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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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 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.     

Quantification of conidial density of Aspergillus flavus and A. parasiticus in soil from almond orchards using real-time PCR

Aims:  To design the Aspergillus flavus and Aspergillus parasiticus -specific primers and a real-time PCR assay for quantification of the conidial density in soil.
Methods and Results:  Aspergillus flavus and A. parasiticus -specific DNA primers were designed based on internal transcribed spacer sequences to distinguish these two species and from other Aspergillus and other fungal species. A method of pathogen DNA extraction directly from soil samples was developed. Using the designed primers, a real-time PCR assay was developed to quantitatively determine the conidial density of each A. flavus and A. parasiticus in soil, after generating corresponding standard curves. Known conidial densities of each A. flavus or A. parasiticus in soil significantly correlated with those tested with the real-time PCR.
Conclusions:  This study demonstrated the applicability of the real-time PCR assay in studies of quantifying A. flavus and A. parasiticus in soil as inoculum sources.
Significance and Impact of the Study:  The A. flacus and A. parasitic -specific primers can be widely used in aflatoxin research. The real-time PCR assay developed in this study provides a potential approach to quantify the plant pathogen density from not only soil but also other sources in relation to aflatoxin contamination from environment, food and feed commodities.     

Growth, sclerotia and aflatoxin production by Aspergillus parasiticus: influence of food preservatives

The influence of six food preservatives on control of aflatoxin production by Aspergillus parasiticus was tested in SMKY and defined media at three concentrations, viz., 0.1, 0.5 and 1.0%. Propionic acid completely inhibited the yield of mycelia and sclerotia, and aflatoxin production in culture medium, mycelia and sclerotia of A. parasiticus at all concentrations, whereas citric acid showed inhibition only at 0.5 and 1.0% concentrations. Sodium metabisulphite did not permit mycelial growth and aflatoxin biosynthesis in SMKY liquid medium but allowed production of sclerotia and aflatoxin on solid media, while the rest of the food preservatives had only marginal inhibitory effects.     

Effect of corn and peanut cultivation on soil populations of Aspergillus flavus and A. parasiticus in southwestern Georgia.

The effect of corn and peanut cultivation on the proportion of Aspergillus flavus to A. parasiticus in soil was examined. Soil populations were monitored in three fields during three different years in southwestern Georgia. Each field was planted in both peanuts and corn, and soil was sampled within plots for each crop. A. flavus and A. parasiticus were present in similar proportions in plots from all fields at the beginning of the growing season. A. terreus, A. niger, and A. fumigatus were the other dominant aspergilli in soil. Fields A and B did not show drought stress in peanut or corn plants, and soil populations of A. flavus and A. parasiticus remained stable during the course of the year. In field C, drought stress in corn plants with associated A. flavus infection and aflatoxin contamination greatly increased soil populations of A. flavus relative to A. parasiticus upon dispersal of corn debris to the soil surface by a combine harvester. Colonization of organic debris after it has been added to the soil may maintain soil populations of A. parasiticus despite lower crop infection.     

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.     

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.     

Characterization and population analysis of the mating-type genes in Aspergillus flavus and Aspergillus parasiticus

We characterize the mating-type genes in Aspergillus flavus,Aspergillus parasiticus and Petromyces alliaceus. A single MAT1-1 or MAT1-2 gene was detected in the genomes of A. flavus and A. parasiticus, which is consistent with a potential heterothallic organization of MAT genes in these species. In contrast, the only known, functionally homothallic species in Aspergillus section Flavi, P. alliaceus, has tightly linked (<2kb) MAT1-1 and MAT1-2 genes, typical of other self-fertile homothallic euascomycetes. This is the first example of linked MAT genes within a homothallic species of Aspergillus. We tested the null hypothesis of no significant difference in the frequency of MAT1-1 and MAT1-2 in A. flavus and A. parasiticus sampled from a single peanut field in Georgia. For each species, mating-type frequencies were determined for the total population samples and for samples that were clone-corrected based on vegetative compatibility groups (VCGs) and aflatoxin gene cluster haplotypes. There was no significant difference in the frequency of the two mating types for A. flavus and A. parasiticus in either VCG or haplotype clone-corrected samples. The existence of both mating-type genes in equal proportions in A. flavus and A. parasiticus populations, coupled with their expression at the mRNA level and the high amino acid sequence identity of MAT1-1 (77%) and MAT1-2 (83%) with corresponding homologs in P. alliaceus, indicates the potential functionality of these genes and the possible existence of a sexual state in these agriculturally important species.     

Cloning of a sugar utilization gene cluster in Aspergillus parasiticus

At one end of the 70 kb aflatoxin biosynthetic pathway gene cluster in Aspergillus parasiticus and Aspergillus flavus reported earlier, we have cloned a group of four genes that constitute a well-defined gene cluster related to sugar utilization in A. parasiticus: (1) sugR, (2) hxtA, (3) glcA and (4) nadA. No similar well-defined sugar gene cluster has been reported so far in any other related Aspergillus species such as A. flavus, A. nidulans, A. sojae, A. niger, A. oryzae and A. fumigatus. The expression of the hxtA gene, encoding a hexose transporter protein, was found to be concurrent with the aflatoxin pathway cluster genes, in aflatoxin-conducive medium. This is significant since a close linkage between the two gene clusters could potentially explain the induction of aflatoxin biosynthesis by simple sugars such as glucose or sucrose.