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Muhammad Kamran Haider

09-NUAS-141

Supervisory Committee

Dr. Iftikhar Ahmed (Supervisor)

Dr. Shazia Erum (Member)

Dr. Muhammad Zakria (Member)

Isolation, Identification and Phylogenetic

Analysis ofBacterial Population Associated with

Citrus Canker Infected Leaves


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INTRODUCTIONINTRODUCTION 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 fruitand ~ 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.


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The citrus many disorders/factors Results in huge economic losses.

Diseases the major factors. Among diseases,

citrus cankerhas its great importance, and adverselyaffects the plant health resulting in low yield and poor quality fruit.

Cankeris occurs through out the citrus growing countries of theworld

(Koizumi, 1985)

The disease, caused by the bacteria: Xanthomonas axonopodis pv. citri

Xanthomonas compestris pv. citri

ISSUES / PROBLEMSISSUES / PROBLEMS


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by appearance of lesions on fruit, foliage, and young stems ofsusceptible cultivars of citrus.

Defoliation and premature abscission of affected fruit occurs onheavily infected trees.

ISSUES / PROBLEMSISSUES / PROBLEMSCitrus cankerCitrus canker

the production ofabundant extracellularpolysaccharides (EPS)

in host tissues.

MechanismMechanism


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Projects implemented on Citrus Canker in Pakistan

Dr. Afzal Akhtar, NARC

Dr. Shahbaz Talib Sahi, UAF

However, there are insufficient reports on theisolation and characterization of the causal agent.

Intensive identification of bacteria based on

16S rRNA gene sequence analysis has beengrossly neglected.

RATIONALERATIONALE


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OBJECTIVESOBJECTIVES

To isolate the bacterial population associatedwith citrus leaves infected with citrus canker

disease.

To identify the bacterial population using16S rRNA gene sequence

To characterize the strains for biochemicaltraits using API Kits

To determine the pathogenicity ofbacterial isolates.


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Material and MethodsMaterial and Methods

Infected citrus leaves

Different regions of Punjab like Sargodha, Multan,

Jhang, Faisalabad and Toba Tek sing.

Different susceptible varieties of citrus.

Sampling

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)

Surface sterilization of infected leaves


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Isolation ofBacteriaIsolation ofBacteria

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 ofIsolates

Screening - on the basis ofmorphological characters.

Purification with streak plate method.

Preservation of bacterial cultures

Preservation in 35% glycerol stock supplemented withnutrients required for bacterial growth


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Identification ofBacteriaIdentification ofBacteria

Polymerase chain reaction

(PCR)

Gel Electophoresis

PCR

product purification

16S rRNA

Gene Sequencing

Genomic DNA extraction &

amplification of 16S rRNA gene

Confirmation of Amplified PCR

product

Purification using PCR purification

KIT

Determine the order of the

nucleotides for gene


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Phylogenetic Analysis ofBacterial Strains

Phylogenetic analysis using bioinformatics (Tamura et al.,

2007) Phylogenetic tree using the Phylip (version 3.69) package

and NeighbourJoining 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, CLUSTALX, BIOEDIT , NJ PLOT software


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API ZYM KIT

API tests for C utilization/acid production

API 2OE KITAPI (ZYM) tests for enzyme analyses

Biochemical Characterization

of Bacterial Isolates from Citrus Cankar

Biochemical Characterization

of Bacterial Isolates from Citrus Cankar


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VALIDATION of a Novel Candidate SpeciesVALIDATION 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

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 characterization

Morphology Phenotypic

Genotypic

Chemotaxonomic

Morphology Phenotypic

Genotypic

Chemotaxonomic


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Pathogenecity Test

Pathogenecity test to confirm

which strain is pathogenic and whichis not.

Soaking

Wounds at leaves

Inoculation

Incubation

Observation for the development oftissue hyperplasia after 7-21 days


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EXPECTEDOUTCOMEEXPECTEDOUTCOME

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.


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THANK YOUTHANK YOU

Suggestions Are Welcome!

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