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

Abstract

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.

FIG. 1.

AcH 505-related streptomycetes based on a neighbor-joining analysis of 16S rRNA gene sequences. The sequence data for five AcH 505-related streptomycete-like cultures were obtained from the GenBank database and included in the tree. Scale bar = 0.01 estimated mutation per sequence position. The accession numbers for the sequences are as follows: Streptomyces sp. strain AcH505, DQ231567; Streptomyces aureus, AY094368; S. laceyi, AY094367; Streptomyces pulveraceus, AJ781377; S. sanglieri, AY094363; and S. setonii, D63872.

FIG. 2.

Structural formulas for auxofuran and the antibiotics WS-5995 B and C, produced by Streptomyces strain AcH 505.

FIG. 3.

Growth-promoting activity of auxofuran. (A) Radial growth of A. muscaria mycelia after 2 weeks of incubation on solid media. The P values (as determined by Student's t test) for pairwise comparisons with the control treatments (C) were <0.01 for 150 nM to 50 μM auxofuran. (B) Comparison of the effects of auxofuran (A) and its synthetic analog 7-dehydroxy-auxofuran (dOH-A) on A. muscaria mycelia on solid media. Both substances were added to the culture medium at a concentration of 15 μM. The P values (as determined by Student's t test) for pairwise comparisons were <0.001 for all times. The P value for the comparison of control and auxofuran treatments after 6 weeks of culture was 0.02.

FIG. 4.

Growth-suppressing effects of WS-5995 B and C on A. muscaria hyphae on solid media. Substances were added to MMN culture medium in 100 μl methanol. Control treatments without and with methanol are indicated by Me− and Me+, respectively. Bars with different letters are significantly different according to a one-way analysis of variance and the Tukey test (P < 0.01).

FIG. 5.

Response of H. cylindrosporum to auxofuran and WS-5995 B. (A) Promotion of H. cylindrosporum growth with 1.5 μM auxofuran (A 1,5 μM). (B) Activity of WS-5995 B against the ectomycorrhizal fungi A. muscaria and H. cylindrosporum. The radial growth of 2-week-old mycelia was measured. Note the different scales.

FIG. 6.

(A) Levels of transcripts of growth- and stress-related A. muscaria genes under the influence of auxofuran and WS-5995 B. Cultures were harvested after 3 h of incubation with MeOH, 15 μM auxofuran (A), or 30 μM WS-5995 B (B). The levels of expression of A. muscaria acetoacyl-CoA synthetase (Aacs), cyclophilin 40 (Cyp40), and γ-aminobutyric acid permease (Uga4) genes were analyzed by Northern analysis. (B) WS-5995 B-based induction of the GABA concentration in A. muscaria mycelia. Changes in the GABA concentration during fungal growth in a submerged culture were investigated. Water (A), MeOH (B), 15 μM auxofuran (C), or 30 μM WS-5995 B (D) was added to actively growing mycelia. The levels of GABA were determined after 3 h of incubation. The means (±standard deviations) for three independent experiments are indicated.