Abstract
Watermelon (Citrullus lanatus Thunb.) is considered as a popular and nutritious fruit crop worldwide. Watermelon blood flesh disease caused by Cucumber green mottle mosaic virus (CGMMV) and bacterial fruit blotch caused by Acidovorax citrulli, 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 A. citrulli 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 A. citrulli, respectively. Transcriptional elongation factor-1α 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 A. citrulli as little as 102 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 A. citrulli, 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.
Highlights
Watermelon (Citrullus lanatus Thunb.) is an economically important fruit crop worldwide (Guo et al, 2015)
Specificity of multiplex reversetranscription polymerase chain reaction (RT-PCR) Total nucleic acids extracted from the watermelon leaves infected by Cucumber mosaic virus (CMV), Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV), Squash mosaic virus (SqMV), P. syringae pv. lachrymans, and X. campestris pv. cucurbitae were performed to assess the specificity of the primers used for multiplex RT-PCR (Fig. 1)
The corresponding amplification segment of 756 or 246 bp in length was showed in watermelon samples infected by Cucumber green mottle mosaic virus (CGMMV) or A. citrulli, while the segments of watermelon elongation factor-1α (EF-1α) were observed only in the watermelon leaves infected by other pathogens (e.g., CMV, WMV, ZYMV, SqMV, P. syringae pv. lachrymans, and X. campestris pv. cucurbitae) or healthy watermelon leaves (Fig. 1)
Summary
Watermelon (Citrullus lanatus Thunb.) is an economically important fruit crop worldwide (Guo et al, 2015). China has become the largest watermelon producing country, accounting for almost 67.1% of the total global watermelon production in 2017. Development of a multiplex RT-PCR assay for simultaneous detection of Cucumber green mottle mosaic virus and Acidovorax citrulli in watermelon. As with many other crops, watermelon production is threatened by pathogens, pests and abiotic stresses. Two plant diseases, blood flesh caused by Cucumber green mottle mosaic virus (CGMMV) and bacterial fruit blotch (BFB) caused by Acidovorax citrulli, have become predominant limiting factors in the yield and quality of watermelon in China (Li et al, 2017; Wu et al, 2016)
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