Location: Northwest Irrigation and Soils Research
2021 Annual Report
Objectives
1. Develop genetic markers that will allow for marker-assisted breeding; develop superior sugar beet germplasm with priority traits, such as high sucrose and resistance to various diseases; and release improved breeding materials, including doubled haploid lines, inbred lines, and genetic mapping populations.
1.1. Develop elite germplasm with curly top, Rhizoctonia crown and root rot (RCRR), Cercospora leaf spot (CLS), and storage rot resistance, and high sucrose and low impurities. (Eujayl, Strausbaugh)
1.2. Conduct whole genome sequencing of elite germplasm lines for genetic variation analysis for RCRR resistance. (Eujayl, Strausbaugh)
1.3. Establish a large complement of single nucleotide polymorphism (SNP) markers for genotyping mapping populations and germplasm for curly top and RCRR resistance. (Eujayl, Strausbaugh)
2. Dissect disease development pathways and host-pathogen interactions, and design improved disease management strategies and screening procedures in sugar beet.
2.1. Investigate the interaction between the most common Leuconostoc van Tiegham haplotypes and the various genetic subgroups of R. solani. (Strausbaugh)
2.2. Investigate the use of RNA interference (RNAi) for the control of Beet curly top virus (BCTV). (Strausbaugh, Eujayl)
2.3. Develop additional management strategies for curly top and pest control in sugar beet. (Strausbaugh)
Approach
The proposed research is a coordinated cooperative effort between USDA-ARS, university scientists, and industry partners which will improve sucrose yield in sugar beet production. Elite sugar beet germplasm will be developed to increase sucrose content, while reducing impurities and improving disease resistance and management options for Beet curly top virus (BCTV), Rhizoctonia solani, and storage rot fungi. The first objective is non-hypothesis research focused on improving or identifying novel traits of interest, releasing elite germplasm with these traits, and sequencing lines to map and develop markers for these traits. Genetic markers will allow for marker-assisted breeding and release of superior sugar beet germplasm. Backcrossing, mass selection, and recurrent selection will be used to produce populations and lines with disease resistance, low impurities, and high sucrose content. Doubled haploid lines from this germplasm will be used to produce hybrids and segregating populations for genetic mapping. Whole genome sequencing will be conducted using PacBio technology and optical mapping. This effort will be complemented with gene expression profiling via RNA-Seq and Iso-Seq to identify differentially expressed genes caused by R. solani infection. A large complement of single nucleotide polymorphism (SNP) markers for genotyping mapping populations and germplasm for curly top and Rhizoctonia crown and root rot resistance will be developed. If additional sources of high sucrose or disease resistance are needed, additional high sucrose parental lines and plant introduction accessions will be screened. The second objective is hypothesis driven research which advances our knowledge of disease development and interactions to improve disease management strategies and screening procedures in sugar beet production. The interaction between Leuconostoc and R. solani will be investigated, since Leuconostoc haplotypes will possibility vary in their ability to create more root rot through a synergistic interaction with genetic subgroups of R. solani. Root inoculations in field studies will be conducted with bacterial isolates representing the predominant haplotypes for L. mensenteroides and L. pseudomesenteroides and R. solani isolates representative of the diversity present in anastomosis groups found in sugar beet. Five weeks after inoculation, rotted tissue will be measured and the pH associated with that tissue will be established. Isolations from the leading edge of the rot from randomly selected roots will be conducted to complete Koch’s postulates. Based on the results from the interaction studies, fungal-bacterial combinations exhibiting the synergistic interaction will be evaluated further through inhibition and enzyme assays. To improve management options for BCTV, the use of RNA interference (RNAi) and foliar insecticides will be investigated. If RNAi proves successful, RNAi will also be investigated for the control of R. solani.
Progress Report
Significant progress was made on all sub-objectives under Objective 1, which addresses the development of genetic markers and superior sugar beet germplasm. The testing of germplasm for heat stress, water deficit, non-bolting, and overwintering was conducted. Two advanced breeding lines (USKPS25 and USK944-6-68) were identified as heat and drought tolerant after three seasons of testing in Egypt. Three mutant breeding lines (KEMS08, KEMS06, KEMS06-600) were confirmed to be highly resistant to Cercospora leaf spot. Sugar beet germplasms highly resistant to Beet curly top virus (BCTV) were characterized using an mRNA sequencing (mRNAseq) approach together with metabolite analysis (untargeted metabolomics). Novel or differentially expressed genes in the BCTV resistant sugar beet genotypes (vs. susceptible line) were identified. A global small RNAseq analysis identified BCTV-derived small non-coding RNAs and their potential role in downregulating the expression of sugar beet defense related genes during plant infection. This work has helped us identify potential viral target genes for future RNAi-based mitigation strategies. A manuscript describing the small RNA research has been drafted.
Significant progress was also made on all sub-objectives under Objective 2, which addresses dissecting disease development pathways, host-pathogen interactions, and designing improved disease management strategies. Under Sub-objective 2.1, the interactions of various Leuconostoc haplotypes versus a genetically diverse range of R. solani strains were investigated. This research area was described in detail in the 2020 annual report. In summary, both isolations and tissue pH suggest late season sugar beet root rot is primarily associated with Leuconostoc and secondary organisms. However, damage was minor without both R. solani AG-2-2 and Leuconostoc strains present when internal rot initiates. The research was published as a refereed journal article. Investigations into the enzymes associated with this interaction were conducted. Results suggest that L. mesenteroides strain L12311 leads to rot when combined with the enzymes cellulase, polygalacturonase, and pectin lyase, but caused little to no rot when combined with xylanase, pectate lyase, and pectin methylesterase. Leuconostoc is not known to produce cellulase, polygalacturonase, and pectin lyase, but R. solani AG-2-2 IIIB strains are capable of producing these enzymes. The preliminary data suggest that R. solani produces these three enzymes and when combined with L. mesenteroides in sugar beet root tissue, considerable rot develops. Higher degradation of complex cell wall carbohydrates was reflected by the increase in simpler sugars such as glucose, galactose, and fructose in the R. solani + Leuconostoc infected roots in comparison to mock control or Leuconostoc-only infected samples. Total carbon and nitrogen analysis of sugar beet roots revealed an increase in nitrogen content and decrease in carbon:nitrogen ratio in the infected samples (vs. control) with the highest increase or decrease in R. solani + Leuconostoc infected roots. Analysis of global gene expression (mRNAseq) in sugar beet during early infection stages (one day, two days, and three days post-infection) by R. solani, and Leuconostoc confirmed the relative contribution of R. solani cellulase, polygalacturonase, and pectin lyase genes during interaction with Leuconostoc. We also identified potential candidate genes associated with pathogenicity in both the pathogens along with sugar beet genes that are differentially regulated based upon exposure to R. solani and Leuconostoc separately or together.
Under Subobjective 2.2, primers and double-strand RNA (dsRNA) protocols were established to investigate the use of RNA interference (RNAi) for the control of BCTV. After conducting an initial small experiment, we decided to wait for the new greenhouse to be completed so agro-inoculation could be used before conducting additional experiments. Through both a Non-Assistance Cooperative Agreement (NACA) and in-house work, agro-inoculation clones of the BCTV strains are being developed, which will allow more targeted and precise work. Candidate target genes associated with pathogenicity in BCTV (e.g. V2/V3 gene/s) along with fungal pathogens including R. solani and Cercospora beticola (e.g. Spds gene) have been identified. RNAi constructs optimizing both transient and stable expression of RNAi are being developed to target critical pathogen genes and reduce disease symptoms during host plant-pathogen interactions. Experiments using BCTV resistant sugar beet germplasms to understand the role of carbon and nitrogen metabolism were performed along with investigations into the host plant microbiome involved in BCTV resistance. In addition, the role of sugar beet topping (prior to harvesting) on post-harvest sugar retention was investigated. Using gene expression and carbohydrate analysis on high sugar-containing sugar beet genotypes, we found that a slight delay in digging roots from the soil after topping significantly increased sugar content, rather than decreasing sugar content. This research is expected to be published next year. A new project was also established to track beet leafhopper populations in southern Idaho and the virus strains associated with them. Preliminary results suggest Elmore County had the most beet leafhoppers and the highest frequency of BCTV. The predominant BCTV strain was Worland.
Under Subobjective 2.3, different insecticide chemistries were investigated for the management of curly top and pests on sugar beet. Six insecticide foliar treatments were compared for their ability to manage curly top versus the Poncho Beta neonicotinoid seed treatment and a non-treated check. The non-treated check was severely infected based on curly top ratings and yield variables, even though a commercial cultivar approved for production was utilized for the study. Three treatments (Poncho Beta seed treatment and the foliar treatments Asana and Venom) provided a similar level of control and allowed for similar yield. The five other foliar insecticide treatments evaluated in the study provided no control of BCTV, since all variables had values similar to the non-treated check. Additional evaluations with other insecticides will be needed if alternatives to neonicotinoid (Poncho and Venom) and pyrethroid (Asana) chemical classes for BCTV control are to be identified.
Accomplishments
1. Enzymes involved in the Leuconostoc-Rhizoctonia interaction were established. Late season Rhizoctonia root rot in sugar beet is a serious yield-limiting disease problem in Idaho caused by an interaction between Rhizoctonia solani and Leuconostoc spp. To aid in the development of additional control measures, ARS researchers at Kimberly, Idaho, investigated the enzymes that initiate the rot process. Three enzymes involved in establishing root rot in sugar beet roots were identified. This information will allow researchers to target these enzymes when developing new disease control measures, which will limit losses for sugar beet growers and the sugar industry.
Review Publications
Strausbaugh, C.A. 2021. Commercial sugar beet cultivars evaluated for rhizomania resistance and storability in Idaho, 2019. Plant Disease Management Reports. 15:V017.
Strausbaugh, C.A. 2021. Experimental sugar beet cultivars evaluated for rhizomania resistance and storability in Idaho, 2019. Plant Disease Management Reports. 15:V016.
Strausbaugh, C.A., Wenninger, E.J. 2021. Foliar insecticides for the control of curly top in Idaho sugar beet, 2020. Plant Disease Management Reports. 15:V015.
Eujayl, I.A., Strausbaugh, C.A. 2021. Beet curly top resistance in USDA-ARS Kimberly germplasm, 2020. Plant Disease Management Reports. 15:V013.
Majumdar, R., Kandel, S.L., Cary, J.W., Rajasekaran, K. 2021. Changes in bacterial endophyte community following aspergillus flavus infection in resistant and susceptible maize kernels. International Journal of Molecular Sciences. 22(7). Article 3747. https://doi.org/10.3390/ijms22073747.
Galewski, P.J., McGrath, J.M. 2020. Genetic diversity among cultivated beets (Beta vulgaris) assessed via population-based whole genome sequences. BMC Genomics. 21:189. https://doi.org/10.1186/s12864-020-6451-1.
Strausbaugh, C.A., Dorn, K.M., Fenwick, A.L. 2020. Ft. Collins sugar beet germplasm evaluated for rhizomania and storage rot resistance in Idaho, 2019. Plant Disease Management Reports. 14:V138.
Dorn, K.M., Fenwick, A.L., Strausbaugh, C.A. 2021. Beet curly top resistance in USDA-ARS Ft. Collins germplasm, 2020. Plant Disease Management Reports. 15.
Dorn, K.M., Fenwick, A.L., Strausbaugh, C.A. 2021. Beet curly top resistance in USDA-ARS plant introduction lines of sugar beet, 2020. Plant Disease Management Reports. 15.
Eujayl, I.A., Strausbaugh, C.A. 2020. Kimberly sugar beet germplasm evaluated for rhizomania and storage rot resistance in Idaho, 2019. Plant Disease Management Reports. 14:V139.
Abuo-Elwafa, S.F., Amin, A.A., Eujayl, I.A. 2020. Genetic diversity of sugar beet under heat stress and deficit irrigation. Agronomy Journal. 112(5):3579-3590. https://doi.org/10.1002/agj2.20356.
