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Applied and Environmental Microbiology, November 2000, p. 4758-4763, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Sergey A. Bulat,1 Mette Lübeck,2,* Irina A. Alekhina,1 Dan Funck Jensen,2 Inge M. B. Knudsen,2 and Peter Stephensen Lübeck2,dagger

Laboratory of Eukaryote Genetics, Petersburg Nuclear Physics Institute, Gatchina 188350, Russia,1 and Plant Pathology Section, Department of Plant Biology, The Royal Veterinary and Agricultural University, DK-1871 Frederiksberg C, Denmark2

Received 17 March 2000/Accepted 11 August 2000

We developed a PCR detection method that selectively recognizes a single biological control agent and demonstrated that universally primed PCR (UP-PCR) can identify strain-specific markers. Antagonistic strains of Clonostachys rosea (syn. Gliocladium roseum) were screened by UP-PCR, and a strain-specific marker was identified for strain GR5. No significant sequence homology was found between this marker and any other sequences in the databases. Southern blot analysis of the PCR product revealed that the marker represented 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 Danish soils, and 31 soil samples, originating from different localities, were tested, and this specificity was confirmed. Two strains responded to the SCAR primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil assays in which total DNA was extracted from GR5-infested and noninoculated field soils showed that the SCAR primers could detect GR5 in a pool of mixed DNA and that no other soil microorganisms present contained sequences amplified by the primers. The assay developed will be useful for monitoring biological control agents released into natural field soil.

* Corresponding author. Mailing address: Plant Pathology Section, Department of Plant Biology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark. Phone: (45) 3528 3304. Fax: (45) 3528 3310. E-mail: met{at}kvl.dk.

dagger Present address: The Danish Veterinary Laboratory, DK-1790 Copenhagen, Denmark.

Applied and Environmental Microbiology, November 2000, p. 4758-4763, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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Copyright © 2000 by the American Society for Microbiology. All rights reserved.