Mollisin

An antifungal protein was isolated from black turtle bean, Phaseolus vulgaris cv. "Black Turtle". The purified protein displayed an N-terminal amino acid sequence with 60-80% homology to chitinases. The isolated protein specifically inhibited two species of fungi, namely Fusarium oxysporum and Mycosphaerella arachidicola , among several phytopathogenic fungi tested. Its antifungal activity was retained after incubation at 60 degrees C for 15 minutes, diminished after exposure to 80 degrees C, and was completely abrogated after treatment at 100 degrees C. The antifungal protein had higher thermostability than the antifungal protein mollisin from chestnut. It stimulated nitric oxide production by murine peritoneal macrophages, but exerted neither mitogenic nor cytotoxic activity on murine splenocytes, activities that may be related to allergic responses.

The antibiotic and fungicidal deuteromycete Mollisia caesia Sacc. was cultivated on a large scale. Mollisin (1; = 8-(dichloroacetyl)-5-hydroxy-2,7-dimethylnaphthalene-1,4-dione) and two new tri- and tetrahalogenated metabolites, mollisin A (2) and mollisin B (3) were isolated from M. caesia. The formation of 2 and 3 indicates that the biosynthesis of these compounds starts from a C(16) polyketide (Scheme). Mollisin (1) shows strong fungicidal activities against Sclerophoma pityophila (Corda) v. Höhn and Heterobasidion annosum (Fr.) Bref., which is one of the most-destructive basidiomycetes in coniferous forests. The metabolites 1-3 possess interesting pharmacological activities in assays in search of anti-inflammatory and antiproliferative drugs.

The mycorrhization helper bacterium Streptomyces sp. AcH 505 inhibits Norway spruce root infection and colonisation by the root and butt rot fungus Heterobasidion annosum 005 but not by the congeneric strain Heterobasidion abietinum 331 because of higher sensitivity of H. annosum 005 towards the AcH 505-derived naphthoquinone antibiotic WS-5995 B. Differences in antibiotic sensitivity between two isolates belonging to two species, H. annosum 005 and H. abietinum 331, were investigated by comparative gene expression analysis using macroarrays and quantitative RT-PCR after WS-5995 B, structurally related mollisin and unrelated cycloheximide application. Treatment with 25 microM WS-5995 B for 2 h resulted in a significant up-regulation of expression of inosine-5'-monophosphate dehydrogenase, phosphoglucomutase and GTPase genes, while the expression of genes encoding for thioredoxin and glutathione dependent formaldehyde dehydrogenase was down-regulated in the sensitive fungal strain. No differential expression in the tolerant strain was detected. Application of WS-5995 B at higher concentrations over a time course experiment revealed that H. annosum 005 and H. abietinum 331 responded differently to WS-5995 B. The fungal gene expression levels depended on both the concentration of WS-5995 B and the duration of its application. The WS-5995 B-unrelated cycloheximide caused highly specific changes in patterns of gene expression. Our findings indicate considerable variations in response to bacterial metabolites by the isolates of the conifer pathogen.