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A new peptaibol, RK-026A (1) was isolated from a fungus, Trichoderma sp. RK10-F026, along with atroviridin B (2), alamethicin II (3), and polysporin B (4) as a cytotoxic compound, which was selected by principal component analysis of the MS data from 5 different culture conditions. The structure of 1 was determined as a new atroviridin B derivative containing Glu at the 18th residue instead of Gln by NMR and HR-MS analyses including the investigation of detailed MS/MS fragmentations. 1 showed cytotoxicity toward K562 leukemia cells at an IC50 value of 4.1 µm., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Trichoderma species are widely used as biofungicides for the control of fungal plant pathogens. Several studies have been performed to identify the main genes and compounds involved in Trichoderma -plant-microbial pathogen cross-talks. However, there is not much information about the exact mechanism of this profitable interaction. Peptaibols secreted mainly by Trichoderma species are linear, 5-20 amino acid residue long, non-ribosomally synthesized peptides rich in α-amino isobutyric acid, which seem to be effective in Trichoderma -plant pathogenic fungus interactions. In the present study, reversed phase (RP) high-performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) mass spectrometry (MS) was used to detect peptaibol profiles of Trichoderma strains during interactions with fungal plant pathogens. MS investigations of the crude extracts deriving from in vitro confrontations of Trichoderma asperellum and T. longibrachiatum with different plant pathogenic fungi ( Fusarium moniliforme , F. culmorum , F. graminearum , F. oxysporum species complex, Alternaria solani and Rhizoctonia solani ) were performed to get a better insight into the role of these non-ribosomal antimicrobial peptides. The results revealed an increase in the total amount of peptaibols produced during the interactions, as well as some differences in the peptaibol profiles between the confrontational and control tests. Detection of the expression level of the peptaibol synthetase tex1 by qRT-PCR showed a significant increase in T. asperellum /R. solani interaction in comparison to the control. In conclusion, the interaction with plant pathogens highly influenced the peptaibol production of the examined Trichoderma strains., Competing Interests: The authors declare no conflict of interest.

Aims: The production of peptaibols, toxic secondary metabolites of Trichoderma, in the indoor environment is not well-documented. Here, we investigated the toxicity of peptaibols in the guttation droplets and biomass of Trichoderma strains isolated from problematic buildings., Methods and Results: Seven indoor-isolated strains of T. atroviride, T. trixiae, T. paraviridescens and T. citrinoviride were cultivated on malt extract agar, gypsum boards and paperboards. Their biomass extracts and guttation droplets were highly cytotoxic in resting and motile boar sperm cell assays and in inhibition of somatic cell proliferation assays. The toxins were identified with HPLC/ESI-MS/MS as trichorzianines, trilongins, trichostrigocins and trichostrigocin-like peptaibols. They exhibited toxicity profiles similar to the reference peptaibols alamethicin, trilongins, and trichorzianine TA IIIc purified from T. atroviride H1/226. Particular Trichoderma strains emitted the same peptaibols in both their biomasses and exudate droplets. The trilongin-producing T. citrinoviride SJ40 strain grew at 37°C., Conclusions: To our knowledge, this is the first report of indoor-isolated Trichoderma strains producing toxic peptaibols in their guttation droplets., Significance and Impact of the Study: 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., (© 2018 The Society for Applied Microbiology.)

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., (© 2017 Wiley-VHCA AG, Zurich, Switzerland.)

A cork oak endophytic strain of Trichoderma citrinoviride, previously selected for its antagonistic potential against various fungal pathogens involved in oak decline, was screened for the production of bioactive secondary metabolites. From liquid culture a mixture of polypeptide antibiotics (peptaibols) belonging to the paracelsin family was isolated and characterized. This peptide mixture was purified by column chromatography and preparative TLC on silica gel, and separated by analytical HPLC. It was analysed by MALDI-TOF MS and nano-ESI-QTOF MS. Tandem mass experiments were performed to determine the amino acid sequences based on the fragmentation pattern of selected parent ions. The mixture comprised 20-residue peptides with C-terminal phenylalaninol and N-terminal acetylation. Twenty-eight amino acid sequences were identified, and amino acid exchanges were located in positions 6, 9, 12 and 17. Among them, seven sequences are new as compared to those reported in the database specifically for peptaibols and in the literature. In addition, we obtained experimental evidence suggesting the existence of non-covalent dimeric forms (homo- and hetero-) of the various peptaibol species. The peptide mixture showed strong antifungal activity toward seven important forest tree pathogens, and it was highly toxic in an Artemia salina (brine shrimp) bioassay. These results emphasize the cryptic role of endophytic fungi as a source of novel bioactive natural products and biocontrol agents.

A novel mycotoxin named acrebol, consisting of two closely similar peptaibols (1726 and 1740 Da), was isolated from an indoor strain of the mitosporic ascomycete fungus Acremonium exuviarum. This paper describes the unique mitochondrial toxicity of acrebol, not earlier described for any peptaibol. Acrebol inhibited complex III of the respiratory chain of isolated rat liver mitochondria (1 mg of protein mL(-1)) with an IC(50) of approximately 80 ng mL(-1) (50 nM) after a short preincubation, and 350 ng mL(-1) caused immediate and complete inhibition. Acrebol thus is a complex III inhibitor almost as potent as antimycin A and myxothiazol but completely different in structure. Similarly to myxothiazol but in contrast to antimycin A, acrebol decreased the level of mitochondrial superoxide anion detectable by chemiluminescent probe 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one. Unlike other peptaibols, acrebol in toxic concentrations did not increase the ionic and solute permeability of membranes of isolated rat liver mitochondria, did not induce disturbance of the ionic homeostasis or the osmotic balance of mitochondria, and did not release apoptogenic proteins like cytochrome c from the intermembrane space of mitochondria. In boar spermatozoa, acrebol inhibited the respiratory chain and caused ATP depletion by activation of the oligomycin-sensitive F(0)F(1)-ATPase, which resulted in the inhibition of the progressive movement. In mouse insulinoma MIN-6 cells, whose energy supply solely depends on oxidative phosphorylation, acrebol induced necrosis-like death. The pathophysiological relevance of these findings is discussed.