Reduced Aflatoxin Accumulation Research Articles

Tissue-Specific Components of Resistance to Aspergillus Ear Rot of Maize

Aspergillus flavus and other Aspergillus spp. infect maize and produce aflatoxins. An important control measure is the use of resistant maize hybrids. There are several reports of maize lines that are resistant to aflatoxin accumulation but the mechanisms of resistance remain unknown. To gain a better understanding of resistance, we dissected the phenotype into 10 components: 4 pertaining to the response of silk, 4 pertaining to the response of developing kernels, and 2 pertaining to the response of mature kernels to inoculation with A. flavus. In order to challenge different tissues and to evaluate multiple components of resistance, various inoculation methods were used in experiments in vitro and under field conditions on a panel of diverse maize inbred lines over 3 years. As is typical for this trait, significant genotype-environment interactions were found for all the components of resistance studied. There was, however, significant variation in maize germplasm for susceptibility to silk and kernel colonization by A. flavus as measured in field assays. Resistance to silk colonization has not previously been reported. A significant correlation of resistance to aflatoxin accumulation with flowering time and kernel composition traits (fiber, ash, carbohydrate, and seed weight) was detected. In addition, correlation analyses with data available in the literature indicated that lines that flower later in the season tend to be more resistant. We were not able to demonstrate that components identified in vitro were associated with reduced aflatoxin accumulation in the field.

Registration of Maize Germplasm Lines Tx736, Tx739, and Tx740 for Reducing Preharvest Aflatoxin Accumulation

Maize (Zea mays L.) production in Texas, the southern United States, and much of the developing world is constrained by preharvest contamination from aflatoxins—potent mycotoxins produced by the fungus Aspergillus flavus. When consumed, aflatoxin can lead to impaired growth, liver cancer, or death of both humans and livestock. Because of these effects, the presence of this mycotoxin is tightly regulated in the U.S. food supply. No complete resistance to A. flavus or aflatoxin is known to exist in maize; however, multiple quantitative traits and some sources of resistance have been identified. We propose the release of three maize lines that demonstrate reduced preharvest aflatoxin accumulation. Tx736 (Reg. No. GP‐578, PI 662937) is a germplasm line derived from modified pedigree selection of a cross between Tx772, a line with reduced aflatoxin accumulation, and T246, a temperate line with good yield and Texas adaptation, backcrossed to Tx772 once. Lines Tx739 (Reg. No. GP‐579, PI 662938) and Tx740 (Reg. No. GP‐580, PI 662939) were selected by pedigree methods from S3 plants selfed out of heterotic groups A, C, and E from Agricomseeds (Santa Cruz, Bolivia). These three lines were field tested as lines per se and as testcross hybrids with introduced inoculation in multienvironmental field trials. In trials, these lines and hybrids had between 30 and 73% lower aflatoxin content than commercial checks. These germplasm lines will serve as unique sources for novel traits and alleles that reduce aflatoxin in elite temperate and subtropical maize.

Laboratory tests for assessing efficacy of atoxigenic Aspergillus flavus strains as biocontrol agents

Biocontrol by competitive inhibition using atoxigenic Aspergillus flavus strains has been shown to be an effective method for controlling aflatoxin production in peanuts, maize and cottonseed. Selecting biocontrol strains is not straightforward, as it is difficult to assess fitness for the task without expensive field trials. Reconstruction experiments have been generally performed under laboratory conditions to investigate the biological mechanisms underlying the efficacy of atoxigenic strains in preventing aflatoxin production and/or to give a preliminary indication of strain performance when released in the field. The study here described was conducted in order to evaluate the potential of the different atoxigenic A. flavus strains, colonizing the corn fields of the Po Valley, in reducing aflatoxin accumulation when grown in mixed cultures together with atoxigenic strains; additionally, we developed a simple and inexpensive procedure that may be used to scale-up the screening process and to increase knowledge on the mechanisms interfering with mycotoxin production during co-infection.

Ear rot, aflatoxin accumulation, and fungal biomass in maize after inoculation with Aspergillus flavus

Aflatoxin, a toxin produced by the fungus Aspergillus flavus Link: Fries, occurs naturally in maize (Z ea mays L.). Aflatoxin is a potent human carcinogen and is also toxic to livestock, pets, and wildlife. When contaminated with aflatoxin, the value of maize grain is markedly reduced. This investigation was conducted to compare ear rot, aflatoxin accumulation, and fungal biomass in maize single crosses with varying degrees of resistance to aflatoxin accumulation and to determine the relative importance of general combining ability (GCA) and specific combining ability (SCA) in the inheritance of resistance to ear rot, aflatoxin accumulation, and fungal biomass. Eight germplasm lines with different levels of resistance to aflatoxin accumulation were used as parents of a diallel cross. The cross was evaluated for visible ear rot, aflatoxin accumulation, and A. flavus infection in the grain. A. flavus infection was determined by quantitative real-time polymerase chain reaction (qRT-PCR) assays. Both GCA and SCA were significant sources of variation in the inheritance of the three traits although GCA accounted for a greater portion of the variation among single crosses. The interactions of GCA and SCA with years were highly significant for aflatoxin accumulation, but not significant for A. flavus infection. Estimates of GCA effects were highly significant for both reduced A. flavus infection and reduced aflatoxin accumulation for Mp313E, Mp715, and Mp717. Conversely, GCA effects associated with GA209 were significant for reduced levels of A. flavus infection and ear rot, but high levels of aflatoxin accumulation. Mp313E, Mp715, and Mp717 should be useful in breeding programs targeting both reduced levels of fungal infection and aflatoxin accumulation.

Health economic impacts and cost-effectiveness of aflatoxin-reduction strategies in Africa: case studies in biocontrol and post-harvest interventions

Advances in health economics have proven useful in evaluating the cost-effectiveness of interventions, where the benefit usually takes the form of improved health outcomes rather than market outcomes. The paper performs health-based cost-effectiveness analyses of two potential aflatoxin control strategies in Africa: (1) pre-harvest biocontrol, using atoxigenic strains of Aspergillus flavus competitively to exclude toxigenic strains from colonizing maize in Nigeria; and (2) post-harvest interventions in a package to reduce aflatoxin accumulation in groundnuts in Guinea. It is described how health benefits gained from each intervention, in terms of fewer aflatoxin-induced hepatocellular carcinoma cases, can be compared with the costs of implementing the interventions. It is found that both interventions would be extremely cost-effective if applied widely in African agriculture. That is, the monetized value of lives saved and quality of life gained by reducing aflatoxin-induced hepatocellular carcinoma far exceeds the cost of either biocontrol or the post-harvest intervention package to achieve those health benefits. The estimated cost-effectiveness ratio (CER; gross domestic product multiplied by disability-adjusted life years saved per unit cost) for biocontrol in Nigerian maize ranged from 5.10 to 24.8; while the estimated CER for the post-harvest intervention package in Guinean groundnuts ranged from 0.21 to 2.08. Any intervention with a CER > 1 is considered by the World Health Organization (WHO) to be ‘very cost-effective’, while an intervention with a CER > 0.33 is considered ‘cost-effective’. Aside from cost-effectiveness, public health interventions must be readily accepted by the public, and must have financial and infrastructural support to be feasible in the parts of the world where they are most needed.

Food-grade antioxidants for control of Aspergillus section Flavi and interrelated mycoflora of stored peanuts with different water activities

The inhibitory effect of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) mixture on fungal populations, Aspergillus section Flavi and aflatoxins accumulation in in-pod peanuts during storage in big bags was investigated. In-pod peanuts were previously conditioned at different water activities (0.94, 0.88, 0.84 and 0.76 aw) and treated with food-grade antioxidants. Both control and treated peanuts were stored for 6 months and sampled at monthly intervals. BHA-BHT mixture reduced the incidence of peanut fungal populations between 0.6-20.4% and between 1.2-33.1% during 1-3 and 4-5 storage months, respectively. Aspergillus section Flavi counts decreased with 36.5%, 46.3% and 77.4% in peanuts conditioned at 0.94, 0.84 and 0.76 aw levels and treated with antioxidants. At the above peanut aw conditions, the treatment applied reduced aflatoxin accumulation by 72.1%, while any effect on this metabolite production was observed at 0.88 aw. The antioxidant formulation used in this study has the potential to control aflatoxigenic populations in in-pod peanuts stored in half-permeable silos at ≤0.84 aw level.

Diallel analysis of aflatoxin accumulation in maize

Since its discovery in numerous feedstuffs, aflatoxin, a carcinogenic compound produced by the fungus Aspergillus flavus Link ex Fries, has caused much concern among consumers and producers alike. This toxin poses a serious economic threat to maize ( Zea mays L.) producers of the southeastern and midwestern regions of the United States. Efforts to identify maize germplasm that is resistant to aflatoxin accumulation and to investigate the genetic basis of this resistance have been undertaken at numerous research institutions. The objectives of this study were to (1) evaluate aflatoxin accumulation in grain harvested from maize inbred lines and a diallel cross among these lines, (2) determine the importance of general and specific combining abilities in inheritance of resistance to aflatoxin accumulation, and (3) estimate general and specific combining ability effects associated with resistance to aflatoxin accumulation in the inbred lines and crosses among them. Eight inbred lines and a diallel cross of the maize lines were inoculated with an A. flavus spore suspension 12–14 d after silk emergence. Following harvest, aflatoxin content was determined from samples of grain. Statistical analyses performed using SAS general linear models (GLM) and DIALLEL-SAS indicated that general and specific combining ability were significant sources of variation in the inheritance of resistance to aflatoxin accumulation. The inbred line Mp313E, which was developed and released as a source of resistance to aflatoxin contamination, had significantly lower aflatoxin accumulation than other lines. Mo18W exhibited excellent general combining ability for reduced aflatoxin accumulation when crossed with the other lines. Both Mp313E and Mo18W could be useful in breeding programs to develop aflatoxin-resistant maize hybrids. Mp339, SC212M, and Ab24E demonstrated aflatoxin susceptibility as both inbreds and in single crosses.

Nonaflatoxigenic Aspergillus flavus TX9-8 competitively prevents aflatoxin accumulation by A. flavus isolates of large and small sclerotial morphotypes

Aflatoxins are a family of highly toxic and carcinogenic toxins produced by several Aspergillus species. Aflatoxin contamination of agricultural commodities both pre- and postharvest is a serious food safety issue and a significant economic concern. Using nonaflatoxigenic A. flavus isolates to competitively exclude toxigenic A. flavus isolates in agricultural fields has become an adopted approach to reduce aflatoxin contamination. From screening subgroups of nonaflatoxigenic A. flavus, we identified an A. flavus isolate, TX9-8, which competed well with three A. flavus isolates producing low, intermediate, and high levels of aflatoxins, respectively. TX9-8 has a defective polyketide synthase gene ( pksA), which is necessary for aflatoxin biosynthesis. Co-inoculating TX9-8 at the same time with large sclerotial (L strain) A. flavus isolates at a ratio of 1:1 or 1:10 (TX9-8:toxigenic) prevented aflatoxin accumulation. The intervention of TX9-8 on small sclerotial (S strain) A. flavus isolates varied and depended on isolate and ratio of co-inoculation. At a ratio of 1:1 TX9-8 prevented aflatoxin accumulation by A. flavus CA28 and reduced aflatoxin accumulation 10-fold by A. flavus CA43. No decrease in aflatoxin accumulation was apparent when TX9-8 was inoculated 24 h after toxigenic L- or S strain A. flavus isolates started growing. The competitive effect likely is due to TX9-8 outgrowing toxigenic A. flavus isolates.

β-1,3-Glucanase Activity in Peanut Seed (Arachis hypogaea) is Induced by Inoculation with Aspergillus flavus and Copurifies with a Conglutin-Like Protein

ABSTRACT Infection of peanut (Arachis hypogaea) seed by Aspergillus flavus and A. parasiticus is a serious problem that can result in aflatoxin contamination in the seed. Breeding resistant cultivars would be an effective approach to reduce aflatoxin accumulation. The objective of this study was to investigate the expression of the pathogenesis-related (PR) protein beta-1,3-glucanase and the isoform patterns in peanut seed inoculated with A. flavus. Peanut genotypes GT-YY9 and GT-YY20 (both resistant to A. flavus infection) and Georgia Green and A100 (both susceptible to A. flavus infection) were used in this study. The activities of beta-1,3-glucanase were similar in the uninfected seed of all genotypes, but increased significantly in the resistant genotypes after inoculation in comparison with the susceptible genotypes. An in-gel (native polyacrylamide gel electrophoresis [PAGE]) enzymatic activity assay of beta-1,3-glucanase revealed that there were more protein bands corresponding to beta-1,3-glucanase isoforms in the infected seed of resistant genotypes than in the infected seed of susceptible genotypes. Both acidic and basic beta-1,3-glucanase isoforms were detected in the isoelectric focusing gels. Thin-layer chromatography analysis of the hydrolytic products from the reaction mixtures of the substrate with the total protein extract or individual band of native PAGE revealed the presence of enzymatic hydrolytic oligomer products. The individual bands corresponding to the bands of beta-1,3-glucanase isoforms Glu 1 to 5 were separated on the sodium dodecyl sulfate-PAGE, resulting in two bands of 10 and 13 kDa, respectively. The sequences of fragments of the 13-kDa major protein band showed a high degree of homology to conglutin, a storage protein in peanut seed. Conglutin is reported as a peanut allergen, Ara h2. Our data provide the first evidences for peanut having beta-1,3-glucanase activities and the association with the resistance to A. flavus colonization in peanut seed. We have not directly demonstrated that conglutin has beta-1,3-glucanase activity.

Influence of Planting Date on Aflatoxin Accumulation in Bt, non‐Bt, and Tropical non‐Bt Hybrids

Aflatoxin, produced by the fungus Aspergillus flavus Link, reduces the value of corn (Zea mays L.) and is usually associated with high temperatures, water stress, and insect damage. The objective of this study was to determine if Bt (Bacillus thuringiensis) corn hybrids or “tropical” germplasm could reduce aflatoxin accumulation with later planting dates. Aflatoxin accumulation (B1, B2, G1, and G2) in corn grain was evaluated on Bt, non‐Bt, and tropical non‐Bt hybrids and four planting dates (March, April, May, and June) from 1998 to 2000. Aflatoxin concentration in corn varied across years but generally decreased with later planting date. In 1998, aflatoxin accumulation was lower in Bt (314 ng g−1) than non‐Bt hybrids (634 ng g−1) but not different than tropical non‐Bt hybrid (470 ng g−1). However, aflatoxin contamination was lower from Bt hybrids (70 ng g−1) than from the tropical non‐Bt hybrid (259 ng g−1) but not different in non‐Bt hybrids (86 ng g−1) in 1999. In 2000, aflatoxin levels were low, and hybrid had no effect on aflatoxin concentration. Temperature and irrigation effects on aflatoxin accumulation were not consistent. Increased temperature and delayed harvest may lead to aflatoxin accumulation before harvest. However, precipitation may influence aflatoxin levels in some years. The results of this study indicate that aflatoxin accumulation in corn may be decreased with later planting date, and less accumulation in Bt than non‐Bt or tropical non‐Bt hybrids may be indirectly explained by insect reductions.

Interactions of saprophytic yeasts with a nor mutant of Aspergillus flavus.

The nor mutant of Aspergillus flavus has a defective norsolorinic acid reductase, and thus the aflatoxin biosynthetic pathway is blocked, resulting in the accumulation of norsolorinic acid, a bright red-orange pigment. We developed a visual agar plate assay to monitor yeast strains for their ability to inhibit aflatoxin production by visually scoring the accumulation of this pigment of the nor mutant. We identified yeast strains that reduced the red-orange pigment accumulation in the nor mutant. These yeasts also reduced aflatoxin accumulation by a toxigenic strain of A. flavus. These yeasts may be useful for reducing aflatoxin contamination of food commodities.

Combining Ability for Resistance to Field Aflatoxin Accumulation in Maize Grain*

AbstractPrevious genetic studies on resistance to aflatoxin production in maize (Zea mays L.) only used a limited amount of germplasm. The objective of this study was to determine general (GCA) and specific combining abilities (SCA) for resistance to prehar‐vest aflatoxin accumulation in grain, utilizing A619 Lfy syn, A632 Lfy syn, B73 Lfy syn, Hy Lfy syn, Mol7 Lfy syn, Wf9 Lfy syn, and 914 Lfy syn. We evaluated 21 F1 crosses (diallel) for aflatoxin accumulation in grain in three environments. Twenty‐one days after mid‐silk, ears were slash‐inoculated with Aspergillus parasiticus (Speare). Samples were analyzed for aflatoxins Bl B2, G1 and G2. Significant differences for aflatoxin accumulation were detected among environments, with the environment that experienced drought stress showing the highest concentration for all four aflatoxins. The GCA and crosses mean squares were not significant for any aflatoxin and the SCA mean square was significant (P = 0.1) only for aflatoxin G2. Relatively small quantities of aflatoxin accumulation on, and nonsignificant differences among the 21 F1 crosses might be indicative of their inherently high resistance levels. The SCA sums of squares constituted two thirds or more of crosses sums of squares, which indicated a preponderance of dominance and/or epistatic effects for aflatoxin accumulation. The Hy Lfy syn GCA effect tended to increase aflatoxin Bl and Wf9 Lfy syn tended to reduce aflatoxin accumulation as it showed a significant (P = 0.1), negative GCA effect for aflatoxin G1. The F1 cross Wf9 Lfy syn × 914 Lfysyn showed significant, negative SCA effect for af‐latoxins B1 and G1, whereas A619 Lfy syn × 914 Lfy syn and Mo17 Lfy syn × Wf9 Lfy syn tended to increase one or more aflatoxins. For aflatoxin Br, a significant environments (E) × crosses (C) interaction was largely due to E × GCA interaction, which, in turn, was due to an interaction of the B73 Lfy syn GCA effect with E. This interaction effect tended to reduce aflatoxin BS. The E × C interaction for aflatoxin G2 was due to both E × GCA and E × SCA interactions. B73 Lfy syn GCA × E tended to reduce aflatoxin G, and Mo17 Lfy syn GCA × E tended to increase aflatoxin G. Additive genetic correlations based on GCA effects among the four aflatoxins were significant (0.76 to 0.96), except the correlation between aflatoxins B, and G2 (0.43), suggesting that, in general, increasing resistance to one toxin may lead to resistance to the other three toxins.