4. 621 Characterization (Seti... Characterization and Phylogenetic Analysis of Soybean Rhizobial Strains from Java and Sumatra SETIYO HADI WALUYO Center for the Application of Isotopes and Radiation Technology, National Nuclear Energy Agency (BATAN), Jalan Lebak Bulus Raya Pasar Jum'at, Jakarta 12070, Indonesia Bradyrhizobium japonicum B. japonicum B. japonicum Bradyrhizobium elkanii Bradyrizobium japonicum Sinorhizobium fredii B. elkanii B. japonicum S. fredii cross- inoculation cross-inoculation” promiscuous Bradyrhizobium japonicum B. japonicum promiscuous distinct Bradyrhizobium japonicum Bradyrhizobium elkanii Bradyrhizobium japonicum Sinorhizobium fredii. B. elkanii B. elkanii B. japonicum S. fredii ARDRA cross-inoculation sequencing Twenty-seven and twenty-four soybean rhizobial isolates from Java and Sumatra, respectively,were characterized. Based on cross-inoculation, eight isolates from Java and nine from Sumatra could be grouped as soybean specific rhizobial species , while 19 isolates from Java and 15 from Sumatra were promiscuous. ARDRA of intergenic spacer region of 16S-23S rDNA showed that the isolates from Java were different from those from Sumatra. Six soybean specific isolates from Java and one from Sumatra were in the same cluster with the reference strain, USDA 110, thus could be classified as . One soybean specific isolate from Java's has a distinct position, while the other soybean specific isolate from Java was placed in another group dominated by isolates from Sumatra. The nineteen promiscuous isolates from Java were clustered in a different group. This group, together with the isolate with distinct position and the other group that were dominated by isolates from Sumatra, were distinct from USDA 110. Therefore it is tempting to speculate that they represent indigenous soybean rhizobial strains. Based on complete sequencing of the amplified 16S rDNA of 21 selected isolates, these isolates could be divided into three groups consisting of twelve , eight and one . Most of the strains were isolated from acid soils at Sitiung, West-Sumatra, while only two isolates were obtained from Java. Four isolates from Java, two isolates from Sitiung, and two isolates from Bukit Tinggi were identified as . One isolate from Java with a distinct position on the ARDRA was identified as . Key words: Amplified Ribosomal DNA Restriction Analysis (ARDRA), cross-inoculation, rhizobia, soybean, sequencing Dua puluh tujuh isolat bakteri rhizobia kedelai dari Jawa dan 24 isolat dari Sumatra telah diidentifikasi secara pada tanaman kedelai dan kacang hijau serta secara genetika molekuler (ARDRA dan pemetaan). Dengan metode “ , 8 isolat Jawa dan 9 isolat Sumatra dapat membentuk bintil akar efektif hanya pada tanaman kedelai (spesifik), sedangkan sisanya 19 isolat Jawa dan 15 isolat Sumatra dapat membentuk bintil akar efektif pada tanaman kedelai dan kacang hijau ( ). Metode ARDRA dapat membedakan antara isolat dari Jawa dan Sumatra. Enam isolat Jawa dan tiga isolat Sumatra berada dalam satu grup dengan USDA 110, dan diklasifikasikan sebagai . Satu isolat Jawa spesifik kedelai menempati posisi tersendiri, dan sisanya 1 isolat berada dalam grup yang didominasi oleh isolat-isolat Sumatra. Sembilan belas isolat Jawa membentuk grup tersendiri, bersama dengan satu isolat yang posisinya dan grup yang didominasi oleh isolat-isolat Sumatra. Grup ini berbeda nyata dengan USDA 110. Oleh karena itu, diduga kuat bahwa isolat-isolat tersebut adalah bakteri rhizobia kedelai asli Indonesia. Pemetaan deret ribosomal DNA 16S dari 21 isolat pilihan menunjukkan bahwa isolat-isolat tersebut dapat dibagi menjadi tiga grup, yaitu grup 12 isolat, grup delapan isolat, dan satu isolat yang khusus tersebut masuk dalam grup Sebagian besar galur di peroleh dari tanah asam di Sitiung, Sumatra Barat. Hanya dua strain yang berasal dari tanah Jawa. Empat isolat Jawa, dua isolat Bukit Tinggi dan dua isolat Sitiung diidentifikasi sebagai . Satu isolat yang khusus diidentifikasi sebagai . Kata kunci: , , kedelai, rhizobia, Leguminous plants nodulating bacteria were first described in 1888 (Beijerinck 1888) and their initial classification as rhizobia was based on their host range specificity. Their symbiotic relations with the host plants remains very important, since this is the most conspicuous feature of rhizobia and has an important practical value. However, the symbiotic and physiological properties of soybean nodulating rhizobia have been found to be more diverse than originally anticipated. The discovery of fast-growing soybean nodulating rhizobia belonging to the genus ( ) (Keyser . 1982; SchollaSinorhizobium Ensifer et al and Elkan 1984; Young 2003, Young 2010) underlines the need to consider the symbiotic properties, particularly since representatives of this genus often fail to nodulate modern soybean cultivars. In addition, there are some rhizobia outside the genus and ( ), now designated spp., that were reported to form nodules on soybean (Chen . 1995; Jarvis . 1997). The ambiguous results often found with host- range nodulation tests have driven the development of DNA based determination techniques (Laguerre . 1994; Massol-Deya . 1995; Vandamme . 1996; Rademaker and De Bruijn 1997). In recent years, other methods, including phenotypic traits, DNA:DNA relatedness and Bradyrhizobium Sinorhizobium Ensifer Mesorhizobium et al et al et al et al et al *Corresponding author, Phone : +62-21-7690709, Fax : +62-21-7513270, E-mail: shwaluyo@yahoo.com ISSN 1978-3477, eISSN 2087-8575 Vol 5, No 4, December 2011, p 170-181 I N D O N E S I A Available online at: http://www.permi.or.id/journal/index.php/mionline DOI: 10.5454/mi.5.4.4 molecular techniques based on Polymerase Chain Reaction (PCR), have been included in rhizobial classification (Sikora and Redzepovic 2003; Kwon . 2005; El-Fiki 2006, Martens . 2007). Presently, there is a great variety of phenotypic and genotypic methods available that permit a different degree of phylogenetic classification varying from genus, species, subspecies, biovar to the strain level. However, the description of new genera and species of root-nodulating rhizobia should fulfill a minimal standard, as has been proposed by Graham (1991) and Novikova (1996). The development and implementation of these molecular techniques have accelerated the taxonomic evaluation of rhizobia. The current classification of rhizobia, which is mainly based on the nucleotide sequences of the small sub- unit ribosomal RNA (rRNA), are ( ), , ( ) , , , , , , and (Hungria . 2006; Willems 2006; Raychaudhuri . 2007; Rivas . 2009, Lu . 2011). The importance of rhizobial strains for soybean cultivation in Indonesia had already been shown since a long time ago (Toxopeus 1938). Meanwhile, the taxonomy of and genera is of particular interest as they consist of strains known to nodulate soybean, which is an important food crop. Most of soybean nodulating rhizobial strains from the primary gene centers for soybean in China or Japan have been well studied and characterised. However, while Indonesia is supposed to be a second gene-centre of soybean plants, (Hymowitz and Newell 1981) there is only a limited information on the diversity of rhizobial strains from Indonesia. There is a general lack of information on the population structure of the indigenous soybean rhizobia native to Indonesian soils. However, insight in the structure of indigenous soybean rhizobia populations is one of the important aspects for the success of Biological Nitrogen Fixation (BNF). Particularly in the areas where indigenous rhizobia are present abundantly (Saono 1988). As a consequence, a thorough survey is needed to study the occurrence of the bacteria in different locations in Indonesia. Furthermore, it is essential to characterise the isolates using reliable molecular methods. It opens the possibility to select elite indigenous soybean rhizobia under favourable conditions, such as those in Java, where they are abundantly present in most soils. In addition, under acidic condition such as in Sumatra, et al et al et al Rhizobium Agrobacterium Bradyrhizobium Sinorhizobium E n s i f e r , M e s o r h i z o b i u m , A z o r h i z o b i u m Methylobacterium Burkholderia Cupriavidus Devosia Ochrobacterium Phyllobacterium Shinella et al et al et al et al Bradyrhizobium Sinorhizobium where the condition is unfavourable for rhizobia, the survey may be important to select rhizobial strains that are well adapted to stress conditions. In this study, therefore, a comparison was made between indigenous soybean rhizobia isolated from traditional soybean areas in Java and a variety of soils in Sumatra. The bacterial isolates were characterised for their symbiotic properties and classified based on Amplified Ribosomal DNA Restriction Analysis (ARDRA) of PCR-amplified 16S rDNA and 16S-23S rDNA intergenic spacer region. A selected number of isolates were characterised in more detail by the amplification, cloning, and sequence analysis of the major part of their 16S rDNA genes. Fifty-one soybean rhizobial isolates from Java and Sumatra, USDA 110 and CB1809 and C B 7 5 6 ( o b t a i n e d f r o m t h e L a b o r a t o r y o f Microbiology, Wageningen University, Netherlands) were maintained in Yeast Extract Mannitol Broth (YEMB) solidified with 1.0% agar and grown for inoculum preparation in YEMB at 30 °C for 4 - 7 days (Somasegaran and Hoben 1995). To obtain indigenous soybean nodulating rhizobial strains, local varieties of soybean seeds ( cv. Tidar) obtained from the Bogor Research Centre for Food Crops, Bogor, Indonesia and mungbean ( cv. Manyar) seeds obtained from the Centre for the Application of Isotopes and Radiation Technology, National Nuclear Energy Agency, Jakarta, Indonesia were used to study the host specificity of the isolates. To characterize the symbiotic properties, the rhizobial isolates were used to inoculate soybean and mungbean plants grown on a modified Hoagland-N free medium as described previously (Winarno and Lie 1979). Plants were harvested 20 days after inoculation. Colour and weight of shoots, as well as weight and number of nodules were determined. The capacity to fix N was deduced by comparing the colour and weight of the control and inoculated plants, all were grown in a N-free medium. Genomic DNA to be used as template for PCR amplification of the 16S rRNA gene and the 16S-23S rRNA intergenic spacer regions was extracted from YEMB-grown bacterial cells as follows. Four ml of bacterial culture containing 10 cfu ml was harvested by centrifugation and suspended in 450 µL TE buffer (1 mM Tris-HCl, 0.1 mM EDTA, pH 7.4) , containing 0.5% SDS, followed by incubation at 37 °C with gentle shaking for 30 minutes. DNA was extracted by the addition of an equal volume of phenol MATERIALS AND METHODS Rhizobial Strains and Media. Agronomic Analysis. Genetic Analysis. B. japonicum Bradyrhizobium spp. Glycine max Vigna radiata 9 -1 Volume 5, 2011 Microbiol Indones 171 buffered in 10 mM Tris-HCl and 1 mM EDTA, pH 7.0 to the mixture. This extraction was repeated once using an equal volume of phenol and chloroform mixture. Subsequently, nucleic acid was precipitated by the addition of equal volume of ethanol 90% (-20 °C) and 10% volume of sodium acetate (3 M, pH 5.2) . The precipitate was collected by centrifugation, dried and dissolved in 50 µL sterile water. The amount of the isolated DNA was determined by electrophoresis on a 1% agarose gel containing ethidium bromide with 0.1 g of DNA digested with III (GibcoBRL LifeTechnology, Breda, Netherlands) as a reference. Ribosomal DNA fragments were amplified using a set of primers as described previously, primer 8f [ 5'-CAC GGA TCC AGA GTT TGAT (C/T) (A/C) TAG TCC AG-3'] an primer 1510r [5'-GTT AA GTT ACTG (C/T) TAC GTT GTT ACG ACTT-3'] for 16S rDNA PCR reaction (Lane 1991; Laguerre et al. 1994) and primer pHr [5'-TGCGGCTGGATCACCTCCTT-3'] and primer p23SROI [5'-GGCTGCTTCTAAGCCAAC- 3'] for 16S-23S rDNA intergenic spacer PCR reaction (Massol-Deya . 1995). DNA amplification using approximately 50 ng of genomic DNA as a template in a 100 l PCR reaction volume was performed using a UNOII Thermocycler, Biometra, Göttingen, Germany as described previously (Massol-Deya et al. 1995). The size and amount of the amplified DNA was examined by electrophoresing 5.0 µL of PCR product on a 0.7% agarose gel containing ethidium bromide and DNA digested with dIII as a reference. Approximately 400 ng of the amplified DNA was digested using restriction endonucleases I, I, III, and I (5 units per 25 µL reaction) following instruction of the manufacturer (GibcoBRL LifeTechnology, Breda, The Netherlands) and the generated fragments were separated by electrophoresis on 3% agarose gels containing ethidium bromide at 100 V for 2 hours. ARDRA fingerprints of 16S rDNA and 23S-16S rDNA (intergenic spacer region) PCR- products were recorded as TIFF files, analysed and used to prepare a dendogram based on predictions by Unweighted Pair Group Method using Arithmetic averages (UPGMA) by Molecular Analysist Software (BioRad 1995). Based on the effectiveness and the efficiency of the soybean nodulating rhizobia strains in biological nitrogen fixation of soybean, seven isolates from Java and 14 isolates from Sumatra were chosen for further examination by 16S rDNA sequencing. The 16S rDNA PCR products from the selected strains, were purified using Qiaquick PCR purification kit following instructions of the Hind et al Hin Cfo Dde Hae Msp Cloning of the 16S rDNA PCR Products and Plasmid DNA Isolation. manufacturer (Qiagen, Hilden, Germany), and were quantified by electrophoresis on 1.2% agarose gel with known amount of DNA digested with dIII as a standard (GibcoBRL Life Technology, Breda, Netherlands). Then the PCR-amplified 16S rDNA fragments were cloned in JM109 using the pGEM -T Vector system following a procedure provided by the supplier (Promega, Leiden, Netherlands). For plasmid DNA isolation, one colony of ampicillin-resistant transformant was used to inoculate to Luria-Bertani (LB) broth medium containing ampicillin (100 g mL ), and incubated at 37 °C for 24 hours (Manniatis . 1982). Plasmid DNA was isolated using a column Wizard Plus Minipreps DNA Purification System (Promega, Leiden, Netherlands). Purified plasmid DNA was quantified by electrophoresis on a 1.2% agarose gel with known amount of DNA digested with dIII as a standard (GibcoBRL Life Technology, Breda, Netherlands). The purified plasmid DNA (250 ng) was used as template for sequencing reaction. Infrared-labelled primers were used for reaction, for the forward reaction primer SP6 IRD800 (5' -GAT TTA GGT GAC ACT ATA G-3') and for the reverse reaction primer T7 IRD800 (5'- TAA TAC GAC TCA CTA TAG GG-3')(MWG Biotech, Ebersberg, Germany). PCR sequencing reactions were performed with reagents provided by the supplier (Amersham Pharmacia Biotech, Freiburg, Germany) and the following temperature setting: 93 °C for 3 minutes; 30 cycles of 93 °C for 30 seconds, 45 °C for 30 seconds, 70 °C for 15 seconds; and storage at 4 °C. The products were separated and analysed on a Li-Cor DNA sequencer 4000L (LiCor, Lincoln, Nebraska, USA). Before loading the samples (1.8 µL), the gel was pre-run for 30 minutes at 1000V. After loading samples, electrophoresis was carried out at 1000 V constant voltage while the gel was heated at 50 °C. The sequences of approximately 1000-1200 nucleotides both in the forward and reverse directions were obtained, corrected manually, and combined into a single contig of 1200 1500 of unambiguous sequence by using SeqMan II DNA Star Software (DNASTAR Inc., Madison, Wisconsin, USA). The obtained 16S rDNA sequence data were analysed for their homology with the blastn program from The GenBank Network (http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/). A rooted phylogenetic tree (neighbour joining) was calculated using the programmes and database from Ribosomal RNA Alignment (ARB), an environment Sequence Analysis. - Identification of the Soybean Rhizobia on the Basis of 16S rDNA Homology. Hin Escherichia coli et al Hin R -1 TM 172 WALUYO ET AL. Microbiol Indones for 16S/18S/23S ribosomal RNA sequence data, and position 150 1114 (Felsenstein correction). The ARB Package is a software containing combination of alignment and dendrogram tools, allowing alignment to a comprehensive 16S rDNA database and detailed phylogenetic analysis. In general, rhizobial strains obtained from Java were as effective as the soybean- nodulating reference strains USDA 110 and CB 1809. The number of nodules were 43, 29, and 46 for USDA 110, CB 1809 and isolates from Java, respectively, while the total weights of nodules were 0.288, 0.25, and 0.263 g for USDA 110, CB 1809 and isolates from Java, respectively. The average shoot weight of soybean inoculated with USDA 110, CB 1809 and isolates from Java were 3.36, 3.7, and 3.75 g, respectively. On the other hand, the average number and weight of nodules, as well as shoots weight, of plants inoculated with the rhizobial strains from Sumatra were 37, 0.225, and 2.43 g, respectively. This is significantly lower than those from Java. The difference was most significant in the weight of shoots (= 0.01, MSTAT-C 1988, Table 3). It was observed that the first two leaves of all soybean plants inoculated with rhizobial isolates from Sumatra were yellow and in most cases were lost very quickly, indicating poor N fixing capacity. The ability of the isolated rhizobial strains to form nodule on mungbean plant was also tested (Fig 1). This analysis revealed that most of the strains from both Java and Sumatra showed promiscuous nodulation properties that were not observed with the well-known inoculant strains of USDA - RESULTS Agronomic Aspects. E. coli B. japonicum B. japonicum B. japonicum B. japonicum B. japonicum 110 and CB 1809. Genomic DNA of the isolated rhizobial strains (27 from Java and 24 from Sumatra) and that of the reference strain USDA 110 were isolated and used as templates for PCR amplifications of the 16S rDNA and the 16S-23S rDNAs spacer regions (Fig 1). The amplified 16S rDNA's all showed the expected size of approximately 1.6 kb and upon digestion with four different restriction enzymes ( I, I, III and I) a variety (up to 19) of restriction length polymorphism patterns were observed. This ARDRA approach allowed the grouping of the rhizobial isolates into two main clusters, while Java isolate J-TGS50 and Sumatra isolates S-ST123 and S- ST414 showed rather unique position (Fig 2). One cluster consists of most (25) of the rhizobial isolates from Java, four isolates from Sumatra and also USDA 110. The other cluster consists of most (18) of the rhizobial isolates from Sumatra and only one strain isolated from Java (J-YG49). Both clusters contained isolates that showed only nodulation of soybean as well as ones with promiscuous nodulation properties. The PCR products of the 16S-23S spacer rDNA were all of the same size of approximately 2 kb. Differentiation of these 16S-23S rDNA amplicons by ARDRA using the same restriction enzymes as used for the 16S rDNA amplicons, revealed that these large DNA fragments showed significant sequence variations (Fig 3, Fig 4, Fig 5, Fig 6). Twenty-seven composite restriction pattern types were obtained by combining data from the digestion patterns (Table 1). I was the most discriminating enzyme, with twenty-one (21) genotypes detected Amplified Ribosomal (16S and 16S-23S) DNA Restriction Analysis (ARDRA). B. japonicum Cfo Dde Hae Msp B. japonicum Dde 2322 2027 564 2322 2322 2322 2027 2027 2027 bpbp 2000 1600 564 Fig 1 16S rDNA (above) and 16S-23S rDNA (below) PCR-products. Volume 5, 2011 Microbiol Indones 173 among the rhizobial isolates. Based on these complex ARDRA fingerprints, the rhizobial isolates were grouped into four main clusters (Fig 7). All strains isolated from Java, except for J-YG49, could be grouped into two clusters, one includes USDA 110. Again the Java isolate J-TGS50 showed a unique position. Similarly, all strains isolated from Sumatra, except for strains S-ST224, S-ST325 and S-ST123, could be grouped in one very large cluster, while two strains (S-BT221 and S-BT322) formed a cluster that was distantly related to that of USDA 110. The obtained 16S rDNA sequences of 21 selected, soybean-nodulating rhizobial strains from those B. japonicum B. japonicum Indonesian isolates were compared to those of reference strains obtained from the GenBank and ARB databases (Table 2). The high homology of the rDNA sequences indicated that all the isolates, except for J-TGS50, could be assigned to genus and are related to either . or (Table 2). Strain TGS50, the unique isolate from Java, was found to belong to genus and its 16S rDNA sequence showed 97.7 % homology to that of . A rooted phylogenetic tree was constructed,showing that all rhizobial isolates from Java and Sumatra, except for strain J-TGS50, could be grouped into 2 species, and (Fig 8). Bradyrhizobium B elkanii B. japonicum Sinorhizobium S. fredii B. elkanii B. japonicum 10090807060504030 Java Java Sumatra Sumatra Java Java Java Java Java Java Java Java Java Java Java Java Java Sumatra Sumatra Sumatra Sumatra Sumatra Sumatra Sumatra Sumatra Sumatra Sumatra Sumatra Sumatra Sumatra Sumatra Sumatra Sumatra Sumatra Sumatra Sumatra Sumatra Java Origin Strain Java Java USDA Java Java Java Java Java Java Java Java Sumatra Sumatra +/+4 Effectiveness soybean mungbean -/-0+/+3 +/+4+/+3 -/-0+/+2 +/+4+/+4 +/+5+/+4 -/-0+/+4 +/+3+/+3 +/+4+/+5 +/+3+/+4 +/+4+/+4 +/+4+/+3 +/+5+/+3 +/+4 -/-0+/+4 -/-0+/+4 -/-0+/+4 +/+4+/+4 -/-0+/-0 +/-0+/+4 +/+2+/+3 +/+4+/+3 -/-0+/+2 +/-0+/+2 +/+2+/+2 +/+1+/+2 +/+3+/+4 -/-0+/+4 +/-0+/+3 +/+4+/+1 +/+2+/+1 -/-0+/+2 -/-0+/+1 +/+2+/+2 +/+4+/+4 +/+3+/+3 -/-0+/+2 +/+3 -/-0 -/-0+/+3 -/-0+/+4 -/-0+/+3 -/-0+/+4 +/+4+/+4 +/+3+/+4 +/+4+/+4 +/+2+/+3 +/+4+/+5 +/+4+/+2 +/+5+/+4 +/+4+/+4 +/+3+/+2 -/-0+/+2 +/+5+/+4 J-DLG1 S-ST24 J-TGS50 S-ST325 S-ST224 J-WG2 J-TM3 J-MJ4 J-KH5 J-NG6 J-WK7 J-PLR8 J-SRG9 J-DLG10 J-BGR15 J-CTM20 J-JKT25 J-MJ28 S-ST123 S-ST16 S-ST41 S-ST42 S-ST29 S-ST33 S-ST45 S-ST310 S-ST18 S-ST311 S-ST316 S-ST215 S-ST219 S-ST518 S-ST220 S-ST117 S-ST17 S-ST412 S-ST414 J-YG49 J-PN33 J-YG38 110 J-MDN39 J-MLD40 J-TGS41 J-BSK42 J-PSR43 J-MJ44 J-PLR45 J-MJ47 S-BT221 S-BT322 J-KLT48Java Fig 2 Dendogram derived from 16S rDNA ARDRA fingerprints and symbiotic properties of rhizobial strains isolated from Java and Sumatra soil samples as well as USDA 110. +/+, nodulating and N-fixing; +/-, nodulated and no N-fixed; -/-, not nodulated and no N-fixed. The effectiveness of N fixation is indicated by values ranging from 1 (ineffective) to 5 (effective). B.japonicum 174 WALUYO ET AL. Microbiol Indones Fig 3 Restriction patterns of PCR-amplified fragments of 16S-23S rDNA intergenic spacer digested with I. Lanes 1, 10 and 19 are 100 bp DNA ladder (Life Technology). Lanes 2-9 (S-ST16; S-ST41; S-ST42; S-ST29; S-ST224; S-ST33; S-ST24; S-ST45) were derived from rhizobial isolates from Sumatra. Lanes 11-18 (J-MDN39; J-TGS50; J-BSK42; J-YG49; J-KLT48; J-KH5; J-MLD40; J-PSR43) were derived from rhizobial isolates from Java. Msp Fig 4 Restriction patterns of PCR-amplified fragments of 16S-23S rDNA intergenic spacer digested with I. Lanes 1, 11 and 20 are 100 bp DNA ladder (Life Technology). Lanes 2-10 (S-ST16; S-ST41; S-ST42; S-ST29; S-ST224; S-ST33; S-ST24; S-ST45; S-ST311) were derived from rhizobial isolates from Sumatra. Lanes 12-19 (J-MDN39; J-TGS50; J-BSK42; J-YG49; J-KLT48; J-KH5; J-MLD40; J-PSR43) were derived from rhizobial isolates from Java. Dde Fig 5 Restriction patterns of PCR-amplified fragments of 16S-23S rDNA intergenic spacer digested with I. Lanes 1, 11 and 20 are 100 bp DNA ladder (Life Technology). Lanes 2-10 (S-ST16; S-ST41; S-ST42; S-ST29; S-ST224; S-ST33; S-ST24; S-ST45, S-ST311) were derived from rhizobial isolates from Sumatra. Lanes 12-19 (J-MDN39; J-TGS50; J-BSK42; J-YG49; J-KLT48; J-KH5; J-MLD40; J-PSR43) were derived from rhizobial isolates from Java. Cfo Volume 5, 2011 Microbiol Indones 175 bp 1500 2072 600 100 bp 1500 2072 600 100 1 2 3 4 5 6 7 8 9 10 11 12 19171614 15 1813 bp 1500 2072 600 100 bp 1500 2072 600 100 1 2 3 4 5 6 7 8 9 10 11 12 19171614 15 1813 20 bp 1500 2072 600 100 bp 1500 2072 600 100 1 2 3 4 5 6 7 8 9 10 11 12 20171614 15 1813 19 Fig 6 Restriction patterns of PCR-amplified fragments of 16S-23S rDNA intergenic spacer digested with I. Lanes 1, 11 and 20 are 100 bp DNA ladder (Life Technology). Lanes 2-10 (S-ST16; S-ST41; S-ST42; S-ST29; S-ST224; S-ST33; S-ST24; S-ST45; S-ST311) were derived from rhizobial isolates from Sumatra. Lanes 12-19 (J-MDN39; J-TGS50; J-BSK42; J-YG49; J-KLT48; J-KH5; J-MLD40; J-PSR43) were derived from rhizobial isolates from Java. Table 1 Hae 16S-23S rDNA genotypes and restriction patterns of the isolated rhizobial strains revealed by ARDRA 176 WALUYO ET AL. Microbiol Indones bp 1500 2072 600 100 bp 1500 2072 600 100 1 2 3 4 5 6 7 8 9 10 1112 19171614 15 1813 20 Isolate 16S-23S r DNA genotype Restriction pattern of amplified 16S-23S rDNA digested with MspI DdeI HaeIII CfoI S-ST16 I a* a a a S-ST29 a a a a S-ST18 II a h a a S-ST215 III a q a h S-ST41 IV b b b a S-ST42 b b b a S-ST45 b b b a S-ST33 b b b a S-ST311 b b b a S-ST117 b b b a S-ST17 b b b a J-YG49 V b a b a S-ST414 VI b j b a S-ST224 VII c c c c J-WG2 c c c c J-NG6 c c c c J-WK7 c c c c J-PLR8 c c c c J-KH5 c c c c J-DLG10 c c c c J-DLG1 c c c c J-SRG9 c c c c S-ST24 VIII d d a d S-ST310 IX d k b i S-ST412 X d k b p J-MDN39 XI e e c e J-KLT48 e e c e J-MJ44 e e c e J-PLR45 e e c e J-BSK42 XII e n c g S-ST220 XIII e p h m J-TGS50 XIV f f d f J-MLD40 XV g g c g J-TGS41 g g c g Isolate 16S-23S r DNA genotype Restriction pattern of amplified 16S-23S rDNA digested with MspI DdeI HaeIII CfoI Table 1 Continued 10090807060504030 J-TGS50Java Origin Strains J-WG2Java J-KH5Java J-NG6Java J-DLG10Java J-DLG1Java J-WK7Java J-PLR8Java J-SRG9Java J-MJ28Java S-ST224Sumatra S-ST325Sumatra J-MLD40Java J-TGS41Java J-MJ4Java J-PSR43Java J-BSK42Java J-MJ44Java J-PLR45Java J-MDN39Java J-KLT48Java J-MJ47Java J-YG38Java J-JKT25Java J-CTM20Java 110USDA J-PN33Java J-TM3Java J-BGR15Java S-ST123Sumatra S-BT221Sumatra S-BT322Sumatra S-ST41Sumatra S-ST42Sumatra S-ST33Sumatra S-ST24Sumatra S-ST45Sumatra S-ST16Sumatra S-ST17Sumatra S-ST18Sumatra S-ST29Sumatra S-ST310Sumatra S-ST311Sumatra S-ST412Sumatra S-ST414Sumatra S-ST215Sumatra J-YG49Java S-ST316Sumatra S-ST117Sumatra S-ST518Sumatra S-ST219Sumatra S-ST220Sumatra Fig 7 Dendogram derived from 16S-23S intergenic spacer ARDRA fingerprints of soybean rhizobial strains isolated from Java and Sumatra soil samples as well as USDA 110.Bradyrhizobium japonicum Volume 5, 2011 Microbiol Indones 177 J-PSR43 g g c g S-ST518 XVI h i e h J-TM3 XVII i l f j J-PN33 i l f j J-YG38 i l f j J-JKT25 i l f j J-BGR15 XVIII o l f j J-CTM20 o l f j S-ST123 XIX l o g k S-ST219 XX m p h l S-ST325 XXI n k b o J-MJ4 XXII j m c g S-ST316 XXIII k d a n S-BT221 XXIV p r i q S-BT322 XXV q s j r J-MJ28 XXVI r t k c J-MJ47 XXVII s u e e *Letters represent the pattern of cut DNA that is presented as a finger-prints. Isolates with similar letter are classified in one group within their associated restriction enzymes. Table 2 Numbers of nucleotide differences and % 16S rDNA homologies in the aligned sequences of well-known soybean-nodulating strains (accession number BEU3500), USDA 110 (accession number Z35330) and USDA 205 (accession number D14516) Bradyrhizobium elkanii Bradyrhizobium japonicum Sinorhizobium fredii B. elkanii USDA76 B. japonicum USDA 110 S. fredii USDA 205 Strain Number of nucleotides N 1 %H 2 Strain Number of nucleotides N % H Strain Number of nucleotides N %H S-ST17 1390 6 99.6 J-WG2 1089 7 99.4 J-TGS50 1440 30 97.9 S-ST414 1415 3 99.8 J-DLG10 1265 28 97.8 J-KH5 1199 11 99.1 J-SRG9 1443 15 99.0 J-Y49 1445 7 99.5 J-TM3 1426 3 99.8 S-ST316 1443 9 99.4 S-ST123 1329 23 98.3 S-ST29 1054 9 99.1 S-ST325 1445 11 99.2 S-ST33 1442 10 99.3 S-BT221 1447 21 98.5 S-ST215 1348 3 99.8 S-BT322 1446 16 98.9 S-ST16 1446 25 98.3 S-ST518 1425 18 98.7 S-ST45 1360 6 99.6 S-ST117 1445 9 99.4 1 2 Number of nucleotide differences. % of 16S rDNA homology. speciesB. 36 20 japonicumB. japonicumB. lupiniB. japonicumB. japonicumB. 28 japonicumB. elkaniiB. 64 67 frediiR. elkaniiB. speciesB. xinjiangensisS. frediiS. 62 99 0.10 47 frediiS. 32 frediiS. 43 saheliS. elkaniiB. elkaniiB. 34 elkaniiB. 35 elkaniiB. elkaniiB. elkaniiB. elkaniiB. elkaniiB. elkaniiB. elkaniiB. elkaniiB. 35 japonicumB. japonicumB. japonicumB. japonicumB. japonicumB. japonicumB. japonicumB. japonicumB. Escherichia coli 55S IAM 12608 LMG 6138 T DSM 30140 USDA 136 J-DLG10* J-SRG9* S-ST518** IAM 13625 USDA 76 LMG 10689 IAM 14142 J-TGS50* LMG 6217 USDA 205 LMG 7837 J-YG49* S-ST33** S-ST16** S-ST29** J-KH5* S-ST316** S-ST45** S-ST215** S-ST17** S-ST117** S-ST414** USDA 110 J-WG2* J-TM3* S-ST123** S-ST325** S-BT221** S-BT322** DSM30131T MG1655 Fig 8 A rooted phylogenetic tree based on 16S rDNA sequences of the Indonesian rhizobial isolates (bold, * from Java and ** from Sumatra) and related other and spp. constructed by the ARB Software with 100 times bootstrap. The bar indicates the phylogenetic distance (0.1 knuc). Bradyrhizobium Sinorhizobium 178 WALUYO ET AL. Microbiol Indones Table 3 Efficiency of rhizobial strains isolated from Java and Sumatra compared to reference strains. Number and weight of nodules formed on soybean as well as shoot weight were determined Strain Nodule plant -1 Shoot Weight (g plant -1 ) Number Weight (g) None Control 0 0 0.57±0.32 Bradyrhizobium spp. @ CB756 0 0 0.30±0.03 Bradyrhizobium japonicum @ USDA 110 43±10 0.288±0.07 3.36±0.80 Bradyrhizobium japonicum @ CB1809 29±5 0.250±0.06 3.70±0.28 Java @@ 46±10a* 0.263± 0.07a** 3.75± 0.62a*** Sumatra @@ 37±14 b 0.225±0.07 b 2.43±0.80 b CV # = 33% CV=29% CV=25% @ @@ # For these reference strains means were calculated from 3 replicates A total of 24 randomly choosen bacterial strains used for each set of soil samples. *, **, *** Values followed by a different letter in same column are statistically different, revealed by T-test with confidence levels at = 0.10, 0.05 and 0.01 respectively. CV = Coefficient Variation (MSTAT-C, 1988) . DISCUSSION Fifty-one different rhizobial strains (27 from Java and 24 from Sumatra) were isolated from nodules. Based on their nodulation ability on both soybean and mungbean, these rhizobial strains could be classified as promiscuous strains (34) or strains that show a narrow host-range (17) and only nodulate soybean (Fig. 2). Saono (1988) suggested that the native soybean- rhizobia population in Java appears to be dominated by promiscuous strains, and this is supported here with quantitative data. All of the soybean plants that were inoculated by rhizobial isolates from Java (except J-MJ44) grew vigorously in an N-free medium. In contrast, several ineffective bradyrhizobial strains were found in Sumatra. It is likely that the abundance of these strains would have a negative impact on the inoculation by effective spp. inoculants. ARDRA of PCR-amplified 16S rDNA and 16S-23S rDNA spacer fragments were used to differentiate soybean-nodulating rhizobial strains. Based on ARDRA of 16S rDNA, nearly all of the soybean- nodulating bacteria could be grouped into two separate large clusters comprising either the Java or the Sumatra isolates. ARDRA of 16S-23S rDNA spacer fragments has been reported to be useful for a further sub- classification of bacteria (Jensen . 1993; Masol- Deya . 1995; Gurtler and Stanisich 1996; Scheinert . 1996). This is due to the fact that the 16S-23S rDNA spacer region of all prokaryotes exhibit a high degree of sequence and size variations at the level of the genus and species. The sequence variations of the 16S-23S rDNA spacer region of the soybean- nodulating bacteria confirmed the clustering of most Sumatra isolates and allowed a further classification of the Java isolates into two large distinct clusters. One of Bradyrhizobium et al et al et al these clusters was found to include strains that were closely related to USDA 110, a reference strain which is highly specific for soybean. Based on their grouping and symbiotic properties, we assume that these Java isolates and one isolate from Sumatra (S-ST123) are strains. Although clustered at one group, the isolate S-ST123 is distinct in its symbiotic property to USDA 110. It formed many but ineffective soybean-nodules. One unique isolate from Java, J-TGS50, showed an unique position in comparison with the clustered isolates from Java and Sumatra. This group and the group containing most Sumatra isolates, which were also shown distinct from USDA 110, nodulated both soybean and the indigeneous mungbean. Hence these strains may represent the indigeneous bacterial population capable of nodulating soybean. was found to be dominant in acid soils from Sitiung, West-Sumatra. Ten out of fourteen studied isolates from this location could be assigned to . Apart from isolate S-ST518 they form a homogeneous group, and notably strains S-ST17, S- ST45, S-ST117, S-ST215 and S-ST414 show high similarity to the reference strain USDA 76 (Fig 5). However in spite of their phylogenetic relation, the strains showed considerable variability in their symbiotic properties. Only two out of seven strains from Java (strain J- KH5 and J-YG49) were found to be . In contrast to the isolates from Sumatra, the N fixation capacity of these strains from Java was all very high on soybean. was found dominant in Java. Four isolates (J-DLG10; J-WG2; J-TM3; J-SRG9) from seven analysed strains could be classified as B. japonicum B. japonicum B. japonicum B. japonicum B. elkanii B. elkanii B. elkanii B. elkanii B. elkanii B. elkanii B. japonicum B. Volume 5, 2011 Microbiol Indones 179 japonicum B japonicum B. japonicum B. japonicum B. japonicum Sinorhizobium S. fredii B. japonicum B. japonicum S. fredii S. saheli S. fredii S. fredii S. saheli et al et al S. fredii Aeschynomene et al. et al. . This confirms earlier results based on symbiotic properties and ARDRA 16S rDNA and 16S- 23S rDNA. One of these isolates, strain J-TM3, was very specific and effective on soybean. This could be due to the cultivation of imported soybean seeds contaminated with soils (Toxopeus 1938) or the introduction as inoculant by researchers (Newton 1962). It is not be possible to differentiate between these or other possibilities, but the observation that strain J-TM3 is highly related to USDA 110, a well known inoculant isolated from Japan, suggests strongly that it is not a native strain (Fig 2/Table 1). Remarkably, the other strains (J-DLG10; J- WG2 and J-SRG9) have been shown to nodulate mungbean plants. This is in apparent contrast with the common phenotype of which is known to nodulate only soybean. Several strains (S-ST123; S-ST325; S-BT221; S-BT322) were also found in Sumatra soils. However, while all strains from Java are highly effective, these strains from Sumatra are ineffective. It is interesting to note that a strain belonging to the genus that include fast growing species, was also found in Indonesia. Hence, the growth properties of this strain, J-TGS50, was determined and compared to the other strains and USDA 110. Mean generation time (g) of J-TGS50 was almost 6-fold lower than that of USDA 110 and even lower than that previously reported for . This was also apparent during growth of colonies on YEM agar plates. While colonies of the strain J-TGS50 with a diameter size between 0.10 1.0 mm could already be observed 3 days after inoculation, no USDA 110 colonies were detectable at the same period of time. Analysis of 16S rDNA sequences of strain J-TGS50 showed it to be related to and . Until now, only and not strains have been reported to nodulate soybean (Keyser . 1982; De Lajudie . 1994; Young 1996). However, strains were found to show a broad host-range and formed nodules on soybean as well as many other legumes, including cowpea, pigeon pea and mungbean (Scholla and Elkan 1984; Stowers and Eaglesham 1984; Chamber and Iruthayathas 1988). This contrasts with the restricted nodulation properties of strain J-TGS50 which did not nodulate mungbean. From the data presented here and the earlier results, it can be concluded that there is no relationship between the nodulation phenotype and rRNA traits. This confirms earlier reports for BTAi1, a phototrophic symbiont of the legume (Young 1991) and for peanut bradyrhizobial strains (Zhang 1999). Therefore, besides nodulation phenotype, - other traits, eg. 16S rDNA sequences, should be taken into account for identification and classification of rhizobial strains. This study demonstrates that species known to nodulate soybean, , and most likely also are present in Indonesia. There appeared a great diversity in effectiveness between these brady- and sinorhizobia strains. In Java, many effective strains are already present in the soil, presumably by selection of soybean over a long period of cultivation. This could be the reason for the absence of a response to soybean inoculation practice in Java (Saono 1988). Bradyrhizobia strains are present in low amount in acid soils of Sumatra and are suggested to occupy niches in the acid soils, which are not toxic to these bacteria. It is interesting that there is a large variation in N fixation capacity among the isolates from Sumatra. 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Sinorhizobium versus Ensifer: may a taxonomy subcommittee of the ICSP contradict the Judicial Commission? Int J Syst Evol Microb. 60: 1711-1733. Zhang A, Nick G, Kaijalainen S, Terefework Z, Paulin L, Tighe SW, Graham PH, Lindstrom K .1999. Phylogeny and diversity of strains isolated from root nodules of peanut ( ) in Sichuan, China. System Appl Microbiol. 22: 378-386. Rhizobium fredii Rhizobium Rhizobium japonicum rhizobium Rhizobium Ensifer Sinorhizobium et al Sinorhizobium morelense et al Ensifer adhaerens Sinorhizobium adhaerens et al Bradyrhizobium Arachis hypogaea Volume 5, 2011 Microbiol Indones 181