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

Abstract

Concern has been raised about the percentage of viable cells within rhizobia populations measured by the immunofluorescence direct count method. The purpose of this study was to evaluate a direct viable count technique which is based on the fact that viable bacteria in natural populations undergo cell elongation when they are exposed to a combination of substrate and the inhibitor of DNA gyrase, nalidixic acid. A soil extraction procedure was developed to recover a high proportion of soil bacteria (ca. 109/g of soil) in suspensions with an optical clarity suitable for accurate microscopic enumeration. After incubation for 16 to 20 h at 27°C in the presence of yeast extract (200 mg/liter) and nalidixic acid (10 mg/liter), between 65 and 74% of the bacteria in soil suspension became significantly elongated (≥4.2 μm). In contrast, ≤0.5% of the same population could be cultured, regardless of the medium composition, nutrient concentration, or incubation conditions. The direct viable count method was combined with immunofluorescence to compare the percent viability and kinetics of appearance of elongated cells within serotypes of a soil population of Rhizobium leguminosarum bv. trifolii. Although the majority of these organisms were viable, as observed by immunofluorescence, we obtained evidence that subpopulations within the soil rhizobia community were in different states of competence to respond to substrate. A consistently low percentage (≤30%) of the population of serotype 23 was elongated even after 24 h of incubation and regardless of when the soil was sampled. Although the population densities of serotypes 6 and 36 were similar (0.8 x 106 to 2.0 x 106/g), the appearance of elongated cells of serotype 36 was consistently delayed relative to the appearance of elongated cells of serotype 6, and the maximum percentage of elongated cells of serotype 36 was less than that of serotype 6 when either a low substrate concentration (50 mg/liter) was used or the soil was air-dried before the bacteria were recovered.