Screening, identification and characterization of antagonistic bacteria against Ralstonia solanacearum

Abstract

Tobacco bacterial wilt caused by Ralstonia solanacearum is one of the most destructive bacterial diseases of tobacco. As a recurring soil-borne disease, it is controlled mainly by chemical methods that are not only costly and less efficient but also have food safety and environment safety problems. Consequently, biological control methods that use antagonistic strains have gained strength in tobacco bacterial wilt disease research. To find new antagonist bacteria against R. solanacearum, 238 strains were identified and screened for in vitro antibiosis. By placing each strain on cultured pathogen plates with toothpicks 24 strains showed inhibition zones. Among the 24 strains, the width of the inhibition zones of 7 antagonist strains exceeded 10 mm. Then pathogen inhibitive ingredients of the 7 strains were extracted to determine bacteriostatic efficacy. Finally, 3 promising strains(H19, Y6, H34) were obtained. H19 and Y6 were identified as Bacillus amyloliquefaciens and the other strain(coded H34) identified as B. methylotrophicus. The strain classification was based on phylogenetic analysis of 16S rDNA sequence, gyrB sequence and fatty acid composition. Siderophores secreted by microorganisms to take up iron from the environment had an action mode of disease suppression that was solely based on competition with the pathogens for iron. Besides, indole-3-acetic acid(IAA) is a phytohormone known to be involved in root initiation, cell division and cell enlargement. In follow-up experiments, all the three strains showed the ability to produce siderophores and plant growth promotor IAA. However, productions of siderophores and IAA were different, with H19 and Y6 as the highest and lowest producers under improved CAS and Salkowski colorimetric methods. The three strains were further characterized for plant growth promoting traits and disease control effects. Compared with control treatment, the strains significantly improved the growth of tobacco with increases of 19%-24% in the maximum leaf length, 7%-12% in the maximum leaf width, 70%-115% in plant height, 2%-14% in stem diameter, 40%-49% in fresh weight and 32%-42% in dry weight. H19, H34 and Y6 reduced bacterial wilt incidence by 76.57%, 60.98% and 69.83%, respectively in greenhouse experiments. This was slightly lower than using 40% agricultural streptomycin which had disease incidence control efficacy of 78.66%. In conclusion, the research isolated new efficient antagonistic strains against tobacco bacterial wilt. It also proved the mechanism of disease control and plant growth promotion by H19, H34 and Y6. The study further showed that the three strains had great potential for plant growth promotion and biological control under greenhouse conditions, which developed the needed reference for follow-up studies.