Winter survival in red clover: experimental evidence for interactions among stresses.

BACKGROUND: There is a lack of knowledge on the combined effects of different stresses on plants, in particular different stresses that occur during winter in temperate climates. Perennial herbaceous plants in temperate regions are exposed to many different stresses during winter, but except for the fact that cold temperatures induce resistance to a number of them, very little is known about their interaction effects. Knowledge about stress interactions is needed in order to predict effects of climate change on both agricultural production and natural ecosystems, and to develop adaptation strategies, e.g., through plant breeding. Here, we conducted a series of experiments under controlled conditions to study the interactions between cold (low positive temperature), clover rot infection (caused by Sclerotinia trifoliorum) and freezing, in red clover (Trifolium pratense) accessions. We also compared our results with winter survival in field experiments and studied associations between stress and shoot growth. RESULTS: Exposure to low positive temperatures (cold acclimation) induced resistance to clover rot. There was a clear negative interaction effect between freezing stress and clover rot infection, resulting in up to 37% lower survival rate compared to what would have been expected from the additive effect of freezing and infection alone. Freezing tolerance could continue to improve during incubation under artificial snow cover at 3 °C in spite of darkness, and we observed compensatory shoot growth following freezing after prolonged incubation. At the accession level, resistance to clover rot was negatively correlated with growth in the field during the previous year at a Norwegian location. It was also negatively correlated with the shoot regrowth of control plants after incubation. Clover rot resistance tests under controlled conditions showed limited correlation with clover rot resistance observed in the field, suggesting that they may reveal variation in more specific resistance mechanisms. CONCLUSIONS: We here demonstrate, for the first time, a strong negative interaction between freezing and infection with a winter pathogen. We also characterize the effects of cold acclimation and incubation in darkness at different temperatures on winter stress tolerance, and present data that support the notion that annual cycles of growth and stress resistance are associated at the genetic level.

Role of ectomycorrhizal colonization in enhancement of nutrients for survival of plants collected from mountainous cold stress areas.

BACKGROUND: Ectomycorrhizal (ECM and ECM-like) structures associated with plant root systems are a challenge for scientists. The dispersion pattern of roots within the soil profile and the nutritional conditions are both favourable factors to motivate the plants to make ECM associations. RESULTS: This study discusses the colonization of mycorrhizal associations in Kobresia and Polygonum species including Polygonum viviparum, Kobresia filicina, K. myosuroides, Alnus nitida, Betula pendula, Pinus sylvestris, and Trifolium repens grown naturally in cold stressed soils of Gilgit-Baltistan (high-altitude alpine Deosai plains), Hazara, Swat, Dir, and Bajaur. Sieved soil batches were exposed to +5 °C (control), -10, -20, -30, -40, -50, -125 °C for 5 h, and selected plants were sown to these soils for 10 weeks under favourable conditions for ECM colonization. Ectomycorrhizal associations were examined in the above mentioned plants. Some ECM fungi have dark mycelia that look like the mantle and Hartig net. Examples of these are Kobresia filicina, K. myosuroides, and Polygonum viviparum. Findings of this study revealed that K. myosuroides excelled in ECM root tip length, dry mass, and NH4 concentration at -125 °C. Contrarily, A. nitida demonstrated the lower values, indicated its minimum tolerance. Notably, T. repens boasted the highest nitrogen concentration (18.7 ± 1.31 mg/g), while P. sylvestris led in phosphorus (3.2 ± 0.22 mg/g). The B. pendula showed the highest potassium concentration (9.4 ± 0.66 mg/g), emphasising species-specific nutrient uptake capabilities in extreme cold conditions. The PCA analysis revealed that the parameters, e.g., NH4 in soil mix (NH4), NO3 in soil mix (NO3), phosphorus in soil in species of Polygonum viviparum, Kobresia filicina, K. myosuroides, Alnus nitida, Betula pendula, Pinus sylvestris, and Trifolium repens are most accurately represented in cases of + 5 °C, -10 °C, and -20 °C temperatures. On the other hand, the parameters for ECM root tips (ECM) and Dry Mass (DM) are best described in -40 °C, -50 °C, and - 125 °C temperatures. All parameters have a strong influence on the variability of the system indicated the efficiency of ECM. The heatmap supported the nutrients positively correlated with ECM colonization with the host plants. CONCLUSION: At lower temperatures, hyphae and spores in roots were reduced, while soluble phosphorus concentrations of leaves were increased in cold stress soils. Maximum foliar nutrient concentrations were found in K. myosuroides at the lowest temperature treatments due to efficient functioning and colonization of ECM.

Intricate microbe-plant-metabolic remodeling mediated by intercropping enhances the quality of Panax quinquefolius L.

Improving the cultivation mode and technology for traditional Chinese medicine has become important for its sustainable development. Monoculture enhances plant diseases, which decreases yield and quality. Intercropping is an effective measure to counterbalance that negative effect. In this study, we focused on Panax quinquefolium L. (ginseng) and four treatments were set up: the control without intercropping, P. quinquefolius + ryegrass (Lolium perenne L.), P. quinquefolius + red clover (Trifolium pratense L.), and P. quinquefolius + ryegrass + red clover. An LC-MS/MS system was used to detect the changes in the P. quinquefolius secondary metabolites, and high-throughput sequencing technology was used to determine the changes in the P. quinquefolius' rhizosphere soil microorganisms. Ginsenoside content, soil enzyme activities, and arbuscular mycorrhizal infection rate of P. quinquefolius were also measured using HPLC, ELISA kits, and microscopy, respectively. Co-intertia and Pearson's analysis were performed to explore the relationship between the metabolites and the P. quinquefolius microorganisms. Intercropping significantly increased the content of ginsenoside metabolites and recruited a large number of beneficial bacteria to the P. quinquefolius rhizosphere. The P. quinquefolius secondary metabolites were associated with the rhizosphere microbial community. For example, the dominant microorganisms, such as Acidobacteriota and Chloroflexi, played a key role in promoting the synthesis of ginsenoside Rd and (20R) ginsenoside Rg3 by P. quinquefolius. Intercropping led to changes in the P. quinquefolius secondary metabolites by driving and reshaping the rhizosphere microorganisms. These findings revealed the potential application of intercropping for improving the quality of P. quinquefolius.

Red clover root-associated microbiota is shaped by geographic location and choice of farming system.

AIMS: This study evaluated the red clover (Trifolium pratense) root-associated microbiota to clarify the presence of pathogenic and beneficial microorganisms in 89 Swedish field sites. METHODS AND RESULTS: 16S rRNA and ITS amplicon sequencing analysis were performed on DNA extracted from the red clover root samples collected to determine the composition of the prokaryotic and eukaryotic root-associated microbe communities. Alpha and beta diversities were calculated and relative abundance of various microbial taxa and their co-occurrence were analyzed. Rhizobium was the most prevalent bacterial genus, followed by Sphingomonas, Mucilaginibacter, Flavobacterium, and the unclassified Chloroflexi group KD4-96. The Leptodontidium, Cladosporium, Clonostachys, and Tetracladium fungal genera known for endophytic, saprotrophic, and mycoparasitic lifestyles were also frequently observed in all samples. Sixty-two potential pathogenic fungi were identified with a bias toward grass pathogens and a higher abundance in samples from conventional farms. CONCLUSIONS: We showed that the microbial community was mainly shaped by geographic location and management procedures. Co-occurrence networks revealed that the Rhizobiumleguminosarum bv. trifolii was negatively associated with all fungal pathogenic taxa recognized in this study.

Major effect loci for plant size before onset of nitrogen fixation allow accurate prediction of yield in white clover.

KEY MESSAGE: Accurate genomic prediction of yield within and across generations was achieved by estimating the genetic merit of individual white clover genotypes based on extensive genetic replication using cloned material. White clover is an agriculturally important forage legume grown throughout temperate regions as a mixed clover-grass crop. It is typically cultivated with low nitrogen input, making yield dependent on nitrogen fixation by rhizobia in root nodules. Here, we investigate the effects of clover and rhizobium genetic variation by monitoring plant growth and quantifying dry matter yield of 704 combinations of 145 clover genotypes and 170 rhizobium inocula. We find no significant effect of rhizobium variation. In contrast, we can predict yield based on a few white clover markers strongly associated with plant size prior to nitrogen fixation, and the prediction accuracy for polycross offspring yield is remarkably high. Several of the markers are located near a homolog of Arabidopsis thaliana GIGANTUS 1, which regulates growth rate and biomass accumulation. Our work provides fundamental insight into the genetics of white clover yield and identifies specific candidate genes as breeding targets.

Complete Genome Sequences of Trifolium spp. Inoculant Strains Rhizobium leguminosarum sv. trifolii TA1 and CC275e: Resources for Genomic Study of the Rhizobium-Trifolium Symbiosis.

Rhizobium leguminosarum symbiovar trifolii strains TA1 and CC275e are nitrogen-fixing microsymbionts of Trifolium spp. and have been used as commercial inoculant strains for clovers in pastoral agriculture in Australia and New Zealand. Here we present the complete genome sequences of both strains, resolving their multipartite genome structures and allowing for future studies using genomic approaches.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Diversity of rhizobia and non-rhizobia endophytes isolated from root nodules of Trifolium sp. growing in lead and zinc mine site Guelma, Algeria.

High concentrations of heavy metals in mine soil disturb the interactions between legumes and microorganisms leading to select strains adapted to these specific conditions. In this work, we analyzed the diversity of fifty strains isolated from Trifolium sp. nodules growing on Pb-Zn mine soil, in the Northeastern of Algeria and highlighted their potential symbiotic traits. The phylogeny of the 16S rRNA gene sequences revealed a high bacterial diversity with a predominance of non-rhizobial endophytes. The identified isolates belong to the thirteen following genera Cupriavidus, Pseudomonas, Bacillus, Acinetobacter, Enterobacter, Roseomonas, Paracoccus, Frondihabitans, Microbacterium, Kocuria, Providencia, Micrococcus and Staphylococcus. Regarding rhizobial strains, only isolates affiliated to Rhizobium genus were obtained. The symbiotic gene nodC and the nitrogen fixation gene nifH present showed that Rhizobium isolates belonged to the symbiovar trifolii. In addition to bacterial, one yeast strain was isolated and identified as Rhodotorula mucilaginosa by sequencing the internal transcribed spacer (ITS) region.

Exopolysaccharide Carbohydrate Structure and Biofilm Formation by Rhizobium leguminosarum bv. trifolii Strains Inhabiting Nodules of Trifoliumrepens Growing on an Old Zn-Pb-Cd-Polluted Waste Heap Area.

Heavy metals polluting the 100-year-old waste heap in Boleslaw (Poland) are acting as a natural selection factor and may contribute to adaptations of organisms living in this area, including Trifolium repens and its root nodule microsymbionts-rhizobia. Exopolysaccharides (EPS), exuded extracellularly and associated with bacterial cell walls, possess variable structures depending on environmental conditions; they can bind metals and are involved in biofilm formation. In order to examine the effects of long-term exposure to metal pollution on EPS structure and biofilm formation of rhizobia, Rhizobium leguminosarum bv. trifolii strains originating from the waste heap area and a non-polluted reference site were investigated for the characteristics of the sugar fraction of their EPS using gas chromatography mass-spectrometry and also for biofilm formation and structural characteristics using confocal laser scanning microscopy under control conditions as well as when exposed to toxic concentrations of zinc, lead, and cadmium. Significant differences in EPS structure, biofilm thickness, and ratio of living/dead bacteria in the biofilm were found between strains originating from the waste heap and from the reference site, both without exposure to metals and under metal exposure. Received results indicate that studied rhizobia can be assumed as potentially useful in remediation processes.

Rhizobium leguminosarum bv. trifolii NodD2 Enhances Competitive Nodule Colonization in the Clover-Rhizobium Symbiosis.

Establishment of the symbiotic relationship that develops between rhizobia and their legume hosts is contingent upon an interkingdom signal exchange. In response to host legume flavonoids, NodD proteins from compatible rhizobia activate expression of nodulation genes that produce lipochitin oligosaccharide signaling molecules known as Nod factors. Root nodule formation commences upon legume recognition of compatible Nod factor. Rhizobium leguminosarum was previously considered to contain one copy of nodD; here, we show that some strains of the Trifolium (clover) microsymbiont R. leguminosarum bv. trifolii contain a second copy designated nodD2. nodD2 genes were present in 8 out of 13 strains of R. leguminosarum bv. trifolii, but were absent from the genomes of 16 R. leguminosarum bv. viciae strains. Analysis of single and double nodD1 and nodD2 mutants in R. leguminosarum bv. trifolii strain TA1 revealed that NodD2 was functional and enhanced nodule colonization competitiveness. However, NodD1 showed significantly greater capacity to induce nod gene expression and infection thread formation. Clover species are either annual or perennial and this phenological distinction is rarely crossed by individual R. leguminosarum bv. trifolii microsymbionts for effective symbiosis. Of 13 strains with genome sequences available, 7 of the 8 effective microsymbionts of perennial hosts contained nodD2, whereas the 3 microsymbionts of annual hosts did not. We hypothesize that NodD2 inducer recognition differs from NodD1, and NodD2 functions to enhance competition and effective symbiosis, which may discriminate in favor of perennial hosts.IMPORTANCE Establishment of the rhizobium-legume symbiosis requires a highly specific and complex signal exchange between both participants. Rhizobia perceive legume flavonoid compounds through LysR-type NodD regulators. Often, rhizobia encode multiple copies of nodD, which is one determinant of host specificity. In some species of rhizobia, the presence of multiple copies of NodD extends their symbiotic host-range. Here, we identified and characterized a second copy of nodD present in some strains of the clover microsymbiont Rhizobium leguminosarum bv. trifolii. The second nodD gene contributed to the competitive ability of the strain on white clover, an important forage legume. A screen for strains containing nodD2 could be utilized as one criterion to select strains with enhanced competitive ability for use as inoculants for pasture production.

Responses of microbial communities and metabolic activities in the rhizosphere during phytoremediation of Cd-contaminated soil.

Phytoremediation is an effective way to repair heavy metal contaminated soil and rhizosphere microorganisms play an important role in plant regulation. Nevertheless, little information is known about the variation of microbial metabolic activities and community structure in rhizosphere during phytoremediation. In this study, the rhizosphere soil microbial metabolic activities and community structure of Trifolium repensL. during Cd-contaminated soil phytoremediation, were analyzed by Biolog EcoPlate™ and high-throughput sequencing. The uptake in the roots of Trifolium repensL. grown in 5.68 and 24.23 mg/kg Cd contaminated soil was 33.51 and 84.69 mg/kg respectively, causing the acid-soluble Cd fractions decreased 7.3% and 5.4%. Phytoremediation significantly influenced microbial community and Trifolium repensL. planting significantly increased the rhizosphere microbial population, diversity, the relative abundance of plant growth promoting bacteria (Kaistobacter and Flavisolibacter), and the utilization of difficultly metabolized compounds. The correlation analysis among substrate utilization and microbial communities revealed that the relative abundance increased microorganisms possessed stronger carbon utilization capacity, which was beneficial to regulate the stability of plant-microbial system. Collectively, the results of this study provide fundamental insights into the microbial metabolic activities and community structure during heavy metal contaminated soil phytoremediation, which may aid in the bioregulation of phytoremediation.

PssJ Is a Terminal Galactosyltransferase Involved in the Assembly of the Exopolysaccharide Subunit in Rhizobium Leguminosarum bv. Trifolii.

Rhizobium leguminosarum bv. trifolii produces exopolysaccharide (EPS) composed of glucose, glucuronic acid, and galactose residues at a molar ratio 5:2:1. A majority of genes involved in the synthesis, modification, and export of exopolysaccharide are located in the chromosomal Pss-I region. In the present study, a ΔpssJ deletion mutant was constructed and shown to produce EPS lacking terminal galactose in the side chain of the octasaccharide subunit. The lack of galactose did not block EPS subunit translocation and polymerization. The in trans delivery of the pssJ gene restored the production of galactose-containing exopolysaccharide. The mutant was compromised in several physiological traits, e.g., motility and biofilm production. An impact of the pssJ mutation and changed EPS structure on the symbiotic performance was observed as improper signaling at the stage of molecular recognition, leading to formation of a significant number of non-infected empty nodules. Terminal galactosyltransferase PssJ was shown to display a structure typical for the GT-A class of glycosyltransferases and interact with other GTs and Wzx/Wzy system proteins. The latter, together with PssJ presence in soluble and membrane protein fractions indicated that the protein plays its role at the inner membrane interface and as a component of a larger complex.

Mutualists Stabilize the Coexistence of Congeneric Legumes.

Coexistence requires that stabilizing niche differences, which cause species to limit themselves more than others, outweigh relative fitness differences, which cause competitive exclusion. Interactions with shared mutualists, which can differentially affect host fitness and change in magnitude with host frequency, can satisfy these conditions for coexistence, yet empirical tests of mutualist effects on relative fitness and stabilizing niche differences are largely lacking within the framework of coexistence theory. Here, we show that N-fixing rhizobial mutualists mediate coexistence in four naturally co-occurring congeneric legume (Trifolium) species. Using experimental greenhouse communities, we quantified relative fitness and stabilizing niche differences for each species in the presence of rhizobia originating from conspecific or congeneric hosts. Rhizobia stabilized coexistence by increasing the self-limitation of Trifolium species grown with rhizobia isolated from conspecifics, thus allowing congeners to increase when rare. Greenhouse-measured invasion growth rates predicted natural, unmanipulated coexistence dynamics of Trifolium species over 2 years at our field sites. Our results demonstrate that interactions with shared mutualists can stabilize the coexistence of closely related species.

A neglected alliance in battles against parasitic plants: arbuscular mycorrhizal and rhizobial symbioses alleviate damage to a legume host by root hemiparasitic Pedicularis species.

Despite their ubiquitous distribution and significant ecological roles, soil microorganisms have long been neglected in investigations addressing parasitic plant-host interactions. Because nutrient deprivation is a primary cause of host damage by parasitic plants, we hypothesized that beneficial soil microorganisms conferring nutrient benefits to parasitized hosts may play important roles in alleviating damage. We conducted a pot cultivation experiment to test the inoculation effect of an arbuscular mycorrhizal fungus (Glomus mosseae), a rhizobium (Rhizobium leguminosarum) and their interactive effects, on alleviation of damage to a legume host (Trifolium repens) by two root hemiparasitic plants with different nutrient requirements (N-demanding Pedicularis rex and P-demanding P. tricolor). Strong interactive effects between inoculation regimes and hemiparasite identity were observed. The relative benefits of microbial inoculation were related to hemiparasite nutrient requirements. Dual inoculation with the rhizobium strongly enhanced promotional arbuscular mycorrhizal effects on hosts parasitized by P. rex, but reduced the arbuscular mycorrhizal promotion on hosts parasitized by P. tricolor. Our results demonstrate substantial contribution of arbuscular mycorrhizal and rhizobial symbioses to alleviating damage to the legume host by root hemiparasites, and suggest that soil microorganisms are critical factors regulating host-parasite interactions and should be taken into account in future studies.

Genomic polymorphism of Trifolium repens root nodule symbionts from heavy metal-abundant 100-year-old waste heap in southern Poland.

In total, 77 rhizobial strains isolated from the root nodules of T. repens, inhabiting heavy metal-contaminated waste heap (36 isolates) and control grassland (41 ones) in southern Poland, were analyzed for genome polymorphism and strength of the heavy metals' (mainly Zn, Pb, Cd) selective pressure on bacterial genome polymorphism using two PCR-based techniques, ERIC- (enterobacterial repetitive intergenic consensus) and REP-PCR (repetitive extragenic palindromic) sequences. Both methods of different discriminatory power index (D) (ERIC-PCR D = 0.9737; REP-PCR D = 0.9826) allowed to distinguish 47 and 44 rhizobial strains, respectively. Combined analysis of ERIC-PCR and REP-PCR DNA amplicons differentiated all tested isolates. Both ERIC- and REP-PCR DNA fingerprinting techniques showed significant decline of the genome polymorphism (h) in rhizobial population from metalliferous waste heap (h = 0.89 ± 0.03; h = 0.90 ± 0.02, respectively) compared to rhizobia from control non-metalliferous area (h = 0.99 ± 0.01; h = 0.98 ± 0.02, respectively) as well as substantial differences in the genomic polymorphism between both these populations (FST = 0.162, p = 0.008; FST = 0.170, p = 0.000, respectively).

Morphological and physiological responses of the external mycelium of Rhizophagus intraradices to water stress.

Most studies dealing with mycorrhizal associations and drought have focused on the plants, not on the fungi, and tolerance and adaptations of arbuscular mycorrhizal (AM) fungi to cope with water stress are virtually unknown. This study was conducted to assess how water stress directly affects an AM fungus isolate, particularly through morphological and physiological changes in the external mycelium. We used two-compartment pots separated by mesh and an air gap that allowed us to apply water stress treatments only to the external mycelium. Clover (Trifolium subterraneum L.) plants inoculated with Rhizophagus intraradices grew at high humidity until external mycorrhizal mycelium developed in the mycelium compartment. Then, we started three watering treatments: high (H, 70% of soil water holding capacity), low (L, 10%), and mixed watering (HLHL, 70-10-70-10%) only in the hyphal compartment. The HLHL treatment was rewetted once to 70% after 42 days. We measured total mycelium length, hyphal length in diameter categories, respiration activity, and protoplasm fragmentation 42 and 76 days after starting the treatments. Rhizophagus intraradices mycelium responded to water stress by reducing its length, maintaining larger diameter hyphae, and concentrating protoplasm activity in fragments in the HLHL and L treatments. In both water stress treatments, changes suggested a trade-off between avoiding desiccation and storing resources, and maintaining soil exploration and water uptake capacity.

Allelic Variants for Candidate Nitrogen Fixation Genes Revealed by Sequencing in Red Clover (Trifolium pratense L.).

Plant-rhizobia symbiosis can activate key genes involved in regulating nodulation associated with biological nitrogen fixation (BNF). Although the general molecular basis of the BNF process is frequently studied, little is known about its intraspecific variability and the characteristics of its allelic variants. This study's main goals were to describe phenotypic and genotypic variation in the context of nitrogen fixation in red clover (Trifolium pretense L.) and identify variants in BNF candidate genes associated with BNF efficiency. Acetylene reduction assay validation was the criterion for selecting individual plants with particular BNF rates. Sequences in 86 key candidate genes were obtained by hybridization-based sequence capture target enrichment of plants with alternative phenotypes for nitrogen fixation. Two genes associated with BNF were identified: ethylene response factor required for nodule differentiation (EFD) and molybdate transporter 1 (MOT1). In addition, whole-genome population genotyping by double-digest restriction-site-associated sequencing (ddRADseq) was performed, and BNF was evaluated by the natural 15N abundance method. Polymorphisms associated with BNF and reflecting phenotype variability were identified. The genetic structure of plant accessions was not linked to BNF rate of measured plants. Knowledge of the genetic variation within BNF candidate genes and the characteristics of genetic variants will be beneficial in molecular diagnostics and breeding of red clover.

Molecular diversity and phylogeny of indigenous Rhizobium leguminosarum strains associated with Trifolium repens plants in Romania.

The symbiotic nitrogen fixing legumes play an essential role in sustainable agriculture. White clover (Trifolium repens L.) is one of the most valuable perennial legumes in pastures and meadows of temperate regions. Despite its great agriculture and economic importance, there is no detailed available information on phylogenetic assignation and characterization of rhizobia associated with native white clover plants in South-Eastern Europe. In the present work, the diversity of indigenous white clover rhizobia originating in 11 different natural ecosystems in North-Eastern Romania were assessed by a polyphasic approach. Initial grouping showed that, 73 rhizobial isolates, representing seven distinct phenons were distributed into 12 genotypes, indicating a wide phenotypic and genotypic diversity among the isolates. To clarify their phylogeny, 44 representative strains were used in sequence analysis of 16S rRNA gene and IGS fragments, three housekeeping genes (atpD, glnII and recA) and two symbiosis-related genes (nodA and nifH). Multilocus sequence analysis (MLSA) phylogeny based on concatenated housekeeping genes delineated the clover isolates into five putative genospecies. Despite their diverse chromosomal backgrounds, test strains shared highly similar symbiotic genes closely related to Rhizobium leguminosarum biovar trifolii. Phylogenies inferred from housekeeping genes were incongruent with those of symbiotic genes, probably due to occurrence of lateral transfer events among native strains. This is the first polyphasic taxonomic study to report on the MLSA-based phylogenetic diversity of indigenous rhizobia nodulating white clover plants grown in various soil types in South-Eastern Europe. Our results provide valuable taxonomic data on native clover rhizobia and may increase the pool of genetic material to be used as biofertilizers.

Host-dependent symbiotic efficiency of Rhizobium leguminosarum bv. trifolii strains isolated from nodules of Trifolium rubens.

Trifolium rubens L., commonly known as the red feather clover, is capable of symbiotic interactions with rhizobia. Up to now, no specific symbionts of T. rubens and their symbiotic compatibility with Trifolium spp. have been described. We characterized the genomic diversity of T. rubens symbionts by analyses of plasmid profiles and BOX-PCR. The phylogeny of T. rubens isolates was inferred based on the nucleotide sequences of 16S rRNA and two core genes (atpD, recA). The nodC phylogeny allowed classification of rhizobia nodulating T. rubens as Rhizobium leguminosarum symbiovar trifolii (Rlt). The symbiotic efficiency of the Rlt isolates was determined on four clover species: T. rubens, T. pratense, T. repens and T. resupinatum. We determined that Rlt strains formed mostly inefficient symbiosis with their native host plant T. rubens and weakly effective (sub-optimal) symbiosis with T. repens and T. pratense. The same Rlt strains were fully compatible in the symbiosis with T. resupinatum. T. rubens did not exhibit strict selectivity in regard to the symbionts and rhizobia closely related to Rhizobium grahamii, Rhizobium galegae and Agrobacterium radiobacter, which did not nodulate Trifolium spp., were found amongst T. rubens nodule isolates.

Options of partners improve carbon for phosphorus trade in the arbuscular mycorrhizal mutualism.

The mutualism between plants and arbuscular mycorrhizal fungi (AMF) is widespread and has persisted for over 400 million years. Although this mutualism depends on fair resource exchange between plants and fungi, inequality exists among partners despite mechanisms that regulate trade. Here, we use (33) P and (14) C isotopes and a split-root system to test for preferential allocation and reciprocal rewards in the plant-AMF symbiosis by presenting a plant with two AMF that differ in cooperativeness. We found that plants received more (33) P from less cooperative AMF in the presence of another AMF species. This increase in (33) P resulted in a reduced (14) C cost per unit of (33) P from less cooperative AMF when alternative options were available. Our results indicate that AMF diversity promotes cooperation between plants and AMF, which may be an important mechanism maintaining the evolutionary persistence of and diversity within the plant-AMF mutualism.

Ecological genomics of mutualism decline in nitrogen-fixing bacteria.

Anthropogenic changes can influence mutualism evolution; however, the genomic regions underpinning mutualism that are most affected by environmental change are generally unknown, even in well-studied model mutualisms like the interaction between legumes and their nitrogen (N)-fixing rhizobia. Such genomic information can shed light on the agents and targets of selection maintaining cooperation in nature. We recently demonstrated that N-fertilization has caused an evolutionary decline in mutualistic partner quality in the rhizobia that form symbiosis with clover. Here, population genomic analyses of N-fertilized versus control rhizobium populations indicate that evolutionary differentiation at a key symbiosis gene region on the symbiotic plasmid (pSym) contributes to partner quality decline. Moreover, patterns of genetic variation at selected loci were consistent with recent positive selection within N-fertilized environments, suggesting that N-rich environments might select for less beneficial rhizobia. By studying the molecular population genomics of a natural bacterial population within a long-term ecological field experiment, we find that: (i) the N environment is indeed a potent selective force mediating mutualism evolution in this symbiosis, (ii) natural variation in rhizobium partner quality is mediated in part by key symbiosis genes on the symbiotic plasmid, and (iii) differentiation at selected genes occurred in the context of otherwise recombining genomes, resembling eukaryotic models of adaptation.