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Fungal Diversity

, Volume 69, Issue 1, pp 117–146 | Cite as

Front line defenders of the ecological niche! Screening the structural diversity of peptaibiotics from saprotrophic and fungicolous Trichoderma/Hypocrea species

  • Christian R. Röhrich
  • Walter M. Jaklitsch
  • Hermann Voglmayr
  • Anita Iversen
  • Andreas Vilcinskas
  • Kristian Fog Nielsen
  • Ulf Thrane
  • Hans von Döhren
  • Hans Brückner
  • Thomas Degenkolb
Open Access
Article

Abstract

Approximately 950 individual sequences of non-ribosomally biosynthesised peptides are produced by the genus Trichoderma/Hypocrea that belong to a perpetually growing class of mostly linear antibiotic oligopeptides, which are rich in the non-proteinogenic α-aminoisobutyric acid (Aib). Thus, they are comprehensively named peptaibiotics. Notably, peptaibiotics represent ca. 80 % of the total inventory of secondary metabolites currently known from Trichoderma/Hypocrea. Their unique membrane-modifying bioactivity results from amphipathicity and helicity, thus making them ideal candidates in assisting both colonisation and defence of the natural habitats by their fungal producers. Despite this, reports on the in vivo-detection of peptaibiotics have scarcely been published in the past. In order to evaluate the significance of peptaibiotic production for a broader range of potential producers, we screened nine specimens belonging to seven hitherto uninvestigated fungicolous or saprotrophic Trichoderma/Hypocrea species by liquid chromatography coupled to electrospray high resolution mass spectrometry. Sequences of peptaibiotics found were independently confirmed by analysing the peptaibiome of pure agar cultures obtained by single-ascospore isolation from the specimens. Of the nine species examined, five were screened positive for peptaibiotics. A total of 78 peptaibiotics were sequenced, 56 (= 72 %) of which are new. Notably, dihydroxyphenylalaninol and O-prenylated tyrosinol, two C-terminal residues, which have not been reported for peptaibiotics before, were found as well as new and recurrent sequences carrying the recently described tyrosinol residue at their C-terminus. The majority of peptaibiotics sequenced are 18- or 19-residue peptaibols. Structural homologies with ‘classical representatives’ of subfamily 1 (SF1)-peptaibiotics argue for the formation of transmembrane ion channels, which are prone to facilitate the producer capture and defence of its substratum.

Keywords

HPLC/QTOF-ESI-HRMS Metabolite profiling Peptaibiotics Peptaibols Aib peptides Trichoderma Hypocrea 

Introduction

Currently, the fungal genus Trichoderma/Hypocrea 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

Producing species and strains

Name of new metabolite(s)

Chemical subclass of metabolites

References

T. atroviride G20-12

4′-(4,5-dimethyl-1,3-dioxolan-2-yl)methylphenol

(3′-hydroxybutan-2′-yl)5-oxopyrrolidine-2-carboxylate

Atroviridetide

 

Lu et al. 2012

T. atroviride UB-LMAa

one bicyclic, three tetracyclic diterpenes

Di- and tetraterpenes

Adelin et al. 2014

T. gamsii SQP 79–1

Trichalasin C, D

Cytochalasans

Ding et al. 2012

  

Spiro-cytochalasan

Ding et al. 2014

T. sp. FKI-6626

Cytosporone S

 

Ishii et al. 2013

T. erinaceum AF007

Trichodermaerin

Diterpenoid lactone

Xie et al. 2013

aThe scientific name of the producer has been misspelled as Trichoderma atrovirid ae in Adelin et al. (2014)

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 trichorzianins2 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)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 peptaibiome4 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

Species

Clade

Habitat

Geographic distribution

Hypocrea thelephoricola (Trichoderma thelephoricola)

Chlorospora

On and around basidiomata of Steccherinum ochraceum, on wood and bark

North America (USA), Europe (Austria)

Hypocrea minutispora (Trichoderma minutisporum)

Pachybasium (core group)

Most common hyaline-spored species in temperate zones

Europe (Austria, Czech Republic, Denmark, Estonia, France, Germany, Spain, Sweden, United Kingdom) and North America (USA)

Hypocrea sulphurea (Trichoderma sp.)

Hypocreanum

On basidiomes of Exidia spp.

Europe (Eastern Austria, Ukraine), North America (USA), Japan

Hypocrea citrina (Trichoderma lacteum)

Hypocreanum

Spreading from stumps or tree bases on soil and debris such as small twigs, bark, leaves, dead plants; incorporating also living plants; more rarely on bark of logs on the ground. Most typically in mixed coniferous forest

widespread and locally common, mostly found from the end of August to the beginning of October. Europe (Austria, Belgium, Czech Republic, Netherlands, Sweden, United Kingdom) and North America (USA)

Hypocrea voglmayrii (Trichoderma voglmayrii)

Lone lineage

On dead, mostly corticated branches and small trunks of Alnus alnobetula (= A. viridis) and A. incana standing or lying on the ground

Austria (at elevations of 1,000–1,400 m in the upper montane vegetation zone of the Central Alps)

Hypocrea gelatinosa (Trichoderma gelatinosum)

Lone lineage

On medium- to well-decayed wood, also on bark and overgrowing various fungi

Europe (Austria, France, Germany, Netherlands, Slovenia, Ukraine, United Kingdom)

Hypocrea parmastoi (Trichoderma sp. [sect. Hypocreanum])

Lone lineage

On medium- to well-decayed wood and bark of deciduous trees

Europe (Austria, Estonia, Finland, France, Germany); uncommon

Data were compiled from Chaverri and Samuels (2003), Overton et al. (2006a, b), and Jaklitsch (2009, 2011)

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

aStroma immature, isolation of single germinable ascospores impossible

bThe specimens of H. sulphurea 1 and 2 were collected from two different trees found in the same area

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 16, 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 26 are likely to represent 11-residue peptaibols, which have been described before (Tables 4 and 5, Table S1a and S1b). Compounds 710 are new 18-residue peptaibols, named thelephoricolins 14 sharing some structural similarity (N-terminal dipeptide, [Gln]6/[Aib]7, C-terminal heptapeptide) with trichotoxins A-50H and A-50-J5 (Brückner and Przybylski 1984). The plate culture produced predominantly 11-residue SF4-peptaibols (compounds 1, 2, 5, 6, 1113), but only two 18-residue peptaibols, thelephoricolins 2 and 3 (Fig. 1b).
Fig. 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

Table 4

Sequences of 11- and 18-residue peptaibiotics detected in the specimen of Hypocrea thelephoricola

No.

tR [min]

[M + H]+

 

Residuea

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

1

37.6–37.9

1161.7527

Ac

Aib

Gln

Vxx

Lxx

Aib

Pro

Vxx

Lxx

Aib

Pro

Lxxol

       

2

37.6–37.9

1161.7527

Ac

Aib

Gln

Vxx

Vxx

Aib

Pro

Lxx

Lxx

Aib

Pro

Lxxol

       

3

39.3–39.5

1175.7712

Ac

Aib

Gln

Vxx

Lxx

Aib

Pro

Lxx

Lxx

Aib

Pro

Lxxol

       

4

39.7 –40.0

1175.7712

Ac

Aib

Gln

Lxx

Lxx

Aib

Pro

Vxx

Lxx

Aib

Pro

Lxxol

       

5

41.5–41.7

1189.7836

Ac

Aib

Gln

Lxx

Lxx

Aib

Pro

Lxx

Lxx

Aib

Pro

Lxxol

       

6

42.9–43.0

1203.7981

Ac

Vxx

Gln

Lxx

Lxx

Aib

Pro

Lxx

Lxx

Aib

Pro

Lxxol

       

7

44.2–44.5

1732.0673

Ac

Aib

Ala

Aib

Ala

Vxx

Gln

Aib

Vxx

Aib

Gly

Lxx

Aib

Pro

Lxx

Aib

Vxx

Gln

Vxxol

8

44.8–45.0

1746.0866

Ac

Aib

Ala

Aib

Ala

Vxx

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Lxx

Aib

Vxx

Gln

Vxxol

9

45.2–46.0

1760.1035

Ac

Aib

Ala

Vxx

Ala

Vxx

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Lxx

Aib

Vxx

Gln

Vxxol

10

47.5–47.8

1774.1161

Ac

Aib

Ala

Vxx

Ala

Vxx

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Lxx

Aib

Vxx

Gln

Lxxol

No.

Compound identical or positionally isomeric with

Ref.

                   

1

New

                   

2

Trichorovins: IIIa, IVa

Wada et al. 1995

                   
 

Hypomurocin A-1

Becker et al. 1997

                   
 

Trichobrachins III: 5, 9b

Krause et al. 2007

                   
 

Tv-29-11-III g

Mukherjee et al. 2011

                   
 

Hypojecorin A: 8

Degenkolb et al. 2012

                   

3

Trichobrachins III: 10a, 12a, 15b

Krause et al. 2007

                   
 

Trichorovins: VIII, IXa

Wada et al. 1995

                   
 

Hypomurocin A-3

Becker et al. 1997

                   
 

Tv-29-11-IV g

Mukherjee et al. 2011

                   

4

Tv-29-11-IV e

Mukherjee et al. 2011

                   

5

Trichobrachins III: 16a, 17, 18

Krause et al. 2007

                   
 

Trichorovins: XIII, XIV

Wada et al. 1995

                   
 

Tv-29-11-V b

Mukherjee et al. 2011

                   
 

Hypomurocins: A-5, A-5a

Becker et al. 1997

                   
 

Trichorozin IV

Iida et al. 1995

                   
 

Trichobrachins: C-I, C-II

Ruiz et al. 2007

                   
 

Trilongin A0

Mikkola et al. 2012

                   

6

Trichofumin B

Berg et al. 2003

                   
 

Tv-29-11-VI

Mukherjee et al. 2011

                   

7

Thelephoricolin-1

                    

8

Thelephoricolin-2

                    

9

Thelephoricolin-3

                    

10

Thelephoricolin-4

                    

aVariable residues are underlined in the table header. Minor sequence variants are underlined in the sequences. This applies to all sequence tables

Table 5

Sequences of 11- and 18-residue peptaibiotics detected in the plate culture of Hypocrea thelephoricola

No.

tR [min]

[M + H]+

 

Residuea

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

11

35.6–35.8

1147.7443

Ac

Aib

Gln

Vxx

Vxx

Aib

Pro

Vxx

Lxx

Aib

Pro

Lxxol

       

1

37.2–37.4

1161.7623

Ac

Aib

Gln

Vxx

Lxx

Aib

Pro

Vxx

Lxx

Aib

Pro

Lxxol

       

2

37.7–37.9

1161.7652

Ac

Aib

Gln

Vxx

Vxx

Aib

Pro

Lxx

Lxx

Aib

Pro

Lxxol

       

12

39.8–40.0

1175.7747

Ac

Aib

Gln

Lxx

Vxx

Aib

Pro

Lxx

Lxx

Aib

Pro

Lxxol

       

5

41.5–41.7

1189.7893

Ac

Aib

Gln

Lxx

Lxx

Aib

Pro

Lxx

Lxx

Aib

Pro

Lxxol

       

13

40.6–40.8

1189.7996

Ac

Vxx

Gln

Vxx

Lxx

Aib

Pro

Lxx

Lxx

Aib

Pro

Lxxol

       

6

42.8–43.0

1203.8004

Ac

Vxx

Gln

Lxx

Lxx

Aib

Pro

Lxx

Lxx

Aib

Pro

Lxxol

       

8

44.8–44.9

1746.0955

Ac

Aib

Ala

Aib

Ala

Vxx

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Lxx

Aib

Vxx

Gln

Vxxol

9

45.5–45.7

1760.1104

Ac

Aib

Ala

Vxx

Ala

Vxx

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Lxx

Aib

Vxx

Gln

Vxxol

No.

Compound identical or positionally isomeric with

Ref.

                   

11

Tv-29-11-II h

Mukherjee et al. 2011

                   

1

                     

2

                     

12

Trichobrachin III 11a

Krause et al. 2007

                   
 

Tv-29-11-IV f

Mukherjee et al. 2011

                   
 

Trichorovin Xa

Wada et al. 1995

                   
 

Hypomurocin A-4

Becker et al. 1997

                   

5

cf. 2

                    

13

Tv-29-11-V d

Mukherjee et al. 2011

                   

6

                     

8

Thelephoricolin-2

                    

9

Thelephoricolin-3

                    

aVariable residues are underlined in the table header. Minor sequence variants are underlined in the sequences. This applies to all sequence tables

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 1427, 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 1921, 23, 25, and 27, named gelatinosins B 1−6, resemble hypomurocins6 or neoatroviridins7. 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 1517, 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, 3032, 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

No.

tR [min]

[M + H]+

 

Residuea

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

14

37.1–37.3

1866.0929

Ac

Aib

Ala

Ala

Ala

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Aib

Gln

Gln

Pheol

15

37.7–37.8

1895.1067

Ac

Aib

Ala

Aib

Aib

Phe

Gln

Aib

Aib

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Aib

Glu

Gln

Lxxol

16

38.0–38.2

1908.1358

Ac

Aib

Ala

Aib

Aib

Phe

Gln

Aib

Aib

Aib

Gly

Lxx

Aib

Pro

Lxx

Aib

Aib

Gln

Gln

Lxxol

17

38.8–38.9

1909.1186

Ac

Aib

Ala

Aib

Aib

Phe

Gln

Aib

Aib

Aib

Gly

Lxx

Aib

Pro

Lxx

Aib

Aib

Glu

Gln

Lxxol

18

39.5–39.6

1880.1083

Ac

Aib

Ala

Ala

Ala

Aib

Gln

Aib

Lxx

Aib

Ala

Lxx

Aib

Pro

Vxx

Aib

Aib

Gln

Gln

Pheol

19

40.2–40.4

1762.0856

Ac

Aib

Ser

Ala

Lxx

Aib

Gln

Aib

Lxx

Aib

Gly

Vxx

Aib

Pro

Lxx

Aib

Aib

Gln

Lxxol

20

40.9–41.1

1762.0840

Ac

Aib

Ser

Ala

Lxx

Aib

Gln

Vxx

Lxx

Aib

Gly

Vxx

Aib

Pro

Lxx

Aib

Aib

Gln

Vxxol

21

41.2–41.4

1776.1023

Ac

Aib

Ser

Ala

Lxx

Vxx

Gln

Aib

Lxx

Aib

Gly

Vxx

Aib

Pro

Lxx

Aib

Aib

Gln

Lxxol

22

41.9

1952.1674

Ac

Aib

Ala

Aib

Aib

Phe

Gln

Aib

Aib

Aib

Ser

Lxx

Aib

Pro

Lxx

Vxx

Aib

Gln

Gln

Lxxol

23

42.1–42.3

1776.1023

Ac

Aib

Ser

Ala

Lxx

Vxx

Gln

Vxx

Lxx

Aib

Gly

Vxx

Aib

Pro

Lxx

Aib

Aib

Gln

Vxxol

6

42.3

1203.8117

Ac

Vxx

Gln

Lxx

Lxx

Aib

Pro

Lxx

Lxx

Aib

Pro

Lxxol

        

24

42.9

1953.1515

Ac

Aib

Ala

Aib

Aib

Phe

Gln

Aib

Aib

Aib

Ser

Lxx

Aib

Pro

Lxx

Vxx

Aib

Glu

Gln

Lxxol

25

43.0–43.1

1790.1199

Ac

Aib

Ser

Ala

Lxx

Vxx

Gln

Vxx

Lxx

Aib

Gly

Vxx

Aib

Pro

Lxx

Aib

Aib

Gln

Lxxol

26

44.6

1919.1568

Ac

Aib

Ala

Aib

Aib

Lxx

Gln

Aib

Aib

Aib

Ser

Lxx

Aib

Pro

Vxx

Aib

Lxx

Glu

Gln

Lxxol

27

45.8

1774.1299

Ac

Aib

Ala

Ala

Lxx

Vxx

Gln

Vxx

Lxx

Aib

Gly

Vxx

Aib

Pro

Lxx

Aib

Aib

Gln

Lxxol

No.

Compound identical or positionally isomeric with

Ref.

                    

14

Hypopulvin-9

Röhrich et al. 2012

                    

15

Gelatinosin-A 1 (C-terminal undecapeptide cf. hypelcins B-I and -II)

Matsuura et al. 1994

                    

16

Gelatinosin-A 2 (C-terminal nonapeptide cf. tricholongin B-I)

Rebuffat et al. 1991

                    

17

Gelatinosin-A 3 (cf. 16)

                     

18

Hypopulvin-14

Röhrich et al. 2012

                    

19

Gelatinosin-B 1 (cf. hypomurocin B-5: [Vxx]8 → [Lxx]8)

Becker et al. 1997

                    

20

Gelatinosin-B 2 (cf. hypomurocin B-3b: [Vxx]8 → [Lxx]8, [Aib]11 → [Vxx]11)

Becker et al. 1997

                    

21

Gelatinosin-B 3 (cf. neoatroviridin B: [Gly]2 → [Ser]2)

Oh et al. 2005

                    

22

Gelatinosin-A 4 (cf. 16: [Gly]10 → [Ser]10, [Aib]15 → [Vxx]15)

                     

23

Gelatinosin-B 4 (cf. hypomurocin B-4: [Aib]5,7 → [Vxx]5,7)

Becker et al. 1997

                    

6

See H. thelephoricola

                     

24

Gelatinosin-A 5 (cf. 17: [Gly]10 → [Ser]10, [Aib]15 → [Vxx]15)

                     

25

Gelatinosin-B 5 (cf. neoatroviridin D: [Gly]2 → [Ser]2)

Oh et al. 2005

                    

26

New (cf. trichostrigocin-A and -B: [Lxx]16 → [Vxx]16, [Gln]17 → [Glu]17)

Degenkolb et al. 2006a, b

                    

27

Gelatinosin-B 6 (cf. neoatroviridin D: [Gly]2 → [Ala]2)

Oh et al. 2005

                    

aVariable residues are underlined in the table header. Minor sequence variants are underlined in the sequences. This applies to all sequence tables

Table 7

Sequences of 11- and 18-residue peptaibiotics detected in the plate culture of Hypocrea gelatinosa

No.

tR [min]

[M + H]+

 

Residuea

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

28

38.0–38.1

1748.0789

Ac

Aib

Ser

Ala

Lxx

Aib

Gln

Aib

Lxx

Aib

Gly

Aib

Aib

Pro

Lxx

Aib

Aib

Gln

Lxxol

29

38.8–38.9

1175.7832

Ac

Aib

Gln

Lxx

Lxx

Aib

Pro

Vxx

Lxx

Aib

Pro

Lxxol

       

30

39.2–39.3

1748.0789

Ac

Aib

Ser

Ala

Lxx

Aib

Gln

Aib

Lxx

Aib

Gly

Vxx

Aib

Pro

Lxx

Aib

Aib

Gln

Vxxol

31

39.4–39.7

1762.0802

Ac

Aib

Ser

Ala

Lxx

Aib

Gln

Vxx

Lxx

Aib

Gly

Aib

Aib

Pro

Lxx

Aib

Aib

Gln

Lxxol

19

40.1–40.4

1762.0814

Ac

Aib

Ser

Ala

Lxx

Aib

Gln

Aib

Lxx

Aib

Gly

Vxx

Aib

Pro

Lxx

Aib

Aib

Gln

Lxxol

32

40.5–40.7

1777.0993

Ac

Aib

Ser

Ala

Lxx

Vxx

Gln

Vxx

Lxx

Aib

Gly

Aib

Aib

Pro

Lxx

Aib

Aib

Glu

Lxxol

33

40.8–41.0

1189.8026

Ac

Aib

Gln

Lxx

Lxx

Aib

Pro

Lxx

Lxx

Aib

Pro

Lxxol

       

20

40.9–41.1

1762.0797

Ac

Aib

Ser

Ala

Lxx

Aib

Gln

Vxx

Lxx

Aib

Gly

Vxx

Aib

Pro

Lxx

Aib

Aib

Gln

Vxxol

34

41.8–42.1

1776.1016

Ac

Aib

Ser

Ala

Lxx

Aib

Gln

Vxx

Lxx

Aib

Gly

Vxx

Aib

Pro

Lxx

Aib

Aib

Gln

Lxxol

6

42.7–42.9

1203.8234

Ac

Vxx

Gln

Lxx

Lxx

Aib

Pro

Lxx

Lxx

Aib

Pro

Lxxol

       

25

43.1–43.3

1790.1139

Ac

Aib

Ser

Ala

Lxx

Vxx

Gln

Vxx

Lxx

Aib

Gly

Vxx

Aib

Pro

Lxx

Aib

Aib

Gln

Lxxol

27

45.7–46.0

1774.1162

Ac

Aib

Ala

Ala

Lxx

Vxx

Gln

Vxx

Lxx

Aib

Gly

Vxx

Aib

Pro

Lxx

Aib

Aib

Gln

Lxxol

No.

Compound identical or positionally isomeric with

Ref.

                   

28

Gelatinosin-B 7 (cf. hypomurocin B-2: [Vxx]8 → [Lxx]8)

Becker et al. 1997

                   

29

Tv-29-11-IV e (positional isomer of 4)

Mukherjee et al. 2011

                   

30

Gelatinosin-B 8 (cf. hypomurocin B-4: [Vxx]8 → [Lxx]8)

Becker et al. 1997

                   

31

Gelatinosin-B 9 (cf. hypomurocin B-3b: [Vxx]8 → [Lxx]8, [Vxxol]18 → [Lxxol]18)

Becker et al. 1997

                   

19

Gelatinosin-B 1 (cf. hypomurocin B-5: [Vxx]8 → [Lxx]8)

Becker et al. 1997

                   

32

Gelatinosin-B 10 (cf. 25: [Gln]17 → [Glu]17)

                    

33

See H. thelephoricola (positional isomer of 5)

                    

20

Gelatinosin-B 2 (cf. hypomurocin B-4: [Aib]7 → [Vxx]7, [Vxx]8 → [Lxx]8)

Becker et al. 1997

                   

34

Gelatinosin-B 11 (cf. trichovirin II 6a and neoatroviridin C: [Gly]2 → [Ser]2)

Jaworski et al. 1999; Oh et al. 2005

                

6

See H. thelephoricola

                    

25

Gelatinosin-B 5

                    

27

Gelatinosin-B 6

                    

aVariable residues are underlined in the table header. Minor sequence variants are underlined in the sequences. This applies to all sequence tables

Fig. 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

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 3751 (Tables 8 and 9, Table S3a and S3b). As all of them are new, the names voglmayrins 117 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 (4045) carry a C-terminal phenylalaninol (Pheol) residue, whereas three minor compounds (3739) terminate in tyrosinol (Tyrol) − a residue that has not been described for peptaibiotics until only recently (Röhrich et al. 2013a). Another six major compounds (4651) 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

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

Table 8

Sequences of 18- and 19-residue peptaibiotics detected in the specimen of Hypocrea voglmayrii

No.

tR [min]

[M + H]+

 

Residuea

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

35

30.2–31.1

1762.0125

Ac

Aib

Ala

Aib

Ala

Aib

Gln

Aib

Aib

Aib

Ala

Lxx

Vxx

Pro

Vxx

Aib

Vxx

Glu

Gln

 

36

31.6–32.0

1775.0433

Ac

Aib

Ala

Aib

Aib

Aib

Gln

Aib

Aib

Aib

Ala

Lxx

Vxx

Pro

Vxx

Aib

Vxx

Gln

Gln

 

37

33.6–33.7

1924.1239

Ac

Aib

Ala

Aib

Aib

Aib

Gln

Aib

Aib

Aib

Ala

Lxx

Vxx

Pro

Vxx

Aib

Vxx

Gln

Gln

Tyrol

38

34.1–34.5

1911.1015

Ac

Aib

Ala

Ala

Aib

Aib

Gln

Aib

Aib

Aib

Ala

Lxx

Vxx

Pro

Vxx

Aib

Vxx

Gln

Glu

Tyrol

39

34.5–34.8

1925.1100

Ac

Aib

Ala

Aib

Aib

Aib

Gln

Aib

Aib

Aib

Ala

Lxx

Vxx

Pro

Vxx

Aib

Vxx

Gln

Glu

Tyrol

40

37.3–37.4

1880.1041

Ac

Aib

Ala

Ala

Aib

Aib

Gln

Aib

Aib

Aib

Ala

Lxx

Aib

Pro

Vxx

Aib

Vxx

Gln

Gln

Pheol

41

37.7–37.9

1894.1197

Ac

Aib

Ala

Aib

Aib

Aib

Gln

Aib

Aib

Aib

Ala

Lxx

Aib

Pro

Vxx

Aib

Vxx

Gln

Gln

Pheol

42

38.5–38.7

1881.0933

Ac

Aib

Ala

Ala

Aib

Aib

Gln

Aib

Aib

Aib

Ala

Lxx

Aib

Pro

Vxx

Aib

Vxx

Gln

Glu

Pheol

43

39.5–39.7

1894.1218

Ac

Aib

Ala

Ala

Aib

Aib

Gln

Aib

Aib

Aib

Ala

Lxx

Vxx

Pro

Vxx

Aib

Vxx

Gln

Gln

Pheol

44

39.9–40.1

1908.1391

Ac

Aib

Ala

Aib

Aib

Aib

Gln

Aib

Aib

Aib

Ala

Lxx

Vxx

Pro

Vxx

Aib

Vxx

Gln

Gln

Pheol

45

41.4–41.5

1909.1203

Ac

Aib

Ala

Aib

Aib

Aib

Gln

Aib

Aib

Aib

Ala

Lxx

Vxx

Pro

Vxx

Aib

Vxx

Gln

Glu

Pheol

46

42.8–43.0

1978.1743

Ac

Vxx

Ala

Ala

Aib

Aib

Gln

Aib

Aib

Aib

Ala

Lxx

Vxx

Pro

Vxx

Aib

Aib

Gln

Gln

Tyr(C 5 H 8 )ol b

47

43.4–43.6

1978.1741

Ac

Aib

Ala

Ala

Aib

Aib

Gln

Aib

Aib

Aib

Ala

Lxx

Vxx

Pro

Vxx

Aib

Vxx

Gln

Gln

Tyr(C 5 H 8 )ol

48

43.8–44.0

1992.1924

Ac

Aib

Ala

Aib

Aib

Aib

Gln

Aib

Aib

Aib

Ala

Lxx

Vxx

Pro

Vxx

Aib

Vxx

Gln

Gln

Tyr(C 5 H 8 )ol

49

44.6–44.7

1979.1585

Ac

Aib

Ala

Ala

Aib

Aib

Gln

Aib

Aib

Aib

Ala

Lxx

Vxx

Pro

Vxx

Aib

Vxx

Gln

Glu

Tyr(C 5 H 8 )ol

50

45.0–45.1

1993.1762

Ac

Aib

Ala

Aib

Aib

Aib

Gln

Aib

Aib

Aib

Ala

Lxx

Vxx

Pro

Vxx

Aib

Vxx

Gln

Glu

Tyr(C 5 H 8 )ol

51

45.9–46.1

2007.1881

Ac

Vxx

Ala

Aib

Aib

Aib

Gln

Aib

Aib

Aib

Ala

Lxx

Vxx

Pro

Vxx

Aib

Vxx

Gln

Glu

Tyr(C 5 H 8 )ol

No.

Compound identical or positionally isomeric with

Ref.

                    

35

Voglmayrin-1 (N-terminal heptapeptide, pos. 13–15 and 18 cf. trichokonin V)

Huang et al. 1995

                   

36

Voglmayrin-2 (cf. 35: [Ala]4 → [Aib]4, [Glu]17 → [Gln]17: deletion sequence of 37)

                     

37

Voglmayrin-3 (cf. 36: + C-terminal Tyrol)

                     

38

Voglmayrin-4

                     

39

Voglmayrin-5 (cf. 37: [Gln]18 → [Glu]18)

                     

40

Voglmayrin-6 (N-terminal nonapeptide cf. trichorzianine B-VIb, [Ser]10 → [Ala]10, C-terminal nonapeptide cf. trichorzianine B-VIb, [Ile]16 → [Vxx]16)

Rebuffat et al. 1989

                   

41

Voglmayrin-7

                     

42

Voglmayrin-8 (homologue of 40: [Gln]18 → [Glu]18)

                     

43

Voglmayrin-9 (homologue of 40: [Aib]12 → [Vxx]12)

                     

44

Voglmayrin-10 (homologue of 37: [Tyrol]19 → [Pheol]19)

                     

45

Voglmayrin-11 (homologue of 39: [Tyrol]19 → [Pheol]19)

                     

46

Voglmayrin-12

                     

47

Voglmayrin-13 (homologue of 48: [Aib]3 → [Ala]3)

                     

48

Voglmayrin-14 (homologue of 37 and 44: prenylated [Tyrol]19)

                     

49

Voglmayrin-15 (homologue of 38: prenylated [Tyrol]19)

                     

50

Voglmayrin-16 (homologue of 49: [Ala]3 → [Aib]3)

                     

51

Voglmayrin-17 (homologue of 50: [Aib]1 → [Vxx]1)

                     

aVariable residues are underlined in the table header. Minor sequence variants are underlined in the sequences. This applies to all sequence tables

bC5H8, prenyl (Prn) or isoprenyl residue at OH-group of Tyr postulated. For details, see text

Table 9

Sequences of 11- and 19-residue peptaibiotics detected in the plate culture of Hypocrea voglmayrii

No.

tR [min]

[M + H]+

 

Residuea

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

52

35.2–35.6

1852.0739

Ac

Aib

Ala

Ala

Aib

Aib

Gln

Ala

Aib

Aib

Ala

Lxx

Aib

Pro

Vxx

Aib

Aib

Gln

Gln

Pheol

53

35.6–35.8

1866.0884

Ac

Aib

Ala

Ala

Aib

Aib

Gln

Ala

Aib

Aib

Ala

Lxx

Aib

Pro

Vxx

Aib

Vxx

Gln

Gln

Pheol

40

37.3–37.6

1880.1099

Ac

Aib

Ala

Ala

Aib

Aib

Gln

Aib

Aib

Aib

Ala

Lxx

Aib

Pro

Vxx

Aib

Vxx

Gln

Gln

Pheol

41

37.7–37.8

1894.1237

Ac

Aib

Ala

Aib

Aib

Aib

Gln

Aib

Aib

Aib

Ala

Lxx

Aib

Pro

Vxx

Aib

Vxx

Gln

Gln

Pheol

43

39.6–39.7

1894.1238

Ac

Aib

Ala

Ala

Aib

Aib

Gln

Aib

Aib

Aib

Ala

Lxx

Vxx

Pro

Vxx

Aib

Vxx

Gln

Gln

Pheol

44

40.0

1908.1395

Ac

Aib

Ala

Aib

Aib

Aib

Gln

Aib

Aib

Aib

Ala

Lxx

Vxx

Pro

Vxx

Aib

Vxx

Gln

Gln

Pheol

54

40.7–41.0

1052.7130

Oc

Aib

Gly

Lxx

Aib

Gly

Gly

Vxx

Aib

Gly

Lxx

Lxxol

        

55

42.8–43.1

1066.7288

Oc

Aib

Gly

Lxx

Aib

Gly

Gly

Lxx

Aib

Gly

Lxx

Lxxol

        

No.

Comment (compound identical or positionally isomeric with)

Ref.

                    

52

Voglmayrin-18 (homologue of 53: [Vxx]16 → [Aib]16; N-terminal hexapeptide cf. trichorzianine B-VIb; C-terminal nonapeptide cf. trichosporins B)

Rebuffat et al. 1989

                   
 

Iida et al. 1990

                   

53

Voglmayrin-19 (homologue of 40: [Aib]7 → [Ala]7; C-terminal nonapeptide cf. polysporin D)

New et al. 1996

                   

40

Voglmayrin-20

                     

41

Voglmayrin-21

                     

43

Voglmayrin-22

                     

44

Voglmayrin-23

                     

54

cf. lipostrigocins B-04 and B-05

Degenkolb et al. 2006a

                   

55

cf. trichogin A-IV

Auvin-Guette et al. 1992;

                  
  

Degenkolb et al. 2006a

                   

aVariable residues are underlined in the table header. Minor sequence variants are underlined in the sequences. This applies to all sequence tables

Fig. 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)

Screening of Hypocrea minutispora. The specimen of H. minutispora has been shown to produce a mixture of eight new 19-residue peptaibols, compounds 5663, named minutisporins 18 (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 5961 were recurrently isolated along with another five new 19-residue sequences, minutisporins 913 (compounds 6468).
Table 10

Sequences of 19-residue peptaibiotics detected in the specimen of Hypocrea minutispora

No.

tR [min]

[M + H]+

 

Residuea

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

56

34.5–34.7

1847.1051

Ac

Aib

Ala

Aib

Gly

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Vxx

Glu

Gln

Lxxol

57

37.5–38.1

1846.1192

Ac

Aib

Ala

Aib

Ala

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Aib

Gln

Gln

Lxxol

58

38.5–38.6

1846.1099

Ac

Aib

Ala

Ala

Ala

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Vxx

Gln

Gln

Lxxol

59

39.1–39.4

1860.1278

Ac

Aib

Ala

Aib

Ala

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Vxx

Gln

Gln

Lxxol

60

39.8–40.1

1861.1130

Ac

Aib

Ala

Aib

Ala

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Vxx

Glu

Gln

Lxxol

61

40.9–41.0

1874.1420

Ac

Aib

Ala

Aib

Ala

Vxx

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Vxx

Gln

Gln

Lxxol

62

41.5–41.6

1875.1390

Ac

Aib

Ala

Aib

Aib

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Vxx

Glu

Gln

Lxxol

63

41.9–42.0

1875.1284

Ac

Aib

Ala

Aib

Ala

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Lxx

Aib

Vxx

Glu

Gln

Lxxol

No.

Compound identical or positionally isomeric with

Ref.

                    

56

Minutisporin-1 (pos. 1–3, 6, 7, 11–16, 18 and 19: cf. trichostrigocins A and B)

Degenkolb et al. 2006a

                 

57

Minutisporin-2 (cf. hypophellin-18: [Pheol]19 → [Lxxol]19; pos 1, 6, 7, 9, and the C-terminal nonapeptide: tricholongin B-I)

Rebuffat et al. 1991

                 

58

Minutisporin-3 (cf. hypophellin-19: [Pheol]19 → [Lxxol]19; trichosporin B-IIIb – [Aib]6, [Pheol]19 → [Lxxol]19)

Röhrich et al. 2013a, b; Iida et al. 1990

                 

59

Minutisporin-4 (cf. hypophellin-20: [Pheol]19 → [Lxxol]19; cf. trichosporin B-VIa – [Aib]6, [Aib]16 → [Vxx]16, [Pheol]19 → [Lxxol]19; C-terminal nonapeptide, cf. tricholongin B-II; cf. trichocellin A-5 – [Ala]6, [Pheol]20 → [Lxxol]20)

Rebuffat et al. 1991; Wada et al. 1994

                 

60

Minutisporin-5 (C-terminal octapeptide, cf. hypelcin B-III)

Matsuura et al. 1994

                 

61

Minutisporin-6 (cf. hypophellin-22: [Pheol]19 → [Lxxol]19; trichorzin HA-V: [Vxx]5–[Pro]13 and C-terminus with [Lxx]14 → [Vxx]14)

Hlimi et al. 1995; Röhrich et al. 2013a

                 

62

Minutisporin-7

                     

63

Minutisporin-8

                     

aVariable residues are underlined in the table header. Minor sequence variants are underlined in the sequences. This applies to all sequence tables

Table 11

Sequences of 19-residue peptaibiotics detected in the plate culture of Hypocrea minutispora

No.

tR [min]

[M + H]+

 

Residuea

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

64

36.1–36.3

1832.1060

Ac

Aib

Ala

Aib

Ala

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Aib

Gln

Gln

Vxxol

65

37.3–37.5

1832.1025

Ac

Aib

Ala

Aib

Gly

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Vxx

Gln

Gln

Vxxol

66

37.5–37.9

1846.1196

Ac

Aib

Ala

Aib

Ala

Aib

Gln

Aib

Lxx

Aib

Gly

Vxx

Aib

Pro

Vxx

Aib

Vxx

Gln

Gln

Lxxol

57

37.8–38.0

1846.1199

Ac

Aib

Ala

Aib

Ala

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Aib

Gln

Gln

Lxxol

67

38.6–38.7

1847.1135

Ac

Aib

Ala

Aib

Ala

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Aib

Glu

Gln

Lxxol

59

39.0–39.2

1860.1318

Ac

Aib

Ala

Aib

Ala

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Vxx

Gln

Gln

Lxxol

60

39.8–40.0

1861.1271

Ac

Aib

Ala

Aib

Ala

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Vxx

Glu

Gln

Lxxol

68

40.4–40.6

1874.1492

Ac

Aib

Ala

Aib

Ala

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Lxx

Aib

Vxx

Gln

Gln

Lxxol

61

40.6–40.9

1874.1554

Ac

Aib

Ala

Aib

Ala

Vxx

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Vxx

Gln

Gln

Lxxol

No.

Compound identical or positionally isomeric with

Ref.

                    

64

Minutisporin-9 (pos. 1, 6–10, 12–19; [Pro]2 → [Ala]2, [Aib]11 → [Lxx]11 and deletion of [Aib]5: cf. stilboflavin B-5)

Jaworski and Brückner 2001b

                

65

Minutisporin-10 (positional isomer of 64: [Ala]4 → [Gly]4, [Aib]16 → [Vxx]16)

                     

66

Minutisporin-11 (positional isomer of 57: [Lxx]11 → [Vxx]11, [Aib]16 → [Vxx]16)

                     

57

Minutisporin-2

                     

67

Minutisporin-12 (positional isomer of 57: [Gln]17 → [Glu]17 and of 56: [Ala]4 → [Gly]4, [Aib]16 → [Vxx]16)

                     

59

Minutisporin-4

                     

60

Minutisporin-5

                     

68

Minutisporin-13 (positional isomer of 61: [Aib]5 → [Vxx]5)

                     

61

Minutisporin-6

                     

aVariable residues are underlined in the table header. Minor sequence variants are underlined in the sequences. This applies to all sequence tables

Fig. 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

Screening of Hypocrea citrina. The specimen of H. citrina was shown to be a prolific producer of 19-residue peptaibols, compounds 6978, of which seven are new, viz. compounds 69, 70, 7274, 76, and 78. The names hypocitrins 17 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

No.

tR [min]

[M + H]+

 

Residuea

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

69

31.6–31.7

1926.1036

Ac

Aib

Ala

Aib

Ala

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Vxx

Gln

Gln

di-OH-Pheol

70

32.0–32.1

1896.0937

Ac

Aib

Ala

Aib

Ala

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Aib

Gln

Gln

Tyrol

71

32.9–33.1

1910.1084

Ac

Aib

Ala

Aib

Ala

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Vxx

Gln

Gln

Tyrol

72

33.6–33.9

1880.0971

Ac

Aib

Ala

Aib

Gly

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Vxx

Gln

Gln

Pheol

73

34.6–34.7

1880.0975

Ac

Aib

Ala

Ala

Ala

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Vxx

Gln

Gln

Pheol

74

36.4–36.6

1880.0999

Ac

Aib

Ala

Ala

Ala

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Vxx

Gln

Gln

Pheol

75

37.7–37.9

1880.1050

Ac

Aib

Ala

Aib

Ala

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Aib

Gln

Gln

Pheol

76

38.2–38.4

1880.1018

Ac

Aib

Ala

Ala

Ala

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Vxx

Gln

Gln

Pheol

77

38.8–39.1

1894.1241

Ac

Aib

Ala

Aib

Ala

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Vxx

Gln

Gln

Pheol

78

39.7–39.9

1895.1083

Ac

Aib

Ala

Aib

Ala

Aib

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Vxx

Glu

Gln

Pheol

No.

Compound identical or positionally isomeric with

Ref.

                    

69

Hypocitrin-1 (homologue of hypophellin-15: [Tyrol]19 → [di-OH-Pheol]19)

Röhrich et al. 2013a

                    

70

Hypocitrin-2 (homologue of hypophellin-15: [Vxx]17 → [Aib]17)

Röhrich et al. 2013a

                    

71

Hypophellin-15

Röhrich et al. 2013a

                    

72

Hypocitrin-3 (positional isomer of 73, 74, and 76: [Ala]3 → [Aib]3, [Ala]4 → [Gly]4)

                     

73

Hypocitrin-4 (positional isomer of 75 and 77, homologue of hypophellin-17: [Vxx]17 → [Aib]17)

Röhrich et al. 2013a

                    

74

Hypocitrin-5 (positional isomer of 73 and 77, homologue of hypophellin-17: [Vxx]17 → [Aib]17)

Röhrich et al. 2013a

                    

75

Hypophellin-18

Röhrich et al. 2013a

                    

76

Hypocitrin-6 (positional isomer of 73 and 75, homologue of hypophellin-17: [Vxx]17 → [Aib]17)

Röhrich et al. 2013a

                    

77

Hypophellin-20

Röhrich et al. 2013a

                    

78

Hypocitrin-7 (homologue of 77: [Gln]17 → [Glu]17)

                     

aVariable residues are underlined in the table header. Minor sequence variants are underlined in the sequences. This applies to all sequence tables

Fig. 6

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

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 3739, 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 4651), 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)

 

Residue

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19a

20b

Ac

Aib

Ala

Aib

Ala

Aib

Ala

Gln

Aib

Lxx

Aib

Gly

Lxx

Aib

Pro

Vxx

Aib

Vxx

Gln

Gln

Pheol

 

(Vxx)

(Ser)

Ala

Aib

(Vxx)

(Aib)

 

(Vxx)

Aib

(Ala)

Ala

(Vxx)

(Vxx)

 

Lxx

(Vxx)

Aib

(Glu)

-

Lxxol

  

(Aib)

(Ser)

(Lxx)

(Phe)

  

(Ala)

(Vxx)

 

(Ser)

(Aib)

  

(Aib)

 

(Lxx)

 

(Glu)

(Vxxol)

  

(Lxx)

(Vxx)

(Ser)

(Ala)

              

(Tyrol)

  

(Vxx)

 

(Gly)

(Lxx)

              

(Tyr(C5H8)ol)

                    

(di-OH-Pheol)

Minor sequence variants are parenthesised

aOne of the Gln/Glu residues is deleted in some of the truncated sequences

bThe C-terminal amino alcohol is deleted in some of the truncated sequences

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 peptaibiotics8. 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

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 VI9 (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.

Footnotes

  1. 1.

    Authors are aware of the drastic change of the ICBN (International Code of Botanical Nomenclature), which has been adopted at the IBC in Melbourne in July 2011 (Gams et al. 2012; Rossman et al. 2013). However, all strains used in this study were deposited at CBS in July/August 2012, and practical work for this study was finished in December 2012. For reasons of conformity with recently published contributions in the field of peptaibiotics, dual nomenclature is retained in this chemically focussed article.

  2. 2.

    The trichorzianin-producing strain ATCC 36042 (= CBS 391.92) has originally been identified as T. harzianum (el Hajji et al. 1987) but later shown to belong to T. atroviride (Kuhls et al. 1996).

  3. 3.

    Neither a specimen, nor a culture of the hypelcin producer has been deposited. However, misidentification of H. peltata is impossible due to its cushion-like big stromata and distinctive bicellular ascospores (Samuels and Ismaiel 2011).

  4. 4.

    Defined as the dynamic entirety of peptaibiotics formed by a producing fungus under defined culture conditions (Krause et al. 2006a).

  5. 5.

    The trichotoxin A-producing strain NRRL 5242 (now A-18169 in the ARS culture collection = CBS 361.97 = ATCC 38501) has originally been identified as T. viride but was subsequently reidentified as T. asperellum (Lieckfeldt et al. 1999; Samuels et al. 1999). The trichotoxin B (= trichovirin) producer, strain NRRL 5243 (= ATCC 90200), is not in the ARS catalogue but available as A-18207.

  6. 6.

    Hypomurocins have been isolated from strain IFO 31288 (Becker et al. 1997), originally misidentified as Hypocrea muroiana. The producer belongs, in fact, to T. atroviride (Samuels et al. 2006).

  7. 7.

    The neoatroviridin producer T. atroviride F80317 (Oh et al. 2005) has neither been deposited with an IDA, nor has its identity been verified phylogenetically.

  8. 8.

    Nielsen KF, Samuels GJ (2013) unpublished results.

  9. 9.

    Trichokonin VI is identical to gliodeliquescin A that has been isolated from Gliocladium deliquescens NRRL 1086 (Brückner et al. 1988) and not from NRRL 3091 (Brückner and Przybylski 1984). According to phylogenetic data, G. deliquescens NRRL 1086 (= CBS 228.48 = ATCC 10097) was re-identified as G. viride, see (www.straininfo.net/strains/260309).

Notes

Acknowledgments

This study was supported by the Hessian Ministry for Science and Art by a grant from the LOEWE-Schwerpunkt ‘Insect Biotechnology’ to Andreas Vilcinskas. DTU acknowledges the grant from the Danish Research Council (FI 2136-08-0023) for the maXis QTOF system, and MYCORED (EC KBBE-2007-222690-2) for supporting Anita Iversen. Walter M. Jaklitsch gratefully acknowledges the support by the Austrian Science Fund (project P22081-B17). Thanks to James L. Swezey (USDA-ARS, NCAUR) for his comments on two peptaibol-producing Trichoderma strains, NRRL 5242 and NRRL 5243. Hans Brückner gratefully acknowledges his position as a Visiting Professor at King Saud University (Riyadh, Kingdom of Saudi Arabia).

Supplementary material

13225_2013_276_MOESM1_ESM.doc (648 kb)
ESM 1 (DOC 647 KB)

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© The Author(s) 2014

Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

Authors and Affiliations

  • Christian R. Röhrich
    • 1
    • 7
  • Walter M. Jaklitsch
    • 2
  • Hermann Voglmayr
    • 2
  • Anita Iversen
    • 3
    • 8
  • Andreas Vilcinskas
    • 1
    • 6
  • Kristian Fog Nielsen
    • 3
  • Ulf Thrane
    • 3
  • Hans von Döhren
    • 4
  • Hans Brückner
    • 5
  • Thomas Degenkolb
    • 6
  1. 1.Bioresources Project GroupFraunhofer Institute for Molecular Biology and Applied Ecology (IME)GiessenGermany
  2. 2.Department of Systematic and Evolutionary Botany, Faculty Centre of BiodiversityUniversity of ViennaViennaAustria
  3. 3.Department of Systems BiologyTechnical University of DenmarkLyngbyDenmark
  4. 4.Biochemistry and Molecular Biology OE 2, Institute of ChemistryTechnical University of BerlinBerlinGermany
  5. 5.Interdisciplinary Research Centre for BioSystems, Land Use and Nutrition (IFZ), Department of Food Sciences, Institute of Nutritional ScienceUniversity of GiessenGiessenGermany
  6. 6.Interdisciplinary Research Centre for BioSystems, Land Use and Nutrition (IFZ), Department of Applied Entomology, Institute of Phytopathology and Applied Zoology (IPAZ)University of GiessenGiessenGermany
  7. 7.AB SCIEX Germany GmbHDarmstadtGermany
  8. 8.Danish Emergency Management AgencyCopenhagenDenmark

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