New morphological criteria and molecular characterization of black aspergilli aggregate from corn, sorghum and wheat grains.

Corn, sorghum and wheat grains are used as livestock feed in the world. Identification of black aspergilli associated with these grains is necessary to make sure of the safety of the grains because its occurrence is an indicator of mycotoxin production. Forty-five isolates were isolated from the samples collected from Upper Egypt's markets and identified morphologically based on colony color, conidia, stipe and vesicle size and molecularly by using ß-tubulin and calmodulin genes. Isolates were divided into 30 strains of Aspergillus welwitschiae and 15 strains of A. niger. We have found new criteria in the morphological identification of A. welwitschiae as its colony color was black to brown with yellow edge, but in A. niger was black with white edge, also A. welwitschiae sometimes produced finely-to-distinctly roughened brownish conidia on malt extract agar (MEA) media. Thirteen isolates of A. welwitschiae and six of A. niger were recognized as potential producers for ochratoxin A.

Functional analysis of three putative galactofuranosyltransferases with redundant functions in galactofuranosylation in Aspergillus niger.

Galactofuranose (Galf)-containing glycostructures are important to secure the integrity of the fungal cell wall. Golgi-localized Galf-transferases (Gfs) have been identified in Aspergillus nidulans and Aspergillus fumigatus. BLASTp searches identified three putative Galf-transferases in Aspergillus niger. Phylogenetic analysis showed that they group in three distinct groups. Characterization of the three Galf-transferases in A. niger by constructing single, double, and triple mutants revealed that gfsA is most important for Galf biosynthesis. The growth phenotypes of the ΔgfsA mutant are less severe than that of the ΔgfsAC mutant, indicating that GfsA and GfsC have redundant functions. Deletion of gfsB did not result in any growth defect and combining ΔgfsB with other deletion mutants did not exacerbate the growth phenotype. RT-qPCR experiments showed that induction of the agsA gene was higher in the ΔgfsAC and ΔgfsABC compared to the single mutants, indicating a severe cell wall stress response after multiple gfs gene deletions.

Isolation of a novel strain Aspergillus niger WH-2 for production of L(+)-tartaric acid under acidic condition.

OBJECTIVES: To isolate a novel cis-epoxysuccinate hydrolase (CESH)-producing fungus for production of L( +)-tartaric acid, before this, all strains were selected from bacteria. RESULTS: A CESH-producing fungus was first isolated from soil and identified as Aspergillus niger WH-2 based on its morphological properties and ITS sequence. The maximum activity of hyphaball and fermentation supernatants was 1278 ± 64 U/g and 5.6 ± 0.3 U/mL, respectively, in a 5 L fermenter based on the conditions optimized on the flask. Almost 70% of CESH was present in hyphaball, which maintained 40% residual activity at pH 4.0 and showed a good acid stability (pH 3.0-10.0), high conversion rate (> 98%), and enantioselectivity (EE > 99.6%). However, the reported CESHs from bacteria can't be catalyzed under acidic conditions. CONCLUSIONS: The Aspergillus niger WH-2 was the first reported CESH-producing fungus, which could biosynthesize L ( +)-tartaric acid under acidic conditions and provide an alternative catalyst and process.

Significance of Aspergillus niger aggregate species as contaminants of food products in Spain regarding their occurrence and their ability to produce mycotoxins.

The Aspergillus niger aggregate contains 15 morphologically indistinguishable species which presence is related to ochratoxin A (OTA) and fumonisin B2 (FB2) contamination of foodstuffs. The taxonomy of this group was recently reevaluated and there is a need of new studies regarding the risk that these species might pose to food security. 258 isolates of A. niger aggregate obtained from a variety of products from Spain were classified by molecular methods being A. tubingensis the most frequently occurring (67.5%) followed by A. welwitschiae (19.4%) and A. niger (11.7%). Their potential ability to produce mycotoxins was evaluated by PCR protocols which allow a rapid detection of OTA and FB2 biosynthetic genes in their genomes. OTA production is not widespread in A. niger aggregate since only 17% of A. niger and 6% of A. welwitschiae isolates presented the complete biosynthetic cluster whereas the lack of the cluster was confirmed in all A. tubingensis isolates. On the other hand, A. niger and A. welwitschiae seem to be important FB2 producers with 97% and 29% of the isolates, respectively, presenting the complete cluster. The genes involved in OTA and FB2 were overexpressed in producing isolates and their expression was related to mycotoxin synthesis.

Characterization of Aspergillus niger siderophore that mediates bioleaching of rare earth elements from phosphorites.

Siderophores are extra-cellular inducible compounds produced by aerobic microorganisms and plants to overcome iron insolubility via its chelation and then uptake inside the cell. This work aims to study the characteristics of siderophore that is produced by a rhizosphere-inhabiting fungus. This fungus has been morphologically and molecularly identified as Aspergillus niger with the ability to produce 87% siderophore units. The obtained siderophore in PDB medium gave a positive result with tetrazolium test and a characteristic spectrum with a maximum absorbance at 450 nm in FeCl3 test that did not shift in response to different pH degrees (5-9). This indicates that the obtained siderophore is a trihydroxymate in nature. After purification, the FTIR and NMR analyses showed that the obtained siderophore is considered to be ferrichrome. The purified siderophore has been further evaluated as a tool to extract uranium, thorium and rare earth elements (REEs) from Egyptian phosphorites obtained from Abu Tartur Mine area. The inductively coupled plasma atomic emission spectroscopy analysis showed that the highest removal efficiency percentage was for uranium (69.5%), followed by samarium (66.7%), thorium (55%), lanthanum (51%), and cerium (50.1%). This result confirmed the ability of hydroxymate siderophores to chelate the aforementioned precious elements, a result that paves the way for bioleaching to replace abiotic techniques in order to save the cost of such elements in an environmentally friendly way.

Drug Sensitivity and Resistance Mechanism in Aspergillus Section Nigri Strains from Japan.

Aspergillus niger and its related species, known as Aspergillus section Nigri, are ubiquitously distributed across the globe and are often isolated from clinical specimens. In Japan, Aspergillus section Nigri is second most often isolated from clinical specimens following Aspergillus fumigatus We determined the species of Aspergillus section Nigri isolated in Japan by DNA sequencing of partial ß-tubulin genes and investigated drug susceptibility by the CLSI M38-A2 method. The collection contained 20 Aspergillus niger, 59 Aspergillus welwitschiae, and 39 Aspergillus tubingensis strains. Drug susceptibility testing revealed 30 to 55% of A. niger, 6.8 to 18.6% of A. welwitschiae, and 79.5 to 89.7% of A. tubingensis isolates to be less susceptible (so-called resistant) to itraconazole (ITC) and/or voriconazole (VRC) according to the epidemiologic cutoff values (ECVs) proposed for A. niger previously. MIC distributions of ITC or VRC showed no remarkable differences between clinical and environmental isolates. When the cyp51A sequences were compared between susceptible and resistant strains, 18 amino acid mutations were specific for resistant isolates of A. niger and A. tubingensis; however, none of them were confirmed to be associated with azole resistance. Three nonrelated A. welwitschiae isolates possessed a partial deletion in cyp51A, likely attributable to being more susceptible to azoles than other isolates. One of five ITC-resistant A. tubingensis isolates showed higher expression of cyp51A than did susceptible strains. Our results show that cyp51A point mutations may have no association with azole resistance but that in some cases the overexpression of cyp51A may lead to the azole resistance in these species.

The Dynamic Microbiota Profile During Pepper (Piper nigrum L.) Peeling by Solid-State Fermentation.

White pepper (Piper nigrum L.), a well-known spice, is the main pepper processing product in Hainan province, China. The solid-state method of fermentation can peel pepper in a highly efficient manner and yield high-quality white pepper. In the present study, we used next-generation sequencing to reveal the dynamic changes in the microbiota during pepper peeling by solid-state fermentation. The results suggested that the inoculated Aspergillus niger was dominant throughout the fermentation stage, with its strains constituting more than 95% of the fungi present; thus, the fungal community structure was relatively stable. The bacterial community structure fluctuated across different fermentation periods; among the bacteria present, Pseudomonas, Tatumella, Pantoea, Acinetobacter, Lactococcus, and Enterobacter accounted for more than 95% of all bacteria. Based on the correlations among the microbial community, we found that Pseudomonas and Acinetobacter were significantly positively related with A. niger, which showed strong synergy with them. In view of the microbial functional gene analysis, we found that these three bacteria and fungi were closely related to the production of pectin esterase (COG4677) and acetyl xylan esterase (COG3458), the key enzymes for pepper peeling. The present research clarifies the solid-state fermentation method of pepper peeling and lays a theoretical foundation to promote the development of the pepper peeling process and the production of high-quality white pepper.

Asperpyrone-Type Bis-Naphtho-γ-Pyrones with COX-2-Inhibitory Activities from Marine-Derived Fungus Aspergillus niger.

Bis-naphtho-γ-pyrones (BNPs) are an important group of aromatic polyketides derived from fungi, and asperpyrone-type BNPs are produced primarily by Aspergillus species. The fungal strain Aspergillus niger SCSIO Jcsw6F30, isolated from a marine alga, Sargassum sp., and identified according to its morphological traits and the internal transcribed spacer (ITS) region sequence, was studied for BNPs secondary metabolisms. After HPLC/MS analysis of crude extract of the fermentation broth, 11 asperpyrone-type BNPs were obtained directly and quickly by chromatographic separation in the extract, and those isolated asperpyrone-type BNPs were structurally identified by NMR and MS analyses. All of the BNPs showed weak cytotoxicities against 10 human tumor cells (IC50 > 30 µM). However, three of them, aurasperone F (3), aurasperone C (6) and asperpyrone A (8), exhibited obvious COX-2-inhibitory activities, with the IC50 values being 11.1, 4.2, and 6.4 µM, respectively. This is the first time the COX-2-inhibitory activities of BNPs have been reported.

Characterization of a novel ß-cypermethrin-degrading Aspergillus niger YAT strain and the biochemical degradation pathway of ß-cypermethrin.

Aspergillus niger YAT strain was obtained from Chinese brick tea (Collection number: CGMCC 10,568) and identified on the basis of morphological characteristics and internal transcribed spacer (ITS) sequence. The strain could degrade 54.83 % of ß-cypermethrin (ß-CY; 50 mg L(-1)) in 7 days and 100 % of 3-phenoxybenzoic acid (3-PBA; 100 mg L(-1)) in 22 h. The half-lives of ß-CY and 3-PBA range from 3.573 to 11.748 days and from 5.635 to 12.160 h, respectively. The degradation of ß-CY and 3-PBA was further described using first-order kinetic models. The pathway and mechanism of ß-CY degraded by YAT were investigated by analyzing the degraded metabolites through high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). Relevant enzymatic activities and substrate utilization were also investigated. ß-CY degradation products were analyzed. Results indicated that YAT strain transformed ß-CY into 3-PBA. 3-PBA was then gradually transformed into permethric acid, protocatechuic acid, 3-hydroxy-5-phenoxy benzoic acid, gallic acid, and phenol gradually. The YAT strain can also effectively degrade these metabolites. The results indicated that YAT strain has potential applications in bioremediation of pyrethroid insecticide (PI)-contaminated environments and fermented food.

Detection and discrimination of four Aspergillus section Nigri species by PCR.

Species of Aspergillus section Nigri are not easily distinguished by traditional morphological techniques, and typically are identified by DNA sequencing methods. We developed four PCR primers to distinguish between Aspergillus niger, Aspergillus welwitschiae, Aspergillus carbonarius and Aspergillus tubingensis, based on species-conserved differences in the calmodulin gene sequence. PCR amplification from total DNA using these primers was species specific; no amplification occurred from nontarget species DNA for each primer pair. Species-specific PCR could distinguish between species in mixed DNA templates, indicating a utility in determining culture uniformity of isolated Aspergillus strains. In addition, with these primer sets, each species could be detected in soil following mixed-species inoculation with Aspergillus spores. This indicates that PCR with these species-specific primers may be useful in determining the distribution of Aspergillus species in environmental samples without the need for species identification from isolated strains, as well as detecting species that may be infrequently isolated by culture-based methods.

Molecular characterization of black Aspergillus species from onion and their potential for ochratoxin A and fumonisin B2 production.

Onion bulbs can become contaminated with various molds during the storage period, the most important causal agents being black aspergilli (Aspergillus section Nigri). Taxonomic studies have revealed that this group of Aspergillus contains many species that cannot be reliably identified using standard morphological methods. Therefore, it is necessary to define the fungus causing this problem in the onion exactly, especially since some species assigned to section Nigri are well known as ochratoxin and/or fumonisin producers. Sixty fungal isolates belonging to 10 fungal genera were isolated from 40 onion samples originated from the Taif region in Saudi Arabia. Black aspergilli were detected in 37 onion samples. Using primer pairs (awaspec and Cmd6) designed based on partial calmodulin gene sequence data, 37 isolates were identified as A. welwitschiae. The ochratoxin A and fumonisin B2 contents of the onion samples were examined. No ochratoxins were detected in the collected samples, while fumonisin B2 was detected in 37.5% of the onion samples. Eighteen of 37 isolates of Aspergillus welwitschiae were recognized as potential producers for fumonisin B2. Multiplex polymerase chain reactions designed to detect biosynthetic genes of fumonisins confirmed these results.

Phytase production by Aspergillus niger CFR 335 and Aspergillus ficuum SGA 01 through submerged and solid-state fermentation.

Fermentation is one of the industrially important processes for the development of microbial metabolites that has immense applications in various fields. This has prompted to employ fermentation as a major technique in the production of phytase from microbial source. In this study, a comparison was made between submerged (SmF) and solid-state fermentations (SSF) for the production of phytase from Aspergillus niger CFR 335 and Aspergillus ficuum SGA 01. It was found that both the fungi were capable of producing maximum phytase on 5th day of incubation in both submerged and solid-state fermentation media. Aspergillus niger CFR 335 and A. ficuum produced a maximum of 60.6 U/gds and 38 U/gds of the enzyme, respectively, in wheat bran solid substrate medium. Enhancement in the enzyme level (76 and 50.7 U/gds) was found when grown in a combined solid substrate medium comprising wheat bran, rice bran, and groundnut cake in the ratio of 2 : 1 : 1. A maximum of 9.6 and 8.2 U/mL of enzyme activity was observed in SmF by A. niger CFR 335 and A.ficuum, respectively, when grown in potato dextrose broth.

Identification of ochratoxin A producing Aspergillus carbonarius and A. niger clade isolated from grapes using the loop-mediated isothermal amplification (LAMP) reaction.

AIMS: To develop two assays based on the loop-mediated isothermal amplification (LAMP) of DNA for the quick and specific identification of Aspergillus carbonarius and ochratoxigenic strains of the Aspergillus niger clade isolated from grapes. METHODS AND RESULTS: Two sets of primers were designed based on the polyketide synthase genes involved or putatively involved in ochratoxin A (OTA) biosynthesis in A. carbonarius and A. niger clade. Hydroxynaphthol blue was used as indirect method to indicate DNA amplification. The limit of detection of both assays was comparable to that of a PCR reaction. Specificities of the reactions were tested using DNA from different black aspergilli isolated from grapes. The two LAMP assays were then used to identify A. carbonarius and ochratoxigenic A. niger and A. awamori grown in pure cultures without a prior DNA extraction. CONCLUSIONS: The two LAMP assays permitted to quickly and specifically identify DNA from OTA-producing black aspergilli, as well as isolates grown in pure culture. SIGNIFICANCE AND IMPACT OF THE STUDY: Monitoring vineyards for the presence of OTA-producing strains is part of the measures to minimize the occurrence of OTA in grape products. The two LAMP assays developed here could be potentially used to speed the screening process of vineyards for the presence of OTA-producing black aspergilli.

Bioresolution of (R)-glycidyl azide by Aspergillus niger ZJUTZQ208: a new and concise synthon for chiral vicinal amino alcohols.

A newly isolated Aspergillus niger strain containing epoxide hydrolase was used to resolve racemic glycidyl azide and four derivatives to the (R)-enantiomers. After optimization of the biotransformation conditions, (R)-glycidyl azide was produced with good enantioselectivity (e.e.s > 95 %, E > 20). The substrate structure, pH, and reaction time were found to have profound influences on the catalytic property of A. niger ZJUTZQ208. Enantiopure glycidyl azide was further utilized to synthesize linezolid in good yield, indicating it is a new and concise synthon for chiral vicinal amino alcohols. Enzyme-substrate docking studies were carried out with glycidyl azide to study the selectivity of this strain.

Aspergillus niger P6 and Rhodotorula mucilaginosa CH4 used for olive mill wastewater (OMW) biological treatment in single pure and successive cultures.

The aim of this study was to investigate the Rhodotorula mucilaginosa CH4 and Aspergillus niger P6 abilities to purify olive mill wastewater (OMW) in single pure and mixed cultures during the treatment. Both fungi were molecularly identified. OMW was used at five dilutions from 5% to 30% with chemical oxygen demand (COD) ranging from 11,600 to 24,600 mg L(-1). Firstly, each fungus was used separately, then they were successively used to treat the OMW. In single pure culture, A. niger showed a better efficiency in OMW purification than R. mucilaginosa. Furthermore, when successively used, the two studied strains exhibited improvements in the decrease of COD, polyphenolic compounds concentration and effluent colour. COD removals were 95.68-56.71% by R. mucilaginosa and 98.02-69.51% by A. niger for OMW dilutions varying from 5% to 30%. Both strains showed an important polyphenolic compounds removal of 83-45% by R. mucilaginosa and 94-58% by A. niger, in accordance with the OMW COD initially used. The COD and phenolic compound removals fitted simple equation models, with high regression coefficients. The strains' growth kinetics decreased according to the OMW concentration, but, when successively used, fungal growth was improved, allowing efficient effluent treatment.

Biochemical characterization of Aspergillus niger CfcI, a glycoside hydrolase family 18 chitinase that releases monomers during substrate hydrolysis.

The genome of the industrially important fungus Aspergillus niger encodes a large number of glycoside hydrolase family 18 members annotated as chitinases. We identified one of these putative chitinases, CfcI, as a representative of a distinct phylogenetic clade of homologous enzymes conserved in all sequenced Aspergillus species. Where the catalytic domain of more distantly related chitinases consists of a triosephosphate isomerase barrel in which a small additional (α+ß) domain is inserted, CfcI-like proteins were found to have, in addition, a carbohydrate-binding module (CBM18) that is inserted in the (α+ß) domain next to the substrate-binding cleft. This unusual domain structure and sequence dissimilarity to previously characterized chitinases suggest that CfcI has a novel activity or function different from chitinases investigated so far. Following its heterologous expression and purification, its biochemical characterization showed that CfcI displays optimal activity at pH 4 and 55-65 °C and degrades chitin oligosaccharides by releasing N-acetylglucosamine from the reducing end, possibly via a processive mechanism. This is the first fungal family 18 exochitinase described, to our knowledge, that exclusively releases monomers. The cfcI expression profile suggests that its physiological function is important in processes that take place during the late stages of the aspergillus life cycle, such as autolysis or sporulation.

Species assignment and antifungal susceptibilities of black aspergilli recovered from otomycosis cases in Iran.

Black aspergilli are among the main causative agents of otomycosis worldwide. In this study, the species assignment of black aspergilli isolated from otomycosis cases in Iran was carried out using sequence analysis of part of the calmodulin gene. The results indicate that Aspergillus niger is not the only black Aspergillus species involved in otomycosis cases in Iran: Aspergillus awamori and Aspergillus tubingensis are also able to cause ear infections. Antifungal susceptibility tests were carried out against five antifungal drugs including amphotericin B, fluconazole, itraconazole, ketoconazole and terbinafine. All isolates were highly susceptible to terbinafine, while they exhibited moderate susceptibilities against amphotericin B, fluconazole and ketoconazole. Aspergillus niger and A. awamori were found to have higher minimal inhibitory concentrations for azoles than A. tubingensis, in accordance with previous findings.

[Pathogenesis of invasive fungal infections]. / Patogenia de la infección fúngica invasora.

Invasive fungal infections remain a life-threatening disease. The development of invasive fungal disease is dependent on multiple factors, such us colonization and efficient host immune response. We aimed to review the pathogenesis of invasive fungal infections, in particular, those caused by Candida and Aspergillus. For this we propose, to describe the fungal characteristics, to detail the host defence mechanisms against fungus and to analyse the host risk factors for invasive fungal infection.

Identification and proteomic analysis of a novel gossypol-degrading fungal strain.

BACKGROUND: Cottonseed meal, an important source of feed raw materials, has limited use in the feed industry because of the presence of the highly toxic gossypol. The aim of the current work was to isolate the gossypol-degrading fungus from a soil microcosm and investigate the proteins involved in gossypol degradation. RESULTS: A fungal strain, AN-1, that uses gossypol as its sole carbon source was isolated and identified as Aspergillus niger. A large number of intracellular proteins were detected using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but no significant difference was observed between the glucose-containing and gossypol-containing mycelium extracts. Two-dimensional gel electrophoresis results showed that the protein spots were concentrated in the 25.0-66.2 kDa range and distributed in different pI gradients. PDQuest software showed that 51 protein spots in the gels were differentially expressed. Of these, 20 differential protein spots, including six special spots expressed in gossypol, were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. CONCLUSION: The fungus AN-1 biodegraded gossypol and the proteomic analysis results indicate that some proteins were involved in the gossypol biodegradation during fungus survival, using gossypol as its sole carbon source.

Heterogeneity of Aspergillus niger microcolonies in liquid shaken cultures.

The fungus Aspergillus niger forms (sub)millimeter microcolonies within a liquid shaken culture. Here, we show that such microcolonies are heterogeneous with respect to size and gene expression. Microcolonies of strains expressing green fluorescent protein (GFP) from the promoter of the glucoamlyase gene glaA or the ferulic acid esterase gene faeA were sorted on the basis of diameter and fluorescence using the Complex Object Parametric Analyzer and Sorter (COPAS) technology. Statistical analysis revealed that the liquid shaken culture consisted of two populations of microcolonies that differ by 90 µm in diameter. The population of small microcolonies of strains expressing GFP from the glaA or faeA promoter comprised 39% and 25% of the culture, respectively. Two populations of microcolonies could also be distinguished when the expression of GFP in these strains was analyzed. The population expressing a low level of GFP consisted of 68% and 44% of the culture, respectively. We also show that mRNA accumulation is heterogeneous within microcolonies of A. niger. Central and peripheral parts of the mycelium were isolated with laser microdissection and pressure catapulting (LMPC), and RNA from these samples was used for quantitative PCR analysis. This analysis showed that the RNA content per hypha was about 45 times higher at the periphery than in the center of the microcolony. Our data imply that the protein production of A. niger can be improved in industrial fermentations by reducing the heterogeneity within the culture.