Bi Xinyue, Li Xiaodong, Yu Haibo, An Mengnan, Li Rui, Xia Zihao, Wu Yuanhua
College of Plant Protection, Shenyang Agricultural University, Shenyang, Liaoning, China.
General Station of Forest and Grassland Pest and Diseases Control, National Forestry and Grassland Administration, Shenyang, Liaoning, China.
PeerJ. 2019 Aug 22;7:e7539. doi: 10.7717/peerj.7539. eCollection 2019.
Watermelon ( Thunb.) is considered as a popular and nutritious fruit crop worldwide. Watermelon blood flesh disease caused by (CGMMV) and bacterial fruit blotch caused by , are two major quarantine diseases of watermelon and result in considerable losses to global watermelon production. In this study, a multiplex reverse-transcription polymerase chain reaction (RT-PCR) method was developed for simultaneous detection of CGMMV and in both watermelon leaves and seeds. Two pairs of specific primers were designed based on the conserved sequences of the genomic RNA of CGMMV and the internal transcribed spacer of , respectively. from watermelon was added as an internal reference gene to prevent false negatives. No cross-reactivity was detected with other viral or bacterial pathogens infecting watermelon. Moreover, the multiplex RT-PCR showed high sensitivity and could simultaneously detect CGMMV and as little as 10 copies of plasmid DNA. This method was successfully applied to test field-collected watermelon leaves and stored seeds of cucurbitaceous crops. These results suggested that the developed multiplex RT-PCR technique is a rapid, efficient, and sensitive method for simultaneous detection of CGMMV and , providing technical support for monitoring, predicting, and preventing these two quarantine diseases. To our knowledge, this is the first report on simultaneous detection of a virus and a bacterium by multiplex RT-PCR in watermelon.
西瓜（Citrullus lanatus (Thunb.) Matsum. & Nakai）被认为是全球一种广受欢迎且营养丰富的水果作物。由黄瓜绿斑驳花叶病毒（CGMMV）引起的西瓜果肉红化病和由西瓜嗜酸菌（Acidovorax citrulli）引起的细菌性果斑病，是西瓜的两种主要检疫性病害，给全球西瓜生产造成了相当大的损失。在本研究中，开发了一种多重逆转录聚合酶链反应（RT-PCR）方法，用于同时检测西瓜叶片和种子中的CGMMV和西瓜嗜酸菌。分别基于CGMMV基因组RNA的保守序列和西瓜嗜酸菌的内转录间隔区设计了两对特异性引物。添加西瓜肌动蛋白（Actin）基因作为内参基因以防止假阴性。未检测到与感染西瓜的其他病毒或细菌病原体的交叉反应。此外，多重RT-PCR显示出高灵敏度，能够同时检测低至10个拷贝质粒DNA的CGMMV和西瓜嗜酸菌。该方法成功应用于检测田间采集的西瓜叶片和葫芦科作物的储存种子。这些结果表明，所开发的多重RT-PCR技术是一种快速、高效且灵敏的同时检测CGMMV和西瓜嗜酸菌的方法，为监测、预测和预防这两种检疫性病害提供了技术支持。据我们所知，这是关于在西瓜中通过多重RT-PCR同时检测病毒和细菌的首次报道。
