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.     

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.     

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.     

Toxigenic Aspergillus flavus and aflatoxins in Sri Lankan medicinal plant material

The fungal flora of 6 Asian medicinal plants, Aerva lanata (Linn.) Juss. Alyssicarpus vaginalis D.C., Tribulus terrestris Linn. Adhatoda vasica Nees., Centella asciatica (L.) Urb., Cardiospermum halicacabum Linn. was determined. After surface disinfection Aspergillus spp. were most frequently observed. Aspergillus flavus, isolated from Alyssicarpus vaginalis and Aerva lanata produced aflatoxins in culture. Aflatoxin B₁ was also detected in a sample of Aerra lanata at a level of 0.5 g/g. Plant material destined for medicinal use should be stored carefully prior to its use to prevent growth of naturally occurring toxigenic mold fungi.     

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.     

Conidial movement of nontoxigenic Aspergillus flavus and A. parasiticus in peanut fields following application to soil

The use of nontoxigenic strains of Aspergillus flavus and A. parasiticus in biological control effectively reduces aflatoxin in peanuts when conidium-producing inoculum is applied to the soil surface. In this study, the movement of conidia in soil was examined following natural rainfall and controlled precipitation from a sprinkler irrigation system. Conidia of nontoxigenic A. flavus and A. parasiticus remained near the soil surface despite repeated rainfall and varying amounts of applied water from irrigation. In addition, rainfall washed the conidia along the peanut furrows for up to 100 meters downstream from the experimental plot boundary. The dispersal gradient was otherwise very steep upstream along the furrows and in directions perpendicular to the peanut rows. The retention of biocontrol conidia in the upper soil layers is likely important in reducing aflatoxin contamination of peanuts and aerial crops such as corn and cottonseed. This revised version was published online in August 2006 with corrections to the Cover Date.     

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).     

Aflatoxin-producing potential of Aspergillus flavus strains isolated from Spanish poultry feeds

A total of 126 fungal strains belonging to the Aspergillus flavus group isolated from commercial poultry mixed feeds were studied. One hundred and twenty-five were identified as A. flavus and one as A. parasiticus. Forty nine strains (39%) produced aflatoxins on a crushed moist wheat medium (28 °C/10 days), whereas only sixteen (13%) showed specific fluorescence on Aflatoxin-Producing Ability Medium. In both media, mainly aflatoxins B₁ and B₂ were detected, the average concentration of aflatoxins being 4294+/–1083 g/kg in crushed moist wheat medium, and 877+/–257 g/kg in Aflatoxin-Producing Ability Medium.     

Assessment of aflatoxin and cyclopiazonic acid production by Aspergillus flavus isolates from Hungary

Thirty-two isolates of Aspergillus flavus were obtained from various sources in Hungary. All isolates were morphologically identified as A. flavus and three atypical variants were confirmed as A. flavus by comparing their DNA with an ex type culture of A. flavus. None of these isolates produced aflatoxins when tested on coconut agar or grown on rice medium and culture extracts examined by thin layer chromatography. Also, none of the isolates converted sterigmatocystin, O-methyl sterigmatocystin, norsolorinic acid, or sodium acetate to aflatoxin. However, 59% of the isolates produced cyclopiazonic acid based on thin layer chromatographic analysis of culture extracts. The isolates that lack the ability to produce both aflatoxin and cyclopiazonic acid are potential candidates for use in bicontrol studies.     

Molecular characterization of an atoxigenic Aspergillus flavus strain AF051

An atoxigenic Aspergillus flavus strain AF051 collected from a peanut field in Jiangsu province, P. R. China was characterized by analysis of aflatoxin gene cluster in this study. By using a thermal asymmetric interlaced PCR (TAIL-PCR) and conventional PCR techniques, an 89.59-kb deletion was found in the cluster, and this deletion was replaced by a 3.83-kb insert, which was located at 300-bp upstream ver1 gene and 2594-bp downstream a putative gluconolactone oxidase gene. Based on the DNA sequence at the breakpoint, a nested-PCR method was developed for the rapid and sensitive detection of AF051 strain in soil and peanut samples once the strain is used as a biological agent.     

Decolourization of Black Oxidized Olive-Mill Wastewater by a New Tannase-Producing Aspergillus flavus Strain Isolated from Soil

Summary By contaminating a Tunisian soil with black oxidized and sterilized olive-mill wastewaters (OMW), 30 new indigenous fungal soil strains able to overcome the OMW toxicity could be directly selected. Ten of the fungal strains previously isolated were screened for their capability to grow in a liquid culture medium containing oxidized OMW as the only source of carbon and energy. According to these preliminary tests, strain F2 showed the best capability of removing black colour and COD (chemical oxygen demand) and was further identified as Aspergillus flavus. After optimization of batch-liquid culture conditions in the presence of oxidized OMW, the time course of biomass and enzyme production by A. flavus F2 was followed in relation to colour and COD removal. A. flavus F2 could efficiently decolourize and detoxify the black oxidized OMW (58 and 46% of colour and COD removal, respectively, after 6 days of cultivation), concomitantly with the production of tannase (8000 UI/l on day 3).     

Separate and combined applications of nontoxigenic Aspergillus flavus and A. parasiticus for biocontrol of aflatoxin in peanuts

A 2-year study was carried out to determine the effect of applying nontoxigenic strains of Aspergillus flavus and A. parasiticus to soil separately and in combination on preharvest aflatoxin contamination of peanuts. A naturally occurring, nontoxigenic strain of A. flavus and a UV-induced mutant of A. parasiticus were applied to peanut soils during the middle of each of two growing seasons using a formulation of conidia-coated hulled barley. In addition to an untreated control, treatments included soil inoculated with nontoxigenic A. flavus only, soil inoculated with nontoxigenic A. parasiticus only, and soil inoculated with a mixture of the two nontoxigenic strains. Plants were exposed to late-season drought conditions that were optimal for aflatoxin contamination. Results from year one showed that significant displacement (70%) of toxigenic A. flavus occurred only in peanuts from plots treated with nontoxigenic A. flavus alone; however, displacement did not result in a statistically significant reduction in the mean aflatoxin concentration in peanuts. In year two, soils were re-inoculated as in year one and all treatments resulted in significant reductions in aflatoxin, averaging 91.6%. Regression analyses showed strong correlations between the presence of nontoxigenic strains in peanuts and aflatoxin reduction. It is concluded that treatment with the nontoxigenic A. flavus strain alone is more effective than the A. parasiticus strain alone and equally as effective as the mixture. The U.S. Government’s right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Evaluation of selected geocarposphere bacteria for biological control of Aspergillus flavus in peanut

Selected bacterial strains isolated from the region of peanut pod development (geocarposphere) and two additional bacterial strains were screened as potential biological control agents against Aspergillus flavus invasion and subsequent aflatoxin contamination of peanut in laboratory, greenhouse, and field trials. All 17 geocarposphere strains tested delayed invasion of young roots and reduced colonization by the fungus in a root-radicle assay used as a rapid laboratory prescreen. In a greenhouse study, seven bacterial strains significantly reduced pod colonization by A. flavus compared to the control. In a field trial, conducted similarly to the greenhouse assay, pods sampled at mid-peg from plants seed-treated with suspensions of either 91A-539 or 91A-550 were not colonized by A. flavus, and the incidence of pods invaded from plants treated with either 91A-539 or 91A-599 was consistently lower than nonbacterized plants at each of five sampling dates. At harvest, 8 geocarposphere bacterial strains significantly lowered the percentage of pods colonized (> 51%) compared to the control. Levels of seed colonization ranged from 1.3% to 45% and did not appear related to aflatoxin concentrations in the kernels.     

Antifungal compounds from Bacillus subtilis B-FS06 inhibiting the growth of Aspergillus flavus

The cell-free culture filtrate (CCF) was prepared from a culture of an Aspergillus flavus antagonist, Bacillus subtilis B-FS06. The CCF inhibited the growth and spore germination of A. flavus at a series of concentrations (10, 25, 50%) (v/v). It still retained the activity after treatment at pH values ranging from 2 to 12 for 24 h or at 100 °C for 30 min. The antifungal activity, however, was reduced by 30% after treatment at 121 °C for 20 min. After purification by anion exchange chromatography, gel filtration chromatography and HPLC, the active compounds revealed six ion peaks: [M–H] m/z = 1006.78, 1020.71, 1034.74, 1049.54, 1056.78, and 1071.64 by using electrospray ionization mass spectrometry (ESI-MS) analysis. In the presence of the active compounds at 200 μg/g, the growth of A. flavus on peanuts was completely inhibited. Ting Zhang and Zhi-Qi Shi contributed equally to this work.     

Understanding the genetics of regulation of aflatoxin production and Aspergillus flavus development

Aflatoxins are polyketide-derived, toxic, and carcinogenic secondary metabolites produced primarily by two fungal species, Aspergillus flavus and A. parasiticus, on crops such as corn, peanuts, cottonseed, and treenuts. Regulatory guidelines issued by the U.S. Food and Drug Administration (FDA) prevent sale of commodities if contamination by these toxins exceeds certain levels. The biosynthesis of these toxins has been extensively studied. About 15 stable precursors have been identified. The genes involved in encoding the proteins required for the oxidative and regulatory steps in the biosynthesis are clustered in a 70 kb portion of chromosome 3 in the A. flavus genome. With the characterization of the gene cluster, new insights into the cellular processes that govern the genes involved in aflatoxin biosynthesis have been revealed, but the signaling processes that turn on aflatoxin biosynthesis during fungal contamination of crops are still not well understood. New molecular technologies, such as gene microarray analyses, quantitative polymerase chain reaction (PCR), and chromatin immunoprecipitation are being used to understand how physiological stress, environmental and soil conditions, receptivity of the plant, and fungal virulence lead to episodic outbreaks of aflatoxin contamination in certain commercially important crops. With this fundamental understanding, we will be better able to design improved non-aflatoxigenic biocompetitive Aspergillus strains and develop inhibitors of aflatoxin production (native to affected crops or otherwise) amenable to agricultural application for enhancing host-resistance against fungal invasion or toxin production. Comparisons of aflatoxin-producing species with other fungal species that retain some of the genes required for aflatoxin formation is expected to provide insight into the evolution of the aflatoxin gene cluster, and its role in fungal physiology. Therefore, information on how and why the fungus makes the toxin will be valuable for developing an effective and lasting strategy for control of aflatoxin contamination.     

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.     

Aflatoxigenicity in Aspergillus: molecular genetics, phylogenetic relationships and evolutionary implications

Aflatoxins (AFs) are toxic and carcinogenic secondary metabolites produced by isolates of Aspergillus section Flavi as well as a number of Aspergillus isolates that are classified outside of section Flavi. Characterization of the AF and sterigmatocystin (ST) gene clusters and analysis of factors governing regulation of their biosynthesis has resulted in these two mycotoxins being the most extensively studied of fungal secondary metabolites. This wealth of information has allowed the determination of the molecular basis for non-production of AF in natural isolates of A. flavus and domesticated strains of A. oryzae. This review provides an overview of the molecular analysis of the AF and ST gene clusters as well as new information on an AF gene cluster identified in the non-section Flavi isolate, Aspergillus ochraceoroseus. Additionally, molecular phylogenetic analysis using AF biosynthetic gene sequences as well as ribosomal DNA internal transcribed spacer (ITS) sequences between various section Flavi and non-section Flavi species has enabled determination of the probable evolutionary history of the AF and ST gene clusters. A model for the evolution of the AF and ST gene clusters as well as possible biological roles for AF are discussed.     

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.     

Metabolic behaviour of aflatoxin producing strain and non-toxigenic strain of Aspergillus flavus to different sources of nitrogen and glucose concentration

The effect of different nitrogen sources and varying glucose concentration on aflatoxin production by a toxigenic and non-toxigenic strain of Aspergillus flavus was studied. Greatest production (3.8 ppm) of aflatoxin B₁ was produced in a synthetic medium when casamino acids were supplied as the nitrogen source. Optimum sugar concentration for aflatoxin B₁ production ranged between 3 and 10 g/100 ml. There was no appreciable difference in the metabolic behaviour between toxigenic and non-toxigenic strains of A. flavus when dry mycelial weight, total proteins, non-protein nitrogen and reducing sugar were the criteria.