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Peptaibol, Secondary-Metabolite, and Hydrophobin Pattern of Commercial Biocontrol Agents Formulated with Species of the Trichoderma harzianum Complex by Thomas Degenkolb* a ) 1), Kristian Fog Nielsen b ), Ralf Dieckmann c ) 2 ), Fabiano Branco-Rocha d ), Priscila Chaverri e ) f ), Gary J. Samuels g ) 3 ), Ulf Thrane b ), Hans von Dçhren c ) 4 ), Andreas Vilcinskas h ) i ), and Hans Brîckner a ) a

) Interdisciplinary Research Centre for BioSystems, Land Use and Nutrition (IFZ), Department of Food Sciences, Institute of Nutritional Science, University of Giessen, Heinrich-Buff-Ring 26 – 32, DE-35392 Giessen b ) Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Building 221, DK-2800 Kgs. Lyngby c ) Biochemistry and Molecular Biology OE 2, Institute of Chemistry, Technical University of Berlin, Franklinstrasse 29, DE-10587 Berlin d ) Universidade Federal do Recüncavo da Bahia, Centro de CiÞncias Agr‚rias, Ambientais e Biolýgicas, Cruz das Almas, Bahia, Brazil e ) University of Maryland, Department of Plant Science and Landscape Architecture, 2112 Plant Sciences Building, College Park, MD 20742, USA f ) Universidad de Costa Rica, Escuela de Biolog†a, Apartado 11501 – 2060, San Pedro, San Jos¦, Costa Rica g ) United States Dept. of Agriculture, Agricultural Research Service, Systematic Mycology and Microbiology Lab., B-010, Beltsville, MD 20705, USA h ) Interdisciplinary Research Centre for BioSystems, Land Use and Nutrition (IFZ), Department of Applied Entomology, Institute of Phytopathology and Applied Zoology (IPAZ), University of Giessen, Heinrich-Buff-Ring 26 – 32, DE-35392 Giessen i ) Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), LOEWE Center for Insect Biotechnology and Bioresources (ZIB), Winchesterstrasse 2, DE-35394 Giessen Dedicated to Dr. Harald Bocker, Jena, on the occasion of his 85th birthday.

The production of bioactive polypeptides (peptaibiotics) in vivo is a sophisticated adaptation strategy of both mycoparasitic and saprotrophic Trichoderma species for colonizing and defending their natural habitats. This feature is of major practical importance, as the detection of peptaibiotics in plantprotective Trichoderma species, which are successfully used against economically relevant bacterial and fungal plant pathogens, certainly contributes to a better understanding of these complex antagonistic interactions. We analyzed five commercial biocontrol agents (BCAs), namely CannaÔ, TrichosanÔ,

1)

2) 3)

4)

Present address: Interdisciplinary Research Centre for BioSystems, Land Use and Nutrition (IFZ), Department of Applied Entomology, Institute of Phytopathology and Applied Zoology (IPAZ), University of Giessen, Heinrich-Buff-Ring 26 – 32, DE-35392 Giessen (phone: þ 49-641-99-37651, fax: þ 49-641-99-37609, e-mail: [email protected]). Present address: Federal Institute for Risk Assessment (BfR), Department of Biological Safety, Diedersdorfer Weg 1, DE-12277 Berlin. Present address: 321 Hedgehog Mt. Rd., Deering, NH 03244, USA. Present address: Schillerstrasse 34, DE-10627 Berlin. Õ 2015 Verlag Helvetica Chimica Acta AG, Zîrich

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)

663

VitalinÔ, PromotÔ WP, and TrichoMaxÔ, formulated with recently described species of the Trichoderma harzianum complex, viz. T. afroharzianum, T. simmonsii, and T. guizhouense. By using the wellestablished, HPLC/MS-based peptaibiomics approach, it could unequivocally be demonstrated that all of these formulations contained new and recurrent peptaibols, i.e., peptaibiotics carrying an acetylated N-terminus, the C-terminus of which is reduced to a 1,2-amino alcohol. Their chain lengths, including the amino alcohol, were 11, 14, and 18 residues, respectively. Peptaibols were also to be the dominating secondary metabolites in plate cultures of the four strains obtained from four of the Trichodermabased BCAs, contributing 95% of the UHPLC-UV/VIS peak areas and 99% of the total ion count MS peak area from solid media. Furthermore, species-specific hydrophobins, as well as non-peptaibiotic secondary metabolites, were detected, the latter being known for their antifungal, siderophore, or plant-growth-promoting activities. Notably, none of the isolates produced low-molecular weight mycotoxins.

1. Introduction. – 1.1. Trichoderma Species as Biocontrol and Growth-Promoting Agents. As of summer 2014, ca. 250 validly described and phylogenetically verified species have been included in the genus Trichoderma (Ascomycota, Pezizomycotina, Sordariomycetes, Hypocreales, Hypocreaceae) [1 – 8]. Trichoderma species have a long, successful history as biocontrol agents (BCAs). Such a BCA in a strict sense (sensu stricto, s. s.) contains one or more taxonomically verified, beneficial microorganism(s) antagonizing a defined range of microbial plant pathogens; recent examples include: – the hyperparasite Trichoderma stromaticum, the active agent of TricovabÔ, a recently approved [9] semi-commercial formulation for integrated pest management of Moniliophthora (syn. Crinipellis) perniciosa, the causal agent of Witches broom disease (WBD) of cocoa (Theobroma cacao) in Brazil [10] [11]; – T. paucisporum and T. theobromicola, which displayed promising in vitro activities against the frosty pod rot of pathogen of cocoa, Moniliophthora roreri [12]; and – T. martiale, which, in small-scale in situ field trials, proved highly effective against black pod rot of cocoa caused by Phytophthora palmivora [13]. Even more Trichoderma species and strains are used in so-called Ðplant growthpromoting agentsÏ (in German: ÐPflanzenst•rkungsmittelÏ), which are regarded as BCAs in a broader sense (sensu lato, s. l.). They are applied as either granules to be mixed with soil, or as water-soluble powders. Use is recommended for cultivation of vegetables and ornamental plants, for plant and tree nurseries and should lead to: – prophylactic protection of plants and scions against soil-borne plant pathogenic fungi; (see also Sect. 1.3); – stimulation of root growth, including a higher percentage of root hairs; – increased yield due to improved germination and rooting rates; – increase of plant dry weight and number of flowers; – earlier onset of flowering and shortening of cultivation periods. The following complex mechanisms may account for the plant-protective and plant growth-promoting effects observed: i) production of volatile (e.g., pyrones: [14]; butenolides, terpenes, and isocyanides: [15]) and non-volatile antifungal compounds (e.g., non-ribosomal polypeptides such as peptaibiotics [16] [17]);

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ii) mycoparasitism, formation of siderophores, competition for nutrients and space in the plant rhizosphere, and/or triggering localized or systemic defense mechanisms or induced resistance [18 – 26]. 1.2. Peptaibiotics and Their Possible Contribution to the Plant-Protective Action of Trichoderma-Based BCAs. Recently, it has been shown that a subclass of nonribosomally biosynthesized, Aib-containing linear polypeptide antibiotics, the peptaibiotics 5 ), which represent the overwhelming majority of secondary metabolites described for Trichoderma [16] [17], was readily produced in vivo, i.e., by specimens collected in the natural habitat. Their unique membrane-modifying bioactivity, which results from amphipathicity and helicity, makes peptaibiotics ideal candidates for assisting both colonization and defense of the natural habitats by their fungal producers. Given that potentially plant-protective polypeptides (peptaibiotics) are not only produced under artificial (i.e., laboratory) conditions, their in vivo detection can be discussed as a sophisticated ecophysiological adaptation strategy of both mycoparasitic and saprotrophic Trichoderma species [27 – 29]. 1.3. Trichoderma harzianum and Its Distinguished Role in Biocontrol. Owing to these encouraging results, we decided to screen five established commercial BCAs, which are – according to the information provided by the manufacturers – formulated with strains of ÐT. harzianumÏ, for the presence of peptaibiotics (Table 1). Trichoderma harzianum (s. l.) is generally regarded as a cosmopolitan and ubiquitous yet largely unresolved species complex, which is capable of colonizing a broad range of substrates. Recently, a study was published aimed at revising the systematics of the T. harzianum species complex, including four strains that have been isolated from commercial plant growth-promoting formulations, viz. T. afroharzianum, T. guizhouense, and T. simmonsii (Table 1) [2]. Usually, T. harzianum isolates are obtained from soil, rotting plant material, other fungi, and, recently, as one the most common endophytes in the sapwood of tropical trees [30 – 37]. Despite occasional reports of T. harzianum being a plant saprobe [38] [39], more recent studies imply that species in this complex exhibit a mycoparasitic or fungicolous mode of nutrition [30]. Due to its antagonistic activity towards soil-borne fungal plant pathogens (Botrytis spp., Fusarium sp., Phytophthora infestans, Pythium spp., Rhizoctonia solani, Sclerotium rolfsii), T. harzianum is probably the most commonly reported complex applied in biocontrol of plant diseases. 1.4. Choice of the Model System. Given the persistently high degree of misidentification amongst plant-protective and antibiotic-producing Trichoderma species [2] [40] [41], the identity of the T. harzianum strains obtained from four of the five BCAs investigated herein was scrutinized and recently revised (Table 1). Consequently, the metabolomic study presented here is aimed at complementing our recent taxonomic contribution [2] by a chemical, peptaibiotic-focussed investigation of the secondary metabolites found in BCAs formulated with species of the Trichoderma harzianum complex.

5)

The non-proteinogenic a-aminoisobutyric acid (Aib) is considered as the marker for peptaibiotics. For more detailed information about structural features of peptaibiotics, the reader is referred to [16][17][27 – 29].

JH Biotech Inc., Ventura, CA, USA d )

MykoMax GmbH, D-Krefeld

Green powder Green powder White powder with a hint of blue

Coarse, dark powder f )

Trichosan

Vitalin

Promot WP

Trichomax

‹ 1   107 ( T. harzianum)

4.5

2.4

2   107 ( T. harzianum) 3   107 ( T. koningii) e )

1.8

1   108 ( T. harzianum)

2.2

2.5

1   108 ( T. harzianum) 1   108 ( T. harzianum)

Yield [mg] b )

Inoculum (spores/g) a )

n. i. g )

T. guizhouense

T. simmonsii

T. afroharzianum

Species

CBS 134707 (IBT 41407, G.J.S. 08-135)

CBS 134708 (IBT 41408, G.J.S. 08-136)

CBS 134706 (IBT 41406, G.J.S. 08-134)

CBS 134709 (IBT 41409, G.J.S. 08-137)

Strain accession numbers

a ) Names of species as indicated by the manufacturer. b ) Peptaibiotics-containing fraction as described in the ÐExper. PartÏ. c ) Obtained from Sauter & Stepper GmbH ( D-Ammerbuch). d ) Obtained from Ernst Mack Fellbach GmbH & Co. KG ( D-Fellbach). e ) Not detected. f ) 1 : 1 Mixture of finely sieved peat ( < 8 mm) and WurzelMaxÔ ( Montmorillonite clay < 30 mm); obtained from Dr. Jîrgen Kutscheidt ( D-Tçnisvorst). g ) The ÐT. harzianumÏ strain could not be recovered from the highly unsterile matrix, delivered in a reclosable plastic bag; n. i., not isolated).

Vitalin Pflanzengesundheit GmbH, D-Ober-Ramstadt c )

Vitalin Pflanzengesundheit GmbH, D-Ober-Ramstadt c )

Canna International BV, NL-Breda

Dark brown powder

Canna

Manufacturer

Appearance

BCA

Table 1. Commercial Biocontrol Agents Investigated in This Work

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2. Results and Discussion. – 2.1. Peptaibiotics as the Dominating Secondary Metabolites in Trichoderma-Based BCAs. As will be outlined below, this investigation undoubtedly revealed that peptaibiotics of different chain length were, indeed, present in commercial Trichoderma-based formulations, which have readily been established on the biocontrol market. The production of peptaibiotics by the Trichoderma strain of the respective BCA was further corroborated by analyzing the peptaibiome 6 ) of four strains obtained from the five commercial formulations by dilution plating (see Exper. Part and Table 1). Pure plate cultures were found to exhibit the same pattern of peptaibiotics (data not shown). Peptaibiotics of different chain length are the dominating secondary metabolites in plate cultures of the four strains obtained from the Trichoderma-based BCAs, contributing 95% of the UHPLC-UV/VIS peak areas and 99% of the total ion count (TIC) MS peak area from solid media. MALDI-TOF Mass spectrometry revealed peptaibols of the subfamily 1 (SF1) as well as 11- and 14residue sequences belonging to subfamily 4 (SF4) [43] as Na þ and K þ adducts. The respective masses were identical to those listed in the corresponding Tables 2 – 7, and Tables S1 – S5 (Supplementary Material). As misidentifications of Trichoderma/Hypocrea strains continue to be a problem (not only in the older literature [2] [40] [41] [44]), the authors prefer to introduce new names for the peptaibiotics sequenced in this study. Those new names refer to the epithets of the recently described producing species [2]. Notably, these results are perfectly in line with recent reports on the unequivocal detection of peptaibiotic biosynthesis in the natural habitats of the producing Trichoderma species [27 – 29]. 2.2. General Remarks on Amino-Acid Analysis. Due to the limited specificity of non-ribosomal peptide synthetases (NRPS), a number of modules will also accept building blocks, which are structurally similar to the dominating one typically found in a defined position. This phenomenon, which is referred to as microheterogeneity, leads to the biosynthesis of structurally highly diverse, natural libraries of peptaibiotics (for a review, see [16] [17]). With the LC-(CID)/MS/MS approaches applied here, isobaric amino acids and 1,2-amino alcohols, respectively, cannot be distinguished. Thus, the abbreviation Vxx is used, which may stand for l-Val, and l-, or d-isovaline (Iva), whereas the corresponding 1,2-amino alcohols valinol (Valol) or isovalinol (Ivaol) are abbreviated as Vxxol. Consequently, Lxx may stand for either Leu or Ile, whereas the abbreviation Lxxol accounts for leucinol (Leuol) or isoleucinol (Ileol), respectively 7 ). Val and d-Iva may even be present in one and the same sequence (cf. positions 5, 7, 8, and 16 in trichorzins HA II – VII: [49]; as well as positions 8 and 11 in hypomurocins B4 and B-5 [48]). Four of the trichokindins terminate in Leuol, whereas the remaining seven carry a C-terminal Ileol residue [50]. 6) 7)

Defined as the dynamic entirety of peptaibiotics produced by a fungal strain under defined culture or natural conditions [42]. Complete structure elucidation would require preparative amounts of isolated individual peptaibols and could only be achieved by combined 2D-NMR [45][46] and i) amino-acid analysis in appropriately derivatized total hydrolysates, and ii) partial hydrolysis/methanolysis experiments, followed by GC/SIM-MS of derivatized dipeptides [47][48]. d-Val has not yet been described as a constituent of linear peptaibiotics [17].

1726 1726 1726 1740 1740 1754 1754 1754 1768

57.3 – 57.8 57.9 – 58.3 58.4 – 58.9 59.7 – 60.1 60.2 – 60.3 60.7 – 61.1 60.7 – 61.1 61.2 – 61.6 62.1 – 62.5

62.6 – 63.1 1768

1 2 3 4 5 6 7 8 9

10

new new TZ_HA I TZ_HA II TZ_HA III new new TZ_HA V TZ_HA VI, TZ_HA VII TZ_HA VI, TZ_HA VII

[ M þ Na] þ Remarks

No. tR [min] Ac Ac Ac Ac Ac Ac Ac Ac

Aib Aib Aib Aib Aib Aib Aib Aib

Gly Gly Gly Gly Gly Gly Gly Gly

Ala Ala Ala Ala Ala Ala Ala Ala

Aib Aib Aib Aib Aib Aib Aib Aib

Ala Aib Aib Aib Aib Vxx Vxx Aib

Vxx Vxx Vxx Vxx Vxx Vxx Vxx Vxx

8 Aib Aib Aib Aib Aib Aib Aib Aib

9 Gly Gly Gly Gly Gly Gly Gly Gly

10

Lxx Lxx Lxx Lxx Lxx Lxx Lxx Lxx

11

12

13

Pro

Gln Gln Gln Gln Gln Gln Gln Gln

Aib Ala Aib Aib Vxx Aib Vxx Vxx

7

Ac Aib Gly Ala Aib Vxx Gln Vxx Vxx Aib Gly Lxx Aib

6

5

Pro

4

Ac Aib Gly Ala Aib Vxx Gln Vxx Vxx Aib Gly Lxx Aib

3

Pro Pro Pro Pro Pro Pro Pro Pro

2

Aib Aib Aib Aib Aib Aib Aib Aib

1

Residue

Lxx

Lxx

Lxx Lxx Lxx Lxx Lxx Lxx Lxx Lxx

14

Aib

Aib

Aib Aib Aib Aib Aib Aib Aib Aib

15

Vxx

Vxx

Vxx Vxx Aib Vxx Aib Vxx Aib Vxx

16

Gln

Gln

Gln Gln Gln Gln Gln Gln Gln Gln

17

Lxxol

Lxxol

Lxxol Lxxol Lxxol Lxxol Lxxol Lxxol Lxxol Lxxol

18

Table 2. Sequences of Afroharzianins, 18-Residue Peptaibols, Detected in Extracts of the BCA ÐCannaÏ. Minor sequence variants in exchange positions are underlined in Tables 2 – 6.

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015) 667

58.0 – 58.3 1756

60.9 – 61.2 1770

61.9 – 62.1 1784 62.5 – 62.9 1784

63.5 – 63.8 1798

12

13 14

15

[M þ Na]

11

No. tR [min] þ

new ( HM B1: Ala4 ! Aib4 ) new ( HM B4: Ala4 ! Aib4 ) new new ( HM B4: Ala4 ! Aib4, Aib7 ! Vxx7 ) new

Remarks 2

3

4

5

6

7

8

9

10

11

12

Ac Aib Ser Aib Lxx Vxx Gln Vxx Vxx Aib Gly Vxx Aib

Ac Aib Ser Aib Lxx Vxx Gln Aib Vxx Aib Gly Vxx Aib Ac Aib Ser Aib Lxx Aib Gln Vxx Vxx Aib Gly Vxx Aib

Ac Aib Ser Aib Lxx Aib Gln Aib Vxx Aib Gly Vxx Aib

Ac Aib Ser Aib Lxx Aib Gln Aib Vxx Aib Gly Aib Aib

1

Residue

Pro

Pro Pro

Pro

Pro

13

Lxx

Lxx Lxx

Lxx

Lxx

14

Table 3. Sequences of Trichosimmonsins, 18-Residue Peptaibols, Detected in the BCA ÐTrichosanÏ

Aib

Aib Aib

Aib

Aib

15

Aib

Aib Aib

Aib

Aib

16

Gln

Gln Gln

Gln

Gln

17

Vxxol

Vxxol Vxxol

Vxxol

Vxxol

18

668 CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)

1756 1770 1784 1784 1798

56.6 – 56.9 59.7 – 60.1 60.9 – 61.2 61.6 – 62.1 62.3 – 62.5

62.6 – 63.0 1798 62.8 – 63.0 1768

63.2 – 63.4 1812

63.4 – 64.1 1782

64.2 – 64.4 1796

16 17 18 19 20

21 22

23

24

25

cf. 11 cf. 12 cf. 13 cf. 14 new (cf. HM B5: Ala3 ! Aib3, Aib7 ! Vxx7 ) cf. 15 new (cf. 14, 19: Ser3 ! Ala3 ) new (homolog of 20: Aib5 ! Vxx5 ) new (cf. 15, 21: Ser3 ! Ala3 ) new (homolog of 24: Vxxol18 ! Lxxol18 )

[ M þ Na] þ Remarks

No. tR [min] Aib Aib Aib Aib Aib

Ser Ser Ser Ser Ser

2 Aib Aib Aib Aib Aib

3 Lxx Lxx Lxx Lxx Lxx

4

6 Gln Gln Gln Gln Gln

5 Aib Aib Vxx Aib Aib

Aib Aib Aib Vxx Vxx

7 Vxx Vxx Vxx Vxx Vxx

8 Aib Aib Aib Aib Aib

9 Gly Gly Gly Gly Gly

10

Aib Vxx Vxx Vxx Vxx

11

Aib Aib Aib Aib Aib

12

Pro Pro Pro Pro Pro

13

Lxx Lxx Lxx Lxx Lxx

14

Aib Aib Aib Aib Aib

15

Aib Aib Aib Aib Aib

16

Gln Gln Gln Gln Gln

17

Vxxol Vxxol Vxxol Vxxol Lxxol

18

Ac Aib Ala Aib Lxx Vxx Gln Vxx Vxx Aib Gly Vxx Aib Pro Lxx Aib Aib Gln Lxxol

Ac Aib Ala Aib Lxx Vxx Gln Vxx Vxx Aib Gly Vxx Aib Pro Lxx Aib Aib Gln Vxxol

Ac Aib Ala Aib Lxx Aib Gln Vxx Vxx Aib Gly Vxx Aib Pro Lxx Aib Aib Gln Vxxol Ac Aib Ser Aib Lxx Vxx Gln Vxx Vxx Aib Gly Vxx Aib Pro Lxx Aib Aib Gln Lxxol

Ac Aib Ser Aib Lxx Vxx Gln Vxx Vxx Aib Gly Vxx Aib Pro Lxx Aib Aib Gln Vxxol

Ac Ac Ac Ac Ac

1

Residue

Table 4. Sequences of Trichosimmonsins, 18-Residue Peptaibols, Detected in Extracts of the BCA ÐVitalinÏ CHEMISTRY & BIODIVERSITY – Vol. 12 (2015) 669

58.3 – 58.9 59.5 – 59.8 59.8 – 60.0 60.0 – 60.2 60.9 – 61.1 61.1 – 61.3 61.4 – 61.6 62.1 – 62.5

62.9 – 63.1 1768

62.9 – 63.1 1782

27 28 29 30 31 32 33 34

35

36

1726 1740 1740 1740 1754 1754 1754 1768

57.9 – 58.2 1726

new (cf. TZ_HA III: Lxxol18 ! Vxxol18 ) cf. 3: TZ_HA I new (homolog of 27) cf. 4: TZ_HA II cf. 5: TZ_HA III cf. 6 cf. 7 cf. 8: TZ_HA V cf. 9, 10: TZ_HAVI, TZ HAVII cf. 9, 10: TZ_HAVI, TZ HAVII new (homolog of 35: Vxx7 ! Lxx7 )

[ M þ Na] þ Remarks

26

No. tR [min] 2

3

4

5

6

7

8

9

10

11

12

Aib Aib Aib Aib Aib Aib Aib Aib

Gly Gly Gly Gly Gly Gly Gly Gly

Ala Ala Ala Ala Ala Ala Ala Ala

Aib Aib Aib Aib Aib Aib Aib Aib Aib Aib Aib Vxx Aib Vxx Vxx Vxx

Gln Gln Gln Gln Gln Gln Gln Gln

Aib Vxx Aib Aib Vxx Vxx Aib Vxx

Vxx Vxx Vxx Vxx Vxx Vxx Vxx Vxx

Aib Aib Aib Aib Aib Aib Aib Aib

Gly Gly Gly Gly Gly Gly Gly Gly

Lxx Lxx Lxx Lxx Lxx Lxx Lxx Lxx

Aib Aib Aib Aib Aib Aib Aib Aib

Ac Aib Gly Ala Aib Vxx Gln Lxx Vxx Aib Gly Lxx Aib

Ac Aib Gly Ala Aib Vxx Gln Vxx Vxx Aib Gly Lxx Aib

Ac Ac Ac Ac Ac Ac Ac Ac

Ac Aib Gly Ala Aib Vxx Gln Aib Vxx Aib Gly Lxx Aib

1

Residue

Pro

Pro

Pro Pro Pro Pro Pro Pro Pro Pro

Pro

13

Table 5. Sequences of Trichoguizins, 18-Residue Peptaibols, Detected in the BCA ÐPromot WPÏ

Lxx

Lxx

Lxx Lxx Lxx Lxx Lxx Lxx Lxx Lxx

Lxx

14

Aib

Aib

Aib Aib Aib Aib Aib Aib Aib Aib

Aib

15

Vxx

Vxx

Aib Aib Vxx Aib Vxx Aib Vxx Vxx

Aib

16

Gln

Gln

Gln Gln Gln Gln Gln Gln Gln Gln

Gln

17

Lxxol

Lxxol

Lxxol Lxxol Lxxol Lxxol Lxxol Lxxol Lxxol Lxxol

Vxxol

18

670 CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)

58.2 – 58.6 1726 59.9 – 60.3 1740

60.5 – 60.7 1754

61.0 – 61.3 1754 61.7 – 61.8 1768

39

40 41

new new (homolog of 8, 9, 34, 35, 41: Lxxol18 ! Vxxol18 ) cf. 6, 31: ( TZ_HAII: Aib7 ! Vxx7 ) cf. 8, 33: TZ_HAV cf. 9, 10, 34, 35: TZ_HAVI, TZ HAVII

[ M þ Na] þ Remarks

37 38

No. tR [min] 2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

Ac Aib Gly Ala Aib Vxx Gln Vxx Vxx Aib Gly Lxx Aib Pro Lxx Aib Vxx Gln Lxxol

Ac Aib Gly Ala Aib Vxx Gln Aib Vxx Aib Gly Lxx Aib Pro Lxx Aib Vxx Gln Lxxol

Ac Aib Gly Ala Aib Aib Gln Vxx Vxx Aib Gly Lxx Aib Pro Lxx Aib Vxx Gln Lxxol

Ac Aib Gly Ala Aib Aib Gln Aib Vxx Aib Gly Lxx Aib Pro Lxx Aib Vxx Gln Vxxol Ac Aib Gly Ala Aib Vxx Gln Aib Vxx Aib Gly Lxx Aib Pro Lxx Aib Vxx Gln Vxxol

1

Residue

Table 6. Sequences of 18-Residue Trichorzin Homologs Detected in the BCA ÐTrichomaxÏ

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015) 671

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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)

Table 7. Survey of 11- and 14-Residue Peptaibols ([M þ Na] þ ) Detected in the Five BCAs Investigated Canna

Trichosan

Vitalin

Promot WP

Trichomax

n. d. 1170 1184 1198 1212 1226

n. d. 1170 1184 1198 1212 1226

1156 1170 1184 1198 1212 1226

n. d. 1439 1453 1467 1481 1495

n. d. n. d. 1453 1467 1481 n. d.

n. d. 1439 1453 1467 1481 n. d.

n. d. n. d. 1437 1451 1465 1479

n. d. 1423 1437 1451 1465 n. d.

1409 1423 1437 1451 1465 n. d.

a

11-Residue peptaibols ) 1156 1170 1184 1198 1212 1226

n. d. 1170 1184 1198 1212 1226

14-Residue peptaibols (carrying Ser6 ) 1425 1439 1453 1467 1481 n. d.

1425 1439 1453 1467 1481 1495

14-Residue peptaibols (carrying Ala6 ) 1409 1423 1437 1451 1465 n. d.

1409 1423 1437 1451 1465 1479

a ) 11-Residue peptaibols originate from 14-residue precursors by deletion of the internal tripeptide Aib4Pro5-Ser6, most probably by module skipping [51] n.d., not detected.

2.3. Peptaibols Detected in Commercial Trichoderma-Based BCAs. Three major groups of peptaibols were detected in every formulation, viz. 18-residue sequences belonging to SF1 [43] (see Tables 2 – 6), as well as 11- and 14-residue sequences belonging to SF4 [43] (see Table 7). The 11-residue family is widespread in 20 of 34 Trichoderma species investigated, whereas the 14-residue family has been detected in 11 of 34 Trichoderma species [44]. While SF1 peptaibols originate from 18-module NRPS, SF4 peptaibols are biosynthesized by 14-module NRPS. Trichoderma genomes investigated contain one NRPS gene for SF1 compounds and one 14module NRPS gene for the 11- and 14-residue sequences, respectively [51]. To date, ca. 75 sequences of 14-residue SF4 peptaibols and 250 sequences of 11-residue SF4 peptaibols have been described [17]. Many 11-residue peptaibols represent deletion sequences of corresponding 14-residue peptaibols, the former originating from skipping of modules 4 (Aib), 5 (Pro), and 6 (Ser/Ala), as recently described in [51] [52]. Notably, all of them are produced both by saprotrophic and fungicolous species of Trichoderma/Hypocrea, which are found in different clades [31]. As the value of 11and 14-residue peptaibols for chemotaxonomic approaches is thus rather limited, no sequences and diagnostic fragment ions are given here. The 18-residue peptaibols found in this study can be divided into two subtypes, in Canna (Table 2 and Table S1, Fig., a), Promot WP (Table 5 and Table S4), and

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TrichoMax (Table 6 and Table S5): afroharzianins or trichoguizins, i.e., sequences resembling trichorzin HA 8 ) (TZ_HA) with a glycyl residue in position 2 [49] [53], were produced, whereas trichosimmonsins found in Trichosan (Table 3 and Table S2, and Fig., b) and Vitalin (Table 4 and Table S3, and Fig., c) resemble hypomurocins B [48] and trichokindins [50], all carrying a seryl residue in position 2. A general building scheme for these 18-residue SF1-peptaibols is given in Table 8. Notably, 31 of the 41 18residue peptaibols sequenced are new, including positionally isomeric peptides. 2.4. Possible Bioactivities of Peptaibiotics Detected. Bioactivities of 11-residue SF4peptaibiotics have recently been compiled of (cf. [56 – 58]). Harzianins HC, which have been isolated from two T. harzianum strains (MNHN903614 and MNHN-903603, resp.), are regarded as the classical paradigm for 14residue SF4-peptaibols [55]. They were shown to display antibacterial (towards Gram positive bacteria), antifungal, and membrane-modifying activities. Voltage-gated ion channels were formed in a similar manner [55] [59] as described for long-chain SF1 peptaibiotics with 17 to 20 residues [60]. Some of the harzianins HC were shown to be identical with trichovirins I 9 ) from T. cf. afroharzianum NRRL 5243 10) [62 – 64]. A single sequence, SPF-5506-A4 , from Trichoderma sp. SPF-5506 was reported to inhibit the formation of amyloid b-peptides in primary guinea pig cerebral cortex neuron cell culture [65]. SPF-5506-A4 is a homolog of those 14-residue peptaibols that display Ser6, namely harzianins I, III, XI, XII, and trichovirin Ib. Detailed comments on the importance of different structurally conserved amino acid residues for the bioactivity of SF1peptaibiotics can be found in [27], whereas multiple bioactivities of 20-residue SF1peptaibiotics were reported in [28]. Biological activities of 18-residue SF1 peptaibols, the sequences of which are closely related to those reported in this work, are compiled in Table 9. Currently, the beneficial role of peptaibiotics in plant protection is best explained by the widely accepted model of Schirmbçck et al. [79] and Lorito et al. [80]. Under in vitro conditions, the parallel formation of peptaibiotics such as the 19-residue trichorzianins 11) and of hydrolytic enzymes, above all chitinases and b-1,3-glucanases, could be demonstrated. Thus, a synergistic interaction of peptaibiotics and hydrolases in the course of mycoparasitism of Trichoderma atroviride vs. Botrytis cinerea was postulated. 2.5. Production of Low-Molecular-Weight Secondary Metabolites by BCAs. None of the four strains produced the renounced 6-pentyl-2H-pyran-2-one (6-PAP), but all of them did produce a series of secondary metabolites, with T. afroharzianum G.J.S. 08-137 being the most prolific producer of high amounts of anthraquinones (chrysophanic acid; w-hydroxypachybasin) and related isocoumarins [83]. Besides those, the expected fleephilone and harziphilone were also detected [84] [85]. Finally, 8)

Trichorzins HA were originally published as harzianins HA [53], but later renamed trichorzins HA [49][54][55]. 9) The crystal structure of the first 14-residue peptaibol, trichovirin I-4A, and its tentative conformational change on interaction with lipid bilayer membranes have recently been published [61]. 10) Erroneously reported as NRRL 5234 [55]. 11) The trichorzianin producer ATCC 36042 ( ¼ CBS 391.92) has originally been published as T. harzianum [81]. Subsequent molecular analysis revealed its affiliation to T. atroviride [82].

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Figure. HPLC Elution profile of the peptaibiotic-containing fraction of a) Canna, b) Trichosan, and c) Vitalin. Annotations correspond to the consecutive numbering of peptides used in the text and the Tables. Non-annotated peaks between 30 and 50 min represent 11- and 14-residue peptaibols, which have not been sequenced (for details, see text).

Principal strain T. afroharzianum

T. simmonsii T. simmonsii T. guizhouense

n. i.

BCA

Canna

Trichosan

Vitalin

Promot WP

Trichomax

Ac

Ac

Ac

Ac

Ac

Aib

Aib

Aib

Aib

Aib

1

Aib Aib Ala

Ala

Ser Ala Gly

Gly

Ala

Gly

Ser

3

2

Aib

Aib

Lxx

Lxx

Aib

4

Vxx Aib

Aib Ala Vxx Aib Vxx Aib Vxx Vxx Aib

5

Gln

Gln

Gln

Gln

Gln

6

Module number and specificity profiles Aib Vxx Als Aib Vxx Vxx Aib Vxx Aib Lxx Aib Vxx

7

Vxx

Vxx

Vxx

Vxx

Vxx

8

Aib

Aib

Aib

Aib

Aib

9

Gly

Gly

Gly

Gly

Gly

10

Lxx

Vxx Aib Vxx Aib Lxx

Lxx

11

Aib

Aib

Aib

Aib

Aib

12

Pro

Pro

Pro

Pro

Pro

13

Lxx

Lxx

Lxx

Lxx

Lxx

14

Aib

Aib

Aib

Aib

Aib

15

Vxx

Vxx Aib

Aib

Aib

Vxx Aib

16

Gln

Gln

Gln

Gln

Gln

17

Lxxol Vxxol

Lxxol Vxxol Lxxol Vxxol

Vxxol

Lxxo

18

Table 8. Specificity of Non-Ribosomal Peptide Synthetases Forming 18-Residue Peptaibols in the BCA Strains Investigated. Invariable positions are highlighted in bold, whereas variable positions are underlined.

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015) 675

F 80317

IFO 31288 unspecified soil isolate from Nara ( Japan) CBS 361.97 ( ATCC 38501, NRRL 5242) PC01 T32 CBS 130670 ( ATCC 90200, NRRL 5243, G.J.S. 99-1) CBS 101875 ( BBA 70638, G.J.S. 97-183: holotype); CBS 101730 ( BBA 70636, G.J.S. 97-180)

T. harzianum

T. harzianum T. atroviride T. atroviride b ) T. harzianum T. asperellum c )

T. harzianum T. asperellum d ) T. cf. afroharzianum e ) T. stromaticum

Trichorzins HA Trichorzins MA

Trichorzins PA

Neoatroviridins

Hypomurocins B Trichokindins

Trichotoxins A-40

Trichotoxins A-50 Mixture of neutral and acidic trichotoxins Mixture of neutral and acidic trichotoxins Trichovirins II

Trichostromaticins

[54]

Antibacterial (g ) Antifungal Ion-channel formation in BLM Antimycoplasmic Ion-channel formation in BLM, ionophoric Antibacterial (g þ ), antifungal, cytotoxic against human cancer cell lines Ion-channel formation in BLM Antibacterial (g þ ), hemolysis of rat erythrocytes Induction of Ca2 þ -dependent catecholamine secretion from bovine adrenal medullary chromaffin cells: ion-channel formation in BLM Hemolysis of human erythrocytes; ion-channel formation in BLM Ion-channel formation in BLM Antifungal, plant growth promoting

Antifungal: active ingredient of BCA ÐTricovabÏ

Antibacterial (g þ ), hemolysis of sheep erythrocytes

Antibacterial, active ingredient of BCA

[9] [78]

[47]

[77]

[73 – 75] [76]

[71] [72]

[70] [48] [50]

[53] [66] [67] [68] [69]

Ref.

þ

Bioactivities reported

a ) Phylogenetically verified producing strains are highlighted in bold. b ) Originally misidentified as Hypocrea muroiana. c ) Originally misidentified as T. viride. d ) Originally misidentified as T. harzianum. e ) Originally misidentified as T. viride; currently deposited as T. harzianum with CBS. Compared to T. afroharzianum (G.J.S. 04-197, 04-186, 05-113, 05-93), this strain shows only 97% similarity in tef1, at most.

MNHN-902608

MNHN-903602 MNHN-922835

Producing species

Peptaibols

Producing strains a )

Table 9. Biological Activities of 18-Residue SF1-Peptaibols Structurally Closely Related to New Sequences Reported in this Work

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harzianolides A, B, and D, as well as harzianic acid, and trichoharzin (Table 10), were found in G.J.S. 08-137, while harzianopyridone and ergokonin B could only be detected in the three other strains screened. Notably, T. guizhouense G.J.S. 08-135 and T. afroharzianum G.J.S. 08-137 were identified as producers of the chlorinated dichlorodiaportin (cf. Table 10). All extracts were also scrutinized for mycotoxins (s. s.) known from the genus Trichoderma, viz. trichodermin and harzianum A/B [41], as well as other mycotoxins (s. s.), which have not yet been described from the genus Trichoderma such as T-2 toxin, gliotoxin, aflatoxins, etc. None of these toxins or other toxic compounds were detected, even though using a triple data analysis approach of the UHPLC-DAD-qTOF data files: i) UV/VIS data were compared to our UV/Vis library [86], ii) full scan data were inspected by the aggressive dereplication approach [87] using the accurate mass and isotopic pattern of the toxins, as well as all other Trichoderma-derived metabolites. For those compounds with a reference standard, the retention time was compared, iii) finally, all MS/HR-MS data were matched against the DTU Mycotoxin-Fungal Secondary Metabolite MS/HRMS library [88], which currently contains 1,400 compounds, including all major mycotoxins. Part of the library is freely available to download (277 compounds in Agilent PCDL format) from the homepage of the Technical University of Denmark [88] [89]. 2.6. Fingerprinting of BCA Strains by MALDI-TOF Mass Spectrometry. Fingerprinting of whole fungal cells by MALDI-TOF mass spectrometry has been used to identify and differentiate species and strains [90] [91]. It has been shown that prominent peptides include processed hydrophobins, i.e., Cys-rich hydrophobic surfactant proteins that are unique to the fungal kingdom, in a mass range of 5 to 12 kDa [92]. Comparing the specificity regions of 5 to 12 kDa, it is evident that Trichosan and Vitalin contain identical species, as three major peptidic components of 7293, 7473 and 7585 Da are found in both strains. This finding agrees with recent phylogenetic data [2]. These two strains of T. simmonsii differ in other peptide components in the fingerprint region (cf. Table 11) and in the production of different sets of 18-residue peptaibols. Thus, domains 2 and 18 of the respective peptide synthetase gene show slightly different substrate specificities (Table 8). While domain 2 is strictly Ser-specific in CBS 134706, the domain of CBS 134708 accepts Ala as well. The final domain 18 of CBS 134708 also is less stringent and accepts both Vxx and Lxx, while the one of CBS 134706 is Lxx-specific. 2.7. Safety Aspects of BCAs Formulated with Species of the Trichoderma harzianum Complex. As mentioned above, the four strains isolated from the respective BCAs did not produce any low-molecular-weight secondary metabolites, which meet the criteria for classification as mycotoxins (s. s.) [93]. Notably, simple non-macrocyclic mycotoxins have only been reported from four species of the Brevicompactum clade – far distant from any species of practical importance in biocontrol [41]. The potential ÐtoxicÏ effect of peptaibiotics, which is repeatedly claimed from in vitro models [94 – 97], has been discussed in [40]. Trichoderma infections in humans or animals are rather rare. Notably, the majority of species has never been reported as the causal agent of human infection, although a few of them are infrequently encountered as pathogens of humans, most clinically relevant isolates belonging to T. longibrachiatum (s. s.) or T. orientale [98]. Practical use of those two particular species in agriculture and industry should therefore, be

Producing species T. afroharzianum

T. simmonsii T. simmonsii T. guizhouense

BCA

Canna

Trichosan

Vitalin

Promot WP

CBS 134707

CBS 134708

CBS 134706

CBS 134709

Accession No.

Chrysophanic acid*, w-hydroxypachybasin*, citreoisocoumarin*, 6-methylcitreoisocoumarin*, w-hydroxyemodin*, harzianolide A#, harzianic acid, trichoharzin, fleephilone, harziphilone, dichlorodiaportin*, ferricrocin*, harzianolides B and D, unknown anthraquinones C19 H20O6 and C19 H22O6 , three unidentified monooxygenated sesquiterpenes C15 H24O Harzianopyridone*#, ergokonin B, SC 2051, harzianolides B and D, ferricrocin* Diaportinol*, diaportinic acid*, ergokonin B, harzianopyridone*#, trichodermamide A, ferricrocin* Dichlorodiaportin*, diaportinol*, diaportinic acid*, 6-methylcitreoisocoumarin*, harzianolides B and D, harzianopyridone*#, ferricrocin*

Low-molecular-weight secondary metabolites detected

Table 10. Non-Peptaibiotic, Low-Molecular-Weight Secondary Metabolites Detected in Plate Cultures Analyzed. The identity of the metabolites marked with * was confirmed by authentic reference standards. Antifungal metabolites are highlighted in bold, whereas those compounds acting as siderophores are underlined, and substances exhibiting plant-growth promotion activity are marked with # [15] [85].

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Table 11. MALDI-TOF Mass Spectral Fingerprint Data of Biocontrol Strains Investigated in This Work BCA Species

Peaks [m/z]

Mass range [Da]

Canna T. afroharzianum

6000 – 7000

6689 6966

7000 – 8000

7205

7720 7868 7926 8000 – 9000 Š 9000

8840

Trichosan T. simmonsii 6951

Vitalin T. simmonsii 6951

Promot WP T. guizhouense 6951 7189

7293 7473

7293 7475

7585

7585

8807

8807

10043 10540

10291 10869

7539

8808

10540

restricted or avoided [99]. In this regard, a recommendation was published that every Trichoderma culture to be applied in agriculture should be tested for its ability to grow at 378 before receiving final approval [100]. It should be emphasized here that growth at 378 is not an exclusion criterion for a particular Trichoderma species; Trichoderma reesei, which grows well above 378, has been used commercially and experimentally for decades [101], but it has never been implicated in any medical case. The identity of the isolate from the only case of an alleged T. reesei infection reported in humans remains doubtful, as no details were provided in [102] how this particular culture has been identified. To date, three clinically relevant cases of infections with Trichoderma harzianum (s. l.) have been reported: the first one associated with peritonitis caused by chronic ambulatory peritoneal dialysis in a diabetic patient (CAPD) [103]; the second one, a disseminated infection in a renal transplant recipient [104], and the third one, a leukemic pediatric patient [105]. The severe and, finally, fatal course of all three cases can partially be attributed to i) the resistance of the respective isolates to antifungal therapy, and ii) the predisposition of the respective patients because CAPD, organ transplantation, and haematological disorders were recently listed as the three main risk factors for a Trichoderma infection [100]. Handling of BCAs in agriculture should therefore be restricted to qualified, trained, and healthy individuals in order to minimize the risks of possible allergic reactions, fungal infection, and mycotoxicosis [106]. The discussion in this paragraph further emphasizes the urgent need for disentangling and taxonomic reconsideration of the T. harzianum species complex.

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Reliable species identification cannot be achieved based on sequencing of ITS only, as it requires at least a second marker gene such as tef1 or rpb2 [2]. Such an approach is crucial, not only for clinical isolates of Trichoderma sp., but also for safety precautions applicable to users of BCAs. Experimental Part Extraction of Peptaibiotics from BCAs. Every BCA container was thoroughly surface-sterilized using Fermicidal D2 spray from IC Products (CH-Minusio). Containers were opened and sampled aseptically in a laminar flow cabinet. Five g of powder were suspended in a conical flask containing 100 ml of CH2Cl2/MeOH 1 : 1 (v/v). The resulting suspension was stirred in the closed flask. After 30 min of stirring, the soln. was allowed to settle, and the supernatant was filtered. This extraction procedure was repeated twice. Finally, the combined filtrates were evaporated to dryness in vacuo. The residue was re-dissolved in H2O/MeOH 1 : 1 (v/v) and cleaned up as described in [107]. Isolation of Strains from BCAs. Pure cultures of those Trichoderma species used for manufacturing the commercial powdery formulations Canna AkTRIvatorÔ, PromotÔ WP, TrichosanÔ, and VitalinÔ were obtained by dilution plating on DifcoTM potato dextrose agar (PDA) purchased from Becton Dickinson (BD, D-Heidelberg) [2]. Cultivation and Extraction of Strains for Peptaibiomics. The same medium used for isolation was also used for cultivation of the Trichoderma strains obtained from dilution plating. Cultures were grown and extracted as described in [107]. HPLC/MS for Peptaibiomics. For a detailed description of the conditions for anal. HPLC and mass spectrometry, see [51]. Cultivation for Characterization of Low-Molecular-Weight Secondary Metaboliotes. All fungal strains used were grown on PDA, oatmeal agar, Wickerhams antibiotic test medium, potato-carrot agar, V8-juice agar, Czapek yeast extract agar, and yeast extract sucrose agar media for 9 d in the dark at 258 [41] [86]. Nine plugs from every culture, each 5 mm in diameter, were extracted with acidic AcOEt/CH2Cl2/ MeOH 3 : 2 : 1 (v/v/v), evaporated to dryness, and redissolved in MeOH as described in [86]. UHPLC-DAD-QTOF MS Analysis. Ultra-high performance liquid chromatography¢diode array detection¢quadruple time-of-flight mass spectrometry (UHPLC-DAD-QTOF-MS) was performed on an Agilent Infinity 1290 UHPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a UV/VIS diode array detector scanning from 190 to 640 nm. Separation was done on an Agilent Poroshell 120 phenyl-hexyl column (2.1   150 mm, 2.7 mm) with a linear gradient consisting of H2O (A) and (MeCN (B) both buffered with 20 mm HCOOH, starting at 10% B and increased to 100% in 15 min where it was held for 2 min. The flow rate was set to 0.35 ml/min, and temp. to 608. The injection volume was 1 ml [88]. MS Detection was performed in ESI þ mode on an Agilent 6550 iFunnel QTOF MS equipped with an Agilent Dual Jet Stream ESI source. Mass spectra were recorded as centroid data from m/z 85 – 1700 in MS mode, and from m/z 30 – 1700 in MS/MS mode. Automated data-dependent acquisition MS/HRMS (auto-MS/HRMS) analysis was conducted at three distinct fragmentations energies (10, 20, and 40 eV) [88]. Data files were processed by the Find by Auto MS/MS function in Agilent MassHunter, mass match tolerance m/z 0.05. The MS/HR-MS library was searched using precursor and product-ion expansion of 50 ppm þ 2 mDa, as well as minimal reverse and forward scores of 50 each. For analysis of compounds described in the literature and not necessarily available as reference standards, aggressive dereplication [87] was performed on the ESI þ and ESI ¢ full-scan data using the Find by Formulae function in Agilent MassHunter Qualitative Analysis B06.00 software. The following adducts and common fragments were included: ESI þ , [M þ H ¢ H2O] þ , [M þ H] þ , and [M þ Na] þ . Subsequently, the identity of compounds was tentatively verified by MS/HRMS data, searching for fragile bonds and the UV/VIS data [86] [87] [88]. Chemicals for MALDI-TOF Analysis. 2,5-Dihydroxybenzoic acid (DHB) and sinapinic acid (SA) from AnagnosTec GmbH (D-Luckenwalde) were used as matrix for MALDI-TOF experiments. Trifluoroacetic acid (TFA), EtOH, MeCN, and MeOH were obtained from Merck KGaA (D-

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Darmstadt) or Fluka (D-Steinheim). Solvents were of HPLC grade, and all other chemicals of anal. grade, unless otherwise stated. Bidist. H2O was freshly prepared from demineralized tap water prior to analysis using a quartz distil (Heraeus, D-Kleinostheim). Extraction and Preparation of Mycelia for MALDI-TOF Analysis. Several mg of fungal mycelia were suspended in MeCN/MeOH/H2O 1 : 1 : 1 (v/v/v), and 1 ml of this suspension was directly spotted onto target wells of a 100-position sample plate and immediately mixed with 1 ml of matrix soln. (10 mg/ml DHB in MeCN/MeOH/H2O 1 : 1 : 1 (v/v/v) and 0.3% TFA). The sample/matrix mixture was air-dried prior to analysis. One ml of the protein soln. was spotted onto a MALDI target plate and mixed with matrix. An estimated 106 cells were used per spot. Data obtained from triplicates confirmed that the MS analyses were reproducible with respect to the characteristic biomarkers obtained. MS Analysis of Low-Molecular-Mass Peptides. Recordings were performed with an Ultraflex II (Bruker, D-Bremen) in the delayed extraction mode, allowing the determination of monoisotopic mass values. A low mass gate of m/z 800 improved the measurement by filtering out the most intensive matrix ions. The mass spectrometer was used in the positive-ion detection and reflector mode. T. D. gratefully acknowledges support by a grant of the Erwin Stein-Foundation (D-Gießen). The Authors are indebted to R. Humm (Vitalin Pflanzengesundheit GmbH, D-Ober-Ramstadt) and Dr. Jîrgen Kutscheidt (Sachverst•ndigenbîro ÐDer gesunde Baum!?Ï D-Tçnisvorst), who provided samples of VitalinÔ and TrichoMaxÔ, respectively. K. F. N. and U. T. acknowledge support from the Danish Research Agency for Technology and Production (grant No. 09-064967) and the EEC project MycoRed (KBBE-2007-222690-2). K. F. N. and U. T. are grateful to Agilent Technologies for the Thought Leader Donation of the UHPLC-QTOF system.

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