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Genetic Diversity of pseudomonas Syringae Pv. Syringae Causing Leaf Blight of Litchi in Bangladesh

S. Afrose
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.

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

Abstract/ Executive Summary:

Leaf blight of caused by Pseudomonas syringae pv. syringae is a severe disease of litchi saplings, which is considered as a threat for raising healthy sapling of the fruit in Bangladesh. A survey was conducted to find out incidence and severity of the disease in five regions of Bangladesh namely Rajshahi, Dinajpur, Mymensingh, Barishal and Khagrachari. The highest disease incidence was recorded in Dinajpur and Kagrachari regions and the lowest in Mymensingh region. On the other hand, the highest disease severity was found in Dinajpur and Kagrachari regions and the lowest in Mymensingh region. Another experiment was conducted to determine genetic diversity of the causal bacterium following molecular study. Twenty five diseased samples of bacterial leaf blight of litchi were collected from 25 locations in five regions and one isolate of P. syringae pv. syringae was isolated from each sample. The values of pair-wise comparisons of Nei's (1972) genetic distance among5 different locations of P. syringae pv. syringae were computed. From the UPGMA dendrogram drawn on the basis of Nei's (1972) genetic distances and comparatively the highest genetic distance (0.9163) was observed in Khajrachari vs Barishal, Mymensingh vs Barishal and Rajshahi vs Mymensingh but Dinajpur was close to the Barishal with the least genetic distance (0.3567). Thus, the results indicate that remarkable genetic diversity exists among the isolates of P. syringae pv. syringae from different origins Bangladesh.

Keywords: Genetic diversity, Leaf blight of litchi, Pseudomonas syringae pv. syringae, RAPD marker

Location: Rajshahi, Dinajpur, Mymensingh, Rangpur, Khagrachari.

Start Date: 01-01-2011

End Date: 31-12-2011

Program Area: Conservation and Biodiversity

Commodity: Litchi

Objectives:

1. To find out the incidence and severity of leaf blight of litchi in major litchi growing areas of Bangladesh, for molecular characterization of the pathogen

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

Methodology:

A survey was carried out to find out the incidence and severity of bacterial leaf blight of litchi nurseries in Rajshahi, Dinajpur, Mymensingh, Rangpur and Khagrachari areas. The surveyed nurseries were selected randomly. The incidence of leaf blight was recorded determined following the formulae of Rai and Mamtha (2005). The infected portion of the leaf sample was cut into small pieces, washed with sterile distilled water, and plated on nutrient agar (NA). Plates were incubated at 28°C for 24 hrs. The bacterium was characterized by a series of biochemical test 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 and identified as P. syringae pv. syringae. The bacteria for DNA extraction were cultured in Peptone broth medium at 27°C for 18 hours with shaking on an electrical shaker. Genomic DNA of each isolates were extracted following standard protocol as described by (Ferdous, 2012). The amplification conditions were based on Williams et al. (2000) with some modification. PCR reactions were performed on each DNA sample in a total volume of 10 lu containing 0.6 l, Tag buffer 1.0 ul, dNTPs 1.0 ul, MgC1₂ - 0.6 ul , Ampli Taq DNA polymerase 0.2 ul, ddH20 3.2 ul, Primer 2.0 ul. The primer code and sequence of the selected primer used in the experiment was 62538035 and TGCAGCACCG with 70% GC content. The ladder used in this experiment was 100kb. PCR was programmed with an initial denaturing at 92°c for 4 min. followed by 40 cycles of denaturation at 94°c for 1 min, annealing at 37°c for 1 min. and extension at 72°c for 3 min. and the final extension at 72°c for 5 min. in a thermo cycler (Master Cycler Gradient, Eppendorf, Germany. PCR product was mixed with I lu of bromophenol blue and loaded in 1.4% Agarose gel. Electrophoresis was carried out at 120v for 40 min and soaked in ethidium bromide (9ml water + Iml stock) for 15--20 min. Documentation of the DNA samples and data analysis After electrophoresis the image of the gel was documented through UV transilluminator system. The dendrogram was constructed using the POPGENE (Version 1.31) computer program 30.

Source of Information: SAARC JOURNAL OF AGRICULTURE, Vol. 12, Issue 1, 20114, pp. 150-161, ISSN 1682-8348, SAARC Agricultural Information Centre.

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The results indicate that the lower or higher level of genetic distance exists among the isolates of Pseudomonas syringae pv. syringae causal agent of leaf blight of litchi with their different origin. Leaf blight of litchi caused by Pseudomonas syringae pv. syringae seems to be a great threat for raising quality and healthy litchi saplings in order to get higher fruit production. The findings of the present study would definitely be useful to design a comprehensive molecular based study of biodiversity of the pathogen Pseudomonas syringae pv. syringae and to adopt a proper management strategy suitable for the integrated disease management programs.

Knowledge Management: Journal