymy given by Wiltshire in Mycol. Pap., 20,
2–12 or more; emerging through stomata; usu- 1947)
ally simple, erect or ascending; straight or
curved; occasionally geniculate; more or less Colonies amphigenous, effused, rather pale
cylindrical, but often slightly swollen at the base; olive, hairy; the individual large conidia plainly
septate, pale to mild olivaceous brown; smooth; seen under a x 20 binocular dissecting microscope.
up to 70-μ long; 5–8-μ thick. Conidia mostly in Mycelium immersed; hyphae branched, septate,
chains of up to 20 or more, sometimes branched, hyaline, smooth, 4–8-μ thick. Conidiophores aris-
acropleurogenous, arising through small pores in ing in groups of 2–10 or more from the hyphae;
the conidiophore wall, straight, nearly cylindri- emerging through stomata; usually simple, erect or
cal, usually tapering slightly towards the apex or ascending; straight or flexuous; frequently genicu-
obclavate. The basal cell rounded; the beak usu- late; more or less cylindrical but often slightly
ally almost non-existent; the apical cell being swollen at the base; septate; mid-pale greyish olive;
more or less rectangular or resembling a trun- smooth up to 170-μ long; 6–11-μ thick; bearing
cated cone, occasionally better developed but one to several small but distinct conidial scars.
then always short and thick, with 1–11, mostly Conidia solitary or occasionally in chains of up to 4,
less than 6, transverse septa, often constricted at acropleurogenous, arising through small pores in
the septa; pale to dark olivaceous brown; smooth the conidiophore wall; straight or slightly curved;
or becoming slightly warted with age; 18–130-μ obclavate; rostrate with 16–19 (usually 11–15)
long; 8–30-μ thick in the broadest part, with the transverse septa and 0–8 (usually 0–3) longitudinal
beak 1/6 the length of the conidium and 6–8-μ or oblique septa; pale or very pale olive or greyish
thick (Ellis 1968b, 1971; Holliday 1980). The olive; smooth or infrequently, very inconspicu-
fungus is confined to the Brassicaceae (Fig. 3.3). ously warted; 75–350-μ long and usually 20–30-μ
3.6 Morphology of Alternaria species Pathogenic on Cruciferous Crops 61

Fig. 3.4 Alternaria brassicae (×650) Ellis 1971 Fig. 3.5 Alternaria raphani (×650) Ellis 1971

(sometimes up to 40 μ) thick in the broadest part; celled, round, finally many celled and irregular,
the beak about 1/3 to 1/2 the length of the conidium brown. Conidiophores often develop from them
and 5–9-μ thick (Fig. 3.4) (Ellis 1968a, 1971; (Ellis 1971; Holliday 1980). Unlike A. alternata
Holliday 1980). This species produces chlamydo- and A. brassicicola, this species forms chlamydo-
spores. It is mostly confined to the Brassicaceae. spores and the beak of the conidium is smaller
than that of A. brassicae but longer than the
almost non-existent beak of A. brassicicola
3.6.3 Type Species: Alternaria (Fig. 3.5).
raphani Groves & Skolko

Alternaria raphani Groves and Skolko 1944, 3.6.4 Type Species: Alternaria
Can. J. Res., Sect. C, 22:227 cheiranthi (Lib.) Bolle
= Alternaria matthiolae Neergaard 1945
Alternaria cheiranthi (Lib. Bolle 1924, as ‘(Fr.)
Conidiophores simple or occasionally branched, Bolle’, Meded. Phytopath. Lab. Willie
septate, olivaceous brown, up to 150-μ long, Commelin Scholten, 7:55
3–7-μ thick, sometimes swollen slightly at the tip = Helminthosporium cheiranthi Lib., 1827,
and usually with a single conidial scar; conidia apud Desm., Crypt. Fr. Exsicc., 213
commonly in chains of 2–3, straight or slightly = Macrosporium cheiranthi (Lib.) Fr., 1832,
curved, obclavate or ellipsoidal, generally with a Syst. Mycol., 3: 374
short beak, mid to dark golden brown or oliva-
ceous brown, smooth or sometimes minutely ver- Conidiophores arising singly or in groups,
ruculose, with 3–7 transverse and often a number mostly simple but sometimes branched, straight
of longitudinal or oblique septa, constricted at the or flexuous, septate, rather pale olive, often hya-
septa, 50–130 (70)-μ long, 14–30 (22)-μ thick in the line at the apex, smooth, up to 130-μ long, 5–8-μ
broadest part. Chlamydospores formed abun- thick with a single terminal scar at first but later
dantly in culture, sometimes in chains at first 1 with up to 4 scars, which may be borne close
62 3 Pathogen

together without marked geniculation. Conidia

mostly solitary, rarely in chains of 2, 3 or more;
variously shaped, often pyriform, ovoid or elon-
gate ovoid at first, later becoming irregular,
mostly tapering to the apex, which may be drawn
out into a beak; generally rounded at the base,
with numerous transverse, longitudinal and
oblique septa; light olive to golden brown; trans-
lucent, with the interior walls, which are dark;
often plainly visible; smooth or with the wall pit-
ted from the inside; 20–100-μ long; 13–32-μ
thick in the broadest part (Fig. 3.6). Common on
wallflowers and occasionally on other brassica-
ceous plants (Ellis 1971).
To establish the correct systematic designa-
tion of two common species of Alternaria on
Brassicaceae, Wiltshire (1947) gave the follow-
ing account of synonymy, which had appeared in
the literature up to 1945. However, additional
synonymous are listed by Simmons (2007). Fig. 3.6 Alternaria cheiranthi (×650) Ellis 1971

1. Alternaria brassicae (Berk.) Sacc.

1836. Macrosporium brassicae Berk. In 1897. Macrosporium brassicae Berk. var.
Smith’s Engl. Flora. v, Part II P. 339 macrospora Eliason
Hooker’s British Flora, vol. ii, part II, ‘Fungi’ 1901. Sporidesmium brassicae Massee. In
by M.J. Berkeley Kew. Bull., 1901, P. 153
1836. Puccinia (?) brassicae Montagne. In 1902. Leptosphaeria exitiosa (Kuhn) Rostrup.
Ann. Sci. Nat., Ser. II VI, p. 30 In Plantepatolagi Haandbog i Laeren om
1855. Sporidesmium exitiosum Kuhn. In Plantesygdomme for Landbrugere,
Hedwigia, i p. 91 Havebrugere og Skovbrugere, P. 472
1856. Rhopalidium brassicae Mont. & Fr. In 1902. Alternaria brassicae (Berk.) Sacc. var. exi-
Montagne’s Syll. Crypt., P.297 tiosa (Kuhn) Ferraris. In Flora italica, P. 521
1859. Polydesmus exitiosus (Kuhn) Kuhn. In 1917. Alternaria herculea (Ell. & Mart.)
Kramkh. d. Kulturgew., P.165 Elliott. In Amer. Journ. Bot., IV, P. 472
1880. Alternaria brassicae (Berk.) Sacc. var. 1924. Alternaria brassicae (Berk.) Bolle [nee.
minor Sacc. In Mich. ii, P. 172 Sacc.]
1882. Cercospora bloxami Berk. & Br. In 1944. Alternaria macrospora (Sacc.) Sawada.
Ann. & Mag. Nat. Hist. Ser. V, IX. P. 183, In descriptive catalogue of the Formosan
No.1882, 1879 fungi. Part V, Dept. Agric. Govt. Res. Inst.
1882. Macrosporium herculeum Ellis & Formosa, Rept. 51, p. 123
Martin, In Amer. Nat., XVI, p.1003; exsicc. 1945. Alternaria exitiosa (Kuhn) Jorstad. In
N. Amer. Fungi, 1263 Melding fra Statens. Plantepatogiske
1884. Cercospora lepidii Peck. In 35th Rept. Institut. No.1, P. 594
N.Y. State Mus. 1881, p. 140.
1886. Alternaria brassicae (Berk.) Sacc. var. It is extremely confusing that the name A.
macrospora Sacc. In Syll., IV, p.546 brassicae (Berk.) Sacc. has been applied, in
1891. Sporidesmium annii Karst. In Symb. accordance with Saccardo’s usage in the Sylloge,
Myc. Fenn. XXX, P. 67 to the species A. brassicicola by various authors,
3.7 The Infection Process 63

e.g. Voglino (1902), Ferraris (1912), Sawada spores of 11.5–44 by 7–9 μ by which its identity
(1931), Weber (1932), Yoshii (1933) and Fajardo can readily be established (Wiltshire 1947).
and Palo (1934).

2. Alternaria brassicicola (Schwein) Wiltshire 3.7 The Infection Process

1832. Helminthosporium brassicicola.
Schweinitz. In Syn. Fung. Amer. Bar. No. In general, Alternaria species are foliar pathogens
2632, Trans. Amer. Phil. Soc., N.S., IV P. 279 that cause a relatively slow destruction of host tis-
1855. Sporidesmium exitiosum Kuhn formae sues through the reduction of photosynthetic
(B) Alternarioides and (Y) luxuriosum potential. An infection leads to the formation of
Kuhn. In Hedwigia, I, P. 91 necrotic lesions, which sometimes have a target-
1859. Polydesmum exitiosus (Kuhn) Kuhn like appearance due to growth interruptions
formae (B) Alternarioides and (Y) luxurio- caused by unfavourable conditions. The fungus
sum Kuhn) Kuhn. In Krankh. d. Kulturgew., resides in the centre of the lesion, which is sur-
p. 165 rounded by an un-invaded chlorotic halo, a symp-
1875. Macrosporium cheiranthi Fr. var. circi- tom that is commonly observed for the infection
nans Berk. & Curt. In Grevillea, III, P. 105 process of necrotrophic pathogens. This zone is
1880. Alternaria brassicae (Berk.) Sacc. created by the diffusion of fungal metabolites like
minor Sacc. In Mich., II, P. 172 toxins (Agarwal et al. 1997; Tewari 1983).
1886. Macrosporium commune Rabenh. var. Members of the genus cause quiescent infections
circinans (Berk. & Curt.) Sacc. In Syll., iv, in which the fungus enters the tissue where it
P. 524 remains dormant until conditions become favour-
1886. Alternaria brassicae (Berk.) Sacc. In able for infection. Alternaria species generally do
Syll., IV, p. 546 not affect water or nutrient transport throughout
1897. Alternaria brassicae (Berk.) Sacco var. the plant, because they do not specifically target
microspora Brun. In Act. Soc. linn. roots or vessels (Rotem 1994).
Bordeaux, III, P. 149 Alternaria has no known sexual stage or over-
1902. Helminthosporium brassicae P. Henn. wintering spores, but the fungus can survive as
In Hedwigia, XLI, p. 117 mycelium or spores on decaying plant debris for a
1922. Alternaria oleracea Milbraith. In Bot. considerable time or as a latent infection in seeds
Gaz., LXXIV, p. 320 (Rotem 1994). If seed borne, the fungus can attack
1924. Alternaria circinans (Berk. & Curt.) the seedling once the seed has germinated. In
Bolle. In Meded. Phytopath. Lab. Willie other cases, once the spores are produced, they are
Commelin Scholten; Baarn, VII, P. 26 mainly spread by wind on to plant surfaces where
1933. A. brassicae (Berk.) ‘Lindau’ cited in infection can occur. Typically, weakened tissues,
error by Yoshii for A. brassicae (Berk.) Sacc. either due to stresses, senescence or wounding,
are more susceptible to Alternaria infection than
Reviewing then the names that have been healthy tissues. The observation that saprobic
applied to A. brassicicola, it is evident that Alternaria species can become parasitic when
Schweinitz’s name antedates the others by many they meet a weakened host illustrates that the dis-
years. There is no doubt that A. oleracea and A. tinction between saprophytic and parasitic behav-
circinans are identical with it and must rank as iour is not always evident.
synonyms. The species is quite distinct from the Despite the taxonomic and pathogenic differ-
true A. brassicae (Berk.) Sacc., and though A. ences between Alternaria species, they cause
brassicicola shows considerable variations on the similar infection patterns. Dormant spores have
host, in culture, it always produces the same heavily melanized walls that, under favourable
characteristic, almost cylindrical, short beaked conditions, produce one or more germ tubes.
64 3 Pathogen

Subsequently, the germ tubes penetrate stomata, its pathogenic capacity. On the other hand, the tan-
cuticle or wounds with or without the formation gerine pathotype of A. alternata did not show this
of small appressoria. In less virulent species, dependency, possibly because this particular patho-
wounds and stomata are targeted, while more gen largely depends on toxin production for the
virulent species can also penetrate directly colonization of its host (Isshiki et al. 2001).
(Rotem 1991). Enzymatic processes in Alternaria For a specific A. alternata endoglucanase, it
infections are essentially similar to those in other was demonstrated that its production is triggered
diseases. The cuticle, which consists of a combi- by a pathogen-induced pH increase on the host
nation of cutin (a hydroxyl fatty acid polyester) (Eshel et al. 2002b). Correlation studies between
and waxes, comprises the first line of defence to enzyme production and symptom development
be overcome by directly penetrating fungal suggest that endoglucanases and exoglucanases
pathogen. For A. brassicicola, differential expres- are involved in A. alternata pathogenicity (Eshel
sion of cutinase genes was monitored between et al. 2000, 2002a, b).
saprophytic and pathogenic stages of the fungus
(Yao and Köller 1995). Furthermore, it was found
that different cutinolytic enzymes are sequen- 3.8 Identification
tially induced upon landing on, and penetration of Alternaria Genes
of, the cabbage leaf (Fan and Köller 1998).
Constitutively produced cutinases are expressed In order to identify candidate fungal pathogenicity
during the initial contact of A. brassicicola with genes and characterize a compatible host response,
the cuticle. After reaching subcuticular layers, a suppression subtractive hybridization (SSH)
different cutinases that are active during sapro- cDNA library enriched for A. brassicicola and
phytic growth are induced (Trail and Köller 1993; Brassica oleracea genes expressed during the
Yao and Köller 1994). These cutinases are induc- interaction was created, along with a cDNA library
ible by cutin monomers. This implies a switch representing genes expressed during nitrogen star-
between the parasitic and the saprophytic stage of vation (NS). A total of 3749 and 2352 expressed
the fungal pathogen so far. However, it has not sequence tags (ESTs) were assembled into 2834
been demonstrated that any extracellular hydro- and 1264 unisequence sets for the SSH and NS
lase is crucially involved in fungal pathogenesis. libraries, respectively. Cramer et al. (2006) com-
In addition to cutinases, lipases might also con- pared two methods to identify the origins (plant vs.
tribute to the establishment of infection. A. bras- fungal) of ESTs in the SSH library using different
sicicola found to produce a lipase that acts as a classification procedures, with and without the
virulent factor (Berto et al. 1997). Anti- lipase anti- availability of a database representing the A. bras-
bodies were found to have an inhibitory effect on sicicola whole genome sequence and Brassicaceae-
the in vivo infection of cauliflower leaves, as symp- specific genes. BLASTX analyses of the 2834
tom development was inhibited in a dose-dependent unisequence set using the GenBank nonredundant
manner despite normal germination of spores. database identified 114 fungal genes. Further
However, the antibodies did not have an effect on BLASTN analyses of the genes with unidentifi-
fungal infection of dewaxed leaves, which suggests able origin using a database consisting of the 1264
that this lipase has an early pathogenic activity fungal unisequence set from the nitrogen starved
during penetration (Berto et al. 1999). library identified 94 additional fungal genes. By
About one-third of the total cell wall compo- contrast, BLASTN analyses of the same SS11
nents in dicotyledonous plants are pectic polysac- unisequence set using a partially assembled A.
charides. These components can be hydrolysed by brassicicola whole genome draft sequence identi-
fungal galacturonidases. Alternaria citri was found fied a total of 310 unisequences of fungal origin.
to be dependent on endopolygalacturonidase Even a small number of organism-specific EST
activity for establishing an infection, as a mutant sequences can be very helpful to identify pathogen
lacking this activity was severely compromised in genes in a library derived from infected tissue,
3.9 Nuclear Ribosomal DNA Sequences 65

partially overcoming the limitation of the public the Pleosporaceae have hyphomyceteous ana-
databases for little studied organisms. However, morphs (Barr 1987). The phylogenetic trees
using the whole genome draft sequence of A. bras- (Figs. 3.7 and 3.8) based on rDNA sequences sup-
sicicola, one is able to identify approximately port this taxonomic view. All the Loculoas-
30 % more fungal genes in the SS11 library than comycetes included in the analysis, i.e.
without utilizing this resource (Cramer et al. 2006). Leptosphaeria and Pleospora as well as
The biosynthetic gene cluster of brassicicene Alternaria, formed a phyletic group. Within this
C was identified in A. brassicicola strain ATCC group, there are two major lineages: the
96836 from genome database search. In vivo and Leptosphaeriaceae composed of Leptosphaeria
in vitro studies clearly reveal the function of and the Pleosporaceae composed of Pleospora
Orf 8 and Orf 6 as a fusicoccadiene synthase and and Alternaria. The two Ophiosphaerella
methyltransferase, respectively. The understand- species, which are included only in the analysis
ing towards the biosynthetic pathway promises based on the 5–8s rDNA and flanking internal
construction of this type of diterpene compounds transcribed spacers, fall within the Leptosphaeria
with genetic engineering (Minami et al. 2008). group (Fig. 3.7). Ophiosphaerella korrae (Walker
and Smith) Shoemaker and Babc has been placed
in Leptosphaeria (Walker and Smith 1972), while
3.9 Nuclear Ribosomal DNA O. herpotricha (Fr.; Fr) Walker was previously in
Sequences Phaeosphaeria (Holm 1957), a segregate of
Leptosphaeria (Holm 1957). For further details
The sequences coding for the nuclear 18s rRNA, of the phylogeny of Leptosphaeria, see Morales
5–8s rRNA and the internal transcribed spacers et al. (1995).
(ITS1 and ITS2) were amplified by the poly- Most species of Alternaria including the four
merase chain reaction and sequenced for one iso- species pathogenic to crucifers lack a known sex-
late each of Alternaria brassicae, A. brassicicola, ual stage. Alternaria and Pleospora, although
A. raphani, A. alternata and Pleospora her- distinct genera, are sister taxa (Fig. 3.8) based on
barum. The 5–8s rDNA sequences from the four the 18s rDNA sequence data. Simmons (1967)
Alternaria species are identical and differed at considered the Alternaria, Stemphylium and
only one base pair from that of P. herbarum. The Ulocladium to be closely related on the basis of
internal transcribed spacer sequences especially conidial development, as they all produce
ITS1 are very variable in both base composition dictyoporospores. Pleospora herbarum has a
and length. The 18s rDNA sequence is highly Stemphylium anamorphs and provides the first
conserved, but enough variability is present to molecular evidence that asexual species of
distinguish genera clearly. Phylogenetic analysis Alternaria are indeed related to Pleospora.
of the sequence data sets by both parsimony and Therefore, the anamorphic Alternaria should be
maximum likelihood methods clearly separated included in the Pleosporaceae.
genera and species. All of the Alternaria species Wiltshire (1947) considered A. raphani and A.
closely related to Pleospora also appeared to be brassicae to be closely related. It would appear,
more closely related to Alternaria than to however, that A. raphani is actually more closely
Leptosphaeria (Jasalavich et al. 1995). related to A. brassicicola than to A. brassicae
Currently taxonomists consider Pleospora based on rDNA sequence data. Alternaria alter-
and Leptosphaeria to be in separate Loculo- nata, A. brassicae, A. brassicicola and A. raphani
ascomycetes families, the Pleosporaceae and the formed a strong clade of very closely related sis-
Leptosphae-riaceae, respectively (Barr 1987; ter taxa. The 18s rDNA resolved two subclades
Eriksson and Hawksworth 1991), with the main of species within Alternaria at a level of confi-
differential criterion being the conidiomatal dence of 95 % (Fig. 3.8), based on five informa-
structure of the anamorphs. Leptosphaeria species tive sites contained within the 300 bp at 3′ end of
have coelomycetous anamorphs, while genera in sequence alignment. Complete resolution of the
66 3 Pathogen

Fig. 3.7 The consensus tree generated by global parsi- length (drawn in the horizontal dimension only) is the
mony bootstrap analysis of alignment of the 5–8s rDNA maximum likelihood estimates made by the programme
and flanking internal transcribed spaces (ITS1 and ITS 2) DNAML. When the user tree was defined as the bootstrap
sequences. The percentages represent the proportion of consensus tree, the length of the vertical lines has no
1000 bootstrap replications in which the taxa to the right meaning and was adjusted arbitrarily for ease in labelling
of the node were placed together by the programme termini. Leptosphaeria doliolum was designated as the
DNA PARS with randomization of the sequence input out-group (Jasalavich et al. 1995)
order. The other numbers represent the steps. Branch

Fig. 3.8 The consensus tree generated by global parsi- horizontal dimension only) is the maximum likelihood
mony bootstrap analysis of alignment of the 18s estimates made by the programme DNAML. When the
rDNA. The percentages represent the proportion of 1000 user tree was defined as the bootstrap consensus tree, the
bootstrap replications in which the taxa to the right of the length of the vertical lines has no meaning and was
node were placed together by the programme DNA PARS adjusted arbitrarily for ease in labelling termini.
with randomization of the sequence input order. The other Neurospora crassa was designated as the out-group
numbers represent the steps. Branch length (drawn in the (Jasalavich et al. 1995)
3.11 Identification of Pathogenicity Factors 67

species of Alternaria is achieved with the ITS brassicicola NPS genes, AbNPS2. The predicted
sequence data (Fig. 3.7) which are much more amino acid sequence of AbNPS2 shows high
variable and contained more phylogenetically sequence similarity with A. brassicae AbrePsy1,
informative sites than the 18 s rDNA. The ITS Cochliobolus heterostrophus NPS4 and a
sequence data of Leptosphaeria are even more Stagonospora nodorum NPS. The AbNPS2 open
variable than those of Alternaria. Likewise, the reading frame was predicted to be 22 kb in length
range of genetic distance among the and encodes a large protein (7195 amino acids)
Leptosphaeria species is larger than those seen showing typical NPS modular organization. Gene
among the Pleospora and Alternaria species. expression analysis of AbNPS2 in wild-type fun-
The genetic distances between P. herbarum and gus indicated that it is expressed almost exclu-
any of the four Alternaria species examined are on sively in conidia and conidiophores, broadly in the
the same order of magnitude as that between the reproductive developmental phase. AbNPS2 gene
highly virulent and weakly virulent isolates of L. disruption mutants show abnormal spore cell wall
maculans. These differences suggest that genus morphology and a decreased hydrophobicity phe-
Alternaria encompasses less genetic variability notype. Conidium of abnps2 mutants displays an
than does the genus Leptosphaeria. This may aberrantly inflated cell wall and an increase in
reflect the differences in size and reproductive lipid bodies compared with wild type. Further,
strategies of the two genera. Alternaria comprises phenotypic analyses of abnps2 mutants showed
roughly 60 species (Rao 1969; Rossman et al. decreased spore germination rates both in vitro
1987), while Leptosphaeria includes a heteroge- and in vivo and a marked reduction in sporulation
neous mixture of about 1689 taxa (Crane and in vivo compared with wild-type fungus.
Shearer 1991) and is very likely polyphyletic. Moreover, virulence tests on Brassica with abnps2
Perhaps the four Alternaria species have separated mutants reveal a significant reduction in lesion
from each other only recently in evolutionary time size compared with wild type but only when aged
and so have not yet accumulated many changes in spores are used. Collectively, these results indicate
their rDNA sequences. Alternatively, perhaps that AbNPS2 plays an important role in develop-
plant host association may exert a pressure against ment and virulence (Kim et al. 2007).
further divergence. Along with phylogenetic rela-
tionship among the Alternaria species, portions of
the rDNA sequence can have practical uses, e.g. 3.11 Identification
production of diagnostic tools. The variability of Pathogenicity Factors
present in ITS1 can be exploited to design species-
specific probes and primers in order to identify Functional redundancy of lipases with regard to
rapidly species of Alternaria both in culture and in Alternaria pathogenicity has been observed by
plant materials (Jasalavich et al. 1995). many workers (Yao and Köller 1994, 1995; Berto
et al. 1999; Cho et al. 2006). Interestingly, one of
the factors responsible for the pathogenicity has
3.10 Identification, Cloning been predicted to be secondary metabolite pro-
and Sequencing duction. A non-ribosomal peptide synthase gene
of Virulence Genes (NPS6) in Cochliobolus heterostrophus and A.
brassicicola is found to direct the biosynthesis of
In many plant pathosystems, fungal secondary a siderophores metabolite important for oxidative
metabolites derived from non-ribosomal peptide stress tolerance and pathogenicity (Oide et al.
synthetases (NPSs) are phytotoxic virulence fac- 2006). The secondary metabolite corresponding
tors or are antibiotics thought to be important for to or synthesized via AbNPS2 has to be
niche competition with other microorganisms. characterized. There is a need to further charac-
However, many of the functions of NPS genes and terize secondary metabolite biosynthetic genes
their products are largely unknown. Kim et al. and their role in pathogenicity and fungal
(2007) investigated the function of one of the A. development.
68 3 Pathogen

Another important area of investigation in To study regulatory mechanisms of pathogen-

the Alternaria–Brassicaceae pathosystem is the esis, Cho et al. (2012) mined 421 genes in silico
fungal signal transduction. For example, disrup- encoding putative transcription factors in a
tion of the Fus3/Kss1 MAP kinase homologue machine annotated draft genome sequence of A.
(Amk1) in A. brassicicola resulted in a complete brassicicola. Targeted gene disruption mutants
loss of pathogenicity as observed in other fungi for 117 of the transcription factor genes were
(Cho et al. 2006, 2007). Interestingly, in the lat- produced and screened. Three of these genes are
ter study, it was shown that addition of long associated with pathogenesis. Disruption mutants
polypeptide nutrients partially restored patho- of one gene (AbPacC) are non-pathogenic, and
genicity to the mutants. In addition, two novel another gene (AbVf8) causes lesions less than
virulence factors by Cho et al. (2009) were pre- half the diameter of wild-type lesions.
dicted to encode a transcription factor (AbPro1) Unexpectedly, mutants of the third gene, Amr1,
and a two-component histidine kinase gene cause lesions with twofold larger diameter than
(AbNIK1). Both of these kinases are pathoge- the wild-type and complementation mutants.
nicity factors in phytopathogenic fungi. Slt2 Amr1 is a homologue of Cmr1, a transcription
was found to be associated with cell wall integ- factor that regulates melanin biosynthesis in sev-
rity and HOG with oxidative stress tolerance eral fungi. Cho et al. (2012) created gene deletion
(Xu 2000). The identification of virulence factors mutants of ∆amr1 and characterized their pheno-
is the disruption of Aso-1, a gene required for types. The ∆ amr1 mutants used pectin as carbon
hyphal fusion (anastomosis) which is also found source more efficiently than the wild type, were
to be required for pathogenicity in Alternaria melanin deficient and are more sensitive to UV
species (Craven et al. 2008). Eventually over a light and glucanase digestion. The AMR1 protein
hundred genes have been functionally analysed is localized in the nuclei of hyphae and in highly
through various techniques like gene knockout melanized conidia during the late stage of plant
and overexpression experiments making A. pathogenesis. RNA seq analysis reveals that three
brassicicola the species of choice for functional genes in the melanin biosynthesis pathway, along
genomic research to define conserved virulence with the deleted Amr1 gene, are expressed at low
mechanisms for this important genus of fungi levels in the mutants. In contrast, many hydro-
(Oide et al. 2006; Cho et al. 2006, 2007; Kim lytic enzyme-coding genes are expressed at
et al. 2007; Cho et al. 2009). higher levels in the mutants than in the wild type
With the objective of identification of A. bras- during pathogenesis. A gene important for sur-
sicicola, an attempt was made to examine the vival in nature negatively affects virulence, prob-
role of cutinase genes in A. brassicicola patho- ably by less efficient use of plant cell wall
genesis (Yao and Köller 1994, 1995). In these materials. The functions of the Amr1 gene are
studies, biolistic transformation was used to important to be the success of A. brassicicola as
disrupt the CUTAB1 gene. It affected the sapro- competitive saprophyte and plant parasite.
phytic growth since cutin was no longer able to
be utilized as a sole carbon source, but this
disruption had no significant effect on A. bras- 3.12 Growth and Sporulation
sicicola pathogenicity. An extracellular lipase
was found to be produced by A. brassicicola 3.12.1 Culture Media
in vitro (Berto et al. 1999). In this study, anti-
lipase antibodies were found to significantly A number of culture media have been reported
decrease the ability of A. brassicicola to cause suitable for growth and sporulation of Alternaria
disease on cauliflower leaves. However, disrup- species pathogenic on brassicaceous plants
tion of four predicted A. brassicicola lipase genes (Table 3.2). According to Neergaard (1945), A.
expressed during plant infection did not result in brassicae sporulates profusely on malt extract
reduced virulence on cabbage (Cho et al. 2006). and standard nutrient agar, but very poorly on
3.12 Growth and Sporulation 69

Table 3.2 Culture media for growth and sporulation of sicicola and A. raphani, which grew well on a
Alternaria species pathogenic on Brassicaceae (Verma
wide range of agar media. On PDA, Mukadam
and Saharan 1994)
and Deshpande (1977) found that A. brassicae
Liquid/Solid not only grew and sporulated poorly, but it also
media Reference
lost its ability to grow and sporulate with succes-
Alfalfa McDonald (1959)
decoction sive sub-culturing. Cultural characters of A. bras-
Asthana and Gupta et al. (1969) sicae on different culture media are shown in
Hawker’s Table 3.3 (Prasada et al. 1970).
Brown’s starch Ansari et al. (1988); Gupta et al. Alternaria brassicae from Eruca sativa grows
(1969); Prasada et al. (1970) best on glucose-asparagine agar medium and
Coconut Lapis and Ricaforte (1974) Richard’s liquid medium. Mycelial growth is
Corn meal Ansari et al. (1988); Lapis and maximum in Richard’s medium and least in
Ricaforte (1974); Singh (1980)
Brown’s medium. It is capable of utilizing nitrate,
Czapek-Dox Gupta et al. (1969); Lapis and
Ricaforte (1974); Prasada et al. nitrite, ammoniacal and organic forms of nitro-
(1970) gen (Khandelwal et al. 1970; Prasada et al. 1970).
Eruca sativa Prasada et al. (1970) Single-spore transfers of A. brassicae on 10 %
decoction alfalfa decoction agar produce the largest amount
Glucose Prasada et al. (1970) of spores (McDonald 1959). It grows well on
asparagines pechay decoction agar, corn meal agar, Czapek-
Host decoction Prasada et al. (1970)
Dox agar, coconut agar, Leonian agar, PDA, V-8
Houston’s Singh (1980)
juice agar, oat meal agar, nutrient agar and prune
Kirchoff’s Ansari et al. (1988)
agar, while sporulation is abundant on all but corn
Leonian’s agar Lapis and Ricaforte (1974)
and oat meal agar (Lapis and Ricaforte 1974).
Malt extract Ansari et al. (1988)
Billotte (1963) induced abundant sporulation of
Mustard leaf Ansari et al. (1988)
extract A. brassicae by the slow desiccation of culture in
Oatmeal Prasada et al. (1970); Singh (1980) open Petri dishes preceded by removal of the aer-
Pechay Lapis and Ricaforte (1974) ial mycelium and washing in running water.
decoction According to Degenhardt (1973), V-8 juice agar
Potato dextrose Ansari et al. (1988); Prasada et al. with rose bengal plus streptomycin stimulates
(1970) sporulation of both A. brassicae and A. raphani.
Potato dextrose Ansari et al. (1988) According to Gupta et al. (1969), calcium
asparagine
nitrate and, to a lesser extent, potassium nitrate
Potato sucrose McDonald (1959)
support maximum growth of A. brassicae iso-
Rice meal Singh (1980)
lated from B. oleracea var. botrytis. Increased
Richard’s Ansari et al. (1988); Gupta et al.
(1969); Prasada et al. (1970)
growth is correlated with the carbohydrate con-
Sabouraud’s Ansari et al. (1988); Prasada et al. tent of the medium. Buchwaldt et al. (1984)
(1970) found that on Czapek-Dox medium, singrin
V-8 McDonald (1959) (allylglucosinolate) does not influence growth
Wheat meal Singh (1980) rates of A. brassicae, but at increasing singrin
concentrations, colonies become darker; on PDA,
colonies are dark at all singrin concentrations. On
PDA. Atkinson (1950), on the other hand, found PDA, singrin has no effect on growth rates in
good sporulation on both PDA and malt agar. darkness, but in UV light, the high singrin con-
According to Ansari et al. (1988a), although A. centration increases the mycelial growth slightly.
brassicae grows and sporulates well on a wide Alternaria brassicicola grows and sporulates
range of media, PDA was found to be the best. well on a wide range of agar media (Changsri
Changsri (1960, 1961) found slightly poorer 1960, 1961), but shows marked selectivity in uti-
growth of A. brassicae in comparison to A. bras- lizing different carbon sources. Lactose is the
70 3 Pathogen

best source of carbon for maximum growth fol- more demanding growth requirements, with a
lowed by glycerol. The fungus prefers ammonia- distinct optimal growth peak at 22.5 °C (Gupta
cal nitrogen over nitrate and nitrite. Growth of A. et al. 1972; Singh 1980). The temperature growth
brassicicola is stimulated by DL-phenylalanine, optimum of A. brassicicola is 25–27 °C (Sarkar
DL-valine, asparagine, glycine and DL-serine and Sen Gupta 1978), but growth continues to the
(Jain 1974). The biosynthesis of lipids and phos- extremes of 6 and 37 °C. According to Changsri
pholipids is best on Czapek’s medium (Aizina (1960, 1961) and Changsri and Weber (1960,
et al. 1976). 1963), the optimum temperatures for growth in
Alternaria raphani grows well in culture but culture of A. brassicicola, A. brassicae and A.
sporulation is rather poor (Changsri 1961). raphani are 24–28 °C, 20–24 °C and 24–28 °C,
Spores of A. raphani are produced plentiful in respectively. However, according to Taber et al.
most brassicaceous leaf decoction agar media, in (1968), A. raphani and A. brassicae grow better
which chlamydospores are produced abundantly. between 20 and 25 °C on malt agar, whereas A.
The density of the thallus and sporulation brassicicola grows well over a wider temperature
decreases as the amount of leaf decoction per ranges. Lapis and Ricaforte (1974) report profuse
litre is decreased from 400 to 6.5 g. Atkinson mycelial growth and sporulation of A. brassicae
(1950) also reports abundant sporulation by at 16–24 °C, but according to Ansari et al.
wounding plate cultures and removing the lids of (1989a), the optimum is 23 °C.
the culture plates. Alternaria raphani does not Alternaria brassicae isolated from Crambe
require an exogenous vitamin source for growth, abyssinica grows well on malt agar and nutrient
although early growth is stimulated by the addi- agar at 0.5–33 °C with optimum growth at 23 °C;
tion of vitamins (Taber et al. 1968). Alternaria sporulation is maximum at 20–30 °C. Alternaria
raphani grows faster when glutamic acid is sup- brassicicola grows well between 0.5 and 38 °C
plied as a nitrogen source. Taber et al. (1968) in with an optimum growth at 252C; sporulation is
their extensive nutritional studies report that A. maximum at 17–30 °C. Alternaria alternata
brassicae, A. brassicicola and A. raphani grow grows well from 0.5 to 40 °C with optimum
well on most carbon sources; starch supports the growth and sporulation at 25–27 °C and
most rapid growth, whereas methyl cellulose, fil- 20–30 °C, respectively (Czyzewska 1970).
ter paper strips and mannitol are very poor car- According to Maude (1986), at least 12 h of con-
bon sources. tinuous high relative humidity and temperature of
Although the three species differ strikingly in more than 14 °C are required for abundant sporu-
their rates of utilization of mannitol, both A. lation of A. brassicae and A. brassicicola. Below
raphani and A. brassicicola synthesize this 14 °C, the sporulation is delayed under more
polyol. Phenylalanine supports faster growth of humid conditions; at 8 °C, at least 30 h of high
A. brassicicola than other nitrogen sources, relative humidity is required for sporulation.
whereas glutamic acid and ammonium succinate Both fungi produce viable spores for 12 weeks on
stimulate more rapid growth of A. raphani and A. leaf and 20 weeks on stubble under field condi-
brassicae (Taber 1964). tions. Ansari et al. (1989a) observed 95–100 %
relative humidity optimum for mycelial growth
and sporulation of A. brassicae.
3.12.2 Temperature and Relative
Humidity
3.12.3 Hydrogen Ion
The optimum temperature for maximum sporula- Concentrations (pH)
tion of A. brassicae and A. raphani is between 23
and 25 °C (Changsri and Weber 1963; Degenhardt The optimum pH requirement for growth and
1973; McDonald 1959; Neergaard 1945; Singh sporulation of A. brassicicola, A. brassicae and A.
1980; Taber 1964). Alternaria brassicae has raphani are 6.0–8.0, 7.1–8.0 and 7.1–8.0, respec-
3.13 Perpetuation 71

tively (Changsri 1961; Changsri and Weber 1963). and Deshpande 1979a; Taber 1964). According to
According to Gupta et al. (1969), A. brassicae Sasaki et al. (1985), sporulation of A. brassicae
isolated from B. oleracea var. botrytis tolerates a proceeds normally in darkness, but is inhibited by
wide pH range from 3.0 to 9.0, the optimum being monochromatic radiation from 350 to 520 nm and
5.5. No sporulation occurs at pH levels below 3.0 reduced by continuous irradiation of UV radiation
and above 9.0. Very good sporulation occurs at pH shorter than 350 nm. Light intensity is deleterious
levels between 5.0 and 6.5. According to Taber for A. brassicae spore germination. With the
et al. (1968), A. raphani grows well over a pH increase in light intensity from 0 to 5000 lx, there
range of 4.8–7.2. Mycelial growth occurs in pH is reduction in per cent spore germination, num-
range of 2.9–8.2 with an optimum at 6.5 (Ellis ber and size of lesions per leaf (Table 3.5).
1968a, b; Ansari et al. 1989a). Maximum spore germination with number and
Biomass production and sporulation of A. size of lesions per leaf is observed under dark
brassicae and A. brassicicola at different pH conditions (Kadian and Saharan 1984).
have been assessed. The maximum biomass Zone formation in cultures of A. brassicicola
(495.00 mg) of A. brassicae is observed at pH 5.0 is affected by light. Three minutes of light expo-
followed by 480.65 mg biomass at pH 6.0 and sure or 6 h of darkness alternating from the origi-
minimum biomass of 160.5 mg at pH 2. Abundant nal source of light is required to induce zonation.
sporulation of A. brassicae is at the pH 6.0, mod- Spore development is plentiful under combina-
erate sporulation is at the pH 5.0 and 7.0, and no tions of intermittent light and darkness (Changsri
sporulation at pH 2.0 and 3.0. The pH range 1961; Changsri and Weber 1963). Taber (1964)
of 5.0 to 7.0 favours abundant sporulation (++++) found that the longer the wave length, the greater
in the case of A. brassicicola, whereas moderate is the production of conidia in relation to chla-
sporulation is at pH 8.0, and no sporulation at mydospores by A. raphani.
pH 2.0 and 3.0.
In the case of A. brassicicola, the maximum
biomass (590.3 mg) is observed at pH 6.0 fol- 3.13 Perpetuation
lowed by 530.4 mg biomass at pH 7.0 and the
lowest biomass, 193.5 mg, at pH 2.0. The pH of It is believed that Alternaria species infecting
the medium is also one of the major factors brassicaceous plants survive and perpetuate
responsible for the growth and sporulation of the through infected seeds, diseased plant debris,
pathogen. The best suited pH range for the growth pathogen propagules in the soil and cultivated/
of A. brassicae and A. brassicicola are 5.0–6.0 weed hosts in a particular agroecosystem (Chupp
and 5.0–7.0, respectively (Table 3.4). The pH 1925; Chupp and Sherf 1960; Dixon 1981; Ellis
range from 3.0 to 8.5 is most suitable for both 1968a, b, 1971; Humpherson-Jones 1989; Kolte
mycelial development and conidial formation in 1985a, b; Neergaard 1945; Putnam et al. 1972;
Alternaria spp. including A. brassicae with opti- Saharan 1992a, b; Saharan et al. 2005; Sherf and
mum pH range from 5.0 to 6.5 (Kumar and Macnab 1986; Tsuneda and Skoropad 1977;
Choudhary 2006). Vaartnou and Tewari 1972a, b; Verma and
Saharan 1994; Weber 1973). Seed-borne
Alternaria are capable of surviving as viable
3.12.4 Light and Darkness conidia and/or as internal mycelium for periods
long enough for the seed to be harvested, stored,
Maximum growth and sporulation by A. brassi- transported and finally sown. The proportions of
cae occur with alternating light and darkness. Brassica seed carrying Alternaria spp. are very
Continuous light completely inhibits sporulation. high. Richardson (1970) found 40 and 10 %,
In light, a definite zonation of spore production respectively, of Brassica seed infected with A.
occurs (Ansari et al. 1989a; Changsri and Weber brassicicola and A. brassicae. Infection levels up
1963; Gupta et al. 1972; Mridha 1986; Mukadam to 50 % on B. oleracea var. capitata cv. Houston
72 3 Pathogen

Table 3.3 Cultural characters of Alternaria brassicae on different culture media (Prasada et al. 1970)
Average radial growth mma
Medium 6 days after 12 days after Colony character Sporulationb
Potato dextrose agar 31 55 Colony circular, ++
growth profuse with
concentric rings
cottony, olivaceous
black
Glucose asparagine 48 69 Colony irregular, ++
medium mycelium scanty,
cottony without
concentric rings,
brownish in colour
Oat meal agar 31 65 Colony circular, ++
cottony growth with
numerous concentric
rings buff olive in
colour
Sabouraud’s agar 33 59 Colony circular ++
smooth fluffy without
concentric rings,
brownish in colour
Brown’s agar 23 48 Colony circular, +
cottony growth
without concentric
rings, olivaceous in
colour
Czapek-Dox agar 26 56 Colony circular dense ++
with definite zonation,
olivaceous in colour
Richard’s agar 43 76 Colony circular dense +++
centre raised
surrounded by velvet
light brownish in
colour
Host decoction 40 68 Colony circular rough +++
cottony yellowish in
colour
CD @5 % ±2.68 ±2.06
a
Colony diameter recorded as average of four replications
b
+ = poor sporulation, ++ = good sporulation, +++ = excellent sporulation
Treatments – highly significant treatment No. 5 6 1 3 4 8 7 2 (after 6 days)

Evergreen and up to 90 % on turnip rape (B. sprouts may be infected with A. brassicicola, A.
rapa) are reported from Canada (Petrie 1974). In brassicae has not been detected in the seed of
the UK, infected seed is the primary source of the this crop (Humpherson-Jones and Maude 1982a,
Brassica dark leaf spot fungi A. brassicae and A. b; Humpherson-Jones 1985; Humpherson-Jones
brassicicola in autumn sown oilseed rape and and Phelps 1989). Nevertheless, A. brassicae has
vegetable brassicas (Humpherson-Jones 1985; recently assumed increasing dominance as a
Gladders 1984). In oilseed rape, A. brassicae is pathogen of Brussels sprouts. However, in tropi-
the dominant species, but in vegetable brassica cal countries like India, A. brassicae gets elimi-
seeds, A. brassicicola predominates. Although nated from the seeds of oilseed brassicas during
seed of vegetable brassicas including Brussels storage from April to September at 25–35 °C
3.13 Perpetuation 73

Table 3.4 Biomass and sporulation index of A. brassicae and A. brassicicola at different pH levels (Kumar and
Choudhary 2006)
A. brassicae A. brassicicola
Initial pH of Dry mycelial Sporulation Final pH of Dry mycelial Sporulation Final pH of the
filtrate weight (mg) the filtrate weight (mg) filtrate
2.0 160.0 − 3.5 193.5 − 3.4
3.0 244.6 − 5.2 270.7 − 5.6
4.0 390.4 ++ 6.3 396.4 ++ 6.0
5.0 495.0 +++ 7.2 500.0 ++++ 7.5
6.0 480.6 ++++ 7.5 590.3 ++++ 7.0
7.0 370.2 +++ 7.5 530.4 ++++ 7.5
8.0 280.5 ++ 7.9 386.6 +++ 7.4
Mean 346.0 409.7

SEm± CD (0.05 %)
pH of media 6.41 18.57
Pathogen 2.87 08.31
Interaction 9.07 26.27

Table 3.5 Influence of light intensity on A. brassicae

parts of dormant radish seed (Atkinson 1950).
infection (Kadian and Saharan 1984)
According to Dixon (1981), infected seed is the
Disease intensitya main avenue of transmission of three important
Spore Size of
Alternaria spp. of Brassica. It may be both exter-
Light germination No. of lesions/leaf
intensity after 24 h (%) lesions/ leaf (mm) nally as conidia adhering to the testa and inter-
0 lx 78 7.0 19.40 nally as mycelia present within the seed tissues.
1000 lx 63 6.2 15.50 The latter may lead to complete destruction of the
2000 lx 60 5.9 13.80 embryo. Four phases of seed transmission can be
5000 lx 49 3.8 08.20 distinguished: (1) transmission from seed to
a
Average of 20 leaves developing seedlings, (2) transmission from seed
to adjacent seed, (3) transmission from adult
plant to seed by fungal growth through the green
Table 3.6 Survival of Alternaria brassicae in rapeseed–
mustard seed during storage at Ludhiana and Hisar in siliquae coat into the moist atmosphere with the
1979 (Saharan 1984) seed pod and (4) transmission from adult plant to
Per cent seed infection adult plant. In the Philippines, A. brassicae
Yellow remains viable for 12–14 months in infected
Months RLM-198 Prakash Sarson leaves (Lapis and Ricaforte 1974). According to
April 30.0 11.8 15.5 Tripathi and Kaushik (1984), A. brassicae over-
May 24.0 1.7 1.9 winters with plant debris buried in the field below
June 16.0 0.0 0.0 the depth of 7.5 cm. According to Mehta et al.
July 10.0 0.3 0.0 (2002), incorporation of thoroughly mixed plant
August 0.0 0.0 0.0 debris in soil gives 100 % disease incidence
September 0.0 0.0 0.0 (Table 3.7). The pathogen may survive in temper-
ate condition during winter season and result in
infection to the summer crop. The pathogen may
(Table 3.6) temperature (Chahal 1981; Kolte spread to subtropical regions from temperate
1985a; Saharan 1992a; Mehta et al. 2002). regions with the withdrawal of monsoon as well
Alternaria raphani is internally seed borne in all as change of the wind currents from western to
74 3 Pathogen

Table 3.7 Alternaria blight incidence (%) in rapeseed– teristics after its 5-year period in dry soil, nor
mustard following inoculation with diseased debrisa at dif-
any loss of virulence when plants of radish,
ferent depth (Mehta et al., 2002)
stock and wallflower are inoculated (Atkinson
Disease incidence 1953). It is also possible that A. brassicae and A.
Inoculum depth (%)b
raphani survive through chlamydospores
1” below soil 77.77 (81.98)
(Atkinson 1953; Tsuneda and Skoropad 1977;
2” below soil 92.85 (80.83)
Vaartnou and Tewari 1972a, b). According to
3” below soil 45.23 (42.94)
Thoroughly mixed in soil 100.00 (90.00)
Vaartnou and Tewari (1972a, b), hyphal chla-
CD at 5 % −(28.87)
mydospores of A. raphani are produced on the
a stem, siliquae and seed of infected plants. Such
Stored at −10 °C
b spores have been found to remain viable even
Figures in parenthesis are angular transformed values
after prolonged deep freezing of the infected
material. Tsuneda and Skoropad (1977)
eastern areas. It is evident from the observations observed that conidia of A. brassicae are trans-
that the disease may appear during October– formed into microsclerotia (Fig. 4.2; Plate 3.1).
November on Brassica campestris var. toria Such structures are round and darkly pigmented,
sown in the month of August–September and resist desiccation and function in a similar man-
may further spread to main crop from toria in the ner to those of the sclerotia produced by other
subtropical areas of India (Mehta et al. 2002). fungi. More microsclerotia are formed only on
Ansari et al. (1989b) observed that in temper- the previously affected and partially decayed
ate and subtropical countries, A. brassicae sur- plant tissues suggesting the possibility of sur-
vives and perpetuates through diseased plant vival of the fungus through such structures.
debris. In the UK, infected debris of B. oleracea These structures on germination have been
seed crop is shown to be a major source of A. found to produce numerous new conidiophores
brassicicola spores (Humpherson-Jones and and conidia. Numerous brassicaceous weeds,
Maude 1982a). Infected debris of Brassica crops, forage brassicas and other weed hosts also serve
with A. brassicae and A. brassicicola remaining as sources of primary inoculum of Alternaria
on the ground after harvest, may provide a source spp. infecting several economically important
of dark leaf spot infection, which may be impli- brassicas (Table 2.2).
cated in the spread of the disease within and
between crops (Humpherson-Jones 1989). On
leaves of oilseed rape and cabbage, the fungi pro- 3.14 Spore Germination
duce viable spores for as long as the leaf tissue
survives which is up to 6 weeks in winter oilseed 3.14.1 Effect of Culture Media
rape and up to 8 weeks in summer. On cabbage,
spore viability lasts up to 8 weeks in winter and The germinability of spores of A. brassicae and
up to 12 weeks in summer (Humpherson-Jones A. brassicicola is higher when cultivated on rich
and Hocart 1983). Infected Crambe seed is the media than the spores produced on complex or
main source of primary inoculum of A. circinans poorly nutritive media. The presence of meta-
(Holcomb and Newman 1970). The virulence of bolic inhibitors in the growth media reduces
A. brassicicola, also from Crambe, can be retained spore germinability even in the presence of nutri-
for one year by culturing on filter paper and stor- ents (Czapek-Dox agar) and causes abnormalities
age in Petri dishes at 5QC (Kilpatrick 1975, 1976). in their morphology. Spores of both the species
Alternaria raphani is reported to survive for show maximum germinability irrespective of
5 years in soil culture (Atkinson 1950, 1953). their age (up to 20 days). Increase in the age of
The fungus shows no change in cultural charac- the spores (30 days onwards) either increases
3.14 Spore Germination 75

Plate 3.1 Light micrographs showing the development head and beak of the original conidium ×400, (e) germina-
of microsclerotium from Alternaria brassicae conidium: tion of a mature microsclerotium to form hyphae ×400
(a) conidium, (b) initial stage in the formation of a micro- and (f) germination of a frozen-thawed microsclerotium
sclerotium, (c) half-developed microsclerotium with showing many new conidia ×650 (Tsuneda and Skoropad
about 50 cells, (d) mature microsclerotium still showing 1977)
76 3 Pathogen

their latent period and/or reduces their germina- optimum temperature for spore germination of A.
bility, even in the presence of nutrients (Gupta brassicae. Spore germination commences after 4 h
et al. 1969). and reaches its peak after about 24 h; relative
humidity of >90 % is essential for maximum spore
germination (Fig. 3.9).
3.14.2 Effect of Temperature Conidial germination of A. brassicicola is
and Relative Humidity optimal at 30 °C (Gupta et al. 1972). Sarkar and
Sen Gupta (1978) observed that conidia of A.
For Alternaria brassicae causing brown rot of cau- brassicicola germinate best at 90–100 % relative
liflower, the optimum temperature for spore ger- humidity and a temperature of 22–32 °C. Self-
mination is 32–35 °C with the minimum of inhibition of A. brassicicola occurs when the
<1.5 °C and the maximum of 40–46 °C; as no tem- spore load in the germination medium is 0.2 opti-
perature between 40.5 and 46 °C was tried, the cal density. The self-inhibiting compound is vol-
exact maximum temperature was not determined. atile at 35 °C, and denatures at 90 °C, and is
The optimum temperature for mycelial growth is thought to be a substance of low molecular
25–27 °C (Weimer 1925). Degenhardt et al. (1982) weight (Mukadam 1982). Barton and Fine (1958)
observed excellent germination of A. brassicae obtained 86–90 % spore germination of A. bras-
spores at 15 °C with 90 % relative humidity and sicicola in the presence of 200 ppm gibberellic
6–8 h of host leaf wetness period in contrast to acid. Conidia of A. alternata causing leaf and pod
very poor germination of A. brassicicola spores blight of radish germinate at 15–30 °C with an
under similar conditions. Gupta et al. (1972) found optimum at 20–25 °C (Singh and Suhag 1983).
17–19 °C to be optimal for conidial germination of
A. brassicae. According to Ansari et al. (1988b), A.
brassicae spores germinate over a very wide range 3.14.3 Effect of Host Extract
of 10–28 °C with an optimum of 23 °C. However, and Exudates
Kadian and Saharan (1984) recorded 25 °C as the
Leaf extracts of Brassica spp. are generally
inhibitory to spore germination of A. brassicae.
Leaf extracts and exudates of both susceptible
and resistant cultivars are inhibitory, but inhibi-
tory effects of resistant cultivars like Tower and

Table 3.8 Effect of leaf exudates and leaf extracts from

different cultivars of rapeseed and mustard on spore
germination of A. brassicae (Kadian and Saharan 1984)
% Spore germination after 24 h of
incubationa
Cultivars Leaf exudates Leaf extracts
Tower 40 26
RC-781 49 29
YRT-3 66 47
CSR-448 61 44
CSR-741 58 45
CSR-142 62 46
RH-30 71 49
(Prakash)
Control (water) 88 91
Fig. 3.9 Influence of temperature on spore germination
a
of A. brassicae (Kadian and Saharan 1984) Average of 200 spores incubated at 25 °C
3.15 Seed Infection 77

RC-781 are more pronounced; generally, leaf turnip and radish showed the presence of A. bras-
exudates are more inhibitory than leaf extracts sicicola and A. raphani (Michail et al. 1979). In
(Table 3.8) (Kadian and Saharan 1984). The Finland, 91 % of white cabbage and red cabbage
effect of leaf exudates of Yellow Sarson (B. rapa) seed lots were found to be infected with A. bras-
and Taramira (E. sativa) is reported to vary with sicicola. Alternaria brassicae was found in 4 % of
the host variety, age of host plant and maturity of the cabbage and in 31 % of the rape seed lots.
the leaves (Sharma et al. 1985). Alternaria raphani was found in 30 % of the rad-
ish and black radish seed lots (Tahvonen 1979).
In the UK, between 1981 and 1984, up to
3.14.4 Effect of Light Intensity 25 % of B. napus seeds and 8.5 % of B. rapa
(turnip) seeds yielded A. brassicae, and 55 % and
Direct exposure to strong light intensity is 13 % seeds of B. oleracea, respectively, yielded
deleterious to A. brassicae spores, inhibiting A. brassicicola and A. brassicae (Humpherson-
germination and reducing host infection (Kadian Jones 1985). Alternaria brassicae was detected
and Saharan 1984). Maximum sporulation of at varying levels on seed of rape harvested from
A. brassicae occurs with alternating light and different climatic districts of New South Wales
darkness. Continuous light completely inhibits during 1983. The highest levels were observed on
sporulation (Ansari et al. 1989a; Changsri and seed samples from the South West slopes (North)
Weber 1963; Gupta et al. 1972; Mridha 1986; and Riverina (av. 22.8 and 14.2 %, respectively)
Mukadam and Deshpande 1979; Sasaki et al. with much lower levels of infection (av, <8.0 %)
1985; Taber 1964). on samples from central and northern districts
(Stovold et al. 1987). In Scotland, up to 89 % of
seeds of B. napus were recorded as being infected
3.15 Seed Infection with A. brassicae (Kothanur et al. 1982).
In Australia, samples of Brassica oleracea
Alternaria blight has been reported to be a serious seed showed A. brassicicola infection between
seed-borne disease of several seed-producing 24 and 37 % of seeds, with 4–8 % infection in the
brassicaceous crops in many countries of the world embryo tissues. Inoculation of seed with A. bras-
(Crosier and Patrick 1940; Ellis 1968a, b; Green sicicola results in loss of vigour in germinated
1947; Groves and Skolko 1944; Holcomb and seedlings, followed by death. The fungus retains
Newman 1970; Holtzhausen 1978; Holtzhausen its viability and pathogenicity on seed stored for
and Knox-Davies 1974; Jouan et al. 1972; up to 12 months. This investigation indicates that
Kilpatrick 1975, 1976; Kolte 1985a, b; Kothanur a high proportion of commercially available
et al. 1982; Nipoti 1978; Noble and Richardson Brassica seed is contaminated with A. brassicic-
1968; Petrie 1974; Pound et al. 1951; Richardson ola and may therefore be a primary source of
1970, 1979; Schimmer 1953; Van Schreven 1953; infection in Brassica crops in Australia (Sivapalan
Sivapalan and Browning 1992; Verma and Saharan and Browning 1992).
1994; Wiltshire 1947). High recovery of A. In Lithuania, Alternaria seed infection was
raphani from radish seeds in Michigan (McLean 10–100 % in winter rape and 16.0–93.6 % in
1947), and A. brassicicola from cabbage seeds in spring rape seed samples (Brazauskiene and
Washington (Pound et al. 1951), has been reported. Petraitiene 2006). In Japan, A. brassicicola
In South Africa, all three species of Alternaria, i.e. accounted for 57–95 % in cabbage seeds (Kubota
A. brassicae, A. brassicicola and A. raphani, have et al. 2006). Some other fungi, viz. A. alternata,
been isolated from seeds of cabbage, cauliflower, Rhizopus oryzae, Fusarium avenaceum,
broccoli and Brussels sprouts (Holtzhausen and Aspergillus niger, A. flavus, A. ostianus and
Knox-Davies 1974; Knox-Davies 1980). In Egypt, Penicillium spp., have also been recorded in cab-
testing for seed health in cabbage, cauliflower, bage seed from Japan.
78 3 Pathogen

In Canada, Petrie (1974) reported A. brassicae gus first colonizes the testas of inoculated seeds as
and A. raphani as important seed-borne they rupture and then attacks embryos after a fur-
pathogens of oleiferous brassicas. Brassica rapa ther 24–30 h. Testas of naturally infected cabbage
(turnip rape) had higher levels of A. brassicae seeds are colonized prior to rupture and largely in
than A. raphani; B. napus (rape) seed contained the hilum area. Spores lodge in the cavities and
considerably lower levels of both species. In folds of tissue depressions at the hilum and micro-
heavily infested seed of B. rapa, 73 % of the A. pyle. Spore germination and hyphal development
brassicae and 90 % of the A. raphani occurred on are frequently poor at the hilum, but in some cases
the seed surface. Storage of infected seed for 6–8 mycelial development is extensive over the hilum.
months at 25 °C reduced the levels of infestation Development on the remainder of the testa is gen-
by more than 50 %. In rapeseed–mustard, the fre- erally poor, the only extensive growth being pres-
quency of infection of seed by A. brassicae varies ent on damaged seeds (Plate 3.2). According to
with the number of lesions on the siliquae. Seed Vannacci and Pecchia (1988), A. raphani occurs in
infection diminished with the length of storage all seed parts of the radish, and conidia are present
period at higher temperature in summer months on the seed coat. Similar observations were made
(Chahal 1981). Mustard seeds showing 42 % by Atkinson (1950).
infection of A. brassicae in April became com-
pletely free when stored at 45 and 35 °C for 2 and
3 months, respectively (Shivpuri and Siradhana 3.15.1 Location of Seed-Borne
1989). According to Vishnuavat et al. (1985), A. Infection
brassicae seed infection in mustard was elimi-
nated during storage from March to July at 11.4– Component plating reveals the infection of A. bras-
39.2 °C, but survived at 5 °C in cold storage. sicae in the seeds of rapeseed and mustard mainly
Alternaria species comprised 86 % of the fun- internally in the seed coat and rarely in the embryo
gal flora isolated from samples of Crambe seed (Table 3.9). The range of internal seed coat infec-
from Illinois, Indiana, Maryland and Ohio; A. tion varies between 3 and 16 %, but embryo infec-
tenuis was isolated from 47 % and A. brassicicola tion is only 1 %. In infected seeds, sporulation of A.
from 39 % of the samples (Kilpatrick 1976). The brassicae is first observed in the hilum region of
high percentage of A. brassicicola-infected seed seeds after 48 h of incubation. In infected seeds,
indicates that infection is internal and that the fun- hilum infection is recorded after 96 h, and by this
gus is capable of systemic infection and direct time, sporulation is also recorded on other sites of
penetration of pods. The recovery of Alternaria the seed coat. The fungus continues to develop on
spp. is better through the blotter method than the infected seeds up to 240 h (Table 3.9). The sporula-
agar plate method. Ashraf and Chaudhary (1989) tion of the fungus around the hilum area is signifi-
recovered 20.8 % of A. alternata from rapeseed cantly higher than on the remaining parts of the
seed on blotter and 10.7 % on Agar plate; the seed coat (Shrestha et al. 2000).
recovery of A. brassicae was 10.7 % and 10.5 %,
respectively. Jain et al. (1982) recovered 31.5–
10.5 % A. alternata and 6.0–1.5 % A. brassicae 3.15.2 Disease Transmission
from rapeseed seed through the blotter method but in the Field
none through the agar plate method. In Bangladesh,
Latif et al. (2006) isolated Alternaria, Aspergillus, When infected seeds are sown, symptoms on the
Chaetomium, Curvularia, Fusarium, Penicillium cotyledonary leaves become visible in 2–3 weeks
and Rhizopus from mustard seeds. Domsch (1957) as small round or irregular brownish to black
described the infection of rape and cabbage seeds necrotic spots, particularly on the margins. Under
by A. brassicae and A. brassicicola. The relation- a stereomicroscope, the spots are dark brown in
ship between A. brassicicola and Brassica seeds the centre surrounded by concentric rings of light
was determined by Knox-Davies (1980). The fun- brown tissues followed by light-yellow haloes.
3.15 Seed Infection 79

Plate 3.2 Scanning electron micrographs of the surfaces tube with collapsed tip, (d) profuse development of
of cabbage seeds inoculated with Alternaria brassicicola hyphae on damaged seed, (e) roughened hilum area with
and germinated on water agar for 24 h at 21 °C: (a) germi- broken vascular elements of the funiculus and (f) spiral
nated spores with collapsed germ tube and initials of tur- thickening of xylem vessels of the broken vascular ele-
gid aerial hyphae, (b) germinated spore with collapsed ments of the hilum shown in figure (Courtesy: Knox-
germ tube and turgid aerial hyphae, (c) turgid aerial germ Davis 1979)
80 3 Pathogen

Table 3.9 Location and sporulation of Alternaria brassicae in different parts of Brassica seeds examined after 2, 4, 6,
8 and 10 days of infection (Shrestha et al. 2000)
Per cent infectiona Per cent sporulationb T test between
Remainder of location of
Sample Brassica spp. Cultivar Seed coat Embryo Hilum seed coat sporulation
1 B. campestris var. toria Local 3.0 0 3.7 1.5 3.8a
2 B. campestris var. toria Local 5.0 0 4.5 2.7 3.6
3 B. campestris var. toria Local 16.0 1 15.5 2.2 5.3
4 B. juncea TL-15 8.0 0 6.0 4.0 5.7
5 B. juncea Vaibhav 11.0 1 7.2 5.2 4.9
6 B. juncea Kranti 12.0 0 8.2 3.5 4.6
a
Mean of 100 seeds
b
Mean of 400 seeds
*Significant t(0.05) = 3.2

Table 3.10 Transmission of Alternaria brassicae in the field by infected Brassica seeds in 1991 (Shrestha et al. 2000)
Per cent seed Mean number of Mean % of emerged seedlings having
Sample Brassica spp. Cultivar infectiona emerged seedlings symptoms on cotyledonary leaves
1 B. campestris var. toria Local 10.5 260.0 3.8
2 B. campestris var. toria Local 17.2 262.2 7.2
3 B. campestris var. toria Local 27.7 248.5 8.7
4 B. juncea TL-15 20.7 221.5 8.2
5 B. juncea Vaibhav 26.5 191.0 9.7
6 B. juncea Kranti 39.0 199.0 11.1
a
Mean of four replications; 400 seeds sown in each replicate

Table 3.11 Transmission of Alternaria brassicae in the field by infected Brassica seeds in 1992 (Shrestha et al. 2000)

Mean Mean % of emerged

number of Seedlings having Seedlings having
Per cent seed emerged symptoms on symptoms on
Sample Brassica spp. Cultivar infection seedlingsa cotyledonary leaves cotyledonary leaves
1 B. campestris Vikas 12.0 279.5 6.2 13.9
var. toria
2 B. junceab Krishna 2.0 314.7 08 1.4
3 B. junceac Krishna 28.5 260.5 8.2 20.1
4 B. juncea Pusabold 31.5 211.2 14.2 18.3
5 B. juncea Varuna 54.0 189.7 16.2 28.2
a
Mean of four replications; 400 seeds sown in each replicate
b
Seeds collected from mid-hill
c
seeds collected from terai (plain)

On the underside of cotyledons, opposite to the become detached from the stem or remain hang-
necrotic spots, the areas are slightly shrunken ing from it (Tables 3.10 and 3.11; Fig. 3.10a, b).
forming shallow cavities. Occasionally, olive Symptoms on the first true leaves are small
brown conidia of A. brassicae are seen on sides brownish necrotic spots to distinct concentric
of lesions, but mainly they are on the underside. lesions (1–5 mm diameter) surrounded by faint-
The infected cotyledons soon start withering, yellow haloes (Shrestha et al. 2000).
3.15 Seed Infection 81

Fig. 3.10 (a) Relationship between seed infection and Figures showing the relationship for the year 1992
seedling infection due to A. brassicae in rapeseed and (r = 0.801; P < 0.001). Similarly, the correlation between
mustard in field. Correlation coefficient for both seasons seedling infection and first true leaf infection was also sig-
was highly significant (r = 0.0705; p < 0.001) in 1991. (b) nificant (r = 0.562; P < 0.01) (Shrestha et al. 2000)

Table 3.12 Effect of seed treatment fungicides on seed-borne infection of Alternaria brassicae and on subsequent
seedling infection in mustard (Shrestha et al. 2000)
Per cent seed Average count of Per cent infected
Fungicides Dose infectiona normal seedlingsb seedlingsb
Iprodione (Rovral) 2 g.a.i. /kg seed 0.5 (0.7)d 24.7 (5.0) a 0.5 (0.7) d
Thiram (Thiride) 2 g.a.i. /kg seed 2.5 (1.6)cd 22.7 (4.8) b 1.1 (1.0) c d
Mancozeb (Dithane M45) 2 g.a.i. /kg seed 3.0 (1.7)c 22.7 (4.8) b 1.5 (1.2) c
Captan (captan) 2 g.a.i. /kg seed 9.0 (2.9)b 19.7 (4.4) c 2.7 (1.6) b
Control (untreated) – 20.2 (4.5)a 16.7 (4.0) d 7.5 (2.7) a
LSD (0.05) – (0.75)* (0.18)* (0.18)*
CV (%) – 20.5 2.5 18.2
*Significant at (P < 0.001)
Mean in a column with different letters are significantly different at (P = 0.05)
a
Mean of four replications, 100 seeds per replication
b
Mean of four replicates, 25 seeds per replicate; value in parenthesis is square root transformation
82 3 Pathogen

3.15.3 Effect of Seed Treatment Barton LV, Fine JM (1958) Noninterference between
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 Infection Process, Pathogenesis
and Disease Cycle 4

4.1 Introduction an efficient and successful facultative parasites of

cruciferous plants.
Depending upon the growing season, Alternaria
species in crucifers cause primary infection
through the windborne spores produced from 4.2 Infection and Pathogenesis
over summered or overwintered primary inocu-
lum in infected seeds, plant debris, collateral The primary infection results from the windborne
hosts and weeds. Under congenial environmental spores produced on infected plant debris of the
conditions, spores (conidia) germinate readily in previous crop, weeds and other collateral hosts
the presence of moisture by producing a germ growing in the near vicinity. Infected seed as a
tube, which penetrates the hosts directly or source of primary inoculum is important in tem-
through stomata. Further, the process of infection perate regions. Chlamydospores and microscle-
and pathogenesis is governed by a number of rotia may initiate primary infection of A. raphani
external and internal factors of a specific host– and A. brassicae (Fig. 4.2). Conidia readily ger-
pathogen interaction. Induction of cell wall- minate in the presence of moisture by giving rise
degrading enzymes, secretion of toxins, to a germ tube, which emerges from any cell of
enforcement of fungal cell wall architecture and the spore. Theoretically, only one spore can cause
detoxification of host defence molecules are infection in the brassicaceous seedlings indicat-
essential functions for successful infection and ing a threshold infection value at a very low level
pathogenesis of Alternaria species. Genes impor- (Czyzewska 1971). Germ tubes from germinated
tant for each of these functions have been identi- spores of A. brassicae, A. brassicicola, A. raphani
fied in various fungal species. A transcription and A. alternata generally penetrate the undam-
factor gene, AbVF19, has been discovered from aged tissues of many brassicaceous hosts directly
A. brassicicola, which positively regulates hydro- (Changsri and Weber 1963; Czyzewska 1971;
lytic enzyme-coding gene. Another transcription Minkevicius 1932; Neergaard 1945; Schreven
factor gene, Amr1, negatively regulates a subset 1953; Weimer 1926), although indirect penetra-
of these genes during the late stage of pathogen- tion through stomata has been reported in A.
esis and positively regulates melanin biosynthe- brassicae (Changsri 1961; Tsuneda and Skoropad
sis during conidiogenesis. It seems that the 1978a).
suppressive functions of Amr 1 gene contribute to Alternaria conidia are considered as ‘dry
the specialized adaptation of A. brassicicola as conidia’, which are wind dispersed with the

© Springer Science+Business Media Singapore 2016 87

G.S. Saharan et al., Alternaria Diseases of Crucifers: Biology, Ecology and Disease Management,
DOI 10.1007/978-981-10-0021-8_4
88 4 Infection Process, Pathogenesis and Disease Cycle

maximum release taking place in the mid-after- infection processes of both A. brassicae and A.
noon. Since there is no report on the numerical brassicicola are initiated if water is present for
threshold for infection, lesions may consequently 5–8 h. For initiation of infection in the case of A.
be initiated from a single germinating conidium. brassicae in cauliflower leaves and heads,
Maude and Humpherson-Jones (1977) found that 25–31 °C temperature is reported as optimum
infected marrow-stem kale (B. oleracea var. (Weimer 1924); the disease increases with the
acephala) liberates 50 spores per cubic metre of increase in humidity of the surrounding air. In
air just prior to harvesting (190 spores per cubic rapeseed, A. brassicae establishes itself in less
metre of air at cutting) or 3200 spores per cubic than 6 h at 22 °C (Louvet and Billotte 1964). For
metre of air when windrowed crops are harvested. infection of cabbage plants, both A. brassicae
Spores can be carried up to 1000-m downwind and A. brassicicola require free water with an
during harvesting. Sharma and Gupta (1978) optimum temperature of 25 °C for A. brassicic-
trapped A. brassicae and A. brassicicola spores ola and 15 °C for A. brassicae. At these tempera-
from the air during October to April on Brown tures, a minimum of 16 h is necessary for the
Sarson crop fields under Indian conditions. initiation of infection and 48–72 h for optimal
Conidia of A. brassicae were intercepted at all disease development. Alternating wet and dry
hours of exposure, but conidia of A. brassicicola (70–80 % RH) periods of 16 h and 8 h, respec-
were seen only for a specific period during the tively, restrict infection by both species. The dif-
day. Maximum conidial catch was observed in ferential temperature requirements of the two
March, which coincides with the maximum man- species become most marked at 10 °C and 96 h of
ifestation of disease in the field. The main mode incubation after inoculation; A. brassicicola fails
of perpetuation is on debris of infected plants. to produce significant infection, whereas infec-
Mehta et al. (2002) have reported that diseased tion by A. brassicae produces numerous lesions
debris placed in deep freezer conditions (−10 °C) (Humpherson-Jones and Hocart 1983).
were able to cause 100 % infection in the coming The initial infection process including conid-
season when mixed in the soil as compared to the ial germination and mode of germ-tube penetra-
debris placed in the field and laboratory condi- tion are similar in both susceptible (Varuna) and
tions. It was further confirmed that pathogen sur- resistant (PAB 9511) cultivars, although consid-
vives in field for 2 years in the diseased debris erably slower in PAB 9511 due to tolerance.
collected from the temperate conditions (Canada) Apparent differences between the two cultivars,
as compared to the subtropical conditions of however, are noticeable by 1 day after infection.
India (Mehta and Tewari 2002). Local dispersal At the plant surface, impeded fungal growth,
is through air, splashes and occasional driving poor proliferation of spores and active suppres-
rain drops. The primary infection appears on the sion of dense hyphal growth are the factors
cotyledonary leaves forming the source of sec- involved in the expression of tolerance against A.
ondary infection for the entire crop. For infec- brassicae in cultivar PAB 9511 (Giri et al. 2013).
tion, a minimum period of 4 h of leaf wetness is In A. brassicicola, cuticular penetration is
required. Increase in period of leaf wetness at most frequent and stomatal penetration occurs to
25 °C increases infection and spread of the dis- a lesser extent. Penetration is preceded by the for-
ease rapidly. Under favourable temperature con- mation of appressoria from the tip of the germ
ditions, and the presence of dew, the spores infect tubes, which are either spherical or club shaped
other parts of the plant as well. The infection and 3–8 mm in diameter. Stomatal penetration is
occurs through the stomata, and under favourable more common with A. brassicae. Once within the
climatic conditions, the new lesions arise within host, the epidermal cells are fully invaded, the
4–6 days bearing spores (Verma and Saharan mycelia ramify through and between the meso-
1994). phyll and palisade cells, and the entire leaf is
Frequent infection occurs when water is pres- soon parasitized. Early in the post-penetration
ent on the host surface due to dew formation. The phase, the invaded epidermal cells become
4.3 Transcription Factors Associated with Pathogenesis 89

necrotic, and the parenchyma tissues ahead of the ease of rapeseed–mustard has been observed
advancing hyphae often collapse (Dixon 1981). (Nehemiah and Deshpande 1976; Suri and
On the leaves of B. napus cv. Midas and B. Mandahar 1982). The differential susceptibility
rapa cv. Torch, conidia of A. brassicae germinate of the brassicaceous hosts to A. brassicae, A.
at the rate of 12.1 % and 19.5 %, respectively, at brassicicola and A. raphani has been correlated
9 h after inoculation. Conidia usually germinate with differences in amino acid content (Changsri
by producing either germ tubes or secondary 1961). The population of the pathogens varies
conidia. Penetration of leaves by A. brassicae is with the age, variety of the host, maturity of
abundant at 24 h and occurs either with or with- leaves and climatic conditions. The colonies
out the formation of appressoria. Penetration of increase when the temperature and relative
the cultivar Torch leaves occurs either directly humidity of the field are 21.5 °C and 85 %,
through epidermal cells or indirectly through sto- respectively. Both A. brassicae and A. brassicic-
mata, while Midas leaves are penetrated almost ola occur in greater profusion on leaves of B.
exclusively through the stomata. Black spot rapa var. Yellow Sarson than on leaves of E.
lesions develop within 48 h after inoculation sativa. The younger leaves have lower incidence
(Tsuneda and Skoropad 1978b). According to of disease of either pathogen compared to the
Tewari (1983, 1986), A. brassicae in rapeseed older leaves (Sharma et al. 1986). Species of
becomes subcuticular after direct penetration. Alternaria pathogenic to crucifers are prolific
This is followed by colonization of the epidermal toxin producers, which facilitate their necrotic
and the mesophyll cells. In the leaves of rapeseed lifestyle. The role of toxins in the process of
cultivars Candle and Altex, the pathogen heavily infection has been described in Chap. 10 in Sect.
colonizes the necrotic center and is not present in 10.5. Besides role of toxins in Alternaria patho-
the chlorotic area indicating that a diffusible genesis, relatively few genes and/or gene prod-
metabolite may be directly or indirectly respon- ucts have been identified that contribute to or are
sible for leaf chlorosis. The plasma membrane is required for pathogenicity.
the first target of the diffusible metabolite.
Subsequently, the chloroplasts are either directly
or indirectly affected leading to leaf chlorosis. 4.3 Transcription Factors
Relative to chloroplasts, the effect on mitochon- Associated
dria is seen at a much later stage. The cells in the with Pathogenesis
necrotic area are almost completely devoid of
cellular organelles and reveal electron-dense Out of 138 A. brassicicola transcription factor
lamellar deposits (Plates 4.1 and 4.2; Tewari genes with zinc finger domains, and other regula-
1983). Alternaria infection in the C. abyssinica tory genes analysed to date, only six genes includ-
leaves is preceded by toxic substances secreted ing AbSte12C, AbPacC, AbVf19C, Amr1, AbVf8 and
by the fungus (Czyzewska 1971). AbPro1C are strongly associated with pathogene-
The same may be true for other Brassica. sis (Table 4.1). AbSte12 is a homologue of the
According to Suri and Mandahar (1984, 1985, Ste12 gene with mutants that are either non-
1986) and Suri et al. (1983), cytokinin-like sub- pathogenic or reduced in virulence in several
stances appear to be actively involved in infection fungi (Wong and Dumas 2010). Ste12 is regulated
and pathogenesis of A. brassicicola. However, by a MAP kinase, Fus3/Kss1. Mutants of either
cytokinin, 6-benzyl amino purine, reduces dis- homologous gene in A. brassicicola show defec-
ease symptoms and mycelial growth within plant tive conidial development and are non-pathogenic
tissues. It also inhibits in vitro growth of A. bras- (Cho et al. 2007, 2009). The AbPacC gene has a
sicae (Sharma et al. 2010). Poapst et al. (1979) single copy in the genome sequence of A. bras-
suggested the role of the hormone ethylene in sicicola and shows a high sequence similarity
screening cabbage leaves infected with A. bras- (E = 0.0) with the PacC gene in other fungi. AbVf19
sicicola. Production of cellulases and pectolytic regulates suite of genes that are important for
enzymes by A. brassicae causing leaf blight dis- decomposing and utilizing plant materials during
90 4 Infection Process, Pathogenesis and Disease Cycle

Plate 4.1 Alternaria brassicae on leaves of Brassica (SH), epicuticular wax layer (W) and electron-dense layer
rapa cv. Candle. Bar = 5.0 μm: (a) light micrographs (ED) of cuticle ×7000; and (c) light micrograph showing
showing germinating conidia (broad arrows) and subcu- a hypha penetrating through a stroma (narrow arrow) and
ticular hypha (SH) penetrating (asterisk) into epidermal another one penetrating into a subepidermal position
cell (E) (narrow arrows) of cuticle ×1200; (b) transmis- (broad arrow) without becoming subcuticular ×800
sion electron micrograph showing subcuticular hyphae (Tewari 1986)

the late stage of plant infection. Regardless of the late stationary phase of mycelial growth and
their function, however, all mutants studied to also forms under stressful conditions, including
date except Amr 1 are non-pathogenic or show a ultraviolet irradiation, a hyperosmotic environ-
reduction in virulence (Cho et al. 2012). ment, nutrient deprivation or an accumulation of
toxic wastes during in vitro culture. Overexpression
of a Cmr1 ortholog in Bipolaris oryzae (Bmr1)
4.3.1 Melanin Biosynthesis causes continuous induction of the three down-
and Virulence in A. brassicicola stream genes in the melanin synthesis pathway
and increases melanization of colonies (Kihara
Melanin is an ubiquitous pigment that plays an et al. 2008). The loss of function of Cmr1, its
important role in protecting fungi from the dam- homologues or their downstream genes in other
aging effects of environmental stress. It accumu- fungi result in melanin deficiency. Cho et al.
lates in the cell walls of hyphae and conidia during (2012) identified a transcription factor Amr1,
4.3 Transcription Factors Associated with Pathogenesis 91

Plate 4.2 Transmission electron micrographs of ultra- of the cuticle ×8050; (c) junctional region between the
thin sections of A. brassicae on the leaves of Brassica two epidermal cells (E) showing subcuticular hyphae
napus cv. Altex. Bar = 2.0 μm: (a) a penetrating germ tube (SH) and glancing section of a hypha passing through the
(broad arrow), a subcuticular hypha (SH) and two other cell wall (asterisk). The latter hypha is penetrating
hyphae (H), embedded in the cell wall of the host adaxial between the two cells ×7400; (d) section showing two
leaf epidermal cell. Note the electron-dense material at the subcuticular hyphae (SH), one of which is penetrating into
point of penetration (narrow arrows), the layer of epicu- the cell wall of the epidermal cell (E) ×4200; and (e) sec-
ticular was (W) detached from the electron-dense layer of tion showing subcuticular hyphae (SH) a few other hyphae
the cuticle (ED) ×4200; (b) a penetrating germ tube (H) in the epidermal cells and one hypha (asterisk) below
(broad arrow). Note the electron-dense material at the the epidermis close to the palisade mesophyll tissue
point of penetration (narrow arrows), the detached epicu- ×3000 (Tewari 1986)
ticular wax layer (W) and electron-translucent layer (EL)

which regulates the melanin synthesis pathway in melanin is not associated with virulence in A.
A. brassicicola. The melanin synthesis-associated brassicicola as has been reported in other fungi.
structural genes Bm1 and Pks are located together However, there appears to be a linkage between
in a 30-kb region and their organization, and ori- melanin biosynthesis and reduction in virulence,
entation is the same as in the closely related fun- perhaps due to a lifestyle switch from pathogene-
gus Cochliobolus heterostrophus. Interestingly, sis to reproduction (Cho et al. 2012).
92 4 Infection Process, Pathogenesis and Disease Cycle

Table 4.1 Summary of Alternaria brassicicola tran- ducing larger lesions, and require fewer conidia
scription factor domains based on Pfam scans (Cho et al.
for successful infection. Mutation of Amr1
2012)
homologues in the phytopathogenic fungi,
KO KO Pathogenesis- Magnaporthe grisea and Colletotrichum lage-
Description Gene 1a 2b associated genes
narium, produces melanin-deficient conidia. The
Zn finger 221 4 117 AbVf8, AbVf19C,
domain AbSte12C, AbPacC, increased virulence of the ∆amr1 mutants is
AbPro1C, Amr1 unexpected and unique among melanin-deficient
Fungal- 72 1 0 mutants of pathogenic fungi.
specific TF
PHD finger 1 – –
Helix-turn- 29 1 – 4.4 The Cause of Increased
helix
Virulence in ∆amr1 Mutants
Regulatory 23 5 –
receiver
Leucine 21 2 – The pink pigment secreted by ∆amr1 mutants is
zipper neither toxic to host plants nor beneficial for the
High- 15 3 – wild-type A. brassicicola to infect host plants.
mobility box Furthermore, melanin-deficient mutants of
group another gene, abpks7, show no changes in viru-
Helix-loop- 13 5 – lence. Neither the presence of pink pigment nor
helix
the lack of melanin is associated with the increased
DNA-binding 10 – –
myb virulence of the Δamr1 mutants. Additionally,
Jumonji 7 – – there is no difference in germination or vegetative
Homeobox 4 – – growth between the ∆amr1 mutants and the wild
β-Regulatory 3 – – type in the presence of various chemicals.
protein However, the mutants are more susceptible than
T regulatory 2 – – wild type to UV light and the cell wall-degrading
Total 421 21 117 enzymes, glucanase. The mutant’s ability to
a
Genes of previously screened targeted deletion mutants respond to stress is not associated with their
(15) increased virulence, since plant glucanases are
b
Genes of mutants created and screened in this study
c
Genes whose functions were previously characterized
known to be defence enzymes (Cho et al. 2012).
(15, 20) The ability of the mutants to outgrow the wild
type when citrus pectin was the major carbon
source suggested that the mutants were more effi-
4.3.2 Mutation of the Amr1 Gene cient in utilizing the protein component of plant
Unexpectedly Causes cell walls and middle lamellae. The expression
Increased Virulence profile comparisons identified 30 hydrolytic
enzyme genes expressed more in the mutant than
Amr1 gene knockout mutants, both gene disrup- in the wild-type A. brassicicola during pathogen-
tion (amr1) and gene deletion (∆amr1 and esis. However, they included only two pectate
∆amr1; amr1p-GEP), are easily recognized by lyase genes and one pectin esterase gene. Other
the lack of melanin in their conidia, orange- genes upregulated in the mutant compared to the
coloured colonies and the secretion of a pink pig- wild type included gene coding for two cutinases,
ment. These phenotypes are similar to those a lipolytic enzyme and 20 other various glycoside
found in mutants of its homologues in other fungi hydrolases (GH). Interestingly, 6 of the 22 genes
(Kihara et al. 2008). The ∆amr1 mutants are in the GH61 gene family are expressed at higher
more susceptible to UV light and enzyme diges- levels in the ∆amr1 mutant. The GH61 family
tion. Unexpectedly, the Amr1 gene knockout might have a role in degrading cellulose, ligno-
mutants are more virulent than the wild type, pro- celluloses, chitin or other polysaccharides
4.5 Evolution of Virulence in A. brassicicola 93

(Karkehabadi et al. 2008). The GH61 family may associated genes. This finding may aid in the
be involved in digesting pectin’s side chains, selection of specific targets for future analysis of
which are structurally diverse in constituting sug- their functions in pathogenesis (Cho et al. 2012).
ars and glycosidic linkages. These may digest
other cell wall components, such as cellulose and
hemicelluloses along with other glycoside 4.5 Evolution of Virulence
hydrolases. in A. brassicicola
Induction of the Amr1 gene in A. brassicicola
did not occur until 64-h post inoculation (hpi) The melanin-deficient mutant phenotype and
(Fig. 4.1). Suppression of the Amr1 gene might expression pattern of the Amr1, Brn, Brn2 and
be important during pathogenesis when energy is Scd1 genes in A. brassicicola suggest that the
allocated for colonization of host tissues and function of the Amr1 gene is to regulate melanin
overcoming host defence mechanisms, rather synthesis during conidiogenesis. This function is
than producing reproductive dispersal structures highly conserved in other fungi. In addition to
like conidia. In the wild type, the transcripts of regulating the melanin biosynthesis pathway,
hydrolytic enzyme genes were slightly increased Amr1 negatively regulated virulence_ENREF_1.
compared to the actin transcripts, as previously The evolution of virulence has been a subject of
reported (Karkehabadi et al. 2008). In addition, extensive theoretical analysis. The trade-off
when conidiation was initiated, the induction of hypothesis produced a number of models show-
Amr1 was accompanied by a sharp increase in ing a contested relationship between virulence
transcripts of its downstream genes in the mela- and transmissibility. It challenges the hypothesis
nin synthesis pathway. There was also small that strong virulence can seriously damage or kill
increase in the transcripts of hydrolytic enzyme- host plants and threaten pathogen survival, so
coding genes in the wild type. However, many of parasites will ultimately evolve to be less viru-
these genes mostly expressing glycoside hydro- lent. However, analysis of ecological and epide-
lases, a few cutinases and pectate digestion miological factors related to virulence has
enzymes were expressed at higher levels in the produced inconsistent results. Therefore, a modi-
∆amr1-4 and ∆amr1; Amr1p-GFP mutants did fied trade-off theory predicted that strong para-
not express Amr1. This suggests that Amr1 sup- sites became moderately virulent to their hosts
pressed genes predicted to be involved in the over time but did not completely lose their viru-
digestion of lipids and carbohydrates. This lence. Cho et al. (2012) identified a transcription
occurred during late pathogenesis (88 hpi) when factor suppressing virulence but enhancing the
they were still needed to some extent for either ability of a fungus to preserve itself in space, and
killing (Noda et al. 2010) or digesting host tis- time via melanin synthesis, primarily in conidia.
sues. Whether the Amr1 gene is involved in the This study provides an example of a transcription
regulation of most hydrolytic enzymes sequen- factor gene important for survival in nature that
tially induced in A. brassicicola during pathogen- plays a critical role in negatively regulating viru-
esis has yet to be investigated. Other genes lence in the necrotrophic plant pathogens, A.
encoding a necrosis-inducing protein, two P450s, brassicicola. The suppression of virulence
the aegerolysin peptidase homologue and other observed is unique and might have been acquired
hypothetical proteins might have contributed to by this fungus, as this phenotype has not been
the increased virulence of the amr1 mutant and observed in loss-of-function mutants of Amr1
warrant further investigation. Interestingly, 24 homologues in other species of plant pathogenic
genes expressed at higher levels in Δamr1 were fungi. An ancestral fungal strain of A. brassicic-
expressed at lower levels in the ∆Abvf19 deletion ola without the ability to suppress virulence by
mutants that displayed reduction in virulence, the activity of Amr1 could have been a more viru-
suggesting opposite roles of the two transcription lent pathogen. Negative regulation of virulence is
factor genes in the regulation of pathogenesis- rare but exists in other phytopathogenic fungi.
94 4 Infection Process, Pathogenesis and Disease Cycle

Fig. 4.1 Expression of melanin biosynthesis-associated A total of three biological replicates (N = 3) were used for
genes and four hydrolytic enzyme-coding genes. (a–c) this study. Bars represent standard error. Wt = wild type,
Relative transcription abundance of each gene was deter- ∆a = ∆amr1: Amr1pGFP, GY = glucose yeast extract
mined in comparison to actin gene transcripts in the same broth. SCD1 = scytalone dehydratase, Brn1-13HN reduc-
tissue. Y-axes show relative abundance of the transcript tase, Brn2-14HN reductase, Cbh7 = cellobiohydrolase,
compared to the actin gene. (d) Expression ratio between Amr1 = Alternaria melanin regulation, chymo = chymo-
the ∆amr1 and wild type during the late stage of infection. trypsin (Cho et al. 2012)

Currently, A. brassicicola is a prolific saprophyte 4.6 Identification

and necrotroph, efficiently colonizing suscepti- of Pathogenicity Factors
ble, weakened or dead host plants. Its pathoge-
nicity, however, is inhibited on more resistant The functional redundancy of lipases has been
host plants. Cho et al. (2012) speculated that the revealed by Yao and Koller (1994, 1995), Berto
suppressive functions of Amr1 contribute to the et al. (1999) and Cho et al. (2006) with regard to
specialized adaptation of A. brassicicola as an pathogenicity. Interestingly, one of the factors
efficient and successful facultative parasite. responsible for the pathogenicity has been pre-
4.7 Disease Cycle 95

dicted to be secondary metabolic production. A be utilized as a sole carbon source, but this dis-
non-ribosomal peptide synthase gene (NPS6) in ruption had no significant effect on A. brassicic-
Cochliobolus heterostrophus and A. brassicicola ola pathogenicity. An extracellular lipase was
was found to direct the biosynthesis of a sidero- found to be produced by A. brassicicola in vitro
phore metabolite important for oxidative stress (Berto et al. 1999). In this study, anti-lipase anti-
tolerance and pathogenicity (Oide et al. 2006). bodies were found to decrease the ability of A.
The secondary metabolic corresponding to or brassicicola to cause disease on cauliflower
synthesized via AbNPS2 has yet to be character- leaves significantly. However, disruption of four
ized. However, research is needed to further char- predicted A. brassicicola lipase genes expressed
acterize secondary metabolic biosynthetic genes during plant infection did not result in reduced
and their role in pathogenicity and fungal devel- virulence on cabbage (Cho et al. 2006). Results
opment, in the Alternaria–Brassica pathosystem of a non-ribosomal peptide synthase gene (NPS6)
fungal signal transduction, e.g. disruption of the in Cochliobolus heterostrophus and A. brassicic-
Fus3/Kss1. MAP kinase homologue (Amk1) in A. ola suggest to direct the biosynthesis of a sidero-
brassicicola resulted in a complete loss of patho- phore metabolite important for oxidative stress
genicity as observed in other fungi (Cho et al. tolerance and pathogenicity (Oide et al. 2006). In
2006, 2007). Interestingly, in the later study, it another study, non-ribosomal peptide synthase
was shown that addition of long polypeptide gene (AbNPS2) was found to be important for
nutrients partially restored pathogenicity to the cell wall integrity, conidial viability and viru-
mutants. In addition, two novel virulent factors lence of aged spores of A. brassicicola (Kim
were predicted to encode a transcription factor et al. 2007). The secondary metabolite corre-
(AbPro1) and a two-component histidine kinase sponding to or synthesized via AbNPS2 has yet to
gene (AbN1K1) (Cho et al. 2009). Both of these be characterized.
kinases are pathogenicity factors in phytopatho-
genic fungi. Slt2 was found to be associated with
cell wall integrity and HOG kinase homology 4.7 Disease Cycle
with oxidative stress tolerance (Xu 2000).
Another major work pertaining to the studies It is believed that Alternaria survives through
related to the identification of virulence factors infected seed, plant debris, soil and weed hosts. It
was the disruption of Aso-1, a gene required for has been proved that Alternaria does not survive
hyphal fusion (anastomosis), which was also through seed to cause infection in the coming sea-
found to be required for pathogenicity in son due to high-temperature conditions during
Alternaria species (Craven et al. 2008). summer months in storage houses of North India.
Eventually, over a hundred genes have been func- However, the possibility of its survival through
tionally analysed through various techniques like seed on hills cannot be ruled out. Alternaria was
gene knockout and overexpression experiments not recovered from infected seeds of Yellow
making A. brassicicola the species of choice for Sarson and raya stored under Hisar (Haryana) and
functional genomics research to define conserved Ludhiana (Punjab) conditions after June and July
virulence mechanisms for this important genus of months. Alternaria brassicae survives with plant
fungi (Oide et al. 2006; Cho et al. 2006, 2007, debris buried in the field at 7.5-cm depth in the
2009; Kim et al. 2007). form of microsclerotia and chlamydospore in the
For the identification of A. brassicicola, an infected leaves. Alternaria brassicae is known to
attempt was made to examine the role of cutinase become seed borne although this mode of survival
genes in A. brassicicola pathogenesis (Yao and and dispersal is not very significant. The main
Köller 1994, 1995). In these studies, biolistic mode of perpetuation is on debris of infected
transformation was used to disrupt the CUTAB1 plants. Mehta et al. (2002) have reported that dis-
gene. Disruption of CUTAB1 affected sapro- eased debris placed in deep freezer conditions
phytic growth since cutin was no longer able to (−10 °C) were able to cause 100 % infection in the
96 4 Infection Process, Pathogenesis and Disease Cycle

Fig. 4.2 Disease cycle of Alternaria on crucifers (Saharan et al. 2005)

References 97

coming season when mixed in the soil as com- the expression of hydrolytic enzyme genes in the fungus
Alternaria brassicicola. Fungal Gen Biol 44:543–553
pared to the debris placed in the field and labora-
Cho Y, Kim KH, La Rota M, Scott D, Santopietro G,
tory conditions. It was further confirmed that Callihan M, Mitchell TK (2009) Identification of viru-
pathogen survives in field for 2 years in the dis- lence factors by high throughput targeted gene dele-
eased debris collected from the temperate condi- tion of regulatory genes in Alternaria brassicicola.
Mol Microbiol 72:1316–1333
tions (Canada) as compared to the subtropical
Cho Y, Srivastava A, Ohm RA, Lawrence CB, Wang KH,
conditions of India (Mehta and Tewari 2002). Grigoriev IV, Marahatta SP (2012) Transcription fac-
Local dispersal is through air, splashes and occa- tor Amr1 induces melanin biosynthesis and suppresses
sional driving raindrops. Spores are produced virulence in Alternaria brassicicola. PLoS Pathog 8,
e1002974. doi:10.1371/journal.ppat.1002974
abundantly in wet weather. Spore release, which
Craven KD, Velez H, Cho Y, Lawrence CB, Mitchell TK
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restricted by a high humidity so that air spore con- fungal necrotroph Alternaria brassicicola. Eukaryote
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Czyzewska S (1971) The pathogenicity of Alternaria spp.
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Giri P, Taj G, Meena PD, Kumar A (2013) Microscopic
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in period of leaf wetness at 25 °C increases infec- Afr J Microbiol Res 7:4284–4290
Humpherson-Jones FM, Hocart MJ (1983) Alternaria dis-
tion and spread of the disease rapidly. Under
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ence of dew, the spores infect other parts of the Wellesbourne, Warwick, UK, pp 63–64
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C, Sandgren M (2008) The first structure of a glyco-
stomata, and under favourable climatic condi-
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Arase S (2008) Characterization of the BMR1 gene
of the pathogen is presented in Fig. 4.2.
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sis genes in the phytopathogenic fungus Bipolaris ory-
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Changsri W, Weber GF (1963) Three Alternaria species
Maude RB, Humpherson-Jones FM (1977) Dark leaf spot
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gene disruption method for Alternaria brassicicola
brassicae on diseased plant debris of rapeseed and
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(LME) constructs. Mol Plant Microb Interact 19:7–15
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Minkevicius A (1932) Investigations on the influences of role in pathogenesis. Experientia Basel, Birkhauser
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Stemphylium taxonomy, parasitism, economical signifi- sicae (Berk.) Sacc. Plant Sci 41:105–109
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Nehemiah KMA, Deshpande KB (1976) Cellulase pro- and accumulation of labelled metabolites at infection
duction and decomposition of cotton fabric and filter sites of Alternaria brassicicola. Mycopathologia
paper by Alternaria brassicae. Indian Phytopathol 94:153–156
29:55–57 Suri RA, Mandahar CL, Gill PS (1983) Study of secretion
Noda J, Brito N, Gonzalez C (2010) The Botrytis cinerea of cytokinins by Alternaria brassicicola and their pos-
xylanase Xyn11A contributes to virulence with its sible role in pathogenesis in mustard. Indian J Plant
necrotizing activity, not with its catalytic activity. Pathol 1:117–121
BMC Plant Biol 10:38 Tewari JP (1983) Cellular alterations in the black spot of
Oide S, Moeder W, Krasnoff S, Gibson D, Haas H, rapeseed caused by Alternaria brassicae.
Yoshioka K, Turgeon BG (2006) NPS6, encoding a Phytopathology 73:831. (Abstr.)
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Schreven DAV (1953) Alternaria, Stemphylium en Food Canada, Saskatoon, SK, Canada, 162 p
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vivo and in vitro by Alternaria brassicicola and their brassicicola. Mol Plant-Microbe Interact 8:122–130
 Epidemiology and Forecasting
5

5.1 Introduction 5.2 Disease Development

in Relation to Environmental
Alternaria blight of crucifers occurs and devel- Conditions
ops in the epidemic form under the combination
of a number of factors including availability of Alternaria blight of Brassicaceae caused by A.
susceptible host cultivars, source of virulent brassicae is favoured by low temperature, high
pathogen inoculum in the vicinity, and favourable humidity and splashing rain (Table 5.1) (Dey
environmental and cultural conditions. 1948; Humpherson-Jones and Phelps 1989;
Temperature range of 15–25 °C, relative humid- Louvet 1958; Louvet and Billotte 1964; Jose
ity >90 %, wind velocity of 2–5 km/h, and inter- et al. 2005). In Canada (McDonald 1959) and
mittent rains are the most abiotic conducive Holland (Van Schreven 1953), optimum temper-
conditions for development of Alternaria blight ature for the development of A. brassicae is
under field conditions. In addition, closer spacing reported to be between 20 and 24 °C. In India, a
of 30 × 15 cm, high doses of nitrogen (>80 kg/ temperature range of 15–25 °C, relative humidity
ha), frequent irrigation and growing of suscepti- of 70–90 %, intermittent winter rains and wind
ble cultivars further rapidly increase the disease velocity around 2–5 km/h (Fig. 5.1) have been
severity in rapeseed–mustard. The disease pro- reported to be most conducive to Alternaria
gression under field conditions as influenced by blight development in mustard (Ansari et al.
environmental conditions and dates of sowing 1988; Saharan 1991; Saharan and Kadian 1984;
has been measured through the development of Saharan et al. 1981). In France, Louvet (1958,
models. Disease forecasting models have been 1963) observed severe development of A. bras-
developed taking into account the leaf wetness sicae at a temperature range of 15–22 °C, relative
period; number of rainy days; minimum, maxi- humidity of 80 % for a period of 36 h and under
mum and optimum temperatures and relative stormy and high wind velocity conditions.
humidity; date of sowing; variety and species of Similar conducive conditions have also been
Brassica crops grown under different agro-­ observed in India by Awasthi and Kolte (1989)
ecological conditions. and Chahal and Kang (1979). Domsch (1957)

© Springer Science+Business Media Singapore 2016 99

G.S. Saharan et al., Alternaria Diseases of Crucifers: Biology, Ecology and Disease Management,
DOI 10.1007/978-981-10-0021-8_5
100 5 Epidemiology and Forecasting

reported relative humidity between 95 and 100 % 5–7 h of sunshine/day and a minimum tempera-
at least for 18 h and temperature of 21–27 °C for ture range of 7–10 °C concomitant with the maxi-
three successive days as essential for epidemic mum temperature range of 20–23 °C have been
development of Alternaria disease of rape and positively correlated (r = 0.511–0.805) with the
cabbage. Alternaria brassicae produces abun- severity of disease. Increase in age beyond 30
dant spores after a succession of wet and dry days is also positively correlated (r = 0.9802)
periods, which are probably spread over short with the increase in disease severity. Indian mus-
distances by rain and wind and over greater dis- tard is susceptible to leaf infection phase during
tances by the action of wind alone during dry rosette to flowering stage, when the relative
weather (Louvet and Billotte 1964). To a very humidity 81–94 % is concomitant with total rainy
limited extent, slugs can spread the disease by days of 4–11. Low relative humidity with less
ingesting spores, which remain viable in the ali- frequent rain, irrespective of the other factors,
mentary canal and can infect plants when does not result in high severity of the leaf infec-
excreted (Hasan and Vago 1966). tion phase. For Alternaria blight severity on
According to Awasthi and Kolte (1989), pods, relative humidity shows 0.6 % variation,
Alternaria blight of rapeseed–mustard develops whereas the combined effect of relative humidity
best during rosette to flowering stages; relative and maximum and minimum temperatures
humidity from 67 to 73 %, rainfall >70 mm, accounts for 70.3 % variation (Yadav and Brar

Table 5.1 Weather parameters congenial for Alternaria brassicae under (a) field and (b) laboratory conditions (Jose
et al. 2005)
Maximum Minimum Relative Rainfall Wind velocity
Authors temperature (°C) temperature (°C) humidity (%) TFD (days) (mm) (km/h)
(a) Field conditions
Awasthi and 7–10 >67 >6 >70
Kolte (1994),
Pantnagar, India
Chahal and Kang >80
(1979), Punjab,
India
Dang et al. 16.3–25.7 1.6–9.7 79.6–96.5 2.5–6.0
(1995), Hisar,
India
Kolte and Singh 20–23 7–10 67–73 >70
(1997),
Pantnagar, India
Ansari et al. 16.3–25.7 1.6–9.7 79.6–96.5 2.5–6.0
(1989), Aligarh,
India
Saharan and 18.7–23.14 6.8–10.12 53.8–80.8 4.5–8.2
Kadian (1984),
Hisar, India
(b) Laboratory conditions
Authors Optimum temperature Relative humidity
Humpherson-Jones and Phelps 18–24 91.5–100
(1989), Warwick, UK
Kadian and Saharan (1984), Hisar, 25 90
India
Kennedy and Graham (1995), 18–24 87
Warwick, UK
Ansari et al. (1989), Aligarh, India 21–30 85–100
5.2 Disease Development in Relation to Environmental Conditions 101

Fig. 5.1 Influence of weather variables on development of Alternaria blight (Saharan and Kadian 1984)

2003). Similarly, Chahal (1986) reported that the severity on leaves, while total rainy days, RH and
susceptibility of Brown Sarson to A. brassicae maximum and minimum temperatures have enor-
increases with the age of the host. Maximum sus- mous effect on the disease on pods; RH (67 %),
ceptibility of rapeseed plants has been observed total rain duration (TRD) (>6), RF (>70 mm) and
at 55–85-day-old plants (Sarkar and Sen Gupta minimum temperature (7–10 °C) are more condu-
1978). For maximum infection and disease devel- cive for Alternaria blight severity on leaves,
opment in mustard, a minimum period of 4-h leaf whereas the RH and TRD factors significantly
wetness is essential. Longer periods of leaf wet- influence spread of pod infection.
ness at 25 °C increase the infection frequency Dang et al. (1995) during 1988–1990 crop
(Table 5.2) on the leaves (Kadian and Saharan season at Hisar, India, studied the development
1984; Saharan and Kadian 1984; Saharan 1991). and temporal progression of Alternaria leaf
Reduced light intensity is more favourable for blight and correlated the disease severity with
lesion development. Conversely, spores produced environmental conditions on different rapeseed–
under high light intensities show reduced germi- mustard cultivars. Results indicated that cumula-
nation (Kadian 1982; Kadian and Saharan 1984; tive intensity increase is significant among the
Mukadam and Deshpande 1979; Meena et al years, cultivars and observation intervals. The
2011). Awasthi and Kolte (1994) observed that the maximum increase in Alternaria blight is on
combined effect of rainfall (RF), relative humidity Yellow Sarson (YSPb-24) and Brown Sarson
(RH) and minimum temperature influences blight (BSH-1). However, the progression is least on
 102

Table 5.2 Influence of temperature and leaf wetness on infection of A. brassicae (Saharan and Kadian 1984)
a
Disease intensity after hours of leaf wetness
4 8 12 16 20 24
No. of lesion/ Size No. of lesion/ Size No. of lesion/ Size No. of lesion/ Size No. of lesion/ Size No. of lesion/ Size
Temperature (°C) leaf (mm) leaf (mm) leaf (mm) leaf (mm) leaf (mm) leaf (mm)
15 0.1 0.1 0.3 0.2 0.5 0.6 0.6 0.5 0.7 0.7 0.7 0.7
20 0.9 1.0 1.2 1.4 1.6 1.7 1.5 1.9 1.6 1.6 1.7 1.5
25 3.6 5.0 4.2 8.1 6.0 13.9 6.8 14.3 7.0 14.6 7.4 15.2
30 0.2 1.5 0.4 1.3 0.8 1.9 1.5 1.5 0.7 1.7 0.8 1.6
a
Average of ten leaves
5
Epidemiology and Forecasting
5.2 Disease Development in Relation to Environmental Conditions 103

mustard cultivar RH-8113. The variation can be dates at weekly intervals and revealed that the
explained up to 58–60 % with combined effect of disease’s first appearance on leaves occurred 42
temperature and wind velocity. When the cumu- and 139 days after sowing (DAS). The disease on
lative increase is compared with the prevailing pods appeared later between 67 and 142 DAS and
environmental conditions, the disease progresses being highest at 99 DAS. Severity of Alternaria
fairly well with maximum temperature ranging blight on leaves was positively correlated to a
between 16.3 and 25.7 °C, minimum temperature daily temperature maximum (T max.) of 18–27
between 1.6 and 9.7 °C, maximum RH between C, daily temperature minimum (T min) of
79.6 and 96.5 % and wind velocity between 2.5 8–12 °C, daily temperature mean (T mean) of
and 6.0 km/h. The disease progression curves >10 °C, relative humidity morning (RH mor)
(Fig. 5.2) for different varieties showed linear >92 %, relative humidity evening (RH even.)
relationship between disease intensity and crop >40 % and RH mean of >70 % in the preceding
growth stage (R2 = 0.97 to 0.99). Hong and Fitt week. Disease severity on pods was favoured by
(1996) also reported a strong correlation between a daily T max of 20–30 °C, daily T mean of
Alternaria blight development with leaf wetness >14 °C, RH mor >90 %, daily RH mean of
period and temperature. Meena (2005) and >70 %, sunshine >9 h and leaf wetness >10 h.
Meena et al. (2011) reported that disease severity Progression of Alternaria on cultivar Varuna
increases with delay in date of sowing. The value sown on different dates at Bharatpur (India) is
of AUDPC (area under disease progress curve) presented in Fig. 5.3.
and ‘r’ value (apparent infection rate) are more in Alternaria brassicicola fails to produce sig-
the variety ‘Varuna’ with the delayed sowing. nificant infections at 10 °C after 96 h, while A.
The severity of Alternaria blight is significantly brassicae produces lesions on the host under
lower in October-sown crop (Kumar and Kumar similar conditions (Humpherson-Jones et al.
2006). The spread of the disease is more in the 1983). Alternaria brassicicola lesions on over-
broadcasting method than in the line sowing wintered leaf litter of B. oleracea grown for seed
(45 cm). The disease intensity also decreased production produce high concentrations of spores
with the addition of K (40 kg/ha) along with the in the spring and are able to initiate new infec-
recommended dose of fertilizers. Chattopadhyay tions on foliage and subsequently on inflores-
et al. (2005) analysed the data for Alternaria cence and siliquae. A vertical disease gradient
blight progression and development from eight develops in maturing crops, the lowest siliquae
locations using cultivar ‘Varuna’ sown on ten becoming infected first and infection spreading

Fig. 5.2 Temporal progression of Alternaria

leaf blight on five varieties of rapeseed–
mustard during 1988 to 1990 crop seasons
(pooled data) (Dang et al. 1995)
104 5 Epidemiology and Forecasting

Fig. 5.3 Progress of Alternaria leaf blight severity on cultivar Varuna of Indian mustard in different dates of sowing at
Bharatpur in 2001–2002 (Chattopadhyay et al. 2005)

slowly upwards. Spores are produced abundantly increasing with increasing light intensity
after 20 h leaf wetness at a mean temperature of (Humpherson-Jones and Phelps 1989).
13 °C or more. Their release is stimulated by a The primary infection occurs on the cotyle-
reduction in RH but inhibited at a constant high donary leaves of mustard forming the source of
RH resulting in a daily cycle in air spore concen- secondary infection for the entire crop under
trations with minimum occurring in the early field. A minimum of 4 h of leaf wetness is
morning and maximum in the early afternoon required for infection. Increased leaf wetness
(Figs. 5.4 and 5.5); (Humpherson-Jones and duration at 25 °C increases infection and spreads
Maude 1982). Peaks of high spore concentration the disease rapidly. Under favourable tempera-
are usually associated with dry days, shortly after ture conditions, and presence of dew, the spores
rain, high temperature or high wind speed for infect other parts of the plant. The infection
conidial dispersal of A. brassicicola on Chinese occurs through the stomata, and under favourable
cabbage at 10 h. The number of conidia trapped climatic conditions, the new lesions arise within
at a height of 25 cm above ground level is greater 4–6 days bearing spores. The pathogen penetrates
than that at 50, 75 and 100 cm (Fig. 5.6) (Chen the tissues of the pods and infects the seed
et al. 2003). Sporulation in A. brassicae, and A. (Saharan et al. 2005). The congenial factors for
brassicicola on naturally infected leaf discs of Alternaria spores germination have been reported
oilseed rape, and cabbage requires RH > 91.5 % as darkness or low light intensity (<1000 lx),
and 87 %, respectively. The optimum tempera- 25 °C temperature and more than 90 % RH.
ture for sporulation is 18–24 °C for A. brassicae The favourable environmental factors for dis-
and 20–30 °C for A. brassicicola at which tem- ease progression under field conditions have been
perature both fungi produce spores in 12–14 h., reported at temperature 12–25 °C, RH > 70 %
and in temperature above 24 °C, sporulation in A. with intermittent winter rains or irrigation, wind
brassicae is inhibited. White light inhibits sporu- velocity between 2 and 5 km/h, closer plant spac-
lation in A. brassicae with the degree of i­ nhibition ing (30 × 15 cm) and high doses of nitrogen
5.2 Disease Development in Relation to Environmental Conditions 105

Fig. 5.4 The mean daily concentration of Alternaria brassicicola spores in the air within Brassica oleracea seed pro-
duction crops: (a) 1976 and (b) 1977 study; ● >0.2 mm rain; 0–0 infected pods (Humpherson-Jones and Maude 1982)
106 5 Epidemiology and Forecasting

Fig. 5.5 The hourly concentration of

Alternaria brassicicola spores in the
air within a cabbage seed crop. Mean
values of seven high spore count days
(Humpherson-Jones and Maude
1982)

(80 kg /ha). The temperature maximum of where

26–29 °C with RH Av >65 % favours the disease
development (Sangeetha and Siddaramaiah ALTn = Expected Alternaria blight;
2007). ALTc = Disease intensity in current week;
Under Haryana conditions, a raya crop sown ALTp = Disease intensity of previous week. The
in the last week of October recorded 52 % dis- R2 value recorded was 0.84.
ease, while that sown in the third week of
November had only 15.5 % disease (Saharan Compared to the older plants, younger plants
et al. 2005). Under Hisar conditions, Alternaria of radish are less susceptible to A. raphani. The
spores were trapped in a 7-day volumetric spore disease intensities in 28 days old and younger
trap, 10–11 days before the disease appearance plants were 32.5 % and 17 %, respectively.
and their concentration increased and reached However, increase in age beyond 28 days did not
maximum in March. The spores were trapped have much influence on disease development.
maximum during 10.00 am to 2.00 pm (46 % of There is a direct correlation between the host age
total spores) and minimum during 10.00 pm to and disease intensity (Table 5.3). In case of A.
6.00 am. Alternaria spore concentrations alternata, 30–35-day-old radish plants have also
declined sharply after 2.00 pm (Figs. 5.7 and 5.8) been reported highly susceptible to Alternaria
(Singh 2005). Based on the concentration of blight (Suhag et al. 1985). Disease intensities data
spores trapped, a prediction equation was devel- in Table 5.4 reveal that 10-day-old cultures of A.
oped as follows: raphani are more virulent than the older cultures;
as the age of the culture increases, the disease
ALTn = 21.50 + 1.164ALTc + 0.128ALTp
intensity decreases with least in 30-day-­old cul-
- 0.165 ( T max ) - 0.154 ( T min )
ture. This is also true with several other pathogens
- 0.203 ( RH mor ) - 0.095 ( RH even ) , where young cultures are in more active ­metabolic
5.2 Disease Development in Relation to Environmental Conditions 107

a
100
y = 5.03 + 91.09 exp[–89.6 exp(–0.208t)]
r 2 = 0.9999
Disease incidence
80

60
(%)

40

20

b
300
Spore concentration
[spores m-3]

200

100

c
Rainfall
50 100
RH (%)
40 80

RH (%)
Rainfall

30
(mm)

60
20
40
10

0 20

d
40 Max.
Min.
Temperature

30
(°C)

20
10
0
10 20 30 40
Days after inoculation

25 30 5 10 15 20
Dec. 93 Jan. 94

Fig. 5.6 (a) Disease incidence (%) of dark leaf spot on in the air; (c, d) Meteorological data during the experi-
Chinese cabbage and the Gompertz growth curve and mental period in summer (1993–1994) (Chen et al. 2003)
model; (b) spore concentration of Alternaria brassicicola
108 5 Epidemiology and Forecasting

Fig. 5.7 Spore percentage (A.

brassicae) trapped over crop
canopy during different time
interval of the day (Singh
2005)

Fig. 5.8 Weekly average

number of spores of Alternaria
brassicae (Singh 2005)

stage than the older ones. Disease intensity is According to Singh (1988), the conidia of
directly correlated with the inoculum load. The Alternaria appear in the air 8–13 days prior to
maximum disease intensity (25.1 %) is recorded the first onset of disease on the crop. The high
with A. raphani inoculum load of 2.0 × 103 spores ­percentage of disease incidence and disease
ml−1; further increase in inoculum load did not intensity indices is closely related to the high
cause significant increase in disease intensity percentage of Alternaria spore load in the air.
(Table 5.5) (Sangwan et al. 2002). Maximum incidence of mustard blight depends
The effects of humidity, seed infection, tem- upon high RH (77–94 %) and high tempera-
perature and nutrient stress on development of A. ture of 28–30 °C. Alternaria alternata causing
brassicicola seedling blight of cabbage have been leaf and pod blight of radish develops fast
studied under controlled environment chambers when mean temperature is 20.6–23.8 °C and
by Bassey and Gabrielson (1983a). Cotyledon relative humidity from 57.9 to 67.4 % (Singh
lesions are more severe with extended moist incu- and Suhag 1983; Suhag et al. 1985). Under
bation following wound inoculation. The temper- field conditions, A. raphani infection pro-
ature of 30 °C is optimum for wire-­stem disease gresses rapidly at 22–26 °C. At high soil mois-
from naturally infected seed. Natural wire stem ture content, infection is less at 18 °C
does not occur below 15 °C even with a heavily (Atkinson 1950). Alternaria blight develop-
infected seed lot, and little occurs below 20 °C ment on Crambe is dependent on warm and
with a moderately or lightly infected seed lot. damp weather (Czyzewska 1971).
5.3 Disease Development in Relation to Nutrition and Cultural Conditions 109

Table 5.3 Effect of age of radish plants on blight inten- 1984). In Prague (Czechoslovakia), higher doses
sity caused by Alternaria raphani (Sangwan et al. 2002)
of NPK increased A. brassicae infection in win-
Age of plants at inoculation ter rape pods. Similarly, top dressing with nitro-
(days) Disease intensity (%)
gen in spring significantly increased Alternaria
7 7.3 (1.7)
intensity on rape pods (Stankova 1972). Planting
14 14.4 (6.7)
time has a major influence on the incidence of
21 16.7 (8.7)
disease in mustard crops. In Haryana (India),
28 34.5 (32.5)
52 % disease incidence has been recorded on
35 32.1 (28.7)
mustard crop sown in the last week of October.
42 29.8 (25.0)
However, when the crop is sown in the third
CD (p = 0.05) (12.9)
week of November, infection fell to 15.5 %
Values in the parentheses are arc transformed
(Saharan 1984). Severity of Alternaria blight of
mustard decreases with delay in sowing at
Table 5.4 Effect of age of culture (Alternaria raphani) Bharatpur (Meena et al. 2011). The Alternaria
on blight of radish (Sangwan et al. 2002)
pod blight of radish seed crop is significantly
Age of culture (days) Disease intensity (%) less in the normal season transplanting of 15th
10 28.2 (22.4) December in the Punjab (India), whereas
15 26.6 (20.1) Alternaria twig blight is significantly less in the
20 21.4 (13.2) early transplanting of 15th November (Sandhu
25 19.6 (11.4) et al. 1984, 1985).
30 19.4 (10.1) The dwarfing agent, Cycocel (CCC), sprayed
CD (p = 0.05) (5.1) on rapeseed crop tends to decrease infection of A.
Values in the parentheses are arc transformed brassicae, whereas alar increases the infection
(Strzelczyk and Rozej 1974). The fungicide
Table 5.5 Effect of inoculum (Alternaria raphani) load Calixin (75 % w/w tridemorph) applied at
on blight intensity in radish (Sangwan et al. 2002) 1.125 ml in 4-L water predisposes cabbage plants
Inoculum load (×103 conidia / to A. brassicae and A. brassicicola infection
ml−1) Disease intensity (%) (Munro 1984). Incidence of dark leaf blight (A.
0.0 6.3 (1.2) brassicicola) on Chinese cabbage is higher in
0.5 13.8 (5.7) plots with row orientation parallel to the average
1.0 20.7 (12.5) wind direction than in plots with row orientation
1.5 23.9 (16.5) at right angles to the average wind direction
2.0 30.0 (25.1) (Chen and Price 2002).
2.5 29.8 (24.8) The role of NK (N90 kg ha−1 + K 40 kg ha−1) in
3.0 29.6 (24.5)
decreasing the severity of Alternaria blight is
CD (p = 0.05) (4.9)
more pronounced than PK (P 40 kg ha−1 + K
Values in the parentheses are arc transformed 40 kg ha−1), NP and K (40 kg ha−1) applications.
The decrease in Alternaria blight severity due to
K is probably due to increased production of phe-
5.3 Disease Development nolics in plants, which inhibit conidial germina-
in Relation to Nutrition tion and decrease sporulation of A. brassicae.
and Cultural Conditions The decrease in the severity of Alternaria blight
due to NK application consistently increases seed
Closer spacing (30 × 15 cm), high doses of nitro- yield more than 68 % than those of control and
gen (>80 kg N/ha) and frequent irrigations are other treatments. The K-fertilized plants also
known to rapidly increase severity of Alternaria show increased resistance to lodging, increased
blight of mustard (Kadian and Saharan 1988; 1000 seed weight and decreased seed infection.
Saharan 1991, 1992a; Saharan and Kadian Seeds obtained from K-fertilized plants show
110 5 Epidemiology and Forecasting

good seed germinability and vigorous seedling (Table 5.6). Tower gets 15.6 % of leaf infection
growth (Sharma and Kolte 1994). compared to 38.5 % in Prakash.
The rate of disease development is faster on
varieties belonging to B. juncea (RH-30,
5.4 Disease Development RH-8113, RH-8695, RH-8546) and B. campes-
in Relation to Host tris (YSPb-24, BSH-1, Candle, Shiva) compared
Resistance to B. carinata (HC-2, HC-9001), B. napus (GSH-­
1) and B. alba (Mehta et al. 2008). Kumar (2008)
Alternaria blight progression on highly suscepti- observed that three genotypes, viz. PR-8988,
ble (B. juncea cv. Prakash) and moderately PR-9024 and Kranti, have partial resistance and
­resistant (B. napus cv. Tower) cultivars of rape- show lowest blight severity. The size of the spots
seed–mustard is governed by reduction in infec- is positively and significantly correlated with dis-
tion frequencies coupled with reduction in ease severity, leaf defoliation and sporulation.
number and size of lesion formation due to host
resistance (Figs. 5.9 and 5.10). In highly suscep-
tible cultivar Prakash, the onset of disease occurs 5.5 Disease Development
earlier, with a greater abundance of large lesions. in Relation to Flea Beetle
Such cultivars have a shorter latent period, abun-
dant conidial production per lesion and higher Transmission of A. brassicicola to cabbage by
infection rate (0.46) in comparison to moderately flea beetles (Phyllotreta crucifereae) has been
resistant cultivar Tower (Saharan 1991, 1992b; reported by Dillard et al. (1998). Alternaria leaf
Saharan and Kadian 1983). On cultivar Tower, spot develops on plants that are infested with the
infection is delayed up to 25 days with the occur- contaminated flea beetles. Faeces obtained from
rence of very few lesions of restricted size flea beetles that fed on cabbage infected with

Fig. 5.9 Increase in number of Alternaria 5

lesions on rapeseed–mustard cultivars
(Saharan and Kadian 1983)
.41
=0
– 3 r
RT .36
4 Y =0
3 0 r 0.3
9
– r=
RH AS
H
AK 6
PR 0.2 0
r= 0.3
4 8 r=
–4 –741
No OF LESIGNS/LEAF

R 8
3 CS CSR 0.2
2 r=
14
R–
CS .09
r=0
791
RC–

1
r = 0.05
TOWER

0
JAN. 14 JAN. 19 JAN. 24 JAN. 29 FEB. 3
DATE OF OBSERVATIONS
5.5 Disease Development in Relation to Flee Beetle 111

Fig. 5.10 Increase in size of Alternaria 25

lesions on rapeseed–mustard cultivars
(Saharan and Kadian 1983)

20

YRT–3

LESION LENGTH (mm)

0 H
–3 AS
RH AK
PR
15

1
–74
CSR
48
10 CSR–4

CSR–142
81
RC–7
5

TOWER

0
JAN 14 JAN 19 JAN 24 JAN 29 FEB 3
DATE OF OBSERVATION

Table 5.6 Factors influencing resistance/susceptibility of different cultivars of rapeseed–mustard against Alternaria
brassicae (Saharan and Kadian 1983)
Stomata−1 Lesions/leaflet2 Sporulation3
No. Size No. per lesion Incubation 4 Latent
Leaf (per sq. period period
Cultivars surface cm) (mm) Size (mm) (days) (days)
Tower Upper 145 15.7 1.8 1.9 80 10 16
Lower 210 25.9
RC-781 Upper 166 17.3 3.3 5.7 92 8 12
Lower 245 26.7
CSR-448 Upper 196 19.2 3.7 10.7 120 7 10
Lower 303 27.9
CSR-741 Upper 189 19.3 6.3 14.6 125 7 9
Lower 292 20.0
CSR-142 Upper 199 19.3 4.4 10.6 120 8 11
Lower 306 28.0
YRT-3 Upper 225 22.0 10.2 22.3 250 6 9
Lower 403 30.8
RH-30 Upper 221 22.0 8.1 19.1 240 6 9
Lower 392 30.6
Prakash Upper 229 22.7 8.7 18.9 260 6 8
Lower 413 31.2
*1 and 3 average of 100 observations
**2 and 4 average of 10 leaves
112 5 Epidemiology and Forecasting

A. brassicicola contain intact and broken conidia It has received much attention recently
of A. brassicicola and undigested pieces of cab- because of its flexibility and simplicity
bage leaf. The conidia are viable after passing (Fontem 1986).
through the flea beetles, as evidenced by their 4. Gregory power function: y = axb, in which y is
germination on glass slide used for collecting the the proportion of disease at x units of distance
faeces. Conidia of A. brassicicola are observed from the source, a is the value of y at x = I and
by scanning electron microscopy on all parts of b is the rate of change in y with change in x;
the flea beetle bodies including wings, mouth- i.e. b is an estimate of the slope of the gradi-
parts, antennae and legs. ent, and the value of b is usually negative. The
transformation equation is log (y) = log (a) + b
log(x). Since this model cannot be used to pre-
5.6 Disease Development dict disease at the inoculum source (distance
in Relation to Barrier Crops x = 0), Gregory’s model was modified by
Mundt and Leonard (1985) to include a trans-
When oats were used as barrier crops to control lation factor (m) [y = a(x + m)b], which allows
the secondary spread of Alternaria blight of rape- the estimation of disease at the source when
seed–mustard under Hisar, Indian conditions, the x = o. Values of m are approximately the radii
percent disease intensity decreased in subsequent of the sources.
strips of the crop. The percent reduction in dis- 5. Kiyosawa and Shiyomi (1972) model: [y = a
ease intensity was 23.6 % in first strip, which exp(−bx)].
increased to 66.2 % in the fifth strip over the This model has been used to describe spore
inoculated strip (Table 5.7). The barrier crop dispersal in cultivars with multilines.
(oat) was found superior over three sprays of 6. Lambert et al. (1980) model: [y = a exp (ax^n)]
Dithane M-45 for the control of disease (Singh with mixed power and exponential terms and
2005). a shape parameter n.
7. Maffia (1985) hollow-shaped curves: [y = axb
exp(nx)]. Sometimes 1n[y/(1−y)] or −1n[−1n
5.7  odels to Describe
M (y)] is plotted over log10 (x) or x to linearize
the Progress of the Disease hollow-shaped curves.

Several models have been proposed to describe To illustrate some possibilities, Fontem et al.
the progress of compound interest diseases like (1991) evaluated progress and spread of dark leaf
Alternaria disease of crucifers (Fontem et al. spots in three cabbage cultivars during two sea-
1991). sons by fitting representative curves of eight gra-
dient models (Fig. 5.11).
1. Gompertz equations: Y = - ln éë - ln ( y ) ùû Three growth models (logistic, Gompertz and
2. Logisitic equations: Y = - ln éë y / (1 - y ) ùû Weibull) are fitted to the severity values by non-­
linear regression. The shape parameter (c) for the
These equations were compared by Berger
Weibull function averages 3.61. The average
(1981) to describe plant disease progress.
daily epidemic rates with the logistic model are
(
3. Weibull function: Y = ln éë1 / (1 - y ) ùû} ^ 1 / c K = 0.06 in the winter and K = 0.11 in the spring.

Table 5.7 Effect of barrier crop (oat) on the development of Alternaria blight of rapeseed–mustard (Singh 2005)

Disease intensity in Disease intensity in the subsequent strips intercepted by barrier crop (%)
inoculated strip (%) 1 2 3 4 5
71.6 54.7 (23.6) 46.5 (35.0) 38.9 (45.6) 31.7 (55.7) 24.2 (66.2)
5.7 Models to Describe the Progress of the Disease 113

Fig. 5.11 Representation curves of eight gradient mod- distance x =0.1 and decreased to y = 0.01 at x = 10.0.
els. The curves from the bottom to the top of the figure Different curve shapes are possible with the models of
at x = 1.0 were generated by the following models: Lambert et al. and Hoerl by using other values for the
y = axb(Gregory); 1n[y/(1−y)]vs.log (x); y = a exp (bxn), shape parameter (n). The curve shown for the Gregory
n = 0.2 (Lambert et al.,); Y = axb exp(nx), n = −0.2 model typifies many of the steep gradients observed for
(Hoerl); −1n [−1n(y)] vs. log (x); y = a exp (−bx) Alternaria brassicicola on cabbage in which y = disease
(Kiyosawa and Shiyomi); 1n[y/(1−y)] vs. x; and −1n proportion and x = metres (Fontem et al. 1991; Berger
[−1n(y) vs. x. All curves began with proportion y = 0.6 at 1981)

Final disease severity (yt) at the source averages data that is explained by the model (goodness of
0.52 in the winter and 0.97 in the spring. The fit) (Cornell and Berger 1987; Daniel and Wood
nearly flat gradients from 1 to 6.7 m are fitted sat- 1980). The pattern of scatter when residuals (Y
isfactorily by each of above-mentioned gradient observed – Y predicted) are plotted vs. time is
models, but the total gradient is described ade- used to confirm the suitability of the model
quately only by the modified model of Gregory (Cornell and Berger 1987). The progress of dis-
and the Hoerl function. ease among cultivars is compared with observed
The three models are modified as: initial disease (Yo), epidemic rate (k), final dis-
ease severity (Yf) and the area under the disease
1. Logistic (Berger 1981): Yt = 1/{1 + exp [a + k1t]} progress curve (AUDPC) (Shaner and Finney
2. Gompertz (Berger 1981): Yt = exp[−a exp(−Kgt)] 1977).
3. Weibull (Pennypacker et al. 1980): Yt Similar statistical methods are used to evalu-
=1−exp{−Kw(t−a)]C} ate models for the spread of disease. A non-linear
curve-fitting programme (Berger 1988) is used to
In these models, ‘Yt’ is the disease proportion evaluate five gradient models:
at time t, and k is the rate parameter. The term
“a” in the three models is a parameter for initial 1. Gregory (1968): y = axb
disease; in the logistic model, a = −1n [Yo/(1− 2. Modified Gregory (Mundt and Leonard 1985):
Yo)], and in the Gompertz model, a = −1n (Yo). y = a(x + m)b
In the Weibull function, a positions the curve on 3. Kiyosawa and Shiyomi (1972): y = a exp (−bx)
the time axis, and c is the curve-shape parameter. 4. Lambert et al. (1980): y = a exp (−bxn)
In these analyses, ‘Yo’ is the amount of disease 5. Hoerl (Daniel and Wood 1980; Maffia 1985):
at first observations, and the maximum amount y = axb exp(nx)
of possible disease (Y max) is assumed to be 1.0.
The R2 and the residual sum of squares are used In these models, ‘Y’ is the proportion of dis-
to evaluate the magnitude of variation among the ease severity at x units of distance from the
114 5 Epidemiology and Forecasting

i­noculum source and b is the slope parameter. In The above-mentioned models have been
the model of Gregory (1968), a is the disease pro- applied in modified form by several Brassica sci-
portion at one unit of distance from the source, entists to forecast Alternaria disease of crucifers
whereas in the modified Gregory (Mundt and taking into account numbers of environmental,
Leonard 1985) model, a is the disease propor- host age, variety, species, date of planting, dis-
tions at 1-m units of distance. In the models of ease on leaf and pods, time of first appearance of
Lambert et al. (1980) and Hoerl (Daniel and disease and other variables, which influence the
Wood 1980), n is a parameter that modifies the disease development under field conditions
curve shape, and this parameter increases the (Magarey et al. 2005; Chattopadhyay et al. 2005;
flexibility and applicability of the models if curve Mahapatra and Das 2014; Kumar et al. 2013;
shapes vary. Awasthi and Kolte 1994; Dang et al. 2006; Mehta
Three other gradient models were examined in et al. 2002; Mehta 2014).
their linearized form because the models con-
tained logarithms both to base e (1n) and to the
base 10 (log), and the non-linear equations were 5.8 Disease Forecasting
rather complex.
A simple generic infection model has been devel-
1. Logistic gradient model (Berger and Luke oped for predicting infection periods by fungal
1979): foliar pathogens. The model is designed primar-
éë y / (1 - y ) ùû = ln éë a / (1 - a ) ùû - b log ( x ) ily for use in forecasting pathogens that do not
2. Gompertz gradient model (Danos et al. 1984): have extensive epidemiological data. Most exist-
ing infection models require a background epide-
- ln éë - ln ( y ) ùû = ln éë - ln ( a ) ùû - b log ( x ) .
miological data set, usually laboratory estimates
3. A variant of the logistic gradient model of infection at multiple temperature and wetness
(Minogue and Fry 1983): combinations. The model developed can use
ln éë y / (1 - y ) ùû = ln éë a / (1 - a ) ùû - bx. inputs based on subjective estimates of the cardi-
nal temperatures and wetness duration require-
In these regressions, ‘a’ is the average propor- ment. These inputs are available for many
tion of disease on the plants closest (0.3 m) to the pathogens or may be estimated from related
inoculum source. These transformations are pathogens. The model uses a temperature
compared by the R2 and for significance of the response function, which is scaled to the mini-
slope of the regression by ‘t’ test. mum, and optimum values of the surface wetness
The volume under the disease progress sur- duration requirement. The minimum wetness
face (VUDPS), as suggested by Maffia (1985), duration requirement (W min) is the number of
and the isopathic rate (rate of movement in space hours required to produce 20 % disease incidence
of a given level of disease severity) (Berger and or 5 % disease severity on inoculated plant parts
Luke 1979) are calculated from the progress and at a given temperature. The model was validated
spread characteristics of the disease. The VUDPS with published data from 53 controlled labora-
is calculated from the AUDPC at each point in tory studies, each with at least four combinations
space as: of temperature and wetness. Validation yielded
an average correlation coefficient of 0.83 and a
{
VUDPS = å éë( Ai + 1 + Ai ) / 2 ùû [ X i + 1 - X i ] } root mean square error of 4.9 h., but there was
uncertainty about the value of the input parame-
where Ai = the AUDPC at Xi units of distance ters for some pathogens. The value of W min var-
from the source. The isopathic rates are calcu- ied from 1–48 h. and was relatively uniform for
lated from the source to 6.7 m for the y = 0.1 iso- species in the genera Alternaria. Alternaria bras-
path in the winter season and y = 0.05 isopath in sicae requires a W min of =6–8 h, T opt 18 °C, W
the spring season. max 22 h, T min 26 °C and T max
5.8 Disease Forecasting 115

35 °C. Operationally, infection models may use least 1 week ahead of the first appearance of the
hourly or daily weather inputs. In case of the for- disease on the crop. This will allow making deci-
mer, information also is required to estimate the sion for timely and effective fungicidal sprays
critical dry period interruption value, defined as (Chattopadhyay et al. 2005).
the duration of a dry period at relative humidities The stepwise multiple regression analysis
<95 % that will result in a 50 % reduction in dis- (MRA) was carried out to determine the meteoro-
ease compared with a continuous wetness period. logical parameters influencing variation in dis-
Pathogens are classified into three groups on their ease severity of Alternaria leaf blight (Alternaria
critical dry period interruption value. The infec- brassicae and A. brassicicola) of mustard.
tion model is being used to create risk maps of Disease severity estimates (Y) was considered as
exotic pests for the US Department of dependent variable, whereas other weather
Agriculture’s Animal and Plant Health Inspection parameters like temperature maximum (T max)
Service (Magarey et al. 2005). and temperature minimum (T min), RH maxi-
Experiments were laid out at eight locations, mum (RH max) and RH minimum (RH min),
viz. Bharatpur, Mohanpur, Berhampur, Pantnagar, total rainfall (RT), wind velocity evening (WV
Dholi, Kangra, Faizabad and New Delhi, with evening) and morning (WV morning), vapour
Brassica juncea cultivar ‘Varuna’ and an impor- pressure noon (VP noon) and morning (VP morn-
tant cultivar in the locality sown on ten dates at ing) and bright sunshine hours (BSH) were used
weekly intervals to determine epidemiology and as independent variables. The weather variables
forecasting of Alternaria blight of Brassica in were found to influence the disease severity dif-
India. First appearance of Alternaria blight dis- ferently at five different sowing dates in the two
ease (Alternaria brassicae) on leaves of mustard consecutive years. The Gompertz equation was
occurred between 42 and 139 days after sowing best linearized with the disease progress data fol-
(DAS); 44–72, 42–61, 69–83, 45–60, 67–84 DAS lowed by the logistic and the untransformed data
having higher frequencies in 1999–2000, 2000– sets. The linear prediction equations are (1)
2001, 2001–2002, 2002–2003 and 2003–2004, Y = 3.203−0.356(T min) + 0.015 (RH min) for
respectively, being highest in respective years at 20th October sowing; (2) Y = −1.929 + 1.634(WV
45, 46, 75, 45, 76 DAS. The disease first appeared morning) + 0.067(RT) for 5th November sowing;
on pods between 67 and 142 DAS, being highest (3) Y = −121.91 + 1.57(T min) + 1.083(RH
at 99 DAS. Severity of Alternaria blight disease max) + 0.29(RH min) - 2.27 (VP noon) - 0.61 (VP
on leaves was positively correlated to a maxi- morning) -17.17(WV evening) +17.83(WV
mum daily temperature of 18–27 °C, minimum morning) + 1.65(BSH) + 0.113(RT) for 20th
daily temperature of 8–12 °C, daily mean tem- November sowing; (4) Y = −5.131 + 0.25 (VP
perature >10 °C, morning relative humidity noon) +0.256 (BSH) + 0.057(RT) for 5th.
(RHmor) >92 %, >40 % afternoon RH and mean December sowing and (5) Y = −3.19 + 0.235(T min)
RH of >70 % in the preceding week. Disease for 20th December sowing. Gompertz transfor-
severity on pods was favoured by a maximum of mation is the best for linearizing and prediction
20–30 °C, daily mean temperature of >14 °C, of disease severity on all dates of sowing, and for
morning RH of >90 %, daily mean RH of >70 %, early sowing, T min, RH min and WV morning
>9 h of sunshine and >10 h of leaf wetness. influenced the disease progression (Mahapatra
Temperature and RH conditions favourable to and Das 2014) (Table 5.8).
disease development noted in the field matched Models were developed for forewarning crop
with the laboratory findings. Regional- and culti- age at first appearance of Alternaria blight (Y1),
var- specific models could predict the crop age at crop age at peak severity (Y2) and maximum
which Alternaria blight would first appear on severity of Alternaria blight (Y3) for different
leaves and pods, the highest blight severity on locations. The extent of weather influence on dis-
leaves and pods and the crop age at which blight ease development depends not only on the total
severity would be highest on leaves and pods at magnitude but also on the distribution of weather
116 5 Epidemiology and Forecasting

Table 5.8 Prediction equations of two different growth models and untransformed data and their comparable factors
at five different dates of sowing (Mahapatra and Das 2014)
Prediction equations
1st date of sowing (20th October) R2 Adj. R2 SE (est.) RSS
PDI(%) = 1.089−0.063 Temp (min)** 0.67 0.64 0.13 0.016

PDI (Logit) = 12.46−1.637 VP(noon)** + 0.047 RH (min)* 0.78 0.74 1.99 3.94
PDI (Gompit) = 3.203−0.356 Temp (min)** +0.015RH (min)* 0.84 0.82 0.42 0.18
2nd date of sowing (5th November)
PDI(%) = −1.118 + 0.41WV (morning)** +0.019RT* 0.61 0.54 0.58 0.3
PDI (Logit) = −5.319 + 4.248WV (morning)* 0.37 0.32 1.91 3.64
PDI (Gompit) = −1.929 + 1.634WV (morning)** + 0.067RT* 0.64 0.58 0.14 0.02
3rd date of sowing (20th November)
PDI(%) = −28.757 + 0.412 T(min) * + 0.250 RH(max)* + 0.075 0.95 0.83 0.009 0.009
RH(min)*-0.564 VP(noon)*-0.157VP (morning)**-3.754
WV(evening) + 3.963 WV (morning) +0.451 BSH**
+0.030TR**
PDI (Logit) = −315.514 + 0.361 T(max) +3.487 T(min) + 2.833 0.98 0.91 0.55 0.31
RH(max)** + 0.707 RH(min)**- 5.83 VP(noon)**- 1.48VP
(morning)*- 44.31WV (evening)* + 45.41WV (morning)*
+3.602BSH** + 0.288TR**
PDI (Gompit) = −121.91 + 1.57 T(min)** +1.083 0.97 0.89 0.28 0.078
RH(max)** + 0.29 RH(min)**- 2.27 VP(noon)**- 0.61VP
(morning)- 17.17WV (evening)* + 17.83WV
(morning)* + 1.65BSH** + 0.113TR*
4th date of sowing (5th December)
PDI(%) = −1.272 + 0.079 VP 0.83 0.78 0.11 0.012
(noon)** + 0.084BSH** + 0.018TR*
PDI (Logit) = −5.698 + 0.402VP (noon)** 0.50 0.46 0.94 0.88
PDI (Gompit) = −5.131 + 0.25VP (noon)** + 0.256BSH 0.88 0.78 0.13 0.11
** + 0.057TR*
5th date of sowing (29th December)
PDI(%) = −0.653 + 0.075 T (min)** 0.73 0.71 0.41 0.17
PDI (Logit) = −5.827 + 0.376 T (min)** 0.66 0.63 0.76 0.58
PDI (Gompit) = −3.19 + 0.235 T (min)** 0.73 0.71 0.13 0.017
PDI = percent disease index, R2 = coefficient of determination, Adj. R2 = adjusted coefficient of determination, SE
(est.) = standard error (estimate), RSS = residual sum of square

variables over small time intervals. But, use of ing weight according to their importance in dif-
data in small time intervals will increase the ferent time periods. Two approaches were tried
number of variables in the model, and in turn, a using weekly weather data. In the first approach,
large number of model parameters will have to be unfavourable/favourable/highly favourable
evaluated from the data. This will require a long ranges of weather variables were obtained based
series of data for precise estimation of the param- on experience of experts. This information was
eters, which may not be available in practice. utilized to decide the weights (subjective)
Thus, a technique based on relatively smaller depending on values of the weather variables in
number of model parameters, and inclusion of different weeks pertaining to these ranges.
entire weather distribution, was attempted. The However, the results of this approach were not
simplest way to solve the problem is to take found to be satisfactory. The other approach was
weighted accumulation of weather variables, giv- based on objective weights where correlation
5.8 Disease Forecasting 117

coefficients between variable to forecast and (Yt - Ft )

weather variables were taken as the indicators of MAPE = 1 / nå ´ 100
Yt
relative importance in respective weeks. In this
approach, for each weather variable, two indices where Yt is actual observation, Ft is the forecast
were developed, one as the simple total value of form models and n is the total number of tested
weather variables and the other as weighted total; data.
weights being correlation coefficients between Models along with coefficient of determina-
variable to forecast and weather variables in tion (R2) and mean absolute percentage error
respective weeks. The first index represents the (MAPE) are given in Table 5.7.2, which indicate
total amount of different weather variables that the models fitted well because the coefficient
received by the crop during the period under con- of determination is highly significant in most of
sideration, while the other one takes care of dis- the cases. MAPE are in the affordable range. The
tribution of weather variables with special MAPE is high for maximum severity which may
reference to its importance in different weeks in be due to sample fluctuations. Forecasts from the
relation to the variable to forecast. Similarly, for models were very close to the observed values in
joint effects of weather variables, weather indices subsequent years. Therefore, these models, based
were developed as weighted accumulations of on weekly weather data starting from week of
product of weather variables (taken two at a sowing up to 6 weeks of crop growth, can be used
time), weights being correlation coefficients for reliable forewarning of Alternaria blight. The
between variable to forecast and product of reliable forewarnings through this approach are
weather variables considered in respective weeks. possible well in advance. In general, the models
The form of model was (Agrawal et al. 1986; fitted well with all the coefficients of determina-
Agrawal and Mehta 2007): tion are highly significant. Forecasts from the
p p p 1 models were very close to the observed values in
Y = a0 + ååaij Zij + ååbiij Ziij + e subsequent years. Therefore, these models, based
i =1 j = 0 i =0 j =0 on weekly weather data starting from week of
Where sowing up to 6 weeks of crop growth, can be used
n2 for reliable forewarning of Alternaria blight.
Zij = å rjiw X iw
w = n1
Therefore, reliable forewarning for crop age at
first appearance of disease, crop age at peak
n2
Ziij = å rjiiw X iw X iw
w = n1
severity of disease and maximum severity of dis-
eases in two different mustard varieties for
Alternaria blight is possible well in advance
Y, variable to forecast; Xiw, value of ith (Kumar et al. 2013) (Table 5.9).
weather variable in wth week; riw, correlation Prediction models developed by Awasthi and
coefficient between Y and ith weather variable in Kolte (1994) showed multiple regression analysis
wth week; riiw correlation coefficient between the (MRA) equations between weather parameters as
product of Xi and Xi’ in wth week; p, number of independent factors and prediction of Alternaria
weather variables used; n1, initial week for which blight (AB) severity as dependant factors on the
weather data were included in the model; n2 final most commonly grown susceptible rapeseed
week for which weather data were included in the Yellow Sarson cultivar T-151 and mustard culti-
model; and e, error term. var ‘Varuna’ based on the data obtained over 7
Stepwise regression technique was used for years crop periods (1980–1987). Total rainfall
selecting important variables to be included in and minimum temperature show significant con-
the model. The forecasting performance of vari- tribution in MRA equations, whereas combined
ous models was judged by mean absolute per- effect of relative humidity, rainfall and minimum
centage error (MAPE). temperature accounted for more than 98 %
118 5 Epidemiology and Forecasting

Table 5.9 Models to forecast different characters of Alternaria blight in mustard crop along with coefficient of deter-
mination and MAPE in different varieties (Kumar et al. 2013)
Location Variety Character Model R2 MAPE
Bharatpur Rohini (on Y1 81.16−1.38 Z20 + 0.06 Z121 + 0.001 Z341 0.75 8.9
leaf) Y2 2.95 + 0.75 Z11 0.82 26.2
Y3 7.59 + 0.006 Z231 0.40 196.3
Rohini (on Y1 11.42 + 0.0012 Z340 + 0.026 Z231 0.29 24.7
pod) Y2 87.10−00.01 Z240 + 0.01 Z231 + 0.08 Z241 0.40 17.6
Y3 26.94 + 0.06 Z31 + 0.001 Z241 0.63 229.6
Varuna (on Y1 60.65 + 0.002 Z340 + 0.01 Z121 + 0.006 Z341 0.78 14.7
leaf) Y2 7.82 + 0.54 Z11 + 0.005 Z231 0.83 22.3
Y3 4.68 + 0.34 Z11 0.58 150.1
Varuna (on Y1 36.11 + 0.08 Z31 0.60 14.2
pod) Y2 −222.95 + 4.43 Z20-0.14 Z120 + 3.61Z11 + 2.38 Z31 0.63 5.4
Y3 68.41 -0.02 Z130 + 0.66 Z21 + 0.04 Z131 0.71 132.6
Dholi Pusa Bold Y1 28.79 – 0.01 Z240 + 0.04 Z121 + 0.002 Z131 0.96 10.4
(on leaf) Y2 24.29−0.001 Z140 + 0.01 Z131 0.72 7.0
Y3 87.90 + 17.56 Z20 + 31.64 Z21 + 2.32 Z31 0.52 48.5
Pusa Bold Y1 68.95 + 0.39 Z41 + 0.01 Z121 + 0.007 Z131 0.83 6.9
(on pod) Y2 81.10−0.003 Z240 + 0.01 Z121 + 0.005 Z141 0.87 3.3
Y3 144.65 + 1.87 Z31 + 0.33 Z41 + 0.01 Z131 0.77 91.8
Varuna (on Y1 72.89−2.11 Z21 + 3.35 Z41 + 0.07 Z121-0.02 Z341 0.93 8.4
leaf) Y2 −17.86−0.05 Z240 + 0.01 Z131-0.007 Z 231 + 0.10 Z241 0.84 9.3
Y3 −59.10 + 1.72 Z31 + 0.015 Z121 0.55 51.9
Varuna (on Y1 −206.31 + 0.61 Z30 + 0.02 Z121 + 0.005 Z341 0.79 3.9
pod) Y2 106.97−0.03 Z230 + 0.029 Z121 + 0.03 Z231 0.87 3.0
Y3 121.89 + 0.02 Z130 + 0.045 Z131−0.01 Z141 0.87 88.9
Behrampur Binoy (on Y1 57.38−0.025 Z141 + 0.03 Z241 0.54 12.4
leaf) Y2 92.84 + 5.17 Z21 + 3.94 Z31-0.11 Z121 0.61 9.7
Y3 −93.70 + 4.91 Z10 + 0.06 Z250 + 0.31 Z121 0.62 61.0
Binoy (on Y1 −99.41 + 0.021 Z131 0.62 12.6
pod) Y2 93.91−0.04 Z240 + 0.09 Z241-0.004 Z341 0.61 2.9
Y3 170.01 + 0.01 Z131 + 0.005 Z231 0.50 182.7
Varuna (on Y1 59.21 + 0.56 Z30 + 5.67 Z31 + 0.03 Z121 0.57 22.3
leaf) Y2 213 + 0.01 Z130-0.003 Z140 + 0.09 Z121 0.67 9.7
Y3 125.08 + 9.19 Z10 + 27.51 Z11 0.46 61.9
Varuna (on Y1 188.16 + 1.55 Z31 + 1.15 Z41 + 0.03 Z121 0.56 23.3
pod) Y2 148.25 + 1.66 Z31 + 0.02 Z121 0.60 7.4
Y3 −31.168−0.007 Z240 + 0.01 Z341 0.53 68.1

c­ontribution for prediction of AB severity on both Yellow Sarson and mustard and increase in
leaves of both Yellow Sarson and mustard. Total the age are positively correlated (r2 = 0.777 to
rainy days (TRDs), relative humidity, maximum 0.980) with increase in the susceptibility of the
temperature and minimum temperature show sig- crop, the maximum leaf disease severity being at
nificant contribution in MRA equations in AB rosette to flowering.
severity on pods of mustard, whereas only the The data on stepwise multiple regression anal-
TRD factor shows significant effect for spread of ysis equations for prediction of AB severity on
pod infection in Yellow Sarson. It is also found leaves (Y) and r2 value indicated that a combined
that the disease intensity beyond 30 days age on effect of relative humidity, rainfall and minimum
5.8 Disease Forecasting 119

temperature accounts for more than 98 % varia- Dang et al. (2006) developed prediction equa-
tion in AB severity on leaves of both Yellow tion for the development of Alternaria blight
Sarson and mustard. The equations are: using Gompertz model and two factors can be
AB leaf infection severity on Yellow Sarson explained by the following equation:

Y1 = -73.07 + ( 0.71x3 ) + ( 7.68 x2 ) + ( 0.67 x4 ) r 2

élog A Log ( - B *Time ) ùû
DI = Exp ë + C * Sowing day
= 0.99 + D * Factor 1

where Y1 = Alternaria blight leaf infection sever- where DI = disease intensity (A and B are the two
ity, X2 = mean minimum temperature, X3 = percent parameters of the Gompertz model and C and D
relative humidity, X4 = total rainfall. are the coefficient of the sowing day and factor
AB leaf infection severity on mustard 1). All the varieties are based on different genetic
make-ups, which reacted differentially to the nat-
Y1 = 6.98 + ( 9.09 x2 ) + ( 0.67 x4 ) + ( -0.79 x3 ) r 2 ural inoculum, and factor 1 may be interpreted as
= 0.98 weather index and factor 2 as contrast between
the heating factor and moisture factor. The best-­
where Y1 = Alternaria blight leaf infection sever- fitted models worked out for each variety were
ity, X2 = mean minimum temperature, X3 = percent as:
relative humidity, X4 = total rainfall

RH - 30 : Y = E ë
é Log ( 0.620 )*log ( 4.998*Time ) ùû
- 0.890 * sowing Day + 0.238 factor 1; (R 2
= 0.389 )

RH - 8113 : Y = E ë
é Log ( 2.547 )*log (1.072*Time ) ùû
- 0.561* sowing Day + 0.086 factor 1; (R 2
= 0.482 )

YSPb - 24 : Y = E ë
é Log ( 2.199 )*log ( 4.827*Time ) ùû
- 1.868 * sowing Day + 0.105 factor 1; (R 2
= 0.539 )

BSH - 1 : Y = E ë
é Log ( 3.008)*log (1.335*Time ) ùû
- 1.260 * sowing Day + 0.166 factor 1; (R 2
= 0.482 )

where Y = percent disease intensity Regression equations for the development of

The above equations revealed that for a given Alternaria blight on different varieties of rape-
cultivar, if the sowing time is known, and weather seed–mustard are:
parameters at particular time are known, disease
incidence can be predicted using the above mod- B. juncea (var. RH-30)
els. Mehta et al. (2008) developed a prediction
model for adopting better disease management Y1 = -4.3635 + 0.3488 X 1 (R 2
= 0.66 )
practices where four varieties each of B. juncea Y1 = 4.4927 + 0.2755 X 1 - 0.0789 X 3 (R 2
= 0.71)
(RH-30, RH-8113, RH-8695, RH-8546), B.
campestris (YSPb-24, BSH-1, Candle, Shiva), B. campestris (var. YSPb-24)
two of B. carinata (HC-2, HC-9001), one each of
B. napus (GSH-1) and B. alba (local) were moni- Y1 = -4.2310 + 0.3420 X 1 (R 2
= 0.68 )
tored for the development and progression of Y1 = 4.7131 + 0.2680 X 1 - 0.0797 X 3 (R 2
= 0.73)
Alternaria blight. Each cultivar was inoculated
artificially with A. brassicae spores when the B. carinata (var. HC-1)
crop was about 2-month-old. Studies conducted
revealed that temperature and RH had prominent Y1 = -4.8061 + 0.3584 X 1 (R 2
= 0.64 )
role in disease development in addition to varietal Y1 = 6.4244 + 0.2654 X 1 - 0.1001 X 3 (R 2
= 0.71)
behaviour. The prediction equations so devel-
oped were at par with the observed values. where X1 = T max; X3 = RH mor
120 5 Epidemiology and Forecasting

Sangwan et al. (2002) demonstrated that Factor B: 0.317 × T max + 0.317 × T min +
Gompertz model can be effectively used for pre- 0.933 × RH mor + 0.347 × RH eve − 0.618 ×
diction of Alternaria blight with two factors A Sunshine h.
and B drawn from the analysis of weather param-
eters and disease progress. These two factors (A and B) explained 60.3
and 24.5 % of total variation, respectively, and
Factor A: 0.091 × T max + 0.887 × T min + together explained 85 % of total variation among
0.036 × RH mor + 0.808 × RH eve − 0.644 × the weather variables. The model for each group
Sunshine h. is as follows:

Disease = Exp. ëélog ( a ) X - log ( b ) ´ Time ùû + cX Factors (1) + dX Factors ( 2 )

where a and b are constants of Gompertz The equation for each genotype of Brassica
model. was developed as follows:

B. juncea
Exp.[log ( 2.915 ) ´ log (-0.430 ´ Time] - 0.154 ´ Factors (1) + 0.204 ´ Factors ( 2 )
R 2 = 0.931 ( observed vs predicted )
B. campestris
Exp.[log ( 2.722 ) ´ log(0.470 ´ Time] - 0.203 ´ Factors (1) + 0.167 ´ Factors ( 2 )
R 2 = 0.974 ( observed vs predicted )
B. carinata
Exp.[log ( 2.717 ) ´ log(0.479 ´ Time] - 0.202 ´ Factors (1) + 0.168 ´ Factors ( 2 )
R 2 = 0.982 ( observed vs predicted )

B. napus
Exp.[log ( 2.908 ) ´ log(0.424 ´ Time] - 0.185 ´ Factors (1) + 0.170 ´ Factors ( 2 )
R 2 = 0.975 ( observed vs predicted )

B. alba
Exp.[log ( 4.312 ) ´ log (0.270 ´ Time] - 0.284 ´ Factors (1) + 0.212 ´ Factors ( 2 )
R 2 = 0.970 ( observed vs predicted )

B. oleracea
Exp.[log ( 2.823) ´ log (0.455 ´ Time] - 0.196 ´ Factors (1) + 0.192 ´ Factors ( 2 )
R 2 = 0.984 ( observed vs predicted )

Experiments were conducted at two locations for (YSPb-24). These were monitored for the pro-
development of prediction models for two popu- gression of Alternaria blight where disease pro-
lar varieties of rapeseed–mustard belonging to gression was faster on YSPb-24 compared to
two different Brassica genotypes, viz. B. juncea RH-30. Maximum lesion size, 6.31 mm, was
(RH-30) and B. campestris var. Yellow Sarson recorded on YSPb-24, whereas it was 4.21 mm
References 121

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tions for the progression of the disease were in disease progression in addition to the varietal
observed to be at T max of 20 °C with RH >90 %. factors. The prediction equations developed for
The stepwise regression analysis revealed that leaves and pods for a variety for two locations
the T max and RH mor played significant and were as follows (Mehta et al. 2008). Similar
positive roles in disease progression. The R2 equations were developed for other varieties as
value recorded was >0.9 in all the cases, which well.

A. On leaves

RH - 30 ( location - 1) Y = 1.984 + .196 X 1 + .951 X 5 (R 2

= 0.92 )
RH - 30 ( location - 2 ) Y = 2.156 + .235 X 1 - 5.49 + 10-2 X 4 (R 2
= 0.87 )

B. On pods
RH - 30 ( location - 1) Y = -0.471 + 5.802 + 10-2 X 1 (R 2
= 0.84 )
RH - 30 ( location - 2 ) Y = -0.317 + 1.543 + 10-2 X 4 (R 2
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where X1 = T max; X2 = T min; X4 = RHeve.; correlation coefficient (r) = 0.73 for T min and
X5 = wind speed. r = 0.51 of RH min favoured the disease
development.
Jha et al. (2013) recorded that T max posi- The regression equation developed for leaves
tively correlated with disease index. T max of (Y1) and siliqua (Y2) are:
23.2, RH max of 80 % and RH min of 66 % with

Leaves : Y 1 = -47.388 + 5.114 T min - 2.371Tmax + 1.492 RH min R 2 = 0.7376

Siliqua : Y 2 = 31.524 + 4.225 T min - 1.883 T max . R 2 = 0.69203.

Awasthi RP, Kolte SJ (1989) Effect of some epidemio-

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 Pathogenic Variability
6

6.1 Introduction heterokaryoses, uniform host selection, extensive

dispersal and/or a cryptic sexual stage.
The most dynamic and significant aspect in host–
pathogen interactions is that characteristics of
individuals within a species are not ‘fixed’ in 6.2 Historical Developments
their morphology, physiology, biochemistry and
pathogenicity. During reproduction, all individu- Initially, variations in cultural characteristics and
als are expected to be different from each other pathogenesis of different isolates of three
and from their parents in a number of characteris- Alternaria species infecting Brassicaceae hosts
tics, although they retain most similarities with were observed during 1952–1953 by Stoll (1952)
them and belong to the same species (Agrios in A. brassicicola (vegetables), by Van Schreven
2005). When individuals are produced asexually, (1953) in A. brassicae (Brassica) and by Atkinson
the frequency and degree of variability among (1953) in A. raphani (radish host pathosystem).
the progeny are reduced greatly, but even then, Therefore, it can be considered as the beginning
certain individuals among the progeny will show of research development on pathogenic variabil-
different characteristics. Three categories of such ity in Alternaria–crucifers host pathosystem.
populations are of direct interest to the In A. alternata strains showing differences in
Brassicalogists of the world: their physiological and pathological characteris-
tics have been isolated from Crambe (C. abyssi-
1. Populations that differ in their ability to attack nica). Strain A occurs on leaves, stem and
particular varieties of Brassica hosts siliquae, whereas strains B and C are mainly
2. Populations differing in their physiological found on siliquae and leaves, respectively. In
adaptations to specific environmental pathogenic ability, strain B has been reported to
conditions be most virulent, strain A as moderately virulent
3. Populations differing in their ability to toler- and strain C as least virulent (Czyzewska 1969,
ate the effect of toxicants 1971). These strains have different temperature
optima for sporulation. Strain A sporulates abun-
In Alternaria–Brassica host pathosystem, the dantly at 17–35 °C, whereas B requires 20–30 °C
following categories of variability exist: although and strain C sporulates best at 12 °C (Czyzewska
the genus Alternaria is known as an imperfect 1970).
fungus, it shows genetic variability within a spe- Alternaria brassicae is generally most viru-
cies, and this variability might be due to the exis- lent on all brassicaceous hosts. Preliminary
tence of mutation, somatic hybridization, reports on variability in this species were made
© Springer Science+Business Media Singapore 2016 125
G.S. Saharan et al., Alternaria Diseases of Crucifers: Biology, Ecology and Disease Management,
DOI 10.1007/978-981-10-0021-8_6
126 6 Pathogenic Variability

from Holland (Van Schreven 1953) and the UK frequent (7.48 %) than the moderately aggressive
(Mridha 1983). Isolates of A. brassicae from isolates (56.86 %). Cultural and morphological
rapeseed (colza) showed differences in cultural variations in the isolates of this species show no
growth on cherry agar and differed in their patho- distinction in pathogenic behaviour (Campbell
genesis on seedlings. Similarly, Kolte et al. 1970; Campbell et al. 1968; Changsri and Weber
(1989, 1991) and Awasthi and Kolte (1989) dis- 1963). Spontaneous occurrence of albino mutants
tinguished three A. brassicae isolates, viz. A, C of this species has been observed (Campbell
and D, on the basis of their morphology, sporula- 1970; Campbell et al. 1968).
tion, growth and cultural characteristics. On B. Alternaria raphani is the major pathogen of
carinata, these isolates produce distinct types of radish but also occurs on other brassicaceous
lesions. Among the three isolates, isolate C is the hosts. Atkinson (1953) obtained 312 isolates of
most sporulating and isolate A the least. Unlike this species from different geographical areas in
isolates B and C, isolate A produces chlamydo- Canada, classified them as ‘wild type’ and ‘vari-
spores. In a serological study, Kolte et al. (1991) ant type’ and found the former as being less viru-
indicated that the Pantnagar isolates A, C and D lent than the latter. No differences were observed
resembled the Bihar isolates BHl and BH2 and in their nutritional requirements for growth. In a
the Kanpur isolate K, respectively. None of these later study, Changsri and Weber (1963) also did
workers, however, used different Brassica host not find any variations in the A. raphani isolates
differentials to distinguish A. brassicae isolates from B. nigra, B. napus and B. rapa from differ-
on the basis of their virulence. Saharan and ent geographical areas of Canada.
Kadian (1983) used eight commonly cultivated The last decade of twentieth century and
Brassica species to distinguish isolates of A. twenty-first century can be considered as boom
brassicae in India. In a cross-infection study and period for Alternaria–crucifers pathogenic vari-
differential interactions on different hosts, they ability research. During the first two decades of
distinguished three clearly separable isolates and present century, pathogenic variability in
designated them as RM1, RM2 and V3 races, Alternaria–crucifers system has been determined
which were found to be virulent on rapeseed– on the aspect including pathological, symptom-
mustard group of crops. Race RM1 from rape- atological, morphological, cultural, biochemical,
seed–mustard was avirulent on B. oleracea var. nutritional, thermal and fungicidal sensitivity,
capitata. Race RM2 from B. rapa (B. campestris) proteomic analysis, genetical and molecular
var. Brown and Yellow Sarson and Eruca sativa (Saharan et al. 2015). However, no standard
was avirulent on both B. oleracea var. capitata internationally acceptable parameter for the
and B. oleracea var. botrytis. Race V3 was viru- selection of host differentials (single gene lines,
lent on all eight host species tested including rad-
ish, cabbage and cauliflower (Table 6.1). This
study clearly indicated the existence of distinct Table 6.1 Physiological races of Alternaria brassicae
pathotypes in A. brassicae infecting different (Saharan and Kadian 1983)
Brassica species. According to Mridha (1983), Races/pathotypes
13 UK isolates of A. brassicae tested on selected Host differential RM1 RM2 V3
cultivars of winter oilseed rape differed in their Brassica juncea Sa S S
virulence. B. rapa var. Sarson S S S
Alternaria brassicicola is generally more B. rapa var. dichotoma S S S
common on vegetable crops than on oil-yielding B. rapa var. toria S S S
brassicas. Stoll (1952) characterized three iso- Eruca sativa S S S
lates of this species from siliquae of cauliflower Raphanus sativus S S S
seed crop, showing highly aggressive, less Brassica oleracea var. capitata Ra R S
aggressive and non-pathogenic behaviour. Brassica oleracea var. botrytis S R S
a
However, highly aggressive isolates were less R resistant, S susceptible
6.3 Pathological Variations 127

isogenic lines) and nomenclature of pathotypes GDP, which infected 15 host differentials, but
has been maintained. Each researcher has used isolates could not be differentiated into
different sets of host differential and system of pathotypes.
pathotype nomenclature. Gupta et al. (2004), Using 11 B. juncea genotypes as host differen-
however, attempted to use B. juncea varieties in tials, Gupta et al. (2004) identified four distinct
their set of host differentials and gave nomencla- A. brassicae pathotypes, viz. Bj-4 (BWL), Bj-5
ture to pathotypes as Bj-4, Bj-5, Bj-6 and Bj-7. (HSR), Bj-6 (RTK) and Bj-7 (REW). Pathotypes
They have also tried to maintain parity in the Bj-4 was most virulent infecting all 11 host dif-
order of the discovery of pathotypes of A. bras- ferentials, and Bj-5 was least virulent infecting
sicae by Saharan and Kadian (1983) who reported only six host differentials (Table 6.2). Incubation
three pathotypes, viz. RM-1, RM-2 and V-3. and latent periods also exhibited greater variabil-
ity for host genotype x isolate interactions of A.
brassicae. Minimum incubation period of three
6.3 Pathological Variations days was required for pathotypes Bj-4 (BWL)
and Bj-6 (RTK) on cultivars Varuna and RH-30
Out of four species of Alternaria known to occur (Table 6.3). Vishwanath and Kolte (1997) also
on crucifers, Alternaria brassicae (Berk.) Sacc. recorded differential interactions between
is more severe and variable (Verma and Saharan Brassica species and A. brassicae isolates A and
1994). Preliminary reports on variability in C and avirulent isolate D. Alternaria brassicae
Alternaria species from rapeseed (colza) by Van isolate A showed significantly higher disease
Schreven (1953) in Holland and by Mridha scores than isolate C on B. napus genotype
(1983) in the UK showed differences in cultural PPNS1, B. juncea cv. PR15, B. campestris var.
growth on cherry agar and in their pathogenesis toria cvs. PT 303 and PT 30, B. campestris var.
on seedlings. Although pathogenic variability in Yellow Sarson cv. T-151; isolate C showed sig-
A. brassicae has been observed by various work- nificantly higher disease scores on B. campestris
ers (Verma and Saharan 1994), information on var. Yellow Sarson cv. PYST-6, B. campestris
the existence of distinct pathotypes using stan- ssp. rapifera cv. Turnip Red and B. alba in com-
dard host differentials is rather limited (Saharan parison to isolate A. Alternaria brassicae isolate
1992a, b; Mehta et al. 2005a). According to A is a highly virulent pathotype and isolate C is a
Mridha (1983), 13 isolates of A. brassicae tested moderately virulent pathotype. The toxigenicity
on selected cultivars of winter rape differed in studies of three isolates on leaves of various hosts
their virulence. Similarly, Kolte et al. (1989, showed isolate A causing more severe symptoms
1991) and Awasthi and Kolte (1989) also reported than the isolates C and D at both 1:10 and 1:100
variability in A. brassicae. dilutions. Toxin from isolate D produced maxi-
None of these workers used different Brassica mum symptom severity score on E. sativa, but
species to distinguish A. brassicae isolates on the failed to produce symptoms on leaves of other
basis of their reaction on host differentials. Using host cultivars. Isolate A toxin supports signifi-
eight commonly cultivated Brassica species as cantly less seed germination and minimum plu-
differentials, Saharan and Kadian (1983) distin- mule and radical lengths as compared to isolates
guished three A. brassicae isolates and desig- C and D at 1:10 and 1:100 dilutions. Some differ-
nated them as RM-1, RM-2 and V-3 races, which ences among different genotypes, however, were
were found to be virulent on rapeseed and mus- observed with respect to seed germination and
tard group of Brassicas (Table 6.1). Mehta et al. seedling growth with respect to toxins produced
(2003) collected ten isolates from different agro- by the three isolates.
climatic zones of India and cross-inoculated them Fifteen A. brassicae isolates from rapeseed
on a set of 17 host differentials. Among the ten and mustard collected from different locations in
isolates, isolate DLK was the most virulent Haryana (India) showed pathogenic diversity on
infecting 16 differentials followed by RSR-1 and 17 host differentials under greenhouse conditions
128 6 Pathogenic Variability

Table 6.2 Reaction of different isolates of A. brassicae on B. juncea host differentials (Gupta et al. 2004)
Reaction of isolate collected from various locations
Host differentials Bawal (BWL) Hisar (HSR) Rohtak (RTK) Rewari (REW) Number of VI/HD
EC-129126-1 + − − − 1
EC-322090 + − + + 3
EC-322092 + − + + 3
EC-322093 + − + + 3
Varuna + + + + 4
EC-287711 + − + − 2
ZEM-1 + + + + 4
RC-781 + + + + 4
RH-30 + + + + 4
RH-8113 + + + + 4
Rajat + + + + 4
Infectivity size 11 6 10 9 −
VI/HD = virulent isolates per host differential
+ = denotes compatible interaction; − = denotes incompatible interactions

Table 6.3 Incubation and latent period (in days) of botrytis, B. rapa and B. alba differentiated 15
Alternaria brassicae isolates on host differentials under isolates into eight pathotypes/races. The eight
controlled conditions (Gupta et al. 2004) pathotypes identified, CHR-I, CHR-II, JND-II,
Bawal Hisar Rohtak Rewari JHR and SRS were grouped in the first group;
Host (BWL) (HSR) (RTK) (REW) CHR-III, JND-I, and KTL-I in the second group,
differentials IP LP IP LP IP LP IP LP and BHI and KTL-II in third group. However,
EC-129126-1 4 7 – – – – – – isolates BWL, HSR, REW, RTK and SPT formed
EC-322090 4 7 – – 5 11 7 13 as individual group of pathotypes (Kumar et al.
EC-322092 4 7 – – 5 11 7 13 2003a, b) (Table 6.4).
EC-322093 3 7 – – 4 7 7 13 On differentials, each isolate collected from
Varuna 3 6 4 6 3 9 5 11 different locations of India behaves differentially
EC-287711 4 7 – – 5 11 – – (Table 6.5). Isolate DLK is most virulent as it
ZEM-1 5 7 4 7 4 7 6 12 infects 16 host differentials; RSR-I and GDP on
RC-781 4 7 5 9 5 11 6 12 15 host differentials; HSR-I, HSR-III and RSR-II
RH-30 3 6 4 6 3 9 5 11 on 14 host differentials; and isolates HSR-II,
RH-8113 3 7 4 7 4 9 4 9 GNR, LDH and KNR on only 13 differentials.
Rajat 3 7 4 6 4 9 5 11 The comparative study reveals that all the differ-
IP = incubation period; LP = latent period; – = no infection entials are susceptible to different isolates of A.
brassicae. The genotype B. alba is infected by
only four isolates, whereas genotype B. oleracea
(Kumar et al. 2003a, b). Isolates CHR-I, CHR-II, var. botrytis is infected by five isolates. The range
JND-II, JHR and SRS were most virulent infect- of incubation period varies from 3 to 13 days in
ing all 17 host differentials, followed by REW, isolates, but in general, majority took 3–5 days to
RTK and SPT causing infection on 16 host dif- cause the infection (Mehta et al. 2003).
ferentials. Isolates BWL, BHI and KTL-II Sangwan and Mehta (2007) analysed viru-
infected all differentials except E. sativa, and B. lence pattern of 24 A. brassicae isolates on a set
alba, whereas isolate HSR is least virulent pro- of 17 host differentials consisting of Brassica
ducing symptoms only on 13 host differentials. species. The isolates BTD, BBK, DSA, GNR,
Out of 17 host differentials, six differentials of B. HSR and PNT had wide virulence pattern infect-
juncea, B. carinata, B. nigra, B. oleracea var. ing all the 17 host differentials. Isolates BHP,
6.3 Pathological Variations 129

Table 6.4 Differential reactions of different isolates of A. brassicae when inoculated on a set of host differentials
(Kumar et al. 2003a, b)
Alternaria brassicae isolates
Differentials BWL BHI CHR-III HSR JND-I KTL-I KTL-II REW RTK SPT
Brassica + + + + + + + + + +
campestris var.
toria
B. campestris + + + + + + + + + +
var. Yellow
Sarson
B. campestris + + + + + + + + + +
var. Brown
Sarson
B. juncea + − − − − − − + + −
B. napus + + + + + + + + + +
B. carinata + + − − + + + + + +
B. tournefortii + + + + + + + + + +
B. nigra + + + − + + + + + +
B. chinensis + + + + + + + + + +
B. pekinensis + + + + + + + + + +
Raphanus + + + + + + + + + +
sativus
B. oleracea var. + + + + + + + + + +
capitata
B. oleracea var. − + + + + + + − + +
botrytis
B. rapa + + − − − − + + + +
B. caulorapa + + + + + + + + + +
Eruca sativa + + + + + + + + + +
B. alba − − − − − − − + − +

BRT, GDP, HSRP, JPR, NGN, B. alba and Pathogenic variability of purified 98 isolates
Midas-1 are able to infect 16 host differentials. collected from Brassica species was analysed to
Virulence of isolates B. chin and VRN was con- identify virulent pathotype for screening and
fined to 13 host differentials. The host differential breed resistant genotypes of oilseed Brassica
B. alba var. Local was the least susceptible as (Singh et al. 2013). Eight variants were grouped
only 12 isolates are pathogenic on it. In terms of on the basis of resistant and susceptible reac-
incubation period, majority of the isolates tions, incubation and latent period, lesion size
required 3–5 days to initiate the disease, whereas and Alternaria blight severity. Isolates from
isolates ASM, BHP, FRD, JPR and RSR needed Rewari and Fatehabad districts were able to
longer period to produce symptoms on crucifer- infect all host differentials, followed by Bhiwani
ous vegetables. Eight host differentials differenti- district isolates on ten differentials and Rohtak
ated all the 24 isolates into 14 pathotypes/races isolate with only seven differentials. The Rewari
(Fig. 6.1, Tables 6.5, 6.6 and 6.7). district isolate had the shortest incubation (IP)
Meena et al. (2012) measured aggressiveness and latent period (LP) of 4–5 and 6–7 days,
of A. brassicae isolates on host differentials in respectively, compared to 6–8 and 8–10 days,
the form of lesion size. The host differentials B. respectively, of Rohtak district isolates.
alba, B. juncea (PAB, EC-399299), E. sativa, B. Maximum Alternaria blight severity (24.6 %)
carinata and B. napus expressed minimum lesion and maximum lesion size were also produced by
size. the Rewari district isolates (group 1) compared
130 6 Pathogenic Variability

18
16
Number of host infected

14
12
10
8
6
4
2
0
ASM
BHP
BRT
BTD
BBK

DLH
FRD
GNR
GDP
HSR
HSRP
JPR
JBL
KGR
LCK
NGN
PNT
RSR

BCHIN
RC-
MIDAS
VRN
DSA

BALBA
Name of the isolates

Fig. 6.1 Pathogenic reaction of different isolates of Alternaria brassicae on a set of host differential (Sangwan and
Mehta 2007).

Table 6.5 Reaction of different isolates of Alternaria brassicae on Brassica differentials (Mehta et al. 2003)
Reaction of Alternaria brassicae isolates
Differentials HSR-I HSR-II HSR-III GNR LDH KNR RSR-I RSR-II DLK GDP
B. campestris +(5) − +(5) +(11) +(9) +(9) +(9) +(5) +(11) +(5)
var. toria
B. campestris +(5) +(11) +(5) +(11) +(5) +(5) +(5) +(5) +(5) +(5)
var. Yellow
Sarson
B. campestris +(5) +(5) − +(5) +(5) − +(5) +(5) +(5) +(3)
var. Brown
Sarson
B. juncea +(5) +(5) +(5) +(5) +(5) − +(5) +(5) +(5) +(5)
B. carinata +(13) +(5) +(5) +(5) +(11) +(3) +(9) − +(5) +(5)
B. nigra +(5) +(9) +(8) +(11) +(11) +(9) +(9) +(9) +(5) +(8)
B. napus − +(5) +(11) − +(11) +(9) +(11) +(8) +(6) +(5)
B. chinensis − +(3) +(5) +(3) +(5) +(11) +(3) +(3) +(5) +(5)
B. pekinensis +(11) +(3) +(5) +(3) +(3) − +(11) +(5) +(5) +(5)
B. tournefortii +(5) +(3) +(3) +(3) +(5) +(3) − +(3) +(3) +(3)
B. alba +(5) − +(5) − − +(11) − +(5) − −
Eruca sativa +(5) +(9) +(8) +(3) − +(9) +(9) +(5) +(5) +(8)
B. oleracea var. +(9) +(11) +(3) − +(9) +(9) +(9) +(5) +(5) +(3)
capitata
B. oleracea var. − − +(5) − − − +(11) +(5) +(5) +(5)
botrytis
B. rapa +(9) +(9) − +(5) +(9) +(5) +(9) +(8) +(5) −
B. caulorapa +(5) +(5) − +(5) +(9) +(3) +(9) − +(5) +(3)
Raphanus +(5) − +(6) +(9) − +(9) +(11) − +(3) +(5)
sativus
B Brassica, HSR-I Hisar-I, HSR-II Hisar II, HSR-III Hisar III, GNR Sri Ganganagar, LDH Ludhiana, KNR Kanpur, RSR-
I R.S. Pura, Jammu, DLK Dhaula Kuan, GDP Gurdaspur, () incubation period, + infection, − no infection
 6.3
Pathological Variations

Table 6.6 Pathogenic reaction of various isolates of A. brassicae from India on selected host differentials (Sangwan and Mehta 2007)
Alternaria brassicae isolates
RC-
Differentials ASM BHP BRT DLH FRD GDP HSRP PR JBL KGR LCK NGN RSR B alba B chin 781 Midas-1 VRN
Brassica + + + + + + + + + + + + + + + + + +
juncea
B. napus + + − + + + + + + + + + + + + + + +
B. chinensis − + + + + + + + + + + + + − + + + +
B. alba + + + − + − − + − − − − − + − − − −
B. oleracea + − + + − + + + + + + + + + + + + +
var. capitata
Raphanus − + + + − + + − + + + + + + − + + −
sativus
Eruca sativa + + + + + + − + + − − + + + + − + +
B. rapa + + + − + + + + + + + + + + − + + +
B. caulorapa + + + − + + + + + + − − + + − + + +
131
Table 6.7 Pathogenic behaviour of different isolates of Alternaria brassicae from India on a set of host differentials (Sangwan and Mehta 2007)
Isolates of Alternaria brassicae
Differentials ASM BHP BRT BTD BBK DSA DLH FRD GNR GDP HSR HSRP JPR JBL KGR LCK NGN PNT RSR B alba B chin RC-781 Midas-1 VRN
B. campestris + (3)b + (3) + (4) + (7) + (3) + (3) + (4) + (3) + (3) + (5) + (3) + (5) + (6) + (3) + (4) + (5) + (3) + (3) + (9) + (3) + (3) + (4) + (5) + (3)
var. toria
B. campestris + (4) + (3) + (3) + (6) + (3) + (3) + (4) + (3) + (3) + (5) + (5) + (5) + (3) + (3) + (4) + (8) + (4) + (4) + (5) + (3) + (3) + (4) + (5) + (3)
var. Yellow
Sarson
B. campestris + (4) + (3) + (3) + (7) + (4) + (4) + (4) + (3) + (3) + (3) + (3) + (5) + (4) + (3) + (4) + (5) + (4) + (4) + (5) + (6) + (6) + (4) + (5) + (5)
var. Brown
Sarson
B. juncea + (4) + (4) + (5) + (6) + (4) + (4) + (4) + (6) + (6) + (5) + (5) + (5) + (6) + (4) + (4) + (7) + (3) + (4) + (5) + (4) + (3) + (5) + (4) + (5)
B. napus + (4) + (4) − (0) + (6) + (4) + (7) + (6) + (4) + (4) + (5) + (3) + (5) + (4) + (4) + (9) + (8) + (5) + (4) + (11) + (4) + (3) + (3) + (6) + (3)
B. carinata + (7) + (7) + (3) + (6) + (6) + (7) + (7) − (0) + (4) + (5) + (4) + (5) + (7) − (0) + (4) + (8) + (6) + (5) + (9) + (4) + (6) + (5) + (8) − (0)
B. tournefortii + (4) + (4) + (4) + (7) + (4) + (4) + (4) + (3) + (4) + (3) + (4) + (5) + (4) + (4) + (4) + (6 + (5) + (5) − (0) + (4) + (5) + (3) + (7) + (5)
B. nigra + (4) + (4) + (3) + (3) + (3) + (4) + (6) + (4) + (4) + (8) + (3) + (8) + (7) + (4) + (4) + (5) + (5) + (5) + (9) + (4) + (5) + (5) + (7) + (3)
B. chinensis − (0) + (7) + (6) + (3) + (6) + (7) + (5) + (7) + (4) + (5) + (5) + (6) + (7) + (7) + (5) + (5) + (5) + (6) + (3) − (0) + (6) + (5) + (5) + (3)
B. pekinensis + (4) + (4) + (7) + (8) + (6) + (4) + (5) + (4) + (7) + (5) + (5) + (6) + (7) + (4) + (7) + (5) + (3) + (5) + (11) + (4) + (5) + (3) + (7) + (5)
B. alba + (4) + (7) + (7) + (6) + (7) + (7) − (0) + (7) + (7) − (0) + (6) − (0) + (4) − (0) − (0) − (0) − (0) + (3) − (0) + (3) − (0) − (0) − (0) − (0)
B. oleracea var. + (4) − (0) + (3) + (4) + (3) + (4) + (6) − (0) + (4) + (3) + (3) + (5) + (9) + (4) + (4) + (8) + (5) + (5) + (9) + (4) + (6) + (6) + (5) + (8)
capitata
B. oleracea var. + (4) + (9) + (4) + (5) + + (4) + (6) + (9) + (4) + (5) + (6) + (8) + (7) + (9) + (4) + (7) + (5) + (5) + (11) + (4) + (6) + (6) + (5) + (8)
botrytis (10)
B. rapa + (9) + (9) + (6) + (7) + (7) + (4) − (0) + (4) + (4) + (5) + (3) + (6) + (7) + (9) + (4) + (5) + (5) + (5) + (9) + (7) − (0) + (4) + (5) + (9)
B. caulorapa + (9) + (9) + (7) + (7) + (3) + (6) − (0) + (9) + (4) + (3) + (5) + (9) + (9) + (9) + (6) − (0) − (0) + (5) + (9) + (4) − (0) + (4) + (7) + (6)
Eruca sativa + (4) + (9) + (7) + (6) + (7) + (6) + (6) + (7) + (7) + (8) + (5) − (0) + (9) + (4) − (0) − (0) + (5) + (6) + (9) + (7) + (6) − (0) + (7) + (5)
Raphanus − (0) + (7) + (3) + (6) + (6) + (7) + (5) − (0) + (4) + (5) + (3) + (9) − (0) + (5) + (4) + (9) + (5) + (3) + (11) + (7) − (0) + (5) + (5) − (0)
sativus
a
B. = Brassica
b
Figure in parentheses indicates the incubation period
6.5 Morphological and Cultural Variations 133

to Rohtak district isolates (group 6). Brassica Saharan and Kadian (1983). Kolte et al. (1991)
juncea var. Varuna contracted the highest disease identified three pathotypes, A, C and D, on the
severity (24.6 %) and B. alba the least (2.9 %). basis of virulence and some spot characters, viz.
Rewari district isolates can, therefore, be used spot colour, periphery colour, presence or absence
for screening oilseed Brassica germplasm. of concentric rings and central region of the spot.
Alternaria blight tolerance of B. alba genotypes Goyal et al. (2013) reported pathogenic variabil-
can be harnessed as donor parent for breeding ity among A. brassicae isolates on host geno-
resistance/tolerant variety (Singh et al. 2013). types, on the basis of many qualitative characters,
Seven pathotypes (Abr 1 to Abr 7) of A. brassi- including spot colour and periphery colour, cen-
cae infecting rapeseed–mustard were identified tral point and its colour, presence or absence of
on a set of six Brassica host differentials from concentric rings and yellow halo region, and one
Himachal Pradesh, India. Pathotype Abr 4 was quantitative character, viz. per cent disease sever-
most predominant and virulent infecting all the ity. Three characters, i.e. central point colour,
six test host differentials (Kumar et al. 2014). presence or absence of central point and yellow
halo region of the spot, should be used to study
variability among A. brassicae isolates.
6.4 Symptomatological Pathogenic variability test revealed that all the
Variations isolates from rapeseed–mustard were pathogenic
or aggressive at different rates on all 12 host dif-
The symptom variability exhibited by A. brassi- ferentials and produced different types of spots
cae isolates on leaves of different B. juncea host on different hosts (Figs. 6.2 and 6.3).
differentials is generally in the form of medium-
sized, circular, greyish brown spots, 6–8 mm in
diameter, with three regular concentric raised 6.5 Morphological and Cultural
rings of dark brown colour; no yellow halo Variations
(Plate 6.1, Fig. 2) is produced (BWL). Another
type of symptoms produced by other isolates is in Mehta et al. (2003) identified (Table 6.8) various
the form of large, circular lesions (8–10 mm), isolates from different locations on the basis of
light green in the centre with concentric rings the size of A. brassicae spores by designating
containing dark yellow halo (Plate 6.1, Fig. 1) them with the place of collection:
around the spot (RTK). Third type of isolates
produced large, irregular spots of 10 mm in diam- 1. Hisar: HSR-I, B. juncea (var. RH-30); HSR-II,
eter, dark brown in centre and greyish around the B. campestris var. Yellow Sarson (var. YSPb-
margins with concentric rings (Plate 6.1, Fig. 3) 24); and HSR-III, B. tournefortii (var. Local)
without yellow halo (HSR). In the fourth type, no 2. Sri Ganganagar: GNR, B. juncea (var. Kranti)
yellow halo is produced, having medium-sized 3. Ludhiana: LDH, B. juncea (var. RH-30)
spots, light green in colour with only one concen- 4. Kanpur: KNR, B. juncea (var. RH-30)
tric ring and very light pale ring (Plate 6.1, Fig. 4) 5. Dhaula Kuan: DLK, B. juncea (var. RH-30)
around the spots (REW). The symptoms pro- 6. Gurdaspur: GDP, B. juncea (var. RH-30)
duced by four different isolates are stable and 7. R.S. Pura: RSR-I, B. juncea (var. RH-30), and
distinct on B. juncea host differentials; each dis- RSR-II, B. juncea (var. RH-30)
tinct pathotype produces its own characteristic
symptoms. This shows that symptom variability The morphological characteristics of each isolate
is a function of specific pathotype rather than including size (length and breadth), number of
host differentials’ genetic variations (Gupta et al. septa, beak length, beak septa, etc., were recorded
2004). These pathotypes are designated as Bj-4 from 15-day-old culture.
(BWL), Bj-5 (HSR), Bj-6 (RTK) and Bj-7 (REW) Based on spore length, isolates were catego-
in the order of their discovery as suggested by rized into four groups, i.e. small (<100 μm),
134 6 Pathogenic Variability

Plate 6.1 Symptomatological variations of four isolates of Alternaria brassicae on Brassica juncea. Fig. 1, RTK;
Fig. 2, BWL; Fig. 3, HSR; and Fig. 4, REW (Gupta et al. 2004)

medium (101–150 μm), long (151–200 μm) and GDP (Table 6.8). The maximum beak length was
very long (>200 μm). Group 1 includes GDP; observed in the case of HSR-II and minimum in
group 2 includes HSR-I, HSR-III, GNR, KNR the case of GNR (Mehta et al. 2003). The number
and RSR-I; group 3 contains LDH and DLK; and of septa in beak varied from zero to six (Plate 6.2).
group 4 includes HSR-II and RSR-II isolates. Conidial size variations in the A. brassicae
The longest spore length was observed in the isolates are due to nutrition rather than a charac-
case of HSR-II and RSR-II (>200 μm), and the teristic pathological variation (Saharan and
shortest in the case of GDP (94.45 μm). The Kadian 1983). However, glaring differences in
breadth ranged from 13.5 to 36.0 μm with the conidial size are noticed among the isolates even
maximum spore breadth in GNR and minimum when the same medium is used for the growth of
in the case of HSR-II. The number of horizontal the isolates. It can be assumed that variation in
septa varied from 5 to 13, with maximum being the isolates may be inherent since isolates were
in the case of HSR-II and minimum in the case of collected from diverse agroclimatic zones. It is
6.5 Morphological and Cultural Variations 135

SS-01 Conidial/spore measurement recorded on

SS-02 each isolate from Haryana (India) revealed that
SS-13 isolates differed in their conidial size. The aver-
SS-08 age conidial length varied from 118.62 to
SS-04 194.52 μm, being the maximum of isolate RTK
SS-10 and the minimum of isolate HSR. The range of
SS-07 conidial size varied from 81 to 300 μm. The aver-
SS-11 age breadth of conidia varied from 14 to 23 μm,
SS-03 the thickest being of isolate JND-I and the thin-
SS-06 nest of isolate CHR-III. The horizontal septations
SS-12 varied from 3 to 12 and vertical from 0 to 6
SS-09
(Table 6.9). Some variations in beak size were
also recorded. The average beak length varied
SS-05
from 39.99 to 119.07 μm. The longest beak was
1.00 0.81 0.63 0.44 0.25 of isolate RTK and the smallest of JND-I. The
average beak septations varied from 1.9 to 5.8
Fig. 6.2 Dendrogram showing pathogenic variability (Table 6.9). These observations revealed that
among 13 A. brassicae isolates in respect of five qualita-
variation in the conidial size existed in Haryana,
tive characters, i.e. spot colour, periphery colour, presence
or absence of concentric rings, central point and yellow India (Plate 6.3) (Kumar et al. 2003b).
halo region, and one quantitative character, i.e. per cent Variations in morphology and cultural charac-
disease severity (Goyal et al. 2013) teristics among 13 different geographical A.
brassicae isolates in India were analysed by
SS-01 Goyal et al. (2011). All the isolates showed high
SS-03 level of variability in vitro in respect to conidial
SS-04 length, width, beak length and number of septa.
Conidia of Nazirhat isolate (SS 04) are smallest
SS-07
with lowest number of septa. Substantial varia-
SS-08 tions among the isolates were found in mycelial
SS-02 growth, and sporulation in different nutrient
SS-11 media, and artificial environmental conditions
SS-10
including temperature, relative humidity, light
and hydrogen ion concentration. Different opti-
SS-13
mum temperature ranges were found for myce-
SS-05 lial growth (25–30 °C) and sporulation
SS-06 (15–35 °C). All 13 isolates grew bested at 100 %
SS-09 relative humidity. However, they sporulate the
SS-12
most at different relative humidity (40–100 %).
This reflects the adaptation of the respective iso-
1.00 0.81 0.63 0.44 0.25 lates to the ambient conditions in the different
Fig. 6.3 Dendrogram showing molecular variability
cropping areas, which also may have induced the
among 13 A. brassicae isolates based on RAPD finger- cultural variability. All the isolates did not grow
prints obtained from 100 RAPD primers (Goyal et al. and sporulate abundantly on the same nutrient
2013) medium. Asthana and Hawker’s media were gen-
erally better for all the isolates. Variation in opti-
evident from the data that each isolate differed in mum pH and light conditions for mycelial growth
their conidial size. Hence, these variations in the and sporulation was also observed. Cluster anal-
conidial size indicate the existence of variability ysis of data on cultural variability among 13 A.
in this pathogen in India (Mehta et al. 2003). brassicae isolates found a close relationship
136 6 Pathogenic Variability

Table 6.8 Differences in morphological characters of different isolates of Alternaria brassicae from India (Mehta
et al. 2003)
Beak Length
Length (μm) Breadth (μm) Septation (μm) Beak
Sr. Horizontal Vertical
No Isolates Av. Range Av. Range Av. Range Av. Range Av. Range Av. Range
1 ASM 168.2 109.1– 26.4 18.2– 8.7 6–11 3.5 2–6 54.5 18.2– 3.0 2–4
227.3 36.4 90.9
2 BHP 126.4 100.0– 20.5 18.2– 9.5 5–13 1.4 0–4 19.1 9.1– 1.2 0–2
172.7 31.2 27.3
3 BRT 57.6 45.0– 13.5 9.0– 9.5 5–13 1.4 0–4 19.1 9.1– 1.2 0–2
072.0 13.5 27.3
4 BTD 119.1 90.9– 20.5 13.4– 7.9 5–10 1.4 0–4 53.6 27.3– 3.5 2–6
181.8 27.3 109.1
5 BBK 121.8 90.9– 20.5 13.6– 7.6 6–09 1.7 0–3 51.8 27.3– 2.8 1–4
154.5 27.3 72.7
6 DSA 134.5 90.9– 24.1 18.2– 7.3 6–10 1.7 1–4 77.3 27.3– 2.5 1–4
218.2 27.3 136.4
7 DLH 114.5 90.9– 23.6 18.2– 7.2 6–09 0.7 0–2 46.4 27.3– 2.6 1–5
136.4 27.3 63.6
8 FRD 90.0 72.7– 21.8 18.2– 7.1 6–09 1.2 0–3 32.7 27.3– 1.8 1–3
108.1 27.3 45.5
9 GNR 134.1 99.0– 27.0 18.0– 8.1 7–09 1.0 0–2 26.1 18.0– 1.3 1–3
162.0 36.0 36.0
10 GDP 103.6 90.9– 20.5 18.2– 7.1 6–09 1.2 0–3 35.5 18.2– 1.3 0–3
154.5 27.3 90.9
11 HSR 129.2 90.0– 25.7 18.0– 7.3 5–10 1.4 0–3 49.5 27.0– 1.9 0–3
198.0 36.0 72.0
12 HSRP 194.5 172.7– 20.9 18.2– 10.4 8–14 1.6 0–4 75.5 63.6– 2.8 2–13
245.4 27.3 109.1
13 JPR 130.0 90.9– 20.5 13.6– 6.6 5–08 0.7 0–2 55.4 36.4– 1.5 0–6
145.4 27.3 127.3
14 JBL 128.7 72.7– 23.2 18.2– 6.6 5–08 2.9 1–4 49.1 27.3– 2.3 1–6
181.8 27.3 136.4
15 KGR 134.5 109.1– 13.5 18.2– 10.4 8–14 1.6 0–4 75.5 63.6– 2.8 2–13
172.7 27.3 109.1
16 LCK 129.4 90.9– 20.5 18.2– 7.6 6–09 1.4 0–3 49.5 27.0– 2.8 2–13
136.4 27.3 72.0
17 NGN 72.9 54.0– 15.3 18.0– 9.0 8–11 4.2 2–7 19.8 9.0– 1.4 0–2
103.5 13.5 45.0
18 PNT 172.3 127.3– 22.7 18.2– 9.8 8–12 3.5 2–7 54.2 9.1– 3.4 1–6
243.4 27.3 136.4
19 RSR 190.9 136.4– 25.5 18.2– 10.2 6–13 3.8 0–7 54.5 36.8– 3.4 2–6
218.2 36.4 81.8
20 B alba 100.9 72.8– 16.8 13.6– 7.5 5–09 2.3 1–4 47.7 36.4– 2.5 1–9
163.6 18.1 90.9
21 B chin 104.5 90.9– 16.8 13.6– 7.9 6–09 2.3 1–4 41.8 27.3– 1.7 1–3
181.8 27.3 90.9
22 RC-781 147.7 109.1– 19.1 18.2– 8.1 7–11 1.9 1–4 65.5 27.3– 1.0 0–13
236.3 22.7 136.4
23 Midas-1 170.0 119.2– 18.6 18.2– 9.5 6–12 1.8 0–6 63.6 27.3– 3.3 1–5
200.0 22.7 90.9
24 VRN 271.8 72.7– 18.6 18.2– 8.9 6–13 1.7 0–4 185.4 27.3– 8.5 2.1
459.9 22.7 472.9
6.5 Morphological and Cultural Variations 137

Plate 6.2 Morphological variations in conidia of Alternaria brassicae from India (Mehta et al. 2003)
138 6 Pathogenic Variability

Table 6.9 Conidial size of Alternaria brassicae from different locations (Mehta et al. 2003)
Septation (no.) Beak septation
Length (μm) Breadth (μm) Horizontal Vertical Beak length (μm) (no.)
Isolates Av. Range Av. Range Av. Range Av. Range Av. Range Av. Range
HSR-I 129.1 90– 25.6 18–36 7.3 5–10 1.4 0–3 49.5 27–72 1.9 0–3
198
HSR-II 231.3 189– 20.2 13–27 11.6 11–13 1.7 0–3 105.3 81– 3.6 2–6
270 135
HSR-III 140.8 108– 22.0 18–27 8.4 7–10 1.0 0–2 52.6 36–72 2.3 1–4
180
GNR 134.1 99– 27.0 18–36 8.1 7–9 1.0 0–2 26.1 18–36 1.3 1–3
162
LDH 153.9 135– 25.2 18–27 7.3 5–10 1.6 0–3 75.6 63–99 3.1 2–4
171
KNR 144.9 117– 25.6 18–27 8.9 7–10 1.6 1–2 53.5 27–72 2.6 1–4
180
RSR-I 109.8 90– 22.9 18–36 6.8 6–9 1.3 0–2 33.7 18–45 1.7 0–2
135
RSR-II 218.7 153– 23.4 13–36 11.0 9–13 2.1 0–4 52.2 36–90 2.9 2–4
270
DLK 163.3 121– 23.4 18–27 9.5 7–12 1.1 0–2 30.6 18–40 1.8 2–4
225
GDP 94.4 90– 20.7 13–36 7.2 6–9 1.0 0–2 30.6 18–40 1.8 1–2
117
Av. = Average

among isolates from Uttar Pradesh, Uttaranchal 2013). All the isolates showed high level of vari-
and Haryana, but distantly related to other states ability in vitro with respect to conidial length,
(Goyal et al. 2011). width and number of septa. Conidia of isolates
Morphological characteristic of different A. from Uttar Pradesh (CaABU4) were the smallest
brassicae isolates revealed variation in growth, with lowest number of septa. Substantial varia-
shape and pigmentation of colony, conidial mea- tion among isolates was also observed in myce-
surements and number of septa. Conidial length lial growth and sporulation on different nutrient
varied from 106.7 to 285.9 μm, width from 33.5 media. All the isolates do not grow and sporulate
to 57 μm and beak length from 41.4 to 180.0 μm. abundantly on the same nutrient medium.
The number of horizontal septa varied from 3.2 However, potato dextrose agar, cauliflower (host)
to 8.0 and vertical septa from 0.3 to 1.4. Different agar and carrot potato agar were suitable for all
synthetic media showed profound variation in isolates. Cluster analysis of data on cultural vari-
mycelial growth and sporulation indicating that ability among 32 A. brassicae isolates found a
the degree of sporulation in A. brassicae isolates close relationship among isolates of both hosts
is a function of nutrition (Tables 6.10, 6.11 and cauliflower and mustard. Isolates from Uttar
6.12). Pathogens’ aggressiveness demonstrated Pradesh, Delhi, Haryana and West Bengal are
the existence of considerable variations in the found to be similar to each other, whereas the
level of tolerance of Brassica species to A. bras- Rajasthan isolates along with Tamil Nadu and
sicae (Meena et al. 2012). Kerala isolate are distantly related to others. All
Variations in morphology and cultural charac- the isolates are pathogenic in nature but directly
teristics were observed among 32 representative related to the cultural and morphological charac-
Indian geographical isolates of A. brassicae from teristics. These isolates are further molecularly
cauliflower and rapeseed–mustard (Sharma et al. characterized by using internal transcribed spacer
6.5 Morphological and Cultural Variations 139

Plate 6.3 Morphological variations in conidia of Alternaria brassicae from Haryana (India) (Mehta et al. 2003)
140 6 Pathogenic Variability

Table 6.10 Sporulation index of Alternaria brassicae isolates on different culture media (Meena et al. 2012)
Asthana and
Isolates Brown’s Eilliot’s Hawker’s Czapek’s Richard’s
BAB-02 − + ++ + −
BAB-04 − ++ + ++ +++
BAB-06 − + + + −
BAB-08 − + ++ + −
BAB-18 − ++++ ++ ++ ++
BAB-19 − − + − +
BAB-20 − − ++++ + ++
BAB-23 − − ++ + +
BAB-28 − ++++ ++++ ++++ +++
BAB-29 − + + + −
BAB-30 − − ++ + ++
BAB-39 − − ++ − −
BAB-40 − − ++ + +
BAB-41 − ++ + + −
BAB-42 − − + + +++
BAB-43 − + − − +
BAB-44 − + ++ + +
BAB-45 − + − − +
BAB-47 − + + + +
BAB-48 − + + + −
BAB-49 − − +++ + −
BAB-50 − ++ + ++ +++
Sporulation index (number of spores per microscopic fields): − absent (nil); + trace (1–10); ++ mild (11–30); +++
moderate (31–50); ++++ abundant (more than 50)

region where all the isolates are found 56 % simi- including JD, KA and AS with only 24 % similarity
lar to each other and 99 % similar to the A. bras- (76 % dissimilarity) (Pramila et al. 2014).
sicae isolates present in NCBI database.
Alternaria brassicae colonies varied in their cul-
tural behaviour ranging from cottony, flurry to 6.6 Genetic Variability
feathery, with smooth to rough margins, and
white, off white to light brown in colour. The Genetic variability in nucleotide sequence of ITS
growth rate varied from slow, medium to fast, region of four Alternaria species (A. brassicae,
with fastest being in isolate KM and slowest in A. brassicicola, A. raphani, A. alternata) infect-
isolate JD. Significant morphological variations ing crucifers has not been recorded so far
in conidial length, width (105 to 135 × 10 to (Jasalavich et al. 1995). Cluster analysis of
20 μm) and number of horizontal septa were pathogenic variability data reveals a close rela-
observed. Isolates exhibited variations in disease tionship between Nazirhat (SS 04), Jaipur (SS
severity, number and size of lesions. The dendro- 07), Sacha khera (SS-10) and Samalkha (SS-11)
gram analysis based on molecular (DNA, RAPD) isolates. The use of 100 random amplified poly-
basis reveals two groups at 14 % similarity coef- morphic DNA decamer primers indicates genetic
ficient. Group 1 composed of seven isolates, variability among 13 A. brassicae isolates.
namely VR, DV, P7, LM, P10, KR and ND, with Almost all the isolates show relationship accord-
18 % similarity (82 % dissimilarity), while group ing to their geographical origin except Sacha
2 (Figs. 6.4 and 6.5) contained only three isolates khera (SS-10) and Hatikhuti (SS-05) isolates.
6.7 Molecular Techniques 141

Table 6.11 Conidial size of different geographical isolates of A. brassicae (Meena et al. 2012)

A. brassicae Beak length No. of septa

isolates Length (μm) Width (μm) (μm) Horizontal Vertical
BAB-02 152.3 47.5 047.3 3.8 1.3
BAB-04 185.1 47.3 095.2 3.5 0.9
BAB-06 252.1 33.9 178.0 6.8 0.1
BAB-08 106.7 33.5 048.3 3.8 0.3
BAB-18 285.9 47.5 180.0 6.3 0.5
BAB-19 140.6 44.2 061.4 3.2 0.9
BAB-20 189.7 35.6 111.5 4.2 0.3
BAB-23 211.7 41.8 125.9 3.7 0.7
BAB-28 122.2 34.7 044.7 3.4 0.4
BAB-29 198.4 43.4 104.7 3.8 0.9
BAB-30 193.6 57.0 087.3 3.2 0.9
BAB-39 144.5 37.2 070.9 3.2 0.5
BAB-40 198.2 41.6 100.4 5.6 1.1
BAB-41 206.5 44.2 116.8 4.0 0.8
BAB-42 196.6 33.7 113.9 5.5 0.1
BAB-43 147.5 36.6 066.7 3.3 0.5
BAB-44 140.6 35.4 067.9 3.5 0.4
BAB-45 144.1 48.3 041.4 3.4 1.4
BAB-47 168.1 40.4 076.2 4.1 0.9
BAB-48 151.1 33.9 073.9 4.6 0.4
BAB-49 201.2 44.6 105.9 6.1 1.1
BAB-50 284.3 40.0 172.7 8.0 0.6
Mean 182.8 41.0 095.0 4.4 0.7
CV% 26.1 15.2 043.8 30.8 53.4

Pantnagar isolate (SS-09) was found closely ISSR primers revealed wide genetic diversity
related to Sacha khera (SS-10) isolate. No vari- among 32 isolates of A. brassicae from Himachal
ability could be located among the A. brassicae Pradesh. However, they were grouped into five
isolates by internal transcribed spacer–amplified major clusters independent to pathotypes (Kumar
fragment length polymorphism molecular et al. 2014).
marker. Hence, pathogenic variability does exist
among the isolates at the genomic level, but not
in the highly conserved region of the genome of 6.7 Molecular Techniques
the pathogenic A. brassicae isolates (Goyal et al.
2013). However, internal transcript spacer analy- RAPD analysis is easy, efficient, fast and repro-
sis done by Sharma et al. (2013) shows that all ducible than RFLP analysis in the detection of
isolates are 90–100 % similar to each other, indi- intraspecific variation in A. brassicae, A. bras-
cating genetic similarity among different A. bras- sicicola and A. raphani pathogenic to crucifers.
sicae isolates that vary pathogenically. The Polymorphism within an Alternaria species by
analysis of 26 RAPD primers revealed a high RAPD molecule marker has been described by
level of genetic variability among ten isolates of many workers (Sharma and Tewari 1995, 1998;
A. brassicae from different B. juncea cultivars Kumar et al. 2008). Observing polymorphism
(Pramila et al. 2014). DNA fingerprinting using among A. brassicae isolates from different
142 6 Pathogenic Variability

4
3.5

Colony diameter (cm)

3
2.5
2

1.5
1

0.5
0
VR DV LM P7 P10 KR ND JD KA AS
Alternaria brassicae isolates
Growth of A. brassicae isolates on PDA

a
80
Disease index (%)

60

40 5 DAI
10 DAI
20
15 DAI
0
AS KA LM ND P7 P10 VR
Different A.brassicae isolates
b
Average number of

10
spots/10 cm2

8
6 5 DAI
4
10 DAI
2 15 DAI
0
AS KA LM ND P7 P10 VR
Different A.brassicae isolates
c
Average size os spot (cm)

0.6
0.5
0.4
0.3 5 DAI
10 DAI
0.2
15 DAI
0.1
0
AS KA LM ND P7 P10 VR
Different A.brassicae isolates

Fig. 6.4 (a) Disease index. (b) Average number of spores/10 cm2. (c) Average size of spot (cm) on leaves of Divya
inoculated by isolates grown on the PDA medium on 5, 10 and 15 days after inoculation (DAI) (Pramila et al. 2014)
6.9 Nutritional Variability 143

VR

DV

P7

LM

P10

KR

ND

JD

KA

AS

0.15 0.22 0.29 0.35 0.43

Coefficient

Fig. 6.5 Genetic divergence among ten isolates of Alternaria brassicae based on UPGMA cluster analysis (Pramila
et al. 2014)

geographical regions of the world, Sharma and observations that showed extensive colonization
Tewari (1995, 1998), however, found low intra- of the host tissue by the highly virulent isolate.
regional variations among Indian and Canadian Mycelial protein profiles of the two differentially
isolates with 75 % similarity. However, RAPD virulent A. brassicae isolates were compared
analysis of A. brassicae isolates from different using two dimensional gel electrophoresis (2DE)
geographical regions of India using more than and mass spectrometry (MS) in order to identify
one hundred primers suggested a high degree of proteins that may be responsible for the differ-
polymorphism among isolates. The dendrograms ences. Several differences in the mycelial pro-
from both pathogenic and molecular analyses teomes of the two isolates were recorded. The
seem to indicate that the Pant Nagar (SS-09) and proteins that were significantly abundant in the
Hatikhuti (SS-05) isolates are quite different more virulent isolate included a protein with con-
from the others, and the two dendrograms follow served actin-related protein2/3 domain, enolase,
the same trend (Goyal et al. 2013). BLAST anal- malate dehydrogenase and serine protease. The
ysis of the ITS of 32 A. brassicae isolates con- differential protein expression pattern can be
ducted by Sharma et al. (2013) showed high exploited to identify putative virulence and
similarity among the isolates available at the pathogenicity factors in A. brassicae (Sharma
NCBI database. et al. 2012).

6.8 Proteome Analysis 6.9 Nutritional Variability

Two isolates of A. brassicae with significant dif- Fourteen isolates of Alternaria brassicae causing
ferences in virulence have been characterized at Alternaria blight in rapeseed–mustard were char-
the proteome level. The morphological observa- acterized by their responses to various carbon
tions indicated the Ontario isolate to be more and nitrogen sources, as well as to pH. All the
virulent by virtue of increased disease severity isolates behaved differentially in growth and
score as compared to the UAMH7476 isolate. sporulation in relation to different carbon and
This was further confirmed through histological nitrogen sources. Isolates KTL-I and BWL show
 144

Table 6.12 Mycelial growth of A. brassicae under different temperature and relative humidity conditions (Meena et al. 2012)
Radial growth (mm)a
Alternaria
brassicae Temperature (°C)b Relative humidity (%)c
isolate 15 20 25 30 35 40 50 60 70 75 80 85 90 95 100
BAB-18 15.7 16.0 24.7 24.7 16.3 12.3 10.0 13.3 20.0 22.3 24.3 26.3 24.7 29.3 31.3
BAB-19 11.0 16.3 27.0 25.3 16.0 12.7 10.5 13.5 20.5 21.7 23.5 25.7 27.0 29.0 30.0
BAB-20 15.3 19.0 30.0 27.3 18.0 12.7 10.3 13.0 20.3 21.3 24.0 26.7 30.0 28.7 29.5
BAB-23 11.0 15.3 24.0 21.0 15.0 13.0 11.7 14.0 21.0 23.0 25.0 27.7 24.0 30.7 31.0
BAB-30 16.7 16.7 28.0 23.3 16.3 12.3 9.7 12.7 19.5 21.5 23.7 26.7 28.0 29.3 30.0
BAB-43 14.3 15.0 29.3 29.0 16.0 10.0 10.7 13.0 20.3 22.0 24.7 27.0 29.3 29.0 30.0
BAB-48 15.7 16.3 24.3 27.0 16.0 14.0 11.0 13.7 19.7 22.7 24.5 27.3 24.3 29.3 30.5
LSD temperature = 2.2, isolate = 1.8, relative humidity = 3.2, isolate = 2.1
(P < 0.05): temperature x isolate = 2.1; relative humidity × isolate = 2.2
a
Mean of three replications
b
Temperature 25 °C
c
Relative humidity 100 %
6
Pathogenic Variability
6.11 Fungicidal and Plant Extracts Sensitivity 145

significantly higher growth on all the carbon KTL-II contained the maximum amount of RNA,
sources, whereas isolate CHR-I yielded mini- while SPT the minimum. On the basis of RNA
mum growth and responded differentially to dif- contents, isolates can also be categorized into
ferent carbon sources. Similarly, isolate REW three groups. The protein content also differed
showed more variation in sporulation than SPT significantly among isolates. Isolates CHR-I and
and HSR isolates. Among the nitrogen sources JD-I contained the maximum amount of proteins
evaluated, growth was maximum on sodium and isolate KTL-I the least.
nitrate followed by potassium nitrate, ammonium Similarly, there are significant differences
nitrate and glycine. Irrespective of the nitrogen among the isolates in their free amino acid con-
source, isolates KTL-I and CHR-II produced the tent; isolates JD-I and SPT contained the highest
maximum, whereas SRS and JD-II the minimum and lowest amount of free amino acids, respec-
radial growth. Isolates BWL, CHR-I, CHR-II, tively. Alternaria brassicae isolates containing
RTK, KTL-I and SPT responded best on KNO3- higher amount of proteins generally contain
amended medium, whereas REW poorly on gly- moderate amount of free amino acids and RNA,
cine; isolates BWL, CHR-II, JD-I, SRS and SPT while those containing higher amount of RNA
sporulated best on KNO3 and HSR very poorly often contain moderate amount of proteins and
on glycine. All isolates grew better at pH 7.5, but free amino acids. On the basis of similarity in
sporulated best at 5.0 after 21 days of incubation. biochemical composition, all isolates were
On the basis of nutritional behaviour, all the iso- grouped into three categories: maximum, moder-
lates were placed into two major groups: isolates ate and minimum. Isolates BHI, JHR, KTL-I,
BHI, JD-I, JHR, REW, RTK, SRS, CHR-I, SRS, CHR-I and SPT formed the first group with
CHR-II, JD-II and HSR were placed in group 1, maximum amount of biochemicals; isolates
whereas isolates KTL-I, KTL-II and BWL BWL, RTK, CHR-II, HSR, KTL-II and REW
formed the second group (Mehta et al. 2005b; were in the second group with moderate amount,
Tables 6.13, 6.14, 6.15 and 6.16). while isolates JD-I and JD-II were in the third
group with the minimum amount (Khurana et al.
2005b).
6.10 Biochemical Variability

Biochemical constituents of A. brassicae isolates 6.11 Fungicidal and Plant Extracts

differ significantly (Khurana et al. 2005b). The Sensitivity
isolate BWL contains the maximum ortho-
dihydric phenols and KTL-II the least (O.D. phe- The variation in efficacy of fungicides in control-
nols); isolate REW contains the highest amount ling Alternaria diseases of crucifers may be due
of total phenols (Table 6.17). The differences in to response of pathotypes prevalent in a region.
the amount of total and ortho-dihydric phenols According to Vishwanath and Kolte (1997), iso-
indicate the existence of variation in isolates. The late A of A. brassicae showed more tolerance to
isolate JD-II contains the maximum amount of ziram and Ridomil MZ-72 at 50 and 100 ppm as
both reducing and non-reducing sugars and RTK compared to isolate C. Similarly, isolate A exhib-
the minimum. The amount of reducing sugars ited maximum tolerance followed by isolates C
also varied significantly between isolates. The and D against mancozeb and iprodione. Efficacy
isolates BWL, CHR-II, CHR-I and RTK have of several fungicides and neem products was
significantly lower reducing sugars than other evaluated against 15 A. brassicae isolates col-
isolates. Vishwanath and Kolte (1997) reported lected from different locations in Haryana (India)
that A. brassicae isolate containing high amount (Table 6.18). The fungicide Kitazin was highly
of carbohydrate is an indicator of virulence. The effective against all isolates in inhibiting the
estimation of total RNA content revealed signifi- spore germination, which was followed by
cant differences among the isolates; isolate Dithane M-45 and Ridomil MZ-72. Similarly,
 146

Table 6.13 Effect of various carbon sources on the radial growth (cm) of various isolates of Alternaria brassicae from Haryana, India (Mehta et al. 2005b)
Alternaria brassicae isolates
Carbon sources BHI BWL CHR-I CHR-II HSR JD-I JD-II JHR KTL-I KTL-II REW RTK SRS SPT Mean SEM±
Dextrose 5.3 8.6 3.3 2.9 4.7 3.2 6.3 8.4 8.6 7.3 4.2 4.2 5.9 8.5 5.8 0.17
Fructose 5.2 8.9 1.9 5.4 2.7 4.6 5.4 3.5 8.9 7.1 4.1 3.3 3.2 8.5 5.2 0.19
Lactose 5.2 8.6 2.6 5.3 3.5 5.3 5.3 4.5 9.0 7.3 5.2 4.0 6.4 7.5 5.7 0.18
Mannitol 5.1 8.8 2.4 5.5 4.3 5.6 5.4 6.1 9.0 8.6 5.3 5.8 5.9 7.2 6.1 0.16
Sucrose 2.9 8.6 2.2 5.2 2.7 4.8 8.6 5.9 8.6 6.2 5.1 3.0 3.4 7.3 5.3 0.16
Control 3.1 6.4 1.6 2.1 2.6 5.8 6.3 3.6 6.8 6.6 2.9 4.6 1.9 6.3 4.3 0.17
(without
carbon)
Mean 4.4 8.4 2.3 4.4 3.4 4.9 6.2 5.3 8.5 7.2 4.5 4.1 4.5 7.6
CD (p = 0.05 %): isolates (I) = 0.14; carbon source (C) = 0.09; I × C = 0.34
6
Pathogenic Variability
 6.11

Table 6.14 Effect of various carbon sources on the sporulation of various isolates of Alternaria brassicae from Haryana, India (Mehta et al. 2005b)
Spores/microscopic field of Alternaria brassicae isolates
Carbon sources BHI BWL CHR-I CHR-II HSR JD-I JD-II JHR KTL-I KTL-II REW RTK SRS SPT Mean SEM±
Fungicidal and Plant Extracts Sensitivity

Dextrose 6.5 3.8 4.3 6.3 6.0 5.3 6.3 4.5 3.3 5.8 7.3 7.0 5.3 4.5 5.4 0.9
Fructose 7.0 6.8 5.8 4.5 5.0 6.8 6.3 7.3 4.8 5.0 14.0 6.3 7.0 5.0 6.5 1.0
Lactose 2.8 7.3 10.5 2.8 4.8 7.3 5.0 2.5 4.8 3.0 3.5 4.0 6.0 4.8 4.9 0.9
Mannitol 6.5 6.5 6.8 6.8 6.3 5.0 7.3 4.8 6.3 6.5 16.0 6.8 7.0 4.5 6.9 1.1
Sucrose 5.0 5.8 5.3 4.0 4.8 4.0 2.8 6.5 6.3 4.8 14.3 9.8 4.8 5.5 5.9 0.9
Control 2.5 0.3 1.0 1.3 0.8 0.5 1.0 0.8 0.5 0.3 2.5 2.0 1.5 0.8 1.0 0.5
(without
carbon)
Mean 4.9 5.0 5.6 4.3 4.6 4.8 4.8 4.4 4.3 4.2 9.6 6.0 5.3 4.2
CD (p = 0.05 %); isolates (I) = 0.73; carbon sources (C) = 0.47; I × C = 1
147
 148

Table 6.15 Effect of various nitrogen sources on the radial growth of different isolates of Alternaria brassicae from Haryana, India (Mehta et al. 2005b)

Nitrogen Radial growth (cm) of Alternaria brassicae isolates

sources BHI BWL CHR-I CHR-II HSR JD-I JD-II JHR KTL-I KTL-II REW RTK SRS SPT MEAN SEM±
Glycine 2.5 3.1 2.5 2.9 4.0 3.1 2.8 2.6 3.4 3.2 1.9 2.5 2.9 3.3 2.9 0.07
KNO2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.00
KNO3 5.1 7.2 7.1 7.1 5.5 5.6 4.6 6.4 7.2 6.6 4.5 7.0 4.4 7.1 6.1 0.10
NaNO3 6.1 7.5 7.4 7.4 7.5 6.2 6.0 6.4 7.3 6.6 5.0 6.5 4.0 6.5 6.5 0.09
NH4NO3 3.6 3.8 4.0 3.8 4.0 3.7 2.5 3.7 3.4 3.6 3.1 2.5 3.0 3.6 3.5 0.11
Control 3.5 3.8 4.5 4.5 4.3 4.1 3.3 4.3 4.3 3.4 5.5 3.9 3.7 4.1 4.1 0.08
(without
nitrogen)
Mean 3.6 4.3 4.3 4.4 4.3 3.9 3.3 4.0 4.4 4.0 3.4 3.8 3.1 4.2
CD (p = 0.05 %); isolates (I) = 0.07; nitrogen sources (N) = 0.04; I × N = 0.17
6
Pathogenic Variability
 6.11

Table 6.16 Effect of various nitrogen sources on the sporulation of different isolates of Alternaria brassicae from Haryana, India (Mehta et al. 2005b)

Nitrogen Spores per microscopic field of Alternaria brassicae isolates

sources BHI BWL CHR-I CHR-II HSR JD-I JD-II JHR KTL-I KTL-II REW RTK SRS SPT Mean SEM±
Fungicidal and Plant Extracts Sensitivity

Glycine 1.5 2.2 2.5 2.7 1.0 2.7 2.0 3.5 2.2 1.7 2.2 2.2 3.0 1.7 2.2 0.6
KNO2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.00 0.0 0.0 0.0
KNO3 8.7 10.0 8.5 10.0 9.7 11.5 8.2 9.2 9.7 9.5 7.5 9.2 11.2 10.5 9.5 1.2
NaNO3 7.7 6.7 7.2 8.2 7.5 8.5 7.0 7.5 8.2 8.2 7.5 7.0 8.2 7.2 7.6 0.9
NH4NO3 5.2 5.5 6.2 4.0 4.2 4.2 4.7 3.7 5.5 4.5 3.7 4.0 5.2 4.2 4.7 0.7
Control 1.2 2.2 1.7 1.2 1.5 1.7 0.0 1.5 1.2 0.0 2.5 0.0 0.0 1.0 1.1 0.6
(without
nitrogen)
Mean 4.1 4.4 4.4 4.4 4.2 4.8 3.7 4.2 4.5 4.0 3.9 3.7 4.8 4.1
CD (P = 0.05): isolates (I) = 0.62; nitrogen sources (N) = 0.40; I × N = 1.53
149
150 6 Pathogenic Variability

Table 6.17 Biochemical constituents (mg/g)a in isolates of Alternaria brassicae (Khurana et al. 2005a, b)
Biochemical constituents
Ortho- Non- Free
dihydric Total reducing Reducing Total amino
Isolates phenols phenols sugar sugars sugars RNA Proteins acids
BHI 1.6 9.5 2.5 2.0 4.5 6.8 66.8 49.2
BWL 3.5 10.4 3.7 0.7 4.5 9.4 85.7 56.5
CHR-I 2.0 5.5 2.8 1.0 3.9 6.4 103.0 37.5
CHR-II 2.2 4.9 1.7 1.0 2.7 8.8 74.2 45.7
HSR 1.7 10.5 6.2 3.5 9.7 8.6 59.9 57.7
JD-I 2.1 6.1 6.1 1.4 7.5 9.6 100.4 63.9
JD-II 2.8 13.3 10.5 5.3 15.8 11.8 76.9 38.9
JHR 1.5 9.6 4.4 1.8 6.1 5.8 67.5 40.9
KTL-I 3.4 13.4 4.6 1.5 6.1 7.5 49.2 51.8
KTL-II 1.3 12.0 4.0 1.5 5.5 16.4 81.9 52.8
REW 2.2 13.5 2.2 1.6 3.8 6.9 85.2 50.2
RTK 1.4 9.7 1.5 1.1 2.6 11.6 89.9 41.3
SRS 2.2 7.9 4.8 3.2 8.0 6.5 68.7 40.3
SPT 2.4 8.2 2.8 2.2 5.0 4.5 70.4 25.6
C.D.(5 %) 0.13 0.33 0.49 0.16 0.28 0.72 3.63 8.11
SEM± 0.06 0.15 0.23 0.07 0.13 0.33 1.69 3.78
a
Based on dry mycelial weight in 1000 mg

among four neem products, Achook and Bioneem revealed that garlic and neem leaves extracts
were quite effective compared to Furpume and proved to be quite effective against Alternaria
Nimbecidine. Variations were also observed brassicae in vitro and followed by mint, bougain-
among isolates in their sensitivity against fungi- villaea, Lawsonia and eucalyptus. Among iso-
cides. Isolates BHI, CHR-I and CHR-III were lates, REW and RTK were quite sensitive to all
sensitive to all the fungicides, whereas JHR was the plant extract, whereas isolate SRS was sensi-
sensitive only to Dithane M-45, Kitazin and tive to only neem and garlic extract. Isolates
Bavistin. In the case of neem products, HSR iso- KTL-II, BHI and JHR were not sensitive to neem
late was not sensitive to Achook, whereas extract as compared to other isolates. Similarly in
Bioneem proved to be effective against CHR-I, the case of garlic, isolates BHI, HSR, JD-II,
CHR-III, HSR, KTL-I, KTL-II, REW and SPT KTL-I and SRS did not respond effectively as
isolates. Based on their sensitivity against fungi- compared to other isolates. The eucalyptus and
cides and neem products, all isolates fell more or Lawsonia leaves extract which were ineffective,
less in the same group (Tables 6.18 and 6.19). in general, appeared quite effective against REW
Isolates were categorized into eight groups on the and RTK isolates, clearly indicating the variation
basis of their differential sensitivity to fungicides in the isolates of Alternaria brassicae
and neem products. These groups were desig- (Table 6.20).
nated into eight pathotypes/races present under Evaluation of efficacy of ten fungicides
Haryana, Indian, conditions infecting rapeseed– against A. brassicae isolates from various parts of
mustard. Variations in the sensitivity of 14 A. India showed that Emisan-6, in general, proved
brassicae isolates to extracts of bougainvillaea, most effective in inhibiting spore germination
garlic, Lawsonia, neem, mint and eucalyptus followed by Ridomil MZ-72, while Sulfex and
(Table 6.20) have also been observed (Kumar Blitox proved least effective (Sangwan and
et al. 2004; Khurana et al. 2005a). Results Mehta 2006). Isolates PNT, BHP, CAUL and B.
 6.11

Table 6.18 Differential behaviour of various isolates of Alternaria brassicae against fungicides (Kumar et al. 2004)
Alternaria brassicae isolates
Fungicides BWL BHI CHR-I CHR-II CHR-III HSR JND-I JND-II JHR KTL-I KTL-II REW RTK SRS SPT
Fungicidal and Plant Extracts Sensitivity

Emisan-6 + + + + + + + + − − + + + + +
Wettable − + + + + − − − − + − − − + −
sulphur
Ridomil + + + + + + + + − + + + + + +
MZ-72
Blitox-50 + + + + + + + + − + + + + − +
Dithane M-45 + + + + + + + + + + + + + + +
Kitazin + + + + + + + + + − + + + + +
Bavistin − + + − + + + + + − − + + + −
Baynate − + + + + − + + − + − + + − −
+ = sensitive to fungicide (less than 25 % spore germination); − = not sensitive to fungicide (more than 25 % spore germination)
151
 152

Table 6.19 Differential behaviour of various isolates of Alternaria brassicae against neem products (Kumar et al. 2004)
Alternaria brassicae isolates
Neem products BWL BHI CHR-I CHR-II CHR-III HSR JND-I JND-II JHR KTL-I KTL-II REW RTK SRS SPT
Furpume − − − − − + − − − − − − − − −
Bio-neem − − + − + + − − − + + + − − +
Nimbecidine − − − − − + + + + − + + − − −
Achook − − + + − − + + + − − + + + −
+ = sensitive to neem products (less than 25 % spore germination); − = not sensitive to neem products (more than 25 % spore germination)
6
Pathogenic Variability
 6.11

Table 6.20 Sensitivity of different isolates of Alternaria brassicae collected from Haryana, India, to various plant extracts (Kumar et al. 2004)
Per cent spore germination of Alternaria brassicae isolates
Plant extracts BHI BWL CHR-I CHR-II HSR JD-I JD-II JHR KTL-I KTL-II REW RTK SRS SPT Mean SEM±
Bougainvillaea 31.41 29.00 38.73 29.7 41.7 40.4 34.6 48.8 20.1 36.8 11.2 13.7 60.2 35.8 33.73 1.36
(34.0) (32.3) (38.5) (33.0) (40.2) (39.4) (36.0) (44.3) (26.6) (37.5) (19.5) (21.6) (50.9) (36.8) (35.0)
Garlica 23.8 15.2 19.3 13.3 24.9 23.1 22.0 16.7 22.7 16.6 9.2 7.4 24.2 18.3 18.3 1.97
(29.1) (22.9) (25.9) (21.3) (29.9) (28.6) (29.7) (23.9) (28.3) (24.0) (17.5) (15.7) (29.4) (25.2) (25.1)
Fungicidal and Plant Extracts Sensitivity

Lawsonia 30.1 66.3 30.9 30.1 22.4 38.6 62.6 64.6 46.7 32.1 10.00 8.6 58.8 34.0 38.3 1.40
(33.5) (54.6) (33.8) (33.3) (28.2) (38.4) (52.3) (53.2) (43.1) (34.5) (18.3) (17.0) (50.1) (35.7) (37.6)
Neem 41.0 12.1 13.8 18.6 15.0 20.4 12.1 30.1 12.5 42.4 12.3 8.3 23.5 21.5 20.3 1.59
(39.9) (20.3) (21.7) (25.5) (22.8) (26.8) (20.2) (33.2) (20.6) (40.6) (20.5) (16.7) (28.9) (27.6) (26.1)
Mint 46.4 17.3 17.8 25.2 24.0 34.9 28.8 36.6 23.1 14.4 18.3 8.2 36.2 23.3 25.3 1.53
(42.9) (24.6) (25.0) (30.1) (29.3) (36.2) (32.4) (37.2) (41.2) (22.4) (26.1) (16.6) (37.0) (28.8) (30.7)
Eucalyptus 59.2 31.7 31.8 27.7 35.8 33.0 51.0 35.6 48.5 67.1 20.4 14.0 52.2 30.8 38.5 1.66
(47.6) (34.2) (34.3) (31.8) (36.8) (35.1) (45.6) (36.7) (44.1) (55.0) (26.9) (21.8) (46.3) (33.7) (37.8)
Control 75.5 75.7 76.9 77.2 77.1 81.5 76.5 72.5 71.0 83.2 78.7 79.3 82.6 74.6 77.3 1.75
(water only) (60.3) (60.5) (61.2) (61.8) (61.4) (64.6) (61.0) (58.4) (57.4) (65.8) (62.9) (63.2) (65.4) (59.7) (61.7)
Mean 43.9 35.3 32.7 31.7 34.4 38.8 41.1 43.6 34.9 41.8 22.9 19.9 48.2 34.0
(41.1) (35.6) (34.3) (33.8) (35.5) (38.4) (39.5) (41.0) (37.3) (40.0) (27.4) (24.7) (44.0) (35.4)
CD (p = 0.05 %): isolate (I) = 1.20; plant extract (PE) = 0.85; I × PE = 3.18; figures in the parentheses are arc sine values
a
Cloves were used
153
154 6 Pathogenic Variability

alba were highly sensitive, whereas isolates 90 % spore viability at 50 °C; and isolates KTL-I
FRD, B. chin and ASM were the least sensitive and KTL-II formed the third group, where only
(Table 6.21) . Spore germination among isolates 5 % spores survived at 55 °C.
varied from 17.1 to 43.59 % (Sangwan and Mehta
2006). Differential behaviour of various isolates
also indicated that isolates BHP and B. alba were 6.13 Identification
more sensitive followed by CAUL, TRN, PNT and Nomenclature
and HSR. The isolates RC-781, FRD, B. chin, of Pathotypes
ASM, GRN and GDP responded similarly
(Table 6.22). Physiological races or pathotypes of plant patho-
gens are identified on the basis of infection types
produced by them on specific set of cultivars
6.12 Thermal Sensitivity called Differentials. The procedures and prob-
lems involved in the collection of diseased sam-
Differences among A. brassicae isolates in rela- ples, isolation and purification of cultures,
tion to their sensitivity to different temperatures maintenance of specific isolates, techniques of
have been reported. In general, there are no sig- inoculation and scoring of infection types have
nificant differences in spore germination in the been described (Verma and Saharan 1994). In
temperature range of 20–30 °C. As the tempera- biotrophs, host pathosystem (Puccinia, wheat;
ture rise, the viability of the spores declines, and Melampsora, flax) norm and standards of selec-
in most isolates, except KTL-I and KTL-II, tion of host differentials acceptable at interna-
spores lose their viability at 55 °C. In isolates tional level have been followed, but it has not
KTL-I and KTL-II, 5 % spores remain viable been met in the studies conducted in Alternaria–
even at 55 °C. Based on spore germination, iso- crucifers system. Selection of standard host dif-
lates BWL, CHR-II and JND-II are more resis- ferentials consists of a set of host varieties termed
tant to high temperatures. In most isolates, only Differentials, supplemented differentials (addi-
10 % spores germinated at 45–50 °C as compared tional host varieties), single gene lines and near-
to 35–43 % in KTL-II and SPT isolates. The isogenic lines. The use of such a set of host
drastic reduction in spore germination at 45 °C differentials can clear the picture of presence and
indicates that the pathogen cannot survive during identification of pathotypes in Alternaria spp.
summer months in northern India (Table 6.23). infecting crucifers.
Spores of only two isolates, KTL-I and KTL-II, Nomenclature of a race or pathotype has been
germinate at 55 °C indicating their capability to done earlier as:
withstand high temperature, which can have a
significant implication on their survival. The iso- 1. Arbitrary numbers: races are generally desig-
lates, which withstand highest temperature prob- nated as number or letters in an arbitrary man-
ably, have genetic resistance to high temperature. ner, generally in the order of their discovery,
On the basis of their thermal sensitivity, the iso- e.g. cereal rusts
lates were grouped into three categories by 2. Black’s nomenclature: a race is designated on
Kumar et al. (2003a). Isolates BWL, CHR-II and the basis of its virulence on a host resistance
JND-I formed the first group since they lost 90 % gene, e.g. Phytophthora–potato system, races
spore viability at 45 °C; isolates BHI, CHR-I, R1 and R2, races R1 and R4, etc.
CHR-III, HSR, JND-I, JHR, REW, RTK, SRS 3. Virulence formulae: it is based on a race vir-
and SPT formed the second group as they lost ulent and avirulent of particular gene for
 6.13

Table 6.21 Sensitivity of different isolates of Alternaria brassicae from India to various fungicides (Sangwan and Mehta 2006)
Per cent spore germination in various isolates
Fungicides RC-781 KGR FRD B chin Varuna ASM B alba BHP PNT CAUL GRN HSR GDP TRN Mean
Dithane M-45 29.0 28.4 38.5 33.6 24.6 43.6 27.2 22.0 27.1 40.5 36.1 17.1 35.2 32.7 31.7
Sulfex 56.6 46.5 72.3 50.6 48.4 59.7 23.8 39.2 39.3 25.4 56.4 37.8 44.6 38.2 45.4
Blitox 48.2 33.4 63.3 53.7 40.9 47.8 37.4 50.0 33.5 33.3 44.2 25.6 56.5 45.5 43.8
Captan 30.9 32.8 33.7 43.4 33.1 45.2 25.0 21.6 26.3 20.9 51.9 41.2 40.5 24.8 33.6
Identification and Nomenclature of Pathotypes

Kitazin 36.6 26.8 48.1 50.7 35.8 54.4 33.6 22.9 24.2 27.4 35.2 23.4 40.6 29.7 35.0
Bavistin 38.4 23.3 51.4 43.8 34.8 50.5 21.2 30.9 18.4 21.4 41.6 26.9 34.5 25.0 33.0
Vitavax 29.6 30.4 40.3 38.3 72.7 48.4 23.6 22.0 14.7 25.5 35.8 27.0 44.9 22.1 31.1
Raxil 42.6 64.2 49.0 45.6 36.7 60.9 26.1 23.9 26.0 21.3 54.1 61.3 47.7 42.5 43.0
Emisan-6 06.9 05.7 16.5 16.9 09.8 12.4 12.0 11.9 06.8 9.0 14.5 16.3 17.2 12.7 12.0
Ridomil 11.9 11.7 20.5 22.0 16.3 11.7 06.7 11.1 10.1 16.6 06.4 22.3 14.5 15.8 14.1
MZ-72
Control 87.6 85.4 80.2 84.5 82.8 85.3 88.2 85.2 85.1 84.7 83.7 74.7 78.3 78.6 83.6
Mean 39.8 36.2 46.7 44.8 36.6 47.3 30.4 31.0 28.3 29.6 42.7 34.0 41.3 35.2 -
CD (5 %): isolates (I), 2.46; fungicides (F), 1.27 I × F: 3.58
155
156 6 Pathogenic Variability

Table 6.22 Differential behaviour of various isolates of A. brassicae from India against different fungicides (Sangwan
and Mehta 2006)
A. brassicae isolates
RC- B.
Fungicides 781 KGR Varuna ASM alba BHP PNT CAUL HSR GDP TRN
Dithane + + − + + − + + − + +
M-45
Sulfex + + + + − + + + + + +
Blitox + + + + + + + + + + +
Captan + + + + − − + − + + −
Kitazin + + + + + − + + − + +
Bavistin + − + + − + − − + + −
Vitavax + + + + − − − + + + −
Raxil + + + + + − + − + + +
Emisan-6 − − − − − − − − − − −
Ridomil − − − − − − − − − − −
MZ-72

resistance, e.g. the formulae 6, 7, 10/5, 8, 9a et al. (2004) seem to be logical since it is based
and 11 for a race of Puccinia virulent on Sr 6, on the interaction of pathogen isolates with one
Sr7 and Sr10, but avirulent on Sr5, Sr8, Sr9a specific genotype of a host species. Apparently, it
and Sr11 meets gene for gene hypothesis in the absence of
4. Habgood nomenclature standard monogenic/isogenic host differential
5. Virulence analysis sets. The determinant attributes used by different
workers for the identification of pathotypes of
However, out of these criteria of nomenclature, Alternaria species infecting crucifers are given in
Brassica researchers have adopted the first Table 6.24.
method in isolation (not taken into account ear- The utility and advantages of race/pathotype
lier reports) giving their own arbitrary numbers identification in Alternaria can boost Brassica
not keeping parity with others and order of dis- production through the (1) development of
covery. Even researchers had hesitation in desig- resistant cultivars, (2) identification of new
nating pathotypes of Alternaria species except genes for resistance, (3) development of
Saharan and Kadian (1983) and Gupta et al. multigene-resistant cultivars and (4) identifica-
(2004). The procedure and method of Alternaria tion of favourable gene combinations (Singh
species pathotypes designation adopted by Gupta and Chand 1983).
 6.13

Table 6.23 Variation in thermal sensitivity of different isolates of Alternaria brassicae from Haryana, India (Kumar et al. 2003a, b)
Per cent spore germination of Alternaria brassicae isolates*
Temperature BHI CHR-I CHR-II CHR- HSR JND-I JND-II JHR KTL-I KTL-II REW RTK SRS
(°C) BWL III SPT Mean
30 90.3 93.5 96.4 91.5 100.0 94.7 94.1 91.1 95.9 93.3 98.9 96.8 92.2 94.0 91.9 94.3
(72.2) (77.1) (82.3) (75.5) (90.0) (78.4) (77.8) (71.7) (81.7) (77.1) (86.9) (82.6) (76.0) (77.7) (75.8) (78.9)
35 90.2 87.8 88.1 80.1 85.6 88.8 87.3 85.1 88.5 87.0 91.0 90.8 88.1 90.1 84.6 87.5
(68.7) (69.6) (69.9) (63.5) (68.7) (70.7) (69.2) (66.3) (70.4) (69.2) (74.9) (71.3) (69.9) (71.6) (67.2) (69.4)
40 80.4 79.3 73.6 80.3 81.7 79.8 81.8 76.9 76.8 78.3 80.2 79.3 83.6 75.6 69.4 78.4
Identification and Nomenclature of Pathotypes

(63.7) (62.9) (59.1) (63.9) (65.0) (63.4) (60.4) (61.3) (61.3) (62.3) (63.7) (63.1) (66.1) (60.4) (56.6) (52.2)
45 8.3 23.4 17.3 9.2 15.2 12.8 15.6 9.6 17.0 43.8 36.0 17.7 10.5 18.2 35.4 19.3
(17.9) (28.8) (24.5) (17.7) (22.9) (21.0) (23.3) (17.7) (24.3) (39.6) (38.3) (24.7) (18.8) (25.2) (36.5) (25.4)
50 3.7 15.9 14.5 6.7 14.0 6.6 2.4 4.0 9.2 11.7 11.7 9.2 8.5 10.3 12.4 9.4
(15.6) (23.4) (22.4) (17.8) (23.9) (13.2) (11.8) (11.4) (17.4) (19.8) (20.0) (17.3) (16.8) (18.7) (20.3) (18.0)
55 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.6 5.4 0.0 0.0 0.0 0.0 0.7
(0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (13.5) (13.2) (0.0) (0.0) (0.0) (0.0) (1.8)
Control 87.4 98.0 91.6 99.3 91.5 100.0 90.5 90.7 85.4 97.7 98.3 87.6 98.7 100.0 97.0 94.2
(22 ± 10C) (68.4) (83.1) (73.0) (87.6) (73.2) (90.0) (72.1) (72.2) (67.6) (85.6) (84.8) (69.5) (85.5) (90.0) (80.2) (78.9)
Mean 51.5 56.8 54.5 52.4 55.4 54.7 53.1 51.1 53.3 59.6 60.2 54.5 54.5 55.4 55.8
(43.8) (49.3) (47.3) (46.6) (49.1) (48.1) (44.9) (43.0) (46.1) (52.4) (54.5) (46.9) (47.6) (49.1) (48.1)
CD (P = 0.05): temperature (T) = 1.5, isolates (I) = 2.2, T × I = 5.8
*figures in parentheses are angular transformed values
157
158 6 Pathogenic Variability

Table 6.24 Determinants of variability in Alternaria infecting crucifers (Saharan et al. 2015)
Determinant attributes Alternaria species Host Pathotypes/races Reference
Pathological A. alternata Crambe A, B, C Czyzewska,
(1969, 1971)
A. brassicae Crucifers RM-1, RM-2, V-3 Saharan and
Kadian (1983)
A. brassicae Rape 13 Mridha (1983)
A. brassicicola Cauliflower 3 Stoll (1952)
A. raphani Radish Wild Variants Atkinson (1953)
A. brassicae B. juncea Bj-4, Bj-5,Bj-6, Bj-7 Gupta et al.
(2004)
A. brassicae B. juncea DLK, RSR-1, GDP Mehta et al.
(2003)
A. brassicae B. juncea A,C,D Vishwanath and
Kolte (1997)
A. brassicae B. juncea 8 Kumar et al.
(2003a, b)
A. brassicae Brassica spp. 10 Mehta et al.
(2003)
A. brassicae Brassica spp. 14 Sangwan and
Mehta (2007)
A. brassicae Brassica spp. 8 Singh et al.
(2008)
A. brassicae Brassica spp. Abr 1 to Abr7 Kumar et al.
(2014)
Symptomatological A. brassicae Brassica spp. Bj-4, Bj-5, Bj-6, Bj-7 Gupta et al.
(2004)
A. brassicae Brassica spp. A,C,D Kolte et al.
(1991)
A. brassicae Brassica spp. 12 Goyal et al.
(2013)
Morphological, cultural and A. brassicae B. carinata A,B,C,D Kolte et al.
nutritional (1989, 1991)
A. brassicae Brassica spp. 4 Mehta et al.
(2003)
A. brassicae Brassica spp. 4 Goyal et al.
(2011)
A. brassicae Brassica spp. 5 Meena et al.
(2012)
A. brassicae Cauliflower and 2 Sharma et al.
rapeseed– (2013)
mustard
A. brassicae B. juncea 2 Pramila et al.
(2014)
A. brassicae Colza - Van Schreven
(1953)
A. brassicae Rapeseed– 2 Mehta et al.
mustard (2005b)
Biochemical A. brassicae Rapeseed– 3 Khurana et al.
mustard (2005b)
A. brassicae Rapeseed– 3 Vishwanath and
mustard Kolte (1997)
(continued)
References 159

Table 6.24 (continued)

Determinant attributes Alternaria species Host Pathotypes/races Reference
Genetical A. species (4) Crucifers Genetical similarity in Jasalavich et al.
ITS region (1995), Goyal
et al. (2013)
A. brassicae B. juncea Vary pathogenically Sharma et al.
(2013)
A. brassicae B. juncea Isolates genetically Pramila et al.
variable (2014)
A. brassicae Rapeseed– Isolates genetically Kumar et al.
mustard variable (2014)
Molecular A. brassicae Crucifers Polymorphism in Sharma and
isolate by RAPD Tewari (1995,
analysis 1998)
A. brassicicola Crucifers -do- Goyal et al.
(2013)
A. raphani Crucifers -do- Kumar et al.
(2008)
Proteome level A. brassicae Crucifers Variation in protein Sharma et al.
level of virulent and (2012)
avirulent isolates
Thermo-sensitivity A. alternata Crambe A, B, C Czyzewska
(1970)
A. brassicae B. juncea 3 Kumar et al.
(2003a, b)
Fungicidal sensitivity A. brassicae Crucifers A, C, D Vishwanath and
6 Kolte (1997),
Sangwan and
Mehta (2006)
A. brassicae Crucifers 8 Kumar et al.
(2004), Khurana
et al. (2005a, b)

Czyzewska S (1969) Alternaria blight of Crambe abyssi-

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 Fine Structures and Electron
Microscopy 7

7.1 Introduction through a pore, of the inner wall layer of the

mother cell. Young spores have many small mito-
Among the four Alternaria species infecting cru- chondria, and much vesicular endoplasmic reticu-
ciferous hosts, information on fine structures lum, and as they mature, lipid bodies and an
through electron microscopy has been generated unknown polyglucoside are produced. Mature
on A. brassicicola. The sequence of events in the spores have glycogen but very little, if any, lipid.
spore production and maturation has been The vesicular endoplasmic reticulum, multivesic-
described. Changes in the internal organelles of ular bodies and lomasomes are involved in wall
hypha, conidium and conidiophore have been formation. The plasmalemma has rectangular
observed. Conidiophores have a similar structure grooves in its outer surface and corresponding
to mature hyphae, except that after spore produc- ridges on the inner one; both surfaces bear parti-
tion, they have pore in the tip and an annulus. cles of two distinct sizes. The endoplasmic reticu-
There are variations in the number of nuclei in lum may be lamellated or vesicular and is involved
the cells of the vegetative hyphae, conidiophores in wall formation. The vesicles produced by the
and conidia with six chromosomes in dividing endoplasmic reticulum fuse with the plasma-
nuclei of vegetative hyphae (Campbell 1970a, b, lemma. However, dormant spores (Campbell
1972; Knox- Davis 1979). 1970b) have very thick, heavily pigmented, mela-
nized walls with plugged septal pores. The small
amounts of endoplasmic reticulum and the few
7.2 Fine Structures and Electron mitochondria lie near the plasmalemma. The
Microscopy germ-tube walls arise from the inner layers of the
spore walls. Lomasomes and endoplasmic reticu-
The sequence of events in the spore production lum vesicles are probably involved with this wall
and maturation of A. brassicicola was described formation. During germination, mitochondria and
by Campbell (1964, 1969a, b, 1970a, b, 1972). ribosomes increase in numbers, first in the germi-
Each cell of the mature spore of A. brassicicola nating cell and then in the germ tube. As the
has a two-layered wall, the layer distant from the hyphae age, they produce lipid droplets and vacu-
protoplast being melanized. The mature septa are oles, the latter finally fill most of the cell as the
five layered, having two layers of secondary wall cytoplasm degenerates. Conidiophores have
on either side of the septal partition, which itself structure similar to mature hyphae, except that
is three layered. Each septum has one simple after spore production, they have a pore in the tip
pore. New spores are produced by an outgrowth, and an annulus. In an albino strain of A. brassicicola

© Springer Science+Business Media Singapore 2016 163

G.S. Saharan et al., Alternaria Diseases of Crucifers: Biology, Ecology and Disease Management,
DOI 10.1007/978-981-10-0021-8_7
164 7 Fine Structures and Electron Microscopy

Plate 7.1 Conidiophores of Alternaria brassicicola cells; (d) nucleus wedged in pore of terminal of terminal
showing (a) uni- and binucleate cells and apical cell with cell; (e) developing conidia (only one shown) with nucleus
pore and annulus; (b) typical complement of nuclei in wedged in pore between terminal cell of conidiophore and
basal cells, anucleate terminal cells (right and bottom), basal cell of developing conidium; and (f, g) conidia with
pore and annulus (right) and nuclear material and cyto- thick, roughened wall material which stained intensely
plasm wedged in pore in terminal cell (left); (c) typical with Giemsa (Knox-Davies 1979)
complement of nuclei in subtending, basal and terminal
7.2 Fine Structures and Electron Microscopy 165

Plate 7.2 Conidia of Alternaria brassicicola showing pore within a conidium; (d, e) Giemsa-stained material
(a) small, intensely staining nuclei of the terminal cells wedged in the septal pores between adjacent conidia in a
and wide pores of the basal cells; (b) septal pores, typical conidial chain; (f) Giemsa-stained material wedged in the
distribution of nuclei and small, intensely staining nucleus terminal pore of a conidium; and (g) Giemsa-stained
of the terminal cell; (c) Giemsa-stained material wedged material wedged in basal and interstitial pores of a conid-
in septal pores between adjacent conidia and in a septal ium (Knox–Davies 1979)

(Campbell et al. 1968), when compared with the pore in the conidiophore had no annulus
wild type (Campbell 1969a, b, 1970a), there was (Campbell 1970b).
little difference in the structure of the hyphae and Giemsa-stained preparations of A. brassicicola
the conidiophores, except that there were no elec- show variations in the number of nuclei in the cells
tron dense deposits of melanin in the wall and the of the vegetative hyphae from one to many, with
166 7 Fine Structures and Electron Microscopy

the hyphal tip cells having up to 27 nuclei and Campbell R (1969a) An electron microscope study of
spore structure and development in Alternaria bras-
older cells up to 33. There are fewer nuclei (0–3)
sicicola. J Microbiol Genet 54:381–392
in the mature conidiophore cells, and one or two Campbell R (1969b) Further electron microscope studies
nuclei in most cells of the conidia in 3-day-old cul- of the conidium of Alternaria brassicicola. Arch
tures are grown on potato carrot agar. Nuclei are Mikrobiol 69:60–68
Campbell R (1970a) An electron microscope study of
also seen wedged in the connections between
exogenously dormant spores, spore germination,
conidia and in the basal apical pores of conidia hyphae and conidiophores of Alternaria brassicicola.
after separation. They are less frequently wedged New Phytol 69:287–293
in septal pores in the conidia. Six chromosomes Campbell R (1970b) Ultra structure of an albino strain of
Alternaria brassicicola. Trans Br Mycol Soc
are present in dividing nuclei in the vegetative
54:309–313
hyphae (Plates 7.1 and 7.2; Knox-Davies 1979). Campbell R (1972) Changes in volume of reproductive
structures during spore production by Alternaria bras-
sicicola. Trans Br Mycol Soc 59:153–156
Campbell R, Lamer RW, Madelin MF (1968) Notes on an
References albino mutant of Alternaria brassicicola. Mycologia
60:1122–1125
Campbell CJ (1964) Studies on the leaf spot disease of Knox-Davies PS (1979) The nuclei of Alternaria brassici-
Brassica spp. caused by Alternaria brassicicola cola. Trans Br Mycol Soc 72:81–90
(Schw.) Wilts. M.Sc. thesis, University of Exeter, UK
 Biochemistry of Host–Pathogen
Interaction 8

8.1 Introduction Atwal et al. (2004) have determined biochem-

ical changes in relation to Alternaria leaf blight
A number of biochemical changes take place in Indian mustard. Healthy leaves of B. juncea
during the pathogenesis of Alternaria in the host possess significantly higher amount of total solu-
and the pathogen, which produce various kinds ble sugars than those of different infected leaf
of primary and secondary metabolites, which parts at all growth stages. The depletion of sugars
have a great role in host defence and virulence of is highest in lesion areas of infected leaves
pathogen. (necrotic followed by chlorotic and green areas)
as compared to healthy leaves at the initial stages
of infection. However, the accumulation of sug-
8.2 Biochemical Changes ars is noticed in the lesion as well as lesion-free
in the Host areas of the infected leaves with progress of
infection (Table 8.1). Sugar level is higher in
The effect of A. brassicicola infection on the chlorotic and green areas as compared to necrotic
changes of nucleic acids of B. oleracea var. parts of the infected leaves. Both healthy and
botrytis has been studied by Maitra and Samajpati infected leaves have maximum sugar and starch
(1985). An increase in total nucleic acids (97 %), content at 100 DAS, which declines thereafter.
DNA (44 %) and RNA (150 %) in infected leaves Higher levels of total sugars in chlorotic and
is recorded. The increase in RNA content is due green areas of infected leaves suggest that sugars
to an increase of each of the four nucleotides. are probably one of the factors responsible for
However, guanylic acid increases significantly resistance in B. juncea. They may contribute
more than the other three nucleotides. The towards maintaining healthy flora of saprophytic
increase in G/C ratio is also due to an increase in microbes that help to inhibit pathogen growth
guanylic acid. (Juniper and Jeffree 1983). The lower accumula-
Losses of soluble carbohydrates are high from tion in necrotic area can be due to utilization of
dead or damaged seeds; moderate from immature sugars by the fungus for its own growth and
seeds, or seeds which germinate slowly; and low establishment.
from mature seeds with dark, leathery testas, The chlorotic area of infected leaves possesses
which germinate normally. Loss of electrolytes is significantly higher levels of total and orthodihy-
higher in dead, damaged, immature seeds and droxy phenols, as compared to necrotic area,
seeds that germinate slowly than in mature seeds whereas no significant differences are observed
that germinate normally (Knox-Davies 1980). in the green area of infected leaves as compared

© Springer Science+Business Media Singapore 2016 167

G.S. Saharan et al., Alternaria Diseases of Crucifers: Biology, Ecology and Disease Management,
DOI 10.1007/978-981-10-0021-8_8
 168

Table 8.1 Carbohydrates, phenols and chlorophyll content (mg/g dry weight) in leaves of Indian mustard as influenced by Alternaria blight (Atwal et al. 2004)
Total soluble Reducing Total Orthodihydroxy Total
Status of leaves/DAS Starch sugars sugars phenols phenols Flavonols chlorophyll Chlorophyll a Chlorophyll b
Healthy 80 34.2 ± 2.2 72.1 ± 5.2 35.1 ± 3.0 2.6 ± 1.1 0.6 ± 0.3 2.5 ± 0.1 0.27 ± 1.2 0.11 ± 0.01 0.16 ± 2.3
100 59.4 ± 0.9 134.8 ± 3.8 63.3 ± 1.3 4.9 ± 1.4 1.6 ± 0.1 2.3 ± 0.1 16.28 ± 2.0 5.78 ± 0.04 11.04 ± 0.9
120 58.6 ± 0.6 104.7 ± 3.2 56.4 ± 2.6 7.0 ± 0.9 2.3 ± 0.1 1.5 ± 0.1 13.62 ± 1.4 4.69 ± 0.06 8.94 ± 0.6
Necrotic 80 11.2 ± 0.1 19.6 ± 2.1 11.2 ± 0.3 2.2 ± 0.1 0.5 ± 0.2 0.6 ± 0.03 – – –
100 22.3 ± 0.1 36.7 ± 2.0 22.4 ± 0.9 3.0 ± 0.1 1.1 ± 0.1 0.8 ± 0.01 – – –
120 18.4 ± 0.1 29.3 ± 2.4 18.3 ± 1.3 3.5 ± 0.1 2.3 ± 0.1 0.8 ± 0.02 – – –
Chlorotic 80 18.7 ± 1.1 36.7 ± 1.1 20.1 ± 1.7 3.5 ± 2.1 1.7 ± 0.3 1.9 ± 0.13 0.04 ± 0.3 0.01 ± 0.04 0.03 ± 0.04
100 49.4 ± 2.3 78.9 ± 0.9 39.4 ± 2.9 6.7 ± 1.1 2.8 ± 0.2 2.2 ± 0.11 2.67 ± 0.3 1.04 ± 0.03 1.63 ± 0.07
120 32.8 ± 1.1 66.6 ± 2.3 37.2 ± 1.3 8.6 ± 0.9 3.2 ± 0.3 1.5 ± 0.14 0.75 ± 0.7 0.11 ± 0.1 0.64 ± 0.12
8

Green 80 28.6 ± 0.1 48.2 ± 2.3 26.1 ± 4.9 2.5 ± 0.6 1.1 ± 0.4 2.2 ± 0.9 0.25 ± 4.3 0.10 ± 2.3 0.17 ± 0.01
100 53.4 ± 2.6 99.4 ± 2.1 53.6 ± 1.6 4.7 ± 0.4 1.9 ± 0.1 2.0 ± 0.06 6.87 ± 1.9 6.47 ± 0.01 0.40 ± 0.03
120 49.2 ± 0.2 78.3 ± 2.1 42.3 ± 2.0 6.9 ± 0.4 2.1 ± 0.1 1.1 ± 0.40 2.62 ± 1.5 2.14 ± 0.6 0.48 ± 0.04
CD *DAS 2.91 2.81 2.21 0.10 0.20 0.060 0.10 1.21 0.46
H×I
DAS days after sowing * CD (P < 0.05); H-healthy leaves, I-infected leaves
Biochemistry of Host–Pathogen Interaction
8.2 Biochemical Changes in the Host 169

to healthy leaves (Table 8.1). Total and orthodi- ing sugar, chlorophyll content and flavanol con-
hydroxy phenols increase significantly with the tents are more in resistant (B. alba) than in others.
increase in infection, and age of the plant, and are With progress of infection, total phenol,
maximum at 120 DAS in all the cases. The pres- o-dihydroxy phenol and protein content increased
ence of maximum levels of total and orthodihy- in all three genotypes, while the chlorophyll,
droxy phenols in chlorotic area, the region next total sugar, reducing sugar and flavanol content
to the lesion area (necrotic), signifies their pro- decreased (Mathpal et al. 2011).
tective role to restrict the growth, spread of dis- Green islands were observed around infected
ease and invasion of the pathogen by formation spots of A. brassicicola and A. brassicae on mus-
of lignin and lignin-like substances, which are tard leaves by Mandahar and Suri (1983, 1987).
more toxic to fungi. Singh and Singh (1989) also These green islands have higher content of
found higher levels of total and orthodihydroxy cytokinin-like substances, compared to the sur-
phenols in leaves of chilli pepper after inocula- rounding yellowed and healthy tissues. The pres-
tion with cucumber mosaic virus. The content of ence of starch in green islands is correlated with
flavonols is significantly lower in different the formation of metabolic sinks because of their
infected parts of leaves as compared to the higher content of cytokinin-like substances (Suri
healthy leaves, and this decrease is more promi- et al. 1983). Cytokinin-like substances appear to
nent in necrotic area followed by chlorotic and be actively involved in infection and pathogene-
green, indicating less significant role of flavonols sis of A. brassicicola (Suri and Mandahar 1984,
in the defence mechanism as compared to total 1985, 1986). Production of pectolytic and cellu-
and orthodihydroxy phenols. lolytic enzymes by A. brassicae causing leaf spot
The chlorophyll is found to be completely of B. rapa has been observed (Srivastava and
absent in necrotic areas and significantly low in Srivastava 1982; Suri and Mandahar 1982).
chlorotic as compared to green areas of both Alternaria brassicae produces invertase and
infected and healthy leaves. Similar trend is amylase on cabbage decoctions (Weimer 1924),
observed in chlorophyll a and b content. However, while infection in cauliflower leaves brings
the chlorophyll content of the green areas of the changes in polyphenol oxidase and peroxidase
infected leaves is comparable to that of the (Maitra and Samajpati 1982). The presence of
healthy leaves at 80 DAS. The decline in chloro- large number of enzymes, viz. amylase, inver-
phyll content due to various biotic stresses has tase, pectin methyl esterase, phosphorylase,
also been reported in other crops. Maiti et al. aldolase, amidases, ribonuclease, alkaline phos-
(2000) attributed the decline in chlorophyll phorylase, nucleophosphatases, catalase, dehy-
content due to chloroplast structural modifica- drogenases, deaminases, nucleodeaminases,
tion by the fungus such as dilation of the whole glycerophosphatase, cellulose and β-glucosidase,
chloroplast, separation of grana and accumula- in A. alternata, A. brassicicola and A. raphani
tion of starch granules, which have a direct infecting crucifers has been demonstrated
bearing on the photosynthetic capacity of (Dasgupta and Verma 1961, 1962; Verma 1964,
chloroplast. 1971).
Brassica juncea cv. Varuna, B. juncea cv. Alternaria brassicicola stimulates ethylene
PAB-9534 and B. alba showed susceptible, mod- production in closed culture with floating leaf
erately resistant and tolerant disease reactions, discs from cabbage. Production is increased by
respectively, to A. brassicae at different growth pre-culturing the fungus on media containing
stages. In all stages of pathogen infection, dis- cabbage components, but which contains little or
ease severity and characteristic symptoms are no methionine. It suggests that the nature of para-
more prominent in susceptible than the other two sitism of this pathogen on cabbage is character-
genotypes. The biochemical analysis of leaves of ized by a latent capability to cause the production
different mustard varieties revealed that total of the plant-senescing hormone ethylene (Poapst
phenol, o-dihydroxy phenol, total sugar, reduc- et al. 1979).
170 8 Biochemistry of Host–Pathogen Interaction

8.3 Biochemical Changes The conidia of A. brassicicola are remarkably

in the Pathogen resistant to phytoalexins including phaseollins,
phaseollidin and phaseollinisoflavan, often ger-
Alternaria brassicae and A. brassicicola produce minating in assays using 100 μg/ml (Skipp and
antibiotics, which are active against bacteria, Bailey 1977). Similarly, 3-day-old spores were
fungi and algae (Lindenfelser and Ciegler 1969). able to grow in relatively high levels of these
Alternaria brassicicola strain 13 is reported to phytoalexins. Dahiya and Tewari (1991) identi-
form extracellular lipids like phospholipids, fied three plant growth factors from A. brassicae
mono- and diglycerides, sterines, free fatty acids and treatment of seedlings of canola/rapeseed
(predominant) and triglycerides (Aizina 1977). with two of them, N-methyl-2, 5-dimethyl-N-
The biosynthesis of lipids and phospholipids is cinnamoylpiperazine and 3-carboxy-2-
highest on Czapek-Dox medium (Aizina et al. methylene-4-pentenyl-4-butenolide, reduced
1976). their growth, whereas 2-hydroxy-1′-methylzeatin
A melanoid pigment insoluble in strong alkali increased the growth of seedlings.
has been isolated from A. brassicae mycelium.
Melanins are important for survival and longev-
ity of fungal propagules. Abscisic acid (ABA), a 8.4 Glucosinolates
naturally occurring plant growth regulator
involved in the control of various plant processes, Glucosinolates occur as glucosides throughout
also has been characterized in the mycelia of the the Brassicaceae. These are present in the seed
black spot fungus A. brassicae (Dahiya 1988; and other parts of oilseed rape. The hydrolysis
Dahiya et al. 1988). The germinating spores of A. products of glucosinolates include nitriles,
brassicae and A. brassicicola isolated from mus- isothiocyanates, oxazolidinethiones and
tard are able to hydrolyse the various carbohy- thiocyanates, which can be unpalatable and toxic
drates, which are used in germination media. to non-ruminant animals. Thus, the presence of
Alternaria brassicae has a greater ability to glucosinolates in rape meal has limited its use in
hydrolyze pectin and carboxymethyl cellulose animal feed, necessitating the development of the
than A. brassicicola, whereas the reverse is true so-called ‘double-low’ lines. These produce seed
for the hydrolysis of sodium polypectate low in erucic acid and glucosinolates. A decreased
(Mukadam and Deshpande 1979). content of glucosinolates in oilseed rape tissues
A cytokinin, 6-benzyl amino purine, is able to may have negative consequences for pest and
significantly reduce Alternaria blight disease disease incidence on the crop. The hydrolyzed
symptoms and mycelial growth within plant tis- products of glucosinolates are fungitoxic to
sues. This cytokinin is also able to inhibit the rapeseed pathogens in vitro (Mithen et al. 1986),
in vitro growth of Alternaria brassicae (Sharma and there is also much indirect evidence to
et al. 2010). suggest that their presence contributes to
The composition of amino acids extracted resistance to a range of oilseed rape pathogens
from A. brassicicola and A. brassicae is only (Greenhalgh and Mitchell 1976; Mithen et al.
slightly different from those of A. raphani. This 1987; Rawlinson et al. 1985). Following
similarity of amino acids in the first two-pathogen inoculation with A. brassicae, Doughty et al.
extracts is correlated with the severe to moderate (1991) examined the changes in the glucosinolate
susceptibility of the varieties of crucifers, and the content of leaves in cultivars Bienvenu (single
difference of amino acids contained in A. raphani low) and Cobra (double low) and found that
extract may have accounted for the severe sus- glucosinolate contents increased markedly, but
ceptibility only on radish. One amino acid of Rf the response depended on leaf age and cultivar.
0.66 of A. raphani extract corresponds to that of Glucosinolates accumulated more in the sixth
the radish extract at about the same level leaf of Bienvenu. When B. rapa seedlings are
(Changsri 1961). inoculated with A. brassicae, 3-butenyl and
References 171

4-pentenyl isothiocyanates are reduced, together biotic complex brassicicolin (Ciegler and
with dimethyl disulphide, dimethyl trisulphide, Lindenfelser 1969) and phytotoxic brassicicenes
4-oxoisophoron and a number of sesquiterpenes. (Mac Kinnon et al. 1999). Depudecin is C11 com-
The release of isothiocyanates is an evidence for pound containing two epoxide groups and shows
the catabolism of glucosinolates during infection, antitumor activity (Matsumoto et al. 1992).
which is a prerequisite for their involvement in Phytotoxic brassicicenes, fusicoccane-like diter-
resistance (Doughty et al. 1996). penoids, have been isolated from the culture fil-
trate of canola pathogen A. brassicicola (Mac
Kinnon et al. 1999).
8.5 Metabolites Produced The fungal strain Sw-3 showing antimicrobial
activity and isolated from soil of a Chinese cab-
Alternaria brassicae when grown in liquid still bage patch in Korea was identified as A. brassici-
culture produces host-specific phytotoxins, which cola by its morphological characteristics and
have been shown to be cyclodepsipeptides. It also confirmed by the analysis of the 18S gene and
produces several other metabolites including the ITS region of rDNA. The fungus showed a simi-
new drimane sesquiterpenes deoxyuvidin B, larity of 99 % with A. brassicicola in the 18S
albrassitriol and isoalbrassitriol, as well as bras- rDNA sequence analysis. Alternaria brassicicola
sicadiol, a C15 prenylated pentaketide. These com- has been reported to produce an antitumor com-
pounds show no phytotoxicity on canola (Ayer pound called depudecin. Strain SW-3 produces
et al. 1987; Ayer and Pena-Rodriguez 1987a, b). antimicrobial metabolites, in addition to depude-
The earliest reports on phytotoxin production by cin, during sporulation under different growth
A. brassicae or A. raphani are those of Degenhardt conditions. The metabolite of the isolated fungus
(1973, 1978) and Husain and Thakur (1966). has strong antifungal activity against
Cytotoxicity tests with extracts of culture filtrates Microsporum canis and Trichophyton rubrum
of A. brassicae and A. brassicicola show that both and antibacterial activity against Staphylococcus
species produce compounds, which are toxic to aureus and Pseudomonas aeruginosa. The
human epithelial (HEP2) cells, but there is no amount and kind of metabolites produced by the
clear relationship between Alternaria infection of isolates are affected by growth conditions such as
seed and its cytotoxicity. Metabolites produced by nutrients and growth period (Jung et al. 2002).
the two fungi are also phytotoxic, giving rise to Metabolites and phytotoxins produced by
necrosis of leaf discs and inhibition of seedling Alternaria species infecting crucifers have been
root growth, but extracts of the less specialized discussed in greater detail in Chap. 10.
parasite, A. alternata, cause more severe phyto-
toxic effects on crucifer test plants. Extracts of
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J Sci Technol 4:144–145 Leptosphaeria maculans. Trans Br Mycol Soc
Jung DS, Na YJ, Ryu KH (2002) Phylogenic analysis of 88:525–531
Alternaria brassicicola producing bioactive metabo- Mukadam DS, Deshpande KB (1979) Role of light and
lites. J Microbiol 40:289–294 temperature on growth, sporulation and subsequent
Juniper BE, Jefree CE (1983) Plant surfaces. Edward spore germinability of Alternaria brassicae (Berk.)
Arnold, London Sacc. Sci Cult 45:244–246
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Poapst PA, Ramsoomair BA, Gaurley CO (1979) On the Suri RA, Mandahar CL (1984) Secretion of cytokinins in
promotion of senescence in Brassica oleracea var. vivo and in vitro by Alternaria brassicicola and their
Capitata by Alternaria brassicicola and by Botrytis role in pathogenesis. Experientia Basel Birkhauser
cinerea. Can J Bot 57:2378–2386 40:462–463
Rawlinson CJ, Muthyalu G, Poole VA, Cayley GR, Hulme Suri RA, Mandahar CL (1985) Involvement of
PJ, Pickett JA (1985) Mustard oils, fungicides and dis- cytokinin-like substances in the pathogenesis of
ease. Rothamsted Exp Station Rep 1984:124–125 Alternaria brassicae (Berk.) Sacc. Plant Sci
Sharma N, Rahman MH, Liang Y, Kav NNV (2010) 41:105–109
Cytokinin inhibits the growth of Leptosphaeria macu- Suri RA, Mandahar CL (1986) Cytokinin-like substances
lans and Alternaria brassicae. Can J Plant Pathol and accumulation of labelled metabolites at infection
32:306–314 sites of Alternaria brassicicola. Mycopathologia
Singh MJ, Singh J (1989) Mechanisms of resistance to 94:153–156
cucumber mosaic virus in chilli pepper (Capsicum Suri RA, Mandahar CL, Gill PS (1983) Study of secretion
annuum L.). In: Role of phenols and phenologies. of cytokinins by Alternaria brassicicola and their pos-
Eucarpia VII meeting on genetics and breeding on sible role in pathogenesis in mustard. Indian J Plant
capsicum and eggplant, Yugoslavia, pp 193–203 Pathol 1:117–121
Skipp RA, Bailey JA (1977) The fungitoxicity of isoflava- Verma JP (1964) Studies in the enzyme make-up of
noid phytoalexins measured using different types of Alternaria. V. Aldolase activity. Mycologia
bioassay. Physiol Plant Pathol 11:101–112 56:909–918
Srivastava SK, Srivastava AK (1982) Polygalacturonase Verma JP (1971) Studies in the enzyme make-up of
and cellulase production by Alternaria brassicae Alternaria. IX. Nucleao-phosphatase activity. Indian
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Suri RA, Mandahar CL (1982) Production of pectolytic Weimer JL (1924) Alternaria leaf spot and brown rot of
enzymes by Alternaria brassicae. Indian J Mycol cauliflower. J Agric Res 29:421–441
Plant Pathol 12:351–352
 Resistance
9

has also revealed the sequestration of calcium

9.1 Introduction during pathogenesis by A. brassicae (Saharan
1992, 1997; Saharan et al. 2003, 2005).
Attempts have been made to transfer resistance Genetic amelioration for disease resistance is
from wild Cruciferae to cultivated Brassica. considered to be the most effective, stable and
While transferring the resistance into cultivated economical method of protecting crops for
Brassica, the knowledge of pathogenic variabil- ensuring their full productivity. But before the
ity, sources of resistance and methods to deter- research on breeding for disease resistance is
mine nature, mechanism and stability of initiated, the researchers must fully understand
resistance is essential. Various techniques to regarding the method and rate of growth and
incorporate the resistance in the cultivated multiplication and transmission of the patho-
Brassica as well as to evaluate the resistance in gen. In nature, plants employ a wide range of
the field, in screen house and in the laboratory mechanisms to restrict the growth of the patho-
conditions have been developed. The patho- gen. First mechanism operates before the para-
genic variability, especially in A. brassicae, has sitic contact, while the other mechanisms
been identified and requires special attention. operate after the pathogen has made contact
The mechanisms of resistance in rapeseed and with the host. Selection for resistance implies
mustard due to epicuticular wax, as well as for measurement of plant’s resistance which can be
pre-existing rapid accumulation of biochemical done by measuring the growth and development
components, have been studied. The role of of the pathogen; the higher the reduction, the
phytoalexins in the resistance has been con- more resistant the host plant. This is generally
firmed. The importance of phytotoxic com- not possible because in most cases, the patho-
pounds including destruxin B, destruxin B2, gen is either invisible or partially visible.
homodestruxin B and desmethyldestruxin B in However, one can evaluate the pathogen’s
the pathogenesis of A. brassicae has been deter- effects on the host in the form of symptoms. The
mined. The fact that the resistant Brassica vari- information generated so far regarding the exis-
eties produce other metabolites including tence of many components, its multilayered
sesquiterpenes deoxyuvidin, β-albrassitriol, iso- nature and the mechanism of resistance in the
albrassitriol and brassicadiol has also been con- Alternaria–Brassica pathosystem are presented
firmed. Scanning electron microscopy (SEM) in this chapter.

© Springer Science+Business Media Singapore 2016 175

G.S. Saharan et al., Alternaria Diseases of Crucifers: Biology, Ecology and Disease Management,
DOI 10.1007/978-981-10-0021-8_9
176 9 Resistance

9.2 Genetics of Host–Parasite bility to A. brassicae than their respective F1s.

Interaction Performance of backcross progenies tends to
deviate towards their respective recurrent par-
In Brassica juncea cv. RC781, resistance to A. ents. The X2 values in joint scaling test were
brassicae is reported to be governed by a single highly significant in all the crosses indicating
dominant gene (Tripathi et al. 1980). However, simple additive dominance model to be inade-
Brun et al. (1987a, b, 1989) reported a high quate for all the crosses. The six-parameter model
degree of resistance in Sinapis alba cv. Emergo revealed significant additive gene effects in all
against A. brassicae. Intraspecific crosses six crosses, whereas dominance gene effects only
between B. napus and B. juncea have higher con- in the three crosses, viz. Varuna × EC-322092,
tents of epicuticular wax (Singh et al. 1999). RH 30 × EC-322092 and Varuna × PCC-2.
Saharan and Kadian (1983) analysed different However, there was preponderance of additive
components of horizontal resistance (HR) in gene than dominance gene effects in all the six
Brassica genotypes against A. brassicae and crosses. Non-allelic interaction and addi-
found large differences in the number of lesions, tive × additive (i) were significant in all the
size of lesions, latent period, sporulation capabil- crosses except Pusa Basant × EC-322092 and
ity and infection rate in B. napus cv. Tower and in Kranti × EC-322092, whereas additive × domi-
B. juncea cv. RC-781. In other B. juncea geno- nance epistasis was significant only in Pusa
types like CSR I42 and CSR 448, moderate level Basant × EC-322092, Kranti × EC-322092 and
of HR has been recorded. In Tower, infection is RH 30 × HC-1 crosses. The crosses
delayed up to 25 days with very few lesions Kranti × EC-322092, Varuna × EC-322092 and
(0.95) of restricted size. Both Tower and RC-781 RH 30 × EC-322093 exhibited significant domi-
cultivars have longer latent periods of 18 and nance × dominance (I) type epistasis. Based upon
12 days, respectively, compared to 3 days in cv. these results, it is advocated that such breeding
Prakash. The amount of conidial production in methods should be employed which exploit both
Prakash was higher (265 conidia per lesion) than kinds of gene effects. Therefore, reciprocal recur-
in Tower (92 conidia per lesion) (Saharan 1991, rent selection or diallel selective matings can be
1992). Kolte (1987) suggested that the size of successfully used to develop genotypes resistant
lesions and amount of sporulation may also be to Alternaria blight (Tables 9.1, 9.2 and 9.3)
considered important criteria in determining the (Saharan and Krishania 2001).
degree of resistance against Alternaria blight in The per cent disease intensity (PDI) of
Brassica species. Alternaria blight at leaf stage is less in the prog-
enies of R × R cross families as compared to R
self and R open families in each cross indicating
9.2.1 Inheritance of Resistance that inter-mating between resistant plants helps
in increasing the level of resistance. A similar
Genetics of Alternaria blight resistance in inter- improvement has also been reported for multiple
and intraspecific crosses of B. juncea and B. cari- disease resistance in other crops where the fre-
nata have been determined by Krishnia et al. quency of favourable genes increases in the pop-
(2000). Population of six generations, viz. P1, P2, ulation enhancing the probability of obtaining
F1, F2, BC1 and BC2, of six crosses, viz. Pusa multiple disease resistance (Saharan and
Basant × EC-322092, Kranti × EC-322092, Krishania 2001). The utility of biparental tech-
Varuna × EC-322092, RH 30 × EC-322093, RH niques can be more pronounced if the additive or
30 × HC-1 and Varuna × PCC-2, were evaluated additive × additive types of genetic variations are
for the inheritance of resistance to A. brassicae. coupled with repulsion phase linkage between
The F1 hybrids showed intermediate disease genes (Singh et al. 1986). The selection of resis-
intensity compared to their parents. The F2 popu- tance to Alternaria blight in the inter-mated pop-
lations of all six crosses exhibited more suscepti- ulations rather than in F2 and F3 populations had
9.2 Genetics of Host–Parasite Interaction 177

Table 9.1 Per cent disease intensity (PDI) of Alternaria leaf blight (A. brassicae) on different generations of Brassica
crosses (Krishnia et al. 2000; Saharan and Krishania 2001)
Cross P1 P2 F1 F2 BC1 BC2
B. juncea × B. juncea
Pusa 43.42 5.75 23.55 26.65 34.35 17.52
Basant × EC-322092 (41.21) (13.81) (29.06) (31.05) (35.91) (24.73)
±0.314 ±0.184 ±0.132 ±0.088 ±0.372 ±0.190
Kranti × EC-322902 36.35 4.54 19.68 22.85 30.15 15.12
(37.11) (12.11) (26.35) (28.52) (33.27) (22.87)
±0.290 ±0.121 ±0.143 ±0.128 ±0.218 ±0.160
Varuna × EC-322092 46.95 6.32 28.35 33.33 38.85 16.98
(43.72) (14.54) (32.20) (35.24) (38.59) (24.27)
±0.234 ±0.211 ±0.122 ±0.086 ±0.226 ±0.134
RH 30 × EC-322093 38.25 5.58 20.75 22.26 34.12 16.45
(38.23) (13.69) (27.06) (28.39) (35.73) (24.04)
±0.340 ±0.290 ±0.208 ±0.090 ±0.386 ±0.342
B. juncea × B. carinata
RH 30 × HC-1 41.41 6.58 27.35 30.45 36.35 20.75
(40.05) (15.00) (31.56) (33.52) (37.11) (27.06)
±0.284 ±0.202 ±0.192 ±0.100 ±0.252 ±0.222
Varuna × PCC-1 45.52 7.42 31.12 34.88 40.92 19.76
(42.48) (15.79) (33.90) (36.15) (39.76) (26.42)
±0.384 ±0.282 ±0.204 ±0.114 ±0.306 ±0.238
Figures in parentheses are angular transformed values

Table 9.2 Estimates of components of generation means on three-parameter model for A. brassicae on different
crosses of oilseed Brassica (Krishnia et al. 2000)
Cross m d h X2
B. juncea × B. juncea
Pusa Basant × EC-322092 28.79 13.42** 1.81** 71.93**
±0.31 ±0.32 ±0.50
Kranti × EC-322902 25.68 12.39** 2.53** 86.42**
±0.28 ±0.28 ±0.49
Varuna × EC-322092 30.60 14.68** 3.21** 154.46**
±0.28 ±0.29 ±0.48
RH 30 × EC-322093 27.19 12.21** 1.51* 64.35**
±0.34 ±0.36 ±1.51
B. juncea × B. carinata
RH 30 × HC-1 28.99 12.21** 5.04** 105.87**
±0.31 ±0.31 ±0.55
Varuna × PCC-1 30.87 13.57** 5.16** 105.26**
±0.35 ±0.36 ±0.61
*,**Significant at P = 0.05 and P = 0.01 level, respectively
m general mean, d sum of additive effects of genes, h sum of dominance effects of genes

been advocated by Singh and Singh (1989), since Robinson 1952; Matzinger and Cockerham 1963;
the generations of inter-varietal hybrids would Gates et al. 1957).
obviate the harmful effects of linkages and link- The reduction in Alternaria blight at leaf stage
age disequilibrium and shuffle the desirable is less in four crosses, viz. PCR 3 × Shiva, Pusa
genes in one recombinant (Comstock and Basant × Shiva, Rajat × Shiva and Pusa
178 9 Resistance

Table 9.3 Estimates of gene effects under six-generation mean analysis for A. brassicae in different crosses of oilseed
Brassica (Krishnia et al. 2000; Saharan and Krishania 2001)
Cross m d h i j k
B. juncea × B. juncea
Pusa 31.05 11.18** −1.37 −2.92 −2.52** −5.22
Basant × EC-322092 ±0.30 ±0.75 ±1.97 ±1.91 ±0.83 ±3.38
Kranti × EC-322902 28.52 10.40** −0.06 −1.80 −2.10** −8.56**
±0.36 ±0.61 ±1.95 ±1.69 ±0.69 ±3.01
Varuna × EC-322092 35.24 14.32** −12.17** −15.24** −0.27 12.18**
±0.29 ±0.60 ±1.75 ±1.68 ±0.69 ±2.84
RH 30 × EC-322093 28.39 11.69** 7.08** 5.98** −0.58 −19.48**
±0.30 ±0.85 ±2.17 ±2.09 ±0.94 ±3.81
B. juncea × B. carinata
RH 30 × HC-1 33.52 10.05** 1.71 −5.74** −2.48** −4.43
±0.32 ±0.69 ±1.95 ±1.87 ±0.77 ±3.23
Varuna × PCC-1 36.15 13.34** −7.48** −12.24** −.0.01 5.95
±0.34 ±0.74 ±2.09 ±2.00 ±0.84 ±3.47
*Significant at P = 0.05, and **Significant at P = 0.01 level, m general mean, d sum of additive effects of genes, h sum
of dominance effects of genes, i sum of additive × additive epistatic effects of genes, j sum of additive × dominance
epistatic effects of genes, k sum of dominance × dominance epistatic effects of genes

Bahar × Domo, as compared to others indicating progenies. Almost a similar trend in all the
that the parents involved in these crosses have crosses for Alternaria blight PDI at siliquae
similar degree of susceptibility; only slight phase indicates a high-order correlation in
improvement is made through selection in segre- Alternaria blight resistance at both leaf and sili-
gating generations. In general, the progenies of quae phases (Saharan and Krishania 2001).
R × S families record greater variation in disease
score. The numerical reduction in Alternaria
blight (leaf phase) disease is observed in the 9.2.2 Disease Tolerance
progenies of S × S families in comparison to
S-self plant progenies in each cross indicating Disease stress tolerance index (DSTI) was found
that even susceptible plants possess resistant an effective selection criterion for assessing the
genes at different loci with minor effects. Indian mustard genotypes for their disease stress
The significant ‘j’-type epistasis is observed in tolerance and yield potential. The genotypes
crosses Pusa Basant × EC-322092, Rajat, Kranti and RH-781, under normal sown,
Kranti × EC-322092 and RH 30 × HC-1, whereas and Rajat, RL-1359 and Kranti, under late sown
‘I’-type epistasis is significant in crosses conditions, performed with uniform superiority
Kranti × EC-322092, Varuna × EC-322092 and under both non-disease stress and disease stress
RH 30 × EC-322093. The selection of resistant environments. Potential yield under controlled
plants should be, therefore, done in advanced environment (Yp) was significantly and posi-
segregating generation rather than F2. Therefore, tively correlated with yield under disease stress
the crosses possessing predominantly significant environment (Ys). Potential yield, mean produc-
additive gene effect with additive × additive kinds tivity (MP), disease tolerance (TOL), geometric
of epistasis must be exploited to improve through mean productivity (GMP) and disease stress tol-
simple selections. The highest GCV, PCV, h2 and erance index (DSTI) under normal sown and
GG are recorded in progenies of R × S cross fami- non-disease stress conditions (Yp) had signifi-
lies in most of these crosses. The values of cant positive association with yield under disease
heritability and genetic gain are low in suscepti- stress environment (Ys), under late sown condi-
ble plant progenies as compared to resistant tions (Gupta et al. 2002) (Tables 9.4 and 9.5).
9.2 Genetics of Host–Parasite Interaction 179

Table 9.4 Estimates of disease stress tolerance attributes from the potential yield and yield under disease stress envi-
ronment (DSI = 0.195) in Indian mustard (B. juncea) under normal date of sowing (Gupta et al. 2002)
Genotypes Yp Ys MP TOL DSSI GMP DSTI
EC-129126-1 2508 2036 2272 472 0.96 2260 0.87
RH-781 2635 2063 2349 572 1.11 2332 0.93
PHR-1 2034 1753 1894 281 0.71 1888 0.61
Varuna 2586 2087 2337 499 0.99 2323 0.92
Kranti 2738 2217 2478 521 0.98 2464 1.04
RH 8113 2318 1886 2102 432 0.96 2091 0.75
RL 1359 2680 2025 2353 655 1.25 2330 0.93
Rajat 2807 2234 2524 573 1.05 2504 1.07
RH 30 2769 1897 2333 872 1.61 2292 0.90
Pusa Bold 2463 1856 2160 607 1.26 2138 0.78
PR-8805 2107 1826 1967 281 0.68 1961 0.66
RC-781 1838 1781 1810 57 0.16 1757 0.56
ZEM-1 1973 1564 1769 409 1.06 1757 0.53
Shiva 2413 2027 2220 386 0.82 2212 0.84
Mean 2419 1947 2183 473 1.00 2169 1.00
CD (P = 0.05) 183.07 106.87 139.06 111.92 0.19 136.04 0.09
Significant at P = 0.05; Yp yield under disease-controlled environment (kg/ha), Ys yield under artificially pathogen-
inoculated environment, DSI disease stress intensity, MP mean productivity, TOL disease tolerance, DSSI disease stress
susceptibility index, GMP geometric mean productivity, DSTI disease stress tolerance index

Table 9.5 Estimates of disease stress tolerance attributes from the potential yield and yield under disease stress envi-
ronment (DSI = 0.209) in Indian mustard (B. juncea) under late date of sowing (Gupta et al. 2002)
Genotypes Yp Ys MP TOL DSSI GMP DSTI
EC-129126-1 1691 1149 1420 542 1.53 1393 0.70
RH-781 1656 1281 1469 375 1.08 1456 0.76
PHR-1 1576 1294 1435 282 0.86 1428 0.73
Varuna 1865 1483 1674 382 0.98 1663 0.99
Kranti 1768 1463 1616 305 0.83 1608 0.93
RH 8113 1625 1493 1559 132 0.39 1557 0.87
RL 1359 1865 1481 1673 384 0.98 1662 0.99
Rajat 1849 1160 1727 244 0.63 1723 1.07
RH 30 1796 1452 1624 344 0.92 1615 0.94
Pusa Bold 1699 1358 1529 341 0.96 1519 0.83
PR-8805 1598 1005 1302 593 1.77 1267 0.58
RC-781 1575 1201 1388 374 1.14 1375 0.68
ZEM-1 1440 1125 1283 315 1.05 1272 0.58
Shiva 1357 1094 1226 263 0.93 1218 0.53
Mean 1669 1320 1494 348 1.00 1482 0.80
CD (P = 0.05) 88.97 105.34 91.62 66.40 0.19 93.72 0.10
Significant at P = 0.05, Yp yield under disease-controlled environment (kg/ha), Ys yield under artificially pathogen-
inoculated environment, DSI disease stress intensity, MP mean productivity, TOL disease tolerance, DSSI disease stress
susceptibility index, GMP geometric mean productivity, DSTI disease stress tolerance index
180 9 Resistance

9.2.3 Components of Horizontal 9.3 Morphological Resistance

Resistance
The variations in the number and size of sto-
While analysing different components of hori- mata of resistant and susceptible cultivars have
zontal resistance (HR) in Brassica genotypes been recorded (Saharan and Kadian 1983);
against A. brassicae, Saharan and Kadian numbers of stomata were maximum in suscep-
(1983) reported large differences in the number tible cultivar Prakash and minimum in resistant
and size of lesions, latent period, rate of sporu- cultivar Tower. Stomatal aperture was also nar-
lation and infection rate in B. napus cv. Tower rower in resistant cultivars Tower and RC-781
and B. juncea cv. RC-781; B. juncea genotypes compared to susceptible cultivars Prakash,
CSR 142 and CSR 448 showed moderate level RH-30 and YRT-3 (Table 9.8.). Significantly
of HR. the lower number of stomata per unit area and
The latent periods of Tower and RC-781 the smaller aperture of the stomata are impor-
were 18 and 22 days, respectively, compared to tant morphological resistance factors in reduc-
only 3 days in Prakash. The amount of conidial ing infection of Brassica genotypes by A.
production was also considerably higher in brassicae.
Prakash (265 conidia per lesion) compared to
Tower (92 conidia per lesion). Kolte (1987)
suggested that the size of lesions and amount 9.4 Epicuticular Wax
of sporulation are also important factors in
evaluating resistance against A. brassicae. In The structure of wax in Brassica spp. and its
genotypes PR-8988 and PR-9024, A. brassicae role in host resistance has been studied by a
produced significantly reduced number of number of workers (Conn and Tewari 1989a, b;
spots (4.36–15.89), smaller size of spots (2.12– Holloway et al. 1977; Saharan 1992; Skoropad
6.17 mm), lower sporulation (0.30– and Tewari 1977; Tewari 1991a; Tewari and
1.84 × 103conidia), lower disease index Skoropad 1976). In the canola-type cultivars
(36.51–42.2 %), reduced apparent infection Candle, Tobin and Altex, the wax is organized
rate (r = 0.047–0.080) and lesser values of into an amorphous layer on which is situated a
AUDPC (45.35–126.70) on leaf and pod, along crystalline layer. The crystalline layer consists
with reduced leaf defoliation (38.40–44.40 %) of platelike crystals and a layer of erect fila-
in comparison to on national susceptible geno- mentous and rod-like crystals (Plates 9.1, 9.2
type Varuna (Tables 9.6 and 9.7). Therefore, and 9.3; Conn and Tewari 1985, 1989a, b). The
these genotypes showed higher degree of par- wax in Brassica spp. is complex both structur-
tial resistance or slow blighting. The develop- ally and chemically (Conn 1986; Conn et al.
ment and progression of Alternaria blight was 1984; Holloway et al. 1977). In the canola-type
slower in these two genotypes in comparison to cultivars, the wax has the same nine major con-
the others. The yield (q ha−1) of the genotype stituents (alkanes, esters, ketones, aldehydes,
Kranti was highest (14.01) followed by geno- secondary alcohols, ketols, primary alcohols,
type PR-9024 (11.14); yield day−1 was highest triterpenols and fatty acids) but in varying pro-
in genotype Divya (11.12 kg ha−1 day−1) in portions (Conn 1986; Conn et al. 1984; Tewari
comparison to the others (Kumar and Kolte 1991a, b). In the B. napus ssp. oleifera cv.
2001; Kumar and Singh 2006). Genotypes Altex, the major constituents of wax include
PR-8988 and PR- 9024 exhibit slow blighting C29 alkanes, C29 ketone, C29 secondary alcohol
and have the lowest number of lesions/10 cm2 and C40–C48 esters (Conn 1986; Tewari 1991a,
leaf area, reduced spot size, least number of b).
conidia per spot, and the lowest disease index In providing resistance to the host against
on leaf and pod (Kumar and Kolte 2006). Alternaria spp., wax appears to present only a
 9.4 Epicuticular Wax

Table 9.6 Components of Alternaria blight disease resistance and yield of mustard (B. juncea) genotypes (Kumar and Kolte 2001)
Sporulation/spots
No. of spots per (no. of Apparent infection Leaf Yield
10 cm2 Size of spot (mm) conidia × 103) Disease index (%) rate (r) AUDPC (mm2) defoliation Yield (kg ha−1
Genotypes Leaf Pod Leaf Pod Leaf Pod Leaf Pod Leaf Pod Leaf Pod (%) (q/ha) day−1)
Kranti 4.48 17.78 6.25 2.38 2.14 0.33 46.4 38.7 0.054 0.065 124.8 52.5 38.8 14.01 10.51
Varuna 5.10 25.50 7.71 5.76 3.04 0.63 53.9 52.5 0.063 0.115 153.0 58.5 57.6 10.05 8.21
PR-9650 5.56 23.47 7.74 3.27 3.48 0.63 56.1 53.8 0.065 0.140 155.5 65.0 64.8 7.73 6.64
Krishna 4.75 19.56 6.93 2.46 2.74 0.40 48.2 47.0 0.055 0.090 139.1 55.6 50.6 10.18 8.39
PR-8988 4.42 15.89 6.11 2.12 1.94 0.30 44.4 36.6 0.047 0.075 122.3 45.3 44.4 10.07 8.47
PR-9024 4.36 14.38 6.17 2.13 1.81 0.36 45.2 39.2 0.057 0.080 126.7 45.7 38.4 11.14 8.70
PR-8943 4.60 17.82 6.34 2.38 2.40 0.33 46.7 45.0 0.056 0.090 128.4 52.7 40.0 10.78 8.32
Divya 4.50 21.13 6.60 2.70 2.59 0.36 47.3 49.0 0.059 0.100 131.1 60.1 55.3 10.12 11.12
PR-9301 5.97 29.91 8.58 3.24 3.75 0.66 59.5 57.0 0.073 0.140 172.7 70.5 68.6 7.70 6.72
CD at 5 % 0.284 1.685 0.506 0.223 0.404 0.001 2.934 1.839 NS NS NS NS 3.354 1.710 1.270
181
182 9 Resistance

Table 9.7 Correlation coefficients (R) among different components of Alternaria blight disease resistance and yield of
mustard (B. juncea) genotypes (Kumar and Kolte 2001)
Disease components Disease Leaf Infection
and yield Size of spot index defoliation Sporulation AUDPC rate Yield
No. of spots 0.883** 0.897** 0.812** 0.923** 0.956** 0.864** −0.788*
Size of spots – 0.985** 0.934** 0.982** 0.956** 0.893** −0.668*
Disease index – – 0.905** 0.974** 0.949** 0.876** −0.684*
Leaf defoliation – – – 0.952** 0.884** 0.743* −0.625 NS
Sporulation – – – – 0.973** 0.851** −0.679*
AUDPC – – – – – 0.903** −0.790*
Infection rate – – – – – – −0.770*
*Significant at 5 %; **significant at 1 %; NS non-significant

Table 9.8 Factors influencing resistance/susceptibility of different cultivars of rapeseed–mustard against Alternaria
brassicae (Saharan and Kadian 1983)
Incubation Latent period
Leaf Stomata1 Lesions/leaflet2 period (days)4 (days)4
Cultivars surface No. (per sq. cm) Size (mm) No. Size (mm) Sporulation3
Tower Upper 145 15.7 1.8 1.9 80 10 16
Lower 210 25.9
RC-781 Upper 166 17.3 3.3 5.7 92 8 12
Lower 245 26.7
CSR-448 Upper 196 19.2 3.7 10.7 120 7 10
Lower 303 27.9
CSR-741 Upper 189 19.3 6.3 14.6 125 7 9
Lower 292 20.0
CSR-142 Upper 199 19.3 4.4 10.6 120 8 11
Lower 306 28.0
YRT-3 Upper 225 22.0 10.2 22.3 250 6 9
Lower 403 30.8
RH-30 Upper 221 22.0 8.1 19.1 240 6 9
Lower 392 30.6
Prakash Upper 229 22.7 8.7 18.9 260 6 8
Lower 413 31.2
1
and 3 average of 100 observations; 2 and 4 average of 10 leaves

physical barrier without a direct chemical above two effects by impeding the movement
effect (Conn 1986; Conn and Tewari 1989a, b; of plant exudates. Plants of B. napus ssp. oleif-
Skoropad and Tewari 1977; Tewari and era are very waxy compared to those of B. rapa
Skoropad 1976). The wax forms a hydrophobic ssp. oleifera which is more susceptible to A.
coating and reduces the deposition of water- brassicae. The leaves of cultivars Midas and
borne inoculum. Wax also reduces the rate of Tower (B. napus), resistant to Alternaria, have
conidial germination and the number of germ appreciable amounts of epicuticular wax
tubes produced by each conidium. The crystal- (Skoropad and Tewari 1977). According to
line wax layer is made fluffy by enclosed air Gupta et al. (1987a, b, c), Alternaria-resistant
pockets, which may be responsible for the genotypes, viz. Tower, HNS-3 (B. napus),
9.4 Epicuticular Wax 183

Plate 9.1 Scanning electron micrograph of air-dried, osmium vapour-fixed and gold-coated middle leaves of Brassica
rapa cv. Candle showing wax crystals. Bar = 2 μm (Tewari 1991a, b)

Plate 9.2 Scanning electron micrograph of air-dried, osmium vapour-fixed and gold-coated stem of Brassica rapa cv.
Tobin showing flat and erect wax crystals. Bar = 1 μm (Tewari 1991a, b)

HC-2 (B. carinata) and B. alba, have higher number (n) = 9, long columns (LC), short col-
amounts of wax on their leaves at different umns (SC) and netted columns (NC). The B.
growth stages as compared to susceptible gen- napus and B. carinata seemed to inherit the
otypes, BSH-1, YSPb-24 (B. rapa) and RH-30 NC type of wax present in B. oleracea. Singh
(B. juncea). Gomez-Campo and Prakash (1999) et al. (1999) observed that intraspecific crosses
identified three different epicuticular wax col- between B. napus and B juncea had high con-
umns in Brassica species with a chromosome tent of epicuticular wax.
184 9 Resistance

Plate 9.3 (a) Adaxial surface of an upper leaf of Brassica in wax crystals; (e) adaxial surface of an upper leaf cv.
napus cv. Altex showing platelike wax crystals (arrows); Tobin showing filamentous wax crystals (arrows); and (f)
(b) stem surface of cultivar Altex from the middle of a adaxial surface of a middle leaf cv. Tobin showing a
plant showing rods (arrows); (c) adaxial surface of an branched filamentous wax crystal (arrow). The plant sur-
upper leaf of B. rapa cv. Tobin showing a fused rod faces depicted in a-f were prepared for SEM by air-drying
(arrow); (d) adaxial surface of an upper leaf of B. napus method. Bar = 1 μm (Conn and Tewari 1989a, b)
cv. Westar showing fused rods (arrows) and growth rings
9.6 Proteome-Level Resistance 185

9.5 Biochemical Basis Adjuction of A. brassicae culture filtrate into

of Resistance subcultures of hypocotyl explants callus on MS
medium supplemented with 1 mg l−1 NAA and
The Alternaria-resistant B. juncea genotype 1 mg l−1 BAP (MS N1B1), causing greater accu-
RC-781 is characterized by relatively high con- mulation of total soluble and reducing sugars in
centrations of phenols and low concentrations of susceptible B. juncea cv. Kranti than in resistant
sugars and nitrogen compared to a susceptible B. napus cv. GSH-1 and B. juncea cv. RH-781.
genotype Prakash (Gupta et al. 1984). The leaf Phenolic content and flavonols were higher in
surface constituents including wax, total phenols, GSH-1 and RH-781 calli than that of cultivar
soluble nitrogen, total soluble and reducing sug- Kranti. Protein content, though increased in all
ars of susceptible genotypes BSH-1, YSPB-24 three cultivars, was more in resistant than suscep-
(B. rapa) and RH-30 (B. juncea) and resistant tible cultivars (Kiran et al. 2003). Destruxin B
genotypes Tower, HNS-3 (B. napus), AC-2 (B. detoxification of white mustard (Sinapis alba)
carinata) and local (B. alba) have been deter- through biotransformation can help discover the
mined by Gupta et al. (1987a, b, c), at 30, 50, 70 specific disease resistance enzyme(s)/gene(s). If
and 90 days of plant growth. The concentration the black spot disease resistance of white mus-
of the phenolic compounds is significantly higher tard is related with the presence of specific detox-
in resistant compared to susceptible species at all ifying enzyme(s), this trait can be transferred to
plant growth stages. Total soluble sugars, reduc- susceptible brassicas and may improve their
ing sugars and soluble nitrogen levels are, how- resistance to this destructive fungal disease
ever, lower in resistant genotypes. (Fig. 9.1; Pedras et al. 2003).
The analysis of total phenols and specific activi-
ties of polyphenol oxidase (PPO), peroxidase (PO),
and catalase in A. brassicae- and A. brassicicola- 9.6 Proteome-Level Resistance
infected mustard leaves of tolerant (RC-781,
RH-8113) and susceptible (RH 30, Prakash) culti- Two Brassica lines derived from an interspecific
vars of mustard was done at 20 days interval after cross between B. napus and B. carinata were
40 days of sowing and showed that the total phe- evaluated for tolerance to A. brassicae. Pathogen-
nols increased initially and decreased with the age induced chlorosis and necrosis spread signifi-
of the plant; these were markedly higher in tolerant cantly in one line, whereas it remains localized in
as compared to the susceptible ones at all growth the other. Proteome-level changes in response to
stages. After infection, their amount decreased in the fungal pathogen invasion were investigated
all genotypes. The specific activities of polyphenol using two-dimensional electrophoresis. Levels of
oxidase remained higher, while that of peroxidase 48 proteins were significantly affected at various
lower in tolerant than in the susceptible cultivars. In time points in the tolerant line (41 upregulated
response to infection, although the activities of and 7 downregulated). In contrast, in the suscep-
both the enzymes increased sharply in the cultivars, tible line, the levels of 23 proteins were signifi-
this increase was considerably higher in suscepti- cantly affected with four increasing and 19
ble than in tolerant cultivars. Catalase activity was decreasing. The identities of 38 proteins and
appreciably higher at initial stage and dropped those identified from the tolerant line included
markedly at later stages (Gupta et al. 1990). enzymes involved in the generation of reactive

White mustard Rapeseed

1 4 C–labeled destruxin
A
hydroxydestruxi B
(B. alba)

Fig. 9.1 Metabolism of destruxin B by white mustard and rapeseed

186 9 Resistance

oxygen species (ROS), ROS-mediated signalling, the non-transgenic control. The bioassay under
auxin signal transduction and metabolic path- artificial epiphytotic conditions reveals the delay
ways. The changes in proteome levels suggest a in the onset of disease as well as reduces the
role for ROS-mediated auxin signalling in this lesion number and size in 35 S chitinase Brassica
pathosystem, which was further investigated and as compared to the untransformed control plants
confirmed using quantitative real-time PCR (Mondal et al. 2003).
(Sharma et al. 2007). Chitinase-modifying pro- Genotypes EC-399299, EC-399296,
teins (Cmps) are secreted by fungal pathogens of EC-399313 and PHR-2 are comparatively more
crucifers, which interfere with fungalysin Cmp resistant to infection by A. brassicae. Increased
activity to improve plant resistance to multiple level of PAL, PPO and peroxidase may play an
fungal diseases (Naumann and Wicklow 2013). important role in undertaking the defence mecha-
The Arabidopsis thaliana secretome analysed nisms of B. juncea genotypes against Alternaria
by the proteomic approach led to the identifica- blight pathogenesis (Parihar et al. 2012). With
tion of secreted proteins implicated in many 1 ppm concentration of chitosan sprayed on the
aspects of cell biology. Oh et al. (2005) investi- leaves of B. juncea, superoxide dismutase and
gated the change in the Arabidopsis secretome in 5 ppm chitosan peroxide and phenylalanine
response to salicylic acid and identified several ammonia lyase significantly increased resistance
proteins involved in pathogen response. One of to Alternaria blight with respect to control.
these, a secreted lipase with a GDSL-like motif Phenolic content in the leaves of treated plants
designated as GDSL LIPASE 1(GLIP1), was fur- also increased with both the treatments of chito-
ther characterized for its function in disease san. Defence-related enzymes and phenolic con-
resistance; glip1 plants are markedly more sus- tent showed positive correlations, i.e. increase
ceptible to infection by A. brassicicola compared with the elicitor application. Since these enzymes
with the parental wild plants. The recombinants and phenolics increased resistance to pathogens,
GLIP1 protein possessed lipase and antimicro- they may provide protection against Alternaria
bial activities that directly disrupt fungal spore blight (Neerja and Sohal 2012). After inoculation
integrity. GLIP1 appears to trigger systemic of B. juncea leaves with A. brassicae, activities of
resistance signalling in plants when challenged the cell wall-degrading enzymes, polygalacturo-
with A. brassicicola, because pretreatment of nase (EC-3.2.1.15) and cellulase (EC-3.2.1.4),
glip1 mutant with recombinant GLIP1 protein decreased in leaf blight-resistant cultivar RC-781
inhibited A. brassicicola-induced cell death in and increased in the susceptible cultivar Varuna
both peripheral and distal leaves. Moreover, up to 3 days. In both cultivars, 11 polypeptides
glips1 shows altered expression of defence and are observed in leaves in the absence of A. bras-
ethylene-related genes. GLIP1 transcription was sicae inoculation. After inoculation, although
increased by ethephon, the ethylene releaser, but there was no change in the polypeptide pattern in
not by salicylic acid or jasmonic acid. GLIP1 in the resistant cultivar RC-781, but in the suscepti-
association with ethylene signalling may be a ble cultivar Varuna, four polypeptides (43.7–
critical component in plant resistance to A. 58.8 kDa) disappeared on the 3rd day after
brassicicola. inoculation (Garg et al. 1999). Occurrence of a
Chitinase, capable of degrading the cell walls lipase in A. brassicicola spores plays a crucial
of invading phytopathogenic fungi, plays an role in the infection of cauliflower leaves.
important role in plant defence response, particu- Addition of anti-lipase antibodies into A. bras-
larly when this enzyme is overexpressed through sicicola conidial suspension prior to inoculation
genetic engineering. Brassica plant (B. juncea resulted in reducing black spot lesions by 90 %
L.) was transformed with chitinase gene tagged on intact cauliflower leaves, but not on leaves
with an overexpressing promoter 35 S CaMV. In from which surface wax had been removed.
in vitro fungal growth inhibition assays, chitinase Spore surface-bound lipase is thought to interact
inhibits the fungal colony size by 12–56 % over closely with epicuticular leaf waxes for adhesion
9.7 Induced Resistance 187

Table 9.9 Specific activity of peroxidase enzyme in Table. 9.10 Specific activity of enzyme catalase in
hypocotylar calli of Brassica species raised on MS hypocotylar calli of Brassica species raised on MS
medium supplemented with or without fungal culture fil- medium supplemented with or without fungal culture fil-
trate (FCF) of Alternaria brassicae (Dhingra et al. 2004) trate (FCF) of Alternaria brassicae (Dhingra et al. 2004)
Concentration of FCF (%) Concentration of FCF (%)
Species Control 5 10 Species Control 5 10
Brassica juncea cv. 11.21 13.77 16.88 Brassica juncea cv. 0.79 0.83 0.87
Kranti Kranti
Brassica juncea cv. 3.57 13.41 21.73 Brassica juncea cv. 4.21 3.57 1.72
RH-781 RH-781
Brassica juncea cv. 0.18 0.37 1.89 Brassica juncea cv. 1.43 1.21 0.45
GSH-1 GSH-1
Enzymes unit = change in absorbance/3 min/mg/protein Enzymes unit = Change in absorbance/5 min/mg/protein

Table. 9.11 Specific activity of enzyme polyphenol oxi-

and/or penetration of fungal propagules during dase in hypocotylar calli of Brassica species raised on MS
medium supplemented with or without fungal culture fil-
the early stages of host–parasite interactions
trate (FCF) of Alternaria brassicae (Dhingra et al. 2004)
(Berto et al. 1999).
Concentration of FCF (%)
Dhingra et al. (2004) reported higher peroxi-
Species Control 5 10
dase activities in two B. juncea cultivars than in
Brassica juncea cv. 4.12 5.37 6.54
B. napus cv. GSH-1. Between two B. juncea cul- Kranti
tivars, enzyme activity in Kranti was more than Brassica juncea cv. 5.70 8.49 10.56
three times than in RH-781. Addition of fungal RH-781
culture filtrate (FCF) to the culture medium, Brassica juncea cv. 6.19 10.21 14.63
although increased peroxidase activity in all GSH-1
cases, further increases the FCF concentration; Enzymes unit = change in absorbance/5 min/mg/protein
the peroxidase activity increase was much lesser
in Kranti than in RH-781 and GSH-1 (Table 9.9).
The catalase activity is low in the calli of B. jun- 9.7 Induced Resistance
cea cv. Kranti than in B. juncea cv. RH-781 and
B. napus cv. GSH-1. Between the latter two culti- Plant possesses a very vast array of defence
vars, the former showed 3 times more activity strategies to combat pathogen’s growth and
than the latter. Catalase activity was not affected colonization. One of the most efficient and
by FCF in the calli of Kranti, while activity immediate defence reactions of plants against
decreased in other two cultivars; the decrease was the pathogen is the hypersensitive response,
more in GSH-1 than in RH-781 (Table 9.10). which is defined as ‘the rapid death of plant
Polyphenol oxidase activity was low in the calli cells in association with restriction of pathogen
of B. juncea cv. Kranti than in B. juncea cv. growth’ (Goodman and Novacky 1994). It is
RH-781 and B. napus cv. GSH-1 (Table 9.11). characterized by the presence of brown, dead
Addition of FCF to the culture medium, in gen- cells at the site of infection which limit the
eral, increased PPO activity; the increase was growth of biotrophic pathogens. Hypersensitive
much lesser in cv. Kranti than in the other two reaction is a highly concerted complex defence
cultivars. Calli of GSH-1 showed more than two- response which is followed by the production
fold increase in PPO activity over that of RH-781 of reactive oxygen species (Lamb and Dixon
(Table 9.11). Inoculation of leaves with A. bras- 1997), modification of ion fluxes (Levine et al.
sicae increased PPO activity in both resistant and 1996) and activation of defence through the
susceptible cultivars of B. juncea, but the increase synthesis of signalling molecules such as jas-
was much higher in RC-781 than in Varuna monic acid, salicylic acid and protein kinase
(Gupta 1995). (Dangl et al. 1996; Dixon et al. 1994). Salicylic
188 9 Resistance

acid accumulation leads to the onset of sys- expression of MAPK 4 at early hours of infec-
temic acquired resistance in the distal plant tis- tion. Zeatin upregulates plant immunity via an
sues (Ryals et al. 1996). These events are elevation of MAPK 4 and clearly reflects that it
accompanied by the activation of several plant antagonizes the effect of A. brassicae. The
defence genes, local accumulation of pathogen- cross talk between zeatin and MAPK 4 signal-
esis-related (PR) proteins, activation of tran- ling pathways may help plants fine-tune defence
scription factors, degradation of proteins by the responses against A. brassicae in Alternaria
polyubiquitin system and programmed cell blight.
death (Mishra et al. 2010).
A pre-inoculative foliar application of 5 mM
β-aminobutyric acid (BABA) significantly 9.8 Identification, Cloning
inhibits the colonization of A. brassicae on and Sequencing
leaves of B. carinata susceptible cultivar Car 6. of Resistant Genes
BABA treatment leads to transient but signifi-
cant increase in H2O2 level during early stages The antifungal effect of hevein, the chitin-
of pathogen colonization. A significant binding lectin from rubber plant (Hevea brasil-
increase in superoxide dismutase (SOD), iensis), has been analysed in transgenic plants
ascorbate peroxidase (APX) and guaiacol- for potential control of Alternaria blight in
dependent peroxidase (GDP) is known to Indian mustard. A cDNA encoding hevein was
inhibit the oxidative stress in BABA-treated transferred into Indian mustard (B. juncea cv.
plants in response to pathogen infection. RLM-198). Southern analysis of the putative
BABA treatment also leads to proper balance transgenics shows integration of the transgene.
of oxidant and antioxidants suitable for the Northern and Western analyses proved that the
expression of resistance resulting in curtail of integrated transgene is expressed in the trans-
pathogen ingress during early stages of coloni- genics. In whole plant bioassay under glass-
zation (Chavan et al. 2013). house conditions, transgenics possess
Recent studies have revealed an important parameters that are associated with resistance
role of hormones in plant immunity. Cytokinins such as longer incubation and latent period,
are phytohormones that are involved in various smaller necrotic lesion size, lower disease inten-
regulatory processes including plant defence. sity and delayed senescence (Kanrar et al.
Zeatin, a cytokinin, antagonizes the effect of A. 2002).
brassicae pathotoxin in cell culture of B. jun- β-Aminobutyric acid (BABA) pretreatment
cea. Phytohormones are also the inducers of of Brassica plants provides protection against
MAP kinase signalling pathways, which are the the necrotrophic pathogen A. brassicae. The
important signalling modules in eukaryotic achieved resistance level is much higher than
cells. Marmath et al. (2013) observed the role that seen after salicylic acid (SA) and jasmonic
of exogenous application of zeatin on disease acid (JA) pretreatments. BABA pretreatment to
score, infection behaviour of A. brassicae and leaves, 1 day before inoculation, although it
expression pattern of MARK-4 in the non-host leads to an inhibition of the oxidative burst and
Sinapis alba, susceptible B. juncea cv. Varuna, a decrease in SA levels, neither influences
moderately tolerant B. juncea cv. Divya and lipoxygenase activity nor causes callose deposi-
transgenic Brassica to confer its role in plant tion at the site of inoculation. Expression of two
immunity. High concentration of zeatin leads to marker genes of the SA and JA pathways,
increased defence responses by delaying the namely, PR1 and PDF1.2, enhances in response
infection process as well as significantly reduc- to BABA pretreatment. BABA-induced resis-
ing the disease score. Semi-quantitative tance is mediated through an enhanced expres-
RT-PCR reveals that zeatin also increases the sion of pathogenesis-related protein genes,
9.8 Identification, Cloning and Sequencing of Resistant Genes 189

independent of SA and JA accumulation transformation. The stable integration and

(Kamble and Bhargava 2007). The identifica- expression of transgenes in T0 plants were con-
tion and cloning of hsr203j homologues from firmed by Southern blot and Western analysis.
tolerant and susceptible genotypes of B. juncea The transgenic lines showing inheritance in
through RT-PCR analysis have been accom- Mendelian fashion (3:1) were further evaluated
plished. In silico analysis of the sequences iso- by in vitro studies and under greenhouse condi-
lated from susceptible and tolerant genotypes of tions for resistance against A. brassicae. The
B. juncea shows the presence of conserved transgenic plants showed up to 44 % reduction
abhydrolase domain having a role in cell death. in A. brassicae hyphal growth in in vitro anti-
Motif analysis indicates that motif 19 that func- fungal assays. In greenhouse screening, the
tions in prenylation is found exclusively in tol- transgenic plants sprayed with A. brassicae
erant genotypes and motif 12 having spores showed resistance through delayed onset
myristoylation site is found in susceptible geno- of the disease and restricted number, size and
types. Various defence-related important cis and expansion of lesions as compared to wild-type
trans acting factors are also found in these plants. The expression of chitinase and RIP
homologues. This suggests that these hsr203j from a heterologous source in B. juncea pro-
like homologues of Brassica play an important vides subsequent protection against Alternaria
role in differential defence against Alternaria leaf spot and can be helpful in increasing the
blight – a recalcitrant disease caused by A. bras- production of Indian mustard (Chhikara et al.
sicae (Mishra et al. 2010). 2012).
Cysteine-rich antimicrobial peptides isolated Mutation in the gene PAD3, encoding a cyto-
from plants have emerged as potential resource chrome P450, abolishes the biosynthesis of
for protection of plants against phytopathogens. camalexin (Glazebrook and Ausubel 1994) and
The significance of an antimicrobial peptide, results in enhanced susceptibility to necrotro-
Pm AMPI, isolated from Western white pine phic fungi (Thomma et al. 1999). The phytoan-
(Pinus monticola), in providing canola resis- ticipins glucosinolates (GS) are sulphonated
tance against several phytopathogenic fungi, has thioglycosides comprising a common glycone
been reported by Verma et al. (2012). The cDNA moiety with a variable aglycone side chain and
encoding Pm AMPI was successfully incorpo- are considered the major secondary metabolites
rated into the genome of B. napus and its in of Brassicaceae (Fahey et al. 2001). Upon tis-
planta expression conferred greater protection sue damage, GS come into contact with myrosi-
against A. brassicae, Leptosphaeria maculans nases, a specific class of β-thioglucosidases,
and Sclerotinia sclerotiorum. In vitro experi- which are stored separately in the cell.
ments with proteins extracted from transgenic Hydrolysis of GS by myrosinases yields iso-
canola expressing Pm AMPI demonstrated its thiocyanates (ITC), nitrile and epithionitriles.
inhibitory activity by reducing growth of fungal The most common breakdown products, ITC,
hyphae. In addition, the in vitro synthesized exhibit toxicity towards several plant pathogens
peptide also inhibited the growth of fungi. including bacteria, fungi, insects and nema-
Generating transgenic crops expressing Pm todes (Fahey et al. 2001). It has been demon-
AMPI may be an effective and versatile method strated that ITC produced by Arabidopsis
to protect susceptible crops against multiple thaliana significantly inhibit growth of some
phytopathogens. fungal pathogens in plants (Tierens et al. 2001),
To develop resistance against A. brassicae, although recent results suggest that apart from
the barley antifungal genes, class II chitinase their direct toxic effects, GS breakdown prod-
(AAA56786) and type I ribosome-inactivating ucts may also act by modulation of plant
protein (RIP; AAA32951), were co-expressed in defence signalling (Brader et al. 2006). During
Indian mustard via Agrobacterium-mediated host infection, A. brassicicola is exposed to
190 9 Resistance

high levels of antimicrobial defence compounds membrane integrity, and among the camalexin-
such as indolic phytoalexins and glucosinolate elicited genes, several are involved in sterol and
breakdown products. To investigate the tran- sphingolipid biosynthesis. The transcriptomic
scriptomic response of A. brassicicola when analysis suggests that protection against the
challenged with brassicaceous defence metabo- two tested compounds also involves mecha-
lites, suppression subtractive hybridization nisms aimed at limiting their intracellular accu-
(SSH) was performed to generate two cDNA mulation, such as melanin biosynthesis (in case
libraries from germinated conidia treated either of camalexin exposure only) and drug efflux.
with allyl isothiocyanate (AI-ITC) or with From the AI-ITC and the camalexin differen-
camalexin. Following exposure to AI-ITC, A. tially expressed genes identified, 25 are selected
brassicicola displays a response similar to that to perform time course studies during interac-
experienced during oxidative stress. A substan- tions with brassicaceous hosts. In planta, upreg-
tial subset of differentially expressed genes is ulation of all the selected genes are observed
related to cell protection against oxidative dam- during infection of Raphanus sativus, whereas
age. Treatment of A. brassicicola conidia with only a subset are overexpressed during the
the phytoalexin camalexin appears to activate a incompatible interaction with A. thaliana eco-
compensatory mechanism to preserve cell type Columbia (Sellam et al. 2007a; Fig. 9.2).

Fig. 9.2 Functional classification of

upregulated genes in Alternaria a
brassicicola, AI-ITC (a) and
camalexin; (b) treated conidia
according to their putative biological
function (Sellam et al. 2007a)
37% 35%

5% 16%
7%

oxidative burst, stress and

b defence
9% membrane transporters
9% transcription and cell cycle
4%
35% metabolism

cell wall structure and

function
protein fate
25%
4% miscellaneous
6%
8% hypothetical and unknown
9.9 Elicitation of Phytoalexins 191

9.9 Elicitation of Phytoalexins The relationship between conidial concentra-

tion, germling growth and phytoalexin produc-
The accumulation of phytoalexins in brassicas tion by C. sativa leaves inoculated with A.
after exposure to microorganisms and their role brassicae has been studied by Jejelowo et al.
in disease resistance have been demonstrated by (1991). The rapid rate of phytoalexin accumula-
many workers. These are low molecular weight, tion shortly after inoculation results into inhibi-
antimicrobial compounds that are both synthe- tion of fungal growth on the leaf surface. The
sized by and accumulated in plants after exposure phytoalexin slows germination and inhibits
to microorganisms (Paxton 1981). So far, approx- germ-tube growth of A. brassicae conidia in vitro.
imately a dozen different phytoalexins have been Three phytoalexins, camalexin (C11H8N2S),
reported from crucifers (Bains and Tewari 1985; 6-methoxycamalexin (C12H10N2SO) and
Browne et al. 1991; Devys et al. 1988; Takasugi N-methylcamalexin (C12H10N2S), have been iso-
et al. 1988). All phytoalexins identified from lated from shepherd’s purse (Capsella bursa-
brassicas contain sulphur in their molecules. pastoris) challenged by A. brassicae (Fig. 9.3).
Conn et al. (1987, 1991) and Tewari et al. (1987, Phytoalexin elicitation in shepherd’s purse is
1988) described elicitation of phytoalexins in B. associated with its resistance to A. brassicae
rapa var. oleifera (canola), B. rapa ssp. rapifera (Jimenez et al. 1997).
(an accession of turnip), B. napus, Camelina Capsella bursa-pastoris shares one phyto-
sativa, Eruca sativa and Capsella bursa-pastoris alexin with C. sativa and the other one with rape-
similar to that of cyclobrassinin. However, the seed and is also highly resistant to A. brassicae
quantity produced in turnip is much greater. (Conn et al. 1988). The highly resistant Camelina
Jejelowo et al. (1991) studied the kinetics of phy- sativa elicits large quantities of two novel phyto-
toalexin elicitation in C. sativa. An accession of alexins; it appears that in crucifers, both the
E. sativa was reported to show hypersensitive quantity and quality of phytoalexins are impor-
response to A. brassicae (Conn and Tewari 1986). tant in regulating resistance to A. brassicae.
Browne et al. (1991) described camalexin and These phytoalexins are highly antimicrobial, as
methoxycamalexin phytoalexins from C. sativa, shown by the Cladosporium sp. thin-layer chro-
which are thiazolyl-substituted indole phytoalex- matography bioassay (Tewari 1991a, b).
ins, and showed strong structural resemblance to Antifungal compounds have been found to accu-
the fungicide thiabendazole. mulate when root slices of radish are inoculated
The antifungal effects of camalexin, brassinin
and allyl (AI ITC)- and benzyl (Bz ITC)-
isothiocyanates, at different development stages
of A. brassicae and A. brassicicola, have been
observed by Sellam et al. (2007b). Irrespective of
the isolate, the phytoalexin camalexin exhibits
the greater inhibitory effect with mean EC50 val-
ues ranging from 34 μm (germ-tube elongation)
to 183 μm (mycelial growth). Germ-tube elonga-
tion is more sensitive compared to conidial ger-
mination and mycelial growth, with mean EC50
values of the former of 81 μm, 520 μm and
870 μm for brassinin, Bz ITC and AI ITC, respec-
tively. However, the role of camalexin in disease
resistance varies among different Arabidopsis
population in nature and provides some clue to
other possible determinants of resistance to A. Fig. 9.3 Structures of camalexin (1), 6-methoxycamalexin
brassicicola (Kagan and Hammerschmidt 2002). (2) and N-methylcamaxin (3)
192 9 Resistance

with the spore suspension of A. alternata. resistance to A. brassicae in rapeseed by soil or

Antifungal activity of the diffusate shows inhibi- foliar application of calcium compounds (Tewari
tory action to spore germination, germ tube elon- 1991a, b). Foliar sprays of calcium compounds
gation and radial mycelial growth. The sequester the organic acids at the site of infection,
phytoalexins produced are fungistatic in nature and soil application has the potential of boosting
(Kulshreshtha and Chauhan 1985). Chinese cab- calcium content of the plant.
bage lines/hybrids on A. brassicae inoculation
produce phytoalexins (Saharan et al. 2003).
9.11 Sources of Resistance

9.10 Calcium Sequestration Digenomic Brassica species such as B. napus and

B. juncea have better sources of resistance than
The plant cell walls are rich in calcium, which is the monogenomic (Tables 9.12 and 9.13) species
tightly bound to pectins. Calcium is known to be like B. rapa. Some lines of B. alba, B. carinata, B.
a factor in disease resistance. The insoluble cal- spinescenes, B. maurorum, Eruca sativa,
cium polypectates are resistant to hydrolysis by Camelina sativa, Capsella bursa-pastoris,
pectolytic enzymes produced by the pathogens Diplotaxis sp. and N. paniculata have also shown
(Vidhyasekaran 1988). Oxalic acid and possibly resistance to A. brassicae in different areas of the
other organic acids produced by various patho- world (Brun et al. 1987a, b; Conn et al. 1991;
gens sequester calcium, which may overcome Grontoft 1986; Jejelowo et al. 1991; Saharan
this form of resistance (Punja and Jankins 1984; 1992, 1997; Tewari and Conn 1993; Hansen and
Rao and Tewari 1987). Examination of black spot Earle 1997; Chrungu et al. 1999; Sharma et al.
lesions on rapeseed leaves by scanning electron 2002). Sources of resistance to A. brassicae and
microscopy in conjunction with energy- A. brassicicola in different host species from dif-
dispersive X-ray microanalysis has revealed ferent areas of the world are given in Table 9.13.
sequestration of calcium by A. brassicae. In India, Brassica genotypes, viz. CSR 43, CSR
Therefore, there are possibilities of enhancing I42, CSR- I42-2, CSR 343, CSR 448, CSR 622,

Table 9.12 Brassica germplasm holdings at different organizations and research centres in the world
Germplasm accessions
Sr. no. Name of organization/research centre (Total number) References
1. The National Bureau of Plant Genetic Resources 10301 Radhamani et al.
(NBPGR), New Delhi, India (2013)
2. ICAR-All India Coordinated Research Project on 12778 AICRPRM (2011)
Rapeseed-Mustard, Centres
3. ICAR-Directorate of Rapeseed-Mustard Research, 2452 Nanjundan et al.
Bharatpur (Raj) India (2014)
4. National Plant Germplasm System (USDA) Germplasm 2958 http://www.ars-grin.
Resources Information Network (GRIN) gov/cgi-bin/npgs/html/
taxon.pl?319659
5. Biotechnology Department, Polytechnic University, 150 Nuej et al. (1987)
Valencia, Caminode, Spain
6. The Crucifer Genetics Cooperation (CrGC), Department 70 × 17 stocks in Williams (1988b)
of Plant Pathology, University of Wisconsin, Madison, each Brassica crop
USA
7. Wisconsin Fast Plants (WFP), Department of Plant Rapid cycling of Williams (1988a)
Pathology, University of Wisconsin, Madison, USA Brassica seed stocks
8. The Asian Vegetable Research and Development Centre 822 Anonymous (1984);
(A VRDC), Shanhua, Taiwan Opena and Lo (1981)
9.11 Sources of Resistance 193

Table 9.13 Sources of resistance to Alternaria brassicae and Alternaria brassicicola (Verma and Saharan 1994; to
date)
Host species/genotype Common name References
A. brassicae
Brassica rapa ssp. oleifera Turnip rape AICORPO (1989)
EC-242660-61, EC-242646, EC-253287, EC-253291 Toria
B. rapa var. Yellow Sarson, PYS6, BINA 1,2 Kolte (1987); Rahman et al. (1987)
B. rapa rapifera, Edmonton ACC Turnip Conn et al. (1988)
B. carinata Ethiopian mustard AICORPO (1989);
PPSC1, HC-1, EC-25381, EC-253282, Bhowmik and Munde (1987);
EC-253284, EC-253826, S67, PC3, PC5, CE9 Kidane and Bekele (1986)
B. napus spp. oleifera Oilseed rape AICORPO (1989); Conn et al.
(1987);
Altex, Gulivar 1, Karat, Narde, Midas, Primer Conn and Tewari (1989a); Kumar
Tower, WRGI5, Westar, Wei Bull- 541, 521 and Kumar, (1989); Rozej (1974)
Vuindsok, Regent 1, GS-1-1, GS7027, Vestal Romero-Munoz and Jimenez
GSL-1501, 1506, 1508, 1513, HNS1, HNS3, Vestal (1979); Saharan (1992, 1997);
Stankova
EC-338986-2, EC-338996-1, EC-338997, EC-339000 (1972); Kolte et al. (2008);
AICRPRM (2011)
GS-05-1 Kumar et al. (2014)
B. juncea Indian mustard AICORPO (1989); Kolte (1985a);
BECI07-109, 112, EC-I29126-1, PR8805, PHR 1 Saharan (1992, 1997); Kolte et al.
(2000)
KRV Tall, RC-781, Divya, Kranti, PR- 8999, PR-9024; 2008); Kumar (2008); Pratap et al.
EC-399296, EC-399299, EC-399301, EC-399313, 2014
DRMR-2805
RH-8544, Pusa Swarnim, HC-9605 Kumar et al. (2014)
B. oleracea var. alboglabra Chinese kale Munde and Bhowmik (1985);
Zaman and Biswas (1987)
B. oleracea var. botrytis Cauliflower Singh et al. (1987)
Lines 1-6-1-2, 1-6-1-4, Pusa Shubhra
B. oleracea ssp. gemmifera Brussels sprouts Berry and Lennard (1988);
Williams and Pink (1987)
Cambridge no.5
B. alba White mustard Kolte (1987); Saharan (1992)
B. hirta Brun et al. (1987a, b)
Camelina sativa False flax Tewari and Conn (1993);
Capsella bursa-pastoris Shepherd’s purse Conn et al. (1991);
Neslia paniculata Ball mustard Jejelowo et al. (1991); Grontoft
(1986)
Sinapis alba White mustard Kolte (1985b);
S10001, S1004-10 Rai et al. (1977)
A. brassicicola
B. oleracea var. Botrytis Cauliflower Braverman (1971); Singh et al.
(1987)
PI’S 231208-209, 217934, 231209, 267725,
Pusa Shubhra
B. oleracea var. gemmifera Brussels sprouts Braverman (1976, 1977)
B. rapa, Saori, Edononatsu Doullah et al. (2006)
194 9 Resistance

CSR 741, Gulivar, KRV-Tall, Midas, PHR 1, closely related to the Brassica genus, the highest
RC-781, TMV2, Tower and YRT3, were consis- resistance against A. brassicae is found in white
tently found to have field resistance proven after mustard (Sinapis alba) (Kolte 1985a; Brun et al.
testing for several years at different locations 1987a, b; Ripley et al. 1992; Sharma and Singh
under uniform disease nursery trials (Kolte 1985b, 1992; Verma and Saharan 1994; Hansen and Earle
1987; Saharan 1984, 1992, 1997; Saharan and 1995, 1997). The highest overall Alternaria spp.
Chand 1988; Saharan et al. 2003, 2005; Verma resistance, however, have been identified in the
and Saharan 1994). Brassica coenospecies are crucifers more distant from the Brassica, such as
rich reservoir for genetic resistance to A. brassi- Camelina (Camelina sativa; false flax), shepherd’s
cae (Sharma et al. 2002; Table 9.14). Alternaria purse (Capsella bursa-pastoris), taramira (Eruca
brassicae pathotype-specific resistance has been sativa) and ball mustard (Neslia paniculata) (Conn
recorded in the genotype GS-05-1 of B. napus to and Tewari 1986; Conn et al. 1988; Tewari 1991b).
pathotypes Abr 1 and Abr 5, whereas Resistance against A. brassicae and A. brassicic-
genotypes,RH-8544, Pusa Swarnim and HC-9605 ola has been reported among other wild members
of B. juncea showed moderately resistant reaction of the Brassicaceae family (Table 9.14) (Sharma
to three pathotypes (Kumar et al. 2014). et al. 2002; Tewari and Conn 1993; Warwick
2011), viz. Alliaria petiolata, Barbarea vulgaris,
B. elongata, B. desnottessi, B. fruticulosa, B. mau-
9.11.1 Sources of Resistance rorum, B. nigra, B. souliei, B. spinescens, C.
from Cruciferous Relatives sativa, C. bursa-pastoris; Coincya spp., Diplotaxis
catholica, D. berthautii, D. creacea, D. erucoides,
Sources with high level of resistance against A. D. tenuifolia, Erucastrum gallicum, E. vesicaria
brassicicola and A. brassicae have not been iden- subsp. sativa, Hemicrambe fruticulosa, H. matro-
tified among the cultivated Brassica species nalis, N. paniculata, R. sativus, S. alba and S.
although individual cabbage varieties differ con- arvensis. The completely immune plants remain
siderably in their level of susceptibility to black symptom-free both under natural field infection
spot (Otani et al. 2001). The highest level of and under controlled artificial inoculation (Sharma
Alternaria resistance among the oilseed Brassica et al. 2002). Comparatively, broccoli and cauli-
crops is displayed by the Ethiopian mustard (B. flower varieties exhibit moderate Alternaria resis-
carinata). Among the wild cruciferous plants tance, while the cabbage as susceptible.

Table 9.14 Classification of 38 Brassica coenospecies based on the reaction to Alternaria brassicae under in vitro and
in vivo inoculation conditions (Sharma et al. 2002)
Resistant Moderately resistant Susceptible Highly susceptible
Brassica desnottessi Brassica oleracea B. oleracea (cabbage) B. juncea
(broccoli)
Camelina sativa B. oleracea (cauliflower) B. napus B. rapa
Coincya pseuderucastrum B. nigra B. fruticulosa B. carinata
Diplotaxis berthautii B. barrelieri D. gomez-campoi B. cossoniana
Diplotaxis catholica B. oxyrrhina D. assurgens B. spinescens
Diplotaxis cretacea B. gravinae D. tenuisiliqua Coincya longirostra
Diplotaxis erucoides Coincya rupestris Erucastrum abyssinicum D. viminea
Eruca gallicum D. harra E. canarianse Enarthocarpus lyratus
D. muralis Moricandida arvensis Raphanus sativus
D. siifolia Moricandia moricandioides
Sinapis alba S. flexuosa
S. pubescens
9.13 Relationship between Major Foliar Diseases 195

9.12 Sources of Multiple Disease GSL-1501 and Gulivar 1 of B. napus were found
Resistance resistant to A. candida and E. cruciferarum by
Gupta and Singh (1994). The genotypes Wester (B.
Brassica alba and genotypes, viz. HC-1, PCC-2 napus) and Tobin and Candle (B. rapa) are reported
(B. carinata), GSL-1501, GS-7027, Midas, Tower completely free from A. candida and E. cruci-
(B. napus), DIR-1507 and DIR-1522 (B. juncea), ferarum (Shivpuri et al. 1997). The variations in the
have been identified as sources of multiple disease observations may be attributed to the occurrence of
resistance against white rust, Alternaria blight, new races of the pathogen and congenial weather
downy mildew and powdery mildew diseases factors (Table 9.15; Kumar and Saharan 2002).
(Dang et al. 2000; Kumar and Saharan 2002).
Earlier B. napus genotype GSL-1501 was reported
resistant to white rust and powdery mildew (Gupta 9.13 Relationship between Major
and Singh 1994). The variations in the incidence of Foliar Diseases
powdery mildew scores among different Brassica
genotypes differ considerably due to variations in The increase in level of resistance to different
date of sowing, stage of the crop at the time of pathogens is positively correlated; the improve-
observations and prevailing environmental condi- ment in the accumulation of resistance to multiple
tions. Hence, early maturing genotypes or the gen- diseases may be facilitated easily in agricultural or
otypes planted early in the season would help in natural populations; negative association among
escaping the powdery mildew infection. the level of resistance will hinder plant defence
The genotypes EC-322090, EC-322092, against pathogens’ attack and leads to difficulties
EC-322093 and RC-781 of B. juncea; HC-1 and in the development of multiple disease resistance.
PCC-2 of B. carinata; and GS-7027, GSL-1, GSL- The non-availability of single resistance source
1501, Gulivar 1, HNS-4 and Tower sel.1 of B. napus line with different mode of inheritance further
are resistant to A. candida and A. brassicae, whereas complicates the work of genetic amelioration for
genotypes EC-129126-1, EC-129126-2 and multiple disease resistance. The significant and
PR-8805 of B. juncea; Candle, Tobin and Torch of positive association between disease scores of
B. rapa; DHC-1, HDC-4, DHC-1960, HC-1 and white rust, Alternaria blight and powdery mildew
PCC-2 of B. carinata; and GSL-1501, N 20-12-2 has been recorded except white rust stag head and
and Wester of B. napus are resistant to A. candida powdery mildew infection in genotypes of differ-
and Erysiphe cruciferarum. However, HC-1 and ent Brassica species. A high degree of correlation
PCC-2 of B. carinata and GSL- 1501 (B. napus) are is observed between leaf phases of white rust and
resistant to both A. brassicae and E. cruciferarum. Alternaria blight (Table 9.16). Similarly, the

Table 9.15 Sources of multiple disease resistance in oilseed Brassica (Kumar and Saharan 2002)
Resistant to AB Resistant to WR, AB, PM
Resistant to WR and AB Resistant to WR and PM and PM and DM
B. juncea
EC-322090, EC-322092, EC-129126-1,EC-29126-2, None DIR 1507, DIR 1522,
EC-322093, RC-781 PR-8805 Brassica alba
B. rapa
None Candle, Tobin, Torch None None
B. carinata
HC-1, PCC-2 DHC-1, DHC-4, DHC-9601, HC-1, PCC-2 HC-1, PCC-2
HC-1, PCC-2
B. napus
GS-7027, GSL-1, GSL-1501, GSL-1501, N-20-12-2, GSL-1501 GSL-1501, GS-7027,
Gulivar-1, HNS-4, Towel Sel-1 Wester Midas, Tower
WR white rust, AB Alternaria blight, PM powdery mildew, DM downy mildew
196 9 Resistance

Table 9.16 Relationship among major foliar diseases (Kumar and Saharan 2002)
Disease WR (L) WR (S) AB (L) AB (S) PM ADL ADI
B. juncea
WR (L) 1.00
WR (S) 0.32** 1.00
AB (L) 0.39** 0.12 1.00
AB (S) 0.14 0.09 0.75** 1.00
PM 0.10 0.09 0.35** 0.30** 1.00
ADL 0.81** 0.28* 0.69** 0.44** 0.50** 1.00
ADI 0.13 0.11 0.61** 0.71** 0.82** 0.55** 1.00
B. rapa, B. carinata and B. juncea
WR (L) 1.00
WR (S) 0.19 1.00
AB (L) 0.01 0.30 1.00
AB (S) 0.21 0.30 0.96** 1.00
PM 0.46** 0.05 0.07 0.15 1.00
ADL 0.69** 0.25 0.53** 0.64** 0.79** 1.00
ADI 0.43* 0.31 0.73** 0.79** 0.71** 0.94** 1.00
Brassica species
WR (L) 1.00
WR (S) 0.32** 1.00
AB (L) 0.39** 0.26** 1.00
AB (S) 0.34** 0.27** 0.93** 1.00
PM 0.33** 0.14 0.36** 0.33** 1.00
ADL 0.82** 0.31** 0.68** 0.62** 0.68** 1.00
ADI 0.42** 0.29** 0.75** 0.77** 0.85** 0.81** 1.00
WR (L) white rust at leaf, WR (S) white rust at stag head, AB (L) Alternaria blight at leaf, AB (S) Alternaria blight at
siliquae, PM powdery mildew, ADL average disease on leaf, ADI average disease on inflorescence, *significant at
P = 0.05; **significant at P = 0.01

positive genetic correlation is observed between rium may arise from previous generations of selec-
Hyaloperonospora parasitica and Leptosphaeria tion or small population size), (II) pleiotropy and
maculans and Hyaloperonospora parasitica and (III) tight linkage between resistance genes
Albugo candida in B. rapa (Mitchell-Olds et al. (Martin et al. 1993).
1995). Sharma et al. (1991) reported significant
positive correlation between Alternaria blight and
black rot in cauliflower. The strong evidence for a 9.14 Development of Resistant
positive genetic correlation in the level of resis- Cultivars
tance against A. candida, A. brassicae and
Erysiphe cruciferarum may indicate the presence Various methods including conventional as well
of plant resistance genes that provide defence as biotechnological approaches have been uti-
against some fundamental characteristics common lized either alone or in combination to incorpo-
to different taxonomic orders of fungal pathogens rate the desired traits and to steer the genetic
as observed by Bruns et al. (1991). Genetic corre- variability into new improved cultivars of cruci-
lations may arise from several factors like (I) link- fers. The conventional approach involves the
age disequilibrium, a nonrandom association identification and selection of disease-resistant
among genotypes at several loci, which is rarely genotypes and transfer of the desired traits into
found in cross-pollinated species (Crow and agronomically superior genotypes by hybridiza-
Kimura 1970) (however, the linkage disequilib- tion. Hybridization combined with backcrossing,
9.15 Strategies and Methods of Screening for Resistance 197

either singly or in combination with pedigree selection methods and (b) interspecific
selection, is the most widely used and accepted genome substitution, chromosome substitu-
conventional method to transfer disease resis- tion and gene introgression
tance in crucifers. 5. Transfer of resistance through mutation
Attempts to obtain resistance in agronomi- breeding
cally superior genotypes of B. juncea through 6. Use of biotechnological and genetic engineer-
mutagenic agents such as gamma rays have met ing techniques such as (a) genome manipula-
little success. Verma and Rai (1980) exposed the tion, (b) manipulation of cytoplasmic
seeds of B. juncea cultivars Varuna, PR-5 and genomes, (c) use of transformation and for-
RS-3 to gamma rays and obtained a mutant with eign gene expression techniques and (d)
60 KR doses which is promising in yield and embryo rescue techniques for wide
resistant to A. brassicae. Rahman et al. (1987) hybridization
selected mutant BINA 1 resistant to A. brassi-
cae from B. rapa cultivar YS52 treated with
EMS in Bangladesh. Das and Rahman (1989)
obtained the highest frequency of variation for 9.15 Strategies and Methods
Alternaria blight in M2 by irradiating seeds of of Screening for Resistance
Yellow Sarson cultivar YS52 with 70 KR
gamma rays. Sharma (1990) identified mutants The prerequisites to obtain resistant (R) cultivars
in M2 population of cultivars Varuna and Kranti (cvs) are:
as resistant to Alternaria blight on the basis of
small lesion size under natural epiphytotic con- (a) Knowledge of the pathogenic variability
ditions. MacDonald and Ingram (1985) selected (b) Development of a screening method able to
and regenerated plants from calli resistant to mimic the conditions required by the plants
partially purified culture filtrates of A. brassici- when exposed to natural sources of inoculum
cola. According to Katiyar and Chopra (1990), in the diverse field environment
a somaclonal variant obtained from an exotic (c) Availability of usable sources of resistance
yellow-seeded accession of B. juncea retained
resistance to disease similar to the parental Screening methods for disease resistance (R)
material. The transfer of resistance from differ- should be developed within the framework of a
ent sources in different Brassica crops is possi- general strategy for resistance. The changes in
ble and is being done through the following the frequency of virulence genes among the
conventional and modern techniques (Verma populations of pathogens inciting disease of
and Saharan 1994; Saharan 1992, 1997; Saharan the above-ground parts of plants are very fre-
et al. 2003, 2005): quent. Populations of such pathogens vary in
time and space because of the airborne and
1. Germplasm evaluation for sources of resis- seed-borne nature of inoculum which facili-
tance at National and International levels tates long-distance dispersal of their variants.
2. Selection for disease resistance through (a) As a result of these situations, breeding for
pure line selection, (b) mass selection, (c) resistance to foliar pathogens is, in general,
modified recurrent mass selection and (d) more difficult than in the case of less mobile
recurrent selection pathogens, e.g. soil-borne fungi which are,
3. Breeding for disease resistance by increasing therefore, more stable. Screening as part of a
the level of resistance through (a) multiple strategy for developing R cultivars requires
crosses, (b) recurrent selection, (c) diallel good planning and understanding of the pro-
crossing and (d) selective mating system cesses involved in resistance. A screening pro-
4. Transfer of resistance by (a) intraspecific ped- gramme should be initiated with a clear
igree, backcross and modified recurrent mass statement of the type of resistance, which is
198 9 Resistance

sought, i.e. complete resistance (no sporulation identified only when inoculated singly with
of the pathogen) or partial resistance (reduced each isolate but not if the isolates were used in
sporulation of the pathogen) or both, and with a mixture. The use of a single race provides the
at least some knowledge of pathogenicity and best condition for the selection of partial resis-
virulence patterns in pathogen. The durability tance in the presence of complete resistance,
of resistance can be practically tested only and the selected races should have the broadest
when the R cultivars are widely used in space possible virulence spectrum to suppress the
and time. Multilocation cultivar testing or the expression of as many complete R genes as
challenge of cultivars to a large amount of possible. Genotypes with resistance to one vir-
pathogen population can help verify resistance ulent race should then be systematically tested
and give timely warning of the possibility of with a collection of other isolates. The identifi-
resistance breakdown, but cannot actually be cation of cultivars with complete resistance is a
considered as a test for durability of resistance. first step in the development of effective and
Care must be taken in interpreting results of durable resistance, and genetic analysis of
glasshouse or laboratory tests, as the expres- resistance reaction is essential to reveal simi-
sion of resistance in the field may be consider- larities and differences in the gene(s) that con-
ably different because of interaction between fer resistance in each genotype. Several genes
microorganism, pathogens and environmental known to be effective against a given race or
conditions. genes effective against all prevalent races in a
For foliar pathogens, the plant material must productive region are then recombined in a
be adequately challenged with a single race or single cultivar. Large amount of genetic vari-
a pathotype at a realistic inoculum dose to ability sources are available in cruciferous
allow disease development, but at the same crops in the world, which can be exploited by
time not obscure minor differences in host researchers to develop a resistant cultivar
response required to identify partial resistance. against Alternaria blight in each Brassica crop
The use of inoculum composed of a mixture of (Table 9.17). Figure 9.4 illustrates a scheme to
known races or unknown races/pathotypes in develop resistant cultivars of Brassica (Verma
the naturally infested crop debris will not be and Saharan 1994; Saharan et al. 2003, 2005).
adequate to achieve this objective. Illustrations Methods of testing the resistance and screening
of three cultivars each having a single gene for techniques in the field and glasshouse are given
complete resistance to a given race would be in Chap. 12.

Table 9.17 Sources of cruciferous genetic variability in the world

Exotic Indigenous Adv. breeding Registered Released
Species lines collections lines lines varieties Others Total
Brassica juncea 189 636 656 19 73 263 1836
B. rapa var. Toria 12 67 59 – 14 74 226
B. rapa var. Yellow Sarson 07 78 50 01 12 24 172
B. rapa var. Brown Sarson 01 01 – 02 08 12
B. napus 61 30 09 06 14 120
Eruca sativa 27 19 – 03 49
B. carinata 02 10 03 05 01 21
Others 01 – 15 16
Total 299 782 824 33 115 399 2452
A. rapeseed–mustard germplasm at ICAR-DRMR, Bharatpur
9.16 Bottlenecks in Resistance Breeding 199

A B – SICK PLOT DISEASE - FREE PLOT AB – SICK PLOT

R S
MAKE CROSSES

GROW F1

BULK F2

BULK F3

GROW F4 PROGENIES

GROW F5 PROGENIES

GROW F6 PROGENIES

GROW F7 PROGENIES TEST FOR PREL. YIELD GROW F7 PROGENIES

AB – ALTERNARIA BLIGHT

Fig. 9.4 Scheme to develop high-yielding disease-resistant cultivars (Saharan et al. 2003)

9.16 Bottlenecks in Resistance resistant transgenic plants which overexpress dif-

Breeding ferent antifungal compounds like
pathogenesis-related (PR) proteins (chitinase,
Since resistance against Alternaria black spot is glucanase, osmotin, etc.) and ribosome-inhibiting
generally governed by polygenes, breeding for proteins (RIPs) (thionins, defensins, and phyto-
resistance should involve pyramiding minor alexins) to inhibit growth of the pathogen seem
genes to provide additive/polygene resistance. less efficacious (Zhou et al. 1999).
Rapid advances in techniques of tissue culture, For introduction of Camelina-derived A. bras-
protoplast fusion, embryo rescue and genetic sicicola resistance into commercial varieties,
engineering have made possible the transfer of although somatic hybrids between C. sativa and
disease resistance traits across the otherwise B. carinata have been procured, the researchers
impassable self-incompatibility barriers. Disease- failed to multiply the resulting hybrids
200 9 Resistance

(Narasimhulu et al. 1994). Similarly, strategy of 9.17 Biotechnological

protoplast fusion between C. sativa and B. olera- Approaches
cea with subsequent hybrid regeneration has also
proved unsuccessful (Hansen 1998). Brassica Besides utilizing conventional methods of selec-
research groups, although attempted, did not suc- tion and sexual reproduction, biotechnological
ceed in introducing the E. sativa-derived black techniques involving tissue culture and genetic
spot resistance into various species of cultivated transformation-based methods have revolutionized
crucifers (Fahleson et al. 1988; Sikdar et al. 1990; the search and development of economically
Sigareva and Earle 1997). First somatic hybrids to promising disease-resistant crops. These tools
be obtained as a result of protoplast fusion were have enabled the researchers to combine the traits
those of B. napus (rapeseed) and S. alba (Primard from distantly related Brassica species for the
et al. 1988). None of the hybrids procured that development of superior Alternaria blight-
way showed A. brassicae resistance comparable tolerant varieties of Brassica (Agnihotri et al.
to that exhibited by S. alba. Chevre et al. (1991) 2009; Aneja et al. 2012; Aneja and Agnihotri
used these species for interspecies crosses through 2013; Saharan et al. 2003)
somatic hybridization and biodirectional crosses
using the embryo rescue technique; these
researchers have succeeded in regeneration of B. 9.17.1 In Vitro Embryo Rescue
napus plants carrying 38 chromosomes and dis-
played A. brassicae resistance at levels close to Embryo rescue, documented for the first time in
these of S. alba, B. oleracea var. botrytis or B. the eighteenth century, is one of the most success-
carinata (Ryschka et al. 1996). Seeds of devel- ful techniques for the production of interspecific
oped intertribal somatic hybrids between B. napus and intergeneric hybrids from naturally incompat-
and C. sativa (by means of protoplast electrofu- ible crosses that do not result in the production of
sion) exhibited phenotype intermediate to that of viable offspring. The technique involves excision
the parental species. Although the hybrid plants of the ovary/ovule/embryo resulting from an
exhibited higher levels of linolenic and eicosanoic interspecific cross and its culturing and maturing
acids, their resistance against Alternaria still on suitable media for the production of a hybrid
awaits confirmation (Jiang et al. 2009). plant with desired traits. The technique has proven
Researchers, in general, now realize that intro- very efficient in transferring Alternaria blight tol-
duction of Alternaria resistance genes into com- erance in oilseed Brassicas. In vitro ovary culture
mercial crucifer cultivars is dependent on was used to transfer the resistance against
cumulation of horizontal resistance genes Alternaria blight from B. tournefortii to B. juncea
(Sharma et al. 2002). It is, therefore, imperative cv. RH 30 (Yadav et al. 1991). Agnihotri et al.
to identify sources of horizontal resistance among (1991) developed intergeneric hybrids of B.
the various Brassica species and subsequently campestris and B. spinescens through sequential
combine them to increase durable Alternaria ovary, ovule and embryo culture. The hybrid
resistance. Strong cross-incompatibility, poly- plants were then multiplied using somatic
genic background of the resistance (additive and embryogenesis. The resistance trait from Sinapis
dominant gene interactions) and the differences alba cv. Carine was transferred to B. napus cv.
in ploidy (differing number of chromosomes) Brutor using in vitro fertilized ovary culture
between different Brassicaceae species render (Chevre et al. 1994). Intergeneric hybrids with
the Alternaria resistance transfer from the wild to Alternaria blight resistance were developed
the cultivated species difficult. Additionally, between Erucastrum cardaminoides and B. olera-
researchers often need to employ advanced cea var. alboglabra using sequential ovary and
in vitro hybridization techniques, including ovule culture (Mohanty et al. 2009). Gupta et al.
somatic hybridization, embryo and ovary rescue (2010) also used interspecific hybridization in
or protoplast fusion (Nowicki et al. 2012). combination with in vitro ovule culture to incor-
9.17 Biotechnological Approaches 201

porate high tolerance to Alternaria blight and ture experiments. Sharma and Singh (1995) used
white rust from B. carinata cv. Kiran to low eru- somaclonal variations for incorporation of dis-
cic acid TERl (OE) M21 lines; hybrids with high ease resistance/tolerance against Alternaria
tolerance to Alternaria blight and white rust con- blight. Resistance in Alternaria blight-susceptible
tained negligible amount of erucic acid (1.3 %) B. juncea varieties was achieved by inducing
and high oleic acid (42.5 %). variations through mutagenesis (exposing seeds
to gamma radiations) (Verma and Rai 1980) and
by treating the microspore-derived embryos with
9.17.2 Somatic Hybridization chemical mutagens (Agnihotri et al. 2009)
including ethyl methane-sulphonate (EMS) and
Somatic hybridization refers to the enzymatic ethyl nitrosourea (ENU).
removal of the cell wall and isolation of proto-
plasts which are then fused together to produce
hybrids. Due to the absence of the cell wall, there 9.17.4 Genetic Transformation
occurs a non-specific fusion between the two pro-
toplasts without any barrier to intergeneric/inter- Production of transgenic plants through genetic
specific crosses. It also bypasses both the pre- and transformation forms another biotechnological
postfertilization barriers. This technique has been approach to integrate the disease-resistant genes
utilized effectively in Brassica, but the studies are from resistant/tolerant genotypes to the econom-
limited to B. napus and B. oleracea. Efforts have ically important susceptible ones. The genetic
been made to transfer Alternaria resistant trait transformations in B. juncea have been reported
from Moricandida arvensis to B. oleracea to achieve delayed onset of the Alternaria blight.
(Toriyama et al. 1987) and from S. alba to B. Kanrar et al. (2002) isolated cDNA sequence for
napus (Primard et al. 1988). Sharma and Singh ‘hevein-rubber tree lectin’ from Hevea brasilien-
(1992) attempted the transfer of Alternaria resis- sis and transferred it into B. juncea cv. RLM-198
tance trait from B. carinata to B. juncea. Leaf through binary vector pBinAR. The transgenic
mesophyll protoplasts from M. arvensis and B. plants thus produced supported delayed onset of
napus were hybridized to produce disease-resistant Alternaria blight with considerably low disease
hybrid plants (O’Neill et al. 1996). Jouradan and intensity. Mondal et al. (2003) identified the role
Salazar (1993) resynthesized B. carinata by proto- of ‘chitinase’ in plant defence responses and
plast fusion between B. nigra and B. oleracea, and transformed B. juncea (cv. RLMI98) with chitin-
the hybrids thus obtained were fertile and grew ase gene tagged with overexpressing promoter
into robust plants. Hybridization between S. alba 35S CaMV. The transformed plants resulted in
and B. oleracea and between Camelina sativa and delayed disease onset and about 12–56 % reduc-
B. oleracea has also shown to produce resistant tion in fungal colony size. Osmotin protein,
hybrids (Hansen and Earle 1997; Hansen 1998). known for its role in signal transduction, when
Sigareva and Earle (1999) developed somatic transferred to B. juncea, also showed tolerance
hybrids between S. alba and B. oleracea by proto- to A. brassicae in transgenic plants (Taj et al.
plast fusion followed by embryo rescue and recov- 2004). In another study, transgenic Indian mus-
ered four highly resistant hybrid progenies after tard expressing ‘class I glucanase’ gene under
repeated back crosses. the control of CaMV 35S promoter was devel-
oped (Mondal et al. 2007). For undertaking the
genetic transformation, cDNA encoding ‘class I
9.17.3 Somaclonal Variations glucanase’ gene was isolated from tomato and
cloned into BamHI-Sal I restriction site of binary
These are regarded as the impulsive, inheritable vector, pBinAR, flanking CaMV 35S constitutive
changes in gene expression which often appear promoter and an ocs terminator region. The
under in vitro conditions especially in tissue cul- resulting recombinant pBinGB clone was
202 9 Resistance

transferred into Agrobacterium tumefaciens by the use of molecular markers has come to the
strain GV 2260, which was further used to trans- aid of plant breeders and pathologists. The main
form B. juncea (cv. RLM 198). Recent study by advantage of using these fingerprinting tech-
Verma et al. 2012 reported the introgression of niques is that they do not require any prior
cysteine-rich antimicrobial peptide, PmAMP1, knowledge or availability of variable sequences
from Pinus monticola into B. napus, thus confer- in the database, as they deal with the genome-
ring enhanced protection against A. brassicae, wide biodiversity.
Leptosphaeria maculans and Sclerotinia sclero- Various molecular markers being used for
tiorum. Combined expression of type I ribosome- assessing variability include internal tran-
inactivating protein (RIP; AAA32951) and barley scribed spacer (ITS) regions, restriction frag-
‘class II chitinase’ gene in B. juncea not only ment length polymorphism (RFLP), randomly
reduced the growth of fungal hyphae by 44 % amplified polymorphic DNA (RAPD), ampli-
but also affected the onset of disease (Chhikara fied fragment length polymorphism (AFLP),
et al. 2012). Efforts have also been made to study inter-simple sequence repeats (ISSRs), micro-
the signal transduction pathways in Brassica satellite, sequence-tagged sites (STS) and
species. Taj et al. (2011) reported interaction of single-nucleotide polymorphisms (SNPs). Due
MAPK 3 and Lox genes during pathogenesis of to the presumed dependency on traditional
A. brassicae, which is suggested to play a role in characterization, only a few studies are reported
biosynthesis of jasmonic acid and jasmonic acid- on molecular characterization of Alternaria
induced expression of defence genes in B. species pathogenic to oilseed brassicas. The
juncea. ITS regions (also called the conserved regions)
in the fungal genome are considered the most
popular locus for DNA-based mycological
9.17.5 Molecular Markers studies at species level. Chou and Wu (2002)
and Berbee et al. (2003) studied the ITS region
As the efficient management of plant diseases of rDNA of A. brassicae and A. brassicicola to
relies on the accurate identification of the plant evaluate the phylogeny of the pathogen.
pathogen, knowledge of the variability in the Molecular techniques, like RAPD, that do not
pathogen population, both at the pathological require any prior knowledge of the DNA
and genetic levels, is very important. sequences have been efficiently used to analyse
Characterization of variability in the pathogens the genetic variations in Alternaria species
including Alternaria spp. has been done earlier (Cooke et al. 1998; Sharma and Tewari 1995,
using traditional methods of morphological 1998; Gherbawy 2005). However, due to the
markers (Goyal et al. 2011), biochemical tests limitation of reproducibility in RAPD-
(Vishwanath and Kolte 1997) and cultural and generated data, the assaying of genetic variabil-
pathogenic assays (Goyal et al. 2011; Singh ity in Alternaria species has shifted to more
et al. 2011). These markers differentiate the sensitive techniques like AFLP (Bock et al.
pathogens on the basis of their growth behav- 2002) and microsatellite markers. Twelve poly-
iour, physiological characters and microscopic morphic microsatellite markers were isolated
appearance. However, these criteria are greatly from A. brassicicola. The number of alleles
influenced by different factors including age detected in 12 loci ranges from 2 to 10 (mean
and quality of the inoculum, age and type of 3.5). Cross-species amplification shows that the
host, culture media used and incubation condi- designated primers are specific to the pathogen
tions. Also, these techniques are more laborious (Avenot et al. 2005). Though these molecular
and time-consuming and often give non-repro- tools have revealed huge amount of genetic
ducible results. To overcome these limitations variations in Alternaria at the subgeneric as
of traditional markers, detection of genetic vari- well as subspecies level, this field still remains
ability through DNA fingerprinting technology largely unexplored and demands an extensive
9.18 Factors Affecting Plant Disease Resistance 203

study into the variability at different geographic molecular characterization of the pathogen, the
levels among different hosts. clear outcome is still awaited.
Variations in A. brassicae isolates have been
recorded in traits including colony morphology,
9.17.6 Induction of Systemic colony diameter ranging from 32 to 68 mm, sur-
Resistance face texture from velvety to woolly, colour from
cream to dark olive green and sporulation from
Induced host resistance refers to the activation very less to intense. The host–pathogen interac-
of resistance in otherwise susceptible plants, tion studies using four varieties of B. juncea and
through biotic as well as abiotic agents (Stitcher two of B. rapa have shown pathological variation
et al. 1997; Kessman et al. 1994), without among the isolates that differ in their
changing their basic genetic make-up. Foliar aggressiveness from avirulent (few) to highly
application of β-aminobutyric acid induces virulent (Aneja et al. 2012; Aneja and Agnihotri
resistance against A. brassicae in B. carinata 2013). A thorough knowledge of diversity in the
(Chavan et al. 2013). GLIP1 protein in associa- pathogen will not only help in characterizing and
tion with ethylene signalling is a critical compo- analysing it phylogenetically but also in inducing
nent in plant resistance to A. brassicicola (Oh durable resistance against Alternaria blight,
et al. 2005). The resistance/defence-related which can act as an environment-friendly substi-
genes in the vulnerable plants can be activated tute to the traditional use of fungicides.
by inoculating the plant either by an avirulent
form of the pathogen or by limited inoculation
with the pathogen (Deverall 1995). The infect- 9.17.7 Genetic Engineering
ing avirulent pathogen triggers natural defence
responses in the plant through the release of the Development of Alternaria-resistant genetically
elicitors, which then result in the expression of modified plants (GM) is an excellent approach to
novel anti-pathogenic proteins. In order to harp manage Alternaria diseases of crucifers.
the benefits of induced host resistance and build Resistant genes from other plants or bacteria
up a stable, long-term resistance mechanism in encoding enzymes like chitinase or glucanase
the host plant against the pathogen, there is a can be introduced in crucifers. Chitinase and glu-
need to identify the pathogen and understand its canase, respectively, breakdown chitin and glu-
behaviour under diversified conditions. In case can, which are essential components of fungal
of Alternaria blight, considerable morphologi- cell wall. Introduction of plant genes to enhance
cal and pathological variations have been innate plant defence mechanisms like activating
observed in A. brassicae under different envi- phytoalexins, inhibiting proteinase and detoxify-
ronmental conditions (Goyal et al. 2011; Kaur ing phytotoxins is another useful way of develop-
et al. 2007; Singh et al. 2007; Patni et al. 2005; ing resistant varieties. Invoking hypersensitive
Meena et al. 2005, 2012; Ansari et al. 1989; reaction when a fungal pathogen is attacking can
Awasthi and Kolte 1989; Saharan et al. 2015). also be an innovative approach.
Vishwanath et al. (1999) reported the induction
of resistance in susceptible B. juncea cv. PR-15
against extremely virulent A. brassicae isolate 9.18 Factors Affecting Plant
A (AbA) and moderately virulent isolate C Disease Resistance
(AbC) from B. carinata cv. PPCS-1 by inoculat-
ing with an avirulent isolate D (AbD) isolated Different biology of the several types of plant
from the same B. carinata cultivar. About 60 % pathogens also presents a substantial problem in
reduction in the disease severity was observed developing GM-resistant plants. Firstly, the kind
against AbA and AbC by the induction of resis- of organisms causing disease is taxonomically
tance. However, due to the limited studies in highly diverse; the major groups include cellular
204 9 Resistance

pathogens (bacteria, fungi and the algal oomyce- clustered and have undergone duplication and
tes) and molecular pathogens (viruses). These are evolution due to diversifying selection. Functional
physiologically very different from each other, analysis of these entire candidate genes is a
and therefore, no single gene product can be demanding task, but improvement in plant trans-
expected to have a direct toxic effect on all types formation protocols and high-throughput gene
of pathogens. Secondly, pathogens use two major attenuation methods, such as RNA interference
life strategies, namely, biotrophic and necrotro- (RNAi) and virus-induced gene silencing (VIGS),
phic. Biotrophic pathogens essentially act as a should accelerate the identification of novel
sink for the host’s anabolic assimilates and there- genes of practical utility.
fore keep it alive. Meanwhile, necrotrophic
pathogens consume the host tissues on invasion.
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 Phytotoxins
10

an understanding of the pathological roles of tox-

10.1 Introduction ins in relation to epidemiology of diseases as
well as successful pathogenesis appears to be the
The long search for a ‘toxin’ produced by plant critical need and should be the ultimate objective
pathogens met with disappointment until atten- of toxin study for plant pathologists (Nishimura
tion was centred on a host-specific toxin (HST). and Kohmoto 1983).
The disappointment that spanned almost half a Many pathogenic Alternaria species produce
century resulted mostly from the study of toxins toxins, which facilitate their necrotrophic life-
that function in various types of symptom devel- style. Prior to colonization, necrotrophs must kill
opment, but are not the initial inciting agents of their host cells at a distance by producing both
disease. ‘Microorganism X (but not others) pro- toxins and lytic enzymes often by triggering
duces substance Y which damages plant or plant genetically programmed apoptotic pathways or
group A (but not others), and only A is parasit- by directly causing cell damage resulting in
ized’. This statement by Wood (1973) showed necrosis. Many species of Alternaria produce
serious need for toxin study. The finding of a toxins with broad ranges, but some agronomi-
group of highly host selective HSTs, valuable cally important species produce very host-
both as tools for academic research on plant specific toxins with a narrow range often to the
host–parasite interactions and as markers for epi- cultivar level. Alternaria is an important patho-
demiological survey of pathogens in fields, con- gen of many crucifers, and because several of its
firms that the search for toxins is an important species produce carcinogenic, teratogenic and
part of modern plant pathology. mutagenic mycotoxins, researchers around the
The present knowledge of HSTs has come globe are using this necrotrophic pathogen to
almost entirely from the so-called saprophytic study molecular mechanisms of plant defence
pathogens. This group includes familiar fungi, (Oliver and Ipcho 2004; Thaler et al. 2004; Rawe
such as Alternaria and Helminthosporium, and and Kliebenstein 2010). Using A. brassicicola as
others less well known. It is just within the last 30 model representative for their basic research on
years (1985 to date) that we have really begun to virulence, Lawrence et al. (2008) generalized the
work with HSTs in Alternaria species infecting role of toxins in the pathogenesis of genus
crucifers. Until recently, our attention for the tox- Alternaria, and Chung (2012) studied the stress
ins was biased towards the physiological or bio- response and pathogenicity of the necrotrophic
chemical mechanisms of their selectivity and fungal pathogen Alternaria alternata. This chap-
elucidation of their chemical structures. However, ter deals with nature and structure of Alternaria

© Springer Science+Business Media Singapore 2016 211

G.S. Saharan et al., Alternaria Diseases of Crucifers: Biology, Ecology and Disease Management,
DOI 10.1007/978-981-10-0021-8_10
212 10 Phytotoxins

toxins, other metabolites and their effect on destruxin B are similar to A. brassicae on various
pathogenesis of Alternaria spp. at physiological, brassicas (Plates 10.1 and 10.2; Tewari 1991).
biochemical and molecular level along with the Destruxin B is also produced by Metarhizium
role in host resistance. anisopliae and some other fungi and has insecti-
cidal properties as well (Gupta et al. 1989).
Destruxin B is a tripeptide based on isoleucine,
10.2 Historical Developments proline and β-alanine. The second phytotoxin,
homodestruxin B with molecular formula C31 H53
The present knowledge of phytotoxins from N5 O7 (Ayer and Pena-Rodriguez 1987a, b),
Alternaria species infecting crucifers received affects both hosts and non-hosts of A. brassicae
impetus from the age-old discovery of non- (Bains 1989).
specific toxins (tab toxins, fusaric acid, pyricula- Reversed-phase chromatography on Sep-Pak
rin) and host-specific toxins (victorin, Periconia CI8 cartridges followed by high-performance liq-
circinata toxin, Alternaria kikuchiana toxin). Out uid chromatography (HPLC) has successfully
of the four species of Alternaria infecting cruci- been used for the separation and detection of
fers, much of the information on phytotoxins has destruxins from M. anisopliae (Samuels et al.
been generated on A. alternata pathogenesis, that 1988). Using a modification of this method,
too with other than crucifers host pathosystem. three destruxins, viz. destruxin B, destruxin B2
However, research on phytotoxins in the and homodestruxin B, have been isolated from
Alternaria–crucifers host pathosystem was initi- A. brassicae (Buchwaldt and Jensen 1991;
ated during 1980–1990. Credit goes to Tewari Buchwaldt et al. 1991). According to Buchwaldt
and Pedras from Canada who along with their and Green (1992), destruxin B, the major phyto-
associates did excellent work on toxins in toxin produced by A. brassicae, is not host-
Alternaria–crucifers host pathosystem. specific as reported earlier (Bains and Tewari
Alternaria brassicae has a multitoxin system 1987). This toxin causes necrosis and chlorosis
and produces at least three phytotoxins. One phy- on 30 species of host and non-host plants. Two
totoxin is the cyclodepsipeptide, destruxin B, minor destruxins, homodestruxin B and
having molecular formula C30 H51 N5 O7 and a destruxin B2, are phytotoxic to leaves of B.
molecular weight of 593 (Ayer et al. 1987; Ayer napus. However, there are significant differences
and Pena-Rodriguez 1987a, b; Bains 1989; Bains between taxonomic plant groups in their sensi-
and Tewari 1986, 1987, 1989). The effects of tivity to destruxin B. Brassica species are most

Plate 10.1 Symptoms caused

by inoculation of Brassica
napus cv. Altex leaf with
Alternaria brassicae (left leaf).
The right leaf is the control
(Tewari 1991)
10.2 Historical Developments 213

Plate 10.2 Symptoms caused

by the application of destruxin
B on Brassica napus cv. Altex
leaf (left leaf). Compare with
Plate 10.1. The right leaf is the
control (Tewari 1991)

sensitive to the toxin, and sensitivity decreases host systems with cell walls would be suitable
as relatedness of plant groups become more dis- for this purpose because the protoplasts are
tant. The dilution endpoint of destruxin B is insensitive to the toxin (Bains and Tewari 1989).
0.2–3.8 μg/ml for the most sensitive host species In the presence of destruxin B, leaf discs of
and 15–120 μg/ml for the least sensitive. The the susceptible host B. rapa var. Yellow Sarson
sensitivity of non-host species is between 15 and lose significantly more electrolytes than the mod-
750 μg/ml; destruxin B shows a high degree of erately resistant B. rapa var. rapifera (turnip).
biological activity (Bains 1989; Bains and The effect takes up to 48 h to become apparent,
Tewari 1989). In detached rapeseed leaf bioas- and the lowest concentration of toxin detectable
say, the limit of detection of destruxin B lies by this bioassay method is 10 μg/ml. The delayed
between 15 and 30 μg/ml, and it is possible to electrolyte leakage indicates that perhaps the tar-
distinguish pathogen susceptibility differences get site of the toxin in the host cell is not situated
among host species (Bains and Tewari 1987). directly on the cell membrane, because of the
The symptoms caused by destruxin B appear to delay for the permeability change to occur and
be light dependent. Destruxin B appears to be a the electrolyte leakage to start. The mesophyll
virulence factor, contributing to the aggressive- protoplasts of both susceptible (B. rapa var.
ness of A. brassicae by conditioning the host tis- Yellow Sarson) and resistant (Camelina sativa)
sue and, thereby, determining the susceptibility hosts are not affected by destruxin B, even at a
of the host. Homodestruxin B is able to affect concentration of 100 μg/ml. This indicates that
both the host and non-hosts of A. brassicae and, the target site of the toxin must be in the cell wall
therefore, functions as a non-host-specific toxin. or the periplasmic space. Germination and tube
Homodestruxin B differs from destruxin B by growth of pollen grains of the susceptible host (B.
the replacement of a methyl with an ethyl group rapa var. Yellow Sarson) have been found to be
(Bains et al. 1993). Somaclonal variation pro- very sensitive to destruxin B concentration as low
duced in cultured cells and tissues provides a as 2.5 μg/ml for 30 min. Complete inhibition of
good system for selection of new disease- these processes occurs at a concentration of
resistant genotypes of brassicas using destruxin 10.0 μg/ml (Bains and Tewari 1989). These pro-
B. As a host-specific toxin, destruxin B is the cesses are not completely inhibited in the resis-
primary determinant of the black spot disease of tant host (Camelina sativa), even at a toxin
the Brassicaceae. It should be possible to use concentration of 100 μg/ml. Past 1990, phyto-
destruxin B as a selection agent for disease resis- toxin research mushroomed to reach to the pres-
tance in tissue culture systems. Tissue culture ent status.
214 10 Phytotoxins

10.3 Metabolites from Alternaria Polygonum cuspidatum and Glycine max (Lou
et al. 2013).
Some metabolites from Alternaria fungi are toxic The Brassica pathogen, Alternaria brassicae
to plants and animals and are designated as phy- when grown in liquid-still culture produces host-
totoxins and mycotoxins, respectively. These specific phytotoxins; cyclodepsipeptides; drimane
mainly include nitrogen-containing metabolites, sesquiterpenes deoxyuvidin B (4), alberassitriol
steroids, terpenoids, pyranones, quinones and (8) and isoalbrassitriol (9); brassicadiol (10); and
phenolics. Alternaria metabolites exhibit a vari- a C15 prenylated pentaketide. These compounds
ety of biological activities such as phytotoxic, are not phytotoxic to canola (Ayer and Pena-
cytotoxic and antimicrobial properties, which Rodriguez 1987a, b). Alternaria brassicae pro-
have drawn the attention of many chemists, phar- duces host-specific, ABR toxins (protein) from
macologists and plant pathologists in research germinating spores on host leaves (Otani et al.
programmes as well as in application studies. 1998; Parada et al. 2008). AB toxin from A. bras-
Porritoxin 24 from endophytic Alternaria species sicicola germinating spores is induced by a host-
has been reported as the candidate of cancer che- derived oligosaccharide (Oka et al. 2005).
motherapeutic agent. Depudecin, an inhibitor of Metabolites from cultures of A. brassicicola
histone deacetylase (HDAC) from A. brassicic- are phomapyrone F (4.1 mg), phomapyrone A
ola, also shows its antitumor potency. Some (3.30 mg), phomapyrone G (5, 1 mg), brassicico-
Alternaria metabolites such as tenuazonic acid, lin A (1, 30 mg), brassicicene H (8, <1 mg),
maculosin and tentoxin act as the herbicide can- brassicicene G (7, 4 mg), brassicicene I (9, 1 mg)
didate. In the early 1990s, about 70 metabolites and infectopyrone G (6, 2 mg). The three new
from Alternaria fungi were reviewed. Several fusicoccane diterpenes, brassicicenes G (7), H
reviews on Alternaria phytotoxins have been (8) and I (9), were named by analogy to previous
published over the last few decades. In recent designations (MacKinnon et al. 1999).
years, more than 268 metabolites with bio- From the least polar and non-phytotoxic frac-
activities from Alternaria fungi have been iso- tions, phomapyrones A (3), F (4), G (5) and
lated and structurally characterized (Lou et al. infectopyrone (6) were isolated and structures
2013). were determined by comparison of their chro-
matographic and spectroscopic data with those of
authentic samples isolated from Leptosphaeria
10.3.1 Classification and Occurrence maculans and from L. biglobosa (Pedras et al.
1994; Pedras and Biesenthal 2001; Pedras and
The metabolites from Alternaria fungi can be Chumala 2005).
grouped into several categories which include From the most phytotoxic fractions, F4 and
nitrogen-containing compounds, steroids, terpe- F5, brassicicolin A (1) was obtained as a colour-
noids, pyranones (pyrones), quinones and pheno- less and chromatographically homogeneous oil
lics (Table 10.1). Several metabolites are unique (HPLC Rt, 27.3 min); HR ESI-MS analysis indi-
to one Alternaria species, but most metabolites cated a molecular formula of C32H48N2O14, and
are produced by more than one species. The most the FT IR spectrum contained strong bands at
widespread metabolite is alternariol which has 3400, 2145 and 1760 cm−1, suggesting the pres-
been isolated from a few Alternaria species. ence of hydroxyl, isonitrile and carbonyl groups,
Some metabolites have also been isolated from respectively.
other fungal genera and even from higher plants. The 1H NMR spectrum was complex suggest-
Typical examples include AAL toxins from ing that the sample was actually a mixture of
Fusarium species, helvolic acid from Aspergillus closely related compounds. Likewise, the 13C
species and Pichia species, paclitaxel from yew NMR spectral data showed that each cluster of
trees (Taxus spp.) and resveratrol from a variety signals contained four resonances indicating the
of plant species including Vitis vinifera, presence of four isomers. Comparison of these
10.3 Metabolites from Alternaria 215

Table 10.1 Isolated metabolites from Alternaria pathogenic to crucifers (Lou et al. 2013)
Metabolic class Metabolite name Alternaria species
Terpenoids Brassicicene A (106) A. brassicicola
Brassicicene B (107) A. brassicicola
Brassicicene C (108) A. brassicicola
Brassicicene D (109) A. brassicicola
Brassicicene E (110) A. brassicicola
Brassicicene F (111) A. brassicicola
Brassicicene G (112) A. brassicicola
Brassicicene H (113) A. brassicicola
Brassicicene I (114) A. brassicicola
Abscisic acid = ABA (115) A. brassicae
Tricycloalternarenes (TCAs) A. alternata
Pyranones Phomapyrone A A. brassicicola
Phomapyrone G A. brassicae
Nitrogen-containing metabolites Destruxin B (50) A. brassicae
Homodestruxin B (51) A. brassicae
Demethyldestruxin B (52) A. brassicae
Brassicicolin A (58) A. brassicae
Steroids 3β-Hydroxy-ergosta 5,8 (9), 22-trien-7-one (66) A. brassicicolaML-P08
3β,5α-Dihydroxy-ergosta 7, 22-dien-6-one (67) A. brassicicola ML-P08
Cerevisterol (68) A. brassicicola ML-P08
Pyranones Herbarin A (132) A. brassicicola ML P08
Altechromone A (151), 2,5-dimethyl-7 A. brassicicola ML P08
Hydroxychromone (152) Alternaria sp.
Phomapyrone F (153) A. brassicicola
Miscellaneous (polyketide) Depudecin (257) A. brassicicola

spectroscopic data and specific optical activity Three related metabolites were isolated from
([α] D = +16 (c 1.0, CH2Cl2); lit.[α] D =16.1(c, fractions displaying low phytotoxicity. The 1H
1.86 CH2 Cl2)) with those previously reported NMR spectrum of each of these metabolites
(Gloer et al. 1988), along with chemical hydroly- showed the presence of an isopropyl, a methoxy-
sis results, indicated that this metabolite was methylene and two methyl substituents suggest-
brassicicolin A (1), a derivative of mannitol con- ing that these metabolites had fusicoccane
taining two acetyl, two structures, similar to those previously reported
2-isocyano-3-methylbutanoyl and two 2 hydroxy- for brassicicenes A-F (MacKinnon et al. 1999).
3-methylbutanoyl substituents. Fraction F5 was The most oxidized metabolite was obtained as a
further separated to yield a chromatographically white solid whose molecular formula was deter-
homogenous material (C26H41NO13) shown to mined to be C21H32O5 (six-double bond equiva-
contain a mixture of nonsymmetrical mannitol lent) based on NMR and HRE1-MS data. The 1H
derivates in which one of the NMR spectrum displayed four methyl doublets, a
2-isocyano-3-methylbutanoyl groups was miss- methoxyl group and one olefinic proton.
ing. The 13C NMR spectrum displayed clusters of Metabolites from leaves of B. juncea inocu-
four to six peaks, suggesting that the fraction lated with A. brassicicola were detected by
contained a mixture of at least four compounds. Pedras et al. (2009). No phytoalexins were
Given the complexity of this fraction and the detected 24 h post inoculation; four phytoalexins
presence of inseparable epimers, no structures were detected 48 h post inoculation and were
were assigned to each specific component. quantified using calibration curves constructed
216 10 Phytotoxins

with synthetic samples (Pedras et al. 2007). The ity factors, which the pathogenic fungi producing
phytoalexin spirobrassinin was produced in them require to invade tissue and induce disease.
larger amounts than brassicic, cyclobrassinin or All isolates of the pathogen that produce an HST
rutalexin. As well, indolyl-3-acetonitrile was are pathogenic to the specific host. All isolates
detected in higher concentration in leaves (0.8– that fail to produce HSTs lose their pathogenicity
4.0 nmol/g fresh weight) of infected plants than to the host plants. Plants that are susceptible to
in control leaves (<0.1 nmol/g fresh weight). the pathogen are sensitive to the toxin. Such cor-
Furthermore, HPLC-MS analysis of leaf extracts relations between HST production and pathoge-
did not detect any of the metabolites produced by nicity in the pathogens, and between toxin
A. brassicicola in liquid cultures. sensitivity and disease-producing ability in
Nine novel compounds closely related to plants, provide persuasive evidence that HSTs
ACTG toxin, termed as tricycloalternarenes, can be responsible for host-specific infection and
were isolated from a strain of A. alternata, from disease development. Johnson et al. (2000, 2001)
B. sinensis, which was earlier used for the pro- revealed that the genus involved in HST synthe-
duction of non-specific phytotoxin, tentoxin sis such as the cyclopeptide synthetase gene,
(Nussbaum et al. 1999). Liebermann et al. (2000) whose product catalysed AM toxin production in
also reported isolation of 11 new bicycloalter- A. alternata apple pathotype, might reside on a
narenes as well as ACTG toxins A and B. conditionally dispensable (CD) chromosome.
Alternaria brassicae (Berk.) Sacc. produces four The loss of the CD chromosome led to loss of
cyclic depsipeptide phytotoxins including both toxin production and pathogenicity without
destruxin B, homodestruxin, destruxin B2 and affecting fungal growth. On the other hand, the
desmethyldestruxin B host-specific protein- exact roles of non-specific toxins in pathogenesis
aceous ABR toxin (Ayer and Pena-Rodriguez are largely unknown, but some are thought to
1987a, b; Buchwaldt and Green 1992; contribute to the features of virulence, such as the
Montemurro and Visconti 1992; Agarwal et al. symptom development, and in plant pathogen
1994; Parada et al. 2008). In addition to protein- propagation. The virulence and host specificity of
aceous AB toxin (Otani et al. 1998) and brassicil- these pathogens are based on the production of
lin A, a very selective phytotoxin metabolite in the distinctive HSTs. Most of Alternaria HSTs
liquid cultures (Pedras et al. 2009), A. brassicic- are nitrogen-containing metabolites causing
ola also produces depsipeptides and fusicoccin- black spot disease in brassicas, destruxin A,
like toxin compounds (Cooke et al. 1997; destruxin B, homodestruxin B and desmethyl-
MacKinnon et al. 1999). destruxin B. Destruxins are group of HSTs pro-
duced both in vitro and in planta by A. brassicae,
the causal agent of Alternaria black spot disease
10.4 Effect on Plants of rapeseed and canola. These cyclodepsipep-
at Physiological, Biochemical tides exhibit a wide variety of biological activi-
and Molecular Level ties such as antitumor, antiviral, insecticidal,
cytotoxic, immunosuppressant and anti-
The metabolites from plant pathogenic fungi are proliferative effect except their phytotoxicity.
usually toxic to plants and are called phytotoxins. Interaction between Alternaria species and cruci-
They are further divided into host-specific and ferous plants was studied in detail by Pedras et al.
non-host-specific toxins. The host-specific toxins (2009). Necrotrophic phytopathogens such as A.
(HSTs) are toxic only to host plants of the fungus alternata and A. brassicae are known to synthe-
that produces the toxin. Another definition seems size phytotoxins that damage plant tissues and
to be more acceptable that the host-specific tox- facilitate colonization, while in response to
ins are toxic to plants that host the pathogen, but pathogens’ attack, crucifers biosynthesize phyto-
have lower phytotoxicity on non-host plants. anticipins and phytoalexins. Phytoalexins are
Most HSTs are considered to be the pathogenic- secondary metabolites produced de novo by
10.4 Effect on Plants at Physiological, Biochemical and Molecular Level 217

plants in response to diverse forms of stress detoxified into 3-indolyl-methanamine and

including microbial infection, UV irradiation and N″-acetyl-3-indolylmethanamine by the patho-
heavy metal salts, whereas phytoanticipins are gen A. brassicae (Fig. 10.1). Very interestingly,
constitutive defences whose concentrations can cruciferous plants (B. napus and Sinapis alba)
increase upon stress. To the detriment of crucifer- can convert host-specific toxins, destruxin B and
ous plants, the phytopathogens can overcome homodestruxin B, into less phytotoxins,
phytoanticipins and phytoalexins by producing hydrodestruxin B and hydroxyl-homodestruxin
detoxifying enzymes; phytoalexin brassinin is B, respectively (Fig. 10.2; Lou et al. 2013).

H H
N N
SCH3 R

S A. brassicicola

N N
H H
3-Indolylmethanamine, R = H
Brassinin
N ⬙-Acetyl-3-indolylmethanamine, R = Ac

Fig. 10.1 Detoxification pathway of the phytoalexin brassinin by the pathogen A. brassicicola (Lou et al. 2013)

HO

N
O N
O
O Brassica napus
O O NH O
O O NH
O Sinapis alba
HN O O
HN O
N
N N
N

Destruxin B Hydroxydestruxin B

HO

N
O N
O
O Brassica napus
O O NH O
O O NH
O Sinapis alba
HN O O
HN O
N
N N
N

Homodestruxin B Hydroxyhomodestruxin B

Fig. 10.2 Detoxification pathway of the phytotoxin B and homodestruxin B by the hosts Brassica napus and Sinapis
alba (Lou et al. 2013)
218 10 Phytotoxins

Non-host-specific Alternaria phytotoxins can the least sensitive to the toxin. Effects of toxin on
affect many plants regardless of whether they are leaves of these species and the degree of sensitiv-
a host or non-host of pathogen. Non-host-specific ity of leaves of different species are comparable
nitrogen-containing phytotoxins include tenua- to that of their pollen grains. The results on the
zonic acid, porritoxin and tentoxin. Tentoxin, a responses of pollen grains as well as leaves to the
cyclic tetrapeptide from A. alternata, inhibits toxin are in agreement with the degree of suscep-
chloroplast development, which phenotypically tibility/resistance of these species to A. brassi-
manifests as chlorotic tissue. Tentoxin was sug- cae, indicating that the genes imparting
gested to exert its effect on chlorophyll accumu- susceptibility/resistance are expressed in the pol-
lation through over-energization of thylakoids len, a prerequisite for pollen selection. The toxin
(Lou et al. 2013). fed to the cut end of isolated inflorescence axis is
Destruxin B purified from culture filtrates readily taken up by the developing pollen and
(CFs) of A. brassicae induces chlorosis on host results in the inhibition of germination of
leaves at 50–100 μg ml−1 and chlorosis or necro- susceptible pollen (Hodgkin and Macdonald
sis on non-host leaves at 250–500 μg ml−1 (Bains 1986; Shivanna and Sawhney 1993).
and Tewari 1987). Destruxin B was detected in
spore germination fluids (SGFs) on host and non-
host leaves, but not in sufficient amount to exert 10.4.1 Physiological Level
toxicity to host plants. When spores of non-
pathogenic A. alternata are combined with The effect of phytotoxins on plants at the physi-
destruxin B at 100 μg ml−1 and inoculated on ological level is characterized by the malfunc-
leaves, destruxin does not affect the infection tioning of many physiological processes
behaviour of the spores. Interestingly, SGF on including respiration, transpiration, photosynthe-
host leaves allows non-pathogenic spores to colo- sis, translocation and growth and development. It
nize host leaves (Bains 1989). Moreover, a high is also characterized by the appearance of spe-
molecular weight fraction (>5 kDa) without cific symptoms including wilting, growth sup-
destruxin B obtained by ultrafiltration of SGF has pression, chlorosis, necrosis and spotting of
host-specific toxic activity and infection-inducing aerial portions.
activity. According to Parada et al. (2007), Alternaria HSTs are classified into three
destruxin B is not an HST and does not induce groups in terms of primary site of action. AM
the accessibility of the host plant, which is essen- toxin targets the chloroplast and plasma mem-
tial for colonization of the pathogen. In addition, brane, ACR (L) toxin targets mitochondria, and
the results with SGF imply that a high molecular ACT, ACTG, AK and AF target the plasma mem-
weight HST(s) is involved in the host–pathogen brane. Toxins produced by A. brassicae cause a
interaction. brown necrotic spots on leaves and brown streaks
According to Khandelwal et al. (2002), P53- on stem leading to yield losses. In general, respi-
like protein is overexpressed in toxin-treated and ration increases once parasitic relationship is
nutritionally depleted calli. Similar changes are established. Alternaria pathogens infect green
seen in senescent damaged Brassica species indi- aerial tissues and reduce photosynthetic activity;
cating involvement of P53-dependent pathways. produce cytokinins, which lead to the formation
The effects of destruxin B, a host-specific toxin of Green Island below the germinating conidia on
of A. brassicae that causes black spot disease in senescing tissues; and cause deformation in chlo-
oilseed brassicas, were studied on in vitro pollen roplast and mitochondria (Chaturvedi 1972;
germination and pollen tube growth of Brassica Zheng et al. 2006). In B. juncea, A. brassicae,
campestris var. Brown Sarson, B. juncea cvs. HST destruxin B, inhibits almost all macromo-
Westar and Cresor, B. nigra and Sinapis alba. lecular biosynthesis, promotes ion leaching and
Pollen grains of B. nigra, B. juncea and B. camp- causes aberrations in mitochondria and
estris were the most sensitive and those of S. alba chloroplast.
10.4 Effect on Plants at Physiological, Biochemical and Molecular Level 219

10.4.2 Biochemical Level Results of a transcript profiling study of

hsr203J, a known marker gene for HR, showed
Infection of plants by pathogenic fungi may trig- that the expression of hsr203J gene is more in A.
ger several biochemical defence responses includ- brassicae-tolerant than in A. brassicae-
ing enzyme synthesis, cell wall deposition of susceptible genotype of B. juncea (Mishra et al.
lignin and suberin and accumulation of specific 2011). In Chinese cabbage, A. brassicae infec-
metabolites (Daayf and Platt 2000; Abdel-Farid tion significantly increases glucosinolates (ali-
et al. 2009). The mechanisms of host-selective phatic and indole) and anthocyanins, but
pathogenesis are not well understood at the bio- decreases sucrose levels (Ludwig-Müller et al.
chemical level, even in cases where the toxin site 1999; Rostás et al. 2002; Abdel-Farid et al. 2009).
of action is known. The first physical barrier Chitin, an important component of fungal cell
between plants and pathogen invasion is the cuti- wall, is absent in plants, but chitin-degrading chi-
cle (Schweizer et al. 1996; Fan and Köller 1998) tinase enzymes are present in abundant quality
and cell wall, which inhibit both initiation and which emphasizes that chitinase can directly
spread of infection. One characteristic feature of affect the viability of fungal pathogens (Boller
many phytopathogenic organisms is their ability to 1995; Stacey and Shibuya 1997; Shibuya and
produce an array of extracellular and highly stable Minami 2001). This is proved by overexpression
enzymes capable of degrading the complex poly- of chitinase in transgenic plants, which are gener-
saccharides of the plant cell wall and membrane ally more resistant to fungal pathogens. Further,
constituents. These enzymes usually are produced it was observed that more chitinase is produced
inductively in infected tissues; generally, they are and accumulates at the site of fungal infection
extracellular and highly stable. Inoculation of A. (Majeau et al. 1990; Roby et al. 1990; Wubben
brassicae on the leaves of B. juncea blight resis- et al. 1992). It is reported that co-expression of
tant cultivar RC-781 decreases the activities of cell chitinase and ribosome-inactivating protein in B.
wall-degrading polygalacturonase and cellulose juncea provides more protection against A. bras-
enzymes, but increases their activities in a suscep- sicae (Chhikara et al. 2012).
tible cultivar Varuna up to 3 days of infection AAL toxin, structurally related to sphinganine,
(Garg et al. 1999). Infection of A. brassicicola a member of sphinganine-analogue mycotoxin
shows a differential and sequential induction of (SAMS), is an inhibitor of sphinganine-N-acyl-
two classes of cutinolytic esterases; one class is transferase (Gilchrist et al. 1992; Abbas et al. 1994)
expressed only in short duration contact (24 h) enzyme important in sphingolipid biosynthesis,
with intact cutin, but not induced by cutin mono- leading to the accumulation of free sphingoid bases
mer. The second class, however, is induced by (Brandwagt et al. 2000; Spassieva et al. 2002;
cutin monomers only in prolonged exposure with Gechev and Hille 2005). These long-chain sphin-
intact cutin. This differential behaviour indicates a goid bases (LCBs) are determinant in the occur-
sequential recognition of cutin as a barrier to be rence of programmed cell death (PCD) in
penetrated and utilized as a carbon source in sap- susceptible plants (Shi et al. 2007; de Zelicourt
rophytic stages (Yao and Köller 1995; Fan and et al. 2009). Sphinganine-analogue mycotoxin
Koller 1998; Bakar et al. 2005). The small (7–10 (SAMS) inhibits ceramide biosynthesis.
KD) lipid transfer proteins (LTPs) are expected to Biochemical and molecular data demonstrated that
be involved in wax transport because of their PCD triggered by AAL toxin is also associated
increased expression during drought condition with H2O2 (Gechev et al. 2004) as atr mutant of
(Beisson et al. 2003; Cameron et al. 2006). During Arabidopsis which shows enhanced tolerance to
A. brassicicola infection, the ltpgl (GPI-anchored H2O2 and ROS-induced cell death (Gechev and
LTP) knockout mutant showed increased suscepti- Hille 2005; Gechev et al. 2008). Interaction with
bility (Lee et al. 2009), but the genetically uniden- ROS regulates PCD (Gechev and Hille 2005).
tified cutinase-deficient mutants were Brassinin hydrolase, a detoxifying enzyme (BHAb)
non-pathogenic (Tanabe et al. 1988). of a crucifer phytoalexin brassinin, also plays an
220 10 Phytotoxins

important role in the development of Alternaria almost all chito-oligosaccharide-responsive genes

blight caused by A. brassicicola in Brassica (Pedras and leads to increased susceptibility to fungal
et al. 2009). Imazaki et al. (2010) reported that A. pathogens indicating that LysM RLK1 is essential
alternata pathotypes contain abundant peroxi- for chitin signalling in plants (Wan et al. 2008).
somes, which are very important in both tissue Another receptor gene, PSK receptor, PSK2 and
colonization and pathogenesis. Other functions of PSKR I (phytosulphokine receptor), also induced
peroxisomes include fatty acid metabolism, acetyl- in Arabidopsis leaves following A. brassicicola
CoA generation, secondary metabolism, cell wall inoculation, also shows the role of PSK receptor in
biogenesis and peroxisome homeostasis. pathogenesis (Loivamaki et al. 2010).
Molecular mechanism of Alternaria blight in
Brassica shows involvement of chlorotic and
10.4.3 Molecular Level necrotic toxins and phytohormone. Alternaria
brassicae produces a chlorotic toxin destruxin B
The interaction between plants and pathogens at that plays an important role in signal transduction
molecular level is specific, complex and dynamic. leading to programmed cell death and suppressing
Many responses are evoked in plants upon encoun- the defence system (Taj et al. 2004). Differential
tering pathogens, but relatively very few have been expression of cell cycle proteins in toxin-treated
studied in detail. Key events in plant–pathogen leaves and calli and overexpression of p53 suggest
interaction include perception of pathogen on the that the toxin-mediated perturbations in cell cycle
plant or cell surface by receptors/sensors and eventually cause p53-induced programmed cell
transduction of these perceptions through various death (PCD) (Khandelwal et al. 2002). Interestingly,
transcription factors and target genes which are A. brassicae pathotoxin-induced cell death path-
involved in coordination of the appropriate way was antagonized by a phytohormone zeatin, in
responses (Hammond-Kosack and Jones 1996). cell culture of B. juncea (Pandey et al. 2001). The
Different types of both plant and pathogen genes antagonistic effect of these two structurally differ-
have been shown to be involved in plant and necro- ent entities strongly suggests the role of interactive
trophic fungal interaction. These responses range signalling pathways in pathogenesis of Alternaria
from genes that encode proteins like receptor/ blight in Brassica species. Introgression of osmo-
receptor kinase, cell wall-degrading enzymes, tox- tin, a known PR-5 protein, causes perturbation in
ins and transporter proteins to those involved in resistance to biotic and abiotic stresses (Taj et al.
signal transduction cascades such as mitogen-acti- 2004). The effect of toxin and zeatin treatments on
vated protein (MAP) kinases and various tran- the B. juncea leaf proteome was investigated by
scription factors like WRKY and NAC (Lawrence using two-dimensional electrophoresis and LCMS
et al. 2008; Wang et al. 2009; Amselem et al. techniques; results showed that 20 proteins were
2011). Ghose et al. (2008) also studied the differ- uniquely expressed in toxin-treated leaf, while 27
ential profiling of selected defence-related genes proteins were expressed in together with toxin and
induced on challenge by A. brassicicola in resis- zeatin. LCMS technique has also been used to
tant white mustard and their comparative expres- identify a total of 15 proteins with differential
sion pattern in susceptible Indian mustard. These expression in toxin-treated leaves. The proteins
genes have similarity with receptor-like protein identified in response to the toxin are glycosyl
kinase genes, genes involved with calcium-medi- hydrolases, subtilisin-like proteases, P-nitrophenyl
ated signalling, salicylic acid-dependent genes and phosphatase, malate dehydrogenase, heat shock,
other functional genes in Arabidopsis. After the ribulose 1, 5-bisphosphate carboxylase, cucumisin-
attack, the fungal cell wall is hydrolyzed by an like serine proteases, globulin-like protein and ATP
enzyme chitinase, and fragment of cell wall chitin synthase. Sharma et al. (2007) also studied the
is perceived by a receptor known as CERK 1 or proteome-level changes in A. brassicae–B. napus
Lys M RLK 1. A mutation in Lys M receptor-like and suggested role of ROS-mediated auxin signal-
protein (Lys M RLK1) gene blocks the induction of ling in the pathosystem.
10.4 Effect on Plants at Physiological, Biochemical and Molecular Level 221

Mitogen-activated protein kinase cascades are thaliana show increased susceptibility (Thomma
also standard players in the signal transaction lit- et al. 1999b) and resistance against A. brassicic-
erature for diverse organisms (Madhani and Fink ola (Thomma et al. 1999a; Kachroo et al. 2001;
1998; Cobb 1999). Activation of MAPK confers Nandi et al. 2005; Mang et al. 2009); systemic
resistance to both bacterial and fungal pathogens expression of the JA-inducible PDF1.2 gene is
(Sheen et al. 2002a, b). To decipher the MAP also reduced after infection with A. brassicicola.
kinase machinery components in B. juncea, This reflects the necessity of JA-mediated
RT-PCR amplification of all 20 known MAPKs responses for expression of this trait (Glazebrook
has been done. Among the MAP kinases 8, 12 1999). In contrast, the SA-insensitive mutant
and 18 showed no expression, and expression of npr-l and SA-depleted nah-G line have no effect
MAP kinases 3, 10 and 14 was validated with the on the resistance phenotype (Thomma et al.
Northern Blot. MAPK 3 gene is important and 1998), indicating no direct involvement of SA as
directly correlated with the transcription factors, a signalling molecule. In another study, induction
expressed in compatible interaction of A. brassi- of SA signalling marker, PR 1, and enhanced bio-
cae and B. juncea (Taj et al. 2011). Expression of synthesis of the antifungal compound camalexin
MAPKK 4, MAPKK 5, MAPKK 9, MAPKKK upon infection by A. brassicicola in Arabidopsis
1, Wrky 22 and Wrky 29 has also been validated raises the possibility of crosstalk between these
by real-time PCR. Expression analysis of MAP different signalling networks (Doares et al. 1995;
2 K9 and MAPK 6 is also governed during the Kunkel and Brooks 2002; Kachroo et al. 2003a,
pathogenesis of Alternaria blight in Arabidopsis b). Jasmonic acid also helps in the nodulation of
thaliana where simultaneous increased levels up MAP kinase 4 and MAP kinase 6 during phyto-
to middle stage of disease progression were pathogenesis of Alternaria blight in A. thaliana.
observed (Kannan et al. 2011). Furthermore, biosynthesis of antifungal com-
Inculcating the resistance mechanism in pound camalexin which plays a role against
Sinapis alba, against A. brassicicola, the down- Alternaria pathogen has been found to be posi-
stream signalling of MAPK 6 can be shown, tively controlled by SA, ROI and ET. JA-regulated
which was found to be activated after ethylene LOX gene is also known to play a role during A.
treatment (Taj et al. 2010) and might be a signifi- brassicae pathogenesis (Taj et al. 2011). During
cant upregulation step of NDR1/HIN-l-like, incompatible interaction of Arabidopsis, trans-
NHL25 and PR genes reported during S. alba genic plants harbouring CaLOX 1-OX lox1
infection (Varet et al. 2002); these genes were mutant, and A. brassicicola, it was discovered that
downstream targets of MAPK 6 in Arabidopsis lox1 mutant plants are more susceptible than wild
which are controlled by salicylic acid (SA) types, CaLOX1-OX transgenics and CaLOX1
(Desikan et al. 1999; Ghose et al. 2008). plants (Hwang and Hwang 2010). Transcription
Activation of more than one member of MAPK of the plant defence genes PDF1.2 and Thi2.1 is
by Alternaria suggests that MAPK cascades act enhanced in response to Botrytis cinerea and A.
as points of convergence and divergence in sig- brassicicola infection and is dependent on ET, JA
nalling and might play a pivotal role in deciding and OA signals (Epple et al. 1995; Penninckx
whether disease should progress or defence path- et al. 1996; Kachroo et al. 2003a, b). Resurrection
ways defeat the pathogen. I (rst1) mutant plants show more resistance to the
The involvement of phytohormone-dependent necrotrophic fungi B. cinerea and A. brassicicola
pathway is well documented in plant–pathogen by suppressing pathogen growth, sporulation and
interaction. Also well documented is the role of disease symptoms, which might be due to altered
jasmonic acid (JA)-dependent signalling path- cuticular waxes (Chen et al. 2005), because the
way of necrotrophic pathogen and SA-dependent amount of cutin monomers, phytoalexin accumu-
signalling pathway of biotrophic pathogen. lation and basal expression of the PDF1.2 gene
Mutants of JA and/or ethylene (ET) signalling were significantly enhanced in infected leaves
pathways and JA-insensitive coi-l of Arabidopsis (Mang et al. 2009). Unlike rst I mutation, other
222 10 Phytotoxins

mutations including mpk 4, bik 1 and wrky 33 created by the diffusion of fungal metabolites
cause susceptibility to the necrotrophic patho- like toxins (Agarwal et al. 1997; Tewari 1983).
gens, A. brassicicola and B. cinerea (Petersen Members of the genus Alternaria frequently
et al. 2000; Wiermer et al. 2005; Veronese et al. cause quiescent infections in which the fungus
2006; Zheng et al. 2006; Mang et al. 2009). enters the tissue where it remains dormant until
DEAD-box RNA helicase also plays a role during conditions favour infections. Alternaria species
Arabidopsis–A. brassicicola interaction. generally do not affect water or nutrient transport
Transgenic Arabidopsis plant that overexpresses throughout the plant, because they do not specifi-
the OsBIRH1 gene (DEAD-box RNA helicase cally target roots or vessels (Rotem 1994).
protein) also shows enhanced expression of PR-1, Alternaria has no known sexual stage or over-
PR-2, PR-5 and PDF 1.2 and disease resistance wintering spores, but the fungus can survive as
against A. brassicicola (Li et al. 2008). mycelium or spores on decaying plant debris for a
During the course of infection, various genes considerable time, or as a latent infection in seeds
of pathogen are also expressed and play a role in (Rotem 1994). The fungus can attack the seedling
fungal pathogenesis; AaFus3 gene, which encodes once the seed has germinated if seed-borne infec-
for FUS3MAPK in A. alternata, is required for tion exists. In other cases, once the spores are pro-
conidial development and penetration of the fun- duced, they are mainly spread by wind onto plant
gus in plant (Lin et al. 2010). Another study shows surfaces where infection can occur. Typically,
that deletion of AbPro1 (transcription factor) and weakened tissues either due to stresses, senes-
AbNIK 1 (two-component histidine kinase) gene cence or wounding are more susceptible to
of A. brassicicola results in 70 % and complete Alternaria infection than healthy tissues. The
loss of virulence, respectively (Cho et al. 2009). observation that saprobic Alternaria species can
Cyanide hydratase, arsenic ATPase and formate become parasitic when they meet a weakened host
dehydrogenase are some other genes of A. bras- illustrates that the distinction between saprophytic
sicicola, which are being expressed and have a and parasitic behaviour is not always evident.
role in fungal pathogenesis (Cramer and Lawrence Despite the taxonomic and pathogenic differ-
2004). Unfolded protein response (UPR) pathway ences between Alternaria species, these cause sim-
also regulates the fungal pathogenesis, and AbHac ilar infection patterns. Dormant spores have heavily
A gene encodes the major UPR transcriptional melanized walls that, under favourable conditions,
regulator; loss of UPR in mutants of A. brassicic- produce one or more germ tubes. Subsequently, the
ola resulted in complete loss of virulence (Joubert germ tubes penetrate stomata, cuticle or wounds
et al. 2011). with or without the formation of small appressoria.
In less virulent species, wounds and stomata are
targeted, while more virulent species can also pen-
10.5 Role of Toxins etrate directly (Rotem 1994): enzymatic processes
in the Infection Process in Alternaria infections are essentially similar to
those in other diseases. The cuticle, which consists
In general, Alternaria species are foliar patho- of a combination of cutin (a hydroxyl fatty acid
gens that cause relatively slow destruction of host polyester) and waxes, comprises the first line of
tissues through the reduction of photosynthetic defence to be overcome by directly penetrating
potential. An infection leads to the formation of fungal pathogens. In the case of A. brassicicola, the
necrotic lesions, which sometimes have a target- differential expression of cutinase genes was moni-
like appearance due to growth interruptions tored between saprophytic and pathogenic stages
caused by unfavourable conditions. The fungus of the fungus (Yao and Köller 1995), Furthermore,
resides in the centre of the lesion, which is sur- it was found that different cutinolytic enzymes are
rounded by an un-invaded chlorotic halo, a symp- sequentially induced upon landing on and penetra-
tom that is commonly observed for the infection tion of the cabbage leaf (Fan and Köller 1998).
process of necrotrophic pathogens. This zone is Constitutively produced cutinases are expressed
10.6 Toxin Biosynthesis 223

during the initial contact of A. brassicicola with the Often, these toxins are produced as families of
cuticle. After reaching subcuticular layer, different related compounds. Based on selectivity, phyto-
cutinases that are active during saprophytic growth toxins can be divided into two categories:
are induced (Trail and Köller 1993; Yao and Köller non-host-specific toxins and host-specific toxins.
1994). These cutinases are inducible by cutin In general, non-host-specific toxins have rela-
monomers. This implies a switch between the para- tively mild phytotoxic effects, affect a broad
sitic and the saprophytic stage of the fungal patho- spectrum plant species and are thought to be an
gen. So far, however, it has not been demonstrated additional factor of disease for penetration mech-
that any extracellular hydrolase is crucially anisms and enzymatic processes. Although they
involved in fungal pathogenesis. generally act as virulence factors and intensify
In addition to cutinases, lipases might also disease symptom severity, they are not absolutely
contribute to the establishment of infection. A. required for establishing disease, since they are
brassicicola was found to produce a lipase that also toxic to plant species outside the host range
acts as a virulence factor (Berto et al. 1997). of the pathogen. They merely precondition the
Anti-lipase antibodies were found to have an host for disease (Ballio 1991).
inhibitory effect on the in vivo infection of cauli- In Alternaria, many non-host-specific toxins
flower leaves, as symptom development was have been identified, although the precise action
inhibited in a dose-dependent manner despite of only a few has been studied in detail. Brefeldin
normal germination of spores. However, the anti- A (dehydrogenase), curvularin, tenuazonic acid,
bodies did not have any effect on fungal infection tentoxin and zinniol are examples of toxins that
of dewaxed leaves, which suggests that this lipase are produced by several Alternaria species. They
has an early pathogenic activity during penetra- exert their phytotoxic activity through different
tion (Berto et al. 1999). modes. Brefeldin A causes disassembly of the
About one-third of the total cell wall compo- Golgi complex and acts as an inhibitor of secre-
nents in dicotyledonous plants are pectic poly- tion, while curvularin is an inhibitor of cell divi-
saccharides. These components can be sion through its disturbance of the microtubule
hydrolyzed by fungal galacturonidases. On the assembly. Tenuazonic acid inhibits protein syn-
other hand, the tangerine pathotype of A. alter- thesis and zinniol affects membrane permeabili-
nata did not show this dependency, possibly zation (Fujiwara et al. 1988; Meronuck et al.
because this particular pathogen largely depends 1972; Robeson and Strobel 1981; Thuleau et al.
on toxin production for the colonization of its 1988). Tentoxin is produced by A. alternata and
host (Isshiki et al. 2001). acts as a photophosphorylation inhibitor through
For a specific A. alternata endoglucanase, it specific binding to chloroplast ATP synthetase,
was demonstrated that its production is triggered causing the inhibition of ATP hydrolysis and
by a pathogen-induced pH increase on the host ATP synthesis (Steele et al. 1978), because these
(Eshel et al. 2002b). Correlation studies between toxins often target basic cellular processes and
enzyme production and symptom development are often powerful mycotoxins.
suggest that endoglucanases and also exogluca- Host-specific toxins are involved in the devel-
nases are involved in A. alternata pathogenicity opment of a few, destructive diseases. They gen-
(Eshel et al. 2000, 2002a, b). erally display severe effects on a rather narrow
species range that serves as host to the fungus, and
they are indispensable for disease. Along with
10.6 Toxin Biosynthesis species of the genus Cochliobolus
(Helminthosporium), Alternaria species are
Most phytotoxins employed by a fungal invader known to produce host-specific toxins (Markham
are chemically diverse secondary metabolites: and Hille 2001; Wolpert et al. 2002). There are at
low molecular weight components that are not least 12 host-specific toxin-producing plant
required for normal growth or reproduction. pathogenic Alternaria species, most of which
224 10 Phytotoxins

appear to be variants of A. alternata. It was pro- suggests that these homologues are involved in
posed that these variants should be considered the production of common moieties between
pathotypes of A. alternata (Nishimura and these toxins (Hatta et al. 2002; Masunaka et al.
Kohmoto 1983), a hypothesis that is supported by 2000; Tanaka and Tsuge 2000; Tanaka et al.
molecular analysis (Kusaba and Tsuge 1994, 1999). Together with the existence of such homol-
1995, 1997). The toxins that are produced by ogous toxin biosynthesis genes between pathot-
these pathotypes are chemically diverse, ranging ypes, the physical clustering of toxin genes on a
from low molecular weight secondary metabo- single chromosome suggests that these genes are
lites to peptides. In a study on pathogenic (host- acquired through horizontal gene transfer (Tanaka
specific toxin-producing) and non-pathogenic et al. 1999; Walton 2000). This hypothesis is sup-
(non-host-specific toxin-producing) A. alternata ported by the observation that homologues of
species, it was established that all pathotypes toxin biosynthesis genes have not been found in
tested carried (small) extra chromosomes, strains from other pathotypes or non-pathogenic
whereas the non-pathogenic ones did not carry A. alternata isolates, making it unlikely that muta-
these extra chromosomes (Akamatsu et al. 1999). tions or internal genetic rearrangements account
In fungal species, one can regularly find extra for the toxin-producing ability (Hatta et al. 2002;
chromosomes in certain subsets of individuals, Masunaka et al. 2000; Tanaka et al. 1999). There
so-called supernumerary chromosomes. These is no advantage for the clustering of genes during
chromosomes are not essential because they are gene transmission from one generation to the next
not required for normal growth, but they can carry (vertical gene transfer), because the entire genome
extra traits. The supernumerary chromosomes is transferred as a unit. However, during horizon-
confer an adaptive advantage to the individual in tal gene transfer, a relatively small contiguous
some habitats; they are referred to as condition- DNA sequence is generally transferred. Since the
ally dispensable chromosomes. The production of total set of biosynthesis genes should be transmit-
the cyclic AM toxin peptide by the apple patho- ted from one species to the other in order to pro-
gen A. alternata f. sp. mali is determined by such vide the recipient with a complete biosynthesis
a chromosome (Johnson et al. 2000, 2001). The pathway, this chance is higher if genes are clus-
AK and AF toxin biosynthesis genes and their tered. An originally saprophytic fungus like
homologues, identified in the Japanese pear and Alternaria could have acquired pathogenic capac-
strawberry pathotypes of A. alternata, respec- ity in this way (Walton 2000), a hypothesis which
tively, generally clustered together on a small is supported by the finding that single pathotype
chromosome (Hatta et al. 2002; Tanaka and Tsuge populations do not form monophyletic groups
2000; Tanaka et al. 1999). Physical clustering is a (Kusaba and Tsuge 1994, 1995, 1997). The occur-
phenomenon that is commonly found for genes rence of horizontal gene transfer is well accepted
involved in the production of secondary metabo- for prokaryotes, and even for fungal mitochon-
lites in fungi (Keller and Hohn 1997). drial genes, but experimental evidence for the
AK and AF toxins are related to ACT toxin, horizontal gene transfer of fungal nuclear genes is
produced by the tangerine pathotype of A. alter- scarce. However, the difference between patterns
nata, and carry an epoxydecatrienoic ester back- of repeated DNA sequences on certain supernu-
bone. Interestingly, the AF- and ACT-producing merary chromosomes and the rest of the chromo-
strains were also found to be pathogenic on pear somes in the same genome suggests that both
lines that are sensitive to AK toxin, but not vice types have a different evolutionary history (Covert
versa (Kohmoto et al. 1993; Maekawa et al. 1984). 1998). Furthermore, it was demonstrated for the
Four genes involved in the biosynthesis of AK fungus Colletotrichum gloeosporioides that a
toxin have been cloned from the Japanese pear small chromosome could be transferred between
pathotype genome of A. alternata, and homo- two genetically isolated strains, thus demonstrat-
logues of three of these genes have been identified ing that horizontal gene transfer can occur (He
in the strawberry and tangerine pathotypes. This et al. 1998).
10.7 Mode of Action of Host-Specific Toxins 225

10.7 Mode of Action of Host- gesting an active participation of the host in AAL
Specific Toxins toxin-mediated cell death (Wang et al. 1996b).
Indeed, transgenic tomato plants expressing a
Although the site of action of different Alternaria viral anti-apoptotic gene were protected against
toxins varies, in the end, they all trigger host cell AAL toxin-induced cell death and pathogen
death. AF, ACT and ACTG toxins act at the infection (Lincoln et al. 2002). The use of the
plasma membrane and cause permeabilization host’s suicide programme as a compatibility fac-
(Otani et al. 1995). AM toxin not only affects the tor was not only shown for AAL toxin-producing
plasma membrane but also acts on chloroplasts, A. alternata strains, but seems to be a general
while ACT and AT toxins were found to affect strategy of necrotrophic pathogens (Dickman
mitochondria (Otani et al. 1998). ACR toxin et al. 2001; Lincoln et al. 2002).
induces swelling, and other morphological modi- In plants, insensitivity to AAL toxin and resis-
fications of mitochondria, and increases NADH tance to AAL-producing A. alternata f. sp. lycop-
oxidation, which is followed by plasma mem- ersici is determined by the codominant Alternaria
brane disorders leading to electrolyte leakage and stem cancer (Asc-1) gene, a homologue of a yeast
necrosis (Akimitsu et al. 1989). For most of these longevity assurance gene of which homologues
toxins, however, their mechanism of action is are also found in nematodes and humans
barely understood. (Brandwagt et al. 2000, 2002; Spassieva et al.
AAL toxin is an aminopentol ester, an ana- 2002). Although the precise mode of action of
logue of the sphingosine precursor sphinganine, Asc-1 has not yet been determined, based on
which is produced by the tomato pathogen A. studies with human and nematode homologues in
alternata f. sp. lycopersici. This toxin closely yeast, it is believed that Asc-1 relieves a toxin-
resembles the mycotoxin fumonisin B1 that was induced block on sphingolipid synthesis through
identified as a Fusarium moniliforme toxin and is salvage of the ER to Golgi transport of GPI-
also produced by AAL toxin-producing A. alter- anchored proteins (Brandwagt et al. 2000).
nata species (Chen et al. 1992). Interestingly, In addition to a poor knowledge of the mecha-
fumonisin B1 is also selectively toxic to AAL nism of action of different host-specific toxins,
toxin-sensitive tomato genotypes (Gilchrist et al. the basis of host specificity is only understood for
1992). Alternaria alternata mutants affected in a few of them. For destruxin B, a host-selective
AAL toxin production also lose their pathogenic- phytotoxin produced by A. brassicae (Pedras
ity, demonstrating the requirement of this toxin et al. 2002), the mechanism for host selectivity
for pathogenicity (Akamatsu et al. 1997). Like was recently revealed. The selective phytotoxicity
fumonisin B1, AAL toxin is an inhibitor of sphin- appears to be due to a fast and efficient detoxifica-
golipid (ceramide) biosynthesis (Abbas et al. tion, by sequential hydroxylation and glycosyl-
1994; Spassieva et al. 2002). The application of ation reactions, in tissues of resistant species.
AAL toxin leads to an accumulation of sphingoid These reactions were also found to occur in sus-
base precursors, a depletion of complex sphingo- ceptible species, but at a slower rate, providing an
lipids, and subsequently to the cell death of explanation for selective toxicity (Pedras et al.
sensitive tomato species (Abbas et al. 1994; 2001). Similarly, toxin detoxification has been
Wang et al. 1996b). Similar effects of the toxin demonstrated to determine resistance against the
are observed on mammalian cells (Wang et al. host-specific toxin-producing fungus
1996a). In eukaryotic cells, ceramides modulate Cochliobolus carbonum (Multani et al. 1998).
a number of biochemical and cellular stress For ACR toxin, specificity seems to be deter-
responses such as cell cycle arrest and apoptosis mined by differential post-transcriptional pro-
(Hannun 1996). The AAL toxin-induced cell cessing of a mitochondrial gene. This gene is
death observed in plant cells shares characteris- present in the mitochondrial DNA of toxin-
tics with genetically controlled cellular suicide, sensitive as well as toxin-resistant species, but
or apoptosis, as observed in animals, thus sug- the transcript of the gene is shorter in resistant
226 10 Phytotoxins

than in sensitive mitochondria. In the end, an between camalexin production upon A. brassicic-
oligomeric protein is produced in toxin-sensitive ola inoculation on one hand and resistance against
mitochondria, whereas the transcript is not trans- this pathogen on the other (Kagan and
lated in resistant mitochondria. Although the Hammerschmidt 2002) This is perhaps not sur-
gene is likely to encode a mitochondrial mem- prising, since defence against A. brassicicola in
brane protein, no function has been assigned yet Arabidopsis appears to depend on multiple defence
(Ohtani et al. 2002). components, the relative importance of which can
vary between ecotypes. Previously, jasmonate has
also been implicated in defence against Alternaria,
10.8 Role of Toxin in Host Defence as the jasmonate-insensitive Arabidopsis mutant
against Alternaria species coi1 was found to display enhanced susceptibility
to this pathogen (Thomma et al. 1998, 1999a,
Destruxin B is a host-specific Alternaria phyto- 2000). Jasmonate is a plant hormone that, along
toxin as mentioned before, whose selective phy- with salicylic acid and ethylene, plays a role in the
totoxicity is due to the detoxification in tissues of activation of defence responses in plants (Thomma
resistant species (Pedras et al. 2001). In addition, et al. 2001). However, mutants affected in either
the hydroxylation product that is generated dur- salicylic acid or ethylene signalling is as resistant
ing detoxification was found to induce phyto- as wild-type plants, leading to the conclusion that
alexin biosynthesis in resistant, but not in neither salicylic acid nor ethylene is directly
susceptible, plant species. This suggests that involved in resistance against Alternaria in
resistance depends on phytotoxin detoxification Arabidopsis (Thomma et al. 1998, 1999a).
and simultaneous phytoalexin elicitation (Pedras Jasmonate was found not to directly induce cama-
et al. 2001). Phytoalexins have also been impli- lexin biosynthesis, and in addition, jasmonate
cated in pathogen containment for other insensitivity does not lead to camalexin deficiency.
Alternaria/host interactions. In Camelina sativa It was therefore concluded that both camalexin
(false flax), resistance against A. brassicae has and jasmonate mediate host resistance against
been correlated with production of the phyto- Alternaria through separate but parallel pathways
alexin camalexin (Jejelowo et al. 1991). in Arabidopsis (Thomma et al. 1998, 1999b;
Moreover, Arabidopsis synthesizes camalexin 2001). Further, support for this observation came
upon pathogen challenge. Since Arabidopsis is from the finding that the Arabidopsis mutation
amendable to phytopathological studies as well esa1 affects resistance against A. brassicicola
as molecular genetics, it has been established as a through a severe reduction in both camalexin pro-
model for dissecting plant defence mechanisms. duction and jasmonate-dependent gene induction
The Arabidopsis pad3 mutant (phytoalexin defi- (Tierens et al. 2002). It is currently not clear, how-
cient) was found to be deficient in camalexin bio- ever, which jasmonate-inducible effect or mole-
synthesis and to display severely enhanced cules are responsible for containment of Alternaria
susceptibility to A. brassicicola, demonstrating in Arabidopsis.
that camalexin contributes to Alternaria resis- In a cDNA microarray analysis on a set of
tance (Thomma et al. 1999b). Although cama- 2375 Arabidopsis genes, 168 genes were found to
lexin was found to exhibit direct antimicrobial be upregulated and 39 were found to be repressed
activity to this fungus in vitro (Thomma et al. 72 h after inoculation with A. brassicicola. About
1999b), it cannot be excluded that camalexin also 10 % of these 207 genes can be implicated in
contributes to Alternaria resistance in an indirect plant defence and about one-third in cell mainte-
way, as camalexin was found to inhibit produc- nance and development (Schenk et al. 2000).
tion of the host-specific toxin destruxin B in A. However, a precise role for any of the responsive
brassicae (Pedras et al. 1998). genes in the defence of Arabidopsis against A.
In a study of 24 different Arabidopsis ecotypes, brassicicola has not yet been established. Studies,
it was not possible to discover a direct relationship using ethylene- and jasmonate-insensitive
10.8 Role of Toxin in Host Defence against Alternaria species 227

Arabidopsis mutants, concluded that a set of PR harzianum has resulted in enhanced Alternaria
genes encoding plant defensin, and hevein-like resistance in transgenic tobacco, potato and broc-
protein, and basic chitinase are not markedly coli (Lorito et al. 1998; Mora and Earle 2001). In
involved in Alternaria resistance (Thomma et al. addition, the treatment of plants with biocontrol
1998, 1999a). In tomato, PR proteins have been organisms has in some cases resulted in enhanced
implicated in Alternaria resistance through the resistance against Alternaria pathogens (Morita
observation that resistance in tomato against A. et al. 2003; Ton et al. 2002).
solani correlates with high and rapid accumula- Many aspects of the interaction between
tion of PR proteins. However, it is speculated that Alternaria species and their respective hosts are
those PR proteins, many of which possess hydro- currently under investigation. The function and
lytic activity, are not necessarily directly involved biosynthesis of host-specific toxins, melanins in
in arresting the pathogen, but rather release elici- particular, have gained much attention, and con-
tors from the pathogen cell wall, thus triggering a siderable advances have been made. Finally,
HR (Lawrence et al. 2000). although the general strategies employed by the
The production of reactive oxygen species pathogenic strains to attack their hosts are simi-
(ROS) is one of the earliest defence reactions of lar, the responses of the various hosts against
plants against pathogen attack. In the presence of these attacks are very diverse (Thomma 2003).
tobacco plant extracts, A. alternata was found to White mustard cells can transform a very
synthesize mannitol, which can act as a potent toxic compound called destruxin B (14C labelled),
quencher of reactive oxygen species. This sug- produced by the microorganism that causes the
gests that Alternaria uses the active oxygen plant disease, into a non-toxic compound called
quencher mannitol to overcome host defence. On hydroxydestruxin B. Hydroxydestruxin B (14 C
the other hand, pathogen attack was found to labelled) was further biotransformed to the β-d-
induce mannitol dehydrogenase expression in glucosyl derivative at slower rates. The structures
tobacco, suggesting that plants counter this fun- of hydroxydestruxin B and β-d-glucosyl
gal defence system by catabolizing fungal man- hydroxydestruxin B were deduced from their
nitol (Jennings et al. 2002). Support for this spectroscopic data (NMR, HRMS, FTIR) and
hypothesis also came from the fact that the con- confirmed by total chemical synthesis (Fig. 10.3).
stitutive expression of a mannitol dehydrogenase Although these hydroxylation and glucosylation
gene in tobacco confers resistance against A. reactions occurred in both resistant (S. alba) and
alternata, but not against non-mannitol-secreting susceptible (B. napus, B. juncea and B. rapa)
fungi (Jennings et al. 2002). species, hydroxylation was the rate-limiting step
Using a transgenic approach, enhanced levels in the susceptible, whereas glucosylation was the
of resistance against Alternaria species were also rate-limiting step in the resistant species.
obtained in a number of cases. It was previously Remarkably, it was observed that the hydroxyl
discussed that the expression of anti-apoptotic destruxin B induced the biosynthesis of phyto-
genes provides disease resistance, against alexins in black spot resistant, but not in suscep-
Alternaria species, among others, through block- tible species. This appears to be a unique example
ing a programmed cell death response (Dickman of phytotoxin detoxification and simultaneous
et al. 2001; Lincoln et al. 2002). Constitutive phytoalexin elicitation by the detoxification
expression of a radish defensin gene, a rubber product. Sinapis alba can overcome the fungal
tree chitin-binding lectin and a human lysozyme, invader through detoxification of destruxin B
respectively, conferred enhanced resistance coupled with production of phytoalexins
against Alternaria spp. in tobacco, Indian mus- (Figs. 10.4 and 10.5). Since the hydroxylation
tard and carrot (Kanrar et al. 2002; Takaichi and reaction happens at a very fast rate and it leads to
Oeda 2000; Terras et al. 1995). In addition, con- a less toxic product, it may prevent the plant cells
stitutive expression of an endochitinase gene iso- from being killed by the pathogen. Canola plants
lated from the biocontrol fungus Trichoderma can become resistant to the black spot disease
228 10 Phytotoxins

plant pathogen

phytotoxins
are compounds produced by plant
pathogens to damage the plant cells

resistant plant
susceptible plant

Fig. 10.3 Phytotoxin are produced by microbial plant pathogens

Alternaria blackspot

N N
N N
N N
O NH O O
O NH O O
O O
O
HN O
O * HN O
O * HN O
O
O
O white O canola
O
mustard O O
N N
N

HO
H HO
HO O
destruxin B HO-destruxin B HO O
OH

* = 14C b-D-gluc-O-destruxin B

Fig. 10.4 Enzymatic reactions of plants to the pathogen causing Alternaria black spot

upon incorporation of this detoxification trait mechanism for overcoming this chemical by
(Pedras et al. 1999, 2001). adopting phytoalexin biosynthetic pathways oper-
In unique interactions, three different rapeseed ating in planta. This strategy appears quite plau-
pathogens convert the phytoalexin cyclobrassinin sible, especially considering that most fungal
into three different phytoalexins, brassilexin, pathogens have been coevolving with plants for
dioxibrassinin and brassicanal A, and ultimately numerous generations (Pedras and Okanga 1999;
each phytoalexin into non-toxic products. Such Fig. 10.6). Each detoxification pathway is (i) spe-
pathogen may have acquired a more effective cific to a group of pathogens and (ii) the enzymes
10.8 Role of Toxin in Host Defence against Alternaria species 229

Fig. 10.5 Defence responses of plants resistant and susceptible to Alternaria black spot

H H
HO N SMe N
P.lingam SMe O
S S R.solani
O SMe
N N N
H H H
dioxybrassinin cyclobrassinin
brassicanal A

P.wasabiae

N
S
N
H
brassilexin

Fig. 10.6 Transformation of the phytoalexin cyclobrassinin by different pathogens of Brassica

involved in these transformations are quite selec- library (Pedras et al. 2000), none of these phyto-
tive (Pedras 1998; Pedras et al. 2000). toxins could be detected in either extracts of liq-
Pedras et al. (2009) have established that A. uid cultures or in infected leaf tissues. In addition,
brassicicola produces brassicicolin A (1) (a mix- the four polyketides, phomapyrones A (3), F (4)
ture of epimers, Gloer et al. 1988), as the major and G (5) and infectopyrone (6), previously iso-
host-selective phytotoxin. Brassicicolin A (1) lated from Leptosphaeria maculans and L.
appeared to be more phytotoxic to the susceptible biglobosa (Pedras et al. 1994; Pedras and
cruciferous species B. juncea than to the tolerant Biesenthal 2001; Pedras and Chumala 2005),
B. napus. Previous claims that A. brassicicola were also isolated from A. brassicicola. Clones
produced destruxins (Cooke et al. 1997; Evans of A. brassicicola (altr203 x n03 and altrO14 x
et al. 1996) could not be confirmed; in spite of the eO1) were recently reported (Cramer et al. 2006)
availability of several destruxins in metabolite to encode enzymes involved in the biosynthesis
230 10 Phytotoxins

of undetermined polyketides, which could be the 10.9 In Silico Protein–Protein

phomapyrones 3 and 5. In this context, it is perti- Interaction
nent to point out that genome sequences currently
available in public databases have indicated the Since cell perceives the signal through the cell
existence of numerous ‘orphan pathways’, i.e., surface receptors including receptor-like kinases
gene clusters for which the encoded natural prod- (RLK), receptor-like proteins (RLP), and
uct is unknown (Gross 2007). Therefore, it is receptor-like extracellular binding proteins, they
expected that further new metabolites and/or are known to interact with MAMPs (microbe-
phytotoxins could potentially be isolated from associated molecular pattern) or PAMPS
fungal species previously investigated, such as A. (pathogen-associated molecular pattern) and
brassicicola (Pedras et al. 2009). effectors. Receptor-like kinases interact with
Phytoalexin production in leaves of B. juncea pathogen-associated molecular pattern, which
infected with A. brassicicola led to the detection are small peptides like chitin oligosaccharide
and isolation of two known phytoalexins and a (Hamel and Beaudoin 2010) and flagellin
phytoanticipin: cyclobrassinin (11) and spiro- (Pitzschke et al. 2009). Destruxin B, which is a
brassinin (12) and indolyl-3-acetonitrile (15), small peptide, produced by A. brassicae, is found
respectively. Since indolyl-3-acetonitrile (15) to interact with Lys M receptor-like kinase and
was occasionally detected in control leaves, it is WAK receptor through in silico interaction. The
most likely a phytoanticipin. In addition, other results of docking interaction of destruxin B with
minor phytoalexins (13 and 14) and phytoanti- different receptor-like kinases from different spe-
cipins (16 and 17) were detected in fractions of cies indicate that Lys M receptor-like kinase in
leaf extracts. Since the product of detoxification Arabidopsis showed better interaction with it. A
of brassinin (10), and N′-acetyl-3- mutation in Lys M receptor-like protein (Lys M
indolylmethanamine (19), was also isolated RLK I) gene blocked the induction of almost all
from infected leaves, but never detected in con- chito-oligosaccharide-responsive genes and led
trol leaves, it is suspected that brassinin (10) to increased susceptibility to fungal pathogens
does not accumulate in sufficient quality in indicating that Lys M RLK1 is essential for chitin
infected tissues because it is quickly detoxified signalling in plants (Wan et al. 2008).
to 19, similar to the detoxification that occurs in Arabidopsis genome has more than 80
A. brassicicola cultures. To support this hypoth- MAPKKKs, but very few members of MAPKKKs
esis, Pedras et al. (2009) determined that A. have been studied. To continue the downstream
brassicicola detoxifies brassinin (10) to indolyl- cascade and to take part in the defence, docking
3-methanamine (18) and N′-acetyl-3- was performed with MAPKKKs (MAPKKK I,
indolylmethanamine (19) in fungal cultures. MAPKKK 2, MAPKKK I2 and MAPKKK 19)
Although brassinin (10) is not a strong antifun- and receptor kinases which showed better inter-
gal against A. brassicicola, this phytoalexin is a action with destruxin B. It was found that
precursor of brassilexin (14) and other phyto- MAPKKK 19 showed better interaction with Lys
alexins of B. juncea (Pedras et al. 2007). It is M receptor-like kinase (Ricinus communis);
very probable that its detoxification makes the WAK receptor-like kinase (A. thaliana) and
plant more vulnerable to fungal invasion, and MAPKKK 2 were also found to interact better
the brassinin-detoxifying enzymes produced by with CHRK 1 receptor-like kinase (Senecio
A. brassicicola might contribute to its virulence. squalidus).
The comparison of the in vitro antifungal activ- The activity of MAPK 4 has been identified in
ity of the phytoalexins present in infected tis- plant defence against biotic stress (Desikan et al.
sues of B. juncea and that of camalexin (20, not 2001). Out of all 10 MAPKKs known, the activity
produced in Brassica species) suggests that of some MAPKKs in plant defence has already
camalexin might be a more effective plant been identified against biotic stress, viz. MAPKK
defence against A. brassicicola. 3 and MAPKK 5 (Asai et al. 2002). Docking
References 231

results of all 10 MAPKKs with MAPK 4 showed 4 interacts with all above-listed WRKY proteins,
that MAPKK 5, MAPKK 8, MAPKK 3 and except WRKY 29, a downstream partner of
MAPKK 9 have better interaction with MAPK 4. MAPK 3/MAPK6; it can be assumed that this
There are several evidences in the literature that might be due to the specificity provided by the
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 Disease Management
11

Kumar and Kumar 2006; Peruch and Michereff

11.1 Introduction 2007). Seed infection can largely be controlled by
hot water treatment at 50 °C for 18 min (Schimmer
No single method or approach in current use is 1953) or by soaking Brassica seeds in a 0.2 %
feasible, viable, stable, effective and economical aqueous suspension of Thiram for 24 h at 30 °C
in dealing with any host–pathogen system. (Maude 1978; Maude and Shuring 1968; Maude
Therefore, it is necessary to integrate all the et al. 1969). In tropical countries including India,
methods of plant disease control, which are avail- aging of Brassica seeds for 5–6 months at tem-
able, including cultural, chemical, biological and peratures above 35 °C eliminates seed infection
host resistance to effectively manage Alternaria and seed-borne inoculum (Chahal 1981; Kolte
diseases of brassicaceous plants (Eastburn 1989; 1985). Alternaria pod blight of radish is signifi-
Saharan 1992; Verma and Saharan 1994). cantly less in the early transplanting crop (15
November) under Indian conditions (Sandhu et al.
1985). In Sweden, A. brassicae occurs more fre-
11.2 Cultural Measures quently in areas with both summer and winter oil-
seed crops than in areas with mainly summer
Among the common cultural practices, use of crops (Morner 1980). In India, early planting of
clean, large, healthy seed, seed treatment, long toria (B. rapa) in September usually escapes
crop rotation (4 years under Canadian conditions), Alternaria infection. In the Lujaska area of
sanitation, weed control, planting shallow (2 cm) Leningrad (USSR), there is lower incidence of A.
at recommended times; use of balanced nutrients, brassicae on cabbage, if transplanting is done
proper plant density (45 cm row spacing), proper between 20 and 30 June. Application of fertilizers
drainage in the fields, plant debris management; containing humus (45 % peat and 5 % mullcin,
use of tolerant/resistant cultivars and application plus 3.9 g ammonium nitrate, 3.4 g potassium
of chemicals at proper time with adequate foliage chloride, and 8.1 g superphosphate/100), added to
coverage have been advocated to control Alternaria 110 g organic matter, reduces the disease better
diseases of brassicaceous plants (Channon and than the organic manure alone (Kupryanova 1957).
Maude 1971; Chupp 1925; Chupp and Sherf 1960; Alternaria black spot of broccoli can be managed
Daebeler et al. 1981, 1987; Dixon 1981; Kolte by incorporating infected leaf debris in the soil, at
1985; Lapis and Ricaforte 1974; Saharan and least 10 cm depth for no less than a 60 days inter-
Chand 1988; Singh 1987; Thomas 1984; Verma val in subsequent Brassica plantings (Peruch and
and Saharan 1994; Kharbanda and Tewari 1996; Michereff 2007).

© Springer Science+Business Media Singapore 2016 239

G.S. Saharan et al., Alternaria Diseases of Crucifers: Biology, Ecology and Disease Management,
DOI 10.1007/978-981-10-0021-8_11
240 11 Disease Management

To avoid storage, and transit losses in cabbage 1972, 1984; Mridha and Safa 1985; Randhawa
and cauliflower, heads should be handled care- and Aulakh 1982; Verma and Saharan 1994; Wu
fully to avoid bruising and surface moisture and Lu 1984; Latif et al. 2006).
allowed to evaporate before storage. The storage Soaking Brassica seeds in an aqueous suspen-
house should be kept at 33–34 °F and ample ven- sion of Thiram (0.2 %) for 24 h at 30 °C eradi-
tilation provided to reduce humidity (Walker cates Alternaria infection (Maude 1978; Maude
1927). and Shuring 1968; Maude et al. 1969). In New
South Wales, two samples of infected seed were
treated with iprodione at 1.25 g a.i. Kg−1 seed,
11.3 Seed Treatment and the level of seed-borne A. brassicae was
reduced from 21 to 2 % and from 14 to 4.5 %,
11.3.1 Hot Water Treatment respectively. The same treatment eliminates A.
brassicae from pieces of trash mixed with seed
Excellent control of A. brassicae in white cabbage (Stovold et al. 1987). Seed treatment with iprodi-
and cauliflower seeds has been obtained in Danish one, which virtually eradicates seed-borne infec-
experiments from 1934 to 1936 by 30-min immer- tion of Alternaria spp. in the Brassicaceae, is
sion in water heated to 45 °C or for 20 min at now used to treat basic seed stocks in England
50 °C, or even 30 min at 40 °C. This treatment (Maude and Humpherson-Jones 1980a, b). This
improves germination by up to 13 % along with practice is most effective in crops grown in isola-
elimination of other fungi such as Penicillium and tion from maturing brassicaceous seed crops.
Mucor spp. (Nielsen 1936). Hot water seed treat- However, where seed crops are grown succes-
ment at 50 °C for 30 min controls the disease in sively and in close proximity, as it often occurs in
cabbage (Walker 1952). According to Ellis (1968a, the main seed-producing areas, the infection may
b), hot water treatment for 25 min at 50 °C elimi- arise from spores released from diseased seeding
nates Alternaria infection from brassicaceous crops during cutting and threshing and dissemi-
seeds. Seed treatment at 50 °C for 20 min is highly nate by the wind to young crops destined for seed
effective in controlling the seed-borne fungi production in the following year (Humpherson-
including A. brassicae of raya (B. juncea) without Jones and Maude 1982). In such situations, addi-
any pronounced effect on the germination of the tional control measures are necessary.
seeds (Randhawa and Aulakh 1984). According to Maude and Suett (1986), use of
a prototype fluidized-bed seed treater to apply
iprodione in a polymer film coat to cabbage seeds
11.3.2 Chemical Treatment infected with A. brassicae and A. brassicicola
has been found effective. Analysis of seed-to-
For controlling seed-borne A. brassicae, A. bras- seed variability in dosing demonstrated the
sicicola, A. raphani and A. alternata infections in greater accuracy in fungicide application and
brassicaceous plants, seed dressings with Benlate, control of A. brassicae by film coating than by
Dithane M-45, Dithane Z-78, Difolatan, slurry or dust methods. Application of polymer
Dichlofluanid, Delan C, Bristan, Bavistin, film coat slightly reduces superficial inoculum of
Brassicol, Captan, Fenpropimorph, Granosan M, A. brassicicola.
Quinolate, Morpan, Rovral, Thiram, Vitavax, Seed treatment of mustard with Bavistin,
Thiride and Zineb have been found to be very Difolatan (1.5 g/Kg) and Dithane M-45 (2 g/
effective (Atkinson 1950; Chahal et al. 1977; Kg−1) eradicates A. brassicae infection. A mini-
Champion et al. 1979; Chirco and Harman 1979; mum period of 24 h is necessary for obtaining
Ellis 1968a, b; Gupta and Saxena 1984; effective control of the pathogen in seeds (Kumar
Holtzhausen 1978; Humpherson-Jones et al. and Singh 1986). Seed treatment with iprodione
1980, 1981, 1983, 1984; Kanwar and Khanna (1.35–2.5 g a.i./Kg−1) and Fenpropimorph
1979; Kumar and Singh 1986; Maude and (0.625–2.5 g a.i./Kg−1) virtually eliminated A.
Humpherson-Jones 1980a, b, 1984; Maude et al. brassicicola in naturally infected Brassica seeds
11.4 Chemical Control 241

without affecting seed germination in laboratory observed by several workers (Ansari et al. 1990;
and seedling tests (Maude 1983, 1986). In Chand and Jatian 1969; Maude 1976; Prasada
Bangladesh, seed treatment of mustard with et al. 1970; Singh and Bhowmik 1985; Singh and
Rovral (0.25 %) enhances seed germination Rai 1982; Tewari and Skoropad 1979).
remarkably (Latif et al. 2006). In vitro and in vivo evaluation of large number
In Finland, A. brassicicola on Chinese cab- of chemicals (Table 11.1) against Alternaria spe-
bage (B. pekinensis) is effectively controlled by cies of Brassicaceae have been carried out (Verma
dressing seed with Thiram or powdered and Saharan 1994). Some of the fungicides,
Streptomyces griseoviridis (Valkonen and which have been found to be effective in control-
Koponen 1990). According to Crisan and Pall ling the Alternaria diseases and increasing the
(1986), seed treatment with Vitavax, Quinolate, yield under field conditions, are Apron 35 SD
Dichlofluanid, Zineb and Morpan completely (6 g/Kg−1), Antracol (0.02 %), Baycor (0.2 %),
inhibits A. alternata infection in cauliflower seed. Benlate (0.1 %), Blitane (0.2 %), Blitox-50
In India, seed-borne infections of A. alternata in (0.3 %), Bordeaux mixture (0.3 %), Boric acid
Eruca sativa have been controlled by treating the powder (0.53 %), Brestan (0.1 %), Captan
seed with Agrosan GN and Dithane M-45 at (0.2 %), Captafol (0.3 %), Cuman L (0.1 %),
0.3 % (Gupta and Saxena 1984). Difolatan (0.2 %), Dithane M-45 (0.2 %), Dithane

11.3.3 Bioagent Treatment

Seed treatment with Mycostop, a powdery for-

mulation of spores, and mycelium of Streptomyces
griseoviridis controls A. brassicicola damping-
off in cauliflower and cabbage (Fig. 11.1; White
et al. 1990). Biocontrol of seed-borne A. raphani
and A. brassicicola in radish has been obtained
through other antagonists including Chaetomium
globosum, Trichoderma harzianum, T. koningii
and Fusarium spp. (Plate 11.1; Vannacci and
Harman 1987). Garlic extract is most effective in
controlling the seed-borne fungi of mustard
(Latif et al. 2006).

11.4 Chemical Control

Inhibition of A. alternata, A. brassicae and A.

brassicicola growth in culture and spore germi-
nation by Bavistin, Blitane, Blitox-50, Benlate,
Brestan-60, Captan, Captafol, Ceresen dry,
Copper oxychloride, Carbendazim, Cupramar,
Cuman L, Dithane M-45, Dithane Z-78,
Difolatan, Emisan, Fytolan, Fentinhydroxide, Fig. 11.1 The effect of Streptomyces griseoviridis prepa-
Fostyl-al dinobuton, Griseofulvin, Rovral, ration on (a) percentage emergence and (b) percentage
disease-free seedlings of cabbage cultivar Celtic. O….O
Methofuroxan, Mycostatin, Polyoxin B, Polyoxin
untreated seed; □….□ seed treated with S. griseoviridis
D, Thiram, Tridimefon, Wettable sulphur, preparation; and ∆…….∆ seed treated with iprodione
Zincop, Quintozene, and Ziram have been (White et al. 1990)
242 11 Disease Management

Plate 11.1 Biocontrol of seed-borne Alternaria raphani around conidia of A. brassicicola. Bar = 35 μm; (e) scan-
and A. brassicicola: (a) coiling of Trichoderma harzia- ning electron micrographs of C. globosum hyphae coiling
num ATCC 56678 hyphae around A. brassicicola myce- around hyphae of A. brassicicola. Bar = 7 μm; (f) scanning
lium. Bar = 70 μm; (b) hyphae of T. harzianum 420 electron micrographs of C. globosum hyphae coiling
growing towards hyphae of A. raphani. Bar = 80 μm; (c) around A. raphani conidia. Bar = 25 μm; and (g) reaction
parallel growth of hyphae of Fusarium sp. along A. bras- zones (arrows) of A. brassicicola under the stimulus of C.
sicicola hyphae. Bar = 25 μm; (d) scanning electron globosum. Bar = 25 μm (Vannacci and Harman 1987)
micrographs of Chaetomium globosum hyphae coiling
Table 11.1 Chemicals tested against species of Alternaria attacking Brassicaceae (Verma and Saharan 1994; to date)
Fungicides
Acetone Fentin acetate Syllit
Actidione Ferbam Tebuconazole (Nativo)
Agrosan GN Fermate Tetrahydropyrimidine
Alar Folicur (tebuconazole) Thiabendazole
Antracol Folpet Thiophanate methyl
Arasan Flutriafol Thiovit
Azoxystrobin (Amistar) Formaldehyde Thiram
Azadirachtin (Neemarin) Granosan Tillex
Bafin Guazatin Topsin M
BAS 480 F Halogenated derivatives Triademefon
Baycor Imazalil Triapenthenol
Bayleton Indofil M-45 Triarimol
Bavistin Indofil Z-78 Tribasic copper sulphate
Benlate Iprodione (Rovral) Tribasic copper zinc
Benz (1,2) isoxazoles Karbam white Tridemorph
Bioquin Kavach Trifloxystrobin
Blitox Lunasan Trimiltox forte
Bordeaux mixture Malic acid Vinclozolin (Ronilan)
Boric acid Mancozeb Wettable sulphur
Boscalid Maneb Zato 50 WG
Brassicol Manzate Zincop
Brestan Merpan Zineb
Bromosan Metalaxyl Zinc sulphate
Calixin Metiram Ziram
Captaf Miltox Antibiotics
Captafol 4- Nitrosopyrazole Griseofulvin
Captan Nuarimole Mycostatin
Carbendazim Ozone Mycothricin
Carboxiin (Vitavax) Panogen Polyoxin B and D
Ceresen Parzate Insecticides
Chlorothalonil (Daconil, Bravo) Penconazole (Topaz) Cypermethrin
Copper oxychloride (Blitox) Pentachlorophenol Deltamethrin
Copper sulphate Phygon Dimecron
Cuman L P-Methoxytetrachlorophenol Fenvalerate
Cupravit Prochloraz Flucythrinate
Cupric acetate Propiconazole Metasystox
Cuprox Propineb Methyl demeton
Cycloheximide Pyraclostrobin Permethrin
Delan C Pyrena compounds Phosphamedon
Dichlofluanid zQuinolate Rogar
Difolatan Quinone Nutrients
Difenoconazole (Score) Ridomil MZ N, P, K, Ca, Cacl2
Dithane D-14 Ronilan CuSO4, CO(NO3)2, Fe EDTA,
Dithane M-45 (Mancozeb) Semesan Mn SO4, Na2 BO7, Zn SO4
Dithane Z-78 Signum 334WG Thiourea
Duter Sisthane Borax
Edifenphos Sodium fluoride S, SO2
Euparen Spergon Zn
Fenarimol S-triazines
Fenpropimorph Sumilex (Procymidone)
244 11 Disease Management

Z-78 (0.2 %), Daconil (0.2 %), Indofil M-45 in reducing radial growth of the pathogenic fungi
(0.02 %), Duter (0.2 %), Folicur (500 g a.i./ha), (74.2 %). Ridomil MZ reduced disease severity
Kavach (0.02 %), Ridomil MZ (0.2 %), Rovral by 32 % and was followed in effectiveness by
(0.2 %), Procymidone (0.750 Kg a.i./ha), combination of Bavistin + Captaf (26.5 %).
Prochloraz (500 g a.i./ha), TPTH (fentin hydrox- Maximum yield was obtained in plots sprayed
ide and triphenyltin hydroxide) (0.2 %) (Ansari with Bavistin + Captaf (1198 kg ha−1) followed by
et al. 1990; Chahal et al. 1977; Chand and Jatian Bavistin + Indofil Z-78 (1172 kg ha−1). It was
1969; Domsch 1957; Gaur and Ahmed 1980; worth noting that highest net profit as well as the
Gupta et al. 1977, 1985; Howlidar et al. 1985; highest cost–benefit ratio was obtained with
Kaspers and Siebert 1989; Keyworth 1969; Kolte Bavistin + Indofil Z-78 (1:3.2) followed by
1985; Kolte and Tewari 1978; Lapis and Ricaforte Bavistin + Captaf (1:1.3) (Khan et al. 2007).
1974; Maude and Dudley 1972; Noon et al. 1988; Lower concentration of SO2 (143 μg SO2 m−3)
Saharan 1991, 1992; Shivpuri et al. 1988; Singh stimulates Alternaria blight of mustard, but higher
1986; Singh and Bhowmik 1985; Singh and Sobti concentration (214.5 μg SO2 m−3) suppresses the
1980; Singh and Singh 2005; Verma and Saharan disease (Khan and Khan 2010). Six new fungi-
1994; Yadav 2003) (Fig. 11.1). cides, viz. azoxystrobin (Amistar), trifloxys-
Fungicides Mancozeb and Carbendazim trobin + tebuconazole ready-mix compound
caused 100 % reduction in mycelial growth of (Nativo), difenoconazole (Score), penconazole
Alternaria brassicae in vitro, while 1 % (w/v) (Topas), mancozeb (Dithane M-45) and azadi-
aqueous bulb extract of Allium sativum and leaf rachtin (Neemarin) were tested against Alternaria
extract of Acacia nilotica caused significant blight of rapeseed–mustard. Both in vitro and
reductions. In dual culture, GR isolate of in vivo studies showed almost the same order of
Trichoderma viride performed the best among the efficacy of the six different fungicides used. All
test isolates of Trichoderma, causing 82 % reduc- fungicides were significantly effective in reducing
tion in mycelial growth of A. brassicae. disease severity and increased seed yield.
Performance of isolates SI-2, P and SI-1 of T. However, the lowest disease severity (%), highest
viride was at par (P < 0.01) with that of GR isolate seed yield and significant increase in test weight
(Meena et al. 2004). Conidial suspension and cul- were recorded from two sprays of Nativo (1 g l−1)
ture filtrate (3:1) of T. viride cause inhibition of (Mondal et al. 2007b).
radial growth of A. brassicae (74 %) and A. bras- Three to four sprays of dithiocarbamate fungi-
sicicola (77 %) in dual cultures (Reshu and Khan cides, copper oxychloride and Bordeaux mixture
2012). Three systemic fungicides, Topsin-M have been found effective for the control of
(thiophanate methyl, 70%WP), Ridomil MZ Alternaria disease of rapeseed–mustard (Kolte
(mancozeb, 64 % + metalaxyl, 8 % WP) and 1985). The time of first spray, the interval and
Bavistin (carbendazim, 50%WP), alone and in number of sprays would depend upon the type of
combination with four non-systemic fungicides, crop. In B. rapa and B. juncea crops in India, a
Captaf (captan, 50%WP), Indofil M-45 (manco- significant control of the disease was achieved if
zeb, 75 % WP), Indofil Z-78 (zineb-78%WP) and the first spray was given at 60–75 days after sow-
Thiram (thiram 75%WP) were evaluated both ing; 2–4 more sprays at 10–15 days intervals,
in vitro and in vivo for their effectiveness to man- depending on the maturity of the crop, increased
age Alternaria blight of rapeseed–mustard caused the control (Kaushik et al. 1983, 1984; Kolte
by Alternaria brassicae. All the fungicides were 1985; Tripathi et al. 1987). The economics, in
evaluated for their efficacy at various concentra- relation to number of sprays, have been calcu-
tions, viz. 50, 100, 150, 200 and 500 ppm, and lated at Hisar, India, for the most effective five
were sprayed in the field at 0.2 % a.i. l−1. All the fungicides in rapeseed–mustard; it revealed that
fungicides significantly reduced the severity of four sprays each of Dithane M-45, Dithane Z-78,
the disease, but Ridomil MZ was most effective. Difolatan, Duter and Blitox-50 were required to
Topsin-M at conc. of 500 ppm was most effective minimize the disease and increased the yield.
11.4 Chemical Control 245

However, the maximum net profit was obtained 15 days also reduced disease intensity from 69.8 to
with four sprays of Dithane M-45 starting after 15.6 % in B. juncea, 79.6 to 20.5 % in B. rapa
30–45 days of sowing and repeated at an interval variety Yellow Sarson and 74.6 to 19.6 % in B.
of 15 days. Optimum growth stage of B. juncea in rapa variety Brown Sarson; a significant increase
reducing black spot infection with higher yields in yield was also obtained (Saharan 1991). The
through fungicidal sprays is 30–45 days after optimum time for application of fungicides to con-
sowing. In Bangladesh, Alternaria blight of mus- trol Alternaria disease of B. juncea has been found
tard was controlled by spraying Rovral (0.25) at to be 30–45 days after sowing. Fungicidal sprays
30, 40, 50 and 60 days after sowing (Sultana et al. at 60 days after sowing are unable to arrest the
2009) (Tables 11.2, 11.3, and 11.4) (Kaushik disease.
et al. 1983, 1984; Saharan and Chand 1988; In Bangladesh, among the three fungicides
Tripathi et al. 1987; Saharan 1991). tested, Dithane M-45 proved to be the best for
Integrated control of Alternaria and aphid pop- controlling Alternaria blight of B. rapa variety
ulation was obtained with a mixture of three fungi- Sonari Sarisha. Maximum reduction in disease
cides, Dithane M-45, Difolatan, Blitox and three intensity and maximum increase in yield was
insecticides, Rogar, Metasystox and Dimecron obtained with seven sprays starting at 30 days
(Table 11.5). The maximum net profit was obtained after seeding. Disease control by Trimiltox forte
with sprays of Difolatan + Metasystox followed by and Cuprovit was also satisfactory (Meah et al.
Difolatan + Dimecron, Blitox + Metasystox and 1988).
Dithane M-45 + Metasystox (Saharan 1992; In England, iprodione (0.5 Kg a.i./ha) or pro-
Tripathi et al. 1985). According to Thind and cymidone (0.75 Kg a.i./ha) has been shown to
Jhooty (1988), Captafol (0.2 %) provides maxi- protect winter oilseed rape and turnip rape against
mum disease control and persists longer on all A. brassicae for a period of up to 7 weeks follow-
brassicas. Mancozeb, Zineb and Copper oxychlo- ing application at 95 % petal fall (GS 4.9) (Evans
ride at 0.3 % exhibit highly differential efficacy and Gladders 1981; Evans et al. 1983, 1984,
against this disease on test brassicas. In mustard, 1988; Gladders and Rhodes 1985). Cox et al.
Shivpuri et al. (1988) obtained best control of (1981, 1983) in the UK and France also reported
Alternaria blight and a significant increase in yield good control of this disease with a single spray of
with Rovral followed by Captafol and Dithane iprodione at the early pod stage. Control was
M-45 (0.2 %). Sinha and Prasad (1989) found obtained with high-volume sprays and with vol-
Captafol was best, followed by Dithane M-45, for umes as low as 22 L/ha applied by aircraft.
the control of Alternaria blight of cauliflower to Flowable formulations are superior to wetta-
provide maximum yield. Similarly, Captafol ble powder (Humpherson-Jones et al. 1981).
applied at 0.2 % (w/v) four times at an interval of Seed yield increase of up to 20–30 % together

Table 11.2 Optimum time for spraying Difolatan (2 g product/l water) for the control of Alternaria leaf blight of raya
at different locations in 1979–1980 (Saharan 1984)
Hisar Ludhiana Kanpur
Sprays after Disease Disease Disease
sowing (days) intensity (%) Yield (Qtls) intensity (%) Yield (Qtls) intensity (%) Yield (Qtls)
30–45 33.7 21.6 46.0 19.0 20.4 8.4
60 42.9 19.7 46.4 17.8 24.2 7.7
75 50.5 19.5 53.4 19.9 30.7 7.3
82 57.8 19.2 – – 36.7 6.6
90 54.7 19.8 62.3 17.8 43.3 6.4
105 – – 73.8 17.0 – –
120 – – 78.9 14.7 – –
Control 63.4 18.3 84.4 9.1 46.9 6.1
246 11 Disease Management

Table 11.3 The efficacy and economics of fungicidal Procymidone, Iprodione and Prochloraz fungi-
spray on Alternaria leaf spot of raya at Hisar in 1977–
cides have also been reported effective against
1978 (Saharan 1984)
Alternaria spp. on winter oilseed rape in Poland
No. of Disease Yield/ha Net profit /
(Bonin and Fratczak 1987) and Chinese cab-
Fungicides spray intensity (%) (Qtls) ha (Rupees)
bage in Denmark (Anonymous 1988).
Dithane 1 47 12 53
M-45 2 45 13 72
Tebuconazole suppresses the Alternaria blight
3 40 14 372 of rape in Lithuania more effectively than pro-
4 29 15 803 chloraz. The highest efficacy is recorded when
Dithane 1 51 13 212 the fungicides are applied after the first symp-
Z-78 2 42 13 70 toms of the disease are spotted on siliques
3 40 14 312 (BraZauskiene and Petraitiene 2004).
4 33 15 751 In India, iprodione spray (500 g a.i./ha) during
Difolatan 1 42 12 12 the early pod stage is most effective in reducing
2 37 13 152 Alternaria blight of mustard with increase in
3 32 15 639 yield by 24–59 % (Mukherjee et al. 2003). In
4 22 16 792 Czechoslovakia, A. brassicae and A. brassicicola
Duter 1 50 13 248 on cauliflower were successfully controlled, and
2 38 14 444 yield increased by 2–22 times after spraying with
3 33 14 451 a mixture of Thiram + Benlate alternated with
4 28 16 748 Mancozeb + insecticide (Kudela et al. 1978).
Blitox-50 1 53 13 211 For the control of Alternaria blight on radish
2 47 14 357
seed crop, Dithane-M-45 (0.25 %) proved most
3 43 13 187
effective, followed by Bordeaux mixture (0.4 %)
4 32 15 748
in increasing seed weight, improving seed germi-
Control – 62 12 –
nation, and in reducing seed infection (Hussaini
and Singh 1989). However, Mondal et al. (1989)
found Rovral (0.2 %) best followed by Dithane
Table 11.4 Optimum growth stage of B. juncea for reduc-
M-45, Dithane Z-78 and Captan for the control of
ing black spot through fungicidal sprays (Saharan 1991)
Alternaria blight of radish.
Fungicidala sprays after Disease Yield (Q/
In the UK, Channon (1970) found Propineb
days of sowing intensity (%) ha)
30 15.5 21.5
and Mancozeb (0.2 %) sprays consistently effec-
45 19.0 21.3
tive against A. brassicicola on cabbage in glass-
60 40.6 18.2 house. Similarly, three sprays of iprodione at
75 49.5 17.0 0.5–1 Kg a.i./ha rate, at 3 weeks interval starting
90 65.5 15.0 at the young green pod stage till harvesting, con-
Control 75.5 12.0 trolled pod infection of A. brassicicola and
C.D. at 5 % 3.33 – increased yields (Humpherson-Jones and Maude
a
Captafol (0.2 %) was sprayed 1982). In India, Singh et al. (1989) found three
sprays any of Mancozeb, Cuman L and Zineb
found best for the control of A. brassicicola leaf
with low residual levels of inoculum on the seed spot of cabbage.
has been reported following application of ipro- Storage rot of white cabbage due to A. bras-
dione (Ogilvy 1984). Two sprays of Prochloraz sicicola can also be effectively controlled with
at 500 g a.i./ha, the first at stem extension and application of 5 % iprodione mixed in talc pow-
second at mid-late flowering, have also proved der (Kear et al. 1977). Similarly, a post-harvest
effective in controlling this disease in winter iprodione drench on stored winter cabbage con-
oilseed rape together with a significant increase trols Alternaria spp. (Geeson and Browne 1979).
in yield (Marshall and Harris 1984). On the other hand, Tasca and Trandaf (1984)
11.4 Chemical Control 247

Table 11.5 The effect of fungicide and insecticide mixture on Alternaria leaf spot and aphid population on Brown
Sarson (BSH-1) at Hisar in 1977–1978 (Saharan 1984)
Mixture of fungicide and Aphid population/10 Disease intensity Yield/ha Net profit/ha
insecticide twigs (%) (Qtals) (Rupees)
Dithane M-45 + Rogar 72 36 9.0 875
Dithane M-45 + Metasystox 57 25 9.7 1088
Dithane M-45 + Dimecron 75 42 8.8 766
Difolatan + Rogar 75 31 8.4 459
Difolatan + Metasystox 62 17 10.7 1285
Difolatan + Dimecron 72 33 10.4 1279
Blitox + Metasystox 70 48 9.9 1151
Blitox + Rogar 107 49 8.8 748
Blitox + Dimecron 67 56 8.9 836
Control 820 72 5.6 –
C.D. at 5 % 6.8 6.6 – –

reported pre-harvest treatment of cabbage with carbamate) inhibits pollen germination and/or
Mancozeb (0.2 %) or Folpet (0.2 %) for protec- tube elongation in apple and cucumber. He et al.
tion from A. brassicae storage rots. Ronilan- (1996) reported that lower concentration of
smoke, a vinclozolin formulation used as smoke Benomyl has limited effects on pollen germina-
pellets, in Brassica storage rooms also controls tion, and tube elongation, while higher concen-
A. brassicae (Jennrich 1985). tration inhibits germination appreciably and
Foliar diseases of Brussels sprouts in the UK causes greater number of ruptured and swollen
are effectively controlled with the application of tubes in Tradescantia virginiana.
mixtures of iprodione, triadimefon/Carbendazim Application of Dithane M-45 also inhibits
and metalaxyl/Mancozeb. The timing of the both pollen germination and tube growth of B.
sprays is more important than the number of juncea. The germination inhibition is more pro-
applications. Two sprays, one in August and nounced in RH-8812 than RH-30. However, it
another one in September, are enough to reduce inhibits germination completely in RH-30 at
fungicide input (Brokenshire 1987; Davies et al. 0.2 % concentration, while a substantial num-
1986). ber (18.85 %) of pollen germinate in RH-8812
at same concentration. Lower concentration
(0.025 %) of fungicide decreases tube length
11.4.1 Effect of Fungicides on Host significantly (38.84 %) in RH-30, which
Growth remains unaffected in RH-8812 by the same
concentration of fungicide (Table 11.6). Pollen
Fungicides are frequently applied for crop pro- germination also decreases with increasing
tection in Brassicas from flowering to maturity. concentration of Ridomil MZ-72, and it is com-
These fungicides may interfere with microsporo- pletely inhibited at a concentration of 0.15 %
genesis and on pollen production, which are and above. The overall reduction in germina-
sometimes less vigorous/non-viable or may tion is more severe in RH-8812 and RH-30.
affect pollen tube growth through the style and However, converse is more true with regard to
thus interfere with fruit set and economic yield tube length (Table 11.6) (Jain et al. 2000).
adversely (Williams et al. 1987). However, these Spraying Ridomil MZ-72 at 0.0125 % may not
aspects have not received due attention. Available have deleterious effect on pollen biology, and
information indicates that Benomyl (methyl consequently, crop yield as has been suggested
1-(butyl carbamoyl)-2-benzimidazole- by Kolte et al. (1981).
 248

Table 11.6 The effect of Dithane M-45 and Ridomil MZ-72 on in vitro pollen germination (%), and tube length (μm) in two cultivars of B. juncea (Jain et al. 2000)
Pollen germination (transformed values) Pollen tube length
Fungicide Dithane M-45 Ridomil MZ-72 Dithane M-45 Ridomil MZ-72
conc. (%) RH-30 RH-8812 Mean RH-30 RH-8812 Mean RH-30 RH-8812 Mean RH-30 RH-8812 Mean
Control 58.4 55.0 56.7 58.4 55.0 56.7 193.6 105.3 149.5 193.6 105.3 149.5
(72.5) (67.0) (69.7) (72.5) (67.0) (69.7)
0.025 56.2 38.9 47.6 41.2 19.8 30.5 118.4 105.3 111.9 73.2 85.1 79.2
(69.1) (39.4) (54.2) (43.4) (11.7) (27.5)
0.05 37.2 39.0 38.1 40.3 18.1 (9.7) 29.2 114.9 96.9 105.9 62.2 75.5 68.9
(36.7) (39.1) (37.9) (41.9) (25.8)
0.10 32.6 28.2 30.4 28.2 16.8 (8.7) 22.5 114.1 86.0 100.1 48.2 74.6 61.4
(29.4) (22.3) (25.8) (22.3) (15.5)
0.15 32.1 24.3 28.2 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 95.6 72.8 84.2 0.0 0.0 0.0
(28.3) (16.9) (22.6)
0.20 0.0 (0.0) 18.8 9.4 (5.2) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 68.4 34.2 0.0 0.0 0.0
(10.5)
Mean 36.1 34.0 – 28.0 18.3 – 106.1 89.1 – 62.9 56.7 –
(39.3) (32.7) (30.0) (16.2)
C.D. Cultivar (cv.) 2.29 5.7
(P < 0.05) Fungicide (F) 1.87 3.3
Cv × F 4.57 8.1
11

Figures in parentheses are mean of actual values, while others are angular transformed ones of the same
Disease Management
11.5 Biological Control 249

11.5 Biological Control growth and germination of A. brassicicola (Yasmeen

and Saxena 1990; Ahmed and Agnihotri 1977).
11.5.1 Plant Extracts Plant extracts of roots, leaves, stems, inflores-
as Fungitoxicants cence and fruits of several species (Table 11.7)
have shown fungicidal activities in in vitro and
Essential oil from the roots of radish inhibits (1:2500) in vivo conditions against two major Alternaria
A. brassicae (Nehrash 1961). The deproteinized leaf spp., viz. A. brassicae and A. brassicicola caus-
extracts of Acacia nilotica, Enicostema hyssopifo- ing blight or black spot diseases of crucifers. In
lium, Mimosa hamata and Vitis vinifera have shown in vitro conditions, almost all of the plant extracts
fungistatic activity against A. brassicicola (Umalkar restrict mycelial growth, sporulation and conidial
et al. 1977). The extracts prepared from the leaves of germination. Some of them have shown efficacy
Lawsonia alba, roots of Datura stramonium and under field conditions by restricting the infection
inflorescence of Mentha piperita have fungitoxic points, spot size and disease intensity (Khurana
activity against A. brassicae isolated from cauli- et al. 2005; Patni et al. 2005; Muto et al. 2006; Ho
flower leaves. Extracts of the ferns Adiantum cauda- et al. 2006, 2007; Patni and Kolte 2006; Guleria
tum, Diplazium esculentum and Pteris vittata reduce and Kumar 2010; Guleria et al. 2010; Baka 2010;

Table 11.7 Plant extracts tested against Alternaria spp. attacking crucifers
Plant species Concentration (%) References
Acacia nilotica – Umalkar et al. (1977)
Encostema hyssopifolium – Umalkar et al. (1977)
Mimosa hamata – Umalkar et al. (1977)
Vitis vinifera – Umalkar et al. (1977)
Lawsonia alba – Umalkar et al. (1977); Yasmeen and Saxena (1990)
Datura stramonium – Umalkar et al. (1977)
Mentha piperita – Yasmeen and Saxena (1990)
Adiantum caudatum – Ahmed and Agnihotri (1977)
Diplazium esculentum – Ahmed and Agnihotri (1977)
Pteris vittata – Ahmed and Agnihotri (1977)
Allium sativum 5 Khurana et al. (2005)
Azadirachta indica 10 Sasode et al. (2012)
Lawsonia inermis 5 Meena and Sharma (2012)
Erythrina chiaposana 5 Meena and Sharma (2012)
Ricinus communis 5 Meena and Sharma (2012)
Zingiber officinale 5 Meena and Sharma (2012)
Euphobia pulchrima 5 Patni et al. (2005)
Rumax dentatus 5 Patni et al. (2005)
Urtica dioica 5 Patni et al. (2005)
Eucalyptus globules 5 Patni et al. (2005)
Solanum nigrum 5 Muto et al. (2006)
Rhus chinensis – Ho et al. (2006)
Ocimum sanctum 5 Patni and Kolte (2006)
Anagallis arvensis 5 Patni and Kolte (2006)
Polygonum perfoliatum 1 Ho et al. (2007)
Agave americana 1 Guleria and Kumar (2010)
Solanum xanthocarpum 1 Guleria et al. (2010)
Lavandula pubescens 10 Baka (2010)
Calotropis procera 15 Singh et al. (2013)
250 11 Disease Management

Meena and Sharma 2012; Sasode et al. 2012; factors, such as climatic conditions, age of the host
Singh et al. 2013). plant, maturity of the leaves and variety of the
Eucalyptus spray gives significantly lesser host, have profound influence on the deposition of
number of spots per leaf (2.05), minimum size of conidia of both the pathogens and S. rochei
spots (1.28 mm), minimum sporulation intensity (Sharma and Gupta 1980). Jayant and Sinha
(1.22 × 105) and minimum disease index followed (1981) reported that S. hygroscopicus is strongly
by Calotropis, Ocimum and Polyanthai extracts at antagonistic to A. brassicae and A. brassicicola.
5 % (Patni and Kolte 2006). Alternaria blight of When the culture filtrate is sprayed over the Sarson
mustard can be managed by spraying aqueous plants, a week before or a week after spraying the
extract of Azadirachta indica, Allium sativum and spore suspension of A. brassicae and A. brassici-
Zingiber officinale at 5, 10, 15 and 20 % along with cola, germination and disease development are
higher yield. However, spraying of 15 % A. indica reduced. Whenever the spore population of
extract gives highest yield with best cost–benefit Streptomyces arabicus is higher on leaves of
ratio (Mahapatra and Das 2013). While, according Yellow Sarson and Taramira, the conidial popula-
to Meena et al. (2004, 2008), application of bulb tion of A. brassicae and A. brassicicola declines.
extract of Allium sativum at 45 and 75 d.a.s. gave The population of antagonistic S. arabicus was
highest seed yield at Sewar, India. The Allium sati- higher in the young leaves as compared to old ones
vum extract is most effective in controlling seed- (Sharma et al. 1984). The antifungal substance in
borne fungi of mustard also (Latif et al. 2006). the diffusate of the antagonist is thermolabile
(Sharma et al. 1985). A pigmented and xylose-
utilizing strain of S. bobili is found to be active
11.5.2 Antagonists for Biocontrol against A. brassicae, A. brassicicola and A.
raphani (Sharma and Sinha 1989).
Saprophytic phylloplane fungi such as An isolate of Streptomyces spp. obtained from
Aureobasidium pullulans and Epicoccum nigrum light-coloured, Finnish Sphagnum horticultural
are pathogenic to A. brassicicola (Pace and peat has proved effective biological control agent
Campbell 1974). The Verticillium state of Nectria against plant pathogens (Tahvonen 1982a, b).
inventa Pethybridge, a destructive mycoparasite, Treating cauliflower seeds with Trichoderma
is one of the dominant phylloplane fungi of rape- viride and Streptomyces spp. isolates inhibits or
seed (Tsuneda and Skoropad 1978b). Among the reduces damping-off caused by A. brassicicola
leaf surface mycoflora, the most antagonistic (Tahvonen 1982b, 1988). Seed dressing with
fungi are E. purpurascens, A. pullulans and Mycostop, a powdery formulation prepared from
Cladosporium cladosporioides in the case of A. spores and mycelium of Streptomyces, was suc-
brassicae. The metabolites of Acremonium cessful in controlling damping-off (80–90 %)
roseogriseum, Aspergillus terreus and C. clado- from seeds artificially infected with A. brassicic-
sporioides inhibit A. brassicae. The most signifi- ola. The dressing remains effective on seeds
cant effects are observed when the spores of the stored under dry conditions for 5–6 weeks, but
leaf surface fungi, and their metabolites, are subsequently decreases slowly. Streptomyces
sprayed prior to inoculation of pathogen on dressing controls in a manner comparable to
leaves (Rai and Singh 1980). chemical dressing with Thiram, preventing
Pre-treatment application with the spore sus- damping-off caused by Alternaria fungi in seed-
pension of Streptomyces rochei or its diffusate lings, which are grown from commercial seed
results in a marked reduction of leaf blight inten- lots of different origin (Fig. 11.2; Tahvonen 1985;
sity caused by A. brassicae and A. brassicicola on Tahvonen and Avikainen 1987). White et al.
Brown Sarson, B. rapa variety Dichotoma (Sharma (1990) reported that Mycostop, a biofungicide
and Gupta 1978a, b, 1979). With the decline in the based on a selected S. griseoviridis isolate from
population of S. rochei, there is a rise in the popu- Finnish Sphagnum peat, introduced either by
lation of Alternaria from December onwards indi- seed treatment or soil treatment, controls A. bras-
cating the possibility of antagonism. Several sicicola of cauliflower and cabbage (Fig. 11.1).
11.5 Biological Control 251

Fig. 11.2 The biological control of (a) seed-borne with a powdery preparation of Streptomyces sp. (Tahvonen
Alternaria brassicicola and (b) A. brassicicola content of and Avikainen 1987)
seedlings grown from naturally infected Brassica seed

Brassicaceous seeds treated with either of A. brassicicola. Conidia of A. brassicicola

Gliocladium virens-19, Trichoderma harzianum- shrivel and plasmolyze in the presence of antago-
22, T. harzianum-50, Penicillium corylophilum- nistic fungi (Plate 11.2; Wu and Lu 1984). For
36 and P. oxalicum-76 gave significantly higher biocontrol of seed-borne A. raphani and A. bras-
emergence of healthy seedlings. The hyphae of sicicola of radish, antagonists like Chaetomium
antagonistic fungi were able to adhere to conidia globosum, T. harzianum, T. koningii and Fusarium
or coil around or penetrated germ tubes or hyphae spp. treated plants showed consistently increased
252 11 Disease Management

numbers of healthy seedlings (Vannacci and 20–35 °C temperature (Reshu and Khan 2012). In
Harman 1987). Wu and Lu (1984) found Poland, Madej (1986) reported Gonatobotrys
Trichoderma, Gliocladium and Penicillium spp. simplex as a hyperparasite of A. brassicae.
parasitizing A. brassicicola. The application of A.
alternata, prior to inoculation with A. brassicae,
reduced the level of A. brassicae on oilseed rape 11.5.3 Mechanisms of Biocontrol
by about 60 % and subsequent to inoculation by
about 26 % (Mee 1991). Spraying of T. viride Antagonistic mechanism of biocontrol has been
conidial suspension causes reduction in Alternaria studied in a few selected host–parasite systems. The
blight of mustard on leaves (76 %) and pods parasitism of A. brassicae by the Verticillium state
(68 %), respectively. The bioagents survive on of Nectria inventa occurs either by penetration or
phylloplane up to 30 days at 80–90 % RH with contact without penetration. Parasitic hyphae

Plate 11.2 The effectiveness of antagonistic fungi around a conidium on a cabbage seed; and (d) P. citrinum
against Alternaria brassicicola; (a) Penicillium coryloph- coiled around the condial germ tube on a cabbage root
ilum-36 and (b) Trichoderma harzianum-22 coiled around (Wu and Lu 1984)
the conidia on agar medium; (c) T. harzianum-22 coiled
11.5 Biological Control 253

induce abnormal responses in host cells upon con- of N. inventa. As a result, the germination of
tact. A reaction consisting largely of an electron- Alternaria conidia is drastically reduced to less than
dense transparent matrix and dispersed tubule-like 5 %, and they are eventually destroyed by N. inventa
electron-dense material develops between the cell (Tsuneda and Skoropad 1978c). The treatment of
wall and the invaginated plasma membrane. The oilseed rape seeds with Bacillus strains UCMB-
tubule-like elements subsequently aggregate to 5113 and UCMB-5036 results in significant protec-
form electron-dense deposits below the cell wall. tion against all fungal pathogens tested. The
The affected cell forms a septal plug, accumulates protection seems to be a combination of lower
membranes and finally degenerates. Hyphae of N. infection frequency and less severe disease symp-
inventa penetrate the conidial cells of A. brassicae toms on treated plants. Phenological analysis of
mainly by a process, which appears primarily enzy- plants for fitness effects due to Bacillus inoculation
matic in nature. The cytoplasm of the penetrated showed a significantly higher survival rate for the
cell becomes progressively less dense, and the cell seeds treated with UCMB-5033, UCMB-5036 and
eventually appears empty (Tsuneda 1977; Tsuneda UCMB-5113 and providing a growth-promoting
and Skoropad 1980; Tsuneda et al. 1976). effect. No visible signs of infection such as chloro-
Penetration of A. brassicae hyphae causes separa- sis or yellowing are observed on plants that germi-
tion of the cells; penetration of conidia occurs most nated after bacterial treatment. The germination
frequently at the septum or at the basal pore in juve- efficiency was also same as the seed harvested from
nile conidia (Tsuneda and Skoropad 1977a, b). different plants including control (Danielsson et al.
Conidia of N. inventa require at least 24 h to initiate 2006). Bacillus subtilis strain UK-9, an isolate from
germination and 4 days to parasitize A. brassicae on reclaimed soils, was studied for its biological con-
intact leaves (Plates 11.3, 11.4, 11.5, 11.6, and 11.7; trol activity against Alternaria leaf spot of mustard.
Tsuneda and Skoropad 1978a, b). Conidia of A. In dual culture, production of antifungal metabo-
brassicae release various amino acids and sugars lites by the bacteria caused morphological altera-
when they are exposed to an alternate dry–wet con- tions of vegetative cells, and spores, disruption and
ditions; the longer the drying period, the larger the lysis of their cell wall. The antagonists reduced
amount of leakage is. Among the amino acids, glu- spore germination on leaves, and disease incidence
tamine is exuded in the largest amount followed by of the pathogen in plant as well demonstrated plant
aspartic acid and glutamic acid. The sugar fraction growth-promoting ability (Sharma and Sharma
consists mainly of glucose and fructose. These 2008). Many antagonistic species produce antibi-
leaked nutrients stimulate germination and growth otic substances, which have the ability to produce

Plate 11.3 Excised non-dried

rapeseed leaf inoculated with
Alternaria brassicae alone
(left half of leaf) and with the
mixture of A. brassicae and
Nectria inventa (right half of
leaf) at 1 week after
inoculation. In the necrotic
areas (NA) caused by A.
brassicae, note the presence of
a white mycelia mass of A.
brassicae with conidia (AB)
on the left half and its absence
on the right half of the leaf
with abundant conidia of N.
inventa (NI). × 1.5 (Tsuneda
and Skoropad 1978b)
254 11 Disease Management

Plate 11.4 Phase contrast light micrographs of the (b) profuse growth of parasitic hyphae around a host
conidia of Alternaria brassicae parasitized by Nectria conidium. × 1,200. Legend: P = parasite, C = host condio-
inventa: (a) healthy-appearing conidium and infected phore, MC = mature host conidium, JC = juvenile host
mature and juvenile conidia. Note non-infected cells conidium (Tsuneda et al. 1976)
(arrows) in the heavily infected conidium. × 1,200; and

enzymes causing the lysis of cell wall components 2010a). Biochemical analysis reveals that applica-
of the pathogenic fungus. This helps the antagonist tion of biocontrol agents results in increase in lipid
to penetrate the host hypha and grow as a hyper- and protein content in seeds collected from treated
parasite (Tapio and Pohto-Lahdenpera 1989). plants. The proportion of various lipidic fractions,
i.e. phospholipids, glycolipids and sterol contents
in seeds, also increased with a corresponding
11.5.4 Biological Control vs. decrease in total glycerides. The proportion of
Biochemical Changes 18:3, 20:1 and 22:1 fatty acids increased, while
that of 18:1 and 18:2 fatty acids decreased in seeds
Indian mustard seeds were treated separately with with application of biocontrol agents. There are
three bioagents, viz. Trichoderma harzianum, both qualitative and quantitative differences in the
Pseudomonas fluorescens and Bacillus subtilis banding patterns of albumin and globulin proteins
and grown in experimental fields, followed by after application of biocontrol agents. The bio-
spraying with these antibiotics at 30 and 60 days chemical alterations in the host induced by treat-
after sowing (DAS). The use of biocontrol agents ment with biocontrol agents can be associated
showed enhanced growth in comparison to control with defence mechanisms and enhanced growth of
and decreased disease index on leaves as well as the plant (Sharma et al. 2010b).
on pods. These biocontrol agents also enhanced
the content of dry matter, total phenol, ortho-dihy-
droxyphenol, starch, total soluble sugars, reducing 11.6 Host Resistance
sugars, total lipids and different membrane lipids
in the leaves. However, the total protein content Four species of Alternaria, i.e. A. brassicae, A.
decreased. It suggests that treatment with biocon- brassicicola, A. raphani and A. alternata, are
trol agents initiates in the plants a number of bio- pathogenic on brassicas. Out of these, A. brassicae
chemical changes, which can be considered to be a and A. brassicicola are widespread and economi-
part of plant defence responses (Sharma et al. cally most important since they cause losses of
11.6 Host Resistance 255

Plate 11.5 Hyphae of Alternaria brassicae parasitized scanning electron micrographs of the parasite coiling
by Nectria inventa; (a) light micrograph of parasite around host hyphae. ×15,000. Legend: P = parasite,
hyphae parallel to a host hypha. Note the swollen H = host, AB = appressorium-like body of parasite,
appressorium-like body of the parasite. × 4300; and (b, c) HB = hyphal branch of the host (Tsuneda et al. 1976)

more than 70 %. All commercial brassicas are tive germ-plasm screening techniques from diverse
essentially susceptible to these pathogens. sources to broaden the genetic base: availability of
However, there are some differences in their degree usable source of resistance, nature and mechanism
of susceptibility. Brassica napus and B. carinata of resistance. The various techniques are available
are less susceptible than B. rapa and B. juncea. In to screen germ plasm/varieties/lines, etc., against
addition to these, some wild crucifers have high A. brassicae in the field, screen house/glass house
degree of resistance to A. brassicae, and attempts and in the laboratory for resistance. Various studies
are being made to transfer this resistance to culti- have also been conducted which indicate the exis-
vated brassicas. Alternaria blight management tence of A. brassicae pathotypes (Saharan 1992;
strategies are to incorporate the host resistance as Saharan and Kadian 1983b; Saharan et al. 2003).
one of the components into the cultivated brassicas. Inheritance of resistance in inter- and intra-
One should have the knowledge of pathogenic vari- specific crosses of B. juncea x B. carinata genotypes
ability development and standardization of effec- reveals polygenic, additive gene effects, dominance
256 11 Disease Management

Plate 11.6 Scanning electron micrograph of mycopara- like bodies (A) formed on the host conidum. Note pres-
site Nectria inventa hyphae growing on Alternaria bras- ence of adhesive material under these bodies. × 11,000;
sicae; (a) parasite hyphae occurring predominantly in the and (c) appressorium-like body (A) with fibrous adhesive
septal area (arrows) and the basal portions of the germ material (arrow) formed on host hypha (H). × 25,000
tubes (G) of a host conidium. × 1800; (b) appressorium- (Tsuneda and Skoropad 1977a)

gene effects, additive x dominance epistasis and nents of horizontal resistance in B. napus cv. Tower
non-allelic interactions. Inter-mating between resis- and B. juncea cv. RC-781 can be exploited to breed
tance plants helps in increasing the level of resis- for horizontal/slow blighting resistance. Rapid
tance through resistance gene pyramiding. Disease progress has been made in identification, cloning
tolerance attributes in B. juncea genotypes have and sequencing of resistant genes in B. juncea, B.
been identified. Identification of different compo- napus, R. sativus and A. thaliana (Krishnia et al.
11.6 Host Resistance 257

Plate 11.7 The Alternaria brassicae–Nectria inventa The host conidium is penetrated through the basal pore.
host–parasite interface; (a) Mature conidium of A. bras- MC, infected cell of a mature conidium. CP, conidiophore
sicae penetrated by hyphae of N. inventa. Note collapsed produced by the mature conidium × 2500; (d) light micro-
cell wall of the conidium (arrow) × 1.800; (b) enlarged graph of a thin section of a normal juvenile conidium of A.
view of penetration site. A large hole develops in the wall brassicae showing the basal pore and septal pore
of the host cell, and a meshwork of material appears at the (arrow) × 2800; and (e) Scanning electron micrograph of a
penetration site (arrow) × 27,000; (c) penetration of a basal pore in a juvenile conidium of A. brassicae × 3500
juvenile conidium (JC) of A. brassicae by N. inventa (P). (Tsuneda and Skoropad 1977a)

2000; Saharan and Krishnia 2001; Gupta et al. 2008; Mishra et al. 2010; Verma et al. 2012;
2002; Saharan and Kadian 1983a; Kumar and Kolte Chhikara et al. 2012; Sellam et al. 2007).
2001, 2006; Kanrar et al. 2002; Kamble and The effect of host-specific phytotoxins pro-
Bhargava 2007; Mondal et al. 2007a; Minami et al. duced by Alternaria species on cruciferous plants
258 11 Disease Management

at physiological, biochemical and molecular lev- tion with other subpopulations. Only highly fit
els has been determined. The role of phytotoxins, resistant individuals can be maintained in or even
and other metabolites produced during Alternaria dominate the population, causing loss of control of
brassicae interactions on pathogenesis, and host the corresponding fungicides. It has been fre-
defence along with activity of MAPK 4 cascade quently observed that iprodione-resistant isolates
gene in plant defence against biotic stress has of different plant pathogenic fungi have higher
been identified (Bains and Tewari 1989; Verma osmotic sensitivity than do sensitive isolates. This
and Saharan 1994; Lou et al. 2013; Pedras et al. osmotic sensitivity is considered to be responsible
2009; Thomma 2003; Marmath et al. 2013). for the lower pathogenicity and lower fitness of the
The nature and mechanisms of resistance in resistant isolates. Huang and Levy (1995) investi-
cruciferous crops are multilayered. It is in the gated the parasitic and saprophytic fitness, the
form of morphological barriers/features like fewer resistance to other fungicides and the osmotic sen-
numbers of stomata with narrow aperture in some sitivity of 25 laboratory mutant isolates of A. bras-
genotypes of B. napus and B. juncea. The pres- sicicola exhibiting iprodione resistance and the
ence of epicuticular wax layers in Brassica geno- corresponding wild types from which they were
types provides a hydrophobic leaf surface, which isolated. The resistance of Alternaria brassicicola
hinders Alternaria spore germination and infec- isolates to iprodione exhibits a polymodal distribu-
tion. Large number of biochemical constituents tion. Four distinct groups were detected demon-
governs biochemical resistance. Proteome and strating ED50s of 0–7, 10–40, 70–80 and 300–516 μg
enzyme activity in resistant genotypes activates a.i. per ml. Most resistant isolates produce smaller
resistant genes. Hypersensitive response genes for colonies on unamended potato dextrose agar.
induced resistance have been identified. Fewer conidia are produced per colony for some
Expression of MAPK 4 (mitogen-activated pro- resistant isolates than for their corresponding wild
tein kinase) cascade gene antagonizes the effects types, while other resistant isolates produced more
of pathogens in resistant genotypes. Elicitation of conidia per unit of colony area. The resistant iso-
phytoalexins by Brassica species inhibits fungal lates either produce smaller lesions or sporulate
growth on host leaf surface. Calcium sequestra- less or both than the sensitive isolates on untreated
tion character of brassicas gives ample possibili- broccoli leaf discs. Only resistant isolates are able
ties of enhancing resistance to Alternaria spp. to produce lesions on leaf discs sprayed with 50 μg
infection. Sources of resistance in cruciferous a.i. of Iprodione per ml. Conidia of resistant iso-
species (closely as well as distantly related) lates germinated in distilled water containing ipro-
against Alternaria spp. have been identified, but dione at a concentration as high as 250 μg a.i. per
very few have been explored for commercial use ml, while the germination of sensitive isolates was
to develop resistant cultivars. The information greatly inhibited in distilled water containing only
generated so far on parameters of host resistance 5 μg a.i. of Iprodione per ml. Iprodione-resistant
is being utilized through conventional and bio- isolates are cross-resistant to vinclozolin and
technological approaches (Verma and Saharan dichloran. Resistant isolates exhibited a greater
1994; Saharan et al. 2003: Saharan and Kadian osmotic sensitivity than sensitive isolates; however,
1983a; Kumar and Kolte 2006; Gupta et al. 1990; osmotic sensitivity is independent on the degree of
Dhingra et al. 2004; Oh et al. 2005; Mishra et al. resistance (Huang and Levy 1995).
2010; Marmath et al. 2013; Conn et al. 1991; Three sterol biosynthesis inhibiting fungicides,
Sellam et al. 2007; Aneja and Agnihotri 2013). two of the triazole group (flutriafol and difeno-
conazole) and one of the imidazole group (pro-
chloraz), performed equally well in reducing the
11.7 Fungicidal Resistance mycelial growth of A. brassicae, A. brassicicola
and A. japonica. Out of the two selected dicar-
Mathematical and experimental studies have boximides, Iprodione proved to be more effective
shown that parasitic and saprophytic fitness of a than Procymidone. Most of the tested isolates are
resistant subpopulation is crucial for its competi- also very sensitive to fludioxonil (phenylpyrroles
11.8 Integrated Disease Management 259

family). However, in vitro tests for mycelial after the harvesting of the crop to restrict the
growth, conidial germination and germ-tube elon- sources of primary inoculum.
gation revealed the existence of A. brassicicola 2. Deep summer ploughing in May–June should
isolates highly resistant (EC50 > 100 mg/l) to both be done to destroy the resting structures of the
dicarboximides (e.g. iprodione and procymidone) pathogen.
and phenylpyrroles (e.g. fludioxonil). These resis- 3. Crop rotation with non-cruciferous crops for
tant isolates did not exhibit lower sporulation at least 3 years should be followed in the case
capacities or reduced aggressiveness towards host of severe disease epidemic areas.
plants compared to sensitive A. brassicicola iso- 4. Timely sowing; application of recommended
lates (Iacomi-Vasilescu et al. 2004). dosages of fertilizers; use of nutrients like
boron, potassium, phosphorus, sulphur, zinc,
biofertilizers, biocontrol agents; maintaining
11.8 Integrated Disease optimum plant population; timely thinning;
Management timely irrigation, weeding and control of
insect pests should be followed.
It includes all means of disease and crop manage- 5. Sowing time should be adjusted depending
ment, viz. cultural, nutritional, biological, bio- upon weather conditions and disease-
chemical, biotechnological, chemical, host prevailing conditions in a particular area. Early
resistance and genetic engineering in an inte- sowing (up to 20 October) of the crop escapes
grated way including pest management (Mukerji the major diseases including Alternaria blight
et al. 1999; Verma and Saharan 1994; Saharan under North Indian conditions.
and Mehta 2002; Sharma and Kolte 1994; Kolte
2005; Godika et al. 2001; Pathak and Godika The multiple disease control strategy is mainly
2010; Meena et al. 2011, 2012, 2013; Mehta dependent on the balanced fertilizer application
2014). Following cultural practices have been (N100;P40:K40), and available level of tolerance
advocated for better management of Alternaria in host varieties, early sowing time (first week of
disease of cruciferous crops. Cultural control of October) and seed treatment with Apron 35 SD fol-
crucifer diseases is largely a matter of sanitation lowed by use of fungicidal spray (Metalaxyl + man-
and manipulating the environment to the advan- cozeb = Ridomil MZ 72 @0.25 %) for Alternaria
tage of the host and to the detriment of the patho- blight, white rust + downy mildew complex control
gen. In addition, the judicious use of fertilizer or carbendazim spray at 0.05 % for Sclerotinia rot
should be adopted. The various cultural practices and powdery mildew control (Kolte 2005;
help in reducing the diseases are: Tables 11.8, 11.9, and 11.10).
Godika et al. (2001) reported that the combi-
1. Crop debris containing resting structures of nation of boron (0.53 %) with boric acid or zinc
the pathogens should be burnt or destroyed (0.22 %) spray through zinc oxide showed

Table 11.8 Effect of fungicidal seed treatment on plant stand of mustard plants (Kolte 2005)
Plant stand (%)a
Fungicides S. rolfsii R. solani F. oxysporum
Mancozeb 67.1 (84.2) 7.9 (4.0) 63.0 (79.2)
Thiophanate methyl 63.6 (79.5) 35.8 (35.2) 64.6 (81.5)
Thiram 67.3 (85.2) 21.5 (17.5) 62.2 (78.0)
Carbendazim 62.2 (78.0) 29.6 (33.5) 36.3 (69.0)
Metalaxyl (Apron 35SD) 66.4 (83.7) 6.5 (1.70) 50.3 (58.2)
Check (untreated) 23.3 (15.7) 20.6 (12.7) 22.9 (16.2)
CD at P = 0.5 7.8 21.1 11.4
a
Figures in parentheses are actual % plant stand and others are arcsine transformed values (Khan and Kolte 2002)
 260

Table 11.9 Integrated disease management module (seed treatment, spray schedule and fertilizer doses for the control of DM, WR and AB) and its significance in achieving
higher yield of mustard during 1999–2000 to 2001–2002a (Kolte 2005)
DM cotyledons DM leaf 60 WR leaf index AB leaf 100 AB pod 2 DM + WR staghead DM + WR 1000 grain Yield potential Yield increase
Treatments index (%) DAS (%) (%) 100DAS DAS (%) WBM (%) (incidence) staghead (severe) weight (g) (kg/ha) over check %
DIVIP1P1 17.71 33.58 29.34 45.45 46.92 1.92 4.64 3.74 1901.6 72.03
D1V1P0P0 62.28 36.84 40.42 59.41 52.74 2.13 4.71 3.36 1105.1
D2V1P1P1 14.99 22.06 35.47 52.77 39.88 19.01 15.75 3.35 902.7 37.08
D2V1P0P0 44.90 24.06 43.31 58.97 47.17 16.11 21.50 3.04 658.0
D1V2P1P1 17.71 34.13 28.44 46.14 46.65 1.76 3.71 3.76 1918.1 84.06
D1V2P0P0 63.06 37.57 40.64 59.65 51.75 2.64 5.06 3.45 1042.7
D2V2P1P1 14.99 19.10 35.24 52.77 38.95 20.19 16.79 3.33 904.8 112.79
D2V2P0P0 42.03 21.50 – 59.70 49.16 16.85 20.24 2.99 425.2
CD at 5 % 16.06 13.24 10.47 10.59 8.23 19.45 13.77 0.51 900.4
a
Data indicated in the table are averages of 3 years crop seasons 1999–2000 to 2001–2002;
DM downy mildew, WR white rust, AB Alternaria blight
D1 = Sowing date 20 October; D2 = Sowing date 20 November V1 = Varuna, V2 = Kranti
P0P0 = No plant protection chemical treatment
P1 P1 = Recommended plant protection practices i.e. NPK = 100:40; 40 kg ha−1
Seed treatment with Apron 35 SD (6 g/kg)
Ridomil MZ at 0.25spray at 50 DAS followed by mancozeb spray at 0.2 % at 70 and 90 DAS
11
Disease Management
11.8 Integrated Disease Management 261

Table 11.10 Some micronutrients as possible inducer for multiple disease resistance in rapeseed–mustard (Kolte
2005)
Disease index
Under artificial conditions Underfield conditions
Phytoalexin Yield/ 20
Treatments (%) Conc. inhibition zone (mm) DM WR AB WR AB plants (gm)
Fe EDTA 0.2 95.0** 66.9** 77.0* 39.8 36.7** 40.2** 85.6
MnSO4 0.2 95.9** 43.9** 56.3** 64.5 28.6** 50.2 88.9
CaCl2 1.0 90.9** 88.8 56.9** 24.4** 25.6** 46.2* 82.4
ZnSO4 0.5 119.9** 4.1** 37.0** 39.2 48.3 49.7 109.0*
CuSO4 0.1 119.0** 9.5** 4.0** 54.6 21.9** 47.7* 111.3*
CO(NO3)2 0.5 69.2** 59.3** 53.8** 28.3** 29.1** 46.6* 118.5*
Na2BO7 0.5 53.5** 31.8** 18.4** 23.0** 29.3** 50.2 11.4*
Distilled water 8.5 92.6 83.6 36.9 47.7 53.5 77.9 –
**Significant at P < 0.01, *Significant at P < 0.05
DM downy mildew, WR white rust, AB Alternaria blight

synergistic effect in the effectiveness of manco- ments besides providing significant reduction in
zeb and gave 16–20 % improvement in disease disease severity also increased dry seed yield of
control in comparison with such treatments when the crop (mancozeb = 2052 kg ha−1; gar-
used separately. It indicates the importance of lic = 2006 kg ha−1; control = 1561 kg ha−1)
integrating plant nutrients such as boron, potas- (Tables 11.11, 11.12, and 11.13; Meena et al.
sium and zinc with foliar sprays of fungicides for 2011).
management of the Alternaria disease in oilseed Seed treatments with freshly prepared Allium
rape. sativum bulb aqueous extract (1 % w/v) resulted
The use of biofertilizers (Azatobacter and in significantly higher initial plant stands, across
PSB) and Trichoderma along with the recom- locations and years. Seed treatment with A. sati-
mended doses of fertilizers enhances the plant vum bulb extract, followed by its use as a foliar
growth and improves yield along with less inci- spray, results in significantly reduced Alternaria
dence of Alternaria blight and white rust. Organic leaf, and pod blight, white rust, fewer stagheads
manure results better growth, enhanced yield and incidence per plot, reduced downy mildew,
reduced disease incidence in mustard crop. Plant Sclerotinia rot incidence and reduced powdery
growth and yield of mustard is also promoted by mildew severity. It also provides significantly
basal use of elemental sulphur as a nutritional higher seed yields compared to the control and
supplement and spray of thiourea (0.1 %) at 50 % was at par with chemical fungicides. It was also
flowering stage of the crop, along with adoption effective as the combination of seed treatment
of recommended doses of fertilizers (NPK, with Trichoderma harzianum and foliar spraying
80:40;40 kg/ha) (Pathak and Godika 2010). with Pseudomonas fluorescens and T. harzianum.
Mancozeb recorded the lowest mean severity Economic returns were higher when using biora-
(leaf: 33.1 %; pod: 26.3 %) of Alternaria blight tional treatments (A. sativum bulb extract, T. har-
with efficacy of garlic bulb extract alone zianum, P. fluorescens) compared with chemical
(leaf = 34.4 %; pod = 27.3 %) or in combination fungicides. The combination of seed treatments
with cow urine (leaf = 34.2 %; pod = 28.6 %) with T. harzianum followed by its use as a foliar
being statistically at par with the recommended spray (17.22), and the similar combination of
chemical fungicide. Chemicals also proved effec- seed treatments, and foliar spraying with the A.
tive in reducing Alternaria blight severity on sativum bulb extract (17.18) gave higher cost–
leaves and pods of Indian mustard (leaf = 36.3– benefit ratio (Tables 11.14, 11.15, and 11.16;
37.9 %; pod = 27.5–30.1 %). The effective treat- Meena et al. 2013).
 262

Table 11.11 Effect of different chemicals, plant extracts and bioagents on Alternaria leaf blight severity (Meena et al. 2011)
% Alternaria leaf blight severitya
Bharatpur Ludhiana Hisar Dholi Pantnagar Mean Pooled
Treatment 2005 2006 2005 2006 2005 2006 2005 2006 2005 2006 2005 2006 mean
Mancozeb 14.5 11.1 52.6 32.4 22.2 20.1 33.8 20.3 44.9 57.6 33.6 28.3 30.9
(22.4) (19.3) (63.0) (34.7) (27.9) (26.4) (31.0) (26.8) (50.2) (49.4) (34.9) (31.4) (33.1)
Cow urine 16.6 13.8 56.8 45.8 43.3 36.7 35.5 18.7 50.0 65.7 40.4 36.1 38.3
(24.1) (21.7) (70.0) (42.6) (41.0) (37.2) (33.7) (25.6) (58.5) (54.1) (39.1) (36.3) (37.7)
Garlic + cow 16.2 15.6 55.9 45.0 22.3 14.6 29.9 16.0 48.8 64.7 34.6 31.2 32.9
urine (23.7) (23.2) (68.6) (43.1) (27.9) (22.4) (25.0) (23.5) (56.7) (53.6) (35.5) (33.0) (34.3)
Garlic 15.5 12.2 56.1 36.6 27.3 20.7 29.3 20.3 47.6 63.6 35.2 30.7 32.9
(23.2) (20.4) (69.0) (37.2) (31.3) (27.0) (24.0) (26.8) (54.7) (52.9) (35.9) (32.9) (34.4)
P. fluorescens 22.3 19.9 54.9 34.2 32.33 24.1 34.6 13.3 51.0 67.1 39.0 31.7 35.4
(28.2) (26.5) (67.0) (35.8) (34.6) (29.4) (32.3) (21.3) (60.2) (54.9) (38.4) (33.6) (36.0)
E. globosus 21.7 16.5 55.7 39.9 38.3 29.3 32.8 22.3 46.5 61.3 39.0 33.9 36.4
(27.8) (23.9) (68.2) (39.1) (38.2) (32.8) (29.3) (28.2) (52.9) (51.5) (38.4) (35.1) (36.8)
T. harzianum 17.6 11.8 53.5 33.5 35.0 27.3 28.9 15.0 50.0 68.3 37.0 31.2 34.1
(24.8) (20.1) (64.6) (35.4) (36.3) (31.0) (23.3) (22.8) (58.7) (55.7) (37.1) (33.1) (35.1)
Calcium 28.6 17.1 58.2 37.6 25.0 16.7 30.4 24.7 48.1 70.5 38.1 33.3 35.7
sulphate (32.3) (24.4) (72.2) (37.8) (30.0) (24.0) (25.7) (29.8) (55.6) (57.1) (37.9) (34.6) (36.3)
Borax 26.5 15.5 58.8 43.8 33.3 25.1 33.2 21.7 50.6 73.9 40.5 36.0 38.2
(31.0) (23.2) (73.3) (41.4) (35.3) (30.1) (30.0) (27.0) (59.5) (59.3) (39.4) (36.3) (37.9)
Potash + sulphur 23.5 17.3 54.9 38.6 28.7 20.7 34.6 25.3 49.8 73.1 38.3 35.0 36.6
(29.0) (24.5) (67.0) (38.4) (31.9) (27.0) (32.3) (30.2) (58.2) (58.7) (38.0) (35.8) (36.9)
11

Zinc sulphate 21.4 18.3 56.9 35.5 36.7 28.6 35.7 19.3 49.8 73.9 40.1 35.1 37.6
(27.5) (25.3) (70.3) (36.6) (37.3) (32.3) (34.0) (26.1) (58.3) (59.2) (39.1) (35.9) (37.5)
Control 40.6 20.9 68.3 47.6 44.3 36.8 38.4 27.7 56.1 81.3 49.5 42.9 46.2
(39.6) (27.2) (78.4) (43.6) (41.6) (37.3) (38.7) (31.6) (67.4) (64.4) (44.8) (40.9) (42.8)
LSD (P < 0.05) 2.1 5.5 0.5 0.8 7.1 6.6 2.6 2.9 0.9 1.8 3.3 3.2 2.2
a
Figures in parenthesis are angular transformed values and others are actual percent disease severity; mean of three replications 2005 = 2004–2005 season; 2006 = 2005–2006
season
Disease Management
 11.8

Table 11.12 Effect of different chemicals, plant extracts and bioagents on Alternaria pod blight severity (Meena et al. 2011)
% Alternaria pod blight severitya
Pooled
Bharatpur Ludhiana Hisar Dholi Pantnagar Mean mean
Treatments 2005 2005 2006 2005 2006 2005 2006 2005 2006 2005 2006
Mancozeb 7.2 (15.6) 50.3 30.1 5.3 (13.3) 3.7 (10.9) 18.1 (9.7) 20.0 44.9 19.1 25.2 18.2 21.7
(59.2) (33.2) (26.5) (50.2) (25.9) (28.3) (24.4) (26.3)
Cow urine 7.6 (16.0) 52.6 44.2 22.2 18.1 23.2 10.0 50.0 23.5 31.1 23.9 27.5
(63.2) (41.6) (27.9) (25.2) (15.7) (18.4) (58.5) (29.0) (32.9) (28.7) (30.8)
Integrated Disease Management

Garlic + cow 6.1 (14.1) 54.7 44.0 16.7 4.3 (12.0) 14.5 (6.3) 16.7 48.8 22.2 28.2 21.8 24.9
urine (66.7) (41.5) (24.1) (24.0) (56.7) (28.1) (30.6) (26.7) (28.6)
Garlic 5.0 (12.9) 53.3 35.2 8.3 (16.8) 6.7 (14.9) 13.6 (5.7) 19.3 47.6 21.0 25.6 20.5 23.0
(64.4) (36.4) (26.1) (54.7) (27.3) (28.4) (26.3) (27.3)
P. fluorescens 7.4 (15.6) 54.3 33.7 12.7 9.8 (18.2) 19.9 8.3 (16.7) 51.0 29.9 29.1 20.4 24.7
(66.0) (35.4) (20.8) (11.7) (60.2) (33.1) (31.2) (26.1) (28.7)
E. globosus 8.6 (17.0) 54.3 38.7 18.7 12.8 16.4 (8.0) 15.7 52.9 19.5 30.2 21.7 25.9
(66.0) (38.5) (25.6) (21.0) (23.2) (46.5) (26.2) (32.1) (27.3) (29.7)
T. harzianum 5.9 (13.9) 52.8 32.1 16.7 13.7 13.3 (5.3) 10.0 50.0 29.7 27.7 21.4 24.6
(63.5) (39.5) (24.1) (21.6) (18.4) (58.7) (33.0) (30.2) (27.0) (28.6)
Calcium sulphate 6.7 (15.0) 55.8 36.5 6.7 (14.9) 4.3 (12.0) 13. 6 16.0 48.1 27.0 25.1 20.9 23.6
(68.4) (37.2) (5.7) (23.5) (55.6) (31.3) (28.8) (26.2) (27.5)
Borax 7.5 (15.8) 50.9 42.0 13.3 10.5 18.8 12.3 50.6 28.8 28.2 23.4 25.8
(60.3) (40.8) (21.4) (18.9) (10.7) (20.5) (59.5) (32.4) (30.8) (28.2) (29.5)
Potash + sulphur 6.2 (14.3) 55.7 37.5 8.7 (17.1) 5.8 (13.9) 15.6 (7.3) 15.7 49.8 29.0 27.2 22.0 21.6
(68.3) (37.7) (23.3) (58.2) (32.6) (29.6) (27.1) (28.4)
Zinc sulphate 5.1 (12.9) 53.3 34.5 16.7 13.3 24.8 15.0 49.8 30.4 29.9 23.3 26.6
(64.3) (35.9) (24.1) (21.4) (17.7) (22.7) (58.3) (33.5) (31.8) (28.5) (30.1)
Control 15.7 (23.3) 59.9 46.8 26.7 19.3 27.7 27.7 56.1 36.1 37.2 32.5 34.8
(75.0) (43.2) (31.2) (26.1) (21.7) (31.7) (67.4) (37.0) (37.1) (34.5) (35.8)
LSD (P < 0.05) 3.7 0.7 0.3 4.9 4.1 2.9 2.7 0.9 1.5 4.4 4.4 2.6
a
Figures in parenthesis are angular transformed values and others are actual percent disease severity; mean of three replications 2005 = 2004–2005 season; 2006 = 2005–2006
season
263
 264

Table 11.13 Effect of different non-toxic chemicals, plant extracts and bioagents on Alternaria leaf blight severity (Meena et al. 2011)
Yield (kg/ha)a
Bharatpur Ludhiana Hisar Dholi Pantnagar Mean Pooled
Treatments 2005 2006 2005 2006 2005 2006 2005 2006 2005 2006 2005 2006 mean
Mancozeb 1889 4183 2796 1287 1605 1705 1178 1200 2738 1936 2041 2062 2052
Cow urine 1195 3911 2657 730 1520 1635 1000 1333 2264 1480 1727 1817 1773
Garlic + cow urine 1534 3778 2729 737 1595 1715 1356 1488 2412 1675 1925 1879 1902
Garlic 2050 4233 2603 1130 1585 1698 1156 1200 2664 1744 2011 2001 2006
P. fluorescens 1156 3932 2603 1212 1560 1670 822 1266 2338 1478 1695 1911 1803
E. globosus 1578 3856 2544 1060 1530 1645 1178 911 2701 1916 1906 1878 1892
T. harzianum 1645 4278 2739 1257 1550 1660 1556 1377 2206 1503 1939 2015 1977
Calcium sulphate 1489 3711 2657 1110 1610 1720 1356 1000 2309 1501 1884 1808 1846
Borax 1539 3606 2603 775 1570 1685 844 1244 1894 1418 1690 1745 1718
Potash + sulphur 1250 3750 2618 1080 1580 1680 800 866 2005 1427 1650 1761 1706
Zinc sulphate 1445 3694 2704 1175 1545 1655 722 911 2168 1434 1716 1774 1745
Control 1117 3500 2563 725 1510 1650 700 822 1813 1215 1540 1582 1561
LSD (P < 0.05) 274 458 NS 83 49 49 469 30 114 32 211 287 169
a
Figures are average of three replications 2005 = 2004–2005 season; 2006 = 2005–2006 season
11
Disease Management
11.8 Integrated Disease Management 265

Table 11.14 The effect of different treatments on the initial plant stand of Indian mustard (Meena et al. 2013)
Initial plant stand (25 days after sowing)
Treatments 2006–2007a 2007–2008b Pooled mean
Garlic bulb extract 1 % w/v (ST) 270.8pq 267.6p 269.2p
Apron 35 SD 6 g/kg (ST) 261.8pq 249.3qrst 255.5pq
Carbendazim 1 g a.i. (ST) 262.5pq 244.9rst 253.7qr
p pqrs
Apron 35 SD 6 g/kg + carbendazim 1 g a.i. (ST) 274.6 259.1 266.8pq
pq pqr
Trichoderma harzianum 10 g/kg (ST) 267.4 260.5 264.0pq
pq pq
T. harzianum (ST) + P. fluorescens 10 ml/l (FS) 271.2 250.3 260.7pq
qr pqrs
T. harzianum (ST) + T. harzianum 10 ml/l (FS) 259.5 258.2 258.9pq
pq pq
Garlic bulb extract (ST) + garlic bulb extract (FS) 270.3 263.9 267.1pq
pq pqrs
Apron 35 SD (ST) + Ridomil MZ 72 WP 2 g/l (FS) 267.5 255.0 261.3pq
pq pqrs
Carbendazim (ST) + Ridomil MZ 72 WP 2 g/l (FS) 267.0 254.2 260.6pq
qr t
Control 248.3 233.2 240.7r
LSD (P < 0.05) 13.4 16.4 15.2
Cultivar Varuna, ST seed treatment; FS foliar spray; abNumber of plants per plot of 5 m × 3 m
Same letter in superscript indicates no significant difference among data within the column
a
Mean of three replications at each of the following locations: Faizabad, Morena, Navgaon and Kanpur
b
Mean of three replications at each of the following locations: Faizabad, Morena, Navgaon and Jagdalpur

Table 11.15 The effect of different treatments on Alternaria blight severity (percent) in Indian mustard 90 days after
sowing (Meena et al. 2013)
Treatment 2006–2007a 2007–2008b 2008–2009c Pooled mean
Garlic bulb extract 1 % w/v (ST) 30.9q (27.3) 31.4q (25.6) 27.1 (23.0) 30.2q (25.3)
Apron 35 SD 6 g/kg (ST) 31.5q (28.3) 30.9q (26.0) 27.4 (23.4) 30.6pq (25.9)
Carbendazim 1 g a.i. (ST) 30.1q (31.2) 31.2q (26.3) 27.9 (23.8) 30.3q (25.4)
Apron 35 SD 6 g/kg + carbendazim 1 g a.i. (ST) 28.9pq (24.1) 31.1q (26.9) 27.3 (23.1) 29.8q (24.7)
Trichoderma harzianum 10 g/kg (ST) 30.2q (26.4) 31.9q (27.3) 27.5 (23.5) 30.5q (25.7)
T. harzianum (ST) + P. fluorescens 10 ml/ l (FS) 29.2pq (24.8) 30.6q (25.7) 25.8 (20.5) 29.1pq (23.7)
T. harzianum (ST) + T. harzianum 10 ml/l (FS) 29.2pq (24.8) 29.5q (22.7) 25.0 (19.5) 28.2pq (22.3)
Garlic bulb extract (ST) + garlic bulb extract (FS) 26.1p (20.1) 26.3p (19.8) 26.4 (21.7) 26.9p (20.5)
Apron 35 SD (ST) + Ridomil MZ 72 WP 2 g/l (FS) 26.2p (20.3) 26.6p (19.6) 25.5 (19.9) 26.1p (19.3)
Carbendazim (ST) + Ridomil MZ 72 WP 2 g/l (FS) 30.0q (26.5) 29.9q (24.2) 27.1 (22.5) 29.6q (24.4)
Control 36.9r (36.5) 37.5r (36.1) 28.0 (24.3) 34.6r (32.3)
LSD (P < 0.05) 3.4 2.7 NS 3.1
The figures in parentheses are the actual means of disease severity, and the other figures are angular transformed
values
ST seed treatment, FS foliar spray, cv. varuna, NS not significant: same letter in superscript indicates no significant dif-
ference among data within the column
a
Mean of three replications at each of the following locations: Sri Ganganagar, Faizabad, Morena, Pantnagar, Ludhiana,
Navgaon and Kanpur
b
Mean of three replications at each of the following locations: Sri Ganganagar, Faizabad, Morena, Pantnagar, Jagdalpur,
Kanpur, Hisar and Dholi
c
Mean of three replications at each of the following locations: Sri Ganganagar, Faizabad, Morena, Pantnagar, Ludhiana,
Navgaon and Jagdalpur
266 11 Disease Management

Table 11.16 The effect of different treatments on Alternaria blight severity (percent) in Indian mustard 120 days after
sowing (Meena et al. 2013)
Treatment 2006–2007a 2007–2008b 2008–2009c Pooled mean
Garlic bulb extract 1 % w/v (ST) 34.2r (32.4) 30.9r (27.2) 24.4 (19.3) 30.8q (26.3)
Apron 35 SD 6 g/kg (ST) 33.6r (31.5) 30.5r (27.1) 23.8 (18.6) 30.5q (25.7)
Carbendazim 1 g a.i. (ST) 33.4qr (31.1) 31.1r (27.7) 24.3 (19.4) 30.7q (26.1)
Apron 35 SD 6 g/kg + carbendazim 1 g a.i. (ST) 34.4r (32.7) 30.9r (27.7) 24.8 (20.3) 31.1qr (26.9)
Trichoderma harzianum 10 g/kg (ST) 33.4qr (31.2) 30.9r (27.8) 25.3 (20.9) 31.0q (26.9)
T. harzianum (ST) + P. fluorescens 10 ml/ l (FS) 34.1r (32.1) 31.6r (28.6) 25.1 (20.3) 31.3qr (27.0)
T. harzianum (ST) + T. harzianum 10 ml/l (FS) 33.7r (31.4) 28.6q (24.1) 24.0 (19.0) 29.9q (24.8)
Garlic bulb extract (ST) + garlic bulb extract (FS) 30.5pq (27.5) 25.2p (20.1) 23.0 (17.8) 27.8p (21.8)
Apron 35 SD (ST) + Ridomil MZ 72 WP 2 g/l (FS) 29.8p (26.5) 25.6p (20.2) 22.5 (17.3) 27.5p (21.3)
Carbendazim (ST) + Ridomil MZ 72 WP 2 g/l (FS) 34.8r (33.4) 31.3r (28.1) 24.4 (19.4) 31.3qr (27.0)
Control 36.4r (35.8) 35.0s (33.0) 25.4 (20.6) 33.1r (29.8)
LSD (P < 0.05) 3.0 1.4 NS 2.1
The figures in parentheses are the actual means of disease severity, and the other figures are angular transformed
values
ST seed treatment, FS foliar spray, cv. varuna, NS not significant
Same letter in superscript indicates no significant difference among data within the column
a
Mean of three replications at each of the following locations: Sri Ganganagar, Faizabad, Morena, Pantnagar, Ludhiana,
Navgaon and Kanpur
b
Mean of three replications at each of the following locations: Sri Ganganagar, Faizabad, Pantnagar, Jagdalpur, Kanpur,
Hisar and Dholi
c
Mean of three replications at each of the following locations: Sri Ganganagar, Faizabad, Morena, Pantnagar, Ludhiana
and Jagdalpur

Bonin K, Fratczak E (1987) Control of fungal diseases in

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Saharan GS, Krishnia SK (2001) Multiple disease resis- Sharma S, Singh J, Munshi GD, Munshi SK (2010a)
tance in rapeseed and mustard. In: Nagarajan S, Singh Biochemical changes associated with application of
DP (eds) Role of resistance in intensive agriculture. biocontrol agents on Indian mustard leaves from
Kalyani Publication, New Delhi, pp 98–108 plants infected with Alternaria Blight. Arch
Saharan GS, Mehta N (2002) Fungal diseases of rapeseed- Phytopathol Plant Protect 43:315–323
mustard. In: Gupta VK, Paul YS (eds) Diseases of Sharma S, Singh J, Munshi GD, Munshi SK (2010b)
field crops. Indus Publishing Company, New Delhi, Effect of biocontrol agents on lipid and protein com-
pp 193–228 posion of Indian mustard seed from plants infected
Saharan GS, Mehta N, Sangwan MS (2003) Nature and with Alternaria spp. Arch Phytopathol Plant Protect
mechanism of disease resistance to Alternaria blight in 43:589–596
rapeseed-mustard system. Annu Rev Plant Pathol Shivpuri A, Siradhana BS, Bansal RK (1988) Management
2:85–125 of Alternaria blight of mustard with fungicides. Indian
Sandhu KS, Singh H, Kumar R (1985) Effect of different Phytopathol 41:644–646
nitrogen levels and dates of planting on Alternaria Singh DB (1986) Evaluation of fungicides for control of
blight and downy mildew diseases of radish seed crop. Alternaria brassicae and Drechslera graminea.
J Res Punjab Agric Univ Ludhiana 22:285–290 Iranian J Plant Pathol 22:39–43
Sasode RS, Sweta P, Gupta A, Pandya RK, Yadav A Singh RS (1987) Chapter 5: Alternaria leaf spot or black
(2012) In vitro study of some plant extracts against spot of crucifers. In: Diseases of vegetable crops.
Alternaria brassicae and Alternaria Brassicicola. Oxford and IBH Publ. Co, New Delhi, pp 159–163
J Phytol 4:44–46 Singh A, Bhowmik TP (1985) Persistence and efficacy of
Schimmer FC (1953) Alternaria brassicicola on summer some common fungicides against Alternaria brassi-
cauliflower seed. Plant Pathol 2:16–17 cae, the causal agent of leaf blight of rapeseed and
Sellam A, Dongo A, Guillemette T, Hudhomme P, Simoneau mustard. Indian Phytopathol 38:35–38
P (2007) Transcriptional responses to exposure to the Singh DB, Rai B (1982) Effect of leaf extracts of mustard
brassicaceous defence metabolites camalexin and allyl- and barley on growth behavior of some phylloplane
isothiocyanate in the necrotrophic fungus Alternaria microfungi. Bull Torrey Bot Club 108:419–421
brassicicola. Mol Plant Pathol 8:195–208 Singh RB, Singh RN (2005) Fungicide management of
Sharma SK, Gupta JS (1978a) Biological control of leaf foliar disease of mustard in mid-eastern India. Indian
blight disease of brown sarson caused by Alternaria Phytopathol 58:51–56
brassicae and A. brassicicola. Indian Phytopathol Singh HG, Sobti AK (1980) Effect of fungicides on
31:448–449 Alternaria blight of mustard. Indian J Mycol Plant
Sharma SK, Gupta JS (1978b) Effect of brown sarson leaf Pathol 10:49
leachates on the germination of the conidia of Singh BP, Singh SP, Abu M, Mishra GM (1989) Relative
Alternaria brassicae and Alternaria brassicicola. Proc efficacy of different fungicides for the control of leaf
Indian Natl Sci Acad B 44:57–58 spot of cabbage. Veg Sci 16:62–65
Sharma SK, Gupta JS (1979) Role of surface microorgan- Singh S, Godara SL, Gangopadhyay S (2013) Studies on
isms of brown sarson in relation to Alternaria brassi- antifungal properties of plant extracts on mustard
cae and Alternaria brassicicola. Agra Univ J Res Sci blight caused by Alternaria brassicae. Indian
28:109–111 Phytopathol 66:172–176
Sharma SK, Gupta JS (1980) Streptomyces rochei in rela- Sinha PP, Prasad RK (1989) Chemical management of
tion to Alternaria brassicae and A. brassicicola on the Alternaria blight of cauliflower seed crop. Indian
surface of brown sarson. J Indian Bot Soc 59:161–163 J Mycol Plant Pathol 19:204–205
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Stovold GE, Mailer RJ, Francis A (1987) Seed-borne levels, Tsuneda A, Skoropad WP (1978a) Behavior of Alternaria
chemical seed treatment and effects on seed quality fol- brassicae and its mycoparasite Nectria inventa on
lowing a severe outbreak of Alternaria brassicae on rape- intact and exicised leaves of rapeseed. Can J Bot
seed in New South Wales. Plant Prot Quart 2:128–131 56:1333–1340
Sultana NA, Khan MAH, Islam MN, Kamrun N (2009) Tsuneda A, Skoropad WP (1978b) Nutrient leakage from
Evaluation of appropriate time for the application of dried and rewetted conidia of Alternaria brassicae and
rovral against Alternaria blight incidence and yield of its effect on the mycoparasite Nectria inventa. Can
mustard. Int J Sustain Agric 1:20–23 J Bot 56:1341–1345
Tahvonen R (1982a) Preliminary experiments into the use Tsuneda A, Skoropad WP (1978c) Phylloplane fungal
of Streptomyces spp. isolated from peat in the flora of rapeseed. Trans Br Mycol Soc 70:329–334
biological control of soil and seed-borne disease in Tsuneda A, Skoropad WP (1980) Interactions between
peat culture. J Sci Agric Soc Finl 54:357–369 Nectria inventa, a destructive mycoparasite, and four-
Tahvonen R (1982b) The suppressiveness of Finnish light teen fungi associated with rapeseed. Trans Br Mycol
coloured sphagnum peat. J Sci Agric Soc Finl Soc 74:501–507
54:345–356 Tsuneda A, Skoropad WP, Tewari JP (1976) Mode of
Tahvonen R (1985) Mycostop-a biological formulation for parasitism of Alternaria brassicae by Nectria inventa.
control of fungal diseases. Vaxtskyddsnotiser 49:86–90 Phytopathology 66:1056–1064
Tahvonen RT (1988) Microbial control of plant disease Umalkar GV, Mukadam DS, Nehemiah KMA (1977)
with Streptomyces spp. Bull OEPP 18:55–59 Fungistatic properties of some deproteinized leaf
Tahvonen R, Avikainen H (1987) The biological control extracts. Sci Cult 43:437–439
of seed-borne Alternaria brassicicola of cruciferous Valkonen JPT, Koponen H (1990) The seed-borne fungi
plants with a powdery preparation of Streptomyces sp. of Chinese cabbage (Brassica pekinensis), their patho-
J Agric Sc Finl Helsinki 59:199–208 genicity and control. Plant Pathol 39:510–516
Tapio E, Pohto-Lahdenpera A (1989) Interaction between Vannacci G, Harman GE (1987) Biocontrol of seed-borne
antagonists and pathogenic fungi. Vaxtskyddsnotiser Alternaria raphani and A. brassicicola. Can
53:12–18 J Microbiol 33:850–856
Tasca G, Trandaf F (1984) Efficacy of pre-harvest treat- Verma PR, Saharan GS (1994) Monograph on Alternaria
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ing storage. Lucrari stiintifice Institutulde cercetari si Technical Bulletin 1994–6E. Agriculture and Agri-
Proiectari Pentru Valorificarea si Industrializarea Food Canada, Saskatoon. 162p
Legumelor si Fructelor 15:89–93 Verma SS, Yajima WR, Rahman MH, Shah S, Liu JJ,
Tewari JP, Skoropad WP (1979) The effect of polyoxin B Ekramoddoullah AK, Kav NN (2012) A cysteine-
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Thind TS, Jhooty JS (1988) Differential behaviour of fun- pathogens in canola (Brassica napus). Plant Mol
gicides against Alternaria blight of some cruciferous Biol 79:61–74
hosts. Indian J Mycol Plant Pathol 18:122–127 Walker JC (1927) Diseases of cabbage and related plants,
Thomas P (1984) Alternaria blackspot (grey leaf spot). In: US Dept Agric Fanners’ bulletin, 1439. U.S. Dept. of
Canola growers manual. Canola Council of Canada, Agriculture, Washington, DC, 30p
Winnipeg, pp 1056–1058 Walker JC (1952) Chapter 6: Diseases of crucifers. In:
Thomma BPHJ (2003) Pathogen profile- Alternaria spp., Diseases of vegetable crops. McGraw Hill Book Co,
from general saprophyte to specific parasite. Mol New York/London, pp 150–152
Plant Pathol 4:225–236 White JG, Linfield CA, Lahdenpera ML, Uoti J (1990)
Tripathi NN, Singh H, Kaushik CD, Singh M (1985) Mycostop a novel biofungicide based on Streptomyces
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 Techniques
12

disease appear within 4–5 days of inoculation. In

12.1 Introduction 15–20 days, the cut ends of test shoots initiate
root formation with profuse rooting in 30 days.
Techniques are methods, procedures and tools to New shoots may generate from the axillary buds.
be utilized by the Brassicologists to do research Such stem explants can be transferred to soil in
on various aspects of crucifer’s diseases. These pots where pod formation with good seed devel-
techniques are also helpful to the students, exten- opment can be possible. This technique is advan-
sion workers, farmers and all those who wish to tageous over others in its simplicity and obtaining
handle Alternaria–crucifers system. The tech- the seeds of test genotypes in the same season for
niques given in this chapter are standardized, further use (Kolte and Yadav 1990).
reproducible and developed by renowned scien-
tists and practical Brassica workers.
12.3 Leaf Disc Inoculation

12.2 Stem Explant Culture Test plants are grown under controlled condi-
Inoculation tions. From the excised healthy leaves of such
plants, leaf discs 2.5–3.0-cm diameter are cut and
The technique consists of cutting 6–15-cm termi- washed thoroughly by rinsing 5–6 times with dis-
nal shoots bearing leaves and/or flowers and pods tilled water in test tube of wider diameter
at 3–5.3 growth stage of the plants. The cut end (>3 cm). To ensure elimination of surface con-
of such shoots is immediately immersed in tap taminants, fresh aliquots of sterilized water are
water in Erlenmeyer flasks or test tubes. Care is used for each rinse. Surface-sterilized discs are
taken to see that cut ends of the shoots remain placed on a 4-mm-thick layer of 1 % plain steril-
submerged in water. The leaves and/or pods are ized agar Petri dishes containing 60 ppm benz-
spray inoculated with A. brassicae conidial sus- imidazole. The adaxial surface is placed against
pension. Inoculated shoots are incubated in a the agar, and the abaxial surface is drop inocu-
polyethylene moist chamber (90–95 % RH) at lated with 0.01–0.02 ml of conidial suspension
20–30 °C maximum and 6–14 °C minimum tem- (Humpherson-Jones and Phelps 1989). Suitable
peratures in diffused light for 72 h. Special care is controls with distilled sterilized water drop-
taken to make up the loss of water in flasks or test inoculated discs are maintained. Excess moisture
tubes by slowing down the transpiration through from the leaf discs is removed with sterilized
shaded moist conditions. Symptoms of the absorbent paper. The inoculated discs are

© Springer Science+Business Media Singapore 2016 273

G.S. Saharan et al., Alternaria Diseases of Crucifers: Biology, Ecology and Disease Management,
DOI 10.1007/978-981-10-0021-8_12
274 12 Techniques

incubated under continuous cool white fluores- 0.4 mg/l−1 rose bengal at room temperature used
cent light (5.5 μEM2 S−1) at 18–24 °C tempera- 18 photoperiod was used to inoculate fully
ture for A. brassicae and at 20–30 °C for A. expanded detached fourth leaf (ca. 18 days after
brassicicola for 4–5 days. The symptoms in the sowing) placed in a clear plastic container lined
form of lesions are recorded to test the reaction of with water-soaked paper towels. The leaves are
the genotypes. punctured of both sides of the midrib with a num-
ber 24-needle inoculator to induce circular
wounds of ca. 3-mm diameter and inoculated
12.4 Detached Leaf and Pod each wound with 2.5-μl drop of spore suspension
Inoculation with an Eppendorf micropipette. Control leaves
are inoculated with sterilized distilled water. The
Third or fourth leaves of the test plants (at 3.3– plastic container sealed with clear plastic wrap
4.2 growth stages) and pods (at 5.3–6.5 growth was incubated at room temperature under con-
stages) are detached, an approximately 3 mm2 of tinuous light. Four days after inoculation, lesion
the upper surface is gently scratched with the tip diameter, including the chlorotic zone, was
of a Pasteur pipette, and 50-μl drop of spore sus- recorded.
pension (5 × 104 spores ml−1) is placed on the
scratched area (Bains and Tewari 1987); five
leaves/pods from each test genotype are inocu- 12.6 Greenhouse Method
lated at four points at random. Suitable checks for Testing Resistance
with sterilized water droplet inoculation are kept.
The inoculated leaves/pods are incubated for Small differences in infection of A. brassicae on
72 h on a moist filter paper in Petri dishes at Brassica species are difficult to detect in the
25–26 °C temperature under continuous cool field. Therefore, a more detailed method under
white fluorescent light (5.5 μEM2 S−1). The symp- greenhouse conditions was developed by
toms in the form of lesions develop within Grontoft and O’Connor (1990). Plants of test
4–5 days after inoculation. genotypes are grown in pots. When the first true
leaf emerges, the plants are thinned to one
healthy plant per pot. From the time of sowing
12.5 Detached Leaf Inoculation to the time of evaluations of genotypes, the tem-
perature is kept constant at approximately
In a detached leaf inoculation technique under 20 °C. The plants are inoculated at the three-leaf
laboratory study, Bansal et al. (1990) screened stage using Alternaria inoculum grown on PDA
Brassica species against A. brassicae on the basis for 3 weeks at 18 °C. Inoculation is performed
of the size of lesions produced on detached using small agar discs (10 mm) cut from just
wounded leaves. The leaves are wounded artifi- behind the active mycelial growth front with a
cially to eliminate the protective effect of the wax cork borer. The discs are placed with the fungus
layer in order to examine the reaction of the growth sides against the upper side of the sec-
underlying tissues. Ten plants from each geno- ond leaf over a 4-mm hole. A second hole of
type are grown individually in 5 × 5-cm pots con- similar size is cut on the other side of the mid-
taining soil-free growth medium in the greenhouse vein. This hole is used to control if the size of
at 20/16 °C day/night temperature and 18-h pho- necrotic lesions is in any way influenced by leaf
toperiod achieved with supplementary illumina- surface area growth. Immediately after inocula-
tion provided by 400-W high-pressure sodium tion, each plant is covered with plastic bag in
lamps. order to increase relative humidity and enhance
Aqueous spore suspension (4–5 × 105 spores fungal growth. The genotypes are evaluated
−1
ml ) from 10- to 14-day-old cultures of A. bras- 7 days later by measuring the diameter of the
sicae grown on V-8 agar supplemented with necrotic lesions on the leaves.
12.8 Semi-Selective Medium for Detecting Seed-Borne A. brassicicola 275

12.7 Brassica Germ-Plasm 25 °C. Secondary embryoids have exhibited a

Screening for Resistance wide range of reactions, including a number of
through AB Toxin resistant plants (MacDonald and Ingram 1985;
1986; MacDonald et al. 1986).
Sensitivity of test genotypes to the Alternaria In vitro pollen selection (microspore culture)
blight toxin is correlated with the disease reac- in Brassica for resistance to toxins from A. bras-
tion in this technique assuming that the level of sicae and A. brassicicola has been applied
sensitivity of different Brassica species to the (Hodgkin 1990; Tewari and Bains 1988). The AB
toxin is similar to the in vivo susceptibility of toxin from A. brassicae at 12.5 μg/ml completely
these to A. brassicae (Bains and Tewari 1987). It inhibits the germination of pollens of highly sus-
can cause symptoms of varying severity, depend- ceptible B. rapa var. Yellow Sarson, whereas pol-
ing upon the host genotype, which range from lens of highly resistant C. sativa germinate even
severe chlorosis and necrosis to almost no visible at 100 μg/ml of the toxin (Tewari and Bains
chlorosis (Tewari and Bains 1988). Test geno- 1988). Hodgkin (1990) described in vitro pollen
types are grown under controlled conditions, selection in B. napus for resistance to phytotoxic
3 mm2 area is gently scratched with a Pasteur compounds from A. brassicicola. Pollen samples
pipette on third or fourth detached leaf (3.3–4.2 from B. napus cv. ‘Arran’ and ‘Hercules’ are
growth stage), and 20 μl of the purified AB toxin incubated for 1 h in a germination medium or in
from the single-spore culture of A. brassicae is a medium to which 20 mg ml−1 of an A. brassici-
applied (Bains and Tewari 1987). The leaves are cola toxic extract has been added. The pollen
incubated on a moist filter paper in a Petri dish on samples are then used to pollinate cultivar Primar.
laboratory benches under continuous fluorescent A number of plants, obtained from such pollina-
light, and the reaction of genotypes is recorded tions, produce pollen with a significantly
48 h of incubation. increased ability to germinate in medium con-
In vitro screening of protoplasts of different taining 10 mg ml−1 of the extract. Evidence that
Brassica genotypes, or screening of secondary some selection for resistance to the toxic com-
embryoids, originally derived from a single pounds produced by A. brassicicola has been
anther embryoid through AB toxin, has been obtained (Hodgkin and MacDonald 1986).
employed since plant protoplasts are known to be
more sensitive to host-specific toxins (MacDonald
and Ingram 1985; 1986; MacDonald et al. 1986). 12.8 Semi-Selective Medium
Secondary embryogenic culture lines are main- for Detecting Seed-Borne
tained on MS medium (Murashige and Skoog A. brassicicola
1962) containing 0.8 % Difco agar and 2 %
sucrose but without plant growth regulators. A semi-selective medium containing benomyl,
When regenerants are required, secondary sodium propionate, streptomycin sulfate and
embryoids are transferred for 3–7 days to MS chloramphenicol in peptone–dextrose agar
medium supplemented with kinetin and then detected seed-borne A. brassicicola (Wu and Lu
transferred to fresh medium without growth sub- 1984). This medium is more sensitive than the
stances for root development. Plantlets are potted blotter method to detect A. brassicicola in cab-
in peat balls and placed in high humidity in the bage seeds. Seeds incubated on this semi-
greenhouse until they are established. Leaves selective medium at 22 °C for 7 days produced
from such plants are surface sterilized and placed velvety olivaceous colour colonies of A. brassici-
in plastic boxes on moist filter paper. Two drops cola, with abundant sporulation. There is a posi-
(10 μl each of suspension of approximately tive correlation between the detached amount of
5 × 105 spores ml−1 of A. brassicicola) are placed seed-borne A. brassicicola infection on this
on each detached leaf and incubated in low con- medium and the percentage of diseased seedlings
tinuous light (approximately 1.7 m W cm−2) at produced in autoclaved field soil or in peat moss.
276 12 Techniques

12.9 Radish Root Extract Agar mixer for 3 min., and suspension is filtered
for A. brassicae Sporulation through two layers of sterile lens tissue (Whatman
no. 105) and washed further with a 5 ml of 0.01 %
Preparation of a radish root extract filtrate by sterilized Tween 20 solution. The conidial sus-
cooking 200 g of grated radish roots in 500 ml pension is centrifuged for 10 min at 2750 g (rav,
distilled water and 20 g in 500 ml agar solution 7.5 cm), supernatant is discarded, and conidia are
supplemented with 1 % sucrose and 1 % manni- resuspended in 5 ml of 0.01 % sterilized Tween
tol proved very effective in producing large colo- 20 solution and counted using haemocytometer.
nies and profuse sporulation of A. brassicae The mean of six counts is taken for each replicate.
(Thakur and Kolte 1985). Conidial germination is assessed by preparing
spread plates (0.1 ml aliquots of conidial suspen-
sion spread over PDA plates) and incubating them
12.10 Inducing Sporulation for 24 h at 20 °C. For each isolate, 200 conidia are
of A. brassicae examined, and the percentage germination is cal-
culated (Senior et al. 1987).
Single-conidium stock cultures of A. brassicae
stored at room temperature on oxoid potato dex-
trose agar (PDA) slopes under sterile paraffin oil 12.11 Brassica Callus Culture
was used to initiate Petri dish cultures of indi- to Induce Sporulation
vidual isolates for 7 days at 25 °C on PDA; a in Alternaria brassicae
2-cm2 mycelium piece is placed in a sterile 25-ml
masticator with 10 ml sterile distilled water, mac- Twenty-five surface-sterilized B. juncea seeds
erated at maximum speed for 1 min. and 0.2-ml placed into a 250-ml conical flask containing
portion of the macerate is transferred to 9-cm 30 ml of sterilized Murashige–Skoog (MS)
diameter triple vented polystyrene Petri dishes medium (Murashige and Skoog 1962) are incu-
containing 10 ml of diluted autoclaved V-8 juice. bated in the dark for 3 days and in the light
Three replicates of each of eight field isolates are (300 lux) for 2–3 days until seedlings reach to the
incubated separately under near ultraviolet (UV) neck of the flask. Each cotyledon is excised into
radiation (two 40-W Philips 310, 420-nm bulb), two pieces and transferred to a 100-ml conical
white light (two 85-W Sylvania cool white (310, flask containing 30 ml modified MS replacing
330, 350, 750 nm) fluorescent lamps) and dark- IAA and kinetin with 2 mg/l NAA, 3 mg/l 2,4-D
ness at 25 °C for 7 days. Two to 3 days after incu- and 0.2 mg/l of 2-iPr (2-isopentenyl-purine ribo-
bation, the mycelium forms a loosely interwoven side). The A. brassicae inoculated flasks are incu-
mat of hyphae throughout the liquid medium, bated at 25 °C (±2) under continuous light
producing a solid gelatinous matrix. As the cul- (300 lux). Callus cultures are subcultured every
tures slowly dehydrate, the pinkish-white myce- 3 weeks. A non-sporulating isolate of A. brassi-
lia turn dark olivaceous brown, which can be cae is grown in culture tubes in radish–mannitol
correlated with the production of melanized agar (RMA) medium, which is prepared in the
conidiophores and conidia. After 7 days, the cul- same way as PDA medium except that potato and
tures are dehydrated to 90 % of their original dextrose are replaced by radish and mannitol,
mass (Senior et al. 1987). respectively. The fungus is subcultured every
Conidia are removed by flooding individual 15 days. Two-week-old calluses are transferred
plates with 10 ml of a 0.01 % (V/V) sterilized onto 10 g/l plain agar. Further, modified MS
Tween 20 solution while rubbing the mycelial mat medium is supplemented with 100 mg/l Captafol.
with a surface-sterilized glass spreader for 1 min. Petri dishes containing one callus per dish incu-
The loosely interwoven mycelial mat and its con- bated at 25 °C (±2) under continuous light
tents of the Petri dish are transferred to sterilized (300 lux) were used to cut approximately 1-mm
30-ml universal bottles and mixed using a whirli- diameter. Small mycelial disc from the growing
12.14 Ovary and Ovule Culture 277

edge of the fungus culture was placed at the top exudates extracted from apple, vine, tomato,
of each callus (Joshi et al. 1988). potato and wallflower reduced the fungitoxicity
of ETD to A. brassicicola. The spectrum of the
leaf exudate effect is wide, and the magnitude of
12.12 Method of Estimating the effect can vary in each host–parasite–fungi-
Alternaria brassicicola cide relationship. It seems that a large proportion
in Seed of fungicide efficacy may be reduced by antago-
nistic materials, which either occur on the plant
Naturally infected cabbage seeds are incubated for surface or are readily and quickly available from
4 days at 18–21 °C with alternating light and dark intact plant tissue. It is likely that the active prin-
periods on filter papers moistened with 2000 ppm ciple affects the fungal spore, either by reducing
aqueous solution of Dow sodium salt of 2,4-dichlo- its permeability to fungicides or by influencing
rophenoxyacetate (2,4-D). When seeds are placed some part of the metabolism involved in spore
closer than 6 mm apart, or incubated longer than germination. Stimulation of the spore metabo-
4 days, higher pathogen counts occur. Two thou- lism could activate systems capable of detoxify-
sand ppm 2,4-D efficiently inhibits seed germina- ing alien chemicals. The major part of the exudate
tion and does not reduce pathogen count. Incubation effect observed is due to glucose.
temperature above the optimum temperature range
of 20–25 °C increases problem with the contami-
nant fungi. Continuous light or alternate light and 12.14 Ovary and Ovule Culture
dark are preferable to continuous dark. Excessive
moisture is detrimental. Pretreating seeds with eth- Four- to 12-day-old ovaries are excised and steril-
anol (EtOH) or sodium hypochlorite (NaOCl) ized with 0.25 % aqueous HgCl2 for 8–10 min. fol-
effectively reduces rapidly growing contaminant lowed by three washings with sterilized distilled
fungi but significantly reduces pathogen count water. The base of the pedicel is cut off, and ova-
(Bassey and Gabrielson 1983a, b). However, other ries are cultured on MS medium (Murashige and
common methods in use for detection of seed- Skoog 1962) supplemented with various concen-
borne inoculum are (a) deep-freezing blotter trations and combinations of growth regulators.
method, (b) germination test, (c) blotter method Three to five ovaries are cultured in each tube con-
and (d) agar plate method (Vannacci 1981). taining 15 ml of medium solidified with 0.8 %
agar and incubated at 25 °C (±1) in an illuminated
room (700 lux). Thirty-five days after culture, the
12.13 Identification of Fungicide ovaries are cut open to collect hybrid seeds. The F1
Antagonists in Leaf Exudates seeds are germinated in Petri dishes, and the seed-
lings transferred to pots in glass house (Sharma
Attempts have been made to identify the fungi- and Singh 1992). For ovule culture, 12–17 days
cide antagonists in exudate extracts of leaves of after pollination, the ovaries are surface sterilized
wallflower (Cheiranthus cheiri) (Beynon and and cut open aseptically under dissecting micro-
Brown 1969). In extracts, prepared by immersing scope. Ten to 12 excised ovules are cultured in test
wallflower leaves in water, the antagonistic activ- tubes containing MS and modified PC-L2 medium
ity of the fungicide ethylene thiuram disulphide (Phillips and Collins 1979). Observations on seed
(ETD) against the spores of A. brassicicola has formation are recorded 15 days after culture. The
been shown to be due to the presence of glucose seeds are first germinated in Petri dishes and trans-
and fructose. These compounds are present in the ferred to 8″ diameter pots in the glass house. The
extracts at concentrations corresponding to F1 hybrids are evaluated for their reaction to A.
0.015 μg of glucose and 0.002–0.005 μg of fruc- brassicae by inoculating with spore suspension
tose for every square centimetre of leaf extracted. (2000–2500 spores ml−1) in the glass house
Dunn et al. (1969) showed that water-soluble leaf (Sharma and Singh 1992).
278 12 Techniques

12.15 Method for Evaluating trose broth (PDB) and incubated at 22 ± 1 °C for
Partial Resistance 15 days in dark. Fungal mycelium is separated by
to Alternaria brassicicola filtering through sterilized Whatman no. 1 filter
paper, and filtrate further is sterilized by passing
Three inoculation methods (cotyledon, detached through millipore filter of 0.22-μm size; the pH
leaf and seedling) were evaluated by Doullah of the filtrate is adjusted to 5.8 before filter steril-
et al. (2006). The detached leaf inoculation test is ization. The sterilized filtrate is added to the auto-
the most suitable for screening B. rapa genotypes claved MS medium containing 1 mg l−1 NAA and
because clear symptoms are observed on the 1 mg l−1 BAP (MSN1B1) during cooling and dis-
leaves in less than 24 h. There is a significant pensed in sterilized conical flasks. The medium is
positive correlation between the results from the stored at 25 ± 1 °C in dark and used for inocula-
detached leaf inoculation test and seedling inocu- tion within a week of its preparation.
lation test, an established method considered to Seedlings of different species and cultivars are
yield reliable results. In addition, it is very easy raised aseptically on hormone-free MS medium
to screen plants for resistance on a large scale and with 0.5 % sucrose and 0.8 % agar. Hypocotyl
to maintain standard physical conditions using segments measuring 5–6 mm are excised from 6-
detached leaves. For successful infection, inocu- to 7-day-old seedlings and implanted on MSN1B1
lum concentration should be adjusted to 5 × 104 medium with or without fungal CF. The inocu-
conidia ml−1, and incubation temperature should lated flasks are incubated at a temperature
be between 20 °C and 25 °C. The third/fourth 25 ± 1 °C for 1 month under 16-h light (1500 lux)
true leaves from 30-day-old plants are optimal and 8-h dark. After 1 month, per cent callus
for inoculation. induction and fresh and dry weights of individual
For this test, two leaves (third/fourth true callus are recorded (Kiran et al. 2002).
leaves) are collected from 30-day-old plants hav-
ing approximately six leaves. A replicate for the
detached leaf tests consists of thee randomly 12.17 Inoculation Methods
selected leaves from a set of third/fourth true for Pathogenesis
leaves collected from several plants that are of Alternaria brassicae
pooled. Alternaria brassicicola inoculum (5 × 104
conidia ml−l) is sprayed on the upper surface of Five inoculation methods including spraying,
the collected leaves to run-off, and inoculated infiltration, wounding, spore suspension drop and
leaves are placed in a plastic box (length × width spore suspension drop along with agarose method
× height = 32 × 23 × 5 cm) where strips of moist were compared. Among the five inoculation
tissue paper are put on four vertical sides to main- methods compared, spore suspension drop along
tain humidity at 98 %. The plastic box is inocu- with agarose inoculation method proved most
lated at 22 ± 1 °C for 3 days in the dark in an ideal as this fixes the inoculum on the target site.
incubator or controlled temperature room Compared to other four inoculation methods, the
(Doullah et al. 2006). highest mean numbers of initial disease lesions in
drop plus agarose recorded were 312.2, 484.2,
664.2, 734.2 and 799.2, respectively, at 24, 48,
12.16 Tissue Culture 72, 96 and 120 h after inoculation. Besides easy
to handle the inoculated plants, plus agarose
Alternaria brassicae is isolated aseptically from method has the advantage of being accurate and
the diseased Brassica leaves and multiplied on precise.
potato dextrose agar (PDA) for further studies. A single conidium of A. brassicae culture is
About 5-mm-diameter small mycelia of A. bras- derived from a conidia produced on a diseased B.
sicae are transferred to the 250-ml Erlenmeyer juncea leaf and maintained on potato dextrose
flask containing 30 ml of sterilized potato dex- agar (PDA). For conidiation, fungus is grown on
12.18 PCR-Based Assay for Detecting Alternaria brassicae in Cruciferous Seed 279

V-8 agar (10 % V-8 juice, 0.02 % CaCO3 and 2 % detached leaf is carefully inverted, exposing the
agar) at 24 °C for 3–4 days under cool white fluo- abaxial side. A 1-ml needleless syringe contain-
rescent light (2000 lux), followed by further ing a spore suspension is used to pressure infil-
2-day incubation at 18 °C (Aragaki 1964). A trate the leaf intracellular space(s), but the
conidial suspension is prepared by scraping vascular system of the leaf is avoided from
mycelia and conidia from plates of actively grow- injection.
ing cultures into autoclaved water and filtering Detached leaves are kept in sealed Petri dishes
through four layers of cheese cloth to remove with 1 % agar and placed in growth chambers at
most of the mycelia. The filtered spore suspen- 25 °C with 70 % relative humidity. The Petri
sion is centrifuged at 2000 × g for 5 min. and plates are observed for A. brassicae initial symp-
resuspended in to deionized water. This centrifu- toms at intervals of 24 h up until 120 h after inoc-
gation is repeated once more in order to ensure a ulation. The numbers of disease lesions are
clear conidial suspension-free of metabolites. counted on the detached leaves in all the inocula-
After the final wash, supernatant is discarded, tion methods at 24, 48, 72 and 120 h after inocu-
conidia resuspended in water containing 0.05 % lation (Giri et al. 2013).
Tween 20, conidia counted using haemocytome-
ter, and the concentration adjusted to 104 spores/
ml. 12.18 PCR-Based Assay
Seeds of brown mustard (B. juncea) are sown for Detecting Alternaria
in plastic inserts (7.5 × 5 cm; two seeds per insert) brassicae in Cruciferous Seed
consisting a mixture of soil, sand and vermicom-
posts (2:1:1) in the green house (22/18 °C day/ Current detection methods, based on culture, and
night, 16-h photoperiod). Pathogenicity test is morphological identification of the fungus are
carried out with spore suspension on detached time-consuming, laborious and not always reli-
leaves’ seedling. Equal amount of inoculums able. Therefore, a polymerase chain reaction
(500 μl) is used with a sterile micropipette, and (PCR)-based assay was developed with A.
ten replicate plants are used for each method. brassicae-specific primers designed on the basis
In the spore suspension drop method, inocu- of sequence of two clustered genes potentially
lum is placed on each detached leaf in the form of involved in pathogenicity by Guillemette et al.
a drop, whereas in the spore suspension drop plus (2004). Two sets of primers were selected for
agarose method, the inoculum drops are steril- conventional and real-time PCR, respectively. In
ized on a target area to prevent any adverse effect both cases, A. brassicae is specifically detected
on spores due to high temperature; 5 ml of 0.08 % using DNA extracted from seed. The real-time
agarose in a test tube is heated in a microwave PCR-based method can be automated easily, and
oven at high power for 40 s to melt the agarose, preliminary results indicate that it is efficient for
and the test tube is then placed in a water bath at quantitative estimation of seed infection.
about 42 °C in a 250-ml beaker to keep the aga-
rose in the liquid state at this temperature. Each
inoculum site on the leaf is covered with agarose, 12.18.1 Preparation of Seed
and the agarose drop appears as a clear dome on Samples
the plant surface. The agarose solidifies and fixes
the inoculum on the target site within few Seed samples are prepared from contaminated
minutes. seed of radish and cabbage as described by
In the wounding method, inoculation is per- Iacomi-Vasilescu et al. (2002). For artificial
formed by gently wounding the detached leaves contaminations, seeds are disinfected with 1 %
with a pipette tip. In the spraying method, inocu- sodium hypochlorite, rinsed with sterilized
lum is sprayed on detached leaves with the help water, soaked for 1 h in a calibrated spore sus-
of an atomizer. In syringe infiltration method, the pension (106 spores/ml) and dried at room
280 12 Techniques

temperature on sterilized filter paper. ACCCTATAGACC CACGTCGACTA-3′), and

Disinfection and inoculation accuracy is con- 115 rev (5′GATGGTACGCAAGGCTTGGT-3′),
firmed by placing 20 seeds on the surface of complementary to a portion of ORFI, are
malt agar plates for 1 week at 25 °C. Seed designed for the standard and real-time PCR
batches showing A. brassicae 0 % (disinfected assays, respectively, using the appropriate soft-
seed) or 100 % A. brassicae grown (inoculated ware (Primer 3 online and ABI PRISM Primer
seed) are selected and mixed together to pre- express). The two ORFs are deduced from the
pare seed samples with different levels of con- nucleotide sequence of an A. brassicae genomic
tamination (0, 5, 10, 50 and 100 %). Samples DNA fragment identified after screening a
are produced by placing 20 seeds (artificially cosmid library with a probe corresponding to a
contaminated) or 100 seeds (naturally contami- portion of the non-ribosomal peptide synthase
nated) in a sterile tube and covering them with (NRPS) gene. In order to test the specificity of
0.5 and 3 ml, respectively, of liquid culture malt the primer pairs against A. brassicae isolates,
extract–dextrose–peptone (2 % malt extract, amplifications are performed using DNA
2 % dextrose, 0.1 % peptone) (MDP) culture extracted from pure fungal cultures from a range
medium. The tubes are incubated at 25 °C for of Alternaria spp. and other fungi isolated from
48 h with occasional shaking. This incubation seed. Then the PCR procedure is applied to
step is an enrichment phase that allows an opti- detect seed contamination. The universal primer
mal increase of the fungal biomass from seed. pair ITS1/ITS2 is used as a positive control to
The tubes are then vortexed briefly to separate assess the quality of the extracted
mycelia and conidia from seed. The seeds are DNA. Conventional PCR is performed using
discarded, and the fungal structures collected 2 μl of undiluted DNA preparation under the
on microcentrifuge filter (pore size 0.45 μm; following conditions: Tris HCL, pH 9.0, 20 mM
Ultrafree-Mc Millipore) by centrifugation at (HN4)2SO4, 0.01 % (w/v) Tween 20, 1.5 mM
5000 × g for 30 min. MgCl2, 200 μM each deoxyribonucleotide tri-
phosphate and 1 unit of thermostable DNA
polymerase. A thermo jet thermo cycler is used
12.18.2 DNA Manipulation with an initial step of 3 min at 95 °C, followed
by 35 cycles of 30s at 95 °C, 50s at 60 °C and
DNA is extracted from fungal mycelia and 1 min at 72 °C and a final step of 10 min at
conidia by scraping the surface of Petri plate cul- 72 °C. The amplification results are visualized
tures (extraction from fungal culture) or collected after electrophoresis of an aliquot (10 μl) of the
on microcentrifuge filters (extraction from seed reaction mixture on a 1.2 % agarose gel. The
samples). Nucleic acids are isolated according to real-time PCR reactions are carried out in a
microwave miniprep procedure described by 25-μl final volume containing 2.5 μl of DNA,
Goodwin and Lee (1993). Alternatively, DNA is 12.5 μl of SYBR Green PCR Master Mix 2X,
extracted using the NucleoSpin food kit accord- 300 nM forward and reverse primers and H2O
ing to the manufacturer’s instructions. up to 25 μl. An ABI PRISM 700 sequence
Detection System (Applied Biosystems) is used
with the following steps: 2 min at 50 °C, 10 min
12.18.3 PCR-Based Assay at 95 °C and 40 cycles consisting in one step at
95 °C for 15 s followed by one step at 60 °C for
Specific oligonucleotides AB Csens 1 min. Nucleic acids are quantified in unknown
(5′CTGGTGAAAAGGTT GCGATCGT 3′) and samples by direct comparison to a standard. The
AB Crev (5′GTGACTTCATGAAATGACA DNA used as standard is extracted from fungal
TTGATG 3′), complementary to 3′ end of the cultures, with the concentration measured by
open reading frame (ORF) 2 and 115 sens (5′A fluorometric assay before dilution.
12.20 Assessment of Methods of Inoculation for Resistance to Alternaria 281

12.19 Quantitative Inoculation placed in plastic bag moist chamber (50-cm

Method diameter, 90-cm high), each with 100 ml water
on the bottom, and kept on the laboratory floor at
Alternaria brassicae is derived from a conidium 24 °C for observation at different time intervals.
produced on diseased leaf of mustard. Conidiation Three replicate plants are used for each treat-
of A. brassicae is induced by growing the fungus ment, and experiments are repeated at least once
on V-8 agar (10 % V-8 juice, 0.02 % CaCO3 and with similar results (Xu and Ko 1998).
2 % agar) at 24 °C for 3–4 days under cool white
fluorescent light (2000 lux) followed by 2-day
incubation at 18 °C (Aragaki 1964). A conidial 12.20 Assessment of Methods
suspension is prepared by placing three pieces of of Inoculation for Resistance
culture blocks (ca 5 × 5 × 3 mm) in 5 ml wheat to Alternaria
grain extract in a test tube and by agitating the
test tube for 1 min with a vortex mixer. Wheat Out of four methods of inoculation (seed inocula-
grain extract is prepared by autoclaving whole tion, cotyledonary leaf inoculation, detached true
wheat grains in distilled water at the ratio of 1:6 leaf inoculation and detached green pod inocula-
by volume. Only the top layer of clear extract is tion), detached true leaf inoculation is more effi-
used. cient and reliable for screening germ plasm for
Seedlings of black mustard are established resistance to Alternaria blight of rapeseed and
from seeds planted in Supersoil potting mix in mustard.
rectangular plastic pots (6.5 × 6 × 6 cm). All the
test plants are fertilized with Osmocote 14-14-14
and used after 1–2 months. The spore concentra- 12.20.1 Method of Inoculation
tion in the suspensions is adjusted by approxi-
mating the desired number per μl. Five 1-μl drops A conidial suspension of A. brassicae containing
of the spore suspension are then placed on a glass 1.5 × 104 conidia/ml is prepared, and then with
slide and examined under the microscope. Those the help of Eppendorf micropipette, three sepa-
drops with exact number of spores are needed rate 10-μl drops, each containing 4–6 conidia, are
individually, transferred with microlitre pipette to placed on the upper surface of true leaves at three
leaves or stems about 3–5 cm above the pots and equidistant places. Similarly, distilled water
temporarily positioned horizontally during inoc- drops (without conidia) are placed on other coty-
ulation. The emptied spots on the glass slide are ledons, which serve as checks. All the treatments
examined again with microscope to assure that are replicated three times for each cultivar with
all the spores in each drop are transferred. One each isolate. Observations on disease severity are
drop per leaf or stem is used. recorded on 0–5 rating scale as described at
Sea plaque agarose, which gels at about 30 °C, Sect. 12.20.3.
is used to stabilize inoculum drops on target areas
to prevent injuring to spores due to high tempera-
ture. Five ml of 0.8 % agarose in a test tube is 12.20.2 Detached True Leaf
heated in a microwave oven at high power for Inoculation
40 s to melt the agarose. The test tube is then
placed in water bath at 32 °C in a 250-ml beaker True leaves are collected from 30-day-old plants,
to keep the agarose in the liquid state at this tem- surface sterilized with sterilized distilled water
perature. Each inoculum site on the leaf or stem with the help of sterilized cotton, and leaves inoc-
is covered with 10 μl agarose. The agarose drop ulated following the method described above.
appeared as a clear dome on the plant surface. Inoculated leaves are kept in Petri plate moist
The agarose solidifies and fixes the inoculum on chamber and incubated at 20 ± 2 °C for 7 days,
the target site within 5 min. Inoculated plants are and leaves scored for disease severity.
282 12 Techniques

12.20.3 Infection Score and Disease 12.22 ELISA Diagnostic Kits

Index
A number of disease detection kits have been
A 0–5 rating scale is followed, where 0 = no visi- developed for use at the site where a disease is
ble symptom, 1 = 1–10 % leaf or pod area cov- suspected. These kits, which in most cases do not
ered with small spot, 2 = 11–25 % leaf or pod area require laboratory equipment, are especially use-
covered with bigger (more than 3 mm) spot, ful to growers. Some tests only take 5 min. to
3 = 26–50 % leaf or pod area covered with bigger perform. The diagnostic kits are based on a
(more than 3 mm) spots with initiation of coalesc- method that uses proteins called antibodies to
ing on leaves and deep lesions on pods, detect disease causing organisms of plants (plant
4 = 51–75 % leaf or pod area covered with bigger pathogen). The technique used is called ELISA
commonly coalescing spot and 5 = 76–100 % leaf (enzyme-linked immunosorbent assay). This
or pod area covered with very large spots giving assay is based on the ability of an antibody to rec-
blighting appearance (Vishwanath and Kolte ognize, and bind to a specific antigen, a substance
1999). associated with a plant pathogen. The process is
described in Fig. 12.1. The antibodies used in the
diagnostic kits are highly purified proteins pro-
duced by injecting a warm-blooded animal (like
12.21 Image-Based Disease rabbit) with an antigen associated with one par-
Identification ticular plant disease (Fig. 12.1(1)). The animal
proteins react to the antigen and produce antibod-
Image-based rapeseed–mustard disease expert ies (Fig. 12.1(2)). The antibodies produced rec-
system – an effective extension tool – has been ognize and react only with proteins associated
developed by Kumar et al. (2008). The identifica- with the causal agent of plant disease (Reddy
tion of disease is the difficult task. If the disease 2014).
is identified timely, the control measures can be These antibodies are bound to a plastic plate
applied effectively. Nowadays, the use of digital or similar detector unit in a test kit. The individ-
technology can produce high-quality digital ual running the test prepares the plant sample by
images including photos and clips of healthy and grinding it between two pieces of abrasive paper.
infected plants easily that can play very impor- The ground plant sample is placed in a bottle
tant role in disease identification. Digital images filled with an extraction liquid. This liquid is then
can be seen and shared easily among the experts. placed in wells in the plastic plate (Fig. 12.1(3)).
Image can be examined on camera screen liter- If the disease-causing organism is present in the
ally within the second; they are captured and sample, the specific antibodies in the plastic plate
downloaded to a computer for closer inspection will bind to pathogen-associated proteins and
within minutes. A computerized expert tool adhere to the unit (Fig. 12.1(4a)). A second anti-
image-based rapeseed–mustard disease expert body is added that also reacts with the pathogen-
system has been developed to help extension per- associated proteins. This antibody is special
sonals, researchers and farmers in identification because it can react with colour-producing chem-
and management of these diseases. The expert icals called reagents (Fig. 12.1(5a)). Colour
system uses a hierarchical classification and a changes on the unit’s surface indicate a positive
mix of the text description, photographs and reaction. If no pathogen-associated proteins are
artistic pictures. The system involves two main present (Fig. 12.1(4b)), the detector antibodies
subtasks, namely, diagnosis and management. cannot bind to the colour-carrying reagents and
The system designed and developed using Visual are washed away (Fig. 12.1(5b)).
Basic as front end and Microsoft Access 2000 as Presently, a limited number of these onsite
back end software. disease detection kits are available. Many ELISA
12.24 Nucleic Acid Probes 283

Fig. 12.1 ELISA diagnostic kit (Courtesy,

P. Parvatha Reddy 2014)

diagnostic kits are available for use in the labora- that bind only to the disease-causing pathogen in
tory, however, and can test for a wide range of question are then introduced to this paper. A
plant pathogens. colour change indicates a positive result and
shows the location of the pathogen in the hot tis-
sue (Fig. 12.2) (Reddy 2014).
12.23 Direct Tissue Blotting

Another diagnostic assay that also uses specific 12.24 Nucleic Acid Probes
antibodies as a detection tool is known as direct
tissue blotting. With this technique, the location Another set of tools that can be used in plant
of a disease-causing pathogen within the host disease diagnostics is nucleic acid probes.
plant can be determined, allowing earlier detec- These probes are fragments of nucleic acid
tion and a better understanding of how a disease arranged in a sequence complementary to that
progresses through a plant. Host plant tissue is of the DNA or RNA of the disease-causing
placed onto a special piece of paper. Antibodies pathogen. Because the sequences complement
284 12 Techniques

Fig. 12.2 Direct tissue blotting

(Courtesy, P. Parvatha Reddy 2014)

each other, the probes can be used to identify 12.24.2 Polymerase Chain Reaction
specific diseases. (PCR)

A new technology, PCR, has great potential for

12.24.1 Squash Blot Method raising the sensitivity of various assays that use
nucleic acid probes. PCR is used to produce
A method that uses nucleic acid probe technol- enormous numbers of copies of a specified
ogy is known as the squash blot method nucleic acid sequence. This technique can allow
(Fig. 12.3). This technology is similar to direct the detection of very small amounts of a patho-
tissue blotting. Tissue from a plant that is sus- gen in a sample by amplifying the pathogen
pected to be diseased is ‘squashed’ onto a spe- sequences to a detectable level. PCR is especially
cial piece of paper, called a membrane useful in plant quarantine point of view owing to
(Fig. 12.3(1)). This membrane is then treated its fastness.
with a probe (Fig. 12.3(2)) that can bind or The specificity of PCR, be it conventional or
hybridize with nucleic acid of the plant patho- real time, depends upon the designing of proper
gen suspected to be in the plant tissue. PCR primers that are unique to the target organ-
Hybridization or binding will occur when alike ism. Highly conserved gene resigns are often the
sequences are present (Fig. 12.3(3)). After add- target for designing primers. Closely related
ing several more substances to the membrane, a microbial species often differ in a single (single-
colour reaction (Fig. 12.3(4)) indicates that the nucleotide polymorphism (SNPs)) to few bases
probe and pathogen nucleic acid sequences in such genes. PCR allows detection of such
have hybridized and the disease is present. No SNPs (Papp et al. 2003). With the advancements
colour reaction means the test for the disease is in high-throughput DNA sequencing, more and
negative (Reddy 2014). more genomes of plant pathogens are sequenced,
12.24 Nucleic Acid Probes 285

the assay, and proper negative controls must be

included in the test. It is also recommended to
have separate dedicated areas for pre- and post-
PCR handling.
Due to the advancement of fluorogenic chem-
istry, a second-generation PCR known as real-
time PCR has become an emerging technique for
the detection and quantification of microorgan-
isms in the environment. In PCR, the target DNA
sequence is amplified over a number of denatur-
ation annealing extension cycles. In a conven-
tional PCR, only the final concentration of the
amplifications may be monitored using a DNA-
binding fluorescent dye. However, in the quanti-
tative real-time PCR, the concentrations of the
amplicons are monitored throughout the
amplification cycles using a group of fluorescent
reagents. The fluorescence intensity emitted dur-
ing this process reflects the amplifications’ con-
centration in real time. Undoubtedly, most of the
future tests will be quantitative in nature, and the
real-time detection system will be a method of
choice. The real-time data will serve as useful
basis for establishing inoculum threshold levels
and detailed analysis of disease epidemics.

Fig. 12.3 Nucleic acid probe squash blot method 12.24.3 DNA Microarray Technology
(Courtesy, P. Parvatha Reddy 2014)
The DNA microarray technology, originally
and nucleotide sequence data will be available designed to study gene expression and generate
increasing the possibility for designing unique single-nucleotide polymorphism (SNP) profiles,
primers and probes for specific detection of is currently a new and emerging pathogen diag-
pathogens. nostic technology which in theory differs a plat-
PCR is highly sensitive technology. However, form for unlimited multiplexing capability. The
its sensitivity is greatly affected by the presence principle of microarray is the hybridization of
of inhibitors, which prevent or reduce amplifica- fluorescently labeled sequences or targets to their
tion. A wide range of inhibitors are reported. complementary sequences spotted on solid sur-
Although their mode of action is not clear, these face, such as glass slides, serving as probes. Tens
inhibitors are believed to interfere with poly- of thousands of such DNA probes can be spotted
merase activity for amplification of the target in a defined and addressable configuration on the
DNA. On the other hand, it is worth mentioning glass slide forming the chip. The unlimited capa-
that the high sensitivity of PCR also causes one bility for simultaneous detection of pathogen
of the limitations of PCR, i.e. detection sensitiv- makes microarray to be an approach with a
ity exceeding threshold levels or clinical signifi- potential capacity of detecting all relevant patho-
cance and false-positive results from slight DNA gens of a specific crop. Development of microar-
contamination (Yang and Rothman 2004). Hence, ray for diagnostic applications is a recent history.
stringent conditions are necessary in conducting In plant pathology, the method was applied for
286 12 Techniques

identifying oomycetes, nematode and bacterial Grontoft M, O’Connor D (1990) Greenhouse method for
testing of resistance of young Brassica plants to
and fungal DNA from pure cultures (Fessehaie
Alternaria brassicae. Plant Breed 105:160–164
et al. 2003; Lievens et al. 2003). However, for Guillemette T, Iacomi-Vasilescu B, Simoneau P (2004)
application in practice, pathogens should be Conventional and real-time PCR-based assay for
detected from environmental samples (plants, detecting pathogenic Alternaria brassicae in crucifer-
ous seed. Plant Dis 88:490–496
soil, etc.). Recently, the possibilities of parallel
Hodgkin T (1990) In vitro pollen selection in Brassica
detection of pathogens from such environments napus L. for resistance to phytotoxic compounds from
were shown (Nicolaison et al. 2005). In contrast Alternaria brassicicola (Schw.) Wilts. Sex Plant
with studies using pure cultures, microarray- Reprod 3:116–120
Hodgkin T, MacDonald MV (1986) The effect of a phyto-
mediated analysis from environmental samples
toxin from Alternaria brassicicola on Brassica pollen.
presents several challenges that must be addressed New Phytol 104:631–636
(Whittle et al. 2005). Humpherson-Jones FM, Phelps K (1989) Climatic factors
influencing spore production in Alternaria brassicae
and Alternaria brassicicola. Ann Appl Biol
114:449–459
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Murashige T, Skoog F (1962) A revised medium for rapid Tewari JP, Bains PS (1988) Mode of action of the host-
growth and bioassays with tobacco tissue cultures. specific toxins produced by Alternaria brassicae. In:
Physiol Plant 15:473–497 Proceedings of 5th international congress of plant
Nicolaison M, Justesen AF, Thrane V, Skouboe P, pathology, Kyoto, 20–27 August 1988, p 234
Holmstrom K (2005) An oligo-nucleotide microarray Thakur R, Kolte SJ (1985) Radish root extract agar, a suit-
for identification and differentiation of trichothecene able medium for the growth and sporulation of
producing and non-producing Fusarium species Alternaria brassicae. Cruciferae Newsl 10:117–118
occurring on cereal grains. J Microbiol Methods Vannacci G (1981) Seed born Alternaria brassicicola:
62:57–69 detection by means of symptoms on seedlings. Acta
Papp AC, Pinsonneault JK, Cooke G, Sadec W (2003) Horticult 111:123–129
Single nucleotide polymorphism genotyping using Vishwanath K, Kolte SJ (1999) Methods of inoculation
allele-specific PCR and fluorescence melting curves. for resistance to Alternaria blight of rapeseed and
Biotechniques 34:1068–1072 mustard. J Mycol Plant Pathol 29:96–99
Phillps GC, Collins GB (1979) In vitro tissue culture of Whittle IHF, Klammer SH, Insam H (2005) Design and
selected legumes and plant regeneration from callus application of an oligo-nucleotide microarray for
cultures of red clover. Crop Sci 19:59–64 investigation of compost microbial communities.
Reddy PP (2014) Molecular diagnosis of plant diseases. J Microbiol Methods 62:37–56
In: Genetic engineering for crop protection. Studium Wu WS, Lu JH (1984) A semi-selective medium for
Press LLC, Houston, pp 209–215 detecting seed-borne Alternaria brassicicola. Plant
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technique for inducing profuse sporulation of Xu XL, Ko WH (1998) A quantitative confined inocula-
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89:244–246 plants. Bot Bull Acad Sin 39:187–190
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46(4):373–378 4:337–348
 Future Strategies and Priorities
for the Management of Alternaria 13
Diseases of Crucifers

With the increasing demands of a growing global tial diseases. There is an urgent need to carry out
population, there has been a significant change in intensive investigation with regard to the envi-
cropping patterns of edible and industrial oil- ronmental parameters responsible for causation
yielding crucifers as well as vegetables. In the and spread of diseases under field conditions.
absence of host resistance, intensive cultivation Multilocational trials with staggering dates of
has resulted in perpetuation, build-up and dissem- planting could be helpful in analysing disease
ination of Alternaria species on crucifers in areas development in relation to environment by com-
where these crops are grown. The main factors puting disease progression at regular intervals.
responsible for the increase of inoculum of patho- These efforts could be undertaken to study vari-
genic Alternaria to the present level include the ous disease prediction models (geophytopatho-
lack of genetic sources of resistance, cultivation logical, bioclimatic, simulation system analysis,
of susceptible crops with high plant density, use etc.) which could play a meaningful role in devel-
of irrigation with high rates of nitrogen fertilizers, oping an effective disease control strategy.
contiguous areas under monoculture, staggered
sowing dates, poor weed management and poor
plant protection strategies. The information gath- 13.2 Physiological Specialization
ered in this book on Alternaria diseases of cruci-
fers indicates that some gaps and bottlenecks still Identification and standardization of host differ-
exist in our comprehensive understanding of vari- entials are fundamental prerequisites to the gath-
ous dimensions of Alternaria–host pathosystem; ering of meaningful information on races/
some of these are highlighted below to be resolved pathotypes. This is a high-priority area of investi-
priority wise in areas of crucifer production. gation. Designation and nomenclature of pathot-
ypes should be uniform, logical and
internationally acceptable. Virulence genes may
13.1 Disease Epidemiology be identified.
and Forecasting

Factors governing disease development and con- 13.3 Genetics of Resistance

sequent progression are not completely under-
stood. Deeper insight with regard to various Although we have some information on genetic
epidemiological aspects will help to develop sources of resistance, the information on the
strategies to curb the progression of these poten- nature, mechanism and inheritance of resistance

© Springer Science+Business Media Singapore 2016 289

G.S. Saharan et al., Alternaria Diseases of Crucifers: Biology, Ecology and Disease Management,
DOI 10.1007/978-981-10-0021-8_13
290 13 Future Strategies and Priorities for the Management of Alternaria Diseases of Crucifers

are still not satisfactory and/or lacking. The 13.6 Comparative Studies on All
results of this area of investigation are an impor- Aspects of Host–Parasite
tant prerequisite to incorporate stable and effec- Interaction
tive sources of resistance in breeding programmes.
In diseases caused by Alternaria species, where Comparative studies on all aspects of host–para-
major gene resistance is not known, the potential site interaction in crucifers with all the four spe-
of minor genes/horizontal resistance, tolerance cies of Alternaria may reveal some fruitful results
and slow blighting should be exploited. for planning comprehensive management strate-
Feasibility of induced resistance needs attention. gies of these pathogens.
Biotechnology or molecular biology and genetic
engineering techniques can be used to transfer
resistant genes from distantly related sources. 13.7 Phytotoxins
Identification and sequencing of resistant genes
along with identification of suitable gene combi- Relatively few genes and/or gene products have
nations may be taken up. Hybrids and GM culti- been identified that contribute to or are required
vars with multiple disease resistance is the need for pathogenicity. More research is required to
of the hour. further characterize secondary metabolites bio-
synthetic genes and their role in pathogenicity
and fungal development along with fungal signal
13.4 Genetics of Virulence transduction mechanisms.

Characterization of secondary metabolite bio-

synthetic genes along with signal transduction 13.8 Genome Sequencing
and their role in pathogenicity and fungal
development are important areas of investiga- Alternaria species genome sequencing will allow
tion. Disruption of Aso-1, a gene required for the identification of putative secreted pathogen
hyphal fusion (anastomosis) which is important effectors’ proteins in silico that can be used in a
for Alternaria spores to become pathogenic, variety of functional analyses.
needs attention. Studies on the nature and
mechanism of inheritance of virulence genes in
different species of Alternaria are required. 13.9 Disease Control Strategy
Determination of virulence spectrum of pathot-
ypes will reveal use of number of resistance Some useful information is available on chemical
genes in breeding of disease-resistant control of various Alternaria diseases; however,
brassicas. much more is needed in order to develop a viable
disease control strategy under field conditions.
Efforts should also continue to search for low-
13.5 Exploitation cost effective chemicals, which can provide eco-
of Morphological, Structural nomically significant disease control. Active
and Biochemical Basis ingredient of plant extracts should be identified.
of Resistance Control of Alternaria diseases in rapeseed–
mustard by microbial antagonists should also be
Exploitation of morphological, structural and explored. Seed treatment with Trichoderma spp.,
biochemical basis of resistance needs in-depth Gliocladium spp., Penicillium spp. and Mycostop-
study in crucifer since a lot of genetic variability like preparations of Streptomyces spp. should be
is available in related and distantly related cruci- further investigated to determine their level of
fers all over the world. control and economic feasibility under field
13.10 Integrated Management 291

conditions. Similarly, control by foliar applica- host resistance and biotechnology tools for early
tions of Streptomyces rochei, S. hygroscopicus, S. detection of disease should be integrated to man-
arabicus and Nectria inventa should also be eval- age the disease at early stage of plant growth.
uated under field conditions. Considering the role of insects like aphids (virus
transmission) and flea beetles (transmission of A.
brassicicola to cabbage), insecticides should be
13.10 Integrated Management one of the components in integrated disease man-
agement package and practices. Disease forecast-
No single methods or approach can be effective ing system should be strengthened using modern
and economical in dealing with any biological information technology available at village level
systems. Therefore, all means of plant disease to warn farmers in advance for timely action of
control including chemical, biological, cultural, proper control strategy.
Index

A B
Abiotic stresses, 220 Benzimidazole Agar medium, 273
AB toxin, 214, 216, 275 Bioagents treatment, 10, 252, 254, 262–264
Accessions, 192 Bioassay, 186, 188, 213
Acquired resistance, 188 Biochemistry of host pathogen interaction
Aggressiveness, 213 amino acids, 170
Albino strain, 163, 165 carbohydrates, 167, 168
Alternaria alternata, 1, 2, 6, 7, 18, 19, 23, 24, 34, enzymes, 169
39, 53, 58, 59, 64, 65, 70, 76–78, 87, 108, fatty acids, 170
125, 140, 169, 171, 192, 211, 212, 215, 216, glucosinolates, 170–171
218, 220, 222–225, 227 growth substances, 167, 169
Alternaria brassicae, 127, 133, 138, 240 metabolites, 167
Alternaria brassicicola, 2, 6–10, 18, 19, 22, 23, phenolic compounds, 8, 185
28, 37, 38, 53, 54, 59–65, 68–74, 76–79, photosynthesis, 169
87–89, 91–95, 103–110, 112, 113, 115, phytoalexins, 170
125, 126, 140, 158, 159, 163–167, 169–171, proteins, 169
185, 186, 189–194, 202, 203, 211, 214–217, respiration, 218
219–223, 226, 229, 230, 240–242, 246, RNA content, 167
249–252, 255, 258, 259, 274, 275, 277, toxins, 145, 169
278, 291 Biological changes, 254
Alternaria raphani, 2, 6, 7, 18, 19, 22, 23, 30, 31, 33, 34, Biological control agent, 250
54, 61, 65, 69–72, 74, 77, 78, 87, 89, 106, 108, Biosynthesis, 190
109, 126, 141, 171, 240 Biotechnology, 192, 290, 291
Alternaria species, 1, 2, 5–10, 23, 24, 53, 54, Biotic stress, 230
58–60, 63, 65, 67, 68, 71, 78, 87, 95, 163, Brassica crops, 2–5, 10, 18, 23, 74, 99,
171, 202, 211, 212, 214–216, 222, 223, 192, 197, 198
226–230, 241, 289 Brassica species
Alternaria symptoms, 5, 17, 18, 20–22, 39, 78, 80, 89, B. alba, 31, 34, 110, 129, 133, 183, 185, 192, 193,
127–129, 133, 169, 170, 175, 212, 213, 218, 195
221, 273–275, 278 B. alboglabra, 27, 32
Antagonism, 250 B. campestris, 2, 3, 24, 27, 31, 34, 36, 37, 73, 110,
Antibiotics tested 119, 120, 200, 218
Griseofulvin¸, 243 B. campestris var. toria, 80
Mycostatin, 243 B. carinata, 3, 7–9, 28, 31–34, 37, 128, 176–178,
Mycothricin, 243 182, 185, 188, 192–196, 198, 199, 201, 203
Polyoxin B & D, 243 B. hirta, 32, 193
Area Under Disease Progress Curves (AUDPC), 114, B. juncea, 127–129, 133
180–182 B. napus, 2, 3, 5, 9, 24, 26–28, 30–32, 34, 36, 37, 77,
Assessment of losses, 35, 43 91, 110, 176, 180, 182, 184, 185, 187, 189,
Attributes, 7 191–195, 200, 201, 212, 213, 217, 227, 229,
Avirulence, 7, 203 256, 275
Azadirachta indica, 249 B. napus cv, 89

© Springer Science+Business Media Singapore 2016 293

G.S. Saharan et al., Alternaria Diseases of Crucifers: Biology, Ecology and Disease Management,
DOI 10.1007/978-981-10-0021-8
294 Index

Brassica species (cont’d) Asthana and Hawker’s, 69

B. nigra, 3, 128, 194, 201, 218 Brown’s starch agar, 69
B. oleracea, 3, 5, 7, 22, 25, 30, 32–34, 38, 64, 71, 74, Coconut, 69
77, 88, 103, 105, 126, 128, 193, 194, 200, 201 Corn meal agar, 69
B. oleracea (cauliflower), 194 Czapek-Dox, 69, 170
B. pekinensis, 24, 31–34, 241 Eruca sativa decoction, 69
B. rapa, 2, 3, 5, 7, 9, 18, 29, 31–34, 36, 72, 76, 77, glucose asparagine medium, 69, 72
89, 90, 183–185, 191–198, 203, 213, 227, 244, host decoction, 69, 72
275, 278 Houston’s, 69
B. tournefortii, 3 Kirchoff’s, 69
Broccoli, 3–5, 22, 23, 25, 30, 38, 194, 227, 239 Leonian’s agar, 69
Brown Sarson, 3, 18, 31, 34–37, 88, 101, 218 malt extract, 68, 69
Brussels sprouts, 4, 5, 22, 23, 27, 29, 32, 38, 72, 193 mustard leaf extract, 69
oatmeal, 69
pechay decoction, 69
C potato dextrose, 69
Cabbage, 3–5, 7, 22–30, 32–34, 38, 39, 64, 68, 74, potato dextrose asparagine, 69
77–79, 88, 89, 95, 100, 104, 106–110, 112, potato sucrose, 69
113, 126, 169, 194, 222, 239–241, 246, 247, rice meal, 69
250, 252, 277, 279, 291 Richard’s, 69
Calcium sequestration, 9, 192 Sabouraud’s, 69
Callus culture, 276–277 V-8, 69, 274, 276, 279, 281
Camalexin, 9, 189–191, 221, 226, 230 wheat meal, 69
Camelina sativa, 3, 191–194, 213, 226 Curd blight, 17
Candytuft, 26, 28 Cutinases, 64, 68, 92, 93, 95, 222, 223
Canola, 5, 180, 189, 214, 227 Cytokinins, 8, 89, 188, 218
Capsella bursa-pastoris, 191–194
Cauliflower, 3–5, 7, 17, 22–25, 27–30, 32–34, 38, 64, 68,
76, 88, 95, 126, 169, 186, 193, 194, 196, 223, D
240, 249 Damage, 9, 34, 211, 216
Cellular alterations, 256 Disease
Chinese cabbage, 4, 25, 26, 28, 30, 32, 104, 109, 192, disease assessment, 43
219 disease assessment keys, 39–45
Chlamydospores, 6, 61, 70, 71, 74, 87, 95 disease incidence, 17, 37, 43, 73, 74, 107–109,114
Collards, 4, 5, 23, 24 disease index, 39, 43, 180–182, 282
Collateral host disease intensity, 5, 35, 37, 43, 73, 112, 176, 188, 201
Anagallis arvensis, 27 disease score, 178, 188, 195
Convolvulus arvensis, 32, 34 disease severity, 37–41, 43, 101, 113, 203, 244, 281
Colony growth, 54 Disease cycle, 6, 87–97
Colony morphology, 203 Disease development, 88, 115, 121, 198, 216, 250, 289
Colza, 2, 25, 26 Disease distribution, 115–116
Crambe spp. Disease forecasting, 291
C. abyssinica, 3, 5, 19–22, 24, 26, 27, 29, 32–34, 38, Disease forecasting models, 7, 99
70, 89 Disease management
C. hispanica, 3, 5 biological control, 249–254
Crop residues, 53 chemical control, 241–248
Crop rotation, 9, 239 cultural control, 239
Crucifers fungicidal control, 38
Eruca sativa, 3, 18, 19, 24, 27, 30, 31, 33, 34, 36, 69, host resistance, 8–10, 17, 39, 43, 110, 154, 180, 203,
76, 89, 126, 191, 192, 198 212, 226, 239, 254–259, 289, 291
Raphanus sativus, 5, 33, 34, 190, 194 integrated disease management, 259–266
Cultivars, 3, 5, 7, 9, 22, 35, 36, 43, 44, 76, 80, 88, 99, plant extracts, 10, 145–154, 227, 249–250, 262–264,
101, 111, 112, 119, 180, 182, 185–188, 196, 290
197, 200, 211, 219, 275, 278, 281 seed treatment, 80–82, 239–241, 250, 259–261, 290
Cultural characters, 72 Disease plant debris, 6, 71, 74
Cultural conditions, 99 Disease progress models, 19, 22, 39, 43, 103, 115
Cultural practices, 9 Disease stress, 5, 44, 178, 179
Culture filtrates (CFs), 171, 185, 218 Disease symptoms, 19–22
Culture media Disease synonyms, 17–18
alfalfa decoction, 69 Disease tolerance, 44, 178
Index 295

E Chlorothalonil (Daconil, Bravo), 243

Economic importance, 23 Copper oxychloride (Blitox), 243
Efficacy of fungicides, 39, 145 Copper sulphate, 243
Electron microscopy, 8, 163 Cuman L, 241, 243
Epidemiology Cupravit, 243
effect of cultural practices, 109–110, 259 Cupric acetate, 243
effect of date of sowing, 7, 99, 103, 104, 115, 116 Cuprox, 243
effect of environmental conditions, 99–109 Cycloheximide, 243
relative humidity, 99, 100, 103, 108 Delan C, 243
rainfall, 100, 101 Dichlofluanid, 243
temperature, 99–104, 106, 108 Difenoconazole (Score), 243
wind velocity, 99, 100, 103, 104, 106 Difolatan, 241, 243
effect of micronutrients, 259 Dithane D-14, 243
effect of nutrition, 109–110, 259 Dithane M-45 (Mancozeb), 155, 156, 240, 243
effect of flee beetle, 110–112 Dithane Z-78, 240, 241, 243, 246
Epidemiological models, 7, 114, 289 Duter, 243, 244, 246
Estimation of losses, 43 Edifenphos, 243
Exudates, 76, 182, 277 Euparen, 243
Fenarimol, 243
Fenpropimorph, 243
F Fentin acetate, 243
Fern extract, 249 Ferbam, 243
Fertilizers, 2, 103, 239, 259 Fermate, 243
Fine structures flutriafol, 243
Conidia, 163–166 folicur (Tebuconazole), 243
conidiophores, 8, 18, 20, 54, 57–61, 67, 74, 276 folpet, 243
hyphae, 8, 57, 60, 68, 90, 91, 166, 189, 251, 276 formaldehyde, 243
mycelium, 20, 54, 57, 60, 63, 69, 71, 72, 170, 222, Granosan, 243
276, 278 Guazatin, 243
Fungicides tested Halogenated derivatives, 243
Acetone, 243 Imazalil, 243
Actidione, 243 Indofil M-45, 243, 244
Agrosan GN, 241, 243 Indofil Z-78, 243, 244
Alar, 109, 243 Iprodione (Rovral), 81, 243
Antracol, 243 Karbam white, 243
Arasan, 243 Kavach, 243
Azadirachtin (Neemarin), 243 Lunasan, 243
Azoxystrobin (Amistar), 243, 244 Malic acid, 243
Bafin, 243 Mancozeb, 243, 244, 259
BAS 480F, 243 Maneb, 243
Bavistin, 243 Manzate, 243
Baycor, 243 Merpan, 243
Bayleton, 243 Metalaxyl, 243
Benlate, 240, 241, 243, 246 Metiram, 243
Benz (1,2) isoxazoles, 243 Miltox, 243
Bioquin, 243 4- Nitrosopyrazole, 243
Blitox, 150, 155, 156, 243 Nuarimole, 243
Bordeaux mixture, 241, 243, 244, 246 Ozone, 243
Boric acid, 241, 243 Panogen, 243
Boscalid, 243 Parzate, 243
Brassicol, 240, 243 Penconazole (Topaz), 243, 244
Brestan, 241, 243 Pentachlorophenol, 243
Bromosan, 243 Phygon, 243
Calixin, 109, 243 P-Methoxytetrachlorophenol, 243
Captaf, 243 Prochloraz, 243
Captafol, 241, 243, 245 Propiconazole, 243
Captan, 243 Propineb, 243
Carbendazim, 243 Pyraclostrobin, 243
Carboxiin (Vitavax), 243 pyrena compounds, 243
Ceresen, 243 Quinolate, 243
296 Index

Fungicides tested (cont’d) H

Quinone, 243 Historical, 53–54, 125–127, 212–213
Ridomil MZ, 151, 156, 243, 244, 248 Host range
Ronilan, 243 Abyssinian mustard, 3, 32, 33
S- triazines, 243 black mustard, 3, 4, 32, 281
Semesan, 243 brussels sprouts, 4, 5, 22, 23, 27, 29, 32, 38, 72, 77,
Signum 334WG, 243 193, 247
Sisthane, 243 cabbage, 3–5, 7, 21– 30, 32–34, 38, 39, 64, 68,
Sodium fluoride, 243 74, 77–79, 88, 89, 95, 100, 104, 106–110,
Spergon, 243 112, 113, 126, 169, 171, 192, 194, 219,
Sumilex (Procymidone), 243 222, 239–241, 246, 247, 250, 252, 275,
Syllit, 243 277, 279, 291
Tebuconazole (Nativo), 243 Candytuft, 26, 28, 33
Tetrahydropyrimidine, 243 Canola, 5, 32, 170, 171, 180, 189, 214, 216,
Thiabendazole, 243 227, 228
Thiophanate methyl, 243, 259 cauliflower, 3–5, 7, 21–25, 27–30, 33, 34, 38, 64, 68,
Thiovit, 243 76, 77, 88, 95, 126, 158, 169, 186, 193, 194,
Thiram, 243 196, 223, 240, 241, 245, 246, 249, 250
Tillex, 243 Chinese cabbage, 4, 23, 25, 26, 28, 30, 32, 33, 104,
Topsin M, 243, 244 107, 109, 171, 192, 219, 241, 246
Triademefon, 243 Crambe, 3, 5, 7, 19–22, 28, 32–34, 38, 70, 74, 108,
Triapenthenol, 243 128, 158, 194
Triarimol, 243 Garden cress, 33
Tribasic copper sulphate, 243 Guar, 32
Tribasic copper zinc, 243 Hedge mustard, 33
Tridemorph, 243 Hiran khuri, 34
Trifloxystrobin, 243 Honesty, 33
Trimiltox forte, 243 horse radish, 23, 25, 32
Vinclozolin (Ronilan), 243 Indian mustard, 3, 33, 34, 37, 100, 104, 167, 168,
Wettable sulphur, 243 178, 188, 189, 193, 201, 220, 227, 254, 261,
Zato 50 WG, 243 265
Zinc sulphate, 243 Kale, 4, 5, 29, 32, 88, 193
Zincop, 243 kohlrabi, 23
Zineb, 243 Lettuce, 33, 34
Ziram, 243 mustard, 23
Fungistasis, 192 radish, 23
Fungitoxic, 170, 249 Rapeseed, 23
rutabaga, 23
Santhi, 34
G Spinach, 4, 34
Gamma rays, 197 Stink weed, 33, 34
Garden stock, 22, 30 Stock, 2, 22, 30, 33, 34, 74, 192, 240, 276
G:C ratio, 167 swedes, 23
Genetics of Host-Parasite Interaction, 176 Taramira, 3, 19, 27, 33, 34, 76, 194, 250
Genotypes, 5, 8, 17, 18, 37, 43, 44, 110, 120, 127–129, Tomato, 34, 201, 204, 225, 227, 277
133, 156, 169, 176, 178–182,185, 186, 189, Tumbling weed, 33
192–198, 201, 213, 219, 225, 255, 256, 258, turnip, 23
273–275, 278 Wall flower, 33
Geographical distribution, 23 White mustard, 3, 25, 32, 33, 185, 193, 194, 220,
Germ-plasm screening, 255, 275 227, 228
Gliocladium virens, 251 Wild mustard, 28, 30, 32, 33
Glucosinolates, 3, 5, 8, 170, 171, 189, Wormseed mustard, 33
190, 219 Homodestruxin B, 175, 212, 213, 215–217
Green islands, 169, 218 Horizontal resistance (HR), 8, 43, 176, 180, 200
Growth and sporulation, 6, 10, 53, 68–71, 135, Horseradish, 23, 25, 32
138, 143 Host age, 114
Growth in culture, 6, 70, 241 Host differentials, 7, 154, 289
Growth regulators, 275, 277 Host diversity, 127
Growth stage key, 39, 41 Host non specific toxins, 9, 213, 223, 224
Index 297

Host resistance Life cycle of disease, 204

Identification, 1, 6, 53, 54, 64–65, 67–68, 94–95, Life cycle of pathogen, 1
154–156, 186, 188–190, 196, 198, 202, 204, Light and darkness, 6, 71
256, 277, 279, 282, 289, 290 Light intensity, 39, 71, 77, 104
Induction, 203 Losses
Host–pathogen interactions, 8, 17, 87, 125 Fatty acids, 37
Host-specific toxins (HSTs), 58, 211, 212, 216, 218, 223, oil content, 36
225–227, 275 yield, 3, 17, 23–39, 43, 218
Hot water treatment, 239–240 Lunaria annua, 26, 33
Hydrogen ion concentrations (pH), 70–71 Lunaria rediviva, 26, 33
Hydrolysis, 170, 215, 223
Hyper-parasite, 254
Hypersensitive reaction, 187, 203 M
Macrosporium species, 54
Mapping of genes, 204
I Matthiola incana, 22, 25, 33, 34
Iberis species, 28 Metabolites, 8, 9, 63, 67, 89, 95, 171, 175, 189, 190,
Iberis umbellata, 25, 33 214–216, 219, 222–224, 229, 279
Incubation period, 127–132, 182 Micronutrients tested
Infection, 2, 17, 53, 87–97, 100, 126, 167, 176, 216, Borax, 243
239, 274 Ca, 243
Infection grades, 42 Cacl2, 261
Infection, rate, 110, 176, 180–182 CO (NO3)2, 243, 261
Inheritance of resistance, 8, 255 CuSO4, 243, 261
Inhibition of spore germination, 192 Fe EDTA, 243, 261
Inoculation technique, 274 K, 243
Insecticides tested Na2 BO7, 243, 261
Cypermethrin, 243 P, 243
Deltamethrin, 243 S, 243
Dimecron, 243, 245, 247 SO, 243
Fenvalerate, 243 Thiourea, 243, 261
flucythrinate, 243 Zn, 243
Metasystox, 243, 245, 247 Zn SO4, 243, 261
Methyl demeton, 243 Microsclerotia, 6, 53, 87, 95
Permethrin, 243 Mode of infection, 88, 95
Phosphamedon, 243 Molecular biology, 290
Rogar, 243, 245, 247 Molecular formula, 212, 214
Integrated control, 245 Multiple disease resistance sources, 195
In-vitro study, 53, 65, 189 Mutation, 92, 125, 189, 220, 221, 224,
Irrigation, 7, 104 226, 230
Isolates, 7, 10, 18, 55, 65, 126, 133, 134, 143, 145, 150, Mycoparasitism, 250, 256
198, 203, 216, 224, 258, 276, 280, 281 Mycostop, 241, 250, 290
Isolation, 201, 230, 240

N
K Necrosis, 9, 185, 211, 212, 218, 225, 275
Kale, 4, 5, 29, 32, 88 Nectria inventa, 252, 291
Kohlrabi, 4, 5, 22, 23, 25, 29, 30, 32 Nitrogen sources in the medium, 148, 149
Nuclei, 8, 163, 166
Nucleic acid, 167, 283–286
L Nutrition, 5, 7, 9, 10, 17, 70, 109–110, 126, 134, 138,
Laboratory and field techniques, 88, 100, 115, 143–145, 158, 259, 261
255, 282
Latent period, 74, 129, 176, 180, 188
Leaf age, 170 O
Leaf extracts, 76, 216, 230, 244, 249 Oil content, 35–37
Leaf exudates, 76, 277 Oilseed rape, 2, 28, 30, 41, 72, 74, 193
Leaf surface mycoflora, 250 Oil quality, 2, 17, 23, 34, 191, 202
Leaf wetness, 76, 88, 97, 101, 103, 104 Ovule culture, 200
Lepidium spp., 27 Oxalic acid, 192
298 Index

P Resistance nature
Pathogen biochemical basis, 185
A.alternata, 53, 55, 58–61, 64, 65, 70, 76–78 calcium sequestration, 192
A.brassicae, 53, 54, 59–63, 65, 68–78, 80, 81 Components of HR, 180
A.brassicicola, 53, 54, 59–65, 67–74, 76–79 disease tolerance, 178
A.cheiranthi, 61–62 elicitation of phytoalexins, 191–192
A.raphani, 53, 54, 61, 65, 69–72, 74, 77, 78 epicuticular wax, 175, 176, 180–183
Pathogen classification, 58–59 factors affecting resistance, 203
Pathogen morphology genetical basis, 204
nomenclature, 57–58 identification of R genes, 188–191
perpetuation, 71–74 induced resistance, 187–188
phylogeny, 6, 53, 55–56 inheritance of resistance, 176–178
taxonomy, 57–58 morphological resistance, 180
Pathogenesis, 6–8, 53, 64, 68, 87–97, 126, 167, 175, 186, proteome level resistance, 185–187
188, 199, 211, 216, 219–223, 231, 278–279 sequencing of R genes, 188–191
Pathogenic variability, 7–8, 175, 197 Resistance sources
Pathotoxins, 188 closely related, 194
Pathotypes, 5, 7, 8, 17, 194, 198, 216, 220, 223, 224, distantly related, 258, 290
255, 290 multiple disease resistant sources, 195
Pectinases, 61, 92, 93, 169, 170, 192 screening resistant sources, 197–199
Penetration, 64, 78, 87, 88, 91, 187, 222, 223, 253 Resistant cultivars, 9, 10, 76, 186, 196–199, 258
Phenolic compounds, 8, 185 Resistant variety, 203
Phospholipids, 70
Physiological specialization, 289
Phytoalexins, 8, 9, 170, 175, 190–192, 199, 203, S
215–217, 226–228, 230, 258 Sanitation, 9
Phytotoxins, 9, 171, 203 Seed disorder, 18
Plant age, 123 Seed infection, 22, 24, 25, 29, 35, 36, 73, 77–82, 108,
Plant growth stages, 185 109, 239, 279
Pollen selection, 275 Seed quality, 34, 37
Protein content, 254 Seed transmission, 73
Senescence, 63, 188, 222
Sinapus species, 194
Q Slow blighting, 8, 180
Qualitative loss, 5 Slug association, 100
Quantitative loss, 5 Soil culture, 74
Somaclonal variations, 201
Sources of resistance, 9, 175, 192–194, 197, 289
R Spore dispersal, 112
Radish, 3–5, 7, 22–34, 39, 73–78, 106, 108, 109, 125, Spore germination, 6, 67, 71, 73–78, 277
126, 158, 170, 191, 227, 239, 241, 246, 249, Spore structure and development, 82, 166
251, 266, 276, 279 Sporulation, 6, 7, 21–22, 54, 67, 77, 78, 80, 104, 125,
Radish root extract, 276 135, 145, 176, 180–182, 198, 221, 250, 259,
Rainfall, 100, 101, 117 275–277
Rape, 2, 3, 5, 7, 17, 18, 25–30, 38, 39, 41, 71, 72, 74, 77, Spraying of chemicals, 10
78, 100, 104, 109, 126, 127, 158, 170, 193, Starch, 167
245, 246, 252, 253, 261 Stemphylium spp., 1, 2, 53, 54, 58, 65
Rapeseed-mustard, 7, 18–21, 23–38, 43, 53, 54, 73, 78, Storage fungi, 50
89, 99–101, 103, 110–112, 119, 120, 126, 133, Strain, 2, 7, 55, 57, 59, 171, 216, 224, 225, 227
138, 143, 150, 158, 182, 192, 198, 244, Streptomyces spp, 290
261, 282, 290 Subcuticular growth, 98
Regression analysis, 115, 117, 118, 121 Survival of pathogen, 6, 93
Relationship between diseases, 43, 103 Susceptibility of the host, 213
Relative humidity, 6, 7, 70–71, 76, 89, 99, 100, 118, 274, Symptomatology, 19–22
279 Crambe, 19–22
Remote sensing, 17, 43 Garden Stock, 22
Research priorities, 10 Rapeseed–Mustard, 18
Resistance development Taramira, 19
biotechnological approaches, 200–203 Vegetable Crops, 22
conventional approach, 196 weeds, 23
Index 299

Symptoms, 5, 17–22, 39, 63, 64, 78, 80, 89, 175, V

211, 213, 216, 218, 221–223, Variants, 7, 224
273–275, 278, 279, 282 Variability determinants, 158–159, 160, 209
Symptom variability, 133 Verticillium spp., 250, 252
Synonyms, 6, 17–18, 59, 60, 63 Video image, 17, 43–44
Virulence, 6–8, 53, 67, 68, 74, 89, 90, 92–95, 167, 197,
198, 211, 213, 216, 222, 230, 290
T Vitamins, 70
Taxonomy, 1, 2, 6, 53, 55, 57–59
Techniques, 6, 9, 10, 17, 43, 53, 68, 176, 199, 200,
202, 220 W
Temperature effect, 76 Wallflowers, 23, 33, 62, 74, 277
Thlaspi arvensis, 27, 33 Wax crystals, 183, 184
Tolerance, 44, 67, 88, 95, 178, 185, 201, 219 Weeds as host, 5, 17
Toria, 3, 4, 18, 31, 33, 34, 73, 193 White cabbage, 29, 38, 246
Toxins, 6, 9, 58, 63, 87, 89, 127, 211, 212, 214, 216, 217,
220, 222–227, 275
Transfer of resistance, 175, 197, 290 Y
Trichoderma spp, 290 Yellow Sarson, 3, 31, 34–37, 73, 89, 95, 117, 120, 197,
Turnip, 2–4, 22, 23, 25–33, 38, 71, 198, 213, 275
193, 213 Yield components, 35, 36
Yield estimates, 31, 34, 37, 179
Yield increase, 245, 246, 260
U Yield losses, 3, 17, 23–39, 43, 218
Ultra structures, 11, 166, 205, 231 Yield potential, 44–45