Research Project: Increasing Sugar Beet Productivity and Sustainability through Genetic and Physiological Approaches

Location: Sugarbeet and Potato Research

2022 Annual Report

Objectives
Objective 1: Identify genes and metabolic pathways responsible for deterioration of sugar beet root quality in storage, and develop new and more efficient storage protocols for wounded and drought-stressed sugar beet roots. Objective 2: Develop and release superior sugar beet germplasm with improved genetic diversity, resistance to the sugar beet root maggot, and improved processing quality. Objective 3: Develop physiological methods that promote and enhance natural plant defense mechanisms of sugar beet, including manipulation by plant hormones. Objective 4: Develop genomic and transcriptomic resources to better identify fungicide-resistant and fungicide-sensitive strains of Cercospora beticola. Objective 5: Facilitate the development of improved sugarbeet disease resistance to C. beticola through comparative genomics, transcriptomics, and pathogenicity studies on strains isolated from wild sea beet and cultivated sugarbeet germplasm. Objective 6: Develop improved sugarbeet resistance to C. beticola using effector-based screening. Objective 7: Facilitate the development of improved sugar beet disease resistance to Beet necrotic yellow vein virus and other important pathogens through comparative genomics and pathogenicity studies and the use of gene editing to manipulate promising candidate genes.

Approach
The sugarbeet industry is a significant contributor to the U.S. economy and ensures a domestic supply for a staple in the American diet. The industry’s future, however, is challenged by stagnant sugar prices, increasing production costs, and competition from alternative sweeteners, sugarcane and imported sugar. Increased productivity is essential for the industry to remain profitable, competitive and sustainable. Sugarbeet productivity is determined by the quantity of sugar produced after processing. This yield, the extractable sugar yield, depends on sucrose accumulation during production, sucrose retention during storage, and sucrose recovery during processing. Physiological and genetic research is proposed that potentially will lead to new production and storage protocols and new hybrids to improve sucrose accumulation, retention, and recovery during production, storage, and processing. Specifically, the proposed research will (1) increase production yield by (a) generating genetically diverse germplasm with unique disease, pest, and stress resistance genes, (b) creating improved breeding lines with resistance to the sugarbeet root maggot, and (c) utilizing plant hormones to induce native plant defense mechanisms to enhance yield; (2) reduce storage losses by (a) identifying genetic and metabolic pathways responsible for sucrose and quality losses during storage, (b) characterizing temperature effects on postharvest wound-healing, (c) determining preharvest drought effects on storage properties, and (d) evaluating the effects of defense-inducing plant hormones on storage properties; and (3) improve sucrose recovery by creating germplasm lines with reduced concentrations of compounds that prevent the extraction of a portion of the sucrose during processing.

Progress Report
Research conducted during fiscal year (FY) 2022 significantly advanced all project objectives except Objective 5 which was completed in FY 2021. Objective 1: Research to identify and characterize the molecular and pathological events responsible for sugarbeet storage losses advanced with the identification of more than 9000 genes and metabolites that change in sugarbeet roots during storage. The characterization of these genes and metabolites with respect to storage duration, temperature, and root deterioration identified key genes and metabolic pathways that are likely involved in postharvest sucrose loss, root softening, non-sucrose carbohydrate formation, and enhanced susceptibility to storage diseases. Pathogens responsible for storage diseases were also isolated from diseased sugarbeet roots that were collected from commercial storage piles, and these pathogens will be identified in mid-August to September 2022. Additionally, the detrimental impact of preharvest water stress on postharvest storage was established and quantified through research that determined the effect of different levels of pre-harvest drought stress on storage sucrose loss, root respiration rate, susceptibility to storage pathogens, and the accumulation of non-sucrose carbohydrates that reduce sugar recovery during processing. Objective 2: Eight breeding lines that were derived from crosses between cultivated sugarbeet and wild sea beet were planted to two fields with high disease pressure for Rhizoctonia root and crown rot (RRCR) and these lines will be evaluated for yield and RRCR resistance in September 2022. Twenty-three lines derived from crosses between sugarbeet root maggot (SBRM)-resistant and RRCR-resistant germplasm were planted in a field in St. Thomas, North Dakota where SBRM pest pressure is typically high and consistent across years. Resistance of these lines to SBRM will be evaluated in mid-July to early August 2022 and roots with high SBRM resistance and root shapes characteristic of commercial sugarbeet will be selected during the September 2022 harvest. Field trials to determine root and sucrose yield of breeding lines that differ in concentration of root impurities that cause sucrose loss to molasses (LTM) during processing were planted to fields in Crookston, Minnesota and St. Thomas, North Dakota, along with two sugarbeet breeding lines from the previous year that respectively exhibited high and low LTM values. Roots from these trials will be harvested in September and characterized for root yield, sucrose content, and LTM. Objective 3: A repetition of field and storage studies evaluating the effects of methyl jasmonate (MeJA) and salicylic acid (SA) applications on sugarbeet root yield, sucrose content, sucrose yield, and storage properties was completed, generating the final data in a three-year study. Objective 4: Results from a genome wide association study (GWAS) that identified mutations associated with fungicide resistance in the sugarbeet pathogen Cercospora beticola (Cb) were confirmed. The GWAS identified multiple genetic mutations in the CbCYP51 gene that were strongly associated with fungicide resistance and found that different combinations of fungicide resistant mutations within an isolate resulted in varying levels of fungicide resistance. Mutations associated with fungicide resistance included both mutations that changed the structure of the resulting protein and mutations that are not predicted to change the structure of the resulting protein. The identified mutations were used to develop genetic marker probes capable of detecting fungicide resistance with high sensitivity. Objective 6: Candidate effector genes from Cb were infiltrated into leaves of Nicotiana benthamiana to identify genes involved with virulence. This assay identified two genes that induce necrosis and are likely integrally involved with disease. Objective 7: Genomes of rhizomania symptomatic sugarbeet collected from fields in the Red River Valley of Minnesota and North Dakota, Idaho, and the Imperial Valley of California are undergoing sequencing to identify viruses that co-exist and may influence virulence of beet necrotic yellow vein virus (BNYVV), the causal agent of rhizomania. Further, a new method to detect BNYVV and beet curly top virus (BCTV), the causal agent of beet curly top disease, was developed utilizing clustered regularly interspaced short palindromic repeats (CRISPR)-associated technology. Evaluation of the method using greenhouse grown plants produced from seed sown on soil collected from rhizomania-infested fields for BNYVV and field samples with curly top symptoms for BCTV established its ability to detect BNYVV and BCTV with high sensitivity and specificity without the need for sophisticated instrumentation.

Accomplishments
1. New fungicide resistance assays for improved management of sugarbeet Cercospora leaf spot disease. Fungicide resistance in Cercospora beticola (Cb) is a major obstacle in managing Cercospora leaf spot (CLS) disease of sugarbeet and has been associated with significant yield loss and severe CLS epidemics. Methods for detecting fungicide resistance are presently limited to post-season assays that are unable to provide timely information necessary to guide CLS management decisions in the current growing season. ARS researchers in Fargo, North Dakota, identified genetic mutations associated with fungicide resistance and used this information to develop new diagnostic assays that detect mutations associated with fungicide resistance. Since these assays can be used to identify fungicide-resistant Cb strains during the growing season, they provide sugarbeet agronomists and growers a new tool to tailor management practices to the specific Cb isolates present in their fields and discourage the development of fungicide resistance.

2. New method of promoting sugarbeet seed germination using hydrogen peroxide. Poor seed germination prevents plant breeders from utilizing the genetic potential that resides in dormant seeds or seeds that have deteriorated during storage. New methods that improve germination of poor germinating seeds, therefore, would allow breeders to access a wider range of genetic resources. ARS researchers in Fargo, North Dakota, developed a seed treatment protocol using hydrogen peroxide that improves sugarbeet seed germination by as much as 70%. Analysis of the genetic changes caused by the seed treatment revealed that hydrogen peroxide promotes germination by accelerating the degradation of expressed genes that inhibit germination and stimulates the expression of genes involved in cell growth and proliferation. The new seed treatment provides breeders a method to improve germination of sugarbeet lines and wild sugarbeet relatives that otherwise had little or no germination, thereby making these germplasms available for use in breeding programs. The research also provides new insight into the role of hydrogen peroxide as a signaling molecule that regulates gene activities during germination.

3. New tools to study respiration in stored sugarbeet roots. Respiration is responsible for 60 to 80% of the sucrose loss that occurs during sugarbeet root postharvest storage. Although reducing sucrose loss to respiration is a major goal for the sugarbeet industry, progress towards this goal is stymied by a lack of knowledge of the genetic elements and metabolic processes that control root respiration rate. ARS researchers in Fargo, North Dakota, generated two new breeding lines with genetic differences in storage respiration rate and characterized the genes that differ in expression between these two lines. The developed lines are potentially useful for elucidating the genetic and physiological processes that control storage respiration rate and the effect of variations in respiration rate on root metabolism during storage. The research, additionally, identifies genes that potentially contribute to, regulate, or are influenced by sugarbeet storage respiration rate. The new lines and identified genes are the first available genetic tools to be made available to the sugarbeet research community for use in investigating the heritable elements and metabolic processes that influence sugarbeet root respiration rate and sucrose loss during storage.

4. New diagnostic assay to detect the pathogen responsible for rhizomania in sugarbeet. Rhizomania is a viral disease that threatens the viability of the sugarbeet industry worldwide. Although traditionally managed by genetic resistance, rhizomania has re-emerged as a major concern due to the evolution of resistance-breaking viral strains. ARS scientists in Fargo, North Dakota, developed a genome-editing based assay for detecting the virus causing rhizomania. The method utilizes clustered regularly interspaced short palindromic repeats (CRISPR)-associated technology and allows the virus to be detected with high sensitivity, specificity, and accuracy without the need for sophisticated laboratory instruments. The CRISPR-based detection method is a new tool for the sugarbeet industry for assessing the presence of the BNYVV virus and will assist agronomists in formulating rhizomania disease management strategies and breeders in selecting varieties with rhizomania resistance.

Review Publications
Spanner, R., Neubauer, J., Heick, T.M., Grusak, M.A., Hamilton, O., Rivera-Varas, V., De Jonge, R., Pethybridge, S., Webb, K.M., Leubner-Metzger, G., Secor, G.A., Bolton, M.D. 2022. Seed-borne Cercospora beticola can initiate Cercospora leaf spot in sugar beet (Beta vulgaris L.) fruit tissue. Phytopathology. 112:1016-1028. https://doi.org/10.1094/PHYTO-03-21-0113-R.
Chu, C.N., Rudd, J.C., Chen, M., Wang, S., Ibrahim, A.M., Xue, Q., Devkota, R.N., Baker, J.A., Baker, S., Simoneaux, B., Opena, G., Dong, H. 2022. A new strategy for using historical imbalanced yield data to conduct genome-wide association studies and develop genomic prediction models for wheat breeding. Molecular Breeding. 42. Article e18. https://doi.org/10.1007/s11032-022-01287-8.
Dhakal, S., Liu, X., Chu, C.N., Rudd, J.C., Ibrahim, A.M., Xue, Q., Devkota, R., Baker, J., Baker, S., Bryan, S., Gigi, O., Jessup, K.E., Wang, S., Johnson, C.D., Metz, R., Liu, S. 2021. Genome-wide QTL mapping of yield and agronomic traits in two widely adapted winter wheat cultivars from multiple mega-environments. PeerJ. 9. Article e12350. https://doi.org/10.7717/peerj.12350.
Zhang, S., Du, P., Lu, X., Fang, J., Wang, J., Chen, X., Chen, J., Wu, H., Yang, Y., Tsujimoto, H., Chu, C.N., Qi, Z. 2021. Frequent numerical and structural chromosome changes in early generations of synthetic hexaploid wheat. Genome. https://doi.org/10.1139/gen-2021-0074.
Fugate, K.K., Campbell, L.G., Lafta, A.M., Eide, J.D., Khan, M.F., Chu, C.N., Finger, F.L. 2022. Newly developed sugarbeet lines with altered postharvest respiration rates differ in transcription factor and glycolytic enzyme expression. Crop Science. 62(3):1251-1263. https://doi.org/10.1002/csc2.20729.
Kumar, R., Mazakova, J., Ali, A., Sur, V., Sen, M., Bolton, M.D., Manasova, M., Rysanek, P., Zouhar, M. 2021. Characterization of the molecular mechanisms of resistance against DMI fungicides in Cercospora beticola populations from the Czech Republic. The Journal of Fungi. 7(12). Article e1062. https://doi.org/10.3390/jof7121062.
Chu, C.N., Poore, R.C., Bolton, M.D., Fugate, K.K. 2022. Mechanism of sugarbeet seed germination enhanced by hydrogen peroxide. Frontiers in Plant Science. 13. Article e888519. https://doi.org/10.3389/fpls.2022.888519.
Rangel, L., Hamilton, O., De Jonge, R., Bolton, M.D. 2021. Fungal social influencers: Secondary metabolites as a platform for shaping the plant-associated community. The Plant Journal. 108(3):632-645. https://doi.org/10.1111/tpj.15490.
Spanner, R., Taliadoros, D., Richards, J., Rivera-Varas, V., Neubauer, J., Natwick, M.B., Hamilton, O., Vaghefi, N., Pethybridge, S., Secor, G.A., Friesen, T.L., Stukenbrock, E.H., Bolton, M.D. 2021. Genome-wide association and selective sweep studies reveal the complex genetic architecture of DMI fungicide resistance in Cercospora beticola. Genome Biology and Evolution. 13(9). https://doi.org/10.1093/gbe/evab209.
Rangel, L., Bolton, M.D. 2022. The unsung roles of microbial secondary metabolite effectors in the plant disease cacophony. Current Opinion in Plant Biology. 68. Article e102233. https://doi.org/10.1016/j.pbi.2022.102233.
Broccanello, C., Bolton, M.D., Secor, G., Richards, C.M., Ravi, S., Concheri, G., Campagna, G., Squartini, A., Stevanato, P. 2022. Bacterial endophytes as indicators of susceptibility to Cercospora Leaf Spot (CLS) disease in Beta vulgaris L. Scientific Reports. 12. Article e10719. https://doi.org/10.1038/s41598-022-14769-8.