Occurrence of aflatoxin B1 and ochratoxin A in Lebanese cultivated wheat

An extensive survey of filamentous fungi isolated from wheat grown and consumed in Lebanon and their capacity to produce aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) was conducted to assess fungi potential for producing these toxins in wheat. From the 468 samples of wheat kernel, collected at preharvest stage from different locations during 2008 and 2009 cultivation seasons, 3,260 fungi strains were isolated with 49.4% belonging to Penicillium spp. and 31.2% belonging to Aspergillus spp. Penicillium spp. was detected on wheat samples with a high amount of P. verrucosum (37.0%). Among the different Aspergillus spp. isolated, A. niger aggregate was predominant and constituted 37.3%. whereas the isolation rate of A. flavus and A. ochraceus was 32.2 and 25.6%, respectively. The ability to produce OTA and AFB₁ by isolates belonging to Aspergillus spp. and Penicillium spp. was analyzed by high performance liquid chromatography with fluorescence detector (HPLC-FLD). It was found that 57.0% of Penicillium spp. and 80% of A. ochraceus isolates tested produced OTA, respectively, at maximum concentrations of 53 and 65 μg/g CYA. As for the aflatoxinogenic ability, 45.3% of A. flavus produced AFB₁, with maximum concentration of 40 μg/g CYA. A total of 156 wheat samples were analyzed for the levels of OTA and AFB₁ by HPLC-FLD. The results showed that 23.7% were contaminated with OTA, at a concentration higher than 3 μg/kg and 35.2% of these samples were contaminated with AFB₁ at concentration higher than 2 μg/kg. The risks originating from toxin levels in wheat produced in Lebanon should be monitored to prevent their harmful effects on public health.     

First Report on Mould and Mycotoxin Contamination of Pistachios Sampled in Algeria

Mycotoxin contamination of pistachios represents a serious food safety hazard. The aim of this study was to evaluate fungal contamination and aflatoxin (AF) and ochratoxin A (OTA) occurrence in pistachio sampled in Algeria and to study the mycotoxigenic capacities of the isolates. A total of 31 pistachio samples were collected from retail outlets from different regions of Algeria. The most frequently found fungi were Penicillium spp. (38%), Aspergillus section Nigri (30%) and A. flavus (22%). A total of 56.5% of A. flavus isolates were able to produce AFB₁ and AFB₂. No A. section Nigri uniseriate isolate was OTA producer, whereas OTA production capacity was detected in 33.3% of the A. section Nigri biseriate. At least one of the potentially ochratoxigenic species was found in 64.5% of samples. Despite the high number of pistachio samples containing AFs and OTA-producing isolates, only two samples contained AFs (always below the EU maximum tolerable level) and only one sample showed OTA contamination. This is the first report on the occurrence of toxigenic moulds and mycotoxins in pistachios from Algerian market.     

Evaluation of aflatoxin B1 and ochratoxin A in interacting mixed cultures of Aspergillus sections Flavi and Nigri on peanut grains

The influence of inoculum size on the colony-forming units, production of aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) was determined when Aspergillus flavus and A. niger aggregate strains were cultured alone and in pairs on irradiated peanut grains at 28°C and 0.97 water activity (a_W). The results showed a marked influence of inoculum factor on fungal counts, AFB₁ and OTA production in single and paired cultures. Fungal counts of the A. niger aggregate strain in interacting cultures at 7, 14 and 21?days of incubation were significantly higher than those observed in the A. flavus strain, except in the mixed culture with 10² spores/ml of both strains. In all mixed culture assays, the AFB₁ production was significantly reduced in comparison with the accumulation of mycotoxin in single cultures. A total inhibition in AFB₁ production was observed in some interactions as 10² spores/ml of A. flavus and 10³ spores/ml of A. niger aggregate strain at 7 and 14?days, among others. With regard to OTA production, a stimulation in the interacting cultures was observed at all inoculum sizes and incubation period. The highest levels of OTA accumulation were observed at 14?days for all interacting cultures. The maximum level was reach in the culture 10³ spores/ml of A. niger aggregate and 10⁴ spores/ml of A. flavus (p?<?0.001). These results suggest that, under optimal environmental conditions in peanut grains, the interaction between A. flavus and A. niger aggregate strains could result in an inhibition of AFB₁ and in a stimulation of OTA production.     

Mycotoxigenic potential of fungi isolated from freshly harvested Argentinean blueberries

Alternaria alternata, A. tenuissima, Fusarium graminearum, F. semitectum, F. verticillioides, Aspergillus flavus, and Aspergillus section Nigri strains obtained from blueberries during the 2009 and 2010 harvest season from Entre Ríos, Argentina were analyzed to determine their mycotoxigenic potential. Taxonomy status at the specific level was determined both on morphological and molecular grounds. Alternariol (AOH), alternariol monomethyl ether (AME), aflatoxins (AFs), zearalenone (ZEA), fumonisins (FBs), and ochratoxin A (OTA) were analyzed by HPLC and the trichotecenes deoxynivalenol (DON), nivalenol (NIV), HT-2 toxin (HT-2), T-2 toxin (T-2), fusarenone X (FUS-X), 3-acetyl-deoxynivalenol (3-AcDON), and 15-acetyl-deoxynivalenol (15-AcDON) by GC. Twenty-five out of forty two strains were able to produce some of the mycotoxins analyzed. Fifteen strains of Aspergillus section Nigri were capable of producing Fumonisin B₁ (FB₁); two of them also produced Fumonisin B₂ (FB₂) and one Fumonisin B₃ (FB₃). One of the F. graminearum isolated produced ZEA, HT-2, and T-2 and the other one was capable of producing ZEA and DON. Two A. alternata isolates produced AOH and AME. Four A. tenuissima were capable of producing AOH and three of them produced AME as well. One Aspergillu flavus strain produced aflatoxin B₁ (AFB₁), aflatoxin B₂ (AFB₂), and aflatoxin G₁ (AFG₁). To our knowledge, this is the first report showing mycotoxigenic capacity of fungal species isolated from blueberries that include other fungi than Alternaria spp.     

An insight into the distribution,genetic diversity,and mycotoxin production of <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Flavi</Emphasis> in soils of pistachio orchards

In the present study, 193 Aspergillus strains were isolated from a total of 100 soil samples of pistachio orchards, which all of them were identified as Aspergillus flavus as the most abundant species of Aspergillus section Flavi existing in the environment. Approximately 59%, 81%, and 61% of the isolates were capable of producing aflatoxins (AFs), cyclopiazonic acid (CPA), and sclerotia, respectively. The isolates were classified into four chemotypes (I to IV) based on the ability to produce AFs and CPA. The resulting dendrogram of random amplified polymorphic DNA (RAPD) analysis of 24 selected A. flavus isolates demonstrated the formation of two separate clusters. Cluster 1 contained both aflatoxigenic and non-aflatoxigenic isolates (17 isolates), whereas cluster 2 comprised only aflatoxigenic isolates (7 isolates). All the isolates of cluster 2 produced significantly higher levels of AFs than those of cluster 1 and the isolates that produced both AFB₁ and AFB₂ were found only in cluster 2. RAPD genotyping allowed the differentiation of A. flavus from Aspergillus parasiticus as a closely related species within section Flavi. The present study has provided for the first time the relevant information on distribution and genetic diversity of different A. flavus populations from nontoxigenic to highly toxigenic enable to produce hazardous amounts of AFB₁ and CPA in soils of pistachio orchards. These fungi, either toxigenic or not-toxigenic, should be considered as potential threats for agriculture and public health.     

Isolation of Bacterial Antagonists of Aspergillus flavus from Almonds

Bacteria were isolated from California almond orchard samples to evaluate their potential antifungal activity against aflatoxin-producing Aspergillus flavus. Fungal populations from the same samples were examined to determine the incidence of aflatoxigenic Aspergillus species. Antagonistic activities of the isolated bacterial strains were screened against a nonaflatoxigenic nor mutant of A. flavus, which accumulates the pigmented aflatoxin precursor norsolorinic acid (NOR) under conditions conducive to aflatoxin production. Using solid and liquid media in coculture assays, 171 bacteria isolated from almond flowers, immature nut fruits, and mature nut fruits showed inhibition of A. flavus growth and/or inhibition of NOR accumulation. Bacterial isolates were further characterized for production of extracellular enzymes capable of hydrolyzing chitin or yeast cell walls. Molecular and physiological identification of the bacterial strains indicated that the predominant genera isolated were Bacillus, Pseudomonas, Ralstonia, and Burkholderia, as well as several plant-associated enteric and nonenteric bacteria. A set of 20 isolates was selected for further study based on their species identification, antifungal phenotypes, and extracellular enzyme production. Quantitative assays using these isolates in liquid coculture with a wild-type, aflatoxin-producing A. flavus strain showed that a number of strains completely inhibited fungal growth in three different media. These results indicate the potential for development of bacterial antagonists as biological control agents against aflatoxigenic aspergilli on almonds.     

黄曲霉毒素生物防控菌的筛选及鉴定

筛选黄曲霉毒素生物防控菌,为黄曲霉毒素的生物防控提供支持。以花生原产地土壤为材料,采用牛津杯法筛选所需菌株。对筛选出的拮抗菌株进行抑制产毒曲霉菌株的生长、产孢、降解黄曲霉毒素实验。筛选出2株黄曲霉毒素生防细菌,编号21-1-2、17-3,经鉴定,拮抗菌21-1-2为枯草芽胞杆菌,拮抗菌17-3为地衣芽胞杆菌。分别对拮抗菌对曲霉孢子萌发的抑制、抑制黄曲霉的生长和菌丝延长以及减少黄曲霉毒素的产生、对黄曲霉毒素的分解作用等几个方面进行研究,结果表明,拮抗菌可以明显抑制产毒曲霉孢子的萌发、生长、菌丝的延长,减少黄曲霉毒素的产生以及分解黄曲霉毒素。     

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.     

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     

Aspergillus flavus infection and aflatoxin contamination in sorghum seeds and their biological management

In order to establish the current scenario of aflatoxigenic fungal infection and aflatoxin contamination in sorghum seeds across India, 58 seed samples were collected from different agro-climatic regions. Among these, 67.2% samples were infected with Aspergillus spp. and 28% were found contaminated with aflatoxins ranging from 0.0 to 130?μg?kg^?1. Greenhouse studies revealed no correlation between incidence of Aspergillus flavus and aflatoxin content, and its effect on seed quality parameters. Among the 37 A. flavus strains isolated, six were non-aflatoxigenic when analysed through cultural, TLC and ic-ELISA. Seed treatment with biocontrol agents (antagonistic Rhizobacteria and Trichoderma) suppressed the growth of A. flavus under laboratory and significantly enhanced seed quality variables under greenhouse conditions to a various extent. Field trials with selected biocontrol agents showed that talcum powder formulations of Pseudomonas putida Has-1/c, Bacillus spp. 3/a, Trichoderma asperellum M5 and T. asperellum T2 improved seedling emergence, % nutrient accumulation in plants, increased plant biomass and 1000 seed weight. Seeds harvested from treated plants showed significant increase in seed quality variables under laboratory and greenhouse conditions in comparison with control, but there was no significant difference in A. flavus infection and aflatoxin was completely absent in all treatments.     

Effect of acid lactic bacteria isolated from faeces of healthy dogs on growth parameters and aflatoxin B<Subscript>1</Subscript> production by <Emphasis Type="Italic">Aspergillus</Emphasis> species in vitro

The aim of the present study was to evaluate the inhibitory effect of Enterococcus faecium and Lactococcus lactis subsp. lactis isolated from faeces of healthy dogs on (i) lag phase, (ii) growth rate, and (iii) aflatoxin B₁ production by Aspergillus section Flavi on in vitro assays. Thirteen lactic acid bacteria (LAB) isolates were used as antagonist microorganisms. Antagonistic activity was assayed against four potentially aflatoxigenic Aspergillus section Flavi isolates: A. flavus (AF210 and AF281), A. parasiticus (AP245) and A. parasiticus (NRRL 2999). In general, the longest lag phases of Aspergillus isolates were obtained with E. faecium GJ40. Respecting the growth rate, no significant reduction was found in this parameter in the interaction assays with A. flavus and antagonist isolates respecting the control. While in A. parasiticus a significant reduction in growth rate was only observed in the interaction among reference strain and E. faecium MF5 isolate (p < 0.05). In general, AFB₁ production was reduced by most of the LAB isolates assayed, except for E. faecium GJ18, GJ20, MF3 and MF4. This study provides the first data about the antiaflatoxigenic activity of autochthonous LAB isolated from dog faeces.     

Assessment of mycoflora and infestation of insects,vector of <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Flavi</Emphasis>, in stored peanut from Argentina

The occurrence of spoilage fungi and Aspergillus section Flavi populations, the aflatoxins incidence, the role of insects as vectors of mycotoxin-producing fungi and the AFs-producing ability of the isolated species throughout the peanut (Arachis hypogaea L.) storage period were evaluated. Analyses of fungal populations from 95 peanut seed samples did not demonstrate significant differences between the incidences in each sampling period. Aspergillus section Flavi were isolated during all incubation periods. Cryptolestes spp. (Coleoptera: Cucujidae) were collected in August, September and October with 18, 16 and 28% of peanut samples contaminated, respectively. Insects isolated during August showed 69% of Aspergillus section Flavi contamination. A. flavus was the most frequently isolated (79%) from peanut seeds and from insect (59%). The greater levels of AFB₁ were detected in September and October with a mean of 68.86 μg/kg and 69.12 μg/kg respectively. The highest proportion of A. flavus toxigenic strains (87.5%) was obtained in June. The presence of Aspergillus section Flavi and insect vectors of aflatoxigenic fungi presented a potential risk for aflatoxin production during the peanut storage period. Integrated management of fungi and insect vectors is in progress.     

Contamination of Groundnut in South‐Western Nigeria by Aflatoxigenic Fungi and Aflatoxins in Relation to Processing

Groundnut is commonly consumed in its roasted form by many Nigerians. This study was therefore conducted to determine the levels of aflatoxin in roasted groundnut retailed in south‐western Nigeria with a view to assessing the fitness of the processed nut for human consumption. The effects of roasting and de‐coating as alternative methods for reducing the ‘aflatoxin scare’ in the nut were further assessed on aflatoxigenic fungal load and aflatoxin content of the nuts. Forty‐eight samples of retailed raw and roasted groundnut were collected and assessed by mycological and thin‐layer chromatographic analysis for changes in aflatoxigenic fungal population and aflatoxin concentration, respectively. Consequently, 480 isolates of the Aspergillus section Flavi group, A. flavus L strain (n = 410), A. tamarii (n = 56), A. parasiticus (n = 7) and A. parvisclerotigenus (n = 7), were recovered from all samples. Aflatoxigenic isolates of A. flavus L strain (58.8%) had a significantly (P < 0.05) higher incidence than the non‐aflatoxigenic isolates (41.2%). Aflatoxins were detected in 43 (89.6%) of the samples. Approximately 25% of all samples exceeded the 20 ng/g limit for aflatoxin B₁ (AFB₁) adopted by the National Agency for Food and Drug Administration and Control while 83 and 79% of all samples contained AFB₁ and total aflatoxins above the European Union limits of 2 and 4 ng/g, respectively. Aflatoxin concentrations in the raw and coated samples were as much as five times higher than those in the roasted and de‐coated nuts, respectively. However, no significant difference was recorded between aflatoxin levels in the coated and de‐coated samples. This study has shown that roasting of groundnut and testa removal (de‐coating) are effective processing interventions that can significantly lower aflatoxin quantities in the kernels, thus making it fit for human consumption.     

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.     

Characterization of aflatoxigenic and non-aflatoxigenic <Emphasis Type="Italic">Aspergillus flavus</Emphasis> isolates from pistachio

Pistachio is a popular snack food. Aflatoxin contamination of pistachio nuts is a serious problem for many producing countries. The development of biological control methods based on ecological parameters is an environmentally friendly approach. Thirty-eight Aspergillus flavus isolates collected from a pistachio orchard in California (CA) were analyzed for production of aflatoxin (AF), cyclopiazonic acid (CPA), vegetative compatibility groups (VCGs), and mating types. All aflatoxigenic isolates produced both AFB₁ and CPA. The most toxigenic one was CA28 which produced 164 μg AFB₁ per 5 ml PDA fungal culture and small sclerotia (S strain, sclertoium size less than 400 μm). The other aflatoxigenic strains produce AFB₁ ranging from 1.2 μg to 80 μg per 5 ml fungal culture. Twenty-one percent of the CA isolates produced AFB₁, 84% produced CPA and half formed sclerotia on at least one of three tested media. The 38 CA isolates formed 26 VCGs, 6 of which had two or more isolates and 20 contained single isolates. The S strain isolates belong to 4 different VCGs. Genomic profiling by a retrotransposon DNA probe revealed fingerprint patterns that were highly polymorphic. The predicted VCGs (Pred-VCGs) based on a similarity coefficient >80% matched the VCGs of multiple isolates determined by complementation. All isolates within a VCG had the same mating-type gene of either MAT1-1 or MAT1-2. Uncorrected and VCG-corrected MAT1-1 and MAT1-2 among the isolates were equally distributed.     

Chemosensitization of Aflatoxigenic Fungi to Antimycin A and Strobilurin Using Salicylaldehyde,a Volatile Natural Compound Targeting Cellular Antioxidation System

Various species of fungi in the genus Aspergillus are the most common causative agents of invasive aspergillosis and/or producers of hepato-carcinogenic mycotoxins. Salicylaldehyde (SA), a volatile natural compound, exhibited potent antifungal and anti-mycotoxigenic activities to A. flavus and A. parasiticus. By exposure to the volatilized SA, the growth of A. parasiticus was inhibited up to 10–75% at 9.5 mM ≤ SA ≤ 16.0 mM, while complete growth inhibition was achieved at 19.0 mM ≤ SA. Similar trends were also observed with A. flavus. The aflatoxin production, i.e., aflatoxin B₁ and B₂ (AFB₁, AFB₂) for A. flavus and AFB₁, AFB₂, AFG₁, and AFG₂ for A. parasiticus, in the SA-treated (9.5 mM) fungi was reduced by ~13–45% compared with the untreated control. Using gene deletion mutants of the model yeast Saccharomyces cerevisiae, we identified the fungal antioxidation system as the molecular target of SA, where sod1Δ [cytosolic superoxide dismutase (SOD)], sod2Δ (mitochondrial SOD), and glr1Δ (glutathione reductase) mutants showed increased sensitivity to this compound. Also sensitive was the gene deletion mutant, vph2Δ, for the vacuolar ATPase assembly protein, suggesting vacuolar detoxification plays an important role for fungal tolerance to SA. In chemosensitization experiments, co-application of SA with either antimycin A or strobilurin (inhibitors of mitochondrial respiration) resulted in complete growth inhibition of Aspergillus at much lower dose treatment of either agent, alone. Therefore, SA can enhance antifungal activity of commercial antifungal agents required to achieve effective control. SA is a potent antifungal and anti-aflatoxigenic volatile that may have some practical application as a fumigant.     

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.     

Molecular identification of <Emphasis Type="Italic">Aspergillus</Emphasis> and <Emphasis Type="Italic">Eurotium</Emphasis> species isolated from rice and their toxin-producing ability

Thirty milled rice samples were collected from retailers in 4 provinces of Malaysia. These samples were evaluated for Aspergillus spp, infection by direct plating on malt extract salt agar (MESA). All Aspergillus holomorphs were isolated and identified using nucleotide sequences of ITS 1 and ITS 2 of rDNA. Five anamorphs (Aspergillus flavus, A. oryzae, A. tamarii, A. fumigatus and A. nigef) and 5 teleomorphs (Eurotium rubrum, E. amstelodami, E. chevalieri, E. cristatum and E. tonophilum) were identified. The PCR-sequencing based technique for sequences of ITS 1 and ITS 2 is a fast technique for identification of Aspergillus and Eurotium species, although it does not work flawlessly for differentiation of Eurotium species. All Aspergillus and Eurotium isolates were screened for their ability to produce aflatoxin and ochratoxin A (OTA) by HPLC and TLC techniques. Only A. flavus isolate UPM 89 was able to produce aflatoxins B1 and B2.     

Evaluation of different genotypes of nontoxigenic Aspergillus flavus for their ability to reduce aflatoxin contamination in peanuts

Aflatoxins produced by the fungus Aspergillus flavus are potent carcinogens and account for large monetary losses worldwide in peanuts, maize, and cottonseed. Biological control in which a nontoxigenic strain of A. flavus is applied to crops at high concentrations effectively reduces aflatoxins through competition with native aflatoxigenic populations. In this study, eight nontoxigenic strains of A. flavus belonging to different vegetative compatibility groups and differing in deletion patterns within the aflatoxin gene cluster were evaluated for their ability to reduce aflatoxin B₁ when paired with eight aflatoxigenic strains on individual peanut seeds. Inoculation of wounded viable peanut seeds with conidia demonstrated that nontoxigenic strains differed in their ability to reduce aflatoxin B₁. Reductions in aflatoxin B₁ often exceeded expected reductions based on a 50:50 mixture of the two A. flavus strains, although one nontoxigenic strain significantly increased aflatoxin B₁ when paired with an aflatoxigenic strain. Therefore, nontoxigenicity alone is insufficient for selecting a biocontrol agent and it is also necessary to test the effectiveness of a nontoxigenic strain against a variety of aflatoxigenic strains.     

Aflatoxin-producing Aspergillus species from saffron field soils in the South Khorasan province of Iran

The aim of the present study was to isolate and identify Aspergillus species associated with saffron plants in the city of Birjand (South Khorasan Province, Iran) as well as to assess their aflatoxin B1 production. Sampling was performed during 2013–2014 crop season. Aspergillus species were isolated and purified using general and specific culture media. Growth rates and macroscopic and microscopic characteristics of the isolates were determined using yeast extract, Czapek yeast extract, malt extract and creatine sucrose agar media at 25 and 37 °C. DNA was extracted by the modified CTAB method and beta-tubulin, calmodulin and internal transcribed spacer genes were amplified and sequenced. Phylogenetic position of the isolates was determined against other Aspergillus species. Thin layer chromatography was used to investigate the production of aflatoxin B1 by Aspergillus isolates. Based on the morphological characteristics, shape and colour of the colonies, and sequencing results, the isolates belonged to Aspergillus terreus, A. flavus, A. flavipes and A. niger species. Only A. flavus isolates were aflatoxin B1 producers. We concluded that the soil of the studied saffron fields contained several species of Aspergillus, with A. flavus significantly affecting crop production through contamination of the crop by aflatoxin.