Galega orientalis is more diverse than Galega officinalis in Caucasus--whole-genome AFLP analysis and phylogenetics of symbiosis-related genes

Legume plants can obtain combined nitrogen for their growth in an efficient way through symbiosis with specific bacteria. The symbiosis between Rhizobium galegae and its host plant Galega is an interesting case where the plant species G. orientalis and G. officinalis form effective, nitrogen‐fixing, symbioses only with the appropriate rhizobial counterpart, R. galegae bv. orientalis and R. galegae bv. officinalis, respectively. The symbiotic properties of nitrogen‐fixing rhizobia are well studied, but more information is needed on the properties of the host plants. The Caucasus region in Eurasia has been identified as the gene centre (centre of origin) of G. orientalis, although both G. orientalis and G. officinalis can be found in this region. In this study, the diversity of these two Galega species in Caucasus was investigated to test the hypothesis that in this region G. orientalis is more diverse than G. officinalis. The amplified fragment length polymorphism fingerprinting performed here showed that the populations of G. orientalis and R. galegae bv. orientalis are more diverse than those of G. officinalis and R. galegae bv. officinalis, respectively. These results support the centre of origin status of Caucasus for G. orientalis at a genetic level. Analysis of the symbiosis‐related plant genes NORK and Nfr5 reveals remarkable diversity within the Nfr5 sequence, although no evidence of adaptive evolution could be found.     

Symbiotic and Genetic Diversity of Rhizobium galegae Isolates Collected from the Galega orientalis Gene Center in the Caucasus

This paper explores the relationship between the genetic diversity of rhizobia and the morphological diversity of their plant hosts. Rhizobium galegae strains were isolated from nodules of wild Galega orientalis and Galega officinalis in the Caucasus, the center of origin for G. orientalis. All 101 isolates were characterized by genomic amplified fragment length polymorphism fingerprinting and by PCR-restriction fragment length polymorphism (RFLP) of the rRNA intergenic spacer and of five parts of the symbiotic region adjacent to nod box sequences. By all criteria, the R. galegae bv. officinalis and R. galegae bv. orientalis strains form distinct clusters. The nod box regions are highly conserved among strains belonging to each of the two biovars but differ structurally to various degrees between the biovars. The findings suggest varying evolutionary pressures in different parts of the symbiotic genome of closely related R. galegae biovars. Sixteen R. galegae bv. orientalis strains harbored copies of the same insertion sequence element; all were isolated from a particular site and belonged to a limited range of chromosomal genotypes. In all analyses, the Caucasian R. galegae bv. orientalis strains were more diverse than R. galegae bv. officinalis strains, in accordance with the gene center theory.     

Structures of sugar-binding peptides of <Emphasis Type="Italic">Galega orientalis</Emphasis> and <Emphasis Type="Italic">G. officinalis</Emphasis> lectins determine the choice of partner in rhizobium—Legume symbiosis

RAPD and RFEL analyses revealed appreciable genetic heterogeneity of Rhizobium galegae bv. officinalis and R. galegae bv. orientalis, which are nitrogen-fixing symbiosis partners of Galega officinalis and G. orientalis, respectively, and do not form a single cross-inoculation group. Comparison of nucleotide and amino acid sequences for their lectins revealed relatively high general homology, testifying again to their close phylogenetic relationships. Yet the lectin region of the carbohydrate-binding peptide (CBP) proved to differ considerably, being TYCNPGWDPRDR in G. orientalis and TFYNEEWDLVIKDEH in G. officinalis. Conserved positions in the CBP were observed for amino acid residues involved in binding Ca²⁺ and Mn²⁺ and stabilizing the spatial structure of the carbohydrate-binding pocket. These findings confirm the role in Rhizobium— legume symbiosis for lectins and especially for their carbohydrate-binding domains.Translated from Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 103–111.Original Russian Text Copyright © 2005 by Baimiev, Gubaidullin, Chemeris, Vakhitov.     

Soybeans inoculated with root zone soils of Canadian native legumes harbour diverse and novel Bradyrhizobium spp. that possess agricultural potential

An assessment was made of the evolutionary relationships of soybean nodulating bacteria associated with legumes native to eastern Canada to identify potential new sources of soybean inoculant strains.Short season soybeans were used to selectively trap bacteria from root zone soils of four native legume species. Screening of more than 800 bacterial isolates from soybean root nodules by analysis of recA gene sequences followed by analyses of selected genotypes using six core and two symbiosis (nodC and nifH) gene sequences permitted identification of diverse taxa that included eight novel and four named Bradyrhizobium species as well as lineages attributed to the genera Afipia and Tardiphaga.Plant tests showed that symbionts related to four named species as well as a novel Bradyrhizobium lineage were highly efficient with regard to nitrogen fixation on soybeans relative to an inoculant strain.A new symbiovar (sv. septentrionalis) is proposed based on a group of four novel Bradyrhizobium spp. that possess distinctive nodC and nifH gene sequences and symbiotic characteristics.Evidence is provided for horizontal transfer of sv. septentrionalis symbiosis genes between novel Bradyrhizobium spp., a process that rendered recipient bacteria ineffective on soybeans.Diverse lineages of non-symbiotic and symbiotic Bradyrhizobium spp. co-occured within monophyletic clusters in a phylogenetic tree of concatenated core genes, suggesting that loss and/or gain of symbiosis genes has occurred in the evolutionary history of the bacterial genus.Our data suggest that symbiont populations associated with legumes native to eastern Canada harbour elite strains of Bradyrhizobium for soybean inoculation.     

The Mimosoid tree Leucaena leucocephala can be nodulated by the symbiovar genistearum of Bradyrhizobium canariense

Leucaena leucocephala is a Mimosoid legume tree indigenous to America that has spread to other continents, although it is not still present in some European countries such as Portugal. Nevertheless, we found that this legume can be nodulated in this country by slow-growing rhizobial strains which were identified as Bradyrhizobium canariense trough the analysis of the core genes recA and glnII. The analysis of the symbiotic gene nodC showed that these strains belong to the symbiovar genistearum, which commonly nodulates Genistoid legumes. Although two strains nodulating L. leucocephala in China and Brazil were classified within the genus Bradyrhizobium, they belong to undescribed species and to the symbiovars glycinearum and tropici, respectively. Therefore, we report here for the first time the ability of L. leucocephala to establish symbiosis with strains of B. canariense sv genistearum confirming the high promiscuity of L. leucocephala, that allows it to establish symbiosis with rhizobia native to different continents increasing its invasiveness potential.     

Sinorhizobium meliloti succinylated high‐molecular‐weight succinoglycan and the Medicago truncatula LysM receptor‐like kinase MtLYK10 participate independently in symbiotic infection

The formation of nitrogen‐fixing nodules on legume hosts is a finely tuned process involving many components of both symbiotic partners. Production of the exopolysaccharide succinoglycan by the nitrogen‐fixing bacterium Sinorhizobium meliloti 1021 is needed for an effective symbiosis with Medicago spp., and the succinyl modification to this polysaccharide is critical. However, it is not known when succinoglycan intervenes in the symbiotic process, and it is not known whether the plant lysin‐motif receptor‐like kinase MtLYK10 intervenes in recognition of succinoglycan, as might be inferred from work on the Lotus japonicus MtLYK10 ortholog, LjEPR3. We studied the symbiotic infection phenotypes of S. meliloti mutants deficient in succinoglycan production or producing modified succinoglycan, in wild‐type Medicago truncatula plants and in Mtlyk10 mutant plants. On wild‐type plants, S. meliloti strains producing no succinoglycan or only unsuccinylated succinoglycan still induced nodule primordia and epidermal infections, but further progression of the symbiotic process was blocked. These S. meliloti mutants induced a more severe infection phenotype on Mtlyk10 mutant plants. Nodulation by succinoglycan‐defective strains was achieved by in trans rescue with a Nod factor‐deficient S. meliloti mutant. While the Nod factor‐deficient strain was always more abundant inside nodules, the succinoglycan‐deficient strain was more efficient than the strain producing only unsuccinylated succinoglycan. Together, these data show that succinylated succinoglycan is essential for infection thread formation in M. truncatula, and that MtLYK10 plays an important, but different role in this symbiotic process. These data also suggest that succinoglycan is more important than Nod factors for bacterial survival inside nodules.     

<Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">trifolii rosR</Emphasis> is required for interaction with clover,biofilm formation and adaptation to the environment

Background  

Rhizobium leguminosarum bv. trifolii is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants Trifolium spp. Bacterial surface polysaccharides are crucial for establishment of a successful symbiosis with legumes that form indeterminate-type nodules, such as Trifolium, Pisum, Vicia, and Medicago spp. and aid the bacterium in withstanding osmotic and other environmental stresses. Recently, the R. leguminosarum bv. trifolii RosR regulatory protein which controls exopolysaccharide production has been identified and characterized.     

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.     

Alleviation of salt stress of symbiotic Galega officinalis L. (goat's rue) by co-inoculation of Rhizobium with root-colonizing Pseudomonas

Background and aims

Salt stress is an increasing problem in agricultural soils in many parts of the world, and salt tolerant cropping systems are in great demand. We investigated the effect of co-inoculation of Galega officinalis with Rhizobium galegae and two plant growth promoting Pseudomonas species on plant growth, nodulation, and N content under salt stress.

Methods

The effect of inoculation with R. galegae sv. officinalis HAMBI 1141 alone and in combination with the root-colonizing Pseudomonas extremorientalis TSAU20 or Pseudomonas trivialis 3Re27 on the growth of G. officinalis exposed to salt stress (50 and 75 mM NaCl) was studied under gnotobiotic and greenhouse conditions.

Results

The growth of goat’s rue was reduced at 50 and 75 mM NaCl both in the gnotobiotic sand system and in low-fertilized potting soil in the greenhouse. Co-inoculation of unstressed and salt-stressed goat’s rue with R. galegae HAMBI 1141 and either P. extremorientalis TSAU20 or P. trivialis 3Re27 significantly improved root and shoot growth and increased nodulation of plant roots in both growth systems compared with plants inoculated with R. galegae alone. The nitrogen content of co-inoculated plant roots was significantly increased at 75 mM NaCl in potting soil. Co-inoculation of G. officinalis with either of the two plant growth promoting (PGPR) Pseudomonas strains also improved root tip-colonization by R. galegae cells.

Conclusions

The co-inoculation of goat’s rue with Rhizobium and PGPR Pseudomonas strains alleviated salt stress of plants grown in NaCl-amended gnotobiotic sand systems and in potting soil in the greenhouse.     

A Comparative Study on Essential Oil Intraspecific and Seasonal Variations: Melissa romana Mill. and Melissa officinalis L. from Sardinia

Two species of Melissa are currently present in Sardinia: Melissa officinalis L. and Melissa romana Mill . Our research can only count on a few supported evidences (as reported in Flora Italiana and Moris) and some notes on new stations in Sardinia that give us some information about morphology, distribution, bioecological, and ethnobotanical characteristics of both species. In this paper, we present the results of the research about morphological aspects of M. romana vs. M. officinalis, and their essential oils in different stations at different phenological periods. Moreover, we compared the essential oil of M. romana with the one obtained from M. officinalis growing in the few naturalized stations still present in Sardinia. The most evident morphological differences between the two entities are the long‐stalked capitate glandular trichomes, shorts and inclined capitate trichomes, and peltate hairs. The chemical composition of essential oil presents several significant differences between the species. In fact, oils show that in none of the phenological stages, M. romana recalls in its composition M. officinalis. Major distinctions are also evident between dry and fresh plants, and among essential oils distilled in different seasons.     

Description of two biovars in the Rhizobium galegae species: biovar orientalis and biovar officinalis

Twenty-six Rhizobium galegae strains, representing the center of origin of the host plants Galega orientalis and G. officinalis as well as other geographic regions, were used in a polyphasic analysis of the relationships of R. galegae strains. Phage typing, lipopolysaccharide (LPS) profiling, pulsed field gel electrophoresis (PFGE) profiling and rep-PCR (use of repetitive sequences as PCR primers for genomic fingerprinting) with REP and ERIC primers investigated nonsymbiotic properties, whereas plasmid profiling and hybridisation with a nif gene probe, and with nodB, nodD, nod box and an IS sequence from the symbiotic region as probes, were used to reveal the relationships of symbiotic genes. The results were used in pairwise calculations of distances between the strains, and the distances were visualised as a dendrogram. Indexes of association were compared for all tests pooled, and for chromosomal tests and symbiotic markers separately, to display the input of the different categories of tests on the grouping of the strains. Our study shows that symbiosis related genetic traits in R. galegae divide strains belonging to the species into two groups, which correspond to strains forming an effective symbioses with G. orientalis and G. officinalis respectively. We therefore propose that Rhizobium galegae strains forming an effective symbiosis with Galega orientalis are called R. galegae bv. orientalis and strains forming an effective symbiosis with Galega officinalis are called R. galegae bv. officinalis.     

Aerobiological and clinical aspects ofParietaria officinalis in the area of Thessaloniki,Greece

Summary We collected the daily pollen samples during a 3-year period (Febr '87–Dec '89), using a Burkard volumetric trap, located on a high level area in the center of the city.Parietaria officinalis pollen was not differentiated under microscope from the other Urticaceae but through phenological criteria. The patients included in the detection of the sensitivity toP. officinalis pollen came from the Out-patient Clinic of Bronchial Asthma of the General Hospital «G. Papanikolaou». They had a seasonal pollinosis and they were submitted to Pricktest using a battery of 22 groups and aP. officinalis pollen extract. The Urticaceae pollen appears first in the atmosphere of Thessaloniki in the end of March, shows a peak in the beginning of May and continues to be present till the end of August. We detectedP. officinalis pollen sensitivity combined with other allergens in 24.1% of the patients and in 1.4% a monosensitivity toP. officinalis.     

Insights into the history of the legume‐betaproteobacterial symbiosis

The interaction between legumes and rhizobia has been well studied in the context of a mutualistic, nitrogen‐fixing symbiosis. The fitness of legumes, including important agricultural crops, is enhanced by the plants’ ability to develop symbiotic associations with certain soil bacteria that fix atmospheric nitrogen into a utilizable form, namely, ammonia, via a chemical reaction that only bacteria and archaea can perform. Of the bacteria, members of the alpha subclass of the protebacteria are the best‐known nitrogen‐fixing symbionts of legumes. Recently, members of the beta subclass of the proteobacteria that induce nitrogen‐fixing nodules on legume roots in a species‐specific manner have been identified. In this issue, Bontemps et al. reveal that not only are these newly identified rhizobia novel in shifting the paradigm of our understanding of legume symbiosis, but also, based on symbiotic gene phylogenies, have a history that is both ancient and stable. Expanding our understanding of novel plant growth promoting rhizobia will be a valuable resource for incorporating alternative strategies of nitrogen fixation for enhancing plant growth.     

Host plant as an organizer of microbial evolution in the beneficial symbioses

Evolution of beneficial plant–microbe symbioses is presented as a result of selective processes induced by hosts in the associated microbial populations. These processes ensure a success of “genuine mutualists” (which benefit the host, often at the expense of their own fitness) in competition with “symbiotic cheaters” (which consume the resources provided by host without expressing the beneficial traits). Using a mathematical model describing the cyclic microevolution of rhizobia–legume symbiosis, we suggest that the selective pressures in favor of N₂-fixing (Fix⁺) strains operate within the in planta bacterial population due to preferential allocation of C resources into Fix⁺ nodules (positive partners’ feedbacks). Under the clonal infection of nodules, Fix⁺ strains (“genuine mutualists”) are supported by the group (inter-deme, kin) selection while under the mixed infections, this selection is ineffective since the Fix⁺ strains are over-competed by Fix⁻ ones (“symbiotic cheaters”) in the nodular habitats. Nevertheless, under mixed infections, Fix⁺ strains may be supported due to the coevolutionary responses form plant population which induce the mutualism-specific types of natural (group, individual) selection including the frequency dependent selection implemented in rhizobia population during the competition for host infection. Using the model of multi-strain bacterial competition for inoculation of symbiotic (rhizospheric, nodular) habitats, we demonstrate that the individual selection in favor of host-specific mutualist genotypes is more intensive than in favor of non-host-specific genotypes correlating the experimental data on the coordinated increases of symbiotic efficiency and specificity in the rhizobia–legume coevolution. However, an overall efficiency of symbiotic system is maximal when the non-host-specific mutualists are present in rhizobia population, and selection in favor of these mutualists operating at the whole population level is of key importance for improving the symbiosis. Construction of the agronomically valuable plant–microbe systems should provide the optimization of host-specific versus non-host-specific mutualists’ composition in legume inoculants combined with the clonal penetration of these mutualists into the nodules.     

Symbiotic mutants deficient in nodule establishment identified after T-DNA transformation of Lotus japonicus

Nitrogen-fixing root nodules develop on legumes as a result of an interaction between host plants and soil bacteria collectively referred to as rhizobia. The organogenic process resulting in nodule development is triggered by the bacterial microsymbiont, but genetically controlled by the host plant genome. Using T-DNA insertion as a tool to identify novel plant genes that regulate nodule ontogeny, we have identified two putatively tagged symbiotic loci, Ljsym8 and Ljsym13, in the diploid legume Lotus japonicus. The sym8 mutants are arrested during infection by the bacteria early in the developmental process. The sym13 mutants are arrested in the final stages of infection, and ineffective nodules are formed. These two plant mutant lines were identified in progeny from 1112 primary transformants obtained after Agrobacterium tumefaciens T-DNA-mediated transformation of L. japonicus and subsequent screening for defects in the symbiosis with Mesorhizobium loti. Additional nontagged mutants arrested at different developmental stages were also identified and genetic complementation tests assigned all the mutations to 16 monogenic symbiotic loci segregating recessive mutant alleles. In the screen reported here independent symbiotic loci thus appeared with a frequency of ∼1.5%, suggesting that a relatively large set of genes is required for the symbiotic interaction. Received: 12 May 1998 / Accepted: 24 June 1998     

Symbiotic properties of a chimeric Nod-independent photosynthetic Bradyrhizobium strain obtained by conjugative transfer of a symbiotic plasmid

The lateral transfer of symbiotic genes converting a predisposed soil bacteria into a legume symbiont has occurred repeatedly and independently during the evolution of rhizobia. We experimented the transfer of a symbiotic plasmid between Bradyrhizobium strains. The originality of the DOA9 donor is that it harbours a symbiotic mega-plasmid (pDOA9) containing nod, nif and T3SS genes while the ORS278 recipient has the unique property of inducing nodules on some Aeschynomene species in the absence of Nod factors (NFs). We observed that the chimeric strain ORS278-pDOA9* lost its ability to develop a functional symbiosis with Aeschynomene. indica and Aeschynomene evenia. The mutation of rhcN and nodB led to partial restoration of nodule efficiency, indicating that T3SS effectors and NFs block the establishment of the NF-independent symbiosis. Conversely, ORS278-pDOA9* strain acquired the ability to form nodules on Crotalaria juncea and Macroptillium artropurpureum but not on NF-dependent Aeschynomene (A. afraspera and A. americana), suggesting that the ORS278 strain also harbours incompatible factors that block the interaction with these species. These data indicate that the symbiotic properties of a chimeric rhizobia cannot be anticipated due to new combination of symbiotic and non-symbiotic determinants that may interfere during the interaction with the host plant.