Abstract: 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.

Key words: Watermelon anthracnose, Colletetrichum orbiculare, Molecular detection, Duplex PCR