Abstract
Pyricularia oryzae is a devastating pathogen affecting rice and a wide range of cereal crops. Accurate and specific detection of this species is essential for population genetics, epidemiological studies, and the enforcement of quarantine measures. In this study, the specificity of several P. oryzae-specific diagnostic primer pairs, including pfh2a/pfh2b, Pot2a-L2/Pot2a-R2, mif23_01F/mif23_01R, MIF-forward/MIF-reverse, MHP1F/MHP1R, and PoM- 1F/PoM-1R were re-evaluated using in silico and PCR assays. In addition, the efficacy of an ISSR marker was assessed as a cost-effective, species-level screening method for a large set of Pyricularia isolates. The results of in silico analysis revealed that PoM-1F/PoM-1R primer pair was not species-specific, while four other primers showed probable specificity for P. oryzae detection. However, PCR assays revealed that none of the tested P. oryzae-specific primers could reliably distinguish this species from other formally accepted species within the genus Pyricularia. These results highlight the urgent need to develop novel molecular markers that can unambiguously differentiate P. oryzae from related Pyricularia species. Such markers would significantly improve the diagnosis and surveillance of blast diseases. Meanwhile, the ISSR-PCR banding patterns produced from 36 strains representing seven formally accepted Pyricularia species were reproducible and congruent with phylogenetic species recognition. Therefore, in studies with large numbers of isolates, where cost-effective grouping is required prior to multi-gene sequencing, this marker represents a highly beneficial tool for selecting representative isolates corresponding to distinct species.