Molecular identification of a novel inulinolytic fungus isolated from and grown on tubers of Helianthus tuberosus and statistical screening of medium components

Of 24 fungi belonging to more than five genera isolated from tubers of rotten Helianthus tuberosus, 11-inulinolytic active isolates were able to develop halo zones around their fungal colonies, indicating inulinase activity. Alternaria, Aspergillus, Fusarium, Pencillium and Trichoderma were the most common inulinolytic genera, representing more than 90?% of the total positive inulinolytic fungi. Aspergillus tamarii and Pencillium citrinum quantitatively recorded better growth (5.5 and 4.7?mg?ml^?1) and inulinase production (21.53 and 20.15?U?ml^?1) in submerged culture. The enzyme preparation showed also invertase activity. Aspergillus tamarii, as the most potent producer of inulinase, was identified using the Inter Transcribed Spacer marker. The sequence comparisons showed that our molecularly identified strain (GU295949) is related more closely to A. tamarii strains of the gene bank. Statistical screening using the fractional factorial Plackett-Burman design with 12 run was applied for screening ten variables, the low levels of pH (4.8), inoculum size (10³ spore?g^?1), NH₄NO₃ (1.0?mg?g^?1) and MgSO₄ (0.12?mg?g^?1), were the most significant variables on A. tamarii inulinase production. The high inulinase/invertase ratio (1.841?C4.293) classified the enzyme preparation as inulinases, which can be used efficiently in production of fructose syrup from tubers of H. tuberosus.     

Modeling lipid accumulation in oleaginous fungi in chemostat cultures: I. Development and validation of a chemostat model for Umbelopsis isabellina

Lipid-accumulating fungi may be able to produce biodiesel precursors from agricultural wastes. As a first step in understanding and evaluating their potential, a mathematical model was developed to describe growth, lipid accumulation and substrate consumption of the oleaginous fungus Umbelopsis isabellina (also known as Mortierella isabellina) in submerged chemostat cultures. Key points of the model are: (1) if the C-source supply rate is limited, maintenance has a higher priority than growth, which has a higher priority than lipid production; (2) the maximum specific lipid production rate of the fungus is independent of the actual specific growth rate. Model parameters were obtained from chemostat cultures of U. isabellina grown on mineral media with glucose and NH₄ ⁺. The model describes the results of chemostat cultures well for D > 0.04 h⁻¹, but it has not been validated for lower dilution rates because of practical problems with the filamentous fungus. Further validation using literature data for oleaginous yeasts is described in part II of this paper. Our model shows that not only the C/N-ratio of the feed, but also the dilution rate highly influences the lipid yield in chemostat cultures.     

Stereospecific ligands and their complexes. V. Synthesis, characterization and antimicrobial activity of palladium(II) complexes with some alkyl esters of (S,S)-ethylenediamine-N,N′-di-2-propanoic acid

Three new ligands and their palladium(II) complexes of general formula [PdCl₂(R₂-S,S-eddp)] (R = n-propyl, n-butyl and n-pentyl) have been synthesized and characterized by microanalysis, infrared and ¹H and ¹³C NMR spectroscopy. Antimicrobial activity of these ligands and complexes was tested by microdilution method and both minimal inhibitory and microbicidal concentration were determined. These tested complexes demonstrated the significant antifungal activity against pathogenic fungi Aspergillus flavus and Aspergillus fumigatus. On the other hand, these complexes demonstrated moderate antibacterial activity.     

Triazole Susceptibilities in Thermotolerant Fungal Isolates from Outdoor Air in the Seoul Capital Area in South Korea

Emerging fungi resistant to triazoles are a concern because of the increased use of medical triazoles and exposure to agricultural triazoles. However, little is known about the levels of triazole susceptibility in outdoor airborne fungi making it difficult to assess the risks of inhalation exposure to airborne, antifungal-resistant fungi. This study examined triazole susceptibilities of the airborne thermotolerant fungi isolated from the ambient air of the Seoul Capital Area of South Korea. We used impactor air sampling with triazole-containing nutrient agar plates as the collection substrates to screen for airborne fungal isolates based on their triazole susceptibilities. This study estimated that 0.17% of all the culturable fungi belong to the pathogenic thermotolerant taxa, among which each isolate of Aspergillus niger and Aspergillus tubingensis showed a minimum inhibitory concentration (MIC) of 2 μg/mL or greater for itraconazole. Their concentration in air was 0.4 CFU/m³. Seven human pathogenic Paecilomyces variotii isolates had MICs of 32 μg/mL or greater and lower than 2 μg/mL for the agricultural fungicide tebuconazole and the medical triazole itraconazole, respectively. Though the concentration was low, our results confirm the presence of airborne fungi with high MICs for itraconazole in ambient air. Inhalation is an important exposure route because people inhale more than 10 m³ of air each day. Vigilance is preferred over monitoring for the emergence of triazole-resistant fungal pathogens in ambient outdoor air.     

Fungal Metabolism of Toluene: Monitoring of Fluorinated Analogs by 19F Nuclear Magnetic Resonance Spectroscopy

We used isomeric fluorotoluenes as model substrates to study the catabolism of toluene by five deuteromycete fungi and one ascomycete fungus capable of growth on toluene as the sole carbon and energy source, as well as by two fungi (Cunninghamella echinulata and Aspergillus niger) that cometabolize toluene. Whole cells were incubated with 2-, 3-, and 4-fluorotoluene, and metabolites were characterized by ¹⁹F nuclear magnetic resonance. Oxidation of fluorotoluene by C. echinulata was initiated either at the aromatic ring, resulting in fluorinated o-cresol, or at the methyl group to form fluorobenzoate. The initial conversion of the fluorotoluenes by toluene-grown fungi occurred only at the side chain and resulted in fluorinated benzoates. The latter compounds were the substrate for the ring hydroxylation and, depending on the fluorine position, were further metabolized up to catecholic intermediates. From the ¹⁹F nuclear magnetic resonance metabolic profiles, we propose that diverse fungi that grow on toluene assimilate toluene by an initial oxidation of the methyl group.     

Diversity of facultatively anaerobic microscopic mycelial fungi in soils

The numbers of microscopic fungi isolated from soil samples after anaerobic incubation varied from tens to several hundreds of CFU per one gram of soil; a total of 30 species was found. This group is composed primarily of mitotic fungi of the ascomycete affinity belonging to the orders Hypocreales (Fusarium solani, F. oxysporum, Fusarium sp., Clonostachys grammicospora, C. rosea, Acremonium sp., Gliocladium penicilloides, Trichoderma aureoviride, T. harzianum, T. polysporum, T. viride, T. koningii, Lecanicillum lecanii, and Tolypocladium inflatum) and Eurotiales (Aspergillus terreus, A. niger, and Paecilomyces lilacimus), as well as to the phylum Zygomycota, to the order Mucorales (Actinomucor elegans, Absidia glauca, Mucor circinelloides, M. hiemalis, M. racemosus, Mucor sp., Rhizopus oryzae, Zygorrhynchus moelleri, Z. heterogamus, and Umbelopsis isabellina) and the order Mortierellales (Mortierella sp.). As much as 10–30% of the total amount of fungal mycelium remains viable for a long time (one month) under anaerobic conditions.     

Improved enzyme production by co-cultivation of Aspergillus niger and Aspergillus oryzae and with other fungi

Aspergillus niger and Aspergillus oryzae were co-cultivated with each other and with Magnaporthe grisea or Phanerochaete chrysosporium, respectively. Enzyme assays for plant polysaccharide and lignin-degrading enzymes showed that co-cultivation can improve extracellular enzyme production. Highest ??-glucosidase, ??-cellobiohydrolase, ??-galactosidase, and laccase activities were found for A. oryzae in combination with other fungi, in particular with P. chrysosporium. Highest ??-xylosidase activity was obtained when A. niger was co-cultivated with P. chrysosporium. SDS-PAGE protein profiles demonstrated that A. niger and A. oryzae contributed most to the overall enzyme activities found in the culture medium of the mixed cultivations. These data demonstrate that co-cultivation of two major industrial fungi, A. niger and A. oryzae, results in improved production of biotechnologically relevant enzymes.     

Generation of Large Chromosomal Deletions in Koji Molds Aspergillus oryzae and Aspergillus sojae via a Loop-Out Recombination

We established a technique for efficiently generating large chromosomal deletions in the koji molds Aspergillus oryzae and A. sojae by using a ku70-deficient strain and a bidirectional marker. The approach allowed deletion of 200-kb and 100-kb sections of A. oryzae and A. sojae, respectively. The deleted regions contained putative aflatoxin biosynthetic gene clusters. The large genomic deletions generated by a loop-out deletion method (resolution-type recombination) enabled us to construct multiple deletions in the koji molds by marker recycling. No additional sequence remained in the resultant deletion strains, a feature of considerable value for breeding of food-grade microorganisms. Frequencies of chromosomal deletions tended to decrease in proportion to the length of the deletion range. Deletion efficiency was also affected by the location of the deleted region. Further, comparative genome hybridization analysis showed that no unintended deletion or chromosomal rearrangement occurred in the deletion strain. Strains with large deletions that were previously extremely laborious to construct in the wild-type ku70⁺ strain due to the low frequency of homologous recombination were efficiently obtained from Δku70 strains in this study. The technique described here may be broadly applicable for the genomic engineering and molecular breeding of filamentous fungi.     

Especies de Aspergillus en ambientes hospitalarios con pacientes pediátricos en estado crítico

BackgroundAspergillus is a group of opportunistic fungi that cause infections, with high morbimortality in immunosuppressed patients. Aspergillus fumigatus is the most frequent species in these infections, although the incidence of other species has increased in the last few years.AimsTo evaluate the air fungal load and the diversity of Aspergillus species in hospitals with pediatric patients in critical condition.MethodsThe Intensive Care Unit and Burns Unit of a pediatric hospital were sampled every 15 days during the autumn and spring seasons. The air samples were collected with SAS Super 100^® and the surface samples were collected by swab method.ResultsThe UFC/m³ counts found exceeded the acceptable levels. The UFC/m³ and the diversity of Aspergillus species found in the Intensive Care Unit were higher than those found in the Burns Unit. The fungal load and the diversity of species within the units were higher than those in control environments. The use of both methods –SAS and swab– allowed the detection of a higher diversity of species, with 96 strains of Aspergillus being isolated and 12 species identified. The outstanding findings were Aspergillus sydowii, Aspergillus niger, Aspergillus flavus, Aspergillus terreus and Aspergillus parasiticus, due to their high frequency. Aspergillus fumigatus, considered unacceptable in indoor environments, was isolated in both units.ConclusionsAspergillus was present with high frequency in these units. Several species are of interest in public health for being potential pathogenic agents. Air control and monitoring are essential in the prevention of these infections.     

Nests of Marsh harrier (Circus aeruginosus L.) as refuges of potentially phytopathogenic and zoopathogenic fungi

Birds’ nests may be refuges for various species of fungi including that which are potentially phytopathogenic and zoopathogenic. Among the 2449 isolates of fungi obtained from nests of Marsh harriers 96.8% belonged to filamentous fungi. In total, 37 genera were identified from 63 fungi species. Within the mycobiotas of the examined nests populations of fungi which are potentially pathogenic for humans, homoiothermous animals and plants dominated. Among 63 species, 46 (72%) were potentially pathogenic fungi of which 18 species were potentially phytopathogenic and 32 species were pathogenic for homoiothermous animals. Inter alia species of fungi were found in the Marsh harriers nests: Aspergillus fumigatus, Aspergillus flavus, Scopulariopsis brevicaulis, Chrysosporium keratinophilum and Fusarium poae, Fusarium sporotrichioides. In terms of numbers, dominant in Marsh harrier nests were fungi pathogenic to birds, other homoiothermous animals and humans. On that basis it was concluded that Marsh harrier nests are both a source of fungal infections for that species and one of the links in the epidemiological cycle of opportunistic fungi for humans.     

Comparative efficacy and pathogenicity of keratinophilic soil fungi against Culex quinquefasciatus larvae

Out of seven fungal species belonging to four genera isolated from pond and wallow soils using feathers of Pavo cristatus as bait, four species viz., Aspergillus flavus, Aspergillus niger, Chrysosporium pseudomerdarium and Trichophyton ajelloi were most frequent. Chrysosporium and Trichophyton spp. were more pathogenic on Culex quinquefasciatus larvae than Aspergillus and Penicillium. The bioefficacy tests conducted as per the protocol of World Health Organization and the LC₅₀ values calculated by the Probit analysis showed that 3^rd-instar C. quinquefasciatus were more susceptible to the conidia of above fungi. Highest mortality was observed in the larvae of C. quinquefasciatus when exposed to T. ajelloi. The density of fungal conidia was greatest on the ventral brush, palmate hair and anal region of the mosquito larvae after exposing for 72 hours. The potentiality of these fungi for use in the control of C. quinquefasciatus is discussed which can be exploited as a suitable biocontrol agent in the tropics.     

Fungi Isolated from Flue-cured Tobacco at Time of Sale and After Storage

The fungi isolated from 100 samples of flue-cured tobacco from 12 markets in 2 tobacco belts comprised 11 genera, including 10 species of Aspergillus. The mean percentage per sample isolated from 62 samples of tobacco from Middle Belt markets was Alternaria, 40.6%; Aspergillus niger, 47.8%; Aspergillus repens, 38.0%; and Penicillium, 25.8%. The mean percentage per sample isolated from 38 samples of tobacco from Old Belt markets was Alternaria, 74.0%; Penicillium, 52.5%; Aspergillus repens, 38.0%; and Aspergillus ruber, 36.2%. Damaged (74 samples) and nondamaged (26 samples) stored tobacco yielded species of six genera of fungi, including eight species of Aspergillus. Species of Aspergillus and Penicillium were commonly isolated from both damaged and nondamaged tobacco, whereas species of Alternaria, Cladosporium, Fusarium, and Rhizopus were isoalted more frequently from nondamaged tobacco. The fungi that occurred in the highest population in damaged tobacco were Aspergillus repens, A. niger, A. ruber, and Penicillium species.     

Cheese whey as a renewable substrate for microbial lipid and biomass production by Zygomycetes

Three Zygomycetes, Mortierella isabellina, Thamnidium elegans and Mucor sp., were tested for their ability of producing biomass and lipid‐containing γ‐linolenic acid (GLA) during their cultivation on cheese whey. M. isabellina consumed all of the available lactose and a significant amount of the available protein. On the contrary, the two other fungi seemed incapable of consuming lactose after protein exhaustion. In the second series of experiments, for M. isabellina a supplementary quantity of lactose was added into the medium in order to increase the C/N ratio and hence to increase the production of fat. In the case of T. elegans and Mucor sp., a supplementary quantity of ammonium sulfate was added in order to favor the consumption of lactose and the production of biomass. Indeed, enhancement of lipid production was observed for M. isabellina and biomass production for T. elegans and Mucor sp.. Fatty acid analysis of the microbial lipid showed a composition that presented non‐negligible changes in relation with the age of the culture and the C/N molar ratio of the medium. Further analysis of the fat showed that the quantity of neutral lipids was the more abundant. The fatty acid composition of neutral lipids resembled to that of total lipids. Phospholipids were the more unsaturated fraction for Mucor sp. and M. isabellina. GLA was synthesized in all trials but its concentration presented differences related with the utilized strains and the fermentation time. Growth of M. isabellina on lactose‐supplemented whey resulted in a maximum GLA production of 301 mg/L.     

Microbial hydroxylation of non-activated carbon atoms in racemic 2-dodeceno-1-yl-succinic anhydride by <Emphasis Type="Italic">Mortierella isabellina 212</Emphasis>

Microbial hydroxylation of 2-dodecen-1-yl-succinic anhydride at non-activated carbon atoms has been described. A screening with fourteen fungi strains was carried out. Most of the microorganisms transformed the substrate into new products. Biotransformation conditions with Mortierella isabellina 212 were optimized. Optically active products of hydroxylation at C–10 and C–11 were isolated. Chemical structures of the unreacted optically active substrate and the products were determined by spectroscopic methods (¹H NMR, ¹³C NMR, GCMS, IR). Non-enzymatic hydrolysis of the anhydride at different pH was studied and methods of detection of the anhydride and the forming acid were discussed.     

Bioprospecting of Amazon soil fungi with the potential for pigment production

The aim of this study was to isolate fungi able to produce pigments. Fifty strains were isolated from the Amazon soil by the conventional technique of serial dilution. Submerged fermentation was performed in Czapeck broth in order to select strains able to synthesise pigments. Five strains were able to produce pigments and were identified by sequencing the rDNA (ITS regions). These fungi were identified as Penicillium sclerotiorum 2AV2, Penicillium sclerotiorum 2AV6, Aspergillus calidoustus 4BV13, Penicillium citrinum 2AV18 and Penicillium purpurogenum 2BV41. P. sclerotiorum 2AV2 produced intensely coloured pigments and were therefore selected for chemical characterisation. NMR identified the pigment as sclerotiorin. In this work, the influence of nutrients on sclerotiorin yield was also studied and it was verified that rhamnose and peptone increased production when used separately. These results indicate that Amazonian fungi bioprospecting is a viable means to search for new sources of natural dyes.     

Microorganisms associated with production lots of the nucleopolyhedrosis virus of the gypsy moth,Lymantria dispar [Lep.: Lymantriidae]

Samples of a gypsy moth nucleopolyhedrosis virus product, Gypchek^®, were taken each day during a 100-day production run and monitored for the presence of pathogenic bacteria and fungi. The standard plate count/g of product was 5.97±1.51×10⁸ over the 100-day period, while the sporulating bacteria count was 3.81±1.21×10⁶/g. We did not detect obligate anaerobic or fecal coliform bacteria in any of the samples.Bacillus cereus, Staphylococcus epidermidis, B. licheniformis, Streptococcus faecalis, Serratia liquefaciens, andAspergillus niger were the most frequently isolated microorganisms. We did not detect primary pathogenic bacteria or fungi, but the presence of opportunistic pathogens indicated that assiduous monitoring of the virus production facility and rigorous quality control of production batches are necessary.     

A preliminary survey on the occurrence of mycotoxigenic fungi and mycotoxins contaminating red rice at consumer level in Selangor, Malaysia

Red rice is a fermented product of Monascus spp. It is widely consumed by Malaysian Chinese who believe in its pharmacological properties. The traditional method of red rice preparation disregards safety regulation and renders red rice susceptible to fungal infestation and mycotoxin contamination. A preliminary study was undertaken aiming to determine the occurrence of mycotoxigenic fungi and mycotoxins contamination on red rice at consumer level in Selangor, Malaysia. Fifty red rice samples were obtained and subjected to fungal isolation, enumeration, and identification. Citrinin, aflatoxin, and ochratoxin-A were quantitated by ELISA based on the presence of predominant causal fungi. Fungal loads of 1.4?×?10⁴ to 2.1?×?10⁶?CFU/g exceeded Malaysian limits. Monascus spp. as starter fungi were present in 50 samples (100 %), followed by Penicillium chrysogenum (62 %), Aspergillus niger (54 %), and Aspergillus flavus (44 %). Citrinin was present in 100 % samples (0.23–20.65 mg/kg), aflatoxin in 92 % samples (0.61–77.33 μg/kg) and Ochratoxin-A in 100 % samples (0.23–2.48 μg/kg); 100 % citrinin and 76.09 % aflatoxin exceeded Malaysian limits. The presence of mycotoxigenic fungi served as an indicator of mycotoxins contamination and might imply improper production, handling, transportation, and storage of red rice. Further confirmatory analysis (e.g., HPLC) is required to verify the mycotoxins level in red rice samples and to validate the safety status of red rice.     

Identification of Ochratoxin A Producing Fungi Associated with Fresh and Dry Liquorice

The presence of fungi on liquorice could contaminate the crop and result in elevated levels of mycotoxin. In this study, the mycobiota associated with fresh and dry liquorice was investigated in 3 producing regions of China. Potential toxigenic fungi were tested for ochratoxin A (OTA) and aflatoxin B1 (AFB1) production using liquid chromatography/mass spectrometry/mass spectrometry. Based on a polyphasic approach using morphological characters, β-tubulin and RNA polymerase II second largest subunit gene phylogeny, a total of 9 genera consisting of 22 fungal species were identified, including two new Penicillium species (Penicillium glycyrrhizacola sp. nov. and Penicillium xingjiangense sp. nov.). The similarity of fungal communities associated with fresh and dry liquorice was low. Nineteen species belonging to 8 genera were detected from fresh liquorice with populations affiliated with P. glycyrrhizacola, P. chrysogenum and Aspergillus insuetus comprising the majority (78.74%, 33.33% and 47.06% of total) of the community from Gansu, Ningxia and Xinjiang samples, respectively. In contrast, ten species belonging to 4 genera were detected from dry liquorice with populations affiliated with P. chrysogenum, P. crustosum and Aspergillus terreus comprising the majority (64.00%, 52.38% and 90.91% of total) of the community from Gansu, Ningxia and Xinjiang samples, respectively. Subsequent LC/MS/MS analysis indicated that 5 fungal species were able to synthesize OTA in vitro including P. chrysogenum, P. glycyrrhizacola, P. polonicum, Aspergillus ochraceus and A. westerdijkiae, the OTA concentration varied from 12.99 to 39.03 µg/kg. AFB1 was absent in all tested strains. These results demonstrate the presence of OTA producing fungi on fresh liquorice and suggest that these fungi could survive on dry liquorice after traditional sun drying. Penicillium chrysogenum derived from surrounding environments is likely to be a stable contributor to high OTA level in liquorice. The harvesting and processing procedure needs to be monitored in order to keep liquorice free of toxigenic fungi.     

Chemical Forms of Selenium in the Metal-Resistant Bacterium Ralstonia metallidurans CH34 Exposed to Selenite and Selenate

Ralstonia metallidurans CH34, a soil bacterium resistant to a variety of metals, is known to reduce selenite to intracellular granules of elemental selenium (Se⁰). We have studied the kinetics of selenite (Se^IV) and selenate (Se^VI) accumulation and used X-ray absorption spectroscopy to identify the accumulated form of selenate, as well as possible chemical intermediates during the transformation of these two oxyanions. When introduced during the lag phase, the presence of selenite increased the duration of this phase, as previously observed. Selenite introduction was followed by a period of slow uptake, during which the bacteria contained Se⁰ and alkyl selenide in equivalent proportions. This suggests that two reactions with similar kinetics take place: an assimilatory pathway leading to alkyl selenide and a slow detoxification pathway leading to Se⁰. Subsequently, selenite uptake strongly increased (up to 340 mg Se per g of proteins) and Se⁰ was the predominant transformation product, suggesting an activation of selenite transport and reduction systems after several hours of contact. Exposure to selenate did not induce an increase in the lag phase duration, and the bacteria accumulated approximately 25-fold less Se than when exposed to selenite. Se^IV was detected as a transient species in the first 12 h after selenate introduction, Se⁰ also occurred as a minor species, and the major accumulated form was alkyl selenide. Thus, in the present experimental conditions, selenate mostly follows an assimilatory pathway and the reduction pathway is not activated upon selenate exposure. These results show that R. metallidurans CH34 may be suitable for the remediation of selenite-, but not selenate-, contaminated environments.