Multiplex PCR assay for rapid identification of Monilinia rubi, the causal agent of dry-berry disease of caneberries

Abstract

Monilinia rubi is the causal agent of dry-berry disease of raspberry and blackberry in northern Washington state and western Canada. The symptoms are visible on green fruits and include necrotic and dried drupelets with progressive necrosis from the receptacle into the peduncle. Diagnosis is based on symptoms and isolation and identification of the slow-growing fungal pathogen. Diagnosis is slow and difficult with late season tissues because abiotic stresses or other diseases may cause similar symptoms and the slow-growing pathogen is not easily isolated from tissues harboring fast-growing environmental fungi. A multiplex PCR assay with primers to amplify an ITS region and beta-tubulin was designed to provide a rapid method to identify the pathogen in culture and in infected berry tissues. For M. rubi and infected berries, two amplicons that differ in length by 400 bp are visualized on agarose gels. No bands were obtained from fungal outgroups or non-symptomatic berries. For further confirmation of the pathogen and the disease, a single amplicon can be sequenced directly from the multiplex reaction and compared to reference sequences in GenBank. This rapid multiplex assay streamlines diagnosis of dry-berry disease and its application may provide valuable information on the range of the pathogen, especially in other caneberry production regions.