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A quantitative real‐time PCR assay for detection of C olletotrichum lindemuthianum in navy bean seeds
Author(s) -
Chen Y. Y.,
Conner R. L.,
Gillard C. L.,
McLaren D. L.,
Boland G. J.,
Balasubramanian P. M.,
Stasolla C.,
Zhou Q. X.,
Hwang S. F.,
Chang K. F.,
Babcock C.
Publication year - 2013
Publication title -
plant pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.928
H-Index - 85
eISSN - 1365-3059
pISSN - 0032-0862
DOI - 10.1111/j.1365-3059.2012.02692.x
Subject(s) - colletotrichum lindemuthianum , biology , primer (cosmetics) , genomic dna , pathogen , botany , cultivar , gene , microbiology and biotechnology , genetics , chemistry , organic chemistry
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.