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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

Immuno-electron microscopic localization of extracellular metabolites in spruce wood decayed by brown-rot fungus Postia placenta
1990 - IRG/WP 1441
Degradation by Postia placenta in spruce and birch wood was shown to occur not only in the wood cell wall but also in the middle lamellae region. Middle lamellae was often found to be degraded along the centerline so that cells could separate along this line. Extracellular membrane structures were found surrounding the hyphae and this matrix labelled positively with antisera produced to Postia placenta extracellular metabolites. This matrix was also visible in the secondary wall of degraded birch wood. Antisera labelling was also noted in the secondary cell walls of the wood cells, but not in the middle lamellae region.
Y S Kim, B Goodell, J Jellison

Cytoplasmic and extracellular localization of manganese II dependent peroxidase(s) in white rot fungi during degradation of woody materials
1989 - IRG/WP 1416
The manner by which lignin is degraded in-situ in natural substrates by white rot fungi still remains a controversial issue particularly the distribution and role(s) played by lignin degrading enzymes (i.e. manganese II peroxidase and lignin peroxidase). In the present study, use was made of anti-manganese II peroxidase and immunolabelling techniques in conjunction with transmission electron microscopy (TEM) to study the spatial distribution of manganese II peroxidase during degradation of wood and woody fragments by Phanerochaete chrysosporium and Lentinus edodes. Intracellularly, manganese II peroxidase was found localized in the peripheral regions of the fungal cell cytoplasm in association with both the outer cell membrane and membranes within characteristic vesicular bodies. In addition the enzyme was frequently found localized at the interfacial regions of the cell membrane and inner fungal cell wall. Using double immunolabelling procedures and in addition anti-lignin peroxidase, the cytoplasmic distribution of the two lignin degrading enzymes was compared. Both enzymes showed a fairly similar peripheral cytoplasmic localization although manganese II peroxidase tended to be more concentrated compared to lignin peroxidase in peripheral vesicular bodies. Extracellularly, and in solid wood samples manganese II peroxidase was found localized in all wood cell wall regions of either Betula verrucosa, Populus sp. or Fagus sylvatica decayed by either Phanerochaete chrysosporium or Lentinus edodes at both early and late stages of degradation. In particular, manganese II peroxidase was localized in characteristic zones of degradation produced within the secondary wood cell wall regions. These regions displayed a more open structure compared to unattacked wood cell walls and were easily penetrated by lignin degrading enzymes as judged by infiltration and double immunolabelling studies with highly purified and partially purified manganese II and lignin peroxidases. With Lentinus edodes a very characteristic pattern of lignin degradation was noted in which the middle lamella regions between wood cells was selectively degraded. In these regions manganese II peroxidase was found concentrated and associated with its degrading matrix. An extracellular distribution of manganese II peroxidase associated with wood fragments was also observed in liquid cultures of Phanerochaete chrysosporium grown under conditions optimal for peroxidase production. Despite the immersed conditions, similarities between the patterns of attack and extracellular distribution of the enzyme as for solid wood were noted. With both solid wood and wood fragments, manganese II peroxidase penetration was restricted to regions showing structurally modification, and penetration into undecayed cell walls was not observed. The present work suggests a close substrate-enzyme association during wood cell wall and lignin degradation under natural conditions, and in addition, a close correlation between changes in the micromorphology of decay and manganese II peroxidase distribution. Possible reasons for the failure of previous and similar immunolabelling studies to show such a correlation with lignin degrading enzymes are briefly discussed.
G F Daniel, B Pettersson, T Nilsson, J Volc

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

Immunolabelling studies on the detection of enzymes during the degradation of wood by Phanerochaete chrysosporium
1988 - IRG/WP 1364
The degradation of lignin in native lignocellulosic substrates by white rot fungi is poorly understood. Biochemical studies have shown the involvement of a number extracellular ligninolytic enzymes released by white rot fungi which are capable of the oxidative conversion of DHP's (lignin model compounds) in vitro, but to date conclusive evidence for occurrence of these enzymes in wood undergoing degradation is limited. In the present work, the distribution of lignin peroxidase during the degradation of Betula verrucosa and Pinus sylvestris by the ligninolytic fungus Phanerochaete chrysosporium (wild type) was studied by using antisera produced against the purified enzyme in conjunction with appropriate light and electron microscopic immunological detection methods. Light microscopic immunofluorescence and immunocytochemical observations showed the enzyme to be closely associated with both the fungus and exposed sites of erosion decay of the wood fibres. This was confirmed by post-embedding TEM immunolabelling studies using gold labelling methods which showed the enzyme to be localized within the peripheral fungal cell cytoplasm, cell membrane, fungal cell wall regions and occassionally extracellular slime materials. Similar gold-labelling methods showed enzymic localization along areas of lumen wall erosion of birch fibres at various stages of decay. Labelling occurred on all cell wall layers including the middle lamella when exposed. The distribution of the enzyme in degrading pine fibres was also restricted to sites exposed during erosion decay. In contrast comparative cytochemical studies performed with 3' 3' diaminobenzidine for general peroxidase enzymes suggested an intracellular distribution within the S2 cell wall of degrading fibres. The enzyme distribution was seen to have a close association with characteristic zones of decay which radiated out from the inner fibre wall regions during attack. Possible reasons for the disparity in the results are discussed as are the implications for the apparent restriction of lignin peroxidase to exposed surface regions of wood fibres during degradation.
G F Daniel, T Nilsson, B Pettersson