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.

A novel salt-tolerant strain Trichoderma atroviride HN082102.1 isolated from marine habitat alleviates salt stress and diminishes cucumber root rot caused by Fusarium oxysporum.

BACKGROUND: Salt stress threaten the growth of plants, and even aggravate plant disease. In this article, salt-tolerant Trichoderma strain was isolated, and its potential to alleviate salt stress and diminish cucumber root rot caused by Fusarium oxysporum was evaluated. RESULTS: Twenty-seven Trichoderma isolates were isolated from samples of sea muds and algae collected from the South Sea of China. Among these, the isolate HN082102.1 showed the most excellent salt tolerance and antagonistic activity against F. oxysporum causing root rot in cucumber and was identified as T. atroviride. Its antagonism ability may be due to mycoparasitism and inhibition effect of volatile substances. The application of Trichoderma mitigated the adverse effects of salt stress and promoted the growth of cucumber under 100 mM and 200 mM NaCl, especially for the root. When T. atroviride HN082102.1 was applied, root fresh weights increased by 92.55 and 84.86%, respectively, and root dry weights increased by 75.71 and 53.31%, respectively. Meanwhile, the application of HN082102.1 reduced the disease index of cucumber root rot by 63.64 and 71.01% under 100- and 0-mM saline conditions, respectively, indicating that this isolate could inhibit cucumber root rot under salt stress. CONCLUSIONS: This is the first report of salt-tolerant T. atroviride isolated from marine habitat showing antagonistic activity to F. oxysporum, and the results provide evidence for the novel strain T. atroviride HN082102.1 in alleviating salt stress and diminishing cucumber root rot, indicating that T. atroviride strain HN082102.1 can be used as biological control agent in saline alkali land.

Monitoring the volatile language of fungi using gas chromatography-ion mobility spectrometry.

Fusarium oxysporum is a plant pathogenic fungus leading to severe crop losses in agriculture every year. A sustainable way of combating this pathogen is the application of mycoparasites-fungi parasitizing other fungi. The filamentous fungus Trichoderma atroviride is such a mycoparasite that is able to antagonize phytopathogenic fungi. It is therefore frequently applied as a biological pest control agent in agriculture. Given that volatile metabolites play a crucial role in organismic interactions, the major aim of this study was to establish a method for on-line analysis of headspace microbial volatile organic compounds (MVOCs) during cultivation of different fungi. An ion mobility spectrometer with gas chromatographic pre-separation (GC-IMS) enables almost real-time information of volatile emissions with good selectivity. Here we illustrate the successful use of GC-IMS for monitoring the time- and light-dependent release of MVOCs by F. oxysporum and T. atroviride during axenic and co-cultivation. More than 50 spectral peaks were detected, which could be assigned to 14 volatile compounds with the help of parallel gas chromatography-mass spectrometric (GC-MS) measurements. The majority of identified compounds are alcohols, such as ethanol, 1-propanol, 2-methyl propanol, 2-methyl butanol, 3-methyl-1-butanol and 1-octen-3-ol. In addition to four ketones, namely acetone, 2-pentanone, 2-heptanone, 3-octanone, and 2-octanone; two esters, ethyl acetate and 1-butanol-3-methylacetate; and one aldehyde, 3-methyl butanal, showed characteristic profiles during cultivation depending on axenic or co-cultivation, exposure to light, and fungal species. Interestingly, 2-octanone was produced only in co-cultures of F. oxysporum and T. atroviride, but it was not detected in the headspace of their axenic cultures. The concentrations of the measured volatiles were predominantly in the low ppbv range; however, values above 100 ppbv were detected for several alcohols, including ethanol, 2-methylpropanol, 2-methyl butanol, 1- and 3-methyl butanol, and for the ketone 2-heptanone, depending on the cultivation conditions. Our results highlight that GC-IMS analysis can be used as a valuable analytical tool for identifying specific metabolite patterns for chemotaxonomic and metabolomic applications in near-to-real time and hence easily monitor temporal changes in volatile concentrations that take place in minutes.

Combined Use of Trichoderma atroviride CCTCCSBW0199 and Brassinolide to Control Botrytis cinerea Infection in Tomato.

Tomato gray mold caused by Botrytis cinerea is one of the main diseases of tomato and significantly impacts the yield and quality of tomato fruit. The overuse of chemical fungicides has resulted in the development of fungicide-resistant strains. Biological control is becoming an alternative method for the control of plant diseases to replace or decrease the application of traditional synthetic chemical fungicides and genus Trichoderma is widely used as a biological agent for controlling tomato gray mold. Brassinolide (BR) is a plant-growth-promoting steroid. To enhance the efficiency and stability of Trichoderma activity against B. cinerea, an optimal combination of Trichoderma atroviride CCTCCSBW0199 and BR that controls B. cinerea infection in tomato was identified. Strain CCTCCSBW0199 was found to have antagonistic activity against B. cinerea both in vitro and in vivo. In addition, a fermented culture of chlamydospores and metabolites, or metabolites only of strain CCTCCSBW0199 also reduced growth of B. cinerea. BR reduced growth of B. cinerea and had no effect on the sporulation and growth of Trichoderma spp. An application of metabolites of a Trichoderma sp. + BR reduced gray mold on tomato leaves by approximately 70.0%. Furthermore, the activities of induced defense response-related enzyme, such as peroxidase, superoxide dismutase, catalase, and phenylalanine ammonia-lyase were increased in tomato plants treated with a Trichoderma sp. + BR. Our data suggested that applying a mix of metabolites of T. atroviride CCTCCSBW0199 + BR was effective at reducing gray mold of tomato and may lay a theoretical foundation for the development of novel biofungicides.

The Trichoderma atroviride Strains P1 and IMI 206040 Differ in Their Light-Response and VOC Production.

Trichoderma atroviride is a strong necrotrophic mycoparasite antagonizing and feeding on a broad range of fungal phytopathogens. It further beneficially acts on plants by enhancing growth in root and shoot and inducing systemic resistance. Volatile organic compounds (VOCs) are playing a major role in all those processes. Light is an important modulator of secondary metabolite biosynthesis, but its influence has often been neglected in research on fungal volatiles. To date, T. atroviride IMI 206040 and T. atroviride P1 are among the most frequently studied T. atroviride strains and hence are used as model organisms to study mycoparasitism and photoconidiation. However, there are no studies available, which systematically and comparatively analyzed putative differences between these strains regarding their light-dependent behavior and VOC biosynthesis. We therefore explored the influence of light on conidiation and the mycoparasitic interaction as well as the light-dependent production of VOCs in both strains. Our data show that in contrast to T. atroviride IMI 206040 conidiation in strain P1 is independent of light. Furthermore, significant strain- and light-dependent differences in the production of several VOCs between the two strains became evident, indicating that T. atroviride P1 could be a better candidate for plant protection than IMI 206040.

The pathway of 2,2-dichlorovinyl dimethyl phosphate (DDVP) degradation by Trichoderma atroviride strain T23 and characterization of a paraoxonase-like enzyme.

Dichlorvos (DDVP) is widely applied in the agricultural industry, and its residues are considered hazardous to the environment. Microbial bioremediation is an innovative technology with the potential to mitigate such pollution. Trichoderma atroviride strain T23, a filamentous fungus, is very efficient at degrading DDVP. Therefore, we used DDVP as a model organophosphate pesticide to study the mechanism by which Trichoderma degrades organophosphate pesticides, with the aim of attaining a global understanding of the molecular mechanism of enzymatic degradation of organophosphate pesticides by beneficial fungi. DDVP can be biodegraded via two routes, and the primary one involves hydrolysis of the P-O bond, which can result in the production of the novel degradation intermediate trichloroethanol. TaPon1-like showed continuously high expression during 120 h, and deletion of the gene decreased the efficiency of P-O bond hydrolysis. The enzyme produced by TaPon1-like had a low Km for DDVP (0.23 mM) and a high kcat (204.3 s-1). The enzyme was able to hydrolyze broad substrates such as organophosphate oxons and lactone and maintain stable activity in a wide range of pH and temperature values. The TaPon1-like hydrolase played an important role in the first step of DDVP degradation by strain T23 and contributed to a comprehensive understanding of the mechanism of organophosphate pesticide degradation.

Trichoderma based synthesis of anti-pathogenic silver nanoparticles and their characterization, antioxidant and cytotoxicity properties.

Antibacterial silver nanoparticles (AgNPs) are synthesized from various biological resources, but studies on Trichoderma-based synthesis are limited. Hence, the current work synthesized antibacterial silver nanoparticles using Trichoderma atroviride and also tested for their biomedical properties. AgNPs synthesis was confirmed by the occurrence of plasmon resonance at 390-400 nm in the UV-vis spectrum. FTIR spectrum displayed the bands at 1115.4 and 3450 cm-1 indicating the vibrations of the metallic oxides. Transmission electron microscopic and EDX analysis confirmed a high percentage signal of anisotropic structural AgNPs with the size of 15-25 nm. The AgNPs exhibited high antibacterial activity and DPPH scavenging activity in a dosedependent manner. The cytotoxicity assay \\\\ indicated that the AgNPs triggered the cancer cell death at the inhibitory concentration of IC50 16.5 µg/ml. Current work reported for the first time Trichoderma atroviride based synthesis of anisotropic structural AgNPs with the relatively small size of 15-25 nm with remarkable anti-bacterial, antioxidant and cytotoxicity activities, and this deserves future investigations for biomedical applications.

Antibacterial, and antioxidant potentials of non-cytotoxic extract of Trichoderma atroviride.

Trichoderma species are a rich source of metabolites, but less known for biomedical potential. This work deals with antibacterial and antioxidant potentials of intracellular non-cytotoxic metabolites, extracted from Trichoderma atroviride (KNUP001). A total of 53 fractions was collected by column chromatography and tested for cytotoxicity by MTT assay. Only one fraction (F41) was found to be non-toxic to Vero cells with 95.4 ±â€¯0.61% of survival. The F41 was then subjected to chemical analysis, antibacterial and antioxidant assays. The F41 at 500 µg ml-1 showed the total antioxidant of 48.70 ±â€¯2.90%, DPPH radical scavenging activity of 37.25 ±â€¯2.25, nitric oxide (NO) radical scavenging activity of 54.55 ±â€¯1.95 and H2O2 radical scavenging activity of 43.75 ±â€¯3.21. The F41 at 25 µg ml-1 displayed antibacterial activity against E. coli (14.25 ±â€¯0.25 mm), Proteus mirabilis (10.40 ±â€¯0.60 mm), and Enterobacter aerogenes (5.60 ±â€¯0.40 mm). GC-MS analysis revealed the dominant presence of oleic acid C 18.1 (63.18%), n-hexadecanoic acid (6.17%), and ethyl oleate (4.93%) in the F41, and hence these fatty acids are likely responsible for the antioxidant and antibacterial activities of F41. Hence, further investigation deserves on purification and characterization of the active metabolites from T. atroviride strain KNUP001 towards developing molecular leads to effective antibacterial drugs, and non-toxic to host cells.

Characterization of a novel dsRNA mycovirus of Trichoderma atroviride NFCF028.

Molecular characterization of the most common dsRNA element from Trichoderma atroviride indicated that it comprised 8,566 bp and encoded two large open reading frames (ORF1 and 2). The two ORFs were found to overlap by 46 bp with a typical (-1) slippery heptanucelotide sequence. The deduced protein sequences of ORF1 and ORF2 showed significant similarities to those of known mycoviral structural proteins and RNA-dependent RNA polymerases, respectively. Phylogenetic analysis indicated that this dsRNA is a member of a distinct species related to a group of unclassified mycoviruses; therefore, it was named Trichoderma atroviride mycovirus 1 (TaMV1).

Co-inoculation of Glomus intraradices and Trichoderma atroviride acts as a biostimulant to promote growth, yield and nutrient uptake of vegetable crops.

BACKGROUND: The application of beneficial microorganisms at transplanting can promote rapid transplant establishment (starter effect) for achieving early and high yields. The aim of this study was to evaluate the biostimulant effects of Glomus intraradices BEG72 (G) and Trichoderma atroviride MUCL 45632 (T) alone or in combination on plant growth parameters, yield, chlorophyll index (SPAD), chlorophyll fluorescence and mineral composition of several vegetable crops. RESULTS: The T. atroviride strain was capable of producing siderophores and auxin-like compounds under a wide range of substrate pH conditions (5.5-8.0). The highest shoot, root dry weight, SPAD and chlorophyll fluorescence in lettuce, tomato and zucchini was observed in the G + T combination, followed by a single inoculation of G or T, whereas the lowest values were recorded in the uninoculated plants. Under greenhouse conditions, the shoot dry weight was significantly increased by 167%, 56%, 115%, 68% and 58% in lettuce, melon, pepper, tomato and zucchini, respectively, when supplied with both beneficial microorganisms in comparison with the control. This increase in root and shoot weight was associated with an increased level of nutrient uptake (e.g. P, Mg, Fe, Zn and B). Under open field conditions, the lettuce shoot and root dry weight increased by 61% and 57%, respectively, with biostimulant microorganism application in field conditions. For zucchini, early and total yields were significantly increased by 59% and 15%, respectively, when plants were inoculated with both microorganisms. CONCLUSION: The application of the biostimulant tablet containing both G and T can promote transplant establishment and vegetable crop productivity in a sustainable way.

Are1-mediated nitrogen metabolism is associated with iron regulation in the mycoparasite Trichoderma atroviride.

Trichoderma atroviride is a mycoparasitic fungus with antagonistic activity against fungal pathogens and is used as a pathogen control agent alternative to synthetic fungicides. Sensing nutrient availability in the environment and adjusting metabolism for optimal growth, development and reproduction is essential for adaptability and is relevant to its mycoparasitic activity. During mycoparasitism, secondary metabolites are produced to weaken the fungal prey and support the attack. Are1-like proteins act as major GATA-type transcription factors in the activation of genes subject to nitrogen catabolite repression. Since the quality and quantity of nitrogen has been proven particularly relevant in remodeling the biosynthesis of secondary metabolites in fungi, we decided to functionally characterize Are1, the ortholog of Aspergillus nidulans AreA, in T. atroviride. We show that the growth of the T. atroviride ∆are1 mutant is impaired in comparison to the wild type on several nitrogen sources. Deletion of are1 enhanced sensitivity to oxidative and cell-wall stressors and altered the mycoparasitic activity. We were able to identify for the first time a link between Are1 and iron homeostasis via a regulatory mechanism that does not appear to be strictly linked to the nitrogen source, but rather to an independent role of the transcription factor.

Impact of tps1 Deletion and Overexpression on Terpene Metabolites in Trichoderma atroviride.

Terpenoids are structurally diverse natural products that have been widely used in the pharmaceutical, food, and cosmetic industries. Research has shown that fungi produce a variety of terpenoids, yet fungal terpene synthases remain not thoroughly explored. In this study, the tps1 gene, a crucial component of the terpene synthetic pathway, was isolated from Trichoderma atroviride HB20111 through genome mining. The function of this gene in the terpene synthetic pathway was investigated by constructing tps1-gene-deletion- and overexpression-engineered strains and evaluating the expression differences in the tps1 gene at the transcript level. HS-SPME-GC-MS analysis revealed significant variations in terpene metabolites among wild-type, tps1-deleted (Δtps1), and tps1-overexpressed (Otps1) strains; for instance, most sesquiterpene volatile organic compounds (VOCs) were notably reduced or absent in the Δtps1 strain, while nerolidol, ß-acorenol, and guaiene were particularly produced by the Otps1 strain. However, both the Δtps1 and Otps1 strains produced new terpene metabolites compared to the wild-type, which indicated that the tps1 gene played an important role in terpene synthesis but was not the only gene involved in T. atroviride HB20111. The TPS1 protein encoded by the tps1 gene could function as a sesquiterpene cyclase through biological information and evolutionary tree analysis. Additionally, fungal inhibition assay and wheat growth promotion assay results suggested that the deletion or overexpression of the tps1 gene had a minimal impact on fungal inhibitory activity, plant growth promotion, and development, as well as stress response. This implies that these activities of T. atroviride HB20111 might result from a combination of multiple metabolites rather than being solely dependent on one specific metabolite. This study offers theoretical guidance for future investigations into the mechanism of terpenoid synthesis and serves as a foundation for related studies on terpenoid metabolic pathways in fungi.

Impact of the Biocontrol Product, Esquive® WP, on the Indigenous Grapevine Wood Microbiome after a 6-Year Application Period.

Grapevine trunk diseases (GTDs) are currently limiting grapevine productivity in many vineyards worldwide. As no chemical treatments are registered to control GTDs, biocontrol agents are being tested against these diseases. Esquive® WP, based on the fungus Trichoderma atroviride I-1237 strain, is the first biocontrol product registered in France to control GTDs. In this study, we determine whether, following grapevine pruning wound treatments with Esquive® WP, changes occurred or not in the indigenous microbial communities that are colonizing grapevine wood. Over a 6-year period, Esquive® WP was applied annually to pruning wounds on three grapevine cultivars located in three different regions. Wood samples were collected at 2 and 10 months after the Esquive® WP treatments. Based on MiSeq high-throughput sequencing analyses, the results showed that specific microbial communities were linked to each 'region/cultivar' pairing. In certain cases, a significant modification of alpha diversity indexes and the relative abundance of some microbial taxa were observed between treated and non-treated grapevines 2 months after Esquive® WP treatment. However, these modifications disappeared over time, i.e., 10 months post-treatment. This result clearly showed that Esquive® WP pruning wood treatment did not induce significant changes in the grapevine wood's microbiome, even after 6 years of recurrent applications on the plants.

Crosstalk between H2O2 and Ca2+ signaling is involved in root endophyte-enhanced tanshinone biosynthesis of Salvia miltiorrhiza.

Tanshinones are bioactive ingredients derived from the herbal plant Salvia miltiorrhiza and are used for treating diseases of the heart and brain, thus ensuring quality of S. miltiorrhiza is paramount. Applying the endophytic fungus Trichoderma atroviride D16 can significantly increase the content of tanshinones in S. miltiorrhiza, but the potential mechanism remains unknown. In the present study, the colonization of D16 effectively enhanced the levels of Ca2+ and H2O2 in the roots of S. miltiorrhiza, which is positively correlated with increased tanshinones accumulation. Further experiments found that the treatment of plantlets with Ca2+ channel blocker (LaCl3) or H2O2 scavenger (DMTU) blocked D16-promoted tanshinones production. LaCl3 suppressed not only the D16-induced tanshinones accumulation but also the induced Ca2+ and H2O2 generation; nevertheless, DMTU did not significantly inhibit the induced Ca2+ biosynthesis, implying that Ca2+ acted upstream in H2O2 production. These results were confirmed by observations that S. miltiorrhiza treated with D16, CaCl2, and D16+LaCl3 exhibit H2O2 accumulation and influx in the roots. Moreover, H2O2 as a downstream signal of Ca2+ is involved in D16 enhanced tanshinones synthesis by inducing the expression of genes related to the biosynthesis of tanshinones, such as DXR, HMGR, GGPPS, CPS, KSL and CYP76AH1 genes. Transcriptomic analysis further supported that D16 activated the transcriptional responses related to Ca2+ and H2O2 production and tanshinones synthesis in S. miltiorrhiza seedlings. This is the first report that Ca2+ and H2O2 play important roles in regulating fungal-plant interactions thus improving the quality in the D16-S. miltiorrhiza system.

Near real-time quantification of microbial volatile organic compounds from mycoparasitic fungi: Potential for advanced monitoring and pest control.

Microbial volatile organic compounds (MVOCs) are thought to play a key role in the interactions between mycoparasitic fungi, such as the biocontrol agent Trichoderma atroviride (T. atroviride), and their environment. However, the analysis of MVOC emissions from fungal samples is challenging because of low analyte concentrations, typically in the ppbV-range, and the complex chemical nature of biological samples. In a recent study using proton transfer reaction-time of flight-mass spectrometry (PTR-ToF-MS) to determine MVOC emissions from T. atroviride, many product ions were unspecific, as they could arise from a large number of possible analytes. The aim of the present study was to determine whether fast gas chromatography (fast-GC) coupled to PTR-ToF-MS could be used to overcome this issue and constitute a suitable on-line, near real-time method to identify and quantify fungal MVOC emissions in the ppbV-to-ppmV regime. Using gas standards of eleven MVOCs known to be emitted by T. atroviride such as 6-amyl-α-pyrone (6-PP), 2-pentylfuran, 1-octen-3-ol, 2-heptanone, 3-octanone, 2-methyl-1-propanol, 2-pentanone, 3-methyl-1-butanol, 3-methylbutanal, acetone and ethanol, we developed a fast-GC method with a total runtime of 180 s which significantly enhances the analytical specificity of PTR-ToF-MS compared to conventional PTR-ToF-MS without fast-GC separation. Limits of detection were on the order of 0.1-4 ppbV. The increased analytical specificity demonstrated notable benefits, especially for MVOCs having partially overlapping distributions of product ions when analyzed directly using PTR-ToF-MS. In order to demonstrate the applicability of the analytical method, we analysed T. atroviride samples in four biological replicates twice daily over a duration of five days. Using the fast-GC method, nine out of the eleven MVOC species considered in this study in the headspace of T. atroviride could be identified and quantified and their time evolution over the five-day incubation period determined. The measured volume mixing ratios (VMRs) ranged from single-digit ppbV (2-pentylfuran) up to few ppmV (6-PP and ethanol), with the other compounds in the 10-to-100-ppbV range (1-octen-3-ol, 2-heptanone, 2-methyl-1-propanol, 3-methyl-1-butanol, 3-methylbutanal and acetone). Our results suggest that fast-GC-PTR-ToF-MS is a method well-suited for the analysis of gas-phase samples of biological origin, including but not limited to (mycoparasitic) fungi, in a wide range of VMRs from sub-ppbV to few-ppmV.

The histone deacetylase Hda1 affects oxidative and osmotic stress response as well as mycoparasitic activity and secondary metabolite biosynthesis in Trichoderma atroviride.

The mycoparasitic fungus Trichoderma atroviride is applied in agriculture as a biostimulant and biologic control agent against fungal pathogens that infest crop plants. Secondary metabolites are among the main agents determining the strength and progress of the mycoparasitic attack. However, expression of most secondary metabolism-associated genes requires specific cues, as they are silent under routine laboratory conditions due to their maintenance in an inactive heterochromatin state. Therefore, histone modifications are crucial for the regulation of secondary metabolism. Here, we functionally investigated the role of the class II histone deacetylase encoding gene hda1 of T. atroviride by targeted gene deletion, phenotypic characterization, and multi-omics approaches. Deletion of hda1 did not result in obvious phenotypic alterations but led to an enhanced inhibitory activity of secreted metabolites and reduced mycoparasitic abilities of T. atroviride against the plant-pathogenic fungi Botrytis cinerea and Rhizoctonia solani. The ∆hda1 mutants emitted altered amounts of four volatile organic compounds along their development, produced different metabolite profiles upon growth in liquid culture, and showed a higher susceptibility to oxidative and osmotic stress. Moreover, hda1 deletion affected the expression of several notable gene categories such as polyketide synthases, transcription factors, and genes involved in the HOG MAPK pathway.IMPORTANCEHistone deacetylases play crucial roles in regulating chromatin structure and gene transcription. To date, classical-Zn2+ dependent-fungal histone deacetylases are divided into two classes, of which each comprises orthologues of the two sub-groups Rpd3 and Hos2 and Hda1 and Hos3 of yeast, respectively. However, the role of these chromatin remodelers in mycoparasitic fungi is poorly understood. In this study, we provide evidence that Hda1, the class II histone deacetylases of the mycoparasitic fungus Trichoderma atroviride, regulates its mycoparasitic activity, secondary metabolite biosynthesis, and osmotic and oxidative stress tolerance. The function of Hda1 in regulating bioactive metabolite production and mycoparasitism reveals the importance of chromatin-dependent regulation in the ability of T. atroviride to successfully control fungal plant pathogens.

The glyoxylate cycle is involved in pleotropic phenotypes, antagonism and induction of plant defence responses in the fungal biocontrol agent Trichoderma atroviride.

Isocitrate lyase (ICL), a signature enzyme of the glyoxylate cycle, is required for metabolism of non-fermentable carbon compounds like acetate or ethanol, and virulence in bacteria and fungi. In the present study, we investigate the role of the glyoxylate cycle in the fungal biocontrol agent Trichoderma atroviride by generating icl deletion and complementation mutants. Phenotypic analyses of the deletion mutant Δicl suggest that ICL is required for normal growth, conidial pigmentation and germination, and abiotic stress tolerance. The Δicl strain display reduced antagonism towards Botrytis cinerea in plate confrontation assays. Secretion and sandwich assays further show that secreted factors are partly responsible for the reduced antagonism. Furthermore, in vitro root colonization assays shows that the Δicl strain retains the ability to internally colonize Arabidopsis thaliana roots. However, the Δicl strain has a reduced ability to induce systemic defence in A. thaliana leaves that results in reduced protection against B. cinerea. These data shows that ICL and the glyoxylate cycle are important for biocontrol traits in T. atroviride, including direct antagonism and induction of defence responses in plants.

Elicitors from the endophytic fungus Trichoderma atroviride promote Salvia miltiorrhiza hairy root growth and tanshinone biosynthesis.

Biotic elicitors can be used to stimulate the production of secondary metabolites in plants. However, limited information is available on the effects of biotic elicitors from endophytic fungi on their host plant. Trichoderma atroviride D16 is an endophytic fungus isolated from the root of Salvia miltiorrhiza and previously reported to produce tanshinone I (T-I) and tanshinone IIA (T-IIA). Here, the effects of extract of mycelium (EM) and the polysaccharide fraction (PSF), produced by T. atroviride D16, on the growth and secondary metabolism of S. miltiorrhiza hairy roots are reported. The results indicated that both EM and PSF promoted hairy root growth and stimulated the biosynthesis of tanshinones in hairy roots. EM slightly suppressed the accumulation of phenolic acids, while PSF had no significant influence on the accumulation of these compounds. When comparing the effects of EM versus PSF, it was concluded that PSF is one of the main active constituents responsible for promoting hairy root growth, as well as stimulating biosynthesis of tanshinones in the hairy root cultures. Moreover, the transcriptional activity of genes involved in the tanshinone biosynthetic pathway increased significantly with PSF treatment. Thus, PSF from endophytic T. atroviride D16 affected the chemical composition of the host plant by influencing the expression of genes related to the secondary metabolite biosynthetic pathway. Furthermore, treatment with PSF can be effectively utilized for large-scale production of tanshinones in the S. miltiorrhiza hairy root culture system.

Light-Induced Changes in Secondary Metabolite Production of Trichoderma atroviride.

Many studies aim at maximizing fungal secondary metabolite production but the influence of light during cultivation has often been neglected. Here, we combined an untargeted isotope-assisted liquid chromatography-high-resolution mass spectrometry-based metabolomics approach with standardized cultivation of Trichoderma atroviride under three defined light regimes (darkness (PD), reduced light (RL) exposure, and 12/12 h light/dark cycle (LD)) to systematically determine the effect of light on secondary metabolite production. Comparative analyses revealed a similar metabolite profile upon cultivation in PD and RL, whereas LD treatment had an inhibiting effect on both the number and abundance of metabolites. Additionally, the spatial distribution of the detected metabolites for PD and RL was analyzed. From the more than 500 detected metabolites, only 25 were exclusively produced upon fungal growth in darkness and 85 were significantly more abundant in darkness. The majority were detected under both cultivation conditions and annotation revealed a cluster of substances whose production followed the pattern observed for the well-known T. atroviride metabolite 6-pentyl-alpha-pyrone. We conclude that cultivation of T. atroviride under RL can be used to maximize secondary metabolite production.

Identification and evaluation of suitable reference genes for RT-qPCR analyses in Trichoderma atroviride under varying light conditions.

BACKGROUND: Trichoderma atroviride is a competitive soil-borne mycoparasitic fungus with extensive applications as a biocontrol agent in plant protection. Despite its importance and application potential, reference genes for RT-qPCR analysis in T. atroviride have not been evaluated. Light exerts profound effects on physiology, such as growth, conidiation, secondary metabolism, and stress response in T. atroviride, as well as in other fungi. In this study, we aimed to address this gap by identifying stable reference genes for RT-qPCR experiments in T. atroviride under different light conditions, thereby enhancing accurate and reliable gene expression analysis in this model mycoparasite. We measured and compared candidate reference genes using commonly applied statistical algorithms. RESULTS: Under cyclic light-dark cultivation conditions, tbp and rho were identified as the most stably expressed genes, while act1, fis1, btl, and sar1 were found to be the least stable. Similar stability rankings were obtained for cultures grown under complete darkness, with tef1 and vma1 emerging as the most stable genes and act1, rho, fis1, and btl as the least stable genes. Combining the data from both cultivation conditions, gapdh and vma1 were identified as the most stable reference genes, while sar1 and fis1 were the least stable. The selection of different reference genes had a significant impact on the calculation of relative gene expression, as demonstrated by the expression patterns of target genes pks4 and lox1. CONCLUSION: The data emphasize the importance of validating reference genes for different cultivation conditions in fungi to ensure accurate interpretation of gene expression data.