Aspergillus flavus colonization and aflatoxin B1 formation in barley grain during interaction with other fungi

Colonization of barley grain by Aspergillus flavus and formation of aflatoxin B₁ in the presence of Penicillium verrucosum, Fusarium sporotrichioides, and Hyphopichia burtonii were studied over a three-week period in all combinations of 20 or 30 °C and 0.97, 0.95 or 0.90 a_w. Grain colonization was assessed initially by observing hyphal extension on the grain surface, using scanning electron microscopy, and then from the proportion of seeds infected and numbers of colony forming units (cfu) formed. Aflatoxin b¹ concentrations were determined by enzyme linked immunosorbent assay using a monoclonal antibody. These studies showed that interaction between A. flavus and other fungi in paired culture had different effects on both colonization and aflatoxin formation depending on the species involved and environmental conditions. Germination of A. flavus spores was unaffected by the presence of other species on the grain surface. Subsequently, three principal patterns of A. flavus colonization of barley grain were observed through the incubation period in the presence of other fungal species: (a) colonization unaffected by the presence of other species; (b) colonization initially slower in the presence of other species but later differing little from pure cultures; and (c) colonization adversely affected by the presence of other species. Five main patterns of aflatoxin B₁ production were observed relative to pure culture but with no consistent relationship with species, a_w, temperature or incubation period; (a) little changed; (b) increased slowly; (c) decreased; (d) enhanced; and (e, f) increased initially but later decreased to (e) the same level as in pure culture or (f) to less than in pure culture. Generally, production of aflatoxin B₁ by A. flavus was less than in pure culture but sometimes was changed only slightly by the presence of P. verrucosum, F. sporotrichioides or H. burtonii or was temporarily enhanced.     

Behaviour of Aspergillus flavus in presence of Aspergillus niger during biosynthesis of aflatoxin B1

Aspergillus niger or Aspergillus tamarii when grown as mixed cultures with toxigenic A. flavus inhibits biosynthesis of aflatoxin by A. flavus, owing primarily to its ability to produce inhibitors of aflatoxin biosynthesis and to their ability to degrade aflatoxin. Gluconic acid partly prevents aflatoxin production. The other factors such as changes in pH of the medium and the effect on the growth of A. flavus have no role in imparting capabilities to these cultures to inhibit aflatoxin production by A. flavus.     

Influence of formic acid on fungal flora of barley and on aflatoxin production inAspergillus flavus link

In recent yearsAspergillus flavus and aflatoxin production have been noted on several occasions in grain preserved with formic acid. Samples of mouldy barley treated with formic acid and stored in an open bin were investigated for the presence of fungi. In the lower part of the bin there was a clear dominance ofFusarium sporotrichioides, and deoxynivalenol and neosolaniol were detected.A. flavus andA. fumigatus were also present.Paecilomyces variotii occurred, almost as a pure culture, in the upper part of the bin, but no patulin was found. Cultivation of four fungal isolates from these genera on laboratory substrates containing formic acid showedP. variotii to be the most tolerant to formic acid, withstanding 150 mM, but still without patulin production.F. sporotrichioides andA. fumigatus tolerated only 6 mM formic acid. The growth ofA. flavus was reduced and atypical at 60 mM formic acid. Pretreatment ofA. flavus spores with formic acid increased aflatoxin production about 800 times.     

Divergent regulation of aflatoxin production at acidic pH by two <Emphasis Type="Italic">Aspergillus</Emphasis> strains

Production of aflatoxins (AF) by Aspergillus flavus and A. parasiticus is known to occur only at acidic pH. Although typical A. flavus isolates produced more AF as the external pH became increasingly acidic, an atypical strain from West Africa produced less. The lower AF production was not well correlated with decreases in expression of the aflatoxin pathway regulatory gene, aflR, or of two other biosynthesis genes.     

Mycotoxins in Australia: biocontrol of aflatoxin in peanuts

The major mycotoxin problem in Australia is the formation of aflatoxins in peanuts by Aspergillus flavus and A. parasiticus. This is controlled by good farm management practice, segregation into grades on aflatoxin content at intake to shelling facilities, colour sorting and aflatoxin assays. A second problem is the potential presence of ochratoxin A in grapes and grape products, resulting from infection by Aspergillus carbonarius. Good quality control before and during wine making ensures ochratoxin A is kept to very low levels, but in dried vine fruit, ochratoxin A levels may be higher. Biocontrol by competitive exclusion has been developed as the most promising means of controlling aflatoxins in peanuts. Some details of the process are given, including some basic laboratory experiments.     

黄曲霉毒素生物合成的遗传调控研究进展

黄曲霉毒素是一类具有较强毒性和致癌力的次级代谢产物,在小麦、水稻、玉米和花生等多种粮食、油料、饲料和食品中检出率均比较高。因此,黄曲霉毒素不仅给人和动物的健康造成极其严重的威胁,而且也给食品和饲料等行业造成了巨大的经济损失。黄曲霉毒素主要由黄曲霉和寄生曲霉产生。自上个世纪60年代首次发现黄曲霉毒素以来,研究者在黄曲霉毒素合成途径、降解、合成机制和致病机理等方面做了大量研究。本文主要综述近年来国内外以黄曲霉为对象的黄曲霉毒素合成的遗传调控机制研究进展。从转录调控、蛋白翻译后修饰、信号转导途径、参与生长发育和形态建成的蛋白和其他酶等方面对黄曲霉毒素合成机制展开综述,为今后进一步深入系统研究黄曲霉毒素合成机制奠定基础,同时为制定防治黄曲霉及其毒素的策略提供理论基础。     

Evaluation of the mycological status of luncheon meat with special reference to aflatoxigenic moulds and aflatoxin residues

The luncheon meat samples analyzed, which were produced locally by the two main luncheon meat producing companies in Egypt were relatively highly contaminated either by moulds and yeasts in general, aflatoxigenic species and aflatoxin residues in particular. The most frequently encountered fungi from the samples were yeasts, Aspergillus niger, A. flavus, Penicillium chrysogenum, Rhizopus stolonifer, Mucor circinelloides. Less common were Cladosporium sphaerospermum, Alternaria alternata, Mycosphaerella tassiana, P. aurantiogriseum and P. oxalicum. The most important aflatoxigenic species, A. flavus, was isolated frequently. It was 10% of the total fungal isolates from both samples of the two companies. Seven luncheon meat samples out of 50 analyzed were positive for aflatoxin B₁ or B₁ and G₁, while all samples were negative for aflatoxins B₂, G₂, M₁ and M₂. Aflatoxin B₁ was detected only in 4 and 3 samples out of 25 analyzed from each of company A and B, respectively. The highest detectable level, 11.1 ppb, was recorded in a sample from company B and the least, 0.5 ppb, in a sample from company A. Aflatoxin G₁, at concentration of 3.2 ppb, was detected in only one sample of the aflatoxin B₁ – contaminated 3 samples of company B: this sample also had the highest level of aflatoxin B₁. Some luncheon meat samples had higher numbers of aflatoxigenic A. flavus than others, however these samples were negative for aflatoxins. The hazardous potential of such contamination will be discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

A versiconal hemiacetal acetate converting enzyme in aflatoxin biosynthesis

Conversion of the aflatoxin biosynthetic intermediate versiconal hemiacetal acetate (VHA) in a cell free extract ofAspergillus parasiticus ATCC 15517 is investigated. The enzymatic reaction is monitored by a method using high performance liquid chromatography (HPLC). The major product of the enzymatic reaction is a water soluble compound not chloroform-extractable at pH 7.5. The product becomes chloroform extractable upon acidification of the reaction medium and is separated and quantitated by reversed-phase HPLC. It is tentatively identified as versiconal hemiacetal alcohol, which is converted to versicolorin C (VC) upon acid treatment.     

Substrate depletion during formation of aflatoxin and kojic acid on corn inoculated with Aspergillus flavus

During a period of 8 years 300 cases of dermatophytoses involving both hairy areas and the glabrous skin were found to be caused by M. canis. There was scalp involvement in 60%, including 8 infants and 27 adults; most of the adults presented Kerion-like lesions and presented various clinical aspects such as seborrhea capitis, folliculitis and discois lupus erythematosus. In the 21 patients showing invasion of the beard the clinical manifestations included superficial erythematosquamous patches with hyperemic slightly elevated margins, folliculitis or abscess-like lesions and Kerion-like lesions. Among the lesions found on the glabrous skin there were unusual aspects of tinea faciei in 19 adults, mimicking lymphocytic infiltration, granuloma faciale or discoid lupus erythematosus. Some of the cases of tinea corporis found in 70 patients also had lesions simulating various other dermatological entities, including erythema multiforme, psoriasiform eruption, pityriasis rosea and seborrheic dermatitis. The hands were invaded in 5 adults patients, with involvement of the finger nails in one. Repeated mycologic examinations were necessary to establish the true etiology in many of these cases.     

Different media and methodologies for the detection of aflatoxin production by Aspergillus flavus strains isolated from trout feed

We have studied the aflatoxin producing capacity of 41 Aspergillus flavus strains isolated from the mycoflora present of natural media (wheat, rice and mixed feed) synthetic medium (Aflatoxin Producing Ability Medium) and semisynthetic media (Coconut Agar Medium and Glucose Yeast Extract Agar) were compared. Aflatoxins were analysed on days 4 and 8 post-inoculation under an incubation temperature of 28 °C. A total of 30 strains (75.7%) were producers on natural media as detected by Thin Layer Chromatography: 23 strains on wheat, 27 on rice and 12 on mixed feed. The results by qualitative flourescence tests on synthetic and semisynthetic media were: 3 strains positive on Coconut Agar Medium (CAM) 1 on Glucose Yeast Extract Agar (GY + Agar) and none on Aflatoxin Producing Ability Medium (APA).     

Regulatory variability of camalexin biosynthesis

The anthranilate synthase ASA1, CYP79B2 and CYP71B15 (PAD3) are biosynthetic genes of the Arabidopsis phytoalexin camalexin, which are induced after pathogen infection and abiotic treatments like silver nitrate spraying. The natural variation of camalexin biosynthesis in response to Pseudomonas syringae infection was determined in several ecotypes, and differential CYP71B15 regulation as a potential basis for this variation was investigated. The expression of camalexin biosynthetic genes was restricted to the tissue undergoing cell death. After droplet infection with Alternaria alternata, a potent camalexin inducer in the Col-0 ecotype, camalexin formation and the induction of ASA1, CYP79B2 and CYP71B15 were strictly co-localized with the infection site.     

Occurrence of Aspergillus section Flavi and aflatoxin B1 in corn genotypes and corn meal in Argentina

A study has been carried out in Argentina on samples of corn genotypes from a breeding station as well as in commercially available corn meal. All samples were analyzed for fungal infection and aflatoxin B₁.Mycological analysis of corn genotypes showed the presence of three principal genera of filamentous fungi Fusarium (100%), Penicillium (67%) and Aspergillus (60%). In the genus Fusarium three species were identified, F. moniliforme (42%), F. nygamai (56%) andF. proliferatum (1.8%). Eight species ofPenicillium were identified, the predominant species isolated were P. minioluteum, P. funiculosum and P. variabile. In the genus ranked third in isolation frequency, two species were identified, A. flavus and A. parasiticus, the percentage of infection was 78% and 21%, respectively. Only one corn genotype was contaminated with aflatoxin B₁ at a level of 5 ppb. The cornmeal samples showed great differences in fungal contamination, the values ranging from 1 × 10¹ to 7 × 10⁵ cfu g^–1. Fusarium (68%), Aspergillus (35%) and Penicillium (21%) were the most frequent genera isolated. Among the genus, Aspergillus, A. parasiticus (38%) was the most frequent species isolated. All the samples of corn meal were negative to aflatoxin B₁. These results indicate a low degree of human exposure to aflatoxins in Argentina through the ingestion of maize or corn meal.This revised version was published online in October 2005 with corrections to the Cover Date.     

Preharvest seed infection byAspergillus flavus group fungi and subsequent aflatoxin contamination in groundnuts in relation to soil types

Preharvest seed infection byAspergillus flavus and aflatoxin contamination in selected groundnut genotypes (fourA. flavus-resistant and fourA. flavus-susceptible) were examined in different soil types at several locations in India in 1985–1990. Undamaged mature pods were sampled at harvest and seed examined forA. flavus infection and aflatoxin content in two or more trials at ICRISAT Center on light sandy soils and red sandy loam soils (Alfisols), and on Vertisols, at Anantapur on light sandy soils, and at Dharwad and Parbhani on Vertisols. Rainy season trials (1985–1989) were all rainfed. Post-rainy season trials were irrigated; late-season drought stress (90 days after sowing (DAS) until harvest at 125 DAS) was imposed in the 1987/88 and 1989/90 seasons.A. flavus infection and aflatoxin contamination levels were much lower in seed of all genotypes from Vertisols than in seed from Alfisols across locations and seasons. Vertisols also had significantly lower populations ofA. flavus than Alfisols. There were no marked differences between light sandy soils and red sandy loam soils (Alfisols) in respect of seed infection byA. flavus and aflatoxin contamination. Significant interactions between genotypes and soil types were evident, especially in theA. flavus-susceptible genotypes. Irrespective of soil types,A. flavus-resistant genotypes showed lower levels of seed infection byA. flavus and other fungi than didA. flavus-susceptible genotypes. The significance of the low preharvest aflatoxin risk in groundnuts grown on Vertisols is highlighted.ICRISAT Journal Article No. JA 1122     

Considerations on the distribution of aflatoxigenic Aspergillus flavus in feeds

The distribution of aflatoxin producing isolates of the Aspergillus flavus group in feeds was studied. Aflatoxin production was investigated by a sequential method previously reported (fluorescence in Coconut Agar Medium, rapid extraction from a wheat medium, and total extraction from the same wheat medium). Twenty-seven of 32 samples contained A. flavus, and 21 of them had at least one aflatoxicogenic isolate of A. flavus. Of the 115 isolates analysed, 65 produced aflatoxins, mainly B aflatoxins.     

Regulatory potential of nonautonomous mariner elements and subfamily crosstalk

Two naturally occurring nonautonomous mariner elements were tested in vivo for their ability to down-regulate excision of a target element in the presence of functional mariner transposase. The tested elements were the peach element isolated from Drosophila mauritiana which encodes a transposase that differs from the autonomous element Mos1 in four amino acid replacements, and the DTBZ1 element isolated from D. teissieri which encodes a truncated protein consisting of the first 132 residues at the amino end of the normally 345-residue transposase. We provide evidence that the protein from the peach element does interact to down-regulate wildtype transposase, indicating that at least some nonautonomous elements in natural populations that retain their open reading frame may play a regulatory role. In contrast, our tests reveal at most a weak interaction between transposase from the autonomous Mos1 element and the truncated protein from DTBZ1 and none between Mos1 transposase and that from the distantly related mariner-like element Himar1 identified in the horn fly Haematobia irritans. Hence, the extent of regulatory crosstalk between mariner-like elements may be limited to closely related ones. The evolutionary implications of these results are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Time ofAspergillus flavus infection and aflatoxin formation in ripening of figs

Figs in an orchard were inoculated with an aflatoxigenicAspergillus flavus strain in two ways by spore injection or by dusting at three maturation stages: firm ripe, shrivelled, and dried. Fruits were individually examined for fungal development and analyzed for aflatoxin B₁ (AF B₁) after 2, 4, 6, 8 and 10 days. Fruit injected at the first stage showed fungal development and AF B₁ contamination within two days. The toxin level increased sharply to 1 ppm after 10 days. The mean level of AF B₁ (284.75 ng/g) was significantly higher than those observed in other conditions. Figs dusted at the first stage showed only a tiny fungal growth even after 10 days. AF B₁ appeared after 6 days with a low frequency (35%), mean level (7.6 ng/g) and a great variation among figs (0.22–15 ng/g). Among fruits inoculated during the shrivelled fig and dried fruit stages, no fungal growth was observed and AF B₁ was detected with a lower incidence in association with low mean levels (less than 1.25 ng/g). Methods of prevention of aflatoxin contamination at the critical step, the firm ripe stage, are discussed.     

A USA–Africa collaborative strategy for identifying, characterizing, and developing maize germplasm with resistance to aflatoxin contamination

Aflatoxin contamination of maize by Aspergillus flavus poses serious potential economic losses in the US and health hazards to humans, particularly in West Africa. The Southern Regional Research Center of the United States Department of Agriculture, Agricultural Research Service (USDA-ARS-SRRC) and the International Institute of Tropical Agriculture (IITA) initiated a collaborative breeding project to develop maize germplasm with resistance to aflatoxin accumulation. Resistant genotypes from the US and selected inbred lines from IITA were used to generate backcrosses with 75% US germplasm and F₁ crosses with 50% IITA and 50% US germplasm. A total of 65 S₄ lines were developed from the backcross populations and 144 S₄ lines were derived from the F₁ crosses. These lines were separated into groups and screened in SRRC laboratory using a kernel-screening assay. Significant differences in aflatoxin production were detected among the lines within each group. Several promising S₄ lines with aflatoxin values significantly lower than their respective US resistant recurrent parent or their elite tropical inbred parent were selected for resistance-confirmation tests. We found pairs of S₄ lines with 75–94% common genetic backgrounds differing significantly in aflatoxin accumulation. These pairs of lines are currently being used for proteome analysis to identify resistance-associated proteins and the corresponding genes underlying resistance to aflatoxin accumulation. Following confirmation tests in the laboratory, lines with consistently low aflatoxin levels will be inoculated with A. flavus in the field in Nigeria to identify lines resistant to strains specific to both US and West Africa. Maize inbred lines with desirable agronomic traits and low levels of aflatoxin in the field would be released as sources of genes for resistance to aflatoxin production.     

The occurrence of Aspergillus flavus in vegetative tissue of cotton plants and its relation to seed infection

Twenty-seven mature cotton bolls with Aspergillus flavus Link colonies naturally occurring on the surface of the boll or lint were collected in the field in Arizona along with their subtending stems and peduncles. Bolls inoculated through the carpel wall 30 days after anthesis were allowed to mature in the field and were collected in the same manner. The seed and stem and peduncle sections of each boll were surface-sterilized, plated on agar media and observed for A. flavus. Seventy-eight percent of the naturally contaminated bolls with A. flavus in the seed also had the fungus in the stem and peduncle, whereas only 31% of the naturally contaminated bolls with no A. flavus in the seed had the fungus in the stem or peduncle. This difference was significant (P=0.0125), indicating a positive relationship between seed infection and stem and peduncle infection. All of the bolls inoculated through the carpel wall had A. flavus in the seed, but only 11% of the stem and peduncle sections were infected, indicating that the fungus does not readily grow downward from the boll into the supporting stem or peduncle.This unidirectional pattern of movement (upward) was further substantiated in greenhouse experiments where cotton seedlings were inoculated at the cotyledonary leaf scar with A. flavus and plants were sequentially harvested, surface sterilized and plated. Aspergillus flavus was isolated from the cotyledonary leaf scar, flower buds, developing bolls, and stem sections in the upper portion of the plant. It was never isolated from roots or stem sections below the cotyledonary node, again indicating that the fungus does not readily move downward through the plant.