Epidemiology and Control of Ranunculus Bacterial Blight 1998 Proposals
EXECUTIVE SUMMARY
Epidemiology and Control of Ranunculus Bacterial Blight
Donald A. Cooksey University of California, Riverside
We recently published a description of a new Ranunculus disease caused by the bacterium Xanthomonas campestris. In addition to causing symptoms of blight on leaves and stems, the bacterium was shown to move systemically into root tubers and to infest seed. It is likely that the bacterium is spread in the industry in infested seed and tubers, and disinfest ation of these materials should be key to preventing the disease during Ranunculus selection and propagation. Ml seed lots tested were infected with this pathogen, which has become widespread and severe in the major Ranunculu s-growing region of Southern California. Infested seed lots have a lower percentage of viable seeds than noninfested seed lots, and infested tubers also show signs of necrosis. Infested tubers have resulted in diseased plants appearing in homeowners gardens. This proposal is to test seed and tuber treatment methods for eliminating the bacterial pathogen and to follow disease progression during two growing seasons at a commercial Ranunculus production site. The first season is in progress under flinding from the American Floral Endowment. This proposal is to continue that ftinding for the second year.
Information gained from this project will be disseminated through a peer-reviewed journal and Cooperative Extension publications directed toward both the floriculture industry and homeowners. The results are expected to provide practical solutions to this disease problem during Ranunculus selection and propagation, as well as improve the quality of Ranunculus in commercial and home gardens.
DESC~ON AND OBJECTIVES
Most xanthomonads have narrow host ranges, and we expect that the Ranunculus strain primarily infects Ranunculus or related plants As a xanthomonad, it is not likely to survive in soil or elsewhere in the environment, and disruption of the disease cycle through the elimination of seed and tuber infestations should therefore provide effective disease control. The proposed experiments will test this hypothesis through treatment of infested seed and tubers, followed by analysis of disease progression in the field.
Seed treatments and other sanitation practices are economic, safe methods of preventing many bacterial diseases. Sensitive methods of detecting the bacteria in seed and other propagative materials are key to the success of such a treatment program. We have used a selective medium to detect and quantif~r X canipestris in Ranunculus seed and tubers, and we have also used very sensitive polymerase chain reaction (PCR) technology to detect xanthomonad-specific DNA sequences from these bacteria.
Our objectives are to:
- Test the efficacy of seed and tuber treatments for removing Xanthomonas campestris, and measure effects on Ranunculus seed and tuber viability.
- Quanti~ the severity and systemic spread ofXanthomonas campestris in field plots established with treated and nontreated Ranunculus seed and tubers.
PROGRESS TO DATE
Our original proposal was to conduct our experiments during two seasons beginning with a fall planting in 1997. That planting has been made, and the experiments will continue through the growing season until May or June of 1998. In addition, we conducted a preliminary field trial at the commercial Ranunculus production site in Carlsbad, California, during the 1996-1997 growing season. That preliminary trial confirmed the appropriateness of our general plot design and of its placement in the commercial production field. Reviewers of the original proposal expressed concern that bacterial inoculum from the non-treated Ranunculus plants at the commercial site might contaminate our plots. However, the extensive use of border rows of clean plants surrounding the treated and untreated plots prevented contamination between plots and from the surrounding infested fields.
One cultivar was used that had a 3.4% frequency of seed infestation with Xanthomonas campestris. The seed was treated or untreated as described in Materials and Methods, and planted in the commercial site with six replicates for each treatment in a randomized block design. Seed was collected from those plots at the conclusion of the growing season, and we were pleased to find that our seed treatments before planting effectively controlled the development of bacterial inoculum and contamination of seed in plants grown out from the treated seed.
Seed treatment Freciuency of seed infestation after harvest
Control 4.96%
1/10 Chlorox for 15 mm 0.41%
1/10 Chlorox for 30 min 0.08%
1/10 Chlorox + 500C for 15 mm 0.0%
MATERIALS AND METHODS
Seed treatment. Preliminary tests have suggested that various chlorine bleach and hot water treatments were effective in reducing or eliminating the bacterial pathogen from seed.
Further tests will be conducted with seed and tubers to define the best treatments or combination of treatments, that maximize seed and tuber viability and elimination of bacteria. Treatments with chlorine bleach (1/10 dilution) and hot water (45~55O C) will range from 15-30 mm.
Seed and tuber viability and bacterial contamination will be assessed after seed treatments compared with control seed. Individual seeds will be plated directly on selective media to determine the percentage of seeds contaminated with Xanthomoiias campestris, as we have done previously. In addition, larger numbers of seed can be soaked in buffer with a mild detergent to remove bacteria from the seed, and the liquid can then be concentrated and plated on selective media for a more sensitive detection of low numbers of X campestris. Since this bacterium spreads systemically and may also be internal to seeds, we will also extract seed using a blender technique that is used for tomato seed internally contaminated with the bacterial canker pathogen. The blender technique will also be used in detecting the bacterium in treated and nontreated tubers. An additional, potentially more sensitive technique to assess seed and tuber contamination, is the amplification of DNA from X campestris using polymerase chain reaction technology (‘)CR). We have already shown that we can specifically amplif~ DNA from cultured strains ofX campestris from Ranunculus using PCR, and preliminary results show that we can get amplification of DNA directly from washes of infested plant materials. We will determine the sensitivity of this detection method with seed and tubers artificially contaminated with different known concentrations of bacteria. We have also submitted a preproposal to more extensively evaluate PCR technology, including a cost analysis, for routine detection of several bacterial pathogens of floriculture crops. If that proposal is flinded, we will include a much more extensive evaluation of PCR detection for the Ranunculus bacterial blight than planned above.
Field Experiments. Seed and tubers treated under conditions that optimize viability and elimination of bacteria will be planted in the field in replicated plots compared with nontreated plots. The plots will be incorporated into a commercial Ranunculus production site in Carlsbad, California, where have conducted preliminary tests last season and currently have our first tull set of experiments planted for the 1997-1998 season. Six replicates of each treatment will be planted. Each replicate will consist of 15 ft of a commercial bed with two rows of plants and drip irrigation in the center of the bed. Approximately 225 plants will be grown in each replicate plot. Replicates of each treatment will be arranged in a randomized block design, with border rows of plants that are free of the pathogen. The entire experimental design will be replicated with two different cultivars in two separate areas of the commercial Ranunculus production site. The first planting was done in December 1997, and we will sample each month for a total of about 7 months. At each sample date, visual disease ratings will be taken, and 10 plants from each plot will be removed for analysis. The presence ofX campestris will be assessed in leaves, stems, and roots/bulbs. At the end of the season, seeds and bulbs will be assessed for the presence of the pathogen. The experiment is to be repeated over at least two growing seasons, as environmental conditions favoring bacterial disease progression vary considerably from season to season.
During our preliminary trial in 1996-1997, tomato spotted wilt virus symptoms were common and masked some of the bacterial disease symptoms. In collaboration with UC virologists also working on Ranunculus at this site, this problem has hopeft~lly been eliminated by removal of adjacent weed hosts of the virus and thrips vector.
At the suggestion of reviewers of the original proposal, we are also assessing the role of weeds or other plant hosts as reservoirs of inoculum, and we are obtaining tubers from European and other U.S. sources to more generally assess the occurrence of this pathogen in Ranunculus.
The results from these tests will allow us to assess the success of seed and tuber treatment methods in a field environment conducive to the development of this disease. Such direct testing under conducive conditions can sometimes be more sensitive than laboratory tests for the presence of bacterial contaminants. In addition, the data we will collect will allow us to quantif~ disease progression during the growing season, from initial leaf spot symptoms to systemic infection, and movement to bulbs and seed. Such basic information about the biology of this pathogen is unknown, but needs to be considered in developing disease management practices.
BUDGET
Year I (funded)
Supplies (Media, petri dishes, PCR reagents)
Graduate Student Researcher
Travel to field site
Year 2 (proposed)
Supplies (Media, petri dishes, PCR reagents)
Graduate Student Researcher
Travel to field site
$ 5,000
17,662
500
$23,162
$ 5,000
18,436*
500
$23,936
*The increased salary reflects changes in statewide salary scales as of 11/11/97.
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Leader Qualifications
Name: Donald A. Cooksey
Title: Professor and Chair
Address: Department of Plant Pathology
University of California, Riverside
Education: B.A., Albion College, Biology Ph.D., Oregon State University, Botany and Plant Pathology
Employment: 1996-present, Chair, Department of Plant Pathology, University of California, Riverside
1994-present, Professor and Bacteriologist, Department of Plant Pathology, University of California, Riverside
1988-1994, Associate Professor and Associate Bacteriologist, Department of Plant Pathology, University of California, Riverside
1982-1988, Assistant Professor and Assistant Plant Pathologist, Dept. of Plant Pathology, University of California, Riverside
