Agromyces Luteolus Sp. Nov., Agromyces Rhizospherae Sp. Nov

International Journal of Systematic and Evolutionary Microbiology (2001), 51, 1529–1537 Printed in Great Britain

Agromyces luteolus sp. nov., Agromyces rhizospherae sp. nov. and Agromyces bracchium sp. nov., from the mangrove rhizosphere

Institute for Fermentation, Mariko Takeuchi and Kazunori Hatano Osaka, 17-85, Juso- honmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan Author for correspondence: Mariko Takeuchi. Tel: j81 06 6300 6555. Fax: j81 06 6300 6814. e-mail: takeuchi-mariko!ifo.or.jp

The taxonomic positions of four strains isolated from the mangrove rhizosphere were studied by a polyphasic approach using phenotypic, chemotaxonomic and genetic methods. The four isolates contain 2,4- diaminobutyric acid in their peptidoglycan, and rhamnose as the major cell wall sugar. The predominant menaquinones are MK-12 and MK-11. The

predominant cellular fatty acids are iso-C16:0, anteiso-C15:0 and/or anteiso-C17:0. The GMC content of the DNA ranges from 700to733 mol%. The four strains formed a coherent cluster with Agromyces species in a phylogenetic inference based on 16S rDNA sequences. Interestingly, the four isolates grew well in the presence of 5% NaCl. The differences in some phenotypic and chemotaxonomic characteristics, 16S rDNA sequence similarity data and DNA–DNA relatedness data indicate that the four isolates represent three new species in the genus Agromyces, for which are proposed the names Agromyces luteolus for strain 8T (IFO 16235T l VKM Ac-2085T ), Agromyces bracchium for strain 65T (IFO 16238T l VKM Ac-2088T ) and Agromyces rhizospherae for strains 14T (IFO 16236T l VKM Ac-2086T ) and 58(5) (IFO 16237 l VKM Ac-2087).

Keywords: Agromyces luteolus sp. nov., Agromyces rhizospherae sp. nov., Agromyces bracchium sp. nov.

INTRODUCTION Micrococcus, 1 strain Gordonia and 1 strain Rhodo- coccus, and 1 strain which contained -diamino- The diversity and populations of micro-organisms in pimelic acid in the cell wall (Takeuchi & Hatano, 1999) the mangrove rhizosphere have been studied at the was of undetermined taxonomic position. Institute for Fermentation, Osaka (IFO) for several years. It has been suggested that the mangrove The object of the present study was to determine the rhizosphere is a good source for isolating new and exact taxonomic positions of the four strains of diverse actinomycetes (Hatano, 1997; Takeuchi & the genus Agromyces (Casida, 1986), strains 8 (IFO 16235T), 14 (IFO 16236T), 58(5) (IFO 16237) and 65 Hatano, 1998, 1999). We have previously isolated 25 T actinobacteria from the rhizosphere of mangroves in (IFO 16238 ), on the basis of morphological, physio- the estuary of the Shiira River, Iriomote Island, and logical and chemotaxonomic characteristics, together determined their taxonomic positions at the genus with phylogenetic studies based on 16S rDNA level on the basis of phylogenetic and chemotaxonomic sequences and DNA relatedness. traits. Four strains were determined to be members of the genus Agromyces, 13 strains Cellulomonas, 2 strains These strains are yellow, ﬁlamentous, elementary Microbacterium, 2 strains Mycobacterium, 1 strain branching, irregular rod-shaped bacteria with all of the characteristic chemotaxonomic markers of the ...... genus Agromyces, including 2,4-diaminobutyric acid Abbreviation: DAB, 2,4-diaminobutyric acid. (DAB) in their cell walls, DNA GjC contents ranging The DDBJ accession numbers for the 16S rDNA sequences determined in from 70n0to73n3 mol%, and MK-12 with smaller this study are AB023356–AB023359. amounts of MK-11 and\or MK-13 menaquinones.

01776 # 2001 IUMS 1529 M. Takeuchi and K. Hatano

The genus Agromyces was established by Gledhill & Chemotaxonomic characterization. Analyses of cell wall Casida (1969) for the single species Agromyces amino acid and sugar composition and the acyl type of the ramosus, a filamentous, branching, catalase-negative peptidoglycan, menaquinone composition, cellular fatty actinomycete isolated from soil. Since then, two species acid composition and DNA GjC content were performed each with two subspecies, Agromyces cerinus subsp. as described previously (Takeuchi & Hatano, 1998). cerinus and Agromyces cerinus subsp. nitratus, and DNA–DNA hybridization. DNA–DNA hybridization was Agromyces fucosus subsp. fucosus and Agromyces carried out fluorometrically in microdilution wells by using fucosus subsp. hippuratus (Zgurskaya et al., 1992), and biotinylated DNA (Ezaki et al., 1989). later a further species, Agromyces mediolanus (Suzuki 16S rDNA sequence determination and phylogenetic analy- et al., 1996), have been added to the genus. sis. A 16S rDNA fragment corresponding to position 8–1540 in the Escherichia coli numbering system (Brosius et al., By comparing the four isolates from the mangrove 1978) was amplified by PCR, sequenced directly with a rhizosphere with previously described Agromyces ThermoSequenase cycle sequencing kit with 7-deaza-dGTP species, we conclude that the four isolates represent (Amersham), and analysed with a LI-COR 4200L-2 (2-dye three new species in the genus Agromyces. In this system) DNA sequencer, following the manufacturer’s paper, we propose the new species names Agromyces instructions. The 16S rDNA sequences were aligned with T luteolus for strain 8 (IFO 16235 ), Agromyces published sequences available from DDBJ, EMBL and bracchium for strain 65 (IFO 16238T) and Agromyces GenBank. ‘Agromyces succinolyticus’ is a DAB-containing rhizospherae for strains 14 (IFO 16236T) and 58(5) coryneform bacterium isolated from soil (K.-I. Suzuki & (IFO 16237). J. Sasaki, unpublished data). The   version 1.6 (Thompson et al., 1994) software package was used to generate the evolutionary distances (Knuc values) (Kimura, METHODS 1980) and the similarity values. A phylogenetic tree was constructed by the neighbour-joining method (Saitou & Nei, Bacterial strains and cultivation. Soil samples were taken 1987), and the topology of the tree was evaluated by the from the rhizosphere of mangroves in the estuary of the bootstrap resampling method (Felsenstein, 1985) with 1000 Shiira River, Iriomote Island, Japan, in July 1997. Soils were replicates. stored at 5–8 mC, dried in air at 17 mC for 6 d, and then passed through sieves of 20 and 60 mesh to separate soil and fine roots. Strain 8 (IFO 16235T ) was isolated from the RESULTS AND DISCUSSION surface of roots of Sonneratia alba, strain 14 (IFO 16236T ) Morphological and physiological characteristics was isolated from soil of the rhizosphere of S. alba and T strains 58(5) (IFO 16237) and 65 (IFO 16238 ) were isolated The four strains studied in this work were Gram- from soil of the rhizosphere of Bruguera gymnorrhiza by the positive, non-spore-forming, irregular rods. The cells method described by Hayakawa & Nonomura (1987, 1989). measured 0 2–0 4by15–6 0 µm long (Fig. 1a); they A. cerinus subsp. cerinus IFO 15780T, A. cerinus subsp. n n n n nitratus IFO 15783T, A. fucosus subsp. fucosus IFO 15781T, differed in length from strain to strain, and tended to A. fucosus subsp. hippuratus IFO 15782T (Zgurskaya et al., be shorter in old cultures as shown in Fig. 1(b). T Filamentous elongation and branching were observed 1992), A. mediolanus IFO 15704 (Suzuki et al., 1996) and A. T T T ramosus IFO 13899T (Gledhill & Casida, 1969) were used for in strains IFO 16235 , IFO 16236 and IFO 16238 , comparison of physiological properties and for DNA–DNA but were not observed obviously in strain IFO 16237. hybridization tests with the isolates. Each strain was cul- All strains formed yellow to pale yellow, entire, convex tivated aerobically at 30 mC in peptone-yeast extract medium colonies on PY medium agar. Colonies sometimes (PY medium) containing 1% Polypepton (Wako Pure penetrated into the agar. Aerial mycelia were not Chemicals), 0n2% yeast extract and 0n1% MgSO%.7H#O produced. (pH 7n2). Cells used for biochemical tests were harvested by centrifugation during the stationary phase, washed with All strains grew optimally at a temperature of 20– water and lyophilized. 30 mC, but did not grow at 10 or 37 mC. They grew well Morphological and physiological characterization. For in the presence of 5% NaCl, but very little in the investigation of morphological characteristics, cultures presence of 8% NaCl. All strains were positive for grown on PY medium to the early growth phase (approx. catalase, hydrolysis of aesculin, gelatin and starch 16 h) and to stationary phase at 30 mC were observed with a (data not shown). Using the API CORYNE system, light microscope and a scanning electron microscope (model pyrazinamidase and aesculinase were positive, but JSM-5400; JEOL). Samples for scanning electron micro- alkaliphosphatase, nitrate reductase, β-glucuroni- scopy were prepared by fixing in 2% glutaraldehyde in dase, β-galactosidase, α-glucosidase and N-acetyl-β- potassium phosphate buffer for 4 h at 4 mC and 1% osmic glucosaminidase were negative for all strains. Using acid in potassium phosphate buffer for 16 h at 4 mC, the API 50CH system, acid was produced from dehydrating through a graded acetone series, critical point fructose, glucose, maltose and mannose, but was not drying (model HCP-2; Hitachi Koki), and then sputter-   coating with palladium under vacuum. Motility was de- produced from adonitol, -arabinose, -arabitol, termined by the hanging-drop method. Biochemical charac- dulcitol, erythritol, -fucose, N-acetylglucosamine, teristics of the Agromyces strains were examined by using the α-methyl--glucoside, glycerol, inositol, inulin, α- API CORYNE system and API 50CH with AUX Medium. methyl--mannoside, sorbitol, sorbose, -tagatose, Other physiological characteristics were determined as -xylose, β-methyl--xyloside or xylitol. Other bio- described by Cowan & Steel (1965). chemical characteristics that showed different results

1530 International Journal of Systematic and Evolutionary Microbiology 51 Three new species in the genus Agromyces

(a) (b)

(i)

(ii)

(iii)

(iv)

...... Fig. 1. Scanning electron micrographs of A. luteolus, A. rhizospherae and A. bracchium grown on PY medium at 30 mC. A. luteolus IFO 16235T (i), A. rhizospherae IFO 16236T (ii), A. rhizospherae IFO 16237 (iii) and A. bracchium IFO 16238T (iv) in the early growth phase (a) and in the exponential growth phase (b). Bars, 1 µm. among the four strains are shown in Table 1. Two and lactose. Strain IFO 16235T was diﬀerentiated from strains, IFO 16236T and IFO 16237, showed the same strains IFO 16236T and IFO 16237 by acid production results in the API 50CH system with regard to acid from amidon, cellobiose, gentibiose, salicin and tre- production from all carbohydrates except for glycogen halose. Strain IFO 16238T was diﬀerentiated from IFO

International Journal of Systematic and Evolutionary Microbiology 51 1531 M. Takeuchi and K. Hatano

Table 1. Biochemical characteristics that differentiate the four new Agromyces strains ...... j, Positive reaction; j, weakly positive reaction; k, negative reaction.

Characteristic A. luteolus A. rhizospherae A. bracchium IFO 16235T IFO 16238T IFO 16236T IFO 16237

Pyrrolidonyl arylamidase* jjjk Acid production from:† Amidon kjjj Amygdalin kkkj -Arabinose kkkj Arbutin kkkj Cellobiose kjjj Galactose kkkj Gentiobiose kj j j Gluconate kkkj Glycogen kjkj Lactose jkjj Mannitol j kk j Melezitose kkkj Melibiose jw kk j Raﬃnose kkkj Rhamnose jw kkj Ribose kkkj Salicin kjjw j Sucrose jw kk j Trehalose jkkj -Turanose kkkj -Xylose kkkj

* API CORYNE system. † Carbohydrate assimilation reactions: API 50CH and AUX medium, incubated at 30 mC.

T T 16235 , IFO 16236 and IFO 16237 by pyrrolidonyl and anteiso-C"& ! as the second most common type. T : arylamidase activity and by acid production from IFO 16238 contained anteiso-C"&:! as the primary amygdalin, -arabinose, arbutin, galactose, gluconate, cellular fatty acid and iso-C"':! and anteiso-C"(:! as melezitose, raﬃnose, ribose, -turanose and -xylose. the second most common types. All strains except for IFO 16238T contained MK-12 and MK-11 as the T Chemotaxonomic characteristics major menaquinones. IFO 16238 contained MK-12 as the major menaquinone and MK-13 as the second The molar ratio of glycine, alanine and DAB to most common type. glutamic acid in the cell wall peptidoglycan isolated from cells of strains IFO 16235T, IFO 16236T, IFO T 16237 and IFO 16238 was (0n85–1n31):(0n55– Phylogenetic analysis 0n62) :(1n38–1n77) :1n00, as shown in Table 2. This ratio The 16S rDNA sequences of the four strains IFO is the same as that obtained for A. ramosus ATCC T T T T 16235 , IFO 16236 , IFO 16237 and IFO 16238 were 25173 (0n98:0n71:1n73:1n00) (Fiedler & Kandler, compared with representative members of the genus 1973). These cell walls were of an acetyl type. The main Agromyces and related micro-organisms. A phylo- sugar component of the cell wall of all strains was genetic tree (Fig. 2) showed that they formed a rhamnose, and small amounts of glucose and mannose coherent cluster with species of the genus Agromyces were also detected in the cell walls of all the strains (Takeuchi & Hatano, 1999). Strains IFO 16236T and studied. In addition, fructose and galactose were IFO 16237 had a 16S rDNA sequence similarity value present in the cell walls of strains IFO 16235T and IFO T of 99n2% with each other, and they represented a 16238 , respectively. distinct lineage within the genus Agromyces with All the four strains had cellular fatty acids of the iso- similarity values of 96n5–97n8% among the represen- and anteiso-branched type. All except IFO 16238T tative strains of previously described Agromyces T contained iso-C"':! as the primary cellular fatty acid species (data not shown). Strains IFO 16235 and IFO

1532 International Journal of Systematic and Evolutionary Microbiology 51 Three new species in the genus Agromyces

Table 2. Chemotaxonomic characteristics of the four new Agromyces strains

Characteristic A. luteolus A. rhizospherae A. bracchium IFO 16235T IFO 16238T IFO 16236T IFO 16237

Amino acid composition of cell wall (molar ratio to glutamic acid) Glutamic acid 1n00 1n00 1n00 1n00 Glycine 1n15 0n85 1n25 1n31 Alanine 0n55 0n60 0n56 0n62 DAB 1n77 1n38 1n56 1n47 Sugar composition of cell wall (%) Galactose kkk 6 Glucose 2 5 4 7 Fructose 25 kk k Mannose 6 6 7 7 Rhamnose 67 89 89 80 Cellular fatty acid composition (%) iso-C"%:! 4n1 k 1n0 k iso-C"&:! 6n17n79n34n3 iso-C1':! 47n953n343n928n1 anteiso-C"&:! 32n526n127n041n6 anteiso-C"(:! 9n412n918n926n0 Major menaquinone (%) MK-11 35 48 19 8 MK-12 65 52 81 78 MK-13 kkk14

16238T had a 16S rDNA sequence similarity value of new species in the genus Agromyces. Similarly, the low 98n4% with each other. The closest species to these two levels of DNA–DNA relatedness between strains IFO strains was A. mediolanus, but the levels of 16S rDNA 16235T and IFO 15238, and among these strains and sequence similarity between these strains and A. other strains used in this study confirmed that strains T T mediolanus was 97n5 and 97n7%, respectively (data not IFO 16235 and IFO 16238 each represents a new shown). species in the genus Agromyces. From the above results, we propose three new species in the genus DNA base composition and DNA–DNA hybridization Agromyces, Agromyces rhizospherae for strains IFO 16236T and IFO 16237, A. luteolus for strain IFO The DNA base composition of the four strains ranged 16235T and A. bracchium for strain IFO 16238T. from 70n0to73n3 mol%, as shown in Table 3. The levels of DNA–DNA relatedness between IFO 16236T The phenotypic characteristics also distinguish these and IFO 16237 were 69 and 73%, but levels of three new species from the previously described species DNA–DNA relatedness among these two strains and and subspecies in the genus Agromyces, as shown in other strains studied in the genus Agromyces were Table 4. A. mediolanus is the closest species to A. 11–22%. These results show that the two strains bracchium and A. luteolus on the basis of phylogenetic should be considered as a new species in the genus analysis (Fig. 2), but they can be differentiated by Agromyces. In addition, strains IFO 16235T and IFO nitrate reduction, urease production, hydrolysis of 16238T also showed levels of DNA–DNA relatedness starch and acid production from glycerol, fructose, of 13 and 36% with each other, and 8 to 36%, and 13 rhamnose and mannitol. The finding that all strains of to 39%, respectively, with other strains studied. These the new species studied grew well in the presence of 4% results show that strains IFO 16235T and IFO 16238T NaCl, in contrast to almost all Agromyces species also each represent a new species in the genus described previously, is very interesting from the Agromyces. viewpoint of ecology, because the soils of the mangrove rhizosphere have higher salinity than terrestrial soils The high values of 16S rDNA sequence similarity and (Wakushima et al., 1994). DNA–DNA relatedness between strains IFO 16236T and IFO 16237, and the low levels of DNA–DNA The sugar components of the cell walls are also useful relatedness among these strains and previously de- to distinguish the three new species from the previously scribed species in the genus Agromyces, confirmed that described Agromyces species, as shown in Table 4. All the two strains IFO 16236T and IFO 16237 represent a strains of the new species studied contained rhamnose

International Journal of Systematic and Evolutionary Microbiology 51 1533 M. Takeuchi and K. Hatano

...... Fig. 2. Unrooted phylogenetic tree showning the relationship of A. luteolus, A. rhizospherae and A. bracchium to related micro-organisms. The numbers indicate the percentages of bootstrap samplings, derived from 1000 samplings of the internal branches (Felsenstein, 1985).

as the major cell wall sugar; galactose was not found as Agromyces contain galactose as the major cell wall a major component. A. luteolus was also characterized sugar. A. ramosus also contains xylose (Gledhill & by having fructose in the cell wall. In contrast, all the Casida, 1969), and A. cerinus subsp. cerinus also previously described species or subspecies in the genus contains tyvelose in their cell walls, and A. cerinus

1534 International Journal of Systematic and Evolutionary Microbiology 51 Three new species in the genus Agromyces

Table 3. DNA base compositions and levels of DNA–DNA homology of Agromyces species

Species GjC content % DNA–DNA reassociation with: (mol%) IFO 16235T IFO 16236T IFO 16237 IFO 16238T

A. luteolus IFO 16235T 71n1 100 27 27 13 A. rhizospherae IFO 16236T 71n2 13 100 69 31 A. rhizospherae IFO 16237 73n3 8 73 100 39 A. bracchium IFO 16238T 70n0 36 13 14 100 A. mediolanus IFO 15704T 72n321111824 A. ramosus IFO 13899T 68n915191318 A. cerinus subsp. cerinus IFO 15780T 70n524201813 A. cerinus subsp. nitratus IFO 15783T 70n928151726 A. fucosus subsp. fucosus IFO 15781T 70n610181331 A. fucosus subsp. hippuratus IFO 15782T 70n831142224

Table 4. Characteristics that differentiate the species and subspecies of the genus Agromyces ...... 1, A. luteolus;2,A. rhizospherae;3,A. bracchium;4,A. mediolanus;5,A. ramosus;6,A. cerinus subsp. cerinus;7,A. cerinus subsp. nitratus;8,A. fucosus subsp. fucosus;9,A. fucosus subsp. hippuratus. Data for all species except A. luteolus, A. rhizospherae and A. bracchium are from Gledhill & Casida (1969), Zguraskaya et al. (1992) and Suzuki et al. (1996). j,90%or more of strains positive; k, 90% or more of strains negative; j, weakly positive reaction; , variable reaction; , not determined.

Characteristic 1 2 3 4 5 6 7 8 9

Catalase production jjjjk jjjj Nitrate reduction kkkjk kjkj Urease production kkkj kkkk Starch hydrolysis jjjkj jjjj Acid production from: Glycerol kkkjj jkkj Ribose kkjjj kkkk Galactose kkjj jkkk Glucose jjjjj kkkk Fructose jjjk jkkk Mannose jjjj kkkk Rhamnose j kj kj j j k j Mannitol j kjkj kkkk Amygdalin kkjk jkkk Aesculin jjjj jjjk Salicin kjjj jkkk Maltose jjjjj jkkk Sucrose j kjjj kkkk Growth in 4% NaCl jjj  kkk  Growth at 37 mC kkk  kjkk Cell wall sugars* Rha, Fruc, Rha, (Glc, Rha, (Gal,  Gal, Glc, Man, Gal, Rha, Gal, (Glc, Gal, Rha, Gal, Rha, (Glc, Man) Man) Glc, Man) Rha, Xyl Tyv, (Man) Man) (Glc, Man) (Man)

* Rha, rhamnose; Fruc, fructose; Glc, glucose; Man, mannose; Xyl, xylose; Tyv, tyvelose. Parentheses indicate that a compound may or may not be present. subsp. nitratus is characterized by the absence of Description of Agromyces luteolus sp. nov. rhamnose. Agromyces luteolus (lu.teho.lus. N.L. adj. luteus yellow; On the basis of the phenotypic, chemotaxonomic and M.L. dim. adj. luteolus yellowish, somewhat yellow). phylogenetic studies, as well as DNA–DNA relatedness data, we conclude that the four isolates represent Cells are aerobic, Gram-positive, irregular rods that three new species in the genus Agromyces. form ﬁlaments (0n2–0n4by3n0–5n0 µm) or show el-

International Journal of Systematic and Evolutionary Microbiology 51 1535 M. Takeuchi and K. Hatano ementary branching in the early growth phase and Cells are aerobic, Gram-positive, irregular rods that fragment into short rods (0n2–0n4by2n0–3n0 µm) during form filaments (0n2–0n4by5n0–6n0 µm) or show el- exponential growth phase. Colonies on PY agar ementary branching in the early growth phase and medium are yellow to whitish yellow, opaque, entire fragment into short rods (0n2–0n4by1n5–3n0 µm) during and convex, and sometimes penetrate into the agar. exponential growth phase. Colonies on PY agar Grows optimally at 20–30 mC, but does not grow at 10 medium are yellow, opaque, entire and convex, and or 37 mC. Catalase-positive. Acid is produced from sometimes penetrate into the agar. Grows optimally at glucose, fructose, lactose, maltose, mannose and tre- 20–30 mC, but does not grow at 10 or 37 mC. Catalase- halose, but is not produced from adonitol, amidon, positive. Acid is produced from amidon, amygdalin, amygdalin, arabinose, arbutin, cellobiose, dulcitol, -arabinose, arbutin, cellobiose, fructose, galactose, erythritol, fucose, galactose, gentiobiose, gluconate, gentiobiose, gluconate, glucose, glycogen, lactose, glycerol, glycogen, inositol, inulin, melezitose, maltose, mannitol, mannose, melezitose, melibiose, raffinose, ribose, salicin, sorbitol, sorbose, tagatose, raffinose, rhamnose, ribose, salicin, sucrose, trehalose, turanose, xylose or xylitol. Aesculin, gelatin and starch -turanose and -xylose, but is not produced from are hydrolysed. Urease is not produced. DAB is the adonitol, -arabinose, arabitol, dulcitol, erythritol, principal amino acid of the cell wall peptidoglycan. fucose, glycerol, inositol, inulin, sorbitol, sorbose, The cell wall is of an acetyl type. The predominant tagatose, -xylose or xylitol. Aesculin, gelatin and isoprenoid quinone is MK-12, with small amounts of starch are hydrolysed. Urease is not produced. DAB is MK-11. The predominant cellular fatty acids are iso- the principal amino acid of the cell wall peptidoglycan. C"':! and anteiso-C"&:!. The GjC content of the The cell wall is of an acetyl type. The predominant DNA is 71n1 mol%. Isolated from soil of rhizosphere isoprenoid quinone is MK-12 with small amounts of of mangrove on Iriomote Island, Japan. The type MK-13 and MK-11. The predominant cellular fatty T T strain is IFO 16235 (l VKM Ac-2085 ). acids are anteiso-C"&:!, anteiso-C"(:! and iso-C"':!. The GjC content of the DNA is 70n0 mol%. Isolated from soil of rhizosphere of mangrove on Iriomote T Description of Agromyces rhizospherae sp. nov. Island, Japan. The type strain is IFO 16238 (l VKM T Agromyces rhizospherae (rhi.zohspher.ae. M.L. rhizo Ac-2088 ). root; Gr. adj. sphera sphere; M.L. n. rhizospherae within the sphere of the root). ACKNOWLEDGEMENTS Cells are aerobic, Gram-positive, irregular rods that We thank Dr Ludmila Evtushenko, VKM-All-Russian form filaments (0n2–0n4by1n5–3n0 µm) or show el- Collection of Microorganisms, Skryabin Institute of Bio- ementary branching in the early growth phase and chemistry and Physiology of Microorganisms, Russian fragment into short rods (0n2–0n4by0n8–1n5 µm) during Academy of Sciences, for valuable suggestions and dis- exponential growth phase. Colonies on PY agar cussion. We thank Kozaburo Mikata, Senior Research Head medium are yellow, opaque, entire and convex, and of yeast in the Institute for Fermentation, Osaka, for his help sometimes penetrate into the agar. Grows optimally at with scanning electron microscopy. We also thank Yayoi Yamaguchi for her excellent technical assistance. 20–30 mC, but does not grow at 10 or 37 mC. Catalase- positive. 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