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Detection of disease in Cucurbita maxima Duch. ex Lam. caused by a mixed infection of Zucchini yellow mosaic virus, Watermelon mosaic virus, and Cucumber mosaic virus in Southeast China using a novel small RNA sequencing method

The genus Cucurbita comprises many popular vegetable and ornamental plants, including pumpkins, squashes, and gourds, that are highly valued in China as well as in many other countries. During a survey conducted in Zhejiang province, Southeast China in 2016, severe symptoms of viral infection were o...

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Autores principales: Wang, Yi, Zhu, Pu, Zhou, Qin, Zhou, Xiaojun, Guo, Ziqing, Cheng, Linrun, Zhu, Liyan, He, Xiaochan, Zhu, Yidan, Hu, Yang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: PeerJ Inc. 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6815192/
https://www.ncbi.nlm.nih.gov/pubmed/31660276
http://dx.doi.org/10.7717/peerj.7930
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author Wang, Yi
Zhu, Pu
Zhou, Qin
Zhou, Xiaojun
Guo, Ziqing
Cheng, Linrun
Zhu, Liyan
He, Xiaochan
Zhu, Yidan
Hu, Yang
author_facet Wang, Yi
Zhu, Pu
Zhou, Qin
Zhou, Xiaojun
Guo, Ziqing
Cheng, Linrun
Zhu, Liyan
He, Xiaochan
Zhu, Yidan
Hu, Yang
author_sort Wang, Yi
collection PubMed
description The genus Cucurbita comprises many popular vegetable and ornamental plants, including pumpkins, squashes, and gourds, that are highly valued in China as well as in many other countries. During a survey conducted in Zhejiang province, Southeast China in 2016, severe symptoms of viral infection were observed on Cucurbita maxima Duch. ex Lam. Diseased plants showed symptoms such as stunting, mosaicking, Shoe string, blistering, yellowing, leaf deformation, and fruit distortion. Approximately, 50% of Cucurbita crops produced in Jinhua were diseased, causing an estimated yield loss of 35%. In this study, we developed a method using all known virus genomes from the NCBI database as a reference to map small RNAs to develop a diagnostic tool that could be used to diagnose virus diseases of C. maxima. 25 leaf samples from different symptomatic plants and 25 leaf samples from non-symptomatic plants were collected from the experimental field of Jihua National Agricultural Technology Garden for pathogen identification. Small RNAs from each set of three symptomatic and non-symptomatic samples were extracted and sequenced by Illumina sequencing. Twenty-four different viruses were detected in total. However, the majority of the small RNAs were from Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV), and Cucumber mosaic virus (CMV). Mixed infections of these three viruses were diagnosed in leaf samples from diseased plants and confirmed by reverse transcription PCR (RT-PCR) using primers specific to these three viruses. Crude sap extract from symptomatic leaf samples was mechanically inoculated back into healthy C. maxima plants growing under greenhouse conditions. Inoculated plants developed the same disease symptoms as those observed in the diseased plants and a mixed infection of ZYMV, WMV, and CMV was detected again by RT-PCR, thus fulfilling Koch’s postulates. The diagnostic method developed in this study involves fewer bioinformatics processes than other diagnostic methods, does not require complex settings for bioinformatics parameters, provides a high level of sensitivity to rapidly diagnose plant samples with symptoms of virus diseases and can be performed cheaply. This method therefore has the potential to be widely applied as a diagnostic tool for viruses that have genome information in the NCBI database.
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spelling pubmed-68151922019-10-28 Detection of disease in Cucurbita maxima Duch. ex Lam. caused by a mixed infection of Zucchini yellow mosaic virus, Watermelon mosaic virus, and Cucumber mosaic virus in Southeast China using a novel small RNA sequencing method Wang, Yi Zhu, Pu Zhou, Qin Zhou, Xiaojun Guo, Ziqing Cheng, Linrun Zhu, Liyan He, Xiaochan Zhu, Yidan Hu, Yang PeerJ Agricultural Science The genus Cucurbita comprises many popular vegetable and ornamental plants, including pumpkins, squashes, and gourds, that are highly valued in China as well as in many other countries. During a survey conducted in Zhejiang province, Southeast China in 2016, severe symptoms of viral infection were observed on Cucurbita maxima Duch. ex Lam. Diseased plants showed symptoms such as stunting, mosaicking, Shoe string, blistering, yellowing, leaf deformation, and fruit distortion. Approximately, 50% of Cucurbita crops produced in Jinhua were diseased, causing an estimated yield loss of 35%. In this study, we developed a method using all known virus genomes from the NCBI database as a reference to map small RNAs to develop a diagnostic tool that could be used to diagnose virus diseases of C. maxima. 25 leaf samples from different symptomatic plants and 25 leaf samples from non-symptomatic plants were collected from the experimental field of Jihua National Agricultural Technology Garden for pathogen identification. Small RNAs from each set of three symptomatic and non-symptomatic samples were extracted and sequenced by Illumina sequencing. Twenty-four different viruses were detected in total. However, the majority of the small RNAs were from Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV), and Cucumber mosaic virus (CMV). Mixed infections of these three viruses were diagnosed in leaf samples from diseased plants and confirmed by reverse transcription PCR (RT-PCR) using primers specific to these three viruses. Crude sap extract from symptomatic leaf samples was mechanically inoculated back into healthy C. maxima plants growing under greenhouse conditions. Inoculated plants developed the same disease symptoms as those observed in the diseased plants and a mixed infection of ZYMV, WMV, and CMV was detected again by RT-PCR, thus fulfilling Koch’s postulates. The diagnostic method developed in this study involves fewer bioinformatics processes than other diagnostic methods, does not require complex settings for bioinformatics parameters, provides a high level of sensitivity to rapidly diagnose plant samples with symptoms of virus diseases and can be performed cheaply. This method therefore has the potential to be widely applied as a diagnostic tool for viruses that have genome information in the NCBI database. PeerJ Inc. 2019-10-23 /pmc/articles/PMC6815192/ /pubmed/31660276 http://dx.doi.org/10.7717/peerj.7930 Text en ©2019 Wang et al. https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.
spellingShingle Agricultural Science
Wang, Yi
Zhu, Pu
Zhou, Qin
Zhou, Xiaojun
Guo, Ziqing
Cheng, Linrun
Zhu, Liyan
He, Xiaochan
Zhu, Yidan
Hu, Yang
Detection of disease in Cucurbita maxima Duch. ex Lam. caused by a mixed infection of Zucchini yellow mosaic virus, Watermelon mosaic virus, and Cucumber mosaic virus in Southeast China using a novel small RNA sequencing method
title Detection of disease in Cucurbita maxima Duch. ex Lam. caused by a mixed infection of Zucchini yellow mosaic virus, Watermelon mosaic virus, and Cucumber mosaic virus in Southeast China using a novel small RNA sequencing method
title_full Detection of disease in Cucurbita maxima Duch. ex Lam. caused by a mixed infection of Zucchini yellow mosaic virus, Watermelon mosaic virus, and Cucumber mosaic virus in Southeast China using a novel small RNA sequencing method
title_fullStr Detection of disease in Cucurbita maxima Duch. ex Lam. caused by a mixed infection of Zucchini yellow mosaic virus, Watermelon mosaic virus, and Cucumber mosaic virus in Southeast China using a novel small RNA sequencing method
title_full_unstemmed Detection of disease in Cucurbita maxima Duch. ex Lam. caused by a mixed infection of Zucchini yellow mosaic virus, Watermelon mosaic virus, and Cucumber mosaic virus in Southeast China using a novel small RNA sequencing method
title_short Detection of disease in Cucurbita maxima Duch. ex Lam. caused by a mixed infection of Zucchini yellow mosaic virus, Watermelon mosaic virus, and Cucumber mosaic virus in Southeast China using a novel small RNA sequencing method
title_sort detection of disease in cucurbita maxima duch. ex lam. caused by a mixed infection of zucchini yellow mosaic virus, watermelon mosaic virus, and cucumber mosaic virus in southeast china using a novel small rna sequencing method
topic Agricultural Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6815192/
https://www.ncbi.nlm.nih.gov/pubmed/31660276
http://dx.doi.org/10.7717/peerj.7930
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