APPLIEDAND ENVIRONMENTALMICROBIOLOGY, July 1999, p. 3084-3094 Vol. 65, No. 7 0099-2240/99/$04.00+0 Copyright O 1999, American Society for Microbiology. All Rights Reserved. Photosynthetic Bradyrhizobia from Aeschynomene spp. Are Specific to Stem-Nodulated Species and Form a Separate 16s Ribosomal DNA Restriction Fragment Length Polymorphism Group FLORE MOLOUBA,’ JEAN LORQUIN,l ANNE WILLEMS? BART HOSTE,2 ERIC GIRAUD; BERNARD DREYFUS: MONIQUE PHILIPPE DE LAJUDIE,1,2” AND CATHERINE MASSON-BOIVIN3 Laboratoire de Microbiologie, I. R. D., Dakar, Sénégal’; Laboratorium voor Microbiologie, Universiteit Gent, B-9000, Glient, Belgium2; and Laboratoire des Symbioses Tropicales et Méditeranéennes, I. R. D., Canapus de Baillarguet, 34032 Montpellier Cedex, France3 Received 15 January 1999/Accepted 5 May 1999 We obtained nine bacterial isolates from root or collar nodules of the non-stem-nodulated Aeschynomene speciesA. elaphroxyloiz,A. uiz$lora, or A. schiinperi and 69 root or stem nodule isolates from the stem-nodulated Aesclzynoinerie species A. afraspera,A. Ciliata,A. iizdica,A. izilotica,A. sensitiva, and A. tambacouizde~zsisfrom var- ious places in Senegal. These isolates, together with 45 previous isolates from various Aesclzyizomeize species, were studied for host-specific nodulation within the genus Aeschyizomene, also revisiting cross-inoculation groups described previously by D. Alazard (Appl. Environ. Microbiol. 50732-734, 1985). The whole collection of Aeschyrzornene nodule isolates was screened for synthesis of photosynthetic pigments by spectrometry, high- pressure liquid chromatography, and thin-layer chromatography analyses. The presence ofpuf genes in photo- synthetic Aeschynomene isolates was evidenced both by Southern hybridization with a Rhodobacter capsulatus photosynthetic gene probe and by DNA amplification with primers defined from photosynthetic genes. In addi- tion, amplified 16s ribosomal DNA restriction analysis was performed on 45 Aeschynomeize isolates, including strain BTAil, and 19 reference strains from Bradyrlzizobiunzjapoizicuin, Bradyrhizobium elkanii, and other Brady- rhizobium sp. strains of uncertain taxonomic positions. The 16s rRNA gene sequence of the photosynthetic strain ORS278 (LMG 12187) was determined and compared to sequences from databases. Our main conclusion is that photosynthetic Aeschynomerze nodule isolates share the ability to nodulate particular stem-nodulated spe- cies and form a separate subbranch on the Bradyrhizobium rRNA lineage, distinct from B. japonicuin and B. elkanii. Rhizobia symbiotically interact with leguminous plants to onomy, 20 species have been identified and assigned to six form nitrogen-fixing nodules most often exclusively occur- genera, Rhizobium, Sinorhizobizmi, Mesorhizobium,Bradyrhizo- ring on the roots. A few legumes, however, including several biunt and Azorhizobiuni (for a review, see reference 58), and Aeschynomene species, form nodules also on stem-located clllorhizobiunz (10). The unusual presence of a photosynthetic sites. The genus Aesclzynomene includes 22 stem-nodulated system in strain BTAil led to the tentative name “Pltotorhizo- species that readily nodulate all along the stem and many other bium thompsonum” (16) or “Photorhizobiunatlzompsonianuin” species, considered non-stem nodulated, since their nodulation (15) for this strain. However, 16s rRNA gene sequence anal- is restricted to the lower (collar) and submerged part of the ysis showed that strain BTAil was very closely related to both stem (6). A number of stem isolates ofAeschynoniene spp. are Bradyrhizobium japoiaicunz and Rhodopseudomoiaas palustris, of special interest because of their unusual ability to produce suggesting that BTAi1 could be appropriately named Brady- photosynthetic pigments, including both bacteriochlorophyll a rlzizobiimz sp. (A. indica) (59). This was later confirmed by (Bchl a) and carotenoids (15, 16, 34, 46). The well-studied additional 16s rRNA gene sequencing of other photosynthetic strain BTAil, isolated from stem nodules of Aeschynomene rhizobia (47, 55) and by fatty acid analysis (47). Additional indica, was the first bacteriochlorophyll-synthesizing rhizobia1 data came from numerical taxonomy (150 phenotypic charac- strain described (14, 15, 17). When grown aerobically and teristics) indicating that the photosynthetic rhizobia constitute heterotrophically under a light-dark cycle, strain BTAil syn- a unique phenon that could be considered distinct from Bra- thesizes photosynthetic pigments and forms photosynthetic re- dyrliizobiurn (33). The precise taxonomic status of Aeschyno- action centers like those of the purple nonsulfur photosyn- inene photosynthetic rhizobia thus remained unclear. thetic bacteria (17,42). Light-induced CO, and light-decreased Each rhizobium can nodulate only a limited number of le- O, uptakes gave evidence of the photosynthetic activity of this gumes, referred to as its host range. Depending on the extent strain (17, 26, 27). Because of its functional photosynthetic of their host range, rhizobia can be considered specific or apparatus, strain BTAil can be considered photosynthetic, and nonspecific. Aesclaynomeiie symbionts comprise both nonspe- by extension, so can all the rhizobia producing photosynthetic cific rhizobia of the cowpea group and rhizobia specific to the pigments. stem-nodulated species (1). No correlation between symbiotic Rhizobia are taxonomically very diverse. By polyphasic tax- properties and photosynthetic pigment synthesis could be es- tablished (48). * Corresponding author. Mailing address: Laboratoire des Symbio- Since the different reports on Aeschynoinene bradyrhizobia ses Tropicales et Méditerranéennes, I.R.D., Campus de Baillarguet, -generallv studied different rhizobium collections and focused B.P. 5035, 34032 Montpellier Cedex, France. Phone: (33) 04 67 59 38 on either nodulation (I), phylogeny (55), or photosynthesis 24. Fax: (33) 4 67 59 38 02. E-mail: [email protected]. (48), the data aph-. 3 3084 I Ex:LIWD I VOL.65, 1999 PHOTOSYNTHETIC BRADYRHIZOBIUM FROM AESCHINOMENE 3085 TABLE 1. Reference strains used in this study Sp. and strain LMG Original host plant Reference ARDRA group no. Bradyrhizobiiiin japonicum NZP5533 6136 Glycine ma B NZP554gT 613gT Glycine mm B USDA135 8321 Glycine ma B Bradyrhizobium elkanii NZP5531T 6134T Glycine ntm C NZP5532 6135 Glycine mm C Bradyrhizobium sp. ORS348 12200 Aeschynomene sp. 3 D ORS103 10665 Faidherbia albida 13 B ORS110 10666 Faidherbia albida 13 B ORS121 10671 Faidherbia albida 13 C ORS133 10689 Faidherbin albida 13 C ORS162 10705 Faidherbia albida 13 C ORS169 10712 Fnidherbia albida 13 B ORS174 10717 Faidherbia albida 13 C ORS175 10718 Faidherbin albidn 13 C ORS180 10719 Faidherbia albida 13 Sep. ORS187 10726 Faidherbin albida 13 B BR29 9520 Unknown C BR3621 9966 Acacia mangiiim 37 C BR4406 9980 Enterolobiiinz ellipticum 31 C INPA9A 10029 Demk sp. 37 B Abbreviations and designations: ORS, I.R.D. Collection, Institut de Recherche pour le Développement, Montpellier, France; LMG, Collection of Bacteria of the Laboratorium voor Microbiologie, University of Ghent, Ghent, Belgium; BR, strain from the CNPBS-EMBRAPA, Centro Nacional de Pesquisa cm Biologia do Solo, Seropedica, and Emprasa Brasiliera de Pesquisa Agropequaria, Rio de Janeiro, Brazil; INPA, National Institute of Amazonia Research, Manaus, Brazil; NZP, Culture Collection of the Department for Scientific and Industrial Research, Biochemistry Division, Palmerston North, New Zealand; USDA, US. Department of Agriculture, Beltsville, Md.; Sep., separate. prehensive view of the diversity and evolution of Bradyrhizo- were grown under continuous light (20 W/mz) at 28°C. Four to six plants were bium isolates from Aeschynomene species. Our objective was tested for each strain. Plants were observed for nodule formation over 6 to 8 weeks, and effectivenesswas estimated from visual observation of plant vigor and thus to examine possible links among the presence of photo- foliage color. synthetic pigments, nodulation capacity, and 16s rRNA gene- Photosynthetic pigment determination. Cultures were grown at 30°C for 7 based phylogeny among bradyrhizobia from Aeschynomene days under aerobic conditions on a 15-h-9-h light-dark cycle. Bchl was extracted species. These three topics were studied with a large collection under dim light with cold acetone-methanol (72 [vol/vol]) at 4°C for 30 min (35). of isolates. We first enlarged our collection of 45 Aeschyno- The supematant was analyzed with a Beckman DU40 spectrophotometer. Ab- sorption spectra were generated by scanning over a wavelength range from 350 mene strains (1-3a) with 78 new bacterial isolates from nodules to 800 nm. Carotenoids were further purified and analyzed by high-pressure of diverse native stem- and non-stem-nodulatedAeschynomene liquid chromatography (HF'LC) or thin-layer chromatography (TLC) as previ- species from different places in Senegal. We screened the iso- ously described (35). lates for photosynthetic pigment production (Bchl a and caro- Southern hybridization. Genomic DNA was extracted as previously described tenoids), for DNA hybridization with a Rhodobacter capsulatus (36). Total DNA was digested by EcoRI, PstI, or HindIII as specified by the manufacturer (Boehringer Mannheim or Pharmacia). Restricted DNA was run photosynthetic gene probe, and for DNA amplification with in a 0.8% agarose gel and transferred to a nylon membrane under alkaline photosynthetic gene primers. We characterized their host conditions by the Southem blot