Molecular characterization of isolates-causing brown spot of rice [oryza sativa L.] in rainfed ecosystems in the Philippines

The causal pathogen of brown spot in rice was originally classified in the Helminthosporium group, but was subsequently transferred to Bipolaris, which is characterized by bipolarly germinating conidia. However, there are reports that it might be caused by Drechslera due to observance of intercalary germination in some of the isolates. The effect of selection, culture media, and age of culture on the type of germination of the conidia was determined on three isolates from Cavinti, San Pablo, and Palawan obtained from infected leaves showing typical symptoms of brown spot. Selection for bipolarly-germinating conidia had no effect on the number of spores that bipolarly-germinated in the subsequent generation cultures. Among the seven different culture media tested, Rabbit Food Agar (RFA) and Water Agar (WA) increased the percentage of bipolar germination of conidia, but this varied between isolates. Incubation of the cultures for longer periods consistently increased the number of bipolarly-germinating conidia with a maximum for conidia harvested from 20-30 days RFA cultures. This study shows that conidial maturity is the most critical factor in increasing the amount of bipolar germination. To study the genetic diversity of 189 brown spot isolates from Iloilo, Leyte and from adjacent islands, a Variable Number Tandem Repeat Marker was used. Thirty-one haplotypes were observed, where 25 haplotypes, were observed, were 25 haplotypes were composed of isolates from Leyte and Iloilo, and of these 25 haplotypes, five haplotypes composed 84% of all the isolates, which were geographically homogenous indicating the prevalence of asexual reproduction within an individual field resulting in clonal populations in Iloilo and Leyte. No association was established between haplotype and their geographical origin, varietal host or pathogenecity, based on percent diseased leaf area. The identity of the brown spot isolates from Laguna, Palawan, Leyte, Iloilo and North Cotabato were confirmed to be Bipolaris oryzae based on phylogenetic analyses of their ITS1,ITS2 and 5.8S rDNA nucleotide sequences.