Aflatoxin is degraded at different temperatures and pH values by mycella of Aspergillus parasiticus

Summary Blended 9-day-old mycelia of Aspergillus parasiticus NRRL 2999 were tested for their ability to degrade aflatoxins B₁ and G₁ at 7,19,28,36, and 45°C. Rates for degradation of aflatoxin B₁ and G₁ were maximum at 28°C. Intermediate rates of aflatoxin degradation were observed at 19 and 36°C while little aflatoxin was degraded at 7 and 45°C. Five different pH values (2.0, 3.0, 4.0, 5.0, and 6.5) were also tested to determine the effect of pH on ability of blended 9-day-old mycelia of A. parasiticus NRRL 2999 to degrade aflatoxins. The ability of mycelia to degrade aflatoxin was pH-dependent. Of the pH values tested, greatest rates of aflatoxin B₁ and G₁ degradation occurred when pH was in the range of 5 to 6.5. Little aflatoxin was degraded at pH 4.0 and essentially no aflatoxin was degraded by mycelia at pH 2.0 or 3.0 although some aflatoxin was degraded by acid conditions only at pH values of 4 or less.     

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.     

Aflatoxin production by Aspergillus parasiticus in a competitive environment

Aspergillus parasiticus NRRL 2999 was grown in the presence of Rhizopus nigricans, Saccharomyces cerevisiae, Acetobacter aceti, or Brevibacterium linens and aflatoxin concentration was determined after 3,5,7, and 10 days of incubation at 28C. R. nigricans and S. cerevisiae inhibited growth and aflatoxin production by A. parasiticus. B. linens caused slight inhibition and A. aceti stimulated growth and aflatoxin production by A. parasiticus.     

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.     

Aflatoxin B1, zearalenone and deoxynivalenol production by Aspergillus parasiticus and Fusarium graminearum in interactive cultures on irradiated corn kernels

The influence of inoculum size in the production of aflatoxin B1 (AFB1), zearalenone (ZEN) and deoxynivalenol (DON) was determined when Aspergillus parasiticus NRRL 3000 and Fusarium graminearum ITEM 124 were cultured alone and in pairs on irradiated corn kernels at 28 °C and 0.97 water activity (aw). The highest levels of AFB1 produced by A. parasiticus were produced at the lowest levels of the inoculum (103 spores/ml). No significant differences were observed in ZEN and DON production at any inoculum level during the experimental period. When A. parasiticus was co-inoculated with F. graminearum both to the same inocula (106 spores/ml), AFB1 inhibition percentage were 60, 72 and 56% at 10, 20 and 35 days of incubation respectively, while at 106 spores/ml the percentages of inhibition were 34, 84 and 93% at 10, 20 and 35 days. In the mixture cultures A. parasiticus 103 × F. graminearum 106 spores/ml the percentage of inhibition of AFB1 oscillated in 99% during all the incubation. In the interaction A. parasiticus 106 spores/ml × F. graminearum 103 spores/ml the accumulation of AFB1 decreased in 80, 94 and 86% at 10, 20 and 35 days of incubation respectively. In single culture F. graminearum was inoculated with 10³ or 106 spores/ml and the highest levels of ZEN and DON were detected at 35 days of incubation. The levels oscillated in 538–622 μg/kg for ZEN and 870–834 μg/kg for DON respectively. In paired cultures there were no significant differences in the levels regardless of the spore concentrations during the incubation time. This revised version was published online in June 2006 with corrections to the Cover Date.     

Peroxidase activity in mycelia of Aspergillus parasiticus that degrade aflatoxin

Summary Extracts of 9-day-old mycelia of Aspergillus parasiticus NRRL 2999 were assayed for peroxidase activity and for their ability to degrade aflatoxin. A positive relationship existed between rates of aflatoxin degradation and amount of peroxidase activity in these extracts. The supernatant fluid of homogenates from mycelia grown under similar conditions varied in amount of peroxidase present (170 to 2215 U/g). The fraction obtained, by precipitation with (NH₄)₂SO₄ at 45% of saturation, from six different homogenates prepared from three mycelial mats contained peroxidase and degraded aflatoxin. Rates of aflatoxin degradation by and amounts of peroxidase activity in each sample obtained from mycelial homogenates with (NH₄)₂SO₄ at 60% of saturation varied; however, when increased amounts of peroxidase activity were present, more aflatoxin was degraded and vice versa. Relatively little peroxidase activity was present in the fraction obtained with (NH₄)₂SO₄ at 30% of saturation and little or no aflatoxin was degraded by this precipitate. Trends for degradation of aflatoxin when more or less peroxidase activity was present in mycelial preparations suggest that the enzyme may be involved in degradation of aflatoxin by the Aspergillus.     

Some Cultural Conditions That Control Biosynthesis of Lipid and Aflatoxin by Aspergillus parasiticus

Synthesis of total lipid and aflatoxin by Aspergillus parasiticus as affected by various concentrations of glucose and nitrogen in a defined medium and by different incubation temperatures was studied. Maximal yields of lipid and aflatoxin were obtained with 30% glucose, whereas mold growth, expressed as dry weight, was maximal when the medium contained 10% glucose. Maximal mold growth occurred when the medium contained 3% (NH(4))(2)SO(4); however, 1% (NH(4))(2)SO(4) favored maximum accumulation of lipid and aflatoxin. Growth of mold and synthesis of lipid and toxin also varied with the incubation temperature. Maximal mold growth occurred at 35 C, whereas most toxin appeared at 25 C. Maximal production of lipid occurred at 25 and 35 C but production was more rapid at 35 C. Essentially all glucose in the medium (5% initially) was utilized in 3 days at 25 and 35 C but not in 7 days at 15 and 45 C. Patterns for formation of lipid and aflatoxin were similar at 15 and 25 C when a complete growth medium was used and at 28 C when the substrate contained various concentrations of glucose or (NH(4))(2)SO(4). They were dissimilar when the mold grew at 35 or 45 C. At these temperatures lipid was produced preferentially and only small amounts of aflatoxin appeared.     

Aflatoxin Production in Peanut Varieties by aspergillus flavus Link and Aspergillus parasiticus Speare

Levels of aflatoxin produced in peanuts differed with the genetic variety of plant and with the species and strain of invading fungus. Possibilities for identifying groundnut varieties partially resistant to aflatoxin production are discussed.     

Function of the cypX and moxY Genes in Aflatoxin Biosynthesis in Aspergillus parasiticus

The pathway oxoaverantin (OAVN) → averufin (AVR) → hydroxyversicolorone (HVN) → versiconal hemiacetal acetate (VHA) is involved in aflatoxin biosynthesis, and the cypX and moxY genes, which are present in the aflatoxin gene cluster, have been previously suggested to be involved in this pathway. To clarify the function of these two genes in more detail, we disrupted the genes in aflatoxigenic Aspergillus parasiticus NRRL 2999. The cypX-deleted mutant lost aflatoxin productivity and accumulated AVR in the mycelia. Although this mutant converted HVN, versicolorone (VONE), VHA, and versiconol acetate (VOAc) to aflatoxins in feeding experiments, it could not produce aflatoxins from either OAVN or AVR. The moxY-deleted mutant also lost aflatoxin productivity, whereas it newly accumulated HVN and VONE. In feeding experiments, this mutant converted either VHA or VOAc to aflatoxins but did not convert OAVN, AVR, HVN, or VONE to aflatoxins. These results demonstrated that cypX encodes AVR monooxygenase, catalyzing the reaction from AVR to HVN, and moxY encodes HVN monooxygenase, catalyzing a Baeyer-Villiger reaction from HVN to VHA as well as from VONE to VOAc. In this work, we devised a simple and rapid method to extract DNA from many fungi for PCR analyses in which cell disruption with a shaker and phenol extraction were combined.     

Enzymatic Function of the Nor-1 Protein in Aflatoxin Biosynthesis in Aspergillus parasiticus

The nor-1 gene is involved in aflatoxin biosynthesis in Aspergillus parasiticus and was predicted to encode a norsolorinic acid ketoreductase. Recombinant Nor-1 expressed in Escherichia coli converted the 1′ keto group of norsolorinic acid to the 1′ hydroxyl group of averantin in crude E. coli cell extracts in the presence of NADPH. The results confirm that Nor-1 functions as a ketoreductase in vitro.     

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.     

Inhibition of Aflatoxin B1 Production by Aspergillus parasiticus Using Nonaflatoxigenic Strains: Role of Vegetative Compatibility

The effect of vegetative compatibility on the inhibition of aflatoxin B₁ production by Aspergillus parasiticus was examined using nonaflatoxigenic strains. Nonaflatoxigenic white-conidial mutants were paired in different proportions on an agar medium with aflatoxigenic yellow-conidial mutants belonging to the same isolate, to the same vegetative compatibility group but with the original wild types differing in phenotype, and to different vegetative compatibility groups. Heterokaryosis as a result of hyphal anastomosis was detected by the presence of conidiogenous structures with a mixture of green and parental (white and/or yellow) chains of conidia. Sclerotium production (number and dry weight) was significantly greater in pairings of compatible strains that formed heterokaryons than in pairings of strains from different vegetative compatibility groups. In contrast, there were no consistent differences in aflatoxin B₁ inhibition by nonaflatoxigenic strains in pairings from the same vegetative compatibility group and pairings from different groups. Therefore, the composition of vegetative compatibility groups within a population may be of minor importance in predicting the efficacy of a particular nonaflatoxigenic strain for the biological control of aflatoxin contamination of crops.     

Chromosomal Location Plays a Role in Regulation of Aflatoxin Gene Expression in Aspergillus parasiticus

The nor-1 gene in the filamentous fungus Aspergillus parasiticus encodes a ketoreductase involved in aflatoxin biosynthesis. To study environmental influences on nor-1 expression, we generated plasmid pAPGUSNNB containing a nor-1 promoter-β-glucuronidase (GUS) (encoded by uidA) reporter fusion with niaD (encodes nitrate reductase) as a selectable marker. niaD transformants of A. parasiticus strain NR-1 (niaD) carried pAPGUSNNB integrated predominantly at the nor-1 or niaD locus. Expression of the native nor-1 and nor-1::GUS reporter was compared in transformants grown under aflatoxin-inducing conditions by Northern and Western analyses and by qualitative and quantitative GUS activity assays. The timing and level of nor-1 promoter function with pAPGUSNNB integrated at nor-1 was similar to that observed for the native nor-1 gene. In contrast, nor-1 promoter activity in pAPGUSNNB and a second nor-1::GUS reporter construct, pBNG3.0, was not detectable when integration occurred at niaD. Because niaD-dependent regulation could account for the absence of expression at niaD, a third chromosomal location was analyzed using pAPGUSNP, which contained nor-1::GUS plus pyrG (encodes OMP decarboxylase) as a selectable marker. GUS expression was detectable only when pAPGUSNP integrated at nor-1 and was not detectable at pyrG, even under growth conditions that required pyrG expression. nor-1::GUS is regulated similarly to the native nor-1 gene when it is integrated at its homologous site within the aflatoxin gene cluster but is not expressed at native nor-1 levels at two locations outside of the aflatoxin gene cluster. We conclude that the GUS reporter system can be used effectively to measure nor-1 promoter activity and that nor-1 is subject to position-dependent regulation in the A. parasiticus chromosome.     

Hydroxylation of biphenyl by Aspergillus parasiticus: Approaches to yield improvment in fermenter cultures

We carried out experiments designed to increase the rate of production of 4,4'-dihydroxybiphenyl (biphenol) from biphenyl by Aspergillus parasiticus. We show that 0.5 mg/ml biphenyl, the substrate for the reaction, significantly inhibits growth of the organism and that at 0.04 mg/ml, 2-hydroxybiphenyl or 4-hydroxybiphenyl (an intermediate of the reaction) strongly inhibit oxygen uptake, probably by inhibition of mitochondrial electron transport. Both factors may contribute to the low hydroxylation rates observed previously [J. H. Golbeck and J. C. Cox, Biotechnol. Bioeng., 26, 434 (1984)]. We therefore adapted the organism to the presence of 0.08 mg/ml 2- and 4-hydroxybiphenyl in the growth medium and found that cultures of adapted strains hydroxylated biphenyl at rates ca. three-fold faster than control cultures. Once the fungal mycelia were grown, they could be recycled at least twice into fresh fermentation broth. Recycled organisms were capable of hydroxylating biphenyl more rapidly than cells in the primary fermentation culture and there was no lag period between introduction of biphenyl and the onset of hydroxylation. Cell recycle thus results in a considerable saving in carbon costs and fermentation time.     

The Affinity Purification and Characterization of a Dehydrogenase from Aspergillus parasiticus Involved in Aflatoxin B1, Biosynthesis

Abstract

A two step scheme has been developed for the purification of a dehydrogenase from mycelia of 84 hours old Aspergillus parasiticus (1-11-105 Wh 1), which catalyzes the conversion of norsolorinic acid (NA) to averantin (AVN). The dehydrogenase was purified from cell-free extracts using reactive green 19-agarose and norsolorinic acid-agarose affinity chromatography. The latter affinity matrix was synthesised by attaching norsolorinic acid to ω-aminohexylagarose. The purified protein was shown to be homogenous on non-denaturing polyacrylamide gel electrophoresis. A final purification of 215-fold was achieved. Results of gel filtration chromatography indicated the approximate molecular mass of the native protein to be 140 000 daltons. The isoelectric point of the protein was about 5.5 as determined by chromatofocusing. The reaction catalyzed by the dehydrogenase was optimum at pH 8.5 and between 25[ddot] to 35[ddot]C. The Km of the enzyme for NA and NADPH was determined to be 3.45 μM and 103 μM respectively.