Location: Northwest Irrigation and Soils Research
2022 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 Objective 1, which addresses development of genetic markers and superior sugar beet germplasm. Germplasm was tested for heat stress, water deficit, non-bolting, and overwintering. Two advanced breeding lines (USKPS25 and USK944-6-68) were identified as heat and drought tolerant after three seasons of testing in Egypt. Sugar beet germplasms highly resistant to Beet curly top virus (BCTV) were characterized. Three mutant breeding lines (KEMS08, KEMS06, KEMS06-600) were confirmed to be highly resistant to Cercospora leaf spot (CLS). Whole Genome Sequencing (WGS) experiments on these three Cercospora resistant lines have been initiated to identify the molecular markers linked to CLS resistance, which will facilitate future marker-assisted breeding strategies. Through ethyl methanesulfonate (EMS) mutation, sugar beet genotypes were developed that do not bolt under normal conditions. Using genome sequencing and transcriptomic analysis, potential mutations and markers associated with the non-bolting phenotype were identified. These markers will help with future trait introgression (through breeding) in commercially important sugar beet cultivars. Non-bolting beets could be planted in the fall to extend the growing season, resulting in higher root and sucrose yields.
Significant progress was also made on all sub-objectives under Objective 2, which addresses disease development pathways, host-pathogen interactions, and improved disease management strategies. Prior research demonstrated that late season sugar beet root rot is primarily associated with the bacteria Leuconostoc and secondary organisms; however, damage was minor without both the fungus R. solani AG-2-2 and Leuconostoc strains present when internal rot initiates. 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. The data suggest that R. solani produces these three enzymes and when combined with L. mesenteroides in sugar beet root tissue, considerable rot develops. Analysis of global gene expression (mRNAseq) in sugar beet during early infection stages (1 day, 2 days, and 3 days post-infection) confirmed the relative contribution of R. solani cellulase, polygalacturonase, and pectin lyase genes during interaction with Leuconostoc. Potential candidate genes were identified that are associated with pathogenicity in both of the pathogens along with sugar beet genes that are differentially regulated based upon exposure to R. solani and Leuconostoc separately or together. These data were published in a referred journal article.
Through both an extramural agreement and in-house research, agro-inoculation clones of the BCTV strains are being developed which will allow for more targeted, precise identification of curly top resistance. Candidate target genes associated with pathogenicity in BCTV 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.
The regulatory role of BCTV strain specific small non-coding RNAs (sncRNAs) interacting with host plants was investigated. Using BCTV susceptible and resistant genotypes, natural infection, and global RNAseq, we demonstrated differential regulation of sugar beet genes by strain specific BCTV sncRNAs. Among detected sncRNAs, sncRNA_26 was common to all four strains and showed higher negative correlation with the expression of EL10Ac7g16816 (UPF0554 protein) gene in the susceptible line. Whereas CA/Logan specific sncRNAs, namely sncRNA_4, 20 and 21, showed higher negative correlation with the expression of EL10Ac1g01206 (LRR protein), EL10Ac5g12605 (7-deoxyloganetic acid glucosyltransferase), and EL10Ac6g14074 (transmembrane emp24) genes respectively in the susceptible line. This data suggests that genome divergence among BCTV strains differentially affects the production of sncRNA and putative small peptides that could potentially affect pathogenicity and disease symptom development.
Different insecticide chemistries were investigated for the management of curly top and pests on sugar beet. Two treatments (Poncho Beta seed treatment and the Scorpion foliar treatment) provided a similar level of control on the second rating and had similar root yield and estimated recoverable sugar (ERS). The Applaud foliar treatment provided less control based on ratings, but ERS was not significantly different from the Poncho Beta and Scorpion treatments. The foliar insecticide treatments containing Asana or Spear T provided marginal control of BCTV, but these treatments were better than the non-treated check in foliar ratings, root yield, and ERS. The Venom and Ninja + Asana treatments also showed higher root yield and ERS relative to the non-treated check. The remaining treatments were similar to or worse than the non-treated check with respect to percent sucrose, root yield, and ERS. Additional evaluations with other insecticides will be needed if alternatives to the neonicotinoid chemical class (Poncho and Scorpion) for BCTV control are to be identified.
We continued to track beet leafhopper populations in southern Idaho and the virus strains associated with them. Preliminary results from three years of research suggest that Elmore County has the most beet leafhoppers and the highest frequency of BCTV. In southern Idaho, the predominant BCTV strain was Worland alone or co-infected with Colorado. The role of host plant nitrogenous compounds, such as polyamines (PAs) and amino acids (AAs), in sugar beet resistance against BCTV was investigated. Both PAs and AAs are known for their diverse role in stress tolerance in plants. Our results show that there may be a threshold of PAs and AAs in sugar beet beyond which they contribute to susceptibility, rather than resistance to BCTV.
Using 16S rRNA sequencing and BCTV resistant genotypes (KDH13, KDH4-9) along with a susceptible genotype (KDH19-17), the leaf bacteriome changes during BCTV post inoculation (pi) were investigated. Six days post-inoculation (dpi), Cyanobacteria were predominant (approximately 90%), whereas four weeks post-innoculation (wkpi) Firmicutes (11-66%), Bacteroidetes (17-26%), and Verrucomicrobia (12-29%) were predominant and genotype dependent. Both Bacteroidetes and Verrucomicrobia increased post infection only in the resistant lines. Brevibacillus increased at 6 dpi, and Akkermansia and Bacteroides at 4 wkpi in the resistant lines. Linear discriminant analysis Effect Size identified potential biomarkers in the resistant lines vs. susceptible line. Functional profiling revealed bacterial enrichment associated with tricarboxylic acid cycle, polyisoprenoid, and L-methione biosynthesis pathways only in KDH4-9 at 6 dpi. At 4 wkpi, bacteria associated with tryptophan and palmitate biosynthesis in the resistant lines and uridine monophosphate, phosphatidyl glycerol, and phospholipid biosynthesis in the susceptible line, were enriched. Future characterization of bacterial genera with antiviral properties will help establish their use as biocontrol agents or biomarkers against BCTV.
The antimicrobial properties of non-pathogenic bacteria derived natural products (prodigiosin and violacein) were evaluated against major fungal, bacterial, and viral pathogens in sugar beet. In vitro results showed a 98% reduction in growth of fungal and bacterial pathogens and significant improvement in BCTV resistance by prodigiosin at a specific concentration. These compounds are currently being assessed for their efficacies against the pathogens under field conditions.
The genome of beet leafhopper, Circulifer tenellus Baker, was sequenced, assembled (at the chromosome level), and annotated for the first time. Beet leafhopper is a major pest and vector of plant pathogens in sugar beet and other economically important crops grown worldwide.
Accomplishments
1. A high-density marker set was developed to track beet curly top resistance in sugar beet. Beet curly top is a viral disease that can cause serious sugar beet yield losses in semi-arid production areas worldwide. Current commercial sugar beet cultivars have only low to intermediate resistance to Beet curly top virus (BCTV). PacBio sequences were generated and assembled by ARS researchers in Kimberly, Idaho, to identify the specific variation underpinning durable BCTV resistance within the KDH13 genome, which has superior resistance to BCTV. This additional sequencing established a high-density marker dataset distributed globally across the genome. These markers can be used to track genomic segments in populations where KDH13 is used as parental material to improve BCTV resistance in commercial sugar beet cultivars.
2. Discovery of regulatory roles of small non-coding RNAs in sugar beet resistance against Beet curly top virus. Beet curly top virus (BCTV) is a major problem for sugar beet production and other crops in semi-arid areas worldwide. ARS researchers in Kimberly, Idaho, investigated the role of small non-coding RNAs during early infection to determine the molecular mechanisms of viral resistance. Plant-derived small RNAs targeted BCTV capsid protein and replication-related genes. The genes related to small RNA production is useful information for plant breeders when improving BCTV resistance in sugar beet and other susceptible crops.
Review Publications
Majumdar, R., Galewski, P.J., Eujayl, I.A., Minocha, R., Vincill, E.D., Strausbaugh, C.A. 2022. Regulatory roles of small non-coding RNAs in sugar beet resistance against beet curly top virus. Frontiers in Plant Science. 12. Article 780877. https://doi.org/10.3389/fpls.2021.780877.
Strausbaugh, C.A. 2022. Commercial sugar beet cultivars evaluated for rhizomania resistance and storability in Idaho, 2020. Plant Disease Management Reports. 16. Article V038.
Strausbaugh, C.A. 2022. Experimental sugar beet cultivars evaluated for rhizomania resistance and storability in Idaho, 2020. Plant Disease Management Reports. 16. Article 037.
Strausbaugh, C.A., Wenninger, E.J. 2022. Foliar insecticides for the control of curly top in Idaho sugar beet, 2021. Plant Disease Management Reports. 16. Article V036.
Majumdar, R., Strausbaugh, C.A., Galewski, P.J., Minocha, R., Rogers, C.W. 2022. Cell wall degrading enzymes originating from Rhizoctonia solani increase sugar beet root damage in the presence of Leuconostoc mesenteroides. International Journal of Molecular Sciences. 23(3). Article 1366. https://doi.org/10.3390/ijms23031366.
Strausbaugh, C.A., Chu, C.N. 2021. Fargo sugar beet germplasm evaluated for rhizomania and storage rot resistance in Idaho, 2020. Plant Disease Management Reports. 15. Article V160.
