柑橘黑点病菌(Diaporthe citri)快速分子检测技术  

Rapid molecular detection of Diaporthe citri,the pathogen of citrus melanose

作　　者：曾雅婷 熊桃 李红叶[1] ZENG Yating;XIONG Tao;LI Hongye(Institute of Biotechnology,Zhejiang University,Hangzhou 310058,China)

机构地区：[1]浙江大学生物技术研究所,浙江杭州310058

出　　处：《浙江农业学报》2022年第7期1457-1465,共9页Acta Agriculturae Zhejiangensis

基　　金：浙江省重点研发计划(2019C02022);现代农业(柑橘)产业技术体系(CARS-26)。

摘　　要：柑橘黑点病是一种严重危害柑橘生产的世界性真菌病害,快速及时地检测出柑橘黑点病病菌(Diaporthe citri)对柑橘黑点病早期诊断和科学防控具有重要的实践意义。本研究基于β-微管蛋白序列,设计柑橘黑点病病菌的特异引物对,基于常规PCR和SYBR Green I实时荧光定量PCR(qRT-PCR)建立了快速分子检测体系,对柑橘黑点病病菌的菌丝和典型带病叶片进行检测验证。结果发现,在常规PCR检测中,该引物可从柑橘黑点病病菌获得244 bp的特异性扩增片段,检测灵敏度达0.78 ng·μL^(-1);在实时qRT-PCR中,该引物也只有在柑橘黑点病病菌中获得唯一的产物吸收峰,检测灵敏度达0.35 ng·μL^(-1)。利用PCR检测体系对发病黑点叶片进行检测,结果显示,常规PCR的阳性检出率为30%,qRT-PCR的阳性检出率为60%,表明qRT-PCR比常规PCR的灵敏度更高。因此,研究设计的柑橘黑点病病菌特异性引物,以及所建立的常规和实时qRT-PCR检测方法具有速度快、特异性强等特点,可以用于柑橘黑点病病菌的分子鉴定,对病害诊断具有参考价值。Citrus melanose is a worldwide fungal disease which seriously harms citrus production.Rapid detection of Diaporthe citri,the pathogen of citrus melanose,is of great practical significance for early diagnosis and scientific prevention and control of citrus melanose.In this study,a specific primer pair for D.citri was designed based onβ-tubulin gene sequence,and a rapid molecular detection system was established based on conventional PCR and SYBR Green I Real-time PCR(qRT-PCR)to detect and verify the mycelia and typical infected leaves of D.citri.The results showed that in conventional PCR,the primers could obtain a 244 bp specific amplification fragment from D.citri,and the detection sensitivity was up to 0.78 ng·μL^(-1).In qRT-PCR,the primers could only obtained a unique product absorpation peak from D.citri,and the detection sensitivity was up to 0.35 ng·μL^(-1).The typical infected leaves were detected by PCR detection system.The results showed that the positive detection rate of conventional PCR was 30%,and that of qRT-PCR was 60%,indicating that qRT-PCR was more sensitive than conventional PCR.Therefore,the specific primers designed in this study and the established conventional and qRT-PCR detection method have the advantages of fast speed,strong specificity and high sensitivity,which can be used for molecular identification of D.citri and also have reference value for disease diagnosis.

关 键 词：柑橘黑点病菌 β-微管蛋白序列 SYBR GreenⅠ实时荧光定量PCR 常规PCR检测 

分 类 号：S436.66[农业科学—农业昆虫与害虫防治]