Rapid Detection of Wheat Blast Pathogen Magnaporthe oryzae Triticum Pathotype Using Genome-Specific Primers and Cas12a-mediated Technology

文献类型： 外文期刊

第一作者： Kang, Houxiang

作者： Kang, Houxiang;Peng, Ye;Deng, Yufei;Zhou, Yilin;Zhou, Xueping;Hua, Kangyu;Bellizzi, Maria;Wang, Guo-Liang;Gupta, Dipali Rani;Mahmud, Nur Uddin;Paul, Sanjoy Kumar;Islam, Md Tofazzal;Urashima, Alfredo S.;Peterson, Gary

作者机构：

关键词： Wheat blast; Magnaporthe oryzae Triticum; Cas12a; Nucleic acid rapid lateral flow immunoassay; Field detection

期刊名称：ENGINEERING （ 影响因子：7.553； 五年影响因子：8.977 ）

ISSN： 2095-8099

年卷期： 2021 年 7 卷 9 期

页码：

收录情况： SCI

摘要： 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. (C) 2021 THE AUTHORS. Published by Elsevier LTD on behalf of Chinese Academy of Engineering and Higher Education Press Limited Company.

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