Development of PCR-Based Assays for Detecting and Differentiating Three Species of Botrytis Infecting Broad Bean.

ABSTRACT

Botrytis cinerea, B. fabae, and B. fabiopsis are known to cause chocolate spot on broad bean. This study was conducted to develop polymerase chain reaction (PCR)-based assays to detect and differentiate these three species. Two sets of primers, Bc-f/Bc-r for B. cinerea and Bfab-f/Bfab-r for B. fabiopsis, were designed based on two sequence-characterized amplified region markers derived from two random amplified polymorphic DNA assays. The other primer set, Bfa-f/Bfa-r for B. fabae, was designed based on the necrosis and ethylene-inducing protein 1 gene sequence. The three primer sets were highly specific for the corresponding species of Botrytis in both single and multiplex PCR assays. The PCR detection limit was 40, 40, and 400 pg of DNA per 25-µl reaction mixture for B. fabae, B. fabiopsis, and B. cinerea, respectively. Presence of the broad bean DNA in the PCR reactions at 11000 (Botrytis DNA/broad bean DNA [wt/wt]) had negligible effects on detection of the targeted Botrytis spp. The multiplex PCR assay was able to detect three Botrytis spp. in artificially infected and naturally infected broad bean leaves. These results suggest that the multiplex PCR assay developed in this study could be used to monitor the epidemics of chocolate spot of broad bean in the field.