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.     

Host-Controlled Restriction of Nodulation by Bradyrhizobium japonicum Strains in Serogroup 110

We previously reported the identification of a soybean plant introduction (PI) genotype, PI 417566, which restricts nodulation by Bradyrhizobium japonicum MN1-1c (USDA 430), strains in serogroup 129, and USDA 110 (P. B. Cregan, H. H. Keyser, and M. J. Sadowsky, Appl. Environ. Microbiol. 55:2532-2536, 1989, and Crop Sci. 29:307-312, 1989). In this study, we further characterized nodulation restriction by PI 417566. Twenty-four serogroup 110 isolates were tested for restricted nodulation on PI 417566. Of the 24 strains examined, 62.5% were restricted in nodulation by the PI genotype. The remainder of the serogroup 110 strains tested (37.5%), however, formed significant numbers of nodules on PI 417566, suggesting that host-controlled restriction of nodulation by members of serogroup 110 is strain dependent. Analysis of allelic variation at seven enzyme-encoding loci by multilocus enzyme electrophoresis indicated that the serogroup 110 isolates can be divided into two major groups. The majority of serogroup 110 isolates which nodulated PI 417566 belonged to the same multilocus enzyme electrophoresis group. B. japonicum USDA 110 and USDA 123 were used as coinoculants in competition-for-nodulation studies using PI 417566. Over 98% of the nodules formed on PI 417566 contained USDA 123, whereas less than 2% contained USDA 110. We also report the isolation of a Tn5 mutant of USDA 110 which has overcome nodulation restriction conditioned by PI 417566. This mutant, D4.2-5, contained a single Tn5 insertion and nodulated PI 417566 to an extent equal to that seen with the unrestricted strain USDA 123. The host range of D4.2-5 on soybean plants and other legumes was unchanged relative to that of USDA 110, except that the mutant nodulated Glycine max cv. Hill more efficiently. While strain USDA 110 has the ability to block nodulation by D4.2-5 on PI 417566, the nodulation-blocking phenomenon was not seen unless strain USDA 110 was inoculated at a 100-fold greater concentration than the mutant strain.     

Lysogeny in Bradyrhizobium japonicum and Its Effect on Soybean Nodulation

Rhizobiophage V, isolated from soil in the vicinity of soybean roots, was strongly lytic on Bradyrhizobium japonicum 123B (USDA 123) but only mildly lytic on strain L4-4, a chemically induced small-colony mutant of 123. Numerous bacteriophage-resistant variants were isolated from L4-4 infected with phage V; two were studied in detail and shown to be lysogenic. The two, L4-4 (V5) and L4-4 (V12), are the first reported examples of temperate-phage infection in B. japonicum. Phage V and its derivative phages V5 and V12 were closely related on the basis of common sensitivity to 0.01 M sodium citrate and phage V antiserum, phage immunity tests, and apparently identical morphology when examined by electron microscopy. However, the three phages differed in host range and in virulence. Lysogens L4-4 (V5) and L4-4 (V12) were immune to infection by phages V and V5 but not to infection by V12. Southern hybridization analysis confirmed the incorporation of phage V into the genomes of strains L4-4(V5) and L4-4(V12) and also demonstrated the incorporation of phage V into the genome of a phage V-resistant derivative of USDA 123 designated 123 (V2). None of the three lysogens, L4-4(V5), L4-4(V12), or 123B(V2), was able to nodulate soybean plants. However, Southern hybridization profile data indicated that phage V had not incorporated into any of the known B. japonicum nodulation genes.     

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.     

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.     

Nodulation of Glycine max by Six Bradyrhizobium japonicum Strains with Different Competitive Abilities

The root nodule locations of six Bradyrhizobium japonicum strains were examined to determine if there were any differences which might explain their varying competitiveness for nodule occupancy on Glycine max. When five strains were added to soybeans in plastic growth pouches in equal proportions with a reference strain (U.S. Department of Agriculture, strain 110), North Carolina strain 1028 and strain 110 were the most competitive for nodule occupancy, followed by U.S. Department of Agriculture strains 122, 76, and 31 and Brazil strain 587. Among all strains, nodule double occupancy was 17% at a high inoculum level (10⁷ CFU pouch⁻¹) and 2% at a low inoculum level (10⁴ CFU pouch⁻¹). The less competitive strains increased their nodule representation by an increase in the doubly occupied nodules at the high inoculum level. Among all strains, the number of taproot and lateral root nodules was inversely related at both the high and low inoculum levels (r = −0.62 and −0.69, respectively; P = 0.0001). This inverse relationship appeared to be a result of the plant host control of bacterial infection. Among each of the six strains, greater than 95% of the taproot nodules formed at the high inoculum density were located on 25% of the taproot length, the nodules centering on the position of the root tip at the time of inoculation. No differences among the six strains were observed in nodule initiation rates as measured by taproot nodule position. Taproot nodules were formed in the symbiosis before lateral root nodules. One of the poorly competitive strains (strain 76) occupied three times as many taproot nodules as lateral root nodules when competing with strain 110 (nodules were harvested from 4-week-old plants). Among these six wild-type strains of B. japonicum, competitive ability evidently is not related to nodule initiation rates.     

The Soybean Rj4 Allele Restricts Nodulation by Bradyrhizobium japonicum Serogroup 123 Strains

Of nine Bradyrhizobium japonicum serogroup 123 strains examined, 44% were found to be restricted for nodulation by cultivar Hill. Nodulation studies with soybean isoline BARC-2 confirmed that the soybean Rj4 allele restricts nodulation by the same serogroup 123 isolates. Immunological analyses indicated that B. japonicum strains in serogroups 123 and 31 share at least one surface somatic antigen.     

Cloning and Mapping of a Novel Nodulation Region from Bradyrhizobium japonicum by Genetic Complementation of a Deletion Mutant

The phenotypes of a set of Bradyrhizobium japonicum 110 mutants with large deletions in the region of symbiotic gene cluster I were tested. The majority of the mutants showed a delayed nodulation on soybean and, by mixed-infection experiments, were found to be strongly reduced in their competitiveness. Phenotypic comparison of mutants with different deletion endpoints allowed a preliminary localization of two genomic regions, called nod-1 and nod-2, which were required for normal nodulation on soybean. Loss of nod-1 was found to result in a Nod⁻ phenotype on cowpea, mung bean, and siratro. A recombinant cosmid was identified which fully restored nodulation ability of a mutant lacking nod-1. Using Tn5-containing derivatives and subclones of this cosmid for complementation, we delimited the nod-1 region to a DNA segment of 3.1 to 3.5 kilobase pairs.     

Restriction Endonuclease and nif Homology Patterns of Bradyrhizobium japonicum USDA 110 Derivatives With and Without Nitrogen Fixation Competence

DNAs from Bradyrhizobium japonicum USDA 110 derivatives that differ in nitrogen-fixing ability produced similar electrophoretic patterns with five different restriction enzymes. Our data support the hypothesis of common ancestry for these derivatives. Derivatives I-110 and L1-110 differed as much as 100-fold in acetylene reduction activity when they were tested with several soybean cultivars in both greenhouse and field experiments. While possessing nodulating ability, derivative L1-110 is deficient in symbiotic nitrogen-fixing ability, whereas derivative I-110 is symbiotically competent. Hybridization of nifDK and nifH probes from B. japonicum to Southern blots of restricted DNAs from strain USDA 110 derivatives produced similar patterns. This finding indicates similar structural gene organization for both derivative I-110 and derivative L1-110 and implies that the difference in symbiotic nitrogen fixation is probably not due to structural gene rearrangements. However, our hybridization data do not rule out the possibility of differences in expression of structural nif genes or alterations in the structure or expression of other genes required for symbiotic nitrogen fixation.     

Functional Role of Bradyrhizobium japonicum Trehalose Biosynthesis and Metabolism Genes during Physiological Stress and Nodulation

Trehalose, a disaccharide accumulated by many microorganisms, acts as a protectant during periods of physiological stress, such as salinity and desiccation. Previous studies reported that the trehalose biosynthetic genes (otsA, treS, and treY) in Bradyrhizobium japonicum were induced by salinity and desiccation stresses. Functional mutational analyses indicated that disruption of otsA decreased trehalose accumulation in cells and that an otsA treY double mutant accumulated an extremely low level of trehalose. In contrast, trehalose accumulated to a greater extent in a treS mutant, and maltose levels decreased relative to that seen with the wild-type strain. Mutant strains lacking the OtsA pathway, including the single, double, and triple ΔotsA, ΔotsA ΔtreS and ΔotsA ΔtreY, and ΔotsA ΔtreS ΔtreY mutants, were inhibited for growth on 60 mM NaCl. While mutants lacking functional OtsAB and TreYZ pathways failed to grow on complex medium containing 60 mM NaCl, there was no difference in the viability of the double mutant strain when cells were grown under conditions of desiccation stress. In contrast, mutants lacking a functional TreS pathway were less tolerant of desiccation stress than the wild-type strain. Soybean plants inoculated with mutants lacking the OtsAB and TreYZ pathways produced fewer mature nodules and a greater number of immature nodules relative to those produced by the wild-type strain. Taken together, results of these studies indicate that stress-induced trehalose biosynthesis in B. japonicum is due mainly to the OtsAB pathway and that the TreS pathway is likely involved in the degradation of trehalose to maltose. Trehalose accumulation in B. japonicum enhances survival under conditions of salinity stress and plays a role in the development of symbiotic nitrogen-fixing root nodules on soybean plants.Rhizobia induce the formation of nodules on the roots of legume plants, in which atmospheric nitrogen is fixed and supplied to the host plant, thereby enhancing growth under nitrogen-limiting conditions. The symbiotic interaction between rhizobia and their cognate leguminous plants is important for agricultural productivity, especially in less developed countries. However, physiological stresses, such as desiccation and salinity, negatively affect these symbiotic interactions by limiting nitrogen fixation (44). The osmotic environment within the rhizosphere may affect root colonization, infection thread development, nodule development, and the formation of effective N₂-fixing nodules (21). Moreover, when legume seeds are inoculated with appropriate rhizobial strains prior to planting in the field, the vast majority of nodules produced are often not formed by the inoculant bacteria but rather by indigenous strains in the soil (36). This is in part due to the death of inoculant strains from rapid seed coat-mediated desiccation. Therefore, improvement of the survival of rhizobia under conditions of physiological stresses may promote biological nitrogen fixation and enhance plant growth.Rhizobia synthesize and accumulate compatible solutes, including trehalose, in response to desiccation and solute-mediated physiological stresses (5, 21, 42). Trehalose, a nonreducing disaccharide with an α,α-1,1 linkage between the two glucose molecules, has been shown to protect cell membranes and proteins from stress-induced inactivation and denaturation (8, 23, 24). The relationship between trehalose accumulation and symbiotic phenotype is dependent on rhizobial species and host genotype. Suarez et al. (39) reported an increase in root nodule number and nitrogen fixation by Phaseolus vulgaris inoculated with a trehalose-6-phosphate synthase-overexpressing strain of Rhizobium etli. In contrast, trehalose accumulation in Rhizobium leguminosarum and Sinorhizobium meliloti cells did not result in an increase in nitrogen-fixing nodules but led to enhancement of competitiveness on clover and on certain alfalfa genotypes, respectively (1, 16, 20).Four trehalose biosynthetic pathways, mediated by OtsAB, TreS, TreYZ, and TreT, have been reported thus far for prokaryotes (8, 25). The OtsAB pathway results in the condensation of glucose-6-phosphate with UDP-glucose by trehalose-6-phosphate synthase (OtsA) to form trehalose-6-phosphate. Trehalose is subsequently formed from trehalose-6-phosphate by the action of trehalose-6-phosphate phosphatase (OtsB). The TreS pathway involves a reversible transglycosylation reaction in which trehalose synthase (TreS) converts maltose, a disaccharide with α,α-1,4 linkage between the two glucose molecules, to trehalose. The third pathway, mediated by TreYZ, involves the conversion of maltodextrins into trehalose. The terminal α-1,1-glycosylic bond at the end of the maltodextrin polymer is hydrolyzed by maltooligosyltrehalose synthase (TreY), and trehalose is subsequently released from the end of the polymer via hydrolysis by maltooligosyltrehalose trehalohydrolase (TreZ). More recently, a trehalose glycosyltransferring synthase (TreT) was shown to catalyze the reversible formation of trehalose from ADP-glucose and glucose (25).In addition to biosynthesis, Gram-negative bacteria have also been reported to have trehalose degradation systems. Typically, trehalose is hydrolyzed into two glucose moieties by periplasmic and cytoplasmic trehalase enzymes, TreA and TreF, respectively (13, 15). However, Sinorhizobium meliloti also uses ThuA and ThuB for trehalose utilization (16).Bradyrhizobium japonicum, the root nodule symbiont of soybeans, accumulates trehalose in cultured cells and bacteroids (34, 35). Biochemical studies indicated that B. japonicum has three independent trehalose biosynthetic pathways involving trehalose synthase (TreS), maltooligosyltrehalose synthase (TreYZ), and trehalose-6-phosphate synthetase (OtsAB) (38). Sequence analysis of the B. japonicum USDA 110 genome identified the genes that encode these biosynthetic pathways: otsAB (bll0322 to bll0323), two homologs of treS (blr6767 and bll0902), and treYZ (blr6770 to blr6771), but not treT (17). Orthologous gene sequences to the trehalose degradation genes treA, treF, and thuAB have not been found in the genome of B. japonicum USDA 110. Cytryn et al. (6) reported that expression of otsA, treS (blr6767), and treY genes were highly induced by desiccation stress. Moreover, the concentrations of these three enzymes increased when B. japonicum was cultured in the presence of salt (38). Trehalose concentration in B. japonicum has been reported to increase due to desiccation stress (6), and this sugar is purported to act as an osmoprotectant. The addition of exogenously supplied trehalose has been reported to enhance the survival of B. japonicum in response to desiccation and salinity stresses (9, 37). Despite this information, little is known about how the various trehalose biosynthetic pathways modulate stress tolerance and symbiotic performance in B. japonicum.The purpose of this study was to examine the functional role(s) of the B. japonicum trehalose biosynthetic pathways on stress survival by constructing single, double, and triple mutants and by producing strains that overexpress the trehalose biosynthesis enzymes. Here we report on the relationship between trehalose accumulation and physiological responses to salinity and desiccation stresses in mutant and overexpression strains and that mutations in the trehalose biosynthesis pathways altered the symbiotic performance of B. japonicum USDA 110 on soybeans. Results of these studies indicate that trehalose accumulation in B. japonicum plays a prominent role in the saprophytic and symbiotic competence of this agriculturally important soil bacterium.     

Enhanced Nodulation and Nitrogen Fixation in the Abscisic Acid Low-Sensitive Mutant enhanced nitrogen fixation1 of Lotus japonicus

The phytohormone abscisic acid (ABA) is known to be a negative regulator of legume root nodule formation. By screening Lotus japonicus seedlings for survival on an agar medium containing 70 μm ABA, we obtained mutants that not only showed increased root nodule number but also enhanced nitrogen fixation. The mutant was designated enhanced nitrogen fixation1 (enf1) and was confirmed to be monogenic and incompletely dominant. The low sensitivity to ABA phenotype was thought to result from either a decrease in the concentration of the plant''s endogenous ABA or from a disruption in ABA signaling. We determined that the endogenous ABA concentration of enf1 was lower than that of wild-type seedlings, and furthermore, when wild-type plants were treated with abamine, a specific inhibitor of 9-cis-epoxycarotenoid dioxygenase, which results in reduced ABA content, the nitrogen fixation activity of abamine-treated plants was elevated to the same levels as enf1. We also determined that production of nitric oxide in enf1 nodules was decreased. We conclude that endogenous ABA concentration not only regulates nodulation but also nitrogen fixation activity by decreasing nitric oxide production in nodules.Many legumes establish nitrogen-fixing root nodules following reciprocal signal exchange between the plant and rhizobia (Hayashi et al., 2000; Hirsch et al., 2003). The host plant produces chemical compounds, frequently flavonoids, which induce rhizobial nod genes, whose products are involved in the synthesis and secretion of Nod factor. Perception of this chitolipooligosaccharide by the host plant results in the triggering of a signal transduction cascade that leads to root hair deformation and curling and subsequent cortical cell divisions, which establish the nodule primordium. The rhizobia enter the curled root hair cell and nodule primordial cells through an infection thread. Eventually, the rhizobia are released into nodule cells, enclosed within a membrane, and differentiate into nitrogen-fixing bacteroids that reduce atmospheric nitrogen into ammonia. In return, the host plant supplies photosynthetic products, to be used as carbon sources, to the rhizobia (Zuanazzi et al., 1998; Hayashi et al., 2000).The host plant is known to be important for regulating the number of nodules established on its roots. For example, hypernodulating mutants such as nitrate-tolerant symbiotic1 (nts1; Glycine max), hypernodulation aberrant root formation1 (har1; Lotus japonicus), super numeric nodules (sunn; Medicago truncatula), and symbiosis29 (sym29; Pisum sativum) disrupt the balance between supply and demand by developing excessive root nodules (Oka-Kira and Kawaguchi, 2006). Grafting experiments demonstrated that leaf tissue is a principal source of the systemic signals contributing to the autoregulation of nodulation (Pierce and Bauer, 1983; Kosslak and Bohlool, 1984; Krusell et al., 2002; Nishimura et al., 2002b; van Brussel et al., 2002; Searle et al., 2003; Schnabel et al., 2005). The Nts1, Har1, Sunn, and Sym29 genes encode a receptor-like kinase similar to CLAVATA1, which regulates meristem cell number and differentiation (Krusell et al., 2002; Nishimura et al., 2002a; Searle et al., 2003; Schnabel et al., 2005).Phytohormones are also known to regulate nodulation (Hirsch and Fang, 1994). For example, ethylene is a well-known negative regulator of nodulation, influencing the earliest stages from the perception of Nod factor to the growth of infection threads (Nukui et al., 2000; Oldroyd et al., 2001; Ma et al., 2003). The ethylene-insensitive mutant sickle1 (skl1) of M. truncatula has a hypernodulating phenotype (Penmetsa and Cook, 1997). Skl1 is homologous to Ethylene insensitive2 of Arabidopsis (Arabidopsis thaliana), which is part of the ethylene-signaling pathway (Alonso et al., 1999; Penmetsa et al., 2008). In contrast, cytokinin is a positive regulator of nodulation. The cytokinin-insensitive mutant hyperinfected1 (loss of function) of L. japonicus and the spontaneous nodule formation2 (gain of function) mutants of M. truncatula provide genetic evidence demonstrating that cytokinin plays a critical role in the activation of nodule primordia (Gonzalez-Rizzo et al., 2006; Murray et al., 2007; Tirichine et al., 2007).Abscisic acid (ABA), added at concentrations that do not affect plant growth, also negatively regulates nodulation in some legumes (Phillips, 1971; Cho and Harper, 1993; Bano et al., 2002; Bano and Harper, 2002; Suzuki et al., 2004; Nakatsukasa-Akune et al., 2005; Liang et al., 2007). Recently, M. truncatula overexpressing abscisic acid insensitive1-1, a gene that encodes a mutated protein phosphatase of the type IIC class derived from Arabidopsis and that suppresses the ABA-signaling pathway (Leung et al., 1994; Hagenbeek et al., 2000; Gampala et al., 2001; Wu et al., 2003), was shown to exhibit ABA insensitivity as well as a hypernodulating phenotype (Ding et al., 2008).In this study, we isolated a L. japonicus (Miyakojima MG20) mutant that showed an increased root nodule phenotype and proceeded to carry out its characterization. This mutant, named enhanced nitrogen fixation1 (enf1), exhibits enhanced symbiotic nitrogen fixation activity. Most legume nitrogen fixation activity mutants, such as ineffective greenish nodules1 (ign1), stationary endosymbiont nodule1, and symbiotic sulfate transporter1 (sst1), are Fix⁻ (Suganuma et al., 2003; Krusell et al., 2005; Kumagai et al., 2007).     

Cytokinin Production by Bradyrhizobium japonicum

Although there is considerable circumstantial evidence for the involvement of cytokinins in legume nodulation, the cytokinins produced by rhizobia have not been well characterized. Bradyrhizobium japonicum 61A68, a bacterium which nodulates soybean (Glycine max [L.] Merr.), was grown in defined medium. Cytokinins were purified from the culture medium by Amberlite XAD-2 chromatography and fractionated by column chromatography on Sephadex LH-20 in 35% ethanol. Pooled fractions from the Sephadex column were analyzed for cytokinin activity with the tobacco callus bioassay. Cytokinin activity was observed in fractions corresponding to the elution volumes of zeatin, ribosylzeatin, and methylthiozeatin. No activity corresponding to the elution volumes of isopentenyladenine or its riboside was found. Total cytokinin activity in the B. japonicum culture filtrate was equivalent to approximately 1 microgram of kinetin per liter. Transfer RNA was isolated from B. japonicum cells by phenol extraction, followed by potassium acetate extraction, cetyltrimethylammonium bromide precipitation, and DEAE cellulose chromatography. Transfer RNA was enzymically hydrolyzed to nucleosides. High performance liquid chromatographic analysis of cytokinin nucleosides showed peaks corresponding to the retention times of trans-ribosylzeatin, methylthioribosylzeatin, isopentenyladenosine, and methylthioisopentenyladenosine. Analysis of the tRNA hydrolysate by Sephadex LH-20 chromatography and tobacco bioassay showed cytokinin activity in fractions corresponding to ribosylzeatin, methylthioribosylzeatin, and isopentenyladenosine. The presence of the trans isomer of ribosylzeatin was also determined by enzyme immunoassay.     

Genetic Diversity in Bradyrhizobium japonicum Serogroup 123 and Its Relation to Genotype-Specific Nodulation of Soybean

The genetic diversity among 20 field isolates of Bradyrhizobium japonicum serogroup 123 was examined by using restriction endonuclease digestions, one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cell proteins, Southern hybridization analysis of nif and nod genes, and intrinsic antibiotic resistance profiles. All of the isolates were previously separated into three broad nodulation classes (low, medium, and high) based on their ability to form symbioses with specific soybean genotypes. Results of our studies indicate that there is a relationship between these three genotype-specific nodulation classes and groupings that have been made based on genomic DNA digestion patterns, sodium dodecyl sulfate-protein profiles, and Southern hybridizations to a nifHD gene probe. Intrinsic antibiotic resistance profiles and nodAB gene hybridizations were not useful in determining interrelationships between isolates and nodulation classes. Southern hybridizations revealed that two of the isolates had reiterated nod genes; however, there was no correlation between the presence of extra nodAB genes and the nodulation classes or symbiotic performance on permissive soybean genotypes. Hybridizations with the nif gene probe indicated that there is a relationship among serogroup, nodulation class, and the physical organization of the genome.     

Enhanced competitiveness of a Bradyrhizobium japonicum mutant strain improved for nodulation and nitrogen fixation

Bradyrhizobium japonicum strain TA-11NOD⁺, with altered indole biosynthesis, exhibited enhanced nodulation and nitrogen fixation on soybean in previous greenhouse studies. In this study, field experiments were conducted at Upper Marlboro, Maryland, in the summers of 1988 and 1993. In 1988, the site used was essentially free of soybean-nodulating bacteria and seed yield in plots inoculated with either I-110ARS or TA-11NOD⁺ was significantly higher by 12 or 20%, respectively, than that of the uninoculated controls. The 1993 site had an indigenous soil population (about 10⁴ cells g^-1) of symbiotically ineffective soybean-nodulating bacteria. Nevertheless, six-week-old Morgan soybean plants inoculated with strain TA-11NOD⁺ had 44% more nodules and exhibited 50% more nitrogen fixation by acetylene reduction when compared with plants that received the parental strain I-110ARS. Nodule occupancy, as determined using genetic markers for rifampicin and streptomycin resistance, was significantly higher for strain TA-11NOD⁺ than for strain I-110ARS. Overall, for the two years and the two soybean genotypes, the yield obtained with TA-11NOD⁺ was 6% higher than that obtained with I-110ARS. Competition experiments were conducted in the greenhouse and strain TA-11NOD⁺ was significantly more competitive than strain I-110ARS in competition with strains USDA 6 or USDA 438.     

Degradation of catechin by Bradyrhizobium japonicum

Rhizobia utilize phenolic substances as sole carbonsource. Bradyrhizobium japonicum utilizescatechin, a unit of condensed tannin as carbonsource. To establish the degradative pathway ofcatechin, the products of catechin degradation wereisolated by paper chromatography and TLC andidentified by HPLC, UV, IR and NMR spectra. B.japonicum cleaves catechin through catechinoxygenase. Phloroglucinolcarboxylic acid andprotocatechuic acid were identified as the initialproducts of degradation. Phloroglucinolcarboxylicacid is further decarboxylated to phloroglucinolwhich is dehydroxylated to resorcinol. Resorcinolis hydroxylated to hydroxyquinol. Protocatechuicacid and hydroxyquinol undergo intradiol cleavagethrough protocatechuate 3,4-dioxygenase andhydroxyquinol 1,2-dioxygenase to form-carboxy cis, cis-muconic acidand maleylacetate respectively. The enzymes ofcatechin degradative pathway are inducible. Estimation of all the enzymes involved in thecatabolism of catechin reveals the existence of acatechin degradative pathway in B. japonicum.     

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.     

Nodulation and Nitrogen Fixation Efficacy of Rhizobium fredii with Phaseolus vulgaris Genotypes

Phaseolus plant introduction (PI) genotypes (consisting of 684 P. vulgaris, 26 P. acutifolius, 39 P. lunatus, and 5 P. coccineus accessions) were evaluated for their ability to form effective symbioses with strains of six slow-growing (Bradyrhizobium) and four fast-growing (Rhizobium fredii) soybean rhizobia. Of the 684 P. vulgaris genotypes examined, three PIs were found to form effective nitrogen-fixing symbioses with the R. fredii strains. While none of the Bradyrhizobium strains nodulated any of the genotypes tested, some produced large numbers of undifferentiated root proliferations (hypertrophies). A symbiotic plasmid-cured R. fredii strain failed to nodulate the P. vulgaris PIs and cultivars, suggesting that P. vulgaris host range genes are Sym plasmid borne in the fast-growing soybean rhizobia.