Location: Sunflower and Plant Biology Research
2018 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.
Common Bean Studies: A new small red bean variety ‘Cayenne’ was released in 2017 as an upright cultivar that possesses excellent canning quality, tolerance to common bacterial blight and white mold. Cayenne combines high yield potential with resistance to lodging and high pod placement within the canopy making it suitable for direct harvest under narrow row production systems.
Efforts were carried out to define the white-mold (WM) WM2.2 quantitative trait loci (QTL) position in common bean through a meta-QTL analysis. The QTL region was saturated with 60 indel markers. Another population was developed to map WM2.2 along with WM8.3. Development of five recombinant inbred pinto bean populations was continued to validate the effects of major genes/QTL WM2.2, WM3.1, and WM5.4.
To transfer and validate WM resistance factors from Phaseolus coccineus and Phaseolus vulgaris in common bean breeding lines, nested association mapping, genome wide association study (GWAS) and recombinant inbred lines (RILs) were used for QTL mapping. A greenhouse seedling straw test was developed and validated that allows inoculation approximately one week after planting and the test proceeds quickly with readings in about 4-8 days.
Coordinated multi-site field and greenhouse screening of dry and snap bean breeding lines were continued. In 2017/2018, 95 Sclerotinia sclerotiorum isolates were genotyped from geographically divergent grower fields adding to a previous survey of 366 isolates. Aggressiveness data obtained with the straw test method was completed. A preliminary set of 120 isolates was used to characterize sensitivity to eight fungicides.
Soybean Studies: New advanced breeding lines were evaluated for soybean stem rot (SSR) resistance in a naturally infected field. The top 10 lines showed high resistance. F1 plants from 8 crosses between new resistance sources and elite cultivars were grown and about 1000 F2 lines were genotyped with SNPs for disease resistance. Seeds of a new cultivar (E12076T) with enhanced resistance to SSR was released to a seed company to increase seed for commercial production.
Studies were executed with the parents and progeny of a Glycine latifolia (soybean wild relative) RIL population to standardize inoculation and assay conditions for evaluation of sensitivity to S. sclerotiorum. Stem cuttings were evaluated for sensitivity to oxalic acid. Leaf samples from the evaluated RILs were collected for marker analysis.
Pea, Chickpea, Lentil Studies: Newly acquired cool season grain legume germplasm was propagated to generate enough seeds for pathogenicity screening. Thus far, 69 accessions have been tested for resistance to white mold and three accessions showed slower disease onset or progression, relative to a moderately resistant cultivar.
Next generation sequencing technologies were used to investigate the host-pathogen interaction between Pisum sativum and S. sclerotiorum. An expressed sequence tag (EST) data set was developed and SSR markers were identified to screen for polymorphism across 23 pea individuals including parents from 4 recombinant inbred lines. On the pathogen side, de novo assembly and analysis of RNA-seq data from S. sclerotiorum has been conducted.
Canola Studies: CAPS markers for four SNPs linked to SSR resistance QTL were developed and have been validated using 15 North Dakota State University (NDSU) Brassica napus breeding lines. Knock-out mutants for four candidate genes were evaluated using detached leaf assays and stem inoculations. Deletion of cytochrome P450 and SsBGT1 resulted in significant reduction in colony diameter and mycelial density on synthetic media. Deletion of cytochrome P450 also resulted in no lesion formation on stems of canola plants.
Crosses between NEP63 and elite breeding line 14B17 were made. F1 seeds have been planted and F2 seeds will be harvested in 2018. Three disease tolerant breeding lines (NDC-E15234, NDC-E15174, NDC-E15275) were identified using the petiole inoculation method and were crossed with existing canola cultivars. Also, 20 tolerant/resistant lines were identified in a group of 229 accessions. Lines PI633119, PI436554, PI458940, and PI169080 were crossed with cultivar Westar.
A newly identified Arabidopsis thaliana gene, hypersusceptible to S. sclerotiorum (HSS1), was tested to engineer resistance to white mold. The BnHSS1 gene was expressed in the Arabidopsis hss1 mutant and several lines were found that restored the hypersusceptible phenotype to wild-type levels. A related gene from rice (OsMED16) also complemented the hypersusceptibility phenotype and efforts to screen additional homologs from other nonhost monocot species are underway.
Sunflower Studies: Whole genome sequencing of an open pollinated variety association panel, as well as new inbred and breeding lines in the USDA sunflower breeding program were finished. GWAS will be used to develop an improved sunflower map. A rhizosphere data set was developed that revealed a consortium of 40 rhizosphere bacterial taxa that are negatively associated with Sclerotinia infection in sunflower.
A sunflower RIL population was evaluated for basal stem rot (BSR) resistance and genotyped to discover single nucleotide polymorphism (SNP) markers and six BSR resistance QTL. A highly BSR tolerant germplasm was released. Successful transfer of BSR resistance from wild annual Helianthus species into cultivated sunflower background was achieved. Seven wild Helianthus introgression lines were released with high levels of BSR resistance. Seven Sclerotinia head rot and BSR resistant germplasm bulks derived from five perennial species were released.
Evaluation of the aggressiveness of 253 S. sclerotiorum isolates on stem tissue of two sunflower inbred lines, HA 207 and HA 441, was completed. Aggressiveness differed across sunflower genotypes. Sequencing for 227 isolates was completed and about 6,000 SNP markers were identified. Marker replacement DNA constructs have been developed for evaluation of six candidate virulence genes.
Two inoculated nurseries for sunflower basal stalk rot were established in 2017 to evaluate factors influencing successful white mold disease development. Mulching of plots with straw to improve moisture retention and temperature regulation proved to be beneficial at the Carrington, North Dakota location. A greenhouse-based stalk rot phenotyping procedure was developed and validated on a panel of 30 sunflower genotypes.
A mist-irrigated inoculated Sclerotinia head rot nursery at Staples, Minnesota was used to evaluate 450 rows of sunflower germplasm and wild annual and perennial species, as well as advanced generation breeding lines. An inoculated stalk rot nursery at Staples, Minnesota was also used to evaluate 1900 rows of sunflower germplasm. Stalk rot disease pressure was poor for a second year at this location, possibly due to sandy soil with low organic content.
The relative aggressiveness of a panel of 20 diverse S. sclerotiorum isolates in causing mid-stem lesions on 10 USDA sunflower inbred lines was undertaken. Results indicate that isolates do not necessarily behave in a uniform manner on different sunflower genotypes and suggest putatively resistant sunflower germplasm lines should be evaluated against multiple isolates to determine if resistance is broad-spectrum or limited to one or a few specific isolates.
In 2017, a mist-irrigated inoculated Sclerotinia head rot nursery at Carrington, North Dakota was established to evaluate 500 rows of sunflower germplasm and an inoculated stalk rot nursery at Carrington, North Dakota was established to evaluate 2320 rows of experimental sunflower germplasm. Both head rot and stalk rot disease pressure were excellent, providing useful data for multiple projects.
Management Studies: Activities continued to quantify the impact of Sclerotinia infection timing on seed yield, seed quality, disease severity and optimal fungicide usage in pinto beans (Sclerotinia stem rot), soybeans (Sclerotinia stem rot), and sunflowers (Sclerotinia head rot). Major findings from the sunflower screening nurseries suggest that individually inoculating every sunflower head twice, once as it reaches the R5.4-R5.6 growth stage and again 2-3 days later, confers highly replicable results.
A white mold biocontrol mycovirus strain has been imported from China. Efforts have discovered the best medium for inoculum preparation to ensure high titer of the mycovirus in the fungal mycelium and efficient means to detect the mycovirus. Nutrient-poor medium was found to give the best results.
Sclerotinia Fungal-Specific Studies: Using genetically defined mutants defective at the oxaloacetate acetylhydrolase (oah) gene, studies showed that oah mutants lost oxalate production, but did accumulate fumaric acid in culture and remained pathogenic on some legume plants. The oah mutants also produced higher levels of fumaric acid as ambient pH increased.
A collection of 360 Arabidopsis accessions is being evaluated for S. sclerotiorum resistance and GWAS mapping of Sclerotinia resistance is being conducted. Initial results suggest that resistance is governed by genes involved in defensive secondary metabolite biosynthesis, pathogen detection, regulation of hormone metabolism and signaling, and cell wall modification.
Efforts were undertaken to demonstrate the existence of RNA silencing mechanisms in S. sclerotiorum by infecting wild-type and RNA-silencing-deficient fungal strains with an RNA and a DNA virus. Key silencing-related genes (two dicer genes) were disrupted. Disruption mutants showed that S. sclerotiorum has robust RNA silencing mechanisms that process both DNA and RNA mycoviruses.
Accomplishments
1. Development and validation of a greenhouse-based method for evaluating sunflower basal stalk rot. Progress in breeding for improved resistance to Sclerotinia basal stalk rot, a fungal disease affecting sunflower, is severely limited by available methods to consistently and accurately assess sunflower germplasm for this disease. ARS scientists in Fargo, North Dakota, in collaboration with scientists at North Dakota State University, developed a new greenhouse-based method to evaluate sunflower germplasm resources for Sclerotinia basal stalk rot. This method closely follows field observations and provides reasonable throughput for genetic studies. The method can also potentially be adopted by commercial breeders with modest laboratory and greenhouse facilities to accurately evaluate priority breeding lines and germplasm resources for this disease.
2. Release of sunflower germplasms with enhanced white mold resistance and high yield potential. Sunflower breeders and seed companies require germplasm resources that combine sources of disease resistance with other agronomic traits in high yield-potential genetic backgrounds. Through breeding and field evaluations, ARS scientists in Fargo, North Dakota, in cooperation with collaborators at North Dakota State University Carrington Research Extension Center and Central Lakes College Ag and Energy Center, developed three female and four male sunflower lines with improved white mold (Sclerotinia) resistance and high yield potential, along with other important agronomic traits such. These lines can be used by seed companies to develop new high-yielding commercial hybrids with improved tolerance to white mold and other fungal diseases.
Review Publications
Kelly, J., Varner, G., Miklas, P.N., Cichy, K.A., Wright, E. 2018. Registration of 'Cayenne' small red bean cultivar. Journal of Plant Registrations. 12:194-198. https://doi.org/10.3198/jpr2017.05.0033crc.
Hulke, B.S., Ma, G., Qi, L.L., Gulya, T.J. 2018. Registration of oilseed sunflower germplasms RHA 461, RHA 462, RHA 463, HA 465, HA 466, HA 467, and RHA 468 with diversity in Sclerotinia resistance, yield, and other traits. Journal of Plant Registrations. 12:142-147. https://doi.org/10.3198/jpr2017.04.0023crg.
