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.

Unveiling a Microexon Switch: Novel Regulation of the Activities of Sugar Assimilation and Plant-Cell-Wall-Degrading Xylanases and Cellulases by Xlr2 in Trichoderma virens.

Functional microexons have not previously been described in filamentous fungi. Here, we describe a novel mechanism of transcriptional regulation in Trichoderma requiring the inclusion of a microexon from the Xlr2 gene. In low-glucose environments, a long mRNA including the microexon encodes a protein with a GAL4-like DNA-binding domain (Xlr2-α), whereas in high-glucose environments, a short mRNA that is produced encodes a protein lacking this DNA-binding domain (Xlr2-ß). Interestingly, the protein isoforms differ in their impact on cellulase and xylanase activity. Deleting the Xlr2 gene reduced both xylanase and cellulase activity and growth on different carbon sources, such as carboxymethylcellulose, xylan, glucose, and arabinose. The overexpression of either Xlr2-α or Xlr2-ß in T. virens showed that the short isoform (Xlr2-ß) caused higher xylanase activity than the wild types or the long isoform (Xlr2-α). Conversely, cellulase activity did not increase when overexpressing Xlr2-ß but was increased with the overexpression of Xlr2-α. This is the first report of a novel transcriptional regulation mechanism of plant-cell-wall-degrading enzyme activity in T. virens. This involves the differential expression of a microexon from a gene encoding a transcriptional regulator.

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.

Genotype-by-genotype interkingdom cross-talk between symbiotic nitrogen fixing Sinorhizobium meliloti strains and Trichoderma species.

In the understanding of the molecular interaction between plants and their microbiome, a key point is to identify simplified models of the microbiome including relevant bacterial and fungal partners which could also be effective in plant growth promotion. Here, as proof-of-concept, we aim to identify the possible molecular interactions between symbiotic nitrogen-fixing rhizobia and soil fungi (Trichoderma spp.), hence shed light on synergistic roles rhizospheric fungi could have in the biology of symbiotic nitrogen fixation bacteria. We selected 4 strains of the model rhizobium Sinorhizobium meliloti and 4 Trichoderma species (T. velutinum, T. tomentosum, T. gamsii and T. harzianum). In an experimental scheme of 4 ×4 strains x species combinations, we investigated the rhizobia physiological and transcriptomic responses elicited by fungal spent media, as well as spent media effects on rhizobia-host legume plant (alfalfa, Medicago sativa L.) symbiosis. Fungal spent media had large effects on rhizobia, specific for each fungal species and rhizobial strains combination, indicating a generalized rhizobia genotype x fungal genotype interaction, including synergistic, neutral and antagonistic effects on alfalfa symbiotic phenotypes. Differential expression of a high number of genes was shown in rhizobia strains with up to 25% of total genes differentially expressed upon treatment of cultures with fungal spent media. Percentages over total genes and type of genes differentially expressed changed according to both fungal species and rhizobial strain. To support the hypothesis of a relevant rhizobia genotype x fungal genotype interaction, a nested Likelihood Ratio Test indicated that the model considering the fungus-rhizobium interaction explained 23.4% of differentially expressed genes. Our results provide insights into molecular interactions involving nitrogen-fixing rhizobia and rhizospheric fungi, highlighting the panoply of genes and genotypic interactions (fungus, rhizobium, host plant) which may concur to plant symbiosis.

Genetic diversity and antagonistic properties of Trichoderma strains from the crop rhizospheres in southern Rajasthan, India.

There are fewer studies on Trichoderma diversity in agricultural fields. The rhizosphere of 16 crops was analyzed for Trichoderma species in 7 districts of Rajasthan state of India. Based on DNA sequence of translation elongation factor 1α (tef-1α), and morphological characteristics, 60 isolates were identified as 11 species: Trichoderma brevicompactum, species in Harzianum clade identified as T. afroharzianum, T. inhamatum, T. lentiforme, T. camerunense, T. asperellum, T. asperelloides, T. erinaceum, T. atroviride, T. ghanense, and T. longibrachiatum. T. brevicompactum is the most commonly occurring strain followed by T. afroharzianum. No new species were described in this study. T. lentiforme, showed its first occurrence outside the South American continent. The morphological and cultural characteristics of the major species were observed, described, and illustrated in detail. The isolates were tested for their antagonistic effect against three soilborne plant pathogens fungi: Sclerotium rolfsii, Rhizoctonia solani, and Fusarium verticillioides in plate culture assays. One of the most potent strains was T. afroharzianum BThr29 having a maximum in vitro inhibition of S. rolfsii (76.6%), R. solani (84.8%), and F. verticillioides (85.7%). The potential strain T. afroharzianum BThr29 was also found to be efficient antagonists against soil borne pathogens in in vivo experiment. Such information on crop selectivity, antagonistic properties, and geographic distribution of Trichoderma species will be beneficial for developing efficient Trichoderma-based biocontrol agents.

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.

Plant growth-promoting bacteria potentiate antifungal and plant-beneficial responses of Trichoderma atroviride by upregulating its effector functions.

Trichoderma uses different molecules to establish communication during its interactions with other organisms, such as effector proteins. Effectors modulate plant physiology to colonize plant roots or improve Trichoderma's mycoparasitic capacity. In the soil, these fungi can establish relationships with plant growth-promoting bacteria (PGPBs), thus affecting their overall benefits on the plant or its fungal prey, and possibly, the role of effector proteins. The aim of this study was to determine the induction of Trichoderma atroviride gene expression coding for effector proteins during the interaction with different PGPBs, Arabidopsis or the phytopathogen Fusarium brachygibbosum, and to determine whether PGPBs potentiates the beneficial effects of T. atroviride. During the interaction with F. brachygibbosum and PGPBs, the effector coding genes epl1, tatrx2 and tacfem1 increased their expression, especially during the consortia with the bacteria. During the interaction of T. atroviride with the plant and PGPBs, the expression of epl1 and tatrx2 increased, mainly with the consortium formed with Pseudomonas fluorescens UM270, Bacillus velezensis AF12, or B. halotolerans AF23. Additionally, the consortium formed by T. atroviride and R. badensis SER3 stimulated A. thaliana PR1:GUS and LOX2:GUS for SA- and JA-mediated defence responses. Finally, the consortium of T. atroviride with SER3 was better at inhibiting pathogen growth, but the consortium of T. atroviride with UM270 was better at promoting Arabidopsis growth. These results showed that the biocontrol capacity and plant growth-promoting traits of Trichoderma spp. can be potentiated by PGPBs by stimulating its effector functions.

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.

Potential application of a fungal co-culture crude extract for the conservation of post-harvest fruits.

Fungal plant pathogens are responsible for serious losses in many economically important crop species worldwide. Due to the use of fungicides and the fungi genome plasticity, multi-drug resistant strains are emerging as a new generation of pathogens, causing an expansive range of superficial and systemic plant infections, or new opportunistic fungal pathogens for humans. The group of antagonistic fungi Trichoderma spp. has been widely used to enhance plant growth and for the control of different pathogens affecting crops. Although Neurospora crassa is not a mycoparasitic fungus, its secretion of secondary metabolites with antimicrobial activity has been described. In this work, the effect of crude extract of the monoculture of Trichoderma asperellum T8a or the co-culture with N. crassa as an inhibitory treatment against the fungal pathogens Botrytis cinerea and Fusarium solani was evaluated. The findings demonstrate that the secondary metabolites contained in the T. asperellum crude extract have a clear fungistatic activity against B. cinerea and F. solani. Interestingly, this fungistatic activity highly increases when T. asperellum is co-cultivated with the non-pathogenic fungus N. crassa. Moreover, the co-culture crude extract also showed antifungal activity on post-harvest fruits, and no toxic effects on Murine fibroblast L929 (CCL-1) and murine macrophages RAW 264.7 (TIB-71) were observed. All these results together are solid evidence of the potential of the co-culture crude extract of T. asperellum and N. crassa, as an antifungal agent against phytopathogenic fungi, or post-harvest fruits during the transportation or commercialization time.

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.

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.

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.

Characterization of a novel acidophilic, ethanol tolerant and halophilic GH12 ß-1,4-endoglucanase from Trichoderma asperellum ND-1 and its synergistic hydrolysis of lignocellulosic biomass.

A novel acidophilic GH5 ß-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.

Inferring co-expression networks of Arabidopsis thaliana genes during their interaction with Trichoderma spp.

Fungi of the Trichoderma genus are called "biostimulants" because they promote plant growth and development and induce disease resistance. We used conventional transcriptome and gene co-expression analyses to understand the molecular response of the plant Arabidopsis thaliana to inoculation with Trichoderma atroviride or Trichoderma virens. The transcriptional landscape of the plant during the interaction with these fungi showed a reduction in functions such as reactive oxygen species production, defense mechanisms against pathogens, and hormone signaling. T. virens, as opposed to T. atroviride, was more effective at downregulating genes related to terpenoid metabolism, root development, and chemical homeostasis. Through gene co-expression analysis, we found functional gene modules that closely link plant defense with hypoxia. Notably, we found a transcription factor (locus AT2G47520) with two functional domains of interest: a DNA-binding domain and an N-terminal cysteine needed for protein stability under hypoxia. We hypothesize that the transcription factor can bind to the promoter sequence of the GCC-box that is connected to pathogenesis by positioned weight matrix analysis.

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.

Fungal community dynamics associated with the outbreaks of sugarcane root rot disease.

Sugarcane is a critical sugar and bioenergy crop in China. However, numerous factors, including root rot disease, hamper its yield. Root rot disease is a severe agricultural issue, reducing yield and threatening sustainable crop production. The current study aimed to explore the fungal community structure, identify and characterize the primary pathogen for sugarcane root rot in Guangzhou, China. Eighty-nine samples of sugarcane root, stalk, rhizosphere soil, and irrigation water were collected from five sites in Guangzhou, China. Subsequently, 276 fungal strains were isolated to identify the primary pathogens. The five most common genera identified were Penicillium, Fusarium, Gongronella, Trichoderma, and Cladosporium. Fusarium was more prevalent in the infected soil samples than in healthy ones. Pathogenic assays of the strains revealed that the strain GX4-46 caused 80% of the disease. The strain was confirmed as Fusarium commune through phylogenetic and genome sequence analysis. Rhizosphere soil samples from different regional crops were collected to better understand the fungal community structure and the primary pathogen. We observed a significant presence of Fusarium in irrigation water, indicating that the root rot disease could originate from the irrigation water and then spread as a soil-borne disease. This research is pioneering and one of the most comprehensive investigations on the occurrence and prevalence of sugarcane root rot disease. This study will serve as a reference for expanding the sugarcane industry and a foundation for further exploration and control of root rot.IMPORTANCESugarcane, a significant economic crop, faces challenges due to root rot pathogens that accumulate each year in plants and soil through ratoon planting. This disrupts soil microbial balance and greatly impedes sugarcane industry growth. Symptoms range from wilting and yellowing leaves to stunted growth and reduced seedling tillers. The rhizosphere microbiota plays an important role in plant development and soil health. Little is known about root rot fungal community structure, especially in sugarcane. Here, we focused on exploring the main causative pathogen of root rot in the area alongside a detailed survey of the rhizosphere soil of different severity sugarcane cultivars and rotation crops of the region. To validate the findings, we also investigated the irrigation water of the area. Our study revealed Fusarium commune as the causative pathogen of root rot in the area, primarily originating from water and later as soil-borne. Using Trichoderma can control the disease effectively.

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.

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.

Endophyte genomes support greater metabolic gene cluster diversity compared with non-endophytes in Trichoderma.

Trichoderma is a cosmopolitan genus with diverse lifestyles and nutritional modes, including mycotrophy, saprophytism, and endophytism. Previous research has reported greater metabolic gene repertoires in endophytic fungal species compared to closely-related non-endophytes. However, the extent of this ecological trend and its underlying mechanisms are unclear. Some endophytic fungi may also be mycotrophs and have one or more mycoparasitism mechanisms. Mycotrophic endophytes are prominent in certain genera like Trichoderma, therefore, the mechanisms that enable these fungi to colonize both living plants and fungi may be the result of expanded metabolic gene repertoires. Our objective was to determine what, if any, genomic features are overrepresented in endophytic fungi genomes in order to undercover the genomic underpinning of the fungal endophytic lifestyle. Here we compared metabolic gene cluster and mycoparasitism gene diversity across a dataset of thirty-eight Trichoderma genomes representing the full breadth of environmental Trichoderma's diverse lifestyles and nutritional modes. We generated four new Trichoderma endophyticum genomes to improve the sampling of endophytic isolates from this genus. As predicted, endophytic Trichoderma genomes contained, on average, more total biosynthetic and degradative gene clusters than non-endophytic isolates, suggesting that the ability to create/modify a diversity of metabolites potential is beneficial or necessary to the endophytic fungi. Still, once the phylogenetic signal was taken in consideration, no particular class of metabolic gene cluster was independently associated with the Trichoderma endophytic lifestyle. Several mycoparasitism genes, but no chitinase genes, were associated with endophytic Trichoderma genomes. Most genomic differences between Trichoderma lifestyles and nutritional modes are difficult to disentangle from phylogenetic divergences among species, suggesting that Trichoderma genomes maybe particularly well-equipped for lifestyle plasticity. We also consider the role of endophytism in diversifying secondary metabolism after identifying the horizontal transfer of the ergot alkaloid gene cluster to Trichoderma.