Antifungal compounds produced by chitinolytic bacteria from grain ecosystems.

A chitinolytic strain of Bacillus pabuli (K1) isolated from mouldy grain was evaluated in terms of its potential as a biocontrol agent. This work showed that optimal conditions for chitinase production were: 30 deg C, initial pH 8, initial oxygen concentration 10% and aw greater than 0.99. Chitinase production was induced by N,N'4-diacetylchitiobiose, larger chito-oligosaccharides and chitin and repressed by glucose, starch, laminarin, beta-glucan (barley) and glycerol. Crude extracts from 10 bacterial isolates were used to compare different methods for quantifying chitinase activity. The methods compared were based on the measurements of the release of p-nitrophenol from p-nitrophenyl-N-acetyl glucosamine (pNP-(GlcpNAc)), pNP-(GlcpNAc)2 and pNP-(GlcpNAc)3, the release of reducing sugars from chitin and the formation of clearing zones on chitin agar. No correlation between methods was found when chitinases from different bacterial isolates were compared. The release of reducing sugars from chitin correlated well with the release of p-nitrophenol from pNP-(GlcpNAc)2 when crude chitinase from Bacillus pabuli K1 was used. Chitinolytic bacteria constituted up to 0.5% of the total count of aerobic bacteria in samples of airtight stored grain. Chitinase production and ability to inhibit fungal growth was not correlated. Two strongly antifungal isolates (K139 and K122) were selected for further studies, in which their effects on the growth of a number of potentially harmful fungi were assessed. Mycelial extracts of K139, a Streptomyces coelicolor isolate, contained two antifungal compounds. UV spectroscopy revealed that one of these compounds was a polyene, belonging to the heptaene group. The mycelial extract showed strong similarities to a commercial preparation of candicidin. Chemical instability made identification of the second antifungal compound difficult. Streptomyces halstedii K122 inhibited fungal radial growth and induced drastic changes in hyphal morphology and branching. No antifungal activity was detected in culture filtrates or in mycelial extracts when K122 was cultivated in liquid substrates. When cultivated in thin (1 mm) liquid layers, it was possible to extract antifungal compounds from the mycelium with methanol. The compounds were identified as the bafilomycins B1 and C1, using 2D NMR and FAB-MS. Minimal inhibitory concentrations of the V-ATPase inhibitors, bafilomycins, were determined for Aspergillus fumigatus, Mucor hiemalis, Penicillium roqueforti and Paecilomyces variotii.