Location: National Peanut Research Laboratory2017 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.
This is the final progress report for this project which has been replace by project 6044-42000-011-00D, "Integrated Management of Fungal Pathogens in Peanut to Reduce Mycotoxin Contamination and Yield Losses." Aspergillus (A.) flavus and A. parasiticus that are responsible for aflatoxin contamination of agricultural commodities, were crossed to obtain rare hybrid progeny, which showed extensive recombination throughout the genome. Some of the progeny exhibited enhanced aflatoxin production as well as unique mycotoxin profiles. The research was published. The genetic stability of non-toxigenic strains of A. flavus used in biological control of aflatoxins was examined. Biocontrol strains were crossed with aflatoxin-producing strains. Non-toxigenic strains had the potential to mate with aflatoxin-producing strains, but did not pose a risk for creating high aflatoxin-producing strains. The research was published. In the drought experiments with corn, A. flavus sclerotia were detected in 0.6% of corn ears with no evidence of sexual reproduction, while under laboratory conditions sexual reproduction occurred in up to 6.1% of the sclerotia. For sexual reproduction to occur in the field, A. flavus sclerotia require an additional incubation period on soil. The research was published. Six new phytoalexins were isolated from fungus-challenged seeds of a peanut cultivar that is highly resistant to microbial and nematode infection. The structures of the new compounds were elucidated, and the compounds were investigated for antifungal and antibacterial properties. The results were published. Enzymatic degradation of major phytoalexins was studied systematically. Elucidation of the phytoalexin detoxification system involved in infection of peanut by Aspergillus species (spp.) could provide strategies for preventing plant invasion by the fungi that produce aflatoxins. Two manuscripts were published. Aspergillus showed reduction in aflatoxin accumulation in presence of peanut phytoalexins. For three of those phytoalexins we studied their effect on spore germination, hyphal growth, and transcriptomes. A manuscript is in preparation. In collaboration with other projects, we developed a workflow of populations of Aspergillus in peanuts in different geographic areas; aflatoxin production of several hundreds of isolates was determined for the phenotypic and genotypic characterization of isolates from Georgia and Ethiopia. A manuscript will be published. After the term date of the reporting project, significant progress by a newly appointed ARS Research geneticist was made within a new project: “Integrated Management of Fungal Pathogens in Peanut to Reduce Mycotoxin Contamination and Yield Losses”. Diploid Arachis germplasm collections were mined for resistance to various pathogens; 400 prospective wild germplasm and introgression lines were selected; 250 accessions were grown at research farms (800 plots) and evaluated for resistance to peanut late leaf spot and Tomato Spotted Wilt Virus diseases; 30 accessions were evaluated for resistance to A. flavus and aflatoxin accumulation; 380 Simple Sequence Repeat and Insertion/Deletion markers were developed and used for fingerprinting 18 wild Arachis accessions. We also developed an original workflow that allows for simultaneous determination of single seed viability, genotype, and quantitative phytoalexin and aflatoxin accumulation profiles. The method allowed for the study of genotypic/phenotypic characteristics of wild peanut species and their crosses with cultivars.
1. Development of a method for screening of aflatoxin resistant peanuts. The method for fast screening (one week) of aflatoxin resistance in peanuts developed by ARS researchers in Dawson, Georgia, is being adopted by the peanut community around the world as a method of choice to determine the effectiveness of Ribonucleic Acid Interference (RNAi) mediated control of aflatoxins. We are also using this method to screen wild species of peanut in the search for aflatoxin resistance genes.
2. Discovery of aflatoxin accumulation suppression in Aspergillus species by peanut phytoalexins. Prenylated peanut stilbenoids (phytoalexins) are capable of suppressing aflatoxin production in toxigenic Aspergillus species. Peanuts accumulate numerous defensive phytoalexins in response to the presence of pathogens. ARS researchers in Dawson, Georgia, demonstrated in vivo experiments that different peanut stilbenoid phytoalexins are capable of completely blocking or significantly reducing aflatoxin production by important toxigenic Aspergillus Species (spp). This discovery led to new genetic approaches to control aflatoxin production in toxigenic fungi.
Clevenger, J., Marasigan, K., Liakos, B., Sobolev, V., Vellidis, G., Holbrook, C.C., Ozias-Akins, P. 2016. RNA sequencing of contaminated seeds reveals the state of the seed permissive for pre-harvest aflatoxin contamination and points to a potential susceptibility factor. Toxins. 8:317. https://doi.org/10.3390/toxins8110317.
Lamb, M.C., Sorensen, R.B., Butts, C.L., Nuti, R., Davis, J.P., Dang, P.M., Arias De Ares, R.S., Sobolev, V. 2017. Chemical interruption of flowering to improve harvested peanut maturity. Peanut Science. 44(1):60-65.
Power, I.L., Dang, P.M., Sobolev, V., Orner, V.A., Powell Jr, J.L., Lamb, M.C., Arias De Ares, R.S. 2017. Characterization of small RNA populations in non-transgenic and aflatoxin-reducing-transformed peanut. Plant Science. 257:106-125. doi:10.1016/j.plantsci.2016.12.013.