Download - kamran haider
-
8/6/2019 kamran haider
1/16
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
-
8/6/2019 kamran haider
2/16
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.
-
8/6/2019 kamran haider
3/16
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
-
8/6/2019 kamran haider
4/16
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
-
8/6/2019 kamran haider
5/16
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
-
8/6/2019 kamran haider
6/16
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.
-
8/6/2019 kamran haider
7/16
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
-
8/6/2019 kamran haider
8/16
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
-
8/6/2019 kamran haider
9/16
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
-
8/6/2019 kamran haider
10/16
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
-
8/6/2019 kamran haider
11/16
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
-
8/6/2019 kamran haider
12/16
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
-
8/6/2019 kamran haider
13/16
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
-
8/6/2019 kamran haider
14/16
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.
-
8/6/2019 kamran haider
15/16
-
8/6/2019 kamran haider
16/16
THANK YOUTHANK YOU
Suggestions Are Welcome!