【作者】 高小宁；

【导师】 黄丽丽；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2012， 博士

【摘要】 油菜(Brassica campestris L.)是我国重要的油料作物,由核盘菌(Sclerotinia sclerotiorum (Lib.) de Bary)引起的油菜菌核病是世界范围内的一种重要病害,在我国发生普遍且危害严重,一般年份发病率为10%-30%,严重年份或严重地块的发病率达80%以上,导致严重减产和品质下降。由于目前尚未发现免疫或高抗菌核病的油菜品种,生产上主要采用轮作及化学药剂结合的防治措施,然而连年大量使用化学药剂不仅造成了严重的环境污染,同时已经导致了病原菌抗药性的产生。因此寻找对环境友好的有益微生物进行菌核病的生物防治已经成为研究的热点,受到了越来越多的重视。植物内生细菌作为新挖掘的微生物资源,正在被广泛开发利用。本实验室前期分离纯化了大量植物内生菌,并从中筛选获得了一株对油菜菌核病菌菌丝生长和菌核形成具有强烈抑制作用的内生细菌Em7菌株。本研究在前期工作的基础上,进一步对内生细菌菌株Em7防治油菜菌核病的田间应用及机理进行了系统研究,取得了如下结果：1.通过温室和田间试验明确了内生细菌菌株Em7对油菜菌核病的防治效果。大量温室盆栽试验发现整株喷施菌株Em7的培养滤液和菌悬液在油菜苗期和成株期均可有效减轻菌核病的发生,其中喷施菌株Em7后接种病菌的防效明显高于接种病菌后喷施的防效,防病效果达到90%以上。在陕西汉中地区连续三年(2009-2011年)的田间防治试验结果表明,在油菜初花期连续喷施两次(间隔期7-10d)菌株Em7的培养液(109CFU mL-1)可有效降低菌核病的发病率和严重程度,防治效果分别达到53.8%、71.8%和70.5%,与对照药剂戊唑醇(Tebuconazole)和多菌灵(Carbendazim)防效相当,无显著性差异。2.通过培养特征和形态特点观察,生理生化特性、16S rDNA序列分析以及Biolog微生物自动鉴定系统将Em7菌株鉴定为枯草芽孢杆菌(?)(?)acillus subtilis (Ehrenberg) Cohn。3.采用利福平抗性标记方法揭示了B. subtilis菌株Em7在油菜体内的定殖和消长规律。在油菜幼苗期分别采用整株喷施和灌根的方法研究了Em7Rif标记菌株在油菜不同组织中的定殖规律。整株喷施Em7Rif菌株(1×109CFU mL-1)24h后,在油菜叶片和茎秆组织内检测到的菌量最大,分别达到4.0×105CFU g-1和9.6×105CFU g-1鲜组织；28d后仍然能够从茎秆及叶片组织内检测到,但在根组织内却未检测到标记菌株。灌根施用Em7Rif菌株(109CFU mL-1),24h即可从根和茎秆中检测到标记菌株,施用后3d后标记菌株在根部和茎杆中的数量最大,分别达到5.6×106CFU g-1和4.6×105CFU g-1,此后检测的菌量逐渐减少,但28d后仍可检测到标记菌株。4.利用Biolog代谢指纹方法分析了B. subtilis菌株Em7对油菜根际土壤微生物群落的影响。灌根法于苗期分别施用菌株Em7菌悬液和培养滤液后不同时间取根际土壤,用Biolog ECO板检测不同土样微生物群落的多样性,结果显示施用菌株Em7后的油菜根际土壤微生物群落的活性和多样性明显提高。菌株Em7处理后的土壤微生物群落碳源利用能力明显增强,各种碳源平均颜色变化率AWCD值和Shannon(?)数明显高于对照处理。5.初步明确了诱导抗性可能是B. subtilis菌株Em7对油菜菌核病的生防机制。在油菜苗期和成株期喷施菌株Em7菌悬液和培养滤液后不同时间(1、3、5、7、9、11和15d)接种菌核病菌,油菜植株均表现出良好的抗病效果；并且发现喷施菌株Em7后,油菜叶片内过氧化物酶(POD)、多酚氧化酶(PPO)和苯丙氨酸解氨酶(PAL)的活性在一定时期内有显著的升高,与对照组相比差异显著。6.利用显微镜技术揭示了B. subtilis菌株Em7对菌核病菌抑制的细胞学特征。光学和扫描电镜观察发现受到菌株Em7影响后,病菌菌落边缘的菌丝形态发生异常变化,受抑制菌丝粗细不均匀、皱缩、畸形,并有明显的细胞质外渗现象；进一步的透射电镜观察表明受抑制菌丝细胞的超微结构发生了明显的变化,主要表现为细胞内液泡数量增加,细胞器紊乱,细胞壁溶解和细胞质外渗等。对菌株Em7作用后菌核病菌入侵和扩展过程的观察结果表明：接种病菌前1d喷施菌株Em7可抑制病菌侵染结构(侵染垫)的形成,并可致使病菌菌丝出现膨大、顶端分支增多且分支不规则；接种病菌后1d喷施菌株Em7可导致病菌形成的侵染垫塌陷,菌丝出现皱缩及坏死等现象。此外,皿内试验结果发现菌株Em7不仅能够抑制病菌的菌丝生长,而且可有效抑制菌核萌发,菌核萌发的抑制效果与生防菌浓度和处理时间密切相关。7.在摇瓶条件下优化了B.subtilis菌株Em7产生抗菌物质的条件,并初步明确了其抗菌物质的特性和抑菌谱。采用单因素和正交试验,获得菌株Em7的最佳培养基配方为：麸皮2%,牛肉膏1%,MnS040.15%；当培养基初始pH7,接种量为1%,摇瓶装液量50ml/250ml时,在28℃,150rpm培养获得的培养液抑菌活性最强。进一步通过硫酸铵盐析、凝胶过滤等方法明确了菌株Em7的主要活性物质为蛋白类抗菌物质；硫酸铵饱和度30%提取的粗蛋白对热、酸碱及部分蛋白酶稳定,并且对常见的10余种植物病原菌的菌丝生长和分生孢子萌发具有广谱的抑制作用。更多还原

【Abstract】 Oilseed rape(Brassica campestris L.) is the major oil crop in China, with a production area of approximately70×106ha. Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is one of the important diseases on oilseed rape in the world. In China, the disease incidences were10-20%in general, but reach over80%in serious land or serious year. Conventional methods for controlling Sclerotinia disease such as chemical and cultivating control measures have been applied in different countries for many years. However, conventional breeding programs to increase resistance in rape to Sclerotinia disease have been less effective because of the restricted gene pool; and because of the adverse effects on non-target organisms and environment, the use of fungicides have been limited. Therefore, the relatively unreliable control with traditional methods and concerns about fungicides residues has prompted interest in biological control as an alternative strategy for disease management. Endophytes, living asymptomatically within plant tissues, have beneficial effects on host plants. In recent years, endophytic bacteria have gained increasing scientific interest, but few studies were reported about biocontrol of oilseed rape Sclerotinia stem rot by endophytic bacteria. In preliminary studies, we detected that the endophytic bacterial strain Em7strongly inhibited S. sclerotiorum mycelium growth and sclerotia formation in vitro. Then, the biocontrol effect and mechanism were studied in this paper. The main results were as follows:1. Biocontrol efficacies of strain Em7to S. sclerotiorum were detected in greenhouse and in field trials. In the greenhouse, the culture filtrates and cell suspensions of strain Em7displayed effective biocontrol activities in the seedling and blossom stage. The biocontrol activities were influenced by the time and concentration of spraying strain Em7. The control efficacy was higher when strain Em7was applied prior to inoculation of the pathogen than after inoculation. Three-year field trials (2009-2011) showed that twice sprays of strain Em7cell suspension at blossom stage significantly reduced disease incidence and severity. The control efficacies were53.8%,71.8%and70.5%, respectively in2009,2010, and2011. There was no significant difference in control efficacy among treatments with strain Em7and the fungicides Carbendazim or Tebuconazole.2. Strain Em7was identified as Bacillus subtilis (Ehrenberg) Cohn on the basis of morphology, physiological and biochemical characteristics,16S rDNA sequence analysis and Biolog microbial automatic identification system.3. The strain Em7colonization in rape plant was detected by rifampicin-labeled Em7. The results showed that bacteria could colonize into stems, leaves and roots, but the colonization of mutant strain Em7could be determined by different treatment. Bacteria could largely colonize into stems and leaves after spraying1d, the number of mutant strain Em7were4.0×105CFU·g-1and9.6×105CFU·g-1, respectively, in leaves and stems. Then the quantity gradually decreased. The labelld-strain could be recovered from stems and leaves of plants even after28days, the quantity is up to3.1×102CFU·g-1in stems. The mutant strian could be detected in roots and stems after root drench treatment. The biomass of mutant strain Em7in roots and stems could be reached5.6×106CFU·g-1and4.6×105CFU·g-1after3d. Although the quantity gradually decreased, the labelled strain could be still detected in stems and roots after28d.4. The effects of applying biocontrol agent strain Em7on the oilseed rpae soil microbial communities was evaluated by Biolog metabolic fingerprinting method. The profiles derived from Biolog ECO plates indicated higer substrate richness, average well color development (AWCD, microbial activity) were influenced by strain Em7. The values of AWCD and Shannon functional diversity index were higher treated with strain Em7than untreated.5. It was concluded that the induced resistance is one of the biocontrol mechansim of endophytic bacteria Em7against S. sclerotiorum. The culture filtrate and cell suspensions of strain Em7was applied prior to inoculation of the pathogen in the seedling and blossom stage, the resistance of oilseed rape was induced to S. sclerotiorum. After the treatment of strain Em7, the activity of POD, PPO and PAL had obviously rised in certain period of time, which had significant difference to the contrast.6. Light and scanning electronic microscopy studies revealed that in the presence of strain Em7, morphology of mycelial cell of S. sclerotiorum showed leakage, deformation, and disintegration of hyphal cytoplasm. The ultrastructure of mycelial cell was investigated under transimission electronic microscopy. The results showed that strain Em7caused increasing of vacuole in the cytoplasm, disordered of the orgarelles, rupture of the cell wall, and leakage of the cytoplasma. The effects of strain Em7on the infection and extension of S. sclerotiorum in the rape leaves were observed under scanning and transmission electronic microscopy. Treated with strain Em71d before inoculation, the mycelia couldn’t form the infection structure on the sueface of leaves, and the mycelia were irregularlly swollen and collapsed. Treated with strain Em71d after inoculation, the infected cushion was destroyed, and the hyphae were collapsed and coated by film, the cytoplasm exosmosis. In Petri dish, strain Em7could inhibit not only mycelial growth but also sclerotia germination. However sclerotia germination was obviously influenced by concentration of strain Em7cell suspension.7. The optimum fermentation culture medium of strain Em7was optimized at shake flask. The results of single-factor experiment and the orthogonal test showed that the optimum medium was bran2.0%, beef extract1.0%, and MnSO40.15%. The maximum antibiotic activity was achieved at the initial pH7.0, medium volume of50ml in250ml flasks, rotary speed of150rpm, temperature28℃and inoculation volume of1%. The antifungal substance was studied by ammonium sulfate precipitation and Gel filtration chromatography. The main active substance of strain Em7as protein antibacterial substance had been definited. Crude protein extracted by30%saturation ammonium sulfate was stable to thermal, acid and alkali. Furthermore, the antifungal protein also showed a broad antifungal spectrum on mycelium growth and conidia germination of numerous important plant pathogenic fungi. Light microscopic observations revealed that the antifungal protein caused morphological alterations including increased branching, swelling, and shriveling of hyphae as well as collapse of cytoplasm.更多还原