Mycobiota of ground red pepper and their aflatoxigenic potential

To investigate contamination of ground red pepper with fungi and mycotoxin, we obtained 30 ground red pepper samples from 15 manufacturers in the main chili-pepper-producing areas in Korea. Fungal contamination was evaluated by spreading diluted samples on potato dextrose agar plates. The total fungi counts ranged from 0 to 7.3 × 10³ CFU/g. In the samples, the genus Aspergillus had the highest incidence, while Paecilomyces was isolated most frequently. The next most frequent genera were Rhizopus, Penicillium, Cladosporium, and Alternaria. Within Aspergillus, A. ruber was predominant, followed by A. niger, A. amstelodami, A. ochraceus, A. terreus, A. versicolor, A. flavus, and A. fumigatus. The samples were analyzed for aflatoxins, ochratoxin A, and citrinin by ultra-perfomance liquid chromatography (UPLC) with a fluorescence detector. Ochratoxin A was detected from three samples at 1.03?2.08 μg/kg, whereas no aflatoxins or citrinin were detected. To test the potential of fungal isolates to produce aflatoxin, we performed a PCR assay that screened for the norB-cypA gene for 64 Aspergillus isolates. As a result, a single 800-bp band was amplified from 10 A. flavus isolates, and one Aspergillus sp. isolate. UPLC analyses confirmed aflatoxin production by nine A. flavus isolates and one Aspergillus sp. isolate, which produced total aflatoxins at 146.88?909.53 μg/kg. This indicates that continuous monitoring of ground red pepper for toxigenic fungi is necessary to minimize mycotoxin contamination.     

A Review of Onychomycosis Due to <Emphasis Type="Italic">Aspergillus</Emphasis> Species

Aspergillus spp. are emerging causative agents of non-dermatophyte mould onychomycosis (NDMO). New Aspergillus spp. have recently been described to cause nail infections. The following criteria are required to diagnose onychomycosis due to Aspergillus spp.: (1) positive direct microscopy and (2) repeated culture or molecular detection of Aspergillus spp., provided no dermatophyte was isolated. A review of 42 epidemiological studies showed that onychomycosis due to Aspergillus spp. varies between < 1 and 35% of all cases of onychomycosis in the general population and higher among diabetic populations accounting for up to 71% and the elderly; it is very uncommon among children and adolescence. Aspergillus spp. constitutes 7.7–100% of the proportion of NDMO. The toenails are involved 25 times more frequently than fingernails. A. flavus, A. terreus and A. niger are the most common aetiologic species; other rare and emerging species described include A. tubingensis, A. sydowii, A. alliaceus, A. candidus, A. versicolor, A. unguis, A. persii, A. sclerotiorum, A. uvarum, A. melleus, A. tamarii and A. nomius. The clinical presentation of onychomycosis due to Aspergillus spp. is non-specific but commonly distal–lateral pattern of onychomycosis. A negative culture with a positive KOH may point to a NDM including Aspergillus spp., as the causative agent of onychomycosis. Treatment consists of systemic therapy with terbinafine or itraconazole.     

Diversity,molecular phylogeny and fingerprint profiles of airborne <Emphasis Type="Italic">Aspergillus</Emphasis> species using random amplified polymorphic DNA

In the present study, diversity and phylogenetic relationship of Aspergillus species isolated from Tehran air was studied using random amplified polymorphic DNA (RAPD)–polymerase chain reaction (RAPD-PCR). Thirty-eight Aspergillus isolates belonging to 12 species i.e. A. niger (28.94 %, 11 isolates), A. flavus (18.42 %, 7 isolates), A. tubingensis (13.15 %, 5 isolates), A. japonicus (10.52 %, 4 isolates), A. ochraceus (10.52 %, 4 isolates), and 2.63 %, 1 isolate from each A. nidulans, A. amstelodami, A. oryzae, A. terreus, A. versicolor, A. flavipes and A. fumigatus were obtained by settle plate method which they were distributed in 18 out of 22 sampling sites examined. Fungal DNA was extracted from cultured mycelia of all Aspergillus isolates on Sabouraud Dextrose Agar and used for amplification of gene fragments in RAPD-PCR using 11 primers. RAPD-PCR data was analyzed using UPGMA software. Resulting dendrogram of combined selected primers including PM1, OPW-04, OPW-05, P160, P54, P10 and OPA14 indicated the distribution of 12 Aspergillus species in 8 major clusters. The similarity coefficient of all 38 Aspergillus isolates ranged from 0.02 to 0.40 indicating a wide degree of similarities and differences within and between species. Taken together, our results showed that various Aspergillus species including some important human pathogenic ones exist in the outdoor air of Tehran by different extents in distribution and diversity and suggested inter- and intra-species genetic diversity among Aspergillus species by RAPD-PCR as a rapid, sensitive and reproducible method.     

New resources for functional analysis of omics data for the genus <Emphasis Type="Italic">Aspergillus</Emphasis>

Background

Detailed and comprehensive genome annotation can be considered a prerequisite for effective analysis and interpretation of omics data. As such, Gene Ontology (GO) annotation has become a well accepted framework for functional annotation. The genus Aspergillus comprises fungal species that are important model organisms, plant and human pathogens as well as industrial workhorses. However, GO annotation based on both computational predictions and extended manual curation has so far only been available for one of its species, namely A. nidulans.

Results

Based on protein homology, we mapped 97% of the 3,498 GO annotated A. nidulans genes to at least one of seven other Aspergillus species: A. niger, A. fumigatus, A. flavus, A. clavatus, A. terreus, A. oryzae and Neosartorya fischeri. GO annotation files compatible with diverse publicly available tools have been generated and deposited online. To further improve their accessibility, we developed a web application for GO enrichment analysis named FetGOat and integrated GO annotations for all Aspergillus species with public genome sequences. Both the annotation files and the web application FetGOat are accessible via the Broad Institute's website (http://www.broadinstitute.org/fetgoat/index.html). To demonstrate the value of those new resources for functional analysis of omics data for the genus Aspergillus, we performed two case studies analyzing microarray data recently published for A. nidulans, A. niger and A. oryzae.

Conclusions

We mapped A. nidulans GO annotation to seven other Aspergilli. By depositing the newly mapped GO annotation online as well as integrating it into the web tool FetGOat, we provide new, valuable and easily accessible resources for omics data analysis and interpretation for the genus Aspergillus. Furthermore, we have given a general example of how a well annotated genome can help improving GO annotation of related species to subsequently facilitate the interpretation of omics data.

     

Investigation of stored wheat mycoflora,reporting the<Emphasis Type="Italic">Fusarium</Emphasis> cf.<Emphasis Type="Italic">langsethiae</Emphasis> in three provinces of Iran during 2007

Wheat is the most important cereal produced in Iran. A mycological survey was carried out for the first time, on the stored wheat samples in Tehran, East Azarbayejan and Mazandaran provinces in 2007. Exogenous and endogenous fungi, were isolated by the method of flotation with Malachite green agar (MGA 0.25) and Freeze blotter techniques respectively. In this study, 46 species belonging to 23 different genera were isolated.Cladosporium spp. (57.1–89.2%) andAlternaria spp. (82.4–100%) species were the predominant fungal species identified as endogenous mycoflora. The predominant exogenous fungi werePenicillium spp. (78.4–92.8%) andAspergillus spp. (71.4–85.7%) species.Fusarium proliferatum was the most prevalent species ofFusarium isolates.Aspergillus niger (39.4%) andAspergillus flavus (36.7%) were the predominantAspergillus species identified as exogenous mycoflora.Aspergillus flavus (26.6%) was the predominantAspergillus species identified as endogenous mycoflora. Flotation method with MGA 0.25 recommended for isolating of hyaline fungi from wheat cereals. In this study one isolate fromFusarium species was isolated on the basis of morphology and ribosomal internal transcribed spacer classified asFusarium langsethiae but on the basis of partial translation elongation factor-1alpha gene grouped withFusarium sporotrichioides. To our knowledge, this is the first report aboutF. cf.langsethiae in Iran and Asia.     

Synthesis of α-cyclopiazonic acid by fungi of the genus <Emphasis Type="Italic">Aspergillus</Emphasis>

The presence of α-cyclopiazonic acid has been studied among metabolites of Aspergillus fungi. The study was performed with 138 cultures of 13 species obtained from the All-Russia Collection of Microorganisms and the collection of our institute. α-Cyclopiazonic acid was most frequently encountered among the metabolites of the section Flavi (the ability to synthesize α-cyclopiazonic acid was expressed in 61% of the strains of A. flavus, 83% of the strains of A. oryzae, and all strains of A. tamarii). This expression index for A. versicolor was less than 5%. We showed for the first time that α-cyclopiazonic acid is produced by A. fumigatus and A. phoenicis (expression in 30% of the strains of either species).     

Emerging Antifungal Drug Resistance in <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> and Among Other Species of <Emphasis Type="Italic">Aspergillus</Emphasis>

Purpose of Review

The purpose of this review is to give an overview of recent findings on antifungal resistance in Aspergillus fumigatus (the major causative agent of aspergillosis) and sibling Aspergillus species, which can be hidden agents of aspergillosis.

Recent Findings

Azole resistance by Cyp51A mutation in A. fumigatus is a growing problem worldwide. The resistance can occur in patients or in the environment. The former occurs by drug selection in the host, inducing mutations in Cyp51A. The latter is characterized by a tandem repeat in the promoter region of cyp51A gene and mutation(s) in Cyp51A. Environmental resistant strains are prevailing rapidly and globally. Moreover, efflux pump and biofilm formation are closely related with antifungal resistance of A. fumigatus. Finally, sibling species of Aspergillus are described with regard to antifungal resistance.

Summary

Environmental azole-resistant strains have newly emerged and been dispersed globally, and continuous survey and countermeasures are urgently needed against these strains. Although the contributions of Cyp51A and efflux pumps to antifungal resistance are becoming clear, other resistance mechanisms remain unclear. Further investigations including genome comparisons will help to clarify the novel resistant mechanisms and to develop countermeasures or novel antifungal drugs against resistant strains of A. fumigatus and other Aspergillus species that have low susceptibility to antifungal therapeutics.

     

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

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

LTR retrotransposons in the <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> and <Emphasis Type="Italic">A. nidulans</Emphasis> genomes

Fungi of the genus Aspergillus can infect all tissues and organs, causing invasive mycosis (aspergillosis). This disease can be fatal, especially in immunocompromised patients. Microbiological monitoring of these infectious agents is obligatory in modern medical facilities. Mobile elements can be used as markers to identify the Aspergillus species and strains found indoors as well as to diagnose aspergillosis. Genomic sequences of two Aspergillus species, A. fumigatus and A. nidulans, were analyzed in silico in order to detect LTR retrotransposons. These species were found to considerably differ in the composition of retrotransposon families. One of the families, present in both Aspergillus species, was phylogenetically quite different from all known fungal retrotransposons. The majority of its elements were damaged copies. Nevertheless, allegedly undamaged LTR retrotransposon copies were described that contained intact ORFs and might be active.     

<Emphasis Type="Italic">Acorus calamus</Emphasis> Linn.: phytoconstituents and bactericidal property

Acorus calamus Linn. of the family Araceae (Acoraceae), commonly known as Sweet Flag and Vacha. The rhizome of this plant has medicinal properties against bugs, moths, lice and emetic stomach in dyspepsia. Chemical composition of the hydro-distilled essential oil obtained from the rhizomes of A. calamus was analyzed by gas chromatography equipped with flame ionization detector and gas chromatography coupled with mass spectrometry. The essential oil of A. calamus and its major compound β-asarone were tested against five Gram-positive, eight Gram-negative bacteria, and three fungi by the tube-dilution method at a concentration rang of 5.0–0.009 mg/mL. Forty constituents were identified which comprised 98.3 % of the total oil. The major compound β-asarone (80.6 %) was identified and confirm by NMR (¹H– & ¹³C–) in rhizome oil of A. calamus. The organism Micrococcus luteus was found to be more susceptible to the oil with minimum bactericidal concentration (MBC) value of 0.032 ± 0.004 mg/mL, followed by Aspergillus fumigatus, Aspergillus niger and Micrococcus flavus with MBC values of 0.104 ± 0.016, 0.117 ± 0.017 and 0.143 ± 0.013 mg/mL, respectively. The compound β-asarone was susceptible to the microorganism A. niger with MBC value 0.416 ± 0.065 mg/mL. The present study revealed that tetraploid variety of A. calamus is growing in this region with substantial amount of β-asarone. The oil showed bactericidal property against tested bacteria and fungi. The β-asarone exhibited poorer bactericidal activity against test microorganisms.     

A mini-survey of moulds and mycotoxins in locally grown and imported wheat grains in Nigeria

A preliminary survey involving limited sample size was conducted to determine the spectrum of moulds and mycotoxins in wheat grains from flour mills and local markets in Nigeria. Fourteen wheat samples were analyzed for moulds using standard mycological methods and for toxic fungal metabolites using a liquid chromatography-tandem mass spectrometric method. Fusarium (range of incidence 12.5–61.7%) dominated in the wheat grains though species of Aspergillus (range of incidence 2.24–3.86%) were also recovered from the samples. The identified fungal species were Aspergillus flavus (7.7%), Aspergillus niger clade (2.6%), Fusarium avenaceum (10.9%), Fusarium culmorum (22.4%) and Fusarium graminearum (56.4%). A total of 54 microbial metabolites were detected in the samples at concentration ranging between 0.01 μg/kg for macrosporin and 2560 μg/kg for deoxynivalenol. Among the four mycotoxins addressed by regulations in the European Union (EU) found in the samples, deoxynivalenol (incidence 100%) dominated in the samples and its levels exceeded the maximum acceptable EU limit (750 μg/kg) in 36% of the samples. This report underscores the need for more robust surveys with larger sample sizes and across several agro-ecologies in the country.     

Metabolic activity of <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Fusarium lateritium</Emphasis> induced by ethoxylated oleyl cetyl alcohol and their bioremediation and biotechnological potential

The effect of ethoxylated oleyl cetyl alcohol (Henkel, Serbia) on the growth and metabolic activity of Aspergillus niger and Fusarium lateritium was in the focus of this paper. The fungi were isolated from wastewater of Lepenica River (Kragujevac, Serbia) at a place where municipal wastewater discharged into the river. The fungi were grown in Czapek-Dox liquid nutrient medium without and with addition of 0.5% pollutant. The physico-chemical and biochemical changes of pH, total biomass dry weight, quantity of free and total organic acids, proteolytic activity and quality of carbohydrates were evaluated from 4-th to 19-th day of fungal growth. The capacity of fungi to decrease concentration of pollutant in medium was determined by cobalt thiocyanate method. The pollutant caused an inhibitory effect on biomass dry weight of A. niger and F. lateritium for 8.50 and 30.61%, respectively. Among tested fungi, A. niger had the better biodegradation capacity (83%) than F. lateritium (65%). Alkaline protease activity of A. niger enhanced in the presence of pollutant for 7.6% whereas the enzyme of F. lateritium retained about 62.2% activity. Overall, the obtained results indicate the potential application of tested fungi in wastewater treatment, detergent industry and biotechnology.     

Characterization of the velvet regulators in <Emphasis Type="Italic">Aspergillus flavus</Emphasis>

Fungal development and secondary metabolism are closely associated via the activities of the fungal NK-kB-type velvet regulators that are highly conserved in filamentous fungi. Here, we investigated the roles of the velvet genes in the aflatoxigenic fungus Aspergillus flavus. Distinct from other Aspergillus species, the A. flavus genome contains five velvet genes, veA, velB, velC, velD, and vosA. The deletion of velD blocks the production of aflatoxin B1, but does not affect the formation of sclerotia. Expression analyses revealed that vosA and velB mRNAs accumulated at high levels during the late phase of asexual development and in conidia. The absence of vosA or velB decreased the content of conidial trehalose and the tolerance of conidia to the thermal and UV stresses. In addition, double mutant analyses demonstrated that VosA and VelB play an inter-dependent role in trehalose biosynthesis and conidial stress tolerance. Together with the findings of previous studies, the results of the present study suggest that the velvet regulators play the conserved and vital role in sporogenesis, conidial trehalose biogenesis, stress tolerance, and aflatoxin biosynthesis in A. flavus.     

Survey of Philippine coffee beans for the presence of ochratoxigenic fungi

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

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

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

Probiotic Evaluation of Antimicrobial <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> VJC38 Isolated from the Crop of Broiler Chicken

The aim of this study was to evaluate probiotic properties of antimicrobial Lactobacillus plantarum VJC38 in vitro. L. plantarum VJC38 was isolated from the crop of broiler chicken and characterized using dnaK gene sequence. The inhibitory activities of L. plantarum VJC38 against bacterial and fungal pathogens were evaluated. Antifungal compounds secreted by the strain VJC38 were identified using Gas Chromatography and Mass Spectrometry (GC-MS). The strain was evaluated for its tolerance to low pH, resistance to bile salts, auto-aggregation, co-aggregation with pathogenic Escherichia coli, cell surface hydrophobicity, cholesterol lowering activity, β-galactosidase production, adhesion ability to Caco-2 cells, mucin degradation, hemolytic activity and biogenic amine production. Phylogenetic analysis of dnaK gene of bacterial strain VJC38 showed 99% sequence similarity to Lactobacillus plantarum var. plantarum. It showed effective inhibition against food spoiling and pathogenic organisms like Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, Aspergillus niger, Penicillium expansum and Eurotium species. The antifungal compound phenol- 2,4-bis(1,1-dimethylethyl) (PD) was identified in the culture filtrate of L. plantarum VJC38 and reported to have inhibition against Aspergillus species. L. plantarum VJC38 exhibited tolerance to low pH, resistance to bile salts, bile salt hydrolase activity, auto-aggregation (87.5%), co-aggregation with Escherichia coli (55.7%), cholesterol lowering activity (64%), β-galactosidase production (1206 MU), adherence to Caco-2 cells (11%), negative for mucin degradation, hemolytic activity and biogenic amine production. L. plantarum VJC38 could be a good candidate for further investigation in vivo to elucidate its health benefits and to evaluate its technological properties as a bio-protective strain.     

Are Cystic Fibrosis <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> Isolates Different? Intermicrobial Interactions with <Emphasis Type="Italic">Pseudomonas</Emphasis>

Pseudomonas aeruginosa and Aspergillus fumigatus are the leading bacterial and fungal pathogens in cystic fibrosis (CF). We have shown that Af biofilms are susceptible to Pseudomonas, particularly CF phenotypes. Those studies were performed with a reference virulent non-CF Aspergillus. Pseudomonas resident in CF airways undergo profound genetic and phenotypic adaptations to the abnormal environment. Studies have also indicated Aspergillus from CF patients have unexpected profiles of antifungal susceptibility. This would suggest that Aspergillus isolates from CF patients may be different or altered from other clinical isolates. It is important to know whether Aspergillus may also be altered, as a result of that CF environment, in susceptibility to Pseudomonas. CF Aspergillus proved not different in that susceptibility.     

Sensitivity to 5-fluorocytosine and virulence for mice of some human isolates of Aspergillus

The sensitivity to 5-Fluorocytosine (5-FC) and the virulence for mice was assessed for 16 human isolates of Aspergillus: A. fumigatus 2, A. niger 7, A. flavus 4, A. terreus 2 and A. conicus (A. restrictus group) 1.Fourteen strains were recovered from ear exsudates, 1 from sputum and 1 from the pus of mastoid cells of an autopsy case with meningeal and cerebral involvement.Nine of them, which included strains of A. fumigatus and A. niger were sensitive to 5-FC (M.I.C. 6.4 g/ml) whereas strains of A. flavus and A. terreus were resistant (M.I.C. > 51.2 g/ml).Cultures of A. fumigatus, A. flavus and A. conicus proved to be the most virulent for mice and A. terreusstrains the less virulent. Results observed with the strain of A. conicus deserves further investigations.