Development, validation and utility of conventional and real-time PCR based marker for the detection of Bipolaris oryzae causing brown spot disease of rice

Brown spot, one of the emerging diseases affecting rice production worldwide, has been studied for over a century. A quick and reliable PCR-based diagnostic assay has been developed to detect the causal organism of brown spot disease, Bipolaris oryzae for its rapid monitoring in rice-grown areas. In this study, we designed a set of primers (ssp1RABo-F and ssp1RABo-R) from a hypothetical small-secreted protein (SSP) gene, unique to B. oryzae (XM_007689836.1) that was identified through comparative secretome analysis. This specific marker (ssp1BoRA_278, KU900505.1) amplified a sequence of 278 bp in all the isolates of B. oryzae tested during the study. This novel marker distinguished B. oryzae from other Bipolaris spp. as well as from other fungal pathogens of rice and other crops. The analytical sensitivity of the marker was observed as 1 pg (copy no. 27.91) using conventional PCR assay. To enhance the sensitivity and utility of the marker, a real-time PCR-based (qPCR) assay was also developed using the same primer set as used in conventional PCR. The sensitivity of the marker was enhanced by 10 times to detect as less as 100 fg DNA (copy no. 2.791) of the pathogen through qPCR. The PCR and qPCR-based detection using this marker will provide a rapid and reliable technique for quick and efficient detection, quantification for genotype resistance, and monitoring of B. oryzae in field, seed, and soil. The marker could detect the pathogen in the host before the appearance of the symptoms. Therefore, early detection using this marker will help in better management of brown spot disease of rice.