Identification of Aspergillus species in Central Europe able to produce G-type aflatoxins

The occurrence of potential aflatoxin producing fungi was examined in various agricultural products and indoor air in Central European countries including Hungary, Serbia and Croatia. For species identification, both morphological and sequence based methods were applied. Aspergillus flavus was detected in several samples including maize, cheese, nuts, spices and indoor air, and several isolates were able to produce aflatoxins. Besides, three other species of Aspergillus section Flavi, A. nomius, A. pseudonomius and A. parasiticus were also isolated from cheese, maize and indoor air, respectively. This is the first report on the occurrence of A. nomius and A. pseudonomius in Central Europe. All A. nomius, A. pseudonomius and A. parasiticus isolates were able to produce aflatoxins B1, B2, G1 and G2. The A. nomius isolate came from cheese produced very high amounts of aflatoxins (above 1 mg ml–1). All A. nomius, A. pseudonomius and A. parasiticus isolates produced much higher amounts of aflatoxin G1 then aflatoxin B1. Further studies are in progress to examine the occurrence of producers of these highly carcinogenic mycotoxins in agricultural products and indoor air in Central Europe

Antibacterial potential of marine fungus Aspergillus nomius isolated from green algae Bornetella sp

Isolation of marine fungi symbiont of green algae Bornetella sp as a producer of antibacterial compounds has been carried out. This study aims to obtain symbiont fungi from green algae Bornetella sp which produces antibacterial compounds. The symbiont fungus was isolated using the direct planting method. Screening for the antibacterial activity of pure symbiont fungi isolates against Escheriscia coli and Staphylococcus aureus, and was carried out by the agar diffusion method. The isolation results obtained 4 isolates, but the one with the most potential to inhibit the growth of the tested bacteria was MFALM2. Molecular characterization showed that the MFALM2 isolate was identified as Aspergillus nomius with a 100% closeness level. Keywords: isolation; marine fungi; Aspergillus nomius; green alga; Bornetella sp. AbstrakIsolasi jamur laut yang bersimbion dengan alga hijau Bornetella sp sebagai penghasil senyawa antibakteri telah dilakukan. Penelitian ini bertujuan untuk memperoleh jamur simbion dari alga hijau Bornetella sp yang menghasilkan senyawa antibakteri. Isolasi jamur simbion dilakukan dengan metode direct planting. Skrining aktivitas antibakteri isolat murni jamur simbion terhadap bakteri uji Escheriscia coli dan Staphylococcus aureus dilakukan dengan metode difusi agar.  Hasil isolasi diperoleh 4 isolat, namun yang paling berpotensi menghambat pertumbuhan bakteri uji adalah MFALM2. Karakterisasi molekuler menunjukkan bahwa isolat MFALM2 teridentifikasi sebagai Aspergillus nomius dengan tingkat keeratan sebesar 100%.Kata kunci: isolasi; jamur lau; Aspergillus nomius; alga hijau; Bornetella sp

Two new aflatoxin producing species, and an overview of Aspergillus section Flavi

Aspergillus subgenus Circumdati section Flavi includes species with usually biseriate conidial heads, in shades of yellow-green to brown, and dark sclerotia. Several species assigned to this section are either important mycotoxin producers including aflatoxins, cyclopiazonic acid, ochratoxins and kojic acid, or are used in oriental food fermentation processes and as hosts for heterologous gene expression. A polyphasic approach was applied using morphological characters, extrolite data and partial calmodulin, β-tubulin and ITS sequences to examine the evolutionary relationships within this section. The data indicate that Aspergillus section Flavi involves 22 species, which can be grouped into seven clades. Two new species, A. pseudocaelatus sp. nov. and A. pseudonomius sp. nov. have been discovered, and can be distinguished from other species in this section based on sequence data and extrolite profiles. Aspergillus pseudocaelatus is represented by a single isolate collected from Arachis burkartii leaf in Argentina, is closely related to the non-aflatoxin producing A. caelatus, and produces aflatoxins B & G, cyclopiazonic acid and kojic acid, while A. pseudonomius was isolated from insects and soil in the USA. This species is related to A. nomius, and produces aflatoxin B1 (but not G-type aflatoxins), chrysogine and kojic acid. In order to prove the aflatoxin producing abilities of the isolates, phylogenetic analysis of three genes taking part in aflatoxin biosynthesis, including the transcriptional regulator aflR, norsolonic acid reductase and O-methyltransferase were also carried out. A detailed overview of the species accepted in Aspergillus section Flavi is presented

Characterization of Aspergillus species on Brazil nut from the Brazilian Amazonian region and development of a PCR assay for identification at the genus level.

Brazil nut is a protein-rich extractivist tree crop in the Amazon region. Fungal contamination of shells and kernel material frequently includes the presence of aflatoxigenic Aspergillus species from the section Flavi. Aflatoxins are polyketide secondary metabolites, which are hepatotoxic carcinogens in mammals. The objectives of this study were to identify Aspergillus species occurring on Brazil nut grown in different states in the Brazilian Amazon region and develop a specific PCR method for collective identification of member species of the genus Aspergillus. Results:Polyphasic identification of 137 Aspergillus strains isolated from Brazil nut shell material from cooperatives across the Brazilian Amazon states of Acre, Amapá and Amazonas revealed five species, with Aspergillus section Flavi species A. nomius and A. flavus the most abundant. PCR primers ASP_GEN_MTSSU_F1 and ASP_GEN_MTSSU_R1 were designed for the genus Aspergillus, targeting a portion of the mitochondrial small subunit ribosomal RNA gene. Primer specificity was validated through both electronic PCR against target gene sequences at Genbank and in PCR reactions against DNA from Aspergillus species and other fungal genera common on Brazil nut. Collective differentiation of the observed section Flavi species A. flavus, A. nomius and A. tamarii from other Aspergillus species was possible on the basis of RFLP polymorphism. Conclusions:Given the abundance of Aspergillus section Flavi species A. nomius and A. flavus observed on Brazil nut, and associated risk of mycotoxin accumulation, simple identification methods for such mycotoxigenic species are of importance for Hazard Analysis Critical Control Point system implementation. The assay for the genus Aspergillus represents progress towards specific PCR identification and detection of mycotoxigenic species

Metabolomics Analysis Reveals Specific Novel Tetrapeptide and Potential Anti-Inflammatory Metabolites in Pathogenic Aspergillus species

Infections related to Aspergillus species have emerged to become an important focus in infectious diseases, as a result of the increasing use of immunosuppressive agents and high fatality associated with invasive aspergillosis. However, laboratory diagnosis of Aspergillus infections remains difficult. In this study, by comparing the metabolomic profiles of the culture supernatants of 30 strains of six pathogenic Aspergillus species (A. fumigatus, A. flavus, A. niger, A. terreus, A. nomius and A. tamarii) and 31 strains of 10 non-Aspergillus fungi, eight compounds present in all strains of the six Aspergillus species but not in any strain of the non-Aspergillus fungi were observed. One of the eight compounds, Leu–Glu–Leu–Glu, is a novel tetrapeptide and represents the first linear tetrapeptide observed in Aspergillus species, which we propose to be named aspergitide. Two other closely related Aspergillus-specific compounds, hydroxy-(sulfooxy)benzoic acid and (sulfooxy)benzoic acid, may possess anti-inflammatory properties, as 2-(sulfooxy)benzoic acid possesses a structure similar to those of aspirin [2-(acetoxy)benzoic acid] and salicylic acid (2-hydroxybenzoic acid). Further studies to examine the potentials of these Aspergillus-specific compounds for laboratory diagnosis of aspergillosis are warranted and further experiments will reveal whether Leu–Glu–Leu–Glu, hydroxy-(sulfooxy)benzoic acid and (sulfooxy)benzoic acid are virulent factors of the pathogenic Aspergillus species. © 2015, MDPI AG. All rights Reserved.published_or_final_versio

Identification and characterization of Aspergillus flavus and aflatoxins

Aspergillus flavus is the main producer of the well known carcinogenic aflatoxins. The presence of this fungus and aflatoxins is of huge concern in terms of food safety. The identification of A. flavus is not straightforward due to similarities with closely related species (e.g. A. parasiticus and A. nomius). Also, from the biochemical point of view the closely-related species are able to produce different mycotoxins. In order to clarify the differentiation between species the identification schemes is revisited. Selective media, data from mycotoxins production and molecular biology tools are discussed in order to clarify the concept of A. flavus species.Fundação para a Ciência e a Tecnologia (FCT

The distribution of Aspergillus spp. opportunistic parasites in hives and their pathogenicity to honey bees

Stonebrood is a disease of honey bee larvae caused by fungi from the genus Aspergillus. As very few studies have focused on the epidemiological aspects of stonebrood and diseased brood may be rapidly discarded by worker bees, it is possible that a high number of cases go undetected. Aspergillus spp. fungi are ubiquitous and associated with disease in many insects, plants, animals and man. They are regarded as opportunistic pathogens that require immunocompromised hosts to establish infection. Microbiological studies have shown high prevalences of Aspergillus spp. in apiaries which occur saprophytically on hive substrates. However, the specific conditions required for pathogenicity to develop remain unknown. In this study, an apiary was screened to determine the prevalence and diversity of Aspergillus spp. fungi. A series of dose–response tests were then conducted using laboratory reared larvae to determine the pathogenicity and virulence of frequently occurring isolates. The susceptibility of adult worker bees to Aspergillus flavus was also tested. Three isolates (A. flavus, Aspergillus nomius and Aspergillus phoenicis) of the ten species identified were pathogenic to honey bee larvae. Moreover, adult honey bees were also confirmed to be highly susceptible to A. flavus infection when they ingested conidia. Neither of the two Aspergillus fumigatus strains used in dose–response tests induced mortality in larvae and were the least pathogenic of the isolates tested. These results confirm the ubiquity of Aspergillus spp. in the apiary environment and highlight their potential to infect both larvae and adult bees

Polygalacturonase production by Aspergillus nomius MR103 in solid state fermentation using Agro-industrial wastes

The present study was aimed at polygalacturonase production from Aspergillus nomius MR103 under solid state fermentation. A total of 57 fungal strains were obtained from mangrove soils collected from Gilakaladindi and Malakayalanka of Krishna District Andhra Pradesh. For the isolation of fungi these Soil samples were serially diluted and plated on pectin agar media plates.  Among them, the isolate which showed maximum polygalacturonase activity was selected for this study. This strain was identified as A. nomius MR 103 by 18S rRNA sequences analysis. Pectin rich agro-industrial wastes like apple peel, citrus peel, orange peel, wheat bran, rice bran and sugarcane bagasse were used as substrates for polygalacturonase production by A. nomius MR 103. This strain was inoculated into the nutrient broth containing agro industrial wastes under solid state fermentation and amount of Polygalacturonase production was estimated. Maximum enzyme production of 4.83 IU/mg was recorded at pH 7.0 and temperature 35?C after 7 days of incubation, when orange peels were used as substrate.  Addition of carbon and nitrogen sources to orange peel media improved the Polygalcturonase production. Sucrose as carbon and peptone as nitrogen sources were proved  to be the best for  maximum production of Polygalcturonase by A. nomius MR 103 on orange peel substrate. Utilization of agro-industrial by-products provided the establishment of a cost-efficient and sustainable process for enzyme production.&nbsp

Gene duplication, modularity and adaptation in the evolution of the aflatoxin gene cluster

<p>Abstract</p> <p>Background</p> <p>The biosynthesis of aflatoxin (AF) involves over 20 enzymatic reactions in a complex polyketide pathway that converts acetate and malonate to the intermediates sterigmatocystin (ST) and <it>O</it>-methylsterigmatocystin (OMST), the respective penultimate and ultimate precursors of AF. Although these precursors are chemically and structurally very similar, their accumulation differs at the species level for Aspergilli. Notable examples are <it>A</it>. <it>nidulans </it>that synthesizes only ST, <it>A</it>. <it>flavus </it>that makes predominantly AF, and <it>A</it>. <it>parasiticus </it>that generally produces either AF or OMST. Whether these differences are important in the evolutionary/ecological processes of species adaptation and diversification is unknown. Equally unknown are the specific genomic mechanisms responsible for ordering and clustering of genes in the AF pathway of <it>Aspergillus</it>.</p> <p>Results</p> <p>To elucidate the mechanisms that have driven formation of these clusters, we performed systematic searches of aflatoxin cluster homologs across five <it>Aspergillus </it>genomes. We found a high level of gene duplication and identified seven modules consisting of highly correlated gene pairs (<it>aflA/aflB, aflR/aflS, aflX/aflY</it>, <it>aflF/aflE, aflT/aflQ</it>, <it>aflC/aflW</it>, and <it>aflG/aflL</it>). With the exception of <it>A. nomius</it>, contrasts of mean <it>Ka/Ks </it>values across all cluster genes showed significant differences in selective pressure between section <it>Flavi </it>and non-section <it>Flavi </it>species. <it>A. nomius </it>mean <it>Ka/Ks </it>values were more similar to partial clusters in <it>A. fumigatus </it>and <it>A. terreus</it>. Overall, mean <it>Ka/Ks </it>values were significantly higher for section <it>Flavi </it>than for non-section <it>Flavi </it>species.</p> <p>Conclusion</p> <p>Our results implicate several genomic mechanisms in the evolution of ST, OMST and AF cluster genes. Gene modules may arise from duplications of a single gene, whereby the function of the pre-duplication gene is retained in the copy (<it>aflF</it>/<it>aflE</it>) or the copies may partition the ancestral function (<it>aflA/aflB</it>). In some gene modules, the duplicated copy may simply augment/supplement a specific pathway function (<it>aflR/aflS </it>and <it>aflX/aflY</it>) or the duplicated copy may evolve a completely new function (<it>aflT/aflQ </it>and <it>aflC/aflW</it>). Gene modules that are contiguous in one species and noncontiguous in others point to possible rearrangements of cluster genes in the evolution of these species. Significantly higher mean <it>Ka/Ks </it>values in section <it>Flavi </it>compared to non-section <it>Flavi </it>species indicate increased positive selection acting in the evolution of genes in OMST and AF gene clusters.</p