A method involving ice nucleation for the identification of microorganisms antagonistic to Erwinia amylovora on pear flowers

A method was developed for the rapid selection of antagonists capable of suppressing growth of Erwinia amylovora on pear flowers. The population size of E. amylovora on flowers pretreated with potential antagonists was estimated by a flower-freezing assay. E. amylovora harboring the iceC gene from Pseudomonas syringae on the stable plasmid pVSP61 expressed high levels of ice nucleation activity. The threshold freezing temperature of flowers colonized by the ice nucleation-active (Ice(+)) strain of E. amylovora increased linearly with the logarithm of the population size of the pathogen. Bacterial and yeast strains were selected from the predominant microflora of pear flowers and tested for their ability to lower the threshold freezing temperature of flowers subsequently inoculated with the Ice(+) E. amylovora strain. For each antagonist, the proportion of 20 treated flowers that had frozen was evaluated when approximately 95% of the control (E. amylovora only) had frozen. Among 257 strains tested, 45 (4 yeasts and 41 bacteria) were capable of reducing freezing of inoculated flowers by 70% or more. The reduction in the proportion of pretreated flowers that froze relative to the control for a number of antagonists was correlated (R(2)=0.61) with reduced E. amylovora population sizes. Over 50% of the antagonistic strains that reduced the proportion of frozen flowers by 50% or more during an initial screening conferred a similar reduction in the number of frozen flowers in a second test. Several strains were more antagonistic to E. amylovora in situ than P. fluorescens strain A506, which is used commercially for biological control of fire blight.