Aspergillus Section Nigri Strains Research Articles

Prevalence of Azole-Resistant Aspergillus Section Fumigati Strains Isolated from Romanian Vineyard Soil Samples.

A total of 265 soil samples from various Romanian vineyards were screened for fungi resistant to azoles. Aspergillus section Fumigati isolates exhibited significant resistance to itraconazole and voriconazole, but no azole-resistant Aspergillus fumigatus strains were detected. Six percent of the samples were positive for Aspergillus section Fumigati strains, all of which were azole-resistant. The strains were mainly Aspergillus udagawae (93.75%) and Aspergillus lentulus (6.25%). The predominant azole-resistant Aspergillus species were Aspergillus section Nigri strains, which were found in 75 soil samples. This study highlights the importance of understanding fungal resistance in vineyard soils for both the agricultural and clinical sectors. The presence of resistant strains may affect vine health and wine production while also constituting a challenge in the selection of effective treatments against severe and potentially fatal fungal infections in humans, stressing the importance of species-specific antifungal resistance knowledge.

Molecular Analysis for Potential Hospital-Acquired Infection Caused by Aspergillus Tubingensis Through the Environment.

The identification of Aspergillus species has been performed mainly by morphological classification. In recent years, however, the revelation of the existence of cryptic species has required genetic analysis for accurate identification. The purpose of this study was to investigate five Aspergillus section Nigri strains isolated from a patient and the environment in a university hospital. Species identification by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry identified all five black Aspergillus strains as Aspergillus niger. However, calmodulin gene sequence analysis revealed that all five strains were cryptic species, four of which, including the clinical strain, were Aspergillus tubingensis. Hospital-acquired infection of the patient with the A. tubingensis strain introduced from the environment was suspected, but sequencing of six genes from four A. tubingensis strains revealed no environmental strain that completely matched the patient strain. The amount of in vitro biofilm formation of the four examples of the A. tubingensis strain was comparable to that of Aspergillus fumigatus. An extracellular matrix was observed by electron microscopy of the biofilm of the clinical strain. This study suggests that various types of biofilm-forming A. tubingensis exist in the hospital environment and that appropriate environmental management is required.

Antifungal action of quaternary ammonium compounds against environmental molds isolated from food industries

AbstractMolds are ubiquitously found microorganisms that are usually present as contaminants in food industrial environments. It has been shown that Cladosporium and Aspergillus genera are two of the most abundant and widely distributed in these locations because they are highly resistant species to sanitizing treatments. Hence, the search of antimicrobial compounds that are effective to these types of fungal contamination becomes relevant. The aim of this report was to evaluate the antifungal capacity of two commercial preparations made both with benzalkonium chloride (a first generation QAC) alone and with the addition of glutaraldehyde at different concentrations and contact times. A suspension‐neutralization test was performed employing spores of five strains of both Aspergillus section Nigri and Cladosporium cladosporioides, all isolated from food industries environments. Results have shown that benzalkonium chloride preparation was successful in destroying Cladosporium spores at 5% (v/v) concentration (average 4D value of 9.1 min) but failed in neutralizing Aspergillus propagules at the same conditions. On the other hand, the commercial mixture made of benzalkonium chloride and glutaraldehyde was effective in the inactivation of all food spoilage fungi spores at 1% (v/v) concentration (average 4D value of 1.8 and 8.8 min for Cladosporium and Aspergillus strains, respectively).

Enzyme activity of Aspergillus section Nigri strains isolated from the Korean fermentation starter, nuruk.

Aspergillus section Nigri is a fungus used industrially because of its ability to produce enzymes such as cellulolytic, amylolytic and proteolytic enzymes. In this study, we obtained twenty-eight strains of Aspergillus section Nigri from the traditional Korean fermentation starter, nuruk, which is known as a mixed culture of enzymatic filamentous fungi and yeasts. All strains were identified as Aspergillus section Nigri through combined phylogenetic analysis using partial β-tubulin and calmodulin gene sequences. The cellulase, amylase and protease activities of Korean strains were measured and compared with ten reference strains of Aspergillus niger. Most Korean strains showed higher cellulolytic activity than reference strains, and Aspergillus neoniger KCN5 showed the highest β-glucosidase activity. Two-thirds of the Korean strains showed similar levels of α- and glucoamylase activity as the reference strains. The protease activity of Aspergillus section Nigri strains was the highest at pH 3.0, and A. niger KSJ2 showed the highest acidic protease activity. By comparing ten reference strains and twenty-eight Korean strains, our results suggested useful Aspergillus section Nigri strains from nuruk with high enzyme activity, such as KCN5 and KSJ2, and their potential for industrial applications as enzyme producers.

Advances in understanding the enzymatic potential and production of ochratoxin A of filamentous fungi isolated from cocoa fermented beans

In this work, we determined the degree of filamentous fungi contamination in cocoa fermented seeds and the fungal potential to produce enzymes that could contribute to the cocoa quality. Internal transcribed spacer amplicon sequencing (ITS) showed 11 different species with Byssochlamys spectabilis, Aspergillus niger, Aspergillus flavus, Aspergillus carbonarius, Circinella muscae and Penicillium citrinum as the most abundant species. Our results evidenced intra and inter-species differences in the enzymes production. Cellulases, β-glucosidase, ornithine decarboxylase and phenylalanine decarboxylase were the most diffused enzymes expressed in the 53 strains here studied. Moreover, A. niger (6/12), A. carbonarius (2/3), and P. citrinum (3/3) showed high pectinolytic activity. Remarkable was the amino decarboxylase activity of P. citrinum, and A. flavus strains. For the first time we reported the presence of B. spectabilis in cocoa fermented beans, which could play an important role in the biogenic amines formation. In addition, we explored the capability of the Aspergillus section Nigri strains, to produce ochratoxin A (OTA) in a cacao model system (CPMS) and in malt extract medium (MEL). We observed that CPMS, but not MEL, stimulated the OTA production in 6 out 15 strains of Aspergillus section Nigri, reaching values ranging between 1.70 and 4995 μg OTA kg−1 dry mycelium.

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.

Genetic Diversity, Ochratoxin A and Fumonisin Profiles of Strains of Aspergillus Section Nigri Isolated from Dried Vine Fruits.

We investigated ochratoxin A (OTA) contamination in raisin samples purchased from Slovak markets and determined the diversity of black-spored aspergilli as potential OTA and fumonisin (FB1 and FB2) producers. The taxonomic identification was performed using sequences of the nuclear ITS1-5.8s-ITS2 region, the calmodulin and beta-tubulin genes. We obtained 239 isolates from eight fungal genera, of which 197 belonged to Aspergillus (82%) and 42 strains (18%) to other fungal genera. OTA contamination was evidenced in 75% of the samples and its level ranged from 0.8 to 10.6 µg/kg. The combination of all three markers used enabled unambiguous identification of A. carbonarius, A. luchuensis, A. niger, A. tubingensis and A. welwitschiae. The dominant coloniser, simultaneously having the highest within-species diversity isolated from our raisin samples, was A. tubingensis. Out of all analysed strains, only A. carbonarius was found to produce OTA, but in relatively high quantity (2477–4382 µg/kg). The production of FB1 and FB2 was evidenced in A. niger strains only.

Polyphasic, Including MALDI-TOF MS, Evaluation of Freeze-Drying Long-Term Preservation on Aspergillus (Section Nigri) Strains.

This study aims to evaluate the effect of freeze-drying and long-term storage on the biotechnological potential of Aspergillus section Nigri strains. Twelve selected strains were freeze-dried and aged by accelerated storage, at 37 °C in the dark, for 2 and 4 weeks. To assess possible changes as a consequence of the ageing in the freeze-drying ampoules, morphological characteristics, mycotoxins and enzymes production, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALTI-TOF MS) spectra, and M13 phage probe fingerprinting were used as part of a polyphasic approach. Phenotypical changes were observed; nevertheless, they did not substantially affect the potential biotechnological use of these strains. The activity of hydrolytic enzymes (protease, carboxymethylcellulase, xylanase, pectinase and mannanase) was maintained or increased after freeze-drying. MALDI-TOF MS data originated spectra that grouped, for the majority of samples, according to strain independently of preservation time point. M13 profiles revealed the presence of some genetic polymorphisms after preservation. However, the three studied times still clustered for more than 50% of strains. Our results show that the studied strains maintain their biotechnological potential after preservation, with minimal phenotypic alterations. These findings provide evidence that freeze-drying preservation is a suitable option to preserve biotechnologically relevant aspergilli strains from section Nigri, and one should consider that the observed effects might be species/strain-dependent.

Capsaicin, an inhibitor of Ochratoxin A production by Aspergillus section Nigri strains in grapes (Vitis vinifera L.)

ABSTRACTFood decay by spoilage fungi leads to significant economic losses and hazards to consumers’ health due to the potential of mycotoxin occurrence. Ochratoxin A (OTA) is a mycotoxin known as nephrotoxic and carcinogenic to humans. Natural capsaicin was evaluated for its effectiveness against the growth of five Aspergillus section Nigri strains and accumulation of OTA in inoculated black grapes. Results showed that capsaicin was effective in inhibiting fungal growth and OTA production by new four Aspergillus section Nigri strains (ATHUM 6997, 6998, 6999, 7000) and by Aspergillus carbonarius as well. Moreover, capsaicin addition exhibited maximum inhibition of OTA produced by ATHUM 6997, 6998, 6999, and 7000 in black grapes at 28.9%, 8.6%, 68.4%, and 78.1%, respectively. Inhibition percentage of OTA production by A. carbonarius in grapes treated with capsaicin was estimated at 61.5%. These results suggest that capsaicin influences the OTA biosynthesis pathway of all Aspergillus section Nigri strains and therefore could be used as an effective natural preservative against OTA contamination of vineyards. Risk assessment revealed that when grapes are treated with capsaicin, consumers are less exposed to OTA.

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

IntroductionAspergillus 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. MethodsForty-three black Aspergillus isolates from otomycosis patients were identified by using the PCR-sequencing of the β-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). ResultsThe 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. ConclusionSpecies 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.

Transcriptional factor influence on OTA production and the quelling attribute of Sirna on the OTA producing strains of Aspergillus section Nigri

This study determined the influence of some transcriptional factors on ochratoxin A production as well as investigates the quelling attributes of some designed siRNA on the OTA producing Aspergillus section Nigri using standard recommended techniques. Results obtained following comparison of the pks gene promoter sequences from 15 isolates depicts differences in length and homology with the pks gene ranging from 218bp in a strain of the Aspergillus niger to 700bp in Aspergillus carbonarius. The alignment of the pks gene promoter region revealed that six and two of the aligned genes have Aba A binding site corresponding to CATTCT and CATTCC respectively while Brl A binding site was absent in all the isolates. Pac C binding site corresponding to CCTGGC and GCCAAG was also found in two and three of the pks gene promoter region respectively. The three designed siRNA shows significant impact on OTA inhibitions with no significant statistical differences (80.9, 74.4 and 75.3% for pks_Ia, pks_Ib and pks_Ic respectively) (F= 3.830, p&gt;0.05). It can be concluded that Are A and Aba A are potential enhancers for ochratoxin A biosynthesis and none of the investigated transcriptional factors is enough for the activation of ochratoxin A production. However, pks gene was seen as a good target gene for inactivation in order to develop efficient means for ochratoxin A control using RNA silencing technology.Key words: Transcriptional factors, Ochratoxin A, siRNA, Quelling , Aspergillus section Nigri

Ochratoxin a production by four new Aspergillus section Nigri strains isolated from currants of Greek origin

Ochratoxin a production by four new Aspergillus section Nigri strains isolated from currants of Greek origin

Characterization of Aspergillus section Nigri species populations in vineyard soil using droplet digital PCR.

This is the first study to demonstrate the utility of ddPCR as a means to quantify species of Aspergillus section Nigri in soil. This method eliminates the need for isolation and sequence identification of individual fungal isolates, and allows for greater throughput in measuring relative population sizes of important (i.e. mycotoxigenic) Aspergillus species within a population of morphologically indistinguishable species.

Diversity in Secondary Metabolites Including Mycotoxins from Strains of Aspergillus Section Nigri Isolated from Raw Cashew Nuts from Benin, West Africa.

In a previous study, raw cashew kernels were assayed for the fungal contamination focusing on strains belonging to the genus Aspergillus and on aflatoxins producers. These samples showed high contamination with Aspergillus section Nigri species and absence of aflatoxins. To investigate the diversity of secondary metabolites, including mycotoxins, the species of A. section Nigri may produce and thus threaten to contaminate the raw cashew kernels, 150 strains were isolated from cashew samples and assayed for their production of secondary metabolites using liquid chromatography high resolution mass spectrometry (LC-HRMS). Seven species of black Aspergilli were isolated based on morphological and chemical identification: A. tubingensis (44%), A. niger (32%), A. brasiliensis (10%), A. carbonarius (8.7%), A. luchuensis (2.7%), A. aculeatus (2%) and A. aculeatinus (0.7%). From these, 45 metabolites and their isomers were identified. Aurasperone and pyranonigrin A, produced by all species excluding A. aculeatus and A. aculeatinus, were most prevalent and were encountered in 146 (97.3%) and 145 (95.7%) isolates, respectively. Three mycotoxins groups were detected: fumonisins (B2 and B4) (2.7%) ochratoxin A (13.3%), and secalonic acids (2%), indicating that these mycotoxins could occur in raw cashew nuts. Thirty strains of black Aspergilli were randomly sampled for verification of species identity based on sequences of β-tubulin and calmodulin genes. Among them, 27 isolates were positive to the primers used and 11 were identified as A. niger, 7 as A. tubingensis, 6 as A. carbonarius, 2 as A. luchuensis and 1 as A. welwitschiae confirming the species names as based on morphology and chemical features. These strains clustered in 5 clades in A. section Nigri. Chemical profile clustering also showed also 5 groups confirming the species specific metabolites production.

Variability among strains of Aspergillus section Nigri with capacity to degrade tannic acid isolated from extreme environments

Tannins are polyphenolic compounds that cause astringent flavor and turbidity in food. Tannase is an enzyme that catalyzes the hydrolysis of tannins and is used in food industry. This study was conducted to determine the genetic variability and the tannase alleles variation in fungal strains isolated from soil and plants at five extreme areas of Coahuila, México. Two screening assays under 1 and 20% of tannic acid were performed, with the isolations. In these assays, it was possible to identify 756 and 128 fungal strains, respectively. The major fungal variability was observed in "Cuatro Ciénegas" with 26 strains. The microorganisms were distributed in 11 groups, which correspond to Aspergillus section Nigri. AN7 and AN1 groups showed the major number of isolates from "Paila" and "Cuatro Ciénegas" locations, respectively. In the last location, the major diversity and specific richness were found. But in "Ojo Caliente," tannase allele conservations were observed.

Occurrence of Aspergillus section Flavi and section Nigri and aflatoxins in raw cashew kernels (Anacardium occidentale L.) from Benin

The objective of this study was to evaluate the presence of Aspergillus section Flavi and A. section Nigri in cashew nuts harvested in the Northern Guinea (NG) and Southern Sudanian (SS) agro-ecological zones of Benin. Also, the presence of aflatoxins was investigated. For detection of fungal contamination, a total of 100 kernels/sample (with disinfection) and 40 kernels/sample (without disinfection) were plated. Seventy samples from fourteen villages were used. Aflatoxins occurrence was analysed on 84 samples by ultra–high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). The average water content and the cashew nuts count were respectively 8.6% and 172 nuts/kg in NG and 8.7% and 174 nuts/kg in SS. Significant differences between villages in both zones were found for both water content and nuts count. In disinfected samples, strains of Aspergillus section Nigri were predominant, in NG and SS zones (90.2% and 87.2%) respectively. When non disinfected kernels were plated, A. section Nigri was predominant in both NG and SS zones, with percentages of 89.7% and 93.4%, respectively. None of the 84 nuts samples were positive for natural occurrence of aflatoxins with a detection limit of 0.05–0.2 μg/kg.

FUNGAL CONTAMINATION AND MYCOTOXIN PRODUCTION BY ASPERGILLUS SPP. IN NUTS AND SESAME SEEDS

This work reports the occurrence of the fungal flora and evaluates the mycotoxigenic potential of Aspergillus genera in 63 samples of oil seeds and nuts (almonds, pistachio and sesame seeds). Fungal isolation and identification revealed the presence of 5 genera (Aspergillus, Penicillium, Cladosporium, Mucor and yeasts) with the predominance of Aspergillus section Nigri. A number of 138 strains of Aspergillus section Nigri and 91 of Aspergillus section Flavi were isolated and tested for their ability to produce ochratoxin A (OTA) and Aflatoxin, respectively. The detection of Aflatoxins and OTA production was carried out using thin-layer chromatography (TLC). Our results showed that 93.93% of Aspergillus carbonarius were able to produce OTA, but none of the Aspergillus niger aggregate was found to be an OTA-producer. Among the 91 Aspergillus section Flavi isolates, 88 were identified as Aspergillus flavus and 3 as Aspergillus parasiticus. All A. parasiticus were strong AFB and AFG producers. A percentage of 30.3% of A. flavus isolates produced AFB1, with levels ranging from 0.69 to 44.28 μg.g−1. The frequency of aflatoxigenic A. flavus strains was higher in pistachios (46.3%) than in almonds (30.3%) or sesame seeds (23.52%). Cyclopiazonic acid (CPA) and sclerotia production were carried out on CYA medium. All aflatoxigenic A. flavus strains produced CPA, whereas 29.2% produced sclerotia (L-type).

Mycotoxigenic potential of fungi isolated from freshly harvested Argentinean blueberries.

Alternaria alternata, A. tenuissima, Fusarium graminearum, F. semitectum, F. verticillioides, Aspergillus flavus, and Aspergillus section Nigri strains obtained from blueberries during the 2009 and 2010 harvest season from Entre Ríos, Argentina were analyzed to determine their mycotoxigenic potential. Taxonomy status at the specific level was determined both on morphological and molecular grounds. Alternariol (AOH), alternariol monomethyl ether (AME), aflatoxins (AFs), zearalenone (ZEA), fumonisins (FBs), and ochratoxin A (OTA) were analyzed by HPLC and the trichotecenes deoxynivalenol (DON), nivalenol (NIV), HT-2 toxin (HT-2), T-2 toxin (T-2), fusarenone X (FUS-X), 3-acetyl-deoxynivalenol (3-AcDON), and 15-acetyl-deoxynivalenol (15-AcDON) by GC. Twenty-five out of forty two strains were able to produce some of the mycotoxins analyzed. Fifteen strains of Aspergillus section Nigri were capable of producing Fumonisin B1 (FB1); two of them also produced Fumonisin B2 (FB2) and one Fumonisin B3 (FB3). One of the F. graminearum isolated produced ZEA, HT-2, and T-2 and the other one was capable of producing ZEA and DON. Two A. alternata isolates produced AOH and AME. Four A. tenuissima were capable of producing AOH and three of them produced AME as well. One Aspergillu flavus strain produced aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), and aflatoxin G1 (AFG1). To our knowledge, this is the first report showing mycotoxigenic capacity of fungal species isolated from blueberries that include other fungi than Alternaria spp.

Factors affecting distribution and abundance of Aspergillus section Nigri in vineyard soils from grapevine growing regions of Argentina

Aspergillus species belonging to section Nigri are the main fungi responsible for ochratoxin (OTA) contamination in grapes and wine. These species live as saprophytes in the superficial layer of the vineyard soil. We evaluated the biodiversity of potentially ochratoxigenic strains of Aspergillus section Nigri isolated from vineyard soils from different grapevine growing regions of Argentina. The isolates were characterized by classical and molecular methods. A multiple correspondence analysis was performed to identify the overall correlation of the Aspergillus group distribution with environmental conditions, geographical characteristics and vineyard practices. Aspergillus niger aggregate was the prevalent group (71%) and A. carbonarius made up only 2%. Species discrimination by species-specific primers showed that in A. niger aggregate 89% were A. tubingensis; 97% of the uniseriate were A. japonicus/A. aculeatus. Isolates belonging to these groups were unable to produce OTA. Our results clearly demonstrate a strong association between presence of A. carbonarius, high average temperatures and drip irrigation. Precipitation levels appear as a secondary factor, and altitude, vineyard age, predominant species, grape variety or total fungal count showed no association with A. carbonarius. We demonstrated a low prevalence of ochratoxigenic species in vineyard soil from the grape-growing regions of Argentina.

Aflatoxin B1 production by Aspergillus parasiticus and strains of Aspergillus section Nigri in currants of Greek origin

Abstract Aflatoxin B1 (AFB1) mostly produced by Aspergillus flavus and Aspergillus parasiticus, is an extremely toxic and carcinogenic metabolite. Currants are used in the Mediterranean diet as a food with antioxidant properties. Four strains of Aspergillus section Nigri have been isolated from currants originated from Crete and Corinth. In this study AFB1 production by A. parasiticus and the four strains of Aspergillus section Nigri in Cretan and Corinthian currants (Vitis vinifera L.) is investigated. AFB1 determination was performed by HPLC–FID. Results revealed that the four strains Aspergillus section Nigri, as well as the aflatoxigenic strain A. parasiticus produced AFB1 (0.0052–1.31 μg AFB1 15 g−1, corresponding to 0.0003–0.087 μg AFB1 g−1) in both type of currants (Cretan and Corinthian) on the 12th day of observation. Moreover, AFB1 production, by A. parasiticus in the synthetic Yeast Extract Sucrose (YES) medium was also studied. The ability of AFB1 production has been affected by the special characteristics of each isolate and the currants substrate.