A new species of Devosia that forms a unique nitrogen-fixing root-nodule symbiosis with the aquatic legume Neptunia natans (L.f.) druce

Rhizobia are the common bacterial symbionts that form nitrogen-fixing root nodules in legumes. However, recently other bacteria have been shown to nodulate and fix nitrogen symbiotically with these plants. Neptunia natans is an aquatic legume indigenous to tropical and subtropical regions and in African soils is nodulated by Allorhizobium undicola. This legume develops an unusual root-nodule symbiosis on floating stems in aquatic environments through a unique infection process. Here, we analyzed the low-molecular-weight RNA and 16S ribosomal DNA (rDNA) sequence of the same fast-growing isolates from India that were previously used to define the developmental morphology of the unique infection process in this symbiosis with N. natans and found that they are phylogenetically located in the genus Devosia, not Allorhizobium or RHIZOBIUM: The 16S rDNA sequences of these two Neptunia-nodulating Devosia strains differ from the only species currently described in that genus, Devosia riboflavina. From the same isolated colonies, we also located their nodD and nifH genes involved in nodulation and nitrogen fixation on a plasmid of approximately 170 kb. Sequence analysis showed that their nodD and nifH genes are most closely related to nodD and nifH of Rhizobium tropici, suggesting that this newly described Neptunia-nodulating Devosia species may have acquired these symbiotic genes by horizontal transfer.     

Coculture of Pachyrhizus erosus (L.) hairy roots with Rhizobium spp.

Summary We have established an in vitro system for the induction and study of nodulation in Pachyrhizus erosus (jicama) via a hairy root-Rhizobium coculture. In vitro-grown P. erosus plantlets were infected with Agrobacterium rhizogenes (ATCC No. 15834) and two hairy root lines were established. Hairy roots were grown in a split-plate system in which compartment I (CI) contained MS medium with nitrogen and different sucrose levels (0–6%), while CII held MS medium without nitrogen and sucrose. Nodule-like structures developed in transformed roots grown in CI with 2–3% surcose, inoculated with Rhizobium sp. and transferred to CII. Nodule-like structures that developed from hairy roots lacked the rigid protective cover observed in nodules from plants grown in soil. Western blot analysis of nodules from hairy roots and untransformed roots (of greenhouse-grown jicama) showed expression of glutamine synthetase leghemoglobin and nodulins. Leghemoglobin was expressed at low levels in hairy root nodules.     

TheRhizobium-legume symbiosis Two methods to discriminate between nodules and other root-derived structures

Summary Two methods have been developed in order to discriminate between lateral roots, nodules and root-derived structures which exhibit both root and nodule histological features and which can develop on legumes inoculated with certainRhizobium mutants. The first method, known as the clearing method, allows the observation by light microscopy of cleared undissected root-structures. The second, known as the slicing method, is a complementary technique which provides a greater degree of structural information concerning such structures. The two methods have proved invaluable in defining unequivocally the nature of the interaction between a rhizobial strain and a legume host.     

The root-nodule symbiosis between Sarothamnus scoparius L. and its microsymbionts

When nitrogen fixing root nodules are formed, Sarothamnus scoparius (broom) is inoculated with its microsymbionts. Nodules studied under light and electron microscopy exhibited typical indeterminate nodule histology with apical, persistent meristem, age gradient of nodule tissues, and open vascular bundles, and also with some particular features such as: the presence of mitotic activity in the infected meristematic cells, lack of infection threads, distribution of bacteria by process of host cell division, and occurrence of a large bacteroid zone only with infected cells. The results of cross-inoculation tests have shown a broad host range for S. scoparius microsymbionts including not only the native host but also species such as: Lupinus luteus, Ornithopus sativa, Lotus corniculatus, Genista tinctoria, Chamaecitisus ratisbonensis, Macroptilium atropurpureum, and Phaseolus vulgaris. In addition, our data established a close symbiotic relationship of S. scoparius nodulators to Bradyrhizobium sp. (Lupinus) by comparison of the partial sequence of nodC gene of the strain CYT7, specific for the broom, to those from Bradyrhizobium sp. (Lupinus) strain D1 and others available in the public databases.     

Correlation between infection by Rhizobium leguminosarum and lectin on the surface of Pisum sativum L. roots

The lectin on the surface of 4- and 5-dold pea roots was located by the use of indirect immunofluorescence. Specific antibodies raised in rabbits against pea seed isolectin 2, which crossreact with root lectins, were used as primary immunoglobulins and were visualized with fluorescein- or tetramethylrhodamine-isothiocyanate-labeled goat antirabbit immunoglobulin G. Lectin was observed on the tips of newly formed, growing root hairs and on epidermal cells located just below the young hairs. On both types of cells, lectin was concentrated in dense small patches rather than uniformly distributed. Lectin-positive young hairs were grouped opposite the (proto)xylematic poles. Older but still-elongating root hairs presented only traces of lectin or none at all. A similar pattern of distribution was found in different pea cultivars, as well as in a supernodulating and a non-nodulating pea mutant. Growth in a nitrate concentration which inhibits nodulation did not affect lectin distribution on the surface of pea roots of this age. We tested whether or not the root zones where lectin was observed were susceptible to infection by Rhizobium leguminosarum. When low inoculum doses (consisting of less than 10⁶ bacteria·ml^-1) were placed next to lectin-positive epidermal cells and on newly formed root hairs, nodules on the primary roots were formed in 73% and 90% of the plants, respectively. Only a few plants showed primary root nodulation when the inoculum was placed on the root zone where lectin was scarce or absent. These results show that lectin is present at those sites on the pea root that are susceptible to infection by the bacterial symbiont.Abbreviations FITC fluorescein isothiocyanate - TRIC tetramethylrhodamine isothiocyanate     

Nitrate effects on the nodulation of legumes inoculated with nitrate-reductase-deficient mutants of Rhizobium

The effect of nitrate on the symbiotic properties of nitrate-reductase-deficient mutants of a strain of cowpea rhizobia (32H1), and of a strain of Rhizobium trifolii (TA1), were examined; the host species were Macroptilium atropurpureum (DC.) Urb. and Trifolium subterraneum L. Nitrate retarded initial nodulation by the mutant strains to an extent similar to that found with the parent strains. It is therefore unlikely that nitrite produced from nitrate by the rhizobia, plays a significant role in the inhibition of nodulation by nitrate. Nitrite is an inhibitor of nitrogenase, and its possible production in the nodule tissue by the action of nitrate reductase could be responsible for the observed inhibition of nitrogen fixation when nodulated plants are exposed to nitrate. However, the results of this investigation show that nitrogen fixation by the plants nodulated by parent or mutant strains was depressed by similar amounts in the presence of nitrate. No nitrite was detected in the nodules. Nodule growth, and to a lesser extent, the nitrogenase specific activity of the nodules (mol C₂H₄g^–1 nodule fr. wt. h^–1), were both affected by the added nitrate.     

Relationship between growth,nitrogen fixation and assimilation in a legume (Medicago sativa L.)

Summary Symbiotic N₂ fixation, NO ₃ ⁻ assimilation and protein accumulation in the shoots were measured simultaneously in alfalfa (Medicago sativa L.) grown in the field or in pots, in order to study how the balance between the two modes of nitrogen nutrition could be influenced by agronomic factors, such as harvest, mineral nitrogen supply and drought stress. During periods of rapid growth, fixation and assimilation may function simultaneously; they are antagonistic at the beginning and at the end of the growth cycle, when the nitrogen requirement of the plant is lower. When nitrogen nutrition does not limit growth, mineral nitrogen supply favours assimilation at the expense of fixation, but does not modify the amount of nitrogen accumulated, which is adjusted to the growth capacity of the plant. After cutting, nitrate assimilation compensated for the decrease in fixation and supplied the plant with the nitrogen required by the regrowth, the proliferation of which determined the fixation recovery. Drought stress decreased N₂ fixation much more than NO ₃ ⁻ assimilation. The latter made growth recovery possible when water supply conditions became normal again. These results suggested the existence of an optimum level of nitrate assimilation, which differed depending on the age of the plants and allowed both maximum growth and fixing activity.     

Host genetic control of symbiosis in soybean (Glycine max L.)

Genes controlling nitrogen-fixing symbioses of legumes with specialized bacteria known as rhizobia are presumably the products of many millions of years of evolution. Different adaptative solutions evolved in response to the challenge of survival in highly divergent complexes of symbionts. Whereas efficiency of nitrogen fixation appears to be controlled by quantitative inheritance, genes controlling nodulation are qualitatively inherited. Genes controlling nodulation include those for non-nodulation, those that restrict certain microsymbionts, and those conditioning hypernodulation, or supernodulation. Some genes are naturally occurring polymorphisms, while others were induced or were the result of spontaneous mutations. The geographic patterns of particular alleles indicate the role of coevolution in determining symbiont specificites and compatibilities. For example, the Rj4 allele occurs with higher frequency (over 50%) among the soybean (G. max) from Southeast Asia. DNA homology studies of strains of Bradyrhizobium that nodulate soybean indicated two groups so distinct as to warrant classification as two species. Strains producing rhizobitoxine-induced chlorosis occur only in Group II, now classified as B. elkanii. Unlike B. japonicum, B. elkanii strains are characterized by (1) the ability to nodulate the rj1 genotype, (2) the formation of nodule-like structures on peanut, (3) a relatively high degree of ex planta nitrogenase activity, (4) distinct extracellular polysaccharide composition, (5) distinct fatty acid composition, (6) distinct antibiotic resistance profiles, and (7) low DNA homology with B. japonicum. Analysis with soybean lines near isogenic for the Rj4 versus rj4 alleles indicated that the Rj4 allele excludes a high proportion of B. elkanii strains and certain strains of B. japonicum such as strain USDA62 and three serogroup 123 strains. These groups, relatively inefficient in nitrogen fixation with soybean, tend to predominate in soybean nodules from many US soils. The Rj4 allele, the most common allelic form in the wild species, has a positive value for the host plants in protecting them from nodulation by rhizobia poorly adapted for symbiosis.     

Induced genetic variability in Rhizobium leguminosarum for nitrogen fixation parameters in Vicia faba L.

Sumary The objective of this work was to know the behaviour and variability of Rhizobium leguminosarum after irradiation. The induced variation was tested under greenhouse conditions on the variety JV 3 of broad beans (Vicia faba) in six replications. Induced genetic variabilty was observed for strain, parent and mutant versus parent. Out of 24 irradiated strains, strain 93-32 performed better with a greater number of nodules and higher dry weight of nodules per plant and biological yield. Environment played an important role in the expression of characters observed. High heritability and genetic advance of these traits indicated that the nitrogen fixation ability of Rhizobium can easily be improved by selection.     

Selection for improved nitrogen fixation inGlycine max (L.) Merr. andPhaseolus vulgaris L.

Summary While symbiotic nitrogen (N₂) fixation byG. max andP. vulgaris reduces their need for combined N, N₂ fixation under field conditions is rarely maximized. This paper reviews constraints to N₂ fixation in these species, then examines the genetic variability recorded for traits affecting N₂ fixation and the further work needed in this area. It considers emerging programs for the improvement of N₂ fixation inG. max andP. vulgaris and pays particular attention to methodological considerations.Scientific Journal Series, Minnesota Agri. Exp. Station. No 14190.     

The ethylene-inhibitor aminoethoxyvinylglycine restores normal nodulation by Rhizobium leguminosarum biovar. viciae on Vicia sativa subsp. nigra by suppressing the ‘Thick and short roots’ phenotype

Nodulation of Vicia sativa subsp. nigra L. by Rhizobium bacteria is coupled to the development of thick and short roots (Tsr). This root phenotype as well as root-hair induction (Hai) and root-hair deformation (Had) are caused by a factor(s) produced by the bacteria in response to plant flavonoids. When very low inoculum concentrations (0.5–5 bacteria·ml^-1) were used, V. sativa plants did not develop the Tsr phenotype and became nodulated earlier than plants with Tsr roots. Furthermore, the nodules of these plants were located on the primary root in contrast to nodules on Tsr roots, which were all located at sites of lateral-root emergence. The average numbers of nodules per plant were not significantly different for these two types of nodulation. Root-growth inhibition and Hai, but not Had, could be mimicked by ethephon, and inhibited by aminoethoxyvinylglycine (AVG). Addition of AVG to co-cultures of Vicia sativa and the standard inoculum concentration of 5·10⁵ bacteria·ml^-1 suppressed the development of the Tsr phenotype and restored nodulation to the pattern that was observed with very low concentrations of bacteria (0.5–5 bacteria·ml^-1). The delay in nodulation on Tsr roots appeared to be caused by the fact that nodule meristems did not develop on the primary root, but only on the emerging laterals. The relationship between Tsr, Hai, Had, and nodulation is discussed.Abbreviations AVG aminoethoxyvinylglycine - cfu colonyforming units - Had root-hair deformation - Hai root-hair induction - NB naringenin-bacteria filtrate - Tsr Thick and short roots     

Asymbiotic association of Rhizobium with pea epicotyls treated with a plant hormone

Treatment of epicotyls of dark-grown pea (Pisum sativum L.) seedlings with indole-3-acetic acid causes swelling of the tissue. Application of Rhizobium to the cut surface of the swollen tissue results in the development of an infection. The infection spreads in the cortical cells and proceeds 2–3 mm deep into the stem within 3–4 days. An acetylene reduction assay used for detecting nitrogen-fixation capacity of the infected tissue was negative at 10% [O₂]; however, if [O₂] was reduced to below 1%, some activity could be detected. Ultrastructural observations indicate that the cytoplasmic contents of the infected cells are destroyed and no membrane structure around the bacteria is formed during this infection. Rhizobium does not appear to have developed any symbiotic relationship with the host. Failure to develop symbiosis appears to result in a parasitic or saprophytic association and the nitrogen fixed under such conditions may not be of any use to the plant.     

Lectin-gene expression in pea (Pisum sativum L.) roots

The expression of a lectin gene in pea (Pisum sativum L.) roots has been investigated using the copy DNA of a pea seed lectin as a probe. An mRNA which has the same size as the seed mRNA but which is about 4000 times less abundant has been detected in 21-d-old roots. The probe detected lectin expression as early as 4 d after sowing, with the highest level being reached at 10 d, i.e. just before nodulation. In later stages (16-d- and 21-d-old roots), expression was substantially decreased. The correlation between infection by Rhizobium leguminosarum and lectin expression in pea roots has been investigated by comparing root lectin mRNA levels in inoculated plants and in plants grown under conditions preventing nodulation. Neither growth in a nitrate concentration which inhibited nodulation nor growth in the absence of Rhizobium appreciably affected lectin expression in roots.Abbreviation cDNA copy DNA - poly(A)⁺RNA polyadenylated RNA     

Nodulation,nitrogen fixation and nitrogen uptake in pigeonpea (Cajanus cajan (L.) Millsp) of different maturity groups

Summary The seasonal patterns of nodulation, acetylene reduction, nitrogen uptake and nitrogen fixation were studies for 11 pigeonpea cultivars belonging to different maturity groups grown on an Alfisol at ICRISAT Center, Patancheru, India. In all cultivars the nodule number and mass increased to a maximum around 60–80 days after sowing and then declined. The nodule number and mass of medium- and late-maturing cultivars was greater than that of early-maturing cultivars. The nitrogenase activity per plant increased to 60 days after sowing and declined thereafter, with little activity at 100 days when the crop was flowering. At later stages of plant growth nodules formed down to 90 cm below the soil surface but those at greater depth appeared less active than those near the surface. All the 11 cultivars continued to accumulate dry matter until 140 days, with most biomass production by the late-maturing cultivars (up to 11 t ha⁻¹) and least by the early-maturing determinate cultivars (4 t ha⁻¹). Total nitrogen uptake ranged from 69 to 134 kg ha⁻¹. Nitrogen fixation by pigeonpea was estimated as the difference in total nitrogen uptake between pigeonpea and sorghum and could amount to 69 kg N ha⁻¹ per season, or half the total nitrogen uptake. Fixation by pigeonpea increased with crop duration, but there were differences within each maturity group. The limitations of the methods used for estimating N₂ fixation by pigeonpea are discussed. Submitted as J.A. No. 552 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT).     

Aerial root nodules in the tropical legume,Pentaclethra macroloba

Summary Symbiotic nitrogen fixation in angiosperms normally occurs in buried root nodules and is severely inhibited in flooded soils. A few plant species, however, respond to flooding by forming nodules on stems, or, in one case, submerged roots with aerenchyma. We report here the novel occurrence of aerial rhizobial nodules attached to adventitious roots of the legume,Pentaclethra macroloba, in a lowland tropical rainforest swamp in Costa Rica. Swamp sapdings (1–10 cm diameter) support an average 12 g nodules dry weight per plant on roots 2–300 cm above water, and nodules remain in aerial positions at least 6 months. Collections from four swamp plants maintained linear activity rates (3–14 moles C₂H₄/g nodule dry weight/hr) throughout incubations for 6 and 13 hrs; excised nodule activity in most legumes declines after 1–2 hrs. Preliminary study of the anatomy and physiology suggest aerial nodules possess unusual features associated with tolerance to swamp conditions. High host tree abundance and nodulation in the swamp compared to upland sites indicate the aerial root symbiosis may contribute more fixed nitrogen to the local ecosystem than the more typical buried root symbiosis.     

A New Species of Devosia That Forms a Unique Nitrogen-Fixing Root-Nodule Symbiosis with the Aquatic Legume Neptunia natans (L.f.) Druce

Rhizobia are the common bacterial symbionts that form nitrogen-fixing root nodules in legumes. However, recently other bacteria have been shown to nodulate and fix nitrogen symbiotically with these plants. Neptunia natans is an aquatic legume indigenous to tropical and subtropical regions and in African soils is nodulated by Allorhizobium undicola. This legume develops an unusual root-nodule symbiosis on floating stems in aquatic environments through a unique infection process. Here, we analyzed the low-molecular-weight RNA and 16S ribosomal DNA (rDNA) sequence of the same fast-growing isolates from India that were previously used to define the developmental morphology of the unique infection process in this symbiosis with N. natans and found that they are phylogenetically located in the genus Devosia, not Allorhizobium or Rhizobium. The 16S rDNA sequences of these two Neptunia-nodulating Devosia strains differ from the only species currently described in that genus, Devosia riboflavina. From the same isolated colonies, we also located their nodD and nifH genes involved in nodulation and nitrogen fixation on a plasmid of approximately 170 kb. Sequence analysis showed that their nodD and nifH genes are most closely related to nodD and nifH of Rhizobium tropici, suggesting that this newly described Neptunia-nodulating Devosia species may have acquired these symbiotic genes by horizontal transfer.     

Derivatives of Rhizobium meliloti strains carrying a plasmid of Rhizobium leguminosarum specifying hydrogen uptake and pea-specific symbiotic functions

pIJ1008, a Rhizobium leguminosarum plasmid which determines hydrogen uptake ability and symbiotic functions in pea was transferable to three of seven natural isolates of R. meliloti tested. In these three strains, pIJ1008 was maintained stably with the respective sym megaplasmid indigenous to each R. meliloti strain. These strains carrying both plasmids nodulated alfalfa but not pea. By reisolation and examination of the strains from alfalfa nodule tissue, it was shown that pIJ1008 continued to be maintained but that pea-nodulation ability was suppressed.In one strain of R. meliloti which carries a 200 kb cryptic plasmid (in addition to a megaplasmid), the transfer and selection for pIJ1008 resulted in the loss of the cryptic plasmid.In three separate plant growth experiments, alfalfa nodules induced by each of the R. meliloti strain carrying both sym plasmids were assayed for hydrogen uptake activity. The average activity was 40-, 3.5-and 2-fold higher than with the respective pIJ1008-free strains. However, this higher activity was not accompanied by an increase in plant biomass or nitrogen content of shoots.C.B.R.I. Contribution Number: 1478     

Production of gibberellins and indole-3-acetic acid by Rhizobium phaseoli in relation to nodulation of Phaseolus vulgaris roots

Similar ranges of gibberellins (GAs) were detected by high-performance liquid chromatography (HPLC)-immunoassay procedures in ten cultures of wild-type and mutant strains of Rhizobium phaseoli. The major GAs excreted into the culture medium were GA₁ and GA₄. These identifications were confirmed by combined gas chromatographymass spectrometry. The HPLC-immunoassays also detected smaller amounts of GA₉- as well as GA₂₀-like compounds, the latter being present in some but not all cultures. In addition to GAs, all strains excreted indole-3-acetic acid (IAA) but there was no obvious relationship between the amounts of GA and IAA that accumulated. The Rhizobium strains studied included nod ^– and fix ^– mutants, making it unlikely that the IAA- and GA-biosynthesis genes are closely linked to the genes for nodulation and nitrogen fixation.The HPLC-immunoassay analyses showed also that nodules and non-nodulated roots of Phaseolus vulgaris L. contained similar spectra of GAs to R. phaseoli culture media. The GA pools in roots and nodules were of similar size, indicating that Rhizobium does not make a major contribution to the GA content of the infected tissue.Abbreviations EIA enzyme immunoassay - GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - Me methyl ester - RIA radioimmunoassay - TLC thin-layer chromatography     

Model of gas exchange and diffusion in legume nodules

A mathematical model is described which allows the estimation of rates of O₂, CO₂, N₂, and H₂ exchange from legume nodules under steady state conditions of N₂ fixation. Calculated rates of gas exchange under defined conditions of nodule size, relative growth rate (RGR), specific total nitrogenase activity (TNA), nitrogenase electron allocation coefficient (EAC), uptake-hydrogenase activity (HUP) and nature of the N export product compared favorably with experimentally-obtained rates reported in the literature. Therefore the model was used to predict the effects of varying each of these nodule characteristics on the rates of gas exchange, and on the apparent respiratory cost (CO₂/NH₃) and sucrose cost (sucrose consumed/NH₃) of N₂ fixation.The model predicted that, all other characters being equal, ureide-producing nodules would consume 8% less sucrose per N fixed than asparagine-producing nodules, but would display an apparent respiratory cost which would be 5% higher than that in asparagine-producing nodules. In both ureide-producing and asparagine-producing nodules, the major factor affecting the apparent respiratory cost of N₂ fixation was predicted to be EAC, followed by TNA, nodule RGR and nodule size. The relative importance of HUP in improving the apparent respiratory cost of N₂ fixation was predicted to be largely dependent upon its potential role in the regulation of EAC. Abbreviations: See Appendix 1.