M. Timur Momol

New diversity of Ralstonia solanacearum strains associated with vegetable and ornamental crops in Florida

In 2003 and 2004, 15 isolates of Ralstonia solanacearum were obtained from wilting plants of field-grown pepper (Capsicum annuum) in south Florida and from pot-grown hydrangea (Hydrangea paniculata and H. macrophylla) and geranium (Pelargonium × hortorum) in commercial nurseries and retention ponds in north Florida. Diagnostic immunoassays and polymerase chain reaction (PCR) analyses identified all the isolates as R. solanacearum but not race 3 biovar 2. Pathogenicity studies on tomato, pepper, and tobacco revealed that all 15 strains had similar high virulence on tomato and all caused wilting of tobacco, although there were significant differences among the strains in aggressiveness on tobacco. An indigenous Florida tomato strain, race 1 biovar 1 (Rs5), caused no disease on tobacco and little or none on pepper. The three pepper strains were more aggressive than Rs5 or two hydrangea strains on all three pepper cultivars studied. Phylogenetic analysis based on an endoglucanase gene sequence indicated that these strains had three distinct origins. The three pepper strains belonged to phylotype I biovar 3 and clustered with strains from diverse hosts in Asia belonging to sequevar 13. The six geranium strains and four of the hydrangea strains were closely related to strains in sequevar 5, a distinct subcluster of phylotype II biovar 1 strains isolated from the French West Indies and Brazil. Two other biovar 1 strains from hydrangea and strains K60, AW, and Rs5 belonged to sequevar 7 in phylotype II and probably are native to North America. None of the Florida isolates belong to the highly regulated Select Agent race 3 biovar 2 subgroup, according to both the DNA sequence analysis and the biovar phenotypic test results. However, the race 3 biovar 2-specific B2 primers weakly amplified a product from some race 1 biovar 1 strains in real-time PCR, indicating that this assay may give false positives under some conditions. Given the high cost of a misdiagnosis, it seems advisable to use at least two independent diagnostic methods to confirm that a suspect isolate is R. solanacearum R3B2. This is the first report of the presence of R. solanacearum race 1 biovar 3 or phylotype I strains in North America, and the first report confirming R. solanacearum causing natural infection of hydrangea in Florida. Thus, R. solanacearum strains that are quite distinct from presumably indigenous strains are present and can infect diverse hosts in Florida.

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.

Development of an Integrated Approach for Managing Bacterial Wilt and Root-Knot on Tomato Under Field Conditions

A 2-year field study was conducted to develop a field application method using thymol as a preplant soil treatment for controlling bacterial wilt and root-knot nematode on tomato (Lycopersicon esculentum). In addition, acibenzolar-S-methyl (ASM), which induces plant systemic resistance, was applied in conjunction with thymol to determine whether combining these tactics could improve bacterial wilt management. The test sites were artificially infested with Ralstonia solanacearum and Meloidogyne arenaria, and thymol was applied as preplant fumigation through drip irrigation lines under polyethylene mulch at a rate of 73 kg/ha in both 2004 and 2005. ASM was applied primarily as foliar spray at a concentration of 25 mg/liter. Application of thymol significantly reduced incidence of bacterial wilt on tomato in both years of the trial. In thymol-treated plots, 26.0 and 22.6% of the plants wilted in 2004 and 2005, respectively; whereas, in untreated plots, more than 95% of the plants wilted in each year. Number of root-knot nematode juveniles was significantly reduced in field plots treated with thymol and ASM for both years. The combined use of thymol and ASM provided the greatest reduction of root galling among the treatments. Tomato yield (cv. FL47) was evaluated only in the 2005 trial; thymol-treated plots produced significantly higher marketable yield than untreated plots, and the thymol treatment in combination with ASM significantly increased tomato yield compared with thymol or ASM alone. These results indicate that use of thymol and ASM was beneficial in controlling bacterial wilt and root-knot. We developed an effective method for applying thymol through drip irrigation lines for managing these diseases in tomato production.

Integrating Plant Essential Oils and Kaolin for the Sustainable Management of Thrips and Tomato Spotted Wilt on Tomato

Thrips-vectored Tomato spotted wilt virus is one of the most devastating pest complexes affecting tomato in the southern United States and elsewhere. Field trials were conducted over 2 years to determine the effects of volatile plant essential oils and kaolin-based particle films on the incidence of tomato spotted wilt and population dynamics of Frankliniella spp. thrips. The essential oils, geraniol (a monoterpene constituent of various plant essential oils), lemongrass (Cymbopogon flexuosus) oil, and tea tree (Melaleuca alternifolii) oil, were compared with a standard insecticide treatment and an untreated control. All treatments were applied with and without kaolin, in a 5 × 2 factorial design. Although the treatments did not clearly affect the abundance of vector species, there were treatment differences in the incidence of tomato spotted wilt. When combined with kaolin, the three essential oils reduced tomato spotted wilt incidence by 32 to 51% in 2005 and by 6 to 25% in 2006 compared with the control. In both years, tea tree oil plus kaolin reduced tomato spotted wilt as well as the standard insecticide treatments. Overall, kaolin significantly increased yield by over 26% compared to treatments without kaolin. When applied with kaolin, the three essential oils produced yields similar to the insecticide standard. Therefore, naturally occurring products, such as essential oils and kaolin, could be used successfully to reduce insecticide use on tomatoes.

Diversity among Ralstonia solanacearum strains isolated from the southeastern United States.

This is the first comprehensive study of a collection of Ralstonia solanacearum strains from the southeastern United States to be characterized based on biovar, pathogenicity, hypersensitive reaction on tobacco, and phylogenetic analyses of the egl sequence. Rigorous phylogenetic analysis of the commonly used egl gene produced robust phylogenies that differed significantly from a neighbor-joining tree differed from and previously published phylogenies for R. solanacearum strains. These robust trees placed phylotype IV within the phylotype I clade, which may suggest that phylogenies based solely on egl may be misleading. As a result of phylogenetic analyses in this study, we determined that U.S. strains from Georgia, North Carolina, South Carolina, and older Florida strains isolated from solanaceous crops all belong to phylotype II sequevar 7. However, many strains recently isolated in Florida from tomato and other crops were more diverse than the southeastern United States population. These unique Florida strains grouped with strains mostly originating from the Caribbean and Central America. One of the exotic strains, which in a previous study was determined to be established in northern Florida, was characterized more extensively. Upon using Musa-specific multiplex polymerase chain reaction, this strain produced a unique banding pattern, which has not previously been reported. Inoculation of this strain into Musa spp. did not result in wilt symptoms; however, the plants were stunted and root masses were significantly reduced. Furthermore, following root inoculation, the bacterium, unlike a typical Florida race 1 biovar 1 strain, was recovered from the roots and stems, indicating systemic movement. This is the first report of an R. solanacearum strain isolated in the United States that is deleterious to the growth of Musa plants.

Detection of Ralstonia solanacearum in Irrigation Ponds and Aquatic Weeds Associated with the Ponds in North Florida

The discovery of exotic Ralstonia solanacearum biovar 1 strains on geranium in north Florida led to a concern that this strain may have become established. Therefore, we monitored irrigation ponds and potential alternative aquatic weeds from 2002 to 2005 for the presence of this strain. We report that this strain, possibly originating from the Caribbean, has become established in several ponds in Gadsden County, FL. Cladistic taxonomy was used to subclassify the bacterium at the species level into four groups or phylotypes based on multiplex polymerase chain reaction of the internal transcribed spacer (ITS) region. The bacterium was further divided into sequevars by sequencing the endoglucanase gene (egl). The strains were determined to belong to phylotype II/sequevar 4 NPB (nonpathogenic on banana) that was recently reported in Martinique. Partial sequencing of the egl followed by phylogenetic analysis placed the new Caribbean strains in a different clade than the typical Florida endemic strains. Pulsed-field gel electrophoresis (PFGE) revealed different haplotypes upon comparison of the collected pond strains and the Floridian strains. Based on PFGE polymorphism, egl sequencing, and phylogenetic analysis, the Caribbean strains were shown to be identical to the strain isolated from infected geranium plants. Experiments were undertaken to monitor R. solanacearum in irrigation ponds and associated weeds. R. solanacearum was detected in surface-disinfested common aquatic weeds growing in the irrigation ponds, including Hydrocotyle ranunculoides (dollar weed) and Polygonum pennsylvanicum (Pennsylvania smart weed). Both weeds were latently infected and showed no signs of wilt when collected. Two different Hydrocotyle spp. were artificially inoculated with R. solanacearum under greenhouse conditions and both developed symptoms 14 days post inoculation (dpi) and the bacterium was recovered from the tissues 42 dpi. There was a positive correlation between ambient temperature and R. solanacearum populations in irrigation water, as previously shown by other researchers.

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.

Physiological responses of calcium-efficient and calcium-inefficient tomato genotypes to differential bacterial wilt conditions.

Bacterial wilt (BW) disease, caused by Ralstonia solanacearum, can severely limit tomato (Solanum lycopersicum) production in southern United States. Tomato genotypes display variable responses to BW disease. The physiological mechanism underlying BW resistance is not well understood. In this study, experiments were conducted to compare two tomato genotypes, PI117566 [calcium (CA)–efficient] and PI109315 (Ca-inefficient), for wilting, shoot growth, final fresh weight, and shoot Ca concentrations when inoculated with R. solanacearum. The inoculation concentration of R. solanacearum varied from 0 to 108 colony forming units/ml. Genotype PI109315 appear to be more BW-resistant compared with genotype PI117566 under sufficient Ca conditions. Furthermore, we found that genotype PI109315 had greater shoot growth and final fresh weight than genotype PI117566. These findings implied that Ca-efficient tomato genotypes may not play a key role in BW resistance of tomato under sufficient Ca conditions because Ca efficiency showed no effect on the suppression of BW.

Effect of Ralstonia solanacearum on Mineral Nutrients and Infrared Temperatures in Two Tomato Cultivars

ABSTRACT The objective of this study was to determine how the responses of two tomato cultivars to Ralstonia solanacearum relate to their leaf infrared temperature and acquiring of nutrients from soil. Tomato (Solanum lycopersicum L.) cultivars of disease susceptible-‘FL 47’ and resistant-‘H 7998’ were grown in soil inoculated with R. solanacearum. Bacterial wilt incidence, leaf infrared temperatures, and uptake of nutrients were measured for 28 d. In bacterial wilt-resistant cultivar ‘H 7998’, concentration of sulfur (S; +77%), calcium (Ca; +66%), boron (B; +60%) were found higher and nitrogen (N; −26%) were found lower, compared with susceptible ‘FL 47’. Infrared temperatures were correlated with wilt percentage at 14 d, but not at 7 d. These results provide evidence that there is a correlation between bacterial wilt resistance and translocation of some nutrients in the shoots. Additionally, data indicates that the infrared thermometer could only detect wilting after obvious symptoms were visibly incited by R. solanacearum in tomato.