Location: Sunflower and Plant Biology Research
2020 Annual Report
Objectives
Coordinate the development of a Sclerotinia initiative for expanded research to control this devastating disease which affects canola, sunflowers, soybeans, edible dry beans, lentils, peas and other crops. Research should be coordinated with interested ARS, state, and industry cooperators and administered through specific cooperative agreements. Planning workshops and annual meetings involving interested parties will be organized throughout the funding period.
Approach
Exotic and emerging plant diseases pose severe problems throughout the United States. Their increasing importance may be attributed to the introduction of pathogens into new geographic regions; modification of the environment that favor diseases; change in crop management practices; genetic shifts in the pathogen population; and other processes that may give them a competitive advantage.
Progress Report
This report documents progress for cooperative research performed as part of the National Sclerotinia Initiative and involves researchers at several U.S. universities and USDA-ARS locations, in cooperation with USDA-ARS in Fargo, North Dakota.
Enhancing soybean for resistance to Sclerotinia stem rot. To continue the breeding pipeline for enhancing resistance to Sclerotinia stem rot in soybean, several breeding lines were advanced to 1st year yield trials (preliminary yield test). Of 69 lines, nine were advanced based on yield performance and will be entered in 2020 advance yield test. Moreover, 15 new crossing combinations were made focusing on yield, protein, and the sclerotinia disease package. Parental lines were selected based on both the predicted and observed disease severity index (DSI). Sixty-one hybrid pods were harvested from these combinations. In addition, 130 new advanced breeding lines were evaluated for white mold resistance in a naturally infected field on a research farm. Among those lines, six showed a high level of disease resistance.
White mold resistance QTL: identification, interactions, and fine mapping in common bean. Intercrossing was initiated among snap beans and dry beans with high genomic breeding values for white mold resistance. Two Recombinant Inbred Line (RIL) populations (P2 & P3) were advanced to a fifth generation. Population P2 was genotyped with the new 12K genotyping chip and was characterized for Sclerotinia resistance using the straw test. Three major Quantitative Trait Loci (QTL) were detected that can assist bean breeders. A genome-wide association study with 500 bean lines, evaluated using the seedling straw test, was completed. Thirty genomic regions associated with resistance on several chromosomes were identified.
Improved white mold resistance in dry and snap beans through multi-site screening and pathogen characterization throughout major production areas. Greenhouse and field trials (multiple sites in bean-producing states) were conducted using pinto, navy, black, and small red bean seed classes to identify and/or verify resistance to white mold in wide dry bean crosses and adapted dry and snap bean lines. In greenhouse studies, we also characterized the aggressiveness (ability to cause severe disease) of 366 Sclerotinia isolates on dry bean and showed that aggressiveness and bean genotypes were associated with the region where they originated.
Improving resistance to Sclerotinia sclerotiorum in spring canola. A back-cross population from a cross between NEP63 (winter type) and an elite breeding line NDC-E12027 was developed. A total of 147 spring type and 50 semi-winter type canola lines were evaluated for Sclerotinia resistance under field conditions at two field locations. Results of these trials confirmed the reaction of resistant lines identified in greenhouse inoculations. Crosses between lines NDSU-g151, NDSU-g127, NDSU-g339, NDSU-g149, and NDSU-g27 with advanced canola breeding lines were conducted to start the process of incorporating white mold resistance into these lines.
Screening for resistance sources to Sclerotinia white mold in recently acquired germplasm of cool season grain legumes. Ninety-five lines of the recently acquired pea collection from China were screened for resistance to white mold, using greenhouse tests. The three most resistant lines discovered in 2018 were also retested in 2019 and were found still to be the most resistant lines. Additionally, more lines of the recently acquired germplasm collection of cool season grain legumes were propagated in Central Ferry, Washington. About 90 lines of chickpea and 40 lines of pea were imported from Pakistan and the Global Crop Diversity Trust, respectively, in 2019. These will be tested in 2020.
Validation and characterization of cultivated sunflower lines with resistance to Sclerotinia basal stalk rot. We completed the greenhouse evaluations of 60 cultivated sunflower lines to identify and prioritize germplasm resources exhibiting high levels of physiological resistance to basal stalk rot. The 60 tested lines were selected on the basis of prior field data from trials involving natural infection or artificial inoculation in disease nurseries. We then prioritized 15 lines for molecular marker analysis to determine the likelihood that these lines possess novel resistance QTL that have not been previously mapped.
Targeting essential genes in Sclerotinia sclerotiorum to achieve Sclerotinia stem rot resistance in soybean. We took advantage of our bean pod mottle virus (BPMV) virus-induced gene silencing (VIGS) system to deliver a target sequence into soybean. Studies were conducted to show that sRNAs corresponding to our target fungal gene do accumulate in soybean and this leads to decreased lesion size in Sclerotinia-challenged plants. We also identified additional fungal targets that will be further tested using host-induced gene silencing (HIGS) in the upcoming year. Namely, genes involved in the detoxification of plant secondary metabolites will be explored.
Understanding how sunflower soil microbiome impacts resistance to Sclerotinia stalk rot. We have isolated 62 bacterial strains from sunflower rhizosphere soils. These will be used for assessment of defense against Sclerotinia in in vitro and in vivo assays. Close relatives of taxa-of-interest in the global soil database were identified as common across multiple habitats and soil types. However, these were not universal, suggesting that specific environmental conditions at each field site may play a role in the abundance of each taxa.
Characterizing pathogenicity effectors of Sclerotinia sclerotiorum preferentially expressed under acidic conditions and during plant infection. After showing that the Sclerotinia extracellular effector SsE1 (a protein that helps the fungus cause disease) and its polygalacturonase SsPG1 both interact with plant polygalacturonase-inhibiting protein (AtPGIP1), we used three methods to demonstrate that SsE1 has a higher affinity with PGIP1 than does SsPG1. Higher affinity with AtPGIP1 explains the effectiveness of SsE1 in reducing the defense mechanisms of the plant. Additionally, we tested the functions of SsE1 in transgenic plants. Results showed expression of SsE1 in plants increased susceptibility to infection by three different pathogen genotypes (wildtype strain, SsE1- and SsPG1-deletion mutants).
Biological control of white mold using the mycovirus SsHADV-1-infected hypovirulent strain DT-8 of Sclerotinia sclerotiorum. The SsHADV-1 mycovirus was transmitted to U.S. isolate A1 from pea to produce a derived strain A1V3-1 containing the mycovirus. Strain A1V3-1 was then used as a donor to transmit the virus to additional U.S. isolates (OD29-2V1 from sunflower and 05LD1V2 from lentil). All derived Sclerotinia strains harboring the SsHADV-1 mycovirus became non-pathogenic (hypovirulent) and all enabled white mold resistance in bean plants when co-infected with virulent strains of Sclerotinia. Additionally, we developed two inoculation techniques for delivering SsHADV-1 harboring strains - liquid inoculum for treating the seeds before planting and solid inoculum (colonized millet seeds) for inoculating seeds at planting.
Developing gemycircularvirus-based pesticide for the control of Sclerotinia sclerotiorum. We successfully rescued the mycovirus, determined the host range, and extracted the viral particles for transmission electron microscopy image analysis as the confirmation of viral replication in Sclerotinia. We expressed two viral genes, SlaGemV-1 and SsHADV-1, separately in either Botrytus cinerea or S. sclerotiorum. In B. cinerea, site-directed gene-displacement was achieved due to the availability of expression vector. In S. sclerotiorum, multiple mutants having gene expression were obtained. To test the mycoviruses as natural biopesticides delivered by Sclerotinia in field trials, we produced SlaGemV-1 and SsHADV-1 infected Sclerotinia soil inoculum from autoclaved millet. Soybean seeds treated with soil inoculum at planting showed resistance to the challenge of white mold at an early vegetative stage, using the cut-stem pipet tip agar plug approach.
Role of WRKY transcription factors in quantitative resistance to Sclerotinia sclerotiorum. We completed evaluations of T-DNA insertion mutants impaired in each of five selected transcription factors (from a family of transcription families called WRKY) for their response to Sclerotinia. Leaves were spot inoculated with a mycelial suspension of Sclerotinia isolate 1980 and disease was evaluated at 4 days post-inoculation (DPI) and 7 DPI using a standard rating scale. At both 4 and 7 DPI, wrky4-2 mutant plants were more susceptible to Sclerotinia than the Col-0 parent ecotype, indicating that WRKY4 positively influences resistance to Sclerotinia. Additionally, at both 4 and 7 DPI, wrky27 mutant plants showed significantly increased resistance to Sclerotinia compared to the Col-0 parent line, indicating that WRKY27 negatively affects Arabidopsis resistance to Sclerotinia. We then evaluated gene expression levels of WRKY4 and WRKY27 in resistant and susceptible Arabidopsis ecotypes both in untreated plants and after inoculation with Sclerotinia. WRKY4 transcript levels in untreated plants (steady-state expression levels) showed some variation between ecotypes, but no pattern that would suggest an association with resistance or susceptibility. In contrast, a clear difference between resistant and susceptible ecotypes was observed at 24 hours post-inoculation (hpi) with Sclerotinia isolate 1980. In four resistant ecotypes, WRKY4 expression was upregulated approximately 2-3-fold at 24 hpi compared to mock-inoculated control plants.
Accomplishments
1. Biological control of white mold disease using a fungal-infecting mycovirus. The fungal pathogen Sclerotinia sclerotiorum causes significant disease in over 400 plant species, including several economically important crop plants. Resistance to the pathogen is limited or nonexistent in many crops, so chemical (fungicides) or biological control is needed to prevent significant crop losses. ARS scientists working cooperatively in Pullman, Washington, and Fargo, North Dakota, studied a unique mycovirus that infects Sclerotinia and reduces its ability to cause disease. The mycovirus was transferred to a number of Sclerotinia strains and when bean plants were co-inoculated with these strains, along with pathogenic strains that would normally cause disease, the bean plants showed resistance to white mold disease. The research demonstrates that this mycovirus can now be tested as an environmentally friendly control agent for several crop species where inherent genetic white mold resistance by that crop is limited.
Review Publications
Chiniquy, D., Underwood, W., Corwin, J., Ryan, A., Szemenyei, H., Cherk Lim, C., Stonebloom, S.H., Birdseye, D.S., Vogel, J., Kliebenstein, D., Scheller, H.V., Somerville, S. 2019. PMR5, an acetylation protein at the intersection of pectin biosynthesis and defense against fungal pathogens. Plant Journal. 100(5):1022-1035. https://doi.org/10.1111/tpj.14497.
Smart, B.C., Koehler, B.D., Misar, C.G., Gulya, T.G., Hulke, B.S. 2019. Registration of oilseed sunflower germplasms HA 482, RHA 483, and RHA 484, selected for resistance to Sclerotinia and Phomopsis diseases. Journal of Plant Registrations. 13(3):450-454. https://doi.org/10.3198/jpr2019.07.0030crg.
Koehler, B.D., Gulya, T.J., Hulke, B.S. 2019. Registration of oilseed sunflower germplasms RHA 478, RHA 479, RHA 480, and HA 481, providing diversity in resistance to necrotrophic pathogens of sunflower. Journal of Plant Registrations. 13(3):444-449. https://doi.org/10.3198/jpr2019.04.0017crg.
Attanayake, R.N., Xu, L., Chen, W. 2018. Sclerotinia sclerotiorum populations: Clonal or recombining? Tropical Plant Pathology. 44:23-31. https://doi.org/10.1007/s40858-018-0248-7.
