3,4,5-三羟基苯甲酸甲酯抑制番茄青枯病菌的机制及防病作用研究

发布时间：2018-08-06 13:58

【摘要】：番茄青枯病是由茄青枯拉尔氏菌(Ralstonia salanocearum)引起的一种毁灭性土传病害。目前对该病害的防治仍以使用化学药剂为主,由于防治该病害的药剂种类少,且长期使用后病原菌对目前使用的药剂已产生不同程度的抗性,导致防效降低。因此,农业生产迫切需要开发防治植物青枯病的高效、低毒、低残留的新型药剂。前期的研究表明,从木蜡树叶片提取物中分离得的3,4,5-三羟基苯甲酸甲酯(Methyl Gallate, MG)对茄青枯拉尔氏菌有较强抑制作用。但MG对茄青枯拉尔氏菌抑制机制和对茄青枯病的防治作用研究不够深入。因此,本文测定MG对茄青枯拉尔氏菌生理生化的影响,分析茄青枯拉尔氏菌菌体的蛋白质组在MG的抑制作用下发生的变化情况,测定MG对番茄青枯病的防治作用和在番茄植株的传导特性,取得的主要结果如下：1.MG能够破坏茄青枯拉尔氏菌菌体细胞壁的完整性、抑制纤维素酶和果胶酶的产生、降低病菌的过氧化物酶(POD)和过氧化氢酶(CAT)的活性,可通过促使NADH/NAD+比值的升高,抑制病菌的糖酵解-三羧酸循环。2.在20 μg/mLMG作用下,R. solanacearum菌体蛋白质表达差异明显的蛋白质有29个,采用质谱技术鉴定出22种差异蛋白质,其中新增蛋白11种、缺失蛋白5种、表达上调蛋白1种和表达下降蛋白5种。这些蛋白包括参与能量代谢、信号转导、物质运输和转录修复相关的蛋白。初步推测MG对茄青枯拉尔氏菌应有多个作用靶标,其中抑制菌体的能量代谢可能是MG对茄青枯拉尔氏菌的抑制作用的机制之一。3.将MG作用后茄青枯拉尔氏菌新增蛋白F0F1ATP合酶ε亚基的基因RSc3316进行敲除,获得了缺失突变体△DM3316以及该突变体的互补菌株ACM3316,为研究MG作用茄青枯拉尔氏菌后的新增蛋白FOF1ATP合酶ε亚基的基因功能打下基础。4.MG对番茄青枯病的保护作用有较长的持效期,施药15d后接种青枯病菌,防效仍达52.15%,接种病原菌5d后施用MG的植株,防治效果仅为13.03%,说明该药剂对番茄青枯病的治疗作用较弱。MG可被番茄植株根系快速吸收,番茄根部浸泡MG再接种病原的防效达96.67%。MG在番茄植株内的向顶和向基的传导性则很差,根部浸MG后叶部接种和叶片施MG后根部接种的防效分别为14.44%和9.44%。5.MG处理后番茄根系与生根和抗病相关的酶活性以及次生代谢物质发生变化。与用0.1%甲醇溶剂处理的植株比较,用MG处理后的番茄植株,IAAO活性下降,POD活性没有明显变化,但PPO活性上升。用MG处理并接种病原菌的植株,PAL活性持续增加。苗期和大田期施用MG处理的番茄根系,与抗病相关的香叶醇、豆甾醇、β-谷甾醇和木栓醇等的含量均高于用0.1%甲醇溶剂处理的番茄植株。6.MG对番茄种子的萌发有一定的促进作用。20 μg/mL和50 μg/mL的MG、清水和0.1%甲醇溶剂处理在第4d的萌发率分别为73.38%、66.69%、53.33%、48.89%,20 μg/mL和50 μg/mL的MG处理的种子萌发率均显著高于清水和0.1%甲醇溶剂处理。7.MG对番茄青枯病有较好的防治作用。0.5 g/L的MG和20%噻森铜悬浮剂对番茄青枯病的防效分别为60.22%和48.86%。MG处理的亩产量为2982.4kg,与清水CK处理比较,增产率达153.99%,且对土壤中的细菌种类和数量没有造成影响。
[Abstract]:The bacterial wilt of tomato is a devastating soil borne disease caused by Ralstonia salanocearum of Solanum Solanum. At present, chemical agents are still used in the prevention and control of the disease, because there are few fungicides to prevent and control the disease, and the pathogenic bacteria have produced different degrees of resistance to the current used medicament after long-term use, which leads to the prevention of effect. Therefore, agricultural production urgently needs to develop a new type of high efficiency, low toxicity, and low residual fungicide to prevent and control plant bacterial wilt. Previous studies have shown that the 3,4,5- three hydroxybenzoate (Methyl Gallate, MG) isolated from the leaves of the wood wax leaves has a strong inhibitory effect on Solanum Solanum L., but MG inhibits the Solanum Solanum The mechanism and the study on the prevention and control of Solanum wilt were not thorough enough. Therefore, the effects of MG on the physiological and biochemical characteristics of Solanum Solanum were determined, and the changes of the protein group under the inhibition of MG were analyzed, the control effect of MG on Tomato Bacterial Wilt and the transmission characteristics of the tomato plant were measured. The main results are as follows: 1.MG can destroy the integrity of cell wall of Solanum Solanum mycelium, inhibit the production of cellulase and pectinase, reduce the activity of peroxidase (POD) and catalase (CAT), and inhibit the glycolysis - three carboxylic acid cycle.2. in 20 mu by promoting the increase of the NADH/NAD+ ratio. There are 29 proteins with distinct differences in protein expression in R. solanacearum, and 22 different proteins are identified by mass spectrometry, including 11 new proteins, 5 missing proteins, 1 expressions of up regulation proteins and 5 expression decreasing proteins. These proteins include participation in energy metabolism, signal transduction, material transport and transcription repair. It is preliminarily conjectured that MG should have multiple target targets for Solanum Solanum, in which the inhibition of the energy metabolism of the bacteria may be one of the mechanisms of the inhibitory effect of MG on Solanum Solanum..3. will knock out the gene RSc3316 of the new protein F0F1ATP synthase epsilon of Solanum Solanum after the action of MG and obtain the missing mutant Delta DM33 16 and the complementary strain ACM3316 of the mutant, in order to study the genetic function of the new protein FOF1ATP synthase epsilon subunit after the MG effect of Solanum Solanum,.4.MG has a long holding time for the protection of tomato bacterial wilt. After 15d, inoculated with bacterial wilt, the control effect is still 52.15%. After inoculation of the pathogen 5D, the MG plant is applied to prevent the inoculation. The treatment effect was only 13.03%, indicating that the therapeutic effect of the medicament on tomato bacterial wilt is weak.MG can be absorbed quickly by tomato plant root system, and the control effect of 96.67%.MG in tomato roots for MG inoculating pathogen is very poor in the heading and direction of tomato plant, and the root inoculation after MG and root inoculation after MG after MG are inoculated. The enzyme activities related to the root and disease resistance and the secondary metabolites of tomato roots after treatment with 14.44% and 9.44%.5.MG were changed. Compared with the plants treated with 0.1% methanol solvent, the activity of IAAO decreased and the activity of POD had not changed obviously, but the activity of PPO was not obviously changed, but the viability of PPO was increased. MG was used to treat and inoculate the plant of pathogenic bacteria, PAL The activity continued to increase. The root of tomato treated with MG in seedling and field period was higher than that of disease related geraniol, sterol, beta glutamol and cork alcohol, which were higher than that of tomato plants treated with 0.1% methanol solvent.6.MG, which had a certain promoting effect on the germination of tomato seeds, such as.20 mu g /mL and 50 g/mL MG, clear water and 0.1% methanol solvent. The germination rates of 4D were 73.38%, 66.69%, 53.33%, 48.89%, 20 mu g/mL and 50 g/mL MG respectively. The seed germination rates were significantly higher than that of the clear water and 0.1% methanol solvent treatment.7.MG for tomato bacterial wilt disease,.0.5 g/L MG and 20% thiazen suspension agent for tomato bacterial wilt disease were 60.22% and 48.86%.MG respectively. The yield per mu was 2982.4 kg, which was 153.99% higher than CK treatment, and had no effect on the species and quantity of bacteria in soil.
【学位授予单位】：广西大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S436.412.15

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