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.     

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.     

Estimation of nodulation tendency among Rj-genotype soybeans using the bradyrhizobial community isolated from an Andosol

The nodulation tendency of indigenous soybean bradyrhizobia on Rj -genotype soybean cultivars was investigated using approximately 260 bradyrhizobia isolated from an Andosol with 13 soybean cultivars of five Rj -genotypes (non- Rj , Rj ₂ Rj ₃, Rj ₃, Rj ₄ and Rj ₂ Rj ₃ Rj ₄). A dendrogram was constructed based on restriction fragment length polymorphism analysis of the polymerase chain reaction products (PCR-RFLP) of the 16S–23S rDNA internal transcribed spacer (ITS) region. Bradyrhizobium USDA strains were used as a reference. The dendrogram indicated nine clusters based on similarities among the reference strains. The ratio of beta diversity to gamma diversity ( H' _β/ H' _γ), which represents differences in the bradyrhizobial communities by pair-wise comparison between each cultivar, was obtained from Shannon–Wiener diversity indices. The results showed that bradyrhizobial communities among the same Rj -genotype cultivars were similar to each other, whereas bradyrhizobial communities between the Rj ₂-genotype and non- Rj , Rj ₃ or Rj ₄-genotype cultivars were significantly different. These results suggest that the Rj ₂-gene might not only affect the nodulation compatibility between Rj -genotype soybeans and bradyrhizobia, but also the nodulation tendency of the bradyrhizobia.     

Grouping of Bradyrhizobium USDA strains by sequence analysis of 168 rDNA and 168-238 rDNA internal transcribed spacer region

In order to select appropriate Bradyrhizobium USDA reference strains for primary grouping of indigenous soybean bradyrhizobia, we systematically constructed phylogenetic trees of 20 USDA strains based on DNA sequence analysis and PCR-restriction fragment length polymorphism (RFLP) targeted to 16S rDNA and the internal transcribed spacer (ITS) region between 16S and 23S rDNAs. The phylogenetic trees of 16S rDNA showed 3 major groups, cluster USDA 110 (USDA 62, 110, 122, 125, and 129), cluster USDA 6 (USDA 4, 6^T, 38, 115, 123, 127, 135, and 3622^T) and cluster B. elkanii (USDA 31, 46, 61, 76^T, 94, and 130), as well as the phylogenetically independent strain USDA 124. The topology of the ITS trees was almost similar to that of 16S rDNA, although the positions of two extra-slow-growing strains, USDA 135 and USDA 3622^T were variable among the ITS sequences, PCR-RFLP of the ITS region and 16S rDNA. Only two strains, USDA 110 and USDA 122, harbored hup genes and they fell into the USDA 110 cluster. These results suggest that PCR-RFLP analysis of 16S rDNA and the 16S-23S rDNA ITS region may be useful for the grouping of bradyrhizobia and for the first screening of hup-positive strains. Based on the above results, we propose a minimum set of USDA strains reflecting Bradyrhizobium diversity that includes B. japonicum USDA 6^T, B. japonicum USDA 110, B. japonicum USDA 124, and B. elkanii USDA 76^T. In addition, an extra-slow-growing strain with the serotype USDA 135 might be necessary for genomic diversity analysis of bradyrhizobia, because their phylogenetic positions were variable.     

Genetic diversity and relationship between Bradyrhizobium strains isolated from blackgram and cowpea

The genetic diversity of bradyrhizobial strains associated with blackgram and cowpea grown in two different agricultural soils (non-saline and saline) along the coastline of Tamil Nadu has been analysed. Phenotypically indistinguishable isolates were analysed for DNA polymorphism using random amplification of polymorphic DNA (RAPD) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of 16S rDNA and nifD. Although these bacteria belong to a group with a broad host range, RAPD analysis showed a considerable level of genetic diversity among the strains isolated from different host plants. Soil pH and salinity seem to have an effect on the selection of natural populations as revealed by PCR-RFLP of 16S rDNA. A combination of PCR-RFLP genotyping with nodulation studies indicates that monocropping of blackgram and the salinity of the soil have made ineffective rhizobia the dominant genotype, thereby creating an ecological burden on their other compatible hosts. A group of strains and a type strain sharing three different 16S PCR-RFLP types were shown to have the same set of symbiotic genes as inferred from the PCR-RFLP pattern of nifD. Another group of cowpea rhizobia that were found to be effective nitrogen fixers and sharing distinct 16S profiles were found to have a different set of symbiotic genes.     

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.     

Multiple occupancy of nodules by nodulating rhizobia on field-grown soybeans with attendance of Sinorhizobium spp.

Abstract

This study analyzed the phenotypic and genotypic characters of nodulating rhizobia isolated from two soybean cultivars, Kyushu 151 and Sachiyutaka, in the same field of the Yamaguchi Prefectural Technology Center of Agriculture and Forestry in Japan. The isolates were classified into groups using phenotypic characteristics, such as growth rate, color change on Bromothymol blue-containing yeast extract-mannitol agar (YMA) plates and colony morphology on YMA plates, and by genotypic characteristics, such as polymerase chain reaction–restriction fragment length polymorphism patterns of the 16S ribosomal RNA genes (16S rDNA) and the internal transcribed sequence (ITS) regions. In Kyushu 151, single phenotypic and genotypic groups were isolated from every nodule examined. In Sachiyutaka, plural strains belonging to distinct groups were obtained frequently from single nodules, indicating that multiple occupancy was established at high frequency. No fixed combination of the groups was found in the composition of multiple occupancy. An increase in the relative abundance of isolates belonging to Sinorhizobium fredii (Ensifer fredii) occurred concomitantly with the increase in the proportion of nodules with multiple occupancy. Nearly 60% of the isolates from Sachiyutaka belonged to S. fredii; 75% of them were obtained from nodules with multiple occupancy.     

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.     

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.     

Genetic diversity of rhizobia associated with alfalfa in Serbian soils

We have evaluated the genetic diversity and phylogeny of alfalfa rhizobia, originating from different types of soils in Serbia and their ability to establish an effective symbiosis with alfalfa (Medicago sativa L.). A collection of 65 strains isolated from root nodules of alfalfa were characterized by rep-PCR analysis, partial and complete 16S rDNA gene and recA gene sequencing, as well as atpD gene sequencing and DNA–DNA hybridizations. The results of the sequence analyses revealed that Sinorhizobium meliloti is the dominant species in alfalfa nodules. Only one strain was identified as Sinorhizobium medicae, two strains as Rhizobium tibeticum and one strain as Rhizobium sp. Despite the fact that the majority of strains were identified as S. meliloti, a high genetic diversity at strain level was detected. Almost all isolates shared the ability to nodulate and fix nitrogen with M. sativa, except 11 of them, which were incapable of fixing nitrogen with this species. About 50% of the isolates showed values of symbiotic effectiveness (SE) above 50%, while 10% of the strains were highly effective with SE values above 70%. Some of the strains which were highly effective in nitrogen fixation at the same time could intensively solubilize phosphates, offering a possibility for multipurpose inoculum development. This was the first genetic study of rhizobia isolated from this region and also the first report of natural presence of R. tibeticum in root nodules of M. sativa.     

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.     

两株发光酶基因标记的重组大豆根瘤菌在土壤缩影中的存活研究

研究了发光酶基因 (luxAB)标记的重组费氏中华根瘤菌HN0 1DL、大豆慢生根瘤菌TA113QD与其出发菌株HN0 1、TA11在灭菌和未灭菌土缩影中的存活动态。结果表明 :HN0 1DL和TA113QD在灭菌土缩影和未灭菌土缩影中的存活动态特征显著不同 :标记菌株在灭菌土缩影中的早期存活数量一般有所下降后而保持相对稳定的较高存活水平 ,而在未灭菌土缩影中其存活数量在整个跟踪过程中持续下降至约 4 5logcfug- 1 土。在本实验中未发现供试标记重组菌株和出发菌株在土壤缩影中的存活性存在显著差异     

铅锌矿区重金属污染对微生物数量及放线菌群落结构的影响

采集四川省汉源县富泉乡万顺铅锌矿区5个不同重金属浓度的土壤样品,进行了微生物数量及放线菌多样性的研究。经分离、纯化得到43株不同的放线菌,然后对其进行BOXAIR-PCR和16SrDNAPCR-RFLP分析。结果表明,铅锌矿区重金属复合污染对土壤微生物数量有较大的影响,随着铅锌矿区重金属污染程度的加剧,土壤微生物的总数下降。相关性分析表明,重金属含量与细菌数量呈极显著负相关（P〈0.01）,与放线菌数量、真菌数量呈显著负相关（P〈0.05）。供试菌株的16SrDNA用HaeⅢ、HinfⅠ和TaqⅠ酶切后具有32种遗传图谱类型。BOXAIR-PCR的聚类结果表明在86%的水平上,所有菌株分为10个遗传类型,结果基本与16SrDNAPCR-RFLP聚类差异不大。来源于高重金属的含量样品的菌株基本聚在一起,可能是重金属含量影响了放线菌的分布。同时,16SrDNA序列聚类分析结合系统发育树分析表明链霉菌属是汉源铅锌矿区主要的放线菌属并且具有遗传多样性。     

Effects of Interactions Between Chemical Seed Dressings And Bradyrhizobium Japonicum on Soybean Seeds

Soybean seeds, both untreated and dusted with selected pesticides, were inoculated with Bradyrhizobium japonicum and stored in the laboratory at room temperature. Approximately 1 hour, 24 hours and 48 hours following inoculation samples of the seeds were taken to count the surviving rhizobia and to grow soybean plants in the greenhouse experiment. Numbers of B. japonicum cells surviving on soybean seeds (two cultivars) treated with the tested pesticidal dressings and on the untreated seeds did not differ significantly when analysed within the first hour following seed inoculation with the rhizobia. However, analyses performed 24 hours and 48 hours later showed no B. japonicum cells surviving on the seeds dressed with mancozeb (fungicide), while the numbers of these bacteria on the untreated seeds and on the seeds treated with a mixture of carbendazim + thiram (fungicides) or with carbosulfan (insecticide) were similar and remained on relatively high levels. Only soybean plants that developed from mancozeb treated seeds had significantly less nodules, both in terms of numbers and mass, as well as lower fresh and dry weight of shoots as compared to the plants grown from the control seeds and the seeds treated with the other pesticides tested. The toxicity of mancozeb to the symbiosis was particularly severe when soybean seeds inoculated with B. japonicum were stored for 24-48 hours before planting     

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.     

Distribution and genetic diversity of rhizobia nodulating natural populations of Medicago truncatula in tunisian soils

A collection of 299 isolates of rhizobia nodulating Medicago truncatula was isolated from 10 Tunisian soils and was characterized by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR/RFLP) of 16S rRNA gene. Results showed that 227 and 72 isolates were assigned, respectively, to Sinorhizobium meliloti and Sinorhizobium medicae. In 9 out of 10 soils S. meliloti was detected, whereas S. medicae was recovered from only 5 out of 10 soils. The cross-nodulation of three populations of M. truncatula grown on Bulla Regia soil, which contained naturally the two Sinorhizobium species, showed that M. truncatula population collected from Amra site was selective to S. meliloti at least in soil conditions. Forty-eight isolates of each Sinorhizobium species trapped by M. truncatula populations collected from Bulla Regia, Soliman and Rhayet sites on Bulla Regia soil were characterized by repetitive extragenic palindromic-PCR (REP-PCR) and showed a clear distinction between the two Sinorhizobium species and a higher diversity for S. meliloti.     

西北部分矿区豆科植物根瘤菌重金属抗性及16S rDNA RFLP分析

从生长在西北部分矿区的豆科植物根瘤中分离筛选到对重金属有抗性的38株根瘤菌,采用PCR-RFLP分子技术进行16S rDNA指纹图谱分析,选取每种类型的代表菌株进行16S rDNA全序列测定,建立系统发育树状图,并对38株菌进行Zn、Hg、Cu、Cd和Pb5种重金属的抗性研究。结果表明,供试菌株分别归属于中华根瘤菌属（Sinorhizobium）、根瘤菌属（Rhizobium）和土壤杆菌属（Agrobacterium）。代表菌株CCNWSX1277和CCNWSX1294可耐受2.0mmol·L-1的Zn2＋,CCNWSX1277可耐受0.25mmol·L-1的Hg2＋,多数代表菌株可耐受1.6mmol·L-1的Cu2＋,仅3株代表菌株可以耐受Cd2＋,其中CCNWSX1277能耐受1.4mmol·L-1的Cd2＋,所有代表菌株能耐受2.5mmol·L-1的Pb2＋。Agrobacterium属的2株代表菌株对5种重金属均有较强的耐受性;而Rhizobium属的4株菌和Sinorhizobium属的3株菌对5种重金属的耐受性不同,表现出较大的差异。总体来看,供试菌株对重金属的耐受性顺序为Agrobacterium〉Rhizobium〉Sinorhizobium。     

The diversity of rhizobia nodulating chickpea (Cicer arietinum) under water deficiency as a source of more efficient inoculants

Chickpea is an important pulse crop grown mainly in the arid and semi-arid regions. The effect of water deficiency on nodulation, biomass production, and competition for nodule occupancy was evaluated in three different soils with two chickpea cultivars, Amdoun I and Chetoui. Two watering regimes were considered; a control that was irrigated three times per week and a water-deficient treatment that was irrigated only one time a week. Results showed that water deficiency significantly decreased the nodule number and the shoot dry weight for both cultivars. Root-nodule bacteria were isolated and characterized by PCR-RFLP of 16S rDNA and nifD-K intergenic spacer. The results show that water deficiency affects the diversity of nodulating rhizobia. The nodulation by Mesorhizobium mediterraneum was reduced while inefficient nodulation by Ensifer meliloti was favoured. In both treatments, chickpea was preferentially nodulated by nifD-K type N6. Analysis for NaCl tolerance showed that most of rhizobia nodulating chickpea under water deficiency are NaCl tolerant. Inoculation with the selected salt-tolerant strain of M. mediterraneum LILM10 increased significantly nodule number and grain yield in the field.     

Effects of symbiosis with Rhizobium fredii on transport of fixed nitrogen in the xylem of soybean plants

An experiment was conducted to identify the main nitrogenous compound transported in the xylem sap of soybean plants nodulated with Rhizobium fredii. Soybean (Glycine max L. Merr.) cultivars, wild type Bragg (nod⁺, fix⁺) and its nitrate tolerant, hypernodulating mutant ntsll16 (nod⁺⁺, fix⁺) were used for this experiment. These soybean plants were inoculated with a slowgrowing rhizobium, Bradyrhizobium japonicum USDAllO or fast-growing rhizobia consisting of a mixture of R. fredii USDA191, USDA193, and USDA-194 and grown in a phytotron under natural light and controlled temperature conditions. Xylem sap was collected from Bragg and ntsll16 plants at the flowering and pod elongation stages. Acetylene reduction activity per plant or per nodule weight was not different between soybean lines and inoculums. The composition of the nitrogenous compounds in the xylem sap was compared between the symbionts, with B. japonicum and R. fredii. At the flowering stage, ureide-N and amide-N accounted for 53 to 70% and 20 to 27% respectively of the total N in the sap collected from the plants inoculated either with B. japonicum or R. fredii. At the pod elongation stage, ureide-N and amide-N accounted for 74 to 85%, and 7 to 19% of total sap N. With the growth of the soybean plants, the ratio of ureide-N in the xylem sap increased. These results suggest that in the case of wild soybean and the hypernodulating mutant line nodulated by R. fredii, ureide is transported as the main nitrogenous compound of fixed nitrogen in the xylem sap in the same way as in plants nodulated with B. japonicum.     

Burr medic (Medicago polymorpha L.) selections for improved N2 fixation with naturalised soil rhizobia

Burr medic (Medicago polymorpha L.) is an annual pasture legume that is widely distributed in southern Australian farming systems. Burr medic is nodulated by rhizobia (Sinorhizobium meliloti and Sinorhizobium medicae) that reside in many Australian soils, but the symbioses that develop are often sub-optimal in their rate of N₂ fixation. We attempted to identify burr medic lines, which are able to form effective symbioses with the naturalised Sinorhizobium in Australian field soils, as potential parents for a breeding program. There were three glasshouse experiments. Initially, 222 lines (including the M. polymorpha cvv. Santiago, Serena and Circle Valley) were inoculated with extracts of two soils that had been collected near Waikerie (soil S109) and Lochiel (soil S142) in South Australia. These soils were used because they contained numerically large communities of naturalised Sinorhizobium spp. that produced sub-optimal rates of N₂ fixation with cv. Santiago. None of the 222 lines of burr medic were able to form an effective symbiosis with the rhizobia from soil S109. However, when nodulated by the rhizobia from soil S142, some lines (e.g. SA8194) formed a very effective symbiosis, producing up to double the shoot dry matter (DM) of Santiago and eight times the DM of uninoculated plants. Seven promising lines were selected for further testing (with extracts of nine soils). Subsequently, two lines (SA20056 and SA8194) were selected and their symbiotic performance compared with that of Santiago, using extracts from 28 soils. While soil treatment had a major effect on mean shoot DM (soil N103=120 mg, soil N105=17 mg), the three medic lines performed similarly. Santiago, SA20056 and SA8914 all formed ineffective symbioses with the rhizobia in at least half of the 28 soils, even though >95% of the plants were nodulated. These experiments confirm that ineffective symbioses are common between burr medics and the rhizobia that have become naturalised in many Australian soils. Although some lines of burr medic were identified that were able to form more effective symbioses with the rhizobia in individual soils, none were able to form effective symbioses with a wide range of soil rhizobia. If a plant breeding approach is to be used to improve symbiotic performance of burr medic we propose that its hybridisation with other medic species, that have less specific rhizobial needs, will be required.