瓜果上主要实蝇类害虫PCR-RFLP分子鉴定技术的建立及应用

Establishment and application of PCR-RFLP molecular identification techniques for fruit fly pests of melon and fruit

【目的】建立一种针对实蝇幼虫快速鉴定的技术方法,并应用该技术以明确我国瓜果上主要实蝇类害虫的优势种。【方法】以我国瓜果蔬菜上危害最为严重的瓜实蝇Bactrocera cucurbitae(Coquillett)、南亚果实蝇Bactrocera tau Walker和橘小实蝇Bactrocera dorsalis(Hendel)为对象,基于3种实蝇种间存在线粒体细胞色素氧化酶亚基Ⅰ因子(mt COⅠ)基因的序列差异,利用3种限制性内切酶SapⅠ、BsmⅠ和SacⅠ酶切识别这些差异序列的特异性酶切位点,根据酶切后mtCOⅠ基因片段大小不同,对3种实蝇进行快速的鉴定识别,并进一步应用此技术对来自我国7省11市的7种瓜菜和2种水果上的21份实蝇幼虫样本进行鉴定。【结果】通过单独或组合酶切的方式建立了一套针对瓜实蝇、南亚果实蝇和橘小实蝇的PCR-RFLP快速分子鉴定技术,并将此方法应用于田间监测。田间鉴定结果表明,湖北、湖南、浙江等地区危害瓜菜的实蝇主要是南亚果实蝇,广东、广西、海南等地区瓜菜上的实蝇种类主要是瓜实蝇,且瓜实蝇和南亚果实蝇存在寄主重叠现象,云南危害火龙果和芒果等水果上的实蝇主要是橘小实蝇。【结论】本研究建立的瓜实蝇、南亚果实蝇和橘小实蝇的识别鉴定方法,因不受虫体、虫态的影响,且具有低成本、高效、快速、重复性好的特点,适用于田间瓜果上实蝇样本的快速诊断检测。依据田间鉴定结果,预测近年来瓜实蝇可能有逐渐向北扩散的趋势。

[Objectives]A method for rapid identification of fruit flies was developed and applied to identify the dominant species of these in China.[Methods]Differences in the mitochondrial cytochrome oxidase subunit Ⅰ factor(mtCOⅠ)gene sequences of three fruit fly species;Bactrocera cucurbitae(Coquillett),Bactrocera tau Walker and Bactrocera dorsalis(Hendel),and differences in mtCOⅠ gene fragment size after enzyme digestion(SapⅠ,BsmⅠ and SacⅠ),allowed these three species of fruit flies to be rapidly identified.This technique was further applied to the identification of 21 fruit fly larvae samples from 7 kinds of vegetable and 2 kinds of fruit crops from 7 provinces and 11 cities in China.[Results]Identification methodology based on PCR-RFLP was successfully established and used to identify sample of fruit fly larvae collected in the field.The results show that the main fruit fly pest of melons and vegetables in the Hubei,Hunan and Zhejiang regions is B.tau;whereas in Guangdong,Guangxi and Hainan the main fruit flies are B.cucurbitae,with some B.tau and B.cucurbitae.The main fruit fly pest of pitaya and mango crops in Yunnan is B.dorsalis.[Conclusion]The PCR-RFLP method developed for the identification of B.cucurbitae,B.tau and B.dorsalis is not affected by the developmental stage of fruit flies,and is relatively inexpensive,efficient,fast and repeatable.It is especially suitable,therefore,for identifying fruit fly larvae.Using this method to identify samples of larvae from different provinces and cities suggests that B.cucurbitae may be gradually spreading northwards.