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

Abstract

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.

Insights into the early transcriptomic response against watermelon mosaic virus in melon

“…Viruses in the main melon producing areas have a great yield-limiting potential and are a major economic concern for growers [2]. Different studies carried out recently in Europe [3][4][5], Asia [6][7][8] and America [9,10] indicate that the potyvirus Watermelon mosaic virus (WMV) is the most prevalent virus in cucurbits fields. WMV infection in melon leads to a severe symptomatology, that includes chlorosis, mosaic, leaf distortion, lead tip stunting, as well as the stop of plant growth, which results in yield reduction and fruit quality loss.…”

Development, Validation, and Application of Reverse Transcription Real-Time and Droplet Digital PCR Assays for the Detection of the Potyviruses Watermelon Mosaic Virus and Zucchini Yellow Mosaic Virus in Cucurbits

“…Symptoms of ZYMV and WMV infections are often overlapping because the two potyviruses frequently occur in mixed infection, as reported in several cucurbit crops from the Mediterranean basin to Americas and Asia [ 11 , 12 , 13 ]. Interactions between co-occurring viruses are known to induce either synergistic or antagonistic effects, namely one or both viruses can benefit or suffer from the presence of each other in terms of viral titre, pathogenicity, and transmission by vectors [ 14 , 15 ].…”

“…Both serological and molecular diagnostic methods are available for the detection of WMV and ZYMV in routine diagnosis, such as enzyme-linked immunosorbent analysis (ELISA) [ 17 ], conventional reverse-transcription polymerase chain reaction (RT-PCR) [ 6 , 18 , 19 , 20 ], reverse transcription loop-mediated isothermal amplification (RT-LAMP) [ 21 , 22 ], and small RNA sequencing [ 11 ]. Most of these methods were not validated for their sensitivity and specificity according to the international requirements for plant pest diagnosis.…”