Diagnosis and identification of Didymella bryoniae, causal agent of gummy stem blight of greenhouse cucumbers, using a dot blot technique

Abstract

SummaryThe purpose of this study was to develop a molecular method for rapid diagnosis and identification of Didymella bryoniae, causal agent of gummy stem blight of greenhouse cucumbers. DNA from fungal cultures and from diseased plant tissue was extracted, amplified by PCR using primers specific to septate fungi, and fixed to nylon membranes. Using sequence information obtained from the GenBank database, specific oligonucleotides were designed and after labelling with digoxigenin-d-UTP, were used as probes in a dot-blot hybridization procedure. After preliminary testing, two of these probes were selected for further study. For probe D6, a positive reaction was obtained with all isolates of D. bryoniae and Phoma cucurbitacearum tested, and with fresh or frozen gummy stem blight diseased plant tissue. All other fungi tested gave a negative result. Cucumber stem tissue infected with Botrytis cinerea also gave a negative result. Uninfected, healthy cucumber stem tissue did not yield a PCR product. The other probe (D7) reacted slightly with D. applanata but otherwise showed the same results as D6. This dot blot assay can reliably be used to identify infection by Didymella bryoniae.