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.     

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.     

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.     

Mycotoxins of fungal strains from stored herbal plants and mycotoxin contents of Nigerian crude herbal drugs

The ability of fungi isolated from stored herbal drug plants to produce mycotoxins in semisynthetic media was studied. The results obtained show that aflatoxins and ochratoxin A, were produced by Aspergillus flavus, A. parasiticus and A. ochraceus isolates. The time-production courses of aflatoxins B₁, B₂, ₁ and ochratoxin A in crude herbal drug preparations show that more of these toxins were produced with increase in time of storage of the drugs. The results indicate that the potential exists for the toxigenic strains to elaborate mycotoxins in a large quantity in herbal drug substrates than in semisynthetic media.This revised version was published online in October 2005 with corrections to the Cover Date.     

Amino acid supplementation reveals differential regulation of aflatoxin biosynthesis in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> NRRL 3357 and <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis> SRRC 143

Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. To better understand the molecular mechanisms that regulate aflatoxin production, the biosynthesis of the toxin in A. flavus and A. parasticus grown in yeast extract sucrose media supplemented with 50 mM tryptophan (Trp) were examined. Aspergillus flavus grown in the presence of 50 mM tryptophan was found to have significantly reduced aflatoxin B₁ and B₂ biosynthesis, while A. parasiticus cultures had significantly increased B₁ and G₁ biosynthesis. Microarray analysis of RNA extracted from fungi grown under these conditions revealed 77 genes that are expressed significantly different between A. flavus and A. parasiticus, including the aflatoxin biosynthetic genes aflD (nor-1), aflE (norA), and aflO (omtB). It is clear that the regulatory mechanisms of aflatoxin biosynthesis in response to Trp in A. flavus and A. parasiticus are different. These candidate genes may serve as regulatory factors of aflatoxin biosynthesis.     

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.     

Studies on the ability of mycotoxins to diffuse in bread

Summary The moulds Aspergillus parasiticus (aflatoxins B₁, B₂, G₁, G₂, and M₁), A. ochraceus (ochratoxin A) and Penicillium chrysogenum (citrinin) were grown on whole wheat bread either in the presence or absence of oxygen. In the presence of oxygen, both A. parasiticus and A. ochraceus developed dense colonies and formed considerable amounts of mycotoxins whereas Penicillium chrysogenum only grew and produced citrinin on the surface of the bread. In the absence of oxygen fungal growth did not occur and most of the toxins were undetectable even in regions of bread immediately adjacent to the moulds although a very slight diffusion of the aflatoxins B₁ and G₁ through 1 cm was observed. It is concluded that diffusion of the tested mycotoxins from hyphae into bread is not a problem for food safety.     

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     

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.     

Enzyme reactions and genes in aflatoxin biosynthesis

Aflatoxins are highly toxic and carcinogenic substances mainly produced by Aspergillus flavus and Aspergillus parasiticus. Sterigmatocystin is a penultimate precursor of aflatoxins and also a toxic and carcinogenic substance produced by many species, including Aspergillus nidulans. Recently, the majority of the enzyme reactions involved in aflatoxin/sterigmatocystin biosynthesis have been clarified, and the genes encoding the enzymes have been isolated. Most of the genes constitute a large gene cluster in the fungal genome, and their expression is mostly regulated by a product of the regulatory gene aflR. This review will summarize the enzymatic steps and the genes in aflatoxin/sterigmatocystin biosynthesis.     

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.     

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.     

Aflatoxin is degraded by heated and unheated mycelia,filtrates of homogenized mycelia and filtrates of broth cultures of Aspergillus parasiticus

Steaming one-half of a lot of 9-day-old mycelia of Aspergillus parasiticus NRRL 2999 for 6 min resulted in little or no subsequent degradation of aflatoxin B₁ or G₁ by these mycelia. The other half of these mycelia was not heat-treated and degraded aflatoxins B₁ and G₁ Filtrates of the growth substrate which remained after the mycelium was removed from 8- to 15-day old cultures of A. parasiticus NRRL 2999 did not degrade substantial amounts of aflatoxin B₁ or G₁, whereas mycelia originally produced on these filtrates degraded substantial amounts of both aflatoxins. The supernatant fluid from homogenates of 9-day-old mycelia of A. parasiticus NRRL 2999 degraded aflatoxins B₁ and G₁ when 0.1 M or 1.0 M phosphate buffer, pH 6.5, was used to suspend the homogenate. These data support the hypothesis that the aflatoxin degrading factor(s) present in the mycelium of A. purasiticus is/are enzyme(s) or at least influenced by enzyme(s).     

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.     

Inhibitory effect of hena (Lawsonia inermis leaves) and carrot root on aflatoxin production byAspergillus parasiticus

Experiments were undertaken to evaluate the effect of some natural products (hena, and carrot root) on growth and aflatoxins production byAspergillus parasiticus FRR 2752. Powdered hena (0.5 and 5%) inhibited mycelial growth and delayed 1 sporulation ofA parasiticus during 7 days. The inhibition of growth was increased with increasing the added amount. Aflatoxins production byA parasiticus was reduced with 40–100% in the presence of hena (Lawsonia inermis leaves). Carrot root extract stimulated the fungal growth and aflatoxin production, whereas carrot root fibers slightly enriched fungal growth, inhibited aflatoxins production (B₁, G₁, and G₂), but there was no inhibition of aflatoxin B₂ production byA parasiticus.     

Mycotoxin-producing potential of fungi associated with qat (Catha edulis) leaves in yemen

Forty-four fungal species belonging to 20 genera were isolated from 30 samples of qat leaves. The most frequent genera wereAspergillus, Alternaria, Penicillium, andCladosporium followed byFusarium, Drechslera, Chœtomium, andMucor. The most prevalent species in above genera wereAspergillus niger, A. flavus, A fumigatus, Alternaria alternata, Penicillium chrysogenum, P. citrinum, Cladosporium cladosporioides, andFusarium verticillioides. From these fungi, 17 species (39%) related to 7 genera (35%) proved to be true endophytes. Eleven out of 75 isolates were mycotoxigenic.A. alternata produced alternariol and alternariol monomethyl ether whereasA. flavus produced aflatoxins B₁ and B₂. Ochratoxin A, sterigmatocystin, citrinin and T-2 toxin were produced byA. ochraceus, A. versicolor, P. citrinum andF. oxysporum, respectively. The presence of such toxigenic fungi associated with qat leaves is considered to be a threat to public health.     

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.     

Aflatoxins: Detection,toxicity, and biosynthesis

Aflatoxins are toxic and carcinogenic secondary metabolites produced mainly byAspergillus flavus andAspergillus parasiticus. The aflatoxins present in food and feed are hazardous to both human and animal health. A number of studies have been conducted on the detection, toxicity, biosynthesis, and regulation of aflatoxins due to the discovery of serious aflatoxicosis in farm animals, and the presence of aflatoxins in many food products. There are many reviews that focus on the biosynthesis of aflatoxin, yet there are few examinations of the overall aspects of aflatoxins, including detection, toxicity, and the regulation on biosynthesis. Thus, the goal of this article is to give an overview of the overall aspects of aflatoxins. This review consists of four parts; i) detection methods for aflatoxins, ii) the toxicity mechanism of aflatoxin B1, iii) gene cluster for aflatoxin biosynthesis, and iv) the regulation of aflatoxin biosynthesis.     

Fungal flora and mycotoxins of six kinds of nut seeds for human consumption in Saudi Arabia

A wide range of moulds representing several genera and species, was recorded in this study from 5 seed samples of each almond, cashew nut, chestnut, hazelnut, pistachio nut and walnut collected from different markets in Ar' Ar, Saudi Arabia. The total counts of fungi were widely fluctuated between 1960–7704 and 1948–7434 colonies/g dry seeds on glucose-Czapek's and glycerol agar media at 28°C, respectively, and represented twenty genera, 53 species and 2 varieties of fungi. The prevalent fungi on the 2 agar media wereAspergillus flavus, A. niger andPenicillium chrysogenum. On glucose-Czapek's agar,Rhizopus stolonifer andAspergillus flavus var.columnaris were isolated from all 6 kinds of nut,A. parasiticus from 5 kinds andA. fumigatus from 4 kinds with high frequencies.Eurotium species were completely absent on glucose-Czapek's agar but they were isolated in high frequency from all kinds of nut on glycerol agar medium. The different nut samples were analyzed by thin layer chromatography for the presence of aflatoxins B₁, B₂, G₁ & G₂, citrinin, ochratoxins, patulin, sterigmatocystin, diacetoxyscirpenol, T-2 toxin and zearalenone. Aflatoxins B₁ & G₁ were detected in 3 out of the 5 samples tested of chestnut at concentrations ranging between 20 to 60 µg/kg. All other samples of almond, cashew nut, hazelnut, pistachio nut, and walnut that were analyzed were mycotoxin free.