O INSTITUTPASTEUR/ELSEVIER Res. Microbiol. Paris 1993 1993, 144, 55-67 kc,f Enzyme polymorphism of Azorhizobium strains and other stem- and root-nodulating bacteria isolated from Sesbanìa rostrata G. Rinaudo (l), M.P. Fernandez (2), A. Effosse (2), B. Picard (3) and R. Bardin (2) (I) Institut Français de Recherche Scientifique pour le Développement en Coopération (ORSTOM), (2) Unite‘ de Recherche Associée au Centre National de Recherche Scientifique 1450, (’I 2, Laboratoire d’Ecologie microbienne du Sol, université Claude-Bernard-Lyon I, 69622 Villeurbanne Cedex (France), and (3) Laboratoire de Microbiologie, Hôpital Beaujon, 92110 Clichy (France) SUMMARY Relationships between bacterial groups nodulating Sesbania rostrata were evaluat- ed through examination of electrophoretic polymorphism of esterases and metabolic en- zymes. The following conclusions were drawn: (i)the differentiation of two genomic species within Azorhizobium strains and a group of non-identified strains (probably ßhizo- bium) was strongly supported by enzyme electrophoresis ;(i¡) esterases were more elec- trophoretically polymorphic than metabolic enzymes, since 35 and 11 electrophoretic types, respectively, were detected within the 57 strains studied ; (iii)strains isolated from stem or root nodules were genetically very similar and could not be differentiated; (iv) six Azorhizobium strains isolated from plants growing in saline soils could not be grouped separately from the other strains, which might be attributed to the adaptation of azorhizobia to epiphytic conditions; and (VIa comparative study of esterase patterns of azorhizobia showed that strains isolated in the Philippines probably originated in north- ern Senegal, but did not reveal a clear separation between strains originating from north- ern and central Senegal. Key-words: Nitrogen, Enzyme polymorphism, Azorhizobium; Stem- and root- nodulating bacteria ; Electrophoretic types, Sesbania rostrata. INTRODUCTION um, quite distinct from the genera Rhizobium a9g Bradyrhizobium, and containing one spe- The tropical legume Sesbania rostrata gener- cies, A. caulinodans (type strain ORS 571). ally behaves as a wild annual plant in periodi- These bacteria were unusual in. that they fixed cally flooded soils. The study of bacteria able atmospheric nitrogen both symbiotically and ex to produce N2-fixing nodules, both on the stems planta (Elmerich et al., 1982). DNA-rRNA and on the roots of S. rostrata, led Dreyfus et hybridization studies placed Azorhizobium al. (1988) to propose a new genus Azorhizobi- closer to Xanthobacter and Bradyrhizobium Submitted June 25, 1992, accepted November 30, 1992. 2 O AVR. 1993 -”.-”..-.- - - 56 G. RINAUDO ET AL. than to Rhizobium and Agrobacterium. In ad- azorhizobia, where most strains constitute a nar- dition to azorhizobia, two strains isolated from row cluster which can be grouped into a single root nodules of S. cannabina and S. grandiflora, species, A. caulinodans. respectively, formed effective nodules on both The aim of this study was to evaluate the roots and stems of S. rostrata, but did not fix differences between and within the bacterial N, in culture. The DNA-rRNA hybridization groups found by DNA-DNA hybridization with- results showed that these strains belonged to the in the S. rostrata nodulating strains and to ex- Rhizobium-Agrobacterium complex (Dreyfus et amine the possible correlation between the al., 1988). More recently, Rinaudo et al. (1991) genetic diversity of strains and enh-onmental analysed the genetic diversity of 191 strains iso- factors by comparing the electrophoreticprofiles lated from stem and root nodules of S. rostrata of esterases and metabolic enzymes. Multilocus grown in different geographical areas in Sene- enzyme electrophoresis is probably one of the gal and in the Philippines, by examining DNA best approaches in large-scale studies to estimate base composition, DNA-DNA hybridizations the genetic diversity and structure of related and the ability to fix nitrogen in symbiosis with populations (Selander et al., 1986). It has recent- S. rostrata and/or in the free-living state. The ly been used to study the genetic population following conclusions were drawn : (i) most of structure of soil bacteria involved in symbiotic the isolates (184 strains) belonged to the genus associations with plants, namely the Rhizobia- Azorhizobium; (ii) the seven remaining strains ceae (Engvild and Nielsen, 1984; Young, 1985; exhibited very low levels of DNA binding with Young et al., 1987; Pinero et al., 1988; Azorhizobium strains and probably belonged to Martinez-Romero et al., .1991) and Frankia the genus Rhizobium, since they had G+C strains (Gardes et al., 1987; Prin et al., 1991). values between 59 and 63 mol Yo, were fast- The present'study is the first report on genetic growing and did not fix N, under free-living diversity among bacterial symbionts of S. ros- conditions ; (iii) the Azorhizobium strains could trata, assessed by multilocus enzyme elec- be divided into two groups : genomic species 1, trophoresis. constituting the majior group (175 strains), cor- responds to A. caulinodans since it contains type strain ORS 571, while genomic species 2 contains MATERULS AND METHODS only nine strains. The DNA-DNA hybridization method, which Bacterial strains measures the relatedness of entire genomes, is The 57 bacterial strains included in this investi- relatively unaffected by small variations in DNA gation are listed in table I. They were isolated from sequences (Schleifer and Stackebrandt, 1983). stem and root nodules of S. rostrata plants located Other methods such as protein gel electropho- in various geographical areas in Senegal and The resis, DNA restriction fragment length polymor- Philippines. All of the strains were selected from the collection of strains previously studied by Rinaudo phism or multilocus enzyme electrophoresis, et al. (1991). Fifty strainsrwere found to belong to must be used in order to distinguish differences the genus Azorhizobium: 41 strains belong to geno- between closely related organisms, such as ~ mic species 1¶ which corresponds to A. caulinodans, UNA = u-naphthyl acetate. GDH = glutamate dehydrogenase. uNB = u-naphthyl butyrate. HBD = hydroxybutyrate dehydrogenase. UNP = a-naphthyl propionate. IA = indoxyl acetate. ßNA = ß-naphthyl acetate. LDH = lactate dehydrogenase. ßNB = ß-naphthyl butyrate. MDH = malate dehydrogenase. ßNP = ß-naphthyl propionate. MF = relative mobility value. DIA = diaphorase. NI strains = non-identified strains. ET = electrophor,etic type. .. .. ENZYME POLYMORPHISM OF NODULATING BACTERIA 57 Table I. Origin of the strains tested and DNA relatedness. Vo Reassociation with labelled Strain DNA from Serial no. Registry no. Origin Isolated from strain ORS 571 (*I strain SD 02 (*I Azorhizobium strains : Genomic species 1 (A. caulinodans) 1 ORS 571 North Senegal stem nodule 100 2 sv o1 99 YY 91 3 sv 12 YY 9) 101 4 SV 17 >Y7) Y9 97 5 SV 27 9) 98 6 SV 31 YY root nodule 87 7 sv 33 9) 9) 87 8 BD 05 Y9 97 92 9 RT O1 YY stem nodule 88 10 RT 06 Y9 Y9 91 11 RT 10 Y? YY 104 12 RT 13 YY Y9 87 13 RT 15 Y9 root nodule 84 14 FY O1 Y9 stem nodule 93 15 FY 10 YY 9) 81 16 FY 12 YY Y9 88 17 FY 18 YY Y9 90 18 FY 29 YY root nodule 89 19 SG O1 Central Senegal stem nodule 97 20 SG 03 YY Y9 96 21 SG 10 Y7 YY 94 22 SG 21 Y9 Y9 97 23 SG 23 Y9 YY 83 24 SG 26 YY root nodule 85 25 DP 03 YY stem nodule 88 26 DP 04 YY Y9 92 27 DP 07 Y9 99 97 28 DP 13 ?Y 99 92 29 DP 23 YY root nodule 81 30 KL 03 YY stem nodule 94 31 KL 05 >Y Y> 80 32 KL 08 YY Y9 96 33 KL 14 Y9 9) 90; 34 KL 17 93 root nodule 92 35 SD O1 Y9 stem nodule 104 ?Y Y9 36 SK 02 -=w 99 37 IRG 10 The Philippines 9) 92 38 IRG 13 Y9 YY 1O0 39 IRG 22 Y9 93 40 IRG 23 root nodule 1O0 41 IRG 42 stem nodule 96 Genomic species 2 42 SG 05 Central Senegal stem nodule 44 79 43 SG 06 >Y 9) 46 94 (continued on next page) 58 G. RINAUDO ET AL. To Reassociation with labelled Strain DNA from Serial no. Registry no. Origin Isolated from strain ORS 571 (*) strain SD 02 (*I 44 SG O9 Y, 51 76 45 SG 19 3, 51 83 46 SG 22 Y? 48 89 Y, 47 SG 25 50 J 90 48 SG 28 root nodule 47 92 49 SD 02 stem nodule 53 1o:o 50 SD 04 9, 44 86 NI strains: 51 RT O9 Northern Senegal stem nodule 4 O 52 RT 11 3, Y, 2 -1 53 RT 12 $2 9, O 2 54 RT 14 9, root nodule 4 O 55 DP 21 Central Senegal Y, O 6 56 KL 13 Y, stem nodule -2 4 57 TAL 674 NifTal Culture YY 4 3 Collection (*) From: Rinaudo et al. (1991): ORS 571, type strain of genomic species 1 (A. caulinodans); SD 02, type strain of genomic species 2. and 9 strains to genomic species 2. The seven other tant voltage (7 V/cm) until the bromophenol blue strains probably belong to the genus Rhizobium as marker had run 13 cm (Uriel, 1966; Goullet and previously indicated. They will be designated here as Picard, 1985a). The esterases were stained accord- NI strains (non-identified strains). ing to Uriel (1961) using a-naphthyl acetate (aNA), ß-naphthyl acetate (ßNA), a-naplhthyl propionate (aNP), ß-naphthyl propionate (ßNP), a-naphthyl Preparation of bacterial extracts butyrate (aNB), ß-naphthyl butyrate (ßNB) and in- doxyl acetate (IA) as substrates. The following five Isolates were grown for 24 h on an orbital shaker metabolic enzymes were assayed : glutamate de- at 28°C in 50 ml of yeast extract-lactate medium hydrogenase (GDH), malate dehydrogenase (MDH), (Rinaudo et al., 1991).