INTERNATIONAL,JOURNAL OF SYSTEMATICBACTERIOLOGY, July 1993, p. 574584 Vol. 43, No. 3 0020-7713/93/030574-11$02.00/0 Copyright 0 1993, International Union of Microbiological Societies Azoarcus gen, nov. , Nitrogen-Fixing Proteobacteria Associated with Roots of Kallar Grass (LeptochZoa fusca (L.) Kunth), and Description of Two Species, Azoarcus indigens sp. nov. andhoarcus communis sp. nov. BARBARA REINHOLD-HUREK,lt* THOMAS HUREK,’$ MONIQUE GILLIS,l BART HOSTE,’ MARC VANCANNEYT,’ KAREL KERSTERS,’ AND JOZEF DE LEY’ Laboratorium voor Microbiologie, Universiteit Gent, K. -L. Ledeganckstraat 35, B-9000 Ghent, Belgaum, and Biozentrum der Universitat, Abteilung Mikrobiologie, CH-4056 Basel, Switzerland2 Among the nitrogen-fixing bacteria associated with roots of LeptochZuu fuscu (L.) Kunth in saline-sodic soils in the Puqjab of Pakistan, we repeatedly found yellow-pigmented, straight to curved, gram-negative rods. To group and identify these organisms, we examined morphological, nutritional, and biochemical features and performed polyacrylamide gel electrophoretic analyses of cellular proteins, gas chromatographic analyses of fatty acids, DNA-rRNA hybridizations, and DNA-DNA hybridizations. Our results showed that 11 isolates formed five groups distinct at the species level, with each group containing one to three members. These bacteria constituted a separate rRNA branch in rRNA superfamily 111 (corresponding to the beta subclass of the Proteobucteriu) at a branching T,(,, level of 67.7OC [TmCe,is the temperature at which 50% of a hybrid is denatured under standard conditions]. On this branch, the five groups were located in two clusters with Tmce, values of 79.4 to 80.4OC and around 71.5OC. We propose a new genus, the genus Azourcus, for these strains. Azoarcus indigens is the type species and has a growth factor requirement; its type strain is strain VB32 (= LMG 9092). A second named species, AzoQrcus communis, includes a strain obtained from French refinery oily sludge, strain LMG 5514. Bacteria of this genus have a strictly aerobic type of metabolism, fix nitrogen microaerobically, and grow well on salts of organic acids but not on carbohydrates. Swedish isolates obtained from human sources (E. Falsen group 15 strains LMG 6115 and LMG 6116), as well as “[Pseudomonas] gmotrophu” LMG 75mT, were also located on this rRNA branch at a lower Tm(,,level (70.4 to 71.2”C). In the rhizospheres of tropical, subtropical, and temperate However, the diazotrophs predominating inside the roots grasses, nitrogen-fing bacteria belonging to at least 11 were gram-negative rods which could not be assigned to genera have been found (see reference 15). Often, several previously described taxa on the basis of phenotypic char- species can be isolated from one plant (34, 44). A genus acteristics (44). Indirect evidence for the colonization of the which has been isolated frequently (13) and has been studied root interior by these organisms was confirmed by the results intensively with respect to its associations with grasses is the of immunofluorescence studies (41) and immunogold elec- genus Azospirillum. tron microscopy (23). The higher probability of metabolite Kallar grass (Leptochloafisca (L.) Kunth) is associated transfer between host and diazotrophs inside roots than with several different nitrogen-fixing bacteria (44). This between host and root surface bacteria focused our attention grass, which tolerates soil salinity and waterlogged condi- on the diazotrophs inside roots (39). tions, is widely distributed in tropical to subtropical regions In this paper we describe the repeated isolation of one ranging from Australia to Africa. In the Punjab of Pakistan, group of diazotrophic rods (44) from Kallar grass roots and it is used as a pioneer plant on saline-sodic, alkaline soils the results of a polyphasic taxonomic study that led to having low fertility (47). Because of its luxuriant growth taxonomic description of these organisms. On the basis of without the addition of any nitrogenous fertilizer, giving our results, we propose the new genus Azoarcus, consisting harvests of 20 to 40 metric tons (2 x lo4 to 4 x lo4kg) of hay of two named species, Azoarcus communis and Azoarcus per ha per year (46), we became interested in the nitrogen- indigens, and three additional unnamed groups which are fixing bacteria colonizing Kallar grass roots. also distinct at the species level. Bacteria from other sources Studies of rhizospheric populations were carried out on a could also be assigned to thekoarcus rRNA branch. field in Punjab which had been under Kallar grass cultivation (A preliminary account of this study was presented previ- for more than 10 years. Nitrogen-fixing bacteria were found ously [43].) to form root-zone-specific associations, with different popu- lations colonizing the surface and the interior of roots (44). Two Azospirillum species were dominant on the rhizoplane, MATERIALS AND METHODS and one of these organisms has been described as a new, salt-toleran t species, Azospirillum halopraeferens (42). Isolation of diazotrophic strains. Samples of Kallar grass were collected from the same field in the Punjab region of Pakistan in 1984 and 1988. Kallar grass grew almost exclu- * Corresponding author. sively on the saline-sodic soil at this site. Samples were -f Present address: Max-Planck-Institut fur Terrestrische Mikro- subjected to isolation procedures either immediately (in biologie, Karl-von-Frisch-Strasse, D-35043 Marburg, Germany. 1984) or within 4 days after transport at 4°C to Europe. A $ Present address: Pfarracker 5, DW-35043 Marburg-Bauerbach, sample of Kallar grass from a mangrove-covered, brackish Germany. sandy river bank near Brisbane, Australia, was processed 2 574 VOL.43, 1993 AZOARCUS GEN. NOV. 575 days after collection. Enrichment cultures were prepared by gas-liquid chromatography have been previously described using pieces of roots with adhering rhizosphere soil, surface- (51). FAME fingerprints were identified by using the Micro- sterilized root pieces, and serial dilutions of preparations bial Identification System software package (MIS version containing surface-sterilized, homogenized roots and stem 3.2; Microbial ID, Inc., Newark, Del.). A statistical program bases. Surface sterilization, homogenization, and serial di- developed by Microbial ID, Inc., was used for cluster lution were carried out as described previously (44). Putative analysis of the strains. nitrogen-fixing bacteria were enriched on two semisolid, Preparation of high-molecular-weight DNA. Cells were nitrogen-free, malate-containing media: SM medium supple- grown in Roux flasks on the appropriate growth medium. mented with vitamins (44) and SSM medium (44). SSM DNA was prepared by the method of Marmur (31) or by a medium was modified to contain the salt concentrations slightly modified procedure (42). When available, DNAs commonly found in soil saturation extracts of the saline- obtained from members of our research group were used sodic soils in the Punjab. After two successive enrichment (Table 1). steps of 48 to 72 h, bacteria from subsurface pellicles were DNA base composition. The average guanine-plus-cytosine isolated on the media, which contained 0.8% agar instead of (G+C) contents of the DNAs from isolates were determined 0.2% agar and 20 mg of yeast extract per liter. For up to 7 by the thermal denaturation method and were calculated by days after inoculation, single colonies were transferred to using the equation of Marmur and Doty (32), as modified by semisolid medium. Strains which exhibited growth as sub- De Ley (6). The base composition was determined for one surface pellicles were further purified. Nitrogenase activity representative strain of each group. was assayed by the acetylene reduction test (42) with a DNA-rRNA hybridization. Single-stranded high-molecu- model IGC 120DFL gas chromatograph (Intersmat, lar-weight DNA was fixed onto membrane filters (type SM Suresnes, France). Bacteriological purity was checked on 11358; Sartorius, Gottingen, Germany) as described by De plates containing VM medium, a modification of SM medium Ley and De Smedt (9). 3H-labeled rRNAs from isolates which contains half as much potassium DL-malate, 0.3% beef BH72 and H6a2 were prepared and purified (9) by using extract (Oxoid), 0.1% yeast extract (Oxoid), 0.1% NaCl, and [3H]adenine and [3H]uracil as precursors; the specific activ- 0.05% NH4Cl. The growth temperature used for enrichment ities of the 23s rRNA preparations were 21 x lo3 and 11 X and isolation was 30"C, and the temperature used for further lo3 cpm wg-', respectively. Labeled rRNAs from the fol- cultivation was 37°C unless stated otherwise. Except for lowing reference organisms were available from members of strains BH72 and Bb6c1, the designations for strains that our research group: Alteromonas haloplanktis LMG 2852T were isolated from salt-enriched (SSM) medium begin with (T = type strain) (SO), Xanthomonas cam estris LMG 56gT S. Pure cultures of N,-fixing strains were maintained at 30°C (4), Pseudomonas Jluorescens LMG 1794.p (1 l), Oceanospir- in semisolid SM medium supplemented with vitamins or illum linum LMG 5414T (36), Azorhizobium caulinodans SSM medium with biweekly transfers. Strains were also kept LMG 6465T (14), Aquaspirillum sevens biovar bengal strain for 1to 2 months at 4°C on slants of VM medium after 2 days LMG 6234T (37), [PseudomonasJ rubrisubalbicans LMG of growth at 37°C. Long-term preservation was achieved by 2286T (18), Hydrogenophaga palleronii LMG 2366T (52), and placing the organisms in liquid nitrogen as described previ- Oligella urethralis LMG 5304 (45). Hybridizations between ously (40) or by lyophilization. filter-fixed DNA and labeled 23s rRNA were carried out as Bacterial strains. The strains used in this study are listed in described previously (9). As a measure of the thermal Table 1. In addition to our own isolates, other free-living N, stability of each hybrid, we determined its T,+) (the temper- fixers and reference organisms belonging to rRNA superfam- ature at which 50% of the hybrid was denatured under ilies I1 and I11 were included.