Germination and post-germination development of Colletotrichum dematium f. circinans on Allium cepa

p6e production of aflatoxins by Aspergillus flavus SRRC-1000 growing on soybean (Forrest) and glandless cottonseed (Deltapine 16) meals was examined with respect to effects of zinc and phytate. Aflatoxins were not produced on unautoclaved soybean meal. Addition of zinc (as zinc sulfate) to autoclaved meal inhibited aflatoxin production and supplementation with sodium phytate relieved this inhibition. Addition of sodium phytate alone promoted production. When cottonseed meal was treated to release native phytate into the meal from phytate-sequestering globoids, aflatoxin production increased. However, the largest production on cottonseed meal occurred upon dialysis of the meal without releasing phytate, implying removal of a small molecular weight inhibitor.     

Effect of phytate on aflatoxin formation by Aspergillus parasiticus and Aspergillus flavus in synthetic media

The effect of phytate on the production of aflatoxins by Aspergillus parasiticus and Aspergillus flavus grown on synthetic media was examined. In the absence of pH control (initial pH 4.5–6.5) for A. parasiticus, phytate (14.3 mM) caused a six-fold decrease in aflatoxins in the medium and a ten-fold decrease in those retained by the mycelia. When the initial pH of the medium was adjusted to 4.5 no effect on aflatoxin production was observed. With A. flavus or A. parasiticus grown on media with a higher initial pH value (6 to 7), the presence of phytate in the media caused an increase in aflatoxin production. These results are inconsistent with previous studies which indicated that phytate depresses aflatoxin production by rendering zinc, a necessary co-factor for aflatoxin biosynthesis, unavailable to the mold.     

Incidence of aflatoxins and aflatoxin producing fungi in animal feedstuffs

Fifty four samples including 5 of broken rice, 8 of corn grains, 8 of corn gluten feed, 13 of cottonseed cake and 4 each of rice polish, corn gluten, sesame oil cake, guar meal and wheat bran were screened for the presence of aflatoxins. Among all the samples, 14 were damaged and 40 apparently undamaged. The incidende of aflatoxins was found to be 60, 25, 25, and 23 per cent in broken rice, corn grains, corn gluten feed and cottonseed cake. Aflatoxins were not detected from rice polish, corn gluten, sesame oil cake, guar meal and wheat bran. Damaged sample revealed a much higher incidence i.e. 50 per cent as compared to undamaged ones i.e. 7.5 per cent. Mean concentration of aflatoxin B and G was found to be 15.5 and 12.2 ppb respectively.Cultural examination of aflatoxin positive feedstuffs yielded 39 isolates of different fungi including 21 of Aspergillus, 7 of Mucor, 6 of Rhizopus, 4 of Fusarium and one of Penicillium. These strains when tested for aflatoxin producing ability, revealed this property in only one isolate, identified as Aspergillus parasiticus.     

Incidence of aflatoxin producing strains and aflatoxin contamination in dry fruit slices of quinces (Cydonia oblonga Mill.) from the Indian State of Jammu and Kashmir

An investigation was undertaken to obtain data on the occurrence of aflatoxins and the aflatoxin producing potential of Aspergillus flavus strains isolated from dry fruit slices of quinces produced in jammu and Kashmir, India. A total of 147 A. flavus isolates recovered from dr fruit slices were grown in liquid rice flour medium and screened for the production of various aflatoxins by thin layer chromatography. The results showed that 23.14% of the tested isolates were aflatoxigenic, producing aflatoxins B₁and B₂ in varying amounts. Aflatoxins G₁ and G₂ were not detected. All 25 of the investigated market samples were also found to be aflatoxin B₁ positive and the level of contamination ranged from 96 to 8164 g/kg of the dry fruit which is quite high in comparison to the permissible level of 30 ppb. As per these results biochemical composition of dry fruit slices of quinces, along with climatic conditions seem to be very favourable for aflatoxin production by the toxigenic A. flavus strains. Therefore,monitoring of aflatoxins in dry fruit slices of quincesis recommended for this region.This revised version was published online in October 2005 with corrections to the Cover Date.     

Potential pathogenicity of Cladosporium tennuisimum,Phaeoisaria clematidis and Ramichloridium subulatum in a mouse model

In Sri Lanka, rice is the main staple which is mostly processed into parboiled rice. The levels of aflatoxin B₁ (AFB₁) and aflatoxin G₁ (AFG₁) in parboiled and raw milled rice collected from major rice producing areas and rice consuming townships were estimated. In almost all the samples of parboiled rice examined, the AFB₁ and AFG₁ contents were significantly higher than in raw milled rice. The highest AFB₁ content was 185 µg/kg and AFG₁ content 963 g/kg. These samples were collected from a major rice producing/milling district where the mean relative humidity is 78% and mean annual temperature 27 °C which is the highest amongst the rice growing areas in Sri Lanka. Raw rice was either free of aflatoxins or when toxins were detected, they occurred in less than 10% of the samples. The frequency of occurrence of surface fungal flora (Aspergillus/Penicillium) and aflatoxin content in market samples was closely related. Brownish or greenish moldy rice samples with fermented odour contained over 1000 g/kg of AFB₁.     

Effect of sucrose supplementation on aflatoxin production by Aspergillus parasiticus 2999 cultures in soybeans and cashew fruit juice

The aflatoxigenic potential ofAspergillus parasiticus 2999 on soybeans (raw and cooked) and cashew fruit juice (ripe, unripe, raw and cooked) variously supplemented with sucrose (0–20g) has been evaluated. Aflatoxin production showed a dual increase with increasing sucrose (0–20g) and soybeans (0.1–2.0g) concentrations which probably indicates the limited availability of suitable carbon sources in soybeans. This may be partly responsible for its resistance to aflatoxin synthesis. Two to five per cent (w/v) sucrose supplementation was optimal for maximal toxin production in cashew juice. Above this range aflatoxin production dropped steeply. Cooked soybeans supported higher yields of toxin than raw, in direct contrast with cashew fruit juice. Ripe cashew juice produced a greater quantity of toxin than the unripe. More fluorescent metabolites were synthesized in cashew fruit media than in soybeans. These results have been discussed in relation to the limiting role of the carbon source and the resistance to aflatoxin production on natural substrates.     

Phytase production byAspergillus ficuum on semisolid substrate

Summary Phytase production byAspergillus ficuum was studied using solid state cultivation on several cereal grains and legume seeds. The microbial phytase was used to hydrolyze the phytate in soybean meal and cotton seed meal. Wheat bran, soybean meal, cottonseed meal and corn meal supported good fungal growth and yielded a high level of phytase when an adequate amount of moisture was present. The level of phytase production on solid substrate was higher than that obtained by submerged liquid fermentation. Higher levels of phosphorus (more than 10 mg Pi/100 g substrate) in the growth medium (static culture) inhibited phytase synthesis, and the degree of phosphorus inhibition was less apparent in semisolid medium than in liquid medium. A static cultivation on semisolid substrate produced a higher level of phytase (2-20-fold) than that obtained by agitated cultivation. The minimal amount of water required for growth and enzyme production on those substrates was about 15%, while the optimum level for phytase production was between 25 and 35% and that for cell growth was above 50%. Optimum pH for phytase production was between 4 and 6.A ficuum grew well on raw (unheated) substrate containing a minimal amount of water and produced as much phytase as on heated substrate. About half of the phytic acid in soybean meal and cottonseed meal was hydrolyzed by treatment withA. ficuum phytase.     

Factors influencing the inhibition of aflatoxin production in corn by Aspergillus niger

Aspergillus niger, a mold commonly associated with Aspergillus flavus in damaged corn, interferes with the production of aflatoxin when grown with A. flavus on autoclaved corn. The pH of corn-meal disks was adjusted using NaOH-HCl, citric acid-sodium citrate, or a water extract of A. niger fermented corn. Aflatoxin formation was completely inhibited below pH 2.8-3.0, irrespective of the system used for pH adjustment. When grown in association with A. flavus NRRL 6432 on autoclaved corn kernels, A. niger NRRL 6411 lowered substrate pH sufficiently to suppress aflatoxin production. The biodegradation of aflatoxin B1 or its conversion to aflatoxin B2a were eliminated as potential mechanisms by which A. niger reduces aflatoxin contamination. A water extract of corn kernels fermented with A. niger caused an additional inhibition of aflatoxin formation apart from the effects of pH.     

Production of Aflatoxin on Soybeans

Probable factors influencing resistance to aflatoxin synthesis in soybeans have been investigated by using cultures of Aspergillus parasiticus NRRL 3240. Soybeans contain a small amount of zinc (0.01 mug/g) bound to phytic acid. Autoclaving soybeans at 15 pounds (6803.88 g) for 15 min increases the aflatoxin production, probably by making zinc available. Addition of zinc to both autoclaved and nonautoclaved soybeans promotes aflatoxin production. However, addition of varying levels of phytic acid at a constant concentration of zinc depresses aflatoxin synthesis with an increase in the added phytic acid. In a synthetic medium known to give good yields of aflatoxin, the addition of phytic acid (10 mM) decreases aflatoxin synthesis.     

Survey of Vietnamese Peanuts,Corn and Soil for the Presence of <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis>

Aspergillus flavus and Aspergillus parasiticus cause perennial infection of agriculturally important crops in tropical and subtropical areas. Invasion of crops by these fungi may result in contamination of food and feed by potent carcinogenic aflatoxins. Consumption of aflatoxin contaminated foods is a recognised risk factor for human hepatocellular carcinoma (HCC) and may contribute to the high incidence of HCC in Southeast Asia. This study conducted a survey of Vietnamese crops (peanuts and corn) and soil for the presence of aflatoxigenic fungi and used microsatellite markers to investigate the genetic diversity of Vietnamese Aspergillus strains. From a total of 85 samples comprising peanut (25), corn (45) and soil (15), 106 strains were isolated. Identification of strains by colony morphology and aflatoxin production found all Vietnamese strains to be A. flavus with no A. parasiticus isolated. A. flavus was present in 36.0% of peanut samples, 31.1% of corn samples, 27.3% of farmed soil samples and was not found in virgin soil samples. Twenty-five per cent of the strains produced aflatoxins. Microsatellite analysis revealed a high level of genetic diversity in the Vietnamese A. flavus population. Clustering, based on microsatellite genotype, was unrelated to aflatoxin production, geographic origin or substrate origin.     

Phytate dephosphorylation by free and immobilized cells ofSaccharomyces cerevisiae

Summary Saccharomyces cerevisiae in the form of baker's yeast, cells cultivated on a yeast extract-peptone-glucose medium, as well as cells immobilized in 18% (w/v) polyacrylamide gel showed the ability to hydrolyze 1.727 mM sodium phytate solution at 45°C, pH 4.6, in a stirred tank reactor. Seventy percent yield of dephosphorylation was observed after 2 h using a baker's yeast concentration of 5.8 g dry matter per 100 ml. Hydrolytic activity at 1.8–2.0 M Pi min^–1 was observed between 1st and 3rd h of the reaction in cells cultured 24 or 48 h. No inhibition by the substrate was found at sodium phytate concentrations of 0.587–1.727 mM. After 1.5 h of hydrolysis a single, well distinguished peak ofmyo-inositol-triphosphate was the main product found. By means of immobilization the stability of the biocatalyst was enhanced 3.3-fold and reached its half-life at 64 ninety-minute runs.     

Effects of Moisture Content and Temperature on Aflatoxin Production in Corn

Samples of freshly harvested and remoistened corn, of various moisture contents, were stored at different temperatures; analyses for aflatoxin content were made periodically. At moisture levels above 17.5% and at temperatures of 24 C or warmer, aflatoxins were formed by Aspergillus flavus present in the original epiphytic mycoflora. Remoistened dried corn was subject to more rapid fungal deterioration and aflatoxin formation than freshly harvested corn. Screening of the fungi present in the corn revealed aflatoxin production only by A. flavus. The toxigenic strains produced only aflatoxins B(1) and B(2).     

Inhibitory effect of hena (Lawsonia inermis leaves) and carrot root on aflatoxin production byAspergillus parasiticus

Experiments were undertaken to evaluate the effect of some natural products (hena, and carrot root) on growth and aflatoxins production byAspergillus parasiticus FRR 2752. Powdered hena (0.5 and 5%) inhibited mycelial growth and delayed 1 sporulation ofA parasiticus during 7 days. The inhibition of growth was increased with increasing the added amount. Aflatoxins production byA parasiticus was reduced with 40–100% in the presence of hena (Lawsonia inermis leaves). Carrot root extract stimulated the fungal growth and aflatoxin production, whereas carrot root fibers slightly enriched fungal growth, inhibited aflatoxins production (B₁, G₁, and G₂), but there was no inhibition of aflatoxin B₂ production byA parasiticus.     

β-Carotene inhibition of aflatoxin biosynthesis amongAspergillus flavus genotypes from Illinois corn

Thirty-nineAspergillus flavus genotypes (DNA fingerprinting) isolated from corn grown in a field near Kilbourne, Illinois were evaluated for their sensitivity to β-carotene (50 μg/ml) inhibition of aflatoxin B₁ biosynthesis. Inhibition of aflatoxin was greater than 90% for 28 of the genotypes and >70% for 38 of the 39 genotypes. FiveA. flavus strains (4 fingerprint groups) isolated from molded raw peanuts, NRRL 3239, NRRL 3357, NRRL 6514, NRRL 6515 and NRRL 13135, produced greater quantities of aflatoxin than all 39 genotypes isolated from corn, and were less sensitive to β-carotene inhibition.Aspergillus flavus NRRL 3357 is commonly used as inoculum in variety trials for aflatoxin resistance. Isolate identity and sensitivity to potential inhibitors in corn can be critical in assessing corn resistance to aflatoxin.     

The Capacity of Manno-Oligosaccharides,Thermolysed Yeast and Active Yeast to Attenuate Aflatoxicosis

This study assessed the ability of thermolysed, active yeast, Saccharomyces cerevisiae, and manno-oligosaccharides, to reduce the effects of aflatoxins in animals. A basic ration was developed with six different formulations. Each formulation was considered a treatment. The treatments were: an aflatoxin-free formulation, an aflatoxin control (400 g kg^–1) and four aflatoxin-supplemented formulations. The supplements were 0.1 and 0.2% manno-oligosaccharides, 1% thermolysed yeast and 1% dehydrated active yeast. The experiment was randomly designed and had five repetitions per treatment. The feed was contaminated with aflatoxins from naturally contaminated peanuts. A bioassay with Wistar rats was conducted after 28 days. The aflatoxin toxicity was evaluated by weighing body organs (heart, kidneys and liver) and by analysing the liver tissue of the animals. No significant differences were observed for the weights of the body organs from the animals fed with the different rations. However, the analysis of the liver tissue showed animals fed with 400 g of aflatoxin kg^–1, and those fed diets with aflatoxin amended with either manno-oligosaccharides or with thermolysed yeast had clear signs of toxicity and damage, while those fed with dehydrated active yeast showed less intense toxicity and less liver damage. Therefore, the thermolysed yeast and manno-oligosaccharides did not suppress damage to liver tissue caused by aflatoxins, while active yeast reduced the aflatoxin symptoms in the hepatocytes.     

Aflatoxin is degraded by mycelia from toxigenic and nontoxigenic strains of aspergilli grown on different substrates

The ability of 9-day-old mycelia of Aspergillus parasiticus NRRL 2999 to degrade aflatoxin varied depending on the substrate used to grow the mold. Substrates which allowed substantial mycelial growth yielded mycelia which actively degraded aflatoxin. Substrates which allowed minimal growth of mycelia yielded mycelia with little ability to degrade aflatoxin. Biodegradation of aflatoxin was also strain-dependent. A. parasiticus NRRL 2999 and NRRL 3000 actively degraded aflatoxin, A. flavus NRRL 3353 was less active, and A. flavus NRRL 482 and A. parasiticus NRRL 3315 degraded minimal amounts of aflatoxins. Those aspergilli producing greatest amounts of aflatoxin also degraded aflatoxins most rapidly, whereas those strains which produced minimal amounts of aflatoxin generally degraded aflatoxins less effectively. Substrates which allowed maximum aflatoxin production also yielded mycelia which actively degraded aflatoxins, whereas media which allowed limited production of aflatoxin generally yielded mycelia with minimal ability to degrade the toxin. Although exceptions exist, generally as aflatoxin production increased so did the ability of mycelia to degrade the toxin.     

Cyclopiazonic acid production by aflatoxigenic and non-aflatoxigenic strains of aspergillus flavus

Twenty-eight of 54 isolates of Aspergillus flavus grown on autoclaved agricultural commodities such as wheat, rice and corn were found to produce the mycotoxin cyclopiazonic acid. Eighteen of the A. flavus isolates produced aflatoxin, and fourteen isolates produced both cyclopiazonic acid and aflatoxin. A preliminary screening of some aflatoxin-contaminated corn samples revealed for the first time the natural occurrence of cyclopiazonic acid in agricultural commodities.     

Effect of selected inhibitors on growth,pigmentation, and aflatoxin production by Aspergillus parasiticus

We have treated a wild type strain of Aspergillus parasiticus with several known aflatoxin inhibitors in hopes of finding specific metabolic blocks in the aflatoxin biosynthetic pathway. In defined medium, benzole acid (2 and 3 mg/ml), cinnamon (1 mg/ml), and sodium acetate (5 mg/ml) were fungitoxic. Benzoic acid (0.5 and 1 mg/ml), chlorox (5 l/ml), and dimethyl sulfoxide (5 l/ml) did not affect dry weight or mycelial pigmentation. Sodium benzoate (1, 2, 4 and 8 mg/ml) added after 2 days growth inhibited aflatoxin production in two defined media. We were unable to confirm previously published reports that an uncharacterized yellow pigment accumulates with benzoate-inhibition of aflatoxin biosynthesis.     

Effects of various acids and salts on growth and aflatoxin production by Aspergillus flavus NRRL 3145.

The effects of sodium chloride, sodium acetate, benzoic acid, sodium benzoate, malonic acid, and sodium malonate on growth and aflatoxin production by Aspergillus flavus were investigated in synthetic media. Sodium chloride at concentrations equivalent to or greater than 12 g/100 ml inhibited growth and aflatoxin production, while at 8 g or less/100 ml, growth and aflatoxin production were stimulated. At 2 g or less/100 ml, sodium acetate also stimulated growth and aflatoxin production, but reduction occurred with 4 g or more/100 ml. Malonic acid at 10, 20, 40, and 50 mM reduced growth and aflatoxin production (over 50%) while sodium malonate at similar concentrations but different pH values had the opposite effect. Benzoic acid (pH 3.9) and sodium benzoate (pH 5.0) at 0.4 g/100 ml completely inhibited growth and aflatoxin production. Examination of the effect of initial pH indicated that the extent of inhibitory action of malonic acid and sodium acetate was a function of initial pH. The inhibitory action of benzoic acid and sodium benzoate appeared to be a function of undissociated benzoic acid molecules. Aflatoxin reduction was usually accompanied by an unidentified orange pigment, while aflatoxin stimulation was accompanied by unidentified blue and green fluorescent spots but with lower Rf values that aflatoxins B1, G1, B2, and G2 standards.     

Aflatoxins: Production of the Toxins In Vitro in Relation to Temperature

The production or accumulation of aflatoxins in vitro by four isolates on three substrates (acid-delinted cottonseed, shelled Spanish peanut, and rough rice) was studied in relation to temperature in the range of 10 to 40 C. Within the first 10 days after inoculation, the optimal temperature range for aflatoxin production was between 20 and 35 C. Only small amounts of the toxins were produced at 10 and 40 C. Within the optimal temperature range, the time required for toxin production and for significant accumulation decreased as the temperature increased. More aflatoxin G was produced or accumulated in relation to aflatoxin B at low temperatures (within the optimal range), and the G aflatoxins were metabolized more rapidly at the higher temperatures.