Failure to fix nitrogen by non-reproductive symbiotic rhizobia triggers host sanctions that reduce fitness of their reproductive clonemates

The legume-rhizobia symbiosis is a classical mutualism where fixed carbon and nitrogen are exchanged between the species. Nonetheless, the plant carbon that fuels nitrogen (N(2)) fixation could be diverted to rhizobial reproduction by 'cheaters'--rhizobial strains that fix less N(2) but potentially gain the benefit of fixation by other rhizobia. Host sanctions can decrease the relative fitness of less-beneficial reproductive bacteroids and prevent cheaters from breaking down the mutualism. However, in certain legume species, only undifferentiated rhizobia reproduce, while only terminally differentiated rhizobial bacteroids fix nitrogen. Sanctions were, therefore, tested in two legume species that host non-reproductive bacteroids. We demonstrate that even legume species that host non-reproductive bacteroids, specifically pea and alfalfa, can severely sanction undifferentiated rhizobia when bacteroids within the same nodule fail to fix N(2). Hence, host sanctions by a diverse set of legumes play a role in maintaining N(2) fixation.     

Life histories of symbiotic rhizobia and mycorrhizal fungi

Research on life history strategies of microbial symbionts is key to understanding the evolution of cooperation with hosts, but also their survival between hosts. Rhizobia are soil bacteria known for fixing nitrogen inside legume root nodules. Arbuscular mycorrhizal (AM) fungi are ubiquitous root symbionts that provide plants with nutrients and other benefits. Both kinds of symbionts employ strategies to reproduce during symbiosis using host resources; to repopulate the soil; to survive in the soil between hosts; and to find and infect new hosts. Here we focus on the fitness of the microbial symbionts and how interactions at each of these stages has shaped microbial life-history strategies. During symbiosis, microbial fitness could be increased by diverting more resources to individual reproduction, but that may trigger fitness-reducing host sanctions. To survive in the soil, symbionts employ sophisticated strategies, such as persister formation for rhizobia and reversal of spore germination by mycorrhizae. Interactions among symbionts, from rhizobial quorum sensing to fusion of genetically distinct fungal hyphae, increase adaptive plasticity. The evolutionary implications of these interactions and of microbial strategies to repopulate and survive in the soil are largely unexplored.     

Genetic markers for search of rhizobia based on symbiotic genes

The possible application of rhizobial symbiotic genes as markers for the search and primary identification of rhizobia from temperate-zone legumes was studied. It was shown that conservative sym genes nifH and nifD could be used as markers for rapid search of rhizobia among the analyzed isolates, while more variable genes nifK and nodC could be used for their primary identification. Efficiency of the proposed method was shown in analysis of bacterial isolates obtained from Onobrychis arenaria and Astragalus cicer root nodules.     

Adaptation of chickpea to desiccation stress is enhanced by symbiotic rhizobia

This study examined the influence of three inoculant strains of Bradyrhizobium japonicum (Thal-8, Tal 620, Dulawala) on the ability of chickpea (Cicer arietinum (L.) to adapt to drought-stress. Strain Thal-8 was most effective in the root-nodule symbiosis and also partially alleviated decreased growth and yield imposed by drought stress. Strain Thal-8, in pure culture, also produced higher amounts of gibberellic acid (GA) and indole-3-acetic acid (IAA) and lower amounts of abscisic acid (ABA) than the other two test strains. Thal-8 increased the root biomass, GA and IAA contents of leaves of chickpea plants, including ICC 4948NN, a non-nodulating line. These results are consistent with the hypothesis that GA and IAA is produced by the Thal-8 strain and/or elevates levels of these phytohormones in chickpeas. This contributes to its high performance as a nitrogen-fixing microsymbiont. The growth-promoting response evoked by different strains of Bradyrhizobium correlated with higher ratios of GA and IAA relative to ABA phytohormones in the plants.     

Diversity in symbiotic specificity of cowpea rhizobia indigenous to Zimbabwean soils

Tropical cowpea rhizobia are often presumed to be generally promiscuous but poor N fixers. This study was conducted to evaluate symbiotic interactions of 59 indigenous rhizobia isolates (49 of them from cowpea (Vigna unguiculata)), with up to 13 other (mostly tropical) legume species. Host ranges averaged 2.4 and 2.3 legume species each for fast- and slow-growing isolates respectively compared to 4.3 for slow-growing reference cowpea strains. An average of 22% and 19% of fast- and slow-growing cowpea isolates respectively were effective on each of 12 legume species tested. We conclude that the indigenous cowpea rhizobia studied have relatively narrow host ranges. The ready nodulation of different legumes in tropical soils appears due to the diversity of indigenous symbiotic genotypes, each consisting of subgroups compatible with a limited number of legume species.     

Polyphasic taxonomy of symbiotic rhizobia from wild leguminous plants growing in Egypt

About 20 strains of rhizobia from wild legumes were characterized based on numerical analysis of phenotypic characteristics, nodulating ability, fatty acid methyl esters (FAME) and SDS-PAGE profiles of whole cell proteins. FAME analysis revealed that palmitic (16:0), stearic (18:0) and arachidonic (20:0) were detected in most of wild-legume rhizobia, the latter being uncommon in fatty acid profiles of Rhizobium and Sinorhizobium. Numerical analysis of FAME classified strains of wild-legume rhizobia into 9 clusters and one heterogeneous group. There was both agreement and disagreement with the clustering data based on phenotypic analysis and FAME analysis. Four strains were grouped together in the same cluster based on both methods. However, 4 another strains, which were placed in one cluster of phenotypic analysis, were distributed in several clusters after FAME analysis. SDS-PAGE of whole-cell proteins revealed that the rhizobial strains exhibited protein profiles with peptide bands ranging from 5-19 band per profile and showed molar mass of 110-183 kDa. As in the case of FAME analysis, numerical analysis of protein bands was compared with clustering of phenotypic analysis. Agreement of the two methods was obvious when clustering some strains but conflicted in the classification of some other strains. However, integration of the three methods could be the basis of a polyphasic taxonomy. The twenty strains of wild-legume rhizobia were finally classified as follows: 12 strains related to Rhizobium leguminosarum, 5 strains related to Sinorhizobium meliloti and 3 strains to Rhizobium spp. Rhizobia nodulating wild herb legumes are among indigenous strains nodulating crop legumes in cultivated as well as noncultivated lands.     

Quorum-sensing regulation in rhizobia and its role in symbiotic interactions with legumes

Legume-nodulating bacteria (rhizobia) usually produce N-acyl homoserine lactones, which regulate the induction of gene expression in a quorum-sensing (or population-density)-dependent manner. There is significant diversity in the types of quorum-sensing regulatory systems that are present in different rhizobia and no two independent isolates worked on in detail have the same complement of quorum-sensing genes. The genes regulated by quorum sensing appear to be rather diverse and many are associated with adaptive aspects of physiology that are probably important in the rhizosphere. It is evident that some aspects of rhizobial physiology related to the interaction between rhizobia and legumes are influenced by quorum sensing. However, it also appears that the legumes play an active role, both in terms of interfering with the rhizobial quorum-sensing systems and responding to the signalling molecules made by the bacteria. In this article, we review the diversity of quorum-sensing regulation in rhizobia and the potential role of legumes in influencing and responding to this signalling system.     

Expression of symbiotic genes of Rhizobium japonicum USDA 191 in other rhizobia.

A 200-megadalton plasmid was mobilized from Rhizobium japonicum USDA 191 to other Rhizobium strains either that cannot nodulate soybeans or that form Fix- nodules on certain cultivars. The symbiotic properties of the transconjugants indicate that both soybean specificity for nodulation and cultivar specificity for nitrogen fixation are plasmid encoded.     

Genetic diversity and symbiotic effectiveness of Phaseolus vulgaris-nodulating rhizobia in Kenya

Phaseolus vulgaris (common bean) was introduced to Kenya several centuries ago but the rhizobia that nodulate it in the country remain poorly characterised. To address this gap in knowledge, 178 isolates recovered from the root nodules of P. vulgaris cultivated in Kenya were genotyped stepwise by the analysis of genomic DNA fingerprints, PCR-RFLP and 16S rRNA, atpD, recA and nodC gene sequences. Results indicated that P. vulgaris in Kenya is nodulated by at least six Rhizobium genospecies, with most of the isolates belonging to Rhizobium phaseoli and a possibly novel Rhizobium species. Infrequently, isolates belonged to Rhizobium paranaense, Rhizobium leucaenae, Rhizobium sophoriradicis and Rhizobium aegyptiacum. Despite considerable core-gene heterogeneity among the isolates, only four nodC gene alleles were observed indicating conservation within this gene. Testing of the capacity of the isolates to fix nitrogen (N₂) in symbiosis with P. vulgaris revealed wide variations in effectiveness, with ten isolates comparable to Rhizobium tropici CIAT 899, a commercial inoculant strain for P. vulgaris. In addition to unveiling effective native rhizobial strains with potential as inoculants in Kenya, this study demonstrated that Kenyan soils harbour diverse P. vulgaris-nodulating rhizobia, some of which formed phylogenetic clusters distinct from known lineages. The native rhizobia differed by site, suggesting that field inoculation of P. vulgaris may need to be locally optimised.     

Effects of peanut rhizobia on the growth and symbiotic performance ofArachis hypogaea under abiotic stress

The effects of saline and osmotic stress on four peanut rhizobia, plant growth and symbiotic N₂-fixation inArachis hypogaea were studied. Abiotic stress was applied by adding either 100 mM NaCl or 20 mM PEG6000. At the rhizobial level,Bradyrhizobium ATCC10317 and TAL1000 showed stronger tolerance to stress than TAL1371 and SEMIA6144. The effect of salinity on the bacterium-plant association was studied by using the variety Blanco Manfredi M68. In the absence of stresses, all the strains induced a significantly higher number of nodules on the roots, although TAL1371 and SEMIA6144 were more effective. Both stresses affected the interaction process, while TALl371 was the best partner.     

The Lotus japonicus nucleoporin GLE1 is involved in symbiotic association with rhizobia

Nucleoporins are components of the nuclear pore complexes, channels that regulate the transport of macromolecules between the nucleus and cytoplasm. The nucleoporin GLE1 (GLFG lethal1) functions in the export of messenger RNAs containing poly(A) tails from the nucleus into the cytoplasm. Here we investigated a mutant of the model legume Lotus japonicus that was defective in GLE1, which we designated Ljgle1. The growth of Ljgle1 was retarded under symbiotic association with rhizobia, and the nitrogen-fixation activities of the nodules were around one-third of those in the wild-type plant. The growth of Ljgle1 was not substantialy recovered by supplemention of combined nitrogen. Nodules formed on the Ljgle1 were smaller than those on the wild-type and colored faint pink. The numbers of infected cells of nodules on the Ljgle1 were smaller than on the wild-type plant, and the former cells remained undeveloped. Rhizobia in the cells of the Ljgle1 exhibited disordered forms, and the symbiosome membrane was closely attached to the bacterial membrane. These results indicate that GLE1 plays a distinct role in the symbiotic association between legumes and rhizobia.     

Insight into the evolutionary history of symbiotic genes of Robinia pseudoacacia rhizobia deriving from Poland and Japan

The phylogeny of symbiotic genes of Robinia pseudoacacia (black locust) rhizobia derived from Poland and Japan was studied by comparative sequence analysis of nodA, nodC, nodH, and nifH loci. In phylogenetic trees, black locust symbionts formed a branch of their own suggesting that the spread and maintenance of symbiotic genes within Robinia pseudoacacia rhizobia occurred through vertical transmission. There was 99–100% sequence similarity for nodA genes of Robinia pseudoacacia nodulators, 97–98% for nodC, and 97–100% for nodH and nifH loci. A considerable sequence conservation of sym genes shows that the symbiotic apparatus of Robinia pseudoacacia rhizobia might have evolved under strong host plant constraints. In the nodA and nodC gene phylograms, Robinia pseudoacacia rhizobia grouped with Phaseolus sp. symbionts, although they were not closely related to our isolates based on 16S rRNA genes, and with Mesorhizobium amorphae. nifH gene phylogeny of our isolates followed the evolutionary history of 16S rDNA and Robinia pseudoacacia rhizobia grouped with Mesorhizobium genus species. Nodulation assays revealed that Robinia pseudoacacia rhizobia effectively nodulated their native host and also Amorpha fruticosa and Amorpha californica resulting in a significant enhancement of plant growth. The black locust root nodules are shown to be of indeterminate type.     

Genetic diversity and symbiotic evolution of rhizobia from root nodules of Coronilla varia

Ninety symbiotic rhizobial isolates from root nodules of Coronilla varia growing in the Shaanxi province of China were characterized. Combined with the results of RFLP patterns, six genotypes were defined among the rhizobial strains and they were divided into three genomic genera. These included Mesorhizobium sp., M. alhagi, M. amorphae, M. metallidurans/M. gobiense as the dominant group (86.7%), and Rhizobium yanglingense and Agrobacterium tumefaciens as the minor groups, according to analysis of the corresponding 16S rRNA, nodC and nifH genes. Five nodC types, which mainly grouped into the Mesorhizobium genus, were obtained from all the isolates examined, implying that nodC genes probably occurred from the native habitat through lateral transfer and long-term adaptation, finally evolving toward M. alhagi. Four different nifH types, displaying obvious differences compared to those of 16S rRNA and nodC, implied that possible lateral transfer of the symbiotic genes occurred between different genera. The association between soil components and the genetic diversity of the rhizobial population demonstrated that combined genotypes were positively correlated with the pH of soil samples.     

Correction to: Diversity and symbiotic divergence of endophytic and non-endophytic rhizobia of Medicago sativa

The authors wish to clarify that the right Fund No. of National Natural Science Foundation of China (NSFC) is 31560666. The Fund No. in the original article was wrong. The authors apologise for this error.     

Formation of pyridine nucleotides under symbiotic and non-symbiotic conditions between soybean nodules and free-living rhizobia

Enzymatic regulation of pyricline nucleotide formation, under symbiotic and non-symbiotic conditions, was analyzed using soybeans (Glycine max L. cv. 'Akisengoku') and rhizobia (Bradyrhizobia japonicum strain A1017), respectively. It was found that levels of pyridine nucleotides in bacteroids in root nodules were different from those in free-living cells of rhizobia. This difference was associated with differences in activities of enzymes involved in the pathway from L-tryptophan to NAD and NADP. That is, these activities were lower in bacteroids than in free-living bacteria and lower in the nodule cytosol than in root extracts. The optimum pH for NAD synthetase in bacteroids, was 9.0. Additionally, the optimum pH for ATP-nicotinamide mononucleotide (NMN) adenyltransferase, final step enzyme in NAD formation, was estimated to be 7.6. In the bacteroid fraction, the K(m) of NAD synthetase (22 microM) was approximately 1/22 of that of ATP-NMN adenyltransferase (482 microM). Vmax values were estimated to be almost in the same order for both NAD synthetase and ATP-NMN adenyltransferase. This is the first report on the formation of pyridine nucleotides originating from L-tryptophan in bacteroids in soybean nodules and free-living bacteria.     

Different species and symbiotic genotypes of field rhizobia can nodulate Phaseolus vulgaris in Tunisian soils

A collection of 160 isolates of rhizobia nodulating Phaseolus vulgaris in three geographical regions in Tunisia was characterized by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified 16S rDNA, nifH and nodC genes. Nine groups of rhizobia were delineated: Rhizobium gallicum biovar (bv.) gallicum, Rhizobium leguminosarum bv. phaseoli and bv. viciae, Rhizobium etli bv. phaseoli, Rhizobium giardinii bv. giardinii, and four groups related to species of the genus Sinorhizobium, Sinorhizobium meliloti, Sinorhizobium medicae and Sinorhizobium fredii. The most abundant rhizobial species were R. gallicum, R. etli, and R. leguminosarum encompassing 29–20% of the isolates each. Among the isolates assigned to R. leguminosarum, two-thirds were ineffective in nitrogen fixation with P. vulgaris and harbored a symbiotic gene typical of the biovar viciae. The S. fredii-like isolates did not nodulate soybean plants but formed numerous effective nodules on P. vulgaris. Comparison of nodC gene sequences showed that their symbiotic genotype was not related to that of S. fredii, but to that of the S. fredii-like reference strain GR-06, which was isolated from a bean plant grown in a Spanish soil. An additional genotype including 16% of isolates was found to be closely related to species of the genus Agrobacterium. However, when re-examined, these isolates did not nodulate their original host.     

Measuring fitness by means of balancer chromosomes

We present the theoretical background to a new method for measuring genetic variation for total fitness in Drosophila. The method allows heterozygous effects on total fitness of whole wild-type chromosomes to be measured under normal demography with overlapping generations. The wild-type chromosomes are competed against two balancer chromosomes (B1, B2, say), providing a standard genotype B1/B2 against which variation in the fitness effects of the wild-type chromosomes can be assessed. Fitness can be assessed in two ways: (i) at equilibrium of all three chromosomes under heterozygote advantage, and (ii) during displacement of one balancer by the other. Equilibrium with all three chromosomes present will be achieved only if the wild-type homozygote is not too fit, and if the fitnesses of the three heterozygotes are not too unequal. These conditions were not satisfied for any of a sample of 12 lethal-bearing chromosomes isolated from a random-bred laboratory population of Drosophila. At equilibrium, genotypic frequencies show low sensitivity to changes in genotypic fitness. Furthermore, where all four genotypes are viable and fertile, supplementary information from cages with only two chromosomes present and from direct measurements of pre-adult viability are required to estimate fitnesses from frequencies. The invasion method has the advantages of a greater sensitivity and of not requiring further data to estimate fitnesses if the wild-type homozygote is fertile. However, it requires that multiple samples be taken as the invasion progresses. In a discrete generation model, generation time influences fitness estimates from this method and is difficult to estimate accurately from the data. A full age-structured model can also be applied to the data from both types of experiment. For the invasion method, this gives fitness estimates close to those from the discrete generation model.