【作者】 王震；

【导师】 郭爱玲； 冯莉；

【作者基本信息】 华中农业大学 ， 微生物学， 2008， 硕士

【摘要】 青枯病是由青枯雷尔氏菌（Ralstonia solanacearum）引起,严重危害作物生长的一种细菌性土传病害。至今已发现该病菌可侵染44个科近300多种植物。青枯病防治困难的主要原因是与多种因素有关,如:植株品种、土壤管理、栽培管理、施肥状况、气候等。因此,青枯病的防治不能单靠某一种方法的作用,而需采取综合防治措施进行防治。目前在番茄青枯病的多种防治方法中,生物防治对番茄生产的可持续发展具有很好的应用前景。本研究在立足于生物防治的基础上,以水培番茄系统作为青枯病研究的媒介,从番茄防御酶、生防菌及营养元素等三方面对番茄青枯病进行了系统、深入的防治研究,主要研究内容有:1.通过稀释涂布法和菌脓分离法获得强致病力青枯菌株。对两种方法进行对比证明,菌脓分离法有利于获得强致病力菌株,所用时间短,纯化次数少,且所分离菌株的致病性保持时间长。分离时期以植株发病初期分离的青枯菌株致病力最强。2.通过单因素试验和均匀试验设计研究了水培条件下番茄青枯病发生的最佳条件。结果表明,青枯病发生的最佳条件为:番茄苗龄4～5叶期,伤根,营养液pH6.4,温度32℃,环境湿度80%,EC 2.2ms/cm,青枯菌接种浓度6.3×10⁸cfu/mL。3.通过拮抗试验从健康番茄植株根际土壤中获得4株对青枯菌具有强拮抗性的菌株,并用LuxAB和链霉素、四环素进行标记,命名为S59、S8、S57、S43。经进一步拮抗试验、催芽试验、防效测验及在番茄根部的定殖测定试验,筛选出具有强定殖力,可在番茄根际和茎中大量繁殖,且对导管和根表面青枯菌有强的抑制作用的S59菌株。4.通过改变营养液Ca²⁺、K⁺浓度分别与拮抗菌S59的共同作用,对番茄苗青枯病发病率影响进行了研究。结果表明,拮抗菌S59与K⁺结合施用有利于提高番茄植株抗性,增强防病能力。同时,在拮抗菌S59浓度不变的条件下,番茄青枯病发病率随K⁺浓度的升高而下降,从40.1%下降到32.4%,与对照相比降低了52.0%,防效上升9.1%,作用显著。而在水培条件下Ca²⁺浓度的增加对番茄青枯病的防治是不利的,当Ca²⁺浓度由3.5mmol/L上升到9.5mmol/L时,发病率从39.5%上升到48.7%,其防效下降了11.5%。5.通过对番茄生理生化分析研究表明,经不同K⁺浓度与拮抗菌处理的番茄苗,苯丙氨酸酶（PAL）、过氧化物酶（PPO）、多酚氧化酶（POD）活性均在处理后有明显的提高,且随K⁺浓度的升高而升高;Ca²⁺与拮抗菌处理的番茄苗三种酶活性,围绕对照3.5mmol/L浓度处理起伏变化,呈无规律性,各浓度间差异不显著。番茄苗超氧化物歧化酶（SOD）活性在经不同Ca²⁺浓度与拮抗菌S59处理后,具有随Ca²⁺浓度的增加而升高的趋势;不同K⁺浓度与拮抗菌S59处理的番茄苗SOD酶活性无明显变化。从番茄苗四种防御酶活性的动态变化曲线看,拮抗菌的加入使各酶活性的峰值出现时间有所推迟。以上研究结果,对番茄青枯病的持续有效控制、新生防菌剂的开发、番茄栽培管理、施肥管理等均具有十分重要的理论和应用意义,同时对其他植物土传病害的研究也具有十分重要的参考价值。更多还原

【Abstract】 Ralstonia solanacearum can cause serious harm to crops spreading through soil. The bacteria may infect nearly 300 varieties of plants in over 44 families so far. This bacterial disease is difficult to control, which is related to several factors, including plant species, soil management, cultivation and management, fertilization conditions and climate variability. Therefore, integrated control is needed, rather than single way.At present, biocontrol is an promising way to control tomato disease in many methods of disease-contronl. The studies were carried out on hydroponic tomato as a media of R. solanacearum. Three aspects were designed to systematic and deep studied, including the activities of defense enzymes, biocontrol and nutrition elements. The main research results were as follows:1. The highly pathogenic R.solanacearum was obtained by the methods of dilution and bacterial pus separation, and the latter was favorite, which is benefit to obtain pathogenic strains with a shorter time, fewer purification, and much longer pathogenic maintaining. The initial infected tomato was the best source of R.solanacearum.2. The conditions of break-out of tomato bacterial wilt were investigated through Single-factor experiments and Uniform Design in hydroponics. The optimal conditions were at 32℃, pH6.4, EC（electricity conductance） 2.2ms/cm, in 80% relative humidity, by root-wounding method, on tomato seedling in 4 or 5 leaf stages and with inoculation concentration of 6.3×10⁸ cfu/mL.3. 4 strains of antagonistic bacteria, namely S59, S8, S57, S43, were obtained from the rhizosphere soil of healthy tomatos by the antagonize experiment, taking LuxAB and streptomycin, tetracycline as markers. By antagonistic experiment, germination test and disease-control experiment, the results showed that S59 strain could restrain germination of tomato seed, and control plant disease effectively. The colonization study of S59 strain indicated that it could not only colonizate but also maitain a unsiderably population on tomato roots-surface, stems. 4. The incidence of tomato bacterial wilt was studied by the combination of Ca²⁺ or K⁺ in nutrient and S59. The results showed that the integrated application of S59 and K⁺ is beneficial to raising tomato resistance and enhancing the ability of disease prevention. At the same time, the incidence of bacterial wilt decreased along with the increase of K⁺ concentration, decreased from 40.1% to 32.4%, under the same concentration of S59 strain, compared with the control lower 52.0%, the controlling efficiency increased 9.1%. The increase of Ca²⁺ concentration is detrimental to the prevention of tomato bacterial wilt in hydroponics. When Ca²⁺ concentration increased from 3.5 mmol/L to 9.5 mmol/L, the incidence of tomato increased from 39.5% to 48.7% and the controlling efficiency declined 11.5%.5. The results of the physiological and biochemical analysis on tomato showed that the activities of phenylalanine （PAL）, peroxidase （PPO） and polyphenol oxidase （POD） activities has been significantly improved by the treatment of different concentration of K⁺ and S59 strain, proportional to the concentration of K⁺. The combination of Ca²⁺ and S59 showed no any regularity on above three enzymes. The activity of superoxide dismutase （SOD） would be increased with the increase of Ca²⁺ concentration, but SOD activity showed no significant changes after the treatment of K⁺ and S59 strain. Judged from the dynamic curves of the four defense enzyme activities in tomato seedlings, the peak activity would be delayed after the entry of antagonistic bacteria S59.All above research results have important theoretical and practical significances on the sustainability control of tomato bacterial wilt, the development of new anti-microbial agent, tomato cultivation and management, fertilizer management, and also have very important reference value to other plants on soil-borne diseases.更多还原