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Diversity and Pathogenicity of the Rice Brown Spot Pathogen, Bipolaris Oryzae (breda De Haan) Shoem. in Bangladesh Assessed by Genetic Fingerprint Analysis

M. M. KAMAL
Plant Pathology Division, BRRI Regional Station, Barisal, Bangladesh.

M. A. T. MIA
Seed Pathology and Molecular Laboratory, Plant Pathology Division, Bangladesh Rice Research Institute, Gazipur-1701, Bangladesh

Abstract/ Executive Summary:

A survey on the occurrence of rice brown spot pathogen, Bipolaris oryzae (Breda de Haan) Shoem. was carried out across Bangladesh. A representative set of 152 isolates were characterized based on DNA fingerprinting, using variable number tandem repeat analysis and assessed their phylogenetic identity, diversity and pathogenicity. The analysis showed considerable diversity among the isolates, being separated into 12 fingerprint types at 75% similarity level. Majority of the samples possessed one fingerprint type. Pathogenicity test with nine fingerprint types on rice variety BR24, showed 20.5-84.5% germination failure, 2.50-12.50, 5.84-11.67 and 7.50-14.17% post-emergence seedling death at 15, 30 and 45 days after transplanting and 18.75-22.50% yield loss. Significant differences in aggressiveness were observed among the isolates of the same fingerprint types.

Keywords: Bipolaris oryzae; Rice brown spot; Pathogenicity; Agarose-synergel; Fingerprints

Location: Seed Pathology and Molecular Laboratory, Plant Pathology Division, Bangladesh Rice Research Institute

Start Date:

End Date:

Program Area: Pest Management

Commodity: Rice

Objectives:

The present work was undertaken to characterize the fungus following DNA fingerprinting, determine their biological variation, record their diversity and determine the variation in their pathogenicity.

Methodology:

Isolation and purification: The pathogen, Bipolaris oryzae (Breda de Haan) Shoem. was isolated by tissue planting method and purified through transfer of single conidium to potato dextrose agar (PDA) medium. The fungus was grown in potato dextrose broth for three days, mycelia were harvested and blotted to remove excess fluid and stored at -20º C in aluminium foil packet. DNA was extracted from 306 isolates including 2 isolates of IRRI following Raeder and Broda (1985) and Anonymous (2001). DNA concentration was estimated on 0.8% agarose gel and 1ng/µl stocks were prepared for variable number tandem repeat-polymerase chain reaction (VNTR-PCR, Pascual et al. 2000). Agarose-synergel electrophoresis for VNTR analysis: Based on the morphology of the fungus, 152 isolates were selected for VNTR-PCR. An amount of 1ng of fungal DNA was amplified with primer MR (GAGGGTGGCGGTTCT) under the conditions: 94º C for 2 minutes, then 35 cycles of 94º C for 20 seconds, 45º C for 30 seconds and finally 72º C for 2.5 minutes. This was followed by 5 minutes at 72º C and then held at 4º C until the sample was retrieved. The Primer MR was used following Bridge et al. (1997). Amplified DNA of 152 isolates and 1kb ladder was loaded into the gel and resolved by electrophoresis for 7 hours at 100 millivolts. The gel was stained and the fingerprints photographed. DNA fingerprints were scored visually and recorded according to the position of bands for each of the 152 isolates as 1, for the presence and 0, for the absence of a band. A similarity matrix based on Jaccard's similarity coefficient was used and cluster analysis of the matrix was done using unweighted pair group method with arithmetic mean (UPGMA) by numerical taxonomy and multivariate analysis system (NTSYS-PC) version 2.11a (Rohlf 2000). Pathogenicity of Bipolaris oryzae: The effects of Bipolaris oryzae, on seed infection, pre- and post-emergence death of seedlings and subsequent disease development in plants were investigated. The soil was fertilized with 60 kg N + 35 kg P + 40 kg K/ha. Hot water treated truthfully labelled seeds (TLS) were placed on two days old cultures of B. oryzae, representing nine fingerprint types (FPTs) keeping necessary control and observed for 5-7 days and percentage of germination was recorded. Eight replications for each isolate were maintained in completely randomized design (CRD). In a glasshouse, 30 germinated seeds for each replication/isolate were sown in each tray of sterilized soil and the trays were arranged in CRD with four replications. Necessary cultural practices were done as and when needed. Post-emergence death of the seedlings was recorded at 15 days after transplanting (DAT), 30 DAT and 45 DAT. Another set of inoculated and un-inoculated seeds were directly sown in a 1 x 1 m plots with spacing of 20 x 15 cm following CRD with five replications. Number of effective tillers at maturity from each treatment was counted and grain yield was calculated.

Source of Information: Bangladesh J. Bot. 38(2): 119-125, 2009 (December)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Pathogenicity test with nine fingerprint types on rice variety BR24, showed 20.5-84.5% germination failure, 2.50-12.50, 5.84-11.67 and 7.50-14.17% post-emergence seedling death at 15, 30 and 45 days after transplanting and 18.75-22.50% yield loss. Significant differences in aggressiveness were observed among the isolates of the same fingerprint types.

Knowledge Management: Journal