Molecular detection of Colletotrichum lindemuthianum by duplex PCR

A duplex PCR technique was developed to detect the pathogenic fungus Colletotrichum lindemuthianum infection in the tissues of common bean. Based on the differences of 24 internal transcribed spacer, DNA sequences of Colletotrichum spp. retrieved from GeneBank database, one pair of specific primers of CY1/CY2 (CY1: 5'-CTT TGT GAA CAT ACC TAA CC-3'; CY2: 5'-GGT TTT ACG GCA GGA GTG-3'), was designed. The CY1/CY2 primers amplified a single PCR product of 442 bp only from C. lindemuthianum and Colletotrichum orbiculare, not from any other tested species. By using random amplification of polymorphic DNA technique, a product closely associated with C. lindemuthianum was generated. This product was cloned, sequenced and used for designing a species-specific primers of CD1/CD2 (CD1: 5'-ACC TGG ACA CAT AAG TCA AAG-3'; CD2: 5'-CAA CAA TGC CAG TAT CAG AG-3'). The CD1/CD2 primers could distinguish C. lindemuthianum from C. orbiculare by a 638 bp PCR band. A duplex PCR method, combining both primers of CY1/CY2 and CD1/CD2, was used to detect C. lindemuthianum infection. The sensitivity of the detection with this PCR method was 1 pg of pure genomic DNA from the pathogen. Therefore, the PCR-based methods could be used for accurate and rapid detection of C. lindemuthianum from common bean.