This report proves that indoor isolates of Trichoderma release peptaibols in their guttation droplets. The presence of toxins in these types of exudates may serve as a mechanism of aerosol formation for nonvolatile toxins in the indoor air.
This study examined the structural diversity and bioactivity of peptaibol compounds produced by species from the phylogenetically separated Longibrachiatum Clade of the filamentous fungal genus Trichoderma , which contains several biotechnologically, agriculturally and clinically important species. HPLC-ESI-MS investigations of crude extracts from 17 species of the Longibrachiatum Clade ( T. aethiopicum, T. andinense, T. capillare, T. citrinoviride, T. effusum, T. flagellatum, T. ghanense, T. konilangbra, T. longibrachiatum, T. novae-zelandiae, T. pinnatum, T . parareesei, T. pseudokoningii, T. reesei, T. saturnisporum, T. sinensis , and T. orientale ) revealed several new and recurrent 20-residue peptaibols related to trichobrachins, paracelsins, suzukacillins, saturnisporins, trichoaureocins, trichocellins, longibrachins, hyporientalins, trichokonins, trilongins, metanicins, trichosporins, gliodeliquescins, alamethicins and hypophellins, as well as eight 19-residue sequences from a new subfamily of peptaibols named brevicelsins. Non-ribosomal peptide synthetase genes were mined from the available genome sequences of the Longibrachiatum Clade. Their annotation and product prediction were performed in silico and revealed full agreement in 11 out of 20 positions regarding the amino acids predicted based on the signature sequences and the detected amino acids incorporated. Molecular dynamics simulations were performed for structural characterization of four selected peptaibol sequences: paracelsins B, H and their 19-residue counterparts brevicelsins I and IV. Loss of position R6 in brevicelsins resulted in smaller helical structures with higher atomic fluctuation for every residue than the structures formed by paracelsins. We observed the formation of highly bent, almost hairpin-like, helical structures throughout the trajectory, along with linear conformation. Bioactivity tests were performed on the purified peptaibol extract of T . reesei on clinically and phytopathologically important filamentous fungi, mammalian cells, and Arabidopsis thaliana seedlings. Porcine kidney cells and boar spermatozoa proved to be sensitive to the purified peptaibol extract. Peptaibol concentrations ≥0.3 mg ml −1 deterred the growth of A . thaliana . However, negative effects to plants were not detected at concentrations below 0.1 mg ml −1 , which could still inhibit plant pathogenic filamentous fungi, suggesting that those peptaibols reported here may have applications for plant protection.
AimsEmission of toxic metabolites in guttation droplets of common indoor fungi is not well documented. The aims of this study were (i) to compare mycotoxins in biomass and guttation droplets from indoor fungi from a building following health complaints among occupants, (ii) to identify the most toxic strain and to test if mycotoxins in guttation liquids migrated trough air and (iii) to test if toxigenic Penicillium expansum strains grew on gypsum board.Methods and ResultsBiomass suspensions and guttation droplets from individual fungal colonies representing Aspergillus, Chaetomium, Penicillium, Stachybotrys and Paecilomyces were screened toxic to mammalian cells. The most toxic strain, RcP61 (CBS 145620), was identified as Pen. expansum Link by sequence analysis of the ITS region and a calmodulin gene fragment, and confirmed by the Westerdijk Institute based on ITS and beta‐tubulin sequences. The strain was isolated from a cork liner, was able to grow on gypsum board and to produce toxic substances in biomass extracts and guttation droplets inhibiting proliferation of somatic cells (PK‐15, MNA, FL) in up to 20 000‐fold dilutions. Toxic compounds in biomass extracts and/or guttation droplets were determined by HPLC and LC‐MS. Strain RcP61 produced communesins A, B and D, and chaetoglobosins in guttation droplets (the liquid emitted from them) and biomass extracts. The toxins of the guttation droplets migrated c. 1 cm through air and condensed on a cool surface.ConclusionsThe mycotoxin‐containing guttation liquids emitted by Pen. expansum grown on laboratory medium exhibited airborne migration and were >100 times more toxic in bioassays than guttation droplets produced by indoor isolates of the genera Aspergillus, Chaetomium, Stachybotrys and Paecilomyces. Significance and Impact of the StudyToxic exudates produced by Pen. expansum containing communesins A, B and D, and chaetoglobosins were transferable by air. This may represent a novel mechanism of mycotoxin dispersal in indoor environment.
A novel, objective, and rapid computed motility inhibition (CMI) assay was developed to identify and assess sublethal injury in toxin-exposed boar spermatozoa and compared with a subjective visual motility inhibition (VMI) assay. The CMI values were calculated from digital micrographic videos using a custom MATLAB® script by contrasting the motility index values of each experiment with those of the background and control experiments. Following a comparison of the CMI and VMI assays results, it was determined that their agreement depended on the shape of the dose-response curve. Toxins that exhibited a steep slope were indicative of good agreement between the assays. Those depicted by a gentle decline in the slope of the dose-response curve, the CMI assay were shown to be two times more sensitive than the VMI assay. The CMI assay was highly sensitive to the inhibition of mitochondrial function and glucose transport activity by sublethal doses of toxins and to disruption of cellular cation homeostasis by carrier ionophoric toxins, when compared to the cytotoxicity and lethal toxicity assays (i.e., that evaluated the inhibition of cell proliferation in somatic cell lines (FL, PK-15, and MNA cells)) and disruption to spermatozoa membrane integrity. The CMI assay recognized subtle sublethal toxicity changes in metabolism, manifested as a decrease in boar spermatozoa motility. Thus, it was feasible to effectively compare the objectively-measured numerical values for motility inhibition using the CMI assay against those reflecting lethal damage in the spermatozoa cells and somatic cell lines using a cytotoxicity assay.
Certain Trichoderma species are causing serious losses in mushroom production worldwide. Trichoderma aggressivum and Trichoderma pleuroti are among the major causal agents of the green mould diseases affecting Agaricus bisporus and Pleurotus ostreatus, respectively. The genus Trichoderma is well-known for the production of bioactive secondary metabolites, including peptaibols, which are short, linear peptides containing unusual amino acid residues and being synthesised via non-ribosomal peptide synthetases (NRPSs). The aim of this study was to get more insight into the peptaibol production of T. aggressivum and T. pleuroti. HPLC/MS-based methods revealed the production of peptaibols closely related to hypomurocins B by T. aggressivum, while tripleurins representing a new group of 18-residue peptaibols were identified in T. pleuroti. Putative NRPS genes enabling the biosynthesis of the detected peptaibols could be found in the genomes of both Trichoderma species. In vitro experiments revealed that peptaibols are potential growth inhibitors of mushroom mycelia, and that the host mushrooms may have an influence on the peptaibol profiles of green mould agents.
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