Rapid Extracellular Enzyme Assays For Screening Potential Antisapstain Biological Control Agents
Keywords: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.
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