Direct visual detection of aflatoxin synthesis by minicolonies of Aspergillus species

Single-spore colonies of Aspergillus flavus and Aspergillus parasiticus, grown for 4 to 5 days at 25 degrees C on a coconut extract agar containing sodium desoxycholate as a growth inhibitor, produced aflatoxin, readily detectable as blue fluorescent zones under long-wave (365 nm) UV light. Over 100 colonies per standard petri dish were scored for aflatoxin production by this procedure. Progeny from some strains remained consistently stable for toxin production after repeated subculture, whereas instability for toxin synthesis was revealed among progeny from other strains. Spore color markers were used to rule out cross-contamination in monitoring strains. A yellow-spored and nontoxigenic strain of A. flavus, reported previously to produce aflatoxin in response to cycloheximide treatment, proved to be toxin negative even after repeated exposure to cycloheximide. Extended series of progeny from another strain of A. flavus and from a strain of A. parasiticus were each compared by this plating procedure and by fluorometric analysis for aflatoxin when grown in a coconut extract broth. Both of these strains showed variation for toxin synthesis among their respective progeny, and specific progeny showed a good correlation for aflatoxin synthesis when examined by the two procedures.     

Detection of toxigenic isolates of Aspergillus flavus and related species on coconut cream agar

A new readily-prepared medium, coconut cream agar, was developed for the detection of aflatoxin production by isolates of Aspergillus flavus and related species. Coconut cream agar, which comprised coconut cream (50%) and agar (1.5%), detected isolates of A. flavus more effectively than the synthetic media tested and was as effective as media containing desiccated coconut. Fluorescence colouring of colonies grown on coconut cream agar could be used to differentiate A. flavus from A. parasiticus and A. nomius. In addition, conidial colour of A. flavus and A. nomius was quite distinct from that of A. parasiticus.     

Improved method of screening for aflatoxin with a coconut agar medium

Nine isolates of Aspergillus flavus and Aspergillus parasiticus were screened for aflatoxin production on a coconut extract agar medium. Aflatoxin-producing colonies were detected under long-wave UV light (365 nm) by blue fluorescence on the reverse side after 2 to 5 days of growth. Aflatoxin production was verified by chemical analysis. Several types of shredded coconut available in the United States were tested and found to be satisfactory. No additives were required. Various parameters affecting the test were investigated.     

Improved method of screening for aflatoxin with a coconut agar medium.

Nine isolates of Aspergillus flavus and Aspergillus parasiticus were screened for aflatoxin production on a coconut extract agar medium. Aflatoxin-producing colonies were detected under long-wave UV light (365 nm) by blue fluorescence on the reverse side after 2 to 5 days of growth. Aflatoxin production was verified by chemical analysis. Several types of shredded coconut available in the United States were tested and found to be satisfactory. No additives were required. Various parameters affecting the test were investigated.     

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.     

Color mutants of Aspergillus flavus and Aspergillus parasiticus in a study of preharvest invasion of peanuts

A comparison of the invasion of flowers, aerial pegs, and kernels by wild-type and mutant strains of Aspergillus flavus or A. parasiticus along with aflatoxin analyses of kernels from different drought treatments have supported the hypothesis that preharvest contamination with aflatoxin originates mainly from the soil. Evidence in support of soil invasion as opposed to aerial invasion was the following. A greater percentage of invasion of kernels rather than flower or aerial pegs by either wild-type A. flavus or mutants. Significant invasion by an A. parasiticus color mutant occurred only in peanuts from soil supplemented with the mutant, whereas adjacent plants in close proximity but in untreated soil were only invaded by wild-type A. flavus or A. parasiticus. Aflatoxin data from drought-stressed, visibly undamaged peanut kernels showed that samples from soil not supplemented with a mutant strain contained a preponderance of aflatoxin B's (from wild-type A. flavus) whereas adjacent samples from mutant-supplemented soil contained a preponderance of B's plus G's (from wild-type and mutant A. parasiticus). Preliminary data from two air samplings showed an absence of propagules of A. flavus or A. parasiticus in air around the experimental facility.     

Color mutants of Aspergillus flavus and Aspergillus parasiticus in a study of preharvest invasion of peanuts.

A comparison of the invasion of flowers, aerial pegs, and kernels by wild-type and mutant strains of Aspergillus flavus or A. parasiticus along with aflatoxin analyses of kernels from different drought treatments have supported the hypothesis that preharvest contamination with aflatoxin originates mainly from the soil. Evidence in support of soil invasion as opposed to aerial invasion was the following. A greater percentage of invasion of kernels rather than flower or aerial pegs by either wild-type A. flavus or mutants. Significant invasion by an A. parasiticus color mutant occurred only in peanuts from soil supplemented with the mutant, whereas adjacent plants in close proximity but in untreated soil were only invaded by wild-type A. flavus or A. parasiticus. Aflatoxin data from drought-stressed, visibly undamaged peanut kernels showed that samples from soil not supplemented with a mutant strain contained a preponderance of aflatoxin B's (from wild-type A. flavus) whereas adjacent samples from mutant-supplemented soil contained a preponderance of B's plus G's (from wild-type and mutant A. parasiticus). Preliminary data from two air samplings showed an absence of propagules of A. flavus or A. parasiticus in air around the experimental facility.     

Intrafungal distribution of aflatoxins among conidia and sclerotia of Aspergillus flavus and Aspergillus parasiticus

This research examines the distribution of aflatoxins among conidia and sclerotia of toxigenic strains of Aspergillus flavus Link and Aspergillus parasiticus Speare cultured on Czapek agar (21 days, 28 degrees C). Total aflatoxin levels in conidia and sclerotia varied considerably both within (intrafungal) and among strains. Aspergillus flavus NRRL 6554 accumulated the highest levels of aflatoxin (conidia: B1, 84000 ppb; G1, 566000 ppb; sclerotia: B1, 135000 ppb; G1, 968000 ppb). Substantial aflatoxin levels in conidia could place at risk those agricultural workers exposed to dust containing large numbers of A. flavus conidia. Cellular ratios of aflatoxin B1 to aflatoxin G1 were nearly identical in conidia and sclerotia even though levels of total aflatoxins in these propagule types may have differed greatly. Aflatoxin G1 was detected in sclerotia of all A. flavus strains but in the conidia of only one strain. Each of the A. parasiticus strains examined accumulated aflatoxin G1 in both sclerotia and conidia. These results are examined in the context of current evolutionary theory predicting an increase in the chemical defense systems of fungal sclerotia, propagules critical to the survival of these organisms.     

Variation of Aflatoxin Content of Cultures of Aspergillus flavus with Duration of Incubation and its Relation to Studies on Aflatoxin Production

S ummary : Strains of Aspergillus flavus recently isolated from coconut products were cultured on grated coconut. The aflatoxin content of serial cultures was found to vary significantly with duration of incubation and for some strains to show more than one phase of increase of aflatoxin content. The occurrence of these variations indicates that the study of aflatoxigenic capacity of strains or of the capacity of a medium to support toxin production, should be based upon a knowledge of the pattern of variation of toxin content with duration of incubation of the cultures under the experimental conditions used. Assay of toxin level in a culture after one period of incubation could lead to erroneous conclusions about the identity or quantities of toxin components which the strain is able to produce.     

Aflatoxin contamination in soybeans: role of proteinase inhibitors, zinc availability, and seed coat integrity

Soybean trypsin inhibitors are thought to ward off pathogens. Studies with aflatoxigenic strains of Aspergillus flavus and A. parasiticus, frequent soybean contaminants, revealed that trypsin inhibitors do not affect the growth of these fungi and aflatoxin production. Further, the availability of zinc, an essential mineral for aflatoxin synthesis that was thought to explain increased aflatoxin accumulation in cooked compared with raw soybeans, was shown to decrease upon cooking. Seed coat integrity, ensuring limited access and a low moisture content, is responsible for the slow colonization of the seed by A. flavus.     

Aflatoxin contamination in soybeans: role of proteinase inhibitors, zinc availability, and seed coat integrity.

Soybean trypsin inhibitors are thought to ward off pathogens. Studies with aflatoxigenic strains of Aspergillus flavus and A. parasiticus, frequent soybean contaminants, revealed that trypsin inhibitors do not affect the growth of these fungi and aflatoxin production. Further, the availability of zinc, an essential mineral for aflatoxin synthesis that was thought to explain increased aflatoxin accumulation in cooked compared with raw soybeans, was shown to decrease upon cooking. Seed coat integrity, ensuring limited access and a low moisture content, is responsible for the slow colonization of the seed by A. flavus.     

Function of native OmtA in vivo and expression and distribution of this protein in colonies of Aspergillus parasiticus

The activities of two enzymes, a 168-kDa protein and a 40-kDa protein, OmtA, purified from the filamentous fungus Aspergillus parasiticus were reported to convert the aflatoxin pathway intermediate sterigmatocystin to O-methylsterigmatocystin in vitro. Our initial goal was to determine if OmtA is necessary and sufficient to catalyze this reaction in vivo and if this reaction is necessary for aflatoxin synthesis. We generated A. parasiticus omtA-null mutant LW1432 and a maltose binding protein-OmtA fusion protein expressed in Escherichia coli. Enzyme activity analysis of OmtA fusion protein in vitro confirmed the reported catalytic function of OmtA. Feeding studies conducted with LW1432 demonstrated a critical role for OmtA, and the reaction catalyzed by this enzyme in aflatoxin synthesis in vivo. Because of a close regulatory link between aflatoxin synthesis and asexual sporulation (conidiation), we hypothesized a spatial and temporal association between OmtA expression and conidiospore development. We developed a novel time-dependent colony fractionation protocol to analyze the accumulation and distribution of OmtA in fungal colonies grown on a solid medium that supports both toxin synthesis and conidiation. OmtA-specific polyclonal antibodies were purified by affinity chromatography using an LW1432 protein extract. OmtA was not detected in 24-h-old colonies but was detected in 48-h-old colonies using Western blot analysis; the protein accumulated in all fractions of a 72-h-old colony, including cells (0 to 24 h) in which little conidiophore development was observed. OmtA in older fractions of the colony (24 to 72 h) was partly degraded. Fluorescence-based immunohistochemical analysis conducted on thin sections of paraffin-embedded fungal cells from time-fractionated fungal colonies demonstrated that OmtA is evenly distributed among different cell types and is not concentrated in conidiophores. These data suggest that OmtA is present in newly formed fungal tissue and then is proteolytically cleaved as cells in that section of the colony age.     

Aflatoxin: A metabolic product of several fungi

Summary Culture extracts produced by 107 fungi isolated from corn grains were assayed by thin layer chromatography for aflatoxin. Certain isolates ofAspergillus flavus, A. niger, A. parasiticus, A. ruber, A. wentii, Penicillium citrinum, andP. variabile produced aflatoxin.Penicillium frequentans andP. puberulum elaborated this toxin only in trace amounts. Bioassays of extracts from 4 of these fungi showed that only the extract fromA. parasiticus was highly toxic.     

Sequence variability in homologs of the aflatoxin pathway gene aflR distinguishes species in Aspergillus section Flavi.

The Aspergillus parasiticus aflR gene, a gene that may be involved in the regulation of aflatoxin biosynthesis, encodes a putative zinc finger DNA-binding protein. PCR and sequencing were used to examine the presence of aflR homologs in other members of Aspergillus Section Flavi. The predicted amino acid sequences indicated that the same zinc finger domain, CTSCASSKVRCTKEKPACARCIERGLAC, was present in all of the Aspergillus sojae, Aspergillus flavus, and Aspergillus parasiticus isolates examined and in some of the Aspergillus oryzae isolates examined. Unique base substitutions and a specific base deletion were found in the 5' untranslated and zinc finger region; these differences provided distinct fingerprints. A. oryzae and A. flavus had the T-G-A-A-X-C fingerprint, whereas A. parasiticus and A sojae had the C-C-C-C-C-T fingerprint at the corresponding positions. Specific nucleotides at positions -90 (C or T) and -132 (G or A) further distinguished A. flavus from A. oryzae and A. parasiticus from A. sojae, respectively. A sojae ATCC 9362, which was previously designated A. oryzae NRRL 1988, was determined to be a A. sojae strain on the basis of the presence of the characteristic fingerprint, A-C-C-C-C-C-C-T. The DNAs of other members of Aspergillus Section Flavi, such as Aspergillus nomius and Aspergillus tamarii, and some isolates of A. oryzae appeared to exhibit low levels of similarity to the A. parasiticus aflR gene since low amounts of PCR products or no PCR products were obtained when DNAs from these strains were used.     

Stimulation of aflatoxin biosynthesis by lipophilic epoxides

Epoxy fatty acids added to the culture media either with the inoculum or at the end of exponential growth phase stimulated aflatoxin production by toxigenic strains of Aspergillus flavus and Aspergillus parasiticus. This effect did not appear when the unsaturated fatty acids used for the synthesis of the epoxides and the polyhydroxyacids (which can be considered to be derived from the opening of the oxirane ring) replaced the epoxides in the culture media. No significant differences were detected in the lipid fractions (diglycerides, sterols, triglycerides, free fatty acids, sterol esters) extracted from the mycelia grown in the presence of any of the fatty acid derivates.     

Cloning of the Aspergillus parasiticus apa-2 gene associated with the regulation of aflatoxin biosynthesis.

An Aspergillus parasiticus gene, designated apa-2, was identified as a regulatory gene associated with aflatoxin biosynthesis. The apa-2 gene was cloned on the basis of overproduction of pathway intermediates following transformation of fungal strains with cosmid DNA containing the aflatoxin biosynthetic genes nor-1 and ver-1. Transformation of an O-methylsterigmatocystin-accumulating strain, A. parasiticus SRRC 2043, with a 5.5-kb HindIII-XbaI DNA fragment containing apa-2 resulted in overproduction of all aflatoxin pathway intermediates analyzed. Specific enzyme activities associated with the conversion of norsolorinic acid and sterigmatocystin were increased approximately twofold. The apa-2 gene was found to complement an A. flavus afl-2 mutant strain for aflatoxin production, suggesting that apa-2 is functionally homologous to afl-2. Comparison of the A. parasiticus apa-2 gene DNA sequence with that of the A. flavus afl-2 gene (G. A. Payne, G. J. Nystorm, D. Bhatnagar, T. E. Cleveland, and C. P. Woloshuk, Appl. Environ. Microbiol. 59:156-162, 1993) showed that they shared > 95% DNA homology. Physical mapping of cosmid subclones placed apa-2 approximately 8 kb from ver-1.     

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.     

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.