Thomas Degenkolb

The Trichoderma brevicompactum clade: a separate lineage with new species, new peptaibiotics, and mycotoxins

The Brevicompactum clade is recognized as a separate lineage in Trichoderma/Hypocrea. This includes T. brevicompactum and the new species T. arundinaceum, T. turrialbense, T. protrudens and Hypocrea rodmanii. The closest relative of the Brevicompactum clade is the Lutea clade. With the exception of H. rodmanii, all members of this clade produce the simple trichothecene-type toxins harzianum A or trichodermin. All members of the clade produce peptaibiotics, including alamethicins. Strains previously reported as T. harzianum (ATCC 90237), T. viride (NRRL 3199) or Hypocrea sp. (F000527, CBS 113214) to produce trichothecenes are reidentified as T. arundinaceum. The Brevicompactum clade is not closely related to species that have biological application.

Recent advances and future prospects in peptaibiotics, hydrophobin, and mycotoxin research, and their importance for chemotaxonomy of Trichoderma and Hypocrea.

Fungi of the genus Trichoderma with teleomorphs in Hypocrea are abundant producers of a group of amphiphilic, non‐ribosomal peptide antibiotics, which are rich in the non‐proteinogenic amino acid Aib (α‐aminoisobutyric acid). They are referred to as peptaibiotics, or peptaibols, if a 1,2‐amino alcohol is present at the C‐terminus. Trichoderma/Hypocrea, like other ascomycetous fungi, also produce hydrophobins, a class of small, cysteine‐rich proteins. Advanced soft ionization mass spectrometric techniques such as LC‐CID‐MS, LC‐ESI‐MSn, and IC‐MALDI‐TOF‐MS enabled the high‐throughput analysis, simultaneous detection and sequence determination of peptaibiotics and hydrophobins from minute quantities of fungal materials. Some Trichoderma species have been recognized to produce peptaibiotics as well as simple mycotoxins of the trichothecene group. The combination of sequence data of both groups of peptides with the pattern of low‐molecular‐weight secondary metabolites, including trichothecene‐type mycotoxins, independently confirmed the results of morphological, molecular, and phylogenetic analyses. This approach established a new lineage in Trichoderma/Hypocrea, the Brevicompactum clade, comprising four new and one redescribed species. Notably, commercial preparations of single or mixed cultures of Trichoderma species, in particular T. harzianum, and T. koningii, are registered as biocontrol agents for soil and plant pathogens. In this context, it is emphasized that the four mycotoxin‐producing species of the recently established Brevicompactum clade (T. brevicompactum, T. arundinaceum, T. turrialbense, and T. protrudens) are not closely related to any of the Trichoderma species currently used as biocontrol agents. Furthermore, possible health concerns about release of peptaibiotics in the biosphere are discussed with respect to their bioactivities and their use as drugs in human and veterinary medicine. Finally, future prospects regarding novel bioactivities and further research needs, including interdisciplinary taxonomic approaches, are outlined.

Peptaibiomics: Towards a Myriad of Bioactive Peptides Containing Cα-Dialkylamino Acids?

Fungi are generally regarded as a literally infinite resource of bioactive secondary metabolites displaying remarkable structural diversity. Research on a particular group of linear and cyclic peptide antibiotics comprehensively named peptaibiotics – as result of the abundance of the non‐proteinogenic Cα‐dialkylated α‐amino acids α‐aminoisobutyric acid (Aib) and isovaline (Iva) – has been started 50 years ago. These peptides have gained constantly increasing interest because of their unique bioactivities and conformations. This review, reflecting the history of peptaibiotic research from 1958 to 2008, is focussed on introducing both the structural diversity and natural microheterogeneity of the peptaibiotics, as well as the biodiversity of their fungal producers. Recently introduced state‐of‐the‐art methods for rapid screening and sequencing of peptaibiotics, such as peptaibiomics and intact‐cell MALDI‐TOF mass spectrometry, are discussed. Finally, future prospects in peptaibiotic research are presented. Owing to the ubiquity and biodiversity of the fungal producers in the biosphere, the discovery of a myriad of peptaibiotics within the next decade is predicted.

Aib and iva in the biosphere: neither rare nor necessarily extraterrestrial.

Fourty-nine species and strains of filamentous fungi of the genera Acremonium, Bionectria, Clonostachys, Emericellopsis, Hypocrea/Trichoderma, Lecythophora, Monocillium, Nectriopsis, Niesslia, Tolypocladium, and Wardomyces, deposited with the culture collection of the Centraalbureau voor Schimmelcultures (CBS) in Utrecht, The Netherlands, were grown on nutrient agar plates. Organic extracts of mycelia were analyzed after acidic total hydrolysis and derivatization by GC/SIM-MS on Chirasil-L-Val for the presence of Aib (=alpha-aminoisobutyric acid, 2-methylalanine) and DL-Iva (=isovaline, 2-ethylalanine). In 37 of the hydrolysates, Aib was detected, and in several of them D-Iva or mixtures of D- and L-Iva. Non-proteinogenic Aib, in particular, is a highly specific marker for a distinctive group of fungal polypeptides named peptaibols or, comprehensively, peptaibiotics, i.e., peptides containing Aib and displaying (anti)biotic activities. The biotic synthesis of these amino acids by filamentous fungi contradicts the still widespread belief that alpha,alpha-dialkyl-alpha-amino acids do not or rarely occur in the biosphere and, if detected, are of extraterrestrial origin. The abundant production of peptaibiotics by cosmopolitan species of microfungi has also to be considered in the discussion on the occurrence of Aib and Iva in ancient and recent sediments. The detection of trace amounts of Aib in ice samples of Antarctica that are devoid of meteorites might also be related to the presence of Aib-producing microorganisms, being either indigenous psychrophiles, or being transported and localized by mechanisms related to bioaerosols and cryoconites. The presence of microfungi being capable of producing alpha,alpha-dialkyl alpha-amino acids in terrestrial samples, and possible contamination of extraterrestrial materials are pointed out to be of relevance for the reliable interpretation of cosmogeochemical data.

The Comprehensive Peptaibiotics Database

Peptaibiotics are nonribosomally biosynthesized peptides, which – according to definition – contain the marker amino acid α‐aminoisobutyric acid (Aib) and possess antibiotic properties. Being known since 1958, a constantly increasing number of peptaibiotics have been described and investigated with a particular emphasis on hypocrealean fungi. Starting from the existing online ‘Peptaibol Database’, first published in 1997, an exhaustive literature survey of all known peptaibiotics was carried out and resulted in a list of 1043 peptaibiotics. The gathered information was compiled and used to create the new ‘The Comprehensive Peptaibiotics Database’, which is presented here. The database was devised as a software tool based on Microsoft (MS) Access. It is freely available from the internet at http://peptaibiotics‐database.boku.ac.at and can easily be installed and operated on any computer offering a Windows XP/7 environment. It provides useful information on characteristic properties of the peptaibiotics included such as peptide category, group name of the microheterogeneous mixture to which the peptide belongs, amino acid sequence, sequence length, producing fungus, peptide subfamily, molecular formula, and monoisotopic mass. All these characteristics can be used and combined for automated search within the database, which makes The Comprehensive Peptaibiotics Database a versatile tool for the retrieval of valuable information about peptaibiotics. Sequence data have been considered as to December 14, 2012.

Secondary Metabolites Released by The Burying Beetle Nicrophorus vespilloides : Chemical Analyses and Possible Ecological Functions

Burying beetles of the genus Nicrophorus localize small vertebrate carcasses by emitted volatiles. The carcass that serves as reproduction and breeding site is buried in the soil by the beetles. Biparental care for offspring includes both preservation of the carrion and its preparation as diet and nursery. Buried carcasses show no signs of microbial decay, and those experimentally treated with Nicrophorus secretions are known to grow fewer bacteria and fungi. In order to investigate the chemical composition of these secretions, we used GC-MS for analysis of methanolic extracts of anal and oral secretions released by adult N. vespilloides. Furthermore, we analyzed the headspace of adult N. vespilloides by SPME-GC-MS and searched for compounds with known antimicrobial activity. We identified 34 compounds in the headspace, and anal and oral secretions, 26 of which occurred consistently. We discuss the ecological relevance of these compounds with respect to both their antimicrobial activity and ecological relevance.

Systematics of the Trichoderma harzianum species complex and the re-identification of commercial biocontrol strains

Trichoderma harzianum is known as a cosmopolitan, ubiquitous species associated with a wide variety of substrates. It is possibly the most commonly used name in agricultural applications involving Trichoderma, including biological control of plant diseases. While various studies have suggested that T. harzianum is a species complex, only a few cryptic species are named. In the present study the taxonomy of the T. harzianum species complex is revised to include at least 14 species. Previously named species included in the complex are T. guizhouense, T. harzianum, and T. inhamatum. Two new combinations are proposed, T. lentiforme and T. lixii. Nine species are described as new, T. afarasin, T. afroharzianum, T. atrobrunneum, T. camerunense, T. endophyticum, T. neotropicale, T. pyramidale, T. rifaii and T. simmonsii. We isolated Trichoderma cultures from four commercial biocontrol products reported to contain T. harzianum. None of the biocontrol strains were identified as T. harzianum s. str. In addition, the widely applied culture ‘T. harzianum T22’ was determined to be T. afroharzianum. Some species in the T. harzianum complex appear to be exclusively endophytic, while others were only isolated from soil. Sexual states are rare. Descriptions and illustrations are provided. A secondary barcode, nuc translation elongation factor 1-α (TEF1) is needed to identify species in this complex.

The Production of Multiple Small Peptaibol Families by Single 14‐Module Peptide Synthetases in Trichoderma/Hypocrea

The most common sequences of peptaibiotics are 11‐residue peptaibols found widely distributed in the genus Trichoderma/Hypocrea. Frequently associated are 14‐residue peptaibols sharing partial sequence identity. Genome sequencing projects of three Trichoderma strains of the major clades reveal the presence of up to three types of nonribosomal peptide synthetases with 7, 14, or 18–20 amino acid‐adding modules. Here, we provide evidence that the 14‐module NRPS type found in T. virens, T. reesei (teleomorph Hypocrea jecorina), and T. atroviride produces both 11‐ and 14‐residue peptaibols based on the disruption of the respective NRPS gene of T. reesei, and bioinformatic analysis of their amino acid‐activating domains and modules. The sequences of these peptides may be predicted from the gene sequences and have been confirmed by analysis of families of 11‐ and 14‐residue peptaibols from the strain 618, termed hypojecorins A (23 sequences determined, 4 new) and B (3 sequences determined, 2 new), and the recently established trichovirins A from T. virens. The distribution of 11‐ and 14‐residue products is strain‐specific and depends on growth conditions as well. Possible mechanisms of module skipping are discussed.

Harmonine, a defence compound from the harlequin ladybird, inhibits mycobacterial growth and demonstrates multi-stage antimalarial activity

The harlequin ladybird beetle Harmonia axyridis has been introduced in many countries as a biological control agent, but has become an invasive species threatening the biodiversity of native ladybirds. Its invasive success has been attributed to its vigorous resistance against diverse pathogens. This study demonstrates that harmonine ((17R,9Z)-1,17-diaminooctadec-9-ene), which is present in H. axyridis haemolymph, displays broad-spectrum antimicrobial activity that includes human pathogens. Antibacterial activity is most pronounced against fast-growing mycobacteria and Mycobacterium tuberculosis, and the growth of both chloroquine-sensitive and -resistant Plasmodium falciparum strains is inhibited. Harmonine displays gametocytocidal activity, and inhibits the exflagellation of microgametocytes and zygote formation. In an Anopheles stephensi mosquito feeding model, harmonine displays transmission-blocking activity.

Natural cyclopeptaibiotics and related cyclic tetrapeptides: structural diversity and future prospects.

Linearity is not considered a prerequisite anymore, and extension of the current definition of ‘peptaibiotics’ to cyclic, Aib‐containing peptides is proposed. Sequences and bioactivities, together with ecophysiological importance of cyclopeptaibiotics and related cyclic tetrapeptides, and their fungal‐taxonomic relationships, are discussed.