Chitinolytic activity of ectomycorrhizal symbionts
Chitinolytic activities from Pinus sylvestris L., Eucalyptus pilularis Sm. and a number of basidiomycete, ascomycete and oomycete fungi were characterised using a fluorogenic assay based on 4-methylumbelliferyl glycosides of N-acetylglucosamine oligosaccharides. Fungal extracellular chitinolytic activities were induced in the presence of chitin, and subject to catabolite repression by glucose, except those from Armillaria ostoyae Romagn. Activities were not repressed by ammonium phosphate. Addition of chitin to medium containing glucose and ammonium phosphate enhanced the growth of several fungi (e.g. Heterobasidion annosum (Fr.) Karst, Boletinus cavipes (Opat.) Kalch br., Paxillus involutus (Batsch) Fr. and Trichoderma harzianum Rifai) but repressed growth of the non-chitinous fungus, Phytophthora cinnamomi Rands. 14C dilution indicated that a change in carbon allocation within fungal mycelium occurred in the presence of chitin. Constitutive endochitinase activities from both plants and fungi were inhibited by the specific chitinase inhibitor allosamidin, but this was generally ineffective against exochitinase. Only exochitinase from P. involutus was inhibited sufficiently to allow determination of an IC50 value of 270 nM. A synthetic analogue of N-acetylglucosamine, (2-acetamido-2-deoxy-D-glucopyranosylidene) amino phenylcarbamate (PUGNAC), inhibited chitinolytic activities. The effect varied with species and substrate used, but was generally most marked against exochitinase. PUGNAC was used in challenge studies to distinguish between host and fungal endochitinase activities in sterile microcosms containing tree seedlings. Unchallenged P. sylvestris roots produced little N-acetylglucosaminidase activity. When challenged by the pathogen, H.annosum, root N-acetylglucosaminidase activity increased significantly. There was no response by either P. sylvestris or E. pilularis roots to challenge by the pathogen, P. cinnamomi, or the ectomycorrhizal fungus, Pisolithus tinctorius (Pers.) Coker & Couch.