Agrobacterium cucumeris sp. nov. isolated from crazy roots on cucumber (Cucumis sativus).

Three plant rhizogenic strains O132T, O115 and O34 isolated from Cucumis sp. L. were assessed for taxonomic affiliation by using polyphasic taxonomic methods. Based on the results of the sequence analysis of the 16S rRNA and multilocus sequence analysis (MLSA) of the three housekeeping genes atpD, recA and rpoB, all the strains were clustered within the genus Agrobacterium where they form a novel branch. Their closest relative was Agrobacterium tomkonis (genomospecies G3). Moreover, digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) comparisons between strains O132T and O34 and their closest relatives provided evidence that they constitute a new species, because the obtained values were significantly below the threshold considered as a borderline for the species delineation. Whole-genome phylogenomic analysis also indicated that the cucumber strains are located within the separate, well-delineated biovar 1 sub-clade of the genus Agrobacterium. Furthermore, the physiological and biochemical properties of these strains allowed to distinguish them from their closest related species of the genus Agrobacterium. As a result of the performed overall characterization, we propose a new species as Agrobacterium cucumeris sp. nov., with O132T (=CFBP 8997T = LMG 32451T) as the type strain.

Genomic analysis provides novel insights into diversification and taxonomy of Allorhizobium vitis (i.e. Agrobacterium vitis).

BACKGROUND: Allorhizobium vitis (formerly named Agrobacterium vitis or Agrobacterium biovar 3) is the primary causative agent of crown gall disease of grapevine worldwide. We obtained and analyzed whole-genome sequences of diverse All. vitis strains to get insights into their diversification and taxonomy. RESULTS: Pairwise genome comparisons and phylogenomic analysis of various All. vitis strains clearly indicated that All. vitis is not a single species, but represents a species complex composed of several genomic species. Thus, we emended the description of All. vitis, which now refers to a restricted group of strains within the All. vitis species complex (i.e. All. vitis sensu stricto) and proposed a description of a novel species, All. ampelinum sp. nov. The type strain of All. vitis sensu stricto remains the current type strain of All. vitis, K309T. The type strain of All. ampelinum sp. nov. is S4T. We also identified sets of gene clusters specific to the All. vitis species complex, All. vitis sensu stricto and All. ampelinum, respectively, for which we predicted the biological function and infer the role in ecological diversification of these clades, including some we could experimentally validate. All. vitis species complex-specific genes confer tolerance to different stresses, including exposure to aromatic compounds. Similarly, All. vitis sensu stricto-specific genes confer the ability to degrade 4-hydroxyphenylacetate and a putative compound related to gentisic acid. All. ampelinum-specific genes have putative functions related to polyamine metabolism and nickel assimilation. Congruently with the genome-based classification, All. vitis sensu stricto and All. ampelinum were clearly delineated by MALDI-TOF MS analysis. Moreover, our genome-based analysis indicated that Allorhizobium is clearly separated from other genera of the family Rhizobiaceae. CONCLUSIONS: Comparative genomics and phylogenomic analysis provided novel insights into the diversification and taxonomy of Allorhizobium vitis species complex, supporting our redefinition of All. vitis sensu stricto and description of All. ampelinum. Our pan-genome analyses suggest that these species have differentiated ecologies, each relying on specialized nutrient consumption or toxic compound degradation to adapt to their respective niche.

Agrobacterium vaccinii sp. nov. isolated from galls on blueberry plants (Vaccinium corymbosum).

Four non-pathogenic strains isolated from the galls on blueberry plants (Vaccinium corymbosum) were characterized by using polyphasic taxonomic methods. Based on 16S rRNA gene phylogeny, strains were clustered within the genus Agrobacterium. Furthermore, multilocus sequence analysis (MLSA) based on the partial sequences of atpD, recA and rpoB housekeeping genes and whole-genome-based phylogeny indicated that the strains studied form a novel Agrobacterium species. Analyses showed that the strains belong to "rubi" sub-clade of Agrobacterium genus and their closest relatives are Agrobacterium rubi and "Agrobacterium bohemicum". Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) comparisons between genome sequences of representative strains B7.6T and B19.1.4, and their closest relatives, confirmed the distinct phylogenetic position of studied strains, because obtained values were considerably below the proposed thresholds for the species delineation. The four strains studied were phenotypically distinguishable from other species of the genus Agrobacterium. Overall, polyphasic characterization showed that the strains studied represent a novel species of the genus Agrobacterium, for which the name Agrobacterium vaccinii sp. nov. is proposed. The type strain of A. vaccinii is B7.6T (=CFBP 8740T = LMG 31849T).

Epigenetic Modulating Chemicals Significantly Affect the Virulence and Genetic Characteristics of the Bacterial Plant Pathogen Xanthomonas campestris pv. campestris.

Epigenetics is the study of heritable alterations in phenotypes that are not caused by changes in DNA sequence. In the present study, we characterized the genetic and phenotypic alterations of the bacterial plant pathogen Xanthomonas campestris pv. campestris (Xcc) under different treatments with several epigenetic modulating chemicals. The use of DNA demethylating chemicals unambiguously caused a durable decrease in Xcc bacterial virulence, even after its reisolation from infected plants. The first-time use of chemicals to modify the activity of sirtuins also showed some noticeable results in terms of increasing bacterial virulence, but this effect was not typically stable. Changes in treated strains were also confirmed by using methylation sensitive amplification (MSAP), but with respect to registered SNPs induction, it was necessary to consider their contribution to the observed polymorphism. The molecular basis of the altered virulence was deciphered by using dualRNA-seq analysis of treated Xcc strains infecting Brassica rapa plants. The results of the present study should promote more intensive research in the generally understudied field of bacterial epigenetics, where artificially induced modification by epigenetic modulating chemicals can significantly increase the diversity of bacterial properties and potentially contribute to the further development of the fields, such as bacterial ecology and adaptation.

Multilocus Sequence Analysis of Selected Housekeeping- and Pathogenicity-Related Genes in Venturia inaequalis.

The relationship between housekeeping and pathogenicity-related genes and virulence or avirulence towards the primary Malus resistance genes (R) has not been previously studied for Venturia inaequalis fungus, the causal agent of apple scab. In this study, the sequences of two housekeeping genes encoding elongation factor alpha (EF-1α) and ß-tubulin and two previously unstudied effector genes of V. inaequalis from mannosidase and glucosidase families of 100 strains collected from apple cultivars with Rvi6, Rvi1, and Rvi17 and without known scab resistance genes were submitted to the analyses. Based on the phylogenetic and diversity data, as well as recombination analyses of the sequenced regions, we assessed the phylogenetic relationships and genetic structure of the pathogen within the species and the evolutionary forces that are currently acting upon this microorganism. The topology of the obtained phylograms demonstrates the lack of a relationship between the phylogenetic position of the strain and the host cultivar and the geographical origin or race of the strain. The isolates from different hosts were differentiated but did not form diagnosable, distinct phylogenetic groups. These results suggest that the analyzed genes may be too conserved to reflect the adaptation of pathogens to apple genotypes with different R genes; thus, they do not adequately reflect race discrimination. In contrast, based on variation and gene flow estimation, genetic divergence was observed among strains virulent to apple trees containing Rvi6. The results of this study confirmed a lack of free recombination between strains and demonstrated that the analyzed regions are in linkage disequilibrium and contain non-random polymorphisms associated with the strain.

Transcriptome analysis of Xanthomonas fragariae in strawberry leaves.

Xanthomonas fragariae is a quarantine bacterial pathogen that causes angular leaf spot on strawberry. The aim of our study was to analyse the mechanism of interaction of this bacterium with its host plant at the transcriptome level. For this purpose, mRNAs of X. fragariae growing in Wilbrink's medium and from infected strawberry cv. Elsanta plants were isolated and sequenced using the Illumina MiSeq platform. The expression profiles of the bacteria in Wilbrink's medium and in planta were very diverse. Of the 3939 CDSs recorded, 1995 had significantly different expression in planta (966 and 1029 genes were down- and upregulated, respectively). Among the genes showing increased expression in planta, those with eggNOG/COG (evolutionary genealogy of genes: Non-supervised Orthologous Groups/Cluster of Orthologous Groups) categories associated with bacterial cell motility, signal transduction, transport and metabolism of inorganic ions and carbohydrates and transcription were overrepresented. Among the genes with the most increased expression in planta, genes primarily associated with flagella synthesis and chemotaxis were found. It is also interesting to note that out of the 31 genes localized on a plasmid, 16 were expressed differently in planta, which may indicate their potential role in plant-pathogen interactions. Many genes with differentiated expression that were localized on chromosome and plasmid encode proteins of unknown function.

Host-Pathogen Interactions between Xanthomonas fragariae and Its Host Fragaria × ananassa Investigated with a Dual RNA-Seq Analysis.

Strawberry is economically important and widely grown, but susceptible to a large variety of phytopathogenic organisms. Among them, Xanthomonas fragariae is a quarantine bacterial pathogen threatening strawberry productions by causing angular leaf spots. Using whole transcriptome sequencing, the gene expression of both plant and bacteria in planta was analyzed at two time points, 12 and 29 days post inoculation, in order to compare the pathogen and host response between the stages of early visible and of well-developed symptoms. Among 28,588 known genes in strawberry and 4046 known genes in X. fragariae expressed at both time points, a total of 361 plant and 144 bacterial genes were significantly differentially expressed, respectively. The identified higher expressed genes in the plants were pathogen-associated molecular pattern receptors and pathogenesis-related thaumatin encoding genes, whereas the more expressed early genes were related to chloroplast metabolism as well as photosynthesis related coding genes. Most X. fragariae genes involved in host interaction, recognition, and pathogenesis were lower expressed at late-phase infection. This study gives a first insight into the interaction of X. fragariae with its host. The strawberry plant changed gene expression in order to consistently adapt its metabolism with the progression of infection.

mRNA extraction of Xanthomonas fragariae in strawberry and validation of reference genes for the RT-qPCR for study of bacterial gene expression.

This is the first study which describes a unique procedure of isolating of high-quality, intact RNA from strawberry leaves of Xanthomonas fragariae, three most suitable reference genes, crucial for the normalization of RT-qPCR data for this pathogen and accurate expression analysis of target genes. In our study, various mathematic algorithms: NormFinder geNorm, BestKeeper, the delta CT method, RefFinder were adopted for validation of most stable reference genes from nine candidate genes (ffh, glyA, gyrA, gyrB, proC, pykA, recA, rpoB, rpoD). The analyses allowing to select three most suitable pioneer reference genes, gyrB, ffh, and pykA, that we recommend for the normalization of RT-qPCR data and for the study of the expression of target genes in Xf. Moreover, their combination as references allowed for an accurate expression analysis and computation of the fold change of the flhF and iroN2 genes in Xf. These two genes are important for the success of the colonization of plant tissue and pathogenicity and sequences of primers designed to study these genes, are presented.

Two Novel Genomospecies in the Agrobacterium tumefaciens Species Complex Associated with Rose Crown Gall.

In this study, we explored the pathogenicity and phylogenetic position of Agrobacterium spp. strains isolated from crown gall tissues on annual, perennial, and ornamental plants in Iran. Of the 43 strains studied, 10 strains were identified as Allorhizobium vitis (formerly Agrobacterium vitis) using the species-specific primer pair PGF/PGR. Thirty-three remaining strains were studied using multilocus sequence analysis of four housekeeping genes (i.e., atpD, gyrB, recA, and rpoB), from which seven strains were identified as A. larrymoorei and one strain was identified as A. rubi (Rer); the remaining 25 strains were scattered within the A. tumefaciens species complex. Two strains were identified as genomospecies 1 (G1), seven strains were identified as A. radiobacter (G4), seven strains were identified as A. deltaense (G7), two strains were identified as A. nepotum (G14), and one strain was identified as "A. viscosum" (G15). The strains Rnr, Rnw, and Rew as well as the two strains OT33 and R13 all isolated from rose and the strain Ap1 isolated from apple were clustered in three atypical clades within the A. tumefaciens species complex. All but eight strains (i.e., Nec10, Ph38, Ph49, fic9, Fic72, R13, OT33, and Ap1) were pathogenic on tomato and sunflower seedlings in greenhouse conditions, whereas all but three strains (i.e., fic9, Fic72, and OT33) showed tumorigenicity on carrot root discs. The phylogenetic analysis and nucleotide diversity statistics suggested the existence of two novel genomospecies within the A. tumefaciens species complex, which we named "G19" and "G20." Hence, we propose the strains Rew, Rnw, and Rnr as the members of "G19" and the strains R13 and OT33 as the members of G20, whereas the phylogenetic status of the atypical strain Ap1 remains undetermined.

Minimal standards for the description of new genera and species of rhizobia and agrobacteria.

Herein the members of the Subcommittee on Taxonomy of Rhizobia and Agrobacteria of the International Committee on Systematics of Prokaryotes review recent developments in rhizobial and agrobacterial taxonomy and propose updated minimal standards for the description of new species (and genera) in these groups. The essential requirements (minimal standards) for description of a new species are (1) a genome sequence of at least the proposed type strain and (2) evidence for differentiation from other species based on genome sequence comparisons. It is also recommended that (3) genetic variation within the species is documented with sequence data from several clearly different strains and (4) phenotypic features are described, and their variation documented with data from a relevant set of representative strains. Furthermore, it is encouraged that information is provided on (5) nodulation or pathogenicity phenotypes, as appropriate, with relevant gene sequences. These guidelines supplement the current rules of general bacterial taxonomy, which require (6) a name that conforms to the International Code of Nomenclature of Prokaryotes, (7) validation of the name by publication either directly in the International Journal of Systematic and Evolutionary Microbiology or in a validation list when published elsewhere, and (8) deposition of the type strain in two international culture collections in separate countries.

Evolutionary Relatedness and Classification of Tumor-Inducing and Opine-Catabolic Plasmids in Three Rhizobium rhizogenes Strains Isolated from the Same Crown Gall Tumor.

Plasmids play a crucial role in the ecology of agrobacteria. In this study, we sequenced tumor-inducing (Ti) and opine-catabolic (OC) plasmids in three Rhizobium rhizogenes (Agrobacterium biovar 2) strains isolated from the same crown gall tumor on "Colt" cherry rootstock and conducted comparative genomic analyses. Tumorigenic strains C5.7 and C6.5 carry nopaline-type Ti plasmids pTiC5.7/pTiC6.5, whereas the nonpathogenic strain Colt5.8 carries the nopaline-type OC plasmid pOC-Colt5.8. Overall, comparative genomic analysis indicated that pTiC5.7/pTiC6.5 and related Ti plasmids described before (pTiC58 and pTi-SAKURA) originate from a common ancestor, although they have diverged during evolution. On the other hand, plasmid pOC-Colt5.8 was most closely related to the well-known OC plasmid pAtK84b; however, analysis suggested that they had different evolutionary histories and seem to share a more distant common ancestor. Although the reconstruction of the evolutionary history of Ti and OC plasmids is still speculative, we hypothesized that nopaline-type Ti plasmid might originate from the nopaline-type OC plasmid. Our results suggested that OC plasmids are widespread and closely associated with crown gall tumors. Finally, we proposed a thorough scheme for classification of Ti and OC plasmids that is based on separate comparative analysis of each functional element of the plasmid studied.

Rhizobium tumorigenes sp. nov., a novel plant tumorigenic bacterium isolated from cane gall tumors on thornless blackberry.

Four plant tumorigenic strains 932, 1019, 1078T and 1081 isolated from cane gall tumors on thornless blackberry (Rubus sp.) were characterized. They shared low sequence identity with related Rhizobium spp. based on comparisons of 16S rRNA gene (≤98%) and housekeeping genes atpD, recA and rpoB (<90%). Phylogenetic analysis indicated that the strains studied represent a novel species within the genus Rhizobium, with Rhizobium tubonense CCBAU 85046T as their closest relative. Furthermore, obtained average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH) values calculated for whole-genome sequences of strain 1078T and related Rhizobium spp. confirmed the authenticity of the novel species. The ANI-Blast (ANIb), ANI-MUMmer (ANIm) and in silico DDH values between strain 1078T and most closely related R. tubonense CCBAU 85046T were 76.17%, 84.11% and 21.3%, respectively. The novel species can be distinguished from R. tubonense based on phenotypic and chemotaxonomic properties. Here, we demonstrated that four strains studied represent a novel species of the genus Rhizobium, for which the name Rhizobium tumorigenes sp. nov. is proposed (type strain 1078T = DSM 104880T = CFBP 8567T). R. tumorigenes is a new plant tumorigenic species carrying the tumor-inducing (Ti) plasmid.

The Ecology of Agrobacterium vitis and Management of Crown Gall Disease in Vineyards.

Agrobacterium vitis is the primary causal agent of grapevine crown gall worldwide. Symptoms of grapevine crown gall disease include tumor formation on the aerial plant parts, whereas both tumorigenic and nontumorigenic strains of A. vitis cause root necrosis. Genetic and genomic analyses indicated that A. vitis is distinguishable from the members of the Agrobacterium genus and its transfer to the genus Allorhizobium was suggested. A. vitis is genetically diverse, with respect to both chromosomal and plasmid DNA. Its pathogenicity is mainly determined by a large conjugal tumor-inducing (Ti) plasmid characterized by a mosaic structure with conserved and variable regions. Traditionally, A. vitis Ti plasmids and host strains were differentiated into octopine/cucumopine, nopaline, and vitopine groups, based on opine markers. However, tumorigenic and nontumorigenic strains of A. vitis may carry other ecologically important plasmids, such as tartrate- and opine-catabolic plasmids. A. vitis colonizes vines endophytically. It is also able to survive epiphytically on grapevine plants and is detected in soil exclusively in association with grapevine plants. Because A. vitis persists systemically in symptomless grapevine plants, it can be efficiently disseminated to distant geographical areas via international trade of propagation material. The use of healthy planting material in areas with no history of the crown gall represents the crucial measure of disease management. Moreover, biological control and production of resistant grape varieties are encouraging as future control measures.

Agrobacterium rosae sp. nov., isolated from galls on different agricultural crops.

The plant tumorigenic strain NCPPB 1650T isolated from Rosa×hybrida, and four nonpathogenic strains isolated from tumors on grapevine (strain 384), raspberry (strain 839) and blueberry (strains B20.3 and B25.3) were characterized by using polyphasic taxonomic methods. Based on 16S rRNA gene phylogeny, strains were clustered within the genus Agrobacterium. Furthermore, multilocus sequence analysis (MLSA) based on the partial sequences of atpD, recA and rpoB housekeeping genes indicated that five strains studied form a novel Agrobacterium species. Their closest relatives were Agrobacterium sp. R89-1, Agrobacterium rubi and Agrobacterium skierniewicense. Authenticity of the novel species was confirmed by average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH) comparisons between strains NCPPB 1650T and B20.3, and their closest relatives, since obtained values were considerably below the proposed thresholds for the species delineation. Whole-genome-based phylogeny further supported distinctiveness of the novel species, that forms together with A. rubi, A. skierniewicense and Agrobacterium sp. R89-1 a well-delineated sub-clade of Agrobacterium spp. named "rubi". As for other species of the genus Agrobacterium, the major fatty acid of the strains studied was 18:1 w7c (73.42-78.12%). The five strains studied were phenotypically distinguishable from other species of the genus Agrobacterium. Overall, polyphasic characterization showed that the five strains studied represent a novel species of the genus Agrobacterium, for which the name Agrobacterium rosae sp. nov. is proposed. The type strain of A. rosae is NCPPB 1650T (=DSM 30203T=LMG 230T=CFBP 4470T=IAM 13558T=JCM 20915T).

Population structure of Venturia inaequalis, a causal agent of apple scab, in response to heterogeneous apple tree cultivation.

BACKGROUND: Tracking newly emergent virulent populations in agroecosystems provides an opportunity to increase our understanding of the co-evolution dynamics of pathogens and their hosts. On the one hand host plants exert selective pressure on pathogen populations, thus dividing them into subpopulations of different virulence, while on the other hand they create an opportunity for secondary contact between the two divergent populations on one tree. The main objectives of the study were to explore whether the previously reported structure between two Venturia inaequalis population types, virulent or avirulent towards Malus x domestica cultivars carrying Rvi6 gene, is maintained or broken several years after the first emergence of new virulent strains in Poland, and to investigate the relationship between 'new' and 'native' populations derived from the same commercial orchards. For this purpose, we investigated the genetic structure of populations of the apple scab fungus, occurring on apple tree cultivars containing Rvi6, Rvi1 or Rvi17 resistance gene or no resistance at all, based on microsatellite data obtained from 606 strains sampled in 10 orchards composed of various host cultivars. RESULTS: Application of genetic distance inferring and clustering methods allowed us to observe clear genetic distinctness of the populations virulent towards cultivars carrying Rvi6 gene from the Rvi6-avirulent populations and substructures within the Rvi6-group as a consequence of independent immigration events followed by rare, long-distance dispersals. We did not observe such a structuring effect of other genes determining apple scab resistance on any other populations, which in turn were genetically homogenous. However, in two orchards the co-occurrence of strains of different virulence pattern on the same trees was detected, blurring the genetic boundaries between populations. CONCLUSIONS: Among several resistance genes studied, only Rvi6 exerted selective pressure on pathogens populations: those virulent toward Rvi6 hosts show unique and clear genetic and virulence pattern. For the first time in commercial Malus x domestica orchards, we reported secondary contacts between populations virulent and avirulent toward Rvi6 hosts. These two populations, first diverged in allopatry, second came into contact and subsequently began interbreeding, in such way that they show unambiguous footprints of gene flow today.

Comparative transcriptome analysis of a lowly virulent strain of Erwinia amylovora in shoots of two apple cultivars - susceptible and resistant to fire blight.

BACKGROUND: Erwinia amylovora is generally considered to be a homogeneous species in terms of phenotypic and genetic features. However, strains show variation in their virulence, particularly on hosts with different susceptibility to fire blight. We applied the RNA-seq technique to elucidate transcriptome-level changes of the lowly virulent E. amylovora 650 strain during infection of shoots of susceptible (Idared) and resistant (Free Redstar) apple cultivars. RESULTS: The highest number of differentially expressed E. amylovora genes between the two apple genotypes was observed at 24 h after inoculation. Six days after inoculation, only a few bacterial genes were differentially expressed in the susceptible and resistant apple cultivars. The analysis of differentially expressed gene functions showed that generally, higher expression of genes related to stress response and defence against toxic compounds was observed in Free Redstar. Also in this cultivar, higher expression of flagellar genes (FlaI), which are recognized as PAMP (pathogen-associated molecular pattern) by the innate immune systems of plants, was noted. Additionally, several genes that have not yet been proven to play a role in the pathogenic abilities of E. amylovora were found to be differentially expressed in the two apple cultivars. CONCLUSIONS: This RNA-seq analysis generated a novel dataset describing the transcriptional response of the lowly virulent strain of E. amylovora in susceptible and resistant apple cultivar. Most genes were regulated in the same way in both apple cultivars, but there were also some cultivar-specific responses suggesting that the environment in Free Redstar is more stressful for bacteria what can be the reason of their inability to infect of this cultivar. Among genes with the highest fold change in expression between experimental combinations or with the highest transcript abundance, there are many genes without ascribed functions, which have never been tested for their role in pathogenicity. Overall, this study provides the first transcriptional profile by RNA-seq of E. amylovora during infection of a host plant and insights into the transcriptional response of this pathogen in the environments of susceptible and resistant apple plants.

Validation of reference genes for the normalization of the RT-qPCR gene expression of virulence genes of Erwinia amylovora in apple shoots.

To study the expression of pathogenicity-related genes in Erwinia amylovora, seven candidate reference genes (ffh, glyA, gyrA, proC, pykA, recA, rpoB) were selected and validated with the following five different mathematic algorithms: geNorm, NormFinder, BestKeeper, the delta CT method and the RefFinder web-based tool. An overall comprehensive ranking output from each of the selected software programs revealed that proC and recA, followed by ffh and pykA, were the most stably expressed genes and can be recommended for the normalization of RT-qPCR data. A combination of the three reference genes, proC, recA and ffh, allowed for the accurate expression analysis of amsB and hrpN genes and the calculation of their fold change in E. amylovora after its infection of susceptible and resistant apple cultivars. To the best of our knowledge, this is the first study presenting a list of the most suitable reference genes for use in the relative quantification of target gene expression in E. amylovora in planta, selected on the basis of a multi-algorithm analysis.

Pseudomonas cerasi sp. nov. (non Griffin, 1911) isolated from diseased tissue of cherry.

Eight isolates of Gram-negative fluorescent bacteria (58(T), 122, 374, 791, 963, 966, 970a and 1021) were obtained from diseased tissue of cherry trees from different regions of Poland. The symptoms resembled those of bacterial canker. Based on an analysis of 16S rDNA sequences the isolates shared the highest over 99.9% similarity with Pseudomonas ficuserectae JCM 2400(T) and P. congelans DSM 14939(T). Phylogenetic analysis using housekeeping genes gyrB, rpoD and rpoB revealed that they form a separate cluster and confirmed their closest relation to P. syringae NCPPB 281(T) and P. congelans LMG 21466(T). DNA-DNA hybridization between the cherry isolate 58(T) and the type strains of these two closely related species revealed relatedness values of 58.2% and 41.9%, respectively. This was further supported by Average Nucleotide Identity (ANIb) and Genome-to-Genome Distance (GGDC) between the whole genome sequences of strain LMG 28609(T) and closely related Pseudomonas species. The major cellular fatty acids are 16:0 and summed feature 3 (16:1 ω7c/15:0 iso 2OH). Phenotypic characteristics differentiated the novel isolates from other closely related species. The G+C content of the genomic DNA of strain 58(T) was 59%. The diversity was proved by PCR MP and BOX PCR, eliminating the possibility that they constitute a clonal population. Based on the evidence of this polyphasic taxonomic study the eight strains are considered to represent a novel species of the genus Pseudomonas for which the name P. cerasi sp. nov. (non Griffin, 1911) is proposed. The type strain of this species is 58(T) (=LMG 28609(T)=CFBP 8305(T)).

Pararhizobium polonicum sp. nov. isolated from tumors on stone fruit rootstocks.

Five Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from galls on different stone fruit rootstocks in Poland: strains F5.1(T) and F5.3 from Prunus avium F12/1, strains CP3.5 and CP17.2.1 from Prunus avium and strain AL5.1.8 from Prunus cerasifera. On the basis of 16S rDNA phylogeny, the strains cluster together and belong to the genus Pararhizobium with type strain of Pararhizobium herbae (99.6-99.8%) as their closest relative. Phylogenetic analysis of the novel strains using housekeeping genes atpD, recA and rpoB revealed their distinct position separate from other known Rhizobium species and confirmed their relation to P. herbae. DNA-DNA hybridization of strains F5.1(T), with the type strain of P. herbae LMG 25718(T) and Pararhizobium giardinii R-4385(T) revealed 28.3% and 27.9% of DNA-DNA relatedness, respectively. Phenotypic and physiological properties differentiate the novel isolates from other closely related species. On the basis of the results obtained, the five isolates are considered to represent a novel species of the genus Pararhizobium, for which the name Pararhizobium polonicum sp. nov. (type strain F5.1(T)=LMG 28610(T)=CFBP 8359(T)) is proposed.