Fast-growing Rhizobium Strains Research Articles

Variabilité génotypique de l'aptitude à la fixation symbiotique de l'azote chezAcacia cyanophyllaLindl.

Genotypic variability of the symbiotic nitrogen fixation ability on Acacia cyanophylla Lindl. The phenotypic and functional characteristics of four rhizobium strains isolated from nodules of Acacia cyanophylla Lindl., syn. Acacia saligna (Labill.) H. Wendl., and the inoculated host-plant genetic variability were evaluated. The evaluation used permitted the ranking of the tested rhizobium strains and seedlots according to their performance. The growth pattern of these strains indicated that A. cyanophylla was able to nodulate and fix N2with slow- and fast-growing rhizobium strains. The acetylene reduction activity test reflected a variable effectiveness of the strains depending on their site of isolation. Strains RFH383 and RFH483 were more effective than RFH183 and RFH283. The high antibiotic resistance acquisition by RFH183 and RFH283 strains seemed to be inversely related to their effectiveness. Acacia cyanophylla seedlings from five seedlots displayed a high variability regarding their growth, nodulation, and ability to fix nitrogen. The ability to fix nitrogen was greater in the seedlings from KL seedlot than those from EN, KR, and TZ seedlots. The qualitative assessment of the seedlots using correspondence factorial analysis showed that the seedlings from KL seedlot preformed better than those from KR and TZ. The seedlings from TN and EN seedlots displayed an intermediate behaviour pattern. Seed origin seemed to affect the growth, nodulation, and nitrogen fixation of inoculated A. cyanophylla. The behaviour of this species and that of its associated rhizobium strains appeared to be very sensitive to variations in site characteristics. Results suggested that the association between plants derived from KL seedlot and RFH383 strain may represent the best partnership allowing the improvement of symbiotic nitrogen fixation.Key words: Acacia cyanophylla, rhizobium, nodulation, N2fixation, genotypic variability, correspondence factorial analysis.

Diversity of Partial 16S rRNA Sequences Among and Within Strains of African Rhizobia Isolated from Acacia and Prosopis

Summary To assess the phylogenetic diversity of fast-growing rhizobium strains isolated from root nodules of Acacia senegal and Prosopis chilensis trees growing in Sudan and Kenya, a 230-nucleotide segment of the 16S ribosomal RNA gene was sequenced from each of thirty strains. Twelve different sequences were found: four were identical to those of previously described species, eight were novel. Sequence comparisons indicated that one strain belonged to the Rhizobium huakuii phylogenetic branch, while the rest were Sinorhizobium , as they were close to S. (Rhizobium) meliloti, S. f redii, S. teranga and S. saheli . Isolates from Acacia and from Prosopis often had the same sequence, but the sequence type was not very well correlated with the phenotypic characteristics determined previously for these strains. One isolate had two 16S rRNA sequences, differing at six positions, in approximately equal copy number. The type strain of S. saheli was also shown to have two different sequences.

Characterization of Fast-Growing Rhizobia from Nodulated Soybean [ Glycine max (L.) Merr.] in Vietnam

Summary We have examined six fast-growing Rhizobium strains isolated from nodulated soybean [Glycine max (L.) Merr.] plants growing in Vietnam, with special emphasis on two of them, SMX11 and SMH12. The abilities of strains SMX11 and SMH12 to utilize 11 carbon sources were indistinguishable from that of control Sinorhizobium fredii strains USDA257 and HH103, but their growth rates in standard media were considerably more rapid. The Vietnamese strains also were more salt-tolerant and antibiotic-resistant than the S. fredii reference strains, and their LPS banding patterns were less complex. Weak immunological cross-reactivity between the Vietnamese strains and S. fredii was evident. RFLP patterns of a series of nodulation genes and a repetitive sequence from S. fredii matched those of the Vietnamese strains, as did the sequence of a portion of a 16S rRNA gene. Analysis of cellular fatty acids was consistent with the assignment of strain SMH 12 and four other Vietnamese strains to S. fredii, but it aligned strain SMX11 more closely to other related groups of rhizobia. The Vietnamese strains have broad host ranges for nodulation of legumes, and their abilities to fix nitrogen in association with five soybean cultivars are equivalent to or surpass that of S. fredii.

Symbiotic properties and Rhizobium requirements for effective nodulation of five tropical dry zone acacias

Five tropical dry zone acacias were inoculated with three fast-growing Rhizobium strains and four slow-growing Bradyrhizobium strains. After two months of growth in the greenhouse, the plants were harvested and their shoot height and dry weight, nodule number and dry weight, acetylene reduction activity, and total shoot nitrogen were determined. The results showed that: (1) Acacia polyacantha (being evaluated for the first time) was promiscuous, nodulating indiscriminately with Rhizobium and Bradyrhizobium strains. (2) Faidherbia albida and Acacia holosericea (that were previously known to nodulate with Bradyhizobium strains only) nodulated with Rhizobium strains as well and (3) Acacia senegal and Acacia nilotica that were previously known to be nodulated exclusively by Rhizobium strains only were nodulated with both genera.

Long term field study shows increased biomass production in tree legumes inoculated with Rhizobium

Nodulation potential, nitrogen fixation efficiency (nitrogenase activity) and biomass yield response of Leucaena leucocephala and Acacia nilotica to inoculation with 6 selected fast growing Rhizobium strains was explored in long-term (5 year) field trials. All the strains formed nodules and fixed nitrogen in L. leucocephala and A. nilotica. Seasonal effects on nitrogenase activity was observed and in winter (ambient temperature about 20 °C), nitrogenase activity could not be detected. However, with the onset of spring and a rise in temperature, fresh nodulation (renodulation) by all the inoculant rhizobial strains was observed in both the tree legumes. In L. leucocephala, maximum renodulation was exhibited by strain A1 while in A. nilotica, strain AB3 formed the maximum renodulation 24 months after transplantation. Dry matter yield of all the inoculated plants demonstrated a significant increase over that of the uninoculated plants at the end of five years after transplanting. In L. leucocephala, strain NGR8 gave the maximum response (45% more dry matter yield) in dry matter production while in A. nilotica, strain USDA 3325 showed a 25% increase in total dry matter yield five years after transplantation.

Lipopolysaccharide patterns in SDS-PAGE of rhizobia that nodulate leguminous trees

Lipopolysaccharide (LPS) patterns were obtained from fast-growing Rhizobium strains after silver staining of proteinase K treated cells lysates, run in SDS-PAGE. The rhizobia came from root nodules of Acacia senegal and Prosopis chilensis, collected in differents part of the Sudan. The LPS profiles of all strains were typical of rhizobia. Two different LPS region with lower and higher electrophoretic mobility (region I and region II, respectively) coulld be dinguished in the gels, and based on the profiles the strains were divided into three groups. Strains isolated from A. senegal showed a wider range of different profiles than strains isolated from P. chilensis, even though the plants belong to the same cross-infection group. Otherwise there was no clear correlation between the taxonomic relatedness or site of isolation of the strains and their LPS profiles.

Cell surface lipopolysaccharides of different rhizobia

Polyacrylamide gel electrophoresis profiles of various cell surface lipopolysaccharides (LPS) obtained from fast-growing strains and plasmidless mutant strains of Rhizobium ‘hedysari’, R. leguminosarum and from slow-growing Bradyrhizobium japonicum strains have been compared. Correlations were obtained by using serological techniques. Two of the fast-growing strains and one slow-growing strain employed showed several LPS with slow mobility and a few others characterized by fast mobility in the gel. Other fast-growing rhizobium strains and all the other slow-growing bradyrhizobia tested had bands widely distributed in the gels. Location of bands, in these rhizobia, did not correlate with rapidity of growth of the strains. Induced antibiotic resistance did not affect LPS composition for either the fast- or slow-growing rhizobia. The LPS composition was apparently affected by the loss of plasmids. It appears to be associated with the lack of some bands in the region ranging from 30–50 kDa. The LPS composition was also affected by the mutation in the fast mobility region. The mutant strain lacked a low molecular mass band compared with the parental strain. [ 1H]-NMR spectra of LPS extracts from the latter strain also revealed a signal that is missing from the spectrum of the LPS of mutant strain.

Cell surface lipopolysaccharides of different rhizobia

Abstract Polyacrylamide gel electrophoresis profiles of various cell surface lipopolysaccharides (LPS) obtained from fast-growing strains and plasmidless mutant strains of Rhizobium ‘hedysari’, R. leguminosarum and from slow-growing Bradyrhizobium japonicum strains have been compared. Correlations were obtained by using serological techniques. Two of the fast-growing strains and one slow-growing strain employed showed several LPS with slow mobility and a few others characterized by fast mobility in the gel. Other fast-growing rhizobium strains and all the other slow-growing bradyrhizobia tested had bands widely distributed in the gels. Location of bands, in these rhizobia, did not correlate with rapidity of growth of the strains. Induced antibiotic resistance did not affect LPS composition for either the fast- or slow-growing rhizobia. The LPS composition was apparently affected by the loss of plasmids. It appears to be associated with the lack of some bands in the region ranging from 30–50 kDa. The LPS composition was also affected by the mutation in the fast mobility region. The mutant strain lacked a low molecular mass band compared with the parental strain. [ 1 H]-NMR spectra of LPS extracts from the latter strain also revealed a signal that is missing from the spectrum of the LPS of mutant strain.

Leaf curl syndrome of pigeonpea ( Cajanus cajan (L.) Millsp.) is a systemic response to effective nodulation by the Rhizobium strain IC3342

Rhizobium strain IC3342 is an unusual bacterium that causes a leaf curl syndrome in pigeonpea ( Cajanus cajan Millsp.) Growth characteristics, plasmid profile, conserved nif and nod gene sequences, and nodulation host range of this strain resemble that of the fast-growing Rhizobium strain ANU240 (NGR234). Leaf curl induction occurred only in hosts effectively nodulated by this strain. A plasmid-cured, non-nodulating derivative failed to induce leaf curl symptoms. The strain IC3342 competed poorly with fast- and slow-growing root-nodule bacteria, but the observed nodule occupancy of 10% was enough to produce leaf curl symptoms. Suppression of nodule development by added inorganic nitrogen also prevented symptom expression. Approach grafting of a healthy pigeonpea plant and a plant with leaf curl symptoms resulted in the development of leaf curl symptoms on the growing shoots of the healthy plant within 8 days of graft union. Further symptom expression ceased after graft separation. Feeding xylem sap from the leaf curled plant to a healthy plant induced the initial symptom of the syndrome, bending of the growing leaf tip. We conclude that the leaf curl induction is a systemic response for which effective nodulation is an apparent prerequisite.

Repetitive Sequences with Homology toBradyrhizobium japonicumDNA and the T-DNA ofAgrobacterium rhizogenesAre Closely Linked tonodABCofRhizobium frediiUSDA257

We have detected strong homology between a 9.2-kb EcoRI restriction fragment from Rhizobium fredii USDA257 that contains nodABC and eight additional EcoRI fragments in DNA digests from this organism. A series of repetitive sequences responsible for this hybridization lies within a 0.95-kb HindIII/SalI subfragment about 1-kb upstream of nodA. This subfragment also hybridizes to multiple restriction fragments from nine other strains of R. fredii, but only one is common to all strains. The 0.95-kb subfragment does not hybridize to genomic DNA from 17 other strains of fast-growing rhizobia, but there is weak homology to two fragments from Rhizobium sp. strain NGR234. We sequenced 2,432 base pairs (bp) of the region encompassing the repetitive sequences. It contains 65 separate 8- to 11-bp inverted and direct repeats, as well as two large open reading frames (ORFs) that overlap on opposite strands. ORF1 reads in the same direction as nodABC, contains 1,071 bp, and encodes a 40.6-kD protein. It has 74% sequence homology to an ORF within the T-DNA of Agrobacterium rhizogenes and similar homology to a series of repetitive sequences from Bradyrhizobium japonicum. ORF2 (981 bp) reads in the opposite direction, encodes a 34.7-kD protein, and has partial identity with a second ORF from A. rhizogenes. We could detect no poly(A)+ nodule transcripts with homology to ORF1 and ORF2. The eight sets of repetitive sequences found in other EcoRI fragments of the genome were cloned from USDA257 on separate cosmids. Some of these cosmids appear to overlap, and two have fragments with homology to nifKDH.

A chromosomal genetic map of Rhizobium sp. NGR234 generated with Tn5-Mob

Rhizobium sp. NGR234 in a fast-growing Rhizobium strain with a broad host range. The location and role of chromosomal genes involved in cellular metabolism or in the legume symbioses is unknown. We isolated a series of auxotrophic and antibiotic resistant mutants of NGR234 and utilized a chromosome mobilization system based on Tn5-Mob and pJB3JI; Tn5-Mob donor strains behaved like Hfr strains, transferring the chromosome polarly at high frequency from a fixed point of insertion. The use of four different strains with Tn5-Mob located at different nutritional loci in crosses with double auxotrophic recipients, allowed us to build up a circular linkage map of NGR234 based on relative recombination frequencies. Also, symbiotically important genes identified by site-directed mutagenesis, such as hemA and ntrA, could be located and mapped on the chromosome.

Rhizobium Infection of Leucaena leucocephala via the Formation of Infection Threads in Curled Root Hairs

Summary The infection of the tropical legume Leucaena leucocephala by the broad-host-range, fast-growing Rhizobium strain NGR234 was studied using both light and electron microscopy. Rhizobium infection of Leucaena was shown to be via infection threads formed in root hairs. Electron microscopic studies revealed both inter- and intra-cellular infection threads in nitrogen-fixing nodules. Root hair deformation and infection was mostly on lateral roots but occasionally found on the taproot. The lateral roots appeared on plants 3 days after two and half day-old seedlings were transferred to the growth medium in the assay vessel. Root hair curling or branching was first observed 5 days after Rhizobium inoculation. Infection threads were seen on each plant between 10-18 days after inoculation. Nodules were visible on plants 12 days after inoculation and most of them were located on lateral roots.

A note on physiological characteristics and genetic relatedness of a fast-growingRhizobiumfrom stem nodule ofAeschynomene asperaL

A fast-growing Rhizobium strain was isolated from a stem nodule of Aeschynomene aspera and was characterized by standard laboratory tests, nodulation, nutritional and carbohydrate utilization patterns. Its relationship to other members of Rhizobium was studied by DNA homology tests which suggest it cannot be classified as any known species of Rhizobium.

Characterization of DNA sequences homologous to Klebsiella pneumoniae nifH, D, K and E in the tropical Rhizobium ORS571

The fast-growing Rhizobium strain ORS571 isolated from the tropical legume Sesbania rostrata can grow in the free living state utilizing molecular nitrogen. The organization of the nif genes was analyzed by hybridization using Klebsiella pneumoniae nif DNA probes. Homology was limited to nifHDK, the structural genes for the nitrogenase, nifE, which is involved in formation of the ironmolybdenum cofactor and nifJ, which is involved in electron transport. This is the first report of homology in another diazotroph to K. pneumoniae nifE. A cluster containing nifHDKE was identified. The four genes are contiguous on a 6.3 kb SalI-BamHI fragment. They are all in the same orientation and in the same order as in K. pneumoniae. A second copy of nifH unlinked to this cluster was also identified.

Rhizobium nodulation genes involved in root hair curling (Hac) are functionally conserved

Five specific transposon-induced nodulation defective (Nod(-)) mutants from different fast-growing species ofRhizobium were used as the recipients for the transfer of each of several endogenous Sym(biosis) plasmids or for recombinant plasmids that encode early nodulation and host-specificity functions. The Nod(-) mutants were derived fromR. trifolii, R. meliloti and from a broad-host-rangeRhizobium strain which is able to nodulate both cowpea (tropical) legumes and the non-legumeParasponia. These mutants had several common features (a), they were Nod(-) on all their known plant hosts, (b), they could not induce root hair curling (Hac(-)) and (c), the mutations were all located on the endogenous Sym-plasmid of the respective strain. Transfer to these mutants of Sym plasmids (or recombinant plasmids) encoding heterologous information for clover nodulation (pBR1AN, pRt032, pRt038), for pea nodulation (pJB5JI, pRL1JI::Tn1831), for lucerne nodulation (pRmSL26), or for the nodulation of both tropical legumes and non-legumes (pNM4AN), was able to restore root hair curling capacity and in most cases, nodulation capacity of the original plant host(s). This demonstrated a functional conservation of at least some genes involved in root hair curling. Positive hybridization between Nod DNA sequences fromR. trifolii and from a broad-host-rangeRhizobium strain (ANU240) was obtained to other fast-growingRhizobium strains. These results indicate that at least some of the early nodulation functions are common in a broad spectrum ofRhizobium strains.

Studies on growth and symbiotic nitrogen fixation of Rhizobium of Vigna radiata under stress conditions

SummaryOne fast-growing acid-producing Rhizobium strain 995 of Vigna radiata was screened for growth behaviour in acid, saline and alkaline media. It grew well in yeast-extract mannitol broth of wide pH range as well as varying concentrations of NaCl, Na2SO4 and MnCl2. Variation in nodulation and N2-fixation efficiency occurred on different varieties of green gram, while strain characteristics were not affected by soil stress.

Mise en évidence d'une propriété métabolique de Rhizobium meliloti utilisable pour sa classification

Summary Fast-growing Rhizobium and Agrobacterium strains were studied fortheir behaviour towards two carbohydrates (glucose and fructose) according to the following criteria: acidification and soluble exopolysaccharide production by resting cells. R. meliloti and Agrobacterium spp. showed a similar behaviour: in glucose-containing medium, acidification occurred and few exopolysaccharides were produced, whereas in medium supplemented with fructose, a neutral pH was maintained and large amounts of exopolysaccharides were synthesized. Other fast-growing Rhizobium spp. did not acidify these media and produced variable amounts of exopolysaccharides.

Group antigens in slow-growing rhizobia

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.