Detection of the mycoparasite<i>Stachybotrys elegans</i>, using primers with sequence- characterized amplification regions in conventional and real-time PCR

Abstract

A specific and sensitive polymerase chain reaction (PCR) assay was developed for the detection of Stachybotrys elegans, a potential biological control agent and mycoparasite of Rhizoctonia solani. A pair of primers (SE-13F and SE-13R) with sequence-characterized amplification regions was designed and assayed in conventional and real-time PCR to detect S. elegans, in pure cultures and in field soil samples. Following conventional PCR amplification, a product of 880 base pairs was amplified from DNA of all isolates belonging to S. elegans. No product was amplified from DNA of isolates belonging to other species of Stachybotrys, common soil fungi, or bacteria, or from DNA of plant tissue, confirming the specificity of the primers for S. elegans. Stachybotrys elegans was also detected and quantified in field soils, 2 days after its inoculation, using real-time PCR conjugated with the fluorescent SYBR® Green I dye. The assay is reliable and has detected as little as 150 ng of DNA per gram of natural soil. The results of the assay were compared with those of the number of colony-forming units of S. elegans per gram of soil recovered from the same soils. The potential of these specific primers in quantitative PCR assays will facilitate the study of the distribution of the fungus in the soil in field studies of biological control of R. solani.