Relationship between Nif plasmids of fast-growing Rhizobium species and Ti plasmids of Agrobacterium tumefaciens.

By use of the Southern blot hybridization technique the extent of DNA homology was determined between the Nif plasmid of a number of fast-growing Rhizobium species and Ti plasmids of the octopine (pTiAch5) and nopaline (pTiC58) type. DNA sequences common to these plasmids were located on functional maps of the Ti plasmids. No homology between Nif plasmids and the T region of Ti plasmids was detected.     

Involvement of Rhizobium japonicum O antigen in soybean nodulation.

Non-nodulating mutant strains of Rhizobium japonicum lacked a surface antigen that was present on the wild type. This surface antigen is associated with the O antigen portion of the lipopolysaccharide. Paper chromatography of hydrolyzed lipopolysaccharide and O antigen revealed three major component differences between the non-nodulating strains and the wild type.     

R-prime plasmids from Bradyrhizobium japonicum and Rhizobium fredii

The formation of R-prime plasmids was selected in crosses involving soybean microsymbionts with genomic Tn5 insertions and carrying plasmid pJB3JI (with one IS2) copy as donors and Escherichia coli HB101 as recipient. Whereas the parent plasmid was 60 kb, recombinant plasmids between 76 kb and 121 kb were obtained. Restriction and Southern analyses confirmed the mobilization of Tn5 on four R-primes from Bradyrhizobium japonicum I-110 and on an R-prime plasmid from Rhizobium fredii HH303. The largest R-prime plasmid was obtained from the rescue of two symbiotically defective R. fredii mutant strains that required adenosine.Non-standard abbreviation TDP transposon donor poolScientific article number A-4728 and contribution number 7724 of the Maryland Agricultural Experiment Station     

High-frequency induction of nodulation and nitrogen fixation mutants of Rhizobium japonicum.

More than 50 symbiotic mutants of Rhizobium japonicum were isolated by purported plasmid-curing techniques. Wild-type R. japonicum strains were grown in liquid culture at 28 or 36 degrees C in different concentrations of acridine orange, ethidium bromide, or sodium dodecyl sulfate for selection of mutants. The symbiotic traits of 133 isolates from nine treatment groups were determined. Forty-two isolates were Nod- Nif+, seven were Nod+ Nif-, and two were Nod- Nif-. The nifDH genes were deleted in three mutants and consequently showed no hybridization to a nifDH probe. None of these mutants showed any detectable loss of plasmid DNA.     

Nitrogen fixation (nif) genes and large plasmids of Rhizobium japonicum.

The location of structural nitrogen-fixation genes was determined for the slow- and fast-growing types of Rhizobium japonicum. Slow-growing R. japonicum strains do not harbor structural nif genes, homologous to nifD and nifH, on large plasmids (100 to 200 megadaltons). In contrast, all fast-growing R. japonicum strains, except PRC194, contain structural nif genes on large plasmids.     

Studies of symbiotic plasmids in Rhizobium trifolii and fast-growing bacteria that nodulate soybeans

The Rhizobium trifolii symbiotic plasmid pRt5a was transferred to the fast-growing soybean strain USDA 194. Transconjugants carrying pRt5a were not able to nodulate clovers and one of the transconjugants had lost its smallest resident plasmid and did not fix nitrogen in soybean. Transconjugants of USDA 194 carrying pRt5a were able to transfer pRt5a back to a non-nodulating R. trifolii which inherited the symbiotic properties of the R. trifolii strain from which the plasmid was derived.     

Isolation and expression of Rhizobium japonicum cloned DNA encoding an early soybean nodulation function.

A first visible step in the nodulation of legumes by Rhizobium spp. is the deformation and curling of root hairs. We have identified and cloned DNA sequences encoding this function from two strains of Rhizobium japonicum (USDA 122 and USDA 110) with a weakly homologous probe from Rhizobium meliloti. Root hair curling encoded by the cloned DNA fragments was examined on soybeans (Glycine soja ) after conjugative transfer of these sequences in broad-host-range vectors to various bacterial genera. Pseudomonas putida gave unambiguous expression of the root hair curling genes. This enabled us to identify the 8.7-kilobase EcoRI fragments encoding root hair curling from each strain. The phenotypes encoded by the plasmids pBS1 (derived from strain USDA 122) and pBS2 (derived from strain USDA 110) are distinct and represent a phenotype characteristic of their parent R. japonicum strains. Subclones of pBS1 and pBS2 were generated in single and multicopy vectors, and their expression was analyzed in P. putida. We established that a 4.2-kilobase internal Sa/I fragment of pBS1 and a 3.5-kilobase SstI -EcoRI fragment of pBS2 are sufficient to confer root hair curling on soybeans.     

Transfer from Rhizobium japonicum to Azotobacter vinelandii of genes required for nodulation

A mutant strain of Azotobacter vinelandii that is unable to fix N2 (Nif-) was transformed to Nif+ with DNA from Rhizobium japonicum. Of 50 Nif+ transformants tested, 3 contained the O antigen-related polysaccharide that is present on the cell surface of a nodulating R. japonicum strain, but is absent from a non-nodulating mutant strain.     

Competition among Rhizobium leguminosarum bv. phaseoli strains for nodulation of common bean.

Six effective Rhizobium leguminosarum bv. phaseoli strains were examined for nodulation competitiveness on common bean (Phaseolus vulgaris L.), using all possible two-strain combinations of inoculum. Nodule occupancy was determined with strain-specific fluorescent antibodies. The strains were divided into three groups according to their overall competitive abilities on pole bean cv. Kentucky Wonder and bush bean cv. Bountiful. Strains TAL 182 and TAL 1472 were highly competitive (greater than 70% nodule occupancy); strains KIM-5, Viking 1, and CIAT 899 were moderately competitive (approximately 50% nodule occupancy); and strain CIAT 632 was poorly competitive (less than 5% nodule occupancy). The competitiveness of the six strains was similar on the two host cultivars. The proportion of competing strains in the inoculum influenced the nodule occupancy of the highly competitive and moderately competitive strains, but not that of the poorly competitive strain. Two outstanding strains (TAL 182 and TAL 1472) were identified as ideal model strains for molecular and genetic studies on nodulation competitiveness.     

Speed of nodulation and competitive ability among strains of Rhizobium leguminosarum bv phaseoli

This study examines the speed of nodulation of 20 strains of Rhizobium leguminosarum bv phaseoli, and relates this trait to the competitive performance of these strains with Phaseolus vulgaris L. At 25/20°C day/night temperature, and with 10⁷ cells applied per growth pouch, there was a strong positive correlation between the speed of nodulation and the competitiveness of strains with the nod⁺ fix^– reference strain UMR 1116. Strains UMR 1084, 1125, 1165, 1173 and 1384 combined good competitive performance with extensive nodulation in the uppermost root regions. When inoculant levels in the RTM studies were reduced to 10³ cells per pouch no correlation between the apparent competitiveness of strains and their speed in nodulation was evident, presumably because cells had to undergo multiplication before infection. Nodulation was also delayed when growth temperatures were raised to 31/26°C, but a correlation was still evident between competitive performance and nodulation in the region 0.1 to 5.0 mm below the RTM at the time of inoculation. From these results speed of nodulation can be used to estimate the competitive potential of Rhizobium strains, but only under carefully regulated conditions. The effects of inoculation level and temperature on the relationship between speed of nodulation and strain competitiveness could explain the inconsistent results obtained in earlier studies on this topic.Journal paper No. 16962, Agricultural Experiment Station, University of Minnesota, St. Paul, MN 55108, USA     

Growth regulators,Rhizobium and nodulation in peas

The cytokinin content of roots and nodules of pea and the culture supernatants from two strains of Rhizobium leguminosarum has been examined. Roots, nodules and wild-type Rhizobium culture medium contained very little cytokinin as indicated by bioassay. Chemical ionisation gas chromatography-mass spectrometric analysis of the isopentenyladenine content of the culture medium from the Rhizobium strains confirmed that the content of the wild-type was low (approx. 1 ng dm^-3) but that it was increased by the introduction of the Agrobacterium Ti plasmid into the Rhizobium strain.Abbreviations CI chemical ionisation - GCMS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - iPAde isopentenyladenine - MIM multiple ion monitoring     

Uptake hydrogenase in fast-growing strains of Rhizobium sp. (Sesbania) in relation to nitrogen fixation

Eight of 104 fast-growing strains of Rhizobium sp. ( Sesbania ) tested possessed a hydrogen recycling system. Depression of uptake hydrogenase occurred in poor as well as in rich carbon media. A statistically significant increase (> 22%) in total plant nitrogen content and dry matter yield was observed by the inoculation of hup + strains over hup ^- strains.     

Plasmid transfer within and between serologically distinct strains of Rhizobium japonicum, using antibiotic resistance mutants and auxotrophs.

Methionine-requiring and pantothenic acid-requiring auxotrophs of Rhizobium japanicum USDA 31, as well as highly antibiotic-resistant mutants of R. japonicum strains USDA 31, USDA 110, USDA 138, and Webster 48, were isolated. These mutants were used to transfer the P-1 group plasmids R68.45 and RP4 within and between strains USDA 31, USDA 110, and Webster 48. Attempts to demonstrate transfer of either plasmid to strain USDA 138 were unsuccessful.     

nodT,a positively-acting cultivar specificity determinant controlling nodulation of Trifolium subterraneum by Rhizobium leguminosarum biovar trifolii

Rhizobium leguminosarum biovar trifolii strain TA1 nodulates a range of Trifolium plants including red, white and subterranean clovers. Nitrogen-fixing nodules are promptly initiated on the tap roots of these plants at the site of inoculation. In contrast to these associations, strain TA1 has a Nod^- phenotype on a particular cultivar of subterranean clover called Woogenellup (A.H. Gibson, Aust J Agric Sci 19: (1968) 907–918) where it induces rare, poorly developed, slow-to-appear and ineffective lateral root nodules. By comparing the nodulation gene region of strain TA1 with that of another R. leguminosarum bv. trifolii strain ANU843, which is capable of efficiently nodulating cv. Woogenellup, we have shown that the nodT gene (B.P. Surin et al., Mol Microbiol 4: (1990) 245–252) is essential for nodulation on cv. Woogenellup. The nodT gene is naturally absent in strain TA1. A cosmid clone spanning the entire nodulation gene region of strain TA1 was capable of conferring nodulation ability to R.l. bv. trifolii strains deleted for nodulation genes, but only on cultivars of subterranean clovers nodulated by strain TA1. This shows that cultivar recognition events are, in part, determined by genes in the nodulation region of strain TA1. Complementation studies also indicated that strain TA1 contains negatively-acting genes located on the Sym plasmid and elsewhere, which specifically block nodulation of cv. Woogenellup.     

Enhanced growth and nodulation of pigeon pea by co-inoculation of Bacillus strains with Rhizobium spp

Endophytic bacteria which are known to reside in plant tissues have often been shown to promote plant growth. Present study deals with the isolation of putative endophytes from the surface sterilized root nodules of pigeon pea (Cajanus cajan) designated as non-rhizobial (NR) isolates. Three of these non-rhizobial isolates called NR2, NR4 and NR6 showed plant growth promotion with respect to increase in plant fresh weight, chlorophyll content, nodule number and nodule fresh weight when co-inoculated with the rhizobial bioinoculant strain IC3123. The three isolates were neither able to nodulate C. cajan nor did they show significant plant growth promotion when inoculated alone without Rhizobium spp. IC3123. All the three isolates were gram positive rods with NR2 and NR4 showing endospore formation and formed one single cluster in Amplified Ribosomal DNA Restriction Analysis (ARDRA). Partial sequences of 16S rRNA genes of NR4 and NR6 showed 97% similarity to Bacillus megaterium. The Bacillus strains NR4 and NR6 were able to produce siderophores which the rhizobial bioinoculant IC3123 was able to cross-utilize. Under iron starved conditions IC3123 showed enhanced growth in the presence of the Bacillus isolates indicating that siderophore mediated interactions may be underlying mechanism of beneficial effect of the NR isolates on nodulation by IC3123.