Isolation of aminopeptidase from Aspergillus flavus

A mixture of aminopeptidase and neutral protease from the Aspergillus flavus mould obtained by chromatography on DEAE-Sephadex was fractionated by chromatography on the hydroxyalkyl methacrylate gel with chemically bonded 1,6 hexamethylene diamine and d-leucine. Aminopeptidase thus obtained was electrophoretically homogeneous. Conditions for chromatography were worked out allowing a one stage isolation of a highly active aminopeptidase sample directly from the alcoholic precipitate of the culture medium of the Aspergillus flavus mould.     

Degradation of aflatoxin by Aspergillus flavus

Aflatoxin degradative activity was demonstrated in 6- to 12-d-old intact mycelium and cell-free extracts of Aspergillus flavus. The addition of cycloheximide, SKF 525-A or metyrapone to cultures of A. flavus prevented subsequent degradation of the aflatoxins, while in cell-free extracts degradation was inhibited by SKF 525-A, metyrapone and cytochrome c but not by KCN. In cell-free extracts, aflatoxin degradation was enhanced by NADPH and NaIO4. The results suggest the involvement of cytochrome P-450 monooxygenases in the aflatoxin degradative activity of A. flavus.     

5-Azacytidine inhibits aflatoxin biosynthesis in Aspergillus flavus

Aflatoxins, mainly produced by Aspergillus flavus and A. parasiticus, are a group of potent mycotoxins with carcinogenic, hepatotoxic, and immunosuppressive properties. Many studies have been devoted to investigating their biosynthesis mechanism since they were discovered half a century ago. 5-Azacytidine (5-AC), a derivative of the nucleoside cytidine and an inactivator of DNA methyltransferase, is widely used for studies in epigenetics and cancer biology, and has also been used for studying secondary metabolism in fungi. In this study, 5-AC was applied to investigate its effect on the development and aflatoxin biosynthesis of A. flavus. The results indicate that 5-AC inhibits the ability to produce aflatoxin and also causes a fluffy aconidial phenotype. Further studies revealed that 5-AC affects gene expression of A. flavus to a limited degree, and the unique homolog of DNA methyltransferase gene (DmtA) expressed constitutively during different developmental stages of A. flavus irrespective of 5-AC. This work may provide some basic data to elucidate the role of 5-AC in aflatoxin biosynthesis and the development of A. flavus.     

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.     

Viral influences on aflatoxin formation by Aspergillus flavus

Summary A nontoxigenic isolate of Aspergillus flavus (NRRL 5565) contains a viral genome consisting of 3 double-stranded RNA (ds-RNA) components with molecular weights of approximately 3 kb each. It thus shares a characteristical feature with a virus occuring in strains of Penicillium chrysogenum.Application of known inhibitors of doublestranded RNA virus synthesis results in stable aflatoxin formation by this originally nontoxigenic strain and the simultaneous loss of its ds-RNA traits. Since the inhibitor induced toxicity can be completely reverted by incubation with a virus from Penicillium chrysogenum (PcV), it is presumed that PcV or a functional related virus possibly constitutes the aflatoxin repressing determinant in Aspergillus flavus.     

Isolation of Bacterial Antagonists of Aspergillus flavus from Almonds

Bacteria were isolated from California almond orchard samples to evaluate their potential antifungal activity against aflatoxin-producing Aspergillus flavus. Fungal populations from the same samples were examined to determine the incidence of aflatoxigenic Aspergillus species. Antagonistic activities of the isolated bacterial strains were screened against a nonaflatoxigenic nor mutant of A. flavus, which accumulates the pigmented aflatoxin precursor norsolorinic acid (NOR) under conditions conducive to aflatoxin production. Using solid and liquid media in coculture assays, 171 bacteria isolated from almond flowers, immature nut fruits, and mature nut fruits showed inhibition of A. flavus growth and/or inhibition of NOR accumulation. Bacterial isolates were further characterized for production of extracellular enzymes capable of hydrolyzing chitin or yeast cell walls. Molecular and physiological identification of the bacterial strains indicated that the predominant genera isolated were Bacillus, Pseudomonas, Ralstonia, and Burkholderia, as well as several plant-associated enteric and nonenteric bacteria. A set of 20 isolates was selected for further study based on their species identification, antifungal phenotypes, and extracellular enzyme production. Quantitative assays using these isolates in liquid coculture with a wild-type, aflatoxin-producing A. flavus strain showed that a number of strains completely inhibited fungal growth in three different media. These results indicate the potential for development of bacterial antagonists as biological control agents against aflatoxigenic aspergilli on almonds.     

Cloning of the afl-2 gene involved in aflatoxin biosynthesis from Aspergillus flavus.

Aflatoxins are extremely potent carcinogens produced by Aspergillus flavus and Aspergillus parasiticus. Cloning of genes in the aflatoxin pathway provides a specific approach to understanding the regulation of aflatoxin biosynthesis and, subsequently, to the control of aflatoxin contamination of food and feed. This paper reports the isolation of a gene involved in aflatoxin biosynthesis by complementation of an aflatoxin-nonproducing mutant with a wild-type genomic cosmid library of A. flavus. Strain 650-33, blocked in aflatoxin biosynthesis at the afl-2 allele, was complemented by a 32-kb cosmid clone (B9), resulting in the production of aflatoxin. The onset and profile of aflatoxin accumulation was similar for the transformed strain and the wild-type strain (NRRL 3357) of the fungus, indicating that the integrated gene is under the same control as in wild-type strains. Complementation analyses with DNA fragments from B9 indicated that the gene resides within a 2.2-kb fragment. Because this gene complements the mutated afl-2 allele, it was designated afl-2. Genetic evidence obtained from a double mutant showed that afl-2 is involved in aflatoxin biosynthesis before the formation of norsolorinic acid, the first stable intermediate identified in the pathway. Further, metabolite feeding studies with the mutant, transformed, and wild-type cultures and enzymatic activity measurements in cell extracts of these cultures suggest that afl-2 regulates gene expression or the activity of other aflatoxin pathway enzymes. This is the first reported isolation of a gene for aflatoxin biosynthesis in A. flavus.     

Genetics of Aspergillus flavus: linkage of aflatoxin mutants

Eight aflatoxin (afl) mutants of Aspergillus flavus were induced with N-methyl-N'-nitro-N-nitrosoguanidine. Heterozygous diploids formed between afl mutants and tester strains revealed that each afl mutant was recessive. Haploids selected from these heterozygous diploids indicated the linkage of all eight afl mutants to markers on group VII. These include previously mapped arg-7 (arginine), leu (leucine), dominant afl-1, and nor which accumulates norsolorinic acid that is visible as an orange-red pigment. Diploid complementation tests indicated that all but two afl mutants were nonallelic. Diploids homozygous for nor, resulting from crossing-over, were isolated and used to map new afl genes.     

黑曲霉对黄曲霉生长、产毒及黄曲霉毒素B1的影响

目的研究黑曲霉对黄曲霉生长、产毒的抑制作用及对AFB1的降解作用。方法将黑曲霉分别与黄曲霉、AFB1共同培养,定期测定培养液pH、菌丝体干重、黄曲霉孢子数、AFB1含量。结果黑曲霉与黄曲霉混合培养时,黄曲霉孢子数、AFB1含量均比单独培养的低,2组之间差异有统计学意义(P<0.05),抑制率达到68.06%~91.52%;加入黑曲霉后,AFB1含量降低,实验组与对照组之间差异有统计学意义(P<0.05),降解率为46.19%。结论黑曲霉既能抑制黄曲霉生长、产毒,又能降解AFB1。     

Assessment of aflatoxin and cyclopiazonic acid production by Aspergillus flavus isolates from Hungary

Thirty-two isolates of Aspergillus flavus were obtained from various sources in Hungary. All isolates were morphologically identified as A. flavus and three atypical variants were confirmed as A. flavus by comparing their DNA with an ex type culture of A. flavus. None of these isolates produced aflatoxins when tested on coconut agar or grown on rice medium and culture extracts examined by thin layer chromatography. Also, none of the isolates converted sterigmatocystin, O-methyl sterigmatocystin, norsolorinic acid, or sodium acetate to aflatoxin. However, 59% of the isolates produced cyclopiazonic acid based on thin layer chromatographic analysis of culture extracts. The isolates that lack the ability to produce both aflatoxin and cyclopiazonic acid are potential candidates for use in bicontrol studies.     

Identification of genes differentially expressed during aflatoxin biosynthesis in Aspergillus flavus and Aspergillus parasiticus

A complex regulatory network governs the biosynthesis of aflatoxin. While several genes involved in aflatoxin production are known, their action alone cannot account for its regulation. Arrays of clones from an Aspergillus flavus cDNA library and glass slide microarrays of ESTs were screened to identify additional genes. An initial screen of the cDNA clone arrays lead to the identification of 753 unique ESTs. Many showed sequence similarity to known metabolic and regulatory genes; however, no function could be ascribed to over 50% of the ESTs. Gene expression analysis of Aspergillus parasiticus grown under conditions conducive and non-conductive for aflatoxin production was evaluated using glass slide microarrays containing the 753 ESTs. Twenty-four genes were more highly expressed during aflatoxin biosynthesis and 18 genes were more highly expressed prior to aflatoxin biosynthesis. No predicted function could be ascribed to 18 of the 24 genes whose elevated expression was associated with aflatoxin biosynthesis.     

Influence of woodsmoke on aflatoxin production by Aspergillus flavus

Summary Woodsmoke delayed aflatoxius B₁ and G₁ release and significantly exerted inhibitory effects on the toxins production by a toxigenic Asperigillus flavus. The fungistatic efficiency of the woodsmoke increased with reduced moisture content in fish.     

Screening for resistance to Aspergillus flavus and aflatoxin production in groundnut

All the varieties, advanced breeding lines, germplasm lines, and wild species used in the experiments differed significantly for their ability to allow invasion and aflatoxin production by an aflatoxigenicAspergillus flavus strain. Infection and colonisation were strongly correlated (r = 0.82), while there was no relation between infection and aflatoxin content or colonisation and aflatoxin content (r = 0.15). The varieties ICGS11 and S 206 supported less infection and colonisation (range 35 to 40%). Lowest aflatoxin content was recorded in Chitra (3,200 ppb), while it was highest in Kaushal (38,250 ppb). A cross derivative of GAUG1 × NC Ac 17133 R F showed lowest infection and colonisation (86,3 and 25,28%, respectively), and also supported moderate aflatoxin production (4,000 ppb). Among germplasm lines spancross supported lowest aflatoxin production (2,026 ppb) while both the wild species vz. ICG 8127 and ICG 8128 were highly susceptible to infection, colonisation, and aflatoxin production.     

Possible implications of reciprocity between ethylene and aflatoxin biogenesis in Aspergillus flavus and Aspergillus parasiticus

Aspergillus flavus and Aspergillus parasiticus produced ethylene during early growth. However, the onset of toxin biosynthesis was marked by the absence of ethylene evolution. 2-Chloroethyl phosphonic acid, an ethylene-generating compound, inhibited aflatoxin biosynthesis in vivo. The reciprocal relationship between the production of aflatoxin and ethylene by the organism may indicate the involvement of the latter in the regulation of aflatoxin biogenesis.     

Understanding the genetics of regulation of aflatoxin production and Aspergillus flavus development

Aflatoxins are polyketide-derived, toxic, and carcinogenic secondary metabolites produced primarily by two fungal species, Aspergillus flavus and A. parasiticus, on crops such as corn, peanuts, cottonseed, and treenuts. Regulatory guidelines issued by the U.S. Food and Drug Administration (FDA) prevent sale of commodities if contamination by these toxins exceeds certain levels. The biosynthesis of these toxins has been extensively studied. About 15 stable precursors have been identified. The genes involved in encoding the proteins required for the oxidative and regulatory steps in the biosynthesis are clustered in a 70 kb portion of chromosome 3 in the A. flavus genome. With the characterization of the gene cluster, new insights into the cellular processes that govern the genes involved in aflatoxin biosynthesis have been revealed, but the signaling processes that turn on aflatoxin biosynthesis during fungal contamination of crops are still not well understood. New molecular technologies, such as gene microarray analyses, quantitative polymerase chain reaction (PCR), and chromatin immunoprecipitation are being used to understand how physiological stress, environmental and soil conditions, receptivity of the plant, and fungal virulence lead to episodic outbreaks of aflatoxin contamination in certain commercially important crops. With this fundamental understanding, we will be better able to design improved non-aflatoxigenic biocompetitive Aspergillus strains and develop inhibitors of aflatoxin production (native to affected crops or otherwise) amenable to agricultural application for enhancing host-resistance against fungal invasion or toxin production. Comparisons of aflatoxin-producing species with other fungal species that retain some of the genes required for aflatoxin formation is expected to provide insight into the evolution of the aflatoxin gene cluster, and its role in fungal physiology. Therefore, information on how and why the fungus makes the toxin will be valuable for developing an effective and lasting strategy for control of aflatoxin contamination.     

Effect of phytate on aflatoxin formation by Aspergillus parasiticus and Aspergillus flavus in synthetic media

The effect of phytate on the production of aflatoxins by Aspergillus parasiticus and Aspergillus flavus grown on synthetic media was examined. In the absence of pH control (initial pH 4.5–6.5) for A. parasiticus, phytate (14.3 mM) caused a six-fold decrease in aflatoxins in the medium and a ten-fold decrease in those retained by the mycelia. When the initial pH of the medium was adjusted to 4.5 no effect on aflatoxin production was observed. With A. flavus or A. parasiticus grown on media with a higher initial pH value (6 to 7), the presence of phytate in the media caused an increase in aflatoxin production. These results are inconsistent with previous studies which indicated that phytate depresses aflatoxin production by rendering zinc, a necessary co-factor for aflatoxin biosynthesis, unavailable to the mold.