Rapid molecular technique for identification of a biological control agent Rhodosporidium diobovatum

ISSN： 1360-0478

Source： Biocontrol Science and Technology, Vol.15, Iss.8, 2005-12, pp. : 827-834

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Abstract

The purpose of this study was to develop a rapid molecular technique for identification of the biological control agent, Rhodosporidium diobovatum . DNA from all yeast cultures described below was extracted, amplified by PCR using primers specific to septate fungi, and fixed to nylon membranes. Using sequence information obtained from the GenBank database, Rhodosporidium diobovatum- specific oligonucleotides were designed and, after labeling with digoxigenin-d-UTP, were used as probes in a dot-blot hybridization assay. After preliminary testing, two probes were selected for further study. For probe RD3, a positive reaction was obtained at 38, 42 and 48°C with R. diobovatum in pure culture. Other yeast isolates such as Rhodosporidium toruloides , R. fluviale , R. babjevae , R. sphaerocarpum , R. kratochvilovae , R. azoricum , Pichia anomala , and some common greenhouse pathogens tested gave a negative result. The other probe (RD1) reacted with two species, R. diobovatum and R. babjevae at 42°C. The present dot-blot assay can be used reliably to identify the biocontrol agent, Rhodosporidium diobovatum , from pure culture and plant tissue .