Trichoderma spp. can enhance plant resistance against a wide range of biotic stressors. However, the fundamental mechanisms by which Trichoderma enhances plant resistance against Meloidogyne incognita, known as root-knot nematodes (RKNs), are still unclear. Here, we identified a strain of Trichoderma asperellum (T141) that could effectively suppress RKN infestation in tomato (Solanum lycopersicum L.). Nematode infestation led to an increase in the concentrations of reactive oxygen species (ROS) and malondialdehyde (MDA) in roots but pre-inoculation with T141 significantly decreased oxidative stress. The reduction in ROS and MDA was accompanied by an increase in the activity of antioxidant enzymes and the accumulation of flavonoids and phenols. Moreover, split root test-based analysis showed that T141 inoculation in local roots before RKN inoculation increased the concentration of phytohormone jasmonate (JA) and the transcripts of JA synthesis and signaling-related genes in distant roots. UPLC-MS/MS-based metabolomics analysis identified 1051 differentially accumulated metabolites (DAMs) across 4 pairwise comparisons in root division test, including 81 flavonoids. Notably, 180 DAMs were found in comparison between RKN and T141-RKN, whereas KEGG annotation and enrichment analysis showed that the secondary metabolic pathways, especially the flavonoid biosynthesis, played a key role in the T141-induced systemic resistance to RKNs. The role of up-regulated flavonoids in RKN mortality was further verified by in vitro experiments with the exogenous treatment of kaempferol, hesperidin and rutin on J2-stage RKNs. Our results revealed a critical mechanism by which T141 induced resistance of tomato plants against the RKNs by systemically promoting secondary metabolism in distant roots.

Enzymatic degradation of lignocellulosic biomass provides an eco-friendly approach to produce value-added macromolecules, e.g., bioactive polysaccharides. A novel acidophilic GH5 β-1,4-endoglucanase (termed TaCel5) from Trichoderma asperellum ND-1 was efficiently expressed in Komagataella phaffii (∼1.5-fold increase, 38.42 U/mL). TaCel5 displayed both endoglucanase (486.3 U/mg) and alginate lyase (359.5 U/mg) enzyme activities. It had optimal pH 3.0 and strong pH stability (exceed 86 % activity retained over pH range 3.0-5.0). 80 % activity (both endoglucanase and alginate lyase) was retained in the presence of 15 % ethanol or 3.42 M NaCl. Analysis of action mode revealed that hydrolytic activity of TaCel5 required at least three glucose (cellotriose) residues, yielding mainly cellobiose. Glu241 and Glu352 are essential catalytic residues, while Asp106, Asp277 and Asp317 play auxiliary roles in cellulose degradation. TaCel5 displayed high hydrolysis efficiency for glucan and alginate substrates. ESI-MS analysis indicated that the enzymatic hydrolysates of alginate mainly contained disaccharides and heptasaccharides. This is the first detailed report of a bifunctional GH5 endoglucanase/alginate lyase enzyme from T. asperellum. Thus TaCel5 has strong potential in food and feed industries as a catalyst for bioconversion of cellulose- and alginate-containing waste materials into value-added products oligosaccharides, which was of great benefit both for the economy and environment.

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.

A novel acidophilic GH5 β-1,4-endoglucanase (TaCel12) from Trichoderma asperellum ND-1 was efficiently expressed in Pichia pastoris (a 1.5-fold increase). Deglycosylated TaCel12 migrated as a single band (26.5 kDa) in SDS-PAGE. TaCel12 was acidophilic with a pH optimum of 4.0 and displayed great pH stability (>80 % activity over pH 3.0-5.0). TaCel12 exhibited considerable activity towards sodium carboxymethyl cellulose and sodium alginate with Vmax values of 197.97 μmol/min/mg and 119.06 μmol/min/mg, respectively. Moreover, TaCel12 maintained >80 % activity in the presence of 20 % ethanol and 4.28 M NaCl. Additionally, Mn2+, Pb2+ and Cu2+ negatively affected TaCel12 activity, while the presence of 5 mM Co2+ significantly increased the enzyme activity. Analysis of action mode revealed that TaCel12 required at least four glucose (cellotetraose) residues for hydrolysis to yield cellobiose and cellotriose. Site-directed mutagenesis results suggested that Glu133 and Glu217 of TaCel12 are crucial catalytic residues, with Asp116 displaying an auxiliary function. Production of soluble sugars from lignocellulose is a crucial step in bioethanol development, and it is noteworthy that TaCel12 could synergistically yield fermentable sugars from corn stover and bagasse, respectively. Thus TaCel12 with excellent properties will be considered a potential biocatalyst for applications in various industries, especially for lignocellulosic biomass conversion.

A novel aspartic protease gene (TaproA1) from Trichoderma asperellum was successfully expressed in Komagataella phaffii (Pichia pastoris). TaproA1 showed 52.8% amino acid sequence identity with the aspartic protease PEP3 from Coccidioides posadasii C735. TaproA1 was efficiently produced in a 5 L fermenter with a protease activity of 4092 U/mL. It exhibited optimal reaction conditions at pH 3.0 and 50 °C and was stable within pH 3.0-6.0 and at temperatures up to 45 °C. The protease exhibited broad substrate specificity with high hydrolysis activity towards myoglobin and hemoglobin. Furthermore, duck blood proteins (hemoglobin and plasma protein) were hydrolyzed by TaproA1 to prepare bioactive peptides with high ACE inhibitory activity. The IC50 values of hemoglobin and plasma protein hydrolysates from duck blood proteins were 0.105 mg/mL and 0.091 mg/mL, respectively. Thus, the high yield and excellent biochemical characterization of TaproA1 presented here make it a potential candidate for the preparation of duck blood peptides. KEY POINTS: • An aspartic protease (TaproA1) from Trichoderma asperellum was expressed in Komagataella phaffii. • TaproA1 exhibited broad substrate specificity and the highest activity towards myoglobin and hemoglobin. • TaproA1 has great potential for the preparation of bioactive peptides from duck blood proteins.

Trichoderma, a well-known and extensively studied fungal genus, has gained significant attention for its remarkable antagonistic abilities against a wide range of plant pathogens. In this study, a total of 108 Trichoderma isolates were screened through in vitro dual antagonistic assays and culture filtrate inhibition against Fusarium graminearum. Of these, the YNQJ1002 displayed noteworthy inhibitory activities along with thermal stability. To validate the metabolic differences between YNQJ1002 and GZLX3001 (with strong and weak antagonism, respectively), UPLC-TOF-MS/MS mass spectrometry was employed to analyze and compare the metabolite profiles. We identified 12 significantly up-regulated metabolites in YNQJ1002, which include compounds like Trigoneoside, Torvoside, trans,trans-hepta-2,4,6-trienoic acid, and Chamazulene. These metabolites are known for their antimicrobial properties or signaling roles as components of cell membranes. Enriched KEGG analysis revealed a significant enrichment in sphingolipid metabolism and linoleic acid metabolism, as well as autophagy. The results demonstrated that YNQJ1002\'s abundance of antimicrobial substances, resulting from specific metabolic pathways, enhanced its superior antagonistic activity against F. graminearum. Finally, YNQJ1002 was identified using the ITS, tef1-1α, and rpb2 regions, with MIST system sequence matching confirming its classification within the species. Overall, we have obtained a novel strain, T. asperellum YNQJ1002, which is rich in metabolites and shows potential antagonistic activity against F. graminearum. This study has opened promising prospects for the development of innovative Trichoderma-derived antifungal compounds, featuring a unique mechanism against pathogens.

BACKGROUND: Quorum sensing inhibitors (QSIs) are an emerging control tool that inhibits the quorum sensing (QS) system of pathogenic bacteria. We aimed to screen for potential QSIs in the metabolites of Trichoderma and to explore their inhibitory mechanisms.
RESULTS: We screened a strain of Trichoderma asperellum LN004, which demonstrated the ability to inhibit the color development of Chromobacterium subtsugae CV026, primarily attributed to the presence of emodin as its key QSI component. The quantitative polymerase chain reaction with reverse transcription results showed that after emodin treatment of Pectobacterium carotovorum subsp. carotovorum (Pcc), plant cell wall degrading enzyme-related synthetic genes were significantly downregulated, and the exogenous enzyme synthesis gene negative regulator (rsmA) was upregulated 3.5-fold. Docking simulations indicated that emodin could be a potential ligand for ExpI and ExpR proteins because it exhibited stronger competition than the natural ligands in Pcc. In addition, western blotting showed that emodin attenuated the degradation of n-acylhomoserine lactone on the ExpR protein and protected it. Different concentrations of emodin reduced the activity of pectinase, cellulase, and protease in Pcc by 20.81%-72.21%, 8.38%-52.73%, and 3.57%-47.50%. Lesion size in Chinese cabbages, carrots and cherry tomatoes following Pcc infestation was reduced by 10.02%-68.57%, 40.17%-88.56% and 11.36%-86.17%.
CONCLUSIONS: Emodin from T. asperellum LN004 as a QSI can compete to bind both ExpI and ExpR proteins, interfering with the QS of Pcc and reducing the production of virulence factors. The first molecular mechanism reveals the ability of emodin as a QSI to competitively inhibit two QS proteins simultaneously. © 2023 Society of Chemical Industry.

Mannan, a highly abundant and cost-effective natural resource, holds great potential for the generation of high-value compounds such as bioactive polysaccharides and biofuels. In this study, we successfully enhanced the expression of constructed GH5 β-mannanase (TaMan5) from Trichoderma asperellum ND-1 by employing propeptide in Pichia pastoris. By replacing the α-factor with propeptide (MGNRALNSMKFFKSQALALLAATSAVA), TaMan5 activity was significantly increased from 67.5 to 91.7 U/mL. It retained higher activity in the presence of 20% ethanol and 15% NaCl. When incubated with a high concentration of mannotriose or mannotetraose, the transglycosylation action of TaMan5 can be detected, yielding the corresponding production of mannotetraose or mannooligosaccharides. Moreover, the unique mechanism whereby TaMan5 catalyzes the degradation of mannan into mannobiose involves the transglycosylation of mannose to mannotriose or mannotetraose as a substrate to produce a mannotetraose or mannopentose intermediate, respectively. Additionally, the production of soluble sugars from lignocellulose is a crucial step in bioethanol development, and it is noteworthy that TaMan5 could synergistically yield fermentable sugars from corn stover and bagasse. These findings offered valuable insights and strategies for enhancing β-mannanase expression and efficient conversion of lignocellulosic biomass, providing cost-effective and sustainable approaches for high-value biomolecule and biofuel production.

Fusarium oxysporum is the primary pathogen of blueberry root rot; furthermore, we found that Fusarium commune can also cause root rot in blueberries. Trichoderma spp. is widely used to control plant diseases. We isolated Trichoderma asperellum (TM11) from blueberry rhizosphere soil to explore its control effect and mechanism on F. oxysporum and F. commune. We found that the inhibitory effects of TM11 volatiles and broth metabolites on F. oxysporum were significant, but only F. commune volatile metabolites had a significant inhibitory effect on its growth. Twelve known antimicrobial metabolites were detected from the methanol extract of TM11 fermentation broth by HPLC-MS. TM11 lysed and coiled around the hyphae of F. oxysporum and F. commune. The pot experiment showed that TM11 had significant control effects against F. oxysporum and F. commune, and inoculation of TM11 prior to that of F. oxysporum and F. commune was more effective. The TM11, TM11 and F. oxysporum, or F. commune and distilled water treatments had different effects on the activities of superoxide dismutase, peroxidase and catalase, and the enzyme activity levels exhibited the following order: TM11 > TM11 and F. oxysporum or F. commune > distilled water. The results showed that TM11 provided effective control of blueberry root rot.

As the most abundant and renewable natural resource in the world, lignocellulose is a promising alternative to fossil energy to relieve environmental concerns and resource depletion. However, due to its recalcitrant structure, strains with efficient degradation capability still need exploring. In this study, a fungus was successfully isolated from decayed wood and named as Trichoderma asperellum LYS1 by phylogenetic and draft genomic analysis. The further investigations showed that strain LYS1 had an outstanding performance on lignocellulose degradation, especially for hemicellulose-rich biomass. After the analysis of encoded CAZymes, mainly on GH family, a large amount of genes coding β-glucosidase and xylanase may contribute to the high degradation of cellulose and hemicellulose. Collectively, the results generated in this study demonstrated that T. asperellum LYS1 is a potential cell factory for lignocellulose biorefinery.