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ARS Home » Plains Area » Fargo, North Dakota » Edward T. Schafer Agricultural Research Center » Sunflower and Plant Biology Research » Research » Research Project #432211
Research Project: Sclerotinia Initiative

Location: Sunflower and Plant Biology Research

2019 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. Dry bean research: To identify, transfer, and validate white mold (WM) resistance factors from Phaseolus coccineus and Phaseolus vulgaris in common bean breeding lines, a nested association mapping (NAM) population was evaluated for WM in the field for a 2nd year; genotyping is under way. Studies with the Unidor/5630 population (differential WM resistance) identified one quantitative trait locus (QTL) that was mapped to bean chromosome Pv02. Efforts continued to define meta-QTL for resistance to white mold in common bean and to verify their phenotypic effect. Three Andean lines ADP-0014, -0436, and -0734 were studied and continue to perform well in the straw test. Physical regions of the common bean genome associated with major white mold resistance QTL were defined to investigate candidate genes. Work continued toward fine-mapping the WM2.2 QTL. To identify and/or verify resistance to white mold in wide dry bean crosses and adapted dry and snap bean lines, greenhouse and field trials (multiple locations) were conducted using pinto, navy, black, and small red bean seed classes. In FY19, 11 seed lines were received (9 for field and 11 for greenhouse screening) from four breeders. To identify pathogen phenotypic variation that may guide new bean line breeding, data from 366 Sclerotinia sclerotiorum isolates were genotyped and analyzed. Soybean research: A long-term goal of this research is to develop soybean varieties or germplasm with a high level of resistance to soybean white mold (SWM). Marker-assisted selections (MAS) were carried out for screening F2:3 lines, which were developed from 8 cross combinations between new resistance sources and elite cultivars. New advanced breeding lines (132) were evaluated for SWM in a naturally infected field. Among those lines, 16 lines showed high level of resistance with disease severity index (DSI) less than 10. For germplasm release, line E12076T with enhanced resistance to SWM was increased by a seed company to enable release of seeds for commercial production. The reliability of a genomic selection (GS) model was maximized by optimizing statistical methods and the choice of reference individuals in a training set. Eighty advanced breeding lines were evaluated for white mold resistance in a disease nursery and genotyped with SoySNP6K BeadChip. Efforts were carried out to target essential genes in S. sclerotiorum to achieve sclerotinia stem rot (SSR) resistance in soybean. Host Induced Gene Silencing (HIGS) was used to target an important virulence factor, oxalic acid (OA), to debilitate S. sclerotiorum growth and pathogenic development. The RNA silencing machinery of the soybean plant was recruited to target an essential fungal gene for OA biosynthesis, Ssoah1 (oxaloacetate acetyl hydrolase). Targeting this gene in S. sclerotiorum leads to limited lesions on soybean plants, suggesting that HIGS can be a viable strategy to control SSR. Chickpea, lentil, and pea research: Recently acquired germplasm accessions for chickpea, lentil and pea are being screened for new resistance sources to Sclerotinia white mold. In FY19, recently acquired pea germplasm accessions from China were screened for resistance to white mold in the greenhouse, with plant reactions to Sclerotinia inoculation monitored by measuring the lesion length. Ten accessions showed disease severity lower than the tolerant cultivar DPS used as a check. The most tolerant lines were W6 44561 and W6 44593 originally from Inner Mongolia and the Qinghai provinces of China, respectively. Sunflower research: A small graphical user interface (GUI) software package that integrates the best genomic selection models for sunflower is being developed. Many of the R packages typically used for GS are not flexible enough to deal with a hybrid crop where genotypic effects are subdivided into “female” and “male” line effects (GCA and SCA) for single cross hybrid data; However, we found new packages, including ‘BGLR’ and ‘sommer’ that are more flexible in the number of random effects that can be handled. An updated (v.2) GWAS model to infer candidate genes and develop hypotheses on necrotrophic disease resistance architecture was used to find 7 major stalk rot resistance loci, 4 major head rot loci, 7 major Phomopsis stalk canker resistance loci (another important necrotrophic disease) and a sizeable number of low to moderate effect loci. QTL mapping of Sclerotinia basal stalk rot (BSR) resistance derived from sunflower wild species was continued with three advanced backcross (AB) populations. Field screening trials were conducted at Carrington, North Dakota, using randomized incomplete block design with three replications. Highly significant (p<0.001) genetic variations were observed for BSR disease incidence in all three populations in both individual and combined analysis. A genetic linkage map was developed to identify QTL on twelve linkage groups (LGs) associated with BSR resistance for the three data sets. Canola research: To develop effective and durable disease resistance for Sclerotinia stem rot in canola, overexpression of MED16, a subunit of the Mediator protein complex that functions in transcriptional regulation of defense genes, is being used. To determine the molecular basis of OsMED16 resistance, an assay was developed to determine proteolytic sensitivity of MED16 orthologs, because MED16 is shown to be degraded during infection. The MED16 signaling cascade was also being engineered to test for a synergetic increase in resistance. Previous research had identified AtWRKY33 as an interactor of MED16 and thus efforts are underway to identify a WRKY33 ortholog from rice that could be used in combination with the resistance-conferring MED16, also from rice. A mapping population derived from the cross of canola cultivars NEP63 (resistant) x Topas was developed using doubled haploid technique. This population will be evaluated in 2019. A second population derived from NEP32 x Topas is being produced and we expect to have it ready for evaluation late in 2019. These populations will be used for further validating Cleaved Amplified Polymorphic Sequence (CAPS) markers for resistance to Sclerotinia. Sclerotinia pathogen biology research: A previously identified S. sclerotiorum effector protein (SsE1) that interacts with the plant defense protein polygalacturonase-inhibiting protein (PGIP) was studied to verify the specific interaction and to demonstrate its function. SsE1 has the same expression pattern as S. sclerotiorum polygalacturonase 1 (SsPG1) and the importance of SsPG1 in virulence was shown through deletion analyses. This research provides the first example that S. sclerotiorum secretes a small effector protein (SsE1) that specifically interacts with and mitigates the inhibitory effects of plant PGIPs. Disease management research: Studies with mutants of S. sclerotiorum genes encoding disrupted homologues of Dicer-like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerase (RdRp) proteins were conducted to assess alterations in growth, sclerotial development, pathogenicity on plants and sensitivity to infection by mycoviruses. Wild-type and mutant strains were transfected with a single-stranded RNA virus, SsHV2-L, and copies of a single-stranded DNA mycovirus, SsHADV-1, as a synthetic virus constructed in this study. Disruption of dcl-1 or dcl-2 resulted in no changes in phenotype compared to wild-type S. sclerotiorum; however, the double dicer mutant strain exhibited significantly slower growth. Furthermore, the delta dcl-1/dcl-2 double mutant, which was slow growing without virus infection, exhibited much more severe debilitation following virus infections. Deletion of ago-2 gene but not ago-4 resulted in compromised growth and virulence prior to virus infection suggesting the contributions made by ago-2 to physiological and developmental processes. The feasibility of using the hypovirulent strain DT-8 of S. sclerotiorum as an option in managing Sclerotinia white mold is being tested. Several experiments were carried out by spraying the DT-8 mycelium homogenates onto detached leaves before inoculating with a virulent strain, anticipating that the virus particles will enter the virulent strain and render it less virulent. Some trials showed promise, but difficulties with virus titer lead to inconsistencies in the results. New procedures will be developed to improve our culturing of the DT-8 mycovirus before further plant tests are executed.


Accomplishments
1. Soybean varieties developed with resistance to white mold disease. Soybean white mold (SWM) is caused by Sclerotinia sclerotiorum, a fungus capable of infecting a wide range of plants. Resistance mechanisms within a crop plant, like soybean, can play a key role in effective management of SWM. Modern breeding technologies, including the use of molecular markers, have been used to incorporate white mold resistance traits with other agronomic and seed quality traits. Soybean line E14077 is in progress to be released as a new variety in 2019 and line E13268 is under development for future release, both having good resistance to SWM. Another newly developed soybean line, E12076T, exhibiting enhanced resistance to SWM was released to a seed company for commercial production in 2019, such that seeds would be available to farmers in 2020.


Review Publications
Westrick, N.M., Ranjan, A., Jain, S., Grau, C.R., Smith, D.L., Kabbage, M. 2019. Gene regulation of Sclerotinia sclerotiorum during infection of Glycine max: on the road to pathogenesis. BMC Genomics. 20:157. https://doi.org/10.1186/s12864-019-5517-4.
 Mochama, P., Jadhav, P., Neupane, A., Marzano, S.-Y.L. 2018. Mycoviruses as triggers and targets of RNA silencing in white mold fungus Sclerotinia sclerotiorum. Viruses. 10:214. https://doi.org/10.3390/v10040214.
 Kandel, R., Chen, C.Y., Grau, C.R., Dorrance, A.E., Liu, J.Q., Wang, Y., Wang, D. 2018. Soybean resistance to white mold: evaluation of soybean germplasm under different conditions and validation of QTL. Frontiers in Plant Science. 9:505. https://doi.org/10.3389/fpls.2018.00505.
 Wang, C., Zhang, X., Li, J., Zhang, Y., Mou, Z. 2016. The Elongator complex-associated protein DRL1 plays a positive role in immune responses against necrotrophic fungal pathogens in Arabidopsis. Molecular Plant Pathology. 19(2):286-299. https://doi.org/10.1111/mpp.12516.
 Liang, X., Rollins, J.A. 2018. Mechanisms of broad host range necrotrophic pathogenesis in Sclerontinia sclerotiorum. Phytopathology. 108:1128-1140. https://doi.org/10.1094/PHYTO-06-18-0197-RVW.
 Neupane, A., Feng, C., Feng, J., Kafle, A., Bucking, H., Marzano, S.-Y.L. 2018. Metatranscriptomic analysis and in silico approach identified mycoviruses in the arbuscular mycorrhizal fungus Rhizophagus spp. Viruses. 10:707. https://doi.org/10.3390/v10120707.
 Wen, Z., Tan, R., Zhang, S., Collins, P.J., Yuan, J., Du, W., Gu, C., Ou, S., Song, Q., An, Y., Boyse, J.F., Chilvers, M.I., Wang, D. 2018. Integrating GWAS and gene expression data for functional characterization of resistance to white mould in soya bean. Plant Biotechnology. https://doi.org/10.1111/pbi.12918.
 Liu, Q., Chang, S., Hartman, G.L., Domier, L.L. 2018. Assembly and annotation of a draft genome sequence for Glycine latifolia, a perennial wild relative of soybean. Plant Journal. 91:71-85.
 Marzono, S.L., Neupane, A., Domier, L.L. 2018. Transcriptional and small RNA responses of the white mold fungus Sclerotinia sclerotiorum to infection by a virulence-attenuating hypovirus. Viruses. 10:713. https://doi.org/10.3390/v10120713.
 Talukder, Z.I., Long, Y., Seiler, G.J., Underwood, W., Qi, L. 2019. Introgression and monitoring of wild Helianthus praecox alien segments associated with Sclerotinia basal stalk rot resistance in sunflower using genotyping-by sequencing. PLoS One. 14(3):e0213065. https://doi.org/10.1371/journal.pone.0213065.
 Talukder, Z.I., Long, Y.M., Seiler, G.J., Underwood, W., Qi, L.L. 2019. Registration of oilseed sunflower germplasms HA-BSR6, HA-BSR7, and HA-BSR8 highly resistant to sclerotinia basal stalk rot and downy mildew. Journal of Plant Registrations. https://doi.org/10.3198/jpr2018.10.0071crg.
 Money, K., Koehler, B.D., Misar, C.G., Grove, M.S., Underwood, W., Hulke, B.S. 2019. Registration of oilseed sunflower germplasms RHA 485, RHA 486, and HA 487, selected for resistance to Phomopsis stalk canker and Sclerotinia, in a high yielding and high-oil background. Journal of Plant Registrations. 13(3):439-442. https://doi.org/10.3198/jpr2019.02.0008crg.
 Qi, L.L., Talukder, Z.I., Long, Y.M., Seiler, G.J. 2018. Registration of oilseed sunflower germplasms HA-BSR2, HA-BSR3, HA-BSR4, and HA-BSR5 with resistance to sclerotinia basal stalk rot and downy mildew. Journal of Plant Registrations. https://doi.org/10.3198/jpr2017.11.0083crg.