Molecular Differentiation of Bipolaris oryzae and Nigrospora oryzae Associated with Brown Spot Symptoms in Rice

Rice brown spot is primarily associated with Bipolaris oryzae; however, Nigrospora oryzae can also be recovered from rice leaves exhibiting similar symptoms, complicating their differentiation based on field symptoms alone. In this study, the temporal occurrence of these fungi was investigated in symptomatic rice leaves collected during the 2024 and 2025 growing seasons, and molecular assays were developed for their rapid discrimination. B. oryzae predominated during the early to mid-growing season, whereas the relative frequency of N. oryzae increased during later sampling periods. Sequence comparisons were used to develop primer sets targeting ITS, TEF1, and GAPDH for B. oryzae and ITS, TEF1, and TUB2 for N. oryzae. The primers preferentially amplified their corresponding target species in conventional and real-time PCR assays. Validation using additional field isolates revealed weak non-target amplification with some primer sets in conventional PCR, whereas real-time PCR provided clear quantitative separation between target and non-target isolates, with ΔCq values ranging from 9.9 to 15.8. These results demonstrate temporal changes in the relative occurrence of B. oryzae and N. oryzae on symptomatic rice leaves and indicate that real-time PCR provides a useful molecular approach for differentiating the two fungi. The developed assays may facilitate further investigation of their seasonal occurrence and association with brown spot symptoms in rice.