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【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 r DNA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 r DNA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 r DNA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。
【Objective】 It has been found that Hishimonus sellatus (Uhler) and Hishimonus lamellatus Cai in Beijing jujube orchard are confused with each other. Known concave edge leafhopper can spread jujube bingo, and leafhopper leafhopper carrying jujube broom phytoplasma remains to be proved. Proper identification of Zaoyuan Zhongling leafhopper species and determination of its body jujube broom infected with pathogens help explain the epidemic of jujube broom disease in the field, which put forward effective prevention of jujube broom disease and its vector insects appear to be very important. The traditional morphological identification of two species of the leafhopper is limited to male adult genitalia, the purpose of this study is to establish a rapid method of molecular biology, Jujube Park in the distinction between the two jujube leafhoppers at the same time, Detection of parasites jujube broom phytoplasma. 【Method】 The COI gene of Lycium barbatus and Lymantria disparica and the 16S r DNA of jujube witches’ broom were used as primers to design a multiplex PCR system containing 3 pairs of primers . The sensitivity and accuracy of the multiplex PCR system to the total DNA of leafhoppers were tested, and their discrimination ability and the sensitivity to 16S r DNA of the jujube baker’s phytoplasma were analyzed. 【Result】 The multiplex PCR was able to distinguish accurately the diamondback leafhopper and the diamondback leafhopper, and to detect the jujube baker’s phytoplasma in the insect body. The sensitivity to total insect DNA was 0.012 ng, Phytoplasma 16S r DNA template sensitivity of 900 copies. 【Conclusion】 This method is very convenient for the field population dynamics of jujube leafhoppers and the monitoring of jujube broom phytoplasma in juveniles.