Identification of a universally primed-PCR-derived sequence-characterized amplified region marker for an antagonistic strain of Clonostachys rosea and development of a strain-specific PCR detection assay

We developed a PCR detection method that selectively recognizes a single bi ological control agent and demonstrated that universally primed PCR (UP-PCR ) can identify strain-specific markers. Antagonistic strains of Clonostachy s rosea (syn, Gliocladium roseum) were screened by UP-PCR, and a strain-spe cific marker was identified for strain GR5, No significant sequence homolog y was found between this marker and any other sequences in the databases. S outhern blot analysis of the PCR product revealed that the marker represent ed a single-copy sequence specific for strain GR5. The marker was converted into a sequence-characterized amplified region (SCAR), and a specific PCR primer pair was designed. Eighty-two strains, isolated primarily from Danis h soils, and 31 soil samples, originating from different localities, were t ested, and this specificity was confirmed. Two strains responded to the SCA R primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil ass ays in which total DNA was extracted from GR5-infested and noninoculated fi eld soils showed that the SCAR primers could detect GR5 in a pool of mixed DNA and that no other soil microorganisms present contained sequences ampli fied by the primers. The assay developed will be useful for monitoring biol ogical control agents released into natural field soil.