Determining the environmental fate of a filamentous fungus,Trichoderma reesei, in laboratory-contained intact soil-core microcosms using competitive PCR and viability plating

Abstract

Trichoderma spp. are used extensively in industry and are routinely disposed of in landfill sites as spent biomass from fermentation plants. However, little is known regarding the environmental fate of this biomass. We tracked the survival of T. reesei strain QM6A#4 (a derivative of strain QM6A marked with a recombinant construct) over a 6-month period in laboratory-contained, intact soil-core microcosms incubated in a growth chamber. Survival was tested in 3 different soils and the effect of a plant rhizosphere (bush lima beans, Phaseolus limensis) was investigated. Levels and viability of the fungus were determined, respectively, by quantitative competitive polymerase chain reaction analysis of total soil DNA extracts and dilution-plating of soil on a semiselective growth medium. Whereas chemically killed QM6A#4 became undetectable within 3 d, QM6A#4 added as a live inoculum decreased ~4- to ~160-fold over the first 1–3 months and then reached a steady state. After 4 months, soil cores were subjected to a 1.5-month simulated winter period, which did not significantly affect QM6A#4 levels. Throughout the experiment, QM6A#4 remained viable. These results indicate that, following release into the environment, live T. reesei will persist in soil for at least 2 seasons.Key words: competitive PCR, genetically engineered microorganisms (GEMs), genetically modified organism (GMO), survival of microorganisms, microcosm, Trichoderma.