International Journal of Systematic and Evolutionary Microbiology (2000), 50, 371–380 Printed in Great Britain

Leifsonia poae gen. nov., sp. nov., isolated from nematode galls on Poa annua, and reclassification of ‘Corynebacterium aquaticum ’ Leifson 1962 as Leifsonia aquatica (ex Leifson 1962) gen. nov., nom. rev., comb. nov. and Clavibacter xyli Davis et al. 1984 with two subspecies as Leifsonia xyli (Davis et al. 1984) gen. nov., comb. nov.

Lyudmila I. Evtushenko,1 Lubov V. Dorofeeva,1 Sergey A. Subbotin,2 James R. Cole3 and James M. Tiedje3

Author for correspondence: Lyudmila I. Evtushenko. Tel: j7 095 9257448. Fax: j7 095 9233602. e-mail: evtushenko!ibpm.serpukhov.su

1 All-Russian Collection of The new genus Leifsonia gen. nov. with two new species, Leifsonia poae sp. Microorganisms (VKM), nov. (type strain VKM Ac-1401T) and Leifsonia aquatica (ex Leifson 1962) nom. Institute of Biochemistry T T and Physiology of rev., comb. nov. (the type species, with VKM Ac-1400 l DSM 20146 l JCM Microorganisms, Russian 1368T as type strain), is proposed to accommodate found in Poa annua Academy of Sciences, root gall, induced by the nematode Subanguina radicicola, and Pushchino, Moscow Region, 142292, Russia ‘Corynebacterium aquaticum ’ Leifson 1962. Further, it is proposed to reclassify Clavibacter xyli Davis et al. 1984 with two subspecies in the new genus as 2 Institute of Parasitology, Russian Academy of Leifsonia xyli (Davis et al. 1984) comb. nov., Leifsonia xyli subsp. xyli (Davis et Sciences, Leninskii al. 1984) comb. nov. and Leifsonia xyli subsp. cynodontis (Davis et al. 1984) prospect, 33, Moscow, comb. nov. Members of the proposed genus are characterized by coryneform 117071, Russia morphology, peptidoglycans based upon 2,4-diaminobutyric acid, the major 3 Center for Microbial menaquinone MK-11, phosphatidylglycerol and diphosphatidylglycerol as Ecology, Michigan State University, East Lansing, principal phospholipids, the high content of anteiso- and iso-branched MI 55556, USA saturated fatty acids, and a DNA GMC base composition of 66–73 mol%. They form a distinct phylogenetic branch attached to the line of descent of spp. The new and reclassified species of the new genus clearly differ from each other phylogenetically and phenetically and can be recognized by their morphologies, the cell wall sugar composition, the requirement of complex media for growth, and numerous physiological characteristics, including the oxidase reaction.

Keywords: , Leifsonia gen. nov., Clavibacter xyli,‘Corynebacterium aquaticum’, Subanguina radicicola

INTRODUCTION & Casida, 1969), Clavibacter (Davis et al., 1984), (Zgurskaya et al., 1993), Until recently, six genera of Gram-positive bacteria (Groth et al., 1996), (Takeuchi et al., with 2,4-diaminobutyric acid (DAB) in their cell 1996) and (Suzuki et al., 1997). The walls have been described, i.e. Agromyces (Gledhill following salient characteristics were proposed to differentiate the above genera at the phenetic level: ...... morphology, major menaquinones, amino acid com- Abbreviation: DAB, 2,4-diaminobutyric acid. position of the peptidoglycan, phospholipid pattern, The GenBank accession number for the 16S rDNA sequence of strain fatty acid profile and growth temperature. Recently, Leifsonia poae gen. nov., sp. nov. VKM Ac-1401T is AF116342. Sasaki et al. (1998) demonstrated that the - and -

01184 # 2000 IUMS 371 L. I. Evtushenko and others isomers of DAB in the peptidoglycans were also added to this ground gall suspension at a final concentration indicative of these genera: Agromyces, Agrococcus, of 0n1% (w\v) for inhibition of most Gram-negative Cryobacterium, Leucobacter and Rathayibacter were bacteria. One drop of this suspension was plated onto characterized by the -DAB exclusively, whereas corynebacterial (CB) agar (Zgurskaya et al., 1993) and organisms of the genus Clavibacter contained - and - incubated for 3 weeks at room temperature (18–24 mC). Representative colonies were selected and regrown on CB DAB in their peptidoglycan in almost equal pro- agar. The isolated strains were stored at 8 mC or under freeze- portions. Additionally, Altenburger et al. (1997) dried conditions. showed differences in some of the above genera in the Strains and their cultivation. The novel isolate VKM Ac- polyamine pattern. Some other bacteria known to 1401T and the following type and reference strains were used have DAB in their peptidoglycan were misclassified or in this comparative study: ‘Corynebacterium aquaticum’ not assigned to validly described taxa (Leifson, 1962; VKM Ac-1400T (l DSM 20146T l JCM 1368T), Clavi- Iizuka & Komagata, 1964;Yamada & Komagata, bacter xyli subsp. cynodontis VKM Ac-2041T (l ICMP 1970a; Schleifer & Kandler, 1972; Fiedler & Kandler, 8790T l JCM 1376T), Clavibacter xyli subsp. cynodontis 1973; Collins & Jones, 1980, 1981; Hensel, 1984; VKM Ac-2032 (l ICMP 8792), Agromyces mediolanus Bendinger et al., 1992; Evtushenko et al., 1994). VKM Ac-1388T (l DSM 20152T l JCM 3346T), Agromyces cerinus subsp. cerinus VKM Ac-1340T and Agromyces Subsequently, based on comparative analysis of T 16S rDNA, ‘B. helvolum’ and ‘Corynebacterium fucosus subsp. fucosus VKM Ac-1345 (VKM, All-Russian aquaticum’ were shown to form two separate sublines Collection of Microorganisms, Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of of descent within the radiation of actinomycetes Sciences, Pushchino, Russia; DSM, Deutsche Samm- belonging to the family Microbacteriaceae (Rainey et lung von Mikroorganismen und Zellkulturen GmbH, al., 1994; Takeuchi & Yokota, 1994), and they were Braunschweig, Germany; ICMP, International Collection suggested to be nuclei of novel genera (Rainey et al., of Microorganisms from Plants, Manaaki Whenua 1994). Furthermore, ‘Corynebacterium aquaticum’ Landcare Research, Auckland, New Zealand; JCM, Japan was determined to be a phylogenetic neighbour of Collection of Microorganisms, Institute of Physical and Clavibacter xyli subsp. cynodontis, and these organisms Chemical Research, Wako, Japan). All strains were grown on CB agar, except Clavibacter xyli subsp. cynodontis VKM formed an independent cluster positioned close to the T Agromyces branch (Takeuchi & Yokota, 1994; Sasaki Ac-2041 and Clavibacter xyli subsp. cynodontis VKM Ac- 2032, which were cultivated on complex SC medium con- et al., 1998). Based on the phylogenetic data and −" similarity in salient chemotaxonomic characteristics of taining (l ) 17 g cornmeal agar (Difco), 8 g peptone from soy meal, 1 g K HPO ,1gKHPO ,02 g MgSO ;7H O, Corynebacterium aquaticum Clavibacter xyli # % # % n % # ‘ ’ and 0n5 g glucose, 1 g cysteine-free base, 2 g bovine serum subsp. cynodontis, Sasaki et al. (1998) suggested that a albumin and 15 mg bovine haemin chloride, pH to 6n6 new genus should be established to accommodate (Davis et al., 1980). these organisms. In addition, a high level of phylo- T Cell chemistry. Shake cultures of VKM Ac-1401 and genetic similarity of Clavibacter xyli subsp. cynodontis ‘Corynebacterium aquaticum’ used for chemical analysis and Clavibacter xyli subsp. xyli was shown (Lee et al., were grown in liquid CB media. For strains Clavibacter xyli 1997), supporting the latter subspecies to be another subsp. cynodontis, SC medium was used. Cell walls were member of the genus. prepared from wet cells which were disrupted by sonifi- cation, separated from unbroken cells by centrifugation and In the course of studying micro-organisms from root purified using trypsin and 2% SDS as described by galls induced by the grass root gall nematode Sub- Streshinskaya et al. (1979). The cell wall preparations were anguina radicicola in annual meadow grass (Poa hydrolysed with 6 M HCl at 105 mC for 6 h (Schleifer & annua), we found a new set of coryneform bacteria Kandler, 1972). The presence of DAB was detected by TLC which were phenotypically close to ‘Corynebacterium (Bousfield et al., 1985). Quantitative determination of the aquaticum’. In this paper, we present the results of amino acids was performed with an LC 600 amino acid the taxonomic study of a representative of these analyser (Biotronic). Whole-cell sugars were determined by organisms. Based on data published previously and the method of Hasegawa et al. (1983). Cell wall sugars were obtained in this study, we propose a new genus, studied in acid hydrolysates (3 M trifluoroacetic acid, 100 mC, 6 h) as described previously (Maltsev et al., 1992). Leifsonia gen. nov., to accommodate bacteria from P. Menaquinones were extracted and purified as described by annua root galls, ‘Corynebacterium aquaticum’ Leifson Collins et al. (1977), and their composition was determined 1962 and Clavibacter xyli Davis et al. 1984. using a model MAT 8430 mass spectrometer (Finnigan). Isolation and analysis of phospholipids were performed by TLC using the method of Minnikin et al. (1978). Fatty acid METHODS composition was analysed as described by Evtushenko et al. Isolation of bacteria. The plants of Poa annua infected by the (1989). grass root gall nematode Subanguina radicicola were col- Morphology and physiology. Morphology and life cycle lected in Moscow Region in June 1991. The bacteria were were studied in cultures grown on CB or SC agar by phase- isolated from root galls of these herbarium samples in 1993. contrast microscopy. Physiological features were examined Surfaces of the root galls were sterilized with 75% (v\v) as described previously (Zgurskaya et al., 1993). CB agar ethanol for 1 min and dried; the galls were pre-incubated in was used to check the strains for their tolerance to antibiotics sterile tap water at 24 mC for 2 h, cut into pieces, added to and growth inhibitors. Oxidase activity was tested using 1% 2 ml NaCl solution at a concentration of 0n85% (w\v), and (w\v) tetramethyl-p-phenylenediamine solution on filter ground using a pestle. Then, 10% (w\v) KOH solution was paper discs as reported by Groth et al. (1996).

372 International Journal of Systematic and Evolutionary Microbiology 50 Leifsonia gen. nov.

DNA isolation and hybridization. Genomic DNA was iso- filaments fragmented into shorter (about 2n0–2n5 µm) lated from thawed cells resuspended in TE buffer (10 mM elements which were usually motile by peritrichous Tris, 1 mM EDTA; pH 8 0) by the technique of Bradley et $ n flagella. Cells with clubbed ends were frequently seen. al. (1973). [H ]DNAs from reference strains were obtained In old cultures, irregular rods predominated and by in vitro nick translation. DNA–DNA hybridization was occurred singly, in pairs or in short chains with performed by using the membrane filter method (Meyer & Schleifer, 1978; Stackebrandt et al., 1981) as described diphtheroid arrangements. Colonies of ‘Coryne- previously (Evtushenko et al., 1989). bacterium aquaticum’ were yellow, circular, convex, opaque, glistening and butyrous. The young cells Analysis of 16S rDNA sequences. The 16S rRNA gene was amplified by using the PCR method and prokaryotic 16S (16–24 h) were Gram-positive, non-spore-forming, rDNA universal primers fD1 and rP1 (Weisburg et al., 1991) pleomorphic motile rods (1n2–2n5i0n4–0n7 µm) with as described by Zhou et al. (1997). PCR products were long peritrichous flagella (length 10 µm or more), as detected by agarose gel electrophoresis and visualized by UV reported by Leifson (1962). Primary branching was fluorescence after ethidium bromide staining, and were then observed occasionally. In 3–4 d and older cultures, purified and concentrated by using a Wizard PCR Preps shorter pleomorphic rods and coccoid cells were DNA purification system (Promega), according to the predominant and occurred singly, in pairs or in short manufacturer’s instructions. The 16S rDNA sequences were chains with diphtheroid arrangements. The above analysed directly using the purified PCR products as the rapidly growing organisms differed morphologically sequencing template. The sequencing reactions were per- from the slowly growing Clavibacter xyli subsp. formed by automated fluorescent Taq cycle sequencing using the ABI Catalyst 800 and a model ABI 373A automatic cynodontis (SC medium), whose cells were consider- DNA sequencer (Applied Biosystems), according to the ably thinner (0n2–0n3 µm). Both of the Clavibacter xyli subsp. cynodontis strains examined, VKM Ac-2032 manufacturer’s protocol. Nucleotide substitution rates were T calculated as described by Kimura & Ohta (1972), and the and VKM Ac-2041 , were weakly motile in living phylogenetic tree was constructed by the neighbour-joining cultures, although the flagellated cells were not method (Saitou & Nei, 1987) with   software observed previously (Davis et al., 1984). (Thompson et al., 1994). Tree topologies were evaluated by bootstrap analysis of the sequence data with the same software. GenBank, DDBJ and EMBL accession numbers Cell chemistry for reference 16S rDNA sequences of the strains used in this The cell wall of strain VKM Ac-1401T contained analysis are given in Fig. 1. Brevibacterium linens DSM T glycine, glutamic acid, DAB and alanine in a molar 20425 (X77451) was used as an outgroup member. ratio close to 1:1:2:1. The qualitative sugar com- position of the whole cells was similar in all strains RESULTS AND DISCUSSION examined (glucose, galactose, mannose, fucose, ribose, Isolation rhamnose and traces of xylose) except for strain VKM Ac-1401T, which lacked fucose. Quantitative cell wall Most of the colonies that developed from the ground sugar analysis showed a predominant amount of gall suspension plated on CB agar were yellow, rhamnose and minor amounts of glucose, galactose orange–yellow or orange. Numerous coryneform iso- and mannose in strain VKM Ac-1401T. In the cell wall lates forming colonies of different tints of yellow were of ‘Corynebacterium aquaticum’ VKM Ac-1400T and found to be motile. One representative of the last T T Clavibacter xyli subsp. cynodontis VKM Ac-2041 , group, strain VKM Ac-1401 , was studied in detail in fucose was found additionally, in significant or trace this work. amounts (Table 1). In addition, members of Clavi- bacter xyli subsp. cynodontis and Clavibacter xyli Morphology subsp. xyli were previously shown to be similar in the Colonies of strain VKM Ac-1401T on CB agar were composition of cell wall sugars, although fucose was yellowish to deep yellow with age, circular, convex, determined as a main sugar along with rhamnose, and opaque, glistening and butyrous. The young culture mannose was not found (Davis et al., 1984). (16–24 h) consisted of Gram-positive, curved long Cell wall teichoic acids were not found in the two T rods (4–6i0n6–0n9 µm) or filaments with primary tested strains VKM Ac-1401 and ‘Corynebacterium branching. In a 3–4-d-old culture, the rods and aquaticum’ VKM Ac-1400T (I. B. Naumova, personal

Table 1. Cell wall sugars of the strains studied (molar ratio)

Strain Glucose Galactose Mannose Fucose Rhamnose

VKM Ac-1401T 0n71n00n7 k 19n4 ‘Corynebacterium aquaticum’ VKM 1n31n02n73n97n6 Ac-1400T Clavibacter xyli subsp. cynodontis 0n61n0 Trace Trace 0n6 VKM Ac-2041T

International Journal of Systematic and Evolutionary Microbiology 50 373 L. I. Evtushenko and others

Table 2. Phenotypic differences between strains VKM Ac-1401T,‘Corynebacterium aquaticum’ VKM Ac-1400T and Clavibacter xyli subspecies

Character VKM Ac-1401T ‘Corynebacterium Clavibacter xyli Clavibacter xyli aquaticum’ subsp. xyli* subsp. cynodontis* VKM Ac-1400T

Cell width (µm) 0n6–0n90n4–0n70n20n2–0n3 Cell length (µm) 8n0–15n01n2–2n55n03n0–6n0 Visible colonies (day) 2 2 7–10 5–7 Colony colour Yellow Yellow White Yellow Growth on CB agar jj k k Fucose in the cell wall kj j j Oxidase kj k k Production of H#S kj k k Voges–Proskauer test jk k j Methyl red test jk k j Utilization of: Citrate kj k j Gluconate kj k k Propionate jj k k Hydrolysis of: Starch kj k j Aesculin jk k k Gelatin jk k k Growth in 5% NaCl kj k k Acid from: -Arabinose jj k k -Galactose jj k k Salicin jj k k -Sucrose jj k k Used as C-source for growth: Adonitol kj   -Arabinose jk   Inositol kj   Melezitose kj   Raffinose jk   -Sorbose kj   " Tolerance to antibiotics (µgml− ): Chloramphenicol (10) kj   Doxycycline (5) kj   Erycycline (10) kj   Gramicidin (50) kj   Rifampicin (30) jk   Major menaquinones MK-11 MK-11, 10† MK-11, 12† Source of isolation Nematode gall on Distilled water Saccharum, interspecific Cynodon dactylon P. annua roots hybrid

* Data from Davis et al. (1984). † Data from Sasaki et al. (1998).

communication). The predominant menaquinone was acids (16:0, iso-15:0, 15:0, iso-17:0, 18:1 and iso- MK-11; a minor amount of MK-10 and traces of MK- 18:0) were present in small amounts, 1% or less. 12 were also detected. The principal phospholipids Mycolic acids were not found. In ‘Corynebacterium were phosphatidylglycerol and diphosphatidyl- aquaticum’ and Clavibacter xyli subsp. cynodontis, the glycerol. Among cellular fatty acids of strain VKM lipid characteristics were similar as a whole (Collins & Ac-1401T, large proportions of anteiso- and iso- Jones, 1980; Suzuki et al., 1996; Sasaki et al., 1998), branched acids were determined (36n3% anteiso-15:0, including the predominance of anteiso-branched 45n4% anteiso-17:0 and 15n6% iso-16:0). Other fatty fatty acids as reported by Collins & Jones (1980),

374 International Journal of Systematic and Evolutionary Microbiology 50 Leifsonia gen. nov.

...... Fig. 1. Phylogenetic tree showing the position of strain VKM Ac-1401T based on 16S rDNA analysis. The sequence of strain Brevibacterium linens DSM 20425T (X77451) served as an outgroup sequence (not presented). Accession numbers of nucleotide sequences are given in parentheses. Numbers within the dendrogram indicate the percentages of occurrence of the branching order in 1000 bootstrapped trees. Bar, 1 nucleotide substitution per 100 nucleotides.

Henningson & Gudmestad (1991) and Suzuki et al. sequences indicated that our strain was most similar to T (1996). ‘Corynebacterium aquaticum’ DSM 20146 (l VKM Ac-1400T), Clavibacter xyli subsp. cynodontis JCM T T Physiology 9733 (l VKM Ac-2041 ) and Clavibacter xyli subsp. xyli F1A. All above organisms formed a separate The rapidly growing strains VKM Ac-1401T and T phylogenetic branch with a 99n8% bootstrap rep- ‘Corynebacterium aquaticum’ VKM Ac-1400 were lication value attached to the Agromyces cluster (Fig. T aerobic, catalase-positive and mesophilic, with a 1). Strain VKM Ac-1401 exhibited 98n4 and 98n1% growth optimum at 24–28 mC. These strains differed 16S rDNA sequence similarity to the sequences of from each other in the oxidase reaction, utilization of ‘Corynebacterium aquaticum’ and Clavibacter xyli adonitol, -arabinose, inositol, melezitose, raffinose subsp. cynodontis, respectively, whereas the similarity and -sorbose as sole carbon sources, hydrolysis of between ‘Corynebacterium aquaticum’ and Clavibacter aesculin, gelatin and starch, production of H#S, xyli subsp. cynodontis was 98n8%. A high level of Voges–Proskauer and methyl red tests, tolerance to phylogenetic similarity of Clavibacter xyli subsp. xyli 5% NaCl and some antibiotics, utilization of citrate to Clavibacter xyli subsp. cynodontis was shown and gluconate and some other characteristics (Table previously by Lee et al. (1997). 2). Most of these characteristics were previously found to be negative in the slowly growing organisms of the DNA–DNA hybridization study species Clavibacter xyli (Davis et al., 1984). Strains VKM Ac-1401T,‘Corynebacterium aquaticum’ T 16S rDNA sequence analysis VKM Ac-1400 and Clavibacter xyli subsp. cynodontis VKM Ac-2041T were hybridized against reference $ More than 1480 bases of the 16S rDNA sequence were [H ]DNA of strains VKM Ac-1401T and ‘Coryne- determined in strain VKM Ac-1401T. Comparison of bacterium aquaticum’ VKM Ac-1400T. DNA–DNA the sequence of this strain with available reference homology between VKM Ac-1401T and ‘Coryne-

International Journal of Systematic and Evolutionary Microbiology 50 375 L. I. Evtushenko and others

Table 3. DNA relatedness (%) between studied strains

Strain VKM Ac-1401T ‘Corynebacterium aquaticum’ VKM Ac-1400T

VKM Ac-1401T 100n044n6 ‘Corynebacterium aquaticum’ VKM Ac-1400T 43n6 100n0 Clavibacter xyli subsp. cynodontis VKM Ac-2041T 40n940n1 Agromyces cerinus subsp. cerinus VKM Ac-1340T 5n07n7

bacterium aquaticum’ VKM Ac-1400T was on average and that they are responsible for or associated with 44n1%, while each of these organisms exhibited plant diseases (Davis et al., 1980, 1984; Metzler et al., 40n9 and 40n1% DNA relatedness with Clavibacter 1997), while the natural habitat of Agromyces spp. is xyli subsp. cynodontis VKM Ac-2041T, respectively mainly soil (Gledhill & Casida, 1969; Zgurskaya et al., (Table 3). 1992; Suzuki et al., 1996). Thus the evidence at hand suggests that the bacteria Taxonomic affiliation of strains under discussion comprise a new genus of the family As mentioned above, our isolate from P. annua root Microbacteriaceae (Park et al., 1993). This conclusion galls induced by the grass root gall nematode Sub- is in line with the opinion expressed earlier by Rainey anguina radicicola was most similar to ‘Coryne- et al. (1994) and Sasaki et al. (1998). We propose the bacterium aquaticum’ and Clavibacter xyli both phylo- name Leifsonia for this new genus, in recognition of genetically and in salient chemotaxonomic charac- Einar Leifson, who isolated and described ‘Coryne- teristics. These organisms form a distinct phylogenetic bacterium aquaticum’ (Leifson, 1962). The phenotypic branch close to the Agromyces spp. cluster. At the properties differentiating the new genus Leifsonia from chemotaxonomic level, strains VKM Ac-1401T, other genera of the family Microbacteriaceae with ‘Corynebacterium aquaticum’ VKM Ac-1400T and DAB in the cell wall are summarized in Table 4. T Clavibacter xyli subsp. cynodontis VKM Ac-2041 are The DNA–DNA homology between the rapidly grow- distinguished from Agromyces spp. by the major ing ‘Corynebacterium aquaticum’ VKM Ac-1400T and menaquinone, which is the essential differentiating isolate VKM Ac-1401T and their phenotypic dif- characteristic of genera containing DAB in their ferences in cell wall sugar composition (Table 1), the peptidoglycan (Zgurskaya et al., 1992, 1993; Groth et oxidase reaction, the tolerance to 5% NaCl, the al., 1996; Takeuchi et al., 1996; Suzuki et al., 1997; Voges–Proskauer test, the production of H#S and Sasaki et al., 1998). Furthermore, members of the quite a number of other physiological characteristics ‘Corynebacterium aquaticum’ group differ from agro- (Table 2) indicate that these strains belong to different mycetes by their cell wall composition: ‘Coryne- species (Wayne et al., 1987). The names Leifsonia bacterium aquaticum’ and Clavibacter xyli subsp. aquatica (ex Leifson 1962) nom. rev., comb. nov. and cynodontis contained both - and -DAB isomers in Leifsonia poae sp. nov. are proposed. The species their peptidoglycan (Sasaki et al., 1998), whereas Leifsonia aquatica nom. rev., comb. nov. and the Agromyces spp. had -DAB almost exclusively. Also, T T strains Leifsonia aquatica VKM Ac-1400 and our isolate, VKM Ac-1401 , and ‘Corynebacterium Leifsonia poae VKM Ac-1401T are proposed as types aquaticum’ do not contain cell wall teichoic acids (I. B. of the respective taxa. Naumova, personal communication), in contrast to most members of the genus Agromyces, which possess In turn, both slowly growing subspecies of Clavibacter these polymers (Shashkov et al., 1993, 1995; Gnilozub xyli, Clavibacter xyli subsp. cynodontis and Clavibacter et al., 1994). Altenburger et al. (1997) found that the xyli subsp. xyli, causing bermudagrass (Cynodon total polyamine content in strain VKM Ac-1401T dactylon) stunting disease and ratoon stunting disease (designated ‘Agromyces sp.’ DL 89 in the respective of sugar cane (Saccharum, interspecies hybrid), are −" publication) was 0n62 µmol g , while the true markedly distinguished from Leifsonia poae sp. nov. −" Agromyces species contain only 0n21–0n28 µmol g of and Leifsonia aquatica nom. rev., comb. nov. in these compounds. In addition, all members of the morphology, physiological features, including the re- genus Agromyces are non-motile (Gledhill & Casida, quirement of complex media for growth, and in their 1969; Zgurskaya et al., 1992; Suzuki et al., 1996), sources of isolation (Table 2). Furthermore, these whereas our isolate, ‘Corynebacterium aquaticum’ and subspecies have a lower DNA GjC content Clavibacter xyli subsp. cynodontis were motile. It is (66 mol%) (Davis et al., 1984) than Leifsonia aquatica interesting that most of the organisms of the group nom. rev., comb. nov. VKM Ac-1400T (about under consideration, except strain ‘Corynebacterium 70 mol%) (Yamada & Komagata, 1970b; Dopfer et aquaticum’ VKM Ac-1400T, were isolated from plants al., 1982). These differences between Clavibacter xyli

376 International Journal of Systematic and Evolutionary Microbiology 50 Leifsonia gen. nov.

Table 4. Salient phenotypic characteristics that differentiate Leifsonia gen. nov. from other genera containing DAB in their peptidoglycan ...... The table is based on the data obtained in this study and extracted from relevant references (see text). R, Rods; F, filaments; C, cocci; j, positive; k, negative; -DAB,  and  isomers of diaminobutyric acid; GABA, γ-aminobutyric acid; Asp, asparagine; Thr, threonine; PUT, putrescine; SPM, spermine; SPD, spermidine; , no data.

Genus Morphology Motility Major Peptidoglycan Polyamine Total Growth menaquinone amino acid* pattern polyamine at 18 mC (µmol g−1)

Leifsonia gen. nov. R, F j MK-11, 10 -DAB PUT 0n6 j Agromyces F, R k MK-12 -DAB PUT 0n2–0n3 j Clavibacter R k MK-9 -DAB SPD, SPM 2n0–7n0 j Rathayibacter R k MK-10 -DAB SPD, SPM 4n8–18n0 j Cryobacterium R k MK-10 -DAB   k Leucobacter R k MK-11 -DAB, GABA   j Agrococcus C k MK-12, 11 -DAB, Asp, Thr SPM 0n9 j * All organisms also contain alanine, glutamic acid and glycine. subsp. cynodontis and the rapidly growing species trations of polyamines are low; putrescine is the Leifsonia aquatica nom. rev., comb. nov. and Leifsonia predominant compound. The DNA GjC content poae sp. nov. were demonstrated to be of the species ranges from 66n0 to 73 mol%. Some species dis- level by DNA–DNA homology values, 40n1 and tinguished by tolerance to antibiotics. Forms a co- 40n9%, respectively. The phylogenetic similarity of herent phylogenetic cluster attached to the branch of members of different subspecies of Clavibacter xyli was Agromyces spp. The type species of this genus is shown previously (Lee et al., 1997). Based on the Leifsonia aquatica. The genus description is based on above data, we propose to reclassify the species the data cited above and obtained in this study. Clavibacter xyli with two subspecies into the new genus Leifsonia as Leifsonia xyli (Davis et al. 1984) Description of Leifsonia aquatica (ex Leifson 1962) comb. nov., Leifsonia xyli subsp. xyli (Davis et al. nom. rev., comb. nov. 1984) comb. nov. and Leifsonia xyli subsp. cynodontis (Davis et al. 1984) comb. nov. Leifsonia aquatica; basonym, ‘Corynebacterium aquaticum’ Leifson 1962. Description of Leifsonia gen. nov. The colonies on CB agar are yellowish to deep yellow with age, circular, somewhat convex, glistening, Leifsonia (Leif.so’ni.a. M.L. fem. n. Leifsonia named opaque and butyrous. Cells are Gram-positive, non- after Einar Leifson, who isolated and described the spore-forming, pleomorphic rods (length 1 2–2 5 µm, first organism of this genus). n n width 0n4–0n7 µm); motile by long peritrichous flagella The colonies are yellow or white, circular, somewhat (10 µm or more). Primary branching is observed convex, glistening, opaque and butyrous. The cells are occasionally. In older cultures, short rods or coccoid Gram-positive, non-spore-forming, irregular rods or cells predominate and occur singly, in pairs or in short filaments which usually fragment into shorter rods or chains with diphtheroid arrangements. Rod–coccus coccoid elements. Primary branching occurs in young cycle is observed. Aerobic. Catalase- and oxidase- cultures of some species. Usually motile. Non-acid- positive. Growth occurs between 7 and 37 mC; op- fast. Mesophilic. Obligately aerobic. Catalase-positive. timum is 24–28 mC. Adonitol, cellobiose, dulcitol, - Oxidase test is variable among species. Cell wall fructose, -galactose, -glucose, glycerol, inositol, peptidoglycan of B-type contains glycine, glutamic lactose, maltose, -mannitol, -mannose, melezitose, acid, DAB and alanine in a molar ratio close to melibiose, -rhamnose, salicin, -sorbose, sucrose, 1:1:2:1; both isomers, -DAB and -DAB, are trehalose, turanose and -xylose are used for growth usually present in almost equal proportions. The major as carbon sources in mineral medium supplemented menaquinone is MK-11; MK-10 is present. Cell wall with 0n1% (w\v) yeast extract and 0n1% (w\v) sugars consist of a predominant amount of rhamnose casitone. -Arabinose, -fucose, inulin, lyxose, meso- and minor amounts of glucose, galactose and man- erythritol, raffinose, ribose and sorbitol are not used as nose; some species contain fucose. Cell wall teichoic carbon source in the same medium. Acids are produced acids and mycolic acids are not present. The principal from -arabinose, -fructose, -galactose, mannose phospholipids are phosphatidylglycerol and di- and sucrose, but not from melibiose. An alkaline phosphatidylglycerol. Among fatty acids, anteiso- reaction is observed with acetate, citrate, formate, 15:0, anteiso-17:0 and iso-16:0 predominate. Concen- fumarate, gluconate, α-keto-1-glutarate, malonate and

International Journal of Systematic and Evolutionary Microbiology 50 377 L. I. Evtushenko and others propionate, but no reaction occurs with oxalate, but no reaction occurs with citrate, formate, fumarate, succinate or tartrate. Methyl red and Voges–Proskauer gluconate, oxalate, succinate or tartrate. Methyl red tests are negative. H#S is produced. Tween 40 and and Voges–Proskauer tests are positive. H#S is not starch are decomposed; aesculin, gelatin, hypox- produced. Aesculin and gelatin are decomposed. anthine, tyrosine, xanthine, casein, Tween 60, Tween Hypoxanthine, starch, tyrosine, xanthine, casein, 80 and urea are not hydrolysed. Tolerates 5% (w\v) Tween 40, Tween 60 and Tween 80 are not hydrolysed. " NaCl, 0 02% (w\v) potassium tellurite and the fol- Sensitive to 5% (w\v) NaCl, doxycycline (5 µgml− ) n " " lowing antibiotics: ampicillin (50 µgml− ), chloram- and erycycline (10 µgml− ), but tolerant to potassium " " phenicol (10 µgml− ), doxycycline (5 µgml− ), tellurite at 0 02% (w\v) and the following antibiotics: " " n " " erycycline (10 µgml− ), gentamicin (50 µgml− ), gra- ampicillin (50 µgml− ), chloramphenicol (10 µgml− ), " " " " micidin (50 µgml− ), lincomycin (50 µgml− ), neo- gentamicin (50 µgml− ), gramicidin G (10 µgml− ), " " " " mycin (50 µgml− ), penicillin G (50 µgml− ), rifam- lincomycin (50 µgml− ), neomycin (50 µgml− ), peni- " " " " picin (10 µgml− ), streptomycin (50 µgml− ) and cillin G (50 µgml− ), rifampicin (30 µgml− ), strep- " " " tetracycline (10 µgml− ). Sensitive to rifampicin (30 µg tomycin (50 µgml− ) and tetracycline (10 µgml− ). −" ml ). DNA GjC content is about 70 mol%. Cell Cell wall sugars consist of a predominant amount of wall sugars consist of a predominant amount of rhamnose and minor amounts of glucose, galactose rhamnose and minor amounts of fucose, glucose, and mannose. No teichoic acids are present in the cell galactose and mannose. No teichoic acids are present wall. The major menaquinone is MK-11. Other salient in the cell wall. The major menaquinones are MK-11 chemotaxonomic characteristics are as given above in and MK-10. Other salient chemotaxonomic charac- the genus description. The type strain is VKM Ac- teristics are as given above in the genus description. 1401T. Isolated from Poa annua root gall induced by T T The type strain is VKM Ac-1400 l DSM 20146 l the grass root gall nematode Subanguina radicicola. JCM 1368T isolated from distilled water. The above description is based on the data of Leifson (1962), Description of Leifsonia xyli (Davis et al. 1984) comb. Yamada & Komagata (1970b), Collins & Jones (1980), nov. Dopfer et al. (1982) and our study. Leifsonia xyli (Davis et al. 1984); basonym, Clavibacter Description of Leifsonia poae sp. nov. xyli Davis et al. 1984. Leifsonia poae (po’a.e. M.L. gen. n. poae of Poa, The description of this species is as presented by Davis generic name of the annual meadow grass, Poa annua, et al. (1984) and supplemented by Sasaki et al. (1998). the source of the type strain of this species). Phenotypic characteristics differentiating Leifsonia xyli (Davis et al. 1984) comb. nov. from other species The colonies on CB agar are yellowish to deep yellow of the genus Leifsonia are presented in Table 2. The with age, circular, somewhat convex, glistening, type subspecies is Leifsonia xyli subsp. xyli (Davis et opaque and butyrous. The young culture (16–24 h) al. 1984). consists of Gram-positive, curved long cells or filaments (length 8–15 µm, width 0n6–0n9 µm) with primary branching; after 3–4 d cultivation on CB Description of Leifsonia xyli subsp. xyli (Davis et al. agar, they break up into shorter irregular fragments 1984) comb. nov. (length 2n0–2n5 µm), which are usually motile by Leifsonia xyli subsp. xyli (Davis et al. 1984); basonym, peritrichous flagella. Cells with clubbed ends are Clavibacter xyli subsp. xyli Davis et al. 1984. frequently seen. In old cultures, pleomorphic rods are predominant and occur singly, in pairs or in short The description of this subspecies is as presented by chains with diphtheroid arrangements. Growth occurs Davis et al. (1984). The additional phylogenetic charac- between 7 and 37 mC; the optimum is 24–28 mC. teristics of the subspecies are as given by Lee et al. Aerobic. Catalase-positive. Oxidase, tested by using (1997). Phenotypic properties differentiating the sub- 1% (w\v) tetramethyl-p-phenylenediamine solution, species from other intrageneric taxa of the genus Leifsonia are presented in Table 2. The type strain is is negative. -Arabinose, cellobiose, -fructose, - T T galactose, -glucose, glycerol, dulcitol, lactose, malt- ICMP 7127 l LMG 7352 . ose, -mannitol, -mannose, melibiose, methyl α-- glucopyranoside, raffinose, -rhamnose, salicin, su- Description of Leifsonia xyli subsp. cynodontis (Davis crose and -xylose are used as sole carbon sources in et al. 1984) comb. nov. mineral medium supplemented with 0 1% (w\v) yeast n Leifsonia xyli subsp. cynodontis (Davis et al. 1984); extract and 0n1%(w\v) casitone. Adonitol, dextran, - fucose, inositol, inulin, lyxose, melezitose, meso-eryth- basonym, Clavibacter xyli subsp. cynodontis (Davis et ritol, sorbitol, sorbose, ribose and trehalose are not al. 1984). utilized as the only carbon source in the same medium. The description of this subspecies is as presented by Acids are produced from -arabinose, -fructose, - Davis et al. (1984) and supplemented by Sasaki et al. galactose, mannose and sucrose, but not from (1998). Phenotypic characteristics differentiating the melibiose. An alkaline reaction is observed with α- subspecies from other intrageneric taxa of the genus keto-1-glutarate, malate, malonate and propionate, Leifsonia are presented in Table 2. The type strain is

378 International Journal of Systematic and Evolutionary Microbiology 50 Leifsonia gen. nov.

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