Ice nucleation bacteria are among the main factors responsible for frost damage and freezing injury in cold-sensitive plants. Ice nucleation genes in bacteria are typically single chromosomal genes, often classified within the ina gene family, and play a key role in the ice nucleation active (INA+) phenotype. One approach to mitigating frost damage is modifying the genetic structure of the gene encoding the INA protein.
Amplification of the inaZ gene from Pseudomonas syringae was performed using gene-specific primers and the polymerase chain reaction (PCR). The PCR product was cloned into the pTG19-T vector and transferred into Escherichia coli K12. To evaluate and compare the ice nucleation frequency of recombinant E. coli, the droplet freezing assay was used. Ethyl methanesulfonate (EMS) was applied to induce mutations in the ina gene in Pseudomonas syringae. To confirm mutagenesis, in addition to PCR analysis, mutant bacteria lacking the ice nucleation gene were compared with wild-type bacteria using the droplet freezing assay.
The results were determined based on the T50 value (temperature at which 50% of droplets freeze) in each treatment compared with sterile distilled water and wild-type E. coli lacking ice nucleation activity. In all positive treatments, ice nucleation activity ranged from -4 to -7°C depending on the dilution of the suspensions. The results demonstrated successful transfer of the ice nucleation inaZ gene from P. syringae to E. coli. In addition, mutant colonies of P. syringae lacking the ice nucleation gene were obtained. These mutant bacteria represent suitable candidates for competitive exclusion of INA+ bacteria, as they can colonize plant surfaces with similar fitness to wild-type strains and inhibit biological ice nucleation. Therefore, they may serve as a potential alternative to limited plant bactericides.