Aflasafe SN01 is the First Biocontrol Product Approved for Aflatoxin Mitigation in Two Nations, Senegal and The Gambia.

Aflatoxin contamination is caused by Aspergillus flavus and closely related fungi. In The Gambia, aflatoxin contamination of groundnut and maize, two staple and economically important crops, is common. Groundnut and maize consumers are chronically exposed to aflatoxins, sometimes at alarming levels, and this has severe consequences on their health and productivity. Aflatoxin contamination also impedes commercialization in local and international premium markets. In neighboring Senegal, an aflatoxin biocontrol product containing four atoxigenic isolates of A. flavus, Aflasafe SN01, has been registered and is approved for commercial use in groundnut and maize. We detected that the four genotypes composing Aflasafe SN01 are also native to The Gambia. The biocontrol product was tested during two years in 129 maize and groundnut fields and compared with corresponding untreated fields cropped by smallholder farmers in The Gambia. Treated crops contained up to 100% less aflatoxins than untreated crops. A large portion of the crops could have been commercialized in premium markets due to the low aflatoxin content (in many cases no detectable aflatoxins), both at harvest and after storage. Substantial aflatoxin reductions were also achieved when commercially produced groundnut received treatment. Here we report for the first time the use and effectiveness of an aflatoxin biocontrol product registered for use in two nations. With the current scale-out and -up efforts of Aflasafe SN01, a large number of farmers, consumers, and traders in The Gambia and Senegal will obtain health, income, and trade benefits.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

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.

Prevalence and mitigation of aflatoxins in Kenya (1960-to date).

Aflatoxins are highly toxic metabolites of several Aspergillus species widely distributed throughout the environment. These toxins have adverse effects on humans and livestock at a few micrograms per kilogram (µg/kg) concentrations. Strict regulations on the concentrations of aflatoxins allowed in food and feed exist in many nations in the developing world. Loopholes in implementing regulations result in the consumption of dangerous concentrations of aflatoxins. In Kenya, where 'farm-to-mouth' crops become severely contaminated, solutions to the aflatoxins problem are needed. Across the decades, aflatoxins have repeatedly caused loss of human and animal life. A prerequisite to developing viable solutions for managing aflatoxins is understanding the geographical distribution and severity of food and feed contamination, and the impact on lives. This review discusses the scope of the aflatoxins problem and management efforts by various players in Kenya. Economic drivers likely to influence the choice of aflatoxins management options include historical adverse health effects on humans and animals, cost of intervention for mitigation of aflatoxins, knowledge about aflatoxins and their impact, incentives for aflatoxins safe food and intended scope of use of interventions. It also highlights knowledge gaps that can direct future management efforts. These include: sparse documented information on human exposure; few robust tools to accurately measure economic impact in widely unstructured value chains; lack of long-term impact studies on benefits of aflatoxins mitigation; inadequate sampling mechanisms in smallholder farms and grain holding stores/containers; overlooking social learning networks in technology uptake and lack of in-depth studies on an array of aflatoxins control measures followed in households. The review proposes improved linkages between agriculture, nutrition and health sectors to address aflatoxins contamination better. Sustained public awareness at all levels, capacity building and aflatoxins related policies are necessary to support management initiatives.

Frequent Shifts in Aspergillus flavus Populations Associated with Maize Production in Sonora, Mexico.

Aspergillus flavus frequently contaminates maize, a critical staple for billions of people, with aflatoxins. Diversity among A. flavus L morphotype populations associated with maize in Sonora, Mexico was assessed and, in total, 869 isolates from 83 fields were placed into 136 vegetative compatibility groups (VCGs) using nitrate-nonutilizing mutants. VCG diversity indices did not differ in four agroecosystems (AES) but diversity significantly differed among years. Frequencies of certain VCGs changed manyfold over single years in both multiple fields and multiple AES. Certain VCGs were highly frequent (>1%) in 2006 but frequencies declined repeatedly in each of the two subsequent years. Other VCGs that had low frequencies in 2006 increased in 2007 and subsequently declined. None of the VCGs were consistently associated with any AES. Fourteen VCGs were considered dominant in at least a single year. However, frequencies often varied significantly among years. Only 9% of VCGs were detected all 3 years whereas 66% were detected in only 1 year. Results suggest that the most realistic measurements of both genetic diversity and the frequency of A. flavus VCGs are obtained by sampling multiple locations in multiple years. Single-season sampling in many locations should not be substituted for sampling over multiple years.

Prevalence of Aflatoxin Contamination in Maize and Groundnut in Ghana: Population Structure, Distribution, and Toxigenicity of the Causal Agents.

Aflatoxin contamination in maize and groundnut is perennial in Ghana with substantial health and economic burden on the population. The present study examined for the first time the prevalence of aflatoxin contamination in maize and groundnut in major producing regions across three agroecological zones (AEZs) in Ghana. Furthermore, the distribution and aflatoxin-producing potential of Aspergillus species associated with both crops were studied. Out of 509 samples (326 of maize and 183 of groundnut), 35% had detectable levels of aflatoxins. Over 15% of maize and 11% of groundnut samples exceeded the aflatoxin threshold limits set by the Ghana Standards Authority of 15 and 20 ppb, respectively. Mycoflora analyses revealed various species and morphotypes within the Aspergillus section Flavi. A total of 5,083 isolates were recovered from both crops. The L morphotype of Aspergillus flavus dominated communities with 93.3% of the population, followed by Aspergillus spp. with S morphotype (6%), A. tamarii (0.4%), and A. parasiticus (0.3%). Within the L morphotype, the proportion of toxigenic members was significantly (P < 0.05) higher than that of atoxigenic members across AEZs. Observed and potential aflatoxin concentrations indicate that on-field aflatoxin management strategies need to be implemented throughout Ghana. The recovered atoxigenic L morphotype fungi are genetic resources that can be employed as biocontrol agents to limit aflatoxin contamination of maize and groundnut in Ghana. Copyright © 2018 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Aflatoxin contamination of groundnut and maize in Zambia: observed and potential concentrations.

AIMS: The aims of the study were to quantify aflatoxins, the potent carcinogens associated with stunting and immune suppression, in maize and groundnut across Zambia's three agroecologies and to determine the vulnerability to aflatoxin increases after purchase. METHODS AND RESULTS: Aflatoxin concentrations were determined for 334 maize and groundnut samples from 27 districts using lateral-flow immunochromatography. Seventeen per cent of crops from markets contained aflatoxin concentrations above allowable levels in Zambia (10 µg kg-1 ). Proportions of crops unsafe for human consumption differed significantly (P < 0·001) among agroecologies with more contamination (38%) in the warmest (Agroecology I) and the least (8%) in cool, wet Agroecology III. Aflatoxin in groundnut (39 µg kg-1 ) and maize (16 µg kg-1 ) differed (P = 0·032). Poor storage (31°C, 100% RH, 1 week) increased aflatoxin in safe crops by over 1000-fold in both maize and groundnut. The L morphotype of Aspergillus flavus was negatively correlated with postharvest increases in groundnut. CONCLUSIONS: Aflatoxins are common in Zambia's food staples with proportions of unsafe crops dependent on agroecology. Fungal community structure influences contamination suggesting Zambia would benefit from biocontrol with atoxigenic A. flavus. SIGNIFICANCE AND IMPACT OF THE STUDY: Aflatoxin contamination across the three agroecologies of Zambia is detailed and the case for aflatoxin management with atoxigenic biocontrol agents provided. The first method for evaluating the potential for aflatoxin increase after purchase is presented.

The vegetative compatibility group to which the US biocontrol agent Aspergillus flavus AF36 belongs is also endemic to Mexico.

AIMS: To assess frequencies of the Aspergillus flavus atoxigenic vegetative compatibility group (VCG) YV36, to which the biocontrol agent AF36 belongs, in maize-growing regions of Mexico. METHODS AND RESULTS: Over 3500 A. flavus isolates recovered from maize agroecosystems in four states of Mexico during 2005 through 2008 were subjected to vegetative compatibility analyses based on nitrate nonutilizing mutants. Results revealed that 59 (1·6%) isolates belong to VCG YV36. All 59 isolates had the MAT1-2 idiomorph at the mating-type locus and the single nucleotide polymorphism in the polyketide synthase gene that confers atoxigenicity. Additional degradation of the aflatoxin gene cluster was detected in three isolates. Microsatellite loci analyses revealed low levels of genetic diversity and no linkage disequilibrium within VCG YV36. CONCLUSIONS: The VCG to which the biocontrol agent AF36 belongs, YV36, is also native to Mexico. The North American Free Trade Agreement should facilitate adoption of AF36 for use by Mexico in aflatoxin prevention programs. SIGNIFICANCE AND IMPACT OF THE STUDY: An USEPA registered biocontrol agent effective at preventing aflatoxin contamination of crops in the US, is also native to Mexico. This should facilitate the path to registration of AF36 as the first biopesticide for aflatoxin mitigation of maize in Mexico. Economic and health benefits to the population of Mexico should result once aflatoxin mitigation programs based on AF36 applications are implemented.

Method for monitoring deletions in the aflatoxin biosynthesis gene cluster of Aspergillus flavus with multiplex PCR.

UNLABELLED: The report presents a rapid, inexpensive and simple method for monitoring indels with influence on aflatoxin biosynthesis within Aspergillus flavus populations. PCR primers were developed for 32 markers spaced approximately every 5 kb from 20 kb proximal to the aflatoxin biosynthesis gene cluster to the telomere repeat. This region includes gene clusters required for biosynthesis of aflatoxins and cyclopiazonic acid; the resulting data were named cluster amplification patterns (CAPs). CAP markers are amplified in four multiplex PCRs, greatly reducing the cost and time to monitor indels within this region across populations. The method also provides a practical tool for characterizing intraspecific variability in A. flavus not captured with other methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Aflatoxins, potent naturally-occurring carcinogens, cause significant agricultural problems. The most effective method for preventing contamination of crops with aflatoxins is through use of atoxigenic strains of Aspergillus flavus to alter the population structure of this species and reduce incidences of aflatoxin producers. Cluster amplification pattern (CAP) is a rapid multiplex PCR method for identifying and monitoring indels associated with atoxigenicity in A. flavus. Compared to previous techniques, the reported method allows for increased resolution, reduced cost, and greater speed in monitoring the stability of atoxigenic strains, incidences of indel mediated atoxigenicity and the structure of A. flavus populations.

Diversity of aflatoxin-producing fungi and their impact on food safety in sub-Saharan Africa.

Crops frequently contaminated by aflatoxins are important sources of revenue and daily nourishment in many portions of sub-Saharan Africa. In recent years, reports have associated aflatoxins with diminished human health and export opportunities in many African Nations. Aflatoxins are highly carcinogenic metabolites mainly produced by members of Aspergillus sect. Flavi. The current study examined aflatoxin-producing fungi associated with maize grain intended for human consumption in 18 sub-Saharan African countries. 4469 Aspergillus sect. Flavi isolates were obtained from 339 samples. The majority (75%) of isolates belonged to the L strain morphotype of A. flavus. Minor percentages were A. tamarii (6%), A. parasiticus (1%), and isolates with S strain morphology (3%). No A. bombycis or A. nomius isolates were detected. Phylogenetic analyses of partial sequences of the nitrate reductase gene (niaD, 1.3kb) and the aflatoxin pathway transcription factor gene (aflR, 1.7kb) were used to verify isolate assignments into species and lineages. Phylogenetics resolved S strain isolates producing only B aflatoxins into two lineages fully supported by sizes of deletions in the gene region spanning the aflatoxin biosynthesis genes cypA (aflU) and norB (aflF). One lineage was the A. flavus S strain with either 0.9 or 1.5kb deletions. The second lineage, recently described from Kenya, has a 2.2kb deletion. Taxa with S strain morphology differed in distribution with strain SBG limited to West Africa and both A. minisclerotigenes and the new lineage from Kenya in Central and East Africa. African A. flavus L strain isolates formed a single clade with L strain isolates from other continents. The sampled maize frequently tested positive for aflatoxins (65%), fumonisins (81%), and deoxynivalenol (40%) indicating the presence of fungi capable of producing the respective toxins. Percentage of samples exceeding US limits for total aflatoxins (regulatory limit), fumonisins (advisory limit), and deoxynivalenol (advisory limit) were 47%, 49%, 4%, respectively.

Aspergillus parasiticus communities associated with sugarcane in the Rio Grande Valley of Texas: implications of global transport and host association within Aspergillus section Flavi.

In the Rio Grande Valley of Texas (RGV), values of maize and cottonseed crops are significantly reduced by aflatoxin contamination. Aflatoxin contamination of susceptible crops is the product of communities of aflatoxin producers and the average aflatoxin-producing potentials of these communities influence aflatoxin contamination risk. Cropping pattern influences community composition and, thereby, the epidemiology of aflatoxin contamination. In 2004, Aspergillus parasiticus was isolated from two fields previously cropped to sugarcane but not from 23 fields without recent history of sugarcane cultivation. In 2004 and 2005, A. parasiticus composed 18 to 36% of Aspergillus section Flavi resident in agricultural soils within sugarcane-producing counties. A. parasiticus was not detected in counties that do not produce sugarcane. Aspergillus section Flavi soil communities within sugarcane-producing counties differed significantly dependent on sugarcane cropping history. Fields cropped to sugarcane within the previous 5 years had greater quantities of A. parasiticus (mean = 16 CFU/g) than fields not cropped to sugarcane (mean = 0.1 CFU/g). The percentage of Aspergillus section Flavi composed of A. parasiticus increased to 65% under continuous sugarcane cultivation and remained high the first season of rotation out of sugarcane. Section Flavi communities in fields rotated to non-sugarcane crops for 3 to 5 years were composed of <5% A. parasiticus, and fields with no sugarcane history averaged only 0.2% A. parasiticus. The section Flavi community infecting RGV sugarcane stems ranged from 95% A. parasiticus in billets prepared for commercial planting to 52% A. parasiticus in hand-collected sugarcane stems. Vegetative compatibility assays and multilocus phylogenies verified that aflatoxin contamination of raw sugar was previously attributed to similar A. parasiticus in Japan. Association of closely related A. parasiticus genotypes with sugarcane produced in Japan and RGV, frequent infection of billets by these genotypes, and the ephemeral nature of A. parasiticus in RGV soils suggests global transport with sugarcane planting material.

Influence of the host contact sequence on the outcome of competition among aspergillus flavus isolates during host tissue invasion.

Biological control of aflatoxin contamination by Aspergillus flavus is achieved through competitive exclusion of aflatoxin producers by atoxigenic strains. Factors dictating the extent to which competitive displacement occurs during host infection are unknown. The role of initial host contact in competition between pairs of A. flavus isolates coinfecting maize kernels was examined. Isolate success during tissue invasion and reproduction was assessed by quantification of isolate-specific single nucleotide polymorphisms using pyrosequencing. Isolates were inoculated either simultaneously or 1 h apart. Increased success during competition was conferred to the first isolate to contact the host independent of that isolate's innate competitive ability. The first-isolate advantage decreased with the conidial concentration, suggesting capture of limited resources on kernel surfaces contributes to competitive exclusion. Attempts to modify access to putative attachment sites by either coating kernels with dead conidia or washing kernels with solvents did not influence the success of the first isolate, suggesting competition for limited attachment sites on kernel surfaces does not mediate first-isolate advantage. The current study is the first to demonstrate an immediate competitive advantage conferred to A. flavus isolates upon host contact and prior to either germ tube emergence or host colonization. This suggests the timing of host contact is as important to competition during disease cycles as innate competitive ability. Early dispersal to susceptible crop components may allow maintenance within A. flavus populations of genetic types with low competitive ability during host tissue invasion.

Identification of Atoxigenic Aspergillus flavus Isolates to Reduce Aflatoxin Contamination of Maize in Kenya.

Aspergillus flavus has two morphotypes, the S strain and the L strain, that differ in aflatoxin-producing ability and other characteristics. Fungal communities on maize dominated by the S strain of A. flavus have repeatedly been associated with acute aflatoxin poisonings in Kenya, where management tools to reduce aflatoxin levels in maize are needed urgently. A. flavus isolates (n = 290) originating from maize produced in Kenya and belonging to the L strain morphotype were tested for aflatoxin-producing potential. A total of 96 atoxigenic isolates was identified from four provinces sampled. The 96 atoxigenic isolates were placed into 53 vegetative compatibility groups (VCGs) through complementation of nitrate non-utilizing mutants. Isolates from each of 11 VCGs were obtained from more than one maize sample, isolates from 10 of the VCGs were detected in multiple districts, and isolates of four VCGs were found in multiple provinces. Atoxigenic isolates were tested for potential to reduce aflatoxin concentrations in viable maize kernels that were co-inoculated with highly toxigenic S strain isolates. The 12 most effective isolates reduced aflatoxin levels by >80%. Reductions in aflatoxin levels caused by the most effective Kenyan isolates were comparable with those achieved with a United States isolate (NRRL-21882) used commercially for aflatoxin management. This study identified atoxigenic isolates of A. flavus with potential value for biological control within highly toxic Aspergillus communities associated with maize production in Kenya. These atoxigenic isolates have potential value in mitigating aflatoxin outbreaks in Kenya, and should be evaluated under field conditions.

Molecular characterization of atoxigenic strains for biological control of aflatoxins in Nigeria.

Aflatoxins are highly toxic carcinogens produced by several species in Aspergillus section Flavi. Strains of A. flavus that do not produce aflatoxins, called atoxigenic strains, have been used commercially in North America as tools for limiting aflatoxin contamination. A similar aflatoxin management strategy is being pursued in Nigeria. In the current study, loci across the 68 kb aflatoxin biosynthesis gene cluster were compared among 18 atoxigenic and two aflatoxin-producing vegetative compatibility groups (VCGs) from Nigeria and an atoxigenic VCG used commercially in North America. Five of the atoxigenic VCGs had large deletions (37-65 kb) extending from the teleomeric side of the aflatoxin biosynthesis cluster. In one VCG (AV0222) the deletion extended through the cluster to the adjacent sugar cluster. The remaining twelve atoxigenic VCGs, including the VCG used for aflatoxin management in North America, contained all the aflatoxin pathway genes, but with defects. Two observations support the long-term persistence of atoxigenicity within A. flavus: first, a comparison of pathway genes revealed more changes in atoxigenic than in aflatoxin-producing isolates relative to the aflatoxin-producing strain NRRL 3357; and second, several non-synonymous changes are unique to atoxigenics. Atoxigenic VCG diversity was assessed with phylogenetic analyses. Although some atoxigenics share relatively recent ancestry, several are more closely related to aflatoxin producers than to other atoxigenics. The current study demonstrates VCGs of A. flavus in West Africa with diverse mechanisms of atoxigenicity and potential value in aflatoxin management programmes.

Variation in competitive ability among isolates of Aspergillus flavus from different vegetative compatibility groups during maize infection.

ABSTRACT Aspergillus flavus, the primary causal agent of aflatoxin contamination, includes many genetically diverse vegetative compatibility groups (VCGs). Competitive ability during infection of living maize kernels was quantified for isolates from 38 VCGs. Kernels were inoculated with both a common VCG, CG136, and another VCG; after 7 days (31 degrees C), conidia were washed from kernels, and aflatoxins and DNA were extracted from kernels and conidia separately. CG136-specific single-nucleotide polymorphisms were quantified by pyrosequencing; VCGs co-inoculated with CG136 produced 46 to 85 and 51 to 84% of A. flavus DNA from kernels and conidia, respectively. Co-inoculation with atoxigenic isolates reduced aflatoxin up to 90% and, in some cases, more than predicted by competitive exclusion alone. Conidia contained up to 42 ppm aflatoxin B(1), indicating airborne conidia as potentially important sources of environmental exposure. Aflatoxin-producing potential and sporulation were negatively correlated. For some VCGs, sporulation during co-infection was greater than that predicted by kernel infection, suggesting that some VCGs increase dispersal while sacrificing competitive ability during host tissue colonization. The results indicate both life strategy and adaptive differences among A. flavus isolates and provide a basis for selection of biocontrol strains with improved competitive ability, sporulation, and aflatoxin reduction on target hosts.

Impact of Aspergillus section Flavi community structure on the development of lethal levels of aflatoxins in Kenyan maize (Zea mays).

AIMS: To evaluate the potential role of fungal community structure in predisposing Kenyan maize to severe aflatoxin contamination by contrasting aflatoxin-producing fungi resident in the region with repeated outbreaks of lethal aflatoxicosis to those in regions without a history of aflatoxicosis. METHODS AND RESULTS: Fungi belonging to Aspergillus section Flavi were isolated from maize samples from three Kenyan provinces between 2004 and 2006. Frequencies of identified strains and aflatoxin-producing abilities were assessed, and the data were analysed by statistical means. Most aflatoxin-producing fungi belonged to Aspergillus flavus. The two major morphotypes of A. flavus varied greatly between provinces, with the S strain dominant in both soil and maize within aflatoxicosis outbreak regions and the L strain dominant in nonoutbreak regions. CONCLUSIONS: Aspergillus community structure is an important factor in the development of aflatoxins in maize in Kenya and, as such, is a major contributor to the development of aflatoxicosis in the Eastern Province. SIGNIFICANCE AND IMPACT OF THE STUDY: Since 1982, deaths caused by aflatoxin-contaminated maize have repeatedly occurred in the Eastern Province of Kenya. The current study characterized an unusual fungal community structure associated with the lethal contamination events. The results will be helpful in developing aflatoxin management practices to prevent future outbreaks in Kenya.

Genetic isolation among sympatric vegetative compatibility groups of the aflatoxin-producing fungus Aspergillus flavus.

Aspergillus flavus, a fungal pathogen of animals and both wild and economically important plants, is most recognized for producing aflatoxin, a cancer-causing secondary metabolite that contaminates food and animal feed globally. Aspergillus flavus has two self/nonself recognition systems, a sexual compatibility system and a vegetative incompatibility system, and both play a role in directing gene flow in populations. Aspergillus flavus reproduces clonally in wild and agricultural settings, but whether a cryptic sexual stage exists in nature is currently unknown. We investigated the distribution of genetic variation in 243 samples collected over 4 years from three common vegetative compatibility groups (VCGs) in Arizona and Texas from cotton using 24 microsatellite loci and the mating type locus (MAT) to assess population structure and potential gene flow among A. flavus VCGs in sympatric populations. All isolates within a VCG had the same mating type with OD02 having MAT1-2 and both CG136 and MR17 having MAT1-1. Our results support the hypothesis that these three A. flavus VCGs are genetically isolated. We found high levels of genetic differentiation and no evidence of gene flow between VCGs, including VCGs of opposite mating-type. Our results suggest that these VCGs diverged before domestication of agricultural hosts (>10,000 yr bp).

Twenty-four microsatellite markers for the aflatoxin-producing fungus Aspergillus flavus.

Aspergillus flavus infects both plants and humans and contaminates diverse agricultural crops with aflatoxins, highly carcinogenic fungal metabolites. We describe 24 microsatellite markers developed to assess genetic diversity and recombination within and between three vegetative compatibility groups (VCGs) of Aspergillus flavus. These loci are polymorphic within at least one VCG or between VCGs. For loci polymorphic across all three VCGs, the number of alleles ranged from two to 19. These markers will be useful for genetic studies of this economically important pathogen.

Evaluation of atoxigenic isolates of Aspergillus flavus as potential biocontrol agents for aflatoxin in maize.

Aflatoxin contamination resulting from maize infection by Aspergillus flavus is both an economic and a public health concern. Therefore, strategies for controlling aflatoxin contamination in maize are being investigated. The abilities of eleven naturally occurring atoxigenic isolates in Nigeria to reduce aflatoxin contamination in maize were evaluated in grain competition experiments and in field studies during the 2005 and 2006 growing seasons. Treatments consisted of inoculation of either grains in vials or ears at mid-silking stage in field plots, with the toxigenic isolate (La3228) or atoxigenic isolate alone and co-inoculation of each atoxigenic isolate and La3328. Aflatoxin B(1) + B(2) concentrations were significantly (p < 0.05) lower in the co-inoculation treatments compared with the treatment in which the aflatoxin-producing isolate La3228 was inoculated alone. Relative levels of aflatoxin B(1) + B(2) reduction ranged from 70.1% to 99.9%. Among the atoxigenics, two isolates from Lafia, La3279 and La3303, were most effective at reducing aflatoxin B(1) + B(2) concentrations in both laboratory and field trials. These two isolates have potential value as agents for the biocontrol of aflatoxin contamination in maize. Because these isolates are endemic to West Africa, they are both more likely than introduced isolates to be well adapted to West African environments and to meet regulatory concerns over their use throughout that region.

Ecology of aflatoxin producing fungi and biocontrol of aflatoxin contamination.

Aflatoxins, highly toxic and carcinogenic compounds that frequently contaminate foods and feeds, are produced by several genera in the genusAspergillus. Aspergillus flavus, the most common species causing crop contamination, is a common inhabitant of the Sonoran desert of North America where it resides in complex communities composed of diverse individuals. This diversity reflects divergent adaptation to various ecological niches. SomeA. flavus isolates that are well adapted to plant associated niches do not produce aflatoxins yet have the capacity to competitively exclude aflatoxin producers. These atoxigenic strains can serve as biological control agents for management of aflatoxins in crops. Detailed knowledge of the ecology of aflatoxin-producing fungi may lead to novel practical methods for limiting contamination.

Aflatoxin biosynthesis gene clusters and flanking regions.

AIMS: To compare the biosynthetic gene cluster sequences of the main aflatoxin (AF)-producing Aspergillus species. METHODS AND RESULTS: Sequencing was on fosmid clones selected by homology to Aspergillus parasiticus sequence. Alignments revealed that gene order is conserved among AF gene clusters of Aspergillus nomius, A. parasiticus, two sclerotial morphotypes of Aspergillus flavus, and an unnamed Aspergillus sp. Phylogenetic relationships were established using the maximum likelihood method implemented in PAUP. Based on the Eurotiomycete/Sordariomycete divergence time, the A. flavus-type cluster has been maintained for at least 25 million years. Such conservation of the genes and gene order reflects strong selective constraints on rearrangement. Phylogenetic comparison of individual genes in the cluster indicated that ver-1, which has homology to a melanin biosynthesis gene, experienced selective forces distinct from the other pathway genes. Sequences upstream of the polyketide synthase-encoding gene vary among the species, but a four-gene sugar utilization cluster at the distal end is conserved, indicating a functional relationship between the two adjacent clusters. CONCLUSIONS: The high conservation of cluster components needed for AF production suggests there is an adaptive value for AFs in character-shaping niches important to those taxa. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first comparison of the complete nucleotide sequences of gene clusters harbouring the AF biosynthesis genes of the main AF-producing species. Such a comparison will aid in understanding how AF biosynthesis is regulated in experimental and natural environments.