Wheat blast, caused by the fungus Magnaporthe oryzae Triticum (MoT) pathotype, is a devastating disease persistent in South America and Bangladesh. Since MoT generally fails to cause visual symptoms in wheat until the heading stage when the infection would have advanced, disease control by fungicide application solely based on the detection of visual symptoms is ineffective. To develop an accurate and sensitive method to detect MoT at the seedling and vegetative stages for disease control, we sequenced the genomes of two MoT isolates from Brazil and identified two DNA fragments, MoT-6098 and MoT-6099, that are present in the MoT genome but not in the genome of the rice-infecting Magnaporthe oryzae Oryzae (MoO) pathotype. Using polymerase chain reaction (PCR), we confirmed the specificity of the two markers in 53 MoT and MoO isolates from South America and Bangladesh. To test the efficiency of the two markers, we first established a loop-mediated isothermal amplification (LAMP) method to detect MoT at isothermal conditions, without the use of a PCR machine. Following this, we used the Cas12a protein and guide RNAs (gRNAs) to target the MoT-6098 and MoT-6099 sequences. The activated Cas12a showed indiscriminate single-stranded deoxyribonuclease (ssDNase) activity. We then combined target-dependent Cas12a ssDNase activation with recombinase polymerase amplification (RPA) and nucleic acid lateral flow immunoassay (NALFIA) to develop a method that accurately, sensitively, and cost-effectively detects MoT-specific DNA sequences in infected wheat plants. This novel technique can be easily adapted for the rapid detection of wheat blast and other important plant diseases in the field.

小麦瘟是由真菌Magnaporthe oryzae Triticum (MoT) 致病型引起的，是一种持续存在于南美洲和孟加拉国的毁灭性疾病。由于 MoT 通常不会在小麦中引起视觉症状，直到感染进展的抽穗期，因此仅基于视觉症状的检测通过施用杀菌剂来控制病害是无效的。为了开发一种准确灵敏的方法来检测幼苗和营养阶段的 MoT 以控制疾病，我们对来自巴西的两个 MoT 分离株的基因组进行了测序，并鉴定了两个 DNA 片段MoT-6098和MoT-6099，它们存在于 MoT 中基因组但不在感染水稻的Magnaporthe oryzae Oryzae的基因组中(MoO) 病理类型。使用聚合酶链反应 (PCR)，我们确认了来自南美洲和孟加拉国的 53 个 MoT 和 MoO 分离株中的两个标记的特异性。为了测试这两种标记的效率，我们首先建立了一种循环介导的等温扩增 (LAMP) 方法来在等温条件下检测 MoT，而不使用 PCR 机器。在此之后，我们使用 Cas12a 蛋白和引导 RNA (gRNA) 来靶向MoT-6098和MoT-6099序列。激活的 Cas12a 显示出不加选择的单链脱氧核糖核酸酶 (ssDNase) 活性。然后，我们将目标依赖性 Cas12a ssDNase 激活与重组酶聚合酶扩增 (RPA) 和核酸侧流免疫分析 (NALFIA) 相结合，开发出一种方法，可以准确、灵敏且经济高效地检测受感染小麦植株中的 MoT 特异性 DNA 序列。这种新技术很容易适用于小麦瘟疫和田间其他重要植物病害的快速检测。