Diversity and geographical distribution of indigenous soybean-nodulating bradyrhizobia in Japan

Abstract

Genetic diversity and distribution of indigenous soybean-nodulating bradyrhizobia in Japan were investigated based on restriction fragment length polymorphism analysis of PCR product (PCR-RFLP) analysis of the 16S?23S rDNA internal transcribed spacer (ITS) region using Bradyrhizobium USDA strains as reference strains. Soil samples were collected from five field sites in Hokkaido, Fukushima, Kyoto, Miyazaki and Okinawa in Japan. A total of 300 isolates were derived from three Rj-genotype soybean cultivars, Akishirome (non-Rj), CNS (Rj ₂ Rj ₃) and Fukuyutaka (Rj ₄), and five field site combinations. The PCR products of the ITS region were digested with HaeIII, HhaI, MspI and XspI. Electrophoresed patterns were analyzed for phylogenetic relationship using Bradyrhizobium reference strains. Results revealed 22 RFLP patterns and 11 clusters. The RFLP patterns of the seven clusters were similar or identical to Bradyrhizobium japonicum USDA 6, 38, 110, 115, 123 and Bradyrhizobium elkanii USDA 76 and 94. Four minor clusters were independent from the clusters of the reference strains. The isolation ratio revealed the major clusters at each field site. These results suggested that major clusters of indigenous bradyrhizobia might be in the order Bj123, Bj38, Bj110, Bj6 and Be76 from the northern to southern regions in Japan.     

Polyphasic characteristics of bradyrhizobia isolated from nodules of peanut (Arachis hypogaea) in China

Peanuts (Arachis hypogaea L.) were introduced to China about 500 years ago. However, the diversity of Rhizobial strains in China that can nodulate peanut was poorly understand. Diversity and phylogeny of 50 slow-growing strains, isolated from root nodules of peanut in different geographical regions of China, were studied using polyphasic techniques. All stains were clustered by phenotypic tests into two distinct groups: Group I: 16S rRNA RFLP genotype 3, and Group II, which divided into 16S rRNA RFLP genotypes 1 and 2. Genotype 1 shares the same genotype with USDA110, USDA122 and USDA127 of Bradyrhizobium japonicum, and genotype 2 solely consisted of extra-slow growing bradyrhizobia isolated from Hongan, China. Results of 16S rRNA sequencing revealed that peanut bradyrhizobia were phylogenetically related to B. japonicum and their sequence divergence was less than 1.1%. Based upon the size of the internally transcribed spacer (ITS) between the16S and 23S RNA genes, strains were classified into ITS-I, ITS-II and ITS-III genotypes. Strains could be further divided into sub-clusters IA, IB, IIa, IIb and IIc five sub-clusters through ITS PCR-RFLP and repetitive extragenic palindromic PCR (REP-PCR) analysis. Host specificity test revealed that all peanut bradyrhizobia tested nodulated Phaseolus vulgaris and strains of clusters IIb and IIc nodulated Glycine soja efficiently. Bradyrhizobia isolated from peanut were related, but still exhibited phylogenetical divergence with B. japonicum.     

Phylogenetic Analysis of Soybean-Nodulating Rhizobia Isolated from Alkaline Soils in Vietnam

In order to analyze the phylogeny of soybean-nodulating bacteria in alkaline soils in Vietnam, indigenous soybean-nodulating bacteria were isolated from root nodules by cultivating three kinds of Rj -soybean cultivars on two alkaline soils in Vietnam. The 120 isolates were classified into two major genera of soybean-nodulating rhizobia, namely Bradyrhizobium and Sinorhizobium genera, based on a growth analysis on medium and PCR-RFLP analyses of 16S rDNA and of the 16S–23S rDNA internal transcribed spacer (ITS) region. Most of the isolates of B. japonicum were extra-slow-growing and their ITS types were similar to that of B. japonicum USDA 135. They were not isolated from the soybean cultivar CNS used as Rj₂Rj₃ genotype. Isolates of Sinorhizobium were divided into two groups, S. fredii and S. sp., based on a PCR-RFLP analysis of 16S rDNA. Furthermore, PCR-RFLP analysis of the 16S–23S rDNA ITS region enabled to separate them into five types, three ITS types associated with S. fredii and two with S. sp. Sinorhizobium was frequently isolated from the three soybean cultivars on two soils. From the isolate ratio, it was suggested that B. japonicum strains similar to B. japonicum USDA 135 and S. fredii predominated in the alkaline soils of Vietnam. Additionally, our findings indicated that the Rj -genotypes affected not only the compatibility, but also the preference for nodulation between the host soybean and rhizobia.     

Nodulation competition among Bradyrhizobium japonicum strains as influenced by rhizosphere bacteria and iron availability

Summary Bacteria isolated from the root zones of field-grown soybean plants [Glycine max (L.) Merr.] were examined in a series of glasshouse experiments for an ability to affect nodulation competition among three strains of Bradyrhizobium japonicum (USDA 31, USDA 110, and USDA 123). Inocula applied at planting contained competing strains of B. japonicum with or without one of eleven isolates of rhizosphere bacteria. Tap-root nodules were harvested 28 days after planting, and nodule occupancies were determined for the bradyrhizobia strains originally applied. Under conditions of low iron availability, five isolates (four Pseudomonas spp. plus one Serratia sp.) caused significant changes in nodule occupancy relative to the corresponding control which was not inoculated with rhizosphere bacteria. During subsequent glasshouse experiments designed to verify and further characterize these effects, three fluorescent Pseudomonas spp. consistently altered nodulation competition among certain combinations of bradyrhizobia strains when the rooting medium did not contain added iron. This alteration typically reflected enhanced nodulation by USDA 110. Two of these isolates produced similar, although less pronounced, effects when ferric hydroxide was added to the rooting medium. The results suggest that certain rhizosphere bacteria, particularly fluorescent Pseudomonas spp., can affect nodulation competition among strains of R. japonicum. An additional implication is that iron availability may be an important factor modifying interactions involving the soybean plant, B. japonicum, and associated microorganisms in the host rhizosphere.Paper No. 10648 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601, USA     

Genetic diversity and phylogeny of indigenous rhizobia from cowpea [<Emphasis Type="Italic">Vigna unguiculata</Emphasis> (L.) Walp.]

16S rRNA RFLP, 16S rRNA sequencing, 16S-23S rRNA Intergenetic Spacer (IGS) RFLP and G-C rich random amplified polymorphic DNA (RAPD) assays were conducted to genetically characterise indigenous cowpea [Vigna unguiculata (L.) Walp.] rhizobia from different geographic regions of China. Isolated cowpea rhizobia comprised six 16S rRNA genospecies. Genotype I was composed of 14 isolated strains and the reference strains of B. japonicum and B. liaoningense. This group was divided into two sub-groups respectively related to B. japonicum and B. liaoningense by 16S rRNA sequencing, IGS restriction fragment length polymorphism and RAPD assays. Genotype II composed of 27 isolates from a variety of geographic regions. Four different assays confirmed this group was genetically distinct from B. japonicum and B. liaoningense and probably represent an uncharacterised species. Strains isolated from Hongan, Central China and B. elkanii were grouped to genotype III. Strain DdE4 was solely clustered into genotype IV and related to Rhizobium leguminosarum. Genotypes V and VI consisted of six fast-growing isolates and clustered with reference strain of Sinorhizobium fredii. Comparing with the miscellaneous slow-growing isolates, fast-growing isolates mainly isolated from cowpea cultivar Egang I exhibited strict microbe–host specificity except SjzZ4. Nucleotide sequences reported were deposited in the GenBank with the accession numbers DQ786795–DQ786804.     

Serological properties and intrinsic antibiotic resistance of soybean bradyrhizobia isolated from Thailand

A group of Bradyrhizobium strains isolated from soybean plants in Thailand did not correspond to any known DNA homology groups of Bradyrhizobium japonicum and Bradyrhizobium elkanii reported by Hollis et al. (J. Gen. Microbiol., 123, 215–222, 1981). To clarify the phenotypic characteristics of the group, serological properties and intrinsic antibiotic resistance (IAR) profile of 94 Thai strains were compared with those of USDA and Japanese strains. Indirect ELISA tests for each Thai strain were performed agaiIl.st polyclonal antisera prepared against 15 USDA standard serotype strains of B. japonicum and B. elkanii. Among the 94 Thai strains tested, 36 which were previously identified as B. elkanii, with the exception of one strain, were strongly responsive to an antiserum prepared against USDA 31. The remaining 58 strains, with the exception of two strains, showed multiple cross reactions which were peculiar to the Thai strains. These serological reaction patterns did not correspond to any known serogroups labeled as B. japonicum and B. elkanii. In the IAR test, the taxonomically unknown Thai soybean bradyrhizobia exhibited a high level of resistance to neomycin (50 µg/mL), polymyxin (50 µg/mL), nalidixic acid (15 µg/mL), and kanamycin (15 µg/mL). Kanamycin could thus be useful in combination with neomycine and nalidixic acid for distinguishing between the unknown Thai strains and strains of B. japonicum and B. elkanii. Our results demonstrated that the unknown Thai strains were serologically and IAR-phenotypically remote from both B. japonicum and B. elkanii.     

Genetic variability of soybean bradyrhizobia populations under different soil managements

Due to their ecologic and economic importance, bradyrhizobia have been extensively studied in recent years. Since 1992, Bradyrhizobium elkanii SEMIA 587 and SEMIA 5019 and Bradyrhizobium japonicum SEMIA 5079 and SEMIA 5080 have been widely used in most Brazilian soybean fields. The objective of this work was to estimate the genetic variability of bradyrhizobial isolates recovered from soils under rhizobial inoculation and different soil managements. Only 25% of the isolates demonstrated high similarities to the original strains, and a strong correlation was obtained between the bradyrhizobial genetic variability and soil management. A high level of genetic diversity was observed both within isolates (H = 5.46) as well as among the different soil practices. Soil under no-tillage presented a higher bradyrhizobia diversity compared with bradyrhizobia isolated from soil under conventional tillage. Serological characterization also indicated that B. elkanii strains SEMIA 587 and SEMIA 5019 were more competitive and presented a higher nodular occupancy capacity than strains belonging to B. japonicum species in Southern Brazilian soils.     

Distribution and biodiversity of soybean rhizobia in the soils of Shennongjia forest reserve,China

Soil samples were collected at an altitude of 500, 1,060, 1,500, 1,950, 2,400 and 3,100 m, respectively, from Shennongjia, a forest reserve in Hubei province (central China). Their corresponding pHs were 5.50, 4.91, 5.64, 5.28, 5.49 and 4.60. By using a plant trap method, a total of 25 soybean rhizobia were isolated from the soil above an altitude of 1,500 m and all identified to be Sinorhizobium fredii. Their genetic biodiversity was characterized by 16S–23S rDNA internally transcribed spacer (ITS) region polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and random amplification DNA (RAPD) analysis. All the tested strains produced a 2.1 kb 16S–23S rDNA ITS fragment. After digestion with three restriction endonucleases (HaeIII, MspI and CfoI), respectively, great variations in 16S–23S rDNA ITS PCR-RFLP patterns were observed. The tested strains could be differentiated into 11 ITS genotypes. The genotypes of rhizobia were not related to geographical location. Twelve primers were applied to RAPD analysis and a dendrogram was obtained, showing that all the strains (including reference strain S. fredii USDA205) were divided into two diverging groups. Moreover, each group could be further divided into two subgroups. Both RAPD and 16S–23S rDNA ITS PCR-RFLP analysis indicated that a high degree of genetic diversity existed among S. fredii strains isolated from Shennongjia virgin soils. Since Shennongjia is an unexploited forest region in central China and the gene centre of soybean is located in China, the symbiotic genes harboured by these strains may be of great importance and the rich diversity of these strains might contribute to the adaptation of soybean to an alpine environment.     

Diversity,phylogeny and host specificity of soybean and peanut bradyrhizobia

A study on the diversity, phylogeny, and host specificity of soybean (Glycine max L.) and peanut (Arachis hypogaea L.) bradyrhizobia was conducted based on the 16S ribosomal RNA (rRNA) restriction fragment length polymorphisms (RFLPs), 16S rRNA sequencing, and 16S–23S rRNA intergenetic spacer (IGS) RFLP assays. Based on 16S rRNA RFLP assay, tested bradyrhizobia were divided into five genotypes, which could be further clustered into five groups by IGS RFLP assays. According to the 16S rRNA sequencing, strains of IGS-II, IV, and V were phylogenetically related to Bradyrhizobium liaoningense, Bradyrhizobium japonicum, and Bradyrhizobium elkanii, while strains of IGS-Ic and IGS-III related to Bradyrhizobium yuanmingense and Bradyrhizobium canariense, respectively. All isolates could crossly nodulate Phaseolus vulgaris, forming small white nodules. Strains of IGS-II originally isolated from peanut could efficiently nodulate Glycine soja, and two strains isolated from soybean could also nodulate peanut.     

Multiphase characterization of wild Vigna associated root nodule bacteria from Japanese subtropical islands unveiled novel high temperature resistant Bradyrhizobium strains having high symbiotic compatibility with soybean and mungbean

ABSTRACT

Vigna riukiuensis plant – a rare type of vigna, found only in Taiwan and the islands of Okinawa prefecture, Japan – possesses intrinsic property of high level of salt and heat tolerance. To understand the diversity and identify suitable rhizobia, multiphase characterization of root nodule bacteria associated with V. riukiuensis grown in Ishigaki and Iriomote Islands of Okinawa prefecture was performed. Multigene phylogenetic analysis of housekeeping genes based on 16S rRNA gene sequences, 16S-23S rRNA gene internal transcribed spacer (ITS) and 23S rRNA gene sequences identified three main groups closely similar to Bradyrhizobium japonicum, B. elkanii and B. jicamae family. However, analysis of symbiotic nifH and nodD1 genes and their phylogenetic trees showed similar topology, having only few discrepancies in comparison to the housekeeping gene phylogeny. Interestingly, for some of the isolates having similarity with B. elkanii, growth was observed at 40°C, which exceed the highest record for B. elkanii to the best of our knowledge. All the isolates were observed to have the capability of forming root nodules and fix nitrogen in their original host plant V. riukiuensis and two other crops: soybean and mungbean. Most of the isolates showed similar or higher nitrogen-fixing capability in comparison with B. diazoefficiens USDA110 in V. riukiuensis and V. radiata (mungbean), and Iri 5/6 in V. riukiuensis, Iri 5/12 in soybean and Ishi 7/2 in mungbean showed highest acetylene reduction assay (in µmol/h/gm nodule dry weight) activity, which was significantly higher than B. diazoefficiens USDA110. In addition, six isolates attained higher soybean biomass production compared with B. diazoefficiens USDA110, suggesting high symbiotic compatibility with soybean. Among them, Iri 5/7 of B. elkanii group contributed 29% higher soybean biomass production than B. diazoefficiens USDA110 and could grow at 40°C, hence it could be a promising soybean inoculant in the tropics.     

Rhizobiophage effects on nodulation,nitrogen fixation,and yield of field-grown soybeans (Glycine max L. Merr.)

Summary Previous laboratory and greenhouse studies have shown that phages significantly reduce soil populations of homologous rhizobia. Reductions in nodulation and N₂ fixation have also been observed. The purpose of the current study was to examine the effect of a phage specific ofBradyrhizobium japonicum USDA 117 on nodulation, nodule occupancy, N₂ fixation and soybean growth and yield under field conditions. The phage was inoculated in combination withB. japonicum USDA 117 and/orB. japonicum USDA 110 (resistant strain) into a rhizobia-free sandy loam soil and planted toGlycine max (L.) Merr. Williams. When the phage was applied to soil inoculated withB. japonicum USDA 117 alone, significant reductions in nodule weight and number, shoot weight, foliar N, nitrogenase activity, and seed index were observed. When, however, the soil also contained the non-homologous strain,B. japonicum USDA 110, no significant effects on any of these parameters were found. Nodule occupancy by competing strains ofB. japonicum USDA 110 and USDA 117 was also affected by the phage. In soil which did not contain the phage, 46% and 44% of the identified nodules were occupied by USDA 110 and 117, respectively. When the phage was present in the soil, nodule occupancy byB. japonicum USDA 117 was reduced to 23%, while occupancy byB. japonicum USDA 110 was increased to 71%. These results suggest that nodulation by selected strains of rhizobia can be restricted and nodulation by more effective, inoculated strains can be increased through the introduction of a homologous phage to soils.     

Repeated sequence RSα is diagnostic for Bradyhizobium japonicum and Bradyrhizobium elkanii

The genome of Bradyrhizobium japonicum and B. elkanii contains multiple copies of the repeated DNA sequence RSα. A collection of 18 B. japonicum, 4 B. elkanii and 72 other bacterial strains was screened by polymerase chain reaction (PCR) using a pair of primers specific for RSα. Only strains of B. japonicum and B. elkanii gave the predicted amplification product. Restriction analysis of PCR products obtained from different strains of B. japonicum showed that the RSα sequence was generally conserved. The usefulness of RSα as a specific probe for Bradyrhizobium strains capable of nodulating soybean was also demonstrated. Received: 11 May 1995     

Nitrogen fixation capacity and nodule occupancy by Bradyrhizobium japonicum and B. elkanii strains

 In a previous study soybean Bradyrhizobium strains, used in Brazilian studies and inoculants over the last 30 years, and strains adapted to the Brazilian Cerrados, a region frequently submitted to environmental and nutritional stresses, were analyzed for 32 morphological and physiological parameters in vivo and in vitro. A cluster analysis allowed the subdivision of these strains into species Bradyrhizobium japonicum, Bradyrhizobium elkanii and a mixed genotype. In this study, the bacteria were analyzed for nodulation, N₂ fixation capacity, nodule occupancy and the ability to increase yield. The goal was to find a relationship between the strain groups and the symbiotic performance. Two strains of Brazilian B. japonicum showed higher rates of N₂ fixation and nodule efficiency (mg of N mg^–1 of nodules) under axenic conditions. These strains also showed greater yield increases in field experiments when compared to B. elkanii strains. However, no differences were detected between B. japonicum and B. elkanii strains when comparing nodule occupancy capacity. The adapted strains belonging to the serogroup B. elkanii SEMIA 566, most clustered in a mixed genotype, were more competitive than the parental strain, and some showed a higher capacity of N₂ fixation. Some of the adapted strains, such as S-370 and S-372, have shown similar N₂ fixation rates and nodulation competitiveness to two Brazilian strains of B. japonicum. This similarity demonstrates the possibility of enhancing N₂ fixing ability, after local adaptation, even within B. elkanii species. Differences in the DNA profiles were also detected between the parental SEMIA 566 and the adapted strains by analyses with the ERIC and REP-PCR techniques. Consequently, genetic, morphological and physiological changes can be a result of adaptation of rhizobia to the soil. This variability can be used to select strains capable of increasing the contribution of N₂ fixation to soybean nutrition. Received: 28 May 1997     

Ability of free-living cells ofBradyrhizobium japonicum to denitrify in soils

Summary Experiments to assess the ability of free-living cells of six strains of soybean rhizobia (Bradyrhizobium japonicum USDA 76, 94, 110, 122, 123, and 135) to denitrify nitrate in five soils showed that although some strains ofB. japonicum have the capacity to rapidly denitrify nitrate in soils under anaerobic conditions, it is unlikely that the numbers of soybean rhizobia commonly found under field conditions are sufficient to significantly influence either the extent or the products of denitrification in soil. It is our general conclusion that the advantages, if any, that the ability to denitrify conveys to rhizobia or to the rhizobia-legume symbiosis are not offset by increased losses of plant-available N when denitrifying strains of rhizobia are present as free-living cells in soil.     

Characterization of local rhizobia in Thailand and distribution of malic enzymes

TWenty-six isolates were obtained from nodules of various legume plants (Glycine max, Vigna sinensis, Arachis hypogaea, Desmanthus virgatus, Acacia mangium, Centrosema pascuorum, Pterocarpus indicus, Xylia xylocarpa, and Sesbania rostrata) in Thailand. After confirming their nodulation and nitrogen-fixing abilities, they were identified by 16S rRNA gene analysis as Bradyrhizobium japonicum, Bradyrhizobium elkanii, Rhizobium leguminosarum, Rhizobium gallicum, and Rhizobium galegae. Using these local isolates, the distribution of the activities of both NAD⁺-dependent (DME: EC 1.1.1.39) and NADP⁺-dependent (TME: EC 1.1.1.40) malic enzymes was surveyed. The malic enzyme activities were present in all the isolated rhizobia and in other 17 local Bradyrhizobium strains in Thailand. In almost all the rhizobia, the DME activity predominated whereas the TME activity predominated only in the Rhizobium gallicum strains that were major symbionts of Sesbania rostrata. Southern hybridization analysis was performed to survey the distribution of the malic enzyme genes among the local rhizobia, which are similar to those of B. japonicum. DNA probes (ME1 for DME and ME2 for TME) were prepared by polymerase chain reaction (PCR) using degenerated primers from conserved regions of the protein sequences of bacterial malic enzymes. Southern blot analysis with ME1 as a probe showed a single band in about half of the isolates, especially in B. japonicum and R. leguminosarum strains, suggesting the wide distribution of such DME genes among local rhizobia. In contrast, Southern blot analysis with ME2 as a probe detected a single band only in five B. japonicum strains, suggesting that the TME genes, which are similar to those of B. japonicum, would be unique in a group of B. japonicum.     

Occurrence and nature of mixed infections in nodules of field-grown soybeans (Glycine max)

Summary Mixed infections of Bradyrhizobium japonicum strains in early and late nodules of four soybean cultivars were studied in a field soil. Nodule occupants were identified by immunofluorescence using serogroup specific antibodies prepared against B. japonicum strains USDA 110, USDA 123, and USDA 138. Double infection was determined directly by combined examination of the same microscopic field by fluorescence and phase contrast microscopy. Double strain occupancy was observed consistently, and its occurrence did not differ substantially in pouch, soil pot, and field experiments, ranging in incidence from 12% to 32%. No significant differences in the incidence or nature of double infection could be attributed to cultivar, seed inoculation, or plant maturity. Strains reactive to strain USDA 123-fluorescent antibody were dominant in both singly and doubly infected nodules irrespective of cultivar, plant age, or seed inoculation with strain USDA 110.Paper no. 15092 in the Scientific Journal Series of the Minnesota Agricultural Experiment Station, St. Paul     

Yield and N accumulation responses of late‐season determinate soybean to irrigation and inoculation with various strains ofBradyrhizobium japonicum

Abstract

Cobb and Coker 488, late‐season (maturity group VIII) cultivars of soybean [Glycine max(L.) Merr], were grovn under irrigated and non‐irrigated conditions on a Norfolk loamy sand in a two‐year field experiment. Each cultivar was inoculated withBradyrhizobium japonicumstrains [USDA 3I1b110; Brazil 587; NifTAL 184 and 102 (NifTAL cultures of Brazil 587 and USDA 110, respectively); and North Carolina 1001, 1004, 1005, 1010, and 1029). Drought conditions were present both years, and irrigation significantly increased the overall yield (2.49 vs 1.92 Mg ha^‐1). Coker 488 was significantly higher in seed yield than Cobb (2.55 vs 2.02 Mg ha^‐1). Strain ofB.japonicumalso affected seed yields. NC1010‐inoculated soybean was significantly higher in seed yield rank than all other soybean at the P<0.01 level, when compared by single degree of freedom contrast (sdfc). The yield ranking of soybean inoculated with NC1001 was significantly lower than soybean inoculated with all other strains, when compared by sdfc (P<0.10). Other strains differed in responses which ranged from good to poor inoculants under specific water management conditions. For instance, under nonirrigated conditions, soybean inoculated with strains ofB.japonicumfrom North Carolina was significantly higher in seed yield than those inoculated with the cultures of USDA 110, B587, or the control, when compared by sdfc (P>0.03, 0.05, 0.06, respectively). Since soybean inoculated with either strain of USDA 110 was generally high in yield rank under irrigated conditions, their response to irrigation was large relative to soybean inoculated with the NC strain (P<0.04). Neither seed nitrogen nor xylem water potential was highly correlated to seed yield. Since seed yield and N content were not highly correlated, the amount of N accumulated in soybean dry mass and that removed in seed were not highly correlated. Thus, the amount of N returned to the soil would be affected by management combinations of late‐season determinate soybean cultivar,B.japonicumstrain, and irrigation     

Intragenomic variation in the internal transcribed spacer regions between 16S–23S rRNA genes among the three copies of Sinorhizobium fredii strains

The soybean-nodulating Sinorhizobium fredii strain has been reported to possess three copies of rRNA gene operons. In the present study, we investigated the diversity of the 16S–23S rDNA internal transcribed spacer (ITS) regions of S. fredii strains. Based on the sequences of the ITS regions, we divided the sequences of the S.   fredii strains into two groups, type A and type B. A dot-matrix analysis indicated that the region flanked by tRNA-Ile and tRNA-Ala is longer in type A than in type B, whereas type B sequences possess longer regions upstream of tRNA-Ile and downstream of tRNA-Ala than those of the type A sequence. Restriction fragment length polymorphism of polymerase chain reaction product (PCR-RFLP) of the ITS region in the cloned plasmids as templates could reconstruct the PCR-RFLP pattern from the total DNA as a template. The results of Southern hybridization using the insert sequence between tRNA-Ile and tRNA-Ala in type A as a probe indicated differences in the copy numbers of the type A ITS regions among the strains tested. These results indicated that S. fredii strains possess the type A and type B sequences of the ITS regions at ratios of 3:0, 2:1, 1:2 or 0:3. These S. fredii strains may be useful biological materials for the study of intraspecific variations.     

Phylogeny and nitrogen fixation potential of Bradyrhizobium species isolated from the legume cover crop Pueraria phaseoloides (Roxb.) Benth. in Eastern Cameroon

Pueraria is an herbaceous, perennial legume crop originating in Asia. Pueraria phaseoloides (Roxb.) Benth. (tropical kudzu) is frequently introduced into production systems and is used as green manure, a cover crop and a forage plant, making it important economically. We used P. phaseoloides as a trap crop to study and characterize soil rhizobia in Eastern Cameroon. Bacteria were isolated from fresh nodules collected from field-grown P. phaseoloides roots. The 16S-23S rRNA internal transcribed spacer (ITS) sequences from 30 bacterial isolates were amplified by polymerase chain reaction (PCR) and the reaction products were sequenced. Phylogenetic analysis revealed that all isolates were ascribed to the genus Bradyrhizobium and were grouped into three clusters of Bradyrhizobium sp. strains, one cluster of B. yuanmingense strains, and one cluster of B. elkanii strains. Acetylene reduction assay (ARA) results indicated that the B. yuanmingense strains had significantly higher nitrogen fixation potential and that they could be used as inoculants to enhance nitrogen fixation in Pueraria grown in Eastern Cameroon.     

Establishment of <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> and <Emphasis Type="Italic">B. elkanii</Emphasis> strains in a Brazilian Cerrado oxisol

The competition with established soil populations of Bradyrhizobium able to nodulate soybean has been one of the major constraints to the introduction of more efficient strains in Cerrados soils. The effects of nodulation establishment and persistence of four serologically distinct strains of Bradyrhizobium japonicum (CPAC 15 and CPAC 7, belonging to serogroups USDA 123 and CB 1,809) and B. elkanii (29 W and SEMIA 587, belonging to serogroups 29 W and 587) were examined. These strains were introduced in a dark-red oxisol, without indigenous populations of soybean bradyrhizobia, and were evaluated for 6 years. The experimental design was a completely randomized block with four replicates. In the first year, besides the inoculation treatments, there was also an uninoculated control. In the second year, the main plots were split into three sub-plots and treatments consisted of an uninoculated control, CPAC 7 and CPAC 15. In the third year, the entire area was inoculated with CPAC 7. In the fourth and sixth years, the plots were planted with soybean without inoculation, and in the fifth year the plots were left fallow. The strains introduced in the first year influenced nodule occupancy by strain CPAC 7 until the third successive growing season. By the fourth and sixth years, as a consequence of the dispersal of strains serologically related to serocluster 123 in the entire experimental area, this serogroup dominated the nodulation, occurring, on average, in more than 50% of the nodules of the treatments where it had never been inoculated.