Mycoparasitism is a key feature of Trichoderma (Hypocreales, Ascomycota) biocontrol agents. Recent studies of intracellular signal transduction pathways of the potent mycoparasite Trichoderma atroviride revealed the involvement of Tmk1, a mitogen-activated protein kinase (MAPK), in triggering the mycoparasitic response. We previously showed that mutants missing Tmk1 exhibit reduced mycoparasitic activity against several plant pathogenic fungi. In this study, we identified the most robustly regulated targets that were governed by Tmk1 during mycoparasitism using transcriptome and proteome profiling. Tmk1 mainly exerts a stimulating function for T. atroviride during its mycoparasitic interaction with the fungal plant pathogen Rhizoctonia solani, as reflected by 89% of strongly differently responding genes in the ∆tmk1 mutant compared to the wild type. Specifically, 54% of these genes showed strong downregulation in the response with a deletion of the tmk1 gene, whereas in the wild type the same genes were strongly upregulated during the interaction with the fungal host. These included the gene encoding the mycoparasitism-related proteinase Prb1; genes involved in signal transduction pathways such as a candidate coding for a conserved 14-3-3 protein, and a gene coding for Tmk2, the T. atroviride cell-wall integrity MAP kinase; genes encoding a specific siderophore synthetase, and multiple FAD-dependent oxidoreductases and aminotransferases. Due to the phosphorylating activity of Tmk1, different (phospho-)proteomics approaches were applied and identified proteins associated with cellular metabolism, energy production, protein synthesis and fate, and cell organization. Members of FAD- and NAD/NADP-binding-domain proteins, vesicular trafficking of molecules between cellular organelles, fungal translational, as well as protein folding apparatus were among others found to be phosphorylated by Tmk1 during mycoparasitism. Outstanding downregulation in the response of the ∆tmk1 mutant to the fungal host compared to the wild type at both the transcriptome and the proteome levels was observed for nitrilase, indicating that its defense and detoxification functions might be greatly dependent on Tmk1 during T. atroviride mycoparasitism. An intersection network analysis between the identified transcripts and proteins revealed a strong involvement of Tmk1 in molecular functions with GTPase and oxidoreductase activity. These data suggest that during T. atroviride mycoparasitism this MAPK mainly governs processes regulating cell responses to extracellular signals and those involved in reactive oxygen stress.
- MeSH
- Hypocreales * metabolismus MeSH
- mitogenem aktivované proteinkinasy genetika metabolismus MeSH
- proteom metabolismus MeSH
- regulace genové exprese u hub MeSH
- signální transdukce MeSH
- Trichoderma * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- mitogenem aktivované proteinkinasy MeSH
- proteom MeSH
Fungal metabolic carbon acquisition and its subsequent partitioning between biomass production and respiration, i.e. the carbon-use efficiency (CUE), are central parameters in biogeochemical modeling. However, current available techniques for estimating these parameters are all associated with practical and theoretical shortcomings, making assessments unreliable. Gene expression analyses hold the prospect of phenotype prediction by indirect means, providing new opportunities to obtain information about metabolic priorities. We cultured four different fungal isolates (Chalara longipes, Laccaria bicolor, Serpula lacrymans and Trichoderma harzianum) in liquid media with contrasting nitrogen availability and measured growth rates and respiration to calculate CUE. By relating gene expression markers to measured carbon fluxes, we identified genes coding for 1,3-β-glucan synthase and 2-oxoglutarate dehydrogenase as suitable markers for growth and respiration, respectively, capturing both intraspecific variation as well as within-strain variation dependent on growth medium. A transcript index based on these markers correlated significantly with differences in CUE between the fungal isolates. Our study paves the way for the use of these markers to assess differences in growth, respiration and CUE in natural fungal communities, using metatranscriptomic or the RT-qPCR approach.
- Klíčová slova
- carbon-use efficiency, fungi, gene markers, growth, metatranscriptomics, respiration,
- MeSH
- Ascomycota genetika metabolismus MeSH
- Basidiomycota genetika MeSH
- biologické markery * analýza MeSH
- fungální proteiny * genetika metabolismus MeSH
- houby * genetika metabolismus MeSH
- Hypocreales genetika metabolismus MeSH
- Laccaria genetika metabolismus MeSH
- transkriptom * MeSH
- Trichoderma genetika metabolismus MeSH
- uhlík * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- biologické markery * MeSH
- fungální proteiny * MeSH
- uhlík * MeSH
There are increasing efforts to identify biocontrol-active microbial metabolites in order to improve strategies for biocontrol of phytopathogens. In this work, Fusarium oxysporum f. sp. conglutinans was confronted with three different biocontrol agents: Trichoderma harzianum, Bacillus amyloliquefaciens, and Pseudomonas aeruginosa in dual culture bioassays. Metabolites produced during the microbial interactions were screened by a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). T. harzianum exhibited the strongest inhibition of growth of F. oxysporum resulting in overlay of the pathogen colony with its mycelium. Recorded metabolite profiles suggested a direct attack of F. oxysporum mycelium by T. harzianum and B. amyloliquefaciens by means of membrane-attacking peptaibols and a set of antimicrobial lipopeptides and siderophores, respectively. The direct mode of the biocontrol activity of T. harzianum and B. amyloliquefaciens corresponded to their ability to suppress F. oxysporum production of mycotoxin beauvericin suggesting that this ability is not specific only for Trichoderma species. In the case of P. aeruginosa, siderophores pyoverdine E/D and two rhamnolipids were produced as major bacterial metabolites; the rhamnolipid production was blocked by F. oxysporum. The results showed that this type of biocontrol activity was the least effective against F. oxysporum. The effective application of MALDI-MS profiling to the screening of nonvolatile microbial metabolites produced during the interaction of the phytopathogen and the biocontrol microorganisms was demonstrated.
- MeSH
- Bacillus amyloliquefaciens metabolismus fyziologie MeSH
- biologická ochrana * MeSH
- druhová specificita MeSH
- Fusarium růst a vývoj metabolismus MeSH
- glykolipidy metabolismus MeSH
- kokultivační techniky MeSH
- metabolomika MeSH
- mikrobiální interakce MeSH
- mycelium růst a vývoj metabolismus MeSH
- mykotoxiny metabolismus MeSH
- nemoci rostlin mikrobiologie prevence a kontrola MeSH
- Pseudomonas aeruginosa metabolismus fyziologie MeSH
- siderofory metabolismus MeSH
- Trichoderma metabolismus fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- biologická ochrana * MeSH
- glykolipidy MeSH
- mykotoxiny MeSH
- rhamnolipid MeSH Prohlížeč
- siderofory MeSH
The study of the soil microbial community represents an important step in better understanding the environmental context. Therefore, biological characterisation and physicochemical integration are keys when defining contaminated sites. Fungi play a fundamental role in the soil, by providing and supporting ecological services for ecosystems and human wellbeing. In this research, 52 soil fungal taxa were isolated from in situ pilot reactors installed to a contaminated site in Czech Republic with a high concentration of hexachlorocyclohexane (HCH). Among the identified isolates, 12 strains were selected to evaluate their tolerance to different isomers of HCH by using specific indices (Rt:Rc; T.I.) and to test their potential in xenobiotic biotransformation. Most of the selected taxa was not significantly affected by exposure to HCH, underlining the elevated tolerance of all the tested fungal taxa, and different metabolic intermediates of HCH dechlorination were observed. The oxidative stress responses to HCH for two selected species, Penicillium simplicissimum and Trichoderma harzianum, were investigated in order to explore their toxic responses and to evaluate their potential functioning in bioremediation of contaminated environments. This research suggests that the isolated fungal species may provide opportunities for new eco-friendly, integrated and cost-effective solutions for environmental management and remediation, considering their efficient adaptation to stressful conditions.
- Klíčová slova
- Bioremediation, Contaminated sites, HCH, Oxidative stress responses, Persistent organic pollutants, Soil fungi, Tolerance,
- MeSH
- biodegradace MeSH
- biotransformace * MeSH
- ekosystém MeSH
- hexachlorcyklohexan analýza metabolismus MeSH
- houby metabolismus MeSH
- isomerie MeSH
- látky znečišťující půdu analýza metabolismus MeSH
- oxidační stres MeSH
- Penicillium metabolismus MeSH
- půda MeSH
- půdní mikrobiologie * MeSH
- tolerance léku MeSH
- Trichoderma metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
- Názvy látek
- hexachlorcyklohexan MeSH
- látky znečišťující půdu MeSH
- půda MeSH
Efficient hydrolysis of holocellulose depends on a proper balance between cellulase (endoglucanase, exoglucanase, β-glucosidase) and xylanase activities. The present study aimed to induce the production of cellulases and xylanases using liquid cultures (one, two, three, and four fungal strains on the same bioreactor) of wild strains of Trichoderma harzianum, Aspergillus niger, and Fusarium oxysporum. The strains were identified by amplification and analysis of the ITS rDNA region and the obtained sequences were deposited in Genbank. Enzymes (endoglucanase, exoglucansae, β-glucosidase, and xylanase activities) and the profile of extracellular protein isoforms (SDS-PAGE) produced by different fungal combinations (N = 14) were analyzed by Pearson's correlation matrix and principal component analysis (PCA). According to our results, induction of endoglucanase (19.02%) and β-glucosidase (6.35%) were obtained after 4 days when A. niger and F. oxysporum were cocultured. The combination of A. niger-T. harzianum produced higher endoglucanase in a shorter time than monocultures. On the contrary, when more than two strains were cultured in the same reactor, the relationships of competition were established, trending to diminish the amount of enzymes and the extracellular protein isoforms produced. The xylanase production was sensible to stress produced by mixed cultures, decreasing their activity. This is important when the aim is to produce cellulase-free xylanase. In addition, exoglucanase activity did not change in the combinations tested.
- Klíčová slova
- Aspergillus, Endoglucanase, Exoglucanase, Filamentous fungi, Fusarium, Trichoderma, β-Glucosidase,
- MeSH
- Ascomycota enzymologie růst a vývoj izolace a purifikace metabolismus MeSH
- Aspergillus niger enzymologie růst a vývoj izolace a purifikace metabolismus MeSH
- biomasa MeSH
- bioreaktory mikrobiologie MeSH
- celulasy biosyntéza metabolismus MeSH
- celulosa metabolismus MeSH
- fermentace MeSH
- fungální proteiny biosyntéza metabolismus MeSH
- Fusarium enzymologie růst a vývoj izolace a purifikace metabolismus MeSH
- kokultivační techniky * MeSH
- mikrobiální interakce fyziologie MeSH
- průmyslová mikrobiologie metody MeSH
- Trichoderma enzymologie růst a vývoj izolace a purifikace metabolismus MeSH
- xylosidasy biosyntéza metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- celulasy MeSH
- celulosa MeSH
- fungální proteiny MeSH
- xylosidasy MeSH
Xylanase III (Xyn III), a specific endoxylanase that belongs to family 10 of the glycoside hydrolases, was overexpressed in Trichoderma reesei QM9414 using a constitutive strong promoter of the gene encoding pyruvate decarboxylase (pdc). The maximum recombinant xylanase activity achieved was 817.2 ± 65.2 U/mL in the transformant fermentation liquid. The productivities of Xyn III accounted for approximately 53% of the total protein secreted by the recombinant. The enzyme was optimally active at 60 °C and pH 6. The recombinant Xyn III was stable at pH 5-8. This is the first report on the homologous expression of xyn3 in T. reesei QM9414. The properties of Xyn III make it promising in a variety of industrial use.
- MeSH
- aktivace enzymů MeSH
- endo-1,4-beta-xylanasy genetika metabolismus MeSH
- exprese genu * MeSH
- klonování DNA MeSH
- koncentrace vodíkových iontů MeSH
- stabilita enzymů MeSH
- teplota MeSH
- Trichoderma genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- endo-1,4-beta-xylanasy MeSH
Puerarin-7-O-fructoside was transformed by Trichoderma harzianum CGMCC 1523 into 3'-hydroxypuerarin-7-O-fructoside; this was identified by MS and NMR. However, puerarin-7-O-glucoside was not directly hydroxylated but hydrolyzed back into puerarin, which was transformed into 3'-hydroxypuerarin by the same fungi. Comparative analysis of free radical scavenging activity of DPPH showed that the free radical scavenging activity of puerarin-7-O-glucoside was reduced to approximately 1/2 of that of puerarin, while the free radical scavenging activity of puerarin-7-O-fructoside was increased to approximately 1.5 times of that of puerarin. The free radical scavenging activity of 3'-hydroxypuerarin-7-O-fructoside was further increased by 2.2 times of that of puerarin-7-O-fructoside, which was close to that of 3'-hydroxypuerarin.
- MeSH
- biotransformace MeSH
- fruktosa analogy a deriváty chemie metabolismus MeSH
- glykosylace MeSH
- hydroxylace MeSH
- isoflavony chemie metabolismus MeSH
- scavengery volných radikálů chemie metabolismus MeSH
- Trichoderma metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fruktosa MeSH
- isoflavony MeSH
- puerarin-7-O-fructoside MeSH Prohlížeč
- scavengery volných radikálů MeSH
Conditions for conidiation of a natural isolate of Trichoderma atroviride during submerged cultivation in Erlenmeyer flasks and in a laboratory stirred-tank fermenter were optimized. From the simple sugars tested, cellobiose was the best substrate for conidia production while cellulose fines from paper mill waste proved to be a suitable cheap complex carbon source. Optimum temperature for conidiation was 24-26 degrees C, and the required dissolved oxygen level was > 40% saturation. After initial slight decrease during the 1st d after inoculation, the pH of the culture medium constantly increased throughout the sporulation period. Attempts to regulate the pH during fermentation did not improve the spore yields. The most intense formation of conidia took place between 2nd and 3rd d of growth and the overall volumetric productivity of conidia was 4.1-8.2 x 10(9) conidia per L/h.
- MeSH
- bioreaktory mikrobiologie MeSH
- fermentace fyziologie MeSH
- koncentrace vodíkových iontů MeSH
- metabolismus sacharidů fyziologie MeSH
- mykologie metody MeSH
- osmotický tlak MeSH
- spory hub fyziologie MeSH
- Trichoderma růst a vývoj metabolismus MeSH
- vysoká teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Thirty-three isolates belonging to six species of the genus Trichoderma were tested for the ability to hydroxylate progesterone to 11alpha-, 11beta-, 11alpha,17alpha- and 6beta, 17alpha-derivatives, and epicortisol. T. aureoviride, T. harzianum, T. polysporum and T. pseudokoningii produced 11alpha-hydroxyprogesterone. T. harzianum and T. hamatum can form only the 11beta-isomer. T. koningii and T. hamatum produced 11alpha-, 11beta-, 11alpha,17alpha- and 6beta,11alpha-hydroxy derivatives. 11alpha, 11beta, 6beta,11alpha- and 11alpha,17alpha-hydroxyprogesterones and epicortisol are produced by T. aureoviride and T. pseudokoningii. Cortisol was produced only when the medium was fortified by 10 g/L peptone. This is the first record of conversion of progesterone to mono-, di- and trihydroxyprogesterones by these Trichoderma species.
- MeSH
- biotransformace MeSH
- hydroxylace MeSH
- progesteron chemie metabolismus MeSH
- Trichoderma růst a vývoj izolace a purifikace metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- progesteron MeSH
Using indirect methods based on uptake of [3H]tetraphenylphosphonium cation and [14C]benzoic acid by cells of the fungus Trichoderma viride we found that the illumination-induced transient hyperpolarization of the plasma membrane is followed immediately by a rapid temporary decrease in intracellular pH. Hyperpolarization and intracellular acidification were completely suppressed by 150 mM-KCl and by the K(+)-ionophore valinomycin. The light-induced acidification of the cytoplasm was not observed in the presence of the cytochrome respiratory chain inhibitors antimycin A and mucidin. Based on these results, we hypothesize that the hyperpolarization of the cells is the consequence of an efflux of K+ through a light-activated K(+)-channel in the plasma membrane. The loss of positive charge in the cytoplasm caused by this efflux of cations is counterbalanced by H+ originating from the light-activated mitochondrial respiratory chain.
- MeSH
- draslík metabolismus MeSH
- kinetika MeSH
- koncentrace vodíkových iontů MeSH
- membránové potenciály účinky záření MeSH
- světlo * MeSH
- Trichoderma metabolismus účinky záření MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- draslík MeSH
