| Tobacco bacterial wilt caused by Ralstonia solanacearum is one of the most destructive bacterial diseases on tobacco, causing huge economic loss every year. In order to improve the biocontrol efficacy and stability, many overseas researchers have been working on mixing of biocontrol bacteria with active materials of plants in recent years, while less study has been done in China. In this research rhizospheric and endophytic bacteria were isolated from Hubei province and Shandong province. Firstly, the antagonistic bacteria were screened via antagonizing action and anti-disease test. Secondly, anti-bacteria activity of ethanol extracts from40species of Chinese herbal medicines were determined in vitro against Ralstonia solanacearum. Finally the plant extracts with the best anti-bacteria activity was selected to mix with antagonistic bacteria. We then obtained the formula that has the best control efficacy against Ralstonia solanacearum. Meanwhile, the extracts of antagonistic bacteria and plant were analyzed via gas chromatography-mass spectrometry (GC-MS) to identify their active components, and to provide a reference for future research. The results are as following:(1) Two hundred and thirty eight bacterial strains were isolated from different tobacco fields. By placing each strain on cultured pathogen plates with toothpicks and extracting pathogen inhibitive ingredients to determine bacteriostatic efficacy,3candidate strains (H19, Y6, H34) were obtained. H19and Y6were identified as Bacillus amyloliquefaciens and strain H34was identified as B. methylotrophicus. The strains classification were based on morphological observation, phylogenetic analysis of16S rDNA sequence, gyrB sequence and fatty acid composition. 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 between the strains, with H19and Y6being the highest and lowest producers when measured with improved CAS and Salkowski colorimetric methods. The3strains were further characterized for plant growth enhancing traits and disease control effects. Compared with control treatments, the strains significantly improved the growth of tobacco with70%-115%increase in plant height,40%-49%increase in fresh weight and32%-42%increase in dry weight. H19, H34and Y6reduced bacterial wilt incidence by76.57%,60.98%and69.83%, respectively in greenhouse experiments. This was slightly lower than using agricultural streptomycin which had a control efficacy of78.66%.(2) Forty species of Chinese herbal medicines were selected, and their ethanol extracts were obtained through microwave extraction, infusion method and ultrasonic wave extraction method successively. Then oxford cup method was used to determine their bacteriostasis in vitro against Ralstonia solanacearum. The results showed that32different herbal medicines have antibacterial activity, and clove extract showed stronger activity against Ralstonia solanacearum than other medicinal extracts. The diameter of inhibition zone against Ralstonia solanacearum of clove extract was19.53mm.(3) The extracts of H19and clove were analyzed via gas chromatography-mass spectrometry(GC-MS), and24,12chemical components were identified, respectively. Analytic quality reagents of4chemical components with higher relative content from extract were used to test their bacteriostasis against t Ralstonia solanacearum. The result showed that inhibition zone against Ralstonia solanacearum of2,3-Butanediol,[S-(R*,R*)]-of H19was10.23mm,. Eugenol of clove possesses the highest antibacterial activity, and its inhibition zone against Ralstonia solanacearum was18.5mm, while other compotents showed no bacteriostatic effect. According to a preliminary inspection,2,3-Butanediol,[S-(R*,R*)]-may be the active ingredients of strain H19fermentation, while eugenol and eugenol acetate may be the active ingredients of clove extract.(4) Toxicity test using the extracts of H19and clove showed that the50%effective concentration (EC50) was0.0509mg/mL for H19and1.4042mg/mL for clove. The joint effect of the extracts of H19and clove with different proportions were determined, and their synergistic ratio(SR) were calculated. The results showed that when the volume ratio of H19extract to clove extract was7:3, their SR was up to18.60. The control effect of this formula was64.29%, while the control effect of H19and clove were50.00%and42.86%, respectively. All of them were lower than agricultural streptomycin which had a control efficacy of71.43%. |
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