Aflatoxin contamination of corn is a major threat to the safe food and feed. The United States Federal Grain Inspection Service (FGIS) monitors commercial grain shipments for the presence of aflatoxin. A total of 146 Aspergillus flavus were isolated from 29 highly contaminated grain samples to characterize the visual phenotypes, aflatoxin-producing potential, and genotypes to explore the etiological cause of high aflatoxin contamination of US corn. Five of the isolates had reduced sensitivity (43-49% resistant) to the fungicide azoxystrobin, with the remainder all being over 50% resistant to azoxystrobin at the discriminating dose of 2.5 µg/mL. Only six isolates of the highly aflatoxigenic S morphotype were found, and 48 isolates were non-aflatoxigenic. Analysis of the mating type locus revealed 45% MAT 1-1 and 55% MAT 1-2. The A. flavus population originating from the highly aflatoxin contaminated grain samples was compared to a randomly selected subset of isolates originating from commercial corn samples with typical levels of aflatoxin contamination (average < 50 ppb). Use of simple sequence repeat (SSR) genotyping followed by principal component analysis (PCoA) revealed a similar pattern of genotypic distribution in the two populations, but greater diversity in the FGIS-derived population. The noticeable difference between the two populations was that genotypes identical to strain NRRL 21882, the active component of the aflatoxin biocontrol product Afla-Guard™, were ten times more common in the commercial corn population of A. flavus compared to the population from the high-aflatoxin corn samples. The other similarities between the two populations suggest that high aflatoxin concentrations in corn grain are generally the result of infection with common A. flavus genotypes.
Humans and animals are exposed to aflatoxins, toxic carcinogenic fungal metabolites, through consumption of contaminated food and feed. Aspergillus flavus , the primary causal agent of crop aflatoxin contamination, is composed of phenotypically and genotypically diverse vegetative compatibility groups (VCGs). Molecular data suggest that VCGs largely behave as clones with certain VCGs exhibiting niche preference. VCGs vary in aflatoxin‐producing ability, ranging from highly aflatoxigenic to atoxigenic. The prevalence of individual VCGs is dictated by competition during growth and reproduction under variable biotic and abiotic conditions. Agronomic practices influence structures and average aflatoxin‐producing potentials of A. flavus populations and, as a result, incidences and severities of crop contamination. Application of atoxigenic strains has successfully reduced crop aflatoxin contamination across large areas in the United States. This strategy uses components of the endemic diversity to alter structures of A. flavus populations and improve safety of food, feed, and the overall environment.
Frequency and numbers of Campylobacter spp. were assessed per freshly processed, contaminated broiler carcass. Campylobacter-positive flocks were identified by cecal sample analysis at slaughter. These flocks had been tested as Campylobacter negative at 4.1 ± 0.9 d prior to slaughter. Levels of contamination were estimated using 2 sampling approaches per carcass: (1) free weep fluids and (2) whole-carcass, 100 mL of distilled water rinses. Estimations of counts were determined by directly plating dilutions of weeps and rinses onto Campy-Cefex agar and incubating the plates at 41.5°C under microaerobic atmosphere. Confirmation was provided by latex agglutination to quantify levels per milliliter of weep and per 100 mL of rinse. Thirty-two slaughter groups (∼20 carcasses per group) were compared from 2003 to 2004. The Campylobacter-positive weep frequency was 84.8%, whereas the frequency for rinse samples was 74.4% (P < 0.001). Enumeration of Campylobacter spp. on positive samples ranged from 0.70 to 6.13 log10 cfu/mL of weep (geometric mean of 2.84) and from 2.30 to 7.72 log10 cfu/100 mL of rinse (geometric mean of 4.38). The correlations between weep and rinse were 0.814 with 0.5 mL of rinse and 0.6294 when applying 0.1 mL of rinse The quantitative regression analyses for these 2 corresponding tests were log10 rinse (for 0.5 mL of inoculum) = 1.1965 log10 weep + 0.4979, and log10 rinse (for 0.1 mL of inoculum) = 1.322 log10 weep − 0.1521. FlaA SVR sequencing of isolates indicated that the same genotypes were found in weep and rinse samples. Weep and rinse sampling led to different proportions of Campylobacter-positive carcasses detection, but we demonstrated that this difference was reduced by increasing the amount of rinse fluid used for plating.
Summary 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.
Aflatoxin (AF) contamination occurs throughout sub-Saharan Africa reducing trade opportunities and exposing populations to a potent carcinogen that causes liver cirrhosis, stunting, and reduced immune function.Use of atoxigenic strains of Aspergillus flavus to competitively exclude AF producers is an established tool for AF prevention in the US.Utilizing the same principles, highly effective biocontrol products were developed for several African nations.Each product uses 4 genetically distinct atoxigenic isolates of A. flavus as active ingredients.The isolates are endemic to target nations to ensure no introduction of exotics and adaptation to target agroecosystems.Adaptation to Africa began in Nigeria where the resulting product aflasafe™ was evaluated in farmer's fields for 5 seasons on over 500 fields.Treatments reduced AF by 82-95% in maize and peanut.Also in West Africa, products for Senegal (aflasafe SN01) and Burkina Faso (aflasafe BF01) were evaluated in farmer's fields for multiple seasons with reductions exceeding 75%.Aflasafe KE01, developed for Kenya in East Africa, where lethal aflatoxicoses has been repeatedly reported had excellent efficacy on farm for two seasons.In one area, untreated controls averaged >1,100 ppb and treated fields <75 ppb.The aflasafe biocontrol products have area-wide and long-term influences that offer real promise for relieving human populations in Africa of the health effects caused by chronic AF exposure. Role of plant elicitor peptides and phytoalexins in enhancing maize resistance to Aspergillus flavus infectionA. HUFFAKER (1), J. Sims (1), S. Christensen (1), E. A. Schmelz (1) (1) USDA-ARS CMAVE, Gainesville, FL, U.S.A. Phytopathology 104(Suppl.3):S3.139Maize responds to pests and pathogens with complex defense responses.To facilitate effective breeding for pest and pathogen resistance, we're elucidating cellular and molecular functions of regulatory and metabolic components of these maize defense responses.Our studies of regulatory components have focused on a family of peptide signals (ZmPeps) and their cognate receptors (ZmPEPRs) that regulate maize immunity.One of these, ZmPep1, triggers synthesis of plant defense phytohormones and induces expression of genes encoding pathogenesis-related proteins.ZmPep1 also promotes accumulation of the maize defense chemical HDMBOA-Glc.Treatment of maize plants with ZmPep1 prior to inoculation enhances resistance to fungal pathogens.A second peptide, ZmPep3, induces plant resistance responses against Lepidopteran herbivores associated with spread of mycotoxin-producing fungal pathogens.ZmPep3 stimulates expression of proteinase inhibitor genes and emission of volatiles that attract natural enemies of herbivorous pests.We've also discovered two families of fungalinduced terpenoid phytoalexins that accumulate at the plant pathogen interface, the kauralexins and zealexins.Several of these terpenoids have antimicrobial activity and we're examining their effects on aflatoxin production.We aim to provide understanding of molecular processes regulating maize defense and new strategies for enhancing resistance to pests, disease and mycotoxin accumulation. Genomic approaches to characterize the regulatory circuits ofAspergillus flavus controlling aflatoxin biosynthesis G. PAYNE (1), X. Shu (1), G. OBrian (1), B. Musungu (2), M. Geisler (2), A. M. Fakhoury (2) (
