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.
- 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
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
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
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
