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Fungal nonribosomal peptide synthetases (NRPSs) are responsible for the biosynthesis of numerous metabolites which serve as virulence factors in several plant-pathogen interactions. The aim of our work was to investigate the diversity of these genes in a Fusarium graminearum sequence database using bioinformatic techniques. Our search identified 15 NRPS sequences, among which two were found to be closely related to peptide synthetases of various fungi taking part in ferrichrome biosynthesis. Another peptide synthetase gene was similar to that identified in Aspergillus oryzae which is possibly responsible for the biosynthesis of fusarinine, an extracellular iron-chelating siderophore. To our knowledge, this is the first report on the identification of a putative NRPS gene possibly responsible for the biosynthesis of fusarinine-type siderophores. The other NRPSs were found to be related to peptide synthetases taking part in the biosynthesis of various peptides in other fungi. Transcription factors carrying ankyrin repeats were observed in the vicinity of four of the identified peptide synthetase genes. Additionally, NRPS related genes similar to putative long-chain fatty acid CoA ligases, acyl CoA ligases, ABC transport proteins, a highly conserved putative transmembrane protein of Aspergillus nidulans, and a-aminoadipate reductases have also been identified. Further studies are in progress to clarify the role of some of the identified NRPS genes in plant pathogenesis.
Ochratoxin A is a mycotoxin produced by Aspergillus and Penicillium species. This mycotoxin is a common contaminant of various foods including cereal products, spices, dried fruits, coffee, beer and wine. Besides cereal products, goods of grape origin contribute significantly to ochratoxin exposure of humans. The ochratoxin content and mycobiota of raisins purchased in Hungarian outlets were examined in this study. Ochratoxin A content was examined by an immunochemical technique, and the results were confirmed by HPLC analysis using fluorescent detection. Altogether 20 raisin samples were analyzed. Ochratoxin A was detected in all but two samples with ochratoxin concentrations ranging from 0 to 6.2 mg kg-1. The most heavily contaminated raisin sample came from Iran. However, none of the raisins contained ochratoxin A above 10 mg kg-1, the European Community maximum allowable limit in raisins. The mycobiota of raisin samples was also examined to clarify which species could be responsible for ochratoxin A contamination. All except three raisin samples were contaminated with black aspergilli, some of which produced ochratoxin A. Besides A. carbonarius, ochratoxin producing A. tubingensis isolates dominated in the samples.
In order to identify a specific marker for T. harzianum AS12-2, a strain capable of controlling rice sheath blight caused by Rhizoctonia solani, UP-PCR was performed using five universal primers (UP) both separately and in pairwise combinations. The application of two UP primers resulted in the amplification of unique fragments from the genomic DNA of T. harzianum AS12-2, clearly distinguishing it from other Trichoderma strains. The unique fragments had no significant sequence homology with any other known sequence available in databases. Based on the sequences of the unique fragments, 14 oligonucleotide primers were designed. Two primer sets amplified a fragment of expected size from the DNA of strain T. harzianum AS12-2 but not from any other examined strains belonging to T. harzianum, to other Trichoderma species assayed, or to other common fungi present in paddy fields of Mazandaran province, Iran. In conclusion, SCAR (sequence characterized amplified regions) markers were successfully identified and rapid, reliable tools were provided for the detection of an effective biocontrol Trichoderma strain, which can facilitate studies of its population dynamics and establishment after release into the natural environment.
Ochratoxin A is a mycotoxin produced by Aspergillus and Penicillium species. This mycotoxin is a common contaminant of various foods including cereal products, spices, dried fruits, coffee, beer and wine. Besides species assigned to Aspergillus section Circumdati, black Aspergilli including A. niger, A. carbonarius and A. sclerotioniger are also able to produce this mycotoxin. Black Aspergilli have been found to be the predominant fungi contaminating pistachio nuts worldwide. We examined the species distribution of black Aspergilli on Iranian pistachio nuts. Sequence-based identifications have been carried out using partial calmodulin sequence data. Our data indicate that instead of the potential ochratoxin and fumonisin producing A. niger species, A. tubingensis dominates on Iranian pistachio nuts. This species is unable to produce either of these mycotoxins, consequently do not contribute to mycotoxin contamination of pistachio nuts in Iran. Further studies are in progress to clarify the role of other Aspergilli in ochratoxin contamination of pistachio in Iran.
Black mould rot caused by black Aspergilli is an important post-harvest disease of onion worldwide. Usually Aspergillus niger is cited as the causative agent based on morphological criteria. In this study, the mycobiota and fumonisin contamination of mouldy onion bulbs purchased in Hungary were examined. All except one of the examined mouldy samples were found to be contaminated with black Aspergilli, which could be isolated both from the outer dry and the inner fleshy scales of onion bulbs. Species assignment of the isolates was carried out using sequence analysis of part of the calmodulin gene. Sequence data revealed that all 35 black Aspergilli isolated from onions belong to the Aspergillus awamori species. The range of fumonisin isomers present in the onion samples was also examined using reversed-phase high-performance liquid chromatography/electrospray ionization-ion trap mass spectrometry. Two of the examined onion samples were found to be contaminated with fumonisins at a rate of about 0.3 mg kg−1. This is the first report on fumonisin contamination of onion bulbs. The fumonisin isomers observed include fumonisins B2–4, 3-epi-FB4, iso-FB1 (FB6) and an iso-FB2,3 form. The range of fumonisin isomers detected in the onion bulbs indicates that probably A. awamori is responsible both for mould rot and fumonisin contamination of onions in Hungary.
Ochratoxin A is a mycotoxin produced by Aspergillus and Penicillium species. This mycotoxin is a common contaminant of various foods including cereal products, spices, dried vine fruits, coffee, cocoa, beer and wine. Apart from cereal products, beer and wine contribute significantly to ochratoxin exposure of humans. In the Mediterranean region of Europe, the black Aspergillus species are the sources of ochratoxin contamination of grape products. In this study, we examined the source of ochratoxin contamination of grapes in Hungary and the Czech Republic. The mycobiota of grape berries from 25 Hungarian and Czech vineyards was examined. Potential ochratoxin producing fungi were only identified in grapes from Southern Hungary. Among the 16 black Aspergillus strains isolated, 12 belong to the A. niger species, and 10 produced small amounts (1.5–10 μg kg −1 ) of ochratoxin A in a liquid medium. We could also identify an A. tubingensis isolate which produced 3.5 μg kg −1 ochratoxin A in a liquid medium at pH 6.0. However, the amount of ochratoxin A produced was very low even in a medium which is favourable for mycotoxin production, and ochratoxin A was not detected in any of the grape juice, must and wine samples examined, indicating the absence of health hazard to costumers. Other potentially toxigenic fungi including Aspergillus flavus, Penicillium expansum and Alternaria species were also isolated. Further studies are in progress to evaluate the importance of these fungi in food safety.
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.
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.
