Effect of peanut tannin extracts on growth of Aspergillus parasiticus and aflatoxin production

Twenty-three peanut (Arachis hypogaea L.) genotypes were evaluated for kernel resistance to Aspergillus parasiticus Spear. colonization and aflatoxin contamination when incubated under high relative humidity. Also, tannin-containing extracts from kernel coats (testae) and cotyledons of these genotypes were prepared and tested for their effect on A. parasiticus growth and aflatoxin production in vitro. The lowest degree of colonization, less than 30% was noted in kernels from the genotypes, Toalson x UF 73-4022 (selections TX-798731 and TX-798736), A72118, SN 55-437, PI337409, and Florunner. Genotypes with low levels of colonization also had the lowest aflatoxin contamination. The coefficient of correlation between infection frequency and aflatoxin contamination was 0.66. Higher levels of tannins were detected in the testae (23.9–97.2 mg g tissue) compared to the cotyledons (0.17–0.82 mg g tissue). Some of the methanol-extracted and water-soluble tannin extracts from testae and cotyledons, when incorporated in yeast extract sucrose liquid medium (100 mg l), significantly inhibited A. parasiticus growth and reduced the levels of aflatoxin produced. There was no overall correlation between the peanut genotypes and the influence of tannin extracts on A. parasiticus growth and aflatoxin production. However, correlations were higher for specific genotypes. For example, the coefficient of correlation between the ability of tannin extracts from testae of genotypes PI337409 and TX-798736 to inhibit aflatoxin production was 0.93 and 0.85 respectively.     

Effect of miconazole on growth and aflatoxin production by Aspergillus parasiticus

At 5 M, miconazole prevented the growth of Aspergillus parasiticus Speare in a number of media. Sensitivity to miconazole was increased approximately 10-fold in a medium containing glycerol. At sub-inhibitory concentrations, miconazole stimulated aflatoxin synthesis on media which normally support toxin formation. Miconazole inhibited respiration and altered mitochondrial ultrastructure, suggesting that miconazole inhibits growth and stimulates aflatoxin production by depressing mitochondrial activity.     

Chemoprevention by thyme oils of Aspergillus parasiticus growth and aflatoxin production

The essential oils from Thymus eriocalyx and Thymus X-porlock obtained by hydrodistillation were analyzed by GC/MS. The major components of T. eriocalyx and T. X-porlock oils were thymol (63.8, 31.7%), beta-phellandrene (13.30, 38.7%), cis-sabinene hydroxide (8.1, 9.6%), 1,8-cineole (2, 1.7%), and beta-pinene (1.31, 2%), respectively. Antifungal activities of the oils were studied with special reference to the inhibition of Aspergillus parasiticus growth and aflatoxin production. Minimal inhibitory (MIC) and minimal fungicidal (MFC) concentrations of the oils were determined. Static effects of the above oils against A. parasiticus were at 250 ppm and lethal effects of T. eriocalyx and T. X-porlock were 500 and 1000 ppm of the oils, respectively. Aflatoxin production was inhibited at 250 ppm of both oils with that of T. eriocalyx being stronger inhibitor. Transmission electron microscopy (TEM) of A. parasiticus exposed to MIC level (250 ppm) of the oils showed irreversible damage to cell wall, cell membrane, and cellular organelles. It is concluded that the essential oils could be safely used as preservative materials on some kinds of foods at low concentrations to protect them from fungal infections.     

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.     

Effect of specific amino acids on growth and aflatoxin production by Aspergillus parasiticus and Aspergillus flavus in defined media.

Four amino acids were used as sole nitrogen sources or as supplements to ammonium sulfate, and casein and ammonium sulfate were used as sole nitrogen sources to examine their effects on aflatoxin production by Aspergillus parasiticus NRRL 2999 and Aspergillus flavus 3357 grown on synthetic liquid media. In general, when proline, asparagine, casein, and ammonium sulfate were used as sole nitrogen sources, they supported more growth and toxin production than tryptophan or methionine. However, proline stimulated more toxin production per gram of mycelium in stationary cultures than the other nitrogen sources, including the amino acid asparagine, which is generally recognized as supporting good aflatoxin production. The exact responses to individual nitrogen sources were influenced by the species of fungus and whether cultures were stationary or shaken. In shake cultures, but not in stationary cultures, increased growth was generally associated with increased toxin production.     

Effect of pyridazinone herbicides on growth and aflatoxin release by Aspergillus flavus and Aspergillus parasiticus.

The influence of pyridazinone herbicides on aflatoxin production by Aspergillus flavus and A. parasiticus was studied in liquid media. Mycelia production was not affected by 20, 40, or 60 micrograms of herbicide per ml; however, aflatoxin production by A. parasiticus was higher in media with herbicide, whereas A. flavus produced lower aflatoxin levels.     

Reduced aflatoxin production by Aspergillus parasiticus after growth on a caffeine-containing medium

Previous observations that the presence of caffeine could cause a reduction in the specific productivity of aflatoxins by Aspergillus parasiticus were confirmed. However, it was also shown that the reisolation of A. parasiticus from media containing caffeine as the sole source of nitrogen, provided a strain with a significantly reduced specific productivity even when grown in the absence of caffeine. This property has remained stable over several subcultures. The specific productivity of this strain is further reduced when it is grown in the presence of sub-inhibitory concentrations of caffeine. Under normal culture conditions it appears to be indistinguishable from the parent strain except in the reduced specific productivity of aflatoxins.     

Decrease of growth and aflatoxin production in Aspergillus parasiticus caused by spices

Non-commercial spices and herbs Tetrapleura tetrapetra, Triumfetta cordifolia, Garcina kola, Monodora myristica and Xylopia aethiopica at 0.08 to 0.32% (w/v) decreased the mycelial weight of Aspergillus parasiticus NRRL 2999 in yeast extract/sucrose broth by up to 68%. Aflatoxin production, monitored with ELISA, was most effectively decreased, from 97 to 23 g/ml, when the extract of G. kola was added at 0.32% (w/v).     

Effect of pyridazinone herbicides on growth and aflatoxin release by Aspergillus flavus and Aspergillus parasiticus

The influence of pyridazinone herbicides on aflatoxin production by Aspergillus flavus and A. parasiticus was studied in liquid media. Mycelia production was not affected by 20, 40, or 60 micrograms of herbicide per ml; however, aflatoxin production by A. parasiticus was higher in media with herbicide, whereas A. flavus produced lower aflatoxin levels.     

Effect of specific amino acids on growth and aflatoxin production by Aspergillus parasiticus and Aspergillus flavus in defined media

Four amino acids were used as sole nitrogen sources or as supplements to ammonium sulfate, and casein and ammonium sulfate were used as sole nitrogen sources to examine their effects on aflatoxin production by Aspergillus parasiticus NRRL 2999 and Aspergillus flavus 3357 grown on synthetic liquid media. In general, when proline, asparagine, casein, and ammonium sulfate were used as sole nitrogen sources, they supported more growth and toxin production than tryptophan or methionine. However, proline stimulated more toxin production per gram of mycelium in stationary cultures than the other nitrogen sources, including the amino acid asparagine, which is generally recognized as supporting good aflatoxin production. The exact responses to individual nitrogen sources were influenced by the species of fungus and whether cultures were stationary or shaken. In shake cultures, but not in stationary cultures, increased growth was generally associated with increased toxin production.     

Effect of ultraviolet light irradiation on viability and aflatoxin production by Aspergillus parasiticus

The effect of ultraviolet light irradiation (254 nm) on both the viability and the aflatoxin-producing ability of the fungus Aspergillus parasiticus NRRL 2999, a good aflatoxin-producing strain, was studied. This strain showed noticeable resistance and irradiation for more than 10 min was necessary to reduce survival to under 10%, while the white mutants were more susceptible (5 min of irradiation reduced survival to under 1%). Induction of mutants with complete loss of aflatoxigenicity was rare and only 3 of the 1463 survivors tested were aflatoxinless.     

Minimal moisture content for growth and aflatoxin production by Aspergillus parasiticus in mixed feeds

Minimal moisture content for growth and aflatoxin production by Aspergillus parasiticus in mixed feeds was studied. Minimal moisture content for growth is 16.51%+/–0.45. Very low amounts of aflatoxins are accumulated at days 1 or 2 after the growth started when the initial moisture content of the mixed feed was 17% or lower; on the other hand, significant amounts of aflatoxin are detected when it was 18% or higher.     

Influence of inoculum size of Aspergillus parasiticus spores on aflatoxin production.

The influence of the inoculum size on growth and aflatoxin production was examined in Aspergillus parasiticus (NRRL 3145) by using a synthetic medium. The reduction in the number of spores by 4 to 5 log cycles either by serial dilution or by gamma irradiation caused a two fold increase in the toxin production. The decrease in the inoculum size induced a lag in growth of the culture, though the final yield of the mycelium over the 28-day experimental period was the same. The maximal accumulation of aflatoxin was observed on day 14 of incubation. A transition from the biphasic to monophasic pattern in aflatoxin production could be correlated with the size of the inoculum. The enhanced toxin production from dilute inocula was similar to that obtained with the surviving fraction of the spores after gamma irradiation (0 to 150 krads).     

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.     

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.     

Effect on aflatoxin production of competition between wild-type and mutant strains of Aspergillus parasiticus

Co-cultivation of a strain of Aspergillus parasiticus, capable of making aflatoxins, with blocked mutant strains, capable of producing none or only a low level of aflatoxins, reduced the net yield of aflatoxins more than that expected based on spore recovery. Yields of aflatoxins were 8-fold less for a norsolorinic acid-producing strain, 14-fold less for an averantin-producing strain, 6-fold less for an averufin-producing strain, and 21-fold less for a versicolorin A-producing strain when co-cultured in equal amounts with a wild-type strain of Aspergillus parasiticus. Even when the wild-type strain was initially present in 100-fold excess, with two of the mutant strains, reduced aflatoxin production was still observed.     

Enzymatic conversion of sterigmatocystin into aflatoxin B1 by cell-free extracts of Aspergillus parasiticus.

A cell-free extract, prepared from Aspergillus parasiticus ATCC 15517 grown in synthetic medium, was active in converting [14C]sterigmatocystin into aflatoxin B1 in the presence of reduced nicotinamide adenine dinucleotide phosphate. The activity was demonstrated by the time course of conversion and the linear dependence of the yield of product on enzyme concentrations. Optimum activity was obtained at pH 7.5 to 7.8 at 27 C. The results confirm sterigmatocystin as a biogenetic precursor of aflatoxin B1. Techniques were developed for enzymatic studies on aflatoxin biosynthesis.