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.
- 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
The present work aimed at purifying the intracellular fungal metabolites, such as 16-methylheptadecanoic acid methyl ester (HDA) and 9,12-octadecadienoic acid (ODA) from marine Trichoderma, Hypocrea lixii TSK8, Hypocrea rufa SKS2 respectively, and investigating their anticancer and antioxidant effects. The two fungal metabolites were tested against two human cancer cell lines, namely oral cancer (KB) and skin carcinoma (A431) by using MTT assay. The inhibitory concentrations (IC50) against KB oral cancer cells were found to be 18.75 ± 0.12 μg/mL for HDA and 75.50 ± 0.42 μg/mL for ODA. Whereas IC50 values of HDA and ODA against A431 were found 37.5 ± 0.42 μg/mL and 72.89 ± 0.15 μg/mL, respectively. In addition, the down-regulation of heat shock protein 90 kDa (HSP90) was confirmed by using SDS-PAGE and Western blot analysis. The effect of HDA induced apoptosis via ROS-dependent internucleosomal DNA fragmentation was confirmed by AGE analysis. We further evaluated the in vivo anti-skin cancer activity of HDA in Swiss albino mice induced with skin cancer by 7,12-dimethylbenz(a)anthracene (DMBA) and croton oil (CO). The in vivo hematological, biochemical and histopathological results revealed that the fungal metabolite HDA was a highly potent anticancer compound against the skin cancer.
- Klíčová slova
- methyl ester 16-methylheptadekanové kyseliny (HDA) a 9, 12-oktodekanové kyseliny (ODA),
- MeSH
- antioxidancia izolace a purifikace terapeutické užití MeSH
- apoptóza genetika imunologie účinky léků MeSH
- fragmentace DNA MeSH
- houby cytologie izolace a purifikace metabolismus MeSH
- kyseliny dekanové * izolace a purifikace metabolismus MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- myši MeSH
- nádorové buněčné linie cytologie metabolismus účinky léků MeSH
- nádory kůže farmakoterapie MeSH
- oxidační stres genetika imunologie účinky léků MeSH
- proteiny tepelného šoku HSP90 imunologie izolace a purifikace metabolismus MeSH
- protinádorové látky * MeSH
- statistika jako téma MeSH
- Trichoderma * genetika izolace a purifikace účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
Molecular markers that enable monitoring of fungi in their natural environment or assist in the identification of specific strains would facilitate Trichoderma utilization, particularly as an agricultural biocontrol agent (BCA). In this study, sequence analysis of internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) of the ribosomal RNA (rRNA) gene cluster, a fragment of the translation elongation factor 1-alpha (tef1) gene, and random amplified polymorphic DNA (RAPD) markers were applied to determine the genetic diversity of Trichoderma atroviride strains collected in Poland, and also in order to identify loci and PCR-based molecular markers useful in genetic variation assessment of that fungus. Although tef1 and RAPD analysis showed limited genetic diversity among T. atroviride strains collected in Poland, it was possible to distinguish major groups that clustered most of the analyzed strains. Polymorphic RAPD amplicons were cloned and sequenced, yielding sequences representing 13 T. atroviride loci. Based on these sequences, a set of PCR-based markers specific to T. atroviride was developed and examined. Three cleaved amplified polymorphic sequence (CAPS) markers could assist in distinguishing T. atroviride strains. The genomic regions identified may be useful for further exploration and development of more precise markers suitable for T. atroviride identification and monitoring, especially in environmental samples.
- MeSH
- DNA fungální chemie genetika MeSH
- elongační faktor 1 genetika MeSH
- fylogeneze MeSH
- genetická variace * MeSH
- genetické lokusy * MeSH
- genetické markery MeSH
- mezerníky ribozomální DNA chemie genetika MeSH
- molekulární sekvence - údaje MeSH
- molekulární typizace MeSH
- mykologické určovací techniky MeSH
- sekvenční analýza DNA MeSH
- shluková analýza MeSH
- technika náhodné amplifikace polymorfní DNA MeSH
- Trichoderma klasifikace genetika izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Polsko 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.
We aimed to establish an efficient RNA interference (RNAi) system in the industrially important filamentous fungus Trichoderma koningii using the DsRed protein as a reporter of the silencing process. To accomplish this, a DsRed expression cassette was transformed into T. koningii, and a recombinant strain that stably expressed DsRed was obtained. Next, a vector-directing expression of a DsRed hairpin RNA was constructed and transformed into the T. koningii recipient strain. Approximately 79 % of transformants displayed a decrease in DsRed fluorescence, and expression of DsRed in some transformants appeared to be fully suppressed. Characterization of randomly selected transformants by genomic DNA PCR analysis, real-time PCR quantification, and western blot confirmed downregulation of gene expression at different levels. The RNA silencing approach described here for T. koningii is effective, and the DsRed reporter gene provides a convenient tool for identification of silenced fungal transformants by their DsRed fluorescence compared to the control strain. The results of this study demonstrate the power of RNAi in T. koningii, which supports the use of this technology for strain development programs and functional genomics studies in industrial fungal strains.
- MeSH
- fluorescence MeSH
- genový knockdown metody MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- luminescentní proteiny analýza genetika MeSH
- malá interferující RNA genetika MeSH
- mikrobiální genetika metody MeSH
- rekombinace genetická MeSH
- reportérové geny MeSH
- RNA interference * MeSH
- stanovení celkové genové exprese MeSH
- transformace genetická MeSH
- Trichoderma genetika MeSH
- western blotting MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The present study includes the molecular characteristics of Trichoderma pleurotum and Trichoderma pleuroticola isolates collected from green moulded cereal straw substrates at 47 oyster mushroom farms in Poland. The screening of the 80 Trichoderma isolates was performed by morphological observation and by using the multiplex PCR assay. This approach enabled specific detection of 47 strains of T. pleurotum and 2 strains of T. pleuroticola. Initial identifications were confirmed by sequencing the fragment of internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) of the rRNA gene cluster and the fragment including the fourth and fifth introns and the last long exon of the translation-elongation factor 1-alpha (tef1) gene. ITS and tef1 sequence information was also used to establish the intra- and interspecies relationship of T. pleurotum and T. pleuroticola originating from the oyster mushroom farms in Poland and from other countries. Comparative analysis of the ITS sequences showed that all T. pleurotum isolates from Poland represent one haplotype, identical to that of T. pleurotum strains from Hungary and Romania. Sequence analysis of the tef1 locus revealed two haplotypes ("T" and "N") of Polish T. pleurotum isolates. The "T" type isolates of T. pleurotum were identical to those of strains from Hungary and Romania. The "N" type isolates possessed a unique tef1 allele. Detailed analysis of the ITS and tef1 sequences of two T. pleuroticola isolates showed their identicalness to Italian strain C.P.K. 1540.
- MeSH
- DNA fungální chemie genetika MeSH
- elongační faktor 1 genetika MeSH
- genetická variace * MeSH
- haplotypy MeSH
- mezerníky ribozomální DNA chemie genetika MeSH
- mikroskopie MeSH
- molekulární sekvence - údaje MeSH
- multiplexová polymerázová řetězová reakce MeSH
- Pleurotus * MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza DNA MeSH
- Trichoderma klasifikace cytologie genetika izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Polsko MeSH
Biofilms represent mixed communities present in a diverse range of environments; however, their utility as inoculants is less investigated. Our investigation was aimed towards in vitro development of biofilms using fungal mycelia (Trichoderma viride) as matrices and nitrogen-fixing and P-solubilizing bacteria as partners, as a prelude to their use as biofertilizers (biofilmed biofertilizers, BBs) and biocontrol agents for different crops. The most suitable media in terms of population counts, fresh mass and dry biomass for Trichoderma and Bacillus subtilis/Pseudomonas fluorescens was found to be Pikovskaya broth ± 1 % CaCO(3), while for Trichoderma and Azotobacter chroococcum, Jensen's medium was most optimal. The respective media were then used for optimization of the inoculation rate of the partners in terms of sequence of addition of partners, fresh/dry mass of biofilms and population counts of partners for efficient film formation. Microscopic observations revealed significant differences in the progress of growth of biofilms and dual cultures. In the biofilms, the bacteria were observed growing intermingled within the fungal mycelia mat. Further, biofilm formation was compared under static and shaking conditions and the fresh mass of biofilms was higher in the former. Such biofilms are being further characterized under in vitro conditions, before using them as inoculants with crops.
Twenty-eight isolates of Trichoderma belonging to four different species were screened in vitro for their antagonistic ability against Fusarium oxysporum f.sp. dianthi causing carnation wilt. Three different levels of antagonism observed in dual plate assay were further confirmed by cell-free culture filtrate experiments. Isolates showing class I level of antagonism produced maximum lytic enzymes, chitinases and beta-1,3-glucanases. Genetic variability of 25 selected isolates was assessed by random amplified polymorphic DNA technique and the amplified products were correlated for their level of antagonism. Unweighed pair-group method with arithmetical averages cluster analysis revealed prominent inter-and intraspecific genetic variation among the isolates. Based on their genetic relationship, the isolates were mainly distributed into 3 major groups representing T. atroviride, T. pseudokoningii and T. harzianum, with 20-35% interspecific dissimilarity. However, the polymorphism shown by the isolates did not correlate to their level of antagonism.
- MeSH
- antibióza MeSH
- chitinasy genetika metabolismus MeSH
- fungální proteiny genetika metabolismus MeSH
- Fusarium fyziologie MeSH
- fylogeneze MeSH
- genetická variace MeSH
- nemoci rostlin mikrobiologie MeSH
- polymorfismus délky restrikčních fragmentů MeSH
- půdní mikrobiologie MeSH
- Trichoderma fyziologie genetika izolace a purifikace klasifikace MeSH
