2009 Annual Report
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.