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

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

机构地区：[1]State Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193,China [2]Department of Plant Pathology,The Ohio State University,Columbus,OH 43210,USA [3]Institute of Biotechnology and Genetic Engineering(IBGE),Bangabandhu Sheikh Mujibur Rahman Agricultural University,Dhaka 1706,Bangladesh [4]Centro de Ciências Agrárias,Universidade Federal de São Carlos,Araras,SP CEP 13600-000,Brazil [5]Foreign Disease-Weed Science Research Unit(FDWSRU),United States Department of Agriculture-Agricultural Research Service(USDA-ARS),Ft.Detrick,MD 21702,USA

出　　处：《Engineering》2021年第9期1326-1335,共10页工程（英文）

基　　金：The data that support the findings of this study have being submitted to GenBank and the accession numbers are JAAXMV000000000 and JAAXMU000000000.

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 targetdependent 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.

关 键 词：Wheat blast Magnaporthe oryzae Triticum Cas12a Nucleic acid rapid lateral flow immunoassay Field detection 

分 类 号：S435.121[农业科学—农业昆虫与害虫防治]