Progress 09/15/07 to 08/31/10

Outputs
OUTPUTS: Objective 1: Synergistic interactions between copper (Cu) and garlic-derived compounds were characterized in depth. Cu, allyl disulfide (AD), diallyl sulfide mixture (DADS), or garlic extract (GAE) were applied to nutrient broth either alone or in combination at their respective ED10, ED25, and ED50 values. A suspension of X. arboricola pv. pruni was added to give a final concentration of 50,000 CFU/ml. Bacterial growth inhibition was measured after 48 h, and synergism in the mixtures was determined using the Gowing equation. In a second set of experiments ("killing curve method"), Cu, the three garlic-derived compounds, and X. arboricola pv. pruni were applied to nutrient broth as described above; aliquots of the suspensions were dilution-plated onto nutrient agar after 48 h; and CFUs were counted 48 h later. Mixtures that reduced log(CFU) by more than 2-fold compared with the more effective singular compound were considered to act synergistically. Objective 2: A total of seven field trials was conducted in Georgia and Pennsylvania between 2008 and 2010 to evaluate the performance of Cu-DADS mixtures and other oxytetracycline alternatives applied during the cover sprays in reducing leaf and fruit infection by X. arboricola pv. pruni across a range of peach or nectarine cultivars. Early-season (dormant through bloom) copper sprays were omitted to allow build-up of bacterial inoculum for the test. In addition, plots were spray-inoculated at bloom, petal fall, and/or shuck-split with a bacterial suspension of the pathogen. The trials also included Cu alone, DADS alone, an oxytetracycline (OTC) standard, and an untreated check. Furthermore, an oxytetracycline-Cu mixture as well as the experimental antibiotics gentamicin or kasugamycin (the latter in mixture with either Cu, captan, or Penncozeb) were evaluated in select trials. There were between 6 and 8 cover spray applications for each treatment. In three trials, between one and five soil drench applications (without foliar sprays) of the resistance inducer acibenzolar-S-methyl were evaluated as an alternative type of bactericide treatment. These applications were made in a volume of 0.5 or 0.75 L/tree with a concentration of active ingredient between 0.01 and 0.04%. Leaf and fruit disease incidence or severity as well as phytotoxicity were assessed in each trial. In untreated plots in 2009 and 2010, custom shelters were used to protect immature fruit from bacterial splash and infection starting at shuck-split. Developing fruit were exposed by removing the shelters at various stages of fruit development. In separate experiments on peach seedlings outdoors, the effect of temperature, natural sunlight, simulated rainfall, and three ultraviolet (UV) protectants and one sticker-extender (lignin, titanium oxide, oxybenzone, and Nu Film-17) on the longevity of OTC on the leaf surface were quantified. Numerous grower presentations and field visits related to bacterial spot management were made in the two participating states. An MS student and a postdoc working on the disease were mentored at Penn State and the Univ. of Georgia, respectively. PARTICIPANTS: Harald Scherm, University of Georgia, Athens (PI; overall project coordination, participation in field trials in Georgia); Phillip M. Brannen, University of Georgia, Athens (co-PI; conducted outreach in Georgia); Henry K. Ngugi, Penn State University, Biglerville (co-PI; conducted field trials and outreach in Pennsylvania). PARTNER ORGANIZATIONS: USDA-ARS Fruit and Tree Nut Research Lab, Byron, GA (provided access to field sites). COLLABORATORS: M. Jefferson Cook, IV, Taylor County Extension Coordinator, Butler, GA (assisted in field trials in Georgia). TRAINING/ PROFESSIONAL DEVELOPMENT: Rock S.C. Christiano, University of Georgia, Athens (postdoc; planned, conducted, and analyzed all research under objective 1); Sarah J. Bardsley, Penn State University, Biglerville (MS student; assisted in field trials in Pennsylvania). TARGET AUDIENCES: The primary target audience for this project consists of commercial stone fruit growers in the Southeast and the mid-Atlantic region of the United States. Knowledge transfer has occurred annually in educational sessions at the Cumberland-Shenandoah Fruit Workers Conference; the Mid-Atlantic Fruit and Vegetable Convention; the FREC field day in Biglerville, PA; grower meetings in Adams, Franklin and Lancaster Counties in Pennsylvania; the Middle Georgia and North Florida-South Georgia Peach Updates; and numerous farm and packinghouse visits in both states between 2008 and 2010. Overall, at least 150 fruit growers were educated on bacterial spot management and specifically on use of low rates of Cu and other OTC alternatives during cover sprays. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Objective 1: The observed antibacterial activities in the Cu-garlic mixtures were greater than the expected antibacterial activities (calculated based on the assumption of additive action of Cu and the garlic product) in every single case (Cu at 3 concentrations combined with each garlic product at 3 concentrations), indicating synergism. In 19 of these 27 combinations, synergism was statistically significant. The relative levels of synergism between the three garlic derivatives were similar and ranged from 18 to 42%. Mixtures of Cu with AD, DADS, and GAE reduced log(CFU) by 7.3, 2.0, and 2.1, respectively. These results are expected to spark further research to determine the chemical or physiological mechanism(s) of the synergistic interaction with regard to bacterial disease control. Objective 2: Although disease levels and the ranking of different treatments varied across the seven trials, several robust conclusions could be drawn with regard to the efficacy of OTC alternatives. Cu at low rates provided disease suppression equivalent to or better than OTC. Furthermore, gentamicin alone as well as kasugamycin in mixture with Cu or captan had significant activity against X. arboricola pv. pruni. This suggests that Cu compounds and the fungicide captan may have a synergistic activity on kasugamycin since this antibiotic is generally not effective against bacterial spot when applied alone. Results with the Cu-DADS tank-mix were inconsistent across trials, with no significant improvement in disease suppression (compared with Cu alone) in most cases. Acibenzolar-S-methyl drench applications also performed inconsistently, with significant disease suppression in one trial and limited efficacy in two trials. In the shelter experiments, fruit exposed continuously had the highest disease severity while fruit sheltered from infection throughout most of the trial had the least amount of disease. From the beginning of the season, disease severity and fruit susceptibility declined exponentially. Thus, it should be possible to scale back bacterial spot sprays starting early June. In the study on OTC longevity on the leaf surface, constant temperatures up to 40C did not affect OTC degradation on leaves. In contrast, OTC residue decreased rapidly in natural sunlight in the absence of rain, declining by 43.8, 77.8, and 92.1% within 1, 2, and 4 days after application, respectively. Use of shade fabric, simulating overcast sky, reduced OTC degradation significantly but did not extend OTC persistence beyond 7 days. OTC residues during the 7-day exposure period were negatively correlated with solar radiation, but not with temperature. UV protectants and Nu Film-17 were ineffective in improving OTC persistence outdoors. Simulated rainfall drastically lowered OTC residue after 2 min, and levels were near the detection limit after 60 min of continuous rain. Over the past 3 years, data collected from this project have led to the major revision of the sections on bacterial spot management in the Pennsylvania Tree Fruit Production Guide and the Southern Peach, Nectarine, and Plum Pest Management and Culture Guide.

Publications

• Christiano, R.S.C., Reilly, C.C., Miller, W.P., and Scherm, H. 2010. Oxytetracycline dynamics on peach leaves in relation to temperature, sunlight, and simulated rain. Plant Dis. 94:1213-1218.
• Ngugi, H.K., Bardsley, S.J., Lehman, B.L., and Jarjour, B. 2010. Evaluation of alternatives to oxytetracycline for control of bacterial spot of peach during cover sprays, 2009. Plant Disease Management Reports 4:STF023.

Progress 09/15/08 to 09/14/09

Outputs
OUTPUTS: Objective 1: The laboratory experiments to characterize and quantify synergistic interactions between copper (Cu) and garlic-derived compounds (such as diallyl disulfide, DADS) have been completed and the results are being prepared for publication. Objective 2: Field trials were conducted in Georgia (cultivar O'Henry) and Pennsylvania (cultivars Redhaven, Easternglo, and Beekman) to evaluate the performance of Cu-DADS mixtures applied during the cover sprays in reducing leaf and fruit infection by X. arboricola pv. pruni. At both sites, early-season (dormant through bloom) copper sprays were omitted to allow build-up of bacterial inoculum for the test. Furthermore, the plots were spray-inoculated at full bloom (Georgia) or at shuck-split (Pennsylvania) with a bacterial suspension of the pathogen. In addition to the Cu-DADS mixture, bactericide treatments included Cu alone, DADS alone (at a concentration of 1.0 and 0.1% in Georgia and Pennsylvania, respectively), an oxytetracycline standard, and an untreated check. The trial in Georgia further included an oxytetracycline-Cu mixture and the experimental antibiotic gentamicin (GWN-9350), whereas that in Pennsylvania included the experimental antibiotic kasugamycin (Kasumin) in mixture with either Cu or Captan 80 WDG. There were 8 and 7 cover spray applications for each treatment in Georgia and Pennsylvania, respectively. Furthermore, soil drench applications (without foliar sprays) of the resistance inducer acibenzolar-S-methyl (Actigard 50WG) were evaluated as a second type of bactericide treatment in Georgia. The drench applications were made at 10-20% bloom and again during pit-hardening by pouring 0.5 L of a 0.01 or 0.04% suspension of the formulated product around the trunk of each tree on each application date. Leaf and fruit disease incidence or severity were assessed at both sites. Several grower presentations and visits related to bacterial spot management were made in the two participating states. Furthermore, an MS student and a postdoc working on the disease were mentored at Penn State and the Univ. of Georgia, respectively, as part of the project. PARTICIPANTS: Harald Scherm, University of Georgia, Athens (PI; overall project coordination, participation in field trials in Georgia); Phillip M. Brannen, University of Georgia, Athens (co-PI; conducted outreach in Georgia); Henry K. Ngugi, Penn State University, Biglerville (co-PI; conducted field trials and outreach in Pennsylvania). PARTNER ORGANIZATIONS: USDA-ARS Fruit and Tree Nut Research Lab, Byron, GA (provided access to field sites). COLLABORATORS: M. Jefferson Cook, IV, Taylor County Extension Coordinator, Butler, GA (assisted in field trials in Georgia). TRAINING/ PROFESSIONAL DEVELOPMENT: Rock S.C. Christiano, University of Georgia, Athens (postdoc; planned, conducted, and analyzed all research under objective 1); Sarah J. Bardsley, Penn State University, Biglerville (MS student; assisted in field trials in Pennsylvania). TARGET AUDIENCES: The primary target audience for this project consists of commercial stone fruit growers in the Southeast and the mid-Atlantic region of the United States. Knowledge transfer has occurred in educational sessions at the Cumberland-Shenandoah Fruit Workers Conference; the Mid-Atlantic Fruit and Vegetable Convention; the FREC field day in Biglerville, PA; grower meetings in Adams, Franklin and Lancaster Counties in Pennsylvania; the Middle Georgia and North Florida-South Georgia Peach Updates; and numerous farm and packinghouse visits in both states in 2008 and 2009. Overall, at least 150 fruit growers were educated on bacterial spot management and specifically on use of low rates of Cu during cover sprays. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Objective 1: Model experiments have documented repeatedly and reproducibly the presence of an intermediate level of synergism (ranging from 18 to 42%) in mixtures of Cu with several garlic-derived compounds such as DADS, allyl disulfide, or garlic extract. Publication of these results is expected to spark further research to determine the chemical or physiological mechanism(s) of the synergistic interaction with regard to bacterial disease control. Objective 2: Moderately severe levels of fruit infection developed In the Georgia orchard trial, and significant treatment effects were observed for the overall incidence of affected fruit, the incidence of severely affected fruit (with disease-induced cracking and extensive gumming), and percent marketable fruit. Only gentamicin and the Cu-DADS tank-mix reduced overall fruit disease incidence and increased marketable yield compared with the untreated check. These two treatments also reduced the incidence of severe symptoms significantly, as did Cu alone, the tank-mix of Cu + oxytetracycline, and the acibenzolar-S-methyl drench at the higher of the two rates. Oxytetracycline, the commercial standard for bacterial spot control, was ineffective when applied alone. In the Pennsylvania trial, all treatments (except DADS applied alone at the low rate of 0.1%) were effective in reducing disease severity on fruit across all cultivars with the exception of Redhaven, which is considered resistant and had the lowest disease levels overall. Treatments containing Cu or kasugamycin + captan typically provided as good as or better fruit protection than the oxytetracycline standard. The results from the two states confirm observations made in 2008 that Cu at low rates provides better disease suppression than oxytetracycline, and that gentamicin alone as well as kasugamycin in mixture with copper or captan has significant activity against X. arboricola pv. pruni. The latter observation suggests that Cu compounds and the fungicide captan may have a synergistic activity on kasugamycin since this antibiotic is generally not effective on bacterial spot when applied alone. Results with the Cu-DADS tank-mix were inconsistent across the two states, most likely because of the different concentrations used in the trials. Further testing is also warranted for the acibenzolar-S-methyl drench application, where earlier timing of the second application and combination with standard foliar bactericide sprays should be evaluated. Over the past 2 years, data collected from this project have led to the major revision of the sections on bacterial spot management in the Pennsylvania Tree Fruit Production Guide and the Southern Peach, Nectarine, and Plum Pest Management and Culture Guide.

Publications

• Cook, M.J., Christiano, R.S.C., Savelle, A.T., Reilly, C.C., Hotchkiss, M.W., and Scherm, H. 2010. Evaluation of alternatives to oxytetracycline for control of bacterial spot of peach during the cover sprays, 2009. Plant Disease Management Reports 4:STF017.
• Ngugi, H.K., Bardsley, S.J., Halbrendt, N.O., and Jarjour, B. 2009. Evaluation of alternatives to oxytetracycline for control of bacterial spot of peach during cover sprays, 2008. Plant Disease Management Reports 3:STF022.
• Ngugi, H.K., Bardsley, S.J., Halbrendt, N.O., and Kauffman, K. 2009. Evaluation of low rates of copper bactericides for control of bacterial spot of peach during cover sprays, 2008. Plant Disease Management Reports 3:STF021.
• Ngugi, H.K., Bardsley, S.J., and Halbrendt, N.O. 2009. Refining management strategies for bacterial spot of peach. Pennsylvania Fruit News 89 (2):18-24.

Progress 09/15/07 to 09/14/08

Outputs
OUTPUTS: Objective 1: Synergistic interactions between copper (Cu) and garlic-derived compounds were characterized further in laboratory experiments. In one set of experiments, Cu, allyl disulfide (AD), diallyl sulfide mixture (DADS), or garlic extract (GAE) were applied to nutrient broth either alone or in combination at their respective ED10, ED25, and ED50 values. A suspension of X. arboricola pv. pruni was added to give a final concentration of 50,000 CFU/ml. Bacterial growth inhibition (absorbance at 600 nm) was measured after 48 h, and synergism in the mixtures was determined using the Gowing equation. In a second set of experiments ("killing curve method"), Cu, the three garlic-derived compounds, and X. arboricola pv. pruni were applied to nutrient broth as described above; aliquots of the suspensions were dilution-plated onto nutrient agar after 48 h; and CFUs were counted 48 h later. Mixtures that reduced log(CFU) by more than 2-fold compared with the more effective singular compound were considered to act synergistically. Objective 2: Field trials were conducted in Georgia (cultivar O'Henry) and Pennsylvania (cultivars Easternglo, Beekman, Snow King, and Sweet Dream) to evaluate the performance of Cu-DADS mixtures applied during the cover sprays in reducing leaf and fruit infection by X. arboricola pv. pruni. At both sites, early-season (dormant through bloom) copper sprays were omitted to allow build-up of bacterial inoculum for the test. Furthermore, the plots were spray-inoculated at bloom and petal fall (Georgia) or at shuck-split (Pennsylvania) with a bacterial suspension of the pathogen. In addition to the Cu-DADS mixture, treatments included Cu alone (Kocide 2000 in Georgia, Kocide 3000 in Pennsylvania), DADS alone, an oxytetracycline standard (Flameout in Georgia, Mycoshield in Pennsylvania), and an untreated check. The trial in Georgia further included an oxytetracycline-Cu mixture and the experimental antibiotic gentamicin (GWN-9350), whereas that in Pennsylvania included the experimental antibiotic kasugamycin (Kasumin) in mixture with either Cu or Captan 80 WP. There were 7 and 6 cover spray applications for each treatment in Georgia and Pennsylvania, respectively. Leaf and fruit disease incidence or severity were assessed. At Penn State Univ., an MS student was recruited and mentored to work on bacterial spot disease. The student was involved in disease assessment in the field trial. PI Ngugi presented five talks on bacterial spot management related to this project. PARTICIPANTS: INDIVIDUALS: Harald Scherm, University of Georgia, Athens (PI; overall project coordination, participation in field trials in Georgia); Phillip M. Brannen, University of Georgia, Athens (co-PI; conducted outreach in Georgia); Henry K. Ngugi, Penn State University, Biglerville (co-PI; conducted field trials and outreach in Pennsylvania). PARTNER ORGANIZATIONS: USDA-ARS Fruit and Tree Nut Research Lab, Byron, GA (provided access to field sites). COLLABORATORS: M. Jefferson Cook, IV, Taylor County Extension Coordinator, Butler, GA (assisted in field trials in Georgia). TRAINING/ PROFESSIONAL DEVELOPMENT: Rock S.C. Christiano, University of Georgia, Athens (postdoc; planned, conducted, and analyzed all research under objective 1); Sarah J. Bardsley, Penn State University, Biglerville (MS student; assisted in field trials in Pennsylvania). TARGET AUDIENCES: The primary target audience for this project are stone fruit growers in the Southeast and in the mid-Atlantic region of the United States. Knowledge transfer occurred in educational sessions at the Cumberland-Shenandoah Fruit Workers Conference (November 2007); producer meetings in Adams, Franklin and Lancaster Counties in Pennsylvania (Feb. 2008); the Middle-Georgia Peach Update (Feb. 2008); and the FREC field day in Biglerville, PA (Sept. 2008). Overall, at least 150 fruit growers were educated on bacterial spot management and specifically on use of low rates of Cu during cover sprays. As a result, at least 100 acres of peach were treated at least twice with low rates of Cu as an alternative to the antibiotic oxytetracyline. This action could have saved the growers a minimum of $25 on cost of bactericides alone. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Objective 1: In the first experiment, the observed antibacterial activities in the Cu-garlic mixtures were greater than the expected antibacterial activities (calculated based on the assumption of additive action of Cu and the garlic product) in every single case (Cu at 3 concentrations combined with each of three garlic products at 3 concentrations), indicating synergism. In 19 of these 27 combinations, synergism was statistically significant at P=0.05. The relative levels of synergism between the three garlic derivatives were similar and ranged from 18 to 42%. In the second experiment, mixtures of Cu with AD, DADS, and GAE reduced log(CFU) by 7.3, 2.0, and 2.1, respectively, indicating significant synergism which was most pronounced for the Cu-AD mixture. Objective 2: In the Georgia trial, only gentamicin reduced the incidence of infected leaves compared with the untreated check, whereas no treatment reduced the overall incidence of infected fruit. However, the incidence of fruit with severe symptoms was lowered significantly by gentamicin and the treatments containing Cu (either alone or in combination with DADS or oxytetracycline). The Cu-DADS mixture did not improve disease control compared with Cu alone. In the Pennsylvania trial, all treatments, except DADS applied alone, were effective at reducing fruit disease severity across all cultivars. In all cultivars except Snow King, the Cu-DADS mixture resulted in numerically lower disease severity levels than Cu applied alone; however, this effect was not statistically significant. Also in Pennsylvania, at least 150 fruit growers were educated on bacterial spot management and specifically on use of low rates of Cu during cover sprays. As a result, at least 100 acres of peach were treated at least twice with low rates of Cu as an alternative to the antibiotic oxytetracyline. This action could have saved the growers a minimum of $25 on cost of bactericides alone.

Publications

• No publications reported this period