Aspergillus nomius,a new aflatoxin-producing species related to Aspergillus flavus and Aspergillus tamarii

Aspergillus nomius is described and represents a new aflatoxigenic species phenotypically similar to A. flavus. Strains examined were isolated from insects and agricultural commodities. Separation from A. flavus is based on the presence of indeterminate sclerotia and a lower growth temperature. Comparisons of DNA relatedness show A. nomius to have only relatively recently evolved from A. flavus and A. tamarii.     

Yellow Pigments of Aspergillus niger and Aspergillus awamori

Alkaline degradation of Aurasperone A, C₃₂H₂₆O₁₀, gave a binaphthyl (IIa), m.p. 255°C and acetone. (IIa) afforded a tetraacetate (IIb), C₃₂H₃₀O₁₂ m.p. 219°C and a tetramethyl ether (IId), C₂₈H₃₀O₈, m.p. 188°C. These facts along with the NMR spectra of aurasperone A and (IIb) confirm that aurasperone A is a dimeric 2-methyl-5-hydroxy-6,8-dimethoxy-4H-naphtho[2,3-b]pyran-4-one with asymmetric C-C linkage (7-10′ or 9-10′). The ether (IId) is not identical with 1,1′ ,3,3′ ?6,6′ ,8,8′-octamethoxy-4,4′-binaphthyl. Thus, it follows that (IId) is a 2,4′-binaphthyl and hence aurasperone A is 2,2′-dimethyl-5,5′- dihydroxy-6,6′,8,8′-tetrahydroxy-7,10′-bi[4H-naphtho[2,3-b]pyran-4-one] (I).     

Intracellular hydrolases of Aspergillus parasiticus and Aspergillus flavus

When the distribution profile of hydrolases in mycelial homogenates and culture filtrates of A. parasiticus and A. flavus was examined, six hydrolytic enzymes viz. N-acetyl-beta-glucosaminidase, aryl sulfatase, alkaline proteinase, cathepsin B, cathepsin D and aminopeptidase were detected in homogenate. The culture filtrates were devoid of any activity of these enzymes. The enzyme levels varied with the stage of incubation. The most abundant fungal exopeptidase showing preference for basic amino acid naphthylamides seems to be an aminopeptidase B. Incorporation of CEPA, an ethylene generating compound, stimulated the amino peptidase activity in the mycelium but inhibited the enzyme in vitro. The enzyme was also inhibited by different aflatoxins to varying degree. While aminopeptidase B was located intracellularly, a non-dialysable, heat-stable inhibitor of the enzyme was found to be secreted in the culture filtrate. This peptide inhibitor was however ineffective on the other enzymes.     

Bidirectional gene transfer between Aspergillus fumigatus and Aspergillus nidulans

Abstract The rpmF-plsX-fabH gene cluster of Rhodobacter capsulatus homologous to that of Escherichia coli was identified. rpmF encodes ribosomal protein L32, plsX plays an undefined role in membrane lipid synthesis, and fabH encodes β-ketoacyl-acyl carrier protein synthase III. The R. capsulatus plsX gene complemented a defect in an E. coli strain with the plsX50 mutation. Overproduction of the fabH gene product of R. capsulatus in E. coli resulted in dramatically increased β-ketoacyl-acyl carrier protein synthase III activity. These results indicate that plsX and fabH apparently function the same in R. capsulatus as in E. coli .     

Ochratoxin production by the Aspergillus ochraceus group and Aspergillus alliaceus

Ochratoxin A is a toxic and carcinogenic fungal secondary metabolite; its presence in foods is increasingly regulated. Various fungi are known to produce ochratoxins, but it is not known which species produce ochratoxins consistently and which species cause ochratoxin contamination of various crops. We isolated fungi in the Aspergillus ochraceus group (section Circumdati) and Aspergillus alliaceus from tree nut orchards, nuts, and figs in California. A total of 72 isolates were grown in potato dextrose broth and yeast extract-sucrose broth for 10 days at 30 degrees C and tested for production of ochratoxin A in vitro by high-pressure liquid chromatography. Among isolates from California figs, tree nuts, and orchards, A. ochraceus and Aspergillus melleus were the most common species. No field isolates of A. ochraceus or A. melleus produced ochratoxin A above the level of detection (0.01 microg/ml). All A. alliaceus isolates produced ochratoxin A, up to 30 microg/ml. We examined 50,000 figs for fungal infections and measured ochratoxin content in figs with visible fungal colonies. Pooled figs infected with A. alliaceus contained ochratoxin A, figs infected with the A. ochraceus group had little or none, and figs infected with Penicillium had none. These results suggest that the little-known species A. alliaceus is an important ochratoxin-producing fungus in California and that it may be responsible for the ochratoxin contamination occasionally observed in figs.     

New taxa of Neosartorya and Aspergillus in Aspergillus section Fumigati

Three new species of Neosartorya and one new Aspergillus of section Fumigati are proposed using a polyphasic approach based on morphology, extrolite production and partial β-tubulin, calmodulin, and actin gene sequences. The phylogenetic analyses using the three genes clearly show that the taxa grouped separately from the known species and confirmed the phenotypic differences. Neosartorya denticulata is characterized by its unique denticulate ascospores with a prominent equatorial furrow; N. assulata by well developed flaps on the convex surface of the ascospores which in addition have two distinct equatorial crests and N. galapagensis by a funiculose colony morphology, short and narrow conidiophores and ascospores with two wide equatorial crests with a microtuberculate convex surface. Aspergillus turcosus can be distinguished by velvety, gray turquoise colonies and short, loosely columnar conidial heads. The four new taxa also have unique extrolite profiles, which contain the mycotoxins gliotoxin and viriditoxin in N. denticulate; apolar compounds provisionally named NEPS in N. assulata and gregatins in N. galapagensis. A. turcosus produced kotanins. N. denticulata sp. nov., N. assulata sp. nov., N. galapagensis sp. nov., and A. turcosus sp. nov. are described and illustrated. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ochratoxin Production by the Aspergillus ochraceus Group and Aspergillus alliaceus

Ochratoxin A is a toxic and carcinogenic fungal secondary metabolite; its presence in foods is increasingly regulated. Various fungi are known to produce ochratoxins, but it is not known which species produce ochratoxins consistently and which species cause ochratoxin contamination of various crops. We isolated fungi in the Aspergillus ochraceus group (section Circumdati) and Aspergillus alliaceus from tree nut orchards, nuts, and figs in California. A total of 72 isolates were grown in potato dextrose broth and yeast extract-sucrose broth for 10 days at 30°C and tested for production of ochratoxin A in vitro by high-pressure liquid chromatography. Among isolates from California figs, tree nuts, and orchards, A. ochraceus and Aspergillus melleus were the most common species. No field isolates of A. ochraceus or A. melleus produced ochratoxin A above the level of detection (0.01 μg/ml). All A. alliaceus isolates produced ochratoxin A, up to 30 μg/ml. We examined 50,000 figs for fungal infections and measured ochratoxin content in figs with visible fungal colonies. Pooled figs infected with A. alliaceus contained ochratoxin A, figs infected with the A. ochraceus group had little or none, and figs infected with Penicillium had none. These results suggest that the little-known species A. alliaceus is an important ochratoxin-producing fungus in California and that it may be responsible for the ochratoxin contamination occasionally observed in figs.     

Mycotoxins in Aspergillus

The toxigenicity of 392 strains ofAspergillus, representing 132 species and 19 varieties, was assessed on chicks and mice fed iso-caloric 50 % basal feed mixes of wheat and soybeans molded by these strains. On the basis of death numbers (3 or more of 6 on test), low weight gain of survivors, and low feed consumption over a 4-week period, 166 of the strains, representing 73 species and 9 varieties, were toxigenic. Ten species (A. alliaceus, A. avenaceus, A. clavato-flavus, A. janus, A. melleus, A. ochraceus, A. parasiticus, A. quercinus, A. sclerotiorum, andA. sulphureus) had strains that were markedly toxic to both animal types on one or both feeds; the other 63 species and 9 varieties had strains that were moderately to mildly toxic to one or both animal types on one or both feeds. In retests of some strains, toxigenicity varied between successive preparation of molded feed.This is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, United States Department of Agriculture.South Dakota Agricultural Experiment Station Journal Series No. 954.     

Allergic Aspergillus sinusitis

The condition of allergic aspergillosis of the paranasal sinuses is presented. Two cases are added to the seven previously described in the world literature. The clinical, radiologic, and pathologic features are outlined, and a careful follow-up regime is advised.     

Substrate Specificity of Alkaline Proteinases from Aspergillus sydowi and Aspergillus sulphureus

l-Fucose (l-galactose) dehydrogenase was isolated to homogeneity from a cell-free extract of Pseudomonas sp. No 1143 and purified about 380-fold with a yield of 23 %. The purification procedures were: treatment with polyethyleneimine, ammonium sulfate fractionation, chromatographies on phenyl-Sepharose and DEAE-Sephadex, preparative polyacrylamide gel electrophoresis, and gel filtration on Sephadex G-100. The enzyme had a molecular weight of about 34,000. The optimum pH was at 9 — 10.5 and the isoelectric point was at pH 5.1. l-Fucose and l-galactose were effective substrates for the enzyme reaction, but d-arabinose was not so much. The anomeric requirement of the enzyme to l-fucose was the β-pyranose form, and the reaction product from l-fucose was l-fucono- lactone. The hydrogen acceptor for the enzyme reaction wasNADP⁺, and NAD ⁺ could be substituted for it to a very small degree. Km values were 1.9mm, 19mm, 0.016mm, and 5.6mm for l-fucose, l- galactose, NADP⁺, and NAD⁺, respectively. The enzyme activity was strongly inhibited by Hg^{2 +}, Cd^{2 +}, and PCMB, but metal-chelating reagents had almost no effect. In a preliminary experiment, it was indicated that the enzyme may be usable for the measurement of l-fucose.     

Aflatoxin production by entomopathogenic isolates of Aspergillus parasiticus and Aspergillus flavus

Fourteen isolates of Aspergillus parasiticus and 2 isolates of Aspergillus flavus isolated from the mealybug Saccharicoccus sacchari were analyzed for production of aflatoxins B1, B2, G1, and G2 in liquid culture over a 20-day period. Twelve Aspergillus isolates including 11 A. parasiticus and 1 A. flavus produced aflatoxins which were extracted from both the mycelium and culture filtrate. Aflatoxin production was detected at day 3 and was detected continually for up to day 20. Aflatoxin B1 production was greatest between 7 and 10 days and significantly higher quantities were produced by A. flavus compared to A. parasiticus. Aflatoxin production was not a stable trait in 1 A. parasiticus isolate passaged 50 times on agar. In addition to loss of aflatoxin production, an associated loss in sporulation ability was also observed in this passaged isolate, although it did maintain pathogenicity against S. sacchari. An aflatoxin B1 concentration of 0.16 micrograms/mealybug (14.2 micrograms/g wet wt) was detected within the tissues of infected mealybugs 7 days after inoculation. In conclusion, the ability of Aspergillus isolates to produce aflatoxins was not essential to the entomopathogenic activity of this fungus against its host S. sacchari.     

Galactosaminogalactan secreted from Aspergillus fumigatus and Aspergillus flavus induces platelet activation

Platelets are meanwhile recognized as versatile elements within the immune system and appear to play a key role in the innate immune response to pathogens including fungi. Previous experiments revealed platelet activation by direct contact with the hyphal-associated polysaccharide galactosaminogalactan (GAG). Since secreted fungal products may also be relevant and trigger immune reactions or thrombosis, we screened culture supernatants (SN) of human-pathogenic fungi for their capacity to activate platelets. For that purpose, platelets were incubated with SN from various fungal species; platelet activation and GAG deposition on the surface of platelets were detected by flow cytometry and electron and confocal microscopy, Culture supernatants of Aspergillus fumigatus and flavus isolates were potent platelet stimulators in a dose- and time-dependent manner, while SN of other Aspergillus species and all tested mucormycete species did not significantly induce platelet activation. The capacity of culture SN to activate platelets was dependent on fungal production of GAG and deposition of secreted GAG on the platelet surface; supernatants from mucormycetes or mutants of A. fumigatus lacking GAG secretion did not affect platelet activity. These results suggest that invading fungi can stimulate platelets not only locally through direct interactions with fungal hyphae, but can also act over a certain distance through secreted GAG.     

Transformation of Aspergillus niger using the argB gene of Aspergillus nidulans

A mutant of Aspergillus niger defective in ornithine transcarbamylase function was transformed with plasmids carrying a functional copy of the argB gene of Aspergillus nidulans after treatment of spheroplasts in the presence of polyethylene glycol and calcium ions. The plasmid pDG3 gave stable transformants at a frequency of 4 per microgram of input DNA. Southern blot analysis of DNA from transformants showed that pDG3 DNA had integrated into the A. niger chromosomes at a variety of locations. The transformants were phenotypically stable for many mitotic divisions. This procedure may potentially be used to insert any gene into the genome of A. niger. A cosmid shuttle vector, pDG1, for cloning in Aspergillus was also constructed.     

Transformation of Aspergillus parasiticus using autonomously replicating plasmids from Aspergillus nidulans

Abstract A genetic transformation system for the aflatoxin-producing fungus Aspergillus parasiticus using two autonomously replicating plasmids from A. nidulans (ARp1 and pDHG25) is reported. Transformation frequencies using the plasmid pDHG25 were from 5 × 10² to 2.5 × 10⁴ transformants per 10⁶ viable protoplasts and μg DNA. The stability of the plasmids in the transformants was also studied. This transformation system offers a new opportunity to clone genes related to aflatoxin production using appropriate aflatoxin-defective mutants.