Location: Sunflower and Plant Biology Research2008 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 FY2008 by components of the 35 specific cooperative agreements and the numerous additional 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.
0. The National Sclerotinia Initiative (NSI) contributes to the goals of ARS National Program 303 – Plant Diseases, which is comprised of four components a) Disease Diagnosis: Detection, Identification and Characterization of Plant Pathogens, b) Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and their Relationships with Hosts and Vectors; c) Plant Disease Resistance; and d) Biological and Cultural Strategies for Sustainable Disease Management. In this continuing project, 5442-21220-023-00D, each of the milestones listed will be addressed during the next 4 years and anticipated progress is currently outlined in the Sclerotinia Initiative Strategic Plan. Definitive plans will evolve depending on cooperators involved in the research projects, subject to the availability of research funds each year.1. 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. The challenge in developing transformation systems is the coordination of transformation and regeneration; being able to target cells/tissues for DNA introduction into tissues that are also capable of regenerating into whole plants. The main objective of this research is to develop a consistent and reliable transformation method for sunflower, using a line, recently identified as exceptionally responsive to tissue culture manipulation. At this early point in the research, we have been able to successfully demonstrate shoot production from sunflower cotyledonary tissues using a confection sunflower line, RHA280. The most responsive tissues were excised from dry cotyledons, with a decline in shoot induction if the seeds were permitted to germinate. We have also recently demonstrated, as anticipated, that cotyledonary tissues of sunflower are quite responsive to Agrobacterium-mediated transformation. Transient expression of the green fluorescent protein gene has been observed, with expression mostly localized to the internal cotyledonary tissue. Lastly, we have been able to successfully obtain and modify the retrotransposon construct, which will be used in our reverse genetics approaches to understand Sclerotinia resistance in 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. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
2. Mapping QTL for White Mold Resistance in an Interspecific Dry Bean Backcross Population: We developed an interspecific inbred backcross inbred line (IBL) population derived from a cross between the most resistant RIL from the G122/CO72548 population and P. coccineus accession PI 255956 to introgress QTL for resistance from scarlet runner bean with common bean. High levels of resistance to white mold were found in the WM67/PI255956 IBL population. Four lines were more resistant than check cultivar G 122. Three of the four resistant lines possessed levels of genetic background from scarlet runner bean from 0 to 26%. The line that possessed the highest percentage (70%) of scarlet runner bean alleles, had an ASI value of 3.63 and the line with the second highest ASI had 55% of PI 255956 alleles. All lines with scarlet runner alleles were partially sterile, late maturing in the field, and not agronomically suitable, but by developing these lines we demonstrated the combination of resistance genes from common bean with resistance from scarlet runner bean and validated the effect of QTL found in separate studies for each species. These lines are currently being increased for release as germplasm. In the WM67/PI255956 IBL population, six reported QTL linked to white mold resistance from common bean RIL population G 122/CO72548 and one from scarlet runner bean RIL population Wolven Pole/PI 255956 were tested for their effect. Markers linked to the B2b QTL, B7 QTL, B8 QTL, and the 1c QTL in P. coccineus were associated with resistance in the WM67/PI 255956 population and accounted for 9.7, 12.8, 10.8, and 7.0% of the phenotypic variation in resistance, respectively. The results and germplasm from this research will contribute to marker assisted selection to broaden the genetic base of white mold resistance in commercial dry bean cultivars produced worldwide. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
3. Identifying Virulence Factors of Sclerotinia sclerotiorum: Very little is known about the genetics of Sclerotinia sclerotiorum pathogenicity factors, in spite of its devastating effects in many cropping systems. This has been attributed to a lack of an efficient transformation system for functional analyses of the genes. With the genome of S. sclerotiorum being sequenced, development of an efficient transformation system for S. sclerotiorum will be timely for functional analyses of predicated pathogenicity genes. We developed a protocol to efficiently transform S. sclerotirum through Agrobacterium-mediated transformation and generated 137 stable transformants, and through screening transformants for loss or reduced virulence on lentil, we identified 16 transformants with reduced virulence and one transformant that lost pathogenicity. During 2007-2008, we identified four putative genes of pathogenicity factors through sequencing disrupted genes in selected non-pathogenic transformants. The four putative genes are a haloacid dehalogenase-like hydrolase gene in transformant Y22, a nucleoside phosphatase genes in transformant M139.2, a gene coding for esterase of the alpha-beta hydrolase in transformant M-2, and a GTPase gene in the transformant M-69. These putative genes need to be confirmed for their roles in pathogenicity through complementation tests and targeted mutagenesis. Completion of the proposed research will allow us to determine and demonstrate the functions of the identified putative genes in pathogenicity of Sclerotinia sclerotiorum. Results of the research will enhance our understanding of pathogenic mechanisms of S. sclerotiorum, providing better knowledge for devising disease control strategies such as novel approaches to engineering resistant crops. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
4. 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 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. In order to examine genetic variation and population structure of S. sclerotiorum from different cropping systems in the PNW, isolates of S. sclerotiorum were obtained from five fields of different crops across the region of Pacific Northwest. 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. The significance of our 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, our results also provide insights to emergence of pathogen virulence and fungicide resistance, and genotypic selection through cultural practices. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
5. Sclerotinia stem rot dynamics in canola: Sclerotinia stem rot inoculum does not move much within a canola field and inoculum produced within the field is far more important than inoculum produced outside. In 2007, experiments were installed in Cando and Langdon (ND), as during previous years, and the existence of a disease gradient, likewise detected in previous years, was confirmed. Similarly, a close association was detected between inoculum concentrations in the air and stem rot incidence. Analysis of environmental conditions that promote disease development indicated that during 2005 and 2007 the daily number of hours with relative humidity higher than 90% under the canopy was on average 10. This finding confirms a previous report that indicated that a minimum of 10 hours per day may be required for disease development. In 2006, the daily number of hours with relative humidity higher than 90% was 3 and disease did not develop in the fields. The results of this project will contribute to the development and improvement of field-scale risk maps for Sclerotinia stem rot in canola. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
6. Evaluation of Wild Helianthus Species for Resistance to Sclerotinia Stalk Rot: The two most important diseases affecting U.S. sunflowers are stalk rot and head rot caused by Sclerotinia sclerotiorum. In attempts to achieve higher levels of resistance, wild progenitors of a crop are a likely source of different and potentially unique genes. Because the Helianthus genus is native to North America, sunflower researchers have a valuable opportunity to discover new genes. In 2007, we placed five H. resinosus accessions and one Helianthus hybrid (H. annuus x argophyllus, PI 597912) in field trials. The H. resinosus accessions as a group had 92.5% plant survival (no wilt) in the field vs. 94% in the greenhouse and PI 597912 had 50% plant survival in the field vs. 72% in the greenhouse. The most resistant commercial check in the study had 17% survival in the field vs. 56% in the greenhouse. One-hundred and fifty accessions (~9,000 plants) were tested in the greenhouse between June-2007 and May-2008. This included all available accessions of Helianthus argophyllus (18 acc.), H. debilis (43 acc.) and H. praecox (41 acc.) species. These species were identified in previous tests as containing accessions with resistance superior to that of the best commercial hybrids. In addition, 46 accessions of H. annuus (23 from the U.S. and 23 from Australia) and two accessions of the perennial H. resinosus were evaluated. From these greenhouse screening results, 20 accessions have been selected for testing in a 2008 field trial along with resistant and susceptible commercial checks. This included H. annuus (6 acc.), H. argophyllus (3 acc.), H. debilis (9 acc.) and H. resinosus (2 acc). 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. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
7. Screening for improved Sclerotinia resistance in canola: A total of 406 Brassica rapa plant introductions have been screened for their reaction to Sclerotinia sclerotiorum using the petiole inoculation technique (PIT) under greenhouse and field conditions. Four accessions were identified as the best materials based on greenhouse and field evaluations. Of these accessions, individual plants that survived the greenhouse inoculation in this stage were then self-pollinated. Of these, S1 seeds from accessions 426281, 163497, 175050, and 21738 were advanced to the final screening stage. In the third and final stage PI 426281 had the highest survival rate at 17% compared to 7% survival rate for Hyola 357 Magnum, the resistant commercial control. Plants from this accession that survived the inoculation will produce S2 seeds, and this procedure will be continued until S4 seeds are produced. Progeny from elite crosses between B. napus accessions identified as resistant to S. sclerotiorum are being evaluated under greenhouse conditions using the PIT. Field trials are ongoing to evaluate 35 elite NDSU breeding lines with herbicide tolerance. These lines were planted in Langdon (ND) and will be rated for Sclerotinia stem rot during the 2008 growing season, followed by greenhouse evaluations later in the year. Elite B. napus materials previously identified as having superior levels of resistance to S. sclerotiorum were used in crosses. F2 seeds from 4 of these crosses have been evaluated and surviving plants are being self-pollinated. Seeds produced by these lines will be inoculated in the winter months of 2009. This project will contribute to developing canola breeding lines with herbicide tolerance in addition to improved resistance to Sclerotinia sclerotiorum. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
8. Disease-warning system for Sclerotinia stem rot in canola: We have developed a model that estimates the probability of Sclerotinia stem rot apothecia formation as expression of risk and have made this model available to canola growers via the internet. A new model which estimates the risk of incidence of Sclerotinia stem rot on canola was also developed. The new model uses temperature, precipitation, and solar radiation measurements to estimate risk of disease development. This new model has an accuracy >75% (estimated using an independent data set) and when compared to independent data sets proved to be significantly better than the model based on probability of apothecia formation. During 2008, risk maps created using the model based on weather conditions were produced twice weekly between June 24 and July 21, the period during which most ND canola fields were flowering. Maps were also made available to growers via internet at more than one location, including the Northern Canola Growers Association website. Maps depicting areas of high, intermediate or low risk were presented at the (ND) state level and an interactive program 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. Field-level risk estimates were obtained using an algorithm that combines weather patterns with information related to cultural practices, previous history of Sclerotinia incidence in their fields, and other information provided by growers. Field surveys to validate model predictions will be completed during the 2008 growing season. Risk maps contribute to on farm profitability by improving spray application efficiencies. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
9. 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. We 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. Our 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. The 19 lines were evaluated in yield tests during 2007, as well as 12 replications of the detached leaf test. Initial yield test results indicate that lines with improved resistance and similar yield will be recovered. Our second goal is to improve the use of calcium cyanamide as a control option for S. sclerotiorum. Our previous research results indicated that the cah gene showed no negative effects on yield in the transgenic lines vs. the non-transgenic control. Furthermore, Perlka™ (granular Ca-cyanamide) application reduced germination of sclerotia and increased yield. If Perlka application also significantly reduces soybean cyst nematode (SCN) populations, it could make an attractive disease management package for producers. Results from yield tests over four environments during 2006 and 2007 show some possible reduction in SCN counts for the 100 kg ha-1 Perlka application at planting. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
10. The genetics of Sclerotinia resistance in soybean: Our research has focused on selection methods to identify Sclerotinia stem rot (SSR) resistance in soybean. We have investigated preformed stem lignin as a stable soybean trait that can be used to select for resistance to SSR in years not conducive to disease. We have also investigated light intensity as an environmental variable that, when controlled in a growth chamber setting can provide disease data that closely mimics what is observed in a natural environment. Observations of the soybean-S. sclerotiorum interaction suggested a role for preformed stem lignin content in disease resistance. We hypothesized that plants with low stem lignin are more susceptible and exhibit greater SSR severity than plants with high lignin concentrations. Six soybean accessions that varied in response to S. sclerotiorum were selected for study in a series of field experiments. Soybean stems were sampled at reproductive developmental stages that correspond to specific events in both soybean plant development and the SSR disease cycle. The lignin concentration of stem component samples was quantified. Soybean accessions expressed statistically different disease phenotypes in both 2004 and 2006. Lignin concentrations differed among accessions, growth stages and plant parts. Results were contrary to our hypothesis, with positively ranked correlations observed between accession Sclerotinia stem rot severity and lignin concentration for all nodes and internodes assayed. For the R3 growth stage, lignin concentration of the internode between the fourth and fifth trifoliate leaves correlated best to disease severity data. These results indicate that resistance is related to low stem lignin concentration and that soybean stem lignin concentration can be used as a biological marker to select for resistance to S. sclerotiorum. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
11. 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 will collect disease response data of 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. The test was planted on 21 May 2007 and plants were thinned in mid-June when at approx. the V3 growth stage. A total of 79 single-row plots were inoculated in early August across three replicates. The test was misted until 1 September. A total of 75 hybrid entries were tested as well as one known resistant check. Plant heads were rated twice for Sclerotinia head rot disease severity using a 0 to 5 scale where 0 = healthy and 5 = completely diseased. Entry means varied from a disease rating low of 0.9 for Proseed 6481 to a high of 5.0 for Triumph 7449. Disease incidence data varied from a low of 20% for a known resistant check to a high of 98% for Triumph 7442 and Triumph 7449. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
12. Sclerotinia disease resistant sunflower germplasm: Sclerotinia is the most serious sunflower disease in the world. None of the available sunflower hybrids have adequate resistance to Sclerotinia, and the best released USDA lines, HA 441 and HA 410, are only moderately resistant to head and stalk, respectively. 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 BC1F1 progenies with 2n=34 chromosomes were self-pollinated. Crosses between HA 410 and stalk rot resistant hexaploids H. schweinitzii and H. californicus have been backcrossed with HA 410 three times, and a large portion of BC4F2 plants have 2n=34, which will provide BC4F3 families for the 2009 field evaluation. Crosses of interspecific amphiploids with stalk rot tolerant line HA 410 have been advanced to BC2F2 and BC3F1 generation, and 2n=34 plants were identified for seed increase for 2009 field evaluation. 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 BC1F3 families are currently being evaluated in replicated. In sum, we have identified sources highly resistant to Sclerotinia stalk rot and head rot in diploid and hexaploid wild perennial Helianthus accessions, and in interspecific amphiploids involving diploid and tetraploid wild Helianthus species. Results from this project will provide the foundation for the production of sunflower germplasm with resistance to both head and stalk rot at a level that precludes economic loss of sunflower from Sclerotinia attack. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
13. 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. We identified novel QTL associated with white mold resistance from undomesticated wild bean germplasm in a population of 89 BC2F3:4 inbred backcross lines (IBL) derived from the cross between the Mexican black bean ‘Tacana,’ the recurrent parent, and the wild Mexican accession PI 318695. In 2007, two recombinant inbred line (RIL) mapping populations (AP630 and AP647) developed from the cross of MSU pinto breeding lines P02630 and P02647 to common pinto parent, AN-37 were evaluated in the field for reaction to white mold. Data were also collected on agronomic traits such as yield, maturity, lodging and seed size in both populations, consisting of over 100 RILs each. Twenty-two RILs in the AP630 population exceeded yield of 40 cwt/acre and the top entry, P07863, had the lowest white mold incidence rating (11%) in the trial. The AP630 population was selected, based on superior performance, for QTL analysis of resistance to white mold in pinto bean and more in depth field and greenhouse studies on reactions to Sclerotinia. The top yielding entry, B07104 (among 64 lines) in the national white mold trials conducted in Michigan in 2007 was derived from a cross with a wild bean (above). Identification of QTL for white mold resistance in a well adapted pinto background suitable for direct harvest will serve as a valuable bridge to develop resistance in other medium-seeded classes and further our understanding of white mold resistance in dry beans. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
14. Improved resistance to S. sclerotiorum in pea and lentil through breeding and biotechnology: Genetic resistance is the most economic means of control for Sclerotinia sclerotiorum, the causal organism of white mold, which causes severe crop losses annually on many broadleaf crop plants including pea and lentil. Natural genetic resistance is limiting in pea germplasm. A set of 36 varieties and 291 germplasm accessions were evaluated in the field at Carrington, ND, under high disease pressure to identify resistant genotypes. In 2008, 260 germplasm accessions were evaluated for a second year to verify results from the first evaluation. The project has taken a two-fold approach to improve the level of resistance in pea to Sclerotinia sclerotiorum, 1) genetic transformation and 2) discovery of natural genetic resistance in diverse germplasm. Initial transformation efforts generated more than 1000 transformation events; however, no transformed plantlets were recovered. A revised binary plasmid was developed to deliver the selectable marker and the oxalate oxidase gene in independent transfer DNA fragments. This will allow the two introduced genes to be separated based on Mendelian segregation. This will have significant implications where retention of the selectable marker is not desired. The second component of the project has identified individual germplasm accessions and varieties with moderate levels of resistance. Thirty-six cultivars were evaluated in replicated trials over three years. Disease incidence scores varied between years due to environmental conditions and generally ranged from 1 to 7 on a scale of 1 to 9, where 1 = no disease and 9 = dead plants. Varieties and germplasm accessions with improved resistance have been used in the breeding program to improve resistance to Sclerotinia white mold. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
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. F2 progeny from two hybrid populations were grown in the greenhouse to produce F3 family seed. 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 F2 plant and will be used to establish a skeletal genetic map to identify putative QTL based on F3 family disease reactions. Genetic resistance, albeit partial, is a significant step toward developing agronomically suitable varieties of pea with improved resistance to Sclerotinia sclerotiorum. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
16. Genetic characterization derived resistance to white mold in common bean: Scarlet Runner Bean is an important source of partial resistance for improving dry bean resistance to white mold (Sclerotinia) disease. Dry bean lines with white mold resistance introgressed from Scarlet Runner Bean exist, but the inheritance of this resistance is unknown. Researchers at USDA-ARS Prosser have developed genetic populations which enable the study and characterization of the partial resistance to white mold derived from Scarlet Runner Bean. Three separate genetic populations have been developed and evaluated for white mold reaction in greenhouse and field trials. Replicated field and greenhouse evaluation of 140 lines for reaction to white mold disease for one population was successfully conducted in 2007. For another population, two genes (QTL) were identified, tagged with DNA markers, and chromosomal position of the genes on the genetic linkage map was determined. These novel genes will be beneficial for the continued improvement of white mold resistance in dry bean cultivars. Markers linked with the genes can be used to monitor and expedite transfer of the genes into new cultivars. Dry bean cultivars with partial resistance to white mold improve yield potential and reduce fungicide dependency. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
17. Interspecific transfer of white mold resistance in common bean: White mold (Sclerotinia sclerotiorum) is a serious disease of common bean (Phaseolus vulgaris). One of the best sources of resistance to white mold is P. coccineus (runner bean). Two interspecific populations developed for white mold resistance, 91G/PI 433251B and MO162/PI 433251B, were advanced to the BC2F5 generation in the greenhouse during 2007-08. The PI accession is a Phaseolus coccineus accession previously found to be one of the most resistant in our testing efforts. DNA was extracted from all lines at the BC2F4 generation. Parental lines are being screened for polymorphism with SSR, RAPD and EST primers. Fifty-one SSR primers have been identified that amplify, 25 of which are polymorphic in both populations. Both populations are being advanced to BC2F6 in the field in 2008. Plots for white mold resistance evaluation were planted in the field on 30 June 2008 of both populations. Due to seed limitations, a nearest neighbor experimental design was used. The remnant seed for all lines in the 91G/PI 433251B and MO162/PI 433251B population was planted in the greenhouse for evaluation in a straw test. Our research continues with the screening of additional backcross-inbred interspecific populations to characterize resistance and discover molecular markers linked to resistance. Increasingly, our research is focused on identifying candidate genes in Phaseolus that may underlie QTL for resistance. To increase our chances of transferring all of the resistance genes, we 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. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
18. Genes regulating interactions between diseases and plants: Sclerotinia white mold, caused by Sclerotinia sclerotiorum, can cause significant economic losses in dry pea (Pisum sativum) production systems. The interaction between pathogen and host and the expression of host resistance may depend strongly on specific interactions between S. sclerotiorum and the pea host. Currently, nothing is known about the genetic mechanisms that control the basic biology and pathology of S. sclerotiorum interacting with pea. We used a novel approach to generating Expressed Sequence Tag (EST) data for the interaction between Sclerotinia and pea. Briefly, the pea cv. ‘Lifter’, identified as having partial resistance to S. sclerotiorum, was inoculated with a selected S. sclerotiorum isolate, confirmed to be representative of S. sclerotiorum genotypes infecting pea. The sequencing of the resulting normalized cDNA pool will likely yield a substantial number of genes involved in pathogenicity and resistance responses. The plant and fungal transcripts will be sorted by mapping the reads to the S. sclerotiorum genome and where possible ESTs will be assembled into larger contiguous reads. All EST sequences will be submitted to the public domain by entry into dbEST, the NCBI expressed sequence tag database. ESTs resulting from this research will provide a basis for future functional genomic projects, such as generation of gene expression profiling and functional analysis of selected genes. 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 the control of white mold disease of pea. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
19. Developing rapid screening field techniques for developing Sclerotinia resistance in sunflower: Each year we cooperate to screen sunflower germplasm with USDA-ARS breeders and geneticists for improved resistance to head rot with the coordination of stalk rot effort, screen commercial and experimental sunflower hybrids for improved resistance to head rot at multiple sites, study novel methodologies methods to improve the efficiency and efficacy of the Sclerotinia misting nursery. Head rot screening nurseries using the misting system procedures developed within this project were utilized across all cooperating research sites. The screening of commercial and experimental sunflower hybrids for improved resistance to head rot is a major component of this project. Sunflower companies with breeding programs submit hybrids to project investigators who select a group of at least 75 hybrids for screening in the “Initial Test” that is planted at two locations. A series of smaller screening nurseries referenced as the “Repeat Test” were established at four additional sites. The “Repeat Test” is designed to include a group of 20 hybrids that exhibited the best resistance to both Sclerotinia head and stalk rot based on the results from the previous year’s evaluations. In 2007, head rot disease pressure in the “Initial Test” varied widely between sites. Likewise, disease pressure varied among the four locations where the ‘Repeat Test’ was conducted. However, a number of hybrids showed improved resistance to both head rot and stalk rot at the Carrington site in 2007. In 2008, germplasm and fungicide evaluations have all been established and are being coordinated so that the primary hybrids being evaluated are assessed for both head rot and stalk rot resistance. Results from this project will yield significant advances in sunflower resistance to Sclerotinia. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
20. Integrated Pest Management (IPM) of white mold in common bean: White mold caused by Sclerotinia sclerotiorum is a major concern to common bean (Phaseolus vulgaris L.) growers throughout the U.S. Various integrated pest management (IPM) strategies have been proposed to the dry bean (and other crop) industries, but adoption has been slow. We have 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 in the central High Plains and adjacent regions of North America. Preliminary results suggest a % increase in yield when comparing 1 to 2 lines (planted) that was 74% and 55% in 2006, but only 14% and 0% in 2007 for cultivars Montrose and Vision, respectively. Increased plant density did not provide an increase in yield, regardless of the fungicide program, for any entry tested in 2007. Assuming this modest disease control and associated yield gain with an upright, susceptible cultivar like Vision, 1 fungicide application, and 2 lines per bed, a grower could net an additional $190/A or $512/ha at a cost of $30/A or $74/ha for the fungicide application in the presence of white mold. The main experiment is being repeated in Colorado and Idaho during 2008, and we are evaluating the performance of six experimental breeding lines, developed by National Sclerotinia Initiative collaborators, with physiological resistance and/or plant avoidance to infection under varying plant populations. Results from this project will support the development of improved germplasm, modified production practices, and effective integrated pest management strategies against white mold in bean production throughout the U.S. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
21. Pyramiding and introgressing white mold resistance into pinto bean: 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) were selected based on the repeated evaluation of white mold resistant germplasm and breeding lines in the field and greenhouse over time. 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 have pinto seed. 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. Subsequently, these nine single crosses will he 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. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
22. Increasing white mold resistance in common bean: Introgressing the high levels of white mold resistance from Phaseolus species of the common bean’s secondary gene pool is important to the rapid development of improved germplasm. Recently, four interspecific breeding lines (IBL) out of 433 derived from crosses of ICA Pijao with Phaseolus species of the secondary gene pool survived the repeated greenhouse and field screening from 2002 to 2007. Three IBL derived from P. coccineus G 35172 that had moderate to high levels of white mold resistance, and an additional 81 IBL involving G 35172 developed by Dr. James Beaver at the University of Puerto Rico, Mayaguez were introduced and screened for white mold resistance in the greenhouse during early 2008. Of these five IBL were found to be variable for resistance and all others were susceptible. Twenty-three single plant selections were harvested from five IBL for further screening. Single plant selections from three IBL that were variable for white mold reaction were screened in the greenhouse under severe disease pressure (2-3 inoculations plant-1 and delaying evaluations until =28 days post inoculation). Twenty-four white mold resistant plants were harvested from the three IBL. A total of 1,375 plants from 157 IBL, derived from crosses of pinto Othello and UI 320 with P. coccineus PI 433246 and PI 439534 were screened in the greenhouse. Eighty-two plants (6%) from 23 of 157 (15%) IBL were resistant in this first screening. There was higher frequency of resistant plants in a single-cross than in recurrent backcrosses. Progenies of single plants harvested from variable IBL will be screened for white mold reaction. Subsequently, seed of resistant IBL will he multiplied, genetic gains from introgression of resistance from the secondary gene pool will he measured, and all IBL will be released for further use. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
23. Developing regional-scale white mold resistance in common bean: The development of bean cultivars with partial physiological resistance and/or architectural avoidance to white mold would reduce disease losses for growers. We tested putative sources of resistance at multiple sites located in most of the major bean production areas of the U.S. Since the inception of multi-site testing, a resistant snap bean and three resistant dry beans have been released. Seventeen field-greenhouse tests will be conducted at 11 locations by cooperators in 2008-09. The use of consistent rating scales in field nurseries has provided more informative results for bean breeders and pathologists, and it has allowed fields with low infection to be identified. Field disease severity at screening sites has been highly variable from state-to-state; this supports the need for multi-site testing. The number of bean lines with putative white mold resistance submitted for testing has tripled due to the SI support of this and bean germplasm-breeding projects. The multi-site field tests combined with greenhouse results helped to identify disease escape or avoidance in bean breeding line B95055 that is similar to that previously identified in the navy bean variety Bunsi. B05055 ranked among the susceptible lines in the greenhouse but was more resistant in the field. Five lines were identified with partial resistance in the greenhouse tests, and six lines were identified from field tests in 2007. We have demonstrated that screening difficulties can be reduced by using multiple location screening sites and understanding the role of pathogen variation in the screening system. The multi-site approach using standardized methods and isolates to search for resistance in common bean will be applicable for canola, sunflower, soybean, pulse crops and other agronomic hosts. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
24. Standardized methods improve Sclerotinia resistance screening: Past inconsistent correlations between field, lab and greenhouse screening methods that made identification of sources of Sclerotinia resistance difficult may have been due to different test methods and/or genetic variation of the pathogen. No previous study on pathogen variation in bean-production areas in the U.S. or in any crop has been published. Mycelial compatibility groupings (MCGs) and aggressiveness (virulence) were used to identify genetic and phenotypic isolate variation that can influence resistance evaluation studies. The use of isolates from specific host lines and replications at each resistance screening site permitted us to sample a broad range of bean production regions. High variability of pathogen isolates was found within and between fields and greenhouse test isolates using MCGs. In tests of isolate aggressiveness using the straw test, significant differences were found between some isolates. However, when the isolate aggressiveness was compared to the MCGs the isolates came from, only isolates compared between MCGs were significantly different, while the isolates within MCGs did not differ in aggressiveness. Thus, the differences in aggressiveness are associated with the MCG that the screening isolates form. We have demonstrated that screening difficulties can be reduced by using multiple location screening sites and understanding the role of pathogen variation in the screening system. The multi-site approach using standardized methods and isolates to search for resistance in common bean will be applicable for canola, sunflower, soybean, pulse crops and other agronomic hosts. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
25. Identifying molecular markers linked to white mold resistance in lentil: Lentil is an essential rotational crop in the Pacific Northwest and Northern Plains states where it is grown in predominantly cereal based cropping systems. Unfortunately, lentil yields are often reduced due to the effects of diseases, including Sclerotinia wilt. Currently, no commercial lentil varieties have acceptable levels of field resistance. It is difficult to screen for disease resistance in the field because sources of inoculum and environmental conditions that promote disease expression are not consistent. The identification of molecular markers associated with resistance to Sclerotinia wilt would accelerate the development of resistant lentil varieties and provide genetic landmarks for the subsequent isolation of genetic domains conditioning disease resistance. We have a developed a mapping population of approximately 220 recombinant inbred lines (RILs) derived from a cross between two commercial lentil varieties, Pennell and Pardina. The two parents have been screened with 115 different sequence related amplified polymorphism (SRAP) primer pairs, and this analysis has detected 70 polymorphic SRAP markers. We have also extracted DNA from all 220 RILs and are currently screening these lines with polymorphic SRAP markers to detect linkage arrangements. We will continue to screen the parents with additional SRAP markers to identify more polymorphic markers and will also screen the RILs for resistance to Sclerotinia wilt to detect markers that are associated with disease resistance. The identification of molecular markers associated with disease resistance has relevance to the priority need of conducting research on host/pathogen genomics including genome-wide gene expression, gene profiling of susceptible and resistant hosts, and marker development. The National Sclerotinia Initiative contributes to the goals of ARS National Program 303 – Plant Diseases.
5. Significant Activities that Support Special Target Populations