Genetic Diversity of Plant Growth Promoting Rhizobacteria of Bacillus sp. Based on 16S rRNA Sequence and Amplified rDNA Restriction Analysis

Abstract

Plant-growth promoting rhizobacteria (PGPR) are rhizosphere associated soil-borne bacteria that can enhance plant growth and inhibit the development of root pathogens. Many soil bacteria have been used as PGPR, and one of them is Bacillus sp. The implementation of PGPR is constrained by genotype fluctuation that makes it inactive on the rhizosphere. Our previous study had characterized and revealed that 11 Bacillus sp. isolated from the soybean plant rhizosphere were PGPR. To asses and compare the genetic diversity of these isolates, Amplified Ribosomal DNA Restriction Analysis (ARDRA) and DNA sequence analysis of 16S rRNA were conducted. The construction of Neighbor-joining trees and bootstrap analysis of 100 resamples of ARDRA and 16S rRNA gene sequences were performed using Treecon software for windows ver. 1.3b. ARDRA analysis was done by using four restriction enzymes (RsaI, HaeIII, CfrI and HinfI), resulting in four phylotypes, respectively phylotype I (Bacillus sp. Cr24, Cr33, Cr64 and Cr68), phylotype II (Bacillus sp. Cr 31 and Cr66), phylotype III (Bacillus sp. Cr44 and Cr71) and phylotype IV (Bacillus sp. Cr67, Cr28 and Cr69). Results of BLASTN from 16S rRNA gene sequences showed that these isolates are genetically diversed. The evolution relationship of Bacillus sp. could be shown by the 16S rRNA gene sequences analysis, while ARDRA based on the digestion sites showed their variability.Key words: Bacillus sp., plant-growth promoting rhizobacteria, 16S rDNA, ARDRA, genetic diversity * Corresponding author, Phone/Fax: +62-251-8622833, E-mail: aristri2003@yahoo.comThe slow growth of plant is a major handicap that can decrease food production and ecosystem stability. Synthetic fertilizers have been used to solve this problem. However, it can give negative effects on human and environment, such as pollution of the surrounding ecosystem and the death of non-target microbes. Therefore, efforts to find alternative solutions, among others by using plant-growth promoting rhizobacteria (PGPR), i.e. associated soil-borne bacteria on the rizosphere that can enhance plant growth and inhibit the growth of root pathogens, such as Bacillus sp.Bacillus is a motile, catalase-positive, Gram-positive rod, with 40-60% GC content. It forms endospore that is very resistant to extreme environmental conditions. Bacillus has been known for the production of phytohormone such as indole acetic acid (Glick 1995), siderophores (Compant et al. 2005) and antibiotics such as zwittermicin A (Silo-suh et al. 1994), bacilin, clorotetain and Iturin A (Phister et al. 2004). These compounds are natural and beneficial to promote plant growth. Hence, it is potential to utilize those mechanisms in agriculture. Its implementation, however, is constrained by the genotype fluctuation.The 16S rRNA gene has been routinely used as a reliable molecular marker for phylogeny identification. It contains conserved region, a unique array of sequences that are relative among species or different species (Woose 1987;Moyer et al. 1994). It is the basis of m...