Metabolic properties, maximum growth temperature and phage sensitivity of Rhizobium sp. (Galega) compared with other fast-growing rhizobia

Abstract Rhizobium strains nodulating Galega species were characterized by metabolic tests, maximum growth temperature determinations in a temperature gradient incubator and phage typing, and compared with other fast-growing rhizobia. By numerical taxonomy it was shown that the Galega Rhizobium strains are closely related to each other and unrelated to the recognized species of Rhizobium . The maximum growth temperature of rhizobia nodulating G. orientalis was 33.0–34.0°C and of rhizobia nodulating G. officinalis 35.0–37.0°C. The Galega rhizobia were only lysed by their own phages, and not by typing phages for other Rhizobium species. The G + C% of Rhizobium sp. ( Galega ) strainswas 63%.44 previously unclassified fast-growing rhizobia from tropical plants, which were included in the experiments, were shown to form a heterogenous group with diverse properties. The results confirm and extend previous findings, and suggest that Rhizobum sp. ( Galega ) should be considered a new species of Rhizobium .     

Binding of Pseudomonas aeruginosa Lectin to Rhizobium sp.

Binding sites for the mannosephilic lectin of Pseudomonas aeruginosa and for concanavalin A were found on the nitrogen-fixing symbiont Rhizobium sp. which infects Phaseolus lathyroides , and on the root cells of its host. The interaction between these lectins and the Rhizobium or the root cells was demonstrated by adsorption tests and by mannose-specific peroxidase-binding to the lectin-coated bacteria. Treatment of the bacteria with formaldehyde, phenol, ethanol or boiling did not alter their ability to adsorb the lectins. The growth-rate of the Rhizobium was unaffected when the Pseudomonas lectin was added to the culture medium. Addition of these lectins to the Rhizobium cells used for seedling infection led to an increased bacterial adsorption to seedling roots and to enlargement of nodule size, but did not increase nodulation.     

Effects of some organophosphate and carbamate insecticides on nitrification and legume growth

Summary Nine insecticides (six organophosphates and three carbamates) were tested for their effects on soil nitrification, growth of legume seedlings, and growth of four species of rhizobia bacteria. No inhibition of nitrification was found at normal field rates (5 ppm) of application. Some instances of inhibition were observed at 50 ppm and at 500 ppm. Similarly, 5 ppm applications did not inhibit growth of alfalfa or sweetclover seedlings ... with one exception. Disc inhibition tests of the rhizobia bacteria showed thatRhizobium leguminosarum andRhizobium trifolii were most sensitive to the pesticides.Rhizobium meliloti, and particularlyRhizobium japonicum, were resistant to the insecticides. No consistent correlation was observed between tests on the nodulating bacteria and the tests on legume growth.Published with the approval of the Director of the North Dakota Agricultural Experiment Station as Journal Article No.309. Portion of a thesis presented by the senior author in partial fulfillment of the requirements for the M.S. degree in bacteriology at North Dakota State University.     

Numerical taxonomic analysis of cross-inoculation patterns of legumes and Rhizobium

Summary A survey was made of published results of tests of the capacity of Rhizobium derived from one legume genus to nodulate plants from other genera. The data were derived from more than 14,000 separate cross-inoculation trials involving species from 165 genera of legumes. Numerical taxonomic techniques were applied to 113 of the genera for which results of substantial cross-infection tests were available. The data were examined using mean character difference coefficients re-expressed as total and positive-only similarity coefficients. The resulting similarity matrices were clustered by the unweighted pair-group method using arithmetic averages. Eighteen affinity groups were defined at the 70% similarity level. With few exceptions, the physiological and cultural behavior of the rhizobia was consistent within the defined groups. Two broad categories were suggested in the numerical taxonomic analysis, and their validity is discussed in regard to the geographic, physiological and cultural characteristics of the legumes and their Rhizobium microsymbionts. The taxonomic and agronomic value of this approach and the new groupings are discussed.     

Colonization of Phaseolus vulgaris nodules by Agrobacterium-like strains

Non-nodulating Agrobacterium-like strains identified among root nodule isolates of common bean were labeled with gusA, a reporter gene encoding beta-glucuronidase (GUS). Bean plants were then co-inoculated with an infective Rhizobium strain and labeled transconjugants of Agrobacterium-like strains. Blue staining of nodules showed that Agrobacterium-like strains were able to colonize these symbiotic organs. Isolation and characterization by restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes revealed a mixed population of Rhizobium and Agrobacterium-like strains in all nodules showing GUS activity. PCR amplification of the nifH gene and nodulation tests did not show any evidence of acquisition of symbiotic gene by lateral transfer from Rhizobium to Agrobacterium-like strains. Moreover, these strains were able to invade mature nodules. Based on sequencing of the 16S rRNA gene, one of these Agrobacterium-like strains showed 99.4% sequence similarity with Agrobacterium bv. 1 reference strains and 99% similarity with an Agrobacterium bv. 1 strain isolated from Acacia mollisima in Senegal. Agrobacterium tumefaciens C58 and the disarmed variant AT123 did not show any ability to colonize nodules. Co-inoculation of bean seeds with Agrobacterium and Rhizobium strains did not enhance nodulation and plant yield under controlled conditions.     

Strain Identification in Rhizobium Using Intrinsic Antibiotic Resistance

The variation in intrinsic resistance to low levels of eight antibiotics was used as an identifying characteristic for 26 Rhizobium leguminosarum strains. The pattern of antibiotic resistance of each strain was a stable property by which rhizobia isolated from root nodules of inoculated Pisum sativum could be recognized. The antibiotic tests for strain identification with R. leguminosarum were applied to R. phaseoli . It was necessary to include reference cultures in tests with this species, as the tests most suitable for the R. leguminosarum strains showed some variability with R. phaseoli .     

Filter Mud as a Carrier for Rhizobium Inoculants

Laboratory tests indicated that filter mud, a waste product from sugar cane mills, proved to be a suitable carrier for Rhizobium inoculants, provided it was not dried by heating. Autoclaving air dried material was not detrimental and ensured high survival when held at room temperature. The adverse effect of oven drying, which was increased by subsequent autoclaving or gamma irradiation, lessened with time.     

Phylogenetic analysis of symbiotic genes of nodule bacteria in the plants of the genus Lathyrus (L.) (Fabaceae)

The comparative analysis of the symbiotic genes nifD, nifH, nodA of wild-growing Lathyrus L. species (Fabaceae) connected by genes sequences of 16S aRNA to Rhizobium leguminosarum bv. viceae, Rhizobium tropici, Agrobacterium sp., and Phyllobacterium sp. was carried out. It was demonstrated that all tested genes of strains taken for analysis had high degree of homology with analogous genes of Rhizobium leguminosarum bv. viceae. It was suggested that symbiotic genes were introduced into Rhizobium tropici, Agrobacterium sp., and Phyllobacterium sp. strains by means of horizontal gene transfer over from Rhizobium leguminosarum bv. viceae strain. The recombinant strains were formed, capable to nodulate Lathyrus L. species that earlier was not considered characteristic for these plants.     

Fructose 1,6-bisphosphate aldolase activity ofRhizobium species

FDP aldolase was found to be present in the cell-free extracts of Rhizobium leguminosarum, Rhizobium phaseoli, Rhizobium trifolii, Rhizobium meliloti, Rhizobium lupini, Rhizobium japonicum and Rhizobium species from Arachis hypogaea and Sesbania cannabina. The enzyme in 3 representative species has optimal activity at pH 8.4 in 0.2M veronal buffer. The enzyme activity was completely lost by treatment at 60 degrees C for 15 min. The Km values were in the range from 2.38 to 4.55 X 10(-6)M FDP. Metal chelating agents inhibited enzyme activity, but monovalent or bivalent metal ions failed to stimulate the activity. Bivalent metal ions in general were rather inhibitory.     

Parasitic origins of nitrogen-mixing Rhizobium-legume symbioses. A review of the evidence

This paper is divided into two sections. The first part (I) reviews the literature on the legume-Rhizobium association with emphasis on the processes leading to the establishment of the association. In the second part (II) it is proposed that the legume-Rhizobium association was originally necrotrophic , beginning when the free-living, nitrogen-fixing, saprotrophic Rhizobium developed the ability to infect the plant. The pre-infection events, infection processes and nodulation in the colonization of the legumes by the Rhizobium are similar to those of other parasitic associations. Likewise, the host responses to the Rhizobium entry, infection thread synthesis and bacteroid formation are comparable to those of other plants when they encounter phytopathogens . Evolutionary processes acted in the selection of biotrophy , the fine control and regulation of the extracellular enzymes of the necrotrophic Rhizobium converted the association into biotrophy . The nutritional dependence of the Rhizobium on the legume, the requirement of the plant for combined nitrogen and the Rhizobium potential to meet this requirement drove the biotrophic association into mutualism . This became possible when regulation of the nitrogen-fixing system of the Rhizobium was modified and the oxygen carrying protein leghemoglobin was acquired or evolved by the legume to enhance nitrogen fixation.     

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.     

An Evaluation of the Nile Blue Test for Differentiating Rhizobia from Agrobacteria

The ability of agrobacteria to reduce Nile Blue more strongly than do rhizobia is the basis of a test for separating these two groups (Hamdi 1969). In a modified test using only 35 parts 10° of Nile Blue in the medium, 89 of 90 rhizobia ( Rhizobium japonicum, R. leguminosarum, R. lupini, R. phaseoli, R. trifolii , cowpea, groundnut and Lotus rhizobia) failed to reduce the dye whereas all 24 strains of agrobacteria ( Agrobacterium radiobacter var. radiobacter, A. r. var. tumefaciens and A. r. var. rhizogenes ) reduced it to the colourless state. Only one Rhizobium strain formed 3-ketolactose from lactose, but 13 agrobacteria produced it. Rhizobium meliloti strains (12) gave variable reactions in both tests. The Nile Blue Test detected rapidly, but not slowly growing, strains of agrobacteria present as contaminants of rhizobia cultures even when their initial numbers were small.     

Adsorption and selection of rhizobia with ion-exchange papers

Ion exchange papers were used to study the adsorption of 32P-labelled rhizobia on defined surfaces. Two strains of Rhizobium japonicum and one each of R. leguminosarum and R. lupini were compared with Escherichia coli and Bacillus subtilis. The ratio of adsorption to strong and to weak acid papers/strong and weak basic papers was consistantly higher for all rhizobial strains compared to the other bacteria. The process of desorption by increasing the ion-concentration causes about 35% desorption between 0.02 and 0.1 M MgCl2, however, an increase to 1 M does not desorb more labelled Rhizobium japonicum or E. coli cells. The ratio of adsorbed cpm to colony formers, desorbed by 0.1 M NaCl was similar with Rhizobium japonicum for all six ion exchange papers. For E. coli this ratio varied widely for the different papers. The selection of Rhizobium against a more closely related bacterium by this adsorption/desorption procedure was demonstrated with mixed cultures of Rhizobium japonicum and Chromobacterium violaceum giving a more than 80 fold enrichment of the former. Rhizobium japonicum cells, ad/desorbed from all ion exchange papers kept their infectivity and formed nodules on Glycine max with an activity of 20-40 nM C2H4-hr(-1)-mg nodule(-1). A desorption of Rhizobium japonicum from soybean roots also occurred by increasing the ion concentration. 2-3 times as many cells were removed in this way compared to washing with water.     

AMF和饭豆根瘤菌对饭豆、玉米套种促生作用的研究

将从饭且根瘤中分离的饭豆根瘤菌(Rhizobium sp.CYY3302,Rhizobium sp.HCY9101,Rhizobium SP.JMC1402)与ANF(Arbuscular Mycorrltizal Fungi)共同接种于饭豆,进行饭豆、玉米田间小区各种试验。结果表明,接种饭豆根瘤菌和AMF的处理与未接种的处理相比,饭豆的结瘸率比对照提高52％-134％;饭豆及玉米的菌根感染率比对照分别增加43．1％-80％和46．8％-97．6％;饭豆的产量提高了54％-67％,而玉米的产量提高了2．4％-19．5％。研究结果还表明：豆科作物接种根瘸菌,即使在种过豆科作物的老区,也是有效的。     

Phylogenetic diversity of non-nodulating Rhizobium associated with pine ectomycorrhizae

Most Rhizobium species described are symbionts that form nodules on legume roots; however, non-nodulating strains of Rhizobium are also widespread in nature. Unfortunately, knowledge of non-nodulating Rhizobium is quite limited compared with nodulating Rhizobium . Here, we studied the phylogenetic diversity of Rhizobium species that inhabit Japanese red pine roots ( Pinus densiflora ). Because fine roots of pine trees are usually colonized by ectomycorrhizal fungi in nature, we mainly used ectomycorrhizal root tips for bacterial isolation. Out of 1195 bacteria isolated from 75 independent root samples from the field and greenhouse experiments, 102 isolates were confirmed to be Rhizobium following partial 16S rRNA gene analysis. Rhizobium species were occasionally dominant in culturable bacterial communities, whereas no Rhizobium species were isolated from the soil itself. Molecular phylogenetic analyses using 16S rRNA, atpD , and recA gene sequences revealed that isolated Rhizobium strains were phylogenetically diverse and that several were distantly related to known Rhizobium species. Considering that a single species of pine is associated with unique and phylogenetically diverse Rhizobium populations, we should pay more attention to non-nodulating strains to better understand the diversity, ecology, and evolution of the genus Rhizobium and plant– Rhizobium associations.     

Nitrate reductase activities of rhizobia and the correlation between nitrate reduction and nitrogen fixation.

All species of Rhizobium except R. lupini had nitrate reductase activity. Only R. lupini was incapable of growth with nitrate as the sole source of nitrogen. However, the conditions necessary for the induction of nitrate reductase varied among species of Rhizobium. Rhizobium japonicum and some Rhizobium species of the cowpea strains expressed nitrate reductase activities both in the root nodules of appropriate leguminous hosts and when grown in the presence of nitrate. Rhizobium trifolii, R. phaseoli, and R. leguminosarum did not express nitrate reductase activities in the root nodules, but they did express them when grown in the presence of nitrate. In bacteroids of R. japonicum and some strains of cowpea Rhizobium, high N2 fixation activities were accompanied by high nitrate reductase activities. In bacteroids of R. trifolii, R. leguminosarum, and R. phaseoli, high N2 fixation activities were not accompanied by high nitrate reductase activities.     

Isolation of unique nucleic acid sequences from rhizobia by genomic subtraction: Applications in microbial ecology and symbiotic gene analysis

A subtraction hybridization and PCR amplification procedure was used to isolate two Rhizobium DNA probes which exhibited high degrees of specificity at different levels of taxonomic organization and which could be used as tools for detection of rhizobia in ecological studies. First, a probe was isolated from Rhizobium leguminosarum bv. trifolii strain P3 by removing those Sau3A restriction fragments from a P3 DNA digest which cross hybridized with pooled DNA from seven other strains of the same biovar. The remaining restriction fragments hybridized to DNA from strain P3 but not to DNA from any of the seven other strains. In a similar experiment another DNA probe, specific for the Rhizobium leguminosarum bv. phaseoli and Rhizobium tropici group, was generated by removing sequences from R. leguminosarum bv phaseoli strain Kim 5s with pooled subtracter DNA from eight other Rhizobium, Bradyrhizobium and Agrobacterium species. The same subtraction hybridization technique was also used to isolate symbiotic genes from a Rhizobium species. Results from a 1:1 subtractive DNA hybridization of the broad host range Rhizobium sp NGR234 against highly homologous S. fredii USDA257, combined with those from competitive RNA hybridizations to cosmid digests of the NGR234 symbiotic plasmid, allowed the identification of several NGR234 loci which were flavonoid-inducible and not present in S. fredii USDA257. One of these, ORF-1, was highly homologous to the leucine responsive regulatory protein of E. coli.     

Physiology of the Rhizobium-legume association: I—The presence of bacterial DNA within the plant root

Summary Rhizobium deoxyribonucleic acid has been detected within Vicia faba root cells by in situ hybridization and autoradiography after exposure of root apexes to Rhizobium viable cells. Reannealed regions are localized into the cortex cells; the presence of bacterial DNA is specific for the root tissue; labelled regions were not detectable within apexes exposed to non-nodulating strains or to bacteria other than Rhizobium; Rhizobium DNA was not detectable in tissues of plants other than its leguminous host. re]19750313     

The relation between nitrogen percent and dry weight of inoculated legumes

Summary Results of numerous tests of tropical and temperate legume hosts and Rhizobium strains accumulated between 1952 and 1966 were examined for a relation between N percent (y) and dry weight per plant (x). The data fitted the equation y=A–be^–cx. The most effective Rhizobium strains can be selected on the basis of dry matter yields of whole plants or plant tops only, without the need for N analyses, using statistical analyses. A previously proposed method which employs the linear relation between N-yield and dry matter yield was shown to apply only when data for strains which are not fully effective are excluded.It is postulated that symbiotically fixed N forms a different compound from that resulting from applied mineral N and that this compound cannot be remobilised before flowering.deceased     

Isolation of the replication DNA region from a Rhizobium plasmid and examination of its potential as a replicon for Rhizobiaceae cloning vectors

The DNA region essential for replication and stability of a native plasmid (pTM5) from Rhizobium sp. (Hedysarum) has been identified and isolated within a 5.4-kb PstI restriction fragment. The isolation of this region was accomplished by cloning endonuclease-restricted pTM5 DNA into a ColE1-type replicon and selecting the recombinant plasmids containing the pTM5 replicator (pTM5 derivative plasmids) by their ability to replicate in Rhizobium. DNA homology studies revealed that pTM5-like replicons are present in cryptic plasmids from some Rhizobium sp. (Hedysarum) strains but not in plasmids from strains of other Rhizobium species or Agrobacterium tumefaciens. The pTM5 derivative plasmids were able to replicate in Escherichia coli and A. tumefaciens and in a wide range of Rhizobium species. On the basis of stability assays in the absence of antibiotic selective pressure, the pTM5 derivative plasmids were shown to be highly stable in both free-living and symbiotic cells of Rhizobium sp. (Hedysarum). The stability of these plasmids in other species of Rhizobium and in A. tumefaciens varied depending on the host and on the plasmid. Most pTM5 derivative plasmids tested showed significantly higher symbiotic stability than RK2 derivative plasmids pRK290 and pAL618 in Rhizobium sp. (Hedysarum), R. meliloti, and R. leguminosarum by. phaseoli. Consequently, we consider that the constructed pTM5 derivative plasmids are potentially useful as cloning vectors for Rhizobiaceae.