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1 UMR de Pathologie Végétale INRA-INH-UNIVERSITE, BP 57, 42 rue G. Morel, 49071 Beaucouzé, France
2 UMR 6543 CNRS and Université de Nice Sophia Antipolis, Centre de biochimie, Parc Valrose, 06108 Nice cedex 2, France
3 CEA/Cadarache, DSV-DEVM, LEMIR, UMR 163 CNRS-CEA-Univ. Méditerranée, Saint Paul-lez-Durance, France
4 INRA, Station de Pathologie Végétale, Domaine St Maurice, BP 94, 84143, Montfavet, France
Correspondence
Louis Gardan
gardan{at}angers.inra.fr
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Published online ahead of print on 4 July 2003 as DOI 10.1099/ijs.0.02718-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of CFBP 5189T is AY131237.
A full phylogenetic tree is available as supplementary material in IJSEM Online.
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) was identified; this bacterium was further typed to the genus Erwinia by Magrou (1937). Nelson & Alvarez (1980) described a purple stain of Carica papaya, due to Erwinia herbicola. More recently, Trujillo & Schroth (1982) reported a bacterial decline of papaya caused by two Erwinia species in the Mariana Islands. Finally, reports from Webb (1985) in the US Virgin Islands, from Frossard et al. (1985) and Prior et al. (1985) in the French West Indies and from Guevara et al. (1993) in Venezuela, all consistently described this disease, which was named ‘bacterial stem canker’. Typical symptoms of bacterial stem cankers on Solo-type cultivars include greasy, water-soaked lesions and spots on leaves, which evolve into foliar, angular lesions. Firm, water-soaked cankers develop on the stem, sometimes leading to the destruction of papaya trees (Prior et al., 1985; Webb, 1985).
Preliminary identification of this novel bacterium by classical phenotypic tests led Trujillo & Schroth (1982), Frossard et al. (1985), Prior et al. (1985) and Webb (1985) to place this bacterium in the genus Erwinia and in the Amylovora group according to Dye (1968). In order to clarify the taxonomic status of this phytopathogenic bacterium, we developed a polyphasic taxonomic study.
Gram-negative bacteria were isolated from water-soaked lesions and were typically slow-growing, giving colonies of 2–3 mm diameter after 3–4 days incubation. Colonies were hyaline on YBGA (0·7 % yeast extract, 0·7 % bactopeptone, 0·7 % glucose and 1·5 % agar, pH 7·2) or on King's medium B at 25 °C, with a mucoid aspect and white to creamy white colour after 2–3 days. A non-diffusible blue pigment was observed on King's medium B.
This study included a selection of 19 strains from a collection of 72 strains that were isolated in 1982–1991 from Carica papaya in different islands of the Caribbean (Table 1) and 38 type or reference strains of phytopathogenic Enterobacteriaceae that belong to the genera Erwinia, Enterobacter, Brenneria, Pantoea, Pectobacterium and Samsonia.
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We used 121 phenotypic tests, including 22 conventional tests and assimilation of 99 carbon sources by using Biotype 100 strips (bioMérieux), as described previously (Gardan et al., 2002). A distance matrix was calculated by using the Jaccard coefficient and cluster analysis was done by using UPGMA (Sneath & Sokal, 1973). Discriminative tests were selected by using a diagnostic ability coefficient that was deduced from the numerical analysis (Descamp & Véron, 1981).
Selection of related sequences was performed according to previous phylogenetic analyses of a database of 62 000 already aligned bacterial 16S rRNA gene sequences and BLAST searches against the latest release of EBI (European Bioinformatics Institute). The new sequence was aligned manually by using SeaView (Galtier et al., 1996). In a preliminary analysis, 150 sequences were selected. A phylogenetic tree of 32 sequences was then built, including a sequence for each type strain of species of the genera Erwinia and Pantoea that were available, as well as representatives of the genera Brenneria, Pectobacterium and Samsonia. Sequences from three Pantoea species that do not yet have validly published names (‘Pantoea oleae’, ‘Pantoea cedenensis’ and ‘Pantoea toletana’) were also included for completeness of the analysis. Phylogenetic trees were then constructed by using three different methods: neighbour-joining (NJ) by using BIONJ (Gascuel, 1997), maximum-likelihood (ML) and maximum-parsimony (MP). For the BIONJ analysis, distance matrices were calculated by using Kimura's two-parameter correction. ML and MP were from PHYLIP (Phylogeny Inference Package, version 3.573c; distributed by J. Felsenstein, Department of Genetics, UW, Seattle, WA, USA). Almost-entire sequences that corresponded to positions 74–1436 of strain CFBP 5189T were used for these analyses (shown in Fig. 1 and Supplementary Fig. A in IJSEM Online). Phylogenetic trees were drawn by using NJPLOT (Perrière & Gouy, 1996). The topology shown is that of the bootstrap tree, as it has been demonstrated that this topology is often better than that of a simple tree (Berry & Gascuel, 1996). There is no distance bar in Fig. 1; it would be meaningless as (i) distances are corrected (see above) and (ii) this is a bootstrap tree.
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. Native DNA of strain CFBP 5189T was labelled in vitro by nick-translation with tritium-labelled nucleotides (Amersham Biosciences). The S1 nuclease/trichloroacetic method was used for DNA–DNA hybridization (Crosa et al., 1973). DNA reassociation was performed at 65 °C.
The DNA G+C content of strain CFBP 5189T was determined by the thermal denaturation method of Marmur & Doty (1962) and calculated by using the equation of Owen & Lapage (1976).
Isolates that gave a positive reaction induced water-soaked, greasy spots around the inoculation point, 5–7 days after inoculation. These water-soaked lesions developed into stem cankers, mainly due to secondary invaders. Death occurred about 3 weeks after inoculation. Thus, all symptoms observed under field conditions were reproduced and the original bacteria could be reisolated consistently as pure cultures from water-soaked necroses.
A dendrogram of phenotypic distances among the 57 strains is shown in Fig. 2. At a distance of 0·419, six phenons and four isolated strains were delineated. Phenon 4 corresponded to the 19 strains isolated from papaya and Erwinia mallotivora CFBP 2503T. Phenons 1, 2, 3, 5 and 6, each of which contained between two and nine strains, corresponded to type and reference strains of phytopathogenic Enterobacteriaceae. Phenotypic characteristics that differentiate the papaya strains from other reference strains are shown in Table 2. Numerous tests are available to identify the papaya strains, as they used only some carbon sources in comparison with most other strains characterized in this study (an exception is Erwinia tracheiphila). Like Erwinia amylovora, this novel bacterium requires growth factors; Prior et al. (1985) supplemented growth media with yeast extract. The papaya strains are differentiated clearly from E. mallotivora by their ability to assimilate L-(+)-arabinose and D-glucosamine, but not mannitol or DL-lactate, and to reduce sucrose compounds; the reverse reactions were obtained for E. mallotivora (Table 2). Strain-dependent reactions for papaya strains were observed for utilization of cis-aconitate (four strains), mucate (five strains), galacturonate (11 strains) and citrate (five strains). Phenon 4, which contained the papaya strains, was split into two subphena by E. mallotivora. Strains of different origins (several islands) are clustered in the same subphenon and, conversely, strains of one origin (one island, e.g. Guadeloupe, France) are clustered in different subphena; thus, we did not observe a correlation between geographical origin and clustering in the two subphena. Papaya strains evolved all over the Caribbean, which is also the diversification centre for Carica papaya; such restricted environments are well-known to develop particular traits in terms of population genetics, but we have no likely hypothesis to explain why the papaya strains were split into two subphena.
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) demonstrated that this species had to be reassigned as Pantoea agglomerans, as supported by our phylogenetic analysis. In conclusion, the novel bacterium belongs phylogenetically to the genus Erwinia; DNA–DNA hybridization data are necessary to decide whether or not it constitutes a distinct species.
DNA–DNA reassociation results are shown in Table 3. The 19 papaya strains were 67–100 % related to strain CFBP 5189T (mean, 82·3±10·2 %). E. mallotivora CFBP 2503T was 44 % related to strain CFBP 5189T and was the most closely related organism to the papaya strains. The rest of the type and reference strains of phytopathogenic Enterobacteriaceae were 2–18 % related to strain CFBP 5189T. Thus, the papaya strains constitute a homogeneous genomic group at the species level.
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).
All these results correspond to the definition of bacterial species according to Wayne et al. (1987) and we propose the name Erwinia papayae sp. nov. for this novel bacterium that is pathogenic to Carica papaya.
Description of Erwinia papayae sp. nov.
Erwinia papayae (pa.pa'yae. N.L. gen. n. papayae of papaya, the host plant from which the organism was isolated).
This species has the characteristics of the genus Erwinia. Colonies on YBGA are hyaline and 2–3 mm in size after 3–4 days incubation at 25 °C. A non-diffusible blue pigment is produced on King's medium B. Growth occurs at 28 °C, but not at 36 or 39 °C; sucrose-reducing compounds are produced. In addition to the characteristics mentioned in Table 2
, 
-D-(+)-fructose, D-(+)-galactose, D-(+)-mannose, sucrose, D-(-)-ribose, glycerol, N-acetylglucosamine and D-gluconate are utilized; strains do not assimilate 73 of 100 carbon sources of the Biotype 100 gallery (bioMérieux). DNA G+C content of the type strain is 52·5 mol%.
The type strain is CFBP 5189T (=NCPPB 4294T). Strains of Erwinia papayae cause bacterial canker of papaya.
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Brenner, D. J., McWhorter, A. C., Knutson, J. K. & Steigerwalt, A. G. (1982). Escherichia vulneris: a new species of Enterobacteriaceae associated with human wounds. J Clin Microbiol 15, 1133–1140.
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Gardan, L., Gouy, C., Christen, R. & Samson, R. (2002). Elevation of three subspecies of Pectobacterium carotovorum to species level: Pectobacterium atrosepticum sp. nov., Pectobacterium betavasculorum sp. nov. and Pectobacterium wasabiae sp. nov. Int J Syst Evol Microbiol 53, 381–391.
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Webb, R. R. (1985). Epidemiology and control of bacterial canker of papaya caused by an Erwinia sp. on St Croix, US Virgin Islands. Plant Dis 69, 305–309.
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