Optimizing microbioreactor cultivation strategies for Trichoderma reesei: from batch to fed-batch operations.

BACKGROUND: Filamentous fungi have long been recognized for their exceptional enzyme production capabilities. Among these, Trichoderma reesei has emerged as a key producer of various industrially relevant enzymes and is particularly known for the production of cellulases. Despite the availability of advanced gene editing techniques for T. reesei, the cultivation and characterization of resulting strain libraries remain challenging, necessitating well-defined and controlled conditions with higher throughput. Small-scale cultivation devices are popular for screening bacterial strain libraries. However, their current use for filamentous fungi is limited due to their complex morphology. RESULTS: This study addresses this research gap through the development of a batch cultivation protocol using a microbioreactor for cellulase-producing T. reesei strains (wild type, RutC30 and RutC30 TR3158) with offline cellulase activity analysis. Additionally, the feasibility of a microscale fed-batch cultivation workflow is explored, crucial for mimicking industrial cellulase production conditions. A batch cultivation protocol was developed and validated using the BioLector microbioreactor, a Round Well Plate, adapted medium and a shaking frequency of 1000 rpm. A strong correlation between scattered light intensity and cell dry weight underscores the reliability of this method in reflecting fungal biomass formation, even in the context of complex fungal morphology. Building on the batch results, a fed-batch strategy was established for T. reesei RutC30. Starting with a glucose concentration of 2.5 g l - 1 in the batch phase, we introduced a dual-purpose lactose feed to induce cellulase production and prevent carbon catabolite repression. Investigating lactose feeding rates from 0.3 to 0.75 g (l h) - 1 , the lowest rate of 0.3 g (l h) - 1 revealed a threefold increase in cellobiohydrolase and a fivefold increase in ß -glucosidase activity compared to batch processes using the same type and amount of carbon sources. CONCLUSION: We successfully established a robust microbioreactor batch cultivation protocol for T. reesei wild type, RutC30 and RutC30 TR3158, overcoming challenges associated with complex fungal morphologies. The study highlights the effectiveness of microbioreactor workflows in optimizing cellulase production with T. reesei, providing a valuable tool for simultaneous assessment of critical bioprocess parameters and facilitating efficient strain screening. The findings underscore the potential of microscale fed-batch strategies for enhancing enzyme production capabilities, revealing insights for future industrial applications in biotechnology.

Extracellular vesicles from the mycoparasitic fungus Trichoderma harzianum.

Trichoderma harzianum is a filamentous fungus that can act as a mycoparasite, saprophyte, or a plant symbiotic. It is widely used as a biological control agent against phytopathogenic fungi and can also be used for plant growth promotion and biofortification. Interaction between T. harzianum and phytopathogenic fungi involves mycoparasitism, competition, and antibiosis. Extracellular vesicles (EVs) have been described as presenting a central role in mechanisms of communication and interaction among fungus and their hosts. In this study, we characterized extracellular vesicles of T. harzianum produced during growth in the presence of glucose or S. sclerotiorum mycelia. A set of vesicular proteins was identified using proteomic approach, mainly presenting predicted signal peptides.

Performance evaluation of Trichoderma reseei in tolerance and biodegradation of diuron herbicide in agar plate, liquid culture and solid-state fermentation.

The present study evaluated the performance of the fungus Trichoderma reesei to tolerate and biodegrade the herbicide diuron in its agrochemical presentation in agar plates, liquid culture, and solid-state fermentation. The tolerance of T. reesei to diuron was characterized through a non-competitive inhibition model of the fungal radial growth on the PDA agar plate and growth in liquid culture with glucose and ammonium nitrate, showing a higher tolerance to diuron on the PDA agar plate (inhibition constant 98.63 mg L-1) than in liquid culture (inhibition constant 39.4 mg L-1). Diuron biodegradation by T. reesei was characterized through model inhibition by the substrate on agar plate and liquid culture. In liquid culture, the fungus biotransformed diuron into 3,4-dichloroaniline using the amide group from the diuron structure as a carbon and nitrogen source, yielding 0.154 mg of biomass per mg of diuron. A mixture of barley straw and agrolite was used as the support and substrate for solid-state fermentation. The diuron removal percentage in solid-state fermentation was fitted by non-multiple linear regression to a parabolic surface response model and reached the higher removal (97.26%) with a specific aeration rate of 1.0 vkgm and inoculum of 2.6 × 108 spores g-1. The diuron removal in solid-state fermentation by sorption on barley straw and agrolite was discarded compared to the removal magnitude of the biosorption and biodegradation mechanisms of Trichoderma reesei. The findings in this work about the tolerance and capability of Trichoderma reesei to remove diuron in liquid and solid culture media demonstrate the potential of the fungus to be implemented in bioremediation technologies of herbicide-polluted sites.

Specific activity and utility of recombinant cellobiohydrolase II (Cel6A) produced in maize endosperm.

Cellobiohydrolase II (CBH II) is an exo-glucanase that is part of a fungal mixture of enzymes from a wood-rot fungus, Trichoderma reesei. It is therefore difficult to purify and to establish a specific activity assay. The gene for this enzyme, driven by the rice Os glutelin promoter, was transformed into High II tissue culture competent corn, and the enzyme accumulated in the endosperm of the seed. The transgenic line recovered from tissue culture was bred into male and female elite Stine inbred corn lines, stiff stalk 16083-025 (female) and Lancaster MSO411 (male), for future production in their hybrid. The enzyme increases its accumulation throughout its 6 generations of back crosses, 27-266-fold between T1 and T2, and 2-10-fold between T2 and T3 generations with lesser increases in T4-T6. The germplasm of the inbred lines replaces the tissue culture corn variety germplasm with each generation, with the ultimate goal of producing a high-yielding hybrid with the transgene. The CBH II enzyme was purified from T5 inbred male grain 10-fold to homogeneity with 47.5% recovery. The specific activity was determined to be 1.544 units per µg protein. The corn-derived CBH II works in biopolishing of cotton by removing surface fibers to improve dyeability and increasing glucose from corn flour for increasing ethanol yield from starch-based first-generation processes.

Exploiting Systematic Engineering of the Expression Cassette as a Powerful Tool to Enhance Heterologous Gene Expression in Trichoderma reesei.

Many endeavors in expressing a heterologous gene in microbial hosts rely on simply placing the gene of interest between a selected pair of promoters and terminator. However, although the expression efficiency could be improved by engineering the host cell, how modifying the expression cassette itself systematically would affect heterologous gene expression remains largely unknown. As the promoter and terminator bear plentiful cis-elements, herein using the Aspergillus niger mannanase with high application value in animal feeds and the eukaryotic filamentous fungus workhorse Trichoderma reesei as a model gene/host, systematic engineering of an expression cassette was investigated to decipher the effect of its mutagenesis on heterologous gene expression. Modifying the promoter, signal peptide, the eukaryotic-specific Kozak sequence, and the 3'-UTR could stepwise improve extracellular mannanase production from 17 U/mL to an ultimate 471 U/mL, representing a 27.7-fold increase in expression. The strategies can be generally applied in improving the production of heterologous proteins in eukaryotic microbial hosts.

Antagonistic properties against Fusarium sporotrichioides and glycosylation of HT-2 and T-2 toxins by selected Trichoderma strains.

The present study assessed the ability of Trichoderma to combat F. sporotrichioides, focusing on their antagonistic properties. Tests showed that Trichoderma effectively inhibited F. sporotrichioides mycelial growth, particularly with T. atroviride strains. In co-cultures on rice grains, Trichoderma almost completely reduced the biosynthesis of T-2 and HT-2 toxins by Fusarium. T-2 toxin-α-glucoside (T-2-3α-G), HT-2 toxin-α-glucoside (HT-2-3α-G), and HT-2 toxin-ß-glucoside (HT-2-3ß-G) were observed in the common culture medium, while these substances were not present in the control medium. The study also revealed unique metabolites and varying metabolomic profiles in joint cultures of Trichoderma and Fusarium, suggesting complex interactions. This research offers insights into the processes of biocontrol by Trichoderma, highlighting its potential as a sustainable solution for managing cereal plant pathogens and ensuring food safety.

Synthesis of nanoparticles using Trichoderma Harzianum, characterization, antifungal activity and impact on Plant Growth promoting Bacteria.

Globally cultivated cereals are frequently threatened by various plant pathogenic agents such as Fusarium fungi. To combat these pathogens, researchers have made nanoparticles as potential agricultural pesticides. In this study, selenium and titanium dioxide NPs were synthesized using Trichoderma harzianum metabolites. Characterization of the NPs indicated varying size and shapes of both NPs and functional groups existence to constitute both NPs. The evaluation of antifungal activity of NPs against plant pathogenic fungi, Fusarium culmorum, indicated both NPs maximum antifungal activity at concentration of 100 mg/L. The impacts of nanoparticles on some beneficial plant growth promoting bacteria (PGPB) were evaluated and showed their inhibition effect on optical density of PGPB at a concentration of 100 mg/L but they did not have any impact on nitrogen fixation by bacteria. Existence of TiO2NPs reduced the intensity of color change to pink compared to the control indicating auxin production. Both NPs demonstrated different impact on phosphate solubilization index. This study suggests that the synthesized nanoparticles have the potential to serve as antifungal compounds at special concentration against plant diseases without significantly reducing the potential of PGPB at low concentrations.

The regulatory pathway of transcription factor MYB36 from Trichoderma asperellum Tas653 resistant to poplar leaf blight pathogen Alternaria alternata Aal004.

In fungi, MYB transcription factors (TFs) mainly regulate growth, development, and resistance to stress. However, as major disease-resistance TFs, they have rarely been studied in biocontrol fungi. In this study, MYB36 of Trichoderma asperellum Tas653 (Ta) was shown to respond strongly to the stress caused by Alternaria alternata Aa1004. Compared with wild-type Ta (Ta-Wt), the inhibition rate of the MYB36 knockout strain (Ta-Kn) on Aa1004 decreased by 11.06%; the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities decreased by 82.15 U/g, 0.19 OD470/min/g, and 1631.2 µmol/min/g, respectively. The MYB36 overexpression strain (Ta-Oe) not only enhanced hyperparasitism on Aa1004, caused its hyphae to swell, deform, or even rupture, but also reduced the incidence rate of poplar leaf blight. MYB36 regulates downstream (TFs, detoxification genes, defense genes, and other antifungal-related genes by binding to the cis-acting elements "ACAT" and "ATCG". Zinc finger TFs, as the main antifungal TFs, account for 90% of the total TFs, and Zn37.5 (23.24-) and Zn83.7 (23.18-fold) showed the greatest expression difference when regulated directly by MYB36. The detoxification genes mainly comprised 11 major major facilitator superfamily (MFS) genes, among which MYB36 directly increased the expression levels of three genes by more than 2-3.44-fold. The defense genes mainly encoded cytochrome P450 (P450) and hydrolases. e.g., P45061.3 (2-10.95-), P45060.2 (2-7.07-), and Hyd44.6 (2-2.30-fold). This study revealed the molecular mechanism of MYB36 regulation of the resistance of T. asperellum to A. alternata and provides theoretical guidance for the biocontrol of poplar leaf blight and the anti-disease mechanism of biocontrol fungi.

Engineering of glycoside hydrolase family 7 cellobiohydrolases directed by natural diversity screening.

Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, glycoside hydrolase family 7 (GH7) CBHs are critically important for the bioeconomy and typically difficult to engineer. Here, we target the discovery of highly active natural GH7 CBHs and engineering of variants with improved activity. Using experimentally assayed activities of genome mined CBHs, we applied sequence and structural alignments to top performers to identify key point mutations linked to improved activity. From ∼1500 known GH7 sequences, an evolutionarily diverse subset of 57 GH7 CBH genes was expressed in Trichoderma reesei and screened using a multiplexed activity screening assay. Ten catalytically enhanced natural variants were identified, produced, purified, and tested for efficacy using industrially relevant conditions and substrates. Three key amino acids in CBHs with performance comparable or superior to Penicillium funiculosum Cel7A were identified and combinatorially engineered into P. funiculosum cel7a, expressed in T. reesei, and assayed on lignocellulosic biomass. The top performer generated using this combined approach of natural diversity genome mining, experimental assays, and computational modeling produced a 41% increase in conversion extent over native P. funiculosum Cel7A, a 55% increase over the current industrial standard T. reesei Cel7A, and 10% improvement over Aspergillus oryzae Cel7C, the best natural GH7 CBH previously identified in our laboratory.

Sulfur assimilation using gaseous carbonyl sulfide by the soil fungus Trichoderma harzianum.

Fungi have the capacity to assimilate a diverse range of both inorganic and organic sulfur compounds. It has been recognized that all sulfur sources taken up by fungi are in soluble forms. In this study, we present evidence that fungi can utilize gaseous carbonyl sulfide (COS) for the assimilation of a sulfur compound. We found that the filamentous fungus Trichoderma harzianum strain THIF08, which has constitutively high COS-degrading activity, was able to grow with COS as the sole sulfur source. Cultivation with 34S-labeled COS revealed that sulfur atom from COS was incorporated into intracellular metabolites such as glutathione and ergothioneine. COS degradation by strain THIF08, in which as much of the moisture derived from the agar medium as possible was removed, indicated that gaseous COS was taken up directly into the cell. Escherichia coli transformed with a COS hydrolase (COSase) gene, which is clade D of the ß-class carbonic anhydrase subfamily enzyme with high specificity for COS but low activity for CO2 hydration, showed that the COSase is involved in COS assimilation. Comparison of sulfur metabolites of strain THIF08 revealed a higher relative abundance of reduced sulfur compounds under the COS-supplemented condition than the sulfate-supplemented condition, suggesting that sulfur assimilation is more energetically efficient with COS than with sulfate because there is no redox change of sulfur. Phylogenetic analysis of the genes encoding COSase, which are distributed in a wide range of fungal taxa, suggests that the common ancestor of Ascomycota, Basidiomycota, and Mucoromycota acquired COSase at about 790-670 Ma.IMPORTANCEThe biological assimilation of gaseous CO2 and N2 involves essential processes known as carbon fixation and nitrogen fixation, respectively. In this study, we found that the fungus Trichoderma harzianum strain THIF08 can grow with gaseous carbonyl sulfide (COS), the most abundant and ubiquitous gaseous sulfur compound, as a sulfur source. When the fungus grew in these conditions, COS was assimilated into sulfur metabolites, and the key enzyme of this assimilation process is COS hydrolase (COSase), which specifically degrades COS. Moreover, the pathway was more energy efficient than the typical sulfate assimilation pathway. COSase genes are widely distributed in Ascomycota, Basidiomycota, and Mucoromycota and also occur in some Chytridiomycota, indicating that COS assimilation is widespread in fungi. Phylogenetic analysis of these genes revealed that the acquisition of COSase in filamentous fungi was estimated to have occurred at about 790-670 Ma, around the time that filamentous fungi transitioned to a terrestrial environment.

Mechanisms for plant growth promotion activated by Trichoderma in natural and managed terrestrial ecosystems.

Trichoderma spp. are free-living fungi present in virtually all terrestrial ecosystems. These soil fungi can stimulate plant growth and increase plant nutrient acquisition of macro- and micronutrients and water uptake. Generally, plant growth promotion by Trichoderma is a consequence of the activity of potent fungal signaling metabolites diffused in soil with hormone-like activity, including indolic compounds as indole-3-acetic acid (IAA) produced at concentrations ranging from 14 to 234 µg l-1, and volatile organic compounds such as sesquiterpene isoprenoids (C15), 6-pentyl-2H-pyran-2-one (6-PP) and ethylene (ET) produced at levels from 10 to 120 ng over a period of six days, which in turn, might impact plant endogenous signaling mechanisms orchestrated by plant hormones. Plant growth stimulation occurs without the need of physical contact between both organisms and/or during root colonization. When associated with plants Trichoderma may cause significant biochemical changes in plant content of carbohydrates, amino acids, organic acids and lipids, as detected in Arabidopsis thaliana, maize (Zea mays), tomato (Lycopersicon esculentum) and barley (Hordeum vulgare), which may improve the plant health status during the complete life cycle. Trichoderma-induced plant beneficial effects such as mechanisms of defense and growth are likely to be inherited to the next generations. Depending on the environmental conditions perceived by the fungus during its interaction with plants, Trichoderma can reprogram and/or activate molecular mechanisms commonly modulated by IAA, ET and abscisic acid (ABA) to induce an adaptative physiological response to abiotic stress, including drought, salinity, or environmental pollution. This review, provides a state of the art overview focused on the canonical mechanisms of these beneficial fungi involved in plant growth promotion traits under different environmental scenarios and shows new insights on Trichoderma metabolites from different chemical classes that can modulate specific plant growth aspects. Also, we suggest new research directions on Trichoderma spp. and their secondary metabolites with biological activity on plant growth.

Molecular insights into the mutualism that induces iron deficiency tolerance in sorghum inoculated with Trichoderma harzianum.

Iron (Fe) deficiency is a common mineral stress in plants, including sorghum. Although the soil fungus Trichoderma harzianum has been shown to mitigate Fe deficiency in some circumstances, neither the range nor mechanism(s) of this process are well understood. In this study, high pH-induced Fe deficiency in sorghum cultivated in pots with natural field soil exhibited a significant decrease in biomass, photosynthetic rate, transpiration rate, stomatal conductance, water use efficiency, and Fe-uptake in both the root and shoot. However, the establishment of T. harzianum colonization in roots of Fe-deprived sorghum showed significant improvements in morpho-physiological traits, Fe levels, and redox status. Molecular detection of the fungal ThAOX1 (L-aminoacid oxidase) gene showed the highest colonization of T. harzianum in the root tips of Fe-deficient sorghum, a location thus targeted for further analysis. Expression studies by RNA-seq and qPCR in sorghum root tips revealed a significant upregulation of several genes associated with Fe uptake (SbTOM2), auxin synthesis (SbSAURX15), nicotianamine synthase 3 (SbNAS3), and a phytosiderophore transporter (SbYS1). Also induced was the siderophore synthesis gene (ThSIT1) in T. harzianum, a result supported by biochemical evidence for elevated siderophore and IAA (indole acetic acid) levels in roots. Given the high affinity of fungal siderophore to chelate insoluble Fe3+ ions, it is likely that elevated siderophore released by T. harzianum led to Fe(III)-siderophore complexes in the rhizosphere that were then transported into roots by the induced SbYS1 (yellow-stripe 1) transporter. In addition, the observed induction of several plant peroxidase genes and ABA (abscisic acid) under Fe deficiency after inoculation with T. harzianum may have helped induce tolerance to Fe-deficiency-induced oxidative stress and adaptive responses. This is the first mechanistic explanation for T. harzianum's role in helping alleviate Fe deficiency in sorghum and suggests that biofertilizers using T. harzianum will improve Fe availability to crops in high pH environments.

Proteome profiling of enriched membrane-associated proteins unraveled a novel sophorose and cello-oligosaccharide transporter in Trichoderma reesei.

BACKGROUND: Trichoderma reesei is an organism extensively used in the bioethanol industry, owing to its capability to produce enzymes capable of breaking down holocellulose into simple sugars. The uptake of carbohydrates generated from cellulose breakdown is crucial to induce the signaling cascade that triggers cellulase production. However, the sugar transporters involved in this process in T. reesei remain poorly identified and characterized. RESULTS: To address this gap, this study used temporal membrane proteomics analysis to identify five known and nine putative sugar transporters that may be involved in cellulose degradation by T. reesei. Docking analysis pointed out potential ligands for the putative sugar transporter Tr44175. Further functional validation of this transporter was carried out in Saccharomyces cerevisiae. The results showed that Tr44175 transports a variety of sugar molecules, including cellobiose, cellotriose, cellotetraose, and sophorose. CONCLUSION: This study has unveiled a transporter Tr44175 capable of transporting cellobiose, cellotriose, cellotetraose, and sophorose. Our study represents the first inventory of T. reesei sugar transportome once exposed to cellulose, offering promising potential targets for strain engineering in the context of bioethanol production.

Transcriptomics integrated with metabolomics reveal the competitive relationship between co-cultured Trichoderma asperellum HG1 and Bacillus subtilis Tpb55.

Microbial co-culture has proven to be an effective way to improve the ability of microorganisms to biocontrol. However, the interactive mechanisms of co-cultural microbes, especially between fungi and bacteria, have rarely been studied. By comparative analysis of morphology, transcriptomics and metabolomics, the interactive mechanisms of a sequential co-culture system of Trichoderma asperellum HG1 and Bacillus subtilis Tpb55 was explored in this study. The results revealed that co- culture has no significant effect on the growth and cell morphology of the two strains, but lead to mycelium wrinkling of HG1. RNA-seq analysis showed that co-culture significantly upregulated the HG1 genes concerning amino acid degradation and metabolism, proteolysis, resisting environmental stress, cell homeostasis, glycolysis, the glyoxylate cycle, and the citric acid (TCA) cycle, while Tpb55 genes related to cell homeostasis, spore formation and membrane fluidization were significantly upregulated, but genes associating to TCA, glycolytic cycles and fatty acid ß-oxidation were significantly downregulated. Metabolomic results revealed that some amino acids related to energy metabolism were significantly altered in HG1, whereas palmitic acid, which is related to cell membrane functions, was upregulated in Tpb55. These results indicated that HG1 could interfere with carbon metabolism and cell membrane fluidity, but accelerate spore formation of Tpb55. Biophysical assays further convinced that co-culture could decrease ATP content and inhibit ATPase activity in HG1, and could promote spore formation and reduce the cell membrane fluidity of Tpb55. In addition, co-culture also accelerated the production of intracellular anti-oomycete compound octhilinone. The above results indicate that HG1 and Tpb55 are mainly in a competitive relationship in the co culture system. These findings provide new insights for understanding the interaction mechanism between co cultured microbes.

Regulation of cellulase production via calcium signaling in Trichoderma reesei under PEG8000 stress.

In this study, the effect of polyethylene glycol 8000 (PEG8000) stress on cellulase biosynthesis in Trichoderma reesei CICC2626 via calcium signaling was investigated, and a plausible mechanism by which intracellular Ca2+ regulates the transcription of cellulase genes was proposed. The results indicated that the total cellulase (filter paper-hydrolyzing activity [FPase]), endoglucanase (carboxymethyl cellulase activity [CMCase]), and ß-glucosidase activities of the strain were 1.3-, 1.2-, and 1.3-fold higher than those of the control (no PEG8000 addition) at a final concentration of 1.5% (w/v) PEG8000. Moreover, the transcriptional levels of cellulase genes, protein concentrations, and biomass increased. With the synergistic use of commercial cellulase and T. reesei CICC2626 cellulase to hydrolyze alkali-pretreated rice straw, the released reducing sugar concentration reached 372.7 mg/g, and the cellulose content (22.7%, 0.32 g) was significantly lower than the initial content (62.5%, 1.88 g). Transcriptome data showed that 12 lignocellulose degradation-related genes were significantly upregulated in the presence of 1.5% PEG8000. Furthermore, the addition of Ca2+ inhibitors and deletion of crz1 (calcineurin-responsive zinc finger 1-encoding gene, which is related to the calcium signaling pathway) demonstrated that calcium signaling plays a dominant role in PEG8000-induced cellulase genes overexpression. These results revealed a link between PEG8000 induction and calcium signaling transduction in T. reesei CICC2626. Moreover, this study also provides a novel inducer for enhanced cellulase production. KEY POINTS: • Cellulase biosynthesis in Trichoderma reesei could be enhanced by PEG8000 • PEG8000 could induce a cytosolic Ca2+ burst in Trichoderma reesei • The activated calcium signaling was involved in cellulase biosynthesis.

Insights into the molecular mechanism of Trichoderma stimulating plant growth and immunity against phytopathogens.

Trichoderma species have received significant interest as beneficial fungi for boosting plant growth and immunity against phytopathogens. By establishing a mutualistic relationship with plants, Trichoderma causes a series of intricate signaling events that eventually promote plant growth and improve disease resistance. The mechanisms contain the indirect or direct involvement of Trichoderma in enhancing plant growth by modulating phytohormones signaling pathways, improving uptake and accumulation of nutrients, and increasing soil bioavailability of nutrients. They contribute to plant resistance by stimulating systemic acquired resistance through salicylic acid, jasmonic acid, and ethylene signaling. A cascade of signal transduction processes initiated by the interaction of Trichoderma and plants regulate the expression of defense-related genes, resulting in the synthesis of defense hormones and pathogenesis-related proteins (PRPs), which collectively improve plant resistance. Additionally, advancements in omics technologies has led to the identification of key pathways, their regulating genes, and molecular interactions in the plant defense and growth promotion responses induced by Trichoderma. Deciphering the molecular mechanism behind Trichoderma's induction of plant defense and immunity is essential for harnessing the full plant beneficial potential of Trichoderma. This review article sheds light on the molecular mechanisms that underlie the positive effects of Trichoderma-induced plant immunity and growth and opens new opportunities for developing environmentally friendly and innovative approaches to improve plant immunity and growth.

Mycoparasitism related targets of Tmk1 indicate stimulating regulatory functions of this MAP kinase in Trichoderma atroviride.

Mycoparasitism is a key feature of Trichoderma (Hypocreales, Ascomycota) biocontrol agents. Recent studies of intracellular signal transduction pathways of the potent mycoparasite Trichoderma atroviride revealed the involvement of Tmk1, a mitogen-activated protein kinase (MAPK), in triggering the mycoparasitic response. We previously showed that mutants missing Tmk1 exhibit reduced mycoparasitic activity against several plant pathogenic fungi. In this study, we identified the most robustly regulated targets that were governed by Tmk1 during mycoparasitism using transcriptome and proteome profiling. Tmk1 mainly exerts a stimulating function for T. atroviride during its mycoparasitic interaction with the fungal plant pathogen Rhizoctonia solani, as reflected by 89% of strongly differently responding genes in the ∆tmk1 mutant compared to the wild type. Specifically, 54% of these genes showed strong downregulation in the response with a deletion of the tmk1 gene, whereas in the wild type the same genes were strongly upregulated during the interaction with the fungal host. These included the gene encoding the mycoparasitism-related proteinase Prb1; genes involved in signal transduction pathways such as a candidate coding for a conserved 14-3-3 protein, and a gene coding for Tmk2, the T. atroviride cell-wall integrity MAP kinase; genes encoding a specific siderophore synthetase, and multiple FAD-dependent oxidoreductases and aminotransferases. Due to the phosphorylating activity of Tmk1, different (phospho-)proteomics approaches were applied and identified proteins associated with cellular metabolism, energy production, protein synthesis and fate, and cell organization. Members of FAD- and NAD/NADP-binding-domain proteins, vesicular trafficking of molecules between cellular organelles, fungal translational, as well as protein folding apparatus were among others found to be phosphorylated by Tmk1 during mycoparasitism. Outstanding downregulation in the response of the ∆tmk1 mutant to the fungal host compared to the wild type at both the transcriptome and the proteome levels was observed for nitrilase, indicating that its defense and detoxification functions might be greatly dependent on Tmk1 during T. atroviride mycoparasitism. An intersection network analysis between the identified transcripts and proteins revealed a strong involvement of Tmk1 in molecular functions with GTPase and oxidoreductase activity. These data suggest that during T. atroviride mycoparasitism this MAPK mainly governs processes regulating cell responses to extracellular signals and those involved in reactive oxygen stress.

Thirteen new peptaibols with antimicrobial activities from Trichoderma sp.

From the fungus Trichoderma sp., we isolated seven novel 18-residue peptaibols, neoatroviridins E-K (1-7), and six new 14-residue peptaibols, harzianins NPDG J-O (8-13). Additionally, four previously characterized 18-residue peptaibols neoatroviridins A-D (14-17) were also identified. The structural configurations of the newly identified peptaibols (1-13) were determined by comprehensive nuclear magnetic resonance (NMR) and high-resolution electrospray ionization tandem mass spectrometry (HR-ESI-MS/MS) data. Their absolute configurations were further determined using Marfey's method. Notably, compounds 12 and 13 represent the first 14-residue peptaibols containing an acidic amino acid residue. In antimicrobial assessments, all 18-residue peptaibols (1-7, 14-17) exhibited moderate inhibitory activities against Staphylococcus aureus 209P, with minimum inhibitory concentration (MIC) values ranging from 8-32 µg·mL-1. Moreover, compound 9 exhibited moderate inhibitory effect on Candida albicans FIM709, with a MIC value of 16 µg·mL-1.

Metabolomics reveal metabolic variation caused by co-culture of Arthrobacter ureafaciens and Trichoderma harzianum and their impacts on wheat germination / La metabolómica revela la variación metabólica causada por el cocultivo de Arthrobacter ureafaciens y Trichoderma harzianum y sus impactos en la germinación del trigo

Arthrobacter ureafaciens DnL1-1 is a bacterium used for atrazine degradation, while Trichoderma harzianum LTR-2 is a widely used biocontrol fungus. In this study, a liquid co-cultivation of these two organisms was initially tested. The significant changes in the metabolome of fermentation liquors were investigated based on cultivation techniques (single-cultured and co-cultured DnL1-1 and LTR-2) using an UPLC-QTOF-MS in an untargeted metabolomic approach. Principle components analysis (PCA) and partial least squares discriminant analysis (PLS-DA) supervised modelling revealed modifications of the metabolic profiles in fermentation liquors as a function of interactions between different strains. Compared with pure-cultivation of DnL1-1, 51 compounds were altered during the cocultivation, with unique and significant differences in the abundance of organic nitrogen compounds (e.g. carnitine, acylcarnitine 4:0, acylcarnitine 5:0, 3-dehydroxycarnitine and O-acetyl-L-carnitine) and trans-zeatin riboside. Nevertheless, compared with pure-cultivation of LTR-2, the abundance of 157 compounds, including amino acids, soluble sugars, organic acids, indoles and derivatives, nucleosides, and others, changed significantly in the cocultivation. Among them, the concentration of tryptophan, which is a precursor to indoleacetic acid, indoleacetic acid, aspartic acid, and L-glutamic acid increased while that of most soluble sugars decreased upon cocultivation. The fermentation filtrates of co-cultivation of LTR-2 and DnL1-1 showed significant promoting effects on germination and radicle length of wheat. A subsequent experiment demonstrated synergistic effects of differential metabolites caused by co-cultivation of DnL1-1 and LTR-2 on wheat germination. Comprehensive metabolic profiling may provide valuable information on the effects of DnL1-1 and LTR-2 on wheat growth.(AU)

N -acylhomoserine lactone-degrading activity of Trichoderma species and its application in the inhibition of bacterial quorum sensing.

Many gram-negative pathogens can activate virulence factors under the control of N-acylhomoserine lactone (AHL）-mediated quorum sensing. AHL-degrading enzymes have been investigated for their application in disease control. Trichoderma is a genus of fungi inhabiting various types of soil and are widely used as biocontrol agents for plant pathogens. When the AHL-degrading activity of 33 strains belonging to Trichoderma species was investigated, most strains can degrade AHL. AHL lactonase catalyzes AHL ring opening by hydrolyzing lactone. Two model strains, Trichoderma atroviride MAFF 242473 and MAFF 242475, degrade AHL using their AHL lactonase activity and rapidly metabolize ring-opening AHL. Moreover, co-inoculation with MAFF 242473 and MAFF 242475 effectively inhibited AHL production by the plant pathogens, Pantoea ananatis and Pectobacterium carotovorum subsp. carotovorum. Our study suggested that Trichoderma might be an effective biocontrol agent to inhibit the expression of virulence factors via AHL-mediated quorum sensing.