Detection and Identification of a Biocontrol Agent, Rhodotorula mucilaginosa, by a Dot Blot Hybridization Technique

The purpose of this study was to develop a molecular technique for rapid identification of biological control agent, strain S-33 of Rhodotorula mucilaginosa, of diseases of greenhouse crops. DNA from yeast cultures and from diseased plant tissue was extracted, amplified by PCR using primers specific to fungi and fixed to nylon membranes. Using sequence information obtained from the GenBank database, specific oligonucleotides were designed and after labeling with digoxigenin-d-UTP, were used as probes in a dot-blot hybridization assay. After preliminary testing with 5 probes, two of these probes were selected for further study. With probe RD5, a positive reaction was obtained with strain S-33 of R. mucilaginosa in pure culture and with fresh gummy stem blight (Didymella bryoniae) diseased plant tissue sprayed with R. mucilaginosa. One yeast isolate each of Rhodosporidium toruloides, R. fluviale, R. babjevae, R. sphaerocarpum, R. kratochvilovae, R. azoricum, one isolate of Pichia anomala and some common greenhouse pathogens were also tested using this assay. All other yeasts and pathogenic fungi tested gave a negative result. Cucumber stem tissue infected with Didymella bryoniae also gave a negative result. Uninfected, healthy cucumber stem tissue did not yield a positive result. The other probe (RD4) reacted with two species, Rhodotorula mucilaginosa and Rhodotorula dairenensis. The present dot blot assay can be used to identify biocontrol agent stain S-33 of Rhodotorula mucilaginosa in pure culture and in plant tissue.