© 2002 Plant Health Progress.
Managing Bacterial Speck and Spot of Tomato with Acibenzolar-S-Methyl in Virginia
A. S. Graves and S. A. Alexander, Eastern Shore Agricultural Research and Extension Center, Department of Plant Pathology, Physiology and Weed Science, Virginia Polytechnic Institute and State University, Painter, Virginia 23420
Tomato (Lycopersicon esculentum Mill.) production on the Eastern Shore of Virginia in 2000 was approximately 1,659 ha with an estimated value of $30.9 million (17). Plastic mulch is a very effective and efficient method for producing fresh market tomatoes (7); however, because of its impermeability, the potential for rainfall run-off is increased. Agriculture and aquaculture often exist immediately adjacent to each other and are inextricably connected by fingers of the Chesapeake Bay and Atlantic Ocean, which intrude into the agro-ecosystem of the Eastern Shore of Virginia. Recent data collected from some estuaries showed copper at levels potentially toxic to clams and other inhabitants of these estuaries (6,14).
Bacterial diseases are difficult to control and can be a limiting factor on tomato production in Virginia. Bacterial spot of tomato, caused by Xanthomonas axonopodis pv. vesicatoria (Doidge) Dye, is extremely difficult to control under moist, warm conditions, which can occur on the Eastern Shore of Virginia. The bacterial spot pathogen has a higher optimum growth temperature (24 to 30°C) than the optimum growth temperature of the bacterial speck pathogen (18 to 24°C) (9,10). When the weather conditions become moist and cooler than normal, bacterial speck, caused by Pseudomonas syringae pv. tomato (Okabe 1933) Young, Dye & Wilkie 1978, becomes the predominant bacterial disease. The bacterial spot pathogen has a higher optimum growth temperature (24 to 30°C) than the optimum growth temperature of the bacterial speck pathogen (18 to 24°C) (10,11). Bacterial disease development is favored by high rainfall, and by low-pressure disturbances with high wind velocity. The bacteria penetrate plant tissue through wounds created by wind-driven sand, insects, or other mechanical means such as stringing, pruning, and harvesting. Severe defoliation and fruit spotting result in significant yield loss (21). Three races of X. axonopodis pv. vesicatoria (T1, T2, and T3) have been described (11) and two have been identified on the Eastern Shore of Virginia (T1 and T3).
Copper combined with mancozeb is the standard pesticide treatment used for managing bacterial diseases on tomato (2). When environmental conditions are marginal for disease development, copper + mancozeb provides adequate control, but when environmental conditions favor disease development and inoculum levels are high, the standard treatment becomes considerably less effective (5,8). With the presence of copper-resistant strains of these bacterial pathogens (1), copper-based control measures become less effective (Fig. 1). Area growers averaged 17 applications of copper + mancozeb resulting in an estimated 34,000 kg copper being applied on the Eastern Shore of Virginia in 2000.
Plant elicitors represent a new approach to integrated disease management strategies (16). Ross (19) coined the term “systemic acquired resistance” (SAR) to describe long-lasting resistance induced by a pathogen to subsequent infections. To be a SAR-inducing compound, the compound must elicit protection to the same spectrum of pathogens as the biological inducer, cause the expression of the same biochemical markers as the biological inducer, and not have any anti-microbial activity (13). The plant elicitor acibenzolar-S-methyl (acibenzolar) is in the benzothiadiazole chemical class and complies with the definition of a SAR-inducer. Acibenzolar is currently marketed in the U. S. as Actigard (Syngenta Crop Protection, Greensboro, NC) and is formulated as a 50% water dispersible granule (12). The induction time of acibenzolar is thought to be 2 to 4 days suggesting that acibenzolar should be used as a preventative means of disease control. During SAR expression, several mechanisms appear to be activated simultaneously against pathogen establishment. This may reduce the risk of developing SAR-insensitive strains of the bacterial pathogens (20).
In an environmentally sensitive area such as the Eastern Shore of Virginia, the most effective way to reduce copper run-off from plasticulture is to reduce the amount of copper-based pesticides required for disease management. The objective of this study was to determine the efficacy of acibenzolar-S-methyl as a replacement for copper-based pesticides in the management of bacterial spot and bacterial speck diseases in tomato production on the Eastern Shore of Virginia.
General Field Procedures for the Bacterial Experiments
Experiments were carried out in commercial plasticulture tomato fields located in Bloxom, VA, (98A); Modestown, VA, (99A); Painter, VA, (99B); Parksley, VA, (00A); and Tasley, VA, (00B) on Virginia’s Eastern Shore during the growing seasons of 1998, 1999 and 2000. Experiment 99C was established prior to disease development at the Eastern Shore Agricultural Research and Extension Center (ESAREC).
In commercial fields, experiments 98A, 99A, 99B, 00A, and 00B were established in areas where bacterial disease was already present. Plots were established in single rows, 3.0 to 4.6 m long spaced 1.8 m apart with a plant spacing of 0.6 m. Table 1 shows pathogen, cultivars, transplant, and harvest dates for all experiments. The experiments were maintained with conventional fertilization, and disease, weed, and insect control. General field preparations for the experiments at the ESAREC (99C) were the same as used in commercial fields. Trickle irrigation tubing was laid at the same time that methyl bromide (281 kg/ha) was applied, and the beds were covered with plastic mulch. Tomato seedlings were transplanted into the beds 5 days later. Rows were staked at a 45-cm spacing and plants trellised with string 3 to 4 times per season depending on rate of growth. Plots consisted of single rows; 9.2 m in length spaced 1.8 m apart, with a plant spacing of 0.5 m. Experimental plots were laid out in a randomized complete block design with four replications per treatment.
* Pseudomonas syringae pv. tomato
** Xanthomonas axonopodis pv. vesicatoria
Treatments were initiated when plants were 30 to 61 cm high (pre-bloom), and reapplied in all tests on a 7- to 10-day interval. Names, formulations, and rates for all treatments are shown in Table 2. Treatments for experiments 98A, 99A, 99B, 00A, and 00B were applied with a propane-pressurized backpack sprayer, which delivered 486 liters/ha at 276 kPa. Plant height ranged from 60 cm at initiation of the experiment to approximately 120 cm at the end, with water volume kept constant for all applications. The boom was held parallel to the staked tomato row and both sides of the row were sprayed for uniform coverage. Experiment 99C was sprayed with a multi-boom sprayer mounted on a high-clearance Hagie tractor that delivered 373.5 liters/ha at 276 kPa. Water volume was kept constant for all applications.
Disease ratings of percent foliar infection consisted of an estimate of percent leaf area affected, and these estimates were used to calculate the Area Under the Disease Progress Curve (AUDPC) for each treatment (4). Research sites in commercial fields were selected based on the presence and uniformity of bacterial infection. Initial assessment of foliar infection was taken before any treatments were applied. Disease ratings were taken four times in 1998, three times in 1999, and five times in 2000 (Figs 2A to 2F).
Fruit was harvested from six plants per plot in experiments 98A and 99C, from four plants per plot in experiments 99A and 99B, and from two plants per plot in experiments 00A and 00B. Fruits were sized, weighed, and graded for infection. Mean separation was calculated by Duncan’s new multiple range test, Agricultural Research Manager software (Gyllings Data Management, Brookings, SD). Mean comparisons were performed only when the Analysis of Variance P (F) was significant at the mean comparison observed significance level.
Bacterial Speck Experiments
Actigard (acibenzolar-S-methyl) applied at 10.5 g a.i./ha significantly (P < 0.05) reduced infected leaf area and the AUDPC compared to the standard treatment (copper hydroxide + mancozeb) and untreated control in all experiments (Figs. 2A to C). In experiment 98A (Fig. 2A), chemicals were applied six times before the second disease rating and a total of nine times before the last rating. In experiments 00A (Fig. 2B) and 00B (Fig. 2C), treatments were applied once before the second disease rating, once before the third, twice before the fourth, and once before the last disease rating for a total of five treatment applications. Actigard alone or Actigard + Quadris (azoxystrobin) or Kocide (copper hydroxide) provided significantly (P < 0.05) better control than the standard treatment. The addition of copper hydroxide or azoxystrobin to Actigard did not significantly (P < 0.05) increase the level of foliar disease control as compared to Actigard alone. Dithane (mancozeb) alone provided significantly (P < 0.05) better foliar disease control than the untreated control (Figs. 2B and C).
There were no significant differences (P < 0.05) in marketable yield (tomatoes showing no symptoms of bacterial infection) between treatments in any of the six experiments (Table 3). There was no significant difference (P < 0.05) between treatments in fruit showing symptoms of bacterial speck in experiments 98A, 00A, and 00B.
* Fruit not showing symptoms of bacterial spot or speck. Experiment 99A was only harvested one time.
** Quadris in the 99C study was applied four times according to Tomcast, 25DSV+15DSV,with Actigard applied on the last three applications after detection of bacterial spot, all other treatments applied at 7-day interval. All means within each column were not significantly different (P<0.05).
Bacterial Spot Experiments
Actigard provided significantly (P < 0.05) better foliar bacterial spot protection than the untreated control (Fig. 2D, E, and F). Actigard alone did not significantly (P < 0.05) differ from the standard in disease severity, although numerically, Actigard averaged slightly better control. In experiment 99C (Fig 2F), applications were started before the development of disease with eight applications made before the second disease rating and one more before the last rating. The Actigard + Kocide or Actigard + Quadris treatments in experiment 99C (Fig 2F) provided significantly (P < 0.05) better control at the last rating than the standard treatment. In experiment 99B (Fig 2E), the Actigard treatment with Kocide provided 46% less foliar infection than the standard treatment at the last rating. The Actigard + Kocide or Actigard + Quadris treatments in experiment 99C (Fig 2F) provided significantly (P < 0.05) better control at the last rating than the standard treatment. In experiment 99B (Fig 2E) at the last rating, only Actigard and Actigard + Kocide had significantly (P < 0.05) better disease control than the untreated control. In experiments 99A (Fig 2D) and 99C (Fig 2F), the standard treatment provided significantly (P < 0.05) better protection against foliar infection than the untreated control. As in the bacterial speck experiments, the Dithane alone treatment had less bacterial foliage infection (P < 0.05) than the untreated control in experiment 99A (Fig 2D), which indicates that Dithane may be affecting bacterial infection. Marketable yields were not significantly different (P < 0.05) between treatments (Table 3). In experiment 99A, the Actigard + Kocide and Actigard + Quadris treatments resulted in numerically higher marketable yields than the untreated control. In experiments 99B and 99C, Actigard in combination with Kocide or Quadris and Dithane alone produced the highest numerical yields.
Bacterial Pathogen Identification and Copper Sensitivity
To identify the bacterial pathogens present in each experimental plot, 10 to 20 symptomatic leaves were randomly collected and removed to the laboratory where isolations were made. Leaves were washed for 20 minutes in running water with several drops of Tween 20, then dipped in 0.5 % NaOCl for 15 seconds, followed by two 15-second washes in sterile distilled water. Sections of leaf tissue were transferred to approximately 3 ml sterile saline (0.85%) in a sterile petri plate, covered and allowed to sit for 1 hour, then streaked with an inoculating loop onto tryptic soy agar (TSA) in 10 cm diameter petri plates (Difco). After 48 hr incubation at 27°C, single well-isolated colonies that were either pale yellow, shiny, circular colonies typical of Xanthomonas axonopodis pv. vesicatoria or pale white, flat colonies typical of Pseudomonas syringae pv. tomato on TSA were selected for further testing. Colonies were tested for oxidase reaction by transferring to BBL DrySlide Oxidase slides (Becton Dickinson and Co., MD), and streaked onto Bacto Pseudomonas Agar F plates (Becton Dickinson and Co., MD) to check for fluorescein production. Colonies were then transferred to blood agar plates (Gibson Laboratories, Inc., KY), which contained 5% sheep’s blood in TSA. Bacteria were then allowed to grow for 24 hours before identification. Bacteria were identified using the Biolog Microlog System (Biolog, Inc., CA). Only P. syringae pv. tomato was found in experimental plots 98A, 00A, and 00B, and only X. axonopodis pv. vesicatoria was found in experimental plots 99A, 99B, and 99C.
A bacterial culture identified as P. syringae p v. tomato isolated from experiment 98A, and bacterial cultures identified as X. axonopodis pv. vesicatoria from experiments 99A, 99B, and 99C were sent to Dr. Seong Hwan Kim, Pennsylvania Department of Agriculture, for confirmation and pathogenicity testing using Koch’s postulates, and copper-sensitivity testing. Bacterial cultures of P. syringae pv. tomato from the experiments 00A and 00B were tested for pathogencity and for copper-sensitivity, one representative isolate from each experiment, using the procedure reported by Bender and Cooksey (3). Two copper-sensitive isolates (PDDCC 3357 and CNBP 1323) of P. syringae pv. tomato provided by Dr. Diane Cuppels, Agriculture and Agri-Food Canada, London, ON were used for comparison.
The P. syringae pv. tomato strain collected from experiment 98A was inhibited by 368 mg/ml copper sulfate as compared to a sensitive strain that was inhibited by 175 mg/ml copper (1). The P. syringae pv. tomato strain from experiments 00A and 00B grew on MGY plates amended with 0.75-mM copper sulfate as compared to the sensitive strains that grew at 0.5-mM copper sulfate. Growth of strains of X. axonopodis pv. vesicatoria from experiments 99A, 99B, and 99C was inhibited at a concentration of 406 mg/ml copper as compared to a copper-sensitive strain that was inhibited at 88 mg/ml copper.
Summary and Conclusions
Managing diseases, reducing pesticide usage and addressing environmental concerns are important issues in modern agriculture. These factors all come to the forefront in a high value crop such as fresh market tomatoes grown in an area with environmental concerns, such as the Eastern Shore of Virginia. Copper + mancozeb has been the best bactericide available for managing foliar bacterial pathogens for more than 30 years. However, when conditions are highly favorable for disease development, standard copper bactericides cannot provide adequate control of foliar bacterial diseases in tomato (5,8). The existence of copper-tolerant and/or copper-resistant populations further complicates bacterial disease management (1). Applying copper and mancozeb every 3 to 5 days has been needed to manage severe bacterial infections because no other effective alternative was available. The objective of this study was to identify and evaluate a replacement for copper that was safer for the environment and could provide effective management of bacterial diseases of tomato.
Actigard effectively controlled bacterial speck and bacterial spot on the Eastern Shore of Virginia. Actigard treatments generally provided equivalent or superior control of foliar bacterial disease when compared to the standard treatment, which was Kocide + Dithane. There were no significant yield differences between treatments found in this study. Actigard treatments were not associated with yield reductions.
Growers’ fields provided an excellent opportunity to evaluate the efficacy of Actigard in an agricultural production system. Copper was not as effective as Actigard for controlling P. syringae pv. tomato, but was somewhat more effective against X. axonopodis pv. vesicatoria. This may have been due to the presence of more copper-sensitive strains of X. axonopodis pv. vesicatoria. Some differences in disease severity may be associated with the presence of copper-tolerant bacteria when copper is applied or simply different environmental and cropping conditions for each growing season. Indications of copper-tolerant strains were found in all study areas. Among all three growing seasons and six fields, the best control resulted from the use of Actigard or tank mixes of fungicides in combination with Actigard. Considering other diseases that need to be controlled, especially early blight, Actigard in combination with Quadris alternated with a chlorothalonil or mancozeb fungicide is most likely the best recommendation.
Yield reduction has been reported in bell pepper treated with Actigard at rates of 17 and 35 g a.i./ha (18). Louws et al. (15) observed reduced plant growth in greenhouse tomato experiments in Ohio and reduced yield of extra large tomato fruit in a field trial in Florida. Actigard rates used in these field evaluations were 35 g a.i./ha and higher. Louws et al. (15) reported no consistent or significant tomato yield reduction at rates of 35 g a.i./ha. In our study, Actigard controlled bacterial diseases at a rate of 10.5 g a.i./ha with no signs of phytotoxicity or yield loss. In the study conducted by Louws et al. (15), Actigard was initially applied 1 to 30 days after transplanting to the field and a minimum of 7 days prior to bacterial infection. In five of the six experiments in this study, Actigard was initially applied after bacterial infection had reached 3 to 20% and 30 to 60 days after transplanting. These data suggest that rates of 35 g a.i./ha and the initiation of treatments before infection, although recommended to growers, may not be essential to manage bacterial diseases. This provides considerably more flexibility for use in an integrated disease management program.
Actigard used at the rate of 10.5 g a.i./ha per application requires significantly less pesticide input than the standard Kocide 1.2 kg a.i./ha + Dithane 1.7 kg a.i./ha per application. Based on data collected from these experiments, Actigard can effectively replace copper for bacteria control. An average of 17 applications of copper + mancozeb are made per crop during a normal weather pattern. Economically, even though Actigard is more expensive than the copper + mancozeb combination, the reduction in the number of applications from 17 to 8 makes Actigard cost effective, especially at the 10.5 g a.i./ha used in this study.
This study shows that Actigard is comparable to or more effective than copper + mancozeb in controlling bacterial speck and spot diseases on tomatoes. Also, the existence of copper-resistant strains of these bacterial pathogens favors the use of Actigard. The results of this study support the general findings reported by Louws et al. (15) favoring the use of acibenzolar-S-methyl over copper + mancozeb; however, the effective rate of 10.5g a.i./ha is substantially lower in this study. Although Actigard applications began after infection in this study, in an operational sense it would probably be more effective to initiate Actigard treatments before bacterial disease symptoms appeared. The risk of a pathogen developing SAR-insensitive strains of bacteria is substantially reduced, since acibenzolar-S-methyl has no direct activity on the pathogen (20). The use of Actigard for managing bacterial diseases of tomatoes is an essential tool for meeting the objectives of providing effective disease management and environmental compatibility within a sensitive ecosystem. The results from this study show that Actigard, at a rate of 10.5 g a.i./ha, can provide producers on the Eastern Shore of Virginia and other environmentally sensitive areas with an effective alternative to copper-based pesticides that is better for the environment and is effective in managing bacterial diseases in fresh market tomato production.
This research was supported in part by a grant from the Virginia Department of Agriculture and Consumer Services project #98-008-3, and was used for the partial fulfillment of the requirements for the Master of Science degree for the senior author. The authors wish to thank Ms. Christine Waldenmaier for her technical assistance and support.
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