Location: Sunflower and Plant Biology Research
2017 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 Project Number 3060-21220-031-00D, which started at the beginning of February 2017 and continues research from Project Number 3060-21220-028-00D, entitled “Sclerotinia Initiative.” Research conducted in the past reporting period was focused specifically on the pathogen, Sclerotinia sclerotiorum, or on one of the following susceptible crops: sunflower, canola, soybeans, edible dry beans, or peas.
Sclerotinia progress. Genotype-by-sequencing (GBS) was used to genotype a population of Sclerotinia sclerotiorum collected from diverse crops and the data were utilized to conduct an association mapping study on aggressiveness. Our initial goal was to develop ~10,000 single nucleotide polymorphism (SNP) markers at unique loci across the 38 Mb genome of S. sclerotiorum, placing a SNP marker at about 4 kb intervals across the genome. We did not achieve this level of saturation; however, the density of markers that we did achieve was adequate for association mapping to identify genes underlying loci contributing to phenotypic variation for pathogen aggressiveness. We also provided preliminary validation of candidate virulence genes by RNAseq and are well on our way to functional analysis in-planta using host induced gene silencing.
In other Sclerotinia-focused studies, isolates sampled from replicated screening nurseries across bean-production areas over several years allowed us to determine if pathogen variation in test plots influence the identification of bean resistance between sites. Genotyping of 366 S. sclerotiorum isolates was performed using microsatellite (SSR) markers. Aggressiveness was assessed for 366 isolates genotyped and ongoing fungicide sensitivity tests to help identify phenotypic variation. We are testing the hypothesis that isolates collected from screening nursery sites and those used in greenhouse tests show similar phenotypic and genotypic variability when compared to isolates collected from grower fields in the same region.
Sunflower progress. In the past year, we finished whole genome sequencing of 150 inbred sunflower lines and several important parental lines that have contributed to the development of new sunflower lines in the USDA breeding program since 2008. These results, combined with other public data and the over 1000 lines for which we have generated genotype-by-sequencing data provides us the basis for calling genotype variants and filling in missing data with trio imputation. We have finished development of an improved bioinformatic pipeline to do this over the last year, including development of our own imputation algorithm that takes into account whole genome parental data and data on multiple, adjacent SNP sites as haplotypes.
In sunflower breeding work, wild perennial Helianthus species that are highly resistant to Sclerotinia stalk and head rot are being employed. Six interspecific amphiploids have shown excellent stalk and head rot resistance. In order to introgress the resistance genes from wild Helianthus species, crosses and backcrosses have been conducted between amphiploid, hexaploid, tetraploid and diploid perennials with cultivated sunflower.
Field trials seeded to sunflowers were established at the North Dakota State University (NDSU) Carrington Research Extension Center and at the NDSU Robert Titus Research Farm in Oakes. At each location, two sets of research trials were established: (1) Studies evaluating the impact of the timing of Sclerotinia head rot disease development on the effectiveness of fungicides for control of Sclerotinia head rot and (2) studies screening commercial sunflower breeding lines and hybrids for susceptibility to Sclerotinia head rot. The goals of the fungicide studies were to optimize fungicide applications relative to the timing of weather conditions favorable for Sclerotinia head rot and to quantify the impact of early, intermediate, and late disease onset on Sclerotinia head rot disease development and seed yield and quality of sunflowers under head rot disease pressure. The goals of the resistance screening nurseries were to develop screening methodologies that improved the replicability of results.
Under heavy Sclerotinia head rot disease pressure, applications of a prothioconazole fungicide at 5.7 fl oz/ac conferred an average yield increase of 374 lbs/ac with fungicide applications over the top of the sunflower canopy and an average yield increase of 623 lbs/ac with fungicide applications through drop nozzles. Fungicides were applied at mid-bloom (average R5.4-R5.7) and similar yield responses were observed irrespective of whether the sunflowers were inoculated 2 to 4 days earlier or 1 to 3 days later. End-of-season disease levels were not always reduced, suggesting that the fungicide application may have slowed disease development (thereby permitting additional seeds to fill, increasing yields), but did not stop it.
Canola progress. Activities reported here are associated with the development and validation of the use of molecular markers associated with genes conferring partial resistance to S. sclerotiorum to canola lines; and to the identification and functional characterization of S. sclerotiorum genes associated with pathogenicity in canola. Cleaved Amplified Polymorphic Sequence (CAPS) markers for four SNPs linked to SSR resistance QTL were developed. Quantitative PCR-based high resolution melt (HRM) assays were also developed to readily identify the polymorphisms.
In canola breeding work, crosses between NEP63 and lines WC1417 and WC1421 were made in December 2016. F1 seeds from these crosses were planted in the greenhouse earlier this summer and the F1 plants will be allowed to self-pollinate to produce F2 seeds. The F2 seeds will be planted in the fall to be screened for their reaction to S. sclerotiorum. Resistant materials will be identified and backcrossed to the susceptible parent.
In order to engineer high levels of disease resistance in canola, we are exploiting a newly identified Arabidopsis thaliana gene hypersusceptible to S. sclerotiorum that encodes the HSS1/Med16 protein. Arabidopsis transgenic lines co-expressing HSS1 and ODC2 for resistance to S. sclerotiorum have been developed for resistance characterization studies.
Soybean progress. In the last reporting period, two soybean cultivars with partial Sclerotinia stem rot (SSR) resistance were released. One of them, E12076T, was licensed by a seed company for commercial production. Three additional seed companies requested seeds of E12076T for pre-commercialization evaluations.
One hundred ten new advanced breeding lines and sixty four soybean plant introductions (PIs) were evaluated for SSR in a naturally infected field. Among the advanced breeding lines, 20 showed a high level of resistance with a disease severity index (DSI) rating less than 10. Sixty-one PIs showed a high level of resistance to white mold with a DSI less than 10 and confirmed as new sources of resistance to SSR.
Edible dry bean progress. Studies were conducted to identify and characterize quantitative trait loci (QTL) for resistance to white mold from scarlet runner bean (P. coccineus) and common bean. QTL analyses were conducted in two bi-parental populations A195/OSU6137 (AO) and G122/WMG904-20-3 (GW) for disease reaction in the field at the Oregon State University (OSU)-Vegetable Research Farm in 2015 and 2016, and greenhouse (seedling straw test). Six QTL associated with white mold resistance were detected in the AO population. Three QTL on linkage groups Pv01 (WM1.1), Pv03 (WM3.3) and Pv09 (WM9.3) were detected in the field and three others on Pv01 (WM1.3), Pv05 (WM5.5), and Pv07 (WM7.4) were detected in the straw test. One significant QTL was identified in GW population on Pv08 (WM8.1) for both field and seedling straw test. More QTL were detected in AO because it was a resistant x susceptible, while GW was a resistant x resistant cross.
A genome wide association study (GWAS) was conducted on the 376 cultivar Snap Bean Association Panel (SnAP). The panel was evaluated for white mold resistance using seedling straw test and genotyped using genotyping by sequencing (GBS). From 40,000 SNP markers generated, 20 SNPs were statistically associated with white mold resistance. Some SNPs align with known QTL for resistance and others appear novel. The SnAP GWAS identified Unidor wax bean cultivar as highly resistant in two years of field tests.
Work was performed to complete the development of the WM-MAGIC population (White Mold - Multi-parent Advanced Generation Inter-Cross). The individual F2:5 bean plants are currently growing in the greenhouse and seed from each plant will be bulk harvested. More than 1000 lines are expected in this population, which will provide excellent resolution for genetic studies. The bulked seed will be tested for reaction to white mold in the straw test in 2017/2018 and further increased for seed for white mold field nurseries in 2018.
Pea progress. We have been utilizing next generation sequencing technologies to investigate the host-pathogen interaction between P. sativum and S. sclerotiorum. In initial experiments, an expressed sequence tag (EST) data set was developed with massively parallel sequencing on a 454 Roche platform. This data set has been analyzed and a manuscript has been published. Also, SSR (microsatellite) markers were identified in the 454 sequence data from pea and were utilized to develop markers that were screened for polymorphism across 23 pea individuals including parents from 4 recombinant inbred lines. A manuscript describing these markers was published. Additionally, we analyzed the gene expression profiling via RNAseq, on the interaction between two different lines of pea and S. sclerotiorum, specifically during the early stages of infection. We are using these results to map potential resistance genes to QTL’s, as well as to identify S. sclerotiorum genes involved in pathogenicity.
Accomplishments
1. Meta-analysis of Sclerotinia resistance gene locations in common bean. Sclerotinia sclerotiorum, a fungal pathogen that causes white mold, is a major disease that limits common bean production and quality worldwide. The interaction between the plant and pathogen is complex, with partial resistance in the plant requiring multiple resistance genes that confer a suite of resistance traits. Previous studies have identified several locations in the bean genome where these genes reside. In this study, cooperating scientists at North Dakota State University, Oregon State University, and ARS in Prosser, Washington conducted a meta-analysis of these genomic locations from different bean populations to identify regions with high confidence for Sclerotinia resistance. The candidate regions found in this meta-analysis are recommended as potential targets to breed for partial resistance to white mold in common bean.
Review Publications
Foley, M.E., Dogramaci, M., West, M.S., Underwood, W.R. 2016. Environmental factors for germination of Sclerotinia sclerotiorum sclerotia. Journal of Plant Pathology & Microbiology. doi:10.4172/2157-7471.1000379.
Tock, A., Fourie, D., Walley, P.G., Holub, E.B., Soler, A., Cichy, K.A., Pastor Corrales, M.A., Song, Q., Porch, T.G., Hart, J.P., Vaconcellos, R., Vicente, J.G., Barker, G.C., Miklas, P.N. 2017. Genome-wide linkage and association mapping of halo blight resistance in common bean to race 6 of the globally important bacterial pathogen. Frontiers in Plant Science. 8:1170. https://doi.org/10.3389/fpls.2017.01170.
Zahirul, T.I., Seiler, G.J., Song, Q., Ma, G., Qi, L. 2016. SNP discovery and QTL mapping of Sclerotinia basal stalk rot resistance in sunflower using genotyping-by-sequencing (GBS). The Plant Genome. 9(3). doi:10.3835/plantgenome 2016.03.0035.
Qi, L., Long, Y., Talukder, Z., Block, C., Gulya, T.J. 2016. Genotyping-by-sequencing uncovers the introgression alien segments associated with Sclerotinia basal stalk rot resistance from wild species—I. Helianthus argophyllus and H. petiolaris. Frontiers in Genetics. doi:10.3389/gene.2016.00219.
Underwood, W. 2016. Contributions of host cellular trafficking and organization to the outcomes of plant-pathogen interactions. Seminars in Cell and Developmental Biology. 56:163-173.
