Production and immobilization of ß-glucanase from Aspergillus niger with its applications in bioethanol production and biocontrol of phytopathogenic fungi.

ß-Glucanase has received great attention in recent years regarding their potential biotechnological applications and antifungal activities. Herein, the specific objectives of the present study were to purify, characterize and immobilize ß-glucanase from Aspergillus niger using covalent binding and cross linking techniques. The evaluation of ß-glucanase in hydrolysis of different lignocellulosic wastes with subsequent bioethanol production and its capability in biocontrol of pathogenic fungi was investigated. Upon nutritional bioprocessing, ß-glucanase production from A. niger EG-RE (MW390925.1) preferred ammonium nitrate and CMC as the best nitrogen and carbon sources, respectively. The soluble enzyme was purified by (NH4)2SO4, DEAE-Cellulose and Sephadex G200 with 10.33-fold and specific activity of 379.1 U/mg protein. Tyrosyl, sulfhydryl, tryptophanyl and arginyl were essential residues for enzyme catalysis. The purified ß-glucanase was immobilized on carrageenan and chitosan with appreciable yield. However, the cross-linked enzyme exhibited superior activity along with remarkable improved thermostability and operational stability. Remarkably, the application of the above biocatalyst proved to be a promising candidate in liberating the associate lignocellulosic reducing sugars, which was utilized for ethanol production by Saccharomyces cerevisiae. The purified ß-glucanase revealed an inhibitory effect on the growth of two tested phytopathogens Fusarium oxysporum and Penicillium digitatum.

Molecular analysis of Aspergillus section Nigri isolated from onion samples reveals the prevalence of A. welwitschiae.

The aim of this study was to isolate Aspergillus section Nigri from onion samples bought in supermarkets and to analyze the fungal isolates by means of molecular data in order to differentiate A. niger and A. welwitschiae species from the other non-toxigenic species of black aspergilli, and detect genes involved in the biosynthesis of ochratoxin A and fumonisin B2. Aspergillus section Nigri were found in 98% (94/96) of the onion samples. Based on the results of multiplex PCR (performed on 500 randomly selected strains), 97.4% of the Aspergillus section Nigri strains were recognized as A. niger/A. welwitschiae. Around half of them were subjected to partial sequencing of the CaM gene to distinguish one from the other. A total of 97.9% of the isolates were identified as A. welwitschiae and only 2.1% as A. niger. The fum8 gene, involved in fumonisin B2 biosynthesis, was found in 36% of A. welwitschiae isolates, but radH and pks genes, involved in ochratoxin A biosynthesis, were found in only 2.8%. The presence/absence of fum8 gene in the A. welwitschiae genome is closely associated with ability/inability of the isolates to produce fumonisin in vitro. Based on these results, we suggest that in-depth studies are conducted to investigate the presence of fumonisins in onion bulbs.

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.

Label-free surface enhanced Raman scattering spectroscopy for discrimination and detection of dominant apple spoilage fungus.

Fungal infection is one of the main causes of apple corruption. The main dominant spoilage fungi in causing apple spoilage are storage mainly include Penicillium Paecilomyces paecilomyces (P. paecilomyces), penicillium chrysanthemum (P. chrysogenum), expanded Penicillium expansum (P. expansum), Aspergillus niger (Asp. niger) and Alternaria. In this study, surface-enhanced Raman spectroscopy (SERS) based on gold nanorod (AuNRs) substrate method was developed to collect and examine the Raman fingerprints of dominant apple spoilage fungus spores. Standard normal variable (SNV) was used to pretreat the obtained spectra to improve signal-to-noise ratio. Principal component analysis (PCA) was applied to extract useful spectral information. Linear discriminant analysis (LDA) and non-linear pattern recognition methods including K nearest neighbor (KNN), Support vector machine (SVM) and back propagation artificial neural networks (BPANN) were used to identify fungal species. As the comparison of modeling results shown, the BPANN model established based on the characteristic spectra variables have achieved the satisfactory result with discrimination accuracy of 98.23%; while the PCA-LDA model built using principal component variables achieved the best distinguish result with discrimination accuracy of 98.31%. It was concluded that SERS has the potential to be an inexpensive, rapid and effective method to detect and identify fungal species.

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.

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.

Discrimination of the microbial subspecies using the ribosomal protein spectra coupled with the metabolite high resolution mass spectra.

Routine microbial identification by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has been achieved based on the spectra of ribosomal proteins with molecular masses between 2000 and 20000Da. It is a rapid, cost-effective, and simple method to characterize different species of microorganisms. But for some subspecies of molds, there are high similarities between their spectra in 2000-20000Da, it makes them indistinguishable in this mass range. Based on the specialized metabolite production, there are obvious differences between the high resolution spectra of the same samples in 600-2000Da. It allows the rapid discrimination of these microbial subspecies. The ability of the method to discriminate microbial subspecies was demonstrated by characterizing three different Aspergillus niger strains. Furthermore, this approach has been applied to discriminate two different Acremonium alternatum strains which were collected from mildew plants. It demonstrated the applicability of the method to the actual samples. The high resolution MS in the range of 600-2000Da was presented as a complementary approach for the routine method in 2000-20000Da. The molds could be identified into species-level group by the spectra between 2000 and 20000Da and the strains within each group could be further discriminated based on differences in metabolites. The spectra between 2000 and 20000Da and the spectra between 600 and 2000Da were obtained from the same samples, which extracted with the same method. There is no need of additional pre-processing to obtain the high resolution spectra. It potentially provides a powerful tool for the fast and accurate identification of microbial subspecies.

Aspartic protease from Aspergillus niger: Molecular characterization and interaction with pepstatin A.

In the pursuit of industrial aspartic proteases, aspergillopepsin A-like endopeptidase from the fungi Aspergillus niger, was identified and cultured by solid state fermentation. Conventional chromatographic techniques were employed to purify the extracellular aspartic protease to apparent homogeneity. The enzyme was found to have single polypeptide chain with a molecular mass of 50 ±â€¯0.5 kDa. The optimum pH and temperature for the purified aspartic protease was found to be 3.5 and 60 °C respectively. The enzyme was stable for 60 min at 50 °C. The purified enzyme had specific activity of 40,000 ±â€¯1800 U/mg. The enzyme had 85% homology with the reported aspergillopepsin A-like aspartic endopeptidase from Aspergillus niger CBS 513.88, based on tryptic digestion and peptide analysis. Pepstatin A reversibly inhibited the enzyme with a Ki value of 0.045 µM. Based on homology modeling and predicted secondary structure, it was inferred that the aspartic protease is rich in ß-structures, which was also confirmed by CD measurements. Interaction of pepstatin A with the enzyme did not affect the conformation of the enzyme as evidenced by CD and fluorescence measurements. Degree of hydrolysis of commercial substrates indicated the order of cleaving ability of the enzyme to be hemoglobin > defatted soya flour > gluten > gelatin > skim milk powder. The enzyme also improved the functional characteristics of defatted soya flour. This aspartic protease was found to be an excellent candidate for genetic manipulation for biotechnological application in food and feed industries, due to its high catalytic turn over number and thermostability.

The multivariate statistical selection of fungal strains isolated from Neoteredo reynei, with the high hydrolytic potential to deconstruct cellulose.

The aim of this study was to select isolated filamentous fungi, naturally occurring in the digestive tract of Neoteredo reynei, with high potential to hydrolyze cellulolytic biomass. The selection of the fungi strains, which produce cellulases, was performed by adding carboxymethylcellulose (CMC), microcrystalline cellulose (MCC) or glucose in the media containing peptone and yeast-extract. In this case, the glucose was used as a reference standard for the growth of the mycelia. The abilities of the fungal strains, to hydrolyze cellulose, on the solid media (MCC or CMC), were evaluated. Two methods were used: the congo-red and the speed of mycelial growth. The measurements of the diameters were performed at 24 h intervals, and the speed of the mycelial growth was calculated after 72 h of cultivation. The molecular and morphological identification of the fungi were applied to the isolated strains. Statistical analysis, including ANOVA and Tukey test, and multivariate analysis were applied as tools to select the strains. Twelve strains were isolated and the results of the identification were 2 strains of Hypocrea lutea, 2 strains of Trichoderma reesei, 2 strains of Aspergillus niger; 2 genera of Aspergillus sp., 2 genera of Trichoderma sp., 1 genus of Fusarium sp. and 1 genus of Gliocadium sp. The discrimination analysis methods such as HCA (Hierarchical Cluster Analysis) and PCA (Principal Component Analysis) indicated three strains with the highest potential to hydrolyze cellulose. In this study, the selection strategy was successful, resulting in the classification of strains from the genera Trichoderma and Hypocrea. This is the first time that this kind of studying was applied to select the potential of the cellulolytic fungal strains, isolated from N. reynei, using the methods of the growth of the mycelial diameter and the statistical discrimination.

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.

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.

Fungal Stress Database (FSD)--a repository of fungal stress physiological data.

Abstract: The construction of the Fungal Stress Database (FSD) was initiated and fueled by two major goals. At first, some outstandingly important groups of filamentous fungi including the aspergilli possess remarkable capabilities to adapt to a wide spectrum of environmental stress conditions but the underlying mechanisms of this stress tolerance have remained yet to be elucidated. Furthermore, the lack of any satisfactory interlaboratory standardization of stress assays, e.g. the widely used stress agar plate experiments, often hinders the direct comparison and discussion of stress physiological data gained for various fungal species by different research groups. In order to overcome these difficulties and to promote multilevel, e.g. combined comparative physiology-based and comparative genomics-based, stress research in filamentous fungi, we constructed FSD, which currently stores 1412 photos taken on Aspergillus colonies grown under precisely defined stress conditions. This study involved altogether 18 Aspergillus strains representing 17 species with two different strains for Aspergillus niger and covered six different stress conditions. Stress treatments were selected considering the frequency of various stress tolerance studies published in the last decade in the aspergilli and included oxidative (H2O2, menadione sodium bisulphite), high-osmolarity (NaCl, sorbitol), cell wall integrity (Congo Red) and heavy metal (CdCl2) stress exposures. In the future, we would like to expand this database to accommodate further fungal species and stress treatments. URL: http://www.fung-stress.org/

Species identification and in vitro antifungal susceptibility testing of Aspergillus section Nigri strains isolated from otomycosis patients.

INTRODUCTION: Aspergillus niger is the most commonly reported etiology of otomycosis based on morphological characteristics. This fungus is a member of Aspergillus section Nigri, a set of morphologically indistinguishable species that can harbor various antifungal susceptibility patterns. The aim of this study was to accurately identify and determine the susceptibility pattern of a set of black aspergilli isolated from otomycosis patients. METHODS: Forty-three black Aspergillus isolates from otomycosis patients were identified by using the PCR-sequencing of the ß-tubulin gene. Furthermore, the susceptibility of isolates to three antifungal drugs, including fluconazole (FLU), clotrimazole (CLT) and nystatin (NS), were tested according to CLSI M38-A2. The data were analyzed using the SPSS software (version 15). RESULTS: The majority of isolates were identified as A. tubingensis (32/43, 74.42%) followed by A. niger (11/43, 25.58%). The lowest minimum inhibitory concentration (MIC) values were observed for NS with geometric means (GM) of 4.65µg/mL and 4.83µg/mL against A. tubingensis and A. niger isolates, respectively. CLT showed wide MIC ranges and a statistically significant inter-species difference was observed between A. tubingensis and A. niger isolates (P<0.05). FLU was inactive against both species with GMs>64µg/mL. CONCLUSION: Species other than A. niger can be more frequent as observed in our study. In addition, considering the low and variable activity of tested antifungal drugs, empirical treatment can result in treatment failure. Accurate identification and antifungal susceptibility testing of isolates is, however, recommended.

Differentiation and identification of grape-associated black aspergilli using Fourier transform infrared (FT-IR) spectroscopic analysis of mycelia.

The purpose of this study was to evaluate the potential of FT-IR spectroscopy as a high-throughput method for rapid differentiation among the ochratoxigenic species of Aspergillus carbonarius and the non-ochratoxigenic or low toxigenic species of Aspergillus niger aggregate, namely A. tubingensis and A. niger isolated previously from grapes of Greek vineyards. A total of 182 isolates of A. carbonarius, A. tubingensis, and A. niger were analyzed using FT-IR spectroscopy. The first derivative of specific spectral regions (3002-2801cm-1, 1773-1550cm-1, and 1286-952cm-1) were chosen and evaluated with respect to absorbance values. The average spectra of 130 fungal isolates were used for model calibration based on Discriminant analysis and the remaining 52 spectra were used for external model validation. This methodology was able to differentiate correctly 98.8% in total accuracy in both model calibration and validation. The per class accuracy for A. carbonarius was 95.3% and 100% for model calibration and validation, respectively, whereas for A. niger aggregate the per class accuracy amounted to 100% in both cases. The obtained results indicated that FT-IR could become a promising, fast, reliable and low-cost tool for the discrimination and differentiation of closely related fungal species.

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.

Molecular Cloning and Characteristic Features of a Novel Extracellular Tyrosinase from Aspergillus niger PA2.

Aspergillus niger PA2, a novel strain isolated from waste effluents of food industry, is a potential extracellular tyrosinase producer. Enzyme activity and L-DOPA production were maximum when glucose and peptone were employed as C source and nitrogen source respectively in the medium and enhanced notably when the copper was supplemented, thus depicting the significance of copper in tyrosinase activity. Tyrosinase-encoding gene from the fungus was cloned, and amplification of the tyrosinase gene yielded a 1127-bp DNA fragment and 374 amino acid residue long product that encoded for a predicted protein of 42.3 kDa with an isoelectric point of 4.8. Primary sequence analysis of A. niger PA2 tyrosinase had shown that it had approximately 99% identity with that of A. niger CBS 513.88, which was further confirmed by phylogenetic analysis. The inferred amino acid sequence of A. niger tyrosinase contained two putative copper-binding sites comprising of six histidines, a characteristic feature for type-3 copper proteins, which were highly conserved in all tyrosinases throughout the Aspergillus species. When superimposed onto the tertiary structure of A. oryzae tyrosinase, the conserved residues from both the organisms occupied same spatial positions to provide a di-copper-binding peptide groove.

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.

A study of organic acid production in contrasts between two phosphate solubilizing fungi: Penicillium oxalicum and Aspergillus niger.

Phosphate solubilizing fungi (PSF) have huge potentials in enhancing release of phosphorus from fertilizer. Two PSF (NJDL-03 and NJDL-12) were isolated and identified as Penicillium oxalicum and Aspergillus niger respectively in this study. The quantification and identification of organic acids were performed by HPLC. Total concentrations of organic acids secreted by NJDL-03 and NJDL-12 are ~4000 and ~10,000 mg/L with pH values of 3.6 and 2.4 respectively after five-days culture. Oxalic acid dominates acidity in the medium due to its high concentration and high acidity constant. The two fungi were also cultured for five days with the initial pH values of the medium varied from 6.5 to 1.5. The biomass reached the maximum when the initial pH values are 4.5 for NJDL-03 and 2.5 for NJDL-12. The organic acids for NJDL-12 reach the maximum at the initial pH = 5.5. However, the acids by NJDL-03 continue to decrease and proliferation of the fungus terminates at pH = 2.5. The citric acid production increases significantly for NJDL-12 at acidic environment, whereas formic and oxalic acids decrease sharply for both two fungi. This study shows that NJDL-12 has higher ability in acid production and has stronger adaptability to acidic environment than NJDL-03.

Aspergillus tubingensis and Aspergillus niger as the dominant black Aspergillus, use of simple PCR-RFLP for preliminary differentiation.

This work aimed to identify the species distribution of common clinical and environmental isolates of black Aspergilli based on simple restriction fragment length polymorphism (RFLP) analysis of the ß-tubulin gene. A total of 149 clinical and environmental strains of black Aspergilli were collected and subjected to preliminary morphological examination. Total genomic DNAs were extracted, and PCR was performed to amplify part of the ß-tubulin gene. At first, 52 randomly selected samples were species-delineated by sequence analysis. In order to distinguish the most common species, PCR amplicons of 117 black Aspergillus strains were identified by simple PCR-RFLP analysis using the enzyme TasI. Among 52 sequenced isolates, 28 were Aspergillus tubingensis, 21 Aspergillus niger, and the three remaining isolates included Aspergillus uvarum, Aspergillus awamori, and Aspergillus acidus. All 100 environmental and 17 BAL samples subjected to TasI-RFLP analysis of the ß-tubulin gene, fell into two groups, consisting of about 59% (n=69) A. tubingensis and 41% (n=48) A. niger. Therefore, the method successfully and rapidly distinguished A. tubingensis and A. niger as the most common species among the clinical and environmental isolates. Although tardy, the Ehrlich test was also able to differentiate A. tubingensis and A. niger according to the yellow color reaction specific to A. niger. A. tubingensis and A. niger are the most common black Aspergillus in both clinical and environmental isolates in Iran. PCR-RFLP using TasI digestion of ß-tubulin DNA enables rapid screening for these common species.