【作者】 戚玮真；

【导师】 赵蕾；

【作者基本信息】 山东师范大学 ， 微生物学， 2012， 硕士

【摘要】 随着全球环境的恶化，土壤的沙漠化、盐渍化问题日益威胁着人类赖以生存的有限的土地资源，据不完全统计，世界盐碱土面积约占总土地面积的7%左右，并有逐年增加的趋势。此外，设施土壤的次生盐渍化也在影响着农业生产与生态环境。在我国，由于温室栽培灌溉措施不当以及过度施用农药化肥造成的土壤板结、积盐加重、耕地退化的土壤次生盐渍化问题，已成为农业生产中的重要限制因子。土壤的盐渍化不仅影响植物代谢和光合作用，还会降低植物对土壤养分及微量元素的摄入，由于土壤中的铁主要以难溶性的高价氧化物等形式存在，而盐碱土又会进一步降低土壤可溶性铁的含量，从而导致植物的缺绿症。因此，生长在盐渍化土壤中的植物往往同时受到盐胁迫的危害以及铁缺乏的威胁。木霉菌（Trichoderma spp.）是一类能够刺激作物生长并增强其抗生物和非生物胁迫的多功能益生真菌。但目前国内外对于生防木霉菌在增强植物耐盐性及其作用机理方面尚缺乏深入、系统的研究。本文结合生产实际，从分离、筛选具有解盐促生特性的生防木霉菌入手，通过盆栽及水培试验对分离并鉴定的木霉菌株在盐胁迫条件下对黄瓜幼苗的促生作用及其嗜铁素在解盐促生中的作用进行初步研究，利用亲和层析法对该菌产生的嗜铁素进行分离纯化，并对其类型进行鉴定。具体结果如下：1.从大棚作物根际土中分离得到一株具有多种促生特性的生防木霉菌Q1，此株木霉菌对黄瓜枯萎病菌（F. oxysporum f. sp. Cucumerinum）、西瓜枯萎病菌（F. oxysporum f. sp. Niveum）、茄镰孢菌（Fusarium solani）等多种植物病原菌具有良好的生物拮抗作用。同时，此株木霉菌还具有溶磷、产生植物激素、嗜铁素及ACC脱氨酶活性的植物促生特性，经形态学和分子生物学方法将其鉴定为棘孢木霉（Trichoderma asperellum）。结果表明，木霉菌Q1是一株兼具多种促生特性的生防菌。2.通过盆栽试验系统研究了棘孢木霉（Trichoderma asperellum）菌Q1对黄瓜幼苗的解盐促生作用，并分别测定了添加菌(Q1)、盐(salt)、盐+菌(Q1+salt)、清水(control)四种处理的生理生化反应和光合效率的变化。结果发现，无论在有无盐胁迫条件下，接种木霉菌株Q1孢子悬浮液均可促进黄瓜幼苗的生长，与对照相比，处理25d后，黄瓜幼苗的株高、叶绿素含量，叶片中渗透分子（可溶性糖和可溶性蛋白）含量、抗氧化酶活性（SOD和POD）以及净光合速率（PN）、蒸腾速率（Evap）、气孔导度（GS）、胞间CO2浓度（C Int）等光合参数均有不同程度的提高，有效降低了黄瓜幼苗在盐胁迫条件下受到的生理损伤。3.为进一步完善棘孢木霉（Trichoderma asperellum）菌Q1对植物解盐促生机制的研究，本文针对该菌产生的大量嗜铁素，通过水培试验研究了嗜铁素在提高植物抗盐中的作用。采用含NaCl 60 mmol l-1的缺铁水培液对黄瓜幼苗进行盐胁迫，在有无难溶性铁（Fe3+）以及有无菌株Q1嗜铁素发酵滤液（SCF）的4组处理（SCF+Fe）、（SCF-Fe）、（Non-SCF+Fe）、（Non-SCF-Fe）中，添加难溶性Fe3+及嗜铁素发酵滤液（SCF+Fe）的黄瓜幼苗获得了最大的生物量，植株的茎/根长以及第一片真叶叶面积增加，植株的萎蔫率及缺绿症明显降低，说明棘孢木霉嗜铁素缓解了盐胁迫及缺铁条件下对植物产生的负面影响。4.为确定棘孢木霉（Trichoderma asperellum）嗜铁素的类型，本文利用固相化金属离子亲和层析法（Immobilized Metal Affinity Chromatography, IMAC）对木霉菌株Q1嗜铁素发酵滤液进行分离纯化，并通过四唑盐实验将菌株Q1分泌的嗜铁素鉴定为无荧光的异羟肟酸型。这一研究结果是对棘孢木霉（Trichodermaasperellum）嗜铁素类型研究的有效补充，为今后深入了解其化学结构及嗜铁素对铁的转运机制提供了一定的理论依据。更多还原

【Abstract】 With the global environmental deterioration, soil desertification and salinizationproblems are increasingly threats to the limited land resources for human survival.Approximately 7% of the global land surface is covered with saline soil. Moreover,there exist wide areas of secondary soil salinization in China, which effects theagricultural production and the ecological environment, due to poor irrigation systemwithout proper drainage and inorganic fertilizer management. These cultivated landsaffected by salts may adversely cause oxidative damage to the soil which in turnaffects the plant growth by reducing plant’s nutrient uptake and metabolic andphotosynthetic activities. In addition, most of the saline soils have alkaline pH valueand most iron exists in an insoluble form (Fe3+), which prevents plants from optimalgrowth and even leads to leaf chlorosis. Thus plants growing on this kind of soils areoften simultaneously subject to salinity and iron deficiency.Trichoderma spp. are versatile beneficial fungi which can stimulate growth andplant resistance to biotic and abiotic stresses. They have long been recognized asagents for their plant disease control and plant growth promotion, but there are fewreports on their ability to improve salt tolerance of plant and the specific knowledgeabout the underlying mechanisms remain to be explored. For further research of thepotential of Trichoderma in promoting the cucumber growth under salt stress and itspossible mechanisms, we isolated a biocontrol fungus Trichoderma isolate Q1 withplant growth-promoting activities and evaluated its potential in promoting cucumbergrowth and the role of siderophore produced by the strain in alleviating negativeeffect of salinity. In addition, the siderophore was purified by using immobilizedmetal affinity chromatography (IMAC) and identified the type. The specific results srepresented as follows:1. Strain Q1 was isolated from the rhizosphere in greenhouse and identified as Trichoderma asperellum based on its morphological features and the molecularphylogenetic analyses. The Trichoderma asperellum Q1 showed antagonism againstmany phytopathogenic fungi, such as F. oxysporum f. sp. Cucumerinum, F. oxysporumf. sp. Niveum and Fusarium solani. In addition, it exhibited some plantgrowth-promoting attributes of phosphate solubilization,1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, auxin and siderophoreproduction. These results indicated that Trichoderma asperellum Q1 as biologicalcontrol agents are able to produce potential growth-promoting metabolites tostimulate plant growth.2. The pot trial was conducted to study the effects of Trichoderma asperellum Q1on the growth of cucumber seedlings by assaying the changes in physiological andbiochemical parameters and photosynthetic efficiency under salt stress. Fourtreatments were set up as follows: cucumber seedlings inoculated with strain Q1 (Q1),strain Q1 and NaCl (Q1+salt), uninoculated but NaCl-treated (salt), uninoculated andwithout salt stress (control). Cucumber seedlings were inoculated with sporesuspension of strain Q1 with or without exposure to salt conditions, and changes ingrowth and biochemical parameters such as plant height, chlorophyll content, theosmosis molecules soluble sugar, soluble protein, malondialdehyde (MDA) and theactivities of antioxidant enzyme superoxide dismutase (SOD), peroxidase (POD) incucumber leaves as well as several plant physiological parameters like netphotosynthesis (PN), transpiration rate (Evap), stomatal conductance (GS),intercellular CO2concentration (C Int) were evaluated at 25 days after inoculation.The results indicated that application of strain Q1 could significantly promoteseedlings growth and alleviated growth suppression induced by salt stress comparedwith the uninoculated and salt stressed controls of cucumber seedlings.3. Furthermore, the role of siderophores was proved through solution cultureexperiments. In culture solution with limiting iron and 60 mmol l-1NaCl stress, fourtreatments were set up as follows: SCF with and without 30μmol l–1Fe3+(SCF±Fe),uninoculated MM9 medium with and without 30μmol l–1Fe3+(Non-SCF±Fe), andthe treatment of Non-SCF-Fe was used as control. The result indicted that the biomass of cucumber seedlings is maximum under the treatment of siderophore culture filtrate(SCF) of strain Q1 with insoluble Fe3+in salt stress, compared to the other treatments.Meanwhile, cucumber shoot or root length and first euphylla leaf area were increased,and the percentage of wilted cucumber seedlings was decreased greatly. This clearlydemonstrated that siderophores may play an important role in alleviating thedeleterious effects of salinity to cucumber seedlings, especially under iron deficientconditions.4. For identified the type of siderophore produced by Trichoderma asperellumstrain Q1, the siderophore-containing culture filtrate is purificated by usingimmobilized metal affinity chromatography (IMAC). It was identified as hydroxamatetype siderophore and no fluorescence based on MTT test. This result supplementedeffectively study of siderophore types, and may provided theoretical basis for thechemistry structure study and its role in iron transport.更多还原