System. Appl. Microbiol. 23, 418-425 (2000) SYSTEM4TIC AND © Urban & Fischer Verlag _htt-,-p:_Ilw_w_w_.ur_ba_nf_isc_h_er._de-,-/jo_u_rna_ls_/s_am____________ APPLIED MICROBIOLOGY Restriction Fragment Length Polymorphism Analysis of 165 rONA and Low Molecular Weight RNA Profiling of Rhizobial Isolates from Shrubby Legumes Endemic to the Canary Islands ADRIANAjARABO-LoRENZOI, ENCARNA VELAzQUEZ2, RICARDO PEREZ-GALDONAI, MARfA c. VEGA-HERNANDEZ\ EUSTOQUIO MARTINEZ-MoLINA2, PEDRO F. MATEOS2, PABLO VINUESA3, ESPERANZA MARTINEZ-RoMERO\ and MILAGROS LEON-BARRIOS! I Departamento de Microbiologia y Biologia Celular, Facultad de Farmacia, Universidad de La Laguna, Tenerife, Spain 2 Departamento de Microbiologia y Genetica, Universidad de Salamanca, Spain 3 Fachgebiet fiir Angewandte Botanik und Zellbiologie, Philipps-Universitat Marburg, Germany 4 Centro de Investigaci6n sobre Fijaci6n de Nitr6geno, Universidad Nacional Aut6noma de Mexico, Cuernavaca, Morelos, Mexico Received June 16,2000 Summary Thirty-six strains of slow-growing rhizobia isolated from nodules of four woody legumes endemic to the Canary islands were characterised by 165 rDNA PCR-RFLP analyses (ARDRA) and LMW RNA profil­ ing, and compared with reference strains representing Bradyrhizobium japonicum, B. eikanii, B. iiaonin­ gense, and two unclassified Bradyrhizobium sp. (Lupinus) strains. Both techniques showed similar re­ sults, indicating the existence of three genotypes among the Canarian isolates. Analysis of the combined RFLP patterns obtained with four endonucleases, showed the existence of predominant genotype com­ prising 75% of the Canarian isolates (BTA-1 group) and the Bradyrhizobium sp. (Lupinus) strains. A second genotype was shared by nine Canarian isolates (BGA-1 group) and the B. japonicum and B. iiaoningense reference strains. The BE5-5 strain formed an independent group, as also did the B. elka­ nii reference strains. LMW RNA profile analysis consistently resolved the same three genotypes detected by 165 ARDRA among the Canarian isolates, and suggested that all these isolates are genotypically more related to B. japonicum than to B. elkanii or B. iiaoningense. Cluster analysis of the combined 165 ARDRA and LMW RNA profiles resolved the BTA-1 group with the Bradyrhizobium sp. (Lupinus) strains, and the BES-5 isolate, as a well separated sub-branch of the B. japonicum cluster. Thus, the two types of analyses indicated that the isolates related to BTA-1 conform a group of bradyrhizobial strains that can be clearly distinguishable from representants of the tree currently described Bradyrhizobium species. No correlation between genotypes, host legumes, and geographic location was found. Key words: 165 rDNA RFLP analysis - LMW RNA profiles - Bradyrhizobium - Canary Islands - tagasaste (Chamaecytisus proliferus) - genetic diversity Introduction The rhizobia are a group of alpha proteobacteria ca­ ed, Rhizobium, Azorhizobium, Sinorhizobium, Mesorhi­ pable of inducing nitrogen-fixing nodules on the roots or zobium (for reviews see MARTINEZ-RoMEO and CA­ stems of legumes. Despite the fact that the Leguminosae BALLERO-MELLADO, 1996; YOUNG and HAUKKA, 1996; is one of the largest families of plants with more than VAN BERKUM and EARDLY, 1998), and Allorhizobium (DE 19,000 species, only a small number have been studied LAJUDIE et al., 1998). A polyphasic approach has been re­ with regard to the rhizobia nodulating them. The isola­ sponsible for the new taxonomy in the fast-growing rhi­ tion of bacteria from nodules of previously uninvestigat­ zobia, but the most relevant studies for developing the ed legumes has revealed the existence of large rhizobia I current rhizobia classification have been 165 rDNA se- diversity, leading to deep changes in the taxonomy of the Rhizobiaceae. Among the fast-growing rhizobia a large number of new species and genera have been described in Abbreviations: LMW RNA - Low Molecular Weight RNA; recent years, and currently five genera are widely accept- ARDRA - amplified ribosomal DNA restriction analysis 0723-2020/00/23/03-418 $ 15.00/0 16S ARDRA and LMW RNAs of Canarian Bradyrhizobia 419 quence analyses. The taxonomy of slow-growing rhizo­ markers because their ubiquitous distribution in living bia, on the other hand, is less refined, due in part to the beings and their conserved structure and homologous conflictive results obtained in the analyses of phenotypic biochemical functions in protein synthesis (HbFLE, and genotypic traits (So et aI., 1994; VAN ROSSUM et aI., 1988). Recently, a new single dimension electrophoretic 1995). The number of Bradyrhizobium species currently technique in polyacrylamide gels - Staircase Electro­ admitted has increased from one (B. japonicum, JORDAN, phoresis (SCE) - has permitted optimum separation of 1982) to three, with the descriptions of B. elkani (KUYK­ these molecules (CRuz-SANCHEZ, et aI., 1997), and has ENDALL et aI., 1991) and B. liaoningense (Xu et aI., been successfully applied in the identification of prokary­ 1995). But, as in the case of fast-growing rhizobia, the otes (VELAZQUEZ et aI., 1998a; 198b) and eukaryotes number of Bradyrhizobium species is expected to in­ (VELAZQUEZ et aI., 2000). This technique has already crease as more slow-growing strains isolated from previ­ been applied to genera and species of the Rhizobiaceae, ously unstudied legumes are characterised. yielding a different profile for each bacterial species anal­ In the Canary Islands flora a large number of endemic ysed (VELAZQUEZ et aI., 1998a). The LMW RNA profiles legumes is found (HANSEN and SUNDING, 1993). Among comprise the 5S rRNA, class 1 tRNA and class 2 tRNA. them, some shrub legumes are of great agricultural and The 5S rRNA zone is characteristic for each genus while ecological interest. Tagasaste (Chamaecytisus proliferus the tRNA profiles are characteristic for each of the L. fil. Link ssp. proliferus var. palmensis (Christ) Hans. species analysed. Therefore, LMW RNA profiles produce and Sund. (ACEBES-GINOVES et aI., 1991) is the most a molecular fingerprint that can be used as a valuable valuable native fodder shrub found in the archipelago. Its tool for taxonomic and diversity studies (HbFLE, 1990; high nutritive value and palatability for grazing animals, VELAZQUEZ et aI., 1998a; 1998b, 2000). pigs and poultry is well documented (PEREZ DE PAZ et aI., In this work we have analysed the genetic diversity 1986; BORENS and POPPI, 1990; SNOOK, 1996). Tagasaste found among nodule isolates from endemic woody has been used as forage in the canaries for centuries legumes of the Canary islands. The results of 16S rDNA (PEREZ DE PAZ et aI., 1986). In the last century, it was in­ PCR-RFLPs and LMW RNA profiles are compared. troduced in other parts of the world such as Australia and New Zealand (FRANCISCO-ORTEGA et aI., 1991; SNOOK, 1996), where it has been widely cultivated, and Materials and Methods where it has deserved preparation of an specific inocu­ lant (ROUGHLEY, 1990). In addition to its value as fodder Bacterial strains and growth conditions plant, tagasaste plantations have been used as a shelter The strains used in this study are presented in Table 1. Those for other crops to reduce erosion in expossed slops, for not previously described (indicated in Table 1) were isolated redemption of wasteland, firebreaks, or salinity control from root nodules following the procedure described by VIN­ (SNOOK, 1996). CENT (1970). The bacteria were grown at 28°C in yeast manni­ In previous works it was shown that tagasaste and tol (YM) medium (VINCENT, 1970). other endemic legumes are nodulated in the Canaries by a group of Bradyrhizobium strains phenotypically and Plant infection tests genetically diverse (LEON-BARRIOS et aI., 1991; SANTA­ Isolates were tested for nodulation on tagasaste and Glycine max. Seed surface sterilisation was carried out with 5% com­ MARIA et aI., 1997; VINUESA et aI., 1998). In this work we mercial hypochlorite as described (VINCENT, 1970). Germina­ have used a relatively large collection of isolates (36 tion of tagasaste seeds required a previous scarification with strains) obtained from root nodules of several genera of concentrated sulphuric acid. endemic woody legumes of the Canary Islands, collected in different islands and locations. Our aim was to study DNA isolation the diversity of the rhizobia isolates nodulating woody Total genomic DNA was obtained from bacterial batch cul­ endemic legumes growing in four different Islands of the tures grown until late exponential phase using a standard archipelago, to determine if a specific group of bradyrhi­ cetyltrimethylammonium bromide (CTAB) protocol (WILSON, zobial strains associated with these shrubs legumes are K., 1994). The DNA concentration of the preparations was esti­ mated by visually comparing the DNA samples with samples of confined to these Islands (as suggested from our previous known concentrations of lambda-DNA in agarose gels. works, VINUESA, et aI., 1999, 2000), and to find out the distribution and representation of different genotypes de­ peR amplification of 165 rRNA genes tected in the Canarian Archipelago. The universal primers fDl and rDl were used to amplify Here we have used two different approaches to detect nearly full-length 16S rRNA gene (WEISBURG et aI., 1991). The diversity: RFLP analysis of PCR-amplified 16S rDNA primers, Taq polymerase, dNTPs and buffers were purchased fragments and Low Molecular Weight RNA (LMW from Amersham-Pharmacia Biotech. The PCRs were performed RNA) profiles. Amplified ribosomal