Novel Multiplex and Loop-Mediated Isothermal Amplification Assays for Rapid Species and Mating-Type Identification of Oculimacula acuformis and O. yallundae (Causal Agents of Cereal Eyespot), and Application for Detection of Ascospore Dispersal and In Planta Use

Abstract

Eyespot, caused by the related fungal pathogens Oculimacula acuformis and O. yallundae, is an important cereal stem-base disease in temperate parts of the world. Both species are dispersed mainly by splash-dispersed conidia but are also known to undergo sexual reproduction, yielding apothecia containing ascospores. Field diagnosis of eyespot can be challenging, with other pathogens causing similar symptoms, which complicates eyespot management strategies. Differences between O. acuformis and O. yallundae (e.g., host pathogenicity and fungicide sensitivity) require that both be targeted for effective disease management. Here, we develop and apply two molecular methods for species-specific and mating-type (MAT1-1 or MAT1-2) discrimination of O. acuformis and O. yallundae isolates. First, a multiplex PCR-based diagnostic assay targeting the MAT idiomorph region was developed, allowing simultaneous determination of both species and mating type. This multiplex PCR assay was successfully applied to type a global collection of isolates. Second, the development of loop-mediated isothermal amplification (LAMP) assays targeting β-tubulin sequences, which allow fast (<9 min) species-specific discrimination of global O. acuformis and O. yallundae isolates, is described. The LAMP assay can detect very small amounts of target DNA (1 pg) and was successfully applied in planta. In addition, mating-type-specific LAMP assays were also developed for rapid (<12 min) genotyping of O. acuformis and O. yallundae isolates. Finally, the multiplex PCR-based diagnostic was applied, in conjunction with spore trapping in field experiments, to provide evidence of the wind dispersal of ascospores from a diseased crop. The results indicate an important role of the sexual cycle in the dispersal of eyespot.