Biofertilizante 2020 Leer

Environmental and Microbial Biotechnology
Manoj Kumar
Vivek Kumar
Ram Prasad Editors
Phyto-
Microbiome
in Stress
Regulation
Environmental and Microbial
Biotechnology
Series Editor
Ram Prasad, Department of Botany, Mahatma Gandhi Central University,
Motihari, Bihar, India
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Manoj Kumar • Vivek Kumar • Ram Prasad
Editors
Phyto-Microbiome
in Stress Regulation
Editors
Manoj Kumar Vivek Kumar
Department of Life Sciences Swami Rama Himalayan University
Central University of Jharkhand Himalayan School of Biosciences
Ranchi, Jharkhand, India Dehradun, Uttarakhand, India
Ram Prasad
Department of Botany
Mahatma Gandhi Central University
Motihari, Bihar, India
ISSN 2662-1681 ISSN 2662-169X (electronic)

Environmental and Microbial Biotechnology
ISBN 978-981-15-2575-9 ISBN 978-981-15-2576-6 (eBook)
https://doi.org/10.1007/978-981-15-2576-6
© Springer Nature Singapore Pte Ltd. 2020

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Singapore
Preface
This book presents state-of-the-art research on the many facets of the plant
microbiome, including survivability aspects of plants based on ecology, physiol-
ogy and genomics, as well as molecular mechanisms of plant-microbe interac-
tions. Topics are well thought out which include the significance of microbial
assisted plant growth, induced systemic resistance, tolerance to abiotic stress and
biological control of plant pathogens.
The respective contributions show how microbes help plants to cope with abiotic
stresses and represent significant progress towards understanding the complex
regulatory networks critical to host-microbe interaction and plant adaptation in
extreme environments. New insights into the mechanisms of microbial actions in
inducing plant stress tolerance open new doors for improving the efficacy of micro-
bial strategies and could produce new ways of economically increasing crop yields
without harming the environment. As such, this book offers an essential resource for
students and researchers with an interest in plant-microbe interaction, as well as
several possibilities for employing the plant microbiome for the enhancement of
crop productivity under future climate change scenarios.
Phyto-stimulation and bio-control by plant-associated microbes set a revolution-
ary paradigm in modern findings towards farming culture in developing countries.
At the same time, genetically modified (GM) crops harbouring few significant
strains, i.e. Bacillus thuringiensis (BT) gene(s) to combat biotic stress caused by
insect pests, endorse the contemporary theme of bridging research for the agricul-
ture sector. The book captures the cumulative efforts of contributors in a review
format, which can be a meaningful addition for modern researchers.
It not only provides details on existing challenges but also offers deeper insights
into the possibility of solving problems, compiled as follows, but not limited to:
• Applications of plant-microbe interactions in contaminated agro-ecosystem

management.
• Development of future bio-formulations for sustainable environment.
• Heavy metals, hydrocarbons, radioactive materials and bioremediation of dyes.
• Microbes and soil and water toxicants management.
• Role of plants, genetically modified organisms (GMOs) and earthworms in
bioremediation or any topic on bioremediation.
v
vi Preface
Emphasis is given on the most neglected part of scientific exploration that is

rare micro-flora of earth where scientists are understanding the mechanism behind
survivals; (b) targeted that the efficient plant microbiome and their benefits in agro-
ecosystem, water bodies, human health and targeting global community in plant,
soil, water and rare microbe synergism field; (c) highly specialized book on biotic
and abiotic stresses and its management through microbial technological inputs,
will help the academicians, researchers, environmentalists, industrialists, agricul-
turists and graduate students and (d) experts across the globe expressed the prac-
ticed knowledge in the shape of chapters which deal with zest of specific research
on microbiome mediated stress tolerance.
Ranchi, Jharkhand, India Manoj Kumar

Dehradun, Uttarakhand, India Vivek Kumar
Motihari, Bihar, India Ram Prasad
Contents
1 Phytostimulation and Biocontrol by the Plant-Associated

Bacillus amyloliquefaciens FZB42: An Update�� 1
Rainer Borriss
2 Genetically Modified (GM) Crops Harbouring
Bacillus thuringiensis (BT) Gene(S) to Combat Biotic Stress
Caused by Insect Pests�� 21
Bhupendra Koul
3 Characterization and Efficiency of Rhizobial Isolates
Nodulating Cytisus monspessulanus in the Northwest
of Morocco: In Relation to Environmental Stresses �� 63
Taoufik Belechheb, Mohammed Bakkali, Amin Laglaoui,
and Abdelhay Arakrak
4 Isolation and Characterization of the Roots and Soil
Endomycorrhizae of Hedysarum pallidum Desf.
in the Northeast of Morocco�� 73
Rachid M’saouar, Mohammed Bakkali, Amin Laglaoui,
5 Friends and Foes: Phyto-Microbial Interactions
in Molecular Perspective �� 81
Shyam Solanki, Gazala Ameen, Debankur Sanyal, Shalu Jain,
Ammar Elakhdar, Shwetank Lall, Kishore Chittem,
Leah Brueggeman, Ajay Kumar, and Robert Brueggeman
6 Isolation and Screening of Inorganic Phosphate Solubilizing
Pseudomonas Strains from the Lotus creticus Rhizosphere Soil
from the Northwest of Morocco�� 99
Imane Achkouk, Saida Aarab, Amin Laglaoui, Mohammed Bakkali,
vii
viii Contents
7 Screening and Characterization of Phosphate-Solubilizing

Rhizobia Isolated from Hedysarum pallidum
in the Northeast of Morocco�� 113
Samia Hamane, Mounir Hassani Zerrouk, Anass E. Lyemlahi,
Saida Aarab, Amin Laglaoui, Mohammed Bakkali,
8 Development of Abiotic Stress Tolerance in Crops
by Plant Growth-Promoting Rhizobacteria (PGPR)�� 125
Sivakumar Subiramani, Sathishkumar Ramalingam,
Thiruvengadam Muthu, Shivraj Hariram Nile,
and Baskar Venkidasamy
9 Plant Growth-Promoting Rhizobacteria (PGPR)
and Their Action Mechanisms in Availability of Nutrients
to Plants�� 147
Hassan Etesami and Sina M. Adl
10 Plant Growth and Development Under Suboptimal
Light Conditions�� 205
Ravinderjit Kaur, Gaganpreet Kaur, Kashmir Singh,
and Baljinder Singh
11 Microbial Biotechnology: A Key to Sustainable Agriculture�� 219
S. K. Gosal, Jaspreet Kaur, and Jupinder Kaur
12 Stress Signalling in the Phytomicrobiome: Breadth
and Potential�� 245
Sahana Basu and Gautam Kumar
13 A Simple Procedure for Isolation, Culture of Protoplast,
and Plant Regeneration �� 269
Indu Kumari
14 Plant Antimicrobial Peptides: Next-Generation Bioactive
Molecules for Plant Protection�� 281
Paomipem Phazang, Neelam Prabha Negi, Meenakshi Raina,
and Deepak Kumar
15 Nitrogen Stress in Plants and the Role of Phytomicrobiome �� 295
Garima Malik, Navneet Singh, and Sunila Hooda
16 Halotolerant Microbes for Amelioration of Salt-Affected
Soils for Sustainable Agriculture�� 323
Sanjay Arora
About the Editors
Manoj Kumar is working as Associate Professor and

Head of in the Department of Life Sciences, Central
University of Jharkhand, Ranchi, India. He is a scientist
with sanguine behavior who is adoring about research and
development, with a commitment to lifelong learning. Dr.
Kumar has pursued his PhD in Plant Biotechnology and
continuing his career in multidisciplinary research in
Plant Developmental Biology, Plant-Microbe Interaction
and Forest Molecular Genetics. He is a referee for many
journals, including Phytoremediation, Journal of Soil
Sediments etc. He has guided several research scholars at
doctoral and masters level, also he is leading a multidisci-
plinary research group comprised of diverse research
background in biological sciences.
Vivek Kumar is an agricultural microbiologist hav-

ing 20 Years of experiences in teaching, research and
guidance, with a pledge to enduring knowledge. Dr.
Kumar is working as Associate Professor Department
of Microbiology, Himalayan School of Biosciences,
Swami Rama Himalayan University, India.
He is serving in Editorial board of reputed interna-
tional journals and also a reviewer of peer journals. He
has published 61 research papers, 19 book chapters, six
review articles and two books. Dr. Kumar has also
served as Microbiologist for eight years in Department
of Soil and Water Research, Public Authority of
Agricultural Affairs & Fish Resources, Kuwait.
ix
x About the Editors
Ram Prasad is an Assistant Professor at the Amity

Institute of Microbial Technology, Amity University,
Uttar Pradesh, India. His research interests include
plant-microbe interactions, sustainable agriculture, and
microbial nanobiotechnology. Dr. Prasad has published
more than a hundred research papers, reviews, and book
chapters and has five patents issued or pending and
edited or authored several books. Dr. Prasad has 12
years of teaching experience and was awarded the
Young Scientist Award (2007) and Prof. J.S. Datta
Munshi Gold Medal (2009) by the International Society
for Ecological Communications, FSAB Fellowship
(2010) by the Society for Applied Biotechnology,
Outstanding Scientist Award (2015) in the field of
Microbiology by Venus International Foundation,
BRICPL Science Investigator Award (2017), Award for
Excellence in Research from 6th Academic Brilliance
Awards-2018, and the American Cancer Society UICC
International Fellowship for Beginning Investigators
(USA, 2014). In 2014–2015, Dr. Prasad served as a
Visiting Assistant Professor at the Department of
Mechanical Engineering at Johns Hopkins University,
USA, and since 2017 has served as a Research Associate
Professor at the School of Environmental Science and
Engineering, Sun Yat-Sen University, China.
Phytostimulation and Biocontrol
by the Plant-Associated Bacillus 1
amyloliquefaciens FZB42: An Update
Rainer Borriss
Abstract
Bacillus amyloliquefaciens FZB42, the type strain for representatives of the
plant-associated subspecies plantarum, stimulates plant growth and suppresses
soil-borne plant pathogens. The strain has been sequenced in 2007. The B. amy-
loliquefaciens FZB42 genome reveals an unexpected potential to produce sec-
ondary metabolites. In total, 11 gene clusters representing nearly 10% of the
genome are devoted to synthesizing antimicrobial metabolites and/or to confer
immunity against them. Ability to synthesize nonribosomally, the antibacterial
polyketides macrolactin and difficidin and the antifungal lipopeptide bacillomy-
cin D is an unique feature of the subspecies plantarum. However, according to
latest research, most of the secondary metabolites are not expressed in plant rhi-
zosphere suggesting that the antibiome expressed during the plant-associated
state of PGPR Bacilli does not reflect the vast genetic arsenal devoted to the
formation of secondary metabolites. There is now strong evidence that plant-
associated Bacilli trigger pathways of induced systemic resistance, which protect
plants against attacks of pathogenic microbes, viruses, and nematodes.
Keywords
Bacillus amyloliquefaciens · Macrolactin · Antimicrobial–metabolites ·
plantarum
R. Borriss (*)
Humboldt-Universität zu Berlin, Institut für Biologie, Berlin, Germany
Nord Reet UG, Greifswald, Germany
e-mail: [email protected]
© Springer Nature Singapore Pte Ltd. 2020 1

M. Kumar et al. (eds.), Phyto-Microbiome in Stress Regulation, Environmental
and Microbial Biotechnology, https://doi.org/10.1007/978-981-15-2576-6_1
2 R. Borriss
1.1 Introduction
Environmental-friendly biotechnological approaches, such as the use of microbial

biopesticides, offer alternatives to chemical control of plant diseases and pests.
Among these alternatives, the use of bioformulations, which are manufactured from
plant growth-promoting rhizobacteria (PGPR) with biocontrol activity (BC)
(Lugtenberg et al. 2013), is steadily increasing. At present, due to the long-term
shelf life of their endospores, bacilli are the most widely used bacteria on the biopes-
ticide market. Their use in agriculture has been previously reviewed (Borriss 2011).
An update of Bacillus-based bioformulations, currently available for the farmer
interested on sustainable agriculture, is presented in Table 1.1.
Plant rhizosphere is a highly competitive environment in which bacteria are
abundantly present due to availability of nutrients actively secreted by the plant root
and mucilage. Some of these bacteria which are living within or in the vicinity of
plant roots and supporting plant growth are generally referred to as PGPR (Kloepper
et al. 1980). In many cases, the plant growth-promoting activity is linked with their
ability to suppress soil-borne plant pathogens (bacteria and microfungi), occurring
in the competing microflora. Different mechanisms are discussed in this context.
Besides production of antimicrobial (“antibiotics”) and nematicidal compounds,
also stimulation of plant-induced systemic resistance (ISR, Doornbos et al. 2012),
and a beneficial effect on the composition of the host-plant microbiome might con-
tribute to their suppressive effect (Erlacher et al. 2014). In other PGPR, termed
“biofertilizer”, plant growth promotion by hormone-like compounds and increased
accessibility of nutrients dominates. The mechanisms that are involved in this pro-
cess can include nitrogen fixation, phosphate and mineral solubilization, and the
production of macromolecule degrading enzymes (amylases, proteases, hemicellu-
lases), phytohormones (auxin, cytokinin, and gibberellins), and volatile growth
stimulants (such as acetoin and 2,3 butanediol) (Borriss 2011).
Bacillus amyloliquefaciens FZB42 is the type strain for a group of plant-
associated Bacillus spp. classified as B. amyloliquefaciens subsp. plantarum
(Borriss et al. 2011). Its 3918-kb genome, containing an estimated 3693 protein-
coding sequences, lacks extended phage insertions, which occur ubiquitously in the
closely related but nonplant-associated Bacillus subtilis 168 genome. Further analy-
sis revealed that FZB42 is a bacterium with impressive capacity to produce metabo-
lites with antimicrobial activity (Chen et al. 2007). Its antifungal activity is due to
nonribosomal synthesis of the cyclic lipopeptides bacillomycin D and fengycin
(Koumoutsi et al. 2004), whilst its antibacterial activity is mainly due to nonribo-
somally synthesized polyketides (Chen et al. 2006), bacilysin (Chen et al. 2009a),
and ribosomally synthesized bacteriocins (Scholz et al. 2011, 2014). Recent pro-
teome and transcriptome studies revealed that plant root exudates stimulate expres-
sion of genes involved in root colonization and plant–bacteria interactions (Borriss
2015a, b; Fan et al. 2012a, b, 2015; Kierul et al. 2015). Its plant colonizing ability
was demonstrated with a GFP-labeled FZB42 strain on maize and Arabidopsis
using confocal laser scanning microscopy (Fan et al. 2011). Beneficial effects on
plant growth and disease suppression were documented for B. amyloliquefaciens
1 Phytostimulation and Biocontrol by the Plant-Associated Bacillus 3
Table 1.1 Examples for commercial use of Bacillus-based bioformulations in agriculture

Trade name Bacillus strain Known properties Company
Kodiak™ Bacillus subtilis EPA-registered (71065–2) biological Bayer Crop
GB03 and seed treatment fungicide Science, former
Gustafsson
LLC
Companion Bacillus subtilis EPA-registered (71065–2) Growth
GB03 biofungicide, prevent and control Products Ltd.,
plant diseases. It produces a White Plains,
broad-spectrum Iturin antibiotic that NY 10603
disrupts the cell-wall formation of
pathogens, and it triggers an
advantageous induced systemic
resistance (ISR) in plants, whereby a
plant’s natural immune system is
activated to fight plant diseases
Yield Shield Bacillus pumilus EPA-registered biofungicide Bayer Crop
GB34 (=INR7) (264–985), suppression of root Science,
diseases caused by Rhizoctonia and previously
Fusarium Gustafsson
BioYield™ B. Combination of strong ISR activity Bayer Crop
amyloliquefaciens (GB99) with phytostimulaton Science,
GB99 + Bacillus (GB122) previously
subtilis GB122 Gustafsson
Subtilex®, Bacillus subtilis EPA-registered (71840–8.) Becker
INTEGRAL® MBI600 biofungicide, provides protection Underwood,
against soil-borne pathogens such as Saskatoon,
Rhizoctonia solani, Pythium spp. and Canada
Fusarium spp. to help prevent acquired by
damping-off and other root diseases BASF
VAULT® Bacillus subtilis Produced by “BioStacked®” Becker
MBI600 technology, enhancing growth of soy Underwood,
beans and pea nuts Saskatoon,
Canada
Bacillus pumilus EPA-registered (71840-RG, −RE, Becker
BU F-33 2013) plant growth stimulator, Underwood,
induced systemic resistance Saskatoon,
Canada
SERENADE Bacillus subtilis EPA-registered (69592–11) Bayer Crop
Max QST713 biofungicide, Annex 1 listing of the Science,
EU agrochemical registration previously
directive (91/414) AgraQuest
SERENADE Bacillus subtilis EPA-registered (69592-EI, 2012) Bayer Crop
SOIL(R) QST713 biofungicide for food crops Science,
previously
AgraQuest
(continued)
4 R. Borriss
Table 1.1 (continued)
SERENADE Bacillus subtilis EPA-registered (2013) biofungicide/ Bayer Crop
Optimum® QST713 bactericide for prevention. It works Science,
by stopping spore germination, previously
disrupting cell membrane, and AgraQuest
inhibiting attachment of the
pathogen to leaves. For use in leafy
and fruiting vegetables, strawberries
and potatoes. Active against fungal
(Botrytis, Sclerotinia), and bacterial
pathogens (Xanthomonas and
Erwinia)
CEASE® Bacillus subtilis Aqueous suspension biofungicide, BioWorks, Inc.,
QST713 recommended for leafy and fruiting Victor,
vegetables, herbs and spices, and New York,
ornamentals USA
SONATA® Bacillus pumilus EPA-registered (69592–13) Bayer Crop
QST2808 biofungicide, powdery mildew Science,
control previously
AgraQuest Inc.
RhizoVital® Bacillus Biofertilizer, plant growth promoting ABiTEP
amyloliquefaciens activity, provides protection against GmbH, Berlin
FZB42 various soil-borne diseases,
stimulation of ISR
RhizoPlus® Bacillus subtilis Plant growth-promoting ABiTEP
rhizobacterium and biocontrol agent. GmbH, Berlin
It can be used for potatoes, corn,
vegetables, fruits, and also turf
Taegro® Bacillus subtilis EPA-registered biofungicide. FZB24 Syngenta,
FZB24 has been originally isolated by FZB Basel,
Berlin, the forerunner of ABiTEP previously
GmbH. Registration as a Novozyme,
biofungicide for the United States Davis,
was performed by Taegro Inc. and California and
then sold to Novozymes without Earth
agreement with ABiTEP GmbH Biosciences
where the product is still offered
POMEX Bacillus subtilis Microbial fungicide, control and NIN Co. Ltd.,
CMB26 inhibition germination effect on
powdery mildew, Cladosporium
fulvum and Botrytis cinerea
Bacillus subtilis EPA-registered 71840-RG-RE Certis
CX9060 (2012) fungicide, bactericide for Columbia, MD
food crops, turf and ornamentals USA
(continued)
Easy Start® Bacillus subtilis Rhizosphere bacterium that COMPO
TE-Max E4-CDX competes with harmful pathogens Expert GmbH,
for space around the roots of the Münster,
grass plant. Once established, this Germany
unique strain physically protects the
roots and inhibits the advance of
soil-borne fungi
Double Nickel B. EPA-registered (70051-RNI, 2011) a Certis
55™ amyloliquefaciens broad spectrum preventive biofungi- Columbia, MD
D747 cide for control or suppression of USA
fungal and bacterial plant diseases
(Powdery mildew, Sclerotinia,
Botrytis, Alternaria, bacterial leaf
spot, bacterial spot and speck, Fire
blight, Xanthomonas, Monilinia
Amylo-X® B. Annex 1 listing of the EU Certis
amyloliquefaciens agrochemical registration directive. Columbia, MD
D747 Launched in Italy by Intrachem Bio USA/Intrachem
Italia SpA for control of Botrytis and Bio Italia SpA
other fungal diseases of grapes,
strawberries, and vegetables, and
bacterial diseases such as fire blight
in pome fruit and PSA in kiwi fruit
BmJ WG Bacillus mycoides It works entirely as a microbial SAR Certis
BmJ activator with no direct effect on the Columbia, MD
plant pathogen itself. Under USA
development
Bacillus pumilus EPA-registered fungicide (2012), Premier
GHA 181 food crops, seeds, ground cover, and Horticulture
ornamentals
BioNem Bacillus firmus EPA-registered (2008), suppressing AgroGreen,
GB-126 plant pathogenic nematodes, Israel acquired
Bacillus firmus creates a living by Bayer Crop
barrier that prevents nematodes from Science
reaching the roots
Note: The U.S. governmental EPA registration does not depend on successful field trials; it is only
necessary to demonstrate that no negative effects are connected with the use of the biofungicide.
The table is taken from Borriss (2015b)
FZB42 on tomato, cucumber, cotton, tobacco, and lettuce, for example (Grosch
et al. 1999; Idriss et al. 2004; Yao et al. 2006; Guel et al. 2008; Wang et al. 2009;
Chowdhury et al. 2013). Two review articles published in open access journals in
2015 (Chowdhury et al. 2015b; Wu et al. 2015b) cover the aspects stressed in this
contribution in more detail and are recommended for further reading.
6 R. Borriss
1.2 oot Colonization by FZB42 and Its Impact on the Host

R
Plant Microbiome
The ability of FZB42 to colonize the rhizoplane is a precondition for plant growth
promotion. Using a GFP-tagged derivative (Fan et al. 2011, 2012a, b), the fate of
bacterial root colonization was recently studied. It ruled out that the bacterium
behaves distinctly in colonizing root surfaces of different plants. In contrast to
maize, FZB42 colonized preferentially root tips when colonizing Arabidopsis thali-
ana (Dietel et al. 2013). On duckweed, Lemna minor, FZB42 accumulated prefer-
ably along the grooves between epidermal cells of roots and in the concave spaces
on ventral sides of fronds. In vitro studies performed with maize seedlings revealed
that the segment within 2–8 cm distant from the basal site of the primary root was a
most colonized region by FZB42. On the contrary, few bacterial cells could be
observed within the range of 2 cm of root tip. In general, the green fluorescent
FZB42 cells were decreasingly observed from the upper part of a root down to the
root tip. Scanning electron microscopy confirmed the presence of FZB42 on root
hairs, where the bacterial cells were usually associated with a wealth of presumed
root exudates (Fan et al. 2012a, b). In lettuce, Lactuca sativa, seedlings, bacterial
colonization occurred mainly on primary roots and root hairs, as well as on root tips
and adjacent border cells. Occurrence of labeled bacteria decreased towards the root
tips of the lateral roots, and no colonization of the finer roots could be observed
(Chowdhury et al. 2015a).
The rhizosphere competence of FZB42 was recently studied using a combination
of field and greenhouse trials. FZB42 is able to effectively colonize the rhizosphere
(7.45 to 6.61 Log 10 CFU g−1 root dry mass) within the growth period of lettuce in
the field. Our results demonstrated that FZB42 is able to effectively reduce the dis-
ease severity of bottom rot caused by soil-borne pathogen Rhizoctonia solani on
lettuce (Chowdhury et al. 2013).
From a practical point of view, it is interesting to note that the application mode
of the biocontrol agent is a key factor for efficacy of FZB42. An effective suppres-
sion of R. solani was found only after two times application of FZB42, before and
after transplanting. For the settlement of the inoculated strain in the rhizosphere in
a sufficient high number, it might be important that the microflora in the rhizosphere
of young plants is not yet stabilized (Berendsen et al. 2012).
As revealed by T-RFLP, application of FZB42, independent of its mode of
application, did not shift the composition of rhizosphere bacterial community in a
measurable extent—as also shown for B. amyloliquefaciens BNM122 on soybean
(Correa et al. 2009). By contrast, inoculation with the pathogen did change the rhi-
zosphere microbial community structure. In complementing that study, the effect of
FZB42 and the pathogen R. solani on the microbial community of lettuce was more
deeply analyzed by 454-amplicon sequencing focusing on the presence of gamma-
proteobacteria (Erlacher et al. 2014). Clear differences between plants infected by
R. solani compared to noninoculated healthy plants were found, corroborating the
results obtained by T-RFLP. A significant increase in gamma-proteobacterial diver-
sity was detected in samples inoculated with the pathogen. However, together with
FZB42, this increase was less distinct, suggesting a selective compensation of the
impact of a pathogen on the indigenous plant-associated microbiome by FZB42.
The number of DNA fragments corresponding to FZB42 in samples taken in vicin-
ity of plant roots was steadily decreasing. After five weeks, only 55% of the initial
number of FZB42 DNA was traceable (Kröber et al. 2014).
1.3 Plant Growth Promotion
Although the ability of FZB42 to support growth of potatoes, maize, cotton, tobacco,
leafy and fruiting vegetables, and ornamentals is well documented (Bochow et al.
2001; Yao et al. 2006; Guel et al. 2008; Burkett-Cadena et al. 2008; Chowdhury et al.
2013), the molecular reasons for the “biofertilizer” effect of beneficial plant-associated
Bacilli are still not completely understood. However, we know that several factors are
involved in the complex interplay between root-colonizing bacteria and plant:
1. Ability to colonize and to persist at plant roots (see previous section). Their abil-
ity to suppress soil-borne pathogens might positively affect the indigenous
microbiome of the rhizosphere.
2. Stimulation of plant growth by tryptophan-dependent synthesis of indole-3-
acetic acid. Inactivation of genes involved in tryptophan biosynthesis and in a
putative tryptophan-dependent IAA biosynthesis pathway led to reduction of
both IAA concentration and plant growth-promoting activity in the respective
mutant strains (Idris et al. 2007).
3. Volatiles, as 2,3-butanediol and 3-hydroxy-2-butanone (acetoin), released by
Bacillus subtilis GB03 and Bacillus amyloliquefaciens IN937a were reported as
enhancing plant growth (Ryu et al. 2003). To synthesize 2,3-butanediol, pyruvate
is converted to acetolactate by the acetolactate synthase (AlsS), which is subse-
quently converted to acetoin by the acetolactate decarboxylase (AlsD) (Fig. 1.1).
FZB42 mutant strains, deficient in synthesis of volatiles due to mutations
interrupting the alsD and alsS genes, were found impaired in plant growth pro-
motion (Borriss 2011).
Fig. 1.1 Anaerobic and aerobic formation of 2,3-butanediol via acetoin involves acetolactate syn-
thase and decarboxylase encoded by the alsSD operon. The alsS insertion mutation abolishes syn-
thesis of 2,3-butandiol (Renna et al. 1993; Cruz Ramos et al. 2000). The figure is taken from
Chowdhury et al. (2015b)
8 R. Borriss
4. Enhancement of nutrient availability by phytase-producing bacteria. Soil phos-

phorous is an important macronutrient for plants. Improved phosphorous nutri-
tion is achievable by “mobilization” of phosphorous fixed as insoluble organic
phosphate in phytate (myo-inositol-hexakisphosphate) by soil bacteria (Singh
and Satyanarayana 2011). The extracellular 3(1)-phytase of the plant growth-
promoting B. amyloliquefaciens FZB45 hydrolyzed phytate to D/L-Ins(1,2,4,5,6)
P5 in vitro. A phytase-negative mutant strain, whose phyA gene was disrupted,
did not stimulate plant growth under phosphate limitation (Idriss et al. 2002).
Further experiments under field conditions revealed that FZB45 can only stimu-
late plant growth when phytate is present in soils, which are poor in soluble
phosphate, suggesting that phytase acts only under certain conditions as a plant
growth stimulator (Ramírez and Kloepper 2010).
1.4 Biocontrol
Genome analysis revealed that nearly 10% of the genome is devoted to synthesizing
antimicrobial metabolites and their corresponding immunity genes (Chen et al.
2009b). FZB42 harbors 11 gene clusters involved in synthesis of antimicrobial com-
pounds. Nine of them are involved in nonribosomal synthesis of lipopeptides and
polyketides and two in conventional synthesis and modification of bacteriocin pep-
tides. In addition, three further gene clusters contain genes mediating immunity
against antimicrobial compounds produced by other related Bacillus strains
(Table 1.2). This antibiotic arsenal makes B. amyloliquefaciens FZB42 and related
B. amyloliquefaciens plantarum strains to an efficient microbial biopesticides,
developed to control plant diseases (Borriss 2011).
For a long time, the plant protective activity of PGPR has been correlated with
the potential to secrete a wide array of antibiotic compounds upon growth as plank-
tonic cells in isolated cultures under laboratory conditions. We determined expres-
sion of the corresponding secondary metabolites by MALDI TOF mass spectrometry
from FZB42 cultures grown in liquid Landy medium under laboratory conditions.
Except the orphan nrs gene cluster, all expected bioactive compounds were synthe-
sized in reasonable amounts, but the iron siderophore bacillibactin was detected
only under iron-deprived conditions. In recent years, it became doubtful that synthe-
sis of metabolites by the planktonic cells grown under laboratory conditions does
correspond to their capability to produce those compounds also when grown in
biofilm-related structures on the surface of plant tissues.
1.4.1 Lipopeptides, Bacillibactin, and Antifungal Activity
Five gene cluster involved in nonribosomal synthesis of cyclic lipopeptides and the
iron-siderophore bacillibactin were identified in the genome of FZB42 (Table 1.2).
Three of the respective gene clusters were assigned for synthesis of surfactin,
Table 1.2 Genes and gene cluster encoding for secondary metabolites and immunity against bacteriocin in FZB42
Gene cluster From To Size Metabolite Effect against Reference
Sfp-dependent nonribosomal synthesis of lipopeptides
srfABCD, aat,334,ycx,CycxD,sfp,yczE 342,618 368,776 32.0 kb Surfactin Virus Koumoutsi et al. (2004)
bmyCBAD 1,871,172 1,908,422 39.7 kb Bacillomycin D Fungi Koumoutsi et al. (2004)
fenABCDE 1,931,328 1,968,997 38.2 kb Fengycin Fungi Koumoutsi et al. (2004)
nrsABCDEF 2,868,410 2,885,927 15.0 kb Orphan NRP1 Unknown, siderophore? Chen et al. (2007)
dhbABCDEF 3,021,250 3,032,970 12.8 kb Bacillibactin Iron deficiency, siderophore Chen et al. (2007)
Sfp-dependent nonribosomal synthesis of polyketides
mlnABCDEFGHI 1,391,841 1,445,094 53.9 kb Macrolactin Bacteria Schneider et al. (2007)
baeBCDE,acpK,baeGHIJLMNRS 1,700,345 1,701,022 74.3 kb Bacillaene Bacteria Chen et al. (2006)
dfnAYXBCDEFGHIJKLM 2,276,743 2,346,266 71.1 kb Difficidin Bacteria Chen et al. (2006)
Sfp-independent nonribosomal synthesis
bacABCDE,ywfG 3,593,877 3,599,784 6.9 kb Bacilysin Bacteria Chen et al. (2009a, b, c)
Ribosomal synthesis of modified peptides (bacteriocins)
pznFKGHIAJCDBEL 726,469 736,360 9.96 kb Plantazolicin Gram-positive bacteria Scholz et al. (2011)
acnBACDEF 3,044,506 3,048,678 4.49 kb Amylocyclicin Closely related bacteria Scholz et al. (2014)
1 Phytostimulation and Biocontrol by the Plant-Associated Bacillus
Immunity, but no synthesis genes

mrsK2R2FGE 3,769,734 3,774,552 4.82 kb Mersacidin Resistance against Y2 He et al. (2012)
bceBASR 2,856,835 2,861,322 4.49 kb Bacitracin Resistance against B. cereus Unpubl. Results
spaKREF 3,210,423 3,214,712 4.29 kb Subtilin Resistance against B. subtilis Unpubl. Results
The table is taken from Chowdhury et al. (2015b) with modifications
9
10 R. Borriss
Fig. 1.2 Effect of FZB42 on Rhizoctonia solani. A clear inhibition zone indicating growth sup-
pression of the fungal pathogen is visible on agar plates simultaneously inoculated with both
microbes. Bacillomycin D was detected as the only prominent compound by MALDI TOF mass
spectrometry of samples taken from the surface of the agar plate within the inhibition zone. The
figure is taken from Chowdhury et al. (2015b) with slight modifications
fengycin, and bacillomycin D. Bacillomycin D was identified as being the most
powerful antifungal metabolite produced by FZB42 (Fig. 1.2).
The heptapeptide moiety of bacillomycinD, belonging to the iturin family of
cyclic lipopeptides (LP), is attached to a β-amino fatty acid chain of variable length
(C14–C17). The peptide moiety of the heptapeptide surfactin is linked to a β-hydroxyl
fatty acid (C12–C16), whilst the fengycin decapeptides are linked to a β–hydroxyl
fatty acid chain (C 14–C18). Their synthesis is performed by multimodular peptide
synthetases and depends on a functional phospho-pantheinyl transferase (Sfp),
which transfers 4′- phosphopanthetheine from coenzyme A to the carrier proteins
during nonribosomal synthesis.
Within last few years, Ongena and coworkers performed pioneering work for
elucidating antibiotic production in planta using Matrix-Assisted Laser Desorption/
Ionization Mass Spectrometry Imaging (MALDI MSI). They investigated antibiotic
production in a gnotobiotic system in which the plantlet and the associated B. amy-
loliquefaciens S499, a close relative of FZB42, were growing on a gelified medium
covering the MALDI target plate. Under these conditions, S499 grows as biofilm on
the surface of the plant roots, allowing exact assays of secondary metabolites in the
vicinity of root surface. Surfactins were detected in the root environment in much
higher relative amounts, which are representing more than 90% of the whole LP
production, and their synthesis is rapidly progressing during early biofilm forma-
tion. By contrast, synthesis of iturin and fengycin was delayed until the end of the
aggressive phase of colonization (Nihorimbere et al. 2012; Debois et al. 2014).
Earlier experiments performed with FZB42 colonizing duckweed (Lemna minor)
plantlets corroborated that surfactin is the most prominent compound which could
be detected by MALDI TOF MS in the plant-bacteria system (Idris et al. 2007).
Using a gnotobiotic quartz sand system consisting of lettuce plants, the beneficial
bacterium FZB42, and the pathogen R. solani, it was demonstrated by using alterna-
tive techniques (e.g., Fourier Transform Ionen-Cyclotron Massenspectrometry) that
lipopeptides were detectable in the order surfactin > bacillomycinD > fengycin at
the plant–bacteria interface (Chowdhury et al. 2015a).
An early surfactin secretion could be of biological relevance since this lipopep-
tide, although less fungitoxic then iturins and fengycins, is essential for moving on
tissues (Kinsinger et al. 2003) and for matrix formation in biofilms (Hofemeister
et al. 2004; Lopez et al. 2009a, b). Considering the relative low amounts of the fun-
gitoxic iturins and fengycins in vicinity of plant roots, it might be concluded that
their biocontrol effect is possibly less important than expected. The same is true for
the iron siderophore bacillibactin, which could not be detected under the conditions
of the artificial plant–bacteria associations applied in these studies.
1.4.1.1 Polyketides, Bacilysin, and Bacteriocins Direct Antibacterial

Activity
The polyketides, nonribosomally synthesized by FZB42 (Chen et al. 2006;
Schneider et al. 2007), have been extensively reviewed previously (Chen et al.
2009b, 2009c; Borriss 2013). The three gene clusters encoding the modularly orga-
nized polyketide synthases (PKS) for synthesis bacillaene, macrolactin, and diffici-
din cover nearly 200 kb and are the largest ones, which are occurring in the FZB42
genome (Table 1.2). Difficidin is the most effective antibacterial compound pro-
duced by FZB42T, but also macrolactin and bacillaene possess antibacterial activity.
Difficidin is efficient in suppressing plant pathogenic bacterium Erwinia amylov-
ora, which causes fire blight disease in orchard trees (Chen et al. 2009a).
Another product of nonribosomal synthesis, the dipeptide bacilysin consisting of
anticapsin and alanine moieties, was found to be also involved in suppression of
Erwinia amylovora. By contrast to the lipopeptides and polyketides mentioned
above, bacilysin synthesis is not dependent on the Sfp PP-transferase. A mutant
strain CH3, with a disruption of the sfp gene and unable to produce any polyketide
or lipopeptide, was still able to synthesize bacilysin and to suppress E. amylovora
(Chen et al. 2009a). Recent experiments, performed by the group of Xuewen Gao,
Nanjing Agriculture University, demonstrated that bacilysin, despite difficidin, is
efficient in suppressing Microcystis aeruginosa: the main causative agent of cyano-
bacterial bloom in lakes and rivers (Wu et al. 2015a). However, corroborating these
results in field trials has to be done. Until now, polyketides and bacilysin have not
been detected in plants colonized by B. amyloliquefaciens (Debois et al. 2014).
Antimicrobial peptides, ribosomally synthesized as linear precursor peptides,
remained unknown in B. amyloliquefaciens plantarum for a long time with one
remarkable exception: mersacidin, a B-type lantibiotic, was detected in Bacillus sp.
HIL Y85 (Chatterjee et al. 1992). The strain HIL Y85 was later classified as being
B. amyloliquefaciens plantarum (Herzner et al. 2011). Nowadays, mersacidin pro-
duction was also detected in B. amyloliquefaciens B9601-Y2 (He et al. 2012).
Genes involved in mersacidin self-protection reside also in the genome of FZB42.
Transfer of mersacidin biosynthesis genes from HIL Y85 resulted in efficient mer-
sacidin production by the surrogate strain constructed from the FZB42 host (Herzner
et al. 2011).
12 R. Borriss
Another representative of the type B lantibiotics, amylolysin from B.

amyloliquefa-ciens GA1, was recently described. These lantibiotics are active on an
array of Gram-positive bacteria, including Listeria spp. and methicillin-resistant S.
aureus by interacting with the membrane lipid II (Arguelles Arias et al. 2013).
The driving force in our search for ribosomally synthesized peptides in FZB42
was the finding that the FZB42 mutant RS06, which is deficient in the Sfp-dependent
synthesis of lipopeptides, polyketides, and in the Sfp-independent bacilysin produc-
tion (Chen et al. 2009a), still produced an antibacterial substance active against
Bacillus subtilis HB0042. In fact, a metabolite (cpd1335) with a molecular mass of
[M + H]+ = 1336 Da was assigned by MALDI TOF MS in FZB42 and in RS06, as
well. The compound was named plantazolicin, and the respective gene cluster pzn
consisting of 12 genes was identified by cassette mutagenesis. Plantazolicin was
characterized as a highly modified peptide undergoing several steps of modification
after synthesis. It ruled out that it is a thiazole/oxazole-modified microcin (TOMM)
resembling microcin B17 and streptolysin S. Plantazolicin displayed antibacterial
activity toward closely related gram-positive bacteria. Due to its extensive degree of
modification, Pzn is highly protected from premature degradation by peptidases
within the plant rhizosphere (Scholz et al. 2011). Remarkably, human pathogen
Bacillus anthracis was found sensitive against PZN and underwent massive lysis at
4 μg mL−1 (Molohon et al. 2011). The exact structures of plantazolicin A and B were
Fig. 1.3 The structure of the mature bacteriocin amylocyclicin bearing a head-to-tail cyclization
of L1 and W64. Amylocyclicin effect on a related B. subtilis strain without immunity against the
bacteriocin was demonstrated by a spot-on-lawn test performed with an amylocyclicin-producing
(top) and -nonproducing strain (bottom). The figure is taken from Chowdhury et al. (2015b)
elucidated, unveiling a hitherto unusual number of thiazoles and oxazoles formed

from a linear 14mer precursor peptide (Kalyon et al. 2011).
By transposon mutagenesis of the FZB42 mutant strain RS06, we identified a
hitherto unknown gene cluster involved in synthesis and post-translational process-
ing of a novel circular bacteriocin, named amylocyclicin (Fig. 1.3). It ruled out that
amylocyclicin inhibits growth of bacterial strains closely related to FZB42, suggest-
ing that this bacteriocin might have a function in competing with other Bacillus
strains attracted to the plant rhizosphere (Scholz et al. 2014).
1.4.1.2 Nematicidal Activity Is Directed by Plantazolicin

Parasitic nematodes of plants are important plant pathogens that represent a signifi-
cant financial burden on agriculture. The annual losses in agriculture resulting from
this pest amounted to $125 billion worldwide in past years (Sasser and Freckman
1987; Oka et al. 2000). Chemical insecticides of nonselective nature possessing
rapid nematicidal effects are widely used as control measures against these patho-
gens. However, the potential negative impact on the environment and ineffective-
ness after prolonged use have led to banning or restricting of the use of most
chemical nematicides. Therefore, identification of safe and effective nematicides is
urgently needed, and biocontrol measures have recently been given much attention
as viable options (Xia et al. 2011). BioNem® prepared from Bacillus firmus GB-126
(Table 1.1) was proven for its efficiency in greenhouse and field trials. The numbers
of nematode females, eggs, and vermiform life stages at the end of the growing
season decreased in the presence of the biocontrol agent, and the cotton yields were
similar to those from Aldicarb, the chemical nematicide standard; however, the
molecular reason for this effect remained unknown (Castillo et al. 2013).
FZB42 has been shown to reduce nematode eggs in roots, juvenile worms in soil,
and plant galls on tomato (Burkett-Cadena et al. 2008). In order to identify specific
nematicide-related genes, a random transposon insertion library of FZB42 was
screened for relevant genes involved in nematicidal activity and, surprisingly, a gene
within the pzn gene cluster was identified as a pathogenic factor against nematodes.
Further experiments revealed that PZN displayed a moderate nematicidal activity
(Liu et al. 2013).
1.5 I nduced Systemic Resistance Is Triggered by Plant

Growth-Promoting Bacilli
Except surfactin, concentration of antifungal lipopeptides determined in planta was

found relatively low. Moreover, antibacterial polyketides were not detected so far in
vicinity of plant roots colonized by PGPR Bacilli (Debois et al. 2014). Therefore, it
is tempting to speculate that induced systemic resistance (ISR) is a main factor for
suppressing plant pathogens by PGPR Bacilli. ISR occurs when the plant’s defense
mechanisms are stimulated and primed to resist infection by pathogens (Van Loon
1997). This activation is distinct from systemic acquired resistance (SAR) in which
the response is triggered by pathogenic microorganisms associated with the aerial
14 R. Borriss
portions of the plant. Selected Bacillus PGPR strains emit volatiles (VOCs) that can
elicit plant defenses. Exposure to VOCs consisting of 2,3-butanediol and acetoin
(3-hydroxy-2-butanone) from PGPR Bacillus amyloliquefaciens activates ISR in
Arabidopsis seedlings (Ryu et al. 2004). Arabidopsis thaliana plants exposed to
Bacillus subtilis strain FB17 results in reduced disease severity against Pseudomonas
syringae compared to plants without FB17 treatment. Exogenous application of
acetoin triggers ISR and protects plants against the pathogen in the aerial parts
whilst 2,3-butanediol did not (Rudrappa et al. 2010). In this context, it is worth to
mention that expression of AlsS of FZB42 involved in synthesis of acetoin (Fig. 1.1)
was triggered in the presence of maize root exudate (Kierul et al. unpublished), sug-
gesting that root exudates play a role in eliciting of acetoin biosynthesis in FZB42.
It is known that some of the plant metabolites present in root exudates, such as
organic acids, trigger the alsSD operon (Rudrappa et al. 2010). B. amyloliquefa-
ciens FZB24 and FZB42 applied to tobacco roots led to a reduction of tobacco
mosaic virus symptoms visible on tobacco leaves and led to decreasing amounts of
virus proteins present in leaf tissues. Due to spatial distance between beneficial
bacterium and the pathogen, plant ISR, stimulated by the rhizobacterium, might be
responsible for this effect (Wang et al. 2009).
The induction of ISR when treated with PGPRs is mediated primarily through
plant-signaling molecules such as jasmonic acid (JA), a lipoxygenase pathway
product, and ethylene (ET). Salicylic acid (SA) appears to be a critical plant mes-
senger of pathogen exposure and disease resistance in systemic acquired resistance
(SAR) (Durner et al. 1997). ISR restricts pathogen multiplication and disease pro-
gression through a SA/ET and NPR1-dependent mechanism. In order to determine
the signaling pathways triggered by FZB42, the expression of several marker genes
in lettuce plants, exposed to FZB42 and the pathogenic fungus Rhizoctonia solani,
was analyzed by quantitative real-time (RT)-PCR (S. Paul Chowdhury et al.:
´Systemic resistance of Lactuca sativa against R. solani and secondary metabolite
production by FZB42 in an axenic model system´, unpublished). In absence of the
pathogen, FZB42 increased expression of PR1 (pathogenesis protein 1, SA marker
gene) and PDF1.2 (defensin, JA/ET marker gene), suggesting that SA and ET path-
ways are involved in upregulating defense response by ISR in lettuce. A similar
result was obtained previously, when Arabidopsis plantlets were inoculated with
Bacillus subtilis FB17 and acetoin (Rudrappa et al. 2010). In simultaneous presence
of FZB42 and the pathogen R. solani, PDF1.2 expression was dramatically
enhanced, suggesting a synergistic activation of the JA/ET pathway, whilst the SA
pathway—as indicated by a decreased expression of PR-1—was suppressed in pres-
ence of both antagonists.
It was found that the circular lipopeptides surfactin and fengycin can act as elici-
tors of host plant immunity and contribute to increased resistance toward further
pathogenesis ingress in bean and tomato plants (Raaijmakers et al. 2010). In bean,
purified fengycins and surfactins provided a significant ISR-mediated protective
effect on bean plants against the fungal pathogen Botrytis cinerea, similar to the one
induced by living cells of the producing strain B. amyloliquefaciens S499 (Ongena
et al. 2007).
We found (Chowdhury et al. 2015a) that the dramatic increase of the defensin 1.2
gene (PDF1.2) expression in simultaneous presence of both antagonists occurred
only when wild-type cells of FZB42 were applied. Mutant strains deficient in syn-
thesis of surfactin, fengycin, or acetoin did not stimulate expression of the JA/ET
pathway, suggesting that cyclic lipopeptides and acetoin contribute together to the
ISR plant response triggered by FZB42.
1.6 Conclusion
An increasing amount of data have been accumulated in course of the last years,
suggesting that the antibiome expressed during the plant-associated state of PGPR
Bacilli does not necessarily reflect the vast genetic arsenal devoted to the formation
of lipopeptides, polyketides, and bacteriocins, which has been elucidated, for exam-
ple, in the B. amyloliquefaciens plantarum FZB42 genome. Obviously, there is a
large discrepancy in gene expression of the planktonic cells growing in liquid labo-
ratory cultures and cells growing as biofilms on plant tissue surfaces. Except cyclic
lipopeptides no other bioactive compounds such as polyketides were detected in
samples taken from the vicinity of plant roots colonized by PGPR B. amyloliquefa-
ciens (Debois et al. 2014). Interestingly, surfactin has multiple biological functions
in motility, biofilm formation, and cell-to-cell signaling, but is less efficient in direct
suppressing of other competing microbes than other lipopeptides or polyketides; it
was by far the most prominent compound occurring in the plant rhizosphere, previ-
ously being inoculated by PGPR B. amyloliquefaciens. For this reason, I conclude
that the direct effects exerted by the array of secondary metabolites encoded by the
Bacillus genome might not be as important and that the biocontrol effects exerted
by the Gram-positive bacteria are mainly due to other more indirect effects. I assume
that under field conditions, the stimulating effects on plant ISR are more important
than direct biocontrol by secreted secondary metabolites. In case of Bacilli, it is
very likely that ISR stimulation is a multifactorial process dependent on several
compounds produced by the rhizobacteria. Candidate compounds are surfactin, and
volatiles, especially acetoin and 2,3 butanediol (Fig. 1.4), since mutants of FZB42,
deficient in synthesis of these compounds, were found unable to protect plants from
pathogens. Moreover, high expression of defensin, indicating the JA/ET pathway in
ISR, was not found when the mutant strains were applied to the plant.
These findings are important for future strategies for screening of powerful
PGPR and BC strains. It is known for long time that high efficiency in suppressing
fungal or bacterial pathogens do not necessarily reflect the potential of these selected
strains for their performance under field conditions. Novel screening procedures
have to be developed for functional tests under more appropriate conditions, either
directly on plants or at least under conditions allowing biofilm formation on artifi-
cial surfaces. However, performance under field conditions remains the most impor-
tant criterion.
Taken together, the beneficial effect of Bacillus PGPR depends, besides their
rhizosphere competence, on at least three main factors:
16 R. Borriss
Fig. 1.4 Scanning electron microscopy of FZB42 cells colonizing Arabidopsis thaliana roots.
Important compounds as surfactin, indole-3-acetic acid, and 2,3-butanediol, which are formed
when growing on root surfaces (in planta), are indicated
1. Stimulation of plant ISR by bacterial metabolites produced in vicinity of plant

roots. Volatiles, such as acetoin and 2,3 butanediol, contribute not only to ISR,
but have a direct plant growth-promoting effect, whilst surfactin is important in
the early stage of colonization and biofilm formation. In addition, surfactin
strengthens the plant ISR response, which suppresses growth of fungal, bacte-
rial, viral, and other plant pathogens.
2. Direct antifungal action by secondary metabolites, such as iturins (e.g., bacillo-
mycin D) and fengycins, secreted into the rhizosphere. However, the suppressing
effect exerted by such compounds might be relatively weak, since the amount of
such compounds in vicinity of plant root was found relatively low. Until now,
antibacterial compounds, such as polyketides, were not detected in this
environment.
3. Application of PGPR Bacilli, as FZB42, might compensate, at least in part,
undesired changes in the composition of the plant microbiome, caused by the
presence of pathogens, as R. solani.
Without doubt, other features of PGPR, as production of plant hormones and

making available fixed macro- and micro nutrients for plant nutrition, contribute
also to the beneficial effect exerted by these microbes, but could not be appropri-
ately treated in this review due to space limitation.
Acknowledgements Many of the recent data, reported in this review, have been obtained in close
collaboration with the Helmholtz Center in Munich in frame of the PATHCONTROL project, and
the laboratory of Yuewen Gao, Nanjing Agricultural University, China, in frame of a Chinese
Collaborative project, financially supported by the BMBF, the German Ministry of Education and
Research. I thank especially Soumitra Paul Chowdhury, Anton Hartmann, Joachim Vater, Liming
Wu, Xuewen Gao, and Ben Fan (Nanjing Forestry University) for their fruitful collaboration dur-
ing the last years.
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Genetically Modified (GM) Crops
Harbouring Bacillus thuringiensis (BT) 2
Gene(S) to Combat Biotic Stress Caused
by Insect Pests
Bhupendra Koul
Abstract
Insect pests are a menace to the crop plants as they cause 15–22% annual crop
loss. Bacillus thuringiensis (Bt) crystal protein toxin(s) have been observed to be
effective against lepidopteran, coleopteran, dipteran and hemipteran insect pests.
With the emergence of recombinant DNA technology, computational biology
and plant transformation procedures, it is now possible to design, modify and
transfer any gene (natural or synthetic) into crop plants especially, to cope with
insect pests, herbicide tolerance, various abiotic stresses and to enhance the
expression level and nutritional quality. Bt-based biopesticides are an alternative
to synthetic pesticides and are insect- specific, effective, eco-friendly and cost-
effective. Agrobacterium-mediated plant transformation technique utilizes the
natural genetic engineering property of Agrobacterium tumefaciens which has
played a pivotal role in plant genetic engineering and development of stable
transgenics, over conventional breeding procedures. Several stable Bt-transgenics
(potato, maize, cotton, soybean, canola, squash, rice, etc.) developed by various
companies (Monsanto, Dow AgroSciences, Syngenta, Bayer cropScience, etc.)
have been approved by Genetic Engineering Appraisal Committee (GEAC),
Environment Protection Agency (EPA), and commercialized. The most success-
ful story of Bt-transgenics is that of Bt-cotton (Bollgard: trade name) harbouring
Bt-cry1Ac like gene. In order to avoid the development of insect resistance, vari-
ous strategies such as use of hybrid gene, Bt-gene pyramiding, refugia strategies,
enhanced expression of Bt-gene(s) and use of sterile insects are followed as and
when required for maintaining the sustainability of Bt-technology. In the last few
years, after analysing the effectiveness and promising future of this ‘green
B. Koul (*)
Lovely Faculty of Technology and Sciences, Department of Biotechnology, School of
Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India

22 B. Koul
t echnology,’ there has been a remarkable progress in the list of countries accept-
ing the Bt-GM crops.
Keywords
Bacillus thuringiensis · cry gene · Agrobacterium tumefaciens · Plant transforma-
tion · Bt-GM crops
2.1 Introduction
The sustainable plant productivity and crop yield(s) in coming years is the major
constrain for food and nutritional security for the human population in develop-
ing countries, where arable land per capita is shrinking, while human and live-
stock population is steadily increasing. Plant and crop productivity and yield are
the result of interaction of several physiological, biochemical and metabolic pro-
cesses over a defined period of time, reflected in gain of total biomass or con-
verted harvestable commodity like seeds, fruits or edible plant parts under a set
of environmental conditions that consist of several physical, geo-chemical and
biological components. Therefore, besides the genetic potential of plant species,
the phenotypic performance of crop plants in field profoundly depends on and is
influenced by several physical, abiotic and biotic parameters and is highly vari-
able. Hence, plant yield or harvest index is dependent on several factors and
several of them are beyond human control and are part of climate change and
environment. Among biotic components that influence plant/crop yield perhaps
infestation of plant pathogens and insect pest are major issues after the agro-
nomic inputs and practices. The infestation of insect pests alone during field and
storage condition may affect up to from 24 to 65 ± 5% loss in grain yield of major
crops (Ronald 2011). Control of agricultural insect pests under field and storage
conditions largely depend on the wide spread use of synthetic insecticides and
pesticides which are harmful to the ecosystem and human population (Hilder and
Boulter 1999; Wahab 2009). Alternative to conventional chemical insecticides,
application of microbial insecticides containing different microbial preparations
and delta endotoxins (Cry proteins) from Bacillus thuringiensis (Bt) have
emerged as ecofriendly and sustainable method for control of agricultural insect
pests in the last 50–60 years (Sanahuja et al. 2011). Attempts are being made to
use alternative bioinsecticides in field as well as in storage conditions to mini-
mize the losses in grain yield. In recent past, with the development of diverse
biotechnological tools and techniques of recombinant DNA and genetic engi-
neering, it is now possible to transfer and express a desired gene in its native or
modified form into the identified organism including plants, animals and
microbes. Among the battery of genetically modified organisms (GMOs), the
transgenic plants, expressing genes from either trans- or cis-origin, are the latest
introduction for sustainable crop and plant yield (Park et al. 2011).
2 Genetically Modified (GM) Crops Harbouring Bacillus thuringiensis (BT… 23
The most widely used and well-documented example of transgenic plant in

agriculture practice is the Bt-cotton, where Bt-toxin crystalline proteins of Cry1A
family are expressed starting from the native wild cry1Ab and cry1Ac genes of
Bacillus thuringiensis to highly modified synthetic version that are expressed in
cotton followed by maize and soybean which are released for commercial cultiva-
tion (Perlak et al. 2001; James 2012). Since then, transgenics of major crop plants
like cotton, maize, soybean, canola, tomato, rice, squash, potato, papaya, sugar-
cane and mustard have been developed for insect-pest resistance, herbicide toler-
ance and resistance to viruses and have been grown in more than 30 countries over
181.5 million hectares in 2016. About 17.3 million farmers over the world have
been benefited by transgenic technology and are growing biotech crops.
Interestingly, recently, five conservative European countries, namely Spain,
Portugal, Czechia, Romania and Slovakia, have agreed to cultivate Bt-maize.
Therefore, the transgenic technology has been adopted by both developed and
developing countries like the United States, China, Brazil, Argentina, Canada and
India and African countries, for different traits.
Bacillus thuringiensis (Bt) is a gram-positive soil bacterium which can produce
crystalline inclusions during the second phase of sporulation. These inclusions
eventually develop into hydrophobic crystalline structures consisting of several
toxin proteins that are of insecticidal nature against a wide spectrum of agricultural
insect pests (Whiteley and Schnepf 1986). Most of the crystal proteins are protox-
ins of proteinaceous nature and are proteolytically converted into smaller toxic
polypeptides in the midgut region of corresponding agricultural insect. This acti-
vated toxin interacts with the midgut epithelial cells of susceptible insects
(Hofmann et al. 1988; Bravo et al. 2007, 2011; Vachon et al. 2012; Pardo Lopez
et al. 2013) and biochemically generate the pores in the cells of brush border mem-
brane, thus disturbing the osmotic balance and eventually the septicemia in the
target insect leading into death of the insect (Knowles and Ellar 1988; Bravo et al.
2007, 2011). Several specific high-affinity binding sites on insect membranes to B.
thuringiensis toxins have been documented for specificity of different toxin pep-
tides generated by different strains/species/isolates of B. thuringiensis owing to
different genes coding for the corresponding crystal protein (Schnepf et al. 1998;
Hofte and Whiteley 1989).
Since the first cloning of an insecticidal crystal protein (ICP) gene (cry) from B.
thuringiensis by Schnepf and Whiteley (1981), a large number of cry genes from
different strains/species of Bt have been cloned, identified and characterized
(Crickmore et al. 1998; deMaagd et al. 2001). Till date, more than 500 different cry
genes from B. thuringiensis have been characterized and systematically documented
in the literature and enlisted in website maintained by Crickmore and his group
(www. glfc.cfs.nrcan.gc.ca/bacillus). These insecticidal genes code specific toxins
effective against insect orders belonging to Lepidoptera, Diptera and Coleoptera.
Some are effective against other insect orders like Hymenoptera, Homoptera,
Orthoptera, and Mallophaga, nematodes, mites and protozoa as well (Feitelson
et al. 1992; Bravo et al. 2007). B. thuringiensis strains have a genome size of 2.4–
5.7 million bp, and most of these bacterial strains possess both circular and
24 B. Koul
sometimes linear extra chromosal elements; however, the cry genes are mostly
located on the large plasmid (Gonzalez et al. 1981; Gonzalez et al. 1982). A large
number of cry genes producing insecticidal toxins effective against common agri-
cultural insect pests have been identified, cloned and expressed in different plant
species to develop insect pest resistance genetically modified transgenic plants
(James 2012).
Since the first introduction of cry gene into model plant tobacco for expressing
insect-resistant trait (Barton et al. 1987; Vaeck et al. 1987), several major crop spe-
cies have been genetically modified for expression of different insecticidal cry
genes affective against different order of insects (Fischhoff et al. 1987; Perlak et al.
1990; Perlak and Fischhoff 1993; Fujimoto et al. 1993; Koziel et al. 1993; Adang
et al. 1993; Nayak et al. 1997; Sanyal et al. 2005). The initial studies with introduc-
tion and expression of native full-length cry genes from B. thuringiensis into plants
have shown very poor expression of toxin production, and the produced toxin was
unstable in the plant system (Perlak et al. 1990; Schnepf et al. 1998). Several bio-
chemical and genetical reasons have been attributed for poor stability and low
expression of Bt-toxins in transgenic plants.
The earlier studies with transfer of Bt-cry genes showing poor expression were
attributed to silencing of foreign gene, instability of RNA transcripts of insecticidal
crystal protein genes (Murray et al. 1989), early termination of the transcript due to
existence of polyadenylation at multiple sites in coding region of native Bt-cry
genes (Diehn et al. 1996, 1998) and rapid degradation of mRNA (Perlak et al. 1991;
Adang et al. 1993; DeRocher et al. 1998). The evidence to these factors was associ-
ated to earlier reports for lack of a correlation between promoter activity and mRNA
accumulation (Fischhoff et al. 1987; Vaeck et al. 1987). The analytical results of
tobacco transgenics expressing full-length native cry1Ac showed majority of tran-
script shorter than anticipated full length of the gene (Barton et al. 1987). These
studies based on expression of full-length native cry1Ac and cry1Ab insecticidal
genes lead to characterization of several polyadenylation sequences along with
cryptic termination sequences in native Bt-cry genes.
These early reports suggested reinvesting the Bt-cry gene for its structure and
functioning in the plant system. Subsequently, by analysing the nucleotide
sequences of several cry genes, it was evident that crystal protein genes of B.
thuringiensis were destined for expression in prokaryotic cell and of typical pro-
karyotic architecture in having codon sequences preferable to prokaryotes and
gene length for optimum expression and stability of toxin in hydrophobic state
and nucleotide sequences and GC content suitable to prokaryotes. These observa-
tion lead to several modifications in Bt-cry genes which included truncation of 3′
end of gene to eliminate hydrophobicity of the endotoxin, removable of polyade-
nylation, mRNA instability and criptic termination sequences, for higher expres-
sion of Bt-cry genes in plants (Fischhoff et al. 1987; Vaeck et al. 1987; Perlak
et al. 1991). A major modification in the cry gene was incorporated to modify and
introduce plant-preferred codons in the truncated version of Bt-cry genes
(Delannay et al. 1989; Perlak et al. 1990, 1991).
These studies eventually led to major modification in designing of synthetic ver-

sion of truncated cry1Ac and cry1Ab genes comprising of about ~1845 bp, where
maximum care was taken to possibly use plant-preferred codons, elimination of all
the termination sequences and mRNA instability components (Perlak et al. 1991;
Sardana et al. 1996; Cheng et al. 1998). The designed genes were successfully
shown to express the Cry1Ab and Cry1Ac toxins in different plant species, and
promising transgenic plants of various species were developed (Perlak et al. 1991;
Stewart Jr et al. 1996; Singsit et al. 1997; Perlak et al. 2001; Sanyal et al. 2005).
Based on these developments and further molecular investigation of cry toxin and
its interaction with different receptor on susceptible insect resulted in development
of hybrid and fusion cry genes for wider host range and enhanced toxicity against
agriculturally important target insects (Datta et al. 1998; Wu et al. 2000; Naimov
et al. 2003; Singh et al. 2004; Ho et al. 2006; Rajamohan et al. 2006). To enhance
host range of Cry toxin and to address the growing resistance development in target
insects against these toxins, several mutations have been incorporated in the toxin
for effective binding to receptor (Bravo and Soberon 2008; Soberon et al. 2013).
Similarly, translation fusions of two cry genes or additional sequences for wider
host range have been designed (Bohorova et al. 2001; Mehlo et al. 2005).
Lepidoptera is the most devastating group of field insects causing significant
damages to large number of crop plants. Among them, Helicoverpa armigera,
Heliothis virescens, Ostrinia nubilalis, Spodoptera spp., Plutella xylostella and
Pectinovophora gossypiella are the important insects infesting several important
crops like cotton, cabbage, okra, tomato, cauliflower, chickpea, maize and soybean.
Two Bt-genes, cry1Ab and cry1Ac, have been documented for coding most effective
toxin showing maximum mortality in range of 20–80 ng toxin/mg of fresh weight.
Both these genes are most widely used for developing insect-resistant phenotype.
To make these two toxins highly effective and efficient against target insect pests,
several modifications have been incorporated including truncation, codon optimiza-
tion, point mutations and application of 5′ regulatory sequences for over expression
of the toxins at desired level in different plant species. The mechanism for pore
formation and recognition of different receptors and their affinity to these toxins
have been well documented. The native and modified versions of full length cry1Ab
(3.5 kb) and cry1Ac (3.5 kb) and their synthetic modified truncated versions of
1.8 kb size have been widely used for developing the transgenic plants of different
species exhibiting resistance against a number of insect pests (Cheng et al. 1992;
Koziel et al. 1993; Stewart Jr et al. 1996; Alam et al. 1998; Cheng et al. 1998; Perlak
et al. 2001; Sanyal et al. 2005; Mehrotra et al. 2011; Sanahuja et al. 2011). Except
for selection of a unique event of transgenic cotton expressing a full-length native
cry1Ac gene with few modifications and transgenic maize expressing cry1Ab, which
have gone for commercial cultivation (Koziel et al. 1993; Perlak et al. 2001; Ferry
et al. 2004; James 2012), most of the transgenics of different plant species are lim-
ited to demonstration under laboratory conditions. Despite several modifications
incorporated in native wild type cry1Ab and cry1Ac genes which share more than
94 ± 0.5% sequence homology, their over-expression, however, in different plant
species to recover promising transgenic plants with sufficient level of Bt-toxin(s)
26 B. Koul
have been a matter of concern (Diehn et al. 1996; DeRocher et al. 1998). The most
widely used successful transgenic event of Bt-cotton (Monsanto to 531) resistant to
bollworm complex of Heliothis virescens/Helicoverpa armigera, Pectinophora gos-
sypiella and Helicoverpa zea was developed with native full-length cry1Ac gene
having some specific minor modification (Perlak et al. 2001). The event has been
designated as Bollgard I and been grown commercially in large areas in several
countries (James 2012). Subsequently, to check the possibility of insect developing
resistance against Bt-cotton technology, a second version of transgenic cotton plant
designated as Bollgard II, expressing two different cry genes such as cry1Ac and
cry2Ab, has been developed and released for commercial cultivation (Purcell et al.
2004; Ferry et al. 2004). Interestingly, native cry1Ac coding gene was documented
for very poor expression in higher plants owing to high AT content and presence of
several pre-termination sequences. This situation necessitated the truncation and
enrichment of GC content, since plants in general have a higher GC content than
that found in bacterial genes (Murray et al. 1989), and particularly delta-endotoxin
cry genes have higher AT content. Modifying the coding sequences to increase GC
content, 3′ truncation and possible elimination of polyadenylation or termination
sequences of the native cry genes resulted into dramatic increase in the expression
of the insecticidal toxin proteins (Delannay et al. 1989; Perlak et al. 1991; Carozzi
et al. 1992). A highly modified cry1Ab gene-coding toxin protein of 648 amino acid
of the native proto-toxin of 1155 amino acids was expressed in maize to develop
resistance against European corn borer (ECB), Ostrinia nubilalis (Hubner), a major
pest of maize (Carozzi et al. 1992; Koziel et al. 1993). Comparative nucleotide and
amino acid sequences of prominent cry1A group of genes (cry1Aa, Ab and Ac) cod-
ing insecticidal crystal proteins affective against large number of Lepidopteran
insects showed distinct homology and similarities in 5′ coding sequences for toxin
molecules comprising of pore forming and receptor-binding domains except for the
specific changes in the sequences coding for the receptor-recognizing domains of
the toxin molecules (Haider and Ellar 1987; Schnepf et al. 1998; Bravo and Soberon
2008). This comparative and exhaustive sequence analysis was further executed to
other group of insecticidal crystal protein genes to reflect the diversity and evolution
of different cry genes coding different insecticidal toxin proteins effective against
specific insects (Feitelson et al. 1992; DeMaagd et al. 2001; Sanahuja et al. 2011).
Among cry1A group of genes, the response of the toxins against lepidopteran
insects has been found in the order cry1Ac > cry1Ab and least in cry1Aa gene. This
is further attributed to the molecular structure of insecticidal Cry toxin and its affin-
ity to bind with a different receptor on the midgut of susceptible insects and attach-
ment of toxin molecules with different epitopes of same or different receptors on
BBMV cells (Estela et al. 2004; Bravo et al. 2007). Considering the close resem-
blance and high homology of nucleotide sequences of cry1Ab and cry1Ac gene and
based on the architecture of toxin-coding sequences, completely synthetic version
of both the 1.8 kb genes has been developed and extensively used for optimal
expression in higher plants (Sardana et al. 1996; Cheng et al. 1998). The compara-
tive sequence analyses of both cry1Ab and cry1Ac genes have shown three blocks of
668, 403 and 279 bp which are identical in both the case while the fourth block of
495 bp comprises sequence variations that seem to code for the receptor-binding
domain of the toxin protein and may be the possible reason for differential toxicity.
Considering the high homology and similarities between modified synthetic cry1Ab
and cry1Ac genes for enhanced expression of toxin in higher plant, achieving prom-
ising number of transgenic with high level of toxin expression is not a routine pro-
cess. Despite the successful commercial release of Bt-cotton expressing cry1Ac
gene but recovery of stable transgenic plant with high level of Cry1Ac toxin is still
confined to laboratory level around the globe. Only restricted plant species have
been documented for high level expression of Cry1Ac toxin compared to number of
transgenic plants developed with Cry1Ab toxin. The modified-cry1Ab gene has
been successfully introduced and expressed to sufficient level in several plant spe-
cies like maize (Koziel et al. 1993; Singh et al. 2005), rice (Fujimoto et al. 1993;
Wunn et al. 1996; Wu et al. 1997; Cheng et al. 1998; Alam et al. 1998, 1999; Tu
et al. 2000; Marfà et al. 2002), cotton (Perlak et al. 1990), brinjal (Kumar et al.
1998), soybean (Parrott and Clemente 2004), tomato (Kumar and Kumar 2004),
sugar beet (Jafari et al. 2009) and chickpea (Mehrotra et al. 2011). However,
restricted plant species have been transformed with cry1Ac gene to develop stable
transgenic plants of cotton (Perlak et al. 1990, 2001; Rawat et al. 2011), tobacco
(Barton et al. 1987; Vaeck et al. 1987), tomato (Fischhoff et al. 1987; Mandaokar
et al. 2000), chickpea (Kar et al. 1997; Sanyal et al. 2005), peanut (Singsit et al.
1997) and canola (Stewart Jr et al. 1996).
2.2 Genetic Transformation
Genetic transformation is the deliberate alternation and modification of the genome

of an organism (bacteria, plant, animal) by introduction of one or few specific for-
eign genes using other than conventional procedures, and the modified organism is
termed as transformed or transgenic organism. Genetic transformation of plants is
becoming an indispensable aid to plant physiologists, biochemists and biotechnolo-
gists in understanding the role of individual and application of these procedures for
crop improvement with newer traits. Scientists of Calgene Inc. of Davis, California,
used the antisense RNA technology to inactivate the gene (polgalacturonase [PG])
responsible for softening the tomato to produce first genetically modified tomato
‘Flavr-Savr’ in 1991 and was approved by the US FDA in 1994.
2.3 Gene Transfer Method
2.3.1 Direct DNA Transfer Methods
The direct DNA transfer method has been proved to be simple and effective for
introducing foreign DNA into plant genomes (Fig. 2.1). Among these methods, the
most frequently used one is the microprojectile bombardment procedure where
28 B. Koul
Gene Transfer Methods
Vector mediated/ Vectorless/

Indirect DNA transfer Direct DNA transfer
Agrobacterium- mediated
transformation
Agrobacterium- mediated
virus infection (Agroinfection)
Physical gene Chemical gene DNA imbibition by cell,

transfer methods transfer methods tissues, embroys and seeds
(DMGT)
Electroportion
Not reliable
DNA transfer via pollen PEG mediated gene transfer
Ultrasound mediated DNA

transformation/ sonication
Calcium phosphate coprecipitation
Particle bombardment
microprojectile/ biolistics
Macroinjection The polycation DMSO technique
Microinjection
DEAE dextran procedure
Liposome mediated
transformation
Silicon carbide fiber- mediated

transformation (SCF)
Fig 2.1 Schematic representation of the various gene transfer strategies
transforming DNA is coated onto metal microcarriers like tungsten or gold that are
accelerated with high velocity either by gun powder device or through compressed
inert gases. The microcarriers acquire sufficient kinetic energy to allow them to
penetrate to the intact plant, animal or bacterial cell wall and plasma membrane
without killing the cells.
2.3.2 Indirect DNA Transfer Method
As with other dicotyledonous crops, Agrobacterium-mediated transformation is the

most widely used method for gene transfer. Among the various vectors used in plant
transformation, the Ti plasmid of Agrobacterium tumefaciens has been widely used.
This bacteria is known as ‘natural genetic engineer’ of plants, because these bacte-
ria have natural ability to transfer T-DNA of their plasmids into plant genome upon
infection of cells at the wound site and cause an unorganized growth of a cell mass
known as crown gall. Ti plasmids are used as gene vectors for delivering useful
foreign genes into target plant cells and tissues. The foreign gene is cloned in the
T-DNA region of Ti plasmid in place of unwanted sequences.
2.4 Agrobacterium-Mediated Genetic Transformation
Agrobacterium tumefaciens is a gram-negative, soil phytopathogen of family

Rhizobiaceae that causes the disease ‘crown gall’ in a wide variety of dicotyledon-
ous plants (Fig. 2.2). Crown gall is a plant tumour, a lump of undifferentiated tissue,
which often forms at the area of crown, the junction between the root and the stem
of the infected plants. The pathogenic property of this bacterium was recognized
much earlier (Smith and Townsend 1907).
A. tumefaciens: induces crown gall disease.
A. rhizogenes: induces hairy root disease.
A. radiobacter: an avirulent strain.
During the infection at wound site, the bacterium transfers a small part of its own
plasmid DNA called T-DNA (transfer DNA) into the plant cell that results in two
key events.
1. The plant cell begins to proliferate and form tumours and receive the ability to
grow in cultures, which even do not have any growth regulator.
2. They begin to synthesize an unusual arginine derivative called opines (octopine,
nopaline, etc.) which are not found in normal tissues.
Bacteria can be classified as octopine, nopaline, agropine, succinamopine or

chrysopine strains (octopine is condensation product of arginine and pyruvic acid).
The metabolism of opines is a central feature of crown gall disease. The type of
opine produced is not determined by the host plant but by the bacterial strain. In
general, the bacterium induces the synthesis of an opine, which it can catabolize and
Fig. 2.2 (a) Electron micrograph of A. tumefaciens (b) A plant root with crown galls, (c) A plant
showing symptoms of hairy roots
30 B. Koul
use as its sole energy source for carbon and nitrogen. Clearly, an interesting inter-
relationship is evolved, where A. tumefaciens subvert the plant’s metabolism to
make amino acids, which can be utilized only by the bacteria as a food and energy
source.
2.4.1 Ti Plasmid of Agrobacterium
The ability of Agrobacterium tumefaciens to induce crown gall disease in plants is

controlled by genetic information carried on a large conjugative plasmid (of about
200 kb size) called Ti plasmid for its tumour-inducing capacity. Virulence is lost
when the bacterium is cured for the plasmid, and cured strains have lost the capacity
to utilize octopine or nopaline. Ti plasmids have temperature-sensitive replication,
i.e. high temperature (more than 30 °C) leads to curing of plasmids. Ti plasmids
have regions for replication (origin of replication), conjugal transfer, virulence and
T-DNA.
Three bacterial genetic elements are required for T-DNA transfer to plants.
1 . 25 bp direct repeated flanking and defining the T-DNA

2. Virulence (vir) genes encoded by the Ti plasmid in a region outside of the
T-DNA.
3. Number of chromosomal genes, of which some are important for attachment to
the bacterium to the plant cell
2.4.2 Organization of T-DNA
T-DNA (transfer DNA) is about 23 kb segment of Ti plasmid, which is transferred

into the plant genome during Agrobacterium infection. T-DNA contains the gene for
constitutive synthesis of auxins, cytokinins and opines and is defined on both the
sides by 24 bp direct inverted repeat called border sequences, which are required for
T-DNA excision and transfer. The deletion of either border sequence completely
blocks the transfer of T-DNA into the plant cell. However, mutational analysis
shows that only the right repeat is absolutely required for T-DNA transfer and they
function in cis and polar fashion. The T-DNA is organized into two distinct regions
called TL (left T-DNA) and TR (right T-DNA). Both TL and TR are always trans-
ferred together in nopaline plasmids and integrated into the plant genome as a single
segment. But in octopine plasmids, the TL and TR are transferred independently so
that a single cell may contain one or both of these segments. T-DNA has three
genes, which are involved in crown gall formation. Two of these genes, iaaM and
iaaH encodes tryptophan 2-monooxygenase and indoleacetamide hydrolase,
respectively, which together convert tryptophan into indole 3-acetic acid (IAA); the
locus was earlier called ‘shooty’ locus, and the genes were designated as tms1
(tumour with shoots) and tms2. The third gene, ipt, encodes a zeatin-type cytokinin,
isopentenyl transferase; the locus was earlier designated as ‘rooty’ locus and
designated as tmr (tumour having roots). T-DNA also contains genes involved in
opine biosynthesis near the right border. All the genes present in T-DNA contain
eukaryotic regulatory sequences. As a result, these genes are expressed only in plant
cells, and they are not expressed either in Agrobacterium or in E. coli.
2.4.3 Organization of vir Region
The vir region of the Ti plasmid contains 8 operons, which together span to about
40 kb of DNA and possesses 25 genes. This region mediates the transfer of T-DNA
in both cis and trans fashion into plant genome, and hence is essential for virulence
and transfer of T-DNA (Hooykaas and Mozo 1994). Among the eight vir operons,
four operons, viz., virA, virB, virD and virG are essential for virulence, while the
remaining four operons play an accessory role in transfer of T-DNA. VirA and virG,
which are constitutively expressed, regulate the expression of other vir loci. Signal
transduction proceeds via activation of virG by virA, in response to the activation of
virA by plant phenolics like acetosyringone and α-hydroxy acetosyringone. After
activation, virG dimerizes and activates the transcription of other vir genes
(Zambryski et al. 1989). The functions of different vir genes are given in Table 2.1.
2.4.4 T-DNA Transfer Process
T-DNA transfer begins with the introduction of bacteria into a plant wound
(Fig. 2.3). Wounding is a necessary event in the process and may, at least is part, be
required for the synthesis by the plant, certain compounds that induce the expres-
sion of the vir genes. Two of the most active substances identified are acetosysin-
gone and β-hydroxy acetosysingone. T-DNA transfer process starts by binding of
virD1 gene product to the right border (RB) sequence, virD1 has the topoisomerase
activity that facilitates the action of protein virD2, as endonuclease; in nicking, at
the right border and covalently binds to the 5′ end. The 3′ end produced at the site
Table 2.1 Functions of different vir genes

Vir No. of
region genes Function
virA 1 Encodes a sensor protein; receptor for acetosyringone and functions as an
autokinase; also phosphorylates virG protein; constitutive expression
virB 11 Membrane proteins; role in conjugal tube formation
virC 2 Helicase activity
virD 4 VirD1, has topoisomerase activity and virD2 is an endonuclease
virE 2 Single strand binding protein (SSBP)
virF 1 Not well understood
virG 2 DNA binding protein, induces the expression of all vir operon; constitutive
expression
virH 2 Not well known
32 B. Koul
Fig. 2.3 Model for Agrobacterium-mediated genetic transformation of plants (Tzfira and Citovsky
2006). The transformation process comprises of 10 major steps and begins with recognition and
attachment of the Agrobacterium to the host cells (1). Sensing of specific plant signals by the
Agrobacterium VirA/VirG two-component signal-transduction system (2). Following activation of
the vir gene region (3), a mobile copy of the T-DNA is generated by the VirD1/D2 protein complex
(4) and delivered as a VirD2–DNA complex (immature T-complex), together with several other Vir
proteins, into the host-cell cytoplasm (5). Following the association of VirE2 with the T-strand, the
mature T-complex forms, travels through the host-cell cytoplasm (6) and is actively imported into
the host-cell nucleus (7). Once inside the nucleus, the T-DNA is recruited to the point of integration
(8), stripped of its escorting proteins (9) and integrated into the host genome (10)
of nick serves as a primer for replacement synthesis of DNA in the 5′ → 3′ direction
as a result of which the T-strand is displaced from the DNA duplex.
The virE2 protein is a single-strand DNA-binding protein and about 600 copies
of it binds to the single-stranded T-DNA, thus protecting it from nuclease action.
VirB operon encodes membrane-bound proteins, which participate in conjugal tube
formation between the bacterial and plant cells to provide a channel for T-DNA
transfer, whereas virB11 has ATPase activity, which generates energy needed for the
delivery of T-DNA into the plant cells (Zambryski et al. 1989). The nuclear
localization signals present on the virD2 and virE2 proteins drive the T-DNA
towards the nucleus of the plant cell. This mechanism accounts for the polarity; cis-
acting nature of the border repeat sequences also explains the importance of right
border repeat in T-DNA transfer. Apart from Ti plasmid, chromosomal virulence
genes (chv) are also involved in T-DNA transfer from Agrobacterium to plants. The
chv genes are required for the synthesis of cyclic glucans, which are involved in
plant cell-binding Chv A, chvB and psc A that are involved in the synthesis and
export of cyclic β-1,2-glucan. A more direct role in attachment has been demon-
strated for rhicadhesin, a calcium-binding protein located on bacterial cell surface.
The induction of Agrobacterium vir genes in response to plant wound-specific com-
pounds implies that a bacterial recognition system must detect the plant signal and
transmit the information inside the bacterial cells. This process is mediated by prod-
ucts of vir A and vir G.
2.4.5 Vectors Derived from Ti Plasmids
Large size, absence of unique restriction sites and tumourigenic properties of Ti

plasmids precluded the use of wild-type Ti plasmids as vectors. Presently, plant
transformation vectors have been produced by replacing tumour-including genes
with dominant selectable markers and desired traits. These types of vectors are
known as disarmed vectors; with functional vir genes and T-DNA border
sequences. Such non-oncogenic plant transformation vectors are either co-inte-
grated or binary types.
2.4.6 Co-integrate Vector System
Vectors that recombine via DNA homology into a resident Ti plasmid are often
referred to as integrative or cointegrative vectors. In this type of vector systems,
both T-DNA and vir regions are present in the same Ti plasmids. Gene of interest
can be inserted in between T-DNA borders by a co-integration event between the
homologous sequences present in the cloning vector and T-DNA region of Ti plas-
mid. Efficiency of co-integrate system relies on the frequency of conjugal transfer
and homologous recombination.
2.4.7 Binary Vector System
The binary vector system consists of two autonomously replicating plasmids within
A. tumefaciens a shuttle (more commonly referred to as binary) vector that contains
gene of interest between the T-DNA border and a helper Ti plasmid that provides the
vir gene products. The vir gene can act in trans and encode proteins, which are
required for the transfer of T-DNA. The standard components of binary vector are:
34 B. Koul
1 . Multiple cloning site

2. A broad host range origin of replication functional in both E. coli and A.
tumefaciens
3. Selectable markers for both bacteria and plants
4. T-DNA border sequences (although only right border is absolutely essential)
2.4.8 Selectable Markers
Selection of transformed cells is a key factor in developing successful methods for

genetic transformation. This is done by certain selectable marker genes that are
present in the vector along with the gene of interest. Selectable markers are an inte-
gral part of plant transformation strategies (Table 2.2).
Each selectable marker presents some favourable and some unfavourable fea-
tures. Therefore, the choice of a marker should be based on the plant species and
other considerations in the study. The NPT II gene from transposon Tn5 confers
resistance to the amino glycoside antibiotics kanamycin, neomycin and G 418. The
NPT II gene product, neomycin phosphotransferase, inactivates these antibiotics
through its phosphorylation (Bevan et al. 1983). This marker is a most widely used
system for plant selection and screening as no endogenous level is reported so far in
green plants.
2.4.9 Advantages of Agrobacterium-Mediated Plant

Transformation
It is a natural means of DNA transfer and is perceived as a more acceptable tech-

nique over long conventional breeding procedures. It is capable of infecting intact
plant cells, tissues and organs. Transformed plants can be regenerated more rapidly.
It is capable of transferring large fragments of DNA very efficiently without sub-
stantial rearrangements of the transgene. Integration of DNA is relatively a precise
Table 2.2 Selectable markers genes used for gene transfer

Selectable marker genes Substrates used for selection
Neomycin phospotransferase (nptII) G 418, kanamycin, neomycin,
paromycin
Hygromycin phospotransferase (npt) Hygromycin B.
Gentamycin acetyl transferase Gentamycin
Streptomycin phospotransferase Streptomycin
Dihydrofolate reductase (dhfr) Methotrexate
Phosphinothricin acetyl transferase L-Phosphinothricin (PPT)
5-Enolpyruvyl shikimate 3 phosphate (EPSP) syhthase Glyphosafe
(aroA)
Acetolactate synthase mutant form (als) Sulphonyl urea, imidazolinones
Bromoxynil nitrilase (bxn) Bromoxynil
process; it serves as an ideal insertional mutagenesis vehicle as it introduces one to

several copies of the transferred DNA into the intact genome at one or few loci. The
integrated DNA gives consistent maps and appropriate segregation ratios. The sta-
bility of the gene(s) and the respective trait(s) have been found to be stable over
many generations. All of these features make this technique reliable for commer-
cialization of transgenic plants. A wide range of explants have been successfully
transformed using Agrobacterium, although cotyledons have been most commonly
used (McCormick et al. 1986). Other explants like vegetative leaves and hypocotyl
(McCormick et al. 1986) stem have also been used with high transformation fre-
quency both with binary as well as co-integrate Ti plasmid vectors used in these
experiments.
2.4.10 Disadvantages of Agrobacterium-Mediated Plant

Transformation
There is limitation of host range as it cannot transform many important food

crops. Cells and tissues that are able to regenerate are difficult to transform. The
embryogenic cells are placed in deeper layers and are thus not amenable to T-DNA
transfer.
2.5 Factors Affecting Plant Transformation
A successful gene transfer procedure is mainly dependant on the following factors:

(1) simple, reproducible, genotype-independent and cost-effective regeneration pro-
tocol for (2) target tissues, which are both competent for transformation and regen-
eration, (3) an efficient DNA delivery method, (4) procedure to select for transgenic
tissues and (5) the ability to recover fertile plants avoiding somaclonal variation in
transgenic plants (Velcheva et al. 2005; Thi Van et al. 2010).
Availability of high-frequency genotype-independent in vitro regeneration sys-
tem amenable to Agrobacterium-mediated transformation is the major pre-requisite
for developing transgenic lines (Birch 1997). A number of factors influencing
genetic transformation such as genotype, type of explant, explant orientation,
wounding procedure, co-cultivation duration, the role of phenolic compounds,
Agrobacterium strain, bacterial cell density, etc. play an important role in determin-
ing overall transformation efficiency. The optimization of selection and screening
procedures are crucial for improving transformation efficiency and most impor-
tantly developing non-chimeric transgenic plants.
36 B. Koul
2.6 acillus thuringiensis (Bt) Endotoxin Crystal Protein

B
Genes for Insect Resistance
Agricultural pests are mostly controlled by the use of synthetic pesticides and rarely
by cultural practices. Therefore, the excessive and reckless use of agrochemicals
has been a subject of public concern as it has led to harmful consequences on the
environment and carcinogenicity to non-targets organisms.
The reliance on gene transfer technology to transfer insect-resistance genes of
diverse origin into crop plants provides an economical, feasible and eco-friendly
alternative to the extensive use of chemicals pesticides. Insect-resistant transgenic
plants may be raised by introducing foreign genes encoding either δ-endotoxin,
protease inhibitors (PI), lectins, amylase inhibitors, etc. (Boulter 1993; Gatehouse
et al. 1997). The most widely used, well-documented and reliable approach in this
context is the insecticidal crystal protein (ICP) genes of Bacillus thuringiensis (Bt)
which code for δ-endotoxin (Whiteley and Schnepf 1986). Gram-positive spore-
forming entomopathogenic bacteria of Bacillaceae family particularly Bacillus
thuringiensis produce a large variety of protein toxins to aid them to invade, infect
and kill their hosts. This bacterium produces an insecticidal crystal protein which
forms inclusion bodies of bipyramidal, cuboidal, flat rhomboid or a composite with
two or more crystal types during sporulation (Bajwa and Kogan 2001). ICPs are one
of the several classes of endotoxins produced during sporulation, and δ-endotoxins
(delta endotoxins) are the most effective than other classes of α-, β- and γ-endotoxins
(alpha, beta and gamma) to agricultural insect pests. The genes coding these toxins
are called cry genes.
Although the Cry proteins exhibit diversity, they are specific to the target insect
orders: lepidoptera (moths and butterflies), diptera (mosquitoes and flies) and cole-
opteran (weevils and beetles), and few new toxins have been identified to kill
hymenopterans (bees and wasps) and nematodes (Schnepf et al. 1998; Pigott and
Ellar 2007; Bravo et al. 2007). Considering a large number of cry genes and diver-
sity of encoded toxins against different groups of insects and microbes, several
nomenclatures and classification of ICP genes have been proposed (Hofte and
Whiteley 1989; Sanchis et al. 1988; Crickmore et al. 1998; Crickmore et al. 2011).
Table 2.3 Classification of cry genes on the basis of their activity spectruma
Protoxin/active
Protein Subspecies (strain) Activity spectrum molecular mass in kDa
Cry I CryI Kurstaki (HD-1), Lepidopteran 130–160/ca.60
aizawai, sotto
CryII CryII Kurstaki (HD-1), Lepidopteran and dipteran 70–71/ca.65
Kurstaki (HD-263) (mosquito)
CryIIIA Tenebrionsis Coleopteran (chrusomelids) 73/ca.65
CryIIIB Japonicus Coleopteran (scrarabaeids) 73/ca.55
CryIV Israelensis Diptera (mosquito, black 72–134/ca.46–48
flies and nematodes)
a
Hofte and Whiteley (1989); Rukmini et al. 2000)
However, new toxin-encoding genes are being identified and the number is increas-
ing therefore, nomenclature and name of the new cry genes is assigned according to
the extent of evolutionary divergence, as projected by phylogenetic tree algorithms.
The large and variable family of insecticidal proteins of BT was earlier classified on
the basis of their activity, into five major classes, as shown in Table 2.3. Later,
Crickmore et al. (1998) suggested a common platform for nomenclature of Bt-cry
genes and broadly classified them into 22 groups of cry genes and two groups of
cytolytic (cyt) parasporal inclusion protein genes that exhibited hemolytic activity.
According to Crickmore et al. (2011), Cry toxins have been classified on the
basis of their primary amino acid sequence and more than 500 different cry gene
sequences have been classified into 70 subgroups. These cry gene sequences
have been divided into four phylogentically unrelated protein families with dif-
ferent modes of action: three domain Cry toxins (3D), mosquitocidal Cry toxins
(Mtx), binary-like (Bin) and the Cyt toxins. Among these toxins, the family of
three-domain Cry toxins represents the largest group with more than 53 different
subgroups.
As mentioned before, Bt-toxins are extremely specific to the target insect pests,
non-toxic to animals including non-target insects and human beings, non-hazardous
and eco-friendly (DeMaagd et al. 2001). These characteristics led to the advance-
ment of bioinsecticides, and formulations based on Bt-spores to control agricultural
insects have been developed and used extensively. Besides production of insecti-
cidal δ-endotoxins by B. thuringiensis, some of the bacterial species are documented
to express toxins during the non-sporulating state called ‘Vip,’ or vegetative insec-
ticidal protein, which are toxic to insects and microbes (Gatehouse 2008). Both Cry
and Cyt toxins interact with very specific receptors on susceptible insect pests. The
primary mode of Cry protein is to recognize the receptor on insect midgut epithelial
cells and lyse the cells by inserting the domain I and resulting into pore formation.
The three-domain Cry toxins are globular molecules harbouring three distinct
domains connected by single linkers. The domain I at the N-terminal end com-
prises a series of α-helices arranged in a cylindrical formation while domain II
comprises a triple β-sandwich for receptor binding. Most of the Bt-toxins are
expressed as protoxin of higher molecular weight and are non-toxic; however,
their proteolytic products are of smaller size and are highly toxic to the suscep-
tible insects. The main difference between the 65 and 130-kDa three-domain Cry
toxin is a C-terminal extension that is found in the 130-kDa protoxins, which is
cleaved by proteases present in the larval midgut and is therefore dispensable for
toxicity (DeMaagd et al. 2001). The N-terminal region of all three-domain cry
genes codes for the N-terminal fragment of protoxin which comprises 20–60
residues, while the active toxin is composed of approximately 600–620 amino
acid residues. The X-ray crystallographic studies of different trypsin-activated
Cry toxins, such as Cry1Aa (Lepidopteran specific), Cry3Aa, Cry3Bb and
Cry8Ea (Coleopteran specific), Cry4Aa and Cry4Ba (Dipteran specific) and
Cry2Aa protoxin (Dipteran-lepidopteran specific), have been determined (Li
et al. 1991; Grochulski et al. 1995; Galitsky et al. 2001; Morse et al. 2001;
Boonserm et al. 2005, 2006; Guo et al. 2009).
38 B. Koul
Cry proteins are modular in structure, consisting of three different functional

domains as I, II and III (Schnepf et al. 1998). N-terminal part of the toxin fragment
comprising six amphipathic helices (α-1, 2, 3, 4, 6, 7) with a central hydrophobic
helix (α-5) makes the domain I of δ-endotoxins (Li et al. 1991; Grochulski et al.
1995). Two alternative models, viz. ‘Penknife Model’ (Hodgman and Ellar 1990)
and ‘umbrella model’ (Li et al. 1991), were proposed to explain the pore-forming
mechanism of domain I of δ-endotoxins. Following insertion of the toxin, helix α-1
is removed due to protease digestion, and it is the only helix that does not bind to
BBMV vesicles as synthetic peptide mimicking studies show that α-5 helix and α-4-
α-5 helix loop is important for toxin aggregation and ion channel formation (Gerber
and Shai 2000). It has been proposed that after the toxin binds to the receptor, there
occurs a change in the conformation of this domain allowing the hydrophobic sur-
faces of the helices to face the exterior of the bundle, leading to insertion into the
membrane and the formation of ion channels (Knowles 1994). Domain II is made of
three antiparallel β-sheets, oriented parallel to the α-helices of domain I. Domain III
is made of two antiparallel β-sheets into β-sandwich structure which is involved in
several functions such as stability, as receptor binding, specificity determination and
ion channel gating (Schnepf et al. 1998). Arginine-rich block in domain III of
δ-endotoxin is called ‘arg face,’ through which domain III makes contact with
domain I and regulates ion channel conductance (Saraswathy and Kumar 2004).
The results of phylogenetic analysis suggest that domain I sequences seem com-
mon only for a subgroup of toxin proteins. Shuffling of the functional domains was
observed only for domain II and III in some toxins. Toxins with dual specificity for
lepidopteran and coleopteran insects are examples of domain III shuffling among
coleopteran and lepidopteran-specific toxins. The phylogenetic analysis of the Cry
toxin family shows that the great variability in the biocidal activity has resulted
from two fundamental evolutionary processes: (i) independent evolution of the
three functional domains and (ii) domain swapping among different toxins. These
two processes have generated toxin proteins with similar modes of action but with
diverse specificities. It is suggested that sequence divergence in combination with
domains swapping by homologous recombination might have caused extensive
range of specificities and evolution of different Bt-toxins (DeMaagd et al. 2001;
Bravo et al. 2007).
After ingestion of Bt-ICP by a target insect, Bt-protoxin first passes through the
peritrophic matrix (PM) diffusing into the midgut brush border, where it is digested
to yield toxin of smaller molecular mass that mediates insect death (Gill et al. 1992;
Knowles 1994). The PM is a single semiporous tube consisting of several layers of
mucin like glycoproteins and chitin microfibrils (Nation 2002; Ma 2005). It serves as
a barrier against the entry of virus, bacteria and bacterial products, such as Bt-protoxin
(Nation 2002). Receptor binding is a key factor for specificity, specific binding
involves two steps: one that is reversible and other is irreversible. Recent data sug-
gested that toxicity correlates with irreversible binding (Aronson and Shai 2001).
Irreversible binding might be related to insertion of the toxin into the membrane but
could also reflect a tighter interaction of the toxin with the receptor. The delta endo-
toxin-binding receptors in the larval midgut are identified as glycoprotein. Domain II
loops showed immunoglobulin-like structural folds, and carbohydrates are used as

recognition epitopes by these folds (Li et al. 1991). CryIAc toxin specifically binds
to a 120 kDa aminopeptidase-N (APN) receptor and binding interaction is mediated
by Gal NAc, presumably covalently attached to the APN. Knight et al. (1994) have
shown that O-glycans associated with a C-terminal O-glycosylated ‘Stalk’ structure
in the APN molecule are the most likely site for CryIAc toxin binding determined by
lectin binding and carbohydrate compositional analysis.
Cadherin-like proteins also serve as receptors for CryIAc toxins in lepidopteran
insects. Cadherin is critical for initial binding with toxin followed by further proteo-
lytic changes, oligomerization, binding to APNs in lipid rafts and insertion into the
cell membrane for forming pores (Hua et al. 2004). Regions of domain II of CryIA
toxins bind to specific sites on Bt-R1 Cadherin-like protein. Three CryIAb toxin-
binding regions in Manduca sexta Bt-R1 have been mapped to aa865–aa875 (Site 1),
aa1331–aa1342 (Site 2) and aa1363–aa1464 (Site 3). The first site 865NITIHITDTNN875 is
involved in binding loop 2 and second site 1331 IPLPASILTVTV1342 binds to loop α-8
located on CR11. Ectodomain CR12 (Site 3) is a critical Cry1Ab receptor epitope
and is the minimum region found to be crucial to confer cell susceptibility to
Cry1Ab to the same level as full-length Bt-R1 (Hua et al. 2004; Xie et al. 2005).
2.7 echanism of Action of Three-Domain Cry Toxins

M
in Lepidoptera
The activated toxin of 60 kDa goes through a complex sequence of binding events
with different insect gut Cry-binding proteins (receptors), leading to membrane
insertion and pore formation (Bravo et al. 2004; Pigott and Ellar 2007; Pacheco
et al. 2009). Two models have been proposed which demonstrate the series of events
that occur during receptor–Bt-protein interaction: [A] pore formation model and [B]
signal transduction model.
2.7.1 Pore Formation Model
According to the pore formation model, binding to Bt-R1 (receptor) is possibly the
first event in the interaction with the microvilli membrane. This initial binding
promotes a conformational change in the toxin-facilitating proteolytic cleavage of
helix α-1, by a membrane-bound protease followed by formation of pre-pore oligo-
meric structure. The oligomeric toxin then binds to the APN which induces a con-
formation change and a molten globule state of the toxin which is inserted into
lipid rafts inducing pore formation and cell swelling (Bravo et al. 2007). After
insertion into the membrane bilayers, the toxin inhibits k+ transport and amino acid
assimilation in the gut lumen, causing imbalance in pH, ion and other macro mol-
ecules and culminate into insect death (Ma 2005). According to a recent report of
Pardo Lopez et al. (2013), which is an extension of pore-formation model, the first
binding/interaction of activated Cry1A toxins is a low-affinity interaction with
40 B. Koul
Fig. 2.4 Schematic representation of the mechanism of action of three-domain Cry toxins in
Lepidoptera at the molecular level. (A) the larvae ingest the three domain-Cry protoxin, which is
solubilized in the midgut lumen due to high pH and reducing conditions and get activated by gut
proteases, thus generating the toxin fragment. (B) the monomeric three domain-Cry toxin binds
ALP and APN receptors, in a low-affinity interaction, the toxin is then located in close proximity
to the membrane (C) the monomeric three domain-Cry toxin binds the cadherin receptor in a high-
affinity interaction and this interaction induces proteolytic cleavage of the N-terminal end of the
toxin, including helix α-1 of domain I (D) the cleaved three domain-Cry toxin is then able to
oligomerize in a toxin pre-pore oligomer (E) the oligomeric three domain-Cry structure binds to
ALP and APN receptors with high affinity (F) the pre-pore inserts into the membrane causing pore
formation
ALP (alkaline phosphatase) and APN receptors (aminopeptidase-N) (Kd = 101 nM

for APN and 287 nM for ALP). The interaction with APN occurs through exposed
loop 3 of domain II and interaction with ALP through strand β-16 of domain III
(Masson et al. 1995; Pacheco et al. 2009; Arenas et al. 2010). ALP and APN are
highly abundant proteins anchored to the membrane by a glycosyl phosphati-
dylinositol anchor (Upadhyay and Singh 2011). The interaction with ALP and
APN concentrates the activated toxin in the microvilli of the midgut cells due to
which the toxin is able to bind in a high affinity interaction to the cadherin receptor
(Kd = 1 nm; Vadlamudi et al. 1995; Gómez et al. 2006, Pacheco et al. 2009; Arenas
et al. 2010). A schematic representation of mechanism of action of three-domain
Cry toxin in Lepidopterans at the molecular level has been shown in Fig. 2.4.
2.7.2 Signal Transduction Model
Another model which is signal transduction suggests that Bt-toxicity could be

related to G-protein-mediated apoptosis following the receptor binding (Zhang
et al. 2006). Binding of Cry toxin to Bt-R1 mediates cell death by activating a signal-
ling pathway involving stimulation of the stimulatory G-protein-α-subunit (G-αs)
and adenylyl cyclase (AC), which increases the cyclic adenosine monophosphate
(AMP) levels, and activation of protein kinase A (PKA). Activation of AC/PKA
signalling pathway initiates a series of cytological events that include membrane
blebbing, appearing of nuclear ghosts and cell swelling followed by cell lysis
(Zhang et al. 2006). Diagrammatic view of the two models of Cry toxin action has
been shown in Fig. 2.5.
Broderick et al. (2006) have put up an interesting observation that B. thuringien-
sis toxicity depends on the interaction with microorganisms of the normal gut com-
munity. Elimination of gut microbial community by oral administration of antibodies
abolished insecticidal toxicity, and re-establishment of an enterobacter sp., that nor-
mally resides in the midgut microbial community has restored B. thuringiensis-
mediated killing.
Transgenic plants expressing B. thuringiensis toxins have been used successfully
to provide resistance against selected agricultural insects. Since the development of
first transgenic tobacco and tomato plants with native Bt-cry gene (Vaeck et al.
1987; Fischhoff et al. 1987; Barton et al. 1987) considerable progress has been
made to develop promising transgenic plants with highly modified Bt-cry genes for
stability of mRNA and high-level expression (Gatehouse 2008). A large number of
stable transgenic plants of different families, expressing various Bt-cry genes have
been developed which exhibit significant protection to insect damages in lab and
field (Hilder and Boulter 1999; Sharma et al. 2000; Tabashnik et al. 2003).
2.8 BT-GM Crops
A large number of crop plants expressing Bt-insecticidal endotoxin have been suc-
cessfully transformed by Agrobacterium-mediated approach. Major reports on
development of insect-resistant plants are summarized in Table 2.4.
Stable transgenic plants of tobacco (Barton et al. 1987), tomato (Delannay et al.
1989; Gordon-Kamm et al. 1990), rice (Koziel et al. 1993; Datta et al. 1998;), soy-
bean (Parrott et al. 1994; Stewart Jr et al. 1996), groundnut (Singsit et al. 1997),
pigeonpea (Surekha et al. 2005) and chickpea (Kar et al. 1997; Sanyal et al. 2005)
have been developed. Recently, very high level of expression of Bt-cry2Aa2 protein
in chloroplast up to 35.5% of total protein (DeCosa et al. 2001) and expression and
inheritance of multiple transgenes (gene pyramiding) in rice (Cheng et al. 1998;
Maqbool et al. 2001) and cabbage (Cao et al. 2001; Zhao et al. 2003) have been
documented, for efficient management of insects and as insect-resistance manage-
ment strategy. The global status of approved and commercially available Bt-GM
crops is shown in Table 2.5.
42
Fig. 2.5 Models of the mode of action of Cry toxins and resulting mechanism for resistance (Bravo and Soberon 2008). Two different mechanisms can be
distinguished: the pore formation model (top) and the signal transduction model (bottom), which both include similar initial steps for toxin solubilization in
midgut lumen (1) activation by midgut proteases (2) and binding to primary receptor cadherin. In the pore formation model (top), step (3) induces the cleavage
of helix α-1 and triggers toxin oligomerization (4) the toxin oligomer then binds to a secondary receptor, such as aminopeptidase or alkaline phosphatase, which
are anchored by a glycosylphosphatidylinositol anchor in the membrane, and (5) finally, the toxin inserts itself into the membrane, thereby forming a pore that
kills the insect cells. (6) The signal transduction model (bottom) proposes that the interaction of the Cry toxin with a cadherin receptor triggers an intracellular
B. Koul
cascade pathway that is mediated by activation of protein G (4a) which, in a subsequent step (5a), activates adenylyl cyclase. This signal then increases the
levels of cyclic adenosine monophosphate, which activates protein kinase A and leads to cell death
Table 2.4 Agriculturally important plants transformed with Bt-genes for insect resistance
Crop Botanical name Gene Useful trait Expression Reference(s)

Arachis cry1Ac Efficacy against 0.18% Singsit et al.
hypogea lesser cornstalk (1997)
borer
Brassica napus cry1Ac Resistance to H. 0.4% Stewart Jr
zea Boddie and S. et al. (1996)
exigua Hubner
Brassica cry1Ab Resistance to 0.5.ng g−1 f.w. Cao et al.
oleracea diamond back moth (2001),
larvae Bhattacharya
et al. (2002)
cry1C Plutella xylostella – Zhao et al.
(2001)
Cajanus cajan cry1EC Resistance to – Surekha et al.
Spodoptera litura (2005)
cry1Ab Protection from – Verma and
Helicoverpa Chand (2005)
armigera
cry1Ab Protection from H. – Sharma et al.
armigera (2006)
Cicer arietinum cry1Ac Resistance against 0.003% Kar et al.
pod borer Heliothis (1997)
armigera
cry1Ac Pod borer insect H. 14.5–23.5 ng. Sanyal et al.
armigera Mg−1 (2005)
cry1Ac Protection from H. 6–20 ng.Mg−1 Indurker et al.
armigera and S. (2010)
litura
Coffea cry1Ac Resistance to leaf >0.1% Leroy et al.
canephera/ miner (2000)
Coffea arabica
Glycine max cry1Ac Resistance to 0.02% Stewart Jr
bollworm (H. zea et al. (1996)
Boddie Boddie) and
bud worm (H.
virescens F.)
Gossypium cry1Ab Resistance to 0.05–0.1% Perlak et al.
hirsutum cry1Ac cotton bollworm (1990)
(H. armigera
Hubner)
cry2Ab Resistance to – Tabashnik
pinkboll worm et al. (2002)
(Pectinophora
gossypiella)
(continued)
44 B. Koul

Ipomoea cryIIIA Resistance against – Morán et al.
batatas δ-endotoxin sweet potato weevil (1998)
(Cylas formicarius)
Lycopersicon cry1Ac Resistance to 0.001% Fischhoff et al.
esculentum tobacco hornworm (1987)
(Manduca sexta L.)
Bt(k) Resistance to 1 ng mg−1 TSP Delannay et al.
tobacco hornworm (1989)
(M. sexta L.),
tomato pinworm
(Keiferia
lycopersicella) and
tomato fruit worm
(Heliothis zea)
cry1Ac Resistance to 0.06–0.42% Mandaokar
fruitworm (H. et al. (2000)
armigera Hubner)
cry1Ab Protection against – Kumar and
fruitborer (H. Kumar (2004)
armigera Hubner)
Meidcago cryIC Resistance to S. 0.01–2% Strizhov et al.
sativa litura and S. exigua (1996)
Nicotiana cry1Aa Resistance to – Barton et al.
tabacum tobacco hornworm (1987)
(M. sexta L.)
δ-Endotoxin Resistance to – Barton et al.
var. kurstaki lepidopteran insects (1987)
HD1
cry1Ab Resistance to 0.001% Vaeck et al.
(M. sexta L.) and
budworm (H.
virescens Fabricius)
cry1Ac Resistance to 0.03% Perlak et al.
(M. sexta L.)
cry1Ab Resistance to 400 ng−1μg Carozzi et al.
lepidopteran pests g−1f.w. (1992)
cry1Ac Resistance to 3–5% McBride et al.
tobacco budworm (1995)
(H. virescens
Fabricius)
cry1C Resistance to S. 0.01–0.2% Strizhov et al.
litura and S. exigua (1996)
(continued)

cry1Ia5 Protection against 0.06% Selvapandiyan
Heliothis armigera et al. (1998)
cry1Aa2 Resistance to H. 2–3% Kota et al.
virescens, H. zea, S. (1999)
exigua
cry2Aa2 Resistance to 35.5% DeCosa et al.
cotton bollworm (2001)
(H. zea Boddie)
δ-Endotoxin Control of – Singh et al.
polyphagous pest S. (2004)
litura
cry2Aa2 Effective control of 0.21% Zaidi et al.
H. virescens (2005)
cry1Ac Control of H. 0.083% Gulbitti-
virescens and M. Onarici et al.
sexta (2009)
Oryza sativa cry1Ab Resistance to 0.05% Fujimoto et al.
striped stem borer (1993)
(Chilo suppressalis
Walker), and leaf
folder
(Cnaphalocrocis
medinalis Guenee)
cry1Ac Resistance to – Nayak et al.
yellow stem borer (1997)
(S. incertulas
Walker)
cry1Ab Resistance to – Wu et al.
(S. incertulas)
cry1Ab/Ac Resistance to 3% Cheng et al.
striped stemborer & (1998)
yellow stem borer
cry1Ab Resistance to – Datta et al.
(S. incertulas
Walker)
cry2A Effective control of 5% Maqbool et al.
yellow stemborer (1998)
and rice leaf folder
cry1B Resistance to 0.01–0.4% Breitler et al.
striped stem borer (2004)
(continued)
46 B. Koul

cry1Ab Resistance to 8 1% Shu et al. 2000
lepidopteran rice
pests
cry1Ab Resistance to leaf 0.01–0.2% Tu et al.
cry1Ac folder (C. medinalis (2000)
Hybrid Guenee) and yellow
Stem borer (S.
incertulas Walker)
cry1Ab Resistance to eight – Shu et al.
lepidopteran rice (2000)
pests
cry1Ac Resistance to 0.1% Khanna and
yellow stem borer Raina (2002)
(S. incertulas)
cry1Ab Resistance to rice – Ye et al.

leaffolder C. (2003)
medinalis
cry1Ab/Ac Resistance to stem – Ramesh et al.
borer (2004)
cry2A Resistance to 9.65– Chen et al.
lepidopteran rice 12.11 μg g−1f.w. (2005)
pest
Populus cry1Aa Resistance to forest – McCown et al.
tremuloides tent caterpillar (1991)
(Malacosoma
disstria
Lasiocampidae)
and gypsy moth
cry3A Chrysomela – Cornu et al.
tremulae F. (Col.) (1996)
Saccharum cry1Ab Resistance to stem – Arencibia
officinarum borer (Diatraea et al. (1997)
saccharalis F.)
cry1Ac Control against 50 ng mg−1 TSP Weng et al.
stemborer in field (2010)
trials
Solanum cryIIIb Resistance to – Arencibia
melongena Colorado potato et al. (1997)
beetle (Leptinotarsa
decemlineata Say)
cryIIIA Resistance to – Jelenkovic
Colorado potato et al. (1998)
beetle (L.
decemlineata Say)
(continued)

cry1Ab Significant 0.02% Kumar et al.
insecticidal activity (1998)
against Leucinodes
orbonalis
Solanum cry1Ab Resistance to tuber – Peferoen et al.
tuberosum moth (Phthorimaea (1990); Rico
operculella Zeller) et al. (1998)
cryIIIA Tolerance to – Adang et al.
Colorado beetle (L. (1993); Perlak
decemlineata Say) and Fischhoff
(1993);
Coombs et al.
(2002)
cryV Bt Resistance to potato – Douches et al.
cry1Ab tuber moth (P. (1998)
operculella Zeller)
cry9Aa2 Resistance to – Gleave et al.
potato tuber moth (1998)
cry1Ab Resistance to H. 0.005–0.04% Chakrabarti
armigera et al. (2000)
cry1Ac Resistance to Tecia 0.02–17 μg g−1 Valderrama
solanivora f.w. et al. (2007)
Vigna cry1Ac Protection from H. – Kamble et al.
aconitifolia armigera (2003)
Zea mays cry1Ab Resistance to 0.4% Koziel et al.
European corn (1993)
borer (Ostrinia
nubilalis Hubner)
cry1Ab Resistance to O. 14–213 ng g−1 Fearing et al.
nubialis f.w. (1997)
cry1Ab Protection against 46.8–85.3 Dutton et al.
S. littoralis ngcm−2 (2005)
Table 2.5 Global status of approved and commercially available Bt-crops
48
Crop Transgenic event(s) Trade name Company Trait genes Trait targets
Cotton LLCotton25, MON FiberMax Bayer cropScience bar, cry 1Ac, cry2Ab Lepidopteran pests,
15985 Libertylink weeds
Bollguard II∗
Cotton DAS-21023-5, Wide strike∗ Dow AgroSciences pat, cry1Ac, cry1Fa Lepidopteran pests,
DAS-24236-5 weeds
Cotton DAS-21023-5, Wide strike∗ Dow AgroSciences pat, cry1Ac, cry1Fa, Lepidopteran pests,
DAS-24236-5, Roundup/Ready∗ CP4EPSPS weeds
MONO1445-2
Cotton DAS-21023-5, Wide strike∗/ Dow AgroSciences pat, cry1Ac, cry1Fa, Lepidopteran pests,
DAS-24236-5, Roundup Ready∗ CP4EPSPS weeds
MON88913-8 Flex
Cotton MON531, Roundup Ready∗, Monsanto cry1Ac, CP4EPSPS Lepidopteran pests,
MON1445-2 Bollguard weeds
Cotton MON88913-8, Bollguard II∗, Monsanto CP4EPSPS, cry1Ac, cry1Ab Lepidopteran pests,
MON15985 Roundup/Ready∗ weeds
Flex
Maize TC1507 Herculex CB Dow AgroSciences and cry1Fa, pat Lepidopteran pests
Pioneer Hi Bred (European corn
borer), weeds
Maize TC1507 Herculex CB Dow AgroSciences and cry1Fa, pat Lepidopteran pests
Pioneer Hi Bred (European corn
borer), weeds
Maize DAS-59122-7 Herculex RW Dow AgroSciences and cry34Ab/cry35Ab1, pat Coleopteran pests
Pioneer Hi Bred (corm rootworms),
weeds
B. Koul
Crop Transgenic event(s) Trade name Company Trait genes Trait targets
Maize TC1507, Herculex XTRA Dow AgroSciences and cry1Fa, cry34Ab1, cry35Ab1, Lepidopteran and
DAS-59122-7 Pioneer Hi Bred pat coleopteran pests,
weeds
Maize DAS-59122-7, Herculex XTRA/ Dow AgroSciences and pat, CP4EPSPS, cry34Ab1, Lepidopteran and
TC1507, NK603 Roundup/Ready∗ Pioneer Hi Bred cry35Ab1, cry1Fa 2 coleopteran pests,
2 weeds
Maize MON89034 Yield guard VT Monsanto cry1A105, cry2Ab2 Lepidopteran pests
pro
Maize MON88017 Yield guard VT Monsanto CP4EPSPS, cry3Bb1 Coleopteran pests
(corm rootworms),
weeds
Maize MON810, Yield guard VT Monsanto cry1A105, cry2Ab2, cry3Bb Lepidopteran and
MON88017 Triple coleopteran pests,
weeds
Maize MON89034, Genuity VT Triple Monsanto cry1A105, cry2Ab2, cry3Bb Lepidopteran and
MON88017 Pro coleopteran pests,
weeds
Maize MON89034, Genuity Smartstax Monsanto and Dow pat, CP4EPSPS, cry 1Fa2, Lepidopteran and
TC1507, TM AgroSciences cry1A105, cry2Ab, cry3Bb1, coleopteran pests,
MON88017, cry34Ab1, cry35Ab1 weeds
DAS-59122-7
Maize BtII, GA21 Agrisure GT/CB/ Syngenta cry1Ab, pat, mutant maize Lepidopteran pests
LL EPSPS (European corn
2 Genetically Modified (GM) Crops Harbouring Bacillus thuringiensis (BT…
borer), weeds
Maize BtII, MIR604 Agrisure CB/LL/ Syngenta cry1Ab, mcry3Aa, pat Lepidopteran and
RW coleopteran pests,
weeds
Maize GA21, BtII, Agrisure 3000GT Syngenta pat, cry1Ab, mcry3Aa, mutant Lepidopteran and
MIR604 (GT/CB/LL/RW) maize EPSPS coleopteran pests,
49
weeds
50 B. Koul
2.9 Management of Resistance Development
Since most of the insect-resistant transgenic plants released for commercial cultiva-
tion harbour single insecticidal Bt-cry gene and the target insect populations are
consistently being exposed to the single toxin protein, the possibility of insects
evolving resistance to single Bt-toxin is high (Zhao et al. 2005; Gunning et al.
2005). There are reports on development of resistance to cry1Ab in open field popu-
lations of the diamond black moth, Plutella xylostella (Tabashnik et al. 1993;
Ballester et al. 1994) and resistance to cry1Aa, cry1Ab, cry1Ac and cry1F have been
reported in laboratory selection experiments (Tabashnik et al. 1997). In recent years,
several Bt-cotton hybrid lines expressing cry1Ac have been approved for commer-
cial cultivation in India, and due to small farm holdings, diverse cropping system
and immigration of insects to alternative hosts, the possibility of developing hetero-
geneous insect population is very high (James 2012). Moreover, pink bollworm
resistant to Bt-cotton harbouring the Bt-cry1Ac gene has been reported in the fields
in India, where farmers rarely follow the refugia strategy (Tabashnik et al. 2010).
Several strategies have been proposed for the management of resistance develop-
ment in field insects, including the application of diverse mixture of toxins, high
expression of Bt-toxin, weedy refugia, hybrid and pyramiding of different Bt-toxin
genes and use of sterile insect (Gatehouse 2008; Tabashnik et al. 2010). Few key
reports that have demonstrated the beneficial aspect of gene pyramiding in trans-
genic plants have been summarized in Fig. 2.6.
Cry1Ab
Cheng et al. (1998),
Mehrotra et al. (2011) &
Yang et al. (2011)
Yang et al.
Gajendra Cry1Ac (2011)
Sivasupramaniam et al. Babu et al.
(2000) (2002)
&
Yang et al. Yang et al.
(2011) (2011)
Cry2Aa Cao et al.
(2002),
Zhao et al.
(2003)
&
Cry2Ab2 Yang et al. Malwar and Glimer
Yang et al. (2011) (2000)
(2011)
Cry1C
Cry1Fa
Fig. 2.6 Bt-gene pyramiding as a preventive and resistance management strategy

In recent years, transgenic plants expressing two dissimilar insect toxins have been
developed, and the most successful example is Bt-cotton ‘Bollgard II’ expressing
cry1Ac and cry2Ab2 genes (Perlak et al. 2001; Zhao et al. 2005). The efficacy and
sustainability of transgenic plants towards prevention of resistance development in
insects rely on the pyramiding and co-expression of two or more diverse transgenes,
without affecting the yield parameters (Zhao et al. 2003; Gatehouse 2008).
2.10 Conclusions
The transfer of Bt-cry gene(s) into plants has provided potentially powerful alterna-
tive strategies for the protection of crops against major agricultural field insects. The
toxin encoded by cry1A gene(s) is highly effective against Lepidopteran group of
insects that causes major damages to crop plants. A comparative interaction of
Bt-toxins Cry1Aa, Cry1Ab and Cry1Ac encoded by corresponding genes with lar-
val midgut binding sites (receptors) of Helicoverpa armigera has shown their com-
petition for common binding sites to different epitopes of the receptors in the order
of Cry1Ac > 1Ab > 1Aa and exerting corresponding toxicity (Estela et al. 2004;
Bravo et al. 2007).
The cry1Ac gene has been extensively modified and codon optimized along with
other modifications for over-expression in different plant species like tobacco, cot-
ton, tomato, potato, chickpea and rice (Sharma et al. 2004; Ferry et al. 2004). The
most successful story is the commercialization of transgenic cotton expressing the
cry1Ac gene as Bollgard I in 1996 and Bollgard II with cry1Ac and cry2Ab in the
year 2000 that has offered significant benefits over the application of synthetic insec-
ticides and yield to the farmers (Perlak et al. 2001). The expression of native (wild
type) full-length cry1Ac gene in plants was very low due to instability and premature
termination of transcript (DeRocher et al. 1998; Perlak et al. 1990). Several modifi-
cations have been incorporated in the cry gene for over-expression, and the major
breakthrough has been in the designing of synthetic versions of the gene with codon
modifications to remove the putative polyadenylation sequences and use of plant
preferred codons for high-level expression in plants (Perlak et al. 1990).
The 5′ and 3′ UTR leader sequences play an important role in transgene expres-
sion by regulating transcription and translation initiation of the foreign gene (Tyc
et al. 1984; Lu et al. 2008). In particular, the use of viral leaders 5′ UTR has shown
to greatly increase the accumulation of recombinant proteins (Dowson Day et al.
1993). The most preferred are tobacco mosaic virus Ω sequence (TMV), tobacco
etch virus (TEV) and alfalfa mosaic virus (AMV) leader sequences (Datla et al.
1993; Gallie et al. 1995; Wang et al. 2001) which have been used for optimization
of expression of several foreign proteins in plants (Haq et al. 1995; Agarwal et al.
2008; Wang et al. 2008). The 3′ UTR contains message for transcript polyadenyl-
ation that directly affects mRNA stability (Chan and Yu 1998). Heterologous 3′
UTR from plant or plant viruses have been used to stabilize the transcript formation
(Hood et al. 1997; Staub et al. 2000; Ko et al. 2003).
52 B. Koul
The use of a synthetic truncated version (1.85 kb) of the cry genes coding toxin
portion has been demonstrated to be most effective for Bt-transgenics against
Lepidopteran insects (Perlak et al. 1990; Sardana et al. 1996). However, the most
promising transgenic event of cotton (Monsanto 531) which has been commercial-
ized is developed with full-length modified cry1Ac gene (Perlak et al. 2001; Purcell
et al. 2004). All insect-resistant transgenic cotton varieties derived from this single
event are performing well under field conditions in different agroclimatic regions
across the globe (James 2012). Interestingly, development of stable transgenic
plants of tomato expressing Cry1Ab toxin has been documented for insect protec-
tion (Kumar and Kumar 2004; Fischhoff et al. 1987; Srivastava 2007). Moreover,
frequency and recovery of promising transgenic plant expressing Bt-toxin coded by
full-length gene is also extremely low. But a question arises as to why the full-length
synthetic gene was used, while the initial trials were performed with its truncated
version? The answer to this is the modified full-length Cry1Ac toxin, although
exhibits lower expression levels, efficiently induces oligomerization, prepore for-
mation and insecticidal activity compared to modified truncated Cry1Ac toxin, at
higher expression levels. These results suggest the importance of modified full-
length cry1Ac gene for stability and integrity of the insect-resistance trait compared
to truncated version of cry1Ab or cry1Ac gene(s) alone (Koul 2013). In reality, the
commercially released Bt-cotton was developed with full-length cry1Ac-like gene
whose nucleotide alignment study revealed that ‘Monsanto 531’ cry gene sequence
is a hybrid gene where the sequence 1–1398 bp is that of cry1Ab gene. It was done
in order to provide a blend of binding characteristics offered by Cry1Ab as well as
pore formation characteristics offered by Cry1Ac, in the aforementioned successful
cry1Ac-like gene, for raising transgenic cotton and its commercialization.
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Characterization and Efficiency
of Rhizobial Isolates Nodulating Cytisus 3
monspessulanus in the Northwest
of Morocco: In Relation to Environmental
Stresses
Taoufik Belechheb, Mohammed Bakkali, Amin Laglaoui,

Abstract
Phenotypic characteristics of 69 rhizobial strains isolated from root nodules of
Cytisus monspessulanus leguminous shrub growing in soils collected from the
northwest of Morocco were studied. Tolerance to salinity, high temperatures,
acid and alkaline pH, heavy metals, carbon and nitrogen source assimilation, and
symbiotic and cultural characteristics allowed the description of a wide physio-
logical diversity among tested isolates. Numerical analysis of the phenotypic
characteristics showed that below the boundary level of 48, 91% of average simi-
larity isolates fell into at least four distinct groups. A number of potential isolates
have been identified for inoculation trials. They were efficient and able to form
nodules with indigenous rhizobia in this region.
Keywords
Rhizobia · Cytisus monspessulanus · Biological nitrogen fixation (BNF)
3.1 Introduction
Cytisus monspessulanus is a legume shrub native to the Mediterranean region very

promising for regeneration of degraded soils in semiarid regions. A method of reha-
bilitating a degraded land is the establishment of agroforestry systems (Le Houérou
1993), where shrub legumes play an important role (Lefroy et al. 1992; Douglas et al.
1996). One of its advantages is good forage quality during winter because it can
T. Belechheb · M. Bakkali · A. Laglaoui · A. Arakrak (*)

Equipe de Recherche de Biotechnologies et Génie des Biomolécules (ERBGB), Faculté des
Sciences et Techniques de Tanger, Tanger, Morocco

64 T. Belechheb et al.
produce large amounts of biomass in the arid continental climate zones (González-
Andrés and Ortiz 1996a). In addition, the quality of the grass produced by Cytisus
monspessulanus is similar to that of alfalfa (González-Andrés and Ortiz 1996b).
Also, it is characterized by its high seed germination and plant survival in the field,
its high dry matter production potential, and its favorable and consistent chemical
composition during the year. Moreover, Cytisus monspessulanus can invade a wide
range of habitats including roadsides, fields, logged areas, bluffs, and coastal areas;
it can adapt well to open, sunny, and well-drained sites; and it has been introduced
for use as ornamentals or hedge plants (Parsons and Cuthbertson 2001).
This perennial leguminous shrub fixes atmospheric nitrogen (N2) via symbiotic
bacteria (general term “rhizobia”) in root nodules and converts it into ammonia
(NH3) to provide nitrogen nutrients for both rhizobia and the host plant (Howard
and Rees 1994; Dixon and Kahn 2004). This can give them an advantage under low
soil nitrogen (N) conditions if other factors are favorable for growth (Raven 2010;
Andrews et al. 2013). Furthermore, N2 fixation by legumes can be a major input of
N into natural and agricultural ecosystems (Andrews et al. 2007; Vitousek et al.
2013), which plays a key role in the nitrogen cycle. Biological nitrogen fixation
(BNF) accounted for 65% of global nitrogen resources (in term of mineral, not N2
gas), while chemical syntheses made about 30% (Dixon and Kahn 2004). Three-
quarters of BNF are generated through symbiosis between rhizobia and legume
plants. However, knowledge about rhizobia and Cytisus monspessulanus is still
unknown that is needed to enhance this symbiosis within this shrub conservation
plans, particularly in the northwest of Morocco.
The aim of this study is to assess the characterization, efficiency, and diversity of
the rhizobia that nodulates Cytisus monspessulanus in the region of Tangier, north-
west of Morocco.
First, plant samples were collected from different locations, and the bacteria
were evaluated based on their response to various physiological conditions such as
salinity stresses, extreme pH, high temperature, and heavy metal tolerance. Then,
the bacteria were assessed for their infectivity, focusing only on rhizobial strains
that are very competitive and can adapt well to extreme environmental conditions.
3.2 Materials and Methods
3.2.1 Location of Nodule Collection
The collection of root nodule was set up in three sites at Tangier City, northwest of
Morocco. This place is characterized by the following geographic coordinates: lati-
tude 35°46′02″ N, longitude 5°47′59″ W, and altitude of sites 150 m (Fig. 3.1).
3.2.1.1 Rhizobia Isolation and Purification

The bacterial strains used in this study were isolated from the root of Cytisus mon-
spessulanus plant collected according to the method described by Vincent (1970)
and Somasegaran and Hoben (1994). The nodules were sterilized with 0.1%
3 Characterization and Efficiency of Rhizobial Isolates Nodulating Cytisus 65
Fig. 3.1 Geographical location of sampling sites
acidified mercuric chloride and ethanol and then washed thoroughly in at least ten
changes of sterile distilled water. The isolation of bacteria was achieved by the
method of Vincent (1970). The colonies growing on three media, yeast extract
mannitol + Congo red (YEM+CR), glucose peptone agar + bromocresol purple
(GPA+BCP), and YEM + bromothymol blue (YEM+BTB), were ascertained to cor-
respond to the phenotypic descriptions recommended by Vincent, depending on the
shape and color of the colonies.
3.2.2 Response to Environmental Stress Factors
Isolates were examined for growth under different stress conditions of high tem-
perature, high salinity, and extreme pH. In the case of temperature tolerance, iso-
lates were kept at 28 (as a control), 38, 40, 42, and 45 °C on YEM plates for 4–5 days.
The ability of the isolates to grow in different concentrations of salt was tested by
streaking isolates on YEM medium containing 0.5%, 1%, 2%, and 5% (w/v) NaCl.
Similarly, the growth of rhizobial strains was compared at different pH (3.2, 5.7, 9,
and 10) on YEM medium.
3.2.3 Utilization of Carbon and Nitrogen Sources
Isolates were tested for their ability to utilize some carbohydrate as a sole carbon
source. For the analysis of carbohydrate utilization, a modified YEM agar was used,
where yeast extract was reduced to 0.05 g/L (Somasegaran and Hoben 1994) and
0.01% NH4NO3 was utilized as a source of nitrogen. Mannitol was replaced by
another carbohydrate to a final concentration of 1% w/v. Two control media were

used for comparison: YEM containing mannitol as a positive control, and the
medium without any carbon source as a negative control. A modified mannitol
medium, in which yeast extract was replaced by 0.1% w/v of the tested amino acid
and mineral salts, was used to investigate the utilization of nitrogen compounds.
N-free modified mannitol medium (devoid of any nitrogen source) was used as a
control. All the plates were incubated at 28 °C for 2–7 days.
3.2.4 Heavy Metal Tolerance
All isolates were tested for their sensitivity to five heavy metals salts, namely CuCl2,
ZnCl2, MnCl2, AlCl3, and PbCl2. Sensitivity pattern was studied on YEM agar plate
containing graded concentration of heavy metals. The stock solution of heavy met-
als was prepared in distilled water, and solution was added to the YEM medium
after filtration through Millipore membrane (0.2 μm porosity). In all experiments,
growth was recorded after 3 days of incubation at 28 °C in triplicate.
3.2.5 Authentication of Isolates
All the rhizobial isolates were evaluated as pure cultures that could form nodules on
their respective host plants. Seeds of the leguminous plants were pre-germinated in
petri dish after scarification with H2SO4. The pre-germinated seeds were planted in
growth pouches (Somasegaran and Hoben 1994) and inoculated with 1 ml YEM
broth culture of each isolate with each treatment replicated three times. Seven days
after planting, the growth pouches were inoculated a second time with 1 ml YEM
broth culture of each isolate. Uninoculated pouches received weekly 0.5% (w/v)
KNO3 as nitrogen source and served as a positive control. The pouches were placed
in racks and kept in the greenhouse. Plants were harvested 14 weeks after planting,
and a nodule scoring chart was applied to evaluate the infectivity of strains using the
chart proposed by (Howieson and Dilworth 2016). Effectiveness of strains in nitro-
gen fixation was evaluated by scoring the total fresh/dry matter weight, plant height,
and nodule number.
3.2.6 Numerical Analysis
The unweighted pair group method with arithmetic mean (UPGMA) (Sneath and
Sokal 1973) was used for cluster analysis of phenotypic features. The similarity
coefficient was computed, and the results are shown as a dendrogram using the
XLSTAT software (2014). The data obtained will be subject to statistical analysis
using the SAS software (2002) followed by mean comparison using Duncan’s test.
The values are means of three replicates.
3.3 Results and Discussion
3.3.1 Phenotypic Evaluation
A total of 69 bacteria were recovered from the root nodules of Cytisus monspessulanus
collected from three sites (Boubana, R’milat, and Sloukia) in the region of Tangier and
were examined for 50 phenotypic characteristics.
The majority of the isolates (85%) were creamy or white opaque with little to
moderate exopolysaccharide (EPS) production and differed slightly in their absorp-
tion of Congo red dye similar to other bacteria hosted in the root nodules of three
Mediterranean wild legume species Hedysarum carnosum, Hedysarum spinosissi-
mum subsp. capitatum, and Hedysarum pallidum (Benhizia et al. 2004). Other inter-
esting and useful characteristics of rhizobia are other growth reactions in the
standard YEM medium containing bromothymol blue (BTB) as the pH indicator. In
our study, all colonies produce an acid reaction on YEM+BTB plates and changed
to yellow after 3 days of incubation at 28 °C. These rhizobia can be classified as
fast-growing rhizobia according to Somasegaran and Hoben (1994). Unlike the ear-
lier belief that rhizobia have no ability to grow on glucose peptone agar medium
(Somasegaran and Hoben 1994; Vincent 1970), in this study, some isolates grew on
this medium and turn the medium to yellow (Fig. 3.2). Finally, all retrieved strains
were Gram negative. These characteristics are the first clues to the identification of
rhizobia, according to Vincent (1970) and Somasegaran and Hoben (1994).
3.3.2 T
he Numbers Are the Number of Isolates Giving Positive
Reaction
The isolated strains showed a large diversity among rhizobia and formed heteroge-
neous group based on physiological properties such as carbon/nitrogen substrate
assimilation; tolerance to pH, salt, temperature, and heavy metal; and their geographic
origin (Table 3.1). This geographic diversity in rhizobial species composition has been
shown to be related to local environmental conditions (Yang et al. 2013; Li et al. 2012).
Growth on YEM+CR Growth on GPA+BCP Growth on YEM
Fig. 3.2 Growth of isolates in different media

Table 3.1 Physiological characteristics of root-nodule isolates

Sites
Boubana R’milat Sloukia
Characteristic n = 21 n = 32 n = 16
Growth at temperature °C 28 21 32 16
40 11 8 9
45 0 1 2
Growth at pH 3.2 18 0 12
9 21 29 16
10 21 29 16
NaCl tolerance % 0.5 21 32 16
2 21 11 16
5 16 0 16
Carbohydrate assimilation Sucrose 21 13 14
Raffinose 21 6 16
Rhamnose 18 18 14
Maltose 21 10 16
Galactose 21 9 16
Glucose 18 18 14
Utilization of nitrogen source His 21 3 16
Pro 21 28 16
Arg 21 30 16
Asp 21 32 16
KNO3 3 0 7
NH4Cl 21 32 13
Resistance to heavy metals μg/ Aluminum 100 21 29 16
ml 200 21 26 13
400 5 0 0
Zinc 50 21 17 16
100 21 14 11
200 21 6 9
Manganese 200 21 32 16
300 21 28 16
400 21 26 13
Lead 400 21 28 16
600 21 28 14
1000 10 7 2
Copper 100 12 4 3
200 0 2 0
400 0 0 0
n = number of isolates
Our isolates have a better assimilation which exceeds 80% of the nitrogen
c ompounds tested, with the exception of ammonium chloride, which appears to be
unusable as a source of nitrogen. On the other hand, it was noted that all isolates can
use the various carbohydrates tested and do not require mannitol as a carbon source.
The osmotolerance was found to be very high, with up to 5% of NaCl for all
isolates from the Sloukia site and the majority from Boubana site, while bacteria
harvested from the R’milat site are inhibited by salinity. Sloukia isolates are also
able to grow over a fairly wide range of pH from 3.2 to 10, while those of R’milat
cannot withstand the acidity of the environment.
As for the temperature, the total tolerance ranges from 28 °C to 45 °C, except for
the CmS4, CmS11, and CmR17 isolates, which have been shown to be resistant to
high temperature.
In soil, salinity and pH changes are generally accompanied by mineral toxicity.
We therefore assessed the degree of inhibition of the growth of the isolates using five
different heavy metals. Our results showed the sensitivity of isolates to high concen-
trations of heavy metals in the order Cu > Al > Pb > Zn > Mg, with the exception of
isolates from Boubana (CmB21, CmB22), Sloukia (CmS9), and R’milat (CmR21)
which conform to the results of Parvaze and Mohammad (2012) and Luo et al.
(2011), respectively. The RL9 strain isolated from the lentil nodules was able to
withstand high lead concentrations of up to 1400 μg/ml, and the LRE07 strain iso-
lated from the plant Solanum nigrum resisted toxic concentrations of heavy metals.
The selection of strains resistant to heavy metals is of great practical interest.
Several studies are currently investigating the use of symbiosis between legumes
and resistant rhizobia as an effective means of “bioremediation” against soil
contamination by heavy metals (Gianfreda and Rao 2004a). In addition, the use of
rhizobium as a preventive agent in contaminated soils has been reported by Abbas
and Kamel (2004).
The analysis using UPGMA (unweighted pair group method with arithmetic
mean) allowed us to highlight phenotypic groups that contain a wide diversity among
the four clusters (Fig. 3.3) determined within each cluster. Indeed, the isolates of
each cluster exhibit great variability in the phenotypic tests examined. Cluster I con-
sists of heat-resistant isolates that have demonstrated basic pH tolerance and resis-
tance to heavy metals (isolates belonging to the site of R’milat, CmR). Cluster II is
the most heterogeneous with isolates belonging to the three different nodule-harvest-
ing sites, with resistance to most of the factors tested. In addition, isolates belonging
to the same geographical site can be grouped in different clusters, as is the case for
the CmB and CmS isolates that are distributed to clusters II, III, and IV.
This phenotypic diversity allows us, however, to select the isolates which are the
right candidates for any inoculation trial of Cytisus monspessulanus, depending on the
different pedoclimatic conditions considered (salt stress, extreme temperatures, etc.)
3.3.3 Plant Assay
The capacity of the rhizobia to produce an infection on the roots of legumes and to
induce the formation of the nodules is called infectivity. This property is limited to
a specific group of rhizobium and host, where the infection is induced. It is impor-
tant to test the ability of strains to produce nodules with the original plant from
which they were isolated (Beck et al. 1993).
Fig. 3.3 Phenogram showing phenotypic relatedness among 69 isolates from Cytisus monspessu
lanus nodules based on average-linkage cluster analysis of 50 characteristics
In our study, the infectivity was tested with the isolates which have shown an
ability to grow under extreme environmental conditions (CmR6, CmR31, CmB3,
CmB12, CmS3, CmS9) and showed a large diversity in their capacity to infect the
host plant and to fix atmospheric nitrogen. The mean comparison showed several
overlapping groups with significant differences between the isolates of the three sites.
Among these isolates, it would appear that CmS9 and CmS3 isolates represented
the most infectious and gave the best results with respect to stem height
(59.25 cm/55.75 cm), fresh matter weight (12.47 g/10.18 g), dry matter weight
(5.29 g/4.44 g), and nodules (33.5 g/27.5 g), compared to positive control and other
isolates, while CmR31 was the least efficient with only 8.92 g of fresh matter
weight, 2.31 g of dry matter weight, and 17.5 g of nodules (Table 3.2). Also, we
noted that the stem height and the weight of fresh and dry matter increase with the
number of nodules. This is confirmed by the study of Rupela and Dart (1980) that
showed a significant correlation between the increase of dry matter and the number
or the dry weight of nodules. And in another study, it has been shown that the dry
matter yield was rather correlated with the nodule leghemoglobin concentration
than with the number or the dry weight of nodules (Dudeja et al. 1981).
Table 3.2 Nodulation and efficiency of Cytisus monspessulanus evaluated at three sites in
Tangier, Morocco
Stem height Weight of fresh Weight of dry Nodule
Isolate (cm) matter (g) matter (g) number
CmS9 59.25a ± 2.86 12.47a ± 0.53 5.29 a ± 0.33 33.5a ± 1.92
CmS3 55.75a,b ± 1.58 10.18b ± 0.53 4.44b ± 0.31 27.75b ± 3.65
Positive 54.50a-c ± 1.77 9.25b,c ± 0.59 3.57c ± 0.43 0
control
CmR31 48.5b-d ± 2.87 8.92c ± 0.87 2.41d ± 0.16 17.5d ± 2.44
CmR6 47.5a ± 8.05 9.96b,c ± 0.36 3.27c ± 0.21 22.5c ± 2.32
CmB3 47b-d ± 2.13 9.31b,c ± 0.74 3.2c ± 0.40 21.25c,d ± 3.15
CmB12 44d ± 6.50 9.33b,c ± 0.60 2.79c,d ± 0.48 22.5c ± 2.4
SEM 1.267 0.243 0.191 1.053
Sig. ∗∗ ∗∗∗ ∗∗∗ ∗∗∗
SEM standard error of the mean
Values with different letters (a–d) in the same column are significantly different (P < 0.0001)
Sig.: Level of significance: (**): p < 0.01; (***): p < 0.001
3.4 Conclusion
To conclude, this study is the first work that phenotypically characterizes root-nodule
microsymbionts of Cytisus monspessulanus in Morocco.
Combined analysis of all phenotypic data allowed us to identify high-performing
isolates. These were justified by the differences between the clusters defined here,
based on the behavior of the isolates in relation to the extreme environmental condi-
tions (carbon/nitrogen source utilization, heavy metal tolerance, salinity, pH, and
maximum temperature). Moreover, our results show that among the six isolates, two
showed their effectiveness and infectivity by the high intensity of nodule formation
on the roots of the host plant and consequently the fixation of nitrogen.
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Isolation and Characterization
of the Roots and Soil Endomycorrhizae 4
of Hedysarum pallidum Desf.
in the Northeast of Morocco
Rachid M’saouar, Mohammed Bakkali, Amin Laglaoui,

Abstract
This study aims to describe the endomycorrhizal status of Hedysarum pallidum
Desf. in the northeast of Morocco. A detailed description of the mycorrhizal
associations in this species soil and roots is reported for the first time in this
study. To achieve this goal, some tests were run on soil and root sample collected
from the rhizosphere of Hedysarum pallidum Desf. The microscopic examina-
tion of the roots revealed the presence of typical endomycorrhizal structures. The
mycorrhizal frequency was greater than 98%, the mycorrhizal intensity was high
(50.9%), and the arbuscular intensity reached 34.76%. The spore number of the
arbuscular mycorrhizal fungi (AMF) isolated from the soil was 1200 spores/100 g
of soil. These spores presented three AMF genera: Glomus, Scutellospora, and
Septoglomus. The diversity of arbuscular mycorrhizal fungi present in the rhizo-
sphere can be selected and used in improving the production of vigorous plants.
Keywords
Arbuscular mycorrhizal fungi · Diversity · Hedysarum pallidum Desf. · Morocco
4.1 Introduction
Hedysarum pallidum Desf. (Fig. 4.1), a Mediterranean herbaceous legume, partici-

pates in the soil conservation and has the ability to fix its own nitrogen. However, its
potential use as a forage plant was emphasized.
R. M’saouar · M. Bakkali · A. Laglaoui · A. Arakrak (*)

Research Team of Biotechnology and Biomolecular Engineering (ERBGB) Faculty of
Sciences and Techniques, Tangier, Morocco

74 R. M’saouar et al.
Fig. 4.1 Hedysarum pallidum Desf. from the site of Touissit (northeast of Morocco)
This wild steppic plant was able to survive on the most polluted soils in the
a bandoned antimony mining area of DjebelHamimat (Algeria) (Rached-Mosbah
1997); it also tolerates the salty soils (Foury 1954).
Mycorrhizal is a symbiotic association between plant root and specialized soil
fungi, with evidence that it helps plants in the acquisition of immobile nutrients
such as P, N, Zn, and Cu in deficient soils (Ibiremo and Fagbola 2008). Arbuscular
mycorrhizal fungi (AMF) provide plants with a higher protection against pathogens
and toxic elements in the soil (Herrman et al. 2004). The host plants are strongly
affected by AMF, and this association has been classified as vital in the structuring
among plant species (Wardle 2002).
Presently, the mycorrhizal association and its beneficial role toward plants are
accepted as a universal phenomenon. Mycorrhizal associations are so prevalent that
the non-mycorrhizal plant is more of an exception than the rule. In addition, plants
growing under natural conditions differ in their ability to enrich the soil by the
mycorrhizal propagules; the effectiveness of AMF depends on the native host spe-
cies (Azcon-Aguilar et al. 2003) (Caravaca et al. 2005).
However, there are no studies on the potential of Hedysarum pallidum Desf. to
produce arbuscular mycorrhizal inoculum to be used in revegetation strategies. So,
the present work was undertaken to study the arbuscular mycorrhizal fungi (AMF)
of Hedysarum pallidum Desf. in the northeast of Morocco. It provides the basis for
any project aiming the improvement of this plant growth through the AMF as these
symbiotic fungi are the most favorable in this context. For this purpose, specific
tests were carried out: extraction, counting, and identification of endomycorrhizal
spores of soils collected from the northeast of Morocco and measurement of the
root’s mycorrhization rate.
4 Isolation and Characterization of the Roots and Soil Endomycorrhizae… 75
The study site

(Twissit)
Fig. 4.2 Geographic location of the sample site
4.2.1 Soil Samples and Plant Material
Sampling of soil is performed in the first 20 cm deep on the site of Touissit (Cardinal
direction: northeast/latitude: 34.470020°N/longitude: 1.773897°W) in the northeast
of Morocco (Fig. 4.2). Three individual plants of Hedysarum pallidum Desf. were
randomly chosen, and three soil samples were taken. The soil was air-dried, sieved
on 2-mm mesh sieves, and placed in favorable conditions throughout the duration of
the study.
4.2.2 Methods
4.2.2.1 AMF Spore Extraction

Spores were extracted following the wet sieving method described by Gerdemann and
Nicolson (1963). In a 1-L beaker, 100 g of each composite sample of soil is submerged
in 0.5 L of tap water and stirred for 1 minute with a spatula. After 10 to 30 s of settling,
the supernatant is passed through a sieve of four bunks with decreasing mesh size (500,
200, 80, and 50 μm). This operation was repeated twice. The content retained by the
sieves of 200, 80, and 50 μm was divided into two tubes and centrifuged for 5 min at
2000 rev/min. The supernatant was discarded, and a viscosity gradient is created by
adding 20 ml of sucrose solution at 60% in each centrifuge tube (Walker 1982). The
mixture is rapidly stirred, and the tube provided is centrifuged again for 1 min at
3000 rpm/min. Unlike the first centrifuging, the supernatant is poured onto the sieve of
50 μm; the resulting substrate was rinsed with distilled water to remove sucrose. The
spores were then recovered with a little distilled water in an Erlenmeyer flask and
counted, and their density (spore number per 100 g dry soil) was determined.
4.2.2.2 Measuring of the Root Mycorrhization Rate

As described by Phillips and Hayman (1970), roots were first washed with water,
and the finest ones were cut into a length of 1 cm, immersed in a solution of 10%
potassium hydroxide (KOH), and placed in a water bath at 90 °C for 2 h. Fragments
were rinsed with distilled water and stained with a solution of cresyl blue for 15 min
at 90 °C in water bath. They were finally rinsed with distilled water and observed
under a microscope, each fragment being carefully checked along its entire length,
at magnifications of 100 and 400 to record mycorrhizal structures, such as arbus-
cules, hyphal walls, vesicles, and intra- and intercellular hyphae. The AMF arbus-
cule and vesicle frequency and levels inside the root bark were measured by the
method of Trouvelot et al. (1986) and expressed as frequency of AMF colonization
(F%), intensity of AMF colonization (M%), and arbuscule abundance (A).
Parameters of mycorrhization were calculated with the MYCOCALC software
(available at: http://www.dijon.inra.fr/mychintec/Mycocalc-prg/download.html.)
4.3.1 Richness, Diversity, and Identification of AMF Spores
Concerning the estimation of the spore density in the soil, the average recorded was
1200 spores/100 g of soil, and extracted spores had generally a spherical shape. A
detailed analysis of the morphological characteristics of the spore’s community
revealed the presence of three genera (Fig. 4.3) in the order of Glomales:
Scutellospora, which was the most abundant, Glomus, and Septoglomus.
The largest proportion belongs to the family of Gigasporaceae, when it comes to
Scutellospora sp. The family of Glomeraceae is represented by Glomus sp. and
Septoglomus (Fig. 4.4).
Analysis of AMF spores found in the soil of H. pallidum Desf. showed that on
average their number was high and reached 1200 spores/100 g soil, which was more
important than some results in the literature. It was reported that about 170
spores/100 g dry soil were found in the association Quercus ilex-Tetraclinis articu-
lata (Bakkali Yakhlef et al. 2009), about 63–98 spores/100 g soil in the coastal
dunes of the Souss-Massa (Hatimi and Tahrouch 2007), and about 2–22 spores/100 g
soil in the rhizosphere of Casuarina sp. (Tellal et al. 2008).
However, about 5384 spores were obtained in 100 g of soil cultivated previously
by peanut (Bouhraoua et al. 2015).
Our study has shown a high density of spores of mycorrhizal fungi in soil under
H. pallidum Desf., indicating a significant mycorrhizal potential infection; counting
AMF spores from soils showed that the plants are capable to enrich the soil with
mycorrhizal propagules. The result reflects the biological soil fertility.
Spore walls
Scutellospora Septoglomus Glomus.

Hyphae
Suspensor bulbus
Glomus Scutellospora
Fig. 4.3 Showing genera of Glomales: Scutellospora that was the most abundant, Glomus, and
Septoglomus
700
600
500
400
300
200
100
0
Scutellospora Septoglomus Glomus
Fig. 4.4 Abundance of the endomycorrhizal genera spores isolated from the rhizosphere of
Hedysarum pallidum Desf.
4.3.2 Evaluation of Root Mycorrhization
The examination of the roots of Hedysarum pallidum Desf. showed that all of them
were mycorrhized and densely colonized, and the mycorrhizal frequency of the roots
measured in the studied site was important reaching 98.89% (Fig. 4.5). Also, the
mycorrhization intensity attained 50.9%, and the arbuscular intensity was 34.76%.
The observation of Hedysarum pallidum Desf. root fragments collected from the
studied site helped to demonstrate the presence of mycorrhizal structures: intra- and
extraradical hyphae, vesicles, and arbuscules (Fig. 4.6).
The mycorrhizal roots rate of H. pallidum Desf. observed in this study appeared
higher compared with those of Tetraclinis articulata (between 27 and 57%) and
Ceratonia siliqua (40%) recorded in Morocco by Abbas et al. (2006).
120
100
80
Mycorrhizal frequency
60 Mycorrhizal intensity
Arbuscular intensity
40
20
Fig. 4.5 Mycorrhizal parameters of Hedysarum pallidum Desf. on the soil of the studied site
Arbuscules. (G: 400) Intraradical hyphae(G: 400) Extraradical hyphae(G: 100)
Vesicles(G: 400)
Fig. 4.6 Six roots with mycorrhizal infection of Hedysarum pallidum Desf.
The mycorrhizal infection observed high soil propagule pressure on the roots of
Hedysarum pallidum Desf., also indicating a relatively large abundance of arbus-
cules and vesicles in the roots. These parameters indicate the ability of fungi to
spread into the root system of the plant and to establish exchanges through the fine
arbuscular ramifications. Hedysarum pallidum Desf. was able to provide a high
number of infective propagules per unit of soil weight. Previous reports have already
described that many plants from the Mediterranean area form arbuscular mycorrhi-
zae association and have been classified as “obligatory mycorrhizal” or as “highly
dependent on mycorrhiza (Caravaca et al. 2002).
4.4 Conclusion
By integrating particular parameters (richness and diversity of AMF spores, high

mycorrhizal frequency, and different mycorrhizal structures in the roots), Hedysarum
pallidum Desf. is regarded as a mycotrophic legume establishing a close symbiosis
between the endomycorrhizae of the rhizosphere. This species promotes the growth
of the propagules in the soil which is biologically fertile. For this reason, autochtho-
nous plant species are widely used for reclaiming degraded lands in Mediterranean
areas (Caravaca et al. 2002). Therefore, the diversity of arbuscular mycorrhizal fungi
naturally present in the soils can be selected and used in (a) the restoration of
degraded ecosystems, (b) the improvement of the production of vigorous forage
plants, and (c) the valuation of the fallows and their enrichment in organic nitrogen.
References
Abbas Y, Ducousso M, Abourough M, Azcon R, Duponnois R (2006) Diversity of arbuscular
mycorrhizal fungi in Tetraclinisarticulata (Vahl) masters woodlands in Morocco. Ann For Sci
63:285–291
Azcon-Aguilar C et al (2003) Analysis of the mycorrhizal potential in the rhizosphere of represen-
tative plant from desertifcation-threatened Mediterranean shrublands. Appl Soil Ecol 22:29–37
Bakkali Yakhlef S Abbas Y, Abourouh M, Hafidi M, Duponnois R 2009 Diversité phénotypique et
moléculaire des champignons mycorhiziens de l’association Quercus ilex-Tetraclinisarticulata.
Actes du congrès international “Biotechnologie microbienne au service du développement”
(MICROBIOD) Marrakech, MAROC 02–05 Novembre 2009. www.ucam.ac.ma/micro
Bouhraoua D, Aarab S, Laglaoui A, Bakkali M, Arakrak A (2015) Effect of PGPR on growth
and mycorrhization of KT22’s peanut variety (Arachishypogaea L.) grown in the northwest of
Morocco. Am J Res Commun 3(2):12–24. www.usa-journals.com, ISSN: 2325-4076
Caravaca F, Barea JM, Figueroa D, Roldán A (2002) Assessing the effectiveness of mycorrhi-
zal inoculation and soil compost addition for enhancing reafforestation with Olea europaea
subsp. sylvestris through changes in soil biological and physical parameters. Appl Soil Ecol
20:107–118
Caravaca F, Alguacil MM, Barea JM, Roldán A (2005) Survival of inocula and native AM fungi
species associated with shrubs in a degraded Mediterranean ecosystem. Soil Biol Biochem
37:227–233
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Friends and Foes: Phyto-Microbial
Interactions in Molecular Perspective 5
Shyam Solanki, Gazala Ameen, Debankur Sanyal,
Shalu Jain, Ammar Elakhdar, Shwetank Lall,
Kishore Chittem, Leah Brueggeman, Ajay Kumar,
and Robert Brueggeman
Abstract
Soil acts as an natural abode for plants as well as for diverse micro/macroflora
and fauna, and thus provides the rhizosphere environment, the fraction of soil
surrounding the root system, where plants interact with their root microbiomes.
Plants maintain a continuum of interactions with associated microbes that have
effects on their cellular physiology resulting in changes in development and
function, which can have both positive and negative outcomes. Plant-microbial
or microbial-microbial interactions that occur at the plant root-soil interface can
also have dynamic effects on the rhizosphere microbiome that greatly affects the
overall health and vigor of plants, a key metric in agricultural productivity. This
chapter reviews the current understanding of the range of interactions happening
in the rhizosphere and the recent advancements that next generation sequenc-
ing technologies have had on the ability to identify and classify the rhizosphere
microbiome.
S. Solanki (*) · G. Ameen · L. Brueggeman · R. Brueggeman

Department of Crop and Soil Sciences, Washington State University,
Pullman, Washington, DC, USA
D. Sanyal
Department of Soil Science, North Dakota State University, Fargo, ND, USA
S. Jain · K. Chittem
Department of Plant Pathology, North Dakota State University, Fargo, USA
A. Elakhdar
Institute of Plant Genetic Resources, Kyushu University, Fukuoka, Japan
S. Lall
ICAR-Indian Agricultural Statistics Research Institute, New Delhi, India
A. Kumar
Department of Plant Sciences, North Dakota State University, Fargo, ND, USA

82 S. Solanki et al.
Keywords
Rhizosphere · Microbiome · Next generation sequencing (NGS) · PhyloChip ·
16S rRNA · Shotgun metagenomics
5.1 Introduction
“We know more about the movement of celestial bodies than about the soil
underfoot.”
This remark from Leonardo da Vinci can still be used to describe the current
knowledge gaps in the understanding of soil microbiomes. Although scientific per-
spectives and approaches have accelerated our understanding in recent generations,
we have just begun to break the shackles in this field of science as characterizing
and understanding the diversity underlying the biology and ecology of the soil
microbiome are still one of the great challenges in science. Soils act as the natural
biological support with a complex group of micro- and macrofauna that represents
one of the most important and richest microbial communities on earth. This com-
plex ecosystem that harbors vast genetic diversity, which has only just begun to be
detected, let alone harnessed, is hypothesized to contain new forms of antibiotics,
catalysts, and metabolites that could be utilized for yet to be imagined purposes in
human medicine, agriculture, and beyond (Gans et al. 2005; Wagg et al. 2014).
As the home of many forms of life, mediating diverse and multiphyletic interac-
tions, the soil acts as the factory of life. Soil organisms consist of harmful, benefi-
cial, and neutral living components in the perspective of plant health and productivity.
The rhizosphere, defined as all soil under plant root influence, acts as a hotspot
between soil inhabiting organisms and plant roots, thus providing the temporal and
special cues that drive the dynamic interactions that occur in it (Hinsinger and
Marschner 2006; Raaijmakers et al. 2009). The rhizosphere communities are largely
made up of organisms such as archaea, bacteria, oomycetes, fungi, algae, arthro-
pods, and their associated viruses as well as other classes of living organisms (Buée
et al. 2009; Mendes et al. 2013).
Many biotic and abiotic factors including but not limited to soil type, soil prop-
erties, prevailing environmental conditions, inhabiting flora and fauna, and agricul-
tural management practices have strong influence on the structural and functional
diversity of rhizosphere communities (Berg and Smalla 2009). Also, the impor-
tance of plant species and how their root properties play a large role in the function
of soil-phyto-microbial interactions cannot be understated. Plants in their natural
environment are surrounded by a remarkable diversity of plant associated microbes,
termed as “the plant microbiota.” Establishment of microbiota in and around the
plant depends on multiple factors that facilitate niche colonization, a term used for
establishment of a population of species in a community, taking account of totality
of biological and environmental factors affecting the species and utilization of
these factors in the species establishment (Bulgarelli et al. 2013; Vandermeer
1972). Clearly, the majority of bacterial diversity in the soil is dominated by a few
5 Friends and Foes: Phyto-Microbial Interactions in Molecular Perspective 83
bacterial phyla such as Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi,

Firmicutes, and Proteobacteria (Fierer et al. 2009). However, it has been shown
that the presence of the host plant drives the diversity in the rhizosphere microbi-
ome filtering few classes of bacteria and selecting for Actinobacteria and
Proteobacteria classes. Understanding this complex biophysicochemical interplay
and its components is imperative to use soil biodiversity for protective agriculture.
Next generation omics such as metagenomics and metabolomics approaches pro-
vides a remarkable tool to understand the complex plant microbiota and their inter-
twined complex interactions.
To understand microbial population, the use of next generation sequencing
(NGS) provides an excellent tool for detection of genetic diversity represented by
16S rRNA coding gene sequencing, shotgun metagenome (whole genome) or tran-
scriptome profiling. Classification of microbial groups can be achieved by analyz-
ing similarity of sequenced reads to a known reference which has predetermined
taxonomical units for phylotyping, or by similarity of sequenced reads within a
microbial community and assigning operational taxonomic units (OTUs) (Chen
et al. 2013; Schloss and Westcott 2011). Here we will attempt to sketch the rhizo-
sphere microbial interaction in detail and the genomics approaches utilized to clas-
sify the microbial community involved in such interactions.
5.1.1 Friends and Foes: A Side to Pick
Rhizosphere acts as a battlefield between plants and microbes, full of microbial

warriors, few acting as foe, few as friends, and the rest not directly taking part in this
battle but just being part of the dynamic microbiome interactions with microbiome
structure and community, deciding the fate of such interactions around the plant
roots. Many bacterial and fungal communities help plants to derive micro- and mac-
ronutrients essential for plant growth and development. Plant reciprocates the favor
by providing habitat and carbon sources to the bacterial and fungal communities in
its rhizosphere. Such mutualistic relationships benefit both interacting partners and
are also utilized in agricultural practices. However, pathogenic microbes within the
microbiome are greedy and seek out opportunities to utilize the plant carbon sources
without reciprocating any energy. This invasion and infection of the plant system to
use its nutrient source for its establishment, colonization, and reproduction are the
foundation of a host-pathogen relationship and once established as a specialized
pathogen begin a molecular arms race where both constantly evolve to gain the
upper hand in the interaction.
5.1.2 Microbiome Communities as a Friend for Plant
Root microbiomes provide a remarkable pool of friends for plants, helping them in the
nutrient cycling and maintenance of rhizosphere activity essential for proper plant
health and vigor. Many bacterial communities majorly belonging to Actinobacteria,
Bacteroidetes, Firmicutes, and Proteobacteria inhabit a soil zone that is in close prox-
imity to the plant root interface, making up an active site of the soil biome. Few of
these bacterial species help in N2 fixation symbiotically or nonsymbiotically. Legumes
fix atmospheric N2 symbiotically with a specific group of bacteria in the genus
Rhizobium (Phillips 1980). Rhizobia, a group of gram-positive, spore-forming bacte-
ria, belong to the α-subclass of Proteobacteria (Rhizobiaceae) and nodulate legumes
(Hong et al. 2012; Peters et al. 1995). The Proteobacteria have been classified into six
different genera, namely, Rhizobium, Bradyrhizobium, Mesorhizobium, Allorhizobium,
Azorhizobium, and Sinorhizobium, which are phylogenetically separated from each
other (De Lajudie et al. 1998). The Rhizobia reduce atmospheric N2 inside specialized
organs, “nodules,” which are formed as an outcome of a multistep process initiated
with infection of plant roots by Rhizobia due to positive chemotaxis to root exudates
(Caetano-Anollés et al. 1992). The Rhizobia bacteria are attracted to amino acids, the
dicarboxylic acids present in the root exudates, and even to very low concentrations of
excreted flavonoids, which are exudates from the secretory part of rhizodeposition,
and the bacteria once in contact subsequently attach to the plant root surface. The
infection proceeds with specific attachment of bacterial polysaccharides (lipochitooli-
gosaccharides) to specific lectin proteins of host plants, followed by root hair curling,
deforming, and branching (Bohlool and Schmidt 1974; Van Rhijn and Vanderleyden
1995; Yuan et al. 2017). Infection threads are formed, cortical cells divide to form
nodules, and Rhizobia are released in the plant cell cytoplasm through the infection
thread (Wood and Newcomb 1989) and colonize and initiate biological nitrogen fixa-
tion, a symbiotic interaction between the plant and the transformed bacteriod.
Biological nitrogen fixation (BNF) is an important input of nitrogen (N) in the global
agricultural systems. BNF is catalyzed by a complex metalloenzyme, nitrogenase,
which is composed of two main components: a heterotetrameric core and a homodi-
meric dinitrogenase reductase subunit (Kim and Rees 1994). The dinitrogenase reduc-
tase is an iron (Fe) protein containing 4 Fe atoms (Fe4S4), and the heterotetramer is a
molybdenum-iron (MoFe) protein containing 2 Mo and 30 Fe atoms (Peters et al.
1995). This enzyme system helps the reduction of dinitrogen (N2) that involves reduc-
tion of Fe binding protein by different electron carriers (like ferredoxin), followed by
transfer of one electron from Fe protein to Mo-Fe protein, and subsequently from
Mo-Fe protein to the N2 (the substrate) in the last step of reduction (Kim and Rees
1994). This conversion (reduction) of atmospheric N2 to ammonia (NH3) can only be
carried out by a small group of microorganisms like Rhizobia, Frankia and
Azospirillum, known as “diazotrophs” (Burns and Hardy 2012).
The first discovered Rhizobium genes related to nitrogen fixation were nif
(fixation) and nod (nodulation) genes, and it was found that the nod genes and
genes related to host specificity were tightly clustered with the nif genes(Long
2001). Dinitrogenase reductase subunit of the nitrogenase complex is encoded by
the nifH gene, while the heterotetrameric core is encoded by nifD and nifK (Yates
et al. 1992). The nifH gene is a universally accepted biomarker for BNF as it is
found to be highly conserved among N2-fixing organisms in different natural
environments (such as marine, terrestrial, and hydrothermal sites) and is widely
used to study the ecological and evolutionary aspects of N2-fixing bacteria
(Izquierdo and Nüsslein 2006; Mehta et al. 2003; Wartiainen et al. 2008). A pre-
vious study by Hennecke et al. (1985) reported that the phylogeny of the nifH
genes is closely related to the 16S rRNA gene and the nifH genes and might have
evolved with the bacterial evolution. This finding is in contrast with the conven-
tion of “lateral gene transfer” among microorganisms. However, a comprehen-
sive study focused on sequence analysis of a large number of nifH genes was
carried out and found that the phylogenetic trees of nifH genes were inconsistent
with the phylogenetic tree of 16S rRNA; instead, a phylogenetic similarity was
found with nodA genes (Haukka et al. 1998). The nifH genes were mostly stud-
ied by culture-independent approaches because these provide better understand-
ing of the bacterial community than the culture-dependent method microbiome
community studies (Poly et al. 2001). Along with PCR amplification followed by
denaturing gradient gel electrophoresis (DGGE), other approaches like DNA
hybridization analysis, restriction fragment analysis, and cloning and sequencing
approaches were used to study the diversity and abundance of nifH genes (Freitag
and Prosser 2003; Hamelin et al. 2002; Neufeld et al. 2001; Stres et al. 2004).
Composition of nifH gene pools were also studied in various environments using
various techniques, such as polymerase chain reaction (PCR)-restriction frag-
ment length polymorphism (RFLP), PCR cloning, DGGE, and fluorescently
labeled terminal (FLT)-RFLP (Chelius and Lepo 1999; Piceno and Lovell 2000;
Zehr et al. 1995).
Different soil physicochemical properties like texture, total carbon (C), and total
N influence the variation in nifH gene pools, and the nifH genetic structures are the
outcomes of the adaptation of the changing or constant soil environmental condi-
tions (Poly et al. 2001). In a gene expression study on Azotobacter vinelandii, it was
revealed that the nifH gene expression is positively correlated with N2-fixation
(Bürgmann et al. 2003). Again, different dry bean cultivars showed differential nifH
gene expressions, depending on their symbiotic efficiencies with the rhizobia
strains, correlated with their N2-fixation potentials (Sanyal et al. 2020, Akter et al.
2013).
The diversity and abundance of nifH genes have been associated with N2-fixation
rates and thus depend on the structure and dynamics of bacterial communities (Hsu
and Buckley 2009; Reed et al. 2010). PCR and qPCR are considered as powerful
tools to study specific gene expression and soil microbiome diversity (Bürgmann
et al. 2003; Zehr and McReynolds 1989). Zehr and McReynolds (1989) designed
degenerate universal PCR primers to target a wide range of variable nifH gene
sequences in diverse bacterial communities and for the first time were able to study
community composition. Later, many universal primers were designed to target the
nifH gene in the most conserved portions of amino acid sequences (Bürgmann et al.
2004). In an “in silico” analysis, 51 universal and 35 group specific nifH primers
were assayed using more than 20,000 nifH gene sequences, and 15 universal mark-
ers were identified that successfully targeted more than 90% of the N2-fixers (Gaby
and Buckley 2017). However, this study also revealed that many of these primers
targeted genes that are not related to N2-fixation, and were often found to miss sig-
nificant variants of nifH genes (Gaby and Buckley 2017).
5.1.3 Microbial Foes: A Concern for Plant Health
Soil-borne pathogens are major yield limiting factor in most agriculture crops
worldwide. Soil-borne pathogens can survive in the bulk soil as a part of the soil
microbiome for several years. When a suitable host plant is available, the patho-
gens identify the host plant by perceiving cues from the rhizosphere and initiate
infection to cause disease. The rhizosphere acts as a playground to the complex
microbial community, which can also affect the host pathogen interaction influenc-
ing the outcome of the pathogen infection process, resulting in compatible (suscep-
tibility and disease) or incompatible (resistant) interactions. Bacterial pathogens
such as Erwinia and Pectobacterium species cause disease in plants and reduce
their health and vigor and are important soilborne pathogens. Fungi and oomycetes
make up one of the most important groups of plant pathogens accounting for more
than 70% of all the major crop diseases (Agrios 2005). Fungi are eukaryotic, fila-
mentous, multicellular, and heterotrophic organisms. They produce a network of
hyphae called the mycelium and absorb nutrients from their host (Alexopoulos
et al. 1996). Most of host-pathogen associations are very specific with a special-
ized host range. The host-limiting fungi are termed as biotrophs (feed from living
host cells without killing them) or necrotrophs (kill the host tissues to derive nutri-
ents as part of the colonization process). Some of the fungal pathogens overwinter
in soil and may survive for years in soil even in the absence of their host.
Nevertheless, controlling root fungal pathogens is a major challenge compared to
foliar pathogens that attack the aboveground portions of the plant (Bruehl 1987).
Fusarium root rot is a prominent root disease and is caused by a complex of several
Fusarium species. For example, four species of the Fusarium (F. graminearum, F.
culmorum, F. poae, and F. sporotrichioides) are mainly associated with the foot
and crown rot disease in wheat (Kuzdraliński et al. 2014). Another devastating
disease of wheat and barley is Fusarium head blight (FHB), which also causes
grain contamination by secreting harmful mycotoxins, leaving them useless for
consumption as well as planting material (McMullen et al. 2012). To date, at least
15 known species of the F. graminearum species complex have been reported as
the causal agent of FHB (O’Donnell et al. 2008; Wang and Cheng 2017). Specific
species were more prevalent than others in FHB disease complex to wheat, and
such variations are related to the ecological preference of the pathogens. One of the
most common species, F. oxysporum, causes vascular wilt disease in a wide variety
of economically important crops (Beckman 1987). Sudden death syndrome, a
major yield-limiting soybean disease, is caused by F. virguliforme (previously
named Fusaium solani f. sp. glycines).
Signaling processes are the first and most critical step in defining either success
or failure in establishing disease. Fungal and oomycete pathogens depend on sev-
eral different signaling molecules from the host plant to germinate, grow, and per-
sist in the rhizosphere. Many cues such as amino acids, organic acids, flavonols,
glucosinolates, indole compounds, fatty acids, and polysaccharides are secreted
from microbial community and plant roots in the rhizosphere to establish commu-
nication. This wide range of secreted compounds is collectively known as
rhizodeposition (Dakora and Phillips 2002; De-la-Peña and Loyola-Vargas 2014).

The type and composition of root secretion are dependent on plant species and
cultivars, growth stage, and stress factors, which all greatly influence the rhizo-
sphere microbiome communities. Root exudates can affect the microbial commu-
nity in the soil favoring beneficial microbes while preventing the growth of harmful
microbes (Huang et al. 2014; Szoboszlay et al. 2016). Chemical analysis of vege-
table root exudates revealed the presence of major organic acids such as citric,
succinic, and malic acid and major sugars such as fructose and glucose (Badri et al.
2012; Kamilova et al. 2006). Low concentrations of phenolic compounds like
p-hydroxybenzoic, gallic, coumaric, cinnamic, ferulic, salicylic, and sinamic acids
in root exudates stimulated conidial germination of F. oxysporum f. sp. niveum,
while inhibitory effects were observed at higher concentrations (Wu et al. 2008a,
b) indicating roles of rhizodeposition in the species dynamics of rhizosphere
microbiota. A study by Wasmann and VanEtten (1996) showed that transformation-
mediated chromosome loss and pisatin demethylase gene (PDA1) disruption can
decrease the virulence of the pea pathogen Nectria haematococca (anamorph F.
solani f. sp. pisi). Pisatin (a phytoalexin) can be detoxified by cytochrome P-450-
mediated demethylation. Recently, a cluster of five pea pathogenicity (PEP) genes,
which are expressed during N. haematococca infection, were identified in close
proximity to PDA1 on a supernumerary chromosome (Han et al. 2001). The PEP
gene cluster have differences in GC-content and codon bias compared to genes on
essential chromosomes indicating that horizontal transfer occurred for these patho-
genicity gene clusters.
5.2 etagenomics Approaches for Taxonomical

M
and Functional Classification of Microbiomes
To understand and unravel the components of microbiomes, it is required to adopt a

comprehensive approach rather than studying each component separately. Use of
molecular techniques such as PCR, qPCR, and DGGE provides valuable tools to
study individual elements of the microbiome, but they are insufficient to undertake
large-scale analyses to understand the system as a whole (Sharpton 2014). The
advent of next generation sequencing techniques and recent high-throughput
sequencing technologies (HTST) coupled with state-of-the-art bioinformatics pipe-
lines to assemble sequencing reads provided a boom for soil microbiome studies to
explore the diverse taxonomical inhabitants in the rhizosphere. Reduced sequencing
cost of per mega base of DNA sequence (Fig. 5.1) further made technology avail-
able to all, and genome sequencing became more common specially for small
genomes such as fungal, bacterial, and viral whole genome sequencing and pan-
genome sequencing (Wetterstrand 2016). Today, many approaches are available for
microbiome species identification and classification in conjunction with NGS
platforms.
Fig. 5.1 An estimation of raw cost of per megabase (Mb) of DNA sequence over a period of time
(reproduced from the National Institutes of Health (Wetterstrand 2016))
5.2.1 P
hyloChip and Amplicon-Based Classification
of Microbiomes
Identification and phylogenetic classification of fungal species largely rely on inter-

nal transcribed spacer 1 (ITS1) and ITS2 region sequencing of the ribosomal RNA
(rRNA) cistron as a universal DNA barcode (Schoch et al. 2012; Stielow et al.
2015). However, ITS2 is considered as a region of choice for fungal classification
due to better representation in databases and less variability in length (Nilsson et al.
2009; Op De Beeck et al. 2014). For fungal and oomycete genomics and functional
data analysis, FungiDB provides an excellent database for the design of experimen-
tal primers to carry out phylogenetic analysis (Stajich et al. 2012). Nonetheless, the
major portion of the soil microbiome is encompassed by prokaryotes such as bacte-
rial species; thus, we will limit our discussion to the perspective of bacterial micro-
biome. Nuclear 16S rRNA (ribosomal RNA) gene sequencing has been successfully
utilized to identify prokaryotic microbial diversity in natural and agricultural eco-
systems (Fierer et al. 2012; Pace et al. 1986). The ubiquitous prokaryotic 16S rRNA
cistron is comprised of nine V1-V9 hypervariable regions flanked by conserved
DNA sequences suitable for universal primer binding and amplification. Thus, the
diversity in variable region is utilized to classify the organisms based on sequence
dissimilarities (Chakravorty et al. 2007; Ree et al. 1989). The 16S rRNA gene
sequences for bacterial microbiome studies are available from the National Center
for Biotechnology Information (NCBI) or other secondary datasets such as
EzBioCloud (Yoon et al. 2017). The recent interest of the scientific community to
study the soil microbiome in agricultural and non-agricultural ecosystems has led
to the development and utilization of the high-density 16S rRNA gene oligonucle-
otide microarray popularly known as the PhyloChip (Brodie et al. 2007). In the
study by Weinert et al. (2011), a high-density 16S rRNA probe-based PhyloChip
array capable of detecting 8741 known OTUs was utilized to study and characterize
the bacterial microbiota of two different field sites with different soil properties
from the rhizosphere of three different potato cultivars. Some 2432 OTUs were
detected and classified into 43 phyla in which only one phyla comprised of less than
10 OTUs. Interestingly, differences in soil between two sites influence root bacterial
microbiota describing 28% divergence in detected bacterial OTUs, whereas only
9% of the OTUs showed cultivar dependency. Approximately 4% of the OTUs were
dependent on both cultivar and site, belonging to Proteobacteria, Bacteroidetes,
Firmicutes, Chloroflexi, and Streptomycetaceae phyla. Thus, these phyla dominated
the bacterial community in each site irrespective of cultivars grown. This study
concludes that historical profiling of soil plays a major role in determining the
potato rhizosphere bacterial microbiota composition rather than the effect of the
cultivar genotype itself. However, such probe-based studies relied on dominant
ribotypes in the sample profile and were limited to the number of known probe
OTUs on the PhyloChip. The latest generation of PhyloChip arrays includes 60,000
OTUs capable of classifying147 phyla and 1123 classes of bacterial and archaeal
domains, thus enormously increasing its capacity (Hazen et al. 2010). Using the
high density PhyloChip, Mendes et al. (2011) identified 33,346 bacterial and
archaeal OTUs in the rhizosphere of sugar beet plants grown in disease suppressive
soil for Rhizoctonia solani, a fungal pathogen (Mendes et al. 2011). Interestingly,
Proteobacteria, Firmicutes, and Actinobacteria were found associated with disease
suppression in the study samples (Mendes et al. 2011). Thus, this study implicates
a role of natural modification in microbial communities to provide disease suppres-
sion, a remarkable finding useful for agricultural crop settings. A study carried out
on wild and domesticated barley (Bulgarelli et al. 2015) to understand associated
bacterial root microbiota indicated a large share of microbiota is comprised of
Actinobacteria, Bacteroidetes, and Proteobacteria using the 16S rRNA survey with
an NGS approach. Interesting inferences were made upon annotation and functional
classification of sample sequences acquired from bulk soil and host inhabited rhizo-
sphere and root samples. Root and rhizosphere bacterial taxas were significantly
enriched for the 12 functional categories of proteins considered to be important for
adaptation of microbes to interact with the host root along with protein families for
sugar transport and iron mobilization. Thus, the presence of the host barley plants
drives the diversity of the rhizosphere for protein functional categories considered
to be important for host-microbe interactions either pathogenic or mutualistic
(Bulgarelli et al. 2015).
5.2.2 Shotgun Metagenomics for Microbiome Studies
Despite the majority of studies utilizing DNA barcodes and 16S rRNA amplicon-
based pyrosequencing, shotgun sequencing approaches for metagenome analyses
provide a remarkable upgrade to classify and understand the structure and function
of rhizosphere microbiome interactions. PCR-based sequencing approaches can
introduce amplification bias (Hong et al. 2009) and require availability of known
taxonomically informative genetic markers and thus make analysis difficult for
novel or highly diversified bacterial taxa. Further, horizontal gene transfer between
distantly related taxons, especially for 16S rRNA genetic locus, can result in over-
representation of diversity (Acinas et al. 2004). Thus, shotgun approaches enables
researchers to study uncultured microbiota and also overcome the majority of the
aforementioned concerns for PCR-based sequencing approaches. The shotgun
sequencing approach was successfully utilized in a study conducted by Castañeda
and Barbosa (2017) to investigate the taxonomic diversity of bacterial and fungal
species and their metabolic function in the forest and vineyard soils in Chile. They
found bacterial OTU abundance in both habitats, i.e., forest and vineyard, and
Proteobacteria accounted for the majority of bacterial phyla and most of the fungal
communities were assigned to the Ascomycota. Thus, using shotgun approaches
enabled researchers to study the soil from both the bacterial and fungal perspective
using a single DNA library, whereas in amplicon-based sequencing, it is required to
make fungal and bacterial amplicon libraries separately based on ITS and 16S
rRNA markers, respectively.
5.2.3 Approaches to Acquire and Analyze Metagenomics Data
Various large-scale coordinated efforts were initiated to investigate and characterize

the soil microbial taxonomy and functional diversity such as the Earth Microbiome
Project (Gilbert et al. 2014), ECOFINDERS (http://projects.au.dk/ecofinders/), and
TerraGenome (Vogel et al. 2009). These initiatives provide a good resource of infor-
mation for any interested novice laboratories that decide to make the leap into soil
microbiome studies. However, many practical roadblocks still prevail before carry-
ing out a microbiome analyses. Since the majority of environmental microbes are
unculturable, this is a major hurdle to acquiring and studying representative natural
environment samples. For example, using soil samples from a single experimental
site, Delmont et al. found that the DNA extraction methods can produce larger vari-
ations in the metagenomic composition of the microbiome than either the season of
sampling or the soil depth (Delmont et al. 2012). Thus, just DNA extraction meth-
ods used can have a profound effect on confounding the data for analysis of metage-
nomic variation. Since the requirement of a high-quality DNA sample is the first
step and depending on the sequencing platform, DNA amplification steps may be
required for low DNA yielding soil samples (permafrost soil), these PCR steps fur-
ther add bias to the samples (Mackelprang et al. 2011). Thus, to adopt a common
efficient method although unlikely to develop is required for a true representation of
sample.
One of the important steps to exploring the complexity of soil microbiomes is the
analysis of large amount of sequencing data after the NGS run. Many bioinformat-
ics pipelines and methods have been described. The recent availability of many
online analysis tools such as Galaxy and KBase that can perform sequence quality
analysis and assembly has made this task a bit less complex and has provided an
excellent alternative for setting up a computing facility and expertise in “big data”
analysis. Open-source pipelines such as Quantitative Insights Into Microbial

Ecology (QIIME2) (Caporaso et al. 2010) or MEGAN6 (Huson et al. 2007, 2016)
are very useful tools, which can be installed locally to analyze data offline. EBI
metagenomics provides a cloud-based automated pipeline for analysis and archiving
of metagenomic data with tools to determine the phylogenetic diversity as well as
functional and metabolic analysis of submitted datasets (Mitchell et al. 2016)
(Fig. 5.2). Many informative web resources and literature are also available to walk
through the process of data analysis, yet researchers must pave their own paths
when utilizing these publicly available tools (Table 5.1) (Clooney et al. 2016; Kunin
et al. 2008; Oulas et al. 2015; Quince et al. 2017; Thomas et al. 2012).
5.3 Concluding Remarks
Biodiversity loss is a major concern affecting ecosystem homeostasis and stability

among its inhabitants. Soil biodiversity comprised of micro- and macrofauna is still
largely understudied; thus, we have insufficient information on the effects that cli-
mate change and environmental pollution are having on the soil ecosystems world-
wide. The majority of these soil inhabitants or the microbiome components play a
crucial role in soil health, nutrient cycle, rhizosphere interactions, and ecosystem
function; thus, mass extinction and loss of ecosystem homeostasis could be happen-
ing under foot, and we may never be aware of it or its consequences until it is too
late.
On the rhizosphere frontline between plant and microbes, many microbial
friends, foes, and spectators stand tall in the arena presenting numerous possibilities
for interaction. The microbiome structure and community decide the fate of such
interactions in the rhizosphere. Many bacterial and fungal pathogen communities
prevail as a part of soil microbiome, yet introduction of the agricultural systems
sufficiently modify the microbiome structure of particular soils leading to the estab-
lishment of a unique microbial community. In a study conducted by Costa et al.
(2007) comparing uncultured Pseudomonas in bulk and rhizosphere soil, it was
observed that strawberries infected with Verticillium dahliae attract fluorescent
pseudomonads having close relatedness to P. fluorescens strain F113, a biocontrol
agent. This strain produces DAPG (2,4-diacetylphloroglucinol), a phenolic com-
pound having antiphytopathogenic properties (Bangera and Thomashow 1999;
Costa et al. 2007). Thus, such examples indicate the summoning of beneficial bac-
teria from microbial pools by plant for their own benefits. On the other hand, patho-
genic microbes act as foes continuously invading plants as a host to gain nutrients
required for their pathogenic lifestyle. Many fungal pathogens such as Sclerotinia
sclerotiorum, Fusarium oxysporum, and Pythium spp. cause root disease in a wide
plant host range adversely affecting the plant health as previously discussed.
Symbiotic microbes live in a mutualistic relationship with plants and thus work as a
friend in the soil microbiome fraction. The use of Bacillus and Trichoderma strains
(Benítez et al. 2004; Chowdhury et al. 2015) is common as a biocontrol agent to
augment the soil microbial population to keep check on pathogenic fungal strains,
Fig. 5.2 An illustration and schematic overview describing the rhizosphere microbiome and its interac-
tions. PCR amplicon-based or shotgun metagenomics approaches can be adopted using next generation
sequencing (NGS) approaches. Various bioinformatics pipelines can be adopted for sequence assembly
and alignment for microbiome identification, taxonomical classification, and functional annotation
Table 5.1 Few of the Web-based graphical user interface (GUI) and command line interface
(CLI) bioinformatics pipelines for microbiome metagenomics data analysis and visualization
Resource Type Link
EBI metagenomics Web https://www.ebi.ac.uk/metagenomics/
IMG/M Web https://img.jgi.doe.gov/cgi-bin/m/main.cgi
MG-RAST Web http://metagenomics.anl.gov/
Metavir Web http://metavir-meb.univ-bpclermont.fr/
LEfSe (GALAXY) Web https://bitbucket.org/biobakery/biobakery/wiki/
lefse
KBase Web https://kbase.us/
MicrobesOnline Web http://www.microbesonline.org/
MEGAN6 GUI https://ab.inf.uni-tuebingen.de/software/megan6/
welcome
QIIME2 CLI http://qiime.org/index.html
PATRIC Web https://www.patricbrc.org/
and they are extensively studied. Pseudomonas-based formulations are also popular
for soil augmentation and biocontrol (Weller 2007). Interaction among microbial
communities, plant roots, and rhizodeposition to establish rhizosphere microbiota is
a complex mechanism and needed to be understood for use for improved agricul-
tural practices and soil health.
The integration of modern genetic tools is allowing for the dissection of the com-
plex structure and makeup of the rhizosphere microbiome in relation to plant-
microbe and microbe-microbe interactions and for the study of their intertwined
molecular signaling pathways. Next generation sequencing approaches for metage-
nomics and metabolomics analysis coupled with conventional phenotype-dependent
approaches to study the structure and function of microbial life in the plant rhizo-
sphere illuminated and enhanced our limited understanding of these interactions
and its components. This information will help us to make better use of the currently
underutilized and vastly untapped resources of the soil microbiome. Shotgun-based
NGS approaches will not only enable us to classify different known soil microbes
but will also help to identify rare species prevailing in certain ecosystem important
for its maintenance. It is important to establish a larger comprehensive catalog of
microbial communities in different soil types for future comparative studies and
taxonomical and functional classification. Characterizing the specific inhabitants of
soil microbiome communities will allow us to pinpoint their contributions and
cooperation in natural ecosystems and bring them under the purview of biotechno-
logical manipulations to work as microbial factories. The microbiome acts as a
dynamic entity and requires its largest to smallest community fractions to work
properly as a system of ‘fellowship or disunity’ for its maintenance. Individual
microbial contribution to its microbiome entity can be sketched by the famous
remark quoted by the fictional character Galadriel in J.R.R Tolkien’s novel Lord of
the Rings—“Even the smallest person can change the course of the future.”
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Isolation and Screening of Inorganic
Phosphate Solubilizing Pseudomonas 6
Strains from the Lotus creticus
Rhizosphere Soil from the Northwest
of Morocco
Imane Achkouk, Saida Aarab, Amin Laglaoui,

Mohammed Bakkali, and Abdelhay Arakrak
Abstract
In vitro screening of plant growth-promoting (PGP) traits was carried out using
63 Pseudomonas, isolated from the rhizosphere of Lotus creticus collected from
the northwest region of Morocco. The isolates were tested for their capacity of
solubilizing tricalcium phosphate (TCP) on Pikovskaya (PVK) solid medium,
and 20 Pseudomonas could solubilize TCP. We then selected five phosphate
solubilizing bacteria (PSB) based on their halo diameters to undergo more tests.
As a result, all five isolates were positive for hydrogen cyanide (HCN) produc-
tion, and the amount of indole acetic acid (IAA) produced by them fluctuated
between 5.72 ± 0.09 mg/L and 195.16 ± 0.38 mg/L. All the selected strains could
produce siderophores. Except urease activity, all isolates possessed at least two
enzyme activities such as cellulase, chitinase, protease, and amylase activities. In
addition, all these PSB were able to produce ammonia. Thus, the strains were
evaluated by TCP solubilizing quantitative assay in PVK liquid medium. The
concentrations of solubilized P were between 20.80 mg/L and 159.55 mg/L. This
solubilization was accompanied by a pH decrease of the medium from 7 to 4.16.
Furthermore, the five PSB were tested in vitro for antagonism against phyto-
pathogenic fungus Fusarium oxysporum, and all the PSB except LCP18 strain
were capable of inhibiting its growth.
I. Achkouk · A. Laglaoui · M. Bakkali · A. Arakrak (*)

S. Aarab
Faculté des Sciences et Techniques d’Al Hoceima, Ajdir Al Hoceima, Morocco

100 I. Achkouk et al.
Keywords
Plant growth-promoting (PGP) · Phosphate solubilizing bacteria (PSB) ·
Fusarium oxysporum
6.1 Introduction
One of the most frequently encountered problems is the low availability of phospho-
rus (P) for plants, which is one of the essential components for the plants. This low
availability is due to the fact that the vast majority of P in the soil is present in
insoluble forms, while the plants absorb it only in two soluble forms: the monobasic
(H2PO4−) and dibasic (HPO42−) ions (Glass 1989). In calcareous soils, which is the
case of the majority of Moroccan soils (FAO 2006), the P-Ca forms represent
80–90% of total mineral phosphorus (Arakrak et al. 2006).
Therefore, there is a growing awareness on the use of environment-friendly, sustain-
able nutrient management practices that lay emphasis on restoration and maintenance
of soil fertility both in the short- and long-term. Thus, effective biological technologies
like the use of plant growth-promoting rhizobacteria (PGPR) are being exploited for
enhancing crop yields. Among PGPR, Pseudomonas are considered to be the most
promising group of PGPR involved in growth promotion and biocontrol of plant patho-
gens (Singh et al. 2010). In previous studies, Pseudomonas spp. have shown increased
production of indole acetic acid (IAA) (Patten and Glick 2002), the production of sid-
erophores (Dey et al. 2004), phosphate solubilization (Wu et al. 2005), ACC deami-
nase, root elongation, degradation of toxic compound (Bano and Musarrat 2003), and
as biological control agent against phytophatogens (Dey et al. 2004).
According to the remarkable PGPR characters of Pseudomonas sp., we isolated
bacteria from the rhizosphere of Lotus creticus belonging to this genus. The isolates
were evaluated for phytostimulator and biocontrol capabilities in order to gain
potential inoculants to enhance crop yields.
6.2.1 Isolation of Pseudomonas from Lotus creticus Rhizosphere
Soil samples were collected from the rhizosphere soil of Lotus creticus growing in
Achakar and Tahadartregion (Tangier). The rhizosphere was dug out with intact root
system. The samples were placed in plastic bags and stored immediately at 4 °C. The
rhizospheric soil was suspended in 9 mL of physiological saline solution, centri-
fuged for 1 h (200 rpm, 28 °C), and 1 ml sample was diluted from 10−1 to 10−7.
Aliquots of 100 μL of each dilution were plated on a King’s B solid medium. The
plates were incubated at 28 °C for 3 days (King et al. 1954). After incubation, mor-
phologically variable colonies were picked up and purified on the same medium,
King’s B plates.
6 Isolation and Screening of Inorganic Phosphate Solubilizing Pseudomonas… 101
6.2.2 Phosphate Solubilization
The isolates were screened for phosphate solubilization; the colonies were trans-
ferred to the Pikovskya medium and then incubated at 28 °C. The plates were exam-
ined after 7 days of incubation, and data were recorded; visual detection of phosphate
solubilizing ability of bacteria is possible by plate screening methods that show
clear zone around the colonies in media containing insoluble mineral phosphate
(tricalcium phosphate) as P source. The diameter of solubilization was calculated by
subtracting the total diameter (diameter halo + colony diameter) of the diameter of
colony. Isolates with solubilizing diameters ≥0.4 cm were conserved.
6.2.3 Production of Hydrogen Cyanide (HCN)
To estimate HCN production, 100 μL of bacterial culture were streaked on TSA

supplemented with 4.4 g/L glycine. Filter paper discs (9 cm diameter) were soaked
in 2% sodium carbonate in 0.5% picric acid solution and were placed in the lid of
each Petri dish (Bakker and Schippers 1987). The plates were sealed with parafilm
and incubated at 28 °C. Change in color from yellow to orange or brown indicated
the synthesis of HCN production.
6.2.4 Determination of Indole Acetic Acid (IAA) Production
The tested bacterial strains were cultured for 2 days in sucrose-minimal salts (SMS)
medium (sucrose 1%; (NH4)2SO4 0.1%; K2HPO4 0.2%; MgSO4 0.05%; NaCl
0.01%; yeast extract 0.05%; CaCO3 0.05%; pH 7.2) supplemented with 0.05% of
L-tryptophan. After incubation, 1 mL of supernatant was mixed with 2 mL of
Salkowski reagent, and the development of a pinkish color indicated the production
of IAA (Gordon and Weber 1951). The absorbance of pink color developed after
25 min of incubation at room temperature was read at 535 nm.
6.2.5 Production of Siderophores
The bacteria were spot inoculated on King’s B medium, and the plates were incubated
for 3 days at 28 °C. A layer of chrome azurol S (CAS) medium (Schwyn and Neilands
1997) was poured on the surface of these plates. After 24 h in the dark, change in color
of CAS medium from blue to orange indicated the production of siderophores.
6.2.6 Production of Hydrolytic Enzymes
6.2.6.1 Cellulase
The strains were grown on carboxyl methyl cellulose (CMC) agar containing (g/L)
KH2PO4 1, MgSO4.7H2O 0.5, NaCl 0.5, FeSO4.7H2O 0.01, MnSO4.H2O 0.01,
NH4NO3 0.3, CMC 10, and Agar 15. The pH was adjusted to 7 with 1 M NaOH. The
plates were incubated at 30 °C for 5 days. At the end of incubation, agar medium
was flooded with an aqueous solution of Congo red (0.1%) and then washed with
NaCl solution (1 M). Formation of clear zone indicated cellulose degradation
(Miller 1959).
6.2.6.2 Chitinase
In order to demonstrate the production of the chitinase enzyme that degrades chitin,
colloidal chitin was used as carbon source. The selective medium was prepared
using (g/L) colloidal chitin 0.8, K2HPO4 2.7, KH2PO4 0.3, MgSO4.7H2O, NaCl 0.5,
KCl 0.5, yeast extract 0.13, and agar 20. The plates were incubated, and the produc-
tion of chitinase was indicated with the formation of transparent halos around the
colonies.
6.2.6.3 Protease
The protease production is tested using an agar medium prepared by mixing two
fractions A and B. The fraction A contains (g/L) dried milk 50 and ionized water
500 mL, and it is autoclaved at 115 °C for 10 min. The fraction B contains (g/L)
agar 20 and ionized water 500 mL and is autoclaved at 120 °C for 20 min. After
sterilization, and at a temperature of 55–60 °C, the two fractions were mixed asepti-
cally and poured on the Petri dishes. Then, 100 μL of fresh culture of each isolate
was then deposited separately on the dishes which were incubated at 28 °C for 48 h.
The formation of a clear halo around the colony indicates the degradation of the
protein.
6.2.6.4 Amylase
Amylase production was evaluated on starch agar plates containing (g/L) peptone 5,
beef extract 3, soluble starch 2, and agar 15. The isolates were streaked on starch
agar plates and incubated for 48 h at 30 °C. Amylase production was detected by
flooding the plates with iodine solution (Cappuccino and Sherman 1992).
6.2.6.5 Urease
Testing the production of urease consists in the alkalinization of the medium con-
taining urea as the sole nitrogen source, and phenol red is used as a pH indicator.
The medium is prepared using (g/L) glucose 1, peptone 1, NaCl 0.5, KH2PO4 2,
Congo red 0.012, and agar 20. The pH was adjusted to 7 with 1 M NaOH. After
sterilization of the medium, and at a temperature of 45 °C, 50 mL of 40% urea steril-
ized by filtration was added. The plates were inoculated with bacteria to assess.
After incubation, the production of urease is indicated by the development of a
purplish pink color around the colonies on a yellow background.
6.2.7 Production of Ammonia
All the bacterial isolates were tested for the production of ammonia as described by
Cappuccino and Sherman (1992). Twelve-hour-old bacterial cultures were inocu-
lated in peptone water (10 mL) and incubated for 48–72 h at 36 ± 2 °C. Development
of brown to yellow color after addition of Nesseler’s reagent indicates a positive test
for ammonia; no color change indicates negative test.
6.2.8 Qualitative Phosphate Solubilization Assay
Isolates were tested for their ability to solubilize the phosphorus in the liquid
medium; this is realized by inoculating 50 mL of PVK liquid medium by 500 μL of
bacterial culture. Autoclaved and not inoculated media were used as controls. The
inoculated media and controls were incubated for 7 days at 28 °C on shaker
(180 rpm). The media were centrifuged at 13.000 rpm for 20 min. The soluble P of
the supernatant was determined by the colorimetric method of Ames (1966), and the
pH of the medium was also determined.
6.2.9 Antifungal Activity
The antifungal activity was tested using potato dextrose agar (PDA) (Rabindran and
Vidyasekaran 1996). Bacterial isolates were tested for their ability to inhibit the
growth of the plant pathogenic fungus Fusarium oxysporum isolated and character-
ized by El Aaraj et al. (2015). A 5-mm agar disk of the fungus was deposited in the
center of the PDA Petri plates. A volume of 20 μL of each bacterial culture was
seeded in 3 cm spot of the fungal strain. A negative control of the fungal strain is
tested in the absence of bacteria. Plates were incubated for 7 days at 25 °C and
examined for evidence of fungal growth inhibition. The zone of inhibition was
determined using the following formula:
% Inhibition of radial growth = 100 × (r1–r2/r1)
where r1 is the radial growth of the mycelium in control and r2 is the radial growth
of the mycelium in treatment. The results represent the average of three replicates.
6.2.10 Statistical Analysis
The data are reported as means ± SD (standard deviation) for three replicates. The
results were compared by analysis of variance (ANOVA) according to Fisher’s pro-
tected LSD test (p < 0.05). Data analysis was made on the rate of produced IAA,
solubilized P, and percentage of inhibition.
The rhizosphere of plants is known to be an environmentally preferred area by soil

microorganisms due to its rich nutrient availability. PGPR colonize plant roots and
improve its growth by a variety of mechanisms. The exact mechanism by which
PGPR stimulate the plant growth is not clearly known, although several activities
such as phosphate solubilization production of hormones and inhibiting the growth
of phytopathogens are generally thought to be involved in promoting plant growth.
6.3.1 I solation and Selection of Phosphate Solubilizing

Bacteria (PSB)
A total of 63 isolates were tested for their inorganic phosphate solubilizing activity
on solid medium. The solubilization of TCP is indicated by the formation of a clear
halo around the bacterial colony on solid PVK medium. A total of 20 isolates out of
63 tested (31.74%) in PVK solid medium were able to form a transparent halo indi-
cating the phosphate solubilization, and halo diameters were between 0.2 and
1.2 cm. Zaidi et al. (2009) explained that the solubilization of inorganic phosphorus
usually occurs following the action of low molecular weight organic acids which are
synthesized by various soil bacteria. These organic acids can react as ion-mineral
chelating agents or can cause a decrease in the pH to acidify the medium. This acidi-
fication leads to the release of the phosphate ions from the mineral P by substituting
the Ca2+with H+. So the TCP solubilization in solid medium depends primarily on
the concentration and the type of organic substances produced by the rhizobacteria.
At the end of this test, five best isolates that formed halo diameters ⩾0.4 cm were
selected for further tests of PGP activities.
6.3.2 Screening of PGP Activities of Isolated Pseudomonas
The PSB retained, namely, five isolates, underwent the qualitative test of HCN pro-
duction, in which the gas production is indicated by a color change of filter paper
from yellow to orange/brown. Indeed, all of the PSB were able to produce this gas,
which is involved in the biocontrol mechanism of plants (Table 6.1). In fact, Voisard
Table 6.1 PGP activities of tested Pseudomonas

Isolates Concentration of IAA (mg/L) Halo diameters (cm) HCN Siderophores
LCP18 5.72 ± 0.09a 1.2 +++ +
LCP25 6.04 ± 0.88a 0.8 ++ +
LCP79 9.01 ± 0.93a 0.7 ++ +
LCP88 195.16 ± 0.38b 0.4 +++ ++
LCP90 5.83 ± 0.12a 0.5 ++ +
The letters on the bars of the same parameter indicate significant differences according to Fisher’s
protected LSD test (p < 0.05)
et al. (1989) showed that the production of HCN by Pseudomonas strains suppresses
infectious diseases of plants while the mutant strain defective in the synthesis of
HCN loses the ability to protect plants against diseases. This shows the crucial
antagonist role played by this compound in the biocontrol mechanism. However, it
was shown by Bakker and Schippers in 1987 that this compound may also cause
negative effects on plant growth by inhibiting growth of the roots. Blumer and Haas
(2000) reported that the HCN is formed from glycine by the action of HCN synthase
enzyme, which is associated with the plasma membrane of certain rhizobacteria. We
can say then that the presence of this enzyme is essential for the formation of HCN
in which its quantity controls the concentration.
The results showed that LCP88 isolate produced significantly the highest con-
centration (195.16 ± 0.38 mg/L) (Table 6.1). The amounts produced by LCP18,
LCP25, LCP79, and LCP90 were not significantly different. Patten and Glick (1996)
explained that IAA is the most common plant hormone and one of the most charac-
terized hormones. In addition, Mohite (2013) showed in a study of isolation and
characterization of IAA producing bacteria from the rhizosphere soils of several
plants, banana, corn, cotton, and wheat, that five retained strains br1, br2, br3, wr2,
and mr2 produced more IAA when incubated in the medium provided with the
tryptophan than when incubated in medium without L-tryptophan. The production
of auxin by the PGPR may cause positive and negative effects on plant growth
(Vacheron et al. 2013). The effectiveness of the auxin depends on its concentration;
at a low concentration, it improves plant growth (Patten and Glick 2002), while at a
high concentration, it inhibits the root growth (Xie et al. 1996).
Iron plays an important role in nutrition and plant protection. Therefore, the abil-
ity to synthesize siderophores was evaluated for the retained PSB that were all posi-
tive by forming an orange halo around the colony (Table 6.1). Thus, our results are
in agreement with those obtained by Castagno et al. (2011) in which all the PSB
isolated from the rhizosphere of Lotus tenuis were positive for the production of
siderophores. Similarly, Vansuyt et al. (2007) showed that the iron-pyoverdine syn-
thesized by P. fluorescens strain C7 and tested on Arabidopsis thaliana plants
increased iron levels in the plant and improved their growth.
6.3.3 Production of Lytic Enzymes
The PSB were tested for their capacity of producing some lytic enzymes, and
Table 6.2 shows the results: two of the five isolates showed positive cellulase activ-
ity and were able to form a halo around the bacterial colony. Hence, four of the
isolates produced chitinase during this qualitative test, and the highest chitinolytic
activity was observed for the LCP90 isolate. In addition, the evaluation of the capac-
ity of the isolates to hydrolyze starch allowed us to identify three isolates with
amylase activity. The protease production test showed that four PSB were positive.
These three enzymes have the ability to degrade component of the fungal cell wall,
which is an important mechanism of fungal inhibition (Reetha et al. 2014).
Table 6.2 The results of the qualitative tests for cellulase, chitinase, protease, amylase, and ure-
ase and ammonia production of the five isolates selected
Isolates Cellulase Chitinase Protease Amylase Urease Ammonia
LCP18 − + + − − ++
LCP25 + + − − − +
LCP79 ++ ++ +++ ++ − +
LCP88 − − + + − +++
LCP90 − ++++ ++ ++ − +++
(−) absence of production; (+) low production; (++) average production; (+++) strong production
Based on the change in color of the culture medium by means of the pH indicator
(phenol red), none of the PSB were able to hydrolyze the urea. Indeed, Someya
et al. (2007) demonstrated that bacteria producing a single lytic enzyme may well
be used in combination with other biocontrol agents, thus leading to a synergistic
inhibitory effect against phytopathogens.
6.3.4 Ammonia Production
The addition of the Nessler’s reagent during the qualitative test of the production of
ammonia causes a change in the color of the medium from yellow to orange/brown.
This allowed us to identify the capacity of the five strains to produce this compound.
It has been demonstrated that volatile substances are involved in the biocontrol
mechanism against various phytopathogens.
6.3.5 Q
uantitative Test of Phosphate Solubilization in Liquid
Medium
The solubilized P assay was carried out according to Ames method (Ames 1966).
The P concentrations obtained during this test were between 20.80 mg/L and
159.55 mg/L (Fig. 6.1). Similar results were obtained in a study of tricalcium phos-
phate solubilization by Rhizobium isolated from the nodules of three legumes
(Hydesarum coronarium, Vicia sativa, and Lupinus angustifolius) carried out by
Aarab et al. (2009). They tested six best isolates for their ability to solubilize TCP
in liquid medium. The results obtained in this study ranged from 9.62 mg/L to
158.64 mg/L. Besides, another study by Park et al. (2010) who isolated PSB from
different rhizospheric samples showed that six isolates tested solubilized quantities
higher than 250 mg/L.
Indeed, the concentrations of solubilized P by the isolates LCP79 and LCP90
were significantly higher than the other isolates. It should be noted that the isolates
LCP18 and LCP25 formed important solubilization diameters in PVK solid
medium: 1.2 cm and 0.8 cm, respectively, while they solubilized less P in liquid
medium 71.91 mg/L and 59.38 mg/L. Conversely, the isolate LCP90 and LCP79
were less efficient in the qualitative test than the quantitative one; they formed
200
Mean concentraons of
b
solubilized P in mg/L
b
150
100 a a
50 a
0
LCP18 LCP25 LCP79 LCP88 LCP90
Isolates
Fig. 6.1 Concentrations of solubilized P by the five PSB retained after 7 days of incubation. The
letters on the bars of the same parameter indicate significant differences according to Fisher’s
protected LSD test (p < 0.05)
8
7 d
e
6 c
b
5 a
4
PH
3
2
1
0
Isolates
Fig. 6.2 Final pH of the five PSB incubation media after 7 days of incubation. The letters on the
bars of the same parameter indicate significant differences according to Fisher’s protected LSD test
(p < 0.05)
modest diameters in the solid PVK medium (0.5 and 0.7 cm, respectively); however,
they solubilized higher concentrations of P (175.46 and 159.55 mg/L, respectively).
These results are in agreement with those obtained by Cherif (2014) who showed
that the analysis of the correlation between P solubilization on solid and liquid PVK
medium is not significant. Also, Alam et al. (2002) obtained a significantly negative
correlation between the concentration of solubilized P from the tricalcium phos-
phate and the solubilization halo diameter. It should be noted that the solubilization
of the TCP is accompanied by a decrease in pH value after 7 days of incubation of
the five isolates (Fig. 6.2). The initial pH of the medium (7) decreased to a value of
4.16. The decrease of the pH of the medium was significantly different between the
five isolates.
The analysis of the dependence of the amount of solubilized P and the final pH
of the medium revealed the existence of a significantly negative correlation
(r = −0.93) (Fig. 6.3). This shows that the more the pH of the medium is acid, the
more the phosphate is solubilized.
8 y = -0.0164x + 7
R² = 0.8151
6
4
PH
0
0 50 100 150 200
[P] (mg/L)
Fig. 6.3 Correlation between solubilized P and pH
This is in agreement with Vinodrai and Vyas (2014) who tested the ability of four
isolates to solubilize P in liquid medium, in which the correlation between these two
parameters was negative. Moreover, Wani et al. (2008) reported that the P solubiliza-
tion is very often associated with a decrease in the pH of the medium, and they showed
the existence of a significant negative correlation between the pH values and the inor-
ganic P concentrations, in particular calcium phosphate. According to the literature,
several mechanisms can be adapted by the PSB to solubilize inorganic P; Hwangbo
et al. (2003) have shown that the production of organic acids by PSB plays an impor-
tant role in the acidification of the medium facilitating the P solubilization. Further,
Vassilev and Medina (2006) showed that there are other mechanisms by which PSB
can solubilize inorganic P than the secretion of organic acids, for example, via the pro-
duction of siderophores. Patel et al. (2008) demonstrated that this bio-solubilization
can even occur following the secretion of phenolic compounds and humic substances.
6.3.6 Antagonism Test
The antifungal activity of the strains was tested on PDA medium toward the fungus
Fusarium oxysporum, which is known for its phytopathogenicity. As a result, all the
isolates, except LCP18, were able to inhibit the growth of the fungus by contact
inhibition in the Petri plate after 7 days of incubation. The inhibition percentages of
the isolate LCP79 and LCP90 were significantly the highest, while the inhibition
percentages of the isolate LCP25 were significantly the lowest. Similarly, Tarun
et al. (2012) tested ten PGPR isolated from the rice rhizosphere in the Tarai region
of India for their inhibitory potency, and they found only three isolates (PGB5,
PGB6, and PGB10) of these PGPR tested that were able to inhibit the growth of
Fusarium oxysporum. They later suggested that the siderophores produced by these
three bacteria act as suppressors of the growth of phytopathogenic fungi, which is
consistent with our results since all the isolates were positive for siderophore pro-
duction except the isolate LCP18 (Fig. 6.4).
According to Geetha et al. (2014), several mechanisms may explain the inhibi-
tion of phytopathogenic fungi by Bacillus and Pseudomonas spp., such as the pro-
duction of antibiotics, synthesis of hydrolytic enzymes, competition for nutrients, or
70
d
Pourcentage of inhibion %
60 d c
50
40 b
30
20
10 a
0
The isolates
Fig. 6.4 Antagonist effect of the isolates retained against the phytopathogenic fungus Fusarium
oxysporum. The letters on the bars of the same parameter indicate significant differences according
to Fisher’s protected LSD test (p < 0.05)
a combination of these mechanisms in synergy. Besides, Toyoda and Utsumi (1991)

reported that some PGPR degrade fusaric acid produced by Fusarium sp., which is
the causative agent of wilting, and therefore prevent pathogenesis. In addition,
Prashar et al. (2013) observed that the production of compounds such as ammonia
and HCN is the main source of inhibition of the fungus tested. It was reported that
HCN is produced in various quantities by different species of Pseudomonas, as well
as by other bacteria such as Chromobacterium violaceum (Blom et al. 2011).
Finally, it can be said that the inhibition of fungal growth by the isolates is prob-
ably associated with HCN production, ammonia production, siderophore produc-
tion, lytic enzymes, or even other activities that were not researched in our case.
These evaluated activities can act either separately or in synergy.
6.4 Conclusion
The use of PGPR falls within the context of the fertilization of saline and arid soils
and stimulates the growth and natural defenses of plants, of which the purpose is to
reduce the application of phytosanitary products and to mitigate the inhibitory
effects of poor soils. In this context, this study confirms the importance of some
phosphate solubilizing Pseudomonas isolated from the rhizosphere of Lotus creti-
cus. The selected isolates have different phytobenificial properties and activities
in vitro. All isolates produced IAA, and this makes it possible to classify the isolates
as phytostimulators. The production of lytic enzymes, HCN, and ammonia demon-
strates the biocontrol activity that these isolates may possess against other types of
fungi and phytopathogenic bacteria. The results obtained are extremely encourag-
ing in view of the use of phosphate solubilizing bacteria in agriculture as biofertil-
izers for the improvement growth and development of plants.
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Screening and Characterization
of Phosphate-Solubilizing Rhizobia 7
Isolated from Hedysarum pallidum
in the Northeast of Morocco
Samia Hamane, Mounir Hassani Zerrouk,
Anass E. Lyemlahi, Saida Aarab, Amin Laglaoui,
Mohammed Bakkali, and Abdelhay Arakrak
Abstract
Due to high cost of chemical fertilizers and negative environmental effects, the
use of rhizobia as plant growth-promoting rhizobacteria (PGPR) has shown
potentials to be a promising technique to assure a sustainable agriculture. Our
goal was to select rhizobia strains isolated from nodules of Hydesarum pallidum
plants present in Touissit, northeast of Morocco, exhibiting different activities
that can stimulate directly and/or indirectly plant growth. A total of 37 bacteria
were isolated, of which 19 were capable of solubilizing tricalcium phosphate
(TCP). Based on the diameter of solubilization halos (diameter ≥0.4 cm), 15
strains were selected and evaluated for more PGP activities in vitro for selected
isolates. As a result, 11 bacteria were proved to be able to synthesize hydrogen
cyanide (HCN). Amounts of indole acetic acid (IAA) produced by these bacte-
ria ranged between 1.04 and 3.43 mg L−1. Their ability to secrete siderophores
was also evaluated; 80% of the strains were positive for these compounds’ pro-
duction. We also looked for extracellular enzymes such as cellulase, amylase,
protease, chitinase, and urease. The percentages of bacteria that were positive
for the production of these hydrolytic enzymes were, respectively, 73.3%,
93.3%, 40%, 26.67%, and 33.3%. Eight of selected bacteria were checked for
S. Hamane · A. E. Lyemlahi · A. Laglaoui · M. Bakkali · A. Arakrak (*)

M. H. Zerrouk
Faculté polydisciplinaire de Larache, Larache, Morocco
S. Aarab
Faculté des Sciences et Techniquesd’Alhoceima, Ctre Ait Youssef Ou Ali, Morocco

114 S. Hamane et al.
quantitative assay of TCP solubilization, and soluble P concentrations were

between 2 and 137 mg/L, accompanied by a drop in media pH from 5.67 to
3.87. This study reveals the potential of some rhizobia to be used as efficient
biofertilizers.
Keywords
Hydesarum pallidum · Siderophores · Plant growth-promoting rhizobacteria
(PGPR)
7.1 Introduction
Agriculture today faces challenges, such as imbalance in nutritional status, biologi-

cal and physical properties of soil, and fluctuating climatic factors. All these issues
are the interlinked contributing factors for reduced agricultural productivity.
Sustainability and environmental safety of agricultural production rely on eco-
friendly approaches like biofertilizers. Microbes influencing the plant growth posi-
tively by any mechanism are referred as plant growth-promoting rhizobacteria
(PGPR) (Kloepper et al. 1986). PGPR are important for sustainable agriculture in
order to promote the growth and yield of plants and circulation of plant nutrients.
Also they deem to be the best alternative to chemical fertilizers due to the fact that
application of PGPR as biofertilizers reduces the cost of crop production. Phosphorus
(P), the most limiting nutrient for plant growth, is required by the plants for funda-
mental functions. Consequently, acquisition of sufficient concentration of P
enhances the growth and development of plants in different production systems
(Hayat et al. 2010; Ahemad et al. 2009; Vikram and Hamzehzarghani 2008). The
use of phosphate-solubilizing bacteria as inoculants simultaneously increases P
uptake by the plant and crop yield. Several species of phosphate-solubilizing bacte-
ria have been identified so far including Pseudomonas, Bacillus, Rhizobium,
Bradyrhizobium, and Berkholderia (Rodriguez et al. 1999) having both wide and
limited host ranges. These bacteria are usually associated with rhizosphere, rhizo-
plane, or root tissues (Khan et al. 2009b). Besides phosphate solubilizing, they can
also improve plant growth by utilizing diverse mechanisms, phytohormone produc-
tion, nitrogen fixation, and biocontrol of plant pathogens (Vessey 2003;
Bhattacharyya and Jha 2011). A single PGPR may possess one or more than one of
these plant beneficial traits (Vessey 2003). Keeping in view the importance of
Rhizobium as PGPR, the present study was undertaken to characterize and study
different strains of Rhizobium isolated from nodules of Hydesarum pallidum plants
for optimum biofertilizer production.
7 Screening and Characterization of Phosphate-Solubilizing Rhizobia Isolated… 115
7.2.1 Bacterial Isolates
The method of isolating root-nodulating bacteria from nodules was described by

Vincent (1970). After incubation for 3 days at 28 °C, single colonies were picked
and checked for purity by repeated streaking on to YEM plate containing Congo red
(25 mg/mL) and Gram stain reaction. The pure isolates were stored in 25% (v/v)
glycerol at −20 °C.
7.2.2 Inorganic Phosphate Solubilization
A method of spotting of isolates on Pikovskaya (1948)) agar plates was applied to

confirm their ability to dissolve inorganic phosphate. The inoculated plates were
incubated at 28 °C for 7 days and then observed on solid plates for halo formation.
The diameter of the halo of solubilization was calculated by subtracting the colony
diameter from the total diameter (diameter of the halo + colony diameter).
7.2.3 Hydrogen Cyanide (HCN) Production
To estimate HCN production, a spot of bacterial cultures was streaked on YEM

plate amended with 4.4 g/L glycine. A Whatman filter paper no. 1 soaked in 2%
sodium carbonate in 0.5% picric acid solution was placed on the top of each plate
(Bakker and Schippers 1987). Plates were sealed with parafilm and incubated at
28 ± 2 °C. Development of orange to red color indicated HCN production.
7.2.4 Siderophores Production
The plates of YEM were spot-inoculated with test bacteria and incubated at 28 ± 2 °C
for 3 days. A layer of chrome azurol S (CAS) medium (Schwyn and Neilands 1997)
was poured on the surface of each plate. After 24 h in the dark, development of orange
halo around the bacteria was considered as positive for siderophore production.
7.2.5 Quantitative Assay of Indole Acetic Acid (IAA) Production
Bacterial cultures grown in SMS medium were used to inoculate other tubes con-
taining the same SMS medium supplemented with 0.05% tryptophan and incubated
at 28 °C for 2 days.
All media were centrifuged at 13.000 rpm for 3 min. The supernatant (1 mL) was
mixed with 2 mL of Salkowski reagent, and the development of a pinkish color
indicated the production of IAA (Gordon and Weber 1951). The optical density was
taken at 535 nm after 20 min of incubation at room temperature. Concentrations of

produced IAA were measured with the help of its standard curve.
7.2.6 Cellulase Production
Carboxymethyl cellulose (CMC) agar plates were prepared by screening for cellu-
lose enzyme production according to the method by Miller (1959). After incubation,
the plates were flooded with Congo red solution for 15 min, followed by destaining
with the salt solution (1 M) for 15 min. The colonies developing a yellow halo
reflect the production of the cellulase enzyme.
7.2.7 Urease Production
Urease test was performed by inoculating the isolates on urease medium. The plates
were inoculated with the selected strains and incubated for 5 days at 30 °C.
7.2.8 Amylase Production
For the qualitative determination of amylase production, starch agar medium (SAM)
was used. About 10 μL of fresh bacterial culture of each isolate was deposited on the
SAM medium. After 48 h of incubated at 37 °C, the reading is carried out by impreg-
nating a 1% iodine solution for 5 min. The clear zone surrounding the bacterial
colony indicates the production of amylase (Marx et al. 2004).
7.2.9 Protease Production
The production of protease is carried out using a skimmed milk agar. The formation
of a transparent halo around the colony indicates the degradation of the protein.
7.2.10 Chitinase Production
The method of Renwick et al. (1991) was followed for the qualitative determina-
tion of chitinase production. The cultures of bacterial isolates were spot-inoculated
on minimal medium agar plates containing chitin as a sole source of carbon, fol-
lowed by incubation for 7 days at 30 ± 2 °C. The plates were observed for the
development of clear halo zones around the developed colonies, confirming chitin-
ase production.
7.2.11 Quantitative Assay of P Solubilization
The test isolates were inoculated in 50 mL PVK broth and negative control con-
sisted of uninoculated broth. All flasks were incubated at 28 ± 2 °C with shaking for
7 days. The cultures were centrifuged at 13,000 rpm for 20 min, and the P of super-
natant was determined by the colorimetric method as described by Ames (1966)).
The amount of soluble P was detected from the standard curve of KH2PO4. The pH
was determined using a pH meter.
7.2.12 Statistical Analysis
The data are reported as means ± SD (standard deviation) for three replicates or
more. The results were subjected to analysis of variance (ANOVA) according to
Fisher’s protected LSD test (p < 0.05) using the Statgraphics Plus version 4.0.
7.3.1 Selection of PSB
In the present study, the selected strains were confirmed as phosphate-solubilizing

bacteria (PSB) by observing halo zone around the bacterial colony (Fig. 7.1). A total
of 37 isolates were obtained from H. pallidum nodules. Based on halo’s solubilization
(diameter ≥0.4 cm), 15 PSB were selected for identification and characterization for
plant growth-promoting (PGP) activities. Solubilization index (SI) of these bacteria
on solid media ranged between 2.4 and 3 (Table 7.1). The highest SI was observed for
HPT10. These results are consistent with literature studying phosphate-solubilizing
Fig. 7.1 Solubilization of

TCP on Pikovskaya agar
medium by rhizobium
isolates
Table 7.1 Solubilization of Isolates SI D.H (cm)

TCP by selected rhizobia HPT 24 2.56 0.50
HPT 23 2.88 0.70
HPT 22 2.50 0.40
HPT 26 2.50 0.40
HPT 29 2.63 0.50
HPT 16 2.56 0.50
HPT 11 2.75 0.60
HPT 35 2.75 0.60
HPT 2 2.50 0.40
HPT 33 2.67 0.60
HPT 10 3.00 0.70
HPT 15 2.44 0.40
HPT 9 2.67 0.60
HPT 8 2.40 0.40
HPT 7 2.63 0.50
Solubilization index (SI) = diame-
ter of the colony + halo zone / diam-
eter of the colony
bacteria by similar tests of clear halo formation around colonies on agar medium con-
taining insoluble phosphate (Alikhani et al. 2006; Khan et al. 2007; Babana et al.
2013). These halos could be due to the production of organic acids or due to the activ-
ity of phosphatase enzymes of phosphate-solubilizing bacterial strains (Khan et al.
2009a).
7.3.2 In Vitro Screening of Isolates for Multiple PGP Activities
7.3.2.1 Hydrogen Cyanide (HCN) Production

In the present study, 15 isolates of rhizobia were screened in vitro for HCN produc-
tion. Eleven isolates produced HCN in varied amounts based on color intensity of
the reaction. Hydrogen cyanide is a secondary metabolite produced during the early
stationary growth phase (Knowles and Bunch 1986) by several PGPR, notably
Pseudomonas spp. and Bacillus (Wani et al. 2007) and Rhizobium spp. (Wani et al.
2008a, b). It is released by microbial communities in solution, acts as a secondary
metabolite, confers a selective advantage onto the producer strains (Vining 1990),
and provides protection to plants against weeds and various pathogens, therefore
serving as a biocontrol agent (Sahin et al. 2004).
7.3.2.2 Siderophore Production

Another important trait of PGPR, which may indirectly influence the plant growth,
is the production of siderophores. They bind to the available form of iron Fe3+ in
the rhizosphere, thus making it unavailable to the phytopathogens and protecting
the plant health. In this present investigation, 12 rhizobia strains were siderophore
producers. The current results are dissimilar with Abdul et al. (2014) were they
5.00
4.50 d
4.00
Concentraon of AIA (mg/l)
3.50
3.00
c
2.50 ab abc
abc bc
2.00
ab ab
1.50 a
a a
1.00
0.50
0.00
HPT 23
HPT 35
HPT 2
HPT 10
HPT 24
HPT 33
HPT 11
HPT 26
HPT 8
HPT 15
HPT 9
Fig. 7.2 Amount of AIA released by the PSB selected. Values are means of three replications.
Means followed by the same letter are not significantly different at P < 0.05 (Fisher’s least signifi-
cant difference (LSD) test); ± values indicate standard errors of the means.
found that out of eight rhizobia isolated from nodules of Swabi pea, only four were
able to produce siderophores. The antifungal activity of the test isolates indicated a
close relationship between production of HCN and siderophores.
7.3.2.3 Quantification of Phytohormone (IAA)

All of the 15 Rhizobium strains were subjected to IAA production test. IAA was mea-
sured in the presence of L-tryptophan (Asghar et al. 2002). Eleven of the strains pro-
duced low concentrations of IAA ranging from 3.43 mg/L to 1.04 mg/L. Among these
isolates, HPT 26 and HPT 24 produced maximum amounts of IAA (3.43–2.35 mg/L).
The lowest amount was detected in the presence of HPT 10 (1.04 mg/L) (Fig. 7.2).
The current results are different to Ahmad et al. (2008) who isolated 72 rhizobac-
teria, which produced IAA at significant concentrations. The ability of bacteria to
produce IAA in the rhizosphere depends on the availability of precursors and uptake
of microbial IAA by plant. Growth promotion may be attributed to other mecha-
nisms such as production of plant growth-promoting hormones in the rhizosphere
and other PGP activities (Glick 1995).
7.3.3 C
haracterization for Extracellular Hydrolytic Enzyme
Production
In the present work, we investigated in vitro the production of extracellular hydro-

lytic enzymes by the 15 rhizobia selected. All the PSB chosen were found positive
for amylase and cellulase production. Only four rhizobia showed chitinase activity
and five strains for urease activity developing a red halo around the colonies, while
six strains were protease positive (Table 7.2). These bacterial traits not only confer
significant advantage in the presence of phytopathogens, since their cell walls will
be degraded by the extracellular enzymes and their deleterious effects suppressed
but also help in the process of P mineralization in soils. It was reported that PSB
with cellulolytic activity enhanced the mineralization and decomposition of crop
residues (Hameeda et al. 2006). Production of hydrolytic enzymes by PGPR is an
important mechanism against phytopathogens for sustainable plant disease manage-
ment. Bacterial strains with the ability of hydrolytic enzyme production could able
to lysis the cell wall of pathogenic fungi and protect the host from pathogens
(Moataza 2006). In another study, Naher et al. (2009) reported that some diazo-
trophs isolated from rice can produce hydrolytic enzymes.
7.3.4 Quantitative Assay of P Solubilization
After evaluating the phosphate-solubilizing ability on solid medium, the quantita-

tive assay of this activity was carried out in PVK liquid medium. The results show
Table 7.2 Biochemical tests of selected isolates

Isolates HCN Siderophore Urease Cellulase Protease Chitinase Amylase
HPT − + − − − − +
24
HPT + + + + + − +
23
HPT − − − − − + +
22
HPT + ++ − + + − +
26
HPT ++ + − + − − +
29
HPT ++ + − + − − +
16
HPT + − − + − ++ −
11
HPT + + + + + + +
35
HPT 2 + − + + ++ − +
HPT + ++ − + − − +
33
HPT ++ + − − + − +
10
HPT ++ + + + + − +
15
HPT 9 ++ ++ + + − − +
HPT 8 − ++ − − − ++ +
HPT 7 − + − + − − +
that the solubilization ability varied among the eight PSB (Table 7.2). The highest P
solubilized from 0.5% TCP was found in HPT 33 (137.39 mg/L), HPT 10
(119.39 mg/L), and HPT 24 (100.49 mg/L) compared to all the other PSB strains,
while HPT 11 (2.99 mg/L) released low amount of P. The highest among of P solu-
ble released by Bacillus and Pseudomonas with 373.07 mg/L and 368.58 mg/L,
respectively, isolated from repeatedly chemical pesticide used agriculture soil from
Dhanbad area (Tripti et al. 2012). In another study, Sridevi and Mallaiah (2007)
shows that maximum solubilization was recorded with Rhizobium isolated from
Cassia absus (620 mg/L) followed by Rhizobium sp. strain 19 (391 mg/L), and least
in Rhizobium sp. strain 26 (156 mg/L) from Sesbania sesban.
The final pH of the culture filtrate ranged from 5.6 to 3.8 starting at initial pH of
6.8–7.0 after 7 days of incubation, indicating acidic nature. The maximum drop of
pH was observed in HPT 33 (pH = 3.8) followed by HPT 10 (pH = 4.1). In Table 7.3,
both the strains showed almost similar trend in increase in P solubilization as the pH
decreases. The relationship between final culture pH and concentrations of solubi-
lized P in the culture released by PSB was proved (Fig. 7.3). Significant negative
correlation between final culture pH and concentrations of solubilized P was found
(r = −0.95). If the final culture pH decreased, concentrations of solubilized P
increased. Our findings are very well supported by the work done by Collavino et al.
(2010) that showed that acidification of the broth medium coincided with phospho-
rus solubilization. Another study supports the previous reports. Meanwhile, a group
of researchers from China has also shown that P released from insoluble form of
phosphate was negatively correlated to the solution pH (Liu et al. 2012). Furthermore,
Table 7.3 Solubilization of Isolates [P] (mg/L) pHf

0.5% TCP by selected HPT 100.49(±15.42)c 4.26(±0.11)b
rhizobia 24
HPT 20.99 (±2.95)b 5.78 (±0.04)d
23
HPT 21.89 (±2.51)b 5.79 (±0.14)d
29
HPT 2.99 (±2.34)a 5.67 (±0.11)d
11
HPT 137.39 (±4.98)e 3.87 (±0.08)a
33
HPT 119.39 (±8.57)d 4.11 (±0.19)ab
10
HPT 18.89 (±1.45)b 5.77 (±0.04)d
15
HPT 7 95.74 (±5.01)c 3.81 (±0.10)a
Values are means of three replications. Means
in the same column followed by the same letter
are not significantly different at P < 0.05
(Fisher’s least significant difference (LSD) test;
± values indicate standard errors of the means)
7
6
5
Final culture pH
4
y = -0.0168x + 5.9695
3 R² = 0.9182
2
1
0
0 50 100 150
P-solubilized ( mg/l)
Fig. 7.3 Correlation analysis between final culture pH and soluble P
Pérez et al. (2007) also suggested that acidification of culture supernatants can be
the main mechanism for P solubilization. It is well known in the literature that PSB
solubilize insoluble phosphate in soil by secreting acid; this may indicate that our
isolates might have used the same mechanism to solubilize TCP, which ultimately
caused decline in the pH of final culture.
7.4 Conclusion
In our study, the characterization and screening of rhizobia from nodules of H. pal-
lidum that helped in the selection of various phosphate-solubilizing bacteria have
the potential to increase the available phosphate in the soil, which in turn will help
to minimize the chemical fertilizer and promote sustainable agriculture. Some of
the above-tested isolates could exhibit more than two or three PGP traits, which
may promote plant growth directly or indirectly or synergistically.
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Development of Abiotic Stress Tolerance
in Crops by Plant Growth-Promoting 8
Rhizobacteria (PGPR)
Sivakumar Subiramani, Sathishkumar Ramalingam,
Thiruvengadam Muthu, Shivraj Hariram Nile,
and Baskar Venkidasamy
Abstract
Agriculture production was effectively decreased by abiotic and biotic stresses,
which affect the plant growth by ion toxicity, hormonal and nutritional imbal-
ance, and physiological and metabolic changes. Plant growth-promoting rhizo-
bacteria (PGPR) are the root-colonizing non-pathogenic bacterium, which helps
in plant growth promotion and alleviation of the stress-induced changes to result
in the maintenance of agricultural productivity. Plants inoculated with the PGPR
provide resistance to various abiotic stresses such as salt, drought, and heavy
metal toxicity. Some PGPR strains protect both the biotic and abiotic stresses. In
addition, several PGPR contribute to multiple abiotic stress tolerance in plants.
PGPR produce phytohormones, siderophores, organic acids, and stress-induced
metabolites such as osmotic solutes, prolines, and antioxidant enzymes and up-
and downregulates the expression of various stress-responsive genes that provide
resistance to the plants under stressful conditions. The use of PGPR is a simple
and effective alternative approach to genetic engineering and breeding methods
for crop improvement, since breeding and genetic engineering are time-
consuming, expensive, and laborious procedures. In this chapter, we described
the potential role of PGPR in the abiotic stress tolerance in plants. Moreover, the
mechanism of PGPR in drought, salt, and heavy metal stress alleviation was
described briefly.
S. Subiramani · S. Ramalingam · B. Venkidasamy (*)

Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University,
Coimbatore, Tamil Nadu, India
T. Muthu
Department of Applied Bioscience, College of Life and Environmental Sciences, Konkuk
University, Seoul, Republic of Korea
S. H. Nile
Department of Bioresources and Food Science, College of Life and Environmental Sciences,
Konkuk University, Seoul, Republic of Korea

126 S. Subiramani et al.
Keywords
PGPR · Biotic stress · Abiotic stress · Salt · Drought · Heavy metal · Alleviation
8.1 Introduction
The soil zone surrounding the plant roots is called rhizosphere, where the interac-
tions between the soil, root, and other biotic factors can occur, which is influenced
by the plant roots. The organic carbon in the form of root exudates released from the
plant roots provides nutrition for microbial growth, and therefore the rhizosphere
soil exhibits maximal microbial population (Burdman et al. 2000). These microbes
can interact with the plant roots, and these interactions might serve as favorable,
deleterious, and neutral, therefore effectively influencing the plant growth and
development (Ahmad et al. 2011a; Lau and Lennon 2011). Various types of microbes
include bacteria, actinomycetes, fungi, algae, and protozoa were often colonized
with the plant roots. The growth and development of the plants were enhanced by
the application of these microbes are well documented. Among the different micro-
biota, bacteria constitute the major and dominant in rhizosphere soil (Kaymak et al.
2009). The microbial interaction specificity is influenced by the amount and consti-
tution of the root exudates as well as the soil condition. The exudates released from
the plant roots act as a signal for rhizobacteria to reach the root surface and interac-
tion though a process called chemotaxis. Several studies hypothesized the role of
PGPR in plant growth promotion, whereas their precise mechanism was still largely
unrevealed. This growth-promoting activity differed between the bacterial strains
and the compounds secreted by the microorganisms.
A diverse group of bacterial populations dwelling in the zonal root area referred
as plant growth-promoting rhizobacteria (PGPR). PGPR include several genera
such as Arthrobacter, Azotobacter, Azospirillum, Bacillus, Enterobacter,
Pseudomonas, Serratia, and Streptomyces spp. (Ma et al. 2011a, b). On the basis of
relationship to plants, PGPR are divided into symbiotic (intracellular) and a
free-living (extracellular) bacterium. Rhizobia are the well-known group belonging
to the intracellular PGPR, and they produce nodules in legume plants.
PGPR are associated with several plants and offer various favorable functions
including enhanced plant growth and decreased sensitivity to diseases caused by
plant pathogens (Shahzadi et al. 2012). The enhanced root hair branches, efficient
seed germination, more leaf area per plant, liberation of phytohormones, enhanced
availability of nutrients to plants, and enhanced plant biomass are the mechanisms
of PGPR in plant growth promotion (Podile and Kishore 2006). Some studies
showed that the production of the plant hormone, biocontrol agents, and enhanced
uptake of nutrients by the plants are facilitated by the PGPR. Several investigations
described the mechanisms of PGPR in plant growth promotion. Plant growth-pro-
moting activity of PGPR was achieved through the production of siderophores and
organic acids for nutrient mobilization and phytohormones (auxin, cytokinin,
abscisic acid, gibberellic acid, and ethylene) for plant growth regulation (Dimkpa
8 Development of Abiotic Stress Tolerance in Crops by Plant Growth-Promoting… 127
et al. 2009). Modern agriculture faces several stresses including biotic (living) and
abiotic (nonliving) stresses. Plant pathogens (virus, bacteria, and fungi), herbivores
(insects, nematodes), and parasitic weeds are the major biotic stressors imposed a
severe reduction in crop production. Similarly, the abiotic stress factors include
excess water, drought, heat, and chilling also cause a major reduction in agriculture
productivity, and this contributes >50% reduction in the yield (Tardieu and Tuberosa
2010). The oxidative stress induced by drought and salinity stresses causes meta-
bolic and biochemical changes which in turn affects the crop yield (Shafi et al.
2010). Several studies reported the enhanced plant tolerance to abiotic stresses by
PGPR treatment, and it can generate the “induced systemic tolerance” in plants to
salt and drought stresses (Yang et al. 2009; Grichko and Glick 2001; Yuwono et al.
2005; Egamberdiyeva 2007; Sziderics et al. 2007; Belimov et al. 2009; Dimkpa
et al. 2009a). In addition, PGPR facilitates the uptake of nutrients from the soil,
which leads to decreased accumulation of fertilizers (nitrates and phosphates) in the
cultivation field. Figure 8.1 showed the potential role of PGPR in the alleviation of
abiotic stress tolerance in plants. It was reported that microbes isolated from one
host plant root grown in desert farming environment improved the growth of another
distinct host plant species cultivated under drought condition (Marasco et al. 2013).
PGPR can be applied to plants for growth promotion through various methods.
Biopriming is a common method of seed treatment with the bacterial cells for
improving plant growth. The foliar spray also employed the application of PGPR in
A/Biotic stresses
Drought Decreased plant growth
Salt Hormonal imbalance
Pathogen Susceptibility to diseases
Heavy metal Toxicity
Ethylene production
PGPR
Phytohormones production
Siderophore production
EPS production
Fig. 8.1 Potential role of plant growth-promoting rhizobacteria (PGPR) in abiotic stress tolerance
in plants. EPS exopolysaccharide
plants. Thus, the application of PGPR reduced the inclusion of fertilizer and
decreased the environmental pollution which comes through the fertilizer runoff
and contaminated the aquatic ecosystem.
8.2 Tolerance to Drought Stress
Similar to salt stress, drought is the growing risk factor that decreased the world-
wide agricultural productivity. Decreased assimilation of photosynthetically active
radiation, reduced use of radiation capability, and diminished harvest index are the
main factors of crop yield loss due to drought stress (Farooq et al. 2009). Plants
needed a various adaptation and alleviation strategies to survive under drought con-
ditions. PGPR are effectively involved in the alleviation of drought stresses in
plants. The plant rhizosphere/endorhizosphere-residing PGPR involved in the pro-
duction of phytohormones, exopolysaccharides (EPS), volatile compounds, and
ACC deaminase stimulates the production of osmolytes, antioxidants, regulation of
stress-responsive genes, and alteration of root morphology, which results in drought
tolerance in plants. The PGPR application enhanced shoot growth of the plants.
Therefore, the PGPR maintain the shoot growth rates under drought stress, which
leads to enhanced agricultural productivity. Improved shoot growth was observed in
the PGPR (Bacillus spp.)-treated corn plants (Vardharajula et al. 2011). Stimulation
of osmotic and stress-responsive genes by PGPR is essential for the plants to grow
under drought stress. PGPR plays an important role in providing resistance and
adaptation of plants to drought stresses and has the significant role in deciphering
the future food security problems. In addition to the metabolic and osmotic changes
induced in the plants by the PGPR treatment, it also alters the soil properties to
facilitate the plant growth. Although the PGPR stimulates the plant growth and
yield, the type of inoculation affects their activity. For instance, in species of
Brassica oilseed rape, the application of either Pseudomonas fluorescens or
Pseudomonas putida results in the better growth, yield, and alleviation of drought
stress (Arvin et al. 2012), whereas the co-inoculation of Pseudomonas fluorescens
or Pseudomonas putida leads to antagonistic effects which indicates that the inocu-
lation methods also determine the mitigation of stress. List of studies was carried
out for the drought stress resistance induced by the PGPR treatment in various
plants was shown in Table 8.1. Another major advantage of PGPR application is the
inclusion of resistant to multiple types (more than one stress) of stresses (abiotic/
biotic). In the arid and semiarid lands, crops often faced multiple stresses, and the
application of PGPR would provide resistant to multiple stresses (Mayak et al.
2004; Rodriguez et al. 2008). For example, the enhanced resistance to drought and
pathogen attack was observed in the Arabidopsis plant symbiosis with the
Paenibacillus polymyxa bacterium (Timmusk and Wagner 1999). Genetic engineer-
ing and plant breeding are commonly employed for the development of drought-
resistant crop varieties, whereas it is an expensive and time-consuming process.
However, the PGPR application could effectively alleviate the drought stress in
Table. 8.1 List of studies that used the PGPR strains for abiotic stress tolerance in plants
Crop PGPR Stress References
Triticum aestivum Bacillus safensis Salt Chakraborty et al.
Ochrobactrum pseudogregnonense (2013)
Triticum aestivum Pseudomonas putida Salt Nadeem et al.
Enterobacter cloacae (2013)
Serratia ficaria
P. fluorescens
Oryza sativa Alcaligenes faecalis Salt Bal et al. (2013)
Bacillus pumilus
Ochrobactrum sp.
Oryza sativa P. pseudoalcaligenes Salt Jha et al. (2013)
B. pumilus
Triticum aestivum B. subtilis Salt Upadhyay et al.
Arthrobacter sp. (2012)
Triticum aestivum Azospirillum sp. Salt Zarea et al. (2012)
Triticum aestivum Streptomyces sp. Salt Sadeghi et al.
(2012)
Persea gratissima Pseudomonas sp. Salt Nadeem et al.
Bacillus sp. (2012)
Variovorax sp
Zea mays Azotobacter chroococcum Salt Rojas-Tapias et al.
(2012)
Cicer arietinum P. pseudoalcaligenes Salt Patel et al. (2012)
P. putida
Arachis hypogaea Brachybacterium saurashtrense Salt Shukla et al.
Brevibacterium casei (2012)
Haererohalobacter sp
Phaseolus P. extremorientalis Salt Egamberdieva
vulgaris P. ehlororaphis (2011)
Triticum aestivum Bacillus Salt Upadhyay et al.
Burkholderia (2011)
Enterobacter
Microbacterium
Paenibacillus
Lycopersicon P. fluorescens Salt Tank and Saraf
esculentum P. aeruginosa, (2010)
P. stutzeri
Solanum Pseudomonas sp. Salt Fu et al. (2010)
melongena
Triticum durum Azospirillum sp. Salt Nabti et al. (2010)
Gossypium P. putida Salt Yao et al. (2010)
hirsutum
Zea maize B. megaterium Salt Marulanda et al.
(2010)
(continued)
Table. 8.1 (continued)
Raphanus sativus Agrobacterium rubi Salt Kaymak et al.
Burkholderia gladii (2009)
P. putida
B. subtilis
B. megaterium
Hordeum vulgare A. brasilense Salt Omar et al. (1994)
L. sativa L. cv. P. mendocina Salt Kohler et al. (2009,
Tafalla 2010)
Arabidopsis B. subtilis Salt Zhang et al. (2008)
thaliana
Phaseolus A. brasilense Salt Dardanelli et al.
vulgaris (2008)
Zea mays Bacillus sp. Salt Principe et al.
Ochrobactrum sp. (2007)
Zea mays P. syringae Salt Nadeem et al.
P. fluorescens (2007)
E. aerogenes
Arachis hypogaea P. fluorescens Salt Saravanakumar
and Samiyappan
(2007)
L. sativa Azospirillum Salt Barassi et al.
(2006)
Piper nigrum P. fluorescence Salt Paul et al. (2006)
Oryza sativa P. pseudoalcaligenes Salt Diby et al. (2005)
Lycopersicon Achromobacter piechaudii Salt Mayak et al. (2004)
esculentum
Triticum aestivum Aeromonas hydrophila Salt Ashraf et al. (2004)
B. insolitus
Bacillus sp.
Zea mays Azospirillum Salt Hamdia et al.
(2004)
C. arietinum A. brasilense Salt Hamaoui et al.
Vicia faba (2001)
Leptochloa fusca A. lipoferum Salt Malik et al. (1997)
A. brasilense
Azoarcus
Pseudomonas sp.
Solanum Glomus mosseae Salt He et al. (2007)
lycopersicum
Glycine max Glomus etunicatum Salt Sharifi et al. (2007)
Oryza sativa Osmotolerant bacteria Drought Yuwono et al.
(2005)
Lycopersicon Achromobacter piechaudii Drought Mayak et al.
esculentum (2004)
Capsicum annuum
Triticum aestivum Azospirillum Drought Creus et al. (2004,
2005)
(continued)
Zea mays A. brasilense Drought Casanovas et al.
(2002)
P. vulgaris A. brasilense Drought German et al.
(2000)
Lycopersicon A. brasilense Drought Creus et al. (2005),
esculentum Molina-Favero
et al. (2008)
Zea mays Azospirillum lipoferum Drought Cohen et al. (2009)
Triticum aestivum Azospirillum sp. Drought Arzanesh et al.
(2011)
Arabidopsis Phyllobacterium brassicacearum strain Drought Bresson et al.
thaliana STM196 (2013)
Platycladus Bacillus subtilis Drought Liu et al. (2013)
orientalis
Glycine max P. putida H-2–3 Drought Sang-Mo et al.
(2014)
Lavandula dentate B. thuringiensis Drought Ahmad et al.
(2014)
Triticum aestivum Rhizobium leguminosarum (LR-30), Drought Hussain et al.
Mesorhizobium ciceri (CR-30, CR-39) (2014)
Rhizobium phaseoli (MR-2)
Lycopersicon Achromobacter piechaudii ARV8 Drought Mayak et al.
esculentum (2004)
Capsicum annuum
Pisum sativum Variovorax paradoxus 5C-2 Drought Dodd et al. (2005)
Pisum sativum Pseudomonas fluorescens Drought Zahir et al. (2008)
Biotype G (ACC-5)
Pisum sativum V. paradoxus 5C-2 Drought Belimov et al.
(2009)
Triticum aestivum ACC deaminase-producing rhizobacteria Drought Shakir et al. (2012)
Cicer arietinum Consortia of Bacillus isolate 23-B Drought Sharma et al.
Pseudomonas 6-P (2013)
Mesorhizobium ciceris
Capsicum annuum Bacillus licheniformis K11 Drought Hui and Kim
(2013)
Triticum aestivum Bacillus thuringiensis AZP2 Drought Timmusk et al.
(2014)
Zea mays Pseudomonas putida GAP-P45 Drought Sandhya et al.
(2010)
Zea mays P. fluorescens Drought Ansary et al.
(2012)
Lavandula dentate B. thuringiensis Drought Ahmad et al.
(2014)
Lycopersicon Bacillus polymyxa Drought Shintu and
esculentum Jayaram (2015)
(continued)
Oryza sativa Consortia containing P. jessenii, R62, P. Drought Gusain et al.
synxantha, R81 (2015)
A. nitroguajacolicus strainYB3, strain
YB5
Zea mays Azospirillum lipoferum Drought Bano et al. (2013)
Phaseolus Rhizobium etli Drought Suarez et al. (2008)
vulgaris
Zea mays A. brasilense Drought Rodriguez et al.
(2009)
Arabidopsis Bacillus subtilis GB03 Drought Zhang et al. (2010)
thaliana
Zea mays Klebsiella variicola F2, Pseudomonas Drought Gou et al. (2015)
fluorescens YX2
Raoultella planticola YL2
Oryza sativa Azoapirillum brasilense Az39 Drought Cassan et al.
(2009)
Arabidopsis Paenibacillus polymyxa B2 Drought Timmusk and
thaliana Wagner (1999)
Capsicum annuum B. licheniformis K11 Drought Lim and Kim
(2013)
Triticum aestivum Bacillus amyloliquefaciens 511 Drought Kasim et al. (2013)
Azospirillum brasilense NO40
Arabidopsis Pseudomonas chlororaphis O6 Drought Cho et al. (2013)
thaliana
Sugarcane Gluconacetobacter diazotrophicus PAL5 Drought Vargas et al. (2014)
Arabidopsis Azospirillum brasilense sp. 245strain Drought Cohen et al. (2015)
thaliana
Cucumis sativa Bacillus cereus strain AR156 Drought Wang et al. (2012)
cucumber B. subtilis strain SM21
Serratia sp. strain XY21
Helianthus Achromobacter xylosoxidans (SF2) Drought Castillo et al.
annuus Bacillus pumilis (SF3 and SF4) (2013)
Hyoscyamus niger Pseudomonas putida strain (PP), Drought Ghorbanpour et al.
Pseudomonas fluorescens strain (PF) (2013)
Sorghum bicolor Bacillus spp. Drought Grover et al.
Strains KB122, KB129, KB133, KB142 (2014)
Solanum Bacillus pumilus strain DH-11 Drought Gururani et al.
tuberosum Bacillus firmus strain 40 (2013)
Vigna radiata Pseudomonas fluorescens strain Pf1 Drought Saravanakumar
Bacillus subtilis EPB5, EPB22, and EPB et al. (2011)
31
Zea mays PGPR isolate 1 K, 9 K and KB Drought Yasmin et al.
(2013)
Zea mays Proteus penneri strain(Pp1) Drought Naseem and Bano
Pseudomonas aeruginosa strain (Pa2) (2014)
Alcaligenes faecalis strain (AF3)
(continued)
Zea mays Burkholderia phytofirmans strain PsJN Drought Naveed et al.
Enterobacter sp. strain FD17 (2014)
Zea mays Bacillus amyloliquefaciens strain Drought Vardharajula et al.
HYD-B17 B. licheniformis strain (2011)
HYTAPB18 B. Thuringiensis strain
HYDGRFB19 Paenibacillus favisporus
strain BKB30 B. Subtilis strain
RMPB44
Zea mays Burkholderia sp. strainLD-11 Drought Fan et al. (2015)
Oryza sativa Bacillus subtilis Heavy
Bacillus megaterium metal
Bacillus sp.
Brassica juncea Rhodococcus sp. Heavy Belimov et al.,
Variovorax paradoxus metal (2005)
Pisum sativum P. brassicacearum AM3, P. marginalis Heavy Safronova et al.,
Dp1 metal (2006)
Trifolium pratense Brevibacillus spp. Heavy Vivas et al., (2003)
metal
Zea mays Soil bacteria AN8, AN12 Heavy Hassan et al.
metal (2014)
Cicer aritenum Acinetobacter sp. nbri05 Heavy Srivastava and
metal Singh (2014)
Zea mays Acinetobacter sp. RG30 Heavy Rojas-Tapias et al.
Pseudomonas putida GN04 metal (2014)
Zea mays Pseudomonas sp. TLC 6–6.5-4 Heavy Li and
Helianthus metal Ramakrishna
annuus (2011)
Lolium Bradyrhizobium sp. YL-6 Heavy Guo and Chi
multiflorum metal (2014)
Glycine max
Brassica napus Rahnella sp. JN6 Heavy He et al. (2013)
metal
Alyssum Arthrobacter nicotinovorans SA40 Heavy Cabello-Conejo
pintodasilvae metal et al. (2014)
Medicago Sinorhizobium meliloti CCNWSX0020 Heavy Kong et al. (2015)
lupulina metal
Triticum aestivum Klebsiella sp. CIK-502 Heavy Ahmad et al.
Zea mays metal (2014)
Zea mays Ralstonia eutropha Heavy Moreira et al.
Chryseobacterium humi metal (2014)
Brassica juncea Bacillus sp. MN3–4 Heavy Shin et al. (2012)
metal
Salix caprea Burkholeria sp. RX232 Heavy Kuffner et al.
metal (2010)
Salix caprea Microbacterium sp. EX72 Heavy Kuffner et al.
metal (2010)
(continued)
Lens culinaris var. Rhizobium sp. RL9 Heavy Wani and Khan
Malka metal (2013)
Solanum nigrum Rahnella sp. JN27 Heavy
Zea mays metal
Amaranthus
hypochondriacus
Amaranthus
mangostanus
Sorghum bicolour Bacillus sp. SLS18 Heavy Luo et al. (2012)
Phytolacca metal
acinosa
Solanum nigrum
Brassica napus Cu-resistant isolates belonged to Heavy Sun et al. (2010)
Firmicutes, b metal
Actinobacteria
Proteobacteria
Alyssum Pseudomonas sp. A3R3 Heavy Ma et al. (2011a, b)
serpyllifolium metal
Brassica juncea
plants, and it is a rapid and cheap method for the drought stress management in
dryland agriculture.
8.3 Protection Against Salt Stress
Among the various abiotic stresses, salinity was the major factor that decreased the
crop yield through affecting the germination, plant phase transition, plant vigor, and
yield (Munns and Tester 2008). Saline soils are often considered as barren lands,
which are not suitable for plant cultivation. Previous reports indicated that salinity
affects ~800 million hectares of land all over the world. Salt stress reduced the crop
yield through suppression of photosynthesis, protein synthesis, and metabolism of
lipids. The application of PGPR could be useful to resolve this issue. Various benefi-
cial bacteria such as Azospirillum, Azotobacter, Rhizobium, Bradyrhizobium,
Bacillus, and Pseudomonas have been isolated from the unfavorable stressful eco-
systems (acid soils, saline, alkaline, desert environments and eroded hill slopes, and
they could participate in reclamation of the soil (Selvakumar et al. 2009; Upadhyay
et al. 2009). Salinity-resistant PGPR attribute osmotolerance to the plants inhabiting
in the saline soil that in turn provide various beneficiary actions such as improved
growth (enhanced root and shoot growth), nutrient uptake, chlorophyll content,
vigor, and yield. The mobilization of nutrients and regulation of phytopathogens in
the rhizosphere soil, as well as the synthesis of 1-aminocyclopropane-1-carboxylate
deaminase (ACC deaminase) and phytohormones, contribute to plant tolerance.
Moreover, nutrient circulation in the rhizosphere soil and osmolyte accumulation in
plants were also achieved by the PGPR treatment. Higher accumulation of K+ ion in
the PGPR treatment causes the enhanced K+/Na+ ratio, which in turn facilitates the
salinity tolerance. Salt stress-mediated oxidative stresses was scavenged by the rhi-
zobacterial antioxidative enzymes. ACC deaminase produced by the PGPR
enhanced the plant growth under adverse environmental conditions (Belimov et al.
2001). Furthermore, the production of ACC deaminase by the PGPR plays a role in
the regulation of ethylene production. In line with this, a recent study demonstrated
that Distichlis spicata (halophilic grass)-associated rhizobacteria (Bacillus sp. and
Pseudomonas lini) effectively stimulates the growth of Arabidopsis seedlings in
saline conditions (Palacio-Rodríguez et al. 2017). The salt tolerance is associated
with the phosphate solubilization and production of IAA and siderophore. In addi-
tion, ACC deaminase expression and enhanced auxin redistribution in the roots of
Arabidopsis were also observed. This is emphasized that salt resistant is rendered by
the rhizobacterial strains associated with the saline-tolerant crops. It was reported
that survival of the seedlings was improved by suppressing the ethylene level in the
first few days after sowing and enhanced the root formation (Glick et al. 1998).
Wang et al. (2016) demonstrated that ACC deaminase producing rhizobacterial
(Variovorax paradoxus 5C-2) inoculation in pea plants decreased the Na+ flow and
increased the K+ uptake and root to shoot K flow under salt stress. PGPR inoculation
causes enhanced photosynthetic efficiency and improves the plant growth.
Furthermore, multiple stress tolerance was also reported for the microbes inocu-
lated with the plants. For instance, the enhanced resistance to salt and Fusarium and
Blumeria infections was found in the barley plants applied with the Piriformospora
indica (Waller et al. (2005). Several studies showed the potential application of
PGPR in the salt stress tolerance in various plants as represented in Table. 8.1. The
use of PGPR as microbial inoculants for crop improvement in saline ecosystems is
a powerful strategy for saline agriculture management.
8.4 Protection Against Heavy Metal Stress
Apart from the salt and drought stress, plants are continuously exposed to heavy
metal stress, which mainly arises from the industrial and other environmental pollu-
tion. In addition to having plant growth-promoting traits, certain bacterial strains are
also very important in the alleviation of the heavy metal toxicity in plants. In the
metal-accumulated soil, PGPR could help in the plant growth and survival (Rajkumar
and Freitas 2008). PGPR and mycorrhizal fungi are involved in the reduction of
harmful heavy metals which in turn protects the plants from toxicity induced by
heavy metals (Denton 2007). Soil microbes secrete acids, phytoantibiotics, proteins,
and other chemicals that help in the alleviation of toxic heavy metal-induced stresses
in plants (Denton 2007). The facilitation of heavy metal stress tolerance in plants is
provided by some PGPR through the increased plant growth by enhanced phytore-
mediation, mitigation of metal toxicity, altered metal accumulation capability, and
enhanced translocation of metals within the plant. PGPR aided the improved plant
growth via the decreased metal phytotoxicity and the altered phytoavailability of
heavy metals in polluted soil through the detoxification, accumulation,
transformation, and sequestration (Ma et al. 2016). Phytoremediation was strongly

influenced by the metal phytotoxicity. The reduction of phytotoxicity by the action
of Bacillus sp. MN3–4 (lead-resistant bacterium) isolated from the Alnus firma
(metal hyperaccumulator) through extracellular sequestration and intracellular
accumulation, which results in the improved phytoremediation (Shin et al. 2012).
Furthermore, the secretion of organic acids and siderophores by PGPR facilitates
the increased metal and mineral mobilization and enhanced uptake of heavy metal
that leads to increased metal phytoextraction potential of host plants (Chen et al.
2014). For instance, the synthesis of ACC deaminase, IAA production, P solubiliza-
tion, siderophore production, and plant polymer-hydrolyzing enzymes by the action
of Pseudomonas sp. A3R3 (Ni-resistant bacteria) isolated from the hyperaccumula-
tor (A. serpyllifolium) host plant enhanced the growth of Brassica juncea cultivated
in the Ni-contaminated soil (Ma et al. 2011a, b). Certain bacterial species include
Pseudomonas sp. can produce biosurfactants, which are involved in heavy metal
mobilization in the contaminated soil (Braud et al. 2006). Sun et al. (2010) reported
that improved plant growth and translocation of Cu in B. napus with the help of
copper-resistant PGPR via the production of ACC deaminase, siderophore synthe-
sis, and arginine decarboxylase activity. The reduction of Cd bioavailability in rhi-
zosphere soils by the action of B. megaterium H3 and Neorhizobium huautlense
T1–17 leads to decreased accumulation of Cd in the polished rice (Li et al. 2017).
The multibeneficial action of PGPR included the enhanced plant growth and devel-
opment and decreased the heavy metal accumulation in plants. Various studies
showed the potential application of PGPR in the heavy metal stress tolerance in
plants, as represented in Table. 8.1. Moreover, the application of microbial commu-
nities was more effective in plant growth and development as well as heavy metal
degradation as compared to the single microorganism.
8.5 Harmful Aspects of PGPR
PGPR functions in maintaining the soil fertility as well as plant growth and develop-
ment. Most of the studies emphasized the positive aspects (growth enhancement) of
PGPR, whereas some reports indicated the negative action of PGPR (Alstrom and
Burns 1989; Saharan and Nehra 2011; Suslow and Schroth 1982). For instance,
certain Pseudomonas species produces cyanide, which acts as growth promotor as
well as suppressor activities. Cyanide acts as a biocontrol agent against certain plant
pathogens, and it can also cause adverse effects on plant growth (Martínez-Viveros
et al. 2010; Bakker and Schippers 1987). PGPR is also known to produce auxin,
which functions as positive and negative fashion depending on their concentration
(Eliasson et al. 1989; Vacheron et al. 2013; Young and Mulkey 1997). For example,
at low concentration, it enhances plant growth, whereas at a high level, it inhibits
root growth (Patten and Glick 2002; Xie et al. 1996). Similar dual roles of PGPR
products were reported including the rhizobitoxine produced by Bradyrhizobium
elkanii. It acts as a suppressor of ethylene synthesis and thus mitigates the harmful
effect of stress-stimulated ethylene synthesis on nodulation (Vijayan et al. 2013).
On the other hand, it can induce foliar chlorosis in soybeans (Xiong and Fuhrmann
1996). Although PGPR is very effective for promoting plant growth and develop-
ment, certain bacterial species may function as growth inhibitory action. However,
such a negative role may occur under certain specific conditions and also for some
particular traits. Therefore, the selection of a suitable strain is crucial for obtaining
maximum benefits regarding improved plant growth and development.
8.6 Concluding Perspectives
Plant growth-promoting rhizobacteria are enriched in the rhizosphere soil (root

zones) and serve in the enhancement of plant growth and development. PGPR gives
protection to plants against salt, drought, and heavy metal stresses. In general,
PGPR was shown to produce growth promoting (phytohormones, siderophores,
organic acids, and volatile compounds) and stress-induced metabolites (osmotic
solutes, proline, nitric oxides, and induction of antioxidant enzymes) that function
in the plant growth promotion and stress mitigation activities. These potential ben-
eficial functions of PGPR indicate that their role in the sustainable agriculture pro-
duction. Although the genetic engineering and breeding approaches were helpful to
generate the abiotic stress-tolerant crops, it required more time and considered as
expensive as compared to the PGPR application. The phenotypical, physiological,
and molecular mechanism of biotic stress tolerance attributed by the bacterium has
been conducted widely (Van Loon et al. 1998). Several studies reported the PGPR-
mediated abiotic stress tolerance, whereas details on the mode of action in abiotic
stress tolerance were still lacking. Moreover, the detailed molecular mechanisms
articulate their role and are still understudied. Therefore, it is warranted that the
molecular mechanism behind the stress alleviation and growth promotion is essen-
tial to unravel their specific functions.
Acknowledgments This study was supported by a grant (Sanction No.: PDF/2016/002258LS)

from the National Post Doctoral Fellowship, Department of Science and Technology, Science and
Engineering Research Board, Government of India.
Conflict of interest: The authors declare that they have no conflict of interest.
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Plant Growth-Promoting Rhizobacteria
(PGPR) and Their Action Mechanisms 9
in Availability of Nutrients to Plants
Hassan Etesami and Sina M. Adl
Abstract
One of the main obstacles to plant growth is the lack of the availability of nutri-
ent elements in many agricultural environments in the world, especially the
tropics where soils can be extremely low in nutrients. Using different mecha-
nisms of action, plant growth-promoting rhizobacteria (PGPR) participate in
geochemical nutrition cycles and determine their access to plants and the
microbial community of the soil. Use of these bacteria as bio-inoculants will
increase the availability of nutrient elements in soil, help to minimize the
chemical fertilizer application, reduce environmental pollution, and promote
sustainable agriculture. Considering comprehensive reviews previously pub-
lished on plant growth enhancement mechanisms, this review focuses on what
is known about the action mechanisms underlying the increase of the availabil-
ity of nutrient elements as an effect of microbial colonization especially
PGPR. In this chapter, some of the most important mechanisms and processes
regarding the effects of PGPR on the availability and hence uptake of nutrient
elements by plant are reviewed. The awareness of such mechanisms can be
important for the selection and hence production of microbial inoculums,
which are appropriate for biological fertilization as substituting or decreasing
the need of using chemical fertilizers in crops. In this review, special consider-
ation is given to the role of PGPR in the availability of nitrogen (N), phospho-
rus (P), potassium (K), and sulfur (S) as macronutrients and iron (Fe) and
manganese (Mn) as micronutrients.
H. Etesami (*)
Department of Soil Science, Faculty of Agricultural Engineering & Technology, University
College of Agriculture & Natural Resources, University of Tehran, Tehran, Iran
S. Adl
Department of Soil Sciences, University of Saskatchewan, Saskatoon, Canada

148 H. Etesami and S. Adl
Keywords
PGPR · Nutrient elements · Availability of nutrients · Action mechanisms
9.1 Introduction
Many of the plant-related microorganisms are known for their ability to promote
plant growth (Compant et al. 2010). Plants produce a wide range of organic com-
pounds between 6% and 21% of the carbon fixed including sugars (such as glu-
cose, xylose, fructose, maltose, sucrose, and ribose), organic acids (such as citric,
malic, lactic, succinic, oxalic, and pyruvic acids), amino acids, fatty acids, nucleo-
tides, putrescine, and vitamins, which can be used as nutrients or signals by micro-
bial populations. These signal molecules can also be used to link plants and
microbes (Lugtenberg 2015). Plant-associated microorganisms, on the other hand,
regulate the growth and morphogenesis of plant or activate plant immunity by
releasing small molecules or volatile compounds and phytohormones (Ortíz-Castro
et al. 2009). Symbionts, pathogens, epiphytes, or endophytes are four ways in
which microorganisms are associated with plants (Iniguez et al. 2005). The micro-
organisms can colonize different parts of the plant, which are grouped into three
groups based on their colonization area: rhizosphere (in the vicinity of root) micro-
organisms, rhizoplane (on the surface of root) microorganisms, and endophytic
microorganisms. Endophytes are plant-associated microorganisms that are isolated
from the tissues that reside without damage to the host (Andrews and Harris 2000),
while those isolated from rhizoplane and phylloplane surfaces are called epiphytes
(Azevedo et al. 2000; Petrini et al. 1989; Sturz et al. 2000). There are three basic
types of ecological based microbial interactions: the neutral, negative, and positive
interaction that is commonly found between microorganisms and plants (Whipps
2001). Most microorganisms are commensals in which the microorganisms inter-
act safely with host plants that have no significant effects on the overall host’s
growth and physiology (Beattie 2007). In negative interactions, phytopathogenic
microorganisms produce phytotoxic substances such as hydrogen cyanide (HCN)
or ethylene, which negatively affects plant growth and physiology (Khalid et al.
2004). In contrast to these deleterious microorganisms, some of these microorgan-
isms can promote plant growth and development either directly or indirectly (Glick
2012a, 2014, 2015a, b). Soil bacteria that are useful for plant growth by colonizing
the plant root are commonly referred to as plant growth-promoting rhizobacteria
(PGPR) (Hayat et al. 2010). Majority of credible group of PGPRs belong to genera
Frankia, Acinetobacter, Arthrobacter, Azotobacter, Azospirillum Streptomyces
spp., Bacillus, Enterobacter, Burkholderia, Bradyrhizobium, Rhizobium, Serratia,
Thiobacillus, and Pseudomonas (Dimkpa et al. 2009; Gray and Smith 2005; Vessey
2003). It has been reported that PGPRs are beneficial to plants in various ways
(Hayat et al. 2010; Lugtenberg and Kamilova 2009; Paul 2012). Although the pre-
cise mechanisms of stimulating plant growth remain largely speculative, a possible
explanation includes (i) improving soil structure and bioremediating the polluted
9 Plant Growth-Promoting Rhizobacteria (PGPR) and Their Action Mechanisms… 149
soils by sequestering toxic heavy metal species and degrading xenobiotic com-
pounds; (ii) improving abiotic stress resistance; (iii) biological nitrogen fixation
(BNF); (iv) producing numerous plant growth regulators, like abscisic acid (ABA),
gibberellic acid (GA), cytokinins (CK), and auxin, i.e., indole-3-acetic acid (IAA);
(v) solubilization and mineralization of nutrients, particularly mineral phosphate;
(vi) protecting plants from phytopathogens by controlling or inhibiting them like
antibiotic production, production of siderophores, induction of systemic resistance,
chelation of available Fe in the rhizosphere, synthesis of extracellular enzymes to
hydrolyze the fungal cell wall, and competition for niches within the rhizosphere;
(vii) producing siderophores; and (viii) reducing the level of ethylene in the root of
developing plants by production of 1-aminocyclopropane-1-carboxylate (ACC)
deaminase (Braud et al. 2009; Hayat et al. 2010).
In all, plants require 17 essential elements, 14 of which are taken up in inorganic
forms by the roots. The absence or paucity of any one of these essential elements
will commonly lead to plant death or inability to complete its life cycle. In the pres-
ence of nutrient deficiencies, even at asymptomatic levels, performance of crop,
yield, and quality of crop are often at risk (Jewell et al. 2010). Since the nutrients in
soils are generally bound to inorganic and organic soil constituents, or alternatively
present as insoluble precipitates, plenty of nutrients are not available to plants for
root absorption. PGPR play an essential role in the environment by contributing to
the release of key nutrients from primary minerals that are required not only for
their own nutrition but also for that of plants (Uroz et al. 2009). Use of these bacteria
as bio-inoculants will increase the availability of nutrients in soil, help to minimize
the chemical fertilizer application, reduce environmental pollution, and promote
sustainable agriculture. PGPR have been proved to be vital for circulation of plant
nutrients in many ways. Researchers are studying these microbes for the past
30 years to understand the action mechanisms employed by PGPR to support plant
growth. Awareness of the mechanisms operated by these bacteria in promoting plant
growth is a prerequisite for the development of new management strategies for sus-
tainable agriculture. In the following, some of the most important mechanisms and
processes regarding the effects of PGPR on the availability and hence uptake of
nutrient elements are reviewed.
9.2 ction Mechanisms of PGPR of Providing Nutrients

A
for Plants
PGPR enhance plant growth and health by the beneficial mechanisms which are
direct or indirect. Any mechanism that protects the plant against infections (biologi-
cal stress) or helps the plant grow healthy under abiotic stress is considered as indi-
rect mechanics, whereas any mechanism that directly increases plant growth through
the provision of nutrients or the production of growth regulators is considered as a
direct mechanism (Goswami et al. 2016). This section focuses on plant growth pro-
motion by PGPR directly. Generally, modes of action of PGPR of providing nutri-
ents for plants are as below.
9.2.1 Increasing Nutrient Supply of Plants
In the absence of a nutrient in the soil, PGPR can provide the nutrient for plants,
such as nitrogen (N) by fixing atmospheric nitrogen (N2) (Fig. 9.1a). In the rhizo-
sphere, there are microorganisms able to fix N2 forming specialized structures (e.g.,
Rhizobium and related genera) or simply establishing associative relationships (e.g.,
Azospirillum and Acetobacter). Furthermore, some bacteria (e.g., ammonifiers and
nitrifiers) convert organic N compounds into inorganic forms (i.e., NH4+ and NO3−)
that are available for root uptake.
9.2.2 Increasing Nutrient Availability to Plants
A large proportion of nutrients are unavailable for the root uptake by plants because
the nutrients in soils are generally bound to organic and inorganic soil constituents,
or alternatively present as insoluble precipitates. Therefore, in these conditions,
there are these nutrients in soil but their solubility is low and PGPR enhance the
availability of these nutrients to plants by different mechanisms such as enhancing
the solubility of phosphorus (P) and iron (Fe) (Fig. 9.1b). On the other hand, the
increase of PGPR-derived ion concentration would help the uptake of nutrients by
roots because one of the mechanisms of ion transport to plant roots is diffusion
movement, which is caused by differences in concentration.
9.2.3 Enhancing Plant’s Greater Access to Soil Nutrients
In these conditions, there is nutrient in soil and its solubility is also high but plants
do not have any or more access to it. Therefore, PGPR enhance the access of plants
to the nutrient and more uptake of it by increasing the root growth of plant by dif-
ferent mechanisms. The most important mechanisms involved in root elongation by
bacteria are the production of IAA and ACC deaminase (Fig. 9.1c). Since one of the
mechanisms of ion transport to plant roots is root interception (growth of roots
throughout the soil mass), which is a physical contact resulted by root growth, it
may be concluded that PGPR by IAA production and subsequently increased root
length can enhance plant’s greater access to soil nutrients. Therefore, a good root
system is a prerequisite for nutrient acquisition. It is a commonplace that the contact
between the nutrient and the root of the plant may be necessary before it may be
taken up. However, both availability and efficiency largely depend on the contact
between nutrients and the root. In general, it has well been known that many PGPR
may reduce the growth rate of the primary root (Dobbelaere et al. 1999), increase
the number and/or length of lateral roots (Chamam et al. 2013; Combes-Meynet
et al. 2011), and stimulate root hair elongation in vitro (Contesto et al. 2008;
Dobbelaere et al. 1999). Consequently, the uptake of minerals and water, and thus
the growth of the whole plant, can be increased. Some of these effects, including
increased root and shoot biomass, are also documented for PGPR-inoculated plants
9 Plant Growth-Promoting Rhizobacteria (PGPR) and Their Action Mechanisms…
Fig. 9.1 Modes of action of PGPR of providing nutrients for plants. (a) Bacteria can provide the nutrients in soil which is lacking, (b) bacteria can increase
insoluble nutrients availability to plants, and (c) bacteria can enhance plant greater access to soil nutrients
151
growing in soil (El Zemrany et al. 2006; Veresoglou and Menexes 2010; Walker
et al. 2012).
9.3 Essential Plant Nutrients
Only 17 elements have been found to be absolutely essential for plant growth and
metabolism that plants require to complete in their life cycle, based upon the criteria
for essentiality of an element. These elements are further divided into two broad
categories on the basis of their quantitative requirements: (i) macronutrients includ-
ing carbon (C), hydrogen (H), oxygen (O2), nitrogen (N), phosphorus (P), potas-
sium (K), sulfur (S), calcium (Ca), and magnesium (Mg) and (ii) micronutrients or
trace elements including manganese (Mn), iron (Fe), copper (Cu), molybdenum
(Mo), zinc (Zn), nickel (Ni), chlorine (Cl), and boron (B). Among the essential ele-
ments mentioned above, O2, H, and C are mainly obtained from CO2 and H2O, while
the others are absorbed from the soil as mineral nutrition. Crop nutrition is affected
by several factors. These factors can be internal or genetic factors (plant factors) and
external factors (soil factors). Both types play significant roles in the nutrition pro-
cesses that we can observe in crops. Availability of the nutrients is the resultant of a
complex of soil factors. Among soil factors, soil pH is one of the most important
factors affecting nutrient availability in the soil, which may either increase or
decrease nutrient availability (Fig. 9.2). As shown in Fig. 9.2, maximum availability
for the majority of nutrients is at pH = 6.5 (soils with pH levels higher or lower than
6.0 and 7.0), i.e., under slightly acidic conditions. Among nutrient elements, N, K,
and S solubility are less affected by pH, but still are to some extent. P, however, is
affected. For example, at acidic pH, phosphate ions react with aluminum (Al) and
Fe and they become less soluble compounds and these ions also react rapidly with
Ca and Mg to form the same less soluble compounds at alkaline pH greater than pH
7.5. Availability of microelements increases with acidity, with the exception of
molybdenum. The effect of soil pH on chloride availability is also neutral. In addi-
tion to pH, the availability of S, Fe, and Mn is also affected by redox reactions. In
this review, special consideration is given to the role of PGPR in the availability of
N, P, K, and S as macronutrients and Fe and Mn as micronutrients.
9.3.1 Nitrogen (N)
N is an essential element in plant development and a limiting nutrient for both natu-
ral and agricultural ecosystems. Although there are about 78% N2 in the atmosphere,
this form of N is not available to plants. Since there is a triple bond between the two
N atoms, making the molecule almost inert, N2 cannot be directly assimilated by
living cells. However, certain bacteria genera acquired an enzyme complex that uses
N2 and converts it into organic N-containing molecules in the cytoplasm. Ammonium
(NH4+) and nitrate (NO3–) are the predominant inorganic forms of N in soils. Plants
absorb the available N in the soil through their roots in the form of NH4+ and NO3−.
Fig. 9.2 Availability ranges of nutrient elements depending on soil pH
9.3.1.1 N2-Fixing Bacteria (NFB)

Biological nitrogen fixation (BNF) is the process by which N2 is reduced to ammo-
nia by a specialized group of microorganisms called diazotrophs. All the nitrogen-
fixing organisms are prokaryotes (archaebacteria and eubacteria). Diazotrophic
bacteria possessing the trait of N2 fixation are classified into three subgroups: sym-
biotic, free living, and associative. Root/legume-associated symbiotic bacteria pos-
sess the specificity and infect the roots to produce nodule. Several types of symbiotic
biological N2-fixing associations are known. The most prominent among them is the
legume-bacteria (strains of Rhizobium) relationship. The amount of N2 fixed by
legumes into usable N can be substantial (176 × 1012 g year−1). The legume host
plant provides the bacteria with their necessary carbohydrates and possibly all the
other nutrients they require in the exchange for the fixed N2 by the bacteria. In this
biological process, nodule-forming rhizobia inhabit the roots of leguminous plants
and through a symbiotic relationship convert atmospheric N2 to a form the plant can
use (Fig. 9.1a). The total BNF is estimated to be twice as much as the total nitrogen
fixation by nonbiological processes (80 × 1012 g year−1). Associative nitrogen-fixing
bacteria are a wide variety of the diazotrophs that form associative and/or endo-
phytic relationships with a wide variety of plant roots including those of cereals and
colonize the root surface of nonleguminous plants but do not inhabit specialized
growth structures on their host plants (Franche et al. 2009). This relationship is
described as a nonspecific and loose symbiosis. In other words, associative nitrogen

fixation is commonly defined as nitrogen fixation by a free-living diazotroph under
the direct influence of a host (Dalton and Kramer 2006). These bacteria do not pos-
sess specificity to plants such as Azospirillum, Burkholderia, Enterobacter,
Gluconoacetobacter, Herbaspirillum, Azoarcus spp., and Klebsiella (Dalton and
Kramer 2006). Due to a very close relationship established between associative
NFB and plants, the fixed N2 (some excess N) by these bacteria can also be taken up
by the plant and the microbes can utilize plant-derived carbon compounds to fuel
the nitrogen fixation reaction. Furthermore, plants may provide suitable conditions
for protecting the nitrogenase complex from exposure to oxygen. Generally, these
bacteria can make only a small contribution to the nitrogen nutrition of the plant
because nitrogen fixation is an energy-expensive process, and large amounts of
organic nutrients are not continuously available to microbes in the rhizosphere.
Bacterial genera such as Klebsiella, Azotobacter, Azoarcus, Bacillus, Enterobacter,
Xanthobacter, Beijerinckia, Achromobacter spp., Arthrobacter spp., Clostridium
spp., Corynebacterium spp., Herbaspirillum spp., Pseudomonas spp.,
Rhodopseudomonas, Rhodospirillum, Azomonas, and Derxia (Saharan 2011;
Saxena and Tilak 1998) are examples of the NFB that live independently of other
organisms (any plant species). These bacteria are also named as free-living nitrogen-
fixing bacteria. Almost all of the nitrogen fixed by free-living NFB is used by these
bacteria.
9.3.1.2 Action Mechanisms of Bacteria in Providing N for Plant
Biological N2 Fixation (BNF)

Many associated bacteria can fix N2 so that they could provide N to the plant. N2-
fixing PGPR can increase plant N uptake by different processes. The N cycle is
biologically influenced. PGPR have a central role in almost all aspects of N avail-
ability. In terms of availability of N to plants, some bacteria (diazotrophs) can con-
vert N2 into ammonia by the process termed biological nitrogen fixation (BNF) and
using a complex enzyme system known as nitrogenase. Mechanism of BNF has
been well known and documented (Franche et al. 2009; Santi et al. 2013). In nitro-
gen fixation process, 16 moles of ATP and a supply of electrons and protons (hydro-
gen ions) are needed to produce two ammonia molecules from a mole of N2 gas (the
equation below). Nitrogenase enzyme catalyzes the nitrogen fixation reaction:
N 2 + 8H + + 8e - + 16Mg　 ATP ® 2 NH 3 + H 2 + 16Mg　 ADP + 16Pi

In addition to rhizobial bacteria associated with legume plants, numerous nitrogen-
fixing species have also been identified that are able to colonize the root surface and,
in some cases, the root interior of a variety of pasture grasses and cereal crops (non-
leguminous plants) (Franche et al. 2009).
Mineralization of Organic Nitrogenous Compounds

Another mechanism of bacteria in making N available to plant is mineralization of
organic forms of N in soil (Fig. 9.3). Many bacteria degrade organic matter and
release fixed N for reuse by other organisms (nonlegume plant). Each part of a
legume crop that remains after harvest (e.g., roots, leaves, and nodules) can supply
N to the soil system during the decomposition of plant materials. In addition, these
plants do not use the entire N they receive from the atmosphere and return extra N
to the soil. During the decomposition of plant matter, dead bacteria, and root exu-
dates including N, organic N is once again converted to inorganic ammonium and
released into the soil. The process that converts organic N to ammonium is called
mineralization (conversion of organic N to inorganic forms) and plays a significant
role in the management of N. The first step of mineralization is called aminization,
in which microorganisms including bacteria break down complex proteins to sim-
pler amino acids, amides, and amines. Ammonification is the second step of
Fig. 9.3 Mechanisms increasing the availability of N in the rhizosphere to leguminous and nonle-
guminous plants. In legumes and a few other plants, the bacteria live in small growths on the roots
called nodules. Within these nodules, nitrogen fixation is done by the bacteria, and the NH3 pro-
duced is absorbed by the plant. Almost all of the nitrogen fixed goes directly into the leguminous
plant. Little leaks into the soil for a neighboring nonlegume plant. However, other plants (nonlegu-
minous plants) benefit from nitrogen fixing bacteria when the bacteria die and release nitrogen to
the environment, or when the bacteria (associative and free living) live in close association with the
plant, or by the release of ammonium or simple organic nitrogen compounds through the decom-
position of organic matter obtained from vegetation (roots, leaves, fruits) of leguminous plants
mineralization in which amino (NH2) groups are converted to ammonium. Again,

microorganisms, including bacteria, do this. The two steps of nitrification (conver-
sion of ammonium to nitrate) are also performed by microbial activity. Nitrosomonas
(obligate autotrophic bacteria) convert ammonium to nitrite. Nitrobacter species
perform the second step of nitrification, which converts nitrite to nitrate. This step
quickly converts ammonium into nitrite, and thus nitrite concentration in soils is
usually low.
Immobilization of Soluble Inorganic N

Immobilization, or the temporary tying up of inorganic N by soil microorganisms
decomposing plant residues, is not strictly a loss process. A large proportion of the
total fixed N will be locked up in the biomass or in the dead remains of organisms.
Immobilized N will be unavailable to plants for a time, but will eventually become
available as residue decomposition proceeds and populations of microorganisms
decline. Therefore, it may be concluded that PGPR by immobilization of inorganic
N (NO3−) can make more N available for plants because immobilization decreases
the loss of soluble NO3−, which is highly mobile and is easily lost from the soil
system by leaching and denitrification (conversion of NO3− to N gases).
Increased Root System of Plant

Nutrient presence in soil and its solubility may be high, but still plants do not have
any access to it due to limitations in root growth or activities. Since essential nutri-
ents are absorbed from the soil by the root, a good root growth is a prerequisite for
increasing plant growth (Mills et al. 1996). Root hairs, along with the rest of the root
surface, are the major sites of water and nutrient uptake. In an important analysis
and review of the literature, Kuzyakov and Xu (2013) argued that microorganisms
were more effective than roots at obtaining nutrients from the soil. Thus microor-
ganisms win in the competition for nitrogen against roots. However, over the dura-
tion of the growing season, as root biomass increases it will outcompete
microorganisms. Key to this dynamic is the high turnover rate of microbes con-
sumed by the soil food web, in contrast to the continuously increasing root surface
for membrane transport and overall biomass. PGPR increase root system of plants
by production of IAA and ACC deaminase. Rhizobacterium-mediated root prolif-
eration has been well proved (Diby et al. 2005). Plants treated with PGPR have
better root with a subsequent increase of nutrient and water uptake. Promotion of
root growth results in a larger root surface and can, therefore, have positive effects
on water acquisition and nutrient uptake (Diby et al. 2005b; Paul and Sarma 2006)
that is expected to move nutrient (e.g., N) from soil to root (mechanism of mass flow
in ion transport to plant roots). Phytohormone IAA, whose biosynthesis requires
L-tryptophan (L-Trp) as a precursor, is primarily involved in stimulating the prolif-
eration of lateral roots in plants; thereby root surface area is increased and they
absorb more water and soil minerals (Egamberdieva and Kucharova 2009;
Lugtenberg and Kamilova 2009). Under both biotic (e.g., phytopathogen attacks)
and abiotic (e.g., heavy metals, flooding, and salinity) stresses, plant produces eth-
ylene up to the level that is inhibitory to root growth (Arshad et al. 2007; Chen et al.
2013; Khalid et al. 2006; Nadeem et al. 2009). An enzyme ACC deaminase pro-
duced by many soil microflora including PGPR (He et al. 2010; Kumar et al. 2009)
degrades ACC (an immediate precursor for ethylene in plants) and decreases the
ethylene biosynthesis in plant tissues (Saleem et al. 2007; Shaharoona et al. 2007;
Zahir et al. 2009). Many PGPR produce IAA and enzyme ACC deaminase that
undoubtedly affect root growth, leading to the formation of root systems with
increased exploratory capacity. Plant growth-promoting non-rhizobial bacteria can
help the fixation of N by enhancing the capacity of rhizobial bacteria to colonize
plant roots and increasing the number of nodules (Masciarelli et al. 2014). In addi-
tion, IAA-producing PGPRs by increasing root exudates can have a positive role in
N2 fixation. It has been reported that the phenolics and aldonic acids that are directly
secreted by the roots of N2-fixing legumes act as the main signal for the bacteria that
form root nodules where N2 is reduced to ammonia (Dakora and Phillips 2002).
Overall, IAA and ACC deaminase-producing PGPR increase root surface area and
length (Potters et al. 2009, 2007; Ryan et al. 2008; Vessey 2003) and thus increase
the access of plants to nutrients and water absorption.
IAA Production
A member of the group of phytohormones, IAA is usually considered to be the most
important native auxin. Almost most rhizospheric bacteria (usually more than 80%
of bacteria) have the ability to produce this hormone (Khalid et al. 2004). At pres-
ent, IAA-producing PGPR are the most well-studied phytohormone producers
(Spaepen et al. 2007; Tsavkelova et al. 2006). The majority of root-related bacteria,
which have a positive effect on plant growth, produce IAA (Hayat et al. 2010). An
increase in the number of lateral roots and root hairs causes addition of root surface
available for nutrients and water uptake (Fig. 9.1c). Higher water and nutrient
uptake by inoculated roots causes an improved water status of plant, which in turn
could be the main factor enhancing plant growth (Dalla Santa et al. 2004;
Egamberdieva 2009; Egamberdieva and Kucharova 2009; Mostajeran et al. 2002).
Inoculation of various plant species with such bacteria leads to increased root
growth and/or enhanced formation of lateral roots and root hairs (Dimkpa et al.
2009) that can result in enhanced uptake of nutrients such as N. In addition to the
production of IAA, GA and other growth regulators produced by PGPR can support
increased root length, root surface area, and number of root tips, leading to enhanced
uptake of nutrients (Egamberdieva and Kucharova 2009). By increasing nutrient
availability via mechanisms such as producing plant growth-promoting (PGP) prod-
ucts, the symbiotic, free-living, and associative NFB and other PGPR can also
enhance plant growth directly. The production of IAA appears to be widespread in
associative NFB and has since been confirmed in a number of other genera includ-
ing Azospirillum, Herbaspirillum, and Pseudomonas (Pedraza et al. 2004). Although
the growth-promoting effects of Azospirillum have been well documented, the exact
mechanism of growth promotion goes beyond nitrogen fixation to include nitrate
reduction, phytohormone production, production of undefined signal molecules that
can interfere with plant metabolism, and enhancement of mineral uptake by plants
in response to root elongation (Okon and Itzigsohn 1995). Morphological plant root
changes have been observed repeatedly upon Azospirillum inoculation and have
been attributed to the production of PGP substances, CK and GA, with auxin pro-
duction being quantitatively the most important (Spaepen et al. 2008). Specific evi-
dence for the interference of IAA produced by Azospirillum in root development
was obtained in many cases. In a study (El-Khawas and Adachi 1999), the inocula-
tion of IAA-producing A. brasilense to the roots of rice resulted in an increase in
root length, root surface, root dry matter, and development of lateral roots and root
hair in comparison with uninoculated roots. Similarly, IAA-producing A. brasilense
Cd induced many roots and increased root length of soybean plants (Molla et al.
2001). More direct evidence for the importance of IAA was provided when several
IAA-attenuated mutants were compared with their parental wild types for their
effect on plant growth. A mutant of A. brasilense with low production of phytohor-
mones, but high N2-fixing activity, did not enhance root growth over uninoculated
controls (Kundu et al. 1997).
Bacterial IAA, by loosening plant cell walls (Chaintreuil et al. 2000; Chi et al.
2005; James et al. 2002; Sevilla et al. 2001), can also promote an increase in root
exudation (carbon exudation) that provides additional nutrients to support the
growth of rhizosphere bacteria. Due to IAA bacterial derived root exudation, the
increased microbial population enhances microbial respiration and subsequently
reduces oxygen. Reduced oxygen supply in the root zone has been shown to enhance
nitrogenase activity in rhizosphere organisms (Döbereiner et al. 1972). In addition,
the correlation of nitrogenase activity and photosynthate flux indicates that carbon
exudates are a major regulatory factor in diazotrophic activity in the rhizosphere
(Dalton and Kramer 2006). Bacterial IAA is also involved in many processes of
nodule formation by rhizobia in legume plants. Founder cell specification, nodule
initiation and differentiation (IAA accumulation), nodule numbers, vascular bundle
formation, and cell division and differentiation are some of the processes of nodule
formation mediated by bacterial IAA. These three latter events are more necessary
for nodule formation (Glick 2012b; Theunis 2005). In addition, IAA-producing
bacteria, by increasing the root system, provide more active sites for more bacteria
colonization. As an example, Parmar and Dadarwal (1999) reported that increased
root growth provides more active sites and provides access to nodulation for rhizo-
bia in chickpea plant. In another study, the presence of PGPR in the vicinity of the
root can improve the ability of rhizobia to compete with indigenous populations to
nodulation. Therefore, it is suggested to pay more attention in selecting microbial
inoculants with high phytohormone production to potentially increase the uptake of
N. In addition to hormone production, associative fixing bacteria may also benefit
hosts plants in a variety of ways including improved nutrient cycling or uptake
(especially through production of siderophores for iron uptake) (Dobbelaere et al.
2003). Bacterial IAA production also stimulates the activity of the enzyme ACC
deaminase involved in the degradation of the ethylene precursor ACC (Glick 2005).
In general, IAA and ACC deaminase work in concert to stimulate root elongation
(Etesami et al. 2015a, 2014).
ACC Deaminase Activity

PGPR contain the enzyme ACC deaminase; it can act to modulate the level of ethyl-
ene in plants (Glick 2014; Singh et al. 2011). This enzyme is responsible for the
Fig. 9.4 Mechanisms by which PGPR may affect nodule number and nitrogen fixation in a
legume plant
cleavage of the plant ethylene precursor, ACC, into ammonia and α-ketobutyrate
(Glick et al. 2007). Plants that are inoculated with bacteria that produce enzyme ACC
deaminase can adjust their ethylene levels and thus help the wider root system
(Arshad et al. 2007; Safronova et al. 2006; Stearns et al. 2005). The ACC deaminase
trait has been extensively studied in PGPR (Glick 2005) such as the genera
Achromobacter, Acidovorax, Alcaligenes, Enterobacter, Klebsiella,
Methylobacterium, Pseudomonas, Rhizobium, and Variovorax (Esquivel-Cote et al.
2010). In general, ACC deaminase-containing PGPRs may act as a sink for ACC. It
has been well known that under stressful conditions, nodulation, nitrogenase activity,
N2 fixation, and total N content in legume plants are reduced. One of the main rea-
sons for this decrease may be due to the production of stress-induced ethylene.
Ethylene inhibits the elongation of infection threads and, consequently, the forma-
tion of nodules in most legumes (Sugawara et al. 2006). Extra ethylene production
can also inhibit root prolongation, growth of lateral roots, and root hair growth
(Belimov et al. 2009; Mayak et al. 2004; Saleem et al. 2007), which subsequently
result in decrease in the nodule number of root. Fe deficiency also decreases nodule
mass and particularly leghemoglobin content, number of bacteroids, and nitrogenase
activity (Garcia et al. 2015; Tang et al. 1990). The deficiency of P supply and avail-
ability also remains a severe limitation of N2 fixation and symbiotic interactions
(Pereira and Bliss 1989). It has been well known that PGPR can alleviate the effect
of these stresses on legume plant and increase N2 fixation by different ways (Fig. 9.4).
9.3.2 Phosphorus (P)
After N, the essential mineral element that most frequently limits the growth of
plants is phosphorus (P), which is taken up only in monobasic (H2PO4−) or diba-
sic (HPO42−) soluble forms. P is found mainly in inorganic fractions, which are
either adsorbed into the soil’s inorganic surfaces or found as sparingly available
precipitates, and in organic forms that are either adsorbed, incorporated within
biomass, or associated with soil organic matter (Richardson and Simpson 2011).
Even in soils with abundant P ranging from 400 to 1200 mg kg-1 of soil, usually
only about 1% of the soil P is actually in a readily available, soluble form, and
over 90% is generally bound tightly to soil particles and inorganic minerals such
as apatite, hydroxyapatite, and oxyapatite or appear as one of several organic
forms including inositol phosphate (soil phytate), phosphomonoesters, and phos-
photriesters (Khan et al. 2007b), which require mineralization before they
become plant available (Jewell et al. 2010). P is an integral part of various bio-
chemical substances such as nucleic acids, phospholipids, nucleotides, and phos-
phoproteins. Calcium concentration, soil pH, proportion of organic matter, type
and proportion of clay, soil moisture, soil texture, root density, and exudates are
among the parameters that have been able to influence the availability of soil P to
the plant (Barber 1995). Parameters including high soil pH, high soil CaCO3, low
soil organic matter, and drought decrease P availability to plants in the calcare-
ous soils of Iran, with arid and semiarid climates. As previously mentioned, the
soil pH for optimum P availability is 6.5. P reactions in soil are pH dependent. At
high or neutral pH, phosphate is converted to less soluble compounds such as
dicalcium phosphate dihydrate or octacalcium phosphate. In some cases it may
eventually convert to hydroxyapatite. P may react with Al and Fe to form low-
solubility Fe- and Al-phosphates such as strengite and varescite under acidic
conditions. The limitation in bioavailability of P from the soil along with the fact
that this element is essential for plant growth suggests that the inability to obtain
sufficient amount of P restricts plant growth (Khan et al. 2007b). Plants are well
adapted to the uptake of P from low-concentration soil solution under low-P
conditions (Jungk 2001). Plants have been demonstrated to alter the rhizosphere
with specific exudates, commonly organic acids or enzymes, to improve the
availability of nutrients such as phosphate (Hong et al. 2008; Park et al. 2007;
Xiao et al. 2007). Furthermore, by inhibiting primary root growth, promoting
lateral root growth, and enhancing root hair development and cluster root forma-
tion, which all promote P acquisition by plants, plants adjust their root architec-
ture to low-P conditions (Jain et al. 2007; Ma et al. 2003; Niu et al. 2013; Osmont
et al. 2007). Lateral roots have been known to play an important role in the
absorption of P via different ways such as solubilizing insoluble P (Lynch 2007)
and increasing the absorptive surface of the root system (Pérez-Torres et al.
2008) and soil exploration (Zhu et al. 2005).
9.3.2.1 Phosphate-Solubilizing Bacteria (PSB)

It has been known that strategies mentioned above are often not efficient enough to
meet the needs of the plants growing especially in calcareous and alkaline soils.
Therefore, using phosphate-solubilizing bacteria (PSB) for providing accessible
forms of P for plants is necessary when it is scant or unavailable in soils. The con-
version of insoluble phosphate compounds (both organic and inorganic) in a form
accessible to the plant is an important trait of PSB. Solubilization of insoluble P by
microorganisms was reported by Pikovskaya (1948). In soil, PSB constitute 1–50%
of the total respective population. PSB have been isolated, using serial plate dilution
method or by enrichment culture technique, from almost all areas, including from
rhizosphere and non-rhizosphere soils, rhizoplane, phyllosphere, and rock P deposit
area soil and even from stressed soils (Zaidi et al. 2009).
The ability to solubilize insoluble inorganic phosphate compounds such as
hydroxyapatite, tricalcium phosphate, rock phosphate, and dicalcium phosphate has
been reported in PGPR strains belonging to various genera (Khan et al. 2009b;
Ramaekers et al. 2010). A significant number of microbial species show the capacity
of solubilizing P; these include actinobacteria, bacteria, fungi, and even algae. The
solubilization of insoluble phosphates has been reported in most known bacterial
genera (e.g., Streptomyces sp., Agrobacterium sp., Azospirillum brasilense, Bacillus
sp., B. circulans, B. cereus, B. fusiformis, B. pumilus, B. megaterium, B. mycoides, B.
polymyxa, B. coagulans, B. subtilis, Rhodococcus, Klebsiella, Vibrio proteolyticus,
Alcaligenes sp., Aerobacter aerogenes, Achromobacter sp., Enterobacter,
Thiobacillus ferrooxidans, T. thiooxidans, Xanthomonas sp., Actinomadura oligos-
pora, Brevibacterium sp., Citrobacter sp., Arthrobacter, Serratia, Chryseobacterium,
Gordonia, Phyllobacterium, Xanthobacter agilis, Delftia sp., Azotobacter,
Xanthomonas, Pantoea, Pseudomonas sp., P putida, P. striata, P. fluorescens, P. cal-
cis, Flavobacterium sp., Nitrosomonas sp., Erwinia sp., Micrococcus sp., and
Nitrobacter sp.) (Sharma et al. 2013). By mobilizing inorganic and organic P, symbi-
otic nitrogenous rhizobia like Rhizobium leguminosarum bv. Trifolii and Rhizobium
species nodulating Crotalaria species also improved plant P nutrition (Abril et al.
2007; Sridevi et al. 2007; Zaidi et al. 2009). Of the bacterial genera mentioned above,
Pseudomonas and Bacillus were reported as the most important bacterial genera that
were able to effectively solubilize insoluble phosphates.
9.3.2.2 Action Mechanisms of P Solubilization by PSB

Solubilization and mineralization of P in rhizosphere are the most common modes
of action implicated in PGPR that increase the nutrient availability to the host plant
(Glick 2012a; Rashid et al. 2004b). PGPR play an important role in all three major
components of the soil P cycle (i.e., dissolution–precipitation, sorption–desorption,
and mineralization–immobilization). An example is the PSB, which dissolve vari-
ous sparingly soluble P sources such as Ca3(PO4)2 (Rodriguez et al. 2004) and
Zn3(PO4)2 (Saravanan et al. 2007) by lowering pH of the rhizosphere soil and mak-
ing P available for plant uptake. By solubilizing and mineralizing reactions, and
immobilizing P into microbial biomass and/or forming sparingly available forms of
inorganic and organic soil P, PSB and their interactions in soil play a critical role in
mediating the distribution of P between the available pool in soil solution and the
total soil P. Overall, phosphate-solubilizing PGPRs can either convert these insolu-
ble phosphates into available forms through acidification, chelation, exchange reac-
tions, release of complexing or mineral dissolving compounds (e.g., organic acid
anions, protons, hydroxyl ions, CO2), secretion of siderophores, IAA production,
ACC deaminase activity, and release of organic acids (Chung et al. 2005; Glick
2012a) or mineralize organic phosphates by secreting a variety of different extracel-
lular phosphatases, catalyzing the hydrolysis of phosphoric esters (Gyaneshwar
et al. 2002; Van Der Heijden et al. 2008). Each organism can act in one or more than
one way to bring about the solubilization of insoluble P. Though it is difficult to
pinpoint a single mechanism, production of organic acids and consequent pH reduc-
tion appear to be of great importance. In the following section, different mecha-
nisms involved in the solubilization and mineralization of insoluble P by PSB are
discussed.
Production of Organic Acids

One of the most known mechanisms of P solubilization by PSB is associated with
the production of organic and inorganic acids and proton excretion. H+ excretion is
originated from NH4+ assimilation by plant and PSB (Parks et al. 1990). For some
microorganisms, NH4+-driven proton release seems to be the sole mechanism to
promote P solubilization. An HPLC analysis of the culture solution of Pseudomonas
sp., in contrast to the expectation, did not detect any organic acid while solubiliza-
tion occurred (Illmer and Schinner 1995). According to these authors, probable
cause for the dissolution of phosphate without acid production is the release of
protons originated from assimilation of NH4+.
Organic acids (e.g., acid, oxalic acid, citric acid, lactic acid, tartaric acid, and
aspartic) are the product of the microbial metabolism, mostly by oxidative respira-
tion or by fermentation of organic carbon sources (e.g., glucose) (Trolove et al.
2003) or by oxidation of the soil organic matter or being added as manure.
Furthermore, PGPR (e.g., IAA producers) can enhance the amount of root exudates.
Root exudates include a huge diversity of organic nutrients (i.e., organic acids, phy-
tosiderophores, sugars, vitamins, amino acids, nucleosides, mucilage) and signals
that attract microbial populations, especially PSB able to metabolize plant-exuded
compounds and proliferate in this microbial habitat (Badri and Vivanco 2009;
Drogue et al. 2013; Khan et al. 2007a; Pothier et al. 2007; Sharma et al. 2013).
Organic acids produced by PSB and plant decrease the rhizosphere pH favoring
the solubility of precipitated P forms. Organic anions produced by PSB can also
compete with phosphates for fixation sites or even replace phosphate (anion
exchange of phosphate) sorbed on the surfaces of soil clays (kaolinite, goethite,
montmorillonite, and amorphous Al oxides). They also can enhance the chelation of
the cations (Al3+, Fe3+, and Ca2+) bound to P or the formation of soluble complexes
with metal ions associated with insoluble P avoiding thus the precipitation of phos-
phate (Osorio Vega 2007; Rashid et al. 2004a; Whitelaw 1999) and thus P is released.
The monovalent anion phosphate H2PO4− is a major soluble form of inorganic phos-
phate, which usually occurs at lower pH. However as the pH of the soil environment
increases the divalent and trivalent forms of Pi (HPO4−2 and HPO4−3, respectively)
occur. Thus, the synthesis and discharge of organic acid by the phosphate-
solubilizing PGPR strains into the surrounding environment acidify the cells and
their surrounding environment that ultimately leads to the release of P ions from the
P mineral by H+ substitution for the cation bound to phosphate (Goldstein 1994).
When phosphate-solubilizing PGPRs are inoculated to neutral or alkaline soils, the
acid production decreases the rhizosphere pH, favoring thus the solubility of cal-
cium phosphates and apatites (Fig. 9.5a). If the activity of H+ increases in the reac-
tants of the reactions of the solubility of dicalcium phosphate and hydroxyapatite,
these reactions proceed. In addition, the sequestering of Ca2+ by organic anions or
other chelating agents such as siderophores favors the reactions. In acid soils, the
minerals variscite and strengite control the solubility of phosphate. The presence of
organic acids assists the formation of complexes with Al3+ and Fe3+ ions, which in
turn facilitates the dissolution of these minerals. If Fe3+ and Al3+ are sequestered via
chelation with organic anions, the reactants of the reactions of the solubility of
strengite and variscite proceed to the right (Fig. 9.5b).
Production of Inorganic Acids

PSB have also been shown to solubilize insoluble phosphates by producing inorganic
acids (e.g., HCl) (Kim et al. 1997). Bacteria of the genera Nitrosomonas and
Thiobacillus species (microbial sulfur oxidation) and other bacteria can also dissolve
phosphate compounds by producing nitric and sulfuric acids, and carbonic acid
formed as a result of the decomposition of organic residues, respectively (Azam and
Memon 1996), decreasing soil pH. Therefore, elemental sulfur can be inoculated
with Thiobacillus to enhance the P solubility of apatite, and hence plant biomass
(Stamford 2003). However, the effectiveness of inorganic acids is lower than that of
organic acids in solubilizing insoluble phosphates (Kim et al. 1997). The other mech-
anism is the production of H2S, which reacts with ferric phosphate to yield ferrous
sulfate with concomitant release of phosphate (Swaby and Sperber 1958). Overall,
acidification does not seem to be the only mechanism of solubilizing insoluble phos-
phates by phosphate-solubilizing PGPRs because the ability to reduce PH in some
cases is not related to the ability to solubilize P minerals (SubbaRao 1982).
Production of IAA and ACC Deaminase

PGPR can enhance the capacity of plants to acquire P from soil through increased
root growth either by hormonal stimulation of root growth, branching, or root hair
development (e.g., production enzymes that alter plant ethylene precursors, such as
ACC deaminase or production of IAA) or by an extension of existing root systems
(Hayat et al. 2010; Richardson et al. 2009). ACC deaminase can affect plant root
growth by degrading the precursor for the production of the stress hormone, ethyl-
ene. Increased level of ethylene production in plant can decrease root growth. As a
consequence, the enzymes can also indirectly influence P effect on root growth as
well as its uptake by plant, because ethylene can adjust root architectural response
to P availability in the soil. Under stresses such as P deficiency, the increased
Fig. 9.5 (a) Role of PGPR in enhancing the capacity of plants to acquire P from soil through
alteration of sorption equilibria that may result in increased net transfer of orthophosphate ions into
soil solution. Organic anions and protons are particularly effective in solubilizing precipitated
forms of P (e.g., Ca phosphates under alkaline conditions and (b) Fe and Al under acidic condi-
tions), chelating metal ions that are commonly associated with complexed forms of soil P (as is for
the role of siderophores in mediating Fe availability)
production of stress can adversely affect plant response to P and decrease the num-
ber of root hairs (Borch et al. 1999).
Bacterial IAA can increase the root exudates and root system. Organic acids
(e.g., gluconic and citric acid) found in the root exudates in turn result in acidifica-
tion of the rhizosphere (Amir and Pineau 2003; Dakora and Phillips 2002; Jones
et al. 2003). In addition, production of CO2 by respiration processes (due to release
of carbohydrates, amino acids, lipids, and vitamins by roots and subsequently stim-
ulation of microorganisms in the soil), pump of H+ in nutrient uptake by plant and
microbes, organic matter decomposition, and N2 fixation by the symbiosis Rhizobium
legume (Marschner and Rimmington 1988) are some of the responsible mecha-
nisms for acidification of rhizosphere than the bulk soil. By the complexation of
essential ions, the organic acids play an important role in the increase of mobility of
the elements for plant uptake. Acid pH is common for the rhizosphere environment
due to proton extrusion through the root cell membrane (Spaepen et al. 2007). The
acidification can also contribute to plant growth by mobilizing nutrients such as P
and micronutrient. Increase in the acidity of the surrounding soil can occur by
releasing proton and organic acids from the seeds and roots and absorbing nutrient
ions by the plant (Hartmann et al. 2008). Altered root morphology of inoculated
plants may enhance P uptake. In addition, the prevalence of root hair and lengths is
also associated with an increase in the absorption of relatively immobile elements
such as P. A large number of phosphate-solubilizing PGPR (Ahemad 2012; Ahemad
and Khan 2010; He et al. 2010; Misra et al. 2012; Oves et al. 2013) in soils have
been reported to secrete IAA that is absorbed by plant roots to increase the endog-
enous pool of plant IAA (Glick et al. 2007). Datta et al. (1982) reported that a
P-solubilizing and IAA-producing strain of B. firmus increased the grain yield and
P uptake of rice in a P-deficient soil amended with rock phosphate.
In general, stimulation of root growth or greater elongation of root hairs by spe-
cific microorganisms may enhance plant P nutrition indirectly by allowing greater
exploration of soil, rather than by direct increase in the availability of soil P. It is
presumed that the supply and availability of P to the root surface are influenced by
the root and microbial processes. According to the materials listed above, it may be
suggested that IAA-producing PGPR (due to having a role in enhancing root exu-
dates and root surface area) can also solubilize insoluble phosphates similar to phos-
phate-solubilizing bacteria (Fig. 9.6) (Dobbelaere et al. 1999; Lambrecht et al.
2000; Steenhoudt and Vanderleyden 2000).
Production of Siderophores
Siderophores are complexing agents that have a high affinity for Fe(III) and are
produced by almost all microorganisms in response to Fe deficiency. Siderophores,
in the case of iron deficiency, act as a solubilizing agent for Fe from organic com-
pounds or minerals. Some of the produced siderophores (~500 known siderophores)
are exclusively used by microbial species and strains that produce them and some of
them are used by a wide variety of plants and microorganisms (Crowley 2006). The
ability to produce siderophores by phosphate-solubilizing PGPR is well established
(Caballero-Mellado et al. 2007; Hamdali et al. 2008; Vassilev et al. 2006). The
Fig. 9.6 The schematic representation of role of IAA-producing PGPR in the availability of nutri-
ent elements (e.g., P) to plant by affecting plant (root) growth and hence plant root exudates
siderophores can increase the availability of P for plants either by chelating cations
(e.g., Ca2+, Fe+3, and Al3+) forming precipitations with P or by exchange of ligands.
Considering the dominance of mineral dissolution over ligand exchange by organic
acid anions as a P-solubilizing mechanism (Parker et al. 2005), the potential role of
siderophores in enhancing the availability of P should be clear (Sharma et al. 2013).
Production of Exopolysaccharides (EPS)

The role of exopolysaccharides (EPS) in the microbial mediated solubilization of P
has also been confirmed (Yi et al. 2008). Microbial EPS, produced by some bacteria
and fungi, are polymers composed mainly of carbohydrates and secreted outside the
cell wall of microbes. However, these organic compounds may be homo- or hetero-
polysaccharides and may also contain a number of different organic and inorganic
substituents. In general, the composition and structures of EPS are very diverse
(Sutherland 2001). It has been known that EPS-producing PGPR (e.g., Enterobacter
sp., Arthrobacter sp., and Azotobacter sp.) have the ability to solubilize tricalcium
phosphate (TCP) (Yi et al. 2008). However, more studies are needed to understand
the relationship between phosphate solubilization and EPS production (Sharma
et al. 2013).
Mineralization of Organic P
In addition to mechanisms involved in releasing P from inorganic compounds, the
release of phosphatase enzymes that mineralize organic P compounds has also been
Fig. 9.7 The role of PGPR in the release of phosphatase enzymes mineralizing organic P com-
pounds and releasing inorganic P (HPO42−)
suggested as another mechanism involved (Fig. 9.7). Organic P solubilization is

also called mineralization of organic P. Mineralization of soil organic P plays an
imperative role in P cycling of a farming system. Organic P may constitute 4–90%
of the total soil P (Khan et al. 2009b). Organic P of organic compounds can be
released in soil by enzymes of phosphatase. Phosphate-solubilizing PGPR similarly
produce a range of phosphatases and when cultured in laboratory media have the
capacity to utilize P from various forms of organic P that occur in soil. These
enzymes, depending on their pH, are divided into acid and alkaline phosphatase,
both of which can be produced by phosphate-solubilizing PGPR depending on the
external conditions (Jorquera et al. 2008; Kim et al. 1998). So it is clear that alkaline
phosphatases usually dominate in neutral and alkaline soils, while acid phospha-
tases are abundant in acidic soils (Renella et al. 2006). Although the roots of plants
can produce acid phosphatase, they rarely produce large quantities of alkaline phos-
phatase, suggesting that this is a potential niche for phosphate-solubilizing PGPR
(Criquet et al. 2004). It is also difficult to differentiate between root- and phosphate-
solubilizing PGPR-produced phosphatases (Richardson et al. 2009); however, some
evidence suggests that microbial phosphatases have a higher affinity for phosphorus
compounds than plant phosphates and are also effective in releasing orthophosphate
from soil organic P (Tarafdar et al. 2001). NSAPs (nonspecific acid phosphatases)
dephosphorylate phospho-ester or phosphoanhydride bonds of organic matter.
Among the variety of phosphatase enzyme classes released by phosphate-
solubilizing PGPR, phosphomonoesterases (often just called phosphatases) are the
most abundant and best studied (Nannipieri et al. 2011). Inositol phosphate is a
dominant form of organic phosphorus found in many soils (Turner 2006). Phytases
specifically cause release of P from phytate degradation. Phytate in its original form
is the main source of the inositol and the main form of P stored in plant seeds and
pollen, and the main component of P is organic matter (Richardson et al. 1994).
Bünemann (2008) reported that up to 60% of the total organic P may typically be
hydrolyzed by phosphatases with highest amounts being released by phytases
(monoester phosphatases active against phytate). Phosphonatases and C–P lyases
also can cleave the C–P bond of organophosphonates (Rodríguez et al. 2006).
Immobilization of Inorganic P
Phosphate-solubilizing PGPR decompose organic amendments added to soil (e.g.,
manures and plant residues) and mineralize organic P along with that of soil organic
matter. However, in the long run, all of the microbial phosphorus is potentially
available to plants, and it has been suggested that the immobilization of phosphorus
in biomass is an important mechanism for regulating the supply of P in soil solution
(Seeling and Zasoski 1993) and for maintaining it in labile forms that are protected
(in a temporal sense) from reactions with soil (Olander and Vitousek 2004). In gen-
eral, PGPR in the presence of labile C serve as a sink for P, by rapidly immobilizing
it even in low-P soils; therefore phosphate-solubilizing PGPR become a source of P
to plants upon its release from their cells. Release of P immobilized by phosphate-
solubilizing PGPR primarily occurs when cells die due to changes in environmental
conditions, starvation, or predation. Environmental changes, such as drying–rewet-
ting or freezing–thawing, can result in so-called flush events, a sudden increase in
available P in the solution due to an unusually high proportion of microbial cell lysis
(Butterly et al. 2009). According to the theory of sink, phosphate-solubilizing PGPR
remove and assimilate phosphorus from the liquid and thus activate the indirect dis-
solution of calcium phosphate compounds by sequentially removing P from the
liquid medium. For instance, the P content in the biomass of Pseudomonas sp. and
P. aurantiogriseum was similar to that observed in non-phosphate-solubilizing
PGPR (Illmer et al. 1995) which can be explained by the fact that the P content in
biomass of organisms is consistently correlated with the decomposition of P con-
taining organic substrates (Dighton and Boddy 1989).
9.3.2.3 Promotion of Plant Growth by PSB

Besides making soluble P accessible for uptake by plants, there have been a number
of reports on plant growth promotion by these microorganisms (Sharma et al. 2013).
There are studies showing that phosphate-solubilizing microorganisms under con-
trolled conditions and, in some cases, in field conditions have increased plant P nutri-
tion and subsequently plant growth (Gyaneshwar et al. 2002; Harvey et al. 2009;
Jakobsen et al. 2005; Khan et al. 2009a, 2007a; Whitelaw 1999; Zaidi et al. 2009).
Following inoculation of Ricinus communis and Helianthus annuus with
P-solubilizing Psychrobacter sp. SRS8 (Ma et al. 2010), wheat with P-solubilizing
Pseudomonas sp. (Babana and Antoun 2006), and peanut with P-solubilizing Pantoea
J49 (Taurian et al. 2010), an increase in growth and P uptake of these plants over
Table 9.1 Plant growth-promoting substances released by PSB

Bacterial isolates PGP traits References
Pseudomonas aeruginosa Production of IAA and Oves et al. (2013)
strain OSG41 siderophores
Pseudomonas sp. Production of IAA Singh et al. (2013)
Acinetobacter haemolyticus Production of IAA Misra et al. (2012)
RP19
Pseudomonas putida Production of IAA and Ahemad and Khan (2011b,
siderophores 2012c, d)
Pseudomonas fluorescens Production of IAA and Upadhyay and Srivastava
strain Psd siderophores (2010)
Bacillus thuringiensis Production of IAA Sandip et al. (2011)
Pseudomonas aeruginosa Production of IAA and Ahemad and Khan (2010b,
siderophores 2011a, d, 2012a)
Pseudomonas sp. TLC Production of IAA and Li and Ramakrishna (2011)
6-6.5-4 siderophore
Bacillus sp. Production of IAA Karuppiah and Rajaram
(2011)
Klebsiella sp. Production of IAA and Ahemad and Khan (2011c, e,
siderophores 2012b)
Enterobacter asburiae Production of IAA and Ahemad and Khan (2010a)
siderophores
Bacillus species PSB10 Production of IAA and Wani and Khan (2010)
siderophores
Arthrobacter sp. MT16, Production of ACC deaminase, He et al. (2010)
Microbacterium sp. JYC17, IAA, and siderophore
Pseudomonas chlororaphis
SZY6, Azotobacter vinelandii
GZC24, and Microbacterium
lactium YJ7
Pseudomonas sp. Production of IAA and Tank and Saraf (2009)
siderophore
Enterobacter aerogenes Production of ACC deaminase, Kumar et al. (2009)
NBRI K24 and Rahnella IAA, and siderophore
aquatilis NBRI K3
Enterobacter sp. Production of ACC deaminase, Kumar et al. (2008)
IAA, and siderophore
Burkholderia Production of ACC deaminase, Jiang et al. (2008)
Pseudomonas aeruginosa Production of ACC deaminase, Ganesan (2008)
uninoculated plants was observed. In addition to solubilizing P, phosphate-

solubilizing PGPR also promote plant growth through N2 fixation (He et al. 2010),
lowering ethylene levels (Jiang et al. 2008; Kumar et al. 2009), siderophore produc-
tion (Ahemad and Khan 2012a, b), and phytohormone secretion (Misra et al. 2012;
Oves et al. 2013) (Table 9.1).
Fig. 9.8 Forms of potassium (K) in the soil and their plant availability
9.3.3 Potassium (K)
Potassium (K), one of the most important macronutrients, plays an important role in
plant growth that is required in adequate quantities for all crops to achieve their
maximum yield. K together with N and P forms the NPK chemical fertilizer used in
both intensive and extensive agriculture. Non-exchangeable K, exchangeable K,
mineral non-exchangeable K, and K in soil solution (water-soluble K) are four
forms of K in the soil (Fig. 9.8). Although K deposits are generally large in soil,
most soil K is not directly available for plant capture (Zörb et al. 2014). Mineral
form makes up more than 90–98% of soil K (Sparks 1987), which is tightly bound,
and most of it is unavailable for plant uptake. The potassium present in the soil solu-
tion is absorbed by the plants. Owing to soil erosion, introduction of high-yielding
crop varieties and hybrids during green revolution, low application of K fertilizer,
imbalanced fertilizer application, intensive cropping, runoff, leaching, and presence
of insoluble K sources, the K availability to plants is decreasing (Xiafang and Weiyi
2002; Zörb et al. 2014). As a consequence, K deficiency is becoming one of the
major constraints in crop production, and therefore many crops do respond to K
fertilization in soils. In this situation, the role of PGPR is gaining importance in
modern agriculture for sustainable crop production, which can enhance K availabil-
ity in soil by their activities. The use of K-solubilizing microorganisms is one of the
effective technologies to fulfill the K requirement of crops.
9.3.3.1 KSB (Potassium-Solubilizing Bacteria)

Soil bacteria, fungi, and actinobacteria are important in the cycling of mineral ele-
ments. Among these microbes, bacteria are the important players in this system. The
bacteria involved in the solubilization of K from K-bearing minerals are called KSB
(potassium-solubilizing bacteria). Potassium-solubilizing PGPRs have the ability to
convert insoluble/mineral K into available K in soil making them available to the
plants (Diep and Hieu 2013; Gundala et al. 2013; Keshavarz Zarjani et al. 2013; Zeng
et al. 2012). KSB play an important role in the natural K cycle (Meena et al. 2014;
Parmar and Sindhu 2013; Sindhu et al. 2014b). A wide range of bacteria including
Pseudomonas, Burkholderia, Acidithiobacillus ferrooxidans, Enterobacter hormae-
chei, Paenibacillus glucanolyticus, Arthrobacter spp., Paenibacillus mucilaginosus,
P. glucanolyticus, Bacillus mucilaginosus, B. edaphicus, and B. circulans have been
reported to release K from K-bearing minerals (Basak and Biswas 2009; Keshavarz
Zarjani et al. 2013; Li et al. 2006; Lian et al. 2002; Prajapati et al. 2013; Sangeeth
et al. 2012; Sheng 2005; Sheng and He 2006; Uroz et al. 2007; Zhang et al. 2013).
9.3.3.2 Action Mechanisms of KSB in the Availability of K

The K-bearing minerals are a major source of insoluble K in soils (Mengel and
Kirkby 2001). The minerals are biotite, feldspar, mica, vermiculite, muscovite,
orthoclase, illite/smectite, etc. These minerals supply slowly available K to plants.
Clay minerals are selective for K ions and release K slowly from the lattice wedge
sites (Mengel and Kirkby 2001). It has been known that KSB by converting min-
eral K into available K have a significant role in providing K to plants. At this
time, there is little information on the mechanisms used by KSB to solubilize
K. The K solubilization is a complex phenomenon affected by many factors, e.g.,
amount of mineral, microorganisms involved, nutritional status of soil, soil min-
eral type, size of mineral, and environmental factors (Sindhu et al. 2016). Like the
basic mechanism of PGPR for solubilizing P, potassium-solubilizing PGPR also
solubilize K through the production of organic acids. Extracellular polysaccha-
rides, production of capsular polysaccharides, hydroxyl anion, siderophores,
organic ligands, extracellular enzymes, and formation of biofilms on the rhizo-
spheric mineral surfaces are also involved in dissolution of minerals to release K
(Balogh-Brunstad et al. 2008; Barker et al. 1998; Basak and Biswas 2012; Das
and Pradhan 2016; Keshavarz Zarjani et al. 2013; Liermann et al. 2000; Liu et al.
2006; Meena et al. 2015; Parmar and Sindhu 2013; Sheng and He 2006; Singh
et al. 2015; Uroz et al. 2009; Vandevivere et al. 1994). In general, some of the
direct mechanisms used by KSB (Fig. 9.9) include (i) acidolysis, (ii) chelation,
(iii) oxidation, and (iv) production of carbon dioxide (CO2), explained separately
in the following sections.
Acidolysis
Acidolysis is defined as decomposition resulting from the interaction of a com-
pound and an acid. The major mechanism involved in mineral weathering is acidifi-
cation. Although K appears to be less affected directly by soil pH, lowering the pH
is one of the mechanisms for KSB to solubilize K. Minerals are known to be
Fig. 9.9 The role of IAA producing and K solubilizing PGPR in the availability of K to plant by
different mechanisms
susceptible to various biological by-products of bacterial metabolism, including

protons, organic acids, and more complex molecules (Uroz et al. 2009). K-bearing
mineral weathering in the rhizosphere is a proton attack as a result of microbial
production of organic and inorganic acids, followed by removal of the products of
weathering (cations dissolved from the mineral) by the production of complex-
forming agents (organic acids, extracellular polymeric substances, siderophores,
etc.) and/or by cellular cation uptake (Shelobolina et al. 2012), which induces the
releasing of K. According to Le Chatelier’s principle, after removal of the products
of weathering by the production of complex-forming agents or by cellular cation
uptake, the equilibrium shifts to the left to produce more K (Fig. 9.9).
Acidity in soils can be generated from several sources. K-solubilizing PGPR can
also have a significant role in acid production. CO2 is released from decomposition
of soil organic matter (SOM) by soil microorganisms and respiration from plant
roots and soil fauna. After being hydrated, CO2 is converted into carbonic acid
(CO2 + H2O ↔ H2CO3) (Fig. 9.9). In addition, decomposition of organic materials
and sulfide oxidation by microorganisms result in the production of organic acids
and sulfuric acid, respectively. Nitric and nitrous acids are also produced by nitrify-
ing bacteria. The hydrogen ion released by these acids can react with aluminosili-
cate minerals (feldspars, micas, clays, etc.). For example, hydrogen ion can convert
K feldspar (a primary mineral) into kaolinite, a secondary mineral (Fig. 9.9). As a
result of this reaction and the breakdown of K-feldspar, H+ is used up and K+ is
released to solution, a kind of ion-exchange reaction.
In general, such acidolysis by organic acids produced by KSB can either directly
dissolve the mineral K as a result of slow releases of exchangeable K or readily
available exchangeable K or chelate by both Al and Si ions associated with K min-
eral (Römheld and Kirkby 2010). For example, KSB had the ability to weather
phlogopite through acidic dissolution and aluminum chelation of the crystal net-
work (Abou-el-Seoud and Abdel-Megeed 2012; Meena et al. 2014). Increasing evi-
dence also exists for a mechanism of direct silicate precipitation by bacteria via
metal sorption at the cell membrane (Konhauser and Ferris 1996; Urrutia and
Beveridge 1994).
As mentioned above, bacterial IAA increases root system and promotes an
increasing amount of root exudation. The IAA-derived root system increase
enhances the bacterial effect on mineral mobilization due to increased surface area
for reactivity and helps improve nutrient uptake and mobilization of minerals
(Gahoonia et al. 1997). Some of the roles of bacterial mediated root exudation in
weathering K-bearing minerals include the following: (i) root exudation of high
concentrations of organic acid anions can lower rhizosphere pH (Dakora and
Phillips 2002); (ii) root exudates help by indirectly providing the substrates for the
production of weathering metabolites by bacteria (Gahoonia et al. 1997); and (iii)
root exudates include complex-forming agents (organic acids, extracellular poly-
meric substances, siderophores, etc.) (Shelobolina et al. 2012).
It has been reported that solubilization of K-bearing minerals by KSB is due to
the production of organic acids like citric acid, tartaric acids, 2-ketogluconic acid,
oxalic acid, gluconic acid, malic acid, propionic, fumaric, glycolic, and succinic
acid (Keshavarz Zarjani et al. 2013; Prajapati and Modi 2012; Prajapati et al. 2012;
Sheng and He 2006; Wu et al. 2005), which convert insoluble K (i.e., mica, musco-
vite, biotite, feldspar) to soluble form of K (soil solution form) with the net result of
increasing the availability of the nutrients to the plants. Gluconic, oxalic acids,
α-ketogluconic, and succinic citric are the most efficient acids released by
K-solubilizing PGPR strains that are effectively involved in the solubilization of
insoluble K. In addition, the various types of organic acids produced by KSB dif-
fered with different organisms (Table 9.2) (Maurya et al. 2014; Verma et al. 2014;
Zhang and Kong 2014).
Chelation Process
Chelation is a type of bonding of ions and molecules to metal ions. It involves the
formation or presence of two or more separate coordinate bonds between a polyden-
tate (multiple bonded) ligand and a single central atom. Usually these ligands are
organic compounds. Chelating molecules might increase the dissolution rates of
cations by forming strong bonds with them or with mineral surfaces (Uroz et al.
2009). Complex-forming agents (e.g., organic acids, extracellular polymeric sub-
stances, siderophores) produced by K-solubilizing PGPR or in root exudates form a
complex with cations dissolved from K-bearing minerals, removing the products of
weathering. According to Le Chatelier’s principle, with the uptake of K by plant or
K-solubilizing PGPRs and/or removal of K by forming complex, the equilibrium is
disturbed and K will be drawn upon from the non-exchangeable and soil mineral
Table 9.2 Mechanisms used by KSB for K solubilization

KSB Action mechanism References
Enterobacter hormaechei Organic acids Prajapati et al. (2013)
Paenibacillus mucilaginosus Tartaric, citric, and oxalic acids Liu et al. (2012)
Bacillus mucilaginosus Acidification Abou-el-Seoud and
Abdel-Megeed (2012)
Burkholderia glathei Siderophores and organic ligands Calvaruso et al. (2007)
Burkholderia Acidification and complexation Uroz et al. (2007)
Bacillus circulans Lipo-chitooligosaccharides Lian et al. (2002)
production
Bacillus mucilaginosus IAA production Han and Lee (2005)
Bacillus mucilaginosus Production of citric, tartaric, and Sheng (2005)
Bacillus edaphicus oxalic acid
Bacillus spp. Gluconic acid Gundala et al. (2013)
Bacillus mucilaginosus Polysaccharides Liu et al. (2006)
Bacillus edaphicus Production of organic acids like Sheng and He (2006)
oxalic acid and tartaric acids and
production of capsular
polysaccharides (CPS)
Rhizobium tropici Production of tartaric acids and Wang et al. (2015)
extracellular polysaccharides
Pseudomonas aeruginosa Acetic, citric, and oxalic acids Badr et al. (2006)
Bacillus, Clostridium, and production of mucilaginous Groudev (1987)
Thiobacillus capsules containing of
exopolysaccharides
Cladosporium Production of protons, organic Kumar et al. (2015),
cladosporioides and acids, siderophores, and organic Meena et al. (2014,
Penicillium sp. ligands as chelating agents 2015)
fraction (Fig. 9.9). K-solubilizing PGPR secrete organic acids which act as chelat-
ing agents and quickly dissolve rock and silicon ions, ultimately releasing the K
ions into the soil (Bennett et al. 1998).
Štyriakova et al. (2003) showed that K solubilization occurred by buildup of
complex between organic acids and metal ions such as Fe2+, Al3+, and Ca2+. Organic
acids can either directly enhance dissolution by either a proton- or a ligand-mediated
mechanism or indirectly enhance dissolution by the formation of complexes in solu-
tion with reaction products (Ullman and Welch 2002). Liu et al. (2006) demon-
strated that polysaccharides strongly adsorbed the organic acids and attached to the
surface of the mineral, resulting in an area of high concentration of organic acids
near the mineral. It was suggested that the extracellular polysaccharides adsorbed
SiO2 and this affected the equilibrium between the mineral and fluid phases and led
to the reaction toward SiO2 and K+ solubilization. Adhering to mineral surfaces and
extracting nutrients from mineral particles by electron transfer, breaking the oxygen
links, and chelating ions present in solution through their carboxyl and hydroxyl
groups are some of the action mechanisms of organic acids and chelating molecules
on mineral weathering (Welch et al. 2002). The latter indirectly accelerates the dis-
solution rate of the mineral by creating an imbalance between cation and anion
concentrations in the solution. Bacterial organic acids have been found to facilitate
the weathering of minerals through the formation of metal organic complexes with
silicon ions to bring the K into solution, in addition to directly dissolving K from
rocks (Bennett et al. 1998). According to previous discoveries, acidification does
not seem to be the only solubilization mechanism, so that the ability to reduce pH in
some cases is not consistent with the ability to solubilize K minerals by K-solubilizing
PGPR (Liu et al. 2006; Sheng and Huang 2001; Subhashini and Kumar 2014; Zhang
and Kong 2014). In general, acidolysis and complexolysis processes can be simul-
taneously used by K-solubilizing PGPR to impact mineral stability. Agrobacterium
and Bacillus strains were described for their ability to weather phlogopite via alu-
minum chelation and acidic dissolution of the crystal network (Leyval and Berthelin
1989). Some of the selected examples about mechanisms used by KSB to solubilize
K have been shown in Table 9.2.
Oxidation
Microbial Fe(II) oxidation as an additional mechanism of microbial weathering of
K-bearing minerals having Fe (II) (e.g., biotite) in the rhizosphere has been reported,
contributing to soil formation and providing K and Fe for plant nutrition (Shelobolina
et al. 2012). Microbial oxidation of structural Fe (II) led to biotite changes similar
to those found in nature, including a decrease in the unit cell b dimension toward
dioctahedral levels and Fe and K release. Structural Fe (II) oxidation can entangle
either direct enzymatic oxidation, followed by solid-state mineral transformation, or
indirect oxidation as a result of forming aqueous Fe, followed by electron transfer
from Fe (II) in the mineral to Fe (III) in solution. These cells indirectly attack biotite
through oxidation of the sorbed Fe (II) in indirect oxidation that was generated
because of electron exchange between structural Fe (II) and surface Fe (III) in the
biotite (Shelobolina et al. 2012).
Production of CO2
The weathering of K-bearing minerals may be the result of carbonic acid formation
from the respiratory CO2 release of the microorganisms and its subsequent reaction
with water (Barker et al. 1998). Bacterial IAA can attract more rhizosphere bacteria
in the rhizosphere by increasing more amount of root exudation, resulting in more
production of CO2. In addition, CO2 can directly release K from K-bearing minerals
(Rosenqvist et al. 2014). For example, CO2 can convert K feldspar into muscovite
and release K (Fig. 9.10). In general, K-solubilizing PGPR can dissolve K-bearing
minerals by production of organic acids, IAA, siderophore, and polysaccharides.
Previous studies showed K-solubilizing PGPR with other PGP traits. For example,
Zhao et al. (2008) isolated bacterial strains with multiple beneficial activities such
as IAA production, K solubilization, and siderophore production. Prajapati et al.
(2013) reported that inoculation with IAA-producing PGPR strain Enterobacter
hormaechei enhanced the root and shoot length of okra and mobilized K efficiently
in plant when feldspar was added to the soil. The plants with more fibrous, branch-
ing roots increase nutrient (K)-absorbing surface. Nadeem et al. (2009) reported
that ACC deaminase-containing rhizobacteria improved the uptake of K in maize
Fig. 9.10 The direct role of CO2 in releasing K from K-bearing minerals
under salinity stress. They observed that these strains were strong colonizers of
plant roots. The increased colonization by the K-solubilizing PGPR made the plants
capable to explore more soil that might have improved the uptake of K indirectly.
Previous studies showed that the solubilization and release of K through organic
acids by K-solubilizing PGPR resulted in plant growth promotion leading to
enhanced yield and production, which have been well reviewed (Ahmad et al. 2016;
Bahadur et al. 2014; Das and Pradhan 2016; Meena et al. 2016; Sharma et al. 2016;
Sindhu et al. 2014a, 2016; Velázquez et al. 2016; Zahedi 2016). The above discus-
sion shows that bacterial strains have a number of potential mechanisms to solubi-
lize K from insoluble sources and the contribution of these mechanisms in
weathering of K-bearing minerals is variable. Among these mechanisms, the pro-
duction of organic acids is one of the major mechanisms used by K-solubilizing
PGPR. It has also been observed that K solubilization by the bacteria is affected by
a large number of soil and environmental factors (Uroz et al. 2009).
9.3.3.3 KSB and Increased Availability of K and Other Nutrients

As discussed above, the production of organic acids is one of the major mechanisms
used by KSB to solubilize K-bearing minerals and release K. The availability of
some plant nutrients is also greatly affected by soil pH. Therefore, these bacteria
can also provide the availability of nutrients such as P, Fe, Zn, Cu, and Mn.
According to Prajapati et al. (2013), K-solubilizing PGPR can not only activate the
insoluble K mineral but also change that into available P, Zn, and Fe. Sheng (2005)
and Sheng et al. (2008) showed that inoculation of cotton and rape plants with the
K-solubilizing PGPR strain Paenibacillus edaphicus NBT enhanced the N and P
content in both plants and soil. Increases ranging from 26% to 30% were found in
both plants when illite was added to soil as a source of insoluble K. The plant dry
weight and the uptake of both K and N by tobacco seedlings enhanced significantly
with respect to uninoculated controls mainly when the strain inoculated was
Klebsiella variicola XF11 (Zhang and Kong 2014). Lin et al. (2002) indicated sig-
nificant increases in K and P uptake in tomato plants inoculated with silicate-
dissolving bacterium (B. mucilaginosus) compared with the non-inoculated plants.
K-solubilizing PGPR also resulted in increased biomass and enhanced contents of
P and K in sorghum plants than uninoculated control (Zheng and Tu 2005).
Inoculation of K-solubilizing PGPR combined with K- and P-bearing minerals
caused increase in dry matter yield of sorghum plants along with P and K uptake
and also improved fertility in three different soils, i.e., clay, sandy, and calcareous
soils (Badr et al. 2006). The organic acids and siderophores generated by PGPR
could play a crucial role in the solubilization of elements such as K, Si, and Fe from
the liquid medium containing acid-leached soil, muscovite, and biotite (Liu et al.
2006). In general, the K-solubilizing PGPR contribute to exudation of soluble com-
pounds, decomposition of soil organic matter, and mobilization and mineralization
of other nutrients (Abhilash et al. 2013; Archana et al. 2013; Diep and Hieu 2013;
Rajawat et al. 2012; Zeng et al. 2012).
9.3.4 Sulfur (S)
Sulfur (S), an essential macronutrient required for growth, is increasingly becoming a

limitation to crop yield and quality as a result of a reduction in atmospheric S levels
due to the increasing use of low-S fuels and enhanced emission controls and crop
varieties removing S from soil more rapidly (Fowler et al. 2005; Irwin et al. 2002).
Since crop plants have become increasingly dependent on the soil to supply the S,
these changes have had an important effect on agriculture. S is needed for the synthe-
sis of proteins and a number of essential vitamins and cofactors and also is a constitu-
ent of the essential amino acids cysteine, cystine, and methionine. In agricultural soils,
most of the soil S (>95%) is present in an organic form (Gahan and Schmalenberger
2014) as a heterogeneous mixture of forms, partly included in microbial biomass and
partly in the soil organic matter. In addition, S present in soil is approximately 95%
organically bound largely in one of the two major forms: sulfate esters and carbon-
bonded S (sulfonates or amino acid sulfur) (Kertesz and Mirleau 2004). Sulfonates
and sulfate esters are not directly available to plants which depend upon microorgan-
isms in rhizosphere and soil for mobilizing these organic forms (Kertesz et al. 2007).
S occurs in a variety of oxidation states with three oxidation states of −2 (sulfide and
reduced organic sulfur), 0 (elemental sulfur), and +6 (sulfate) being the most signifi-
cant in nature. Plants obtain S in the form of sulfate (SO42−), which is the dominant
plant-available source of S and constitutes less than 5% of the total soil S (Autry and
Fitzgerald 1990), while to a lesser extent atmospheric reduced S may be utilized
(Leustek et al. 2000). Chemical or biological agents help transformation of S from one
state to another. A biogeochemical cycle which characterizes these transformations
includes many oxidation-reduction reactions.
Fig. 9.11 The major processes of transformation involved in the cycling of S in soil
9.3.4.1 Action Mechanisms of Sulfur (S) Availability by PGPR

Similar to some other nutrients, S is also subjected to biological alterations in soil
by the soil bacteria. The major processes of transformation involved in the cycling
of S in soil are (i) mineralization of organic S to the inorganic form (H2S), (ii)
immobilization or assimilation of S into organic compounds by plants or microor-
ganisms, (iii) oxidation of S and inorganic S compounds, and (iv) reduction of S and
incompletely oxidized inorganic compounds of S (Fig. 9.11). Due to having indirect
and direct functions in these processes, microorganisms especially bacteria can
increase the availability of S to plants, which are explained as follows.
Mineralization of Sulfur (S)

Plant S nutrition depends primarily on the uptake of inorganic sulfate. Due to inter-
conversion of sulfate ester-S and carbon-bonded S to inorganic sulfate by soil
microorganisms, it has been shown that the sulfate ester and sulfonate pools of soil
S are also plant bioavailable (Kertesz and Mirleau 2004). Aerobic and anaerobic
heterotrophic bacteria (Pseudomonas and Clostridium) release S from sulfate-esters
using sulfatases (R-O-SO3− + H2O → ROH + H+ + SO42−); however, release of S
from sulfonates is catalyzed by a bacterial multicomponent monooxygenase system
(Gahan and Schmalenberger 2014). Splitting of the C-O bond of aliphatic sulfate
esters and the O-S bond of aromatic sulfate esters is performed by the enzymes of
alkylsulfatase and arylsulfatase, respectively (Kertesz 2000). Some bacteria such as
Pseudomonas, Klebsiella, Salmonella, Enterobacter, Serratia, and Comamonas are
able to mobilize sulfate esters (Hummerjohann et al. 2000). Sulfide can be produced
by anaerobic bacteria as a result of the breakdown of proteins to amino acids and

further degradation of amino acids to sulfide (R-SH → R + H2S) (desulfurization).
Immobilization of Sulfur (S)

Sulfur (S) immobilization is microbial conversion of inorganic S compounds to
organic S compounds, first to sulfate esters and subsequently to carbon-bound sul-
fur (Kertesz and Mirleau 2004). S is used for the biosynthesis of amino acids and
proteins by bacteria (assimilatory sulfate reduction) in this process. Bacteria reduce
only enough sulfates to meet their nutritional requirements for S. During the study
period, soil microorganisms were capable of binding all the available sulfate into
microbial biomass. In general, PGPR in the presence of labile C serve as a sink for
S, by rapidly immobilizing it; therefore PGPR become a source of S to plants upon
its release from their cells. Release of S immobilized by PGPR primarily occurs
when cells die.
Oxidation of Sulfur (S)

Sulfur (S) oxidation is the process by which a variety of microorganisms convert
hydrogen sulfide (H2S) into elemental sulfur (S0) by partial oxidation, or sulfate
(SO42−), which can be used by the plants, while the acidity produced by oxidation
helps to solubilize plant nutrients and improve alkali soils (Wainwright 1984).
Instead of H2S, also other sulfur compounds like thiosulfate (S2O32−) and tetrathion-
ate (S4O62−) can be converted to sulfate (S0 → H2S → S2O32− → S4O62− → S3O62− →
SO32− → SO42). Thiobacilli (e.g., bacteria of the genus Thiobacillus) play an impor-
tant role in S oxidation in soil. Beggiatoa, Sulfolobus, Thermothrix, Thiobacillus,
and Thiothrix, known as colorless sulfur-oxidizing bacteria (Janssen et al. 1998),
are the most important chemolithotrophic sulfur-oxidizing bacteria (SOB). S oxida-
tion is the most important step of S cycle, which improves soil fertility and decreases
pH soil and subsequently increases the availability of micronutrients and
P. Photoautotrophic or chemolithotrophic sulfide-oxidizing bacteria use sulfide as
an electron donor and convert it to S or sulfate (Robertson and Kuenen 2006). The
CO2 and oxygen (aerobic species) or nitrate and nitrite (anaerobic species) are used
as the terminal electron acceptors in photoautotrophic and chemolithotrophic
sulfide-oxidizing bacteria, respectively. Light energy and oxidizing reactions supply
directly energy needed for metabolism of photoautotrophic SOB and chemoli-
thotrophic SOB, respectively. Among photoautotrophic sulfide-oxidizing bacteria,
the green SOB like Chlorobium, Prosthecochloris, Chloroherpeton, Pelodictyon,
and Ancalochloris and purple SOB such as Chromatium, Allochromatium,
Thioalkalicoccus, Thiococcus, Thiorhodococcus, Thiocystys, and Thiospirillum
(Tang et al. 2009) are the most frequent. In addition to being oxidized biologically,
oxidation of the reduced S compound is also carried out chemically by ferric iron as
the oxidizing agent, and iron-oxidizing bacteria are utilized to regenerate the ferric
iron (Fe3+) for further use (Pagella and De Faveri 2000).
Reduction of Sulfur (S)

In addition to reductive reactions, bacteria have also a major role in reductive reac-
tions of the biological S cycle (Kertesz and Mirleau 2004). Sulfate, in turn, can be
reduced back to sulfide by sulfate-reducing bacteria such as Desulfovibrio and
Desulfatomaculum (Tang et al. 2009). Sulfate in the absence of oxygen functions as
a terminal electron acceptor in metabolic pathways for anaerobic respiration and is
converted to sulfide (dissimilatory sulfate reduction). Although dissimilatory sulfate
reduction results in decrease of plant-available sulfate, H2S produced by the reduc-
tion of sulfate is further oxidized by some of the green and purple phototrophic
bacteria to release elemental sulfur (S0). The latter can also be oxidized to sulfate by
SOB once again.
9.4 ction Mechanisms of PGPR in Availability

A
of Micronutrients
9.4.1 Production of Organic and Inorganic Acids
Since most of the nutrients (micronutrients especially) tend to be less available

when soil pH is above 7.5 (Fig. 9.2), it seems that decreasing soil pH (a slightly
acidic pH, e.g., 6.5–6.8) is one of the action mechanisms of PGPR in the availability
of these nutrients to plants. Due to strongly being adhered to soil particles, these
metals are not easily available for uptake by plants in most of the metalliferous soils
(Gamalero and Glick 2012). In this context, PGPR producing acid such as PSB,
sulfur-oxidizing bacteria (SOB), and nitrifying bacteria are very promising agents.
By secreting low-molecular-weight organic acids, the PGPR listed above can solu-
bilize the insoluble and biologically unavailable metals and subsequently facilitate
the bioavailability of these nutrients for plant uptake (Fig. 9.12) (Becerra-Castro
et al. 2011, 2011; Li and Ramakrishna 2011; He et al. 2013).
9.4.2 Production of Chelating Agents
In addition to soil pH, chelation process can increase nutrient availability to plants
by different ways (Fig. 9.13). Hence, the natural chelating agents produced by
PGPR may be considered as the other action mechanism of PGPR in the availability
of nutrients to plants. Hydroxamate siderophores, organic acids, and amino acids
are some of the most important substances possessing this nature, which are natu-
rally produced by soil microorganisms. These substances are essential in natural
ecosystems to solubilize and transport these nutrients to plant roots. For example,
iron occurs mainly as Fe3+ and forms insoluble hydroxides and oxyhydroxides, and
thus is not easily available to both plants and microorganisms (Ahemad and Kibret
2014). Under iron-limiting conditions to acquire Fe, PGPR secrete low-molecular-
weight siderophores, which are iron chelators with exceptionally strong affinity for
ferric iron (Fe3+) (Schalk et al. 2011). Despite their preferential affinity for Fe3+, they
Fig. 9.12 The role of PGPR in increasing the availability of micronutrients to plant by different
mechanisms
can also chelate several other metals such as Mg2+, Mn2+, chromium (Cr3+), gallium
(Ga3+), cadmium (Cd), Zn2+, Cu2+, Ni, arsenic (As) and lead (Pb), and radionuclides,
including plutonium (Pu4+) with variable affinities (Nair et al. 2007; Rajkumar et al.
2010; Schalk et al. 2011). Glycolic, oxalic, malonic, tartaric, lactic, citric,
α-ketogluconic, piscidic, succinic, malic, valeric, and formic are some of the known
organic acids with chelating properties similar to siderophores (Panhwar et al.
2013). In addition to producing chelating agents, PGPR such as K-solubilizing
PGPR can increase metal bioavailability in metal-stressed soils by producing bio-
surfactant, which aids in metal release from soil particles (Gamalero and Glick
2012; Singh and Cameotra 2013).
9.4.3 Production of IAA
As mentioned above, phytohormone (IAA)-producing PGPR can enhance indi-

rectly the availability of micronutrients by improving root development and growth
and root exudates. The exudates released by roots of plant also attract the wide
range of PGPR with other plant growth-promoting traits such as siderophore pro-
duction, phosphate solubilizing, and ACC deaminase production. These bacteria
Fig. 9.13 The role of chelating agents produced by PGPR in increasing nutrient availability to
plants by different ways
can also enhance the availability of micronutrients by siderophore as a chelating

agent, production of acid, and decrease of ethylene and subsequent increase of root
system (Fig. 9.6). Root exudates can also act as binding material/cementing agents
of soil and, thus, improve soil structure and regulate and maintain the microbial
population near the root surface. Microbial activity near the root surface plays an
important role in the development and rooting pattern of the plant. In addition,
attracted PGPR themselves also produce many exudates, which are very helpful in
plant nutrition and growth. The presence of various composites of amino acids,
organic acids, sugars, vitamins, purines, adenine, guanine, cytidine, uridine,
enzymes (e.g., phosphatase), and some gaseous molecules (e.g., CO2) in root exu-
dates (Dakora and Phillips 2002) enhances the availability of micronutrients. A
fraction of these exudates are further metabolized by PGPR in the vicinity as C and
N sources, and some bacterium-oriented molecules are subsequently retaken up by
plants for growth and development (Sheng and He 2006). Another nutritional effect
that organic acids have in root exudates is acidification of the rhizosphere, which
enhances the availability of micronutrients. In general, PGPR, especially IAA-
producing bacteria, can increase the availability of micronutrients in the soil directly
by the production of different compounds (such as carboxylates, phenolic com-
pounds, etc.) or indirectly through affecting plant growth and hence the production
of root exudates (Fig. 9.14) (Badri and Vivanco 2009). To understand the action
Fig. 9.14 Impact of PGPR on micronutrients acquisition and root functioning. PGPRs can modu-
late root development and growth through the production of phytohormones such as IAA, second-
ary metabolites. PGPR also influence plant nutrition via ACC deaminase, solubilization of
phosphorus, or siderophore production, and modify root physiology by changing gene transcrip-
tion and metabolite biosynthesis in plant cells
mechanism of PGPR in the availability of micronutrients to plant, two nutrient ele-

ments Fe and Mn are explained in more detail as follows.
9.5 Iron (Fe)
As an essential nutrient for plants, iron (Fe) is crucial for a variety of cellular func-
tions and essential physiological processes, including respiration and photosynthe-
sis and a necessary cofactor for many enzymatic reactions (Zuo and Zhang 2010).
Under aerobic conditions, Fe exists predominantly as Fe3+ and reacts to form highly
insoluble hydroxides and oxyhydroxides that are basically unavailable to plants and
microorganisms (Desai and Archana 2011; Zuo and Zhang 2010). High soil pH
reduces while acidic soil conditions increase Fe availability. As pH increases by one
unit, activity of Fe3+ decreases by 1000-fold. In most soils, Fe is present in large
quantities, but mainly in forms that are not available to plants (Schmidt 2003;
Wintergerst et al. 2007). It has been reported that most plants need the concentra-
tions of soluble Fe at 10−4 to 10−8 M (optimal soils usually slightly acidic) for their
optimal growth. However, 10−9 M or lower concentrations of soluble Fe (calcareous
or alkaline soils with low bioavailable Fe) are insufficient for plant growth and
plants may develop a Fe deficiency-associated leaf chlorosis as a disease symptom.
There are many factors that affect the availability of Fe in the soil. The availability
of Fe strongly depends on soil redox potential (redox change) and pH. When
decreasing redox potential and pH, availability of Fe increases. PGPR can increase
the availability of Fe by decreasing soil redox potential and pH.
9.5.1 Fe Acquisition Strategies by Plants
Despite the abundance of Fe in soils, its availability for plants and microbes is low.
Plants and microorganisms have evolved active strategies of Fe uptake. Mobilization
of Fe is the prerequisite for uptake of Fe into the roots, which is achieved by two
different strategies in the plant kingdom. These strategies are named as strategy I
and strategy II. In strategy I, all plant species (monocotyledonous and dicotyledon-
ous plants), except grasses, acidify the rhizosphere and produce organic products. In
addition, Fe3+ is reduced into Fe2+ by a Fe chelate reductase enzyme, converting Fe
(III) to Fe (II) (Hartmann et al. 2008). Subsequently, Fe2+ can be taken up by a
membrane-bound Fe (II) transporter. However, strategy II grasses handle Fe defi-
ciency by the synthesis and secretion of siderophores and uptake of them by the
activation of the Fe3+ siderophore transporter in the plasmalemma of root cells
(Altomare and Tringovska 2011; Charlson and Shoemaker 2006; Curie et al. 2001;
Guerinot 2010; Wintergerst et al. 2007).
9.5.2 Action Mechanisms of PGPR in Fe Availability
It has been known that strategies I and II are often not efficient enough to meet the
needs of the plants growing especially in calcareous and alkaline soils. Studies show
that many IAA- and siderophore-producing PGPR strains could improve iron nutri-
tion (Etesami et al. 2015a; Jin et al. 2006; Ramos-Solano et al. 2010).
9.5.2.1 Production of Siderophores

Most plant-associated bacteria can produce iron chelators called siderophores in
response to low iron levels in the rhizosphere. Siderophores are low-molecular-
weight organic compounds, which have high affinity to bind some elements such as
Fe3+ as well as other metal ions and increase their availability (Boukhalfa and
Crumbliss 2002). Several examples of increased Fe uptake in plants with concurrent
stimulation of plant growth as a result of PGPR inoculations have been reported
(Barzanti et al. 2007; Burd et al. 2000; Carrillo-Castañeda et al. 2002; Lemanceau
et al. 2009). Many studies have demonstrated that microbial siderophores can be
used as Fe source for plants with strategies I and II (Fernández and Winkelmann
2005; Jin et al. 2006; Johnson et al. 2002; Rasouli-Sadaghiani et al. 2014; Robin
et al. 2008; Siebner-Freibach et al. 2003; Vansuyt et al. 2007).
According to results of previous studies, it is most probable that the bacterial
siderophore is not absorbed by the plant, and iron is obtained through a reduction-
based mechanism (Johnson et al. 2002). Ferric siderophores are transported into
cells via specific Fe-siderophore membrane receptors, allowing siderophore release

for subsequent reuse (Lemanceau et al. 2009) (Fig. 9.15). It has been found that the
ability of siderophores in supplying Fe+3 to the root surfaces and in the intracellular
spaces of root cells is the most important function of these chelating compounds in
the gramineous plant nutrition. Accordingly, the higher concentrations of Fe+3 ions,
which are available to the root phytosiderophores, enhance their subsequent absorp-
tion by plants (Sharma et al. 2003). It has been shown that ligand exchange is
another theory on the supply of Fe by siderophores (Latour et al. 2009; Yehuda et al.
1996). This theory has been suggested for iron, showing that Fe supplied by sidero-
phores interacts with phytosiderophores in a ligand exchange reaction and is finally
absorbed by the phytosiderophores. This theory confirms the indirect role of sidero-
phore in Fe uptake (Shweta et al. 2008). Among different known microbial sidero-
phores, plants are capable of using hydroxamates, ferrichrome, rhodotorulic acid,
desferrioxamine B, agrobactin, as well as catecholate-hydroxamate (Fernández and
Winkelmann 2005). Generally, compared to most microorganisms, most plants can
grow at very low concentrations of Fe (O'Sullivan and O'Gara 1992); therefore
many plants can take up iron in the presence of siderophore-producing microorgan-
isms. In addition to high affinity for Fe (III) ions, siderophores can also form com-
plexes with other bivalent ions (and including phosphates and other micronutrients)
that can be assimilated by the plant (Ramos-Solano et al. 2010).
9.5.2.2 Production of IAA

As previously mentioned, the production of root and microbial exudates is an
important parameter determining the availability of nutrients in the rhizosphere.
Organic acids present in root and microbial exudates result in acidification of the
rhizosphere (Dakora and Phillips 2002), which can, in turn, contribute to plant
growth by mobilizing nutrients such as Fe. Because of the function of IAA in secret-
ing root exudates (rhizodeposits) and increasing rooting system and since these exu-
dates are involved in acidifying rhizosphere and in providing a reducing condition
required for converting Fe3+ to Fe2+, it may be suggested that IAA-producing PGPR
can also solubilize insoluble Fe sources and induce plant growth and iron uptake in
a similar manner to strategy I in plants (Fig. 9.15). For example, protons and elec-
trons are secreted within carbon compounds as undissociated acids or compounds
with reducing capabilities. Some of the compounds in root exudates are able to form
Fe complexes that improve availability. High-molecular-weight components (i.e.,
proteins, mucilage) and low-molecular-weight compounds (i.e., secondary metabo-
lites, organic acids, carbohydrates, amino acids, phenolics) are typically the domi-
nant soluble-reduced carbon compounds in rhizodeposits (Badri and Vivanco 2009;
Wen et al. 2007). The consumption of O2, due to respiration by the root (increase of
root system due to bacterial IAA) and associated microflora (increase of microflora
activity due to production of more root exudates), can also result in steep redox
gradients in the rhizosphere (Etesami et al. 2015a, b; Hartmann et al. 2008).
Likewise, chelating agents present in root and microbial exudates such as organic
acids are capable of chelating Fe3+ and making it available to plant roots in a similar
manner to strategy II in plants (Fig. 9.15).
Fig. 9.15 Schematic representations of role of siderophore (Sid)-producing PGPR in enhancing

iron availability for plant. In non-grass species (Strategy I), acidification of the rhizosphere occurs
in part through the activity of a plasma membrane H+-ATPase. This H+ excretion contributes to the
solubilization of Fe+3, which is reduced to Fe+2 by the FRO2 ferric chelate reductase, transferring
electrons (e−) from NADPH to Fe+3 (Lemanceau et al. 2009). In grasses, Strategy II involves the
synthesis of phytosiderophores (P-Sid). P-Sid is secreted from the roots by an uncharacterized
mechanism into the rhizosphere where it chelates Fe+3. The Fe+3–P-Sid complex is then transported
into the epidermal cells of the roots. PGPRs do not take up Fe+3-Sid complexes, but rather obtain
iron through a reduction-based mechanism involving Fe-Sid membrane receptors, acquiring Fe2+
while releasing Sid for subsequent reuse. Sid increases the Fe+3 pools in the rhizosphere, increasing
Fe+3 available to the root P-Sid. P-Sid that has a higher affinity for Fe3+ than Sid may acquire it via
ligand exchange
9.6 Manganese (Mn)
As a micronutrient, manganese (Mn) is essential for many plant functions (as a

component of enzymes) and is also involved in photosynthesis and root growth. Mn
as free Mn2+ in the soil is readily available to plants, and as oxides is of low solubil-
ity. The proportion of Mn in various forms in the soil is dependent both on chemical
reactions and on microbial activity. As previously mentioned (Fig. 9.2), high soil pH
greatly reduces the solubility of soil Mn, and therefore its availability to roots. Thus,
Mn deficiency is most likely to occur in soils that are alkaline or have been limed.
Mn is a nutrient element and its availability in the rhizosphere is affected by two
major factors, namely redox condition and pH. In oxidized soils, Mn is present in its
oxidized form, Mn4+, in the low-soluble mineral pyrolusite. It has been known that
Fig. 9.16 The schematic representation of role of PGPR in the availability of Mn and Fe to plant
by affecting plant (root) growth and hence plant root exudates. The electrons and protons required
to the reduction of Mn and Fe in reactions (1), (2), and (3) are supplied by the decomposition of
carbonaceous compounds and the proton excretion system of root cells, respectively. The roots and
PGPR by producing chelating agents (phenolic compounds, organic acids) can form soluble com-
plex with Mn, Fe, and other elements avoiding the reprecipitation of them
some PGPR can increase the availability of this element to plants. For example,
PGPR such as Bacillus, Pseudomonas, and Geobacter could reduce oxidized Mn4+
to Mn2+, which is the chemical form that is metabolically useful for plants (Osorio
Vega 2007). These bacteria can affect Mn availability in the soil mostly by affecting
plant growth and hence plant root exudates (Dutta and Podile 2010; Miransari
2011). Increased root exudates originated from bacterial activities in turn supply
electrons (by the decomposition of organic molecules present in root exudates) and
protons (by the proton excretion system of root cells) required for the reduction of
Mn in the following reaction:
MnO2 + 4H+ + 2e− ↔ Mn2+ + 2H2O
Consequently, the activity of Mn reducers is highly favored in the rhizosphere
(Osorio Vega 2007). By producing electron and H+, applications of organic matter
(OM) can also favor the reduction of Mn (Hue et al. 2001). Therefore, in alkaline
soils where Mn usually is insoluble the rhizosphere effect and application of OM
can be beneficial. In addition, roots and PGPR can produce chelating agents (phe-
nolic compounds, organic acids) that form soluble complex with Mn, Fe, and other
elements avoiding the reprecipitation of them (Fig. 9.16).
9.7 Concluding Remarks and Future Perspectives
It is evident that PGPR have a high potential to be used in the management of nutri-
ent-deficient soils. Using PGPR to increase the availability of nutrition elements in
soil is an attractive proposition for developing a more sustainable agriculture. These
PGPR have an important role in the cycling of nutrient elements in soil-plant systems
and it is anticipated that better understanding of their contribution to mobilizing soil
nutrients and plant nutrient nutrition will provide an opportunity for developing more
nutrient-efficient and sustainable agricultural systems and improved knowledge of
ecosystem function. Developing the proper formulation and delivery systems to
ensure survival and effective establishment of target PGPR within the rhizosphere is
a main requirement for prosperous deployment of bacterial inoculants (Richardson
and Simpson 2011). Increased knowledge concerning the beneficial interactions of
PGPR with plants and a proper screening will be of special importance for sustain-
able agriculture that depends on biological processes and resources, rather than on
the use of agrochemicals for maintaining soil fertility and plant health. Previous stud-
ies clearly demonstrate the presence of one or more than one type of PGP character
in majority of the bacterial strains. A bacterial strain possessing multiple PGP traits
is expected to indicate better response than those having single PGP characteristic.
However, it would be desirable to examine whether all the traits of PGP are expressed
concurrently or at different phases of growth of the bacteria. In other words, since
many PGPR possess several of PGP traits simultaneously, different mechanisms at
various times during the life cycle of the plant can be used. However, the exact modes
by which PGPR promote plant growth at a specific step in life cycle are not fully
understood (Bhattacharyya and Jha 2012), which need further studies in the future.
In addition, certain issues, such as what should be an ideal and universal delivery
system, how to improve the efficacy of biofertilizers, how to stabilize PGPR in soil
systems, and how nutritional and root exudation aspects could be controlled in order
to get maximum benefits from PGPR application, have not been well known until
this moment, which are needed to be addressed by scientists in the future.
Biotechnological and molecular approaches may possibly develop more understand-
ing about PGPRs’ mode of actions that will result in more successful plant–microbe
interaction and prosperous application of the beneficial bacteria (Khalid et al. 2009).
Acknowledgments We wish to thank University of Tehran and University of Saskatchewan for

providing the necessary facilities for this study.
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Plant Growth and Development Under
Suboptimal Light Conditions 10
Ravinderjit Kaur, Gaganpreet Kaur, Kashmir Singh,
and Baljinder Singh
Abstract
Light regulates various processes throughout the plant life from seed germination
to flowering. Photoreceptors (phytochromes and cryptochromes) sense the changes
in light conditions that trigger various signaling mechanisms resulting in upregula-
tion and downregulation of several genes and transcription factors. Therefore,
genetic and physiological responses, i.e., seedling growth and development, skoto-
morphogenesis, photomorphogenesis, shade avoidance, and flowering, are regu-
lated by the changes in gene expression mediated by light. Phytohormones are also
involved in controlling these developmental changes. Light also plays an important
role in plant defense against various pathogens by inducing the jasmonic acid and
salicylic acid pathways that trigger SAR (systemic acquired response). Once the
plant becomes reproductively competent, light regulates the complex process of
floral initiation by activation of floral genes and flowering hormones.
Keywords
Photoreceptors · Cryptochromes · Skotomorphogenesis · Jasmonic acid
10.1 Introduction
Plants being immobile have acquired intrinsic properties to adapt themselves accord-
ing to the changing environmental conditions for their survival. Various abiotic fac-
tors like wind, water, temperature, and light influence the growth and development of
Ravinderjit Kaur and Gaganpreet Kaur contributed equally to this work.
R. Kaur · G. Kaur · K. Singh · B. Singh (*)

Department of Biotechnology, Panjab University, Chandigarh, India

206 R. Kaur et al.
plants. Light being a major environmental factor plays a vital role in overall growth
and development of plants. It is responsible for induction of massive reprogramming
of gene expression in plants (Petrillo et al. 2014). The change in the quantity, quality,
and duration of light leads to alteration in various basic processes in plants like seed
germination, photomorphogenesis, and transition to flowering which are regulated
by expression of various genes.
10.1.1 Light Quantity, Quality, and Duration
Light quantity is the intensity or concentration of sunlight which increases or

decreases according to seasonal variations. Basically, light quality is the color or
wavelength of light that reaches to the plant surface. Light duration is the amount of
time that a plant is exposed to sunlight, i.e., photoperiodism. It mainly controls the
floral development. Photoperiodic regulation is controlled by various genes which
are either activated or inhibited depending upon the duration of exposure to light
(Thomas 2006).
10.1.2 Phytohormones
Plant hormones like auxins, cytokinins, gibberellins, abscisic acid, ethylene, and
brassinosteroids regulate the developmental translations and are crucial for growth
regulations. In plants, various light responses control the changes in hormonal
metabolism and distribution. Phytohormones like abscisic acid, gibberellic acid,
and ethylene respond to varying light duration and thus regulate the process of seed
germination (de Wit et al. 2016).
Global gene expression is altered in response to changes in light conditions.
Light is perceived by photoreceptors triggering many of the biological processes in
plants by affecting gene expression (Rossel et al. 2002). Cryptochromes and phyto-
chromes are the main photoreceptors that can localize in the nucleus and control
light-regulated nuclear gene expression by transducing these signals to chromatin,
influencing the process of transcription, post-transcription, alternative splicing, and
translation that ultimately leads to adaptive changes at the cellular and organismic
levels (Petrillo et al. 2014). One of the important organelle in plants, chloroplast,
can also act as a light sensor and is involved primarily in the process of photosyn-
thesis (Godoy Herz et al. 2014).
10.2 Seed Germination
The sprouting of a seedling from a seed is the beginning of a plant life which is
regulated by two major environmental factors, i.e., water and light. During unfavor-
able conditions, seeds are present in a dormant state where metabolic activity is very
low. When ample amount of water is present in the surroundings, the seed uptakes
10 Plant Growth and Development Under Suboptimal Light Conditions 207
water by the process of imbibition and gets filled with water, which plays a vital role
in the activation of various proteins and enzymes that are involved in the process of
plant growth.
The seedling establishment and its further growth are regulated by phytochromes
(photochromic proteins) which are light-sensitive proteins called photoreceptors.
Various photoreceptor proteins of different families perceive the light spectrum rang-
ing from near UV-B (280–315 nm) to far-red (750 nm) light (de Wit et al. 2016).
10.2.1 Phytochromes and Their Role in Seed Germination
Phytochromes are cytoplasmic serine/threonine kinases which sense both red and
far-red light. It is a homodimer (MW 250 kDa) consisting of a polypeptide chain
called apoprotein (MW 124 kDa) with a covalently attached linear tetrapyrrole
light-absorbing pigment molecule called chromophore also called phytochromobi-
lin via a thioether linkage to an invariant cysteine residue (Rockwell et al. 2006).
The five members of phytochrome gene family are PHYA, PHYB, PHYC,
PHYD, and PHYE (Devlin and Kay 2000). These phytochromes are divided into
two categories: TYPE I which is light labile and TYPE II which is light stable.
TYPE I includes PHYA, which encodes for the protein phytochromeA (phyA)
which mainly perceives far-red light, whereas TYPE 2 includes the PHYB, PHYC,
PHYD, and PHYE, and this family of phytochromes perceive red light (Neff et al.
2000). The blue light and UV-A wavelength of the spectrum is sensed by another
group of photoreceptors called cryptochromes and phototropins, whereas UVR8
photoreceptor receives UV-B light (Petrillo et al. 2014).
10.2.2 Photoreversibility
In etiolated seedlings, phytochromes are present in a red-light-absorbing form, Pr

(λmax = 660 nm) which upon exposure to red light gets photoconverted into far-red-
light-absorbing form, Pfr (λmax = 730 nm). When Pfr is exposed to far-red light, it is
photoconverted to Pr. This process is known as photoreversibility. During the con-
version of Pr to Pfr, both chromophore and protein moieties undergo conforma-
tional changes. The Pr chromophore undergoes a cis-trans isomerization of the
double bond between C15 and C16 and rotation of C14–C15 single bond (Fig. 10.1).
Plant hormones like gibberellic acid and abscisic acid play an important role in
seed germination. During seed germination and seedling establishment, gibberellins
stimulate the production of α-amylase and other hydrolytic enzymes in the aleurone
layer surrounding the endosperms of grains which help in the breakdown of com-
plex food resources. It also plays a major role in stem elongation. Abscisic acid acts
as an antagonist to gibberellins during seed germination thus inhibiting this process.
This phytohormone also plays an important role during stress conditions and pro-
motes the process of seed dormancy.
208 R. Kaur et al.
Fig. 10.1 Photoreversibility. Pr (inactive) changes to Pfr (active) upon exposure to red light, and
Pfr reverts back to Pr form when exposed to far-red light. Prolonged darkness can also convert Pfr
to Pr or mediate its degradation. The degraded product is referred as Pd
10.2.3 Gene Signaling Mechanism During Seed Germination
In model plant Arabidopsis thaliana, light-regulated germination signaling process

is well understood. This process is regulated by abscisic acid that inhibits germina-
tion, whereas gibberellins promote the process of seed germination. In dark condi-
tions, phyB resides in the cytosol in an inactivated form; thus PIF1
(phytochrome-interacting factor 1) which is a transcription factor gets accumulated
in the nucleus and regulates transcription of various genes that leads to accumula-
tion of abscisic acid, whereas the biosynthesis of gibberellic acid is inhibited.
In contrast, red light activates phyB, stabilizing its Pfr form which migrates into the
nucleus. PIF1 a transcription factor interacts with phytochrome in the nucleus where
its phosphorylation and degradation occur via ubiquitin ligase. Further its activity is
inhibited by the heterodimer formation along with long hypocotyl in far-red 1 (HFR1).
Hence, the genes involved in the process of gibberellic acid biosynthesis are activated
(GA3 oxidase1 and GA3 oxidase2). Also the CYP707A2 gene involved in abscisic
acid catabolism is activated. Thus, all the events during light conditions lead to the
accumulation of gibberellic acid and inhibition of abscisic acid which triggers the
process of seed germination (de Wit et al. 2016) (Fig. 10.2 and Table 10.1).
Therefore, it is the light quality (red and far-red light) that regulates seed germi-
nation. We conclude that the phenomenon of seed germination is affected by vary-
ing light conditions. It is a highly complex process which is governed by the plant
pigment phytochrome within the seed. Red light sensed by phytochrome induces
seed germination, whereas far-red and blue light inhibits the growth and its germi-
nation. So, the amount of light either continuous or brief exposure effects the pro-
cess of seed germination.
Fig. 10.2 In dark conditions, inactive phyB resides in the cytosol, due to which PIF1 is accumu-
lated in the nucleus. Thus, genes RGA, GAI, DAG1, SOM, ABI3, and ABI5 are transcribed, as a
result of which ABA is accumulated and GA biosynthesis is inhibited. Hence, the seed does not
germinate in dark. In contrast, red light activates phyB and translocated into the nucleus and medi-
ates the degradation PIF1. HFR1 forms a heterodimer with PIF1, which lowers down its activity
further lowering the levels of ABA concentration and accumulation of GA concentrations. Hence,
seed germination takes place in light
10.3 Seedling Growth and Development
The growth and development of the seedling start with the process of skotomorpho-
genesis followed by photomorphogenesis. During these developmental changes,
seedling emerges from the seed and reaches the soil surface.
10.3.1 Skotomorphogenesis
After the process of seed germination has occurred, the process of etiolated growth
of the seedling, i.e., skotomorphogenesis (growth in the dark), begins from the bur-
ied seed towards the soil surface in the upward direction (Toledo-Ortiz et al. 2010).
In this process, hypocotyl elongation occurs in the dark at a very fast rate, such that
its tip reaches the soil surface where it is exposed to light before the seed resources
are exhausted. The cotyledons in the dark are tightly closed and are underdeveloped.
The energy is derived from the reserve food material present in the seed as the pho-
tosynthetic machinery is inactive in this stage. The etioplasts are undifferentiated in
210 R. Kaur et al.
Table 10.1 Genes involved in seed germination in Arabidopsis thaliana induced by light via
phytochrome B (phyB)
Genes involved in seed germination in Hormone
Arabidopsis effected Gene function
RGA (Repressor of GAI-3) Gibberellic acid Inhibition of GA biosynthesis
(GA)
GAI (Gibberellic acid insensitive) Gibberellic acid Inhibition of GA biosynthesis
(GA)
DAG (DOF affected germination) Gibberellic acid Inhibition of GA3ox1 and GAox2
(GA)
SOM (Somnus) Gibberellic acid Inhibition of GA3ox1 and GAox2
(GA)
GA3ox1 (Gibberellin3-oxidase 1) Gibberellic acid Activation of GA biosynthesis
(GA)
GA3ox2 (Gibberellin3-oxidase 2) Gibberellic acid Activation of GA biosynthesis
(GA)
GA2ox2 (Gibberellin2-oxidase 2) Gibberellic acid Inhibition of GA biosynthesis
(GA)
ABI3 (Abscisic acid insensitive 3) Abscisic acid Activation of ABA biosynthesis
(ABA)
ABI5 (Abscisic acid insensitive 5) Abscisic acid Activation of ABA biosynthesis
(ABA)
ABA1 (Abscisic acid 1) Abscisic acid Activation of ABA biosynthesis
(ABA)
NCED6 (9-cis-epoxycarotenoid Abscisic acid Activation of ABA biosynthesis
dioxygenase 6) (ABA)
NCED9 (9-cis-epoxycarotenoid Abscisic acid Activation of ABA biosynthesis
dioxygenase 9) (ABA)
CYP707A2 Abscisic acid Inhibition of ABA synthesis
(ABA) (catabolism of ABA)
which the precursor of chlorophyll, protochlorophyllide, accumulates which gives

it a yellowish appearance.
This type of etiolated growth, i.e., growth in the absence of light, is governed by
various transcription regulators which regulate the expression of various genes.
Most of them are bHLH TFs. Among these, HY5 plays an important role in hypo-
cotyl elongation. In the absence of light, HY5 is degraded by ubiquitinylation via
COP1 as HY5 is a negative regulator of hypocotyl elongation; as a result, the
absence of HY5 leads to hypocotyl elongation. COP1 also degrades HFR1 (long
hypocotyl in far-red 1) in the dark, which is an atypical bHLH TF and is involved in
phytochrome- and cryptochrome-dependent signal transduction (Mancini et al.
2016; Toledo-Ortiz et al. 2014).
PIFs (phytochrome-interacting factors) on the other hand indirectly regulate
hypocotyl elongation by degrading PhyB. Hormones involved in etiolated growth
conditions are auxins, cytokinins, ethylene, gibberellins, and brassinosteroids.
Gibberellins destabilize DELLA protein (growth repressing transcription regulator)
and thus lead to hypocotyl elongation (Achard et al. 2004). Thus, the process of
hypocotyl elongation is a characteristic of skotomorphogenesis, i.e., the growth of a

seedling under the soil in the dark conditions.
10.3.2 Photomorphogenesis
Once the seedling has emerged out from the soil, further development is mediated
by light which has been termed as photomorphogenesis (Godoy Herz et al. 2014;
Toledo-Ortiz et al. 2010; Arsovski et al. 2012). After germination, the light-grown
seedlings have a different morphology than those grown in the dark, as seedlings in
the dark conditions do not express light-inducible genes. Upon exposure to light, the
light-responsive genes are induced; therefore the seedling undergoes rapid light-
mediated morphological changes.
In the presence of light, transcription regulators HY5 and HFR1 are accumulated
in the nucleus because their degradation by COP1-mediated ubiquitin ligase is pre-
vented, as a result of which the elongation of hypocotyls is inhibited. Upon expo-
sure to light, the underdeveloped cotyledons start expanding; thus the light capturing
surface is increased. The process of de-etiolation begins with the greening of coty-
ledons in which chloroplasts start accumulating the chlorophyll pigment. Hence, the
green chloroplasts are photosynthetically active, and therefore now the energy is
derived by the process of photosynthesis which is utilized for further growth and
development.
10.4 Shade Avoidance
Once the seedling becomes a photoautotroph, both the external and the internal
environments regulate the plant growth and help it to enter in the juvenile phase.
Many factors like pathogens and shade due to neighboring vegetation (canopy) act
as obstacles during this phase transition. Light is often a limiting factor in dense
forests where canopy blocks the light from reaching the plants growing below it.
These plants hence respond by the phenomenon of shade avoidance, i.e., changes
which occur in response to the enrichment of far-red light under a leaf canopy
(Casal 2012; Franklin and Whitelam 2004). These varying light conditions are
sensed by phytochromes, and such responses include elevation of leaf angles (hypo-
nasty), enhanced hypocotyls and petiole growth, early flowering, abundant PIF4
and PIF5 proteins, degradation of DELLA proteins, etc. collectively known as shade
avoidance syndrome (Leivar et al. 2008). The R:FR ratio decreases in the shade
conditions leading to alterations in the growth of the plant.
212 R. Kaur et al.
10.5 Role of Light in Plant Defense
In the initial phase of growth, the plant utilizes most of the energy fixed by the pro-
cess of photosynthesis for its growth. When surplus amounts of carbohydrates are
available, they are converted into secondary metabolites like terpenes, alkaloids,
nitrogen-containing compounds, etc., which play an important role in plant defense.
Thus, in growing plants, these compounds are produced in a very less amount
because the major proportion of carbon fixed is diverted for its growth and develop-
ment. Due to the lower levels of secondary metabolites in the new emerging seed-
ling, the growing plant is vulnerable to attack by pathogens. Thus, in this phase of
growth, the plant needs to grow at a very fast rate in order to compete with its
neighboring plants and also needs to defend itself from the microbial pathogens
(Ballare 2014). As the plant is still growing in its juvenile phase, the intact barrier
consisting of the bark or waxy cuticle is not available as a first line of defense to
protect the plant against the microbial attack, as a result of which, the pathogen can
easily enter inside the plant body.
Light sensed by the phytochrome B (phyB) photoreceptor induces signal trans-
duction pathways that lead to the production of various compounds like jasmonic
acid, salicylic acid, etc., which are involved in plant defense mechanism. These
compounds trigger the induction of SAR (systemic acquired response) (Bolton
2009; Ryals et al. 1996). Hence, if the plant is growing under suboptimal condi-
tions, i.e., shade (low R:FR), it leads to inactivation of phyB that downregulates the
jasmonic acid and salicylic acid signaling.
10.6 Light-Mediated Floral Induction
With time, various developmental changes cause alterations in the plant growth due
to which the plant enters from juvenile stage to a mature adult plant. In juvenile
phase, vegetative meristem does not respond to internal and external signals that
initiate flowering, i.e., incompetence. Under internal optimal conditions, the devel-
opmental changes allow the plant to become reproductively competent. These con-
ditions include factors like plant size, number of vegetative nodes, and amount of
sucrose, the main energy source which fuels the plant to begin the complex process
of floral initiation. Along with these factors, hormones such as gibberellic acid,
cytokinins, and “florigen” also known as the flowering hormone play a major role in
gaining internal competence (Corbesier and Coupland 2006). Once the plant
becomes internally competent to reproduce, external environmental conditions like
light (photoperiod, light quality, and quantity), temperature (vernalization, i.e.,
exposure to long cold conditions), nutrient, and water availability determine the
process of floral induction.
10.6.1 Photoperiodism
Photoperiodism plays a crucial role in regulating floral development. Different

plants respond to light conditions depending upon the duration of light exposure to
that respective plant. Based on this condition, these plants are divided into three
basic categories, namely short-day plants, long-day plants, and day-neutral plants.
The short-day plants are those in which floral initiation takes place when exposure
to light is less than the critical duration, for example chrysanthemum and soybeans.
These plants usually flower in late summer. The long-day plants require exposure to
light for a period more than a well-defined critical duration, i.e., these plants require
a long duration of light exposure, for example lettuce and spinach. These plants
normally flower in spring and early summer. The third category includes day-neutral
plants in which there is no relation between photoperiodism and floral induction.
Initiation of flowering occurs independent of light duration. For example,
Sorghastrum nutans commonly known as Indian grass is a day-neutral plant
(Lumsden 2002; Andreas and Coupland 2012).
The shoot apical meristem (SAM) in the initial phases of plant growth leads to
the formation of vegetative organs like leaves, but once the plant becomes internally
competent to reproduce and all the external conditions are favorable and optimal,
SAM makes transition to the reproductive development, and the production of
flower is initiated (Benlloch et al. 2007). These changes are mediated by signals that
turn on various genes that lead to morphological changes specifying the location of
floral organs like sepals, petals, stamens, and carpels.
10.6.2 Photoreceptor Proteins Regulating Floral Formation
Various photoreceptor proteins are present in the leaf of the plant which play a vital
role in the process of gene activation that leads to the initiation of flowering. In
Arabidopsis thaliana, a facultative long-day plant, the far-red and blue light is per-
ceived by phytochrome (phyA) and cryptochrome (cry1 and cry2) (Zuo et al. 2011)
that promote floral initiation, whereas the red light perceived by phyB inhibits flow-
ering. Florigen (flowering hormone) production takes place in leaves which is regu-
lated by the duration of light exposure, i.e., photoperiodism, and later on this
signaling hormone is translocated via phloem to SAM where the process of flower
formation is induced (Tsuji and Taoka 2014).
10.6.3 Genes Involved in Floral Formation
As a result of the abovementioned events, various genes are activated that initiate
the process of flowering. These genes are divided into two groups, namely floral
meristem identity genes that are responsible for the transition of vegetative meri-
stem to floral meristem, and they include AGAMOUS-LIKE 20, LEAFY, APETALA1
(AP1), etc. The second group is floral organ identity genes or floral homeotic genes
214 R. Kaur et al.
that lead to the formation of the floral parts, and these include APETALA2 (AP2),
APETALA3 (AP3)/PISTILATA (PI), and AGAMOUS (AG). These two groups of
genes act in a sequential manner to initiate the process of flowering. The signaling
hormone “florigen” is responsible for the activation of CONSTANS gene which is
expressed in long days, and it encodes a transcription factor that further initiates the
signaling cascade by activating AGAMOUS-LIKE 20, a floral meristem identity
gene (Achard et al. 2007). It further activates LEAFY which is the most important
gene for the production of flower. LEAFY in turn activates floral homeotic genes
that are responsible for the formation of various floral organs (Benlloch et al. 2007;
Balasubramanian et al. 2006). AGAMOUS-LIKE 20 is inhibited by another gene
FLOWERING LOCUS C. Therefore, to initiate the process of floral induction, this
inhibition needs to be relieved which is done upon exposure to low-temperature
conditions, i.e., the process of vernalization (Figs. 10.3 and 10.4).
Along with low temperature, gibberellins also play an important role in control-
ling this complex process of flowering. Thus, in most of the plants, both light and
temperature play a very important role in controlling the rate of flowering.
Fig. 10.3 Various genes are activated or inhibited by different floral developmental processes
including photoperiodism, autonomous/vernalization pathway, energy pathway, and gibberellin
pathway that eventually lead to the production of various floral organs
Fig. 10.4 ABC
Whorl 1 2 3 4
MODEL. The formation of
floral organs depends upon
the interaction between
three floral homeotic genes B
AP2 (type A), AP3/PI (type Activity
B), and AG (type C). AP2 type A C
results in the formation of
Structure Sepal Petal Stamen Carpel
sepals. The interaction of
AP3/PI with AP2 leads to
APETALA3/
the formation of petals,
Genes PISTILATA
whereas the interaction of
AP3/PI with AG forms APETALA2 AGAMOUS
stamens, and AG alone
forms carpels in the last Structure
Sepal Petal Stamen Carpel
whorl
10.7 Summary
Light plays a very important role in overall growth and development of a plant start-
ing from the seed germination till it becomes a full-fledged adult reproductive plant.
Plants respond to the external light conditions via photoreceptors. Phytochromes
and cryptochromes are the main photoreceptors that sense red/far-red and blue light,
respectively, and trigger various signaling cascades, as a result of which, various
metabolic processes are either initiated or repressed. The processes like seed germi-
nation, seedling growth and development, formation of secondary metabolites, and
floral initiation are the most important events in the plant life. Light basically regu-
lates various phytohormones like auxins, gibberellins, abscisic acid, ethylene, cyto-
kinins, brassinosteroids, etc. which in turn regulate the overall growth and
development of the plant. Photoperiodism and vernalization are the two main pro-
cesses that control the process of flower formation in adult plants. Light perceived
by photoreceptors upregulates and downregulates the transcription of various genes
which are involved in the plant developmental process. Thus, without light, there is
no life.
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cub.2013.03.048
Microbial Biotechnology: A Key
to Sustainable Agriculture 11
S. K. Gosal, Jaspreet Kaur, and Jupinder Kaur
Abstract
The exploitive and improper agricultural practices lead to degradative processes
such as nutrient depletion, loss in soil fertility, and soil organic matter. These
processes contribute to a serious decline in soil productivity. The degraded soils
can be restored and rehabilitated by alternative agricultural practices such as use
of potential microbial inoculants to provide favorable environment for optimum
crop production and protection. The use of bioinoculant is one of the important
components of integrated nutrient management as they facilitate a cost-effective
renewable source of plant nutrients which supplement chemical fertilizers con-
tributing to sustainable agriculture. Several microorganisms are currently being
marketed commercially as biofertilizers for crop plants. Unfortunately, these
microorganisms are not always as efficient in the field as they are in laboratory
or greenhouse experiments. The use of microbial biotechnology has manipulated
the microorganism at their genetic level which leads to increase in their survival
and efficiency in soil. The genetically modified microorganisms can be used as
potent bioinoculants in agriculture, but their undesirable effects and ethical
implications still remain a major problem whether they should be accepted or
not. The presence of antibiotic resistance gene, horizontal transfer of genes, and
unstable vector in modified microorganism made them unsuitable for environ-
mental application as these characteristics can get transferred to indigenous
microorganisms which lead to mutations. More intense research is required to
assess the stability of genetically modified microorganism and their effect on
indigenous microflora. These studies can open the way to the production of more
effective, stable, and reliable recombinant inoculants for maintaining sustainable
agriculture.
S. K. Gosal (*) · J. Kaur · J. Kaur

Department of Microbiology, Punjab Agricultural University, Ludhiana, India

220 S. K. Gosal et al.
Keywords
Bioinoculants · Green revolution · Microbial communities · Bioinoculants
11.1 Introduction
The intensive agricultural technologies led to a “green revolution” which resulted in

enhanced global pollution, unfavorable climatic changes, and loss of biodiversity.
The first two decades of the “green revolution” increased the productivity of crops
immensely. Afterwards, the crop productivity began to decline due to the indis-
criminate use of agrochemicals along with deficiencies of essential nutrients (major
and minor nutrients) in the soil. In response to this particular situation, the develop-
ment of sustainable agriculture is required that results in high productivities of crop
with a minimal imbalance in the environment (Noble and Ruaysoongnern 2010).
The most promising strategy to reach this goal is to substitute hazardous agrochemi-
cals with environment-friendly microbes. This biological approach is based on
exploiting the role of soil microbial communities for a sustainable and healthy crop
production while preserving the biosphere. The microbial communities present in
the soil can be used as potential bioinoculants. Their application can improve the
soil health, soil fertility as well as protect crops from biotic (pathogens, pests), and
abiotic (including pollution and climatic change) stresses. Use of these microbial
inoculants facilitates nutrient availability to agricultural crops which in turn
increases growth as well as crop productivity.
The efficiency of bioinoculants is affected by various environmental factors
which lead to uncertainty in their performance under greenhouse and field condi-
tion. The remedy to this particular situation can be “microbial biotechnology”
which is a synthetic research field to exploit the genetic information of microbes to
generate more efficient microbial strains. This approach can increase reliability of
the bioinoculants in different environmental conditions. The regular movement of
microorganisms between plant, animal, and soilborne niches is analyzed to recon-
struct the microbiota in natural and agricultural ecosystems, so that the genetic
information about a particular gene can be used to improve the microorganism
potential. Accordingly, several strategies have been designed for effective exploita-
tion of beneficial microbial services for creating sustainable and environmental
friendly agroecosystem. In this chapter, we elucidate the application of genetic
engineered microbes on the improvement in potential of microorganism by genetic
modification for the specific traits and their survival in soil.
11.2 Role of Microbes in Sustainable Agriculture
The present agricultural system has faced major challenges in recent years, which
include overdependence on agrochemicals, global climate change, population
explosion, and increased economic and environmental costs of nonrenewable
11 Microbial Biotechnology: A Key to Sustainable Agriculture 221
resources. The present agricultural practices used to get higher productivity lead to
major problems associated with pollution of soil, surface, and groundwater due to
enhanced usage of agrochemicals. Consequently, these agrochemicals have adverse
impacts on the environment, safety, and quality of food. So, there is a need to reduce
the excessive use of agrochemicals (fertilizers and pesticides) in food crop produc-
tion. This leads to increased interest in sustainable agriculture, which can be defined
as maintenance of biological diversity, productivity, regeneration capacity, vitality,
and ability of agricultural ecosystem by managing and utilizing the indigenous bio-
logical resources. The concept of sustainable agriculture emphasizes on the use of
microbial inoculants in shifting the soil microbial equilibrium to get higher produc-
tivity along with the protection of crops. Microorganisms can be harnessed to pro-
duce biofertilizers, to decompose organic wastes more efficiently and combat
plant-pest diseases with greater efficiency than ever before. In agricultural ecosys-
tem, microorganisms have a critical role in transformation, mineralization, and
solubilization of nutrients. The population of microorganisms in their natural habi-
tat is often scanty, and it can be enhanced by addition of microbial inoculants with
specific properties. The microbial inoculants can be represented as potential “green”
alternative to the intensive use of agrochemicals (artificial fertilizers and pesticides)
in agricultural systems. A wide spectrum of preparations of diverse microbial spe-
cies may be used as substituent nutrients to enhance crop production and disease
suppression (Andrews et al. 2010). However, nutrient substitution by microbes is
usually partial and only sometimes can be complete. The balance between microbial
inoculants, organic manure, and inorganic nutrition may lead to improvement in
sustainable agroecosystems (Hedin et al. 2009). This integrated approach is most
promising in all agricultural systems as the strong correlations between the micro-
organism and crops. Presence of microbes increases the nutrient availability such as
nitrogen fixation and phosphorus solubilization and mobilization hence increasing
the nutrient availability. Microbes also secrete secondary metabolites which include
plant growth-promoting hormones, metal chelators, antibiotics, and exopolysaccha-
rides. These secondary metabolites increase the plant growth, suppress disease inci-
dence, and improve metal scavenging as well as improve the soil texture by
increasing soil organic carbon. The improved soil texture enhances the physiochem-
ical properties of soil such as high water-holding capacity and pH buffering. So,
microbes have an important role in soil nutrient transformation, soil physicochemi-
cal properties, and consequently in maintaining sustainability in agricultural eco-
system which is lacking in the case of conventional practices (Fig. 11.1).
11.3 Role of Biotechnology in Sustainable Agriculture
Biotechnology is defined as “any technological application that uses biological sys-

tems, living organisms, or derivatives thereof, to make or modify products or pro-
cesses for specific use” (UN Convention on Biological Diversity, Art. 2, 2017). By
implementing genetic engineering, it is possible to get specific properties in host
that cannot be produced by means of traditional method. All of the means of genome
Fig. 11.1 Role of soil microorganism in nutrient transformation. —available nitrogen,

metal-bound insoluble phosphorus, —available phosphorus, —organic carbon,
—plant growth-promoting metabolites, and , , —rhizospheric microorganisms
manipulation used in biotechnology belong to genetic engineering and rDNA tech-

nology. The genetic engineering differs from genetic changes that occur in nature as
in this it is possible to combine the characteristics of evolutionally different and
unrelated species. In this way, molecular methods are replacing conventional genetic
methods used in agriculture. The use of biotechnology increased food production as
well as made agriculture more sustainable from an environmental point of view.
Biotechnology has been contributing important role in sustainable agriculture
through various ways as shown in Fig. 11.2.
Biotechnology contributes to sustainable agriculture by reducing the dependence
on agrochemicals, particularly pesticides, through the employment of genetically
modifications that promotes plant growth, improves nutrient availability, and
increases resistance to biotic and abiotic stresses.
Fig. 11.2 Contribution of biotechnology in sustainable agriculture
11.4 Microbial Biotechnology and Agriculture
Soil microbes are composed of prokaryotic (eubacteria, actinomycetes, and archaea)

and eukaryotic (fungi, yeasts, protozoa, and algae) organisms, whose populations
vary from soil to soil. Their population in soil is influenced by variety of soil and
environmental parameters including cultivar, soil texture, soil structure, soil pH, soil
moisture content, soil temperature, etc. There are enormous ways in which microbes
have been used over the past years. These include exploiting microbes for the ben-
efits of agriculture, medicine industry, food industry, genetic engineering, and envi-
ronmental protection. The uniqueness of microorganisms, their uncertain nature,
and biosynthetic capabilities in particular set of conditions made them the potent
candidates for solving various problems of agroecosystem. For increasing their bio-
logical capabilities and certainty, one of the technological advances is to increase
the microbial potential by application of “biotechnology” which is an integral appli-
cation of biochemistry, microbiology, and genetic engineering (Fig. 11.3).
Microbial biotechnology deals with the manipulation of microbes or their compo-
nents to produce useful products for various applications in biological science. This
technique permitted breakthroughs in agriculture to produce healthier foods, safer
pesticides, innovative environmental technologies, and new energy sources. Microbial
biotechnology can improve the agroecosystem by employing microorganisms to
increase the soil nutrient content, enhance crop productivity, and enhance resistance
to specific plant pathogens and to produce safer herbicides. Complete genome
sequence of a microbe provides crucial information about its biology, which leads to
the first step toward understanding a microbe’s biological potentials and modifying
them according to agricultural needs. The microbes which are genetically manipu-
lated with gene of interest by using biotechnological techniques are referred to as
genetically modified microorganism (GMM). Microbes are engineered to (1) improve
expression of beneficial traits and (2) be traceable in plant and soil systems (Fig. 11.4).
Fig. 11.3 Biotechnology of an integrated branch of various applied sciences
Fig. 11.4 Schematic representation to generate genetic modification in microorganism

Application of microbes in biotechnology (microbial biotechnology) allows the

genomic studies, which lead to breakthrough in improved bacterial inoculants.
These genetically modified bacterial inoculants are efficient in their functional char-
acteristics such as biological control of pest, reduced virulence for pathogens, and
bioremediation of environment. Therefore, generation of genetically modified
microorganism by the application of biotechnology is getting better attention in
advancing sustainable development of agroecosystem and environmental health.
11.5 Molecular Tools for Manipulation of Microorganisms
Genetic engineering is a modern technology which allows manipulation in genetic

makeup of microorganisms to achieve planned and desired results to perform spe-
cific functions. This technique involves isolation, introduction, integration, and sta-
ble expression of the foreign gene into an unrelated organism. It offers opportunity to
create artificial combination of desirable genes that do not exist together in nature.
The microorganisms can be manipulated by engineering single gene or operon, mod-
ification in biochemical pathway, and alterations in the existing genes sequences
(Dale and Park 2007). The microorganisms in which genetic manipulations are stable
and fully expressed are referred to as genetically modified microorganisms (GMM).
The steps for constructing genetically modified microorganisms are the following:
1. Gene isolation and excision: Insertion of exogenous gene into a non-native host
requires selection and physical excision of desired DNA so that it can be trans-
ferred to host for proper gene expression. The isolation of desired DNA requires
three basic steps:
(a) Opening the target cells: The target cell will be disrupted using gentle
methods, preferably utilizing enzymatic degradation of cell wall material (if
present) and detergent-mediated lysis of cell membrane. Lysis of cells yields
mixture of DNA, proteins, and lipids with other cell components.
(b) Separation and recovery of the DNA: The lysed product obtained from the
opening of cell will be treated with deproteinases and lipases following one
or more extractions using phenol or phenol/chloroform mixtures. Then, sub-
sequent centrifugation results into separation of the protein molecules into
the phenol phase and nucleic acids in the aqueous phase. The nucleic acid
(DNA and RNA) obtained in aqueous phase will be precipitated from solu-
tion using isopropanol or ethanol. For preparation of pure DNA, the nucleic
acids will be further treated with enzyme ribonuclease (RNase) which
digests the RNA in the preparation. If the target gene is located in the plas-
mid DNA, then further gradient centrifugation will be required. As an alter-
native to gradient centrifugation, size-exclusion chromatography (gel
filtration) or similar techniques may be used.
(c) Excision of target gene: Pure isolated DNA will be treated with restriction
endonucleases (RE) type II to excise target gene. These enzymes hydrolyzed
the sugar-phosphate backbone of DNA molecules at specific site; this
s pecific site is called as restriction site. Excision with the desired gene should
be excised with its promoter sequences so that the genes can be expressed
into the host cell.
2. Vector preparation and ligation: Cloning vector can be defined as “a DNA
molecule originating from a virus, a plasmid, or the cell of other organism into
which the desired DNA fragment of appropriate size can be integrated without
the loss of its self-replication property.” Vectors act as gene vehicle which intro-
duces foreign gene into host cells, where it clones to reproduce in large quanti-
ties. There are various types of vectors which include plasmids, cosmids, yeast
artificial chromosomes, transposons, etc. The selection of cloning vector depends
upon various factors like the method of gene transfer, the desired outcome of the
modification, and the application of the modified microorganisms (laboratory or
environmental). For example, replicating vectors (plasmid vectors with high or
low copy numbers) are commonly used to express the desired genes in heterolo-
gous hosts for manufacturing expressed proteins. These vectors are also used to
increase the copies of gene in cell to enhance the production of the metabolite.
Cosmid and yeast artificial chromosome vectors can accept DNA fragments as
large as 100 kb. These are necessary to clone a large piece of DNA into host for
high-level metabolite production (Sosio et al. 2000). Conjugal vectors are alter-
native approach to transfer the gene which is usually difficult to transform. These
vectors can transfer the genes into indigenous microorganism by horizontal
transfer. Many genes have been transferred by integrating the gene with transpo-
sons. Transposons can move from one genomic position to another by a cut-and-
paste mechanism. They are powerful method for genetic change which can
significantly modify many genomes. As genetic tools, DNA transposons can
introduce a piece of foreign DNA into a genome. For example, plasmid-born
catabolic genes for the degradation of toxic substances can be often located in
transposons. It was reported that pUTK21 plasmid of P. fluorescens HK44 was
made by transposon Tn4431 insertion into NAH7 plasmid from P. fluorescens
5R. This transposon originated from Vibrio fischeri and carried luxCDABE gene
cassette. This genes cassette was expressed under a common promoter which
resulted in simultaneous degradation of naphthalene and luminescent signal.
The desired gene can be ligated into selected vector DNA using specific
enzymes called as DNA ligases. These are important cellular enzymes which
seal discontinuities in the sugar-phosphate backbone chains that arise during
joining the target DNA with vector DNA. The most commonly used T4 DNA
ligase enzyme is purified from E. coli cells infected with bacteriophage T4. This
enzyme works the best at 37 °C, but is often used at much lower temperatures
(4–15 °C) to prevent thermal denaturation.
3 . Gene transfer method: Microorganisms can be manipulated by transferring
specific genes from one microorganism to other. Genes can be transferred by
various genetic recombination methods mainly by transformation, conjugation,
and transduction. The techniques of transformation and transduction represent
the simplest methods available for getting recombinant DNA. In the context of
cloning in E. coli cells, transformation refers to the uptake of plasmid DNA and
Table 11.1 Examples of gene transfer methods in microorganisms

Gene transfer
Microorganism to modify Gene transferred method References
P. putida mt-2 strain B13 TOL plasmid pWW0 Conjugation
Alcaligenes sp. A7 Benzoate and phenol Conjugation Schwien and

degrader genes Schmidt (1982)
P. putida strain B13 xylXYZ (toluate-1,2- Conjugation Lehrbach et al.
dioxygenase) and xylL (1984)
P. putida strain B13 nahG (salicylate Conjugation Lehrbach et al.
hydroxylase) (1984)
E. coli JM109 Plasmids bphA1 Electroporation Kumamaru et al.
(shuffled) and (1998)
bphA2A3A4BC (KF707)
Sinorhizobium meliloti plasmid pE43 Electro- Chen et al.
transformation (2005)
B. cepacia BU0072 pTOM-Bu61 plasmid Conjugation Taghavi et al.
from B. cepacia BU61 (2005)
Mycobacterium sp Plasmid pNC950 Electroporation Matsui et al.
(2006)
P. putida KT2442, P. Plasmid pKST11 Conjugation Ouyang et al.
stutzeri 1317, and A. (2007)
hydrophila 4AK4
E. coli M15 (pREP4). Functional gcd Phage P1 mediated Tripura and
transduction Podile (2007)
E. coli S17-1 pASF101 Transformation Menn et al.
(2009)
P. protegens Pf-5 Cosmid X940 Transformation Ayub et al.
(2009)
A. vinelandii AvOP pGDEGS1 and Transformation Shashidhar and
pGDEPS1 Rao (2009)
R. leguminosarum vktA gene Conjugation Orikassa et al.
(2010)
A. brasilense Sp245-Rif Plasmids pFAJ0526, Conjugation Baudoin et al.
pFAJ0529 and pFAJ0535 (2010)
Pseudomonas A1501 Plasmid pUC4K Transformation Setten et al.
(X06404) (2013)
transduction to the uptake of phage DNA. However, the most frequently used

method is transformation. This process includes the microbial uptake of the
exogenous genetic material through the cell membrane. This uptake can occur
only at specific physiological stage of competence. Natural bacterial competence
is inefficient to uptake DNA significantly, so, competence is induced in bacterial
cells by chemical treatment (treating with calcium chloride). Escherichia coli is
the most commonly used cloning host for generating competent cell by chemical
treatments. Some of the examples to transfer the gene for modification in micro-
organisms are shown in Table 11.1.
Transformation involves preparation of protoplasts using lysosome enzyme in

the presence of polyethylene glycol to promote the uptake of DNA. Growth
medium, growth phase, ionic composition of transformation buffers, and d uration
of treatment are the important factors that should be optimized before starting
the process. However, the process of transduction involves packaging of phage
with copies of desired genes which are in the form of concatemers linked through
cos sites. Afterward, phage infects the host cell to release the DNA inside the
bacteria through the injectisome apparatus. This process is used where the com-
petent cells are inefficient to take up the DNA.
Conjugation is another method used to introduce plasmid DNA into microor-
ganisms. In this process the donor strain contains both the gene of interest and
the origin of replication on a plasmid which is transferred to recipient cell upon
transient contact between them through the formation of conjugation bridge.
After the transfer of genetic material, donor cells are eliminated with an antibi-
otic to which the recipient cells are resistant. There is horizontal gene transfer
between the two strains which results in host cell with foreign gene. Moreover,
it does not involve the tedious processes such as protoplast formation and regen-
eration as in the case of transformation. There are many other alternative methods
to transfer the gene in organisms such as electroporation, liposome-mediated
transformation, microinjection, etc., but electroporation is widely used for gene
transfer method in the case of microorganism. The electroporation process
involves the application of electric impulse for specific time interval to generate
transient pores in the cell membrane, thereby allowing DNA transfer. Growth
phase, cell density, electroporation parameters, and growth medium must be
optimized to achieve desirable efficiency. Electroporation is often used when
protoplast transformation is insufficient or ineffective. So, the methods available
for getting recombinant DNA into cells depend on the type of host/vector system
which ranges from very simple procedures to much more complicated ones.
4 . Selection and target gene expression: Selection of recombinant cells from the
large number of cells in which gene has been transferred is very critical process.
Some cells get genetically modified, but majority of cells remain unmodified.
The selection process is necessary as the number of recombinant cells is often
significantly less than the number of nonrecombinant cells. To select the recom-
binant cells, various methods are used such as selectable marker, gene disruption
method, positive selection method, colony PCR, DNA sequencing, etc. The
selectable marker genes are important part of cloning vectors and are required
for identification of transformed cells. Selectable marker gene usually codes for
the antibiotic resistance or toxin resistance which gives the selective advantage
to recombinant cells over the others. Usually, high-level expression of a select-
able marker gene is required for complete elimination of nonrecombinant cells.
Antibiotic resistance genes are routinely used as selectable markers, but these
genes are not generally acceptable for the construction of recombinant microor-
ganisms for environmental application (Akada et al. 2002). Gene disruption
method is another method which involves insertion of target gene in the middle
of previously present gene, thus disrupting the expression of the gene. A classic
example of gene disruption is the insertion of gene between the lacZα encoding
sequence in plasmid DNA. The gene insertion disrupts the production of
β-glucosidase enzyme (encoded by lacZα); as a result, recombinant microorgan-
ism is unable to utilize X-gal (substrate analog) in the presence of inducer
isopropyl β-D-1-thiogalactopyranoside (IPTG) in growth media thus appears
white on the medium, whereas nonrecombinants appear blue (commonly known
as blue white colony selection). Another similar method is positive selection of
recombinant vectors which is an effective and simple method for screening the
presence of target gene. In this type of selection, the vector contains a lethal
gene, and the target gene was inserted in multiple cloning sites present inside that
lethal gene. The expression of the lethal gene is disrupted by ligation of a target
DNA; as a result, only cells with recombinant plasmids can survive. This
approach can save time and cost of selection since it typically yields more than
99% recombinant clones.
Genetically modified microorganisms should completely express the target gene in
environmental condition which is necessary for the improvement in functional
characteristics of microorganism. Expression of gene depends upon the presence
of other gene sequence such as promoter sequence. This sequence is about 100–
1000 base pairs long, present on genome near the transcription start sites of
genes where the RNA polymerase will bind and initiate transcription. Thus, pro-
moter sequence regulates the expression of the gene. There are two types of
promoters—constitutive and inducible promoters. Constitutive promoters are
continuously active and known as housekeeping genes, whereas inducible
promoters become activated only under certain conditions such as the presence
of an inducer. Promoter selection is very important to optimize the target gene’s
expression. The constitutive promoters are used for the continuous expression of
a target gene. However, inducible promoters are used for expression of gene
under specific set of environmental and biochemical conditions.
11.6 A
pplications of Genetically Modified Microbes
in Agriculture
The potential of microbes is analyzed from their ecological impacts such as

increased availability of nutrients in soil, improved crop productivity, plant growth
promotion, and disease suppression. Their ecological impacts can be intensified by
addressing the mechanisms for mutual interaction between microbial partners and
crop plant. Most of the microbial interactions are regulated by specific molecules/
signals which are responsible for key environmental processes, such as the biogeo-
chemical cycling of nutrients and the maintenance of plant health as well as soil
quality (Barea et al. 2004). The microbial interaction can be of two types:
(a) Dead plant material-based interaction: The microbial interactions with plant
debris which lead to the degradation of detritus material and recycling of nutri-
ents in the ecosystem, thus, affect energy and nutrient flows.
Fig. 11.5 Role of microbial interaction in maintaining sustainable agroecosystem
(b) Living plant roots-based interactions: The interaction between microorganisms

and plant roots increases the production of beneficial secondary metabolites, avail-
ability of unavailable nutrients, and soil nutrient level and fertility, thus increasing
the fitness of agroecosystem.
Both types of interactions are relevant to agronomy as well as ecology (Fig. 11.5).

These interactions can be targeted by microbial biotechnology to produce geneti-
cally modified microbes which can improve the interaction between the microbes
and crop plants leading to increased microbial ecological efficiencies.
These enhanced ecological efficiencies improve the communication between
genetically modified microbes and their environment. As a result, genetically
improved microbial population will have enhanced survival and better function and
provide improved ecological services (Fig. 11.6).
11.6.1 Improvement in Phytostimulation Activity
Successful crop growth depends on the genetic makeup of the plant, adequate avail-
ability of nutrients, presence of beneficial microbes, and absence of phytopatho-
gens. The presence of beneficial microorganisms increases availability of nutrients
and growth-promoting metabolites in vicinity of plant roots. Plant growth-promoting
rhizobacteria (PGPR) are such beneficial and obligate rhizoplane as well as rhizo-
spheric bacteria which promote plant growth by a wide range of mechanisms such
Fig. 11.6 Applications of genetic modified microorganism in sustainable agriculture
as release of beneficial metabolites and bioactive substances that have measurable

impact on growth. Bioactive substances secreted by microbes include vitamins,
phytohormones, and amino acids. These metabolites contribute to the host root res-
piration, root metabolism, root abundance, suppression of soilborne pathogens, etc.
which result in improved mineral water uptake and disease suppression in inocu-
lated plants.
The efficacy of PGPR depends upon microbial inoculant-crop compatibility, soil
composition, moisture content, and most importantly mechanisms (direct or indi-
rect) employed by PGPRs to facilitate plant growth. Various studies have shown that
PGPR strains typically harbor more than one plant growth-promoting property. So,
the genes encoding for these properties contribute to plant beneficial traits and can
be selected by the interaction of these bacteria with plants. These genes include
nifHDK (nitrogenase-encoding genes), pqqBCDEFG (pyrroloquinoline quinone-
encoding genes), ipdC/ppdC (gene of the indole-3-pyruvate synthesis pathway),
nirK (copper nitrite reductase gene), acdS (1-aminocyclopropane-1-carboxylate
(ACC) deaminase gene), hcnABC (hydrogen cyanide genes), phlACBD
(2,4-diacetylphloroglucinol), etc. for synthesis of various plant beneficiary metabo-
lites as well as activities. Such information would bring fundamental insights into
the associations of PGPR bacteria with plants which can be exploited for genetic
manipulations to increased phytobeneficial potential (Bruto et al. 2009).
Indigenous plant growth-promoting bacteria associated with plants do not have
uniform and expected effects on the growth and fitness of all host plants in different
conditions (Long et al. 2008). It may be due to non-efficient colonization of microbes
in rhizospheric area. The root colonization activity of plant growth-promoting rhizo-
bacteria is a very important feature. Identification of genes associated in root coloni-
zation and other plant-microbe interactions are useful techniques for generating
strains with enhanced competence for the rhizosphere. By this, the efficiency of
plant-associated bacteria increased to such a level that can produce reliable results
under the presence of variable environmental factors. There are enormous plant
growth-promoting traits which can be targeted in genetic engineering to improve the
performance of plant growth-promoting rhizobacteria in soil. Root-associated rhizo-
bacteria can produce the plant growth hormone indole-3-acetic acid (IAA) which
triggers the growth of the meristematic tissues of the root and shoot. The insertion of
multiple copies of ipdC (coding an indole-3-pyruvate decarboxylase/phenylpyruvate
decarboxylase) to rhizobacteria can improve the plant growth-promoting activity of
recipient microorganism. The presence of ipdC construct in microorganism causes
increased shoot biomass which may be due to increased early nutrient acquisition
and improved functioning of the root system in terms of nutrient acquisition (Bertrand
et al. 2000). The potential to exploit soil microorganisms as plant growth promoters
may be achieved by the development of more effective microbial inoculants through
the genetic manipulation of microorganisms. However, genetic manipulation appears
to offer the reliable and reproducible results in the field.
11.6.2 Enhanced Biofertilization Capability
Plant production confides on biogeochemical cycling, which is primarily driven by

microbes and causes release of essential nutrients in soil ecosystem. Nutrients are
important for the growth and development of plants as well as microorganisms. The
rhizosphere harbors abundant and diverse species of bacteria and fungi that inter-
play between plant and soil nutrient (C, N, & P) dynamics. Functional activities of
the beneficial rhizospheric microbes are related to plant nutrition, organic matter
decomposition, nitrogen fixation, nutrient solubilization, mobilization, and trans-
port (Philippot et al. 2013). Modification of characteristics of microorganisms by
genetic manipulation can increase the availability of major nutrients in the soil.
Enhanced Nitrogen Fixation Nitrogen is a major component in building up of

amino acids and proteins and usually is the most limiting nutrient. Nitrogen (N2)
fixation is the ability to fix atmospheric nitrogen and supply it in a usable form to
the host plant. The process of conversion of atmospheric nitrogen into available
forms that can be assimilated by plants is a unique process driven by prokaryotes.
Some nitrogen-fixing organisms such as Azospirillum (associative N2 fixer) and
Azotobacter (free-living N2 fixer), commonly associated with cereals in temperate
zones, are able to improve cereal crop yields and are used as a biofertilizer for vari-
ous crops. The nitrogen-fixing capability of microorganisms depends on the pres-
ence of nif genes which codes for nitrogenase enzyme. Nitrogenase enzyme is the
key enzyme in nitrogen fixation which provides the site for reduction of dinitrogen
to ammonia (available form). Genetic modification of microorganism by transfer-
ring the whole set of genes is required for the expression and functioning of nitro-
genase enzyme which lead to release of significant amount of ammonia into
medium, and hence, these modified microorganisms act as biofertilizers.
Transformation of Pseudomonas protegens Pf-5 with genes encoding the nitroge-
nase of Pseudomonas stutzeri A1501 via the X940 cosmid is an example of increase
in the biofertilizing capacity. The transformed strain showed high nitrogenase activ-
ity and released significant quantities of ammonium in medium. Similar to
Pseudomonas protegens Pf-5, Pseudomonas putida, Pseudomonas veronii, and
Pseudomonas taetrolens can be transformed with same cosmid which can cause the
constitutive expression of nitrogenase activity and high ammonium production.
Inoculation of Arabidopsis, alfalfa, tall fescue, and maize with Pf-5 X940 enhanced
the soil ammonium concentration and plant productivity under nitrogen-deficient
conditions (Setten et al. 2013).
Root-nodulating bacteria should have high competitiveness which is very critical

for the successful use of rhizobial inoculants. Therefore, it is desirable that the inoc-
ulant strain be genetically modified to ensure that it will occupy a sufficient number
of root nodules in the plant host. Nodulation competitiveness of several strains can
be enhanced by genetic manipulation. This genetic manipulation consists of the
modified expression of the regulatory nifA gene. The expression of nif genes, nod
genes, and ntr operon in the microorganism which regulates nitrogen fixation, nitro-
gen uptake, and root nodulation, respectively, is regulated by Nif A gene (Germaine
et al. 2013).
Enhanced Phosphorus Solubilization Phosphorus is the second major nutrient

for crop plants which is present in soil in relatively larger amounts but in unavail-
able form. The release of microbial enzymes (phosphatases and phytases) and acids
(formic acid, gluconic acid, succinic acid, etc.) causes breakdown of metal-bound
insoluble phosphorus into plant available form. Molecular techniques are advanta-
geous approach for obtaining and characterizing improved PSB strains (Igual et al.
2001). The direct oxidation of glucose to gluconic acid in the periplasmic space of
gram-negative bacteria is catalyzed by quinoprotein glucose dehydrogenase (GDH).
The genes for glucose dehydrogenase (gcd) along with glutamine synthetase (glnA)
and phosphate transport system (pts) gene promoters have been mobilized into
indigenous microorganism. The expression of these genes in transformed strains
improved the biofertilizing potential in terms of mineral phosphate solubilization
and plant growth-promoting activity with a small decrease in nitrogen fixation abil-
ity (Sashidhar and Rao 2009).
The metabolic pathways present in microorganisms have direct and indirect rela-
tions with other metabolic pathways. The little variation in one pathway can improve
or diminish the production of other metabolites. The phosphorus-solubilizing activ-
ity and the production of the auxin indole-3-acetic acid (IAA) were co-expressed in
some of the microorganisms although a direct correlation linking IAA production to
P solubilization is not established. It has been reported that overproduction of IAA
(with insertion of ipcC/ipcD gene) triggered the TCA cycle enzymes and caused
increase in the excretion of malic, succinic, and fumaric acids (TCA cycle interme-
diates) which resulted in positive effects in both P-sufficient and P-limiting condi-
tions (Bianco and Defez 2010). The presence of soil microorganisms causes
enhanced utilization of inositol phosphate by plants (Richardson et al. 2001). It was
observed that transformation of pyrroloquinoline quinone (PQQ) synthesizing
genes into bacterial strains can increase the efficiency to solubilize more amount of
insoluble phosphate as compared to parental strains (Rodriguez 2000). Hence, tar-
geting the specific enzyme or biochemical pathway to genetically improve the
microbes results in increased functional ability and biofertilization capacity of
microorganism.
11.6.3 Improvement in Stress Tolerance Activity
The intense agricultural practices cause detrimental effects on soil ecology, soil
irrigation needs, and human health, as well as posing threat to the environment
which results in altered environmental conditions such as drought, temperature
extremes, and soil salinity. These environmental conditions induce stress in plants.
In addition to these environmental stresses, the presence of pathogenic microorgan-
ism/plant pests also induces stress condition in plants. The efficiency of plant growth
and productivity decreases in stress conditions as most of the plant metabolism is
focused to de-stress. Therefore, environmentally amiable approaches have to be
employed to maintain proper growth in such adverse conditions. Microorganisms
can provide alternative approach to cope the situation by reducing damage caused
by environmental or pathogenic stress. So, basically stress can be of two types:
1. Abiotic stress
2. Biotic stress
11.6.3.1 Abiotic Stress Tolerance

Abiotic stress is induced in plants under the harsh and unfavorable environmental
conditions. This stress affects plants throughout their life span, and plants have to
develop mechanisms for increased survival in the presence of these stresses. The
environmental changes such as extreme temperature, flooding, drought, freezing,
salinity, strong light, change in pH, exposure to radiation, and heavy metals signifi-
cantly affect the agriculture. Such adverse environmental conditions exert negative
impact on crop productivity and pose a major problem for food security especially in
tropical regions. So, to cope with the problem, there is an utmost need to develop the
environmental friendly and cost-effective techniques. It involves the utilization of
multifunctional microorganisms with an established role in stress management (pro-
vides induced systemic resistance to the plant by microbial growth). The stress toler-
ance response can be alleviated by inoculating crop seeds and seedlings with plant
growth-promoting bacteria (PGPB). The microorganisms have to continually develop
a complex stress tolerance system to survive with the changes in their external envi-
ronment. Genetic engineering techniques help in improving stress tolerance potential
of microbes in accordance to changing environment. It is believed that stress-induced
responses are directly related with action of auxins (growth hormone). Variation in
auxin metabolism (transport and catabolism) is induced by abiotic stresses which
may be due to alteration in the expression of auxin-encoding genes (Carmen and
Roberto 2012). The expression of PIN genes gets altered during drought or salinity
which affects the auxin transport (Potters et al. 2009). This leads to increases in free
auxin, which in turn inhibits root elongation and provides protection to plant against
stresses. Targeting PIN gene by genetic engineering techniques can increase the sur-
vival of the plant in stressed conditions. Another enzyme, catalase (encoded by Kat
gene), plays a very important role in degrading reactive oxygen species such as per-
oxides (H2O2) as well as significantly affects nodulation and nitrogen-fixing activi-
ties of root-nodulating bacteria. The root-nodulating bacteria had lower catalase
activity than the other genera of aerobic or facultative anaerobic bacteria; as a result,
they have higher vulnerability to H2O2. The gene encoding for the catalase activity
can improve the survival and nitrogen-fixing capability of root-nodulating bacteria in
stress condition due to high oxygen concentration. So, the increase of catalase activ-
ity in rhizobial cells could be a valuable way to improve the nodulation, nitrogen-
fixing ability, and biofertilizing ability in high oxygen conditions (Orikasa et al.
2010). During the stress period, 1- aminocyclopropane- 1-carboxylate (ACC) is
released by plants which is a precursor for ethylene hormone production. However,
ethylene accumulation in the roots intensifies the plant stress (Babalola et al. 2007).
Microbial degradation of ACC seems to be particularly important under stress such
as cold, drought, saline soils, or heavy metals in contaminated flooded soils. The
cloning of ACC deaminase genes and inserting them into plant-associated bacteria
resulted in increased plant growth (Davoud et al. 2010).
11.6.3.2 Biotic Stress Tolerance

Pathogenic microorganisms are a major threat to plant growth, development, and
productivity. Traditional methods such as crop rotation, breeding for resistant plant
cultivars, and application of chemical pesticides seem to be insufficient to control
plant diseases of important crops. The presence of pathogens/plant pests induces
stress in crop plants. This leads to increased use of pesticides to control the diseases
which become threat to environment. In the future, the greater reliance will be laid
on biotechnological applications including the use of microorganisms as antago-
nists to control the pathogenic microorganisms. Therefore, interest in biological
control has been increased in the past few years due to environmental concern over
the use of chemicals. Biological control is generally defined as “any practice which
can decrease the survival as well as activity of a pathogen through any other living
organism (except man) and thus reduces the stress induced by the pathogen.” After
colonizing the rhizosphere, biocontrol agents must be able to defend the nutrient-
rich sites under conditions of intense competition with indigenous/pathogenic
microorganisms. Application of biocontrol agents to suppress diseases is often
unsuccessful because of inconsistent performance under field conditions. This may
be due to variable plant root colonization by the biocontrol agent, nutrient starvation
due to competition, instability or degradation of the antibiotic, insufficient concen-
tration of the released antibiotic, and genetic diversity of the pathogen. One approach
to overcome some of these problems is to genetically modify biocontrol strains for
enhanced and/or constitutive expression of biocontrol properties.
Pseudomonas fluorescens strain genetically modified for Cry1Ac and Cry1C
genes encoding the δ-endotoxin proteins from Bacillus thuringiensis (Panetta
1993). This endotoxin persists longer in the environment when expressed inside
Pseudomonas as compared to original host B. thuringiensis. This occurred due to
the fact that after death of Pseudomonas, cell wall remained intact, whereas the
Bacillus cell wall disintegrates (Soares and Quick 1990). The production of
phenazine-1-carboxylic acid (PCA) or DAPG is an important trait in biological con-
trol agents and can lead to suppression of plant root diseases. It was reported that
genetic modification of root-colonizing bacterium Pseudomonas putida WCS358r
by insertion of phz gene encoding for PCA enhances the ecological fitness of the
plant and environment (Viebahn et al. 2003). The cloning of phlACBDE operon,
encoding 2,4 DAPG production, into a number of Pseudomonas strains, can result
into enhanced biocontrol activity against plant microbial pathogens such as
Gaeumannomyces graminis and Ralstonia solanacearum (Zhou et al. 2005).
Agrobacterium radiobacter produces an antibiotic agrocin 84 which is normally
toxic to agrobacteria carrying a nopaline/agrocinopine-type Ti plasmid, but it can
spread the plasmid by horizontal transfer method to other species. So, a mutant
designated A. radiobacter strain K1026 was constructed which cannot transfer the
modified agrocin plasmid to pathogenic agrobacteria, thereby retaining its capabil-
ity to act as a biocontrol agent (Jones et al. 1988). This strain is particularly effective
against Agrobacterium tumefaciens, the causative agent of crown gall disease of
stone fruit trees and almonds. Another mechanism to control the growth of pathogen
is to produce lytic enzymes such as chitinases, glucanases, and xylanase.
Pseudomonas putida strain 101-9 was genetically engineered to chitinase produc-
tion. It harbors the chitinase-expression vector pKAC9-p07 (degrade chitin, a major
component of fungal cell walls) which resulted in overproduction of enzyme chitin-
ase and suppressed the fungal pathogens (Ohno et al. 2011).
Genetic enhancement of biocontrol strains has mainly emphasized on the intro-
duction of new biocontrol traits into plant-associated strains. However, to be a
successful biocontrol agent, the microbe must have the ability to survive, compete,
and maintain a high population in the target crop. Future research should also focus
not only those traits involving colonization and effectiveness but also traits involved
in stress responses such as desiccation and nutrient starvation. Also the genetic
manipulation of root-associated traits to enhance establishment and proliferation
will help biocontrol agents in the plant biosphere (Singh et al. 2011).
11.6.4 Genetic Engineering to Improve Bioremediation Potential

of Microbes
Unprecedented growth of population, anthropogenic activities, and urbanization has

increased pollutants in soil to critical levels. In day-to-day life, we use thousands of
chemicals in the form of fuels, consumer products, industrial solvents, drugs, pesti-
cides, fertilizers, and food additives. Traditional methods routinely used for the
remediation of contaminated environmental soil include excavation, transport to
specialized landfills, incineration, stabilization, and vitrification. Much interest is
growing to use microorganism in bioremediation technologies to degrade toxic con-
taminants in environmental soil into less-toxic and/or nontoxic substances. The
microorganisms are particularly effective for degradation of pollutants. Metabolic
potential of microorganisms provide an effective mechanism for eliminating envi-
ronmental pollutants. Using biotechnological techniques, plant-associated bacteria
(rhizospheric and/or endophytic) can be engineered to produce specific enzymes
which are capable of degrading toxic organic pollutants present in the environment.
Genetic engineering of endophytic and rhizospheric bacteria is considered one of the
Table 11.2 Genetically modified microorganisms for degradation of organic compounds

Target gene for
Microorganism modification Contaminant References
Pseudomonas sp. dmpN Phenol Selvaratnam
et al. (1997)
Escherichia coli AtzA Atrazine Atrazine Strong et al.
chlorohydrolase (2000)
Pseudomonas fluorescens luxCDABE Naphthalene Sayler and Ripp
HK44 (2000)
Cycloclasticus sp. phnA1, phnA2, PAH Kasai et al.
phnA3, and phnA4 (2002)
Pseudomonas sp., Bordetella nccA Nickel, cobalt, Abou-shanab
sp. cadmium et al. (2003)
Burkholderia cepacia L.S.2.4 pTOD plasmid Toluene Barac et al.
(2004)
Pseudomonas fluorescens Operon bph, gfp Chlorinated biphenyls Boldt et al.
F113rifpcbrrnBPI::GFP- (2004)
MUT3
Pseudomonas putida KT2442 pNF142 plasmid, Naphthalene Filnov et al.
gfp (2005)
Burkholderia cepacia VM1468 Toluene Taghavi et al.
VM1468 pTOM-Bu61 (2005)
plasmid
Rhodococcus sp. fcbABC operon 2(4)-chlorobenzoate Rodrigues et al.
RHA1 2(4)-chlorobiphenyl (2006)
Staphylococcus aureus chrB Chromate Aguilar-barajas
et al. (2008)
Escherichia coli JM109 Escherichia coli Decolorize azo dyes, Jin et al. (2009)
JM109 C.i. direct blue 71
Pseudomonas putida pDH5 plasmid 4-chlorobenzoic acid
PaW340(pDH5)
Bacillus subtilis, Bacillus czcD Cobalt, zinc, cadmium
cereus
Pseudomonas aeruginosa merA, merB Organic and inorganic Dash and Das
mercury (2012)
most promising new technologies for remediation of contaminated environmental

sites (Dzantor 2007). In order to increase the bioremediation potential and/or meta-
bolic activity of any microorganism, the insertion of certain functional genes into
their genome is necessary. This phenomenon can be achieved by the insertion of new
genes into the genomic complexion, insertion of new plasmid, alteration of meta-
bolic pathways, and most importantly adaption of features toward the environmental
conditions. Due to significant developments in the field of microbial biotechnology,
there are numerous developments in genetically engineered microbes for bioreme-
diation of toxic substances. The examples of selected GMMs degrading toxic organic
compounds are listed in Table 11.2.
Improvement in existing catabolic pathways or extension of these pathways can

be targeted to degrade some compounds which are not possible to degrade by using
wild strain. The whole catabolic pathway may be encoded by a single microorgan-
ism or by a consortium of microorganisms, each performing one or more of the
stages of bioremediation of xenobiotics. In this way, genetically modified microor-
ganisms with improved degradation capabilities can be constructed. Alteration of
gene sequences during the process of construction further improves the efficiency of
the catabolic pathways. Genetic modification of rhizospheric bacteria with the bph
operon could enable the bacteria to degrade polychlorinated biphenyls (PCBs) more
efficiently than the wild-type rhizospheric bacteria. The genetic engineered
Pseudomonas fluorescens HK44 was the first strain used for bioremediation because
of special characteristics such as the absence of antibiotic resistance gene in vector,
stable bph element, and nontransferable bph element making it more suitable for
environment. Similarly, the insertion of naphthalene gene into microorganism is
responsible for naphthalene degradation pathway (Sayler and Ripp 2000).
The enzymes mediate various metabolic pathways which are produced by the
transcription and translation of specific genes. Microorganisms can be developed by
hybrid gene clusters which alter the activity and substrate specificities of enzyme.
Deinococcus radiodurans, the most radio-resistant organism, has been modified
genetically to consume and digest toluene and the ionic form of mercury from
nuclear wastes. A recombinant Rhizobium tropici strain expressing 1,9-α-dioxygenase
enzyme was used for the degradation of dioxine-like compounds. Similarly various
other degradative enzymes such as orthomonooxygenase are cloned for degradation
of toluene in rhizospheric region of soil.
Genetic engineering offers a great scope for the use of natural ability of bacteria
in construction of GMMs. Unfortunately, they are applicable mainly in laboratory
conditions. The new approach to use plant-associated endophytic bacteria seems to
be a very promising solution in remediation of contaminated areas. However, this
field of study requires still much work in laboratory scale.
11.7 Achievements
Some examples of currently use genetically engineered microbes are listed below:
1. The first commercially available genetically modified biocontrol agent was a

modified strain of Agrobacterium radiobacter strain K84 which has been mar-
keted in Australia since 1989. This strain is used as a means of controlling
Agrobacterium tumefaciens, the causative agent of crown gall disease which
affects stone fruit trees and almonds.
2. Agrobacterium radiobacter K1026 (Jones and Kerr 1989) is a Tra- (transfer neg-
ative) derivative of A. radiobacter K84 which is a naturally occurring bacterium
effective against crown gall. This mutant designated A. radiobacter strain K1026
was constructed so that it can no longer transfer the modified agrocin plasmid to
pathogenic agrobacteria, thereby retaining its effectiveness as a biocontrol agent
(Jones et al. 1988). This strain is commercially available under the trade name
“NoGall” and is also a registered biocontrol agent in the United States.
3. Biocontrol microbe that has been genetically modified and is commercially
available is a Pseudomonas fluorescens strain. The Cry1Ac and Cry1C genes
encoding the δ-endotoxin proteins from Bacillus thuringiensis were transferred
into this Pseudomonas strain (Panetta 1993). This product is sold under the trade
name “MVP bioinsecticide” and used to reduce crop damage from the diamond-
back moth (Plutella xylostella).
4. There was a transient commercialization of Sinorhizobium (Rhizobium) meliloti
RMSPC-2 (EPA 1998) as seed inoculants for alfalfa. The strain had genes to
enhance nitrogen fixation and nutrient utilization, as well as an antibiotic resis-
tant marker gene.
5. The commercial transfer of the Bt delta endotoxin gene to the endophyte

Clavibacter xyli for control of the corn earworm (Tomasino et al. 1995) lost to
competition with the development of Bt genes transformed as integral parts of
the plant cell.
6. P. fluorescens HK44 contained pUTK21 plasmid, the genes responsible for
naphthalene degradation pathway. Pseudomonas fluorescens HK44 was the first
strain used in the field experiment because of special characteristics such as the
absence of antibiotic resistance gene in vector, stable bph element, and nontrans-
ferable bph element making it more suitable for environment.
11.8 Future
Genetic engineering is used to modify microorganisms capable to carry out unique

processes. This becomes popular way of increasing the efficiency of microorganism
in laboratory studies. Techniques which are used for improvement of microbial
strains include engineering with single genes, alteration of the sequences of existing
genes (both coding and controlling sequences), and pathway construction. But, the
researchers should emphasize the ethical responsibilities before releasing a geneti-
cally modified microorganism into the environment.
The ethical problem raised by genetically modified microorganisms (GMMs) is
mainly related to the impact they have on the Earth’s biosphere. As microorganisms
can be found everywhere, including in the ecological niches that are the most
unsuited to life forms, the repercussions can be substantial and irreversible. The
danger posed by these genetically modified organisms is therefore related both to
their dispersal into the environment and to their potential for adaptation to a new
environment. So, genetically modified microorganisms can alter the animal and
plant microbial ecological balance hence disturb the environment to a greater or
lesser extent. However, they may potentially transfer their modified genetic material
to other organisms. This will lead to the appearance of new variants which may have
the capability to significantly disrupt the environment. The studies carried out so far
have shown that following appropriate regulations, genetically modified microor-
ganisms can be applied safely in agriculture. Nowadays, the application and release
of genetically engineered bacteria directly in the environment are not accepted by

the public and government. One reason behind this rejection is the presence of
antibiotic resistant genes in transgenic strains which have been introduced in the
bacteria during the allelic replacement process. Another reason may be the impact
of recombinant microorganism on the indigenous soil microbial communities.
Therefore, it is very difficult to predict the influence on environment which may be
generated by the dispersal of genetically modified microorganisms. Several studies
related to gene transfer will be necessary over the coming years in order to give an
accurate assessment of the risks and consequently to develop GMMs which do not
pose a risk to humans and their environment.
However, in view of the real technical difficulties relating to the detection of
these microorganisms, their unlawful use, in particular by industrialists, is a cause
for concern. As regards fundamental research on microorganisms which gives rise
to new modified strains, the question of the real need for such research must be
considered in the same way as with all research in all disciplines.
11.9 Conclusion
Microbes have immense potential to contribute to sustainable agriculture. Microbial

inoculants are widely used for plant growth promotion, biocontrol, or bioremedia-
tion, but the results are inconsistent with low levels of success in varying field con-
ditions. Genetic engineering paves a novel way to increase the reliability and
performance of microbes to accomplish the particular target, but this will require
further discovery of novel bacterial strains, genetic improvement of microbes, and
improved inoculum delivery systems. In this context, genomic analysis and genetic
engineering are helpful for obtaining improved bioformulations of promising ben-
eficial microbes. This strategy will enable us to save considerable amounts of agro-
chemicals, especially chemical fertilizers and chemical pesticides, and hence our
environment.
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Stress Signalling
in the Phytomicrobiome: Breadth 12
and Potential
Sahana Basu and Gautam Kumar
Abstract
Higher plants continually compete with microbes to conserve their predominance
within the particular niche. Plants have evolved constant relationships with a suite
of microbes, known as the phytomicrobiome. The associates of phytomicrobiome
exhibit symbiotic relationship. Plant-microbe and microbe-microbe interactions
within the phytomicrobiome are carried out through the release of signalling com-
pounds. Bacterial community within the phytomicrobiome communicates among
themselves through quorum-sensing mechanism. Diversity, stability and resil-
ience of microbial community in phytomicrobiome are the major determinants of
plant health and productivity. Therefore, phytomicrobiome is under intensive
investigation to improve our understanding of its strong effects on plant develop-
ment, health and resistance to parasites. Exploration of the plant-microbe interac-
tions within the phytomicrobiome is a promising avenue to improve crop
productivity and agricultural sustainability. Understanding the regulation and
relatedness of plant and microbial community may aid in engineering plants with
improved pathogen resistance and novel symbiotic interactions. Comprehensive
study of phytomicrobiome, especially the molecular signalling pathway, may pro-
vide new insights into the mechanism of plant disease management, successively
inspiring new plant breeding strategies.
Keywords
Crop · Molecular signal · Phytomicrobiome · Symbiosis · Quorum sensing
S. Basu
Department of Biotechnology, Assam University, Silchar, Assam, India
G. Kumar (*)
Department of Life Science, Central University of South Bihar, Gaya, Bihar, India

246 S. Basu and G. Kumar
12.1 Introduction: Phytomicrobiome
Phytomicrobiome is defined as the intimate, complex and subtle association between

higher plants and the community of microbial organisms (such as bacteria, fungi and
viruses), which can adhere to the external surfaces of plants or may colonize internal
plant tissues (Smith and Zhou 2014). This symbiotic association leads to the formation
of holobiont (Hartmann et al. 2014). Plant is a meta-organism having a persistent and
regulated relationship with its phytomicrobiome (Berg et al. 2013). Phytomicrobiome
includes three components—rhizomicrobiome, the community of microbes associated
with plant root (Lundberg et al. 2012); phyllomicrobiome, above ground associated
(Kembel et al. 2014); and endosphere, colonizing internal plant tissues (Berg et al.
2014). The lower group of plants have also been reported to form complex phytomicro-
biomes, including highly specific associations (Bragina et al. 2013).
Phytomicrobiome has been estimated to be evolved about half a billion years
ago, since plants colonized the terrestrial ecosystem (Knack et al. 2015). First ter-
restrial plants supposed to have poorly developed roots, making these plants requir-
ing microbial support (Smith et al. 2015). As plants adapted to and dispersed through
diverse terrestrial environments, evolving to grow under a range of conditions, it is
probable that their associations with microbes also evolved. Higher plants and their
associated phytomicrobiome affect each other in several ways (Berendsen et al.
2012). Microbial cohort of the phytomicrobiome employs inter-organismal signal
compounds to alter the behaviour of the plants they associate with and the behaviour
of the phytomicrobiome is regulated by the signal compounds from the plants.
Thus, one organism within the phytomicrobiome alters the behaviour of another for
its own benefit but often to the benefit of the other organism as well, leading to
mutualistic symbiosis. Occasionally, the symbioses lead to the development of root
nodules (Riely et al. 2006). Several symbioses require specific signalling pathways
for intracellular accommodation of microbes (endosymbioses). Analysis of plant-
microbe interactions within the phytomicrobiome can be exploited to (1) develop
new approaches to crop growth promotion and (2) develop novel and more consis-
tent biocontrol mechanisms for field crops (East 2013).
12.2 Different Types of Plant-Microbe Interactions
As plants evolved to acclimatize themselves to diverse terrestrial environments, the

microbial community associated with the plants also evolved.
12.2.1 Mycorrhizal Symbiosis
Mycorrhiza (Greek ‘mykos’, fungus and ‘rhiza’, root) is a symbiotic association

between roots of vascular plant and fungus (Smith and Read 2008). The term mycor-
rhiza refers to the intracellular (arbuscular mycorrhizal fungi) or extracellular (ecto-
mycorrhizal fungi) fungal colonization in the plants’ rhizosphere (root system).
12 Stress Signalling in the Phytomicrobiome: Breadth and Potential 247
Arbuscular mycorrhizae form symbioses with more than 80% of the angiospermic
plant species (Newman and Reddell 1987). Mycorrhizal fungi have existed more
than 460 million years ago, since the first terrestrial plants appeared on this planet
(Redeker et al. 2000). Fossil endomycorrhizal association occurred in the early
Devonian period, demonstrating the association of plant roots with fungal elements
of the rhizomicrobiome (Bonfante and Genre 2008).
Mycorrhizal fungi form an obligate symbiotic association with the host plants
and assist the plants with the acquisition of essential mineral nutrients, particularly
phosphorus (Smith et al. 2003). They form a network of fine filaments associated
with plant roots to draw water and nutrients from the soil that the root system would
not be able to access. Mycorrhizae play important roles in soil aggregation through
hyphae networking (Rillig et al. 2003). The plant provides carbohydrates and other
nutrients to the fungi. The carbohydrates are being utilized by the fungi for their
growth and synthesis of glomalin (glycoprotein) molecules. These molecules result
in better soil structure and increase soil organic matter content (Rillig et al. 2003).
Arbuscular mycorrhizae also protect the host plants from herbivores and different
environmental stresses including drought and salinity (Brem and Leuchtmann 2002;
Miransari 2009; Liu et al. 2017).
There are two major groups of mycorrhizal fungi:
1. Endomycorrhizal fungi
2. Ectomycorrhizal fungi
12.2.1.1 Endomycorrhizal Fungi

Members of this group of fungi penetrate the plant cells where direct metabolic
exchanges can occur. These fungi colonize trees, shrubs and herbaceous plants but
do not form visible structures. Arbuscular mycorrhizal (AM) fungi are the most
predominant endomycorrhizal fungi. They form finely branched structures called
‘arbuscules’ within the plant root cell and serve as a major metabolic exchange site
between the plant and the fungus (Harrison 2005). Sac-like vesicular structures are
also found in some species of AM fungi, emerging from hyphae, which serve as
storage organs for lipids.
12.2.1.2 Ectomycorrhizal Fungi

These fungi are essentially found on trees and develop visible structures exclusively
on the exterior of root cells without penetrating them. Their hyphae grow externally
between root cells, forming dense growth known as a fungal mantle. These fungi
form symbiotic relationships with most pines, spruces and some hardwood trees
including beech, birch, oak and willow. They also form visible reproductive struc-
tures (mushrooms) at the feet of trees they colonize.
12.2.2 Nitrogen-Fixing Microorganisms
Leguminous plants develop a symbiotic relationship with nitrogen-fixing bacteria

called rhizobia, allowing the bacteria to infect them within special structures known
as nodules that are located along their roots. The nitrogen-fixing rhizobia include
the genera Allorhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium,
Rhizobium and Sinorhizobium which enter into symbiotic associations with their
legume host plants and fix atmospheric dinitrogen inside root nodules differentiated
for this purpose (Mabood et al. 2014). Plants utilize the nitrogen fixed inside the
nodule and provide photosynthetically fixed carbon to the rhizobia. There has been
an increase in rhizobia-based commercial inoculants as biofertilizers (Vessey 2003),
and given the large quantities of fossil fuels used to produce nitrogen fertilizers and
the steep rise in fossil fuel prices over recent years, a significant expansion in the use
of biofertilizers is most likely to continue.
Besides rhizobia, there are other rhizobacteria that live and fix nitrogen outside
of formal symbioses, and these are referred to as free-living or associative nitrogen-
fixing bacteria; among them are Azospirillum, Acetobacter, Herbaspirillum,
Azoarcus and Azotobacter (Steenhoudt and Vanderleyden 2000). These rhizobacte-
ria have been getting a great deal of attention as biofertilizers as they are able to fix
nitrogen in association with nonlegume plants and can be used in marginal lands as
a low-input way to provide nutrients to crop plants (Boddey et al. 1991).
12.2.3 Cycad-Cyanobacterial Symbiosis
In all genera of cycads, in addition to normal geotropic roots, dichotomously

branched apogeotropic roots are formed. These modified perennial lateral roots are
referred to as coralloid because of their irregular, beady appearance (Chamberlain
1935). The coralloid roots are infected with endophytic cyanobacteria (blue-green
algae). These endophytic algae associated with root tissues have been described as
different species of Anabaena or Nostoc (Costa et al. 1999). The filamentous hetero-
cystous are located in the intercellular spaces of a specialised, hollow, cylindrical
zone within the cortex (Lindblad 2009). These symbiotic algae fix atmospheric
nitrogen and supply it to the host plant in the form of nitrates. The blue-green algae
in association with root tissues produce beneficial amino acids like asparagine and
citrulline from the fixed nitrogen.
12.3 Molecular Signalling in Phytomicrobiome
The composition of phytomicrobiome is influenced by the complex matrix of plant-

microbe and microbe-microbe communications, which are carried out through the
release of signalling compounds. The social network of plants with major root endo-
symbionts—rhizobia and AM fungi—is decided by the genetic composition of host
plants. The major beneficial plant-microbe associations—the rhizobium-legume
symbiosis (RLS) and arbuscular mycorrhiza (AM)—are regulated by a common set

of signalling components that act downstream of both fungal and rhizobial signal
perception and upstream of the activation of the appropriate response to either sym-
biont (Oldroyd, 2013). Secondary messengers, reactive oxygen and nitrogen species
have also been found to be associated with RLS and AM signalling, but their actual
role in relation to the common symbiotic pathway is unclear (Calcagno et al. 2012;
Zhang et al. 2013).
12.3.1 Signalling in the Legume-Rhizobia and Mycorrhizal

Symbioses: The Common Symbiotic Pathway
The gene regulation of common symbiotic pathway (CSP) has been extensively
studied in Medicago truncatula and Lotus japonicus (Kevei et al. 2007; Shimoda
et al. 2012). Early development of rhizobial symbiosis occurs through a chemical
communication between the plant and rhizobium, present in the rhizosphere. Host
plant roots release flavonoids as a signal to rhizobia. Rhizobia produce nodulation
factors (Nod factors) that are recognized by the host plant to activate a CSP required
for mycorrhizal and rhizobial symbioses (Fig. 12.1).
Fig. 12.1 Common signalling pathway in root-nodule symbiosis and arbuscular mycorrhiza (AM)
The first set of CSP proteins includes membrane-bound two LysM (lysin motives)
receptor kinases NFR1/LYK3 (nod factor receptor 1/ lysin motif receptor-like kinase
3), NFR5/NFP (nod factor receptor5/ nod factor perception), a leucine-rich receptor
kinase DMI2/SYMRK (does not make infections2/symbiosis receptor kinase) and the
enzyme HMGR1 (HMGR1, 3-hydroxy-3-methylglutaryl-coenzyme A reductase1)
(Madsen et al. 2003; Kevei et al. 2007; Lefebvre et al. 2010). NFR1/LYK3, a new com-
mon symbiotic gene 13, has also been found to be involved in mycorrhizal symbiosis.
Signal transduction pathways are triggered by Nod and Myc factors in legume root
cells. Nod- and contact-dependent Myc factors are recognized at the cell surface by
receptor-like kinases (NFR1/LYK3, NFR5/NFP and SYMRK/DMI2). As a conse-
quence of HMGR1 activation, mevalonate production can also be localized in the
vicinity of the cytoplasmic face of the plasma membrane (Venkateshwaran et al. 2015).
A second cluster of CSP proteins is located on the nuclear membrane, comprising
two cation channels (Castor and Pollux) and three nucleoporins (NENA, NUP85 and
NUP133) at the core of the nuclear pore (Saito et al. 2007; Charpentier et al. 2008;
Groth et al. 2010). These components are required for Nod factor-induced calcium
oscillations (Oldroyd 2013). ATP-powered Ca2+ pump MCA8 is also bound to the
nuclear envelope (Capoen et al. 2011). Intense oscillations in nuclear Ca2+ concentra-
tion (spiking) are observed during both AM and RLS establishment (Chabaud et al.
2016; Sieberer et al. 2012). Protein phosphorylation leads to Ca2+ influxes into the
cytoplasm and Ca2+ spiking in the perinuclear region, mediated by Castor/Pollux and
NUP gene products. Some unidentified channels have been assumed to release Ca2+
from the nuclear envelope lumen, which is sustained by the opposite flow of potas-
sium ions (K+) through Castor/Pollux (Venkateshwaran et al. 2012). MCA8 activity
contributes to the restoration of basic nuclear Ca2+ concentration. Cytoplasmic Ca2+
oscillation has also been found to activate mycorrhiza-specific calcium-dependent
protein kinases (MSCaPKs), which activate specific transcription factors (TF2)
involved in the regulation of gene expression in AM (Lambais 2006).
The last group of CSP proteins are located in the nucleoplasm. Calcium oscilla-
tions are perceived through a nuclear-localized calcium and calmodulin-dependent
protein kinase (CCaMK, known as DMI3 in M. truncatula) (Shimoda et al. 2012;
Poovaiah et al. 2013). Activation of CCaMK induces symbiotic processes and initi-
ates nodule organogenesis (Tirichine et al. 2006; Takeda et al. 2012). The enzyme,
CCaMK, associates with and phosphorylates CYCLOPS (known as IPD3 in M.
truncatula), indispensible for rhizobial and mycorrhizal colonization (Yano et al.
2008). Phosphorylated CYCLOPS regulates gene expression either directly, like in
the NIN promoter (Nodule Inception) and induce nodulation in the absence of rhi-
zobia (Singh et al. 2014), or through other transcription factors like NSP1 (nodula-
tion signalling pathway 1), NSP2 (nodulation signalling pathway 2) and RAM1
(required for arbuscular mycorrhization 1) (Oldroyd, 2013).
DELLA proteins have been reported to promote nodule development and infec-
tion thread formation during root nodule symbiosis (Jin et al. 2016). These proteins
can promote CCaMK–IPD3/CYCLOPS complex formation and increase the
phosphorylation of IPD3/CYCLOPS. DELLAs can form a protein complex with

NSP2–NSP1 and are able to bridge a protein complex containing IPD3/CYCLOPS
and NSP2. These particular proteins represent a missing link in the common symbi-
otic signalling pathway required for both rhizobial and mycorrhizal symbioses.
12.3.2 Communication Among Bacterial Community
Bacterial community within the phytomicrobiome communicates among them-

selves through a molecular signalling pathway, known as quorum sensing (QS).
This mechanism is essential for within-species communication as well as for the
crosstalk between species which defines the synergistic or antagonistic relation-
ship of bacteria with their host plants (Straight and Kolter 2009). The QS signals in
the phytomicrobiome can trigger immune responses and change the hormone pro-
files in plants, leading to growth responses and disease resistance (Hartmann and
Schikora 2012; Hartmann et al. 2014). The QS mechanism regulates distinct
microbial activities, including mobility, biofilm formation, virulence, symbiosis,
antibiotic production and conjugation (Hartmann et al. 2009). Biofilm formation
allows bacteria to adhere to host tissues, improves plant growth and root prolifera-
tion (Azospirillum in wheat) and acts as a biocontrol agent (Bacillus subtilis)
(Farrar et al. 2014).
Plant-associated bacteria produce and utilize diffusible QS molecules (e.g.
N-acyl homoserine lactones, AHLs) to signal to each other and to regulate their
gene expression (Berendsen et al. 2012). Bacterial AHLs have been shown to affect
the root development of Arabidopsis (Ortiz-Castro et al. 2008) and have been sug-
gested to elicit induced systemic resistance, which allows the plants to resist patho-
gen attacks. Quorum-sensing circuits of plant-associated bacteria contain
homologues of two Vibrio fischeri regulatory proteins—LuxI and LuxR. The LuxI-
like proteins are responsible for the biosynthesis of AHLs that act as autoinducer
and increase with the increasing cell-population density. At high concentrations,
AHL binds to cognate cytoplasmic LuxR-like proteins and activates target gene
transcription (Zhu and Winans 2001). Plant can also exploit this microbial commu-
nication system to manipulate gene expression in their associated microbial com-
munities. LuxR-like proteins in plant-associated bacteria are stimulated by plant
signals (Ferluga and Venturi, 2009). Some of the bacteria have the capacity to
quench signals by degrading various plant and microbe-produced compounds in the
rhizosphere that can negatively affect plants (Quiza et al. 2015).
12.4 Phytomicrobiome Signalling and Plant Growth
Microbial community has been found as one of the significant contributors for plant
growth (Schmidt et al. 2014). The commonly recognized mechanisms of plant
growth promotion are as follows:
1. Biofertilization
2. Phytohormone production
3. Biocontrol for disease suppression
4. Volatile signal compound production for disease control
5. Induction of plant disease resistance to phytopathogens
12.4.1 Biofertilization
Biofertilizer is the substance containing living microorganisms that promotes

growth of host plants by increasing the availability of primary nutrients (Vessey
2003). When biofertilizer is applied to plant surfaces or soil, the microorganisms
colonize the rhizosphere of the plant. Plant growth-promoting rhizobacteria (PGPR)
act as biofertilizers and protect them from various forms of abiotic stress and soil-
borne diseases (Yang et al. 2009). PGPR play a major role in improving plant health
by (1) increasing nutrient availability for plants, (2) biological nitrogen fixation and
(3) enhancing symbioses (Mabood et al. 2014). Nitrogen-fixing symbiotic rhizobia
and nitrogen-fixing bacteria (free-living or associative) are the most commonly used
bacterial biofertilizers. PGPR also stimulate plant growth by solubilizing plant
growth-promoting nutrients, making it substantially more available to plants. For
instance, phosphate-solubilizing bacteria mobilize phosphorus that is one of the
essential nutrients in plant growth (Kim et al. 1998; Rodriguez and Fraga 1999).
Some rhizobacteria produce siderophores that enhance iron availability to plants
(Bloemberg and Lugtenberg 2001). Several other bacteria play a significant role in
micronutrient availability to plants (Fasim et al. 2002).
12.4.2 Phytohormone Production
Phytohormones play a significant role in regulating the physiology and growth of

crop plants. Therefore, the production of phytohormones by PGPR has extreme
agricultural importance. Production of phytohormone—indole-3-acetic acid (IAA)
by rhizobacteria—causes plant growth promotion. Selected strains of Pseudomonas
spp. have been found to promote plant growth and drive developmental plasticity
in plant roots by producing IAA (Zamioudis et al. 2013). PGPR also produce cyto-
kinins, important in the control of cell division, chloroplast development and bud
formation (Serdyuk et al. 1995). A range of bacterial species including Proteus
mirabilis, P. vulgaris, Klebsiella pneumoniae, Bacillus megaterium, B. cereus and
Azospirillum spp. have been reported to produce IAA, gibberellin, cytokinin
(zeatin) and abscisic acid (Karadeniz et al. 2006). Rhizobium leguminosarum
strains promote early plant growth and development of canola and lettuce by pro-
ducing IAA and cytokinins (Noel et al. 1996). Rhizobacteria can also regulate
ethylene biosynthesis in plants. Some rhizobacteria produce the enzyme
1-aminocyclopropane-1-carboxylate (ACC) deaminase that lowers ethylene level
in plants by cleaving and hydrolysing plant-produced ACC. Thus, the plants
become more resistant to a wide variety of environmental stresses (Glick 2005).

Moreover, PGPR produce salicylates, which are able to induce systemic acquired
resistance in colonized plants (Ryals et al. 1996).
12.4.3 Biocontrol for Disease Suppression
Plant growth and development are also enhanced by PGPR. These rhizobacteria
control harmful microorganisms through a range of complex mechanisms.
1 . Production of broad-spectrum antibiotics

2. Production of narrow-spectrum bacteriocins
3. Production of extracellular lytic enzymes
12.4.3.1 Production of Broad-Spectrum Antibiotics

Antibiotics are compounds recognized by the host plant that activate an immune
response via high-affinity cell surface pattern-recognition receptors (Dang et al.
2013). The role of antibiosis in rhizobacteria-mediated control of plant pathogens
has been an area of extensive research during the past several decades (Whipps
2001). Pseudomonads produce a wide range of antibiotics including
2,4-diacetylphloroglucinol (DAPG), amphisin, oomycin A, phenazine, hydrogen
cyanide, pyoluteorin, pyrrolnitrin and cyclic lipopeptides (Raaijmakers et al.
2002; de Souza et al. 2003; Nielsen and Sorensen 2003). Besides pseudomonads,
Streptomyces, Bacillus and Stenotrophomonas spp. have also been reported to
produce antibiotic substances, such as oligomycin A, zwittermicin A, kanosamine
and xanthobaccin (Milner et al. 1995, 1996; Kim et al. 1999; Nakayama et al.
1999). Antibiotics synthesis by bacteria is affected by several factors, like carbon
source, pH of the growth medium, growth temperature and availability of trace
elements (Ownley et al. 1992; Duffy and Defago 1997). The presence of particu-
lar nutrient types and the age of the host plants also affect the biosynthesis of
antibiotics by selected bacterial strain (Picard et al. 2000). The addition of glu-
cose, as a carbon source to P. fluorescens, stimulates the biosynthesis of DAPG,
while it inhibits the biosynthesis of pyoluteorin, a dominant antimicrobial com-
pound (Duffy and Defago 1999).
12.4.3.2 Production of Narrow-Spectrum Bacteriocins

Bacteriocins are proteinaceous antimicrobial peptides, produced by some rhizobac-
teria, which show bactericidal or bacteriostatic effects against bacteria closely
related to the producer strain (Jack et al. 1995). Recently, it has been shown that
Bacillus thuringiensis produces some bacteriocins, like nisin, thuricin 17 (T17) and
bacthuricin F4 (BF4) that show antimicrobial activity against a broad range of
closely related bacterial species (Jung et al. 2011).
12.4.3.3 Production of Extracellular Lytic Enzymes

Biocontrol of plant pathogens is also employed through the production of extracel-
lular lytic enzymes. These cell wall-degrading enzymes exert antifungal activities,
contributing to the biocontol activity of the rhizobacteria. The enzymes include chi-
tinases, glucanases, cellulases and proteases that cause lysis and degradation of the
fungal cell wall. Some actinomycete isolates have been reported to cause lysis of
Phytophthora cell walls by hydrolysing cell wall glucans. Cell wall glucan is hydro-
lysed by producing ß-1,3-glucanase, ß-1,4-glucanase and ß-1,6-glucanase. Thus,
causing Phytophthora fragariae var. rubi, a pathogen causing raspberry root rot is
being controlled (Valois et al. 1996). Chitinase-producing bacteria like Enterobacter
agglomerans (Chernin et al. 1995), Bacillus cereus (Pleban et al. 1997) and
Paenibacillus illinoisensis (Jung et al. 2003) have decreased the incidence of dis-
ease symptoms caused by Rhizoctonia solani in cotton and cucumber. Paenibacillus
illinoisensis also controls the blight disease in pepper (Capsicum annuum L.) caused
by Phytophthora capsici (Jung et al. 2005). Extracellular proteases are also impor-
tant biocontrol agents. Proteases produced by Stenotrophomonas maltophilia W81
are involved in the biocontrol of Pythium ultimum in sugar beet (Dunne et al. 1997).
Micromonospora carbonacea produces cellulase and shows biocontrol activity
against Phytophthora cinnamomi, the causal organism of root rot in Banksia gran-
dis (El-Tarabily et al. 1996).
Lytic enzymes produced by rhizobacteria are of great importance in the biologi-
cal control of fungal plant pathogens. Paenibacillus sp. 300 and Streptomyces sp.
385 produce chitinase and ß-1,3-glucanase, which play a crucial role in the biologi-
cal control of Fusarium oxysporum f. sp. cucumerinum causing Fusarium wilt of
cucumber (Cucumis sativus) (Singh et al. 1999). Pseudomonas cepacia also pro-
duces ß-1,3 glucanase that lyses fungal cell walls and decreases the incidence of
diseases caused by Rhizoctonia solani, Sclerotium rolfsii and Pythium ultimum
(Fridlender et al. 1993).
12.4.4 Volatile Signal Compound Production for Disease Control
Volatile signal molecules such as methyl salicylate, methyl jasmonate (MeJA) and
ethylene play a crucial role in plant communication. These compounds are released
at very low concentrations by plants and act as signals to neighbouring plants
(Baldwin et al. 2006). Some of the PGPR strains have also been found to communi-
cate with plants by producing volatile compounds that are responsible for plant
growth and systemic resistance (Ryu et al. 2003, 2004). The PGPR strains Bacillus
subtilis GB03 and Bacillus amyloliquefaciens IN937a release two volatile com-
pounds, 3-hydroxy-2-butanone (acetoin) and 2,3-butanediol (2,3-B), which pro-
mote growth of Arabidopsis plants (Ping and Boland 2004).
12.4.5 Induction of Plant Disease Resistance to Phytopathogens
Growth and yield of plants are affected by several biotic stresses, including fungal,
bacterial or viral infections. Plants activate diverse defensive pathways to encounter
the deleterious effect of stress.
12.4.5.1 Systemic Acquired Resistance

Systemic acquired resistance (SAR) refers to a distinct signal transduction pathway
that plays an important role in the ability of plants to defend themselves against patho-
gens. Activation of SAR pathway is associated with numerous cellular defence
responses, such as synthesis of PR (pathogenesis-related) proteins, phytoalexins, accu-
mulation of active oxygen species and rapid alterations in cell wall, which result in the
development of a broad-spectrum, systemic resistance (Ryals et al. 1996). Salicylic
acid (SA) plays a central role in SAR and its induction of pathogenesis-related proteins
in plants (Gaffney et al. 1993). Several rhizobacteria have been described to synthesize
SA under iron-limiting conditions, triggering the SAR pathway and inducing systemic
resistance in plants (Press et al. 1997). SA produced by Pseudomonas aeruginosa
7NSK2 has been reported to activate SAR pathway in bean plants (De Meyer et al.
1999). The SAR pathway acts as a modulator of disease resistance mechanisms by
converting a compatible plant-pathogen interaction into an incompatible one.
12.4.5.2 Induced Systemic Resistance

Induced systemic resistance (ISR) is the resistance mechanism activated by biotic or
abiotic factors. ISR allows the plants to endure pathogen attack that could be lethal,
without the presence of these bacterial factors. Selected strains of PGPRs suppress
diseases by antagonism between the bacteria and soil-borne pathogens by inducing
a systemic resistance in plants (Choudhary et al. 2007). ISR protects plants that are
able to resist subsequent pathogen infections (van Loon 1997). ISR does not pro-
voke a visible hypersensitive response, as it is induced by PGPR (Wei et al. 1991).
Rhizobacteria-mediated stimulation of ISR is considered different from SAR, as
ISR is not SA dependent and involves jasmonic acid and ethylene in a signal trans-
duction cascade leading to improved disease resistance (Pieterse et al. 1998).
12.5 P
lant-Microbe Interaction for Improving the Efficiency
of Ecosystem
Abiotic stresses, including drought, extreme temperatures, soil salinity, acidity, alka-
linity and heavy metals, cause severe loss in crop yield. Response of leguminous
plants to various stresses depends on the host plant reaction, which can be influenced
by the rhizobia and the symbiotic process (Yang et al. 2009). Tolerance and nodulat-
ing capacity of Rhizobium and Bradyrhizobium to different abiotic stresses have been
elaborately studied (Grover et al. 2010). Mycorrhiza also plays a significant role in
the alleviation of soil stresses (Miransari 2010). AM fungi lead to enhanced plant
growth and production under different stress conditions (Daei et al. 2009).
12.5.1 Drought
Drought is one of the major abiotic stresses limiting plant growth and productivity
under arid and semi-arid conditions (Feng et al. 2002). Plants have acquired various
strategies for drought tolerance (Turner et al. 2000). Plants usually overexpress
zeatin for delayed leaf senescence as a drought tolerance mechanism (Xu et al.
2012). Production of extracellular polymeric substances or exo-polysaccharides
(EPS) by the soil microbes has been reported as one of the drought tolerance mecha-
nisms (Vanderlinde et al. 2010). Microbial intraspecies difference was observed
under different water conditions. Bradyrhizobium ORS 3257 was found to grow
well under favourable water conditions, while Bradyrhizobium ORS 3260 grows
well under water-stressed conditions (Krasova-Wade et al. 2006).
Mycorrhizal symbiosis also plays a significant role in alleviating drought stress.
Mycorrhizal plants can grow much better under drought conditions (Subramanian
et al. 2006). AM influence plant growth under water stress by stabilizing soil struc-
ture through the binding of soil particles with glomalin that alters soil structure and
moisture retention capability (Auge 2001). Moreover, higher uptake of nutrient,
enhancement of root surface area and dense root growth improve the drought toler-
ance of mycorrhizal plants (Subramanian et al. 2006). Under drought stress, mycor-
rhiza contributes in plant water movement, influencing hydration and physiological
processes of plants (Auge 2001). Mycorrhizal plants under stress conditions can
absorb several forms of nitrogen that are unavailable to plants, causing higher plant
growth (Subramanian et al. 2006). Under drought conditions, mycorrhizal plants
increase biomass through the higher accumulation of organic products, like proline,
glycine-betaine, carbohydrates (sucrose, mannitol) and inorganic ions (K+, Cl-)
(Ruiz-Lozano et al. 2006). AM symbiosis enhances drought tolerance of host plant
through altering plant physiology and gene expression (Ruiz-Lozano et al. 2006).
Mycorrhizal plants also produce antioxidant enzymes under drought stress that
reduce the effect of stress and sustain plant growth (Ruiz-Lozano 2003).
12.5.2 Extreme Temperatures
Agricultural cultivation is often exposed to different abiotic stresses including

extreme temperatures affecting crop productivity. High temperatures cause
enhanced transpirational water loss leading to drought stress, which results in
delay in nodulation, reduced nodule number, rhizobial growth, rate of colonization
and infection events. The optimum temperature for rhizobial growth has been
reported to be 28–31oC. Rhizobia grown under high temperature have been found
to lose their infectiveness and symbiotic properties (Hartel and Alexander 1984).
Some of the N2 fixing Rhizobium strains have been reported to be heat-tolerant
(Hungria and Franco 1993). Stress adaptation of microorganisms involves the lipo-
polysaccharide (LPS) and extracellular polymeric substances/exo-polysaccharides
(EPS) (Nandal et al. 2005). Small heat-shock proteins have also been found to play
significant roles in heat resistance of microbial associates of the phytomicrobiome
(Michiels et al. 1994; Munchbach et al. 1999). Higher induction of chaperone
genes have also been observed in heat-tolerant isolates than in heat-sensitive iso-
lates of the same species, which help the hydrophobic domains of the target protein
to regain their native structure since they get denatured upon stress (Hartl and
Hayer-Hartl 2009; Alexandre and Oliveira 2011).
Mycorrhizal fungi have been found to ameliorate temperature stress in thermo-
philic plants (Bunn et al. 2009). Elevated temperatures limit plants’ available habitat
by inhibiting their root growth. AM fungi can extend extra-radical hyphae into the
soils that increase host plants’ access to water and nutrients by extending the growth
of extra-radical hyphae into the soils.
12.5.3 Salinity
Salinity is the most detrimental abiotic stress for sustainable agricultural produc-
tion in the arid and semi-arid tropical ecosystems and accounts for about 40% of
the world’s land surface (Zhan et al. 1991). Salinity disturbs ion homeostasis of
plants and interferes with internal solute balance (Kumar et al. 2009). Salinity
affects bacterial infection process, nodule growth and biological nitrogen fixation
(Zhang et al. 1991). Rhizobial species have been found to exhibit variation in their
salt sensitivity, such as R. meliloti (Zhang et al. 1991) and R. japonicum (Yelton
et al. 1983) which are salt tolerant, whereas R. leguminosarum is salt sensitive
(Chein et al. 1992). Salinity tolerance in rhizobia is associated with the production
of compatible solutes [trehalose, N-acetyl glutaminyl glutamine amide (NAGGN)
and glutamate], osmoprotectants [betaine, glycine-betaine, proline-betaine, glu-
cans, trehalose, sucrose, ectoine, 3-dimethyl sulfoniopropionate (3-dimethylpro-
piothetin or DMSP), 2-dimethyl sulfonioacetate (2-dimethyl thetin or DMSA)] and
pipecolic acid and cations (Ca2+, K+) (Chen 2011; Streeter 2003; Sugawara et al.
2010). Salt tolerance also comprises a number of gene families, including the pro-
duction of glycine-betaine (AraC) and proline-betaine (betS/prb) (Boscari et al.
2004), sucrose (zwf) and trehalose (zwf) (Barra et al. 2003) and cation efflux
(phaA2/phaD2/phaF2/phaG2) (Jiang et al. 2004).
Arbuscular mycorrhizal fungi have significant contribution in alleviation of salt
stress. Increased salinity increases the dependency of plants on AM symbiosis indi-
cating the influence of AM to reduce the effect of salinity stress (Tian et al. 2004).
AM biologically enhance plant growth and crop production under salinity stress
(Daei et al. 2009). AM enhance salt tolerance in host plants by improving of nutrient
(N and P) uptake, improved leaf respiration and transpiration that increases the gas-
eous (carbon dioxide and water vapour) exchange through stomata and eventually
affect water use efficiency of host plants. AM also increase the concentration of
osmolytes (carbohydrates and electrolytes) in plant roots that can alleviate salinity
stress on plant (Daei et al. 2009). AM improve leaf chlorophyll content under salin-
ity stress by minimizing the inhibitory effect of Na on magnesium (Mg) absorption
photosynthesis and increase Mg uptake which is necessary for chlorophyll forma-
tion (Miransari 2009). AM also stimulate root development and enhance nutrient
uptake by increasing root hydraulic conductivity (Giri et al. 2003). AM also stabi-
lize the K+/Na+ ratio under saline conditions and increase K+ uptake resulting in
sustainable plant growth (Giri et al. 2003; Daei et al. 2009).
12.5.4 Soil Acidity
Soil acidity (low pH) is another abiotic stress that leads to crop failures by limiting
plant growth and productivity. Soil pH reduces the mobilization and availability of
nutrients to plants (Haynes 1990; Marschner 1995). High concentration of protons
in the acid soil results in the failure of rhizobia-legume symbiosis (Richardson et al.
1988). Some strains of Rhizobium, Azorhizobium and Bradyrhizobium are low pH
tolerant due to the production of extracellular polysaccharide or polyamines (gluta-
mate) concentration in the cell (Graham et al. 1994). Acid tolerant R. leguminosa-
rum bv. trifolii has been reported to accumulate higher level of potassium and
phosphorus than an acid-sensitive strain (Watkin et al. 2003).
Mycorrhizal fungi modify the pH of their root environment. For instance,
Scutellospora calospora formed detectable extra-radical mycelium at lower pH
(van Aarle et al. 2002). Hayman and Tavares (1985) have reported AM fungi Glomus
clarum to stimulate plant growth at considerably low pH (pH 4). The AM-colonized
plants grown on acidic soils have been observed to accumulate increased K, Ca and
Mg as compared to neutral or alkaline soils (Harrier and Watson 2003). Low soil pH
reduces phosphorus (P) availability for plants which is a major element for plant
growth and development. AM fungi increase host plants’ access to limiting nutri-
ents P in acid soils (Seguel et al. 2013).
12.5.5 Heavy Metals
Industrial revolution has increased the environmental pollution by the toxic metals
due to the dumping of solid wastes and use of industrial waste waters for irrigation.
Heavy metals damage plant growth and productivity. Heavy metals are non-
degradable in nature and thus often enter the food chain causing serious illness to
human beings. Bioremediation is one of the popular practices to control the heavy
metal accumulation in the soil (Gianfreda and Rao 2004). Microorganisms including
different species of Bacillus, Pseudomonas, Azotobacter, Enterobacter and
Rhizobium have been found to accelerate the process of phytoremediation (Ma et al.
2011). Increasing concentrations of heavy metals have been found to alter the expres-
sion of symbiotic nod genes in Rhizobium sp. (Stan et al. 2011). Involvement of EPS
and LPS was also found in Rhizobia for influencing heavy metal resistance by form-
ing complexes with metal ions through electrostatic interactions (Liu et al. 2001).
Mycorrhiza plays a crucial role in reducing the heavy metal concentrations of the
soil. The AM hyphae allow the fungus to absorb high levels of heavy metals from
the rhizosphere and consequently decrease its translocation from plant roots to
shoots (phytostabilization) (Leyval et al. 2002). Symbiotic association of
metallophytes (plants that can grow under heavy metals stress) with AM reduces
heavy metal uptake by the plants and improves their ability to grow under heavy
metal stress (Berreck and Haselwandter 2001). AM keep heavy metals out of plants
or reduce concentrations in plants through immobilization of heavy metals by bind-
ing with insoluble glycoprotein (glomalin) produced by AM hyphae (Hildebrandt
et al. 2007). The AM colonization with root of metallophytes leads to the expression
of metallothionein proteins producing genes that increase the heavy metal tolerance
of plants (Rivera-Becerril et al. 2005). Different species of AM, including Glomus
intraradices, are able to enhance tolerance of plants such as tomato, corn and
Medicago truncatula to heavy metal stress (Wulf et al. 2003; Hildebrandt et al.
2007). The AM symbiosis can regulate the transcription of a number of genes
involved in heavy metal tolerance, including metal transporter genes (Hildebrandt
et al. 2007). However, AM also produce antioxidant enzymes, including glutathione
S-transferase, superoxide dismutase, cytochrome P450 and thioredoxin that allevi-
ate the stress of reactive oxygen species (ROS), thus decreasing the oxidative stress
of heavy metals on plants (Hildebrandt et al. 2007). Thus, the combined effects of
bacteria and AM can enhance plant tolerance to heavy metals by promoting plant
growth (through phytohormone IAA production) and increasing AM activity in
heavy metal containing soils (Vivas et al. 2003).
12.5.6 Pesticides
Frequent application of pesticide and their slow degradation rate lead to their accu-
mulation in the soil that affects plant growth by altering morpho-physiological fea-
tures of plants. Use of pesticide beyond the recommended level reduces microbial
density on soil and also affects the growth of nitrogen-fixing bacteria (Martinez-
Toledo et al. 1996; Mathur 1999). Several microorganisms have been reported to
have the ability to degrade the pesticides due to the presence of degradative genes in
their plasmids/transposons/chromosomes (Kumar et al. 1996).
The chemical residues of several pesticides used worldwide have exceeded the
food safety standards as they create public health risk. AM fungi play a significant
role in organic uptake and translocation by plants, dissipation and degradation of
organics in soils including atrazine, PAHs (polycyclic aromatic hydrocarbon), DDT
and weathered p,p-DDE in soils (Joner et al. 2001; White et al. 2006; Wu et al.
2008; Huang et al. 2009). The extra-radical hyphae of G. intraradices have been
reported to hydrolyse 5-bromo-4-chloro-3-indolyl phosphate and phenolphthalein
diphosphate (Koide and Kabir 2000). AM colonization of Cynara cardunculus has
been found to be unaffected by phoxim (Marin et al. 2002). Thus, AM fungi contrib-
ute to enhanced biodegradation of organophosphorus pesticides in soils (Wang et al.
2011). Furthermore, AM fungi are widely used as natural fertilizers, as they provide
mineral nutrients and water to their host plants (Smith and Read 2008). AM fungal
hyphae colonize the host plant’s root cortex and form highly branched structures
that mediate the nutrient exchange (Balestrini et al. 2015). AM fungi also improve
the soil structure and aggregation (Rillig et al. 2015)
12.6 Conclusion
Phytomicrobiome is a complex, structured and dynamic community that can

improve the overall efficiency of an ecosystem by influencing plant growth, health,
productivity and functions. Therefore, symbiotic plant-microbe associations can be
successfully applied for improving the agricultural strategies to attain food security.
Exploration of novel ‘omics tools will certainly contribute in more sustainable
agriculture.
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A Simple Procedure for Isolation, Culture
of Protoplast, and Plant Regeneration 13
Indu Kumari
Abstract
This chapter presents different steps involved in isolation and culture of proto-
plast; discusses basic steps involved in the developmental sequence from proto-
plast to plant; and overviews a simple procedure for protoplast isolation, culture,
and plant regeneration. Protoplasts are naked plant cells without the cell wall, but
they possess plasma membrane and other components of cell. Protoplasts are
potentially totipotent individuals at the single-cell level. They have the potential
to regenerate a cell wall, dedifferentiate, divide mitotically and form unlimited
growing cell clones, and differentiate shoot and root meristems or embryos
which regenerate whole plant.
Keywords
Isolation · Culture · Embryos · Cell clones · Protoplast
13.1 Introduction
The protoplasts are isolated, single, and naked plant cells. Protoplast is the content
which is enclosed by plasma membrane. The term protoplast means spherical plas-
molysed content of plant cell covered by plasma membranes or naked cell without
cell wall. Protoplasts are naked plant cells without the cell wall, but they possess
plasma membrane and all other cellular components. Protoplast represents the func-
tional plant cells but for the lack of the barrier cell wall. Protoplasts of different
species can be fused to generate a hybrid and this process is known as protoplast
fusion or somatic hybridization.
I. Kumari (*)
Department of Botany, Nirmala College, Doranda, Ranchi, India

270 I. Kumari
The term protoplast was introduced by Hanstein in 1880. The first isolation of
protoplasts was achieved by Klercker employing a mechanical method. A real
research in protoplast was started by Cocking (1960). He used an enzymatic method
for the removal of cell wall. Protoplasts can be isolated directly from the different
parts of whole plant which contains parenchymatous tissue or indirectly from the
in vitro-grown plant tissue or callus tissue.
Takebe et al. (1971) regenerated complete plants from leaf protoplasts of tobacco
that increased the potential of protoplast culture methods. Several species of plants
have been regenerated by using protoplasts of different plants (Bajaj 1974; Davey
and Short 1973; Frearson et al. 1973; Grun and Chu 1978; Hess et al. 1973; Sink
and Power 1977; Wilson et al. 1980; Wallin and Eriksson 1972).
Protoplast cultures have been used in genetic engineering for the transfer of
DNA and extrachromosomal bodies like chloroplast, mitochondria, plasmids, and
nif (nitrogen fixing) genes.
13.2 Sources of Explant for Protoplast Isolation
The protoplasts can be isolated from a variety of tissues including leaves, roots, in vitro
shoot cultures, callus, cell suspension, and pollen. Among these, the mesophyll tissue
of fully expanded leaves of young plants or new shoots is used most frequently. In addi-
tion, callus and suspension cultures also serve as good sources for protoplast isolation.
Young cell suspensions are ideal for isolation of protoplasts in large quantities. Cell
suspension cultures may provide a very good source of protoplast.
13.3 T
echniques of Isolation, Culture and Regeneration
of Protoplast
The basic techniques of isolation, culture, and regeneration of protoplast are

following:
1. Sterilization of Samples: Samples like mature leaves are collected from healthy
plants which are washed in tap water to remove adhering soil particles and steril-
ized with sodium hypochlorite solution.
2. Rinsing in Suitable Osmoticum: After 10 min, sample is properly washed with
sterile distilled water or MS medium adjusted to a suitable pH and buffer to
maintain osmotic pressure. Washing should be done for about six times to remove
the traces of sodium hypochlorite.
3. Plasmolysis of Cells: The lower epidermis covered by thin wax cuticle is
removed with a forceps. Stripping should be done from midrib to margin of
lamina. The stripped surface of leaf is kept in mannitol solution (13% W/V) for
3 h to allow plasmolysis of cells.
4. Peeling of Lower Epidermis: Thereafter, leaves of plant are peeled off and used
for isolation of protoplasts.
13 A Simple Procedure for Isolation, Culture of Protoplast, and Plant Regeneration 271
Fig. 13.1 Isolated
protoplasts (source:
Bhojwani and Rajdan)
Fig. 13.2 Protoplasts obtained from Trichoderma harzianum by means of enzymatic action
(source: Bhojwani and Rajdan)
5. Isolation of Protoplast: Each plant cell is enclosed by cellulose wall which is

attached with each other by middle lamella ,made up of pectin. Protoplast is the
content which is enclosed by plasma membrane. It is essential to get isolated
protoplast, to remove the pectin material to obtain the single cells and then to
remove cell wall (Figs. 13.1 and 13.2). Protoplasts may be isolated by any one of
the two following ways: (a) mechanical method and (b) enzymatic method.
13.3.1 Mechanical Method
In mechanical method, cells are kept in a suitable plasmolyticum (in plasmolysed

cells, protoplasts shrink away from cell wall) and cut with a fine knife, so that pro-
toplasts are released from cells cut through the cell wall, when the tissue is again
deplasmolysed. Mechanical method is suitable for isolation of protoplasts from
272 I. Kumari
vacuolated cells such as onion bulbs, scales, and radish roots. The mechanical
method was used as early as 1892. This technique involves the following stages:
Step 1. A small piece of epidermis from a plant is selected.

Step 2. The cells are subjected to plasmolysis. This causes protoplasts to shrink
away from the cell walls.
Step 3. The tissue is dissected to release the protoplasts.
13.3.1.1 Limitation of Mechanical Method

Mechanical method for isolation of protoplasts is restricted to certain tissues with
vacuolated cells. This method is not suitable for isolating protoplast from meriste-
matic and less vacuolated cells. It gives poor yield of protoplasts and also the viabil-
ity of protoplasts is low. It is laborious and tedious.
13.3.2 Enzymatic Method
This method involves the use of enzymes to dissolve the cell wall for releasing pro-
toplasts. The enzymatic method is almost invariably used now for the isolation of
protoplasts. Protoplasts can be isolated from plant tissues or cultured cells by enzy-
matic digestion to remove the cell walls. It gives large quantities of protoplasts,
where cells are not broken and osmotic shrinkage is minimum. Sometimes mechan-
ical and enzymatic methods are combined, where cells are first separated mechani-
cally and later protoplast is isolated through enzymatic method. The credit of
developing high-yield protoplast isolation through enzymatic method goes to
Cocking (1960). He used crude cellulase from the fungus Myrothecium verrucaria
to dissolve cell wall and release the protoplasts from tomato roots. Later this method
with suitable modifications and using purified enzymes has been extensively used
by other scientists.
13.3.2.1 Enzymes for Protoplast Isolation

The enzymes that can digest the cell walls are required for protoplast isolation.
Chemically, the plant cell wall is mainly composed of cellulose, hemicellulose, and
pectin which can be, respectively, degraded by the enzymes cellulase, hemicellu-
lase, and pectinase. The enzymes are usually used at a pH 4.5–6.0 and temperature
25–30 °C with a wide variation in incubation period that may range from half an
hour to 20 h. The success of protoplast isolation depends especially on the condition
of the tissue and the combination of enzymes being used. After release of protoplast
into the suspension, for removal of enzymes the protoplasts are collected in centri-
fuge tube as pellet and washed several times with the osmoticum.
The enzymatic method could be used as a one-step method (direct method), or as
a two-step method (sequential method).
13.3.2.2 The One-Step Method

Protoplasts are isolated directly from the tissue by using two enzymes, cellulase and
pectinase, simultaneously.
13.3.2.3 The Two-Step Method

Cells are first isolated from callus or tissue by using pectinase and to this cell sus-
pension cellulase is added to digest the cell wall and release protoplasts.
An enzymatic solution used for protoplast isolation contains the enzymes and a
sugar as an osmoticum to prevent the plasma membrane from rupturing. Some salts
and nutrients are also used as osmoticum. Generally 50 mM CaCl2 is added to
increase the stability of released protoplasts.
13.4 Advantages of Enzymatic Method
Enzymatic method is a very widely used technique for the isolation of protoplasts.
It includes good yield of viable cells and minimal or no damage to the protoplasts.
13.5 Purification of Protoplasts
Leaf debris is removed with forceps, and enzyme solution containing protoplasts is
filtered with a steel or a nylon mesh (45 mm). The cell clumps and undigested tis-
sues can be removed by filtration. This is followed by centrifugation and washing of
the protoplasts (Gregory and Cocking, 1965; Power and Cocking, 1969; Evans et al.
1980a, b; Schenk and Hilderbrandt 1969). Filtrate is centrifuged at 75 × g for 5 min
and supernatant is decanted. Again a fresh MS medium plus 13% mannitol is added
Fig. 13.3 Protoplast purification and regeneration (source: Bhojwani and Rajdan)
274 I. Kumari
to centrifuge. The protoplasts settle to the bottom of the centrifuge tube while the
supernatant is removed with the help of a pipette. Repeated washing with nutrient
medium, centrifugation, and decantation are done for about three times. Traces of
enzyme are removed by washing the protoplasts twice or thrice with the medium
(Fig. 13.3).
13.6 Viability of Protoplasts
It is essential to ensure that the isolated protoplasts are healthy and viable so that
they are capable of undergoing sustained cell divisions and regeneration.
13.6.1 There Are Several Methods to Assess the Protoplast

Viability
• Fluorescein diacetate (FDA) staining method—Fluorescence microscopy detects

the dye accumulates inside viable protoplasts.
• Phenosafranine stain is used which is specific for dead protoplasts that show red
color, while the viable cells remain unstained.
• Exclusion of Evans blue dye by intact membranes.
• Cell wall formation can be detected by staining with 0.1% calcofluor white
(CPW) fluorescent stain. Calcofluor white (CFW) stain binds to the newly
formed cell walls which emit fluorescence.
• Oxygen uptake by protoplasts can be measured by oxygen electrode.
• The ability of protoplasts to undergo continuous mitotic divisions (direct measure).
• Photosynthetic activity of protoplasts.
13.7 Protoplast Culture and Regeneration
Protoplast regeneration which may also be regarded as protoplast development

occurs in two stages: first formation of cell wall and second development of callus/
whole plant.
13.7.1 Formation of Cell Wall
The process of cell wall formation in cultured protoplasts starts within a few hours
after isolation. From the protoplast solution of known density (about 105 protoplast/
mL) about 1 mL suspension is poured on sterile and cooled-down nutrient medium
in Petri dishes. The plates are incubated at 25 °C in a dim white light. Protoplast
develops cell wall within two to several days under suitable conditions. The process
of cell wall development requires continuous supply of nutrients, particularly car-
bon source such as sucrose.
As the cell wall development occurs, the protoplasts lose their characteristic
spherical shape. The newly developed cell wall by protoplasts can be detected by
staining with 0.1% calcofluor white (CPW) fluorescent stain, while the presence of
a proper wall is essential for a regular division. On the other hand, protoplasts with
poorly regenerated cell wall show budding and fail to undergo normal mitosis.
13.7.2 Development of Callus/Whole Plant
Protoplasts are cultured either in semisolid agar or liquid medium. Sometimes, pro-
toplasts are first allowed to develop cell wall in liquid medium, and then transferred
to agar medium. Agarose is the most frequently used agar to solidify the culture
media. In agar cultures, the protoplasts remain in a fixed position, divide, and form
cell clones. The advantage with agar culture is that clumping of protoplasts is
avoided. But liquid culture is easy to dilute and transfer. Density of the cells can be
manipulated as desired. For some plant species, the cells cannot divide in agar
medium, and therefore liquid medium is the only choice. Osmotic pressure of liquid
medium can be altered as desired.
13.7.2.1 Culture Media

The culture media for protoplast culture contains nutritional components and
osmoticum.
13.7.2.2 Nutritional Components

The nutritional requirements of protoplasts are similar to callus and suspension cul-
tures. Mostly, MS and B5 media with suitable modifications are used. The medium
for protoplast culture should be devoid of ammonium, and the quantities of iron and
zinc should be less. The concentration of calcium should be 2–4 times higher than
used for cell cultures. This is needed for membrane stability. The vitamins used for
protoplast cultures are the same as used in standard tissue culture media. Glucose is
the preferred carbon source by protoplasts although a combination of sugars such as
glucose and sucrose can be used. High auxin/kinetin ratio is suitable to induce cell
divisions while high kinetin/auxin ratio is required for regeneration.
13.7.3 Osmoticum
Osmoticum broadly refers to the reagents or chemicals that are added to increase the
osmotic pressure of a liquid. The isolation and culture of protoplasts require osmotic
protection until they develop a strong cell wall. In fact, if the freshly isolated proto-
plasts are directly added to the normal culture medium, they will burst. Thus, addi-
tion of an osmoticum is essential for both isolation and culture media of protoplast
to prevent their rupture. The osmotica are of two types—nonionic and ionic.
276 I. Kumari
13.7.3.1 Nonionic Osmotica

The nonionic substances most commonly used are soluble carbohydrates such as
mannitol, sorbitol, glucose, fructose, galactose, and sucrose. Mannitol, being meta-
bolically inert, is most frequently used.
13.7.3.2 Ionic Osmotica

Potassium chloride, calcium chloride, and magnesium phosphate are the ionic sub-
stances in use to maintain osmotic pressure. When the protoplasts are transferred to
a culture medium, the use of metabolically active osmotic stabilizers (e.g., glucose,
sucrose) along with metabolically inert osmotic stabilizers (mannitol) is advanta-
geous. As the growth of protoplasts and cell wall regeneration occurs, the metaboli-
cally active compounds are utilized, and this results in the reduced osmotic pressure
so that proper osmolarity is maintained.
13.8 Culture and Regeneration of Protoplast
As the cell wall formation around protoplasts is complete, the cells increase in size.
The protoplasts, which are capable of dividing, undergo first division within 2–7
days and form small cell colonies after 2–3 weeks. With suitable manipulations of
nutritional and physiological conditions, the cell colonies may be grown continu-
ously, and form visible colonies (macroscopic colonies). These colonies are then
transferred to an osmotic-free (mannitol- or sorbitol-free) medium for further devel-
opment to form callus. The callus can also be subcultured. With induction and
appropriate manipulations, the callus can undergo organogenic or embryo genic
differentiation to finally form the whole plant. A general view of the protoplast iso-
lation, culture, and regeneration is represented in Fig. 13.4.
Plant regeneration can be done from the callus obtained either from protoplasts
or from the culture of plant organs. There are however certain differences in these
two calluses. The callus derived from plant organs carries preformed buds or orga-
nized structures, while the callus from protoplast culture does not have such
structures.
The first success of regeneration of plants from protoplast cultures of Nicotiana
tabacum was achieved by Takebe et al. (1971). Since then, several species of plants
have been regenerated by using protoplasts (Binding et al., 1978; Cocking and
Pojnar 1969; Cocking 1972; Dos Santos et al. 1980; Harada 1973; Vasil and Vasil
1974, 1980; Zieg and Outka 1980). Some examples of plant species regenerated
from protoplasts are listed in Table 13.1.
13.9 Conclusions
The isolation, culture, and fusion of protoplasts are a fascinating field in plant
research. Protoplasts have a wide range of applications. A whole plant can be regen-
erated by protoplast culture. It is easy to perform single-cell cloning with protoplasts.
Callus differentiation Leaf sterilization
Epidermis peeling
Callus tissue
Peeled leaf segment

Regenerated
plantlet
Plasmolysed cells in an
enzyme mixture
Colony formation
Regenerated plant transferred
from test tube to soil
Partial wall digestion
Fusion
Clump of cells
NaNO3
treatment
Fused protoplasts
First division Pellet of protoplasts

Wall Isolated
regeneration protoplasts
Plating of protoplasts
Fig. 13.4 Major steps of protoplast isolation, culture, and regeneration of plant
Protoplast fusion or somatic hybridization can produce hybrids. Protoplasts are

excellent materials for studies of ultrastructure of cell. Protoplasts are useful for stud-
ies of membrane function like transport and uptake processes. Isolation of cell organ-
elles and chromosomes is easy from protoplasts. Genetic transformations can be
achieved through genetic engineering of protoplast DNA. It is easy to isolate mutants
from protoplast cultures.
278 I. Kumari
Table 13.1 Selected examples of plant species regenerated from protoplasts

Category Plant species
Cereals Oryza sativa, Zea mays
Vegetables Brassica oleracea, Capsicum annuum
Ornamentals Chrysanthemum sp., Rosa sp.
Woody trees Prunus avium, Larix eurolepsis
Oil crops Brassica napus, Helianthus annuus
Legumes Glycine max
References
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protoplasts of tobacco. Naturwissenschaften 58:318–320
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Plant Sci Lett 18:105–114
Plant Antimicrobial Peptides: Next-
Generation Bioactive Molecules for Plant 14
Protection
Paomipem Phazang, Neelam Prabha Negi,
Meenakshi Raina, and Deepak Kumar
Abstract
Plants are under constant attack by diverse groups of pathogenic microorganisms
but are able to survive and thrive harmoniously with these organisms. To accom-
plish this, they produce bioactive molecules called antimicrobial peptides
(AMPs) constitutively or after receiving chemical cues and downstream process-
ing of the signals. The AMPs are low-molecular-weight molecules with broad-
spectrum activity and less cytotoxicity affecting not only pathogenic microbes
but also neoplastic cells. They play an important role in the innate immunity of
plants. Studies have shown that heterologous expression of AMPs in plants con-
ferred disease resistance. In this chapter we have discussed two major families of
plant AMPs, elaborating their mode of action and their use in plant protection.
We have also highlighted the plant-based expression systems for AMPs in brief
and addressed its application in agriculture and therapeutic purposes.
Keywords
Antimicrobial peptides · Plant defense · Pathogenesis-related proteins
P. Phazang
School of Life Sciences, Jawaharlal Nehru University, New Delhi, India
N. P. Negi
University Institute of Biotechnology, Chandigarh University, Mohali, Punjab, India
M. Raina · D. Kumar (*)
Department of Botany, Central University of Jammu, Jammu, India

282 P. Phazang et al.
14.1 Introduction
Antimicrobial peptides (AMPs) are short sequence peptides, with usually fewer
than 50 amino acid residues reported in living systems. AMPs are an important part
of the innate immune system and these small molecules possess antifungal, anti-
parasitic, antibacterial, and antiviral activity (Sels et al. 2008). Hence, antimicrobial
peptides are considered a first line of defense in plants and animals against pathogen
attack (Egorov et al. 2005). AMPs are ubiquitous and are found from microbes to
plants to animals. These peptides can be circular, linear, or polycyclic. Circular
peptides include bacteriocins from bacteria, cyclotides from plants, and theta-
defensins from animals. Lantibiotics from bacteria are polycyclic peptides (Egorov
et al. 2005; Tam et al. 2015). Plants produce antimicrobial peptides as host defense
molecules. In general, plants fight infections using their two-branched innate
immune system. The first branch consists of the transmembrane pattern recognition
receptors (PRRs) recognizing the pathogen-associated molecular patterns (PAMPs)
common to many types of microbes. This branch is known as PAMP-triggered
immunity (PTI). The second branch is the effector-triggered immunity (ETI). ETI
gets activated upon the release of virulence factors by successful pathogens that
have “outdone” the PTI. A characteristic feature of ETI is the induction of systemic
acquired resistance (SAR), a hallmark of “plant memory.” Downstream signaling of
both pathways leads to ROS production, hormone biosynthesis, and signaling and
generation of pathogenesis-related proteins (PR) (Jones and Dangl 2006; Egorov
et al. 2005).
PR proteins were first discovered in tobacco leaves upon infection by tobacco
mosaic virus. They were later defined as induced proteins upon pathogenic chal-
lenge. They play an important role in the systemic acquired resistance. Until
recently, 17 PR families have been identified (Table 14.1). They have antimicrobial
or insecticidal properties. This chapter discusses about the PR proteins that have
antimicrobial activities. They are also known as antimicrobial peptides (AMPs).
Major plant AMP families are PR-13/thionins, PR-12/defensins, PR-14/lipid trans-
fer proteins, hevein-like peptides, knottins, and snakins (Van Loon and Van Strien
1999; Epand and Vogel 1999).
Plant AMPs are Cys rich that enable them to form disulfide bonds, thereby con-
tributing to their compact structures. In general they share many common character-
istics with microbes, animals, and insects and they include molecular forms,
amphipathic nature, and positive charge (Tam et al. 2015; Hammami et al. 2009).
Plant AMPs are expressed constitutively or upon microbial challenge and are
often tissue specific also. They are similar to the vertebrate immunoglobulin as they
also have hypervariable sequences encased and exhibit molecular diversity. The
defining features of plant AMPs include moderate size with molecular weight rang-
ing from 2 kDa to 6 kDa, being cationic, and presence of 2–6 intramolecular disul-
fide bonds; “peptide promiscuity” as a single peptide displays multiple functions in
addition to being antimicrobial. They are all ribosomally derived and bioprocessed
from precursors containing three domains, viz. N-terminal, C-terminal, and AMP
domain. The presence of the disulfide bonds not only allows them to have compact
14 Plant Antimicrobial Peptides: Next-Generation Bioactive Molecules for Plant… 283
Table 14.1 List of pathogenesis-related (PR) proteins with their properties

Family Representative member(s) Size (kDa) Properties
PR-1 Tobacco PR-1a 15 Antifungal
PR-2 Tobacco PR-2 30 β-1,3-Glucanase
PR-3 Tobacco P, Q 25–30 Chitinase I, II, IV, V, VI
PR-4 Tobacco R 15–20 Chitinase I, II
PR-5 Tobacco S 25 Thaumatin-like
PR-6 Tomato/potato inhibitor I 8 Proteinase inhibitor
PR-7 Tomato P69 75 Endoproteinase
PR-8 Cucumber chitinase 28 Chitinase III
PR-9 Tobacco lignin-forming peroxidase 35 Peroxidase
PR-10 Parsley PR-1 17 Ribonuclease-like
PR-11 Tobacco class V chitinase 40 Chitinase I
PR-12 Radish Rs-AFP3 5 Defensin
PR-13 Arabidopsis THI2.1 5 Thionin
PR-14 Barley LTP4 9 Lipid transfer protein
PR-15 Barley OxOa 20 Oxalate oxidase
PR-16 Barley OxOLP 20 Oxalo oxidase-like
PR-17 Tobacco PRp27 27 Unknown
(Source: Van Loon and Van Strien 1999; Epand and Vogel 1999)
secondary and tertiary structures but also offers high chemical, thermal, and enzy-
matic stability (Tam et al. 2015; Harris et al. 2009). This chapter gives an insight
into the major classes of plant AMPs and their applications in agriculture and for
diverse therapeutic purposes.
14.2 Antimicrobial Microbial Peptides (AMPs) from Plants
Plants made their innate immune system besides microbial attack through numerous
lines of defense, with native and general creation of secondary metabolites, pro-
teins, and ROS with antimicrobial activity. Plants prevent dispersion of pathogens
by hypersensitive response. There are six major families of plant AMPs, namely
thionins, defensins, hevein-like peptides, knottin-type peptides (linear and cyclic),
lipid transfer proteins, and snakins. These families are classified based on their Cys
motifs and their tertiary structures as a result of the distinctive disulfide bonds. The
major families of plant AMPs discussed in the chapter are represented in Table 14.2.
14.2.1 Thionins
Thionins are peptides with molecular size ranging from 45 to 48 amino acids. Eight-
cysteine-type (8-C) and six-cysteine-type (6-C) thionins are the two main subgroups
of this peptide family. The 8-C and 6-C thionins are characterized by the presence
of 3- and 4-disulfide bonds, respectively. All thionins have common 3D structures:
Table 14.2 List of major families of plant AMPs

Disulfide
bonds Some
Peptide (No.) Disulfide motif Structural motif representative
6C 3 Gamma (Г) fold Crambin,
Thionin β1-α1-α2-β2- viscotoxin
coil motif
8C 4 αl-Purothionin,
Thionin β-purothionin
8C 4 CSαβ motif NaD1
Defensin β1-coil-α-β2-β3
10C 5 PhDs
Defensin
LTP1 4 Hydrophobic Ace-AMP1
cavity (onion)
α1-α2-α3-α4-
coil
LTP2 4 DIR1
(Arabidopsis)
Knottins 6 Cystine knot PAFP-S
short β strand
and coil
6C hevein 3 Gly&Cys-rich Ac-AMP1
central β-strands
and (short
helical) side
coils
8C hevein 4 Hevein
10C 5 EAFP1
hevein
Snakins 6 α-Helices Snakin-1
(Source: Tam et al. 2015)
they all exist as L-shape. Two antiparallel α-helices and two antiparallel β-strands
form the long and short arms, respectively. The L-shape molecular structure has the
hydrophobic residues clustered on the outer surface and the hydrophilic residues at
the inner surface, thus imparting the amphipathic property to the peptides. Examples
of 8-C thionins include αl-purothionin and β-purothionin. Crambin and viscotoxin
are few examples of 6-C thionins (Rao 2015; Stec 2006; Clore et al. 1987).
Thionins have been isolated from both monocots and dicots. They are expressed
in leaves, roots, and endosperm of seeds. It has been reported in barley, Arabidopsis
thaliana, and certain dicots that different genes control the expression of thionins in
different tissues and hence the tissue-specific expression is observed (Ponz et al.
1983; Steinmuller et al. 1986). It has also been shown that the plant hormone methyl
jasmonate plays an important role in signal transduction for thionin expression
(Gausing 1987; Epple et al. 1995).
Thionins are ribosomally derived and are synthesized as preproproteins. The

p reproproteins have a mature thionin domain between two domains, i.e., the signal
sequence domain at the N-terminus and the prodomain at the C-terminus. Studies
have shown that the proprotein domain at the C-terminus plays an important role as
chaperones in the intravesicular trafficking of the proteins (Ponz et al. 1983;
Pelegrini and Franco 2005).
14.2.2 Defensins
Plant defensins are the most abundant plant AMPs. They have molecular size rang-
ing from 45 to 54 amino acids (Gao et al. 2000; Terras et al. 1992). Because of the
size similarity they share with thionins they were mistaken as one of them and
named them as γ-thionin subgroups. But the molecular structure analysis revealed
that they are structurally unrelated. Defensins showed the presence of an α-helix
and triple-stranded antiparallel β-sheets. Another characteristic feature defining
defensins is the formation of two disulfide bonds between two cysteines of CXXXC
segment of α-helix to the CXC segment of the β-strand. This structure is known as
cysteine-stabilized α-helix motif and is found in insect defensins as well (Terras
et al. 1995; Bruix et al. 1993).
Defensins are found in almost all plant species. Like thionins they also show
tissue-specific expression. Localization studies showed that they are expressed in
the peripheral cell layer of most tissues like leaves, seeds, pods, and flower organs
(Bruix et al. 1993-Finkina et al. 2008). These peptides are derived from two precur-
sors: (a) precursor containing an N-terminal signal peptide and mature defensin
domain and (b) precursor with additional propeptide at the C-terminal and only few
defensins like the flower specific are synthesized from this precursor (Kader 1996).
14.2.3 Lipid Transfer Proteins (LTP)
Lipid transfer proteins (LTP) were first identified in an in vitro bioassay for measur-
ing the transfer of phospholipids from artificial liposomes to mitochondria. Thus,
this class of peptides derived their name from their ability to transfer phospholipids
from a donor to an acceptor (Bloj and Zilversmit 1977; Gomar et al. 1998). They are
classified into two subgroups, LTP1 and LTP2, based on their molecular sizes and
also their tertiary structures. LTP1 subfamily consists of peptides with molecular
size ranging from 90 to 95aa. They have four α-helices stabilized by four disulfide
bonds (between CysI and CysVI, CysII and CysIII, CysIV and CysVII, and CysV
and CysVIII) and a flexible C-terminal coil. LTP2 subfamily is made of peptides
with molecular size of 70aa. These peptides have three extended helices, two single-
turn helices, and four disulfide bonds between CysI and CysV, CysII and CysIII,
CysIV and CysVII, and CysVI and CysVIII. The helices in LTP1 and LTP2 form a
hydrophobic cavity, which accommodates a variety of lipids. The presence of 8
cysteine residues forming four disulfide bridges and the hydrophobic and aromatic
residues at 12 invariable positions of all the peptides are the characteristic features
of this class of peptides (Tassin et al. 1998; Pallaghy et al. 1994).
LTP expression has been observed in various plant species. Studies in Arabidopsis,
barley, maize, and broccoli have shown the expression of LTPs in a variety of plant
organs including embryos, cotyledons, leaves, stems, siliques, and various flower
organs. The expression levels were highest in the epidermal layer of the organs and
all around abscised regions (Bloj and Zilversmit 1977; Tassin et al. 1998). Synthesis
of LTPs also occurs via precursors containing a N-terminal with signal peptide of
20–25aa and a mature domain with 8 cysteine residues at the C-terminal (Tassin
et al. 1998; Pallaghy et al. 1994).
14.2.4 Knottin-Type Peptides
Knottins are the smallest but the most diverse in functions among the plant AMPs.
They have a molecular size of 30 amino acids. The prototypic tertiary structure of
knottins is defined by the presence of six cysteine residues forming a cysteine knot
because of the disulfide bonds between CysI and CysIV, CysII and CysV, and CysIII
and CysVI. The characteristic structural motif is a compact triple-stranded β-sheet
and a long loop connecting the first to the second β-strand. They mostly exist in two
molecular forms, i.e., linear or cyclic. Inhibitors of α-amylase, trypsin, and carboxy-
peptidase families and cyclotides are representatives of this class of AMPs (Tam
et al. 2015; Franco 2011; Craik et al. 1999).
Their bioactive functions include hormone-like functions, and enzyme-inhibitory,
cytotoxic, antimicrobial, insecticidal, and anti-HIV activities (Franco 2011). This
diversity in function also known as “peptide promiscuity” is another characteristic
of knottin-type peptides (Franco 2011; Heitz et al. 1999).
They are found in dicot plants of the Rubiaceae, Violaceae, Cucurbitaceae,
Fabaceae, and Solanaceae families to a monocot plant of the Poaceae family
(Gruber et al. 2008; DeBolle et al. 1993). Studies in transgenic tobacco overex-
pressing knottin-type peptide from Mirabilis jalapa showed extracellular expres-
sion in leaves (De Bolle et al. 1995; Nguyen et al. 2011). They are mostly
synthesized from a precursor having a signal peptide, a prodomain, a mature pro-
tein domain, and a short C-terminal tail. However, variations in synthesis have
been identified. Cliotides identified from Clitoria ternatea showed that they origi-
nate from chimeric precursors consisting of albumin-1 chain A and cyclotide
domains (Ireland et al. 2006). Violacin A, a naturally occurring linear cyclotide
from Viola odorata, lacks the essential bioprocessing signal, the C-terminal Asn
residue required for cyclization due to the presence of a stop codon earlier in the
C-terminal sequence (Archer 1960).
14.2.5 Hevein-Like Peptides
Hevein was first identified from the latex of the rubber tree, Hevea brasiliensis. It
showed antifungal activity and is attributed to the presence of the chitin-binding
domain (Van Parijs et al. 1991; Lipkin et al. 2005). Peptides identified to contain the
hevein domain were later named as hevein-like peptides. They are basic peptides
with molecular size of 25–45aa. They have 3–5 disulfide bonds based on the num-
ber of cysteine residues. Thus they are subclassified as 6C, 8C, and 10C hevein-like
peptides. They are also characterized by the presence of cysteine knot but differ in
structures from those of knottins. The defining structural motif is the coil-β1-β2-
coil-β3 and a short arm that separates the β1 and β2 coils although variations are
observed in the length of the arm. They are rich in Gly and aromatic residues and
these residues are conserved across the peptides. Like hevein, hevein-like peptides
showed toxicity to chitin-containing fungi and other plant fungal pathogens
(Andreev et al. 2012; Segura et al. 1999).
Very little is known about the expression patterns of hevein-like peptides. The
rubber seedlings showed expression of hevein in the leaves and stem but not in the
roots. Wounding and application of abscisic acid also triggered the accumulation of
hevein transcripts in the leaves and stem. Hevein and hevein-like peptides are pro-
cessed from precursors containing three domains: a signal peptide of 25aa at the
N-terminal, a mature peptide of 33aa, and a 34aa C-terminal region, which undergo
cleavage during posttranslational modification (Segura et al. 1999).
14.2.6 Snakins
This class of peptides was first isolated from potato tubers and was named snakins
by Sengura and coworkers (1999) because of the similarity in sequence to the hemo-
toxin of snakes (Berrocal-Lobo et al. 2002). Two subtypes of snakins have been
identified from potato, Solanum tuberosum: snakin-1 having 63aa and snakin-2
having 66aa residues. They are cationic peptides and contain 12 cysteine residues
and are predicted to have 2 α-helices with disulfide bonds between CysI and CysIX,
CysII and CysVII, CysIII and CysIV, CysV and CysXI, CysVI and CysXII, and
CysVIII and CysX. Snakins show both antibacterial and antifungal activities and
are recognized components of constitutive and inducible plant defense systems
(Berrocal-Lobo et al. 2002; Van Loon et al. 1994).
14.2.7 Mode of Action of Plant AMPs
The cationic charge and the amphipathic nature play an important role in the anti-
microbial activities of the plant AMPs. In general, the cationic peptides bind to the
negatively charged phospholipids and liposaccharides of Gram-negative bacteria
and teichoic acid of Gram-positive bacteria and cause disruption of the lipid mem-
brane bilayer. This in turn causes membrane collapse and cell lyses. Intrinsic factors
such as the peptides’ ability to self-assemble and oligomerize and extrinsic factors
like that of the phospholipid composition of the membrane, head group size, and
membrane fluidity play a critical role in exerting their antimicrobial activities
(Sitaram and Nagaraj 1999; Carmona et al. 1993).
The mode of action of plant AMPs can be broadly categorized into three types: (a)
the barrel-stave mechanism, (b) the toroidal pore or wormhole mechanism, and (c)
the carpet mechanism (Yeamn and Yount 2003; Carmona et al. 1993). In the barrel-
stave model, the cationic peptides initially aggregate and interact with each other
forming a barrel ring. After reaching the threshold concentration, the peptides orient
perpendicularly to the membrane of the microbes. This orientation allows the inter-
action of the hydrophobic portions of the peptides with the head group of the phos-
pholipids of the bilayer membrane of the bacteria, thus forming a pore in the
membrane. The barrel-stave complex formed thus acts as a hydrophilic channel on
the membrane allowing cell lyses to occur (Yeamn and Yount 2003). The toroidal
pore or the wormhole model takes place in two stages. In the first step, when the
peptide concentration is low also known as the inactive state, the peptides align par-
allel to the membrane and the polar regions interact with the lipid head groups of the
membrane. The second step occurs when the peptide concentration reaches the
threshold level. At this stage the peptides reorient themselves perpendicular to the
membrane causing the formation of the hydrophilic transmembrane pore. The barrel-
stave and the toroidal pore models are very similar except that the pore formation in
the latter takes place in two stages depending on the peptide concentration (Yeamn
and Yount 2003; Carmona et al. 1993). The carpet model mode of action of the cat-
ionic peptides aggregates by electrostatically binding themselves forming a carpet-
like structure on top of the microbial membrane. The peptide aggregate then binds to
the phospholipids of the bilayer membrane, causing disturbance on the membrane
fluidity. The membrane damage of the pathogenic microbes occurs in a dispersion-
like manner unlike the channel formation observed in the other two models (Carmona
et al. 1993).
14.2.8 Plant AMPs as Plant Protection Agents: A Plausible

Application in Agriculture
The primary objective of plant biotechnology is to enroot crop plants with superior
ability to resist disease caused by bacterial and fungal pathogens, thereby increasing
production. Genetic transformation of plants has helped in establishing the roles of
various plant AMPs. Antimicrobial peptide genes taken from plants and overex-
pressing them in model as well as crop plants have shown the potential of the pep-
tides for protection against phytopathogens (Oard and Enright 2006). Tobacco
plants expressing a barley thionin gene under the CaMV 35S promoter showed
reduced lesion symptoms when challenged with either Pseudomonas syringae pv.
Tabaci or Pseudomonas syringae pv. Syringae (Oard and Enright 2006).
β-Purothionin isolated from wheat endosperm conferred increased resistance to
Pseudomonas syringae pv. Tomato and F. oxysporum in Arabidopsis (Oard and
Enright 2006). Another thionin from wheat, puroindolin genes pinA and pinB, was
overexpressed in rice and was found to give increased tolerance to Magnaporthe
grisae and Rhizoctonia solani (Krisnamurthy et al. 2001). Defensins isolated from
various plant sources have also been investigated. Transgenic tobacco plants
expressing the cDNA of radish defensin, Rs-AFP2, showed sevenfold reduction in
lesion size on the leaves upon infection by Alternaria longipes, a foliar fungal
pathogen. The alfalfa defensin, Alf-AFP expressed in potato protected against
Verticillium dahliae (Terras et al. 1992), and pea defensin, DRR206 expressed in
canola and tobacco, showed protection against Leptosphaeria maculans (Wang
et al. 1999). BSD1 cabbage defensin expressed in tobacco conferred resistance to
Phytophthora parasitica (Park et al. 2002a, b). WT1 wasabi defensin expressed in
rice protected against Magnaporthe grisea (Kanzaki et al. 2002), eggplant express-
ing the Dm-AMP1 dahlia defensin was able to show resistance to pathogens like
Botrytis cinerea and Verticillium alboatrum (Turrini et al. 2004), and Mj-AMP1
jalapa defensin expressed in tomato protected against Alternaria solani (Schaefer
et al. 2005).
Overexpression of the PR-14 gene from barley coding for a lipid transfer protein
in tobacco and Arabidopsis thaliana conferred enhanced resistance against
Pseudomonas syringae pv. Tabaci (Chassot et al. 2007). It has also been reported of
the resistance provided to Arabidopsis thaliana against Botrytis cinerea by overex-
pression of three endogenous LTP (PR-14)-like genes (Chassot et al. 2007).
In addition, it may also be mentioned that some synthetic antimicrobial peptides
have gained similar status as plant-protecting agents. Overexpression of MsrA1, a
synthetic AMP, a cecropin-melittin cationic chimera peptide in potato, showed pro-
tection against Phytophthora cactotrum, Fusarium solani, and Erwinia carotovora
(Osusky et al. 2000). In Brassica juncea overexpression of MsrA1 has shown resis-
tance to Alternaria brassicae and Sclerotinia sclerotiorum, two of the most devas-
tating pathogens of B. juncea crops (Rustagi et al. 2014). Another synthetic peptide
D4E1 also showed its antimicrobial property against Colletotrichum destructivum
in transgenic tobacco (Cary et al. 2000). Poplar plants also expressing the D4E1
showed resistance against Agrobacterium tumefaciens and Xanthomonas populi pv.
populi (Mentag et al. 2003).
These reports of transgenic model and crop plants expressing the plant AMPs
showing enhanced resistance to pathogens are paving way for their large-scale
application in agriculture. Today, the world is challenged with issues related to food
security. Farmers across the globe are not able to produce enough food for the ever-
increasing population. Mass destruction of crops by phytopathogens and abiotic
stress factors is the main reason attributing to the shortage of food. Thus, one of the
major goals of plant biotechnology is to develop crop plants that would show resis-
tance to these stress factors and at the same time bring about sustainable agricultural
developments. There is an urgent need to come up with new plant-protecting agents
to replace the chemical pesticides that have been used extensively and have caused
enormous damage on our agricultural lands. Plant AMPs thus seem to be one of the
potential alternative biocontrol agents discovered so far. The various facets of the
antimicrobial properties displayed by them not only highlight the inbuilt
mechanism and strategies adapted by plants but also give us a status on “plant
health.” If plant molecular biotechnology and plant breeding as well as other inter-
disciplinary science collaborate, we would be able to further tap the AMPs of plant
sources for beneficial use in agriculture.
14.3 Conclusion
The diversity displayed by plant AMPs in terms of structure and function is spec-
tacular. Their constitutive and inducible expression patterns reveal that they func-
tion not only to fight against pathogens but also as part of the basal plant machineries.
Though many of them have been isolated/discovered and extensively investigated,
we still need novel strategies to understand the structure-function relationships to be
able to fully understand their mechanisms for beneficial exploitation. There is no
denial that they are advantageous as they are of plant origin and are essential com-
ponents of the plant innate immune system and have been used by them to fight
back against the constant attack by microbes. In addition, they are also effective
against plant-feeding insects. Thus, the antimicrobial property (antibacterial, antivi-
ral, and antifungal) and insecticidal property confer plant AMPs as potential plant-
protecting agents.
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Nitrogen Stress in Plants and the Role
of Phytomicrobiome 15
Garima Malik, Navneet Singh, and Sunila Hooda
Abstract
Nitrogen (N) being an important macronutrient for plants is a major factor that
determines its growth and yield. Nitrogen uptake, nutrition, and signaling have
received a lot of attention in the last few decades. More recently, the focus of the
research has shifted to regulatory networks within or outside the N metabolism.
We know that N is not just the essential nutrient required to support the optimal
plant growth and yield, but is also an important signal involved in a wide array of
plant responses to a broad range of biotic and abiotic stresses including nutrient
deficiency, light, salinity, and drought. The recent progress in the genome
sequencing data has allowed us to draw a more comprehensive picture of the
molecular and structural diversities of the genes and the encoded proteins
involved in morphological and physiological responses to N. Most plants have
the ability to enhance nutrient acquisition through symbiosis—close and long-
term relationship of microbes with plants. The current review focuses on the
most exciting developments in the field of microbes and its role in N stress.
Keywords
Nitrogen · Nitrate · Diazotrophs · Biological nitrogen fixation · Sustainable
agriculture
G. Malik
R.G. (PG) College, C.C.S University, Meerut, Uttar Pradesh, India
N. Singh
John Curtin School of Medical Research, Australian National University,
Canberra, ACT, Australia
S. Hooda (*)
Ram Lal Anand College, University of Delhi, New Delhi, India

296 G. Malik et al.
15.1 Introduction
Nitrogen (N) is an essential macronutrient required in large amount for proper plant
development (Marschner 1995; Epstein and Bloom 2005; Miller et al. 2007). Its
accessibility is a major factor that governs plant growth and vigor in both natural
and agricultural ecosystems (Galloway and Cowling 2002). N plays an important
role in diverse aspects of the plant’s life cycle and affects all levels of plant function,
from metabolism to resource allocation, growth, and development including plant
morphology, nutrient availability, net photosynthesis, root and shoot biomass pro-
duction, and synthesis of essential biomolecules such as nucleic acids, proteins,
phospholipids, chlorophyll, growth hormones, many cofactors, and secondary
metabolites (Crawford 1995, Marschner 1995, Tschoep et al. 2009). N is a funda-
mental constituent of nucleotides and amino acids, primary building block of
nucleic acids and proteins, respectively, present in each and every cell of the plant
body. It is also an essential part of the chlorophyll molecule, which imparts green
color to the plant leaves and is involved in the synthesis of food material for the
plant via photosynthesis. Apart from photosynthesis, it performs a crucial role in
various other metabolic and physiological processes of plants—it promotes growth
and differentiation of different vegetative and reproductive parts of plants.
Furthermore, it stimulates rapid early growth in plant seedlings, enhances dry mat-
ter of leafy vegetables, improves quality and quantity of fruit set along with seed
production, and increases the protein content of fodder and forage crops (Marschner
2011). It also facilitates the uptake and utilization of other essential nutrients such
as potassium (K) and phosphorous (P) and thereby controls overall growth and sur-
vival of plants (Bloom 2015). Deficiency of N in plants leads to chlorosis (appear-
ing first on older leaves, usually starting at the tips), stunted growth (due to reduction
in cell division), restricted growth of lateral buds (from which leaves, stem, and
branches develop), lowered protein content of vegetative parts and seeds, reduced
flower set, and overall reduction in yield and quality (Uchida 2000). Likewise,
excessive application of N has negative effects on plant and adversely affects plant
growth, promotes extra dark-green color on the leaves, reduces the fruit quantity
and quality, and accelerates senescence (Uchida 2000).
N is present in the biosphere in various chemical forms and the earth’s atmo-
sphere is composed of 80% of molecular nitrogen (N2) (Sanhueza 1982). However,
plants cannot directly use an unreactive molecular form of N. N2 enters into the
global N cycle in three predominant ways (Kraiser et al. 2011): (a) through biologi-
cal nitrogen fixation (BNF), i.e., conversion of N2 to ammonia by microorganisms/
prokaryotes; (b) by atmospheric nitrogen fixation (ANF), i.e., conversion of N2 to
nitrate (NO3−) by means of lightning and photochemical reactions; and (c) by the
Haber–Bosch industrial N fixation, i.e., conversion of N2 to ammonia (Marschner
1995). Without human interference, BNF and ANF would be the only sources of
new N in the environment. Fritz Haber in 1908 discovered how ammonia (a chemi-
cally reactive and highly usable form of N) could be synthesized artificially by
reacting atmospheric N2 with hydrogen in the presence of iron at high pressures and
15 Nitrogen Stress in Plants and the Role of Phytomicrobiome 297
temperatures (Erisman et al. 2008). His discovery transformed the world and later
in 1918 he was awarded the Nobel Prize in Chemistry.
Once N is fixed in the form of NO3− and ammonium, it can be used for the bio-
synthesis of N-containing metabolites (e.g., nucleotides, amino acids, small poly-
peptides, urea) by various life forms, and finally returned back to the atmosphere as
elemental form (Marschner 1995, Miller et al. 2007). The ability of a plant to cap-
ture fixed N from the soil depends on plant species, physical and chemical proper-
ties of soil, and various environmental factors. The use of N by plants involves
various steps inclusive of uptake, assimilation, translocation, recycling, and remobi-
lization. Proper growth and development of plants require an optimum supply of
N. The N supply is indispensable for plants and increases in N supply have been
exploited to increase the yield of agricultural crops in order to provide food for the
fast-growing global human population. However, N is one of the most expensive
nutrients to supply and chemical fertilizers signify the major cost in crop production
for farmers. It has been estimated that more than 100 million metric tonnes of
nitrogenous fertilizers are added annually to the soil worldwide and almost half of
the human population depends on N fertilizer for their food supply (Good et al.
2004, Erisman et al. 2008). However, the excessive use of these chemical fertilizers
is also the main source of soil and water pollution. In order to overcome these prob-
lems, various plant scientists are now focusing on developing plant varieties with
better inherent N use efficiency (NUE), thus reducing environmental degradation
and paving a path for sustainable agriculture production (Hirel et al. 2007).
15.1.1 Biological Nitrogen Fixation (BNF)
Biologically available N or fixed N is essential to sustain life on earth. The lack of

biologically available N is often the limiting factor in the productivity of crop plants
and thus food production. In order for N to be assimilated by plants, it must be fixed
or combined or reduced in the form of ammonium (NH4+) or NO3− ions (Mancinelli
1996). The global biogeochemical N cycle allows the interconversions of N com-
pounds to keep a comparatively small amount of fixed N in a constant exchange
with atmospheric N2 (Cabello et al. 2004). BNF, a process where molecular N2 is
reduced in multiple electron transfer reactions resulting in the synthesis of ammonia
and the release of hydrogen, is carried out by a group of prokaryotic microorgan-
isms called nitrogen-fixing organisms (diazatrophs), distributed across the archaeal
and eubacterial domains (Kim and Rees 1992; Raymond et al. 2004). Due to their
prominent function in the global N cycle, diazotrophs are present in virtually all
ecosystems ranging from rhizosphere which includes the ocean to specialized root
nodules in legume plants. Diazotrophs are capable of N fixation due to the presence
of a nitrogenase enzyme complex that carries out one of the most metabolically
expensive processes of N2 fixation in biology, requiring large amounts of both
reducing power and high-energy phosphate (ATP) (Simpson and Burris 1984).
Nitrogenase is an ATP-dependent enzyme complex that consists of two metallopro-
teins, a molybdenum-iron (MoFe) protein heterotetramer (dinitrogenase) and an
298 G. Malik et al.
iron-protein (Fe-protein) homodimer (dinitrogenase reductase) encoded by the

nitrogen fixation (nif) genes (Hageman and Burris 1978; Peters et al. 2011).
Supplementary genes in the nif operon code for proteins are involved in the biosyn-
thesis of nitrogenase cofactor, along with proteins required for electron transport to
nitrogenase and regulation, and some proteins of unfamiliar functions (Dean and
Jacobsen 1992; Rubio and Ludden 2008). On the basis of the composition of their
metal centers, nitrogenase can be categorized into three broad categories: iron and
molybdenum (Fe/Mo), iron and vanadium (Fe/V), or iron only (Fe), of which Fe/
Mo type is the most common one (Mus et al. 2016). Nitrogenase catalyzes the con-
version of N2 to NH4+ as represented by the following equation (Halbleib and
Ludden 2000):
N 2 + 10H + + 8e − + 16MgATP → 2 NH 4 + + H 2 + 16MgADP + 16Pi

The nitrogenase enzyme complex is sensitive to O2, which irreversibly inacti-
vates the enzyme. Thus N-fixing microorganisms have evolved various mechanisms
which concomitantly permit the supply of O2 required for obtaining chemical
energy while protecting nitrogenase from its poisonous effect (Rubio and Ludden
2008; Peters et al. 2011). The requirement of high energy for N fixation and the
oxygen sensitivity of nitrogenase impose considerable physiological constraints on
N-fixing organisms (Argudo et al. 2005).
15.1.2 Phytomicrobiome and Its Role in N Fixation
A plant sustains an ecosystem. It would be ideal to consider it from a systems per-

spective. The term “phytomicrobiome” refers to the microbial community associ-
ated with the plant system. There is an intricate relationship of a plant with its
relatively constant type of microorganisms. The plant-beneficial microbiome con-
fers growth-promoting effects on plants by mobilizing nutrients in the soil, support-
ing under abiotic stress and biotic stress and bioremediating the soil polluted with
heavy metals (Miransari 2011; Richardson et al. 2009; Ahemad and Malik 2011).
The indispensable role of microorganisms in building and conserving soil fertility
has been recognized ever since the commencement of agriculture.
Different plant compartments harbor different niches of bacteria. Generally,
these niches are divided into three broad habitats based on the plant parts they
inhabit—phyllosphere (habitat defined by aerial plant parts, particularly stem and
leaves), endosphere (encompassing habitat inside plant organs both above- and
belowground), and rhizosphere (microbial habitat around the roots, both inside and
outside soil). Evident loss of N from aerial parts of the plants in the form of gaseous
ammonia and unavailability of a suitable form of N in the soil for plant uptake are
reasons for N stress in plants. Phyllosphere is constantly exposed to changes in light
intensity, humidity, temperature, etc. directly affecting the nutrient composition
whereas there is assured higher abundance of nutrients in rhizosphere due to uncom-
mon changes in light and temperature conditions. Thus, numerous species of bacte-
ria, nematodes, fungi, and protozoa colonize the rhizosphere. Mendes and colleagues
have summarized past studies that focused on identification of diversity of bacterial

taxa present in different rhizosphere samples highlighting the presence of
Proteobacteria as dominant phylum followed by Firmicutes (Mendes et al. 2013).
Among various interactions of plants with microorganisms, the one beneficial inter-
action is that of N-fixing plant growth-promoting rhizobacteria (PGPR), which
colonize rhizosphere. Some of these are diazotrophic bacteria.
Many microbial species capable of fixing N, both as free-living soil microorgan-
isms (e.g., bacteria and blue-green algae (BGA)) or in symbiotic association with
plants (e.g., rhizobia-legume; angiosperm-actinomycetes; and BGA associative sys-
tems, including rhizosphere, phyllosphere, and lichens), have been identified.
N-fixing microorganisms are found in several phyla and representatives from the
majority of these phyla are known to fix N either in nonsymbiotic or symbiotic
association with plants (Boyd and Peters 2013; Hardoim et al. 2015). Plants have
developed various ways to achieve a mutualistic relationship with diazotrophs to
obtain atmospheric N (Mus et al. 2016). In all these associations and symbioses, the
host plants get the benefit of the fixed N provided by the symbiotic partner, which,
in turn, receives reduced C along with other nutrients it requires. Also, the symbiotic
or endophytic plant structure colonized by the N-fixing microorganisms may offer
the suitable conditions for shielding the nitrogenase enzyme complex from O2 expo-
sure (Santi et al. 2013).
15.1.2.1 Nonsymbiotic N Fixation

Free-living soil N-fixing microorganisms include Archaea, Proteobacteria,
Firmicutes, and Cyanobacteria (Zhan and Sun 2012). Archaea form a monophyletic
domain of remarkably diverse and successful organisms distinct from bacteria and
eukarya. Archaea inhabits some of the most extreme environments on the planet,
distributed over two main subdivisions: crenarchaeota (primarily hyperthermo-
philes and anaerobic respirers) and euryarchaeota (includes methanogens and
extreme halophiles) based on 16S rRNA sequence comparisons (Woese et al. 1990;
Brown and Doolittle 1997; Cabello et al. 2004). N2 fixation in Archaea was first
discovered in 1984 in Methanosarcina barkeri and Methanococcus thermolithotro-
phicus (Murray and Zinder 1984; Belay et al. 1984). Within the Archaea, N fixation
has been found exclusively in the methanogenic euryarchaeota. However, N fixation
is widespread within the methanogens, extending to all three orders, viz.
Methanococcales (e.g., Methanococcus thermolithotrophicus, Methanococcus mar-
ipaludis), Methanomicrobiales (e.g., Methanosarcina barkeri, Methanospirillum
hungatei), and Methanobacteriales (e.g., Methanobacterium bryantii) (Leigh 2000,
Offre et al. 2013). Many bacteria reside in the soil and fix significant levels of N
without the direct interaction with other organisms including Azotobacter (aerobic),
Klebsiella, Beijerinckia (aerobic), Bacillus, Clostridium (anaerobic and non-
photosynthetic), and Rhodospirillum (anaerobic and photosynthetic). Sergei
Winogradsky in 1983 discovered the first N-fixing bacterium, Clostridium pasteur-
ianum, followed by Martinus Beijerinck’s description of Azotobacter in 1901.
Following the discovery of Clostridium and Azotobacter an increasing number of
N-fixing microorganisms were isolated. Within the domain bacteria, the phylum
300 G. Malik et al.
Proteobacteria comprises the largest and phenotypically most diverse phylogenetic

lineage scattered over five classes at present, viz. Alphaproteobacteria,
Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, and
Epsilonproteobacteria (Kersters et al. 2006). The Proteobacteria include both free-
living and symbiotic N-fixing bacteria. The genus Azotobacter belongs to the
gamma-subclass of the Proteobacteria (Becking 2006). Azotobacter spp. are free-
living, Gram-negative, aerobic bacteria primarily found in neutral to alkaline soils
along with rhizosphere of many plants. They are nonsymbiotic heterotrophic bacte-
ria capable of fixing an average 20 kg N/ha/per year and also produce plant growth-
promoting substances. Azotobacter chroococcum is the most prevalent species
found. The genus Beijerinckia belongs to the alpha-subclass of the Proteobacteria.
Beijerinckia spp. are free-living, aerobic, chemoheterotrophic bacteria commonly
found in acidic soils and within rhizosphere and phyllosphere environments of
many higher plants (Kennedy 2005). Certain Bacilli comprise the known faculta-
tive, non-photosynthetic N-fixing bacteria. The genus Klebsiella belongs to the
gamma-subclass of the Proteobacteria. Klebsiella spp. are Gram-negative, faculta-
tive anaerobic bacteria. Among the anaerobes, the most ubiquitous N-fixing genus
is Clostridium, found in almost all kinds of soils. About 15 genera of O2-evolving
photosynthetic BGA (cyanobacteria) are found freely in the soil where they fix free
atmospheric N2 including Nostoc, Anabaena, and Aulosira. N2-fixing cyanobacteria
can be broadly divided into two groups: heterocystous forms and non-heterocystous
forms. Heterocysts are specialized thick-walled cells, which occur at intervals along
the cyanobacterial filaments and provide an environment suitable for the function-
ing of nitrogenase enzyme complex. In comparison to heterocystous N-fixing BGA,
non-heterocystous N-fixing BGA are less in number, e.g., Oscillatoria, Phormidium,
and Gloeothece.
15.1.2.2 Symbiotic N Fixation

On the basis of the level of proximity and interdependency of the plants and N-fixing
microbes, their association could be broadly divided into three categories: loose
associations with free-living N fixers, intercellular endophytic associations, and
endosymbioses (Mus et al. 2016). Many free-living microorganisms are loosely
associated with a variety of plants. These associations do not engage any kind of
structural or morphological accommodation of the N-fixing organisms but involve
more of close physical contacts in which reciprocal influence can be exerted between
the symbionts (Hamdi 1982). N-fixing bacteria that live in loose associations with
plant roots are usually designated as “associative” N-fixing bacteria (Elmerich
2007). An interaction between plant and associative N-fixing bacteria is one of the
simplest types of N-fixing symbiosis. Azospirillum (α-subclass of proteobacteria), a
Gram-negative free-living N-fixing bacteria, is one of the best studied associative
diazotrophs that colonize the rhizosphere of many plants, but do not invade plant
tissues. These PGPR positively influence the acquisition of N, P, and other essential
minerals and thereby improve the yield and growth of several crops, such as wheat,
maize, and rice (Steenhoudt and Vanderleyden 2000). The plant scientists are
exploring PGPR and investigating their modes of action so as to use them commer-
cially as biofertilizers. Leaf surfaces (phyllosphere) have also been shown to
support extensive surface populations of a range of microorganisms, including diaz-

otrophic bacteria. Ruinen (1975) mentioned the common genera of microorganisms
detected in the phyllosphere in different regions of the globe, including Pseudomonas,
Xanthomonas, Bacillus, Clostridium, Cyanophyceae, Chlorophyceae, and lichens.
In tropical forests, N-fixing cyanobacteria belonging to the genera Scytonema,
Oscillatoria, Microcoleus, and Stigonema have been reported to colonize the phyl-
losphere of plants (Bentley 1987; Fürnkranz et al. 2008). Azolla, an aquatic fern, has
been used by farmers as biofertilizers in rice fields since ages as heterocystous cya-
nobacterium Nostoc azollae is present within the specialized cavities on the under-
side of Azolla leaves.
In more developed associations, diazotrophic bacteria spread and multiply within
plant tissues without causing any visible harmful effects, classified as endophytic
N-fixing bacteria due to their firm association with plant tissues (Mus et al. 2016).
Several diazotrophic endophytes have been identified in grass species (e.g., maize,
rice, sorghum, sugarcane, and wheat) that are members of the alpha-, beta-, and
gamma-subclasses of the proteobacteria including Azoarcus, Herbaspirillum,
Acetobacter diazotrophicus, Burkholderia, Serratia marcesens, and
Gluconacetobacter (Triplett 1996; Reinhold-Hurek and Hurek 1998; Pedraza
2008). Endophytic bacteria invade plant tissues; however, they do not dwell within
living plant cells and does not stimulate the development of any differentiated plant
structure like that of endosymbionts (Carvalho et al. 2014). Endophytic bacteria
usually reside in root intercellular spaces, xylem vessels, and lignified xylem paren-
chyma, as well as in dead cells (James 2000). In order to uphold stable symbiosis,
apart from N-fixation diazotrophic endophytes produce numerous compounds that
support plant growth and help them acclimatize better to the environment. The most
well-studied diazotrophic endophytes are Gluconacetobacter diazotrophicus and
Herbaspirillum spp. in sugarcane; Azoarcus spp. in Kallar grass; and Bacillus,
Enterobacter, Herbaspirillum, and Pseudomonas in rice and maize (James 2000).
Inoculation experiments on sugarcane plants with different strains of endophytic
diazotrophic bacteria reported an approximately 30% contribution of BNF, along
with a 39% increase in total plant biomass (Oliveira et al. 2002). Nostoc is able to
endophytically colonize the thallus of two genera of liverworts, viz. Blasia and
Cavicularia (in dome-shaped auricles), and all hornworts (in slime cavities or
mucilage-filled canals) along with the cortical layer of the specialized coralloid
roots of cycads that arise from the lateral roots (Adams and Duggan 2008; Costa and
Lindblad 2002). Cycads are the only known gymnosperms so far that have shown
the ability to develop a N-fixing symbiosis with cyanobacteria through an endo-
phytic association. Calothrix has also been reported to form endosymbiotic associa-
tion with Cycadaceae occasionally (Costa et al. 1999; Thajuddin et al. 2010).
The highly specific, intimate, and most efficient form of N-fixing plant-microbe
association is endosymbiosis. In some plants, diazotrophs elicit the formation of
root nodules, specialized 2–5 mm diameter organs that are usually developed on
roots to house N-fixing bacteria (Gage 2004). Diazotrophic endophytic bacteria and
endosymbionts have an advantage over associative bacteria as they are in direct
contact with plant cells, a better niche for N fixation and assimilation of fixed N by
plants. The most well-studied plant endosymbioses are those between legumes
302 G. Malik et al.
(family Fabaceae) and rhizobia, Gram-negative members of the alpha-subgroup of

the phylum proteobacterial Rhizobiaceae family (including the genera Rhizobium,
Mesorhizobium, Sinorhizobium, Bradyrhizobium, and Azorhizobium), and between
actinorhizal plants and Frankia bacteria (Santi et al. 2013). Frankia is able to fix N
both under symbiotic and free-living conditions, unlike rhizobia.
In order to begin a fruitful mutualistic relationship with the host plant, rhizobia
must first identify and then respond to the presence of host plant roots. Plants secrete
certain flavonoids in the soil to attract the bacteria. These flavonoids induce the
expression of signaling molecules called nodulation (Nod) factors which are impor-
tant for nodule formation on the plant roots. The particular chemical structure of
these Nod factors recognized by plant varies among bacterial species adding to
host-symbiont specificity. Rhizobia enter the roots of most legume plants via root
hair infection, which grows through the root cortex and branch repeatedly (Sprent
et al. 2013). During growth in the rhizosphere, rhizobia recognize compounds
secreted by the host root (e.g., flavonoids and betaines) and in turn respond by
inducing nod genes (Gage 2004). The nod genes then encode around 25 proteins
necessary for the bacterial synthesis and export of Nod factor, a lipo-oligosaccharide
nodulation signal molecule that initiates many developmental changes in the host
plant including root hair deformation, membrane depolarization, initiation of cell
division in the root cortex, and initiation of nodule primordia (Gage 2004). The
formation of infection thread is initiated when a deformed root hair quickly curls,
and traps the bacteria bound to it between appressed cell walls. Afterwards, degra-
dation of the cell wall and invagination of the cell membrane, followed by tip growth
of the invagination, lead to extension of infection thread that grows down the inside
of the root hair and into the body of the epidermal cell (Callaham and Torrey 1981).
The rhizobia keep on dividing and growing inside the thread, thereby keeping the
tubule filled with bacteria and infection thread propagated further towards the root
interior. Rhizobia eventually enter cortical cells of the roots via endocytosis. In
cortical cells, they differentiate into N-fixing bacteroides within a unique plant
organelle known as symbiosome and continue to produce proteins essential for
N-fixation and for the maintenance of the symbiotic relationship (van Spronsen
et al. 1994; Andrews and Andrews 2017). The symbiosome is delimited by a plant-
derived membrane that controls the nutrient exchange between the symbionts (Mus
et al. 2016). Other than the usual hosts of rhizobia–legumes (family Fabaceae), the
only non-legume host known is Parasponia species (family Cannabaceae)
(Akkermans et al. 1978). A similar Nod-dependent symbiotic interaction has been
observed for Parasponia.
Frankia, a filamentous Gram-positive actinomycete, induces root nodules on a
variety of woody plants belonging to eight families distributed over three orders,
viz. Fagales (Betulaceae, Casuarinaceae, and Myricaceae), Rosales (Rosaceae,
Elaeagnaceae, and Rhamnaceae), and Cucurbitales (Datiscaceae and Coriariaceae),
collectively called actinorhizal plants (Wall 2000; Pawlowski 2009; Franche and
Bogusz 2011; Berry et al. 2011). The strategies used by Frankia spp. to infect acti-
norhizal plants are somewhat alike to those used by rhizobia. Frankia infects the
root cells of actinorhizal plants either through intercellular root invasion or through
intracellular root-hair infection (Benson and Silvester 1993). Although factors
responsible for host-symbiont specificity and preliminary plant response induction

are poorly understood for Frankia, it is known that nodABC-like genes are present
with other genes such as nif (nitrogenase), suf (sulfur-iron cofactor synthesis), and
hup (hydrogenase uptake), all known to be involved in symbiosis (Alloisio et al.
2010). In temperate climates, the actinorhizal plants inhabited by Frankia have been
reported to fix N at comparable rates to those of legume symbioses, i.e., ~300 kg/ha/
year (Hibbs and Cromack 1990; Wheeler and Miller 1990). Many actinorhizal
plants are also competent of forming mycorrhizal associations, and this tripartite
symbiosis (host plant–Frankia–mycorrhiza) helps actinorhizal plants to grow in
degraded and disturbed soils and play important ecological roles to prevent land
desertification (Dawson 2008).
N-fixing cyanobacteria Nostoc have also been found to colonize plants in the
family Gunneraceae. The intimacy of the symbiosis Gunnera-Nostoc is greater than
in any other cyanobacterial associations as here the cyanobacteria are hosted intra-
cellularly. The cyanobiont enters nonlegume angiosperm Gunnera plants through
specialized glands located on the stem in the axis of the leaves, which secrete
carbohydrate-rich mucilage and attract specific symbiotic cyanobacteria (Silvester
and McNamara 1976; Towata 1985; Bonnett 1990). After entering the gland, cyano-
bacteria induce divisions in the host cells and are accommodated within inner cortex
cells (Bergman et al. 1992). Nostoc filaments are surrounded by the host’s plasma
membrane, which acts as the interface for nutrient exchange. Other diazotrophic
bacteria with the common ability to fix N are mentioned in Table 15.1. Recent
research and advances in the understanding of associative, endophytic, and endo-
symbiotic N-fixation with legumes and nonlegume plants may lead to the develop-
ment of novel techniques for delivering N to cereals and other nonlegume crops
which is crucial for the future of sustainable agriculture, including the reduction of
soil pollution and ensuring of food security.
15.1.3 N Uptake and Its Regulation in N-Deficit Conditions
N is acquired by plants in both inorganic (ammonium and NO3-) and organic (amino
acids, urea, etc.) forms; however, ammonium and NO3− are universally favored and
found in most soils. As a general practice, chemical fertilizers rich in N are used as
additives for crops. This has significantly impacted the global N balance in a nega-
tive way contributing to pollution of lakes and rivers, accumulation of greenhouse
gases, acidification of soil, and ozone-layer depletion. Most of these mentioned
concerns root from the fact that plants utilize only a fraction of available N. The
availability of N is low and variable in natural soils which create N-depletion zones
(Jackson and Caldwell 1993). In response to low N availability plants enhance NUE
(Nacry et al. 2013). NUE has been defined in many ways. To sum, it is the ratio of
the amount of fertilizer used by the crop to the amount of fertilizer applied. N uptake
and N utilization together contribute to plant NUE. The former is the N-acquiring
capacity of the roots and the latter is the fraction of the acquired N that is converted
to plant biomass (Xu et al. 2012). Achieving synchrony between the two under lim-
iting N conditions is a challenge which plants face to survive.
304 G. Malik et al.
Table 15.1 Non-nodulating and nodule-inducing diazotrophic bacteria from legume root nodules
(source: Martínez-Hidalgo and Hirsch 2017)
Phylum/ Nodule inducing/
class Genus Host non-nodulating
Alpha-proteobacteria
Agrobacterium Sebania, Glycine Nodule inducing
Aminobacter Anthyllis Nodule inducing
Bosea Ononis, Lupinus Nodule inducing
Devosia Neptunia Nodule inducing
Methylobacterium Crotalaria, Listia, Lotononis Nodule inducing
Microvirga Listia, Lupinus, Vigna Nodule inducing
Ochrobactrum Cytisus, Lupinus Nodule inducing
Phyllobacterium Ononis, Sophora Nodule inducing
Shinella Kummerowia Nodule inducing
Azospirillum Trifolium, Phaseolus, Vicia, Medicago Non-nodulating
Gluconacetobacter Glycine Non-nodulating
Ochrobactrum Cicer, Glycyrrhiza, Pisum, Lupinus, Non-nodulating
Vigna
Methylobacterium Arachis Non-nodulating
Beta-proteobacteria
Burkholderia Papilionoid, Mimosoid Nodule inducing
Cupriavidus Mimosa Nodule inducing
Burkholderia Mimosa, Glycine, Arachis, Lespedeza Non-nodulating
Variovorax Crotalaria, Acacia Non-nodulating
Gamma-proteobacteria
Klebsiella Arachis, Glycine, Vicia Nodule inducing
Pseudomonas Hedysarum, Robinia Nodule inducing
Klebsiella Vigna, Arachis Non-nodulating
Pseudomonas Vigna, Arachis Non-nodulating
Pantoea Mimosa, Lathyrus, Lotus, Medicago, Non-nodulating
Melilotus, Robinia, Trifolium, Vicia,
Phaseolus
Actinobacteria
Rhodococcus Anthyllis, Lotus Nodule inducing
Arthrobacter Lespedeza, Pisum, Trifolium Non-nodulating
Brevibacterium Cicer, Cajanus Non-nodulating
Micromonospora Lupinus, Pisum, Medicago, Casuarina Non-nodulating
Streptomyces Pisum, Cicer Non-nodulating
Firmicutes
Bacillus Calycotome, Cicer, Glycine, Oxytropis, Non-nodulating
Pisum, Sophora
Paenibacillus Cicer, Medicago, Lupinus, Prosopis Non-nodulating
15.1.3.1 Root Architecture

Due to the heterogeneous availability of resources, plant roots modulate its physi-
ological structure to maximize resource capture. Root proliferation, that is, produc-
tion of new roots (lateral initiation in particular), occurs in/towards nutrient-rich
patches (Hodge 2004). This phenomenon is enhanced when there is a scarcity of a

particular nutrient. This holds true for NO3−, one of the most important growth-
limiting nutrients. The strength of the N limitation and varied environmental cues
depict the root modifications. For example, under the mild N limitation, the length
of primary and lateral roots is increased, whereas during severe N limitation total
root development is affected, the outcome being shortened primary roots (Lopez-
Bucio et al. 2003). The heterogeneity in resource distribution in soil is a result of
microbial decomposition. Microbial decomposition yields inorganic nutrients read-
ily available for plant capture from both organic and inorganic sources. Root prolif-
eration albeit a slow process eventually outcompetes the microbial population.
NO3− is the major form of N present in aerobic soils and ammonium is the major
form in flooded wetlands or waterlogged soils. NO3 and ammonium are taken up by
the roots of plants via their specific promoters.
15.1.3.2 Nitrate Transporters

Nitrate transporter system can be divided into low-affinity transport system (LATS)
and high-affinity transport system (HATS) based on the supply of cellular energy
(Miller et al. 2007). There are four gene families of nitrate transporters, of which
nitrate transporter 1 (NRT1)/peptide transporter (PTR) family (now renamed as
NPF family) and nitrate transporter 2 (NRT2) are involved in NO3− uptake (Nacry
et al. 2013; Krapp et al. 2014; Fan et al. 2017). NPF family comprises 53 members
in Arabidopsis and 93 members in rice (Oryza sativa L.) (Léran et al. 2014; von
Wittgenstein et al. 2014). Except for AtNPF6.3 (NRT1.1) in Arabidopsis (Ho et al.
2009) and MtNRT1.3 in Medicago truncatula (Morere-Le Paven et al. 2011), which
function as dual-affinity transporters, NPF members suggestively belong to LATS
(Léran et al. 2014). On the other hand, all the NRT2 family members belong to
HATS and generally require a partner protein, namely NAR2 (nitrate assimilation-
related protein), to transport NO3− (Fan et al. 2017). Notably, there are about seven
members of the NRT2 family in Arabidopsis (Miller et al. 2007) and five in rice
genome (Feng et al. 2011).
Under high-NO3− conditions, both the dual-affinity transporters and low-affinity
transporters have been shown to function. However, under N-limiting conditions,
high-affinity transporters of NRT2 family play a major role in NO3− influx through
roots (Fan et al. 2016a, b; O’Brien et al. 2016). Among the seven genes of NRT2
family in Arabidopsis, four, namely AtNRT2.1, AtNRT2.2, AtNRT2.4, andAtNRT2.5,
are predominantly expressed under N-deprived conditions. These transporters con-
tribute to 95% of NO3− influx (Lezhneva et al. 2014). AtNPF6.3, a dual-affinity
transporter, is also expressed under N-limiting conditions; however, its contribution
towards NO3− uptake is almost nonexistent (Glass and Kotur 2013).
The role of nitrate transceptor (transporter/sensor) NRT1.1 is well established in
NO3− sensing (Remans 2006; Ho et al. 2009; Gojon et al. 2011). It works as a dual
transporter of NO3− and auxin in NO3−-rich conditions. There are numerous other
regulatory molecules that are involved in NO3− perception and transportation and
control root developmental activity accordingly. For example, in calcineurin B-like
306 G. Malik et al.
(CBL) interacting protein kinase (CIPK) gene, namely CIPK8, the expression is
NO3− induced. CIPK8 induces downstream nitrate transporter genes, genes required
for nitrate assimilation and also nitrate-induced primary root growth. Another
kinase, CIPK23, phosphorylates NRT1.1, which acts as a high-affinity transporter
when phosphorylated and as a low-affinity transporter when dephosphorylated,
contributing to its dual nature. It is also responsible for localized root proliferation.
This highlights a faint corner of the picture, giving us an idea of the complex nature
of NO3− sensing and how plants are able to actively forage NO3− in a heterogeneous
N environment by transcriptome reprogramming.
15.1.3.3 Ammonium Transporters

Uptake of ammonium is mediated by ammonium transporters (AMTs) or methyl-
ammonium permeases (MEPs). In Arabidopsis, AtAMT family comprises six mem-
bers, all of which are high-affinity transporters (Loque et al. 2006), and in rice, at
least ten AMT homologues are present designated as OsAMT-like transporters fur-
ther subgrouped into two families—OsAMT1 and OsAMT2 (Sonoda et al. 2003;
Suenaga et al. 2003).
In Arabidopsis, under N-limiting conditions, it has been observed that the tran-
script levels of five out of six AMT genes are upregulated. Of these, the first three
genes mentioned account for 90% of high-affinity ammonium uptake from soil
under the same conditions (Yuan et al. 2007). While AMT expression is activated
under N-limited conditions, in rice, maize, and tomato an “ammonium-inducible”
expression is observed (Sonoda et al. 2004; Gu et al. 2013).
15.1.4 Nitrate Sensing and Signaling
Plants often grow in N-deficient soils and they must sense changes in external and
internal concentrations of metabolites and adjust to the nutrient availability for sur-
vival in such nutrient-limited environments. NO3− is the most common form of
nitrogen available to plants; sensing and signal transduction networks that control
plant responses to nutrient deprivation are not well characterized for N. The N and
carbon (C) metabolisms are linked and therefore cross talk between the signal trans-
duction pathways that regulate N assimilation and C metabolism is expected.
Though many studies have been done in the past few decades, the mechanisms by
which N and C signaling is integrated into plants are still not clear. NO3− sensing is
very complex, necessitating the study of the coordinated regulation of multiple met-
abolic and regulatory pathways (including N and C) by NO3− as a signal (Zheng
2009; Nunes-Nesi et al. 2010). Recently, HY5 has been identified as a novel regula-
tory molecule with the ability to maintain C/N status in the plants (Chen et al. 2016).
15.1.4.1 How Plants Perceive N Stress

Nitrate absorbed by NO3− transporters in plants is transported to other organs and it
is also involved in sensing as well as integrated plant responses. The role of NO3− in
modulating a wide range of processes including plant growth, root system
architecture, leaf development, seed dormancy, and flowering time is very well doc-
umented in literature (Alvarez et al. 2012; Krouk et al. 2010; Rahayu et al. 2005;
Alboresi et al. 2005; Marín et al. 2011). Under N deficiency, activities of nitrate/
ammonium transporters can be modulated at both the transcriptional and posttrans-
lational levels by signaling molecules such as Ca2+, ROS, and phytohormones in
plants (Schachtman and Shin 2007). In addition, light, regulatory proteins, down-
stream N metabolites, and epigenetic mechanisms are also shown to be involved in
NO3− sensing and regulation as discussed below. The comprehensive network estab-
lished by the integration of various signaling pathways and the mechanism of regu-
lation need to be investigated further.
15.1.4.2 Candidates in Nitrate Sensing and Signaling

Nitrate as a signal has been proposed for a long time, yet there is still much less
clarity about how the plant monitors its N status and the downstream signaling
events associated with it. Much work has focused on the immediate response of the
addition of nitrate and the induction of nitrogen metabolism. The root responses to
nitrate are well documented, and the transporter NRT2.1 was reported to be involved
in nitrate sensing or transduction in roots. The activity of the enzyme nitrate reduc-
tase (NR), involved in conversion of nitrate to nitrite, is controlled by both its
steady-state levels and reversible protein phosphorylation by the action of protein
phosphatases and kinases. NR activity responds rapidly to light/dark signals. NR is
active and dephosphorylated in the light by protein phosphatases, while it is inactive
and phosphorylated in the dark by sucrose nonfermenting-related kinase (SnRK)
and calcium-dependent protein kinase (CDPK). The light-mediated regulation of
NR gene expression has been studied in depth by Raghuram and Sopory (1995a);
and Lillo and Appenroth (2001). More studies on the phytochrome-mediated regu-
lation of NR gene expression in maize and rice mediated through G-protein
(Raghuram et al. 1999), PI cycle, and protein kinase C (Raghuram and Sopory
1995b) have been reported. The posttranslational regulation of metabolic activity of
nitrate reductase by 14-3-3 is studied in detail (Shin et al. 2011,). Moreover, the
central role of 14-3-3 in maintaining metabolic coordination between multiple
enzymes such as glutamine synthetase, sucrose-phosphate synthase, trehalose-
phosphate synthase, glutamyl-tRNA synthetase, and various signaling molecules of
C/N metabolism also needs to be investigated further (Pathak et al. 2008, 2011).
Similarly, much work has been done on the downstream metabolites of nitrate
assimilation pathway such as nitrite, ammonium ions, glutamine, and other amino
acids, which is beyond the scope of this review. Numerous nitrogen metabolites
have been shown to regulate carbon metabolism to meet the demands of increasing
N flux through 2-OG. Glutamine is another key metabolite linked to nitrogen
metabolism that plays a role in metabolic regulation and possibly signal transduc-
tion (Fallon and Weng 2014). In addition to sensing nitrate, plant cells may also
sense carbon status, which leads to the regulation of key nitrate transporters NRT2.1
and NRT1. Another key metabolite involved in nitrogen metabolism is glutamate
and the presence of multiple glutamate receptors in plant genomes (Price et al.
2012) may indicate the potential importance of this metabolite in initiating signal
transduction cascades.
308 G. Malik et al.
Nitrogen sensing and plant response are also mediated through plant hormones,
though the signaling pathways need to be characterized in detail further. The levels
of ABA decrease under nitrogen sufficiency, while nitrogen deficiency leads to
decreased cytokinin levels to facilitate biomass allocation between roots and shoots.
Auxin synergistically affects cytokinin activity on cell division and organogenesis
(Soni et al. 1995), while ABA antagonizes the cytokinin-mediated nitrogen signal-
ing by negatively regulating cytokinin-inducible response regulator genes.
With the recent whole-transcriptomics studies, more than a thousand genes are
shown to be nitrate responsive and the presence of nitrate response elements (NRE)
has been proposed in nitrate reductase and nitrite reductase from Arabidopsis thali-
ana and birch (Hwang et al. 1997; Warning and Hatchel, 2000). But later they were
shown to be randomly distributed throughout the genome (Raghuram et al. 2006).
There are no further reports of new cis-regulatory elements working independently
or in integration with carbon metabolism (Pathak et al. 2011).
Global transcriptomic studies have also revealed several MADS-box transcrip-
tion factors like ANR1 and a few Myb transcription factors which were found to be
upregulated by nitrogen deprivation and are thought to play important roles in the
signaling of nitrogen deprivation (Schachtman and Shin 2007) but their targets are
yet unknown. There are few other candidates that have been proposed in N signaling
such as Ca2+ and protein kinases/phosphatases in the expression of NR, NiR, and
GS2 transcripts (Sakakibara et al. 1998; Sueyoshi et al. 1999); the target of rapamy-
cin (TOR) signaling pathway; the general control non-derepressible 2 (GCN2) path-
way; the plastidic PII-dependent pathway; and family of plant glutamate receptors
(GLR); however, their specific role in N sensing is not elucidated yet. GLR is pro-
posed to act upstream of a Ca2+ regulated signaling pathway to regulate the plant’s
response to changes in N status (Gent and Forde 2017).
15.1.5 Epigenetic Regulation in N Homeostasis
Many miRNAs are constantly being implicated in novel cellular responses, includ-
ing nutrient response in plants. These are a part of complex epigenetic mechanisms
involved in sensing nutrient limitation and maintaining homeostasis (Paul et al.
2015; Chiou 2007). Till now, very few studies have focused on the role of chromatin
regulation in response to changes in nutrient availability, and thus its potential role
in regulating nutrient homeostasis mainly in P, N, and sulfur (S). Kou et al. (2011)
showed enhanced tolerance to the N-deficiency stress in rice by DNA methylation.
Recently, two nitrate-responsive miRNA, miR393 and miRNA167, along with their
target, auxin receptors, AFB3 and ARF8, respectively, have been shown to regulate
root growth in response to NO3− availability in soils (Vidal et al. 2010). Moreover,
the role of posttranslational chromatin modification in N acquisition in Arabidopsis
has been reported by Widiez et al. (2011) in case of high N-insensitive 9 (HNI9)
which represses NRT2.1 expression by DNA methylation in roots.
The signaling mechanisms also synchronize the mineral status of the plant in
response to the nutrient availability at the root level by altering the expression and/
or activity of the membrane transporters (Liu et al. 2009). Epigenetic mechanisms
involved in this adaptive response definitely will provide new targets for plant
breeding and gene editing.
15.1.6 Cross Talk Between Nitrate and Other Mineral Nutrients
Recently, many reports have indicated that nitrate signaling has a close relationship
with other nutrients at the transcriptional as well as posttranslational level. Initially,
ROS signaling may be induced in any of the nutrient deficiencies involving N, P, K,
or S (Schachtman and Shin 2007). Lin et al. (2008) have reported that in Arabidopsis,
K+ deficiency not only induces the expression of K+ transporter genes but also alters
the transcription of nitrate transporter genes. This indicates that a cross talk may
exist among different signaling pathways for plant responses to different nutrient
deficiencies.
15.1.7 Use of Microbiome as Bioinoculants or Biofertilizers
There has been a constant demand to increase agricultural productivity due to

increase in human population and environmental pressure. Green revolution paved
the road to modern practices involving the use of N fertilizers. The use of N fertil-
izers has increased from 10 to 100 million metric tons from 1960 to 2005 in devel-
oped and developing nations (Ogburn 2010). Despite evident advantages of N
fertilizers, today a situation has come where this abuse has influenced the global N
balance and placed our ecosystems into a precarious condition. It is a threat to cli-
mate, human health, fish, and other wildlife through run off, and not only this, there
are associated risks of explosion with storage of N fertilizers. A lot of money and
energy are invested each year in research to minimize the use or get rid of these
fertilizers and there are not many alternatives. Improving plant N uptake and assimi-
lation by genetic improvement (through conventional breeding and indirect manipu-
lation of quantitative trait loci) seemingly over simplistic is rather unrealistic as not
every plant can be modified and there is a threshold limit to improvement. The other
suitable way is to modify soil microflora.
There are many reports where beneficial effects of PGPR have been shown in
improving plant growth and yield, even though the mechanisms used to directly or
indirectly promote plant growth are not fully understood yet. PGPR can be used as
either biofertilizers, phytostimulants, or biopesticides based on their mode of action.
It is known that many PGPR often use more than one mode for improving plant
growth and the utilization of microbial inoculants can play an important role in
sustainable agriculture (Bhattacharyya and Jha 2012; Glick 2012; Nehra and
Choudhary 2015). Many of the PGPRs like Azospirillum, Azotobacter, Bacillus,
Burkholderia, Pseudomonas, Rhizobium, and Serratia have been used for large
scale commercially yet successful commercial-scale production of numerous new
PGPR like Azoarcus, Exiguobacterium, Methylobacterium, Paenibacillus, and
Pantoea with substantial beneficial activities is yet to be achieved. These PGPR can
offer excellent solutions for enhanced yields and production of staple food crops.
The use of PGPR for enhanced plant growth is summarized in Table 15.2.
310 G. Malik et al.
Table 15.2 Effects of addition of PGPPR as biofertilizers, phytostimulants, and biopesticides

S. Bacterial Effect on plant
no. Plant consortium Role/abilities growth References
1. M. truncatula Pseudomonas Biofertilizer Significantly Kisiel and
brassicacearum enhance fresh and Kępczyńska
KK 5 and dry weight of root, (2016)
consortium of shoots, and whole
Pseudomonads, 5-week-old
Bacillus sp, seedlings
Xanthomonas
sp.
2. Maize Serratia Biofertilization Increase in maize Lalande et al.
liquefaciens, (N), biocontrol yield by 14% (dry (1989)
Bacillus sp. (several root wt.) when
Pseudomonas pathogens) inoculated as
sp. (individual consortia, S.
and consortia liquefaciens
experiments) increased the dry
weight of maize
by more than
10%, Bacillus sp.
by more than 7%
and Pseudomonas
sp. by more than
10%
3. Cotton Klebsiella Seeds, In addition to Yue et al.
oxytoca Rhizosphere significant (2007)
increase in height
and dry weight of
cotton plants,
inoculation with
PGPR uptake of
major nutrients
like N, P, K, and
Ca increased
while Na deceased
4. Wheat Pseudomonas Rhizosphere Significant Zahir et al.
putida, P. increase in plant (2009)
aeruginosa, S. height, root
proteamaculans length, and
chlorophyll
content
5. Glycine max, B. japonicum, Rhizosphere BNF, promote Bai et al.
Zea mays L. Azospirillum seed germination (2003)
brasilense, and early seedling
Pseudomonas, growth
Bacillus
subtilis, B.
thuringiensis,
Aeromonas sp,
Serratia sp
(continued)
6. Pinus AM fungi, Roots Stimulates root Kohler et al.
sabiniana, free-living N2 colonization, (2010)
Solanum fixing bacteria BNF, increases
lycopersicum, like biomass, limits
Lactuca Azospirillum soil salinity stress,
sativa, and brasilense and affects plant
Zea mays or Azotobacter yield
7 Zea mays Pseudomonas Roots Stimulates plant Egamberdiyeva
sp, Bacillus, growth, N, P, and (2007)
Mycobacterium K uptake in
nutrient-deficient
soil
8 Lycopersicon Bacillus subtilis Rhizosphere Co-inoculation Felici et al.
esculentum strain 101 shows more plant (2008)
Azospirillum height, node
brasilense number, and total
sp. 245 biomass
9. Artichoke Pseudomonas Rhizosphere Phosphate- Jahanian et al.
(Cynara putida, solubilizing (2012)
scolymus) Azospirillum, bacteria along
Azotobacter with nitrogen-
fixing bacteria led
to significant
increase in radicle
and shoot length,
shoot weight,
coefficient of
velocity of
germination,
seedling, vigority
index, and
significant
decrease in mean
time of
germination
10. Brassica Pseudomonas Rhizosphere Increased Ma et al.
juncea, sp. A3R3 significantly the (2011)
Alyssum biomass (B.
serpyllifolium juncea) and Ni
content (A.
serpyllifolium) in
plants grown in
Ni-stressed soil
(continued)
312 G. Malik et al.
11. Lupinus Bradyrhizobium Rhizosphere Increased both Dary et al.
luteus sp. 750 biomass and N (2010)
Pseudomonas content,
sp., accumulation of
Ochrobactrum metals
cytisi (phytostabilization
potential)
12. Mung bean Ochrobactrum Rhizosphere Lower the toxicity Faisal and
Bacillus cereus of cadmium to Hasnain (2005)
seedlings by
reducing Cr (VI)
to Cr (III)
13. Brassica Xanthomonas Rhizosphere Stimulated plant Sheng and Xia
napus sp. RJ3, growth and (2006)
Azomonas sp. increased
RJ4 cadmium
Pseudomonas accumulation
sp. RJ10
Bacillus RJ31
14. Peanut P. fluorescens Rhizosphere Enhanced pod Dey et al.
(Arachis strains PGPR1, yield (23–26%), (2004)
hypogaea L.) PGPR2, PGPR4 haulm yield and
nodule dry weight,
increased root
length, pod
number
15. Brassica Pseudomonas Rhizosphere Increased the Ma et al.
juncea, B. sp. SRI2, biomass of the test (2009a)
oxyrrhina Psychrobacter plants and
sp. SRS8, enhanced Ni
Bacillus sp. accumulation in
SN9 plant tissues
16. B. juncea, B. Psychrobacter Rhizosphere Enhance the metal Ma et al.
oxyrrhina sp. SRA1, accumulation in (2009b)
Bacillus cereus plant tissue by
SRA10 facilitating the
release of Ni from
non-soluble phase
in the soil
17. Maize Pseudomonas Rhizosphere Promoted plant Braud et al.
aeruginosa, P. growth, facilitated (2009)
fluorescens, soil metal
Ralstonia mobilization,
metallidurans enhanced Cr and
Pb uptake
(continued)
18. Lupinus Bradyhizobium Rhizosphere Increased both Dary et al.
luteus sp. 750 biomass and N (2010)
Pseudomonas content,
sp., accumulation of
Ochrobactrum metals
cytisi
19. L. sativa P. putida Rhizosphere Significant Rekha et al.
CCR2–4, increase in shoot (2007)
Bacillus subtilis length and root
CC-pg104 length achieved
through
encapsulated
inoculant
20. Maize P. putida strain Rhizosphere Significant Gholami et al.
R-168, P. increase in plant (2009)
fluorescens height, shoot dry
strain R-93, P. weight, seed
fluorescens, weight, number of
DSM 50090, P. seeds per ear and
putida leaf area
DSM291,
Azospirillum
lipoferum DSM
1691, A.
brasilense
DSM 1690
21. Cicer Escherichia Rhizosphere Increased plant Dasgupta et al.
arietinum L. coli, P. height and leaf (2015)
fluorescens, numbers and
Burkholderia sp biomass
22. Solanum Bacillus Rhizosphere Dry biomass Adesemoye
lycopersicum, subtilis, increased 31% for et al. (2008)
Abelmoschus Pseudomonas tomato, 36% for
esculentus okra, and 83% for
African spinach
23. Cotton Azotobacter, Rhizosphere Seed yield (21%), Anjum et al.
Chroococum, plant height (5%), (2007)
Azospirillum and microbial
lipoferum population in soil
(41%) increased
over their
respective controls
while boll weight
and staple length
remained
statistically
unaffected
(continued)
314 G. Malik et al.
24. Cajanus Bacillus Rhizosphere Increase in plant Rajendran
cajan subtilis, fresh weight, et al. (2008)
Bacillus chlorophyll
pumilus, content, nodule
Rhizobium sp.
IC3 123
25. Rice Bacillus sp. Roots Promote root and Beneduzi et al.
Paenibacillus shoot growth (2008)
sp. significantly
26. Wheat, Bacillus cereus Roots All bacterial Cakmakci
spinach RS18 B. strains were et al. (2007)
licheniformis effective in IAA
RC08 production
27. Mustard Pseudomonas Rhizosphere Rhizosphere Rajkumar et al.
sp., Bacillus sp. stimulated plant (2006)
growth and
decreased CR (VI)
content
28. O. sativa Azospirillum Rhizosphere Increased rice Thakuria et al.
brasilense, grain yield, (2004)
Bacillus maximum up to
pantothenticus 76.9%
29 T. aestivum K. pneumonia Rhizosphere Significantly Sachdev et al.
strains K11 and increased root (2009)
K42 length and shoot
height of
inoculated wheat
seedlings over the
control
30. Achyranthes P. aeruginosa Endophytic Increased N, P, Devi et al.
aspera L. AL2-14B and K contents in (2017)
plant by 3.8,
12.59, and
19.15%,
respectively.
Significant
enhancement of
shoot and root
length, dry leaf,
dry shoot and dry
root weight, and
leaf surface area
as compared to
control
15.2 Conclusion
The knowledge of phytomicrobiome is essential and is necessary to enhance sus-

tainable food production and mitigate environmental challenges. The excessive use
of fertilizers is disturbing the natural balance between phytomicrobiome and plants.
Beneficial effects of phytomicrobiome in improving plant growth and yield have
been shown, though the mechanisms are not fully understood yet. The immediate
challenge is implementing sustainable cropping systems and practices with man-
agement of phytomicrobiome along with plant breeding efforts to support a new
Green Revolution that ensures food security for future generations. The agronomic
practices should focus on the management of soil microbes adjusted to specific
plant genotypes in specific environments. The focus should be on designing new
strategies to utilize beneficial contribution of phytomicrobiomes to improve the
crop yields and also exhaustive screening of plant varieties which can be optimized
for higher yield using defined microbial composition. A deeper understanding on
the role of phytomicrobiomes of crops in nitrogen and other mineral stresses along
with suitable crop as well as soil management practices will offer solutions for
increased agricultural production.
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Halotolerant Microbes for Amelioration
of Salt-Affected Soils for Sustainable 16
Agriculture
Sanjay Arora
Abstract
Soil salinity is one of the major abiotic stresses that adversely affect the sustainable
agricultural production globally. About 20% of the total land area is affected by
salinity, and the area is increasing at an alarming rate. There is a damaging effect
of salinity on soil microbial communities, and their activities have been reported in
majority of the studies. Excess accumulation of salts in the root zone often deterio-
rates the soil properties, viz. physical, chemical and biological to such an extent
that crop production is adversely affected. Also, salt-affected soils are poor in
organic matter content and thus the biomass as well as microbial activity, thereby
affecting the microbiologically mediated processes required for plant growth. The
methods available for reclamation of salt-affected soils are not cost effective, and
further the availability of good-quality waters required for leaching salts in saline
soils and mineral gypsum or organic amendments for sodic soils is scarce.
Halotolerant and halophilic microorganisms having plant growth-promoting (PGP)
traits have the potential to assuage salt stress and enhance plant growth and produc-
tion in salt-affected soils. These plant growth-promoting rhizobacteria (PGPR) tol-
erate wide range of salt stress and thus enable plants to withstand salinity by
different mechanisms such as hydraulic conductance, osmotic accumulation,
sequestering toxic Na+ ions, maintaining the higher osmotic conductance and pho-
tosynthetic activities. The halophilic microbes have the potential to influence direct
growth promotion of plants by fixing atmospheric nitrogen, solubilizing insoluble
nutrients and secreting hormones such as IAA, GAs and kinetins besides ACC
deaminase production, which helps in regulation of ethylene. Some of the recent
researchers have confirmed the possibility of using halophiles in recovery of salt-
affected soils and sustain agricultural production in degraded lands. We also
S. Arora (*)
ICAR-Central Soil Salinity Research Institute, Lucknow, Uttar Pradesh, India

324 S. Arora
observed beneficial effects of using PGP halophilic bacteria isolated from the
native salty soils for enhancing crop production under salt stress conditions. For
easy application in agriculture, liquid bioformulations have been prepared for effi-
cient strains, and their use has enhanced the yield of rice and wheat by 11–14% and
also for other crops like mustard, vegetables and fodder crops under salt stress
conditions. Therefore, the bioremediation approach being cheap and eco-friendly
is being promoted to optimize crop yields under sodic and saline-sodic soils of the
Indo-Gangetic plains of north India.
Keywords
Soil salinity · Plant growth-promoting rhizobacteria (PGPR) · Halophiles
16.1 Introduction
The productivity of agricultural crops is constrained by several environmental biotic

and abiotic stresses that have adversely affected the area under cultivation, crop
productivity and quality. Amongst the different abiotic stresses, soil salinization,
soil pH, acidification, drought and temperatures are the major restrictive factors in
sustaining crop production. Soil salinity is a major issue for agriculture because
high concentration of salt turns useful lands into unproductive areas. Thus, soil sali-
nization has been recognized as one of the most devastating soil degradation threats
on the Earth. Globally, soil salinity has affected almost 1 billion ha of land area,
representing about 7% of Earth continental extent, with about 1–3 million ha land
area in Europe, about 850 million ha in Asia and 104 million ha in Pacific subregion
(Rengasamy 2006; Ladeiro 2012). In India, about 6.73 million ha of land is salt
affected which spreads in 194 districts and represents 2.1% of the total geographical
area of the country (Mandal et al. 2009). Out of these, 2.8 million ha of area is sodic
in nature and mainly occurring in the Indo-Gangetic alluvial plains of north India.
It has also been estimated that globally, about 20% of total agricultural land is
affected by high salinity, and these saline areas are increasing at the rate of 10%
annually due to low rainfall, high surface evaporation, weathering of native rocks,
irrigation with poor (saline) quality water and poor cultural practices, especially in
arid and semi-arid regions. Furthermore, it is expected that about more than 50% of
the arable lands would turn salinized by 2050 (Jamil et al. 2011).
The agricultural crops under salinity exhibit a variety of responses that range from
decline in crop yields due to alterations in physicochemical properties of the soil to the
disturbance in ecological balance of the region. Salinity is thus the major cause of land
abandonment and aquifers for agricultural purposes and a foremost factor for reduc-
ing agricultural productivity. The impacts include poor crop productivity, low eco-
nomic returns and erosion of soils (Hu and Schmidhalter 2002). The poor crop
productivities are due to complex interactions amongst morphological, physiological
and biochemical processes because of salinity-induced limited water and nutrient
uptakes throughout the crop growth cycle (Akbarimoghaddam et al. 2011; Singh and
16 Halotolerant Microbes for Amelioration of Salt-Affected Soils for Sustainable… 325
Chatrath 2001). The limited uptake of water and nutrients affects almost every devel-
opmental stage of the crop plant with osmotic and oxidative stress, nutrient (N, Ca, K,
P, Fe and Zn) deficiency and toxicity of ions (Munns 2002). Ion toxicity through the
accumulation of excessive salt ions in the cell walls leads to osmotic stress, causing
replacement of K+ by Na+ in biochemical reactions inducing conformational changes
in proteins. Likewise, enzyme activities during the developmental stages get affected
as K+ ions, which act as cofactors and are required for binding tRNA to ribosomes.
Substitution of K+ by Na+ also adversely affects the protein synthesis (Zhu 2002).
Metabolic imbalance, caused by ion toxicity and osmotic stress, in turn, leads to oxi-
dative stress (Chinnusamy et al. 2006), resulting in osmotic balance failure, loss of
turgidity and dehydration of cell, and eventually culminating in cell death.
The major causes of naturally induced salinity are salt water intrusion and salt depo-
sition through salt-laden winds. Salts also originate from weathering of minerals and
anthropogenic factors like irrigation of crops with salty waters through which salt accu-
mulates in soil and salinization gets accelerated. The other factors include injudicious
use of inorganic fertilizers and soil amendments like gypsum, composts, manures, etc.
Salinization results in inhibition of plant growth that results due to accumulation of
dissolved salts in soil water. The major water-soluble salts that accumulate in the soil
include potassium (K+), magnesium (Mg 2+), calcium (Ca 2+), chloride (Cl−), sulphate
(SO42−), carbonate (CO32−), bicarbonate (HCO3−) and sodium (Na+) ions. The soil
solutions differ in dissolved salt contents, and when the concentration of salts in terms
of electrical conductivity (ECe) exceeds 4 dSm−1 in the soil, these are categorized as
salt affected (Abrol et al. 1988). A saline soil is defined as the soil having a high con-
centration of soluble salts (ECe > 4 dSm−1) that are enough to affect the growth and
development of plants. However, many crops are affected by soil with an ECe < 4 dSm−1.
Excessive sodium (Na+) salt accumulation destroys soil structure, deteriorates soil
hydraulic properties, increases soil pH and reduces water infiltration and soil aeration,
leading to compaction of soil and thereby increasing erosion and higher water run-off.
Furthermore, sodium, being the most prominent destructor of secondary clay minerals
by dispersion, replaces calcium (Ca2+) and other coagulators like Mg2+ and gets
adsorbed on the surface and/or interlayers of soil aggregates (Ondrasek et al. 2010).
Dispersion of clay particles undergoes leaching through the soil to accumulate and
block pore spaces, especially in fine-textured soil horizons. These lands are thus
degraded in structural, chemical, nutritional, hydrological and microbiological char-
acteristics. The degraded sodic soil thus becomes unsuitable for proper root growth
and plant development. The secondary result is salinity-induced sodicity, where leach-
ing either through natural or human-induced processes washes away the soluble salts
into the subsoil and leaves negative charges of sodium bound to the clay.
16.2 Reclamation and Management of Salt-Affected Soils
To improve crop growth in saline soils, the excess salts need to be removed from the
root zone. Leaching is one of the most effective methods for removing salts beyond
the root zone. Leaching is accomplished by ponding fresh water on the soil surface
326 S. Arora
and allowing it to infiltrate, and it is effective only when the salty drainage water is
discharged through drains out of the area under reclamation. The process of leach-
ing may reduce salinity levels in the absence of artificial drains when there is suffi-
cient natural drainage, i.e. the ponded water drains without raising the water table.
It is preferred to leach when the soil moisture content is low and the groundwater
table is deep.
Sodic or alkali soils are generally reclaimed using mineral gypsum along with
organic amendments/manures. The availability of mineral gypsum and also manures
is scarce these days. Also the estimation of gypsum requirement of sodic soil is
tedious, and most of the soil testing laboratories either do not have facility for gyp-
sum requirement or lack expertise in estimation, so a model was developed to esti-
mate gypsum requirements based on soil pH value. The mobile application
“GypCal” in Hindi and English was developed to promote judicious use of chemical
amendment gypsum for reclamation of sodic soil using soil pH as input, and this
application is made freely available for download through Google Play Store.
Both physical and chemical methods for reclamation of saline and sodic soils
are not cost-effective, and on the other hand, organic crop production is being
promoted. The microbial strains available as bio-fertilizers for different crops do
not perform effectively under salt stress, and their activity decreases when used in
salt-affected soils due to osmolysis. The soils of vast areas of Indo-Gangetic
plains in north India are sodic or saline-sodic. The halophilic plant growth-pro-
moting microbes have potential to ameliorate these soils. The halophilic bacterial
strains can help in recovery of salt-affected soils by directly supporting vegetation
growth thus indirectly increasing crop yields under salt stress conditions.
Halophilic plant growth-promoting bacteria have high potential for remediation
of salt-affected soils and enhancing productivity of crops especially paddy, mus-
tard and wheat.
16.3 The Effect of Salinity on the Soil Microorganisms
The soil microbial communities perform a fundamental role in nutrient cycling,

decomposition of organic matter in the soil and in maintaining plant productivity. It
is therefore important to understand the microbial response to environmental stress.
Stress can be damaging for sensitive microorganisms and decrease the activity of
surviving cells, due to the metabolic load imposed by the need for stress tolerance
mechanisms. In a dry hot climatic condition, the low humidity and soil salinity are
the major stressful factors for the soil microbial communities, and these stresses
frequently occur simultaneously. Excessive accumulation of salts in soil hampers
the growth and activity of soil microflora thereby affecting the population of N2-
fixing and phosphate-solubilizing bacteria which lead to low soil fertility. Due to
increased quantity of salts, the microbial flora is worst affected; this also interfered
with nitrogen-fixing and phosphate-solubilizing ability of bacteria. The salinity
effect is always more pronounced in the rhizosphere according to the increase in
water absorption by the plants due to transpiration. This is so as life in high salt
concentrations has a high bioenergetic taxation, because the microorganisms need

to maintain osmotic equilibrium between the cytoplasm and the contiguous medium,
excluding sodium ions from inside the cell which require sufficient energy for
osmo-adaptation.
16.3.1 Salinity Impacts on Rhizosphere Microbes
Soluble ion concentrations (especially sodium ion) greater than about 0.15 M ions
in soil lead to hyperosmotic conditions which force water to diffuse out of a micro-
bial cell. The cells will then shrink or plasmolyse. In addition, the high sodium ion
concentration also causes the water associated with such solutes to become unavail-
able to microorganisms. Basically, the effect of sodium ion on the growth of micro-
organisms of different species will differ due to growing water activity of each
microorganism.
Bacteria are adsorbed onto soil particles by ion exchange, and a soil is consid-
ered to be naturally fertile when the soil organisms are releasing inorganic nutrients
from the organic reserves at a rate sufficient to sustain rapid plant growth. Since the
soil organic matter and consequently the biomass and microbial activity are gener-
ally more relevant in the surface layer of the soil, salinization close to the surface
significantly affects a series of microbiologically mediated processes. Along with it
disturbs the natural ecosystem functioning and plant health. For rhizobacteria, life
in high salt concentrations is difficult as they need to maintain an osmotic balance
between their cytoplasm and the surrounding medium while excluding Na+ ions for
which sufficient energy is required for adaptation. Depletion of potassium ions by
plants under saline conditions further reduces the ability of rhizobacteria to use
potassium ions as a primary osmoregulator. Plant use of osmolytes under salt stress
deprives rhizobacteria of osmolytes, which finally limits the bacterial growth. The
salinity level above 5% thus reduces the total population of bacteria and actinobac-
teria drastically. In addition, it inhibits nitrogen fixation, root exudation and decom-
position of organic matter. Negative correlations between soil electrical conductivity
and carbon dioxide emission or microbial biomass C suggested that it has a severe
adverse effect on microbial biomass and activities. Naturally occurring soil organic
matter decomposers thus become sensitive to salt-induced stress, and the effect is
always more pronounced in the rhizosphere pursuant to increased water uptake by
the plants due to transpiration. Alteration of proteins, exo-polysaccharide and lipo-
polysaccharide composition of the bacterial cell surface, impairment of molecular
signal exchange between bacteria and their plant host due to the alteration of mem-
brane glucan contents and inhibition of bacterial mobility and chemotaxis towards
plant roots significantly affect microbial diversity in the rhizosphere, under saline
conditions. Overall, salinity has a negative impact on microbial abundance, diver-
sity, composition and functions.
328 S. Arora
16.4 Soil Salinity Effects on Plant Growth and Development
All the major plant growth processes such as germination, cell division and elonga-
tion, leaf growth, leaf expansion, photosynthesis, protein synthesis, energy and lipid
metabolism are adversely affected under salt stress. During the vegetative stages,
salt stress induces stomatal closure, leading to reduction in CO2 assimilation and
transpiration. The reduced turgor potentials affect the leaf expansion and leaf area,
which in turn reduces the light interception and photosynthetic rates, coupled with
spurt in respiration resulting into reduced biomass accumulation. Water potential of
the soil is reduced due to excessive salts, thus, making the soil solution unavailable
to the plants and creating physiological drought. Further, osmotic pressure in the
rhizosphere solution exceeds in root cells thereby reducing water and nutrient
uptake. Salinity further creates nutritional imbalance through increase in uptake of
Na+ or decrease in uptake of Ca2+ and K+ in leaves. Excess Na+ causes metabolic
disturbances in processes where low Na+ and high K+ or Ca2+ are required for opti-
mum growth and developmental functions. Excess sodium and more importantly
chlorides affect plant enzymes and cause cell swelling, resulting in reduced energy
production and other physiological changes. The uptake and accumulation of Cl−
disrupt the photosynthetic function through inhibition of nitrate reductase activity.
Under excessive Na+ and Cl− rhizosphere concentrations, competitive interactions
with other nutrient ions (K+, NO3−and H2PO4−) occur for binding sites and transport
proteins in root cells that have adverse effects on translocation, deposition and par-
titioning within the plant. Once the capacity of cells to store salts is exhausted, salt
build-up in intercellular space leads to cell dehydration and death. Plants suffer
from membrane destabilization and a general nutrient imbalance. All micro- and
macronutrient contents decrease in roots and shoots with increasing NaCl concen-
tration in the soil. Osmotic stress decreases cell growth and development, reduces
leaf area and chlorophyll content, accelerates defoliation and senescence and
reduces the yields. The primary salinity effects give rise to numerous secondary
ones such as oxidative stress, characterized by accumulation of reactive oxygen spe-
cies potentially harmful to bio-membranes, proteins, nucleic acids and enzymes.
The plants with perturbed nutrients relations are more susceptible to invasion of
different pathogenic microorganisms and physiological dysfunctions, whereas their
edible parts have markedly less economic and nutritional value due to reduced fruit
size and shelf life, non-uniform fruit shape and decreased vitamin contents.
16.5 Halotolerant Microbes
The halotolerant and halophilic microorganisms are those which can grow in hyper-
saline environments, while only halophiles specifically require at least 0.2 M of salt
for their growth. Halotolerant microorganisms can only tolerate media containing
<0.2 M of salt. Distinctions between different kinds of halophilic microorganisms
are based on their level of salt requirement and salt tolerance. The halotolerant
microorganisms grow best in media containing <0.2 M (∼1%) salt and may also
tolerate high salt concentrations. This definition is widely referred to in many
reports (Arahal and Ventosa 2002; Ventosa et al. 1998; Yoon et al. 2003).
Bacteria inhabiting soil play a role in conservation and restoration biology of
higher organisms. The domain bacteria contain many types of halophilic and halo-
tolerant microorganisms, spread over a large number of phylogenetic groups
(Ventosa et al. 1998). The different branches of the Proteobacteria contain halo-
philic representatives often having close relatives that are nonhalophilic. Similarly,
halophiles are also found amongst the cyanobacteria (Oren 1999), the
Flavobacterium-Cytophaga branch, the Spirochetes and the Actinomycetes. Within
the lineages of Gram-positive bacteria (Firmicutes), halophiles are found both
within the aerobic branches (Bacillus and related organisms) and within the anaero-
bic branches. Most halophiles within the domain bacteria are moderate rather than
extreme halophiles in general. However, there are a few types that resemble the
archaeal halophiles of the family Halobacteriaceae in their salt requirements and
tolerance. There is abundance of halophilic bacteria in saline soil, and the dominant
types encountered in saline soil belong to genera of Alcaligenes, Bacillus,
Micrococcus and Pseudomonas (Rodriguez-Valera 1988). It was reported that halo-
tolerant Gram-positive endospore-forming rods isolated from saline soils and sedi-
ments of salterns located in different areas were assigned to the genus Bacillus. The
majority of them were classified as extremely halotolerant microorganisms as they
are able to grow in most cases in up to 20% or 25% salts (Garabito et al. 1998).
Many of the halotolerant microbial species have been isolated and identified
such as Azotobacter, Azospirillum, Phosphobacter and blue-green algae from
marine aquatic sediments. The bacterial sequences were assigned into 5784 opera-
tional taxonomic units (OTUs, based on ≥97% sequence identity), representing 24
known bacterial phyla, with maximum of Proteobacteria (44.9%) followed by
Actinobacteria (12.3%), Firmicutes (10.4%), Acidobacteria (9.0%), Bacteroidetes
(6.8%) and Chloroflexi (5.9%) being predominant. Bacterial genus Lysobacter
(12.8%) was the dominant in saline soils followed by Sphingomonas (4.5%),
Halomonas (2.5%) and Gemmatimonas (2.5%). Archaeal sequences were assigned
to 602 OTUs, primarily from the phyla Euryarchaeota (88.7%) and Crenarchaeota
(11.3%). Halorubrum and Thermofilum were the dominant archaeal genera in saline
soils. Rarefaction analysis indicated less than 25% of bacterial diversity and approx-
imately 50% of archaeal diversity, in saline soil.
These microorganisms have developed mechanisms to survive in such adverse
media and many endemisms. The halophilic microorganisms are thus also called
“salt-loving” microorganisms living in environments with high salt concentration
that would kill most other microbes. Microorganisms under hypertonic environ-
ments (low water activity) either die or remain dormant except halotolerant and
halophilic microorganisms that can overcome this problem. Generally, high salt
concentration can interfere with the growth and activity of soil microbes; hence it
indirectly affects the availability of nutrients to plants under salt stress.
330 S. Arora
16.5.1 Mechanisms for Halotolerance
Halotolerance is the adaptation of living organisms to conditions of high salinity.

High osmolarity in hypersaline conditions can be deleterious to cells since water is
lost to the external medium until osmotic equilibrium is achieved. Many microor-
ganisms respond to enhanced osmolarity by accumulating osmotica in their cytosol,
which protects them from cytoplasmic dehydration (Yancey et al. 1982). All micro-
organisms have to keep their cytoplasm at least isoosmotic with their environment
to prevent loss of cellular water; when a turgor pressure is to be maintained, the
cytoplasm should even be slightly hyperosmotic. Adaptation to conditions of high
salinity has an evolutionary significance. The concentration of brines during prebi-
otic evolution suggests haloadaptation at earliest evolutionary times (Dundas 1998).
Osmophily is related to the osmotic aspects of life at high salt concentrations, espe-
cially turgor pressure, cellular dehydration and desiccation. Halophily refers to the
ionic requirements for life at high salt concentrations.
Salt-tolerant or halophilic microorganisms usually adopt either of the two strate-
gies of survival in saline environments: ‘compatible solute’ strategy and ‘salt-in’ strat-
egy (Ventosa et al. 1998). The cell volume is maintained when an isoosmotic balance
with the medium is achieved. The majority of moderately halophilic and halotolerant
bacteria, some yeasts, algae and fungi, employ the compatible solute strategy wherein
the cells maintain low concentrations of salt in their cytoplasm by balancing osmotic
potential through the synthesis or uptake of organic compatible solutes and exclusion
of salts from the cytoplasm as much as possible. The compatible solutes or osmolytes,
small organic molecules that are soluble in water to molar concentrations, that accu-
mulate in halophiles are available in great spectrum and used in all three domains of
life. These are assigned in two classes of chemicals, i.e. (1) the amino acids and their
derivatives, such as glycine betaine, glutamine, glutamate, proline, ectoine or N-acetyl-
β-lysine, and (2) polyols, e.g. glycine betaine, ectoine, sucrose, trehalose and glycerol,
which do not disrupt metabolic processes and have no net charge at physiological
pH. The accumulation can be accomplished either by uptake from the medium or by
de novo synthesis (Shivanand and Mugeraya 2011).
The salt-in strategy is generally employed by true halophiles, including halo-
philic archaea and extremely halophilic bacteria. These microorganisms are adapted
to high salt concentrations and cannot survive when the salinity of the medium is
lowered (Arora et al. 2014a). They generally do not synthesize organic solutes to
maintain the osmotic equilibrium. In this adaptation, the intracellular K+ concentra-
tion is generally higher than that of the outside, the intracellular Na+ concentration
is generally lower than that in the medium, and the intracellular K+ concentration
increases with increasing external concentration of NaCl in a non-linear pattern. All
the halophilic microorganisms contain potent transport mechanisms, which is gen-
erally based on Na+/H+ antiporters (Oren 1999).
Halobacillus is one of the first chloride-dependent bacteria reported, and several
cellular functions depend on chloride ion for maximal activities, the most important
being the activation of solute accumulation. Halobacillus switches its osmolyte
strategy with the environmental salinity by the production of different compatible
solutes. Glutamate and glutamine dominate at intermediate salinities, and proline

and ectoine dominate at high level of salinities. Chloride ion stimulates the gluta-
mine synthetase thereby activating the enzyme and the product glutamate and then
turns on the biosynthesis of proline by inducing the expression of the proline bio-
synthetic genes. Halobacillus dabanensis is used as a model organism to know
about the genes involved in halotolerance, including genes encoding Na+/H+ anti-
porters as well as enzymes involved in osmotic solute metabolism and stress
proteins.
16.5.2 Vesicular Arbuscular Mycorrhiza (VAM)
There are reports of common occurrence of vesicular arbuscular mycorrhizal fungi

commonly called as VAM in natural and saline environment. Relationships between
soil salinity and the presence of VAM have been investigated by several researchers.
It has been reported that the number of VAM spores or infectivity of VAM fungi
changed with change in salt concentration (Juniper and Abbott 1993). The stresses
due to saline soils effect the growth of plants, fungus or both.
VA mycorrhizal fungi species that most commonly observed in saline soils are
Glomus spp. (Juniper and Abbott 1993), and this suggests that this may be adapted
to grow in saline conditions. There is evidence that the distribution of VAM species
is markedly changed with increase in salt concentration (Stahl and Williams 1986).
It has been observed by Aliasgharzadeh et al. (2001) that the most predominant spe-
cies of arbuscular mycorrhizal fungi (AMF) in the severely saline soils of the Tabriz
plains were Glomus intraradices, G. versiform and G. etunicatum. Few studies have
indicated that the mycorrhizal fungi can increase growth of plants growing in saline
habitats (Yadav et al. 2017). The VA mycorrhizal fungi have the ability to guard
plants from salt stress although the mechanism is not fully known. At present it is
suggested that fungi do have a potential to enhance plant growth by increasing nutri-
ent uptake. The efficacy of three species of AMF—Glomus mosseae, G. intraradi-
ces and G. claroideum—was evaluated to alleviate salt stress in nursery of olive
trees (Porras-Soriano et al. 2009). It was observed that G. mosseae was the most
efficient fungus in terms of olive tree performance and protection against the detri-
mental effects of salinity. These findings suggest that the capability of AMF in pro-
tecting plants from the detrimental effects of salt stress may depend on the behaviour
of the species.
16.6 Applications of Halophilic Bacteria
There is high potential for biotechnological applications of halophilic bacteria for

two main reasons: (1) their participation in biogeochemical processes of C, N, S and
P, the formation and dissolution of carbonates, immobilization of phosphate and
production of growth factors and nutrients (Rodriguez-Valera et al. 1985) and (2)
their simple nutritional requirements. The majority of halophilic bacteria can use a
332 S. Arora
wide range of compounds as their sole carbon and energy source. Most of them can
grow at high salt concentrations, minimizing the risk of contamination. Moreover,
several genetic tools developed for the nonhalophilic bacteria can be applied to the
halophiles, and thus their genetic manipulation seems feasible (Ventosa et al. 1998).
Halophilic bacteria have the ability to produce compatible solutes, which are
useful for the biotechnological production of the osmolytes. Some compatible sol-
utes, especially glycine, betaines and ectoines, may be used as stress protectants
(against high salinity, thermal denaturation, desiccation and freezing) and stabiliz-
ers of enzymes, nucleic acids, membranes and whole cells. There are many indus-
trial applications of these compounds in enzyme technology. The other compatible
solutes such as trehalose, glycerol, proline, ectoines, sugars and hydroxyectoine
from halophilic bacteria showed the highest efficiency of protection of lactate dehy-
drogenase against freeze-thaw treatment and thermal stress. Halophilic bacteria can
produce enzymes that have optimal activity at high salinity, which is advantageous
for harsh industrial processes.
Also, halophilic bacteria produce a number of extra- and intracellular enzymes
and antimicrobial compounds that are currently of commercial interest (Kamekura
and Seno 1990). The application of halophilic bacteria in environmental biotechnol-
ogy is possible for the (1) recovery of saline soil, (2) decontamination of saline or
alkaline industrial wastewater and (3) degradation of toxic compounds in hypersa-
line environments. The use of halophilic bacteria in the recovery of saline soils is
covered by the following hypotheses (Arora et al. 2014a, 2014b). The first hypoth-
esis is that in saline soil, microbial activities may favour the growth of plants resis-
tant to soil salinity. The second hypothesis is based on the utilization of these
bacteria as bio-indicators as these microorganisms can be selected by their abilities
to grow at different salt concentrations. These organisms could indicate that well
water could be used with producing low saline contamination of plants or soils
which could be alleviated by the desertification of soil. The last hypothesis is the
application of halophilic bacteria using genetic manipulation technique to aid wild-
type plants to adapt to grow in saline soil by giving them the genes for vital enzymes
that are taken from halophiles (Arora et al. 2017).
16.7 Microbial Bioremediation
Utilization of microorganisms to the metabolically mediated desired chemical reac-

tions or physical processes is a useful general definition of bioremediation (Skladany
and Metting 1993). The use of selected symbiotic soil microorganisms to enhance
plant growth, widely practised for some organisms and widely researched for oth-
ers, fits at one end of the spectrum. These organisms include mycorrhizal fungi,
Rhizobium and Frankia, which can enhance plant growth by increasing the supply
of growth-limiting nutrients.
Bioremediation has been proposed as an economical, sustainable, effective and
environmentally friendly alternative to conventional remediation technologies.
Bioremediation is an expanding area of environmental biotechnology and can be
simply considered as the application of biological processes to the treatment of pol-

lution. The metabolic usefulness of microorganisms underpins practically all biore-
mediation applications, and most work to date has concentrated on organic
pollutants.
16.7.1 Plant Growth-Promoting Rhizobacteria (PGPR)

for Bioremediation
The plant growth-promoting rhizobacteria (PGPR) can affect plant growth by dif-
ferent direct and indirect mechanisms (Glick 1995). PGPR influence direct growth
promotion of plants by fixing atmospheric nitrogen, solubilizing insoluble
phosphates and secreting hormones such as IAA, GAs and kinetins besides ACC
deaminase production, which helps in regulation of ethylene (Glick et al. 2007;
Glick 2014). Induced systemic resistance, antibiosis, competition for nutrients, par-
asitism and production of metabolites (hydrogen cyanide, siderophores) suppres-
sive to deleterious rhizobacteria are some of the mechanisms that indirectly benefit
plant growth. Numerous species of soil bacteria which flourish in the rhizosphere of
plants may stimulate plant growth by a plethora of mechanisms (Vessey 2003). Soil
bacteria are very important in biogeochemical cycles and have been used for crop
production for decades. Plant bacterial interactions in the rhizosphere are the deter-
minants of plant health and soil fertility (Vivekanandan et al. 2015). Interaction of
plant growth-promoting rhizobacteria (PGPR) with host plants is an intricate and
interdependent relationship involving not only the two partners but other biotic and
abiotic factors of the rhizosphere region (Dutta and Podile 2010). PGPR bacteria are
free-living soil bacteria that can either directly or indirectly facilitate root growth
and development (Mayak et al. 1999) as well as overall growth of plants (Glick
1995). It is believed that generally about 2–5% of rhizosphere bacteria are PGPR
(Antoun and Prevost 2005). PGPRs are the potential tools for sustainable crop pro-
duction and trend for the future agriculture. The mechanism by which bacteria are
adsorbed onto soil particles is by simple ion exchange, and a soil is said to be natu-
rally fertile when the soil organisms are releasing inorganic nutrients from the
organic reserves at a rate sufficient to sustain rapid plant growth.
In agriculture, the use of PGPR as inoculums to alleviate salt stress is the most
promising approach to enhance production and yield in saline soils (Arora et al.
2012). These PGPR tolerate wide range of salt stresses and enable plants to tolerate
salinity by hydraulic conductance, osmotic accumulation, sequestering toxic Na+
ions, maintaining the higher osmotic conductance and photosynthetic activities
(Dodd and Perez-Alfocea 2012). The bacteria isolated from saline environment
(Moral et al. 1988) include Flavobacterium, Azospirillum, Alcaligenes,
Acinetobacterium, Pseudomonas (Rodriguez-Valera et al. 1985; Reinhold et al.
1987; Moral et al. 1988; Ilyas et al. 2012), Sporosarcina, Planococcus (Ventosa
et al. 1998), Bacillus (Upadhyay et al. 2009), Thalassobacillus, Halomonas,
Brevibacterium, Oceanobacillus, Terribacillus, Enterobacter, Halobacillus,
334 S. Arora
Staphylococcus and Virgibacillus (Roohi et al. 2012). Halophilic bacteria strain
(CSSRO2 Planococcus maritimus) and CSSRY1 (Nesterenkonia alba) having plant
growth promotion properties were isolated from the rhizosphere of dominant halo-
phytes from coastal ecosystem (Arora et al. 2012). Salt-tolerant Rhizobium species
were isolated from the rhizosphere of legumes in coastal saline soils of India
(Trivedi and Arora 2013).
The plant growth regulating hormone ethylene is produced in response to water-
logging (Grichko and Glick 2001), salinity and/or drought (Kausar and Shahzad
2006; Nadeem et al. 2007; Zahir et al. 2007). In the stressed environment, PGPR
exhibit 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity (Arshad
et al. 2007), and this reduces the level of ACC and endogenous ethylene (Glick et al.
1998; Yuhashi et al. 2000) thereby mitigating the deleterious effects of stress on
overall plant growth (Ligero et al. 1991; Hirch and Fang 1994). Inoculation of plants
with PGPR having ACC deaminase is relatively more tolerant to environmental
stress (Singh and Jha 2015).
The inoculation with halophilic strains of PGPR will help to improve the plants
tolerance in stress environment especially salinity and promote their growth particu-
larly in food crops which is essentially required to meet the national food demands.
Plant growth-promoting rhizobacteria (PGPR) assist in diminishing the accumu-
lation of ethylene levels and re-establish a healthy root system needed to cope with
environmental stress. The primary mechanism includes the destruction of ethylene
through the production of enzyme ACC deaminase. There are number of reports
(Ghosh et al. 2003; Govindasamy et al. 2008; Duan et al. 2009) mentioning rhizo-
sphere bacteria like Achromobacter, Azospirillum, Bacillus, Enterobacter,
Pseudomonas and Rhizobium that exhibit ACC deaminase activity. Most of the
researches have demonstrated the production of ACC deaminase gene in the plants
treated with PGPR under environmental stress. It has been reported by Grichko and
Glick (2001) that tomato seeds inoculated with Enterobacter cloacae and
Pseudomonas putida express ACC deaminase activity and register an increase in
plant resistance. Similarly, Ghosh et al. (2003) recorded ACC deaminase activity in
three Bacillus species, namely, Bacillus circulans DUC1, Bacillus firmus DUC2
and Bacillus globisporus DUC3 that encouraged root elongation in Brassica camp-
estris. Mayak et al. (2004) observed significant increase in fresh and dry weight of
tomato plants inoculated with the bacterium Achromobacter piechaudii under water
and salinity stress conditions.
Researchers have also demonstrated the feasibility of Azospirillum inoculation to
mitigate negative effects of salt (NaCl) on plant growth parameters. This beneficial
mitigating effect of Azospirillum inoculation that was previously observed in wheat
(Triticum aestivum) seeds under salt stress was also evident (Creus et al. 1997).
Azospirillum inoculated wheat (T. aestivum) seedlings subjected to osmotic stress
developed significant higher coleoptiles, with higher fresh weight and better water
status than non-inoculated seedlings (Alvarez et al. 1996; Creus et al. 1998).
16.8 P
lant-Microbiome Interactions for Salt Stress
Alleviation
The stress factors including salinity, drought, nutrient deficits, contamination, _dis-
eases, pests, etc. can alter plant-microbe interactions in the plant rhizosphere.
Researches evidencing that plant perception of environmental stress cues triggers
the activation of signalling molecules, phytohormones play a key role (Barea 2015).
This signal input is followed by a signal processing and finally by a signal output,
which enables plants to respond to these environmental constraints. As the plants
are exposed to multiple stresses simultaneously, appropriate meta-analyses reveal a
complex regulation of the plant growth and immunity (Dimkpa et al. 2009).
Understanding how phytohormones interact in the signalling network is fundamen-
tal to learn how plant-microbiome systems thrive and survive in stressed environ-
ments. This understanding is relevant to frame biotechnological strategies to
optimize plant adaptation mechanisms and to improve the capability of soil microbes
for stress alleviation in crop plants (Pozo et al. 2015), although mechanisms involved
in plant-microbe interactions under stress situations are poorly understood. However,
ongoing research is evidencing the involvement of changes in plant morphology,
physiology, transporter activity and root exudation profiles, changes that can induce
the plant to employ microbes with stress-alleviating capacities, a strategy that can
help crop productivity under stress (Zolla et al. 2013).
As stress factors cause detrimental impacts on the functionality or productivity
of agricultural systems, the role of rhizosphere microorganisms to enable plants to
thrive in adverse conditions is important (Barea et al. 2013). Plants have highly
beneficial interactions with their more mobile companions, microbes; and some of
these interactions involve highly sophisticated symbioses that confer stress toler-
ance, such as with mycorrhizae and rhizobia that help ameliorate nutritional and
water deficiencies under stress, while others are more transitory (Etesami and
Beattie 2017).
The biotic and abiotic plant factors that shape the plant-associated microbiome
through biasing the rhizosphere offer many challenges that current research is trying
to envisage. The future work on plants must focus on reprogramming transport
functions, while those on microorganisms have to focus on the uptake secreted
nutrients and the time-course changes in the microbial community structure. A
combination of these approaches can improve the understanding on how to enhance
the competitiveness and persistence of bacteria in the influenced rhizosphere to
finally improve plant growth and agroecosystem productivity (Savka et al. 2013).
16.9 I solation of Halophilic Microbes from Rhizospheric Soils

of Halophytes
The rhizospheric soil samples from halophyte plant species were collected in dupli-
cate from coastal Gujarat, India. The area is affected by soil salinity due to seawater
ingress. The soil pH of the rhizospheric soil varied from 7.3 to 8.8, and salinity
336 S. Arora
(electrical conductivity) varied from 2.7 to 39.6 dS m−1. The isolation of bacteria
was carried on nutrient agar medium and studied for colony and morphological
characteristics in relation to soil biochemical properties. Salt tolerance of isolates
was determined with varying NaCl concentrations of 0.5–20%. It was found that 7
out of 44 isolates were able to tolerate salt concentration up to 10%, while 29 iso-
lates were able to tolerate salt concentration up to 5% NaCl. Thus, from the rhizo-
sphere of dominant halophytes and other salt-tolerant plant species, various
halotolerant bacteria, which were able to tolerate salt concentrations up to 10%
NaCl, have been isolated. Out of 13 isolates that were able to tolerate salt concentra-
tion up to 15% NaCl, 3 were from the rhizospheric soil of Capparis decidua, 2 each
from both rhizospheric soil of plants of Capparis decidua and Salvadora oleoides
and 1 each from the rhizospheric soil of Cressa cretica, Aeluropus lagopoides and
Suaeda maritima (Arora et al. 2014a).
16.9.1 Isolation of Halophilic Rhizobia spp.
Few Gram-negative bacteria, known as rhizobia, have been isolated from the saline
soil samples. Rhizobium spp. can tolerate up to 500 mM of NaCl. It has been found
out that some species of rhizobia adapt to saline conditions through the intracellular
accumulation of low-molecular-weight organic solutes called osmolytes, such as
glutamate, trehalose, glycine betaine and polyamines, or an accumulation of K+
(Trivedi and Arora 2013). The ability of the isolates to grow in different concentra-
tions of salt was tested by streaking isolates on YEM media containing 0.5%, 1%,
2%, 3%, 3.5%, 4% and 5% (w/v) NaCl. Twenty Rhizobium isolates could tolerate u
pto 2% NaCl, while only 10 could tolerate up to 4% NaCl concentration.
16.9.2 Isolation of Halophilic Endophytic Bacteria
From the leaves of four dominant halophytes or salt-tolerant plants from coastal
Gujarat, isolated halophilic bacteria. Twenty isolates were screened based on salt
tolerance and their fast growth. All of the 20 endophytes showed superior growth at
2.5% NaCl concentration, while 18 (90%) endophytes sustain up to 5% NaCl, 17
(85%) isolates survived at 7.5% NaCl, and 15 (75%) tolerated up to 10% NaCl con-
centration (Arora et al. 2014a). Bacillus foraminis and Bacillus gibsonii were able
to tolerate salt concentration up to 7.5% of NaCl, while Acinetobacter baumannii
and Paenibacillus xylanisolvens could tolerate only up to 2.5% NaCl concentration
and Pseudomonas fluorescens up to 5% NaCl level (Table 16.1). The other isolates
were capable of tolerating 10% NaCl concentration in the media. Overall, the
growth rate of endophytes showed decline with increasing NaCl concentration in
the media. The bacterial counts were found maximum in Sphaeranthus indicus
(40%) and were minimum in Salicornia brachiata (10%). Of the 20 endophyte iso-
lates selected, 3 were pigmented, and 17 were non-pigmented isolates. Regarding
cell shape and Gram’s staining, seven were Gram-negative cocci, two Gram-positive
Table 16.1 Salt tolerance of endophytic bacteria from leaves of halophytes and salt-tolerant plant
species
Salt tolerance (NaCl %)
Isolate ID Endophytic bacteria 2.5% 5.0% 7.5% 10%
EB1 Acinetobacter baumannii + − − −
EB2 Kocuria flavus + + + +
EB3 Bacillus cereus + + + +
EB4 Bacillus firmus + + + +
EB5 Staphylococcus pasteuri + + + +
EB6 Paenibacillus xylanisolvens + − − −
EB7 Bacillus horneckiae + + + +
EB8 Paenibacillus xylanisolvens + + + +
EB9 Bacillus licheniformis + + + +
EB10 Bacillus foraminis + + + −
EB11 Virgibacillus picturae + + + +
EB12 Oceanobacillus picturae + + + +
EB13 Bacillus subtilis + + + +
EB14 Bacillus aerius + + + +
EB15 Pseudomonas fluorescens + + − −
EB16 Bacillus subtilis + + + +
EB17 Bacillus aryabhattai /megaterium + + + +
EB18 Arthrobacter luteolus + + + +
EB19 Bacillus gibsonii + + + −
EB20 Paenibacillus sp. + + + +
cocci, four Gram-negative bacilli and seven Gram-positive bacilli. Motility test
results depicted that 18 isolates were motile, while only 2 isolates were nonmotile.
In total, 11 isolates showed positive results for oxidase test, whereas all endophytic
bacterial cultures showed negative catalase test. The enzymatic activity of endo-
phytic isolates revealed that 50% isolates exhibited amylase activity, and only 15%
isolates showed urease activity (Arora et al. 2014a).
Of the 20 endophytic bacteria screened for plant growth-promoting substances,
six (30%) and two (10%) isolates showed positive test for ammonia production and
phosphate solubilization activity. Only four (20%) were mixed acid fermenters, five
(25%) showed the production of acetoin, and none of the isolates exhibited IAA
production (Arora et al. 2014a). The selected bacterial isolates were submitted for
16S rRNA gene sequencing, and it was observed that Acinetobacter baumannii,
Bacillus cereus, Bacillus firmus, Bacillus aerius, Pseudomonas fluorescens and
Bacillus subtilis were positive for ammonia production, while phosphate solubiliza-
tion was positive for Acinetobacter baumannii and Pseudomonas fluorescens (Arora
and Vanza 2017).
338 S. Arora
16.10 L
iquid Bioformulations Developed for Enhancing Crop
Production in Salt-Affected Soils
Salt-tolerant (halophilic) bacterial strains of N-fixers and phosphate solubilization bac-

teria (PSB) were isolated from the salt-affected soils of Indo-Gangetic plains at ICAR-
CSSRI, Regional Research Station, Lucknow (UP). These strains were characterized
for plant growth promotion and tested for their efficacy under different levels of salt
stress, Figs. 16.1 and 16.2. For seed application of these promising selected strains of
beneficial soil microorganisms, these were cultured in laboratory and prepared in suit-
able standardized media as liquid bioformulations, viz. Halo-Azo and Halo-PSB. These
can be used either for seed/seedling root treatment or soil application. These bioformu-
lations when applied help to mobilize plant nutrients like nitrogen and phosphorous
through their activities in the soil or rhizosphere and make available to plants in a
gradual manner under salt stress. Also liquid formulations ‘Halo-Zinc’ and ‘Halo-
Rhizo’ having salt-tolerant strains of zinc solubilizers and Rhizobium species, respec-
tively, were developed and found to be effective under salt stress. These shall also help
in maintenance of soil health, minimize environmental pollution and cut down on the
use of chemicals in agriculture. The bioformulations are affordable for most of the
small and marginal farmers. These bioformulations are also an ideal input for reducing
the cost of cultivation and for promoting organic farming on salt-affected soils.
In sodic and saline-sodic soils, the bioformulation has been tested at farm, vali-
dated at different farmers’ fields in five salt-affected districts of Indo-Gangetic
plains. The seedling dip or seed inoculation with the bioformulation resulted in
enhanced crop yields, management of soil health and stress regulation.
These liquid bioformulations are very beneficial for enhancing the production of
cereal crops mainly rice and wheat as well as vegetable crops. These can be easily
used as seed treatment, seedling dip and soil application with farmyard manure or
compost. The packing of 100 ml bottle is sufficient for treating seeds of 1 acre land
or root dip. It has been found to be very effective in sodic soil, and multilocation
testing of these bioformulations was done in diverse sodic soils of Indo-Gangetic
plains. There was increase in rice and wheat yield by 11.5–14% under salt stress
conditions in Indo-Gangetic plains (Fig. 16.1). This is the cheap and eco-friendly
approach for bioremediation of salt-affected soils and to optimize agricultural crop
yields in the degraded lands.
Fig. 16.1 Liquid bioformulations including salt-tolerant bacterial strains

Fig. 16.2 Efficacy of liquid bioformulations of halophilic PGP strains on wheat and rice on sodic
soils
Table 16.2 Effect of bioformulation use on sodic soil properties after harvest (initial soil
pH = 9.42)
Exch. Av
Na N Av P DHA
pH EC OC (mg/ (kg/ (kg/ MBC (μgTPF/
Treatment (1:2) (dS/m) (%) kg) ESP ha) ha) (μg/g) g/d)
Control (FYM) 9.24 0.432 0.28 338 44 103 10.8 44 10
FYM + halo azo 8.94 0.318 0.35 266 42 119 11.4 55 13.9
FYM + halo 9.12 0.364 0.33 272 43 113 15.1 52 12.2
PSB
FYM + halo 9.18 0.385 0.31 282 43 121 14.4 58 13.2
Azosp
FYM + consortia 8.91 0.322 0.38 238 41 123 15.6 61 14.8
The application of liquid bioformulations has the potential to improve the growth
and yield of crops under salt stress, and they were also found to play a role in soil
health improvement as observed in soil after harvest of the crop (Table 16.2).
16.11 Ameliorative Potential of Halophilic Microbes
There was substantial improvement in soil pH and exchangeable sodium content. It

was observed that after 2 years of continuous rice-wheat with inoculation of halo-
philic plant growth-promoting microbial formulations, soil pH reduced from initial
value of 9.42–8.91 and 8.94. Similarly reduction of exchangeable sodium from 416
to 238 mg/kg was noticed. The build-up of soil organic C and available N apart from
340 S. Arora
improvement in soil microbial biomass C and dehydrogenase activity was observed

with application of liquid bioformulations. Soil microbial biomass carbon enhanced
to 61 μg/g with the application of consortia over 41 μg/g where no bioformulations
were used (Table 16.2).
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Environmental and Microbial Biotechnology

More information about this series at http://www.springer.com/series/16324

ISSN 2662-1681 ISSN 2662-169X (electronic)

© Springer Nature Singapore Pte Ltd. 2020

• Applications of plant-microbe interactions in contaminated agro-ecosystem

Emphasis is given on the most neglected part of scientific exploration that is

Ranchi, Jharkhand, India Manoj Kumar

1 Phytostimulation and Biocontrol by the Plant-Associated

7 Screening and Characterization of Phosphate-Solubilizing

Manoj Kumar is working as Associate Professor and

Vivek Kumar is an agricultural microbiologist hav-

Ram Prasad is an Assistant Professor at the Amity

© Springer Nature Singapore Pte Ltd. 2020 1

Environmental-friendly biotechnological approaches, such as the use of microbial

Table 1.1 Examples for commercial use of Bacillus-based bioformulations in agriculture

1.2  oot Colonization by FZB42 and Its Impact on the Host

1.3 Plant Growth Promotion

4. Enhancement of nutrient availability by phytase-producing bacteria. Soil phos-

1.4.1 Lipopeptides, Bacillibactin, and Antifungal Activity

Immunity, but no synthesis genes

1.4.1.1 Polyketides, Bacilysin, and Bacteriocins Direct Antibacterial

Another representative of the type B lantibiotics, amylolysin from B.

elucidated, unveiling a hitherto unusual number of thiazoles and oxazoles formed

1.4.1.2 Nematicidal Activity Is Directed by Plantazolicin

1.5 I nduced Systemic Resistance Is Triggered by Plant

Except surfactin, concentration of antifungal lipopeptides determined in planta was

1. Stimulation of plant ISR by bacterial metabolites produced in vicinity of plant

Without doubt, other features of PGPR, as production of plant hormones and

© Springer Nature Singapore Pte Ltd. 2020 21

The most widely used and well-documented example of transgenic plant in

These studies eventually led to major modification in designing of synthetic ver-

2.2 Genetic Transformation

Genetic transformation is the deliberate alternation and modification of the genome

2.3 Gene Transfer Method

2.3.1 Direct DNA Transfer Methods

Gene Transfer Methods

Vector mediated/ Vectorless/

Physical gene Chemical gene DNA imbibition by cell,

Ultrasound mediated DNA

Macroinjection The polycation DMSO technique

Silicon carbide fiber- mediated

Fig 2.1 Schematic representation of the various gene transfer strategies

2.3.2 Indirect DNA Transfer Method

As with other dicotyledonous crops, Agrobacterium-mediated transformation is the

2.4 Agrobacterium-Mediated Genetic Transformation

Agrobacterium tumefaciens is a gram-negative, soil phytopathogen of family

Bacteria can be classified as octopine, nopaline, agropine, succinamopine or

2.4.1 Ti Plasmid of Agrobacterium

The ability of Agrobacterium tumefaciens to induce crown gall disease in plants is

1 . 25 bp direct repeated flanking and defining the T-DNA

2.4.2 Organization of T-DNA

T-DNA (transfer DNA) is about 23 kb segment of Ti plasmid, which is transferred

2.4.3 Organization of vir Region

2.4.4 T-DNA Transfer Process

Table 2.1 Functions of different vir genes

2.4.5 Vectors Derived from Ti Plasmids

Large size, absence of unique restriction sites and tumourigenic properties of Ti

2.4.6 Co-integrate Vector System

2.4.7 Binary Vector System

1 . Multiple cloning site

Ranchi, Jharkhand, India Manoj Kumar

1 Phytostimulation and Biocontrol by the Plant-Associated

7 Screening and Characterization of Phosphate-Solubilizing

1.2 oot Colonization by FZB42 and Its Impact on the Host

1.3 Plant Growth Promotion

1.4.1 Lipopeptides, Bacillibactin, and Antifungal Activity

1.4.1.1 Polyketides, Bacilysin, and Bacteriocins Direct Antibacterial

1.4.1.2 Nematicidal Activity Is Directed by Plantazolicin

1.5 I nduced Systemic Resistance Is Triggered by Plant

2.2 Genetic Transformation

2.3 Gene Transfer Method

2.3.1 Direct DNA Transfer Methods

2.3.2 Indirect DNA Transfer Method

2.4 Agrobacterium-Mediated Genetic Transformation

2.4.1 Ti Plasmid of Agrobacterium

2.4.2 Organization of T-DNA

2.4.3 Organization of vir Region

2.4.4 T-DNA Transfer Process

2.4.5 Vectors Derived from Ti Plasmids

2.4.6 Co-integrate Vector System

2.4.7 Binary Vector System

2.4.8 Selectable Markers

2.4.9 Advantages of Agrobacterium-Mediated Plant

2.4.10 Disadvantages of Agrobacterium-Mediated Plant

2.5 Factors Affecting Plant Transformation

2.6 acillus thuringiensis (Bt) Endotoxin Crystal Protein

2.7 echanism of Action of Three-Domain Cry Toxins

2.7.1 Pore Formation Model

2.7.2 Signal Transduction Model

2.8 BT-GM Crops

2.9 Management of Resistance Development

3.2 Materials and Methods

3.2.1 Location of Nodule Collection

3.2.1.1 Rhizobia Isolation and Purification

3.2.2 Response to Environmental Stress Factors

3.2.3 Utilization of Carbon and Nitrogen Sources

3.2.4 Heavy Metal Tolerance

3.2.5 Authentication of Isolates

3.2.6 Numerical Analysis

3.3 Results and Discussion

3.3.1 Phenotypic Evaluation

3.3.3 Plant Assay

4.2 Materials and Methods

4.2.1 Soil Samples and Plant Material

4.2.2.1 AMF Spore Extraction

4.2.2.2 Measuring of the Root Mycorrhization Rate

4.3 Results and Discussion

4.3.1 Richness, Diversity, and Identification of AMF Spores

4.3.2 Evaluation of Root Mycorrhization

5.1.1 Friends and Foes: A Side to Pick