Seed-borne fungi of date-palm,Phoenix dactylifera L. from Saudi Arabia

Six different varieties of date-palm viz. Sukhari, Saggae, Rotana, Kholasi, Rashoodia and Nabtat Ali, were screened for seed-borne fungi. Eleven species belonging to nine different genera of fungi were isolated. The genera isolated were Alternaria, Eurotium, and Fusarium (two species), Aspergillus, Drechslera, Penicillium, Rhizopus, and Curvularia (one species each). This is the first record of seed-borne fungi from Phoenix dactyliera L. in Saudi Arabia.     

Effect of water activity and temperature on the growth of Eurotium species isolated from animal feeds

Background

Xerophilic fungi represent a serious problem due to their ability to grow at low water activities causing the spoiling of low and intermediate moisture foods, stored goods and animal feeds, with the consequent economic losses.

Additional notes on species ofAspergillus,Eurotium andEmericella from Egyptian desert soil

Aspergillus floriformis, A. pseudodeflectus, Eurotium xerophilum (st. con.A. xerophilus) andEmericella purpurea (st. con.A. purpureus) are described and illustrated as new species. In addition the morphology of strains identified asAspergillus melleus, A. caespitosus andA. versicolor is discussed.     

分离自黑茶的散囊菌属真菌中的NRPS基因的检测和分布

【目的】检测源自黑茶的散囊菌属分离菌株中的NRPS基因,推测它们相应的次级代谢产物合成能力,并考察了基于NRPS基因分布特征进行散囊菌属真菌遗传多样性分析的可能性。【方法】运用IQS法提取来自湖南、四川和湖北等地黑茶样品的14株散囊菌(Eurotium spp.)菌株的总DNA,利用特异性引物对菌株中11种NRPS基因片段进行PCR检测和序列分析,并结合UPGMA法分析了NRPS基因的分布。【结果】各菌株分别含有4 10个NRPS基因片段。NRPS7、CPS1基因在所有菌株中都可以检测到,而NRPS2、NRPS6基因则仅在个别菌株中检测到;其它NRPS基因的分布则呈现出多样性。来自同一块砖茶的菌株Fw-30813-7、Fw-30813-4、Fw-30813-1和Fw-30925-5之间,NRPS1、NRPS2、NRPS3、NRPS4和NRPS8五个基因的分布存在差异,显示出黑茶内散囊菌的遗传多样性。来自湖南白沙溪茶厂的茯砖样品的菌株Fa-20719-3不含有NRPS5和NRPS8基因;而从湖南益阳茶厂茯砖茶7个分离菌株中均含有这两种基因。不同地点加工出来的黑茶样品中的散囊菌菌株之间存在一些明显的遗传差异。【结论】首次从我国黑茶中的散囊菌属分离菌株中检测到NRPS基因,并推测了NRPS产物的多样性,展示了其多样化分布特征,提示NRPS基因的分析有可能成为衡量散囊菌遗传多样性的另一个尺度。     

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.     

Purification and Characterization of Novel Antifungal Compounds from the Sourdough Lactobacillus plantarum Strain 21B

Sourdough lactic acid bacteria were selected for antifungal activity by a conidial germination assay. The 10-fold-concentrated culture filtrate of Lactobacillus plantarum 21B grown in wheat flour hydrolysate almost completely inhibited Eurotium repens IBT18000, Eurotium rubrum FTDC3228, Penicillium corylophilum IBT6978, Penicillium roqueforti IBT18687, Penicillium expansum IDM/FS2, Endomyces fibuliger IBT605 and IDM3812, Aspergillus niger FTDC3227 and IDM1, Aspergillus flavus FTDC3226, Monilia sitophila IDM/FS5, and Fusarium graminearum IDM623. The nonconcentrated culture filtrate of L. plantarum 21B grown in whole wheat flour hydrolysate had similar inhibitory activity. The activity was fungicidal. Calcium propionate at 3 mg ml⁻¹ was not effective under the same assay conditions, while sodium benzoate caused inhibition similar to L. plantarum 21B. After extraction with ethyl acetate, preparative silica gel thin-layer chromatography, and chromatographic and spectroscopic analyses, novel antifungal compounds such as phenyllactic and 4-hydroxy-phenyllactic acids were identified in the culture filtrate of L. plantarum 21B. Phenyllactic acid was contained at the highest concentration in the bacterial culture filtrate and had the highest activity. It inhibited all the fungi tested at a concentration of 50 mg ml⁻¹ except for P. roqueforti IBT18687 and P. corylophilum IBT6978 (inhibitory concentration, 166 mg ml⁻¹). L. plantarum 20B, which showed high antimold activity, was also selected. Preliminary studies showed that phenyllactic and 4-hydroxy-phenyllactic acids were also contained in the bacterial culture filtrate of strain 20B. Growth of A. niger FTDC3227 occurred after 2 days in breads started with Saccharomyces cerevisiae 141 alone or with S. cerevisiae and Lactobacillus brevis 1D, an unselected but acidifying lactic acid bacterium, while the onset of fungal growth was delayed for 7 days in bread started with S. cerevisiae and selected L. plantarum 21B.     

Evaluation of Chemicals for Restricting Colony Spreading by a Xerophilic Mold, Eurotium amstelodami, on Dichloran-18% Glycerol Agar

Twenty chemicals were screened for their effectiveness in restricting colony spreading by four strains of a xerophilic mold, Eurotium amstelodami, on dichloran-18% glycerol agar. Triton X-100, Triton X-301, Tergitol NP-7, and Tergitol 15-S-3 (each at 200 μg/ml) and 1,000 μg of sodium deoxycholate, 1 μg of iprodione, 0.1 μg of propiconazole, and 0.01 μg of Maxim per ml were judged to be most effective for restricting the rate of colony spreading.     

New and interesting species ofEurotium from Chinese soil

Eurotium taklimakanense, a new species isolated from desert soil in the Taklimakan desert, Xinjiang Province, China, is described and illustrated. It is characterized by light yellow to reddish yellow colonies on Czapek's agar with 70% (w/v) sucrose, yellow ascomata, broadly lenticular ascospores with conspicuously irregular equatorial crests and tuberculate or verrucose convex surfaces, and anAspergillus anamorph.Eurotium cristatum is also described as a new record from China. It is characterized by light yellow colonies on Czapek's agar with 70% sucrose, yellow ascomata, broadly lenticular ascospores with two equatorial crests and echinulate convex surfaces, and small tuberculate conidia.     

Eurotium spp. and echinulin in feed refused by swine.

Feed refused by swine contained a high-propagule density of Eurotium chevalieri Mangin (anamorph, Aspergillus chevalieri (Mangin) Thom and Church), Eurotium amstelodami Mangin (anamorph, Aspergillus amstelodami (Mangin Thom and Church), and Aspergillus candidus Link. Echinulin (8 micrograms/g of feed) was detected in the feed. Isolates of E. chevalieri and E. amstelodami but not A. candidus produced echinulin on rice or cracked corn. Mice refused to drink water containing 90 micrograms of echinulin per ml. This is the first report of the alkaloid echinulin in feed refused by swine.     

Ketonic rancidity in coconut due to xerophilic fungi

A type of deterioration called ketonic rancidity occurred in coconut after inoculation with four xerophilic fungi, Eurotium amstelodami, E. chevalieri, E. herbariorum and Penicillium citrinum. The fungi were incubated at low water activity and oxygen tension. A homologous series of aliphatic methyl ketones and secondary alcohols C₅C₁₁ were isolated and identified in the rancid samples after fungal growth. Evidence is presented that odd numbered methyl ketones (C₅C₁₁) are derived from even numbered short chain fatty acids with one more carbon atoms than the ketones via a modified β-oxidation of the parent fatty acid. Heptan-2-one and heptan-2-ol are the main reaction products except in the case of E. herbariorum where even numbered hexan-2-one, hexan-2-ol, octan-2-one and octan-2-ol were produced. Other moulds grown on coconut under similar conditions—Aspergillus flavus Link and Chrysosporium farinicola (Burnside) Skou (C. fastidium Pitt)—did not cause ketonic deterioration.     

Evaluation of molecular identification of Aspergillus species causing fungal keratitis

Fungal keratitis caused by the species of Aspergillus is a common and leading problem in developing countries like India. In this study, a total of 135 isolates from Aspergillus keratitis were studied by sequence analyses of the internal transcribed spacer (ITS) region performed by nucleotide-nucleotide BLAST analysis followed by the initial identification of the isolates based on conidial and colony morphology. The sequence analysis revealed several unusual species which were never reported in eye infections such as A. tamrii, A. tubingensis, A. braslliensis, A. nomius, A. pseudonomius, A. sydowii, Eurotium amstelodami. The sequence analysis of the ITS region; the β-tubulin and calmodulin genes brought out the genetic diversity among the isolates as the study intended to locate a more sensitive target sequence to study genetic diversity among a set of test fungal isolates. The PCR amplified sequences of the test isolates of the study as well as sequences belonging to section Flavi obtained from Genbank database were compared and analyzed along with three standard isolates by phylogenetic tree (Neighbor-joining) as to find out a target region/gene that could produce a better resolution to differentiate the isolates. Accordingly, the calmodulin gene had provided better resolution compared to ITS and β-tubulin to study the diversity among the test Aspergillus species isolated from fungal corneal ulcer.     

Environmental factors and preservatives affect carbon utilization patterns and niche overlap of food spoilage fungi

Nutrient assimilation by two mycotoxigenic spoilage fungi (Penicillium verrucosum, Aspergillus ochraceus) and four other food spoilage fungi (Penicillium coryolophilum, P. roqueforti, Cladosporium herbarum, Eurotium repens), of 32 key C-sources in wheat bread were examined in relation to abiotic factors of water availability, pH, temperature and the presence/absence of a preservative, potassium sorbate. These studies were to understand the relative potential co-existence, nutritional partitioning and niche exclusion in bread-based matrices. The niche size decreased significantly with decrease in water availability, temperature and pH. There were also significant interactive effects between pH and the preservative. The data were used to determine the niche overlap indices (NOI) of competing fungi relative to the two ochratoxigenic species. These showed that P. verrucosum and A. ochraceus were nutritionally dominant over the other species. The NOIs showed that potential co-existence and/or niche exclusion was influenced by environmental factors. Overall, the addition of the preservative increased the interspecific competition for nutrients. The level of co-existence and nutritional partitioning was significantly modified by abiotic factors, and this can help explain the dominance of specific fungal species in food matrices. This approach could also be useful for understanding competitiveness and dominance of fungal species in other ecosystems, especially in relation to impacts of abiotic and biotic factors.     

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。     

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.     

Disclosing a crypt: Microbial diversity and degradation activity of the microflora isolated from funeral clothes of Cardinal Peter Pázmány

A crypt can be considered as a particular environment where different microbial communities contribute to decomposition of organic materials present inside during a long interval of time. The textile remains of the funeral clothes (biretta and tunic) of Cardinal Pázmány, an important historic figure dead in Bratislava the 19th March 1637, conserved in this kind of environment were subjected to microbial investigation. The sampling comprised three different approaches and the use of various kinds of cultivation media. Two different PCR-based clustering methods, f-ITS and f-CBH, were employed in order to select the bacterial and fungal microfloras which were identified in a second step by the 16S rRNA and ITS sequencing respectively. The isolated microflora was tested for its proteolytic, keratinolytic and cellulolytic activities and for its ability to grow on Fibroin agar medium. The combination of cultural, molecular and biodegradative assays was able to isolate and characterize a bacterial community composed mainly by members of the phyla Firmicutes and Actinobacteria. The fungal community appeared more diversified, together with several Penicillium and Aspergillus strains, members belonging to the species Beauveria bassiana, Eurotium cristatum, Xenochalara juniperi, Phialosimplex caninus and Myriodontium keratinophilum were isolated. Bacteria, especially the Bacillus members, showed their strong ability to degrade keratin and gelatin and a large portion of them was able to growth on Fibroin agar. The fungal isolates displayed a widespread cellulolytic activity and fibroin utilization, although they possessed a weaker and slower proteolytic and keratinolytic properties respect to bacterial counterpart. The present study can be considered perhaps as the first or among the few microbial investigations which treated the textile biodegradation from such unusual environment.     

Analyzing the relation between the microbial diversity of DaQu and the turbidity spoilage of traditional Chinese vinegar

Vinegar is a traditional fermented condiment, and the microbial diversity of DaQu makes the quality of vinegar products. Recently, turbidity spoilage of vinegar sharply tampered with the quality of vinegar. In this study, the relation between the microbial diversity of DaQu and turbidity spoilage of vinegar was analyzed by plating technique, PCR–denaturing gradient gel electrophoresis (DGGE), and high-performance liquid chromatography (HPLC). The 16S rRNA sequencing and DGGE analysis indicated that Bacillus (Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus thuringiensis) and Lactobacillus (including Lactobacillus acidipiscis and Lactobacillus pobuzihii) species were the dominant contaminants in vinegar products. Meanwhile, DGGE analysis showed that the dominant bacteria in DaQu belonged to genera Bacillus, Lactobacillus, Pseudomonas, Weissella, Saccharopolyspora, Enterobacter, and Pantoea. However, only two yeast species (Pichia kudriavzevii and Saccharomycopsis fibuligera) and seven mold species including Aspergillus oryzae, Aspergillus niger, Aspergillus candidus, Rhizopus microspores, Eurotium herbariorum, Absidia corymbifera, and Eupenicillium javanicum were detected in the DaQu. The population level of fungi was below 5 log CFU/g in DaQu. The chemical and physical properties of vinegar and sediments were also determined. On the basis of a combined microbial diversity-chemical analysis, we demonstrated that turbidity spoilage of vinegar was a result of cooperation among the low population level and abundance of fungal species in DaQu, the suitable climate conditions, and the contaminants in vinegar. This is the first report to analyze the relation between the microbial diversity of DaQu and turbidity spoilage of vinegar.     

Water activity and temperature effects on germination and growth of Eurotium amstelodami, E. chevalieri and E. herbariorum isolates from bakery products

This study investigated the effect of temperature (5-30 degrees C), water activity (0.775-0.90 aw) and their interactions on the temporal rates of germination and mycelial growth of three species of Eurotium on flour wheat sucrose medium. Germination was quite rapid at aw >0.85, with an almost linear increase with time for all isolates. However, under more extreme water stress, germination was slower. The aw minima for germination were usually lower than those for growth and varied with temperature. The effect of aw x temperature interactions on the lag phases (h) prior to germination and on the germination rates (h-1) were predicted using the Gompertz model modified by Zwietering. Eurotium spp. had shown short lag times at 0.90 aw over a wide range of temperatures. At marginal temperatures, lag phases were significantly longer, especially at >15 degrees C. The temperature x aw profiles for mycelial growth varied between species in terms of rates (mm d(-1)). Predictions of the effect of important environmental factors, such as temperature, aw and their interactions on lag times to germination, germination rates and mycelial growth, are important in the development of hurdle technology approaches to predict fungal spoilage in food products.     

Prediction of Toxigenic Fungal Growth in Buildings by Using a Novel Modelling System

There is growing concern about the adverse effects of fungal bioaerosols on the occupants of damp dwellings. Based on an extensive analysis of previously published data and on experiments carried out within this study, critical limits for the growth of the indoor fungi Eurotium herbariorum, Aspergillus versicolor, and Stachybotrys chartarum were mathematically described in terms of growth limit curves (isopleths) which define the minimum combination of temperature (T) and relative humidity (RH) at which growth will occur. Each growth limit curve was generated from a series of data points on a T-RH plot and mathematically fitted by using a third-order polynomial equation of the form RH = a₃T³ + a₂T² + a₁T + a₀. This fungal growth prediction model was incorporated within the ESP-r (Environmental Systems Performance [r stands for “research”]) computer-based program for transient simulation of the energy and environmental performance of buildings. For any specified location, the ESP-r system is able to predict the time series evolution of local surface temperature and relative humidity, taking explicit account of constructional moisture flow, moisture generation sources, and air movement. This allows the predicted local conditions to be superimposed directly onto fungal growth curves. The concentration of plotted points relative to the curves allows an assessment of the risk of fungal growth. The system’s predictive capability was tested via laboratory experiments and by comparison with monitored data from a fungus-contaminated house.     

Optimization of physico‐chemical and nutritional parameters for a novel pullulan‐producing fungus,Eurotium chevalieri

Aims: To isolate the novel nonmelanin pullulan‐producing fungi from soil and to optimize the physico‐chemical and nutritional parameters for pullulan production. Methods and Results: A selective enrichment method was followed for the isolation, along with development of a suitable medium for pullulan production, using shake flask experiments. Pullulan content was confirmed using pure pullulan and pullulanase hydrolysate. Eurotium chevalieri was able to produce maximum pullulan (38 ± 1·0 g l^?1) at 35°C, pH 5·5, 2·5% sucrose, 0·3% ammonium sulfate and 0·2% yeast extract in a shake flash culture medium with an agitation rate of 30 rev min^?1 for 65 h. Conclusions: The novel pullulan‐producing fungus was identified as E. chevalieri (MTCC no. 9614), which was able to produce nonmelanin pullulan at from poorer carbon and nitrogen sources than Aureobasidium pullulans and may therefore be useful for the commercial production of pullulan. Significance and Impact of the Study: Eurotium chevalieri could produce pullulan in similar amounts to A. pullulans. Therefore, in future, this fungus could also be used for commercial pullulan production, because it is neither polymorphic nor melanin producing, hence its handling during pullulan fermentation will be easier and more economical.