Isolation, Identificationand
Isolation,Identification andPhylogenetic
Phylogenetic
Analysis
Analysisof
of
Bacterial
BacterialPopulation
PopulationAssociated
Associatedwith
with
Citrus
CitrusCanker
CankerInfected
InfectedLeaves
Leaves
Muhammad
Supervisory Kamran Haider
Supervisory Committee
Committee
09-NUAS-141
Dr. Iftikhar Ahmed (Supervisor)
Dr. Iftikhar Ahmed (Supervisor)
Dr.
Dr.Shazia
ShaziaErum
Erum (Member)
(Member)
Dr.
Dr.Muhammad
MuhammadZakria
Zakria (Member)
(Member)
INTRODUCTION
• Citrus – Second most important fruit worldwide after
grapes in terms of
• area and
• production.
• In Pakistan
• citrus production is 2,203000 tonnes
• exported 5,33000 tonnes
• worth 16.554 billion Rs.
(Economic Survey of Pakistan, 2009-10)
• Citrus contributes to ~ 50 % of the total production of fruit
and ~ 25% of the total fruit export in Pakistan (Ali, 2004).
• Pakistan – the largest producer of 'Citrus Reticula' variety (Kinnow)
• ~ 95 % of the total Kinnow is grown in Pakistan.
ISSUES / PROBLEMS
• The citrus – many disorders/factors
– Results in huge economic losses.
• Diseases – the major factors. Among diseases,
citrus canker has its great importance, and adversely
affects the plant health resulting in low yield and poor quality fruit.
Canker is occurs through out the citrus growing countries of the
world
(Koizumi, 1985)
• The disease, caused by the bacteria:
– Xanthomonas axonopodis pv. citri
– Xanthomonas compestris pv. citri
ISSUES / PROBLEMS
Citrus canker
– by appearance of lesions on fruit, foliage, and young stems of
susceptible cultivars of citrus.
– Defoliation and premature abscission of affected fruit occurs on heavily
infected trees.
Mechanism
the production of
abundant extracellular
polysaccharides (EPS)
in host tissues.
RATIONALE
• Projects implemented on Citrus Canker in Pakistan
– Dr. Afzal Akhtar, NARC
– Dr. Shahbaz Talib Sahi, UAF
However, there are insufficient reports on the
isolation and characterization of the causal agent.
Intensive identification of bacteria based on
16S rRNA gene sequence analysis has been
grossly neglected.
OBJECTIVES
• To isolate the bacterial population associated with
citrus leaves infected with citrus canker disease.
• To identify the bacterial population using 16S
rRNA gene sequence
• To characterize the strains for biochemical traits
using API Kits
• To determine the pathogenicity of bacterial
isolates.
Material and Methods
Sampling
Infected citrus leaves
Different regions of Punjab like Sargodha, Multan, Jhang, Faisalabad and Toba Tek sing.
Different susceptible varieties of citrus.
Surface sterilization of infected leaves
Cut and collected infected leaf part
Shaken with 70% ethanol for 1 minute.
Shaken with 2% sodium hypochlorite solution for 5
minutes.
Washing with sterilized distilled water (~ 2-3 times)
Isolation of Bacteria
• Isolation of the bacterium- Dilution Plate Technique
Crushing the infected part of leaves
Dilution using PBS buffer
Spread the suspension on suitable media
Incubation at 28 oC for 24-48 hours
Screening of Isolates
• Screening - on the basis of morphological characters.
• Purification with streak plate method.
Preservation of bacterial cultures
• Preservation in 35% glycerol stock supplemented with
nutrients required for bacterial growth
Identification of Bacteria
Genomic
GenomicDNADNAextraction
extraction&& Polymerase chain reaction
amplification
amplificationof
of16S
16SrRNA
rRNAgene
gene (PCR)
Confirmation
Confirmationof
ofAmplified
AmplifiedPCR
PCR Gel Electophoresis
product
product
Purification PCR
Purificationusing
usingPCR
PCRpurification
purification
KIT product purification
KIT
Determine
Determinethe
theorder
orderof
ofthe
the 16S rRNA
nucleotides
nucleotidesfor
forgene
gene
Gene Sequencing
Phylogenetic Analysis of Bacterial Strains
• Phylogenetic analysis using bioinformatics (Tamura et al.,
2007)
• Phylogenetic tree using the Phylip (version 3.69) package
and Neighbour Joining Plot software (Saitou and Nei,.1987)
• Comparisons of more than two sequences
• Analysis of gene families, including functional predictions
• Estimation of evolutionary relationships among organisms
Phylogenetic analysis using Bioinformatics: DDBJ/NCBI data base, CLUSTAL
X, BIOEDIT , NJ PLOT software
Biochemical Characterization
of Bacterial Isolates from Citrus Cankar
API ZYM KIT
API tests for C utilization/acid production
API 2OE KIT
API (ZYM) tests for enzyme analyses
VALIDATION of a Novel Candidate Species
Identification
16S rRNA gene sequencing…..
similarity < 97%.....NOVEL species
Similarity > 97%.....needs DNA-DNA hybridization
DNA-DNA Hybridization value……< 70%.....NOVEL species
DNA-DNA Hybridization value……> 70%.....not a novel species
Taxonomic
Taxonomiccharacterization
characterization
Morphology
Phenotypic
Genotypic
Chemotaxonomic
Pathogenecity Test
Pathogenecity test to confirm
which strain is pathogenic and which
is not.
Soaking
Wounds at leaves
Inoculation
Incubation
Observation for the development of
tissue hyperplasia after 7-21 days
EXPECTED OUTCOME
• Biodiversity of bacterial Population associated
with Citrus Canker lesions from Pakistani
ecology.
• Characterization of bacterial population may
provide information on other associated
disease-causing organisms in citrus.
• Novel strains will also be expected.
References
• Akhtar MA, Rafi A, Hameed A (2008). Comparison of methods of
inoculation of Xanthomonas oryzea pv. Oryzea in rice cultivars.
Pakistan Journal of Botany. 40(5): 2171-2175.
• Ali T (2004). ‘Marketing of citrus fruit in Pakistan’ (Thesis), Pakistan
Research Respiratory, HEC, Pakistan,
http://eprints.hec.gov.pk/1198/918.html.htm, date accessed: 20th
February 2010.
• Koizumi M (1985). Citrus canker: The world situation. In: Timer, L.W.
(ed.), Citrus Canker: An International Perspective, Pp: 2–7. Proc.
Symptoms Institute of Food Agriculture Sciences University of
Florida, P: 28
• Saitou N and Nei M (1987). The neighbor-joining method: a new
mwethod for reconstructing phylogenetic trees. Mol Biol Evol.4:406-
425.
• Tamura K, Dudley J, Nei M and Kumar S (2007). MEGA4: Molecular
Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol.
Biol. Evol., 24:1596-1599.
THANK YOU
Suggestions Are Welcome!
