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- Author or Editor: J. Kukolya x
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Abstract
The present work aimed to study the yeast communities of whole crop corn silages (CS) that were previously contaminated with aflatoxin-producing Aspergillus flavus (CSCA). In addition, the effect of lactic acid bacterium (LAB) inoculation on the aflatoxin B1 (AFB1) content, genotoxicity, yeast load, and diversity of yeast communities were also investigated. In A. flavus contaminated silages, after two months, the AFB1 content was 40% lower with LAB inoculation, also a lower level of genotoxicity was determined. The number of yeasts cultured from the initial mixture of chopped whole crop corn was 4.8 × 107 CFU g−1 wet mass, while only 2.4 × 106 CFU g−1 from the CSCA and 7.1 × 105 CFU g−1 from the LAB-inoculated CSCA could be cultured. Based on 144 randomly isolated strains, the yeast community of the initial mixture consisted of 8 species. In contrast, the yeast community of CSCA consisted only of 4 species determined by 132 randomly selected isolates. LAB-inoculated CSCA consisted also of 4 species based on 158 randomly isolated strains. Saccharomyces cerevisiae and Pichia kudriavzevii proved to be predominant in the CSCA, while S. cerevisiae and Meyerozyma guilliermondii were the most abundant in the LAB-inoculated CSCA. The species richness was also confirmed by alpha diversity values (1.827, 1.188, and 1.123 as Shannon's indices for CS, CSCA, and LAB-inoculated CSCA, respectively). In response to LAB inoculation, the species diversity decreased considerably.
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.
Abstract
Thermobifida alba is the mesophilic member of the Thermobifida genus, the genome and enzyme sets of which have not been described and published yet. Thermobifida strains are thermotolerant actinomycete, which possess wide sets of cellulose and hemicellulose hydrolysing enzymes. Previously, three endomannanases (Man5ATh, Man5ATc, and Man5AThf) of thermobifidas were cloned and investigated, and hereby the endomannanase of T. alba DSM 43795 is described. All four endomannanases belong to the glycoside hydrolase family 5, their sizes are around 50–55 kDa. Their structure consists of a catalytic domain and a carbohydrate binding module, while there is an interdomain linker region in-between consisting repetitive tetrapeptide motifs (eg.: PPTEPTD-Ta, PTDP-Tc, TEEP-Tf, DPGT-Th). The pH optima of Man5A enzymes from T. alba, Thermobifida halotolerans, Thermobifida cellulosilytica, and Thermobifida fusca are slightly different (6.5, 7.0, 7.5, and 8.0, respectively), however, the temperature optima of the enzymes were detected within a wider range of 65–75 °C. In this research, Man5ATa exhibited the lowest Michaelis-Menten constant (KM) (0.13 mM) on LBG-mannan substrate, while others shared similar kinetic parameters: 0.9–1.7 mM of KM. Despite the high sequence similarity of the investigated mannanases, they exhibit different temperature stability parameters. These different functional characteristics can be advantageous for industrial applications producing biologically active, oligomannan prebiotics under different conditions.
Climate change affects the occurrence of fungi and their mycotoxins in foods and feeds. A shift has recently been observed in the presence of aflatoxin producer Aspergillus spp. in Europe, with consequent aflatoxin contamination in agricultural commodities including maize in several European countries that have not faced with this problem before, including, e.g. Northern Italy, Serbia, Slovenia, Croatia and Romania. Although aflatoxin contamination of agricultural products including maize is not treated as a serious threat to Hungarian agriculture due to climatic conditions, these observations led us to examine the mycobiota of maize kernels collected from Hungarian maize fields. Using a calmodulin sequence-based approach, A. flavus isolates have been identified in 63.5% of the maize fields examined in 2009 and 2010, and 18.8% of these isolates were found to be able to produce aflatoxins above 5 μg kg−1 on maize kernels as determined by ELISA, HPLC-FL, HPLC-MS analyses and SOS-Chromotest. These data indicate that aflatoxin producing Aspergilli are present in Hungarian agricultural fields, consequently climate change with elevated temperatures could lead to aflatoxin contamination of Hungarian agricultural products, too.
Thermobifidas are thermophilic, aerobic, lignocellulose decomposing actinomycetes. The Thermobifida genus includes four species: T. fusca, T. alba, T. cellulosilytica, and T. halotolerans. T. fusca YX is the far best characterized strain of this taxon and several cellulases and hemicellulases have been cloned from it for industrial purposes targeting paper industry, biofuel, and feed applications. Unfortunately, sequence data of such enzymes are almost exclusively restricted to this single species; however, we demonstrated earlier by zymography that other T. alba and T. cellulosilytica strains encode the same enzyme sets. Recently, the advances in whole genome sequencing by the use of next generation genomics platforms accelerated the selection process of valuable hydrolases from uncharacterized bacterial species for cloning purposes. For this purpose T. cellulosilytica TB100T type strain was chosen for de novo genome sequencing. We have assembled the genome of T. cellulosilytica strain TB100T into 168 contigs and 19 scaffolds, with reference length of 4 327 869 bps, 3 589 putative coding sequences, 53 tRNAs, and 4 rRNAs. The analysis of the annotated genome revealed the existence of 27 putative hydrolases belonging to 14 different glycoside hydrolase (GH) families. The investigation of identified, cloned, and heterologously multiple cellulases, mannanases, xylanases, and amylases may result in industrial applications beside gaining useful basic research related information.
