Integrating resistance from wild relatives against downy mildew in Impatiens

Integrating resistance from wild relatives against downy mildew in Impatiens

Summary

     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 appears 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, preliminary data suggests that other, compatible species may be resistant or highly tolerant, and useful for breeding new, more diverse forms.  We have begun screening a wide range of impatiens species for their reaction to downy mildew, and have also attempted hybrids between these and the common species.  Concurrently, we are also investigating the potential of 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 attempted hybrids between these species, as well as having induced diversity through mutation and changes in ploidy, with the hopes of developing more commercially appropriate forms.  In addition, we have been collecting seeds and plants from native jewelweed impatiens populations, and looking at their degree of susceptibility to assess the risk that they might become reservoirs for the disease. 

Objectives/Performance Targets

Objectives/ Performance Targets – Original objectives are in bold, while actions taken towards them are shown as bullet points (•).

1. Screen approximately 30 species for reaction to impatiens downy mildew

• • Acquired approximately 60 impatiens species from commercial sources, private collections, and wild populations.

• • Collected local isolates of impatiens downy mildew on I. walleriana and I. balsamina.

• • Propagated downy mildew on tissue cultured plants.

• • Developed a downy mildew screening method for tissue-cultured plants.

• • Conducted the first round of screening of non-tissue-cultured plants, using 15 different species.

2. Observe reaction of different populations of native jewelweed species to downy mildew

• • Collected or acquired native jewelweeds from 12 locations in 8 states,

• • Collected 2 naturalized species from 3 different locations.

• • Stratified one batch of seeds, and have another one currently stratifying.

 3. Hybridize identified resistant species with common impatiens to introgress resistance

• •  Attempted over 100 crosses involving I. walleriana & obtained several putative hybrids

 4. Asses the success of hybridization process and the resistance and consumer appeal of interspecific hybrids.

• • Learned about different crossing and emasculation procedures

• • Informal discussions about desirable/undesirable qualities in cultivated and underutilized species

5. Hybridize various impatiens species to identify hybrids with potential for seed-propagation as an alternative to common impatiens.

• • Attempted over 120 crosses not involving I. walleriana & obtained several putative hybrids

 6. Identify and create mutants for various impatiens species and evaluate how they may expand and benefit cultivation of the species.

• • Treated 15 species with colchicine

• • Decided on which species to mutate

 7. Present information on resistant species, and their use as landscaping alternatives, to gardeners, growers, extension personnel, and representatives from industry.

• • Presented preliminary background and results to:

1. 1. An undergraduate course

1. 1. A local garden club

1. 1. The Plant Sciences Department at the Pontifical University of Chile

• • Discussed project with commercial growers and researchers

Accomplishments/Milestones

     As this was the first year of the project, our priority was on laying the groundwork for the project through germplasm acquisition and foundational experiments.  One of the first, and most important, steps was creating and cultivating a contact with a major, private collector of impatiens.  Interacting with him has provided access to a wide range of germplasm, good foundational information about the crop and its idiosyncrasies, and access to a wide range of other private collectors and enthusiasts.  He has been very generous in sharing material from his collection, in the expectation of future collaborations from our side, and we now have cuttings of many of his plants growing in our greenhouse.  Several trips to distinct geographical locations provided plants or seeds of several native jewelweeds, which we transplanted to our greenhouses.  These species have a strongly annual growth habit and the seeds require a four month stratification treatment, something which has slowed work with them, but which we are working on circumventing.  Other species, or forms of species, have also been purchased or requested from a range of commercial sources across the United States.  Our pathologist collaborator has also been an essential source of germplasm.  The collection work is ongoing, and with the recent acquisition of a Small Seed Lots Permit from APHIS, we are hoping to gain access to more species from other countries as well.
     After an initial collection of species and cultivars was set up, our next priority has been attempting hybrids between the different plants and, especially, between the different species.  Instructions on impatiens pollination from the aforementioned collector guided our initial crosses but, after consulting with a horticulturalist who conducted previous research on impatiens interspecific hybridization, we have increased the stringency of our emasculation process.  This has been supported by reviewing older literature on compatibility between different impatiens species. 
     To facilitate interspecific crossing, we have also been running several comparisons of embryo rescue protocols and media.  There is a diverse, sometimes contradictory, range of publications covering this, and we have been implementing the changes suggested in individual publications one at a time.  One issue not covered in the publications has been phenolic browning of embryos, especially when rescued from white-flowered females.  Consultation with the PI suggested the addition of antioxidants to the media, and we have seen good results with calcium ascorbate (a pH neutral, more stable form of Vitamin C).  Another change suggested by the literature was changing the carbon source from sucrose to glucose, documented as reducing the mortality rate of embryos.  This change was more problematic, as initial substitutions did not retain the liquid with the standard amount of agarose or gellan gum, and bags of the media ended up contaminated after releasing liquid.  Increasing the concentration of gelling agent, while keeping it within a standard range, seems to have ameliorated this problem.  We continue to experiment with other additives.
     Similarly, we have also been working on getting various species of the collection to grow in tissue culture.  This has been both for reasons of germplasm preservation, in case of contamination or greenhouse mortality, as well as providing insights for how to propagate future interspecific hybrids produced in vitro.  Several surface sterilization techniques were attempted to introduce tissue from greenhouse-grown plants, and we have successfully grown callus or plantlets of approximately five species.  Other plants have been introduced into culture through rescue of spontaneously-formed seed pods.  Four published media preparations from other researchers were also compared, in order to see which might be best for maintenance versus for plant formation.

Figure 1. Growth of one individual of I. balsamina on four previously published media for tissue culture. Two replicates shown for each media.

There appears to be some differences in responses of different genotypes to these media, but the preliminary differences in response to the media suggest some useful ways to maintain and manipulate germplasm in vitro.
     In an effort to get a head start on the chromosome doubling we suspect will be necessary for the interspecific hybrids, we have also been experimenting with different colchicine application methods.  Research published in the 1970’s showed a low concentration of colchicine applied approximately 10 days to plant cuttings would result in useable rates of transformation.  Based on my experience using colchicine in industry, I had added DMSO to the solution as a carrier for better tissue penetration and to reduce chimaerism.  However, plants treated with this modified solution showed necrosis after four days of treatment, and so the treatment was discontinued and the plants were allowed to grow and recover.  A review of more literature on colchicine application suggested that the addition of DMSO reduced the treatment time needed, but was not always uniform.  We have taken modifications from additional literature, and the most recent treatment involved colchicine and DMSO in a glycerin base for a short period, followed by rinsing off of the solution.  After recovery and regrowth, we hope to examine the ploidy of all of the plants both directly, but tediously, through chromosome counts and indirectly, but more rapidly, through stomata guard cell observation.
     The main end goal of this project is to identify species resistant to impatiens downy mildew, and integrate this resistance to other species.  Since the start of this grant in October, we have collected isolates of the pathogen off of two species growing locally: I. walleriana and I. balsamina.  Mycelia from infected tissue was used to inoculate plants of I. walleriana and I. balsamina growing in vitro.  This has produced an undoubtedly impure culture, as no species isolation techniques have been executed on the samples, but the treated plants showed symptoms mimicking those found on naturally infected plants.  As had been suggested from observations from other researchers, I. balsamina showed infection, but reduced symptoms compared to I. walleriana.  While the original intent was to screen species for infection by treating cuttings, this has proven unfeasible due to the large amount of space required, potential danger of cross-contamination with stock plants, and inability of several species to grow from cuttings.   Instead, and with the consultation of a collaborating pathologist, we have been attempting to develop an assay done on leaves detached from the larger plants.  Initially this was done with great success on leaves from plants grown in vitro.

Figure 2. Screening results of plants grown in vitro, two weeks after inoculation with the pathogen. Infection on I. balsamina is indicated by an arrow.

However, we are now attempting to expand this to leaves from plants growing in the greenhouse or locally, in situ.  Surface sterilizing the leaves with hydrogen peroxide appeared to work better for removing contaminating microorganisms while retaining leaf vigor than bleach, ethanol, or combinations thereof.  Slight contamination was still present, but we are hoping that changing the substrate from water agar to sterilized filter paper will retain leaf viability while reducing the growth of other, opportunistic pathogens.  We had also used a standardized spore suspension, but growth seemed to be erratic; we are hoping that using younger, sporulating cultures will fix this.  Another change we plan to implement for the next step is taking leaf discs rather than whole leaves, as has been published in screens with a related pathogen of grapes.  This will standardize the amount of tissue being tested from species-to-species.  Despite the changes planned, we are excited with the successes we have had creating and implementing this screening methodology.

     To summarize, during this first year of the project, we have made good progress towards meeting our objectives, and have laid the groundwork for the next year of experiments.  A wide range of germplasm has been collected, most of it currently growing in our greenhouse or in vitro, and we are working on producing hybrids between the species in our collection.  In the laboratory, we have developed preliminary methodologies for doubling chromosomes, embryo rescue, tissue culture, and pathogen screening of our impatiens species.  Collections of both local populations of native species and local strains of the pathogen have helped to ensure that our work remains relevant to areas served by the grant.  We are working to refine our experimental processes, and have made good contacts with professionals who are aiding us in these endeavors.  Discussions with commercial growers, other researchers, and even passionate hobbyists have also given us an opportunity to share both our experimental mission and hone our goals.

• Figure 1. Growth of one individual of I. balsamina on four previously published media for tissue culture. Two replicates shown for each media.
• Figure 2. Screening results of plants grown in vitro, two weeks after inoculation with the pathogen. Infection on I. balsamina is indicated by an arrow.

Impacts and Contributions/Outcomes

     As the project was just started this year, we suspect our impacts and contributions will be greatest after more research and data collection.  That being said, we have had modest effects on spreading information related to the basis of our experiments, and received constructive input from people across a wide range of specializations.  Perhaps the most useful exchanges have been the talks with commercial impatiens growers.  Often conducted in tandem with germplasm acquisition, these have been excellent opportunities to clear up misconceptions (such as the supposed seed transmission of the pathogen in I. walleriana), hear the latest grower concerns, and share the progress on our project.  We have also tried to include an overview of our work in presentations to other groups.  During this past year, this included talks to faculty and students at the Pontifica Universidad Católica de Chile, the Liberty Hyde Bailey Garden Club, and the ‘Plants, Genes, and World Food Production’ course at Cornell University.  In each case, we used an overview of our project and its progress as an addition to the requested talk, and each event generated helpful questions and commentary.  Interaction with the private hobbyist who has provided the bulk of our collection has also put us in touch with social media connections, such as the ‘Planet Impatiens’ Facebook group.  This allows us to easily share our work internationally, and get advice from experts across a wide range of backgrounds; a lot of interest has been expressed there in seeing the hybrids from our program, as well as resistance data.  As we accumulate more results and produce more germplasm, we hope to be able to share and reach out even more.

Collaborators: