Isolation and characterization of Aspergillus parasiticus mutants with impaired aflatoxin production by a novel tip culture method.

A convenient procedure consisting of UV photography (K. Yabe, Y. Ando, M. Ito, and N. Terakado, Appl. Environ. Microbiol. 53:230-234, 1987) and a tip culture method has been devised for the isolation and characterization of Aspergillus parasiticus mutants relating to aflatoxin production. With the latter procedure, the production of aflatoxins excreted into the culture medium and precursors in the mycelium were easily measured quantitatively or semiquantitatively. A total of 38 mutants in which the aflatoxigenicity was decreased or lost were obtained by UV radiation; 3 were found to be blocked mutants, which accumulated the aflatoxin precursors versicolorin A or averantin.     

Biochemical analysis of oxidative stress in the production of aflatoxin and its precursor intermediates

The relevance of oxidative stress in the production of aflatoxin and its precursors was examined in different mutants of Aspergillus parasiticus, which produce aflatoxin or its precursor intermediates, and compared with results obtained from a non-toxigenic strain. In comparison to the non-toxigenic strain (SRRC 255), an aflatoxin producing strain (NRRL 2999) or mutants that accumulate aflatoxin precursors such as norsolorinic acid (by SRRC 162) or versicolorin (by NRRL 6196) or O-methyl sterigmatocystin (by SRRC 2043) had greater oxygen requirements and higher contents of reactive oxygen species. These changes were in the graded order of NRRL 2999 > SRRC 2043 > NRRL 6196 > SRRC 162 > SRRC 255, indicating incremental accumulation of reactive oxygen species, being least in the non-toxigenic strain and increasing progressively during the ternary steps of aflatoxin formation. Oxidative stress in these strains was evident by increased activities of xanthine oxidase and free radical scavenging enzymes (superoxide dismutase and glutathione peroxidase) as compared to the non-toxigenic strain (SRRC 255). Culturing the toxigenic strain in presence of 0.1–10 μM H₂O₂ in the medium resulted in enhanced aflatoxin production, which could be related to dose-dependent increase in [¹⁴C]-acetate incorporation into aflatoxin B₁ and increased acetyl CoA carboxylase activity. The combined results suggest that formation of secondary metabolites such as aflatoxin and its precursors by A. parasiticus may occur as a compensatory response to reactive oxygen species accumulation.     

Development of a simple and high-throughput method for detecting aflatoxins production in culture media

Aims: To develop a simple, high‐throughput and inexpensive procedure to detect and quantify aflatoxins into the culture media of growing mycelia. Methods and Results: Fungal conidia (Aspergillus flavus) were inoculated into the wells of a microplate containing 200 μl of different formulations of coconut‐derived liquid medium. Time‐dependent production of aflatoxins in the culture media was evaluated by a procedure relying on the UV‐induced fluorescence emission by the toxin, using a microplate reader. These data were validated by comparison with the outputs of a conventional HPLC‐based procedure. Determinations of aflatoxin concentration, according to the fluorimetric procedure, were performed either by withdrawing samples from the plates or by direct ‘in situ’ readings, the latter method reinforcing the high‐throughput feature of the procedure. Fluorescence enhancers (cyclodextrins) did not ameliorate the sensitivity of the procedure to low concentrations of the toxin into the medium. The efficacy of the procedure was also validated by testing the effect on toxin yield of adding an antioxidant agent (α‐lipoic acid) to the medium. Conclusions: We give evidence that our improved procedure is reliable and suitable to analyse aflatoxin accumulation time course in coconut‐derived culture medium. Significance and Impact of the Study: This study shows that our procedure may profitably be used to give insights into the mechanisms of regulation of mycotoxin production and, consequently, to implement different strategies for the containment of aflatoxin contamination of food and feed commodities.     

Direct visual detection of aflatoxin synthesis by minicolonies of Aspergillus species

Single-spore colonies of Aspergillus flavus and Aspergillus parasiticus, grown for 4 to 5 days at 25 degrees C on a coconut extract agar containing sodium desoxycholate as a growth inhibitor, produced aflatoxin, readily detectable as blue fluorescent zones under long-wave (365 nm) UV light. Over 100 colonies per standard petri dish were scored for aflatoxin production by this procedure. Progeny from some strains remained consistently stable for toxin production after repeated subculture, whereas instability for toxin synthesis was revealed among progeny from other strains. Spore color markers were used to rule out cross-contamination in monitoring strains. A yellow-spored and nontoxigenic strain of A. flavus, reported previously to produce aflatoxin in response to cycloheximide treatment, proved to be toxin negative even after repeated exposure to cycloheximide. Extended series of progeny from another strain of A. flavus and from a strain of A. parasiticus were each compared by this plating procedure and by fluorometric analysis for aflatoxin when grown in a coconut extract broth. Both of these strains showed variation for toxin synthesis among their respective progeny, and specific progeny showed a good correlation for aflatoxin synthesis when examined by the two procedures.     

Direct visual detection of aflatoxin synthesis by minicolonies of Aspergillus species.

Single-spore colonies of Aspergillus flavus and Aspergillus parasiticus, grown for 4 to 5 days at 25 degrees C on a coconut extract agar containing sodium desoxycholate as a growth inhibitor, produced aflatoxin, readily detectable as blue fluorescent zones under long-wave (365 nm) UV light. Over 100 colonies per standard petri dish were scored for aflatoxin production by this procedure. Progeny from some strains remained consistently stable for toxin production after repeated subculture, whereas instability for toxin synthesis was revealed among progeny from other strains. Spore color markers were used to rule out cross-contamination in monitoring strains. A yellow-spored and nontoxigenic strain of A. flavus, reported previously to produce aflatoxin in response to cycloheximide treatment, proved to be toxin negative even after repeated exposure to cycloheximide. Extended series of progeny from another strain of A. flavus and from a strain of A. parasiticus were each compared by this plating procedure and by fluorometric analysis for aflatoxin when grown in a coconut extract broth. Both of these strains showed variation for toxin synthesis among their respective progeny, and specific progeny showed a good correlation for aflatoxin synthesis when examined by the two procedures.     

Effect of branch frequency in Aspergillus oryzae on protein secretion and culture viscosity.

Highly branched mutants of two strains of Aspergillus oryzae (IFO4177, which produces alpha-amylase, and a transformant of IFO4177 [AMG#13], which produces heterologous glucoamylase in addition to alpha-amylase) were generated by UV or nitrous acid mutagenesis. Four mutants of the parental strain (IFO4177), which were 10 to 50% more branched than the parental strain, were studied in stirred batch culture and no differences were observed in either the amount or the rate of enzyme production. Five mutants of the transformed parental strain (AMG#13), which were 20 to 58% more branched than the parental strain, were studied in either batch, fed-batch or continuous culture. In batch culture, three of the mutants produced more glucoamylase than the transformed parental strain, although only two mutants produced more glucoamylase and alpha-amylase combined. No increase in enzyme production was observed in either chemostat or fed-batch culture. Cultures of highly branched mutants were less viscous than those of the parental and transformed parental strains. A linear relationship was found between the degree of branching (measured as hyphal growth unit length) and culture viscosity (measured as the torque exerted on the rheometer impeller) for these strains. DOT-controlled fed-batch cultures (in which the medium feed rate was determined by the DOT) were thus inoculated with either the transformed parent or highly branched mutants of the transformed parent to determine whether the reduced viscosity would improve aeration and give higher enzyme yields. The average rate of medium addition was higher for the two highly branched mutants (ca. 8.3 g medium h(-1)) than for the parental strain (5.7 g medium h(-1)). Specific enzyme production in the DOT controlled fed-batch cultures was similar for all three strains (approx. 0.24 g alpha-amylase and glucoamylase [g of biomass](-1)), but one of the highly branched mutants made more total enzyme (24.3 +/- 0.2 g alpha-amylase and glucoamylase) than the parental strain (21.7 +/- 0.4 g alpha-amylase and glucoamylase).     

Sensitivity and single-strand DNA break repair in Chinese hamster mutants exposed to the carcinogen aflatoxin B1 epoxide and its dichloride model.

Aflatoxin B1 (AFB1) is a potent carcinogen and mutagen. It requires metabolic activation to be converted to the DNA-binding product aflatoxin B1 epoxide (AFB1-epoxide). A model of this epoxide is aflatoxin B1 dichloride (AFB1Cl2). Both react at the N7 position of guanine to form large adducts. The major adduct formed can either be rapidly removed to leave an apurinic site or can undergo ring opening of the imidazole ring to form a chemically stable adduct. A number of Chinese hamster DNA repair-deficient mutants have been screened for their sensitivity to AFB1-epoxide and AFB1Cl2. Some of the mutants screened belong to different UV complementation groups. Human genes involved in nucleotide excision-repair correct deficiencies found in these complementation groups. The mutants which were found to be most sensitive to AFB1 (V-C4 and V-H1) were further investigated. Alkaline elution was used to measure AFB1-induced DNA single-strand break repair in the mutants. V-H1 repaired completely in 24 h whereas V-C4 displayed only partial repair.     

Association of aflatoxin biosynthesis and sclerotialdevelopment in Aspergillus parasiticus

Secondary metabolism in fungi is frequently associated with asexual and sexual development. Aspergillus parasiticus produces aflatoxins known to contaminate a variety of agricultural commodities. This strictly mitotic fungus, besides producing conidia asexually, produces sclerotia, structures resistant to harsh conditions and for propagation. Sclerotia are considered to be derived from the sexual structure, cleistothecia, and may represent a vestige of ascospore production. Introduction of the aflatoxin pathway-specific regulatory gene, aflR, and aflJ, which encoded a putative co-activator, into an O-methylsterigmatocystin (OMST)-accumulating strain,A. parasiticus SRRC 2043, resulted in elevated levels of accumulation of major aflatoxin precursors, including norsolorinic acid (NOR), averantin (AVN), versicolorin A (VERA) and OMST. The total amount of these aflatoxin precursors, NOR, VERA, AVN and OMST, produced by the aflR plus aflJ transformants was two to three-fold that produced by the aflR transformants. This increase indicated a synergisticeffect of aflR and aflJ on the synthesis of aflatoxin precursors. Increased production of the aflatoxin precursors was associated with progressive decrease in sclerotial size, alteration in sclerotial shape and weakening in the sclerotial structure of the transformants. The results showed that sclerotial development and aflatoxin biosynthesis are closely related. We proposed that competition for a common substrate, such as acetate, by the aflatoxin biosynthetic pathway could adversely affect sclerotial development in A. parasiticus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Association between vegetative compatibility and aflatoxin production by <Emphasis Type="Italic">Aspergillus</Emphasis> species during intraspecific competition

Intraspecific competition is the basis for biological control of aflatoxins, but there is little understanding of the mechanism(s) by which competing strains inhibit toxin production. Evidence is presented that demonstrates a relationship between strength of the vegetative compatibility reaction and aflatoxin production in Aspergillus flavus and A. parasiticus using the suspended disk culture method. Combining wild-type aflatoxin-producing isolates belonging to different vegetative compatibility groups (VCGs) resulted in a substantial reduction in aflatoxin yield. Pairs of aflatoxin-producing isolates within the same VCG, but showing weak compatibility reactions using complementary nitrate-nonutilizing mutants, also were associated with reduced levels of aflatoxin B₁. In contrast, pairings of isolates displaying a strong compatibility reaction typically produced high levels of aflatoxins. These results suggest that interactions between vegetatively compatible wild-type isolates of A. flavus and A. parasiticus are cooperative and result in more aflatoxin B₁ than pairings between isolates that are incompatible. Successful hyphal fusions among spore germlings produce a common mycelial network with a larger resource base to support aflatoxin biosynthesis. By comparison, vegetative incompatibility reactions might result in the death of those heterokaryotic cells composed of incompatible nuclei and thereby disrupt the formation of mycelial networks at the expense of aflatoxin biosynthesis. The content of this paper was presented at the 50th Anniversary Meeting of the Mycological Society of Japan, June 3–4, 2006, Chiba, Japan     

Variability among atoxigenic Aspergillus flavus strains in ability to prevent aflatoxin contamination and production of aflatoxin biosynthetic pathway enzymes.

Five strains of Aspergillus flavus lacking the ability to produce aflatoxins were examined in greenhouse tests for the ability to prevent a toxigenic strain from contaminating developing cottonseed with aflatoxins. All atoxigenic strains reduced contamination when inoculated into developing bolls 24 h prior to the toxigenic strain. However, only one strain, AF36, was highly effective when inoculated simultaneously with the toxigenic strain. All five strains were able to inhibit aflatoxin production by the toxigenic strain in liquid fermentation. Thus, in vitro activity did not predict the ability of an atoxigenic strain to prevent contamination of developing bolls. Therefore, strain selection for competitive exclusion to prevent aflatoxin contamination should include evaluation of efficacy in developing crops prior to field release. Atoxigenic strains were also characterized by the ability to convert several aflatoxin precursors into aflatoxin B1. Four atoxigenic strains failed to convert any of the aflatoxin biosynthetic precursors to aflatoxins. However, the strain (AF36) most effective in preventing aflatoxin contamination in developing bolls converted all tested precursors into aflatoxin B1, indicating that this strain made enzymes in the aflatoxin biosynthetic pathway.     

Molasses supplementation promotes conidiation but suppresses aflatoxin production by small sclerotial Aspergillus flavus

AIMS: To find a supplemental ingredient that can be added to routinely used growth media to increase conidial production and decrease aflatoxin biosynthesis in small sclerotial (S strain) isolates of Aspergillus flavus. METHODS AND RESULTS: Molasses was added to three commonly used culture media: coconut agar (CAM), potato dextrose agar (PDA), and vegetable juice agar (V8) and production of conidia, sclerotia, and aflatoxins by A. flavus isolate CA43 was determined. The effect of nitrogen sources in molasses medium (MM) on production of conidia, sclerotia and aflatoxins was examined. Water activity and medium pH were also measured. Conidia harvested from agar plates were counted using a haemocytometer. Sclerotia were weighed after drying at 45 degrees C for 5 days. Aflatoxins B(1) and B(2) were quantified by high-performance liquid chromatography. Addition of molasses to the media did not change water activity or the pH significantly. Supplementing CAM and PDA with molasses increased conidial production and decreased aflatoxins. Two-fold increased yield of conidia was found on MM, which, like V8, did not support aflatoxin production. Adding ammonium to MM significantly increased the production of sclerotia and aflatoxins, but slightly decreased conidial production. Adding urea to MM significantly increased the production of conidia, sclerotia and aflatoxins. CONCLUSIONS: Molasses stimulated conidial production and inhibited aflatoxin production. Its effect on sclerotial production was medium-dependent. Water activity and medium pH were not related to changes in conidial, sclerotial or aflatoxin production. Medium containing molasses alone or molasses plus V8 juice were ideal for conidial production by S strain A. flavus. SIGNIFICANCE AND IMPACT OF THE STUDY: Insight into molecular events associated with the utilization of molasses may help to elucidate the mechanism(s) that decreases aflatoxin biosynthesis. Targeting genetic parameters in S strain A. flavus isolates may reduce aflatoxin contamination of crops by reducing the survival and toxigenicity of these strains.     

A Polyketide Synthase Gene Required for Biosynthesis of the Aflatoxin-like Toxin, Dothistromin

Dothistromin is a polyketide toxin, produced by a fungal forest pathogen, with structural similarity to the aflatoxin precursor versicolorin B. Biochemical and genetic studies suggested that there are common steps in the biosynthetic pathways for these metabolites and showed similarities between some of the genes. A polyketide synthase gene (pksA) was isolated from dothistromin-producing Dothistroma septosporum by hybridization with an aflatoxin ortholog from Aspergillus parasiticus. Inactivation of this gene in D. septosporum resulted in mutants that could not produce dothistromin but that could convert exogenous aflatoxin precursors, including norsolorinic acid, into dothistromin. The mutants also had reduced asexual sporulation compared to the wild type. So far four other genes are known to be clustered immediately alongside pksA. Three of these (cypA, moxA, avfA) are predicted to be orthologs of aflatoxin biosynthetic genes. The other gene (epoA), located between avfA and moxA, is predicted to encode an epoxide hydrolase, for which there is no homolog in either the aflatoxin or sterigmatocystin gene clusters. The pksA gene is located on a small chromosome of ~1.3 Mb in size, along with the dothistromin ketoreductase (dotA) gene.     

Sodium bicarbonate reduces viability and alters aflatoxin distribution of Aspergillus parasiticus in Czapek's agar

The potential of sodium bicarbonate to inhibit growth of and aflatoxin synthesis by Aspergillus parasiticus was examined in Czapek's agar (CA), a medium in which fluorescence under UV light indicates aflatoxin production. Incorporation of sodium bicarbonate (SB) into CA at 0.011, 0.022, and 0.033 mol% reduced cell viability 63-, 10(3)-, and greater than 10(7)-fold, respectively. Colonies resulting from surviving cells did not fluoresce under UV light, but thin-layer chromatography analysis of culture extracts detected aflatoxins. Potassium bicarbonate (KB) at 0.011 and 0.022 mol% produced inhibitory effects similar to those of SB, but NaCl and silica had no effect. After 7 days, control cultures had the normal aflatoxin distribution (B1 greater than G1 greater than B2 greater than G2), but this distribution shifted to B2 greater than B1 approximately equal to G2 greater than G1 during prolonged incubation. Cultures supplemented with SB and KB contained mostly aflatoxins B1 and G1 after 28 days. Both SB and KB raised the pH of CA to 7.5 to 8.5 at the time of growth. Culture growth on CA adjusted to pH 7.5 to 8.5 with NaOH was not inhibited but exhibited reduced fluorescence and elevated levels of aflatoxins B1 and G1. Thus, while bicarbonate inhibition of growth could not be attributed to pH elevation, the lack of culture fluorescence on CA-SB and CA-KB and the altered aflatoxin distribution were caused by the ability of SB and KB to elevate pH.     

Sodium bicarbonate reduces viability and alters aflatoxin distribution of Aspergillus parasiticus in Czapek's agar.

The potential of sodium bicarbonate to inhibit growth of and aflatoxin synthesis by Aspergillus parasiticus was examined in Czapek's agar (CA), a medium in which fluorescence under UV light indicates aflatoxin production. Incorporation of sodium bicarbonate (SB) into CA at 0.011, 0.022, and 0.033 mol% reduced cell viability 63-, 10(3)-, and greater than 10(7)-fold, respectively. Colonies resulting from surviving cells did not fluoresce under UV light, but thin-layer chromatography analysis of culture extracts detected aflatoxins. Potassium bicarbonate (KB) at 0.011 and 0.022 mol% produced inhibitory effects similar to those of SB, but NaCl and silica had no effect. After 7 days, control cultures had the normal aflatoxin distribution (B1 greater than G1 greater than B2 greater than G2), but this distribution shifted to B2 greater than B1 approximately equal to G2 greater than G1 during prolonged incubation. Cultures supplemented with SB and KB contained mostly aflatoxins B1 and G1 after 28 days. Both SB and KB raised the pH of CA to 7.5 to 8.5 at the time of growth. Culture growth on CA adjusted to pH 7.5 to 8.5 with NaOH was not inhibited but exhibited reduced fluorescence and elevated levels of aflatoxins B1 and G1. Thus, while bicarbonate inhibition of growth could not be attributed to pH elevation, the lack of culture fluorescence on CA-SB and CA-KB and the altered aflatoxin distribution were caused by the ability of SB and KB to elevate pH.     

Screening and characterization of astaxanthin-hyperproducing mutants of Haematococcus pluvialis

Haematococcus pluvialis was mutated by UV or ethyl methanesulphonate. Mutants resistant to nicotine, diphenylamine, fluridone or norflurazon were then selected. Several nicotine-resistant mutants showed increased (1.9% to 2.5% vs. 1.2% w/w) astaxanthin production. Mutants maintained high astaxanthin production over 4 months of repeated culture.     

N-carboxymethylchitosan inhibition of aflatoxin production: Role of zinc

Aqueous Solutions of N-carboxymethylchitosan (NCMC) suppressed both growth and aflatoxin production byAspergillus flavus andA. parasiticus in submerged culture (Adye and Mateles A&M). Test media were amended with various concentrations of zinc (15, 30, 45, 60 uM), and NCMC solution (0.62 uM). After 8 days incubation NCMC-treated cultures showed marked reduction of aflatoxin production and fungal growth. Enhanced levels of zinc did not overcome the NCMC-mediated inhibition of fungal growth or aflatoxin production.     

高产洛蒙真菌素洛蒙德链霉菌高通量筛选方法的建立与复合育种

[背景]洛蒙德链霉菌S015能生物合成具有广谱抗菌活性的吩嗪类化合物洛蒙真菌素。[目的]因S015菌株的洛蒙真菌素产量较低,将S015菌株经复合诱变育种和基因工程改造,提高洛蒙真菌素产量。[方法]建立洛蒙真菌素产生菌的高通量筛选方法,对出发菌株S0 15进行常压室温等离子体(atmospheric and room temperature plasma,ARTP)技术和紫外复合诱变,筛选得到高产菌株;并在高产菌株上敲除洛蒙真菌素的前体分支酸竟争途径中的关键基因trpE1、trpE2,再过表达全局调控基因afsR。[结果]利用洛蒙真菌素在紫外波长375 nm处的特征吸收峰,以及洛蒙真菌素浓度和375 nm处吸光度值的正相关关系,建立了基于24孔深孔板发酵和酶标仪快速检测的高通量筛选方法。经过6轮ARTP和紫外复合诱变及高通量筛选,从4 320株突变株中筛选得到遗传稳定的高产菌株M6,其洛蒙真菌素的产量为61.33 mg/L,是S015菌株的7.35倍;M6菌株的分支途径基因trpE1、trpE2双敲株的洛蒙真菌素产量为81.89 mg/L,是S015菌株的9.82倍;在该基因工程菌株中过表达全局调控基因afsR,产量为109.53 mg/L,是S015菌株的13.13倍。[结论]建立的高通量筛选方法可以有效筛选高产洛蒙真菌素的突变株,并且操作简单快速。通过ARTP和紫外复合诱变,结合高产株M6的基因工程改造,能进一步提升洛蒙真菌素的产量。     

Characteristics of Insertional Mutants of Pseudomonas syringae with Reduced Epiphytic Fitness

Random Tn5 mutagenesis was used to identify genes ir. Pseudomonas syringae which contribute to epiphytic fitness. Mutants were selected on the basis of deficiencies in epiphytic growth or survival on plants rather than deficiencies in predetermined phenotypes exhibited in culture. A sample freezing procedure was used to measure the population sizes of 5,300 mutants of P. syringae exposed to alternating wet and dry conditions on bean leaves in growth chambers. Eighty-two mutants exhibited reduced population sizes. Of these mutants, over half exhibited a reduced ability to survive the stresses associated with dry leaves, while others grew more slowly or attained reduced stationary-phase population sizes on leaves. While some epiphytic fitness mutants were altered in phenotypes that could be measured in culture, many mutants were not altered in any in vitro phenotype examined. Only three of the epiphytic fitness mutants were auxotrophs, and none had catabolic deficiencies for any of 31 organic compounds tested. Other mutants that exhibited reductions in one or more of the following were identified: motility, osmotolerance, desiccation tolerance, growth rate in batch culture, and extracellular polysaccharide production. All of the mutants retained the abilities to produce disease symptoms on the compatible host plant, bean, to incite a hypersensitive response on the non-host plant, tobacco, and to produce a fluorescent pyoverdine siderophore.     

Dry-column chromatographic purification of aflatoxin precursors

The toxic and carcinogenic properties of aflatoxins are well understood, but little is known about the biological activity of the anthraquinone precursors of aflatoxin. This paper describes a dry column chromatographic method for preparing averantin, averufin, norsolorinic acid and versicolorin A from mycelial extracts of blocked mutants of Aspergillus parasiticus in quantities suitable for toxicological testing.