Rhizobium soli sp. nov., isolated from soil

The genus Rhizobium was first proposed by Frank (1889) and its description was emended by Young et al. (2001). Phylogenetically, the genus forms an evolutionary lineage within the family Rhizobiaceae of the Alphaproteobacteria (Lee et al., 2005). Recent descriptions of novel species, e.g. eight species in 2008 and 2009 (Berge et al., 2009; Gu et al., 2008; Han et al., 2008; Hunter et al., 2007, 2008; Peng et al., 2008; Ramírez-Bahena et al., 2008; Tian et al., 2008), have increased considerably the number of species belonging to the genus Rhizobium. At the time of writing, the genus Rhizobium comprises 31 species with validly published names (Euzéby, 1997). Members of the genus Rhizobium have generally been isolated from nodules on leguminous plants (Peng et al., 2008; Wei et al., 2003). Recently, some Rhizobium species have been isolated from other sources (García-Fraile et al., 2007; Hunter et al., 2007; Quan et al., 2005). In this study, we report on the taxonomic characterization of a Rhizobium-like bacterial strain, DS-42^T, which was isolated from a soil from Korea. The aim of the present work was to determine the exact taxonomic position of strain DS-42^T by using a polyphasic characterization that included determination of phenotypic properties, phylogenetic investigations based on 16S rRNA, atpD and recA gene sequences and genetic analysis.

Strain DS-42^T was isolated by means of standard dilution plating technique at 25 °C on 10-fold-diluted nutrient agar (Difco). The type strains of four Rhizobium species were used as reference strains for DNA–DNA hybridization, phenotypic characterization and fatty acid analysis: Rhizobium huautlense LMG 18254^T, R. galegae LMG 6214^T, R. loessense CIP 108030^T and R. cellulosilyticum DSM 18291^T. These reference strains were cultivated under the culture conditions recommended by the culture collections. The morphological, physiological and biochemical characteristics of strain DS-42^T were investigated using routine cultivation on trypticase soy agar (TSA; Difco) at 25 °C. Cell morphology was examined by light microscopy (Nikon E600) and transmission electron microscopy. Flagellation was determined by using a Philips CM-20 transmission electron microscope with cells from exponentially growing cultures: for this purpose, the cells were negatively stained with 1 % (w/v) phosphotungstic acid and the grids were examined after being air-dried. The Gram reaction was determined by using the bioMérieux Gram stain kit according to the manufacturer’s instructions. Growth at 4, 10, 15 and 20–32 °C (at intervals of 1 °C) was measured on TSA. Growth in the absence of NaCl and at 0.5 % and 1.0–10.0 % (in increments of 1.0 %) (w/v) NaCl was investigated in trypticase soy broth prepared according to the formula of the Difco medium except that NaCl was omitted. The pH range for growth was determined in nutrient broth (Difco) that was adjusted to pH 4.5–10.5 (at intervals of 0.5 pH units) by using sodium acetate/acetic acid and Na₂CO₃ buffers. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber on TSA and on TSA supplemented with potassium nitrate (0.1 %, w/v), both of which had been prepared anaerobically using nitrogen. Catalase and oxidase activities and hydrolysis of casein, gelatin, hypoxanthine, starch, Tweens 20, 40, 60 and 80, tyrosine, urea and xanthine were determined as described by Cowan & Steel (1965). Aesculin hydrolysis and nitrate reduction were studied as described previously (Lányí, 1987). Antibiotic susceptibility was tested on TSA plates using discs containing the following amounts of antibiotic: polymyxin B (100 U), streptomycin (50 μg), penicillin G (20 U), chloramphenicol (100 μg), ampicillin (10 μg), cephalothin (30 μg), gentamicin (30 μg), novobiocin (5 μg), tetracycline (30 μg), kanamycin (30 μg), lincomycin (15 μg), oleandomycin (15 μg), neomycin (30 μg) and carbenicillin (100 μg). Utilization of various substrates, enzyme activities and other physiological and biochemical properties were tested by using the API 20E, API 20NE, API 50CH and API ZYM systems (bioMérieux); utilization of various substrates was determined by inoculating the API 50CH strip with cells suspended in AUX medium (bioMérieux).

A nodulation test was performed by using the common leguminous species Medicago truncatula (barrel medick, a relative of alfalfa), Phaseolus lunatus (lima bean) and Glycine max (soybean). Seeds of M. truncatula and P. lunatus were surface-sterilized by soaking for 1 min in 100 % ethanol and seeds of G. max were surface-sterilized by soaking for 10 min in 1 % (w/v) sodium hypochlorite solution. The seeds were then washed five times with sterile distilled water. After germination, 2-day-old seedlings were transferred to round plastic Petri dishes (150 mm in diameter) containing solid nitrogen-free Murashige and Skoog (MS) salt medium (Gibco-BRL), which includes 0.8 % (w/v) plant agar and 1.5 % (w/v) sucrose. After incubation for 2 days, primary roots were inoculated by dripping 200 μl bacterial suspension at 10⁸ c.f.u. ml⁻¹ onto the root from the tip to the base. The positions of the root tips and the smallest emergent root hairs were marked on the plastic pouches immediately after inoculation with the aid of a dissecting microscope at ×12 magnification. The plants were cultured vertically for 3–4 weeks in a growth chamber at 25 °C with a 12 h photoperiod. Sinorhizobium meliloti 1021 was used as a positive control for nodulation on M. truncatula, and water treatment was used as a negative control.

Cell biomass for DNA extraction and for analysis of isoprenoid quinones was obtained from cultures grown with shaking at 150 r.p.m. for 3 days in trypticase soy broth (Difco, pH 7.3) at 25 °C. Chromosomal DNA was isolated and purified according to the method described by Yoon et al. (1996), with the exception that RNase T1 was used in combination with RNase A to minimize contamination with RNA. The 16S rRNA gene was amplified by PCR using two universal primers as described previously (Yoon et al., 1998). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed as described by Yoon et al. (2003). PCR amplifications of atpD and recA genes were performed under the conditions described by Yoon et al. (1998) using primers described by Tian et al. (2008). The PCR products were purified with the QIAquick PCR purification kit (Qiagen). The amplified atpD and recA genes were cloned into the pGEM T-easy vector (Promega) according to the manufacturer’s instructions. Sequences of the atpD and recA genes were determined for both strands by extension from vector-specific priming sites (primers T7 and SP-6 from the pGEM T-easy vector). PCR amplifications of nodD and nifH genes were performed by using primers and conditions described by Poly et al. (2001) or Rivas et al. (2002). The DNA G+C content was determined by the method of Tamaoka & Komagata (1984) with the modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC. Isoprenoid quinones were extracted according to the method of Komagata & Suzuki (1987) and analysed using reversed-phase HPLC and a YMC ODS-A (250×4.6 mm) column. For fatty acid methyl ester analysis, cell mass of strain DS-42^T, R. huautlense LMG 18254^T, R. galegae LMG 6214^T, R. loessense CIP 108030^T and R. cellulosilyticum DSM 18291^T was harvested after incubation for 3 days at 25 °C on solid medium (CIP medium no. 57) that contained (l⁻¹) 4 g glucose, 4 g yeast extract, 10 g malt extract, 2 g CaCO₃ and 15 g agar (pH 7.2). Fatty acid methyl esters were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). DNA–DNA hybridization was performed fluorometrically by the method of Ezaki et al. (1989) using photobiotin-labelled DNA probes and microdilution wells. Hybridization was performed with five replications for each sample. The highest and lowest values obtained in each sample were excluded, and the means of the remaining three values are quoted as DNA–DNA relatedness values.

Cells of strain DS-42^T were Gram-negative, non-motile, aerobic rods, 0.4–0.7 μm wide and 1.0–4.5 μm long. The strain grew at 4 and 31 °C, with optimum growth at 25 °C. Strain DS-42^T grew optimally at pH 7.0–8.0 and in the presence of 0.5–1.0 % (w/v) NaCl. Morphological, cultural, physiological and biochemical characteristics of strain DS-42^T are given in the species description and in Table 1⇓. Nodulation tests were performed by investigating the ability of strain DS-42^T to form nodules on three different leguminous species, M. truncatula, P. lunatus and G. max. No nodule formation was observed in three repeated experiments (not shown). Even when high-dosage inoculation of strain DS-42^T was used on the root hairs, no swollen tissues appeared. The nodD and nifH genes were also not detected by PCR in strain DS-42^T.

The almost-complete 16S rRNA gene sequence of strain DS-42^T determined in this study comprised 1436 nt (approx. 96 % of the Escherichia coli 16S rRNA sequence). Sequence analyses of the 16S rRNA, atpD and recA genes showed that strain DS-42^T was phylogenetically most closely related to the genus Rhizobium. In the neighbour-joining tree based on 16S rRNA gene sequences, strain DS-42^T fell within the clade comprising Rhizobium species, particularly forming a cluster with R. huautlense, R. galegae, R. cellulosilyticum and R. loessense (Fig. 1⇓). In phylogenetic trees constructed using the maximum-likelihood and maximum-parsimony algorithms, strain DS-42^T fell within the clade encompassed by the genus Rhizobium. Strain DS-42^T exhibited 16S rRNA gene sequence similarity of 97.7, 97.1, 96.9 and 96.7 % to the type strains of R. huautlense, R. galegae, R. cellulosilyticum and R. loessense, respectively, and of 94.1–96.9 % to the type strains of the other Rhizobium species. In neighbour-joining trees based on atpD and recA gene sequences, strain DS-42^T formed distinct phylogenetic lineages within the clade comprising Rhizobium species (Supplementary Figs S1 and S2, available in IJSEM Online). Strain DS-42^T exhibited 85.3–91.3 % atpD gene sequence similarity and 84.2–88.0 % recA gene sequence similarity to the Rhizobium type strains used in this study.

Strain DS-42^T contained ubiquinone-10 (Q-10), at a peak area ratio of approximately 95 %, as the predominant isoprenoid quinone. The cellular fatty acid profile of strain DS-42^T is shown in Table 2⇓, together with those of R. huautlense LMG 18254^T, R. galegae LMG 6214^T, R. loessense CIP 108030^T and R. cellulosilyticum DSM 18291^T, also analysed in this study. The fatty acid profiles of the five strains were essentially similar in that C_18 : 1ω7c is the major fatty acid, although there were differences in the proportions of some fatty acids (Table 2⇓). The DNA G+C content of strain DS-42^T was 60.8 mol%, which is in the range reported for known Rhizobium species (Table 1⇑).

Strain DS-42^T exhibited mean DNA–DNA relatedness of 13–19 % to the type strains of phylogenetically related Rhizobium species [R. huautlense LMG 18254^T (15 %), R. galegae LMG 6214^T (19 %), R. loessense CIP 108030^T (19 %) and R. cellulosilyticum DSM 18291^T (13 %)]. These values indicate that strain DS-42^T represents a genomic species distinct from these four Rhizobium species (Wayne et al., 1987). Strain DS-42^T is clearly distinguishable from these four Rhizobium species by differences in some phenotypic characteristics, including enzyme activities, utilization of substrates and susceptibility to antibiotics (Table 1⇑). The phylogenetic distinctiveness, together with the DNA–DNA relatedness data and differential phenotypic properties, is sufficient to allocate strain DS-42^T to a species that is separate from recognized Rhizobium species (Stackebrandt & Goebel, 1994). Therefore, on the basis of the data presented, strain DS-42^T is considered to represent a novel species within the genus Rhizobium, for which the name Rhizobium soli sp. nov. is proposed.