Phenotypic and Genotypic Characterization of Newly Isolated Xanthomonas euvesicatoria-Specific Bacteriophages and Evaluation of Their Biocontrol Potential.

Bacteriophages have greatly engaged the attention of scientists worldwide due to the continuously increasing resistance of phytopathogenic bacteria to commercially used chemical pesticides. However, the knowledge regarding phages is still very insufficient and must be continuously expanded. This paper presents the results of the isolation, characterization, and evaluation of the potential of 11 phage isolates as natural predators of a severe phytopathogenic bacterium-Xanthomonas euvesicatoria. Phages were isolated from the rhizosphere of tomato plants with symptoms of bacterial spot. The plaque morphology of all isolates was determined on a X. euvesicatoria lawn via a plaque assay. Three of the isolates were attributed to the family Myoviridae based on TEM micrographs. All phages showed good long-term viability when stored at 4 °C and -20 °C. Three of the phage isolates possessed high stability at very low pH values. Fifty-five-day persistence in a soil sample without the presence of the specific host and a lack of lytic activity on beneficial rhizosphere bacteria were found for the phage isolate BsXeu269p/3. The complete genome of the same isolate was sequenced and analyzed, and, for the first time in this paper, we report a circular representation of a linear but circularly permuted phage genome among known X. euvesicatoria phage genomes.

Molecular Epidemiology of Xanthomonas euvesicatoria Strains from the Balkan Peninsula Revealed by a New Multiple-Locus Variable-Number Tandem-Repeat Analysis Scheme.

Bacterial spot of pepper and tomato is caused by at least three species of Xanthomonas, among them two pathovars of Xanthomonas euvesicatoria, which are responsible for significant yield losses on all continents. In order to trace back the spread of bacterial spot pathogens within and among countries, we developed the first multilocus variable number of tandem repeat analyses (MLVA) scheme for pepper- and tomato-pathogenic strains of X. euvesicatoria. In this work, we assessed the repeat numbers by DNA sequencing of 16 tandem repeat loci and applied this new tool to analyse a representative set of 88 X. euvesicatoria pepper strains from Bulgaria and North Macedonia. The MLVA-16 scheme resulted in a Hunter-Gaston Discriminatory Index (HGDI) score of 0.944 and allowed to resolve 36 MLVA haplotypes (MTs), thus demonstrating its suitability for high-resolution molecular typing. Strains from the different regions of Bulgaria and North Macedonia were found to be widespread in genetically distant clonal complexes or singletons. Sequence types of the variable number of tandem repeats (VNTR) amplicons revealed cases of size homoplasy and suggested the coexistence of different populations and different introduction events. The large geographical distribution of MTs and the existence of epidemiologically closely related strains in different regions and countries suggest long dispersal of strains on pepper in this area.

Molecular methods for diversity assessment among xanthomonads of Bulgarian and Macedonian pepper.

Bacterial spot is an important disease of pepper in Bulgaria and Macedonia. For characterization of Xanthomonas species associated with bacterial spot, 161 strains were collected from various field pepper-growing regions. Among them, 131 strains were identified as Xanthomonas euvesicatoria and 30 as Xanthomonas vesicatoria using species-specific primers and polymerase chain reaction followed by restriction fragment length polymorphism analysis. To assess the genetic diversity of the strains, two methods (Random Amplified Polymorphic DNA and Repetitive Element Palindromic-Polymerase Chain Reaction) were applied. Discriminatory index was calculated and analysis of molecular variance was carried out. Combined random amplified polymorphic DNA analysis of the X. euvesicatoria strains with primers CUGEA-4 and CUGEA-6 had greater discriminative power (0.60) than repetitive element palindromic-polymerase chain reaction with ERIC and BOX A1R primers, which makes this method applicable for strain diversity evaluation. Discrimination among the X. vesicatoria strains was achieved by the use of ERIC primers and only for the Bulgarian strains. The results demonstrated that X. euvesicatoria was more diverse than X. vesicatoria and heterogeneity was observed mainly in the Bulgarian populations. According to the analysis of molecular variance, genetic variations in X. euvesicatoria were observed among and within populations from different regions, while the differences between the two countries were minor. Following the principal coordinates analysis, a relation between the climatic conditions of the regions and a genetic distance of the populations may be suggested.

PFGE: a tool for examination of heterogeneity between the bacterial spot-causing xanthomonads of tomato plants in Bulgaria.

Pulsed-field gel electrophoresis (PFGE) is a highly discriminative molecular typing method that is used for epidemiological studies and investigation of outbreaks caused by different pathogens, including phytopathogenic Xanthomonas species. Bacterial spot (BS) is the most common and one of the most destructive diseases of tomato and pepper plants in Bulgaria. Several Xanthomonas species are known to cause BS, but the global distribution and genetic diversity of these species are not well understood. A collection of 100 BS-causing strains, isolated during the period of 1985-2012 from different tomato cultivars and weeds associated with tomato production areas from 11 geographic regions in Bulgaria, were screened for genetic diversity by genomic DNA restriction with rare-cutting endonucleases (XbaI and SpeI) subsequently resolved by PFGE. Two haplotypes for Xanthomonas vesicatoria and one haplotype for Xanthomonas gardneri strains were found.

Molecular methods for diversity assessment among xanthomonads of Bulgarian and Macedonian pepper

ABSTRACT Bacterial spot is an important disease of pepper in Bulgaria and Macedonia. For characterization of Xanthomonas species associated with bacterial spot, 161 strains were collected from various field pepper-growing regions. Among them, 131 strains were identified as Xanthomonas euvesicatoria and 30 as Xanthomonas vesicatoria using species-specific primers and polymerase chain reaction followed by restriction fragment length polymorphism analysis. To assess the genetic diversity of the strains, two methods (Random Amplified Polymorphic DNA and Repetitive Element Palindromic-Polymerase Chain Reaction) were applied. Discriminatory index was calculated and analysis of molecular variance was carried out.Combined random amplified polymorphic DNA analysis of the X. euvesicatoria strains with primers CUGEA-4 and CUGEA-6 had greater discriminative power (0.60) than repetitive element palindromic-polymerase chain reaction with ERIC and BOX A1R primers, which makes this method applicable for strain diversity evaluation. Discrimination among the X. vesicatoria strains was achieved by the use of ERIC primers and only for the Bulgarian strains. The results demonstrated that X. euvesicatoria was more diverse than X. vesicatoria and heterogeneity was observed mainly in the Bulgarian populations. According to the analysis of molecular variance, genetic variations in X. euvesicatoria were observed among and within populations from different regions, while the differences between the two countries were minor. Following the principal coordinates analysis, a relation between the climatic conditions of the regions and a genetic distance of the populations may be suggested.

Molecular methods for diversity assessment among xanthomonads of Bulgarian and Macedonian pepper

ABSTRACT Bacterial spot is an important disease of pepper in Bulgaria and Macedonia. For characterization of Xanthomonas species associated with bacterial spot, 161 strains were collected from various field pepper-growing regions. Among them, 131 strains were identified as Xanthomonas euvesicatoria and 30 as Xanthomonas vesicatoria using species-specific primers and polymerase chain reaction followed by restriction fragment length polymorphism analysis. To assess the genetic diversity of the strains, two methods (Random Amplified Polymorphic DNA and Repetitive Element Palindromic-Polymerase Chain Reaction) were applied. Discriminatory index was calculated and analysis of molecular variance was carried out.Combined random amplified polymorphic DNA analysis of the X. euvesicatoria strains with primers CUGEA-4 and CUGEA-6 had greater discriminative power (0.60) than repetitive element palindromic-polymerase chain reaction with ERIC and BOX A1R primers, which makes this method applicable for strain diversity evaluation. Discrimination among the X. vesicatoria strains was achieved by the use of ERIC primers and only for the Bulgarian strains. The results demonstrated that X. euvesicatoria was more diverse than X. vesicatoria and heterogeneity was observed mainly in the Bulgarian populations. According to the analysis of molecular variance, genetic variations in X. euvesicatoria were observed among and within populations from different regions, while the differences between the two countries were minor. Following the principal coordinates analysis, a relation between the climatic conditions of the regions and a genetic distance of the populations may be suggested.

Draft Genome Sequences of Two Xanthomonas vesicatoria Strains from the Balkan Peninsula.

Xanthomonas vesicatoria causes bacterial spot disease of pepper and tomato plants. We report here the first genome sequences of X. vesicatoria strains that have been isolated from pepper plants. These data will be used for comparative genomics and will allow the development of new detection and typing tools for epidemiological surveillance.

Draft Genome Sequences of Two Xanthomonas euvesicatoria Strains from the Balkan Peninsula.

We report the draft genome sequences of two Xanthomonas euvesicatoria strains from the Balkan Peninsula, which were isolated from symptomatic pepper plants. The availability of these genome sequences will facilitate the development of modern genotyping assays for X. euvesicatoria strains and to define targets for resistance breeding.

Differentiation of Xanthomonas spp. Causing Bacterial Spot in Bulgaria Based on Biolog System.

During the last 20 years, the causative agents of bacterial spot of tomato and pepper have been subjected to many studies and reclassifications. According to the current data, the species are four (X. euvesicatoria, X. vesicatoria, X. gardneri, and X. perforans) and cause similar symptoms in plants but possess different phenotypic properties. This work provides the full metabolic characteristics obtained by Biolog system of bacterial spot's xanthomonads based on a large selection of strains from different vegetable-producing regions of Bulgaria with accent on their major differentiating properties which could be used for species differentiation by metabolic profiles. The results are compared to the data available in the literature in order to clarify the strong features of each species and distinguish the variable ones. Simple characteristics like amylase activity and utilization of cis-aconitate cannot serve alone for differentiation.

Sub-species diversity of Xanthomonas euvesicatoria Bulgarian and Macedonian strains from pepper.

Sub-species diversity of pepper populations of Xanthomonas euvesicatoria in Bulgaria and Macedonia in 2012 was the object of this study. Species determination of 44 strains was performed by molecular methods using two pairs of species-specific primers and RFLP (restriction fragment length polymorphism) analysis of the 16S-23S ITS region with HpaII. The populations were characterized by genotypic and phenotypic properties. The genotypic diversity of the strains was evaluated by RAPD (random amplified polymorphic DNA) technique. Primer CUGEA-6 differentiated the strains in two groups, one of which included only Bulgarian strains and revealed a mixed profile of the type strain. BiologTM metabolite profiles separated the strains in four groups: two of which were composed only of Bulgarian or Macedonian strains. Correlation between the RAPD and the metabolic profiles was observed. Twelve antibiotics and copper ions in five concentrations (1-5 g kg-1) were tested for biological activity. The inhibition zones of the Bulgarian strains were statistically proven to be considerably larger than the Macedonian ones in the tests with kanamycin, streptomycin, polymyxin B sulphate, tetracycline and vankomycin. The inhibition zones of the Bulgarian strains were statistically proven to be relatively larger than the Macedonian ones in the copper tests. Based on our studies the Macedonian population of X. euvesicatoria manifested a relative homogeneity while a greater diversity was observed in the Bulgarian population.

Characterization of Erwinia amylovora strains from Bulgaria by pulsed-field gel electrophoresis.

The aim of this study was to characterize genetically Bulgarian Erwinia amylovora strains using pulsed-field gel electrophoresis (PFGE) analysis. Fifty E. amylovora strains isolated from different hosts, locations, as well as in different years were analysed by PFGE after XbaI, SpeI, and XhoI digestion of the genomic DNA. The strains were distributed into four groups according to their XbaI-generated profile. About 82% of the strains displayed a PFGE profile identical to that of type Pt2. Three strains belonged to the Central Europe Pt1 type. Two new PFGE profiles, not reported so far, were established--one for a strain isolated from Malus domestica and another for all Fragaria spp. strains. The same grouping of the strains was obtained after analysis of the SpeI digestion patterns. On the basis of PFGE profiles, after XbaI and SpeI digestion, a genetic differentiation between the strains associated with subfamily Maloideae and subfamily Rosoideae was revealed. The presence of more than one PFGE profile in the population of E. amylovora in Bulgaria suggests a multiple source of inoculum.

Phenotypic diversity of Erwinia amylovora in Bulgaria.

Fifty-one strains of Erwinia amylovora isolated from nine host plants in Bulgaria were characterized phenotypically and identified by the API 20E and BIOLOG system. The identification was confirmed by PCR amplification of a specific region of the plasmid pEA29 and the genome ams region. The phenotypic diversity of the strains was studied on the basis of their API 20E and BIOLOG metabolic profiles, as well as of their SDS-PAGE protein profile. Metabolic diversity among the strains was established, but no connection with the origin of the strains was revealed. The Bulgarian strains showed API 20E metabolic profiles not found in previous studies of E. amylovora. The strains formed a homogenous group on the basis of their protein profiles. All the strains were sensitive to the antibiotics streptomycin, tetracycline and oxytetracycline. This study was an initial step towards an investigation of the diversity and evolution in the Bulgarian population of E. amylovora, and it was the first characterization of E. amylovora strains isolated from different host plants in the period 1995-2005 in Bulgaria.

New host plants of Erwinia amylovora in Bulgaria.

Nine strains of Erwinia amylovora were isolated from new host plants in Bulgaria--chokeberry and strawberry. The strains were characterized morphologically and biochemically using the API 20E and BIOLOG system. It was established that they showed three different API 20E metabolic profiles, not found by previous studies of E. amylovora. All strains were identified as E. amylovora due to their metabolic fingerprint patterns obtained by the BIOLOG system. The identification was confirmed by PCR amplification of a specific region of plasmid pEA29 and genome ams-region. This study is the first characterization and identification of E. amylovora strains isolated from chokeberry and strawberry by the API 20E and BIOLOG system and by polymerase chain reaction.