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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Autores principales:	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
Formato:	article
Lenguaje:	EN
Publicado:	Elsevier 2021
Materias:	Wheat blast Magnaporthe oryzae Triticum Cas12a Nucleic acid rapid lateral flow immunoassay Field detection Engineering (General). Civil engineering (General) TA1-2040
Acceso en línea:	https://doaj.org/article/32f08025f02b4e44a1f44c8bfd900103
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spelling	oai:doaj.org-article:32f08025f02b4e44a1f44c8bfd9001032021-11-14T04:32:10ZRapid Detection of Wheat Blast Pathogen Magnaporthe oryzae Triticum Pathotype Using Genome-Specific Primers and Cas12a-mediated Technology2095-809910.1016/j.eng.2020.07.016https://doaj.org/article/32f08025f02b4e44a1f44c8bfd9001032021-09-01T00:00:00Zhttp://www.sciencedirect.com/science/article/pii/S209580992030240Xhttps://doaj.org/toc/2095-8099Wheat 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.Houxiang KangYe PengKangyu HuaYufei DengMaria BellizziDipali Rani GuptaNur Uddin MahmudAlfredo S. UrashimaSanjoy Kumar PaulGary PetersonYilin ZhouXueping ZhouMd Tofazzal IslamGuo-Liang WangElsevierarticleWheat blastMagnaporthe oryzae TriticumCas12aNucleic acid rapid lateral flow immunoassayField detectionEngineering (General). Civil engineering (General)TA1-2040ENEngineering, Vol 7, Iss 9, Pp 1326-1335 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Wheat blast Magnaporthe oryzae Triticum Cas12a Nucleic acid rapid lateral flow immunoassay Field detection Engineering (General). Civil engineering (General) TA1-2040
spellingShingle	Wheat blast Magnaporthe oryzae Triticum Cas12a Nucleic acid rapid lateral flow immunoassay Field detection Engineering (General). Civil engineering (General) TA1-2040 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
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.
format	article
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
title	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_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_sort	rapid detection of wheat blast pathogen magnaporthe oryzae triticum pathotype using genome-specific primers and cas12a-mediated technology
publisher	Elsevier
publishDate	2021
url	https://doaj.org/article/32f08025f02b4e44a1f44c8bfd900103
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Rapid Detection of Wheat Blast Pathogen Magnaporthe oryzae Triticum Pathotype Using Genome-Specific Primers and Cas12a-mediated Technology

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