Final Report for GNC08-090
Project Information
We confirmed that spray of low concentrations of Natrium acetate (NaAc) significantly compromised bacterial growth of syringe-inoculated Pseudomonas syringae pv. tomato, the causal agent of bacterial speck disease on tomato, suggesting the potential of NaAc as a new method to control Pseudomonas pathogens. The best concentration of NaAc to suppress bacterial growth of syringe-inoculated Pseudomonas has been identified as 1mM. However, the spray of 1mM NaAc did not significantly suppress the bacterial growth of surface-inoculated (dip-inoculated) Pseudomonas syringae pv. tomato. The possible application of NaAc as an effective bactericide for vegetables and other crops needs further investigation in diseased fields.
Introduction:
Pseudomonas syringae bacteria are causal agents of bacterial speck diseases on many crops such as tomato and bean. Conventional control methods such as various bactericide compounds, biological control strains and breeding approaches are not very effective due to the limitations of their uses. Organic growers need better options for dealing with bacterial diseases. Our preliminary results showed that spray of low concentrations of Natrium acetate (NaAc, the sodium salt of vinegar) significantly compromised bacterial growth and disease symptom development of Pseudomonas syringae pv. tomato, the causal agent of bacterial speck disease on tomato, suggesting the potential of NaAc as a new method to control Pseudomonas pathogens.
To further test the potential of NaAc, there are two objectives in this project:
1. We plan to assess and to optimize the NaAc recipes, which include NaAc concentrations, timing of spray and species of pathogens targeted. Preliminary investigation of efficacy of tested recipes will be conducted in the laboratory and greenhouse facilities on the Kansas State University campus in Manhattan, KS.
2. With the conclusions drawn from assessment of greenhouse trials, we will test the optimized NaAc recipes on field trials at the Rocky Ford Research Center Farm in Manhattan and the K-State Horticulture Research and Extension Center in Olathe, KS. Ultimately, we will be able to determine which tested recipes work best against diseases of interest. The success of this research will provide farmers more options in controlling bacterial speck or other diseases and serve as a basis for further research on NaAc recipe as an effective organic bactericide.
Cooperators
Research
We used tomato plants and Pseudomonas syringae pv. tomato bacterium in this study. Experiments include assessment and optimization of NaAc recipes against bacterial speck on tomato and bean by testing different concentrations of spray (from 0.1mM to 100mM), examining the timing of spray (days and times before or after bacterial inoculation), and testing NaAc application against other bacterial pathogens (such as Xanthomonas on tomato and cabbage). This was done in laboratory and greenhouse facilities on the Kansas State University campus in Manhattan, KS. Inputs were graduate student labor, laboratory, and greenhouse expense for proposed experiments.
Syringe inoculation of Pseudomonas syringae pv. tomato on tomato plants: Pseudomonas syringae pv. tomato was grown in King’B (KB) medium to OD600 = 2.5. Bacterial cells were collected by centrifugation, washed twice with water, and resuspended in water. Bacteria at 2 × 104 CFU/ml (Colony Forming Units) were syringe-injected into leaves of 3-week-old tomato plants. For bacterial growth assays, leaf disks (1 cm2) were removed at 0 and 6 days after inoculation and ground in sterile water. Bacteria were diluted to proper concentration and plated on a KB plate containing rifampicin at 30 mg/liter for bacterial count.
Surface inoculation of Pseudomonas syringae pv. tomato on tomato plants: Leaves of three-week-old tomato plants were dipped into a bacterial suspension (1 × 106 CFU/ml in water) for 1 min. For bacterial growth assays, leaf disks (1 cm2) were removed at 0 and 6 days after inoculation and ground in sterile water. Bacteria were diluted to proper concentration and plated on a KB plate containing rifampicin at 30 mg/liter for bacterial count.
1. Spraying 1mM NaAc solution reduced bacterial growth of syringe-inoculated Pseudomonas syringae pv. tomato about 20 folds compared to water control after 6 days of inoculation, suggesting the potential of NaAc as a new method to control Pseudomonas pathogens.
2. Of concentrations from 0.1mM to 100mM, the best concentration of NaAc for reducing bacterial growth of syringe-inoculated Pseudomonas syringae pv. tomato on tomato has been determined as 1mM. Spraying 100mM NaAc wilted the plants in a couple of hours, indicating that high concentration of NaAc is toxic to tomato plant. Spray of 10mM, 1mM, or 0.1mM NaAc did not show any toxic effects on tomato. We used 1mM NaAc for the following experiments.
3. Unlike the spray of NaAc, syringe-injection of 1mM NaAc solution into leave did not display clear suppression of bacterial growth of syringe-inoculated Pseudomonas syringae pv. tomato in tomato plants. This result further supported the potential of NaAc as a new method to control Pseudomonas pathogens by simply spraying on the leaf surface.
4. For the timing of spray, spraying 1mM NaAc solution 1 day before or at the same day of bacterial inoculation (syringe-injection) showed the best result to reduce bacterial growth. This result suggested that the NaAc can help to prevent tomato from Pseudomonas prior to infection, but may be not very efficient to cure the disease once the bacteria start infection.
5. NaAc assay failed to compromise bacterial growth of Pseudomonas syringae pv. phaseolicola on bean plants and Xanthomonas campestrispv pv. campestris on cabbage plants.
6. The spray of 1mM NaAc did not significantly suppress the bacterial growth of surface-inoculated (dip-inoculated) Pseudomonas syringae pv. tomato on tomato plants. Considering that the surface-inoculation mimicked the natural infection of Pseudomonas on host plants, this result suggested that the best NaAc recipe still remained to be studied in the future. The possible application of NaAc as an effective bactericide for vegetables and other crops needs further investigation in natural diseased fields.
Educational & Outreach Activities
Participation Summary:
1. Deng X. Identification and characterization of Pseudomonas syringae mutants altering type III secretion system induction. PhD dissertation. Department of Plant Pathology, Kansas State University, 2009.
2. Deng X, Lan L, Xiao Y, Kennelly M, Tang X. Pseudomonas syringae two component response regulator RhpR regulates genes carrying an inverted repeat element promoter. Submitted to Mol Plant Microbe Interact.
3. Deng X, Lan L, Xiao Y, Zhou JM, Tang X. 2009. Pseudomonas syringae pv. phaseolicola mutants compromising type III secretion system. Mol Plant Microbe Interact. 22(8):964-976.
Project Outcomes
Our results suggested the good potential of NaAc as a new method to control Pseudomonas pathogens. However, the possible application of NaAc as an effective bactericide for vegetables and other crops needs further investigation in diseased fields.
Areas needing additional study
Because the spray of 1mM NaAc did not significantly suppress the bacterial growth of surface-inoculated (dip-inoculated) Pseudomonas syringae pv. tomato on tomato plants, the possible application of NaAc as an effective bactericide for vegetables and other crops needs further investigation in diseased fields.