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