The in vitm production of different hydrolytic enzyme activities by Verticillium fungicola, a mycoparasite of Agaricus bisponrs fruit bodies, was examined in cultures grown with various carbon sources (glucose, fructose, sucrose and A. bisporus cell walls). Several of the identified enzymes were influenced by the carbon source. The results of A. bisporus cell wall digestion by these enzymes in witro have been compared with V. fingicola infection of A. bisporus fruit bodies in vivo. Evidence supporting mycoparasite enzymic action on the host cell walls is presented.

The hyphal walls of Agaricus bisporus and Agaricus campestris fruiting bodies were isolated and purified. Quantitative analyses revealed that these walls consisted mainly of carbohydrates (78.3-79.2%), lipids (9.9-10.1%), and proteins (8.7-10.2%). The major components of carbohydrate polymers were glucose, N-acetylglucosamine, and glucosamine. In addition, small quantities of galactose and mannose have been found. N-Acetylglucosamine and glucosamine were identified chemically and enzymatically, and also by infrared spectrum and X-ray diffraction analyses, as chitin and chitosan. Neutral polysaccharides include an alkali-soluble glucan with alpha (1-3) linkages and a beta (1-3)- and beta (1-6)-linked glucan. The lipid fractions in both hyphal walls contained precursors of melanin, this pigment being largely represented in Agaricus spore walls. Amino acids analyses indicate that structural wall proteins were very similar in both organisms. In electron micrographs of ultrathin sections of hyphae no distinct layering was apparent in contrast with spore walls of the same organism, which show two wide well-defined layers.

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Agaricus bisporus H 25 produced extracellular endo-1, 3-beta-glucanase when grown in a static culture at 25 degrees C in a minimal synthetic medium supplemented with A. bisporus cell walls plus fructose. Endo-1,3-beta-glucanase was purified 17.85-fold from 20-day-old culture filtrates by precipitation at 80% ammonium sulfate saturation, Sephadex G-75 gel filtration, and preparative PAGE followed by electroelution. The purified enzyme yielded a single band in both native and SDS-polyacrylamide gels with a molecular mass of 32 kDa (SDS-PAGE) and 33.7 kDa (MALDI-MS), showing an isoelectric point of 3.7. The enzyme was active against beta-1,3- linkages and, to a lesser extent, against beta-1,6-, exhibiting an endohydrolytic mode of action and a glycoprotein nature. Significant activities of the endo-glucanase against laminarin and pustulan were observed between pH 4 and 5.5, and between 40 degrees and 50 degrees C for laminarin, and between 30 degrees and 50 degrees C for pustulan. The optimum pH and temperature were 4.5 and 45 degrees C for both substrates.