Rapid Extracellular Enzyme Assays For Screening Potential Antisapstain Biological Control Agents


  • Brenda J. McAfee
  • Benjamin E. Dawson-Andoh
  • Maria Chan
  • Roger Sutcliffe
  • Rawle Lovell


Bioprotection, sapstain and biological control fungi, extracellular enzymes, rapid agar plate assays for enzyme screening


A Rapid Agar Plate Screening Assay (RAPSA) was developed and optimized for assaying individual extracellular enzymes produced by potential biological control agents and sapstain fungi. The RAPSA, which uses culture filtrates rather than agar plugs inoculated with actively growing fungi as used in the classical screening method, was more sensitive in detecting activity, for all extracellular enzymes screened, with the exception of chitinase, for the majority of the fungi tested. The assay was used to screen potential biological control fungi based on comparison of extracellular enzyme profiles of ten potential antisapstain biological control fungi and three sapstain fungi, grown in liquid cultures containing either glucose, hemlock sawdust, or cell wall of the sapstain fungus Ophiostoma piceae as a carbon source. Based on extracellular enzymes profiles, biological control fungi and sapstain fungi were classified into three groups. Group I fungi produced the greatest enzyme activity when glucose was included in the medium. Group II fungi produced equally good activity with sawdust and glucose, while Group III produced high activity with both sawdust and cell wall while enzyme activity with glucose was not consistent.

Five biological control candidates, Gliocladium viride 623E, G. roseum 784A, G. virens 258C, G. roseum 321M, G. virens 258D, in descending order, demonstrated the full spectrum of extracellular enzyme activity screened, irrespective of the growth medium. Production of extracellular enzymes in a minimal medium augmented with sawdust or cell wall is an indicator of secondary resource capability. Gliocladium viride 623E and G. virens 258C demonstrated high extracellular enzyme production in both of these media. On this basis, these two fungi were judged to show the greatest potential as biological control agents. Mariannea elegans 386E and G. solani 810A showed the least potential.


Aronson, J. M. 1965. The cell wall. In G. C. Ainsworth and A. S. Sussman, eds. The fungi, vol. 1. The fungal cell. Academic Press. New York, NY.nDawson-Andoh, B. E., and J. J. Morrell. 1997. Biological protection of freshly sawn sapwood from biological discolorations. Pages 3-9 in Prevention of discolorations in hardwoods and softwood logs and lumber. Forest Prod. Soc. Pub. No. 7283.nDel Ray, F., I. Garcia-Acha, and C. Nombela. 1979. The regulation of β-glucanase synthesis in fungi and yeast. J. Gen. Microb.110:83-89.nDonly, B. C., and A. W. Day. 1984. A survey of extracellular enzymes in the smut fungi. Bot. Gaz.145:483-486.nFravel, D. R. 1988. Role of antibiosis in the biocontrol of plant diseases. Ann. Rev. Phytopathol.26:75-91.nHagerman, A. E., D. M. Blau, and A. L. McClure. 1985. Plate assay for determining the time of production of protease, cellulase, and pectinase by germinating fungal spores. Anal. Biochem.151:334-342.nHankin, L., and S. L. Anagnostakis. 1975. The use of solid media for the detection of enzyme production by fungi. Mycologia67:597-607.nHarney, S., and P. Widden. 1991. Physiological properties of the entomopathogenic hyphomycete Paecilomyces farinosus in relation to its role in the forest ecosystem. Can. J. Bot.69:1-5.nHearn, V. M. and D. W. R. MacKenzie. 1980. The preparation and partial purification of fractions from mycelial fungi with antigenic activity. Molecular Immun.17: 1097-1103.nKjoller, A., and S. Struwe. 1980. Microfungi of decomposing red alder leaves and their substrate utilization. Soil Biol. Biochem.12:425-431.nLewis, J. A., and C. C. Papavizas. 1991. Biocontrol of plant diseases: The approach for tomorrow. Crop Protect.10:95-105.nLewis, K., J. M. Whipps, and R. C. Cooke. 1989. Mechanisms of biological disease control with special reference to the case of Pythiurn oligandrum as an antagonist. Pages 191-217 in J. M. Whipps and R. D. Lumsden, eds. Biotechnology of fungi for improving plant growth. Cambridge University Press, UK.nLumsden, R. D., and J. A. Lewis. 1989. Selection, production, formulation, and commercial use of plant disease biocontrol fungi: Problems and progress. Pages 170-190 in J. M. Whipps and R. D. Lumsden, eds. Biotechnology of fungi for improving plant growth. Cambridge University Press, UK.nLynch, J. M. 1990. Fungi as antagonists. Pages 243-253 in New Directions in biological control: Alternatives for suppressing agricultural pests and diseases. Alan R. Liss, Inc.nMcAfee, B. J., and M. M. Gignac. 1997. Biological control of fungal growth on unseasoned lumber following pasteurization. Mater, und Org.31:45-61.nPan, S. W., X. S. Ye, and J. Kuc. 1991. A technique for detection of chitinase, β-1,3-glucanases and protein pattern after a single separation using polyacrylamide gel electrophoresis of IEF. Phytopathol.81:970-974.nPapavizas, G. C. 1985. Trichoderma and Gliocladium: Biology, ecology, and potential for biocontrol. Ann. Rev. Phytopathol.23:23-54.nPitson, S. R., J. Seviour, J. Bott, and S. J. Stansino-Poulos. 1991. Production of β-glucanases in Acremonium and Cephalosporium isolates. Mycol. Res.95: 352-356.nRayner, A. D. M., and N. K. Todd. 1979. Population and community structure and dynamics of fungi in decaying wood. Advan. Botan. Res.7:333-420.nRidout, C. J., J. R. Coley-Smith, and J. M. Lynch. 1988. Fracionation of extra-cellular enzymes from a mycoparasitic strain of Trichoderma harzianum. Enzyme Microb. Technol.10:180-186.nSaksirirat, W., and H. H. Hope. 1991. Secretion of extracellular enzymes by Verticillium psalliotae Treschow and Verticillium lecanii (Zimm.) Viegas during growth on uredospores of soybean rust fungus (Phakospora pachyrhzi Syd.) in liquid cultures. J. Phytopathol.131: 161-173.nVillanueva, J. R. 1966. The protoplast. in C. G. Ainsworth and A. S. Sussman, eds. The fungi, vol. II. The fungal organism. Academic Press. New York and London.nWood, P. J., and J. Weisz. 1987. Detection and assay of (1-4)β-D-Glucanase, (1-3)(1-4)-β-D-Glucanase and xylanase based complex of substrate with congo red. Cereal Chem.64:8-15.nYang, D. Q., and L. Rossignol. 1999. Evaluation of Gliocladium roseum against wood-degrading fungi in vitro and on major Canadian wood species. Biocontrol Sci. Technol.9:409-420.n






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