Mold flora and aflatoxin contamination of stored and cooked samples of pearl millet in the Paharia tribal belt of Santhal paragana, Bihar, India

Stored and cooked samples of pearl millet (Pennesetum typhoides), which is regularly consumed as food by the Paharia tribe in the hilly regions of Santhal Pargana, Bihar State, India, that were harvested in January 1989 were analyzed for mold flora, natural occurrence of Aspergillus flavus and A. parasiticus, and incidence and levels of aflatoxin B1. Of the 22 fungal species isolated, A. flavus and A. parasiticus were the predominant species (63.8%) during the rainy season, followed by other species of Aspergillus, Penicillium, Fusarium, Rhizopus, Helminthosporium, and Curvularia. Screening of 169 A. flavus and A. parasiticus strains showed that 59 of them were toxigenic, producing various combinations of aflatoxins B1, B2, G1, and G2. The amounts of aflatoxin B1 ranged between 4 and 30 mg/100 ml of liquid medium. Analysis of stored and cooked samples also revealed a high incidence and alarming levels of naturally produced aflatoxin B1. Forty-nine of 75 stored and 16 of 38 cooked samples contained various combinations of aflatoxins. The levels of aflatoxin B1 ranged between 17 and 2,110 ppb in stored samples and 18 and 549 ppb in cooked samples. The correlation of insect damage with A. flavus and A. parasiticus incidence and quantity of aflatoxin B1 was found to be insignificant.     

Mold flora and aflatoxin contamination of stored and cooked samples of pearl millet in the Paharia tribal belt of Santhal paragana, Bihar, India.

Stored and cooked samples of pearl millet (Pennesetum typhoides), which is regularly consumed as food by the Paharia tribe in the hilly regions of Santhal Pargana, Bihar State, India, that were harvested in January 1989 were analyzed for mold flora, natural occurrence of Aspergillus flavus and A. parasiticus, and incidence and levels of aflatoxin B1. Of the 22 fungal species isolated, A. flavus and A. parasiticus were the predominant species (63.8%) during the rainy season, followed by other species of Aspergillus, Penicillium, Fusarium, Rhizopus, Helminthosporium, and Curvularia. Screening of 169 A. flavus and A. parasiticus strains showed that 59 of them were toxigenic, producing various combinations of aflatoxins B1, B2, G1, and G2. The amounts of aflatoxin B1 ranged between 4 and 30 mg/100 ml of liquid medium. Analysis of stored and cooked samples also revealed a high incidence and alarming levels of naturally produced aflatoxin B1. Forty-nine of 75 stored and 16 of 38 cooked samples contained various combinations of aflatoxins. The levels of aflatoxin B1 ranged between 17 and 2,110 ppb in stored samples and 18 and 549 ppb in cooked samples. The correlation of insect damage with A. flavus and A. parasiticus incidence and quantity of aflatoxin B1 was found to be insignificant.     

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.     

Taxonomic comparison of three different groups of aflatoxin producers and a new efficient producer of aflatoxin B1, sterigmatocystin and 3-O-methylsterigmatocystin, Aspergillus rambellii sp. nov

Accumulation of the carcinogenic mycotoxin aflatoxin B, has been reported from members of three different groups of Aspergilli (4) Aspergillus flavus, A. flavus var. parvisclerotigenus, A. parasiticus, A. toxicarius, A. nomius, A. pseudotamarii, A. zhaoqingensis, A. bombycis and from the ascomycete genus Petromyces (Aspergillus section Flavi), (2) Emericella astellata and E. venezuelensis from the ascomycete genus Emericella (Aspergillus section Nidulantes) and (3) Aspergillus ochraceoroseus from a new section proposed here: Aspergillus section Ochraceorosei. We here describe a new species, A. rambellii referable to Ochraceorosei, that accumulates very large amounts of sterigmatocystin, 3-O-methylsterigmatocystin and aflatoxin B1, but not any of the other known extrolites produced by members of Aspergillus section Flavi or Nidulantes. G type aflatoxins were only found in some of the species in Aspergillus section Flavi, while the B type aflatoxins are common in all three groups. Based on the cladistic analysis of nucleotide sequences of ITS1 and 2 and 5.8S, it appears that type G aflatoxin producers are paraphyletic and that section Ochraceorosei is a sister group to the sections Flavi, Circumdati and Cervini, with Emericella species being an outgroup to these sister groups. All aflatoxin producing members of section Flavi produce kojic acid and most species, except A. bombycis and A. pseudotamarii, produce aspergillic acid. Species in Flavi, that produce B type aflatoxins, but not G type aflatoxins, often produced cyclopiazonic acid. No strain was found which produce both G type aflatoxins and cyclopiazonic acid. It was confirmed that some strains of A. flavus var. columnaris produce aflatoxin B2, but this extrolite was not detected in the ex type strain of that variety. A. flavus var. parvisclerotigenus is raised to species level based on the specific combination of small sclerotia, profile of extrolites and rDNA sequence differences. A. zhaoqingensis is regarded as a synonym of A. nomius, while A. toxicarius resembles A. parasiticus but differs with at least three base pair differences. At least 10 Aspergillus species can be recognized which are able to biosynthesize aflatoxins, and they are placed in three very different clades.     

Molasses supplementation promotes conidiation but suppresses aflatoxin production by small sclerotial Aspergillus flavus

AIMS: To find a supplemental ingredient that can be added to routinely used growth media to increase conidial production and decrease aflatoxin biosynthesis in small sclerotial (S strain) isolates of Aspergillus flavus. METHODS AND RESULTS: Molasses was added to three commonly used culture media: coconut agar (CAM), potato dextrose agar (PDA), and vegetable juice agar (V8) and production of conidia, sclerotia, and aflatoxins by A. flavus isolate CA43 was determined. The effect of nitrogen sources in molasses medium (MM) on production of conidia, sclerotia and aflatoxins was examined. Water activity and medium pH were also measured. Conidia harvested from agar plates were counted using a haemocytometer. Sclerotia were weighed after drying at 45 degrees C for 5 days. Aflatoxins B(1) and B(2) were quantified by high-performance liquid chromatography. Addition of molasses to the media did not change water activity or the pH significantly. Supplementing CAM and PDA with molasses increased conidial production and decreased aflatoxins. Two-fold increased yield of conidia was found on MM, which, like V8, did not support aflatoxin production. Adding ammonium to MM significantly increased the production of sclerotia and aflatoxins, but slightly decreased conidial production. Adding urea to MM significantly increased the production of conidia, sclerotia and aflatoxins. CONCLUSIONS: Molasses stimulated conidial production and inhibited aflatoxin production. Its effect on sclerotial production was medium-dependent. Water activity and medium pH were not related to changes in conidial, sclerotial or aflatoxin production. Medium containing molasses alone or molasses plus V8 juice were ideal for conidial production by S strain A. flavus. SIGNIFICANCE AND IMPACT OF THE STUDY: Insight into molecular events associated with the utilization of molasses may help to elucidate the mechanism(s) that decreases aflatoxin biosynthesis. Targeting genetic parameters in S strain A. flavus isolates may reduce aflatoxin contamination of crops by reducing the survival and toxigenicity of these strains.     

The phylogenetics of mycotoxin and sclerotium production in Aspergillus flavus and Aspergillus oryzae

Aspergillus flavus is a common filamentous fungus that produces aflatoxins and presents a major threat to agriculture and human health. Previous phylogenetic studies of A. flavus have shown that it consists of two subgroups, called groups I and II, and morphological studies indicated that it consists of two morphological groups based on sclerotium size, called "S" and "L." The industrially important non-aflatoxin-producing fungus A. oryzae is nested within group I. Three different gene regions, including part of a gene involved in aflatoxin biosynthesis (omt12), were sequenced in 33 S and L strains of A. flavus collected from various regions around the world, along with three isolates of A. oryzae and two isolates of A. parasiticus that were used as outgroups. The production of B and G aflatoxins and cyclopiazonic acid was analyzed in the A. flavus isolates, and each isolate was identified as "S" or "L" based on sclerotium size. Phylogenetic analysis of all three genes confirmed the inference that group I and group II represent a deep divergence within A. flavus. Most group I strains produced B aflatoxins to some degree, and none produced G aflatoxins. Four of six group II strains produced both B and G aflatoxins. All group II isolates were of the "S" sclerotium phenotype, whereas group I strains consisted of both "S" and "L" isolates. Based on the omt12 gene region, phylogenetic structure in sclerotium phenotype and aflatoxin production was evident within group I. Some non-aflatoxin-producing isolates of group I had an omt12 allele that was identical to that found in isolates of A. oryzae.     

Biosynthetic origin of aflatoxin G1: confirmation of sterigmatocystin and lack of confirmation of aflatoxin B1 as precursors

The origin of aflatoxin G1 was studied using mutant strains of Aspergillus parasiticus blocked early in the pathway and by tracing 14C-labelled aflatoxin B1 (AFB1) in wild-type A. flavus and A. parasiticus strains. Sterigmatocystin (ST) was a precursor of AFB1, AFG1 and AFG2 in the four mutants examined. The identity of AFG1 was confirmed by mass spectrometry. No evidence for conversion of AFB1 to AFG1 was found. A rigorously controlled study of conversions of radioactivity based on preparative thin-layer chromatography of aflatoxins demonstrated that low levels of aflatoxin interconversions previously reported in the literature might actually be artifacts.     

Bioremediation of aflatoxins by some reference fungal strains.

Aspergillus parasiticus RCMB 002001 (2) producing four types of aflatoxins B1, B2, G1, and G2 was used in this study as an aflatoxin-producer. Penicillium griseofulvum, P. urticae, Paecilomyces lilacinus, Trichoderma viride, Candida utilis, Saccharomyces cerevisiae as well as a non-toxigenic strain of Aspergillus flavus were found to be able to exhibit growth on aflatoxin B1-containing medium up to a concentration of 500 ppb. It was also found that several fungal strains exhibited the growth in co-culture with A. parasiticus, natural aflatoxins producer, and were able to decreased the total aflatoxin concentration, resulting in the highest inhibition percentage of 67.2% by T viride, followed by P. lilacinus, P. griseofulvum, S. cerevisiae, C. utilis, P. urticae, Rhizopus nigricans and Mucor rouxii with total aflatoxin inhibition percentage of 53.9, 52.4, 52, 51.7, 44, 38.2 and 35.4%, respectively. The separation of bioremediation products using GC/MS revealed that the toxins were degraded into furan moieties.     

Aflatoxin biosynthesis cluster gene cypA is required for G aflatoxin formation

Aspergillus flavus isolates produce only aflatoxins B1 and B2, while Aspergillus parasiticus and Aspergillus nomius produce aflatoxins B1, B2, G1, and G2. Sequence comparison of the aflatoxin biosynthesis pathway gene cluster upstream from the polyketide synthase gene, pksA, revealed that A. flavus isolates are missing portions of genes (cypA and norB) predicted to encode, respectively, a cytochrome P450 monooxygenase and an aryl alcohol dehydrogenase. Insertional disruption of cypA in A. parasiticus yielded transformants that lack the ability to produce G aflatoxins but not B aflatoxins. The enzyme encoded by cypA has highest amino acid identity to Gibberella zeae Tri4 (38%), a P450 monooxygenase previously shown to be involved in trichodiene epoxidation. The substrate for CypA may be an intermediate formed by oxidative cleavage of the A ring of O-methylsterigmatocystin by OrdA, the P450 monooxygenase required for formation of aflatoxins B1 and B2.     

Identification of O-methylsterigmatocystin as an aflatoxin B1 and G1 precursor in Aspergillus parasiticus.

An isolate of Aspergillus parasiticus CP461 (SRRC 2043) produced no detectable aflatoxins, but accumulated O-methylsterigmatocystin (OMST). When sterigmatocystin (ST) was fed to this isolate in a low-sugar medium, there was an increase in the accumulation of OMST, without aflatoxin synthesis. When radiolabeled [14C]OMST was fed to resting mycelia of a non-aflatoxin-, non-ST-, and non-OMST-producing mutant of A. parasiticus AVN-1 (SRRC 163), 14C-labeled aflatoxins B1 and G1 were produced; 10 nmol of OMST produced 7.8 nmol of B1 and 1.0 nmol of G1, while 10 nmol of ST produced 6.4 nmol of B1 and 0.6 nmol of G1. A time course study of aflatoxin synthesis in ST feeding experiments with AVN-1 revealed that OMST is synthesized by the mold during the onset of aflatoxin synthesis. The total amount of aflatoxins recovered from OMST feeding experiments was higher than from experiments in which ST was fed to the resting mycelia. These results suggest that OMST is a true metabolite in the aflatoxin biosynthetic pathway between sterigmatocystin and aflatoxins B1 and G1 and is not a shunt metabolite, as thought previously.     

Incidence of aflatoxin producing strains and aflatoxin contamination in dry fruit slices of quinces (Cydonia oblonga Mill.) from the Indian state of Jammu and Kashmir

An investigation was undertaken to obtain data on the occurrence of aflatoxins and the aflatoxin producing potential of Aspergillus flavus strains isolated from dry fruit slices of quinces produced in jammu and Kashmir, India. A total of 147 A. flavus isolates recovered from dr fruit slices were grown in liquid rice flour medium and screened for the production of various aflatoxins by thin layer chromatography. The results showed that 23.14% of the tested isolates were aflatoxigenic, producing aflatoxins B1 and B2 in varying amounts. Aflatoxins G1 and G2 were not detected. All 25 of the investigated market samples were also found to be aflatoxin B1 positive and the level of contamination ranged from 96 to 8164 micrograms/kg of the dry fruit which is quite high in comparison to the permissible level of 30 ppb. As per these results biochemical composition of dry fruit slices of quinces, along with climatic conditions seem to be very favourable for aflatoxin production by the toxigenic A. flavus strains. Therefore, monitoring of aflatoxins in dry fruit slices of quinces is recommended for this region.     

Genetic diversity in Aspergillus parasiticus population from the peanut agroecosystem in Argentina

AIMS: The aims of this work were to identify potential sources of Aspergillus parasiticus inoculum and to evaluate the sclerotial and toxigenic profiles of this species from the peanut agroecosystem in Argentina. Likewise, the genetic diversity of A. parasiticus population was analysed using vegetative compatibility group (VCG) analysis. METHODS AND RESULTS: The A. parasiticus strains were isolated from soil, debris and peanut seeds in Córdoba Province, Argentina. A. parasiticus was recovered from the three sources analysed. Only 11 of 185 A. parasiticus isolates (5.9%) did not produce aflatoxins, while 57% produced sclerotia. Twenty-four VCG were identified from 63 isolates. The VCG diversity index for A. parasiticus, expressed as the number of groups divided by the total number of isolates, was 0.31. In general, there were significant differences among VCG in aflatoxin production. CONCLUSIONS: The presence of aflatoxigenic strains of A. parasiticus in the three substrates suggests that they may be an important source of aflatoxin in Argentina's peanut agroecosystem. The A. parasiticus population shows a low genetic diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study showed data on inoculum distribution, aflatoxin and sclerotia production and genetic diversity in an A. parasiticus population isolated from the peanut agroecosystem in Argentina.     

Precursor recognition by kinetic pulse-labeling in a toxigenic aflatoxin B1-producing strain of Aspergillus.

Kinetic pulse-labeling of aflatoxin pathway compounds was carried out in Aspergillus parasiticus, beginning with radioactive acetate. Norsolorinic acid, averufin, versicolorin A, and sterigmatocystin (all known as compounds which can be incorporated into the aflatoxin molecule) were radiotraced to follow their order of appearance. Aflatoxin species B1, B2, G1, and G2 were included. Norsolorinic acid and averufin appeared as early transient intermediates followed in order by versicolorin A, aflatoxins, and sterigmatocystin. To date, a mutually confirming array of results has been obtained with established precursors in wild-type strains of A. parasiticus and A. versicolor (as well as with an aflatoxin pathway mutant of A. parasiticus), which together establish a practical methodology for recognition of new pathway intermediates. The kinetic of pulse-labeling for sterigmatocystin in relation to aflatoxins suggests that duel branchlets may exist to flatoxins; i.e., sterigmatocystin may not be an obligatory aflatoxin precursor.     

Aflatoxin Production in Meats. I. Stored Meats

Aflatoxins were produced on fresh beef (in which bacterial spoilage was delayed with antibiotics), ham, and bacon inoculated with toxinogenic fungi and stored at 15, 20 and 30 C. Meats stored at 10 C were spoiled by bacteria and yeast before detectable levels of aflatoxins were produced. High levels of aflatoxins were formed in meats stored at 20 C; one sample supported the production of 630 mug of aflatoxins per g of meat, the major portion (580 mug) of which was aflatoxin G(1). Meats stored below 30 C developed higher levels of aflatoxin G(1) than B(1), but at 30 C Aspergillus flavus produced equal amounts of B(1) and G(1), whereas A. parasiticus continued to produce more G(1) than B(1).     

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.     

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.     

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.     

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.     

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.