Physiological studies and phylogenetic analysis of a root-inducing bacterium

Studies have been carried out with the root-stimulating bacterium (RSB) isolated by Burns (1990) to better understand the mechanism of RSB-stimulated root induction in in-vitro cultures of hypocotylary explants of slash pine (Pinus elliottii Engelm.) and to understand the phylogenetic affiliations of the bacterium through the analysis of its 165 RNA sequence. When inoculated with a culture of the bacterium, seedling explants of 10 half-sib genotypes of slash pine were found to root at different levels depending upon the genotype. Rooting percentages ranged from 8.33% through 66.67% showing genotype-dependent variation in the level of root induction. A putative root-inducing signaling substance (RISS) was maximally extracted at a pH of 2.8 from RSB liquid culture medium. The extract at pH 2.8 induced a level of rooting in the slash pine explants that was not significantly different from the level of induction seen in explants inoculated with a living culture of RSB. The spectral profile of the methanol extract of the bacterium was found to be similar to the profile of carotenoid pigments. The 16S rRNA gene of the bacterium was amplified from its genomic DNA using the polymerase chain reaction with oligonucleotide primers that correspond to conserved eubacterial sequences that flank the majority of the 16S rRNA gene. The amplified product was cloned into the Smal site of the vector pBluescript KS and subsequently sequenced using the Sanger dideoxynucleotide sequencing technique. A phylogenetic analysis of the sequence was carried out using the sequence of 16S rRNA gene of 17 members of the a subdivision of Proteobacteria obtained from GenBank and the Ribosomal Database (Ribosomal Database Project). The analysis revealed that the bacterium is closely related to Sphingomonas paucimobilis, Flavobacterium devorans and Caulobacter subvibrioides in terms of the sequence of its 16S rDNA. The RSB matched most closely with Caulobacter subvibrioides. Comparative analyses of an alignment of the sequence of the four taxa revealed that the RSB differed from Caulobacter subvibrioides by only 45 nucleotides. A study of the segment of the 16S rRNA gene defined by Yabuuchi et al.. (1990) revealed that the two taxa differed by only 4 mismatches suggesting that both might belong to the same genus.