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.
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.
复制标题
新型多重和环介导等温扩增测定，用于快速识别尖眼眼斑和 O. yallundae（谷物眼斑的致病因子）的物种和交配型鉴定，以及用于检测子囊孢子传播和植物用途的应用。
DOI：
10.1094/phyto-04-20-0116-r
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发表时间：
2021
期刊：
影响因子：
3.2
通讯作者：
King KM
中科院分区：
文献类型：
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作者：
King KM
眼斑病是由眼斑菌（OculimaculaacuformisandO.）稻瘟病菌是世界温带地区的一种重要的禾谷类茎基病害。这两个物种主要通过飞溅分散的分生孢子传播，但也已知进行有性生殖，产生含有子囊孢子的孢子囊。眼斑的现场诊断可能具有挑战性，其他病原体引起类似症状，这使眼斑管理策略复杂化。O.尖形藻属O. Yallundae（例如，宿主致病性和杀真菌剂敏感性）要求两者都是有效疾病管理的目标。在这里，我们开发和应用两种分子方法，种特异性和交配型（MAT 1 - 1或MAT 1 -2）的歧视O。尖形藻属O.耶尔森氏菌分离物首先，开发了一种基于多重PCR的诊断检测方法，该方法以MATidiomorph区域为靶点，可以同时确定物种和交配型。该多重PCR检测方法已成功应用于全球分离株的分型。其次，开发了靶向β-微管蛋白序列的环介导等温扩增（LAMP）测定，其允许快速（<9分钟）物种特异性区分globalO。尖形藻属O. Yallundae isolates，被描述。LAMP检测可以检测到非常少量的靶DNA（1 pg），并成功地应用于植物。此外，还开发了交配型特异性LAMP测定法用于快速（<12 min）基因分型。尖形藻属O.耶尔森氏菌分离物最后，多重PCR为基础的诊断，结合孢子诱捕在田间试验中，提供证据的风传播的子囊孢子从患病作物。结果表明，性周期在眼斑的扩散中起着重要作用。
Eyespot, caused by the related fungal pathogensOculimacula acuformisandO. 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 betweenO. acuformisandO. 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 (MAT1-1orMAT1-2) discrimination ofO. acuformisandO. yallundaeisolates. First, a multiplex PCR-based diagnostic assay targeting theMATidiomorph 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 globalO. acuformisandO. yallundaeisolates, 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 ofO. acuformisandO. yallundaeisolates. 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.