Location: National Peanut Research Laboratory
2020 Annual Report
Objectives
1. Evaluate pathogen-host interactions, including enzyme production by host and pathogen during aflatoxin accumulation, and identify potential resistance genes for aflatoxin control.
2. Mine the diploid Arachis germplasm collections in peanut to identify resistance to various pathogens, characterize novel sources of resistance to important fungal pathogens, and introgress genes into cultivated peanuts.
2a. Screen wild peanut germplasm collection to identify useful germplsm for resistance to important fungal pathogens (e.g. Aspergillus, Cercospora, Cercosporidium, and Sclerotinia spp.).
2b. Transfer economically important genetic traits from wild Arachis species to cultivated peanuts.
3. Develop integrated strategies for management of fungus-associated peanut diseases.
Approach
Mycotoxins are toxic secondary fungal metabolites. Contamination of crops with mycotoxins, particularly aflatoxins, is an important food safety issue and threatens the competitiveness of United States agriculture in the world market. Aflatoxins are strong carcinogens produced in crops by the fungus Aspergillus (A.) flavus. Contamination of crops with aflatoxins is an important food safety issue. The purpose of this project is to develop effective integrated strategies for controlling mycotoxin accumulation and fungal diseases that cause yield losses in peanut. One strategy for reducing aflatoxins is to prevent Aspergillus from invading crops. To achieve this goal, the first objective will evaluate fungus-plant interactions, gene expression and chemical profiling of host and pathogen during aflatoxin accumulation. Another strategy for aflatoxin reduction is to prevent its formation by the fungus. This strategy is based on data from our recent research showing that selected peanut stilbenoids significantly reduce or completely block aflatoxin biosynthesis in A. flavus. The second objective will explore wild Arachis germplasm collections to identify resistance to A. flavus, determine and characterize novel sources of resistance to important fungal pathogens, including species causing early and late leaf spot and white mold diseases, and introgress genes into cultivated peanuts. The third objective, which is related to the first and second objectives, is to combine knowledge and methodology obtained from these objectives on the reduction of aflatoxin in peanut. The ultimate goal of this objective is to establish new peanut germplasm with increased resistance to toxigenic A. flavus.
Progress Report
Substantial progress has been made toward Objective 1, a better understanding of the role of selected highly labile polyphenolic phytoalexins in fungus-host interactions and the inhibition of aflatoxin formation. The second stage of the study was completed by ARS researchers at Dawson, Georgia, and the results are being submitted in a peer-reviewed journal. Significant progress was also made in method development for the quantitative determination of aflatoxins in individual seeds and small-size (up to one-digit-milligram) plant samples. The method is not destructive from a breeding standpoint, fast, accurate, sensitive, and inexpensive. The cost of the suggested minicolumn, the “core” of the method, is 10-15 times less than that of commercial proprietary cleanup columns, which is a substantial saving. Such a method has not been reported in literature. Knowledge on aflatoxin content in individual seeds is crucial for the success of the entire project; thousands of samples were analyzed at a minimum cost. The study was completed, and a manuscript is in preparation to be submitted to a peer reviewed journal.
Significant progress has been made toward Sub-objectives 2a and 2b. One manuscript is under review and another manuscript is in preparation to be submitted to a peer reviewed Journal. Prospective peanut accessions with resistance to early leaf spot (Cercospora arachidicola), late leaf spot (Cercosporidium personatum), and aflatoxin accumulation entered the pre-breeding pipeline developed at the NPRL, and consisting of the development of inter-specific F1 hybrids and amphidiploids, evaluation of pre-harvest aflatoxin resistance under controlled environmental conditions, high-throughput genotyping, transcriptome analysis, trait introgression, and marker development. Transcriptome sequence and analysis of Aspergillus-challenged seeds was completed for another set of ten aflatoxin-resistant and susceptible peanut genotypes. Combined with genome-wide SNP genotyping data, aflatoxin formation, and phytoalexin response, this information is being utilized to advance ongoing trait introgression, genetic studies, marker development, and gene/QTL discovery. Research progress was presented at the International Plant and Animal Genome XXVIII Conference, January 11-15, 2020, San Diego, CA and the 52th Virtual Annual Meeting of the American Peanut Research and Education Society, July 14-16, 2020. In a continued effort to identify new sources of resistance to important peanut pathogens, scientists at Dawson, Georgia, in collaboration with other peanut researchers genotyped and characterized a recombinant inbred line (RIL) population derived from a cross between a peanut smut (Thecaphora frezzi) susceptible Arachis hypogaea elite line and a resistant amphidiploid. A genetic mapping and QTL analysis was conducted in the RIL population to identify the genetic basis of resistance to peanut smut. The study was completed and the results are being submitted to a peer reviewed journal.
Accomplishments
1. Inhibition of aflatoxin formation in toxigenic Aspergillus species and the dynamics of peanut polyphenol production during the peanut-host interaction (2nd year research). Aspergillus flavus, common fungal species, are widely distributed in nature and, under certain conditions, invade preharvest peanut seeds. These fungi often produce highly toxic and carcinogenic aflatoxins that pose a threat to human and animal health and are responsible for the multimillion dollar loses in the peanut industry and private farms. Under favorable conditions, the fungus-challenged peanut seeds produce polyphenols, structurally related stilbenoids, capable of retarding fungal vitality and ability to produce aflatoxins. Understanding and potential manipulation of this mechanism may help to resolve the preharvest aflatoxin contamination problem. The present research revealed the unique properties and role of one unsaturated peanut polyphenol in aflatoxin suppression in mature seeds. At present, the findings on the dynamics of peanut polyphenol production are successfully used by ARS researchers, at Dawson, Georgia for discovery and evaluation of resistant peanut species.
2. Identifying the mechanisms of pre-harvest aflatoxin resistance in peanut. The genetics of resistance to aflatoxin accumulation in peanut seeds is quantitative in nature. Current breeding methods for the development of aflatoxin-resistant peanut cultivars are genetically inefficient due to limited understanding of the molecular mechanisms underlying aflatoxin formation in peanut seeds. Using a novel non-destructive method of screening, developed by ARS researchers, in Dawson, Georgia, aflatoxin-resistant peanut genotypes were identified and validated. This germplasm was further utilized to characterize gene expression changes in the peanut-Aspergillus interaction of susceptible vs. resistant genotypes using RNA sequencing. Gene expression of plant genes and fungal genes were analyzed separately. Phytoalexin biosynthesis genes, including those encoding stilbenes and isoflavonoids, were among the plant genes that differentially responded to Aspergillus infection. Results further indicated that peanuts regulate transcription of the aflatoxin-biosynthesis gene cluster in A. flavus at early stages of the infection. These findings are expected to have immediate application in candidate gene studies, genome association analysis, molecular marker development, pre-breeding/breeding, and assessment of resistant candidates at pre- and post-harvest. This research has led to a funded grant for additional research on the identification of mechanisms of pre-harvest aflatoxin resistance in peanut.
3. Importance of hypotheses in planning and interpreting experiments. The hypotheses on the potential enhancement of the use of induced phytoalexins to prevent fungal invasion in peanut seeds was elaborated by ARS researchers and tested in vivo, in Dawson, Georgia. The hypotheses on the role of healing mechanical wounds was confirmed in the laboratory experiments. In vitro experiments, one of the unsaturated peanut stilbenoids demonstrated the ability for prompt polymerization and formation of a film on the wound. The in vitro part of the study was completed by researchers at Dawson, Georgia, and a manuscript is in preparation to be submitted to a peer reviewed journal.
Review Publications
Arias De Ares, R.S., Cazon, I., Massa, A.N., Scheffler, B.E., Sobolev, V., Lamb, M.C., Duke, M.V., Simpson, S.A., Conforto, C., Paredes, J., Soave, J., Buteler, M., Rago, A.M. 2019. Mitogenome and nuclear-encoded fungicide-target genes of Thecaphora frezii- causal agent of peanut smut. Fungal Genomics and Biology. (9)1:160. https://doi.org/10.35248/2165-8056.19.9.160.
Arias De Ares, R.S., Ballard, L.L., Duke, M.V., Simpson, S.A., Liu, X.F., Orner, V.A., Sobolev, V., Scheffler, B.E., Martinez-Castillo, J. 2020. Development of nuclear microsatellite markers to facilitate germplasm conservation and population genetics studies of five groups of tropical perennial plants with edible fruits and shoots: ranbutan (Nephelium lappaceumt). Genetic Resources and Crop Evolution. https://doi.org/10.1007/s10722-020-00965-w.
Arias De Ares, R.S., Mohammed, A., Orner, V.A., Faustinelli, P.C., Lamb, M.C., Sobolev, V. 2020. Sixteen draft genomes sequences representing the genetic diversity of Aspergillus flavus and Aspergillus parasiticus colonizing peanut seeds in Ethiopia. Microbiology Resource Announcements. 9(30):e00591-20. https://doi.org/10.1128/MRA.00591-20.
