Three methods of strain identification were used to determine the composition of the Rhizobium trifolii population in nodules formed on subterranean clover (Trifolium subterraneum L. cv. Mt. Barker) inoculated with a soil suspension. Sodium dodecyl sulfate polyacrylamide gradient gel electrophoresis (SDS‐PAGGE) was used in a microslab system to elucidate the protein profiles of 22 isolates of R. trifolii. Tube agglutination serological tests carried out with antisera raised to four isolates showed that four groups of isolates could be recognized. Only four isolates out of 22 agglutinated with more than one antiserum, although they reacted with only one antiserum in the gel‐immune‐diffusion test. One isolate did not react with any of the four antisera. No group of isolates dominated the nodule population. Gel‐immune‐diffusion analysis showed that isolates from two of the four groups were serologically identical whereas isolates from the other two groups were serologically heterogeneous. Isolates within all four serogroups were subdivided further according to their protein profiles. Many of the isolates within individual groups had very closely similar or identical profiles whereas others were very distinct. Isolates identified as very similar or identical by SDS‐PAGGE had the same symbiotic effectiveness on T. subterraneum. The data illustrate the need for multiple methods of identification for the best delineation of the composition of the nodule population.

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.

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Acidity affected which members of an indigenous soil population of Rhizobiulm trifolii nodulated Trifolium subterraneum L. cv. Mt. Barker. In three experiments involving plants grown either in mineral salts agar adjusted to pH 4.8 or 6.8 and inoculated with a soil suspension or grown directly in samples of unamended soil (pH 4.8) or soil amended with CaCO3 (pH 6.4), 121 of 151 isolates of R. trifolii were placed into four serogroups. Seventy-nine of these isolates were placed into two serogroups (6 and 36) whose nodulating ability was affected by the pH of the plant root environment. Representatives of serogroup 6 occupied the greatest percentage of the nodules at the low pH in both mineral salts agar (77%) and in unlimed soil (47 and 57%). The same serogroup was a minor nodule occupant at the higher pH in mineral salts agar (0%) and in limed soil (0 and 10%). In contrast, serogroup 36 was virtually absent in nodules formed at the low pH, whereas it was the dominant serogroup at the higher pH in both mineral salts agar (32%) and in limed soil (35 and 49%). Despite the isolates from within each serogroup being antigenically identical, separation of cellular proteins by sodium dodecyl sulfatepolyacrylamide gradient gel electrophoresis revealed four and six different gel types within serogroups 6 and 36, respectively. Isolates represented by one or two gel types dominated the contribution of each serogroup to the nodule population. Further evidence for differences between isolates within each gel type were revealed from measurements of symbiotic effectiveness.

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