**2. Non-ribosomal peptides**

nutrients from the remains of their prey [2]. Mycoparasitic fungi are prolific producers of a plethora of volatile and non-volatile secondary metabolites, favoring their ecological fitness and survival under certain environmental conditions. For example, the excretion of siderophores – affecting high affinity iron chelation – is strongly up-regulated under iron-limiting conditions [3] and several antimicrobial metabolites empower the successful perseverance within the ecological niche [4]. The mycoparasitic lifestyle obviously substantiates the overrepresentation of secondary metabolism-associated genes and the extensive excretion of a variety of secondary metabolites [2] enabling the fungus´ successful access to its prey as well as its thriving persistence in or assassination of the host. Furthermore, selected fungal secondary metabolites are known to exhibit beneficial effects on plants: They may promote vitality and growth of roots and shoots, enhance the resilience against abiotic stress factors and prime the plants immune system (induced systemic resistance; ISR) thereby enhancing its resistance and survival in case of prospective infections with pathogens [5]. In recent times, evidence accumulated that some secondary metabolites also act as communication molecules over spe-

A great diversity of mycoparasitic species exists in the fungal kingdom, especially within the order *Hypocreales* [8]. In this aspect *Trichoderma* (teleomorph *Hypocrea*), a worldwide abundant, diverse fungal genus, is one of the best-studied examples [2]. *Trichoderma* comprises necrotrophic mycoparasitic species like *Trichoderma atroviride* or *Trichoderma virens*, which are successfully applied in agriculture as biocontrol agents against plant pathogenic fungi of crop plants. Further they are reported to promote plant growth, vitality and systemic resistance via priming the plants' immune system. The genomes of several *Trichoderma* species have been sequenced and analyzed revealing the ancestral mycoparasitic lifestyle of these fungi [9, 10]. The second largest lineage of mycorparasites within the *Hypocreales* is the genus *Tolypocladium*. *Tolypocladium* comprises, besides of some entomopathogenic species like *Tolypocladium inflatum,* mostly mycoparasitic species like the widespread on northern hemisphere *Tolypocladium ophioglossoides* which mycoparasitizes with a narrow host range on truffles of the genus *Elaphomyces* [11, 12]. In contrast to *Trichoderma*, the genus *Tolypocladium* exhibits an ancestral entomopathogenic lifestyle and developed to a mycoparasite by host jumping. The genomes of *Tolypocladium* species are rich in secondary metabolite gene clusters of which some, like the clusters for the production of peptaibiotics, seem to be exclusive to mycoparisitic lineages [12]. A further well-investigated mycoparasitic fungus is *Escovopsis weberi*. *E. weberi* is a contact necrotrophic mycoparasite on *Leucoagaricus* sp. in leaf-cutting ant agriculture [13]. As foraging for leaves causes considerable economic damages in neotropic agriculture by defoliation of a wide variety of crop plants, *E. weberi* would be a suitable biocontrol agent as it causes a breakdown of the fungal feeding structures, thereby starving out the ant colony leading to a collapse of the whole system [14]. Like other very specialized mycoparasites, the *E. weberi* genome exhibits a reduced size and content, but very unique secondary metabolite clusters for host attack, facilitating the excretion of fungicidal substances which can break down the host mycelia even without contact [15, 16]. Further examples of secondary metabolite analysis on mycoparasitic species include *Stachybotrys elegans,* a potential biocontrol agent against plant

cies boundaries [6, 7].

38 Secondary Metabolites - Sources and Applications

Non-ribosomal peptides (NRPs) are synthesized by NRPSs, enzymes that characteristically consist of multiple domains synthesizing the peptide in one by one steps. Characteristic for NRPSs are the core domains for adenylation, thiolation and condensation. The generated NRPs are very diverse: they mostly comprise of proteinogenic and non-proteinogenic amino acids, can be linear or branched to cyclic with a varying length. After their synthesis outside of the ribosome, they frequently pass extensive secondary modifications. Many fungal NRPs have high economic and/or ecologic value like β-lactam antibiotics, the immunosuppressant cyclosporine A but also mycotoxins like gliotoxin.

The occurrence of NRPS genes is enriched within the genome of mycoparasitic *T. atroviride*  for 60% and in *T. virens* for 180% to the wood-degrading *T. reesei* [21]. Further, the functional involvement of NRPS and PKS in the mycoparasitic interaction was supported by deletion experiments of the *T. virens* 4-phosphopanteteinyl transferase-encoding gene (*ppt*) – which is essential for NRPs and PKSs activation – resulting in mutant strains defective in mycoparasitism and induction of systemic resistance (ISR) in plants [22]. The main NRPSs derived metabolites in *Trichoderma* species are peptaibiotics, epipolythiodioxypiperazines and siderophores.

#### **2.1. Peptaibiotics**

Peptaibiotics are mostly linear to rarely cyclic polypeptides, with a size of 0.5–2.1 kDa consisting of 4–21 residues. Characteristic for peptaibiotics is the inclusion of the non-proteinogenic amino acid α-aminobutyric acid (Aib). By module-skipping one NRPS is frequently capable of synthesizing a whole set of peptaibiotics [23, 24]. According to their sequence alignment and structure, peptaibiotics can be divided into several sub-clades: peptaibols, lipopeptaibols, lipoaminopeptides, cyclic peptaibiotics and two very special, small categories [25]. Because of their unusual synthesis and appearance, they are not included in regular protein databases, but in the "Comprehensive Peptaibiotics Database" [25].

from *T. virens* Q-strains [37]. *T. virens* P-strains antagonize *Pythium ultimum* and do not produce gliotoxin but the terpenoid gliovirin, whereas Q-strains affect *R. solani* but not vice versa [38]. *C. rosea* also produces ETPs like verticillin A and gliocladines involved in the antagonism on nematodes [39] and glioperazine exhibiting antibacterial properties [40]. Whereas the role of gliotoxin (cluster comprising of 12 genes) as a virulence factor in human *Aspergillus fumigatus* infections and the self-protection via the *gliT* gene product of the biosynthetic gliotoxin cluster is well investigated, there is little and partially adverse information on the role of ETPs

Secondary Metabolites of Mycoparasitic Fungi http://dx.doi.org/10.5772/intechopen.75133 41

The weak mycoparasitic *T. reesei* exhibits an incomplete gliotoxin cluster whose genes were not expressed during confrontation with *R. solani* [4], whereas highly mycoparasitic *T. atroviride* does not contain a gliotoxin cluster [2]. The gliotoxin gene cluster of *T. virens* Q-strains consists of eight genes encoding the core enzyme *gliP* – an NRPS dioxypiperazine synthase – whose expression was induced during confrontation with *R. solani* [4]. Deletion of *gliP* resulted in gliotoxin production-deficient mutants, going hand in hand with a significantly reduced induction of ISR in cotton seedlings and antagonistic action against *P. ultimum* and *S. sclerotiorum.* Adversely, the mutants' antifungal activity against *R. solani* remained unaltered [41]. The involvement of ETPs in mycoparasitic interactions stays unresolved and seems to depend on the combination of several – largely unknown – factors like synergistic interactions with other metabolites or enzymes [42], environmental

Siderophores of fungal origin are high affinity iron chelating, linear to cylic oligomeric secondary metabolites mostly characterized by a N5-acyl-N5-hydroxyornithine basic unit [3]. Several siderophores are derived by one NRPS and post-synthetic subsequent modification [43]. As bio-available iron is rare in natural habitats, but an essential trace element to most organisms, efficient chelation, uptake and storage mechanisms for iron play an important role in competition and perseverance, especially within dense microbial communities like in soil [44]. Siderophores are important metabolites in the response against oxidative stress in several fungi like *Aspergillus nidulans, A. fumigatus, Cochliobolus heterostrophus, Gibberella zeae* and *T. virens;* furthermore, they play a role in conidial germination and sexual development

Evidences accumulate that siderophores act in biocontrol as virulence factors against other microbes during iron competition. Further, they promote plant growth by the reduction of oxidative stress: in biocontrol of *Fusarium* wilt disease by *Trichoderma asperellum* strain T34, the tomato plants exhibited reduced numbers of infected roots and a decrease in iron-associated abiotic stress [48]. The over-representation of siderophores in *Trichoderma hamatum* strain GD12 was reported to beneficially influence the biocontrol of *S. sclerotiorum* and plant growth promotion in lettuce [49]. More direct evidence for an involvement of siderophores in mycoparasitic interactions exists in *C. minitans:* the expression of *CmSIT1,* a gene-mediating siderophore-iron transport, not only enhanced antifungal abilities but also reduced growth [50].

conditions, the species, strain and even the host organism.

in biocontrol [36].

**2.3. Siderophores**

[4, 45–47].

Peptaibols are solely described for filamentous fungi exhibiting a mycoparasitic lifestyle, with a high abundance of over 80% of all known substances being derived from *Trichoderma* species [25]. Peptaibols are linear peptides, which besides of Aib include a characteristic acetylation of the N-terminus and a 1.2-amino-alcohol at the C-terminus. The first peptaibols, suzukacillin and alamethicin, have been described in the 1960s [26]. As demonstrated for alamethicin, the amphipathic character of peptaibols allows the voltage-dependent formation of helical structures acting as ion channels, thus spanning and permeabilizing the cell membrane and leading to cytoplasmic leakage and cellular breakdown [27].

Whereas all *Trichoderma* strains produce peptaibols, some substances are synthesized in a species- or even strain-specific manner [25]. For instance, in five different biocontrol agents containing species from the *Trichoderma harzianum* complex, peptaibols were the dominant secondary metabolites including three new and recurrent major groups present in all formulations [28]. Peptaibols play an important role in the mycoparasitic interactions as well as in induction of ISR in plants via up-regulation of the jasmonic acid and salicylic acid synthesis [29, 30]. In *T. harzianum*, peptaibols synergistically act together with hydrolytic, cell wall degrading enzymes on the cell wall destruction of the host fungus [31, 32]. Other mycoparasites such as *T. ophioglossoides* and *E. weberi* also comprise peptaibiotics-associated gene clusters, which are absent in plant- and entomopathogenic lineages of *Hypocreales,* suggesting the restriction of these genes to mycoparasitic species, further indicating their importance in the mycoparasitic interaction [11, 15]. The antifungal activity of the peptaibol trichokonin from *Trichoderma pseudokongii* caused extensive apoptosis by loss of the mitochondrial transmembrane potential resulting in apoptotic cell death in *Fusarium oxysporum* [33]. Similar evidences suggest a major involvement of peptaibiotics in mycoparasitism, substantiated by reports of antifungal action of peptaibols secreted by *Clonostachys rosea* against *S. sclerotiorum* [34], or *Sepedonium tulasneanum* against *Botrytis cinerea* and *Phytophthora infestans* [35].

#### **2.2. Epipolythiodioxypiperazines**

Epipolythiodioxypiperazines (ETPs) are characterized by the presence of an inter- or intramolecular disulfide bridge and a diketopiperazine core. The toxicity of ETPs lies in the disulfide bridging which is facilitating the inactivation of proteins by conjugation and the elicitation of reactive oxygen species (ROS) [36]. The best known substance of this class is gliotoxin derived from *T. virens* Q-strains [37]. *T. virens* P-strains antagonize *Pythium ultimum* and do not produce gliotoxin but the terpenoid gliovirin, whereas Q-strains affect *R. solani* but not vice versa [38]. *C. rosea* also produces ETPs like verticillin A and gliocladines involved in the antagonism on nematodes [39] and glioperazine exhibiting antibacterial properties [40]. Whereas the role of gliotoxin (cluster comprising of 12 genes) as a virulence factor in human *Aspergillus fumigatus* infections and the self-protection via the *gliT* gene product of the biosynthetic gliotoxin cluster is well investigated, there is little and partially adverse information on the role of ETPs in biocontrol [36].

The weak mycoparasitic *T. reesei* exhibits an incomplete gliotoxin cluster whose genes were not expressed during confrontation with *R. solani* [4], whereas highly mycoparasitic *T. atroviride* does not contain a gliotoxin cluster [2]. The gliotoxin gene cluster of *T. virens* Q-strains consists of eight genes encoding the core enzyme *gliP* – an NRPS dioxypiperazine synthase – whose expression was induced during confrontation with *R. solani* [4]. Deletion of *gliP* resulted in gliotoxin production-deficient mutants, going hand in hand with a significantly reduced induction of ISR in cotton seedlings and antagonistic action against *P. ultimum* and *S. sclerotiorum.* Adversely, the mutants' antifungal activity against *R. solani* remained unaltered [41]. The involvement of ETPs in mycoparasitic interactions stays unresolved and seems to depend on the combination of several – largely unknown – factors like synergistic interactions with other metabolites or enzymes [42], environmental conditions, the species, strain and even the host organism.

### **2.3. Siderophores**

**2.1. Peptaibiotics**

40 Secondary Metabolites - Sources and Applications

Peptaibiotics are mostly linear to rarely cyclic polypeptides, with a size of 0.5–2.1 kDa consisting of 4–21 residues. Characteristic for peptaibiotics is the inclusion of the non-proteinogenic amino acid α-aminobutyric acid (Aib). By module-skipping one NRPS is frequently capable of synthesizing a whole set of peptaibiotics [23, 24]. According to their sequence alignment and structure, peptaibiotics can be divided into several sub-clades: peptaibols, lipopeptaibols, lipoaminopeptides, cyclic peptaibiotics and two very special, small categories [25]. Because of their unusual synthesis and appearance, they are not included in regular protein databases,

Peptaibols are solely described for filamentous fungi exhibiting a mycoparasitic lifestyle, with a high abundance of over 80% of all known substances being derived from *Trichoderma* species [25]. Peptaibols are linear peptides, which besides of Aib include a characteristic acetylation of the N-terminus and a 1.2-amino-alcohol at the C-terminus. The first peptaibols, suzukacillin and alamethicin, have been described in the 1960s [26]. As demonstrated for alamethicin, the amphipathic character of peptaibols allows the voltage-dependent formation of helical structures acting as ion channels, thus spanning and permeabilizing the cell membrane and

Whereas all *Trichoderma* strains produce peptaibols, some substances are synthesized in a species- or even strain-specific manner [25]. For instance, in five different biocontrol agents containing species from the *Trichoderma harzianum* complex, peptaibols were the dominant secondary metabolites including three new and recurrent major groups present in all formulations [28]. Peptaibols play an important role in the mycoparasitic interactions as well as in induction of ISR in plants via up-regulation of the jasmonic acid and salicylic acid synthesis [29, 30]. In *T. harzianum*, peptaibols synergistically act together with hydrolytic, cell wall degrading enzymes on the cell wall destruction of the host fungus [31, 32]. Other mycoparasites such as *T. ophioglossoides* and *E. weberi* also comprise peptaibiotics-associated gene clusters, which are absent in plant- and entomopathogenic lineages of *Hypocreales,* suggesting the restriction of these genes to mycoparasitic species, further indicating their importance in the mycoparasitic interaction [11, 15]. The antifungal activity of the peptaibol trichokonin from *Trichoderma pseudokongii* caused extensive apoptosis by loss of the mitochondrial transmembrane potential resulting in apoptotic cell death in *Fusarium oxysporum* [33]. Similar evidences suggest a major involvement of peptaibiotics in mycoparasitism, substantiated by reports of antifungal action of peptaibols secreted by *Clonostachys rosea* against *S. sclerotiorum* [34], or

*Sepedonium tulasneanum* against *Botrytis cinerea* and *Phytophthora infestans* [35].

Epipolythiodioxypiperazines (ETPs) are characterized by the presence of an inter- or intramolecular disulfide bridge and a diketopiperazine core. The toxicity of ETPs lies in the disulfide bridging which is facilitating the inactivation of proteins by conjugation and the elicitation of reactive oxygen species (ROS) [36]. The best known substance of this class is gliotoxin derived

**2.2. Epipolythiodioxypiperazines**

but in the "Comprehensive Peptaibiotics Database" [25].

leading to cytoplasmic leakage and cellular breakdown [27].

Siderophores of fungal origin are high affinity iron chelating, linear to cylic oligomeric secondary metabolites mostly characterized by a N5-acyl-N5-hydroxyornithine basic unit [3]. Several siderophores are derived by one NRPS and post-synthetic subsequent modification [43]. As bio-available iron is rare in natural habitats, but an essential trace element to most organisms, efficient chelation, uptake and storage mechanisms for iron play an important role in competition and perseverance, especially within dense microbial communities like in soil [44]. Siderophores are important metabolites in the response against oxidative stress in several fungi like *Aspergillus nidulans, A. fumigatus, Cochliobolus heterostrophus, Gibberella zeae* and *T. virens;* furthermore, they play a role in conidial germination and sexual development [4, 45–47].

Evidences accumulate that siderophores act in biocontrol as virulence factors against other microbes during iron competition. Further, they promote plant growth by the reduction of oxidative stress: in biocontrol of *Fusarium* wilt disease by *Trichoderma asperellum* strain T34, the tomato plants exhibited reduced numbers of infected roots and a decrease in iron-associated abiotic stress [48]. The over-representation of siderophores in *Trichoderma hamatum* strain GD12 was reported to beneficially influence the biocontrol of *S. sclerotiorum* and plant growth promotion in lettuce [49]. More direct evidence for an involvement of siderophores in mycoparasitic interactions exists in *C. minitans:* the expression of *CmSIT1,* a gene-mediating siderophore-iron transport, not only enhanced antifungal abilities but also reduced growth [50].
