Population Size and Distribution of Rhizobium leguminosarum bv. trifolii in Relation to Total Soil Bacteria and Soil Depth

Abstract

Bacterial cells small enough to pass through 0.4-,um-pore-size filters made up 5 to 9% of the indigenous bacterial population in 0to 20-cm-depth samples of Abiqua silty clay loam. Within the same soil samples, cells of a similar dimension were stained with fluorescent antibodies specific to each of four antigenically distinct indigenous serogroups of Rhizobium leguminosarum bv. trifolii and made up 22 to 34% of the soil population of the four serogroups. Despite the extensive contribution of small cells to these soil populations, no evidence of their being capable of either growth or nodulation was obtained. The density of soil bacteria which could be cultured ranged between 0.5 and 8.5% of the >0.4-p.m direct count regardless of media, season of sampling, or soil depth. In the same soil samples, the viable nodulating populations of biovar trifolii determined by the plant infection soil dilution technique ranged between 1 and 10% of the >0.4-p.m direct-immunofluorescence count of biovar trifolii. The <0.4-p.m cell populations of both total soil bacteria and biovar trifolii changed abruptly between the 10to 15-cm and 15to 20-cm soil depth increments, increasing from 5 to 20% and from 20 to 50%, respectively, of their direct-count totals. The increase in density of the small-cell population corresponded to a significant increase in soil bulk density (1.07 to 1.21 g cm-3). The percent contribution of the <0.4-p.m direct count to individual serogroup totals increased with soil depth by approximately 2-fold (39 to 87%) for serogroups 17 and 21 and by 12-fold (6 to 75%) for serogroups 6 and 36.

Determination of viability within serotypes of a soil population of Rhizobium leguminosarum bv. trifolii

“…Soil samples were collected from a depth of 0 to 5 cm from a silty clay loam of the Abiqua series (Cumulic Ultic Haploxeroll) under permanent pasture. The physical and chemical characteristics of the soil have been described elsewhere (1,7). By using a soil tube auger, 20 soil cores were removed from within each of six grids (1 by 1 m) spaced 20 m apart along a transect running diagonally across the pasture.…”

“…The samples were passed through 25-mm-diameter, 0.4-p.m-poresize Sudan black-stained polycarbonate membranes (Nuclepore Corp., Pleasanton, Calif.). Filters were destained with citrate buffers and prepared for epifluorescence microscopy as described elsewhere (7).…”

“…A portion (0.2 ml) of the nalidixic acid stock solution was immediately added to each growth tube to achieve a final concentration of 10 mg/liter. Over a period of 24 h, replicate tubes were sacrificed at 4-h intervals by adding Formalin (final concentration, 2% [wt/ vol]) and were then cooled to 4°C and processed for microscopy as described elsewhere (7,12). Included in every experiment were extra tubes containing filtered soil suspension supplemented with yeast extract and incubated in the presence or absence of nalidixic acid.…”

“…Although immunofluorescence has allowed researchers to determine the densities of individual serotypes within indigenous soil populations of Bradyrhizobium japonicum (31,(36)(37)(38) and Rhizobium leguminosarum bv. trifolii (1,7,45), concern has been expressed about the proportion of cells which are viable within populations of soil rhizobia observed by immunofluorescence (3, 14,16,22,35). Even though there is evidence that nonviable rhizobia cannot persist for significant lengths of time in soil (5, 10), it is a longestablished fact that only a small fraction of the total bacterial population in soil enumerated by microscopy can be cultured (13,28,29,34,40,42).…”

Genotypic and Phenotypic Comparisons of Chromosomal Types within an Indigenous Soil Population of Rhizobium leguminosarum bv. trifolii

“…For 70 years serotyping has been used to monitor root nodule occupancy by antigenically distinct strains of Bradyrhizobium and Rhizobium species (1,5,6,18,22,39). Serotyping allows researchers to gain a superficial picture of the diversity within native rhizobial populations at specific sites, to track the occurrence and distribution of antigenically related bacteria across states, countries, and continents, and to study the autecology of these bacteria in soil (4,7,13,15,17,30,33,36,38,40,42,49,53). Studies carried out with Bradyrhizobium japonicum (24,34,35,43,50,51,54) and Rhizobium leguminosarum (8,19,55) have shown that individual serotypes can be composed of nonidentical strains.…”