Aflatoxigenic Strains Research Articles

Genome sequence and comparative analyses of atoxigenic Aspergillus flavus WRRL 1519

ABSTRACTAflatoxins are toxic secondary metabolites produced by Aspergillus flavus and a few other closely related species of Aspergillus. These highly toxigenic and carcinogenic mycotoxins contaminate global food and feed supplies, posing widespread health risks to humans and domestic animals. Field application of nonaflatoxigenic strains of A. flavus to compete against aflatoxigenic strains has emerged as one of the best management practices for reducing aflatoxins contamination, yielding successful commercial products for corn, cotton seed, and peanuts. In this study, we sequenced the genome and transcriptome of atoxigenic (does not produce aflatoxin or cyclopiazonic acid) A. flavus strain WRRL 1519 isolated from a tree nut orchard to define the genetic characteristics of the strain in relation to aflatoxigenic and other nonaflatoxigenic A. flavus strains. WRRL 1519 strain was similar to other strains in size (38.0 Mb), GC content (47.2%), number of predicted secondary metabolite gene clusters (46), and number of putative proteins (12 121). About 87.4% of the predicted proteome had high shared identity with protein sequences derived from other A. flavus genomes. However, the atoxigenic A. flavus strain WRRL 1519 had deletions, or low shared identity, for many genes in the clusters required for aflatoxins and cyclopiazonic acid (CPA) synthesis. Over half of the aflatoxin synthesis gene cluster was missing, and none of the components of the CPA gene cluster were identified with high sequence similarity. Importantly, the strain appeared to maintain functional sequences of several genes thought to be required for high infectivity. Since the ability to grow on target crop is an important attribute for a successful biocontrol agent, these results indicate that the nonaflatoxigenic A. flavus strain WRRL 1519 would be a good candidate as a biocontrol agent for reducing aflatoxin and CPA accumulation in high-value nut crops.

Characterization and competitive ability of non-aflatoxigenic Aspergillus flavus isolated from the maize agro-ecosystem in Argentina as potential aflatoxin biocontrol agents

Aspergillus flavus is an opportunistic pathogen and may produce aflatoxins in maize, one of the most important crops in Argentina. A promising strategy to reduce aflatoxin accumulation is the biological control based on competitive exclusion. In order to select potential biocontrol agents among isolates from the maize growing region in Argentina, a total of 512 A. flavus strains were isolated from maize kernels and soil samples. Thirty-six per cent of the isolates from maize kernels did not produce detectable levels of aflatoxins, while 73% of the isolates from soil were characterized as non-aflatoxin producers. Forty percent and 49% of the isolates from maize kernels and soil samples, respectively, were not producers of cyclopiazonic acid (CPA). Sclerotia morphology was evaluated using Czapek Dox media. Eighty-six per cent of the isolates from maize kernels and 85% of the isolates from soil samples were L sclerotia morphotype (average diameter > 400 μm). The remaining isolates did not produce sclerotia. All isolates had MAT 1-1 idiomorph. The competitive ability of 9 non aflatoxigenic strains, 4 CPA(+) and 5 CPA(−), was evaluated in co-inoculations of maize kernels with an aflatoxigenic strain. All evaluated strains significantly (p < 0.05) reduced aflatoxin contamination in maize kernels. The aflatoxin B1 (AFB1) reduction ranged from 6 to 60%. The strain A. flavus ARG5/30 isolated from maize kernels would be a good candidate as a potential biocontrol agent to be used in maize, since it was characterized as neither aflatoxin nor CPA producer, morphotype L, MAT 1-1 idiomorph, and reduced AFB1 content in maize kernels by 59%. This study showed the competitive ability of potential aflatoxin biocontrol agents to be evaluated under field trials in a maize agro-ecosystem in Argentina.

Development of a droplet digital PCR assay for population analysis of aflatoxigenic and atoxigenic Aspergillus flavus mixtures in soil.

Aflatoxin B1 is a potent hepatotoxin and carcinogen that poses a serious safety hazard to both humans and animals. Aspergillus flavus is the most common aflatoxin-producing species on corn, cotton, peanuts, and tree nuts. Application of atoxigenic strains to compete against aflatoxigenic strains of A. flavus has emerged as one of the most practical strategies for ameliorating aflatoxin contamination in food. Genes directly involved in aflatoxin biosynthesis are clustered on an 82-kb region of the genome. Three atoxigenic strains (CA12, M34, and AF123) were each paired with each of four aflatoxigenic strains (CA28, CA42, CA90, and M52), inoculated into soil and incubated at 28°C for 2weeks and 1month. TaqMan probes, omtA-FAM, and norA-HEX were designed for developing a droplet digital PCR (ddPCR) assay to analyze the soil population of mixtures of A. flavus strains. DNA was extracted from each soil sample and used for ddPCR assays. The data indicated that competition between atoxigenic and aflatoxigenic was strain dependent. Variation in competitive ability among different strains of A. flavus influenced the population reduction of the aflatoxigenic strain by the atoxigenic strain. Higher ratios of atoxigenic to aflatoxigenic strains increased soil population of atoxigenic strains. This is the first study to demonstrate the utility of ddPCR to quantify mixtures of both atoxigenic and aflatoxigenic A. flavus strains in soil and allows for rapid and accurate determination of population sizes of atoxigenic and aflatoxigenic strains. This method eliminates the need for isolation and identification of individual fungal isolates from experimental soil samples.

First report of an atypical new Aspergillus parasiticus isolates with nucleotide insertion in aflR gene resembling to A. sojae.

Aflatoxins are toxic and carcinogenic secondary metabolites produced primarily by the filamentous fungi Aspergillus flavus and Aspergillus parasiticus and cause toxin contamination in food chain worldwide. Aspergillus oryzae and Aspergillus sojae are highly valued as koji molds in the traditional preparation of fermented foods, such as miso, sake, and shoyu. Koji mold species are generally perceived of as being nontoxigenic and are generally recognized as safe (GRAS). Fungal isolates were collected from a California orchard and a few were initially identified to be A. sojae using β-tubulin gene sequences blasted against NCBI data base. These new isolates all produced aflatoxins B1, B2, G1, and G2 and were named as Pistachio Winter Experiment (PWE) strains. Thus, it is very important to further characterize these strains for food safety purposes. The full length of aflR gene of these new isolates was sequenced. Comparison of aflR DNA sequences of PWE, A. parasiticus and A. sojae, showed that the aflatoxigenic PWE strains had the six base insertion (CTCATG) similar to domesticated A. sojae, but a pre-termination codon TGA at nucleotide positions 1153-1155 was absent due to a nucleotide codon change from T to C. Colony morphology and scanning microscopic imaging of spore surfaces showed similarity of PWE strains to both A. parasiticus and A. sojae. Concordance analysis of multi locus DNA sequences indicated that PWE strains were closely linked between A. parasiticus and A. sojae. The finding documented the first report that such unique strains have been found in North America and in the world.

Detection of aflatoxigenicity of Aspergillus flavus, based on potential gene marker, from food and feed samples

AbstractIn this study, the incidence of aflatoxigenic strains of Aspergillus flavus was evaluated in cattle feed, water, and milk from three districts of Northern Punjab region of Pakistan. In total, 39 of the hundred samples collected were found contaminated by A. flavus. Specifically, the incidence of A. flavus was 54.28 %, 53.3 %, and 11.42 % in feed (n = 35), water (n = 30), and milk (n = 35) samples, respectively. The UV method indicated aflatoxigenic potential in 62 % of strains, and the ammonia vapor test showed 54 % of samples to be positive for aflatoxin production. Polymerase chain reaction detection of aflatoxin producing cluster of A. flavus was done by identifying four structural genes, that is, nor‐1, ver‐1, omt‐A, afl‐R. It is inferred that omt‐A and afl‐R genes were regarded as potential markers for aflatoxins production. Therefore, this study proved that omt‐A could be used for rapid detection of potential aflatoxigenic strains in feed, water and food products.Practical applicationsIn this study, the isolates of Aspergillus flavus were investigated for their aflatoxigenic nature using cultural (fluorescence under UV‐light and NH4OH vapor induced color change test) and molecular (PCR) methods. The UV test is more sensitive than the ammonia test; however, comparable results from both methods strengthened our confidence in the findings. PCR results indicated the omt‐A and afl‐R genes as potential markers for aflatoxins biosynthesis because these genes were amplified in all those strains that were regarded as aflatoxigenic based on cultural methods for aflatoxin detection. Thus, the combined use of cultural and molecular methods used in this study can provide a cheaper and faster way to detect aflatoxigenic A. flavus in food and feed samples in developing countries.

A risk assessment study of the occurrence and distribution of aflatoxigenic Aspergillus flavus and aflatoxin B1 in dairy cattle feeds in a central northern state, Nigeria.

Nigeria, being a tropical nation, characterized by favorable climatic conditions, may display high chances of feed contaminations due to aflatoxigenic Aspergillus flavus with the consequences of health risks associated with the consumption of dairy products. A cross-sectional study was conducted to examine the risks of occurrence and distribution of aflatoxigenic Aspergillus flavus (A. flavus) and aflatoxin B1 (AFB1) contamination levels based on the European Union (EU) and United States Food and Drug Administration (USFDA) set limits. Feeds (n = 144) were collected from selected conventional and traditional dairy herds; prepared and analyzed using immuno-affinity column (IAC) and high performance liquid chromatography (HPLC) respectively. Forty eight (55.8%) isolates were identified as A. flavus of the isolated Aspergilli (n = 86). Of this proportion, 12 (25.0%) were aflatoxigenic strains. An outrageous number of the tested feeds (86.8%, n = 144) were positive for AFB1 contamination. Detectable AFB1 concentrations ranged between (0.5 and 24.8) μgKg-1 and were distributed variously according to many factors of distribution. Eighty three (66.4%, n = 125) of the AFB1 contaminated feed samples showed AFB1 concentrations between 5 and ≥20 μgKg-1. All-inclusive policies are key to reducing the health risks posed to the consumers of dairy products.

Assessment of chemically characterised Gaultheria fragrantissima Wall. essential oil and its major component as safe plant based preservative for millets against fungal, aflatoxin contamination and lipid peroxidation during storage.

The present study aims survey of fungi causing deterioration of millets during storage, detection of aflatoxigenic fungal strains ans assessment of chemically characterized Gaultheria fragrantissiuma Wall essential oil (EO) and its major component methyl salicylate (MS) as plant based preservative. Essential oil (EO) and its major component methyl salicylate (MS) as plant based preservative was evaluated. In this study a total of 13 fungal species along with toxigenic strain of Aspergillus flavus were isolated from the millets. Chemical characterization of G. fragrantissima EO through GC-MS analysis revealed Methyl salicylate (98.04%) as major component. The EO significantly inhibited growth and aflatoxin B1 production by toxigenic strain of A. flavus LHP (B)-7 at 1.0 and 0.7µlml-1 respectively. In addition, EO exhibited remarkable antioxidant activity (IC50 7.5µl ml-1). EO and MS showed non phytotoxic nature on germination of millets. The LD50 of the EO was 3833.33µlkg-1 for mice showing favourable safety profile. In view of side effects of synthetic preservatives, the study recommends G. fragrantissima EO as a safe plant based preservative to enhance shelf-life of food commodities during storage.

Nanoencapsulated Illicium verum Hook.f. essential oil as an effective novel plant-based preservative against aflatoxin B1 production and free radical generation

The study reports efficacy of Illicium verum essential oil (IvEO) against food borne moudls and its nanoencapsulation for enhancing antifungal and antiaflatoxigenic potency. Chemical characterization of the IvEO showed anethole (89.12%) as major compound followed by estragole (4.859%). The IvEO showed broad fungitoxic spectrum against common food borne moulds. It's minimum inhibitory concentration (MIC) and minimum aflatoxin B1 inhibitory concentration (MAIC) against aflatoxigenic strain Aspergillus flavus LHP-PV-1 were 0.7, and 0.5 μL/mL respectively. Morphological observations of treatment sets by SEM and TEM along with decrease in ergosterol content and enhanced leakage of Ca2+, K+ and Mg2+ ions denoted fungal cell membrane as site of action. The IvEO showed promising free radical scavenging activity and favourable safety profile with high LD50 value on mice. The IvEO also exhibited considerable protection of Pistacia vera from fungal contamination and complete protection from aflatoxin B1 contamination in storage containers. Nanoencapsulated IvEO in gel form and lyophilized form exhibited enhanced efficacy as fungal inhibitor and aflatoxin suppressor. The chemically characterised IvEO may be recommended as plant based preservative having favourable safety and its nanocapsules may be of industrial significance as shelf life enhancer of food items. This is the first report on in situ antiaflatoxigenic efficacy and nanoencapsulation of IvEO.

Antimicrobial and enzymatic activity of anemophilous fungi of a public university in Brazil.

To the fungal microbiota the UFPE and biotechnological potential enzymatic and antimicrobial production. Air conditioned environments were sampled using a passive sedimentation technique, the air I ratio and the presence of aflatoxigenic strains evaluated for ANVISA. Icelles were to determine the enzymatic activity of lipase, amylase and protease metabolic liquids to determine antimicrobial activity. Diversity was observed in all CAV environments, CFU/m3 ranged from 14 to 290 and I/E ratio from 0.1 to 1.5. The of the fungal genera were: Aspergillus (50%), Penicillium (21%), Talaromyces (14%), Curvularia and Paecilomyces (7% each). Aspergillus sydowii (Bainier & Sartory) Thom & Church presented enzymatic activity and the Talaromyces purpureogenus Samson, Yilmaz, Houbraken, Spierenb., Seifert, Peterson, Varga & Frisvad presented antibacterial activity against all bacteria that all environments present fungal species biodiversity no toxigenic or pathogenic fungi were found, according to ANVISA legislation for conditioned environments and airborne filamentous fungi present potential for enzymatic and antimicrobial activity.

MYCOLOGICAL QUALITY OF IMPORTED FROZEN LIVER FOR HUMAN CONSUMPTION

A total of 100 samples of imported frozen liver were randomly collected from different markets at El-Gharbiah governorate, Egypt.The samples were examined for detection of molds and determination of aflatoxins produced by their aflatoxigenic strains. The results showed that 33 % of the samples were contaminated with different mold species and the average mold count (/g)was 3.8 × 104± 8.2× 103/g. Genus Aspergilluswasthe predominant species detected in the positive samples (54.55 %) followed by Genus Penicillium(51.52%). The positive samples were examined for extraction and estimation of aflatoxins B1, B2, G1 and G2 using HPLC. The results indicated that aflatoxins B1, B2, G1 and G2 were detected in 24.2, 21.2, 18.2 and 15.1% of such liver samples, respectively. These results showed that imported frozen liver can represent a potential source of variousmold species and their toxinsconstituting, at times, a public health hazard.

Occurrence of mycotoxigenic fungi in poultry feeds at live-bird markets, Zaria, Nigeria

Contamination of poultry feeds with mycotoxin-producing fungi such as Aspergillus spp is a major threat to animal and human food. This study was conducted to determine the occurrence of aflatoxigenic strain of fungi in feeds, fed to birds in live-bird markets. Feed samples were collected from feeding troughs and feeder in cages of birds and were inoculated on Sabouraud dextrose agar and Czypeck dox agar. Of 300 feed samples, 283 yielded various fungal growth belonging to seven genera, four of them known to be mycotoxigenic. Aspergillus, Rhizopus, Mucor, Dermatophyte, Yeast, Fusarium and Penicillium, whose isolation frequencies were 78%, 6%, 5.67%, 2%, 2%, 0.33% and 0.33% respectively. The aflatoxin producing Aspergillus spp isolated were A. flavus, A. parasiticus and A. nomius 126 (42%), 27 (9%) and 3(1%) respectively. In conclusion A. flavus was the most frequently isolated, and it is a known aflatoxin producer. It is recommended that mycotoxin binders should be added to poultry feed to mitigate the effect of aflatoxin contamination of feed in live-bird market.

Control of aflatoxigenic strains by Cinnamomum porrectum essential oil.

Essential oil from the bark of Cinnamomum porrectum (Roxb.) Kosterm was obtained by hydrodistillation using a Clevenger-type system for 3h and identified by gas chromatography-mass spectrometry. The compounds were safrole (93.9%), elemicine (4.3%) and methyl eugenol (1.7%). The effect of essential oil on mycelial growth, sporulation, and aflatoxin B1 production of Aspergillus parasiticus IMI 283883 and Aspergillus flavus IMI 242684 was evaluated using contact and vapor treatments. Aflatoxin B1 was determined using the Enzyme-linked immunosorbent assay. The results showed that C. porrectum (Roxb.) Kosterm essential oil at concentrations more than 200ppm exhibited inhibition effect on mycelial growth, sporulation, and aflatoxin B1 production of both Aspergillus strains as compared with control. The fumigation activities via vapor treatment showed higher inhibition than contact treatment. This study suggests that C. porrectum (Roxb.) Kosterm essential oil represents a good alternative in eco-friendly control of aflatoxigenic strain on food and agricultural commodities.

Fullerol C60(OH)24 nanoparticles and mycotoxigenic fungi: a preliminary investigation into modulation of mycotoxin production.

Increased use of fullerols in various fields and expected increase of their environmental spread impose the necessity for testing fullerol nanoparticles (FNP) effects on microbiota. There is little information available on the interaction of mycotoxigenic fungi and FNP, despite FNP having a great potential of modifying mycotoxin production. Namely, FNP exhibit both ROS-quenching and ROS-producing properties, while oxidative stress stimulates mycotoxin synthesis in the fungi. In order to shed some light on the extent of interaction between FNP and mycotoxigenic fungi, the effects of fullerol C60(OH)24 nanoparticles (10, 100, 1000ng/mL) on mycelial growth, aflatoxin production and oxidative stress modulation in an aflatoxigenic strain of Aspergillus flavus (NRRL 3251) during 168h of incubation in a liquid culture medium were examined. FNP slightly reduced mycelial biomass weight, but significantly decreased aflatoxin concentration in media. Lipid peroxide content, superoxide dismutase, catalase and glutathione peroxidase activities suggest that FNP treatments hormetically reduced oxidative stress within fungal cells in turn suppressing aflatoxin production. These findings contribute to the assessment of environmental risk and application potential of fullerols.

Application of newly-developed dichlorvos–ammonia (DV–AM) method to direct isolation of aflatoxigenic fungi from field soils

We recently devised the dichlorvos–ammonia (DV–AM) method, a sensitive and simple visual method for detecting aflatoxigenic strains. The DV–AM method enables a prominent distinction of red (positive; aflatoxin-producing) colony from pale-yellow (negative; non-aflatoxigenic) colony via brilliant purple-red color of the backside of positive colonies. We used the DV–AM method to screen aflatoxigenic fungi from soil samples in Tsukuba, Ibaraki, Japan. We herein isolated seven positive (red) colonies and four negative (pale-yellow) colonies from one of the 100 independent samples. Chemical analyses demonstrated that these red isolates produced aflatoxins B1 and B2 without aflatoxins G1 nor G2. In contrast, 4 pale-yellow colonies with similar phenotypes to the Aspergillus flavus group were confirmed to be non-aflatoxigenic fungi. Morphological and phylogenic analyses of one positive strain (HA9-S1-1) indicated that it was A. flavus or its closely related species of Aspergillus. These aflatoxigenic strains showed differences in aflatoxin production, morphological characteristics, and sclerotia formation, suggesting that different types of aflatoxigenic strains were present in the same soil. These results demonstrated that the DV–AM method is practically useful for the direct isolation of aflatoxigenic and non-aflatoxigenic fungi from field samples.

Aspergillus flavus growth and aflatoxin production as influenced by total lipid content during growth and development of cottonseed

ABSTRACTAspergillus flavus infects several food and feed crops, such as corn (Zea mays L.), cotton (Gossypium hirsutum L.), peanuts (Arachis hypogaea L.), and tree nut crops and contaminates the seed with carcinogenic aflatoxins. These susceptible crops contain rich reserves of lipids and fatty acids. The nature of relationship between lipids and the ability of the fungus to infect and produce aflatoxins in mature cottonseed, a protein-rich animal feed, has been addressed previously. In this study, we tracked lipid accumulation in developing cottonseed (15–35 days post-anthesis [DPA]) and also the ability of an aflatoxigenic strain and an isogenic non-aflatoxigenic strain to grow and produce aflatoxins in planta. The aflatoxigenic strain Af-70 green fluorescent protein (GFP) and the isogenic, non-aflatoxigenic strain SRRC 1500 (fungal collections maintained at the Southern Regional Research Center) did not differ much in infection and colonization of cottonseed. The non-aflatoxigenic strain did not produce aflatoxin at all stages of cottonseed development, whereas the aflatoxigenic strain Af-70 GFP produced copious amounts of aflatoxin and it coincided with the increasing levels of lipids, especially in mature cottonseed (30–35 DPA). Fungal growth, as quantified by the GFP expression in the fungus, was highly correlated with toxin production.

Evaluating Aflatoxin Gene Expression in Aspergillus Section Flavi.

The determination of aflatoxin production ability and differentiation of aflatoxigenic strains can be assessed by monitoring the expression of one or several key genes using reverse transcription polymerase chain reaction (RT-PCR). We herein describe the methods for RNA induction, extraction, and quality determination, and the RT-PCR conditions used to evaluate the ability of a given Aspergillus strain to produce aflatoxins.

Aflatoxigenic Aspergillus flavus and Aspergillus parasiticus strains in Hungarian maize fields.

Due to the climate change, aflatoxigenic Aspergillus species and strains have appeared in several European countries, contaminating different agricultural commodities with aflatoxin. Our aim was to screen the presence of aflatoxigenic fungi in maize fields throughout the seven geographic regions of Hungary. Fungi belonging to Aspergillus section Flavi were isolated in the ratio of 26.9% and 42.3% from soil and maize samples in 2013, and these ratios decreased to 16.1% and 34.7% in 2014. Based on morphological characteristics and the sequence analysis of the partial calmodulin gene, all isolates proved to be Aspergillus flavus, except four strains, which were identified as Aspergillus parasiticus. About half of the A. flavus strains and all the A. parasiticus strains were able to synthesize aflatoxins. Aflatoxigenic Aspergillus strains were isolated from all the seven regions of Hungary. A. parasiticus strains were found in the soil of the regions Southern Great Plain and Southern Transdanubia and in a maize sample of the region Western Transdanubia. In spite of the fact that aflatoxins have rarely been detected in feeds and foods in Hungary, aflatoxigenic A. flavus and A. parasiticus strains are present in the maize culture throughout Hungary posing a potential threat to food safety.

Molecular and mycotoxigenic biodiversity of Aspergillus flavus isolated from Brazil nuts

The objective of this study was to carry out a transcription analysis of eight genes belonging to the aflatoxin (AF) and cyclopiazonic acid (CPA) biosynthesis pathway, and to detect aflatoxin B1 (AFB1) and CPA production in Aspergillus flavus strains isolated from Brazil nuts. Additionally, these genes were correlated with the different mycotoxigenic profiles of the same strains. Four previously identified A. flavus strains (ICB-01, ICB-151, ICB-161, and ICB-165) were grown on Brazil nut agar at 25°C for 10days. Mycotoxins were separated by high-performance liquid chromatography. Transcriptional analysis was performed by real-time RT-PCR using specific primers designed based on the conserved regions of two regulatory genes (aflR and aflS), three structural genes of the AFB1 biosynthesis pathway (aflH, aflJ and aflP), and three structural genes of the CPA biosynthesis pathway (maoA, dmaT and pks-nrps). The expression of most genes in the A. flavus isolates varied according to the mycotoxin profile of each strain. The most expressed genes in the aflatoxigenic strain ICB-151 were aflJ (77.11%) and aflH (32.75%), while the CPA-producing strain ICB-161 mainly expressed dmaT (100%), maoA (63.72%), aflS (43.52%), and aflR (42.63%). The ICB-01 isolate was a producer of AFB1 and CPA and the most expressed genes were aflS (47.79%), dmaT (42.77%), aflP (39.5%), and aflR (38.02%). ICB-198 did not produce any mycotoxin and exhibited lower expression of almost all genes analyzed. Furthermore, the ratio of aflS/aflR expression was correlated with the biosynthesis of AF and CPA in A. flavus strains producing exclusively AF or CPA or producing both AF and CPA. The ratio of aflS/aflR expression therefore seems to be related to the production of mycotoxins in Brazil nuts. Our results provide important data for the development of innovative and more cost-effective strategies to reduce and prevent AFB and CPA contamination in Brazil nuts.

Expression of Genes by Aflatoxigenic and Nonaflatoxigenic Strains of Aspergillus flavus Isolated from Brazil Nuts.

The aims of the present study were to monitor the production of aflatoxin B1 (AFB1) and mycelial growth, and to evaluate the expression of genes directly and indirectly involved in the biosynthesis of aflatoxins by Aspergillus flavus isolated from Brazil nuts. Six previously identified A. flavus strains were grown on coconut agar at 25°C for up to 10 days. Mycotoxins were separated by high-performance liquid chromatography and fungal growth was measured daily using the diametric mycelial growth rate. Transcriptional analysis was performed by real-time polymerase chain reaction (PCR) after 2 and 7 d of incubation using specific primers (aflR, aflD, aflP, lipase, metalloprotease, and LaeA). Three (50%) of the six A. flavus isolates produced AFB1 (ICB-1, ICB-12, and ICB-54) and three (50%) were not aflatoxigenic (ICB-141, ICB-161, and ICB-198). Aflatoxin production was observed from d 2 of incubation (1.5 ng/g for ICB-54) and increased gradually with time of incubation until d 10 (15,803.6 ng/g for ICB-54). Almost all A. flavus isolates exhibited a similar gene expression pattern after 2 d of incubation (p > 0.10). After 7 d of incubation, the LaeA (p < 0.05) and metalloprotease (p < 0.05) genes were the most expressed by nonaflatoxigenic strains, whereas aflatoxigenic isolates exhibited higher expression of the aflR (p < 0.05) and aflD genes (p < 0.05). Our results suggest that the expression of aflR and aflD is correlated with aflatoxin production in A. flavus and that overexpression of aflR could affect the transcriptional and aflatoxigenic pattern (ICB-54). Elucidation of the molecular mechanisms that regulate the secondary metabolism of toxigenic fungi may permit the rational silencing of the genes involved and consequently the programmed inhibition of aflatoxin production. Knowledge of the conditions, under which aflatoxin genes are expressed, should contribute to the development of innovative and more cost-effective strategies to reduce and prevent aflatoxin contamination in Brazil nuts.

Non-aflatoxigenic Aspergillus flavus as potential biocontrol agents to reduce aflatoxin contamination in peanuts harvested in Northern Argentina

Biological control is one of the most promising strategies for preventing aflatoxin contamination in peanuts at field stage. A population of 46 native Aspergillus flavus nonaflatoxin producers were analysed based on phenotypic, physiological and genetic characteristics. Thirty-three isolates were characterized as L strain morphotype, 3 isolates as S strain morphotype, and 10 isolates did not produce sclerotia. Only 11 of 46 non-aflatoxigenic isolates did not produce cyclopiazonic acid. The vegetative compatibility group (VCG) diversity index for the population was 0.37. For field trials we selected the non-aflatoxigenic A. flavus AR27, AR100G and AFCHG2 strains. The efficacy of single and mixed inocula as potential biocontrol agents in Northern Argentina was evaluated through a 2-year study (2014–2015). During the 2014 peanut growing season, most of the treatments reduced the incidence of aflatoxigenic strains in both soil and peanut kernel samples, and no aflatoxin was detected in kernels. During the 2015 growing season, there was a reduction of aflatoxigenic strains in kernel samples from the plots treated with the potential biocontrol agents. Reductions of aflatoxin contamination between 78.36% and 89.55% were observed in treated plots in comparison with the un-inoculated control plots. This study provides the first data on aflatoxin biocontrol based on competitive exclusion in the peanut growing region of Northern Argentina, and proposes bioproducts with potential use as biocontrol agents.