DIRECT AMPLIFICATION OF TRICHODERMA HAMATUM 382 DNA FROM COMPOST-AMENDED SUBSTRATES WITH RAPD-DERIVED ISOLATE-SPECIFIC PRIMERS
PA ABBASI, SA MILLER and HAJ HOITINK
Department of Plant Pathology, The Ohio State University, OARDC, Wooster, OH 44691, USA
Background and objectives
Trichoderma hamatum isolate 382 is utilized as a biocontrol agent in composted pine bark (CPB)-amended potting mixes to induce suppression to diseases caused by several plant pathogens. For purposes of registration and assurance of efficacy, it is necessary to confirm the presence of T. hamatum 382 in such mixes. Previously we identified random amplified polymorphic DNA (RAPD) markers that distinguish T. hamatum 382 from other isolates of T. hamatum and Trichoderma spp. These markers were converted into sequence characterized amplified region (SCAR) markers. Finally, the RAPD and/or SCAR markers in combination with a dilution plating technique were used successfully to verify the presence of T. hamatum 382 in compost-amended potting mixes fortified with this biocontrol agent . While this method is effective, the dilution plating/culturing step is time-consuming, and a more rapid method is needed. The objective of this study was to develop a simple procedure for direct amplification of DNA of T. hamatum 382 from the fortified mixes.
Material and methods
Results and conclusions
The three sequence characterized markers, SCH-19588, SCE-16347, SCH-20624, were identified in agarose gels as 588, 347 and 624 bp size DNA fragments, respectively. All five batches of the fortified potting mixes analyzed in the direct PCR assay were positive for T. hamatum 382. Identity of T. hamatum 382 was also confirmed by PCR analysis of individual colonies purified from dilution plates . T. hamatum 382 was not detected in non-fortified mixes. These markers, therefore, facilitated the direct amplification of DNA of T. hamatum 382 from compost-amended substrates and allowed us to verify the presence of the biocontrol agent in the mixes. The procedure was consistent and results were reproducible. Most of the inhibitors of the PCR reaction in the composted material were eliminated in the first DNA extraction step. Dilution of template DNA in the final extraction step further reduced inhibition of PCR amplification. In conclusion, a simplified PCR assay for T. hamatum 382 was developed that can be used to analyze a large number of samples and confirm the presence of T. hamatum 382 in less than 12 hours. We also intend to continue development of this assay for quantitation of T. hamatum 382 populations in compost-amended mixes.