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Genetic Diversity of Pseudomonas Syringae Pv. Syringae Causing Leaf Blight of Mango in Bangladesh

N. Jannat
Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh, Bangladesh.

I. Hossain
Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh, Bangladesh.

M. D. Hossain
Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh, Bangladesh.

P. Dey
Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh, Bangladesh.

M. A. H. khan
Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh, Bangladesh.

Abstract/ Executive Summary:

Mango (Mangifera indica L.) belonging to the family Anacardiaceae is the king of fruits. The genetic variations of 25 isolates of Pseudomonas syringae pv. syringae obtained fro m five regions of Bangladesh were analyzed using RAPD markers by Polymerase Chain Reaction (PCR). The percentage of polymorphic loci was found different from one region to another. Mean diversity across all population for all the loci studied was 0.31. The co-efficient of gene differentiation (G_st) was 1.00 reflecting the existence of high level of genetic variations among the genotypes. Comparatively the highest genetic distance (0.6931) was observed in Isolates of Rangpur vs. Isolates of Dinajpur; the low est genetic distance (0.2877) was estimated in Isolates of Rangpur vs. Isolates of Bogra. Considering the genetic distance values; the results indicated that the isolates of five regions were genetically different from each other. The dendrogram (UPGMA) co nstructed from Nei’s (1972) genetic distance produced 2 main clusters of 25 isolates from five regions. UPGMA dendrogram revealed that isolates of Rangpur, Bogra and Mymensingh form same cluster with least genetic distance. Dinajpur and Rajshahi produce an other cluster with least genetic distance. Genetic distance among the isolates of Rangpur, Bogra and Mymensingh was found very near and genetic distance among the isolates of Dinajpur and Rajshahi was found very near which means these isolates may be virulent and their genetic variation is minimum. The genetic variation among these isolates of different regions was low indicating geographical variation among the isolates collected from different regions.

Keywords: Genetic diversity, Leaf blight of mango, RAPD marker

Location: Rajshahi, Dinajpur, Mymensingh, Rangpur and Bogra in Bangladesh.

Start Date:

End Date:

Program Area: Pest Management

Commodity: Mango

Objectives:

1. To study the incidence and severity of leaf blight in major mango growing areas of Bangladesh, molecular characterization of Pseudomonas syringae pv. syringae, the causal organism of leaf blight of mango, and

2. To determine the genetic variation among the isolates of Pseudomonas syringae pv. syringae collected from different regions of the country.

Methodology:

A survey was carried out to know the status of bacterial leaf blight of mango in nurseries of major mango growing regions viz. Rajshahi, Dinajpur, Mymensingh, Rangpur and Bogra in Bangladesh. The surveyed nurseries were selected randomly and the incidence and severity of leaf blight recorded. The infected leaf samples from different nurseries were brought to the laboratory for the isolation of the pathogen. The infected portion of the leaf was surface disinfected by immersion in sterile distilled water, and plated on nutrient agar (NA). Plates were incubated at 28°C for 24 hrs. Cream or off white colored colony of bacteria was appeared after incubation on NA medium.The bacterium, P. syringae pv. syringae was characterized by a series of biochemical tests viz. KOH solubility test, Gram staining test, Kovac’s oxidase test, Temperature sensitivity test, Levan test, Sugar utilization test, Arginine dihydrolase activity, Catalase test and Pectolytic test. Then the bacterial isolates were multiplied by using NA medium. The petridishes containing NA media were inoculated by the colony of bacteria with the help of sterilized platinum wire dipping in rectified spirit and flaming over a spirit lamp. All the inoculated plates were incubated at room temperature for 24 hours. In case of contamination, the bacterial cultures were purified by streaking a single colony of each isolate by sub culturing on NA medium. DNA samples of each isolates were extracted following chloroform-isoamyle alcohol extraction and ethanol precipitation method from the pure culture of bacteria. Bacteria were grown in 5 ml liquid broth medium at 27°C for 18-24 hours. Cells were collected in 1.5 ml eppendorf tubes, after centrifuging the pellets were left and resuspended in 1 ml of 2M NaCl. The primer code used in this experiment was 62538038 and the sequence was TGCAGCACCG with 70% GC content. The Ladder used in the experiment was 100 bp. The amplification conditions were based with some modification. PCR reactions were performed on each DNA sample containing NH₄ buffer= 1.0 µl, dNTPs= 1.0 µl, Primer= 2.0µl, MgCl₂ =0.6 µl, Template DNA=2.0 µl, AmpliTaq DNA polymerase=0.2 µl, ddH₂O=3.2 µl (Total= 10 µl). During the experiment, PCR buffer, dNTPs, primer and DNA samples solutions were thawed from frozen stocks, mixed by vortexing and kept on ice. DNA template was pipetted first into PCR tubes compatible with the thermo cycler used. A pre-mix was then prepared in course of following order: reaction buffer, dNTPs, DNA template and sterile distilled water. Taq polymerase enzyme was then added to the pre-mix. The pre-mix was then mixed up well and aliquoited into the tubes that already contain primer. The tubes were then sealed and placed in a thermo cycle and the cycling was started immediately. DNA amplification was performed in an oil-free thermal cycler (MasterCycler Gradient Eppendorf). Since RAPD markers are dominant, we assumed that each band represented the phenotype at a single allelic locus. One molecular weight marker, 100 bp DNA ladder, was used to estimate the size of the amplification products by comparing the distance traveled by each fragment with that of the known sized fragments of molecular weight markers. All distinct bands or fragments (RAPD markers) were thereby given identical numbers according to their position on gel and scored visually on the basis of their presence (I) or absence (0), separately for each isolates of mango and primer. The scores obtained using all primers in the RAPD analysis were then pooled to create-a single data matrix. Nei’s genetic distance and identity values were computed from frequencies of polymorphic markers to estimate genetic relationship between the studied 12 cultivars using the Unweighted Pair-Group Method of Arithmetic means (UPGMA). The dendrogram was constructed using the POPGENE (Version 1.31) computer program 30.

Source of Information: International Journal of Agriculture Innovations and Research Volume 2, Issue 4, ISSN (Online) 2319-1473

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Achievement/Findings:

The genetic variation among these isolates of different regions is low and indicating some degrees of geographical variation among the isolates collected from different regions. These isolates are emerging as a great threat of mango cultivation and production. The findings of the present molecular based analysis of the pathogen P. syringae pv. syringae would definitely be useful to adopt a proper management strategy suitable for integrated disease management programs for the eco-friendly management of the leaf blight disease in Bangladesh.

Knowledge Management: Journal