Genetic diversity and symbiotic compatibility among rhizobial strains and Desmodium incanum and Lotus spp. plants

This work aimed to evaluate the symbiotic compatibility and nodulation efficiency of rhizobia isolated from Desmodium incanum, Lotus corniculatus, L. subbiflorus, L. uliginosus and L. glaber plants by cross-inoculation. Twelve reference strains and 21 native isolates of rhizobia were genetically analyzed by the BOX-PCR technique, which showed a high genetic diversity among the rhizobia studied. The isolates were also characterized based on their production of indolic compounds and siderophores, as well as on their tolerance to salinity. Fifteen of the 33 rhizobia analyzed were able to produce indolic compounds, whereas 13 produced siderophores. All the tested rhizobia were sensitive to high salinity, although some were able to grow in solutions of up to 2% NaCl. Most of the native rhizobia isolated from L. uliginosus were able to induce nodulation in all plant species studied. In a greenhouse experiment using both D. incanum and L. corniculatus plants, the rhizobia isolate UFRGS Lu2 promoted the greatest plant growth. The results demonstrate that there are native rhizobia in the soils of southern Brazil that have low host specificity and are able to induce nodulation and form active nodules in several plant species.     

Genome relationships among Lotus species based on random amplified polymorphic DNA (RAPD)

The ability of random amplified polymorphic DNA (RAPD) to distinguish among different taxa of Lotus was evaluated for several geographically dispersed accessions of four diploid Lotus species, L. tennis Waldst. et Kit, L. alpinus Schleich., L. japonicus (Regel) Larsen, and L. uliginosus Schkuhr and for the tetraploid L. corniculatus L., in order to ascertain whether RAPD data could offer additional evidence concerning the origin of the tetraploid L. corniculatus. Clear bands and several polymorphisms were obtained for 20 primers used for each species/accession. The evolutionary pathways among the species/accessions presented in a cladogram were expressed in terms of treelengths giving the most parsimonious reconstructions. Accessions within the same species grouped closely together. It is considered that L. uliginosus which is most distantly related to L. corniculatus, may be excluded as a direct progenitor of L. corniculatus, confirming previous results from isoenzyme studies. Lotus alpinus is grouped with accessions of L. corniculatus, which differs from previous studies. With this exception, these findings are in agreement with previous experimental studies in the L. corniculatus group. The value of the RAPD data to theories on the origin of L. corniculatus is discussed.     

Selecting improved Lotus nodulating rhizobia to expedite the development of new forage species

Aims

In the past decades the increasing focus by Australian pasture development programs on the genus Lotus has seen the evaluation of many species previously untested in Australia. In field trials, nodulation failure was commonplace. This work was undertaken to select effective symbionts for Lotus to ensure further agronomic evaluation of the genus was not compromised. The symbiotic needs of Lotus ornithopodioides were a particular focus of the studies.

Methods

Glasshouse experiments were undertaken to evaluate symbiotic relationships between 15 Lotus spp and 23 strains of nodulating Mesorhizobium loti. This was followed by evaluation of elite rhizobial strains for their ability to persist and form nodules under field conditions.

Results

Complex symbiotic interactions were recorded between strains of lotus rhizobia and the different species of Lotus. Notably, the rhizobia that are currently provided commercially in Australia for the inoculation of Lotus corniculatus (strain SU343) and Lotus uliginosus (strain CC829) did not form effective symbioses with the promising species L. ornithopodioides and L. maroccanus. No strain we evaluated was compatible with all the Lotus species, however several strains with a broad host range were identified. WSM1293 and WSM1348 were the most effective strains on L. ornithopodioides and L. peregrinus.These strains were also moderately effective on L. corniculatus (79 and 52% of SU343), less effective on L. maroccanus (26 and 49% of SRDI110) but were ineffective on L. uliginosus. The latter species overall had very specific rhizobial needs. Both WSM1293 and WSM1348 produced adequate levels of nodulation when inoculated on L. ornithopodioides, over two seasons at three field sites.

Conclusions

Effective and persistent strains are now available that should allow the un-compromised evaluation of many of the contemporary Lotus species in the field. Selecting a strain for use in commercial inoculants will be more problematic, given the very large host-strain interactions for nitrogen fixation. Here, the balance of Lotus species which are adopted by farmers will have a strong bearing on which rhizobial strains are progressed to commerce.     

Effect of Root Temperature on the Infection Processes and Nodulation in Lotus and Stylosanthes

Infection threads were observed abundantly in the root hairsof Lotus corniculatus L., but very rarely in L. hispidus, Desf.,in response to infection by Rhizobium strains 3001 and 3002.Numbers of infections differed between species and strains andwere also affected by temperature. In L. corniculatus all thenodules originated from infection threads, but in L. hispidusmost nodules appeared to originate by direct bacterial penetrationthrough the epidermis, and infected root hairs were very rarelyseen. Both species of Lotus were tolerant to cold temperatures,the minimum temperature for nodulation being 10 ?C. The optimumtemperature for nodulation of L. corniculatus was 20 ?C with3001 and between 27 and 30 ?C with 3002, a few nodules beingformed with both strains at 35 ?C. L. hispidus formed more nodulesthan L. corniculatus and the optimum temperature for both thestrains was between 25 and 27 ?C. No infection threads were seen in root hairs or nodules of Stylosanthesguyanensis (Aubl.) S. W. and S. humilis H.B.K. infected withRhizobium strain CB1552, and all the nodules were formed inthe axils of lateral roots. Optimum temperature for nodulationin S. guyanensis and S. humilis was around 27 ?C; nodulationwas completely inhibited at 15 ?C and very few nodules wereformed at 35 ?C. Both in Lotus and Stylosanthes the transfer of plants from suboptimalto optimal and supraoptimal temperatures increased nodulation.Delayed inoculation and excision of root tips increased nodulation.     

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.     

Evaluation of hypotheses concerning the origin of Lotus corniculatus (Fabaceae) using isoenzyme data

Summary An isoenzyme survey was conducted for several geographically dispersed accessions of four diploid Lotus species, L. alpinus Schleich., L. japonicus (Regel) Larsen, L. tenuis Waldst. et Kit and L. uliginosus Schkuhr, and for the tetraploid L. corniculatus L., in order to ascertain whether isoenzyme data could offer additional evidence concerning the origin of L. corniculatus. Seven enzyme systems were examined using horizontal starch gel electrophoresis. These were PGI, TPI, MDH, IDH, PGM, 6-PGDH, and ME. Lotus uliginosus had monomorphic unique alleles, that were not found within L. corniculatus, at 7 loci. These loci and alleles are: Tpi1-112, Pgm1,2-110, Pgm3-82, Mdh3-68, 6-Pgdh1-110, 6-Pgdh2-98,95, and Me2-100. Other diploid taxa contained alleles found in L. corniculatus for these and other loci. The implications of the isoenzyme data to theories on the origin of L. corniculatus are discussed.Communicated by H. F. Linskens     

Chloroplast-DNA variation in the genus Lotus (Fabaceae) and further evidence regarding the maternal parentage of Lotus corniculatus L.

 To resolve the maternal parentage of the tetraploid Lotus corniculatus, restriction-site variation of chloroplast DNA (cpDNA) was studied in several accessions of that species, in the four putative parental diploid species, L. tenuis, L. alpinus, L. japonicus and L. uliginosus, and in four phylogenetically more distant diploid species, L. hispidus, L. edulis, L. ornithopodoides and Tetragonolobus maritimus var. siliquosus. Evidence of cpDNA maternal inheritance was obtained by using reciprocal controlled crosses between plants of L. corniculatus and natural tetraploid individuals of L. alpinus showing very distinct restriction patterns. Interspecific cpDNA variation in the eight Lotus species and T. siliquosus was analysed by comparing cpDNA fragment patterns produced by five restriction endonucleases and totalling 304 distinct fragments. Genetic differentiation in cpDNA was very high between the L. corniculatus group and L. hispidus on the one hand, and the three other species on the other hand. Sixteen restriction-site mutations and eight length polymorphisms were identified among the five species of the L. corniculatus group and L. hispidus, Lotus uliginosus, L. alpinus and L. japonicus showed at least six DNA changes with regard to the molecule of L. corniculatus. Accordingly, these species should be excluded as maternal progenitors of L. corniculatus. Conversely, the cpDNA of L. tenuis differed from that of L. corniculatus by only two small-length mutations. As also suggested previously from an analysis of several nuclear markers, the results reported here show decisively that L. tenuis may be considered as the most probable maternal ancestor of L. corniculatus. Received: 23 February 1997/Accepted: 28 February 1997     

Chemical defense production in Lotus corniculatus L. I. The effects of nitrogen source on growth,reproduction and defense

Summary The carbon to nitrogen balance theory was examined for a legume, Lotus corniculatus L., which allocates carbon to nitrogen fixation. N-fixation can influence the ratio of carbon to nitrogen in legumes by providing nitrogen in nutrient-poor habitats, and by consuming carbon for support of symbiotic N-fixation. L. corniculatus clones (genotypes) were grown under two levels of nitrogen fertilization: a treatment which suppressed nodulation with fertilization and a treatment which received no additional fertilization. These plants relied solely on symbiotic N-fixation. Plants which supported symbionts had lower biomass and lower tannin concentrations than fertilized plants; this appears to be a result of the large carbon demand on N-fixation. Plants supporting symbionts often had relatively lower protein concentrations than fertilized plants. Cyanide concentration was influenced by plant genotype but not by nitrogen source. Although symbiotic N-fixing plants were smaller, they had three times the reproductive output of fertilized plants.     

The rhizobia of lupinus densiflorus Benth., with some remarks on the classification of root nodule bacteria

Summary Four strains of rhizobia from Lupinus densiflorus Benth. were found to differ from the normal slow-growing strains of Rhizobium lupini by a rapid growth on agar medium, a somewhat different pattern of carbon metabolism, good growth in simple synthetic media, and also in their host plant relationships. Three strains had subpolar flagella like other lupine rhizobia, and the same was found to be predominant in a fourth strain previously described as having peritrichous flagellation.Two strains formed effectively nitrogen-fixing root nodules in Lotus corniculatus and Anthyllis vulneraria where the other two formed semieffective or ineffective nodules. All four strains formed ineffective nodules in Lotus uliginosus and Ornithopus sativus. The slow-growing strains of Rh. lupini mostly produce ineffective nodules in Lotus corniculatus but have now been seen to be effective in Lotus uliginosus.Instead of trying to define Rh. lupini as a cross-inoculation group it seems preferable to abandon it as a species and to transfer the fastgrowing strains to Rhizobium leguminosarum sensu Graham (1964) and De Ley and Rassel (1965), in spite of their predominantly subpolar flagellation. The familiar slow-growing strains would remain in the broad group of slow-growing root nodule bacteria with purely subpolar flagellation, called Phytomyxa japonica by Graham (1964) and Rhizobium japonicum by De Ley and Rassel (1965).     

Effect of plant nutrient supply on nodule effectiveness and rhizobium strain competition for nodulation ofLotus pedunculatus

Summary The effect of nutrient supply on nodule formation and competition between Rhizobium strains for nodulation ofLotus pedunculatus was studied. Limiting plant growth by decreasing the supply of nutrients in an otherwise nitrogen-free medium, increased the size but decreased the number and the nitrogenase activity of nodules formed by a fast-growing strain of Lotus Rhizobium (NZP2037). In contrast decreasing nutrient supply caused only a small decline in the size, number and nitrogenase activity of nodules formed by a slow-growing strain (CC814s). Providing small quantities of NH₄NO₃ (50 to 250 g N) to plants grown with a normal supply of other nutrients stimulated nodule development by both Rhizobium strains and increased the nitrogenase activity of the NZP2037 nodules. Differences in the level of effectiveness (nitrogen-fixing ability) of nodules formed by different Rhizobium strains on plants grown with a normal supply of nutrients were less apparent when the plants were grown with decreased nutrient supply or when the plants were supplied with low levels of inorganic N.Inter-strain competition for nodulation ofL. pedunculatus between the highly effective slow-growing strain CC814s and 7 other fast- and slow-growing strains, showed CC814s to form 42 to 100% of the nodules in all associations. The greater nodulating competitiveness of strain CC814s prevailed despite changes in the nutrient supply to the host plant. A tendency was observed for partially effective Lotus Rhizobium strains to become more competitive in nodule formation when plant growth was supplemented with low levels of inorganic nitrogen.     

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).     

Proteomic mapping for legume nodule organogenesis

While genetic screens have identified mutants of the model legume Lotus japonicus that can nodulate in the absence of rhizobia, the lack of a proteome map is a major hindrance to understanding the functional protein networks associated with this nodulation process. In this issue of Proteomics, Dam et al. (Proteomics 2014, 14, 230–240) developed 2D gel‐based reference maps of nodules and roots of Lotus and a spontaneous nodule formation mutant (snf1). Comparative proteomic analysis of roots and two developmental stages of nodules provide useful insights into tissue‐specific mechanisms underlying nodule organogenesis. Additionally, a comparison of interspecies nodule proteomes displays that overlapping and individual mechanisms are associated with legume nodulation.     

The origin of Lotus corniculatus

Summary Earlier students of the origin of Lotus corniculatus suggested that this tetraploid species arose as an autotetraploid of the closely related diploid species L. tenuis or L. alpinus. More recent studies suggested that L. alpinus and L. japonicus could be ancestral forms. The present study of tannin content, phenolic content, cyanide production, morphology, cytogenetics, Rhizobium specificity and self-incompatibility in the corniculatus group virtually excludes the possibility that L. corniculatus could have arisen through autopolyploidy of L. tenuis or L. alpinus, and suggests that L. corniculatus arose through hybridization of L. alpinus and/or L. tenuis (probably as female parent) with L. uliginosus (probably as male parent), followed by chromosome doubling in the hybrid.     

The proanthocyanidin content as a tool to differentiate between Lotus tenuis and L. corniculatus individuals

Lotus tenuis Wald et Kit and Lotus corniculatus L. are conspicuous elements of the agricultural landscape for cattle production. In South America, commercial L. tenuis stocks usually present contaminations with L. corniculatus, what brings about an important economical injure to the forage producers. A way to reduce or avoid loses is to assess the purity degree of L. tenuis seed lots before seeding. Methods so far described for the diagnosis of Lotus species when flowers are not available are lengthy, time consuming, need the implementation of sophisticated laboratories and are relatively expensive. It has been shown that Lotus species accumulate variable proanthocyanidins amounts, which can be easily visualized by a simple and rapid staining method. In this work, we demonstrate that the leaf PA content is a specific trait of L. tenuis and L. corniculatus and hence, it allows the unambiguous differentiation between both species, even at an early phenological stage.     

Temporal Relationships between Nitrogenase and Intercellular Glycoprotein in Developing White Lupin Nodules

The development of the N₂-fixing symbiosis between white lupin (Lupinus albusL.) cv. Multolupa andBradyrhizobiumstrain ISLU16 was followed using the acetylene reduction assay (ARA), immunoblots of protein extracts, and microscopy/immunogold labelling at 0, 8, 12, 17 and 20 d after infection. There was no ARA at 0, 8 and 12 d, although macroscopically visible nodule primordia had formed on roots by 8 d. The lack of nitrogenase at these times was confirmed by a negative signal to immunogold labelling with nitrogenase-specific antibodies. At 17 d three out of six plants had ARA, and nodules from these gave a positive signal with the nitrogenase antibody. By contrast, ARA⁻(fix⁻) nodules at 17 d were smaller (mean radius of 0.49 mm compared to 1.01 mm with fix⁺nodules) and gave a negative signal with the nitrogenase antibody. Western blots of nodule protein extracts using the monoclonal antibodies MAC236 and MAC265 (which recognize two epitopes on a glycoprotein which is considered to be involved in both rhizobial infection and the regulation of nodule oxygen diffusion) gave a strong signal with nodules (fix⁺) from 20 d plants and with 17 d fix⁺plants. The signal with MAC236/MAC265 was substantially weaker with nodules from 17 d fix⁻plants, and there was no signal apparent from nodules/nodulated roots from the 0, 8 and 12 d harvests. However, further investigation using immunogold labelling revealed that not only were MAC236 and MAC265 expressed within cortical intercellular spaces in 20 d and 17 d fix⁺/fix⁻nodules, but they were also strongly expressed in the developing cortex surrounding the newly-infected tissue in 8 d nodules, as well as in intercellular spaces within the cortex and infected tissue of 12 d nodules. These data demonstrate that the glycoprotein recognized by MAC236 and MAC265 is present before the onset of nitrogenase expression and function, but expression of the epitopes appears to be enhanced from the onset of N₂fixation. Nodules at all harvests were investigated for the presence of infection threads, as the MAC236/MAC265-recognized glycoprotein is also a component of the infection thread matrix in nodules from other legumes. Infection threads were not seen in nodules from any of the harvests except for the 20 d nodules, and then only after serial sectioning. The latter revealed occasional short wide infection threads entering and releasing rhizobia into small pockets of uninfected cells, within the infected tissue, but not within the meristems. The matrix of these infection threads labelled weakly, or not at all, with MAC236 and MAC265, and it was concluded that the majority of the MAC236/MAC265 detected in lupin nodule extracts originated from glycoprotein within cortical intercellular spaces.     

The effect of source,concentration and time of application of nitrogen on the growth,nodulation and nitrogen fixation ofLotus pedunculatus andTrifolium repens

Summary Studies under growth cabinet conditions investigated the effect of source and concentration of nitrogen and timing of nitrogen application on the growth and nitrogen fixation byLotus pedunculatus cv. Maku andTrifolium repens cv. S184. KNO₃, NaNO₃ and NH₄NO₃ were added at transplanting at the following rates: 3.33, 7.78 and 13.33 mg N/plant. KNO₃ was added at 3.33 and 7.78 mg N/plant at 0, 6, 12, 18, 24 or 30 days after transplanting.Lotus shoot weight increased with all increasing nitrogen sources but clover only responded to KNO₃ and NaNO₃. The root weight of both species increased with increasing KNO₃ and NH₄NO₃. The percentage increase in lotus and clover shoot growth was greater than that of root growth when KNO₃ was added within a week of transplanting. Increases in growth by both species resulted from added nitrogen except with lotus when NaNO₃ was applied where increased nitrogen fixation also contributed to increased growth.Weight and number of effective nodules on both species were increased with 3.33 mg N per plant as KNO₃ but nitrogen fixation was not affected. Addition of 13.33 mg N as NaNO₃ reduced weight and number of effective nodules in both species and also nitrogen fixation by lotus.KNO₃ increased growth and nodulation of both species when applied within one week after transplanting. Nodulated lotus plants responded to KNO₃ by increasing growth but not nodulation.KNO₃ appeared to affect infection and development of nodules on lotus and may affect the growth of existing nodules on clover.     

Subcellular localization of glycoprotein epitopes during the development of lupin root nodules

Summary The monoclonal antibodies MAC236 and MAC265, raised against a soluble component of pea nodules, were used to elucidate the presence and subcellular localization of glycoprotein epitopes during the development of lupin (Lupinus albus L. cv. Multolupa) nodules, by means of immunocytochemistry and Western blot analysis. These antibodies recognize a single band of 95 kDa in pea, soybean and bean nodules, whilst two different bands of 240 and 135 kDa cross-react with MAC236 and MAC265 respectively in lupin nodules. This fact may indicate that the recognized epitopes can be present in different subcellular compartments and/or play different roles through the development of functional nodules. The results show that MAC265 is mainly associated with Bradyrhizobium infection and with the development of nodule primordium, in the first stages of nodulation. MAC265 is also detected when glycoprotein transport takes place across the cytoplasm and the cell wall, and also in the intercellular spaces of the middle cortex, attached to cell walls. The amount of MAC265 remains constant through nodule development. In contrast the amount of MAC236 increases with nodule age, parallel to the establishment of nitrogenase activity. This antibody is localized in cytoplasmic globules attached to the inner side of cell walls in the middle cortex, and mainly in the matrix filling the intercellular spaces of the middle and inner cortex. This main site of localization of MAC236 may indicate a role in the functioning of the oxygen diffusion barrier.     

Isoflavan phytoalexins from Anthyllis,lotus and Tetragonolobus

Two previously unreported phytoalexins, 7,4′dihydroxy-2′-methoxy- and 7,2′,4′-trihydroxyisoflavan, have been isolated from the fungus-inoculated leaves of Anthyllis vulneraria and 5 Tetragonolobus species. Examination of Lotus corniculatus revealed the co-occurrence of the latter with the known isoflavans, vestitol and sativan. Only 7,2′4′-trihydroxyisoflavan and vestitol were produced by the closely related L. uliginosus.     

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}     

Growth and ion relations in response to combined salinity and waterlogging in the perennial forage legumes Lotus corniculatus and Lotus tenuis

Lotus tenuis (Wadst. & Kit.) is a perennial legume widely grown for pasture in the flood-prone and salt affected Pampa region of Argentina. The physiology of salt and waterlogging tolerance in L. tenuis (four cultivars) was evaluated, and compared with Lotus corniculatus (three cultivars); the most widely cultivated Lotus species. Overall, L. tenuis cultivars accumulated less Na⁺ and Cl⁻, and more K⁺ in shoots than L. corniculatus cultivars, when exposed to 200 mM NaCl for 28 days in aerated or in stagnant solutions. Root porosity was higher in L. tenuis cultivars due to greater aerenchyma formation. In a NaCl dose–response experiment (0–400 mM NaCl in aerated solution), L. tenuis (cv. Chaja) accumulated half as much Cl⁻ in its shoots than L. corniculatus (cv. San Gabriel) at all external NaCl concentrations, and about 30% less shoot Na⁺ in treatments above 250 mM NaCl. Ion distributions in shoots were determined for plants at 200 mM NaCl. L. tenuis (cv. Chaja) again accumulated about half as much Cl⁻ in old leaves, young leaves and stems, compared with concentrations in L. corniculatus (cv. San Gabriel). There were not, however, significant differences between the two species for Na⁺ concentrations in the various shoot tissues. The higher root porosity, and maintenance of lower shoot Cl⁻ and Na⁺ concentrations in L. tenuis, compared with L. corniculatus, contributes to the greater tolerance to combined salt and waterlogging stress in L. tenuis. Moreover, significant variation for tolerance to combined salinity and waterlogging stress was identified within both L. tenuis and L. corniculatus.