Location: National Peanut Research Laboratory2016 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.
A method for fast screening (one week) of aflatoxin resistance in peanuts was developed. About 150 peanut breeding lines went through the process of transformation with ribonucleic acid interference (RNAi), a biological process, in which RNA molecules inhibit gene expression. Such transformation was aimed at the potential silencing of fungal genes responsible for aflatoxin production. This method was used to determine the effective RNAi-mediated reduction of aflatoxin accumulation in peanut seeds, and the method was published. Real time polymerase chain reaction (RT-PCR) was used and fragments of the RNAi transcript were detected in RNAi peanut plants. An RNAi molecular construct is currently being used at Kenyatta University, Kenya, by our collaborators to transform 3 local peanut cultivars used in Sub-Saharan Africa. In addition, a total of 22 Aspergillus genes showed significant either up or down-regulation when exposed to peanut phytoalexins. A manuscript will be soon submitted for publication. Four new phytoalexins were isolated from fungus-challenged peanut seeds. The structures of the new compounds were elucidated using modern instrumentation. Study of antifungal properties of the new compounds is in progress. Important Aspergillus species with known genome were fed with eight major stilbene phytoalexins to determine their ability to detoxify the phytoalexins. At the same time, the toxigenic potential of the selected fungi was estimated. Enzymatic degradation of major phytoalexins (the arachidins 1 and 3, chiricanine A, resveratrol, SB-1) was studied systematically. While successfully degrading the phytoalexins, at the same time, fungi from this section did not produce aflatoxins in dose-dependent experiments. In addition, in similar experiments, 16 biologically active compounds were tested to study their potential suppression of aflatoxin production in 4 important Aspergillus spp. Elucidation of the phytoalexin detoxification system involved in infection of peanut by important Aspergillus spp. could provide strategies for preventing plant invasion by the fungi that produce aflatoxins.
1. New tool for improving strains of Aspergillus. A method for fast screening (one week) of aflatoxin resistance in peanuts was developed. This method was used to detect the effective RNAi-mediated reduction of aflatoxin accumulation in peanut seeds, and the method was published. RT-PCR was used and fragments of the RNAi transcript were detected in RNAi peanut plants. An RNAi molecular construct is currently being used at Kenyatta University, Kenya, by our collaborators to transform 3 local peanut cultivars used in Sub-Saharan Africa.
2. New tool for determination of the ability of fungi to detoxify peanut defensive phytoalexins. Plants accumulate defensive phytoalexins in response to the presence of pathogens, which in turn produce phytoalexin-detoxification enzymes for successfully invading the plant host. ARS scientists at Dawson, Georgia demonstrated in feeding and in vivo experiments that in parallel with this process major phytoalexins are capable of significantly reducing, or completely blocking aflatoxin production by toxigenic Aspergillus spp. This discovery may lead to new genetic approaches to control aflatoxin production in toxigenic fungi.
1. Participated as Co-PI in obtaining the grant “Silencing of aflatoxin synthesis through RNA interference (RNAi) in peanut plants” from Norman Borlaug Commemorative Research Initiative (“Feed the Future”), USAID (Kenya). The money was granted ($450,000). 2. Participated as Co-PI in obtaining the grant “Genetic diversity of aflatoxigenic Aspergillus species” from Peanut Mycotoxin Innovation Laboratory, USAID (Uganda, Zambia, Ethiopia Kenya, Malawi). The money was granted ($500,000). The goal of this project is to generate a genomic database that will allow scientists to design RNAi strategies to control aflatoxins for each specific geographical region. An undergraduate and a PostDoc, both minority females were hired under this grant. A Ph.D. from Ethiopia has received a 9-month of training at the NPRL on aflatoxin determinations and genetic diversity of Aspergillus. 3. Continued the Cooperative Agreement with Albany State University, an 1890's historically black institution, through which students participate in research while receiving training at the National Peanut Research Laboratory.
Horn, B.W., Gell, R.M., Singh, R., Sorensen, R.B., Carbone, I. 2016. Sexual reproduction in Aspergillus flavus sclerotia: acquisition of novel alleles from soil populations and uniparental mitochondrial inheritance. PLoS One. 11(1): e0146169.
Sobolev, V., Krausert, N.M., Gloer, J.B. 2016. New monomeric stilbenoids from peanut (Arachis hypogaea) seeds challenged by an Aspergillus flavus strain. Journal of Agricultural and Food Chemistry. 64:579-584.