Production of sterigmatocystin by Aspergillus versicolor isolated from roughage

A total of 69 samples of hay and straw collected during the winter period of 1984/85 were surveyed for their contamination by Aspergillus versicolor. The percentage of A. versicolor-positive samples was 14.5%. Nineteen A. versicolor strains mainly isolated from roughage were tested for the production of sterigmatocystin. All of the isolates examined were capable of producing different levels of sterigmatocystin on a cracked corn substrate. The majority of these strains were highly toxigenic; 53% of the isolates produced more than 500 mg/kg of sterigmatocystin. These findings suggest that corn is a very suitable substrate for sterigmatocystin production and that particularly in the surface layers of feed stocks and corn silos such toxigenic strains of A. versicolor can produce considerable growth and possibly sterigmatocystin, too.     

Anthraquinones in the biosynthesis of sterigmatocystin by Aspergillus versicolor

14C-labeled averufin, versiconal hemiacetal acetate, and versicolorin A were efficiently converted to sterigmatocystin by Aspergillus versicolor, thus providing experimental evidence that these anthraquinones are biosynthetic precursors of sterigmatocystin, a xanthone.     

Three new sterigmatocystin analogues from marine-derived fungus Aspergillus versicolor MF359

During the systematic screening of active compounds from marine-derived fungi, the extract of a strain of Aspergillus versicolor MF359 isolated from a marine sponge of Hymeniacidon perleve was identified for detailed chemical investigation. Three new secondary metabolites, named hemiacetal sterigmatocystin (1), acyl-hemiacetal sterigmatocystin (2), and 5-methoxydihydrosterigmatocystin (3), together with a known compound, aversin (4), were characterized. 1 represents a first structure of sterigmatocystin hemiacetal from nature. The antibacterial activities of these identified compounds were evaluated against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa. Compound 3 showed activity against S. aureus and B. subtilis with MIC values of 12.5 and 3.125 μg/mL, respectively.     

Anthraquinones in the biosynthesis of sterigmatocystin by Aspergillus versicolor.

14C-labeled averufin, versiconal hemiacetal acetate, and versicolorin A were efficiently converted to sterigmatocystin by Aspergillus versicolor, thus providing experimental evidence that these anthraquinones are biosynthetic precursors of sterigmatocystin, a xanthone.     

Production of sterigmatocystin by Aspergillus versicolor and Bipolaris sorokiniana on semisynthetic liquid and solid media.

Higher yields of sterigmatocystin were obtained with Aspergillus versicolor than with Bipolaris sorokiniana both in liquid and on solid media. The optimum temperature for sterigmatocystin production by A. versicolor was 27 to 29 degrees C and 23 degrees C for B. sorokiniana. In liquid shake cultures, production of sterigmatocystin by B. sorokiniana was negligible, whereas maximal production by A. versicolor was 210 mg/liter. On solid substrates, the highest yields (8 g/kg) were obtained with A. versicolor on still cultures of whole corn supplemented with Soytone.     

Molecular characterization of toxigenic and atoxigenic Aspergillus flavus isolates,collected from peanut fields in China

Aims: The objectives of this study were to assess the genetic relationships between toxigenic and atoxigenic isolates of Aspergillus flavus collected from peanut fields in China, and to analyse deletions within the aflatoxin biosynthetic gene cluster for the atoxigenic isolates. Methods and Results: Analysis of random‐amplified polymorphic DNA and microsatellite‐primed PCR data showed that the toxigenic and atoxigenic isolates of A. flavus were not clustered based on their regions and their ability of aflatoxin and sclerotial production. These results were further supported by DNA sequence of ITS, pksA and omtA genes. PCR assays showed that 24 of 35 isolates containing no detectable aflatoxins had the entire aflatoxin gene cluster. Eleven atoxigenic isolates had five different deletion patterns in the cluster. Conclusions: Toxigenic and atoxigenic isolates of A. flavus are genetically similar, but some atoxigenic isolates having deletions within the aflatoxin gene cluster can be identified readily by PCR assays. Significance and Impact of the Study: Because the extensive deletions within the aflatoxin gene cluster are not rare in the atoxigenic isolates, analysis of deletion within the cluster would be an effective method for the rapid screening of atoxigenic isolates for developing biocontrol agents.     

Occurrence of Toxigenic Aspergillus versicolor Isolates and Sterigmatocystin in Carpet Dust from Damp Indoor Environments

Over the past decade, there has been growing concern regarding the role of toxigenic fungi in damp indoor environments; however, there is still a lack of field investigations on exposure to mycotoxins. The goal of our pilot study was to quantify the proportion of toxigenic Aspergillus versicolor isolates in native carpet dust from damp dwellings with mold problems and to determine whether sterigmatocystin can be detected in this matrix. Carpet dust samples (n = 11) contained from <2.5 × 10¹ to 3.6 × 10⁵ (median, 3.1 × 10⁴) A. versicolor CFU/g of dust, and the median proportion of A. versicolor from total culturable fungi was 18%. Based on thin-layer chromatography detection of sterigmatocystin, 49 of 50 A. versicolor isolates (98%) were found to be toxigenic in vitro. By using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry, sterigmatocystin could be detected in low concentrations (2 to 4 ng/g of dust) in 2 of 11 native carpet dust samples. From this preliminary study, we conclude that most strains of A. versicolor isolated from carpet dust are able to produce sterigmatocystin in vitro and that sterigmatocystin may occasionally occur in carpet dust from damp indoor environments. Further research and systematic field investigation are needed to confirm our results and to provide an understanding of the health implications of mycotoxins in indoor environments.     

New application of Aspergillus versicolor in promoting plant growth after suppressing sterigmatocystin production via genome mining and engineering

Aspergillus genus is a key component in fermentation and food processing. However, sterigmatocystin (STE)—a mycotoxin produced by several species of Aspergillus—limits the use of some Aspergillus species (such as Aspergillus versicolor, Aspergillus inflatus, and Aspergillus parasiticus) because of its toxicity and carcinogenicity. Here, we engineered an STE-free Aspergillus versicolor strain based on genome mining techniques. We sequenced and assembled the Aspergillus versicolor D5 genome (34.52 Mb), in which we identified 16 scaffolds and 54 biosynthetic gene clusters (BGCs). We silenced cytochrome P450 coding genes STC17 and STC27 by insertional inactivation. The production of STE in the Δstc17 mutant strain was increased by 282% but no STE was detected in the Δstc27 mutant. Metabolites of Δstc27 mutant exhibited growth-promoting effect on plants. Our study makes significant progress in improving the application of some Aspergillus strains by restricting their production of toxic and carcinogenic compounds.     

Relationship between mycobiota in wall-to-wall carpet dust and age of carpet

The objective of this study was to evaluate thefungal concentrations within carpets of the same typeand manufacture during their first 2 years of life.The carpets tested came from several different branchoffices of a bank.A total of 60 dust samples from 20 carpets (3samples per carpet) was analysed. The age of thecarpets varied from 50 to 666 days. Analysis of therelationship between fungal concentration and time wasmade by the Pearson test.Fungal concentrations were very different withina same carpet. Moreover, species distribution was alsoheterogeneous; thus the fungi in 1 m² carpet didnot reflect mycobiota of the whole carpet. Very localfungal contaminations (>10 CFU/mg/dust) were builtup in a relatively short time (138 days). Thehighest concentrations (maximum: 200 CFU/mg dust) weremainly due to species of coelomycetes (mitosporicfungi forming conidia in closed bodies) and to variousyeasts. Comparisons of the 20 carpets sampled did notshow significant differences; the fungal level did notincrease with time (P > 0.05). A decrease waseven noticed. The age of carpets had no significanteffect either on the number of CFU/mg dust of the mainfungal taxa (P > 0.10).It was concluded that these carpets, which wereregularly cleaned, in this particular environment, donot favour important fungal growth. Moreover, sincethe species responsible for the highest contaminationobserved produce conidia or cells not easilytransported through the air, their role in respiratoryallergy would be relatively limited.     

Production of sterigmatocystin by Aspergillus versicolor QM 432 used in fungus resistance tests for United States military specifications

A study was made to determine whether Aspergillus flavus (QM 380; NRRL 3537; ATCC 9643) and Aspergillus versicolor (QM 432; NRRL 573; ATCC 16,020), mold strains routinely combined in fungus resistance tests for United States military specification (MIL-STD-810B; MIL-STD-331A), were capable of producing aflatoxins or sterigmatocystin when grown separately or in mixed culture fermentations of cracked corn (11 days at 25 °C or 28 °C). Substantial quantities of sterigmatocystin (average of 3 replicates= 1,895 ppb) were detected when A. versicolor was the sole colonist. No sterigmatocystin was detected when A. versicolor was simultaneously inoculated with other molds. Aflatoxins were not detected in any of the treatment combinations.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Detection of toxigenic isolates of Aspergillus flavus and related species on coconut cream agar

A new readily-prepared medium, coconut cream agar, was developed for the detection of aflatoxin production by isolates of Aspergillus flavus and related species. Coconut cream agar, which comprised coconut cream (50%) and agar (1.5%), detected isolates of A. flavus more effectively than the synthetic media tested and was as effective as media containing desiccated coconut. Fluorescence colouring of colonies grown on coconut cream agar could be used to differentiate A. flavus from A. parasiticus and A. nomius. In addition, conidial colour of A. flavus and A. nomius was quite distinct from that of A. parasiticus.     

Transformation of sterigmatocystin and O-methylsterigmatocystin by aflatoxigenic and nonaflatoxigenic field isolates of the Aspergillus flavus group

Transformation of sterigmatocystin and O-methylsterigmatocystin (two metabolic aflatoxin precursors) to aflatoxins by aflatoxigenic and nonaflatoxigenic field isolates of Aspergillus flavus was studied. The 24 nonaflatoxigenic isolates investigated failed to transform both precursors. Among the 8 aflatoxin-producing isolates used, 7 transformed both precursors whereas the remaining failed to transform both. According to these results, the usefulness of the measurement of enzymatic activities related to aflatoxin production in understanding the true status of conflictive field isolates is discussed.Abbreviations ST sterigmatocystin - OMST O-methylsterigmatocystin - AFB₁ aflatoxin B₁ - AFB₂ aflatoxin B₂ - AFG₁ aflatoxin G₁ - AFG₂ aflatoxin G₂ - GM growth medium of Adye and Mateles - RM replacement medium of Adye and Mateles     

Structure of versicolorone isolated from Aspergillus versicolor

A new anthraquinone metabolite, versicolorone, has been isolated from Aspergillus versicolor.     

Dihydrosterigmatocystin and Dihydrodemethylsterigmatocystin,New Metabolites from Aspergillus versicolor

L-Arabinose isomerase (L-arabinose ketol-isomerase, EC 5.3.1.4) was demonstrated from the L-arabinose-grown cells of Streptomyces sp. which was isolated from sea water. The enzyme was purified by MnCl₂ treatment, fractionation by polyethylene glycol and by column chromatographies on Sephadex G-150 and DEAE-cellulose. The purified enzyme was specific only for L-arabinose and the Michaelis constant for L-arabinose was 40 mM at pH 7.5. Manganese or cobalt ions were effective for the enzyme activity after dialysis against EDTA. The enzyme activity was inhibited competitively by L-arabitoI, ribitol and xylitol, of which inhibition constants were 1.1, 1.0, and 15 mM, respectively.     

Purification and biochemical characterization of an endoxylanase from Aspergillus versicolor

An endoxylanase (β-1,4-xylan xylanohydrolase, EC 3.2.1.8) was purified from the culture filtrate of a strain of Aspergillus versicolor grown on oat wheat. The enzyme was purified to homogeneity by chromatography on DEAE-cellulose and Sephadex G-75. The purified enzyme was a monomer of molecular mass estimated to be 19 kDa by SDS-PAGE and gel filtration. The enzyme was glycoprotein with 71% carbohydrate content and exhibited a pI of 5.4. The purified xylanase was specific for xylan hydrolysis. The enzyme had a K _m of 6.5 mg ml⁻¹ and a V _max of 1440 U (mg protein)⁻¹.     

The Structure of a New Metabolite from Aspergillus versicolor

α-Glucosidase was purified and crystallized from the mycelia of Mucor javanicus, by procedures including extraction with urea, fractionations with acetone and polyethylene glycol 6000, successive separation on columns of Sephadex G-200 and DEAE-cellulose, and crystallization by the addition of ampholine reagent. The crystalline enzyme was homogeneous in ultracentrifugal analysis and gel electrophoresis. The purified enzyme showed a sedimentation constant of 5.6 S and isoelectric point of pH 8.6. Some properties of the purified enzyme were also investigated. It was recognized that the synthesis of riboflavin α-glucoside was catalyzed by the transglucosylation activity of this α-glucosidase.     

Genotypic variation in Penicillium chysogenum from indoor environments

We examined 198 isolates of P. chysogenum recovered from 109 houses in Wallaceburg, Ontario, and 25 culture collection isolates including seven ex-type strains. Multilocus genotypes were determined by heteroduplex mobility assay of regions spanning introns in acetyl co-enzyme A synthase, beta-tubulin, thioredoxin reductase and the internal transcribed spacer regions of the nuclear ribosomal subrepeat. Five unique multilocus haplotypes were revealed without evidence of recombination, indicating strictly clonal population structures. Phylogenetic analysis of allele sequences using maximum parsimony resolved three strongly supported lineages. The dominant clade included more than 90% of house isolates in addition to the notable laboratory contaminant isolated by Alexander Fleming in 1929 in Britain. A second clade contained more than 5% of house isolates clustered with the ex-type strains of P. chysogenum and P. notatum. Follow-up sampling of outdoor air in the locality failed to reveal P. chysogenum, confirming the rarity of this fungus in outdoor air.     

Absence of trichothecenes in toxigenic isolates of Fusarium moniliforme

Thirty-four isolates of Fusarium moniliforme were obtained from cereal grains collected in various parts of the world. The isolates were grown on rice and tested as a diet for toxicity to rats. Of these isolates, 53% caused death, 12% caused congestion and hemorrhage of the stomach and intestine as well as hematuria, 21% caused diarrhea, 38% caused weight loss, and 9% were nontoxic. The cultures were tested to T-2, HT-2, neosolaniol, acetyl-T-2, T-2-tetraol, iso-T-2, diacetoxyscirpenol, monoacetoxyscirpenol, deoxynivalenol, nivalenol, fusarenone-X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, zearalenone, moniliformin, fusarochromanone, fusarin-C, and wortmannin; all were negative. In addition, F. moniliforme NRRL A25820 was grown on corn and banana fruit as solid substrates as well as on a defined liquid medium; none of the above toxins were found. When F. moniliforme NRRL A25820 was incorporated into a rat diet, no toxicity was noted. Twenty-eight additional isolates of F. moniliforme, isolated from feed associated with equine leukoencephalomalacia, were grown on cracked corn for 2 weeks. The cultures were negative when tested for deoxynivalenol, 15-acetyldeoxynivalenol, diacetoxyscirpenol, monoacetoxyscirpenol, nivalenol, and fusarenone X. Seventy-five percent of the isolates were toxic to ducklings, indicating the presence of a toxin other than trichothecenes. Our results support the conclusion that F. moniliforme does not produce trichothecenes.     

Identification and Biological Evaluation of Secondary Metabolites from the Endolichenic Fungus Aspergillus versicolor

A chemical investigation of the endolichenic fungus Aspergillus versicolor (125a), which was found in the lichen Lobaria quercizans, resulted in the isolation of four novel diphenyl ethers, named diorcinols F–H ( 1 – 3 , resp.) and 3‐methoxyviolaceol‐II ( 4 ), eight new bisabolane sesquiterpenoids, named (?)‐(R)‐cyclo‐hydroxysydonic acid ( 5 ), (?)‐(7S,8R)‐8‐hydroxysydowic acid ( 6 ), (?)‐(7R,10S)‐10‐hydroxysydowic acid ( 7 ), (?)‐(7R,10R)‐iso‐10‐hydroxysydowic acid ( 8 ), (?)‐12‐acetoxy‐1‐deoxysydonic acid ( 9 ), (?)‐12‐acetoxysydonic acid ( 10 ), (?)‐12‐hydroxysydonic acid ( 11 ), and (?)‐(R)‐11‐dehydrosydonic acid ( 12 ), two new tris(pyrogallol ethers), named sydowiols D ( 13 ) and E ( 14 ), and fifteen known compounds, 15 – 29 . All of the structures were determined by spectroscopic analyses, and a number of them were further identified through chemical transformations and electronic circular dichroism (ECD) calculations. Preliminary bioassays of these isolates for the determination of their inhibitory activities against the fungus Candida albicans, and their cytotoxicities against the human cancer cell lines PC3, A549, A2780, MDA‐MB‐231, and HEPG2 were also evaluated.