Rapid Detection of Wheat Blast Pathogen Magnaporthe oryzae Triticum Pathotype Using Genome-Specific Primers and Cas12a-mediated Technology
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...
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| Format: | Article |
| Language: | English |
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Elsevier
2021-09-01
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| Series: | Engineering |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S209580992030240X |
| _version_ | 1818989003617075200 |
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| author | 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 |
| author_facet | 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 |
| author_sort | Houxiang Kang |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-20T19:31:34Z |
| format | Article |
| id | doaj.art-32f08025f02b4e44a1f44c8bfd900103 |
| institution | Directory Open Access Journal |
| issn | 2095-8099 |
| language | English |
| last_indexed | 2024-12-20T19:31:34Z |
| publishDate | 2021-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Engineering |
| spelling | doaj.art-32f08025f02b4e44a1f44c8bfd9001032022-12-21T19:28:46ZengElsevierEngineering2095-80992021-09-017913261335Rapid Detection of Wheat Blast Pathogen Magnaporthe oryzae Triticum Pathotype Using Genome-Specific Primers and Cas12a-mediated TechnologyHouxiang Kang0Ye Peng1Kangyu Hua2Yufei Deng3Maria Bellizzi4Dipali Rani Gupta5Nur Uddin Mahmud6Alfredo S. Urashima7Sanjoy Kumar Paul8Gary Peterson9Yilin Zhou10Xueping Zhou11Md Tofazzal Islam12Guo-Liang Wang13State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China; Corresponding authors.State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaDepartment of Plant Pathology, The Ohio State University, Columbus, OH 43210, USAState Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaDepartment of Plant Pathology, The Ohio State University, Columbus, OH 43210, USAInstitute of Biotechnology and Genetic Engineering (IBGE), Bangabandhu Sheikh Mujibur Rahman Agricultural University, Dhaka 1706, BangladeshInstitute of Biotechnology and Genetic Engineering (IBGE), Bangabandhu Sheikh Mujibur Rahman Agricultural University, Dhaka 1706, BangladeshCentro de Ciências Agrárias, Universidade Federal de São Carlos, Araras, SP CEP 13600-000, BrazilInstitute of Biotechnology and Genetic Engineering (IBGE), Bangabandhu Sheikh Mujibur Rahman Agricultural University, Dhaka 1706, BangladeshForeign Disease-Weed Science Research Unit (FDWSRU), United States Department of Agriculture-Agricultural Research Service (USDA-ARS), Ft. Detrick, MD 21702, USAState Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaState Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaInstitute of Biotechnology and Genetic Engineering (IBGE), Bangabandhu Sheikh Mujibur Rahman Agricultural University, Dhaka 1706, Bangladesh; Corresponding authors.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.http://www.sciencedirect.com/science/article/pii/S209580992030240XWheat blastMagnaporthe oryzae TriticumCas12aNucleic acid rapid lateral flow immunoassayField detection |
| spellingShingle | 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 Rapid Detection of Wheat Blast Pathogen Magnaporthe oryzae Triticum Pathotype Using Genome-Specific Primers and Cas12a-mediated Technology Engineering Wheat blast Magnaporthe oryzae Triticum Cas12a Nucleic acid rapid lateral flow immunoassay Field detection |
| title | Rapid Detection of Wheat Blast Pathogen Magnaporthe oryzae Triticum Pathotype Using Genome-Specific Primers and Cas12a-mediated Technology |
| title_full | Rapid Detection of Wheat Blast Pathogen Magnaporthe oryzae Triticum Pathotype Using Genome-Specific Primers and Cas12a-mediated Technology |
| title_fullStr | Rapid Detection of Wheat Blast Pathogen Magnaporthe oryzae Triticum Pathotype Using Genome-Specific Primers and Cas12a-mediated Technology |
| title_full_unstemmed | Rapid Detection of Wheat Blast Pathogen Magnaporthe oryzae Triticum Pathotype Using Genome-Specific Primers and Cas12a-mediated Technology |
| title_short | Rapid Detection of Wheat Blast Pathogen Magnaporthe oryzae Triticum Pathotype Using Genome-Specific Primers and Cas12a-mediated Technology |
| title_sort | rapid detection of wheat blast pathogen magnaporthe oryzae triticum pathotype using genome specific primers and cas12a mediated technology |
| topic | Wheat blast Magnaporthe oryzae Triticum Cas12a Nucleic acid rapid lateral flow immunoassay Field detection |
| url | http://www.sciencedirect.com/science/article/pii/S209580992030240X |
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