Application of green fluorescent protein (GFP) for studying interactions between Ophiostoma piceae and Trichoderma harzianum in freshly sawn Douglas-fir sapwood

While microbial colonization of wood is presumed to be characterized by a myriad of interactions between numerous organisms, studying these processes is often difficult owing to the opaque nature of the wood and the inability to readily distinguish among the many species colonizing the material. One method for enhancing the ability to distinguish organisms is to induce specific proteins in one or more organisms that can be detected using fluorescence or other light microscopic techniques. The insertion of genes for the production of green fluorescent proteins produced by the jellyfish, Aequora victoria , has been widely used to visualize a variety of organisms. In this study, an important wood sapstain fungus, Ophiostoma piceae; and its biocontrol agent, Trichoderma harzianum were transformed using a green fluorescent protein (SGFP) gene under the control of the ToxA promoter of Pyrenophora tritici-repentis. Spore germination and growth developments of these fungi on freshly sawn Douglas-fir sapwood were examined using fluorescence microscopy. The expression of gfp was particularly useful for studying the spatial distribution of young hyphae in wood.; The gfp transformants were used to study interactions between T. harzianum and O. piceae in freshly sawn Douglas-fir sapwood. O. piceae growth decreased with increasing spore ratios of T. harzianum to O. piceae . Prior establishment of T. harzianum was effective against O. piceae growth on Douglas-fir sapwood, but killing established T. harzianum by gamma-irradiation eliminated this effect. Killing T. harzianum by autoclaving after prior establishment afforded partial protection against O. piceae growth. The results illustrate the potential role of active growth in biocontrol against stain fungi.