Polyphasic characterization of Aspergillus section Flavi isolated from animal feeds in Algeria

In Algeria, little information is available on the population structure of Aspergillus section Flavi in raw materials and resultant animal feeds. A total of 172 isolates belonging to Aspergillus section Flavi were recovered from 57 animal feeds and identified on the basis of macro and micro-morphological characters, mycotoxin production and genetic relatedness. For the molecular analysis, sequencing of the calmodulin gene (CaM) and the internal transcribed spacer (ITS) regions were performed for representative isolates. Four distinct morphotypes were distinguished: Aspergillus flavus (78.5%), Aspergillus tamarii (19.2%), Aspergillus parasiticus (1.7%), and Aspergillus alliaceus (0.6%). All A. flavus isolates were of the L type and no correlation between sclerotia production and aflatoxigenicity was observed. Our results showed that 68% of the A. flavus strains produced aflatoxins B (AFB), and 72.7% were cyclopiazonic acid (CPA) producers. The three isolates of A. parasiticus were able to produce AFB and aflatoxins G but not CPA whereas, all the strains of A. tamarii produced only CPA. The obtained results revealed the presence of different species of Aspergillus section Flavi, among which were aflatoxin producers. This study provides evidence useful for considerations in aflatoxin control strategies.     

Aflatoxin and Cyclopiazonic Acid Production by Aspergillus flavus Isolated from Contaminated Maize

Seven truck-loads of maize were tested for mycotoxin contamination. Aflatoxin was identified in all 7 at concentrations from 3 ng/g-501 ng/g (aflatoxin B₁+ B₂). Cyclopiazonic acid was identified in 4 loads with concentrations from 25-250 ng/g. Deoxynivalenol was found in 4 of 5 loads tested, over a range of 46-676 ng/g. Ninteeen isolates of Aspergillus flavus from the samples were tested for ability to accumulate cyclopiazonic acid and aflatoxin in liquid culture. Fourteen produced cyclopiazonic acid (0.5-135 μg/mL), 12 produced aflatoxin (0.01-0.70 μg/mL, aflatoxin B₁+ B₂), and one aflatoxin-producing isolate did not produce cyclopiazonic acid.     

Cyclopiazonic acid and aflatoxin production by cultures of Aspergillus flavus isolated from dried beans and maize

From the storerooms of individual households 150 samples of dried beans and 90 samples of stored maize were collected for mycological analyses. Two of 27 isolates of A. flavus grown on malt extract agar (MEA) were found to produce the mycotoxin cyclopiazonic acid (25–36 μg/g). Three of the A. flavus isolates grown on crushed moist wheat produced aflatoxin B₁ (0.72–1.6 μg/g) and 6 of 26 A. ochraceus isolates were OA positive (0.5–10.4 μg/g). None of 25 bean samples were contaminated with CPA, AF or OA, while 4 samples of 30 tested maize samples were OA positive with level of OA 0.4–400 μg/g. Toxins were determined by thin layer chromatography and colorimetric method was used for quantitations of CPA.     

Production of cyclopiazonic acid by aflatoxigenic and non-aflatoxigenic strains of Aspergillus flavus

96 strains of Aspergillus flavus isolated from samples of stored grain and smoke-dried meat products were examined for ability to produce cyclopiazonic acid and aflatoxins, grown on mycological broth medium and malt extract agar. Five strains produced cyclopiazonic acid in the range of 0.5 — 30 mg/kg and 9 produced aflatoxin B1 (0.1 — 14.8 mg/kg) but none of them produced both cyclopiazonic acid and aflatoxins.     

Fungi associated with post-harvest rot of sweet orange (Citrus sinensis) and aflatoxin B1 production by isolates of Aspergillus flavus on plain and supplemented orange juice

Samples of rotting sweet orange (Citrus sinensis) were obtained from the depots, sales counters and waste baskets. Fungi associated with rotting fruits were isolated and identified. Out of 12 species of fungi isolated, 8 are known to be producers of toxins. The 7 isolates of Aspergillus flavus obtained were screened for aflatoxin production in a nutrient solution, and 4 were found to be aflatoxigenic, producing primarily aflatoxin B₁. Aflatoxin B₁ production of the toxigenic isolates were further studied on plain juice and juice separately supplemented with 2.0% yeast extract and 2.0% sucrose. The highest yield of aflatoxin B₁ was produced on juice supplemented with yeast extract by the 4 toxigenic A. flavus isolates, followed by sucrose supplementation while the lowest amount of aflatoxin B₁ was detected on plain juice. Optimum temperature for aflatoxin B₁ production by A. flavus isolate (IBA-O1) was 25 °C to 30 °C, for an incubation period of 7–11 days on plain and supplemented juice media.     

Chemical composition of Ocimum basilicum L. essential oil and its efficacy as a preservative against fungal and aflatoxin contamination of dry fruits

The study presents fungal and aflatoxin contamination of some dry fruits and Ocimum basilicum essential oil (EO) as a plant‐based preservative. During mycoflora analysis, 2045 fungal isolates were recorded from dry fruits and 40% isolates of Aspergillus flavus were toxigenic in nature. The EO of O. basilicum exhibited strong fungitoxicity against toxigenic strain of A. flavus. Its minimum inhibitory concentration (MIC) was recorded at 1.0 μL ml^?1, and it completely inhibited aflatoxin B₁ production at 0.5 μL ml^?1. The oil exhibited broad fungitoxic spectrum and considerably reduced A. flavus isolates from dry fruits when used as fumigant in closed storage containers at 1.0 μL ml^?1. The chemical profile of the EO was standardised through GC–MS analysis. Based on antifungal potency, antiaflatoxigenicity and efficacy as fumigant during storage conditions, O. basilicum EO may be recommended as a botanical preservative for enhancing the shelf life of dry fruits and edible products during storage.     

Laboratory tests for assessing efficacy of atoxigenic Aspergillus flavus strains as biocontrol agents

Biocontrol by competitive inhibition using atoxigenic Aspergillus flavus strains has been shown to be an effective method for controlling aflatoxin production in peanuts, maize and cottonseed. Selecting biocontrol strains is not straightforward, as it is difficult to assess fitness for the task without expensive field trials. Reconstruction experiments have been generally performed under laboratory conditions to investigate the biological mechanisms underlying the efficacy of atoxigenic strains in preventing aflatoxin production and/or to give a preliminary indication of strain performance when released in the field. The study here described was conducted in order to evaluate the potential of the different atoxigenic A. flavus strains, colonizing the corn fields of the Po Valley, in reducing aflatoxin accumulation when grown in mixed cultures together with atoxigenic strains; additionally, we developed a simple and inexpensive procedure that may be used to scale-up the screening process and to increase knowledge on the mechanisms interfering with mycotoxin production during co-infection.     

Effect of synthetic antioxidants on stored maize grain mycoflora in situ

BACKGROUND: The influence of a mixture of butylated hydroxyanisole (BHA) and propyl paraben (PP) (each at a concentration of 20 mmol L^?1) on mycoflora and Aspergillus section Flavi populations in stored maize grain was evaluated. A survey of 120 maize samples was carried out from June to November 2005. RESULTS: The predominant populations in non‐treated (control) maize between the first and sixth sampling periods were Aspergillus section Flavi and Penicillium. Aspergillus flavus was the fungus most frequently isolated from both control and antioxidant‐treated kernels. All samples of control and antioxidant‐treated maize kernels were negative for aflatoxins during the 6 month storage period. Aspergillus flavus and A. parasiticus strains showed a variable ability to produce aflatoxins. The contribution of the strains to silo community toxigenicity was higher for A. flavus L (large) and S (small) strains in the fourth sampling period. CONCLUSION: Antioxidant treatment negatively affected natural maize mycoflora and Aspergillus section Flavi populations between the second and sixth months of storage. Copyright © 2007 Society of Chemical Industry     

Identification of genetic defects in the atoxigenic biocontrol strain Aspergillus flavus K49 reveals the presence of a competitive recombinant group in field populations

Contamination of corn, cotton, peanuts and tree nuts by aflatoxins is a severe economic burden for growers. A current biocontrol strategy is to use non-aflatoxigenic Aspergillus flavus strains to competitively exclude field toxigenic Aspergillus species. A. flavus K49 does not produce aflatoxins and cyclopiazonic acid (CPA) and is currently being tested in corn-growing fields in Mississippi. We found that its lack of production of aflatoxins and CPA resulted from single nucleotide mutations in the polyketide synthase gene and hybrid polyketide-nonribosomal peptide synthase gene, respectively. Furthermore, based on single nucleotide polymorphisms of the aflatoxin biosynthesis omtA gene and the CPA biosynthesis dmaT gene, we conclude that K49, AF36 and previously characterized TX9-8 form a biocontrol group. These isolates appear to be derived from recombinants of typical large and small sclerotial morphotype strains. This finding provides an easy way to select future biocontrol strains from the reservoir of non-aflatoxigenic populations in agricultural fields.     

Mycotoxin production by molds isolated from ‘Greek-style’ black olives

Seventeen mold strains were isolated from ‘Greek-style’ black olives produced in Morocco. Eight of these isolates were identified as Aspergillus flavus, seven as Aspergillus petrakii, and two as Aspergillus ocharaceus Wilhelm. The A. flavus strains were tested for production of aflatoxins B₁, B₂, G₁, and G₂; and A. ochraceus and A. petrakii strains were tested for production of ochratoxin, penicillic acid, patulin, and citrinin. The organisms were tested for mycotoxin production on five different substrates, including rice powder-corn steep agar, autoclaved rice, yeast-extract sucrose broth (YES), potato dextrose agar (PDA), and fresh olive paste. All strains of A. flavus produced aflatoxins on all substrates except olive paste and PDA. In PDA, only two strains produced Aflatoxin B₁. Five A. ochraceus group isolates produced penicillic acid on one or more of the substrates, but only two out of the five produced penicillic acid on olive paste. None produced ochratoxin, patulin or citrinin. Quantities of aflatoxin B₁ produced in rice ranged from 5 to 14 μg/g of rice, and of penicillic acid 15–32 μg/g of rice. In olive paste, the concentrations of penicillic acid were 11.4 and 30.2 μg/g. Biological toxicity of extracts of mold cultures was confirmed using chicken embryos and a microbiological test. Crude extracts of cultures were also tested for mutagenicity using the Salmonella mutagenicity (Ames) Test, and some gave positive mutagenic responses.     

Delivery systems for biological control agents to manage aflatoxin contamination of pre-harvest maize

While soil application of a competitive non-toxigenic Aspergillus flavus strains is successful in reducing aflatoxin contamination in certain crops, direct application to aerial reproductive structures could be more effective for maize. A sprayable, clay-based water-dispersible granule formulation was developed to deliver non-toxigenic A. flavus strain K49 directly to maize ears. The efficacy of the K49 water-dispersible granule in mitigating aflatoxin in maize (Zea mays L.) was evaluated. Field studies were conducted to compare K49 colonization and effectiveness in reducing aflatoxin contamination when applied either as a soil inoculant or as a directed spray in plots infested with toxigenic strain F3W4. Fifty percent of non-toxigenic A. flavus was recovered from non-treated controls and from plots soil inoculated with K49 on wheat. In spray treatments with formulated or unformulated K49 conidia, over 90% of A. flavus recovered was non-toxigenic. Soil-applied K49 reduced aflatoxin contamination by 65% and spray applications reduced contamination by 97%. These findings suggest direct spray application of non-toxigenic A. flavus strains may be better than soil inoculation at controlling maize aflatoxin contamination and that a water-dispersible granule is a viable delivery system for maintaining viability and efficacy of the biological control agent, K49.     

Potential of aflatoxin B1 production by Aspergillus flavus strains on commercially important food grains

In this study, we investigated the potential of aflatoxin B₁ (AFB1) production by five Aspergillus flavus strains previously isolated from sorghum grains on cereals (barley, maize, rice, wheat and sorghum), oilseeds (peanuts and sesame) and pulses (greengram and horsegram). Five strains of A. flavus were inoculated on all food grains and incubated at 25 °C for 7 days; AFB1 was extracted and estimated by enzyme‐linked immunosorbent assay. All A. flavus strains produced AFB1 on all food grains ranging from 245.4 to 15 645.2 μg kg^?1. Of the five strains tested, strain Af 003 produced the highest amount of AFB1 on all commodities ranging from 2245.2 to 15 645.2 μg kg^?1. Comparatively, the AFB1 accumulation was high on rice grains ranging from 3125.2 to 15 645.2 μg kg^?1, followed by peanuts ranging from 2206.2 to 12 466.5 μg kg^?1. Less AFB1 accumulation was observed in greengram and sesame seeds ranging from 645.8 to 2245.2 and 245.4 to 2890.6 μg kg^?1, respectively. Our results showed that all food grains tested are susceptible to A. flavus growth and subsequent AFB1 production.     

Phenolic compounds in peanut seeds: Enhanced elicitation by chitosan and effects on growth and aflatoxin B1 production by Aspergillus flavus

Effects of chitosan and Aspergillus flavus to enhance elicitation of phenolic compounds in viable peanut seeds were conducted at two water activity levels. In vitro effects of phenolic acids on A. flavus growth and aflatoxin B₁ production were also studied. Chitosan enhanced elicitation of free phenolic compounds (FPC) at Aw .85 and .95 levels. A. flavus initially decreased and subsequently increased FPC content, but bound phenolic compounds (BPC) decreased during incubation. Chitosan + A. flavus treatment caused an increase in FPC reaching a plateau between 24–48 h at Aw .85 while BPC levels increased over the same period at both Aw levels. Major free and bound phenolic acids detected were p‐coumaric, ferulic and an unknown phenolic acid eluting at a retention time of 22 min. Generally, chitosan significantly enhanced elicitation of free ferulic and p‐coumaric acids and bound p‐coumaric acid at Aw .95. Free unknown phenolic and bound ferulic acids at Aw .85 were enhanced by chitosan. A. flavus caused significant induction of bound p‐coumaric and ferulic acids and free unknown phenol at Aw .85. Chitosan + A. flavus enhanced free p‐coumaric (3 h) and unknown phenolic acids and bound p‐coumaric acid at Aw .95 while bound ferulic acid was enhanced at Aw .85. Chitosan limited A. flavus growth and subsequent aflatoxin production by inducing susceptible tissues to produce more preformed phenolic compounds.

Analysis of liquid cultures of A. flavus revealed that p‐coumaric, ferulic, and vanillic acids and a mixture of these phenolic acids slightly inhibited mycelial growth. Production of aflatoxin B₁ by A. flavus was completely inhibited at 1 mM and 10 mM concentrations of the phenolic acids and their mixture on four days of incubation. Mode of action of phenolic acids is likely on the secondary pathway for aflatoxin B₁ production and not on the primary metabolism for fungal growth.     

Assessment of Pelargonium graveolens oil as plant‐based antimicrobial and aflatoxin suppressor in food preservation

BACKGROUND: Contamination of stored food commodities by moulds and mycotoxins results in qualitative as well as quantitative losses. Most of the synthetic antimicrobials used for preservation of stored food items produce side effects in the form of residual and mammalian toxicity. Recently some higher plant products have been recommended as safe alternatives of such synthetic antimicrobials. In the present investigation antifungal efficacy of some essential oils was evaluated against two toxigenic strains of Aspergillus flavus with special reference to the oil of Pelargonium graveolens to investigate its potential to inhibit aflatoxin B₁ secretion. RESULTS: Essential oil of P. graveolens exhibited absolute fungitoxicity against both the toxigenic strains of A. flavus. The minimum inhibitory concentration of the oil was found to be 0.75 g L^?1 and exhibited a fungistatic nature. It was found superior over the synthetic fungicides tested and exhibited a broad fungitoxic spectrum. The oil showed excellent anti‐aflatoxigenic efficacy as it completely inhibited aflatoxin B₁ production even at 0.50 g L^?1. CONCLUSION: This is the first report on the aflatoxin B₁ inhibitory nature of P. graveolens oil. It may be recommended as a novel plant‐based antimicrobial as well as aflatoxin B₁ suppressor over synthetic preservatives in food protection. Copyright © 2008 Society of Chemical Industry     

Moisture-equilibrium relative humidity relationships in pistachio nuts with particular regard to control of aflatoxin formation

Adsorption and desorption isotherms have been determined both manometrically and by weight equilibration for Turkish pistachio nut kernel, shell and hull. Comparison of the calculated and experimentally determined adsorption isotherms for whole nuts showed good correlation. Nuts inoculated with Aspergillus flavus conidia were equilibrated to various ERH levels and stored in controlled environment cabinets at 28°C. Competitive growth of xerophilic strains of A. amstelodami prevented growth and aflatoxin production by the A. flavus at ERHs of 86% and below. At 88% ERH marked aflatoxin production occurred but competition was observed between the A. flavus and A. niger. In sealed containers metabolic moisture from growth of A. amstelodami raised the ERH from the initial 85% and permitted toxin production by A. flavus. The results are discussed in relation to post-harvest handling and storage of pistachio nuts.     

Phenolic compounds in peanut seeds: Enhanced elicitation by chitosan and effects on growth and aflatoxin b1 production by aspergillus flavus

Effects of chitosan and Aspergillus flavus to enhance elicitation of phenolic compounds in viable peanut seeds were conducted at two water activity levels. In vitro effects of phenolic acids on A. flavus growth and aflatoxin B₁ production were also studied. Chitosan enhanced elicitation of free phenolic compounds (FPC) at Aw .85 and .95 levels. A. flavus treatment initially decreased and subsequently increased FPC content, but bound phenolic compounds (BPC) decreased during incubation. Chitosan + A. flavus treatments caused an increase in FPC that reached a plateau between 24–48 h at Aw .85 while BPC levels increased over the same time period at both Aw levels. The major free and bound phenolic acids detected were p‐coumaric and ferulic acids and an unknown phenol that eluted at a retention time of 22 min. Generally, chitosan treatment significantly enhanced elicitation of free ferulic and p‐coumaric acids and bound p‐coumaric acid at Aw .95. Free unknown phenolic and bound ferulic acids at Aw .85 were enhanced by chitosan. A. flavus treatment caused significant induction of bound p‐coumaric and ferulic acids and free unknown phenol at Aw .85. Chitosan + A. flavus treatment measure to reduce or eliminate pre‐harvest contamination by A. flavus and aflatoxins contributes to sustainable agriculture, especially to developing countries.

The enhanced elicitation of preformed phenolic compounds by chitosan may provide seed tissues an additive or synergistic effect in controlling aflatoxin‐producing fungi and preventing aflatoxin contamination. Further, such investigation will help elucidate the biochemical basis of elicitor‐host interaction that contribute to defensive responses of host tissues. Identification of biochemical factors in induced resistance involves a refinement in the separation and identification of induced phenolic compounds. Methodologies such as spectrophotometric assay or reverse‐phase high performance liquid chromatography (HPLC) may be used to evaluate phenolic compound induction by these elicitors. In addition, these compounds can be tested on their effects on A. flavus mycelial growth and subsequent aflatoxin production in vitro.

Hence, a study on the possible role of phenols on the natural resistance of peanuts to A. flavus invasion was conducted with the following objectives: 1) to quantitate changes in free and bound phenolic compounds influenced by chitosan, A. flavus, and water activity (Aw) levels by Folin‐Ciocalteu assay; 2) to separate, identify, and quantitate free and bound phenolic acids influenced by elicitors and Aw levels; and 3) to determine the effects of phenolic acids in liquid cultures at different concentrations on mycelial growth and aflatoxin B₁ production by A. flavus.     

Aflatoxin-producing potential of Aspergillus flavus and Aspergillus parasiticus isolated from samples of smoked-dried meat

Over a period of three years 420 samples of various smoke-dried meat products, collected from individual households in different region of Croatia were analysed for the presence of aflatoxigenic strains of the Aspergillus flavus group. Strains of A. flavus and A. parasiticus were present in 17,8% of the samples, and aflatoxin-producing ability was tested in 75 strains. In relation to sequential method of aflatoxin detection, 5 of 8 isolates were found in the first step (fluorescence in aflatoxin-producing ability medium - APA) and all of them in the second step (extraction method from syntheses on moist shredded wheat - SW). A. flavus strains produced mainly aflatoxin B₁, and had various levels of toxigenicity (1.4–3.12 mg/kg). Some strains of A. parasiticus produced all four aflatoxins B₁ B₂ G₁ G₂, while the other ones produced AF B₁ + G₁ only, with concentrations of aflatoxins from 0.1 to 450 mg/kg.     

Effect of γ‐irradiation on aflatoxin B1 production by Aspergillus flavus and chemical composition of three crop seeds

The effect of γ‐irradiation on aflatoxin B₁ production by Aspergillus flavus, and the chemical composition of some different crop seeds were investigated. A. flavus infected seeds behaved differently according to their principal constituents. A. flavus caused an increase in protein and decrease in lipids and carbohydrate contents of wheat, soyabean and fababean seeds. Growth of A. flavus and production of aflatoxin B₁ was inhibited at a dose level of 5 kGy. A. flavus utilizes carbohydrates of seeds for its growth and aflatoxin production. Crops were arranged, in descending order, according to aflatoxin produced in seeds as wheat > soyabean > fababean. There were no changes in chemical constituents of irradiated seeds, such as protein, lipids, and carbohydrates.     

Molecular characterization of atoxigenic strains for biological control of aflatoxins in Nigeria

Aflatoxins are highly toxic carcinogens produced by several species in Aspergillus section Flavi. Strains of A. flavus that do not produce aflatoxins, called atoxigenic strains, have been used commercially in North America as tools for limiting aflatoxin contamination. A similar aflatoxin management strategy is being pursued in Nigeria. In the current study, loci across the 68 kb aflatoxin biosynthesis gene cluster were compared among 18 atoxigenic and two aflatoxin-producing vegetative compatibility groups (VCGs) from Nigeria and an atoxigenic VCG used commercially in North America. Five of the atoxigenic VCGs had large deletions (37–65 kb) extending from the teleomeric side of the aflatoxin biosynthesis cluster. In one VCG (AV0222) the deletion extended through the cluster to the adjacent sugar cluster. The remaining twelve atoxigenic VCGs, including the VCG used for aflatoxin management in North America, contained all the aflatoxin pathway genes, but with defects. Two observations support the long-term persistence of atoxigenicity within A. flavus: first, a comparison of pathway genes revealed more changes in atoxigenic than in aflatoxin-producing isolates relative to the aflatoxin-producing strain NRRL 3357; and second, several non-synonymous changes are unique to atoxigenics. Atoxigenic VCG diversity was assessed with phylogenetic analyses. Although some atoxigenics share relatively recent ancestry, several are more closely related to aflatoxin producers than to other atoxigenics. The current study demonstrates VCGs of A. flavus in West Africa with diverse mechanisms of atoxigenicity and potential value in aflatoxin management programmes.