多重PCR法区分枣园两种菱纹叶蝉及检测其体内枣疯病植原体

›› 2015, Vol. 58 ›› Issue (3): 264-270.

多重PCR法区分枣园两种菱纹叶蝉及检测其体内枣疯病植原体

郝少东^1，#, 陈昱圻^1，#, 王进忠^1,*, 王合², 陶万强², 张志勇¹, 石小玉¹, 周赛¹

（1. 北京农学院植物科学技术学院, 农业应用新技术北京市重点实验室, 北京 102206; 2. 北京市林业保护站, 北京100029）

出版日期:2015-03-20 发布日期:2015-03-20
作者简介:郝少东, 男, 1987年12月生, 内蒙古乌兰察布市人, 科研助理, 研究方向为媒介昆虫与植物病原互作, E-mail: haosd@bua.edu.cn; 陈昱圻, 女, 1986年11月生, 黑龙江大兴安岭人, 硕士研究生, 研究方向为媒介昆虫与植物病原互作, E-mail: chenqiberg@163.com

Multiplex-PCR for identification of two Hishimonus species (Hemiptera: Cicadellidae) in jujube orchards and detection of jujube witches’ broom (JWB) phytoplasma in their bodies

HAO Shao-Dong^1,#, CHEN Yu-Qi^1,#, WANG Jin-Zhong ^1,* , WANG He², TAO Wan-Qiang², ZHANG Zhi-Yong¹, SHI Xiao-Yu¹, ZHOU Sai¹

(1. Beijing Key Laboratory of New Technology in Agricultural Application, College of Plant Science and Technology, Beijing University of Agriculture, Beijing 102206, China; 2. Beijing Forest Protection Station, Beijing 100029, China)

Online:2015-03-20 Published:2015-03-20

摘要/Abstract

摘要： 【目的】目前发现，北京枣园中的凹缘菱纹叶蝉Hishimonus sellatus (Uhler)和片突菱纹叶蝉 Hishimonus lamellatus Cai混同发生。已知凹缘菱纹叶蝉可以传播枣疯病，而片突菱纹叶蝉是否携带枣疯病植原体尚待证明。正确鉴别区分枣园中菱纹叶蝉的种类并测定其体内感染枣疯病植原体情况有助于阐明田间枣疯病的流行规律，从而提出有效的预防枣疯病及其媒介昆虫措施显得十分重要。传统形态学鉴定两种菱纹叶蝉种类的方法局限于雄性成虫外生殖器，本研究目的在于建立一种快速的分子生物学方法，在区分枣园中两种枣菱纹叶蝉的同时，可检测虫体内的枣疯病植原体。【方法】以凹缘菱纹叶蝉和片突菱纹叶蝉的COI基因以及枣疯病植原体的16S rDNA为扩增目标，分别设计引物，建立一种包含3对引物的多重PCR体系。测试该多重PCR体系对叶蝉总DNA的灵敏度、准确性，以及当两种叶蝉DNA同时存在时的辨别能力和对枣疯病植原体16S rDNA的灵敏度。【结果】该多重PCR可以准确区分凹缘菱纹叶蝉和片突菱纹叶蝉，并对虫体内枣疯病植原体实现检测，其对昆虫总DNA的灵敏度达到0.012 ng，对枣疯病植原体16S rDNA模板的灵敏度达到900拷贝。【结论】该方法极大方便了对枣菱纹叶蝉的田间种群发生动态及虫体中枣疯病植原体感染的监测。

关键词: 凹缘菱纹叶蝉, 片突菱纹叶蝉, 枣疯病, 植原体, 多重PCR, 分子鉴定

Abstract: 【Aim】 Hishimonus sellatus (Uhler) transmits jujube witches’ broom (JWB). Currently, the Hishimonus leafhoppers occurring in jujube orchards are a mixed population of H. sellatus and H. lamellatus Cai. The latter is now suspected to be a JWB vector. As such, correct identification of Hishimonus species present in jujube orchards is essential for epidemiological surveys. However, the traditional identification of Hishimonus species by morphology is limited to the external genitalia of male adults. This study aims to develop and validate a rapid and inexpensive molecular method to discriminate betwee H. sellatus and H. lamellatus occurring in jujube orchards and to detect JWB phytoplasma in their bodies meantime. 【Methods】 A multiplex PCR method was designed for identification of two species and detection of JWB phytoplasma in their bodies. Three sequences, i.e. 16S rDNA of JWB phytoplasma, and COI genes of both H. sellatus and H. lamellatus, were used as the amplification targets. After preliminarily testing the PCR result, we further tested the accuracy and sensitivity of this multiplex PCR method to the total DNA solutions of H. sellatus and H. lamellatus. We also tested the sensitivity of this method to 16S rDNA of JWB phytoplasma. 【Results】 The results showed that the multiplex PCR method established in this study could identify species of H. sellatus and H. lamellatus accurately, and could detect JWB phytoplasma in H. sellatus or H. lamellatus sensitively. The detection limits of the total DNA of both leafhoppers were approximately 0.12 ng, and the sensitivity of the method to the JWB phytoplasma 16S rDNA template approximately reached 900 copies. The validation results with 48 field collections of male individuals pre-checked by morphological observation showed that there was no mismatch between multiplex PCR and morphological observation in all samples, indicating the 100% accuracy of the multiplex PCR for identification of H. sellatu and H. lamellatus. 【Conclusion】 The new multiplex PCR method developed in this study is greatly helpful for monitoring the dynamics of Hishimonus species occurring in jujube orchards and the infection of JWB phytoplasma in their bodies.

Key words: Hishmonus sellatus, Hishimonus lamellatus, jujube witches&rsquo, broom, phytoplasma, multiplex-PCR, molecular identification

郝少东, 陈昱圻, 王进忠, 王合, 陶万强, 张志勇, 石小玉, 周赛. 多重PCR法区分枣园两种菱纹叶蝉及检测其体内枣疯病植原体[J]. , 2015, 58(3): 264-270.

HAO Shao-Dong, CHEN Yu-Qi, WANG Jin-Zhong , WANG He, TAO Wan-Qiang, ZHANG Zhi-Yong, SHI Xiao-Yu, ZHOU Sai. Multiplex-PCR for identification of two Hishimonus species (Hemiptera: Cicadellidae) in jujube orchards and detection of jujube witches’ broom (JWB) phytoplasma in their bodies[J]. , 2015, 58(3): 264-270.