Introduction

Currently, the fungal genus Trichoderma/Hypocrea Footnote 1 comprises more than 200 validly described species, which have been recognised by molecular phylogenetic analysis (Atanasova et al. 2013). This high taxonomic diversity in Trichoderma/Hypocrea is not only reflected in a permanently increasing number of species (Jaklitsch 2009, 2011; Jaklitsch and Voglmayr 2012; Jaklitsch et al. 2012, 2013; Chaverri et al. 2011; Samuels and Ismaiel 2011, Samuels et al. 2012a,b; Kim et al. 2012, 2013; Yamaguchi et al. 2012; Li et al. 2013; López-Quintero et al. 2013, Yabuki et al. 2014), but also in a fast-growing number of secondary metabolites of remarkable structural diversity. The latter include low-molecular-weight compounds such as pyrones (Jeleń et al. 2013), butenolides, terpenes, and steroids, but also N-heterocyclic compounds and isocyanides. In addition to these relatively nonpolar and often partly volatile compounds, an impressive inventory of non-volatile compounds, comprising some alkaloids and an imposing number of peptide antibiotics, is produced. Reino et al. (2008) reviewed 186 compounds; however, peptaibiotics (see below) were treated only marginally and incomprehensively. As of August 2013, a total of 501 entries are recorded for Trichoderma (461) and Hypocrea (40) in AntiBase, more than 300 of which are N-containing, including less than 100 in the range of 50–800 Da (Laatsch 2013).

Considering recent publications in this field, which have not yet been included into AntiBase 2013 (Table 1), an estimate of 225 to 250 non-peptaibiotic secondary metabolites from Trichoderma/Hypocrea seems appropriate. However, the overwhelming majority of secondary metabolites obtained from this genus so far belong to a perpetually growing family of non-ribosomally biosynthesised, linear or, in a few cases, cyclic peptide antibiotics of exclusively fungal origin, comprehensively named peptaibiotics:

Table 1 Recently described, non-peptaibiotic secondary metabolites from Trichoderma/Hypocrea species not yet listed in AntiBase 2013
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According to the definition, the members of this peptide family show, besides proteinogenic amino acids, i) a relatively high content of the marker α-aminoisobutyric acid (Aib), which is often accompanied by other α,α-dialkyl α-amino acids such as D- and/or L-isovaline (Iva) or, occasionally, α-ethylnorvaline (EtNva), or 1-aminocyclopropane-1-carboxylic acid (Acc); ii) have a molecular weight between 500 and 2,100 Da, thus containing 4–21 residues; iii) are characterised by the presence of other non-proteinogenic amino acids and/or lipoamino acids; iv) possess an acylated N-terminus, and v) in the case of linear peptides, have a C-terminal residue that most frequently consists of an amide-bonded β-amino alcohol, thus defining the largest subfamily of peptaibiotics, named peptaibols. Alternatively, the C-terminus might also be a polyamine, amide, free amino acid, 2,5-diketopiperazine, or a sugar alcohol (Degenkolb and Brückner 2008; Stoppacher et al. 2013).

Of the approximately 1,250 to 1,300 individual sequences of peptaibiotics known as of autumn 2013 (Ayers et al. 2012; Carroux et al. 2013; Figueroa et al. 2013; Kimonyo and Brückner 2013; Röhrich et al. 2012; Röhrich et al. 2013a, b; Chen et al. 2013; Panizel et al. 2013; Ren et al. 2013; Stoppacher et al. 2013), about 950 have been obtained from Trichoderma/Hypocrea species, thus confirming the genus as the most prolific source of this group of non-ribosomal peptide antibiotics (Brückner et al. 1991; Degenkolb and Brückner 2008; Brückner et al. 2009).

Both the taxonomic and metabolic diversity of Trichoderma/Hypocrea are hypothesised to originate from mycoparasitism or hyperparasitism, which may represent the ancestral life style of this genus (Kubicek et al. 2011). The unique bioactivities of peptaibiotics, resulting from their amphipathicity and helicity, make them ideal candidates to support the parasitic life style of their fungal producers:

Under in vitro-conditions, the parallel formation of peptaibiotics such as the 19-residue trichorzianinsFootnote 2 and of hydrolytic enzymes, above all chitinases and β-1,3-glucanases (Schirmböck et al. 1994), could be demonstrated. This observation led to a widely accepted model describing the synergistic interaction of peptaibiotics and hydrolases in the course of mycoparasitism of Trichoderma atroviride towards Botrytis cinerea (Lorito et al. 1996). Despite this, reports on in vivo-detection of peptaibiotics have scarcely been published in the past. Examples include the isolation of hypelcins A and B obtained from ca. 2 kg of dried, crushed stromata of the mycoparasite Hypocrea peltata (Fujita et al. 1984; Matsuura et al. 1993, 1994)Footnote 3 as well as the detection of antiamoebins in herbivore dung, which have been produced by the coprophilous Stilbella fimetaria (syn. S. erythrocephala) (Lehr et al. 2006).

In order to close this gap, we initiated a screening project aimed at resolving the question as to whether peptaibiotic production in vivo is a common adaptation strategy of Trichoderma/Hypocrea species for colonising and defending ecological niches:

Several Hypocrea specimens were freshly collected in the natural habitat and analysed for the presence of peptaibiotics. Sequences of peptaibiotics found were independently confirmed by analysing the peptaibiomeFootnote 4 of pure agar cultures obtained by single-ascospore isolation from the specimens. Using liquid chromatography coupled to electrospray high resolution mass spectrometry we succeeded in detecting 28 peptaibiotics from the polyporicolous Hypocrea pulvinata (Röhrich et al. 2012). Another 49 peptaibiotics were sequenced in Hypocrea phellinicola, a parasite of Phellinus sp., especially Ph. ferruginosus (Röhrich et al. 2013a).

Due to these encouraging results, our screening programme was extended to another nine specimens belonging to seven hitherto uninvestigated mycoparasitic or saprotrophic Trichoderma/Hypocrea species, respectively (Table 2).

Table 2 Habitat and geographic distribution of Hypocrea species included in this study
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Materials and methods

Specimens of Hypocrea teleomorphs were collected from four different locations in Austria (Table 3). Pure agar cultures were obtained by single-ascospore isolations from the respective, freshly collected specimens as previously described by Jaklitsch (2009):

Table 3 Habitat and geographic origin of Hypocrea isolates included in this study
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Parts of stromata were crushed in sterile distilled water. The resulting suspension was transferred to cornmeal agar plates (Sigma, St. Louis, Missouri) supplemented with 2 % (w/v) D(+)-glucose-monohydrate (CMD), and 1 % (v/v) of an aqueous solution of 0.2 % (w/v) streptomycin sulfate (Sigma) and 0.2 % (w/v) neomycin sulfate (Sigma). Plates were incubated overnight at 25 °C. In order to exclude possible contamination by spores of other fungal species, few germinated ascospores from within an ascus were transferred to fresh plates of CMD using a thin platinum wire. The plates were sealed with Parafilm (Pechiney, Chicago, Illinois) and incubated at 25 °C. As all species listed in Table 2 could unambiguously be identified by their morphological and growth characteristics (Jaklitsch 2009, 2011), no molecular phylogenetic analyses needed to be performed.

Detailed descriptions of chemicals, extraction and work-up procedures for specimens and agar plate cultures, cultivation methods, as well as comprehensive protocols for HPLC/QTOF-ESI-HRMS were given by Röhrich et al. (2012, 2013a). For routine screening, a high-resolution micrOTOF Q-II mass spectrometer with orthogonal ESI source (Bruker Daltonic, Bremen, Germany), coupled to an UltiMate 3000 HPLC (Dionex, Idstein, Germany), was used. Samples, which have been screened negative with the above HPLC/MS system, were re-examined using a maXis 3G QTOF mass spectrometer with orthogonal ESI source (Bruker Daltonic, Bremen, Germany), coupled to an UltiMate 3000 UHPLC (Dionex, Idstein, Germany) as previously described (Röhrich et al. 2012, 2013a).

Results and discussion

General considerations. All strains investigated in this study represent phylogenetically well-defined species (Tables 2 and 3). This is in contrast to most of the reports published until the end of the 1990s, when peptaibiotic production by the genus Trichoderma/Hypocrea was − according to Rifai’s classification (1969) − mostly attributed to one of the four common species T. viride, T. koningii, T. harzianum, T. longibrachiatum, and sometimes to T. pseudokoningii and T. aureoviride. Careful inspection of the literature published prior to the turn of the millennium revealed that only three of the Trichoderma strains, reported as sources of ‘classical’ peptaibiotics have correctly been identified and appropriately been deposited, viz. the paracelsin-producing T. reesei QM 9414 (Brückner and Graf 1983; Brückner et al. 1984), the trichosporin/trichopolyn producer T. polysporum TMI 60146 (Iida et al. 1990, 1993, 1999), and the paracelsin E-producing T. saturnisporum CBS 330.70 (Ritieni et al. 1995). Furthermore, none of the numerous peptaibiotic-producing strains, reported to belong to those six Trichoderma species mentioned above, has subsequently been verified by phylogenetic analyses. Statements on the identity of the producers must therefore be regarded with great caution, unless it is being described how isolates were identified (Degenkolb et al. 2008). Unfortunately, most of the peptaibiotic-producing Trichoderma/Hypocrea strains investigated prior to 2000 have never been appropriately deposited either i) in a publicly accessible culture collection or ii) in an International Depositary Authority (IDA) under the conditions of the Budapest Treaty; thus, they are not available to independent academic research. As misidentifications persist to be a continuous problem, not only in the older literature (Neuhof et al. 2007), the authors prefer to introduce new names for the peptaibiotics sequenced in this study. Those new names refer to the epithets of the producing species.

Screening of Hypocrea thelephoricola. Ten peptaibols from the specimen of H. thelephoricola were sequenced (Fig. 1a). Six of them, compounds 1−6, are 11-residue sequences displaying the classical building scheme of subfamily 4 (SF4) peptaibols (Chugh and Wallace 2001; Degenkolb et al. 2012; Röhrich et al. 2013b). Compound 1 is new, whereas compounds 2−6 are likely to represent 11-residue peptaibols, which have been described before (Tables 4 and 5, Table S1a and S1b). Compounds 7−10 are new 18-residue peptaibols, named thelephoricolins 1−4 sharing some structural similarity (N-terminal dipeptide, [Gln]6/[Aib]7, C-terminal heptapeptide) with trichotoxins A-50H and A-50-JFootnote 5 (Brückner and Przybylski 1984). The plate culture produced predominantly 11-residue SF4-peptaibols (compounds 1, 2, 5, 6, 11−13), but only two 18-residue peptaibols, thelephoricolins 2 and 3 (Fig. 1b).

Fig. 1
figure 1

Base-peak chromatograms (BPCs) analysed with the micrOTOF-Q II. a specimen of H. thelephoricola; b plate culture of H. thelephoricola on PDA. †, non-peptaibiotic metabolite(s); ‡, co-eluting peptaibiotics, not sequenced. The y-axis of all BPC chromatograms in this publication refers to relative ion intensities

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Table 4 Sequences of 11- and 18-residue peptaibiotics detected in the specimen of Hypocrea thelephoricola
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Table 5 Sequences of 11- and 18-residue peptaibiotics detected in the plate culture of Hypocrea thelephoricola
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Screening of Hypocrea gelatinosa. A single strain (ICMP 5417) of this species has previously been screened positive Aib and Iva by a GC/MS-based approach (Brückner et al. 1991). From the specimen of H. gelatinosa, 14 compounds 14−27, six 18-residue and eight 19-residue peptaibols, were sequenced. All of them but compounds 14 and 18 are new (Tables 6 and 7, Table S2a and S2b; Fig. 2a). The 18-residue sequences, compounds 19−21, 23, 25, and 27, named gelatinosins B 1−6, resemble hypomurocinsFootnote 6 or neoatroviridinsFootnote 7. Two of the 19-residue sequences, compounds 14 and 18, are identical with the recently described hypopulvins from H. pulvinata (Röhrich et al. 2012). The new compounds 15−17, 22, and 24, named gelatinosins A 1−5, exhibit a partially new building scheme − the residue in position 5 of the peptide chain was assigned as Phe, based upon HR-MS/MS data. In contrast to this, the new 19-residue compound 26 displays a different building scheme, resembling trichostrigocinsA/B (Degenkolb et al. 2006a). The plate culture of H. gelatinosa was shown to produce three minor 11-residue SF4-peptaibols, compounds 6, 29, and 33, and nine gelatinosins B (compounds, 19, 20, 25, 27, 28, 30−32, and 34), 18-residue peptaibols of the hypomurocin/neoatroviridin-type. However, 19-residue peptaibols have not been detected (Tables 6 and 7, Table S2a and S2b; Fig. 2b).

Table 6 Sequences of 11-, 18, and 19-residue peptaibiotics detected in the specimen of Hypocrea gelatinosa
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Table 7 Sequences of 11- and 18-residue peptaibiotics detected in the plate culture of Hypocrea gelatinosa
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Fig. 2
figure 2

Base-peak chromatograms (BPCs) analysed with the micrOTOF-Q II. a specimen of H. gelatinosa; b plate culture of H. gelatinosa on PDA. †, non-peptaibiotic metabolites, not sequenced; ‡, co-eluting peptaibiotics, not sequenced

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Compound 6 is likely to represent the second one of the partial sequences reported by Krause et al. (2006a) for H. gelatinosa CBS 724.87. In contrast, the first one, for which an unknown N-terminal residue m/z 157 was claimed (Krause et al. 2006a), could not be detected in this screening.

Screening of Hypocrea voglmayrii. The most notable species screened is by far H. voglmayrii (Fig. 3), the specimen of which produced two 18-residue deletion sequences, compounds 35 and 36, which lack the C-terminal amino alcohol, as well as 15 19-residue peptaibols, compounds 37−51 (Tables 8 and 9, Table S3a and S3b). As all of them are new, the names voglmayrins 1−17 are introduced. They partly resemble the building schemes of trichokonin V (Huang et al. 1995) and of trichorzianins B (Rebuffat et al. 1989). Six of the major compounds (40−45) carry a C-terminal phenylalaninol (Pheol) residue, whereas three minor compounds (37−39) terminate in tyrosinol (Tyrol) − a residue that has not been described for peptaibiotics until only recently (Röhrich et al. 2013a). Another six major compounds (46−51) display an additional fragment ion 68.0628 ± 2.3 mDa at their C-terminus (Fig. 4). Thus, the p-OH group of their Tyrol residue is hypothesised to be substituted by a prenyl or isoprenyl residue (C5H8, for details see paragraph below). In contrast to this, major 19-residue peptaibols produced by the plate culture, compounds 40, 41, 43, 44, and two additional compounds, 52 and 53, voglmayrins-18 and -19, terminate in Pheol. HR-MS data clearly confirm the presence of additional minor components carrying a C-terminal Tyrol or prenylated Tyrol residue, respectively. Unfortunately, the intensities were too low for MS/MS sequencing of the respective y 6 ions. Two 11-residue lipopeptaibols, compound 54 and 55, resembling lipostrigocin B-04/B-05 (Degenkolb et al. 2006a) and trichogin A IV (Auvin-Guette et al. 1992), have also been sequenced.

Fig. 3
figure 3

Base-peak chromatograms (BPCs) analysed with the micrOTOF-Q II. a specimen of H. voglmayrii; b plate culture of H. voglmayrii on PDA. †, non-peptaibiotic metabolite(s); ‡, co-eluting peptaibiotics, not sequenced; Ħ, minor peptabiotics containing O-prenylated tyrosinol (Tyr(C5H8)ol), the C-terminus of which could not be sequenced

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Table 8 Sequences of 18- and 19-residue peptaibiotics detected in the specimen of Hypocrea voglmayrii
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Table 9 Sequences of 11- and 19-residue peptaibiotics detected in the plate culture of Hypocrea voglmayrii
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Fig. 4
figure 4

HR-MS/MS sequencing of diagnostic, C-terminal y-ions, displaying novel and recurrent residues of β-amino alcohols. a phenylalaninol (Pheol); b tyrosinol (Tyrol); c O-prenylated tyrosinol (Tyr(C5H8)ol); d dihydroxyphenylalaninol (DOPAol)

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Screening of Hypocrea minutispora. The specimen of H. minutispora has been shown to produce a mixture of eight new 19-residue peptaibols, compounds 56−63, named minutisporins 1−8 (Tables 10 and 11, Table S4a and S4b; Fig. 5a), resembling the recently described hypophellins (Röhrich et al. 2013a). Analysis of the plate culture (Fig. 5b) revealed that compounds 59−61 were recurrently isolated along with another five new 19-residue sequences, minutisporins 9−13 (compounds 64−68).

Table 10 Sequences of 19-residue peptaibiotics detected in the specimen of Hypocrea minutispora
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Table 11 Sequences of 19-residue peptaibiotics detected in the plate culture of Hypocrea minutispora
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Fig. 5
figure 5

Base-peak chromatograms (BPCs) analysed with the micrOTOF-Q II. a specimen of H. minutispora; b plate culture of H. minutispora on PDA. †, non-peptaibiotic metabolite(s); ‡, co-eluting peptaibiotics, not sequenced

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Screening of Hypocrea citrina. The specimen of H. citrina was shown to be a prolific producer of 19-residue peptaibols, compounds 69−78, of which seven are new, viz. compounds 69, 70, 72−74, 76, and 78. The names hypocitrins 1−7 were selected in order to avoid possible confusion with the mycotoxin citrinin and its derivatives. The remaining three were identified as hypophellin-15, −18, and −20, respectively (Röhrich et al. 2013a). Notably, compound 69, hypocitrin-1, exhibits a C-terminal substituent, which is novel to peptaibiotics, dihydroxyphenylalaninol (Table 12 and Table S5; Fig. 6). Compound 70, hypocitrin-2, a homologue of hypophellin-15 (compound 73), also terminates in Tyrol (Fig. 4). Due to exceptionally high background noise of unknown origin, the methanolic extract of the well-grown H. citrina plate culture could not be interpreted appropriately.

Table 12 Sequences of 19-residue peptaibiotics detected in the specimen of Hypocrea citrina
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Fig. 6
figure 6

Base-peak chromatograms (BPCs) of the specimen of H. citrina analysed with the micrOTOF-Q II. ‡, co-eluting peptaibiotics, not sequenced

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Screening of Hypocrea sulphurea. All three specimens of H. sulphurea were negatively screened for peptaibiotics. From two of them, plate cultures could be obtained; however, those were also screened negatively (data not shown).

Screening of Hypocrea parmastoi. Neither specimen, nor plate culture of H. parmastoi displayed the presence of peptaibiotics (data not shown).

Screening of specimens collected in the natural habitat(s) corroborated the distinguished importance of the genus Trichoderma/Hypocrea as the currently richest source of peptaibiotics. Five of the nine specimens were screened positively, and the results of this screening confirmed by the sequences obtained from screening of the plate cultures. Notably, 56 of the 78 peptaibiotics (72 %) detected represent new sequences.

Screening of H. voglmayrii and H. citrina revealed five peptaibols (compounds 37−39, 70, and 73) carrying a C-terminal Tyrol, a residue quite recently described for H. phellinicola (Röhrich et al. 2013a), which is considered comparatively rare. The additional substituent of the C-terminal Tyrol of voglmayrins 12−17 (compounds 46−51), which has tentatively been assigned as a prenyl or isoprenyl (C5H8) residue, is hypothesised to be located at the p-hydroxy group. A regiospecific O-prenylation at the 4-position of the aromatic ring has recently been demonstrated for SirD (Zou et al. 2011), a tyrosine O-prenyltranferase (Kremer and Li 2010) catalysing the first pathway-specific step in the biosynthesis of the phytotoxin sirodesmin PL. The latter is produced by Leptosphaeria maculans (anamorph: Phoma lingam), the causal agent of blackleg of canola (Brassica napus). Recently, O-prenyltyrosine diketopiperazines have been described from Fusarium sp. and Penicillium crustosum (Guimarães et al. 2010).

Another notable structural element, dihydroxy-Pheol was found at the C-terminus of hypocitrin-1 (compound 69). While the presence of either Pheol or Tyrol may be assumed to originate from the relaxed substrate specificity in the terminal adenylate domain of the respective peptaibol synthetase, the direct incorporation of dihydroxy-Phe, presumably 3,4-dihydroxy-L-Phe (DOPA), is one possible biosynthetic route. Fungal tyrosinases are known to oxidise not only Tyr and various other monophenols, e.g. in the route to melanins, but also act on tyrosyl residues within peptides and proteins, leading to the formation of inter- and intra-molecular crosslinks (Selinheimo et al. 2007). Thus, Tyrol-containing peptaibols could be further oxidised by tyrosinases, and even become attached to components of the fungal cell wall (Mattinen et al. 2008).

Considering the sequences of all species screened, including those of H. pulvinata and H. phellinicola, a general building scheme for those SF1-peptaibiotics can be given (Table 13):

Table 13 General building scheme of the sequences of Hypocrea/Trichoderma SF1-peptaibiotics screened (Röhrich et al. 2012, 2013a, this study)
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As can be seen from above, all structural features (Röhrich et al. 2012) required for ion channel formation (Grigoriev et al. 2003), are present in the 17-, 18-, 19-, and 20-residue peptaibiotics sequenced. Multiple bioactivities of pore-forming 20-residue SF1-peptaibiotics (Röhrich et al. 2013a) and of 11-residue SF4-peptaibiotics (Bobone et al. 2013; Röhrich et al. 2013b) have recently been compiled.

The results of our screening programme further extend the list of peptaibiotic-producing species of Trichoderma/Hypocrea compiled in Table 14. Most notably, the sequences of peptaibiotics produced by the freshly collected specimens are either identical to those found in the plate cultures, or represent – at least – closely related homologues and positional isomers of the latter. Thus, our LC-MS/MS screening approach confirmed that all peptaibiotic-producing specimens and plate cultures obtained thereof represent one and the same species. Consequently, the same type (= subfamily) of peptaibiotics is produced both in the natural habitat and under artificial (= laboratory) conditions − a fact, which is important for the application of Trichoderma formulations in biocontrol and integrated pest management schemes. A Trichoderma/Hypocrea species capable of producing peptaibiotics under the conditions of its natural habitat may defend its ecological niche more effectively compared to a non-producing species, as will be outlined below. At present, ca. 15 % of the phylogenetically verified Trichoderma/Hypocrea species have been positively screened for peptaibiotics; however, it appears that the inventory of peptaibiotics of the remaining 85 % is still waiting to be scrutinised by state-of-the-art bioanalytical – particularly mass spectrometric – methods. Of approximately 130 Trichoderma/Hypocrea species pre-screened by LC/HRMS (Nielsen et al. 2011), ca. 60 were found to produce peptaibioticsFootnote 8. Thus, the production of peptaibiotics in the natural habitat seems to be independent of the habitat preference, i.e. mycoparasitism vs. saprotrophy (Chaverri and Samuels 2013), but neither predictable per se nor universal.

Table 14 Phylogenetically verified peptaibiotic-producing strains and species of Trichoderma/Hypocrea. NB: Species and strains for which only MALDI-TOF-MS screening data have been published are not considered for inclusion
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Given that peptaibiotics are readily biosynthesised in the natural habitat of the producers, they could significantly contribute to the complex interactions of phytoprotective Trichoderma species, which are used in commercial or semi-commercial biocontrol agents (BCAs) against plant pathogenic fungi (Harman et al. 2004; Viterbo et al. 2007; Vinale et al. 2008a, b). Examples of successful biocontrol approaches using Trichoderma strains include ‘Tricovab’, a Brazilian formulation recently approved (Anonymous 2012) for integrated management of Crinipellis (syn. Moniliophthora) perniciosa, the causal agent of Witches’ broom of cacao (Pomella et al. 2007; Loguercio et al. 2009; Medeiros et al. 2010). Notably, ‘Tricovab’ contains a peptaibiotic-producing strain (Degenkolb et al. 2006a) of the hyperparasitic endophyte Trichoderma stromaticum. Moreover, the in vivo-detection of peptaibiotics corroborates the recently demonstrated pro-apoptotic in vitro-activities of the 19-residue peptaibols trichokonin VIFootnote 9 (Huang et al. 1995) from Trichoderma pseudokoningii SMF2 towards plant fungal pathogens such as Fusarium oxysporum (Shi et al. 2012).

The value of peptaibiotics for chemotaxonomy of Trichoderma/Hypocrea has scarcely been scrutinised in the past (Neuhof et al. 2007; Degenkolb et al. 2008). To exhaustively answer this question, a larger number of strains, belonging to recently described species, are required to be included in an LC-MS/MS-based study aimed at analysing the peptaibiome of strains and species within different clades of Trichoderma/Hypocrea. However, statements on peptaibiotic production by a particular Trichoderma/Hypocrea species must always be treated with great caution as they are highly habitat-, isolate-, and/or cultivation-dependent. Furthermore, ‘peptaibol subfamilies’ were introduced at a time when the total number of peptaibiotics described did not exceed 200 (Chugh and Wallace 2001) − less than a sixth of the currently known sequences. Notably, the additional 1,000−1,100 individual peptaibiotics published since then exhibit both new building schemes and constituents. This issue becomes even more complex as ‘peptaibol subfamilies’ were published when phylogenetic methods have not yet been recognised as an indispensable tool in fungal taxonomy. Thus, a considerable number of peptaibiotics, the sequences of which have been elucidated correctly, cannot be linked to an unambiguously identified producer that is deposited in a publicly accessible culture collection. These facts illustrate the urgent need to reconsider the classification into the nine subfamilies − a task that has to be completed before the aforementioned study can be performed.

Currently, any approach for a peptaibiotics-based chemotaxonomy of Trichoderma/Hypocrea must be regarded as extremely complicated − even within a defined clade −, because i) peptaibiotics only represent one single class of secondary metabolites produced by Trichoderma/Hypocrea, ii) most of the producers reported in literature have never been deposited appropriately, and iii) the persistently high degree of misidentification makes any comparison between members of different clades problematic and challenging. This is illustrated by the following examples (references are compiled in Table 14):

  1. i)

    The 20-residue alamethicins (ALMs) have hitherto been found in four species belonging to the Brevicompactum clade of Trichoderma; however, it is not yet possible to estimate if the Pro2 residue of the ALMs could be regarded as a structurally highly conserved position, comparable to the Pro14 residue. Chemotaxonomy of the Brevicompactum clade encompassed the comparison of hydrophobins, peptaibiotics, and low-molecular weight secondary metabolites, including simple trichothecene-type mycotoxins.

  2. ii)

    The 18-residue trichotoxins (TXT) A-50 and A-40, for example, have been obtained from Trichoderma asperellum NRRL 5242, whereas Trichoderma asperellum Y 19-07 did not produce TXTs but 9- and 10-residue peptaibols instead (and vice versa).

  3. iii)

    Trichoderma citrinoviride strains S 25 and IMI 91968 are rich sources of 20-residue peptaibols of the paracelsin/saturnisporin/trichocellin/suzukacillin/trichoaureocin-type. These are the only two strains of T. citrinoviride that have been investigated for peptaibiotics. Hypocrea schweinitzii ICMP 5421, which has also been verified phylogenetically (Réblová and Seifert 2004), had only been screened positive for Aib by GC/MS; but − to the best of the authors’ knowledge − specimens of that species have never been investigated for its inventory of peptaibiotics. Parcelsins, which have been isolated from T. reesei QM 9414, are also produced by a member of the Longibrachiatum clade. However, the producer of saturnisporin (T. saturnisporum MNHN 903578: Rebuffat et al. 1993) has never been made publicly available, nor has its identity been verified phylogenetically. The producers of both trichocellins and suzukacillins A (Krause et al. 2006b) have not been deposited in a publicly available culture collection; thus, their identification as T. ‘viride’ is highly questionable.

  4. iv)

    T. flavofuscum CBS 248.59 is the only species of Trichoderma/Hypocrea, which produces 13-residue sequences − notably trichofumins C and D are the only two peptaibols of that chain length reported to date. They display the rare Gln-Gln motif in positions 5 and 6. Looking at the sequences, their biosynthesis seems to be distantly related to that one of trichofumins A and B (and positional isomers thereof). The latter are 11-residue SF4-peptaibols and widespread amongst Trichoderma/Hypocrea species.

  5. v)

    T. virens strain Tv29-8 produces common 11- and 14-residue peptaibols, and it is the only phylogenetically verified source of 18-residue peptaibols of the trichorzin-type.

However, the results of our LC-MS/MS screening are also of interest for analysis of environmental samples as well as extraterrestrial materials such as carbonaceous meteorites as their contamination by propagules of soil- or airborne peptaibiotic-producing fungi has to be taken into account (Brückner et al. 2009; Elsila et al. 2011).

To sum up, production of peptaibiotics may generally be regarded as a sophisticated ecological adaptation for the producing fungus providing it with an obvious advantage over non-producing fungal and other competitors. This group of ‘chemical weapons’ in their ‘armoury’ may effectively assist a remarkable number of strains currently identified as belonging to ca. 30 Trichoderma/Hypocrea species in colonising and defending their ecological niches.