Detection of Sclerotinia sclerotiorum in oilseed rape using Oxford Nanopore sequencing and qPCR

Abstract: Sclerotinia sclerotiorum is a notorious phytopathogenic fungus and is the causal agent of the disease Sclerotinia stem rot (SSR) of rapeseed (Brassica napus). SSR is one of the main diseases affecting the yield and oil quality of rapeseed crops worldwide. This disease is very hard to predict and control due to all the different factors that are involved in the development of the disease. Successful disease management depends on accurate identification and early detection of plant pathogens. qPCR is a fast, specific, reproducible, and reliable technique for plant pathogen diagnostics. However, one limitation of qPCR is that it is unsuitable to identify and study unknown species, other than those intended, making the detection of unknown pathogens very difficult. An alternative solution is to apply single molecule sequencing, which can provide information at species and strain level. In this study, a total sample of 15 rapeseed leaves coming from three different fields in Sweden with known incidence of SSR disease were analyzed using qPCR and other 15 leaves, coming from the same fields, were analyzed using Oxford Nanopore sequencing to attempt to identify pathogens, S. sclerotiorum being the main target. S. sclerotiorum was not identified with none of the previous mentioned techniques in any of the samples. Perhaps, S. sclerotiorum was not present on the samples at the time of the collection, due to the unfavorable weather conditions for the release of the spores. However, some issues were present during the development of the qPCR assays that also could have affected the results. Regarding Oxford Nanopore sequencing, other fungal species were identified instead.