Considerations on the distribution of aflatoxigenic Aspergillus flavus in feeds

The distribution of aflatoxin producing isolates of the Aspergillus flavus group in feeds was studied. Aflatoxin production was investigated by a sequential method previously reported (fluorescence in Coconut Agar Medium, rapid extraction from a wheat medium, and total extraction from the same wheat medium). Twenty-seven of 32 samples contained A. flavus, and 21 of them had at least one aflatoxicogenic isolate of A. flavus. Of the 115 isolates analysed, 65 produced aflatoxins, mainly B aflatoxins.     

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.     

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.     

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.     

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.     

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.     

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     

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.     

The Phylogenetics of Mycotoxin and Sclerotium Production in Aspergillus flavus and Aspergillus oryzae

Aspergillus flavus is a common filamentous fungus that produces aflatoxins and presents a major threat to agriculture and human health. Previous phylogenetic studies of A. flavus have shown that it consists of two subgroups, called groups I and II, and morphological studies indicated that it consists of two morphological groups based on sclerotium size, called “S” and “L.” The industrially important non-aflatoxin-producing fungus A. oryzae is nested within group I. Three different gene regions, including part of a gene involved in aflatoxin biosynthesis (omt12), were sequenced in 33 S and L strains of A. flavus collected from various regions around the world, along with three isolates of A. oryzae and two isolates of A. parasiticus that were used as outgroups. The production of B and G aflatoxins and cyclopiazonic acid was analyzed in the A. flavus isolates, and each isolate was identified as “S” or “L” based on sclerotium size. Phylogenetic analysis of all three genes confirmed the inference that group I and group II represent a deep divergence within A. flavus. Most group I strains produced B aflatoxins to some degree, and none produced G aflatoxins. Four of six group II strains produced both B and G aflatoxins. All group II isolates were of the “S” sclerotium phenotype, whereas group I strains consisted of both “S” and “L” isolates. Based on the omt12 gene region, phylogenetic structure in sclerotium phenotype and aflatoxin production was evident within group I. Some non-aflatoxin-producing isolates of group I had an omt12 allele that was identical to that found in isolates of A. oryzae.     

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.     

Aflatoxin contamination of groundnuts in Sudan

Groundnut samples, collected soon after harvest, from different districts in the irrigated region (Central Sudan) were free from aflatoxins with the method used. Samples collected from the rainfed region (Western Sudan) showed variable levels of aflatoxin ranging from 100% sample contamination in El Hamdi to only 10% in Casgeal.Damaged pods were highly contaminated with A. flavus and accumulated large amounts of aflatoxins. However, sound intact pods, recorded lower fungal contamination and were almost free of aflatoxins. Groundnut products collected from Khartoum North (Bahri) have higher levels of aflatoxins than those collected from Khartoum and Umdorman. Gray and red roasted pods showed higher amounts of aflatoxins, while the groundnut paste was the least contaminated.None of the three varieties of groundnuts tested in this work was completely resistant to aflatoxin production. A temperature of 30°C and 86.3% relative humidy (RH) are the optimum conditions for both A. flavus growth and aflatoxin production in groundnuts.     

Study on pectinase and sclerotium producing abilities of two kinds ofAspergillus flavus isolates from Zhejiang

Pectinase and sclerotium production by strains ofAspergillus flavus were determined with a pectinase culture plate assay and a Cz 3% NaNO₃ medium plate assay. In theA. flavus population, 51% of isolates produced sclerotia, the toxigenic strains showing a tendency to have smaller sclerotia. Strains producing both abundant small sclerotia and a large quantity of aflatoxin were not found. There was no linear correlation between the amount of aflatoxin produced and the number of sclerotia. Levels of pectinase produced by the toxigenic strains were higher than that of the non-toxigenic strains, and this character was more obvious in the sclerotium-producing strains than in the non-sclerotium-prodcing strains. In theA. flavus population from Zhejiang in which the toxigenic strain rate was low, toxigenic strains may require higher levels of pectinase to compete with the non-toxigenic strains when infecting foodstuffs.     

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.     

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.     

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.     

Behaviour of Aspergillus flavus in presence of Aspergillus niger during biosynthesis of aflatoxin B1

Aspergillus niger or Aspergillus tamarii when grown as mixed cultures with toxigenic A. flavus inhibits biosynthesis of aflatoxin by A. flavus, owing primarily to its ability to produce inhibitors of aflatoxin biosynthesis and to their ability to degrade aflatoxin. Gluconic acid partly prevents aflatoxin production. The other factors such as changes in pH of the medium and the effect on the growth of A. flavus have no role in imparting capabilities to these cultures to inhibit aflatoxin production by A. flavus.     

Aflatoxin production by Aspergillus flavus isolates pathogenic to coconut insect pests

Aspergillus flavus isolated from naturally infected leaf-eating caterpillar (Opisina arenosella W.), lace bug (Stephanitis typica D.) and plant hopper (Proutista moesta Westwood), insect pests of the coconut palm, were tested for aflatoxin (AT) production by employing various media formulations. These A. flavus isolates were earlier found to be entomopathogenic in laboratory bioassays. A laboratory contaminant and four standard aflatoxigenic A. flavus isolates were also included in this study as reference strains. All A. flavus isolates were tested on seven AT detection media: coconut extract agar, coconut extract-sodium desoxycholate agar, coconut extract-ascorbic acid agar, coconut extract-Czapek Dox agar, coconut extract-milk powder agar, 10% commercial coconut milk powder agar (CCMPA) and 20% CCMPA. Only two isolates of A. flavus, originally isolated from O. arenosella and P. moesta, produced ATs. AT production was detected within 48 h of incubation and was detected continually up to 1 month. These AT-producing A. flavus isolates also produced bright yellow pigmentation in the medium. Of all the seven media used for AT detection, CCMPA (10%) was found to be the best one, followed by 20% CCMPA, for direct and rapid AT detection. AT production was not necessary for pathogenicity in the insects. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inhibition of Aspergillus flavus growth and detoxification of aflatoxin B1 by the medicinal plant zimmu (Allium sativum L. × Allium cepa L.)

Aflatoxins are carcinogenic, teratogenic and immunosuppressive secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus. Aflatoxin contamination of peanut is one of the most important constraints to peanut production worldwide. In order to develop an eco-friendly method of prevention of A. flavus infection and aflatoxin contamination in peanut, aqueous extracts obtained from leaves of 30 medicinal plants belonging to different families were evaluated for their ability to inhibit the growth of A. flavus in vitro. Among them the leaf extract of zimmu (Allium sativum L. × Allium cepa L.) was the only one that showed antifungal activity against A. flavus and recorded 73% inhibition of A. flavus growth. The antifungal activity of the zimmu extract was significantly decreased upon dialysis with a dialysis membrane having molecular cut off 12 kDa or autoclaving at 121°C for 20 min or boiling at 100°C for 10 min and recorded inhibition of 52, 16 and 21%, respectively. When A. flavus was grown in medium containing zimmu extract the production of aflatoxin B₁ (AFB₁) was completely inhibited even at a concentration of 0.5%. When AFB₁ was incubated with zimmu extract a complete degradation of AFB₁ was observed 5 days after incubation. When the roots of zimmu were incubated in water containing 70 ng of AFB₁/ml, a reduction (by 58.5%) in AFB₁ concentration was observed 5 days after incubation. A significant reduction in the population of A. flavus in the soil, kernel infection by A. flavus and aflatoxin contamination in kernels was observed when peanut was intercropped with zimmu. The population of the fungal antagonist, Trichoderma viride in the zimmu-intercropped field increased approximately twofold.     

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.