Location: Sunflower and Plant Biology Research2009 Annual Report
1a. Objectives (from AD-416)
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.
1b. Approach (from AD-416)
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.
3. Progress Report
All milestones listed in question 2 comprise the Sclerotinia Initiative Strategic Plan. Each milestone was addressed in FY2009 by components of the 34 specific cooperative agreements and ARS projects funded from the Initiative. Research is ongoing in all areas and substantial progress has been made in Epidemiology and Disease Management and Variety Development & Germplasm Enhancement. Pathogen and Host Genomics research resulted in significant progress, but substantial efforts are still needed in genome sequencing of the pathogen and gene profiling of susceptible and resistant crops. Pathogen Biology and Development research is ongoing, but additional efforts are needed to identify disease infection processes and to characterize virulence among disease genotypes.
1. Evaluation of Wild Helianthus Species for Resistance to Sclerotinia Stalk Rot: The two most important diseases affecting sunflower in the U.S. are stalk rot and head rot caused by Sclerotinia sclerotiorum and genetic resistance is the most effective and practical means for managing this disease. Because the entire Helianthus genus is native to North America, researchers have a valuable opportunity to discover new genes. From June 2008 to May 2009, 235 wild sunflower accessions were evaluated in the greenhouse, including all available accessions of Helianthus argophyllus, H. debilis, H. exilis, H. neglectus, and H. praecox plus 45 accessions of H. annuus. Accessions with superior wilt resistance were found in all species except for H. exilis. Twenty accessions were entered into a 2008 ND field trial. Entries included susceptible and resistant germplasm from nine H. debilis accessions, three H. argophyllus, six H. annuus and two H. resinosus (perennial) accessions. The three most Sclerotinia-resistant entries were two H. resinosus accessions, PI 650079 and PI 650082, both with 100% survival and one H. argophyllus accession, PI 649863, with 94% survival. The resistant check, Croplan 305, had 88% survival while the susceptible check, Cargill 270, had 59% survival. Three of the H. annuus accessions had survival percentages equivalent to Croplan 305, PI 653604 (87%), PI 435434 (86%) and PI 435417 (85%). The excellent wilt resistance (near immunity) confirmed in the hexaploid species Helianthus resinosus deserves further exploration. In 2009, 25 accessions were entered into replicated field trials at Staples, MN and at Oakes, ND. Twelve accessions were repeats from 2008 and 13 accessions were new. Finding new Sclerotinia resistance genes is achievable in the wild species, and these genes, pyramided with downy mildew and rust resistance genes, will be transferred to cultivated sunflower.
2. Fungicide alternatives for Sclerotinia stem rot in canola. There is urgent need for developing a base line of fungicide sensitivity to common fungicides used for control of S. sclerotiorum, identifying fungicide mixtures that provide improved control on canola, and characterizing the value of new biological control compounds available in the U.S. market. A total of 113 isolates of Sclerotinia sclerotiorum, collected from ND between 1998 and 2008, have been evaluated for their sensitivity to thiophanate methyl and prothioconazole using mycelial growth as indicator. Six isolates collected before 1999 had a mean EC50 of 1.17 parts per million (ppm). Mean EC50 for isolates collected until 2004 (55 isolates evaluated) was 1.53 ppm and 1.56 for isolates collected between 2005 and 2007 – a nearly 30% increase in tolerance. Field trials were installed in Langdon and Carrington (ND) early in the summer of 2008 to evaluate the efficacy of fungicide mixtures as well as to evaluate new biological compounds for control of SSR. No disease developed in Carrington, but in Langdon scientists detected significant differences among treatments. A single application of the biofungicide Polyversum (3 oz/A) provided levels of control similar to Endura applied at a rate of 6 oz/A. Endura is a fungicide newly registered for use in canola. Trials that evaluated fungicide tank mixes were also conducted to compare not only levels of disease control but to examine the economic aspect of disease management. Scientists compared the levels of control provided by tiophanate methyl (Topsin), prothioconazole (Proline), and boscalid (Endura) alone or in combination with other fungicides. Tank mixes of Topsin + Endura and Topsin + Proline provided the best levels of control, although other combinations also provided acceptable levels of control compared to the untreated plots. By tank mixing compounds, the cost of the fungicide could be reduced by up to $6 per acre.
3. Sclerotinia stem rot dynamics in canola: This project is directed toward understanding key environmental factors affecting development of Sclerotinia stem rot in canola. Scientists installed a series of experiments in Langdon and Fargo (ND) to compare the impact of supplemental water as irrigation to the soil vs. misting and natural precipitation. These treatments were superimposed on plots that had overwintered sclerotia or that were inoculated with lab-produced ascospores (and did not have sclerotia in the ground). No statistical differences in disease intensity could be detected between plots that had sclerotia in the ground and those that did not. Misted plots developed significantly higher amounts of disease compared to other treatments (mean incidence of 35%), although plots that depended on natural precipitation had an average of 27%. Supplemental ground irrigation, intended to simulate apothecia formation, did not increase disease incidence compared to natural precipitation and had an average incidence of 25%. In a separate experiment three ascospore concentrations were applied at different times during the flowering period. Ascospores at concentrations of 103, 2.5x103, and 5x103 were applied once at 50% bloom, 100% bloom, four days past 100% bloom, or twice at 50% and 100% bloom or at 100% bloom and 4 days past that stage. A range of disease intensities were observed in this study; however, the highest disease intensity obtained with the lowest concentration occurred when ascospores were inoculated at 100% bloom stage (40%). Inoculations performed at the latest stage (four days past 100% bloom) produced acceptable levels of disease (40% vs. an average of 26% for 103 and 2.5x103 ascospores ml-1) only when the highest concentration (5x103 ascospores ml-1) was used. These results contribute to the development of novel management alternatives through increased knowledge of the Sclerotinia disease cycle in canola.
4. Transformation system will speed improved Sclerotinia resistance in sunflower: The main objective of this research is the development of a consistent and reliable transformation method for sunflower. Successful completion of this objective will lead to the production of transgenic sunflower with altered susceptibility to Sclerotinia. Introduction of an oxalate oxidase gene may provide enhanced resistance directly while introduction of a Tnt retrotransposon will allow mapping and rapid identification of native genes which provide base levels of resistance. Scientists have demonstrated high efficiency shoot production from sunflower cotyledonary tissues using a confection sunflower line, RHA280. The variability in response that was at first observed has been largely reduced by using cotyledon pieces from freshly-harvested seeds of greenhouse-grown plants. Response of the explant is consistently about 75% of the explants forming multiple shoots. Scientists further demonstrated that an explant size of 2mm is optimal for shoot production. Using optimized regeneration and explant preparation, scientists also observed that a previously unresponsive sunflower line (RHA810) remains unresponsive. A combination of use of a responsive cultivar with optimized plant regeneration is apparently needed. Plants regenerated from RHA280 shoots flower prematurely and are tremendously reduced in height but plant regeneration has been shown. Using Agrobacterium-mediated transformation, scientists recovered transformed tissue (not plants), demonstrating that the sunflower line is responsive to Agrobacterium. Sonication treatments increase Agrobacterium-mediated gene introduction in target tissues but reduce shoot production. Inclusion of antioxidants during Agrobacterium co-culture have not reduced tissue browning or led to recovery of transgenic shoots. A sunflower polyubiquitin promoter has been recovered from sunflower genomic DNA and will be evaluated for regulation of gene expression.
5. Enhancing Sclerotinia resistance in soybean: Management strategies for Sclerotinia stem rot or white mold of soybean have limitations. Crop culture modifications often compromise high yield, fungicides add to production costs, and resistance found to date is partial, multigenic, and complex. Scientists are working toward increasing the level of resistance to Sclerotinia sclerotiorum in soybean cultivars and to develop and evaluate improved disease control and resistance options for soybean producers. The first goal is to combine quantitative trait loci (QTL) that were previously mapped and identified with the resistance phenotype into single breeding lines. The QTL come from different sources and represent 8 QTL on 7 different linkage groups. Nineteen soybean lines with resistance alleles at multiple QTL were identified as having a level of resistance equal to or better than the resistant check NKS19-90 and the parental QTL lines from three populations. Initial yield test results indicate that lines with improved resistance and similar yield will be recovered. They will be useful as parents to develop higher yielding soybean lines with improved resistance to Sclerotinia stem rot. The second goal is to determine if a novel antifungal synthetic peptide expressed in soybean will confer resistance to S. sclerotiorum. Some reports indicated broader antimicrobial activity of such a peptide, including other fungal and some bacterial pathogens. Scientists developed transformed plants with a codon-optimized gene-expression cassette for the antifungal peptide that contains the barley alpha-amylase signal sequence to export the peptide to the apoplast. Results indicated no significant difference between plants with lytic peptide and those without the inserted gene expression cassette. Because of the cost of commercializing genetically modified organisms, the conclusion is that the effect of the antifungal peptide is not significant enough to warrant further development effort.
6. White mold resistance for pinto bean: Pinto beans are the most widely grown commercial class of dry beans in the U.S. and are among the most susceptible to white mold. The goal of this project is to identify quantitative trait loci (QTL–a region of DNA associated with a phenotypic trait, like resistance to a particular disease) that are associated with resistance to white mold in pinto beans. FY08 was the second season for the evaluation of a recombinant inbred line (RIL) mapping population (AP630) developed from the cross of MSU pinto breeding line P02630 with pinto breeding line AN-37, reported to exhibit tolerance to white mold. The population, consisting of 94 RILs, was evaluated in the field for reaction to white mold and data were also collected on other agronomic traits such as yield, maturity, height, lodging and seed size. The 96-entry test ranged in yield from 30 to 52 cwt/acre with a test mean over 40 cwt/acre and white mold ranged from 12 to 36% with a mean of 20%. The highest yielding entry P07863 exceeded the test mean by 130% repeating as the same top-yielding entry in 2007 trials. P07863 was entered in the national white mold trials where it topped that 64-entry trial at 53.7 cwt/acre, equivalent to 136% of the test mean. Across all locations P07863 rated 4.0 for white mold on a scale that ranged from 3.0 to 6.7 for the most susceptible genotypes. Combining yield with tolerance to white mold is critical as many of the most resistant entries yielded less than 32 cwt/acre in our trial in 2008. The RIL population will be used for QTL analysis of resistance to white mold in pinto bean and more in depth field and greenhouse studies on reaction to Sclerotinia. In addition, a new pinto bean variety Santa Fe was released based on superior yield performance under white mold pressure. Over three years of testing under white mold pressure, Santa Fe yielded 28.8 cwt/acre compared with yield of 14.3 cwt/acre for the white mold susceptible variety Beryl.
7. Interspecific transfer of white mold resistance in common bean: White mold disease caused by Sclerotinia sclerotiorum is a serious disease of common bean (Phaseolus vulgaris) which has little physiological resistance. Interspecific crosses were made between common bean and scarlet runner bean in attempts to introgress resistance from the scarlet runner bean into the common bean background. This project has been focused on characterizing the resistance in two backcross inbred interspecific populations: 91G/PI433251B and MO162/PI433251B. A total of 50 SSRs were screened on the entire population of 91G/PI433251B and 35 SSRs on the MO162/PI433251B. Markers for candidate genes for white mold resistance have been identified and amplification and polymorphism in parental lines has been confirmed for EIN2\EagI, PP2C (phosphatase-2-C), WRKY (transcription factor), CHIT (chitinase) and NPR1\AciI. Primers for CHI (chalcone isomerase), EST (esterase enzyme) and CHS (chalcone synthase) show potential for use as markers. Due to seed limitations, a single replicate–repeated checks experimental design field trial was planted during the summer of 2008. Both populations and all plots were scored for disease incidence (%) and severity (%). The 91G/PI433251B population was read a single time and MO162/PI433251B population was read twice in order to capture differences among the lines that weren’t evident at the first reading. The results of the field trial showed that the 91G/PI433251B population distribution was skewed toward the greater susceptibility compared to the distribution of the MO162/PI433251B population, which was skewed towards resistance. Once molecular marker data are completed, it will be combined with the phenotypic data and QTL analysis will be performed to detect QTL present in the populations for white mold resistance. The impact of this research is that bean growers will need fewer (or no) applications of fungicide to control white mold.
8. Genetic variation and virulence of Sclerotinia on six crops in the North Central Region: Sclerotinia sclerotiorum is one of the most important pathogens of field crops in the North Central USA, and this project examines genetic variation and virulence for a set of isolates from the current population of the pathogen on canola, dry bean, field pea, lentil, soybean and sunflower. The first objective is to use mycelial compatibility groups (MCGs) and microsatellite markers to characterize genetic diversity of the pathogen from six common, susceptible crops. The second objective is to compare virulence of isolates collected from these crops (in fourteen States) on susceptible and partially resistant cultivars of all six crops when grown in the greenhouse. Scientists screened 98 isolates from canola, dry bean, soybean, and sunflower in the Red River Valley region of ND and MN. For objective 2, scientists completed pilot studies to compare lesion formation rates for pairs of isolates on cultivars of the six crop species (susceptible lines of canola, dry bean, field pea, lentil, soybean, and sunflower, and partially resistant lines of canola and soybean). Scientists plan to establish an experiment comparing virulence of 20 isolates collected from each of four crops (canola, dry bean, soybean, and sunflower) on the six crops in fall of 2009, when conditions are best for inoculation in the greenhouse. Scientists will use the information obtained from Objective 1 to select isolates with rare vs. common genotypes as well as any genotypes that appear to be associated with particular crop species. Using this unique large collection of isolates from four crop species across the North Central Region scientists are beginning to document the extent of genetic variation with respect to both geography and crop and link this variation to measurements of virulence across the six crop species.
9. Genes regulating interactions between diseases and plants in dry pea: Sclerotinia white mold, caused by Sclerotinia sclerotiorum, is a devastating disease of many crop plants and can cause significant economic losses in dry pea (Pisum sativum) production systems. Currently, nothing is known about the genetic mechanisms that control the basic biology and pathology of S. sclerotiorum interacting with pea. The original objective of this investigation was to develop expressed sequence tags (ESTs) for the Sclerotinia sclerotiorum and Pisum sativum interaction. Initial efforts focused on ensuring that a representative S. sclerotiorum isolate for EST development had been chosen. This was achieved by fingerprinting a number of isolates to determine that the select isolate was representative of isolates causing white mold of legumes in the field. Subsequent studies focused on EST library development for a number of different growth stages, including growth in liquid medium, and bacterial sequences were detected in the EST library preparations. Follow up PCR detection and cytology studies suggested that the Sclerotinia isolate employed may have contained a bacterial endosymbiont, so investigations were re-focused on the development of fungal ESTs. EST data generated from this project will be used in expression profiling studies to further understand the genetics of the Sclerotinia sclerotiorum-Pisum sativum interaction. By identifying genes involved in resistance scientists will be able to develop markers for marker assisted breeding of resistant pea lines, and identify target genes for future functional research using reverse/forward genetics and proteomics, that will eventually facilitate development of novel management strategies for the control of white mold disease of pea.
10. Understanding the Sclerotinia disease cycle in plants: This research has three main objectives: 1) microscopically determine the ability and mechanism used to penetrate leaf and stem tissues of multiple crops; 2) determine if the nutritional status of inoculum has an effect on the ability to cause disease; and 3) determine if the physiological status of the host has an effect on susceptibility. Scientists completed specific objectives one and three and are currently repeating the experiments of specific objective two. The significant findings from specific objective one were that mutants deficient in oxalic acid were capable of infecting all host plants examined: sunflower, canola, pinto bean, peas and chickpea. Significantly, hyphae were observed to directly penetrate the cuticles and cell walls of these hosts. The presence of multicellular infection cushions on the surface of infected host plants was observed microscopically with infectious hyphae within the plant tissues. Infectious hyphae were also observed in mesophyll tissue and vascular bundles distant from any obvious penetration sites indicating that the oxalate minus mutants were capable of infecting and colonizing the host. Detailed microscopic observations were documented with thin sections of pinto bean leaves. Specific objective three was also completed. Although variation is sometimes observed with age of cultures, no correlation between leaf age and susceptibility was observed. These finding are significant in the context of understanding pathogen biology and development. Sclerotinia mutants deficient in the ability to produce oxalic acid are capable of infecting and colonizing host plants, although to a much limited degree relative to the wild type. This attenuation in virulence does not appear to be due to an inability to form functional infection cushions.
11. Increasing white mold resistance in common bean: Introgression techniques in plant genetics allow for the movement of a gene from one species into the gene pool of another by backcrossing an interspecific hybrid with one of its parents. These methods are being used in this research to obtain high levels of white mold resistance from Phaseolus species of the common bean’s secondary gene pool, and this is important to the rapid development of improved germplasm. Approximately 10 kg seed of each of two white mold resistant interspecific breeding lines (IBL, VCW 54 and VCW 55) derived from a congruity-backcrossing between ‘ICA Pijao’ and P. coccineus accession G 35172 was produced between June to September 2008 at Parma (ID). Both IBL were released to the public by the Idaho and Colorado Agricultural Experiment Stations in December 2008 and both were registered in the Journal of Plant Registration. A small quantity of seed of another white mold resistant IBL VRW 32 derived from recurrent backcrossing of ICA Pijao with P. costaricensis also was produced. However, it may take two or three more increases to produce sufficient quantity of seed for its public release and registration. An additional 81 IBL derived from P. coccineus accession G 35172 introduced from the University of Puerto Rico were screened in the greenhouse. Thus far two IBL look promising, but they are still variable and may take two or three additional sequential screenings in the greenhouse. Of the new group of IBL developed by backcrossing pinto ‘Othello’ to P. coccineus PI 433246 and pinto ‘UI 320’ to P. coccineus PI 439534, two IBL from the former and 31 IBL from the latter have exhibited resistance to white mold. However, these are still variable for their white mold reactions and may require three to four additional sequential screenings in the greenhouse.
12. Pyramiding and introgressing white mold resistance into pinto bean: Introgression and pyramiding methods, common to plant genetics studies, are being used in this study to move desirable genes for white mold resistance from wild relatives to breeding lines which can be accessed by plant breeders interested in improving white mold resistance in pinto bean. Scientists have selected twelve genotypes (A 195. G 122. 'Chase', CORN 501, CORN 601, 19365-25. USPT-WM-1, VCW 54, VCW 55, 92BG-7, 0785-220-1, and 0785-127-1) based on the repeated evaluation of white mold resistant germplasm and breeding lines in the field and greenhouse. In addition, ABL 15 and USPT-CBB-1 with upright growth habit Type II were selected as the contributors of pinto seed and architectural avoidance characteristics. Chase and USPT-WM-1 with partial white mold resistance also show promise. A 195 and G 122 are large-seed Andean; CORN 501 and CORN 601 are breeding lines with pyramided white mold resistance developed at Cornell University: 19365-25, VCW 54, VCW 55, 92BG-7, 0785-220-1, and 0785-127-1 derive their white mold resistance from P. coccineus, and USPT-WM-1 derives white mold resistance from small white Middle American 'ICA Bunsi'. These 14 genotypes currently are being used to make nine single crosses. These nine single crosses were used to make six or more double-crosses in the fall of 2008 and approximately the same number of multiple-parent crosses in the spring of 2009. Results from this research will be central to the development of pinto bean varieties with resistance to white mold.
13. Developing regional-scale white mold resistance in common bean: Complete resistance to Sclerotinia sclerotiorum, cause of white mold (WM), has not been found in common bean, Phaseolus vulgaris. Variation observed in WM screening results can be due to variability of the screening sites, the screening methods used, and/or the variability of the pathogen. To identify bean germplasm with broad partial resistance, putative sources of resistance developed by breeders were evaluated by field plot and greenhouse screening methods at multiple locations annually. In 2003, collaborating breeders and plant pathologists re-evaluated the WM multi-site screening system. S. sclerotiorum greenhouse screening isolates and isolates collected from the field test sites from each location were characterized. Differences in isolate aggressiveness and genetic variation were identified within and between field locations. Different greenhouse screening methods were compared, and the straw test was identified for common use across locations. Experimental design, screening method, breeding lines, evaluation rating and analysis are now consistent across all screening locations each year. Based on the data collected from test sites, each location uses the indigenous isolates. Utilization of multi-site screening and understanding the role of pathogen variation in the screening system has resulted in consistent results across WM screening locations and is a model for white mold screening in other crops. Six adapted germplasm lines with improved WM resistance were field tested in 2008. The development of bean cultivars with partial physiological resistance and/or architectural avoidance to white mold would reduce disease losses and require no input costs for growers.
14. Enhancing soybean resistance to Sclerotinia stem rot: Sclerotinia stem rot is an important soybean disease in the northern soybean production states of the U.S. The disease was estimated to cause a total yield loss of over 175 million bushels from 1996 to 2006. The best way to control this disease is to use soybean varieties with partial resistance to the disease. The long-term goal of this research is to develop soybean varieties or germplasm with high level of resistance to Sclerotinia stem rot. Six lines derived from crosses with Sclerotinia stem rot resistant parents were selected based on their yield and other agronomic traits. These six lines were evaluated in the Preliminary Test IIA in the Uniform Soybean Tests - Northern Region in 2008. The 392 lines from seven populations segregating for resistance from five new PIs were evaluated once in 2008 for resistance to Sclerotinia stem rot in the greenhouse with the spray-mycelium method we developed. Fifty four lines were selected and were evaluated for yield in 2008 at two locations with two replications at each location. The parents of the 392 lines were tested with DNA markers linked with reported QTLs (QTL–a region of DNA associated with a phenotypic trait, like resistance to a particular disease) to identify markers that are polymorphic among the parents of each cross. A manuscript reporting the results in identification of QTLs for resistance to Sclerotinia stem rot in PI 391589A and PI 391589B was published in 2008.
15. Characterization of the genetic basis for partial resistance to Sclerotinia sclerotiorum in pea: Genetic resistance to plant pathogens is the most economical means of improving crop productivity and delivering improved germplasm to producers. Partial resistance in pea to Sclerotinia sclerotiorum has been identified and genetic populations have been developed using susceptible and the newly identified resistant lines. Individual lines from two mapping populations, PRIL-17 and PRIL-19 have been advanced to the F3. The F4 is currently being increased toward development of recombinant inbred lines via single seed decent. A single seed from each family will be grown to develop recombinant inbred line (RIL) populations and the remaining seed will be used to assess the reaction of each family to Sclerotinia infection. Leaf tissue has been collected from each F3 plant and will be used to establish a skeletal genetic map to identify putative QTL based on F4 family disease reactions. Genetic resistance, albeit partial, is a significant step toward developing agronomically suitable varieties of pea with improved resistance to Sclerotinia sclerotiorum.
16. Screening for improved Sclerotinia head rot resistance in Upper Midwest sunflowers: Sclerotinia head rot disease of sunflower is a significant production issue in the Upper Midwest. Identifying germplasm with resistance is a critical step in managing the disease in an environmentally and economically sound manner. This research effort allows the comparison of disease response data among hybrids following Sclerotinia sclerotiorum ascospore inoculation of sunflower heads under regular misting. Modifying the environment in such a manner promotes disease development and challenges germplasm resistance capabilities, allowing researchers to identify hybrids with varying levels of disease resistance. The purpose of this trial was to test experimental and commercially-available sunflower entries for resistance to Sclerotinia head rot. These are critical data for breeders when developing sunflower hybrids with resistance to the disease. Scientists found that damaging levels of Sclerotinia head rot developed uniformly across the experiment on susceptible sunflower entries. Statistical separations were easily identified between hybrids. Sclerotinia head rot resistant hybrids were clearly delineated from susceptible lines. For example, Proseed 7052 had the lowest disease mean, but the hybrid wasn't significantly different from PANNAR PEX 3426, Seeds2000 X4994, Proseed 6007, and Proseed 7016. At the disease susceptible end of the continuum, six entries showed 100% rotted heads (susceptible check, CHS 08 EX 1, CHS 08-EX5, CHS 08-EX6, Mycogen 8N358c], Red River Commodities 2215), but this group was not significantly more diseased than 34 other hybrids tested. Hybrid resistance is on the increase, and several hybrids are now more resistant than the resistant check entry used. As a general rule of thumb, confection-type hybrids continue to express more susceptibility to Sclerotinia head rot than non-confection types.
17. Integrated Pest Management (IPM) of white mold in common bean: White mold is a major concern to common bean growers throughout the U.S. While plant breeders are seeking better forms of genetic resistance to white mold, there is a critical need for improved cultural and other control practices to reduce yield losses. Beginning in 2006, scientists demonstrated the effectiveness of individual IPM components such as fungicides, cultural practices and cultivar selection, including the incorporation of advanced breeding lines developed by two germplasm improvement projects, for collectively managing this important pathogen. Scientists demonstrated that there was a variable effect on plot yield when the population of commercially-grown pinto cultivars was increased 50% from 1 to 2 lines per bed. The percentage increase in yield when comparing 1 or 2 lines with fungicide protection was + 21% or + 12% for pinto Montrose which is a susceptible cultivar. Vision, also a susceptible cultivar yielded + 27% or + 23% with fungicide protection. The white mold intensity did not vary (85 to 98%) for Montrose at either plant population, with or without fungicide protection; however, the fungicide did contribute to increased yield as noted above. Disease intensity in Vision was lower (43% vs 62% and 78% vs 87%) for 1 vs 2 lines with and without fungicide protection, respectively, illustrating that its more upright plant architecture did contribute to reduced disease pressure in dry bean production systems in semiarid regions of the western U.S. and the fungicide treatment provided additional protection for this genetically susceptible cultivar. With an average yield of 20 cwtlA and average grower price of $0.25/pound, the increased plant population (2 rows) could provide an additional gross return of 10% up to 20% for currently grown commercial cultivars like Montrose and Vision in the absence of white mold or if protected from damage by white mold by a fungicide.
18. Disease-warning system for Sclerotinia stem rot in canola: A model which estimates the risk of incidence of Sclerotinia stem rot on canola was improved to include temperature, precipitation, and solar radiation data. This new model formulation now has an accuracy of >75% (estimated using an independent data set). Risk maps created using the model based on weather conditions are produced twice weekly during the growing season and are made available via internet. Maps depicting areas of high, intermediate or low risk were presented at the state level and an interactive program, the Sclerotinia risk calculator, provided the opportunity to obtain an estimation of risk at field level. Growers visiting the interactive site were asked to provide information on cultural practices conducted on their fields. Estimation of risk at field level was obtained using an algorithm that combines weather patterns with information related to cultural practices, previous history of Sclerotinia incidence, and other information provided by growers. Field surveys conducted at the end of the 2008 indicated the model accurately predicted higher disease prevalence in areas where more than three high risk warnings were made during the flowering period. In 2008, 1876 hits were registered at the risk map site between late June and early August and 46 visitors used the risk calculator. In 2009, the flowering period was delayed by unusual cool weather at the beginning of the growing season. High risk periods were registered throughout the season, especially in the northeastern corner of North Dakota. Disease surveys will be conducted later in the year to validate the forecast of 2009. Leaf wetness data has been collected from four fields located in North Dakota–Langdon, Cando, Devils Lake, and Carrington. Data will be analyzed later this year following disease surveys. Risk maps contribute to on farm profitability by improving spray application efficiencies.
19. Screening for improved Sclerotinia resistance in canola: A microspore culture system was developed to produce double haploid plants from B. napus accessions Ames 26628 (Ca01) and PI458939 (Ca02), and F1 lines PI458940 × PI458940 (Ca03); PI458939 × Ames26628 (Ca04); and PI458940 × Ames26628 (Ca05). Effects of genotype, donor plant condition, flower stage/bud size, and culture temperature on embryogenesis were determined. A total of 89 haploid plants from Ca01 were inoculated in a greenhouse. Of these, 36 survived the inoculation, had their chromosome numbers doubled using colchicine, and will be planted in greenhouse for seed production. Plants from Ca05 will be evaluated for their reaction to S. sclerotiorum; other B. napus and B. rapa elite accessions are being considered . Of 35 NDSU breeding lines evaluated in Langdon, three (0330416, 305457, 0427681) showed statistically higher levels of resistance than commercial controls although incidence in these lines was one third of that of the controls. Two other lines (0427649, and 30522) were superior to only one of the controls. Forty five additional lines were tested in the greenhouse using the PIT. Two lines (427761 and 427765) had significantly similar levels of resistance as Pioneer 45H26 the commercial control used. This information has been shared with the breeder. Phenotypic reaction of 230 F2 plants from a cross between PI458939 and Ames 26628 (B. napus accessions previously identified as having superior levels of resistance to S. sclerotiorum) were evaluated in greenhouse conditions using the PIT. Approximately one third of the plants survived the inoculation and the group was taken to seed production by self pollination. Seeds from these plants will be evaluated in the greenhouse again later this year and plant tissue samples will be taken to extract DNA. This project will contribute to developing canola breeding lines with herbicide tolerance in addition to improved resistance to Sclerotinia.
20. Sclerotinia sclerotiorum pathogenicity and fungicide sensitivity variation: Sclerotinia sclerotiorum causes white mold on more than 400 plant species including many economically important crops like beans, canola, soybean, sunflowers, peas, lentils and chickpeas. Extensive studies have been conducted on molecular marker loci variation, population structure, and the phylogenetic relationships of this plant pathogen. Phenotypic variation of this pathogen is also well documented. However, little information is available on the relationship between variation of molecular marker loci and variation of quantitative traits, especially those relevant to agricultural practices, such as pathogenicity and fungicide sensitivity. In order to examine genetic variation and population structure of S. sclerotiorum from different cropping systems in the PNW, mycelial compatibility grouping was used to measure genetic diversity of 88 isolates of S. sclerotiorum from five fields in three states (Idaho, Oregon and Washington). Techniques were established to detect genetic variation using microsatellite markers. Populations of Sclerotinia sclerotiorum collected from lentil, pea and potato were assessed for variation in virulence and in microsatellite markers, and significant variations were found in virulence among isolates within populations. Fungicide sensitivity of isolates from two populations was compared. All available microsatellite markers were screened on representative isolates. The significance of the results is two fold: First, the novel combined analysis of genetic differentiation and quantitative traits will broaden our current knowledge regarding evolutionary significance of quantitative traits of S. sclerotiorum. Second, the results also provide insights to emergence of pathogen virulence and fungicide resistance, and genotypic selection through cultural practices.
21. Mapping QTL for White Mold Resistance in an Interspecific Dry Bean Backcross Population: This project represents the final step toward the long-term goal of pyramiding and mapping white mold resistance genes introgressed from P. coccineus into common bean. Scientists developed an interspecific inbred backcross population from the cross WM67/PI 255956 which contained lines that possessed high levels of resistance, and found four molecular markers associated with resistance QTL. Two of the molecular markers were contributed by scarlet runner bean PI 255956 and two by common bean WM67. Results suggest that QTL associated with white mold resistance can be introgressed from common and scarlet runner bean in an interspecific population. Segregation distortion at thirty markers and sterility problems were observed in the interspecific common by scarlet runner bean cross. Additional backcrossing to common bean will enable mapping of QTL in the interspecific cross. Due to the low heritability of white mold resistance in common bean, MAS for resistance QTL from scarlet runner bean into common bean may be a viable method to improve overall white mold resistance in common bean. Recently, scientists used two highly resistant IBL lines from the interspecific population to develop recombinant inbred lines derived from a commercially valuable Middle American germplasm (USPT-WM1) and the IBL lines. The two resistant IBL (75A and 25B) were utilized as male parents to cross to Middle American commercial pinto line USPT-WM1. Two hundred lines were developed from each IBL parent between the resistant IBL and USPT-WM1 and advanced to the F4 by spring 2009. These lines will be phenotyped and genotyped during 2009-2010 research year using molecular markers (RAPD, AFLP, and SSR) to identify commercially useful germplasm to release to the public.
22. Genetic characterization of resistance to white mold in dry bean: Dry bean cultivars with partial resistance to white mold improve yield potential and reduce fungicide dependency. Scientists identified two genes (QTL) in VA19 light red kidney bean and five genes in I9365-31 black bean which appear connected to white mold resistance. The QTL were integrated on the Phaseolus core linkage map. Genetic markers for marker-assisted selection of the QTL were developed and validated. A comprehensive linkage map of white mold resistance QTL in common bean was constructed from the QTL identified in this work plan, earlier funded work plans and work plans conducted by other researchers. This comprehensive linkage map is a useful tool for interpreting inheritance studies and coordinating genomic research, serves as a repository for accumulation of resistance genes and markers, and provides a framework to plant breeders from which to generate trait-linked markers for marker-assisted selection and combine specific resistance traits. The linkage map will serve to expand marker-assisted selection in dry bean and facilitate development of germplasm lines and cultivars with improved white mold resistance. The map contains 37 white mold resistance-QTL identified across multiple studies that coalesce into 16 regions across nine linkage groups.
23. Sclerotinia disease resistant sunflower germplasm: Sclerotinia, the most serious sunflower disease worldwide, attacks sunflower heads, stems and roots, and the best released USDA hybrid lines, HA 441 and HA 410, are only moderately resistant to head and stalk infections. Wild Helianthus accessions immune to Sclerotinia infection have been identified in recent years, and the overall goal of this project is to produce highly resistant germplasms by pyramiding resistance genes into HA 441 and HA 410, utilizing new immune sources of wild Helianthus species. Crosses between NMS HA 89 and stalk rot resistant wild perennial diploids H. maximiliani, H. giganteus, and H. grosseserratus were backcrossed with stalk rot tolerant line HA 410 and BC1F3, BC2F2 and BC2F3 progenies with 2n=34 chromosomes were produced. Crosses between HA 410 and stalk rot resistant hexaploid H. californicus have been backcrossed with HA 410 three times, and a large portion of BC4F3 and BC4F4 progeny families have been produced for further seed increase in the field. Crosses between HA 410 and stalk rot resistant hexaploid H. schweinitzii have been backcrossed with HA 410 once and BC1F1 progenies with 2n=51 have been established. Crosses of stalk rot resistant 4x and 6x interspecific amphiploids with stalk rot tolerant line HA 410 have been advanced to BC2F3 and BC3F2 generation, and 2n=34 plants were identified for seed increase in the field. Crosses of NMS HA 89 with head rot resistant diploid perennial accessions H. maximiliani and H. nuttallii have been backcrossed with head rot tolerant HA 441. Their BC1F4 to BC3F2 families are available for field evaluation with two replications at Carrington, ND. We are currently evaluating 166 progeny families for head rot and 326 families for stalk rot in replicated field trials. Results from this project will provide the foundation for the production of sunflower germplasm with resistance to both head and stalk rot.
24. Identifying molecular markers linked to white mold resistance in lentil: White mold disease, caused by Sclerotinia sclerotiorum (Lib.) de Bary is a destructive disease of lentils in the U.S. Difficulties for variety improvement due to quantitative inheritance of resistance and the effects of environment on disease expression have stimulated efforts to develop molecular markers linked to genes conferring resistance to S. sclerotiorum. The objective of this project is to identify molecular markers linked to white mold resistance in a lentil population derived from a cross between the commercial varieties Pennell and Pardina. Scientists are developing an efficient and reproducible method for screening lentils in the greenhouse for resistance to white mold. Six lines of lentils (ILL 6002; ILL 5888; Richlea; Brewer; Eston; Pennell and Pardina) were screened for resistance to white mold in two environments; a greenhouse and a growth chamber. Replicated plants were inoculated by attaching a mycelial plug of S. sclerotiorum to a stem internode. The effect of media composition of the mycelial plug on disease severity was evaluated using five different media formulations. Disease in all lines was more severe when plants were inoculated in a growth chamber versus greenhouse. Disease severity was most severe in both environments when plants were inoculated with a mycelial plug of the fungus grown on 100% PDA and least severe when inoculated with a plug of the fungus grown on water agar. Results suggest that the most tolerant of the liens tested are Eston and Richlea while the most susceptible are ILL 6002 and ILL 5888. Scientists identified inoculation conditions that most consistently cause severe disease in lentils, and these will be used to screen for resistance to white mold. This, in turn, will allow scientists to identify molecular markers associated with disease resistance in lentil.
25. Developing rapid screening field techniques for developing Sclerotinia resistance in sunflower: The objective of this project is to screen sunflower germplasm for improved resistance to head rot and stalk rot and study novel methods to improve the efficiency and efficacy of Sclerotinia misting nurseries. Seventy five hybrids are selected each year, from a pool of private company submissions, for screening in the “Initial Test” at two locations, Carrington, ND and Morden, MB. A series of smaller “Repeat Test” screening nurseries are planted each year at Carrington, Langdon, and Oakes, ND and Morden, MB, each consisting of 20 hybrids exhibiting the best resistance to both Sclerotinia head and stalk rot. In 2008, both the Carrington and Morden sites achieved very good head rot disease pressure for the evaluation of 75 hybrids in the “Initial Test”, a group of hybrids not previously screened. The sunflower hybrids in the Carrington site had head rot incidence levels of 26% to 97%, while the same hybrids at Morden ranged from 11% to 54% infection. The combined analysis indicates that 7 hybrids had head rot infection levels that were numerically lower than the best resistant check. A total of 43 hybrids had head rot incidence levels statistically equal to the resistant check hybrids. These results show that seed companies continue to develop hybrids that reflect improved tolerance to Sclerotinia head rot. Although disease pressure was variable among the four locations where the ‘Repeat Test’ was conducted in 2008, all had adequate head rot incidence to effectively evaluate hybrid differences. The average head rot incidence at the four test locations was 68%, 44%, 27%, and 31% for the Carrington, Langdon, Morden, and Oakes sites respectively. The average incidence of head rot among hybrids in the “Repeat Test” across all four test sites indicate that a number of hybrids (selected in 2007) continued to provide a high level of tolerance to head rot infections.