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

IF 1.8 2区 农林科学 Q2 ENTOMOLOGY
Experimental and Applied Acarology Pub Date : 2024-04-01 Epub Date: 2024-03-15 DOI:10.1007/s10493-023-00900-5
Can Lu, Shao-Dong Hao, Pa-Zi Ha, Li-Bin Huang, Li-Zhen Dai, Jian-Wen Wang, Long Wang, Zhi-Yong Zhang, Zheng-Guang Ren, Jin-Zhong Wang
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引用次数: 0

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.

Abstract Image

快速准确鉴别北京果园中三种蜘蛛螨(Acari: Tetranychidae)的多重直接 PCR 方法。
蜘蛛螨(Acari: Tetranychidae)是一种多食性害虫,在农业中具有重要的经济价值,其中双斑蜘蛛螨(Tetranychus urticae Koch)作为入侵物种已广泛传播到世界各地,对包括北京在内的中国果树生产构成严重威胁。山楂蜘蛛螨(Amphitetranychus viennensis Zacher)也是果树和木本观赏植物的世界性害虫。木薯螨(Tetranychus truncatus Ehara)主要分布在中国、韩国和日本等亚洲国家,主要危害果树和农作物。这三种蜘蛛螨是北京地区普遍存在的严重果树害虫。快速准确地鉴定蜘蛛螨对北京果园田间有效的害虫和植物检疫至关重要。由于蜘蛛螨的形态特征有限,因此很难识别蜘蛛螨的种类。虽然基于 PCR 和实时聚合酶链式反应 TaqMan 的昆虫和螨类鉴定越来越普遍,但由于蜘蛛螨体型微小,DNA 提取困难、昂贵且耗时。因此,本研究旨在建立一种同时鉴定果园中三种常见蜘蛛螨A. viennensis、T. truncatus和T. urticae的直接多重PCR方法,为北京地区果园蜘蛛螨种类的区分和植物检疫措施提供技术支持。以二斑蜘蛛螨和木薯螨的线粒体细胞色素c氧化酶亚单位I(COI)和山楂蜘蛛螨的18S基因序列为扩增靶标,设计了三对特异性引物,优化了引物浓度,建立了直接多重PCR系统,无需DNA提取和纯化,即可快速、准确地区分三种蜘蛛螨。该方法对 T. truncatus 和 T. urticae DNA 的灵敏度高达 0.047 ng,对 A. viennensis 的灵敏度为 0.0002 ng。该方法省去了对蜘蛛螨样本进行 DNA 提取和测序的步骤,为在一个综合微阵列实验室系统中快速监测多种蜘蛛螨提供了可能,减少了叶螨鉴定和田间检疫监测的时间和成本。
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来源期刊
CiteScore
3.90
自引率
9.10%
发文量
81
审稿时长
4-8 weeks
期刊介绍: Experimental and Applied Acarology publishes peer-reviewed original papers describing advances in basic and applied research on mites and ticks. Coverage encompasses all Acari, including those of environmental, agricultural, medical and veterinary importance, and all the ways in which they interact with other organisms (plants, arthropods and other animals). The subject matter draws upon a wide variety of disciplines, including evolutionary biology, ecology, epidemiology, physiology, biochemistry, toxicology, immunology, genetics, molecular biology and pest management sciences.
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