Abstract
Brown spot disease, caused by Bipolaris oryzae is one of the major fungal diseases of rice distributed world wide. An efficient large scale screening protocol was developed at seedling stage under greenhouse conditions against B. oryzae to identify the resistant source and selection in segregating population. Multiplication of B. oryzae was better on Potato Dextrose Agar involving a combination of Near Ultra Violet and fluorescent light. Conidial concentration of 10⁴/ml was significantly (P<0.0001) effective for spray inoculation on 14-day-old rice seedlings followed by 40h incubation time in humid chamber. The second leaf from the top was significantly (P<0.0001) appropriate to estimate brown spot. Significant differences were obtained between rice cultivars from diverse germplasm against 18 monoconidial isolates of B. oryzae collected from Laguna, Philippines. Estimation of brown spot as lesion type ratio (LTR) based on lesion number, lesion size and leaf area was more appropriate and had significant correlation with other disease rating system. Dinorado (accession IRTP 12568), a tall traditional japonica cultivar of the Philippines was resistant and a semi dwarf modern indica cultivar IR 36 was highly susceptible. B. oryzae isolates exhibited significant variation for morphological features, pathogenicity with host-pathogen interactions and its DNA finger printing. The isolates differed significantly for growth rate (P<.0031), conidial concentration (P<.0001), conidial germination (P<.0221) and bipolar germination pattern (P<.0027). Significant cultivar x isolate interaction indicated evidence for the presence of physiological races, but not sufficient to designate into distinct races of the fungus. Analysis of VNTR-PCR DNA fingerprints identified six genetic groups at 72% similarity, which supports the evidence of physiological races in pathogenicity. Genotype IR 36, IR 64, UPLRi 7, N22, MAAL 6, IR 69726-29-2-2 (restorer) could be the source of differentials. The F₁, F₂, F₃, BC₁F₁ and BC₂F₁ population from Dinorado and IR 36 were screened with a highly virulent isolate. Phenotypic segregation of 200 F₃ progenies showed that two recessive genes from Dinorado confer resistance to brown spot. Bulk segregant analysis was applied to analyze corresponding 186 F₂ lines with 160 SSR markers. Four markers on chromosome 12 showed significant (<.0001) association with brown spot resistance. Molecular analysis of homozygous F₃ progenies confirmed the results of F₂ lines. The interval analysis suggested that gene for resistance is located between 8.7 to 18.2 MB on chromosome 12. The two genes were with larger effect of markers RM 1261 and RM 277, smaller effect of markers RM 101 and RM 3264 with at least one QTL. The two genes imparting resistance to brown spot in Dinorado were designated as bs1 and bs2. To the best of our knowledge this is the first report on tagging of resistant genes for brown spot of rice. The present findings would provide guidelines to incorporate resistance from Dinorado to susceptible but otherwise high yielding cultivars of rice. The closely linked DNA markers would be suitable for use in marker-assisted selection in rice breeding programs.