Competition among<Emphasis Type="Italic">Bradyrhizobium</Emphasis> strains for nodulation of green gram (<Emphasis Type="Italic">Vigna radiata</Emphasis>): Use of dark-nodule strain

The competitiveness of dual-strain inoculum of Bradyrhizobium strains S24 and GR4 was demonstrated for nodulation of green gram (Vigna radiata). Strain S24 formed pink nodules, GR4 produced visually distinguishable dark-brown nodules. When a mixture of these Bradyrhizobium strains was applied as inoculum, nodules of both pink and dark-brown types were formed on the same root. The strain GR4, which was less competitive than strain S24, was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine to obtain pigment-diverse mutants and six selected mutants were screened for symbiotic parameters. One mutant produced pink nodules and appreciably increased plant dry mass. The competitive ability of this mutant lacking brown pigment was compared with that of strain S24 by using antibiotic resistance markers; it showed increased nodulation competitiveness than its parent strain GR4. The dark-brown nodule-phenotype could be useful in evaluating nodulation competitiveness of "cowpea miscellany" bradyrhizobia in soil where dark-brown nodule-forming strains are not indigenous.     

Competition between inoculum and native rhizobia for nodulation of cowpea (Vigna unguiculata (L) Walp): Use of a dark-nodule strain

Summary Cowpea rhizobia strains were examined with indigenous populations in nodulating cowpea (Vigna unguiculata (L) Walp) cv. Laura B. strain IRC256 formed dark nodules on cowpea, and were used as the standard against orthodox pink-nodule strains in evaluating nodulating competitiveness. The dark nodule phenotype and intrinsic antibiotic resistance pattern were used to identify the strains in the nodules. Our results showed the usefulness of the dark-nodule strain in evaluating nodulating competitiveness of cowpea rhizobia in soils where dark-nodule strains were not indigenous.     

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.     

Use of the gusA gene marker in a competition study of the Rhizobium strains nodulating the common bean (Phaseolus vulgaris) in Senegal soils

Indigenous rhizobial population is among the factors which influence increased crop yield through inoculation with elite strains. In this study, we compared in greenhouse conditions the competitiveness of Rhizobium strain ISRA 355 for nodulation of the common bean (Phaseolus vulgaris) cultivated in different unsterile Senegal soils in terms of pH, N and C contents. The strain ISRA 355 produced a stable GUS+ transconjugant which was used for competition with indigenous soil rhizobia in six localities. At Bayakh, the transconjugant ISRA 355gusA was less competitive than the indigenous rhizobial strains, whereas in the other localities, it was more competitive since it occupied more than 90% of the nodules. Thus the Rhizobium strain ISRA 355 should be used for successfully inoculating the common bean in Senegal soils.     

Competitive Ability and Efficiency in Nodule Formation of Strains of Bradyrhizobium japonicum

In the American Midwest, superior N₂-fixing inoculant strains of Bradyrhizobium japonicum consistently fail to produce the majority of nodules on the roots of field-grown soybean. Poor nodulation by inoculant strains is partly due to their inability to stay abreast of the expanding soybean root system in numbers sufficient for them to be competitive with indigenous bradyrhizobia. However, certain strains are noncompetitive even when numerical dominance is not a factor. In this study, we tested the hypothesis that the nodule occupancy achieved by strains is related to their nodule-forming efficiency. The nodulation characteristics and competitiveness of nine strains of B. japonicum were compared at both 20 and 30°C. The root tip marking technique was used, with the nodule-forming efficiency of each strain estimated from the average position of the uppermost nodule and the number of nodules formed above the root tip mark. The competitiveness of the nine strains relative to B. japonicum USDA 110 was determined by using immunofluorescence to identify nodule occupants. The strains differed significantly in competitiveness with USDA 110 and in nodulation characteristics, strains that were poor competitors usually proving to be inferior in both the average position of the uppermost root nodule and the number of nodules formed above the root tip mark. Thus, competitiveness was correlated with both the average position of the uppermost nodule (r = 0.5; P = 0.036) and the number of nodules formed above the root tip mark (r = 0.64; P = 0.005), while the position of the uppermost nodule was also correlated to the percentage of plants nodulated above the root tip mark (r = 0.81; P < 0.001) and the percentage of plants nodulated on the taproot (r = 0.67; P = 0.002).     

Unaltered Nodulation Competitiveness of a Strain of Bradyrhizobium sp. (Lotus) after a Decade in Soil

A Bradyrhizobium sp. (Lotus) strain that formed a soil population that was highly competitive for nodulation of Lotus pedunculatus 11 years after its introduction into a field soil and a culture of the same strain stored lyophilized were compared with an antibiotic-resistant mutant in respect of their nodulation competitiveness. The mutant was less competitive than the wild-type strain it was isolated from and had to be present at a cell ratio of 5.76:1 in mixed inoculum in sand culture to form 50% of the nodules on L. pedunculatus (50% nodulation value, 5.76). The 50% nodulation values for a soil population of the mutant mixed with soil populations of the lyophilized and field soil strain were, respectively, 6.83 and 5.77, indicating that the field soil strain was not significantly different from the lyophilized strain in nodulation competitiveness. A 50% nodulation value of 11.18 obtained when soil containing a recently established mutant population was mixed with the field soil containing the population established 11 years before, indicating that the plant infection technique underestimated cell numbers of the field soil population by 100%. Nodulation competitiveness was unaffected by the size of the strain populations in the range of 100 to 1,000 cells per g of soil; at 10 cells per g a significant correlation between strain ratios in nodules and in soil was still evident. The results indicated that apparently superior nodulation competitiveness of a well-established soil population relative to that of a subsequently introduced strain may not necessarily reflect the intrinsic competitive abilites of the strain(s) involved. The soil strain did not differ from laboratory-maintained cultures in antigenic properties, effectiveness, or whole cell protein electrophoresis profiles.     

Mixed Inoculations with Effective and Ineffective Strains of Rhizobium leguminosarum

An ineffective Rhizobium leguminosarum strain capable of forming green nodules of similar size and number as normally effective strains was tested for its ability to compete with an effective strain in nodule formation on the pea. The ineffective strain was found to be more competitive and influenced the pattern of nodulation by the effective strain on the same root system. Nodules containing both strains were pink and able to reduce acetylene.     

Trifolitoxin Production and Nodulation Are Necessary for the Expression of Superior Nodulation Competitiveness by Rhizobium leguminosarum bv. trifolii Strain T24 on Clover

Rhizobium leguminosarum bv. trifolii T24 is ineffective in symbiotic nitrogen fixation, produces a potent antibiotic (referred to here as trifolitoxin) that is bacteriostatic to certain Rhizobium strains, and is very competitive for clover root nodulation (EA Schwinghamer, RP Belkengren 1968 Arch Mikrobiol 64: 130-145). The primary objective of this work was to demonstrate the roles of nodulation and trifolitoxin production in the expression of nodulation competitiveness by T24. Unlike wildtype T24, transposon mutants of T24 lacking trifolitoxin production were unable to decrease clover nodulation by an effective, trifolitoxin-sensitive strain of R. leguminosarum bv. trifolii. A non-nodulating transposon mutant of T24 prevented clover nodulation by a trifolitoxin-sensitive R. leguminosarum bv. trifolii when co-inoculated with a T24 mutant lacking trifolitoxin production. Neither mutant alone prevented nodulation by the trifolitoxin-sensitive strain. These results demonstrate that trifolitoxin production and nodulation are required for the expression of nodulation competitiveness by strain T24. A trifolitoxin-sensitive strain of R. meliloti did not nodulate alfalfa when co-inoculated with T24 and a trifolitoxin-resistant strain of R. meliloti. Thus, a trifolitoxin-producing strain was useful in regulating nodule occupancy on a legume host other than clover. Trifolitoxin production was constitutive in both minimal and enriched media. Trifolitoxin was found to inhibit the growth of 95% of all strains of R. leguminosarum bvs. trifolii, viceae, and phaseoli tested. Strains of all 13 biotypes of R. leguminosarum bv. trifolii were inhibited by trifolitoxin. Three strains of R. fredii were also inhibited. Strain T24 ineffectively nodulated 46 clover species, did not nodulate Trifolium ambiguum, and induced partially effective nodules on Trifolium micranthum. Since T24 produced partially effective nodules on T. micranthum and since a trifolitoxin-minus mutant of T24 induced ineffective nodules, trifolitoxin production is not the cause of the symbiotic ineffectiveness of T24.     

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     

Nodulation and growth ofLeucaena leucocephala (Lam.) de Wit as affected by inoculation and N fertilizer

Leonard jar, pot and field experiments examined the effects of inoculation and the influence of nitrogen fertilizer on nodulation, nitrogen fixation and growth ofLeucaena leucocephala (Lam.) de Wit at IITA, Ibadan, Nigeria. Leucaena responded to both inoculation and/or nitrogen application. Shoot growth and total N and P of inoculated plants were comparable to those of the highest N treatment, and the values were about 55% greater than those of uninoculated ones. Field data indicated that toal N yields of inoculated leucaena were increased by 50% with 40 or 80 kg ha^–1 of N fertilizer. However, N fertilizer depressed N fixation by 56% as was expected from nodule mass data. N-fixation was delayed for about 8 weeks in the plots without N. Application of small amounts of N starter (20 ppm) proved to be beneficial to satisfy the plant need during the early stage of leucaena growth. The rhizobial strains IRc 1045 and IRc 1050 were effective, competitive and survived well in the field one year after their establishment.     

Soybean cultivars affect nodulation competition of Bradyrhizobium japonicum strains

The influence of five Thai soybean cultivars on nodulation competitiveness of four Bradyrhizobium japonicum strains was investigated. Cultures of B. japonicum strains THA5, THA6, USDA110 and SEMIA5019 were mixed with each other prior to inoculating germinated soybean seeds growing in Leonard jars with nitrogen-free nutrient solution. At harvest, nodule occupancy by each strain was determined by a fluorescent antibody technique. The term ‘general competitive ability’ was introduced to describe the average competitive nodule occupancy of a strain in paired co-inoculation with a number of strains on soybean. The nodule occupancies by an individual strain were directly correlated with the proportions of that strain in the inoculum mixtures. USDA110 showed higher nodulation competitiveness than the other strains on three of the five cultivars. The Thai strain THA6 appeared to be more competitive than USDA110 on cultivar SJ5. Thus, nodulation competitiveness of the B. japonicum strains was affected by the cultivars of soybean used. This revised version was published online in August 2006 with corrections to the Cover Date.     

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 by Bradyrhizobium Strains for Nodulation of the Nonlegume Parasponia andersonii

Bradyrhizobium strains isolated from the nonlegume Parasponia spp. formed a group of strains that were highly competitive for nodulation of P. andersonii when paired with strains isolated from legumes. Strains from legumes, including those of similar effectiveness to NGR231 and CP283, were not able to form nodules as single occupants on P. andersonii in the presence of Parasponia strains. However, NGR86, an isolate from Macroptilium lathyroides, jointly occupied one-third of the nodules formed with each of the three strains isolated from Parasponia spp. Time taken for nodules to appear may have influenced the outcome of competition, since CP283 and all isolates from legumes were slow to nodulate P. andersonii. Among the Parasponia strains, competitiveness for nodulation of P. andersonii was not associated with effectiveness of nitrogen fixation. The highly effective strain CP299 was a poor competitor when paired with the least effective strain NGR231. CP283 was the least competitive of the Parasponia strains but was still able to dominate nodules when paired with legume isolates. Dual occupancy was high, up to 67% when the inoculum contained CP299 and CP273. Both the Muc⁺ and Muc^- types of CP283 form a symbiosis of similar effectiveness and were similarly competitive at high inoculation densities, but the Muc^- form was more competitive at low inoculum densities. Both forms frequently occupied the same nodule. Bradyrhizobium strains isolated from Parasponia spp. may have specific genetic information that favor their ability to competitively and effectively infect plants in the genus Parasponia (Ulmaceae) outside the Leguminosae.     

Discrete differences between strains of different Rhizobium spp. for competitive nodule occupancy on beans

Rhizobium etli strain TAL182 and R. leguminosarum bv phaseoli strain 8002, both of which produce melanin pigment, were tested for their nodulation competitiveness on beans by paired inoculation with two strains which do not produce melanin: R. tropici strain CIAT899 and Rhizobium sp. strain TAL1145. An assay was developed to distinguish nodules formed by the melanin-producing and non-producing strains. Strain TAL182 had discrete competitive superiority over CIAT899 and TAL1145 for nodulation of beans. Nodulation competitiveness was not correlated with the ability to produce melanin pigment or the host range of the Rhizobium strains tested.The authors are with the Department of Plant Molecular Physiology, University of Hawaii, 3050 Maile Way, Gillmore 402, Honolulu, HI 96822, USA     

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.     

Competitiveness of a native Rhizobium leguminosarum biovar trifolii strain for nodule occupancy is manifested during infection

The stages in the nodulation process that determined the competitiveness of R. leguminosarum bv. trifolii (Rlt) strain 20–15, which proved to be highly competitive for nodulation in Iceland fields tests over several years, is analysed. White clover (Trifolium repens L.) roots were inoculated with inoculum mixtures containing three strains (Rlt 20-15, Rlt 8-9 and Rlt 32-28) in different proportions and cell densities. Competitiveness in root colonization, formation of infection threads and nodule development was assessed for Rlt 20-15 and its weakest competitor, Rlt 32-28. ERIC-polymerase chain reaction (PCR) DNA fingerprinting was used to identify inoculated strains recovered from root surfaces and individual nodules. GFP or DsRed tagged strains were used to determine identity in root hairs and nodules. Both strains colonized the root equally at all inoculum ratios tested. But, Rlt 20-15 initiated significantly more infection threads and formed more nodules than Rlt 32-28. These results show that Rlt 20-15 expresses its nodulation competitiveness during infection, either at infection thread initiation or during successive growth in the infection threads. The data presented support earlier observations that this strain competed well in the field in spite of its inferior ability to survive in the soil.     

Application of immunodiffusion to the identification of Rhizobium meliloti strains competing for nodulation on Medicago sativa

The immunodiffusion technique was successfully used to unambiguously recognize four strains of Rhizobium meliloti in a study of competition for nodulation with Medicago sativa cv. Apollo inoculated with two-, three- and fourstrain mixtures. The serological reactions of all R. meliloti strains revealed no significant changes following plant passage indicating that the antigens involved in immunodiffusion were stable. R. meliloti 102F70 formed 50% or more of the nodules on M. sativa inoculated with two-, three- and four-strain mixtures. The remaining three strains were less competitive and produced similar proportions of nodules (14–20%) on plants inoculated with three- and four-strain mixtures. Cases of mixed-strain occupancy of nodules involving either two of three strains were detected in a sub-sample of nodules. The data also indicated considerable variation in the proportions of strains in the nodules of individual plants.     

Competitiveness of indigenous populations of Bradyrhizobium japonicum serocluster 123 as determined using a root-tip marking procedure in growth pouches

Soil Bradyrhizobium populations limit nodule occupancy of soybean by symbiotically-superior inoculant strains throughout much of the American midwest. In this study, the competitiveness of indigenous populations of B. japonicum serocluster 123 from Waukegan and Webster soils was evaluated in growth pouches using a root-tip marking procedure. The native rhizobia were from soils incubated 0–8 h in soybean root exudate (SRE) or plant nutrient solution (PNS) prior to inoculation. Populations of serocluster 123 strains in soil and nodule occupancy by these strains were assessed using fluorescent antibodies prepared against B. japonicum USDA 123. There were no significant differences in populations that came from SRE or PNS incubated soils: both populations increased in number over the incubation period. Nodule occupancy by both populations in growth pouches was similar to that previously encountered in field studies with these two soils. With the Waukegan soil, the serocluster 123 population dominated nodulation forming 69 and 62% of taproot nodules above and below the root tip mark, respectively. However, for the more alkaline Webster soil, serocluster 123 strains were much less competitive, producing only 9 and 13%, respectively, of the nodules formed above and below the root tip mark. In growth pouches, soil populations of bradyrhizobia from the Webster soil produced significantly more nodules than those from the Waukegan soil, but both strains and a pure culture of USDA 110 had a similar distribution of nodules.     

Increased metabolic potential of Rhizobium spp. is associated with bacterial competitiveness

Of 105 rhizobial isolates obtained from nodules of commonly cultivated legumes, we selected 19 strains on the basis of a high rate of symbiotic plant growth promotion. Individual strains within the species Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viciae, and Rhizobium etli displayed variation not only in plasmid sizes and numbers but also in the chromosomal 16S-23S internal transcribed spacer. The strains were tagged with gusA gene and their competitiveness was examined in relation to an indigenous population of rhizobia under greenhouse conditions. A group of 9 strains was thus isolated that were competitive in relation to native rhizobia in pot experiments. Nineteen selected competitive and uncompetitive strains were examined with respect to their ability to utilize various carbon and energy sources by means of commercial Biolog GN2 microplate test. The ability of the selected strains to metabolize a wide range of nutrients differed markedly and the competitive strains were able to utilize more carbon and energy sources than uncompetitive ones. A major difference concerned the utilization of amino and organic acids, which were metabolized by most of the competitive and only a few uncompetitive strains, whereas sugars and their derivatives were commonly utilized by both groups of strains. A statistically significant correlation between the ability to metabolize a broad range of substrates and nodulation competitiveness was found, indicating that metabolic properties may be an essential trait in determining the competitiveness of rhizobia.     

Host Plant Effects on Nodulation and Competitiveness of the Bradyrhizobium japonicum Serotype Strains Constituting Serocluster 123

Strains in Bradyrhizobium japonicum serocluster 123 are the major indigenous competitors for nodulation in a large portion of the soybean production area of the United States. Serocluster 123 is defined by the serotype strains USDA 123, USDA 127, and USDA 129. The objective of the work reported here was to evaluate the ability of two soybean genotypes, PI 377578 and PI 417566, to restrict the nodulation and reduce the competitiveness of serotype strains USDA 123, USDA 127, and USDA 129 in favor of the highly effective strain CB1809 and to determine how these soybean genotypes alter the competitive relationships among the three serotype strains in the serocluster. The soybean genotypes PI 377578 and PI 417566 along with the commonly grown cultivar Williams were planted in soil essentially free of soybean rhizobia and inoculated with single-strain treatments of USDA 123, USDA 127, USDA 129, or CB1809 and six dual-strain competition treatments of USDA 123, USDA 127, or USDA 129 versus CB1809, USDA 123 versus USDA 127, USDA 123 versus USDA 129, and USDA 127 versus USDA 129. PI 377578 severely reduced the nodulation and competitiveness of USDA 123 and USDA 127, while PI 417566 similarly affected the nodulation and competitiveness of USDA 129. Thus, the two soybean genotypes can reduce the nodulation and competitiveness of each of the three serocluster 123 serotype strains. Our results indicate that host control of restricted nodulation and reduced competitiveness is quite specific and effectively discriminates between B. japonicum strains which are serologically related.