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1.
Molecules ; 26(18)2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34577165

RESUMO

Endophytic fungal isolates Hypocrea lixii F3ST1 and Beauveria bassiana G1LU3 were evaluated for their potential to endophytically colonize and induce active compounds in Phaseolus vulgaris, as a defense mechanism against pea leafminer (Liriomyza huidobrensis) and fall armyworm (Spodoptera frugiperda). Endophytic colonization was achieved through seed inoculation with the volatile emissions from P. vulgaris plants being analyzed using GC-MS. The crude extracts of P. vulgaris obtained using methanol and dichloromethane were assayed against leafminer and fall armyworm larvae using leaf dipping and topical application, respectively. The two isolates successfully colonized the entire host plant (roots, stems, and leaves) with significant variation (p < 0.001) between fungal isolates and the controls. The results showed qualitative differences in the volatile profiles between the control plants, endophytically colonized and insect-damaged plants attributed to fungal inoculation and leafminer damage. The crude methanol extracts significantly reduced the percentage pupation of 2nd instar leafminer larvae (p < 0.001) and adult-flies emergence (p < 0.05). The survival of the 1st instar fall armyworm larvae was also significantly reduced (p < 0.001) compared to the controls. This study demonstrated the high potential of endophytic fungi H. lixii and B. bassiana in inducing mainly specific defense compounds in the common bean P. vulgaris that can be used against pea leafminer and fall armyworm.


Assuntos
Beauveria/metabolismo , Agentes de Controle Biológico/farmacologia , Dípteros/efeitos dos fármacos , Hypocreales/metabolismo , Phaseolus/metabolismo , Extratos Vegetais/farmacologia , Spodoptera/efeitos dos fármacos , Animais , Dípteros/crescimento & desenvolvimento , Endófitos/metabolismo , Larva/efeitos dos fármacos , Metanol/química , Cloreto de Metileno/química , Controle Biológico de Vetores/métodos , Phaseolus/microbiologia , Doenças das Plantas/prevenção & controle , Pupa/efeitos dos fármacos , Spodoptera/crescimento & desenvolvimento , Compostos Orgânicos Voláteis/análise , Compostos Orgânicos Voláteis/química
2.
Sci Rep ; 11(1): 14872, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34290261

RESUMO

Date pits are nutritious by-products, containing high levels of indigestible carbohydrates and polyphenols. To maximize the biological effects of the active ingredients, the hard shell of the polysaccharide must be degraded. Therefore, the current study aimed to assess the protective potentials of date pits extract (DP) and fungal degraded date pits extract (FDDP) against scopolamine (SCO)-induced neurodegeneration in male rats. Date pits were subjected to fungal degradation and extraction, followed by the measurement of phytochemicals and free radical scavenging activities. Forty-two adult Sprague-Dawley male rats were divided into seven groups: three control groups administered with either saline, DP or FDDP; four groups with neurodegeneration receiving SCO (ip 2 mg/kg/day, SCO group) with no treatment, SCO with DP (oral 100 mg/kg/day, DP + SCO group), SCO with FDDP (oral, 100 mg/kg/day, FDDP + SCO group), and SCO with donepezil (DON, oral, 2.25 mg/kg/day, DON + SCO group). The treatment duration was 28 days, and in the last 14 days, SCO was administered daily. Morris water maze test, acetylcholine esterase activity, oxidative stress, markers of inflammation and amyloidogenesis, and brain histopathology were assessed.


Assuntos
Hypocreales/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/etiologia , Síndromes Neurotóxicas/tratamento farmacológico , Síndromes Neurotóxicas/etiologia , Phoeniceae/química , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Escopolamina/antagonistas & inibidores , Escopolamina/toxicidade , Sementes/química , Animais , Sinergismo Farmacológico , Sequestradores de Radicais Livres , Masculino , Compostos Fitoquímicos/análise , Extratos Vegetais/administração & dosagem , Extratos Vegetais/metabolismo , Ratos Sprague-Dawley
3.
Microb Cell Fact ; 20(1): 136, 2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34281536

RESUMO

BACKGROUND: Trichoderma reesei is currently the main strain for the commercial production of cellulase. Cellulose induced protein 1 (Cip1) is one of the most abundant proteins in extracellular proteins of T. reesei. Reported literatures about Cip1 mainly focused on the regulation of Cip1 and its possible enzyme activities, but the effect of Cip1 on the enzymatic hydrolysis of lignocellulose and possible mechanism have not still been reported. RESULTS: In this study, Cip1 from T. reesei was cloned, expressed and purified, and its effects on enzymatic hydrolysis of several different pretreated lignocellulose were investigated. It was found that Cip1 could promote the enzymatic hydrolysis of pretreated lignocellulose, and the promoting effect was significantly better than that of bovine serum albumin (BSA). And especially for the lignocellulosic substrate with high lignin content such as liquid hot water pretreated corn stover and corncob residue, the promoting effect of Cip1 was even better than that of the commercial cellulase when adding equal amount protein. It was also showed that the metal ions Zn2+ and Cu2+ influenced the promoting effect on enzymatic hydrolysis. The Cip1 protein had no lyase activity, but it could destroy the crystal structure of cellulose and reduce the non-productive adsorption of cellulase on lignin, which partly interpreted the promoting effect of Cip1 on enzymatic hydrolysis of lignocellulose. CONCLUSION: The Cip1 from T. reesei could significantly promote the enzymatic hydrolysis of pretreated lignocellulose, and the promotion of Cip1 was even higher than that of commercial cellulase in the enzymatic hydrolysis of the substrates with high lignin content. This study will help us to better optimize cellulase to improve its ability to degrade lignocellulose, thereby reducing the cost of enzymes required for enzymatic hydrolysis.


Assuntos
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hypocreales/química , Hypocreales/metabolismo , Lignina/metabolismo , Celulase/metabolismo , Clonagem Molecular , Proteínas Fúngicas/isolamento & purificação , Hidrólise , Hypocreales/genética
4.
PLoS One ; 16(6): e0251556, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34086701

RESUMO

A diverse range of monocot and dicot grains and their by-products are commonly used in the animal feed industry. They all come with complex and variable cell wall structures which in turn contribute significant fiber to the complete feed. The cell wall is a highly interconnected matrix of various polysaccharides, proteins and lignin and, as such, requires a collaborative effort of different enzymes for its degradation. In this regard, we investigated the potential of a commercial multicomponent carbohydrase product from a wild type fermentation of Trichoderma reesei (T. reesei) (RONOZYME® MultiGrain) in degrading cell wall components of wheat, barley, rye, de-oiled rice bran, sunflower, rapeseed and cassava. A total of thirty-one different enzyme proteins were identified in the T. Reesei carbohydrase product using liquid chromatography with tandem mass spectrometry LC-MS/MS including glycosyl hydrolases and carbohydrate esterases. As measured by in vitro incubations and non-starch polysaccharide component analysis, and visualization by immunocytochemistry and confocal microscopy imaging of immuno-labeled samples with confocal microscopy, the carbohydrase product effectively solubilized cellulolytic and hemicellulolytic polysaccharides present in the cell walls of all the feed ingredients evaluated. The T. reesei fermentation also decreased viscosity of arabinoxylan, xyloglucan, galactomannan and ß-glucan substrates. Combination of several debranching enzymes including arabinofuranosidase, xylosidase, α-galactosidase, acetyl xylan esterase, and 4-O-methyl-glucuronoyl methylesterase with both GH10 and GH11 xylanases in the carbohydrase product resulted in effective hydrolyzation of heavily branched glucuronoarabinoxylans. The different ß-glucanases (both endo-ß-1,3(4)-glucanase and endo-ß-1,3-glucanase), cellulases and a ß-glucosidase in the T. reesei fermentation effectively reduced polymerization of both ß-glucans and cellulose polysaccharides of viscous cereals grains (wheat, barley, rye and oat). Interestingly, the secretome of T. reesei contained significant amounts of an exceptional direct chain-cutting enzyme from the GH74 family (Cel74A, xyloglucan-specific ß-1,4-endoglucanase), that strictly cleaves the xyloglucan backbone at the substituted regions. Here, we demonstrated that the balance of enzymes present in the T. reesei secretome is capable of degrading various cell wall components in both monocot and dicot plant raw material used as animal feed.


Assuntos
Ração Animal/análise , Parede Celular/metabolismo , Glicosídeo Hidrolases/metabolismo , Hypocreales/metabolismo , Acetilesterase/metabolismo , Celulases/metabolismo , Cromatografia Líquida/métodos , Endo-1,4-beta-Xilanases/metabolismo , Glucanos/metabolismo , Espectrometria de Massas em Tandem/métodos , Xilanos/metabolismo , Xilosidases/metabolismo
5.
Microbiol Res ; 249: 126770, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33932742

RESUMO

Trichoderma species are well known for producing various secondary metabolites in response to different fungal pathogens. This paper reports the effects of the metabolites produced during one-day cultivation of Trichoderma harzianum on the growth and development of the popular pathogen Fusarium culmorum. Inhibition of the growth of the pathogen and production of secondary metabolites including zearalenone was observed on Petri dishes. The presence of proteins such as cytochrome c oxidase subunit 4, glutathione-independent glyoxalase HSP31, and putative peroxiredoxin pmp20 in the extract-treated culture indicated oxidative stress, which was confirmed by the presence of a higher amount of catalase and dismutase in the later hours of the culture. A larger amount of enolase and glyceraldehyde 3-phosphate dehydrogenase resulted in faster growth, and the overexpression of stress protein and Woronin body major protein indicated the activation of defense mechanisms. In addition, a cardinal reduction in major mycotoxin production was noted.


Assuntos
Antibiose , Proteínas Fúngicas/metabolismo , Fusarium/crescimento & desenvolvimento , Fusarium/metabolismo , Hypocreales/metabolismo , Meios de Cultura , Metaboloma , Micotoxinas/metabolismo , Estresse Oxidativo , Pigmentos Biológicos/metabolismo , Proteoma , Metabolismo Secundário , Zearalenona/metabolismo
6.
Microbiol Res ; 249: 126773, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33940365

RESUMO

Purpureocillium lilacinum (formerly Paecilomyces lilacinus) is widely commercialized for controlling plant-parasitic nematodes and represents a potential cell factory for enzyme production. This nematicidal fungus is intrinsically resistant to common antifungal agents used for genetic transformation. Therefore, molecular investigations in P. lilacinum are still limited so far. In the present study, we have established a new Agrobacterium tumefaciens-mediated transformation (ATMT) system in P. lilacinum based on the uridine/uracil auxotrophic mechanism. Here, uridine/uracil auxotrophic mutants were simply generated via UV irradiation instead of a complicated genetic approach for the pyrG gene deletion. A stable uridine/uracil auxotrophic mutant was then selected as a recipient for fungal transformation. We further indicated that the pyrG gene from Aspergillus niger can be used as a selectable marker for genetic transformation of P. lilacinum. Under optimized conditions for ATMT, the transformation efficiency reached 2873 ± 224 transformants per 106 spores. Using the constructed ATMT system, we succeeded in expressing the DsRed reporter gene in P. lilacinum. Additionally, we have identified a very promising mutant for chitinase production from a collection of T-DNA insertion transformants. This mutant possesses a special phenotype of hyper-branching mycelium and produces more conidia in comparison to the wild strain. Conclusively, our ATMT system can be exploited for overexpression of target genes or for T-DNA insertion mutagenesis in the agriculturally important fungus P. lilacinum. The genetic approach in the present work may also be applied for developing similar ATMT systems in other fungi, especially for fungi that their genome databases are currently not available.


Assuntos
Agrobacterium tumefaciens/genética , Hypocreales/genética , Transformação Genética , Antifúngicos/farmacologia , Quitinases/genética , Quitinases/metabolismo , DNA Bacteriano/genética , Genes Fúngicos , Genes Reporter , Hypocreales/efeitos dos fármacos , Hypocreales/metabolismo , Mutagênese Insercional , Mutação , Uracila/metabolismo , Uridina/metabolismo
7.
mBio ; 12(3)2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33975944

RESUMO

Trichoderma reesei has 11 putative ß-glucosidases in its genome, playing key parts in the induction and production of cellulase. Nevertheless, the reason why the T. reesei genome encodes so many ß-glucosidases and the distinct role each ß-glucosidase plays in cellulase production remain unknown. In the present study, the cellular function and distribution of 10 known ß-glucosidases (CEL3B, CEL3E, CEL3F, CEL3H, CEL3J, CEL1A, CEL3C, CEL1B, CEL3G, and CEL3D) were explored in T. reesei, leaving out BGL1 (CEL3A), which has been well investigated. We found that the overexpression of cel3b or cel3g significantly enhanced extracellular ß-glucosidase production, whereas the overexpression of cel1b severely inhibited cellulase production by cellulose, resulting in nearly no growth of T. reesei Four types of cellular distribution patterns were observed for ß-glucosidases in T. reesei: (i) CEL3B, CEL3E, CEL3F, and CEL3G forming clearly separated protein secretion vesicles in the cytoplasm; (ii) CEL3H and CEL3J diffusing the whole endomembrane as well as the cell membrane with protein aggregation, like a reticular network; (iii) CEL1A and CEL3D in vacuoles; (iv) and CEL3C in the nucleus. ß-glucosidases CEL1A, CEL3B, CEL3E, CEL3F, CEL3G, CEL3H, and CEL3J were identified as extracellular, CEL3C and CEL3D as intracellular, and CEL1B as unknown. The extracellular ß-glucosidases CEL3B, CEL3E, CEL3F, CEL3H, and CEL3G were secreted through a tip-directed conventional secretion pathway, and CEL1A, via a vacuole-mediated pathway that was achieved without any signal peptide, while CEL3J was secreted via an unconventional protein pathway bypassing the endoplasmic reticulum (ER) and Golgi.IMPORTANCE Although ß-glucosidases play an important role in fungal cellulase induction and production, our current understanding does not provide a global perspective on ß-glucosidase function. This work comprehensively studies all the ß-glucosidases regarding their effect on cellulase production and their cellular distribution and secretion. Overexpression of cel3b or cel3g significantly enhanced ß-glucosidase production, whereas overexpression of cel1b severely inhibited cellulase production on cellulose. In addition, overexpression of cel3b, cel3e, cel3f, cel3h, cel3j, cel3c, or cel3g delayed endoglucanase (EG) production. We first identified four cellular distribution patterns of ß-glucosidases in Trichoderma reesei Specially, CEL3C was located in the nucleus. CEL3J was secreted through the nonclassical protein secretion pathway bypassing endoplasmic reticulum (ER) and Golgi. CEL1A was secreted via a vacuole-mediated conventional secretion route without a signal peptide. These findings advance our understanding of ß-glucosidase properties and secretory pathways in filamentous fungi, holding key clues for future study.


Assuntos
Proteínas Fúngicas/metabolismo , Expressão Gênica , Hypocreales/enzimologia , Hypocreales/genética , beta-Glucosidase/metabolismo , Celobiose/metabolismo , Celulase/biossíntese , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Hypocreales/metabolismo , beta-Glucosidase/biossíntese , beta-Glucosidase/classificação , beta-Glucosidase/genética
8.
J Am Chem Soc ; 143(15): 5605-5609, 2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33834778

RESUMO

Hirsutellones are fungal natural products containing a macrocyclic para-cyclophane connected to a decahydrofluorene ring system. We have elucidated the biosynthetic pathway for pyrrocidine B (3) and GKK1032 A2 (4). Two small hypothetical proteins, an oxidoreductase and a lipocalin-like protein, function cooperatively in the oxidative cyclization of the cyclophane, while an additional hypothetical protein in the pyrrocidine pathway catalyzes the exo-specific cycloaddition to form the cis-fused decahydrofluorene.


Assuntos
Produtos Biológicos/metabolismo , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Fungos/química , Compostos Heterocíclicos de 4 ou mais Anéis/metabolismo , Pirrolidinonas/metabolismo , Acremonium/química , Acremonium/metabolismo , Produtos Biológicos/química , Hidrocarbonetos Aromáticos com Pontes/química , Catálise , Reação de Cicloadição , Fungos/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/química , Hypocreales/química , Hypocreales/metabolismo , Conformação Molecular , Oxirredução , Oxirredutases/metabolismo , Pirrolidinonas/química , Estereoisomerismo
9.
Int J Mol Sci ; 22(9)2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33925273

RESUMO

BACKGROUND: The filamentous fungus Trichoderma reesei is used on an industrial scale to produce enzymes of biotechnological interest. This fungus has a complex cellulolytic system involved in the degradation of lignocellulosic biomass. However, several aspects related to the regulation of the expression of holocellulolytic genes and the production of cellulases by this fungus are still understood. METHODS: Here, we constructed a null mutant strain for the xyloglucanase cel74a gene and performed the characterization of the Δcel74a strain to evaluate the genetic regulation of the holocellulases during sugarcane bagasse (SCB) cultivation. RESULTS: Our results demonstrate that the deletion of xyloglucanase cel74a may impact the regulation of holocellulase expression during SCB cultivation. The expression of cellulases cel7a, cel7b, and cel6a was reduced in Δcel74a strain, while the hemicellulases xyn1 and xyn2 were increased in the presence of SCB. The cel74a mutation also affected the xyloglucan hydrolysis patterns. In addition, CEL74A activity was modulated in the presence of calcium, suggesting that this ion may be required for efficient degradation of xyloglucan. CONCLUSIONS: CEL74A affects the regulation of holocellulolytic genes and the efficient degradation of SCB in T. reesei. This data makes a significant contribution to our understanding of the carbon utilization of fungal strains as a whole.


Assuntos
Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Hypocreales/genética , Biomassa , Celulases/genética , Celulases/metabolismo , Celulose/metabolismo , Proteínas Fúngicas/metabolismo , Hidrólise , Hypocreales/metabolismo , Saccharum/metabolismo , Trichoderma/genética , Trichoderma/metabolismo
10.
Int J Mol Sci ; 22(7)2021 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-33800567

RESUMO

Plastic pollution is one of the crucial global challenges nowadays, and biodegradation is a promising approach to manage plastic waste in an environment-friendly and cost-effective way. In this study we identified the strain of fungus Trichoderma viride GZ1, which was characterized by particularly high pectinolytic activity. Using differential scanning calorimetry, Fourier-transform infrared spectroscopy techniques, and viscosity measurements we showed that three-month incubation of polylactide and polyethylene terephthalate in the presence of the fungus lead to significant changes of the surface of polylactide. Further, to gain insight into molecular mechanisms underneath the biodegradation process, western blot hybridization was used to show that in the presence of poly(ethylene terephthalate) (PET) in laboratory conditions the fungus produced hydrophobin proteins. The mycelium adhered to the plastic surface, which was confirmed by scanning electron microscopy, possibly due to the presence of hydrophobins. Further, using atomic force microscopy we demonstrated for the first time the formation of hydrophobin film on the surface of aliphatic polylactide (PLA) and PET by T. viride GZ1. This is the first stage of research that will be continued under environmental conditions, potentially leading to a practical application.


Assuntos
Hypocreales/metabolismo , Plásticos/química , Poliésteres/química , Polietilenotereftalatos/química , Biodegradação Ambiental , Varredura Diferencial de Calorimetria , Microscopia de Força Atômica , Polímeros/química , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície , Viscosidade , Microbiologia da Água , Poluentes Químicos da Água
11.
Bioorg Chem ; 111: 104874, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33887585

RESUMO

Seven previously undescribed trichothecenes, named trichothecrotocins M-S (1-7), along with five known compounds, were isolated from rice cultures of the potato-associated fungus Trichothecium crotocinigenum. Their structures and absolute configurations were determined through spectroscopic methods, single-crystal X-ray diffraction, and quantum chemistry calculations on ECD. Compound 1 possesses a rare 6,11-epoxy moiety in the trichothecene family. Compound 6 exhibited strong cytotoxic activity against MCF-7 cancer cell lines with an IC50 value of 2.34 ± 0.45 µM. It promoted apoptosis induction in MCF-7 cells. Moreover, cell cycle analysis showed cell cycle arrest caused by compound 6 at the G2/M phase which resulted to cell proliferation inhibition and pro-apoptotic activity. Further quantitative real-time PCR (qRT-PCR) analysis confirmed that the G2/M arrest was accompanied by upregulation of p21 and down regulation of cyclins B1 in 6-treated MCF-7 cells.


Assuntos
Antineoplásicos/farmacologia , Hypocreales/química , Solanum tuberosum/química , Tricotecenos/farmacologia , Antineoplásicos/química , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Hypocreales/metabolismo , Células MCF-7 , Simulação de Acoplamento Molecular , Estrutura Molecular , Solanum tuberosum/metabolismo , Relação Estrutura-Atividade , Tricotecenos/química , Tricotecenos/metabolismo
12.
Int J Mol Sci ; 22(8)2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33920773

RESUMO

Rice false smut is a fungal disease distributed worldwide and caused by Ustilaginoidea virens. In this study, we identified a putative ester cyclase (named as UvEC1) as being significantly upregulated during U. virens infection. UvEC1 contained a SnoaL-like polyketide cyclase domain, but the functions of ketone cyclases such as SnoaL in plant fungal pathogens remain unclear. Deletion of UvEC1 caused defects in vegetative growth and conidiation. UvEC1 was also required for response to hyperosmotic and oxidative stresses and for maintenance of cell wall integrity. Importantly, ΔUvEC1 mutants exhibited reduced virulence. We performed a tandem mass tag (TMT)-based quantitative proteomic analysis to identify differentially accumulating proteins (DAPs) between the ΔUvEC1-1 mutant and the wild-type isolate HWD-2. Proteomics data revealed that UvEC1 has a variety of effects on metabolism, protein localization, catalytic activity, binding, toxin biosynthesis and the spliceosome. Taken together, our findings suggest that UvEC1 is critical for the development and virulence of U. virens.


Assuntos
Proteínas Fúngicas/metabolismo , Hypocreales/metabolismo , Hypocreales/patogenicidade , Isomerases/metabolismo , Oryza/microbiologia , Doenças das Plantas/microbiologia , Proteômica , Sequência de Aminoácidos , Proteínas Fúngicas/química , Deleção de Genes , Genoma Fúngico , Hypocreales/genética , Hypocreales/crescimento & desenvolvimento , Isomerases/química , Micotoxinas/genética , Micotoxinas/metabolismo , Proteoma/metabolismo , Spliceossomos/metabolismo , Esporos Fúngicos/metabolismo , Estresse Fisiológico , Frações Subcelulares/metabolismo
13.
J Chem Ecol ; 47(4-5): 455-462, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33713251

RESUMO

Plant growth-promoting fungi belonging to genus Trichoderma are known to help plants when dealing with biotic stressors by enhancing plant defenses. While beneficial effects of Trichoderma spp. against plant pathogens have long been documented, fewer studies have investigated their effect on insect pests. Here, we studied the impact of Trichoderma root colonization on the plant defense responses against stink bug feeding attack. For this purpose, a model system consisting of tomato plant, Solanum lycopersicum cv Dwarf San Marzano, Trichoderma harzianum strain T22 and the southern green stink bug, Nezara viridula, was used. We firstly determined stink bug performance in terms of relative growth rate and survival on tomato plants inoculated by T. harzianum T22. Then, we evaluated relative expression of plant defense-related genes on inoculated plants induced by stink bug feeding. We found evidence that T. harzianum T22 affects tomato defense responses against N. viridula nymphs leading to reduction of growth rate. Our results also showed that T. harzianum T22 enhances plant direct defenses by an early increase of transcript levels of jasmonic acid marker genes. Yet this effect was time-dependent and only detected 8 h after herbivore induction. Taken together, our findings provide better understanding on the mechanisms underlying tomato induced resistance against herbivorous stink bugs.


Assuntos
Ciclopentanos/metabolismo , Heterópteros/metabolismo , Hypocreales/metabolismo , Lycopersicon esculentum/metabolismo , Oxilipinas/metabolismo , Animais , Feminino , Regulação da Expressão Gênica de Plantas , Genes de Plantas/fisiologia , Herbivoria , Lycopersicon esculentum/genética , Lycopersicon esculentum/microbiologia , Raízes de Plantas/metabolismo , Sementes/metabolismo , Transdução de Sinais , Simbiose , Fatores de Tempo , Transcrição Genética
14.
Anal Bioanal Chem ; 413(11): 3055-3067, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33675374

RESUMO

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


Assuntos
Fusarium/metabolismo , Cromatografia Gasosa-Espectrometria de Massas/métodos , Hypocreales/metabolismo , Espectrometria de Mobilidade Iônica/métodos , Compostos Orgânicos Voláteis/metabolismo
15.
J Ind Microbiol Biotechnol ; 48(1-2)2021 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-33693788

RESUMO

Hyphal morphology is considered to have a close relationship with the production level of secreted proteins by filamentous fungi. In this study, the gul1 gene, which encodes a putative mRNA-binding protein, was disrupted in cellulase-producing fungus Trichoderma reesei. The hyphae of Δgul1 strain produced more lateral branches than the parent strain. Under the condition for cellulase production, disruption of gul1 resulted in smaller mycelial clumps and significantly lower viscosity of fermentation broth. In addition, cellulase production was improved by 22% relative to the parent strain. Transcriptome analysis revealed that a set of genes encoding cell wall remodeling enzymes as well as hydrophobins were differentially expressed in the Δgul1 strain. The results suggest that the regulatory role of gul1 in cell morphogenesis is likely conserved in filamentous fungi. To our knowledge, this is the first report on the engineering of gul1 in an industrially important fungus.


Assuntos
Celulase/biossíntese , Proteínas Fúngicas/metabolismo , Hifas/metabolismo , Hypocreales/metabolismo , Parede Celular/metabolismo , Fermentação , Proteínas Fúngicas/genética , Hypocreales/genética , Viscosidade
16.
J Sci Food Agric ; 101(14): 5861-5871, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33788973

RESUMO

BACKGROUND: Two Fusarium fungi, F. oxysporum and F. proliferatum, have been recognized as major pathogenic fungi that cause postharvest decay of chili fruits. Ozone and some toxic chemicals are used to control pathogenic infections, leading to longer storage lives of agricultural commodities. However, these chemicals may pose some risks to the applicators and the environment. Therefore, alternative, easy-to-use fumigants for effective control of Fusarium infections in harvested fresh chilies are needed. RESULTS: Two endophytic fungi, Trichoderma afroharzianum strain MFLUCC19-0090 and T. afroharzianum strain MFLUCC19-0091, were isolated from Schefflera leucantha leaves. Their volatile compounds were investigated for antifungal activities against F. oxysporum and F. proliferatum. In vitro results showed that the volatile compounds produced by each strain inhibited pathogen growth. Additionally, the Trichoderma-derived volatile compounds significantly reduced Fusarium-related disease severity and incidence percentages in the inoculated fresh chilies. Antifungal properties of the volatile compounds were found to be specific to the species of the tested pathogens (MFLUCC19-0090 greatly suppressed F. oxysporum and MFLUCC19-0091 greatly suppressed F. proliferatum). Seventy-three volatile compounds were detected from both strains. Among the major volatile compounds detected, phenyl ethyl alcohol was found to possess the strongest antifungal activity against both pathogens. CONCLUSION: These Trichoderma-derived volatile compounds may be used as alternative fumigants for controlling Fusarium rot in harvested fresh chilies. The successful use of volatile compounds as biofumigants can prevent significant market losses and, more importantly, may reduce the health hazards caused by Fusarium-associated mycotoxin exposures among consumers. © 2021 Society of Chemical Industry.


Assuntos
Antifúngicos/farmacologia , Capsicum/microbiologia , Fusarium/efeitos dos fármacos , Doenças das Plantas/prevenção & controle , Trichoderma/química , Compostos Orgânicos Voláteis/farmacologia , Antifúngicos/química , Antifúngicos/metabolismo , Araliaceae/microbiologia , Benzoquinonas , Cicloexanonas , Endófitos/química , Endófitos/isolamento & purificação , Endófitos/metabolismo , Fusarium/fisiologia , Hypocreales/química , Hypocreales/isolamento & purificação , Hypocreales/metabolismo , Doenças das Plantas/microbiologia , Trichoderma/isolamento & purificação , Trichoderma/metabolismo , Compostos Orgânicos Voláteis/química , Compostos Orgânicos Voláteis/metabolismo
17.
Braz J Microbiol ; 52(2): 491-501, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33651333

RESUMO

Filamentous fungus Purpureocillium lilacinum is an emerging pathogen that infects immunocompromised and immunocompetent individuals and is resistant to several azole molecules. Although azole resistance mechanisms are well studied in Aspergillus sp. and Candida sp., there are no studies to date reporting P. lilacinum molecular response to these molecules. The aim of this study was to describe P. lilacinum molecular mechanisms involved in antifungal response against fluconazole and itraconazole. Transcriptomic analyses showed that gene expression modulation takes place when P. lilacinum is challenged for 12 h with fluconazole (64 µg/mL) or itraconazole (16 µg/mL). The antifungals acted on the ergosterol biosynthesis pathway, and two homologous genes coding for cytochrome P450 51 enzymes were upregulated. Genes coding for efflux pumps, such as the major facilitator superfamily transporter, also displayed increased expression in the treated samples. We propose that P. lilacinum develops antifungal responses by raising the expression levels of cytochrome P450 enzymes and efflux pumps. Such modulation could confer P. lilacinum high levels of target enzymes and could lead to the constant withdrawal of antifungals, which would force an increase in the administration of antifungal medications to achieve fungal morbidity or mortality. The findings in this work could aid in the decision-making for treatment strategies in cases of P. lilacinum infection.


Assuntos
Antifúngicos/farmacologia , Fluconazol/farmacologia , Hypocreales/efeitos dos fármacos , Hypocreales/genética , Itraconazol/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Farmacorresistência Fúngica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Humanos , Hypocreales/metabolismo , Testes de Sensibilidade Microbiana , Micoses/tratamento farmacológico , Micoses/microbiologia , Transcriptoma/efeitos dos fármacos
18.
Biomolecules ; 11(2)2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33672420

RESUMO

The major intrinsic protein (MIP) superfamily is a key part of the fungal transmembrane transport network. It facilitates the transport of water and low molecular weight solutes across biomembranes. The fungal uncharacterized X-Intrinsic Protein (XIP) subfamily includes the full protein diversity of MIP. Their biological functions still remain fully hypothetical. The aim of this study is still to deepen the diversity and the structure of the XIP subfamily in light of the MIP counterparts-the aquaporins (AQPs) and aquaglyceroporins (AQGPs)-and to describe for the first time their function in the development, biomass accumulation, and mycoparasitic aptitudes of the fungal bioagent Trichoderma atroviride. The fungus-XIP clade, with one member (TriatXIP), is one of the three clades of MIPs that make up the diversity of T. atroviride MIPs, along with the AQPs (three members) and the AQGPs (three members). TriatXIP resembles those of strict aquaporins, predicting water diffusion and possibly other small polar solutes due to particularly wider ar/R constriction with a Lysine substitution at the LE2 position. The XIP loss of function in ∆TriatXIP mutants slightly delays biomass accumulation but does not impact mycoparasitic activities. ∆TriatMIP forms colonies similar to wild type; however, the hyphae are slightly thinner and colonies produce rare chlamydospores in PDA and specific media, most of which are relatively small and exhibit abnormal morphologies. To better understand the molecular causes of these deviant phenotypes, a wide-metabolic survey of the ∆TriatXIPs demonstrates that the delayed growth kinetic, correlated to a decrease in respiration rate, is caused by perturbations in the pentose phosphate pathway. Furthermore, the null expression of the XIP gene strongly impacts the expression of four expressed MIP-encoding genes of T. atroviride, a plausible compensating effect which safeguards the physiological integrity and life cycle of the fungus. This paper offers an overview of the fungal XIP family in the biocontrol agent T. atroviride which will be useful for further functional analysis of this particular MIP subfamily in vegetative growth and the environmental stress response in fungi. Ultimately, these findings have implications for the ecophysiology of Trichoderma spp. in natural, agronomic, and industrial systems.


Assuntos
Aquaporinas/química , Aquaporinas/fisiologia , Proteínas Fúngicas/química , Proteínas Fúngicas/fisiologia , Hypocreales/metabolismo , Biomassa , Carbono/química , Simulação por Computador , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Hifas , Cinética , Modelos Biológicos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Via de Pentose Fosfato , Fenótipo , Filogenia , Conformação Proteica , Água/química
19.
Parasit Vectors ; 14(1): 177, 2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33761961

RESUMO

BACKGROUND: Malaria transmission depends on infected mosquitoes and can be controlled by transmission-blocking drugs. The recently discovered FREP1-mediated malaria transmission pathway is an excellent target to screen drugs for limiting transmission. METHODS: To identify candidate small molecules, we used an ELISA-based approach to analyze extracts from a fungal library for inhibition of the FREP1-parasite interaction. We isolated and determined one active compound by chromatography and crystallography, respectively. We measured the effects of the bioactive compound on malaria transmission to mosquitoes through standard membrane-feeding assays (SMFA) and on parasite proliferation in blood by culturing. RESULTS: We discovered the ethyl acetate extract of the fungus Purpureocillium lilacinum that inhibited Plasmodium falciparum transmission to mosquitoes. Pre-exposure to the extract rendered Anopheles gambiae resistant to Plasmodium infection. Furthermore, we isolated one novel active compound from the extract and identified it as 3-amino-7,9-dihydroxy-1-methyl-6H-benzo[c]chromen-6-one, or "pulixin." Pulixin prevented FREP1 from binding to P. falciparum-infected cell lysate. Pulixin blocked the transmission of the parasite to mosquitoes with an EC50 (the concentration that gave half-maximal response) of 11 µM based on SMFA. Notably, pulixin also inhibited the proliferation of asexual-stage P. falciparum with an EC50 of 47 nM. The compound did not show cytotoxic effects at a concentration of 116 µM or lower. CONCLUSION: By targeting the FREP1-Plasmodium interaction, we discovered that Purpureocillium lilacinum extract blocked malaria transmission. We isolated and identified the bioactive agent pulixin as a new compound capable of stopping malaria transmission to mosquitoes and inhibiting parasite proliferation in blood culture.


Assuntos
Antimaláricos/farmacologia , Fungos/química , Fungos/metabolismo , Malária/prevenção & controle , Malária/transmissão , Plasmodium falciparum/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Hypocreales/química , Hypocreales/metabolismo , Redes e Vias Metabólicas
20.
ACS Synth Biol ; 10(4): 698-706, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33720696

RESUMO

Fungal natural products are rich sources of clinical drugs. Particularly, the fungicolous fungi have a large number of biosynthetic gene clusters (BGCs) to produce numerous bioactive natural products, but most BGCs are silent in the laboratory. We have shown that a fungicolous fungus Calcarisporium arbuscula NRRL 3705 predominantly produces the highly reduced polyketide-type mycotoxins aurovertins. Here after evaluation of the aurovertin-null mutant ΔaurA as an efficient host, we further screened two strong promoters aurBp and A07068p based on RNA-Seq, and successfully activated an endogenous gene cluster from C. arbuscula as well as three additional exogenous BGCs from other fungi to produce polyketide-type natural products. Thus, we showed an efficient expression system from the fungicolous fungus C. arbuscula, which will be highly beneficial and complementary to the conventional Aspergillus and Penicillium fungal cell factories, and provides a useful toolkit for genome-wide mining of bioactive natural products from fungicolous fungi.


Assuntos
Produtos Biológicos/metabolismo , Hypocreales/metabolismo , Aspergillus/genética , Hypocreales/genética , Família Multigênica/genética , Família Multigênica/fisiologia , Penicillium/genética
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