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Detection of a brown-rot fungus using serological assays
1986 - IRG/WP 1305
Polyclonal antisera produced to Poria placenta (Fr.) Cooke were used in two tests to qualitatively assay for the fungus. Fungal hyphae were fixed to slides and fluorescent antibody (FA) techniques used to visualize the hyphae under the microscope. Fluorescence of non-Poria fungi, when present, could be reduced but not eliminated by cross-absorbing the sera with these fungi. The antisera was also used in an enzyme-linked immunosorbent assay (ELISA) to distinguish between Poria placenta and a non-Poria control fungus Rhizoctonia solani.
B Goodell, J Jellison

Use of fluorescent-coupled lectins as probes for studying fungal degradation of wood
1986 - IRG/WP 1288
The ability of the fluorescent-coupled lectins wheat germ agglutin (WGA) and Concanavalin A (Con A) to react with selected Basidiomycetes, Ascomycetes, and Fungi Imperfecti was evaluated using pure cultures of 35 fungi grown on malt extract agar. WGA, which binds specifically to the n-acetylglucosamine residues found in fungal chitin, reacted with nearly all hyaline fungal structures but did not react with dematiaceous (dark) structures. Several reasons are suggested for this variation. Con A, which is specific for a-D-mannosyl and a-D-glucosyl residues, reacted with about one half of the fungi that reacted with WGA. This variation in reactivity may be useful for studying simultaneous degradation by morphologically similar fungi having different lectin specificities. The results indicate that WGA is a useful probe for studying fungal degradation by non-dematiaceous fungi particularly at the early stages of decay.
J J Morrell, R L Krahmer, L C Lin

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

Visualising Bacteria in Wood Using Confocal Laser Scanning Microscopy (CLSM)
1998 - IRG/WP 98-10272
A fluorescent phospholipid probe was used in conjunction with confocal laser scanning microscopy (CLSM), to visualise bacteria which inhabit in radiata pine wood and degrade pit membranes. CLSM has the ability to collect fluorescent images through different emission filters at the same time, so it is possible to distinguish gram-positive and gram-negative bacteria in infected wood by counterstaining wood sections with specific fluorescent stains. Images obtained using CLSM were compared with those acquired using light microscopy (LM) and scanning electron microscopy (SEM). Strong fluorescence of the phospholipid probe made it possible to visualise bacteria in wood even when present in numbers too small to detect by LM or SEM.
Ying Xiao, A P Singh, R N Wakeling

Colonisation and detection of New Zealand sapstain fungi
2000 - IRG/WP 00-10329
Sapstain is the bluish black discolouration in logs and timber caused in New Zealand by Sphaeropsis sapinea (Diploida pinea) and more than twenty different species of the Ophiostomataceae Family. Laboratory and field trials were conducted by inoculating radiata pine to establish staining colour and extent of colonisation with various New Zealand sapstaining fungal species. Colonisation patterns, competitive ability and the amount of stain development of the staining fungi were followed over time. Ophiostoma floccosum and O. piceae were most dominant colonisers in this field trial, showing persistence against other sapstaining species trying to colonise the logs. There is considerable confusion to customers concerning what constitutes sapstain and particularly confusion of sapstain with mould species. Development of an antibody assay for Sphaeropsis sapinea and the major Ophiostomataceae strains could be used by industry and customers to differentiate between moulds and decay fungi and the presence or absence of sapstain even before staining hyphae are apparent. This presentation discussed the practicalities of sapstain antibody detection for use in forest, mill sites and for marketing purposes.
J M Thwaites, R L Farrell

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

Production of monoclonal antibodies to Serpula lacrymans and their application in immunodetection systems
1993 - IRG/WP 93-10004
Monoclonal antibodies were produced against both whole cell mycelial extracts and exo-antigen extracts of the causative organism of dry rot, Serpula lacrymans. The antibodies were tested for cross-reactivity against fifteen strains of Serpula lacrymans and nine other fungal species. These species represented either other wood decay fungi or non-decay fungi commonly co-isolated with Serpula lacrymans. The antibodies exhibited a range of specificities with the majority cross-reacting with most of the Serpula lacrymans strains. Although a number of relatively non-specific antibodies were produced, it was possible to obtain antibodies which were specific for Serpula lacrymans. Monoclonal antibodies were subsequently used in an enzyme immunoassay (EIA) detection system to screen antigen samples. Non-specific interaction between Serpula lacrymans and enzymelabelled reagents resulted in higher than expected levels of background staining. The incorporation of detergents in buffers reduced but failed to eliminate this prohlem. Using the EIA system many of the antibodies reacted positively with extracts from pine (Pinus sylvestris. L) wood blocks artificially infected with Serpula lacrymans, with greatest reactivity being observed with extracts from blocks showing weight losses of 10-20%. Field samples of different morphological forms of Serpula lacrymans and decayed wood were tested for reactivity with Serpula lacrymans mouse polyclonal antiserum and several monoclonal antibodies. The polyclonal antiserum reacted with all samples tested except a spore extract. The monoclonal antibodies tested exhibited a range of reactions with the different morphological forms of Serpula lacrymans and none reacted with either the spore extract or decayed wood extracts. Future developments of the work and potential applications are discusssed.
H Glancy, J W Palfreyman

Production of monoclonal antibodies to fungal metabolites
1986 - IRG/WP 1306
The role of fungal extracellular enzymes in wood biodegradation is incompletely understood. Our lab is beginning a project utilizing monoclonal antibodies to characterize extracellular metabolites of the brown rot fungus Poria placenta Fr. (Cooke). Monoclonal antibody technology takes advantage of the ability of antibody secreting spleen cells from immunized mice to fuse in the presence of polyethylene glycol (PEG) with myeloma cells, which do not produce antibodies but do have the ability to grow in culture. The resultant hybrid cell or hybridoma has the capacity to produce antibodies of predetermined specificity and to grow "immortally" in culture. These hybridomas can be grown on a selective media, cloned, and the highly specific antibodies they produce purified. Monoclonals can be produced to fungal enzymes or other metabolites of interest. Monoclonal antibodies are capable of being more specific for a particular antigen than polyclonal antibodies because each B-lymphocyte (removed from the spleen) produces only one specific antibody to an antigen fraction. In our research, injection of extracellular fungal filtrates into an animal presents the immune system with a variety of antigen sites to produce antibodies to not only the target antigen (a glucosidase, for example) but also to extraneous materials injected with the extracellular filtrate. Two approaches exist to implement production of antisera to the desired antigen. One is to purify the antigen prior to injection. This solution has obvious advantages but it also has disadvantages.
J Jellison, B Goodell