Assessment of aflatoxin and cyclopiazonic acid production by Aspergillus flavus isolates from Hungary

Thirty-two isolates of Aspergillus flavus were obtained from various sources in Hungary. All isolates were morphologically identified as A. flavus and three atypical variants were confirmed as A. flavus by comparing their DNA with an ex type culture of A. flavus. None of these isolates produced aflatoxins when tested on coconut agar or grown on rice medium and culture extracts examined by thin layer chromatography. Also, none of the isolates converted sterigmatocystin, O-methyl sterigmatocystin, norsolorinic acid, or sodium acetate to aflatoxin. However, 59% of the isolates produced cyclopiazonic acid based on thin layer chromatographic analysis of culture extracts. The isolates that lack the ability to produce both aflatoxin and cyclopiazonic acid are potential candidates for use in bicontrol studies.     

Aflatoxin 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.     

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.     

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.     

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.     

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.     

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.     

Fungi and aflatoxins associated with spices in the Sultanate of Oman

One hundred and five samples of seven spices (cumin, cinnamon, clove, black pepper, cardamom, ginger, and coriander) were purchased from five popular companies in the Sultanate of Oman. The spices were surveyed for the mycoflora and aflatoxins. Twenty fungal species were isolated in which Aspergillus flavus , A. niger . Penicillium , Rhizopus , and Syncephalastrum racemosum were the most dominant. When colony forming units per gm (cfu/gm) of fungi were compared, significant differences were found among spices and companies. Of the seven spices studied, clove was found to be the least contaminated, while cumin was the most contaminated. None of the 15 selected samples of the spices contaminated by A. flavus were found to contain aflatoxins. Nevertheless, nine isolates (45%) of the twenty A. flavus strains screened for aflatoxins were aflatoxigenic. The moisture content of most of the spices was below the maximum standard limit. The results showed that the spices were contaminated by some fungi that might constitute health hazards for humans. This revised version was published online in June 2006 with corrections to the Cover Date.     

A rapid extraction method for detecting aflatoxin producing isolates

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

Mycotoxigenic infestation in samples of cereal straw from Bihar, India

A survey of different types of cereal straw samples viz. paddy, maize and wheat, from Bihar State, India, was conducted in order to examine the mould flora and mycotoxin contamination. Out of 170 samples examined for mould flora,Aspergillus flavus group of fungi had highest level of incidence followed byA niger. Isolates ofA flavus, A ochraceus, Fusarium verticillioides andPenicillium citrinum were screened for their mycotoxins producing abilities. Out of 75, 63 and 68 isolates ofA flavus group obtained from stored straw of paddy, maize and wheat samples, respectively, 27 (36%), 14 (22%) and 24 (35%) were found to be toxigenic which produced different combinations of aflatoxins in different concentrations. The percentage toxigenicity was comparatively lower in the isolates of other mycotoxigenic fungi from all types of samples. Out of 222 samples of straw analysed for natural occurrence of different mycotoxins, besides the aflatoxins present, zearalenone, ochratoxin A and citrinin were also recorded alone or as co-contaminants. A conducive climate together with the socioeconomic conditions of this region are important determinants for the high incidence of mycotoxins in cereal straw samples.     

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.     

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     

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.     

Winged bean (Psophocarpus tetragonolobus (L.) DC) as a substrate for growth and aflatoxin production by aflatoxigenic strains of Aspergillus spp.

The mold flora of seeds of twelve varieties of winged beans were determined both before and after surface disinfections. When seeds were surface disinfected, molds were detected in 73% of the seeds whereas 81% of the seed that was not disinfected produced molds. Aspergillus spp. were most frequently present while Penicillium spp. occurred in seed of 4 varieties and in less than 4% of the seed. Twelve isolates of A. flavus and A. parasiticus were examined for their ability to produce aflatoxins. Whether aflatoxins were produced and the amount of each varied according to the origin of the isolate and the species of Aspergillus. For example all A. flavus isolates produced at least 2 aflatoxins whereas 4 of the A. parasiticus isolates were nontoxigenic. When ground seeds of winged beans were inoculated with an aflatoxigenic strain of A. parasiticus the level of aflatoxins that occurred varied with the variety. All of the varieties supported greater aflatoxin production than peanuts and 6 of the 12 winged bean varieties gave higher levels of aflatoxins than rice.     

Improved methodology for detecting aflatoxin production quantitatively in natural media

This report describes a simple, rapid, and quantitative method for screening the aflatoxin production by moulds of theAspergillus flavus group, using a natural media (moist wheat or rice), and a single chloroform extraction for aflatoxins. The aflatoxins were detected and quantified by thin layer chromatography. The methodology proposed was useful in detecting aflatoxin production on the 3rd day of incubation in more than 85 % of the strains studied (both culture collection strains and field isolates).     

Production of extracellular enzymes and aflatoxins in solid substrate fermentation with aflatoxigenic<Emphasis Type="Italic">Aspergillus</Emphasis> spp.

AflatoxigenicAspergillus flavus andAspergillus parasiticus were subjected to solid substrate fermentation process for 6 days to determine the formation of aflatoxins and production of extracellular enzymes (amyloglucosidase, cellulase, invertase and proteinase). Both organisms produced enzymes which generally increased with fermentation.Aspergillus flavus produced four enzymes whereasA. parasiticus produced three with no proteinase activity.Aspergillus parasiticus produced aflatoxins B₁, B₂ and G₁ but no G₂ andA. flavus produced aflatoxins B₁ and B₂. Invertase showed the highest activity withA. parasiticus and that corresponded with the highest total toxin produced. The enzyme activities were higher withA. parasiticus thanA. flavus although total toxins produced byA. parasiticus were lower than total toxins produced byA. flavus under the same environmental conditions.     

Aflatoxin Production by Aspergillus flavus as Related to Various Temperatures

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

Molecular characterisation and biological control of Aspergillus flavus isolates from Saudi Arabia

Abstract

Aspergillus flavus is a phytopathogenic fungus that produces toxic compounds, aflatoxins, in infected plant tissues which harm human and animal health. In this study, 57 food/feed products were collected from 9 locations in Aseer, KSA. A total of 93 isolates were recovered from the samples and were identified as Aspergillus spp. based on their cultural and microscopic characteristics. Six isolates (Af3, Af23, Af24, Af26, Af45 and Af48) were selected and confirmed as A. flavus using polymerase chain reaction (PCR), sequencing of ITS1-5.8s-ITS2 rDNA region and phylogenetic analyses. The six sequences were deposited in the GenBank under the accession numbers of KU561932, KU561934, KU561935, KU561936, KU561937 and KU561938, respectively. Random amplification of polymorphic DNA (RAPD-PCR) of the six isolates using five primers (OPA-2, OPA-3, OPA-9, OPA-11 and OPA-15), produced polymorphic DNA bands of 12, 36, 25, 1 and 1, respectively. The band sizes ranged from 130 to 1600?bp, whereas no monomorphic bands were observed. The bio-control of the six selected A. flavus isolates using three locally isolated yeasts (Candida davisiana, Rhodotorula graminis and Exophiala dermatitidis) was assessed. On solid media, the three yeast strains inhibited all tested A. flavus isolates. The most effective yeast strain was R. graminis. In liquid media, both yeast strains C. davisiana and R. graminis inhibited the dry weights of the six A. flavus isolates. Bio-control approaches of A. flavus could help controlling the pathogen, ultimately, reduce the risk of aflatoxins in human and animal supplies and reduce the use of chemicals that affect the environment and health.     

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     

FT-IR Spectroscopy for Rapid Differentiation of Aspergillus flavus, Aspergillus fumigatus, Aspergillus parasiticus and Characterization of Aflatoxigenic Isolates Collected from Agricultural Environments

In agricultural areas, Aspergillus flavus, Aspergillus fumigatus and Aspergillus parasiticus are commonly identified in various feedstuffs and bioaerosols originated from feed handling. Some isolates belonging to these fungal species could produce mycotoxins and constitute a risk factor for human and animal health. In this study, Fourier-transform infrared spectroscopy was used for a rapid detection and characterization of 99 isolates collected from agricultural areas. The results showed a first cluster corresponding to strains previously attributed to the A. fumigatus group according to current taxonomic concepts, and a second cluster divided in 2 groups around reference strains of A. flavus and A. parasiticus species. The toxigenic capacity of isolates was evaluated by high performance liquid chromatography coupled to mass spectrometry. In the A. flavus group, only 6 strains of A. parasiticus and 4 strains of A. flavus were able to produce aflatoxins on culture media. FT-IR spectroscopy, respectively, allowed the differentiation of non-toxigenic and toxigenic A. flavus and A. parasiticus isolates at 75 and 100%. Discrimination between toxigenic and non-toxigenic A. fumigatus was not possible because all of the isolates produced at least one mycotoxin.