Metabolomics reveal metabolic variation caused by co-culture of Arthrobacter ureafaciens and Trichoderma harzianum and their impacts on wheat germination / La metabolómica revela la variación metabólica causada por el cocultivo de Arthrobacter ureafaciens y Trichoderma harzianum y sus impactos en la germinación del trigo

Arthrobacter ureafaciens DnL1-1 is a bacterium used for atrazine degradation, while Trichoderma harzianum LTR-2 is a widely used biocontrol fungus. In this study, a liquid co-cultivation of these two organisms was initially tested. The significant changes in the metabolome of fermentation liquors were investigated based on cultivation techniques (single-cultured and co-cultured DnL1-1 and LTR-2) using an UPLC-QTOF-MS in an untargeted metabolomic approach. Principle components analysis (PCA) and partial least squares discriminant analysis (PLS-DA) supervised modelling revealed modifications of the metabolic profiles in fermentation liquors as a function of interactions between different strains. Compared with pure-cultivation of DnL1-1, 51 compounds were altered during the cocultivation, with unique and significant differences in the abundance of organic nitrogen compounds (e.g. carnitine, acylcarnitine 4:0, acylcarnitine 5:0, 3-dehydroxycarnitine and O-acetyl-L-carnitine) and trans-zeatin riboside. Nevertheless, compared with pure-cultivation of LTR-2, the abundance of 157 compounds, including amino acids, soluble sugars, organic acids, indoles and derivatives, nucleosides, and others, changed significantly in the cocultivation. Among them, the concentration of tryptophan, which is a precursor to indoleacetic acid, indoleacetic acid, aspartic acid, and L-glutamic acid increased while that of most soluble sugars decreased upon cocultivation. The fermentation filtrates of co-cultivation of LTR-2 and DnL1-1 showed significant promoting effects on germination and radicle length of wheat. A subsequent experiment demonstrated synergistic effects of differential metabolites caused by co-cultivation of DnL1-1 and LTR-2 on wheat germination. Comprehensive metabolic profiling may provide valuable information on the effects of DnL1-1 and LTR-2 on wheat growth.(AU)

Complete Genome Sequences and Genome-Wide Characterization of Trichoderma Biocontrol Agents Provide New Insights into their Evolution and Variation in Genome Organization, Sexual Development, and Fungal-Plant Interactions.

Trichoderma spp. represent one of the most important fungal genera to mankind and in natural environments. The genus harbors prolific producers of wood-decaying enzymes, biocontrol agents against plant pathogens, plant-growth-promoting biofertilizers, as well as model organisms for studying fungal-plant-plant pathogen interactions. Pursuing highly accurate, contiguous, and chromosome-level reference genomes has become a primary goal of fungal research communities. Here, we report the chromosome-level genomic sequences and whole-genome annotation data sets of four strains used as biocontrol agents or biofertilizers (Trichoderma virens Gv29-8, Trichoderma virens FT-333, Trichoderma asperellum FT-101, and Trichoderma atroviride P1). Our results provide comprehensive categorization, correct positioning, and evolutionary detail of both nuclear and mitochondrial genomes, including telomeres, AT-rich blocks, centromeres, transposons, mating-type loci, nuclear-encoded mitochondrial sequences, as well as many new secondary metabolic and carbohydrate-active enzyme gene clusters. We have also identified evolutionarily conserved core genes contributing to plant-fungal interactions, as well as variations potentially linked to key behavioral traits such as sex, genome defense, secondary metabolism, and mycoparasitism. The genomic resources we provide herein significantly extend our knowledge not only of this economically important fungal genus, but also fungal evolution and basic biology in general. IMPORTANCE Telomere-to-telomere and gapless reference genome assemblies are necessary to ensure that all genomic variants are studied and discovered, including centromeres, telomeres, AT-rich blocks, mating type loci, biosynthetic, and metabolic gene clusters. Here, we applied long-range sequencing technologies to determine the near-completed genome sequences of four widely used biocontrol agents or biofertilizers: Trichoderma virens Gv29-8 and FT-333, Trichoderma asperellum FT-101, and Trichoderma atroviride P1. Like those of three Trichoderma reesei wild isolates [QM6a, CBS999.97(MAT1-1) and CBS999.97(MAT1-2)] we reported previously, these four biocontrol agent genomes each contain seven nuclear chromosomes and a circular mitochondrial genome. Substantial intraspecies and intragenus diversities are also discovered, including single nucleotide polymorphisms, chromosome shuffling, as well as genomic relics derived from historical transposition events and repeat-induced point (RIP) mutations.

The pleiotropic functions of intracellular hydrophobins in aerial hyphae and fungal spores.

Higher fungi can rapidly produce large numbers of spores suitable for aerial dispersal. The efficiency of the dispersal and spore resilience to abiotic stresses correlate with their hydrophobicity provided by the unique amphiphilic and superior surface-active proteins-hydrophobins (HFBs)-that self-assemble at hydrophobic/hydrophilic interfaces and thus modulate surface properties. Using the HFB-enriched mold Trichoderma (Hypocreales, Ascomycota) and the HFB-free yeast Pichia pastoris (Saccharomycetales, Ascomycota), we revealed that the rapid release of HFBs by aerial hyphae shortly prior to conidiation is associated with their intracellular accumulation in vacuoles and/or lipid-enriched organelles. The occasional internalization of the latter organelles in vacuoles can provide the hydrophobic/hydrophilic interface for the assembly of HFB layers and thus result in the formation of HFB-enriched vesicles and vacuolar multicisternal structures (VMSs) putatively lined up by HFBs. These HFB-enriched vesicles and VMSs can become fused in large tonoplast-like organelles or move to the periplasm for secretion. The tonoplast-like structures can contribute to the maintenance of turgor pressure in aerial hyphae supporting the erection of sporogenic structures (e.g., conidiophores) and provide intracellular force to squeeze out HFB-enriched vesicles and VMSs from the periplasm through the cell wall. We also show that the secretion of HFBs occurs prior to the conidiation and reveal that the even spore coating of HFBs deposited in the extracellular matrix requires microscopic water droplets that can be either guttated by the hyphae or obtained from the environment. Furthermore, we demonstrate that at least one HFB, HFB4 in T. guizhouense, is produced and secreted by wetted spores. We show that this protein possibly controls spore dormancy and contributes to the water sensing mechanism required for the detection of germination conditions. Thus, intracellular HFBs have a range of pleiotropic functions in aerial hyphae and spores and are essential for fungal development and fitness.

Production of Polysaccharides and Single-Cell Protein by Some Local Isolates of Trichoderma spp.

<b>Background and Objective:</b> Polysaccharides and Single-cell protein are one of the best essential natural products of microorganisms, they are excreted by different microorganisms such as yeast, fungi, bacteria and algae. This study was carried out to detect the ability of four local fungal isolates of <i>Trichoderma </i>spp. to produce polysaccharides and Single-cell protein. <b>Materials and Methods:</b> Standard Czapek Dox Broth Medium was used to detect the ability of fungal isolates to produce polysaccharides and Single-cell protein, with modified the components of medium for improved production using banana peels as a source of carbon and different nitrogen sources at different concentrations and the factorial experiment was carried out using a completely randomized design <b>Results:</b> The highest dry weight and polysaccharides production and protein content have been achieved for the fungus <i>T. reesei</i> with rates of (2.15, 0.276 and 0.94) g/100 mL, respectively, in comparison with the other treatments, the use of ammonium phosphate at concentration 0.6 g L¹ has given the highest dry weight and production of polysaccharides and protein content with rates of (3.75, 0.364 and 2.77) g/100 mL, respectively, also the use of banana peels extract at concentration 40 mL L¹ has given the highest dry weight and production of polysaccharides and protein content with rates of (5.21, 0.539 and 3.63) g/100 mL, respectively. <b>Conclusion:</b> The possibility of using the local isolate of <i>T. reesei</i> in the production of polysaccharides and Single-cell protein using some cheap agricultural waste such as banana peels as a carbon source instead of throwing them as waste and pollutants for the environment.

Trichoderma reesei ACE4, a Novel Transcriptional Activator Involved in the Regulation of Cellulase Genes during Growth on Cellulose.

The filamentous fungus Trichoderma reesei is a model strain for cellulase production. Cellulase gene expression in T. reesei is controlled by multiple transcription factors. Here, we identified by comparative genomic screening a novel transcriptional activator, ACE4 (activator of cellulase expression 4), that positively regulates cellulase gene expression on cellulose in T. reesei. Disruption of the ace4 gene significantly decreased expression of four main cellulase genes and the essential cellulase transcription factor-encoding gene ace3. Overexpression of ace4 increased cellulase production by approximately 22% compared to that in the parental strain. Further investigations using electrophoretic mobility shift assays, DNase I footprinting assays, and chromatin immunoprecipitation assays indicated that ACE4 directly binds to the promoter of cellulase genes by recognizing the two adjacent 5'-GGCC-3' sequences. Additionally, ACE4 directly binds to the promoter of ace3 and, in turn, regulates the expression of ACE3 to facilitate cellulase production. Collectively, these results demonstrate an important role for ACE4 in regulating cellulase gene expression, which will contribute to understanding the mechanism underlying cellulase expression in T. reesei. IMPORTANCET. reesei is commonly utilized in industry to produce cellulases, enzymes that degrade lignocellulosic biomass for the production of bioethanol and bio-based products. T. reesei is capable of rapidly initiating the biosynthesis of cellulases in the presence of cellulose, which has made it useful as a model fungus for studying gene expression in eukaryotes. Cellulase gene expression is controlled through multiple transcription factors at the transcriptional level. However, the molecular mechanisms by which transcription is controlled remain unclear. In the present study, we identified a novel transcription factor, ACE4, which regulates cellulase expression on cellulose by binding to the promoters of cellulase genes and the cellulase activator gene ace3. Our study not only expands the general functional understanding of the novel transcription factor ACE4 but also provides evidence for the regulatory mechanism mediating gene expression in T. reesei.

A new peptaibol, RK-026A, from the soil fungus Trichoderma sp. RK10-F026 by culture condition-dependent screening.

A new peptaibol, RK-026A (1) was isolated from a fungus, Trichoderma sp. RK10-F026, along with atroviridin B (2), alamethicin II (3), and polysporin B (4) as a cytotoxic compound, which was selected by principal component analysis of the MS data from 5 different culture conditions. The structure of 1 was determined as a new atroviridin B derivative containing Glu at the 18th residue instead of Gln by NMR and HR-MS analyses including the investigation of detailed MS/MS fragmentations. 1 showed cytotoxicity toward K562 leukemia cells at an IC50 value of 4.1 µm.

Integration of Nitrogen and Biofertilizer on the Agronomic Efficiency and Quality of Shallot (Allium ascalonicum L.) Plant in the Rainy Season.

BACKGROUND AND OBJECTIVE: In the rainy season farmers don't interest to cultivate shallot because in addition to providing a high dosage of fertilizer they are also sensitive to pathogenic attacks so they are afraid of crop failure and cause low shallot production. This study aimed to knew effect of agronomic component and quality of shallot under different concentrations of biofertilizer and Ammonium Sulphate (AS) fertilizer dose in the rainy season. MATERIALS AND METHODS: The study was conducted in Cangkring, Srandakan, Bantul, Special Region of Yogyakarta Indonesia from August to October 2019. The study was arranged in RCBD factorial with three replications. The first factor was a various dose of ammonium sulphate (100, 200 and 300 kg ha-1). The second factor was various concentrations of biofertilizer (2, 3 and 4%), and control. The observed variables were the analysis of growth yield and quality component of shallot plant. The analyzed using analysis of variance at 5% of significance then continued by DMRT at 5% of significance. RESULTS: There was the interaction between the application of AS dosage and biofertilizer concentration on all of variable observations. There was a significant difference between treatment with control on all of the observation variables. CONCLUSION: The combination of AS fertilizer 200 kg ha-1 dose and 3% biofertilizer concentration increased agronomic efficiency, growth, bulbs yields, and quality of bulbs include provitamin A, oleoresin compounds.

Anatomical changes induced by isolates of Trichoderma spp. in soybean plants.

In the current work we evaluated the anatomical changes induced by T. harzianum and T. asperellum in two soybean cultivars, BRSGO Caiaponia and NA 5909 RG. Soybean production represents a growing market worldwide, and new methods aimed at increasing its productivity and yield are constantly being sought. Fungi of the genus Trichoderma have been widely used in agriculture as a promising alternative for the promotion of plant growth and for biological control of various pathogens. It is known that Trichoderma spp. colonize plant roots, but the anatomical changes that this fungus can cause are still less studied. Experiment was conducted in a greenhouse to collect leaves and soybean roots to perform analysis of growth parameters, enzymatic activity of defense-related enzymes and anatomical changes. It was observed that inoculation of Trichoderma spp. caused anatomical alterations, among them, increase in stomatal index at the abaxial leaf surface, thickness of the root cortex, thickness of adaxial epidermis, mean diameter of the vascular cylinder, thickness of the mesophyll, and thickness of the spongy parenchyma of the soybean plants. These results indicate that the alterations in these factors may be related to the process of plant resistance to pathogens, and better performance against adverse conditions. This study demonstrates that the anatomical study of plants is an important tool to show the effects that are induced by biological control agents.

Plant Cell Wall Changes in Common Wheat Roots as a Result of Their Interaction with Beneficial Fungi of Trichoderma.

Plant cell walls play an important role in shaping the defense strategies of plants. This research demonstrates the influence of two differentiators: the lifestyle and properties of the Trichoderma species on cell wall changes in common wheat seedlings. The methodologies used in this investigation include microscopy observations and immunodetection. In this study was shown that the plant cell wall was altered due to its interaction with Trichoderma. The accumulation of lignins and reorganization of pectin were observed. The immunocytochemistry indicated that low methyl-esterified pectins appeared in intercellular spaces. Moreover, it was found that the arabinogalactan protein epitope JIM14 can play a role in the interaction of wheat roots with both the tested Trichoderma strains. Nevertheless, we postulate that modifications, such as the appearance of lignins, rearrangement of low methyl-esterified pectins, and arabinogalactan proteins due to the interaction with Trichoderma show that tested strains can be potentially used in wheat seedlings protection to pathogens.

Multiple heavy metal tolerance and removal by an earthworm gut fungus Trichoderma brevicompactum QYCD-6.

Fungal bioremediation is a promising approach to remove heavy-metal from contaminated water. Present study examined the ability of an earthworm gut fungus Trichoderma brevicompactum QYCD-6 to tolerate and remove both individual and multi-metals. The minimum inhibitory concentration (MIC) of heavy metals [Cu(II), Cr(VI), Cd(II) and Zn(II)] against the fungus was ranged 150-200 mg L-1 on composite medium, and MIC of Pb(II) was the highest with 1600 mg L-1 on potato dextrose (PD) medium. The Pb(II) presented the highest metal removal rate (97.5%) which mostly dependent on bioaccumulation with 80.0%, and synchronized with max biomass (6.13 g L-1) in PD medium. However, on the composite medium, the highest removal rate was observed for Cu(II) (64.5%). Cellular changes in fungus were reflected by TEM analysis. FTIR and solid-state NMR analyses indicated the involvement of different functional groups (amino, carbonyl, hydroxyl, et al.) in metallic biosorption. These results established that the earthworm-associated T. brevicompactum QYCD-6 was a promising fungus for the remediation of heavy-metal wastewater.

Trichoderma virens Gl006 and Bacillus velezensis Bs006: a compatible interaction controlling Fusarium wilt of cape gooseberry.

The combination of Trichoderma virens Gl006 and B. velezensis Bs006 as a consortium has high potential to control Fusarium wilt (FW) of cape gooseberry (Physalis peruviana) caused by Fusarium oxysporum f. sp. physali (Foph). However, the interactions between these two microorganisms that influence the biocontrol activity as a consortium have not been studied. Here, we studied the interactions between Gl006 and Bs006 that keep their compatibility under in vitro and greenhouse conditions. Antagonism tests between Gl006 and Bs006 inoculated both individually and in consortium against Foph strain Map5 was carried out on several solid media. The effect of supernatant of each selected microorganism on growth, conidia germination, biofilm formation and antagonistic activity on its partner was also studied. Biocontrol activity by different combinations of cells and supernatants from both microorganisms against Fusarium wilt was evaluated under greenhouse conditions. In vitro antagonism of the consortium against Foph showed a differential response among culture media and showed compatibility among BCA under nutritional conditions close to those of the rhizosphere. The supernatant of Bs006 did not affect the antagonistic activity of Gl006 and vice versa. However, the supernatant of Bs006 promoted the biocontrol activity of Gl006 in a synergistic way under greenhouse, reducing the disease severity by 71%. These results prove the compatibility between T. virens Gl006 and B. velezensis Bs006 as a potential tool to control Fusarium wilt of cape gooseberry.

Comprehensive Optimization of Culture Conditions for Production of Biomass-Hydrolyzing Enzymes of Trichoderma SG2 in Submerged and Solid-State Fermentation.

Lignocellulose biomass contain large macromolecules especially cellulose and hemicelluloses that can be converted to fuel and chemicals using microbial biocatalysts. This study presents comprehensive optimization of production of biomass-hydrolyzing enzymes (BHE) by a high ß-glucosidase-producing Trichoderma SG2 for bioconversion of lignocellulose biomass. Overall, a mixture of paper powder and switchgrass was most suited for production of BHE in submerged fermentation (SmF). BHE production was significantly different for various organic and inorganic nitrogen sources. The combination of peptone, yeast extract, and ammonium sulfate resulted in the highest activities (Units/mL) of BHE: 9.85 ± 0.55 cellulase, 38.91 ± 0.31 xylanase, 21.19 ± 1.35 ß-glucosidase, and 7.63 ± 0.31 ß-xylosidase. Surfactants comparably enhanced BHE production. The highest cellulase activity (4.86 ± 0.55) was at 25 °C, whereas 35 °C supported the highest activities of xylanase, ß-glucosidase, and ß-xylosidase. A broad initial culture pH (4-7) supported BHE production. The Topt for cellulase and xylanase was 50 °C. ß-xylosidase and ß-glucosidase were optimally active at 40 and 70 °C, respectively; pH 5 resulted in highest cellulase, ß-glucosidase, and ß-xylosidase activities; and pH 6 resulted in highest xylanase activity. Response surface methodology (RSM) was used to optimize major medium ingredients. BHE activities were several orders of magnitude higher in solid-state fermentation (SSF) than in SmF. Therefore, SSF can be deployed for one-step production of complete mixture of Trichoderma SG2 BHE for bioconversion of biomass to saccharide feedstock.

Bioremediation of Explosive TNT by Trichoderma viride.

Nitroaromatic and nitroamine compounds such as 2,4,6-trinitrotoluene (TNT) are teratogenic, cytotoxic, and may cause cellular mutations in humans, animals, plants, and microorganisms. Microbial-based bioremediation technologies have been shown to offer several advantages against the cellular toxicity of nitro-organic compounds. Thus, the current study was designed to evaluate the ability of Trichoderma viride to degrade nitrogenous explosives, such as TNT, by microbiological assay and Gas chromatography-mass spectrometry (GC-MS) analysis. In this study, T. viride fungus was shown to have the ability to decompose, and TNT explosives were used at doses of 50 and 100 ppm on the respective growth media as a nitrogenous source needed for normal growth. The GC/MS analysis confirmed the biodegradable efficiency of TNT, whereas the initial retention peak of the TNT compounds disappeared, and another two peaks appeared at the retention times of 9.31 and 13.14 min. Mass spectrum analysis identified 5-(hydroxymethyl)-2-furancarboxaldehyde with the molecular formula C6H6O3 and a molecular weight of 126 g·mol-1 as the major compound, and 4-propyl benzaldehyde with a formula of C10H12O and a molecular weight of 148 g mol-1 as the minor compound, both resulting from the biodegradation of TNT by T. viride. In conclusion, T. viride could be used in microbial-based bioremediation technologies as a biological agent to eradicate the toxicity of the TNT explosive. In addition, future molecular-based studies should be conducted to clearly identify the enzymes and the corresponding genes that give T. viride the ability to degrade and remediate TNT explosives. This could help in the eradication of soils contaminated with explosives or other toxic biohazards.

Synergistic effect of Fusarium lateritium LP7 and Trichoderma viride LP5 promotes ethoxylated oleyl-cetyl alcohol biodegradation.

This study evaluated the growth and metabolic activity of consortium and pure cultures Fusarium lateritium LP7 and Trichoderma viride LP5 in response to the presence of 0.5% ethoxylated oleyl-cetyl alcohol (EOCA) in the liquid Czapek-Dox medium. The effectiveness of mentioned cultures was monitored according to the following parameters: biomass dry weight (BDW), pH, quantity of free and total organic acids, proteolytic activity and the qualitative composition of carbohydrates, during 19 days. The biodegrading efficiency was determined spectrophotometrically. The BDW of consortium was significantly stimulated by EOCA (16.59%) whereas biomass of LP7 was significantly inhibited (30.61%). The EOCA had influence on decrease in pH value of the media of LP5 and consortium, and pH changes were correlated with the amount of excreted organic acids. The alkaline protease activities of consortium, LP7 and LP5 retained 73%, 62.2% and 49.5% activity respectively in the presence of EOCA. Consortium has shown the best biodegradation capacity up to 82% of EOCA. The pure cultures were less effective in biodegradation and removed approximately 65% (LP7) and 60% (LP5) of EOCA after 19 days. In brief, the synergistic interaction between pure cultures enhances capacity to reduce EOCA in environment and influences production of some biotechnological useful metabolites.

The enhancement of plant secondary metabolites content in Lactuca sativa L. by encapsulated bioactive agents.

Encapsulated bioactive agents applied to the Lactuca sativa L. present an innovative approach to stimulate the production of plant secondary metabolites increasing its nutritive value. Calcium and copper ions were encapsulated in biopolymeric microparticles (microspheres and microcapsules) either as single agents or in combination with biocontrol agents, Trichoderma viride spores, a fungal plant growth mediator. Both, calcium and copper ions are directly involved in the synthesis of plant secondary metabolites and alongside, Trichoderma viride can provide indirect stimulation and higher uptake of nutrients. All treatments with microparticles had a positive effect on the enhancement of plant secondary metabolites content in Lactuca sativa L. The highest increase of chlorophylls, antioxidant activity and phenolic was obtained by calcium-based microparticles in both, conventionally and hydroponically grown lettuces. Non-encapsulated fungus Trichoderma viride enhanced the synthesis of plant secondary metabolites only in hydroponics cultivation signifying the importance of its encapsulation. Encapsulation proved to be simple, sustainable and environmentally favorable for the production of lettuce with increased nutritional quality, which is lettuce fortified with important bioactive compounds.

Silver nanoparticle from whole cells of the fungi Trichoderma spp. isolated from Brazilian Amazon.

Metal nanoparticles are a promising approach for the development of new antimicrobial systems. Silver nanoparticles (AgNP) have a significant antibacterial activity through bacterial surface adsorption and oxidative stress induction, as indicated by recent observations. This research aimed to use endophytic fungi from the genus Trichoderma spp. isolated from the Bertholletia excelsa (Brazil-nut) seeds and the soil to biosynthesize AgNPs and also test their antibacterial activity. The use of these fungi for this purpose not only valorizes the Amazon biodiversity but it also uses cleaner and cheaper processes, being part of the Green Chemistry concept. The particles were analyzed through Ultraviolet-Visible Spectroscopy and ZetaSizer and the band of absorption at 420 nm was analyzed through Localized Surface Plasmon Resonance. After characterization, the AgNP were tested for antibacterial activity against several bacterial strains, when it was observed that their antibacterial activity was superior in Gram-negative bacteria.

Chitin and chitosan remodeling defines vegetative development and Trichoderma biocontrol.

Fungal parasitism depends on the ability to invade host organisms and mandates adaptive cell wall remodeling to avoid detection and defense reactions by the host. All plant and human pathogens share invasive strategies, which aid to escape the chitin-triggered and chitin-targeted host immune system. Here we describe the full spectrum of the chitin/chitosan-modifying enzymes in the mycoparasite Trichoderma atroviride with a central role in cell wall remodeling. Rapid adaption to a variety of growth conditions, environmental stresses and host defense mechanisms such as oxidative stress depend on the concerted interplay of these enzymes and, ultimately, are necessary for the success of the mycoparasitic attack. To our knowledge, we provide the first in class description of chitin and associated glycopolymer synthesis in a mycoparasite and demonstrate that they are essential for biocontrol. Eight chitin synthases, six chitin deacetylases, additional chitinolytic enzymes, including six chitosanases, transglycosylases as well as accessory proteins are involved in this intricately regulated process. Systematic and biochemical classification, phenotypic characterization and mycoparasitic confrontation assays emphasize the importance of chitin and chitosan assembly in vegetative development and biocontrol in T. atroviride. Our findings critically contribute to understanding the molecular mechanism of chitin synthesis in filamentous fungi and mycoparasites with the overarching goal to selectively exploit the discovered biocontrol strategies.

Molecular tagging of biocontrol fungus Trichoderma asperellum and its colonization in soil.

AIMS: To conduct molecular tagging of the biocontrol fungus Trichoderma asperellum strain T4 and elucidate its colonization patterns in soil. METHODS AND RESULTS: We constructed an expression vector harbouring a hygromycin B-resistant gene (hph) and an efficient green fluorescent protein (egfp) gene. By applying Agrobacterium AGL-1-mediated genetic transformation technology, we conducted molecular tagging of T. asperellum and monitored the colonization dynamics of T. asperellum in soil. The results of tracking five independent transformants of T. asperellum indicated that its expansion rates ranged from 4·7 to 6·8 cm week-1 . After inoculation in soil, the quantities of T. asperellum could be maintained at over 10 × 104  CFU per gram soil in the first year. In the third year after inoculation, the quantities of T. asperellum in soil were still higher than 1 × 103  CFU per gram soil. In addition, molecularly tagged T. asperellum in soil in the second year (i.e. 12 months) after inoculation could still reach the biocontrol effect on cucumber Rhizoctonia rot by more than 74%. CONCLUSION: Trichoderma asperellum strain T4 is capable of effectively colonizing in soil and surviving for more than 1 year. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has provided the scientific basis for applying T. asperellum as the biocontrol fungus for prevention and control of plant diseases.

Trichothioneic acid, a new antioxidant compound produced by the fungal strain Trichoderma virens FKI-7573.

A new nitrogen-containing compound, trichothioneic acid, was discovered from the metabolites of fungal strain FKI-7573 using a mass spectrometry screening method guided by odd number of molecular weights, which indicates compounds that contain an odd number of nitrogen atoms. Strain FKI-7573 was isolated from soil collected in Obihiro, Hokkaido, Japan, and identified as Trichoderma virens by a sequence analysis of the internal transcribed spacer region, including 5.8S ribosomal RNA. The structure of trichothioneic acid was determined by mass spectrometry, nuclear magnetic resonance spectroscopy, electronic circular dichroism spectra, and chemical degradation analyses. These analyses revealed that trichothioneic acid consists of heptelidic acid and l-ergothioneine, and contains three nitrogen atoms. Trichothioneic acid exhibited hydroxyl radical-scavenging and singlet oxygen-quenching activities.

RNAi-Mediated Gene Silencing of Trcot1 Induces a Hyperbranching Phenotype in Trichoderma reesei.

Trichoderma reesei is the major filamentous fungus used to produce cellulase and there is huge interest in promoting its ability to produce higher titers of cellulase. Among the many factors affecting cellulase production in T. reesei, the mycelial phenotype is important but seldom studied. Herein, a close homolog of the Neurospora crassa COT1 kinase was discovered in T. reesei and designated TrCOT1, which is of 83.3% amino acid sequence identity. Functional disruption of Trcot1 in T. reesei by RNAi-mediated gene silencing resulted in retarded sporulation on potato dextrose agar and dwarfed colonies on minimal medium agar plates containing glucose, xylan, lactose, xylose, or glycerol as the sole carbon source. The representative mutant strain, SUS2/Trcot1i, also displayed reduced mycelia accumulation but hyperbranching in the MM glucose liquid medium, with hyphal growth unit length values decreased to 73.0 µm/tip compared to 239.8 µm/tip for the parent strain SUS2. The hyperbranching phenotype led to slightly but significantly increased cellulase secretion from 24 to 72 h in a batch culture. However, the cellulase production per unit of mycelial biomass was much more profoundly improved from 24 to 96 h.