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Development of one-step multiplex RT-PCR assay for rapid simultaneous detection of five RNA viruses and Acidovorax citrulli in major cucurbitaceous crops in China

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Abstract

Cucurbitaceous fruits and vegetables are important crops. Viral and bacterial diseases cause substantial economic losses to cucurbit crops globally. For rapid detection of these pathogens and improved disease control, a one-step multiplex reverse-transcription polymerase chain reaction (mRT-PCR) system was created. This method allowed for the concurrent detection of Tobacco mosaic virus (TMV), Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV), Cucumber green mottle mosaic virus (CGMMV), Cucumber mosaic virus (CMV), and Acidovorax citrulli. Five pairs of specific primers were created according to the conserved regions around the coat protein (CP) genes of each virus, and one pair was based on the A. citrulli internal transcribed spacer (ITS). To limit false negatives, one pair of primers, created based on the Transcriptional elongation factor 1-α (EF1-α) from the major cucurbitaceous crop species, was put into the mRT-PCR reaction system. Primer concentrations, annealing temperature, extension time, and amplification cycles were optimized. Anticipated fragments of 152 bp (TMV), 205 bp (ZYMV), 318 bp (WMV), 419 bp (CGMMV), 529 bp (CMV), 662 bp (A. citrulli), and 821 bp (EF1-α) were amplified by the multiplex RT-PCR system, and their origin was established via DNA sequencing. This method was successfully used to examine field-collected seed samples of cucurbitaceous crops from China. The results demonstrated that the one-step mRT-PCR technique is a quick, efficient, and sensitive assay for the concurrent detection of six pathogens of cucurbits. It provides a method for monitoring and preventing these diseases.

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All data generated or analyzed during this study are included in this published article (and its supplementary information files).

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Acknowledgements

We would like to thank Ms. Guifen Li and Mr. Meisheng Wei (Institute of Plant Quarantine, Chinese Academy of Inspection and Quarantine) for providing MMV, PRSV, SqMV, and TRSV.

Funding

This work was supported by the Basic Scientific Research Foundation of the Chinese Academy of Inspection and Quarantine (2020JK017), the National Key R&D Program of China (No. 2021YFD1400100, 2021YFD1400103), and the Fellowship of China Postdoc Research Foundation (2021M693037).

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Authors and Affiliations

  1. Institute of Plant Quarantine, Chinese Academy of Inspection and Quarantine, Beijing, 100176, China

    Zhenxing Zhao, Jun Xiang, Qian Tian, Wenjun Zhao & Yongjiang Zhang

  2. Laboratory of Landscaping Agriculture, Ministry of Agriculture and Rural Affairs/Key Laboratory of Biology of Ornamental Plants in East China, National Forestry and Grassland Administration, College of Horticulture, Nanjing Agricultural University, Nanjing, 210095, China

    Jun Xiang

  3. State Key Laboratory of Agrobiotechnology and Key Laboratory of Pest Monitoring and Green Management-MOA, Department of Plant Pathology, China Agricultural University, Beijing, 100193, China

    Tao Zhou

  4. State Key Laboratory of Environmental Chemistry and Eco-Toxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China

    Lixia Zhao

  5. School of Environment, Hangzhou Institute for Advanced Study, UCAS, Hangzhou, 310000, China

    Lixia Zhao

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  1. Zhenxing Zhao
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  2. Jun Xiang
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  3. Qian Tian
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Contributions

ZZ, ZZ, TZ, LZ, and YZ conceived and designed the experiments. ZX and JX performed the experiments. ZX, QT, WZ, LZ, and YZ analyzed the data. ZZ and YZ wrote the paper.

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Correspondence to Lixia Zhao or Yongjiang Zhang.

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Zhao, Z., Xiang, J., Tian, Q. et al. Development of one-step multiplex RT-PCR assay for rapid simultaneous detection of five RNA viruses and Acidovorax citrulli in major cucurbitaceous crops in China. Arch Microbiol 204, 696 (2022). https://doi.org/10.1007/s00203-022-03304-y

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