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.     

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.     

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).     

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.     

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.     

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.     

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.     

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.     

Survey of aflatoxins and ochratoxin A in stored tubers ofCyperus esculentus L.

The mold incidence, moisture contents, pH and levels of mycotoxins (aflatoxins B₁, G₁ and ochratoxin A) on/of/in rootstock snack (tubers ofCyperus esculentus L.) samples were monitored during a 150-day storage period. Whereas the mold incidence, moisture and mycotoxin levels increased with storage time, the pH declined during the same period. Altogether, 12 fungal species, mostly toxigenic, includingAspergillus flavus, A. parasiticus andA. ochraceus were isolated. At collection period only 3 of the 9 snack samples analysed contained trace amounts of aflatoxins. By 120th day, all the 9 samples were contaminated and the average levels were 454 and 80 ppb for aflatoxin B₁ and aflatoxin G₁ respectively on the 150th day. Ochratoxin A was not detected before 120th day and then only at low levels, occuring in a maximum of four samples and ranging between 10 and 80 ppb.     

Effects of Moisture Content and Temperature on Aflatoxin Production in Corn

Samples of freshly harvested and remoistened corn, of various moisture contents, were stored at different temperatures; analyses for aflatoxin content were made periodically. At moisture levels above 17.5% and at temperatures of 24 C or warmer, aflatoxins were formed by Aspergillus flavus present in the original epiphytic mycoflora. Remoistened dried corn was subject to more rapid fungal deterioration and aflatoxin formation than freshly harvested corn. Screening of the fungi present in the corn revealed aflatoxin production only by A. flavus. The toxigenic strains produced only aflatoxins B(1) and B(2).     

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.     

Mycotoxicological control on raw material and tablets of cascara sagrada (Rhamnus purshiana)

Among phytotherapic medicines, tablets of cascara sagrada (Rhamnus purshiana) dried bark, usually used as laxative, are commercially widespread in our market. Taking into account natural origin and/or inappropriate procedures that may allow the occurrence of toxinogenic Aspergillus flavus group, a study on susceptibility to aflatoxin contamination and natural aflatoxin incidence was performed by TLC and HPLC methods. This survey allows one to conclude that bark of Cascara Sagrada is a good substrate for the growth of A. parasiticus NRRL 2999 and for aflatoxins production. Natural anatoxins presence was detected on 2 from 9 raw material samples. One of them (irradiated sample) had only aflatoxin B1 (10 μg/kg) and the other (pasteurized) was positive for aflatoxin B1 (19 μg/kg); G1 (6 μg/kg) and B2 (1.46 μg/kg). Only one from 10 lots of tablets analyzed was positive for aflatoxin B1 (5.42 μg/kg) and B2 (0.32 μg/kg).Therefore, adequate quality control including an aflatoxins assay must be performed to guarantee the harmlessness of natural drugs.     

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.     

Mycotoxins and mycotoxigenic moulds in nuts and sunflower seeds for human consumption

A survey was carried out to obtain data on the occurrence of mycotoxins and the mycotoxin-producing potential of fungi isolated from nuts (almonds, peanuts, hazelnuts, pistachio nuts) and sunflower seeds in Spain. Thin-layer chromatography was used to separate the toxins. Aflatoxins were detected in one sample of almonds (95 ppb aflatoxin B₁ and 15 ppb aflaxtoxin B₂) and in one sample of peanuts at a level below 10 ppb of aflatoxin B1. 100% of samples showed variable incidence of fungal contamination. The predominant fungi present in samples were Penicillium spp, Aspergillus niger, A. flavus, A. glaucus and Rhizopus spp. The results showed that isolates of different species were able to produce aflatoxins B₁, B₂, G₁, and G₂, sterigmatocystin, ochratoxin A, patulin, citrinin, penicillic acid, zearalenone, and griseofulvin.     

Influence of temperature cycling on the production of aflatoxins B1 and G1 by Aspergillus parasiticus.

The effect of temperature cycling on the relative productions of aflatoxins B1 and G1 by Aspergillus parasiticus NRRL 2999 was studied. The cycling of temperature between 33 and 15 degrees C favored aflatoxin B1 accumulation, whereas cycling between 35 and 15 degrees C favored aflatoxin G1 production. Cultures subjected to temperature cycling between 33 and 25 degrees C at various time intervals changed the relative productions of aflatoxins B1 and G1 drastically. Results obtained with temperature cycling and yeast extract-sucrose medium with ethoxyquin to decrease aflatoxin G1 production suggest that the enzyme system responsible for the conversion of aflatoxin B1 to G1 might be more efficient at 25 degrees C than at 33 degrees C. The possible explanation of the effect of both constant and cycling temperatures on the relative accumulations of aflatoxins B1 and G2 might be through the control of the above enzyme system. The study also showed that greater than 57% of aflatoxin B1, greater than 47% of aflatoxin G1, and greater than 50% of total aflatoxins (B1 plus G1) were in the mycelium by day 10 under both constant and cyclic temperature conditions.     

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.     

Mycoflora and incidence of aflatoxin B1, zearalenone and deoxynivalenol in poultry feeds in Argentina

In Argentina, there is rather little information about the natural occurrence of mycotoxins in feedstuffs. The aim of this work was to determine the fungal flora and natural incidence of aflatoxin B1 (AFB1), zearalenone (ZEA) and deoxynivalenol (DON) in poultry feeds from 5 factories of Río Cuarto, Córdoba. Three hundred samples were taken from May 1995 to May 1996. Fungal counts of poultry feeds ranged 104 to 106 CFU g-1. The lowest counts were obtained on the first months from the sampling (May to September 1995) with mean values significantly different from those found at the last of the sampling (October 1995 to April 1996). The most prevalent species isolated of poultry feed samples belonged to the genera Penicillium that was present in 98% of the samples, Fusarium (87%) and Aspergillus (52%). Fusarium species isolated were: F moniliforme in 73% of the samples, F subglutinans (35%), F graminearum (20%) and within Aspergillus species: A. parasiticus (33%) and A. flavus (8%) were identified. In poultry feeds aflatoxin B1 (AFB1) was the most significant mycotoxin with levels ranging from 17 to 197 ng/g. For deoxynivalenol (DON) the levels ranged from 240 to 410 ng/g. Only three out of 300 samples were contaminated with zearalenone (ZEA) in concentrations of 30, 120 and 280 ng/g. These are preliminary data on this subject in our region. This revised version was published online in June 2006 with corrections to the Cover Date.     

Aflatoxin Production of Species and Strains of the Aspergillus flavus Group Isolated from Field Crops

Peanuts, cottonseed, rice, and sorghum from Texas were sampled over a 3-year period. To insure adequate isolation of alfatoxin-producing species of fungi, low-quality lots were sampled at a rate greater than their respective proportional representation. Aflatoxins were found each year in peanut and cottonseed and were found in 2 of 3 years in rice and sorghum. Aflatoxins were detected in all four crops. The Aspergillus flavus group was much more prevalent in peanut and rice than in cottonseed and sorghum. Of the isolates of the A. flavus group, 96% from peanuts, 79% from cottonseed, 49% from sorghum, and 35% from rice produced aflatoxins. The average toxin production of isolates from rice was much less than that from peanuts, cottonseed, or sorghum. More than 90% of all isolates of the A. flavus group were identified as the species A. flavus. A. parasiticus was isolated from all four crops. Only A. parasiticus produced aflatoxin G.     

Mycoflora and Occurrence of Aflatoxin B 1 in Dried Yam Chips from Markets in Ogun and Oyo States,Nigeria

Seventy-six samples of dried yam chips locally called elubo isu were purchased in 2000 from markets in Ogun and Oyo States of southwestern Nigeria. The samples were assessed for pH, moisture content, associated fungi and aflatoxin B1 contamination. The pH of samples ranged from 5.6 to 6.1,while the moisture contents varied from 6.8 to 14.5% in Ogun samples, and 7.1 to 13.6% in samples from Oyo. Aspergillus and Penicillium were the two prevalent genera of fungi, and the number of colony forming units per gram of these two genera in the yam chips studied exceeded the tolerance limit in foodstuffs. The other fungal genera isolated included Botryodiplodia, Cladosporium, Fusarium, Rhizopus, Mucor, Aureobasidium and Paecilomyces. The two most frequent fungal species were A. niger and A. flavus. Thin layer chromatographic analysis showed that 17 samples or 22% contained aflatoxin B1 beyond the detection limit (5 ppb), but only three samples or 4% had toxin level above 30 ppb, the tolerance level in food for human consumption. The mean concentration of aflatoxin B1 in positive samples was 27.1 ppb.