PCR diagnostics for rapid detection of fungi associated with black root rot of strawberries

Abstract

Black root rot is a disease of the strawberry with increasing importance for Germany. Until the early years of the twenty-first century, very different pathogens, including fungi and nematodes, were thought to cause this disease. However, based on substantial research between 2007 and 2014 at the fruit-growing center Jork (Esteburg, Germany) fungi with Cylindrocarpon-like anamorphs, i.e., Dactylonectria torresensis and Ilyonectria spp. were identified as causal agents. Both fungi are soil-borne pathogens and able to infect young strawberry plants in nurseries. Therefore, it is crucial to test plants prior to further propagation for the presence of these organisms. Although speed of diagnosis is a well-known and important factor, until today only time-consuming microbiological tests are available. To improve the situation, we aimed at developing a rapid, PCR-based, assay for the detection of fungi associated with black root rot. Most challenging was the design of specific primers that recognize the pathogen(s) in question while being unspecific for other fungi and Oomycetes routinely found on strawberries. Here, we report on a pair of primers that fulfill these rigorous criteria and present data on their specificity and sensitivity. A concentration of as low as 1.25 pg µl−1 template DNA from target pathogens could be detected in infected tissue. Significantly, the same primers were successfully used in standard and quantitative PCR assays. Based on our results, diagnostic laboratories are now enabled to rapidly survey lots of strawberry plants and provide growers with precise information on the presence of fungi associated with black root rot even in asymptomatic plants.