The effect of the fungicides tridemorph, fenpropimorph and fenarimol on growth and aflatoxin production by Aspergillus parasiticus Speare

The effect of three systemic fungicides, tridemorph, fenpropimorph and fenarimol, on growth and aflatoxin production by Aspergillus parasiticus was studied in a chemically defined medium. Each compound inhibited growth and at the same time gave increased information of aflatoxin. Fenarimol, which is considered to be an inhibitor of cytochrome P₄₅₀, not only affects total aflatoxin production but may also alter the ratio of aflatoxin B1 to G1 in the culture filtrate.     

Effect of Corn Steep Liquor on Mycelial Growth and Aflatoxin Production in Aspergillus parasiticus

Total aflatoxin concentrations produced by Aspergillus parasiticus, isolate 64-R8, in Czapek's broth fortified with corn steep liquor increased proportionately as the concentration of corn steep was increased from 0.5 to 8.0% (v/v) until maximal growth, as measured by dry mycelial weight, was reached. Thereafter, aflatoxin concentrations declined more rapidly than the rate of autolysis of mycelial material. Data are presented which indicate that the concentration of corn steep liquor also affects the ratio of production of aflatoxin B(1) and B(2) to that of aflatoxin G(1) and G(2). Further, this ratio also varies with time of incubation. Although both growth of the fungus and aflatoxin production are stimulated by the addition of corn steep to the basic medium, the stimulation of toxin production is much greater than fungus growth.     

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.     

The effect of aqueous plant extracts on growth and aflatoxin production by Aspergillus flavus

The effect of aqueous leaf extracts of four plants, Argemone mexicana, Cyperus rotundus, Euphorbia hirta and Solanum nigrum , on growth and aflatoxin production by Aspergillus flavus was studied in SMKY liquid medium. All the plants inhibited aflatoxin production. No correlation between the growth of the fungus and aflatoxin synthesis was observed. The influence of these plants on the ratio of aflatoxin B₁ to G₁ is discussed.     

Control of Aspergillus growth and aflatoxin production using antioxidants at different conditions of water activity and pH

AIMS: The effect of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), trihydroxybutyrophenone (THB) and propyl paraben (PP) (at concentrations of 1, 10 and 20 mmol l(-1)) on germination, growth and aflatoxin B1 production by Aspergillus section Flavi was evaluated. METHODS AND RESULTS: Studies on the percentage of spore germination, elongation rate, growth rate and aflatoxin B1 production were carried out in vitro in relation to water activity (aw) at 0.982, 0.937, 0.809 and 0.747 values. At 0.809 and 0.747aw values none of the isolates was able to germinate. Overall, PP and BHA were the antioxidants most effective at inhibiting germination of both species. In the presence of the lowest concentration of BHA and PP (1 mmol l(-1)) the conidial germination percentage ranged from 2 to 19% after 15 h of incubation at the highest water activity tested. BHA and PP at 10-20 mmol l(-1) completely inhibited conidial germination. The antioxidants more efficient in controlling Aspergillus elongation rate were PP, BHT and BHA. All strains were much more sensitive to all antioxidants tested on the percentage of spore germination and growth rate at 0.937aw. The antioxidants PP and BHA completely inhibited aflatoxin B1 production by all strains when added at 1 mmol l(-1). Decreased aflatoxin B1 levels in comparison with the control, were observed with BHT at 1, 10 and 20 mmol(-1) with the strain T20 at 0.982aw. In contrast, stimulation was observed with the antioxidant THB at 10 and 20 mmol l(-1) at 0.937aw with the strains T20 and T23. The effect of BHA and PP at 1 mmol l(-1) on lag phase and growth rate was maintained in the pH range between 6 and 8. At all pH values the inhibitory effect of BHA was higher than PP. No aflatoxin B1 was detected at all pH values. CONCLUSIONS: The data show that BHA and PP could be considered as effective fungitoxicants for A. flavus and A. parasiticus. SIGNIFICANCE AND IMPACT OF THE STUDY: The information obtained show promise for controlling growth and aflatoxin B1 in stored maize. Futher studies should be carried out to examine the potential for antioxidants, such as BHA and PP to effectively control both growth and aflatoxin production.     

Inhibitory effect of molybdenum and vanadium salts on aflatoxin B1 synthesis by Aspergillus flavus

The effects of the elements zinc, manganese, iron, copper, molybdenum, and vanadium, added in various salt forms, on mycelial weights and aflatoxin B1 accumulation in the mycelium of Aspergillus flavus were investigated in liquid shake cultures. Ammonium heptamolybdate, when added to a complete medium at concentrations of 50-100 mg/L, appreciably reduced aflatoxin B1 accumulation without affecting growth of the fungus. Sodium molybdate and sodium monovanadate also reduced aflatoxin B1 yields without affecting mycelial growth but to a lesser extent. The addition of zinc sulphate stimulated aflatoxin B1 production in all media used. The influence of the other trace elements on aflatoxin production depended on the level of trace elements present in the basal medium. In general, manganese chloride had a stimulatory effect, whereas copper sulphate depressed yields. Mycelial levels of aflatoxin had peaked and then declined before mycelial dry weights had reached maximum. High yields of aflatoxin B1 were obtained in media having a final pH as low as pH 2.8.     

Action of phosphine on production of aflatoxins by various Aspergillus strains isolated from foodstuffs.

Phosphine is a food fumigant, used until now as an insecticide and rodenticide. The present work researches the action of phosphine treatment on growth and aflatoxin production of 23 Aspergillus strains. Production of aflatoxins B1, B2, G1, and G2 decreased in almost all cases by a ratio of 10 to 100. Phosphine treatment therefore seems favorable to prevent growth of various Aspergillus strains, in the context of keeping food safe.     

Action of phosphine on production of aflatoxins by various Aspergillus strains isolated from foodstuffs

Phosphine is a food fumigant, used until now as an insecticide and rodenticide. The present work researches the action of phosphine treatment on growth and aflatoxin production of 23 Aspergillus strains. Production of aflatoxins B1, B2, G1, and G2 decreased in almost all cases by a ratio of 10 to 100. Phosphine treatment therefore seems favorable to prevent growth of various Aspergillus strains, in the context of keeping food safe.     

Effect of initial pH on aflatoxin production.

The effect of initial pH on aflatoxin production by Aspergillus parasiticus NRRL 2999 was examined in a semisynthetic medium. Maximal growth, aflatoxin production, and aflatoxin production per unit of growth occurred at initial pH levels of 5.0, 6.0, and 7.0 respectively. Initial pH levels less than pH 6.0 favored production of the B toxins, whereas levels greater than pH 6.0 favored production of the G toxins.     

Sodium bicarbonate reduces viability and alters aflatoxin distribution of Aspergillus parasiticus in Czapek's agar

The potential of sodium bicarbonate to inhibit growth of and aflatoxin synthesis by Aspergillus parasiticus was examined in Czapek's agar (CA), a medium in which fluorescence under UV light indicates aflatoxin production. Incorporation of sodium bicarbonate (SB) into CA at 0.011, 0.022, and 0.033 mol% reduced cell viability 63-, 10(3)-, and greater than 10(7)-fold, respectively. Colonies resulting from surviving cells did not fluoresce under UV light, but thin-layer chromatography analysis of culture extracts detected aflatoxins. Potassium bicarbonate (KB) at 0.011 and 0.022 mol% produced inhibitory effects similar to those of SB, but NaCl and silica had no effect. After 7 days, control cultures had the normal aflatoxin distribution (B1 greater than G1 greater than B2 greater than G2), but this distribution shifted to B2 greater than B1 approximately equal to G2 greater than G1 during prolonged incubation. Cultures supplemented with SB and KB contained mostly aflatoxins B1 and G1 after 28 days. Both SB and KB raised the pH of CA to 7.5 to 8.5 at the time of growth. Culture growth on CA adjusted to pH 7.5 to 8.5 with NaOH was not inhibited but exhibited reduced fluorescence and elevated levels of aflatoxins B1 and G1. Thus, while bicarbonate inhibition of growth could not be attributed to pH elevation, the lack of culture fluorescence on CA-SB and CA-KB and the altered aflatoxin distribution were caused by the ability of SB and KB to elevate pH.     

Sodium bicarbonate reduces viability and alters aflatoxin distribution of Aspergillus parasiticus in Czapek's agar.

The potential of sodium bicarbonate to inhibit growth of and aflatoxin synthesis by Aspergillus parasiticus was examined in Czapek's agar (CA), a medium in which fluorescence under UV light indicates aflatoxin production. Incorporation of sodium bicarbonate (SB) into CA at 0.011, 0.022, and 0.033 mol% reduced cell viability 63-, 10(3)-, and greater than 10(7)-fold, respectively. Colonies resulting from surviving cells did not fluoresce under UV light, but thin-layer chromatography analysis of culture extracts detected aflatoxins. Potassium bicarbonate (KB) at 0.011 and 0.022 mol% produced inhibitory effects similar to those of SB, but NaCl and silica had no effect. After 7 days, control cultures had the normal aflatoxin distribution (B1 greater than G1 greater than B2 greater than G2), but this distribution shifted to B2 greater than B1 approximately equal to G2 greater than G1 during prolonged incubation. Cultures supplemented with SB and KB contained mostly aflatoxins B1 and G1 after 28 days. Both SB and KB raised the pH of CA to 7.5 to 8.5 at the time of growth. Culture growth on CA adjusted to pH 7.5 to 8.5 with NaOH was not inhibited but exhibited reduced fluorescence and elevated levels of aflatoxins B1 and G1. Thus, while bicarbonate inhibition of growth could not be attributed to pH elevation, the lack of culture fluorescence on CA-SB and CA-KB and the altered aflatoxin distribution were caused by the ability of SB and KB to elevate pH.     

Biosynthetic relationship among aflatoxins B1, B2, M1, and M2

Aflatoxins are a family of toxic, acetate-derived decaketides that arise biosynthetically through polyhydroxyanthraquinone intermediates. Most studies have assumed that aflatoxin B1 is the biosynthetic precursor of the other aflatoxins. We used a strain of Aspergillus flavus which accumulates aflatoxin B2 to investigate the later stages of aflatoxin biosynthesis. This strain produced aflatoxins B2 and M2 but no detectable aflatoxin B1 when grown over 12 days in a low-salt, defined growth medium containing asparagine. Addition of dichlorvos to this growth medium inhibited aflatoxin production with concomitant accumulation of versiconal hemiacetal acetate. When mycelial pellets were grown for 24, 48, and 72 h in growth medium and then transferred to a replacement medium, only aflatoxin B2 and M2 were recovered after 96 h of incubation. Addition of sterigmatocystin to the replacement medium led to the recovery of higher levels of aflatoxins B2 and M2 than were detected in control cultures, as well as to the formation of aflatoxins B1 and M1 and O-methylsterigmatocystin. These results support the hypothesis that aflatoxins B1 and B2 can arise independently via a branched pathway.     

Biosynthetic relationship among aflatoxins B1, B2, M1, and M2.

Aflatoxins are a family of toxic, acetate-derived decaketides that arise biosynthetically through polyhydroxyanthraquinone intermediates. Most studies have assumed that aflatoxin B1 is the biosynthetic precursor of the other aflatoxins. We used a strain of Aspergillus flavus which accumulates aflatoxin B2 to investigate the later stages of aflatoxin biosynthesis. This strain produced aflatoxins B2 and M2 but no detectable aflatoxin B1 when grown over 12 days in a low-salt, defined growth medium containing asparagine. Addition of dichlorvos to this growth medium inhibited aflatoxin production with concomitant accumulation of versiconal hemiacetal acetate. When mycelial pellets were grown for 24, 48, and 72 h in growth medium and then transferred to a replacement medium, only aflatoxin B2 and M2 were recovered after 96 h of incubation. Addition of sterigmatocystin to the replacement medium led to the recovery of higher levels of aflatoxins B2 and M2 than were detected in control cultures, as well as to the formation of aflatoxins B1 and M1 and O-methylsterigmatocystin. These results support the hypothesis that aflatoxins B1 and B2 can arise independently via a branched pathway.     

Influence of gamma irradiation of aflatoxin B1 production by Aspergillus flavus growing on some Nigerian foodstuffs

Spores of Aspergillus flavus (UI, 81) inoculated into some local food materials were irradiated at 62.5, 125.0, 250.0 and 500.0 krad, and the effect on aflatoxin B1 production on subsequent incubation was measured. The results show that aflatoxin B1 production decreased with increasing gamma irradiation dose in soya bean, groundnut, palm juice, while paw paw mash showed a relatively high yield of aflatoxin at 125.0 krad as compared to other irradiation levels tested except for the control. Irradiation of soya bean and groundnut inoculated with spores of Aspergillus flavus at 500.0 krad (pre-irradiation incubation period of 2 h) inhibited aflatoxin B1 production. Analysis of variance showed that media, pre-irradiation incubation periods and irradiation levels affected the total amounts of aflatoxin produced.     

Influence of temperature cycling on the production of aflatoxins B1 and G1 by Aspergillus parasiticus.

The effect of temperature cycling on the relative productions of aflatoxins B1 and G1 by Aspergillus parasiticus NRRL 2999 was studied. The cycling of temperature between 33 and 15 degrees C favored aflatoxin B1 accumulation, whereas cycling between 35 and 15 degrees C favored aflatoxin G1 production. Cultures subjected to temperature cycling between 33 and 25 degrees C at various time intervals changed the relative productions of aflatoxins B1 and G1 drastically. Results obtained with temperature cycling and yeast extract-sucrose medium with ethoxyquin to decrease aflatoxin G1 production suggest that the enzyme system responsible for the conversion of aflatoxin B1 to G1 might be more efficient at 25 degrees C than at 33 degrees C. The possible explanation of the effect of both constant and cycling temperatures on the relative accumulations of aflatoxins B1 and G2 might be through the control of the above enzyme system. The study also showed that greater than 57% of aflatoxin B1, greater than 47% of aflatoxin G1, and greater than 50% of total aflatoxins (B1 plus G1) were in the mycelium by day 10 under both constant and cyclic temperature conditions.     

Effect of Diurnal Temperature Cycles on the Production of Aflatoxin

Exposures to short periods of high temperature (40 to 50 C) in each 24-hr diurnal temperature cycle (average temperature ca. 25 C) reduced growth of Aspergillus parasiticus and production and accumulation of the aflatoxins when compared with cultures held continuously at 25 C. In contrast, diurnal cycles with an average temperature of ca. 25 C but with minima as low as 10 C did not appreciably affect either growth or toxin production. The ratio of production of aflatoxin B to aflatoxin G increased as the maximal temperature was raised but remained essentially unchanged with decreasing minimal temperatures.     

Dillapiol and Apiol as specific inhibitors of the biosynthesis of aflatoxin G1 in Aspergillus parasiticus

Dillapiol was isolated from the essential oil of dill as a specific inhibitor of aflatoxin G1 production. It inhibited aflatoxin G1 production by Aspergillus parasiticus with an IC50 value of 0.15 microM without inhibiting aflatoxin B1 production or fungal growth. Apiol and myristicin, congeners of dillapiol, showed similar activity with IC50 values of 0.24 and 3.5 microM, respectively.     

Production of aflatoxin byAspergillus parasiticus and its control

The aim of the present work was to investigate the production of aflatoxin byAspergillus parasiticus and to find out the possible ways to control it. Of 40 food samples collected from Abha region, Saudi Arabia, only 25% were contaminated with aflatoxins. Oil-rich commodities had the highly contaminated commodities by fungi and aflatoxins while spices were free from aflatoxins.Bacillus megatertum andB cereus were suitable for microbiological assay of aflatoxins. Czapek’s-Dox medium was found a suitable medium for isolation of fungi from food samples. The optimal pH for the growth ofA. parasiticus and its productivity of aflatoxin B₁ was found at 6.0, while the best incubation conditions were found at 30°C for 10 days. D-glucose was the best carbon source for fungal growth, as well as aflatoxin production. Corn steep liquor, yeast extract and peptone were the best nitrogen sources for both fungal growth and toxin production (NH₄)₂HPO₄ (1.55 gL^-1) and NaNO₂ (1.6 gL^-1) reduced fungal growth and toxin production with 37.7% and 85%, respectively. Of ten amino acids tested, asparagine was the best for aflatoxin B₁ production. Zn²⁺ and Co²⁺ supported significantly both fungal growth, as well as, aflatoxin B₁ production at the different tested concentrations. Zn²⁺ was effective when added toA. parasiticus growth medium at the first two days of the culture age. The other tested metal ions expressed variable effects depending on the type of ion and its concentration. Water activity (a_w) was an important factor controlling the growth ofA. parasiticus and toxin production. The minimum a_w for the fungal growth was 0.8 on both coffee beans and rice grains, while a_w of 0.70 caused complete inhibition for the growth and aflatoxin B₁ production. H₂O₂ is a potent inhibitor for growth ofA. parasiticus and its productivity of toxins. NaHCO₃ and C₆H₅COONa converted aflatoxin B₁ to water-soluble form which returned to aflatoxin B₁ by acidity. Black pepper, ciliated heath, cuminum and curcuma were the most inhibitory spices on toxin production. Glutathione, quinine, EDTA, sodium azide, indole acetic acid, 2,4-dichlorophenoxy acetic acid, phenol and catechol were inhibitory for both growth, as well as, aflatoxin B₁ production. Stearic acid supported the fungal growth and decreased the productivity of AFB₁ gradually. Lauric acid is the most suppressive fatty acid for both fungal growth and aflatoxin production, but oleic acid was the most potent supporter. Vitamin A supported the growth but inhibited aflatoxin B₁ production. Vitamins C and D₂ were also repressive particularly for aflatoxin production The present study included studying the activities of some enzymes in relation to aflatoxin production during 20-days ofA. parasiticus age in 2-days intervals. Glycolytic enzymes and pyruvate-generating enzymes seems to be linked with aflatoxin B₁ production. Also, pentose-phosphate pathway enzymes may provide NADPH for aflatoxin B₁ synthesis. The decreased activities of TCA cycle enzymes particularly from 4th day of growth up to 10th day were associated with the increase of aflatoxin B₁ production. All the tested enzymes as well as aflatoxin B₁ production were inhibited by either catechol or phenol.     

Mechanism of action of aflatoxin B1 in Bacillus megaterium

Bacillus megaterium cells from various growth phases were equally susceptible to the lethal effects of aflatoxin B1. Known surfactants (EDTA and Tween-80) accentuated the effects of aflatoxin B1. Viability and inulin uptake in aflatoxin B1-exposed cells decreased considerably. The effect was concentration dependent. A straight-line relationship observed in the death curve indicated a single target for aflatoxin B1 action in B. megaterium. Leakage of intracellular constituents in B. megaterium was also concentration dependent, and this can be related to the extent of cell membrane damage.     

Production of aflatoxin by Aspergillus parasiticus NRRL-2999 during growth in the presence of curing salts

Aspergillus parasiticus NRRL-2999 was inoculated into meat mixtures with curing salts and into yeast extractsucrose (YES) and sucrose-ammonium salts (SAS) broth with and without curing salts to determine if the presence of curing salts significantly affected growth and aflatoxin production by the mold. The effect of individual curing salts or curing salt mixtures on growth and toxin elaboration by the aspergillus was substrate dependent. When YES broth contained 100 ppm of NaNO₂, 2% NaCl, or 1 or 2% NaCl plus 200 ppm of NaNO₂ or 200 ppm of NaNO₃, growth and/or aflatoxin production was depressed. Biosynthesis of aflatoxin B₁ was enhanced by presence of 1 and 4% NaCl in YES broth. The SAS broth containing only NaCl or NaCl combined with nitrite or nitrate yielded less aflatoxin than did control broth or no aflatoxin at all. When compared to the control, an increase in growth and amount of aflatoxin occurred in SAS broth which contained 200 ppm of NaNO₃. Sausages containing 100 and 200 ppm NaNO₂ and no NaCl supported more mold growth and aflatoxin production than did control sausage with 3 % NaCl and 100 ppm of NaNO₂. Addition of 2 and 3 % NaCl and no nitrite to sausage resulted in less aflatoxin than in control sausage.