Final Report for GNE13-063
Project Information
The advent of a virulent new race of downy mildew has defoliated and decimated impatiens across the United States, as well as worldwide. Often a key early season crop for small greenhouse growers and nurseries, and a fixture in landscapes and home gardens, susceptibility appeared to be near-universal and severe in the common species, I. walleriana. While the New Guinea types (I. hawkeri) appear resistant, they have drastically different cultivation requirements and methods. Unfortunately, they also do not form viable hybrids with the common species. However, our data suggest that other, compatible species show significantly higher levels of resistance, and may be useful for breeding new, more diverse forms. During the course of this project, we screened over 30 impatiens species for their reaction to downy mildew, and also created hybrids between some of these and the common species. Concurrently, we also created hybrids between other, easy-growing species of impatiens to fill the garden niche held by I. walleriana, diversifying the range of species cultivated and providing more options for growers and gardeners. We have also induced diversity through mutation and changes in ploidy, with the hopes of developing more commercially appropriate forms in the future.
Introduction:
Prior to 2011, impatiens were one of the most popular annual flowers for gardens and landscaping. Their ability to grow and flower in full shade or sun, as well as the presence of a wide range of color forms, lead to a following among consumers and nurseries. Economically, they also formed an important part of the early season economy for greenhouse growers, due to the markup they could charge for buying the flowers as seeds and then selling them as seedlings. However, in 2004 Wegulo et al. documented the presence of impatiens downy mildew (IDM), Plasmopara obducens, on vegetatively propagated impatiens cultivars which had been commercially distributed over a wide area. By 2011, the infection had spread and had been reported across the nation and worldwide. While New York State had reported over $10,027,000 in sales of common impatiens in 2009, by 2014 this had dwindled to only $1,052,000 (USDA NASS).
The genus Impatiens contains over 1000 documented species. Early on, reports of IDM noted that New Guinea impatiens, I. hawkeri, did not appear to become infected by the pathogen. Margery Daughtrey, a Cornell University pathologist of ornamental plants at the Long Island Horticultural Research and Extension Center, performed a field trial of other impatiens species and observed that while some species were susceptible, others appeared completely resistant (Catlin & Daughtrey, 2013). We decided to expand this screen, with her help, in order to investigate the breadth of resistance in the genus and to identify resistant species which could form the basis of our breeding efforts.
Historically, extensive work on impatiens hybridization was carried out at the USDA by Toru Arisumi. Starting in the early 1970's, he developed an extensive project to domesticate various impatiens species through interspecific hybridization, ploidy manipulation, and elucidation of the genetics. His publications (such as: Arisumi, 1973; Arisumi, 1980; Arisumi, 1987), detail many of the techniques to create interspecific hybrid populations between particular species, as well as methods to restore fertility and propagate the plants aseptically. Unfortunately, due to competing interests with the commercial floriculture industry, a decision was made to discontinue his work after his retirement in 1986 and his material was destroyed or neglected (Benjamin, 1990). However, his publications provide a roadmap for impatiens improvement and introgression of traits among impatiens species. We have used these to create breeding strategies and methodologies for our own impatiens improvement program.
In addition to interspecific hybridization, we have also been investigating mutagenesis as a method of impatiens improvement. Many impatiens species are known from small wild populations (Fischer, personal communication), which could be endangered by further collection. At least one species, I. repens has already been classified as 'extinct in the wild', although a surviving population was later discovered. Therefore, rather than expanding the genetic base of impatiens through mining native diversity, we have been looking at induced mutations to create novel phenotypes that could aid in domestication. This work has been strongly influenced by Weigle & Butler (1983), who described a method for inducing mutagenesis. Their work produced a dwarf form of a wild species, a trait that would otherwise take extensive breeding effort to achieve. We hope to apply this to other impatiens species.
One issue that was raised by nursery owners during our preliminary interviews was a question over what could vector IDM to their plants. While many had heard that New Guinea impatiens had not been infected, the realization that our North American native jewelweed species are impatiens produced some concern that these might exacerbate the epidemic. One grower went so far as to say that he had wiped-out all native jewelweed plants from his location. We made sure to include representatives of the two main jewelweed species in our trials, to see whether this concern had merit and, if so, how much.
Cited References
Arisumi, Toru. 1973. “Morphology and Breeding Behavior of Colchicine-Induced Polyploid Impatiens Spp L.” Journal of the American Society for Horticultural Science. 98(6):599–601.
Arisumi, Toru. 1980. “In Vitro Culture of Embryos and Ovules of Certain Incompatible Selfs and Crosses among Impatiens Species.” Journal of the American Society for Horticultural Science. 105(5):629–31.
Arisumi, Toru. 1987. “Cytology and Morphology of Ovule Culture-Derived Interspecific Impatiens Hybrids.” Journal of the American Society for Horticultural Science. 112(6):1026–31.
Benjamin, Joan. 1990. “The History and Development of New Guinea Impatiens.” Thesis, University of Delaware. 91 pages. http://udspace.udel.edu/handle/19716/3717
Catlin, Nora and Margery Daughtrey. 2012. “Downy Mildew on Impatiens: What Is the Host Range of the Disease That Ravaged Plants in Long Island Landscapes in 2011?” Long Island Horticultural Research and Extension Center.
Fischer, Eberhard. 2015. Communication during the First International Impatiens Symposium in Bonn, Germany.
USDA NASS. Accessed 2015. http://usda.mannlib.cornell.edu/MannUsda/viewDocumentInfo.do?documentID=1072
Wegulo, S. N., S. T. Koike, M. Vilchez, and P. Santos. 2004. “First Report of Downy Mildew Caused by Plasmopara Obducens on Impatiens in California.” Plant Disease 88(8):909–909.
Weigle, Jack L., and Judith K. Butler. 1983. "Induced dwarf mutant in Impatiens platypetala." Journal of Heredity 74(3):200-200.
1. Screen approximately 30 species for reaction to impatiens downy mildew
"Use a modified version of the methods described by Catlin & Daughtrey, 2012. Replicated blocks of each species will be inoculated with the pathogen, allowed to grow for approximately two weeks, and then observed. Previous results suggest that some plants will have sporulating patches, while others will not, and that the symptomatic plants will vary in their extent of pathogen growth and sporulation. We will measure observed patches of the pathogen on each species, as well as photograph and describe the extent of sporulation. I. walleriana will be used as a susceptible control, and I. hawkeri as a resistant control. This experiment will be repeated multiple times to avoid accidental escapes from infection."
Achieved, as described
2. Observe reaction of different populations of native jewelweed species to downy mildew
"Using the methods described in 1, we will also test populations of I. capensis, I. pallida, and I. glandulifera from various parts of the United States and possibly Canada. Populations will be obtained from wild collection at sites along the East Coast, samples held at public institutions, and commercial sources. Differences in extent of sporulation or pathogen growth will be measured and photographed. Observations will also be made of species growing in situ, to determine the penetrance of the pathogen into wild stands of the species."
Achieved, with modifications:
While we collected and observed multiple populations of the native species, the lack of disease symptoms made comparison difficult. Challenges associated with propagation also impaired our ability to replicate populations within our screen, so single representative populations from each species were used instead.
3. Hybridize identified resistant species with common impatiens to introgress resistance
"Crossing will be done using normal pollination procedures for impatiens. Crosses which spontaneously abort before seed maturity will be aided by embryo rescue, as described by Arisumi (1985). Application of exogenous hormones, as needed, will help delay the senescent abortive response. Hybrids will be grown to maturity and backcrosses to each parent will be attempted. In cases of sterility, we will induce polyploidy, as described by Arisumi (1973), and make bridge crosses to try to recover fertility."
Achieved, as described
4. Asses the success of hybridization process and the resistance and consumer appeal of interspecific hybrids.
"The methods described in 1 will be applied to the hybrids and populations developed in 3, in order to ensure that the resistance is sustained and to elucidate its method of inheritance. In addition, meetings with growers, Master Gardener groups, and representatives from industry will be organized to identify characteristics important for selection in these populations. While much of the information will be gathered through discussion, a survey may be conducted later based on insights from the meetings."
Achieved, as described
5. Hybridize various impatiens species to identify hybrids with potential for seed-propagation as an alternative to common impatiens.
"In addition to crosses with the common species of impatiens, as detailed in 3, crosses will also be conducted among various other resistant and susceptible species. This will harness a wider range of diversity, as well as provide bridging populations for 3. Information from 4 will be used to aid in identifying species which fill in deficiencies of the common species, or could serve to fill similar niches. Ability to set seed, a trait commonly prized in impatiens production, will be measured in resultant hybrids by comparing their number of seeds per pod or plant to the numbers produced by their parent species, as a calculated proportion of ancestry."
Achieved, with modifications:
While we created several interspecific hybrid populations of impatiens, many carried a double-flowered trait that makes seed propagation difficult. This also made comparing seed-set ability impossible. Instead, we have decided to pursue vegetative propagation for these lines: a common method for introducing new cultivars.
6. Identify and create mutants for various impatiens species and evaluate how they may expand and benefit cultivation of the species.
"Commercial and hobbyist collections of common species will be queried and obtained in order to catalog the variation present, especially for readily identifiable differences between otherwise similar populations. Qualitative traits will be prioritized and studied, via test crosses, in comparison to know inheritance ratios. Species with limited diversity among cultivated forms will be mutagenized in order to expand the genetic diversity and identify potentially useful variants."
Achieved, with modifications:
We created one viable population of mutated plants and also identified a spontaneously-occurring mutant. Multiple other attempts at mutagenesis were made, but did not produce viable plant material. The mutant population necessary to perform the described test crosses is under development.
7. Present information on resistant species, and their use as landscaping alternatives, to gardeners, growers, extension personnel, and representatives from industry.
"Data from 1, 2, 4, & 5 will be written up for publication in popular press outlets (eg. newspapers, growers’ magazines, extension bulletins, etc.) and websites, in addition to the peer-reviewed journal process used to present the more detailed data. We will also work with various outreach groups and individuals, such as extension agents and the Master Gardener groups, in order to make sure that the information is getting spread to anyone interested. This will be supplemented with presentations and posters at professional and non-professional conferences (eg. flower or garden shows), industry events, garden clubs, and other relevant venues. Pertinent information on the effect on native impatiens species will be conveyed to relevant environmental groups."
Achieved, with modifications:
We have drafted several professional manuscripts (see 'Publications/Outreach' below) and are in the process of submitting them for publication. In order to be eligible to publish, we are unable to to release detailed information to other outlets (such as the popular press) until our articles are accepted, which has delayed this aspect of the objective. We have instead done multiple presentations and poster sessions at a wide range of venues to share our work (again, see below), as this is acccepted within the publishing restrictions. After professional publication, we intend to expand our outreach to additional printed sources.
Cooperators
Research
Our primary activity was screening a wide range of impatiens accessions for resistance to impatiens downy mildew (IDM). We acquired accessions from commercial sources, wild populations, and private collectors, eventually obtaining a population that encompassed species from several continents as well as multiple commercial varieties. This population was propagated by vegetative cuttings or stratified seed, depending on the requirements of each species. Plants of each species were then placed in replicated trials, with five plants of an accession screened during each of the three trials. The trials were carried out in a shade house and plants were grown in 6 inch pots using standard potting media supplemented with slow-release fertilizer, and irrigated twice daily using an overhead sprinkler system. Blocking consisted of one plant per accession, placed in a random order in rows within the shadehouse. , Previously-infected plants were placed between and among blocks to act as inoculum. We took data on the number of leaves infected, total number of leaves (used with the number of infected leaves to calculate a percent of infection), as well as the average percent of leaf area infected on each plant.
Plants that showed IDM resistance superior to that found in the common impatiens, I. walleriana, were incorporated into our breeding program. We performed hundreds of crosses between susceptible, but commercially important, plants of the common impatiens and species that we identified as resistant. We also crossed other species amongst themselves to develop novel forms. In order to get viable hybrids, we performed ovule rescue after pollination and cultured the ovules on growth media, modeling our methods after the publications of Toru Arisumi at the USDA. Resulting hybrids were transferred to the greenhouse, assessed, and then grown in a field trial. The field trial was conducted in a shade-cloth high-tunnel with five plants of each of our hybrids interspersed in a randomized order with various commercial impatiens cultivars. Observations were made on growth habit, floriferousness, and survival at the end of the growing season.
We also identified species which were resistant and ornamental, but which had characteristics which interfered with cultivation. These species were put into our mutagenesis program. We treated seed, seedlings, cuttings, or cultures with a mutagen (using methods modified from Weigle & Butler, 1983), in order to increase diversity within the populations of these species. Surviving plants were grown out and then compared to non-mutated forms. We also searched for off-types of these species in non-mutated populations.
Through this project, we discovered several species which did not get infected with IDM, and were able to differentiate levels of susceptibility within the species which did (eg. Figure 1). The specifics of this information will be published in an upcoming publication, and so have not been included in this report. Further information will be released to public sources, as well as the private publication, at that time. Generally though, it appears that resistance is common in a range of other species, but that low levels of infection can still occur. This is visible as a lack of symtpoms on the majority of species tested, with only occasional and not significant spots of infection. Some difficulties in propagation, such as an inability to grow from cuttings or long stratification periods, initially proved challenging but we were able to overcome this by using plants from well-established and morpologically homogenous wild populations instead.
By using the data from the IDM screen, we were able to design a breeding project to introgress resistance into the common species through interspecific hybridization and ovule rescue. Our efforts produced four hybrid populations involving the common impatiens species, and two which did not include it but involved other ornamental species. Despite the existence of previous literature on impatiens interspecific hybridization, we ran into several obstacles, both in culturing the ovules and getting the crosses to survive on the plant. Ovule browning and under-developed ovules were particular problems.We modified our growth media to improve ovule development and slow browning, using information gleaned from a range of other pertinent literature. Our cross survival improved as we tried different pollination techniques and also studied how long the pods should stay on the plant (Figure 2). Plants that survived are under evaluation for commercial release, and are also being progressed through a range of breeding techniques to recover more diverse forms and types. One interesting characteristic that appeared is double-flowers, not present in either of the parent populations but uniform in the offspring and backcrosses to a non-double parent.
Our mutation work yielded a population of plants with phenotypes noticeably different from the control population. The survival was low (only six plants survived out of 120 seeds treated), but the resulting plants were each individually unique (Figure 3). Other treated populations did not survive, likely either due to poor germination (of the control and the treated seeds) or high seedling mortality (likely coupled with reduced vigor from the treatment). Some accessions also failed to respond to the treatment (particularly the in vitro cultures which were treated). One mutant plant identified in a commercial population has also proven to be interesting material for future breeding and research studies, and we are in the process of testing its inheritance and incorporating it into our breeding program.
- Figure 1. Comparison of similar-stage leaves on a susceptible (Left) versus putatively resistant (Right) species. Note the sporulation on the underside of the leaf from the susceptible species.
- Figure 2. Differences in development of ovules rescued from the same plant, but at different stages of maturity. The ovules in the culture on the right are younger.
- Figure 3. Comparison of a seedlot of plants untreated (Top) versus treated (Bottom) with a mutagen.
The results of our research and the products generated for it have major implications for the ornamental nursery industry. Our screen of impatiens species, hybrids, and commercial cultivars identified which varieties being sold are hosts for IDM, as well as the role native species might play in its spread. By distributing this information to growers via publication, we can help them make better decisions about what varieties to grow which do not require additional inputs for disease control. Or, if they choose to grow susceptible varieties, which ones might be better choices. Likewise, gardeners, landscapers, and extension agents can use this information when planning their planting choices or advising others.
Our IDM-resistant hybrids between the susceptible common impatiens and a resistant species also have great potential to improve ornamental horticulture. When we have released these varieties, they will be able to fill the void left by susceptible common impatiens. Currently, growers and people planting ornamentals only have a limited number of choices for the niche previously held by common impatiens, and many of these differ from it in major ways. Having a resistant version of a plant with an established role in the landscape should allow rapid distribution and acceptance within the floriculture world.
The mutagenesis project demonstrates how applied mutation breeding can be used to expand the genetic base of a species for the purpose of domestication, instead of having to pillage fragile native populations. Our results show that in a relatively small population of treated plants, extreme phenotypes can be visible even in the first generation. We anticipate that even more diverse phenotypes will be visible in subsequent generations, and that these can form a basis for future improvement within the species. This work functions as a proof-of-concept for breeding in the face of germplasm conservation, as well as producing material for future breeding and genetics research.
Education & Outreach Activities and Participation Summary
Participation Summary:
Presentations
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Two undergraduate courses at Cornell University
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Presented as a portion of the lecture materials for two consecutive years
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A local garden club
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An invited presentation
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The Plant Sciences Department at the Pontifical University of Chile
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Presentation to Faculty & Students
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The Horticulture Department of Cornell University
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Presentation to Faculty & Students
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The Plant Breeding Department of Cornell University
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Presentation to Faculty & Students
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First International Impatiens Symposium in Bonn, Germany
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An invited presentation
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Long Island Horticultural Research and Extension Center Field Day
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Presented to floriculture growers and professionals
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Posters
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The National Association of Plant Breeders Conference
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The Independent Plant Breeders Conference
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The International Plant Propagators Society Conference (2014 & 2015 [see Figure 4])
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Distributed posters on the pathogen, from the USDA Ornamental Pathology Lab, to interested growers, Master Gardeners, and extension agents (see Figure 5)
Drafted Publications
Towards Improvement of Impatiens
James Keach* & Mark Bridgen, Cornell University
Submitted: Proceeding of the International Plant Propagators Society Conference
Resistance to Impatiens Downy Mildew among Diverse Impatiens Species
James Keach*; Margery Daughtrey; Mark Bridgen, Cornell University
Under revision
'Cherry Bomb' - Madagascar Impatiens: A new double-flowered, mounding shade plant series
James Keach* & Mark Bridgen, Cornell University
Under revision
‘Ironpatiens’ - Common Impatiens Series
James Keach* & Mark Bridgen, Cornell University
Under revision
Additional Outreach
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Discussed project with commercial growers and researchers
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Four undergraduate independent study projects developed from project material
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Results have led to a collaboration on impatiens pharmacology and a travel grant with a research group in Thailand
Project Outcomes
Not applicable
Farmer Adoption
Nursery feedback was received at field day events, during conferences, and in informal discussions. By far, the response was positive and it was reiterated several times that there was a major need for continued research on IDM. Several growers asked to receive information when our breeding lines are released, and there has also been some interest from commercial industry and breeding programs.
This project has major implications for commercial ornamental growers, as it would restore the ability to grow one of their key crops. Current control methods involve continual spraying before releasing the product to the consumer, or selling the material with the understanding that it will succumb to the disease. The resistance demonstrated in our hybrids could reduce farmer inputs, restore a revenue stream, and also safeguard against future issues by broadening the genetic base of the crop.
In addition, the knowledge of which species can be potential hosts for the pathogen will allow farmers to make more informed decisions about their growing choices and practices. Especially, information on the role native impatiens species can play in disease progression will be valuable for growers trying to make decisions that impact the local ecosystem, as well as their own plants.
Areas needing additional study
The work started under this grant will be continued within our laboratory. One major area of research which would be useful is how resistance is inherited within the interspecific hybrid populations, and how best to quantify it. These populations also require some stabilization and testing before release at a commercial level, which may involve more breeding work. We would also like to expand the number of species included in our disease screen, and also come up with more effective and less labor-intensive ways to measure the disease. The mutant populations we developed are also being progressed, and we hope to study inheritance of the induced traits, as well as treating other species.