【作者】 孙权；

【导师】 何振立； 杨肖娥；

【作者基本信息】 浙江大学 ， 农业资源利用， 2008， 博士

【摘要】 本文以"水稻-青菜"轮作制土壤生态系统为对象,综合运用室内外培养、作物盆栽试验、模型模拟,借助微生物代谢指纹图谱方法（Biolog）、磷脂脂肪酸（PLFA）等生物技术并结合传统微生物学方法,研究了黄红壤、青紫泥和小粉土等三种水稻土粮-菜轮作系统主要污染重金属铜对水稻和青菜的毒性效应,并重点探讨了铜胁迫下土壤微生物生态学特征（微生物生物量、呼吸、酶活性、区系组成、代谢剖面、群落结构等）。主要结论如下:1.铜污染对植物生长的毒害作用受植物种类和土壤理化性质的双重影响。铜对水稻和青菜生长的剂量效应表现为低铜浓度(50 mg kg^-1)促进作物的生长,地上、地下部分生物量增加（小粉土除外）;100 mgkg^-1铜显著增加了水稻的根系长度,增加了水稻的分蘖数、株高、穗长、从而显著增加总茎数,产量增加。更高的铜浓度则产生显著的抑制作用。黄红壤和青紫泥水稻铜致死浓度分别为1200 mg kg^-1和1600 mg kg ^-1,而小粉土为50 mgkg^-1;三种土壤改种青菜后的表观致死浓度都为800 mg kg^-1。铜对作物毒害的生理基础为,50 mg kg^-1Cu²⁺促进青菜根系体积、直径和叶绿素增加及水稻产量构成因素,青菜根系长度和表面积及水稻总茎数及单茎成穗数显著增加,显著增产。更高的铜浓度则抑制了SOD活性、叶绿素含量及光合作用,丙二醛MDA含量下降,脯氨酸含量显著下降,造成青菜减产。外源铜浓度大于400mg kg^-1则极显著抑制水稻叶绿素合成,减少穗数、穗粒数和千粒重,产量下降。2.水稻茎叶对铜的吸收随土壤铜污染浓度的升高而呈指数式增长,水稻根系和籽粒中的铜浓度随外源铜浓度的增加而极显著线性增加。水稻不同的器官对土壤铜吸收的顺序为:根》茎叶>糙米。但外源铜浓度超过400 mg kg^-1能够导致糙米铜含量超过食品安全标准。青菜根系的铜含量高达77.77%-80.41%,随铜浓度的增加,青菜地上部分和根系中铜的累积量相应增长,但青菜地上部分铜的相对累积量随铜浓度的增加而下降,从而大大减少高铜污染下铜在青菜可食部位的积累。3.三种土壤中的铜均是以Fe-Mn氧化物结合态为主。四种提取剂提取的有效铜含量和外源铜总量均呈极显著的正相关。各种提取态铜之间以及各种形态铜与水稻产量、青菜生物量之间呈显著或极显著负相关,与水稻及青菜组织体内累积的铜呈极显著正相关。其中,以0.5 mol L^-1DTPA提取铜与外源铜的相关性最好,可以作为土壤有效性铜的最佳化学提取剂。4.因溶解性能源物质数量的消耗,三种水稻土的微生物生物量碳、基础呼吸速率及微生物商随培养时间的延长而显著下降;而在轮作中因根系不断分泌有机物及残根分解的刺激作用而显著增加。三种土壤微生物生物量碳及基础呼吸速率对铜胁迫的剂量效应表现为在低铜浓度下(＜400 mg kg^-1)增加或显著增加,更高铜浓度因毒害作用而显著下降。土壤微生物代谢商随铜污染浓度的提高及培养时间的延长而升高。但在粮.菜轮作系统中稻作淹水下,随铜污染浓度的提高,黄红壤的微生物代谢商和呼吸速率增加,青紫泥和小粉土的微生物代谢商降低。改青菜旱作后,土壤的微生物代谢商和呼吸速率都随铜污染浓度的提高而降低。5.低铜浓度(50-100 mg kg^-1)显著促进了过氧化氢酶、脲酶和蔗糖酶活性,高铜污染浓度则抑制了酶活性。四种酶对铜污染敏感的顺序为:脲酶＞蔗糖酶＞磷酸酶＞过氧化氢酶;粮-菜轮作体系中,根系及其分泌物的共同作用促进土壤酶活性,尤其是水作改旱作后,酶活性进一步极显著提高。6.铜污染胁迫下,三种土壤可培养微生物总量在低铜浓度下随铜污染浓度的逐步提高而显著提高;但当铜浓度超过400 mg kg^-1及随培养时间的延长,铜污染的抑制作用增强。肥沃土壤青紫泥和黄红壤可培养微生物总量随培养时间的延长而显著下降,而贫瘠土壤小粉土可培养微生物总量则因环境条件的改善而随培养时间的延长而提高。铜胁迫使土壤微生物区系组成结构也发生显著变化。三种土壤培养过程中因耐性细菌出现而维持细菌的相对比例不降反升;真菌比例随铜污染浓度的提高而先升后降;而放线菌的比例随铜污染浓度的提高而显著降低。7.铜胁迫导致土壤微生物群落结构或功能多样性发生改变。三种土壤之间的C₁₂-C₂₀磷脂脂肪酸（PLFA）的含量达到显著差异;每种土壤内所含有的C₁₂-C₂₀不同碳结构PLFA则达极显著差异。这种差异首先表现在可检测PLFA的类型变化。培养8周时,100-400 mg kg^-1铜显著促进了可以命名的微生物类型,800 mg kg^-1铜则显著抑制,以贫瘠的小粉土表现最典型。随培养时间延长至52周,各土壤可命名磷脂脂肪酸类型增加,且低铜浓度促进磷脂脂肪酸类型增加,高铜浓度则显著抑制。可见,铜胁迫并非改变被测土壤LFA结构的唯一影响因素。其次,铜污染显著改变了微生物群落主要构成成分的结构比例。三种土壤PLFA随培养时间而变化。黄红壤和青紫泥微生物群落都以细菌占绝大多数,真菌和放线菌类型与数量比例都很低,与培养计数法获得的结果非常一致。小粉土对照土壤培养8周时真菌占比例高达58.58%,细菌只占27.51%,放线菌未能测出;而培养52周时,细菌比例高达69.5%,真菌比例下降到6.85%,放线菌仅占0.69%。即,小粉土即使在无铜污染的情况下,微生物群落结构也发生了剧烈改变。当然,铜胁迫进一步促进了土壤微生物群落结构的改变。细菌和放线菌的比例随铜污染浓度的提高而显著提高,真菌的比例和真菌/细菌的比例都随铜污染浓度的提高而显著下降。此外,铜污染导致土壤微生物产生适应性或耐性反应,一些特征磷脂脂肪酸消失或涌现。铜胁迫使甲烷营养菌Ⅰ型16∶1w5c和好氧细菌15∶0 3OH、16∶0 2OH及硫酸盐还原细菌17∶1 w8c消失,厌氧细菌17∶0 ISO 30H和真菌20∶2W6,9C受激发作为新种出现,革兰氏阴性细菌（17∶0 CYCLO和19∶0CYCLO w8c）和革兰氏阳性细菌（4∶0 iso,15∶0 anteiso,15∶0 iso,15∶0 iso 3OH,16∶0 iso,17∶0 anteiso,17∶0iso）大量涌现。8.铜胁迫降低了土壤的微生物群落代谢剖面（AWCD）,且AWCD值与测试时间之间呈非线性关系。三种土壤Biolog生态盘碳源利用的铜浓度效应受培养时间的影响而存在一定差异。随着铜污染浓度增加到800 mg kg^-1时,土壤的AWCD值下降,微生物群落生理代谢功能下降。三种土壤在31种碳源构建的主成分分析坐标体系中存在明显的空间分异,表明微生物群落结构对铜污染非常敏感。三种土壤铜污染微生物群落的功能多样性指数在同一时间的不同浓度和同一浓度的不同污染时间下存在差异。高铜浓度(＞800 mg kg^-1)导致丰富度显著下降,均匀度和Shannon指数也下降,微生物种群变得单一。9.根据铜污染对水稻、青菜生长的抑制效应,以及粮.菜轮作系统中土壤微生物生物量、微生物商、酶活性等生态指标的铜胁迫剂量效应,以水稻、青菜减产10%,水稻籽粒或青菜可食部位铜含量不超过10 mg kg^-1,特别是运用生态剂量(ED₁₀)的概念,根据上述各项指标与铜浓度对数值的模拟模型,计算了黄红壤、青紫泥和小粉土的铜临界指标分别为198、185和71 mg kg^-1。更多还原

【Abstract】 Three paddy soils namely yellowish red soil（YRS）,purplish clayey soil（PCS） and silt loam soil（SLS）within ’rice-Chinese cabbage’ rotation system were collected from Jiaxin county,Deqing County,and Xiasha District of Hangzhou City,Zhejiang province.Laboratory and greenhouse pot experiments combined with soil biochemical measurements,Biolog and Phospholipids fatty acids analysis were adopted to investigate the toxic effects of copper on both rice and cabbage growth. The research also focus on studying the toxic effects of Cu²⁺on soil microbial biological characteristics such as soil microbial biomass,soil basal respiration,soil enzyme activities,and soil microbial community structures.The results are summarized as follows:1.Toxic effect of Cu on plant growth depends on either vegetative types or soil basic physical-chemical characteristics.Lower Cu loading(50 mg kg^-1)promote cabbage growth except SLS.100 mg kg^-1Cu loading promote rice yield mainly because the increase of root length,tillers,spike length,as well as the number of total stems.Yet higher concentration of Cu has restrictive effect.The lethal concentration of Cu is 1200 mg kg^-1and 1600 mg kg^-1for YRS and PCS respectively,whereas it is 50 mg kg^-1for SLS under rice planting system.However,the lethal concentration is 800 mg kg^-1for all the three paddy soils under cabbage growing system.There are some physiological differences of Cu toxicity between rice and cabbage.50 mg kg^-1Cu loading boost root volume,root diameter,SPAD value,root length,and root surface area.Yet higher Cu loading restrict SPAD and photosynthesis, superoxide dismutase（SOD）activity,Malondialdehyde（MDA）levels,and proline content.The result is cabbage yield declined.Yet 50 mg kg^-1Cu loading prominently boost total stems and spikes,which increase the grain yield.However,higher Cu loading(＞400 mg kg^-1)extremely restrict chlorophyll synthesis,decrease spikes, grain number per panicle,as well as 1000-grain weight,led to grain yield dropped.2 The absorption of Cu by rice stalks and leafs is exponentially promoted along with the increased Cu loading.Cu content in rice roots and seeds is extremely enhanced along with the increased Cu loading.Cu absorption order within different rice organs is:roots＞stalks and leafs＞seeds.Cu accumulative amount both in cabbage upper and under ground part is rise along with increased Cu loading.Cu content in roots of cabbage is 77.77%-80.41%.Much more Cu was stored in roots than in leafs, which is benefit to reduce Cu accumulation in edible part of cabbage.3.The main fractionated Cu conponent in three soils were Fe-Mn oxide-fraction. The amount of available Cu²⁺was notably positive correlated with extracted Cu²⁺by four extractants(water,0.1 mol L^-1HCl、0.5 mol L^-1DTPA、and 1 mol L^-1NH₄OAc （pH7.0）.All kind of extracted Cu was prominently negative correlated with rice yield, fresh cabbage leaf weight,fresh cabbage root weight,and dry leaf weight,but notably positive correlated with accumulated Cu within rice and cabbage organs.The quantity of 0.5 mol L^-1DTPA extracted Cu has the highest correlations shown that it is the best extractant for available Cu extraction.4.Both soil microbial biomass carbon,basal respiration and microbial quotient （MQ）was decreased along with prolonged incubation time mainly because easy usable available C resource provided by organic C mineralization was decreased. However,they were increased because root residual and its exudation provide available C resource for microbial biomass usage when soil planting rice and cabbage. The dose-effect of Cu on microbial biomass C is notably increased less than 400 mg kg^-1Cu loading.Yet higher Cu loading lead to extremely restriction effect on it.Soil microbial metabolic quotient（MMQ）was increased along with increased Cu loading and prolonged incubation time for rice growth.Yet it was decreased in all three paddy soils when planting cabbage.5.Catalase,urease,invertase,and phosphatase activities were notably promoted when Cu loading less than 100 mg kg^-1.Yet restrict effect were observed when Cu loading higher than 100 mg kg^-1.Sensitivity of the four enzymes to Cu contamination is urease＞invertase＞phophatase＞catalase.Enzymes activities were remarkable increased by planting rice and cabbage by decomposition of residual rice root and their exudation.6.Total microorganism was increased along with enhanced Cu loading less than 400 mg kg^-1.Yet extremely restriction effect were observed for PCS and SLS when Cu loading higher than 400 mg kg^-1.Total microorganism was reduced along with incubation time for fertile YRS and PCS.However,opposite effect was observed for SLS.Microorganism structures were also changed under Cu pollution.Bacteria ratio was notably increased along with enhanced Cu loading which may caused by the appearance of endurance bacteria.Fungal ratio was augmented because it’s high resistance capability for threatening environment.Actinomycete ratio was decreased along with increased Cu loading.7.Cu contamination leads to the changes of both microbial biological structures and functions.Notable differences of C₁₂-C₂₀phospholipids fatty acid（PLFA）were measured among three paddy soils and within each of them.The first difference was measurable types of PLFA.In 8^thweek incubation time under 100-400 mg kg^-1Cu loading,the types of PLFA were.However,800 mg kg^-1Cu loading prominently restricted the PLFA types especially for SLS.But in 52^thweek incubation time,the types of PLFA were increased.Great changes were happened not only caused by increased Cu loading,but also caused by prolonged incubation time.The second changes were the ratios of main microorganisms.Bacteria account for absolutely most of the microorganism,whereas quite low percentage of fungal and actinomycetes in YRS and PCS.The result is coincides with the traditional culture method.Very simple PLFA structure was measured for non polluted SLS at 8^thweek incubation time which fungal account for 58.58%of total PLFA,bacteria account for 27.51%,0%for actinomycetes.But the percentage is changed to 6.85%,69.50%,and 0.69%in 52th week incubation time which shown that Cu loading is not the only factor to affect microorganism structure.The ratio of bacteria/total PLFA, actinomycetes/total PLFA were promoted,while the ratio of fungal/total PLFA and fungal/bacteria was decreased along with enhanced Cu loading.In addition,Cu pollution causes flexible or forbearing reaction of microorganisms.Some indicative PLFAs were appeared or disappeared.For example, Cu loading causes methane nourish bacteria-16:1w5c,aerobiosis bacteria-15:0 3OH, 16:0 2OH,and sulphatedeoxidize bacteria-17:1 w8c totally vanish,whereas anaerobicbacteria-17:0 ISO 3OH,and fungal-20:2W6,9C emergence as new PLFA types by Cu pollution inspiring in YRS.The G-bacteria（17:0 CYCLO and 19:0CYCLO w8c）and G⁺-bacteria（4:0 iso,15:0 anteiso,15:0 iso,15:0 iso 3OH,16:0 iso,17:0 anteiso,17:0 iso）was increased greatly.8.Cu pollution decreased microbial community metabolic profile（AWCD）.No linear relationship exists between AWCD and test time.Some differences were observed for carbon resource utility by microorganisms under different Cu loading. 100 mg kg^-1Cu loading promotes AWCD,yet further increased Cu loading to 800 mg kg^-1cause decrease of AWCD.Main components analysis showed that AWCD among 31 carbon resource utilization rate were distribute widely which indicate the sensibility of microbial community structures to Cu contamination.Differences also exist among soil microbial functional diversities under changed Cu loading levels.Shannon index,Shannon Richness and Evenness of microbial community were decreased under higher Cu loading(＞800 mg kg^-1)indicated that the microbial community structure trends to singularity.The usage ability of C resource by microbial community in all three paddy soils was affected by Cu loading rate.100-400 mg kg^-1Cu loading promote C resource utility,but 800 mg kg^-1Cu loading notably restrict microbial community metabolic activity as their structures were simplified.These results demonstrated that both microbial community structure and microbial functional diversity can be used as sensitive bio-indicator for reflecting heavy metal caused environment changes.9.According to the restriction effect of Cu pollution to rice and cabbage growth, and to soil microbial ecological characteristics,we were modeling regression equations to calculate the critical concentration for these three paddy soils by using 10%yield reduction,food security sanitary criteria,and ecological dose as indexes. The critical levels of Cu are 198,185,and 71 mg kg^-1for YRS,PCS and SLS respectively.更多还原