Final Report for GNC13-173
A series of experiments to explore the impacts of temperature, inoculum load, and delayed inoculation on re-isolation of Pseudomonas syringae pv. syringae (PSS)from pruning wounds of sweet cherry. Report on progress of the repeatable infection system to test potential control products for infection of PSS. Temperatures 10ºC and 20ºC have no impact on pruning wound resistance development for at least 10 days after pruning. Recommended inoculum loads for a repeatable infection system are 105 and 103 cfu/mL. Preliminary report on potential efficacy of Kasumin as a PSS control.
Bacterial canker (caused by Pseudomonas syringae pv. syringae, PSS) can cause death of spurs, loss of limbs, decreased yields, and tree mortality. Once the bacteria enter the tree, the infection may become systemic making treatment impossible. The only way to overcome the disease is to prevent the initial infection. Late spring frosts, after a warm March in 2012, subjected many sweet cherry trees in Michigan to damage and subsequent bacterial canker infection which killed the infected spurs. Sweet cherry spurs bear fruit for several years, and the loss of these spurs will severely reduce yields for 2-3 years as the trees replace lost fruiting area with new growth. In a canopy training system trial that was infected with bacterial canker in Clarksville, Michigan, the number of dead meristems (which includes shoot and spur meristem death) ranged from 30 to 120 dead meristems per tree across all training systems (Lillrose & Lang, unpublished). Similar damage was seen throughout Michigan and has influenced grower yield in subsequent years. Damage is not limited to growing points; when spur infections migrate into the woody tissues, they create cankers that can girdle branches causing loss of limbs. These infections may become systemic, ultimately causing tree mortality. Young trees are more susceptible to bacterial canker, creating a concern for the establishment of new orchards because of the high investment cost of planting trees and the 3-4 years of orchard maintenance before marketable yields are attained.
The future uncertainties of climate change (i.e., an increasing incidence of temperature extremes) raise concerns that infections of this magnitude might become more prevalent and possibly cripple the sweet cherry industry. This problem is particularly important in the Midwest region where cool, wet weather in spring favors PSS inoculum growth. It is essential that we understand the factors contributing to PSS growth and infection of sweet cherry so growers can reduce inoculum during critical times when the tree is susceptible to infection.
Only using sprays (i.e., copper) to target bacteria when trees are most susceptible may help to slow the development of resistance in PSS, which can occur with overuse of pesticides. It can also reduce costs for growers and impacts on the environment.
Successful bacterial canker infections result from the convergence of three parameters: 1) a sufficient bacterial population, 2) an infectious environment, and 3) a susceptible host. Bacterial population sizes are influenced by many factors, including temperature and free moisture. Population sizes rise in response to rain events (Latorre et al., 1985). In controlled environments, free moisture increases infection when bacterial populations are low, but doesn't impact infection under higher bacterial pressure. In the field, bacterial populations are under moisture and nutrient stress, and free water enables them to increase. Temperature increases infection rates for twigs up to 20?C (Latorre et al., 2002). In the lab, bacteria grow faster with increasing temperatures between 0-33?C, then growth slows at higher temperatures (Young et al., 1977). PSS have been isolated from tree cankers in the winter and early spring, but were undetectable during summer (Latorre et al., 1985). These higher seasonal populations were probably in response to the lower temperatures and the free moisture at that time of year.
Environment interacts with both the bacterial population size and tree susceptibility. It is hypothesized that wound healing rate is influenced by temperature (under investigation by Lillrose & Lang). Trees become susceptible through wounding events such as pruning, leaf scars in autumn, and freeze-damage of blossoms. More research is needed to understand the interactions of environment, inoculum load, and host susceptibility in bacterial canker infection.
Two SARE projects have relevance to this proposal. One examined the commercial viability of organic sweet cherry production using insect-exclusion netting (Project number LNE03-182). Success was limited by wind-caused branch abrasions that led to bacterial canker infections (Jentsch, 2007). This illustrates the importance of understanding the environmental and horticultural factors that make wounds susceptible to infection, so growers
can focus on strategies to reduce inoculum at those periods.
Another SARE project began in 2012 to study Pseudomonas syringae in blueberry (Project number FW12-074). This research examines the affect of wind turbines on frost-induced bacterial blight in blueberry, while monitoring environmental conditions (Uppal, 2012). Uppal’s research complements the proposed study because sweet cherry infections also occur from freeze damage. Data from the blueberry study could be applied to use of wind turbines in sweet cherry. Uppal’s study does not address woody tissue infections caused by pruning or leaf scar invasion, which is important for overcoming sweet cherry bacterial canker in the North Central Region.
It is essential to understand the environment’s influence on bacterial canker infections to help growers target the bacterium at crucial infection points. This will reduce spray use, save growers time and money, and reduce selection for resistance. The development of a repeatable infection system will enable rapid testing of potential control measures for bacterial canker that will increase sustainability for an expanding North Central (and Northeast) region sweet cherry industry.
Jentsch, P. 2007. Final report for: Determining the commercial viability of an exclusionary production system using disease-resistant columnar apple and sweet cherry cultivars. Available at:
Latorre, B., Gonzalez, J., Cox, J., and Vial, F. (1985). Isolation of Pseudomonas-syringae pv. syringae from cankers and effect of free moisture on its epiphytic populations on sweet cherry trees. PLANT DISEASE 69, 409–412.
Latorre, B., Lillo, C., and Rioja, M. (2002). Effects of temperature, free moisture duration and inoculum concentration on infection of sweet cherry by Pseudomonas syringae pv. syringae. PHYTOPARASITICA 30, 410–419.
Uppal, P. 2012. Proposal: Study and Control of Pseudomonas Syringae on Blueberry Plants. Available at:
Young, J., Luketina, R., and Marshall, A. (1977). Effects on temperature on growth in vitro of Pseudomonas syringae and Xanthamonas-pruni. JOURNAL OF APPLIED BACTERIOLOGY 42, 345–354.
Objective one: To develop a rapid repeatable infection system for leaf scar and pruning infections which will allow for rapid testing of new potential controls.
Objective two: To help determine the conditions that make sweet cherry more susceptible to bacterial canker infection to allow growers to try to prune when trees are less susceptible or spray to reduce leaf scar infection at key infectious periods.
Objective three: Test hypothesis from previous pruning experiments in the field to test pruning timing and effectiveness of a biocontrol for reducing infection.
Leaf scar chamber infection
To develop a repeatable infection assay for leaf scars, three types of leaf removal were tested; pulling of green leaves, clipping lamina from petioles a week before removal, and environmentally-induced senescence simulated by cooling greenhouse trees to 7.5?C in the day and 4.5?C at night with a 10:14 hr light:dark photoperiod. Trees were inoculated with a mixture of 3 rifampicin-resistant strains of Pseudomonas syringae pv. syringae (PSS) bacteria at 107 cfu/ml. Controls were inoculated with de-ionized water. Inoculation was done by misting the entire tree to run off with an atomizer. The goal was to determine if pulling leaf scars or clipping petioles would be an appropriate substitute for environmentally-induced senescence. After 2 months, branches were removed, surface sterilized, and bacteria were extracted by mincing bud tissues adjacent to leaf scars in Phosphate Buffered Saliene (PBS) and plating on Rifampicin (Rif) amended Kings B media (KB).
General Protocol for Pruning Experiments
The following experiments were inoculated with 3 or 4 strains of Rif-resistant PSS and then re-isolated on KB amended with Rif at a rate of 75 micrograms/mL. All pruning sites were surface sterilized with 70% alcohol wipes and pruners were sterilized between cuts with 10% bleach and then sprayed with 75% ethanol. Cut surfaces were soon after inoculated with 50 µL bacteria (at concentrations specified in experiments below) or control (either culturing broth of KB or PBS) and kept at constant temperatures with a 12:12 hr light:dark photoperiod. Trees were maintained in growth chambers for two weeks before attempted recovery and re-isolation of bacteria. Branches were surface sterilized, and shavings of tissue from the pruning site were minced in half strength PBS which was then plated out on Rif-amended KB.
Tissue stage and infection
Greenhouse-grown nursery trees were pruned while endodormant or during summer after leaves were present. Endodormant or summer-pruned trees were pruned and inoculated with 0 or 107 cfu/ml as described above.
Effect of temperature on infection by Pseudomonas syringae pv. syringae
Greenhouse-grown nursery trees of ‘Bing’ or ‘Rainier’ on ‘Gisela 6’, were overwintered outside the greenhouse. Trees were maintained in growth chambers at 20?C and 10?C, and pruned and inoculated as described above with a concentration of 0 or 107 cfu/ml.
Temperature and inoculum load
Freshly-pruned greenhouse-grown nursery ‘Bing’ on ‘Gisela 6’ trees. Trees were put in growth chambers at 10ºC and 20ºC and pruned and inoculated with 0, 10, 103, and 105 cfu/ml as described above.
Testing of pruning wounds with Kasumin and Blossom Protect
Greenhouse-grown nursery trees were ‘Bing’ on ‘Gisela 6’. Treatments imposed were Kasumin, Blossom Protect, and no treatment, spraying either before or before and after inoculation. Pruning and inoculation as described above except that inoculation was delayed about an hour after spraying. Inoculum load was 0, 105, or 107 cfu/ml. Branches were cut and sprayed with product. About an hour later surfaces were inoculated. The “before and after” treatment was sprayed again about an hour after inoculation.
Kasumin testing at different inoculum concentrations
Greenhouse-grown ‘Bing’ on ‘Gisela 6’ nursery trees were used for testing of products. Kasumin (2L) was tested at a rate of 18.9 ml/gal and it and the water control were sprayed one hour before and one hour after inoculation. Pruning and inoculation were performed as described above except that the inoculation was delayed by one hour to allow for spraying. The first spray was completed right after pruning. Inoculation was done with 0, 10, 103, or 105 cfu/ml.
Days to inoculation
Greenhouse-grown nursery trees ‘Bing’ on Gisela 6. Factors were pruning 0, 2, or 5 days before inoculation, inoculum levels of 0, 105, or 107 cfu/ml, and trunk diameter ~1cm or ~1.5cm. Pruning and inoculation were done as described above except branches were pruned on the appropriate days and then inoculated with bacteria or control on day 0.
Time to inoculation and Temperature
Greenhouse-grown nursery trees ‘Bing’ on Gisela 6. Factors were Pruning 0, 10, 17, or 24 days before inoculation, temperature at either 10?C or 20?C, and inoculum levels of 0 or 105 cfu/ml. Branches were pruned and inoculated as described above except that branches were pruned on the appropriate days and then inoculated with bacteria or control on day 0.
Some of these experiments were only recently completed, and statistics are still in progress.
Leaf scar chamber infection
Determining a method for simulating fall leaf scar infections is key for rapid assessment of products that could reduce bacterial canker infection. The goal was to find a way to simulate leaf scars that could be used throughout the year and preferably without a long preconditioning phase to mimic a fall environment. Environmentally-induced senescence and pulled leaf scars yielded 100% re-isolation of inoculated PSS, while clipped petioles only yielded 60% re-isolation of PSS (Fig. 1). Environmentally-induced senescence yielded infections with higher re-isolatable populations (personal observation) while pulled leaves were consistently infected but with lower re-isolatable populations. In a repeatable infection system, maximum infection occurs with environmentally-induced senescence or pulling leaves to create scars directly. Pruning wounds are easier to target with controls by growers because the infection point is easier to identify. Due to the time and resource demands of the pruning experiments, focus was placed on developing a system for pruning and this was the only experiment for leaf scars.
Tissue stage and infection
Plant phenological stage can impact processes such as gene regulation and plant responses. Plants may up-regulate defense mechanisms at different phenological stages, allowing conservation of costly secondary metabolites. Our goal was to observe if different tissue stages were more susceptible to infection. Tissue stage had no effect on susceptibility to infection at 107 cfu/ml (Fig. 2). If there was any difference in plant resistance, the inoculum load might have been too high and overcame any innate resistance. It is unknown whether, at a lower bacterial concentration, the defense mechanisms of the tree may have had an effect.
Effect of temperature on infection
Higher temperature was reported to increase disease incidence (Latorre et al., 2002) in twig explants and we wanted to replicate that study in an entire plant system. All inoculated wounds became infected. Only one uninoculated branch had re-isolation of bacteria. Temperatures studied did not have an impact on re-isolation success at 107 cfu/ml (Fig. 3) and because all became infected, it may be that such a high concentration of bacteria overcame any effect of these temperatures.
Temperature and inoculum load
We further studied the effects of temperature and inoculum load. Inoculum load had a greater impact than temperature, reducing re-isolation by 25% at 103 cfu/mL, compared to 105 cfu/mL at 10?C. There was a 25% reduction in re-isolation at 103 cfu/ml at 10?C compared to 20?C but it may not be statistically significant. 105 cfu/ml bacteria was sufficient to cause infection 100% of the time (Fig. 4). This suggests that 105 cfu/ml may be sufficient for a repeatable infection system and may be a better choice than the extremely high concentrations used in previous studies. Using 103 cfu/ml would also help identify products or environmental influences that may have a more subtle impact on infection. Statistics are needed to determine if the observed results are significant.
Testing of Kasumin and Blossom Protect for pruning wounds
To test the utility of the infection system, some initial product testing was done. The treatments only seemed to affect infection when inoculum level was 105cfu/ml and only when sprayed before and after inoculation (Fig. 5). This provided further evidence that 107 cfu/ml is too high for detecting effectiveness of products that might prevent infection. It is possible that some of these products may be effective at lower concentrations. Spraying before and after increased the effectiveness of the products and further work could be done to determine if only spraying after would be sufficient.
Kasumin testing at different inoculum concentrations
Kasumin was tested further to see if it might be effective at lower inoculum concentrations. Kasumin was not effective at 105 cfu/ml, but a 25-50% reduction in infection was found at 103 and 10 cfu/ml (Fig. 6). The infection of the uninoculated controls is assumed to result from either cross-contamination or insufficient Rifampicin in the re-isolation media which could have allowed growth of additional bacteria. Further study is needed to clarify the impact of inoculum load on Kasumin efficacy.
Days to inoculation, inoculum load, and pruning cut size
To explore the option of delayed inoculation (which a cherry grower could manage by pruning during dry weather), delaying inoculation 0, 2, and 5 days after pruning was studied at different inoculum loads. All inoculated large wounds were infected 100% of the time. There was a 20% reduction in infection on inoculation at Day 2 at 105 cfu/mL and a 40% reduction on Day 5 at 105 cfu/mL (Fig. 7). These results, however, are probably not statistically significant and it might take longer for the tree to resist infection. Suberin accumulation in cherry periderm after wounding starts to appear as early as 7 days and continues increasing for at least 24 days (BIGGS, 1985). This suggests that resistance of wounds to bacterial canker infection could take longer than the 5 day period studied in this experiment.
Temperature and days to inoculation
To investigate the effect of days to inoculation and temperature, an experiment was performed with extremely delayed inoculation. Temperatures 10?C and 20?C were not statistically significant, but the 20?C treatments appeared to resist infection sooner than the 10?C. There was a 33-50% reduction in infection by waiting 17-24 days before inoculating, depending on the temperature at which the trees were kept (Fig. 8). Days to inoculation was significant, and branch resistance to infection took a long time to occur. Therefore, simply waiting for a brief warm, dry period of weather in the spring for pruning will be insufficient to prevent PSS infection when there are high PSS populations. This work becomes very important for a repeatable infection system because it shows that temperature in this range does not impact plant ability to resist infection for at least 10 days. When evaluating products where immediate inoculation would be used, temperatures in this range should have no effect. Further work should address the impact of more extreme temperatures such as those that would be present during winter pruning or pruning after harvest.
Recommendations and discussion for repeatable infection system
Based on the results from this project, we recommend using an inoculum load of 105 and maybe 103 cfu/ml. 105 cfu/ml yields consistent infections that are susceptible to some typical doses of antibiotics. Infections created with 103 cfu/mL could also be helpful because they should be more sensitive for testing of potential control treatments with less efficacy. 107 cfu/ml yields repeatable infections, but under controlled conditions for testing of potential treatments, it is often too high to detect treatment efficacy. Field inoculations at 107 cfu/mL may be needed because of competing microfauna and environmental stresses, such as UV radiation. More research is needed to assess the influence of tissue stage at lower inoculum loads.
Temperatures from 10?C to 20?C had little effect on percent re-isolation within the repeatable infection system. Increased resistance to bacterial canker infection did not occur until after 10 days at 10?C and 20?C. Thus, inoculations made in this temperature range should give consistent results when inoculated soon after pruning. More research is needed to determine the impact of lower temperatures that might occur with winter dormant pruning or pruning after harvest. Appropriate tissue stage should also be taken into consideration in those studies. In the orchard, temperature will likely play a role in native PSS populations and this should be considered when making pruning decisions. When choosing when to prune in spring, growers should be more concerned about PSS population than temperature between 10?C and 20?C, because inoculum load had a greater impact on infection success.
The repeatable infection system showed some effect of Kasumin on reducing bacterial canker infection; however, the experiment should be repeated due to the high level of contamination in the controls.
Pruning wound resistance to infection was reduced by 33 and 50% when inoculation did not occur until 17 and 24 days, respectively, at 20?C. This is important to growers because there was no reduction in infection when 10 days elapsed between pruning and inoculation. Infectious conditions are generally wet weather when PSS populations are high. A previous recommendation has been to wait for several days of dry weather to reduce PSS infection potential. However, that recommendation is unlikely to reduce bacterial canker infection unless there is a warm dry period of over 10 days. In Michigan conditions, that is unlikely to occur and growers should pursue other options such as reducing PSS populations during infectious periods, or pruning in a prolonged dry spell during the summer.
Educational & Outreach Activities
This work will be included in my dissertation. No other publications are currently in progress. Due to the recent completion of some of these experiments, they have not yet been presented.
To date the research has not had a significant impact. Previous recommendations advised pruning with several days of warm, dry weather. This project demonstrated resistance to PSS takes at least 10 days to develop at 10?C to 20?C. As growers are made aware of the time required for pruning wounds to resist infection, it will allow them to make informed pruning decisions. Infectious periods to avoid would be wet weather when PSS populations would be high. Realizing that resistance takes time to develop, will allow growers to focus their attention on reducing PSS populations or changing time of pruning to a time when there will be a prolonged dry spell such as after harvest.
To date, no grower adoption has taken place. However, associated work on bacterial canker and trellis wire wounding has been adopted by growers testing new training systems requiring trellis wires. They are taking precautions of using wire made or covered with plastic.
Areas needing additional study
The repeatable infection system was tested with two control products, but further testing would clarify their usefulness because of contamination in the samples.
Time to pruning wound infection resistance as been documented in spring temperatures, it would be important to test the influence of colder temperatures that growers would encounter with dormant pruning. Tissue stage should be addressed in those experiments because orchard trees would be dormant which could impact infection success.