Viability of Rhizobium bacteroids isolated from soybean nodule protoplasts

Bacteriods isolated from protoplasts taken from Rhizobium japonicum induced root nodule of Glycine max L. showed complete viability when plated onto a conventional rhizobial growth medium supplemented with 0.2 M Mannitol. The same medium but without extra mannitol resulted in the absence of colony formation. The protoplast isolation method eliminated the possibility of contaminant bacteria from infection threads to be scored. The redifferentiated bacteroid clones have the same genetical characteristics as the orginal inoculum strain. This and other recent findings of bacteroid viability are discussed in the light of the existing belief that bacteroids are non-viable.     

Growth regulators,Rhizobium and nodulation in peas

The cytokinin content of roots and nodules of pea and the culture supernatants from two strains of Rhizobium leguminosarum has been examined. Roots, nodules and wild-type Rhizobium culture medium contained very little cytokinin as indicated by bioassay. Chemical ionisation gas chromatography-mass spectrometric analysis of the isopentenyladenine content of the culture medium from the Rhizobium strains confirmed that the content of the wild-type was low (approx. 1 ng dm^-3) but that it was increased by the introduction of the Agrobacterium Ti plasmid into the Rhizobium strain.Abbreviations CI chemical ionisation - GCMS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - iPAde isopentenyladenine - MIM multiple ion monitoring     

Ultrastructure of infection-thread development during the infection of soybean by Rhizobium japonicum

The location and topography of infection sites in soybean (Glycine max (L.) Merr.) root hairs spot-inoculated with Rhizobium japonicum have been studied at the ultrastructural level. Infections commonly developed at sites created when the induced deformation of an emerging root hair caused a portion of the root-hair cell wall to press against an adjacent epidermal cell, entrapping rhizobia within the pocket between the two host cells. Infections were initiated by bacteria which became embedded in the mucigel in the enclosed groove. Infection-thread formation in soybean appears to involve degradation of mucigel material and localized disruption of the outer layer of the folded hair cell wall by one or more entrapped rhizobia. Rhizobia at the site of penetration are separated from the host cytoplasm by the host plasmalemma and by a layer of wall material that appears similar or identical to the normal inner layer of the hair cell wall. Proliferation of the bacteria results in an irregular, wall-bound sac near the site of penetration. Tubular infection threads, bounded by wall material of the same appearance as that surrounding the sac, emerge from the sac to carry rhizobia roughly single-file into the hair cell. Growing regions of the infection sac or thread are surrounded by host cytoplasm with high concentrations of organelles associated with synthesis and deposition of membrane and cell-wall material. The threads follow a highly irregular path toward the base of the hair cell. Threads commonly run along the base of the hair cell for some distance, and may branch and penetrate into subjacent cortical cells at several points in a manner analagous to the initial penetration of the root hair.     

Isolation and oxidative properties of mitochondria and bacteroids from soybean root nodules

Summary A method for the separation and purification of bacteroids and mitochondria from nodules of soybean roots is described. Cross contamination between these two oxidative fractions was easily assessible by using NADH oxidase and -hydroxybutyrate dehydrogenase respectively as specific mitochondrial and bacteroid markers. Bacteroid respiration was characterized by substantial endogenous respiration which could be reduced by keeping plants in the dark prior to isolation, and stimulated by uncoupler or organic acids. Nodule mitochondria readily oxidized external NADH and a range of tricarboxylic acid cycle intermediates, with good respiratory control. A major difference between nodule and root mitochondria was the former's high sensitivity to the inhibitors rotenone and cyanide. This indicates a reduced capacity for non-phosphorylating electron transport in nodule mitochondria, which may be related to the large energy demand during ammonia assimilation in nodule cells.     

Microscopic studies of cell divisions induced in alfalfa roots by Rhizobium meliloti

We have used spot-inoculation and new cytological procedures to observe the earliest events stimulated in alfalfa (Medicago sativa L.) roots by Rhizobium meliloti. Roots were inoculated with 1–10 nl of concentrated bacteria, fixed in paraformaldehyde, and after embedding and sectioning stained with a combination of acridine orange and DAPI (4-6-diamidino-2-phenylindole hydrochloride). Normal R. meliloti provoke cell dedifferentiation and mitosis in the inner cortex of the root within 21–24 h after inoculation. This activation of root cells spreads progressively, leading to nodule formation. In contrast, the R. meliloti nodA and nodC mutants do not stimulate any activation or mitosis. Thus the primary and earliest effect of Rhizobium nod gene action is plant cellular activation. A rapid, whole-mount visualization by lactic acid shows that the pattern of nodule form varies widely. Some R. meliloti strains were found to be capable of stimulating on alfalfa roots both normal nodules and a hybrid structure intermediate between a nodule and a lateral root.     

Carbon catabolism in continuous cultures and bacteroids of Rhizobium leguminosarum MNF 3841

Bacteroids of R. leguminosarum MNF3841 isolated from pea nodules using Percoll gradients had activities of TCA cycle enzymes up to 6-fold higher than those measured in free-living cells grown on fumarate or sucrose. Activities of sugar catabolic enzymes on the other hand were 2–14-fold lower in isolated bacteroids than in sucrose-grown free-living cells. In continuous culture, cells of strain MNF3841 grown on sucrose under P _i limitation had 2–3-fold higher activities of invertase, glucose-6-phosphate dehydrogenase, the Entner-Doudoroff enzymes and 6-phosphogluconate dehydrogenase, than cells grown on fumarate. With one exception O₂ limited cultures had similar activities of the carbon catabolic enzymes to P _i-limited cultures grown in the same substrate. Glucose-6-phosphate dehydrogenase in O₂-limited cells grown of fumarate was 50% lower than in P _i-limited cells. Co-utilization of fumarate and sucrose occurred with chemostat cultures supplied with both under a variety of conditions.Abbreviations E-D Entner-Doudoroff - EMP Embden-Meyerhof-Parnas - PEPCK phosphoenolpyruvate carboxy kinase - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid]     

Lysis of bacterioids in the vicinity of the host cell nucleus in an ineffective (fix-) root nodule of soybean (Glycine max)

In nodules of Glycine max cv. Mandarin infected with a nod ⁺fix^- mutant of Rhizobium japonicum (RH 31-Marburg), lysis of bacteroids was observed 20 d after infection, but occurred in the region around the host cell nucleus, where lytic compartments were formed. Bacteroids, and peribacteroid membranes in other parts of the host cell remained stable until senescence (40d after infection). With two other nod⁺ fix^- mutants of R. japonicum either stable bacteroids and peribacteroid membranes were observed throughout the cell (strain 61-A-165) or a rapid degeneration of bacteroids without an apparent lysis (strain USDA 24) occurred. The size distribution of RH 31-Marburg-infected nodules exhibited only two maxima compared with four in wild-type nodules and nodule leghaemoglobin content was found to be reduced to about one half that of the wild type. The RH 31-Marburg-nodule type is discussed in relation to the stability of the bacteroids and the peribacteroid membrane system in soybean.     

Superoxide dismutase activities in Rhizobium phaseoli bacteria and bacteroids

Superoxide dismutase activity in free-living Rhizobium phaseoli is due to the presence of two different enzymes containing manganese or iron. Under usual culture conditions, the manganese-enzyme appears largely predominant but the induction of the iron-superoxide dismutase can be obtained by addition of methyl viologen to the culture media. The corresponding bacteroid, extracted from French-bean nodules, contains only a manganese-superoxide dismutase whose characteristics are similar to those of the bacterial enzyme. However, the activity of the microsymbiont is slightly lower than that of free-living cells. The presence of an active superoxide dismutase in the bacteroids suggests a significant formation of superoxide anion by their metabolism; this can be correlated with the existence of a large oxygen demand by the microsymbionts within the nodule, as suggested by their important oxygen uptake in vitro.     

Influence of host cultivars and Rhizobium species on the growth and symbiotic performance of Phaseolus vulgaris under salt stress

In order to study the effect of salt stress on the Rhizobium-common bean symbiosis, we investigated the response of both partners, separately and in symbiosis. The comparison of the behaviour of five cultivars of Phaseolus vulgaris differing in seed colour, growing on nitrates and different concentrations of NaCl, showed genotypic variation with respect to salt tolerance. Coco Blanc was the most sensitive cultivar, whereas SMV 29-21 was the most tolerant one. At the Rhizobium level, two strains previously selected for their salt tolerance were used: Rhizobium tropici strain RP163 and Rhizobium giardinii strain RP161. Their relative growth was moderately decreased at 250mM NaCl, but they were able to grow at a low rate in the presence of 342 mM NaCl. Their viability at the minimal inhibitory concentration was slightly affected. The effect of salinity on Rhizobium-plant association was studied by using the tolerant variety SMV 29-21 and the sensitive one Coco Blanc inoculated separately with both strains. In the absence of salinity, the strains induced a significantly higher number of nodules on the roots of the cultivar SMV 29-21 compared to those of Coco Blanc. Concerning effectiveness, both strains were similarly effective with SMV 29-21, but not with Coco Blanc. In the presence of salinity, Coco Blanc was more severely affected when associated with RP163 than with RP161. Salinity affected the nodulation development more than it affected the infection steps. Neither of the two strains was able to nodulate SMV 29-21 under saline conditions, in spite of the fact that this was considered the most salt-tolerant variety. The unsuccessful nodulation of SMV 29-21 could be related to the inhibition by salt of one or more steps of the early events of the infection process. In conclusion, N-fixing plants were found to be more sensitive to salt stress than those depending on mineral nitrogen. Evidence presented here suggests that a best symbiotic N2 fixation under salinity conditions could be achieved if both symbiotic partners, as well as the different steps of their interaction (early events, nodule formation, activity, etc.), are all tolerant to this stress.     

A comparison of cultural characteristics and infectivity ofFrankia isolates from root nodules ofCasuarina species

Summary The isolations of three new strains ofFrankia were made from root nodules ofCasuarina cunninghamiana growing aeroponically. Two strains, HFPCCI1 and HFPCcI2 isolated by Lopez are typicalFrankia strains, producing sporangia among filamentous mats in culture and, in the absence of combined nitrogen, forming vesicles and showing acetylene reduction. They are red-pigmented and, although failing to nodulateCasuarina hosts, effectively nodulatedElaeagnus andHippophae. A third strain HFPCcI3 isolated by Zhang from the same source, also a typicalFrankia, can form sporangia and vesicles in culture and reduce acetylene, is unpigmented, fails to nodulateElaeagnus but effectively nodulatesC. cunninghamiana andC. equisetifolia. Comparisons are made among all of theCasuarina isolates in our collection from around the world (twelve in all) with regard to their cultural characteristics and capacity to infect host plant species. Questions are raised about the specificity of the various isolates and their possible affinities. Opportunities are suggested for inoculation of seedlings for forestry and field application using the infective, effective strains now available.     

Rhizobium leguminosarum genes required for expression and transfer of host specific nodulation

The contributions of various nod genes from Rhizobium leguminosarum biovar viceae to host-specific nodulation have been assessed by transferring specific genes and groups of genes to R. leguminosarum bv. trifolii and testing the levels of nodulation on Pisum sativum (peas) and Vicia hirsuta. Many of the nod genes are important in determination of host-specificity; the nodE gene plays a key (but not essential) role and the efficiency of transfer of host specific nodulation increased with additional genes such that nodFE < nodFEL < nodFELMN. In addition the nodD gene was shown to play an important role in host-specific nodulation of peas and Vicia whilst other genes in the nodABCIJ gene region also appeared to be important. In a reciprocal series of experiments involving nod genes cloned from R. leguminosarum bv. trifolii it was found that the nodD gene enabled bv. viciae to nodulate Trifolium pratense (red clover) but the nodFEL gene region did not. The bv. trifolii nodD or nodFEL genes did significantly increase nodulation of Trifolium subterraneum (sub-clover) by R. leguminosarum bv. viciae. It is concluded that host specificity determinants are encoded by several different nod genes.     

Selection of salt-tolerant Rhizobium isolates of Acacia nilotica

Among 35 Rhizobium isolates of Acacia nilotica, from different agro-climatic zones, two, ANG4 and ANG5, tolerated up to 850 mm NaCl and one, ANG3, was sensitive to NaCl above 250 mm. Nodulation and nitrogenase activity of the three isolates decreased with increasing concentration of salt up to 150 mm. Nodulation by ANG3 was 15% at 75 mm NaCl and nil at 100 mm. With ANG4 and ANG5, nodulation was only slightly decreased at 150 mm NaCl. Nitrogenase activity associated with plants inoculated with ANG3 was halved at 25 mm NaCl compared with salt-free controls, whereas isolates ANG4 and ANG5 retained 25% and 15% activity, respectively, even at 100 mm NaCl. Salt-tolerant Rhizobium isolates can therefore nodulate and fix N₂ in saline soils.     

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.     

4-aminobutyrate is not available to bacteroids of cowpea Rhizobium MNF2030 in snake bean nodules

Laboratory cultures of cowpea Rhizobium MNF2030 grew on 4-aminobutyrate (GABA) as sole source of carbon and nitrogen. GABA transport was active since it was inhibited by carbonyl cyanide mchlorophenyl hydrazone and 2,4-dinitrophenol and cells developed a 400-fold concentration gradient across the cell membrane. Arsenite treatment of GABA-grown cells revealed stoichiometric conversion of GABA to pyruvate, indicating that 2-oxoglutarate is not an intermediate in GABA catabolism. GABA catabolism by cells of strain MNF2030 grown on GABA appreared to involve GABA transaminase, succinic semialdehyde dehydrogenase and malic enzyme; the first two enzymes were specifically induced by growth on GABA. The deamination process and removal of NH₃ in cells catabolizing GABA involved GABA: 2-oxoglutarate transaminase; glutamate: oxaloacetate aminotransferase; asparate: pyruvate aminotransferase and alanine dehydrogenase.Isolated snakebean bacteroids of strain MNF2030 transported only small amounts of GABA and had uninduced levels of GABA catabolic enzymes, even though the nodules contained significant levels of GABA. The data suggest that GABA is not available to snakebean nodule bacteroids, presumably because of a control imposed by the peribacteroid membrane.Abbreviations CCCP Carbonyl cyanide m-chlorophenyl hydrazone - HEPES N-hydroxyethylpiperazine-N-2-ethanesulphonic acid - DTT dithiothreitol - SSAD succinic semialdehyde dehydrogenase - GABAT 4-aminobutyrate transaminase - GABA 4-aminobutyrate     

Chemotaxis by Rhizobium meliloti

Summary Chemotaxis by Rhizobium meliloti strain Ve 26 has been studied and conditions required for chemotaxis have been defined, using the Adler capillary assay technique. Several sugars and amino-acids were shown to be attractants with varying effectiveness for this organism: sugars are weak attractants (except gluconate) and amino-acids are good attractants (except unpolar amino-acids).     

Development of nodules of Glycine max infected with an ineffective strain of Rhizobium japonicum

Bacteroids in ineffective (nitrogenase negative) nodules of Glycine max, infected with Rhizobium japonicum 61-A-24, as compared to those in effective nodules are characterized by reduced specific activities of alanine dehydrogenase to 15%, of 3-hydroxybutyrate dehydrogenase to 50%, and an increase of glutamine synthetase to 400%. In the plant cytoplasm of ineffective nodules, glutamine synthetase activity is reduced to 10–30%, glutamate dehydrogenase to 50–70%, and the aspartate aminotransferase and alanine aminotransferase are enhanced to 120–200%, depending on the age of the nodules. The total pool of soluble amino acids is reduced to 52 mol per g nodule fresh weight, as compared to 186 mol in effective nodules, with a replacement of asparagine (42 mol% of the amino acids) by an unknown amino compound. This compound is absent in nitrogenase, repressed and derepressed, free-living Rhizobium japonicum cells and in the uninfected root tissue. In nitrogenase derepressed, as compared to the repressed free-living cells of Rhizobium japonicum 61-A-101, arginine shows the most obvious change with a reduction to less than one tenth. The ultrastructure of the ineffective nodule is different from the effective organ even in the early stages. The membrane envelopes of the infection vacuoles are decomposing in heavily infected cells within 18 to 20 d after infection. In lightly infected cells very large vacuoles develop with only a few bacteroids inside. No close associations of cristae-rich mitochondria with amyloplasts are observed as in effective nodules. The uninfected cells keep their large starch granules even 40 d after infection. Some poly--hydroxybutyrate accumulation in the bacteroids is observed but only in the early stages, and it is almost absent in old nodules (40 d). At this age the infected cells are obviously compressed by uninfected cells, whereas in effective nodules with nitrogenase activity and leghaemoglobin formation, the infected cells have a much higher osmotic pressure than the neighbouring uninfected cells.Abbreviations PHBA poly--hydroxybutyric acid Prof. Dr. A. Pirson on the occasion of his 70th birthday     

Plant defence and delayed infection of alfalfa pseudonodules induced by an exopolysaccharide (EPS I)-deficient Rhizobium meliloti mutant

Mutants of the symbiotic soil bacterium Rhizobium meliloti that fail to synthesize the acidic exopolysaccharide EPS I were unable to induce infected root nodules on Medicago sativa L. (alfalfa). These strains, however, elicited pseudonodules that contained no infection threads or bacteroids. The cortical cell walls of the pseudonodules were abnormally thick and incrusted with an autofluorescent material. Parts of these cell walls and wall appositions contained callose. Biochemical analysis of nodules induced by the EPS I-deficient R. meliloti mutant revealed an increase of phenolic compounds bound to the nodule cell walls when compared with the wild-type strain. These microscopic and biochemical data indicated that a general plant defence response against the EPS I-deficient mutant of R. meliloti was induced in alfalfa pseudonodules. Following prolonged incubation with the EPS I-deficient R. meliloti mutant, the defence system of the alfalfa plant could be overcome by the rhizobium mutant. In the case of the delayed infections, the mutants colonized lobes of the pseudonodules, but the infection threads in these nodules had an abnormal morphology. They were greatly enlarged and did not contain the typical gum-like matrix inside. The bacteria were tightly packed. Based on the mechanism of phytopathogenic interactions, we propose that EPS I or a related compound may act as a suppressor of the alfalfa plant defence system, enabling R. meliloti to infect the plant.     

Isolation and characterisation of catechol-like siderophore from cowpea Rhizobium RA-1

Cowpea Rhizobium RA-1 produced a catechol-like siderophore. Secondary hydroxamic acids were not detected. Bioassay of the siderophore exhibited a distinct zone of growth of cowpea rhizobia. One litre of culture filtrate gave 6.2 mg of catechol-like siderophore. Glycine and threonine were detected in the siderophore. Maximum production of siderophore was found at 36 h of growth of cowpea Rhizobium RA-1.Abbreviations 2,3-DHBA 2,3-dihydroxy benzoic acid - EDTA ethylenediamine tetraacetic acid     

The synthesis of aspartic acid inRhizobium lupini bacteroids

Summary InRhizobium lupini bacterioids enzymes catalysing biosynthesis of aspartic acid have been found. The first enzyme termed aspartate dehydrogenase catalyses synthesis of aspartate from oxaloacetic acid and ammonia in the presence of NADH. The second enzyme, aspartase (L-aspartate ammonialyase, EC 4.3.1.1.), catalyses synthesis of aspartate from fumaric acid and ammonia. These data show that ammonia can be assimilated not only in the plant part of nodules but also in bacteroids. Biosynthesis of aspartate plays a very important role in the assimilation of ammonia in nodules.     

Phage sensitivity and host range of Rhizobium strains isolated from root nodules of temperate legumes

Twenty-five Rhizobium strains were isolated from root nodules of Astragalus spp. (10), Hedysarum alpinum (7), Glycyrrhiza pallidiflora (3) and Ononis arvensis (5). The sensitivity of these strains to bacteriophages of Rhizobium loti, R. meliloti, R. galegae and R. leguminosarum was studied. Phages specific to R. loti strains were shown to induce the phage lysis of several Astragalus, Hedysarum and Ononis rhizobia. Ten R. loti strains tested for nodulation abilities on the plant hosts under investigation were able to develop nitrogen-fixing nodules on the Ononis arvensis roots. On the other hand, rhizobia from Ononis and Glycyrrhiza could form an effective symbiosis with Lotus corniculatus plants, so these bacteria are considered to belong to the Rhizobium loti taxon. Bacterial strains isolated from Astragalus and Hedysarum were observed to cross-nodulate their plant hosts as well as Oxytropis campestris, Glycyrrhiza uralensis and Ononis arvensis plants, whereas they could not nodulate Lotus plants. It is concluded that these Rhizobium strains comprise a cross-inoculation group related to Rhizobium loti. ei]{gnR O D}{fnDixon}