Botond Balogh

Identification of Open Reading Frames Unique to a Select Agent: Ralstonia solanacearum Race 3 Biovar 2

An 8x draft genome was obtained and annotated for Ralstonia solanacearum race 3 biovar 2 (R3B2) strain UW551, a United States Department of Agriculture Select Agent isolated from geranium. The draft UW551 genome consisted of 80,169 reads resulting in 582 contigs containing 5,925,491 base pairs, with an average 64.5% GC content. Annotation revealed a predicted 4,454 protein coding open reading frames (ORFs), 43 tRNAs, and 5 rRNAs; 2,793 (or 62%) of the ORFs had a functional assignment. The UW551 genome was compared with the published genome of R. solanacearum race 1 biovar 3 tropical tomato strain GMI1000. The two phylogenetically distinct strains were at least 71% syntenic in gene organization. Most genes encoding known pathogenicity determinants, including predicted type III secreted effectors, appeared to be common to both strains. A total of 402 unique UW551 ORFs were identified, none of which had a best hit or >45% amino acid sequence identity with any R. solanacearum predicted protein; 16 had strong (E < 10(-13)) best hits to ORFs found in other bacterial plant pathogens. Many of the 402 unique genes were clustered, including 5 found in the hrp region and 38 contiguous, potential prophage genes. Conservation of some UW551 unique genes among R3B2 strains was examined by polymerase chain reaction among a group of 58 strains from different races and biovars, resulting in the identification of genes that may be potentially useful for diagnostic detection and identification of R3B2 strains. One 22-kb region that appears to be present in GMI1000 as a result of horizontal gene transfer is absent from UW551 and encodes enzymes that likely are essential for utilization of the three sugar alcohols that distinguish biovars 3 and 4 from biovars 1 and 2.

Management of Tomato Bacterial Spot in the Field by Foliar Applications of Bacteriophages and SAR Inducers

Various combinations of the harpin protein, acibenzolar-S-methyl, and bacteriophages were compared for controlling tomato bacterial spot in field experiments. Harpin protein and aciben-zolar-S-methyl were applied every 14 days beginning twice before transplanting and then an additional four applications throughout the season. Formulated bacteriophages were applied prior to inoculation followed by twice a week at dusk. A standard bactericide treatment, consisting of copper hydroxide plus mancozeb, was applied once prior to inoculation and then every 7 days, while untreated plants served as an untreated control. Experiments were conducted in north and central Florida fields during fall 2001, spring 2002, and fall 2002. In three consecutive seasons, acibenzolar-S-methyl applied in combination with bacteriophage or bacteriophage and harpin significantly reduced bacterial spot compared with the other treatments. However, it did not significantly affect the total yield compared with the standard or untreated control. Application of host-specific bacteriophages was effective against the bacterial spot pathogen in all three experiments, providing better disease control than copper-mancozeb or untreated control. When results of the disease severity assessments or harvested yield from the bacteriophage-treated plots were grouped and compared with the results of the corresponding nonbacteriophage group, the former provided significantly better disease control and yield of total marketable fruit.

Phage Therapy for Plant Disease Control

Bacteria cause a number of economically important plant diseases. Bacterial outbreaks are generally problematic to control due to lack of effective bactericides and to resistance development. Bacteriophages have recently been evaluated for controlling a number of phytobacteria and are now commercially available for some diseases. Major challenges of agricultural use of phages arise from the inherent diversity of target bacteria, high probability of resistance development, and weak phage persistence in the plant environment. Approaches for resistance management--by applying phage mixtures and host-range mutant phages and, for increasing residual activity, by employing protective formulations, avoiding sunlight, and utilizing propagating bacterial strains--resulted in better efficacy and reliability. Deployment of phage therapy as part of an integrated disease management strategy, which includes the use of genetic control, cultural control, biological control, and chemical control, also has been investigated and will likely increase in the future.

Improved efficacy of newly formulated bacteriophages for management of bacterial spot on tomato

Bacteriophages are currently used as an alternative method for controlling bacterial spot disease on tomato incited by Xanthomonas campestris pv. vesicatoria. However, the efficacy of phage is greatly reduced due to its short residual activity on plant foliage. Three formulations that significantly increased phage longevity on the plant surface were tested in field and greenhouse trials: (i) PCF, 0.5% pregelatinized corn flour (PCF) + 0.5% sucrose; (ii) Casecrete, 0.5% Casecrete NH-400 + 0.5% sucrose + 0.25% PCF; and (iii) skim milk, 0.75% powdered skim milk + 0.5% sucrose. In greenhouse experiments, the nonformulated, PCF-, Casecrete-, and skim milk-formulated phage mixtures reduced disease severity on plants compared with the control by 1, 30, 51, and 62%, respectively. In three consecutive field trials, nonformulated phage caused 15, 20, and 9% reduction in disease on treated plants compared with untreated control plants, whereas plants treated with PCF- and Casecrete-formulated phage had 27, 32, and 12% and 30, 43, and 24% disease reduction, respectively. Plants receiving copper-mancozeb treatments were included in two field trials and had a 20% decrease in disease in the first trial and a 13% increase in the second one. Skim milk-formulated phage was tested only once and caused an 18% disease reduction. PCF-formulated phage was more effective when applied in the evening than in the morning, reducing disease on plants by 27 and 13%, respectively. The Casecrete-formulated phage populations were over 1,000-fold higher than the nonformulated phage populations 36 h after phage application.

Integration of Biological Control Agents and Systemic Acquired Resistance Inducers Against Bacterial Spot on Tomato

Two strains of plant growth-promoting rhizobacteria, two systemic acquired resistance inducers (harpin and acibenzolar-S-methyl), host-specific unformulated bacteriophages, and two antagonistic bacteria were evaluated for control of tomato bacterial spot incited by Xanthomonas campestris pv. vesicatoria in greenhouse experiments. Untreated plants and plants treated with copper hydroxide were used as controls. The plant growth-promoting rhizobacteria or a tap water control were applied as a drench to the potting mix containing the seedlings, while the other treatments were applied to the foliage using a handheld sprayer. The plant growth-promoting rhizobacteria strains, when applied alone or in combination with other treatments, had no significant effect on bacterial spot intensity. Messenger and the antagonistic bacterial strains, when applied alone, had negligible effects on disease intensity. Unformulated phage or copper bactericide applications were inconsistent in performance under greenhouse conditions against bacterial spot. Although acibenzolar-S-methyl completely prevented occurrence of typical symptoms of the disease, necrotic spots typical of a hypersensitive reaction (HR) were observed on plants treated with acibenzolar-S-methyl alone. Electrolyte leakage and population dynamics experiments confirmed that acibenzolar-S-methyl-treated plants responded to inoculation by eliciting an HR. Application of bacteriophages in combination with acibenzolar-S-methyl suppressed a visible HR and provided excellent disease control. Although we were unable to quantify populations of the bacterium on the leaf surface, indirectly we determined that bacteriophages specific to the target bacterium reduced populations of a tomato race 3 strain of the pathogen on the leaf surface of acibenzolar-S-methyl-treated plants to levels that did not induce a visible HR. Integrated use of acibenzolar-S-methyl and phages may complement each other as an alternative management strategy against bacterial spot on tomato.

Control of Citrus Canker and Citrus Bacterial Spot with Bacteriophages

Bacteriophages, alone or in combination with copper bactericides, were evaluated for managing Asiatic citrus canker and citrus bacterial spot incited by Xanthomonas axonopodis pathovars citri and citrumelo, respectively. In a set of five greenhouse experiments, phage treatment provided consistent control of citrus canker, causing an average of 59% reduction in disease severity. However, treatment with phage was ineffective if applied with skim milk, a protective formulation, which increases phage residual activity. In nursery settings, phage treatment also reduced disease but was less effective than copper-mancozeb, a chemical bactericide. The integration of phage and copper-mancozeb resulted in equal or less control than copper-mancozeb application alone. Phage treatments were evaluated in a commercial citrus nursery for reducing citrus bacterial spot caused by natural inoculum. Phage treatment provided significant disease reduction on moderately sensitive Valencia oranges in two trials (48 and 35%); however, on the highly susceptible grapefruit host it was ineffective. In an experimental citrus nursery, phage treatment provided significant control of citrus bacterial spot caused by a phage-sensitive strain, but was equally or less effective than copper-mancozeb. The combination of phage and copper-mancozeb did not increase control compared with copper-mancozeb alone.

Considerations for using bacteriophages for plant disease control

The use of bacteriophages as an effective phage therapy strategy faces significant challenges for controlling plant diseases in the phyllosphere. A number of factors must be taken into account when considering phage therapy for bacterial plant pathogens. Given that effective mitigation requires high populations of phage be present in close proximity to the pathogen at critical times in the disease cycle, the single biggest impediment that affects the efficacy of bacteriophages is their inability to persist on plant surfaces over time due to environmental factors. Inactivation by UV light is the biggest factor reducing bacteriophage persistence on plant surfaces. Therefore, designing strategies that minimize this effect are critical. For instance, application timing can be altered: instead of morning or afternoon application, phages can be applied late in the day to minimize the adverse effects of UV and extend the time high populations of phage persist on leaf surfaces. Protective formulations have been identified which prolong phage viability on the leaf surface; however, UV inactivation continues to be the major limiting factor in developing more effective bacteriophage treatments for bacterial plant pathogens. Other strategies, which have been developed to potentially increase persistence of phages on leaf surfaces, rely on establishing non-pathogenic or attenuated bacterial strains in the phyllosphere that are sensitive to the phage(s) specific to the target bacterium. We have also learned that selecting the correct phages for disease control is critical. This requires careful monitoring of bacterial strains in the field to minimize development of bacterial strains with resistance to the deployed bacteriophages. We also have data that indicate that selecting the phages based on in vivo assays may also be important when developing use for field application. Although bacteriophages have potential in biological control for plant disease control, there are major obstacles, which must be considered.

Effect of Application Frequency and Reduced Rates of Acibenzolar-S-Methyl on the Field Efficacy of Induced Resistance Against Bacterial Spot on Tomato

Acibenzolar-S-methyl (ASM), a plant activator known to induce systemic acquired resistance, has demonstrated an ability to manage a number of plant diseases, including bacterial spot on tomato caused by four distinct Xanthomonas spp. The aim of this study was to evaluate application rate and frequency of ASM in order to optimize field efficacy against bacterial spot in Florida, while minimizing its impact on marketable yields. ASM was applied biweekly (once every 2 weeks) as a foliar spray at a constant concentration of 12.9, 64.5, and 129 μM throughout four field experiments during 2007-08. A standard copper program and an untreated control were also included. Overall, biweekly applications of ASM did not significantly reduce disease development or the final disease severity of bacterial spot compared with the copper-mancozeb standard or the untreated control. Only one experiment showed a significant reduction in the final disease severity on plants treated with ASM at 129 μM compared with the untreated control. Three additional field trials conducted during 2009-10 to evaluate the effects of weekly and biweekly applications of ASM at concentrations of 30.3 to 200 μM found that weekly applications provided significantly better disease control than biweekly applications. The tomato yields were not statistically improved with the use of ASM relative to the untreated control and standard copper program. Weekly ASM applications at rates as low as 75 μM (equivalent to 1.58 g a.i./ha in 100 liters of water or 0.21 oz. a.i./acre in 100 gallons of water) to 200 μM (equivalent to 4.20 g a.i./ha in 100 liters of water or 0.56 oz. a.i./acre in 100 gallons of water) were statistically equivalent in managing bacterial spot of tomato without significantly reducing yield compared with the untreated control.

Soil-based systemic delivery and phyllosphere in vivo propagation of bacteriophages: Two possible strategies for improving bacteriophage persistence for plant disease control

Soil-based root applications and attenuated bacterial strains were evaluated as means to enhance bacteriophage persistence on plants for bacterial disease control. In addition, the systemic nature of phage applied to tomato roots was also evaluated. Several experiments were conducted applying either single phages or phage mixtures specific for Ralstonia solanacearum, Xanthomonas perforans or X. euvesicatoria to soil surrounding tomato plants and measuring the persistence and translocation of the phages over time. In general, all phages persisted in the roots of treated plants and were detected in stems and leaves; although phage level varied and persistence in stems and leaves was at a much lower level compared with persistence in roots. Bacterial wilt control was typically best if the phage or phage mixtures were applied to the soil surrounding tomatoes at the time of inoculation, less effective if applied 3 days before inoculation, and ineffective if applied 3 days after inoculation. The use of an attenuated X. perforans strain was also evaluated to improve the persistence of phage populations on tomato leaf surfaces. In greenhouse and field experiments, foliar applications of an attenuated mutant X. perforans 91-118:∆OPGH strain prior to phage applications significantly improved phage persistence on tomato foliage compared with untreated tomato foliage. Both the soil-based bacteriophage delivery and the use of attenuated bacterial strains improved bacteriophage persistence on respective root and foliar tissues, with evidence of translocation with soil-based bacteriophage applications. Both strategies could lead to improved control of bacterial pathogens on plants.

Integrated Management of Tomato Bacterial Spot

Bacterial diseases of plants play an important role in the world’s agriculture by reducing yield and marketability of particular crops or by limiting their production in areas with environmental conditions conducive for disease development. Plant pathogenic bacteria present many obstacles in developing efficient plant protection practices. In spite of technological advances, there is no bactericide that can be efficiently used for control of plant bacterial diseases. Due to lack of adequate chemical based bactericides plant pathologists constantly search for alternatives and possibility for their integration with preventive measures in order to develop sustainable disease control strategy. Tomato bacterial spot management currently relies on use of pathogen-free seed and transplants, elimination of volunteer tomato plants, resistant cultivars, and frequent application of a copper-based bactericides. However, these practices are ineffective in regions where hot and humid weather favor spread of the pathogen and development of the disease. Novel technologies, such as application of systemic acquired resistance inducers and use of biocontrol agents integrated with conventional practices, represent new quality in plant protection and provide increase in efficiency of the disease management.