Isolation, identification, and detection of ACC deaminase gene-encoding rhizobacteria from rhizosphere of stressed pineapple

ACC deaminase is a microbial cytoplasmic enzyme that cleaves ACC, a precursor of ethylene, in stressed plant. The aims of this study are to isolate, identify, and detect the presence of ACC deaminase gene-encoding rhizobacteria from rhizospheric soil sample of pineapple which have been exposured by abiotic and biotic stress; herbicide, flooding, and Phytopthora spp. stress. A total of 49 rhizobacterial isolates were obtained and 7 of them were observed for their growth on DF medium containing 3 mM L -1 ACC. Four best growth isolates were selected for genomic DNA extraction. They were molecularly identified as Stenotrophomonas maltophilia (3), Burkholderia territorii (2A), Pseudomonas oryzihabitans (5B), and Bacillus tropicus (1E). A set of primers were constructed which are 105F- acd S 5’-TGCCAAGCGTGAAGACTGC-3’and 244R- acd S 5’-GGGTCTGGTTCGACTGGAT-3’ to amplify the ACC deaminase gene ( acd S). Based on melt peak curve analysis, four products appeared specific single peak at 86, 89, 87, and 89.5 o C indicating a single product was produced. In addition, Blast search showed that four products meet ACC deaminase feature and their acd S sequences are clustered into an ancestral group compared to those of many bacterial strains deposited in the GenBank. This result indicates that ACC deaminase gene-encoding rhizobacteria from pineapple plantation of tropical origin may affect the acd S sequences and may contribute to the stress tolerance for plant host.