Hydrolytic enzymes produced by a bioherbicidal strain of Myrothecium verrucaria

Robert E. Hoagland, Clyde D. Boyette and Mark A. Weaver, USDA-ARS, Southern Weed Science Research Unit, Stoneville, MS 38776,  bob.hoagland@aars.usda.gov

The involvement of cell wall degrading enzymes in infection processes of phytopathogenic microorganisms has been well documented.  Thus far attention has generally focused on hydrolytic enzymes that degrade polysaccharides, but more recently other lytic enzymes such as proteases have been implicated as virulence factors in certain host:pathogen interactions.  We are developing a fungus, Myrothecium verrucaria (MV), as a bioherbicide for kudzu and some other invasive weeds.  Spore and mycelial formulations of MV exhibit relatively rapid bioherbicidal activity when applied to the foliage of these weeds.  Presently, little is known about the biochemical processes involved during infection and the development of necrosis in host weeds treated with MV propagules.  Knowledge of such events and their biochemical mechanisms would be useful to regulate and/or improve bioherbicidal efficacy.  We have recently discovered the presence of various hydrolytic enzymes in the mycelium of MV.  Crude enzyme extracts, prepared using sonicated mycelium (obtained via fermentation), were found to contain proteolytic and polysaccharidase activities.  Proteolytic activity was exhibited on two synthetic substrates (N-benzoyl-arginine-p-nitroanilide and azocoll), with optimal activity occurring between pH values of about 6.8 - 9.0 and 5.0 - 9.0, respectively.  Polysaccharide hydrolytic activity was detected using p-nitrophenyl-ß-galactoside and p-nitrophenyl-ß-glucoside with optimal activity occurring at pH 6.8 - 9.0.  The enzyme preparation also possessed hydrolytic activity on fluorescein diacetate, a general substrate for the assay of lipases, esterases, and proteases.  Further characterization of these enzymes and their roles in the development of infectivity, necrosis, and death of target weeds will be subjects of future research.