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In prokaryotic taxonomy, a set of criteria is commonly used to delineate species. These criteria are generally based on cohesion at the phylogenetic, phenotypic and genomic levels. One such criterion shown to have promise in the genomic era is average nucleotide identity (ANI), which provides an average measure of similarity across homologous regions shared by a pair of genomes. However, despite the popularity and relative ease of using this metric, ANI has undergone numerous refinements, with variations in genome fragmentation, homologue detection parameters and search algorithms. To test the robustness of a 95-96 % species cut-off range across all the commonly used ANI approaches, seven different methods were used to calculate ANI values for intra- and interspecies datasets representing three classes in the Proteobacteria . As a reference point, these methods were all compared to the widely used blast-based ANI (i.e. ANIb as implemented in JSpecies), and regression analyses were performed to investigate the correlation of these methods to ANIb with more than 130000 individual data points. From these analyses, it was clear that ANI methods did not provide consistent results regarding the conspecificity of isolates. Most of the methods investigated did not correlate perfectly with ANIb, particularly between 90 and 100% identity, which includes the proposed species boundary. There was also a difference in the correlation of methods for the different taxon sets. Our study thus suggests that the specific approach employed needs to be considered when ANI is used to delineate prokaryotic species. We furthermore suggest that one would first need to determine an appropriate cut-off value for a specific taxon set, based on the intraspecific diversity of that group, before conclusions on conspecificity of isolates can be made, and that the resulting species hypotheses be confirmed with analyses based on evolutionary history as part of the polyphasic approach to taxonomy.

The Erwiniaceae contain many species of agricultural and clinical importance. Although relationships among most of the genera in this family are relatively well resolved, the phylogenetic placement of several taxa remains ambiguous. In this study, we aimed to address these uncertainties by using a combination of phylogenetic and genomic approaches. Our multilocus sequence analysis and genome-based maximum-likelihood phylogenies revealed that the arsenate-reducing strain IMH and plant-associated strain ATCC 700886, both previously presumptively identified as members of Pantoea, represent novel species of Erwinia. Our data also showed that the taxonomy of Erwinia teleogrylli requires revision as it is clearly excluded from Erwinia and the other genera of the family. Most strikingly, however, five species of Pantoea formed a distinct clade within the Erwiniaceae, where it had a sister group relationship with the Pantoea + Tatumella clade. By making use of gene content comparisons, this new clade is further predicted to encode a range of characters that it shares with or distinguishes it from related genera. We thus propose recognition of this clade as a distinct genus and suggest the name Mixta in reference to the diverse habitats from which its species were obtained, including plants, humans and food products. Accordingly, a description for Mixta gen. nov. is provided to accommodate the four species Mixta calida comb. nov., M. gaviniae comb. nov., M. intestinalis comb. nov. and M. theicola comb. nov., with M. calida as the type species for the genus.

Bacterial isolates from oak trees in Spain and Britain, showing symptoms of bark canker and Acute Oak Decline (AOD), respectively, were examined by a polyphasic approach. Both 16S rRNA gene sequencing and multilocus sequence analysis (MLSA), based on partial sequences of gyrB, rpoB, infB and atpD genes, revealed that the isolates were separated into two genetic groups according to their origin. Their closest phylogenetic relative was Brenneria quercina, the causal agent of drippy nut disease of oak, which clustered distant to the other species of the genus Brenneria. MLSA data for species of the genera Brenneria, Pectobacterium, Dickeya, Erwinia, Pantoea and Samsonia confirmed the polyphyletic nature of the genus Brenneria and indicated synonymy of Dickeya dadantii and Dickeya dieffenbachiae. DNA-DNA hybridization experiments confirmed this synonymy and also revealed DNA-DNA relatedness values of 58-73% between the new oak isolates and B. quercina. Phenotypic and/or chemotaxonomic methods allowed B. quercina and the two genetic groups of new oak isolates to be discriminated from other recognized species of the genus Brenneria and from members of the closely related genera Dickeya, Pectobacterium and Samsonia. Based on the data obtained, the following taxonomic proposals are made: (1) reclassification of B. quercina as the type species of a novel genus, Lonsdalea gen. nov., as Lonsdalea quercina comb. nov. (type strain LMG 2724(T)=ATCC 29281(T)=CCUG 48867(T)=CFBP 3617(T)=CIP 105201(T)=DSM 4561(T)=ICMP 1845(T)), (2) classification of the oak isolates as Lonsdalea quercina subsp. iberica subsp. nov. (type strain LMG26264(T)=NCPPB 4490(T)) and Lonsdalea quercina subsp. britannica subsp. nov. (type strain LMG 26267(T)=NCPPB 4481(T)) and leading to the automatic creation of Lonsdalea quercina subsp. quercina subsp. nov. (type strain LMG 2724(T)=ATCC 29281(T)), (3) emendation of the description of the genus Brenneria, and (4) reclassification of Dickeya dieffenbachiae as Dickeya dadantii subsp. dieffenbachiae comb. nov. (type strain LMG 25992(T)=CFBP 2051(T)), with the automatic creation of Dickeya dadantii subsp. dadantii subsp. nov. (type strain LMG 25991(T)=CFBP 1269(T)).

Several Gram-negative-staining, facultatively anaerobic bacterial isolates were obtained from Eucalyptus seedlings showing symptoms of bacterial blight and dieback in Colombia, Rwanda and South Africa. Partial 16S rRNA gene sequencing, together with partial gyrB sequencing, placed the isolates in the genus Pantoea and indicated that they constituted three novel species. Multilocus sequence analysis (MLSA) based on partial sequences of gyrB, rpoB, infB and atpD revealed Pantoea dispersa, Pantoea eucrina and Pantoea cypripedii as their closest phylogenetic relatives. DNA-DNA hybridization studies confirmed the classification of the new isolates as three novel species and phenotypic tests allowed them to be differentiated from their closest phylogenetic neighbours. The names Pantoea rodasii sp. nov. [type strain LMG 26273(T)=BD 943(T) (deposited with the Plant Pathogenic and Plant Protecting Bacteria Collection, South Africa)=BCC 581(T) (deposited with the Bacterial Culture Collection, Forestry and Agricultural Institute, South Africa)], Pantoea rwandensis sp. nov. (type strain LMG 26275(T)=BD 944(T)=BCC 571(T)) and Pantoea wallisii sp. nov. (type strain LMG 26277(T)=BD 946(T)=BCC 682(T)) are proposed.

Eight yellow-pigmented, Gram-negative, rod-shaped, oxidase-negative, motile, facultatively anaerobic bacteria were isolated from onion seed in South Africa and from an onion plant exhibiting centre rot symptoms in the USA. The isolates were assigned to the genus Pantoea on the basis of phenotypic and biochemical tests. 16S rRNA gene sequence analysis and multilocus sequence analysis (MLSA), based on gyrB, rpoB, infB and atpD sequences, confirmed the allocation of the isolates to the genus Pantoea. MLSA further indicated that the isolates represented a novel species, which was phylogenetically most closely related to Pantoea ananatis and Pantoea stewartii. Amplified fragment length polymorphism analysis also placed the isolates into a cluster separate from P. ananatis and P. stewartii. Compared with type strains of species of the genus Pantoea that showed >97 % 16S rRNA gene sequence similarity with strain BD 390(T), the isolates exhibited 11-55 % whole-genome DNA-DNA relatedness, which confirmed the classification of the isolates in a novel species. The most useful phenotypic characteristics for the differentiation of the isolates from their closest phylogenetic neighbours are production of acid from amygdalin and utilization of adonitol and sorbitol. A novel species, Pantoea allii sp. nov., is proposed, with type strain BD 390(T) ( = LMG 24248(T)).

Bacterial strains belonging to DNA hybridization groups (HG) II, IV and V, in the Erwinia herbicola-Enterobacter agglomerans complex, of Brenner et al. [Int J Syst Bacteriol 34 (1984), 45-55] were suggested previously to belong to the genus Pantoea, but have never been formally described and classified. Additionally, it has been shown in several studies that Pectobacterium cypripedii is more closely related to species of Pantoea than to those of Pectobacterium. In this study, the phylogenetic positions of Brenner's DNA HG II, IV and V and Pectobacterium cypripedii were re-examined by both 16S rRNA gene sequencing and multilocus sequence analyses (MLSA) based on the gyrB, rpoB, atpD and infB genes. The analyses revealed that DNA HG II, IV and V and Pectobacterium cypripedii form five separate branches within the genus Pantoea (strains from HG V were split into two branches). DNA-DNA hybridization data further confirmed that DNA HG II, IV and V constitute four separate species. Pectobacterium cypripedii was shown to be a close phylogenetic relative of Pantoea dispersa and DNA HG IV by both 16S rRNA gene sequence and MLSA analyses. Biochemical analyses performed on strains from DNA HG II, IV and V and Pectobacterium cypripedii confirmed their taxonomic position within the genus Pantoea and revealed phenotypic characteristics that allow the differentiation of these species from each other and from their closest phylogenetic neighbours. It is proposed to emend the description of the genus Pantoea and to describe Pantoea septica sp. nov. for DNA HG II (type strain LMG 5345(T) =BD 874(T) =CDC 3123-70(T)), Pantoea eucrina sp. nov. for DNA HG IV (type strain LMG 2781(T) =BD 872(T) =CDC 1741-71(T) =LMG 5346(T)), Pantoea brenneri sp. nov. for strains of DNA HG V excluding LMG 24534 (type strain LMG 5343(T) =BD 873(T) =CDC 3482-71(T)) and Pantoea conspicua sp. nov. for the remaining strain of DNA HG V (type strain LMG 24534(T) =BD 805(T) =CDC 3527-71(T)) and to transfer Pectobacterium cypripedii to the genus as Pantoea cypripedii comb. nov. (type strain LMG 2657(T) =ATCC 29267(T) =DSM 3873(T) =LMG 2655(T)).

Pantoea citrea, Pantoea punctata and Pantoea terrea were described for strains isolated from fruit and soil originating in Japan. These three 'Japanese' species have been shown to be phylogenetically distant from other species of the genus Pantoea. It has been observed previously that, using multilocus sequence analysis (MLSA), the 'Japanese' species consistently formed a distinct clade with an extended branch length, casting doubt on the inclusion of these species within the genus Pantoea. Furthermore, the 'Japanese' species are closely related to Tatumella ptyseos, strains of which originate from human clinical specimens. DNA-DNA hybridization and phenotypic tests confirmed the observed phylogenetic distance of P. citrea, P. punctata and P. terrea from the genus Pantoea and the affiliation of these species with Tatumella. In addition, strains causing pink disease of pineapple, identified previously as P. citrea , were shown to represent a separate species by using 16S rRNA gene sequence analysis, and MLSA and DNA-DNA hybridization data. The name Tatumella morbirosei sp. nov. with the type strain LMG 23360(T) (=BD 878(T)=NCPPB 4036(T)=CMC6(T)) is proposed to accommodate these strains. The new combinations Tatumella citrea (Kageyama et al. 1992) comb. nov. (type strain, SHS 2003(T)=ATCC 31623(T)=BD 875( T)=CCUG 30156(T)=CIP 105599(T)=DSM 13699(T)=JCM 8882(T)=LMG 22049(T)), Tatumella punctata (Kageyama et al. 1992) comb. nov. (type strain, SHS 2006(T)=ATCC 31626(T)=BD 876( T)=CCUG 30159(T)=CIP 105598(T)=DSM 13700(T)=JCM 8885(T)=LMG 22050(T)) and Tatumella terrea (Kageyama et al. 1992) comb. nov. (type strain, SHS 2008(T)=ATCC 31628(T)=BD 877(T)=CCUG 30161(T)=CIP 105600(T)=DSM 13701(T)=JCM 8887(T)=LMG 22051(T)) are proposed for P. citrea, P. punctata and P. terrea , respectively.

Bacteria isolated from eucalyptus leaves and shoots showing symptoms of blight and die-back collected in Uganda, Uruguay and Argentina and from maize displaying brown stalk rot symptoms in South Africa were tentatively placed in the genus Pantoea on the basis of phenotypic and biochemical tests. These isolates, together with two strains (LMG 2558 and LMG 2560) previously assigned to Pantoea agglomerans based on protein electrophoregrams but later excluded from this species, were further investigated using molecular techniques. 16S rRNA gene sequencing and multilocus sequence analyses (MLSA) revealed that the strains were phylogenetically closely related to Pantoea agglomerans, Pantoea stewartii and Pantoea ananatis. MLSA and amplified fragment length polymorphism analysis placed the strains into four separate clusters, not containing any of the type strains of species of the genus Pantoea. DNA-DNA hybridization confirmed the classification of the isolates into four novel species, for which the names Pantoea vagans sp. nov. (type strain R-21566T=LMG 24199T=BCC 105T=BD 765T), Pantoea eucalypti sp. nov. (type strain R-25678T=LMG 24197T=BCC 076T=BD 769T), Pantoea deleyi sp. nov. (type strain R-31523T=LMG 24200T=BCC 109T=BD 767T) and Pantoea anthophila sp. nov. (type strain LMG 2558T=BD 871T=NCPPB 1682T) are proposed.