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

Authors: WELDON, WILLIAM - Valent Biosciences-Usa; McGhee, Gayle; Delong, Jeffery - Jeff; Stockwell, Virginia

Submitted to: Plant Health Progress
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/30/2022
Publication Date: 4/15/2023
Citation: Weldon, W., McGhee, G., DeLong, J.A., Stockwell, V.O. 2023. Multiplex PCR assay for rapid identification of Monilinia rubi, the causal agent of dry-berry disease of caneberries. Plant Health Progress. https://doi.org/10.1094/PHP-09-22-0095-BR.
DOI: https://doi.org/10.1094/PHP-09-22-0095-BR

Interpretive Summary: Dry-berry is a fungal disease that kills developing berries of raspberries and blackberries. The disease is present in northern Washington state, which is the major production area for individually frozen red raspberry berries. It is not known if the disease is present in other berry growing regions of the USA. Dry-berry disease can be seen as the sudden death of green berries just after flowering. By harvest, it is difficult to determine if berries died of dry-berry, infection by a different fungus, or environmental stresses. Diagnosis of plant diseases often relies on isolating the pathogen from infected tissues. Unfortunately, the dry-berry fungus (Monilinia rubi) grows very slowly in culture media, making diagnosis a slow and labor-intensive process. We developed a PCR assay that simultaneously detects two DNA markers specific to M. rubi in culture or in diseased berries using standard equipment available in laboratories and plant disease clinics. This method will provide a rapid and accurate method to identify the pathogen M. rubi and diagnose dry-berry disease.

Technical 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 the 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.