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United States Department of Agriculture

Agricultural Research Service

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Location: Sunflower and Plant Biology Research

2010 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
Milestones listed in question 2 comprise the National Sclerotinia Initiative Strategic Plan. Each milestone was addressed in FY2010 by components of the 32 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.

4. Accomplishments
1. Improving Sclerotinia resistance in sunflower. The overall goal of this project is to create sunflower breeding lines with higher levels of resistances to both Sclerotinia head rot and Sclerotinia stalk rot by exploiting the available molecular technology of marker-assisted selection in combination with traditional breeding methods. In marker assisted backcrossing experiments, ARS scientists in Fargo, ND have concentrated on two pedigrees of interest to customers, one with RHA 464 background and one with CONFSCL R5 background. These genetic backgrounds were identified by customers and stakeholders as desirable for traits other than Sclerotinia resistance, and needed additional resistance to Sclerotinia. Researchers are interrogating the populations with known TRAP markers associated with major head rot resistance quantitative trail loci(QTL), and will backcross again in summer 2010 with plants predicted to be the most resistant based on marker profile. They are also developing a set of defense-related and random markers for use in association mapping for stalk rot resistance. Phenotypic evaluation of the 260 plant introductions for stalk rot and subsequent statistical analysis have already been completed. Results from this research will contribute to the rapid development of sunflower germplasm with improved resistance to Sclerotinia.

2. Transformation system will speed improved Sclerotinia resistance in sunflower. Plant biotechnology has had a tremendous impact on crop production in the U.S., as well as world-wide. In collaberation with the National Sclerotinia Initiative, investigators at Ohio State University are developing a transformation system for sunflower which will permit the wider use of biotechnological approaches for basic research directed toward the improvement of this crop. Although sunflower transformation has been previously reported by a few different groups, it remains inefficient and largely unusable by most laboratories. Based on preliminary results, a sunflower line has been identified which has been proven to be very responsive to shoot induction in tissue culture. Using this line, regeneration and transformation methodologies will be further optimized using the green fluorescent protein reporter gene. Transformation efforts will be extended to introduce genes of interest, using two different approaches. For the first approach, an oxalate oxidase gene will be introduced into sunflower, which will be evaluated for Sclerotinia resistance. The oxalate oxidase gene will be under regulatory control of different promoters, including a constitutive sunflower ubiquitin promoter. For the second approach, the retrotransposon Tnt will be introduced into sunflower, to evaluate the use of this transposable element as a reverse genetics tool. Tissue culture induction of transposon replication and insertion should generate large number of insertion mutations, which will allow gene tagging, for the first time, in sunflower. The transformation tools being developed will shorten the time required to improve disease resistance in commercial sunflower lines.

3. Transferring Sclerotinia resistance from wild sources. Sclerotinia is the most serious sunflower disease in the world. It attacks sunflower heads, stems and roots, and genes responsible for their resistance are independent from each other and the resistances are multigenic. None of the currently available sunflower hybrids have adequate resistance to Sclerotinia, and the best (released) USDA lines, HA 441 and HA 410, are only moderately resistant to Sclerotinia head and stalk, respectively. Wild Helianthus accessions immune to Sclerotinia infection have been identified in recent years and providing the foundation for this project. ARS scientists at Fargo, ND are conducting research to produce highly resistant sunflower germplasm by utilizing new immune sources of wild Helianthus species and pyramiding resistance genes into HA 441 and HA 410. Both HA 441 and HA 410 had the lowest disease incidence in field tests in recent years. Eight Sclerotinia-resistant diploid accessions, one hexaploid, and five interspecific amphiploids have been successfully crossed with Sclerotinia-tolerant cultivated lines, backcrossed and selfed to produce progeny families for field evaluation. Replicated field screening of 163 and 316 progeny families for head and stalk rot resistance, respectively, indicated good-to-excellent resistance among the progeny families suggesting successful gene introgression. A protocol using genomic in situ hybridization (GISH) to distinguish between chromosomes of perennial Helianthus species and cultivated sunflower has been established providing a new tool for studying gene transfer. A molecular tracking study utilizing molecular markers indicated a higher frequency of gene introgression from diploid perennials than from hexaploid or interspecific amphiploids, suggesting an advantage in using diploid perennials. This project is central to the production of sunflower germplasm with resistance to both Sclerotinia head and stalk rot.

4. Developing rapid screening field techniques for developing Sclerotinia resistance in sunflower. Incidence of Sclerotinia sclerotiorum in commercial sunflower production is a common occurrence that often results in serious loss in crop yield and quality. In a collaboration between ARS, North Dakota State University, University of Minnesota, and Agriculture & Agri-Food Canada, scientists are developing resistance to Sclerotinia among commercial sunflower hybrids and germplasm used in public and private breeding programs. Achieving reliable identification of resistance or good tolerance among commercial sunflower hybrids will result in an effective tool for sunflower producers to lower their risk of infection and the associated yield and quality losses. An additional aspect of this project is to develop and refine misting systems that create the environment conducive for Sclerotinia development. This aspect will generate reproducible and reliable data that identifies genetic resistance. The experiences and data collected will also create a better understanding of the epidemiology of head rot form of Sclerotinia. The specific objectives of this project are: Evaluate commercial sunflower hybrids and experimental lines for improved resistance to Sclerotinia head rot; Implement and manage germplasm screening nurseries for USDA-ARS breeders and geneticists to evaluate sunflower germplasm and breeding lines for improved resistance to Sclerotinia head rot and stalk rot; and Refine management of Sclerotinia misting systems to improve their effectiveness in creating a micro-climate favorable for Sclerotinia infections and thereby the knowledge of Sclerotinia epidemiology. The project objectives are designed to generate new knowledge that will directly benefit sunflower producers, seed companies, and processors.

5. Development of sunflower germplasm with Sclerotinia head and stalk rot resistance. The most important diseases affecting sunflower production in the United States are stalk rot and head rot caused by Sclerotinia sclerotiorum. Control of both Sclerotinia diseases is most effective through the use of genetic resistance. Resistance to Sclerotinia head rot and stalk rot has gradually improved in commercial cultivars, due in large part to the releases made by the Fargo-based ARS Sunflower Research Unit. The germplasm development effort of this Unit strives to move desired traits from both cultivated sunflower and wild Helianthus, providing as diverse a genetic background as possible. Past germplasm releases from this unit used Sclerotinia resistance derived from foreign hybrids or germplasm from foreign public research programs. The USDA’s Plant Introductions sunflower collection (maintained in Ames, IA by ARS scientists) has a large number of newly available accessions and these have not been evaluated for Sclerotinia resistance. Multiple field trials of 250 cultivated accessions in 2008 and 2009 have identified germplasm with stalk rot resistance, and now this same group of germplasm needs to be evaluated for head rot. The effort to evaluate for both head and stalk rot is also generating data to be used in an association mapping project in a companion project. Association mapping of a large group of diverse cultivated sunflower will provide a tool to allow researchers to more efficiently identify and transfer Sclerotinia resistance, and will provide a framework to evaluate both wild and cultivated sunflower in future studies.

6. 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. This project brings together expertise and results from identification of genes involved in resistance to Sclerotinia sclerotiorum in soybean and basic research on plant defense mechanisms in response to fungal pathogen infection. The LysM-encoding genes are known to be involved in reaction to fungal infection, and some of these have been mapped to regions in the soybean genome previously identified as quantitative trait loci (QTL) for resistance to sclerotinia stem rot. This project is focused on the LysM domain encoding genes and mapping them in two soybean recombinant inbred line populations that were used previously to identify quantitative trait loci for resistance to sclerotinia stem rot. In a collaboration between the University of Nebraska and the National Sclerotinia Initiative, scientists are correlating the map locations of the LysM-encoding genes with those of the identified QTL. In addition, they are developing polymorphic markers for the LysM genes that map close to existing QTL. These genes represent promising candidate genes that would explain the resistance to white mold in selected cultivars. Genetic markers developed against these LysM-domain encoding genes should be useful for molecular assisted selection to move the favorable QTL loci into elite lines of soybean.

7. Novel loci for partial resistance to Sclerotinia stem rot in perennial soybean. This research, conducted by ARS scientists at Urbana, IL, is focused on identifying novel loci in segregating populations derived from crosses within two perennial Glycine species, G. tabacina and G. tomentella, for resistance to Sclerotinia stem rot. Large populations (~500 lines each) of recombinant inbred lines with segregating genes for resistance to Sclerotinia stem rot are being developed from crosses between resistant and susceptible accessions within two perennial Glycine species (G. tabacina and G. tomentella). Individuals within the populations will be assayed for response to Sclerotinia sclerotiorum infection using a modified cut-stem assay. Since only a small fraction of the markers developed for G. max are informative in perennial Glycines species, sets of new single nucleotide polymorphism (SNP) markers will be identified by massively parallel transcriptome sequencing. High-through-put Goldengate SNP assays are being developed and used to genotype the populations. The phenotypic and SNP marker data will be combined and used to detect quantiative trait loci for resistance to S. sclerotiorum. Because the SNP markers will be based on coding sequences, it will be possible to rapdily locate homologues on the soybean genome to determine whether or not the identfied loci represent novel genes. Genetic intervals containing novel loci will be narrowed using additional SNP markers, synteny will be compared to soybean. If syntenic, candidate gens from withing region will be evaluated by virus induced gene silencing. If silenced alters response to S. sclerotiorum infection, the genes will be cloned and moved to G. max. The goal of this project is to introduce new genes from perennial soybean into soybean to increase the level of resistance in soybean to Sclerotinia stem rot.

8. Interspecific transfer of white mold resistance in common bean. Common beans such as pinto and kidney beans are highly susceptible to white mold. A related species, the scarlet runner bean has the highest levels of resistance to white mold, and can be crossed to common bean. Resistance in scarlet runner bean is controlled by several genes with strong environmental influence. In collaboration between the University of Idaho and the National Sclerotinia Initiative, scientists are backcrossing runner bean (twice) to the common bean to recover lines with runner bean genes in a common bean background. These lines have been inbred to achieve genetic uniformity, and tested to determine which carry white mold resistance genes. From one cross, scientists have identified seven lines with high levels of white mold resistance. To increase the chances of transferring all of the resistance genes, they are identifying DNA markers that are associated with resistance genes. The impact of this research is that bean growers will need fewer (or no) applications of fungicide to control white mold.

9. White mold resistance for pinto bean. This research is focused on the identification of novel quantitative trait loci (QTL) associated with white mold resistance in pinto beans (Phaseolus vulgaris). Two mapping populations were developed that share a common resistant parent. The second parent of each population is one of two high yielding pinto breeding lines with upright plant architecture that likely contribute to white mold avoidance. In collaboration between Michigan State University and the National Sclerotinia Initiative, scientist are evaluating one population in a naturally infested white mold field nursery and also in the greenhouse using the straw test, and oxalate test. Each mapping population will be genotyped with a diverse group of molecular markers. These data will be used to generate a genetic linkage map where regions of the genome (QTL) associated with resistance to white mold will be identified. These QTL will be validated in the second population and the most robust QTL should be valuable to breeders interested in incorporating these regions into breeding material using marker assisted selection. The populations will also serve as valuable sources of advanced high-yielding pinto bean breeding lines that combine white mold resistance with desirable seed quality, erect architecture, and favorable agronomic characteristics.

10. Mapping quantitative trait loci (QTL) for white mold resistance in an interspecific dry bean backcross population. Genetic resistance to white mold has been reported in both common and scarlet runner beans. In collaboration between Colorado State University and the National Sclerotinia Initiative, scientists are combining resistance from scarlet runner and common beans. Because resistance is unique in common and scarlet runner bean, these lines are valuable to plant breeders to provide genetic resistance to white mold, however the germplasm had low fertility and was un-adapted to temperate zones. This project will complete the process to develop lines in the pinto market class that will be useful as parents in a commercial breeding program in North America. Scientists anticipate releasing common bean lines that possess high levels of resistance to white mold from multiple genetic sources as an outcome of this project. This represents the final step toward our long-term goals of combining and tagging resistance genes from multiple sources of common and scarlet runner beans into useful plant germplasm. These lines will allow plant breeders to broaden the genetic base of commercial dry bean cultivars produced in the USA and enhance crop germplasm and genetic resources.

11. Integrated Pest Management (IPM) of white mold in common bean. In a collaboration between Colorado State University and the National Sclerotinia Initiative are investigating the roles of cultural practices (fertilizer rate, promising varieties) and timely fungicide applications in reducing damage from Sclerotinia sclerotiorum to Phaseolus vulgaris cultivars, with varying degrees of resistance (plant architecture – disease avoidance, interspecific resistance), grown under differing irrigation systems (furrow and sprinkler). Results from this research will help define production and IPM systems that will minimize losses from white mold, while maximizing production and economic returns to growers when confronted with different disease pressure, and input options or limitations (e.g., water, fertilizer, fungicides).

12. Developing regional-scale white mold resistance in common bean. Complete resistance to Sclerotinia sclerotiorum, cause of white mold, has not been found in common bean, Phaseolus vulgaris. Yet the disease causes periodic heavy losses in dry and green beans as well as many oil seed and vegetable crops. 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 to growers. A major goal of research conducted at the University of Nebraska, in collaboration with the National Sclerotinia Initiative, is to test putative sources of resistance in adapted backgrounds at multiple sites located in most of the major bean-production areas of the U.S. In addition, a disease screening method (straw test) is being compared with a number of other methods and chosen because it can consistently identify sources of resistance in adapted and un-adapted common bean lines. This standardized direct screening method will be used to evaluate new sources of resistance that once in adapted backgrounds can be field tested in a standardized nursery design with standard evaluation criteria. To date, ten lines with partial resistance to white mold have been released. There have been inconsistencies in the past between screening methods and test sites and difficulty in verifying sources of resistance. The standardized testing has helped but there are remaining inconsistencies that may be due to genetic variation of the pathogen. Mycelial compatibility (determines clones), aggressiveness (virulence) and molecular markers are being used to identify S. sclerotiorum isolate variation that might influence resistance evaluation studies. The approach we are using is applicable to finding white mold resistance in other crops such as canola, pulses, soybean and sunflower throughout the U.S.

13. Conferring partial resistance to white mold in dry bean. ARS scientists at Prosser, WA are identifying and validating white mold resistance quantitative trait loci (QTL) from scarlet runner bean and transfering them into common bean, examining the interaction among QTL conferring partial resistance to white mold in common bean, and using correlation between common bean and soybean genomes and gene expression studies to leverage the soybean whole genome sequence (and the soon to be available common bean whole genome sequence) for fine-mapping white mold resistance QTL, and candidate gene discovery. Interspecific populations that introgress the scarlet runner genome into a common bean background are being used to create linkage maps, where resistance QTL will be located. The QTL will be validated, environmental effect on expression will be studied, and germplasm incorporating resistance will be developed. Existing and newly discovered QTL will be combined in various genetic combinations to examine QTL interactions. Inbred line populations from bi-parental crosses designed to combine two or more QTL will be tested for white mold reaction in replicated field and greenhouse environments and assayed for presence absence of QTL-linked markers. Fine mapping of regions with resistance QTL will add molecular marker to the region surrounding the QTL and will better define their location and give breeders better tools for moving resistance traits into elite material. Expression studies will help identify potential candidate genes controlling resistance QTL. The basic research on candidate gene discovery and applied research examining the effect of combining QTL on level of resistance proposed herein, build upon previous QTL inheritance, identification and verification studies.

14. Increasing white mold resistance in common bean. In collaboration between the National Sclerotinia Initiative and the University of Idaho, scientists are using introgression techniques in plant genetics to facilitate 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. Seed of white mold (WM) resistant interspecific breeding line (IBL) VRW 32 derived from recurrent backcrossing of ICA Pijao with P. costaricensis was produced in the greenhouse. The first field increase was planted at Kimberly, Idaho on May 21, 2010. After harvest at the end of the summer they will know if additional increases will be required to obtain sufficient quantity of seed for its public release and registration. Additionally, twenty new IBL derived from three different accessions of P. coccineus along with the three previously developed IBL (VCW 54, VCW 55, VRW 32) and eight partially resistant and susceptible checks were compared in the greenhouse in Colorado and Idaho. The mean WM score of all genotypes was lower at 14 d compared to 28 d evaluation. Also, the scores were lower in Colorado than in Idaho. The IBL derived from P. coccineus and P. costaricensis, in general, had lower WM scores than other resistant checks. Furthermore, six of 20 IBL had lower WM scores than previously developed IBL. But, all new IBL were still variable for their WM reaction, and will require additional screenings in the greenhouse. The results of this research will contribute to obtaining high levels of white mold resistance from Phaseolus species of the common bean’s secondary gene pool, which is important to the rapid development of improved germplasm.

15. Pyramiding and introgressing white mold resistance into pinto bean. Partial resistance to white mold (WM) is found in few large-seeded Andean and small-seeded Middle American dry and green bean and in interspecific breeding lines (IBL) derived from the Phaseolus species of the secondary gene pool of the common bean (Phaseolus vulgaris L.). In collaboration between the National Sclerotinia Initiative and the University of Idaho, scientists are conducting research to pyramid the highest levels of WM resistance from Phaseolus species of the primary and secondary gene pools, and to simultaneously introgress the highest levels of pyramided WM resistance (PWMR) into pinto bean, the largest market class in the US. All available contemporary WM resistant large-seeded Andean, small-seeded Middle American dry and green bean and IBL derived from Phaseolus species of the secondary gene pool were screened in the greenhouse in Colorado and Idaho in 2008. Five Andean (A 195, G 122, MO 162, PC 50, VA 19) and one Middle American (ICA Bunsi) dry beans, one green bean (Cornell 501), three IBL derived from P. coccineus (VCW 54, 92BG-7, 0785-220-1), and one IBL derived from P. costaricensis (VRW 32) were crossed within and between groups to test for complementation for WM resistance. The representative WM resistant Andean and Middle American dry and green bean and IBL derived from P. coccineus and P. costaricensis are being used to develop broad-based multiple-parent populations for gamete selection (GS). White mold resistant F1:3 to F5:6 families and breeding lines with high levels of PWMR are also being developed. In each generation from F1 to F5, selected PWMR plants are crossed with elite pinto breeding lines and cultivars. The selected PWMR genotypes of pinto and other seed types are being compared to measure gains from the GS and tested in the national Bean White Mold Nursery (BWMN) to determine their stability and usefulness for combating WM in the US.

16. Characterization of the genetic basis for partial resistance to Sclerotinia sclerotiorum in pea. Sclerotinia sclerotiorum is an important disease pest of many crops including pea (Pisum sativum L.) and crop losses have been significant when environmental conditions were conducive to disease development. Limitation regarding available germplasm with resistance has hampered development of resistant pea cultivars. The recent description of partial genetic resistance to S. sclerotiorum among pea accessions provides an opportunity to study the genetic control of resistance in a collaboration between the National Sclerotinia Intiative and scientists at North Dakota State University. Four sources of resistance, PI 103709, PI 169603, PI 240515 and ICI 1204-3 represent two distinct mechanisms of resistance (inhibited lesion expansion and nodal resistance). Genetic mapping populations involving these resistant sources are in development and will be used to place the genetic factors controlling resistance on the pea map. DNA markers associated with the resistance will be made available to breeding programs to aid selection of resistant progeny. In addition, hybridizations aimed at combining the resistance mechanisms will be made to provide an increased level of durable resistance. Results from this project will reduce the economic impact of the white mold pathogen in pea through 1) the identification of genetic factors (QTL) controlling partial resistance to white mold on the pea map and 2) the pyramiding of available mechanisms of resistance in an effort to develop durable resistance.

17. Support for genomics assisted breeding for Sclerotinia white mold. The fungus Sclerotinia sclerotiorum is a devastating disease of many crop plants and has the potential to cause significant economic losses in pea. In collaboration between the National Sclerotinia Initiative and Michigan State University, scientists are examining the mRNA expression profile of pea-Sclerotinia interaction between two pea genotypes (susceptible and partially resistant) over three time points. They are using the Illumina GA2 platform, a new massively parallel sequencing platform capable of sequencing a total of 160-170 reads per run. The sequencing reactions are divided into 8 lanes, each lane is capable of reading in excess of 24 million reads. This is the first attempt to utilize the Illumina GA2 massively parallel sequencing platform for mRNA expression profiling in Sclerotinia and one of few studies to simultaneously examine host and plant pathogen mRNA profiles using massively parallel sequencing. Scientists currently have a large (~35Mb) EST data set generated on the P. sativum-S. sclerotiorum interaction, funded previously by the National Sclerotinia Initiative, and are utilizing in-house EST data set and other publicly available EST data sets to interpret the mRNA expression profiles that generate on the Illumina GA2. Research on gene expression can provide insights into the molecular mechanisms controlling pathogenicity and host resistance. This research will identify genes and pathways involved in the pathogenicity of S. sclerotiorum on pea and genes and signaling pathways involved in the partial resistance of pea to Sclerotinia. By identifying genes involved in resistance we 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 control of white mold disease of pea.

18. EST library for interactions between diseases and plants in dry pea. Little is known about the genetic mechanisms that control the basic biology and pathology of S. sclerotiorum interacting with pea. In a collaboration between the National Sclerotinia Initiative, Washington State University, and Michigan State University scientists are developing expressed sequence tags (ESTs) for the Sclerotinia sclerotiorum and Pisum sativum interaction. 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. During this time high numbers of bacterial sequences were detected in the EST library preparations. Follow up polymerase chain reaction (PCR) detection and cytology studies suggested that the Sclerotinia isolate employed may have contained a bacterial endosymbiont. This line of investigation was suspended in order to focus on development of fungal ESTs. The adoption of next generation sequencing technologies has enabled scientists to surpass initial estimates of the quantity of EST data that were possible to generate. Future analysis of the data set will include assembly of contiguous sequences prior to sorting reads to species. Contiguous sequences should be longer than initial reads and provide more information on gene expression at the time for which mRNA was sampled from this host-pathogen interaction. This research is a necessary step toward utilizing massively parallel sequencing technologies in other Sclerotinia-host systems particularly those that lack genomic resources, such as bean, sunflower and lentil.

19. Transgenic white mold resistance in lentil. ARS scientists at Pullman, WA are using transgenic approaches as part of the effort to develop lentil lines with enhanced tolerance to white mold, which involves expressing the barley oxalate oxidase gene and evaluate these plants for resistance to S. sclerotiorum. Agrobacterium tumefaciens will be used to transform two advanced ARS lentil breeding lines, LC0360114P and LC03602062T, which are high yielding Pardina and Turkish Red type lentils, respectively. Transgenic T1 progeny will be examined for levels of expression of the oxalate oxidase gene and their reaction to white mold disease will be observed. The relationship between disease reaction and levels of expression of oxalate oxidase will be determined. They are currently optimizing methods for developing transgenic lentils and have determined that cotyledonary nodes are the best explant material for lentil transformation. They have also completed a materials transfer agreement with the University of Kentucky and have received the KYRT strain of Agrobacterium tumefaciens, which is the strain that has shown the greatest success for introducing transgenes into lentil, and have also determined baseline levels of sensitivity of the Turkish Red lentil advanced breeding LC01602062T to two different selectable markers, kanamycin resistance and chlorsulfuron resistance. They have also amplified the oxalate oxidase gene from barley using polymerase chain reaction (PCR) and the gene has been sequenced to confirm the fidelity of the amplification product. Currently they are cloning the gene into a plant transformation vector for subsequent expression in transgenic lentil lines. This research will yield cost effective and sustainable approaches for controlling losses in lentil due to white mold through the development of new lentil varieties that have genetic resistance.

20. Genetic variation and virulence of S. sclerotiorum. There is limited information on the genetic variation of S. sclerotiorum isolates across the U. S. that includes information on isolate virulence across crops such as soybean, sunflower, canola, dry bean, field pea and lentil. In collaboration between the National Sclerotinia Initiative and North Dakota State University, scientists are making a collection of isolates of the pathogen from these crops, identifying mycelial compatibility groups (MCG’s), using microsatellite markers to characterize the genetic variation and then characterizing virulence of isolates that have common and unique haplotypes on six crops grown in the North Central Region. Investigators have completed screening on 149 isolates which form 46 MCGs. A few MCGs appear to be quite common although most are relatively rare and represented by 1-2 isolates: four MCGs contain 10 or more isolates while 36 MCGs contain a single isolate. To date, no differences in MCGs have been detected across crops. The most common was MCG 9 found in nine states and on all four crops. Six of the most common MCGs represented 58% of the isolates and were found across crops. The frequency at which MCG’s occur varies with crop and geographic region. Investigators tested a selection of 30 isolates for virulence on the 6 crops in the greenhouse; these isolates represented the diversity of isolates across crops and geographic areas of the collection. The entire experiment consisted of 900 five week-old plants inoculated with the cut-stem/straw technique, and 72 hours after inoculation all lesions were measured with electronic calipers. Analyses are ongoing, but there are obvious differences between isolates and a repeat of the experiment is currently in progress. Understanding the genetic variation and virulence of the current isolates of S. sclerotiorum in the United States is a fundamental part of the overall strategy of using resistance or other controls for this pathogen.

21. Variation in pathogenicity and fungicide sensitivity in Sclerotinia sclerotiorum. 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 phylogenetic relationship of the plant pathogen Sclerotinia sclerotiorum. 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 quantitative traits relevant to agricultural practices, such as pathogenicity and fungicide sensitivity. The study of phenotypic variation and its underlying genetics is virtually unexplored in Sclerotinia sclerotiorum despite its enormous economical importance. ARS scientists at Pullman, WA are addressing the information gap in our understanding of the economically important pathogen Sclerotinia sclerotiorum. They are collecting isolates to determine genetic variation and levels of variation at three quantitative traits: pathogenicity, fungicide sensitivity, and growth rate at different temperature conditions. Significance of this research 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 study will also provide insights to emergence of pathogen virulence and fungicide resistance, and genotypic selection through cultural practices.

22. Understanding the Sclerotinia disease cycle in plants. Sclerotinia sclerotiorum is a broad host range plant pathogen capable of infecting and causing economically significant diseases on numerous crop plants including canola, bean, sunflower, and pulse crops. The control of Sclerotinia diseases through cultural and chemical strategies have met with little success and the identification and utilization of host resistance to this fungus remains elusive. In collaboration between the National Sclerotinia Initiative and the University of FLorida, scientists are working to develop diverse, effective, durable, and ecologically safe disease control methods for Sclerotinia diseases. They are pursuing this goal through the characterization of a genetically-defined oxalate minus mutant and compounds produced by this mutant. Determining whether factors other than oxalate are required for diseases will reveal whether other strategies for blocking basic–host-pathogen compatibility and controlling disease exists in this system.

23. 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 has been estimated to cause a total yield loss of over 192 million bushels from 1996 to 2008. The best way to control this disease is though the use of soybean varieties with partial resistance to the disease. This continuing research, a collaboration between the National Sclerotinia Initiative and the University of Nebraska, is directed toward: 1) further evaluating the advanced lines selected from a previous project and releasing the best lines as germplasm or varieties; 2) evaluating seven populations of F4 derived lines with resistance from five new resistant plant introductions (PIs) for resistance to Sclerotinia stem rot and agronomic traits; and, 3) determining if reported QTLs associated with resistance to Sclerotinia stem rot are also associated with the resistance in the five new resistance sources. Several promising lines are currently being evaluated for agronomic traits in multiple locations and in multiple states. These lines will also be evaluated for level of resistance to Sclerotinia stem rot in the field and in the greenhouse. About 400 new breeding lines, derived from five new resistant sources, will be evaluated for agronomic performance and for resistance to Sclerotinia stem rot. DNA markers linked to Quantitative Trait Loci (QTLs) associated with resistance to Sclerotinia stem rot will be used to test the 400 derived lines to determine whether these markers are also associated with the disease resistance in the five new PIs. Results from this research will contribute to the development of soybean varieties or germplasm with high level of resistance to Sclerotinia stem rot.

24. Sclerotinia stem rot dynamics in canola. This research, a collaboration between the National Sclerotinia Initiative and North Dakota State University, is directed toward improving the ability to predict when Sclerotinia stem rot (SSR) epiphytotics will develop. Specifically, scientists studied the impact of discontinuous soil moisture conditions on apothecia formation and the effect of inoculum concentration and plant age at time of inoculation on development of SSR epiphytotics. Scientists have added soil temperature as a factor affecting apothecia production in controlled condition studies and field trials. An additional year of field studies is necessary because the unusually cold summer in 2009 reduced disease development significantly in their trials, and did not allow them to obtain significant data. Results of this project will help improve the accuracy of the SSR forecasting system and their ability to reduce the impact of adverse weather conditions on disease development when conducting field studies. At the same time, it will help develop better SSR disease management practices.

25. Fungicide alternatives for Sclerotinia stem rot (SSR) in canola. This research, a collaboration between the National Sclerotinia Initiative and North Dakota State University, will help optimize the control of diseases caused by Sclerotinia in dry bean, canola, and soybean in three major areas: 1) Reduction of risk of development of fungicide-resistant strains of S. sclerotiorum. Some of the fungicides most commonly used in these three crops in the region have a history of promoting development of fungicide resistance in other pathogens. Developing a regional baseline for fungicide sensitivity will help us monitor S. sclerotiorum reaction to these compounds and to sound the alert in case resistance is detected. Research results with North Dakota isolates indicate that a reduced sensitivity against thiophanate methyl is building; 2) Improving efficacy of chemical control of Sclerotinia stem rot in canola through use of fungicide tank mixtures. This would not only help reduce the possibility of fungicide-resistant populations to build up but could also lead to improved SSR control due to synergism among fungicides. Promising mixes include combinations of thiophanate methyl and boscalid or prothioconazole. These mixes would help reduce risk of resistance. Information generated by this project could be validated on soybean and dry bean trials; 3) Evaluation of new chemistries and biological fungicides alone or in mixture with conventional fungicides for control of SSR in canola. As with activities described in the second area, the information generated here could be extended to the other two crops in the region. Research results will provide growers with additional alternatives to manage this important disease.

26. Screening for improved Sclerotinia resistance in canola. In collaboration between the National Sclerotinia Initiative and North Dakota State University, scientists are characterizing sources of resistance to Sclerotinia stem rot (SSR) of canola (SSR) identified in previous years by the PIs of this project in the USDA-ARS Brassica rapa collection. In collaboration with canola breeder, they are examining the reaction of Brassica napus breeding lines with herbicide tolerance to Sclerotinia sclerotiorum and incorporating new resistance genes into breeding lines by developing double haploid populations from crosses between elite not herbicide-tolerant B. napus lines and other breeding lines.

27. Disease-warning system for Sclerotinia stem rot in canola. In collaboration between the National Sclerotinia Initiative and North Dakota State University, scientists have identified weather variables that are very influential in Sclerotinia disease development and experiments under controlled conditions indicate that leaf wetness duration is critical for disease development. Using logistic regression analysis, they have developed a predictive model for disease onset, are currently validating this model, and have placed it on a web site platform that will be accessible to growers during 2010. Additional efforts will be directed toward characterizing the relationship between leaf wetness duration and weather variables. This warning system will help growers make spraying decisions and help reduce the number of unnecessary fungicide applications.

Last Modified: 2/23/2016
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