Phylogeny based on 16S rDNA and nifH sequences of Ralstonia taiwanensis strains isolated from nitrogen-fixing nodules of Mimosa pudica, in India

Bacterial symbionts present in the indeterminate-type nitrogen (N)-fixing nodules of Mimosa pudica grown in North and South India showed maximum similarity to Ralstonia taiwanensis on the basis of carbon-source utilization patterns and 16S rDNA sequence. Isolates from the nodules of M. pudica from North India and South India showed identical ARDRA (Amplified Ribosomal DNA Restriction Analysis) patterns with Sau3AI and RsaI, but AluI revealed dimorphy between the North Indian and South Indian isolates. Alignment of 16S rDNA sequences revealed similarity of North Indian isolates with an R. taiwanensis strain isolated from M. pudica in Taiwan, whereas South Indian isolates showed closer relatedness with the isolates from Mimosa diplotricha. Alignment of nifH sequences from both North Indian and South Indian isolates with that of the related isolates revealed their closer affinity to alpha-rhizobia, suggesting that nif genes in the beta-rhizobia might have been acquired from alpha-rhizobia via lateral transfer during co-occupancy of nodules by alpha-rhizobia and progenitors of R. taiwanensis, members of the beta-subclass of Proteobacteria. Immunological cross-reaction of the bacteroid preparation of M. pudica nodules showed strong a positive signal with anti-dinitrogenase reductase antibody, whereas a weak positive cross-reaction was observed with free-living R. taiwanensis grown microaerobically in minimal medium with and without NH4Cl. In spite of the expression of dinitrogenase reductase under free-living conditions, acetylene reduction was not observed under N-free conditions even after prolonged incubation.     

Burkholderia and Cupriavidus spp. are the preferred symbionts of Mimosa spp. in southern China

Rhizobia were isolated from invasive Mimosa spp. (M.?diplotricha and M.?pudica) in Dehong district of the province of Yunnan in subtropical southern China. Almost all of the 98 isolates were β-rhizobia in the genera Burkholderia and Cupriavidus. These strains were analysed for their distribution characteristics together with strains from a previous study from Sishuangbanna. The proportion of nodules containing each β-rhizobial genus varied between Mimosa species, with Cupriavidus being predominant in M.?diplotricha nodules (63.3% compared to 36.7% occupation with Burkholderia), but with M.?pudica showing a slight preference for Burkholderia over Cupriavidus, with them occupying 56.5% and 43.5% of nodules, respectively. The symbiosis-essential genes nodA and nifH were present in all the Burkholderia and Cupriavidus strains tested, and their phylogenies indicated that these Mimosa symbionts share symbiotic genes with native South American rhizobia. The evolutionary discrepancies among 16S rRNA genes, nodA and nifH of Mimosa spp. symbionts, suggests that the nod and nif genes of β-rhizobia evolved independently.     

Nodulation of Cyclopia spp. (Leguminosae, Papilionoideae) by Burkholderia tuberum

BACKGROUND AND AIMS: Species of the genus Burkholderia, from the Betaproteobacteria, have been isolated from legume nodules, but so far they have only been shown to form symbioses with species of Mimosa, sub-family Mimosoideae. This work investigates whether Burkholderia tuberum strains STM678 (isolated from Aspalathus carnosa) and DUS833 (from Aspalathus callosa) can nodulate species of the South African endemic papilionoid genera Cyclopia (tribe Podalyrieae) and Aspalathus (Crotalarieae) as well as the promiscuous legume Macroptilium atropurpureum (Phaseoleae). METHODS: Bacterial strains and the phylogeny of their symbiosis-related (nod) genes were examined via 16S rRNA gene sequencing. Seedlings were grown in liquid culture and inoculated with one of the two strains of B. tuberum or with Sinorhizobium strain NGR 234 (from Lablab purpureus), Mesorhizobium strain DUS835 (from Aspalathus linearis) or Methylobacterium nodulans (from Crotalaria podocarpa). Some nodules, inoculated with green fluorescence protein (GFP)-tagged strains, were examined by light and electron microscopy coupled with immunogold labelling with a Burkholderia-specific antibody. The presence of active nitrogenase was checked by immunolabelling of nitrogenase and by the acetylene reduction assay. B. tuberum STM678 was also tested on a wide range of legumes from all three sub-families. KEY RESULTS: Nodules were not formed on any of the Aspalathus spp. Only B. tuberum nodulated Cyclopia falcata, C. galioides, C. genistoides, C. intermedia and C. pubescens. It also effectively nodulated M. atropurpureum but no other species tested. GFP-expressing inoculant strains were located inside infected cells of C. genistoides, and bacteroids in both Cyclopia spp. and M. atropurpureum were immunogold-labelled with antibodies against Burkholderia and nitrogenase. Nitrogenase activity was also shown using the acetylene reduction assay. This is the first demonstration that a beta-rhizobial strain can effectively nodulate papilioinoid legumes. CONCLUSIONS: Papilionoid legumes from widely different tribes can be nodulated by beta-rhizobia, forming both indeterminate (Cyclopia) and determinate (Macroptilium) nodules.     

Stem and Root Nodulation in Aeschynomene spp

Nodulation ability of 15 Rhizobium strains isolated from root and stem nodules of tropical Aeschynomene species was studied on 20 different Aeschynomene species and four other legumes—Arachis hypogaea, Stylosanthes guianensis, Macroptilium atropurpureum, and Sesbania rostrata. The results of this investigation showed that Aeschynomene species could be divided into three groups according to the cross-inoculation group concept.     

Burkholderia spp. are the most competitive symbionts of Mimosa, particularly under N-limited conditions

Bacteria isolated from Mimosa nodules in Taiwan, Papua New Guinea, Mexico and Puerto Rico were identified as belonging to either the α- or β-proteobacteria. The β-proteobacterial Burkholderia and Cupriavidus strains formed effective symbioses with the common invasive species Mimosa diplotricha , M. pigra and M. pudica , but the α-proteobacterial Rhizobium etli and R. tropici strains produced a range of symbiotic phenotypes from no nodulation through ineffective to effective nodulation, depending on Mimosa species. Competition studies were performed between three of the α-proteobacteria ( R. etli TJ167, R. tropici NGR181 and UPRM8021) and two of the β-rhizobial symbionts ( Burkholderia mimosarum PAS44 and Cupriavidus taiwanensis LMG19424) for nodulation of these invasive Mimosa species. Under flooded conditions, B. mimosarum PAS44 out-competed LMG19424 and all three α-proteobacteria to the point of exclusion. This advantage was not explained by initial inoculum levels, rates of bacterial growth, rhizobia-rhizobia growth inhibition or individual nodulation rate. However, the competitive domination of PAS44 over LMG19424 was reduced in the presence of nitrate for all three plant hosts. The largest significant effect was for M. pudica , in which LMG19424 formed 57% of the nodules in the presence of 0.5 mM potassium nitrate. In this host, ammonium also had a similar, but lesser, effect. Comparable results were also found using an N-containing soil mixture, and environmental N levels are therefore suggested as a factor in the competitive success of the bacterial symbiont in vivo .     

Competition Among Rhizobium spp. for Nodulation of Leucaena leucocephala in Two Tropical Soils

The successful nodulation of legumes by a Rhizobium strain is determined by the competitive ability of that strain against the mixture of other native and inoculant rhizobia. Competition among six Leucaena rhizobial strains in single and multistrain inoculants were studied. Field inoculation trials were conducted in an oxisol and a mollisol soil, both of which contained indigenous Leucaena-nodulating rhizobia. Strain-specific fluorescent antibodies were used for the identification of the strains in Leucaena nodules. Mixtures of three recommended inoculum strains for Leucaena spp. (TAL82, TAL582, and TAL1145) were used in peat-based inocula either alone or with one of the three other strains isolated from the sites, B213, B214, and B215. Each of these latter three strains was also used as single-strain inocula to study their competition with the native rhizobia in the two soil systems. In the oxisol soil, strains B213 and B215, when used as single-strain inocula, outcompeted the native rhizobia and formed 92 and 62% of the nodules, respectively. Strain B214 was the least competitive in oxisol soil, where it formed 30% of the nodules, and the best in mollisol soil, where it formed 70% of the nodules. The most successful competitor for nodulation in multistrain inocula was strain TAL1145, which outcompeted native and other inoculum Leucaena rhizobia in both soils. None of the strains in single or multistrain inoculants was capable of completely overcoming the resident rhizobia, which formed 4 to 70% of the total nodules in oxisol soil and 12 to 72% in mollisol soil. No strong relationship was detected between the size of the rhizosphere population of a strain and its successful occupation of nodules.     

分离自杭子梢等宿主根瘤的30株菌的结瘤特性的研究

对分离自杭子梢、菜豆和决明等宿主根瘤、处于Agrobacterium系统发育分支、DNA-DNA杂交与A.rubi的相似性达到100%的30株土壤杆菌,分属于Agrobacterium、Bradyrhizobium、Mesorhizobium、Rhizobium和Sinorhizobium 5个属的12个参比菌株。nodA PCR的结果表明,30株供试菌中扩增不出nodA,即没有结瘤性。以Sinorhizobium meliloti USDA1002T的nodA做探针对所提取的细菌总DNA进行斑点杂交,在65℃-68℃严谨洗膜条件下,该探针只能与同种的根瘤菌进行杂交,不能与其它属的根瘤菌或土壤杆菌杂交,初步推测共同结瘤基因nodA探针只能对种内根瘤菌的结瘤性进行鉴定。     

云南省德宏州含羞草β-根瘤菌多样性及系统发育研究

【目的】通过对分离自云南德宏州的含羞草β-根瘤菌进行遗传与表型多样性研究,揭示我国含羞草β-根瘤菌的物种多样性。【方法】应用16S rDNA PCR-RFLP、全细胞蛋白SDS-PAGE电泳及16S rDNA全序列分析对分离得到的60株含羞草根瘤菌进行多样性研究。【结果】16S rDNA PCR-RFLP及全细胞蛋白SDS-PAGE图谱分析将供试菌株分为2个遗传型群和2个表型群,分别与贪铜菌属(Cupriavidus)和伯克霍尔德菌属(Burkholderia)参比菌株聚群。经16S rDNA全序列分析,供试菌株被归到台湾贪铜菌(Cupriavidus taiwanensis)、含羞草伯克霍尔德菌(Burkholderia mimosarum)及结瘤伯克霍尔德菌(Burkholderia phymatum)等3个种群。【结论】云南德宏州的含羞草β-根瘤菌主要为贪铜菌及伯克霍尔德菌类群,其中贪铜菌占绝对优势,且存在遗传和表型的丰富多样性,该研究揭示了含羞草β-根瘤菌的物种多样性并丰富了我国β-根瘤菌菌种资源。     

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

Background and aims

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

Methods

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

Results

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

Conclusions

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

Biodegradation of cis-dichloroethene as the sole carbon source by a beta-proteobacterium

An aerobic bacterium capable of growth on cis-dichloroethene (cDCE) as a sole carbon and energy source was isolated by enrichment culture. The 16S ribosomal DNA sequence of the isolate (strain JS666) had 97.9% identity to the sequence from Polaromonas vacuolata, indicating that the isolate was a beta-proteobacterium. At 20 degrees C, strain JS666 grew on cDCE with a minimum doubling time of 73 +/- 7 h and a growth yield of 6.1 g of protein/mol of cDCE. Chloride analysis indicated that complete dechlorination of cDCE occurred during growth. The half-velocity constant for cDCE transformation was 1.6 +/- 0.2 microM, and the maximum specific substrate utilization rate ranged from 12.6 to 16.8 nmol/min/mg of protein. Resting cells grown on cDCE could transform cDCE, ethene, vinyl chloride, trans-dichloroethene, trichloroethene, and 1,2-dichloroethane. Epoxyethane was produced from ethene by cDCE-grown cells, suggesting that an epoxidation reaction is the first step in cDCE degradation.     

Burkholderia phymatum is a highly effective nitrogen-fixing symbiont of Mimosa spp. and fixes nitrogen ex planta

* The ability of Burkholderia phymatum STM815 to effectively nodulate Mimosa spp., and to fix nitrogen ex planta, was compared with that of the known Mimosa symbiont Cupriavidus taiwanensis LMG19424. * Both strains were equally effective symbionts of M. pudica, but nodules formed by STM815 had greater nitrogenase activity. STM815 was shown to have a broader host range across the genus Mimosa than LMG19424, nodulating 30 out of 31 species, 21 of these effectively. LMG19424 effectively nodulated only nine species. GFP-marked variants were used to visualise symbiont presence within nodules. * STM815 gave significant acetylene reduction assay (ARA) activity in semisolid JMV medium ex planta, but no ARA activity was detected with LMG19424. 16S rDNA sequences of two isolates originally from Mimosa nodules in Papua New Guinea (NGR114 and NGR195A) identified them as Burkholderia phymatum also, with nodA, nodC and nifH genes of NGR195A identical to those of STM815. * B. phymatum is therefore an effective Mimosa symbiont with a broad host range, and is the first reported beta-rhizobial strain to fix nitrogen in free-living culture.     

A Duplex PCR Assay for the Detection of Ralstonia solanacearum Phylotype II Strains in Musa spp.

Banana wilt outbreaks that are attributable to Moko disease-causing strains of the pathogen Ralstonia solanacearum (Rs) remain a social and economic burden for both multinational corporations and subsistence farmers. All known Moko strains belong to the phylotype II lineage, which has been previously recognized for its broad genetic basis. Moko strains are paraphyletic and are distributed among seven related but distinct phylogenetic clusters (sequevars) that are potentially major threats to Musaceae, Solanaceae, and ornamental crops in many countries. Although clustered within the Moko IIB-4 sequevar, strains of the epidemiologically variant IIB-4NPB do not cause wilt on Cavendish or plantain bananas; instead, they establish a latent infection in the vascular tissues of plantains and demonstrate an expanded host range and high aggressiveness toward Solanaceae and Cucurbitaceae. Although most molecular diagnostic methods focus on strains that wilt Solanaceae (particularly potato), no relevant protocol has been described that universally detects strains of the Musaceae-infecting Rs phylotype II. Thus, a duplex PCR assay targeting Moko and IIB-4NPB variant strains was developed, and its performance was assessed using an extensive collection of 111 strains representing the known diversity of Rs Moko-related strains and IIB-4NPB variant strains along with certain related strains and families. The proposed diagnostic protocol demonstrated both high accuracy (inclusivity and exclusivity) and high repeatability, detected targets on either pure culture or spiked plant extracts. Although they did not belong to the Moko clusters described at the time of the study, recently discovered banana-infecting strains from Brazil were also detected. According to our comprehensive evaluation, this duplex PCR assay appears suitable for both research and diagnostic laboratories and provides reliable detection of phylotype II Rs strains that infect Musaceae.     

Coexistence of Burkholderia, Cupriavidus, and Rhizobium sp. Nodule Bacteria on two Mimosa spp. in Costa Rica

rRNA gene sequencing and PCR assays indicated that 215 isolates of root nodule bacteria from two Mimosa species at three sites in Costa Rica belonged to the genera Burkholderia, Cupriavidus, and Rhizobium. This is the first report of Cupriavidus sp. nodule symbionts for Mimosa populations within their native geographic range in the neotropics. Burkholderia spp. predominated among samples from Mimosa pigra (86% of isolates), while there was a more even distribution of Cupriavidus, Burkholderia, and Rhizobium spp. on Mimosa pudica (38, 37, and 25% of isolates, respectively). All Cupriavidus and Burkholderia genotypes tested formed root nodules and fixed nitrogen on both M. pigra and M. pudica, and sequencing of rRNA genes in strains reisolated from nodules verified identity with inoculant strains. Inoculation tests further indicated that both Cupriavidus and Burkholderia spp. resulted in significantly higher plant growth and nodule nitrogenase activity (as measured by acetylene reduction assays) relative to plant performance with strains of Rhizobium. Given the prevalence of Burkholderia and Cupriavidus spp. on these Mimosa legumes and the widespread distribution of these plants both within and outside the neotropics, it is likely that both β-proteobacterial genera are more ubiquitous as root nodule symbionts than previously believed.     

On the Phloem of Mimosa pudica L.

The phloem of Mimosa pudica L. was examined in view of somereports that the sieve elements in this plant show featuresnot previously described for these cells in Leguminosae. Inthe present study only a usual dicotyledonous type of sieveelement was recognized. The sieve elements pass through stagesof differentiation involving development and dispersal of P-proteinbodies, disintegration of nuclei, and appearance of plastidsstoring a starch staining red with iodine. Callose occurs onthe transverse or moderately oblique sieve plates. The phloemcontains secretory cells. They are wide and long and have transverseor more or less inclined end walls. In younger cells the endwall bears in the centre a conspicuous pit to which the protoplastsof the superimposed cells are firmly attached. In many oldercells the pit region is replaced by a perforation so that thecontents of superimposed cells may be completely merged. Itremains to be determined whether the perforation is presentin an intact plant or results from a rupture during sampling.The secretory cells accumulate material that gives a positivetest for carbohydrates and a negative test for proteins.     

Coexistence of Burkholderia, Cupriavidus, and Rhizobium sp. nodule bacteria on two Mimosa spp. in Costa Rica

rRNA gene sequencing and PCR assays indicated that 215 isolates of root nodule bacteria from two Mimosa species at three sites in Costa Rica belonged to the genera Burkholderia, Cupriavidus, and Rhizobium. This is the first report of Cupriavidus sp. nodule symbionts for Mimosa populations within their native geographic range in the neotropics. Burkholderia spp. predominated among samples from Mimosa pigra (86% of isolates), while there was a more even distribution of Cupriavidus, Burkholderia, and Rhizobium spp. on Mimosa pudica (38, 37, and 25% of isolates, respectively). All Cupriavidus and Burkholderia genotypes tested formed root nodules and fixed nitrogen on both M. pigra and M. pudica, and sequencing of rRNA genes in strains reisolated from nodules verified identity with inoculant strains. Inoculation tests further indicated that both Cupriavidus and Burkholderia spp. resulted in significantly higher plant growth and nodule nitrogenase activity (as measured by acetylene reduction assays) relative to plant performance with strains of Rhizobium. Given the prevalence of Burkholderia and Cupriavidus spp. on these Mimosa legumes and the widespread distribution of these plants both within and outside the neotropics, it is likely that both beta-proteobacterial genera are more ubiquitous as root nodule symbionts than previously believed.     

Nodulation of legumes by rhizobium

Abstract. The formation of nitrogen-fixing nodules on leguminous plants is the culmination of an integrated development involving many plant and bacterial genes. This review focuses on the structure, function and regulation of the bacterial genes involved in the nodulation process. We attempt to interpret recent observations on these genes in the context of signal exchanges which occur between the macro-and micro-symbionts.     

Triterpenoid glycosides from the bark of Mimosa tenuiflora.

Two new saponins were isolated from Mimosa tenuiflora and their structures established as 3-O-[alpha-L-rhamnopyranosyl(1----2)-beta-D-glucopyranosyl-(1----3]-(alp ha-L- arabinopyranosyl-(1----4]-beta-D-xylopyranosyl-(1----2)]-[beta-D- xylopyranosyl-(1----4)]-beta-D-glucopyranosyl)-28-O-alpha-L-rhamnopyrano syl oleanolic acid and 3-O-[alpha-L-rhamnopyranosyl-(1----2)-beta-D-glucopyranosyl-(1----3]-(al pha- L-arabinopyranosyl-(1----4]beta-D-xylopyranosyl-(1----2)]-[beta-D- xylopyranosyl-(1----4)]-beta-D-glucopyranosyl) oleanolic acid.     

Biocidal activity in plant pathogenic Acidovorax, Burkholderia, Herbaspirillum, Ralstonia and Xanthomonas spp.

Antibacterial and antifungal activity was investigated for strains of Acidovorax spp., Burkholderia spp., Herbaspirillum rubrisubalbicans and Ralstonia solanacearum ; strains representing 118 species and pathovars of Xanthomonas were also tested for phytotoxic capacity. Antibacterial activity was present in all Burkholderia spp. except B. andropogonis , in biovars II and III of R. solanacearum but not in biovars I and IV, and in two strains of Xanthomonas. Little antibacterial activity was recorded for Acidovorax spp. Antifungal activity was expressed by most strains of A. avenae ssp. avenae and A. avenae ssp. cattleyae. Weak or variable antifungal reactions were given by strains of A. avenae ssp. citrulli and no activity was expressed by A. konjaci. Most strains of B. caryophylli, B. cepacia, B. gladioli pv. agaricicola, B. gladioli pv. alliicola, B. gladioli pv. gladioli , B. glumae and B. plantari produced extensive inhibition zones against Rhodotorula mucilaginosa. Strains of H. rubrisubalbicans and R. solanacearum gave negative, weak or variable reactions. Strains of Xanthomonas spp. exhibited no antifungal activity. In all cases antifungal activity was caused by a low molecular weight toxin. Three Xanthomonas strains exhibited phytotoxic activity. The ecological implications of these data are discussed.     

A new species of Mimosa sect. Mimosa (Leguminosae, Mimosoideae) from Southern Brazil

A new species of Mimosa section Mimosa, Mimosa eurystegia, which is apparently endemic to Paraná in southern Brazil, is described and illustrated. This species is distinguished by plurinerved leaflets and papyraceous, plurinerved, orbicular, amplexicaul stipules. Morphological distinctiveness of this new Mimosa and its relationships with allied species are discussed. Phenological, geographical and ecological data are presented, in addition to a key to the species of Mimosa subser. Sparsae.