A quantitative real‐time <scp>PCR</scp> assay for detection of <i> <scp>C</scp> olletotrichum lindemuthianum </i> in navy bean seeds

Abstract

Bean anthracnose is a seedborne disease of common bean ( P haseolus vulgaris ) caused by the fungal pathogen C olletotrichum lindemuthianum . Using seed that did not test positive for the pathogen has been proven to be an effective strategy for bean anthracnose control. To quantify the extent of anthracnose seed infection, a real‐time PCR ‐based diagnostic assay was developed for detecting C . lindemuthianum in seeds of the commercial bean class navy bean. The ribosomal DNA ( rDNA ) region consisting of part of the18 S rDNA , 5 . 8S rDNA , internal transcribed spacers ( ITS ) 1, 2 and part of the 28 S rDNA of seven races of C . lindemuthianum , 21 isolates of C olletotrichum species and nine other bean pathogens were sequenced with the universal primer set ITS 5/ ITS 4. Based on the aligned sequence matrix, one primer set and a probe were designed for a SYBR Green dye assay and a T aq M an MGB (minor groove binder) assay. The primer set was demonstrated to be specific for C . lindemuthianum and showed a high sensitivity for the target pathogen. The detection limit of both assays was 5 fg of C . lindemuthianum genomic DNA . To explore the correlation between the lesion area and the DNA amount of C . lindemuthianum in bean seed, seeds of the navy bean cultivar N avigator with lesions of different sizes, as well as symptomless seeds, were used in both real‐time PCR assays.