Aflatoxin Formation Research Articles

Effect of intraspecific competition by Aspergillus flavus on aflatoxin formation in suspended disc culture

The ability of two non-aflatoxin producing strains of Aspergillus flavus to interfere with aflatoxin production by a toxigenic A. flavus strain was examined using a replacement series with suspended disc culture method. Individual glass fiber discs, affixed to a pin suspended from the caps of scintillation vials, were inoculated with medium containing A. flavus conidial mixtures in different proportions (aflatoxin producer:non-producer = 100:0, 80:20, 60:40, 20:80 and 0:100 by vol) at a constant total density (1 x 10(5) spores ml(-1)). Reductions in the total conidial density of these strains when grown alone, had little effect on fungal growth (mycelium dry weight) or aflatoxin production. Significant (P < 0.0001) reductions in aflatoxin B1 were recorded when non-toxigenic strains represented any proportion of the inoculum mixture. Aflatoxin yield values were less than (P < 0.0001) expected from the input ratios for toxigenic vs. non-toxigenic conidial inoculum within the replacement series. Aflatoxin yields were also reduced (P < 0.001), with a corresponding increase in fungal growth (P < 0.001), when conidia from aflatoxin producing strains were mixed in equal proportions. This suggests that the substantial inhibition of aflatoxin yield for inoculum mixtures results from the failure of spore germlings to establish a cooperative mycelial network.

Resistance of ‘Tulare’ Walnut (Juglans regia cv. Tulare) to Aflatoxigenesis

ABSTRACT: The ability of tree nuts to inhibit production of aflatoxins by Aspergillus flavus was investigated in vitro. Walnuts were much more resistant to aflatoxin formation than almonds or pistachios. The walnut cultivar ‘Tulare’ showed complete suppression of toxin production. Kernel without seed coat had no activity and the inhibitory factor(s) were shown to be specifically located in seed coat tissue. When seed coat was added back to ‘Tulare’ kernel media, aflatoxin was decreased to the detection limit or below. Aflatoxin production declined rapidly with seed coat maturity and was independent of growing location and rootstock. Breeding programs could increase resistance, enhancing marketability and safety of walnuts for human consumption.

Control of preharvest aflatoxin contamination in maize by pyramiding QTL involved in resistance to ear-feeding insects and invasion by Aspergillus spp.

Several resistance sources and resistance mechanisms to aflatoxin formation and corn earworm ( Helicoverpa zea Boddie) damage to maize ( Zea mays L.) have been identified. Based on this knowledge, experiments were initiated toward achievement of the following objectives: (1) to confirm earlier determinations on resistance traits of germplasm sources and to identify quantitative trait loci (QTL) associated with each of the traits, and (2) upon estimation of the degree of QTL effects on each trait, to generate a maize population, with chemical and physical resistance to Aspergillus spp. and ear-feeding insects, for inbred development. A 2-year field experiment to evaluate selected genotypes inoculated with A. flavus and infested with corn earworm revealed that significant variation exists among the genotypes for aflatoxin contamination and corn earworm damage. The protection of maize ears against aflatoxin contamination was primarily dependent on resistance to fungal infection and ear-feeding insects, and excellent husk coverage and tightness. A major QTL ( p1) identified on chromosome 1S had effects of 54.0, 42.1, and 28.3% on the phenotypic variability for concentrations of silk maysin, 3′-methoxymaysin+apimaysin, and chlorogenic acid, respectively. Markers/QTLs for husk phenotypic traits and total aflatoxin concentrations have been determined, but more detailed mapping of these chromosomic regions will be necessary to locate precise markers/QTLs for husk traits and aflatoxin production. Realizing the complexity of the Aspergillus–aflatoxin-maize system and the factors affecting aflatoxin contamination, we are directing our program toward marker-assisted breeding to enhance or improve general genetic resistance to ear-feeding insects and invasion by Aspergillus spp.

Molecular genetic analysis and regulation of aflatoxin biosynthesis.

Aflatoxins, produced by some Aspergillus species, are toxic and extremely carcinogenic furanocoumarins. Recent investigations of the molecular mechanism of AFB biosynthesis showed that the genes required for biosynthesis are in a 70 kb gene cluster. They encode a DNA-binding protein functioning in aflatoxin pathway gene regulation, and other enzymes such as cytochrome p450-type monooxygenases, dehydrogenases, methyltransferases, and polyketide and fatty acid synthases. Information gained from these studies has led to a better understanding of aflatoxin biosynthesis by these fungi. The characterization of genes involved in aflatoxin formation affords the opportunity to examine the mechanism of molecular regulation of the aflatoxin biosynthetic pathway, particularly during the interaction between aflatoxin-producing fungi and plants.

The production of aflatoxins in fresh date fruits and under simulated storage conditions

AbstractSixteen varieties of date fruit (Phoenix dactylifera) at three stages of maturation (Kimri, Rutab and Tamr) were examined for the presence of fungi and analysed for aflatoxins B1, B2, G1 and G2 and sterigmatocystin. Single samples of each variety were used in the study. Samples as received were initially examined for mycoflora and toxin levels and then stored at 98% relative humidity and 30 °C for 14 days to investigate the effects of possible adverse storage conditions on mycoflora and, in particular, aflatoxin formation. All samples showed an absence of aflatoxins and their precusor, sterigmatocystin, after adverse storage for 14 days, although aflatoxin‐producing Aspergillus flavus isolates were identified in 10 varieties at the first stage of maturation (Kimri). High fungal counts were associated with the Rutab stage and low counts with the Tamr stage. The counts of A flavus ranged from 5.00 to 8.16 log10(cfu g−1) under simulated storage conditions, and three varieties contained significant levels of aflatoxin B1 or B2 ranging from 35 to 11 610 µg kg−1. Sterigmatocystin was not detected in any of the samples as received or under simulated storage conditions.© 2002 Society of Chemical Industry

Synthesis of 11-Hydroxyl O-Methylsterigmatocystin and the Role of a Cytochrome P-450 in the Final Step of Aflatoxin Biosynthesis

The major skeletal rearrangements (anthraquinone --> xanthone --> coumarin) that occur in the complex biosynthesis of aflatoxin B(1) are mediated by cytochromes P-450. Previous experiments have suggested that two successive monooxygenase reactions are required to convert the xanthone O-methylsterigmatocystin (OMST) to aflatoxin, a process we demonstrate is mediated by a single P-450, OrdA, in Aspergillus parasiticus in accord with findings in A. flavus. The first oxidative cycle is proposed to result in the formation of 11-hydroxy O-methylsterigmatocystin (HOMST), while the second entails aryl ring cleavage, demethylation, dehydration, decarboxylation, and rearrangement to give aflatoxin - a remarkable sequence of transformations. To test this hypothesis, HOMST has been synthesized by an alkylnitrilium variant of the Houben-Hoesch reaction. The troublesome xanthone carbonyl was protected as a butylene to allow further elaboration of the molecule, and then the product xanthone was restored in a uniquely facile peracid deprotection. Methods were devised to construct the sensitive dihydrobisfuran and to maintain the oxidation state of the partially methylated hydroquinone. Expression of ordA in a yeast membrane preparation enabled the intermediacy of HOMST both to be detected in the conversion of OMST to aflatoxin and to be established directly in the biosynthesis of the mycotoxin. Having secured the role of HOMST in aflatoxin formation, the mechanism of the second oxidative cycle of this P-450 is considered.

Spice Oils for the Control of Co-Occurring Mycotoxin-Producing Fungi

The effect of nine different oils was evaluated on the growth of Aspergillus parasiticus and Fusarium moniliforme. The experimental design to examine the inhibition of mycotoxins involved the incorporation of each of seven oils into broth and patty cultures. The fungal mycotoxin was identified by high-pressure liquid chromatography. Clove oil (eugenol) was the most inhibitory to the growth of A. parasiticus and F. moniliforme, followed by cinnamon (cinnamic aldehyde), oregano (thymol and carvacol) and mace oils (myristin). Neem and eucalyptus oil (cineole) did not affect fungal growth. The feasibility of implementing the results of this study to control mycotoxin toxicity was examined by costoring whole and ground cloves with mycotoxin-infected grain. Addition of both whole and ground cloves markedly reduced the aflatoxin contamination of the grain. These results clearly suggest that commonly occurring mycotoxigenic fungi can be controlled with clove oil (eugenol), thus spice oil successfully inhibited the growth of A. parasiticus and F. moniliforme, regulated the production of fumonisins, and prevented the formation of aflatoxins. The social implication of this finding is that rural communities can prevent the formation of fungal toxins in contaminated grain by simple measures.

Inhibitory Effects of Naturally Occurring Compounds on Aflatoxin B1 Biotransformation

Effects of naturally occurring compounds from plants on biotransformation of a mycotoxin, aflatoxin B(1), were evaluated. Among 77 naturally occurring compounds tested, anthraquinones, coumarins, and flavone-type flavonoids were shown to be potent inhibitors of aflatoxin B(1)-8,9-epoxide formation. Addition of the flavonoids galangin, rhamnetin, and flavone strongly inhibited mouse liver microsomal conversion of aflatoxin B(1) to aflatoxin B(1)-8,9-epoxide, a metabolically activated mutagenic product. In contrast to these results, addition of isoflavonoids, catechins, terpenes, alkaloids, and quinones to mouse liver microsomes did not inhibit formation of aflatoxin B(1)-8,9-epoxide. Formation of the aflatoxin B(1) reductase product, aflatoxicol, by chicken liver cytosols was strongly inhibited by curcumin, the diferuloylmethane present in turmeric and other Curcuma species. Curcumin analogues also showed inhibitory effects, and a structure-activity study established that beta-diketone groups linked with two benzyl moieties were essential for inhibition of aflatoxicol formation. An additional 37 naturally occurring compounds tested did not inhibit formation of aflatoxicol. These results demonstrate that dietary constituents in certain fruits, vegetables, and spices may have significant inhibitory effects on metabolic transformation of aflatoxins to their hepatotoxic or carcinogenic derivatives or, alternatively, may promote their transformation into nontoxic products.

Growth of fungi and mycotoxin production on cheese under modified atmospheres

The use of modified atmospheres to prevent fungal growth and mycotoxin production in cheese was evaluated. Eight fungal species: Mucor plumbeus, Fusarium oxysporum, Byssochlamys fulva, B. nivea, Penicillium commune, P. roqueforti, Aspergillus flavus and Eurotium chevalieri were inoculated onto cheese and incubated under conditions of decreasing concentrations of O 2 (5% to <0.5%) and increasing concentrations of CO 2 (20–40%). Fungal growth was measured by colony diameter and ergosterol content. All fungi examined grew in atmospheres containing 20% and 40% CO 2 with 1% or 5% O 2, but growth was reduced by 20–80%, depending on species, compared with growth in air. The formation of aflatoxins B 1 and B 2, roquerfortine C and cyclopiazonic acid was greatly decreased but not totally inhibited in these atmospheres. At 20% or 40% CO 2 with <0.5% O 2, only B. nivea exhibited growth, which was very slow. Growth of F. oxysporum, B. fulva, P. commune and A. flavus showed good correlations between colony diameter and ergosterol content. However, for the other species correlations were inconsistent.

Calmodulin mediated activation of acetyl-CoA carboxylase during aflatoxin production by Aspergillus parasiticus.

The relevance of Ca2+-calmodulin-mediated processes in channelling acetate for aflatoxin formation was investigated by studying the influence of trifluoperazine (an anticalmodulin agent) on [14C]-acetate incorporation and activity of acetyl-CoA carboxylase in Aspergillus parasiticus NRRL 2999. Culturing the organism in presence of 0.14 mmol l-1 trifluoperazine resulted in 55% decrease of [14C]-acetate incorporation into aflatoxin B1, along with an 80% decrease in acetyl-CoA carboxylase activity at periods corresponding to maximal aflatoxin production. Concomitant decrement (35%) in the activity of glucose-6-phosphate dehydrogenase indicated decreased availability of reduction potential (NADPH) required for aflatoxin biosynthesis. The ability of calmodulin to activate and trifluoperazine to inhibit acetyl-CoA carboxylase activity in a dose-dependent manner was also noted under in vitro conditions. The combined results suggest calmodulin-mediated activation of acetyl-CoA carboxylase as an important event for aflatoxin production.

Inhibition of growth and aflatoxin biosynthesis of Aspergillus flavus by extracts of some Egyptian plants.

The effect of five different concentrations (2, 4, 6, 8 and 10 mg ml-1) of an aqueous extracts of Lupinus albus, Ammi visnaga and Xanthium pungens were tested on growth and aflatoxin production by Aspergillus flavus in a chemically defined medium. All the plants inhibited mycelial growth and aflatoxin formation. The inhibitory effect was proportional with the applied concentration. Growth and aflatoxin production appeared to be correlated processes. The nature of the plant extract also affected the ratio of B1 to B2, and there was no correlation between the inhibition of aflatoxins or growth of the fungus and the resultant B1: B2 ratio.

Mode of action of pesticides on aflatoxin biosynthesis and oxidase system activity

The effects of nine pesticides on the biosynthesis of aflatoxin and oxidase activity in wild-type Aspergillusflavus and mutant strains of A. parasiticus avr-1 (w 49) and A. parasiticus ver-1 (wh 1) were investigated. In A. parasiticus, phosphonic acid derivative (lancer) reduced the formation of aflatoxin B 2 but B 1, G 1 and G 2 and anthraquinones (versicolorin A, versiconal hemiacetal acetat and averufin) accumulated. Phosphorothioic acid derivatives (pirimiphos-methyl and pyrazophos) reduced the formation of aflatoxin B 2 and G 2 but B 1 and G 1 and anthra- quinones accumulated. Phosphorodithioic acid derivatives (dimethoate and malathion) blocked aflatoxin B 2, reduced B 1 and G 2 but G 1 and anthraquinones accumulated. Phosphoric acid derivative (profenfos) inhibited the formation of all afla-toxins, versicolorin A and versiconal hemiacetal acetate but averufin accumulated. The phenylurea derivatives (linuron and pencycuron) at concentrations of 500 and 1000 ppm inhibited all aflatoxin but anthraquinones accumulated. On the other hand, the dicarboximide derivative (iprodione) inhibited the whole pathway in the mutant strains of A. parasiticus. The oxidase system in wild-type A. flavus was active in the conversion of averufin and versicolorin A into aflatoxin B 1. Most organophosphate and phenylurea derivatives may competitively increase or decrease the oxidase enzymes, however, profenfos and iprodione blocked the enzymes between averufin and versicolorin A.

Mechanism of biochemical action of substituted 4-methylbenzopyran-2-ones. Part II: Mechanism-based inhibition of rat liver microsome-mediated aflatoxin B1–DNA binding by the candidate antimutagen 7,8-diacetoxy-4-methylcoumarin

7,8-Diacetoxy-4-methylcoumarin (DAMC), with no prerequisite for oxidative biotransformation has been reported to produce suicide inactivation of microsomal cytochrome P-450-catalysed formation of aflatoxin B1-8,9-oxide that binds to DNA. Parenteral administration of DAMC to rats caused significant inhibition of AFB1 binding to hepatic DNA in vivo as well as AFB1-induced micronuclei formation in bone marrow cells. These results highlight the antimutagenic potential of DAMC.

β‐l,3‐Glucanase and Resistance to Aspergillus flavus Infection in Maize

Studies of the activity levels of the fungal cell wall degradative enzyme, β‐l,3‐glucanase, were carried out with Zea mays L. embryogenie callus cultures and kernels of genotypes with different abilities to resist aflatoxin formation on the ears. Dual‐culture experiments of the aflatoxin forming fungus, Aspergillus flavus Link: Fr. with maize callus, demonstrated that the growth of A. flavus on plates was inhibited more by callus of a resistant genotype (Tex 6 ✕ Mol7 denoted T✕M) than by a sensitive genotype (Pa91). The inhibition correlated with the activity levels of β‐1,3‐glucanase in the callus and in the culture medium. The presence of the fungus caused an increase in enzyme activity in T✕M but not in Pa91 callus. Direct treatment of callus of both genotypes with A. flavus spores caused the amount of callose to increase within 30 min. An elevated β‐l,3‐glucanase activity in maize kernels was also correlated with lower A. flavus infection observed in the resistant genotype (Tex 6) compared with a susceptible one (B73). Fungal inoculation of the ears increased the enzyme activities both in kernels in contact with and not in contact with the fungus. These studies indicate that β‐l,3‐glucanase activity may have a role in the inhibition of growth of the A. flavus fungus, that maize embryogenic callus and kernels do respond to the presence of this nonpathogenic fungus, and that these systems might be useful in further plant‐fungal interaction studies.

Genetics and physiology of aflatoxin biosynthesis.

Aflatoxins are the most thoroughly studied mycotoxins. Elegant early research on the biosynthetic scheme of the pathway has allowed a molecular characterization of aflatoxin biosynthesis and its regulation. Genetic studies on aflatoxin biosynthesis in Aspergillus flavus and A. parasiticus, and sterigmatocystin biosynthesis in A. nidulans, led to the cloning of 17 genes responsible for 12 enzymatic conversions in the AF/ST pathways. Pathway-specific regulation is by a Zn(II)2Cys6 DNA-binding protein that regulates the transcription of all pathway genes. Less is known about the global factors that regulate aflatoxin biosynthesis, but there is a clear link between development and aflatoxin biosynthesis. There is also a large body of information on physiological factors involved in aflatoxin biosynthesis, but it has been difficult to understand their role in the regulation of this pathway. This chapter discusses current knowledge on the molecular biology and genetics of the pathway, and provides a summary of the physiological factors known to influence aflatoxin formation.

Immunochemical identification of AFLR, a regulatory protein, involved in aflatoxin biosynthesis

Polyclonal antibodies against AFLR, the aflR gene product of Aspergillus flavus and A. parasiticus, were generated by immunizing a rabbit with the Escherichia coli‐expressed recombinant AFLR protein of A. flavus. Immunoblot analysis revealed that the antibodies not only reacted with the recombinant AFLR protein of A. flavus or A. parasiticus but also with native 47‐kDa AFLR in A. flavus and A. parasiticus. Immunoblot analysis revealed that accumulation of the 47‐kDa AFLR in cultures of A. flavus and A. parasiticus correlated well with the production of aflatoxin under various culture conditions that regulate aflatoxin formation. Neither AFLR nor aflatoxin was found when A. parasiticus NRRL 2999 was grown in peptone mineral salts (PMS) medium; however, both were detected after the culture was transferred to glucose mineral salts (GMS) medium. The AFLR protein was absent in the non‐aflatoxigenic Penicillium and Fusarium species grown in GMS medium. The data indicate that the antibodies obtained in the present studies are specific for AFLR and could be used in various studies to monitor the role of AFLR in regulating aflatoxin biosynthesis.

Differential inhibition of aflatoxin B1 oxidation by gestodene action on human liver microsomes.

Human cytochrome P450 (P450) 3A is known to be involved in the formation of both aflatoxin B1-exo-8,9-epoxide (exo-epoxidation) and aflatoxin Q1 (3 alpha-hydroxylation). Gestodene, a known inactivator of P450 3A4, inhibited the formation of AFB1 metabolites in a variety of ways depending on the incubation condition. Preincubation of gestodene with human liver microsomes prior to the addition of AFB1 inhibited both exo-epoxidation and 3 alpha-hydroxylation whereas simultaneous incubation of gestodene with AFB1 only inhibited 3 alpha-hydroxylation. These results suggest that two independent substrate binding sites exist in P450 3A4, and AFB1 binds to both of the binding sites. Gestodene selectively binds to one of the binding sites leading to the formation of AFQ1, whereas it does not affect the formation of exo-epoxide via the other binding site.

Formation of Sclerotia and Aflatoxins in Developing Cotton Bolls Infected by the S Strain of Aspergillus flavus and Potential for Biocontrol with an Atoxigenic Strain

ABSTRACT Aspergillus flavus can be divided into the S and L strains on the basis of sclerotial morphology. On average, S strain isolates produce greater quantities of aflatoxins than do L strain isolates. Sclerotia of the S strain were observed in commercial seed cotton from western Arizona. Greenhouse tests were performed to better define sclerotial formation in developing bolls. Eight S strain isolates were inoculated into developing bolls via simulated pink bollworm exit holes. All eight isolates formed sclerotia on locule surfaces, and seven of eight isolates produced sclerotia within developing seed. Boll age at inoculation influences formation of sclerotia. More sclerotia formed within bolls that were less than 31 days old at inoculation than in bolls older than 30 days at inoculation. Frequent formation of sclerotia during boll infection may both favor S strain success within cotton fields and increase toxicity of A. flavus-infected cottonseed. Atoxigenic A. flavus L strain isolate AF36 reduced formation of both sclerotia and aflatoxin when coinoculated with S strain isolates. AF36 formed no sclerotia in developing bolls and was more effective at preventing S strain isolates than L strain isolates from contaminating developing cottonseed with aflatoxins. The use of atoxigenic L strain isolates to prevent contamination through competitive exclusion may be particularly effective where S strain isolates are common. In addition to aflatoxin reduction, competitive exclusion of S strain isolates by L strain isolates may result in reduced overwintering by S strain isolates and lower toxicity resulting from sclerotial metabolites.

Corn Seed Proteins Inhibitory to Aspergillus flavus and Aflatoxin Biosynthesis

ABSTRACT This study reports the presence of two fractions from corn seeds inhibitory to aflatoxin formation. Using a sensitive laboratory assay that can measure both inhibition of fungal growth and inhibition of aflatoxin biosynthesis, we examined aqueous extracts from seeds of Tex6, a corn inbred shown to be highly resistant to aflatoxin accumulation in field and laboratory evaluations. In these extracts, we identified two biologically active fractions. One inhibited growth of Aspergillus flavus and, thus, aflatoxin accumulation, and the other inhibited aflatoxin formation with little effect on fungal growth. The compounds responsible for these activities appear to be proteaceous, as they are water soluble, heat labile, and sensitive to proteinase K treatment. The compounds were partially purified by ultrafiltration and chromatography. The estimated molecular mass of the growth inhibitor is approximately 28 kDa, and that of the aflatoxin biosynthesis inhibitor appears to be greater than 100 kDa. Partially purified preparations of the growth inhibitor and aflatoxin biosynthesis inhibitor cause 50% inhibition at 26 and 75 mug of protein/ml, respectively. The presence of these compounds in Tex6 may explain its resistance to aflatoxin accumulation.

Relationship between Aflatoxin Biosynthesis and Sporulation inAspergillus parasiticus

Regulation of aflatoxin biosynthesis during differentiation ofAspergillus parasiticuswas analyzed by using a drug that inhibits the development of the fungus and mutants affected in sporulation. Diaminobutanone, a competitive inhibitor of ornithine decarboxylase, repressed spore germination. If added after spore germination had occurred, it blocked sporulation completely and suppressed aflatoxin biosynthesis, but was only partially inhibitory of mycelial growth. Putrescine partially counteracted the inhibitory effect of the drug on both sporulation and aflatoxin biosynthesis. Analysis of mutants affected in sporulation confirmed the existence of a relationship between sporulation and aflatoxin formation. A nonsporulating mutant was unable to synthesize aflatoxins. In a sectorial mutant, the sporulating sector synthesized aflatoxins normally, whereas the asporogenous sector was unable to do so. It is suggested that regulation of aflatoxin biosynthesis is correlated with the sporulation process.