Biological control of grapevine crown gall: purification and partial characterisation of an antibacterial substance produced by Rahnella aquatilis strain HX2

Abstract

Strain HX2 of Rahnella aquatilis has been previously reported as a potential biological control agent (BCA) of grapevine crown gall. The production of an antibacterial substance (ABS) was suggested to be an important factor in the biocontrol process. This study was undertaken to determine the antibacterial properties and mode of action of ABS. Isolation and purification of ABS from culture broth of strain HX2 was achieved by methanol exaction, chromatography on macroporous resin and silica gel, and high-performance liquid chromatography (HPLC). Physical and chemical characteristic analysis revealed that ABS may be a thermostable and alkali-sensitive substance containing sugar(s) and an unknown 285-nm absorbed substance. Antibacterial activity assays revealed that ABS displayed a broad activity spectrum against all the 13 test isolates of phytopathogenic bacteria including the genera of Agrobacterium, Clavibacter, Pectobacterium, Pseudomonas, and Xanthomonas. Agrobacterium spp. strains were more sensitive to ABS than other tested strains, with larger inhibition zones and lower minimal inhibitory concentration (MIC). ABS exhibited a bactericidal effect against A. vitis both in vitro and in vivo. This compound did not cause bacterial cell lysis, as determined by morphological observation with an electron microscope. Also, no leakage of cytoplasmic materials from cells of A. vitis occurred after treatment with ABS at concentrations equivalent to the MIC. However, an inhibition of the incorporation of radiolabelled precursors into RNA and protein was observed after treatment with ABS. These results suggest that ABS inhibits RNA and protein syntheses in tumorigenic A. vitis.