Inhibition of aflatoxin production by selected insecticides

The insecticide naled completed inhibition production of aflatoxins B1, B2, G1, and G2 by and growth of Aspergillus parasiticus at a 100-ppm (100 microgram/ml) concentration. The insecticides dichlorvos, Landrin, pyrethrum, Sevin, malathion, and Diazinon significantly (P = 0.05) inhibited production of aflatoxins at a 100-ppm concentration. However, at a concentration of 10 ppm, significant inhibition in production of aflatoxins was found only with naled, dichlorvos, Sevin, Landrin, and pyrethrum. Dichlorvos, Landrin, Sevin, and naled inhibited growth of A. parasiticus by 28.9 , 18.9, 15.7, and 100%, respectively, at 100 ppm. Stimulation of growth was observed when diazinon was added to cultures. Aflatoxin B1 was most resistant to inhibition by insecticides, followed by G1, G2, and B2, respectively.     

Effects of naled and dichlorvos on growth and production of Luteoskyrin byPenicillium islandicum

Two organophosphorus insecticides, 1, 2-dibromo ?2, 2-dichloroethyl dimethyl phosphate (naled) and dimethyl 2, 2 -dichlorovinyl phosphate (dichlorvos) were used for investigation of their effects on growth and production of luteoskyrin mycotoxin byPenicillium islandicum in various cultural media at 25°C for 30 days or 60 days. When the concentration of naled and dichlorvos in Czapek solution broth reached 5mg/50mL, growth and production of luteoskyrin by the fungus was completely inhibited. In unpolished rice medium, 15mg/50g of naled was required to retard fungal growth, while the concentration to inhibit the biosynthesis of luteoskyrin was 10mg/50g. On the other hand, if the medium contained dichlorvos at the level of 30mg/50g, the ability to produce luteoskyrin byP islandicum was significantly reduced. In the unhulled rice case, both naled and dichlorvos at the concentration of 15mg/50g were necessary to retard the fungal growth, and 1 mg/50g of each compound exhibits its ability to inhibit the toxin production. Furthermore, it was also found when the cultural medium contained only small amounts of naled and dichlorvos [0.5mg/50g (mL)] the capability to synthesize luteoskyrin by the fungus was drastically reduced. These data strongly suggest that both naled and dichlorvos have similar ability to inhibit luteoskyrin biosynthesis byP islandicum and are also able to retard the fungal growth.     

Aflatoxin production by entomopathogenic isolates of Aspergillus parasiticus and Aspergillus flavus

Fourteen isolates of Aspergillus parasiticus and 2 isolates of Aspergillus flavus isolated from the mealybug Saccharicoccus sacchari were analyzed for production of aflatoxins B1, B2, G1, and G2 in liquid culture over a 20-day period. Twelve Aspergillus isolates including 11 A. parasiticus and 1 A. flavus produced aflatoxins which were extracted from both the mycelium and culture filtrate. Aflatoxin production was detected at day 3 and was detected continually for up to day 20. Aflatoxin B1 production was greatest between 7 and 10 days and significantly higher quantities were produced by A. flavus compared to A. parasiticus. Aflatoxin production was not a stable trait in 1 A. parasiticus isolate passaged 50 times on agar. In addition to loss of aflatoxin production, an associated loss in sporulation ability was also observed in this passaged isolate, although it did maintain pathogenicity against S. sacchari. An aflatoxin B1 concentration of 0.16 micrograms/mealybug (14.2 micrograms/g wet wt) was detected within the tissues of infected mealybugs 7 days after inoculation. In conclusion, the ability of Aspergillus isolates to produce aflatoxins was not essential to the entomopathogenic activity of this fungus against its host S. sacchari.     

Bioremediation of aflatoxins by some reference fungal strains.

Aspergillus parasiticus RCMB 002001 (2) producing four types of aflatoxins B1, B2, G1, and G2 was used in this study as an aflatoxin-producer. Penicillium griseofulvum, P. urticae, Paecilomyces lilacinus, Trichoderma viride, Candida utilis, Saccharomyces cerevisiae as well as a non-toxigenic strain of Aspergillus flavus were found to be able to exhibit growth on aflatoxin B1-containing medium up to a concentration of 500 ppb. It was also found that several fungal strains exhibited the growth in co-culture with A. parasiticus, natural aflatoxins producer, and were able to decreased the total aflatoxin concentration, resulting in the highest inhibition percentage of 67.2% by T viride, followed by P. lilacinus, P. griseofulvum, S. cerevisiae, C. utilis, P. urticae, Rhizopus nigricans and Mucor rouxii with total aflatoxin inhibition percentage of 53.9, 52.4, 52, 51.7, 44, 38.2 and 35.4%, respectively. The separation of bioremediation products using GC/MS revealed that the toxins were degraded into furan moieties.     

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.     

Field trials of spinosad as a replacement for naled, DDVP, and malathion in methyl eugenol and cue-lure bucket traps to attract and kill male oriental fruit flies and melon flies (Diptera: Tephritidae) in Hawaii

Spinosad was evaluated in Hawaii as a replacement for organophosphate insecticides (naled, dichlorvos [DDVP], and malathion) in methyl eugenol and cue-lure bucket traps to attract and kill oriental fruit fly, Bactrocera dorsalis Hendel, and melon fly, B. cucurbitae Coquillett, respectively. In the first and second methyl eugenol trials with B. dorsalis, naled was in the highest rated group for all evaluation periods (at 5, 10, 15, and 20 wk). Spinosad was equal to naled at 5 and 10 wk during both trials 1 and 2, and compared favorably with malathion during trial 2. During the first cue-lure trial with B. cucurbitae, naled and malathion were in the top rated group at 5, 10, 15, and 20 wk. Spinosad was equal to naled at 5 wk. During the second cue-lure trial, spinosad and naled were both in the top rated group at 10, 15, and 20 wk. Use of male lure traps with methyl eugenol or cue-lure had no effect on attraction of females into test areas. Our results suggest that spinosad, although not as persistent as naled or malathion, is safer to handle and a more environmentally friendly substitute for organophosphate insecticides in methyl eugenol and cue-lure traps for use in B. dorsalis and B. cucurbitae areawide integrated pest management programs in Hawaii.     

Aflatoxin Production in Meats. I. Stored Meats

Aflatoxins were produced on fresh beef (in which bacterial spoilage was delayed with antibiotics), ham, and bacon inoculated with toxinogenic fungi and stored at 15, 20 and 30 C. Meats stored at 10 C were spoiled by bacteria and yeast before detectable levels of aflatoxins were produced. High levels of aflatoxins were formed in meats stored at 20 C; one sample supported the production of 630 mug of aflatoxins per g of meat, the major portion (580 mug) of which was aflatoxin G(1). Meats stored below 30 C developed higher levels of aflatoxin G(1) than B(1), but at 30 C Aspergillus flavus produced equal amounts of B(1) and G(1), whereas A. parasiticus continued to produce more G(1) than B(1).     

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.     

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.     

Aflatoxin biosynthesis cluster gene cypA is required for G aflatoxin formation

Aspergillus flavus isolates produce only aflatoxins B1 and B2, while Aspergillus parasiticus and Aspergillus nomius produce aflatoxins B1, B2, G1, and G2. Sequence comparison of the aflatoxin biosynthesis pathway gene cluster upstream from the polyketide synthase gene, pksA, revealed that A. flavus isolates are missing portions of genes (cypA and norB) predicted to encode, respectively, a cytochrome P450 monooxygenase and an aryl alcohol dehydrogenase. Insertional disruption of cypA in A. parasiticus yielded transformants that lack the ability to produce G aflatoxins but not B aflatoxins. The enzyme encoded by cypA has highest amino acid identity to Gibberella zeae Tri4 (38%), a P450 monooxygenase previously shown to be involved in trichodiene epoxidation. The substrate for CypA may be an intermediate formed by oxidative cleavage of the A ring of O-methylsterigmatocystin by OrdA, the P450 monooxygenase required for formation of aflatoxins B1 and B2.     

Inhibition of aflatoxin formation by 2-mercaptoethanol.

2-Mercaptoethanol inhibits growth of Aspergillus parasiticus NRRL 3240 and aflatoxin formation by the fungus. When added to the resuspended medium, 2-mercaptoethanol inhibited [1-14C]acetate incorporation into both aflatoxins and neutral lipids, thereby showing that it acts at an early stage of aflatoxin biosynthesis. The inhibition is probably due to its chelating action on zinc, which is essential for aflatoxin production. It is proposed that any chelating agent that selectively binds to zinc will inhibit aflatoxin formation.     

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.     

Inhibition of aflatoxin production by trifluoperazine in Aspergillus parasiticus NRRL 2999

Trifluoperazine, an anti-calmodulin agent, inhibited aflatoxin production by Aspergillus parasiticus NRRL 2999, without affecting the growth significantly. Culturing the organism for 3 days in the presence of 0.14mm trifluoperazine resulted in a generalized decrease in the production of all aflatoxins; the production of aflatoxin B1, a potent hepatocarcinogen, was inhibited to 88% under such conditions. Culturing 7-day-old preformed cultures in the presence of higher concentrations of trifluoperazine (>1mm) completely abolished production of all aflatoxins including AFB1. The inhibitory influence of trifluoperazine on aflatoxin production was accompanied by calmodulin-dependent phosphorylation of an 85kDa cytoplasmic calmodulin-binding protein. While the functions of calmodulin in mediating primary events of germination, growth and differentiation in fungi have earlier been reported, the present results indicate a possible role for calmodulin in the production of fungal toxins.     

A metabolic grid among versiconal hemiacetal acetate, versiconol acetate, versiconol and versiconal during aflatoxin biosynthesis.

Dichlorvos treatment of aflatoxigenic Aspergillus parasiticus SYS-4 (NRRL 2999) or a verscolorin A-accumulating mutant, NIAH-9, resulted in accumulation of versiconol acetate (VOAc) and versiconal hemiacetal acetate (VHA), whereas the production of aflatoxins, versicolorin A (VA), and versiconol (VOH) decreased. In feeding experiments using another non-aflatoxigenic mutant, NIAH-26, aflatoxins were newly produced from each of VHA, VOAc, VOH, versicolorin B (VB) and versicolorin C (VC). In these experiments, aflatoxin production from VHA or VOAc was inhibited by dichlorvos, whereas that from each of VOH, VB and VC was insensitive to dichlorvos. In cell-free experiments using the cytosol fraction of NIAH-26, VHA was converted to VC (or VB) and a substance tentatively identified as versiconal (VHOH). By further addition of NADH or NADPH to the same reaction mixture, VOAc and VOH were also formed together with VC (VB) and VHOH. VOH was produced from VOAc irrespective of nicotinamide adenine nucleotide. Also, the incubation of VOH in the presence of NAD or NADP led to the formation of VC (VB). The production of VC (VB) and VHOH from VHA, and that of VOH from VOAc was inhibited by dichlorvos, whereas the production of VOAc from VHA, and that of VC (VB) from VOH, was insensitive to dichlorvos. These results indicate that a metabolic grid catalysed by dehydrogenase and esterase among VHA, VOAc, VOH and VHOH, and a reaction from VHOH to VC (VB) are involved in aflatoxin biosynthesis. These enzyme activities were also detected when yeast extract peptone medium was used, or when A. oryzae SYS-2 was examined.     

Mold flora and aflatoxin contamination of stored and cooked samples of pearl millet in the Paharia tribal belt of Santhal paragana, Bihar, India

Stored and cooked samples of pearl millet (Pennesetum typhoides), which is regularly consumed as food by the Paharia tribe in the hilly regions of Santhal Pargana, Bihar State, India, that were harvested in January 1989 were analyzed for mold flora, natural occurrence of Aspergillus flavus and A. parasiticus, and incidence and levels of aflatoxin B1. Of the 22 fungal species isolated, A. flavus and A. parasiticus were the predominant species (63.8%) during the rainy season, followed by other species of Aspergillus, Penicillium, Fusarium, Rhizopus, Helminthosporium, and Curvularia. Screening of 169 A. flavus and A. parasiticus strains showed that 59 of them were toxigenic, producing various combinations of aflatoxins B1, B2, G1, and G2. The amounts of aflatoxin B1 ranged between 4 and 30 mg/100 ml of liquid medium. Analysis of stored and cooked samples also revealed a high incidence and alarming levels of naturally produced aflatoxin B1. Forty-nine of 75 stored and 16 of 38 cooked samples contained various combinations of aflatoxins. The levels of aflatoxin B1 ranged between 17 and 2,110 ppb in stored samples and 18 and 549 ppb in cooked samples. The correlation of insect damage with A. flavus and A. parasiticus incidence and quantity of aflatoxin B1 was found to be insignificant.     

Mold flora and aflatoxin contamination of stored and cooked samples of pearl millet in the Paharia tribal belt of Santhal paragana, Bihar, India.

Stored and cooked samples of pearl millet (Pennesetum typhoides), which is regularly consumed as food by the Paharia tribe in the hilly regions of Santhal Pargana, Bihar State, India, that were harvested in January 1989 were analyzed for mold flora, natural occurrence of Aspergillus flavus and A. parasiticus, and incidence and levels of aflatoxin B1. Of the 22 fungal species isolated, A. flavus and A. parasiticus were the predominant species (63.8%) during the rainy season, followed by other species of Aspergillus, Penicillium, Fusarium, Rhizopus, Helminthosporium, and Curvularia. Screening of 169 A. flavus and A. parasiticus strains showed that 59 of them were toxigenic, producing various combinations of aflatoxins B1, B2, G1, and G2. The amounts of aflatoxin B1 ranged between 4 and 30 mg/100 ml of liquid medium. Analysis of stored and cooked samples also revealed a high incidence and alarming levels of naturally produced aflatoxin B1. Forty-nine of 75 stored and 16 of 38 cooked samples contained various combinations of aflatoxins. The levels of aflatoxin B1 ranged between 17 and 2,110 ppb in stored samples and 18 and 549 ppb in cooked samples. The correlation of insect damage with A. flavus and A. parasiticus incidence and quantity of aflatoxin B1 was found to be insignificant.     

Inhibition of aflatoxin production by trifluoperazine in Aspergillus parasiticus NRRL 2999

Trifluoperazine, an anti-calmodulin agent, inhibited aflatoxin production by Aspergillus parasiticus NRRL 2999, without affecting the growth significantly. Culturing the organism for 3 days in the presence of 0.14mm trifluoperazine resulted in a generalized decrease in the production of all aflatoxins; the production of aflatoxin B1, a potent hepatocarcinogen, was inhibited to 88% under such conditions. Culturing 7-day-old preformed cultures in the presence of higher concentrations of trifluoperazine (>1mm) completely abolished production of all aflatoxins including AFB1. The inhibitory influence of trifluoperazine on aflatoxin production was accompanied by calmodulin-dependent phosphorylation of an 85kDa cytoplasmic calmodulin-binding protein. While the functions of calmodulin in mediating primary events of germination, growth and differentiation in fungi have earlier been reported, the present results indicate a possible role for calmodulin in the production of fungal toxins.     

Role of mycoferritin from Aspergillus parasiticus (255) in secondary metabolism (aflatoxin production)

Aspergillus parasiticus (255), a non-toxigenic isolate showed the presence of secondary metabolites-aflatoxins (B1, B2, G1, G2) when grown in yeast extract sucrose media but not in basal media, thus demonstrating its toxigenic potential. Native PAGE of the crude protein isolated at different growth periods of A. parasiticus in yeast extract sucrose media containing iron showed prominent expression of mycoferritin from day four onwards. The production of aflatoxins was also maximal on day four, both in the presence and absence of iron. Indicators of oxidative stress metabolites such as reactive oxygen species, thiobarbituric acid reactive species, reduced and oxidized glutathione and antioxidant enzymes like superoxide dismutase and glutathione peroxidase were analyzed both in the presence and absence of iron and the experimental data suggest oxidative stress as a pre-requisite for aflatoxin production. The pro-oxidant role of iron was minimized by induction of mycoferritin and the concomitant alterations in oxidative stress parameters imply an antioxidant role to mycoferritin in secondary metabolism, a finding of significance that has not been reported previously in fungal systems.     

Inhibition of aflatoxin biosynthesis by tolnaftate.

Tolnaftate [2-napthyl-N-methyl-N-(m-tolyl)thionocarbamate], an antifungal drug, is widely used to control superficial fungal infections in humans and other animals. In this study the effect of tolnaftate on aflatoxin biosynthesis by Aspergillus parasiticus NRRL 3240 was investigated. Tolnaftate changed the morphology of A. parasiticus to yeastlike forms and inhibited aflatoxin formation. The formation of aflatoxin G was blocked considerably, indicating a metabolic block in the conversion of aflatoxin B to aflatoxin G. The incorporation of [1-14C]acetate into aflatoxin was significantly inhibited at a concentration of 1 mM tolnaftate. The presence of zinc in the resuspension buffer resulted in reversal of the tolnaftate-induced inhibition of aflatoxin G1 biosynthesis.