西瓜炭疽病菌Colletetrichum orbiculare的分子检测

【研究目的】探索西瓜炭疽病菌(Colletetrichum orbiculare )的分子检测技术，为西瓜、甜瓜等生长期和采后炭疽病的快速、准确鉴定和检测提供技术和方法。【采用的主要方法和研究结果】根据GeneBank中Colletetrichum属的24个种的ITS系列，比较设计出一对引物CY1/CY2，可以特异的从西瓜炭疽病菌C. orbiculare和菜豆炭疽病菌C. lindemuthianum菌株中扩增到一条442bp的条带，将这两个种的炭疽菌和其它菌分开。进一步利用RAPD随机引物扩增C. orbiculare和C. lindemuthianum，找出在C. orbiculare中的特异性条带，经过克隆、测序后，设计出一对SCAR引物RB/RC，可以特异的在C. orbiculare中扩增出一条216bp的条带，将C. orbiculare和C.lindemuthianum分开。采用两对引物组成双重PCR，能够将C. orbiculare和其它相关和近似菌株分开。【主要结论】利用上述两对引物组成双重PCR检测体系，可以快速鉴定C. orbiculare，并且能够直接在植物组织中将西瓜炭疽病菌C. orbiculare检测出来，灵敏度可以达到1pg/μl。

Molecular Detection of Colletotrichum orbiculare

A duplex PCR was developed to detect the pathogenic fungus Colletetrichum orbiculare caused watermelon anthracnose in diseased leaves and fruits. Based on the differences of 24 ITS DNA sequences of Colletetrichum spp gained from GeneBank, a couple of specific primers of CY1/CY2 (CY1: 5’-CTTTGTGAACATACCTAACC-3’; CY2: 5’-GGTTTTACGGCAGGAGTG-3’) was synthesized. The CY1/CY2 primers amplified only a single PCR band of 442bp from C. orbiculare and C. lindemuthianum and no PCR band from other species. And then, using RAPD patterns, a C. orbiculare associated RAPD band was cloned, sequenced and used to design specific primers of RB/RC (RB：5’-GCTGTCACTTTGTGGTGTG-3’; RC：5’-TGTCGTAGCCCATCTTGTC-3’) for this species. The RB/RC primers can identify C. orbiculare from C. lindemuthianum with the 216bp PCR band. A duplex PCR method, combining primers CY1/CY2 and RB/RC, was used to detect Colletetrichum orbiculare. The detection sensitivity with the two couples of primers was 1pg of genomic DNA. The PCR-based methods could be used for the accurate identification of C. orbiculare and its rapid detection on the watermelon in growth stage and postharvest.