Aflatoxins in mutants of Aspergillus flavus

Mutants ofAspergillus flavus were recovered following the irradiation of conidia with ultraviolet light. Analysis of the mutants for aflatoxins B₁, B₂, G₁, and G₂ indicated a wide range of variability in aflatoxin levels. None of the isolates produced the G toxins, and four produced little or no aflatoxin B₂. Production of B₁ and B₂ by the mutants ranged from 1.3 µ;g/ml to 967 µg/ml and zero to 30 µg/ml, respectively. The correlation between production of B₁ and B₂ was statistically significant. There was no apparent correlation between nutritional requirement or conidial color and aflatoxin production.     

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.     

Association between vegetative compatibility and aflatoxin production by <Emphasis Type="Italic">Aspergillus</Emphasis> species during intraspecific competition

Intraspecific competition is the basis for biological control of aflatoxins, but there is little understanding of the mechanism(s) by which competing strains inhibit toxin production. Evidence is presented that demonstrates a relationship between strength of the vegetative compatibility reaction and aflatoxin production in Aspergillus flavus and A. parasiticus using the suspended disk culture method. Combining wild-type aflatoxin-producing isolates belonging to different vegetative compatibility groups (VCGs) resulted in a substantial reduction in aflatoxin yield. Pairs of aflatoxin-producing isolates within the same VCG, but showing weak compatibility reactions using complementary nitrate-nonutilizing mutants, also were associated with reduced levels of aflatoxin B₁. In contrast, pairings of isolates displaying a strong compatibility reaction typically produced high levels of aflatoxins. These results suggest that interactions between vegetatively compatible wild-type isolates of A. flavus and A. parasiticus are cooperative and result in more aflatoxin B₁ than pairings between isolates that are incompatible. Successful hyphal fusions among spore germlings produce a common mycelial network with a larger resource base to support aflatoxin biosynthesis. By comparison, vegetative incompatibility reactions might result in the death of those heterokaryotic cells composed of incompatible nuclei and thereby disrupt the formation of mycelial networks at the expense of aflatoxin biosynthesis. The content of this paper was presented at the 50th Anniversary Meeting of the Mycological Society of Japan, June 3–4, 2006, Chiba, Japan     

Mechanism of action of aflatoxin B1

The inhibitory effects of aflatoxin B₁ were found to be related to the gram character in procaryotes, used in this study. Ethylene diamine tetra chloroacetic acid (0.05% w/v) or Tween-80 (0.05 % v/v) addition accentuated the aflatoxin B₁ growth inhibition inSalmonella typhi andEscherichia coli at different pH values. The inhibition of lipase production was only 5–20 % inPseudomonas fluorescence ca. 25–48% inStaphylococcus aureus andBacillus cereus at different aflatoxin B₁ concentrations (4–16μg/ml).However, inhibition of α-amylase induction was complete in1Bacillus megaterium whereas the inhibition was partial inPseudomonas fluorescence (27–40%) at 32μg aflatoxin B₁ concentration. An increase in leakage of cell contents and decreased inulin uptake were observed in toxin incubated sheep red blood cell suspension (1 %) with increased aflatoxin B₁ concentration     

β-Carotene inhibition of aflatoxin biosynthesis amongAspergillus flavus genotypes from Illinois corn

Thirty-nineAspergillus flavus genotypes (DNA fingerprinting) isolated from corn grown in a field near Kilbourne, Illinois were evaluated for their sensitivity to β-carotene (50 μg/ml) inhibition of aflatoxin B₁ biosynthesis. Inhibition of aflatoxin was greater than 90% for 28 of the genotypes and >70% for 38 of the 39 genotypes. FiveA. flavus strains (4 fingerprint groups) isolated from molded raw peanuts, NRRL 3239, NRRL 3357, NRRL 6514, NRRL 6515 and NRRL 13135, produced greater quantities of aflatoxin than all 39 genotypes isolated from corn, and were less sensitive to β-carotene inhibition.Aspergillus flavus NRRL 3357 is commonly used as inoculum in variety trials for aflatoxin resistance. Isolate identity and sensitivity to potential inhibitors in corn can be critical in assessing corn resistance to aflatoxin.     

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 B₁and 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 g/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 quincesis recommended for this region.This revised version was published online in October 2005 with corrections to the Cover Date.     

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.     

The relationship betweenPleurotus ostreatus andAspergillus flavus and the production of aflatoxin

The relationship betweenPleurotus ostreatus andAspergillus flavus in common mixed culture on various substrates was investigated. It was found thatP. ostreatus, similarly to some other higher fungi, can liquidate coloniesof A. flavus. This fungus does not produce aflatoxin and chromatographically similar compounds. On straw, corn cobs, millet and wheatA. flavus produced aflatoxin after a 3-week cultivation. A subsequent cultivation of P.ostreatus led to detoxication of straw and corn cobs but millet and wheat were not detoxicated. Cultivation of P.ostreatus in the presence of 40–100 μg of aflatoxin B₁ per g substrate did not result in detoxication of the material even after 34 d but the results showed that the aflatoxin concentration decreased to about one-fourth of the added amount.     

Incidence of mycoflora and mycotoxins in some edible fruits and seeds of forest origin

Twelve fungi namelyAlternaria alternata, Aspergillus flavus, A niger, A ochraceus, Actinomucor repens, Capnodoium spp., Curvularia lunata, Fusarium pallidoroseum, F solani, F verticillioides, Penicillium citrinum and Rhizopus stolonifer were recorded from samples ofAegle marmelos, Aesculus indica, Buchanania lanzan andPinus gerardiana. In case ofPrunus amygdalus only Rstolonifer was recorded. A significant variation in pattern of mycoflora incidence was observed in terms of source and season. Fungal infestation in most of the substrates was found to be highest during monsoon. Aflatoxins were the most common mycotoxins elaborated by different isolates ofA flavus obtained fromA marmelos, B lanzan andP gerardiana. The amount of aflatoxins produced by the toxigenic isolates ofA flavus was in the range of traces to 0.9–26.0 μg/ml inA marmelos, 0.8–17.5 μg/ml inP gerardiana and 0.65–13.2 μg/ml inB lanzan. The percentage toxigenicity was comparatively lower in the isolates of other mycotoxigenic fungi. Aflatoxins were detected almost in all the samples analyzed for mycotoxin contamination. However, traces of zearalenone were detected inA marmelos. The concentration of aflatoxin B₁ was in the range of 0.13–0.75 μg/g inA marmelos, 0.09–0.60 μg/g inP gerardiana and 0.01–0.20 ug/g inB lanzan. Mycotoxins were not detected inAesculus indica andPrunus amygdalus.     

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.     

Evaluation of the mycological status of luncheon meat with special reference to aflatoxigenic moulds and aflatoxin residues

The luncheon meat samples analyzed, which were produced locally by the two main luncheon meat producing companies in Egypt were relatively highly contaminated either by moulds and yeasts in general, aflatoxigenic species and aflatoxin residues in particular. The most frequently encountered fungi from the samples were yeasts, Aspergillus niger, A. flavus, Penicillium chrysogenum, Rhizopus stolonifer, Mucor circinelloides. Less common were Cladosporium sphaerospermum, Alternaria alternata, Mycosphaerella tassiana, P. aurantiogriseum and P. oxalicum. The most important aflatoxigenic species, A. flavus, was isolated frequently. It was 10% of the total fungal isolates from both samples of the two companies. Seven luncheon meat samples out of 50 analyzed were positive for aflatoxin B₁ or B₁ and G₁, while all samples were negative for aflatoxins B₂, G₂, M₁ and M₂. Aflatoxin B₁ was detected only in 4 and 3 samples out of 25 analyzed from each of company A and B, respectively. The highest detectable level, 11.1 ppb, was recorded in a sample from company B and the least, 0.5 ppb, in a sample from company A. Aflatoxin G₁, at concentration of 3.2 ppb, was detected in only one sample of the aflatoxin B₁ – contaminated 3 samples of company B: this sample also had the highest level of aflatoxin B₁. Some luncheon meat samples had higher numbers of aflatoxigenic A. flavus than others, however these samples were negative for aflatoxins. The hazardous potential of such contamination will be discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genetic variability of aflatoxin B<Subscript>1</Subscript> producing <Emphasis Type="Italic">Aspergillus flavus</Emphasis> strains isolated from discolored rice grains

Twenty-two aflatoxin B₁ (AFB1) producing Aspergillus flavus strains were isolated from 1,200 discolored rice grain samples collected from 20 states across India and tested their potential to produce AFB1 on different agar media. Further these isolates were characterized through randomly amplified polymorphic DNA method. All the strains of A. flavus were produced AFB1 on yeast extract sucrose agar media and none of the strains on A. flavus and A. parasiticus agar. Among the 22 strains, two strains from Tamil Nadu (DRAf 009) and Maharashtra (DRAf 015) produced high amount of AFB1 in all the media tested. To assess the genetic variability in A. flavus, the isolates were analyzed by using random amplified polymorphic DNA markers. Isolates showed 17–80% similarity with standard culture of A. flavus (MTCC 2799).     

Contamination of Groundnut in South‐Western Nigeria by Aflatoxigenic Fungi and Aflatoxins in Relation to Processing

Groundnut is commonly consumed in its roasted form by many Nigerians. This study was therefore conducted to determine the levels of aflatoxin in roasted groundnut retailed in south‐western Nigeria with a view to assessing the fitness of the processed nut for human consumption. The effects of roasting and de‐coating as alternative methods for reducing the ‘aflatoxin scare’ in the nut were further assessed on aflatoxigenic fungal load and aflatoxin content of the nuts. Forty‐eight samples of retailed raw and roasted groundnut were collected and assessed by mycological and thin‐layer chromatographic analysis for changes in aflatoxigenic fungal population and aflatoxin concentration, respectively. Consequently, 480 isolates of the Aspergillus section Flavi group, A. flavus L strain (n = 410), A. tamarii (n = 56), A. parasiticus (n = 7) and A. parvisclerotigenus (n = 7), were recovered from all samples. Aflatoxigenic isolates of A. flavus L strain (58.8%) had a significantly (P < 0.05) higher incidence than the non‐aflatoxigenic isolates (41.2%). Aflatoxins were detected in 43 (89.6%) of the samples. Approximately 25% of all samples exceeded the 20 ng/g limit for aflatoxin B₁ (AFB₁) adopted by the National Agency for Food and Drug Administration and Control while 83 and 79% of all samples contained AFB₁ and total aflatoxins above the European Union limits of 2 and 4 ng/g, respectively. Aflatoxin concentrations in the raw and coated samples were as much as five times higher than those in the roasted and de‐coated nuts, respectively. However, no significant difference was recorded between aflatoxin levels in the coated and de‐coated samples. This study has shown that roasting of groundnut and testa removal (de‐coating) are effective processing interventions that can significantly lower aflatoxin quantities in the kernels, thus making it fit for human consumption.     

Metabolic behaviour of aflatoxin producing strain and non-toxigenic strain of Aspergillus flavus to different sources of nitrogen and glucose concentration

The effect of different nitrogen sources and varying glucose concentration on aflatoxin production by a toxigenic and non-toxigenic strain of Aspergillus flavus was studied. Greatest production (3.8 ppm) of aflatoxin B₁ was produced in a synthetic medium when casamino acids were supplied as the nitrogen source. Optimum sugar concentration for aflatoxin B₁ production ranged between 3 and 10 g/100 ml. There was no appreciable difference in the metabolic behaviour between toxigenic and non-toxigenic strains of A. flavus when dry mycelial weight, total proteins, non-protein nitrogen and reducing sugar were the criteria.     

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.     

A survey on distribution and toxigenicity of <Emphasis Type="Italic">Aspergillus flavus</Emphasis> from indoor and outdoor hospital environments

In the present study, genetic diversity and mycotoxin profiles of Aspergillus flavus isolated from air (indoors and outdoors), levels (surfaces), and soils of five hospitals in Southwest Iran were examined. From a total of 146 Aspergillus colonies, 63 isolates were finally identified as A. flavus by a combination of colony morphology, microscopic criteria, and mycotoxin profiles. No Aspergillus parasiticus was isolated from examined samples. Chromatographic analyses of A. flavus isolates cultured on yeast extract–sucrose broth by tip culture method showed that approximately 10% and 45% of the isolates were able to produce aflatoxin B₁ (AFB₁) and cyclopiazonic acid (CPA), respectively. Around 40% of the isolates produced sclerotia on Czapek–Dox agar. The isolates were classified into four chemotypes based on the ability to produce AF and CPA that majority of them (55.5%) belonged to chemotype IV comprising non-mycotoxigenic isolates. Random amplified polymorphic DNA (RAPD) profiles generated by a combination of four selected primers were used to assess genetic relatedness of 16 selected toxigenic and non-toxigenic isolates. The resulting dendrogram demonstrated the formation of two separate clusters for the A. flavus comprised both mycotoxigenic and non-toxigenic isolates in a random distribution. The obtained results in this study showed that RAPD profiling is a promising and efficient tool to determine intra-specific genetic variation among A. flavus populations from hospital environments. A. flavus isolates, either toxigenic or non-toxigenic, should be considered as potential threats for hospitalized patients due to their obvious role in the etiology of nosocomial aspergillosis.     

The effect of <Emphasis Type="Italic">Baccharis glutinosa</Emphasis> extract on the growth of mycotoxigenic fungi and fumonisin B<Subscript>1</Subscript> and aflatoxin B<Subscript>1</Subscript> production

The aim of this study was to evaluate the effect of Baccharis glutinosa isolated extract on the growth of Aspergillus flavus and Aspergillus parasiticus, and their aflatoxin B₁ production; and growth of Fusarium verticillioides, and their fumonisin B₁ production. The three fungi were exposed to an antifungal fraction, designated as fraction F6-1, isolated from B. glutinosa by methanolic extraction followed by silica gel chromatography. The growth of the fungi was evaluated in kinetics of radial extension growth, kinetics of spores germination, length and diameter of hyphae, spores diameter, as well as in aflatoxin B₁ and fumonisin B₁ production. Fraction F6-1 caused radial growth inhibition of the three fungi mainly F. verticillioides. Spores germination of A. flavus and A. parasiticus was delayed in the early stage of the incubation time, although they completely germinated at 27 h. In contrast, spore germination of F. verticillioides was inhibited 87.7% up to 96 h. The lengths and diameters of hyphae, and spore diameters of the three fungi, were significantly smaller in comparison with those of the controls, and several morphological alterations were observed. Concerning aflatoxin B₁ and fumonisin B₁, fraction F6-1 did not show any inhibition effect at the concentration used. Fraction F6-1 was able to significantly inhibit the development of the three fungi, mainly F. verticillioides. The strong inhibitory effect of F6-1 on hyphae and spores suggests that it interacted with the fungi cell walls, which caused severe deformities. Nevertheless, this fraction was unable in inhibiting mycotoxin production from the three fungi at the concentration tested.     

Transformation of sterigmatocystin and O-methylsterigmatocystin by aflatoxigenic and nonaflatoxigenic field isolates of the Aspergillus flavus group

Transformation of sterigmatocystin and O-methylsterigmatocystin (two metabolic aflatoxin precursors) to aflatoxins by aflatoxigenic and nonaflatoxigenic field isolates of Aspergillus flavus was studied. The 24 nonaflatoxigenic isolates investigated failed to transform both precursors. Among the 8 aflatoxin-producing isolates used, 7 transformed both precursors whereas the remaining failed to transform both. According to these results, the usefulness of the measurement of enzymatic activities related to aflatoxin production in understanding the true status of conflictive field isolates is discussed.Abbreviations ST sterigmatocystin - OMST O-methylsterigmatocystin - AFB₁ aflatoxin B₁ - AFB₂ aflatoxin B₂ - AFG₁ aflatoxin G₁ - AFG₂ aflatoxin G₂ - GM growth medium of Adye and Mateles - RM replacement medium of Adye and Mateles     

Evaluation of different genotypes of nontoxigenic Aspergillus flavus for their ability to reduce aflatoxin contamination in peanuts

Aflatoxins produced by the fungus Aspergillus flavus are potent carcinogens and account for large monetary losses worldwide in peanuts, maize, and cottonseed. Biological control in which a nontoxigenic strain of A. flavus is applied to crops at high concentrations effectively reduces aflatoxins through competition with native aflatoxigenic populations. In this study, eight nontoxigenic strains of A. flavus belonging to different vegetative compatibility groups and differing in deletion patterns within the aflatoxin gene cluster were evaluated for their ability to reduce aflatoxin B₁ when paired with eight aflatoxigenic strains on individual peanut seeds. Inoculation of wounded viable peanut seeds with conidia demonstrated that nontoxigenic strains differed in their ability to reduce aflatoxin B₁. Reductions in aflatoxin B₁ often exceeded expected reductions based on a 50:50 mixture of the two A. flavus strains, although one nontoxigenic strain significantly increased aflatoxin B₁ when paired with an aflatoxigenic strain. Therefore, nontoxigenicity alone is insufficient for selecting a biocontrol agent and it is also necessary to test the effectiveness of a nontoxigenic strain against a variety of aflatoxigenic strains.     

Fungal and aflatoxin contamination of medicinal herbs

Since the consumption of aromatic and medicinal herbs has been increasing in the last years, the Argentinian Health Authorities are concerned to control the quality and security of them. Fungal and aflatoxin contamination are two parameters to be taken into account, to ensure the harmlessness of the phytomedicinal products. In 81 different samples, grouped in end products (EP), raw material (RM) and at harvest (SH), fungal flora (enumeration and identification) as well as naturalAspergillus flavus and aflatoxin occurrence were investigated. In all samples fungal counts fulfilled the international general recommendation limits (maximum 10⁵ cfu/g). Predominant flora was made up by xerophilic species ofAspergillus(100%), byPeniciIlium (< 50%) and in less percentage byFusarium (5.6%). Among the Aspergilli, A.flavus was present in all the three groups of samples. Using a TLC method, 47% of A. flavus isolates were toxinogenic, producing aflatoxin B₁ and B₂. In herbs, 4.7% of RM samples were naturally contaminated with aflatoxins B₁ and B₂. Considering the carcinogenic activity of aflatoxins it is essential to regulate them in the raw material (vegetal drug).