Characterisation and differentiation of indigenous rhizobia isolated from Canarian shrub legumes of agricultural and ecological interest

In the course of a study on rhizobia nodulating six indigenous legume shrubs from the Canary Islands, one Rhizobium and 27 Bradyrhizobium Canarian isolates were characterised. It was found that those ascribed to Bradyrhizobium were promiscuous and formed effective nodules not only in their original host but on Chamecytisus proliferus subsp. proliferus (Tagasaste) as well. However, Rhizobium isolate RES-1 was more specific and only nodulated on its host (Teline canariensis). The serotyping of these isolates required a broad antisera panel due to the great antigenic diversity of these rhizobia, that appeared to be due to differences in their lipopolysaccharides, the main antigenic determinants, that showed great structural diversity. The 28 isolates studied produced 22 easily distinguishable electrophoretic profiles of lipopolysaccharides. Protein or plasmid electrophoretic profiles were equally or less discriminating than the lipopolysaccharides profiles and were more difficult to compare. The comparison of the lipopolysaccharide electrophoretic patterns is a more reliable and discriminating method than serotyping or electrophoretic protein and plasmid profile analysis for the identification of Bradyrhizobium strains. No correlation between the lipopolysaccharide profiles of the isolates and the plant from which they were obtained or their geographical origin was observed.     

A Study on the Prevalence of Bacteria that Occupy Nodules within Single Peanut Plants

In this study, bacteria hosted in root nodules of single plants of legume Arachis hypogaea L. (peanut) cv Tegua Runner growing at field were isolated. The collection of nodule isolates included both fast and slow growing strains. Their genetic diversity was assessed in order to identify the more frequently rhizobial strain associated to nodules from single plants. Molecular fingerprinting of 213 nodular isolates indicated heterogeneity, absence of a dominant genotype and, therefore, of a unique strains highly competitive. Efficient nitrogen-fixing isolates were identified as Bradyrhizobium sp. by phylogenetic analysis of the sequences of their 16S rRNA genes. The genetic diversity of 68 peanut nodulating isolates from all the collected plants was also analyzed. Considering their ERIC-PCR profiles, they were grouped in eighteen different OTUs for 60% similarity cut-off. Results obtained in this study indicate that the genetic diversity of rhizobia occupying nodules from single plant is very high, without the presence of a dominant strain. Therefore, the identification of useful peanut rhizobia for agricultural purposes requires strongly the selection, among the diverse population, of a very competitive genotype in combination with a high-symbiotic performance.     

Rhizobium Population Genetics: Enzyme Polymorphism in Rhizobium leguminosarum from Plants and Soil in a Pea Crop

A population of Rhizobium leguminosarum biovar viceae symbiotic on the roots of a commercial pea (Pisum sativum cv. Maro) crop was sampled by extracting a total of 249 isolates from root nodules on nine plants. Another 104 isolates were obtained by using soil from the same site to inoculate test plants, and a further 86 isolates were similarly obtained from soil 20 m distant within the crop. Each isolate was characterized for mobility variants of the enzymes glucose-6-phosphate dehydrogenase, superoxide dismutase, and β-galactosidase by polyacrylamide gel electrophoresis. All three enzymes were polymorphic, and there was a strong disequilibrium among them. Of the 15 observed combinations of alleles (electrophoretic types [ETs]), 12 were indistinguishable from those previously described for isolates from a site 25 km distant. ET frequencies were significantly different among isolates from nodules on primary roots as opposed to lateral roots. The population on each individual plant was very diverse, but ET frequencies were similar from plant to plant. The ETs nodulating the primary roots were almost, although not perfectly, mixed, since the incidence of the same ETs in adjacent nodules was only about twice that expected by chance. The two samples derived from soil had the same ET frequencies but were significantly different from the field nodule sample, although the level of diversity was similar and there were no new ETs.     

Sucrose synthase and enolase expression in actinorhizal nodules ofAlnus glutinosa: comparison with legume nodules

Two different types of nitrogen-fixing root nodules are known — actinorhizal nodules induced byFrankia and legume nodules induced by rhizobia. While legume nodules show a stem-like structure with peripheral vascular bundles, actinorhizal nodule lobes resemble modified lateral roots with a central vascular bundle. To compare carbon metabolism in legume and actinorhizal nodules, sucrose synthase and enolase cDNA clones were isolated from a cDNA library, obtained from actinorhizal nodules ofAlnus glutinosa. The expression of the corresponding genes was markedly enhanced in nodules compared to roots. In situ hybridization showed that, in nodules, both sucrose synthase and enolase were expressed at high levels in the infected cortical cells as well as in the pericycle of the central vascular bundle of a nodule lobe. Legume sucrose synthase expression was studied in indeterminate nodules from pea and determinate nodules fromPhaseolus vulgaris by usingin situ hybridization.     

Nitrogen-fixing nodules from rose wood legume trees (Dalbergia spp.) endemic to Madagascar host seven different genera belonging to alpha- and beta-Proteobacteria

Although legume biodiversity is concentrated in tropical regions, the majority of studies on legume nodulating bacteria (LNB) are focused on cultivated leguminous plants from temperate regions. However, recent works on tropical regions tend to indicate that the actual diversity of LNB is largely underestimated. In this study, we report the isolation and characterization of 68 nitrogen-fixing root nodule bacteria collected from eight endemic tree species of Dalbergia in Madagascar. The isolates were characterized by (i) restriction fragment length polymorphism (RFLP) analysis of 16S-IGS rDNA, (ii) 16S rDNA gene sequencing and (iii) nodulation tests. Results revealed a wide diversity of bacteria present in the nodules of Dalbergia. Among the 68 isolated bacteria, 65 belonged to Bradyrhizobium, Mesorhizobium, Rhizobium, Azorhizobium and Phyllobacterium from the alpha-class of Proteobacteria, and three isolates belonged to Burkholderia and Ralstonia from the beta-class of Proteobacteria. Our results also show for the first time that a strain belonging to the Burkholderia cepacia complex is able to induce efficient nodules on a legume plant.     

Is the Legume Nodule a Modified Root or Stem or an Organ sui generis?

The legume nodule, which houses nitrogen-fixing rhizobia, is a unique plant organ. Its homology with lateral roots has been inferred by a comparison with other nitrogen-fixing nodules, especially those formed on actinorhizal plants in response to Frankia inoculation or on Parasponia roots following inoculation with Bradyrhizobium species. These nodules are clearly modified lateral roots in terms of their structure and development. However, legume nodules differ from lateral roots and these other nodules in their developmental origin, anatomy, and patterns of gene expression, and, consequently, several other evolutionary derivations, including from stems, wound or defense responses, or the more ancient vesicular-arbuscular mycorrhizal symbiosis, have been postulated for the legume nodule. In this review, we first present a broad view of the legume family showing the diversity of nodulation occurrence and types in the different subfamilies and particularly within the subfamily Papilionoideae. We then define the typological and molecular criteria used to discriminate the basic organs — root, stem, leaf— of the plant. Finally, we discuss the possible origins of the legume nodule in terms of these typological and molecular bases.     

Symbiotic Characteristics of Rhizobium leguminosarum bv. trifolii Isolates Which Represent Major and Minor Nodule-Occupying Chromosomal Types of Field-Grown Subclover (Trifolium subterraneum L.)

The symbiotic effectiveness and nodulation competitiveness of Rhizobium leguminosarum bv. trifolii soil isolates were evaluated under nonsoil greenhouse conditions. The isolates which we used represented both major and minor nodule-occupying chromosomal types (electrophoretic types [ETs]) recovered from field-grown subclover (Trifolium subterraneum L.). Isolates representing four ETs (ETs 2, 3, 7, and 8) that were highly successful field nodule occupants fixed between 2- and 10-fold less nitrogen and produced lower herbage dry weights and first-harvest herbage protein concentrations than isolates that were minor nodule occupants of field-grown plants. Despite their equivalent levels of abundance in nodules on field-grown subclover plants, ET 2 and 3 isolates exhibited different competitive nodulation potentials under nonsoil greenhouse conditions. ET 3 isolates generally occupied more subclover nodules than isolates belonging to other ETs when the isolates were mixed in 1:1 inoculant ratios and inoculated onto seedlings. In contrast, ET 2 isolates were less successful at nodulating under these conditions. In many cases, ET 2 isolates required a numerical advantage of at least 6:1 to 11:1 to occupy significantly more nodules than their competitors. We identified highly effective isolates that were as competitive as the ET 3 isolates despite representing serotypes that were rarely recovered from nodules of field-grown plants. When one of the suboptimally effective isolates (ET2-1) competed with an effective and competitive isolate (ET31-5) at several different inoculant ratios, the percentages of nodules occupied by the former increased as its numerical advantage increased. Although subclover yields declined as nodule occupancy by ET2-1 increased, surprisingly, this occurred at inoculant ratios at which large percentages of nodules were still occupied by ET31-5.     

Novel Plant-Microbe Rhizosphere Interaction Involving Streptomyces lydicus WYEC108 and the Pea Plant (Pisum sativum)

A previously undescribed plant-microbe interaction between a root-colonizing Streptomyces species, S. lydicus WYEC108, and the legume Pisum sativum is described. The interaction is potentially of great importance to the health and growth in nature of this nodulating legume. The root-colonizing soil actinomycete S. lydicus WYEC108 influences pea root nodulation by increasing root nodulation frequency, possibly at the level of infection by Rhizobium spp. S. lydicus also colonizes and then sporulates within the surface cell layers of the nodules. Colonization leads to an increase in the average size of the nodules that form and improves the vigor of bacteroids within the nodules by enhancing nodular assimilation of iron and possibly other soil nutrients. Bacteroid accumulation of the carbon storage polymer, poly-β-hydroxybutyrate, is reduced in colonized nodules. Root nodules of peas taken from agricultural fields in the Palouse hills of northern Idaho were also found to be colonized by actinomycete hyphae. We hypothesize that root and nodule colonization is one of several mechanisms by which Streptomyces acts as a naturally occurring plant growth-promoting bacterium in pea and possibly other leguminous plants.     

Spontaneous root-nodule formation in the model legume Lotus japonicus: a novel class of mutants nodulates in the absence of rhizobia

Root-nodule development in legumes is an inducible developmental process initially triggered by perception of lipochitin-oligosaccharide signals secreted by the bacterial microsymbiont. In nature, rhizobial colonization and invasion of the legume root is therefore a prerequisite for formation of nitrogen-fixing root nodules. Here, we report isolation and characterization of chemically induced spontaneously nodulating mutants in a model legume amenable to molecular genetics. Six mutant lines of Lotus japonicus were identified in a screen for spontaneous nodule development under axenic conditions, i.e., in the absence of rhizobia. Spontaneous nodules do not contain rhizobia, bacteroids, or infection threads. Phenotypically, they resemble ineffective white nodules formed by some bacterial mutants on wild-type plants or certain plant mutants inoculated with wild-type Mesorhizobium loti. Spontaneous nodules formed on mutant lines show the ontogeny and characteristic histological features described for rhizobia-induced nodules on wild-type plants. Physiological responses to nitrate and ethylene are also maintained, as elevated levels inhibit spontaneous nodulation. Activation of the nodule developmental program in spontaneous nodules was shown for the early nodulin genes Enod2 and Nin, which are both upregulated in spontaneous nodules as well as in rhizobial nodules. Both monogenic recessive and dominant spontaneous nodule formation (snf) mutations were isolated in this mutant screen, and map positions were determined for three loci. We suggest that future molecular characterization of these mutants will identify key plant determinants involved in regulating nodulation and provide new insight into plant organ development.     

Sucrose synthase and enolase expression in actinorhizal nodules ofAlnus glutinosa: comparison with legume nodules

Two different types of nitrogen-fixing root nodules are known — actinorhizal nodules induced byFrankia and legume nodules induced by rhizobia. While legume nodules show a stem-like structure with peripheral vascular bundles, actinorhizal nodule lobes resemble modified lateral roots with a central vascular bundle. To compare carbon metabolism in legume and actinorhizal nodules, sucrose synthase and enolase cDNA clones were isolated from a cDNA library, obtained from actinorhizal nodules ofAlnus glutinosa. The expression of the corresponding genes was markedly enhanced in nodules compared to roots. In situ hybridization showed that, in nodules, both sucrose synthase and enolase were expressed at high levels in the infected cortical cells as well as in the pericycle of the central vascular bundle of a nodule lobe. Legume sucrose synthase expression was studied in indeterminate nodules from pea and determinate nodules fromPhaseolus vulgaris by usingin situ hybridization.     

The evolution of nodulation

In this review we will first describe the different steps leading to nodule formation, and these will be compared with processes of non-symbiotic plant development and growth. In general, aspects of both actinorhizal as well as rhizobial symbiosis are described, but in several cases, the emphasis will be on the Rhizobium-legume symbiosis because more knowledge of this system is available. Subsequently, the phylogeny of nodulating plants is described and a comparison is made between several aspects of legume and actinorhizal nodulation. At the end of this paper the relationship between nodule symbiosis and endomycorrhizal symbiosis is described, and it is discussed to what extent the development of root nodules involves unique properties, or whether processes and genes have been recruited from common plant development and the endomycorrhizal symbiosis.     

Phylogenetic diversity of non-nodulating Rhizobium associated with pine ectomycorrhizae

Most Rhizobium species described are symbionts that form nodules on legume roots; however, non-nodulating strains of Rhizobium are also widespread in nature. Unfortunately, knowledge of non-nodulating Rhizobium is quite limited compared with nodulating Rhizobium . Here, we studied the phylogenetic diversity of Rhizobium species that inhabit Japanese red pine roots ( Pinus densiflora ). Because fine roots of pine trees are usually colonized by ectomycorrhizal fungi in nature, we mainly used ectomycorrhizal root tips for bacterial isolation. Out of 1195 bacteria isolated from 75 independent root samples from the field and greenhouse experiments, 102 isolates were confirmed to be Rhizobium following partial 16S rRNA gene analysis. Rhizobium species were occasionally dominant in culturable bacterial communities, whereas no Rhizobium species were isolated from the soil itself. Molecular phylogenetic analyses using 16S rRNA, atpD , and recA gene sequences revealed that isolated Rhizobium strains were phylogenetically diverse and that several were distantly related to known Rhizobium species. Considering that a single species of pine is associated with unique and phylogenetically diverse Rhizobium populations, we should pay more attention to non-nodulating strains to better understand the diversity, ecology, and evolution of the genus Rhizobium and plant– Rhizobium associations.     

Molecular Diversity of Rhizobia Occurring on Native Shrubby Legumes in Southeastern Australia

The structure of rhizobial communities nodulating native shrubby legumes in open eucalypt forest of southeastern Australia was investigated by a molecular approach. Twenty-one genomic species were characterized by small-subunit ribosomal DNA PCR-restriction fragment length polymorphism and phylogenetic analyses, among 745 rhizobial strains isolated from nodules sampled on 32 different legume host species at 12 sites. Among these rhizobial genomic species, 16 belonged to the Bradyrhizobium subgroup, 2 to the Rhizobium leguminosarum subgroup, and 3 to the Mesorhizobium subgroup. Only one genomic species corresponded to a known species (Rhizobium tropici). The distribution of the various genomic species was highly unbalanced among the 745 isolates, legume hosts, and sites. Bradyrhizobium species were by far the most abundant, and Rhizobium tropici dominated among the Rhizobium and Mesorhizobium isolates in the generally acid soils where nodules were collected. Although a statistically significant association occurred between the eight most common genomic species and the 32 hosts, there was sufficient overlap in distributions that no clear specificity between rhizobial genomic species and legume taxa was observed. However, for three legume species, some preference for particular genomic species was suggested. Similarly, no geographical partitioning was found.     

Population structure of the clover rhizobia Rhizobium leguminosarum bv. trifolii upon transition from soil into the nodular niche

High-throughput sequencing of the amplicon gene library revealed variations in the population structure of clover rhizobia (Rhizobium leguminosarum bv. trifolii) upon transition from soil into the root nodules of the host plant (Trifolium hybridum). Analysis of rhizobial diversity using the nodA gene revealed 3258 and 1449 nucleotide sequences (allelic variants) for the soil and root nodule population, respectively. They were combined into 29 operational taxonomic units (OTU) according to the 97% identity level; 24 OTU were found in the soil population, 12 were present in the root nodule population, and 7 were common. The predominant OTE13 (77.4 and 91.5% of the soil and root nodule populations, respectively) contained 155 and 200 variants of the soil and root nodule populations, respectively, with the nucleotide diversity increasing significantly upon the “soil → root” transition. The “moving window” approach was used to reveal the sites of the nodA gene in which polymorphism, including that associated with increased frequency of non-synonymous substitution frequency, increased sharply upon transition from soil into root nodules. PCR analysis of the IGS genotypes of individual strains revealed insignificant changes in rhizobial diversity upon transition from soil into root nodules. These results indicate that acceleration of rhizobial evolution in the course of symbiosis may be associated with development of highly polymorphic virulent subpopulations subjected to directional selection in the “plant-soil” system.     

Improvement of biological nitrogen fixation in Egyptian winter legumes through better management of Rhizobium

The diversity of rhizobia nodulating common bean ( Phaseolus vulgaris), berseem clover (Trifolium alexanderinum) and lentil (Lens culinaris) was assessed using several characterization techniques, including nitrogen fixation efficiency, intrinsic antibiotic-resistance patterns (IAR), plasmid profiles, serological markers and rep-PCR fingerprinting. Wide diversity among indigenous rhizobial populations of the isolates from lentil, bean and clover was found. Strikingly, a large percentage of the indigenous rhizobial population was extremely poor at fixing nitrogen. This emphasizes the need to increase the balance of highly efficient strains within the rhizobial population. Use of high-quality inocula strains that survive and compete with other less-desired and less-efficient N2-fixing rhizobia represents the best approach to increase biological nitrogen fixation of the target legume. In field-grown lentils, the inoculant strains were not able to outcompete the indigenous rhizobia and the native lentil rhizobia occupied 76–88% of the total nodules formed on inoculated plants. Nitrogen fixation by lentils, estimated using the ¹⁵N isotope dilution technique, ranged between 127 to 139 kg ha-1 in both inoculated and un-inoculated plants. With berseem clover, the inoculant strains were highly competitive against indigenous rhizobia and occupied 52–79% of all nodules. Inoculation with selected inocula improved N2 fixation by clover from 162 to 205 kg ha-1 in the three cuts as compared with 118 kg ha-1 in the un-inoculated treatment. The results also indicated the potential for improvement of N2 fixation by beans through the application of efficient N2-fixing rhizobia.     

Using amplicon sequencing of rpoB for identification of inoculant rhizobia from peanut nodules

To improve the nitrogen fixation, legume crops are often inoculated with selected effective rhizobia. However, there is large variation in how well the inoculant strains compete with the indigenous microflora in soil. To assess the success of the inoculant, it is necessary to distinguish it from other, closely related strains. Methods used until now have generally been based either on fingerprinting methods or on the use of reporter genes. Nevertheless, these methods have their shortcomings, either because they do not provide sufficiently specific information on the identity of the inoculant strain, or because they use genetically modified organisms that need prior authorization to be applied in the field or other uncontained environments. Another possibility is to target a gene that is naturally present in the bacterial genomes. Here we have developed a method that is based on amplicon sequencing of the bacterial housekeeping gene rpoB, encoding the beta-subunit of the RNA polymerase, which has been proposed as an alternative to the 16S rRNA gene to study the diversity of rhizobial populations in soils. We evaluated the method under laboratory and field conditions. Peanut seeds were inoculated with various Bradyrhizobium strains. After nodule development, DNA was extracted from selected nodules and the nodulating rhizobia were analysed by amplicon sequencing of the rpoB gene. The analyses of the sequence data showed that the method reliably identified bradyrhizobial strains in nodules, at least at the species level, and could be used to assess the competitiveness of the inoculant compared to other bradyrhizobia.     

Auxin distribution in Lotus japonicus during root nodule development

For this work, Lotus japonicus transgenic plants were constructed expressing a fusion reporter gene consisting of the genes beta-glucuronidase (gus) and green fluorescent protein (gfp) under control of the soybean auxin-responsive promoter GH3. These plants expressed GUS and GFP in the vascular bundle of shoots, roots and leafs. Root sections showed that in mature parts of the roots GUS is mainly expressed in phloem and vascular parenchyma of the vascular cylinder. By detecting GUS activity, we describe the auxin distribution pattern in the root of the determinate nodulating legume L. japonicus during the development of nodulation and also after inoculation with purified Nod factors, N-naphthylphthalamic acid (NPA) and indoleacetic acid (IAA). Differently than white clover, which forms indeterminate nodules, L. japonicus presented a strong GUS activity at the dividing outer cortical cells during the first nodule cell divisions. This suggests different auxin distribution pattern between the determinate and indeterminate nodulating legumes that may be responsible of the differences in nodule development between these groups. By measuring of the GFP fluorescence expressed 21 days after treatment with Nod factors or bacteria we were able to quantify the differences in GH3 expression levels in single living roots. In order to correlate these data with auxin transport capacity we measured the auxin transport levels by a previously described radioactive method. At 48 h after inoculation with Nod factors, auxin transport showed to be increased in the middle root segment. The results obtained indicate that L. japonicus transformed lines expressing the GFP and GUS reporters under the control of the GH3 promoter are suitable for the study of auxin distribution in this legume.     

Competition between inoculum and native rhizobia for nodulation of cowpea (Vigna unguiculata (L) Walp): Use of a dark-nodule strain

Summary Cowpea rhizobia strains were examined with indigenous populations in nodulating cowpea (Vigna unguiculata (L) Walp) cv. Laura B. strain IRC256 formed dark nodules on cowpea, and were used as the standard against orthodox pink-nodule strains in evaluating nodulating competitiveness. The dark nodule phenotype and intrinsic antibiotic resistance pattern were used to identify the strains in the nodules. Our results showed the usefulness of the dark-nodule strain in evaluating nodulating competitiveness of cowpea rhizobia in soils where dark-nodule strains were not indigenous.     

The competition between Rhizobium leguminosarum bv. viciae strains progresses until late stages of symbiosis

The competition potential of 14 Rhizobium leguminosarum bv. viciae isolates originating from nodules of Pisum sativum was estimated. Genotypic analyses of the isolates revealed a high level of chromosomal and plasmid content diversity. The isolates tagged with a plasmid-bearing constitutively expressed gusA gene were used to inoculate vetch (Vicia villosa) in competition experiments carried out under laboratory conditions. Soil extract containing autochthonous rhizobial population was used as competitor for gus-tagged strains, and the competition was studied by: (i) estimation of Gus⁺ root nodules on whole root systems, (ii) the pattern of individual nodule colonization by Gus⁺/Gus^? rhizobia, and (iii) the number of Gus⁺/Gus^? bacteria recovered from individual nodules. Several patterns of nodule colonization by Gus⁺/Gus^? bacteria were found. Some nodules identified as Gus⁺ contained gus-tagged bacteria only in the young and saprophytic zones, while the symbiotic zone was occupied by unmarked soil rhizobia. In other Gus⁺ nodules, despite the visible colonization of the entire nodule by gus-marked bacteria, a high number of Gus^? soil-derived rhizobia were recovered. The results suggest that rhizobial strains compete with each other also in the late stage of nodule development. Therefore, they may use different strategies to reach the late saprophytic zone of the nodule, which serves as an optimal environment for massive proliferation.     

Nonlegumes, Legumes, and Root Nodules Harbor Different Arbuscular Mycorrhizal Fungal Communities

Legumes are an important plant functional group since they can form a tripartite symbiosis with nitrogen-fixing Rhizobium bacteria and phosphorus-acquiring arbuscular mycorrhizal fungi (AMF). However, not much is known about AMF community composition in legumes and their root nodules. In this study, we analyzed the AMF community composition in the roots of three nonlegumes and in the roots and root nodules of three legumes growing in a natural dune grassland. We amplified a portion of the small-subunit ribosomal DNA and analyzed it by using restriction fragment length polymorphism and direct sequencing. We found differences in AMF communities between legumes and nonlegumes and between legume roots and root nodules. Different plant species also contained different AMF communities, with different AMF diversity. One AMF sequence type was much more abundant in legumes than in nonlegumes (39 and 13%, respectively). Root nodules contained characteristic AMF communities that were different from those in legume roots, even though the communities were similar in nodules from different legume species. One AMF sequence type was found almost exclusively in root nodules. Legumes and root nodules have relatively high nitrogen concentrations and high phosphorus demands. Accordingly, the presence of legume- and nodule-related AMF can be explained by the specific nutritional requirements of legumes or by host-specific interactions among legumes, root nodules, and AMF. In summary, we found that AMF communities vary between plant functional groups (legumes and nonlegumes), between plant species, and between parts of a root system (roots and root nodules).