The ice nucleation active (INA⁺) bacteria are one of the most important biological factors in fields and orchards frost damage. Most of these bacteria usually accelerate the freezing process at- 5°C and higher temperatures. In order to identify INA⁺ bacteria, many plant samples were screened and one positive Ice bacterium associated with onion bulb was selected. The bacterium was then tentatively identified using some key bacteriological methods. Accurate identification of the strain A at species level was made by PCR amplification of 16SrRNA followed by blast at NCBI. Ice nucleation activity of the bacterium was tested by tube INA assay and droplet freezing method. Due to the absence of a nucleus-producing gene for representative isolate at the NCBI site, a primer set similar to inaZ gene of Pseudomonas syringae was designed and PCR analysis was performed. PCR-amplicons were further analyzed by plasmid DNA analysis as well as cloning and sequencing. The results were indicated that there might be different types of ice gene in Bacillus cereus strain A. This is the first report of an Ice-plus Bacillus cereus from plant samples.

Keywords: INA, Bacillus cereus, Cloning, Ice nucleation bacteria

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