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Transformation of Ophiostoma picea and Trichoderma harzianum with green fluorescent protein (GFP)
2003 - IRG/WP 03-10477
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 report, we describe transformation of two fungi, Ophiostoma picea and Trichoderma harzianum using a green fluorescent protein (SGFP) gene under the control of the ToxA promoter of Pyrenophora tritici-repentis. The growth and wood colonization by two transformed fungi were compared to their non-transformed strains. The expression of gfp was particularly useful for studying the spatial distribution of young hyphae in wood.
Ying Xiao, L M Ciuffetti, J J Morrell


Effects of Prior Establishment of Trichoderma harzianum on Ophiostoma picea Growth in Freshly Sawn Douglas-fir Sapwood
2003 - IRG/WP 03-10476
Trichoderma harzianum has been shown to be an effective biocontrol agent against a number of wood inhabiting fungi under laboratory conditions, but this fungus has performed poorly in field trials. Understanding the interactions between biocontrol agents and their intended targets in wood may provide important clues for developing improved approaches to biocontrol, potentially reducing our reliance on pesticides. One particularly difficult problem with studying biocontrol in wood is the inability to accurately resolve individual organisms as they interact. The opaque nature of wood makes it difficult to observe interactions more than a few cells from the surface. Wood can be cut into thin sections, but the structural similarity of many fungal hyphae makes it difficult to determine which fungus is present. Transformation using genes for the synthesis of green fluorescent protein (GFP) has proven useful for visualizing specific microorganisms in their hosts and could be useful for biocontrol studies. In this study, interactions between gfp transformed Ophiostoma picea and non-transformed T. harzianum were studied on freshly sawn Douglas-fir sapwood blocks. Growth of O. picea was significantly retarded when applied to wood blocks where T. harzianum was well-established. Interestingly the Graphium state of O. picea was still observed on surfaces of the blocks, but the fungus was unable to penetrate deeply into the wood. The results suggest that failure of the biocontrol may not represent an inability to protect the wood beneath the surface and implies that a more detailed study of the causes of previous failures would be useful.
Ying Xiao, J J Morrell, L M Ciuffetti