Location: National Peanut Research Laboratory2014 Annual Report
1. Determine the population dynamics of sexually reproducing Aspergillus flavus under field conditions. 2. Identify genes in Aspergillus flavus responsible for virulence during the infection process and elucidate the role of fungal gene products for overcoming peanut resistance mechanisms. 3. Determine the role of defensive peanut phytoalexins in mediating natural crop resistance against Aspergillus flavus.
Sclerotia of A. flavus will be collected from corn grown in a randomized complete block design consisting of four overhead irrigation treatments to provide different degrees of drought stress. Experiments involving natural field populations of A. flavus will be conducted during years 1 and 2. The same procedure will be repeated for years 3 and 4, except that corn ears will be sprayed with a conidial suspension of a non-toxigenic biocontrol strain (NRRL 21882 [from Afla-Guard® ] or AF36). Sclerotia will incubated on the surface of nonsterile soil (100% relative humidity) for 5-7 months. Ascospores from fertile sclerotia will be germinated to obtain progeny strains. To detect genetic recombination, total genomic DNA will be isolated from progeny strains. Recombination events due to independent assortment of chromosomes and crossing over will be detected by multilocus sequence typing (MLST) and linkage disequilibrium/compatibility analyses. Genes encoding putative phytoalexin-detoxification enzymes (PDEs) will be cloned from pathogenic A. flavus strains. PDE production by A. flavus will be induced in culture by the presence of purified peanut phytoalexins or peanut seeds. cDNA libraries will be generated and used as templates to amplify candidate genes by Polymerase Chain Reaction (PCR). Native in vitro-expressed proteins will be purified and their activity will be tested against a variety of purified peanut phytoalexins. Liquid chromatographic-tandem mass spectrometric (LC-MS) analysis of the phytoalexin samples after exposure to the various purified proteins will be used to detect potential enzymatic modifications of the phytoalexin compounds. Target PDEs will be analyzed from different genotypes of A. flavus and A. parasiticus to assess the genetic variability of these enzymes and thus predict the potential effectiveness of PDE inhibitors. A model system will be developed to screen PDE inhibitors. Pathogenicity tests will be conducted on single peanut seeds inoculated with A. flavus after the application of inhibitory compounds. The bioactivity of phytoalexins will be assayed against economically important plant pathogenic fungi grown on micro-plates. The dynamics of phytoalexin formation will be studied by first determining the most fungal-resistant (high phytoalexin producers) and fungal-susceptible (low phytoalexin producers) peanut genotypes from a core collection of 108 genotypes. Peanut seeds from genotypes will be subjected to different biotic and abiotic elicitors to elucidate changes in the composition of phytoalexins and to detect possible degradation products due to detoxification. The embryos and cotyledons from seeds will be wounded and inoculated with fungi and bacteria, then extracted and analyzed with high performance liquid chromatography (HPLC)/MS. Data obtained from analyses of the core collection of peanut genotypes will be used to identify peanut germplasm with disease resistance. To further examine phytoalexin detoxification (degradation) products, feeding experiments will be conducted in which fungi and bacteria are fed peanut phytoalexins followed by HPLC/MS/Nuclear Magnetic Resonance analysis.
Corn was grown at Shellman, Georgia, for the third and final year of the project. Treatments consisted of different levels of drought stress that included 0% irrigation (dry land cultivation) and 33%, 66% and 100% of the overhead irrigation recommended by Irrigation Pro for Corn software. Aspergillus flavus sclerotia, resistant structures produced by the fungus, were detected in 0.6% of corn ears at harvest and showed no evidence of sexual reproduction. Sclerotia were then incubated under laboratory conditions on the surface of soil containing natural microbial populations. Sexual reproduction occurred in up to 6.1% of the sclerotia for each year and resulted in the formation of viable sexual spores. Therefore, for sexual reproduction to occur in the field, A. flavus sclerotia require an additional incubation period on soil following dispersal at crop harvest. Sexual reproduction is likely responsible for the high genetic variation in A. flavus populations. The research was published in Phytopathology. Four new putative phytoalexins were isolated from fungus-challenged seeds of Tifguard, a peanut cultivar that is highly resistant to microbial and nematode infection. The structures of the new compounds were elucidated using state-of-the-art instrumentation. Since the new compounds may play an important role in natural protection of the peanut plant, investigation of their antifungal and antibacterial properties is now in progress. In addition, two Tifguard varieties that demonstrate a significant difference in nematode resistance were analyzed for nematode-active compounds by comparison of their whole (unfractionated) matrix composition. Analyses at this stage of the research did not reveal any low-molecular compounds responsible for resistance to nematodes. Fractions of the matrices are now being examined for nematode-active compounds.
1. Sexual reproduction in aflatoxin-producing Aspergillus flavus from a cornfield. Aflatoxin produced by the mold A. flavus is a potent carcinogen that contaminates crops worldwide and greatly impacts human health and crop profitability. Sexual reproduction in A. flavus was recently shown to occur in the laboratory but had not been demonstrated in crops. ARS researchers at Dawson, Georgia, in collaboration with scientists at North Carolina State University discovered that A. flavus reproduces sexually after dispersal onto the soil surface at corn harvest. Therefore, the high genetic diversity of A. flavus in fields can be explained by sexual reproduction. Strategies for controlling aflatoxin in crops will need to take sexual reproduction into account, along with the generation of genetically new strains of A. flavus.
Horn, B.W., Sorensen, R.B., Lamb, M.C., Sobolev, V., Olarte, R.A., Worthington, C.J., Carbone, I. 2014. Sexual reproduction in Aspergillus flavus sclerotia naturally produced in corn. Phytopathology. 104(10:75-85.
Moore, G.G., Elliott, J.L., Singh, R., Horn, B.W., Dorner, J.W., Stone, E.A., Chulze, S.N., Barros, G.G., Naik, M.K., Wright, G.C., Hell, K., Carbone, I. 2013. Sexuality generates diversity in the aflatoxin gene cluster: evidence on a global scale. PLoS Pathogens. 9(8):e1003574. doi:10.1371/journal.ppat.1003574.
White, B.L., Oakes, A.J., Shi, X., Price, K.M., Lamb, M.C., Sobolev, V., Sanders, T.H., Davis, J.P. 2013. Development of a Pilot Scale Process to Sequester Aflatoxin and Release Bioactive Peptides from Highly Contaminated Peanut Meal. LWT - Food Science and Technology. 51(2)492-499.