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A multiplex direct PCR method for the rapid and accurate discrimination of three species of spider mites (Acari: Tetranychidae) in fruit orchards in Beijing

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Abstract

Spider mites (Acari: Tetranychidae) are polyphagous pests of economic importance in agriculture, among which the two-spotted spider mite Tetranychus urticae Koch has spread widely worldwide as an invasive species, posing a serious threat to fruit tree production in China, including Beijing. The hawthorn spider mite, Amphitetranychus viennensis Zacher, is also a worldwide pest of fruit trees and woody ornamental plants. The cassava mite, Tetranychus truncatus Ehara, is mainly found in Asian countries, including China, Korea and Japan, and mainly affects fruit trees and agricultural crops. These three species of spider mites are widespread and serious fruit tree pests in Beijing. Rapid and accurate identification of spider mites is essential for effective pest and plant quarantine in Beijing orchard fields. The identification of spider mite species is difficult due to their limited morphological characteristics. Although the identification of insect and mite species based on PCR and real-time polymerase chain reaction TaqMan is becoming increasingly common, DNA extraction is difficult, expensive and time-consuming due to the minute size of spider mites. Therefore, the objective of this study was to establish a direct multiplex PCR method for the simultaneous identification of three common species of spider mites in orchards, A. viennensis, T. truncatus and T. urticae, to provide technical support for the differentiation of spider mite species and phytosanitary measures in orchards in Beijing. Based on the mitochondrial cytochrome c oxidase subunit I (COI) of the two-spotted spider mite and the cassava mite and the 18S gene sequence of the hawthorn spider mite as the amplification target, three pairs of specific primers were designed, and the primer concentrations were optimized to establish a direct multiplex PCR system for the rapid and accurate discrimination of the three spider mites without the need for DNA extraction and purification. The method showed a high sensitivity of 0.047 ng for T. truncatus and T. urticae DNA and 0.0002 ng for A. viennensis. This method eliminates the DNA extraction and sequencing procedures of spider mite samples, offers a possibility for rapid monitoring of multiple spider mites in an integrated microarray laboratory system, reducing the time and cost of leaf mite identification and quarantine monitoring in the field.

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All data is made available in tables in the main text/manuscript and in the Supplementary Materials file.

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Acknowledgements

We sincerely appreciate Yang Aizhen at Key Laboratory of Urban Agriculture in North China, Ministry of Agriculture And Rural Affairs for her help with partial sample testing. And also appreciate Su Hongtian, Chief Animal Breeder of the National Animal Husbandry Station, Thanks for his dedication and hard work on this work. This research was supported by grants from the Beijing Municipal Natural Science Foundation, and the Beijing Municipal Education Commission Science and Technology Plan Key Project (KZ201810020026), the Beijing Municipal Natural Science Foundation (6182002), the National Natural Science Foundation of China (31272099).

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Authors and Affiliations

  1. Key Laboratory of Urban Agriculture, Ministry of Agriculture, College of Bioscience and Resource Environment, Beijing University of Agriculture, Beijing, 102206, North China, China

    Can Lu, Shao-dong Hao, Li-bin Huang, Li-zhen Dai, Jian-wen Wang, Long Wang, Zhi-yong Zhang, Zheng-guang Ren & Jin-zhong Wang

  2. Agriculture Promotion Center of Qinghe County, Xinjiang, 836200, China

    Pa-Zi Ha

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  1. Can Lu
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  2. Shao-dong Hao
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  10. Jin-zhong Wang
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Contributions

Conception and experimental design: WJZ, HSD; Acquisition of data: HPZ, RZG; Statistical analysis: LC, WJW, HLB, DLZ; Results interpretation: LC, ZZY, WL; Manuscript writing: LC, WJZ. All authors read and approved the final version of the manuscript.

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Correspondence to Jin-zhong Wang.

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No ethical approval or specific permit was needed for rearing and experimental use of T. truncatus, T urticae, A. viennensis which are neither protected nor endangered.

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Lu, C., Hao, Sd., Ha, PZ. et al. A multiplex direct PCR method for the rapid and accurate discrimination of three species of spider mites (Acari: Tetranychidae) in fruit orchards in Beijing. Exp Appl Acarol 92, 403–421 (2024). https://doi.org/10.1007/s10493-023-00900-5

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