Competition of Rhizobium japonicum Strains in Early Stages of Soybean Nodulation

The effects of preexposure of soybean (Glycine max L. Merrill) roots to Rhizobium japonicum strains and subsequent establishment of other strains in the nodules were investigated by using combinations of effective strains (USDA 110 and USDA 138) and effective-ineffective strains (USDA 110 and SM-5). Strain USDA 110 was a better competitor than either USDA 138 or SM-5 on cultivars Lee and Peking. However, when either of the two less-competitive strains was inoculated into 2-day-old seedlings before USDA 110 was, their nodule occupancy increased significantly on both cultivars. With USDA 138 as the primary inoculum and USDA 110 delayed for 6, 48, and 168 h, the incidence of USDA 138 nodules increased on cultivar Peking from 6% (at zero time) to 28, 70, and 82% and on cultivar Lee from 17% (at zero time) to 32, 88, and 95% for the three time delays, respectively. Preexposure of 2-week-old roots of cultivar Lee to USDA 138 had essentially the same effect: the incidence of USDA 138 nodules increased from 23% at zero time to 89 and 97% when USDA 110 was delayed for 24 and 72 h, respectively. When the ineffective strain SM-5 was used as the primary inoculum, followed by USDA 110 72 h later, the percentage of nodules containing SM-5 increased from 7 to 76%. These results indicate that the early events in the nodulation process of soybeans are perhaps the most critical for competition among R. japonicum strains.     

Competition Among Rhizobium leguminosarum Strains for Nodulation of Lentils (Lens esculenta)

Thirty-one cultures of Rhizobium leguminosarum were screened for effectiveness (C₂H₂ reduction) on lentils (Lens esculenta). Fluorescent antibodies prepared against three of the most effective strains (Hawaii 5-0, Nitragin 92A3, and Nitragin 128A12) exhibited a high degree of strain specificity; the antibodies reacted strongly with their homologous rhizobia in culture and with bacteroids in nodules. They did not cross-react with one another, and only weakly with 5 of the 47 other R. leguminosarum cultures tested. In competition studies in the growth chamber, whenever strain Nitragin 92A3 was included in the inoculum mixture, it consistently (but not always significantly, P = 0.05) occupied the majority of nodules on all four cultivars used. However, some degree of strain X cultivar interaction was apparent: Hawaii 5-0 was of equal competitiveness (P = 0.05) with Nitragin 92A3 on three of the varieties (Commercial, Tekoa, and Benewah), but inferior (P = 0.01) on the Chilean variety; Nitragin 92A3 completely dominated (P = 0.01) Nitragin 128A12 on all cultivars; and Hawaii 5-0 was of equal competitiveness (P = 0.05) to Nitragin 128A12 on the Chilean variety and more competitive (P = 0.01) on the commercial variety and less so on the other two varieties. In field experiments, Hawaii 5-0 proved of equal competitiveness (P = 0.01) with Nitragin 92A3 in one soil (an Inceptisol) and superior (P ≤ 0.05) to it in another (an Oxisol). Incidence of double-strain occupancy of nodules varied from 0 to 36% in vermiculite, depending on the strains in the mixture and the host variety, and from 0 to 38% in the field, depending on the strains in the mixture and the soil type. The results suggest a close relationship between the competitiveness of a strain and its occurrence in doubly infected nodules.     

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.     

哈茨木霉发酵液中肽类物质对豇豆土著根瘤菌竞争结瘤的影响

通过树脂吸附、离子交换、薄层层析、高效液相色谱系统以及紫外、质谱等分离、纯化和鉴别的方法,从哈茨木霉T2-16菌株发酵液中分离得到一种对豆科作物生长具促进作用的肽类物质。用该肽类物质对豇豆土著根瘤菌进行处理后,用AFLP技术研究了该物质对供试根瘤菌遗传稳定性和在土壤中竞争结瘤能力的影响。结果表明,传代次数和培养温度(28℃~36℃范围)对供试根瘤菌的遗传性状无明显影响,其AFLP指纹未发生明显变化;但经木霉肽类代谢产物处理后,根瘤菌竞争结瘤能力得到提高,为对照根瘤菌的1.53倍。     

Nodulation and Competition for Nodulation of Selected Soybean Genotypes among Bradyrhizobium japonicum Serogroup 123 Isolates

Twenty recently obtained field isolates of Bradyrhizobium japonicum serogroup 123 were tested for their nodule mass production on the standard commercial soybean (Glycine max (L.) Merr. cv. Williams) and on two soybean plant introduction (PI) genotypes previously determined to restrict nodulation by strain USDA 123. Four of the field isolates showed similar restricted nodulation on the two genotypes, while all 20 isolates produced a normal amount of nodules on G. max cv. Williams. Serological analyses with adsorbed fluorescent antibodies showed that members of the 123 serotype ranked low in nodulation of the two PIs, in contrast to members of serotypes 127 and 129. Competition studies on the PIs indicated that isolates which were restricted were not competitive for nodule occupancy against strain USDA 110. However, unrestricted isolates of serogroup 123 were very competitive against USDA 110. On G. max cv. Williams, all serogroup 123 isolates tested were very competitive against USDA 110.     

Influence of Bradyrhizobium japonicum Location and Movement on Nodulation and Nitrogen Fixation in Soybeans

The influence of seed and soil inoculation on bradyrhizobial migration, nodulation, and N₂ fixation was examined by using two Bradyrhizobium japonicum strains of contrasting effectiveness in N₂ fixation. Seed-inoculated strains formed fewer nodules on soybeans (mostly restricted to the tap and crown roots within 0 to 5 cm from the stem base) than did bradyrhizobia distributed throughout the soil or inoculated at specific depths. Nodulation was greater below the depths at which bradyrhizobial cells were located rather than above, even though watering was done from below to minimize passive bradyrhizobial migration with percolating water. The most profuse nodulation occurred within approximately 5 cm below the point of placement and was generally negligible below 10 cm. These and other results suggest that bradyrhizobial migration from the initial point of placement was very limited. Nevertheless, the more competitive strain, effective strain THA 7, migrated into soil to a greater extent than the ineffective strain THA 1 did. Nitrogen fixation resulting from the dual-strain inoculations differed depending on the method of inoculation. For example, the amount of N₂ fixed when both strains were slurried together onto the seed was about half that obtained from mixing the effective strain into the soil with the ineffective strain on the seed. The results indicate the importance of rhizobial distribution or movement into soil for nodulation, nodule distribution, strain competitiveness, and N₂ fixation in soil-grown legumes.     

Influence of Environmental Factors on Interstrain Competition in Rhizobium japonicum

The effect of several biotic and abiotic factors on the pattern of competition between two strains of Rhizobium japonicum was examined. In two Minnesota soils, Waseca and Waukegan, strain USDA 123 occupied 69% (Waseca) and 24% (Waukegan) of the root nodules on Glycine max L. Merrill cv. Chippewa. USDA 110 occupied 2% of the root nodules in the Waseca soil and 12% of the nodules in the Waukegan soil. Under a variety of other growth conditions—vermiculite, vermiculite amended with Waseca soil, and two Hawaiian soils devoid of naturalized Rhizobium japonicum strains—USDA 110 was more competitive than USDA 123. The addition of nitrate to or the presence of antibiotic-producing actinomycetes in the rhizosphere of soybeans did not affect the pattern of competition between the two strains. However, preexposure of young seedings to USDA 110 or USDA 123 before transplantation into soil altered the pattern of competition between the two strains significantly. In the Waseca soil, preexposure of cv. Chippewa to USDA 110 for 72 h increased the percentage of nodules occupied by USDA 110 from 2 to 55%. Similarly, in the Hawaiian soil Waimea, nodule occupancy by USDA 123 increased from 7 to 33% after a 72-h preexposure.     

A Hydrophobic Mutant of Rhizobium etli Altered in Nodulation Competitiveness and Growth in the Rhizosphere

We isolated and characterized CE3003, a Tn5-induced mutant with altered colony morphology derived from Rhizobium etli CE3. CE3003 produced domed colonies and was highly hydrophobic as indicated by its ability to partition into hexadecane, whereas its parent produced flat colonies and was hydrophilic. On bean plants, CE3003 induced nodules and reduced acetylene. CE3003 and CE3 grew at similar rates when they were grown separately or together in culture medium or inoculated singly onto bean seeds. However, when they were mixed at a 1:1 ratio and applied to seeds, CE3003 achieved significantly lower populations than CE3 in the rhizosphere. Five days after coinoculation of CE3 and CE3003, the population of the mutant was less than 10% of the population of CE3 in the bean rhizosphere. To determine the nodulation competitiveness of the mutant, it was coinoculated with CE3 at various ratios at planting, and the ratio of the nodules occupied by each strain was determined 21 days later. A 17,000-fold excess of CE3003 in mixed inocula was required to obtain equal nodule occupancy by the two strains. A genomic library of strain CE3 was mobilized into CE3003, and we identified a cosmid, pRA3003, that restored the parental colony morphology and hydrophilicity to the mutant. Restoration of the parental colony morphology was accompanied by recovery of the ability to grow competitively in the rhizosphere and to compete for nodulation of beans. The data show an association between cell surface hydrophobicity, nodulation competitiveness, and competitive growth in the rhizosphere in mutant CE3003.     

Competition between two somatic serotypes of Rhizobium japonicum used as double-strain inocula in varying proportions

Summary The nodulation response of soybeans (Glycine max, Mukden variety) to single-strain inocula was related to the density of suspensions. The competition between two different somatic serotypes, represented by the strains D 216 and D 344, was also found to be generally related to the ratio of inoculum strain cells in suspensions, but the strain D 216 was more successful in the root-nodule formation, accounting for 100-80 percent of the serotyped root-nodules when the ratio between cells of the strains D 216 and D 344 varied between 3:5 and 1:60.This investigation forms part of a contribution prepared by the Czechoslovak National Committee for the International Biological Programme (Section PP: Production Processes).     

Early Infection and Competition for Nodulation of Soybean by Bradyrhizobium japonicum 123 and 138

Interactions of soybean with Bradyrhizobium japonicum 123 (serogroup 123) and 138 (serogroup c1) were used to examine the relationship between early infection rates, competition for nodulation, and patterns of nodule occupancy. Both strains formed more infections in autoclaved soil (sterile soil) than in untreated soil (unsterile soil). Inoculation did not increase numbers of infection threads in unsterile soil-grown plants, where infection of proximal portions of primary roots was complete by 5 days after planting. Both strains infected and nodulated at similar rates in sterile soil. Nodules were always clustered on the upper root system, regardless of inoculation and soil treatment. Sixty-seven percent of the nodules of uninoculated plants grown in unsterile soil were occupied by rhizobia belonging to serogroups other than 123 or c1. Inoculation with strain 123 or 138 increased occupancy by that strain at the expense of residency by other rhizobia. Eighty-three percent of all nodules on plants dually inoculated with both strains in sterile soil contained strain 138. The corresponding value for plants inoculated in unsterile soil was 31%. Neither inoculum strain dominated occupancy of first-formed nodules in unsterile soil. It appears that north central Missouri soil may not have populations of highly competitive serogroup 123 and that early infection and nodulation rates do not contribute to the competitive success of strain 138.     

Variations in Ability of Rhizobium japonicum Strains To Nodulate Soybeans and Maintain Fixation in the Presence of Nitrate

This study investigated differences in sensitivity to nitrate of soybean (Glycine max cv. Davis) symbioses with 16 different Rhizobium japonicum strains. When nitrate (20 mM) was added to established symbioses, there were no significant differences in the degree of inhibition of acetylene reduction for any of the 16 strains. When nitrate was present during the establishment of nodules, high levels of nitrate (10 mM) were equally inhibitory on all symbioses, whereas specific strain effects appeared at low (0.5 mM) to medium (2.0 mM) levels of nitrate. At 1.5 mM nitrate in solution culture, the days to emergence of nodules varied from less than 10 (CB:1809, Nit61A118) to more than 16 (11 of 16 strains). In a clay-pot trial maintained at the low nitrate level (0.5 mM), symbioses with CB:1809 increased total nodule mass by 30% relative to nitrate-free controls. In the presence of 2.0 mM nitrate, CB:1809 maintained total nodule mass. For the remaining 6 strains tested, total nodule mass decreased to below the levels of the nitrate-free controls. In a separate clay pot trial, CB:1809 increased its competitive ability relative to USDA:110 when nitrate was added. If no nitrate was added, CB:1809 occupied 0.97 times as many nodules as USDA:110; when 10 mM nitrate was added, CB:1809 occupied 1.75 times as many nodules as USDA:110. Attempts to select nitrogen-adapted substrains of R. japonicum through sequential isolation and infection of plants grown on nitrate were not successful.     

Split-Root Assays Using Trifolium subterraneum Show that Rhizobium Infection Induces a Systemic Response That Can Inhibit Nodulation of Another Invasive Rhizobium Strain

Subterranean clover plants possessing two equally infectible and robust lateral root systems (“split roots”) were used in conjunction with several specific mutant strains (derived from Rhizobium trifolii ANU843) to investigate a systemic plant response induced by infective Rhizobium strains. This plant response controls and inhibits subsequent nodulation on the plant. When strain ANU843 was inoculated onto both root systems simultaneously or 24, 48, 72, or 96 h apart, an inhibitory response occurred which retarded nodulation on the root exposed to the delayed inoculum but only when the delay period between inocula was greater than 24 h. Equal numbers of nodules were generated on both roots when ANU843 was inoculated simultaneously or 24 h apart. The ability to infect subterranean clover plants was required to initiate the plant inhibitory response since preexposure of one root system to non-nodulating strains did not retard the ability of the wild-type strain to nodulate the opposing root system (even when the delay period was 96 h). Moreover, the use of specific Tn5-induced mutants subtly impaired in their ability to nodulate demonstrated that the plant could effectively and rapidly discriminate between infections initiated by either the parent or the mutant strains. When inoculated alone onto clover plants, these mutant strains were able to infect the most susceptible plant cells at the time of inoculation and induce nitrogen-fixing nodules. However, the separate but simultaneous inoculation on opposing root systems of the parent and the mutant strains resulted in the almost complete inhibition of the nodulation ability of the mutant strains. We concluded that the mutants were affected in their competitive ability, and this finding was reflected by poor nodule occupancy when the mutants were coinoculated with the parent strain onto a single root system. Thus the split-root system may form the basis of a simple screening method for the ranking of competitiveness of various rhizobia on small seeded legumes.     

Nickel is a component of hydrogenase in Rhizobium japonicum

The derepression of H2-oxidizing activity in free-living Rhizobium japonicum does not require the addition of exogenous metal to the derepression media. However, the addition of EDTA (6 microM) inhibited derepression of H2 uptake activity by 80%. The addition of 5 microM nickel to the derepression medium overcame the EDTA inhibition. The addition of 5 microM Cu or Zn also relieved EDTA inhibition, but to a much lesser extent; 5 microM Fe, Co, Mg, or Mn did not. The kinetics of induction and magnitude of H2 uptake activity in the presence of EDTA plus Ni were similar to those of normally derepressed cells. Nickel also relieved EDTA inhibition of methylene blue-dependent Hup activity, suggesting that nickel is involved directly with the H2-activating hydrogenase enzyme. Adding nickel or EDTA to either whole cells or crude extracts after derepression did not affect the hydrogenase activity. Cells were grown in 63Ni and the hydrogenase was subsequently purified by gel electrophoresis. 63Ni comigrated with the H2-dependent methylene blue reducing activity on native polyacrylamide gels and native isoelectric focusing gels. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the nickel-containing hydrogenase band revealed a single polypeptide with a molecular weight of ca. 67,000. We conclude that the hydrogenase enzyme in R. japonicum is a nickel-containing metalloprotein.     

Possible Involvement of a Megaplasmid in Nodulation of Soybeans by Fast-Growing Rhizobia from China

Several isolates from a newly described group of fast-growing acid-producing soybean rhizobia, Rhizobium japonicum, were analyzed for plasmid content. All contained from one to four plasmids with molecular weights of 100 × 10⁶ or larger. Although most of the isolates shared plasmids of similar size, the restriction endonuclease (BamHI, EcoRI, and HindIII) patterns of the plasmids from three of the isolates were vastly different. Growth in the presence of acridine orange was effective in producing mutants cured of the largest plasmid in one of the strains. These mutants also lost the ability to form nodules on soybeans. High-temperature curing of a smaller plasmid in another strain did not lead to loss of nodulating ability or alteration of symbiotic effectiveness on soybean cultivars. The identities of all of the isolates and mutants were ascertained by immunofluoresence and immunodiffusion. The new fast-growing strains of R. japonicum may provide a better genetic system for the study of the soybean symbiosis than the slow-growing R. japonicum, not all of which can be shown to contain plasmids.     

Procedure for Obtaining Efficient Root Nodulation of a Pea Cultivar by a Desired Rhizobium Strain and Preempting Nodulation by Other Strains

The specificity between the sym-2 gene bred into certain cultivars of pea (Pisum sativum L.) and the nodX gene, present only rarely in isolates of Rhizobium leguminosarum, can be exploited to preempt competition or nodulation blocking by a Rhizobium strain indigenous to a soil environment. The principle is to isolate an R. leguminosarum strain prevalent in a locale, convert it into a strain that will nodulate a desirable pea cultivar carrying sym-2 by establishing nodX in it, and then use the resulting Rhizobium strain with the pea cultivar carrying sym-2. To accomplish this, we first constructed a transposon Tn5 derivative called Tn5nodX and an efficient delivery vehicle that is suicidal in R. leguminosarum. We tested the potential utility of the system in greenhouse experiments. The results are encouraging enough to warrant extensive experiments under field conditions.     

Genes Essential for Nod Factor Production and Nodulation Are Located on a Symbiotic Amplicon (AMPRtrCFN299pc60) in Rhizobium tropici

Amplifiable DNA regions (amplicons) have been identified in the genome of Rhizobium etli. Here we report the isolation and molecular characterization of a symbiotic amplicon of Rhizobium tropici. To search for symbiotic amplicons, a cartridge containing a kanamycin resistance marker that responds to gene dosage and conditional origins of replication and transfer was inserted in the nodulation region of the symbiotic plasmid (pSym) of R. tropici CFN299. Derivatives harboring amplifications were selected by increasing the concentration of kanamycin in the cell culture. The amplified DNA region was mobilized into Escherichia coli and then into Agrobacterium tumefaciens. The 60-kb symbiotic amplicon, which we termed AMPRtrCFN299pc60, contains several nodulation and nitrogen fixation genes and is flanked by a novel insertion sequence ISRtr1. Amplification of AMPRtrCFN299pc60 through homologous recombination between ISRtr1 repeats increased the amount of Nod factors. Strikingly, the conjugal transfer of the amplicon into a plasmidless A. tumefaciens strain confers on the transconjugant the ability to produce R. tropici Nod factors and to nodulate Phaseolus vulgaris, indicating that R. tropici genes essential for the nodulation process are confined to an ampliable DNA region of the pSym.     

Inoculation Response of Legumes in Relation to the Number and Effectiveness of Indigenous Rhizobium Populations

The response of legumes to inoculation with rhizobia can be affected by many factors. Little work has been undertaken to examine how indigenous populations or rhizobia affect this response. We conducted a series of inoculation trials in four Hawaiian soils with six legume species (Glycine max, Vigna unguiculata, Phaseolus lunatus, Leucaena leucocephala, Arachis hypogaea, and Phaseolus vulgaris) and characterized the native rhizobial populations for each species in terms of the number and effectiveness of the population for a particular host. Inoculated plants had, on average, 76% of the nodules formed by the inoculum strain, which effectively eliminated competition from native strains as a variable between soils. Rhizobia populations ranged from less than 6 × 10⁰/g of soil to 1 × 10⁴/g of soil. The concentration of nitrogen in shoots of inoculated plants was not higher than that in uninoculated controls when the most probable number MPN counts of rhizobia were at or above 2 × 10¹/g of soil unless the native population was completely ineffective. Tests of random isolates from nodules of uninoculated plants revealed that within most soil populations there was a wide range of effectiveness for N₂ fixation. All populations had isolates that were ineffective in fixing N₂. The inoculum strains generally did not fix more N₂ than the average isolate from the soil population in single-isolate tests. Even when the inoculum strain proved to be a better symbiont than the soil rhizobia, there was no response to inoculation. Enhanced N₂ fixation after inoculation was related to increased nodule dry weights. Although inoculation generally increased nodule number when there were less than 1 × 10² rhizobia per g of soil, there was no corresponding increase in nodule dry weight when native populations were effective. Most species compensated for reduced nodulation in soils with few rhizobia by increasing the size of nodules and therefore maintaining a nodule dry weight similar to that of inoculated plants with more nodules. Even when competition by native soil strains was overcome with a selected inoculum strain, it was not always possible to enhance N₂ fixation when soil populations were above a threshold number and had some effective strains.