Rothamsted Research, Department of Biointeractions and Crop Protection, Harpenden, Hertfordshire AL5 2JQ, United Kingdom.
School of Life Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom.
Phytopathology. 2021 Mar;111(3):582-592. doi: 10.1094/PHYTO-04-20-0116-R. Epub 2021 Feb 11.
Eyespot, caused by the related fungal pathogens and , 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 and (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 ( or ) discrimination of and isolates. First, a multiplex PCR-based diagnostic assay targeting the 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 and 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 and 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.
眼斑病由相关真菌病原体 和 引起，是世界温带地区重要的谷类茎基病害。这两个物种主要通过飞溅分散的分生孢子传播，但也已知它们会进行有性繁殖，产生含有子囊孢子的子囊果。田间诊断眼斑病具有挑战性，因为其他病原体也会引起类似的症状，这使得眼斑病的管理策略变得复杂。 和 （例如，宿主致病性和杀菌剂敏感性）之间的差异要求针对两者进行有效疾病管理。在这里，我们开发并应用了两种分子方法，用于 和 分离物的种特异性和交配型（或 ）区分。首先，开发了一种基于多重 PCR 的诊断检测方法，靶向 异态区，允许同时确定两个物种和交配型。该多重 PCR 检测方法成功应用于全球分离物的分型。其次，描述了针对 β-微管蛋白序列的环介导等温扩增 (LAMP) 检测方法的开发，该方法允许快速（<9 分钟）对全球 和 分离物进行种特异性区分。LAMP 检测法可以检测到非常少量的靶 DNA（1 pg），并已成功应用于植物体内。此外，还开发了用于快速（<12 分钟）分型 和 分离物的交配型特异性 LAMP 检测法。最后，应用了基于多重 PCR 的诊断检测方法，并结合田间实验中的孢子捕捉，提供了来自患病作物的子囊孢子通过风传播的证据。结果表明，有性循环在眼斑病的传播中起着重要作用。
