Application of Tube-Nucleation Assays to Rapid Population Estimates of Rhizobacteria Expressing Novel Ice-Nucleation Activity

Abstract

The populations of bacteria associated with the surfaces of individual plant parts are best described by a log-normal distribution for both phyllosphere (2) and rhizosphere colonizers (4). This widely varied population density on individual leaves or seedling root systems makes accurate representations of distribution and mean populations highly laborious due to the large number of replicate samples which are necessary for accuracy. The genetic analysis of biological control and other root colonizing bacteria demands a factorial evaluation of how rhizosphere competence in soil is affected by gene mutation, gene replacement, the introduction and expression of novel DNA, or novel regulation. Currently, dilution plating is the only feasible method of differentiating changes in colonization potential.

The characteristic of ice-nucleation activity (INA⁺) of several species of epiphytic bacteria has allowed the development of a rapid “tube nucleation” assay to estimate populations without plating onto agar media (1). This assay uses the knowledge of nucleation-temperature frequencies in a population of cells to determine approximate cell densities from the freezing spectra of plant samples in a known volume of nucleus-free buffer. Although accurate population determinations are not possible, useful comparative values for cell density can be resolved for large numbers of replicate samples.

This paper describes the application of the tube nucleation assay to population studies of rhizosphere colonizing Pseudomonas sp. by the introduction of novel DNA conferring INA⁺ to naturally non-nucleating rhizobacteria. The expression of INA⁺ in vitro and in planta were followed for one strain of P. fluorescens and compared with freezing spectra for greenhouse and field grown seedlings from various soils. A secondary objective was to use this system to study plasmid stability in the rhizosphere and the effects of foreign gene expression on growth and colonization.