Refining the taxonomy of the order Hyphomicrobiales (Rhizobiales) based on whole genome comparisons of over 130 type strains.

The alphaproteobacterial order Hyphomicrobiales consists of 38 families comprising at least 152 validly published genera as of January 2024. The order Hyphomicrobiales was first described in 1957 and underwent important revisions in 2020. However, we show that several inconsistencies in the taxonomy of this order remain and we argue that there is a need for a consistent framework for defining families within the order. We propose a common genome-based framework for defining families within the order Hyphomicrobiales, suggesting that families represent monophyletic groups in core-genome phylogenies that share pairwise average amino acid identity values above ~75 % when calculated from a core set of 59 proteins. Applying this framework, we propose the formation of four new families and to reassign the genera Salaquimonas, Rhodoblastus, and Rhodoligotrophos into Salaquimonadaceae fam. nov., Rhodoblastaceae fam. nov., and Rhodoligotrophaceae fam. nov., respectively, and the genera Albibacter, Chenggangzhangella, Hansschlegelia, and Methylopila into Methylopilaceae fam. nov. We further propose to unify the families Bartonellaceae, Brucellaceae, Phyllobacteriaceae, and Notoacmeibacteraceae as Bartonellaceae; the families Segnochrobactraceae and Pseudoxanthobacteraceae as Segnochrobactraceae; the families Lichenihabitantaceae and Lichenibacteriaceae as Lichenihabitantaceae; and the families Breoghaniaceae and Stappiaceae as Stappiaceae. Lastly, we propose to reassign several genera to existing families. Specifically, we propose to reassign the genus Pseudohoeflea to the family Rhizobiaceae; the genera Oricola, Roseitalea, and Oceaniradius to the family Ahrensiaceae; the genus Limoniibacter to the emended family Bartonellaceae; the genus Faunimonas to the family Afifellaceae; and the genus Pseudochelatococcus to the family Chelatococcaceae. Our data also support the recent proposal to reassign the genus Prosthecomicrobium to the family Kaistiaceae.

Diversity and Evolutionary History of Ti Plasmids of "tumorigenes" Clade of Rhizobium spp. and Their Differentiation from Other Ti and Ri Plasmids.

Agrobacteria are important plant pathogens responsible for crown/cane gall and hairy root diseases. Crown/cane gall disease is associated with strains carrying tumor-inducing (Ti) plasmids, while hairy root disease is caused by strains harboring root-inducing (Ri) plasmids. In this study, we analyzed the sequences of Ti plasmids of the novel "tumorigenes" clade of the family Rhizobiaceae ("tumorigenes" Ti plasmids), which includes two species, Rhizobium tumorigenes and Rhizobium rhododendri. The sequences of reference Ti/Ri plasmids were also included, which was followed by a comparative analysis of their backbone and accessory regions. The "tumorigenes" Ti plasmids have novel opine signatures compared with other Ti/Ri plasmids characterized so far. The first group exemplified by pTi1078 is associated with production of agrocinopine, nopaline, and ridéopine in plant tumors, while the second group comprising pTi6.2 is responsible for synthesis of leucinopine. Bioinformatic and chemical analyses, including opine utilization assays, indicated that leucinopine associated with pTi6.2 most likely has D,L stereochemistry, unlike the L,L-leucinopine produced in tumors induced by reference strains Chry5 and Bo542. Most of the "tumorigenes" Ti plasmids have conjugative transfer system genes that are unusual for Ti plasmids, composed of avhD4/avhB and traA/mobC/parA regions. Next, our results suggested that "tumorigenes" Ti plasmids have a common origin, but they diverged through large-scale recombination events, through recombination with single or multiple distinct Ti/Ri plasmids. Lastly, we showed that Ti/Ri plasmids could be differentiated based on pairwise Mash or average amino-acid identity distance clustering, and we supply a script to facilitate application of the former approach by other researchers.

Genomics of the "tumorigenes" clade of the family Rhizobiaceae and description of Rhizobium rhododendri sp. nov.

Tumorigenic members of the family Rhizobiaceae, known as agrobacteria, are responsible for crown and cane gall diseases of various crops worldwide. Tumorigenic agrobacteria are commonly found in the genera Agrobacterium, Allorhizobium, and Rhizobium. In this study, we analyzed a distinct "tumorigenes" clade of the genus Rhizobium, which includes the tumorigenic species Rhizobium tumorigenes, as well as strains causing crown gall disease on rhododendron. Here, high-quality, closed genomes of representatives of the "tumorigenes" clade were generated, followed by comparative genomic and phylogenomic analyses. Additionally, the phenotypic characteristics of representatives of the "tumorigenes" clade were analyzed. Our results showed that the tumorigenic strains isolated from rhododendron represent a novel species of the genus Rhizobium for which the name Rhizobium rhododendri sp. nov. is proposed. This species also includes additional strains originating from blueberry and Himalayan blackberry in the United States, whose genome sequences were retrieved from GenBank. Both R. tumorigenes and R. rhododendri contain multipartite genomes, including a chromosome, putative chromids, and megaplasmids. Synteny and phylogenetic analyses indicated that a large putative chromid of R. rhododendri resulted from the cointegration of an ancestral megaplasmid and two putative chromids, following its divergence from R. tumorigenes. Moreover, gene clusters specific for both species of the "tumorigenes" clade were identified, and their biological functions and roles in the ecological diversification of R. rhododendri and R. tumorigenes were predicted and discussed.

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.

Characterization of Pseudomonas syringae Strains Associated with Shoot Blight of Raspberry and Blackberry in Serbia.

During May 2016, severe blight symptoms were observed in several raspberry and blackberry fields in Serbia. In total, 22 strains were isolated: 16 from symptomatic raspberry shoots, 2 from asymptomatic raspberry leaves, and 4 from symptomatic blackberry shoots. Additionally, eight raspberry strains, isolated earlier from two similar outbreaks, were included in the study. Pathogenicity of the strains was confirmed on detached raspberry and blackberry shoots by reproducing the symptoms of natural infection. The strains were Gram-negative, fluorescent on King's medium B, ice nucleation positive, and utilized glucose oxidatively. All strains were levan positive, oxidase negative, nonpectolytic, arginine dihydrolase negative, and induced hypersensitivity in tobacco leaves (LOPAT + - - - +, Pseudomonas group Ia). Furthermore, all strains liquefied gelatin and hydrolyzed aesculin but did not show tyrosinase activity or utilize tartrate (GATTa + + - -). Tentative identification using morphology, LOPAT, GATTa, and ice-nucleating ability tests suggested that isolated strains belong to Pseudomonas syringae. The syrB gene associated with syringomycin production was detected in all strains. DNA fingerprints with REP, ERIC, and BOX primers generated identical profiles for 29 strains, except for strain KBI 222, which showed a unique genomic fingerprint. In all, 9 of 10 selected strains exhibited identical sequences of four housekeeping genes: gyrB, rpoD, gapA, and gltA. Five nucleotide polymorphisms were found in strain KBI 222 at the rpoD gene locus only. In the phylogenetic tree based on a concatenated sequence of all four housekeeping genes, strains clustered within phylogroup 2 (i.e., genomospecies 1) of the P. syringae species complex, with pathotype strains of P. syringae pv. aceris and P. syringae pv. solidagae as their closest relatives. There was no correlation between genotype and geographic origin, particular outbreak, host, or cultivar.

Polyphasic study of phytopathogenic bacterial strains associated with deep bark canker of walnut in Serbia revealed a new species, Brenneria izbisi sp. nov.

Serious outbreaks of walnut deep bark canker were observed on young walnut trees (Juglans regia L.) in two localities in the northern part of Serbia during 2020. From the symptomatic walnut tissues, two types of bacterial colonies were isolated, predominantly, light cream, circular and smooth colonies, as well as small, yellowish, mucoid and convex ones. PCR analysis and phenotypic assays suggested that the former group belongs to Brenneria spp., while the latter isolates were identified as Xanthomonas arboricola pv. juglandis. Within the Brenneria group, two strains were identified as Brenneria nigrifluens, while other 15 strains did not belong to any Brenneria species described so far. Therefore, we selected four representative strains of the unknown Brenneria sp. and subjected them to polyphasic analysis. As expected, in a phylogenetic tree based on partial 16S rDNA sequences, four novel strains grouped with other Brenneria representatives, and showed close phylogenetic relationship to Brenneria salicis. Furthermore, multilocus sequence analysis (MLSA) based on the partial sequences of atpD, gyrB, infB and rpoB housekeeping genes and core-genome phylogeny indicated that the studied strains form a novel and a clearly separate Brenneria lineage. Overall genome relatedness indices showed that they represent a new Brenneria species. The new species can be differentiated from the other Brenneria spp. infecting walnut and closely related B. salicis strains based on phenotypic characteristics, as well. Moreover, the pathogenicity tests on two-year-old walnut plants proved the ability of strains to cause necrosis and longitudinal black lesions and cracks on the trunk and branches of walnut trees. Overall, polyphasic characterization showed that the studied strains isolated from walnut with symptoms of deep bark canker represent a novel species of the genus Brenneria for which the name Brenneria izbisi sp. nov. is proposed. The type strain of B. izbisi is KBI 423T (= CFBP 9035T = LMG 32479T). To facilitate rapid identification of newly described species, a conventional PCR protocol and primers targeting the putative gene hrpP, were developed. Further study should reveal the potential role of each pathogen isolated from symptomatic walnut in disease development as well as possible interaction between them.

Two New Rhizobiales Species Isolated from Root Nodules of Common Sainfoin (Onobrychis viciifolia) Show Different Plant Colonization Strategies.

Root nodules of legume plants are primarily inhabited by rhizobial nitrogen-fixing bacteria. Here, we propose two new Rhizobiales species isolated from root nodules of common sainfoin (Onobrychis viciifolia), as shown by core-gene phylogeny, overall genome relatedness indices, and pan-genome analysis. Mesorhizobium onobrychidis sp. nov. actively induces nodules and achieves atmospheric nitrogen and carbon dioxide fixation. This species appears to be depleted in motility genes and is enriched in genes for direct effects on plant growth performance. Its genome reveals functional and plant growth-promoting signatures, like a large unique chromosomal genomic island with high density of symbiotic genetic traits. Onobrychidicola muellerharveyae gen. nov. sp. nov. is described as a type species of the new genus Onobrychidicola in Rhizobiaceae. This species comprises unique genetic features and plant growth-promoting traits (PGPTs), which strongly indicate its function in biotic stress reduction and motility. We applied a newly developed bioinformatics approach for in silico prediction of PGPTs (PGPT-Pred), which supports the different lifestyles of the two new species and the plant growth-promoting performance of M. onobrychidis in the greenhouse trial. IMPORTANCE The intensive use of chemical fertilizers has a variety of negative effects on the environment. Increased utilization of biological nitrogen fixation (BNF) is one way to mitigate those negative impacts. In order to optimize BNF, suitable candidates for different legume species are required. Despite intensive search for new rhizobial bacteria associated with legumes, no new rhizobia have recently been identified from sainfoin (Onobrychis viciifolia). Here, we report on the discovery of two new rhizobial species associated with sainfoin, which are of high importance for the host and may help to increase sustainability in agricultural practices. We employed the combination of in silico prediction and in planta experiments, which is an effective way to detect promising plant growth-promoting bacteria.

Xanthomonas bonasiae sp. nov. and Xanthomonas youngii sp. nov., isolated from crown gall tissues.

The genus Xanthomonas contains a set of diverse bacterial strains, most of which are known for their pathogenicity on annual crops and fruit trees causing economically important plant diseases. Recently, five Xanthomonas strains were isolated from Agrobacterium-induced crown gall tissues of amaranth (Amaranthus sp.) and weeping fig (Ficus benjamina) plants in Iran. Phenotypic characteristics (i.e. biochemical tests and pathogenicity features) and whole genome sequence-based core-genome phylogeny followed by average nucleotide identity and digital DNA-DNA hybridization calculations suggested that these gall-associated strains belong to two new species within the genus Xanthomonas. In this study, we provide a formal species description for these new species where Xanthomonas bonasiae sp. nov. is proposed for the strains isolated from weeping fig with FX4T (=CFBP 8703T=DSM 112530T) as type strain. The name Xanthomonas youngii sp. nov. is proposed for the strains isolated from amaranth with AmX2T (=CFBP 8902T=DSM 112529T) as 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).

Phenotypic and Molecular-Phylogenetic Analyses Reveal Distinct Features of Crown Gall-Associated Xanthomonas Strains.

In summer 2019, widespread occurrence of crown gall disease caused by Agrobacterium spp. was observed on commercially grown ornamental plants in southern Iran. Beside agrobacteria, pale yellow-pigmented Gram-negative strains resembling the members of Xanthomonas were also associated with crown gall tissues on weeping fig (Ficus benjamina) and Amaranthus sp. plants. The purpose of the present study was to characterize the crown gall-associated Xanthomonas strains using plant inoculation assays, molecular-phylogenetic analyses, and comparative genomics approaches. Pathogenicity tests showed that the Xanthomonas strains did not induce disease symptoms on their host of isolation. However, the strains induced hypersensitive reaction on tobacco, geranium, melon, squash, and tomato leaves via leaf infiltration. Multilocus sequence analysis suggested that the strains belong to clade IA of Xanthomonas, phylogenetically close to Xanthomonas translucens, X. theicola, and X. hyacinthi. Average nucleotide identity and digital DNA-DNA hybridization values between the whole-genome sequences of the strains isolated in this study and reference Xanthomonas strains are far below the accepted thresholds for the definition of prokaryotic species, signifying that these strains could be defined as two new species within clade IA of Xanthomonas. Comparative genomics showed that the strains isolated from crown gall tissues are genetically distinct from X. translucens, as almost all the type III secretion system genes and type III effectors are lacking in the former group. The data obtained in this study provide novel insight into the breadth of genetic diversity of crown gall-associated bacteria and pave the way for research on gall-associated Xanthomonas-plant interactions. IMPORTANCE Tumorigenic agrobacteria-members of the bacterial family Rhizobiaceae-cause crown gall and hairy root diseases on a broad range of plant species. These bacteria are responsible for economic losses in nurseries of important fruit trees and ornamental plants. The microclimate of crown gall and their accompanying microorganisms has rarely been studied for the microbial diversity and population dynamics of gall-associated bacteria. Here, we employed a series of biochemical tests, pathogenicity assays, and molecular-phylogenetic analyses, supplemented with comparative genomics, to elucidate the biological features, taxonomic position, and genomic repertories of five crown gall-associated Xanthomonas strains isolated from weeping fig and Amaranthus sp. plants in Iran. The strains investigated in this study induced hypersensitive reactions (HR) on geranium, melon, squash, tobacco, and tomato leaves, while they were nonpathogenic on their host of isolation. Phylogenetic analyses and whole-genome-sequence-based average nucleotide identity (ANI)/digital DNA-DNA hybridization (dDDH) calculations suggested that the Xanthomonas strains isolated from crown gall tissues belong to two taxonomically unique clades closely related to the clade IA species of the genus, i.e., X. translucens, X. hyacinthi, and X. theicola.

Genomic Analyses of Rose Crown Gall-Associated Bacteria Revealed Two New Agrobacterium Species: Agrobacterium burrii sp. nov. and Agrobacterium shirazense sp. nov.

Agrobacterium tumefaciens species complex contains a set of diverse bacterial strains, most of which are well known for their pathogenicity on agricultural plants causing crown gall diseases. Members of A. tumefaciens species complex are classified into several taxonomically distinct lineages called "genomospecies" (13 genomospecies until early 2021). Recently, two genomospecies, G19 (strains RnrT, Rew, and Rnw) and G20 (strains OT33T and R13) infecting Rosa sp. plants in Iran, were described based on biochemical and molecular-phylogenetic data. Whole genome sequence-based core-genome phylogeny followed by average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) calculations performed in this study suggested that genomospecies G19 and G20 could be described as two novel and standalone species. In the phylogenetic tree, these two new genomospecies were clustered separately from other genomospecies/species of A. tumefaciens species complex. Moreover, both ANI and dDDH indices between the G19/G20 strains and other Rhizobiaceae members are clearly below the accepted thresholds for prokaryotic species description. Hence, Agrobacterium burrii sp. nov. is proposed to encompass the G19 strains, with RnrT = CFBP 8705T = DSM 112541T as type strain. Agrobacterium shirazense sp. nov. is also proposed to include G20 strains, with OT33T = CFBP 8901T = DSM 112540T as type strain.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

The chitinolytic activity of the Curtobacterium sp. isolated from field-grown soybean and analysis of its genome sequence.

Curtobacterium sp. GD1 was isolated from leaves of conventionally grown soybean in Brazil. It was noteworthy that among all bacteria previously isolated from the same origin, only Curtobacterium sp. GD1 showed a strong chitinase activity. The enzyme was secreted and its production was induced by the presence of colloidal chitin in the medium. The chitinase was partially purified and characterized: molecular weight was approximately 37 kDa and specific activity 90.8 U/mg. Furthermore, Curtobacterium sp. GD1 genome was sequenced and analyzed. Our isolate formed a phylogenetic cluster with four other Curtobacterium spp. strains, with ANIb/ANIm ≥ 98%, representing a new, still non described Curtobacterium species. The circular genome visualization and comparison of genome sequences of strains forming new cluster indicated that most regions within their genomes were highly conserved. The gene associated with chitinase production was identified and the distribution pattern of glycosyl hydrolases genes was assessed. Also, genes associated with catabolism of structural carbohydrates such as oligosaccharides, mixed polysaccharides, plant and animal polysaccharides, as well as genes or gene clusters associated with resistance to antibiotics, toxic compounds and auxin biosynthesis subsystem products were identified. The abundance of putative glycosyl hydrolases in the genome of Curtobacterium sp. GD1 suggests that it has the tools for the hydrolysis of different polysaccharides. Therefore, Curtobacterium sp. GD1 isolated from soybean might be a bioremediator, biocontrol agent, an elicitor of the plant defense responses or simply degrader.

Occurrence of Crown Gall Disease on Japanese Spindle (Euonymus japonicas var. Green Rocket) Caused by Agrobacterium rosae in Iran.

Crown gall disease caused by diverse Agrobacterium species is one of the main biotic constraints in the ornamental plants industry in Iran (Mafakheri et al. 2017). In August 2019, Japanese spindle (Euonymus japonicus var. Green Rocket) plants showing crown gall symptoms were observed in a commercial greenhouse in Tehran, Iran. Infected plants were characterized by a visible overgrowth on their stems and crown. Bacterial isolation from the gall tissues was performed on nutrient agar (NA) and 1A media as described by Moore et al. (2001). The six resulted bacterial strains (A.E1 to A.E6) were evaluated using PCR primer pair F8360/F8361 amplifying a 453 bp DNA fragment in recA gene and confirmed as Agrobacterium sp. (Shams et al. 2013). Pathogenicity of the strains was evaluated in two independent assays on Japanese spindle plantlets as well as 10-15 day old tomato (Solanum lycopersicum cv. Sunseed 6189) and sunflower (Helianthus annuus cv. Armavirski) plants in greenhouse conditions using the needle prick method as described previously (Mafakheri et al. 2019). The reference strain A. radiobacter ICMP 5856 and sterile distilled water were used as positive and negative controls, respectively. Crown gall symptoms appeared 20-25 days post inoculation on the Japanese spindle plantlets as well as tomato and sunflower plants inoculated with the strains isolated in this study, while the negative control plants remained asymptomatic. Koch's postulates were accomplished by re-isolating on NA medium and PCR-based identification of the inoculated strains from the symptomatic plants. The representative strain A.E1 was subjected to multilocus sequence analysis (MLSA) using the sequences of four housekeeping genes (i.e. atpD, gyrB, recA, and rpoB) as described previously (Mafakheri et al. 2019). MLSA results revealed that the strain A.E1 is phylogenetically closely related to A. rosae. The sequences were deposited into GenBank under the accession numbers MT007962 to MT007965 for atpD, gyrB, recA, and rpoB, respectively. Further, the strain A.E1 was subjected to whole genome sequencing using Illumina HiSeq X platform. DNA extraction was performed using NucleoSpin Microbial DNA kit (Macherey-Nagel, Germany), DNA libraries were obtained with Nextera XT DNA Library Prep Kit (Illumina, USA), and de novo sequence assembly was performed using SPAdes genome assembler. The resulting whole genome sequence was deposited into the GenBank database under the accession number JAFJZW000000000. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values were calculated among all the type strains of Agrobacterium species/genomospecies using standard criteria as detailed previously (Osdaghi et al. 2020; Chen et al. 2021). The strain A.E1 had 97% ANI and 72% dDDH values with A. rosae strain NCPPB 1650, suggesting that the bacterial strains isolated from Japanese spindle in Iran belong to A. rosae. This is the first report of A. rosae causing crown gall disease on Japanese spindle in Iran. The new crown gall disease could negatively affect the ornamental shrub production industry in central Iran unless strict sanitary measures are taken into the account in the nurseries in these areas. Further nationwide surveys and samplings are warranted to elucidate the economic impact of the pathogen on ornamental plant industry in the country.

Isolation, Characterization and Draft Genome Analysis of Bacteriophages Infecting Acidovorax citrulli.

Bacterial fruit blotch and seedling blight, caused by Acidovorax citrulli, is one of the most destructive diseases of melon and watermelon in many countries. Pathogen-free seed and cultural practices are major pillars of the disease control. However, use of bacteriophages as natural biocontrol agents might also contribute to the disease management. Therefore, we isolated 12 bacteriophages specific to A. citrulli, from phyllosphere and rhizosphere of diseased watermelon plants. The phage strains were characterized based on their host range, plaque and virion morphology, thermal inactivation point, adsorption rate, one step growth curve, restriction fragment length polymorphism (RFLP), and genomic analysis. Transmission electron microscopy of three phage strains indicated that they belong to the order Caudovirales, family Siphoviridae. All phages lysed 30 out of 32 tested A. citrulli strains isolated in Serbia, and did not lyse other less related bacterial species. They produced clear plaques, 2 mm in diameter, on bacterial lawns of different A. citrulli strains after 24 h of incubation. The thermal inactivation point was 66 or 67°C. They were stable at pH 5-9, but were sensitive to chloroform and inactivated in either 5 or 10 min exposure to ultraviolet (UV) light. RFLP analysis using EcoRI, BsmI and BamHI enzymes did not show genetic differences among the tested phages. Adsorption rate and one step growth curve were determined for the Acidovorax phage ACF1. Draft genome sequence of the ACF1 phage was 59.377 bp in size, with guanine-cytosine (GC) content 64.5%, including 89 open reading frames. This phage shared a very high genomic identity with Acidovorax phage ACPWH, isolated in South Korea. Evaluation of systemic nature of ACF1 strain showed that it can be absorbed by roots and translocated to upper parts of watermelon plants where it survived up to 10 days.

A Novel Group of Rhizobium tumorigenes-Like Agrobacteria Associated with Crown Gall Disease of Rhododendron and Blueberry.

Crown gall is an economically important and widespread plant disease caused by tumorigenic bacteria that are commonly affiliated within the genera Agrobacterium, Allorhizobium, and Rhizobium. Although crown gall disease was reported to occur on rhododendron, literature data regarding this disease are limited. In this study, an atypical group of tumorigenic agrobacteria belonging to the genus Rhizobium was identified as a causative agent of crown gall on rhododendron. Genome analysis suggested that tumorigenic bacteria isolated from rhododendron tumors are most closely related to Rhizobium tumorigenes, a new tumorigenic bacterium discovered recently on blackberry in Serbia. However, R. tumorigenes and novel rhododendron strains belong to separate species and form a homogenous clade within the genus Rhizobium, which we named the "tumorigenes" clade. Moreover, tumorigenic bacteria isolated from rhododendron seem to carry a distinct tumor-inducing (Ti) plasmid, compared with those carried by R. tumorigenes strains and Ti plasmids described thus far. To facilitate rapid identification of bacteria belonging to the "tumorigenes" clade, regardless of whether they are pathogenic or not, a conventional PCR method targeting putative chromosomal gene-encoding flagellin protein FlaA was developed in this study. Finally, our results suggested that this novel group of tumorigenic agrobacteria occurs on blueberry but it cannot be excluded that it is distributed more widely.

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.

Role of Plasmids in Plant-Bacteria Interactions.

Plants are colonized by diverse microorganisms, which may positively or negatively influence the plant fitness. The positive impact includes nutrient acquisition, enhancement of resistance to biotic and abiotic stresses, both important factors for plant growth and survival, while plant pathogenic bacteria can cause diseases. Plant pathogens are adapted to negate or evade plant defense mechanisms, e.g. by the injection of effector proteins into the host cells or by avoiding the recognition by the host. Plasmids play an important role in the rapid bacterial adaptation to stresses and changing environmental conditions. In the plant environment, plasmids can further provide a selective advantage for the host bacteria, e.g. by carrying genes encoding metabolic pathways, metal and antibiotic resistances, or pathogenicity-related genes. However, we are only beginning to understand the role of mobile genetic elements and horizontal gene transfer for plant-associated bacteria. In this review, we aim to provide a short update on what is known about plasmids and horizontal gene transfer of plant associated bacteria and their role in plant-bacteria interactions. Furthermore, we discuss tools available to study the plant-associated mobilome, its transferability, and its bacterial hosts.