Production of Aflatoxins B1 and G1 by Aspergillus flavus and Aspergillus parasiticus Isolated from Market Pecans

One hundred and forty-eight isolates of Aspergillus flavus and A. parasiticus were isolated from 5,608 pecans obtained from Chicago and Georgia markets. The percentage of internal contamination by these species was 7.3% in the Chicago market pecans and 1.7% in those from markets in Georgia. Of the 148 isolates, 93% of the A. parasiticus, but only 54% of the A. flavus, were capable of producing aflatoxin. Overall, 57% of the isolates were potentially aflatoxigenic. A. parasiticus isolates generally produced a greater amount of aflatoxins than A. flavus.     

Investigation of Reported Aflatoxin Production by Fungi Outside the Aspergillus flavus Group

A screening study of 121 fungus isolates, representing 29 species, for aflatoxin synthesis demonstrated this property only in Aspergillus flavus and A. parasiticus. Eight of the organisms found negative were isolates reported by other investigators to produce aflatoxin. Since similar negative reports have come from several other workers, it is concluded that only the A. flavus group of Aspergillus can presently be certified as sources of these toxins. Reasons for possible false-positive findings are discussed along with precautionary measures and differential analytical procedures useful in aflatoxin screening studies.     

Aflatoxin Production by Aspergillus flavus as Related to Various Temperatures

Two aflatoxin-producing isolates of Aspergillus flavus were grown for 5 days on Wort media at 2, 7, 13, 18, 24, 29, 35, 41, 46, and 52 C. Maximal production of aflatoxins occurred at 24 C. Maximal growth of A. flavus isolates occurred at 29 and 35 C. The ratio of the production of aflatoxin B₁ to aflatoxin G₁ varied with temperature. Aflatoxin production was not related to growth rate of A. flavus; one isolate at 41 C, at almost maximal growth of A. flavus, produced no aflatoxins. At 5 days, no aflatoxins were produced at temperatures lower than 18 C or higher than 35 C. Color of CHCl₃ extracts appeared to be directly correlated with aflatoxin concentrations. A. flavus isolates grown at 2, 7, and 41 C for 12 weeks produced no aflatoxins. At 13 C, both isolates produced aflatoxins in 3 weeks, and one isolate produced increasing amounts with time. The second isolate produced increasing amounts through 6 weeks, but at 12 weeks smaller amounts of aflatoxins were recovered than at 6 weeks.     

Molecular characterization of atoxigenic Aspergillus flavus isolates collected in China

Aspergillus flavus strains were isolated frompeanut fields of Liaoning, Shandong, Hubei and Guangdong Provinces in China, and identified through phenotypic and molecular approaches. Of the 323 A. flavus strains isolated, 76 strains did not produce aflatoxins detectable by UPLC. The incidence of atoxigenic A. flavus strains decreased with increase in temperature and increased with increase in latitude in different geographical locations. Amplification of all the aflatoxin genes in the aflatoxin gene cluster in the atoxigenic isolates showed that there were 25 deletion patterns (A–Y), with 22 deletion patterns identified for the first time. Most of the atoxigenic A. flavus isolates with gene deletions (97%) had deletions in at least one of the four genes (aflT, nor-1, aflR, and hypB), indicating that these four genes could be targeted for rapid identification of atoxigenic strains. The atoxigenic isolates with gene deletions, especially the isolates with large deletions, are potential candidates for aflatoxin control.     

Nutrient Environments Influence Competition among Aspergillus flavus Genotypes

The population dynamics of Aspergillus flavus, shaped in part by intraspecific competition, influence the likelihood and severity of crop aflatoxin contamination. Competition for nutrients may be one factor modulating intraspecific interactions, but the influences of specific types and concentrations of nutrients on competition between genotypes of A. flavus have not been investigated. Competition between paired A. flavus isolates on agar media was affected by varying concentrations of carbon (sucrose or asparagine) and nitrogen (nitrate or asparagine). Cocultivated isolate percentages from conidia and agar-embedded mycelia were quantified by measurements of isolate-specific single-nucleotide polymorphisms with quantitative pyrosequencing. Compositions and concentrations of nutrients influenced conidiation resulting from cocultivation, but the percentages of total conidia from each competing isolate were not predicted by sporulation of isolates grown individually. Success during sporulation did not reflect the outcomes of competition during mycelial growth, and the extents to which isolate percentages from conidia and mycelia differed varied among both isolate pairs and media. Whether varying concentrations of sucrose, nitrate, or asparagine increased, decreased, or had no influence on competitive ability was isolate dependent. Different responses of A. flavus isolates to nutrient variability suggest genotypes are adapted to different nutrient environments that have the potential to influence A. flavus population structure and the epidemiology of aflatoxin contamination.     

Divergence of West African and North American Communities of Aspergillus Section Flavi

West African Aspergillus flavus S isolates differed from North American isolates. Both produced aflatoxin B₁. However, 40 and 100% of West African isolates also produced aflatoxin G₁ in NH₄ medium and urea medium, respectively. No North American S strain isolate produced aflatoxin G₁. This geographical and physiological divergence may influence aflatoxin management.     

Aflatoxin B1 producing potential of isolates of Aspergillus flavus Link ex Fries from cotton,maize and wheat

Twenty-one isolates ofAspergillus flavus Link ex Fries obtained from cotton, maize and wheat were screened for their ability to produce aflatoxins on two liquid media. Of these, sixteen isolates were toxigenic and produced only aflatoxin B₁ as assessed by bioassay on okra seedlings and TLC method. For screening isolates ofA. flavus for aflatoxin formation, 0.7 % YES+ Salt medium was found to be good as also for obtaining higher yields of the toxin. Isolates ofA. flavus produced aflatoxin B₁ ranging from 0.85 to 17.2 mg/50 ml. Maximum yield of aflatoxin was obtained when rice was used as the substrate in case of toxigenic isolates L-27 and C-9, and on maize in isolate M-11.     

A rapid extraction method for detecting aflatoxin producing isolates

A rapid extraction method for screening aflatoxin producing potential ofAspergillus flavus group isolates is described. The method is performed using a moist wheat medium with ca. five infected grains extracted with 2 mL of chloroform, and using thin layer chromatography. This method was proved with 95A. flavus isolates from animal feeds.     

Regional Differences in Production of Aflatoxin B1 and Cyclopiazonic Acid by Soil Isolates of Aspergillus flavus along a Transect within the United States

Soil isolates of Aspergillus flavus from a transect extending from eastern New Mexico through Georgia to eastern Virginia were examined for production of aflatoxin B₁ and cyclopiazonic acid in a liquid medium. Peanut fields from major peanut-growing regions (western Texas; central Texas; Georgia and Alabama; and Virginia and North Carolina) were sampled, and fields with other crops were sampled in regions where peanuts are not commonly grown. The A. flavus isolates were identified as members of either the L strain (n = 774), which produces sclerotia that are >400 μm in diameter, or the S strain (n = 309), which produces numerous small sclerotia that are <400 μm in diameter. The S-strain isolates generally produced high levels of aflatoxin B₁, whereas the L-strain isolates were more variable in aflatoxin production; variation in cyclopiazonic acid production also was greater in the L strain than in the S strain. There was a positive correlation between aflatoxin B₁ production and cyclopiazonic acid production in both strains, although 12% of the L-strain isolates produced only cyclopiazonic acid. Significant differences in production of aflatoxin B₁ and cyclopiazonic acid by the L-strain isolates were detected among regions. In the western half of Texas and the peanut-growing region of Georgia and Alabama, 62 to 94% of the isolates produced >10 μg of aflatoxin B₁ per ml. The percentages of isolates producing >10 μg of aflatoxin B₁ per ml ranged from 0 to 52% in the remaining regions of the transect; other isolates were often nonaflatoxigenic. A total of 53 of the 126 L-strain isolates that did not produce aflatoxin B₁ or cyclopiazonic acid were placed in 17 vegetative compatibility groups. Several of these groups contained isolates from widely separated regions of the transect.     

Cyclopiazonic acid and aflatoxins production byAspergillus flavus isolated from Argentinian corn

Thirty-four isolates ofAspergillus flavus obtained from the main Argentinian corn production area were tested for their ability to produce both cyclopiazonic acid (CPA) on corn and on liquid media and aflatoxins on corn. Aflatoxins and CPA were quantified by comparison with standards. The last one was confirmed by mass spectrometry. All but one of the isolates produced CPA on liquid medium in a range between 3120 to 62500 μg/kg, 27/34 isolates produced CPA on corn at levels ranging from 833 to 10000 μg/kg and 5/34 isolates produced aflatoxin B1 in a range between 29 to 115 μg/kg. According to these findings, the percentage ofAspergillus flavus isolates with CPA production ability and their levels of CPA production were higher than the observed elsewhere. It was observed significant differences (p<0,01) between CPA production on corn (median: 1761 μg/Kg) and in liquid medium (median: 27950 μg/Kg). These data represent the first report of the co-production of CPA and aflatoxin B1 by isolates ofAspergillus flavus obtained from corn in Argentina.     

Clustered genes involved in cyclopiazonic acid production are next to the aflatoxin biosynthesis gene cluster in Aspergillus flavus

Cyclopiazonic acid (CPA), an indole-tetramic acid mycotoxin, is produced by many species of Aspergillus and Penicillium. In addition to CPA Aspergillus flavus produces polyketide-derived carcinogenic aflatoxins. Aflatoxin biosynthesis genes form a gene cluster in a subtelomeric region. Isolates of A. flavus lacking aflatoxin production due to the loss of the entire aflatoxin gene cluster and portions of the subtelomeric region are often unable to produce CPA, which suggests a physical link of genes involved in CPA biosynthesis to the aflatoxin gene cluster. Examining the subtelomeric region in A. flavus isolates of different chemotypes revealed a region possibly associated with CPA production. Disruption of three of the four genes present in this region predicted to encode a monoamine oxidase, a dimethylallyl tryptophan synthase, and a hybrid polyketide non-ribosomal peptide synthase abolished CPA production in an aflatoxigenic A. flavus strain. Therefore, some of the CPA biosynthesis genes are organized in a mini-gene cluster that is next to the aflatoxin gene cluster in A. flavus.     

Molecular characterization of toxigenic and atoxigenic Aspergillus flavus isolates,collected from peanut fields in China

Aims: The objectives of this study were to assess the genetic relationships between toxigenic and atoxigenic isolates of Aspergillus flavus collected from peanut fields in China, and to analyse deletions within the aflatoxin biosynthetic gene cluster for the atoxigenic isolates. Methods and Results: Analysis of random‐amplified polymorphic DNA and microsatellite‐primed PCR data showed that the toxigenic and atoxigenic isolates of A. flavus were not clustered based on their regions and their ability of aflatoxin and sclerotial production. These results were further supported by DNA sequence of ITS, pksA and omtA genes. PCR assays showed that 24 of 35 isolates containing no detectable aflatoxins had the entire aflatoxin gene cluster. Eleven atoxigenic isolates had five different deletion patterns in the cluster. Conclusions: Toxigenic and atoxigenic isolates of A. flavus are genetically similar, but some atoxigenic isolates having deletions within the aflatoxin gene cluster can be identified readily by PCR assays. Significance and Impact of the Study: Because the extensive deletions within the aflatoxin gene cluster are not rare in the atoxigenic isolates, analysis of deletion within the cluster would be an effective method for the rapid screening of atoxigenic isolates for developing biocontrol agents.     

Production of aflatoxin and cyclopiazonic acid by various aspergilli: An ELISA analysis

An enzyme-linked Immunosorbent assay (ELISA) was used to monitor a total of 153 fungi in theAspergillus flavus group, Including 130A. flavus, 15A. parasiticus and 8A. tamarii, for their ability to produce aflatoxins (AFs) and cyclopiazonic acid (CPA) in a mycologlcal broth-sucrose-yeast extract medium. Of 15A. parasiticus isolates, ten produced AFs In a range of 12.4 to 89.3 μg/vial (average 56.9 μg/vial); two isolates produced only trace amounts of AFs and three isolates produced none at all. Production of CPA was not demonstrated in anyA. parasiticus isolate. On the other hand, all A. tamarii isolates produced only CPA with a range of 310 to 1100 gmg/vial. Fifteen percent (14.6%) of theA. flavus isolates (19/130) produced more than 500 μg CPA/vial, but yielded no or little AF (less than 0.1 μg/vial). About 22.3% ofA. flavus (29/130) that produced less than 500 μg of CPA also yielded little or no aflatoxin. MostA. flavus isolates (44.6%) produced both CPA (50 to 300 μg/vial) and AFs (10 to 40 μg/vial). About 9.2% of theA. flavus are low CPA producers (less than 100 μg/vial) but yielded higher amounts of AFs. A small percentage (12/130 or 9.2%) of A. flavus isolates produced neither CPA nor aflatoxin. Excluding the isolates that produced neither AFs nor CPA, there is a negative correlation between the production of CPA and AFs by most A.flavus isolates. Data obtained from ELISA for the production of CPA were consistent with TLC results. Thus, the ELISA method for CPA and AFB could be applied to the screening of toxigenic fungi. Data on the simultaneous production of both toxins by a large percentage of the toxigenicA. flavus isolates suggest that there is a potential health hazard for co-existence of both toxins in foods and feeds.     

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.     

Aflatoxin formation and the dual phenomenon in Aspergillus flavus link

Trials were performed with three aflatoxin-forming isolates of Aspergillus flavus from formic acid-treated materials containing aflatoxin, one A. flavus strain isolated from mouldy barley kept for two months in an anaerobic jar and one non-toxic A. flavus strain from the culture collection at our Department. The nontoxic strain and one aflatoxin producer were cultured in salts-sugar-asparagine substrate (SLM) for aflatoxin production and in a specially prepared grass substrate (GS). Formic acid and ammonium formate were added to both substrates, and sucrose in a low amount was added to the grass substrate. The aflatoxin-forming isolate segregated on the grass substrate into two different lines, one with high aflatoxin production and one with very low aflatoxin-forming ability, higher growth rate and reduced sporulation, on the SLM substrate. When exposed to sucrose in grass substrate and ammonium formate in SLM, one toxic and one non-toxic strain were provoked to increased aflatoxin formation. The A. flavus isolate from the anaerobic jar also segregated on the grass substrate, and these segregants were more sensitive to a high dose of formic acid. In these A. flavus strains there seems to be a continuous variation between different lines, depending on cultivation conditions. In the two aflatoxin-forming isolates left, such segregation tendencies were not very marked on any substrate.     

Sterols and fatty acids of aflatoxin and non-aflatoxin producing isolates of Aspergillus

The fatty acids and sterols present in 5 isolates of Aspergillus flavus and 3 isolates of A. parasiticus were determined; 2 isolates within each species were aflatoxin producers. The 4 major fatty acids were 16:0, 18:0, 18:1 and 18:2 with a trace of 15:0 in one isolate and traces of 17:0 in 3 other isolates. Cholesterol, ergosterol and 5, 7-ergostadienol were present in all isolates; the 5 isolates of A. flavus could be identified on the basis of retention times of minor sterols present. There was no correlation of total lipids, fatty acids or sterols with the production of aflatoxins. Water soluble complexes of sterols were not detected.     

Cyclopiazonic acid production by aflatoxigenic and non-aflatoxigenic strains of aspergillus flavus

Twenty-eight of 54 isolates of Aspergillus flavus grown on autoclaved agricultural commodities such as wheat, rice and corn were found to produce the mycotoxin cyclopiazonic acid. Eighteen of the A. flavus isolates produced aflatoxin, and fourteen isolates produced both cyclopiazonic acid and aflatoxin. A preliminary screening of some aflatoxin-contaminated corn samples revealed for the first time the natural occurrence of cyclopiazonic acid in agricultural commodities.     

Co-occurrence of aflatoxin B<Subscript>1</Subscript> and cyclopiazonic acid in sour lime (<Emphasis Type="Italic">Citrus aurantifolia Swingle</Emphasis>) during post-harvest pathogenesis by <Emphasis Type="Italic">Aspergillus flavus</Emphasis>

During hot and humid seasons, extensive rot of sour lime was observed to be caused by Aspergillus flavus. In view of this, investigations were undertaken to obtain data on the production of various toxins by A. flavus during post harvest pathogenesis of sour lime. Sixty percent of the pathogenic A. flavus isolates were detected to be aflatoxin B₁ producers in sour lime tissue. It was also noted that thirty three percent of aflatoxigenic A. flavus isolates had the potential to coproduce cyclopiazonic acid (CPA). Such aflatoxigenic isolates produced quantitatively more CPA (ranging from 250.0 to 2501.3 g/kg) than aflatoxin B₁ (ranging from 141.3 to 811.7 g/kg) in the affected sour lime. This study demonstrates for the first time that sour lime are a favourable substrate for aflatoxin B₁ and cyclopiazonic acid production by A. flavus isolates. This is of great concern to the health of consumers.     

Sexuality Generates Diversity in the Aflatoxin Gene Cluster: Evidence on a Global Scale

Aflatoxins are produced by Aspergillus flavus and A. parasiticus in oil-rich seed and grain crops and are a serious problem in agriculture, with aflatoxin B₁ being the most carcinogenic natural compound known. Sexual reproduction in these species occurs between individuals belonging to different vegetative compatibility groups (VCGs). We examined natural genetic variation in 758 isolates of A. flavus, A. parasiticus and A. minisclerotigenes sampled from single peanut fields in the United States (Georgia), Africa (Benin), Argentina (Córdoba), Australia (Queensland) and India (Karnataka). Analysis of DNA sequence variation across multiple intergenic regions in the aflatoxin gene clusters of A. flavus, A. parasiticus and A. minisclerotigenes revealed significant linkage disequilibrium (LD) organized into distinct blocks that are conserved across different localities, suggesting that genetic recombination is nonrandom and a global occurrence. To assess the contributions of asexual and sexual reproduction to fixation and maintenance of toxin chemotype diversity in populations from each locality/species, we tested the null hypothesis of an equal number of MAT1-1 and MAT1-2 mating-type individuals, which is indicative of a sexually recombining population. All samples were clone-corrected using multi-locus sequence typing which associates closely with VCG. For both A. flavus and A. parasiticus, when the proportions of MAT1-1 and MAT1-2 were significantly different, there was more extensive LD in the aflatoxin cluster and populations were fixed for specific toxin chemotype classes, either the non-aflatoxigenic class in A. flavus or the B₁-dominant and G₁-dominant classes in A. parasiticus. A mating type ratio close to 1∶1 in A. flavus, A. parasiticus and A. minisclerotigenes was associated with higher recombination rates in the aflatoxin cluster and less pronounced chemotype differences in populations. This work shows that the reproductive nature of the population (more sexual versus more asexual) is predictive of aflatoxin chemotype diversity in these agriculturally important fungi.     

Metabolic precursor regulation of aflatoxin formation in toxigenic and non-toxigenic strains ofAspergillus flavus

Non-aflatoxin-producing isolates ofAspergillus flavus from nature and isolates ofA. flavus that had lost their toxigenic trait following laboratory transfer were compared biochemically. After the addition of aflatoxin B₁ precursors sterigmatocystin or O-methylsterigmatocystin to whole cell cultures, the non-toxin producing isolates from nature remained non-toxigenic while toxigenicity was restored in the nontoxigenic laboratory strains. Results imply a lack of enzymes needed for biochemical conversions of precursors to aflatoxin B₁ in natural non-producers and suppression of these enzymes in the nonproducing laboratory strains.