Suppression of the Bacterial Spot Pathogen Xanthomonas euvesicatoria on Tomato Leaves by an Attenuated Mutant of Xanthomonas perforans

A bacteriocin-producing strain of the bacterial spot of tomato plant pathogen, Xanthomonas perforans, with attenuated pathogenicity was deployed for biocontrol of a bacteriocin-sensitive strain of the genetically closely related bacterial spot of tomato plant pathogen, X. euvesicatoria. The attenuated mutant (91-118ΔopgHΔbcnB) of X. perforans was tested in leaf tissue and shown to significantly inhibit internal populations of the wild-type X. euvesicatoria strain although significantly less than the wild-type 91-118 strain, whereas in a phyllosphere inhibition assay, the mutant strain reduced epiphytic populations comparably to 91-118. Thus, the attenuated mutant limited the sensitive bacterium more efficiently on the leaf surface than inside the leaf. In field experiments, weekly application of 91-118ΔopgHΔbcnB significantly reduced X. euvesicatoria populations compared to the growers’ standard control (copper hydroxide and mancozeb applied weekly and acibenzolar-S-methyl applied every 2 weeks). The biological control agent, 91-118ΔopgHΔbcnB, applied every 2 weeks also significantly reduced X. euvesicatoria populations in one season but was not significantly different from the growers’ standard control. Potentially, attenuated pathogenic strains could be deployed as biological control agents in order to improve disease control of foliar plant pathogens.Bacterial spot of tomato is incited by several Xanthomonas spp., including Xanthomonas euvesicatoria, X. perforans, and X. vesicatoria (13). On tomato plants, three races, designated tomato race 1 (T1), T2, and T3, were originally identified based on their reactions on three tomato genotypes (22, 23, 33, 37). These races, T1, T2, and T3, are principally members of X. euvesicatoria, X. vesicatoria, and X. perforans, respectively. Control of bacterial spot of tomato is extremely difficult when moderate-to-high temperatures and high moisture conditions exist. The disease causes significant damage to the crop, resulting in major losses (27). Management is primarily limited to bactericides, such as fixed coppers (3, 20, 32); however, copper-tolerant strains have become prevalent (31, 32) and chemical control alone is insufficient to control the disease under optimal weather conditions. Additionally, the use of copper compounds has led to soil contamination in some instances (16).Recently, there has been increased interest in integrated biological control strategies for bacterial diseases (5, 7, 11, 19, 23). However, optimization of biocontrol agents for consistent disease suppression for many bacterial diseases has been difficult. Studies are increasing our understanding of the ecology of nonpathogenic saprophytes as biocontrol agents, but their selection is limited to labor-intensive protocols. New biological control strategies are currently being sought, including the use of bacteriocins, bacteriophages, and attenuated plant pathogens (4, 5, 7, 9, 11, 23, 26, 35, 38).For many years, only X. euvesicatoria (T1) was present on tomato plants in Florida. In 1991, X. perforans (T3) appeared in Florida tomato fields (15) and eventually became the prevalent race (14). Following that observation, Jones et al. (12) demonstrated that when X. perforans and X. euvesicatoria were coinoculated onto tomato plants in the field, X. perforans displaced X. euvesicatoria and became the predominant strain. Further studies revealed that the competitive nature of X. perforans was due in large part to its antagonism toward X. euvesicatoria strains (9, 12, 36). Tudor-Nelson et al. (36) identified three different bacteriocins, designated BCN-A, BCN-B, and BCN-C, which were found to confer the ability to inhibit X. euvesicatoria strains in plate assays. Hert et al. (9) determined that these bacteriocins provided X. perforans strains with a competitive advantage in the greenhouse and field and that a mutant X. perforans strain expressing only BCN-A and BCN-C was most effective in displacing X. euvesicatoria and outcompeted wild-type (WT) X. perforans. Field experiments conducted with a nonpathogenic Hrp⁻ strain of X. perforans as a potential biocontrol agent for controlling X. euvesicatoria resulted in marginal control (18). Although the WT X. perforans strain has a competitive advantage over X. euvesicatoria populations, Hrp⁻ mutants such as that used in the study by Liu (18) do not appear to have the necessary competitiveness to suppress X. euvesicatoria populations.Previous research has focused on colonization of the plant by biocontrol agents to determine the relationship between invasion efficiency and biological control efficacy. For example, Frey et al. (8) achieved only low-to-moderate levels of biological control of a WT bacteriocin-sensitive strain of Ralstonia solanacearum when using a bacteriocin-producing nonpathogenic Hrp⁻ mutant strain of R. solanacearum. However, control using a moderately pathogenic hrp mutant (hrcV) capable of higher levels of colonization of the root and stem achieved greater disease suppression (6). Etchebar et al. (6) suggested that there was a positive correlation between colonization of the xylem by the hrp mutant and the level of control of WT R. solanacearum. As a result of previous studies showing that nonpathogenic strains of X. perforans only provide low levels of biological control (18), we hypothesized that using an attenuated pathogenic bacteriocin-producing strain of X. perforans rather than a nonpathogenic strain may increase the efficiency of control under field conditions.In this study, our strategy was to use an attenuated mutant of X. perforans that colonizes leaf tissue more effectively than nonpathogenic strains do and potentially provides more effective colonization and increases the likelihood for interaction between X. perforans and X. euvesicatoria. We selected strain 91-118ΔopgHΔbcnB as the biocontrol agent since it was previously shown that deletion of the osmoregulated periplasmic glucan gene opgH resulted in a pathogenic phenotype with a significantly reduced ability to cause disease and internal colonization in susceptible tomato tissue (22). The selected mutant also lacked BcnB activity based on a previous study in which BcnB appeared to negatively affect competitive ability in that a ΔbcnB mutant was more effective at colonizing tomato leaves in field experiments than WT X. perforans was (9).     

A multilocus sequence analysis of the genus Xanthomonas

A multilocus sequence analysis (MLSA) of strains representing all validly published Xanthomonas spp. (119 strains) was conducted using four genes; dnaK, fyuA, gyrB and rpoD, a total of 440 sequences. Xanthomonas spp. were divided into two groups similar to those indicated in earlier 16S rDNA comparative analyses, and they possibly represent distinct genera. The analysis clearly differentiated most species that have been established by DNA-DNA reassociation. A similarity matrix of the data indicated clear numerical differences that could form the basis for species differentiation in the future, as an alternative to DNA-DNA reassociation. Some species, X. cynarae, X. gardneri and X. hortorum, formed a single heterogeneous group that is in need of further investigation. X. gardneri appeared to be a synonym of X. cynarae. Recently proposed new species, X. alfalfae, X. citri, X. euvesicatoria, X. fuscans and X. perforans, were not clearly differentiated as species from X. axonopodis, and X. euvesicatoria and X. perforans are very probably synonyms. MLSA offers a powerful tool for further investigation of the classification of Xanthomonas. Based on the dataset produced, the method also offers a relatively simple way of identifying strains as members of known species, or of indicating their status as members of new species.     

Xylella and Xanthomonas Mobil'omics

The gamma-proteobacterium Xanthomonadales groups two closely related genera of plant pathogens, Xanthomonas and Xylella. Whole genome sequencing and comparative analyses disclosed a high degree of identity and co-linearity of the chromosome backbone between species and strains. Differences observed are usually clustered into genomic islands, most of which are delimited by genetic mobile elements. Focus is given in this paper to describe which groups of mobile elements are found and what is the relative contribution of these elements to Xanthomonas and Xylella genomes. Insertion sequence (IS) elements have invaded the Xanthomonas genome several times, whereas Xylella is rich in phage-related regions. Also, different plasmids are found inhabiting the bacterial cells studied here. Altogether, these results suggest that the integrative elements such as phages and transposable elements as well as the episomal plasmids are important drivers of the genome evolution of this important group of plant pathogens.     

Cassava diseases caused by Xanthomonas phaseoli pv. manihotis and Xanthomonas cassavae

Xanthomonas phaseoli pv. manihotis (Xpm) and X. cassavae (Xc) are two bacterial pathogens attacking cassava. Cassava bacterial blight (CBB) is a systemic disease caused by Xpm, which might have dramatic effects on plant growth and crop production. Cassava bacterial necrosis is a nonvascular disease caused by Xc with foliar symptoms similar to CBB, but its impacts on the plant vigour and the crop are limited. In this review, we describe the epidemiology and ecology of the two pathogens, the impacts and management of the diseases, and the main research achievements for each pathosystem. Because Xc data are sparse, our main focus is on Xpm and CBB.     

Characterization of Xanthomonas phaseoli Bacteriophages

Ten bacteriophages for Xanthomonas phaseoli were characterized. On the basis of adsorption rates, latent period and burst size, plaque morphology, host range, efficiency of plating, ultrastructure, sensitivity to osmotic shock, streptomycin sensitivity, temperature effects on plating efficiency, and serology, the phages were separated into at least three groups. Some of the phages were infectious for Pseudomonas phaseolicola (four strains) and P. syringae (one strain) in a narrow temperature range. The taxonomic and ecological significance of this finding is discussed.     

Conformation of the extracellular polysaccharide of Xanthomonas campestris.

The solution conformation of the extracellular polysaccharide of the bacterium Xanthomonas campestris is examined by optical rotation, viscometry, and potentiometric titration. Measurements of optical rotation vs. temperature for solutions of the polysaccharide at low ionic strength reveal a sharp transition to a denatured structure which is reversible if sufficient salt is present. The temperature Tm at the transition midpoint increases as log (Na+) or log (Ca2+). Viscosity-temperature profiles substantiate a structural change of the polysaccharide at Tm. The intrinsic viscosity of the native molecule at zero shear rate exceeds 5000 ml/g. This high figure is indicative of a stiff chain. The viscosity of the native molecule is relatively insensitive to salt, whereas the denatured molecule collapses if salt is present. Hydrogen-ion titration shows that the pKapp of the COO- groups of the polymer decreases from 3.2 in 0.01 M NaC1 to 2.6 in 0.2 M NaC1. All these data suggest that the native polysaccharide possesses ordered secondary structure stabilized by nonionic interactions outweighing the repulsion between adjacent COO- groups.     

Structure of the O-polysaccharide of Xanthomonas cassavae GSPB 2437

The following structure of the O-polysaccharide of the phytopathogenic bacterium Xanthomonas cassavae GSPB 2437 was determined by sugar analysis along with 1H and 13C NMR spectroscopy: [structure: see text].     

Deoxyribonucleic acid relatedness of 24 xanthomonad strains representing 23 Xanthomonas campestris pathovars and Xanthomonas fragariae

Twenty Xanthomonas campestris pathotype strains, three non-pathotype strains, and one strain of X. fragariae were studied by S1 DNA:DNA hybridization tests. The results of these tests do not support the retention of X. campestris as a single species. DNA reassociation values among many of the strains were low. Three clusters of closely related strains were observed, but nine strains did not cluster. Xanthomonas campestris pv. secalis was more closely related to X. fragariae than to any other X. campestris pathovar. Mapping the host family upon a three-dimensional genomic distance matrix of the xanthomonads suggested that strains attacking the same plant family usually show some relationship, but only a distant one. Thus, pathogenicity toward members of the same host family is not a measure of the genomic relationships of xanthomonads.     

Fast classification of plant-associated bacteria in the Xanthomonas genus

Abstract A rapid method for identifying Xanthomonas bacteria was developed, based on specific PCR amplification. In all Xanthomonas pathovars tested, a single 16S rDNA fragment of 480 bp was produced. Phytobacteria from other genera showed other fragment patterns on agarose gel electrophoresis. This Xanthomonas identification system was used to screen Xanthomonas -related organisms, newly isolated phytobacteria and bacterial populations in wheat seed extracts.     

The extracellular proteases of the phytopathogenic bacterium Xanthomonas campestris

The culture liquids of three Xanthomonas campestris pv. campestris strains were found to possess proteolytic activity. The culture liquid of strain B-611 with the highest proteolytic activity was fractionated by salting-out with ammonium sulfate, gel filtration, and ion-exchange chromatography. The electrophoretic analysis of active fractions showed the presence of two proteases in the culture liquid of strain B-611, the major of which being serine protease. The treatment of cabbage seedlings with the proteases augmented the activity of peroxidase in the cabbage roots by 28%.     

Computational and biochemical analysis of the Xanthomonas effector AvrBs2 and its role in the modulation of Xanthomonas type three effector delivery

Effectors of the bacterial type III secretion system provide invaluable molecular probes to elucidate the molecular mechanisms of plant immunity and pathogen virulence. In this report, we focus on the AvrBs2 effector protein from the bacterial pathogen Xanthomonas euvesicatoria (Xe), the causal agent of bacterial spot disease of tomato and pepper. Employing homology-based structural analysis, we generate a three-dimensional structural model for the AvrBs2 protein and identify catalytic sites in its putative glycerolphosphodiesterase domain (GDE). We demonstrate that the identified catalytic region of AvrBs2 was able to functionally replace the GDE catalytic site of the bacterial glycerophosphodiesterase BhGlpQ cloned from Borrelia hermsii and is required for AvrBs2 virulence. Mutations in the GDE catalytic domain did not disrupt the recognition of AvrBs2 by the cognate plant resistance gene Bs2. In addition, AvrBs2 activation of Bs2 suppressed subsequent delivery of other Xanthomonas type III effectors into the host plant cells. Investigation of the mechanism underlying this modulation of the type III secretion system may offer new strategies to generate broad-spectrum resistance to bacterial pathogens.     

Non-gamma-proteobacteria gene islands contribute to the Xanthomonas genome

Horizontal gene transfer, a process through which genomes acquire sequences from distantly related organisms, is believed to be a major source of genetic diversity in bacteria. A central question concerning the impact of gene transfer on bacterial genome evolution is the proportion of horizontally transferred sequences within genomes. Through BLAST search, we found that the genomes of two phytopathogens, Xanthomonas campestris pv. campestris and Xanthomonas axonopodis pv. citri, have close to 40% of the genes with the highest similarity to genes from phylogenetically distant organisms (non-gamma-proteobacteria). Most of these genes are found to be contiguous in the genome, forming genome islands, which may have been transferred from other organisms. Overall, the total number of genes within genome islands corresponds to almost one quarter of the entire xanthomonad genomes. Interestingly, many of the genes in these islands are functionally related to plant pathogenesis and virulence. Thus, these results suggest that horizontally transferred genes are clustered in the genome, and may facilitate fitness in new environments, as in the case of plant-bacteria interaction.     

The Extracellular Proteases of the Phytopathogenic Bacterium Xanthomonas campestris

The culture liquids of three Xanthomonas campestris pv. campestris strains were found to possess proteolytic activity. The culture liquid of strain B611 with the highest proteolytic activity was fractionated by salting-out with ammonium sulfate, gel filtration, and ion-exchange chromatography. The electrophoretic analysis of active fractions showed the presence of two proteases in the culture liquid of strain B611, the major of which was serine protease. The treatment of cabbage seedlings with the proteases augmented the activity of peroxidase in the cabbage roots by 28%.     

The type III effectors of Xanthomonas

A review of type III effectors (T3 effectors) from strains of Xanthomonas reveals a growing list of candidate and known effectors based on functional assays and sequence and structural similarity searches of genomic data. We propose that the effectors and suspected effectors should be distributed into 39 so-called Xop groups reflecting sequence similarity. Some groups have structural motifs for putative enzymatic functions, and recent studies have provided considerable insight into the interaction with host factors in their function as mediators of virulence and elicitors of resistance for a few specific T3 effectors. Many groups are related to T3 effectors of plant and animal pathogenic bacteria, and several groups appear to have been exploited primarily by Xanthomonas species based on available data. At the same time, a relatively large number of candidate effectors remain to be examined in more detail with regard to their function within host cells.