[Screening and evaluation of the biocontrol efficacy of a Trichoderma brevicompactum strain and its metabolite trichodermin against banana Fusarium wilt].

The banana Fusarium wilt (BFW) caused by Fusarium oxysporum f. sp. cubense tropical race4 (FocTR4) is difficult to control worldwide, which causes a huge economic losse to banana industry. The purpose of this study was to screen Trichoderma strains with antagonistic activity against FocTR4, to isolate and purify the active compound from the fermentation broth, so as to provide important biocontrol strains and active compound resources. In this work, Trichoderma strains were isolated and screened from the rhizosphere soil of crops, and the strains capable of efficiently inhibiting FocTR4 were screened by plate confrontation, and further confirmed by testing inhibition for the conidial germination and mycelial growth of FocTR4. The phylogenetic tree clarified the taxonomic status of the biocontrol strains. Moreover, the active components in the fermentation broth of the strains were separated and purified by column chromatography, the structure of the most active component was analyzed by nuclear magnetic resonance spectroscopy (NMR), the BFW control effect was tested by pot experiments. We obtained a strain JSHA-CD-1003 with antagonistic activity against FocTR4, and the inhibition rate from plate confrontation was 60.6%. The fermentation broth of JSHA-CD-1003 completely inhibited the germination of FocTR4 conidia within 24 hours. The inhibition rate of FocTR4 hyphae growth was 52.6% within 7 d. A phylogenetic tree was constructed based on the ITS and tef1-α gene tandem sequences, and JSHA-CD-1003 was identified as Trichoderma brevicompactum. Purification and NMR identification showed that the single active compound was trichodermin, and the minimum inhibitory concentration (MIC) was 25 µg/mL. Pot experiments showed that the fermentation broth of strain JSHA-CD-1003 was effective against BFW. The control rate of leaf yellowing was 47.4%, and the rate of bulb browning was 52.0%. Therefore, JSHA-CD-1003 effectively inhibited FocTR4 conidial germination and mycelium growth through producing trichodermin, and showed biocontrol effect on banana wilt caused by FocTR4, thus is a potential biocontrol strain.

Screening and evaluation of the biocontrol efficacy of a Trichoderma brevicompactum strain and its metabolite trichodermin against banana Fusarium wilt / 生物工程学报

The banana Fusarium wilt (BFW) caused by Fusarium oxysporum f. sp. cubense tropical race4 (FocTR4) is difficult to control worldwide, which causes a huge economic losse to banana industry. The purpose of this study was to screen Trichoderma strains with antagonistic activity against FocTR4, to isolate and purify the active compound from the fermentation broth, so as to provide important biocontrol strains and active compound resources. In this work, Trichoderma strains were isolated and screened from the rhizosphere soil of crops, and the strains capable of efficiently inhibiting FocTR4 were screened by plate confrontation, and further confirmed by testing inhibition for the conidial germination and mycelial growth of FocTR4. The phylogenetic tree clarified the taxonomic status of the biocontrol strains. Moreover, the active components in the fermentation broth of the strains were separated and purified by column chromatography, the structure of the most active component was analyzed by nuclear magnetic resonance spectroscopy (NMR), the BFW control effect was tested by pot experiments. We obtained a strain JSHA-CD-1003 with antagonistic activity against FocTR4, and the inhibition rate from plate confrontation was 60.6%. The fermentation broth of JSHA-CD-1003 completely inhibited the germination of FocTR4 conidia within 24 hours. The inhibition rate of FocTR4 hyphae growth was 52.6% within 7 d. A phylogenetic tree was constructed based on the ITS and tef1-α gene tandem sequences, and JSHA-CD-1003 was identified as Trichoderma brevicompactum. Purification and NMR identification showed that the single active compound was trichodermin, and the minimum inhibitory concentration (MIC) was 25 μg/mL. Pot experiments showed that the fermentation broth of strain JSHA-CD-1003 was effective against BFW. The control rate of leaf yellowing was 47.4%, and the rate of bulb browning was 52.0%. Therefore, JSHA-CD-1003 effectively inhibited FocTR4 conidial germination and mycelium growth through producing trichodermin, and showed biocontrol effect on banana wilt caused by FocTR4, thus is a potential biocontrol strain.

Inhibitory Effect and Mechanism of Trichoderma taxi and Its Metabolite on Trichophyton mentagrophyte.

Trichophyton mentagrophytes is an important zoonotic dermatophyte, which seriously harms the skin of humans and animals. Chemical drugs are generally used for the prevention and treatment of the disease caused by T. mentagrophytes. Discovering new compounds from natural products is an important approach for new drug development. Trichoderma includes a variety of fungal species used for biological control of phytopathogenic fungi. However, the antifungal effects of Trichoderma and their metabolites on zoonotic fungal pathogens are largely unknown. Here, the effect of trichodermin, a metabolite derived from the plant endophytic fungus Trichoderma taxi, on T. mentagrophytes was examined, and the underlying mechanism was explored. T. mentagrophytes growth was suppressed significantly by trichodermin and completely inhibited under 1000 µg/mL trichodermin. The production and germination of T. mentagrophytes spores were remarkably reduced upon exposure to trichodermin, in comparison with control samples. Treatment of lesions caused by T. mentagrophytes on the rabbit skin with 1 mg/mL trichodermin prompted the healing process significantly; however, 20 mg/mL trichodermin was likely toxic to the skin. Under trichodermin treatment, the number of mitochondria in T. mentagrophytes increased significantly, while a few mitochondria-related genes decreased, indicating possible mitochondrial damage. In transcriptome analysis, the GO terms enriched by DEGs in the trichodermin-treated group included carbohydrate metabolic process, integral component of membrane, intrinsic component of membrane, and carbohydrate binding, while the enriched KEGG pathways comprised biosynthesis of secondary metabolites, glycolysis/gluconeogenesis, and carbon metabolism. By comparing the wild type and a gene deletion strain of T. mentagrophytes, we found that CDR1, an ABC transporter encoding gene, was involved in T. mentagrophytes sensitivity to trichodermin.

Trichodermin inhibits the growth of oral cancer through apoptosis-induced mitochondrial dysfunction and HDAC-2-mediated signaling.

Trichodermin (TCD), a trichothecene first isolated from marine Trichoderma viride, is an inhibitor of eukaryotic protein synthesis. However, the potential effects of TCD on human oral squamous cell carcinoma (OSCC) cells and the underlying molecular mechanisms remain unknown. In this study, the exposure of OSCC cells (Ca922 and HSC-3 cells) to TCD suppressed cell proliferation assessed using MTT assays and colony formation assays. TCD inhibited the migration and invasion of OSCC cells (Ca922 and HSC-3 cells) through the downregulation of matrix metalloproteinase 9. After treatment of OSCC cells with TCD, the G2/M phase was arrested, caspase-related apoptosis (cleaved caspase-3 and PARP expression) was induced, and the protein level of x-linked inhibitor of apoptosis was reduced. Meanwhile, the TCD-induced cell death was reversed by the pan-caspase inhibitor Z-VAD-FMK. Furthermore, TCD diminished mitochondrial membrane potential, mitochondrial oxidative phosphorylation and glycolytic function in OSCC cells. In addition, TCD decreased the levels of histone deacetylase 2 (HDAC-2) and downstream signaling proteins, including phosphorylated STAT3 and NF-κB. Finally, TCD significantly suppressed tumor growth in a zebrafish OSCC xenotransplantation model. Overall, this evidence demonstrates that TCD is a novel promising strategy for the treatment of OSCCs.

Verification of TRI3 Acetylation of Trichodermol to Trichodermin in the Plant Endophyte Trichoderma taxi.

Trichodermin, a trichothecene first isolated in Trichoderma species, is a sesquiterpenoid antibiotic that exhibits significant inhibitory activity to the growth of many pathogenic fungi such as Candida albicans, Rhizoctonia solani, and Botrytis cinerea by inhibiting the peptidyl transferase involved in eukaryotic protein synthesis. Trichodermin has also been shown to selectively induce cell apoptosis in several cancer cell lines and thus can act as a potential lead compound for developing anticancer therapeutics. The biosynthetic pathway of trichodermin in Trichoderma has been identified, and most of the involved genes have been functionally characterized. An exception is TRI3, which encodes a putative acetyltransferase. Here, we report the identification of a gene cluster that contains seven genes expectedly involved in trichodermin biosynthesis (TRI3, TRI4, TRI6, TRI10, TRI11, TRI12, and TRI14) in the trichodermin-producing endophytic fungus Trichoderma taxi. As in Trichoderma brevicompactum, TRI5 is not included in the cluster. Functional analysis provides evidence that TRI3 acetylates trichodermol, the immediate precursor, to trichodermin. Disruption of TRI3 gene eliminated the inhibition to R. solani by T. taxi culture filtrates and significantly reduced the production of trichodermin but not of trichodermol. Both the inhibitory activity and the trichodermin production were restored when native TRI3 gene was reintroduced into the disruption mutant. Furthermore, a His-tag-purified TRI3 protein, expressed in Escherichia coli, was able to convert trichodermol to trichodermin in the presence of acetyl-CoA. The disruption of TRI3 also resulted in lowered expression of both the upstream biosynthesis TRI genes and the regulator genes. Our data demonstrate that T. taxi TRI3 encodes an acetyltransferase that catalyzes the esterification of the C-4 oxygen atom on trichodermol and thus plays an essential role in trichodermin biosynthesis in this fungus.

Trichodermin Induces G0/G1 Cell Cycle Arrest by Inhibiting c-Myc in Ovarian Cancer Cells and Tumor Xenograft-Bearing Mice.

Ovarian cancer is a fatal gynecological cancer because of a lack of early diagnosis, which often relapses as chemoresistant. Trichodermin, a trichothecene first isolated from Trichoderma viride, is an inhibitor of eukaryotic protein synthesis. However, whether trichodermin is able to suppress ovarian cancer or not was unclear. In this study, trichodermin (0.5 µM or greater) significantly decreased the proliferation of two ovarian cancer cell lines A2780/CP70 and OVCAR-3. Normal ovarian IOSE 346 cells were much less susceptible to trichodermin than the cancer cell lines. Trichodermin predominantly inhibited ovarian cancer cells by inducing G0/G1 cell cycle arrest rather than apoptosis. Trichodermin decreased the expression of cyclin D1, CDK4, CDK2, retinoblastoma protein, Cdc25A, and c-Myc but showed little effect on the expression of p21Waf1/Cip1, p27Kip1, or p16Ink4a. c-Myc was a key target of trichodermin. Trichodermin regulated the expression of Cdc25A and its downstream proteins via c-Myc. Overexpression of c-Myc attenuated trichodermin's anti-ovarian cancer activity. In addition, trichodermin decelerated tumor growth in BALB/c nude mice, proving its effectiveness in vivo. These findings suggested that trichodermin has the potential to contribute to the treatment of ovarian cancer.

Antiproliferative and Antimicrobial Activities of Secondary Metabolites and Phylogenetic Study of Endophytic Trichoderma Species From Vinca Plants.

Endophytic fungi have been recognized as a potential source of bioactive secondary metabolites. The endophytic Trichoderma species were isolated from Vinca plants (Vinca major, Vinca herbacea, and Vinca minor), found in Iran and screened for antimicrobial and anti-proliferative activity. Based on morphological and phylogenetic analyses, four fungal species were identified: T. asperellum, T. brevicompactum, T. koningiopsis, and T. longibrachiatum. In addition, endophytic fungi bioactivity of methanol and ethyl acetate extracts (7.8-250 µgml-1) were assessed against a panel of pathogenic fungi and bacteria and IC80 was calculated. Data showed that both methanol and ethyl acetate extracts from all endophytic isolates had significant cytotoxic effects against the model target fungus Pyricularia oryzae. Further research indicated that they had significant antimicrobial bioactivity against the human pathogenic bacteria Staphylococcus aureus and Escherichia coli, and plant pathogenic bacteria Ralstonia solanacearum and Clavibacter michiganensis as well. According to the bioactivity results, crude ethyl acetate extract of T. koningiopsis VM115 isolate was determined for TLC and GC-MS analysis. An antifungal compound was isolated from ethyl acetate extract of T. koningiopsis VM115 based on bioassay guided fractionation. The 1H-NMR and 13C-NMR spectroscopic data showed that the compound was trichodermin, which exhibited strong fungicidal effects against P. oryzae, Aspergillus fumigatus, and Botrytis cinera with MICs of 31.25 µg ml-1 through in vitro antifungal tests. GC-MS analysis identified six classes of volatile compound produced by T. koningiopsis VM115 (alcohols, esters, pyrones (lactones), acids, furanes and lipids). 6-n-pentyl-6H-pyran-2-one (6PP) was identified as one of the most abundant metabolites in this research. These results indicate that the fungal endophytes from Vinca plants had antibacterial and cytotoxic activities; evidence that endophytes are a good source of biological activity and compounds. This work is the first report of Trichodermin production by T. koningiopsis species.

Tri11, tri3, and tri4 genes are required for trichodermin biosynthesis of Trichoderma brevicompactum.

Trichoderma brevicompactum and T. arundinaceum both can synthesize trichodermin with strong antifungal activity and high biotechnological value. The two Trichoderma species have a tri cluster, which includes seven genes (tri14, tri12, tri11, tri10, tri3, tri4, and tri6) that encode transport and regulatory enzymes required for the biosynthesis of trichodermin. Here, we isolated T. brevicompactum 0248 transformants with disrupted tri11, tri4, or tri3 gene. We also described the effect of tri11, tri3, or tri4 deletion on the expression of other genes in the tri cluster. Targeted Δtri3 knockout mutant exhibited a sharp decline in the production of trichodermin, and trichodermol, which is a substrate for trichodermin production, accumulated. Thus, the results demonstrated that tri3 was responsible for the biosynthesis of trichodermin, and the tri3 gene-encoded enzyme catalyzed the acetylation reaction of the hydroxy group at C-4 of the trichodermin skeleton. In addition, tri4 and tri11 deletion mutants were generated to evaluate the roles of tri4 and tri11 in trichodermin biosynthesis, respectively. Deletion mutant strain Δtri4 or Δtri11 did not produce trichodermin in T. brevicompactum, indicating that tri4 and tri11 are essential for trichodermin biosynthesis. This is the first to report the function of tri3, tri4 and tri11 in T. brevicompactum, although the role of tri4 and tri11 has already been described for T. arundinaceum by Cardoza et al. (Appl Environ Microbiol 77:4867-4877, 2011).

Species specific substrates and products choices of 4-O-acetyltransferase from Trichoderma brevicompactum.

Antagonistic species of Trichoderma such as T. harzianum, T. viride, T. virens and T. koningii are well-known biocontrol agents that have been reported to suppress pathogenic soil microbes and enhance the growth of crop plants. Secondary metabolites (SMs) including trichothecenes are responsible for its biocontrol activities. The trichothecenes, trichodermin and harzianum A (HA) are produced in species dependent manner respectively, by Trichoderma brevicompactum (TB) and Trichoderma arundinaceum (TA). The last step in the pathway involves the conversion of trichodermol into trichodermin or HA alternatively, which is catalyzed by 4-O-acetyltransferase (encoded by tri3 gene). Comparative sequence analysis of acetyltransferase enzyme of TB with other chloramphenicol acetyltransferase (CAT) family proteins revealed the conserved motif involved in the catalysis. Multiple substrate binding studies were carried out to explore the mechanism behind the two different outcomes. His188 was found to have a role in initial substrate binding. In the case of trichodermin synthesis, represented by ternary complex 1, the trichodermol and acetic anhydride (AAn), the two substrates come very close to each other during molecular simulation analysis so that interactions become possible between them and acetyl group may get transferred from AAn to trichodermol, and Tyr476 residue mediates this phenomenon resulting in the formation of trichodermin. However, in case of the HA biosynthesis using the TB version of enzyme, represented by ternary complex 2, the two substrates, trichodermol and octa-2Z,4E,6E-trienedioic acid (OCTA) did not show any such interactions.

Trichodermin induces c-Jun N-terminal kinase-dependent apoptosis caused by mitotic arrest and DNA damage in human p53-mutated pancreatic cancer cells and xenografts.

Pancreatic cancer is an aggressive malignancy, which generally responds poorly to chemotherapy. In this study, trichodermin, an endophytic fungal metabolite from Nalanthamala psidii, was identified as a potent and selective antitumor agent in human pancreatic cancer. Trichodermin exhibited antiproliferative effects against pancreatic cancer cells, especially p53-mutated cells (MIA PaCa-2 and BxPC-3) rather than normal pancreatic epithelial cells. We found that trichodermin induced caspase-dependent and mitochondrial intrinsic apoptosis. Trichodermin also increased apoptosis through mitotic arrest by activating Cdc2/cyclin B1 complex activity. Moreover, trichodermin promoted the activation of c-Jun N-terminal kinase (JNK), and inhibition of JNK by its inhibitor, shRNA, or siRNA significantly reversed trichodermin-mediated caspase-dependent apoptosis. Trichodermin triggered DNA damage stress to activate p53 function for executing apoptosis in p53-mutated cells. Importantly, we demonstrated that trichodermin with efficacy similar to gemcitabine, profoundly suppressed tumor growth through inducing intratumoral DNA damage and JNK activation in orthotopic pancreatic cancer model. Based on these findings, trichodermin is a potential therapeutic agent worthy of further development into a clinical trial candidate for treating cancer, especially the mutant p53 pancreatic cancer.

Deciphering the protein translation inhibition and coping mechanism of trichothecene toxin in resistant fungi.

In modern times for combating the deleterious soil microbes for improved sustainable agricultural practices, there is a need to have a proper understanding of the plant-microbe interactions present in the rhizospheric microbiome of the plant roots. In the present study, the interactions of trichodermin with petidyltransferase centre of ribosomal complex was studied by molecular dynamics and in silico interaction methods to demonstrate its mechanism of action and to decipher the possible reason how it may inhibit protein synthesis at the ribosomal complex. Further we have illustrated how trichodermin resistance protein (60S ribosomal protein L3) helps to overcome the deleterious effects of trichothecene compounds like trichodermin. Normal mode analysis of trichodermin resistance protein and 25S rRNA that constitutes the petidyltransferase centre showed that the W-finger region of the protein moved towards 25S rRNA. Further analysis of molecular dynamics simulation time frames showed that several intermediate states of large motions of the protein molecules towards the 25S rRNA which finally blocks the binding pocket of the trichodermin. It indicated that this protein not only changes the local environment and conformation of the petidyltransferase centre but also restrain trichodermin from binding to the 25S rRNA at the petidyltransferase centre.

Synthesis, antifungal activity, and QSAR study of novel trichodermin derivatives.

In an attempt to discover more potential antifungal agents, in this study, 21 novel trichodermin derivatives containing conjugated oxime ester (5a-5u) were designed and synthesized and were screened for in vitro antifungal activity. The bioassay tests showed that some of them exhibited good inhibitory activity against the tested pathogenic fungi. Compound 5a exhibited better activity against Pyricularia oryzae and Sclerotonia sclerotiorum than trichodermin, and compound 5j showed particular activity against P.oryzae and Botrytis cinerea. The quantitative structure-activity relationship (QSAR) indicated that log P and hardness were two critical parameters for the biological activities. The result suggested that these would be potential lead compounds for the development of fungicides with further structure modification.

Synthesis and biological evaluation of novel trichodermin derivatives as antifungal agents.

To discover more potential antifungal agents, 17 novel trichodermin derivatives were designed and synthesized by modification of 3 and 4a. The structures of all the synthesized compounds were confirmed by (1)H NMR, ESI-MS and HRMS. Their antifungal activities against Ustilaginoidea oryzae and Pyricularia oryzae were evaluated. Most of the target compounds showed potent inhibitory activity, in which 4g showed superior inhibitory effects than 4a and commercial fungicide prochloraz. Furthermore, 4h demonstrated comparable inhibitory activity to 4a. Moreover, 4i and 4l exhibited excellent inhibitory activity for Pyricularia oryzae. Additionally, compound 9 was found to be more active against all tested fungal strains than 3, with EC50 values of 0.47 and 3.71 mg L(-1), respectively.

Antifungal activity of metabolites of the endophytic fungus Trichoderma brevicompactum from garlic.

The endophytic fungus strain 0248, isolated from garlic, was identified as Trichoderma brevicompactum based on morphological characteristics and the nucleotide sequences of ITS1-5.8S- ITS2 and tef1. The bioactive compound T2 was isolated from the culture extracts of this fungus by bioactivity-guided fractionation and identified as 4ß-acetoxy-12,13- epoxy-Δ(9)-trichothecene (trichodermin) by spectral analysis and mass spectrometry. Trichodermin has a marked inhibitory activity on Rhizoctonia solani, with an EC50 of 0.25 µg mL(-1). Strong inhibition by trichodermin was also found for Botrytis cinerea, with an EC50 of 2.02 µg mL(-1). However, a relatively poor inhibitory effect was observed for trichodermin against Colletotrichum lindemuthianum (EC50 = 25.60 µg mL(-1)). Compared with the positive control Carbendazim, trichodermin showed a strong antifungal activity on the above phytopathogens. There is little known about endophytes from garlic. This paper studied in detail the identification of endophytic T. brevicompactum from garlic and the characterization of its active metabolite trichodermin.

Antifungal activity of metabolites of the endophytic fungus Trichoderma brevicompactum from garlic

The endophytic fungus strain 0248, isolated from garlic, was identified as Trichoderma brevicompactum based on morphological characteristics and the nucleotide sequences of ITS1-5.8SITS2 and tef1. The bioactive compound T2 was isolated from the culture extracts of this fungus by bioactivity-guided fractionation and identified as 4β-acetoxy-12,13-epoxy-Δ9-trichothecene (trichodermin) by spectral analysis and mass spectrometry. Trichodermin has a marked inhibitory activity on Rhizoctonia solani, with an EC50 of 0.25 µgmL-1. Strong inhibition by trichodermin was also found for Botrytis cinerea, with an EC50 of 2.02 µgmL-1. However, a relatively poor inhibitory effect was observed for trichodermin against Colletotrichum lindemuthianum (EC50 = 25.60 µgmL-1). Compared with the positive control Carbendazim, trichodermin showed a strong antifungal activity on the above phytopathogens. There is little known about endophytes from garlic. This paper studied in detail the identification of endophytic T. brevicompactum from garlic and the characterization of its active metabolite trichodermin.

Trichodermin induces cell apoptosis through mitochondrial dysfunction and endoplasmic reticulum stress in human chondrosarcoma cells.

Chondrosarcoma is the second most common primary bone tumor, and it responds poorly to both chemotherapy and radiation treatment. Nalanthamala psidii was described originally as Myxosporium in 1926. This is the first study to investigate the anti-tumor activity of trichodermin (trichothec-9-en-4-ol, 12,13-epoxy-, acetate), an endophytic fungal metabolite from N. psidii against human chondrosarcoma cells. We demonstrated that trichodermin induced cell apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353 cells) instead of primary chondrocytes. In addition, trichodermin triggered endoplasmic reticulum (ER) stress protein levels of IRE1, p-PERK, GRP78, and GRP94, which were characterized by changes in cytosolic calcium levels. Furthermore, trichodermin induced the upregulation of Bax and Bid, the downregulation of Bcl-2, and the dysfunction of mitochondria, which released cytochrome c and activated caspase-3 in human chondrosarcoma. In addition, animal experiments illustrated reduced tumor volume, which led to an increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and an increased level of cleaved PARP protein following trichodermin treatment. Together, this study demonstrates that trichodermin is a novel anti-tumor agent against human chondrosarcoma cells both in vitro and in vivo via mitochondrial dysfunction and ER stress.