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A total of 36 UV-induced mutants with altered colony morphology were isolated from strain Trichoderma harzianum T334, a potential biocontrol agent against plant pathogenic fungi with the ability to produce constitutively low levels of chitinases. The level of constitutive β -1,4- N -acetyl-glucosaminidase production in standing and shaken cultures under non-inductive conditions was tested in mutants and compared to that of the parental strain. About 30% of the mutants showed significantly increased levels of enzyme production, with strain T334 col26a being the best producer. This mutant and the parental strain were subjected to in vitro confrontation assays with plant pathogenic Fusarium culmorum, Pythium debaryanum and Rhizoctonia solani strains. The mutant derivative could be characterized by significantly higher biocontrol index values than the parental strain in each experiment, suggesting, that mutants with improved constitutive extracellular chitinase secretion could be applied for biocontrol purposes against fungal plant pathogens.

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Acta Biologica Hungarica
Authors: L. Kredics, Kata Terecskei, Zsuzsanna Antal, A. Szekeres, L. Hatvani, L. Manczinger, and Cs. Vágvölgyi

Eleven cold-tolerant Trichoderma isolates were screened for the production of proteolytic activities at 10 °C. Based on the activity profiles determined with paranitroanilide substrates at 5 °C, strain T221 identified as Trichoderma atroviride was selected for further investigations. The culture broth of the strain grown at 10 °C in casein-containing culture medium was concentrated by lyophilization and subjected to gel filtration, which was followed by chromatofocusing of the fraction showing the highest activity on N -benzoyl-Phe-Val-Arg-paranitroanilide. The purified enzyme had a molecular weight of 24 kDa, an isoelectric point of 7.3 and a pH optimum of 6.2. The temperature optimum of 25 °C and the low thermal stability suggested that it is a true cold-adapted enzyme. Substrate specificity data indicate that the enzyme is a proteinase with a preference for Arg or Lys at the P1 position. The effect of proteinase inhibitors suggests that the enzyme has a binding pocket similar to the one present in trypsin.

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Acta Microbiologica et Immunologica Hungarica
Authors: A. Szekeres, B. Leitgeb, L. Kredics, Zsuzsanna Antal, L. Hatvani, L. Manczinger, and Cs. Vágvölgyi

Peptaibols and the related peptaibiotics are linear, amphipathic polypeptides. More than 300 of these secondary metabolites have been described to date. These compounds are composed of 5-20 amino acids and are generally produced in microheterogeneous mixtures. Peptaibols and peptaibiotics with unusual amino acid content are the result of non-ribosomal biosynthesis. Large multifunctional enzymes known as peptide synthetases assemble these molecules by the multiple carrier thiotemplate mechanism from a remarkable range of precursors, which can be N-methylated, acylated or reduced. Peptaibols and peptaibiotics show interesting physico-chemical and biological properties including the formation of pores in bilayer lipid membranes, as well as antibacterial, antifungal, occasionally antiviral activities, and may elicit plant resistance. The three-dimensional structure of peptaibols and pep­taibiotics is characterized predominantly by one type of the helical motifs a-helix, 310-helix and b-bend ribbon spiral. The aim of this review is to summarize the data available about the biosynthesis, biological activity and conformational properties of peptaibols and peptaibiotics described from Trichoderma species.

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Acta Microbiologica et Immunologica Hungarica
Authors: L. Kredics, Zsuzsanna Antal, A. Szekeres, L. Hatvani, L. Manczinger, Cs. Vágvölgyi, and Erzsébet Nagy

Cellulolytic, xylanolytic, chitinolytic and b-1,3-glucanolytic enzyme systems of species belonging to the filamentous fungal genus Trichoderma have been investigated in details and are well characterised. The ability of Trichoderma strains to produce extracellular proteases has also been known for a long time, however, the proteolytic enzyme system is relatively unknown in this genus. Fortunately, in the recent years more and more attention is focused on the research in this field. The role of Trichoderma proteases in the biological control of plant pathogenic fungi and nematodes has been demonstrated, and it is also suspected that they may be important for the competitive saprophytic ability of green mould isolates and may represent potential virulence factors of Trichoderma strains as emerging fungal pathogens of clinical importance. The aim of this review is to summarize the information available about the extracellular proteases of Trichoderma. Numerous studies are available about the extracellular proteolytic enzyme profiles of Trichoderma strains and about the effect of abiotic environmental factors on protease activities. A number of protease enzymes have been purified to homogeneity and some protease encoding genes have been cloned and characterized. These results will be reviewed and the role of Trichoderma proteases in biological control as well as their advantages and disadvantages in biotechnology will be discussed.

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Acta Microbiologica et Immunologica Hungarica
Authors: L. Kredics, Zsuzsanna Antal, A. Szekeres, L. Manczinger, Ilona Dóczi, F. Kevei, and Elisabeth Nagy

Species belonging to the filamentous fungal genus Trichoderma are well known as potential candidates for the biological control of plant pathogenic fungi and as cellulase producers of biotechnological importance. Several data were published in the last decade also about the clinical importance of this genus, indicating that Trichoderma strains may be potential opportunistic pathogens in immunocompromised patients. However, there is a lack of information about the potential virulence factors of clinical Trichoderma strains. This study was designed to examine the extracellular proteolytic enzymes of six clinical T. longibrachiatum isolates. Supernatants from induced liquid cultures of the examined strains were screened for proteolytic enzyme activities with 11 different chromogenic p-nitroaniline substrates. The production of trypsin-like, chymotrypsin-like and chymoelastase-like protease activities cleaving N-Benzoyl-L-Phe-L-Val-L-Arg-p-nitroanilide, N-Succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide,and N-Succinyl-L- Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide, respectively, was common among the strains examined. Separation of trypsin- and chymotrypsin-like activities by column chromatography revealed, that both systems are complex consisting of several isoenzymes. The pH-dependence of these two protease systems was also studied. Based on the results, the different isoenzymes seem to have different optimal pH values. Extracellular proteolytic enzymes may be involved in the pathogenecity of Trichoderma strains as facultative human pathogens.

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Acta Physiologica Hungarica
Authors: Levente Sára, GyL. Nádasy, P. Antal, M. Szekeres, A. Monori-Kiss, E. Horváth, A. Tőkés, G. Masszi, E. Monos, and Szabolcs Várbíró

To clarify the effects of dihydrotestosterone (DHT)-induced polycystic ovary syndrome (PCOS) on arteriolar biomechanics in a rat model and the possible modulatory role of vitamin D3.Methods and Results The PCOS model was induced in female Wistar rats by ten-weeks DHT treatment. Arteriolar biomechanics was tested in arterioles by pressure arteriography in control as well as DHT- and DHT with vitamin D3-treated animals in contracted and passive conditions. Increased wall stress and distensibility as well as increased vascular lumen were detected after DHT treatment. Concomitant vitamin D3 treatment lowered the mechanical load of the arterioles and restored the vascular diameter.Conclusion The hyperandrogenic state resulted in more rigid, less flexible arteriolar walls with increased vascular lumen compared with controls. DHT treatment caused eutrophic remodelling of gracilis arteriole. These prehypertensive alterations caused by chronic DHT treatment were mostly reversed by concomitant vitamin D3 administration.

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Acta Microbiologica et Immunologica Hungarica
Authors: Zsuzsanna Antal, L. Kredics, J. Pakarinen, Ilona Dóczi, Maria Andersson, Mirja Salkinoja-Salonen, L. Manczinger, A. Szekeres, L. Hatvani, C. Vágvölgyi, and Elisabeth Nagy

Potential virulence factors of 9 saprophytic and 12 clinical Trichoderma longibrachiatum strains were examined in the present study, in order to compare their capacity to cause infection in humans. All of the strains were able to grow at temperatures up to 40 °C and at pH values ranging from 2.0 to 9.0. Carbon and nitrogen source utilization experiments revealed that all of the strains were able to utilize a series of basic amino acids both as sole carbon and nitrogen sources. The MIC values of the tested antifungal drugs were found to be 0.016-8 µg/ml for amphotericin B, 64-256 µg/ml for fluconazole, 0.5-32 µg/ml for itraconazole and 0.008-1 µg/ml for ketoconazole in the case of the examinedis olates. Metabolites of the strains inhibited the growth of different bacteria, furthermore, compounds produced by three clinical isolates reduced the motility of boar spermatozoa, indicating their toxicity to mammalian cells as well. On the whole, there were no significant differences in the examined features between strains derived from clinical or soil samples. The question, however, whether all environmental Trichoderma longibrachiatum strains have the capacity to cause infections or not, remains still unanswered.

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Acta Alimentaria
Authors: Cs. Dobolyi, K. Inotai, I. Bata-Vidács, D. Sárkány, O. Csernus, S. Kocsubé, B. Tóth, A. Szekeres, and J. Kukolya

Abstract

Aspergillus strains were isolated from Hungarian mills in order to get information on the appearance of sterigmatocystin (ST) producing moulds, whose presence has never been demonstrated in Hungary. Fungal isolates were classified into nine morphotypes, sections Nigri, Nidulantes, Versicolores (two morphotypes), Circumdati, Flavi (two morphotypes), Clavati and Terrei by classical mycological assays. ST producing strains could be classified into section Versicolores. ST production of the isolates was assessed by liquid and solid phase growth experiments and compared to ST producing reference strains: Aspergillus pepii SzMC 22332, Aspergillus versicolor SzMC 22333, Aspergillus griseoaurantiacus SzMC 22334 and Aspergillus nidulans RDIT9.32. Four of our isolates marked as Km11, Km14, Km26 and Km31 showed ST production in liquid medium. ST production on solid phase corn grit substrate was measured after three weeks of incubation, and Km26 isolate proved to be the most prominent with a toxin concentration of 277.1 μg g−1, surpassing all reference strains. The toxin-producing ability of Km26 isolate was also tested in a field experiment, where corn was infected. By the end of the experiment, ST level of 19.56 μg kg−1 was measured in infected corn.

Molecular taxonomic identification of the Km26 strain was performed using internal transcribed spacer (ITS), calmodulin and tubulin sequence analyses. Based on these studies, strain Km26 was identified as Aspergillus creber.

Here we report that an ST-producing A. creber strain has appeared in Hungary, and the Km26 strain is the first known extreme ST-producing mould in this country. As a result of climate change, aflatoxin B1 producing Aspergillus flavus strains have appeared in Hungary in the last decade. As strain Km26 is the only A. creber isolate in Hungary so far, there is no sign of mass prevalence, and due to the lower temperature optimum of the species compared to A. flavus, its appearance is probably not related to climate change.

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