The origins of uncooperative rhizobia

Mutualisms are thought to be destabilized by exploitative mutants that receive benefits from partners without reciprocation. Nonetheless, there is surprisingly little evidence for the spread of exploitation in mutualist populations. In particular mutualisms, non‐beneficial partners are commonplace and this raises the question of whether exploitation is invading as an adaptive strategy. Here, we highlight the legume–rhizobium mutualism as a key test case. Rhizobial bacteria fix nitrogen in legume roots in exchange for carbon from their hosts. However, non‐beneficial rhizobia are widespread, including non‐fixing and non‐nodulating strains. Recent research has shown that legumes can punish some uncooperative rhizobia and substantially reduce their fitness, but these sanctions must not be universally effective. Important questions about uncooperative rhizobia remain unresolved. (1) Is it adaptive for rhizobia to be uncooperative with hosts? (2) Do uncooperative rhizobia evolve from cooperative ancestors? (3) What are the mechanisms of rhizobial exploitation? We describe experimental approaches and testable hypotheses that address these gaps in our knowledge.     

Selection for cheating across disparate environments in the legume‐rhizobium mutualism

The primary dilemma in evolutionarily stable mutualisms is that natural selection for cheating could overwhelm selection for cooperation. Cheating need not entail parasitism; selection favours cheating as a quantitative trait whenever less‐cooperative partners are more fit than more‐cooperative partners. Mutualisms might be stabilised by mechanisms that direct benefits to more‐cooperative individuals, which counter selection for cheating; however, empirical evidence that natural selection favours cheating in mutualisms is sparse. We measured selection on cheating in single‐partner pairings of wild legume and rhizobium lineages, which prevented legume choice. Across contrasting environments, selection consistently favoured cheating by rhizobia, but did not favour legumes that provided less benefit to rhizobium partners. This is the first simultaneous measurement of selection on cheating across both host and symbiont lineages from a natural population. We empirically confirm selection for cheating as a source of antagonistic coevolutionary pressure in mutualism and a biological dilemma for models of cooperation.     

Lifestyle alternatives for rhizobia: mutualism, parasitism, and forgoing symbiosis

Strains of rhizobia within a single species can have three different genetically determined strategies. Mutualistic rhizobia provide their legume hosts with nitrogen. Parasitic rhizobia infect legumes, but fix little or no nitrogen. Nonsymbiotic strains are unable to infect legumes at all. Why have rhizobium strains with one of these three strategies not displaced the others? A symbiotic (mutualistic or parasitic) rhizobium that succeeds in founding a nodule may produce many millions of descendants. The chances of success can be so low, however, that nonsymbiotic rhizobia can have greater reproductive success. Legume sanctions against nodules that fix little or no nitrogen favor more mutualistic strains, but parasitic strains that use plant resources only for their own reproduction may do well when they share nodules with mutualistic strains.     

Mutualism as a constraint on invasion success for legumes and rhizobia

Because hereditary symbiont transmission is normally absent in the mutualism of legume plants and root‐nodule bacteria (rhizobia), dispersing plants may often arrive at new habitats where mutualist partners are too rare to provide full benefits. Factors governing invasion success were explored by analysing a system of two coupled pairwise competition models: a legume invader competing with a resident non‐mutualistic plant, and a rhizobial population competing with a resident population of nonsymbiotic bacteria. The non‐linear dependence of benefits on partner abundance in this mutualism creates the possibility of two alternative population size equilibria, so that a threshold density can exist for invasion. If legumes and rhizobia exceed a critical population size, both species achieve rapid population growth, while if initial densities of both species are below their respective thresholds, they remain rare and are thus vulnerable to extinction in the presence of competitors. Overall, the results indicate that legumes may often fail at colonization attempts within habitats where mutualist partners are scarce. Data on legume prevalence in island floras and rates of geographical spread by legume weeds are consistent with this inference. Predictive insights about invasiveness may emerge from comparative research on key traits identified by the model, especially the shape of the function determining the number of nodules formed at low rhizobial density.     

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.     

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.     

Manipulation of Ethylene Synthesis in Roots Through Bacterial ACC Deaminase for Improving Nodulation in Legumes

Symbiotic association between rhizobia and legumes results in the development of unique structures on roots, called nodules. Nodulation is a very complex process involving a variety of genes that control NOD factors (bacterial signaling molecules), which are essential for the establishment, maintenance and regulation of this process and development of root nodules. Ethylene is an established potent plant hormone that is also known for its negative role in nodulation. Ethylene is produced endogenously in all plant tissues, particularly in response to both biotic and abiotic stresses. Exogenous application of ethylene and ethylene-releasing compounds are known to inhibit the formation and functioning of nodules. While inhibitors of ethylene synthesis or its physiological action enhance nodulation in legumes, some rhizobial strains also nodulate the host plant intensively, most likely by lowering endogenous ethylene levels in roots through their 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. Co-inoculation with ACC deaminase containing plant growth promoting rhizobacteria plus rhizobia has been shown to further promote nodulation compared to rhizobia alone. Transgenic rhizobia or legume plants with expression of bacterial ACC deaminase could be another viable option to alleviate the negative effects of ethylene on nodulation. Several studies have well documented the role of ethylene and bacterial ACC deaminase in development of nodules on legume roots and will be the primary focus of this critical review.     

Long‐term nitrogen addition causes the evolution of less‐cooperative mutualists

Human activities have altered the global nitrogen (N) cycle, and as a result, elevated N inputs are causing profound ecological changes in diverse ecosystems. The evolutionary consequences of this global change have been largely ignored even though elevated N inputs are predicted to cause mutualism breakdown and the evolution of decreased cooperation between resource mutualists. Using a long‐term (22 years) N‐addition experiment, we find that elevated N inputs have altered the legume–rhizobium mutualism (where rhizobial bacteria trade N in exchange for photosynthates from legumes), causing the evolution of less‐mutualistic rhizobia. Plants inoculated with rhizobium strains isolated from N‐fertilized treatments produced 17–30% less biomass and had reduced chlorophyll content compared to plants inoculated with strains from unfertilized control plots. Because the legume–rhizobium mutualism is the major contributor of naturally fixed N to terrestrial ecosystems, the evolution of less‐cooperative rhizobia may have important environmental consequences.     

Analysis of the legume-rhizobia symbiosis in shrubs from central western Spain

AIMS: This work analyses the diversity of rhizobia associated with some of the predominant shrubby legumes in central-western Spain. Symbiotic promiscuity and effectiveness were studied using cross-inoculation experiments with shrubby species. MATERIAL AND RESULTS: Six new bradyrhizobia strains were isolated from nodules collected from wild plants of six leguminous species, Cytisus balansae, C. multiflorus, C. scoparius, C. striatus, Genista hystrix and Retama sphaerocarpa. These isolates were genetically characterized by 16S rDNA partial sequencing and random amplification of polymorphic DNA-PCR fingerprinting. The phylogenetic analysis revealed that these isolates could represent three new Bradyrhizobium species. Shrubby legumes and bradyrhizobia displayed a high symbiotic promiscuity both for infectivity and effectiveness. Symbioses were effective in more than 70% of the associations established by four of the six plant species. CONCLUSIONS: Native woody legumes in western Spain are nodulated by Bradyrhizobium strains. The high degree of symbiotic promiscuity and effectiveness highlights the complex dynamics of these communities in wild ecosystems under a Mediterranean-type climate. Furthermore, the results from this study suggest a potential importance of inoculation for these legume species in soil-restoration projects. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study, to our knowledge, that combines both molecular analysis and pot trials to study the rhizobia-legume symbiosis for wild legumes.     

Sanctions and mutualism stability: when should less beneficial mutualists be tolerated?

Why do mutualists perform costly behaviours that benefit individuals of a different species? One of the factors that may stabilize mutualistic interactions is when individuals preferentially reward more mutualistic (beneficial) behaviour and/or punish less mutualistic (more parasitic) behaviour. We develop a model that shows how such sanctions provide a fitness benefit to the individuals that carry them out. Although this approach could be applied to a number of symbioses, we focus on how it could be applied to the legume‐rhizobia interaction. Specifically, we demonstrate how plants can be selected to supply preferentially more resources to (or be less likely to senesce) nodules that are fixing more N₂ (termed plant sanctions). We have previously argued that appreciable levels of N₂ fixation by rhizobia are only likely to be selected for in response to plant sanctions. Therefore, by showing that plant sanctions can also be favoured by natural selection, we are able to provide an explanation for the stability of the plant‐legume mutualism.     

Standing genetic variation in host preference for mutualist microbial symbionts

Many models of mutualisms show that mutualisms are unstable if hosts lack mechanisms enabling preferential associations with mutualistic symbiotic partners over exploitative partners. Despite the theoretical importance of mutualism-stabilizing mechanisms, we have little empirical evidence to infer their evolutionary dynamics in response to exploitation by non-beneficial partners. Using a model mutualism—the interaction between legumes and nitrogen-fixing soil symbionts—we tested for quantitative genetic variation in plant responses to mutualistic and exploitative symbiotic rhizobia in controlled greenhouse conditions. We found significant broad-sense heritability in a legume host''s preferential association with mutualistic over exploitative symbionts and selection to reduce frequency of associations with exploitative partners. We failed to detect evidence that selection will favour the loss of mutualism-stabilizing mechanisms in the absence of exploitation, as we found no evidence for a fitness cost to the host trait or indirect selection on genetically correlated traits. Our results show that genetic variation in the ability to preferentially reduce associations with an exploitative partner exists within mutualisms and is under selection, indicating that micro-evolutionary responses in mutualism-stabilizing traits in the face of rapidly evolving mutualistic and exploitative symbiotic bacteria can occur in natural host populations.     

Nonlegume Parasponia andersonii deploys a broad rhizobium host range strategy resulting in largely variable symbiotic effectiveness

The non-legume genus Parasponia has evolved the rhizobium symbiosis independent from legumes and has done so only recently. We aim to study the promiscuity of such newly evolved symbiotic engagement and determine the symbiotic effectiveness of infecting rhizobium species. It was found that Parasponia andersonii can be nodulated by a broad range of rhizobia belonging to four different genera, and therefore, we conclude that this non-legume is highly promiscuous for rhizobial engagement. A possible drawback of this high promiscuity is that low-efficient strains can infect nodules as well. The strains identified displayed a range in nitrogen-fixation effectiveness, including a very inefficient rhizobium species, Rhizobium tropici WUR1. Because this species is able to make effective nodules on two different legume species, it suggests that the ineffectiveness of P. andersonii nodules is the result of the incompatibility between both partners. In P. andersonii nodules, rhizobia of this strain become embedded in a dense matrix but remain vital. This suggests that sanctions or genetic control against underperforming microsymbionts may not be effective in Parasponia spp. Therefore, we argue that the Parasponia-rhizobium symbiosis is a delicate balance between mutual benefits and parasitic colonization.     

Coexistence of predominantly nonculturable rhizobia with diverse, endophytic bacterial taxa within nodules of wild legumes

A previous analysis showed that Gammaproteobacteria could be the sole recoverable bacteria from surface-sterilized nodules of three wild species of Hedysarum. In this study we extended the analysis to eight Mediterranean native, uninoculated legumes never previously investigated regarding their root-nodule microsymbionts. The structural organization of the nodules was studied by light and electron microscopy, and their bacterial occupants were assessed by combined cultural and molecular approaches. On examination of 100 field-collected nodules, culturable isolates of rhizobia were hardly ever found, whereas over 24 other bacterial taxa were isolated from nodules. None of these nonrhizobial isolates could nodulate the original host when reinoculated in gnotobiotic culture. Despite the inability to culture rhizobial endosymbionts from within the nodules using standard culture media, a direct 16S rRNA gene PCR analysis revealed that most of these nodules contained rhizobia as the predominant population. The presence of nodular endophytes colocalized with rhizobia was verified by immunofluorescence microscopy of nodule sections using an Enterobacter-specific antibody. Hypotheses to explain the nonculturability of rhizobia are presented, and pertinent literature on legume endophytes is discussed.     

Assessing the suitability of antibiotic resistance markers and the indirect ELISA technique for studying the competitive ability of selected Cyclopia Vent. rhizobia under glasshouse and field conditions in South Africa

Background  

Symbiotic N2 fixation in legumes is constrained by many factors, including the paucity of suitable soil rhizobia To maximise growth of legume species therefore often requires the application of effective rhizobia as inoculants. But where native strains out-compete introduced rhizobia for nodule formation, it is important that the competitiveness of selected strains is tested in the field and glasshouse prior to their recommendation as commercial inoculants. However the methodology for strain identification inside nodules has often proved difficult and thus limited this field of research. In this study, the suitability of the antibiotic resistance technique (both intrinsic low-resistance fingerprinting and high-resistance marking) and the serological indirect ELISA method were assessed for their ability to detect selected Cyclopia rhizobia under glasshouse and field conditions. The four rhizobial strains that were used, namely PPRICI3, UCT40a, UCT44b and UCT61a, were isolated from wild Cyclopia species growing in the Western Cape fynbos of South Africa.     

The presence of nodules on legume root systems can alter phenotypic plasticity in response to internal nitrogen independent of nitrogen fixation

All higher plants show developmental plasticity in response to the availability of nitrogen (N) in the soil. In legumes, N starvation causes the formation of root nodules, where symbiotic rhizobacteria fix atmospheric N₂ for the host in exchange for fixed carbon (C) from the shoot. Here, we tested whether plastic responses to internal [N] of legumes are altered by their symbionts. Glasshouse experiments compared root phenotypes of three legumes, Medicago truncatula, Medicago sativa and Trifolium subterraneum, inoculated with their compatible symbiont partners and grown under four nitrate levels. In addition, six strains of rhizobia, differing in their ability to fix N₂ in M. truncatula, were compared to test if plastic responses to internal [N] were dependent on the rhizobia or N₂‐fixing capability of the nodules. We found that the presence of rhizobia affected phenotypic plasticity of the legumes to internal [N], particularly in root length and root mass ratio (RMR), in a plant species‐dependent way. While root length responses of M. truncatula to internal [N] were dependent on the ability of rhizobial symbionts to fix N₂, RMR response to internal [N] was dependent only on initiation of nodules, irrespective of N₂‐fixing ability of the rhizobia strains.     

Legume nodulation: The host controls the party

Global demand to increase food production and simultaneously reduce synthetic nitrogen fertilizer inputs in agriculture are underpinning the need to intensify the use of legume crops. The symbiotic relationship that legume plants establish with nitrogen‐fixing rhizobia bacteria is central to their advantage. This plant–microbe interaction results in newly developed root organs, called nodules, where the rhizobia convert atmospheric nitrogen gas into forms of nitrogen the plant can use. However, the process of developing and maintaining nodules is resource intensive; hence, the plant tightly controls the number of nodules forming. A variety of molecular mechanisms are used to regulate nodule numbers under both favourable and stressful growing conditions, enabling the plant to conserve resources and optimize development in response to a range of circumstances. Using genetic and genomic approaches, many components acting in the regulation of nodulation have now been identified. Discovering and functionally characterizing these components can provide genetic targets and polymorphic markers that aid in the selection of superior legume cultivars and rhizobia strains that benefit agricultural sustainability and food security. This review addresses recent findings in nodulation control, presents detailed models of the molecular mechanisms driving these processes, and identifies gaps in these processes that are not yet fully explained.     

Phosphorus deprivation affects composition and spatial distribution of membrane lipids in legume nodules

In legumes, symbiotic nitrogen (N) fixation (SNF) occurs in specialized organs called nodules after successful interactions between legume hosts and rhizobia. In a nodule, N-fixing rhizobia are surrounded by symbiosome membranes, through which the exchange of nutrients and ammonium occurs between bacteria and the host legume. Phosphorus (P) is an essential macronutrient, and N₂-fixing legumes have a higher requirement for P than legumes grown on mineral N. As in the previous studies, in P deficiency, barrel medic (Medicago truncatula) plants had impaired SNF activity, reduced growth, and accumulated less phosphate in leaves, roots, and nodules compared with the plants grown in P sufficient conditions. Membrane lipids in M. truncatula tissues were assessed using electrospray ionization–mass spectrometry. Galactolipids were found to increase in P deficiency, with declines in phospholipids (PL), especially in leaves. Lower PL losses were found in roots and nodules. Subsequently, matrix-assisted laser desorption/ionization–mass spectrometry imaging was used to spatially map the distribution of the positively charged phosphatidylcholine (PC) species in nodules in both P-replete and P-deficient conditions. Our results reveal heterogeneous distribution of several PC species in nodules, with homogeneous distribution of other PC classes. In P poor conditions, some PC species distributions were observed to change. The results suggest that specific PC species may be differentially important in diverse nodule zones and cell types, and that membrane lipid remodeling during P stress is not uniform across the nodule.

ESI–MS and matrix-assisted laser desorption ionization–mass spectrometry imaging reveal alterations in Medicago truncatula nodules membrane lipid composition and spatial distribution in phosphorus deficiency.