NOVEL METHOD FOR IDENTIFYING BACTERIAL MUTANTS WITH REDUCED EPIPHYTICFITNESS

In order to identify novel traits involved in epiphytic colonization, a technique for the rapid identification of bacterial mutants with qua ntitatively different population sizes in a natural habitat based on m easurements of ice nucleation activity was developed. The threshold fr eezing temperatures of leaves harboring different numbers of cells of ice nucleation-active Pseudomonas syringae B728a differed substantiall y. While few leaves containing less than about 10(6) cells per g (fres h weight) froze at assay temperatures of -2.75-degrees-C or higher, ne arly all leaves froze at these temperatures when population sizes of t his strain increased to about 10(7) cells per g (fresh weight). Presum ptive epiphytic fitness mutants could readily be identified as strains which initiated freezing in fewer leaves than did other strains withi n a given experiment. Most Tn5-induced mutants of strain B728a which c onferred a low frequency of ice nucleation on inoculated bean leaves g enerally had a smaller population size than the parental strain at the time of the leaf freezing assay. The leaf freezing assay was capable of differentiating samples which varied by approximately three- to fiv efold in mean bacterial population size.