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ARS Home Pacific West Area Kimberly, Idaho Northwest Irrigation and Soils Research Research





- Strausbaugh, Carl
- Majumdar, Raj
- Vincill, Eric
- 2023
- Publications
- Plant Genetic Resources, Genomics and Genetic Improvement (301)

Research Project: Decipher Molecular Mechanisms for Genetic Variations in Agronomically Important Traits to Improve Sugar Beet Disease Resistance and Yield **Location:
                            Northwest Irrigation and Soils Research** 2023 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 and continues research from project 2054-21220-005-000D, Development of Elite Sugar Beet Germplasm Enhanced for Disease Resistance and Novel Disease Management Options for Improved Yield.  

In support of Objective 1, 820 commercial plots with sugar beet plants are being screened in the field for resistance to rhizomania (610 plots), rhizoctonia crown and root rot (120 plots), and beet curly top (90 plots) in an effort support of the sugar beet industry in identifying their most resistant and highest yielding cultivars. In addition, there are 1,280 genetic plots with sugar beet Plant Introduction lines or breeding lines being screened in the field for resistance to rhizomania (632 plots), rhizoctonia crown and root rot (198 plots), and beet curly top (450 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.  

In support of Objective 2, researchers have continued germplasm improvement in the C762-17 (PI 560130) population that is resistant to Beet curly top virus (BCTV) and EL57 (PI 663212) that is resistant to Rhizoctonia solani. The F3 and F4 progeny for both of these populations are being screened in disease nurseries for a second year. SNP analysis and genetic linkages to the resistance traits of these populations will be conducted later this year. The ARS researchers are also developing an ethyl methanesulfonate (EMS) mutagenesis pipeline to generate new and novel disease resistance traits. The process of bulking up EL10 germplasm for EMS chemical mutagenesis treatment is under way. The EL10 (PI 689015) population was chosen for this work because it was the basis for the most recently published sugar beet reference genome.

In support of Objective 3, the researchers conducted a degradome analysis of sugar beet plants infected with Beet curly top virus (BCTV). Using experimental and computational approaches, the putative roles of BCTV-derived small non-coding RNAs (sncRNAs) were demonstrated. These sugar beet target genes were further validated using degradome analysis. Our results confirm specific sugar beet gene targets of BCTV derived sncRNAs in a virus strain specific manner, which might have implication for developing future mitigation strategies using cutting-edge functional genomics tools such as CRISPRi.

ARS researchers conducted an untargeted metabolome analysis of sugar beet germplasm. Using sugar beet double haploid lines resistant and susceptible to BCTV, the role of leaf metabolites in BCTV resistance was investigated. When compared versus the susceptible line, results show several times higher levels of flavonoid and iso-flavonoid glycoside derivatives along with specific tri-terpenoids (e.g. ursolic acid) in the leaves of resistant lines. Additional experiments performed along with available information on the role of these compounds in host plant resistance against pathogens indicate the potential role of these compounds in sugar beet resistance to BCTV. 

ARS researchers investigated rhizomania resistance mechanisms in sugar beet germplasm. Using rhizomania susceptible germplasm and EMS mutant sugar beet germplasm that is resistant to rhizomania, a field study was conducted to delineate molecular mechanisms contributing to rhizomania resistance. Global gene expression analysis, accompanied by untargeted metabolome, micronutrient, and microbiome analyses revealed distinct mechanisms of resistance in roots and leaves at early and late infection stages in the resistant lines versus the susceptible line.

ARS researchers investigated the role of microbiome and metabolites to improve the post-harvest storage quality of sugar beet roots. Using sugar beet germplasm developed by ARS researchers in Kimberly, Idaho, that are susceptible or resistant to post-harvest storage pathogens, post-harvest storage experiments were performed. The different genotypes were evaluated for disease resistance, weight loss, sugar content, microbiome, and metabolite related markers. These data are currently being analyzed and will be interpreted for microbiome- and metabolite-related traits of genotypes that affect post-harvest storage quality of sugar beet roots. 

ARS researchers sequenced the beet leafhopper (BLH) genome. The genome sequence and chromosome level assembly of the beet leafhopper has been uploaded to the National Center for Biotechnology Information (NCBI) website as a genome resource to the scientific community. A global gene expression and proteome analyses in response to temperature stresses and developmental stages were conducted to validate BLH genes obtained from genome sequencing work. ARS researchers investigated atmospheric cold plasma to improve post-harvest storage quality of sugar beet roots. The results indicate a significant decrease (16-22 percent (%)) in disease incidence and increase (11-14%) in root biomass retention in plasma-treated (once after harvest) sugar beet roots after five months of storage in a commercial storage facility. Additional research is planned to further investigate and optimize this technology. 

Different insecticide chemistries were investigated for the management of curly top and pests on sugar beet. Seven treatments have been applied to a commercial sugar beet cultivar and are currently being compared versus a non-treated check and two commercial treatments (Poncho Beta seed treatment and an Asana foliar treatment). The plots have been inoculated with viruliferous beet leafhoppers (contain BCTV) and disease ratings and yield will be determined as the season progresses.

To improve management options for curly top, BLH populations in southern Idaho were tracked in five counties during the 2020 and 2021 growing seasons in desert areas and sugar beet and dry bean fields in southern Idaho with yellow sticky cards. Samples were collected on a weekly basis from mid-April through mid-September to assess BLH population levels and identify the curly top virus species/strains and phytoplasmas present. Plants from monitored crop fields were also assessed for the same pathogens. Once BLH populations in Elmore County began increasing in May, they were present in at least double-digit numbers per card through most of the summer at all sites both years. However, the BLH numbers at desert sites in other areas were at or near zero, indicating that local weed populations and not desert areas were the primary source of BLH in crop fields. Two haplotypes (based on cytochrome oxidase gene) dominated the BLH population both years. BCTV strains Worland and Colorado were the primary strains in BLH and plant samples, but Worland was dominate early in the season (May and June samples), while Colorado was dominant in July, August, and September samples. The CA/Logan, Pepper curly top, and Severe strains of BCTV could also be detected in BLH samples along with Spinach curly top Arizona virus. Phytoplasmas were detected in 1% of the BLH samples both years and included both Candidatus Phytoplasma trifolii and pruni. Neither Spinach curly top Arizona virus nor phytoplasmas were detected in plant samples, but the BCTV strains detected in BLH were found in the plant samples except for Pepper curly top. This project established the curly top species/strains for which host plant resistance is needed and the time and areas when sugar beets are at highest risk for BCTV infection.

A collaborative effort with other researchers identified the bacterial endosymbionts in leafhoppers that vector phytoplasmas. Robust markers associated with BCTV resistance in dry beans were established. **Accomplishments**
                                

1. Curly top virus strains in sugar beets, dry beans, and beet leafhoppers confirmed in southern Idaho. Beet curly top caused by Beet curly top virus (BCTV) is a serious yield limiting disease on sugar beet and numerous other crops. In order to improve BCTV management options, beet leafhopper (BLH) populations in southern Idaho were tracked by ARS researchers in Kimberly, Idaho, in desert areas and sugar beet and dry bean fields in five counties during the 2020 and 2021 growing seasons. BLH populations at all sites in one county were present in at least double-digit numbers throughout the summer while BLH numbers at desert sites in other counties were at or near zero, leaving local weed populations as the primary source of BLH in crop fields. Although pathogens not known to be present in the BLH population in Idaho were detected, the plant samples confirmed that the BCTV strains being utilized in the curly top disease nurseries are appropriate for establishing resistance. Researchers and the sugar beet industry are using this information to continue using current BCTV strains to screen for curly top resistance. 2. Identified microbiome and metabolome markers linked to improved post-harvest sugar beet storage. Sugar beet roots stored under ambient conditions can suffer millions of dollars in loses every year. In order to develop better management options for sugar beet storage, post-harvest storage experiments were performed using resistant and susceptible sugar beet germplasm developed by ARS researchers in Kimberly, Idaho. The different genotypes were evaluated for disease resistance, weight loss, sugar content, microbiome, and metabolome. Based on these data, microbiome and metabolite related markers that are genotype specific and affect post-harvest storage quality of sugar beet roots have been identified. Researchers are utilizing these markers to identify germplasm that has lower sucrose losses during storage. **Review Publications**
                            *Strausbaugh, C.A. 2023. Commercial sugar beet cultivars evaluated for rhizomania resistance and storability in Idaho, 2021. Plant Disease Management Reports. 17. Article V041.*
                                *Strausbaugh, C.A. 2023. Experimental sugar beet cultivars evaluated for rhizomania resistance and storability in Idaho, 2021. Plant Disease Management Reports. 17. Article V042.*
                                *Eujayl, I.A., Strausbaugh, C.A., Galewski, P.J. 2023. Beet curly top resistance in USDA-ARS Kimberly germplasm, 2022. Plant Disease Management Reports. 17. Article V043.*
                                *Strausbaugh, C.A., Chu, C.N. 2023. Fargo sugar beet germplasm evaluated for Rhizoctonia crown and root rot resistance in Idaho, 2022. Plant Disease Management Reports. 17. Article V044.*
                                *Strausbaugh, C.A., Majumdar, R., Wenninger, E.J. 2023. Foliar insecticides for the control of curly top in Idaho sugar beet, 2022. Plant Disease Management Reports. 17. Article ST004.*


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