Phylogenetic Position of Phytopathogens within the Enterobacteriaceae

System. App!. Microbia!. 21, 384-397 (1998) SYSTEI\I1ATIC AND _©_G_us_ta_v F_is_ch_er_¥_er_Iag ________________ APPLIED MICROBIOLOGY

Phylogenetic Position of Phytopathogens within the Enterobacteriaceae

LYSIANE HAUBEN1,2, EDWARD R. B. MOORE2, Luc VAUTERINl, MARIJKE STEENACKERS3, JORIS MERGAERT1,

LINDA VERDONCK1, and JEAN SWINGS2

lLaboratorium va or Microbiologie, Universiteit Gent, Belgium 2Division of Microbiology, National Research Centre for Biotechnology (GBF), Braunschweig, Germany 3Institute for Forestry and Game Management (lEW), Geraardsbergen, Belgium

Received February 15, 1998

Summary

The almost complete 16S rDNA sequences of twenty nine plant-associated strains, representing species of the genera Erwinia, Pantoea and Enterobacter were determined and compared with those of other members of the Enterobacteriaceae. The species of the genus Erwinia may be divided into three phylogenetic groups. Cluster I represents the true erwinias and comprises E. amylovora, E. maliotivora, E. persicinus, E. psidii, E. rhapontici and E. tracheiphila. We propose to unite the species of cluster II, E. carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, E. carotovora subsp. carotovora, E. carotovora subsp. odorifera, E. carotovora subsp. wasabiae, E. cacticida, E. chrysanthemi and E. cypripedii in the genus Pectobacterium respectively as P. carotovorum subsp. atrosepticum comb. nov., P. carotovorum subsp. betavasculorum comb. nov., P. carotovorum subsp. carotovorum comb. nov., P. carotovorum subsp. odoriferum comb. nov., P. carotovorum subsp. wasabiae comb. nov., P. cacticidum comb. nov., P. chrysanthemi and P. cypripedii. The species E. alni, E. nigrif/uens, E. paradisiaca, E. quercina, E. rubrifaciens and E. salicis, comprising cluster III, are being classified into a new genus Brenneria gen. nov. respectively as B. alni comb. nov., B. nigrif/uens comb. nov., B. paradisiaca comb. nov., B. quercina comb. nov., B. rubrifaciens comb. nov. and B. salicis comb. nov. The species of the genus Pantoea, included in this study, form a monophyletic unit (cluster IV), closely related with Erwinia, whereas the three phytopathogenic species of the genus Enterobacter are scattered among the genera Citrobacter and Klebsiella.

Keywords: Phytopathogenic Enterobacteriaceae - 16S rDNA - Nucleotide sequence - Phylogeny - Pectobacterium - Brenneria

Introduction

Phytopathogenic Enterobacteriaceae of the genera Erwinia, Pantoea and Enterobacter cause diseases of trees, flowers, potatoes, fruit and vegetables (Table 1), implying quantitative and/or qualitative crop losses.

The genus Erwinia, named after the phytobacteriologist Erwin F. Smith, was founded in 1920 to unite all gram negative, fermentative, non sporulating, peritrichous flagellated plantpathogenic bacteria (WINSLOW et aI., 1920). In Bergey's Manual of Systematic Bacteriology 15 species and three subspecies are described in the genus Erwinia: E. amylovora, E. ananas, E. carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, E. carotovora subsp. carotovora, E. chrysanthemi, E. cypripedii, E. herbicola, E. mallotivora, E. nigri{luens, E. quercina, E. rhapontici, E. rubrifaciens, E. salicis, E. stewartii, E. tracheiphila and E. uredovora (LEL-

LIOTT and DICKEY, 1984). The species incertae sedis E. cancerogena (DICKEY and ZUMOFF, 1988), E. dissolvens and E. nimipressuralis (BRENNER et aI., 1986) have been transferred to the genus Enterobacter, which formerly consisted only of non-plant associated species. The genus Pantoea was established in 1989 and harbours the plant pathogenic species formerly classified as E. herbicola, E. milletiae (GAVINI et aI., 1989), E. ananas, E. uredovora and E. stewartii (MERGAERT et aI., 1993). In the last ten years, four new Erwinia species and two new subspecies were created: E. alni (SURICO et aI., 1996), E. cacticida (ALCORN et aI., 1991), E. persicinus (HAo et aI., 1990), E. psidii (NETO et aI., 1987), E. carotovora subsp. odorifera (GALLOIS et aI., 1992) and E. carotovora subsp. wasabiae (GOTO and MATSUMOTO, 1987).

The genus Pectobacterium was first proposed in 1945 to include the pectolytic Enterobacteriaceae (WALDEE, 1945). In this proposal, the current taxa P. carotovorum subsp. atrosepticum and P. carotovorum subsp. carotovorum together with P. cypripedii belonged to this genus. BRENNER (1973) supported this concept based on the different metabolic patterns exhibited by Erwinia sensu stricto and Pectobacterium. He found that the two genera exhibit different types of disease and show higher intragroup DNA relatedness. Based on DNA hybridization data, he included also E. chrysanthemi and E. rhapontici in the genus Pectobacterium. This proposal was not followed in Bergey's Manual of Systematic Bacteriology (LELLIOTT and DICKEY, 1984). The genus name Pectobacterium, together with the species names P. carotovorum, P. chrysanthemi, P. cypripedii, and P. rhapontici, figure in the Approved Lists of Bacterial Names (SKERMAN et al., 1980).

Phenotypic characteristics of Erwinia, i.e. colony and cell morphology, biochemical and serological characteristics, protein profile patterns and fatty acid profiles have been applied in the classification of the genus (COTHER et al., 1992; VERDONCK et al., 1987; MERGAERT et al., 1984). Besides % (G+C) content and DNA:DNA hybridizations (GOTO and MATSUMOTO, 1987; GAVINI et al., 1989; BRENNER et al., 1973, 1974), little genotypic work has been done, except for the species E. chrysanthemi (COTHER et al., 1992) and E. amylovora (MOMOL et al., 1997), which have been studied in more detail by DNA fingerprinting. Modern taxonomy is situated ideally within a polyphasic framework, with the 16S rDNA sequence-based phylogeny as a backbone. The accessibility towards 16S rDNA sequencing has increased since the application of the polymerase chain reaction (PCR) to amplify the gene before sequencing. Moreover, the rising number of 16S rRNA gene sequences publicly available within databases, provide the necessary reference material for deriving reliable conclusions about phylogeny and for determining nucleotide sequence signature regions for diagnostic purposes. In order to infer an intra- and intergeneric phylogeny of plant-associated Enterobacteriaceae, we sequenced the 165 rRNA genes of 29 representative strains of the genus Erwinia and related plant associated organisms of the genera Pantoea and Enterobacter. After comparison with 16S rDNA sequences of other members of the Enterobacteriaceae, the phylogenetic positions of species of Erwinia, Pantoea and Enterobacter within the Enterobacteriaceae were determined, necessitating an emendation of Erwinia and Pecto bacterium as two separate genera and the foundation of a new genus Brenneria gen. nov. The intergeneric relationships between the genera Erwinia, Pectobacterium and Brenneria are described and sequence data were screened for significant differences.

Materials and Methods

Bacterial strains: The twenty seven bacterial strains used in this study were obtained from the culture collection of the Lab-

Phylogeny of phytopathogenic Enterobacteriaceae 385

oratorium voor Microbiologie (BCCMTM/LMG), Universiteit Gent, Ghent, Belgium. Two additional strains were kindly provided by Dr.]. M. YOUNG of the International Collection of Microorganisms from Plants (ICMP), New Zealand. All strains are listed in Table 1. Strains were grown overnight in 5 ml trypticase soy broth (Difco).

Gas chromatographic analysis of FAMEs: The type strains of the species of Erwinia, Pectobacterium and Brenneria were processed as described previously (VAUTERIN et al., 1991).

DNA preparation, PCR amplification and sequencing of 16S rRNA genes: Cells were grown and DNA was extracted and purified as described previously (HAUBEN et al., 1997). The conditions and primers used for PCR amplification of the 16S rDNA have been described previously (HAUBEN et al., 1997). The PCR products were sequenced using Taq DyeDeoxy Terminator Cycle sequencing (Applied Biosystems Inc., Foster City, California) on an ABI 373A automatic sequencer (Applied Biosystems Inc., Foster City, California). Nearly complete 16S rDNA sequences were determined from overlapping sequence regions using the sequencing primers described before (HAUBEN et al., 1997).

16S rRNA gene sequence comparisons and phylogenetic analysis: The 16S rDNA sequences of the strains investigated in this study have been deposited with the EMBL Data Library under the accession numbers listed in Table 1. The EMBL and Genbank database accession numbers for the sequences used as reference for comparison are shown in Fig. 1. Calculation of similarity values and cluster analysis were done using the GeneCompar software (Applied Maths, Kortrijk, Belgium) taking into account the homologous nucleotide positions after discarding all unknown bases and gaps. Dendrograms were constructed, using the same software package, employing the neighbor joining method (Fig. 1).

Results and discussion

The type strains of 15 validly described species of Erwinia, a representative strain of E. psidii and the type strains of the five subspecies of E. carotovora were selected for 165 rRNA gene sequencing. Five plant-associated Pantoea strains and the type strains of three plantassociated Enterobacter species were sequenced as well. An estimated 95.5% to 97.1 % of the total 16S rDNA primary sequence was determined from these strains, comprising a continuous stretch of 1472 to 1498 bases, ranging from positions 28 to 1524 of the E. coli 16S rRNA gene sequence numbering (BROSIUS et al., 1981). Within the actual genus Erwinia, the overall 165 rDNA sequence similarities range from 92.2 % to 99.7% (Table 2). Three phylogenetic clusters can be distinguished (Figure 1). The dendrogram in figure 1 is based on pairwise aligned sequence similarities. In order to be able to subject the tree to a bootstrap analysis with 100 sampling repeats, the sequences were subsequently aligned to a global consensus (data not shown). The bootstrap values for cluster I, II, III and IV were 69, 43, 60 and 98 respectively.

The type strains of E. amylovora, the type species of the genus, E. persicinus, E. rhapontici, E. tracheiphila, E. mallotivora and a representative strain of E. psidii group together in cluster I, possessing mutual 16S rDNA sequence similarities ranging from 94.9% to 99.7%. A sig-

386 L. HAUBEN et a!.

nificant mean internal genomic DNA similarity of 41 % exists between the species comprising cluster I (BRENNER,

1973; BRENNER et aI., 1974; HAO et aI., 1990). The DNADNA similarities reported between E. persicinus and other species of cluster I show the highest similarity (72 %) between E. persicinus and E. rhapontici (HAO et aI., 1990). These two species have 99.2% 165 rDNA sequence similarity. Phenotypically, they share the production of a rare water-soluble pink pigment (HAO et aI., 1990).

bridizaton value for E. mallotivora is available though, showing 39% relatedness with E. persicinus (HAO et aI., 1990). E. mallotivora shows phenotypic similarities to E. amy/ovora (VERDONCK et aI., 1987). We found a 165 rDNA sequence similarity of 96.8% between these last two species (Table 2).

The type strain of the species E. tracheiphila clusters relatively distant to the other species within cluster I (Fig. 1). Reviewing the literature, this species is often absent or represented by only a few strains in phenotypic and DNA hybridization studies (VERDONCK et aI., 1987; MERGAERT et aI., 1984; BRENNER et aI., 1973, 1974). BRENNER observed 23% DNA relatedness between a strain of E. tracheiphila and a strain of E. amy/ovora. It

The species E. mallotivora and E. psidii constitute a subcluster within cluster I with a mutual 165 rDNA sequence similarity of 97.1 % and 96.6% respectively, on the average, with the other members of cluster 1. Literature data on these two species are scarce. One DNA hy-

Table 1. List of bacterial strains used in this study together with the EMBL accession numbers for their 16S rDNA sequence.

Actualized nomenclature of plant Symptoms and host plants of the species Strains EMBL associated Enterobacteriaceae included in accessIOn

this study number

Enterobacter nimipressuralis wet wood of elm tree LMG 10245T Z96077 Enterobacter cancerogenus canker of poplar LMG2693T Z96078 Enterobacter dissolvens rotting of corn stalks LMG 2683T Z96079 Pantoea stewartii subsp. stewartii vascular wilt of corn LMG2715T Z96080 Pantoea stewartii subsp. indologenes leaf spot disease on grasses, rotting of pineapple LMG2632T Y13251 P antoea ananatis rotting of pineapple and sugarcane LMG2665T Z96081 Pantoea agglomerans black color of flesh of pineapple and grape fruit; LMG2565 Z96082

spot disease and frost damage on corn, soy and clover Pantoea agglomerans galls on Milletia japonica LMG2660 Z96083 Erwinia tracheiphila wilt disease on melon and cucumber LMG2906T Y13250 Erwinia psidii rotting of guava LMG 7034 Z96085 Erwinia mallotivora leaf spot on oak tree LMG2708T Z96084 Erwinia amylovora wilt disease on Rosaceae LMG2024T Z96088 Erwinia persicinus rotting of tomato, banana and cucumber LMG 11254T Z96086 Erwinia rhapontici rotting of rhubarb and wheat LMG 2688T Z96087 [Erwinia rhaponticiJ LMG2691 AJOOl190 Pectobacterium carotovorum subsp. rotting of potato and sugar beet LMG2404T Z96089

carotovorum comb. nov. Pectobacterium carotovorum subsp. black leg disease on potato, tomato, chicory LMG238Gf Z96090

atrosepticum comb. nov. Pectobacterium carotovorum subsp. vascular necrosis of sugar beet LMG 2466T Z96091

betavasculorum comb. nov. Pectobacterium carotovorum subsp. rotting of Japanese horse-radish ICMP 9121T AJ223408

wasabiae comb. nov. Pectobacterium carotovorum subsp. slimy rot of chicory LMG 17566T AJ223407

odoriferum comb. nov. Pectobacterium cacticidum comb. nov. rotting of cactus LMG 17936T AJ223409 Pectobacterium chrysanthemi vascular wilts or parenchymatal necrosis LMG 2804T Z96093

on ornamental plants and corn Pectobacterium cypripedii brown rot of cypripedium orchids LMG2657T Z96094 Brenneria alni comb. nov. ex Erwinia bark canker of alder tree ICMP 12481T AJ223468

alni Brenneria nigrif/uens comb. nov. ex bark canker on Persian walnut LMG2694T Z96095

Erwinia nigrif/uens Brenneria paradisiaca comb. nov. ex rotting of roots of banana LMG2542T Z96096

Erwinia paradisiaca Brenneria salicis comb. nov. ex Erwinia watermark disease of willow LMG 2698T Z96097

salicis Brenneria rubrifaciens comb. nov. ex bark canker of walnut LMG2709T Z96098 Erwinia rubrifaciens Brenneria quercina comb. nov. ex rotting of acorn LMG2724T AJ223469

Erwinia quercina

Table 2. Percentages of sequence similarity for a 1472 to 1498 nucleotide region of 165 rDNAs after removal of all unknown base positions and gaps . The strains correspond with the strains in bold in Fig. 1. Pectobacterium c. stands for Pectobacterium carotovorum subsp. and Pantoea s. stands for Pantoea stewartii subsp.

Enterobacter nimipressuralis Enterobacter cancerogenus Enterobacter dissolvens Pantoea s. indologenes Pan toea s. stewartii Pantoea ananatis Pantoea agglomerans LMG 2660 Pantoea agglomerans LMG 2565 Erwinia amylovora Erwinia rhapontici LMG 2688T

Erwinia persicinus [Erwinia rhaponticiJ LMG 2691 Erwinia tracheiphila Erwinia mallotivora Erwinia psidii Pectobacterium cypripedii Pectobacterium chrysanthemi Pectobacterium c. odoriferum Pectobacterium c. carotovorum Pectobacterium cacticidum Pectobacterium c. betavasculorum Pectobacterium c. wasabiae Pectobacterium c. atrosepticum Brenneria quercina Brenneria alni Brenneria nigrif/uens Brenneria paradisiaca Brenneria salicis Brenneria rubrifaciens

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,------ Hafnia alvei (M59 I 55) ,------- Ewingella americana (X88848) '------ Rahnella aquatilis ATCC 33989 (X79939)

Yersinia enterocolitiea ER·26039·92 (Z49829) Yersinia ruckeri ATCC 29473 (X75275)

'--_--I r---- Yersinia pestis EVpsl+c (Z75317) Yersinia mollaretii ER·2975 (X75280)

,...--- Yersinia bercovieri ER·2937 (X75281) Yersinia intermedia ER-3854 (X75274)

Yersinia rohdei ER-2935 (X75276) Yersinia kristensenii ER·28 I 9 (X75278)

Yersinia aldovae ATCC 35236 (X75277) ~--- Citrobacter freundii (M59291)

Klebsiella planticola DSM 3069' (X93215) Enterobacter sp. HC B (U39556)

,...------- Enterobacter nimipressurtllis Enterobacter ellncerogenus

,------ Serratia marcescens (M59160) '----- Klebsiella pneumoniae (X87276)

Enterobaeter i1issolvens ,------- Salmonella paratyphi (U88547)

Escherichia coli (-, Shigella disenteriae (X80680)

Shigella boydii (X96965) Shigellaflexneri (X80679)

Escherichia vulneris ATCC 33821' (X80734) Shigella sonnei (X96964)

~--- Escherichia hermanii (X80675) ,...----- Salmonella shorn ron (X80878)

Salmonella typhimurium (X80681) Salmonella give (X80683)

Salmonella sojia (X80677) Pantoea stewartii subsp. inilologenes ]

Pan toea stewartii subsp. stewartii r---- Pantoea ananatis Cluster IV

~--- Pan toea agglomerans LMG 2660 Pantoea agglomerans LMG 2565

Erwinia amvlovora ] Erwinill rhapon'tiei LMG 2688T

Erwinitl persieinus /Erwinia rhapontieif LMG 2691 Cluster I

,---------- Erwinia mallotivora '------- Erwinia psiilii

'-------------- Erwinia traeheiphi/II

~----C===~~~;;riu~ Peetobaeterium cypripeilii I" Peetobaeterium cltrysanthemi

,---- Peetobaeterium earotovorum subsp. oilorijerum Peetobacterium earotovorum subsp, carotovorum Cluster II

,-------- Pectobacterium caetieiilum r----- Pectobaeterium carotovorum subsp. betavasculorum

Pectoblleterium cllrotovorum subsp. wasabiae Peetobaeterium earotovorum subsp. (Itroseptieum

Brenneria quercina J ,-------- Brenneria alni '------ Brenneria nigri(luens .. Cluster III

,--------- Brennerw paraillSlaea ,...---------- Brenneria salicis '---------- Brenneria rubrifadens

,-------------------- Arsenophonus nasoniae AfCC 49151 (M9080 I) '--------------- Proteus vulgaris (.f0 1874)

,...----------------- PhotoriJabdus luminesct'ns DSM 3368 (X82248) ,-------- Xenorhabdus nematophillls DSM 3370 (X82251)

r------- Xenorhabdusjaponicus lAM 14265' (078008) ,------- Xenorhabdus bovienii DSM 4766 (X82252) ,------- Xenorhabdus beddingii DSM 4767 (X82254) '------ Xenorhabdus poinarii DSM 4768 (X82253)

'---------------Plesiomonas shigelloides ATCC 14029' (X74688) '-----------------------------------Buchnera aphidicola (L18927)

Fig. 1. Neigbor-joining dendrogram depicting the estimated phylogenetic relationships among the Enterobacteriaceae, based on pairwise comparisons of nearly complete 16S rDNA sequences. 16S rDNA sequences for strains determined in this study are in bold. The distance between two species is obtained by summing the lengths of the connecting horizontal branches using the scale on the top (% sequence difference). Cluster I, II, III and IV represent the genera Erwinia, Pectobacterium, Brenneria and Pantoea respectively. lal: (BROSIUS et al., 1981).

is striking that numerous phenotypic tests yield negative results for E. tracheiphila (LELUOTT and DICKEY, 1984). One of the reasons might be that these strains grow poorly on the standard media. The 16S rDNA sequence of this species shows 95.5% similarity on the average with the other species of cluster 1.

E. rhapontici LMG 2691 represents a number of E. rhapontici strains that were isolated from Triticum aestivum whereas the type strain of E. rhapontici LMG 2688T represents E. rhapontici strains isolated from Rheum rhaponticum. Previous studies have shown strain E. rhapontici LMG 2691 to be part of one homogeneous phenon and a specific protein profile group separate from that of E. rhapontici LMG 2688T. (VERDONCK, 1992). DNA hybridization studies revealed only 26% similarity between LMG 2691 and the type strain of E. rhapontici (MERGAERT, 1989). The 16S rDNA sequence comparisons of this study indicate a closer phylogenetic relationship of strain LMG 2691 with E. persicinus (99.7%) than with E. rhapontici (99.3%). We would therefore suggest strain LMG 2691 to be transferred to the species E. persicinus, but additional DNA-DNA hybridization data are necessary to support the renaming of this strain.

Phylogenetic ally, the species E. amylovora, E. mallotivora, E. persicinus, E. psidii, E. rhapontici and E. tracheiphila, corresponding with the six type strains of cluster I, are to be considered as the authentic erwinias. We propose to restrict the genus Erwinia (sensu stricto) to the species of cluster 1.

The second phylogenetic group, cluster II, contains five subspecies of E. carotovora together with the species E. cacticida, E. chrysanthemi and E. cypripedii (Fig. 1). The species and subspecies comprising this cluster possess a mean internal sequence similarity of 96.8% and a mean sequence similarity of 95.6% with Erwinia (cluster I) (Table 2). The species of cluster II show an average of 27% DNA relatedness with Erwinia (BRENNER, 1973; BRENNER et aI., 1974; HAO et aI., 1990) (Table 3). The DNA similarity values observed between the species of cluster II range from 25% to 62 %, with a mean value of 38% relatedness at 60°C (BRENNER, 1973; BRENNER et aI., 1974), and from 4% to 44% relatedness at 75°C (ALCORN et aI., 1991). Differences in 16S rDNA sequence of 1.2% to 3.0% were noted between the subspecies of E.

Table 3. Genotypic relationships between Erwinia (cluster I), Pectobacterium (cluster II), Brenneria (cluster III) and Pantoea (cluster IV): mean 16S rDNA sequence similarity value (%)/mean DNA similarity value (%) (BRENNER, 1973; BRENNER et ai., 1974; HAO et ai., 1990; GAVIN] et ai., 1989; MERGAERT, 1989).

Erwinia Pectobacterium Brenneria Pantoea

Erwinia Pectobac- Brenneria Pantoea terium

97.0/41 95.6/27 94.5/22 96.1/19

96.8/38 95.4/34 95.3/28

96.0/41 94.6/15 97.5/51


carotovora. The five subspecies of E. carotovora have E. cacticida as closest phylogenetic neighbour. This phylogenetic affininity is also reflected in high phenotypic and genotypic similarities (ALCORN et aI., 1991). E. cacticida constitutes a separate branch and possesses an average of 97.3% sequence similarity with E. carotovora wherein the mean sequence similarity between the subspecies is higher, i.e. 98.1 %. E. carotovora possesses an average of 97.2 % 16S rDNA sequence similarity with E. chrysanthemi (Table 2) and these two species exhibit a close phenotypic (LauoTT and DICKEY, 1984; VERDONCK et aI., 1987; MERGAERT et aI., 1984; WELLS et aI., 1991) and genotypic (WELLS et aI., 1991) resemblance.

The species of cluster II possess only 95.6% sequence similarity, on the average, with Erwinia, whereas the Pantoea cluster (cluster IV) shows an average of 96.1 % 16S rDNA sequence similarity with the Erwinia species (cluster I). In our opinion, it is logical to consider cluster II as a distinct genus, supporting the original genus Pectobacterium, which was proposed to include the pectolytic Enterobacteriaceae (WALDEE, 1945). BRENNER (1973) supported this proposal, but it was not followed in the descriptions contained in Bergey's Manual of Systematic Bacteriology (LELLIOTT and DICKEY, 1984). The 16S rDNA sequence data determined in this study reveal cluster II as a phylogenetic entity and support Pectobacterium as a separate genus. Except for E. rhapontici, cluster II contains the species proposed by WALDEE (1945) and BRENNER (1973) for the genus Pectobacterium, i.e., P. carotovorum subsp. atrosepticum, P. carotovorum subsp. carotovorum, P. chrysanthemi and P. cypripedii. The species P. rhapontici which was included in the genus Pectobacterium (BRENNER, 1973), belongs phylogenetically in Erwinia. The DNA hybridization data of BRENNER (1973), already indicated a lower genotypic relationship between P. rhapontici and the other pectolytic Erwinia species (35%, on the average) but it was included in Pectobacterium because of its biochemical resemblance with the 'soft rot' erwinias. The species E. cacticida, E. carotovora subsp. betavasculorum, E. carotovora subsp. odorifera and E. carotovora subsp. wasabiae which group also in cluster II, were all introduced after BRENNER'S proposal in 1973 (THOMSON et aI., 1981; GOTO and MATSUMOTO, 1987; ALCORN et al., 1991; GALLOIS et aI., 1992). They need to be transferred to the genus Pectobacterium and become P. carotovorum subsp. atrosepticum comb. nov., P. carotovorum subsp. betavasculorum comb. nov, P. carotovorum subsp. carotovorum comb. nov., P. carotovorum subsp. odoriferum comb. nov. , P. carotovorum subsp. wasabiae comb. nov. and P. cacticidum comb. nov. The species P. rhapontici is excluded from the genus Pectobacterium and remains classified as Erwinia rhapontici.

The phylogenetic divergence between Erwinia (cluster I) and Pectobacterium (cluster II) is not only reflected in percentages of 16S rDNA sequence differences, but also in characteristic signature positions within the 16S rRNA gene. Nine base positions, conserved within Erwinia (cluster I) and differing from the sequence of the species of Pectobacterium (cluster II) are shown in Fig. 2. The se-

390 L. HAUBEN et aI.

Erwinia Pectobacterium Brenneria Pantoea

376 -7 G - T (- 387 G-T G-T G - T G- C G - C G-C G - C G- C G-C G-C G-C C - G C - G G-C C - G

"-.J "-.J "-.J "-.J 406-7 G - C (- 436 G -C G - C G-C

T-A T -A T - A T-A A - T/Y G -c G/A - CIT A-T T-G T - G T - G T-G

"-.J \J "-/ \J

593-7C/T - G (- 646 Tty - A T AtG T G

A G T G A G T G

A T A T A T A T

G CA G CA G C A G CA W A A A

T A T A T A T A

C G T A TIC - A/G C G A/G _ TIC G C G/A- CIT A T

G T G T G T GIR- T

A T A T A T A T

T A T A T A T A

G C G C G C G C

T G T G T G T G

G T G - T/A G - T/A G T

"--.) V \..J V

837-7T- G (- 849 T - G T - G T - G GIT- AtTIC G-C G - CIT G-A C- G C-G G - C C-G C - G C-G CIT - A/G C - G

~ "-.J "-.J "-.J

984-7 C G (- 1221 C G C G C G C G C G C G C G/R T A T A T A T A

G- C A/G T A T A T G- C C G C G/A C G C_ G T A T A T A C _ G C G C G T G/C

\J U "--.) "--.)

1308 -7 C - G (- 1329 T-A T-A C- G G-C G-C G-C G- C G-C G-C G-C G- C A-T A-T A-T A- T G-C G-C G-C G- C T-A T-A T-A T- A C _ G C_G C _ G C_ GIR

\J \J \J \J Fig. 2. Regions on the 16S rDNA where differences between Erwinia, Pectobacterium, Brenneria and Pantoea occur. The differentiating nucleotides are in bold. E. coli 16S rRNA gene sequence numbering is indicated (BROSIUS et aI., 1981).

quence differences occur as differences in putative helical secondary structure and are presented as such. They are valid for the selection of sequences used in this study but may not hold in comparison with sequences from other bacterial groups and phyla.

Cluster III comprises the species E. alni, E. nigrifluens, E. paradisiaca, E. quercina, E. rubrifaciens and E. salicis possessing mutual sequence similarities of 94.7% to 97.4% (Fig. 1, Table 2). Phylogenetically, they form a homogeneous group and possess 94.5% and 95.4%, on the average, sequence similarity with the species of Erwinia (cluster I) and Pectobacterium (cluster II), respectively. The mean genomic DNA relatedness of these six species to Erwinia (cluster I) and Pectobacterium (cluster II) is 22% and 34%, respectively (BRENNER, 1973; BRENNER et aL, 1974; HAO et aL, 1990) (Table 3). The phenotypic characteristics of the members of cluster III are comparable with a Gower similarity coefficient of approximately 84% (VERDONCK et aL, 1987) . DNA hybridization data show DNA similarities of 30% to 56% relative binding at 60°C between E. alni, E. nigrifluens, E. quercina, E. rubrifaciens and E. salicis (BRENNER et aL, 1973, 1974) and 13% to 65% relative binding at 40°C (5URICO et aL, 1996). The species E. alni and E. nigrifluens, which show the highest 165 rDNA sequence similarity in this cluster (97.4%), also demonstrate high interspecific DNA hybridization values of 49% to 65% (5URICO et al., 1996). Moreover, within the165 rDNA sequences of the members of cluster III, four signature nucleo tides can be defined which are conserved within these six species and which differ from the homologous sequence positions observed in the sequences of members of Erwinia (cluster I), Pectobacterium (cluster II) and Pantoea (cluster IV) (Fig. 2). We propose to classify the species of cluster III in a new genus Brenneria gen. nov. as B. alni comb nov., B. nigri{luens comb. nov., B. paradisiaca comb. nov., B. quercina comb. nov., B. rubrifaciens comb. nov. and B. salicis comb. nov., respectively.

The data obtained in this study, allowing estimations of phylogenetic relationships, shed a new light on earlier viewpoints, but also raise new perspectives for the taxonomy of the genus Erwinia. Comparing the genotypic data available today, the genera Erwinia, Pectobacterium and Brenneria can, with justification, be delineated (Table 3).

The four Pantoea strains included in this study were the type strains of species classified previously within the genus Erwinia: Erwinia stewartii (LMG 2715), Erwinia ananas (LMG 2665), Erwinia milletiae (LMG 2660) and Erwinia herbicola (LMG 2565). They were reclassified in the genus Pan toea based on phenotypic characterization and DNA:DNA hybridization data (MERGAERT et aL, 1993). All strains of Pantoea species included in this study, cluster together in one distinct branch, cluster IV, with mutual 165 rDNA sequence similarities ranging from 95.9% to 98.8% and an average similarity of 96.1 % with Erwinia (cluster I). DNA hybridization data for the genus Pan toea show an internal DNA similarity of 51 % (GAVINI et al., 1989), 19% with Erwinia (cluster I), 28% with Pectobacterium (cluster II) and 15% with Brenneria (cluster III) (MERGAERT, 1989) (Table 3).


The three plant-associated Enterobacter strains that have been sequenced do not cluster together and do not group near the sequence of the Enterobacter sp. strain included in the database. They differ by 1.6% to 2.1 % in their 16S rDNA sequences and demonstrate 96.1 % similarity, on the average, with the Erwinia species (cluster I) (Table 2). These strains were classified formerly in the genus Erwinia, but have been reclassified within Enterobacter (DICKEY and Z UMOFF, 1988; BRENNER et al., 1986). The 165 rDNA sequence of Enterobacter cancerogenus LMG 2693T is more closely related to that of Citrobacter freundii (98.8 %) than to the sequences of the other Enterobacter strains (98.4% on the average). Enterobacter dissolvens LMG 2683T is, by 16S rDNA sequence comparisons, more related to Klebsiella pneumoniae (98.9%) than to the other Enterobacter strains (98.2 % on the average). Enterobacter nimipressuralis is, phylogenetically, most related to E. cancerogenus with 98.4% 16S rDNA sequence similarity. The genera Enterobactey, Citrobacter and Klebsiella, as they are described currently, overlap with each other and cannot be delineated by means of 165 rDNA sequence comparisons.

Whereas within the Enterobacteriaceae, the genera Pantoea, Erwinia, Pectobacterium and Brenneria form separate monophyletic branches, the plant-associated members of Enterobacter are scattered among the genera Klebsiella and Citrobacter. More 16S rDNA sequences of additional members of Enterobacter are needed in order to infer an encompassing phylogeny of this genus.

The analysis of the 16S rDNA sequences of the phytopathogenic Enterobacteriaceae have indicated new relationships among the different species, which necessitate reevaluation of their taxonomy. An encompassing description of the emended genera Erwinia and Pectobacterium and the new genus Brenneria are listed below.

Description of the genus Erwinia (WINSLOW et al. 1920) emend

The species E. amylovora, E. rhapontici, E. persicinus, E. tracheiphila, E. psidii and E. mallotivora belong to the genus Erwinia. Based on our data and the descriptions of WINSLOW et al. (1920), LELLIOTT and DICKEY (1984), VERDONCK (1992) and RIJCKAERT (1994), the emended description of Erwinia is as follows: the cells are gramnegative rods which are 0.5 to 1.0 by 1.0 to 3.0 ]lm. They occur mostly alone or in pairs, but chains occur as well. Cells are usually motile by means of peritrichous flagella.

Erwinia strains do not produce indole and do not oxidize gluconate. They are facultative anaerobic, oxidase negative and catalase positive. Strains produce acid from fructose, galactose, glucose, mannitol, ~-methylglucoside and sucrose but not from adonitol, arabitol, dextrin, dulcitol, inulin, maltose, starch and tagatose. Strains grow on fructose, fumarate, galactose, gluconate, glucose, glycerol, malate, ~-methylglucoside and succinate but not arabitol, benzoate, butanol, methanol, oxalate, propionate and sorbose as carbon sources. Strains use alanine, glutaminic acid, glycylglycine and serine but not kynureninic acid and trigonelline as nitrogen sources. Erwinias are sensitive to chloramphenicol, furazolidone,


nalidixinic acid, oxytetracycline and tetracycline. Strains do not possess arginine dihydrolase, caseinase, phenylalanine deaminase and urease.

The major fatty acids include 12:0, 14:0 and 16:0. Erwinia strains cause wilt and rotting diseases on

plants or are part of the epiphytic flora. The species of the genus Erwinia comprise a distinct

phylogenetic group, as determined by 16S rRNA gene sequence comparisons, and are characterized by signature nucleotides A, A, C, G, G, C, G, G, G, C, G, C, C, C and G at positions 408,594,598,639,646, 839, 847, 987, 988,989,1216,1217,1218,1308 and 1329, respectively, according to the E. coli 16S rRNA gene sequence numbering (BROSIUS et aI., 1981).

The G+C contents of members of the genus range from 49.8 to 54.1 mol%. The type species is Erwinia amylovora.

Description of Envinia amylovora (WINSLOW et al. 1920) emend

The descriptions of WINSLOW et al. (1920) and LELLIOIT and DICKEY (1984) can be supplemented with the following data of RUCKAERT (1994).

There is no anaerobic growth and strains hydrolyze esculin. All strains produce acid from sorbitol. Strains grow on melibiose and sorbitol as carbon sources. Strains can use isoleucine, methionine and threonine as nitrogen sources. Strains are sensitive to furazolidone.

Erwinia amylovora strains cause wilting on Rosaceae. The G+C contents of members of the species range

from 53.6 to 54.1 mol%. The type strain is LMG 2024T

(= NCPPB 683 = ATCC 15580).

Description of Envinia mallotivora (GOTO 1976) emend

The descriptions of WINSLOW et al. (1920) and LELLIOIT and DICKEY (1984) can be supplemented with the following data ofRI]CKAERT (1994).

Strains possess gelatinase and can not hydrolyze esculin. Strains produce acid from mannose and xylose, but not from arabinose, glycerol, melibiose and sorbitol. Strains grow on maltose but not on arabinose, arbutine, cellobiose, lactulose and melibiose as carbon sources. Strains grow on tryptophane and xanthin but not on anthranilic acid, isoleucine, threonine, thymine, tryptamine and valine as nitrogen sources. Strains are sensitive to ampicillin, carbenicillin, cephalexine, cephaloridine, cephalotin, kanamycin, streptomycin and spectinomycin but not to furazolidone, fusidinic acid, novobiocin and sulfafurazol.

Erwinia mallotivora strains cause wilting on Mallotus Japonicus.

The G+C content of members of the species range from 49.8 to 51.0 mol%. The type strain is LMG 2708T

(= NCPPB 2851 = ATCC 29573).

Description of Envinia tracheiphila (BERGEY et al. 1923) emend

The descriptions of WINSLOW et al. (1920) and LELLIOTT and DICKEY (1984) can be supplemented with the

following data of VERDONCK (1992) and RI]CKAERT (1994).

Strains possess ~-galactosidase but not starch hydrolase, arginine dihydrolase, casein hydrolase, lysine decarboxylase and urease. Strains produce acid from amygdalin, arbutine, n-acetyl glucosamine, glycerol and rhamnose but not from arabinose, cellobiose, esculin, erythritol, fucose, glycogene, inositol, lactose, melibiose, ~-methyl glucoside, ~-methyl mannoside, ~-methyl xyloside, sorbitol, sorbose and xylose. Strains grow on arabinose, arbutine, citrate, maltose, mannose, ~-methyl glucoside, ribose, salicin, sorbitol and sucrose but not on adipate, adipinic acid, adonitol, betaine, cellobiose, dextrin, dulcitol, ethanol, ethylene glycol, galaturonic acid, glycogene, xylose, lyxose, lactose, lactulose, malonic acid, naphtalene, pectinic acid, propanol, propionic acid, sorbinic acid, starch and xylitol as carbon sources. Strains grow on allantoin, ammoniumchloride, arginine, citrulline, glucosamine, glutathion, leucine, phenylalanine and tyrosine but not on betaine, choline, cysteamine, hydroxypyroline, quinolinic acid, sarcosine, spermidine, spermine, thymine, tryptamine and xanthin as nitrogen sources. Strains are sensitive to ampicillin, amoxycillin, carbenicillin, cephaloridine, cephalexine, cephalotin, framycetine and kanamycin but not to fusidinic acid, methicillin and sulfafurazol.

Erwinia tracheiphila strains cause wilting on Cucumis species.

The G+C content of members of the species range from 50.0 to 52.0 mol%. The type strain is LMG 2906T

(= NCPPB 2452 = ATCC 33245).

Description of Envinia rhapontici (BURKHOLDER

1948) emend The descriptions of WINSLOW et al. (1920) and LEL

LIOTT and DICKEY (1984) can be supplemented with the following data of VERDONCK (1992).

Strains can use citrate. Strains produce acid from lactose, melezitose, raffinose, xylitol and lyxose. Strains grow on tyramine and tryptamine as nitrogen sources. Strains are sensitive to ampicillin and amoxycillin.

Erwinia rhapontici strains cause brown rot of Rheum rhaponticum.

The G+C content of the members of the species range from 51.0 to 53.1 mol%. The type strain is LMG 2688T

(= NCPPB 1578 = ATCC 29283).

The descriptions of the species Erwinia perslcmus HAO et al. 1990 and Erwinia psidii NETO et al. 1987 re

, main as given by HAO et al. (1990) and NETO et al. (1987) respectively.

Description of the genus Pectobacterium (WALDEE 1945) emend

The description below is based on the phenotypic description of WALDEE (1945) and our data, as well as the results ofVERDONCK (1992) and RI]CKAERT (1994).

The cells are gram-negative rods which are 0.5 to 1.0 by 1.0 to 3.0 pm with round ends. They occur mostly alone or in pairs, but chains occur as well. Cells are usually motile by means of peritrichous flagella.

Strains are catalase pOSItive and oxidase negative. They apply a fermentative metabolism and are facultative anaerobes. No decarboxylases are formed for arginine, lysine or ornithine. Strains do not possess tryptophane deaminase or urease and hydrolyse esculin but not starch. All strains produce acid from fructose, galactose, glucose, man nose, N-acetylglucosamine, ribose, rhamnose, salicin, sucrose but not from adonitol, arabitol, lyxose, p-methylmannoside, sorbose, starch and tagatose. Pectobacterium strains grow on arabinose, arbutine, p-methylglucoside, citrate, fructose, fumarate, galactose, gluconate, glucose, glycerol, malate, mannitol, mannose, ribose, salicin, succinate and sucrose but not on adipate, arabitol, betaine, benzoate, butanol, gallate, methanol, oxalate, propionate and sorbose as carbon sources. Cells grow on alanine, allantoin, ammoniumchloride, arginine, asparagine, asparaginic acid, citrulline, y-amino butanic acid, glucosamine, glutamine, glutaminic acid, glutathion, glycine, glycylglycine, histidine, leucine, methionine, phenylalanine, serine, tryptophane, tyrosine and ureum but not on anthranilic acid, betaine, choline, cysteamine, hydroxyproline, kynureninic acid, quinolinic acid, sarcosine, spermidine, spermine, trigonelline and trimethylammonium as nitrogen sources.

Pectobacterium strains (except P. cypripedii) possess pectolytic enzymes and cause soft rots, necroses and wilts on food crops and ornamental plants.

The major fatty acids of the type strains include 12:0, 14:0,15:0,16:0,17:1 w8cand 17:0.

The species of the genus Pectobacterium comprise a distinct phylogenetic group, as determined by 16S rRNA gene sequence comparisons, and are characterized by signature nucleotides G, C, T, T, G, C, A, A, C, G, C, C, T, A, G, T, T and A at sequence positions 408, 434, 594, 598,599,638,639,646,839,847,848,988,989,1216, 1217, 1218, 1308 and 1329, respectively, according to the E. coli 16S rRNA gene sequence numbering (Brosius et aI., 1981).

The G+C contents of members of the genus range from 50.5 tot 56.1 mol%. The type species is Pectobacterium carotovorum WALDEE 1945 emend.

Description of Pectobacterium carotovorum (WALDEE 1945) emend

The description of Pectobacterium carotovorum is the same as that of the genus. Strains do not produce indol and do not possess lecithinase activity. Acid is being produced from gentiobiose, mannitol and raffinose but not from meso-inositol, 5-keto-gluconate and sorbitol. Cells grow on cellobiose, a-keto-glutarate and meso-inositol, but not on malonate, sorbitol, tartrate, meso-tartrate, triacetine and xylitol as carbon sources. Cells do not grow on creatine, guanine, nicotinic acid, tryptamine and xanthin as nitrogen sources. Strains are sensitive to amoxycillin and cephalotin.

Pectobacterium carotovorum strains cause rotting diseases on a broad range of hosts.

The G+C contents of members of the speCies range from 50.5 to 54.6 mol%.


Description of Pectobacterium carotovorum subsp. carotovorum (WALDEE 1945) comb. nov.

The description of Pectobacterium carotovorum subsp. carotovorum is the same as that of the species. Cells grow in the presence of 7% NaCI and can use citrate. Strains produce acid from melibiose but not from a-methylglucoside and gluconate. Cells do not grow on hypo xanthin as nitrogen source.

Pectobacterium carotovorum subsp. carotovorum strains cause rotting, particularly of storage tissue, on a wide range of plants.

The G+C contents of members of the subspecies range from 50.5 to 53.1 mol%. The type strain is LMG 2404T

(= NCPPB 312 = ATCC 15713).

Description of Pectobacterium carotovorum subsp. atrosepticum (WALDEE 1945) comb. nov.

The description of Pectobacterium carotovorum subsp. atrosepticum is the same as that of the species. Cells can use citrate and produce acid from gluconate and melibiose. Strains grow on palatinose but not on hypoxanthin and pyrazinamide as carbon sources.

Pectobacterium carotovorum subsp. atrosepticum strains cause black leg disease on potato (Solanum tuberosum) and a storage rot of potato tubers.


(= NCPPB 549 = ATCC 33260).

Description of Pectobacterium carotovorum subsp. betavasculorum (THOMSON, HILDEBRAND and SCHROTH, 1981) comb. nov.

The description of Pectobacterium carotovorum subsp. betavasculorum is the same as that of the species. Cells do not use citrate. Strains produce acid from amethylglucoside and meso-inositol but not from gluconate and melibiose. Strains grow on a-methylglucoside and maltose as carbon sources and on hypoxanthin and pyrazinamide as nitrogen sources.

Pectobacterium carotovorum subsp. betavasculorum strains cause vascular necroses in roots of sugarbeet.


(= NCPPB 2795).

Description of Pectobacterium carotovorum subsp. wasabiae (GOTO and MATSUMOTO, 1987) comb. nov.

The description of Pectobacterium carotovorum subsp. wasabiae is based on the data of GOTO and MATSUMOTO (1987). Cells possess gelatinase activity but no p-galactosidase activity. Strains can use citrate and produce no acid from a-methylglucoside, inuline, maltose, melibiose and raffinose.

Pectobacterium carotovorum subsp. wasabiae strains cause rotting diseases on Japanese horse-radish.

The G+C contents of members of the subspecies range from 51.4 to 51.7 mol%. The type strain is ICMP 912F (= ATCC 43316).


Description of Pectobacterium carotovorum subsp. odoriferum (GALLOIS, SAMSON, AGERON and GRIMONT 1992) comb. nov.

The description of Pectobacterium carotovorum subsp. odoriferum is as given by GALLOIS et al. (1992).

Pectobacterium carotovorum subsp. odoriferum strains cause slimy rot of witloof chicory.

The type strain is LMG 17566T (= CFBP 1878).

Description of Pectobacterium chrysanthemi (BRENNER, STEIGERWALT, MIKLOS and FANNING 1973) emend.

The description of Pectobacterium chrysanthemi is the same as that of the genus. Strains produce acid from amygdalin and arbutine but not from p-methylxyloside, erythritol, fucose, glycogene and xylitol. Strains grow on galacturonate, glycerate, melibiose, mucate, rhamnose, trehalose and xylose as carbon sources. Strains grow on adenine, carnosine, octopine, ornithine, threonine and valine but not on taurine as nitrogen sources.

Pectobacterium chrysanthemi strains cause vascular wilts or parenchymal necroses on a broad range of ornamental plants and food crops like maize.

The G+C contents of members of the species range from 52.6 to 56.1 mol%. The type strain is LMG 2804T

(= NCPPB 402 = ATCC 11663).

Description of Pectobacterium cacticidum (ALCORN, ORUM, STEIGERWALT, FOSTER, FOGLEMAN and BRENNER 1991) comb. nov.

The description of Pectobacterium cacticidum is the same as given by ALCORN et al. (1991).

Pectobacterium cacticidum strains cause rotting diseases on several cactus species.

The G+C contens of members of the species range from 50.8 to 51.7 mol%. The type strain is LMG 17936T

(= ATCC 49481).

Description of Pectobacterium cypripedii (WALDEE 1945) emend.

The description of Pectobacterium cypripedii is the same as that of the genus. Cells do not produce acetoine and are not pectolytic. Strains oxidate gluconate and produce gas from glucose. Cells do not hydrolyse caseine and possess phenylalanine deaminase. Pectobacterium cypripedii strains do not produce indole and are lecithinase negative. They are sensitive to erythromycin. Strains form acid from glycerol, inositol, maltose and trehalose but not from a-methylglucoside, dulcitol, inuline, lactose and raffinose. Cells can use galacturonate and tartrate as carbon sources.

Pectobacterium cypripedii strains cause a brown rot of cypripedium orchids (Cypripedium spp.)


(= NCPPB 3004 = ATCC 29267).

Description of Brenneria gen. nov. Bren.ne'ri.a. M.L. fem.n. Brenneria; named after the

American bacteriologist DON]. BRENNER (Center Disease

Control Prevention, Atlanta). The description below is based on our data and data of WILSON et al. (1957), DYE (1968), LELLIon and DICKEY (1984), VERDONCK (1992), RUCKAERT (1994) and SURICO et al. (1996). Cells are gram negative, oxidase negative, catalase positive and fermentative. The cells are 0.5 to 1.0 by 1.3 to 3.0 ~m, have rounded ends, occur singly or rarely in pairs and are motile by means of peritrichous flagella. They do not produce arginine decarboxlases, arginine dihydrolase, lysine decarboxylases and ornithine decarboxylases. Starch is not hydrolyzed. Acids are produced from galactose, glucose, fructose, mannose, salicin and sucrose but not from adonitol and inositol.

The major fatty acids include 12:0, 14:0, unknown 14.503, 16:0 and 17:0 cyclopropane.

Brenneria strains cause diseases on trees. The species of the genus Brenneria comprise a distinct

phylogenetic group, as determined by 16S rRNA gene sequence comparisons, and are characterized by signature nucleotides G, C T, A, G, C, A, C, T, A, T, T and A at sequence positions 379, 384, 593, 594, 839, 847, 987, 988, 989, 1217, 1219, 1308 and 1329, respectively, according to the E. coli 16S rRNA gene sequence numbering (BROSIUS et aI., 1981).

The G+C contents of members of the genus range from 50.1 to 56.1 mol%. The type species is Brenneria sa/icis.

Description of Brenneria salicis (CHESTER 1939) comb. nov.

The description of Brenneria salicis is the same as that of the genus. Strains possess p-galactosidase and aspartase but not urease, caseine hydrolase and phenylalanine deaminase. Cells do not produce indole. Strains produce acid from amygdalin, arbutine, N-acetyl glucosamine, mannitol, rhamnose, raffinose and ribose but not from arabinose, arabitol, dextrin, dulcitol, erythritol, esculin, fucose, glycogene, inuline, lactose, a-methylmannoside, melibiose, a-d-melezitose, p-methylxyloside, sorbose, starch, tagatose and xylose. Strains grow on arabinose, arbutine, citrate, esculin, fructose, fumarate, gluconate, galactose, glucose, glycerol, malate, mannitol, mannose, melibiose, p-methyl glucoside, raffinose, ribose, salicin, succinate and sucrose but not on adipate, adonitol, arabinose, arabitol, benzoate, betaine, butanol, dextrin, dulcitol, meso-erythritol, ethylene galacturonic acid, glycol, gallate, glutaminic acid, glycogene, lactose, lactulose, lyxose, malonic acid, a-d-melezitose, methanol, naphtalene, oxalate, pectinic acid, propanol, propionate, sorbinic acid, sorbitol, sorbose, starch, xylitol and xylose as carbon sources. Strains grow on alanine, allantoin, ammoniumchloride, anthranilic acid, arginine, asparagine, asparginic acid, citrulline, glucosamine, glutaminic acid, glutathion, glycine, glycylglycine, histidine, isoleucine, leucine, methionine, phenylalanine, serine, tryptophane, tyrosine, ureum, threonine, valine and xanthin but not on betaine, choline, cysteamine, hydroxypyroline, kynureninic acid, quinolinic acid, sarcosine, spermidine, spermine, trigonelline, thymine and tryptamine as nitrogen sources. Strains are sensitive to carbenicillin,

cephaloridin, cephalexin, cephalotin, chloramphenicol, doxycycline, framecytine, furazolidone, kanamycine, nalidixinic acid, nitrofurantoine, oxytetracycline and tetracycline but not to bacitracine, cloxacillin, colistine sulfate, erythromycin, fusidinic acid, gentamycine, lincomycine, methicillin, spectinomycin and sulfafurazol.

Brenneria salicis strains cause watermark disease on willow (Salix spp.).


(= NCPPB 447 = ATCC 15712).

Description of Brenneria nigri{luens (WILSON, STARR and BERGER 1957) comb. nov.

The description of Brenneria nigrif/uens is the same as that of the genus. Strains possess ~-galactosidase and aspartase but not urease and phenylalanine deaminase. Cells do not produce indole. Strains produce acid from N-acetylglucosamine, amygdalin, arabinose, arbutine, esculin, glycerol, mannitol, raffinose, rhamnose, ribose, sorbitol, trehalose and xylose but not from arabitol, dextrin, erythritol, fucose, glycogeen, inuline, lactose, maltose a-methylmannoside, ~-methylxyloside, sorbose, starch, tagatose and xylose. Cells grow on arabinose, arbutine, citrate, esculin, ethanol, fructose, fumarate, gluconate, galactose, glucose, glucuronic acid glycerol, meso-inositol, malate, mannitol, mannose, melibiose, ~-methylglucoside, raffinose, ribose, salicin, sorbitol, succinate, sucrose and trehalose but not on adipate, adonitol, amygdalin, arabinose, arabitol, benzoate, betaine, butanate, cellobiose, dextrin, dulcitol, meso-erythritol, ethylene glycol, galacturonic acid, gallate, glycogen, lactose, lactulose, lyxose, maltose, a-Dmelezitose, methanol, naphtalene, oxalate, propanol, propionate, sorbinic acid, sorbose, starch, xylitol and xylose as carbon sources. Strains grow on alanine, allantoin, ammoniumchloride, arginine, asparagine, asparaginic acid, citrulline, ethanolamine, glucosamine, glutaminic acid, glutathion, glycine, glycocyamine, glycylglycine, histidine, leucine, methionine, phenylalanine, serine, tyrosine, ureum, valine and xanthin but not on acetamide, anthranilic acid, betaine, choline, cysteamine, hydroxypyroline, kynureninic acid, quinolinic acid, sarcosine, spermidine, spermine, threonine, thymine, trigonelline and tryptamine as nitrogen sources. Strains are sensitive to amoxycillin, ampicillin, carbenicillin, cephalorodine, cepahalotin, cephalexin, chloramphenicol, doxycycline, framecytine, furazolidone, nalidixinic acid, nitrofurantoine, oxytetracycline, penicillin, tetracycline and novobiocin but not to bacitracin, cloxacillin, colistine sulfate, erythromycin, fusidinic acid, gentamicine, lincomycin, methicillin, neomycin, polymyxin, spectinomycin, streptomycin and sulfafurazol.

Brenneria nigrif/uens strains cause bark canker on Persian walnut (Juglans regia).

The G+C content of the type strain is 56.1 mol%. The type strain is LMG 2694T (= NCPPB 564 = ATCC 13028).


Description of Brenneria alni (SURICO, MUGNAI, PASTORELLI, GIOVANNETTI and STEAD 1996) comb. nov.

The description of Brenneria alni is the same as given by SURICO et al. (1996).

Brenneria alni strains cause bark canker of alder (Alnus).

The G+C contents of members of the species range from 50.1 to 50.7 mol%. The type strain is ICMP 12481T (= NCPPB 3934).

Description of Brenneria rubrifaciens (WILSON, ZEITOUN and FREDERICKSON 1967) comb. nov.

The description of Brenneria rubrifaciens is the same as that of the genus. Strains possess ~-galactosidase but not urease, caseine hydrolase and phenylalanine deaminase. Cells do not produce indole. Strains produce acid from N-acetyl glucosamine, amygdalin, arabinose, arbutine, mannitol, maltose, a-methylglucoside, ~-methylglucoside, rhamnose and ribose, but not from arabitol, cellobiose dextrin, dulcitol, erythritol, esculin, fucose, glycogen, inuline, lactose, a-D-melezitose, a-methylmannoside, ~methylxyloside, raffinose, sorbitol, sorbose, starch, tagatose and xylose. Strains grow on arabinose, arbutine, citrate, esculin, fructose, fumarate, gluconate, galactose, glucose, glycerol, malate, mannitol, mannose, ~-methyl glucoside, ribose, salicin, succinate and sucrose but not on adipate, adonitol, arabinose, arabitol, benzoate, betaine, butanol, dextrin, dulcitol, meso-erythritol, ethylene glycol, galacturonic acid, gallate, glutaminic acid, glycogene, lactose, lactulose, lyxose, malonic acid, maltose, a-dmelezitose, methanol, naphtalene, oxalate, oxalic acid, pectinic acid, propanol, propionate, raffinose, rhamnose, sorbinic acid, sorbitol, sorbose, starch, trehalose, xylitol and xylose as carbon sources. Strains grow on acetamide, alanine, allantoin, ammoniumchloride, arginine, asparagine, asparginic acid, citrulline, ethanolamine, glucosamine, glutaminic acid, glutathion, glycine, glycocyamine, glycylglycine, histidine, isoleucine, leucine, methionine, pehylalanine, serine, threonine, tryptophane, tyrosine, ureum, valine and xanthin but not on betaine, choline, cysteamine, hydroxypyroline, kynureninic acid, quinolinic acid, sarcosine, spermidine, spermine, trigonelline, thymine and tryptamine as nitrogen sources. Strains are sensitive to amoxycillin, ampicillin, chloramphenicol, carbenicillin, cephaloridine, cephalexin, cephalotin, doxycycline, framecytine, furazolidone, nalidixinic acid, nitrofurantoine, oxytetracycline, penicillin and tetracycline but not to bacitracin, cloxacillin, colistine sulfate, erythromycin, fusidinic acid, gentamicin, lincomycin, methicillin, polymyxin and sulfafurazol.

Brenneria rubrifaciens strains cause necrosis on Persian walnut (Juglans regia).

The G+C contents of members of the species range from 52.0 to 52.6 mol%. The type strain is LMG 27091

(= NCPPB 2020 = ATCC 29291).

Description of Brenneria quercina (HILDEBRAND and SCHROTH 1967) comb. nov.

The description of Brenneria quercina is the same as that of the genus. Strains possess ~-galactosidase but not

396 L. HAUSEN et al.

urease, caseine hydrolase and phenylalanine deaminase. Cells do not produce indole. Strains produce acid from amygdalin, arbutine, N-acetylglucosamine, glycerol, rhamnose, ribose, mannitol and ~-methylglucoside but not from arabinose, arabitol, cellobiose, dextrin, dulcitol, erythritol, esculin, fucose, glycogen, inuline, lactose maltose, a-D-melezitose, melibiose, a-methylmannoside, ~-methylxyloside, raffinose, sorbitol, sorbose, starch, tagatose, trehalose and xylose. Strains grow on arbutine, citrate, esculin, fructose, fumarate, gluconate, galactose, glucose, glycerol, malate, maltose, mannitol, mannose, ~methylglucoside, ribose, salicin, succinate and sucrose but not on adipate, adonitol, arabinose, arabitol, benzoate, betaine, butanol, cellobiose, dextrin, dulcitol, meso-erythritol, ethanol, ethylene glycol, galacturonic acid, gallate, glycogene, lactose, lyxose, malonic acid, ad-melezitose, methanol, naphtalene, oxalate, propanol, propionate, raffinose, sorbinic acid, sorbitol, sorbose, starch, trehalose, xylitol and xylose as carbon sources. Strains grow on alanine, allantoin, ammoniumchloride, arginine, asparagine, asparginic acid, citrulline, ethanolamine, glucosamine, glutaminic acid, glutathion, glycine, glycylglycine, histidine, isoleucine, leucine, methionine, phenylalanine, threonine, tryptophane, tyrosine, ureum and valine but not on acetamide, anthranilic acid, betaine, choline, cysteamine, glycocyamine, hydroxypyroline, kynureninic acid, quinolinic acid, sarcosine, serine, spermidine, spermine, thymine, trigonelline and tryptamine as nitrogen sources. Strains are sensitive to amoxycillin, ampicilline, carbenicillin, cephaloridine, cephalexin, chloramphenicol, doxycyclin, furazolidone, kanamycin, nalidixinic acid, nitrofurantoine, oxytetracycline, penicillin and tetracycline but not to bacitracin, cloxacillin, colis tine sulphate, erythromycine, fusidinic acid, gentamicin, kanamycin, methicillin, neomycin and sulfafurazol.

Brenneria quercina strains cause copious oozing of sap from acorns (Quercus agrifolia and Quercus wislizenii).

The G+C contents of members of the species range from 54.6 to 55.1 mol%. The type strain is LMG 2724T (= NCPPB 1852 = ATCC 29281).

Description of Brenneria paradisiaca (FERNANDEZ

BORRERO and LOPEZ 1970) comb. nov. The description of Brenneria paradisiaca is the same

as that of the genus. Strains possess ~-galactosidase and pectinolase but not urease and caseine hydrolase. Cells produce indole. Strains produce acid from N-acetyl glucosamine, arabinose, arbutine, gentiobiose, gluconate, melibiose, ~-methylglucoside, raffinose, ribose, rhamnose and xylose but not from arabitol, dulcitol, erythritol, fucose, glycogen, lyxose, maltose, a-methylmannoside, ~-methylxyloside, sorbitol, sorbose, starch, tagatose, xylitol and xylose. Strains grow on arabinose, arbutine, citrate, fructose, fumarate, galactose, galacturonate, gluconate, glucose, glycerate, glycerol, a-keto-glutarate, malate, mannitol, mannose, melibiose, ~-methylglucoside, mucate, rhamnose, ribose, salicin, succinate, trehalose and xylose but not on adipate, arabitol, ben-

zoate, betaine, butanol, gallate, meso-inositol, methanol, a-methylglucoside, oxalate, propionate, sorbose, tartrate, triacetine and xylitol as carbon sources. Strains grow on adenine, alanine, allantoin, ammoniumchloride, arginine, asparginic acid, carnosine, citrulline, creatine, glucosamine, glutaminic acid, glutathion, glycine, glycylglycine, histidine, leucine, methionine, octopine, ornithine, phenylalanine, serine, threonine, tryptamine, tryptophane, tyrosine, ureum and valine but not on betaine, choline, cyteamine, guanine, hydroxypyroline, kynureninic acid, nicotinic acid, quinolinic acid, sarcosine, spermidine, spermine, trigonelline, taurine and xanthin as nitrogen sources. Strains are sensitive to amoxycillin, ampicillin, carbenicillin, cephaloridine, cephalexin, cephalotin, chloramphenicol, framecytine, furazolidone, kanamycin, nalidixinic acid, oxytetracycline, streptomycin and tetracycline but not to fusidinic acid, methicillin and sulfafurazol.

Brenneria paradisiaca strains cause rotting of roots of Musa paradisiaca.

The G+C content of the type strain is 54.7%. The type strain is LMG 2542T (= NCPPB 2511 = ATCC 33242).

Acknowledgements Part of the work was carried out for account of the Flemish Government under the authority of the Department Bos en Groen (B&G/3/1995). The authors are grateful to Prof. Dr. J. Young for providing the cultures ICMP 9121 and ICMP 12481. The authors thank C Strompl, A. Kruger and S. Van Eygen for excellent technical assistence.

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Corresponding author: L. HAUBEN, Laboratorium voor Microbiologie, Universiteit Gent, B - 9000 Ghent, Belgium. Tel: +32 (9) 264 5102; fax: +32 (9) 264 5092; e-mail: [email protected]

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Phylogenetic Position of Phytopathogens within the Enterobacteriaceae