Isolation and identification of Ice Nucleation Active (INA) bacteria causing embun upas (frost injury) on leaves of potato plant in the Dieng Plateau, Central Java, Indonesia

Abstract

Abstract. Rajasa AK, Susilowati A, Suranto. 2020. Isolation and identification of Ice Nucleation Active (INA) bacteria causing embun upas (frost injury) on potato plant leaves in the Dieng Plateau, Central Java, Indonesia. Bioteknologi 17: 67-74. Embun upas (frost injury) is one of the symptoms of ice crystal formation that occurs on the leaves of potato plants (Solanum tuberosum L.) in the Dieng area, Central Java, Indonesia. Symptoms of embun upas occur on a large scale and cause a decrease in potato yields of up to 6 tons per hectare. The cause of the symptoms of embun upas on potato plants is suspected to be the activity of ice-core-forming bacteria. This study aimed (i) to obtain isolates of Ice Nucleation Active (INA) bacteria from potato plants in Batur District, Dieng, (ii) to determine the INA bacterial species which can be identified using the gene encoding 16S rRNA, and (iii) to determine the population density of INA bacteria in one gram of plant leaves. Sampling used the purposive sampling technique by taking potato leaves at three different altitudes: ±1,800 m above sea level (Bakal Village), ±2,000 m above sea level (Diengkulon Village), and ±2,200 m above sea level (Sembungan Village) with two repetitions. Bacterial isolation was carried out using the spread plate method on Nutrien Agar media containing 2.5% glycerol (NAG). Colonies with different morphology were taken and purified on slanted agar media. Ice nucleation activity was determined by the tube nucleation test method. INA bacteria were estimated using the multiple-tube nucleation test 3,3,3 method. Identification of INA bacteria was carried out by amplifying the gene encoding 16S rRNA by the PCR method. The gene encoding 16S rRNA amplicon was purified using PCR/DNA Fragments Extraction Kit (Genaid) and continued with determining the concentration and purity of DNA using a biophotometer. Sequencing was carried out by sending samples to 1^st BASE Singapore, and the data from the sequencing results were analyzed using the BLAST Nucleotide device on the NCBI website. Analysis of the data used is descriptive analysis. The isolation results obtained in this study were six isolates of INA bacteria in all sampling villages. The bacterial species obtained were Arthrobacter sulfonivorans (98%), Curtobacterium luteum (98%), and Pseudomonas azotoformans (99%). The total bacterial population on potato leaves was 1.44 x 102 MPN INA/gram in Bakal Village, 3 x 103 MPN INA/gram in Diengkulon Village, and 1.824 x 105 MPN INA/gram in Sembungan Village.