[Culture conditions and analysis of amanitins on Amanita spissa].

Isolate of Amanita spissa was obtained from basidiome stipe material collected from environment. It could utilize a broad range of carbon and nitrogen resources. Study on the influence of different conditions for solid culture was carried out. Optimal culture conditions were at 28 degrees C, pH6, in the dark. A. spissa was then fermentated in liquid culture for more mycelia. In flask and Airlift/ff bioreactor, maximum dry mycelia weight of A. spissa reached 0.893 g/L and 2.33 g/L, respectively. Mycelia obtained from solid culture and Airlift/ff bioreactor were then analyzed by HPLC. The results showed that mycelia from both cultures contained amatoxins but no phallotoxins. alpha-Amanitin in mycelia reached 26.02 microg/DWg under solid culture condition, and 15.25 microg/DWg under liquid culture condition. The amanitins were also confirmed by bud-inhibited assay. The results revealed that the effect of amanitin on mung bean cell was identical to that of authentic amanitins. This work suggests that it is possible to produce amatoxin by liquid culturing of A. spissa.

Auxofuran, a novel metabolite that stimulates the growth of fly agaric, is produced by the mycorrhiza helper bacterium Streptomyces strain AcH 505.

The mycorrhiza helper bacterium Streptomyces strain AcH 505 improves mycelial growth of ectomycorrhizal fungi and formation of ectomycorrhizas between Amanita muscaria and spruce but suppresses the growth of plant-pathogenic fungi, suggesting that it produces both fungal growth-stimulating and -suppressing compounds. The dominant fungal-growth-promoting substance produced by strain AcH 505, auxofuran, was isolated, and its effect on the levels of gene expression of A. muscaria was investigated. Auxofuran and its synthetic analogue 7-dehydroxy-auxofuran were most effective at a concentration of 15 microM, and application of these compounds led to increased lipid metabolism-related gene expression. Cocultivation of strain AcH 505 and A. muscaria stimulated auxofuran production by the streptomycete. The antifungal substances produced by strain AcH 505 were identified as the antibiotics WS-5995 B and C. WS-5995 B completely blocked mycelial growth at a concentration of 60 microM and caused a cell stress-related gene expression response in A. muscaria. Characterization of these compounds provides the foundation for molecular analysis of the fungus-bacterium interaction in the ectomycorrhizal symbiosis between fly agaric and spruce.

Structure elucidation of auxofuran, a metabolite involved in stimulating growth of fly agaric, produced by the mycorrhiza helper bacterium Streptomyces AcH 505.

Mycorrhiza helper bacterium Streptomyces strain AcH 505 stimulates ectomycorrhiza formation between spruce and fly agaric by supporting fungal growth whereas growth of pathogenic fungi is suppressed. A fungal growth promoting substance was isolated and the chemical structure elucidated by mass spectrometry and NMR spectroscopy. The absolute configuration of the novel fungal growth promoting compound auxofuran (1) was deduced from NMR data with the help of Mosher esters.

Influence of copper, manganese and pH on the growth and several enzyme activities in mycorrhizal fungus Amanita muscaria.

The effects of various concentrations of copper, manganese and pH on the growth and several enzyme activities of mycorrhizal fungus Amanita muscaria were investigated. Cu (5-25 mg l-1) and lower pH (3.0-4.0) strongly inhibited the mycelial growth (dry weight), however, the protein content was not affected evidently. Some enzyme activities were lower as the Cu and Mn concentrations were higher and other enzymes had the maximum values at the specified concentration. The activities of the following enzymes were significantly correlated with the fungal growth after the treatment with Cu: G6PDH, MTLDH and trehalase, and with Mn: G6PDH, MTLDH and alpha-mannosidase respectively. Measurement of these enzyme activities might provide a useful biochemical criterion for the evaluation of the fungitoxicity of soil contaminated by copper or manganese.

Unique amanitin resistance of RNA synthesis in isolated nuclei from Amanita species accumulating amanitins.

In order to understand the basis upon which amanitin-accumulating species of Amanita are able to develop in the presence of these specific inhibitors of RNA synthesis, the in vitro RNA synthesizing activities of nuclei isolated from amanitin-accumulating species. Amanita hygroscopica (culture, derived from amanitin-accumulating carpophore) and A. suballiacea (carpophore), and from the non-accumulating species A. solitaria (culture) and A. brunnescens (carpophore) were tested for their sensitivities to alpha-amanitin inhibition. The nuclear RNA synthesizing activities obtained from both carpophores and cultures of non-accumulating organisms displayed significant sensitivities to alpha-amanitin, whereas those obtained from accumulating organisms displayed remarkable resistance to alpha-amanitin. The observed relationship between levels of amanitins in carpophores and resistance of the RNA synthesizing activities to inhibition by alpha-amanitin supports the hypothesis that amanitins may function as regulators of mRNA transcription in Amanita species.