Effect of Postemergence, Supplemental Inoculation on Nodulation and Symbiotic Performance of Soybean (Glycine max (L.) Merrill) at Three Levels of Soil Nitrogen.

The influence of a supplementary bradyrhizobial inoculation after an initial seed slurry inoculation with the same strain on nodulation and N(2) fixation in soybeans was examined in the greenhouse. The plants were grown in a Typic Eutrocrepts soil: sand mixture containing 25, 65, or 83 mg of N per kg (i.e., native soil N plus N-labeled ammonium sulfate). Harvests were made at early flowering and physiological maturity. The supplementary inoculations which were made 14 or 21 days after planting (DAP) caused formation of substantially more nodules than the single slurry inoculation did. Autoregulation was therefore not completely successful in preventing subsequent infections. For the slurry-inoculated plants, at both harvests the proportion of N derived from fixation was greatest in the soil containing the least N, and only slight increases in N(2) fixation resulted from a second inoculation. The inhibition of N(2) fixation at the higher N levels was significantly reduced by a second inoculation at 21 DAP; this treatment resulted in at least a doubling of both the percentage and total amount of N(2) fixed by the single slurry inoculation at physiological maturity. The N(2) fixation increases resulting from the supplementary inoculation at 14 DAP were less pronounced and not significant. Greater N(2) fixation was frequently not reflected by increased total N or dry matter yield, suggesting that the major benefit of the increased fixation was a decreased dependence of plants on soil N for growth.

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 N(2) fixation was examined by using two Bradyrhizobium japonicum strains of contrasting effectiveness in N(2) 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 N(2) 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 N(2) fixation in soil-grown legumes.

Protozoa and the decline of Rhizobium populations added to soil.

A fall in Rhizobium abundance occurred in nonsterile soil inoculated with large numbers of the root-nodule bacteria, but many of the rhizobia still survived. No such decline was evident in sterile soil. Protozoa feeding on these bacteria were isolated from soil and other environments. As the abundance of Rhizobium meliloti and a cowpea Rhizobium strain in soil decreased, the protozoan density increased. The inability of the predators to eliminate their prey from soil was not the result of the presence of organisms feeding on the protozoa because many rhizobia survived in sterile soil inoculated with the prey and cultures of individual protozoa, nor was it the result of the rapid multiplication of the bacteria to replace those consumed because survivors were still numerous in essentially organic matter free soil in which the bacteria did not grow appreciably. The lack of elimination also was not associated with a protective effect of soil particles because survivors were still abundant in solutions inoculated with protozoa and bacteria. It is suggested that the size of the prey population diminishes until a density is attained at which the energy used by the predator in hunting for the survivors equals that obtained from the feeding.

Regulation of predation by prey density: the protozoan-Rhizobium relationship.

Tetramitus rostratus and strains of Hartmanella, Naegleria, and Vahlkampfia consumed large numbers of Rhizobium meliloti cells in a salt solution, but protozoan multiplication and the bacterial decline stopped when the prey density fell to about 10-6 to 10-7 cells/ml. At higher prey densities, the maximum numbers of Hartmanella sp. and Naegleria sp. were proportional to the quantity of R. meliloti initially provided to the amoebas. When supplemental rhizobia were supplied to Hartmanella sp. or Naegleria sp. after their active feeding had terminated, presumably because the remaining 10-6 or 10-7 bacteria/ml could not be captured, replication of the protozoa was initiated. The rate of elimination of rhizobia present in large populations was proportional to the initial abundance of Naegleria sp., but the final numbers of amoebas and surviving R. meliloti cells were independent of initial numbers of predators. The surviving bacteria were not intrinsically resistant to attack because 98% of the survivors, when concentrated, were consumed. It is suggested that large populations of bacteria in nature may be reduced in size by predatory protozoa, but many of the prey cells will not be eliminated.