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}     

Resistance to nodulation of cv. Afghanistan peas is overcome by nodX, which mediates an O-acetylation of the Rhizobium leguminosarum lipo-oligosaccharide nodulation factor

Only some strains of Rhizobium leguminosarum biovar viciae can efficiently nodulate varieties of peas such as cv. Afghanistan, which carry a recessive allele that blocks efficient nodulation by most western isolates of R. I. viciae. One strain (TOM) which can nodulate cv. Afghanistan peas has a gene (nodX) that is required to overcome the nodulation resistance. Strain TOM makes significantly lower amounts of lipo-oligosaccharide nodulation factors than other strains of R. I. viciae. and this effect appears to be due to lower levels of nod gene induction. These nodulation factors are similar to those from other R. I. viciae. strains in that they consist of an oligomer of four or five β1-4-linked N-acetylglucosamine residues in which the terminal non-reducing glucosamine carries an O-acetyl group and a C_18:4 or C_18:1N-acyl group. However, one of the nodulation factors made by strain TOM differs from the factors made by other strains of R. I. viciae. in that it carries an O-acetyl group on the C-6 of the reducing N-acetylglucosamine residue. This acetylation is NodX-dependent and the pentameric nodulation factor is acetylated on the reducing N-acetylglucosamine residue whereas the tetrameric nodulation factor is not. Although the nodL gene product is also an O-acetyl transferase (it O-acetylates the C-6 of the terminal non-reducing glucosamine), there is very little similarity between the amino acid sequences of these two acetyl transferases.     

Structural determination of the lipo-chitin oligosaccharide nodulation signals produced by Rhizobium giardinii bv. giardinii H152

Rhizobium giardinii bv. giardinii is a microsymbiont of plants of the genus Phaseolus and produces extracellular signal molecules that are able to induce deformation of root hairs and nodule organogenesis. We report here the structures of seven lipochitooligosaccharide (LCO) signal molecules secreted by R. giardinii bv. giardinii H152. Six of them are pentamers of GlcNAc carrying C 16:0, C 18:0, C 20:0 and C 18:1 fatty acyl chains on the non-reducing terminal residue. Four are sulfated at C-6 of the reducing terminal residue and one is acetylated in the same position. Six of them are N-methylated on the non-reducing GlcN residue and all the nodulation factors are carbamoylated on C-6 of the non-reducing terminal residue. The structures were determined using monosaccharide composition and methylation analyses, 1D- and 2D-NMR experiments and a range of mass spectrometric techniques. The position of the carbamoyl substituent on the non-reducing glucosamine residue was determined using a CID-MSMS experiment and an HMBC experiment.     

Identification of Lotus Rhizobia by Direct DNA Hybridization of Crushed Root Nodules

Hybridization of crushed Lotus pedunculatus root nodules with ³²P-labeled total genomic DNA probes was used to identify Rhizobium loti and Bradyrhizobium sp. (Lotus rhizobia). Probes always hybridized with homologous target DNA and frequently with DNAs of other strains from the same genus. Intergeneric hybridization did not occur. Results were comparable to those from colony hybridization.     

Rapid identification ofRhizobium species based on cellular fatty acid analysis

As understanding of the evolutionary relationships between strains and species of root nodule bacteria increases the need for a rapid identification method that correlates well with phylogenetic relationships is clear. We have examined 123 strains ofRhizobium: R. fredii (19),R. galegae (20),R. leguminosarum (22),R. loti (17),R. meliloti (21), andR. tropici (18) and six unknowns. All strains were grown on modified tryptone yeast-extract (TY) agar, as log phase cultures, scraped from the agar, lysed, and the released fatty acids derivatized to their corresponding methyl esters. The methyl esters were analysed by gas-chromatography using the MIDI/Hewlett-Packard Microbial Identification System. All species studied contained 16:0, 17:0, 18:0 and 19cyclo_w9C fatty acids but onlyR loti andR tropici produced 12:0 3 OH,13:0 iso 3 OH,18:1_w9C and 15:0 iso 3 OH,17:0 iso 3 OH and 20:2_w6,9C fatty acids respectively. Principal component analysis was used to show that strains could be divided into clusters corresponding to the six species. Fatty acid profiles for each species were developed and these correctly identified at least 95% of the strains belonging to each species. A dendrogram is presented showing the relationships betweenRhizobium species based on fatty acid composition. The data base was used to identify unknown soil isolates as strains ofRhizobium lacking a symbiotic plasmid and a bacterium capable of expressing a symbiotic plasmid fromR. leguminosarum asSphingobacterium spiritovorum.     

Genetic Diversity and Host Range of Rhizobia Nodulating Lotus tenuis in Typical Soils of the Salado River Basin (Argentina)

A total of 103 root nodule isolates were used to estimate the diversity of bacteria nodulating Lotus tenuis in typical soils of the Salado River Basin. A high level of genetic diversity was revealed by repetitive extragenic palindromic PCR, and 77 isolates with unique genomic fingerprints were further differentiated into two clusters, clusters A and B, after 16S rRNA restriction fragment length polymorphism analysis. Cluster A strains appeared to be related to the genus Mesorhizobium, whereas cluster B was related to the genus Rhizobium. 16S rRNA sequence and phylogenetic analysis further supported the distribution of most of the symbiotic isolates in either Rhizobium or Mesorhizobium: the only exception was isolate BA135, whose 16S rRNA gene was closely related to the 16S rRNA gene of the genus Aminobacter. Most Mesorhizobium-like isolates were closely related to Mesorhizobium amorphae, Mesorhizobium mediterraneum, Mesorhizobium tianshanense, or the broad-host-range strain NZP2037, but surprisingly few isolates grouped with Mesorhizobium loti type strain NZP2213. Rhizobium-like strains were related to Rhizobium gallicum, Rhizobium etli, or Rhizobium tropici, for which Phaseolus vulgaris is a common host. However, no nodC or nifH genes could be amplified from the L. tenuis isolates, suggesting that they have rather divergent symbiosis genes. In contrast, nodC genes from the Mesorhizobium and Aminobacter strains were closely related to nodC genes from narrow-host-range M. loti strains. Likewise, nifH gene sequences were very highly conserved among the Argentinian isolates and reference Lotus rhizobia. The high levels of conservation of the nodC and nifH genes suggest that there was a common origin of the symbiosis genes in narrow-host-range Lotus symbionts, supporting the hypothesis that both intrageneric horizontal gene transfer and intergeneric horizontal gene transfer are important mechanisms for the spread of symbiotic capacity in the Salado River Basin.     

Group antigens in slow-growing rhizobia

Summary Internal group antigens of several slow-growing and fast-growing Rhizobium strains were tested by gel-diffusion against antisera to three strains of Rhizobium japonicum. At least one, generally two common antigens were found in 13 strains of R. japonicum, 4 strains of R. lupini, 4 strains isolated from cowpea and two slow-growing strains isolated from Lotus. Forty-six fast-growing rhizobia (including two from Lotus and 4 from Leucaena leucocephala) were clearly distinguished from the slow-growing strains in tests with the same antisera. They were wholly negative (9) or gave a much weaker non-identical line with one antiserum (24 strains), two antisera (8) or three antisera (5). The 5 strains of agrobacteria grouped with the fast-growing rhizobia.     

Induction of nodule primordia on Phaseolus and Acacia by lipo-chitin oligosaccharide nodulation signals from broad-host-range Rhizobium strain GRH2

Rhizobium wild-type strain GRH2 was originally isolated from the tree, Acacia cyanophylla, and has a broad host-range which includes herbaceous legumes, such as Phaseolus and Trifolium species. Here we show that strains of Rhizobium sp. GRH2, into which heterologous nodD alleles have been introduced, produce a large diversity of both sulphated and non-sulphated lipo-chitin oligosaccharides (LCOs). Most of the molecular species contain an N-methyl group on the reducing-terminal N-acetyl-glucosamine. The LCOs vary in the nature of the fatty acyl chain and in the length of the chitin backbone. The majority of the LCOs have an olgosaccharide chain length of five GlcNAc residues, but a few are oligomers having six GlcNAc units. LCOs purified from GRH2 are able to induce root hair formation and deformation on Acacia cyanophylla and A. melanoxylon plants. We show that an N-vaccenoyl-chitopentaose bearing an N-methyl group is able to induce nodule primordia on Phaseolus vulgaris, A. cyanophylla, and A. melanoxylon, indicating that for these plants an N-methyl modification is sufficient for nodule primordia induction.     

Response of Lotus rhizobia to acidity and aluminium in liquid culture and in soil

Measurements of multiplication in liquid culture indicated that fast-growing Lotus rhizobia (Rhizobium loti) were tolerant of acidity and aluminium (at least 50 μM A1 at pH 4.5). Slow-growing Lotus rhizobia (Bradyrhizobium sp. (Lotus)) were less tolerant of acidity but equally tolerant of A1. Both genera were able to nodulateLotus pedunculatus in an acid soil (pH 4.1 in 0.01M CaCl₂) and the slow-growing strains were more effective than the fast-growing strains in this soil over 30 days.     

Identification of enzymes involved in indole-3-acetic acid degradation

Strains of Bradyrhizobium japonicum with the ability to catabolize indole-3-acetic acid (IAA) and strains of B. japonicum, Rhizobium loti, and Rhizobium galegae, unable to catabolize IAA, were analyzed for enzymes involved in the pathway for IAA degradation. Two enzymes having isatin as substrate were detected. An isatin amidohydrolase catalyzing the hydrolysis of isatin into isatinic acid was found in some B. japonicum strains and in two Rhizobium species, R loti and R. galegae. The enzyme was inducible (4–5-fold) by its substrate, isatin, and the partially purified enzyme from R. loti showed an apparent K_M of 11 M for isatin. A NADPH-dependent isatin reductase was measured in extracts from a strain of B. japonicum lacking the isatin amidohydrolase. The structure of the reaction product, dioxindole was verified by NMR spectroscopy. Isatin reductase activity was also detected in extracts of dry pea seeds, and present in at least two isoforms. A low K_M of 10 M for isatin was found with a partially purified preparation of the pea enzyme. The presence of such an enzyme activity in pea indicates dioxindole and isatin as possible intermediates in IAA degradation in pea.     

Ensifer meliloti bv. lancerottense establishes nitrogen-fixing symbiosis with Lotus endemic to the Canary Islands and shows distinctive symbiotic genotypes and host range

Eleven strains were isolated from root nodules of Lotus endemic to the Canary Islands and they belonged to the genus Ensifer, a genus never previously described as a symbiont of Lotus. According to their 16S rRNA and atpD gene sequences, two isolates represented minority genotypes that could belong to previously undescribed Ensifer species, but most of the isolates were classified within the species Ensifer meliloti. These isolates nodulated Lotus lancerottensis, Lotus corniculatus and Lotus japonicus, whereas Lotus tenuis and Lotus uliginosus were more restrictive hosts. However, effective nitrogen fixation only occurred with the endemic L. lancerottensis. The E. meliloti strains did not nodulate Medicago sativa, Medicago laciniata Glycine max or Glycine soja, but induced non-fixing nodules on Phaseolus vulgaris roots. nodC and nifH symbiotic gene phylogenies showed that the E. meliloti symbionts of Lotus markedly diverged from strains of Mesorhizobium loti, the usual symbionts of Lotus, as well as from the three biovars (bv. meliloti, bv. medicaginis, and bv. mediterranense) so far described within E. meliloti. Indeed, the nodC and nifH genes from the E. meliloti isolates from Lotus represented unique symbiotic genotypes. According to their symbiotic gene sequences and host range, the Lotus symbionts would represent a new biovar of E. meliloti for which bv. lancerottense is proposed.     

A Legume Genetic Framework Controls Infection of Nodules by Symbiotic and Endophytic Bacteria

Legumes have an intrinsic capacity to accommodate both symbiotic and endophytic bacteria within root nodules. For the symbionts, a complex genetic mechanism that allows mutual recognition and plant infection has emerged from genetic studies under axenic conditions. In contrast, little is known about the mechanisms controlling the endophytic infection. Here we investigate the contribution of both the host and the symbiotic microbe to endophyte infection and development of mixed colonised nodules in Lotus japonicus. We found that infection threads initiated by Mesorhizobium loti, the natural symbiont of Lotus, can selectively guide endophytic bacteria towards nodule primordia, where competent strains multiply and colonise the nodule together with the nitrogen-fixing symbiotic partner. Further co-inoculation studies with the competent coloniser, Rhizobium mesosinicum strain KAW12, show that endophytic nodule infection depends on functional and efficient M. loti-driven Nod factor signalling. KAW12 exopolysaccharide (EPS) enabled endophyte nodule infection whilst compatible M. loti EPS restricted it. Analysis of plant mutants that control different stages of the symbiotic infection showed that both symbiont and endophyte accommodation within nodules is under host genetic control. This demonstrates that when legume plants are exposed to complex communities they selectively regulate access and accommodation of bacteria occupying this specialized environmental niche, the root nodule.     

Specificity in the symbiotic association of Lotus corniculatus and Rhizobium loti from natural populations

To test whether Rhizobium loti are coadapted to nodulate local plant genotypes, we competed R. loti strains in a common environment with clonally propagated Lotus corniculatus. Both the plants and bacterial strains were originally collected from natural populations in three localities and the R. loti strains used were distinguishable by enzyme electrophoretic markers and differed in geographical origin relative to host plant origin. The proportions of nodules occupied by symbiont strains varied widely and depended on both host plant and symbiont genotype. Nonrandom nodulation patterns resulted primarily from preferential nodulation of host genotypes by the symbiont strain that had been associated with the host in the natural environment. Symbionts nodulating their original hosts were preferentially found in nodules on adventitious tap roots as opposed to the younger, lateral roots (for one host-symbiont pair) or in large nodules, independent of location on the root system (for a second host-symbiont pair). The proportion of nodules occupied by a symbiont on novel host genotypes varied, ranging from nearly random expectation to a significant reduction in the proportion of nodules occupied. The analysis of the bacteria recovered from 994 nodules by multilocus enzyme electrophoresis revealed that 952 (95.8%) nodules were occupied by one of the four inoculant strains and 11 (1.1%) were co-occupied by two inoculant strains. A total of 31 (3.1%) nodules were occupied by strains that did not match the electrophoretic profiles of the original inoculant strains. Based on the comparison of multilocus profiles for 23 enzyme loci, we concluded that these bacteria were foreign strains and not recombinants of the original inoculant strains. Our findings indicate a strong host genotype by strain interaction underlying the outcome of rhizobial competition for nodulation sites and suggest there are distinct mechanisms leading to differential recognition of compatible host and symbiont genotypes.     

Identification of an arsenic resistance mechanism in rhizobial strains

Arsenic (As) is a very toxic metalloid to a great number of organisms. It is one of the most important global environmental pollutants. To resist the arsenate invasion, some microorganisms have developed or acquired genes that permit the cell to neutralize the toxic effects of arsenic through the exclusion of arsenic from the cells. In this work, two arsenic resistance genes, arsA and arsC, were identified in three strains of Rhizobium isolated from nodules of legumes that grew in contaminated soils with effluents from the chemical and fertilizer industry containing heavy-metals, in the industrial area of Estarreja, Portugal. The arsC gene was identified in strains of Sinorhizobium loti [DQ398936], Rhizobium leguminosarum [DQ398938] and Mesorhizobium loti [DQ398939]. This is the first time that arsenic resistance genes, namely arsC, have been identified in Rhizobium leguminosarum strains. The search for the arsA gene revealed that not all the strains with the arsenate reductase gene had a positive result for ArsA, the ATPase for the arsenite-translocating system. Only in Mesorhizobium loti was the arsA gene amplified [DQ398940]. The presence of an arsenate reductase in these strains and the identification of the arsA gene in Mesorhizobium loti, confirm the presence of an ars operon and consequently arsenate resistance.     

Structural identification of metabolites produced by the NodB and NodC proteins of Rhizobium leguminosarum

The Rhizobium nodulation genes nodABC are involved in the synthesis of lipo-chitin oligosac-charides. We have analysed the metabolites which are produced in vivo and in vitro by Rhizobium strains which express the single nodA, nodB and nodC genes or combinations of the three. In vivo radioactive labelling experiments, in which D-[1-¹⁴C]-glucosamine was used as a precursor, followed by mass spectrometric analysis of the purified radiolabelled metabolic products, showed that Rhizobium strains that only express the combination of the nodB and nodC genes do not produce lipo-chitin oligosaccharides but instead produce chitin oligomers (mainly pentamers) which are devoid of the N-acetyl group on the non-reducing terminal sugar residue (designated NodBC metabolites). Using the same procedure we have shown that when the nodL gene is expressed in addition to the nodBC genes the majority of metabolites contain an additional O-acetyl substituent on the non-reducing terminal sugar residue (designated NodBCL metabolites). The NodBC and NodBCL metabolites purified after in vivo labelling were compared with the radiolabelled metabolites produced in vitro by Rhizobium bacterial cell lysates to which UDP-N-acetyl-D-[U-¹⁴C]-glucosamine was added using thin-layer chromatography. The results show that the lysates of strains which expressed the nodBC or nodBCL genes can also produce NodBC and NodBCL metabolites. The same results were obtained when the NodB and NodC proteins were produced separately in two different strains. On the basis of these and other recent results, we propose that NodB is a chitin oilgosaccharide deacetylase, NodC an N-acetylglucosaminyltransferase and, by default, NodA is involved in lipie attachment.     

Characterization of Strains unlike Mesorhizobium loti That Nodulate Lotus spp. in Saline Soils of Granada,Spain

Lotus species are forage legumes with potential as pastures in low-fertility and environmentally constrained soils, owing to their high persistence and yield under those conditions. The aim of this work was the characterization of phenetic and genetic diversity of salt-tolerant bacteria able to establish efficient symbiosis with Lotus spp. A total of 180 isolates able to nodulate Lotus corniculatus and Lotus tenuis from two locations in Granada, Spain, were characterized. Molecular identification of the isolates was performed by repetitive extragenic palindromic PCR (REP-PCR) and 16S rRNA, atpD, and recA gene sequence analyses, showing the presence of bacteria related to different species of the genus Mesorhizobium: Mesorhizobium tarimense/Mesorhizobium tianshanense, Mesorhizobium chacoense/Mesorhizobium albiziae, and the recently described species, Mesorhizobium alhagi. No Mesorhizobium loti-like bacteria were found, although most isolates carried nodC and nifH symbiotic genes closely related to those of M. loti, considered the type species of bacteria nodulating Lotus, and other Lotus rhizobia. A significant portion of the isolates showed both high salt tolerance and good symbiotic performance with L. corniculatus, and many behaved like salt-dependent bacteria, showing faster growth and better symbiotic performance when media were supplemented with Na or Ca salts.Legumes can establish nitrogen-fixing associations with Gram-negative soil bacteria collectively known as rhizobia. Although the symbiotic relationships among rhizobia and many legume species of agricultural importance have been intensively studied, relatively little is known about the symbiotic bacteria of certain plant genera. Lotus is a genus of legumes that includes 125 to 130 species of herbs and small shrubs, mainly distributed in the Northern Hemisphere. Several Lotus species, particularly Lotus corniculatus, Lotus uliginosus, and Lotus tenuis, are used as pasture forage worldwide and are included by phylogenetic studies in the same clade as the model legume Lotus japonicus (4). Until recently, bacteria nodulating Lotus included both intermediate-growing (mesorhizobia) and slow-growing bacteria (12, 16). The mesorhizobia can form effective symbioses with certain Lotus spp. (group I, e.g., L. corniculatus, L. tenuis, or L. japonicus) but form tumor-like structures that do not contain bacteria on L. uliginosus, Lotus subbiflorus, and Lotus angustissimus (group II Lotus spp.) (21, 24). On the other hand, slow-growing strains are usually efficient with Lotus group II species but form no nodules or form inefficient nodules in group I species (12). However, there are rare exceptions to this rule, like strain NZP2037, that can form effective symbioses with both groups of Lotus spp. (23, 25, 28). Furthermore, fast-growing Ensifer meliloti bv. lancerottense strains have been shown to be the symbionts of Lotus lancerottensis but are unable to fix nitrogen with either group I or group II Lotus spp. (19).No apparent relationship exists between the phylogenetic position of Lotus spp. and the type of rhizobia associated. For instance, L. uliginosus and L. angustissimus, which are efficiently nodulated by the bradyrhizobia, are clustered in the same clade as L. corniculatus, L. tenuis, and L. japonicus (clade B) (4), species associated with mesorhizobia. In contrast L. subbiflorus, usually associated with the same rhizobia as L. uliginosus, is clustered in a different clade.The narrow-host-range rhizobia associated with L. corniculatus and other Lotus species were initially classified as Rhizobium loti (13). Later, when the genus Mesorhizobium was created, R. loti was reclassified as Mesorhizobium loti (14), which is considered the type species. Besides the expected differences between the moderate- and the slow-growing Lotus rhizobia, large variabilities in nitrogen-fixing effectiveness (23) as well as in total DNA-DNA hybridization (3, 6) and phylogeny (5, 40) have been shown among the “meso-growing” rhizobia strains classified as M. loti, indicating that they do not form a homogeneous group. Indeed, one of the best-characterized strains of M. loti, strain MAFF303099, has been reclassified as Mesorhizobium huakuii biovar loti (35). In fact, diverse rhizobia have recently been reported to establish symbiosis with Lotus group I species. For instance, bacteria belonging to the newly described species Mesorhizobium gobiense and Mesorhizobium tarimense, were isolated from Lotus frondosus and L. tenuis in China (10). Also, rhizobia assigned to different genera (Rhizobium, Mesorhizobium, Agrobacterium, and Aminobacter) have recently been reported as symbionts of L. tenuis in the Salado River Basin in Argentina (7). While these recent reports indicate that bacteria nodulating Lotus spp. are diverse, their symbiotic genes are rather homogeneous. In fact, most isolates from Argentina and China, regardless their taxonomic assignment, had symbiotic genes closely related to M. loti (7, 10).Soil salinity is a serious and expanding threat to agricultural productivity. Improving crop productivity in saline soils requires selection of well-adapted plant genotypes and, in the case of legumes, highly efficient rhizobial partners adapted to soil conditions. As part of the Euro-South American cooperation project LOTASSA (http://www.lotassa.com/), and aiming to isolate and select for salt-tolerant bacteria able to establish efficient symbiosis with forage Lotus spp., we explored the diversity of Lotus rhizobia in two different locations of Granada province, Spain, where the presence of native Lotus spp. had previously been reported (30).     

Extracellular polysaccharides of the genusRhizobium

This paper reports an investigation of the extracellular polysaccharides produced by 26 strains ofRhizobium andAgrobacterium. Strains ofRhizobium leguminosarum andR. phaseoli produced a water-soluble polysaccharide containing glucose, glucuronic acid and 4-0-methylglucuronic acid. These substances were also identified in the polysaccharide of a single strain fromLotus uliginosus. Glucose was the only detectable component in the polysaccharide produced by strains ofAgrobacterium radiobacter andA. tumefaciens. The polysaccharides obtained from slow-growing rhizobia were not freely water-soluble. Glucose, mannose, rhamnose, galactose and 4-0-methylglucuronic acid were identified as components of this extracellular material.These results are related to previous studies on rhizobial taxonomy and to the infection process in legumes.     

Effect of oxygen availability on nitrogen fixation by two Lotus species under flooded conditions

The pasture legumes Lotus uliginosus (Schk.) and Lotus corniculatus (L.), known to differ in their tolerance to flooding, were inoculated with Rhizobium loti and flooded for 60 d while subjected to two levels of dissolved pO2: 0.241 and 0.094 mol ml^-1. L. uliginosus showed significantly greater growth (shoot and root) and N2 fixation under both pO2s, compared to L. corniculatus, although growth and N2 fixation by L. corniculatus was not affected by the low pO2. Surprisingly, in L. uliginosus., growth, nodulation and N2 fixation were all increased by low pO2 while nodulation of L. corniculatus where low pO2 plants showed a significant increase over that of the higher pO2 plants while L. uliginosus plants showed a decline. Root porosity of L. uliginosus doubled in the low pO2-treatment from a mean of 14.5% in high pO2 roots to 28.5%, whereas that of L. corniculatus was relatively unaffected by pO2, being 7% and 9% for high and low pO2 plants, respectively. The structure of nodules differed little between species and treatments, although nodules/nodulated roots from the L. uliginosus plants had particularly profuse lenticels and aerenchyma. However, L. corniculatus nodules, especially those grown in the lower pO2 showed signs of early senescence with vacuolation of infected cells and green coloration when cut open. Leghaemoglobin (Lb) concentrations in nodules from both species were unaffected by low pO2, although that of L. corniculatus nodules, regardless of pO2, was significantly greater than L. uliginosus. Concentrations of the intercellular glycoprotein recognized by the monoclonal antibody MAC265 were significantly reduced in nodules from the low pO2 treatment in both species. Immunogold labelling showed that the MAC265 antigen was localized primarily within intercellular spaces within nodule cortices from both Lotus species. A marked decrease in deposition of the MAC265 antigen within the cortices of L. uliginosus nodules grown in the lower pO2, is discussed in terms of the relative abilities of the two Lotus spp. to maintain an O2 supply to the N2-fixing bacteroids within submerged nodules.Keywords: Lotus uliginosus, Lotus corniculatus, N2 fixation, flooding, oxygen.      

Effectiveness of Lotus Root Nodules: II. RELATIONSHIP BETWEEN ROOT NODULE EFFECTIVENESS AND 'IN VITRO' SENSITIVITY OF FAST-GROWING LOTUS RHIZOBIA TO FLAVOLANS

An extract from the roots of Lotus pedunculatus plants was foundto contain a compound toxic towards fast-growing Lotus rhizobia.This compound was identified as a flavolan, which has a prodeiphinidin:procyanidin ratio of 75:25. A fast-growing strain of Rhizobium(NZP2213) which forms ineffective root nodules on L. pedunculatuswas four times more sensitive to this flavolan (ED₅₀ = 25 ?gml^–1) than another strain (NZP2037, ED₅₀ = 100 ?g ml^–1)which forms effective root nodules on this species. The rootsof another Lotus species, L. tenuis, on which both strains ofRhizobium form effective root nodules, also contained a flavolan( 95% procyanidin) but both strains were relatively insensitiveto this flavolan (EDED₅₀ = 350 to 500 ?g ml^–1) L. pedunculatusplants bearing ineffective root nodules contained two to threetimes more flavolan in their roots (5–7 mg g^–1 fr.wt.)than uninoculated control plants. Experiments with seven otherLotus species and with hybrid plants developed between L. pedunculatusand L. tenuis showed a relationship between the prodeiphinidin:procyanidin ratio of the flavolan in their roots and the effectivenessof root nodules formed on these plants by NZP2213. Quantitativebinding studies of the flavolan from L. pedunculatus to NZP2037and NZP2213 indicated that, while the affinity constants forbinding were similar for both strains, the surface of strainNZP2037 contained four times more binding sites than NZP2213,possibly correlating with this strain's ability to toleratehigher concentrations of this flavolan. It is suggested thatthe differential sensitivity of these two strains of Rhizobiumto flavolans is related to their ability to form effective rootnodules on Lotus species.