Molecular identification of Aspergillus and Eurotium species isolated from rice and their toxin-producing ability

Thirty milled rice samples were collected from retailers in 4 provinces of Malaysia. These samples were evaluated for Aspergillus spp. infection by direct plating on malt extract salt agar (MESA). All Aspergillus holomorphs were isolated and identified using nucleotide sequences of ITS 1 and ITS 2 of rDNA. Five anamorphs (Aspergillus flavus, A. oryzae, A. tamarii, A. fumigatus and A. niger) and 5 teleomorphs (Eurotium rubrum, E. amstelodami, E. chevalieri, E. cristatum and E. tonophilum) were identified. The PCR-sequencing based technique for sequences of ITS 1 and ITS 2 is a fast technique for identification of Aspergillus and Eurotium species, although it doesn't work flawlessly for differentiation of Eurotium species. All Aspergillus and Eurotium isolates were screened for their ability to produce aflatoxin and ochratoxin A (OTA) by HPLC and TLC techniques. Only A. flavus isolate UPM 89 was able to produce aflatoxins B1 and B2.     

The genus Eurotium - members of indigenous fungal community in hypersaline waters of salterns

Six different species of the known teleomorphic food-borne xerophilic genus Eurotium were repeatedly isolated in a mycodiversity study of hypersaline waters. At salinities above 17% NaCl, E. amstelodami was detected most consistently, followed by E. repens and E. herbariorum, while E. rubrum, E. chevalieri and a potentially new species, "Eurotium halotolerans", were detected only occasionally at lower salinities. The qualitative secondary metabolite profiles produced by Eurotium spp. from salterns were not different from those of Eurotium spp. from foods and other habitats. Spatiotemporal frequency of occurrence and in vitro determined adaptive ability of propagules to survive prolonged exposure to hypersaline conditions indicate that E. amstelodami, E. herbariorum, and E. repens contribute to the indigenous fungal community in hypersaline water environments, while E. rubrum, E. chevalieri and "E. halotolerans" are only temporal inhabitants of brine at lower salinities.     

Tyrosine kinase activation in response to fungal spores is primarily dependent on endogenous reactive oxygen production in macrophages

Studies from our laboratory (Shahan, T. A., Sorenson, W. G., and Lewis, D. M. (1994) Environ. Res. 67, 98-104) demonstrated that spores from different fungal species differentially activate rat alveolar macrophages as detected by the measurement of superoxide anion and cytokine production (Shahan, T. A., Siegel, P. D., Sorenson, W. G., Kuschner, W. G., and Lewis, D. M. (1998) Am. J. Respir. Cell Mol. Biol. 18, 435-441). Spores from Aspergillus candidus stimulated production of the highest levels of superoxide anion (5.2 nmol/1.0 x 10(6) alveolar macrophages (AMs)/30 min), followed by those from Aspergillus niger (2.4 nmol/1.0 x 10(6) AMs/30 min) and Eurotium amstelodami (0.4 nmol/1.0 x 10(6) AMs/30 min). The mechanism of this differential activation was studied. Our data demonstrate that the tyrosine kinases p56(Hck), p72(Syk), p77(Btk), p62(Yes), p56(Lck), and p59(Fyn) were specifically activated in response to spores from A. candidus, whereas spores from either A. niger or E. amstelodami activated p56(Hck), p72(Syk), and p77(Btk). Kinetic analysis of specific tyrosine kinases demonstrated that p56(Hck), p72(Syk), and p77(Btk) were activated faster and to a greater extent by spores from A. candidus as compared with spores from E. amstelodami. These data suggest a relationship between reactive oxygen species and tyrosine kinase activation. Treatment of AMs with H(2)O(2) (1 mM) caused the activation of p72(Syk) only, whereas treatment with superoxide dismutase and catalase before treatment with the spores had no effect on tyrosine kinase activation. Incubation with NADPH oxidase inhibitors inhibited both superoxide anion production and the activation of p56(Hck), p72(Syk), and p77(Btk) in response to fungal spores. These data indicate that endogenous reactive oxygen species are necessary for the activation of p56(Hck), p72(Syk), and p77(Btk) by spores; they also indicate that some species of spores are capable of activating tyrosine kinases independent of superoxide anion.     

三株散囊菌发酵茶叶的初步研究

散囊菌属真菌(Eurotium spp.)能赋予发酵茶独特的口感和香味。本研究利用前期从广西某六堡茶中筛选并鉴定的三株散囊菌属真菌Aspergillus chevalieri E2、Aspergillus chevalieri E3与Aspergillus cristatus E6,探讨在不同温度下以优化察氏液体培养基培养的生长状况,发酵前不同灭菌条件下的茶叶品质,以及所得茶汤中茶多酚含量、总抗氧化能力和DPPH·自由基清除能力。结果显示:三株真菌在优化的察氏液体培养基中31℃～34℃下都能良好生长。茶叶发酵温度为28℃,三株真菌在发酵初始含水量为20%以上生长良好,其中E3和混合发酵组的生长速度最快。E2在茶叶表面生长出大量金黄色子囊果以及大量浅绿色分生孢子梗; E3几乎只有浅绿色分生孢子梗; E6几乎只有金黄色子囊果。发酵茶叶制作的茶汤内茶多酚含量比未发酵低,抗氧化性指标也有所下降,说明本实验真菌发酵促进了茶内抗氧化物质的氧化。本研究对源于六堡茶不同散囊菌属真菌的茶叶发酵有重要参考价值。     

Hypersaline waters - a potential source of foodborne toxigenic aspergilli and penicillia

Previous studies of hypersaline environments have revealed the dominant presence of melanized yeast-like fungi and related Cladosporium spp. In this study, we focused on the genera Aspergillus and Penicillium and their teleomorphic forms. From oligotrophic and eutrophic hypersaline waters around the world, 60 different species were identified, according to their morphological characteristics and extrolite profiles. For the confirmation of five new species, additionally, sequence analysis of the internal transcribed spacer region, the partial large subunit-rDNA and the partial β-tubulin gene was performed. The species Aspergillus niger, Eurotium amstelodami and Penicillium chrysogenum were detected with the highest frequencies at all of the sampled sites; thus, they represent the pan-global stable mycobiota in hypersaline environments. Possible candidates were also Aspergillus sydowii and Eurotium herbariorum, as they were quite evenly distributed among the sampled sites, and Aspergillus candidus, which was abundant, but more locally distributed. These species and their byproducts can accumulate downstream following evaporation of brine, and they can become entrapped in the salt crystals. Consequently, marine salt used for consumption can be a potential source of food-borne fungi and their byproducts. For example, ochratoxin-A-producing species Penicillium nordicum was recovered from brine, salt and salted meat products.     

Taxonomy of Eurotium species isolated from meju

Eurotium strains were isolated from 77 loaves of meju (dried fermented soybeans), in various regions of Korea from 2008 to 2010. Morphological characteristics and DNA sequences of β-tubulin were examined. They were identified as Eurotium amstelodami, E. chevalieri, E. herbariorum, E. repens, E. rubrum, and E. tonophilum. Of these species, E. chevalieri and E. tonophilum had not been previously reported in association with meju. E. chevalieri and E. repens were the species isolated most frequently. This paper summarizes the morphological characteristics of six Eurotium species and provides key to identify the species from meju.     

Molecular identification of <Emphasis Type="Italic">Aspergillus</Emphasis> and <Emphasis Type="Italic">Eurotium</Emphasis> species isolated from rice and their toxin-producing ability

Thirty milled rice samples were collected from retailers in 4 provinces of Malaysia. These samples were evaluated for Aspergillus spp, infection by direct plating on malt extract salt agar (MESA). All Aspergillus holomorphs were isolated and identified using nucleotide sequences of ITS 1 and ITS 2 of rDNA. Five anamorphs (Aspergillus flavus, A. oryzae, A. tamarii, A. fumigatus and A. nigef) and 5 teleomorphs (Eurotium rubrum, E. amstelodami, E. chevalieri, E. cristatum and E. tonophilum) were identified. The PCR-sequencing based technique for sequences of ITS 1 and ITS 2 is a fast technique for identification of Aspergillus and Eurotium species, although it does not work flawlessly for differentiation of Eurotium species. All Aspergillus and Eurotium isolates were screened for their ability to produce aflatoxin and ochratoxin A (OTA) by HPLC and TLC techniques. Only A. flavus isolate UPM 89 was able to produce aflatoxins B1 and B2.     

三株散囊菌属真菌的茶叶液态发酵与其成分分析

散囊菌属真菌(Eurotium spp.)又称“金花菌”,能在六堡茶表面生长出金黄色的子囊果而得名,是一类对发酵茶叶的品质有积极影响的真菌。本研究前期从广西梧州六堡茶中筛选到的三株散囊菌属真菌Aspergillus chevalieri E2(简称E2),Aspergillus chevalieri E3(简称E3)与Aspergillus cristatus E6(简称E6),将菌株接种于自制液体茶粉培养基中培养,检测发酵液中产生的色素物质;测定发酵液中纤维素酶、果胶酶、淀粉酶的酶活力、总糖和还原糖糖含量、氨基酸含量、总抗氧化能力、自由基清除能力和茶多酚含量。结果表明:三株真菌在液体茶粉培养基中生长良好,E3生长尤其迅速。在8 d的培养期限内,E2、E3和E6的最高果胶酶活力分别为2.52、3.44和5.15 U/mL;最高纤维素酶活力分别为0.57、0.82和1.10 U/mL;最高淀粉酶活力分别为0.31、0.35和0.49 U/mL。三株真菌的发酵后培养液中还原糖含量大幅增加,增幅分别达到131.70%、627.02%和459.12%;总糖含量同样呈上升趋势,增幅分别达到6.84%、35.25%和2.07%;E2与E3的氨基酸含量分别下降了17.43%与32.68%,E6则上升了0.52%;E3的总抗氧化活性提升最大,达65.87%;DPPH·自由基清除能力提升最大的是E2,达128.33%;茶多酚含量都显著提升,增幅分别为50.85%、51.58%和53.06%。本研究对改善六堡茶的品质及茶叶的发酵的研究有一定意义和参考价值。     

茯砖茶中优势菌种的鉴定

所谓“黄霉菌”是茯砖茶发酵中的益菌。它实际上是散囊菌属产生的黄色闭囊壳。优势种经鉴定为冠突散囊菌[Eurotium cristatum(Raper ＆ Fennell)Malloch & cain],它的无性型是针刺曲霉(Aspergillus spiculosus Blaser),异名是冠突曲霉(A.cristatus Raper &Fennell)。文中还对有关该菌的命名问题和在我国的分布作了讨论。     

Antifungal activity of volatile compounds generated by essential oils against fungi commonly causing deterioration of bakery products

AIMS: To investigate the volatile fractions of 16 essential oils for activity against the more common fungi causing spoilage of bakery products, Eurotium amstelodami, E. herbariorum, E. repens, E. rubrum, Aspergillus flavus, A. niger and Penicillium corylophilum. METHODS AND RESULTS: The study applied 50 microl of pure essential oils in a sterilized filter paper, were carried out at pH 6 and at different water activity levels (0.80-0.90). First, a wheat flour based agar medium was used, where cinnamon leaf, clove, bay, lemongrass and thyme essential oils where found to totally inhibit all microorganisms tested. These five essential oils were then tested in sponge cake analogues, but the antifungal activity detected was much more limited. CONCLUSION: Five essential oils showed potential antifungal capacity against all species tested, over a wide range of water availability. Their activity, however, seems to be substrate-dependent. More research is needed to make them work in real bakery products, as in the preliminary study limited effectiveness was found. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of the cinnamon leaf, clove, bay, lemongrass and thyme essential oils against species belonging to Eurotium, Aspergillus and Penicillium genus has been demonstrated.     

Biocontrol of mold growth in high-moisture wheat stored under airtight conditions by Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae.

Pichia anomala inhibits the growth of Penicillium roqueforti and Aspergillus candidus on agar. In this investigation, antagonistic activity on agar against 17 mold species was determined. The abilities of Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae to inhibit the growth of the mold Penicillium roqueforti in nonsterile high-moisture wheat were compared by adding 10(3) Penicillium roqueforti spores and different amounts of yeast cells per gram of wheat. Inoculated grain was packed in glass tubes, incubated at 25 degrees C with a restricted air supply, and the numbers of yeast and mold CFU were determined on selective media after 7 and 14 days. Pichia anomala reduced growth on agar plates for all of the mold species tested in a dose-dependent manner. Aspergillus fumigatus and Eurotium amstelodami were the most sensitive, while Penicillium italicum and Penicillium digitatum were the most resistant. Pichia anomala had the strongest antagonistic activity in wheat, with 10(5) and 10(6) CFU/g completely inhibiting the growth of Penicillium roqueforti. Inhibition was least pronounced at the optimum temperature (21 degrees C) and water activity (0.95) for the growth of Penicillium roqueforti. Pichia guilliermondii slightly reduced the growth of Penicillium roqueforti in wheat inoculated with 10(5) and 10(6) yeast CFU/g. S. cerevisiae inhibited mold growth only weakly at the highest inoculum level. Pichia anomala grew from 10(3) to 10(7) CFU/g of wheat in 1 week. To reach the same level, Pichia guilliermondii had to be inoculated at 10(4) CFU while S. cerevisiae required an inoculum of 10(5) CFU to reach 10(7) CFU/g of wheat.     

Identity of the xerophilic species Aspergillus penicillioides: Integrated analysis of the genotypic and phenotypic characters

We examined the identity of Aspergillus penicillioides, the typical xerophilic and strictly anamorphic species, using an integrated analysis of the genotypic and phenotypic characters. Our experimental methods on two genotypic characters, i.e., DNA base composition using the HPLC method and DNA relatedness using the nitrocellulose filter hybridization technique between A. flavus, A. oryzae, and their close relations revealed a good agreement with the values by buoyant density (for DNA base composition) and spectrophotometric determination (for DNA relatedness) reported by Kurtzman et al. in 1986. On the basis of these comparisons, we examined DNA base composition and DNA relatedness of six selected strains of A. penicillioides, including IFO 8155 (originally described as A. vitricola), one strain of A. restrictus, and the respective strains from Eurotium amstelodami, E. repens, and E. rubrum. As a result, five strains within A. penicillioides, including the neotype strain NRRL 4548, had G+C contents of 46 to 49 mol%, whereas IFO 8155 had 50 mol%. A. restrictus had 52 mol%, and three Eurotium species ranged from 46 to 49 mol%. The DNA relatedness between A. penicillioides (five strains), except for IFO 8155, exhibited values greater than 70%, but the DNA complementarity between four strains and IFO 8155 in A. penicillioides revealed values of less than 40%. DNA relatedness values between three species of Eurotium were 65 to 72%. We determined 18S, 5.8S, and ITS rDNA sequences as other genotypic characters from A. penicillioides (six strains), A. restrictus, and related teleomorphic species of Eurotium. In three phylogenetic trees inferred from these sequences, five strains of A. penicillioides, including the neotype strain, were closely related to each other, whereas IFO 8155 was distantly related and grouped with other xerophilic species. Our results have suggested that A. penicillioides typified by NRRL 4548 and A. penicillioides IFO 8155 (ex holotype of A. vitricola) are not conspecific. The enzyme patterns as a genotypic character and general morphology and conidial ornamentation types as phenotypic characters supported this conclusion. Therefore the name A. vitricola Ohtsuki, typified by the holotype strain IFO 8155, should be revived. Evolutionary affinities among Aspergillus species and related teleomorphs, including the xerophilic taxa, are discussed.     

Influence of solute and hydrogen ion concentration on the water relations of some xerophilic fungi.

Germination and growth of six xerophilic fungi, Aspergillus flavus, Aspergillus ochraceus, Eurotium chevalieri, Chrysosphorium fastidium, Wallemia sebi and Xeromyces bisporus were examined on media of a wide range of water activities (aw). The influence of three solutes, NaCl, glycerol and a glucose/fructose mixture, was studied at pH 4-0 and pH 6-5 using a plate-slide technique. Germination times and growth rates were affected by solute type, but the influence of pH was less marked. Except for Wallemia sebi, the fungi grew most strongly on glucose/fructose and were partially or completely inhibited by NaCl. The results showed that a universal isolation medium for xerophilic fungi could be based on glycerol or glucose/fructose but not on NaCl as aw-limiting solute.     

Keratinophilic fungi and other moulds associated with air-dust particles from Egypt

One-hundred and eleven species and three species varieties belonging to 39 genera were collected from 50 dust samples on the five media used at 28°C. Using the hair-baiting technique with horse hair, 10 species ofChrysosporium were isolated:C. asperatum, C. state ofArthroderma tuberculatum, C. indicum, C. inops, C. keratinophilum, C. merdarium, C. pannorum, C. queenslandicum, C. tropicum andC. xerophilum. True dermatophytes were isolated:Trichophyton verrucosum andTrichophyton sp. Also, numerous fungi tolerating high levels of cycloheximide were encountered, such as members ofAcremonium, Aspergillus andPenicillium. On plates of glucose or cellulose Czapek-Dox agar (free from sucrose) the most frequent fungi were:Alternaria alternata, Aspergillus flavus, A. flavus var.columnaris, A. fumigatus, A. niger, A. ochraceus, A. sydowii, A. terreus, Chaetomium globosum, Cladosporium herbarum, Emericella nidulans, Fusarium oxysporum, Mucor hiemalis, Penicillium chrysogenum, P. oxalicum, Scopulariopsis brevicaulis andUlocladium atrum. On plates of 50% sucrose or 10 and 20% NaCl-Czapek's agar, some interesting species were frequently encountered:Eurotium amstelodami, E. chevalieri, E. halophilicum, E. montevidensis, E. repens, E. rubrum andScopulariopsis halophilica. The isolated, fungi have been tested for osmophilicity and halophilicity, they showed different rates of growth on sucrose and sodium chloride-Czapek's medium of various osmotic potential.     

Identification of volatile markers for indoor fungal growth and chemotaxonomic classification of Aspergillus species

Microbial volatile organic compounds (MVOCs) were collected in water-damaged buildings to evaluate their use as possible indicators of indoor fungal growth. Fungal species isolated from contaminated buildings were screened for MVOC production on malt extract agar by means of headspace solid-phase microextraction followed by gas chromatography-mass spectrometry (GC-MS) analysis. Some sesquiterpenes, specifically derived from fungal growth, were detected in the sampled environments and the corresponding fungal producers were identified. Statistical analysis of the detected MVOC profiles allowed the identification of species-specific MVOCs or MVOC patterns for Aspergillus versicolor group, Aspergillus ustus, and Eurotium amstelodami. In addition, Chaetomium spp. and Epicoccum spp. were clearly differentiated by their volatile production from a group of 76 fungal strains belonging to different genera. These results are useful in the chemotaxonomic discrimination of fungal species, in aid to the classical morphological and molecular identification techniques.     

Effect of preharvest fungicides and interacting fungi on Aspergillus carbonarius growth and ochratoxin A synthesis in dehydrating grapes

AIMS: To evaluate the effect of preharvest grape pesticides in Aspergillus section Nigri infection in dehydrating grapes and the final ochratoxin A (OTA) content. Additionally, the effect of coinoculation of moulds frequently isolated from grapes and raisins on Aspergillus section Nigri infection was studied. METHODS AND RESULTS: Fungicide-treated grapes were inoculated with Aspergillus carbonarius, Aspergillus niger aggregate, Eurotium amstelodami and Penicillium janthinellum in different combinations, then dehydrated by reducing a(w) for 20 days. The percentages of colonized grapes treated with fungicides were, in general, lower, but no differences were observed among fungicides. The untreated grapes always showed higher concentrations of OTA, regardless of the inoculum applied. In general, Chorus was the most effective antifungal treatment in reducing OTA accumulation in grapes during dehydration. Penicillium janthinellum reduced Aspergillus section Nigri colonization and OTA accumulation in grapes during dehydration. CONCLUSIONS: The four preharvest fungicides studied reduced the Aspergillus section Nigri growth and OTA production by A. carbonarius during dehydration of grapes. SIGNIFICANCE AND IMPACT OF THE STUDY: The success of these chemical treatments might depend on the mycobiota composition of grapes.     

Eurocristatine,a new diketopiperazine dimer from the marine sponge-associated fungus Eurotium cristatum

A new diketopiperazine dimer, eurocristatine (1), was isolated, in addition to eight known metabolites including the anthraquinones erythroglaucin, physcion, catenarin, emodin and the dioxopiperazine alkaloids echinulin, neoechinulin A, neoechinulin E, variecolorin J, from the culture of the sponge-associated fungus Eurotium cristatum KUFC 7356. The structure of eurocristatine (1) was established based on 1D and 2D NMR spectral analysis as well as HRESIMS, and the absolute configuration of its stereogenic carbons was determined by an X-ray crystallographic analysis. Eurocristatine (1) did not exhibit cytotoxic, antibacterial or antifungal activity.     

Environmental factors and nutritional utilization patterns affect niche overlap indices between Aspergillus ochraceus and other spoilage fungi

The total number, and the type of C source utilized in common by an ochratoxin producing strain of Aspergillus ochraceus and six other spoilage fungi varied with the range used (95, Biolog GN plates, or 18 major C sources found in maize). The niche size and niche overlap index (NOI) were markedly influenced by water availability (water activity, aw) and temperature. With freely available water (0.995 aw) there was ecological similarity between A. ochraceus, A. alternata, A. candidus and A. flavus, all exploiting the same sources at both 18 and 30 degrees C. However, under water stress, A. ochraceus had a low NOI compared with A. niger, A. flavus and A. candidus, and Eurotium spp., suggesting that there was very little niche overlap in utilization of different C sources. The NOIs were not directly related to growth rates. Niche overlap between species was shown to be in a state of flux and significantly influenced by environmental factors. This may have important implications in understanding ecological interactions, and dominance by species in a community, and for pre-emptive exclusion of specific species.     

Microbial populations, ammonification and nitrification in soil treated with urea and inorganic salts

Soil fertilization with urea at rates of 0.2 and 0.5 mg N/g soil was toxic for total counts of bacteria and fungi except with cellulolytic fungi where growth was promoted by addition of urea after 90-d incubation. Also, the population numbers of both bacteria and fungi were significantly decreased when soil was amended with CaCl₂ and K₂SO₄ applied at two levels (50 and 100 μmol/g soil). Some alleviation of the toxic effect of either urea or the inorganic salts was observed when they were applied in combination. Fungal species composition was found to be affected in the treated soil. The most tolerant fungi wereAlternaria alternata, Aspergillus flavus, A. fumigatus, A. niger, A. terreus, Eurotium amstelodami, E. chevalieri, Nectria hœmatococca, Pœcilomyces variotii andStachybotrys chartarum. Other species of fungi were either sensitive to all treatments or sensitive to some and tolerant to others. Ammonium nitrogen was found to be more accumulated in soil treated with urea mixed with inorganic salts compared with soil treated only with urea whereas NO ₃ ⁻ -N (and NO ₂ ⁻ -N) was decreased. The overall effect of the addition of inorganic salts on total mineralized nitrogen, however was promotive. Soil pH was increased in ureatreated soil but was depressed by application of CaCl₂ or K₂SO₄ to values lower than that of untreated soil. The results may be of agronomical interest in overcoming problems encountered with urea application as N fertilizer.     

云南省可培养丝孢真菌资源的调查研究Ⅱ

本文继续报道曲霉属的16个分类群：杂色曲霉原变种,杂色曲霉疣梗变种,聚多曲霉,焦曲霉,土曲霉原变种,土曲霉金色变种,黄柄曲霉,温特曲霉,黄曲霉原变种,黄曲霉柱头变种,溜曲霉,炭黑曲霉,黑曲霉,泡盛曲霉,塔宾曲霉和日本曲霉。