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 Th azad from soil. Two primers (SCAR A1/SCAR A1c) were tested against DNA of 49 isolates of Trichoderma spp. and amplified a 900-bp fragment from T. atroviride 11 and a 1.5-kb fragment from T. harzianum Th azad, 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 Th azad, generating a SCAR marker. To test the specificity of these primers, experiments were conducted using the DNA from 49 Trichoderma spp. strains and 22 field soil samples obtained from different agro-geographical condition of UP. PCR results showed that BR1 and BR2 amplified an 830-bp fragment unique to T. harzianum Th azad. 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 Th azad in a pool of mixed DNA. No other soil-microorganisms containing these sequences were amplified using these primers. To test whether the 830-bp SCAR marker of T. harzianum Th azad could be used in real-time PCR experiments, new primers (CSA-Th azadf and CSA-Th azad) 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.
RT-PCR, Trichoderma, Genomic DNA, SCAR marker.
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