Erwinia piriflorinigrans sp. nov., a novel pathogen that causes necrosis of pear blossoms.

Eight Erwinia strains, isolated from necrotic pear blossoms in València, Spain, were compared with reference strains of Erwinia amylovora and Erwinia pyrifoliae, both of which are pathogenic to species of pear tree, and to other species of the family Enterobacteriaceae using a polyphasic approach. Phenotypic analyses clustered the novel isolates into one phenon, distinct from other species of the genus Erwinia, showing that the novel isolates constituted a homogeneous phenotypic group. Rep-PCR profiles, PCR products obtained with different pairs of primers and plasmid contents determined by restriction analysis showed differences between the novel strains and reference strains of E. amylovora and E. pyrifoliae. Phylogenetic analysis of 16S rRNA, gpd and recA gene sequences showed that the eight novel strains could not be assigned to any recognized species. On the basis of DNA-DNA hybridization studies, the novel isolates constituted a single group with relatedness values of 87-100  % to the designated type strain of the group, CFBP 5888(T). Depending on the method used, strain CFBP 5888(T) showed DNA-DNA relatedness values of between 22.7 and 50  % to strains of the closely related species E. amylovora and E. tasmaniensis. The DNA G+C contents of two of the novel strains, CFBP 5888(T) and CFBP 5883, were 51.1 and 50.5 mol%, respectively. On the basis of these and previous results, the novel isolates represent a novel species of the genus Erwinia, for which the name Erwinia piriflorinigrans sp. nov. is proposed. The type strain is CFBP 5888(T) (=CECT 7348(T)).

BOX-PCR-based identification of bacterial species belonging to Pseudomonas syringae: P. viridiflava group

The phenotypic characteristics and genetic fingerprints of a collection of 120 bacterial strains, belonging to Pseudomonas syringae sensu lato group, P. viridiflava and reference bacteria were evaluated, with the aim of species identification. The numerical analysis of 119 nutritional characteristics did not show patterns that would help with identification. Regarding the genetic fingerprinting, the results of the present study supported the observation that BOX-PCR seems to be able to identify bacterial strains at species level. After numerical analyses of the bar-codes, all pathovars belonging to each one of the nine described genomospecies were clustered together at a distance of 0.72, and could be separated at genomic species level. Two P. syringae strains of unknown pathovars (CFBP 3650 and CFBP 3662) and the three P. syringae pv. actinidiae strains were grouped in two extra clusters and might eventually constitute two new species. This genomic species clustering was particularly evident for genomospecies 4, which gathered P. syringae pvs. atropurpurea, coronafaciens, garçae, oryzae, porri, striafaciens, and zizaniae at a noticeably low distance.

Nitrogen-fixing sinorhizobia with Medicago laciniata constitute a novel biovar (bv. medicaginis) of S. meliloti.

Sixty-eight new rhizobial isolates were obtained from root-nodules of Medicago laciniata and from Mediterranean soils in Tunisia and France. All of them were identified as Sinorhizobium meliloti on the basis of PCR-RFLP analyses of 16S rDNA and the intergenic spacer sequence between 16S and 23S rDNAs. DNA/DNA hybridization, phenotypic characterization and 16S rRNA gene sequencing led to the conclusion that they belong the same taxon. All new isolates shared the ability to nodulate and fix nitrogen with M. laciniata except 11 of them not capable of fixing nitrogen with this plant and originating from French soils containing no efficiently adapted symbionts with M. laciniata. The nitrogen-fixing rhizobia on M. laciniata differed markedly from the other S. meliloti or Sinorhizobium medicae isolates and references in their symbiotic traits such as nifDK RFLP diversity, nodA sequences and nitrogen effectiveness with tree other different annual Medicago species (M. truncatula, M. polymorpha and M. sauvagei). Two infrasubspecific (biovar) divisions are therefore proposed within S. meliloti: bv. medicaginis for Sinorhizobium efficient on M. laciniata and bv. meliloti for the classically known S. meliloti group represented by the strains ATCC9930(T) and RCR 2011 efficient on M. sativa.

Transfer of Pectobacterium chrysanthemi (Burkholder et al. 1953) Brenner et al. 1973 and Brenneria paradisiaca to the genus Dickeya gen. nov. as Dickeya chrysanthemi comb. nov. and Dickeya paradisiaca comb. nov. and delineation of four novel species, Dickeya dadantii sp. nov., Dickeya dianthicola sp. nov., Dickeya dieffenbachiae sp. nov. and Dickeya zeae sp. nov.

A collection of 75 strains of Pectobacterium chrysanthemi (including all biovars and pathovars) and the type strains of Brenneria paradisiaca (CFBP 4178(T)) and Pectobacterium cypripedii (CFBP 3613(T)) were studied by DNA-DNA hybridization, numerical taxonomy of 121 phenotypic characteristics, serology and 16S rRNA gene-based phylogenetic analyses. From analysis of 16S rRNA gene sequences, it was deduced that P. chrysanthemi strains and B. paradisiaca CFBP 4178(T) formed a clade distinct from the genera Pectobacterium and Brenneria; therefore, it is proposed to transfer all the strains to a novel genus, Dickeya gen. nov. By DNA-DNA hybridization, the strains of P. chrysanthemi were distributed among six genomic species: genomospecies 1 harbouring 16 strains of biovar 3 and four strains of biovar 8, genomospecies 2 harbouring 16 strains of biovar 3, genomospecies 3 harbouring two strains of biovar 6 and five strains of biovar 5, genomospecies 4 harbouring five strains of biovar 2, genomospecies 5 harbouring six strains of biovar 1, four strains of biovar 7 and five strains of biovar 9 and genomospecies 6 harbouring five strains of biovar 4 and B. paradisiaca CFBP 4178(T). Two strains of biovar 3 remained unclustered. Biochemical criteria, deduced from a numerical taxonomic study of phenotypic characteristics, and serological reactions allowed discrimination of the strains belonging to the six genomic species. Thus, it is proposed that the strains clustered in these six genomic species be assigned to the species Dickeya zeae sp. nov. (type strain CFBP 2052(T)=NCPPB 2538(T)), Dickeya dadantii sp. nov. (type strain CFBP 1269(T)=NCPPB 898(T)), Dickeya chrysanthemi comb. nov. (subdivided into two biovars, bv. chrysanthemi and bv. parthenii), Dickeya dieffenbachiae sp. nov. (type strain CFBP 2051(T)=NCPPB 2976(T)), Dickeya dianthicola sp. nov. (type strain CFBP 1200(T)=NCPPB 453(T)) and Dickeya paradisiaca comb. nov., respectively.

Erwinia papayae sp. nov., a pathogen of papaya (Carica papaya).

Bacterial canker of papaya (Carica papaya) emerged during the 1980s in different islands of the Caribbean. Nineteen strains of Gram-negative, rod-shaped, non-spore-forming bacteria isolated from papaya were compared to 38 reference and type strains of phytopathogenic Enterobacteriaceae and related bacteria. Phylogenetic analysis of 16S rRNA gene sequences showed that the papaya strains belonged to the genus Erwinia. The DNA G+C content of strain CFBP 5189T, 52.5 mol%, is in the range of the genus Erwinia. The 19 papaya strains were all pathogenic to papaya and were differentiated clearly from type or reference strains of phytopathogenic enterobacteria and related bacteria by phenotypic tests. The papaya strains constituted a discrete DNA hybridization group, indicating that they belonged to a unique genomic species. Thus, strains pathogenic to papaya belong to a novel species for which the name Erwinia papayae sp. nov. is proposed, with the type strain CFBP 5189T (=NCPPB 4294T).

Acidovorax valerianellae sp. nov., a novel pathogen of lamb's lettuce [Valerianella locusta (L.) Laterr].

Bacterial spot disease of lamb's lettuce [Valerianella locusta (L.) Laterr.] was first observed in fields in 1991. This new bacterial disease is localized in western France in high-technology field production of lamb's lettuce for the preparation of ready-to-use salad. Nineteen strains isolated in 1992 and 1993 from typical black leaf spots of naturally infected lamb's lettuce were characterized and compared with reference strains of Acidovorax and Delftia. The pathogenicity of the 19 strains was confirmed by artificial inoculation. Biochemical and physiological tests, fatty acid profiles, DNA-DNA hybridization and other nucleic acid-based tests were performed. A numerical taxonomic analysis of the 19 lamb's lettuce strains showed a single homogeneous phenon closely related to previously described phytopathogenic taxa of the genus Acidovorax. DNA-DNA hybridization studies showed that the lamb's lettuce strains were 91-100% related to a representative strain, strain CFBP 4730(T), and constituted a discrete DNA hybridization group, indicating that they belong to the same novel species. Results from DNA-rRNA hybridization, 16S rRNA sequence analysis and fatty acid analysis studies confirmed that this novel species belongs to the beta-subclass of the Proteobacteria and, more specifically, to the family Comamonadaceae and the genus Acidovorax. The name Acidovorax valerianellae sp. nov. is proposed for this novel taxon of phytopathogenic bacteria. The type strain is strain CFBP 4730(T) (= NCPPB 4283(T)).

Elevation of three subspecies of Pectobacterium carotovorum to species level: Pectobacterium atrosepticum sp. nov., Pectobacterium betavasculorum sp. nov. and Pectobacterium wasabiae sp. nov.

A collection of 42 strains belonging to the five subspecies of Pectobacterium carotovorum (subspecies atrosepticum, betavasculorum, carotovorum, odoriferum and wasabiae) and 11 reference and type strains of biovars of Pectobacterium chrysanthemi, Pectobacterium cacticidum and Brenneria paradisiaca were studied by DNA-DNA hybridization, numerical taxonomy of 120 phenotypic characteristics, serology and new phylogenetic analysis of previously reported sequences from a database of aligned 16S rDNA sequences. The P. carotovorum subspecies formed a clade according to neighbour-joining methods, but they formed two paraphyletic clusters according to maximum-likelihood and maximum-parsimony. However, phylogenetic analysis of 16S rDNA sequences alone is not sufficient to justify generic differentiation and therefore, it is proposed to retain the P. carotovorum subspecies in the genus Pectobacterium. The strains of P. carotovorum were distributed in four genomospecies: genomospecies 1, harbouring all strains of subsp. atrosepticum, genomospecies 2, including the strains of subsp. betavasculorum isolated from sugar beet, sunflower, potato, hyacinth and artichoke, genomospecies 3, clustering all strains of subsp. wasabiae isolated from wasabi in Japan, and genomospecies 4, gathering together strains of subsp. carotovorum and strains of subsp. odoriferum. Four strains of P. carotovorum subsp. carotovorum remained unclustered. Biochemical criteria, deduced from a numerical taxonomy study of phenotypic characteristics and serological reactions, allowed discrimination of strains belonging to the four genomospecies. Thus, it is proposed that three genomospecies be elevated to species level as Pectobacterium atrosepticum sp. nov. (type strain CFBP 1526T =LMG 2386T =NCPPB 549T =ICMP 1526T), Pectobacterium betavasculorum sp. nov. (type strain CFBP 2122T =LMG 2464T =NCPPB 2795T =ICMP 4226T) and Pectobacterium wasabiae sp. nov. (type strain CFBP 3304T =LMG 8404T =NCPPB 3701T =ICMP 9121T). Only two subspecies are maintained within P. carotovorum, subsp. carotovorum (type strain CFBP 2046T =LMG 2404T =NCPPB 312T =ICMP 5702T) and subsp. odoriferum (type strain CFBP 1878T =LMG 5863T =NCPPB 3839T = ICMP 11553T), for which discriminating tests are available.

Phenotypic and genomic evidence for the revision of Pseudomonas corrugata and proposal of Pseudomonas mediterranea sp. nov.

To re-examine the taxonomic status of Pseudomonas corrugata, 27 strains of this species were studied using a polyphasic approach. Numerical analysis of phenotypic data revealed two phena, A (including the P. corrugata type strain) and B, which could be clearly differentiated by the assimilation of mesotartrate, 2-ketogluconate and histamine. The mean DNA reassociation values with labelled DNA of P. corrugata type strain CFBP 2431T (phenon A) and strain CFBP 5447T (phenon B) were high for strains belonging to the same phenon (96.9 and 98.5%, respectively), whereas the DNA relatedness between the two phena was assessed as being close to 70%, which represents the value that is accepted for the definition of a bacterial species. Phena A and B were also differentiated by means of DNA profiles generated by heteroduplex mobility assay of PCR products of 16S rDNA hypervariable region 2, HaeIII restriction of the amplified internal transcribed spacer, REP- and BOX-PCR profiles, and by PCR with two pairs of specific primers. A comparison of the 16S rRNA sequences of strains CFBP 5447T and CFBP 5458 from phenon B with the available sequences of Pseudomonas species showed that these strains formed a cluster distinct from the P. corrugata type strain. Thus, a new species, Pseudomonas mediterranea, is proposed for strains of phenon B. The type strain is strain CFBP 5447T (= ICMP 14184T); its G+C content is 60.2 mol%.

Siderophore typing, a powerful tool for the identification of fluorescent and nonfluorescent pseudomonads.

A total of 301 strains of fluorescent pseudomonads previously characterized by conventional phenotypic and/or genomic taxonomic methods were analyzed through siderotyping, i.e., by the isoelectrophoretic characterization of their main siderophores and pyoverdines and determination of the pyoverdine-mediated iron uptake specificity of the strains. As a general rule, strains within a well-circumscribed taxonomic group, namely the species Pseudomonas brassicacearum, Pseudomonas fuscovaginae, Pseudomonas jessenii, Pseudomonas mandelii, Pseudomonas monteilii, "Pseudomonas mosselii," "Pseudomonas palleronii," Pseudomonas rhodesiae, "Pseudomonas salomonii," Pseudomonas syringae, Pseudomonas thivervalensis, Pseudomonas tolaasii, and Pseudomonas veronii and the genomospecies FP1, FP2, and FP3 produced an identical pyoverdine which, in addition, was characteristic of the group, since it was structurally different from the pyoverdines produced by the other groups. In contrast, 28 strains belonging to the notoriously heterogeneous Pseudomonas fluorescens species were characterized by great heterogeneity at the pyoverdine level. The study of 23 partially characterized phenotypic clusters demonstrated that siderotyping is very useful in suggesting correlations between clusters and well-defined species and in detecting misclassified individual strains, as verified by DNA-DNA hybridization. The usefulness of siderotyping as a determinative tool was extended to the nonfluorescent species Pseudomonas corrugata, Pseudomonas frederiksbergensis, Pseudomonas graminis, and Pseudomonas plecoglossicida, which were seen to have an identical species-specific siderophore system and thus were easily differentiated from one another. Thus, the fast, accurate, and easy-to-perform siderotyping method compares favorably with the usual phenotypic and genomic methods presently necessary for accurate identification of pseudomonads at the species level.

Pseudomonas lini sp. nov., a novel species from bulk and rhizospheric soils.

The taxonomic position of eight fluorescent Pseudomonas strains isolated from bulk and rhizospheric soils, and from water was examined. These eight strains clustered in one phenon together with Pseudomomas mandelii (CFBP 4844T), but could still be differentiated from this type strain by four phenotypic features. The eight stains exhibited internal DNA-DNA hybridization values ranging from 60 to 100%, with deltaTm below 5 degrees C (3.9 and 4.3 degrees C) for the lowest values (60 and 66%). The percentages of hybridization with type or reference strains of other Pseudomonas species tested ranged from 12 to 60% (deltaTm = 5.5 degrees C), indicating that the eight isolates studied constituted a discrete DNA homology group. Comparison of the 16S rDNA sequence of the strain representing this group (CFBP 5737T) with the sequences of other strains belonging to the genus Pseudomonas revealed that strain CFBP 5737T was a member of this genus and that these bacteria did not cluster with any previously described species of the genus Pseudomonas. The eight isolates belonged to two siderovars different from those described so far. On the basis of the results of phenotypic, DNA-DNA and phylogenetic analyses, and of siderotyping, a new species, Pseudomonas lini sp. nov. (type strain CFBP 5737T) is proposed.

Pseudomonas costantinii sp. nov., another causal agent of brown blotch disease, isolated from cultivated mushroom sporophores in Finland.

Pathogenic bacteria are frequently associated with mushroom sporophores exhibiting brown blotch disease symptoms. These bacteria belong mainly to Pseudomonas tolaasii or occasionally to 'Pseudomonas reactans'. Although a group of isolates originating from some Finnish mushroom farms satisfied the two characteristic criteria for diagnosis of infection with P. tolaasii (i.e. yielding a typical brown blotch symptom on Agaricus bisporus sporophores and producing a typical white line in agar when streaked towards the 'P. reactans' LMG 5329 inducing strain), results based on numerical taxonomy, siderotyping, DNA-DNA hybridizations and 16S rDNA phylogenetic analyses supported the view that these isolates constituted a novel species within the genus Pseudomonas, Pseudomonas costantinii. The type strain is PS 3a(T) (= CFBP 5705(T) = HAMBI 2444(T)).

Pseudomonas salomonii sp. nov., pathogenic on garlic, and Pseudomonas palleroniana sp. nov., isolated from rice.

A total of 26 strains, including 15 strains isolated from garlic plants with the typical symptoms of 'Café au lait' disease and 11 strains isolated from diseased or healthy rice seeds and sheaths infested by Pseudomonas fuscovaginae, were compared with 70 type or reference strains of oxidase-positive pathogenic or non-pathogenic fluorescent pseudomonads. The strains were characterized by using a polyphasic taxonomic approach. Numerical taxonomy of phenotypic characteristics showed that the garlic and rice strains were related to each other. However, they clustered into separate phenons, distinct from those of the other strains tested, and were different in several nutritional tests. On the basis of DNA-DNA hybridization, the garlic and rice strains constituted two distinct DNA hybridization groups, indicating that they belonged to separate species. The two groups of strains were also well differentiated by siderotyping. Garlic strains were pathogenic to garlic plants and either weakly pathogenic or non-pathogenic on rice; rice strains were either weakly pathogenic or non-pathogenic on rice and non-pathogenic on garlic. A phylogenetic analysis of 16S rRNA gene sequences confirmed that the two groups of strains belonged to the y-Proteobacteria and to the genus Pseudomonas. The names Pseudomonas salomonii sp. nov. and Pseudomonas palleroniana sp. nov. are respectively proposed for the garlic strains and the rice strains. The type strains are P. salomonii CFBP 2022(T) ( = ICMP 14252(T) = NCPPB 4277(T)) and P. palleroniana CFBP 4389(T) (= ICMP 14253(T) = NCPPB 4278(T)).