Location: Northwest Irrigation and Soils Research
2024 Annual Report
Objectives
Objective 1: Identify traits with the potential to increase productivity and sustainability of sugar beet while reducing economic losses due to beet curly top, Rhizoctonia root rot, rhizomania, postharvest storage losses, Cercospora leaf spot, and frost.
Sub-objective 1.A: Develop elite germplasm with novel sources of resistance to BCTV, C. beticola, R. solani, BNYVV, and storage rots.
Sub-objective 1.B: Develop sugar beet lines with freezing/frost protection.
Objective 2: Develop new sugar beet genetic stocks and breeding lines capable of resolving the genetic determinants of agriculturally important traits, and provide the material and markers to breeders for efficient trait utilization.
Sub-objective 2.A: Generate experimental populations for gene discovery.
Sub-objective 2.B: Generate new and novel agronomically important traits using DNA mutagenesis.
Objective 3: Create in-depth genomic resources that are trait-focused and apply molecular genetics methods to decipher the molecular mechanisms, genes, and gene products influencing important phenotypic variation.
Sub-objective 3.A: Delineate molecular mechanisms related to diverse traits in sugar beet genotypes exhibiting CLS and rhizomania resistance and the non-bolting phenotype.
Sub-objective 3.B: Evaluate the role of sugar beet host-associated bacterial and fungal microbiome in resistance against beet curly top, rhizomania, and post-harvest storage quality.
Sub-objective 3.C: Investigate the interaction between different sugar beet germplasm sources and strains of BCTV found in sugar beet to identify different sources of resistance and how their gene expression and molecular resistance mechanisms may differ.
Sub-objective 3.D: Develop additional management strategies for beet curly top and pest control in sugar beet.
Approach
The Sub-objective 1A research goal is to release elite sugar beet germplasm with novel sources of resistance to Beet curly top virus (BCTV), Rhizoctonia solani, Beet necrotic yellow vein virus (BNYVV), Cercospora beticola,and storage rots. To achieve this goal PI lines and breeding populations will be characterized for their resistance level by screening in disease nurseries. Additional screening will be conducted on germplasm not previously investigated, if novel sources are not found initially.
The Sub-objective 1B research goal is to release elite sugar beet germplasm with freezing/frost tolerance by using a leaf syringe agroinfiltration system to transiently express recombinant proteins in sugar beet leaves. Tobacco or Arabidopsis could be utilized to characterize these proteins, if sugar beet is uncooperative.
The Sub-objective 2A research goal is to discover novel disease resistance genes by screening progeny of F2 derived, synthetic, and recurrent selection populations, and F3 families in disease nurseries. If extreme weather events are encountered, enough seed will always be saved to allow for additional plantings.
The Sub-objective 2B research goal will be to generate new agronomically important traits (such as frost tolerance, non-bolting, etc.) through DNA mutagenesis and link them to specific loci in the sugar beet genome. If no beneficial mutations are identified, the M2 and M3 lines can still be useful for discovery of important genes through loss of function.
Sub-objective 3A will test the hypothesis that mutation-induced changes in gene expression and protein/metabolite production in the sugar beet lines will positively affect pathogen resistance and negatively affect bolting. Genomic analysis of sugar beet EMS mutant lines when combined with RNAseq, proteomics, and metabolite analysis will provide genomic markers associated with the trait of interest. If something unexpected happens with the experiments, we have sufficient seed to replicate the experiments.
Sub-objective 3B will test the hypothesis that host plant specific microbiome can positively affect disease resistance. Sugar beet leaf microbiome changes based on 16S and ITS sequencing in response to BCTV, BNYVV, and post-harvest storage will be investigated in resistant and susceptible lines. If something unexpected happens with the experiments, we have sufficient seed to replicate the experiments.
Sub-objective 3C will test the hypothesis that sugar beet cultivars/lines with different sources of resistance will vary in their response to BCTV strains. BCTV clones will be infiltrated into sugar beet leaves to evaluate their responses in lines previously shown to exhibit differential responses to BCTV strains. If the leaf-infiltration clones prove to be unstable, stab-inoculation clones can be used.
Sub-objective 3D will test the hypothesis that seed and foliar insecticide treatments can be used to supplement or extend the control of BCTV and pests beyond that provided by neonicotinoid seed treatments and host resistance by screening in field plots. If extreme weather events are encountered, enough seed will always be saved to allow for additional plantings.
Progress Report
This report documents the progress for project 2054-21220-006-000D, "Decipher Molecular Mechanisms for Genetic Variations in Agronomically Important Traits to Improve Sugar Beet Disease Resistance and Yield", which started January 2023.
In support of Objective 1, 804 commercial plots with sugar beet plants are being screened in the field for resistance to rhizomania (588 plots), rhizoctonia crown and root rot (126 plots), and beet curly top (90 plots) to support the sugar beet industry in identifying their most resistant and highest yielding cultivars. In addition, 1,188 genetic plots with sugar beet Plant Introduction lines or breeding lines are being screened in the field for resistance to rhizomania (396 plots), rhizoctonia crown and root rot (300 plots), and beet curly top (492 plots) to support the efforts of geneticists in developing breeding lines with novel superior resistance which can be utilized to improve commercial sugar beet cultivars.
For Objective 2, ARS researchers in Kimberly, Idaho, continued to improve the Cercospora leaf spot (CLS) resistance in USDA breeding populations. The sugar beet germplasm KEMS06 (PI 683514), derived from an ethyl methanesulfonate (EMS) mutagenized population, was found to have high CLS resistance and may contain genetic underpinnings that have not been leveraged before in commercial sugar beet cultivars. To date, KEMS06 and hybrid populations derived from the KEMS06 breeding line have been tested in the field for two years and have shown CLS resistance equal to or better than the resistant checks. Single nucleotide polymorphism (SNP) analysis and genetic linkages to the resistance traits of the populations will be conducted later this year. Heritability of the KEMS06 CLS disease resistant trait was tractable into the F2, F3 and F4 filial generations. By following a single plant decent methodology using parental lines with self-fertility traits, complete introgression of the KEMS06 CLS disease resistance trait was observed in the progeny families. Select progeny lines from this study were submitted into the 2024 CLS disease field nursery research plots and showed robust CLS disease resistance in the field. Researchers identified a second sugar beet germplasm from EMS mutagenized population, KEMS08 (PI 683816) that has high CLS resistance in field tests. Hybrid populations of KEMS08 have been created, and the heritability of the KEMS08 CLS resistant trait will be tracked following the methodology used with the KEMS06 germplasm. The KEMS08 line also has genetic male sterility segregating in the population. The is an extremely valuable trait for sugar beet breeding because it allows researchers to generate large quantities of F1 hybrid seed derived from KEMS08 crosses. This is currently being leveraged to determine the dominant/recessive nature of the CLS resistance trait and the compatibility with commercial seed production for our stakeholders when released.
ARS researchers are also continuing to improve beet curly top virus (BCTV) and Rhizoctonia root rot resistance in the USDA sugar beet breeding germplasm. Individual lines from C762-17 (PI 560130) and EL57 (PI 663212) sugar beet germplasm populations that had high resistance to these diseases in last year’s field trials are currently being bulked up for larger scale field trials. Promising lines will be used in generating hybrid crosses to track the heritability of these disease resistance traits in the progeny. Genetic experiments to identify unique and tractable SNPs for use in breeding are currently underway for these lines.
Supporting Objective 3, ARS researchers in Kimberly, Idaho, used comprehensive multi-omics approaches including degradome analysis of sugar beet genes targeted by BCTV derived small non-coding RNAs, transcriptome, and metabolome analyses to understand sugar beet responses during resistance and susceptible interactions. Based on these data, putative molecular mechanisms involved in sugar beet BCTV resistance have been identified. Some of the metabolites identified in the multi-omics approaches, such as jasmonic acid and methyl jasmonate, when used as foliar application in the field increased sugar beet yield and extractable sugar by 30% versus a nontreated commercial cultivar that is moderately susceptible to BCTV. Other metabolites such as ursolic acid are currently being evaluated as a foliar application to protect against BCTV under field conditions. The BCTV genes and other DNA elements identified in the multi-omics approaches have been targeted using clustered regularly interspaced short palindromic repeats (CRISPR) inhibition (CRISPRi). ARS researchers in Kimberly, Idaho, have optimized stable transformation of sugar beets using CRISPRi vectors targeting genes of BCTV and other sugar beet pathogens. Further characterization of the transformants and production of next generation transgenic sugar beet seeds are currently underway.
Graded-pool sequencing of a segregating population demonstrating different levels of resistance are currently being analyzed to identify markers associated with BCTV resistance. A putative single dominant BCTV resistance related gene from dry bean (unlike the recessive multigenic resistance in sugar beet) has been cloned and used to transform Arabidopsis. Production of T2 seeds from transformed Arabidopsis lines is currently underway. Once available, T2 Arabidopsis transgenic lines will be tested for BCTV resistance.
Rhizomania resistance mechanisms were investigated in EMS mutant rhizomania resistant and susceptible sugar beet breeding lines under field conditions. Using whole genome sequencing, global gene expression, and untargeted metabolome analyses, putative candidate genes (such as Bevul.3G055200.1, Bevul.9G034600.3) along with metabolic alterations (e.g., amino acid metabolism) potentially contributing to resistance have been identified. This manuscript is being written for submission this year. Graded-pool sequencing of a segregating population demonstrating resistance or susceptibility is also being analyzed to identify markers associated with rhizomania resistance.
ARS researchers investigated the role of sugar beet root microbiome and metabolites in improving post-harvest storage quality of sugar beet roots. Post-harvest storage experiments were performed using sugar beet breeding lines developed by researchers in Kimberly, Idaho, which are resistant to indoor storage pathogens. The results indicate higher abundance of bacterial phyla, Patescibacteria, and lower abundance of Firmicutes and Desulfobacteria in the resistant lines. The susceptible line had lower abundance of fungal phyla, Ascomycota and Basidiomycota (including Athelia). Metabolites associated with amino acid and butanoate metabolism were higher in the roots of resistant lines. Further correlation analysis between metabolite and microbiome suggests that root specific metabolites might control relative abundance of beneficial microbes protecting against storage pathogens. These results will have implication in future breeding strategies to improve storability of sugar beet roots.
ARS researchers in Kimberly, Idaho, and a university collaborator used the beet leafhopper genome to understand beet leafhopper responses and identify candidate genes putatively involved in virus acquisition and in the detoxification process when exposed to pesticides. Specific beet leafhopper candidate genes identified from this work are being targeted using CRISPRi technology to improve BCTV resistance in sugar beets. The RNAseq and proteome research in response to temperature stresses and developmental stages are being finalized for a peer reviewed publication.
Atmospheric cold plasma application to sugar beet roots for improving post-harvest storage quality was evaluated for a second year. The experiments were conducted using two additional plasma doses on two commercial sugar beet cultivars that are susceptible to storage related diseases. The overall trend of plasma reducing diseases and improving biomass retention were similar to the first year. A 29-54% reduction in disease incidence and 16-25% increase in root biomass retention were observed in plasma-treated sugar beet roots after five months in a commercial storage facility. Microbiome analysis of plasma-treated samples showed a decrease in pathobiome and specific bacterial and fungal genera being sensitive to plasma treatments. Additional research is planned to further investigate and optimize this technology.
DNA barcoding was used to investigate the plants that beet leafhoppers feed on during two growing seasons. The first generation of beet leafhoppers in the spring predominantly fed on pine and dispersed from mountainous areas to crop and sagebrush steppe locations. During July to September, the leafhoppers predominantly fed on Russian thistle and Kochia. Both years the beet leafhoppers that fed on pine had the highest percentage of samples with beet curly top virus. Leafhopper that had fed on Russian thistle and alfalfa had the highest percentage of samples with Spinach curly top Arizonia virus. ARS researchers in Kimberly, Idaho, also found that beet leafhoppers begin dispersing after approximately 130 growing degree days (GDDs) and peak dispersal occurs around 382 GDDs. Based on GDDs and dispersal numbers under optimal conditions, the beet leafhoppers associated with large peaks in May in southern Idaho appear to have originated outside the local area and likely from outside the state.
Accomplishments
1. Beet leafhopper feeding on pine suggests long distance dispersal into crop fields into sugar beet fields. Beet leafhoppers (BLH) carry the beet curly top virus (BCTV) which is a serious yield limiting disease on sugar beet and numerous other crops. Beet leafhopper population dispersal and feeding in southern Idaho were tracked by ARS researchers in Kimberly, Idaho, during the 2020 and 2021 growing seasons. Growing degree day data, feeding, and population numbers suggest that leafhoppers originated outside the study area and likely from outside the state in May and early June. The first generation of BLHs with detectable plant DNA that arrived in crop areas in late June had primarily fed on pine and likely originated in Idaho’s mountains, while later in the season they primarily fed on Russian thistle and Kochia. Leafhoppers that had fed on pine also had the highest percentage of BCTV strains found commonly in the Pacific Northwest. These data establish the information needed for developing forecast models and new management options as well as establish the BCTV strains that need to be emphasized when screening sugar beet and other crops for BCTV resistance.
2. Novel source of Cercospora leaf spot resistance discovered in sugar beet breeding lines. Cercospora leaf spot (CLS) disease is one of the most destructive diseases of sugar beet. Disease prone areas require numerous fungicide applications during the season to keep CLS to a minimum. Recently, the sugar beet germplasm KEMS06 (PI 683514) derived from a mutagenized population was found to have novel CLS disease resistance. The resistant trait was associated with multiple genes which were bred into double-haploid breeding lines. These lines will be released to the public to allow seed companies to improve CLS resistance in the commercial cultivars available to sugar beet growers thereby reducing the number of fungicide applications and improving sugar yield.
3. The first complete beet leafhopper genome sequence was released to the general public. The beet leafhopper carries plant pathogens such as beet curly top virus (BCTV) and phytoplasmas that infect numerous crops. The lack of genomic and transcriptomic resources for the beet leafhopper limits our understanding of the molecular interactions with pathogens. ARS researchers in Kimberly, Idaho, published the first complete genome for the beet leafhopper and released it to the public via the National Center for Biotechnology Information (NCBI). A recent collaboration utilizing the genome revealed the effects of BCTV on leafhopper biological processes. These results revealed the key molecular players in both virus manipulation of vector biology and the vector’s defensive strategies resulting from their long-term association. These findings are being used to discover novel gene targets for functional genomics studies, devise strategies to control the leafhopper populations, and ultimately block the spread of BCTV.
Review Publications
Strausbaugh, C.A., Wenninger, E.J., Jackson, L.K., Vincill, E.D. 2024. Curly top viruses and phytoplasmas in sugar beets, common beans, and beet leafhoppers along with vector population dynamics in southern Idaho. PhytoFrontiers. https://doi.org/10.1094/PHYTOFR-08-23-0115-R.
Strausbaugh, C.A. 2024. Commercial sugar beet cultivars evaluated for rhizomania resistance and storability in Idaho, 2022. Plant Disease Management Reports. 18. Article V012.
Strausbaugh, C.A. 2024. Experimental sugar beet cultivars evaluated for rhizomania resistance and storability in Idaho, 2022. Plant Disease Management Reports. 18. Article V013.
Vincill, E.D., Majumdar, R., Strausbaugh, C.A. 2024. Kimberly sugar beet germplasm evaluated for Rhizoctonia crown and root rot resistance in Idaho, 2023. Plant Disease Management Reports. 18. Article V009.
Strausbaugh, C.A., Majumdar, R., Wenninger, E.J. 2024. Foliar and seed treatment insecticides for the control of beet curly top in Idaho sugar beet, 2023. Plant Disease Management Reports. 18. Article V008.
Galewski, P.J., Majumdar, R., Lebar, M.D., Strausbaugh, C.A., Eujayl, I.A. 2023. Combined omics approaches reveal distinct mechanisms of resistance and/or susceptibility in sugar beet double haploid genotypes at early stages of beet curly top virus infection. International Journal of Molecular Sciences. 24(19). Article 15013. https://doi.org/10.3390/ijms241915013.
