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A total of 34 phytopathogenic strain genomes belonging to the Pseudomonas syringae species complex and related species, including many pathotype strains, were assessed using average nucleotide identity (ANI) analysis. Their taxonomic relationships were consistently confirmed by the tetranucleotide frequency correlation coefficient (TETRA) values, multilocus sequence typing analysis (MLSA) performed with seven housekeeping genes, using both maximum likelihood and Bayesian methods, and split consensus network analyses. The ANI, MLSA, and split consensus analyses provided consistent and identical results. We confirmed the occurrence of the well-demarcated genomospecies inferred sensu Gardan et al. using DNA-DNA hybridization and ribotyping analyses. However, some P. syringae strains of the pathovars morsprunorum and lachrymans were placed in different genomospecies in our analyses. Genomospecies 1, 2, 4, 6, and 9 resulted well demarcated, whereas strains of genomospecies 3 and 8 had ANI values between 95 and 96% in some cases, confirming that this threshold reveals very closely related species that might represent cases of splitting entities or the convergence of different species to the same ecological niche. This study confirms the robustness of the combination of genomic and phylogenetic approaches in revealing taxonomic relationships among closely related bacterial strains and provides the basis for a further reliable demarcation of the phytopathogenic Pseudomonas species. Within each species, the pathovars might represent distinct ecological units. The possibility of performing extensive and standardized host range and phenotypic tests with many strains of different pathovars can assist phytobacteriologists for better determining the boundaries of these ecological units.

The circumscription of bacterial species is a complex task. So far, DNA-DNA hybridization (DDH), 16S rRNA gene sequencing, and multiocus sequence typing analysis (MLSA) are currently the preferred techniques for their genetic determination. However, the average nucleotide identity (ANI) analysis of conserved and shared genes between two bacterial strains based on the pair-wise genome comparisons, with support of the tetranucleotide frequency correlation coefficients (TETRA) value, has recently been proposed as a reliable substitute for DDH. The species demarcation boundary has been set to a value of 95-96% of the ANI identity, with further confirmation through the assessment of the corresponding TETRA value. In this study, we performed a genome-wide MLSA of 14 phytopathogenic pseudomonads genomes, and assessed the ANI and TETRA values of 27 genomes, representing seven out of the nine genomospecies of Pseudomonas spp. sensu Gardan et alii, and their phylogenetic relationships using maximum likelihood and Bayesian approaches. The results demonstrate the existence of a well demarcated genomic cluster that includes strains classified as P. avellanae, P. syringae pv. theae, P. s. pv. actinidiae and one P. s. pv. morsprunorum strain all belonging to the single species P. avellanae. In addition, when compared with P. avellanae, five strains of P. s. pv. tomato, including the model strain DC3000, and one P. s. pv. lachrymans strain, appear as very closely related to P. avellanae, with ANI values of nearly 96% as confirmed by the TETRA analysis. Conversely, one representative strain, previously classified as P. avellanae and isolated in central Italy, is a genuine member of the P. syringae species complex and can be defined as P. s. pv. avellanae. Currently. The core and pan genomes of P. avellanae species consist of 3,995 and 5,410 putative protein-coding genes, respectively.

By applying a coverage-based read selection and filtration through a healthy plant dataset, and a post-assembly contig selection based on homology and linkage, genome sequence drafts were obtained for four phytoplasma strains belonging to the 16SrIII group (X disease clade), namely Vaccinium Witches' Broom phytoplasma (647 754 nt in 272 contigs), Italian Clover Phyllody phytoplasma strain MA (597 245 nt in 197 contigs), Poinsettia branch-inducing phytoplasma strain JR1 (631 440 nt in 185 contigs) and Milkweed Yellows phytoplasma (583 806 nt in 158 contigs). Despite assignment to different 16SrIII subgroups, the genomes of the four strains were similar, comprising a highly conserved core (92-98 % similar in their nucleotide sequence among each other over alignments about 500 kb in length) and a minor strain-specific component. As far as their protein complement was concerned, they did not differ significantly in their basic metabolism potential from the genomes of other wide-host-range phytoplasmas sequenced previously, but were distinct from strains of other species, as well as among each other, in genes encoding functions conceivably related to interactions with the host, such as membrane trafficking components, proteases, DNA methylases, effectors and several hypothetical proteins of unknown function, some of which are likely secreted through the Sec-dependent secretion system. The four genomes displayed a group of genes encoding hypothetical proteins with high similarity to a central domain of IcmE/DotG, a core component of the type IVB secretion system of Gram-negative Legionella spp. Conversely, genes encoding functional GroES/GroEL chaperones were not detected in any of the four drafts. The results also indicated the significant role of horizontal gene transfer among different 'Candidatus Phytoplasma' species in shaping phytoplasma genomes and promoting their diversity.

Background: Hazelnut (Corylus avellana) decline disease in Greece and Italy is caused by the convergent evolution of two distantly related lineages of Pseudomonas syringae pv. avellanae (Pav). We sequenced the genomes of three Pav isolates to determine if their convergent virulence phenotype had a common genetic basis due to either genetic exchange between lineages or parallel evolution., Results: We found little evidence for horizontal transfer (recombination) of genes between Pav lineages, but two large genomic islands (GIs) have been recently acquired by one of the lineages. Evolutionary analyses of the genes encoding type III secreted effectors (T3SEs) that are translocated into host cells and are important for both suppressing and eliciting defense responses show that the two Pav lineages have dramatically different T3SE profiles, with only two shared putatively functional T3SEs. One Pav lineage has undergone unprecedented secretome remodeling, including the acquisition of eleven new T3SEs and the loss or pseudogenization of 15, including five of the six core T3SE families that are present in the other Pav lineage. Molecular dating indicates that divergence within both of the Pav lineages predates their observation in the field. This suggest that both Pav lineages have been cryptically infecting hazelnut trees or wild relatives for many years, and that the emergence of hazelnut decline in the 1970s may have been due to changes in agricultural practice., Conclusions: These data show that divergent lineages of P. syringae can converge on identical disease etiology on the same host plant using different virulence mechanisms and that dramatic shifts in the arsenal of T3SEs can accompany disease emergence.

A recent re-emerging bacterial canker disease incited by Pseudomonas syringae pv. actinidiae (Psa) is causing severe economic losses to Actinidia chinensis and A. deliciosa cultivations in southern Europe, New Zealand, Chile and South Korea. Little is known about the genetic features of this pathovar. We generated genome-wide Illumina sequence data from two Psa strains causing outbreaks of bacterial canker on the A. deliciosa cv. Hayward in Japan (J-Psa, type-strain of the pathovar) and in Italy (I-Psa) in 1984 and 1992, respectively as well as from a Psa strain (I2-Psa) isolated at the beginning of the recent epidemic on A. chinensis cv. Hort16A in Italy. All strains were isolated from typical leaf spot symptoms. The phylogenetic relationships revealed that Psa is more closely related to P. s. pv. theae than to P. avellanae within genomospecies 8. Comparative genomic analyses revealed both relevant intrapathovar variations and putative pathovar-specific genomic regions in Psa. The genomic sequences of J-Psa and I-Psa were very similar. Conversely, the I2-Psa genome encodes four additional effector protein genes, lacks a 50 kb plasmid and the phaseolotoxin gene cluster, argK-tox but has acquired a 160 kb plasmid and putative prophage sequences. Several lines of evidence from the analysis of the genome sequences support the hypothesis that this strain did not evolve from the Psa population that caused the epidemics in 1984-1992 in Japan and Italy but rather is the product of a recent independent evolution of the pathovar actinidiae for infecting Actinidia spp. All Psa strains share the genetic potential for copper resistance, antibiotic detoxification, high affinity iron acquisition and detoxification of nitric oxide of plant origin. Similar to other sequenced phytopathogenic pseudomonads associated with woody plant species, the Psa strains isolated from leaves also display a set of genes involved in the catabolism of plant-derived aromatic compounds.