Partner choice in Medicago Truncatula–Sinorhizobium symbiosis

In nitrogen-fixing symbiosis, plant sanctions against ineffective bacteria have been demonstrated in previous studies performed on soybean and yellow bush lupin, both developing determinate nodules with Bradyrhizobium sp. strains. In this study, we focused on the widely studied symbiotic association Medicago truncatula–Sinorhizobium meliloti, which forms indeterminate nodules. Using two strains isolated from the same soil and displaying different nitrogen fixation phenotypes on the same fixed plant line, we analysed the existence of both partner choice and plant sanctions by performing split-root experiments. By measuring different parameters such as the nodule number, the nodule biomass per nodule and the number of viable rhizobia per nodule, we showed that M. truncatula is able to select rhizobia based on recognition signals, both before and after the nitrogen fixation step. However, no sanction mechanism, described as a decrease in rhizobia fitness inside the nodules, was detected. Consequently, even if partner choice seems to be widespread among legumes, sanction of non-effective rhizobia might not be universal.     

Control of NO level in rhizobium-legume root nodules: Not only a plant globin story

Nitric oxide (NO) is a gaseous signaling molecule which plays both regulatory and defense roles in animals and plants. In the symbiosis between legumes and rhizobia, NO has been shown to be involved in bacterial infection and nodule development steps as well as in mature nodule functioning. We recently showed that an increase in NO level inside Medicago truncatula root nodules also could trigger premature nodule senescence. Here we discuss the importance of the bacterial Sinorhizobium meliloti flavohemoglobin to finely tune the NO level inside nodules and further, we demonstrate that S. meliloti possesses at least two non redundant ways to control NO and that both systems are necessary to maintain efficient nitrogen fixing activity.     

Flavodoxin overexpression reduces cadmium-induced damage in alfalfa root nodules

Flavodoxins are electron carrier flavoproteins that are involved in the response to oxidative stress in bacteria and cyanobacteria. Recently, we obtained Sinorhizobium meliloti bacteria that overexpressed a flavodoxin from the cyanobacterium Anabaena variabilis [Redondo et al. (2009) Plant Physiology 149:1166–1178]. In the present work, tolerance to cadmium was evaluated in free-living transformed S. meliloti and in alfalfa plants nodulated by the flavodoxin-overexpressing rhizobia, in comparison with plants nodulated by wild-type bacteria. Overexpression of flavodoxin protected free-living S. meliloti from cadmium toxicity and had a positive effect on nitrogen fixation of alfalfa plants subjected to cadmium stress. Flavodoxin notably reduced cadmium-induced structural and ultrastructural alterations in alfalfa nodules. Putative protection mechanisms in flavodoxin-overexpressing nodules are discussed. Flavodoxin could have applications as a biotechnological tool to improve the symbiotic performance of alfalfa and other legumes in cadmium polluted soils.     

Inoculation Response of Legumes in Relation to the Number and Effectiveness of Indigenous Rhizobium Populations

The response of legumes to inoculation with rhizobia can be affected by many factors. Little work has been undertaken to examine how indigenous populations or rhizobia affect this response. We conducted a series of inoculation trials in four Hawaiian soils with six legume species (Glycine max, Vigna unguiculata, Phaseolus lunatus, Leucaena leucocephala, Arachis hypogaea, and Phaseolus vulgaris) and characterized the native rhizobial populations for each species in terms of the number and effectiveness of the population for a particular host. Inoculated plants had, on average, 76% of the nodules formed by the inoculum strain, which effectively eliminated competition from native strains as a variable between soils. Rhizobia populations ranged from less than 6 × 10⁰/g of soil to 1 × 10⁴/g of soil. The concentration of nitrogen in shoots of inoculated plants was not higher than that in uninoculated controls when the most probable number MPN counts of rhizobia were at or above 2 × 10¹/g of soil unless the native population was completely ineffective. Tests of random isolates from nodules of uninoculated plants revealed that within most soil populations there was a wide range of effectiveness for N₂ fixation. All populations had isolates that were ineffective in fixing N₂. The inoculum strains generally did not fix more N₂ than the average isolate from the soil population in single-isolate tests. Even when the inoculum strain proved to be a better symbiont than the soil rhizobia, there was no response to inoculation. Enhanced N₂ fixation after inoculation was related to increased nodule dry weights. Although inoculation generally increased nodule number when there were less than 1 × 10² rhizobia per g of soil, there was no corresponding increase in nodule dry weight when native populations were effective. Most species compensated for reduced nodulation in soils with few rhizobia by increasing the size of nodules and therefore maintaining a nodule dry weight similar to that of inoculated plants with more nodules. Even when competition by native soil strains was overcome with a selected inoculum strain, it was not always possible to enhance N₂ fixation when soil populations were above a threshold number and had some effective strains.     

Mixture of endophytic Agrobacterium and Sinorhizobium meliloti strains could induce nonspecific nodulation on some woody legumes

Agrobacterium sp. II CCBAU 21244 isolated from root nodules of Wisteria sinensis was verified as an endophytic bacterium by inoculation and reisolation tests. However, inoculation with a mixture of this strain and a Sinorhizobium meliloti strain could induce root nodules on W. sinensis and two other woody legumes, which do not form a symbiosis with S. meliloti alone. Rod-shaped and irregular nodules were found on the inoculated plants, in which the S. meliloti strain was detected in all of the nodules; while the Agrobacterium strain was inside of the rod-shaped nodules, or occupied only the nodule surface of the irregular globe-shaped nodules. These findings revealed novel interactions among the symbiotic bacteria, endophytic bacteria and the legume plants, although the mechanisms are still unknown.     

Endophytic occupation of peanut root nodules by opportunistic Gammaproteobacteria

Several bacterial isolates were recovered from surface-sterilized root nodules of Arachis hypogaea L. (peanut) plants growing in soils from Córdoba, Argentina. The 16S rDNA sequences of seven fast-growing strains were obtained and the phylogenetic analysis showed that these isolates belonged to the Phylum Proteobacteria, Class Gammaproteobacteria, and included Pseudomonas spp., Enterobacter spp., and Klebsiella spp. After storage, these strains became unable to induce nodule formation in Arachis hypogaea L. plants, but they enhanced plant yield. When the isolates were co-inoculated with an infective Bradyrhizobium strain, they were even found colonizing pre-formed nodules. Analysis of symbiotic genes showed that the nifH gene was only detected for the Klebsiella-like isolates and the nodC gene could not be amplified by PCR or be detected by Southern blotting in any of the isolates. The results obtained support the idea that these isolates are opportunistic bacteria able to colonize nodules induced by rhizobia.     

Wild peanut Arachis duranensis are nodulated by diverse and novel Bradyrhizobium species in acid soils

Aiming at learning the microsymbionts of Arachis duranensis, a diploid ancestor of cultivated peanut, genetic and symbiotic characterization of 32 isolates from root nodules of this plant grown in its new habitat Guangzhou was performed. Based upon the phylogeny of 16S rRNA, atpD and recA genes, diverse bacteria belonging to Bradyrhizobium yuanmingense, Bradyrhizobium elkanii, Bradyrhizobium iriomotense and four new lineages of Bradyrhizobium (19 isolates), Rhizobium/Agrobacterium (9 isolates), Herbaspirillum (2 isolates) and Burkholderia (2 isolates) were defined. In the nodulation test on peanut, only the bradyrhizobial strains were able to induce effective nodules. Phylogeny of nodC divided the Bradyrhizobium isolates into four lineages corresponding to the grouping results in phylogenetic analysis of housekeeping genes, suggesting that this symbiosis gene was mainly maintained by vertical gene transfer. These results demonstrate that A. duranensis is a promiscuous host preferred the Bradyrhizobium species with different symbiotic gene background as microsymbionts, and that it might have selected some native rhizobia, especially the novel lineages Bradyrhizobium sp. I and sp. II, in its new habitat Guangzhou. These findings formed a basis for further study on adaptation and evolution of symbiosis between the introduced legumes and the indigenous rhizobia.     

Spatiotemporal cytokinin response imaging and ISOPENTENYLTRANSFERASE 3 function in Medicago nodule development

Most legumes can establish a symbiotic association with soil rhizobia that trigger the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharides (LCOs) in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Thus, nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) contributes to the coordination of this process, acting as an essential positive regulator of nodule organogenesis. However, the temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In this study, using a fluorescence-based CK sensor (pTCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the sequential activation of CK response during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYLTRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::nls:tdTOMATO and the CK sensor showed that IPT3 induction in the pericycle at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula.

Precise spatial and temporal characterization of cytokinin (CK) responses reveals the function of the CK biosynthesis gene ISOPENTENYLTRANSFERASE 3 during nodule development in Medicago truncatula.     

桂西北喀斯特常见豆科植物根瘤菌的遗传多样性

调查了桂西北喀斯特24种常见豆科植物的结瘤情况及特征,并从15种宿主植物上获得39份根瘤样品,提取根瘤基因组DNA,扩增16S rDNA和nifH基因,构建系统发育树,对根瘤菌遗传多样性进行了研究.结果表明: 有15种豆科植物是结瘤的,其中14种为蝶形花亚科,1种为含羞草亚科,而云实亚科未发现结瘤.一些本应结瘤的植物未发现根瘤,可能与喀斯特土壤的保水性差有关.BLAST和系统发育分析结果均显示,来源于多种豆科植物的根瘤菌均归属于慢生根瘤菌属,仅有2个亮叶崖豆藤样品的根瘤菌归属于中慢生根瘤菌属.在系统发育树上,来源于同一地点或同一宿主植物的根瘤序列均表现出一定的聚集性,说明共生根瘤菌的种类可能受宿主植物及所处生态环境的共同影响.     

Factors affecting co-inoculation with antibiotic-producing bacteria to enhance rhizobial colonization and nodulation

Co-inoculation with antibiotic-producing bacteria and rhizobia resistant to those antibiotics has been proposed as a means of promoting colonization and nodulation of legumes by root-nodule bacteria. A study was conducted to establish some of the factors affecting co-inoculation with antibiotic-producing strains of Bacillus and Streptomyces griseus. The stimulation of Rhizobium meliloti and yield and N uptake by alfalfa was enhanced with increasing inoculum size of Bacillus sp. S. griseus and chitin added to soil increased nodulation of soybeans by Bradyrhizobium japonicum and increased nodulation, yield, and number of pods on a second crop grown in the same soil. Bacillus sp. persisted in soil in sufficient numbers for at least 51 days to increase colonization of soybean roots by B. japonicum. The populations of S. griseus, Bacillus sp., and antibiotic-resistant isolates of R. meliloti and B. japonicum fell after their addition to seeds. Nevertheless, a benefical effect by the antibiotic-producing bacteria was evident on R. meliloti colonization of the rhizosphere, nodulation, and yield of alfalfa grown from seeds stored 94 days and on B. japonicum colonization, nodule number, yield, and seed weight of soybeans grown from seeds stored 90 days. Because non-antibiotic-producing derivatives of Bacillus sp. and S. griseus did not promote colonization or nodulation of alfalfa roots by R. meliloti, the benefit of this co-inoculation is a result of antibiotic formation.     

Queuosine Biosynthesis Is Required for Sinorhizobium meliloti-Induced Cytoskeletal Modifications on HeLa Cells and Symbiosis with Medicago truncatula

Rhizobia are symbiotic soil bacteria able to intracellularly colonize legume nodule cells and form nitrogen-fixing symbiosomes therein. How the plant cell cytoskeleton reorganizes in response to rhizobium colonization has remained poorly understood especially because of the lack of an in vitro infection assay. Here, we report on the use of the heterologous HeLa cell model to experimentally tackle this question. We observed that the model rhizobium Sinorhizobium meliloti, and other rhizobia as well, were able to trigger a major reorganization of actin cytoskeleton of cultured HeLa cells in vitro. Cell deformation was associated with an inhibition of the three major small RhoGTPases Cdc42, RhoA and Rac1. Bacterial entry, cytoskeleton rearrangements and modulation of RhoGTPase activity required an intact S. meliloti biosynthetic pathway for queuosine, a hypermodifed nucleoside regulating protein translation through tRNA, and possibly mRNA, modification. We showed that an intact bacterial queuosine biosynthetic pathway was also required for effective nitrogen-fixing symbiosis of S. meliloti with its host plant Medicago truncatula, thus indicating that one or several key symbiotic functions of S. meliloti are under queuosine control. We discuss whether the symbiotic defect of que mutants may originate, at least in part, from an altered capacity to modify plant cell actin cytoskeleton.     

Bradyrhizobium arachidis sp. nov., isolated from effective nodules of Arachis hypogaea grown in China

Twenty-three bacterial strains isolated from root nodules of Arachis hypogaea and Lablab purpureus grown in five provinces of China were classified as a novel group within the genus Bradyrhizobium by analyses of PCR-based RFLP of the 16S rRNA gene and 16S–23S IGS. To determine their taxonomic position, four representative strains were further characterized. The comparative sequence analyses of 16S rRNA and six housekeeping genes clustered the four strains into a distinctive group closely related to the defined species Bradyrhizobium liaoningense, Bradyrhizobium yuanmingense, Bradyrhizobium huanghuaihaiense, Bradyrhizobium japonicum and Bradyrhizobium daqingense. The DNA–DNA relatedness between the reference strain of the novel group, CCBAU 051107^T, and the corresponding type strains of the five mentioned species varied between 46.05% and 13.64%. The nodC and nifH genes of CCBAU 051107^T were phylogenetically divergent from those of the reference strains for the related species. The four representative strains could nodulate with A. hypogaea and L. purpureus. In addition, some phenotypic features differentiated the novel group from the related species. Based on all the results, we propose a new species Bradyrhizobium arachidis sp. nov. and designate CCBAU 051107^T (=CGMCC 1.12100^T = HAMBI 3281^T = LMG 26795^T) as the type strain, which was isolated from a root nodule of A. hypogaea and had a DNA G + C mol% of 60.1 (Tm).     

Morphogenesis of lucerne root nodules incited by Rhizobium meliloti in the presence of combined nitrogen

Combined light and transmission electron microscopy were used to examine the effect of nitrate on the development of root nodules in lucerne (alfalfa, Medicago sativa L.) following induction by the nitrogen-fixing symbiont, Rhizobium meliloti. The timing of NO ₃ ^- addition was varied in order to study its effect on all of the recognized morphogenetic steps of nodule formation. Roots of plants inoculated in the presence of 18 mM NO ₃ ^- had straight root hairs which were devoid of adherent rhizobia and infection threads, and developed no nodules. However, nodules were formed on roots if 18 mM NO ₃ ^- was added 5 d after inoculation. At this time, the initiation of nodule primordia had already commenced in the root cortex. The histology and ultrastructure of young nodules which had developed for 5 d in the absence of NO ₃ ^- and another 5 d in the presence of 18 mM NO ₃ ^- resembled nodules developing under N-free conditions, except that in the infection threads within the infection zone of the nodule 1) some bacteria tended to loose their normal shape and gain more electron density, indicating premature degradation, and 2) the matrix of the infection threads was abnormally enlarged. In the presence of high NO ₃ ^- levels in the medium, lysis and degeneration of the bacteria released from the infection threads were observed in the infection and bacteroid zones of developing nodules, indicative of premature senescence. On the other hand, the nodule meristems continued to proliferate even after 12 d of exposure of 18 mM NO ₃ ^- . This was the only morphogenetic step of root nodulation which was insensitive to levels of combined nitrogen that completely prevented infection if present at the time of inoculation. These data indicate that all of the recognized steps of root nodule morphogenesis in which the bacteria play a key role are sensitive to the inhibitory effect of combined nitrogen.     

Adaptability to local conditions and phylogenetic differentiation of microsymbionts of TGx soybean genotypes in the semi-arid environments of Ghana and South Africa

The study of the nitrogen fixation and phylogenetic diversity of nodule microsymbionts of grain legumes in many parts of the globe is often carried out in order to identify legume-rhizobia combinations for agricultural sustainability. Several reports have therefore found that rhizobial species diversity is shaped by edapho-climatic conditions that characterize different geographic locations, suggesting that rhizobial communities often possess traits that aid their adaptation to their habitat. In this study, the soybean-nodulating rhizobia from semi-arid savannahs of Ghana and South Africa were evaluated. The authenticated rhizobial isolates were highly diverse based on their colony characteristics, as well as their BOX-PCR profiles and gene sequences. In the 16S rRNA phylogeny, the isolates were placed in the different clades Bradyrhizobium iriomotense and Bradyrhizobium jicamae together with two superclades Bradyrhizobium japonicum and Bradyrhizobium elkanii. The multilocus (atpD, glnII, gyrB, recA) phylogenetic analyses indicated the dominance of Bradyrhizobium diazoefficiens and putative new Bradyrhizobium species in the semi-arid Ghanaian region. The phylogenetic analyses based on the symbiotic genes (nifH and nodC) clustered the test isolates into different symbiovars (sv. glycinearum, sv. retame and sv. sojae). Principal component analysis (PCA) showed that soil factors played a significant role in favoring the occurrence of soybean-nodulating microsymbionts in the tested local conditions. The results suggested that isolates had marked local adaptation to the prevailing conditions in semi-arid regions but further studies are needed to confirm new Bradyrhizobium species.     

Diversity of endemic rhizobia on Christmas Island: Implications for agriculture following phosphate mining

Given that phosphate supplies may diminish and become uneconomic to mine after 2020, there is a compelling need to develop alternative industries to support the population on Christmas Island. Former mine sites could be turned into productive agricultural land, however, large-scale commercial agriculture has never been attempted, and, given the uniqueness of the island, the diversity of rhizobia prior to introducing legumes needed evaluation. Therefore, 84 rhizobia isolates were obtained from nine different hosts, both crop and introduced legumes, located at seven sites across the island. Based on 16S rRNA and recA gene sequence analysis, the isolates grouped into 13 clades clustering within the genus Bradyrhizobium, Ensifer, Cupriavidus and Rhizobium. According to the sequences of their symbiosis genes nodC and nifH, the isolates were classified into 12 and 11 clades, respectively, and clustered closest to tropical or crop legume isolates. Moreover, the symbiosis gene phylogeny and Multi Locus Sequence Analysis gene phylogeny suggested vertical transmission in the Alpha-rhizobia but horizontal transmission within the Beta-rhizobia. Furthermore, this study provides evidence of a large diversity of endemic rhizobia associated with both crop and introduced legumes, and highlights the necessity of inoculation for common bean, chickpea and soybean on the Island.     

Survival and Competitiveness of Bradyrhizobium japonicum Strains 20 Years after Introduction into Field Locations in Poland

It was previously demonstrated that there are no indigenous strains of Bradyrhizobium japonicum forming nitrogen-fixing root nodule symbioses with soybean plants in arable field soils in Poland. However, bacteria currently classified within this species are present (together with Bradyrhizobium canariense) as indigenous populations of strains specific for nodulation of legumes in the Genisteae tribe. These rhizobia, infecting legumes such as lupins, are well established in Polish soils. The studies described here were based on soybean nodulation field experiments, established at the Poznań University of Life Sciences Experiment Station in Gorzyń, Poland, and initiated in the spring of 1994. Long-term research was then conducted in order to study the relation between B. japonicum USDA 110 and USDA 123, introduced together into the same location, where no soybean rhizobia were earlier detected, and nodulation and competitive success were followed over time. Here we report the extra-long-term saprophytic survival of B. japonicum strains nodulating soybeans that were introduced as inoculants 20 years earlier and where soybeans were not grown for the next 17 years. The strains remained viable and symbiotically competent, and molecular and immunochemical methods showed that the strains were undistinguishable from the original inoculum strains USDA 110 and USDA 123. We also show that the strains had balanced numbers and their mobility in soil was low. To our knowledge, this is the first report showing the extra-long-term persistence of soybean-nodulating strains introduced into Polish soils and the first analyzing the long-term competitive relations of USDA 110 and USDA 123 after the two strains, neither of which was native, were introduced into the environment almost 2 decades ago.     

N-acyl-homoserine lactones-producing bacteria protect plants against plant and human pathogens

The implementation of beneficial microorganisms for plant protection has a long history. Many rhizobia bacteria are able to influence the immune system of host plants by inducing resistance towards pathogenic microorganisms. In this report, we present a translational approach in which we demonstrate the resistance-inducing effect of Ensifer meliloti (Sinorhizobium meliloti) on crop plants that have a significant impact on the worldwide economy and on human nutrition. Ensifer meliloti is usually associated with root nodulation in legumes and nitrogen fixation. Here, we suggest that the ability of S. meliloti to induce resistance depends on the production of the quorum-sensing molecule, oxo-C14-HSL. The capacity to enhanced resistance provides a possibility to the use these beneficial bacteria in agriculture. Using the Arabidopsis-Salmonella model, we also demonstrate that the application of N-acyl-homoserine lactones-producing bacteria could be a successful strategy to prevent plant-originated infections with human pathogens.