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.     

Fluorometric analysis of iodinated aflatoxin in minicultures ofAspergillus flavus andAspergillus parasiticus

Summary A convenient miniassay for aflatoxin has been developed for cultures ofAspergillus flavus andA. parasiticus grown for 3–10 days in 10 ml of a coconut extract medium. The sensitivity of the assay, as measured by photofluorometry (365 nm maximum excitation; 445 nm maximum emission), is of the order of 0.01 M (3.12 ng/ml) for aflatoxin B₁ dissolved in aqueous iodine (0.26 mM). High performance liquid chromatography, monitored by fluorometric analysis of both an aflatoxin B₁ standard and selected culture filtrates, confirmed the sensitivity of the assay and indicated specificity for iodine-enhanced fluorescence of aflatoxin in the coconut extract medium. Thin layer chromatography further confirmed the aflatoxin titers and the specificity for enhancement of aflatoxins B₁ and G₁ in culture filtrates.Alabama Agricultural Experiment Station Journal No. 6-871297.     

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.     

Mycobiota and molecular detection of Aspergillus flavus and A. parasiticus aflatoxin contamination of Strawberry (Fragaria ananassa Duch.) fruits

Strawberry fungi were isolated from fresh fruits and juice on the two types of media (Sabouraud dextrose agar, SDA and potato-dextrose agar, PDA) at 28 °C. Nineteen fungal species belong to 12 genera were isolated from fruits and juice on both isolation media. The most common fungal genera and species were Aspergillus flavus, A. niger, Mucor racemosus, Neurospora crassa, Penicillium chrysogenum, Rhizopus stolonifer and Trichoderma harzianum. Twenty A. flavus and A. parasitics isolates were assayed for their abilities to produce aflatoxins. The concentration of aflatoxins ranged between 25.8–75.2 and 23.6–71.1 ng/ml at 350 and 365 nm, respectively. Among A. flavus and A. parasiticus strains tested, aflatoxin B contributed 30–60% of total isolates. However, G type contributed 85–90%. The R_f values of B₁, B₂, G₁ and G₂ were 0.79, 0.61, 0.44 and 0.32, respectively. High-performance liquid chromatography analysis of extracts revealed the presence of aflatoxins with variable levels.     

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     

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     

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.     

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.     

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).     

A Survey on Distribution of Aspergillus Section Flavi in Corn Field Soils in Iran: Population Patterns Based on Aflatoxins, Cyclopiazonic Acid and Sclerotia Production

Soil isolates of Aspergillus section Flavi from Mazandaran and Semnan provinces with totally different climatic conditions in Iran were examined for aflatoxins (AFs; B and G types), cyclopiazonic acid (CPA) and sclerotia production. A total of 66 Aspergillus flavus group strains were identified from three species viz. Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius in both locations. A. flavus (87.9%) was found to be the prominent species followed by A. nomius (9.1%) and A. parasiticus (3.0%). Only 27.5% of A. flavus isolates were aflatoxigenic (B₁ or B₁ and B₂), out of which approximately 75% were capable to producing CPA. All the A. parasiticus and A. nomius isolates produced AFs of both B (B₁ and B₂) and G (G₁ and G₂) types, but did not produce CPA. Sclerotia production was observed in only 4 isolates of A. flavus among all 66 isolates from three identified species. A. flavus isolates were classified into various chemotypes based on the ability to produce aflatoxins and CPA. In this study, a new naturally occurring toxigenic A. flavus chemotype comprising of two strains capable of producing more AFB₂ than AFB₁ has been identified. A relatively larger proportion of aflatoxigenic A. flavus strains were isolated from corn field soils of Mazandaran province which indicate a possible relationship between high levels of relative humidity and the incidence of aflatoxin-producing fungi. The importance of incidence of Aspergillus section Flavi in corn field soils regard to their mycotoxin production profiles and crop contamination with special reference to climatic conditions is discussed.     

Fungal flora and aflatoxin contamination of feedstuff samples in Beida Governorate,Libya

Fungi of 19 genera, 30 species, and one variety were isolated from 25 samples of sheep-, cattle- and camel feedstuffs collected from different farms in the Beida Governorate, Libya.Aspergillus, Penicillium andFusarium were the most common genera in the three substrates tested. TLC was used to establish the identity of aflatoxins in the chloroform extract of all samples and the ability to produce aflatoxins byAspergillus flavus in a synthetic liquid medium. Twenty samples out of 25 tested were naturally contaminated and 21 isolates ofA. flavus out of 30 produced at least one of the following aflatoxins: B₁; B₁, G₁; and B₁, B₂, G₁, G₂. This is the first report about the natural occurrence of aflatoxins and aflatoxin-producers of the genusAspergillus in Libya.     

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.     

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.     

Mycotoxigenic potential ofAspergillus flavus strains isolated from groundnuts growing in Israel

Two hundred strains of the Aspergillus flavus group isolated from groundnuts (peanuts) growing in Israel were examined for their ability to produce mycotoxins in potato dextrose (PD) broth. Almost 77% of the isolates produced aflatoxin; aflatoxins B₁ and B₂ were formed by most of the isolates. Simultaneous production of aflatoxins of groups B and G was detected in only 0.5% of the isolates. Microscopic examination revealed that 98% of the isolates wereA. flavus and only 2%A. parasiticus. Cyclopiazonic acid (CPA) was detected in 22.5% of the isolates, including 3.5% that produced only CPA. Sterigmatocystin was detected in only 2% of the isolates and only one isolate produced aflatoxin simultaneously with CPA and sterigmatocysin. The dry weight (DW) of mycelium, 7 days after inoculating the medium, was between 71–110 mg/30 ml medium in more than 70% of the isolates. A general decrease in the pH was observed and 75% of the isolates reduced the pH to 4.5 or below. After 14 days, a small increase in DW and an increase in the pH toward neutrality was observed. On PD agar, 30% of the isolates produced sclerotia, including 5% that produced them profusely. No correlation between mycelial growth, changes in pH of the medium, sclerotium formation, and aflatoxin accumulation could be observed. The mycotoxigenic potential of theA. flavus strains isolated from groundnuts seems to be relatively high and may present a potential threat to human and animal health.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel. No. 3559-E.     

Examination of fungal growth and aflatoxin production on marihuana

Under favorable growth conditions,Aspergillus flavus andA. parasiticus produced aflatoxins on marihuana. Cultures ofA. flavus ATCC 15548 produced both aflat oxin B₁(AFB₁) and G₁(AFG₁). The production of AFG₁ was substantially greater than that of AFB₁. Cultures ofA. flavus NRRL 3251 andA. parasiticus NRRL 2999 produced only AFB₁. All natural flora cultures tested negative for aflatoxins. NoAspergilli sporulations were observed in these cultures. In the cultures inoculated with known toxigenic fungi, the highest mean level for total aflatoxins was 8.7 g/g of medium. Marihuana appears not to yield large quantities of these mycotoxins but sufficient levels are present to be a potential health hazard for both the user and the forensic analyst who is in daily contact with such plant material. Careful processing, storage, and sanitation procedures should be maintained with marihuana. If these conditions are disregarded due to the illicit status of marihuana, the potential for mycotoxin contamination must be considered.     

Aflatoxin-producing potential of Aspergillus flavus strains isolated from Spanish poultry feeds

A total of 126 fungal strains belonging to the Aspergillus flavus group isolated from commercial poultry mixed feeds were studied. One hundred and twenty-five were identified as A. flavus and one as A. parasiticus. Forty nine strains (39%) produced aflatoxins on a crushed moist wheat medium (28 °C/10 days), whereas only sixteen (13%) showed specific fluorescence on Aflatoxin-Producing Ability Medium. In both media, mainly aflatoxins B₁ and B₂ were detected, the average concentration of aflatoxins being 4294+/–1083 g/kg in crushed moist wheat medium, and 877+/–257 g/kg in Aflatoxin-Producing Ability Medium.     

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.     

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.     

Comparison of the ability of three Aspergillus strains to form aflatoxins on bakery products and on nutrient agar

The growth of Aspergillus parasiticus NRRL 2999, A. parasiticus NRRL 3000 and A. flavus NRRL 3251 on whole wheat bread and on cake (Rührkuchen) was compared and the formation of the aflatoxins B₁, B₂, G₁, G₂ and M₁ on these substrates and, for purpose of comparison, on malt extract agar was determined. On cake the moulds grew better than on bread and formed the highest yields of aflatoxins. Malt extract agar was the most unfavourable substrate for toxin production. The ratio M₁/B₁ on bread and cake was in the order of 0.1–0.4 and was higher than the data reported for grains. The highest yields of aflatoxin B₁ (1.0 g/g) were produced by A. flavus NRRL 3251 on cake.     

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.