The parasitism of the mouldPenicillium purpurogenum onAspergillus niger

The course of the parasitic action of the mouldPenicillium purpurogenum on the mycelium of a plant strain ofAspergillus niger is described in plant and laboratory conditions of surface citric acid fermentation. During the infection, a successive destruction of the mycelium ofAspergillus niger sets in, as well as a decrease in citric acid production, in this case also act the specific inhibitory thermostabile materials, secreted by the parasitic mould into the medium. The main factor is the cell lysis, effected by a specific enzymolytic system of the constitutive type. This system is evolved by the parasitic mould in the nutritions medium, resp. into the mycelium of the infected mouldAspergillus niger and causes the lysis both of entire hyphae, and of isolated cell walls. By this lytic system, up to 85% of the dry weight of the cell walls is transferred into the solution as sugars, mainly as glucose, so that the chief component undergoing the lysis is a polymer carbohydrate. The optimum temperature of the action of the lytic system lies within 30–41°C, optimum pH is 4.2–5.5. In case of several other representatives of the genusPenicillium, no lytic systems have been established, at the same time their inability for the parasitic form of life on the mycelium ofAspergillus niger was proved.     

The parasitism of the mouldPenicillium purpurogenum onAspergillus niger

The causes of the formerly described effect of the limitation of infection of surface citric acid fermentation byPenicillium purpurogenum by the action of an increased air supply have been ascertained. The influence of the increased air supply on the composition ofAspergillus niger mycelium and cell walls was followed, as well as their assailability by the lytic enzyme ofPenicillium purpurogenum. Further the effect of temperature, humidity, oxygen supply and presence of carbon dioxide on the growth of the parasitic mould was evaluated. The main cause of the mentioned effect seems to be the drainage of humidity by increased air supply.     

Synthesis of disaccharides using β-glucosidases from <Emphasis Type="Italic">Aspergillus niger</Emphasis>, <Emphasis Type="Italic">A. awamori</Emphasis> and <Emphasis Type="Italic">Prunus dulcis</Emphasis>

Objective

Glucose conversion into disaccharides was performed with β-glucosidases from Prunus dulcis (β-Pd), Aspergillus niger (β-An) and A. awamori (β-Aa), in reactions containing initial glucose of 700 and 900 g l^?1.

Results

The reactions’ time courses were followed regarding glucose and product concentrations. In all cases, there was a predominant formation of gentiobiose over cellobiose and also of oligosaccharides with a higher molecular mass. For reactions containing 700 g glucose l^?1, the final substrate conversions were 33, 38, and 23.5% for β-An, β-Aa, and β-Pd, respectively. The use of β-An yielded 103 g gentiobiose l^?1 (15.5% yield), which is the highest reported for a fungal β-glucosidase. The increase in glucose concentration to 900 g l^?1 resulted in a significant increase in disaccharide synthesis by β-Pd, reaching 128 g gentiobiose l^?1 (15% yield), while for β-An and β-Aa, there was a shift toward the synthesis of higher oligosaccharides.

Conclusion

β-Pd and the fungal β-An and β-Aa β-glucosidases present quite dissimilar kinetics and selective properties regarding the synthesis of disaccharides; while β-Pd showed the highest productivity for gentiobiose synthesis, β-An presented the highest specificity.

     

Electron Microscopy of Cells of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> infected with Double Stranded RNA Viruses from <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Penicillium stoloniferum</Emphasis>

LHOAS¹ has demonstrated the infection of mating pairs of Saccharomyces cerevisiae with double stranded RNA viruses from Aspergillus niger and Penicillium stoloniferum (preceding communication). I wish to give details of the appearance of the virus particles within the infected yeast cells as determined by electron microscopy.     

Carbohydrate Specificity of Antibodies against Phytopathogenic Fungi of the <Emphasis Type="Italic">Aspergillus</Emphasis> Genus

Brush rabbits were immunized with injections prepared from the fungi Aspergillus fumigatus, Aspergillus niger, and Aspergillus repens. A library of synthetic biotinylated oligosaccharides containing the key fragments of antigenic polysaccharides of the fungal cell wall—galctomannan, α- and β-glucans, mannan, and chitin—was used to analyze carbohydrate specificity. The anticarbohydrate antibodies obtained from animals immunized with preparations from A. fumigatus and A. repens predominantly recognized epitopes containing galactofuranoside residues, while the majority of the antibodies against A. niger bound the chitooligosaccharide ligand. These results are the basis for the identification of specific markers required for the development of immunoenzyme test systems.     

<Emphasis Type="Italic">Streptomyces luozhongensis</Emphasis> sp. nov., a novel actinomycete with antifungal activity and antibacterial activity

A novel actinomycete strain, designated TRM 49605^T, was isolated from a desert soil sample from Lop Nur, Xinjiang, north-west China, and characterised using a polyphasic taxonomic approach. The strain exhibited antifungal activity against the following strains: Saccharomyces cerevisiae, Curvularia lunata, Aspergillus flavus, Aspergillus niger, Fusarium oxysporum, Penicillium citrinum, Candida albicans and Candida tropicalis; Antibacterial activity against Bacillus subtilis, Staphylococcus epidermidis and Micrococcus luteus; and no antibacterial activity against Escherichia coli. Phylogenetic analysis based on 16S rRNA gene sequences affiliated strain TRM 49605^T to the genus Streptomyces. Strain TRM 49605^T shows high sequence similarities to Streptomyces roseolilacinus NBRC 12815^T (98.62 %), Streptomyces flavovariabilis NRRL B-16367^T (98.45 %) and Streptomyces variegatus NRRL B-16380^T (98.45 %). Whole cell hydrolysates of strain TRM 49605^T were found to contain ll-diaminopimelic acid as the diagnostic diamino acid and galactose, glucose, xylose and mannose as the major whole cell sugars. The major fatty acids in strain TRM 49605^T were identified as iso C_16:0, anteiso C_15:0, C_16:0 and Summed Feature 5 as defined by MIDI. The main menaquinones were identified as MK-9(H₄), MK-9(H₆), MK-9(H₈) and MK-10(H₆). The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol and phosphatidylinositol mannoside. The G+C content of the genomic DNA was determined to be 71.2 %. The DNA–DNA relatedness between strain TRM 49605^T and the phylogenetically related strain S. roseolilacinus NBRC 12815^T was 60.12 ± 0.06 %, which is lower than the 70 % threshold value for delineation of genomic prokaryotic species. Based on the phenotypic, chemotaxonomic and phylogenetic data, strain TRM 49605^T (=CCTCC AA2015026^T = KCTC 39666^T) should be designated as the type strain of a novel species of the genus Streptomyces, for which the name Streptomyces luozhongensis sp. nov. is proposed.     

Antifungal activity of 3-acetylbenzamide produced by actinomycete WA23-4-4 from the intestinal tract of <Emphasis Type="Italic">Periplaneta americana</Emphasis>

Actinomycetes are well-known for producing numerous bioactive secondary metabolites. In this study, primary screening by antifungal activity assay found one actinomycete strain WA23-4-4 isolated from the intestinal tract of Periplaneta americana that exhibited broad spectrum antifungal activity. 16S rDNA gene analysis of strain WA23-4-4 revealed close similarity to Streptomyces nogalater (AB045886) with 86.6% sequence similarity. Strain WA23-4-4 was considered as a novel Streptomyces and the 16s rDNA sequence has been submitted to GenBank (accession no. KX291006). The maximum antifungal activity of WA23-4-4 was achieved when culture conditions were optimized to pH 8.0, with 12% inoculum concentration and 210 ml ISP2 medium, which remained stable between the 5^th and the 9^th day. 3-Acetyl benzoyl amide was isolated by ethyl acetate extraction of WA23-4-4 fermentation broth, and its molecular formula was determined as C₉H₉NO₂ based on MS, IR, ¹H, and ¹³C NMR analyses. The compound showed significant antifungal activity against Candida albicans ATCC 10231 (MIC: 31.25 μg/ml) and Aspergillus niger ATCC 16404 (MIC: 31.25 μg/ml). However, the compound had higher MIC values against Trichophyton rubrum ATCC 60836 (MIC: 500 μg/ml) and Aspergillus fumigatus ATCC 96918 (MIC: 1,000 μg/ml). SEM analysis showed damage to the cell membrane of Candida albicans ATCC 10231 and to the mycelium of Aspergillus niger ATCC 16404 after being treatment with 3-acetyl benzoyl amide. In conclusion, this is the first time that 3-acetyl benzoyl amide has been identified from an actinomycete and this compound exhibited antifungal activity against Candida albicans ATCC 10231 and Aspergillus niger ATCC 16404.     

Purification,characterization, gene cloning and sequencing of a new β-glucosidase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> BE-2

The cDNA gene (BgL1), encoding GH3 family β-glucosidase (EC 3.2.1.21) from Aspergillus niger BE-2 (abbreviated to BgL1), was amplified and inserted into the yeast expression pPIC9K vector at the site of Bln I (Avr II) and NotI. The recombinant expression vector, designated as pPIC9K-BgL1, was transformed into Pichia pastoris GS115. The transformants were screened on minimal dextrose plates, which inoculated on geneticin G418-containing yeast extract-peptone-dextrose plates. The transformants expressed the high β-glucosidase activity of 22.6 U/mL. SDS-PAGE demonstrated that the BgL1 was extracellularly expressed with an apparent molecular weight of 90.0 kDa. The purified BgL1 displayed the maximum activity at pH 6.0 and 60°C. It was highly stable at a broad pH range of 4.0–7.5 and temperature of 60°C. The BgL1 displayed high similarity to the β-glucosidases of A. niger FN430671 and A. niger DQ655704, the members of the GH3 family. Its three-dimensional structure was predicted using http://swiss-model.expasy.org/ on-line programs based on the crystal structure of Aspergillus aculeatus β-glucosidase.     

In vitro antifungal efficacy of <Emphasis Type="Italic">Aspergillus niger</Emphasis> ATCC 9642 chitosan-AgNPs composite against post-harvest disease of citrus fruits

Green and blue mold postharvest diseases are the most vital negative components influencing the local market of citrus fruits. Citrus fruits were collected, and fungi were isolated. Among the fungal isolates identified, Penicillium digitatum and Penicillium italicum recorded the highest occurrence of 39.5 and 25.6%, respectively. In this work, we extracted chitosan from Aspergillum niger ATCC 9642. Fourier transform infrared spectroscopy was utilized to confirm the functional groups of the obtained compound, which exhibited the main characteristic bands of O–H stretching at 3302 cm^-1, and C–O–C band at 1125 cm^-1. A. niger ATCC 9642 chitosan had the degree of deacetylation of 88.5%, a molecular weight of 1.8 × 10⁵ Da, and viscosity of 7.3 centipoises; these values were comparable to those for standard shrimp chitosan. Ultraviolet-visible light spectra revealed the presence of A. niger ATCC 9642 chitosan-AgNPs composite. Using antifungal and spore germination assays, it was found that this composite exhibited effective antifungal action against P. digitatum and P. italicum compared with a chitosan standard. In a comet assay, the percentage of tail DNA was considered as a parameter that indicated DNA damage. The comet parameter increased significantly (P < 0.05) with A. niger ATCC 9642 chitosan–AgNPs composite, and the increase was dose-dependent. The increase in the DNA damage positively correlated with the inhibition performance of the A. niger ATCC 9642 chitosan–AgNPs composite.     

Regulatory activity of heterologous gene-activator <Emphasis Type="Italic">xlnR</Emphasis> of <Emphasis Type="Italic">Aspergillus niger</Emphasis> in <Emphasis Type="Italic">Penicillium canescens</Emphasis>

The gene encoding the xlnR xylanolytic activator of the heterologous fungus Aspergillus niger was incorporated into the Penicillium canescens genome. Integration of the xlnR gene resulted in the increase in a number of activities, i.e. endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-galactosidase, and feruloyl esterase, compared to the host P. canescens PCA 10 strain, while β-galactosidase, β-glucosidase, endoglucanase, and CMCase activities remained constant. Two different expression constructs were developed. The first consisted of the nucleotide sequence containing the mature P. canescens phytase gene under control of the axhA promoter region gene encoding A. niger (1,4)-β-D-arabinoxylan-arabinofuranohydrolase. The second construct combined the P. canescens phytase gene and the bgaS promoter region encoding homologous β-galactosidase. Both expression cassettes were transformed into P. canescens host strain containing xlnR. Phytase synthesis was observed only for strains with the bgaS promoter on arabinose-containing culture media. In conclusion, the bgaS and axhA promoters were regulated by different inducers and activators in the P. canescens strain containing a structural tandem of the axhA promoter and the gene of the xlnR xylanolytic activator.     

Survey of Philippine coffee beans for the presence of ochratoxigenic fungi

In 2012 to 2014, Philippine green coffee beans from Coffea arabica in Benguet and Ifugao; Coffea canephora var. Robusta in Abra, Cavite, and Ifugao; and Coffea liberica and Coffea excelsea from Cavite were collected and assessed for the distribution of fungi with the potential to produce ochratoxin A (OTA). The presence of fungal species was evaluated both before and after surface sterilization. There were remarkable ecological and varietal differences in the population of OTA-producing species from the five provinces. Aspergillus ochraceus, A. westerdijkiae, and Penicillium verruculosum were detected from Arabica in Benguet and Ifugao while Aspergillus carbonarius, Aspergillus niger, and Aspergillus japonicus were isolated in Excelsa, Liberica, and Robusta varieties from Abra, Cavite, and Davao. Contamination by Aspergillus and Penicillium species was found on 59 and 19 %, respectively, of the 57 samples from five provinces. After disinfection with 1 % sodium hypochlorite, the levels of infection by Aspergillus and Penicillium fell to 40 and 17 %, respectively. A total of 1184 fungal isolates were identified to species level comprising Aspergillus sections Circumdati (four species), Clavati (one), Flavi (one), Fumigati (one), Nigri (three), and Terrie (one). Within section Circumdati, 70 % of A. ochraceus produced OTA as high as 16238 ng g^?1 while 40 % of A. westerdijkiae produced maximum OTA of 36561 ng g^?1 in solid agar. Within section Nigri, 16.76 % of A. niger produced OTA at the highest 18439 ng g^?1, 10 % of A. japonicus at maximum level of 174 ng g^?1, and 21.21 % of A. carbonarius yielded maximum OTA of 1900 ng g^?1. Of the 12 species of Penicillium isolated, P. verruculosum was ochratoxigenic, with a maximum OTA production of 12 ng g^?1.     

VeA of <Emphasis Type="Italic">Aspergillus niger</Emphasis> increases spore dispersing capacity by impacting conidiophore architecture

Aspergillus species are highly abundant fungi worldwide. Their conidia are among the most dominant fungal spores in the air. Conidia are formed in chains on the vesicle of the asexual reproductive structure called the conidiophore. Here, it is shown that the velvet protein VeA of Aspergillus niger maximizes the diameter of the vesicle and the spore chain length. The length and width of the conidiophore stalk and vesicle were reduced nearly twofold in a ΔveA strain. The latter implies a fourfold reduced surface area to develop chains of spores. Over and above this, the conidial chain length was approximately fivefold reduced. The calculated 20-fold reduction in formation of conidia by ΔveA fits the 8- to 17-fold decrease in counted spore numbers. Notably, morphology of the ΔveA conidiophores of A. niger was very similar to that of wild-type Aspergillus sydowii. This suggests that VeA is key in conidiophore architecture diversity in the fungal kingdom. The finding that biomass formation of the A. niger ΔveA strain was reduced twofold shows that VeA not only impacts dispersion capacity but also colonization capacity of A. niger.     

The survival of micromycetes exposed to space conditions

Original data on the survival of fungal spores exposed to space conditions are presented. The experiment was carried out on the Earth-orbiting Russian satellite Foton-M4. The flight duration of the satellite was 45 days. Thirteen fungal species (hyaline as well as pigmented) from 10 genera recovered from destructed stone materials were studied. Sterile quartz sand was inoculated by the fungal spores and was placed into Eppendorf tubes. During the space flight, the Eppendorf tubes with fungal spores were kept inside the Foton descent capsule in the “Biokont” containers and on the external surface of the capsule in the “Exobiofrost” containers exposed to the open space as well. Spores of ten species (77% of all tested species), i.e. Acremonium charticola, Aspergillus niger, Aspergillus versicolor, Chaetomium globosum, Cladosporium sphaerospermum, Penicillium chrysogenum, Penicillium verrucosum, Purpureocillium lilacinum, Sarocladium kiliense, and Trichoderma harzianum, survived after the flight both inside and outside the descent capsule. Only three species (23% of all tested species), i.e. Acremonium furcatum, Engyodontium album and Verticillium zaregamsianum, failed to survive outside as well as inside the capsule. Spore viability differed depending on the fungal species. Thus, spores of some fungal species are able to survive under the complex of stress factors such as low temperature values, radiation, etc. We have shown that micromycetes can be used as a model group for study of eukaryotic organisms’ resistance to stress factors, due to their high tolerance not only to extreme terrestrial environments, but to the extraterrestrial ones as well.     

Assessment of photosynthetic potential of indoor plants under cold stress

Photosynthetic parameters including net photosynthetic rate (P_N), transpiration rate (E), water-use efficiency (WUE), and stomatal conductance (g_s) were studied in indoor C₃ plants Philodendron domesticum (Pd), Dracaena fragans (Df), Peperomia obtussifolia (Po), Chlorophytum comosum (Cc), and in a CAM plant, Sansevieria trifasciata (St), exposed to various low temperatures (0, 5, 10, 15, 20, and 25°C). All studied plants survived up to 0°C, but only St and Cc endured, while other plants wilted, when the temperature increased back to room temperature (25°C). The P_N declined rapidly with the decrease of temperature in all studied plants. St showed the maximum P_N of 11.9 μmol m^?2 s^?1 at 25°C followed by Cc, Po, Pd, and Df. E also followed a trend almost similar to that of P_N. St showed minimum E (0.1 mmol m^?2 s^?1) as compared to other studied C₃ plants at 25°C. The E decreased up to ≈4-fold at 5 and 0°C. Furthermore, a considerable decline in WUE was observed under cold stress in all C₃ plants, while St showed maximum WUE. Similarly, the g_s also declined gradually with the decrease in the temperature in all plants. Among C₃ plants, Pd and Po showed the maximum g_s of 0.07 mol m^?2 s^?1 at 25°C followed by Df and Cc. However, St showed the minimum g_s that further decreased up to ～4-fold at 0°C. In addition, the content of photosynthetic pigments [chlorophyll a, b, (a+b), and carotenoids] was varying in all studied plants at 0°C. Our findings clearly indicated the best photosynthetic potential of St compared to other studied plants. This species might be recommended for improving air quality in high-altitude closed environments.     

Isolation and properties of fungal β-glucosidases

Using chromatography on different matrixes, three β-glucosidases (120, 116, and 70 kDa) were isolated from enzymatic complexes of the mycelial fungi Aspergillus japonicus, Penicillium verruculosum, and Trichoderma reesei, respectively. The enzymes were identified by MALDI-TOF mass-spectrometry. Substrate specificity, kinetic parameters for hydrolysis of specific substrates, ability to catalyze the transglucosidation reaction, dependence of the enzymatic activity on pH and temperature, stability of the enzymes at different temperatures, adsorption ability on insoluble cellulose, and the influence of glucose on catalytic properties of the enzymes were investigated. According to the substrate specificity, the enzymes were shown to belong to two groups: i) β-glucosidase of A. japonicus exhibiting high specific activity to the low molecular weight substrates cellobiose and pNPG (the specific activity towards cellobiose was higher than towards pNPG) and low activity towards polysaccharide substrates (β-glucan from barley and laminarin); ii) β-glucosidases from P. verruculosum and T. reesei exhibiting relatively high activity to polysaccharide substrates and lower activity to low molecular weight substrates (activity to cellobiose was lower than to pNPG).     

The effects of lead on photosynthetic performance of waxberry seedlings (<Emphasis Type="Italic">Myrica rubra</Emphasis>)

The photosynthesis was investigated 30 d after Pb treatment in Myrica rubra seedlings. The Pb treatment resulted in significantly increased Pb concentrations in shoots. Low Pb concentration exposure (≤2 mM) reduced the net photosynthetic rate (P_N), transpiration rate (E), and stomatal conductance (gs) without affecting the intercellular CO₂ concentration (C_i), chlorophyll (Chl) content, and Chl fluorescence parameters. At 10 d after severe Pb treatment (≥4 mM), P_N was inhibited and accompanied by Chl damage, while at 30 d, the inhibition of P_N was followed by an increase of C_i and a decrease of gs, E, Chl content, and Chl fluorescence parameters. M. rubra showed a promising prospect for use in the soil phytoremediation, when Pb concentration is low, but the remediation efficiency of M. rubra is limited if Pb exceeds 2 mM.     

EEG pattern decoding of rhythmic individual finger imaginary movements of one hand

The results of four-class classification of the motor imagery EEG patterns corresponding to the right hand finger movements (little finger, thumb, index and middle fingers) of eight healthy subjects are presented in this study. The motor imagery of individual right-hand finger movements was executed by the subjects in a prescribed rhythm and the trials contained no external stimuli. Classification was performed by means of a specially developed two-level committee of classifiers on the basis of support vector machine and artificial neural networks at the first level and by generalizing an artificial neural network at the second level. The area under the EEG signal curve and the curve length calculated in a sliding time window for sites F ₃, C ₃, and Cz of the International 10?20 System were selected as the key features of signals from the sensorimotor and adjoining frontal cortical areas contralateral to the movements. The average accuracy of four-class singletrial classification for all subjects was 50 ± 7 [SD] (maximum, 58%) for the pair of sites F ₃–C ₃ and 46 ± 11% [SD] (maximum 62%) for the pair of sites C ₃–Cz with a theoretical guessing level 25%.