Rice blast, caused by Pyricularia oryzae, is one of the most destructive fungal diseases in rice, severely impacting rice production worldwide every year. Rapid, accurate, and visual detection of P. oryzae is essential for more effective prevention and control. In this study, we developed a recombinase polymerase amplification–lateral flow dipstick (RPA-LFD) assay to detect P. oryzae. Species-specific RPA primer pairs and probe were designed based on the target gene MGG_15975. The optimized reaction temperature and time were set at 37°C and 25 min, respectively. Specificity analysis showed that the assay could specifically detect P. oryzae isolates from rice, whereas other fungal species or Pyricularia species from grasses were not detected. Additionally, this assay demonstrated high sensitivity, capable of detecting as low as 10⁻² ng/μl of P. oryzae genomic DNA, which was found to be 100 times more sensitive than conventional PCR. Furthermore, using this assay, P. oryzae was effectively detected in diseased leaves in rice fields and could also be identified at an early stage of infection before obvious lesions appeared in artificially inoculated rice seedlings. Therefore, the RPA-LFD assay developed in our study for the detection of P. oryzae is rapid, highly sensitive, and efficient, which has the potential application for early diagnosis of P. oryzae infection in rice fields.

Rice false smut (RFS), caused by Villosiclava virens, is an important fungal disease in panicles of rice. V. virens is a heterothallic ascomycete controlled by two opposite idiomorphs, MAT1-1 and MAT1-2. Previous study showed that sexual reproduction of V. virens plays an important role in the epidemic of RFS. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay to detect the mating type of V. virens easily and rapidly by using specific primers based on the mating type genes MAT1-1-2 and MAT1-2-1, respectively. The LAMP assay used only a water/dry bath and could recognize the mating type of V. virens in just 45 min. The LAMP assay was so sensitive that it could detect small amounts of V. virens genomic DNA (as low as 2.0 pg of MAT1-1 and 200.0 pg of MAT1-2) and was 10 times more sensitive than PCR. In addition, we demonstrated the application of mating type via LAMP assay by assessing the genomic DNA of V. virens isolated from rice fields. The high efficiency and specificity of this LAMP assay suggest that it can be used as a rapid testing tool in mating type recognition of V. virens isolates in the field.