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Due to the rapid expansion in microbial taxonomy, precise identification of common industrially and agriculturally relevant fungi such as Trichoderma species is challenging. In this study, we introduce the online multilocus identification system (MIST) for automated detection of 349 Trichoderma species based on a set of three DNA barcodes. MIST is based on the reference databases of validated sequences of three commonly used phylogenetic markers collected from public databases. The databases consist of 414 complete sequences of the nuclear rRNA internal transcribed spacers (ITS) 1 and 2, 583 sequence fragments of the gene encoding translation elongation factor 1-alpha ( tef1 ), and 534 sequence fragments of the gene encoding RNA polymerase subunit 2 ( rpb2 ). Through MIST, information from different DNA barcodes can be combined and the identification of Trichoderma species can be achieved based on the integrated parametric sequence similarity search (blastn) performed in the manner of a decision tree classifier. In the verification process, MIST provided correct identification for 44 Trichoderma species based on DNA barcodes consisting of tef1 and rpb2 markers. Thus, MIST can be used to obtain an automated species identification as well as to retrieve sequences required for manual identification by means of phylogenetic analysis. IMPORTANCE The genus Trichoderma is important to humankind, with a wide range of applications in industry, agriculture, and bioremediation. Thus, quick and accurate identification of Trichoderma species is paramount, since it is usually the first step in Trichoderma -based research. However, it frequently becomes a limitation, especially for researchers who lack taxonomic knowledge of fungi. Moreover, as the number of Trichoderma -based studies has increased, a growing number of unidentified sequences have been stored in public databases, which has made the species identification more ambiguous. In this study, we provide an easy-to-use tool, MIST, for automated species identification, a list of Trichoderma species, and corresponding sequences of reference DNA barcodes. Therefore, this study will facilitate the research on the biodiversity and applications of the genus Trichoderma ., (Copyright © 2020 American Society for Microbiology.)

An investigation of Trichoderma biodiversity involving a large-scale environmental gradient was conducted to understand the Trichoderma distribution in China. A total of 3,999 isolates were isolated from forestry, grassland, wetland and agriculture ecosystems, and 50 species were identified based on morphological characteristics and sequence analysis of genetic markers. Trichoderma harzianum showed the largest proportion of isolates and the most extensive distribution. Hypocrea semiorbis, T. epimyces, T. konilangbra, T. piluliferum, T. pleurotum, T. pubescens, T. strictipilis, T. hunua, T. oblongisporum and an unidentified species, Trichoderma sp. MA 3642, were first reported in China. Most Trichoderma species were distributed in Jilin and Heilongjiang Provinces in northeast China and the fewest were distributed in Qinghai Province. Based on the division of ecological and geographic factors, forestry ecosystems and low-altitude regions have the greatest species biodiversity of Trichoderma.

Background: Bacillus spp. are a genus of biocontrol bacteria widely used for antibiosis, while Trichoderma spp. are biocontrol fungi that are abundantly explored. In this study, a liquid co-cultivation of these two organisms was tried firstly., Results and Discussion: Through liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), it was discovered that with an inoculation in the ratio of 1.9:1, the antimicrobial effect of the co-cultured fermentation liquor of Bacillus amyloliquefaciens ACCC11060 and Trichoderma asperellum GDFS1009 was found to be significantly higher than that of pure-cultivation. A raise in the synthesis of antimicrobial substances contributed to this significant increase. Additionally, a co-culture with the inoculation of the two organisms in the ratio of 1:1 was found to enhance the production of specific amino acids. This technique could be further explored for either a large scale production of amino acids or could serve as a theoretical base for the generation of certain rare amino acids., Conclusions: This work clearly demonstrated that co-cultivation of B. amyloliquefaciens ACCC11060 and T. asperellum GDFS1009 could produce more specific biocontrol substances and amino acids.

Due to its efficient broad-spectrum antimicrobial activity, Trichoderma has been established as an internationally recognized biocontrol fungus. In this study, we found and identified a novel strain of Trichoderma asperellum, named GDFS1009. The mycelium of T. asperellum GDFS1009 exhibits a high growth rate, high sporulation capacity, and strong inhibitory effects against pathogens that cause cucumber fusarium wilt and corn stalk rot. T. asperellum GDFS1009 secretes chitinase, glucanase, and protease, which can degrade the cell walls of fungi and contribute to mycoparasitism. The secreted xylanases are good candidates for inducing plant resistance and enhancing plant immunity against pathogens. RNA sequencing (RNA-seq) and gas chromatography-mass spectrometry (GC-MS) showed that T. asperellum GDFS1009 produces primary metabolites that are precursors of antimicrobial compounds; it also produces a variety of antimicrobial secondary metabolites, including polyketides and alkanes. In addition, this study speculated the presence of six antimicrobial peptides via ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-QTOF-MS/MS). Future studies should focus on these antimicrobial metabolites for facilitating widespread application in the field of agricultural bio-control.

Sheath blight, causes by Rhizoctonia spp., threaten maize yield every year throughout the world. Trichoderma could degrade Rhizoctonia solani on maize mainly via competition and hyperparasitism, whereas validamycin A could efficiently inhibit the growth of R. solani via disturbing the energy system. By contrast, validamycin A is efficient but it takes effect in a short period, while Trichoderma takes effect in a long period though time-consuming. To overcome the disadvantages, Trichoderma asperellum GDFS1009 was used together with validamycin A. In vitro tests proved that the combined pathogen-inhibiting efficiency was significantly improved. Furthermore, results based on transcriptome and metabolome showed that validamycin A had no significant effects on growth, basic metabolism and main bio-control mechanisms of T. asperellum GDFS1009. Such few impacts may be attributed to detoxification and tolerance mechanism of T. asperellum GDFS1009. In addition, T. asperellum GDFS1009 has an ability to relieve the stress caused by validaymicn A. Meanwhile, liquid chromatography-mass spectrometry (LC-MS) results showed that only minor degradation (20%) of validamycin A was caused by T. asperellum GDFS1009 during cofermentation. All results together provide solid bases for validamycin A synergy with T. asperellum GDFS1009 in their combined biocontrol application.

To investigate the biodiversity of Trichoderma (Hypocreaceae) and their relation to sediment physical and chemical properties, we collected a total of 491 sediment samples from coastal wetlands (tidal flat and wetland) in Southeast China. Further, we applied two types of molecular approaches such as culture dependent and independent methods for identification of Trichoderma spp. A total of 254 isolates were obtained and identified to 13 species such as T. aureoviride, T. asperellum, T. harzianum, T. atroviride, T. koningiopsis, T. longibrachiatum, T. koningii. T. tawa, T. viridescens, T. virens, T. hamatum, T. viride, and T. velutinum by the culture-dependent (CD) method of these, T. tawa was newly described in China. Subsequently, the culture indepented method of 454 pyrosequencing analysis revealed a total of six species such as T. citrinoviride, T. virens, T. polysporum, T. harzianum/Hypocrea lixii and two unknown species. Notably, T. citrinoviride and T. polysporum were not found by the CD method. Therefore, this work revealed that the combination of these two methods could show the higher biodiversity of Trichoderma spp., than either of this method alone. Among the sampling sites, Hangzhou, Zhejiang Province, exhibited rich biodiversity and low in Fengxian. Correlation and Redundancy discriminant analysis (RDA) revealed that sediment properties of temperature, redox potential (Eh) and pH significantly influenced the biodiversity of Trichoderma spp., Competing Interests: The authors have declared that no competing interests exist.

Lepidoptera is a large order of insects that have major impacts on humans as agriculture pests. The midgut is considered an important target for insect control. In the present study, 10 up-regulated, 18 down-regulated, and one newly emerged protein were identified in the transgenic Trichoderma-treated midgut proteome. Proteins related to stress response, biosynthetic process, and metabolism process were further characterized through quantitative real-time PCR (qPCR). Of all the identified proteins, the glutathione S-transferase sigma 2 (GSTs2) gene displayed enhanced expression when larvae were fed with Trichoderma wild-type or transgenic strains. Down regulation of GSTs2 expression by RNA interference (RNAi) resulted in inhibition of silkworm growth when larvae were fed with mulberry leaves treated with the transgenic Trichoderma strain. Weight per larva decreased by 18.2%, 11.9%, and 10.7% in the untreated control, ddH2O, and GFP dsRNA groups, respectively, at 24h, while the weight decrease was higher at 42.4%, 28.8% and 32.4% at 72 h after treatment. Expression of glutathione S-transferase omega 2 (GSTo2) was also enhanced when larvae were fed with mulberry leaves treated with the transgenic Trichoderma strain. These results indicated that there was indeed correlation between enhanced expression of GSTs2 and the anti-stress response of silkworm larvae against Trichoderma. This study represents the first attempt at understanding the effects of transgenic organisms on the midgut proteomic changes in silkworm larvae. Our findings could not only broaden the biological control targets of insect at the molecular level, but also provide a theoretical foundation for biological safety evaluation of the transgenic Trichoderma strain., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Proteases secreted by fungi belonging to the genus Trichoderma play important roles in biocontrol. In this study, the coding sequence and promoter region of the novel aspartic protease gene Asp55 were cloned from strain Trichoderma asperellum ACCC30536. Many cis-elements involved in phytopathogenic and environmental stress responses were identified in the Asp55 promoter region and may be recognized by MYB or WRKY transcription factors. The expression pattern of Asp55 under eight culture conditions was investigated by RT-qPCR. The expression level of Asp55 was up-regulated by poplar stem powder, Alternaria alternata cell wall fragments and A. alternata fermentation liquid, while it was down-regulated by carbon and nitrogen source starvation, and by powdered poplar leaves and roots. Additionally, the expression patterns of 15 genes encoding MYB transcription factors (Myb1 to Myb15) were also analyzed by RT-qPCR. Myb2 showed the most similar expression pattern with Asp55. The cDNA of Asp55 was expressed in Escherichia coli BL21, and recombinant ASP55 (rASP55) was purified. The purified rASP55 was evaluated for enzymatic activity and showed inhibitory effect on phytopathogenic A. alternata., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

The subtilisin-like serine protease gene ThSS45 has been cloned from Trichoderma harzianum ACCC30371. Its coding region is 1302 bp in length, encoding 433 amino acids, with a predicted protein molecular weight of 44.9 kDa and pI of 5.91. ThSS45 was shown by RT-qPCR analysis to be differentially transcribed in response to eight different treatments. The transcription of ThSS45 was up-regulated when grown in mineral medium, under carbon starvation, and nitrogen starvation, and in the presence of 1% root powder, 1% stem powder, and 1% leaf powder derived from Populus davidiana × P. bolleana (Shanxin poplar) aseptic seedlings. The highest increase in transcription approached 3.5-fold that of the control at 6 h under induction with 1% poplar root powder. The transcription of ThSS45 was also slightly up-regulated by 1% Alternaria alternata cell wall and 5% A. alternata fermentation liquid. Moreover, the analyses of coding and promoter regions of ThSS45 homologs indicated that serine protease may be involved in both mycoparasitism and antibiotic secretion. ThSS45 was cloned into the pGEX-4T-2 vector and then expressed in Escherichia coli BL21. The recombinant protein, with an expected molecular weight of approximately 69 kDa, was then purified. When transformant BL21-ss was induced with 1 mM IPTG for 6 h, the purified protease activity reached a peak of 18.25 U/ml at pH 7.0 and 40°C. In antifungal assays the purified protease obviously inhibited the growth of A. alternata mycelia.

Trichoderma spp. are widely used beneficial microbes in agricultural production; however, the improper carrier choice for Trichoderma agent preparation can alter the effectiveness of Trichoderma fungicides. In this study, the co-culture of four Trichoderma strains produced a large amount of free amino acids, with a content of 392.8414 ug/mL, and significantly improved the production level of γ-aminobutyric acid. A greenhouse experiment further showed that the co-culture of Trichoderma synergistically improved the female flower development and bacterial angular leaf spot resistance. The effects of ten kinds of carriers were compared in terms of water absorption and heat generation, as well as their effects on the seedling emergence rate and the plant growth promotion of maize, cucumber, and pakchoi cabbage. Each carrier was screened to mix with four strains of co-culture metabolites to prepare highly soluble and quality powders. The results showed that there were different effects of the carriers themselves and Trichoderma strain co-culture metabolite powder prepared with the carriers on seedling emergence rate and seedling growth. Β-cyclodextrin performed best in high solubility and low heat generation upon absorbing water and in easy drying in processing operations. Trichoderma strains co-culture metabolite powder with β-cyclodextrin as a carrier provided the most obvious promotion effects on seedling emergence rate and seedling growth. Therefore, β-cyclodextrin was determined to be an ideal carrier to prepare a highly water-soluble Trichoderma agent. Taken together, the study successfully developed a new type of highly soluble powder containing Trichoderma co-culture metabolites that is expected to benefit farming drip irrigation and spraying systems for the promotion of crop growth and disease control. [ABSTRACT FROM AUTHOR]

Abstract This work reports the role of cellulase from Trichoderma harzianum strain Th22 in stimulation of 2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) and defense-related gene expressions in maize root against deoxynivalenol producing Fusarium graminearum (FG) by in vitro, in planta and virtual molecular modeling experiments. The results indicated that cellulase genes such as Thph1 & 2 were involved in biocontrol of FG and its related plant diseases through the activation of JA, ET, SAR, DIMBOA and PII related gene expressions in the maize root as evidenced by qRT-PCR and HPLC analysis. Further, the inhibitory assay also confirms that DIMBOA inhibited the growth of FG at concentrations dependent manner. Furthermore, virtual studies revealed that DIMBOA from maize root metabolizes or inhibit the active site of pathogenicity (FgSwi6) and mycotoxin (FgAtf1) related protein in FG. Finally, this work concluded that cellulase genes Thph1 and Thph2 from T. harzianum was not only limited to control of foliar disease but also involved in biocontrol of Fusarium stalk rot. Graphical abstract Image 1 Highlights • Cellulase genes triggering plant defense against pathogens F. graminearum (FG). • Cellulase involved in synthesis and expression of DIMBOA. • Cellulase activated DIMBOA inhibited the FG. • DIMBOA inhibits pathogencity and mycotoxin related proteins in pathogen FG. • Cellulase from T. harzianum proved as involved in control of Fusarium stalk rot. [ABSTRACT FROM AUTHOR]

The present work was carried out to investigate the application potential of Trichoderma harzianum T-soybean in maize (Zea mays) production. The effects of T-soybean metabolite on pathogen growth, maize growth and resistance response as well as 1-aminocyclopropane-1-carboxylate (ACC) deaminase (ACCD) (EC 4.1.99.4) crude enzyme liquid on germination and seedlings growth of maize were analysed in greenhouse condition. The effects of bio-fertilizer containing T. harzianum T-soybean (BIOT) on maize production were also analysed by determining soil enzymes activities and soil microorganisms counts in field condition. The results suggested that T-soybean significantly inhibited growth of pathogenic Fusarium graminearum and induced resistance response of maize seedlings by increasing antioxidant enzyme activities including peroxidase (POD) (EC 1.11.1.6), polyphenol oxidase (PPO) (EC 1.10.3.2), phenylalanine ammonia-lyase (PAL) (EC 4.3.1.5) and β-1,3-glucanase (EC 3.2.1.39) in leaves. ACCD from T-soybean also was involved in the beneficial effect. Compared with only using chemical fertilizer, BIOT increased microorganism counts and activities of soil enzymes, and then reduced the incidence of maize stalk rot, and increased maize yield. In sum, T-soybean has potential values of applications in sustainable agriculture. • Trichoderma T-soybean promoted growth of maize seedlings and ACCD from T-soybean was involved in the beneficial effect. • T-soybean inhibited growth of pathogen Fusarium graminearum. • T-soybean induced resistance response of maize seedlings by increasing antioxidant enzyme activities including POD, PPO, PAL and β-1,3-glucanase. • BIOT application increased microorganism counts and activities of soil enzymes. • BIOT application reduced the incidence of maize stalk rot and increased maize yield. [ABSTRACT FROM AUTHOR]

Abstract We earlier reported the role of cellulase (Thph1 and Thph2) from Trichoderma harzianum in controlling the Curvularia leaf spot and Fusarium stalk rot through interaction with the maize root. But, the maize root receptor related to cellulase was unknown. Hence, in the present study, we applied label-free proteomics and yeast two-hybrid (Y2H) assay to screen the root receptor that communicates with cellulase by generating a different combination of activation (AD) and binding (bait) domain plasmids such as DDO-SD/-Leu/-Trp QDO/X-SD/-Ade/-His/-Leu/-Trp. The maize roots were treated with T. harzianum strains including knockout Thph1, Thph2 and wild-type and enzyme cellobiohydrolase 1 (CBH1). The treated or untreated maize roots were collected for extraction of RNA and protein for generation of cDNA library and Label-free proteomics assay respectively. The label-free proteomic results indicated that the protein expression in the maize root significantly varied between the treatments (P < 0.05) and the protein clusters were highly expressed in the treatments of T2 (inoculated with one g of conidia of T. harzianum WT Th22), T3 (inoculated with one g of conidia of knockout strains of Thph1 and Thph2), T4 (inoculated with enzyme CBH 1) as compared to T1 (uninoculated control). The Y2H screening results indicated that autophagocytosis associated protein (ZmATG3) and germin-like protein (ZmGLP) from the maize root seemed to communicate with cellulase Thph1 and Thph2 respectively. The present screening results deserve for further cellular localization of ZmATG3 and ZmGLP in the maize root and their single or synergic functioning in induced systemic resistance (ISR) in maize through molecular dialogue between Trichoderma spp. and maize roots. Graphical abstract Image 1 Highlights • Cellulase (Thph1 and Thph2) from Trichoderma regulates the maize roots proteins. • The interactions of Trichoderma elicitor and maize root receptor were screened by Y2H assay. • Autophagocytosis protein from the maize roots interact with cellulase Thph1. • Germin-like protein from the maize roots interact with cellulase Thph 2. [ABSTRACT FROM AUTHOR]

Cucumber ( Cucumis sativus L.) is an important vegetable crop grown worldwide. However, its productivity is largely suppressed the phytopathogen Fusarium oxysporum f. sp. cucumerinum (FOC). We screened 100 isolates of Trichoderma as potential biocontrol agents against FOC. Both in vitro and in vivo methods were used, followed by the analysis of synergistic effect of cell wall degrading enzymes (CWDEs) and its secondary metabolites. In addition, molecular target of growth regulating protein of FOC (Snt2, a BAH/PHD containing transcription factor) was also studied. Of the 100 isolates of Trichoderma , 10 isolates with more than 85% inhibition rate were selected for in vivo antagonism in greenhouse experiment. Trichoderma asperellum strain CCTCC-RW0014 was found to have good biocontrol potential with high disease reduction of 71.67%. The RT-qPCR analysis revealed that the ITS copies of T. asperellum CCTCC-RW0014 were significantly higher in the cucumber roots treated with T. asperellum CCTCC-RW0014. Whereas the ITS copies of FOC declined in plants root in the presence of T . asperellum CCTCC-RW0014 and FOC. T . asperellum CCTCC-RW0014 showed high hydrolytic activity of chitin (87.5 ± 3.21%), gelatin (84.8 ± 2.56%), carboxymethyl cellulose (52.9 ± 1.23%) and pachyman (60.5 ± 2.32%). The crude enzymes; namely chitinase, cellulase, protease, and β (1–3) glucanase; were optimally active at 40 °C and pH 5.0, 30 °C and pH 6.0, 40 °C and pH 7, and 40 °C and pH 6.0 respectively. The secondary metabolites of Trichoderma extracts were determined by GC–MS and further studied for the molecular docking against target protein Snt2. Among the tested compounds, 1, 6-diphenylhexane-1,3,4,6-tetrone showed the highest C dock energy (−27.567 kacl/mol) against the target protein and this may down regulate the expression of protein Snt2. The present work concluded that T . asperellum CCTCC-RW0014 was the highly potential strain to control the FOC, providing scope for a further study on the mechanism of interaction between Trichoderma derived compounds and host plants. [ABSTRACT FROM AUTHOR]