Location: National Peanut Research Laboratory
2023 Annual Report
Objectives
Objective 1. Characterize peanut pathogens, host responses, and host-plant interactions, including diversity of plant invasion and plant health genes, and use genomic and transcriptomic knowledge for discovery and development of novel methods or technologies to control diseases.
Objective 2. Identify, characterize, and evaluate peanut genes involved in disease resistance and drought tolerance, including discovery and elucidation of agriculturally-relevant candidate genes, and work with breeders to facilitate implementation into breeding programs.
Objective 3: Conduct research to develop and assay novel high-throughput pre-harvest aflatoxin resistance screening methods, identify genes, and determine their functions and expression, develop molecular markers with mapping populations, and work with breeders to develop aflatoxin-resistant peanut varieties. (NP 301, C1, PS1A and PS1B).
Objective 4. Determine the physiological mechanisms that link Aspergillus infection with aflatoxin contamination (NP 303, C2, PS 2A).
Objective 5. Understanding the pathway in aspergillus invasion (collaborative effort with FVSU utilizing their scanning electron microscope abilities to understand changes in hull structure under varying environmental conditions), determine impact of Laccase enzymes on hull degradation (NP 303, C2, PS 2A).
Objective 6. Work with breeders to develop varieties with resistance to aflatoxin (NP 301 C1, PS 1A).
Approach
Double strand RNA (dsRNA) that targets aflatoxin synthesis can be used as a therapeutic control of mycotoxins in peanut without genetic transformation. Knowing the genetic makeup of peanut pathogens (Cercospora arachidicola, Cercosporidium personatum, Thecaphora frezzi, Aspergillus (A.) niger, A. flavus and A. parasiticus) allows for a better disease management and longer effectiveness of control. Identification and validation of molecular markers associated with biotic (early and late leaf spot, peanut smut, crown rot disease, mycotoxin producing fungi), and abiotic (drought) stress resistance in wild peanuts and land races will accelerate breeding programs. Analysis and non-GMO manipulation of gene expression, physiology, microRNA expression, and changes in methylation patterns, both of the plant seed as of Aspergillus during the process of infection, can point to resistance genes or other desirable seed response relevant to the control of aflatoxins. Aspergillus Laccase enzymes are a potentially important factor in pathogenicity, studying the activity of these enzymes on hull degradation/penetration in relation to the varying composition of hulls depending on cultivar and maturity, can lead to develop tools to interfere/block the action of laccases and hence seed invasion by Aspergillus, this work will incorporate the assistance of Scientists in our Unit who have expertise in mechanical resistance of hulls. Collaborating with other research projects in our Unit, we will provide the infrastructure support for large scale screening of germplasm to be incorporated in our pre-breeding program.
Progress Report
Unmanned aerial vehicle (UAV)-based hyperspectral image analysis during drought treatment was conducted at the NPRL. High drought tolerance (PI502120, AU-NPL 17, Line 8), moderate drought tolerance (C76-16, TifRunner, and x587), and drought sensitive lines ( AP-3, Ga-Green, and AT3085RO) were used. Three replicates of parent cultivars and F1 descendants were planted in environmental controlled shelter, subjected to drought stress, and UAV images collected at 14, 18, and 29 days after the start of the drought (DAD) treatment. Photosynthesis and stomatal conductance were measured at midday using four LI-6400 systems. Biomass, pod count and pod yield were measured after harvest.
To analyze the physiology peanut genotypes during recovery from drought stress, PI502120, AU-NPL 17, Ga-Green, AP-3, x587, C76-16, AT3085RO, Line 8, and TifRunner were subjected to middle-season drought treatments and re-irrigation. Parameters such as: harvest index, total chlorophyll, gas exchange, and chlorophyll a fluorescence were measured. Pod count and inshell pod weight were evaluated to estimate yield. Leaves from the main stem of three individual plants of each peanut genotype were collected from irrigated controls and drought treatment plots 14 DAD, time when physiological changes among peanut genotypes are expected. Physiological parameter Data are being analyzed, and RNA extractions are in progress to be followed by analysis of gene expression.
A total of 77 hybrid seeds were obtained from 22 crosses in 2022. AU-NPL 17, was used as source of leaf spot resistance and drought tolerance. AT3085RO was also used as source of leaf spot resistance, and lines AU16-28, Line-8, and PI 502120 as sources of drought tolerance were crossed with advanced breeding lines to produce hybrid seeds. AU-NPL 17 x AU16-28 was designed for pyramiding both traits. Hybrid seeds were grown in a winter nursery in Puerto Rico in 2022 and F2 populations have been planted in Headland Experiment Station, AL, to develop recombinant inbred lines (RILs) for further gene discovery.
Few adjustments have been made to our single-seed analysis of aflatoxins on Aspergillus challenged peanut seeds. Time between peanut slicing and inoculation has been shortened, and number of fungal spores reduced when testing wild peanut species that typically have small seeds. Since Aspergillus can frequently harbor genetically diverse nuclei; we sequenced the genomes of five single spore isolates and tested their aflatoxin production in vitro. Based on de novo genome assembly and aflatoxin production, one isolate was selected for its use in challenging experiments.
Breeding peanut for long-lasting resistance to pathogens, requires understanding the genetic makeup and genetic diversity of the pathogens. We have sequenced and published the genomes of the causal agent of peanut smut (Thecaphora frezii), and the causal agent of peanut late leaf spot (Cercosporidium personatum). We are currently analyzing the genetics of morphological variants of C. personatum.
Preliminary work toward the use CRISPR/Cas9 technology has been initiated in collaboration with NP303 at the NPRL. Seeds of 134 accessions from the peanut collection in Griffin, Georgia, are being increased in greenhouse to avoid unwanted cross pollination and to produce genetically homogeneous material for further research. Molecular constructs targeting the phytoene desaturase gene (within the carotenoid pathway) are being prepared to test phenotype changes when changing a single nucleotide in one of the four genomes of peanut.
Accomplishments
1. Identified two strategies of drought tolerance. Defining peanut drought tolerance mechanism(s). Among the eight drought-tolerant peanut cultivars identified in 2022, two different physiological mechanisms were observed. One being minimizing water loss (water saver), and the other being continuation of normal physiology despite drought conditions (water spender). Both strategies observed were associated with high yield. Peanut cultivars Line-8 and AU16-28 were identified as “water savers” due to their early reduction in gas exchange and reduction of photosynthesis rate to minimize water loss during drought stress. Peanut cultivars PI 502120 and AU-NPL 17 were classified as “water spenders” since they continued having high rates of gas exchange and photosynthesis during drought, probably due to acclimation or capacity to extract more water from soil. Both mechanisms are amenable to maintain high yield under limited drought stress observed in the Southeast U.S. This information will assist peanut breeders to develop and select drought tolerant lines with high yield based on physiological measurements and yield quality characteristics.
2. Published the genomes of two destructive peanut pathogens. Two destructive fungal diseases of peanut are late leaf spot and peanut smut, each can cause more than 50% yield losses. Working with these fungi in the laboratory has proven extremely difficult, however, we were able to sequence their genomes and transcriptomes. The data are now available to the public and will be essential to understand their pathogenicity and genetic diversity.
Review Publications
Arias De Ares, R.S., Dobbs, J., Stewart, J., Cantonwine, E., Orner, V.A., Sobolev, V., Lamb, M.C., Massa, A.N. 2023. First draft genome and transcriptome of Cercosporidium personatum, causal agent of late leaf spot disease of peanut. BMC Research Notes. 16:58. https://doi.org/10.1186/s13104-023-06331-0.
Arias De Ares, R.S., Conforto, C., Orner, V.A., Carloni, E.J., Soave, J., Massa, A.N., Lamb, M.C., Bernardi-Lima, N., Rago, A. 2023. First draft genome of Thecaphora frezii, causal agent of peanut smut disease. BMC Genomics. 24:9. https://doi.org/10.1186/s12863-023-01113-w.
Rosso, M.H., De Blas, F.J., Massa, A.N., Oddino, C., Giordano, D.F., Arias De Ares, R.S., Soave, J.H., Soave, S.J., Butteler, M.I., Bressano, M. 2023. Two QTLs govern the resistance to Sclerotinia minor in an interspecific peanut RIL population. Crop Science. 63:613-621. https://doi.org/10.1002/csc2.20875.
Xu, W., Chen, C., Dang, P.M., Carter, J., Zhao, S., Lamb, M.C., Chu, Y., Holbrook Jr, C.C., Ozias-Akins, P., Isleib, T., Feng, Y. 2022. Variabilities in symbiotic nitrogen fixation and carbon isotope discrimination among peanut (Arachis hypogaea L.) genotypes under drought stress. Journal of Agronomy and Crop Science. 1–14. https://doi.org/10.1111/jac.12619.
Zhang, Q., Dang, P.M., Chen, C., Feng, Y., Batchelor, W., Lamb, M.C., Sanz-Saez, A. 2022. Tolerance to mid-season drought in peanut can be achieved by high water use efficiency or high efficient use of water. Crop Science. https://doi.org/10.1002/csc2.20806.
Zhen, X., Zhang, Q., Sanz-Saez, A., Chen, C., Dang, P.M., Batchelor, W. 2022. Simulating drought tolerance of peanut varieties by maintaining photosynthesis under water deficit. Field Crops Research. 287. Article 108650. https://doi.org/10.1016/j.fcr.2022.108650.
