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In April 2019, hydroponically cultivated cucumber plants with characteristic symptoms of crazy root disease were found in two different commercial production cucumber greenhouses in Poland. Due to excessive and inappropriate root growth, this disease led to a reduction in yield and deterioration of the general conditions of infected plants. Bacteria isolated from the roots were subjected to a morphological evaluation, as well as molecular, biochemical and pathogenicity tests. To identify the bacteria causing the disease, Agrobacterium-like colonies were subjected to PCR with primers complementary to the pathogenicity-related genes located on the crazy root-inducing plasmid (Ri-plasmid): the virD2A + virD2E primers complementary to the virD2 gene and the rolBF + rolBR primers complementary to the rolB gene. The pathogenicity of the isolated strains was studied in sunflowers and cucumbers. Twelve strains positive for the Ri plasmid, as determined by PCR, and pathogenic to sunflowers were identified based on sequence analysis of the 16S rRNA and recA genes. One strain was classified as belonging to the genus Pararhizobium, three to Rhizobium, and eight to Agrobacterium biovar 1, with the highest similarity to genomospecies G3. The results of the analyses suggest that these strains may belong to a new, thus far, undescribed species. To confirm this hypothesis, further phylogenetic studies are required.

The results of our previous studies showed that 8 examined bacterial strains originating from the apple phyllosphere or soil environment showed both antagonistic activity towards E. amylovora and the protective ability of apple flowers and terminal shoots against fire blight. Five of the strains represented bacterial species in which this activity was observed for the first time (L16 Pseudomonas vancouverensis, 3M Pseudomonas chlororaphis subsp. aureofaciens, 43M Enterobacter ludwigii, 59M Pseudomonas protegens and 35M Pseudomonas congelans) (Mikiciński et al. 2020). We now present an attempt to explain the potential mechanisms related to the biocontrol capacity of the first four of the above mentioned strains. The studied strains were very effective in protecting pear fruitlet slices against fire blight. The disease severity after preventive treatment of the slices with the tested strains ranged from 0.0 to 0.6, while that of the untreated slices was 4.0 (rating scale: 0.0–4.0). Among the mechanisms studied, the L16 strain, characterized by the highest antagonistic activity, showed the ability to produce siderophores, biosurfactant, hydrogen cyanide (HCN), salicylic acid (SA) and indole-3-acetic acid (IAA). The L16 strain also degraded nicotinic acid. The 43M strain showed the lowest activity, producing only IAA and degrading nicotinic acid. A study of the detection of genes encoding antibiotics characteristic of pseudomonads showed the presence of prnD and gacA in the 3M strain and phlD, pltB, pltC and gacA in 59M. However, none of the genes sought were detected in the L16 strain.

Bacterial canker, caused by Pseudomonas syringae pv. syringae and pv. morsprunorum, is one of the most important diseases of stone fruit trees (Prunus spp.). The pathogen infects buds, flowers, fruitlets, leaves and shoots, from which the disease spreads to the branches, boughs and trunks, causing necrosis and cankers. The efficacy of different chemical and biological products for the control of bacterial canker on stone fruit trees was tested in 2018–2021. The experiments were conducted in sour cherry, plum and sweet cherry orchards in central Poland. Foliar application of the tested preparations was performed three times a season. The biological efficacy of the tested products in the control of bacterial canker was evaluated on sour cherry on the basis of infected leaves and fruits and on plum and sweet cherry on the basis of infected leaves. The highest efficacy was observed for products containing various forms of copper—copper oxide, copper oxychloride and copper hydroxide—as well as fertilizers with copper gluconate and the fungicide Luna Care 71.6 WG (fluopyram and fosetyl-Al). However, the biological preparations were significantly less effective. The conducted studies showed that preparations based on copper gluconate can be a valuable alternative to typical copper fungicides.

When viewing the fire blight disease from the perspective of a disease cycle, it is clear that the pathogen Erwinia amylovora goes through different infection stages on an annual basis. These stages include the initial infection of flowers and shoot tips with ooze as the inoculum; systemic spread through the plant vasculature; production of ooze on plant surfaces as the secondary inoculum, and formation of annual cankers to tolerate winter stresses. Among them, the stage of initial infection of flowers and shoot tips drew most research attention, as in this stage, E. amylovora transits from epiphytic colonization on the plant surface to endophytic infection internally in the plant tissue. Limiting the epiphytic colonization of E. amylovora on flower surfaces is also the focus of fire blight management, as only the epiphytic cells of E. amylovora can be targeted by the antimicrobial sprays. In this review, we focus on some of these “early events” during the initial infection of flowers and shoot tips. We define these “early events” as any events that occur from the production of inoculum to before the systemic spread of the pathogen through the xylem. This includes the production and transfer of the inoculum (ooze) to flowers and shoot tips; epiphytic proliferation of E. amylovora on flower / leaf surfaces; entry of the pathogen cells into host through natural openings or wounds; and initial proliferation immediately after entering the host. In the first half of this review, we summarize findings of some recent pathological investigations of these early events. In the second half of this review, we discuss the pathogenesis of E. amylovora in the context of these early events, particularly how E. amylovora modulates its virulence expression to induce disease symptoms. We focus on recent findings published within the past five years.

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.

This study included eight bacterial isolates originating from the apple phyllosphere or soil environment that were previously selected using the pear fruitlet test (Mikiciński 2017). Identification of these isolates based on phenotypic assays and DNA analysis showed that five of them belonged to species for which an antagonistic activity against Erwinia amylovora and the protective capacity of apple and pear against fire blight were not previously demonstrated. These were L16 identified as Pseudomonas vancouverensis, 3 M as Pseudomonas chlororaphis subsp. aureofaciens, 35 M – Pseudomonas congelans, 43 M – Enterobacter ludwigii, and 59 M – Pseudomonas protegens. Investigation of the biotic relationships between the tested strains and E. amylovora showed that 3 M, 35 M and 59 M inhibited the growth of the pathogen on five out of six media used (NAS, KB, LB, R2A, NAG), but 43 M did not do so on any of these media. Strain L16 did not inhibit the growth of the pathogen on LB or R2A medium. In contrast, all strains grown on medium 925 stimulated the growth of the pathogen, which showed no growth without co-cultivation with these strains. The experiments on apple trees and detached apple branches showed the ability of the tested bacteria to protect flowers at medium to high levels, depending on the experiment (55–93%). In some cases, this protection was even higher than that of the copper product used for comparison. In studies assessing the bacterial ability to protect shoots of M.26, the highest efficacy was observed for strains 35 M (96%) and 43 M (93%) but on ‘Gala Must’ all tested strains showed 100% of efficacy.

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.

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.

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.

Triggering the plant resistance induction phenomenon by chemical compounds, for example acibenzolar-S-methyl ester, has been known and described in scientific literature. Other benzothadiazole derivatives have been also described, however, their properties have not been sufficiently studied. The tested substance, N-methyl-N-methoxyamide-7-carboxybenzo(1.2.3)thiadiazole (BTHWA), is an amide derivative of benzothiadiazole, showing a stimulating effect on plant growth, apart from its plant resistance inducing activity. This article presents the impact of BTHWA, used solo and in the program with fungicides, on the strawberry plants development, fruits health, yield, and quality parameters of the crop. The results show that the combined use of BTHWA and fungicides had a positive impact on the plants health and fruit health and nutraceutical and nutritional composition of compounds when compared to the results obtained when strawberries were treated only with the BTHWA or the fungicide. As a result of BTHWA use, the partitioning of assimilates has changed, which directly translated into the results of the conducted experiments. A reduction in the respiration of the fruit during storage was also observed, possibly due to a reduced disease infestation and a lower dry matter content in the fruit. A correlation between the parameters determined during the experiment was found. The BTHWA mode of action was evidenced to be beneficial to strawberry plants and fruit.

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 β-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.

BackgroundAllorhizobium 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.ResultsPairwise 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.ConclusionsComparative 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.

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.

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.

The effective and rapid detection of Fire Blight, an important bacterial disease caused by the quarantine pest E.amylovora, is crucial for today’s horticulture. This study explored the application of non-invasive proximal hyperspectral remote sensing (RS) in order to differentiate the healthy (H), infected (I) and dry (D) leaves of apple trees. Analysis of variance was employed in order to determine which hyperspectral narrow spectral bands exhibited the most significant differences. Spectral signatures for the range of 400–2500 nm were acquired with Thermo Scientific Evolution 220 and iS50NIR spectrometers. The selected spectral bands were then used to evaluate several RS indices, including ARI (Anthocyanin Reflectance Index), RDVI (Renormalized Difference Vegetation Index), MSR (Modified Simple Ratio) and NRI (Nitrogen Reflectance Index), for Fire Blight detection in apple tree leaves. Furthermore, a new index was proposed, namely QFI. The spectral indices were tested on apple trees infected by Fire Blight in a quarantine greenhouse. Results indicated that the short-wavelength infrared (SWIR) band located at 1450 nm was able to distinguish (I) and (H) leaves, while the SWIR band at 1900 nm differentiated all three leaf types. Moreover, tests using the Pearson correlation indicated that ARI, MSR and QFI exhibited the highest correlations with the infection progress. Our results prove that our hyperspectral remote sensing technique is able to differentiate (H), (I) and (D) leaves of apple trees for the reliable and precise detection of Fire Blight.

For the years 2012–2018, we investigated the occurrence of storage diseases caused by fungi on four cultivars of apples grown in the central part of Poland. The fruits were picked in orchards managed with Integrated Pest Management (IPM) practices and stored in a cold room for 5–7 months. Depending on the season, apple cultivar and localization of orchard, the incidence of diseases was different. On apple cvs “Gala”, “Ligol” and “Golden Delicious”, bull’s eye rot (Neofabraea spp.) was the most frequently observed disease, whereas on apple cv. “Gloster”, gray mold (Botrytis cinerea) predominated. The blue mold (Penicillium expansum), brown rot (Monilinia spp.) and fungi of Alternaria spp. occurred at significantly lower intensity. We detected the occurrence of the new storage diseases of apples caused by Colletotrichum spp., Neonectria ditissima and Diaporthe eres.

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 (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.

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. Electronic supplementary material The online version of this article (10.1186/s12864-017-4251-z) contains supplementary material, which is available to authorized users.

Symptoms of tubercle disease known also as Xanthomonas gall were noticed in Poland in 2014–2017. Roots with disease symptoms showed the reduction of sucrose content compared to the healthy ones. In the literature, there are two gall diseases described: tubercle disease (also known as Xanthomonas gall) and crown gall, which is caused by Rhizobium radiobacter (syn. Agrobacterium tumefaciens). None of the bacterial strains isolated from the malformed tissues were identified as the bacterial pathogen responsible for causing Xanthomonas gall or crown gall. The result was confirmed by searching for the presence of T-DNA, which was not found. Our conclusion is that the tubercle disease observed in Poland is not caused by R. radiobacter.

In this study, we explored the pathogenicity and phylogenetic position of

12 páginas, Herein the members of the Subcommittee on Taxonomy of Rhizobia and Agrobacteria of the International Committee onSystematics of Prokaryotes review recent developments in rhizobial and agrobacterial taxonomy and propose updatedminimal standards for the description of new species (and genera) in these groups. The essential requirements (minimalstandards) for description of a new species are (1) a genome sequence of at least the proposed type strain and (2) evidencefor differentiation from other species based on genome sequence comparisons. It is also recommended that (3) geneticvariation within the species is documented with sequence data from several clearly different strains and (4) phenotypicfeatures 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 relevantgene sequences. These guidelines supplement the current rules of general bacterial taxonomy, which require (6) a namethat conforms to the International Code of Nomenclature of Prokaryotes, (7) validation of the name by publication eitherdirectly in theInternational Journal of Systematic and Evolutionary Microbiologyor in a validation list when publishedelsewhere, and (8) deposition of the type strain in two international culture collections in separate countries

Bull's eye rot is the most important storage disease of apples, causing up to 30-40% crop yield losses on susceptible cultivars in Poland. Due to the intensive plant material exchange, the knowledge about the species composition of causal agent of the disease needs to be updated. In this study, a previously modified multiplex PCR assay (Gariepy et al., 2003) was applied for the identification of the fungi in pure cultures. In years 2014-2015, 497 isolates of Neofabraea spp. from 493 samples of symptomatic fruit collected from 8 cold storages located in different regions of Poland were identified. Neofabraea alba was the predominant species (87% of the isolates) followed by N. perennans and N. kienholzii represented by 10 and 3% of the isolates, respectively. The species N. malicorticis was not detected. For the determination of genetic diversity, representative isolates of N. alba (25), N. perennans (22) and N. kienholzii (10) were analyzed by ISSR PCR with 5 microsatellite markers. The resulting dendrogram comprising results of all five ISSR reactions, segregated all strains according to their species affiliation. No relations were found between the observed amplification pattern and the geographical origin of the strain.

Specific primers were developed to detect the causal agent of stone fruit bacterial canker using conventional and real-time polymerase chain reaction (PCR) methods. PCR melting profile (PCR MP) used for analysis of diversity of Pseudomonas syringae strains, allowed to pinpoint the amplified fragments specific for P. syringae pv. morsprunorum race 1 (Psm1) and race 2 (Psm2), which were sequenced. Using obtained data, specific sequence characterised amplified region (SCAR) primers were designed. Conventional and real-time PCRs, using genomic DNA isolated from different bacterial strains belonging to the Pseudomonas genus, confirmed the specificity of selected primers. Additionally, the specificity of the selected DNA regions for Psm1 and Psm2 was confirmed by dot blot hybridisation. Conventional and real-time PCR assays enabled accurate detection of Psm1 and Psm2 in pure cultures and in plant material. For conventional PCR, the detection limits were the order of magnitude ~10(0) cfu/reaction for Psm1 and 10(1) cfu/reaction for Psm2 in pure cultures, while in plant material were 10(0)-10(1) cfu/reaction using primers for Psm1 and 3 × 10(2) cfu/reaction using primers for Psm2. Real-time PCR assays with SYBR Green I showed a higher limit of detection (LOD) - 10(0) cfu/reaction in both pure culture and in plant material for each primer pairs designed, which corresponds to 30-100 and 10-50 fg of DNA of Psm1 and Psm2, respectively. To our knowledge, this is the first PCR-based method for detection of the causal agents of bacterial canker of stone fruit trees.

The authors wish to make the following corrections to this paper [...]

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.

International audience; The plant tumorigenic strain NCPPB 1650T isolated from Rosa\texttimeshybrida, 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 DNADNA 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\textquotedblright. As for other species of the genus Agrobacterium, the major fatty acid of the strains studied was 18:1 w7c (73.4278.12%). 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).

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.

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. Electronic supplementary material The online version of this article (10.1186/s12862-018-1122-4) contains supplementary material, which is available to authorized users.

Eighty stone fruit nurseries located in different regions of Poland were examined for the presence of crown gall affected plants. The disease was observed in 39 nurseries, and galls were sampled for bacterial isolation. Out of 1213 isolates, 409 were pre-identified as Agrobacterium/Rhizobium spp. with 23S rDNA-based multiplex PCR, and out of these, 315 were pathogenic when tested on sunflowers. Sequence analysis of three housekeeping genes (fusA, recA, rpoD) revealed that 366 strains belonged to Rhizobium rhizogenes, 23 to Agrobacterium tumefaciens species complex, and the rest of the strains were allocated to new phylogenetic lineages. Of these, the most numerous was the lineage allocated in the Pararhizobium genus. Positive results obtained from pathogenicity tests were generally in agreement with results obtained by PCR with primers complementary to T-DNA except for two strains, which were positive for PCR but negative for the pathogenicity test. All detected Ti plasmids were nopaline-type. Independent of their pathogenicity, 59% of tested strains were not sensitive to agrocin 84 in in vitro tests. Analysis of biochemical and physiological features distinguished 50 groups with different phenotypic profiles, but the tested traits were not consistent for strains classified to one taxon. This finding shows limited value of biochemical tests in identification procedures. The bacteria causing tumours were heterogeneous and strains classified to different taxa were found even in a single tumour.

Brown rot caused by fungi belonging to the genus Monilinia is one of the major limiting factors of sour and sweet cherry production. Up to now, three species, M. fructigena, M. laxa and M. fructicola, have been identified as causal agents of brown rot on cherries worldwide. From 2010 to 2013, during the monitoring of cherry orchards in different areas of Poland, a fourth species, M. polystroma, was isolated from brown rot symptoms on sour and sweet cherry fruits. To the best of the authors’ knowledge, this is the first time M. polystroma has been reported as the causal agent of brown rot on cherries. The genetic diversity of M. polystroma isolates from cherries and other hosts was analysed using PCR MP, ISSR and RAPD techniques and showed its clear distinctness from other Monilinia spp. tested. The cluster analysis of fingerprinting data revealed a high similarity of M. polystroma isolates from Poland and their close relationship with the reference strain from Japan, indicating that this species is a recently introduced pathogen. The highest genetic distance between the examined isolates and the highest number of different genotypes was observed in an ISSR assay. Detailed genetic diversity characteristics revealed that M. polystroma isolates from cherries did not create a distinct group but were intermingled with M. polystroma isolates from other hosts. The results of the pathogenicity test conducted on different fruit species indicated a lack of host specificity for M. polystroma isolates.

Based on partial sequence analysis of the β-tubulin gene, 19 isolates of fungi causing bull's eye rot on apple in Poland were classified into species: Neofabraea alba, N. perennans and N. kienholzii. To the authors’ knowledge, the detection of N. kienholzii is the second in Europe and the first in Poland. Species affiliation of these fungi was confirmed by a new species-specific multiplex PCR assay developed on the basis of previously published methods. The new protocol allowed for the specific identification of bull's eye rot-causing species, both from pure cultures and directly from the skin of diseased or apparently healthy apples. In 550 samples of diseased fruits collected from nine cold storage rooms located in three regions of Poland, in 2011 and 2012, N. alba was detected as the predominant species causing bull's eye rot, occurring on average in 94% of the tested samples. Neofabraea perennans was found in a minority of apple samples, N. kienholzii was found only in two apple samples, while N. malicorticis was not detected in any sample tested. In tests on 120 apparently healthy fruits, only N. perennans was detected in a single sample. The results of genetic diversity analyses of bull's eye rot-causing fungi based on the β-tubulin gene sequence and an ISSR (inter-simple sequence repeat) PCR assay with two primers were consistent, showing the expected segregation of tested isolates with respect to their species boundaries. However, the genetic distance between N. perennans and N. malicorticis was very low, as reported previously.

Two plant-tumorigenic strains KFB 330(T) and KFB 335 isolated from galls on raspberry (Rubus idaeus) in Serbia, and a non-pathogenic strain AL51.1 recovered from a cherry plum (Prunus cerasifera) tumor in Poland, were genotypically and phenotypically characterized. Phylogenetic reconstruction based on 16S rDNA placed them within the genus Agrobacterium, with A. nepotum as their closest relative. Multilocus sequence analysis (MLSA) based on the partial sequences of atpD, glnA, gyrB, recA and rpoB housekeeping genes suggested that these three strains represent a new Agrobacterium species, that clustered with type strains of A. nepotum, A. radiobacter, "A. fabrum" and A. pusense. This was further supported by average nucleotide identity values (

Leaves and fruits of walnut trees exhibiting symptoms of bacterial blight were collected from six locations in Poland. Isolations on agar media resulted in 18 bacterial isolates with colony morphology resembling that of the Xanthomonas genus. PCR using X1 and X2 primers specific for Xanthomonas confirmed that all isolates belonged to this genus. In pathogenicity tests on unripe walnut fruits, all isolates caused typical black necrotic lesions covering almost the entire pericarp. Results of selected phenotypic tests indicated that characteristics of all isolates were the same as described for the type strain of Xanthomonas arboricola pv. juglandis. Genetic analyses (PCR MP, ERIC-, BOX-PCR and MLSA) showed similarities between the studied isolates and the reference strain of X. arboricola pv. juglandis CFBP 7179 originating from France. However, reference strains I-391 from Portugal and LMG 746 from the UK were different. MLSA analysis of partial sequences of the fyuA, gyrB and rpoD genes of studied isolates and respective sequences from GenBank of pathotype strains of other pathovars of X. arboricola showed that the X. arboricola pv. juglandis isolates consisted of different phylogenetic lineages. An incongruence among MLSA gene phylogenies and traces of intergenic recombination events were proved. These data suggest that the sequence analysis of several housekeeping genes is necessary for proper identification of X. arboricola pathovars.

Phytosanitary measures to control diseases require sensitive and reliable detection methods. In case of Erwinia amylovora, the causal agent of fire blight, there are several methods developed. We tested six of them: (1) a conventional method; (2) standard PCR; (3) BIO-PCR; (4) Real-time PCR with SYBR Green; (5) Real-time PCR with TaqMan probe; and (6) Loop Mediated Isothermal Amplification (LAMP) in terms of their specificity, sensitivity and repeatability. Of the methods tested, only Real-time PCR with SYBR Green amplified non-specific fragments, which could be however identified by melting curve analysis. Real-time PCRs (both with SYBR Green and TaqMan probe) and LAMP were most sensitive and allowed to detect E. amylovora even in the samples where the concentration of cells was lower than 2 x 101 cfu/reaction. The highest repeatability was obtained using conventional method and Real-time PCRs. Of the methods used, only the LAMP technique was very insensitive to plant impurities.

In 2011, leaf spot disease was observed on the blueberry (Vaccinium corymbosum) cv. Nelson growing on a commercial field located in Central Poland. The disease symptoms could be seen as russet brown, irregular spots. The diameter of the spots was 0.3-0.5 cm, and the spots often coalesced. From these leaf spots, a fluorescent bacterium was repeatedly isolated in almost pure culture. Polymerase chain reaction (PCR) using primers Ps-for and Ps-rev, specific for Pseudomonas spp. confirmed that they belong to this genus. Based on LOPAT tests [levan production from sucrose (L), presence of oxidase (O), pectolytic activity on potato (P), the presence of arginine dihydrolase (A), hypersensitivity reaction on tobacco (T)], 6 isolates were classified to the LOPAT group Ib - group of Pseudomonas syringae subsp. savastanoi and Pseudomonas delphini, and one isolate to group Ia - P. syringae. All isolates caused a hypersensitivity reaction on tobacco plants, and symptoms similar to those under natural conditions, when young leaves of blueberry cv. Nelson were inoculated. Sequence analysis of 16S rRNA and rpoB genes showed the highest similarity of 6 studied strains to the species P. avellanae. Further taxonomic study is necessary to enable definitive classification of these isolates. It is the first time that a bacterial disease caused by the Pseudomonas spp. was observed in Poland.

Standard program of plant protection against fire blight consists of use of management practices and chemical control method. Recently a new, non-conventional possibilities based on application of biocontrol agents (two biopreparations have been already introduced into practice: Bliteban A506 (Pseudomonas fluorescens) and BlossomBless (Pantoea agglomerans), plant extracts active against Erwinia amylovora (AkseBio containing extracts from Thymbra spicata and Biomit Plussz with extracts from various plant species and microelements) and resistance inducers (Regalis, Bion and plant extracts) are of great interest. Also plant transformation with resistance genes such as: hrpN (harpin), dpo (EPS depolymerase) and lytic protein genes (attacin E, cecropin SB-37, T4 lysozyme) is a promising perspective.

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.

Eight isolates of Gram-negative fluorescent bacteria (58T, 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 2400T and Pseudomonas congelans DSM 14939T. 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 281T and P. congelans LMG 21466T. DNA–DNA hybridization between the cherry isolate 58T 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 28609T 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 58T 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 Pseudomonas cerasi sp. nov. (non Griffin, 1911) is proposed. The type strain of this species is 58T (= LMG 28609T = CFBP 8305T).

Based on phenotypic tests, 49 out of 168 isolates of Pseudomonas syringae obtained from various organs of diseased tissue originating from various regions in Poland, were identified as P. syringae pv. morsprunorum race 1 (Psm1), 10 as race 2 of this pathovar (Psm2), 53 as pathovar syringae (Pss) and 56 as an atypical taxon. The pathogenicity test on immature sweet cherry fruits divided the tested strains into two groups: one with isolates causing black brown necrosis and the second containing isolates inducing water soaked superficial lesions. Phenotypic and genetic studies on toxins production by bacteria showed that all Pss produced syringomycin. However, only some Psm1 isolates have the gene for coronatine production. All strains belonging to Psm2 possessed genes encoding yersiniabactin. Results of genetic analyses (rep-PCRs, PCR MP) confirmed the homogeneity of isolates within pathovar morsprunorum and the atypical taxon and revealed diversity within pathovar syringae. A detailed polyphasic approach including phenotypic and genetic characterization applied for eight strains from the atypical taxon showed that the strains represent a novel species of the genus Pseudomonas for which Pseudomonas cerasi sp. nov. (non Griffin, 1911) is proposed.

Three bacterial isolates (CCBAU 101002(T), CCBAU 101000 and CCBAU 101001) originating from root nodules of the herbaceous legume Kummerowia stipulacea grown in the campus lawn of China Agricultural University were characterized with a polyphasic taxonomic approach. Comparative 16S rRNA gene sequence analysis showed that the isolates shared 99.85-99.92% sequence similarities and had the highest similarities to the type strains of Rhizobium mesoamericanum (99.31%), R. endophyticum (98.54%), R. tibeticum (98.38%) and R. grahamii (98.23%). Sequence similarity of four concatenated housekeeping genes (atpD, glnII, recA and rpoB) between CCBAU 101002(T) and its closest neighbor (R. grahamii) was 92.05%. DNA-DNA hybridization values between strain CCBAU 101002(T) and the four type strains of the most closely related Rhizobium species were less than 28.4±0.8%. The G+C mol% of the genomic DNA for strain CCBAU 101002(T) was 58.5% (Tm). The major respiratory quinone was ubiquinone (Q-10). Summed feature 8 (18:1ω7cis/18:1ω6cis) and 16:0 were the predominant fatty acids. Strain CCBAU 101002(T) contained phosphatidylcholine and phosphatidylethanolamine as major polar lipids, and phosphatidylglycerol and cardiolipin as minor ones. No glycolipid was detected. Unlike other strains, this novel species could utilize dulcite or sodium pyruvate as sole carbon sources and it was resistant to 2% (w/v) NaCl. On the basis of the polyphasic study, a new species Rhizobium cauense sp. nov. is proposed, with CCBAU 101002(T) (=LMG 26832(T)=HAMBI 3288(T)) as the type strain.

Five Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from galls on different plant species in Hungary: strain 39/7(T) from Prunus cerasifera Myrobalan, strain 0 from grapevine var. Ezerjó, strain 7/1 from raspberry var. Findus and in Poland, strain C3.4.1 from Colt rootstock (Prunus avium × Prunus pseudocerasus) and strain CP17.2.2 from Prunus avium. Only one of these isolates, strain 0, is able to cause crown gall on different plant species. On the basis of 16S rRNA gene sequence similarity, the strains cluster together and belong to the genus Rhizobium and their closest relative is Rhizobium radiobacter (99.1%). Phylogenetic analysis of the novel strains using housekeeping genes atpD, glnA, gyrB, recA and rpoB revealed their distinct position separate from other known Rhizobium species and confirmed their relation to Rhizobium radiobacter. The major cellular fatty acids are 18:1 w7c, 16:0, 16:0 3OH, summed feature 2 (comprising 12:0 aldehyde, 16:1 iso I and/or 14:0 3OH) and summed feature 3 (comprising 16:1 w7c and/or 15 iso 2OH). DNA-DNA hybridization of strain 39/7(T) with the type strain of R. radiobacter LMG 140(T) revealed 45% DNA-DNA hybridization. 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 Rhizobium, for which the name Rhizobium nepotum sp. nov. (type strain 39/7(T)=LMG 26435(T)=CFBP 7436(T)) is proposed.

The genetic diversity of 47 strains of Agrobacterium originating from different host plants and geographical locations in Poland, together with 12 strains from other countries was investigated. It was analyzed using RFLP of DNA fragment amplified with primers UP-1 and UP-2r flanking part of gyrB and parE genes, gyrB sequencing and randomly amplified polymorphic DNA (RAPD) technique. On the basis of obtained results, we found the majority of agrobacteria isolated in Poland belong to biovar 2. However, among others, three strains distinct from type strains of all the known Agrobacterium species, were discovered. All three methods showed no correlation between genetic diversity and geographical origin or the host plant of all studied strains but they revealed high diversity of the tested agrobacteria. The highest diversity was observed within strains of biovar 1, whereas those of biovar 2 were found to be the more homogenous group. The topology of the constructed gyrB tree corresponds to topologies of 16S and 23S rDNA trees obtained in this and other studies, but the gyrB tree had deeper branching. In the case of RAPD, it was possible to find a unique DNA fingerprint for almost each strain tested. The gyrB gene appeared to be a good phylogenetic marker with high discrimination power allowing better differentiation between species and strains, whereas the RAPD technique can serve as a tool for single strain typing.

Erwinia amylovora is a polyphagous bacterium causing fire blight on apple, pear and over 130 other plant species belonging mainly to the Rosaceae family. Although E. amylovora is regarded as a very homogenous species, the particular strains can differ in pathogenic ability as far as their host range is concerned (e.g. those originating from Rubus or Maloidae plants) as well as by the extent of the disease they cause. It was found that strains originating from North America are generally more genetically heterogeneous than those from Europe. Diversity of E. amylovora is also related to streptomycin resistance as a result of its application to control of fire blight. The level of genetic heterogeneity of E. amylovora is so low (comparative genome analysis revealed a similarity of over 99% for the two genomes tested) that standard DNA-based techniques fail in detection of intra-species variability. Amplified fragment length polymorphism was found to be most useful for differentiation of strains of fire blight causal agent as well as techniques ensuing release of pan-genome sequences of two E. amylovora strains: multi-locus variable number of tandem repeats analysis and clustered regularly interspaced short palindrome repeats.