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

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

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

Targeted Inactivation of the mecB Gene, Encoding Cystathionine-γ-Lyase, Shows that the Reverse Transsulfuration Pathway Is Required for High-Level Cephalosporin Biosynthesis inAcremonium chrysogenum C10 but Not for Methionine Induction of the Cephalosporin Genes

[EN] Targeted gene disruption efficiency in Acremonium chrysogenum was increased 10-fold by applying the double-marker enrichment technique to this filamentous fungus. Disruption of themecB gene by the double-marker technique was achieved in 5% of the transformants screened. Mutants T6 and T24, obtained by gene replacement, showed an inactive mecB gene by Southern blot analysis and no cystathionine-γ-lyase activity. These mutants exhibited lower cephalosporin production than that of the control strain, A. chrysogenum C10, in MDFA medium supplemented with methionine. However, there was no difference in cephalosporin production between parental strain A. chrysogenum C10 and the mutants T6 and T24 in Shen's defined fermentation medium (MDFA) without methionine. These results indicate that the supply of cysteine through the transsulfuration pathway is required for high-level cephalosporin biosynthesis but not for low-level production of this antibiotic in methionine-unsupplemented medium. Therefore, cysteine for cephalosporin biosynthesis in A. chrysogenum derives from the autotrophic (SH2) and the reverse transsulfuration pathways. Levels of methionine induction of the cephalosporin biosynthesis gene pcbC were identical in the parental strain and the mecB mutants, indicating that the induction effect is not mediated by cystathionine-γ-lyase.SIThis work was supported by a grant of the CICYT (BIO97-0289-C02-01). We thank A. Paszewski for providing Aspergillus nidulansstrains M63 and C47 and M. Mediavilla, B. Martı́n, R. Barrientos, and M. Corrales for excellent technical assistance

Vineyard Management and Physicochemical Parameters of Soil Affect Native Trichoderma Populations, Sources of Biocontrol Agents against Phaeoacremonium minimum

[EN] Native strains of Trichoderma in vineyard soil represent an opportunity for reducing the incidence of grapevine trunk diseases (GTDs) in vineyards. Moreover, its relationship with the environment (physicochemical soil characteristics and farming management practices) remains unclear. In the current study, a survey was carried out on farming management used by viticulturists, and soil samples were studied to analyze their physicochemical properties and to isolate Trichoderma strains. Later, statistical analyses were performed to identify possible correlations between Trichoderma populations, soil management and soil characteristics. In addition, in vitro tests, including antibiosis and mycoparasitism, were performed to select those Trichoderma strains able to antagonize Phaeoacremonium minimum. In this study a positive correlation was found between the iron content and pH in the soil, and a lower pH increases Trichoderma populations in soils. Vineyard management also affects Trichoderma populations in the soil, negatively in the case of fertilization and tillage and positively in the case of herbicide spraying. Two Trichoderma native strains were selected as potential biocontrol agents (Trichoderma gamsii T065 and Trichoderma harzianum T087) using antibiosis and mycoparasitism as mechanisms of action. These results led to the conclusion that native Trichoderma strains hold great potential as biological control agents and as producers of secondary metabolites.SIThis research was funded by project GLOBALVITI ‘Solución global para mejorar la’ Centro para el Desarrollo Tecnológico Industrial–CDTI—(Madrid, Spain) Project, GLOBALVITI project (CIEN Program) IDI-20160746, the project LowpHwine (CIEN Program) IDI ‘Estudio de nuevos factores relacionados con el suelo, la planta y la microbiota enológica que influyen en el equilibrio de la acidez de los vinos y en su garantía de calidad y estabilidad en climas cálidos’ and Bodega Pago de Carraovejas S.L.U. Thanks to all the wineries and viticulturists who kindly let us sample their vineyards. Thanks also to the research staff of the GUIIAS group for their technical support

Colonization of Vitis vinifera L. by the Endophyte Trichoderma sp. Strain T154: Biocontrol Activity Against Phaeoacremonium minimum

[EN] Trichoderma strains used in biological control products usually exhibit high efficiency in the control of plant diseases. However, their behavior under field conditions is difficult to predict. In addition, the potential of indigenous strains has been poorly assayed as well as their possible behavior as endophytes. Hence, niche colonization is a key feature for an effective protection. In this study, we aimed to: (i) explore the possibility of using a new Trichoderma strain isolated from vine to control pathogens, (ii) study the in planta interaction with the pathogen Phaeoacremonium minimum W. Gams, Crous, M.J. Wingf. & L. Mugnai (formerly Phaeoacremonium aleophilum), a pioneer fungus involved in Grapevine Trunk Diseases (GTDs) such as esca. For this purpose, fluorescently tagged Trichoderma sp. T154 and a P. minimum strain were used for scanning electron microscopy and confocal scanning laser microscopy analyses. Data showed that the Trichoderma strain is able to colonize plants up to 12 weeks post inoculation and is located in xylem, fibers, as well as in parenchymatic tissues inside the wood. The beneficial fungus reduced colonization of the esca-related pathogen colonizing the same niches. The main observed mechanism involved in biocontrol of Trichoderma against the esca pathogen was spore adhesion, niche exclusion and only few typical hypha coiling was found between Trichoderma and the pathogen. These results suggest that the Trichoderma strain has potential for reducing the colonization of Phaeoacremonium minimum and thus, an inoculation of this biological control agent can protect the plant by limiting the development of GTD, and the strain can behave as an endophyte.SIThe grant awarded to GC-H (FPU15/04681) comes from the Ministry of Education, Culture, and Sport (Spain). We thank Pago de Carraovejas winery for the project “GLOBALVITI IDI- 20120746” “Solució n global para mejorar la producció n vitivinı́ cola frente al cambio climá tico basada en robó tica, tecnologı́ a IT y en estrategias biotecnoló gicas y de manejo del viñedo” (Global solution for enhancing viticulture production against: climatic change based on: robotics, IT technology, biotechnological strategies, and vineyard management) that was granted by the Centro para el Desarrollo Tecnoló gico Industrial –CDTI-. SC and MG received funding via DaFNE Project Nr. 101384 from the Austrian Federal Ministry for Sustainability and Tourism (BMNT)