A quantitative real‐time PCR assay for detection of Colletotrichum lindemuthianum in navy bean seeds |
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| Authors: | Y Y Chen R L Conner C L Gillard D L McLaren G J Boland P M Balasubramanian C Stasolla Q X Zhou S F Hwang K F Chang C Babcock |
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| Institution: | 1. Morden Research Station, Agriculture and Agri‐Food Canada (AAFC), , Morden, MB, R6M 1Y5 Canada;2. Department of Agriculture, Food and Nutritional Science, University of Alberta, , Edmonton, AB, T6G 2P5 Canada;3. Ridgetown College, University of Guelph, , Ridgetown, ON, N0P 2C0 Canada;4. Brandon Research Center, AAFC, , Brandon, MB, R7A 5Y3 Canada;5. Department of Environmental Biology, University of Guelph, , Guelph, ON, N1G 2W1 Canada;6. Lethbridge Research Center, AAFC, , 5403 ‐ 1st Avenue South, Lethbridge, AB, T1J 4B1 Canada;7. Department of Plant Science, University of Manitoba, , Winnipeg, MB, R3T 2N2 Canada;8. Crop Diversification Centre North, Alberta Agriculture and Rural Development (AARD), , Edmonton, AB, T5Y 6H3 Canada;9. Canadian Collection of Fungal Cultures, AAFC, , Ottawa, ON, K1A 0C6 Canada |
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| Abstract: | Bean anthracnose is a seedborne disease of common bean (Phaseolus vulgaris) caused by the fungal pathogen Colletotrichum 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 the18S rDNA, 5.8S rDNA, internal transcribed spacers (ITS) 1, 2 and part of the 28S rDNA of seven races of C. lindemuthianum, 21 isolates of Colletotrichum species and nine other bean pathogens were sequenced with the universal primer set ITS5/ITS4. Based on the aligned sequence matrix, one primer set and a probe were designed for a SYBR Green dye assay and a TaqMan 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 Navigator with lesions of different sizes, as well as symptomless seeds, were used in both real‐time PCR assays. |
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| Keywords: | anthracnose
Colletotrichum lindemuthianum
Phaseolus vulgaris
real‐time PCR seed |
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