A multiplex PCR assay for the detection of six foliar fungal pathogens of faba bean

This research was funded through the Agricultural Development Fund of the Saskatchewan Ministry of Agriculture, the Western Grains Research Foundation and the Saskatchewan Pulse Growers.

Peer Reviewed

ral foliar fungal diseases: ascochyta blight (Ascochyta fabae), alternaria leaf spot (Alternaria sp.), chocolate spot (Botrytis cinerea and Botrytis fabae), anthracnose (Colletotrichum lentis) and stemphylium blight (Stemphylium spp.). Under conducive conditions these diseases can cause significant economic losses. Visual diagnosis based on the appearance of lesions on faba beans is problematic, especially as they progress. The purpose of this study was to develop a multiplex polymerase chain reaction assay to detect the causal organisms of these diseases. Five pairs of primers, AFF7/AFR7 for A. fabae, BCF1/BCR1 for B. cinerea, BFF2/BFR2 for B. fabae, ClF2/ClR2 for C. lentis and Stem_F_S/Stem_R_S for Stemphylium spp. were developed. The previously developed primer pair aagpf1/aagpr1 was used to detect Alternaria sp. The primers were tested for specificity to their target pathogen and primer pairs AFF6/AFR6, aagpf1/aagpr1, BCF1/BCR1, BFF2/BFR2 and ClF2/ClR2 identified their target pathogens. The primer pair Stem_F_S/Stem_R_S identified S. beticola, S. botryosum, S. eturmiunum and S. vesicarium. Identification of pathogens with primers was evaluated using DNA from mycelia and from infected faba bean leaves generated through artificial inoculations under controlled conditions and collected from the field. A multiplex PCR assay with six primer pairs allowed for detection of the target pathogens to the species level for five pathogens, and to the genus level for species causing stemphylium blight. This assay enables quick diagnosis of leaf spots on faba bean, and minimizing time and effort needed to identify the primary cause of the infection through traditional isolation procedures.