Presumptive Differentiation of Phytopathogenic and Non-pathogenic Bacteria by Improved Rapid-Extraction TLC Method

Abstract

TLC profiles of aminolipids extracted from phytopathogenic and non-pathogenic bacteria with chloroform methanol-0.3%NaCl (2:1:0.2, v/v/v) or 2-propanol are useful for discrimination of bacteria. For many bacteria, each TLC profile is genus or species specific and highly reproducible. For most gram-negative bacteria, the uppermost spot (Up) appeared at ca. Rf 0.7 on the chromatograms developed with chloroform- methanol-0.2% CaCl2・2H2O (55:35:8, v/v/v). This spot was absent on the chromatograms of gram-positive bacteria, Clavibacter michiganensis. The profiles of Agrobacterium spp. and Rhizobium spp. were different from other gram-negative bacteria with the uppermost spots at ca. Rf 0.75. For the case of Agrobacterium spp., the chromatograms of the strains belonging to the same biovar were identical. Distinct differences were found among the profiles of Agrobacterium biovar 1, A. biovar 2, A. biovar 3 and A.rubi. The profiles of Rhizobium spp., except for R. tropici, and their relatives such as Bradyrhizobium, Mesorhizobium and Sinorhizobium spp. were quite simple and different from those of Agrobacterium spp. For the case of Burkholderia species, except for B. andropogonis, three spots (designated as S1, S2, S3) appeared under the uppermost spot (Up) and their profiles were species specific for several species such as B. plantarii and B. caryophylli. On the chromatogram of B. andropogonis, the S1 spot (non-phosphorous) was absent and the S3 spot was faint. The profiles of 96 Ralstonia solanacearum strains from various sources were identical. For the case of Erwinia carotovora an intensive benchmark spot appeared at Rf 0.64 but this spot was absent on the chromatograms of pathovars of E. chrysanthemi and E. herbicola. Clear diversity in profiles was observed between Xanthomonas campestris and X. oryzae. The profile of pathovars of Pseudomonas syringae were identical and simple. Substitution of chloroform solvent systems with less hazardous organic solvents was tested. 2-propanol for the lipid extraction and 1-butanol-acetic acid-water (3:1:1 and 5:3:1, v/v/v) for the developing solvents were usable, though development with the butanol systems was highly time-consuming