High hydrostatic pressure shapes the development and production of secondary metabolites of Mariana Trench sediment fungi

Abstract

The hadal biosphere is one of the least understood ecosystems on our planet. Recent studies have revealed diverse and active communities of prokaryotes in hadal sediment. However, there have been few studies on fungi in hadal sediment. Here we report the first isolation and cultivation of 8 fungi from the Mariana Trench sediment. The individual colonies were isolated and identified as Stemphylium sp., Cladosporium sp., Arthrinium sp., Fusarium sp., Alternaria sp., and Aspergillus sp. High hydrostatic pressure (HHP) test was carried out to identify the piezophily of these hadal fungi. Among them, 7 out of the 8 fungal isolates exhibited the ability of germination after incubation under 40 MPa for 7 days. Vegetative growth of the isolates was also affected by HHP. Characterization of secondary metabolites under different pressure conditions was also performed. The production of secondary metabolites was affected by the HHP treatment, improving the potential of discovering novel natural products from hadal fungi. The antibacterial assay revealed the potential of discovering novel natural products. Our results suggest that fungal growth pressure plays an important role in the development and production of secondary metabolites of these hadal fungi under the extreme environment in the Mariana Trench.

Figure 1

Phylogenetic tree of ITS sequences (ITS1/ITS4 primers) of the eight fungi isolated from deep-sea sediments in the Mariana Trench.

Figure 2

The typical spore patterns of 8 strains were obtained in the Mariana Trench sediment under atmospheric pressure. Macroscopic images showed the morphology of colony. Microscopic images showed the morphology of typical spores, taken under × 40 microscopes. The scale bar was 100 μm.

Figure 3

Phenotypes of hadal-derived fungi isolated and cultured in this experiment under the different preset pressures. (a) Colony, mycelium and spore morphology of eight fungi were obtained, respectively, under 0.1, 20 and 40 MPa. (CIEL 1) Alternaria alternata CIEL1, (CIEL 2) Cladosporium sp. CIEL 2, (CIEL 3) Aspergillus sp. CIEL 3, (CIEL 4) Arthrinium sp. CIEL 4, (CIEL 5) Stemphylium vesicarium CIEL 5, (CIEL 6) Alternaria sp. CIEL 6, (CIEL 7) Fusarium. poae CIEL 7, (CIEL 8) Cladosporium sp. CIEL 8. (b) Growth state of Fusarium poae CIEL 7 under 0.1, 20 and 40 MPa. There was no significant difference in the front side of the colonies, but the back side of the colonies showed different colors.

Figure 4

The growth rate of hadal-derived fungi isolated and cultured in this experiment under the different preset pressures. Under the same cultural conditions, the area of the strain changed regularly with the change of pressure. The unit of the area was μm². T-test was used for pairwise comparisons. (NS p > 0.05, ***p < 0.001).

Figure 5

TIC diagrams of metabolites of hadal-derived fungi isolated and cultured under three preset hydrostatic pressures. (a) TIC peak of Cladosporium sp. CIEL 2 at different hydrostatic pressures. In addition to the change of peak intensity, 4 but not 2 appeared in the metabolites at 20 MPa and 40 MPa. (b) TIC peak of Stemphylium vesicarium CIEL 5 at different hydrostatic pressures. A new peak 5 appears at 20 MPa. Each analysis shows the extracted ion track of the corresponding compounds recorded by mass spectrometry. The relative abundance was set at 100.

Figure 6

Antimicrobial activity of metabolites of hadal-derived fungi isolated and cultured under three preset hydrostatic pressures. Among the eight strains of fungi isolated in this experiment, Antimicrobial activity of metabolites produced by Cladosporium sp. CIEL 2, Stemphylium vesicarium CIEL 5 and Cladosporium sp. CIEL 8 against Staphylococcus aureus ATCC25923 and Enterococcus faecalis FA2-2 under 0.1, 20 and 40 MPa. (a) The inhibitory zone of metabolites produced by fungi under different pressures. (b) Antimicrobial zone diameter of the metabolites against S. aureus ATCC25923. The diameter unit was cm. T-test was used for pairwise comparisons. The activity of the Stemphylium vesicarium CIEL 5 metabolic products increases with the increase of pressure. However, the activity of the Cladosporium sp. CIEL 2 and Cladosporium sp. CIEL 8 metabolic products decreases with the increase of pressure. (***p < 0.001). (c) Antimicrobial zone diameter of the metabolites against E. faecalis FA2-2. The diameter unit was cm. T-test was used for pairwise comparisons. Cladosporium sp. CIEL 2 metabolic products are inactive, the activity of the Stemphylium vesicarium CIEL 5 metabolic products increases with the increase of pressure, and the activity of the Cladosporium sp. CIEL 8 metabolic products decreases with the increase of pressure (NS p > 0.05, ***p < 0.001).