Specific PCR Assays for the Detection and Quantification of DNA from the Biocontrol Strain Trichoderma harzianum 2413 in Soil
Strain identification in situ is an important factor in the monitoring of microorganisms used in the field. In this study, we demonstrated the use of sequence-characterized amplified region (SCAR) markers to detect genomic DNA from Trichoderma harzianum 2413 from soil. Two primers (SCAR A1/SCAR A1c) were tested against DNA of 27 isolates of Trichoderma spp. and amplified a 990-bp fragment from T. atroviride 11 and a 1.5-kb fragment from T. harzianum 2413, using an annealing temperature of 68°C. These fragments showed no significant homology to any sequence deposited in the databases. The primer pair, BR1 and BR2, was designed to the 1.5-kb fragment amplified from T. harzianum 2413, generating a SCAR marker. To test the specificity of these primers, experiments were conducted using the DNA from 27 Trichoderma spp. strains and 22 field soil samples obtained from four different countries. PCR results showed that BR1 and BR2 amplified an 837-bp fragment unique to T. harzianum 2413. Assays in which total DNA was extracted from sterile and nonsterile soil samples, inoculated with spore or mycelium combinations of Trichoderma spp. strains, indicated that the BR1 and BR2 primers could specifically detect T. harzianum 2413 in a pool of mixed DNA. No other soil-microorganisms containing these sequences were amplified using these primers. To test whether the 837-bp SCAR marker of T. harzianum 2413 could be used in real-time PCR experiments, new primers (Q2413f and Q2413r) conjugated with a TaqMan fluorogenic probe were designed. Real-time PCR assays were applied using DNA from sterile and nonsterile soil samples inoculated with a known quantity of spores of Trichoderma spp. strains.