The contribution of Trichoderma to balancing the costs of plant growth and defense

Trichoderma is a fungal genus of cosmopolitan distribution and high biotechnological value, with several species currently used as biological control agents. Additionally, the enzyme systems of the fungus are widely applied in industry. Species of Trichoderma protect plants against the attack of soil-borne plant pathogens by competing for nutrients and inhibiting or killing plant pathogenic fungi and oomycetes, through the production of antibiotics and/or hydrolytic enzymes. In addition to the role of Trichoderma spp. as biocontrol agents, they have other benefi cial effects on plants, including the stimulation of plant defenses and the promotion of plant growth. In this review, we focus on the complex plant defense signaling network that allows the recognition of fungi as non-hostile microbes, including microbial-associated molecular patterns (MAMPs), damageassociated molecular patterns (DAMPs) and secreted elicitors. We also examine how fungal interactions with plant receptors can activate induced resistance by priming and balancing plant defense and growth responses. Our observations are integrated into a model describing Trichoderma-plant hormone signaling network interactions. [Int Microbiol 2013; 16(2):69-80]Keywords: Trichoderma spp.; plant–Trichoderma symbiosis; Arabidopsis thaliana; phytohormone networking&nbsp

Organization of the gene cluster for biosynthesis of penicillin in Penicillium nalgiovense and antibiotic production in cured dry sausages

[EN] Several fungal isolates obtained from two cured meat products from Spain were identified as Penicillium nalgiovense by their morphological features and by DNA fingerprinting. All P. nalgiovense isolates showed antibiotic activity in agar diffusion assays, and their penicillin production in liquid complex medium ranged from 6 to 38 μg · ml−1. We constructed a restriction map of the penicillin gene cluster of P. nalgiovense and found that the organization of the penicillin biosynthetic genes (pcbAB, pcbC, andpenDE) is the same as in Penicillium chrysogenum and Aspergillus nidulans. ThepcbAB gene is located in an orientation opposite that of the pcbC and penDE genes in all three species. Significant amounts of penicillin were found in situ in the casing and the outer layer of salami meat during early stages of the curing process, coinciding with fungal colonization, but no penicillin was detected in the cured salami. The antibiotic produced in situ was sensitive to penicillinaseS

Involvement of the Transcriptional Coactivator ThMBF1 in the Biocontrol Activity of Trichoderma harzianum

[EN] Trichoderma harzianum is a filamentous fungus well adapted to different ecological niches. Owing to its ability to antagonize a wide range of plant pathogens, it is used as a biological control agent in agriculture. Selected strains of T. harzianum are also able to increase the tolerance of plants to biotic and abiotic stresses. However, little is known about the regulatory elements of the T. harzianum transcriptional machinery and their role in the biocontrol by this species. We had previously reported the involvement of the transcription factor THCTF1 in the T. harzianum production of the secondary metabolite 6-pentyl-pyrone, an important volatile compound related to interspecies cross-talk. Here, we performed a subtractive hybridization to explore the genes regulated by THCTF1, allowing us to identify a multiprotein bridging factor 1 (mbf1) homolog. The gene from T. harzianum T34 was isolated and characterized, and the generated Thmbf1 overexpressing transformants were used to investigate the role of this gene in the biocontrol abilities of the fungus against two plant pathogens. The transformants showed a reduced antifungal activity against Fusarium oxysporum f. sp. lycopersici race 2 (FO) and Botrytis cinerea (BC) in confrontation assays on discontinuous medium, indicating that the Thmbf1 gene could affect T. harzianum production of volatile organic compounds (VOC) with antifungal activity. Moreover, cellophane and dialysis membrane assays indicated that Thmbf1 overexpression affected the production of low molecular weight secreted compounds with antifungal activity against FO. Intriguingly, no correlation in the expression profiles, either in rich or minimal medium, was observed between Thmbf1 and the master regulator gene cross-pathway control (cpc1). Greenhouse assays allowed us to evaluate the biocontrol potential of T. harzianum strains against BC and FO on susceptible tomato plants. The wild type strain T34 significantly reduced the necrotic leaf lesions caused by BC while plants treated with the Thmbf1-overexpressing transformants exhibited an increased susceptibility to this pathogen. The percentages of Fusarium wilt disease incidence and values of aboveground dry weight showed that T34 did not have biocontrol activity against FO, at least in the 'Moneymaker' tomato variety, and that Thmbf1 overexpression increased the incidence of this disease. Our results show that the Thmbf1 overexpression in T34 negatively affects its biocontrol mechanismsSIThis research project was funded by the Spanish Ministry of Economy and Competitiveness (Project no. AGL2015-70671-C2) and the Junta de Castilla y León (Project no. SA009U16

The importance of chorismate mutase in the biocontrol potential of Trichoderma parareesei

[EN] Species of Trichoderma exert direct biocontrol activity against soil-borne plant pathogens due to their ability to compete for nutrients and to inhibit or kill their targets through the production of antibiotics and/or hydrolytic enzymes. In addition to these abilities, Trichoderma spp. have beneficial effects for plants, including the stimulation of defenses and the promotion of growth. Here we study the role in biocontrol of the T. parareesei Tparo7 gene, encoding a chorismate mutase (CM), a shikimate pathway branch point leading to the production of aromatic amino acids, which are not only essential components of protein synthesis but also the precursors of a wide range of secondary metabolites. We isolated T. parareesei transformants with the Tparo7 gene silenced. Compared with the wild-type, decreased levels of Tparo7 expression in the silenced transformants were accompanied by reduced CM activity, lower growth rates on different culture media, and reduced mycoparasitic behavior against the phytopathogenic fungi Rhizoctonia solani, Fusarium oxysporum and Botrytis cinerea in dual cultures. By contrast, higher amounts of the aromatic metabolites tyrosol, 2-phenylethanol and salicylic acid were detected in supernatants from the silenced transformants, which were able to inhibit the growth of F. oxysporum and B. cinerea. In in vitro plant assays, Tparo7-silenced transformants also showed a reduced capacity to colonize tomato roots. The effect of Tparo7-silencing on tomato plant responses was examined in greenhouse assays. The growth of plants colonized by the silenced transformants was reduced and the plants exhibited an increased susceptibility to B. cinerea in comparison with the responses observed for control plants. In addition, the plants turned yellowish and were defective in jasmonic acid- and ethylene-regulated signaling pathways which was seen by expression analysis of lipoxygenase 1 (LOX1), ethylene-insensitive protein 2 (EIN2) and pathogenesis-related protein 1 (PR-1) genesSIResearch project funding was from the Spanish Ministry of Economy and Competitiveness (Projects no. AGL2012-40041-C01 and AGL2012-40041-C02) and the Junta de Castilla y León (Projects no. LE125A12-2 and LE228U14). Esclaudys Pérez was endowed with a Spanish Foreign Office AECID award and Wagner Bettiol was supported by a fellowship from the Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP (Project no. 2014/03426-1

Involvement of Trichoderma Trichothecenes in the Biocontrol Activity and Induction of Plant Defense-Related Genes

[EN] Trichoderma species produce trichothecenes, most notably trichodermin and harzianum A (HA), by a biosynthetic pathway in which several of the involved proteins have significant differences in functionality compared to their Fusarium orthologues. In addition, the genes encoding these proteins show a genomic organization differing from that of the Fusarium tri clusters. Here we describe the isolation of Trichoderma arundinaceum IBT 40837 transformants which have a disrupted or silenced tri4 gene, a gene encoding a cytochrome P450 monooxygenase that oxygenates trichodiene to give rise to isotrichodiol, and the effect of tri4 gene disruption and silencing on the expression of other tri genes. Our results indicate that the tri4 gene disruption resulted in a reduced antifungal activity against Botrytis cinerea and Rhizoctonia solani and also in a reduced ability to induce the expression of tomato plant defense-related genes belonging to the salicylic acid (SA) and jasmonate (JA) pathways against B. cinerea, in comparison to the wild-type strain, indicating that HA plays an important function in the sensitization of Trichoderma-pretreated plants against this fungal pathogen. Additionally, the effect of the interaction of T. arundinaceum with B. cinerea or R. solani and with tomato seedlings on the expressions of the tri genes was studiedSIResearch project funding was obtained from Junta de Castilla y León (GR67) and the Spanish Ministry of Science and Innovation (AGL2006-05660, AGL2009-13431-C01, and AGL2009-13431-C02). M. G. Malmierca was granted an FPU fellowship by the Spanish Ministry of Science and Innovation (AP2007-02835

Involvement of Trichoderma harzianum Epl-1 Protein in the Regulation of Botrytis Virulence- and Tomato Defense-Related Genes

[EN] Several Trichoderma spp. are well known for their ability to: (i) act as important biocontrol agents against phytopathogenic fungi; (ii) function as biofertilizers; (iii) increase the tolerance of plants to biotic and abiotic stresses; and (iv) induce plant defense responses via the production and secretion of elicitor molecules. In this study, we analyzed the gene-regulation effects of Trichoderma harzianum Epl-1 protein during the interactions of mutant ∆epl-1 or wild-type T. harzianum strains with: (a) the phytopathogen Botrytis cinerea and (b) with tomato plants, on short (24 h hydroponic cultures) and long periods (4-weeks old plants) after Trichoderma inoculation. Our results indicate that T. harzianum Epl-1 protein affects the in vitro expression of B. cinerea virulence genes, especially those involved in the botrydial biosynthesis (BcBOT genes), during the mycoparasitism interaction. The tomato defense-related genes were also affected, indicating that Epl-1 is involved in the elicitation of the salicylic acid pathway. Moreover, Epl-1 also regulates the priming effect in host tomato plants and contributes to enhance the interaction with the host tomato plant during the early stage of root colonizationSIThis work was supported by the State of São Paulo Research Foundation (FAPESP) (Proc. 2012/16895-4, Proc. 2013/24452-8, and Proc. 2016/04274-6). Funding was also obtained from the Junta de Castilla y León, Spain (LE228U14) and the Spanish Government Grants from Ministerio de Economía y Competitividad (AGL2012-40041-C02-02 and AGL2015-70671-C2-2-R

Detection of putative peptide synthetase genes inTrichodermaspecies: Application of this method to the cloning of a gene fromT. harzianumCECT 2413

[EN] Some of the secondary metabolites produced by Trichoderma, such as the peptaibols and other antibiotics, have a peptide structure and in their biosynthesis are involved proteins belonging to the Non-Ribosomal Peptide Synthetase family. In the present work, a PCR-mediated strategy was used to clone a region corresponding to an adenylation domain of a peptide synthetase (PS) gene from 10 different strains of Trichoderma. In addition, and using the fragment isolated by PCR from T. harzianum CECT 2413 as a probe, a fragment of 19.0 kb corresponding to a PS-encoding gene named salps1, including a 1.5 kb fragment of the promoter, was cloned and sequenced. The cloned region of salps1 contains four complete, and a fifth incomplete, modules, in which are found the adenylation, thiolation and condensation domains, but also an additional epimerization domain at the C-terminal end of the first module. The analysis of the Salps1 protein sequence, taking into consideration published data, suggests that it is neither a peptaibol synthetase nor a protein involved in siderophore biosynthesis. The presence of two breaks in the open reading frame and the expression of this gene under nitrogen starvation conditions suggest that salps1 could be a pseudogene. © 2005 Federation of European Microbiological SocietiesSIThe authors acknowledge the financial support of the Spanish Foundations ‘‘Ramo´n Areces’’ (Madrid, Spain) and ‘‘Fundacio´n Andaluza de I+D’’ (Seville, Spain). This research was also supported by the European Commission (project QLK3-CT-2002-02032

Nitrogen Metabolism and Growth Enhancement in Tomato Plants Challenged with Trichoderma harzianum Expressing the Aspergillus nidulans Acetamidase amdS Gene

[EN] Trichoderma is a fungal genus that includes species that are currently being used as biological control agents and/or as biofertilizers. In addition to the direct application of Trichoderma spp. as biocontrol agents in plant protection, recent studies have focused on the beneficial responses exerted on plants, stimulating the growth, activating the defenses, and/or improving nutrient uptake. The amdS gene, encoding an acetamidase of Aspergillus, has been used as a selectable marker for the transformation of filamentous fungi, including Trichoderma spp., but the physiological effects of the introduction of this gene into the genome of these microorganisms still remains unexplored. No evidence of amdS orthologous genes has been detected within the Trichoderma spp. genomes and the amdS heterologous expression in Trichoderma harzianum T34 did not affect the growth of this fungus in media lacking acetamide. However, it did confer the ability for the fungus to use this amide as a nitrogen source. Although a similar antagonistic behavior was observed for T34 and amdS transformants in dual cultures against Rhizoctonia solani, Botrytis cinerea, and Fusarium oxysporum, a significantly higher antifungal activity was detected in amdS transformants against F. oxysporum, compared to that of T34, in membrane assays on media lacking acetamide. In Trichoderma-tomato interaction assays, amdS transformants were able to promote plant growth to a greater extent than the wild-type T34, although compared with this strain the transformants showed similar capability to colonize tomato roots. Gene expression patterns from aerial parts of 3-week-old tomato plants treated with T34 and the amdS transformants have also been investigated using GeneChip Tomato Genome Arrays. The downregulation of defense genes and the upregulation of carbon and nitrogen metabolism genes observed in the microarrays were accompanied by (i) enhanced growth, (ii) increased carbon and nitrogen levels, and (iii) a higher sensitivity to B. cinerea infections in plants treated with amdS transformants as detected in greenhouse assays. These observations suggest that the increased plant development promoted by the amdS transformants was at expense of defensesSIResearch project funding was from the Spanish Ministry of Economy and Competitiveness (Project no. AGL2015-70671-C2) and the Junta de Castilla y León (Projects no. SA230U13 and LE228U14). The grant awarded to Sara Domínguez by Junta de Castilla y León (Spain) and Wagner Bettiol was supported by a fellowship from the Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP (Project no. 2014/03426-1

Silencing of the Aspergillopepsin B (pepB) Gene of Aspergillus awamori by Antisense RNA Expression or Protease Removal by Gene Disruption Results in a Large Increase in Thaumatin Production

[EN] Aspergillopepsin B was identified in culture broths of Aspergillus awamori by in situ detection of its proteolytic activity and by immunodetection with anti-aspergillopepsin B antibodies. Severe thaumatin degradation was observed after in vitro treatment of thaumatin with purified aspergillopepsin B. The pepB gene encoding aspergillopepsin B of A. awamori was cloned and characterized. It is located in chromosome IV of A. awamori, as shown by pulsed-field gel electrophoresis, and encodes a protein of 282 amino acids with high similarity to the aspergillopepsin B of Aspergillus niger var. macrosporus. The pepB gene is expressed at high rates as a monocistronic 1.0-kb transcript in media with casein at acidic pH values. An antisense cassette constructed by inserting the pepB gene in the antisense orientation downstream from the gpdA promoter resulted in a good level of antisense mRNA, as shown by reverse transcription-PCR. Partial silencing of the pepB gene by the antisense mRNA resulted in a 31% increase in thaumatin yield. However, significant residual degradation of thaumatin still occurred. To completely remove aspergillopepsin B, the pepB gene was deleted by double crossover. Two of the selected transformants lacked the endogenous pepB gene and did not form aspergillopepsin B. Thaumatin yields increased by between 45% in transformant APB 7/25 and 125% in transformant 7/36 with respect to the parental strain. Reduction of proteolytic degradation by gene silencing with antisense mRNA or total removal of the aspergillopepsin B by directed gene deletion was a very useful method for improving thaumatin production in A. awamori.S

Identification of plant genes putatively involved in the perception of fungal ergosterol‐squalene

[EN] Trichoderma biocontrol strains establish a complex network of interactions with plants, in which diverse fungal molecules are involved in the recognition of these fungi as nonpathogenic organisms. These molecules act as microbial-associated molecular patterns that trigger plant responses. Previous studies have reported the importance of ergosterol produced by Trichoderma spp. for the ability of these fungi to induce plant growth and defenses. In addition, squalene, a sterol biosynthetic intermediate, seems to play an important role in these interactions. Here, we analyzed the effect of different concentrations of ergosterol and squalene on tomato (Solanum lycopersicum) growth and on the transcription level of defense- and growth-related genes. We used an RNA-seq strategy to identify several tomato genes encoding predicted pattern recognition receptor proteins or WRKY transcription factors, both of which are putatively involved in the perception and response to ergosterol and squalene. Finally, an analysis of Arabidopsis thaliana mutants lacking the genes homologous to these tomato candidates led to the identification of a WRKY40 transcription factor that negatively regulates salicylic acid-related genes and positively regulates ethylene- and jasmonate-related genes in the presence of ergosterol and squaleneSIThis work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO-AGL2015–70671-C2-2-R and MICINN-RTI2018–099600-B-I00 to S.G.), and also by the Junta de Castilla y León (Spain) (LE251P18). L. Lindo was granted a fellowship by the University of León (Spain