Detection and monitoring of fungicide resistance in the rice blast pathogen by innovative molecular methods

Rice blast, caused by Pyricularia oryzae, is one of the most important rice diseases worldwide, causing globally 30% yield losses, threatening thus global food security. Rice blast management relies on fungicides, especially in Europe, as susceptible traditional rice varieties are extensively grown. Often, the first choice among a very limited spectrum of chemical control means are quinone outside inhibitor (QoI) fungicides. However, they are classified as high-risk fungicides as they are prone to resistance development in fungal pathogens. Until now, QoI resistance has been detected in ca. 40 different plant pathogens. Recently, azoxystrobin-resistant strains have been identified in P. oryzae from rice in Japan and Vietnam, which poses concerns about the spread of QoI resistance also in other rice-growing areas. Despite this, no reliable and sensitive detection method of QoI resistance in P. oryzae populations exists at the moment. We developed an allele-specific qPCR-based method for the detection of QoI-resistant strains of P. oryzae. It is based on selective amplification of sensitive (S) or resistant (R) allele of cytochrome b. The method was developed using plasmids with cloned cytochrome b fragment containing or not the single-point mutation and was further verified on genomic DNA mixtures isolated from sensitive and resistant P. oryzae strains. Moreover, the specificity of primers was verified on several non-target fungal pathogens.