Rapid Detection of Wheat Blast Pathogen Magnaporthe oryzae Triticum Pathotype Using Genome-Specific Primers and Cas12a-mediated Technology
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
Affiliations
Houxiang Kang
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China; Corresponding authors.
Ye Peng
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
Kangyu Hua
Department of Plant Pathology, The Ohio State University, Columbus, OH 43210, USA
Yufei Deng
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
Maria Bellizzi
Department of Plant Pathology, The Ohio State University, Columbus, OH 43210, USA
Dipali Rani Gupta
Institute of Biotechnology and Genetic Engineering (IBGE), Bangabandhu Sheikh Mujibur Rahman Agricultural University, Dhaka 1706, Bangladesh
Nur Uddin Mahmud
Institute of Biotechnology and Genetic Engineering (IBGE), Bangabandhu Sheikh Mujibur Rahman Agricultural University, Dhaka 1706, Bangladesh
Alfredo S. Urashima
Centro de Ciências Agrárias, Universidade Federal de São Carlos, Araras, SP CEP 13600-000, Brazil
Sanjoy Kumar Paul
Institute of Biotechnology and Genetic Engineering (IBGE), Bangabandhu Sheikh Mujibur Rahman Agricultural University, Dhaka 1706, Bangladesh
Gary Peterson
Foreign Disease-Weed Science Research Unit (FDWSRU), United States Department of Agriculture-Agricultural Research Service (USDA-ARS), Ft. Detrick, MD 21702, USA
Yilin Zhou
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
Xueping Zhou
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
Md Tofazzal Islam
Institute of Biotechnology and Genetic Engineering (IBGE), Bangabandhu Sheikh Mujibur Rahman Agricultural University, Dhaka 1706, Bangladesh; Corresponding authors.
Guo-Liang Wang
Department of Plant Pathology, The Ohio State University, Columbus, OH 43210, USA; Corresponding authors.
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.
