【作者】 夏娟娟；

【导师】 盛下放；

【作者基本信息】 南京农业大学 ， 微生物学， 2006， 硕士

【摘要】 土壤重金属污染植物修复技术具有绿色廉价、操作简单等优点，但是超积累植物往往生物量低、生长速度慢。而现有的强化植物修复的物理化学等方法又存在修复成本较高，环境风险大等缺点，因此寻求一条大幅度提高植物修复效率，并且环境友好且成本低廉的新的微生物强化植物修复途径，将具有重要的理论意义和现实意义。本研究从生长于污染区的野生油菜、白菜、玉米等植物组织中分离出80株具有重金属抗性的内生细菌，并根据其对重金属的抗性及促生效应筛选到在添加或不添加铅镉的情况下均对油菜及小麦具有较好促生作用的菌株G10、G16和B5。通过形态学观察及生理生化特征测定，初步鉴定G10属于假单胞菌属（Pseudomonas sp．），G16属于短杆菌属（Brevibacterium sp．），B5属于固氮单胞菌属（Azotomonas sp．）。对供试菌株与促生相关的生物学性能进行了测定，结果表明菌株G10具有溶磷、分泌IAA和iPA、产生铁载体和ACC脱氨酶以及拮抗油菜菌核病痛原菌（Sclerotinia sclerotiorum）和小麦纹枯病病原菌（Rhizoctonia cerealis）)的能力；菌株G16具有固氮、分泌IAA和iPA、产生铁载体和ACC脱氨酶的能力；菌株B5具有固氮、溶磷、分泌IAA和iPA、产生铁载体和ACC脱氨酶以及拮抗油菜菌核病病原菌（Sclerotinia sclerotiorum）和小麦纹枯病病原菌（Rhizoctonia cerealis）的能力。研究环境因素对菌株生长的影响，试验结果表明菌株G10和B5在7-37℃和pH5.0-10.0范围内正常生长，G16在17-37℃和pH6.0-10.0范围内生长较好。最适温度为28℃，适宜pH为7.0-9.0左右。供试菌株对高渗环境适应性强，均能在5％的盐浓度下良好生长。菌株对重金属和抗生素具有不同的抗性，G10能在含Pb²⁺ 200mg·L^-1、Cd²⁺20mg·L^-1、Zn²⁺100mg·L^-1、Km 20mg·L^-1、Amp 100mg·L^-1、Str 20mg·L^-1、Spe 20mg·L^-1的固体培养基中良好生长，G16能在含pb2²⁺ 200mg·L^-1、Cd²⁺ 20mg·L^-1、Cu²⁺ 50mg·L^-1、Ni²⁺ 20mg·L^-1、Zn²⁺100 mg·L^-1、Km 20mg·L^-1、Amp 100mg·（-1）、Spe 20 mg·L^-1的固体培养基中生长；B5能耐受pb²⁺ 200mg·L^-1、Cd²⁺ 20mg·L^-1、Cu²⁺ 50mg·L^-1、Zn²⁺ 100mg·L^-1、Km 20mg·L^-1、Amp 100mg·L^-1、Str 20mg·L^-1、Tc 20mg·L^-1和Spe 20mg·L^-1，而对Ni²⁺、Cr⁶⁺和Rif敏感。供试菌株对PbCO₃和CdCO₃的溶解试验表明，菌株能通过代谢产酸使PbCO₃和CdCO₃溶解，G10、G16和B5能分别使含PbCO₃的有氮改良培养基pH值由8.65下降到7.32（G10）、7.28（G16）和7.69（B5），分别使含CdCO₃的有氮改良培齐基pH值由7.20下降到4.68（G10）、5.55（G16）和5.39（B5），有效态Pb比对照高131-328μg·L^-1，有效态Cd比对照高69.95-359.65mg·L^-1。此外，G16还具有较强的产表面活性剂的能力。将利福平标记的菌株用浸种法和浇菌液法对油菜进行回接试验，30d后的检测结果表明G10和G16既能够在植株根际土壤中定殖又能够在油菜根内定殖，属于兼性内生细菌，B5也能够在植株根际较好的存活。研究标记菌株对油菜富集铅镉的影响，结果表明，菌株能促进油菜的生长，使油菜地上部和地下部干重比对照高6.4-77.7％和2.9-149.1％；接菌处理能够使得植株地上部和地下部铅含量比对照增加3.0-81.4％和5.6-43.7％，地上部和地下部镉含量增加3.0-24.8％和11.2-77.7％。在Pb 800mg·kg^-1、Cd 100mg·kg^-1和Pb 400 mg·kg^-1+Cd 100mg·kg^-1的处理中效果显著。接菌处理根际土中有效态重金属的浓度、可溶性糖含量高于不接菌对照，根际土pH值低于对照。菌株在中等程度铅镉污染土壤中的油菜根内和根际能发挥强化植物修复的作用。更多还原

【Abstract】 Phytoremediation is an emerging as a cost-effective and environment-friendlytechnology for remediation of heavymetal-contaminated soils. However, phytoremediators,the plants used for remediation, often have a slow growthrate and produce limited amountsof biomass. Using chemicals to enhance phytoremediation is comparatively available butusually expensive and unfriendly to environment. Therefore, it is very important to searchfor a microbe-strengthened phytoremediation approach to enhance plant remediationeconomically.Bacterial strain G10, G16 and B5 were isolated from root of plants. They were capableof promoting rape and wheat growth either in the presence or absence of Pb²⁺ or Cd²⁺. Thethree strains were primarily identified as Pseudomonas sp.（G10）, Brevibacterium sp.（G16） andAzotomonas sp.（B5）, respectively, based on their morphological and physio-biochemicalcharacteristics.All the strains produced indole acetic acid （IAA）, isopentenyl adenosine （iPA） orsiderophores, and they contained the enzyme 1-aminocyclopropane-1-carboxylate （ACC）deaminase, which hydrolyses ACC （the immediate precursor of plant hormone ethylene） toNH₃ and a-ketobutyrate. Besides, strain G10 exhibited the capability of solubilizinginorganic phosphate and antagonism against Sclerotinia sclerotiorum or Rhizoctoniacerealis. Strain G16 and B5 were able to fix nitrogen. The results of the influence of thetemperature, pH, osmolality, heavy metals and antibiotics on the growth of the strainsshowed that strains G10, B5 grew well at temperature 7℃to 37℃and pH 5.0 to 10.0.The optimum growth conditions were: temperature 28℃, pH 7-9. All strains could growwell in the medium containing 5% NaCl. Strain G10 could grow in the medium added Pb²⁺(200mg·L^-1), Cd²⁺ (20mg·L^-1), Zn²⁺ (100 mg·L^-1), Km (20 mg·L^-1), Amp (100 mg·L^-1),Str (20 mg·L^-1), Spe (20 mg·L^-1) and G16 could grow in the medium added Pb²⁺ (200mg·L^-1), Cd²⁺ (20 mg·L^-1), Cu²⁺ (50 mg·L^-1), Ni²⁺(20 mg·L^-1), Zn²⁺ (100 mg·L^-1), Km (20mg·L^-1), Amp (100 mg·L^-1), Spe (20 mg·L^-1). Strain B5 showed tolerance to Pb²⁺ (200 mg·L^-1), Cd²⁺ (20 mg·L^-1), Cu²⁺ (50 mg·L^-1), Zn²⁺ (100 mg·L^-1), Km (20 mg·L^-1), Amp(100 mg·L^-1), Str (20 mg·L^-1), Tc (20 mg·L^-1) and Spe (20 mg·L^-1). Experiments onactivation of PbCO₃ and CdCO₃ showed that the contents of available Pb in solutionsinoculated with strains were 131-328μg·L^-1more than that of controls while the pH valueswere decreased to 7.32 （G10）, 7.28 （G16） and 7.69 （B5） from 8.65; the contents ofavailable Cd in solutions inoculated with strains were 69.95-359.65 mg·L^-1 more than thatof controls while the pH values were decreased to 4.68 （G10）, 5.55 （G16） and 5.39 （B5）from 7.20. Furthermore, the strains were able to produce suffactant.The three strains labeled by rifampicin were used to inoculate seeds of rape, which weresowed in Pb and Cd-supplemented soils. After 30 days, strain G10 and G16 were isolatedsuccessfully from roots of rape, and the three strains have colonized well aroundrhizosphere. The pot experiments clearly demonstrated the beneficial effects of strains onthe plant growth. Plant dry weights were significantly affected （P＜0.05） by strains, andincreased by 6.4-77.7% （shoot） and 2.9-149.1% （root）. Bacterial inoculations produced upto 3.0-81.4% （shoot） and 5.6-43.7% （root） increase in the efficiency of lead phytoextractionby rape. Cd concentrations within plant tissues were increased by 3.0-24.8% （shoot） and11.2-77.7% （root） afte rinoculated with strains, too. The strains stimulated rape biomassproduction and, hence, its phytoextraction. The contents of available Pb, Cd anddissolubility sugar in the soils inoculated with strains were increased, while the pH valuesof rhizosphere soil were decreased compared with the uninoculation control. Therefore, thethree strains had efficacy in rape rhizosphere and could strengthen phytoremediation inmoderate HM contaminated soils.更多还原