|SALAMONE, A - Washington State University|
Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/2/2020
Publication Date: 4/12/2020
Citation: Salamone, A.L., Okubara, P.A. 2020. Real-time PCR quantification of a Rhizoctonia solani AG-3 variant of potato. Journal of Microbiological Methods. 172. https://doi.org/10.1016/j.mimet.2020.105914.
Interpretive Summary: Potatoes are susceptible to certain soilborne diseases. Management of these diseases are aided by molecular diagnostic assays that can quantify the causal pathogen. Interestingly, assays developed in the Netherlands, Scotland and Australia were not able to recognize our potato pathogen. We report the development of a sensitive and robust diagnostic assay specific to a pathogen found in Pacific Northwest potato production systems.
Technical Abstract: Rhizoctonia solani AG-3 causes various Rhizoctonia diseases including black scurf and stem canker on potatoes, affecting potato-production regions around the world. Accurate identification and detection of R. solani AG-3 is essential for developing and monitoring an integrated management plan. Numerous primers have been developed for molecular detection and quantification of R. solani AG-3 recovered from potato tissue and soil. This study reports that R. solani AG-3 isolate 13-12, from a Yukon Gem tuber with black scurf grown in western Washington, possesses an ITS sequence which does not contain binding sites matching previously-published primers. For that reason, ITS-based primers were developed for the specific detection and quantification of R. solani AG-3 isolate 13-12. ITS sequences for isolate 13-12 and R. solani AG-3 accessions from GenBank were analyzed to identify conserved binding sites.In silico primer-target duplex stability ('G) was used to evaluate the specificity of the developed primers. ITS sequences of AG-4, AG-5 and AG-9 accessions had deltaG values below the default stability cut-off and primer-template binding was not considered to be significant. Our findings demonstrate the necessity of using isolate-specific primers for certain pathosystems.