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[This corrects the article DOI: 10.3389/fmicb.2018.02176.].
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Following analysis of eight phages under in vitro, growth chamber and greenhouse conditions with the bacterial spot of tomato pathogen Xanthomonas perforans, there was no correlation between disease control efficacy and in vitro phage multiplication, in vitro bacterial suppression, or in vivo phage multiplication in the presence of the host, but there was a low correlation between phage persistence on the leaf surface and disease control. Two of the 8 virulent phages (ΦXv3-21 and ΦXp06-02) were selected for in depth analysis with two X. perforans (Xp06-2-1 and Xp17-12) strains. In in vitro experiments, phage ΦXv3-21 was equally effective in infecting the two bacterial strains based on efficiency of plating (EOP). Phage ΦXp06-02, on the other hand, had a high EOP on strain Xp06-2-1 but a lower EOP on strain Xp17-12. In several growth chamber experiments, ΦXv3-21 was less effective than phage ΦXp06-02 in reducing disease caused by strain Xp06-2-1, but provided little or no disease control against strain Xp17-12. Interestingly, ΦXp06-02 could multiply to significantly higher levels on the tomato leaf surface than phage ΦXv3-21. The leaf surface appears to be important in terms of the ability of certain bacteriophages to multiply in the presence of the bacterial host. ΦXv3-21, when applied to grapefruit leaves in combination with a bacterial host, was unable to multiply to high levels, whereas on tomato leaflets the phage multiplied exponentially. One plausible explanation is that the leaf surface may be an important factor for attachment of certain phages to their bacterial host.
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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.
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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.
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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.
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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.
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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.