Molecular identification of ice nucleation active (INA) bacteria causes of frost injury on potatoes crops (Solanum tuberosum L) in Wonosobo, Dieng plateau

Dieng plateau lies at an altitude of more than 1500 meters above sea level (masl), the average temperature is 14°C. In the dry season, the temperature becomes very cold that can reach -2°C, consequently frost injury phenomenon can affect potato plants. Frost injury thought occurs due to the formation of ice by Ice Nucleation Active (INA) bacteria. The purpose of this study was to identify the species and to determine phylogenetic relationship of INA bacteria at Dieng plateau. This research used purposive sampling method by taking potato leaves at two different altitudes that are ± 2000 and ± 2500 masl. Bacteria isolation was done by spread plate method on nutrient agar enriched with 2.5% glycerol. Colonies with different morphology are isolated and cultured on agar slant medium. The nucleation activity of INA bacteria was determined by the tube nucleation test method in the circulating alcohol bath at -4, -7 or -10°C for 10 minutes. Bacterial isolates were identified by the amplification of 16S rRNA encoding gene. The sequencing of 16S rRNA encoding gene was then analyzed by BLAST Nucleotide on the NCBI website. The phylogenetic tree was constructed using MEGA 7.0 program. The result showed that 84 bacterial isolates were found from the potato leaves. There were 20 bacterial isolates that indicate freezing activity. Each of the 5 isolates has similarity with Pseudomonas jessenii 96%, Enterobacter ludwigii 99%, Stenotrophomonas maltophili 94%, Pseudomonas putida 98% and Pseudomonas japonica 99%. INA bacteria found in this study have the close relationship with previously discovered INA bacteria in sub-tropical and tropical regions.