Gallic Acid an Agricultural Byproduct Modulates the Biofilm Matrix Exopolysaccharides of the Phytopathogen Ralstonia solanacearum

Abstract

Ralstonia solanacearum is a soil-borne plant pathogen which causes wilt disease in economically important crops of the Solanaceae family in tropical and temperate regions. As biofilm formation is the major virulence factor in R. solanacearum, research inputs are necessary to identify natural biofilm inhibitors to mitigate virulence of this bacterium. Hence in the present work, the anti-biofilm potential of phytochemical compound gallic acid (GA) isolated from an agricultural byproduct (cashewnut shell) was investigated. Initially the Minimum inhibitory concentration (MIC) of crude extracts of cashewnut shell and coconut shell against R. solanacearum were investigated. The MIC of both the extracts were 400µg/ml and their sub-MIC (200µg/ml) inhibited biofilms in the range of 62-70% and 49-57%, respectively. As the cashewnut shell extract have higher biofilm inhibitory effect compared to coconut shell extract, we proceeded our further study by isolating the major compound GA from cashewnut shell by acid hydrolysate method. The sub-MIC of crude cashewnut shell extract inhibited 85% of young biofilms. The MIC of GA were observed at 3mg/ml and sub-MIC (1.5mg/ml) was found to eradicate 85% of mature biofilms which was confirmed by standard crystal violet assay and the biofilm reduction was further visualized under light microscopy and scanning electron microscopic images. Toxicity of GA was evaluated against R. solanacearum through XTT cell viability assay and found no antibacterial effect at sub-MIC. Additionally, it is confirmed with growth curve and time kill assays. Swimming and twitching motility were considered as an important virulence factors to invade plants and to block the xylem vessels. Therefore, sub-MIC of GA was found to inhibit both swimming and twitching motility of about 93% and 63% respectively. Anti-biofilm efficacy of GA was also worked well with tomato plant model where remarkable biofilm inhibition was found on treatment with GA before and after 24h of infection with R. solanacearum. Hence GA will be an alternative, cheap source which is eco-friendly as well as novel source for the treatment of R. solanacearum biofilms and to prevent wilt disease in important crops.