Red and far-red light improve the antagonistic ability of Trichoderma guizhouense against phytopathogenic fungi by promoting phytochrome-dependent aerial hyphal growth.

Light as a source of information regulates morphological and physiological processes of fungi, including development, primary and secondary metabolism, or the circadian rhythm. Light signaling in fungi depends on photoreceptors and downstream components that amplify the signal to govern the expression of an array of genes. Here, we investigated the effects of red and far-red light in the mycoparasite Trichoderma guizhouense on its mycoparasitic potential. We show that the invasion strategy of T. guizhouense depends on the attacked species and that red and far-red light increased aerial hyphal growth and led to faster overgrowth or invasion of the colonies. Molecular experiments and transcriptome analyses revealed that red and far-red light are sensed by phytochrome FPH1 and further transmitted by the downstream MAPK HOG pathway and the bZIP transcription factor ATF1. Overexpression of the red- and far-red light-induced fluffy gene fluG in the dark resulted in abundant aerial hyphae formation and thereby improvement of its antagonistic ability against phytopathogenic fungi. Hence, light-induced fluG expression is important for the mycoparasitic interaction. The increased aggressiveness of fluG-overexpressing strains was phenocopied by four random mutants obtained after UV mutagenesis. Therefore, aerial hyphae formation appears to be a trait for the antagonistic potential of T. guizhouense.

Trachymyrmex septentrionalis ants promote fungus garden hygiene using Trichoderma-derived metabolite cues.

Fungus-growing ants depend on a fungal mutualist that can fall prey to fungal pathogens. This mutualist is cultivated by these ants in structures called fungus gardens. Ants exhibit weeding behaviors that keep their fungus gardens healthy by physically removing compromised pieces. However, how ants detect diseases of their fungus gardens is unknown. Here, we applied the logic of Koch's postulates using environmental fungal community gene sequencing, fungal isolation, and laboratory infection experiments to establish that Trichoderma spp. can act as previously unrecognized pathogens of Trachymyrmex septentrionalis fungus gardens. Our environmental data showed that Trichoderma are the most abundant noncultivar fungi in wild T. septentrionalis fungus gardens. We further determined that metabolites produced by Trichoderma induce an ant weeding response that mirrors their response to live Trichoderma. Combining ant behavioral experiments with bioactivity-guided fractionation and statistical prioritization of metabolites in Trichoderma extracts demonstrated that T. septentrionalis ants weed in response to peptaibols, a specific class of secondary metabolites known to be produced by Trichoderma fungi. Similar assays conducted using purified peptaibols, including the two previously undescribed peptaibols trichokindins VIII and IX, suggested that weeding is likely induced by peptaibols as a class rather than by a single peptaibol metabolite. In addition to their presence in laboratory experiments, we detected peptaibols in wild fungus gardens. Our combination of environmental data and laboratory infection experiments strongly support that peptaibols act as chemical cues of Trichoderma pathogenesis in T. septentrionalis fungus gardens.

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.

Trichoderma cf. asperellum and plant-based titanium dioxide nanoparticles initiate morphological and biochemical modifications in Hordeum vulgare L. against Bipolaris sorokiniana.

BACKGROUND: Spot blotch is a serious foliar disease of barley (Hordeum vulgare L.) plants caused by Bipolaris sorokiniana, which is a hemibiotrophic ascomycete that has a global impact on productivity. Some Trichoderma spp. is a promising candidate as a biocontrol agent as well as a plant growth stimulant. Also, the application of nanomaterials in agriculture limits the use of harmful agrochemicals and helps improve the yield of different crops. The current study was carried out to evaluate the effectiveness of Trichoderma. cf. asperellum and the biosynthesized titanium dioxide nanoparticles (TiO2 NPs) to manage the spot blotch disease of barley caused by B. sorokiniana and to assess the plant's innate defense response. RESULTS: Aloe vera L. aqueous leaf extract was used to biosynthesize TiO2 NPs by reducing TiCl4 salt into TiO2 NPs and the biosynthesized NPs were detected using SEM and TEM. It was confirmed that the NPs are anatase-crystalline phases and exist in sizes ranging from 10 to 25 nm. The T. cf. asperellum fungus was detected using morphological traits and rDNA ITS analysis. This fungus showed strong antagonistic activity against B. sorokiniana (57.07%). Additionally, T. cf. asperellum cultures that were 5 days old demonstrated the best antagonistic activity against the pathogen in cell-free culture filtrate. Also, B. sorokiniana was unable to grow on PDA supplemented with 25 and 50 mg/L of TiO2 NPs, and the diameter of the inhibitory zone increased with increasing TiO2 NPs concentration. In an in vivo assay, barley plants treated with T. cf. asperellum or TiO2 NPs were used to evaluate their biocontrol efficiency against B. sorokiniana, in which T. cf. asperellum and TiO2 NPs enhanced the growth of the plant without displaying disease symptoms. Furthermore, the physiological and biochemical parameters of barley plants treated with T. cf. asperellum or TiO2 NPs in response to B. sorokiniana treatment were quantitively estimated. Hence, T. cf. asperellum and TiO2 NPs improve the plant's tolerance and reduce the growth inhibitory effect of B. sorokiniana. CONCLUSION: Subsequently, T. cf. asperellum and TiO2 NPs were able to protect barley plants against B. sorokiniana via enhancement of chlorophyll content, improvement of plant health, and induction of the barley innate defense system. The present work emphasizes the major contribution of T. cf. asperellum and the biosynthesized TiO2 NPs to the management of spot blotch disease in barley plants, and ultimately to the enhancement of barley plant quality and productivity.

Decrypting biocontrol functions and application modes by genomes data of three Trichoderma Strains/Species.

Trichoderma is an excellent biocontrol agent, but most Trichoderma genomes remained at the scaffold level, which greatly limits the research of biocontrol mechanism. Here, we reported the chromosome-level genome of Trichoderma harzianum CGMCC20739 (Tha739), T. asperellum CGMCC11653 (Tas653) and T. atroviride CGMCC40488 (Tat488), they were assembled into 7 chromosomes, genome size were 40 Mb (10,611 genes), 37.3 Mb (10,102 genes) and 36.3 Mb (9,896 genes), respectively. The positive selected genes of three strains were associated to response to stimulus, signaling transduction, immune system and localization. Furthermore, the number of transcription factors in Tha739, Tas653 and Tat488 strains had significant difference, which may contribute to the differential biocontrol function and stress tolerance. The genes related to signal transduction and gene clusters related to antimicrobial compounds in Tha739 were more than those in Tas653 and Tat488, which showed Tha739 may keenly sense other fungi and quickly secret antimicrobial compounds to inhibit other fungi. Tha739 also contained more genes associated to detoxification, antioxidant and nutrition utilization, indicating it had higher stress-tolerance to hostile environments. And the substrate for synthesizing IAA in Tha739 was mainly 3-indole acetonitrile and indole acetaldehyde, but in Tat488, it was indole-3-acetamide, moreover, Tha739 secreted more phosphatase and phytase and was more related to soil phosphorus metabolism, Tat488 secreted more urease and was more related to soil nitrogen metabolism. These candidate genes related to biocontrol function and stress-tolerance laid foundations for construction of functional strains. All above proved the difference in biocontrol function of Tha739, Tas653 and Tat488 strains, however, the defects in individual strains could be compensated for through Trichoderma-biome during the commercial application process of biocontrol Trichoderma strains.

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.

Metabolic picture of microbial interaction: chemical crosstalk during co-cultivation between three dominant genera of bacteria and fungi in medicinal plants rhizosphere.

INTRODUCTION: Microbial communities affect several aspects of the earth's ecosystem through their metabolic interaction. The dynamics of this interaction emerge from complex multilevel networks of crosstalk. Elucidation of this interaction could help us to maintain the balance for a sustainable future. OBJECTIVES: To investigate the chemical language among highly abundant microbial genera in the rhizospheres of medicinal plants based on the metabolomic analysis at the interaction level. METHODS: Coculturing experiments involving three microbial species: Aspergillus (A), Trichoderma (T), and Bacillus (B), representing fungi (A, T) and bacteria (B), respectively. These experiments encompassed various interaction levels, including dual cultures (AB, AT, TB) and triple cultures (ATB). Metabolic profiling by LC-QTOFMS revealed the effect of interaction level on the productivity and diversity of microbial specialized metabolites. RESULTS: The ATB interaction had the richest profile, while the bacterial profile in the monoculture condition had the lowest. Two native compounds of the Aspergillus genus, aspergillic acid and the dipeptide asperopiperazine B, exhibited decreased levels in the presence of the AT interaction and were undetectable in the presence of bacteria during the interaction. Trichodermarin N and Trichodermatide D isolated from Trichoderma species exclusively detected during coexistence with bacteria (TB and ATB). These findings indicate that the presence of Bacillus activates cryptic biosynthetic gene clusters in Trichoderma. The antibacterial activity of mixed culture extracts was stronger than that of the monoculture extracts. The TB extract exhibited strong antifungal activity compared to the monoculture extract and other mixed culture treatments. CONCLUSION: The elucidation of medicinal plant microbiome interaction chemistry and its effect on the environment will also be of great interest in the context of medicinal plant health Additionally, it sheds light on the content of bioactive constituents, and facilitating the discovery of novel antimicrobials.

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.

Biocontrol of Diplodia bulgarica, the causal agent of apple canker, using Trichoderma zelobreve.

Apple (Malus domestica Borkh) is one of the most consumed and nutritious fruits. Iran is one of the main producers of the apple in the world. Diplodia bulgarica is the major causal agent of apple tree decline in Iran. Biological control is a nature-friendly approach to plant disease management. Trichoderma zelobreve was isolated from apple trees infected with Diplodia bulgarica in West Azarbaijan province of Iran. The results showed that T. zelobreve strongly inhibited the colony growth of D. bulgarica. In vivo assay on detached branches of apple tree cv. Golden Delicious using T. zelobreve mycelial plug showed that canker length/stem length (CL/SL) and canker perimeter/stem perimeter (CP/SP) indices decreased by 76 and 69%, respectively, 21 days after inoculation. Additionally, wettable powder formulation (WPF) containing the antagonistic fungus "T. zelobreve" decreased CL and CP/SP by 75 and 67%, respectively, 6 months after inoculation. Moreover, canker progress curves and the area under the disease progress curve (AUDPC) supported these findings. The growth temperatures of the antagonist and pathogen were similar, indicating the adaptation of T. zelobreve for biocontrol of apple canker caused by D. bulgarica. The results also showed that T. zelobreve-based WPF stored at 25 °C assure excellent shelf life at least 4 months, allowing the bioproduct to be stored at room temperature, which is a great advantage and cost-effective option.

Sclerotia degradation by Trichoderma-mycoparasitic; an effective and sustainable trend in the drop lettuce disease control caused by Sclerotinia sclerotiorum.

Controlling the hazard of sclerotia produced by the Sclerotinia sclerotiorum is very complex, and it is urgent to adopt an effective method that is harmonious environmentally to control the disease. Among the six isolates isolated from the rhizosphere of lettuce, the isolate HZA84 demonstrated a high activity in its antagonism towards Sclerotinia sclerotiorum in vitro, and produces siderophore. By amplification of internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF1-α), and RNA polymerase II subunit (RPB2) genes, the isolate HZA84 was identified as Trichoderma asperellum, which was confirmed by analysis of phylogenetic tree. The Scanning electron microscope monitoring detected that the isolate HZA84 spread over the sclerotial surface, thus, damaging, decomposing, and distorting the globular cells of the outer cortex of the sclerotia. The Real-time polymerase chain reaction (RT-qPCR) analysis disclosed the overexpression of two genes (chit33 and chit37) encoding the endochitinase in addition to one gene (prb1) encoding the proteinase during 4 and 8 days of the parasitism behavior of isolate HZA84 on the sclerotia surface. These enzymes aligned together in the sclerotia destruction by hyperparasitism. On the other hand, the pots trial revealed that spraying of isolate HZA84 reduced the drop disease symptoms of lettuce. The disease severity was decreased by 19.33 and the biocontrol efficiency was increased by 80.67% within the fourth week of inoculation. These findings magnify the unique role of Trichoderma in disrupting the development of plant diseases in sustainable ways.

Design of Carboxymethylcellulose/Poloxamer-Based Bioformulation Embedding Trichoderma afroharzianum for Agricultural Applications.

Microbial biological control agents are believed to be a potential alternative to classical fertilizers to increase the sustainability of agriculture. In this work, the formulation of Trichoderma afroharzianum T22 (T22) spores with carboxymethyl cellulose (CMC) and Pluronic F-127 (PF-127) solutions was investigated. Rheological and microscopical analysis were performed on T22-based systems at three different CMC/PF-127 concentrations, showing that polymer aggregates tend to surround T22 spores, without viscosity, and the viscoelastic properties of the formulations were affected. Contact angle measurements showed the ability of PF-127 to increase the wettability of the systems, and the effect of the formulations on the viability of the spores was evaluated. The viability of the spores was higher over 21 days in all the formulations, compared to the control in water, at 4 and 25 °C. Finally, the effectiveness of the formulations on sweet basil was estimated by greenhouse tests. The results revealed a beneficial effect of the CMC/PF-127 mixture, but none on the formulation with T22. The data show the potential of CMC/PF-127 mixtures for the future design of microorganism-based formulations.

Mitogenome evolution in Trichoderma afroharzianum strains: for a better understanding of distinguishing genus.

Trichoderma afroharzianum (Hypocreales) is known as an important mycoparasite and biocontrol fungus and feeds on fungal material by parasitizing other fungi. Recent studies indicate that this species is also an ear rot pathogen in Europe. Here, the complete mitochondrial genome of three T. afroharzianum strains was sequenced using next-generation sequencing and comparatively characterized by the reported Trichoderma mitogenomes. T. afroharzianum mitogenomes were varying between 29 511 bp and 29 517 bp in length, with an average A + T content of 72.32%. These mitogenomes contain 14 core protein coding genes (PCGs), 22 tRNAs, two rRNAs, one gene encoding the ribosomal protein S3, and three or four genes including conserved domains for the homing endonucleases (HEGs; GIY-YIG type and LAGLIDADG type). All PCGs are initiated by ATG codons, except for atp8, and all are terminated with TAA. A significant correlation was observed between nucleotide composition and codon preference. Four introns belonging to the group I intron class were predicted, accounting for about 14.54% of the size of the mitogenomes. Phylogenetic analyses confirmed the positions of T. afroharzianum strains within the genus of Trichoderma and supported a sister group relationship between T. afroharzianum and T. simmonsii. The recovered trees also supported the monophyly of all included families and of the genus of Acremonium. The characterization of mitochondrial genome of T. afroharzianum contributes to the understanding of phylogeny and evolution of Hypocreales.

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.

The recombinant L-lysine α-oxidase from the fungus Trichoderma harzianum promotes apoptosis and necrosis of leukemia CD34 + hematopoietic cells.

BACKGROUND: In hematologic cancers, including leukemia, cells depend on amino acids for rapid growth. Anti-metabolites that prevent their synthesis or promote their degradation are considered potential cancer treatment agents. Amino acid deprivation triggers proliferation inhibition, autophagy, and programmed cell death. L-lysine, an essential amino acid, is required for tumor growth and has been investigated for its potential as a target for cancer treatment. L-lysine α-oxidase, a flavoenzyme that degrades L-lysine, has been studied for its ability to induce apoptosis and prevent cancer cell proliferation. In this study, we describe the use of L-lysine α-oxidase (LO) from the filamentous fungus Trichoderma harzianum for cancer treatment. RESULTS: The study identified and characterized a novel LO from T. harzianum and demonstrated that the recombinant protein (rLO) has potent and selective cytotoxic effects on leukemic cells by triggering the apoptotic cascade through mitochondrial dysfunction. CONCLUSIONS: The results support future translational studies using the recombinant LO as a potential drug for the treatment of leukemia.

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.

Microbial organic fertilizer prepared by co-composting of Trichoderma dregs mitigates dissemination of resistance, virulence genes, and bacterial pathogens in soil and rhizosphere.

The use of manure, mycelium dregs and other waste as organic fertilizer is the main source of antibiotic resistance genes (ARGs) and pathogens in farmland. Composting of waste may effectively remove ARGs and pathogens. However, the profiles and drivers of changes in metal resistance genes (MRGs), biocide resistance genes (BRGs), and virulence genes (VGs) in soil-crop rhizosphere systems after compost application remain largely unknown. Here, we prepared two kinds of microbial organic fertilizers (MOF) by using Trichoderma dregs (TDs) and organic fertilizer mixing method (MOF1) and TDs co-composting method (MOF2). The effects of different types and doses of MOF on resistance genes, VGs and pathogens in soil-rhizosphere system and their potential mechanisms were studied. The results showed that co-composting of TDs promoted the decomposition of organic carbon and decreased the absolute abundance of ARGs and mobile genetic elements (MGEs) by 53.4-65.0%. MOF1 application significantly increased the abundance and diversity of soil ARGs, BRGs, and VGs, while low and medium doses of MOF2 significantly decreased their abundance and diversity in soil and rhizosphere. Patterns of positive co-occurrence between MGEs and VGs/MRGs/BRGs/ARGs were observed through statistical analysis and gene arrangements. ARGs/MRGs reductions in MOF2 soil were directly driven by weakened horizontal gene transfer triggered by MGEs. Furthermore, MOF2 reduced soil BRGs/VGs levels by shifting bacterial communities (e.g., reduced bacterial host) or improving soil property. Our study provided new insights into the rational use of waste to minimize the spread of resistomes and VGs in soil.

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.

Characterization of a novel aspartic protease from Trichoderma asperellum for the preparation of duck blood peptides.

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.

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.

Exploring Factors Conditioning the Expression of Botryosphaeria Dieback in Grapevine for Integrated Management of the Disease.

Species from the Botryosphaeriaceae family are the causal agents of Botryosphaeria dieback (BD), a worldwide grapevine trunk disease. Because of their lifestyle and their adaptation to a wide range of temperatures, these fungi constitute a serious threat to vineyards and viticulture, especially in the actual context of climate change. Grapevine plants from both nurseries and vineyards are very susceptible to infections by botryosphaeriaceous fungi due to several cuts and wounds made during their propagation process and their entire life cycle, respectively. When decline becomes chronic or apoplectic, it reduces the longevity of the vineyard and affects the quality of the wine, leading to huge economic losses. Given the environmental impact of fungicides, and their short period of effectiveness in protecting pruning wounds, alternative strategies are being developed to fight BD fungal pathogens and limit their propagation. Among them, biological control has been recognized as a promising and sustainable alternative. However, there is still no effective strategy for combating this complex disease, conditioned by both fungal life traits and host tolerance traits, in relationships with the whole microbiome/microbiota. To provide sound guidance for an effective and sustainable integrated management of BD, by combining the limitation of infection risk, tolerant grapevine cultivars, and biological control, this review explores some of the factors conditioning the expression of BD in grapevine. Among them, the lifestyle of BD-associated pathogens, their pathogenicity factors, the cultivar traits of tolerance or susceptibility, and the biocontrol potential of Bacillus spp. and Trichoderma spp. are discussed.