title: Use of monoclonal antibodies to quantify the dynamics of alpha-galactosidase and endo-1,4-beta-glucanase production by Trichoderma hamatum during saprotrophic growth and sporulation in peat.
abstract: Trichoderma species are ubiquitous soil and peat-borne saprotrophs that have received enormous scientific interest as biocontrol agents of plant diseases caused by destructive root pathogens. Mechanisms of biocontrol such as antibiosis and hyperparasitism are well documented and the biochemistry and molecular genetics of these processes defined. An aspect of biocontrol that has received little attention is the ability of Trichoderma species to compete for nutrients in their natural environments. Trichoderma species are efficient producers of polysaccharide-degrading enzymes that enable them to colonize organic matter thereby preventing the saprotrophic spread of plant pathogens. This study details the use of monoclonal antibodies (mAbs) to quantify the production of two enzymes implicated in the saprotrophic growth of Trichoderma species in peat. Using mAbs specific to the hemicellulase enzyme alpha-galactosidase (AGL) and the cellulase enzyme endo-1,4-beta-glucanase (EG), the relationship between the saprotrophic growth dynamics of a biocontrol strain of Trichoderma hamatum and the concomitant production of these enzymes in peat-based microcosms was studied. Enzyme activity assays and enzyme protein concentrations derived by enzyme-linked immunosorbent assay (ELISA) established the precision and sensitivity of mAb-based assays in quantifying enzyme production during active growth of the fungus. Trends in enzyme activities and protein concentrations were similar for both enzymes, during a 21-day sampling period in which active growth and sporulation of the fungus in peat was quantified using an independent mAb-based assay. There was a sharp increase in active biomass of T. hamatum 3 days after inoculation of microcosms with phialoconidia. After 3 days there was a rapid decline in active biomass which coincided with sporulation of the fungus. A similar trend was witnessed with EG activities and concentrations. This showed that EG production related directly to active growth of the fungus. The trend was not found, however, with AGL. There was a rapid increase in enzyme activities and protein concentrations on day 3, after which they remained static. The reason for the maintenance of elevated AGL probably resulted from secretion of the enzyme from conidia and chlamydospores. ELISA, immunofluoresence and immunogold electron microscopy studies of these cells showed that the enzyme is localized within the cytoplasm and is secreted extracellularly into the surrounding environment. It is postulated that release of oligosaccharides from polymeric hemicellulose by the constitutive spore-bound enzyme leads to AGL induction and could act as an environmental cue for spore germination.
authors.affiliation: Fungal Biology Laboratory, Washington Singer Laboratories, School of Biological and Chemical Sciences, University of Exeter, Perry Road, Exeter, Devon EX4 4QG, UK. C.R.Thornton@ex.ac.uk
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Thornton
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000946:Antigens, Fungal / Q000737:chemistry / Q000276:immunology; D015153:Blotting, Western; D002480:Cellulase / Q000096:biosynthesis*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000378:metabolism; D006825:Hybridomas; D007132:Immunoglobulin Isotypes; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D010504:Periodic Acid / Q000378:metabolism; D011402:Pronase / Q000378:metabolism; D012988:Soil Microbiology; D044003:Sphagnopsida; D013172:Spores, Fungal / Q000201:enzymology; D014242:Trichoderma / Q000201:enzymology* / Q000254:growth & development*; D000519:alpha-Galactosidase / Q000096:biosynthesis*
chemical_list: D000911:Antibodies, Monoclonal; D000946:Antigens, Fungal; D000939:Epitopes; D007132:Immunoglobulin Isotypes; D010504:Periodic Acid; C009288:metaperiodate; D000519:alpha-Galactosidase; D002480:Cellulase; D011402:Pronase