Strains Of Rhizobium Japonicum Research Articles

Competitive growth of slow growingRhizobium japonicum against fast growingEnterobacter andPseudomonas species at low concentrations of succinate and other substrates in dialysis culture

A cultivation system with simultaneous growth of six bacterial cultures in separate bags in dialysis culture was developed. In a medium with no added carbon source (one half concentrated Hoagland solution, water deionized and distilled), cell number ofRhizobium japonicum increased during a 7 day period by a factor of 35, whereas the number ofEnterobacter aerogenes cells decreased to one half. With a concentration of 100 nM succinate as an additional carbon source in the inflow,Rhizobium japonicum 61-A-101 cell number increased by a factor of 50 during an 8 day period, whereas cell number ofEnterobacter cloacae NCTC 10005 only doubled and ofEnterobacter aerogenes NCTC 10006 decreased. At 10 mM concentration of succinate in the inflow, doubling time the twoEnterobacter strains was about 12 h, compared to about 24 h for theRhizobium japonicum strain. Varying the succinate concentration from 10 mM to 100 nM in the inflow,Rhizobium japonicum 61-A-101 surpassed theEnterobacter aerogenes strains in the growth rate between 1 mM and 100 μM succinate in the inflowing medium. Three otherRhizobium japonicum strains (fix+ and fix-) did grow with a similar rate as strain 61-A-101 at very low concentrations of substrate. Growth rates for the strains were confirmed by protein data per culture. Growing in competition with twoPseudomonas strains,Rhizobium japonicum RH 31 Marburg (fix-) did overgrow alsoPseudomonas fluorescens, was however outgrown byPseudomonas putida. In utilizing low concentrations of a14C labelled organic acid (malonate), three strains ofRhizobium japonicum left 2–4 times smaller amounts of14C in the medium than two species ofPseudomonas and two species ofArthrobacter.

Conservation of symbiotic nitrogen fixation gene sequences in Rhizobium japonicum and Bradyrhizobium japonicum

Southern hybridization with nif (nitrogen fixation) and nod (nodulation) DNA probes from Rhizobium meliloti against intact plasmid DNA of Rhizobium japonicum and Bradyrhizobium japonicum strains indicated that both nif and nod sequences are on plasmid DNA in most R. japonicum strains. An exception is found with R. japonicum strain USDA194 and all B. japonicum strains where nif and nod sequences are on the chromosome. In R. japonicum strains, with the exception of strain USDA205, both nif and nod sequences are on the same plasmid. In strain USDA205, the nif genes are on a 112-megadalton plasmid, and nod genes are on a 195-megadalton plasmid. Hybridization to EcoRI digests of total DNA to nif and nod probes from R. meliloti show that the nif and nod sequences are conserved in both R. japonicum and B. japonicum strains regardless of the plasmid or chromosomal location of these genes. In addition, nif DNA hybridization patterns were identical among all R. japonicum strains and with most of the B. japonicum strains examined. Similarly, many of the bands that hybridize to the nodulation probe isolated from R. meliloti were found to be common among R. japonicum strains. Under reduced hybridization stringency conditions, strong conservation of nodulation sequences was observed in strains of B. japonicum. We have also found that the plasmid pRjaUSDA193, which possess nif and nod sequences, does not possess sequence homology with any plasmid of USDA194, but is homologous to parts of the chromosome of USDA194. Strain USDA194 is unique, since nif and nod sequences are present on the chromosome instead of on a plasmid as observed with all other strains examined.

Effect of Phosphorus on the Effectiveness of Strains of Rhizobium japonicum

AbstractWe examined the relationship between P nutrition of soybean [Glycine max (L.) Merr. cv. ‘Davis’] and N2 fixation by five strains of Rhizobium japonicum ranging from an ineffective (SM‐5) to a highly effective (USDA 110) symbiosis with soybean. Phosphorus at 0, 50, 125, and 400 mg P kg−1 soil (P0, P50, P125, P400) as Ca(H2PO4)2 · H2O was applied to pots containing 2.7 kg of a P‐fixing humoxic tropohumult free of R. japonicum. Treatments consisting of + N and five strains of R. japonicum and the four P treatments were arranged in a complete factorial replicated three times. With 400 mg P kg−1 soil treatments ranked + N > USDA 110 > USDA 31> USDA 123> USDA 33>SM‐5 for shoot dry weight and accumulation of N in the shoot. There were no significant strain effects for shoot dry weight, shoot N or nodule activity at Po. Strain rankings were the same at the intermediate levels of P but differences were smaller. Although nodule number was somewhat enhanced by P, it bore no relationship to strain effectiveness and was not a factor limiting N2 fixation at low P. Nodule dry weight and nitrogenase activities significantly increased with P additions. The concentrations of N and P in the shoot also increased with P additions. The concentrations of shoot P and N were higher in plants provided mineral N at the low and intermediate P levels than in the inoculated plants. The results show that P nutrition is important in interpreting the N2 fixation capability of strains of Rhizobium. Synergism between P and Rhizobium inputs requires that to obtain maximal response to the application of one input necessitates employing the other at maximum levels.

A comparison of lectin-binding activity in two strains of Rhizobium japonicum

A comparison of lectin-binding activity in two strains of Rhizobium japonicum

DENITRIFICATION BY INTACT SOYBEAN NODULES IN RELATION TO NATURAL 15N ENRICHMENT OF NODULES

The natural 15N abundance of nodules of soybeans (Glycine max (L.) Merrill) which are actively fixing N2 is considerably higher than other tissues. To investigate the question of whether isotopic fractionation associated with denitrification by bacteroids causes this 15N enrichment, we inoculated soybeans with two strains of Rhizobium japonicum. Free-living cultures of one of these (strain USDA 33) were unable to denitrify or respire NO−3, while free-living cultures of the second (strain USDA 138) were capable of denitrification. USDA 138 formed nodules which fixed N2 very efficiently. The N of these nodules was enriched in 15N and the nodules reduced a substantial amount of NO−3 to NO−2 and N2O. Nodules infected with USDA 33 fixed about half as much N2 as those infected with USDA 138. The former nodules were enriched in 15N (although less so than nodules infected with USDA 138), despite the fact that the nodules formed by USDA 33 did not reduce NO−3. Clearly denitrification could not have been the cause of 15N enrichment of nodules infected with strain USDA 33. Alternative causes of 15N enrichment of soybean nodules and their possible metabolic significance are discussed. Key words: δ15N, nitrogen fixation

Nitrogenase and uptake hydrogenase activities of Rhizobium japonicum during the life cycle of Glycine max (L.) merr.

Uptake hydrogenase and nitrogenase activities of Rhizobium japonicum strains PJ 17.1 and GA in association with Glycine max cvs. Fengshou and Giessen were investigated throughout the life cycles of the hosts.The growth of the two cultivars was greater with PJ 17.1 than with GA. The nitrogenase specific activities were maximum at 37 days after sowing with GA and 48 days after sowing with PJ 17.1 and decreased thereafter, almost similarly on both the cultivars. However, the uptake hydrogenase actities of PJ 17.1 were found to be maximum at 30 days.The relative efficiencies of PJ 17.1 were 1.0 at all stages while those of GA varied similarly on both the cultivars. As plants inoculated with PJ 17.1 were aging, a similar decline was observed in the activities of nitrogenase and hydrogenase. Although relative efficiency of GA varied throughout the life cycle of soybean, no correlation was seen between it and the nitrogenase specific activity.

Mapping of Rhizobium japonicum nifB-,fixBC-, and fixA-like genes and identification of the fixA promoter

New DNA regions likely to be involved in symbiotic nitrogen fixation were identified and mapped, by interspecies hybridization, on cloned DNA from the slowgrowing Rhizobium japonicum strain 110. NifB-and fixBC-like genes were located near the nifH and nifDK operons encoding the nitrogenase polypeptides, whereas a fixA-like gene is not linked to this cluster. NifB was detected by interspecies hybridization with a Klebsiella pneumoniae nifA/nifB probe, and its identity was confirmed due to its homology with the Rhizobium leguminosarum nifB gene. NifB of R. japonicum was located between nifDK and nifH at a distance of 11 kb downstream from the 3′ end of nifK. Using the Rhizobium meliloti fixABC operon as heterologous probe it was found that, in R. japonicum, fixA is distantly separated from fixBC. FixB and fixC are probably contained together in one operon (fixBC). The approximate start of fixB was located 2.8 kb downstream of the 3′ end of nifH. All genes of the nifDK-nifB-nifH-fixBC cluster are transcribed in the same direction. The DNA region harboring fixA was identified in a cosmid clone bank. The promoter and transcription start site of fixA were sequenced and mapped by a nuclease S1 protection experiment. The promoter sequence (5′-ATGGTAC-5bp-TTGCT-3′) is very similar to the nif-type consensus promoter sequence. Hence, the expression of R. japonicum nif and fix genes may be regulated coordinately.

Soybean Root Response to Symbiotic Infection Glyceollin I Accumulation in an Ineffective Type of Soybean Nodules with an Early Loss of the Peribacteroid Membrane

A glyceollin I accumulation of about 6000 pmol · mg dry weight-, a tenfold increase above control root tissue, was found in one type of nodule from Glycine max which had been infected with a fix- strain (61-A-24) of Rhizobium japonicum. In nodules infected with one other ineffective (fix-) strain of Rhizobium japonicum (RH 31-Marburg) or with two fix+ strains of Rhizobium japonicum (61-A-101 and USD A 110) no increase in glyceollin I concentrations above control values was found at either 20 d or 34 d after infection. Nodules infected with Rhizobium japonicum 61-A-24 are distinguished by an early loss of the peribacteroid membrane in the infected host cell, whilst the bacteroids themselves remain stable.

Expression of β-galactosidase inRhizobium japonicum

The plasmid pGC91.14 was used to introduce via conjugation the Escherichia coli lac operon into fast-growing and slow-growing strains of Rhizobium japonicum. Exconjugants now expressed higher levels of β-galactosidase activity which was still inducible by isopropyl-β-d-thiogalactoside (IPTG). The presence of the lac operon allowed the slow-growing strain 61A76 to grow on lactose as the sole carbon source; the fast-growing strains grew poorly on lactose but growth was not inhibited by lactose as had been reported for Rhizobium meliloti. β-galactosidase could be detected in nodule extracts and bacteroid preparations from soybean plants (Glycine max L. Merrill) infected with the strain 61A76 (pGC91.14).

Competition studies with fast-growing Rhizobium japonicum strains

The ability of eight fast-growing strains of Rhizobium japonicum to compete for nodule sites against two slow-growing strains of R. japonicum was measured using different input ratios (1:1, 1:10, and 10:1) on Glycine max cv. Peking and cv. Jacques 130 in growth pouches. The slow growers formed >60% of the nodules on cv. Peking even when the fast growers were added at a 10:1 ratio in their favor. We also measured the competitive ability of 10 fast-growing strains of R. japonicum, including these 8 strains, and ANU240 (a fast-growing broad host range strain) at two inoculum levels, 106 and 109 cells/seed on cv. Peking and cv. Jacques 130 in pots containing two midwestern soils which contained high numbers of indigenous rhizobia. In one soil, 3 of the 10 fast-growing strains occupied >60% of the nodules on cv. Peking at 109 inoculum level. No nodules were formed by the fast-growing strains on cv. Jacques 130 in any of the experiments.

Lack of specific binding of labeledRhizobium japonicum lipopolysaccharides to wild soybean (Glycine soja) roots

Lipopolysaccharides (LPS) were extracted from two strains ofRhizobium japonicum (61A76NS and 3I1b110-I). The extracted LPS was purified by gel filtration column chromatography and the amount of 2-keto-3-deoxyoctonate (KDO) was determined. Column purified LPS from both strains were conjugated to rhodamine isothiocyanate on celite to examine binding of this purified, labeled surface component to aseptically grownGlycine soja (wild soybean) seedlings as a basis for symbiotic specificity using fluorescent microscopy. Rhodamine conjugated LPS from both strains ofRhizobium japonicum did not exhibit specific binding to wild soybean seedling roots.

Nitrate Inhibition of Legume Nodule Growth and Activity

The synthesis and accumulation of nitrite has been suggested as a causative factor in the inhibition of legume nodules supplied with nitrate. Plants were grown in sand culture with a moderate level of nitrate (2.1 to 6.4 millimolar) supplied continuously from seed germination to 30 to 50 days after planting. In a comparison of nitrate treatments, a highly significant negative correlation between nitrite concentration in soybean (Glycine max [L.] Merr.) nodules and nodule fresh weight per shoot dry weight was found even when bacteroids lacked nitrate reductase (NR). However, in a comparison of two Rhizobium japonicum strains, there was only 12% as much nitrite in nodules formed by NR(-)R. japonicum as in nodules formed by NR(+)R. japonicum, and growth and acetylene reduction activity of both types of nodules was about equally inhibited. In a comparison of eight other NR(+) and NR(-)R. japonicum strains, and a comparison of G. max, Phaseolus vulgaris, and Pisum sativum, the concentration of nitrite in nodules was unrelated to nodule weight per plant or to specific acetylene reduction activity. The very small concentration of nitrite found in P. vulgaris nodules (0.05 micrograms NO(2) (-)-N per gram fresh weight) was probably below that required for the inhibition of nitrogenase based on published in vitro experiments, and yet the specific acetylene reduction activity was inhibited 83% by nitrate. The overall results do not support the idea that nitrite plays a role in the inhibition of nodule growth and nitrogenase activity by nitrate.

New culture media to suppress exopolysaccharide production by Rhizobium japonicum

Secretion of exopolysaccharide by Rhizobium japonicum strain USDA 110 ws measured during growth on media comprising 12.5 mM MES [2-(N-morpholino)ethanesulfonic acid] buffer, pH 5.9; 5 mM NH4Cl; trace elements; and 25 or 100 mM of one of 23 different carbon sources. Significant amounts of exopolysaccharide were produced on all aldopentoses, polyols, organic acids, and sugar acids tested, but little or none was secreted during growth on aldohexoses or amino acids. Media suitable for routine slime-free culture of several. R. japonicum strains were made by using 100 mM d-galactose or 25 mM l-aspartate as the carbon source.

Spatial relationships between uninfected and infected cells in root nodules of soybean

In soybean (Glycine max (L.) Merr.) the uninfected cells of the root nodule are responsible for the final steps in ureide production from recently fixed nitrogen. Stereological methods and an original quantitative method were used to investigate the organization of these cells and their spatial relationships to infected cells in the central region of nodules of soybean inoculated with Rhizobium japonicum strain USDA 3I1B110 and grown with and without nitrogen (as nitrate) in the nutrient medium. The volume occupied by the uninfected tissue was 21% of the total volume of the central infected region for nodules of plants grown without nitrate, and 31% for nodules of plants grown with nitrate. Despite their low relative volume, the uninfected cells outnumbered the much larger infected cells in nodules of plants grown both without and with nitrate. The surface density of the interface between the ininfected and infected tissue in the infected region was similar for nodules in both cases also, the total range being from 24 to 26 mm(2)/mm(3). In nodules of plants grown without nitrate, all sampled infected cells were found to be in contact with at least one uninfected cell. The study demonstrates that although the uninfected tissue in soybean nodules occupies a relatively small volume, it is organized so as to produce a large surface area for interaction with the infected tissue.

Mode of infection, nodulation specificity, and indigenous plasmids of 11 fast-growing Rhizobium japonicum strains.

Eleven fast-growing strains of Rhizobium japonicum were characterized with respect to indigenous plasmids and abilities to infect (Inf+) and nodulate (Nod+) cowpea, siratro, wild soybean, and three commercial cultivars of soybean. All strains caused infection via infection threads in root hairs and consistently nodulated cowpea, siratro, and wild soybean in growth pouches. Interactions with commercial cultivars of soybean were strikingly strain specific. Some combinations were Nod-, and infection was delayed in others. The ratios of infections to nodules and the distribution of nodules on primary and lateral roots also varied substantially. A modified in-gel lysis procedure was devised for electrophoretic separation of plasmids from the strains. Plasmids (ranging in size from 35 to greater than 300 megadaltons) were reproducibly detected in all strains.

Defoliation effects on mycorrhizal colonization, nitrogen fixation and photosynthesis in the Glycine‐Glomus‐Rhizobium symbiosis

Soybean [Glycine max (L.) Merr. cv. Wells] plants grown in a greenhouse were inoculated with Rhizobium japonicum strain 61A118 and the vesicular‐arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum (Thaxt. sensu Gerd.) Gerd. & Trappe. Plants were defoliated (26, 48 and 66%) throughout the growth period and evaluated for VAM colonization, N2, fixation and photosynthesis at harvest (six weeks). Photosynthate stress as a result of defoliation affected nodulation and nodule activity most severely. Colonization of the roots by the VAM fungus was little affected in comparison, and the intensity of colonization increased with increasing stress. The CO2‐exchange rate decreased less with defoliation than did leaf mass, and photosynthetic efficiency increased with the severity of defoliation. The increase in photosynthetic efficiency was significantly correlated with increases in leaf P (r = 0.91) and N (r = 0.97) concentrations. The results suggest that the VAM fungus should not be regarded as a simple P source and C sink in the tripartite legume association. Threeway source/sink relationships (VAM‐P, Rhizobium‐N, and host leaf‐C) are discussed.

Reiteration of genes involved in symbiotic nitrogen fixation by fast-growing Rhizobium japonicum.

By using cloned Rhizobium meliloti nodulation (nod) genes and nitrogen fixation (nif) genes, we found that the genes for both nodulation and nitrogen fixation were on a plasmid present in fast-growing Rhizobium japonicum strains. Two EcoRI restriction fragments from a plasmid of fast-growing R. japonicum hybridized with nif structural genes of R. meliloti, and three EcoRI restriction fragments hybridized with the nod clone of R. meliloti. Cross-hybridization between the hybridizing fragments revealed a reiteration of nod and nif DNA sequences in fast-growing R. japonicum. Both nif structural genes D and H were present on 4.2- and 4.9-kilobase EcoRI fragments, whereas nifK was present only on the 4.2-kilobase EcoR2 fragment. These results suggest that the nif gene organizations in fast-growing and in slow-growing R. japonicum strains are different.

Field evaluation of symbiotic nitrogen fixation by rhizobial strains using15N methodology

Small differences in N2 fixation by nodulated soybeans (Glycine max. (L.) Merr.), inoculated with various strains ofRhizobium japonicum, were assessed in field experiments using15N methodology, and compared with yields of plant dry matter and total N. Percentage of plant-N derived from atmospheric N2 and from fertilizer, and values of %15N atom excess had lower coefficients of variation than did total N and dry matter yield. Nevertheless the precision of estimates of kg N/ha fixed were sufficient to differentiate only the extremes of the range of strains tested, and there were discrepancies between ranking of strains based on % N derived from fertilizer and on total N yield.

Isolation of an Asm−mutant ofRhizobium japonicumdefective in symbiotic N2-fixation

Abstract A mutant strain of Rhizobium japonicum (CJ9) unable to assimilate ammonium (Asm − ) was isolated following mutagenesis with N -methyl N -nitro-nitrosoguanidine (NTG). Glutamate synthase activity was not detectable in cell-free extracts of the mutant strain in contrast to the wild type and revertant strains. Although mutant CJ9 induced nitrogenase activity in an ‘in vitro’ assay system under microaerobic conditions, it failed to fix nitrogen (acetylene reduction) in soybean root nodules. These properties of mutant CJ9 constitute a new Asm − mutant class in Rhizobium spp.

Breeding soybeans for the tropics capable of nodulating effectively with indigenousRhizobium spp.

Most soybean varieties fail to nodulate effectively in tropical soils unless inoculated with a competitive strain ofRhizobium japonicum. Developing countries in the tropics, with few exceptions, lack inoculant industries to produce and distribute viable inoculants to small farmers and extension programs to teach them to use inoculant. Several soybean genotypes have been identified that nodulate effectively with many strains of the “cowpea” inoculation group which is ubiquitous in tropical soils of Africa. Soybean genotypes that nodulate and grow well without inoculant application are called “promiscuous”. Methodologies for incorporation of the promiscuity character into high-yielding backgrounds are discussed.