Enhancement of Ice-Nucleating Activity in Pseudomonas fluorescens and Its Effect on Efficacy against Overwintering Colorado Potato Beetles

The application of ice-nucleating bacteria as biological control agents for Colorado potato beetles requires the development of media for mass production that optimize bacterial efficacy against overwintering adults. The production method in our laboratory has been limited to a solid medium, nutrient agar supplemented with 2.5% glycerol (NAG), that is known to enhance ice-nucleating activity. In this study, we report culture conditions that enhance expression of type 1 ice nuclei (i.e., initiate freezing at or above −5°C) in the bacterium Pseudomonas fluorescens F26-4C grown in liquid media. Expression of type 1 ice nuclei was induced during the exponential to the stationary phase of its growth cycle by a low-temperature shift to either 15 or 4°C for at least 1 h. Growth in media with a limiting level of nitrogen and with 5% dextrose or galactose, coupled with a temperature shift to 4°C for 2 h, resulted in the greatest enhancement of type 1 nuclei. Adult Colorado potato beetles fed P. fluorescens cells induced to produce type 1 ice nuclei showed a sustained elevation of supercooling points (i.e., the temperature at which ice formation spontaneously occurs in body fluids) comparable to beetles fed cells grown on NAG. These results suggest that P. fluorescens F26-4C produced in liquid media is as persistent in and as effective against overwintering adults as that grown on a solid medium. Furthermore, the ability to culture in liquid media may facilitate the mass production of bacterial cells needed for field applications.