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Int J Syst Evol Microbiol 55 (2005), 1985-1989; DOI  10.1099/ijs.0.63551-0
© 2005 International Union of Microbiological Societies

Phyllobacterium trifolii sp. nov., nodulating Trifolium and Lupinus in Spanish soils

Angel Valverde1, Encarna Velázquez2, Félix Fernández-Santos2, Nieves Vizcaíno2, Raúl Rivas2, Pedro F. Mateos2, Eustoquio Martínez-Molina2, José Mariano Igual1 and Anne Willems3

1 Departamento de Producción Vegetal, IRNA-CSIC, Salamanca, Spain
2 Departamento de Microbiología y Genética, Lab 209, Edificio Departamental de Biología, Campus Miguel de Unamuno, Universidad de Salamanca, 37007 Salamanca, Spain
3 Laboratorium voor Microbiologie, Vakgroep Biochemie, Fysiologie en Microbiologie, Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium

Correspondence
Encarna Velázquez
evp{at}gugu.usal.es


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Bacterial strain PETP02T was isolated from nodules of Trifolium pratense growing in a Spanish soil. Phylogenetic analysis of the 16S rRNA gene sequence showed that this strain represents a member of the genus Phyllobacterium. However, divergence found with the 16S rRNA gene sequence of the single recognized species of this genus, Phyllobacterium myrsinacearum, indicated that strain PETP02T belongs to a different species. The results of DNA–DNA hybridization, phenotypic tests and fatty acid analyses confirmed that this strain represents a novel species of the genus Phyllobacterium, for which the name Phyllobacterium trifolii sp. nov. is proposed. The type strain is PETP02T (=LMG 22712T=CECT 7015T). This strain was strictly aerobic and used several carbohydrates as carbon source. It was not able to reduce nitrate. Aesculin hydrolysis was negative. It did not produce urease, arginine dihydrolase, gelatinase or {beta}-galactosidase. The DNA G+C content was 56·4 mol%. The nodD gene of this strain showed a sequence closely related to those of strains able to nodulate Lupinus. Infectivity tests showed that this strain is able to produce nodules in both Trifolium repens and Lupinus albus.

Published online ahead of print on 6 May 2005 as DOI 10.1099/ijs.0.63551-0.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and nodD gene sequences of PETP02T are AY786080 and AY786081, respectively.


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Trifolium pratense is a common legume in temperate soils and establishes effective symbioses with Rhizobium strains. The most common endosymbiont of this legume is Rhizobium leguminosarum biovar trifolii, which induces the formation of indeterminate nodules (Jordan, 1984Go). This legume belongs to the natural plant cover of many soils in north-west Spain but there are no studies regarding the diversity of bacteria nodulating Trifolium in these soils. During a study of populations of bacteria nodulating Trifolium in several geographical locations we isolated a strain, designated PETP02T, phylogenetically related to the genus Phyllobacterium. This genus was described by Knösel (1962)Go and currently contains one recognized species, since Phyllobacterium rubiacearum was recently reclassified as Phyllobacterium myrsinacearum (Mergaert et al., 2002Go). The data obtained in the present study show that strain PETP02T belongs to a novel species of Phyllobacterium.

Strain PETP02T was isolated from T. pratense nodules according to the method of Vincent (1970)Go on YMA medium. Colonies are white, mucoid, translucent and convex following growth on this medium. This strain exhibits a growth rate in YMB (Vincent, 1970Go) medium similar to that of Rhizobium species (doubling time of 2 h).

Strain PETP02T was grown on nutrient agar medium for 48 h to check for motility by phase-contrast microscopy. Cells were gently suspended in sterile water, stained with 0·2 % uranyl acetate and examined at 80 kV with a Zeiss EM 209 transmission electron microscope (Peix et al., 2003Go). Gram reaction of cells was ascertained by staining (Doetsch, 1981Go). Cells of strain PETP02T were Gram-negative, rod-shaped, non-sporulating, motile by means of a polar flagellum and commonly observed as single cells.

Strain PETP02T was re-isolated as a pure culture from nodules of Trifolium repens and a single colony was used for all molecular analyses. The nearly complete 16S rRNA gene sequence was analysed as described by Rivas et al. (2002)Go. Comparison with sequences from GenBank using the BLAST program (Altschul et al., 1990Go) indicated that this strain is phylogenetically related to members of the genus Phyllobacterium. Sequences of the new isolate and related bacteria were aligned using CLUSTAL W software (Thompson et al., 1997Go). The distances were calculated according to Kimura's two-parameter method (Kimura, 1980Go). Phylogenetic trees were inferred using the neighbour-joining method (Saitou & Nei, 1987Go). Bootstrap analysis was based on 1000 resamplings. The MEGA2 package (Kumar et al., 2001Go) was used for all analyses. The resulting neighbour-joining tree is shown in Fig. 1Go. The 16S rRNA gene sequence of strain PETP02T showed 98·0 % similarity to that of P. myrsinacearum, suggesting that it belongs to a different species.



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  		Fig. 1. Comparative sequence analysis of the 16S rRNA gene from Phyllobacterium trifolii PETP02T and representative related strains from GenBank. The significance of each branch is indicated by a bootstrap percentage calculated for 1000 subsets. The analysis was based on 1478 nt. Rhizobium leguminosarum ATCC 10004T was used as outgroup. Bar, 5 nt substitutions per 1000 nt.

 
Strain PETP02T was subjected to plasmid profile analysis according to Plazinski et al. (1985)Go, except that electrophoresis was performed at 2 V cm–1 for 90 min, followed by 3 V cm–1 for 60 min and finally at 6 V cm–1 for 3 h. The 175 kb and 205 kb plasmids of Sinorhizobium meliloti GR4 (Toro & Olivares, 1986Go) were used as size markers and as a positive control for Southern analysis. Plasmid DNA was capillary-transferred to a nylon membrane according to Southern (1975)Go and immobilized by baking at 80 °C for 2 h. Oligonucleotide primers were designed to amplify a fragment of the nodD gene conserved among members of the family Rhizobiaceae as described by Rivas et al. (2002)Go. The PCR-amplified fragments of the nodD gene were digoxigenin-labelled with the DIG DNA labelling kit (Roche Diagnostics Corp.) following the manufacturer's instructions and were used as probe. Hybridization was detected with the DIG nucleic acid detection kit (Boehringer Mannheim), using 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) as substrates for alkaline phosphatase, according to the manufacturer's instructions.

Results from the plasmid profile analysis and the Southern hybridization are shown in Fig. 2Go. The technique used revealed three plasmids in strain PETP02T (Fig. 2Go, lane 2). The specific probe detected a nodD gene in the three plasmids of strain PETP02T (Fig. 2Go, lane 4).



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  		Fig. 2. (a) Plasmid profile in horizontal 0·7 % agarose gel: Sinorhizobium meliloti GR4 (lane 1) and strain PETP02T (lane 2). (b) Results of hybridization (marked by arrowheads) using the nodD gene probe: strain GR4 (lane 3) and strain PETP02T (lane 2).

 
A partial sequence of the nodD gene of strain PETP02T was obtained from genomic DNA using the method of Rivas et al. (2002)Go. Phylogenetic analysis (Fig. 3Go) showed that the nodD gene of strain PETP02T is closely related to that of a novel species of the genus Ochrobactrum that is able to nodulate Lupinus albus (nodD gene sequence similarity of 94·9 %; Trujillo et al., 2005Go).



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  		Fig. 3. Comparative sequence analysis of the nodD gene from Phyllobacterium trifolii PETP02T and representative related strains from GenBank. The significance of each branch is indicated by a bootstrap percentage calculated for 1000 subsets. The analysis was based on 486 nt. Rhizobium leguminosarum 384 was used as outgroup. Bar, 5 nt substitutions per 1000 nt.

 
Taking into account the close phylogenetic relationship between the nodD gene present in strain PETP02T and those from strains nodulating Lupinus, we suspected that this strain would also nodulate Lupinus. We therefore tested for nodulation using T. repens and L. albus as described previously (Velázquez et al., 2001Go). The strain R. leguminosarum bv. trifolii ATCC 14480 and Bradyrhizobium sp. ISLU35 (Jarabo-Lorenzo et al., 2003Go) were used as positive controls in T. repens and L. albus, respectively. As expected, strain PETP02T was able to induce nodules in both plants (Fig. 4Go). The morphology of the nodules formed on T. repens was the same as that of those formed by R. leguminosarum bv. trifolii and they were formed along secondary roots in both cases. However, the nodules formed in L. albus were morphologically different from those formed by Bradyrhizobium sp. ISLU35. The nodules induced by strain PETP02T were formed at the intersection of the main and secondary roots, whereas those induced by strain ISLU35 were formed along the secondary roots. Strain PETP02T formed fewer nodules per plant in both Trifolium and Lupinus plants than did R. leguminosarum bv. trifolii ATCC 14480 and Bradyrhizobium sp. ISLU35, used respectively as positive controls (data not shown).



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  		Fig. 4. Nodules induced in Trifolium repens by strain PETP02T (a) and by Rhizobium leguminosarum bv. trifolii ATCC 14480 (b). Bars, 2 cm. Nodules induced in Lupinus albus roots by strain PETP02T (c) and by Bradyrhizobium sp. ISLU35 (d). Bars, 0·2 cm.

 
The DNA G+C content of strain PETP02T as determined by HPLC (Rivas et al., 2003Go) was 56·4 mol%. This value is lower than the range of 60·3–61·3 mol% reported for P. myrsinacearum (de Smedt & de Ley, 1977Go).

DNA–DNA hybridization was performed using a protocol described by Willems et al. (2001)Go and Rivas et al. (2004)Go. Strain PETP02T gave DNA–DNA hybridization levels of 12·0 % with two strains of P. myrsinacearum, LMG 1t1 and LMG 2t2T.

Phenotypic characterization of strain PETP02T was based on growth with different carbon sources (Bergersen, 1961Go) as described previously (Velázquez et al., 2001Go). P. myrsinacearum LMG 2t2T and LMG 1t1 (formerly P. rubiacearum) were used as reference strains. The temperature range for growth was determined by incubating cultures in YMA medium between 4 and 40 °C. The pH range was determined in YMA medium with a final pH between 5·0 and 10·0. Salt tolerance was studied in YMA medium containing 0–5 % (w/v) NaCl. Antibiotic resistance was tested by using the disc diffusion method with the following antibiotics: ampicillin (2 µg), erythromycin (2 µg), ciprofloxacin (5 µg), penicillin (10 IU), polymyxin (300 IU), cloxacillin (1 µg), oxytetracycline (30 µg), gentamicin (10 µg), cefuroxime (30 µg) and neomycin (5 µg) (Becton Dickinson). The basal medium was YMA (Vincent, 1970Go) supplemented with 10 g yeast extract l–1. Strain PETP02T and strains LMG 2t2T and LMG 1t1 were also characterized by using API 20NE tests according to the manufacturer's instructions (bioMérieux).

The results indicated that strain PETP02T differs from strains of P. myrsinacearum in acid production (after 4 days of incubation) from sucrose, trehalose and raffinose, citrate assimilation, and resistance to polymyxin B, oxytetracycline and neomycin (Table 1Go). Acid production from rhamnose and adonitol was positive in P. myrsinacearum but weak in strain PETP02T. Additional phenotypic characteristics of strain PETP02T are given in the species description below.


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  		Table 1. Differential phenotypic characteristics of Phyllobacterium trifolii sp. nov. PETP02T and P. myrsinacearum

+, Positive; –, negative; W, weak.

 
We also compared the fatty acid composition of strain PETP02T with those of P. myrsinacearum strains LMG 1t1 and LMG 2t2T. Cells were grown for 48 h on TY medium (Jarvis et al., 1996Go) and fatty acids were extracted and analysed in duplicate as described by Rivas et al. (2003)Go. The results (Table 2Go) confirmed previous observations for P. myrsinacearum (Mergaert et al., 2002Go). As in the case of P. myrsinacearum, the novel strain contains 18 : 1{omega}7c as the predominant fatty acid. It differs from P. myrsinacearum in that it contains more than 10 % 16 : 0, more than 15 % 18 : 1{omega}7c 11Me, small amounts of 17 : 0 and 20 : 2{omega}6,9c (neither of which was detected in P. myrsinacearum), less than 5 % 18 : 1 2-OH and no 18 : 0 3-OH.


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  		Table 2. Fatty acid methyl ester (FAME) profiles

Values are mean percentages of total FAMEs. Only fatty acids accounting for more than 1·0 % (mean) are indicated. tr, Trace amount (<1·0 %); ND, not detected.

 
Our results showed that strain PETP02T is able to nodulate Trifolium and Lupinus, increasing the number of non-rhizobial species that are able to nodulate legumes. This strain can be differentiated genotypically and phenotypically from previously described species and we therefore consider it to represent a novel species, for which the name Phyllobacterium trifolii sp. nov. is proposed.

Description of Phyllobacterium trifolii sp. nov.
Phyllobacterium trifolii (tri.fo'li.i. L. gen. n. trifolii of clover).

Gram-negative rods, as for the other species of the genus. Colonies are small, pearl white in YMA at 28 °C. Temperature range for growth is 4–37 °C (optimal growth occurs at 28 °C). The pH range for growth is 6–8 (optimal growth occurs at pH 7). Grows in the presence of NaCl concentrations up to 3 % (w/v) although salt is not essential for growth. Isolated from Trifolium pratense, it is able to produce nodules on Trifolium and Lupinus. Nitrate reduction is negative. It does not produce indole gelatinase, {beta}-galactosidase or arginine dihydrolase. Hydrolysis of urea and aesculin was weak. Produces acid from galactose and arabinose. Acid production from rhamnose and arabitol is weak. Uses glucose, L-arabinose, mannose, mannitol, N-acetylglucosamine, maltose and malate as carbon sources. Gentiobiose is weakly used. It does not grow on caproate, adipate, citrate or phenylacetate. Resistant to cloxacillin, penicillin, erythromycin, cefuroxime and ampicillin. Does not grow in the presence of polymyxin B, ciprofloxacin, gentamicin, oxytetracycline or neomycin. The DNA G+C content is 56·4 mol%.

The type strain, PETP02T (=LMG 22712T=CECT 7015T), was isolated from a Trifolium pratense root nodule.


   ACKNOWLEDGEMENTS
 
This work was supported by Junta de Castilla y León and DGCYT (Spanish Government). A. W. is grateful to the Fund for Scientific Research – Flanders for a position as Postdoctoral Researcher. We thank Renata Coopman and Ilse Vandecandelaere for excellent technical assistance.


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