The Antibacterial Effects And Mechanism Of Several Botanical Compounds Against Ralstonia Solanacearum

Ralstonia solanacearum(R. solanacearum) is a soil-borne bacterial pathogen that can cause plant bacterial wilt. The pathogen, widely distributed in many tropical, subtropical and some temperate regions, can infect more than a hundred kinds of crops and cause significant economic losses. In the process of long-term coevolution with host and environment, R. solanacearum forms high levels of complexity in morphology, differentiation, pathogenicity and pathogenesis, etc. which increase the difficulty of controlling plant bacterial wilt. For a long time, there are many strategies to control bacterial wilt mainly including chemical pesticides, resistant varieties, sterile grafting and crop rotation. However, treatment with these agents may cause severe side-effects, especially in resistant strains and environmental pollution problems. Therefore, selecting natural botanical active compounds which are efficient and environmentally friendly is extremely important for controlling the pathogen. In this paper, we took the test of screening for green dead bacteria with antimicrobial activity, a preliminary studying on the botanical compounds, evaluated its antibacterial mechanism, indoor prevention and control effect and impact on pathogenicity in order to obtain the natural botanical active compounds for the antibacterial activity on R. solanacearum, and provide a new idea for the prevention and treatment of bacterial wilt and botanical compounds inhibiting mechanisms and finally a reliable theoretical and practical basis for the development of biological pesticides.1. Evaluating botanical compounds with antibacterial activity to R. solanacearum. After treated with 100 μg/mL and 10 μg/mL drugs, the amount of R. solanacearum was detected. The results showed protocachuic aldehyde, coumarin, carvacrol and resveratrol had inhibitory effect and had significant differences with other compounds. The inhibition rate reached 99.40%,96.04%, 84.66% and 70.27% at 100 μg/mL concentration respectively. Therefore, the test identified protocachuic aldehyde, coumarin, carvacrol and resveratrol as candidates for further research.2. Determining the MIC and MBC of protocachuic aldehyde, coumarin, carvacrol and resveratrol to R. solanacearum and their impact to the growth of R. solanacearum. Results showed that the growth of R. solanacearum was inhibited by protocachuic aldehyde at the concentration of 40μg/mL. Under the concentration of 64 μg/mL, it could the growth of bacterial was inhibited completely. After treated with resveratrol and coumarin, the MIC and MBC respectively were 230 μg/mL,230 μg/mL and 110 μg/mL,110 μg/mL, and the most obvious inhibition to the growth was at a concentration of 128 μg/mL. Inhibitory effect of carvacrol on strains increased with increasing concentration, and MIC and MBC were 210μg/mL and 250μg/mL respectively.3. Defining the four botanical compounds’ influences to the strain cell morphology of R. solanacearum. The strain cell morphology was observed by scanning electron microscopy (SEM) after treated with the four compounds. The results showed that the strain cell morphology was long and slender after treated with protocachuic aldehyde which might inhibit the reproductive division of bacteria. Bacteria morphology showed an incomplete state and the edge blurred after resveratrol treatment, which inferred resveratrol could destroy the integrity of the cell wall or membrane of R. solanacearum, and cause endoplasmic outflow and cell collapse. Coumarin may alter membrane permeability and osmotic pressure of cell surface protrusions and rupture, thereby inhibit the growth of bacteria. Scanning electron microscopy of R. solanacearum cells after treated with carvacrol showed that cell wall was destroyed.4. Studying the effects of the four botanical compounds on the motility and biofihn formation of R. solanacearum to further clarify the impact on early infection stage. These data indicated protocachuic aldehyde stimulated the formation of biofilms and inhibited the motility of R. solanacearum. Resveratrol and carvacrol have significantly inhibited motility and biofihn of strains. After treated with coumarin, the motility and biofilm are less affected.5. Investigating of the expression of virulence genes by using RT-PCR technology after treated with the four compounds. Experimental data showed that, compared with the control group, expression levels of pilT, egl and epsB decreased significantly after resveratrol treatment. The expression of pilT significantly reduced after treated with coumarin. Protocachuic aldehyde can significantly increase gene expression of egl. Carvacrol had a significant role in promoting the expression of pilT and epsB, but it had no significant effect on the egl and peh C.6. Verifying the prevention and control effect of botanical compounds to tobacco bacterial wilt by pot-experiment. Inoculated by hurting the roots and not hurting the roots, the prevention and control effects of botanical compounds to bacterial wilt were evaluated. In the case of not hurting the roots, the effect of strains adhesion and colonization in tobacco roots was detected. The results showed that protocatechuic aldehyde also significantly reduced the disease index of tobacco bacterial wilt, even though it promoted the adhesion of the strains at the root. Resveratrol and coumarin significantly inhibited strains’ pre-adhesion and colonization, and postponed the development of the disease. Carvacrol had no significant effect to the tobacco bacterial wilt. However, the four botanical compounds, in the case of root inoculation, had no effect on tobacco bacterial wilt. Therefore, we can infer the compounds’ effect to tobacco bacterial wilt was at the early infecting stage, and there was no effect after the strains’ invasion to tobacco.In conclusion, the experimental results show coumarin, protocatechuic aldehyde, carvacrol and resveratrol have antibacterial effects on R. solanacearum, and after studying the inhibitory mechanism, we discovered the morphology of R. solanacearumhave different degrees of change after treatment with compounds, and the motility, biofilm and some genes which associated with virulence are affected. The potted experiment show that four botanical compounds have different effect to tobacco bacterial wilt, and the major inhibitory role of compounds to bacterial wilt was at early infection stage. This paper firstly investigates the antimicrobial activity and its inhibitory mechanisms of protocachuic aldehyde, coumarin, carvacrol and resveratrol to plant pathogenic bacterium R. solanacearum, and provides a reference for research and development of new pesticides to control plants bacterial wilt.