Effect of Trichoderma asperellum on Wheat Plants' Biochemical and Molecular Responses, and Yield under Different Water Stress Conditions.

Eight Trichoderma strains were evaluated for their potential to protect wheat seedlings against severe (no irrigation within two weeks) water stress (WS). Considering the plant fresh weight and phenotype, T. asperellum T140, which displays 1-aminocyclopropane-1-carboxylic acid deaminase activity and which is able to produce several phytohormones, was selected. The molecular and biochemical results obtained from 4-week-old wheat seedlings linked T140 application with a downregulation in the WS-response genes, a decrease in antioxidant activities, and a drop in the proline content, as well as low levels of hydrogen peroxide and malondialdehyde in response to severe WS. All of these responses are indicative of T140-primed seedlings having a higher tolerance to drought than those that are left untreated. A greenhouse assay performed under high nitrogen fertilization served to explore the long-term effects of T140 on wheat plants subjected to moderate (halved irrigation) WS. Even though all of the plants showed acclimation to moderate WS regardless of T140 application, there was a positive effect exerted by T. asperellum on the level of tolerance of the wheat plants to this stress. Strain T140 modulated the expression of a plant ABA-dependent WS marker and produced increased plant superoxide dismutase activity, which would explain the positive effect of Trichoderma on increasing crop yields under moderate WS conditions. The results demonstrate the effectiveness of T. asperellum T140 as a biostimulant for wheat plants under WS conditions, making them more tolerant to drought.

Trichoderma and the Plant Heritable Priming Responses.

There is no doubt that Trichoderma is an inhabitant of the rhizosphere that plays an important role in how plants interact with the environment. Beyond the production of cell wall degrading enzymes and metabolites, Trichoderma spp. can protect plants by inducing faster and stronger immune responses, a mechanism known as priming, which involves enhanced accumulation of dormant cellular proteins that function in intracellular signal amplification. One example of these proteins is the mitogen-activated protein kinases (MAPK) that are triggered by the rise of cytosolic calcium levels and cellular redox changes following a stressful challenge. Transcription factors such as WRKYs, MYBs, and MYCs, play important roles in priming as they act as regulatory nodes in the transcriptional network of systemic defence after stress recognition. In terms of long-lasting priming, Trichoderma spp. may be involved in plants epigenetic regulation through histone modifications and replacements, DNA (hypo)methylation, and RNA-directed DNA methylation (RdDM). Inheritance of these epigenetic marks for enhanced resistance and growth promotion, without compromising the level of resistance of the plant's offspring to abiotic or biotic stresses, seems to be an interesting path to be fully explored.

Effect of Inorganic N Top Dressing and Trichoderma harzianum Seed-Inoculation on Crop Yield and the Shaping of Root Microbial Communities of Wheat Plants Cultivated Under High Basal N Fertilization.

Wheat crop production needs nitrogen (N) for ensuring yield and quality. High doses of inorganic N fertilizer are applied to soil before sowing (basal dressing), with additional doses supplied along the cultivation (top dressing). Here, a long-term wheat field trial (12 plots), including four conditions (control, N top dressing, Trichoderma harzianum T34 seed-inoculation, and top dressing plus T34) in triplicate, was performed to assess, under high basal N fertilization, the influence of these treatments on crop yield and root microbial community shaping. Crop yield was not affected by top dressing and T. harzianum T34, but top dressing significantly increased grain protein and gluten contents. Twenty-seven-week old wheat plants were collected at 12 days after top dressing application and sampled as bulk soil, rhizosphere and root endosphere compartments in order to analyze their bacterial and fungal assemblies by 16S rDNA and ITS2 high-throughput sequencing, respectively. Significant differences for bacterial and fungal richness and diversity were detected among the three compartments with a microbial decline from bulk soil to root endosphere. The most abundant wheat root phyla were Proteobacteria and Actinobacteria for bacteria, and Ascomycota and Basidiomycota for fungi. An enrichment of genera commonly associated with soils subjected to chemical N fertilization was observed: Kaistobacter, Mortierella, and Solicoccozyma in bulk soil, Olpidium in rhizosphere, and Janthinobacterium and Pedobacter in root endosphere. Taxa whose abundance significantly differed among conditions within each compartment were identified. Results show that: (i) single or strain T34-combined application of N top dressing affected to a greater extent the bulk soil bacterial levels than the use of T34 alone; (ii) when N top dressing and T34 were applied in combination, the N fertilizer played a more decisive role in the bacterial microbiome than T34; (iii) many genera of plant beneficial bacteria, negatively affected by N top dressing, were increased by the application of T34 alone; (iv) bulk soil and rhizosphere fungal microbiomes were affected by any of the three treatments assayed; and (v) all treatments reduced Claroideoglomus in bulk soil but the single application of T34 raised the rhizosphere levels of this mycorrhizal fungus.

Differential Response of Tomato Plants to the Application of Three Trichoderma Species When Evaluating the Control of Pseudomonas syringae Populations.

Trichoderma species are well known biocontrol agents that are able to induce responses in the host plants against an array of abiotic and biotic stresses. Here, we investigate, when applied to tomato seeds, the potential of Trichoderma strains belonging to three different species, T. parareesei T6, T. asperellum T25, and T. harzianum T34, to control the fully pathogenic strain Pseudomonas syringae pv. tomato (Pst) DC3000, able to produce the coronatine (COR) toxin, and the COR-deficient strain Pst DC3118 in tomato plants, and the molecular mechanisms by which the plant can modulate its systemic defense. Four-week old tomato plants, seed-inoculated, or not, with a Trichoderma strain, were infected, or not, with a Pst strain, and the changes in the expression of nine marker genes representative of salicylic acid (SA) (ICS1 and PAL5) and jasmonic acid (JA) (TomLoxC) biosynthesis, SA- (PR1b1), JA- (PINII and MYC2) and JA/Ethylene (ET)-dependent (ERF-A2) defense pathways, as well as the abscisic acid (ABA)-responsive gene AREB2 and the respiratory burst oxidase gene LERBOH1, were analyzed at 72 hours post-inoculation (hpi) with the bacteria. The significant increase obtained for bacterial population sizes in the leaves, disease index, and the upregulation of tomato genes related to SA, JA, ET and ABA in plants inoculated with Pst DC3000 compared with those obtained with Pst DC3118, confirmed the COR role as a virulence factor, and showed that both Pst and COR synergistically activate the JA- and SA-signaling defense responses, at least at 72 hpi. The three Trichoderma strains tested reduced the DC3118 levels to different extents and were able to control disease symptoms at the same rate. However, a minor protection (9.4%) against DC3000 was only achieved with T. asperellum T25. The gene deregulation detected in Trichoderma-treated plus Pst-inoculated tomato plants illustrates the complex system of a phytohormone-mediated signaling network that is affected by the pathogen and Trichoderma applications but also by their interaction. The expression changes for all nine genes analyzed, excepting LERBOH1, as well as the bacterial populations in the leaves were significantly affected by the interaction. Our results show that Trichoderma spp. are not adequate to control the disease caused by fully pathogenic Pst strains in tomato plants.

Effects on hyphal morphology and development by the putative copper radical oxidase glx1 in Trichoderma virens suggest a novel role as a cell wall associated enzyme.

Trichoderma spp. have been characterized for their capacity to act as biological control agents against several pathogens through the activity of secondary metabolites and cell wall degrading enzymes. However, only T. reesei has been widely studied for the ability to assimilate lignocellulose substrates. Protein analysis by SDS-PAGE of culture filtrate of T. virens revealed the presence of an unknown ∼77 kDa band protein (GLX1) that showed sequence homology to glyoxal-like oxidase genes involved in lignin degradation. The analysis and biochemical characterization of the 1,119 amino acid coded protein showed the presence of five carbohydrate-binding modules (CBMs) with affinity for colloidal chitin, and a functional glyoxal oxidase catalytic domain that is involved in the production of hydrogen peroxide when methylglyoxal was used as a substrate. The silencing of the glx1 gene resulted in mutants with more than 90% expression reduction and the absence of glyoxal oxidase catalytic activity. These mutants showed delayed hyphal growth, reduced colony and conidial hydrophobicity, but showed no changes in their biocontrol ability. Most significantly, mutants exhibited a loss of growth directionality resulting in a curled phenotype that was eliminated in the presence of exogenous H2O2. Here we present evidence that in T. virens, glx1 is not involved in the breakdown of lignin but instead is responsible for normal hyphal growth and morphology and likely does this through free radical production within the fungal cell wall. This is the first time that a glyoxal oxidase protein has been isolated and characterized in ascomycete fungi.

Transcriptomic Analysis of Trichoderma atroviride Overgrowing Plant-Wilting Verticillium dahliae Reveals the Role of a New M14 Metallocarboxypeptidase CPA1 in Biocontrol.

Verticillium dahliae, a vascular-colonizing fungus, causes economically important wilt diseases in many crops, including olive trees. Trichoderma spp. have demonstrated an effective contribution as biocontrol agents against this pathogen through a variety of mechanisms that may involve direct mycoparasitism and antibiosis. However, molecular aspects underlaying Trichoderma-V. dahliae interactions are not well known yet due to the few studies in which this pathogen has been used as a target for Trichoderma. In the present study, Trichoderma atroviride T11 overgrew colonies of V. dahliae on agar plates and inhibited growth of highly virulent defoliating (D) V. dahliae V-138I through diffusible molecules and volatile organic compounds produced before contact. A Trichoderma microarray approach of T11 growing alone (CON), and before contact (NV) or overgrowing (OV) colonies of V-138I, helped to identify 143 genes that differed significantly in their expression level by more than twofold between OV and CON or NV. Functional annotation of these genes indicated a marked up-regulation of hydrolytic, catalytic and transporter activities, and secondary metabolic processes when T11 overgrew V-138I. This transcriptomic analysis identified peptidases as enzymatic activity overrepresented in the OV condition, and the cpa1 gene encoding a putative carboxypeptidase (ID number 301733) was selected to validate this study. The role of cpa1 in strain T11 on antagonism of V-138I was analyzed by a cpa1-overexpression approach. The increased levels of cpa1 expression and protease activity in the cpa1-overexpressed transformants compared to those in wild-type or transformation control strains were followed by significantly higher antifungal activity against V-138I in in vitro assays. The use of Trichoderma spp. for the integrated management of plant diseases caused by V. dahliae requires a better understanding of the molecular mechanisms underlying this interaction that might provide an increase on its efficiency.

What are Microbial-based Biopesticides?

From the ancient civilizations, agriculture has played an important socioeconomic role in the development of our current human society. Modern farming methods developed during the Green Revolution increased the production of food worldwide. Keeping a sustainable production of food supplies will impact the long-term survival of the species as well as the natural resources, so it is important to work on the establishment of new farming methodologies, such as the use of biopesticides that allow the balance between production and preservation of the environment. This introductory chapter will guide the reader through the content of this book providing an overall view of what will be discussed in each chapter.

Development of Biopesticides and Future Opportunities.

Biopesticides, pesticides based on living organisms or their extracts, are increasing in sales around the world, as synthetic pesticides are less available and environmental and health issues drive new approaches. Despite the increasing sales and use, there are still limitations that restrict more widespread uptake, such as slow to kill, cost, difficulties of production, lack of appropriate formulations, and reputation based on previous poor performance of biopesticides. Regulation continues to be problematic in many countries, as the processes are designed for evaluating chemistry rather than live organisms. Biopesticides do have a bright future, given the amount of investment currently in the area, improving products and growing need.

A paralog of the proteinaceous elicitor SM1 is involved in colonization of maize roots by Trichoderma virens.

The biocontrol agent, Trichoderma virens, has the ability to protect plants from pathogens by eliciting plant defense responses, involvement in mycoparasitism, or secreting antagonistic secondary metabolites. SM1, an elicitor of induced systemic resistance (ISR), was found to have three paralogs within the T. virens genome. The paralog sm2 is highly expressed in the presence of plant roots. Gene deletion mutants of sm2 were generated and the mutants were found to overproduce SM1. The ability to elicit ISR in maize against Colletotrichum graminicola was not compromised for the mutants compared to that of wild type isolate. However, the deletion strains had a significantly lowered ability to colonize maize roots. This appears to be the first report on the involvement of an effector-like protein in colonization of roots by Trichoderma.

Host-specific transcriptomic pattern of Trichoderma virens during interaction with maize or tomato roots.

BACKGROUND: Members of the fungal genus Trichoderma directly antagonize soil-borne fungal pathogens, and an increasing number of species are studied for their potential in biocontrol of plant pathogens in agriculture. Some species also colonize plant roots, promoting systemic resistance. The Trichoderma-root interaction is hosted by a wide range of plant species, including monocots and dicots. RESULTS: To test the hypothesis that gene expression by the fungal partner in this beneficial interaction is modulated by the plant, Trichoderma virens was co-cultured with maize or tomato in a hydroponic system allowing interaction with the roots. The transcriptomes for T. virens alone were compared with fungus-inoculated tomato or maize roots by hybridization on microarrays of 11645 unique oligonucleotides designed from the predicted protein-coding gene models. Transcript levels of 210 genes were modulated by interaction with roots. Almost all were up-regulated. Glycoside hydrolases and transporters were highly represented among transcripts induced by co-culture with roots. Of the genes up-regulated on either or both host plants, 35 differed significantly in their expression levels between maize and tomato. Ten of these were expressed higher in the fungus in co-culture with tomato roots than with maize. Average transcript levels for these genes ranged from 1.9 fold higher on tomato than on maize to 60.9 fold for the most tomato-specific gene. The other 25 host-specific transcripts were expressed more strongly in co-culture with maize than with tomato. Average transcript levels for these genes were 2.5 to 196 fold higher on maize than on tomato. CONCLUSIONS: Based on the relevant role of Trichoderma virens as a biological control agent this study provides a better knowledge of its crosstalk with plants in a host-specific manner. The differentially expressed genes encode proteins belonging to several functional classes including enzymes, transporters and small secreted proteins. Among them, glycoside hydrolases and transporters are highlighted by their abundance and suggest an important factor in the metabolism of host cell walls during colonization of the outer root layers. Host-specific gene expression may contribute to the ability of T. virens to colonize the roots of a wide range of plant species.

Role of gliotoxin in the symbiotic and pathogenic interactions of Trichoderma virens.

Using a gene disruption strategy, we generated mutants in the gliP locus of the plant-beneficial fungus Trichoderma virens that were no longer capable of producing gliotoxin. Phenotypic assays demonstrated that the gliP-disrupted mutants grew faster, were more sensitive to oxidative stress and exhibited a sparse colony edge compared with the WT strain. In a plate confrontation assay, the mutants deficient in gliotoxin production were ineffective as mycoparasites against the oomycete, Pythium ultimum, and the necrotrophic fungal pathogen, Sclerotinia sclerotiorum, but retained mycoparasitic ability against Rhizoctonia solani. Biocontrol assays in soil showed that the mutants were incapable of protecting cotton seedlings from attack by P. ultimum, against which the WT strain was highly effective. The mutants, however, were as effective as the WT strain in protecting cotton seedlings against R. solani. Loss of gliotoxin production also resulted in a reduced ability of the mutants to attack the sclerotia of S. sclerotiorum compared with the WT. The addition of exogenous gliotoxin to the sclerotia colonized by the mutants partially restored their degradative abilities. Interestingly, as in Aspergillus fumigatus, an opportunistic human pathogen, gliotoxin was found to be involved in pathogenicity of T. virens against larvae of the wax moth, Galleria mellonella. The loss of gliotoxin production in T. virens was restored by complementation with the gliP gene from A. fumigatus. We have, thus, demonstrated that the putative gliP cluster of T. virens is responsible for the biosynthesis of gliotoxin, and gliotoxin is involved in mycoparasitism and biocontrol properties of this plant-beneficial fungus.

Root-expressed maize lipoxygenase 3 negatively regulates induced systemic resistance to Colletotrichum graminicola in shoots.

We have previously reported that disruption of a maize root-expressed 9-lipoxygenase (9-LOX) gene, ZmLOX3, results in dramatic increase in resistance to diverse leaf and stalk pathogens. Despite evident economic significance of these findings, the mechanism behind this increased resistance remained elusive. In this study, we found that increased resistance of the lox3-4 mutants is due to constitutive activation of induced systemic resistance (ISR) signaling. We showed that ZmLOX3 lacked expression in leaves in response to anthracnose leaf blight pathogen Colletotrichum graminicola, but was expressed constitutively in the roots, thus, prompting our hypothesis: the roots of lox3-4 mutants are the source of increased resistance in leaves. Supporting this hypothesis, treatment of wild-type plants (WT) with xylem sap of lox3-4 mutant induced resistance to C. graminicola to the levels comparable to those observed in lox3-4 mutant. Moreover, treating mutants with the sap collected from WT plants partially restored the susceptibility to C. graminicola. lox3-4 mutants showed primed defense responses upon infection, which included earlier and greater induction of defense-related PAL and GST genes compared to WT. In addition to the greater expression of the octadecanoid pathway genes, lox3-4 mutant responded earlier and with a greater accumulation of H2O2 in response to C. graminicola infection or treatment with alamethicin. These findings suggest that lox3-4 mutants display constitutive ISR-like signaling. In support of this idea, root colonization by Trichoderma virens strain GV29-8 induced the same level of disease resistance in WT as the treatment with the mutant sap, but had no additional resistance effect in lox3-4 mutant. While treatment with T. virens GV29 strongly and rapidly suppressed ZmLOX3 expression in hydroponically grown WT roots, T. virens Δsml mutant, which is deficient in ISR induction, was unable to suppress expression of ZmLOX3, thus, providing genetic evidence that SM1 function in ISR, at least in part, by suppressing host ZmLOX3 gene. This study and the genetic tools generated herein will allow the identification of the signals regulating the induction of resistance to aboveground attackers by beneficial soil microorganisms in the future.

Functional analysis of non-ribosomal peptide synthetases (NRPSs) in Trichoderma virens reveals a polyketide synthase (PKS)/NRPS hybrid enzyme involved in the induced systemic resistance response in maize.

Trichoderma virens genome harbours genes encoding 22 non-ribosomal peptide synthetases (NRPSs) with at least one complete module (containing adenylation, thiolation and condensation domains) and four PKS/NRPS (polyketide synthase/NRPS) hybrid enzymes. After a primary screen for expression of these 26 genes when mycelia of T. virens are in contact with maize roots, seven genes that are upregulated were selected for further study. Using homologous recombination, loss-of-function mutants in six of these were obtained (the seventh, tex2, was acquired from our previous studies). Plant assays in a hydroponics system revealed that all seven mutants retained the ability to internally colonize maize roots. However, a mutation in one of the PKS/NRPS hybrid genes impaired the ability of T. virens to induce the defence response gene pal (phenylalanine ammonia lyase), suggesting a putative role for the associated metabolite product in induced systemic resistance. Interestingly, the mutant retained its ability to induce another defence response gene aos (allene oxide synthase). We thus provide evidence that a PKS/NRPS hybrid enzyme is involved in Trichoderma-plant interactions resulting in induction of defence responses.

Two classes of new peptaibols are synthesized by a single non-ribosomal peptide synthetase of Trichoderma virens.

Peptaibols are a group of small peptides having a high α-aminoisobutyric acid (Aib) content and produced by filamentous fungi, especially by the members of the genus Trichoderma (anamorph Hypocrea). These antibiotics are economically important for their anti-microbial and anti-cancer properties as well as ability to induce systemic resistance in plants against microbial invasion. In this study we present sequences of two classes (11-residue and 14-residue) of peptaibols produced by the biocontrol fungus Trichoderma virens. Of the 35 11-residue peptaibols sequenced, 18 are hitherto not described, and all the 53 14-residue sequences described by us here are new. We have also identified a peptaibol synthetase (non-ribosomal peptide synthetase, NRPS) with 14 complete modules in the genome of this fungus and disruption of this single gene (designated as tex2) resulted in the loss of both the classes of peptaibols. We, thus present here an unprecedented case where a single NRPS encodes for two classes of peptaibols. The new peptaibols identified here could have applications as therapeutic agents for the management of human and plant health.