Competition between rhizobia under different environmental conditions affects the nodulation of a legume

Mutualistic symbiosis and nitrogen fixation of legume rhizobia play a key role in ecological environments. Although many different rhizobial species can form nodules with a specific legume, there is often a dominant microsymbiont, which has the highest nodule occupancy rates, and they are often known as the “most favorable rhizobia”. Shifts in the most favorable rhizobia for a legume in different geographical regions or soil types are not well understood. Therefore, in order to explore the shift model, an experiment was designed using successive inoculations of rhizobia on one legume. The plants were grown in either sterile vermiculite or a sandy soil. Results showed that, depending on the environment, a legume could select its preferential rhizobial partner in order to establish symbiosis. For perennial legumes, nodulation is a continuous and sequential process. In this study, when the most favorable rhizobial strain was available to infect the plant first, it was dominant in the nodules, regardless of the existence of other rhizobial strains in the rhizosphere. Other rhizobial strains had an opportunity to establish symbiosis with the plant when the most favorable rhizobial strain was not present in the rhizosphere. Nodule occupancy rates of the most favorable rhizobial strain depended on the competitiveness of other rhizobial strains in the rhizosphere and the environmental adaptability of the favorable rhizobial strain (in this case, to mild vermiculite or hostile sandy soil). To produce high nodulation and efficient nitrogen fixation, the most favorable rhizobial strain should be selected and inoculated into the rhizosphere of legume plants under optimum environmental conditions.     

根瘤菌生物地理学的研究进展

根瘤菌是一类革兰氏阴性菌,能与特定的宿主植物共生形成根瘤,将空气中的分子态氮转变为植物可以利用的氨态氮。研究根瘤菌的生物地理分布格局及形成机制,不仅具有理论上的意义,还对根瘤菌接种剂的选择具有指导意义。目前,随着分子生物学技术的发展,以及根瘤菌多样性研究数据的积累,根瘤菌生物地理学取得了较大的进展。本文综述了根瘤菌生物地理学的研究方法及现状,并对今后的重点研究方向作了展望。     

Legume growth-promoting rhizobia: An overview on the Mesorhizobium genus

The need for sustainable agricultural practices is revitalizing the interest in biological nitrogen fixation and rhizobia-legumes symbioses, particularly those involving economically important legume crops in terms of food and forage. The genus Mesorhizobium includes species with high geographical dispersion and able to nodulate a wide variety of legumes, including important crop species, like chickpea or biserrula. Some cases of legume-mesorhizobia inoculant introduction represent exceptional opportunities to study the rhizobia genomes evolution and the evolutionary relationships among species. Complete genome sequences revealed that mesorhizobia typically harbour chromosomal symbiosis islands. The phylogenies of symbiosis genes, such as nodC, are not congruent with the phylogenies based on core genes, reflecting rhizobial host range, rather than species affiliation. This agrees with studies showing that Mesorhizobium species are able to exchange symbiosis genes through lateral transfer of chromosomal symbiosis islands, thus acquiring the ability to nodulate new hosts. Phylogenetic analyses of the Mesorhizobium genus based on core and accessory genes reveal complex evolutionary relationships and a high genomic plasticity, rendering the Mesorhizobium genus as a good model to investigate rhizobia genome evolution and adaptation to different host plants. Further investigation of symbiosis genes as well as stress response genes will certainly contribute to understand mesorhizobia-legume symbiosis and to develop more effective mesorhizobia inoculants.     

Genetic resources of nodule bacteria

Nodule bacteria (rhizobia) form highly specific symbiosis with leguminous plants. The efficiency of accumulation of biological nitrogen depends on molecular-genetic interaction between the host plant and rhizobia. Genetic characteristics of microsymbiotic strains are crucial in developing highly productive and stress-resistant symbiotic pairs: rhizobium strain-host plant cultivar (species). The present review considers the issue of studying genetic resources of nodule bacteria to identify genes and their blocks, responsible for the ability of rhizobia to form highly effective symbiosis in various agroecological conditions. The main approaches to investigate of intraspecific and interspecific genetic and genomic diversity of nodule bacteria are considered, from MLEE analysis to the recent methods of genomic DNA analysis using biochips. The data are presented showing that gene centers of host plants are centers of genetic diversification of nodule bacteria, because the intraspecific polymorphism of genetic markers of the core and the accessory rhizobial genomes is extremely high in them. Genotypic features of trapped and nodule subpopulations of alfalfa nodule bacteria are discussed. A survey of literature showed that the genomes of natural strains in alfalfa gene centers exhibit significant differences in genes involved in control of metabolism, replication, recombination, and the formation of defense response (hsd genes). Natural populations of rhizobia are regarded as a huge gene pool serving as a source of evolutionary innovations.     

Influence of different Sinorhizobium meliloti inocula on abundance of genes involved in nitrogen transformations in the rhizosphere of alfalfa (Medicago sativa L.)

Inoculation of leguminous seeds with selected rhizobial strains is practised in agriculture to ameliorate the plant yield by enhanced root nodulation and nitrogen uptake of the plant. However, effective symbiosis between legumes and rhizobia does not only depend on the capacity of nitrogen fixation but also on the entire nitrogen turnover in the rhizosphere. We investigated the influence of seed inoculation with two indigenous Sinorhizobium meliloti strains exhibiting different efficiency concerning plant growth promotion on nitrogen turnover processes in the rhizosphere during the growth of alfalfa. Quantification of six target genes (bacterial amoA, nirK, nirS, nosZ, nifH and archaeal amoA) within the nitrogen cycle was performed in rhizosphere samples before nodule formation, at bud development and at the late flowering stage. The results clearly demonstrated that effectiveness of rhizobial inocula is related to abundance of nifH genes in the late flowering phase of alfalfa. Moreover, other genes involved in nitrogen turnover had been affected by the inocula, e.g. higher numbers of amoA copies were observed during flowering when the more effective strain had been inoculated. However, the respective gene abundances differed overall to a greater extent between the three plant development stages than between the inoculation variants.     

Pseudomonas fluorescens CHA0 can kill subterranean termite Odontotermes obesus by inhibiting cytochrome c oxidase of the termite respiratory chain

In the rhizosphere and their interaction with plants rhizobia encounter many different plant compounds, including phytohormones like auxins. Moreover, some rhizobial strains are capable of producing the auxin, indole-3-acetic acid (IAA). However, the role of IAA for the bacterial partner in the legume– Rhizobium symbiosis is not known. To identify the effect of IAA on rhizobial gene expression, a transposon (mTn 5gusA - oriV ) mutant library of Rhizobium etli , enriched for mutants that show differential gene expression under microaerobiosis and/or addition of nodule extracts as compared with control conditions, was screened for altered gene expression upon IAA addition. Four genes were found to be regulated by IAA. These genes appear to be involved in plant signal processing, motility or attachment to plant roots, clearly demonstrating a distinct role for IAA in legume– Rhizobium interactions.     

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.     

Symbiotic soil microorganisms as players in aboveground plant–herbivore interactions – the role of rhizobia

Rhizobia play a key role for performance of leguminous plants and ecosystem productivity. However, no studies to date have investigated the importance of the rhizobial symbiosis for legume–herbivore interactions. The additional nitrogen provided by the rhizobia improves the nutritional quality of plants, but may also be used for the synthesis of defence compounds. We performed greenhouse experiments with nodulating and non-nodulating, as well as cyanogenic and acyanogenic strains of Trifolium repen s to study the effects of rhizobia Rhizobium leguminosarum on plant growth and the performance of the chewing herbivore Spodoptera littoralis and the phloem-sucking aphid Myzus persicae . We demonstrate that for nodulating strains of T. repens rhizobia increased plant growth and the performance of Spodoptera littoralis . However, this positive effect of rhizobia on the caterpillars did not occur in a cyanogenic clover strain. Reproduction of the phloem-sucking aphid Myzus persicae was inconsistently affected by rhizobia. Our study provides evidence that the additional nitrogen provided by the rhizobia may be used for the production of nitrogen-based defence compounds, thereby counteracting positive effects on the performance of chewing herbivores. The symbiosis with rhizobia is therefore an important driver of legume–herbivore interactions.     

Interactive effects of synthetic nitrogen fertilizer and composted manure on ammonia volatilization from soils

The mutualism between legumes and nitrogen-fixing soil bacteria (rhizobia) is a key feature of many ecological and agricultural systems, yet little is known about how this relationship affects aboveground interactions between plants and herbivores. We investigated the effects of the rhizobia mutualism on the abundance of a specialized legume herbivore on soybean plants. In a field experiment, soybean aphid (Aphis glycines) abundances were measured on plants (Glycine max) that were either (1) treated with a commercial rhizobial inoculant, (2) associating solely with naturally occurring rhizobia, or (3) given nitrogen fertilizer. Plants associating with naturally occurring rhizobia strains exhibited lower aphid population densities compared to those inoculated with a commercial rhizobial preparation or given nitrogen fertilizer. Genetic analyses of rhizobia isolates cultured from field plants revealed that the commercial rhizobia strains were phylogenetically distinct from naturally occurring strains. Plant size, leaf nitrogen concentration, and nodulation density were similar among rhizobia-associated treatments and did not explain the observed differences in aphid abundance. Our results demonstrate that plant–rhizobia interactions influence plant resistance to insect herbivores and that some rhizobia strains confer greater resistance to their mutualist partners than do others.     

Immunity of a leguminous plant infected by nodular bacteria <Emphasis Type="Italic">Rhizobium</Emphasis> spp. F.: Review

Recent studies of the immune system of leguminous plants infected with nodular bacteria (rhizobia) are summarized. The possibility of blocking the invasion of rhizobia into plant organs not affected by the primary infection is discussed. The concept of local and systemic resistance of the leguminous plant to rhizobial infection is introduced. The Nod factors of rhizobia are considered, as well as the plant receptors that interact with these factors upon the formation of symbiosis of the plant and bacteria. The role of bacterial surface exopolysaccharides in the suppression of the protective system of the plants is discussed. The innate immunity of leguminous plant cells is assumed to affect the formation and functioning of the symbiosis of the plant and the bacteria.     

Evolutionary Genetics of Nodule Bacteria: Molecular and Population Aspects

The molecular analysis of the genetic systems controlling the main stages of nodule bacteria (rhizobia) interaction with a legume host (signaling at early stages and symbiotic nitrogen fixation) has shown that the widespread recombination of genetic material in free-living ancestors of rhizobia was an important factor in the evolution of these systems. These recombinations could be conditioned by a high content of repeated DNA sequences and the IS elements in the rhizobial genome. A high recombination activity of rhizobia is manifested in the panmictic structure of their populations, which is associated with frequency-dependent selection favoring rare recombinants. This selection is realized through the competition of virulent strains for the nodule formation and can be controlled by the genes whose expression depends on population density (via the quorum sensing mechanism). A high degree of panmixia in rhizobial populations is associated with their ecotypic polymorphism, manifested as the coexistence of symbiotic and nonsymbiotic strains. This type of polymorphism is caused by individual selection during the periodic changes of ecological niches (soil–plant host) in the rhizobia life cycle. The rhizobia–plant interaction stimulates selection in bacterial populations, which results in the increased levels of their heterogeneity and panmixia. The combination of individual and frequency-dependent selection types resulted in the high rates of symbiosis evolution and polyphyletic origin of diverse rhizobial species.     

Recent development and new insight of diversification and symbiosis specificity of legume rhizobia: mechanism and application

Currently, symbiotic rhizobia (sl., rhizobium) refer to the soil bacteria in α- and β-Proteobacteria that can induce root and/or stem nodules on some legumes and a few of nonlegumes. In the nodules, rhizobia convert the inert dinitrogen gas (N₂) into ammonia (NH₃) and supply them as nitrogen nutrient to the host plant. In general, this symbiotic association presents specificity between rhizobial and leguminous species, and most of the rhizobia use lipochitooligosaccharides, so called Nod factor (NF), for cooperating with their host plant to initiate the formation of nodule primordium and to inhibit the plant immunity. Besides NF, effectors secreted by type III secretion system (T3SS), exopolysaccharides and many microbe-associated molecular patterns in the rhizobia also play important roles in nodulation and immunity response between rhizobia and legumes. However, the promiscuous hosts like Glycine max and Sophora flavescens can nodulate with various rhizobial species harbouring diverse symbiosis genes in different soils, meaning that the nodulation specificity/efficiency might be mainly determined by the host plants and regulated by the soil conditions in a certain cases. Based on previous studies on rhizobial application, we propose a ‘1+n−N’ model to promote the function of symbiotic nitrogen fixation (SNF) in agricultural practice, where ‘1’ refers to appreciate rhizobium; ‘+n’ means the addition of multiple trace elements and PGPR bacteria; and ‘−N’ implies the reduction of chemical nitrogen fertilizer. Finally, open questions in the SNF field are raised to future think deeply and researches.     

Nod genes and Nod signals and the evolution of the Rhizobium legume symbiosis.

The establishment of the nitrogen-fixing symbiosis between rhizobia and legumes requires an exchange of signals between the two partners. In response to flavonoids excreted by the host plant, rhizobia synthesize Nod factors (NFs) which elicit, at very low concentrations and in a specific manner, various symbiotic responses on the roots of the legume hosts. NFs from several rhizobial species have been characterized. They all are lipo-chitooligosaccharides, consisting of a backbone of generally four or five glucosamine residues N-acylated at the non-reducing end, and carrying various O-substituents. The N-acyl chain and the other substituents are important determinants of the rhizobial host specificity. A number of nodulation genes which specify the synthesis of NFs have been identified. All rhizobia, in spite of their diversity, possess conserved nodABC genes responsible for the synthesis of the N-acylated oligosaccharide core of NFs, which suggests that these genes are of a monophyletic origin. Other genes, the host specific nod genes, specify the substitutions of NFs. The central role of NFs and nod genes in the Rhizobium-legume symbiosis suggests that these factors could be used as molecular markers to study the evolution of this symbiosis. We have studied a number of NFs which are N-acylated by alpha,beta-unsaturated fatty acids. We found that the ability to synthesize such NFs does not correlate with taxonomic position of the rhizobia. However, all rhizobia that produce NFs such nodulate plants belonging to related tribes of legumes, the Trifolieae, Vicieae, and Galegeae, all of them being members of the so-called galegoid group. This suggests that the ability to recognize the NFs with alpha-beta-unsaturated fatty acids is limited to this group of legumes, and thus might have appeared only once in the course of legume evolution, in the galegoid phylum.     

Mineral constraints to nitrogen fixation

Mineral nturient defiencies are a major constraint limiting legume nitrogen fixation and yield. In this review general techniques for assessing nutrient involvement in symbiotic nitrogen fixation are described and specific methods are outlined for determining which developmental phase of the symbiosis is most sensitive to nutrient deficiency. The mineral nutrition of the Rhizobium component of the symbiosis is considered both as the free living organism in the soil and as bacteroids in root nodules. Rhizobial growth and survival in soils is not usually limited by nutrient availability. Multiplication of rhizobia in the legume rhizosphere is limited by low Ca availability. Nodule initiation is affected by severe Co deficiency through effects on rhizobia. Nodule development is limited by severe B deficiency via an effect on plant cell growth. Fe deficiency limits nodule development by affecting rhizobia and strains of rhizobia differ widely in their ability to acquire sufficient Fe for their symbiotic development. Nodule function requires more Mo than does the host plant, and in some symbioses nitrogen fixation may be specifically limited by low availability of Ca, Co, Cu and Fe. The importance of the peribacteriod membrane in determining nutrient availability to bacteroids is considered. It is concluded that the whole legume-Rhizobium symbiosis should be considered when improving legume growth and yield under nutrient stress conditions. Differences among rhizobial strains in their ability to obtain mineral nutrients from their environment may be agronomically important.     

Evolutionary history shapes patterns of mutualistic benefit in Acacia–rhizobial interactions

The ecological and evolutionary factors that drive the emergence and maintenance of variation in mutualistic benefit (i.e., the benefits provided by one partner to another) in mutualistic symbioses are not well understood. In this study, we evaluated the role that host and symbiont phylogeny might play in determining patterns of mutualistic benefit for interactions among nine species of Acacia and 31 strains of nitrogen‐fixing rhizobial bacteria. Using phylogenetic comparative methods we compared patterns of variation in mutualistic benefit (host response to inoculation) to rhizobial phylogenies constructed from housekeeping and symbiosis genes; and a multigene host phylogeny. We found widespread genotype‐by‐genotype variation in patterns of plant growth. A relatively large component of this variation (21–28%) was strongly influenced by the interacting evolutionary histories of both partners, such that phylogenetically similar host species had similar growth responses when inoculated with phylogenetically similar rhizobia. We also found a relatively large nonphylogenetic effect for the average mutualistic benefit provided by rhizobia to plants, such that phylogenetic relatedness did not predict the overall benefit provided by rhizobia across all hosts. We conclude that phylogenetic relatedness should frequently predict patterns of mutualistic benefit in acacia‐rhizobial mutualistic interactions; but that some mutualistic traits also evolve independently of the phylogenies.     

Agro-industrial waste materials and wastewater sludge for rhizobial inoculant production: a review

Inoculating legumes with commercial rhizobial inoculants is a common agriculture practice. Generally, inoculants are sold in liquid or in solid forms (mixed with carrier). The production of inoculants involves a step in which a high number of cells are produced, followed by the product formulation. This process is largely governed by the cost related to the medium used for rhizobial growth and by the availability of a carrier source (peat) for production of solid inoculant. Some industrial and agricultural by-products (e.g. cheese whey, malt sprouts) contain growth factors such as nitrogen and carbon, which can support growth of rhizobia. Other agro-industrial wastes (e.g. plant compost, filtermud, fly-ash) can be used as a carrier for rhizobial inoculant. More recently, wastewater sludge, a worldwide recyclable waste, has shown good potential for inoculant production as a growth medium and as a carrier (dehydrated sludge). Sludge usually contains nutrient elements at concentrations sufficient to sustain rhizobial growth and heavy metals are usually below the recommended level. In some cases, growth conditions can be optimized by a sludge pre-treatment or by the addition of nutrients. Inoculants produced in wastewater sludge are efficient for nodulation and nitrogen fixation with legumes as compared to standard inoculants. This new approach described in this review offers a safe environmental alternative for both waste treatment/disposal and inoculant production.     

Two effects of combined nitrogen on the adhesion of Rhizobium etli to bean roots

Combined forms of nitrogen negatively influence rhizobia-legume symbiosis. The effects of combined nitrogen are known for nodulation and dinitrogen (N₂) fixation, but little is known about the effect on preinfection events. Here, we studied the effects of combined nitrogen on the adhesion of Rhizobium etli to common bean (Phaseolus vulgaris L.) roots. When potassium nitrate (KNO₃) or sodium glutamate was added to an incubation mixture of rhizobia and plants that were previously grown in nitrogen-free solution, rhizobial adhesion to roots was stimulated. However, the rhizobial adhesion to bean roots that were previously grown with 10 mM KNO₃ was reduced by half. A fraction of the bean root exudates, which is thermolabile and has molecular mass larger than 12 kDa stimulated rhizobial adhesion, but this stimulatory activity was lost in root exudates obtained with 10 mM KNO₃. Thus, the inhibition of symbiosis in response to combined nitrogen may be controlled by the plant at the preinfection stage as well.     

Residual Impact of the Biocontrol Inoculant Pseudomonas fluorescens F113 on the Resident Population of Rhizobia Nodulating a Red Clover Rotation Crop

A field trial was previously conducted in which sugarbeet seeds were either untreated, inoculated with the biocontrol strain Pseudomonas fluorescens F113Rif, or treated with chemical fungicides. Following harvest of sugarbeet, the field site was sown with uninoculated red clover. The aim of this study was to assess the residual impact of the microbial inoculant (and the fungicide treatment) on the diversity of resident rhizobia nodulating the red clover rotation crop. The percentage of nodules yielding rhizobial isolates after surface disinfection was 67% in the control and 70% in the P. fluorescens F113Rif treatment, but only 23% in the chemical treatment. Isolates were characterized by RAPD analysis. The main RAPD cluster (arbitrarily defined at 70% similarity) was prevalent in all three treatments. In addition, the distribution of RAPD clusters followed a log series model, regardless of the treatment applied, indicating that neither the microbial inoculant nor the fungicide treatment had caused a strong perturbation of the rhizobial population. When the P. fluorescens F113Rif and control treatments were compared using diversity indices, however, it appeared that the genetic diversity of rhizobia was significantly less in the inoculated treatment. The percentage of rhizobia sensitive to 2,4-diacetylphloroglucinol (Phl; the antimicrobial metabolite produced by P. fluorescens F113Rif) fluctuated according to field site heterogeneity, and treatments had no effect on this percentage. Yet, the proportion of Phl-sensitive isolates in the main RAPD cluster was lower in the P. fluorescens F113Rif treatment compared with the control, raising the possibility that the residual impact of the inoculant could have been partly mediated by production of Phl. This impact on the rhizobial population took place without affecting the functioning of the Rhizobium–clover symbiosis.     

Associations Among Rhizobial Chromosomal Background, nod Genes, and Host Plants Based on the Analysis of Symbiosis of Indigenous Rhizobia and Wild Legumes Native to Xinjiang

The associations among rhizobia chromosomal background, nodulation genes, legume plants, and geographical regions are very attractive but still unclear. To address this question, we analyzed the interactions among rhizobia rDNA genotypes, nodC genotypes, legume genera, as well as geographical regions in the present study. Complex relationships were observed among them, which may be the genuine nature of their associations. The statistical analyses indicate that legume plant is the key factor shaping both rhizobia genetic and symbiotic diversity. In the most cases of our results, the nodC lineages are clearly associated with rhizobial genomic species, demonstrating that nodulation genes have co-evolved with chromosomal background, though the lateral transfer of nodulation genes occurred in some cases in a minority. Our results also support the hypothesis that the endemic rhizobial populations to a certain geographical area prefer to have a wide spectrum of hosts, which might be an important event for the success of both legumes and rhizobia in an isolated region.