A Specific and Sensitive Method for the Detection of Colletotrichum lindemuthianum in Dry Bean Tissue

Abstract

To facilitate early diagnosis and improve control of bean anthracnose, a rapid, specific, and sensitive polymerase chain reaction (PCR)-based method was developed to detect the causal agent, Colletotrichum lindemuthianum, in bean (Phaseolus vulgaris) seed. Based on sequence data of the rDNA region consisting of the 5.8S gene and internal transcribed spacers (ITS) 1 and 2 of four C. lindemuthianum races and 17 Colletotrichum species downloaded from GenBank, five forward primers were designed and evaluated for their specificity. Among them, one forward primer was selected for use in combination with ITS4 to specifically detect C. lindemuthianum. A 461-bp specific band was amplified from the genomic DNA template of 16 representative isolates of C. lindemuthianum, but not from 58 representative isolates of 17 other Colletotrichum species or 10 bean pathogens. Moreover, to enhance the sensitivity of detection, nested PCR was applied, which allowed the detection of as little as 10 fg of C. lindemuthianum genomic DNA and 1% infected seed powder, which was mixed with 99% healthy seed powder. The diagnostic analysis can be completed within 24 h, compared with about 2 weeks required for culturing. Furthermore, this method can be performed and interpreted by personnel with no specialized taxonomic expertise.