3.5.10

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 [1]. 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
One g samples of potting mix fortified with T. hamatum 382 taken randomly from commercially prepared mix batches were ground in liquid nitrogen and suspended in sodium phosphate plus sodium dodecyl sulphate (SDS) buffer solution [2] in 1.5 ml microfuge tubes. Tubes were incubated for 30 min at 70 C while mixing occasionally with a vortex and then centrifuged at 12,000 g for 10 min. The supernatant was discarded and the pellet was resuspended in the same buffer solution in tubes incubated at 70 C for another 10 min. DNA was extracted with high salt concentration and chloroform-isoamyl alcohol by mixing and centrifugation. It was precipitated from the aqueous phase with ethanol and the washed pellet was resuspended in TE buffer. DNA was then diluted to 1:200-1:500 and amplified with SCAR primers (SCH-19588, SCE-16347, SCH-20624) in a standard polymerase chain reaction (PCR) assay.

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 [1]. 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.

References
1. Abbasi PA, Meulia T, Hoitink HAJ, Miller SA, 1997. Phytopathology 87, 2.
2. Selenska S, Klingmüller W, 1991. Lett. Appl. Microbiol. 13, 21-24.