Aflatoxigenic Isolates of Aspergillus flavus from Pecans

Of 120 isolates of the Aspergillus flavus group from pecans used in bakery products, 85 were shown to produce aflatoxin on yeast extract sucrose medium. Extracts from moldy sections of raw pecans obtained commercially at the retail level showed aflatoxin-like spots on thin-layer chromatography. Cooked (autoclaved) pecans inoculated with toxigenic isolates of A. flavus were also good substrates for aflatoxin production.     

Investigation of Reported Aflatoxin Production by Fungi Outside the Aspergillus flavus Group

A screening study of 121 fungus isolates, representing 29 species, for aflatoxin synthesis demonstrated this property only in Aspergillus flavus and A. parasiticus. Eight of the organisms found negative were isolates reported by other investigators to produce aflatoxin. Since similar negative reports have come from several other workers, it is concluded that only the A. flavus group of Aspergillus can presently be certified as sources of these toxins. Reasons for possible false-positive findings are discussed along with precautionary measures and differential analytical procedures useful in aflatoxin screening studies.     

Sterols and fatty acids of aflatoxin and non-aflatoxin producing isolates of Aspergillus

The fatty acids and sterols present in 5 isolates of Aspergillus flavus and 3 isolates of A. parasiticus were determined; 2 isolates within each species were aflatoxin producers. The 4 major fatty acids were 16:0, 18:0, 18:1 and 18:2 with a trace of 15:0 in one isolate and traces of 17:0 in 3 other isolates. Cholesterol, ergosterol and 5, 7-ergostadienol were present in all isolates; the 5 isolates of A. flavus could be identified on the basis of retention times of minor sterols present. There was no correlation of total lipids, fatty acids or sterols with the production of aflatoxins. Water soluble complexes of sterols were not detected.     

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.     

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.     

Sexuality Generates Diversity in the Aflatoxin Gene Cluster: Evidence on a Global Scale

Aflatoxins are produced by Aspergillus flavus and A. parasiticus in oil-rich seed and grain crops and are a serious problem in agriculture, with aflatoxin B₁ being the most carcinogenic natural compound known. Sexual reproduction in these species occurs between individuals belonging to different vegetative compatibility groups (VCGs). We examined natural genetic variation in 758 isolates of A. flavus, A. parasiticus and A. minisclerotigenes sampled from single peanut fields in the United States (Georgia), Africa (Benin), Argentina (Córdoba), Australia (Queensland) and India (Karnataka). Analysis of DNA sequence variation across multiple intergenic regions in the aflatoxin gene clusters of A. flavus, A. parasiticus and A. minisclerotigenes revealed significant linkage disequilibrium (LD) organized into distinct blocks that are conserved across different localities, suggesting that genetic recombination is nonrandom and a global occurrence. To assess the contributions of asexual and sexual reproduction to fixation and maintenance of toxin chemotype diversity in populations from each locality/species, we tested the null hypothesis of an equal number of MAT1-1 and MAT1-2 mating-type individuals, which is indicative of a sexually recombining population. All samples were clone-corrected using multi-locus sequence typing which associates closely with VCG. For both A. flavus and A. parasiticus, when the proportions of MAT1-1 and MAT1-2 were significantly different, there was more extensive LD in the aflatoxin cluster and populations were fixed for specific toxin chemotype classes, either the non-aflatoxigenic class in A. flavus or the B₁-dominant and G₁-dominant classes in A. parasiticus. A mating type ratio close to 1∶1 in A. flavus, A. parasiticus and A. minisclerotigenes was associated with higher recombination rates in the aflatoxin cluster and less pronounced chemotype differences in populations. This work shows that the reproductive nature of the population (more sexual versus more asexual) is predictive of aflatoxin chemotype diversity in these agriculturally important fungi.     

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.     

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

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

Mycotoxin-Producing Ability and Chemotype Diversity of Aspergillus Section Flavi from Soils of Peanut-Growing Regions in Iran

Invasion of crops with Aspergillus flavus may result in contamination of food and feed with carcinogenic mycotoxins such as aflatoxins (AF) and cyclopiazonic acid (CPA). In the present study, distribution and toxigenicity of Aspergillus flavus and A. parasiticus in soils of five peanut fields located in Guilan province, Northern Iran was investigated. From a total of 30 soil samples, 53 strains were isolated which all of them were finally identified as A. flavus by a combination of colony morphology, microscopic criteria and mycotoxin profiles. Chromatographic analysis of fungal cultures on yeast extract sucrose broth by tip culture method showed that 45 of the 53 A. flavus isolates (84.9 %) were able to produce either CPA or AFB₁, while eight of the isolates (15.1 %) were non-toxigenic. The amounts of CPA and AFB₁ produced by the isolates were reported in the range of 18.2–403.8 μg/g and 53.3–7446.3 μg/g fungal dry weights, respectively. Chemotype classification of A. flavus isolates based on the ability for producing mycotoxins and sclerotia showed that 43.4 % were producers of CPA, AFB₁ and sclerotia (group I), 13.2 % of CPA and AFB₁ (group II), 9.4 % of AFB₁ and sclerotia (group III), 13.2 % of AFB₁ (group IV), 5.7 % of CPA and sclerotia (group V) and 15.1 % were non-toxigenic with no sclerotia (group VI). No strain was found as producer of only CPA or sclerotia. These results indicate different populations of mycotoxigenic A. flavus strains enable to produce hazardous amounts of AFB₁ and CPA are present in peanuts field soils which can be quite important regard to their potential to contaminate peanuts as a main crop consumed in human and animal nutrition.     

Homologs of Aflatoxin Biosynthesis Genes and Sequence of aflR in Aspergillus oryzae and Aspergillus sojae

The presence, but not expression, of homologs of three structural genes and a regulatory gene necessary for aflatoxin biosynthesis in Aspergillus parasiticus and A. flavus was shown for A. oryzae and A. sojae. Homologs of the regulatory gene aflR were cloned and sequenced from A. oryzae and A. sojae.     

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.     

Regional Differences in Production of Aflatoxin B1 and Cyclopiazonic Acid by Soil Isolates of Aspergillus flavus along a Transect within the United States

Soil isolates of Aspergillus flavus from a transect extending from eastern New Mexico through Georgia to eastern Virginia were examined for production of aflatoxin B₁ and cyclopiazonic acid in a liquid medium. Peanut fields from major peanut-growing regions (western Texas; central Texas; Georgia and Alabama; and Virginia and North Carolina) were sampled, and fields with other crops were sampled in regions where peanuts are not commonly grown. The A. flavus isolates were identified as members of either the L strain (n = 774), which produces sclerotia that are >400 μm in diameter, or the S strain (n = 309), which produces numerous small sclerotia that are <400 μm in diameter. The S-strain isolates generally produced high levels of aflatoxin B₁, whereas the L-strain isolates were more variable in aflatoxin production; variation in cyclopiazonic acid production also was greater in the L strain than in the S strain. There was a positive correlation between aflatoxin B₁ production and cyclopiazonic acid production in both strains, although 12% of the L-strain isolates produced only cyclopiazonic acid. Significant differences in production of aflatoxin B₁ and cyclopiazonic acid by the L-strain isolates were detected among regions. In the western half of Texas and the peanut-growing region of Georgia and Alabama, 62 to 94% of the isolates produced >10 μg of aflatoxin B₁ per ml. The percentages of isolates producing >10 μg of aflatoxin B₁ per ml ranged from 0 to 52% in the remaining regions of the transect; other isolates were often nonaflatoxigenic. A total of 53 of the 126 L-strain isolates that did not produce aflatoxin B₁ or cyclopiazonic acid were placed in 17 vegetative compatibility groups. Several of these groups contained isolates from widely separated regions of the transect.     

Enhanced diversity and aflatoxigenicity in interspecific hybrids of Aspergillus flavus and Aspergillus parasiticus

Aspergillus flavus and A. parasiticus are the two most important aflatoxin‐producing fungi responsible for the contamination of agricultural commodities worldwide. Both species are heterothallic and undergo sexual reproduction in laboratory crosses. Here we examine the possibility of interspecific matings between A. flavus and A. parasiticus. These species can be distinguished morphologically and genetically, as well as by their mycotoxin profiles. Aspergillus flavus produces both B aflatoxins and cyclopiazonic acid (CPA), B aflatoxins or CPA alone, or neither mycotoxin; Aspergillus parasiticus produces B and G aflatoxins or the aflatoxin precursor O‐methylsterigmatocystin, but not CPA. Only four of forty‐five attempted interspecific crosses between opposite mating types of A. flavus and A. parasiticus were fertile and produced viable ascospores. Single ascospore strains from each cross were shown to be recombinant hybrids using multilocus genotyping and array comparative genome hybridization. Conidia of parents and their hybrid progeny were haploid and predominantly monokaryons and dikaryons based on flow cytometry. Multilocus phylogenetic inference showed that experimental hybrid progeny were grouped with naturally occurring A. flavus L strain and A. parasiticus. Higher total aflatoxin concentrations in some F1 progeny strains compared to midpoint parent aflatoxin levels indicate synergism in aflatoxin production; moreover, three progeny strains synthesized G aflatoxins that were not produced by the parents, and there was evidence of allopolyploidization in one strain. These results suggest that hybridization is an important diversifying force resulting in the genesis of novel toxin profiles in these agriculturally important fungi.     

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

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

RAPD analysis of genetic diversity among the isolates of Aspergillus flavus from different hosts and locations

Aflatoxin contamination is a major problem in maize, groundnut, chillies, cotton and tree nuts. These aflatoxins are low molecular weight toxic and carcinogenic secondary metabolites produced by Aspergillus flavus, A. parasiticus and A. nomius. In the present study, a total of 11 isolates of A. flavus isolated from groundnut, maize and chilli collected from different locations of Tamil Nadu, India were tested for their ability to produce aflatoxin B1 (AFB1) in vitro by indirect competitive enzyme-linked immunosorbent assay. The results show that the isolates vary in their level of toxin production. The amount of AFB1 produced by the toxigenic isolates of A. flavus ranged from 6.6 to 108.1?ng?ml^?1. Among the various isolates of A. flavus, the isolate VKR produced the highest amount (108.1?ng?ml^?1) of AFB1. The isolates viz. CBE1, CBE2, BSR1, BSR3 and BSR4 were found to be non-toxigenic. The genetic variability among these isolates was assessed by Random amplified polymorphic DNA (RAPD) analysis. DNA fragments of between 0.15 and 3.0?kb were obtained using 13 random primers, and each isolate differed in the size and number of PCR products indicating considerable polymorphism. Cluster analysis using Unweighted Pair Group Method with Arithmetic Mean clearly separated the isolates into four main clusters confirming the genetic diversity among the isolates of A. flavus. Both toxigenic and non-toxigenic isolates were intermingled in these four groups, indicating that no relationship exists between RAPD profile and the production of aflatoxin by A. flavus.     

Separate and combined applications of nontoxigenic Aspergillus flavus and A. parasiticus for biocontrol of aflatoxin in peanuts

A 2-year study was carried out to determine the effect of applying nontoxigenic strains of Aspergillus flavus and A. parasiticus to soil separately and in combination on preharvest aflatoxin contamination of peanuts. A naturally occurring, nontoxigenic strain of A. flavus and a UV-induced mutant of A. parasiticus were applied to peanut soils during the middle of each of two growing seasons using a formulation of conidia-coated hulled barley. In addition to an untreated control, treatments included soil inoculated with nontoxigenic A. flavus only, soil inoculated with nontoxigenic A. parasiticus only, and soil inoculated with a mixture of the two nontoxigenic strains. Plants were exposed to late-season drought conditions that were optimal for aflatoxin contamination. Results from year one showed that significant displacement (70%) of toxigenic A. flavus occurred only in peanuts from plots treated with nontoxigenic A. flavus alone; however, displacement did not result in a statistically significant reduction in the mean aflatoxin concentration in peanuts. In year two, soils were re-inoculated as in year one and all treatments resulted in significant reductions in aflatoxin, averaging 91.6%. Regression analyses showed strong correlations between the presence of nontoxigenic strains in peanuts and aflatoxin reduction. It is concluded that treatment with the nontoxigenic A. flavus strain alone is more effective than the A. parasiticus strain alone and equally as effective as the mixture. The U.S. Government’s right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

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 was determined both before and after surface disinfections. When seeds were surface disinfected, mold fungi were detected in 73% of the seeds whereas 81% of the seed that was not disinfected produced mold fungi. Aspergillus spp. was most frequently present while Penicillium spp. occurred in seed of 4 varieties and in less than 4% of the seed. Twelve isolates oiA. 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. parasiticus isolates produced at least 2 aflatoxins whereas 4 of the A. parasiticus isolates were non-toxigenic. 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, however, the level of aflatoxin was higher in winged bean than in peanut tissue and 6 of the 12 winged bean varieties contained higher levels of aflatoxins than rice.