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1.
Cells ; 11(19)2022 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-36231012

RESUMO

In the present study, Trichoderma virens TRS 106 decreased grey mould disease caused by Botrytis cinerea in tomato plants (S. lycopersicum L.) by enhancing their defense responses. Generally, plants belonging to the 'Remiz' variety, which were infected more effectively by B. cinerea than 'Perkoz' plants, generated more reactive molecules such as superoxide (O2-) and peroxynitrite (ONOO-), and less hydrogen peroxide (H2O2), S-nitrosothiols (SNO), and green leaf volatiles (GLV). Among the new findings, histochemical analyses revealed that B. cinerea infection caused nitric oxide (NO) accumulation in chloroplasts, which was not detected in plants treated with TRS 106, while treatment of plants with TRS 106 caused systemic spreading of H2O2 and NO accumulation in apoplast and nuclei. SPME-GCxGC TOF-MS analysis revealed 24 volatile organic compounds (VOC) released by tomato plants treated with TRS 106. Some of the hexanol derivatives, e.g., 4-ethyl-2-hexynal and 1,5-hexadien-3-ol, and salicylic acid derivatives, e.g., 4-hepten-2-yl and isoamyl salicylates, are considered in the protection of tomato plants against B. cinerea for the first time. The results are valuable for further studies aiming to further determine the location and function of NO in plants treated with Trichoderma and check the contribution of detected VOC in plant protection against B. cinerea.


Assuntos
Hypocrea , S-Nitrosotióis , Compostos Orgânicos Voláteis , Botrytis/fisiologia , Hexanóis/farmacologia , Peróxido de Hidrogênio/farmacologia , Óxido Nítrico , Nitrogênio , Oxigênio/farmacologia , Ácido Peroxinitroso , Doenças das Plantas , S-Nitrosotióis/farmacologia , Ácido Salicílico/farmacologia , Superóxidos
2.
Phytochemistry ; 203: 113368, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35977601

RESUMO

A document investigation on the fungus Trichoderma virens led to the isolation of thirteen undescribed carotane sesquiterpenes and homologous. All structures were elucidated on the basis of NMR and HRESIMS data, and their absolute configurations were assigned by ECD calculation. Especially, virenscarotins A and B were first ramifications forged by aldol condensation of 4-hydroxy-3-isopentenyl-benzaldehyde with two hydroxyl groups in ring A of traditional carotane sesquiterpenes. Ring rearrangement/expansion and oxidative cleavage of normal carotane sesquiterpenes lead to the six-membered ring A of compound virenscarotin C and the ring A cleavage of compound virenscarotin D. All compounds were evaluated for cytotoxic, anti-inflammatory, and seed germination inhibitory activities.


Assuntos
Hypocrea , Sesquiterpenos , Trichoderma , Benzaldeídos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Sesquiterpenos/química , Trichoderma/química
3.
mBio ; 13(4): e0038922, 2022 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-35862794

RESUMO

Beneficial interaction of members of the fungal genus Trichoderma with plant roots primes the plant immune system, promoting systemic resistance to pathogen infection. Some strains of Trichoderma virens produce gliotoxin, a fungal epidithiodioxopiperazine (ETP)-type secondary metabolite that is toxic to animal cells. It induces apoptosis, prevents NF-κB activation via the inhibition of the proteasome, and has immunosuppressive properties. Gliotoxin is known to be involved in the antagonism of rhizosphere microorganisms. To investigate whether this metabolite has a role in the interaction of Trichoderma with plant roots, we compared gliotoxin-producing and nonproducing T. virens strains. Both colonize the root surface and outer layers, but they have differential effects on root growth and architecture. The responses of tomato plants to a pathogen challenge were followed at several levels: lesion development, levels of ethylene, and reactive oxygen species. The transcriptomic signature of the shoot tissue in response to root interaction with producing and nonproducing T. virens strains was monitored. Gliotoxin producers provided stronger protection against foliar pathogens, compared to nonproducing strains. This was reflected in the transcriptomic signature, which showed the induction of defense-related genes. Two markers of plant defense response, PR1 and Pti-5, were differentially induced in response to pure gliotoxin. Gliotoxin thus acts as a microbial signal, which the plant immune system recognizes, directly or indirectly, to promote a defense response. IMPORTANCE A single fungal metabolite induces far-reaching transcriptomic reprogramming in the plant, priming immune responses and defense, in contrast to its immunosuppressive effect on animal cells. While the negative effects of gliotoxin-producing Trichoderma strains on growth may be observed only under a particular set of laboratory conditions, gliotoxin-linked molecular patterns, including the potential for limited cell death, could strongly prime plant defense, even in mature soil-grown plants in which the same Trichoderma strain promotes growth.


Assuntos
Gliotoxina , Hypocrea , Trichoderma , Animais , Hypocrea/metabolismo , Doenças das Plantas/microbiologia , Imunidade Vegetal , Raízes de Plantas/microbiologia , Trichoderma/genética , Trichoderma/metabolismo
4.
Acta Crystallogr F Struct Biol Commun ; 78(Pt 6): 226-231, 2022 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-35647679

RESUMO

The enzymatic degradation of semi-cellulosic substrates has recently received immense attention. The enzyme endo-1,4-ß-xylanase is essential for the complete digestion of complex and heterogeneous hemicellulose. Here, the purification, crystallization and preliminary X-ray free-electron laser (XFEL) diffraction analysis of endo-1,4-ß-xylanase from the fungus Hypocrea virens (HviGH11) are reported. Codon-optimized HviGH11 was overexpressed in Escherichia coli and spontaneously crystallized after His-tag purification and concentration. Preliminary XFEL diffraction data were collected at the Pohang Accelerator Laboratory XFEL (PAL-XFEL). A total of 1021 images containing Bragg peaks were obtained and indexed. The HviGH11 crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 43.80, b = 51.90, c = 94.90 Å. Using 956 diffraction patterns, the phasing problem was solved and an initial model structure of HviGH11 was obtained.


Assuntos
Endo-1,4-beta-Xilanases , Hypocrea , Cristalização/métodos , Cristalografia por Raios X , Endo-1,4-beta-Xilanases/química , Escherichia coli/genética , Escherichia coli/metabolismo , Hypocrea/genética , Lasers
5.
Folia Microbiol (Praha) ; 67(2): 253-264, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34743285

RESUMO

Production of amylases by fungi under solid-state fermentation is considered the best methodology for commercial scaling that addresses the ever-escalating needs of the worldwide enzyme market. Here response surface methodology (RSM) was used for the optimization of process variables for α-amylase enzyme production from Trichoderma virens using watermelon rinds (WMR) under solid-state fermentation (SSF). The statistical model included four variables, each detected at two levels, followed by model development with partial purification and characterization of α-amylase. The partially purified α-amylase was characterized with regard to optimum pH, temperature, kinetic constant, and substrate specificity. The results indicated that both pH and moisture content had a significant effect (P < 0.05) on α-amylase production (880 U/g) under optimized process conditions at a 3-day incubation time, moisture content of 50%, 30 °C, and pH 6.98. Statistical optimization using RSM showed R2 values of 0.9934, demonstrating the validity of the model. Five α-amylases were separated by using DEAE-Sepharose and characterized with a wide range of optimized pH values (pH 4.5-9.0), temperature optima (40-60 °C), low Km values (2.27-3.3 mg/mL), and high substrate specificity toward large substrates. In conclusion, this study presents an efficient and green approach for utilization of agro-waste for production of the valuable α-amylase enzyme using RSM under SSF. RSM was particularly beneficial for the optimization and analysis of the effective process parameters.


Assuntos
Citrullus , Hypocrea , Amilases , Citrullus/metabolismo , Fermentação , Concentração de Íons de Hidrogênio , Hypocrea/metabolismo , Microbiologia Industrial/métodos , Temperatura , alfa-Amilases/química , alfa-Amilases/metabolismo
6.
Biochemistry ; 60(26): 2116-2129, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34156827

RESUMO

Viperin is a member of the radical S-adenosylmethionine superfamily and has been shown to restrict the replication of a wide range of RNA and DNA viruses. We recently demonstrated that human viperin (HsVip) catalyzes the conversion of CTP to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP or ddh-synthase), which acts as a chain terminator for virally encoded RNA-dependent RNA polymerases from several flaviviruses. Viperin homologues also exist in non-chordate eukaryotes (e.g., Cnidaria and Mollusca), numerous fungi, and members of the archaeal and eubacterial domains. Recently, it was reported that non-chordate and non-eukaryotic viperin-like homologues are also ddh-synthases and generate a diverse range of ddhNTPs, including the newly discovered ddhUTP and ddhGTP. Herein, we expand on the catalytic mechanism of mammalian, fungal, bacterial, and archaeal viperin-like enzymes with a combination of X-ray crystallography and enzymology. We demonstrate that, like mammalian viperins, these recently discovered viperin-like enzymes operate through the same mechanism and can be classified as ddh-synthases. Furthermore, we define the unique chemical and physical determinants supporting ddh-synthase activity and nucleotide selectivity, including the crystallographic characterization of a fungal viperin-like enzyme that utilizes UTP as a substrate and a cnidaria viperin-like enzyme that utilizes CTP as a substrate. Together, these results support the evolutionary conservation of the ddh-synthase activity and its broad phylogenetic role in innate antiviral immunity.


Assuntos
Proteínas Arqueais/química , Proteínas de Bactérias/química , Proteínas Fúngicas/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Sequência de Aminoácidos , Animais , Proteínas Arqueais/metabolismo , Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Biocatálise , Proteínas Fúngicas/metabolismo , Humanos , Hypocrea/enzimologia , Methanomicrobiaceae/enzimologia , Camundongos , Nucleotídeos/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Ligação Proteica , Especificidade por Substrato
7.
Bioorg Chem ; 112: 104959, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33971564

RESUMO

Biotransformation of viridin, an antifungal produced by biocontrol agent, with non-viridin producing microorganisms is studied. The results show that some environmental non-targeted microorganisms are able to reduce it in the known phytotoxin viridiol, and its 3-epimer. Consequently, this reduction, which happens in some cases by detoxification mechanism, could be disastrous for the plant in a biocontrol of plant disease. However, a process fermentation/biotransformation could be an efficient approach for the preparation of this phytotoxin.


Assuntos
Androstenodióis/farmacologia , Androstenos/farmacologia , Antifúngicos/farmacologia , Bacteriocinas/farmacologia , Hypocrea/efeitos dos fármacos , Androstenodióis/química , Androstenodióis/metabolismo , Androstenos/química , Androstenos/metabolismo , Antifúngicos/química , Antifúngicos/metabolismo , Bacteriocinas/química , Bacteriocinas/metabolismo , Biotransformação , Relação Dose-Resposta a Droga , Fermentação/efeitos dos fármacos , Hypocrea/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade
8.
Appl Microbiol Biotechnol ; 105(7): 2867-2875, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33738552

RESUMO

Fungi cause diverse, serious socio-economic problems, including biodeterioration of valuable products and materials that spawns a biocides industry worth ~$11 billion globally. To help combat environmental fungi that commonly colonise material products, this study tested the hypothesis that combination of an approved fungicide with diverse agents approved by the FDA (Food and Drug Administration) could reveal potent combinatorial activities with promise for fungicidal applications. The strategy to use approved compounds lowers potential development risks for any effective combinations. A high-throughput assay of 1280 FDA-approved compounds was conducted to find those that potentiate the effect of iodopropynyl-butyl-carbamate (IPBC) on the growth of Trichoderma virens; IPBC is one of the two most widely used Biocidal Products Regulations-approved fungicides. From this library, 34 compounds in combination with IPBC strongly inhibited fungal growth. Low-cost compounds that gave the most effective growth inhibition were tested against other environmental fungi that are standard biomarkers for resistance of synthetic materials to fungal colonisation. Trifluoperazine (TFZ) in combination with IPBC enhanced growth inhibition of three of the five test fungi. The antifungal hexetidine (HEX) potentiated IPBC action against two of the test organisms. Testable hypotheses on the mechanisms of these combinatorial actions are discussed. Neither IPBC + TFZ nor IPBC + HEX exhibited a combinatorial effect against mammalian cells. These combinations retained strong fungal growth inhibition properties after incorporation to a polymer matrix (alginate) with potential for fungicide delivery. The study reveals the potential of such approved compounds for novel combinatorial applications in the control of fungal environmental opportunists. KEY POINTS: • Search with an approved fungicide to find new fungicidal synergies in drug libraries. • New combinations inhibit growth of key environmental fungi on different matrices. • The approach enables a more rapid response to demand for new biocides.


Assuntos
Desinfetantes , Fungicidas Industriais , Hypocrea , Trichoderma , Animais , Antifúngicos/farmacologia , Desinfetantes/farmacologia , Fungos , Fungicidas Industriais/farmacologia
9.
J Nat Prod ; 84(2): 503-517, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33565879

RESUMO

Malaria remains a worldwide threat, afflicting over 200 million people each year. The emergence of drug resistance against existing therapeutics threatens to destabilize global efforts aimed at controlling Plasmodium spp. parasites, which is expected to leave vast portions of humanity unprotected against the disease. To address this need, systematic testing of a fungal natural product extract library assembled through the University of Oklahoma Citizen Science Soil Collection Program has generated an initial set of bioactive extracts that exhibit potent antiplasmodial activity (EC50 < 0.30 µg/mL) and low levels of toxicity against human cells (less than 50% reduction in HepG2 growth at 25 µg/mL). Analysis of the two top-performing extracts from Trichoderma sp. and Hypocrea sp. isolates revealed both contained chemically diverse assemblages of putative peptaibol-like compounds that were responsible for their antiplasmodial actions. Purification and structure determination efforts yielded 30 new peptaibols and lipopeptaibols (1-14 and 28-43), along with 22 known metabolites (15-27 and 44-52). While several compounds displayed promising activity profiles, one of the new metabolites, harzianin NPDG I (14), stood out from the others due to its noteworthy potency (EC50 = 0.10 µM against multi-drug-resistant P. falciparum line Dd2) and absence of gross toxicity toward HepG2 at the highest concentrations tested (HepG2 EC50 > 25 µM, selectivity index > 250). The unique chemodiversity afforded by these fungal isolates serves to unlock new opportunities for translating peptaibols into a bioactive scaffold worthy of further development.


Assuntos
Antimaláricos/farmacologia , Hypocrea/química , Peptaibols/biossíntese , Trichoderma/química , Produtos Biológicos/farmacologia , Resistência a Medicamentos , Células Hep G2 , Humanos , Estrutura Molecular , Pennsylvania , Peptaibols/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Microbiologia do Solo , Texas
10.
Phytopathology ; 111(10): 1720-1725, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33620234

RESUMO

Sclerotium rolfsii causes destructive soilborne disease in numerous plant species, and biological control may be a promising and sustainable approach for suppressing this widespread pathogen. In this study, the antagonistic effect against S. rolfsii of 10 Trichoderma strains was tested by the dual culture method, and a gliotoxin-producing strain, T. virens T23, was shown to be the most effective, inhibiting growth of S. rolfsii in vitro by 70.2%. To clarify the antagonistic mechanism and gliotoxin biosynthesis regulation of T23, a gliotoxin-deficient mutant was constructed via Agrobacterium tumefaciens-mediated gene knockout in vivo. As expected, disruption of the gene located in the putative gliotoxin biosynthesis gene cluster, gliI-T, resulted in gliotoxin deficiency and attenuation of the antagonistic effect against S. rolfsii, indicating that gliotoxin biosynthesis is regulated by gliI-T and that gliotoxin is an important antifungal metabolite of T23. Transmission electron microscopy revealed that gliotoxin treatment caused marked alterations of the hyphal cells of S. rolfsii depending on the drug concentration, whereby one of the prominent structural alterations was a reduction in the number and length of mitochondrial cristae. When S. rolfsii was exposed to 30 µg/ml of gliotoxin for 12 h, striking plasmolysis and ultrastructural changes were induced. The results demonstrated that gliotoxin is an important secondary metabolite of T. virens T23 in its antagonism against S. rolfsii.


Assuntos
Gliotoxina , Hypocrea , Trichoderma , Basidiomycota , Doenças das Plantas
11.
Microbiol Res ; 253: 126862, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34563853

RESUMO

Trichoderma virens produces viridin/viridiol, heptelidic (koningic) acid, several volatile sesquiterpenes and gliotoxin (Q strains) or gliovirin (P strains). We earlier reported that deletion of the terpene cyclase vir4 and a glyceraldehyde-3-phosphate dehydrogenase (GAPDH, designated as vGPD) associated with the "vir" cluster abrogated the biosynthesis of several volatile sesquiterpene metabolites. Here we show that, the deletion of this GAPDH also impairs the biosynthesis of heptelidic acid (a non-volatile sesquiterpene), viridin (steroid) and gliovirin (non-ribosomal peptide), indicating regulation of non-volatile metabolite biosynthesis by this GAPDH that is associated with a secondary metabolism gene cluster. To gain further insights into the details of this novel form of regulation, we identified the terpene cyclase gene responsible for heptelidic acid biosynthesis (hereafter designated as has1) and prove that the expression of this gene is regulated by vGPD. Interestingly, deletion of has1 impaired biosynthesis of heptelidic acid (HA), viridin and gliovirin, but not of volatile sesquiterpenes. Deletion of the vir cluster associated terpene cyclase gene (vir4), located next to the vGPD gene, did not impair biosynthesis of HA, viridin or gliovirin. We thus unveil a novel circuitry of regulation of secondary metabolism where an HA-tolerant GAPDH isoform (vGPD) regulates HA biosynthesis through the transcriptional regulation of the HA-synthase gene (which is not part of the "vir" cluster). Interestingly, impairment of HA biosynthesis leads to the down-regulation of biosynthesis of other non-volatile secondary metabolites, but not of volatile secondary metabolites. We thus provide evidence that the "vir" cluster associated, HA-tolerant GAPDH in T. virens participates in the biosynthesis of volatile sesquiterpenes as a biosynthetic enzyme, and regulates the production of non-volatile metabolites via regulation of HA biosynthesis. The orthologue of the "vir" cluster in Aspergillus oryzae was earlier reported to synthesize HA by another group. Our study thus proves that the same gene cluster can code for unrelated metabolites in different species.


Assuntos
Gliceraldeído-3-Fosfato Desidrogenases , Hypocrea , Metabolismo Secundário , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Hypocrea/enzimologia , Sesquiterpenos/metabolismo
12.
Int J Biol Macromol ; 191: 267-276, 2021 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-34547313

RESUMO

Trichoderma virens colonizes roots and develops a symbiotic relationship with plants where the fungal partner derives nutrients from plants and offers defence, in return. Tsp1, a small secreted cysteine-rich protein, was earlier found to be upregulated in co-cultivation of T. virens with maize roots. Tsp1 is well conserved in Ascomycota division of fungi, but none of its homologs have been studied yet. We have expressed and purified recombinant Tsp1, and resolved its structure to 1.25 Å resolutions, from two crystal forms, using Se-SAD methods. The Tsp1 adopts a ß barrel fold and forms dimer in structure as well as in solution form. DALI based structure analysis revealed the structure similarity with two known fungal effector proteins: Alt a1 and PevD1. Structure and evolutionary analysis suggested that Tsp1 belongs to a novel effector protein family. Tsp1 acted as an inducer of salicylic acid mediated susceptibility in plants, rendering maize plants more susceptible to a necrotrophic pathogen Cochliobolus heterostrophus, as observed using plant defence assay and RT-qPCR analysis.


Assuntos
Proteínas Fúngicas/química , Interações Hospedeiro-Patógeno , Hypocrea/metabolismo , Evolução Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hypocrea/patogenicidade , Simulação de Dinâmica Molecular , Domínios Proteicos , Homologia de Sequência de Aminoácidos , Zea mays/microbiologia
13.
Mol Plant Microbe Interact ; 34(5): 524-537, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33166203

RESUMO

The interactions of crops with root-colonizing endophytic microorganisms are highly relevant to agriculture, because endophytes can modify plant resistance to pests and increase crop yields. We investigated the interactions between the host plant Zea mays and the endophytic fungus Trichoderma virens at 5 days postinoculation grown in a hydroponic system. Wild-type T. virens and two knockout mutants, with deletion of the genes tv2og1 or vir4 involved in specialized metabolism, were analyzed. Root colonization by the fungal mutants was lower than that by the wild type. All fungal genotypes suppressed root biomass. Metabolic fingerprinting of roots, mycelia, and fungal culture supernatants was performed using ultrahigh performance liquid chromatography coupled to diode array detection and quadrupole time-of-flight tandem mass spectrometry. The metabolic composition of T. virens-colonized roots differed profoundly from that of noncolonized roots, with the effects depending on the fungal genotype. In particular, the concentrations of several metabolites derived from the shikimate pathway, including an amino acid and several flavonoids, were modulated. The expression levels of some genes coding for enzymes involved in these pathways were affected if roots were colonized by the ∆vir4 genotype of T. virens. Furthermore, mycelia and fungal culture supernatants of the different T. virens genotypes showed distinct metabolomes. Our study highlights the fact that colonization by endophytic T. virens leads to far-reaching metabolic changes, partly related to two fungal genes. Both metabolites produced by the fungus and plant metabolites modulated by the interaction probably contribute to these metabolic patterns. The metabolic changes in plant tissues may be interlinked with systemic endophyte effects often observed in later plant developmental stages.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.


Assuntos
Hypocrea , Trichoderma , Endófitos , Raízes de Plantas , Zea mays
14.
Can J Microbiol ; 67(5): 406-414, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33226848

RESUMO

Fungal protoplast fusion is an approach to introduce novel characteristics into industrially important strains. Cellulases, essential enzymes with a wide range of biotechnological applications, are produced by many species of the filamentous fungi Trichoderma. In this study, a collection of 60 natural isolates were screened for Avicel and carboxymethyl cellulose degradation, and two cellulase producers of Trichoderma virens and Trichoderma harzianum were used for protoplast fusion. One of the resulting hybrids with improved cellulase activity, C1-3, was fused with the hyperproducer Trichoderma reesei Rut-C30. A new selected hybrid, F7, was increased in cellulase activity 1.8 and 5 times in comparison with Rut-C30 and C1-3, respectively. The increases in enzyme activity correlated with an upregulation of the cellulolytic genes cbh1, cbh2, egl3, and bgl1 in the parents. The amount of mRNA of cbh1 and cbh2 in F7 resembled that of Rut-C30 while the bgl1 mRNA level was similar to that of C1-3. AFLP (amplified fragment length polymorphism) fingerprinting and GC-MS (gas chromatography - mass spectrometry) analysis represented variations in parental strains and fusants. In conclusion, the results demonstrate that a 3-interspecific hybrid strain was isolated, with improved characteristics for cellulase degradation and showing genetic polymorphisms and differences in the volatile profile, suggesting reorganizations at the genetic level.


Assuntos
Celulase/biossíntese , Hypocreales/enzimologia , Protoplastos/metabolismo , Trichoderma/enzimologia , Trichoderma/genética , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Celulose/metabolismo , Regulação Fúngica da Expressão Gênica , Hypocrea/enzimologia , Hypocrea/genética , Hypocreales/genética , Microbiologia Industrial , Polimorfismo Genético , RNA Fúngico/genética , RNA Mensageiro/genética
15.
Rev Bras Parasitol Vet ; 29(4): e006120, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33084781

RESUMO

The study evaluated the ovicidal activity of enzymatic extracts of Purpureocillium lilacinum and Trichoderma virens against trichostrongylid eggs from sheep. Filtered extract (FE) and macerated crude extract (MCE) were prepared from fungal cultures in minimal broth. In the experiment, 100 trichostrongylid eggs, obtained from the feces of naturally infected sheep, were exposed to fungal extracts for 24 and 48 hours/25°C. In the control group, eggs were incubated in minimal broth. The number of L1 larvae was ascertained. Each treatment consisted of four repetitions and the experiment was repeated five times. It was observed that the effect of FE and MCE of P. lilacinum and T. virens on egg hatchability differed from that of the control group. MCE of T. virens and P. lilacinum showed higher ovicidal activity than FE over both periods and at 48 hours of exposure, respectively. From the percentage reductions in hatchability of the eggs, MCE was shown to be superior to FE for both fungi. This study demonstrated the ovicidal potential of these fungi against trichostrongylid eggs. However, further studies are needed in order to identify the molecules responsible for the ovicidal effects, and to evaluate the behavior of fungal extracts in biotic and abiotic interactions.


Assuntos
Agentes de Controle Biológico , Hypocrea , Hypocreales , Doenças dos Ovinos/prevenção & controle , Ovinos/parasitologia , Tricostrongiloidíase/veterinária , Animais , Larva , Óvulo , Doenças dos Ovinos/parasitologia , Trichostrongyloidea , Tricostrongiloidíase/prevenção & controle
16.
Fitoterapia ; 146: 104715, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32861754

RESUMO

Eight cadinane derivatives, trichocadinins H - N (1-7) and methylhydroheptelidate (8), and two carotane derivatives, 14-O-methyltrichocarotin G (9) and 14-O-methyl CAF-603 (10), including eight new ones (1-6, 9, and 10), were isolated from the culture of Trichoderma virens RR-dl-6-8 obtained from the organohalogen-enriched marine red alga Rhodomela confervoides. Their structures and relative configurations were established by analysis of NMR and mass spectroscopic data, and the absolute configurations were assigned on the basis of ECD curves, highlighted by the ECD diversity of carboxylic acid derivatives. Among the isolates, 1 with a halogen atom and 8, a new naturally occurring compound, are 2,3-seco-cadinane sesquiterpenes, and the epimeric 2 and 3 feature a 2-nor-cadinane skeleton. A commercially-sourced compound with the same planar structure as that of 7 has been reported in a patent, but its configuration was not given. Compounds 1-10 exhibited growth inhibition of some marine phytoplankton species.


Assuntos
Hypocrea/química , Fitoplâncton/efeitos dos fármacos , Sesquiterpenos Policíclicos/farmacologia , Rodófitas/microbiologia , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , China , Testes de Sensibilidade Microbiana , Estrutura Molecular , Sesquiterpenos Policíclicos/isolamento & purificação , Vibrio/efeitos dos fármacos
17.
Protein Expr Purif ; 175: 105697, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32681951

RESUMO

Trichoderma virens genome harbors two isoforms of GAPDH, one (gGPD) involved in glycolysis and the other one (vGPD) in secondary metabolism. vGPD is expressed as part of the "vir" cluster responsible for the biosynthesis of volatile sesquiterpenes. The secondary metabolism-associated GAPDH is tolerant to the anti-cancer metabolite heptelidic acid (HA), produced by T. virens. Characterizing the HA-tolerant form of GAPDH, thus has implications in cancer therapy. In order to get insight into the mechanism of HA-tolerance of vGPD, we have purified recombinant form of this protein. The protein displays biochemical and biophysical characteristics analogous to the gGPD isoform. It exists as a tetramer with Tm of about 56.5 °C, and displays phosphorylation enzyme activity with Km and Kcat of 0.38 mM and 2.55 sec-1, respectively. The protein weakly binds to the sequence upstream of the vir4 gene that codes for the core enzyme (a terpene cyclase) of the "vir" cluster. The EMSA analysis indicates that vGPD may not act as a transcription factor driving the "vir" cluster, at least not by directly binding to the promoter region. We also succeeded in obtaining small crystals of this protein. We have constructed structural models of vGPD and gGPD of T. virens. In silico constrained docking analysis reveals weaker binding of heptelidic acid in vGPD, compared to gGPD protein.


Assuntos
Proteínas Fúngicas , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora) , Hypocrea/genética , Simulação de Acoplamento Molecular , Estabilidade Enzimática , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/biossíntese , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/química , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/isolamento & purificação , Hypocrea/enzimologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Sesquiterpenos/química
18.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 6): 257-262, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32510466

RESUMO

Small secreted cysteine-rich proteins (SSCPs) from fungi play an important role in fungi-host interactions. The plant-beneficial fungi Trichoderma spp. are in use worldwide as biocontrol agents and protect the host plant from soil-borne as well as foliar pathogens. Recently, a novel SSCP, Tsp1, has been identified in the secreted protein pool of T. virens and is overinduced upon its interaction with the roots of the maize plant. The protein was observed to be well conserved in the Ascomycota division of fungi, and its homologs are present in many plant-pathogenic fungi such as Fusarium oxysporum and Magnaporthe oryzae. However, none of these homologs have yet been characterized. Recombinant Tsp1 protein has been expressed and purified using an Escherichia coli expression system. The protein, with four conserved cysteines, forms a dimer in solution as observed by size-exclusion chromatography. The dimerization, however, does not involve disulfide bonds. Circular-dichroism data suggested that the protein has a ß-strand-rich secondary structure that matched well with the secondary structure predicted using bioinformatics methods. The protein was crystallized using sodium malonate as a precipitant. The crystals diffracted X-rays to 1.7 Šresolution and belonged to the orthorhombic space group P212121 (Rmeas = 5.4%), with unit-cell parameters a = 46.3, b = 67.0, c = 173.2 Å. The Matthews coefficient (VM) of the crystal is 2.32 Å3 Da-1, which corresponds to nearly 47% solvent content with four subunits of Tsp1 protein in the asymmetric unit. This is the first report of the structural study of any homolog of the novel Tsp1 protein. These structural studies will help in understanding the classification and function of the protein.


Assuntos
Cisteína/química , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Hypocrea/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Cisteína/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência
19.
Salud Publica Mex ; 62(4): 410-416, 2020.
Artigo em Espanhol | MEDLINE | ID: mdl-32515915

RESUMO

OBJECTIVE: To evaluate the effect of the combination of Metarhizium anisopliae and Gliocladium virens, both with Aqua Reslin Super, on the oviposition, hatching and emergence of Aedes aegypti. MATERIALS AND METHODS: Evaluations were carried out to determine the effect of treatments impregnated on filter paper and exposed within plastic containers on the oviposition, hatching and emergency of Aedes aegypti. RESULTS: The results indicated that the fungus and insecticide combinations did not affect the oviposition behavior, but if the hatching of the eggs and the adult's emergency. CONCLUSIONS: With the results it can be concluded that the combination of fungi + insecticide can be a good option to be applied in oviposition sites with a view to the development of a lethal ovitrap.


OBJETIVO: Evaluar el efecto de la combinación de Metarhizium anisopliae y Gliocladium virens, ambos con Aqua Reslin Super, sobre oviposición, eclosión y emergencia de Aedes aegypti. MATERIAL Y MÉTODOS: Se realizaron evaluaciones para determinar el efecto de los tratamientos impregnados en papel filtro y expuestos dentro de recipientes de plástico sobre la oviposición, eclosión y emergencia de Aedes aegypti. RESULTADOS: Los resultados indicaron que las combinaciones hongo e insecticida no afectaron el comportamiento de oviposición, pero sí la eclosión de los huevos y la emergencia del adulto. CONCLUSIONES: Con los resultados se puede concluir que la combinación de hongos + insecticida puede ser una buena opción para aplicarse en sitios de oviposición con miras al desarrollo de una ovitrampa letal.


Assuntos
Aedes/anatomia & histologia , Hypocrea , Inseticidas , Metarhizium , Oviposição , Butóxido de Piperonila , Piretrinas , Animais , Feminino , Hypocrea/efeitos dos fármacos , Hypocrea/crescimento & desenvolvimento , Metarhizium/efeitos dos fármacos , Metarhizium/crescimento & desenvolvimento , Controle de Mosquitos/métodos , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/crescimento & desenvolvimento
20.
J Biosci Bioeng ; 129(4): 508-513, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31837993

RESUMO

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.


Assuntos
Antioxidantes/isolamento & purificação , Antioxidantes/metabolismo , Trichoderma/metabolismo , Antioxidantes/farmacologia , Relação Dose-Resposta a Droga , Sequestradores de Radicais Livres/isolamento & purificação , Sequestradores de Radicais Livres/metabolismo , Sequestradores de Radicais Livres/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Hypocrea/classificação , Hypocrea/metabolismo , Espectrometria de Massas , Técnicas Microbiológicas , Trichoderma/crescimento & desenvolvimento
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