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.     

Aspergillus flavus Infection and Aflatoxin Production in Fig Fruits

Immature fig fruits did not support colonization and aflatoxin production by Aspergillus flavus Lk. but became susceptible when ripe. While sun-drying on the tree, fruits were particularly vulnerable to fungal infection and colonization. Aflatoxin accumulation equaled levels frequently reported for such seeds as peanuts and cereal grains.     

Production of cyclopiazonic acid by Aspergillus flavus Link.

Production of cyclopiazonic acid by Aspergillus flavus is reported for the first time. A procedure for its production by agitated solid substrate fermentation on red wheat is described along with the isolation procedure and physical and chemical properties of this indole derivative. The compound has been found to exert antibacterial activity.     

Intracellular hydrolases of Aspergillus parasiticus and Aspergillus flavus

When the distribution profile of hydrolases in mycelial homogenates and culture filtrates of A. parasiticus and A. flavus was examined, six hydrolytic enzymes viz. N-acetyl-beta-glucosaminidase, aryl sulfatase, alkaline proteinase, cathepsin B, cathepsin D and aminopeptidase were detected in homogenate. The culture filtrates were devoid of any activity of these enzymes. The enzyme levels varied with the stage of incubation. The most abundant fungal exopeptidase showing preference for basic amino acid naphthylamides seems to be an aminopeptidase B. Incorporation of CEPA, an ethylene generating compound, stimulated the amino peptidase activity in the mycelium but inhibited the enzyme in vitro. The enzyme was also inhibited by different aflatoxins to varying degree. While aminopeptidase B was located intracellularly, a non-dialysable, heat-stable inhibitor of the enzyme was found to be secreted in the culture filtrate. This peptide inhibitor was however ineffective on the other enzymes.     

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.     

Investigation of Reported Aflatoxin Production by Fungi Outside the Aspergillus flavus Group

A screening study of 121 fungus isolates, representing 29 species, for aflatoxin synthesis demonstrated this property only in Aspergillus flavus and A. parasiticus. Eight of the organisms found negative were isolates reported by other investigators to produce aflatoxin. Since similar negative reports have come from several other workers, it is concluded that only the A. flavus group of Aspergillus can presently be certified as sources of these toxins. Reasons for possible false-positive findings are discussed along with precautionary measures and differential analytical procedures useful in aflatoxin screening studies.     

Effect of Aeration on Growth and Aflatoxin Production by Aspergillus flavus in Submerged Culture

Aspergillus flavus ATCC 15517 produced up to 212 mg per liter of total aflatoxin in submerged culture in aerated (3,000, 6,000, 9,000, and 12,000 ml/min) and agitated medium in 14-liter fermentors with 10 liters of medium consisting of 2% yeast extract and 10% sucrose. Aflatoxin production increased with time. A maximum of 212 mg/liter was produced at 9,000 ml/min aeration, whereas the yield decreased substantially at the lower aeration rates. Two other strains of A. flavus synthesized aflatoxin in smaller quantities.     

Aspergillus flavus Infection and Aflatoxin Production in Corn: Influence of Trace Elements

Distribution of trace element levels in corn germ fractions from kernels naturally infected with Aspergillus flavus and from kernels free of the fungus demonstrated an association between the presence of A. flavus and higher levels of metals. A. flavus production of aflatoxin on various autoclaved corn media showed that ground, whole corn was an excellent substrate; similar high levels of toxin were observed on full-fat corn germ but endosperm and defatted corn germ supported reduced yields. The influence of trace elements and their availability in defatted corn germ to A. flavus-mediated aflatoxin biosynthesis were measured. Enrichment of the substrate with 5 to 10 mug of manganese, copper, cadmium, or chromium per g of germ increased toxin yields. Addition of lead or zinc (50 to 250 mug/g) also enhanced toxin accumulation. Aflatoxin elaboration was reduced by the addition of 25 mug of cadmium per g or 500 mug of copper per g of germ.     

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.     

Increased production of aflatoxins by Aspergillus parasiticus Speare in the presence of rubratoxin B.

The influence of rubratoxin B, a metabolite of Penicillium rubrum Stoll, on the growth and aflatoxin production of a strain of Aspergillus parasiticus Speare grown in the chemically defined medium of Reddy et al. (Appl. Microbiol. 22:393-396, 1971) was studied. After 4 days of incubation on a rotary shaker at 25 degrees C, the presence of 10 microgram/ml caused 45 to 50% reduction in dry weight production, although at the same concentration of rubratoxin B, the reduction of growth after 10 days was only 15%. In the presence of 50 microgram/ml there was a reduction in dry weight production of 94% after 4 days of incubation, and it was still 86% after 8 days. Rubratoxin B concentrations of 50 microgram/ml and higher usually caused a reduction in aflatoxin production in the medium comparable with the reduction in biomass, but at concentrations as low as 10 microgram/ml, there was a pronounced increase in the production of aflatoxins, especially of G1, despite the reduction in biomass. The ecological significance of these observations is discussed.     

Substrate-induced lipase gene expression and aflatoxin production in Aspergillus parasiticus and Aspergillus flavus

AIMS: To establish a relationship between lipase gene expression and aflatoxin production by cloning the lipA gene and studying its expression pattern in several aflatoxigenic and nontoxigenic isolates of Aspergillus flavus and A. parasiticus. METHODS AND RESULTS: We have cloned a gene, lipA, that encodes a lipase involved in the breakdown of lipids from aflatoxin-producing A. flavus, A. parasiticus and two nonaflatoxigenic A. flavus isolates, wool-1 and wool-2. The lipA gene was transcribed under diverse media conditions, however, no mature mRNA was detected unless the growth medium was supplemented with 0.5% soya bean or peanut oil or the fungus was grown in lipid-rich medium such as coconut medium. The expression of the lipase gene (mature mRNA) under substrate-induced conditions correlated well with aflatoxin production in aflatoxigenic species A. flavus (SRRC 1007) and A. parasiticus (SRRC 143). CONCLUSIONS: Substrate-induced lipase gene expression might be indirectly related to aflatoxin formation by providing the basic building block 'acetate' for aflatoxin synthesis. No direct relationship between lipid metabolism and aflatoxin production can be ascertained, however, lipase gene expression correlates well with aflatoxin formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Lipid substrate induces and promotes aflatoxin formation. It gives insight into genetic and biochemical aspects of aflatoxin formation.     

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.     

Aspergillus parasiticus Cyclase Catalyzes Two Dehydration Steps in Aflatoxin Biosynthesis

In the aflatoxin biosynthetic pathway, 5′-oxoaverantin (OAVN) cyclase, the cytosolic enzyme, catalyzes the reaction from OAVN to (2′S,5′S)-averufin (AVR) (E. Sakuno, K. Yabe, and H. Nakajima, Appl. Environ. Microbiol. 69:6418-6426, 2003). Interestingly, the N-terminal 25-amino-acid sequence of OAVN cyclase completely matched an internal sequence of the versiconal (VHOH) cyclase that was deduced from its gene (vbs). The purified OAVN cyclase also catalyzed the reaction from VHOH to versicolorin B (VB). In a competition experiment using the cytosol fraction of Aspergillus parasiticus, a high concentration of VHOH inhibited the enzyme reaction from OAVN to AVR, and instead VB was newly formed. The recombinant Vbs protein, which was expressed in Pichia pastoris, showed OAVN cyclase activity, as well as VHOH cyclase activity. A mutant of A. parasiticus SYS-4 (= NRRL 2999) with vbs deleted accumulated large amounts of OAVN, 5′-hydroxyaverantin, averantin, AVR, and averufanin in the mycelium. These results indicated that the cyclase encoded by the vbs gene is also involved in the reaction from OAVN to AVR in aflatoxin biosynthesis. Small amounts of VHOH, VB, and aflatoxins also accumulated in the same mutant, and this accumulation may have been due to an unknown enzyme(s) not involved in aflatoxin biosynthesis. This is the first report of one enzyme catalyzing two different reactions in a pathway of secondary metabolism.