A Chemically Defined Medium That Supports Mycotoxin Production by Stachybotrys chartarum Enabled Analysis of the Impact of Nitrogen and Carbon Sources on the Biosynthesis of Macrocyclic Trichothecenes and Stachybotrylactam

Abstract

Stachybotrys chartarum (Hypocreales, Ascomycota) is a toxigenic fungus that is frequently isolated from water-damaged buildings or improperly stored feed. The secondary metabolites formed by this mold have been associated with health problems in humans and animals. Several authors have studied the influence of environmental conditions on the production of mycotoxins, but these studies focused on undefined or complex substrates, such as building materials and media that impeded investigations of the influence of specific nutrients. In this study, a chemically defined cultivation medium was used to investigate the impact of several nitrogen and carbon sources on growth of S. chartarum and its production of macrocyclic trichothecenes (MTs) and stachybotrylactam (STLAC). Increasing concentrations of sodium nitrate were found to positively affect mycelial growth, the level of sporulation, and MT production, while ammonium nitrate and ammonium chloride had an inhibitory effect. Potato starch was the superior and most reliable carbon source tested. Additionally, we observed that the level of sporulation was correlated with the production of MTs but not with that of STLAC. In this study, we provide a chemically well-defined cultivation medium suitable for standardized in vitro testing of the capacity of S. chartarum isolates to produce macrocyclic trichothecenes. IMPORTANCE Macrocyclic trichothecenes (MTs) are highly toxic secondary metabolites that are produced by certain Stachybotrys chartarum strains, which consequently pose a risk for animals and humans. To identify hazardous, toxin-producing strains by analytical means, it is important to grow them under conditions that support MT production. Nutrients determine growth and development and thus the synthesis of secondary metabolites. Complex rich media are commonly used for diagnostics, but batch differences of supplements pose a risk for inconsistent data. We have established a chemically defined medium for S. chartarum and used it to analyze the impact of nitrogen and carbon sources. A key finding is that nitrate stimulates MT production, whereas ammonium suppresses it. Defining nutrients that support MT production will enable a more reliable identification of hazardous S. chartarum isolates. The new medium will also be instrumental in analyzing the biosynthetic pathways and regulatory mechanisms that control mycotoxin production in S. chartarum.

Keywords: LC-MS/MS; Stachybotrys; carbon source; macrocyclic trichothecenes; nitrogen source; nutrients; stachybotrylactam.

FIG 1

Colonies of S. chartarum genotype S strain ATCC 34916 were grown as three-point cultures on AMM containing glucose (10 g/L) as the sole carbon source. The different panels show cultures without nitrogen addition (a) or supplemented with NaNO₃ (b1 to b7), NH₄NO₃ (c1 to c7), or NH₄Cl (d1 to d7) at the following nitrogen concentrations: 1 mg/L (b1/c1/d1), 5 mg/L (b2/c2/d2), 10 mg/L (b3/c3/d3), 25 mg/L (b4/c4/d4), 50 mg/L (b5/c5/d5), 250 mg/L (b6/c6/d6), and 500 mg/L (b7/c7/d7). Each image is representative of three parallel cultures per condition.

FIG 2

Colonies of S. chartarum genotype S strain ATCC 34916 were grown as three-point cultures on AMM containing fructose (1), maltose (2), potato starch (3), wheat starch (4), or cellulose (5) as the sole carbon source (with concentrations that were normalized to 4 g C/L) and combined with the nitrogen sources NaNO₃ (1a1 to 5a1 and 1a2 to 5a2), NH₄NO₃ (1b1 to 5b1 and 1b2 to 5b2), or NH₄Cl (1c1 to 5c1 and 1c2 to 5c2), each at two concentrations (25 and 250 mg N/L). Each image is representative of three parallel cultures per condition.

FIG 3

Colonies of S. chartarum genotype S strains ATCC 34916 (1), IBT 40293 (2), and DSM 114129 (3) were grown as three-point cultures on AMM containing glucose (1a to 3a), fructose (1b to 3b), maltose (1c to 3c), potato starch (1d to 3d), wheat starch (1e to 3e), or cellulose (1f to 3f) as the carbon source (with concentrations that were normalized to 4 g C/L) and NaNO₃ as the nitrogen source (250 mg N/L). Each image is representative of three parallel cultures per condition.

FIG 4

Accumulated concentrations of the macrocyclic trichothecenes roridin E, roridin L-2, verrucarin J, satratoxin G, satratoxin H, and satratoxin F measured for cultures of S. chartarum genotype S strains ATCC 34916, IBT 40293, and DSM 114129 grown on AMM supplemented with NaNO₃, NH₄NO₃, or NH₄Cl as the nitrogen source at concentrations of 1, 25, or 250 mg N/L. In this figure, the concentrations are normalized to the area of the respective colonies (ng/cm²). For representative images of these corresponding cultures, compare Fig. 1 and Fig. S1 in the supplemental material.

FIG 5

Concentrations of the macrocyclic trichothecenes roridin E, roridin L-2, verrucarin J, satratoxin G, satratoxin H, and satratoxin F measured for cultures of S. chartarum genotype S strains ATCC 34916 (red columns), IBT 40293 (green columns), and DSM 114129 (blue columns) grown on AMM supplemented with NaNO₃, NH₄NO₃, or NH₄Cl at concentrations of 1, 25, or 250 mg N/L. The concentration values are normalized to the area of the respective colonies (ng/cm²).

FIG 6

Accumulated concentrations of the macrocyclic trichothecenes roridin E, roridin L-2, verrucarin J, satratoxin G, satratoxin H, and satratoxin F measured for cultures of S. chartarum genotype S strain ATCC 34916 grown on AMM containing glucose, fructose, maltose, potato starch, wheat starch, or cellulose as the sole carbon source (with concentrations that were normalized to 4 g C/L) and combined with the nitrogen sources NaNO₃, NH₄NO₃, or NH₄Cl (either 25 or 250 mg N/L). The MT concentrations are normalized to the area of the respective colonies (ng/cm²). For representative images of these corresponding cultures, compare Fig. 1 and 2.

FIG 7

Accumulated concentrations of the macrocyclic trichothecenes roridin E, roridin L-2, verrucarin J, satratoxin G, satratoxin H, and satratoxin F measured for cultures of S. chartarum genotype S strains ATCC 34916, IBT 40293, and DSM 114129 grown on AMM containing glucose, fructose, maltose, potato starch, wheat starch, or cellulose as the carbon source (with concentrations that were normalized to 4 g C/L) and NaNO₃ as the nitrogen source (with 250 mg N/L). The data are normalized to the area of the respective colonies (ng/cm²). For representative images of these corresponding cultures, compare with Fig. 3.

FIG 8

Stachybotrylactam concentrations measured for cultures of S. chartarum genotype S strains ATCC 34916, IBT 40293, and DSM 114129 grown on AMM supplemented with NaNO₃, NH₄NO₃, or NH₄Cl as the nitrogen source at concentrations of 1, 25, or 250 mg N/L. The data are normalized to the area of the respective colonies (ng/cm²). For representative images of these corresponding cultures, compare Fig. 1 and Fig. S1 in the supplemental material.

FIG 9

Stachybotrylactam concentrations measured for cultures of S. chartarum genotype S strain ATCC 34916 grown on AMM containing glucose, fructose, maltose, potato starch, wheat starch, or cellulose as the sole carbon source (with concentrations that were normalized to 4 g C/L) and combined with the nitrogen sources NaNO₃, NH₄NO₃, or NH₄Cl (either 25 or 250 mg N/L). The data are normalized to the area of the respective colonies (ng/cm²). For representative images of these corresponding cultures, compare Fig. 1 and 2.

FIG 10

Stachybotrylactam concentrations measured for cultures of S. chartarum genotype S strains ATCC 34916, IBT 40293, and DSM 114129 grown on AMM containing glucose, fructose, maltose, potato starch, wheat starch, or cellulose as the carbon source (with concentrations that were normalized to 4 g C/L) and NaNO₃ as the nitrogen source (with 250 mg N/L). The data are normalized to the area of the respective colonies (ng/cm²). For representative images of these corresponding cultures, compare Fig. 3.