Biocontrol potential of antagonist Bacillus subtilis Tpb55 against tobacco black shank

Abstract

Tobacco black shank caused by Phytophthora nicotianae is a very important oomycete disease of tobacco, and it is widely distributed around the world. In order to develop effective prevention techniques, this study examined the effects of an antagonistic bacterium, Bacillus subtilis Tpb55 strain, on the prevention of tobacco black shank in vitro and in vivo. Dual culture test results showed B. subtilis Tpb55 strain have a strong antagonism to P. nicotianae, inhibit the growth of its hyphae, and produce significant inhibition zones. Scanning electron microscopy showed that the Tpb55 strain can damage the structure of P. nicotianae hyphae, cause hyphae deformity, hyphae rupture, and protoplasm leakage. Control effects of Tpb55 strains on tobacco black shank in pot and field experiment can reach up to 70.66 and 59.34 %, respectively. In this study, Tpb55 strain was also labeled with green fluorescent protein (GFP) in order to monitor their rhizosphere colonization of tobacco. The Tpb55 strain’s colonization on tobacco roots showed a diffused distribution, largely in the root meristem and elongation zone areas. They can gather focally into microcolonies, forming a biofilm like structure. A small number of these bacteria can colonize at the intercellular space and among vascular bundles. After inoculation, Tpb55-GFP was found to colonize tobacco roots for 30 days or more. The number of bacteria peaked on the 4th day at 1.51 × 107 cfu g−1. By the 12th day, it had dropped to 1.1 × 106 cfu g−1. This study shows that the effect of Tpb55 strains on controlling of tobacco black shank is correlated to their ability to inhibit mycelia growth and ability to successfully colonize tobacco roots.