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

Abstract

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.

FIG. 1

UP-PCR banding profiles for C. rosea strains generated with the AS15inv-AA2M2 UP primer combination. Lanes 1 to 14, strains GR3, IK726, GR6, GR10, GR12, GR11, GR9, GR8, GR4, GR5, GR1, GR13, GR7, and GR2, respectively. Lanes M, molecular size markers (λ phage DNA digested with PstI). Arrows shows the marker of interest for strain GR5.

FIG. 2

Southern blot hybridization analysis of the GR5 AS15inv-AA2M2 marker. Lanes 1 to 14, strains IK726, GR12, GR10, GR13, GR5, GR3, GR2, GR4, GR7, GR6, GR11, GR9, GR8, and GR1, respectively. Lanes M, molecular size markers. α-³²P-labeled AS15inv-AA2M2 PCR product from strain GR5 was used as the probe.

FIG. 3

Testing of C. rosea strains for the AS15inv-AA2M2 marker using the SCAR primer set. Lanes 1 to 14, strains IK726, GR12, GR10, GR13, GR5, GR3, GR2, GR4, GR7, GR6, GR11, GR9, GR8, and GR1, respectively. Lanes M, molecular size markers (λ phage DNA digested with PstI).

FIG. 4

Differentiation of GR5 from GR47 and GJS 89-34 by UP-PCR. Lanes 1 to 3, profiles generated with UP primer AS15inv. Lanes 4 to 6, profiles generated with UP primer combination AS15inv-AA2M2. Lanes 7 to 9, profiles generated with UP primer AA2M2. Lanes 1, 4, and 7, strain GR5. Lanes 2, 5, and 8, strain GJS 89-34. Lanes 3, 6, and 9, strain GR47. Lanes M, molecular size markers (λ phage DNA digested with PstI).

FIG. 5

GR5 detection in the B23DK soil using the SCAR primer set. Lane 1, GR5 (positive control); lanes 2 to 5, soil sample with large amount of GR5 (0.1, 0.01, 0.001, and 0.0001 μl of eluted DNA, respectively); lanes 6 to 8, soil sample with a smaller amount of GR5 (0.1, 0.01, and 0.001 μl of eluted DNA, respectively); lanes 9 and 10, noninfested soil sample (negative control) (0.1 and 0.01 μl of eluted DNA, respectively). Lanes M, molecular size markers.