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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

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

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