Efficiency of Nodule Initiation and Autoregulatory Responses in a Supernodulating Soybean Mutant

We compared the formation of nodules on the primary roots of a soybean cultivar (Glycine max (L.) Merr. cv. Bragg) and a supernodulating mutant derivative, nts382. Inoculation with Bradyrhizobium japonicum USDA 110 at different times after seed imbibition showed that the roots acquired full susceptibility to infection only between 3 and 4 days postgermination. When the plants were inoculated with serial dilutions of a bacterial suspension, the number of nodules formed in the initially susceptible region of the roots was linearly dependent on the logarithm of the inoculum dose until an optimum dose was reached. At least 10-fold-lower doses were required to induce half-maximal nodulation responses on nts382 than on the wild type. However, at optimal doses, about six times as many nodules formed in the initially susceptible region of the roots in nts382. Since there was no appreciable difference in the apparent rates of nodule emergence, the increased efficiency of nodule initiation in the supernodulating mutant could have resulted from a lower threshold of response to bacterial symbiotic signals. Two inoculations (24 h apart) of G. max cv. Bragg revealed that there was a host-mediated regulatory response that suppressed nodulation in younger portions of the primary roots, as reported previously for other soybean cultivar-Bradyrhizobium combinations. Similar experiments with nts382 revealed a comparable suppressive response, but this response was not as pronounced as it was in the wild type. This and other results suggest that there are additional control mechanisms for nodulation that are different from the systemic autoregulatory control of nodulation altered in supernodulating mutants.     

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.     

Role of Motility and Chemotaxis in Efficiency of Nodulation by Rhizobium meliloti

Spontaneous mutants of Rhizobium meliloti L5-30 defective in motility or chemotaxis were isolated and compared against the parent with respect to symbiotic competence. Each of the mutants was able to generate normal nodules on the host plant alfalfa (Medicago sativa), but had slightly delayed nodule formation, diminished nodulation in the initially susceptible region of the host root, and relatively low representation in nodules following co-inoculation with equal numbers of the parent. When inoculated in growth pouches with increasing dosages of the parental strain, the number of nodules formed in the initially susceptible region of the root increased sigmoidally, with an optimum concentration of about 10⁵ to 10⁶ bacteria/plant. The dose-response behavior of the nonmotile and nonchemotactic mutants was similar, but they required 10- to 30-fold higher concentrations of bacteria to generate the same number of nodules. The distribution frequencies of nodules at different positions along the primary root were very similar for the mutants and parent, indicating that reduced nodulation by the mutants in dose-response experiments probably reflects reduced efficiency of nodule initiation rather than developmentally delayed nodule initiation. The number of bacteria that firmly adsorbed to the host root surface during several hours of incubation was 5- to 20-fold greater for the parent than the mutants. The mutants were also somewhat less effective than their parent as competitors in root adsorption assays. It appears that motility and chemotaxis are quantitatively important traits that facilitate the initial contact and adsorption of symbiotic rhizobia to the host root surface, increase the efficiency of nodule initiation, and increase the rate of infection development.     

Host recognition in the Rhizobium-soybean symbiosis: detection of a protein factor in soybean root exudate which is involved in the nodulation process

The mechanism of host-symbiont recognition in the soybean-Rhizobium symbiosis was investigated utilizing mutants of R. japonicum defective in nodulation. Soybeans were grown in clear plastic growth pouches allowing the identification of the area on the root most susceptible to Rhizobium nodulation; the area between the root tip (RT) and smallest emergent root hair (SERH). The location of nodules in relation to this developing zone is an indication of the rate of nodule initiation. Nodules were scored as to the distance from the RT mark made at the time of inoculation. Seventy-eight per cent of the plants nodulate above the RT mark when inoculated with the wild type R. japonicum strain 3I1b110 with the average distance of the uppermost nodule being approximately 2 millimeters above the RT mark. These data indicate that the wild type strain initiates nodulation rapidly within the RT-SERH zone following inoculation. However, inoculation with the slow-to-nodulate mutant strain HS111 resulted in 100% of the plants nodulating only below the RT mark with the average distance of the uppermost nodule being approximately 56 millimeters below the RT mark. Thus, mutant strain HS111 is defective in the ability to rapidly initiate infection leading to nodulation within the RT-SERH zone. The location of the nodules suggest that stain HS111 must `adapt' to the root environment before nodulation can occur. To test this, strain HS111 was incubated in soybean root exudate prior to inoculation. In this case, 68% of the plants nodulated above the RT mark with the average distance of the uppermost nodule being approximately 1 millimeter below the RT mark. Experiments indicated that the change in nodule initiation by strain HS111 brought about by incubation in soybean root exudate was due to a phenotypic, rather than a genotypic change. The half-time of root exudate incubation for strain HS111 necessary for optimal nodulation enhancement was less than 6 hours. Heat sensitivity and trypsin sensitivity of the nodulation enhancement factor(s) in soybean root exudate indicate a protein was involved in the reversal of the delay in nodulation by mutant strain HS111.     

Carbohydrate binding activities of Bradyrhizobium japonicum III. Lectin expression, bacterial binding, and nodulation efficiency

In previous studies, evidence that the Bradyrhizobium japonicum lectin, designated BJ38, mediated the observed carbohydrate-specific binding activities of the bacteria, including the saccharide-specific adhesion to soybean root cells was presented. In the present study, it is found that both B. japonicum, as well as the purified BJ38, bind predominantly to young emergent root hairs of soybean roots and, to a much lesser extent, to the root cap, mature root hairs, epicotyl or hypocotyl regions. Thus, the region of preferential binding for both the bacteria and the isolated lectin coincide with the region of the soybean root most susceptible to B. japonicum infection. The importance of bacterial binding for the nodulation process was studied by comparing the nodulation efficiency of binding-deficient mutants N4 and N6 to the wild-type. These mutants had been shown to be defective in carbohydrate recognition, as represented by their diminished ability to bind to soybean roots. BJ38 was immunolocalized to one pole of the cell surface of wild-type B. japonicum, but no surface labeling could be detected on either mutant. Moreover, both N4 and N6 showed a substantial decrease in nodulation activity, relative to the wild-type. These results provide additional evidence that the carbohydrate binding activity of B. japonicum, most probably mediated by BJ38, may play an important role(s) in the initial phases of the infection process.     

Enhanced nodule initiation on alfalfa by wild-typeRhizobium meliloti co-inoculated withnod gene mutants and other bacteria

Nodule formation on alfalfa (Medicago sativa L.) roots was determined at different inoculum dosages for wild-typeRhizobium meliloti strain RCR2011 and for various mutant derivatives with altered nodulation behavior. The number of nodules formed on the whole length of the primary roots was essentially constant regardless of initial inoculum dosage or subsequent bacterial multiplication, indicative of homeostatic regulation of total nodule number. In contrast, the number of nodules formed in just the initially susceptible region of these roots was sigmoidally dependent on the number of wild-type bacteria added, increasing rapidly at dosages above 5·10³ bacteria/plant. This behavior indicates the possible existence of a threshold barrier to nodule initiation in the host which the bacteria must overcome. When low dosages of the parent (10³ cells/plant) were co-inoculated with 10⁶ cells/plant of mutants lacking functionalnodA, nodC, nodE, nodF ornodH genes, nodule initiation was increased 10- to 30-fold. Analysis of nodule occupancy indicated that these mutants were able to help the parent (wild-type) strain initiate nodules without themselves occupying the nodules. Co-inoculation withR. trifolii orAgrobacterium tumefaciens cured of its Ti plasmid also markedly stimulated nodule initiation by theR. meliloti parent strain. Introduction of a segment of the symbiotic megaplasmid fromR. meliloti intoA. tumefaciens abolished this stimulation.Bradyrhizobium japonicum and a chromosomal Tn5 nod^- mutant ofR. meliloti did not significantly stimulate nodule initiation when co-inoculated with wild-typeR. meliloti. These results indicate that certainnod gene mutants and members of theRhizobiaceae may produce extracellular signals that supplement the ability of wild-typeR. meliloti cells to induce crucial responses in the host.Abbreviations EH emergent root hairs - kb kilobase - RDU relative distance unit - RT root tip This is journal article No. 188-87 of the Ohio Agricultural Research and Development Center     

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).     

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.     

Effect of legume seed exudates on the formation of Rhizobium-legume symbiosis

The effect of exudates from germinating lupine and soybean seeds on the development of legumerhizobia symbiosis in the same plants was studied. Treatment with the exudates increased the nodulation activity of Bradyrhizobium sp. (Lupinus) and slowed down the formation of nodules by Bradyrhizobium japonicum 634b. The number of nodules produced by B. japonicum 631 on soybean roots increased when the strain was treated with soybean exudate at a lower concentration. The exudates differently affected nodulation on the primary and secondary roots of the host plant. The formation of symbiosis by B. japonicum 631 incubated with legume seed exudates increased the weight of the green parts of plants at the bud stage.     

Adsorption of slow- and fast-growing rhizobia to soybean and cowpea roots

Roots of soybean (Glycine max [L.] Merr. cv Hardee) and cowpea (Vigna unguiculata [L.] Walp. cv Pink Eye Purple Hull) were immersed in suspensions containing 10⁴Rhizobium cells per milliliter of a nitrogen-free solution. After 30 to 120 minutes the roots were rinsed, and the distal 2-centimeter segments excised and homogenized. Portions of the homogenates then were plated on a yeast-extract mannitol medium for bacterial cell counts. The adsorption capacities of four slow-growing rhizobia and a fast-growing R. meliloti strain varied considerably. Adsorption was independent of plant species and of the abilities of the Rhizobium strains to infect and nodulate. R. lupini 96B9 had the greatest adsorption capacity, and Rhizobium sp. 3G4b16 the least. Rhizobium sp. 229, R. japonicum 138, and R. meliloti 102F51 were intermediate, except on cowpea, where the adsorption of strain 102F51 was similar to that of strain 3G4b16. The initial adsorption rates of bacteria cultured in synthetic media and in the presence of soybean roots were about the same. Addition of soybean lectin to the bacterial inoculum failed to influence initial adsorption rates. Both treatments, however, reduced the numbers of bacteria that bound after incubation with roots for 120 minutes. The relationship between the logarithm of the number of strain 138 cells bound per soybean root segment and the logarithm of the density of bacteria in the inoculum was linear over five orders of magnitude. Binding of strain 138 to soybean roots was greatest at room temperature (27°C) and substantially attenuated at both 4 and 37°C. Although R. lupini 96B9 strongly rejected a model hydrophobic plastic surface, there were no simple correlations between bacterial binding to model hydrophobic and hydrophilic plastic surfaces and bacterial adsorption to roots.     

Host-specificity mutants of Rhizobium meliloti have additive effects in situ on initiation of alfalfa nodules

Pairs of Rhizobium meliloti nod mutants were co-inoculated onto alfalfa (Medicago saliva L.) roots to determine whether one nod mutant could correct, in situ, for defects in nodule initiation of another nod mutant. None of the Tn5 or nod deletion mutants were able to help each other form nodules when co-inoculated together in the absence of the wild-type. However, as previously observed, individual nod mutants significantly increased nodule initiation by low dosages of co-inoculated wild-type cells. Thus, nod mutants do produce certain signal substances or other factors which overcome limits to nodule initiation by the wild-type. When pairs of nod mutants were co-inoculated together with the wild-type, the stimulation of nodulation provided by individual nodABC mutants was not additive. However, clearly additive or synergistic stimulation was observed between pairs of mutants with a defective host-specificity gene (nodE, nodF, or nodH). Each pair of host-specificity mutants stimulated first nodule formation to nearly the maximum levels obtainable with high dosages of the wild-type. Mutant bacteria were recovered from only about 10% of these nodules, whereas the co-inoculated wild-type was present in all these nodules and substantially outnumbered mutant bacteria in nodules occupied by both. Thus, these mutant co-inoculants appeared to help their parent in situ even though they could not help each other. Sterile culture filtrates from wild-type cells stimulated nodule initiation by low dosages of the wild-type, but only when a host-specificity mutant was also present. The results from our studies seem consistent with the possibility that pairs of host-specificity mutants are able to help the wild-type initiate nodule formation by sustained production of complementary signals required for induction of symbiotic host responses.     

Rhizosphere Response as a Factor in Competition Among Three Serogroups of Indigenous Rhizobium japonicum for Nodulation of Field-Grown Soybeans

Rhizosphere response was studied as a factor in competition among indigenous Rhizobium japonicum serogroups for the nodulation of soybeans under field conditions. R. japonicum serogroups 110, 123, and 138 were found to coexist in a Waukegan field soil where they were determined to be the major nodulating rhizobia in soybean nodules. Competitive relationships among the three serogroups in that soil and in rhizospheres were examined during two growing seasons with several host cultivars with and without inoculation and with a nonlegume. Enumeration of each of the three competitors was carried out on inner rhizosphere and nonrhizosphere soil by immunofluorescence with serogroup-specific fluorescent antibodies. Rhizobia present in early- and late-season nodules were identified by fluorescent antibody analysis. Populations of each serogroup increased gradually in host rhizospheres, not exceeding 10⁶/g of rhizosphere soil during the first few weeks after planting, whereas numbers in fallow soil remained at initial levels (10⁴ to 10⁵/g). The rhizosphere effects were minor in host plants during this period of nodule initiation and were about the same for all three serogroups. Although serogroup 123 gave no evidence of dominance in early host rhizospheres, it clearly dominated in nodule composition, occupying 60 to 100% of the nodules. High densities of all three serogroups were observed in host rhizospheres during flowering. Rhizosphere populations, especially of serogroup 123, were still high during pod fill and seed maturation. The rhizosphere responses of the R. japonicum serogroups were much greater with the soybean cultivars than with oats, but even in host rhizospheres the R. japonicum populations were greatly outnumbered by other bacteria. The success of serogroup 123 in achieving nodulation does not appear to be due to superior colonization of the host rhizosphere.     

Influence ofGlycine max nodulation on the persistence in soil of a genetically markedBradyrhizobium japonicum strain

Since competition with indigenous strains limits nodule occupancy by bacteria applied to seeds, the ecology of Bradyrhizobium inoculum strains used for soybean is of concern. A genetically marked strain,B. japonicum I-110 ARS, was directly enumerated from soil on selective medium. A clear long-term positive influence of even limitedGlycine max nodulation was shown by comparisons of population densities obtained with or without plant removal prior to nodule senescence in the first year and with an incompatible as well as a compatible soybean variety after 5 years.     

Factors affecting co-inoculation with antibiotic-producing bacteria to enhance rhizobial colonization and nodulation

Co-inoculation with antibiotic-producing bacteria and rhizobia resistant to those antibiotics has been proposed as a means of promoting colonization and nodulation of legumes by root-nodule bacteria. A study was conducted to establish some of the factors affecting co-inoculation with antibiotic-producing strains of Bacillus and Streptomyces griseus. The stimulation of Rhizobium meliloti and yield and N uptake by alfalfa was enhanced with increasing inoculum size of Bacillus sp. S. griseus and chitin added to soil increased nodulation of soybeans by Bradyrhizobium japonicum and increased nodulation, yield, and number of pods on a second crop grown in the same soil. Bacillus sp. persisted in soil in sufficient numbers for at least 51 days to increase colonization of soybean roots by B. japonicum. The populations of S. griseus, Bacillus sp., and antibiotic-resistant isolates of R. meliloti and B. japonicum fell after their addition to seeds. Nevertheless, a benefical effect by the antibiotic-producing bacteria was evident on R. meliloti colonization of the rhizosphere, nodulation, and yield of alfalfa grown from seeds stored 94 days and on B. japonicum colonization, nodule number, yield, and seed weight of soybeans grown from seeds stored 90 days. Because non-antibiotic-producing derivatives of Bacillus sp. and S. griseus did not promote colonization or nodulation of alfalfa roots by R. meliloti, the benefit of this co-inoculation is a result of antibiotic formation.     

When Does the Self-Regulatory Response Elicited in Soybean Root after Inoculation Occur?

The inoculation of soybean (Glycine max L.) roots with Bradyrhizobium japonicum produces a regulatory response that inhibits nodulation in the younger regions of the roots. By exposing the soybean roots to live homologous bacteria for only a short period of time, the question of whether or not early interactions of rhizobia with root cells, prior to infection, elicit this regulatory response has been explored. B. japonicum cells mixed with infective bacteriophages were applied to the roots and then 6 or 24 hours later roots were again inoculated with phage-resistant rhizobia. Mixing of the rhizobia and bacteriophages caused bacterial lysis in 6 to 8 hours and allowed the bacteria to act as live symbionts on the root for only a few hours. However, the interaction of live homologous bacteria with the soybean roots for a few hours did not cause inhibition of nodulation in the younger regions of the roots. Results of these experiments indicate that the self-regulatory response in soybean is not rapidly produced by the early, pre-infection, interactions between rhizobia and the root cells.     

Feedback regulation of nodule formation in alfalfa

When high dosages of wild-type Rhizobium meliloti RCR2011 were inoculated at two different times, 24 h apart, onto either the primary roots of alfalfa (Medicago sativa L.) seedlings or onto lateral roots on opposite sides of a split-root system, the number of nodules generated by the second inoculum was much smaller than the number generated by the first inoculum. These results provide evidence that alfalfa has an active, systemic mechanism for feedback control of nodulation. Non-nodulating mutants and delayed, weakly nodulating mutants did not elicit a discernable suppression of nodulation by subsequently inoculated wild-type cells. An appreciable number of Rhizobium infections thus seem required to elicit the suppressive response. Mutants in nodulation regions II_b and II_a nodulated extensively in the initially susceptible region of the root, but nodule initiation by these mutants was 100–1000 times less efficient, respectively, than the parent. Nodules formed by these mutants emerged 1 d later than normal. The II_b mutants elicited a relatively strong suppression of nodulation in younger parts of the root, but region-II_a mutants elicited only a weak response. These results indicate that elicitation of the regulatory response need not be proportional to nodule formation and imply that genes in region II_a play an important role in elicitation. At high dosages, the region-II mutants induced the development of thick, short roots in a considerably higher percentage of plants than the wild-type bacteria. Nodules generated by wild-type isolates and region-II mutants did not emerge in strict acropetal sequence, probably because some infections developed more slowly than others. Prior exposure of the root to non-nodulating mutants resulted in nodulation by the parent in regions of the root otherwise too mature to be susceptible, indicating that exposure to these mutants may affect the sequence of root development.Abbreviations RT root tip - EH smallest emergent root hair - Tsr thick, short roots This is contribution No. 79-88 of the Ohio Agricultural Research and Development Center     

Nodule distribution on the roots of soybean and a supernodulating mutant in sand-vermiculite

The distribution of nodules of soybean (Glycine max (L.) Merr.) cultivar Bragg and the supernodulating mutant derivative nts382 was examined on the primary root relative to the first emerging lateral root, and on laterals relative to the base of the roots of plants grown in sand-vermiculite. Mutant nts382 nodulates profusely even in the presence of nitrate and appears defective in a systemic autoregulatory response that regulates nodule number in soybean. Nodules were clustered on primary roots about the first 4 cm down from the first emerging lateral root in both genotypes. Nodulation profiles showed reduced nodulation in younger and older regions of the primary root. Similarly, nodules appeared clustered close to the base of the lateral roots. Decreasing inoculum dose shifted nodule emergence to younger regions of the primary root and to lateral roots emerging in younger portions of the primary root. Our results indicate that the supernodulating mutant is able to regulate nodule number in both primary and lateral roots in the particulate matrix.     

Disparate role of rhizobial ACC deaminase in root-nodule symbioses

The enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase converts ACC, a precursor of the plant hormone ethylene, into ammonia and ??-ketobutyrate. ACC deaminase is widespread among the rhizobia in which it might play a crucial role in protecting rhizobia against inhibitory effects of ethylene synthesized by the host plant in response to the nodulation process. The beneficial action of this enzyme was demonstrated in several rhizobia such as Mesorhizobium loti and Rhizobium leguminosarum where knock-out mutants of the ACC deaminase gene showed nodulation defects. The genome of the slow-growing rhizobial species Bradyrhizobium japonicum also carries an annotated gene for a putative ACC deaminase (blr0241). Here, we tested the possible importance of this enzyme in B. japonicum by constructing an insertion mutant of blr0241 and studying its phenotype. First, the activity of ACC deaminase itself was measured. Unlike the B. japonicum wild type, the blr0241 mutant did not show any enzymatic activity. By contrast, the mutant was not impaired in its ability to nodulate soybean, cowpea, siratro, and mungbean. Likewise, symbiotic nitrogen fixation activity remained unaffected. Furthermore, a co-inoculation assay with the B. japonicum wild type and the blr0241 mutant for soybean and siratro nodulation revealed that the mutant was not affected in its competitiveness for nodulation and nodule occupation. The results show that the role previously ascribed to ACC deaminase in the rhizobia cannot be generalized, and species-specific differences may exist.     

Role of Pili (Fimbriae) in Attachment of Bradyrhizobium japonicum to Soybean Roots

Pili (fimbriae) were observed on cells of each of the five strains of Bradyrhizobium japonicum and the one strain of Rhizobium trifolii examined. Pili on B. japonicum were about 4 nm in diameter and polarly expressed. Piliated cells were estimated by transmission electron microscopy and hydrophobic attachment to polystyrene to constitute only a small percentage of the total population. The proportion of piliated cells in these populations was dependent on culture age in some strains. Piliated B. japonicum cells were selectively and quantitatively removed from suspension when cultures were incubated with either soybean roots or hydrophobic plastic surfaces, indicating that pili were involved in the attachment of the bacteria to these surfaces. Pili from B. japonicum 110 ARS were purified and found to have a subunit molecular weight of approximately 21,000. Treatment of B. japonicum suspensions with antiserum against the isolated pili reduced attachment to soybean roots by about 90% and nodulation by about 80%. Pili appear to be important mediators of attachment of B. japonicum to soybean roots under the conditions examined.