Overcoming non-selective nodulation of Lessertia by soil-borne rhizobium in the presence of inoculant mesorhizobium

Background and aims

Legumes of the South African genus Lessertia, along with their microsymbionts, were introduced into the Western Australia wheatbelt. They achieved poor establishment followed by weak summer survival. This was caused in part by low levels of nodulation with the inoculant strains, and by ineffective nodulation with naturalized strains –an example of non-selective nodulation. The aims of this work were to assess Lessertia spp. symbiotic promiscuity, to study the effect of increased doses of an effective inoculant strain (WSM3565) with L. herbacea, and to study the competitive ability and symbiotic performance of different Mesorhizobium strains nodulating L. diffusa.

Methods

A glasshouse experiment was set up to evaluate the ability of L. diffusa, L. capitata, L. herbacea and L. excisa to nodulate with inoculants under current use in Western Australia. To assess competitive ability two field experiments were set up at Karridale, Western Australia. L. herbacea was inoculated with the strain WSM3565 at different doses and L. diffusa was inoculated with ten different Mesorhizobium strains. Rhizobia were re-isolated from nodules and their identity confirmed through PCR fingerprinting and sequencing of their partial dnaK.

Results

There were differences in promiscuity between different Lessertia spp., where L. herbacea proved to be highly promiscuous under controlled conditions. Increasing the inoculation dose of L. herbacea with WSM3565 did not improve establishment and survival of the legume in the field. Although WSM3565 nodule occupancy improved from 28 to 54 % with higher doses of inoculation, none of the treatments increased L. herbacea yield over the inoculated control. The inoculation of L. diffusa with the strains WSM3598, 3636, 3626 and 3565 resulted in greater biomass production than the uninoculated control. These strains were able to outcompete resident rhizobia and to occupy a high (>60 %) proportion of lateral root nodules. The naturalised strains that achieved nodulation were identified as R. leguminosarum.

Conclusion

The high numbers of resident rhizobia and their ability to rapidly nodulate Lessertia spp. are likely to be the main reasons for the low nodule occupancy achieved by some effective inoculant strains with L. diffusa and L. herbacea. Strains WSM 3636 and 3598 were very competitive on nodule occupancy and together with WSM 3565, WSM 3612 and WSM3626, effective on nodule formation and plant growth of L. diffusa despite the high numbers of resident soil rhizobia. These strains and L. diffusa have potential to be introduced as exotic legumes species and rhizobia strains to Western Australia.     

Does nitrogen transfer between plants confound 15N-based quantifications of N2 fixation?

Background and aims

Transfer of fixed N from legumes to non-legume reference plants may alter the ¹⁵N signature of the reference plant as compared to the soil N available to the legume. This study investigates how N transfer influences the result of ¹⁵N-based N₂ fixation measurements.

Methods

We labelled either legumes or non-legumes with ¹⁵N and performed detailed analyses of ¹⁵N enrichment in mixed plant communities in the field. The results were used in a conceptual model comparing how different N transfer scenarios influenced the ¹⁵N signatures of legumes and reference plants, and how the resulting N₂ fixation estimate was influenced by using reference plants in pure stand or in mixture with the legume.

Results

Based on isotopic signatures, N transfer was detected in all directions: from legume to legume, from legume to non-legume, from non-legume to legume, from non-legume to non-legume. In the scenario of multidirectional N transfer, N₂ fixation was overestimated by using a reference plant in pure stand.

Conclusions

Fixed N transferred to neighbouring reference plants modifies the ¹⁵N signature of the soil N available both to the reference plant and the N₂-fixing legume. This provides strong support for using reference plants growing in mixture with the legumes for reliable quantifications of N₂ fixation.     

Nitrogen contributions from faba bean (Vicia faba L.) reliant on soil rhizobia or inoculation

Background and Aims

Understanding the impact of soil rhizobial populations and inoculant rhizobia in supplying sufficient nodulation is crucial to optimising N₂ fixation by legume crops. This study explored the impact of different rates of inoculant rhizobia and contrasting soil rhizobia on nodulation and N₂ fixation in faba bean (Vicia faba L.).

Methods

Faba beans were inoculated with one of seven rates of rhizobial inoculation, from no inoculant to 100 times the normal rate of inoculation, sown at two field sites, with or without soil rhizobia present, and their nodulation and N₂ fixation assessed.

Results

At the site without soil rhizobia, inoculation increased nodule number and increased N₂ fixation from 21 to 129 kg shoot N ha^?1, while N₂ fixation increased from 132 to 218 kg shoot N ha^?1 at the site with high background soil rhizobia. At the site without soil rhizobia, inoculation increased concentrations of shoot N from 14 to 24 mg g^?1, grain N from 32 to 45 mg g^?1, and grain yields by 1.0 Mg (metric tonne) ha^?1. Differences in nodulation influenced the contributions of fixed N to the system, which varied from the net removal of 20 kg N ha^?1 from the system in the absence of rhizobia, to a net maximum input of 199 kg N ha^?1 from legume shoot and root residues, after accounting for removal of N in grain harvest.

Conclusions

The impact of inoculation and soil rhizobia strongly influenced grain yield, grain N concentration and the potential contributions of legume cropping to soil N fertility. In soil with resident rhizobia, N₂ fixation was improved only with the highest inoculation rate.     

Molecular mechanisms controlling legume autoregulation of nodulation

Background

High input costs and environmental pressures to reduce nitrogen use in agriculture have increased the competitive advantage of legume crops. The symbiotic relationship that legumes form with nitrogen-fixing soil bacteria in root nodules is central to this advantage.

Scope

Understanding how legume plants maintain control of nodulation to balance the nitrogen gains with their energy needs and developmental costs will assist in increasing their productivity and relative advantage. For this reason, the regulation of nodulation has been extensively studied since the first mutants exhibiting increased nodulation were isolated almost three decades ago.

Conclusions

Nodulation is regulated primarily via a systemic mechanism known as the autoregulation of nodulation (AON), which is controlled by a CLAVATA1-like receptor kinase. Multiple components sharing homology with the CLAVATA signalling pathway that maintains control of the shoot apical meristem in arabidopsis have now been identified in AON. This includes the recent identification of several CLE peptides capable of activating nodule inhibition responses, a low molecular weight shoot signal and a role for CLAVATA2 in AON. Efforts are now being focused on directly identifying the interactions of these components and to identify the form that long-distance transport molecules take.     

Nitrogen deposition decreases the benefits of symbiosis in a native legume

Aims

Anthropogenic nitrogen deposition can provide legumes with a cheap source of nitrogen relative to symbiotic nitrogen fixation, leading to the potential breakdown of this critical symbiosis. Here, the effects of nitrogen deposition were tested on a native symbiosis between legumes and rhizobia.

Methods

Deposition rates, soil nitrogen concentration, and plant nitrogen isotopic composition were quantified along a predicted deposition gradient in California. Acmispon strigosus seedlings were exposed to fertilization spanning nitrogen concentrations observed in the plant’s California range. Both wild and experimental plants from pristine and nitrogen polluted sites were tested using rhizobial strains that varied in nitrogen fixation.

Results

Deposition intensity was tightly correlated with nitrogen concentration in soils. The growth benefits of rhizobial nodulation were dramatically reduced by even modest levels of mineral nitrogen, and all Acmispon lines failed to form root nodules at high nitrogen concentrations.

Conclusions

Our dataset suggests that anthropogenic deposition has greatly increased soil nitrogen concentrations in Southern California leading to significantly reduced benefits of rhizobial symbiosis. If nitrogen deposition increases continue, plant host mortality and a total collapse of the symbiosis could result.

     

Improving N management through intercropping alleviates the inhibitory effect of mineral N on nodulation in pea

Background and aims

Symbiotic N₂ fixation is essential in the development of sustainable agriculture, but the nodulation of legumes is usually inhibited by N fertilization. Here, the intercropping of maize and pea in strips under various N managements was used as a means to alleviate the inhibitory effect of mineral N on pea nodulation and N₂ fixation and to improve system performance.

Methods

N natural abundance (δ ¹⁵N) analysis was employed to quantify N₂ fixation in the 3 years (2012 to 2014) of field experiment in Hexi Corridor of Northwestern China. Four N management systems with N rate of 0 kg N ha^?1 (the control), 90?+?45 kg N ha^?1 (base N plus topdressing N), 90?+?90 kg N ha^?1, and 90?+?135 kg N ha^?1 were implemented in the maize/pea strip intercropping to form different ratios of base N to topdressing N.

Results

Intercropped pea improved nodule biomass per plant by 99 %, increased nitrogen derived from the atmosphere (Ndfa) by 35 %, and promoted aboveground plant tissue N accumulation by 35 % as compared with sole pea, averaged across the four N treatments. Compared to the highest N fertilizer treatment, a reduction of topdressing to 45 kg N ha^?1 increased the nodule biomass of intercropped pea by 116 %, Ndfa by 35 %, and grain yield by 6 %.

Conclusions

Adaptation of suitable N management in cereal/legume intercropping systems will allow an effective conversion of atmospheric N₂ into crop available N and thus maximizing the system productivity.

     

Boron deficiency affects rhizobia cell surface polysaccharides important for suppression of plant defense mechanisms during legume recognition and for development of nitrogen-fixing symbiosis

Background and aims

Legumes of the genus Lessertia have recently been introduced to Australia in an attempt to increase the range of forage species available in Australian farming systems capable of dealing with a changing climate. This study assessed the diversity and the nodulation ability of a collection of Lessertia root nodule bacteria isolated from different agro-climatic areas of the Eastern and Western Capes of South Africa.

Methods

The diversity and phylogeny of 43 strains was determined via the partial sequencing of the dnaK, 16srRNA and nodA genes. A glasshouse experiment was undertaken to evaluate symbiotic relationships between six Lessertia species and 17 rhizobia strains.

Results

The dnaK and 16S rRNA genes of the majority of the strains clustered with the genus Mesorhizobium. The position of the strains at the intra-genus level was incongruent between phylogenies with few exceptions. The nodA genes from Lessertia spp. formed a cluster on their own, separate from the previously known Mesorhizobium nodA sequences. Strains showed differences in their nodulation and nitrogen fixation patterns that could be correlated with nodA gene phylogeny. L. diffusa, L. herbacea and L. excisa nodulated with nearly all the strains examined while L. capitata, L. incana and L. pauciflora were more stringent.

Conclusion

Root nodule bacteria from Lessertia spp. were identified mainly as Mesorhizobium spp. Their nodA genes were unique and correlated with the nodulation and nitrogen fixation patterns of the strains. There were marked differences in promiscuity within Lessertia spp. and within strains of root nodule bacteria.     

Biochar application reduces nodulation but increases nitrogenase activity in clover

Background and aims

Biochar is produced from the pyrolysis of organic materials, and when buried in soil can act as a long term soil carbon (C) store. Evidence suggests that biochar can also increase crop yields, reduce nutrient leaching and increase biological nitrogen fixation in leguminous plants. However, the potential for increasing biological N₂ fixation in agroecosystems is poorly understood, with inconsistent reports of root nodulation following biochar application. Therefore, the aim of this study was to determine the effect of biochar application rate and time since application on nodulation and nitrogenase activity in nodules of clover grown in a temperate agricultural soil.

Methods

We used replicated field plots with three biochar application rates (0, 25 and 50 t ha^?1). Three years after biochar amendment, the plots were further split and fresh biochar added at two different rates (25 and 50 t ha^?1) resulting in double-loaded reapplications of 25?+?25 and 50?+?50 t ha^?1.

Results

Three years after biochar application, there was no significant difference in the total number of root nodules between biochar-amended and unamended soil, regardless of the application rate. However, despite clover root nodules being of a similar number and size the level of nitrogenase activity of individual nodules in biochar-amended soil was significantly higher than in unamended soil. Reapplication of biochar resulted in decreased nodulation, although the rate of nitrogenase activity per nodule remained unaffected.

Conclusion

In the short term, biochar influences root nodule number and localised N₂ fixation per nodule; however, total nitrogenase activity for the whole root system remained unaffected by the application rate of biochar or time since its application. These results emphasise the importance of long-term field studies, with a variety of applications rates for determining the influence of biochar applications on N₂-fixing organisms and in providing data that can meaningfully inform agronomic management decisions and climate change mitigation strategies.     

Morphology and anatomy of root nodules of Retama monosperma (L.)Boiss.

Background and aims

In spite of the importance of Retama species for dune stabilization and re-vegetation and the contribution to the bio-fertilization of semi-arid and arid ecosystems, the symbiotic interaction of Retama species with rhizobia remains largely unstudied. In this paper, we aim to provide the first detailed study on nodule morphology and anatomy of Retama monosperma.

Methods

We collected nodules from coastal areas nearby Oran (Algeria) and studied in detail their anatomy and ultrastructure by light and electron microscopy.

Results

First, we confirmed the likely identity of the microsymbiont as B. retamae and found that nodules of R. monosperma belong to the genistoid type of indeterminate nodules. Infection threads, typical for most nodules of legumes, are absent in nodules of R. monosperma and bacterial spread is associated with plant cell division. The nitrogen fixation zone is homogenous with only invaded cells and a network of non-invaded cells found in many nodules, is absent. Moreover, endoreduplication does not take place in bacteroids in nodules of R. monosperma.

Conclusions

The features observed in this study are compared to the morphology and anatomy of nodules of other legumes and the possible consequences for nodule functioning and the mode of infection during the establishment of the interaction are discussed.     

Dry matter yield,root traits,and nodule occupancy of lucerne and Caucasian clover when grown in acidic soil with high aluminium concentrations

Aims and methods

Lucerne and Caucasian clover dry matter were measured in response to recommended lime and capital P inputs for six years in an acidic soil in the New Zealand high country. The initial three years of the field experiment indicated successful establishment and persistence of both legumes. Lucerne dry matter (DM) yield was up to 4 t/ha/yr in this period and higher than Caucasian clover yields. However, a lack of persistence of lucerne was apparent from this point forward compared with Caucasian clover which produced 7.7 t DM/ha in Year 6. An experiment using tubes of soil was used to investigate whether differences in root traits, nodulation and nodule occupancy were responsible for the differences observed in field persistence over time.

Results

These showed that when rhizobia inoculant was added, the fine root length of Caucasian clover was unaffected (R² = 0.14) by aluminium (Al) content of the soil. In contrast, fine root growth of lucerne was suppressed (R² = 0.79) by the soil Al content. Nodulation of Caucasian clover was unaffected by soil pH or Al when the rhizobia inoculant was provided which suggests the viability of the commercial genotype ICC148 in this soil with a pH of 5.5 and Al ca. 7 mg/kg soil. For lucerne, the maximum nodulation score of 7.3 occurred with 2 t/ha of lime added (soil pH ca.6, Al ca. 0.3 mg/kg) plus inoculant.

Conclusions

This suggests an Al toxic threshold of <1.0 mg Al/kg soil for effective lucerne nodulation. From the lucerne nodules, eight naturalized strains of Ensifer meliloti were identified. In contrast, only one R. leguminosarum strain was detected in the Caucasian clover nodules. The competition between those rhizobia genotypes may negatively affect the efficiency of biological nitrogen fixation in lucerne. Therefore, the lack of genetic diversity of R. leguminosarum bv. trifolii in New Zealand soils might be an advantage especially if the commercial strain is acid soil tolerant.

     

Nodulation and effective nitrogen fixation of Macroptilium atropurpureum (siratro) by Burkholderia tuberum, a nodulating and plant growth promoting beta-proteobacterium, are influenced by environmental factors

Background and aims

Burkholderia tuberum STM678^T was isolated from a South African legume, but did not renodulate this plant. Until a reliable host is found, studies on this and other interesting beta-rhizobia cannot advance. We investigated B. tuberum STM678^T’s ability to induce Fix⁺ nodules on a small-seeded, easy-to-propagate legume (Macroptilium atropurpureum). Previous studies demonstrated that B. tuberum elicited either Fix^- or Fix⁺ nodules on siratro, but the reasons for this difference were unexplored.

Methods

Experiments to promote effective siratro nodule formation under different environmental conditions were performed. B. tuberum STM678^T’s ability to withstand high temperatures and desiccation was checked as well as its potential for promoting plant growth via mechanisms in addition to nitrogen fixation, e.g., phosphate solubilization and siderophore production. Potential genes for these activities were found in the sequenced genomes.

Results

Higher temperatures and reduced watering resulted in reliable, effective nodulation on siratro. Burkholderia spp. solubilize phosphate and produce siderophores. Genes encoding proteins potentially involved in these growth-promoting activities were detected and are described.

Conclusions

Siratro is an excellent model plant for B. tuberum STM678^T. We identified genes that might be involved in the ability of diazotrophic Burkholderia species to survive harsh conditions, solubilize phosphate, and produce siderophores.     

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.     

Comparing Symbiotic Efficiency between Swollen versus Nonswollen Rhizobial Bacteroids

Symbiotic rhizobia differentiate physiologically and morphologically into nitrogen-fixing bacteroids inside legume host nodules. The differentiation is apparently terminal in some legume species, such as peas (Pisum sativum) and peanuts (Arachis hypogaea), likely due to extreme cell swelling induced by the host. In other legume species, such as beans (Phaseolus vulgaris) and cowpeas (Vigna unguiculata), differentiation into bacteroids, which are similar in size and shape to free-living rhizobia, is reversible. Bacteroid modification by plants may affect the effectiveness of the symbiosis. Here, we compare symbiotic efficiency of rhizobia in two different hosts where the rhizobia differentiate into swollen nonreproductive bacteroids in one host and remain nonswollen and reproductive in the other. Two such dual-host strains were tested: Rhizobium leguminosarum A34 in peas and beans and Bradyrhizobium sp. 32H1 in peanuts and cowpeas. In both comparisons, swollen bacteroids conferred more net host benefit by two measures: return on nodule construction cost (plant growth per gram nodule growth) and nitrogen fixation efficiency (H₂ production by nitrogenase per CO₂ respired). Terminal bacteroid differentiation among legume species has evolved independently multiple times, perhaps due to the increased host fitness benefits observed in this study.Legume-rhizobia interactions vary widely across a diverse paraphyletic group of soil bacteria known for symbiotic nitrogen fixation inside root nodules of over 18,000 species of legumes throughout the world (Lewis et al., 2005). In several legume species, rhizobial cells are induced to swell during their differentiation into nitrogen-fixing bacteroids (Oono et al., 2010). These legume species belong to five different major papilionoid clades (inverted repeat-lacking clade, genistoids, dalbergioids, mirbelioids, and millettioids), a pattern suggestive of convergent evolution. Swelling apparently leads to terminal differentiation; swollen bacteroids no longer divide normally (Zhou et al., 1985). In other legume host species, bacteroid differentiation is less extreme, leading to nonswollen bacteroids. Nonswollen bacteroids are similar in shape and size to free-living rhizobia and divide normally once outside of their nodules. The proximate mechanisms for host-imposed bacteroid swelling have been investigated (Van de Velde et al., 2010), but what drove the repeated evolution of this trait? The multiple independent origins of host traits causing bacteroids to swell suggest that swollen bacteroids may provide more net benefit to legumes. Could the swelling of bacteroids improve nitrogen fixation efficiency (e.g. nitrogen fixed relative to carbon cost)? In this study, we compare symbiotic efficiencies of rhizobia in legume hosts that are evolutionarily diverged but share a common effective rhizobial strain, whose bacteroids are swollen in one host and nonswollen in the other.Variations among host species in benefits and costs of symbiosis with rhizobia are not commonly explored (Thrall et al., 2000) because legume species typically nodulate with only one group of rhizobia (e.g. Sinorhizobium sp. in Medicago), although some legumes and some rhizobia are more promiscuous. Rhizobium sp. NGR234 has the largest known host range but does not fix nitrogen effectively with any legume species currently recognized to induce swelling of rhizobial bacteroids (Pueppke and Broughton, 1999). Some Sinorhizobium fredii strains apparently fix nitrogen in certain cultivars of soybean (Glycine max; hosting nonswollen bacteroids) and alfalfa (Medicago sativa; hosting swollen bacteroids; Hashem et al., 1997), but our efforts to replicate these results did not lead to successful nodulation. Therefore, we studied two strains, a transgenic strain that nodulates beans (Phaseolus vulgaris) and peas (Pisum sativum) and a second wild strain harvested from cowpeas (Vigna unguiculata) that also nodulates peanuts (Arachis hypogaea). Beans and cowpeas are both within the Phaseolid group and do not induce terminal differentiation of rhizobial bacteroids. Peas and peanuts both host terminally differentiated bacteroids but are in distant clades and likely have different genetic origins for traits that induce terminal differentiation (Oono et al., 2010). Also, the swollen bacteroids in peas are branched while those in peanuts are spherical.Differences in symbiotic qualities between swollen and nonswollen bacteroids have been previously explored in peanuts and cowpeas by Sen and Weaver (1980, 1981, 1984), who also hypothesized that swollen bacteroids are more beneficial to the host plant than nonswollen ones. They found 1.5 to 3 times greater acetylene reduction by nitrogenase (as well as plant nitrogen) per nodule mass in peanuts than in cowpeas at multiple nodule ages (Sen and Weaver, 1980). Acetylene reduction per bacteroid was also greater in peanuts than in cowpeas when measuring whole nodules, but this difference disappeared when isolated bacteroids were assayed (Sen and Weaver, 1984). They concluded that swelling of peanut bacteroids per se was not responsible for the higher rate of nitrogen fixation per bacteroid. They suggested that in cowpea nodules, with greater numbers of smaller bacteroids per nodule volume, availability of oxygen to each bacteroid might be restricted such that the rate of oxidative phosphorylation, necessary for nitrogen fixation, is reduced. Fixation rates per bacteroid may be different between hosts due to nodule gas permeability or bacteroid crowding within nodules. However, fixation efficiency (nitrogen fixed per carbon respired) would not necessarily be affected by these and may be more important for the host than the rate of fixation.Rhizobial performances are often compared by measuring the symbiotic benefits, e.g. rates of acetylene reduction or plant growth (Sen and Weaver, 1984; Hashem et al., 1997; Lodwig et al., 2005), but rarely by measuring the symbiotic costs, e.g. carbon consumed or respired. Up to 25% of a legume’s net photosynthate may be required for nitrogen fixation by rhizobia (Minchin et al., 1981). Faster fixation rates (mol nitrogen per s) can be beneficial for hosts, but carbon costs can also be important. Rhizobia that fix more nitrogen per carbon respired could free more carbon for other functions, including the option of supporting more nodules with the same amount of photosynthate. If legumes are sometimes carbon limited, then improved carbon-use efficiency could enhance plant fitness. Measuring both benefits and costs is therefore key to an accurate understanding of the symbiotic performance of a rhizobial strain.While we recognize the many physiological differences between peas and beans or peanuts and cowpeas, the fact that terminal differentiation induced by host legumes evolved multiple times independently (Oono et al., 2010) suggests there may be some consistent host symbiotic benefit, such as improved fixation efficiency. Here, we measured the efficiency of each of two strains as swollen bacteroids in one host and nonswollen bacteroids in another. We measured nitrogenase activity as hydrogen (H₂) production in an N₂-free atmosphere (Layzell et al., 1984; Witty and Minchin, 1998), and compared it to carbon dioxide (CO₂) respiration to estimate return on nodule operation cost. We also compared host biomass growth per total nodule mass growth to estimate return on nodule construction cost. To further assess carbon allocation to the different types of bacteroids, we also measured the average amounts per bacteroid of polyhydroxybutyrate (PHB), an energy storage compound that can comprise up to 50% of bacteroid dry weight (Trainer and Charles, 2006). A greater PHB accumulation per bacteroid may require a decreased allocation of carbon for nitrogenase activity within the bacteroids, and hence, less plant growth per carbon invested in bacteroids. We demonstrate that peas and peanuts that host swollen bacteroids have higher fixation efficiency as well as greater plant return on nodule construction than beans and cowpeas, respectively, nodulated with the same rhizobial strains. PHB was not consistently correlated with plant:nodule growth efficiency with the tested strains. These findings show that swollen bacteroids can indeed provide greater benefits to their legume hosts.     

Acacia,climate, and geochemistry in Australia

Background

Nitrogen-fixing legumes are key species in grassland ecosystems, as their ability to fix atmospheric nitrogen can facilitate neighboring plants. However, little is known about the fate of this legume effect in the face of extreme weather events, which are increasingly expected to occur.

Methods

Here, we examined experimentally how the presence of a legume modifies above-ground net primary production (ANPP) and nitrogen supply of neighboring non-legumes under annually recurrent pulsed drought and heavy rainfall events by comparing responses of three key species in European grassland versus without legume presence over 4 years.

Results

Legume presence facilitated community productivity of neighboring non-legumes under ambient weather conditions and also under experimental heavy rainfall. However, no facilitation of community productivity by the legume was found under experimental drought. Productivity of the three target species responded species-specifically to legume presence under different weather conditions: Holcus lanatus was facilitated only under control conditions, Plantago lanceolata was facilitated only under heavy rainfall, and Arrhenatherum elatius was facilitated irrespective of climate manipulations. The legume effects on δ ¹⁵N, leaf N concentration, and N uptake were also species-specific, yet irrespective of the climate manipulations. The data suggest that the missing legume effect on community productivity under the pulsed drought was rather caused by reduced N-uptake of the target species than by reduced N-fixation by the legume.

Conclusions

In contrast to heavy rain, the presence of a legume could not effectively buffer community ANPP against the negative effects of extreme drought events in an experimental temperate grassland. Facilitation also depends on the key species that are dominating a grassland community.     

N2 fixation, N transfer, and yield in grassland communities including a deep-rooted legume or non-legume species

Background and aims

Legumes are important components of grassland mixtures due to their ability to sustain high yields with moderate nitrogen inputs. This study investigates nitrogen relationships in mixtures of Trifolium pratense and grasses into which a deep-rooted forb was included, and particularly whether these realtionships differ when the forb is a legume or a non-legume species.

Methods

A field experiment in which mixtures of T. pratense, Phleum pratense, Lolium perenne, and Medicago sativa or Cichorium intybus, and monocropped stands of all species was established in 2007 and harvested in 2008 and 2009. The experiment received a total input of 100 kg?ha^?1?N yearly. Yield and botanical composition were determined in seven harvests. Species were analysed for ¹⁵N abundance, and N₂ fixation and N transfer were calculated. Soil samples were analysed twice for inorganic N.

Results

Non-legumes benefitted from the presence of legumes in terms of N concentration, and the yield of mixtures exceeded that of monocropped non-legumes but not monocropped legumes. The mixture containing M. sativa did not yield more DM or N than did the mixture containing C. intybus. A total of 17.08 kg?N ha^?1 was transferred from T. pratense to the non-legumes in the mixture in which it was the sole legume species.

Conclusions

It is concluded that there was a synergy effect in species mixtures, but the effect did not differ between the two deep-rooted species.     

Nodulation and nitrogen fixation in extreme environments

Biological nitrogen fixation is a phenomenon occurring in all known ecosystems. Symbiotic nitrogen fixation is dependent on the host plant genotype, theRhizobium strain, and the interaction of these symbionts with the pedoclimatic factors and the environmental conditions. Extremes of pH affect nodulation by reducing the colonization of soil and the legume rhizosphere by rhizobia. Highly acidic soils (pH<4.0) frequently have low levels of phosphorus, calcium, and molybdenum and high concentrations of aluminium and manganese which are often toxic for both partners; nodulation is more affected than host-plant growth and nitrogen fixation. Highly alkaline soils (pH>8.0) tend to be high in sodium chloride, bicarbonate, and borate, and are often associated with high salinity which reduce nitrogen fixation. Nodulation and N-fixation are observed under a wide range of temperatures with optima between 20–30°C. Elevated temperatures may delay nodule initiation and development, and interfere with nodule structure and functioning in temperate Iegumes, whereas in tropical legumes nitrogen fixation efficiency is mainly affected. Furthermore, temperature changes affect the competitive ability ofRhizobium strains. Low temperatures reduce nodule formation and nitrogen fixation in temperate legumes; however, in the extreme environment of the high arctic, native legumes can nodulate and fix nitrogen at rates comparable to those observed with legumes in temperate climates, indicating that both the plants and their rhizobia have successfully adapted to arctic conditions. In addition to low temperatures, arctic legumes are exposed to a short growing season, a long photoperiod, low precipitation and low soil nitrogen levels. In this review, we present results on a number of structural and physiological characteristics which allow arctic legumes to function in extreme environments.     

Enhanced chickpea growth-promotion ability of a Mesorhizobium strain expressing an exogenous ACC deaminase gene

Aims

The main goal of the study reported herein was to assess the nodulation performance of a Mesorhizobium strain transformed with an exogenous ACC deaminase gene (acdS), and its subsequent ability to increase chickpea plant growth under normal and waterlogged conditions.

Methods

The Mesorhizobium ciceri strain LMS-1 was transformed with the acdS gene of Pseudomonas putida UW4 by triparental conjugation using plasmid pRKACC. A plant growth assay was conducted to verify the plant growth promotion ability of the LMS-1 (pRKACC) transformed strain under normal and waterlogging conditions. Bacterial ACC deaminase and nitrogenase activity was measured.

Results

By expressing the exogenous acdS gene, the transformed strain LMS-1 showed a 127% increased ability to nodulate chickpea and a 125% promotion of the growth of chickpea compared to the wild-type strain, under normal conditions. Plants inoculated with the LMS-1 wild-type strain showed a higher nodule number under waterlogging stress than under control conditions, suggesting that waterlogging increases nodulation in chickpea. No significant relationship was found between ACC deaminase and nitrogenase activity.

Conclusions

The results obtained in this study show that the use of rhizobial strains with improved ACC deaminase activity might be very important for developing microbial inocula for agricultural purposes.     

Nodulation ability in different genotypes of <Emphasis Type="Italic">Phaseolus lunatus</Emphasis> by rhizobia from California agricultural soils

Phaseolus lunatus is the second economically most important species of the genus Phaseolus. It carries out N fixation through symbiosis with rhizobia. However, it is unclear whether P. lunatus can nodulate with native rhizobia from soils where this legume is not native or was not cultivated previously. Thus, this study assessed the ability of 14 geographically distant lima bean genotypes to nodulate with rhizobia from three California agricultural soils: without a history of legumes or P. lunatus cultivation, with a history of legumes as a cover crop, and with a history of P. lunatus cultivation. Nodulation only occurred on genotypes grown in the soil with a history of P. lunatus planting. The analysis of variance of nodulation traits showed that the genotype effect was highly significant in all the traits measured. Shoot biomass had a higher correlation with nodule size and nodule weight than with nodule number. In addition, shoot biomass and leaf N content were positively correlated with nodule coloration and with nodule position close to the main root of the plant. This study suggests that agricultural soils from California do not appear to have native rhizobia able to nodulate P. lunatus, which suggests the need to inoculate, at least initially, the seeds at planting in order to establish the population of rhizobia. Also, geographically distant lima bean genotypes have different responses to nodulating bacteria and it suggests that future studies to test these genotypes across different environments should be pursued.