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1 Laboratory of Phytopathology and Plant Protection, Katholieke Universiteit Leuven, W. De Croylaan 42, B-3001 Leuven, Belgium
2 BCCM/LMG Bacteria Collection, Laboratory of Microbiology, University of Ghent, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium
Correspondence
Deborah De Clercq
Deborah.declercq{at}biw.kuleuven.be
Jaak Ryckeboer
Jaak.ryckeboer{at}biw.kuleuven.be
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ABSTRACT |
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9c were present. The 16S rRNA gene sequence (1497 bp) of strain B39T showed the highest level of similarity (98.5 %) to that of Rhodanobacter fulvus IAM 15025T, followed by Rhodanobacter lindaniclasticus LMG 18385T (96.0 %; strain no longer extant), Dyella koreensis CCUG 50883T (96.4 %), Dyella japonica DSM 16301T (96.3 %), Frateuria aurantia LMG 1558T (96.2 %) and Fulvimonas soli LMG 19981T (95.9 %). Less than 90 % 16S rRNA gene sequence similarity was observed for other members of the Gammaproteobacteria. The mean DNA–DNA reassociation value for the three strains was 100 % and was 25 % when the strains were compared with DNA from R. fulvus LMG 23003T. The strains had a mean DNA G+C content of 67.6 mol%. On the basis of their phylogenetic, genomic and phenotypic properties, the three strains represent a novel species within the genus Rhodanobacter, for which the name Rhodanobacter spathiphylli sp. nov. is proposed. The type strain is strain B39T (=LMG 23181T=DSM 17631T).
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). The pathogen-suppressive properties of compost are mainly attributable to biological factors, although chemical and physical factors are inextricably involved. In addition to containing the numerous micro-organisms usually observed in compost (reviewed by Ryckeboer et al., 2003), a disease-suppressing compost should also harbour micro-organisms that are beneficial in terms of disease suppression or should be capable of stimulating beneficial edaphic microbial populations through nutritional amendment. However, because of the enormous microbial variability inherent in the composting process, composts are not consistently colonized by the antagonistic micro-organisms (biocontrol agents) necessary for disease suppression (Hoitink & Boehm, 1999). Therefore composts are sometimes artificially amended with one or more of these biological control agents, to increase the overall disease suppressiveness of the material (Hoitink & Boehm, 1999). The organisms described herein were isolated during a screening process carefully designed to select for appropriate biocontrol agents. A polyphasic taxonomic study was performed on three strains isolated from the rhizosphere of Spathiphyllum. These plants were previously grown in a compost-amended substrate with disease-suppressive properties directed against the soil-borne fungal pathogen Cylindrocladium spathiphylli. The three strains exhibit in vivo and in vitro biological control activity towards this pathogen. The results of genotypic and phenotypic analyses showed that the three isolates belong to a novel Rhodanobacter species.
The novel strains, B35, B39T and B42, were isolated at 25 °C on nutrient agar (LAB M) amended with 100 p.p.m. cycloheximide (A.G. Scientific) from the roots and rhizospheres of Spathiphyllum ‘Alpha’ plants. The strains were maintained at –80 °C on beads from Microbank (Pro-Lab Diagnostics). The reference strain Rhodanobacter fulvus LMG 23003T was included in some experiments. The type strain of the type species of the genus, Rhodanobacter lindaniclasticus LMG 18385T, could not be included, as this strain, which is the only known strain of this species, no longer exists (Mergaert et al., 2002).
Small-scale DNA extracts were prepared by using the method of Niemann et al. (1997), and the 16S rRNA gene sequences of strains B35 and B39T were amplified by using a PCR with conserved primers (Coenye et al., 1999). PCR products were purified using the NucleoFast 96PCR Clean-up kit (Macherey-Nagel) according to the instructions of the manufacturer. Sequence analysis was performed as described previously (Van Trappen et al., 2004). Evolutionary distances were calculated using the algorithm of Jukes & Cantor (1969), and a phylogenetic tree was constructed using the neighbour-joining method with the TREECON program (Van de Peer & De Wachter, 1994).
Partial (810 bp) and complete (1497 bp) 16S rRNA gene sequences were determined for strains B35 and B39T, respectively. Comparative sequence analysis revealed that the sequences of these strains were identical over a stretch of 810 bp, except for two unidentified nucleotide positions in the sequence of strain B35. The 16S rRNA gene sequence from B39T revealed 98.5 % similarity with that of R. fulvus IAM 15025T, 96.0 % sequence similarity with that of R. lindaniclasticus LMG 18385T, 96.4 % with that of Dyella koreensis CCUG 50883T, 96.3 % with that of Dyella japonica DSM 16301T, 96.2 % with that of Frateuria aurantia LMG 1558T and 95.9 % with that of Fulvimonas soli LMG 19981T. Less than 90.0 % sequence similarity was observed with respect to other members of the Gammaproteobacteria, such as Xanthomonas sacchari LMG 471T (89.8 %), Stenotrophomonas maltophilia LMG 958T (89.8 %), Lysobacter antibioticus DSM 2044T (89.8 %), Pseudoxanthomonas broegbernensis ATCC BAA-10T (89.7 %), Lysobacter enzymogenes DSM 2043T (89.6 %), Xylella fastidiosa ATCC 35879T (89.6 %), Xanthomonas melonis LMG 8670T (89.3 %), Xanthomonas campestris LMG 568T (89.3 %) and Luteimonas mephitis DSM 12574T (89.2 %). The estimated phylogenetic relationships of strain B39T and the nearest members of the Gammaproteobacteria are shown in a neighbour-joining dendrogram with bootstrap percentages (Fig. 1). The tree obtained with the maximum-likelihood method (TREE-PUZZLE, default parameters with 50 000 puzzling steps) showed essentially the same topography as the neighbour-joining tree (data not shown).
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and DNA–DNA hybridizations were carried out with photobiotin-labelled probes in microplate wells as described by Ezaki et al. (1989), using an HTS7000 Bio Assay Reader (Perkin Elmer) for the fluorescence measurements (mean standard deviation ±7 %; Goris et al., 1998). The hybridization temperature was 50 °C and reciprocal experiments were performed for every pair of strains. The mean hybridization level between strains B39T, B35 and B42 was 100 %, indicating that they belong to a single species (Wayne et al., 1987), whereas the values for DNA–DNA binding with R. fulvus LMG 23003T were low (26 % with B39T, 24 % with B35 and 26 % with B42). These results demonstrate that the three isolates are genotypically distinct from R. fulvus (their nearest neighbour phylogenetically) and constitute a novel species within the genus Rhodanobacter.
The DNA G+C contents of the isolates were determined using an HPLC method, as described by Van Trappen et al. (2003). The G+C contents of strains B39T, B35 and B42 were 67.6, 67.8 and 67.5 mol%, respectively. These values are higher than the G+C contents of the other Rhodanobacter species, which range from 63 to 65.3 mol% (Im et al., 2004).
The cellular fatty acid patterns of the strains were determined as described by Mergaert et al. (2001). The three strains possessed very similar fatty acid profiles: the major fatty acids were 11 : 0 iso (5.6 %), 11 : 0 iso 3-OH (8.2 %), 13 : 0 iso 3-OH (5.1 %), 15 : 0 iso (24.2 %), 17 : 0 iso (9.0 %), 17 : 1 iso 9c (26.9 %) and summed feature 3 (5.7 %), comprising 15 : 0 iso 2-OH, 16 : 17c or both. In Table 1, the fatty acid compositions of the novel strains and R. fulvus are compared: the profiles are very similar but differ in terms of the amounts of 11 : 0 iso, 11 : 0 iso 3-OH, 13 : 0 iso 3-OH, 15 : 0 anteiso and 16 : 0 present.
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). The growth of the strains was investigated under aerobic conditions on nutrient agar, Luria–Bertani agar (Sigma), MacConkey agar no. 3 (Oxoid) and tryptone soy agar (TSA) [tryptone soy broth (TSB; Oxoid) amended with 15 g agar no. 1 l–1 (LAB M)] for 48 h at 25 °C. The temperature range and optimum for growth were assessed through dilution plating and the counting of colony-forming units on TSA incubated at 7, 15, 20, 25, 30, 35, 40 and 45 °C for 48 h (with three replicates for each strain). Strains B35 and B39T were unable to grow at 
7 °C or 
45 °C; strain B42 was unable to grow at 20 °C or 40 °C. The optimum temperature for growth was 30 °C for strains B35 and B42 and 35 °C for strain B39T. Biochemical tests were performed on 2-day-old cultures of the three isolates (grown in TSB at 29 °C) by using the API 20NE (OD550=0.15), API ZYM (OD550=1.5), API 50 CHE (OD550=1) and API ID 32 systems (bioMérieux). API ZYM tests were read after 4 h incubation at 35 °C; all other tests were read after 48 h at 28–29 °C. The API ID 32 system was also applied to R. fulvus LMG 23003T as a positive control. API tests were performed according to the instructions of the manufacturer, with up to three repetitions. The biochemical and physiological characteristics of the three novel strains are detailed in the species description and in Table 2. The three novel strains seem to grow on a narrow range of substrates as sole carbon sources, a property that they seem to have in common with members of the genus Rhodanobacter. The carbon sources utilized by the novel isolates and the Rhodanobacter species differ, however: the isolates use D-sorbitol and glycogen, R. fulvus uses D-glucose, mannose, salicin, D-melibiose, D-sucrose, N-acetylglucosamine and maltose (Im et al., 2004) and the substrates used by R. lindaniclasticus include D-glucose, lactate, caprate, malate and citrate (Nalin et al., 1999). The novel isolates, R. fulvus and R. lindaniclasticus cannot grow on (amongst others) adipic acid, phenylacetic acid, mannitol, L-fucose, L-arabinose, 2-ketogluconate, 5-ketogluconate, D-ribose, inositol or malonate. The Rhodanobacter species and the novel isolates also share the following properties: the presence of oxidase and catalase activities, the absence of indole production and arginine dihydrolase and urease activities and the inability to ferment D-glucose. The three novel strains responded differently from the Rhodanobacter species in some biochemical tests (Table 2), however. Strain B42 shows a positive response for 
-D-glucosidase activity and for the degradation of gelatin, whereas strains B35 and B39T gave negative responses. On the other hand, strains B35 and B39T were positive for N-acetyl--D-glucosaminidase activity and strain B42 was negative. The reactions for the production of acids from D-maltose and starch were negative for strain B42 and weakly positive for strains B35 and B39T.
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Description of Rhodanobacter spathiphylli sp. nov.
Rhodanobacter spathiphylli (spa.thi.phyl'li. N.L. gen. n. spathiphylli of Spathiphyllum, referring to the isolation source of the first strains, the rhizosphere of Spathiphyllum plants).
Cells are Gram-negative, rod-shaped, 0.8x1.5–4.0 µm in size and non-motile. They form smooth, circular, yellow colonies as large as 2–3 mm in diameter on nutrient agar and TSA after 48 h incubation at 30 °C. The strains grow well on nutrient agar, TSA and other common bacteriological media at 25 °C and 35 °C for all strains. Strains B35 and B39T are unable to grow at 7 °C or 45 °C. Strain B42 is unable to grow at 20 °C or 40 °C. The optimum temperature for growth is 30 °C for strains B35 and B42 and 35 °C for strain B39T. All strains give positive results in tests for the following: oxidase, catalase, alkaline phosphatase, C4 esterase, C8 esterase lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, -glucosidase, aesculin, p-nitrophenyl--D-galactopyranosidase, growth on sorbitol and growth on glycogen; weak acid production from aesculin, citrate, salicin and potassium 5-ketogluconate is observed. All strains give negative results in tests for the following: C14 lipase, trypsin, 
-chymotrypsin, -D-galactosidase, -D-galactosidase, -D-glucuronidase, -D-glucosidase, -D-mannosidase, -fucosidase, nitrate reduction, indole production, fermentation of D-glucose, arginine dihydrolase, urease, growth on D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl--D-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, phenylacetic acid, salicin, D-melibiose, L-fucose, propionate, valerate, histidine, 2- and 5-ketogluconate, 3- and 4-hydroxybutyrate, L-proline, rhamnose, D-ribose, inositol, D-sucrose, itaconate, suberate, malonate, acetate, D- and L-lactate, L-alanine, 3-hydroxybenzoate and L-serine and acid production from glycerol, erythritol, D- and L-arabinose, D-ribose, D- and L-xylose, D-adonitol, methyl -D-xylopyranoside, D-galactose, D-glucose, D-fructose, D-mannose, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl -D-mannopyranoside, methyl -D-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, D-cellobiose, D-lactose (bovine origin), D-melibiose, sucrose, D-trehalose, inulin, D-melezitose, D-raffinose, glycogen, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, D- and L-fucose, D- and L-arabitol, potassium gluconate and potassium 2-ketogluconate. The three strains respond differently in tests for some biochemical characteristics. Strain B42 gives a positive response for -D-glucosidase activity and for the degradation of gelatin, whereas strains B35 and B39T give negative responses. Strains B35 and B39T give positive results for N-acetyl--D-glucosaminidase activity whereas strain B42 gives a negative response. The reactions for the production of acids from D-maltose and starch are negative for strain B42 and weakly positive for strains B35 and B39T. The cells contain the fatty acids 15 : 0 iso and 17 : 1 iso 9c as the main constituents. The mean DNA G+C content is 67.6 mol%.
The type strain, B39T (=LMG 23181T=DSM 17631T), was isolated from the rhizospheres of Spathiphyllum plants grown in a compost-amended potting mix.
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ACKNOWLEDGEMENTS |
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