Atoxigenic Aspergillus Flavus Research Articles

An atoxigenic Aspergillus flavus PA67 from Shandong province exhibits potential in biocontrol against toxigenic Aspergillus flavus, Sclerotium rolfsii, and Fusarium proliferatum

Peanuts and corn are susceptible to various soil-borne fungi, leading to significant economic losses. Atoxigenic Aspergillus flavus have been widely used as biocontrol agents for managing aflatoxin contamination because of their minimal environmental impact, strong competitive ability, and sustained inhibition effect. After multiple identifications and cluster amplification pattern (CAP) analysis, three atoxigenic A. flavus PA04, PA10 and PA67 were isolated from peanut samples in Shandong Province, which can reduce aflatoxin levels by up to 90 %. Our study revealed that atoxigenic A. flavus also competed vigorously with Sclerotium rolfsii and Fusarium proliferatum for nutrition and space, achieving notable inhibition rates of up to 90.4 % and 90.6 %, respectively. The supernatants of atoxigenic A. flavus also inhibited the growth of S. rolfsii and F. proliferatum, with PA67 demonstrating the most significant effect. Whole genome sequencing revealed that PA67 contains multiple glycoside hydrolases and metabolites with antifungal activity. The kojic acid production of PA67 was higher than that of PA04 and PA10, reaching 17.48 g/L, which has a significant inhibition on sclerotia germination. PA67 supernatant significantly inhibited the hyphae growth of S. rolfsii and F. proliferatum, and down-regulated genes related to sclerotia and fumonisin formation. This study demonstrates the biocontrol potential of PA67 against three soil-borne fungi and is the first investigation of atoxigenic A. flavus to inhibit S. rolfsii and F. proliferatum.

Efficacy of atoxigenic Aspergillus flavus from southern China as biocontrol agents against aflatoxin contamination in corn and peanuts

Aspergillus flavus is a ubiquitous facultative pathogen that routinely infects important crops leading to formation of aflatoxins during crop development and after harvest. Corn and peanuts in warm and/or drought-prone regions are highly susceptible to aflatoxin contamination. Controlling aflatoxin using atoxigenic A. flavus is a widely adopted strategy. However, no A. flavus genotypes are currently approved for use in China. The current study aimed to select atoxigenic A. flavus endemic to Guangxi Zhuang Autonomous Region with potential as active ingredients of aflatoxin biocontrol products. A total of 204 A. flavus isolates from corn, peanuts, and field soil were evaluated for ability to produce the targeted mycotoxins. Overall, 57.3% could not produce aflatoxins while 17.15% were incapable of producing both aflatoxins and CPA. Atoxigenic germplasm endemic to Guangxi was highly diverse, yielding 8 different gene deletion patterns in the aflatoxin and CPA biosynthesis gene clusters ranging from no deletion to deletion of both clusters. Inoculation of corn and peanuts with both an aflatoxin producer and selected atoxigenic genotypes showed significant reduction (74 to 99%) in aflatoxin B1 (AFB1) formation compared with inoculation with the aflatoxin producer alone. Atoxigenic genotypes also efficiently degraded AFB1 (61%). Furthermore, atoxigenic isolates were also highly efficient at reducing aflatoxin concentrations even when present at lower concentrations than aflatoxin producers. The use of multiple atoxigenics was not always as effective as the use of a single atoxigenic. Effective atoxigenic genotypes of A. flavus with known mechanisms of atoxigenicity are demonstrated to be endemic to Southern China. These A. flavus may be utilized as active ingredients of biocontrol products without concern for detrimental impacts that may result from introduction of exotic fungi. Field efficacy trials in the agroecosystems of Southern China are needed to determine the extent to which such products may allow the production of safer food and feed.

Response of broilers to dietary inclusion of atoxigenic Aspergillus flavus strain as a biocontrol strategy of aflatoxin

ABSTRACT The objective of this trial was to evaluate how broilers responded to Aspergillus flavus strains that are toxigenic and atoxigenic. The study included four treatments in a 2 × 2 factorial design, with six replicates of 10 birds each. As a result of this study measuring feed intake (FI), weight gain (WG), feed conversion ratio (FCR), crude protein, ether extract, and crude fibre, the interaction was insignificant between the toxigenic and atoxigenic diets (P > 0.05). Consumption of toxigenic aflatoxin B1-500 ppb diet decreased FI and WG but increased FCR, and cost to produce live broiler weight (P < 0.05) compared to the control diets. The addition of atoxigenic strains to contaminated diets significantly offset (P < 0.05) the effects. Diets with or without 500 ppb toxigenic and atoxigenic A. flavus did not affect the relative weight g/100gBW of pancreas, gizzard and bursa of Fabricius. Dietary inclusion of 500 ppb toxigenic Aspergillus spp. increased the relative weight (P < 0.05) of the kidney, liver, spleen and thymus while atoxigenic dietary addition reduced the relative weight of the same organs (P < 0.05). Dietary inclusion of toxigenic and atoxigenic Aspergillus spp. did not significantly affect the haematological parameters measured (P < 0.05). Dietary inclusion of 500 ppb toxigenic Aspergillus elevated the urea, creatine, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in the serum of the broilers (P < 0.05). A decrease was observed when atox igenic A. flavus was used in the intervention for urea, creatinine and AST (P < 0.05), whereas an insignificant reduction was observed for ALT and ALP (P ≤ 0.05). This study concluded that dietary atoxigenic strain improved broiler performance, digestibility, and blood parameters.

Displacement of toxigenic Fusarium species by atoxigenic Aspergillus flavus (Aflasafe KE01) application in maize fields in lower Eastern Kenya

Maize is grown in areas with environmental conditions that are ideal for growth of most cereal pathogens such as Fusarium fungi. However, there is lack of effective fungicides to control fumonisin producing Fusarium. This study was conducted to determine the efficacy of atoxigenic Aspergillus flavus (Aflasafe KE01) on the population of Fusarium species in maize fields. The study was carried out in four sub counties in lower Eastern Kenya (Kaiti, Kathiani, Nzambani and Wote). Twenty-four maize fields were selected in each sub county where 12 fields were treated with Aflasafe KE01, while 12 fields comprised the untreated controls. Aflasafe KE01 was applied by hand broadcasting in the maize fields two to three weeks before tasselling of maize. Maize grain samples were collected from each field at harvest and ground using a Bunn coffee mill grinder. Fusarium species were isolated from the ground maize using pour plate method following serial dilution on low strength potato dextrose agar (PDA)and Spezieller Nahrstoffarmer. The results showed that application of Aflasafe KE01 effectively displaced toxigenic Fusarium species in maize fields. Maize samples from Aflasafe KE01 treated maize fields recorded significantly lower incidence (41%) of the Fusarium species compared to untreated maize fields (60%) (p≤0.01). These results indicate that Aflasafe KE01 is a potential biopesticide for the biocontrol of Fusarium species in maize. Key words: Aflasafe KE01, atoxigenic Aspergillus sp, Fusarium species, maize.

Atoxigenic Aspergillus flavus (Aflasafe KE01) application reduces Fumonisin contamination in maize in lower Eastern Kenya

Management of fumonisin contamination in maize has been a challenge largely because there are no effective management measures for Fusarium ear rots. Maize varieties that are resistant to fumonisin contamination are also lacking. This study was conducted to determine the efficacy of atoxigenic Aspergillus flavus (Aflasafe KE01) on fumonisin contamination of maize. The study was carried out in four sub-counties in lower Eastern Kenya. Twenty-four maize fields were selected in each sub-county; where 12 fields were treated with Aflasafe KE01, while 12 fields comprised the untreated controls. Aflasafe KE01 was applied at a rate of 5 kg/ha and 10kg/ha by hand broadcasting in maize fields two to three weeks before tasselling of maize. Fumonisin level in the maize samples was determined using Accuscan Pro-reader enzyme-linked immunosorbent assay (ELISA). The results showed that application of Aflasafe KE01 reduced fumonisin in the maize from the Aflasafe KE01 treated fields by up to 68% compared to samples from untreated fields. About 62.5% of the maize fields treated with 5kg/ha of Aflasafe KE01 met the European Commission regulatory threshold of ≤2ppm for total fumonisin as compared to about 45% from the control fields. This indicates that Aflasafe KE01 is a potential biopesticide for the management of fumonisin in maize. Therefore, efficacy of Aflasafe KE01 to reduce fumonisin contamination of other key staples in Kenya should also be evaluated. Key words: Aflasafe KE01, atoxigenic Aspergillus sp., fumonisins, maize.

The effects of atoxigenic A. flavus co-applied with T. asperellum on the population dynamics of toxigenic A. flavus in the soil

Aspergillus flavus is an important aflatoxigenic fungal specie infecting crops from production to storage. Contamination of foods and their products by these mycotoxins has become a problem worldwide; in Tanzania, related deaths were reported in 2017. This study focused on assessing the effectiveness of atoxigenic Aspergillus flavus, Trichoderma asperellum and a co-inoculated atoxigenic A.flavus and T.asperellum on the population dynamic of toxigenic A.flavus in the soil. A pot experiment was set in the screen house where a known concentration of fungal inoculum was added to the sterile soil. Aspergillus spp. were isolated from soil samples by dilution plate techniques on Modified Dichloran Rose Bengal (MDRB) agar and sub-cultured on potato dextrose agar (PDA). Identification was made using morphological and microscopic characteristics of culture in PDA media. Total colony forming units (CFU g-1 of soil) and incidence percent were derived from colony count data recorded. R-statistical software version 4.0.4 was used to analyse the collected data. There was a significant difference between treatments on reducing the population (CFU/g) of toxigenic A.flavus in soil (p&lt; 2.2×10-16). Co-inoculated T.asperellum and atoxigenic A.flavus significantly reduced the population (CFU g-1 of soil) of toxigenic A.flavus to 1.4×105 CFU g-1 of soil as compared to control treatment 4.5×105 CFU g-1 of soil. There was a strong positive correlation (p&lt;0.001) between the incidence percent and population of toxigenic A.flavus in the soil under different biocontrol treatments. Co-inoculated T. asperellum and atoxigenic A. flavus had the lowest incidence of 36.3% to other treatments. The findings of this study demonstrate that co-inoculated atoxigenic A.flavus and T.asperellum was effective biocontrol treatment for reducing the population of Aspergillus flavus in the soil ecosystem.

Biocontrol efficacy of atoxigenic Aspergillus flavus strains against aflatoxin contamination in peanut field in Guangdong province, South China

ABSTRACT Application of atoxigenic strains of Aspergillus flavusto soils is the most successful aflatoxin biological control approach. The objective of this study was to evaluate the efficacies of native non-aflatoxin producing (atoxigenic) strains as a biocontrol agent in peanut field in China. The competitive atoxigenic A. flavus strains (JS4, SI1and SXN) isolated from different crops, in China were used for field evaluation. The strains applied during the growing season (June – October, 2016) in the field at rate of 25 kg inoculum/hectare. The colonization of these biocontrol agents has been investigated and the population of A. flavus communities in soil were determined. The incidences of toxin producing (toxigenic) A. flavus strains and aflatoxin contamination in peanuts were also determined. Treated plots produced significant reductions in the incidence of toxigenic isolates of A. flavus in soil. However, the total fungal densities were not significantly different (p > 0.05) after treatments. Large percentage of aflatoxin reductions, ranging from 82.8% (SXN) up to 87.2% (JS4) were recorded in treated plots. Generally, the results suggest that the strategy can be used to control aflatoxin contamination and continuous evaluation should be done.

Selection of Atoxigenic Aspergillus flavus for Potential Use in Aflatoxin Prevention in Shandong Province, China.

Aspergillus flavus is a common filamentous fungus widely present in the soil, air, and in crops. This facultative pathogen of both animals and plants produces aflatoxins, a group of mycotoxins with strong teratogenic and carcinogenic properties. Peanuts are highly susceptible to aflatoxin contamination and consumption of contaminated peanuts poses serious threats to the health of humans and domestic animals. Currently, the competitive displacement of aflatoxin-producers from agricultural environments by atoxigenic A. flavus is the most effective method of preventing crop aflatoxin contamination. In the current study, 47 isolates of A. flavus collected from peanut samples originating in Shandong Province were characterized with molecular methods and for aflatoxin-producing ability in laboratory studies. Isolates PA04 and PA10 were found to be atoxigenic members of the L strains morphotype. When co-inoculated with A. flavus NRRL3357 at ratios of 1:10, 1:1, and 10:1 (PA04/PA10: NRRL3357), both atoxigenic strains were able to reduce aflatoxin B1 (AFB1) levels, on both culture media and peanut kernels, by up to 90%. The extent to which atoxigenic strains reduced contamination was correlated with the inoculation ratio. Abilities to compete of PA04 and PA10 were also independently verified against local aflatoxin-producer PA37. The results suggest that the two identified atoxigenic strains are good candidates for active ingredients of biocontrol products for the prevention of aflatoxin contamination of peanuts in Shandong Province.

Degradation of Aflatoxins B1 by Atoxigenic Aspergillus flavus Biocontrol Agents.

Aflatoxins are potent Aspergillus mycotoxins that contaminate food and feed, thereby impacting health and trade. Biopesticides with atoxigenic Aspergillus flavus isolates as active ingredients are used to reduce aflatoxin contamination in crops. The mechanism of aflatoxin biocontrol is primarily attributed to competitive exclusion but, sometimes, aflatoxin is reduced by greater amounts than can be explained by displacement of aflatoxin-producing fungi on the crop. Objectives of this study were to (i) evaluate the ability of atoxigenic A. flavus genotypes to degrade aflatoxin B1 (AFB1) and (ii) characterize impacts of temperature, time, and nutrient availability on AFB1 degradation by atoxigenic A. flavus. Aflatoxin-contaminated maize was inoculated with atoxigenic isolates in three separate experiments that included different atoxigenic genotypes, temperature, and time as variables. Atoxigenic genotypes varied in aflatoxin degradation but all degraded AFB1 >44% after 7 days at 30°C. The optimum temperature for AFB1 degradation was 25 to 30°C, which is similar to the optimum range for AFB1 production. In a time-course experiment, atoxigenics degraded 40% of AFB1 within 3 days, and 80% of aflatoxin was degraded by day 21. Atoxigenic isolates were able to degrade and utilize AFB1 as a sole carbon source in a chemically defined medium but quantities of AFB1 degraded declined as glucose concentrations increased. Degradation may be an additional mechanism through which atoxigenic A. flavus biocontrol products reduce aflatoxin contamination pre- or postharvest. Thus, selection of optimal atoxigenic active ingredients can include assessment of both competitive ability in agricultural fields and their ability to degrade aflatoxins.

Competency of Clove and Cinnamon Essential Oil Fumigation against Toxigenic and Atoxigenic Aspergillus flavus Isolates

Aspergillus flavus is a frequent contaminant of maize grain. We isolated this fungus, determined the colony morphology and species (by internal transcribed spacer sequencing) and measured the aflatoxin content. The selected A. flavus fungi were placed into two groups, toxigenic and atoxigenic; both appeared similar morphologically, except that the atoxigenic group lacked sclerotia. An essential oil fumigation test with clove and cinnamon oils as antifungal products was performed on fungal conidial discs and fungal colonies in Petri plates. Cinnamon oil at 2.5 to 5.0 μL/plate markedly inhibited the mycelial growth from conidial discs of both strains, whereas clove oil showed less activity. The oils had different effects on fungal mycelia. The higher clove fumigation doses of 10.0 to 20.0 μL/plate controlled fungal growth, while cinnamon oil caused less inhibition. Compared with atoxigenic groups, toxigenic A. flavus responded stably. Within abnormal A. flavus hyphae, the essential oils degenerated the hyphal morphology, resulting in exfoliated flakes and shrinkage, which were related to fungal membrane injury and collapse of vacuoles and phialide. The treatments, especially those with cinnamon oil, increased the electroconductivity, which suggested a weak mycelium membrane structure. Moreover, the treatments with essential oils reduced the ergosterol content in mycelia and the aflatoxin accumulation in the culture broth. The fumigations with clove and cinnamon oils inhibited the development of both conidia and colonies of A. flavus in dose-dependent manners.

Distribution of active ingredients of a commercial aflatoxin biocontrol product in naturally occurring fungal communities across Kenya.

Human populations in Kenya are repeatedly exposed to dangerous aflatoxin levels through consumption of contaminated crops. Biocontrol with atoxigenic Aspergillus flavus is an effective method for preventing aflatoxin in crops. Although four atoxigenic A. flavus isolates (C6E, E63I, R7H and R7K) recovered from maize produced in Kenya are registered as active ingredients for a biocontrol product (Aflasafe KE01) directed at preventing contamination, natural distributions of these four genotypes prior to initiation of commercial use have not been reported. Distributions of the active ingredients of KE01 based on haplotypes at 17 SSR loci are reported. Incidences of the active ingredients and closely related haplotypes were determined in soil collected from 629 maize fields in consecutive long and short rains seasons of 2012. The four KE01 haplotypes were among the top ten most frequent. Haplotype H-1467 of active ingredient R7K was the most frequent and widespread haplotype in both seasons and was detected in the most soils (3.8%). The four KE01 haplotypes each belonged to large clonal groups containing 27-46 unique haplotypes distributed across multiple areas and in 21% of soils. Each of the KE01 haplotypes belonged to a distinct vegetative compatibility group (VCG), and all A. flavus with haplotypes matching a KE01 active ingredient belonged to the same VCG as the matching active ingredient as did all A. flavus haplotypes differing at only one SSR locus. Persistence of the KE01 active ingredients in Kenyan agroecosystems is demonstrated by detection of identical SSR haplotypes six years after initial isolation. The data provide baselines for assessing long-term influences of biocontrol applications in highly vulnerable production areas of Kenya.

Soil Microbial Communities in Corn Fields Treated with Atoxigenic Aspergillus flavus

Aspergillus flavus refers to a diverse group of saprophytic soil fungi that includes strains producing aflatoxins (toxigenic strains) in the kernels of corn (Zea mays L.) and other crops, causing pre-harvest and post-harvest aflatoxin contamination. Some A. flavus strains are atoxigenic, and the introduction of such strains into the crop environment helps reduce toxigenic aflatoxin contamination. Corn growers in Texas have used the product FourSure™, which contains four atoxigenic strains of A. flavus; however, effects on soil microbial communities associated with these applications are unknown. We compared soil fungal and bacterial communities in corn fields treated with FourSure™ to nearby untreated (control) corn fields in Texas during the summer of 2019. Analysis of soil microbial community structure showed that total fatty acid methyl esters (FAMEs), fungal, and bacterial populations were not significantly different (p = 0.31) between the FourSure™-treated and control fields, yet corn fields located in the northern counties had more (p &lt; 0.05) Gram—bacteria, actinobacteria, and total bacteria than fields in the southernmost county. The Gram—bacteria and actinobacteria were positively correlated (p = 0.04; r = 0.48 and 0.49, respectively) with soil water content. Similar fungal and bacterial abundances between FourSure™-treated and control fields indicated that atoxigenic A. flavus had no negative effects on soil microbial communities.

Bees Occurring in Corn Production Fields Treated with Atoxigenic Aspergillus flavus (Texas, USA)

A saprophytic soil fungus, Aspergillus flavus, produces aflatoxin (toxigenic strains) in the kernels of corn (Zea mays L.) and seeds of many other crops. Many strains of A. flavus do not produce toxigenic aflatoxin, and soil application of these atoxigenic strains is a suppressive control tactic to assist in controlling toxigenic conspecifics. Effects of atoxigenic A. flavus applications on honey bees (Apis mellifera L.) and other bees are unknown, and basic information on bee occurrences in cornfields treated with and without this biological agent is needed to inform integrated pest management in corn. Fields receiving atoxigenic A. flavus applications of FourSureTM were compared to nearby control fields in three counties in corn production regions in eastern Texas. In each cornfield, 20 bee bowl traps were deployed along four equal transects located between corn rows, with contents of the bowls (i.e., bees) retrieved after 24 h. Eleven bee genera from four families were collected from cornfields, with only two honey bees collected and zero honey bees observed in transects. The sweat bee genus Agapostemon (primarily composed of the Texas striped sweat bee A. texanus) was most abundant in cornfields (44% of the total number of bees collected), followed by long-horned bees (Melissodes spp., 24%). The southernmost county (i.e., San Patricio) produced over 80% of the total number of bees collected. Bee numbers occurring in cornfields with applications of atoxigenic A. flavus applications were not significantly different from those of nearby control fields. Although not statistically significant, total numbers of bees tended to be lower in FourSure-treated fields than in control fields. More extensive research on bee abundances in relation to the effect of atoxigenic A. flavus is warranted.

The Atoxigenic Biocontrol Product Aflasafe SN01 Is a Valuable Tool to Mitigate Aflatoxin Contamination of Both Maize and Groundnut Cultivated in Senegal.

Aflatoxin contamination of groundnut and maize infected by Aspergillus section Flavi fungi is common throughout Senegal. The use of biocontrol products containing atoxigenic Aspergillus flavus strains to reduce crop aflatoxin content has been successful in several regions, but no such products are available in Senegal. The biocontrol product Aflasafe SN01 was developed for use in Senegal. The four active ingredients of Aflasafe SN01 are atoxigenic A. flavus genotypes native to Senegal and distinct from active ingredients used in other biocontrol products. Efficacy tests on groundnut and maize in farmers' fields were carried out in Senegal during the course of 5 years. Active ingredients were monitored with vegetative compatibility analyses. Significant (P < 0.05) displacement of aflatoxin producers occurred in all years, districts, and crops. In addition, crops from Aflasafe SN01-treated fields contained significantly (P < 0.05) fewer aflatoxins both at harvest and after storage. Most crops from treated fields contained aflatoxin concentrations permissible in both local and international markets. Results suggest that Aflasafe SN01 is an effective tool for aflatoxin mitigation in groundnut and maize. Large-scale use of Aflasafe SN01 should provide health, trade, and economic benefits for Senegal.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Potential of Atoxigenic Aspergillus flavus Vegetative Compatibility Groups Associated With Maize and Groundnut in Ghana as Biocontrol Agents for Aflatoxin Management.

Increasing knowledge of the deleterious health and economic impacts of aflatoxin in crop commodities has stimulated global interest in aflatoxin mitigation. Current evidence of the incidence of Aspergillus flavus isolates belonging to vegetative compatibility groups (VCGs) lacking the ability to produce aflatoxins (i.e., atoxigenic) in Ghana may lead to the development of an aflatoxin biocontrol strategy to mitigate crop aflatoxin content. In this study, 12 genetically diverse atoxigenic African A. flavus VCGs (AAVs) were identified from fungal communities associated with maize and groundnut grown in Ghana. Representative isolates of the 12 AAVs were assessed for their ability to inhibit aflatoxin contamination by an aflatoxin-producing isolate in laboratory assays. Then, the 12 isolates were evaluated for their potential as biocontrol agents for aflatoxin mitigation when included in three experimental products (each containing four atoxigenic isolates). The three experimental products were evaluated in 50 maize and 50 groundnut farmers’ fields across three agroecological zones (AEZs) in Ghana during the 2014 cropping season. In laboratory assays, the atoxigenic isolates reduced aflatoxin biosynthesis by 87–98% compared to grains inoculated with the aflatoxin-producing isolate alone. In field trials, the applied isolates moved to the crops and had higher (P < 0.05) frequencies than other A. flavus genotypes. In addition, although at lower frequencies, most atoxigenic genotypes were repeatedly found in untreated crops. Aflatoxin levels in treated crops were lower by 70–100% in groundnut and by 50–100% in maize (P < 0.05) than in untreated crops. Results from the current study indicate that combined use of appropriate, well-adapted isolates of atoxigenic AAVs as active ingredients of biocontrol products effectively displace aflatoxin producers and in so doing limit aflatoxin contamination. A member each of eight atoxigenic AAVs with superior competitive potential and wide adaptation across AEZs were selected for further field efficacy trials in Ghana. A major criterion for selection was the atoxigenic isolate’s ability to colonize soils and grains after release in crop field soils. Use of isolates belonging to atoxigenic AAVs in biocontrol management strategies has the potential to improve food safety, productivity, and income opportunities for smallholder farmers in Ghana.

Aflatoxin in Chili Peppers in Nigeria: Extent of Contamination and Control Using Atoxigenic Aspergillus flavus Genotypes as Biocontrol Agents.

Across sub-Saharan Africa, chili peppers are fundamental ingredients of many traditional dishes. However, chili peppers may contain unsafe aflatoxin concentrations produced by Aspergillus section Flavi fungi. Aflatoxin levels were determined in chili peppers from three states in Nigeria. A total of 70 samples were collected from farmers’ stores and local markets. Over 25% of the samples contained unsafe aflatoxin concentrations. The chili peppers were associated with both aflatoxin producers and atoxigenic Aspergillus flavus genotypes. Efficacy of an atoxigenic biocontrol product, Aflasafe, registered in Nigeria for use on maize and groundnut, was tested for chili peppers grown in three states. Chili peppers treated with Aflasafe accumulated significantly less aflatoxins than nontreated chili peppers. The results suggest that Aflasafe is a valuable tool for the production of safe chili peppers. Use of Aflasafe in chili peppers could reduce human exposure to aflatoxins and increase chances to commercialize chili peppers in premium local and international markets. This is the first report of the efficacy of any atoxigenic biocontrol product for controlling aflatoxin in a spice crop.

Correction: Mauro, A., et al. Biological Control Products for Aflatoxin Prevention in Italy: Commercial Field Evaluation of Atoxigenic Aspergillus flavus Active Ingredients. Toxins 2018, 10, 30.

The authors wish to make the following correction to their paper [...].

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.

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.

Biological Control Products for Aflatoxin Prevention in Italy: Commercial Field Evaluation of Atoxigenic Aspergillus flavus Active Ingredients.

Since 2003, non-compliant aflatoxin concentrations have been detected in maize produced in Italy. The most successful worldwide experiments in aflatoxin prevention resulted from distribution of atoxigenic strains of Aspergillus flavus to displace aflatoxin-producers during crop development. The displacement results in lower aflatoxin concentrations in harvested grain. The current study evaluated in field performances of two atoxigenic strains of A. flavus endemic to Italy in artificially inoculated maize ears and in naturally contaminated maize. Co-inoculation of atoxigenic strains with aflatoxin producers resulted in highly significant reductions in aflatoxin concentrations (>90%) in both years only with atoxigenic strain A2085. The average percent reduction in aflatoxin B1 concentration in naturally contaminated maize fields was 92.3%, without significant differences in fumonisins between treated and control maize. The vegetative compatibility group of A2085 was the most frequently recovered A. flavus in both treated and control plots (average 61.9% and 53.5% of the A. flavus, respectively). A2085 was therefore selected as an active ingredient for biocontrol products and deposited under provisions of the Budapest Treaty in the Belgian Co-Ordinated Collections of Micro-Organisms (BCCM/MUCL) collection (accession MUCL54911). Further work on development of A2085 as a tool for preventing aflatoxin contamination in maize produced in Italy is ongoing with the commercial product named AF-X1™.