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.

Effect of Light Conditions, Trichoderma Fungi and Food Polymers on Growth and Profile of Biologically Active Compounds in Thymus vulgaris and Thymus serpyllum.

The research investigates the influence of different lighting conditions and soil treatments, in particular the application of food polymers separately and in combination with spores of Trichoderma consortium, on the growth and development of herbs-Thymus vulgaris and Thymus serpyllum. The metabolic analysis focuses on detecting changes in the levels of biologically active compounds such as chlorophyll a and b, anthocyanins, carotenoids, phenolic compounds (including flavonoids), terpenoids, and volatile organic compounds with potential health-promoting properties. By investigating these factors, the study aims to provide insights into how environmental conditions affect the growth and chemical composition of selected plants and to shed light on potential strategies for optimising the cultivation of these herbs for the improved quality and production of bioactive compounds. Under the influence of additional lighting, the growth of T. vulgaris and T. serpyllum seedlings was greatly accelerated, resulting in an increase in shoot biomass and length, and in the case of T. vulgaris, an increase in carotenoid and anthocyanin contents. Regarding secondary metabolites, the most pronounced changes were observed in total antioxidant capacity and flavonoid content, which increased significantly under the influence of additional lighting. The simultaneous or separate application of Trichoderma and food polymers resulted in an increase in flavonoid content in the leaves of both Thymus species. The increase in terpenoid content under supplemental light appears to be related to the presence of Trichoderma spores as well as food polymers added to the soil. However, the nature of these changes depends on the thyme species. Volatile compounds were analysed using an electronic nose (E-nose). Eight volatile compounds (VOCs) were tentatively identified in the vapours of T. vulgaris and T. serpyllum: α-pinene, myrcene, α-terpinene, γ-terpinene; 1,8-cineole (eucalyptol), thymol, carvacrol, and eugenol. Tendencies to increase the percentage of thymol and γ-terpinene under supplemental lighting were observed. The results also demonstrate a positive effect of food polymers and, to a lesser extent, Trichoderma fungi on the synthesis of VOCs with health-promoting properties. The effect of Trichoderma and food polymers on individual VOCs was positive in some cases for thymol and γ-terpinene.

Use of Ozone as a Substrate Treatment for the Control of Trichoderma in the Production of Pleurotus ostreatus.

Pleurotus ostreatus is one of the most widely cultivated species in the world. It can be produced in many lignocellulosic substrates after carrying out a treatment to eliminate competing microorganisms. The most commonly used is pasteurization by steam or by immersion in hot water. The aim of this work is to evaluate if ozone can be employed as treatment for decontamination of the substrate used for the production of the edible mushroom P. ostreatus to control of green mold Trichoderma. Wheat straw was employed as a substrate. We used two different methodologies: bubbling ozone into a tank with water and the substrate, and injecting ozone into a closed tank with the substrate inside. Ten treatments were carried out including two treatments with inoculation by a spray of conidia of Trichoderma. The effect of ozone on the conidia was also evaluated. We found that the treatment of the substrate with ozone in immersed water resulted more effective (lower growth of Trichoderma) than injecting ozone into a closed tank. Anyway, we found that the contaminant fungi could grow on the substrate in both treatments with ozone. We observed that although ozone affected the conidia when it was bubbled into water, some of them still managed to survive and could germinate 72 h later. P. ostreatus could grow and produce fruiting bodies on a substrate that was previously treated with ozone and yields were not affected. Based on the results obtained, we conclude that ozone may not be an effective agent to control Trichoderma in highly contaminated substrates, at least in the experimental conditions that we used, for the production of P. ostreatus.

Bio-synthesis and characterization of silver nanoparticles from Trichoderma species against cassava root rot disease.

Cassava root rot disease caused by the fungal pathogens Fusarium solani and Lasiodiplodia theobromae produces severe damages on cassava production. This research was conducted to produce and assess silver nanoparticles (AgNPs) synthesized by Trichoderma harzianum for reducing root rot disease. The results revealed that using the supernatants of T. harzianum on a silver nitrate solution changed it to reddish color at 48 h, indicating the formation of AgNPs. Further characterization was identified using dynamic light scattering (DLS) and scanning electron microscope (SEM). DLS supported that the Z-average size is at 39.79 nm and the mean zeta potential is at - 36.5 mV. SEM revealed the formation of monodispersed spherical shape with a diameter between 60-75 nm. The antibacterial action of AgNPs as an antifungal agent was demonstrated by an observed decrease in the size of the fungal colonies using an increasing concentration of AgNPs until the complete inhibition growth of L. theobromae and F. solani at > 58 µg mL-1 and at ≥ 50 µg mL-1, respectively. At in vitro conditions, the applied AgNPs caused a decrease in the percentage of healthy aerial hyphae of L. theobromae (32.5%) and of F. solani (70.0%) compared to control (100%). The SR-FTIR spectra showed the highest peaks in the first region (3000-2800 cm-1) associated with lipids and fatty acids located at 2962, 2927, and 2854 cm-1 in the AgNPs treated samples. The second region (1700-1450 cm-1) consisting of proteins and peptides revealed the highest peaks at 1658, 1641, and 1548 cm-1 in the AgNPs treated samples. The third region (1300-900 cm-1), which involves nucleic acid, phospholipids, polysaccharides, and carbohydrates, revealed the highest peaks at 1155, 1079, and 1027 cm-1 in the readings from the untreated samples. Finally, the observed root rot severity on cassava roots treated with AgNPs (1.75 ± 0.50) was significantly lower than the control samples (5.00 ± 0.00).

Lignocellulolytic Enzymes Production by Four Wild Filamentous Fungi for Olive Stones Valorization: Comparing Three Fermentation Regimens.

Lignocellulolytic enzymes play a crucial role in efficiently converting lignocellulose into valuable platform molecules in various industries. However, they are limited by their production yields, costs, and stability. Consequently, their production by producers adapted to local environments and the choice of low-cost raw materials can address these limitations. Due to the large amounts of olive stones (OS) generated in Morocco which are still undervalued, Penicillium crustosum, Fusarium nygamai, Trichoderma capillare, and Aspergillus calidoustus, are cultivated under different fermentation techniques using this by-product as a local lignocellulosic substrate. Based on a multilevel factorial design, their potential to produce lignocellulolytic enzymes during 15 days of dark incubation was evaluated. The results revealed that P. crustosum expressed a maximum total cellulase activity of 10.9 IU/ml under sequential fermentation (SF) and 3.6 IU/ml of ß-glucosidase activity under submerged fermentation (SmF). F. nygamai recorded the best laccase activity of 9 IU/ml under solid-state fermentation (SSF). Unlike T. capillare, SF was the inducive culture for the former activity with 7.6 IU/ml. A. calidoustus produced, respectively, 1,009 µg/ml of proteins and 11.5 IU/ml of endoglucanase activity as the best results achieved. Optimum cellulase production took place after the 5th day under SF, while ligninases occurred between the 9th and the 11th days under SSF. This study reports for the first time the lignocellulolytic activities of F. nygamai and A. calidoustus. Furthermore, it underlines the potential of the four fungi as biomass decomposers for environmentally-friendly applications, emphasizing the efficiency of OS as an inducing substrate for enzyme production.

Demonstrating the Applicability of Proton Transfer Reaction Mass Spectrometry to Quantify Volatiles Emitted by the Mycoparasitic Fungus Trichoderma atroviride in Real Time: Monitoring of Trichoderma-Based Biopesticides.

The present study aims to explore the potential application of proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS) for real-time monitoring of microbial volatile organic compounds (MVOCs). This investigation can be broadly divided into two parts. First, a selection of 14 MVOCs was made based on previous research that characterized the MVOC emissions of Trichoderma atroviride, which is a filamentous fungus widely used as a biocontrol agent. The analysis of gas-phase standards using PTR-ToF-MS allowed for the categorization of these 14 MVOCs into two groups: the first group primarily undergoes nondissociative proton transfer, resulting in the formation of protonated parent ions, while the second group mainly undergoes dissociative proton transfer, leading to the formation of fragment ions. In the second part of this investigation, the emission of MVOCs from samples of T. atroviride was continuously monitored over a period of five days using PTR-ToF-MS. This also included the first quantitative online analysis of 6-amyl-α-pyrone (6-PP), a key MVOC emitted by T. atroviride. The 6-PP emissions of T. atroviride cultures were characterized by a gradual increase over the first two days of cultivation, reaching a plateau-like maximum with volume mixing ratios exceeding 600 ppbv on days three and four. This was followed by a marked decrease, where the 6-PP volume mixing ratios plummeted to below 50 ppbv on day five. This observed sudden decrease in 6-PP emissions coincided with the start of sporulation of the T. atroviride cultures as well as increasing intensities of product ions associated with 1-octen-3-ol and 3-octanone, whereas both these MVOCs were previously associated with sporulation in T. atroviride. The study also presents the observations and discussion of further MVOC emissions from the T. atroviride samples and concludes with a critical assessment of the possible applications and limitations of PTR-ToF-MS for the online monitoring of MVOCs from biological samples in real time.

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.

Trichoderma-secreted anthranilic acid promotes lateral root development via auxin signaling and RBOHF-induced endodermal cell wall remodeling.

Trichoderma spp. have evolved the capacity to communicate with plants by producing various secondary metabolites (SMs). Nonhormonal SMs play important roles in plant root development, while specific SMs from rhizosphere microbes and their underlying mechanisms to control plant root branching are still largely unknown. In this study, a compound, anthranilic acid (2-AA), is identified from T. guizhouense NJAU4742 to promote lateral root development. Further studies demonstrate that 2-AA positively regulates auxin signaling and transport in the canonical auxin pathway. 2-AA also partly rescues the lateral root numbers of CASP1pro:shy2-2, which regulates endodermal cell wall remodeling via an RBOHF-induced reactive oxygen species burst. In addition, our work reports another role for microbial 2-AA in the regulation of lateral root development, which is different from its better-known role in plant indole-3-acetic acid biosynthesis. In summary, this study identifies 2-AA from T. guizhouense NJAU4742, which plays versatile roles in regulating plant root development.

Trichoderma koningiopsis fermentation in airlift bioreactor for bioherbicide production.

During scaling of fermentations, choosing a bioreactor is fundamental to ensure the product's quality. This study aims to produce bioherbicides using Trichoderma koningiopsis fermentation, evaluating process parameters in an Airlift bioreactor. As a response, we quantified the production of enzymes involved in the bioherbicide activity (amylase, cellulase, laccase, lipase, and peroxidase). In addition, it evaluated the agronomic efficiency of the fermented extract optimized through tests that promoted soybean growth and nodulation, soybean seed germination, and in vitro phytopathogen control. As a result of optimizing the scaling bioprocess, it was possible to obtain an adequate fermentation condition, which, when applied to soybean seeds, had beneficial effects on their growth. It allowed the production of an enzyme cocktail. These results add a crucial biotechnological potential factor for the success of the optimized formulation in the Airlift bioreactor, in addition to presenting relevant results for the scientific community.

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.

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.

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.

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.

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.