**5. Ecology and regulation of secondary metabolism in mycoparasitic fungi**

Ancestral and recent lifestyles fundamentally influence the existence as well as the expression of secondary metabolite genes and clusters up to the species or even strain level. The transcriptional responses of *T. reesei, T. atroviride* and *T. virens –* which all share an ancestral mycoparasitic lifestyle – to the confrontation with *R. solani* illustrates this fact very well. All three species exhibit few common metabolic responses but autonomous and specific strategies in defeating their opponent. Both potent mycoparasites, *T. atroviride* and *T. virens*, attack their hosts in the stage before physical contact, but with distinct strategies of antibiosis. *T. virens* offends mostly via the NRP biosynthetic pathway for gliotoxin synthesis, whereas *T. atroviride* combats mainly via the PKS biosynthetic pathway, as well as the excretion of 6-pentyl-α-pyrone (6-PP) – a volatile organic compound (VOC) with antifungal and plant growth-promoting properties [2]. Conversely, the transcriptional response of the only slightly mycoparasitic *T. reesei* is more defense-related and targeted on the excretion of cellulases, hemicellulases and ribosomal proteins before hyphal contact [69]. The long-term specialized lifestyle and co-evolution of *E. weberi* with its host in its relatively demarcated ecological niche facilitated the loss of a manifold of genes leading to a more or less obligatory mycoparasitism with limited growth and viability without the presence of the host. Hence, the *E. weberi* genome demonstrates a high degree of specialization, with a unique secretome containing an unusually high content of over 50% of proteins with unknown function. Further, the genome contains only 20% homologs with the closely related *T. atroviride, T. virens* and *T. reesei* and only 12% of the 1066 unique genes exhibit homology with proteins in the whole subdivision of *Pezizomycotina* [15].

Several environmental cues like temperature, light, carbon, nitrogen, pH and competing or synergistic organisms are known to influence the transcriptional regulation of secondary metabolism-associated gene clusters (**Figure 1**). Suboptimal environmental conditions thereby often facilitate and promote transcriptional activation or transcriptional reprogramming events [70]. In media containing chitin or *B. cinerea* cell walls, the predicted cutinase transcription factor 1 encoding gene of *T. harzianum* (*Thctf1*) was up-regulated. *Thctf1* deletion mutants exhibited reduced antagonistic and antifungal ability, and the mutant strain did not synthesize two 6-PP derivatives, indicating a role of *Thctf1* in secondary metabolism of *T. harzianum* [71]. Furthermore, the overexpression of the gene encoding multiprotein bridging factor 1 (*Thmbf1*) of *T. harzianum* – a transcriptional co-activator of *Thctf1* – negatively regulated the antifungal abilities, as well as the expression of VOCs [72].

genes. Further, the mutant exhibited a diminished production of secondary metabolites like viridiol and a reduced expression of secondary metabolism-associated genes [75]. Prior to that, similar results were obtained for *T. atroviride* ∆*tga1* mutants missing the subfamily I Gα protein-encoding gene. Deletion of *tga1* led to a complete loss of overgrowth and mycoparasitism of different preys during direct confrontation and a decrease of 6-PP and sesquiterpene production as well as chitinase gene transcription. Despite the reduction in chitinase and 6-PP accumulation, the ∆*tga1* mutant caused a strong growth inhibition of prey fungi in the interaction zone mediated by yet unidentified low molecular weight antifungal metabolites, thereby evidencing opposite roles of *tga1* in regulating the biosynthesis of different antifungal substances in *T. atroviride* [76]. Similar to ∆*tga1* mutants, transformants bearing a deletion of the subfamily III Gα protein-encoding gene *tga3* were unable to overgrow and lyse prey fungi. However, absence of the adenylyl cyclase-stimulating Tga3 protein led to significantly reduced antifungal activity [77]. The global regulation of secondary metabolism and morphogenesis by the heterotrimeric VELVET protein complex, consisting of the S-adenosylmethionine-dependent methyltransferase LaeA and the velvet proteins VeA and VelB, was first described in *A. nidulans* [78]. Deletion of the *laeA* orthologue *lae1* in *T. atroviride* led to a loss of mycoparasitic abilities in direct confrontation and a major reduction in the synthesis of 6-PP and water-soluble secondary metabolites. Further, the expression of eight mycoparasitism-related genes was decreased in the mutant. The deletion of *vel1 –* the *veA* orthologue – in *T. virens* caused defects in overgrowth and offense against the host in direct confrontation as well as in bioprotective plant interaction, accompanied by a decrease in the

Secondary Metabolites of Mycoparasitic Fungi http://dx.doi.org/10.5772/intechopen.75133 45

expression of several secondary metabolism-associated genes [79, 80].

**6. Cross-talk by and response to secondary metabolites in** 

ing the activation of silent secondary metabolism-associated gene clusters.

In bacteria, it has been shown that at sub-inhibitory concentrations antibiotics serve as mediators of microbial communication and interaction with one of the outcomes being the production of cryptic metabolites [81]. Accordingly, the interaction with other fungi may shape the secondary metabolite profile of a specific fungus, making co-cultures a valuable tool for elicit-

Studies on the mutual effects of secondary metabolites produced during mycoparasitic interactions are rare however. *Trichoderma*-derived 6-PP was shown to suppress the synthesis of the *Fusarium* mycotoxins fusaric acid and deoxynivalenol (DON) [82–85], suggesting that 6-PP acts as communication molecule that elicits biological responses in the interaction partners. On the other hand, fusaric acid and DON modulate 6-PP production as well as chitinase gene expression in *T. atroviride* and recent studies provided evidence that *Fusarium* mycotoxins induce defense mechanisms in mycoparasites such as *T. atroviride* and *C. rosea* which results in mycotoxin detoxification [59, 86]. *C. rosea* was shown to open the ring structure of zearalenone (ZEN), while *Trichoderma* spp. seem to convert ZEN into its reduced and sulfated forms and metabolize DON to deoxynivalenol-3-glucoside, a detoxification product of DON previously identified in plants [87, 88]. In the interaction of the mycoparasite *T. arundinaceum*

**mycoparasitic interactions**

Like known for the production of mycotoxins in non-mycoparasitic species [73], secondary metabolite production in mycoparasitic fungi is governed by heterotrimeric G protein signaling and the associated cAMP-pathway, as well as mitogen-activated protein kinase (MAPK) cascades [74, 75]. *T. atroviride* mutants, lacking the MAPK-encoding gene *tmk1* showed an enhanced production of peptaibols and of 6-PP [74]. First evidence for a positive regulation of the secondary metabolism by cAMP signaling came from *T. virens* ∆*tac1* mutants bearing a deletion of the adenylate cyclase-encoding gene. The mutants were unable to offend *Sclerotium rolfsii* and *R. solani,* but showed a clear inhibition zone in direct confrontation with *Pythium* sp., pointing to a host-dependent expression of secondary metabolism-associated

**Figure 1.** Overview on mycoparasitism-influencing factors and pathways in secondary metabolite biosynthesis of mycoparasitic fungi.

genes. Further, the mutant exhibited a diminished production of secondary metabolites like viridiol and a reduced expression of secondary metabolism-associated genes [75]. Prior to that, similar results were obtained for *T. atroviride* ∆*tga1* mutants missing the subfamily I Gα protein-encoding gene. Deletion of *tga1* led to a complete loss of overgrowth and mycoparasitism of different preys during direct confrontation and a decrease of 6-PP and sesquiterpene production as well as chitinase gene transcription. Despite the reduction in chitinase and 6-PP accumulation, the ∆*tga1* mutant caused a strong growth inhibition of prey fungi in the interaction zone mediated by yet unidentified low molecular weight antifungal metabolites, thereby evidencing opposite roles of *tga1* in regulating the biosynthesis of different antifungal substances in *T. atroviride* [76]. Similar to ∆*tga1* mutants, transformants bearing a deletion of the subfamily III Gα protein-encoding gene *tga3* were unable to overgrow and lyse prey fungi. However, absence of the adenylyl cyclase-stimulating Tga3 protein led to significantly reduced antifungal activity [77]. The global regulation of secondary metabolism and morphogenesis by the heterotrimeric VELVET protein complex, consisting of the S-adenosylmethionine-dependent methyltransferase LaeA and the velvet proteins VeA and VelB, was first described in *A. nidulans* [78]. Deletion of the *laeA* orthologue *lae1* in *T. atroviride* led to a loss of mycoparasitic abilities in direct confrontation and a major reduction in the synthesis of 6-PP and water-soluble secondary metabolites. Further, the expression of eight mycoparasitism-related genes was decreased in the mutant. The deletion of *vel1 –* the *veA* orthologue – in *T. virens* caused defects in overgrowth and offense against the host in direct confrontation as well as in bioprotective plant interaction, accompanied by a decrease in the expression of several secondary metabolism-associated genes [79, 80].
