A duplex-PCR bioassay to detect a Trichoderma virens biocontrol isolate in non-sterile soil

This paper describes the identification and utilisation of a sequence-characterised amplified region (SCAR) marker specific for the Trichoderma virens biocontrol isolate GV4. The marker was developed from a RAPD-PCR amplification product unique to isolate GV4. When used as a hybridisation probe in Southern blot analysis, it hybridised to the DNA of the species T. virens alone and not to that of other Trichoderma species or closely related genera Gliocladium and Verticillium. The marker also produced a GV4-specific RFLP, distinguishing it from other T. virens isolates when probed to blots with HindII, BamHI or PstI genomic DNA digests. Primers designed from the sequence of the RAPD marker produced a diagnostic amplification product of 346 bp for GV4 alone, distinguishing it from all other test isolates. With the exception of one, test isolates did not produce an amplification product with the SCAR primers. The exception was a single Verticillium psalliotae isolate (ICMP5509) that produced a product of approx. 400 bp that was easily distinguished from the 346 bp product of GV4. The reliability of the SCAR-based diagnostic test was further improved with the introduction of a positive PCR reaction control to each test, achieved by converting the test to a duplex PCR system. Two universal primers flanking the two ITS and the 5.8S region of the ribosomal gene complex were introduced to each reaction to provide a test for PCR reaction inhibitors to eliminate false negatives in the diagnosis. Amplification of this multi-copy genomic region did not reduce diagnostic sensitivity of the single copy SCAR marker. To further increase the sensitivity of detecting GV4 propagules while maintaining a fast sample assessment assay, soil was amended with cornmeal, as a nutrient source, and a mix of antibiotics to favour Trichoderma growth. The soil mix was subsequently incubated for 5 d before total DNA was extracted. Under these conditions, the duplex soil PCR assay detected GV4 down to a concentration of 10 spores g−1 soil in non-sterile agricultural field soil. This study is the first to report the use of a duplex-PCR diagnostic bioassay for a species within the Hypocrea/Trichoderma genus.