【作者】 申屠佳丽；

【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】 通过实验室模拟研究了我国长江三角洲地区三种典型土壤（红黄壤、小粉土、青紫泥）中镉的生物化学特性及镉污染对微生物活性的影响;系统研究了青菜-番茄-萝卜轮作体系中镉在不同蔬菜体内的积累、分配规律,并结合水培试验探讨低浓度镉对蔬菜生长及生理特性的影响。应用传统土壤微生物学研究方法及BIOLOG微生物碳源利用（功能）多样性、磷脂脂肪酸（PLFA）等群落结构分析方法研究了蔬菜轮作制下,镉胁迫对红黄壤、小粉土蔬菜根际及非根际土壤微生物生物量、代谢活性及群落结构多样性的影响,并探讨了土壤微生物对蔬菜轮作制及蔬菜根系活动的响应。探索了蔬菜土壤镉污染化学-微生物学诊断方法及蔬菜可食部镉卫生品质安全指标。主要研究结果如下:1.Langmuir方程和Freundlich方程可以很好地拟合镉在三种土壤（红黄壤、小粉土、青紫泥）中的吸附行为。三种土壤中,青紫泥对镉的吸附能力最强,其次为小粉土,红黄壤最弱,根据计算,其对Cd²⁺的最大吸附量分别为6098,4065和3534mg kg^-1;镉吸附能力的差异主要受土壤pH值、粘粒含量、CEC等因素影响。Cd²⁺的吸附导致等温吸附平衡液的pH显著下降,其下降幅度依土壤类型及镉吸附量而变化。被土壤吸附的Cd²⁺大部分不能被解吸,Cd²⁺在三种土壤中的解吸行为差异明显,经过3次连续的解吸,红黄壤吸附态镉0-15%被解吸,小粉土吸附态镉0-9.1%被解吸,而青紫泥只有0-6.8%的吸附镉被解吸,即相同吸附量下,红黄壤镉解吸量最大,小粉土次之,青紫泥最小,说明青紫泥对Cd²⁺的亲和力最大,红黄壤最小。2.镉在蔬菜体内的积累和分配随土壤类型、蔬菜种类及土壤镉含量的不同而不同。青菜体内镉含量的分配为根部＞地上部,番茄体内镉含量的分配为根部＞地上部＞果实。青菜和番茄根系中镉的含量比较高,只有小部分镉可以通过转运在蔬菜地上部积累。青菜和番茄地上部及根系镉含量都随着土壤外源镉含量的增加而增加,但根系的增加速率比地上部快。而萝卜体内镉的积累分配规律和青菜、番茄不同。同一土壤镉处理条件下,地上部镉含量则比根部高,这主要是由于不同植物品种的生理特性不同。不同土壤同一蔬菜体内镉含量也存在差异,红黄壤上生长的蔬菜可食部含量高于小粉土蔬菜。每种蔬菜种植后,土壤NH₄OAc提取态和饱和水提取态镉含量都随着全量的增加而增加,而醋酸铵对镉的提取能力远高于饱和水。同一土壤种植不同蔬菜后可提取态镉含量存在差异,特别是饱和水可提取态。红黄壤对镉的吸附能力较弱,种植同一蔬菜后,红黄壤NH₄OAc和饱和水提取态镉含量高于小粉土。3.土壤中0-7.00mg kg^-1镉对青菜、番茄、萝卜生长都没有明显的抑制作用,且低浓度镉可以刺激蔬菜地上部及根系的生长;而在水培试验中,高浓度镉(＞0.25mg L^-1)显著降低青菜地上部干重。青菜根系各形态指标（除根体积外）对镉含量的变化比较敏感,当土壤中镉浓度为4.00mg kg^-1,培养液中镉浓度为0.05 mg L^-1时,青菜根长,根直径以及根表面积达到最大值。低浓度镉促进青菜叶绿素合成,而高浓度镉则导致青菜叶绿素含量下降,叶片光合特性受抑制。高浓度镉(7.00mg kg^-1（土培）,0.50mg^-1（水培）)增加青菜体内丙二醛含量（MDA）;过氧化物酶（POD）在低镉浓度下(0.70mg kg^-1（土培）,0.05 mg L^-1（水培）)活性增强,随着镉浓度的进一步增加,其活性下降,其抑制作用增强。镉对青菜体内脯氨酸（Pro）积累的影响并不明显。4.采用室内模拟培养试验研究了相对高浓度镉(0-128mg kg^-1)对红黄壤、小粉土、青紫泥微生物生物量及其活性的影响。结果表明,微生物生物量受土壤类型和镉胁迫程度的影响。红黄壤、青紫泥较小粉土有更高的微生物生物量碳,且不同土壤微生物生物量对镉的响应存在差异。低浓度镉对红黄壤、小粉土微生物生物量碳有刺激作用,但在0-128mg kg^-1的处理浓度范围内没有发现镉对微生物生物量的显著抑制作用,而镉对青紫泥微生物生物量碳的影响不显著。镉影响土壤微生物代谢活性,8mg kg^-1镉对土壤基础呼吸作用有刺激作用,从外加镉浓度为16mg kg^-1起,刺激作用减弱,但大多数情况下仍然高于对照。不同土壤的微生物代谢熵在不同的培养时期对镉的响应趋势存在差异,低浓度镉(4-16mgkg^-1)对三种土壤不同培养时期的微生物代谢熵都有促进作用,长时间镉胁迫下(16周,＞16mg kg^-1),红黄壤和青紫泥的微生物代谢熵低于对照。镉对三种土壤的微生物商的影响基本类似,低浓度镉影响下(0-16mg kg^-1),微生物商随镉浓度的增加而增加,而随着镉胁迫的进一步加强,微生物商呈下降趋势。不同土壤的脲酶活性在不同培养时期对镉胁迫的响应不尽相同。在培养初期（2、4周）低浓度镉(＜16mg kg^-1)对红黄壤和小粉土的脲酶活性有刺激作用,而随着培养时间的延长,高浓度镉对脲酶活性有比较明显的抑制作用。5.在青菜-番茄-萝卜轮作系统中,土壤微生物生物量和微生物商受到低浓度镉(＜1 mgkg^-1)的刺激,随着镉浓度的进一步增加而下降,但7.00mg kg^-1镉处理时微生物商仍高于对照。在低浓度镉(＜1.00mg kg^-1)影响下,土壤基础呼吸作用和微生物代谢熵略有减弱,随着外加镉浓度的增加,呼吸作用及微生物代谢熵也随之增强。青菜-番茄-萝卜轮作制不同作物对土壤微生物活动也有影响。种植番茄后的土壤基础呼吸速率、微生物代谢熵高于青菜种植后土壤;而萝卜种植后土壤基础呼吸速率及微生物代谢熵下降。而微生物生物量碳和微生物商随种植蔬菜的变化规律和微生物代谢活性不同,番茄种植后土壤微生物生物量碳和微生物商下降,而萝卜种植后则上升。根际土壤的基础呼吸作用、微生物生物量碳、微生物代谢熵及微生物商等都高于非根际土壤。红黄壤的基础呼吸作用和微生物生物量碳高于小粉土,而红黄壤的微生物代谢熵及微生物商则低于小粉土,这说明红黄壤单位微生物的活性以及对有机质的矿化能力比小粉土弱。6.青菜-番茄-萝卜轮作系统中,土壤微生物群落结构受到外源镉浓度和蔬菜种类的影响。青菜种植后,土壤微生物群落对碳源的利用能力随镉浓度的增加而下降,而萝卜种植后则随镉浓度的增加而升高。同一镉处理条件下,根际土壤微生物对碳源的利用能力强于非根际土壤。萝卜种植后土壤微生物群落更加趋于稳定,对外界胁迫的适应性增强。微生物群落丰富度及Shannon指数随镉浓度的变化规律不稳定,在不同土壤类型,不同蔬菜种植条件下规律不一致,不能较好地反映不同外加镉处理对土壤微生物群落结构的影响。不同微生物类群的脂肪酸含量对镉胁迫的响应不同。真菌、革兰氏阳性菌以及灌木菌根真菌随土壤镉含量的增加而增加,而细菌、革兰氏阴性菌、放线菌则随土壤镉含量的增加而下降。灌木菌根真菌（AMF）的特征脂肪酸16:1ω5c随着镉浓度的增加而增加,而且根际土壤中的AMF相对含量比非根际土壤高:不同土壤类型的微生物群落功能多样性及组成多样性存在较大的差异。根际土壤和非根际土壤微生物群落组成多样性差异明显,根际土壤受镉胁迫的影响较小,群落结构较为稳定,而非根际土壤的微生物群落结构更容易受到环境胁迫的干扰。萝卜种植后土壤微生物群落结构更趋向于稳态系统,微生物较能适应镉污染环境。7.通过生态环境效应法,基于土壤-植物体系和土壤-微生物体系,建立土壤镉污染诊断指标体系。结果表明,基于土壤-微生物体系建立的土壤镉污染临界值远高于基于土壤.植物体系,根据微生物指标建立的镉污染临界值不适于评价土壤镉污染的程度。当蔬菜卫生品质受到影响时,其产量及微生物活性没有受到影响,对于农田土壤镉污染诊断指标体系的建立应重点考虑食品卫生标准临界值。在确定土壤镉污染的农产品安全临界指标时,需要进行细化,考虑蔬菜种类、土壤类型及镉的有效性。本研究中NH₄OAc提取态镉能较好地代表镉在蔬菜中的生物有效性,种植青菜红黄壤和小粉土的NH₄OAc提取态相应临界值分别是0.066和0.116mg kg^-1;种植番茄红黄壤和小粉土的NH₄OAc提取态相应临界值分别是0.089和0.092mg kg^-1;而种植萝卜红黄壤和小粉土中的NH₄OAc提取态临界值分别是0.051和0.045mg kg^-1。更多还原

【Abstract】 Three representative soils（Red yellowish soil,silt loamy soil and purplish clayey soil）, which are widely distributed in Yangtze River Delta of China,were collected from Deqing County,Xiasha District of Hangzhou City,and Jiaxin county,Zhejiang province.Laboratory experiments were conducted to investigate the chemical and biological characteristics of cadmium（Cd）in soils and microbial response to external Cd loading.Greenhouse experiments with three vegetable crops in rotation were conducted using the two soils,i.e.red yellowish soil（RYS）and silt loamy soil（SLS）to study Cd accumulation in pakchoi（Brassica chinensis L.）,tomato（Lycopersicon esculentum）and radish（Raphanus sativus L.）.Root morphology and physiological characteristics of pakchoi were also studied using both soil and hydroponic culture.The effects of increasing Cd loadings on microbial biomass,microbial activity,and microbial community structure were examined and the influences of rotation system and root activity on microbe-Cd interactions were evaluated.Based on these experiments,critical Cd concentrations were established for RYS and SLS by using safety food health standards and microbiological indices.The safety food health standards of Cd criteria are compatible with those obtained from soil microbial response and appear more reliable for assessing Cd pollution in soil.The major findings from this study are summarized as follows:1.Adsorption of Cd²⁺（added as Cd（NO₃）₂）in the three soils was well described by a simple Langmuir equation or a Freundlich model.Behavior of Cd²⁺adsorption-desorption was related to soil properties and the adsorption capacity of Cd²⁺varied among the three soils in the order:PCS＞SLS＞RYS.The maximum adsorption values（X_m）of Cd²⁺obtained from the simple Langmuir model were 6098,4065 and 3534 mg kg^-1,respectively,for PCS,SLS and RYS.The adsorption of Cd²⁺caused a significant decrease in equilibrium solution pH.The extent of pH decrease depended on soil types and the amount of adsorbed Cd²⁺.Most of the adsorbed Cd²⁺couldn’t be released from soils.The behavior of Cd²⁺desorption differed considerably among soil types.After three successive extractions with NaNO₃,only 0 to 15% of the total adsorbed Cd²⁺in the RYS soil was desorbed and the corresponding values were 0 to 9.1%and 0-6.8%,respectively for the SLS and PCS soil,indicating that PCS has a higher affinity for Cd²⁺than RYS or SLS. 2.The accumulation of Cd in vegetables and the change of extractable Cd in soils after vegetable planted are affected by soil type,vegetable species,and external Cd loadings.For pakchoi and tomato,Cd was mainly accumulated in roots,with a portion of absorbed Cd being transported to the aerial parts and only a small proportion of Cd being accumulated in fruit.Cadmium concentrations in both shoots and roots（pakchoi and tomato）increased with increasing Cd loading levels,but root Cd concentration increased faster than shoot Cd. However,contrasting results were obtained with radish.At the same Cd loading rate,Cd concentrations were higher in shoots than roots.Large differences in tissue Cd concentrations were also observed among the three species.Tomato contained higher Cd in roots and shoots than pakchoi and radish,but Cd concentrations in tomato fruits were low.Analysis of variance revealed that soil type significantly affected Cd concentrations in vegetable tissues. Cadmium concentrations in vegetables grown in RYS were significantly greater than those in SLS at the same Cd loading rate.The results also indicated that ammonium acetate-and water-extractable Cd increased with total soil Cd after regardless of vegetable species planted. Ammonium acetate extracted a larger amount of soil Cd than water.Extractable Cd in soil, especially by water,was infuenced by vegetable species planted.Plant availability of Cd was generally higher in RYS than in SLS.3.Shoot growth of vegetables was not inhibited or even stimulated by light Cd pollution (0-7.00 mg kg^-1).Increased Cd²⁺concentration up to 0.25 mg L^-1inhibited the growth of pakchoi in hydroponic culture.The root growth of pakchoi was promoted by Cd addition at the concentrations＜3.50 mg kg^-1in soil culture and 0.05 mg L^-1in hydroponic culture.High Cd concentration(＞0.05 mg L^-1)resulted in the inhibition of root development,as evidenced by reduced root total length,root average diameter,and root surface area.The chlorophyll content of pakchoi increased by low Cd addition,but was reduced by increasing Cd concentration up to 0.25 mg L^-1.Photosynthesis was inhibited at high Cd concentrations (＞3.50 mg kg^-1in soil or＞0.25 mg L^-1in culture solution).The activity of peroxidase（POD） increased initially,but decreased with increasing concentration of Cd from 0.50 to 8.00 mg kg^-1in soil culture or from 0.05 to 0.50 mg L^-1in hydroponic culture.The content of malondialdehyde（MDA）increased when Cd concentration was 0.70 mg kg^-1in soil or 0.05 mg L^-1in culture solution,whereas the content of dissociative proline（Pro）fluctuated with increasing Cd in soil or culture solution. 4.The response of microbial biomass to Cd input is related to external Cd loading rate and soil types.RYS and PCS had higher microbial biomass carbon（MBC）than SLS,and the responses of MBC to Cd were different among soil types.Soil microbial biomass in RYS and SLS was enhanced by 4-8 mg Cd kg^-1soil,and the stimulating effect was less and less with increasing Cd rate up to16 mg kg^-1.The MBC in SLS was stimulated by lower Cd loading rate than RYS,as SLS had lower organic matter and clay content,which resulted in higher Cd bioavailability in the SLS.Soil respiration was enhanced at low Cd loading(＜8 mg kg^-1),but was restrained at higher Cd levels,but still larger than the control,especially in the medium-term of incubation.Although the response of microbial metabolic quotient（MMQ） to Cd was different among soil types or incubation periods,it was generally greater at low Cd loading(4-16 mg kg^-1)for different incubated time in the three soils.Low concentrations of Cd(0-16 mg kg^-1)slightly increased microbial quotient,whereas higher concentrations(＞16 mg kg^-1)of Cd²⁺had inhibitory effects.Urease activities in RYS and SLS were promoted when Cd loading was less than 16 mg kg^-1after being incubated for 2 or 4 weeks,while considerable restricting effect was observed when Cd loading was higher than 32 mg kg^-1 with prolonged incubation time.5.In the rotation system of three vegetables,soil microbial biomass and MQ were enhanced at low Cd levels(＜1.00 mg kg^-1),but was inhibited consistently with increasing Cd rate though the MQ value was still higher than that in control at the Cd rate of 7.00 mg kg^-1. Soil respiration and microbial metabolism quotient（MMQ）were restrained at low Cd loading (＜1 mg kg^-1),and enhanced at higher Cd levels.Microbial activities in three soils were affected by pakchoi-tomato-radish rotation system.The values of basal respiration rate and MMQ in tomato-growing soil were higher than those in pakchoi- or radish-growing soils. Microbial biomass and MQ in tomato-growing soils were lower than those in pakchoi- or radish-growing soil.Root activity of vegetables had a positive effect on soil microbes,the values of basal respiration,microbial biomass carbon,MMQ and MQ in rhizospheric soil were all greater than those in bulk soil.The rhizosphere effect varied between RYS and SLS, and among pakchoi,tomato and radish.Basal respiration rate and microbial biomass were greater in RYS than those in SLS,while SLS had higher values of MMQ and MQ.6.External Cd loading and vegetable species affected microbial community structure in pakchoi-tomato-radish rotation system.Microbial ability of metabolizing different carbon substrates was decreased with increasing Cd loading rate after pakchoi planted,while increased after radish harvest.With same Cd treatment,the ability of microbes in metabolizing carbon substrate in rhizospheric soil was greater than that in bulk soil.Microbial community structure was apt to be more stable after rotation.The responses of microbial community richness and Shannon index to Cd stress were different among soil types or vegetables,and were not suggested as available index to assess Cd pollution in RYS and SLS. The phospholipids fatty acid（PLFA）in soil microbe was sensitive to increased Cd concentrations.The relative content of general PLFAs in fungi,Gram-positive bacteria or arbuscular mycorrhizal fungi（AMF）was all positively correlated with available Cd in soil, while that in bacteria,actinomycetes or Gram-negative was all negatively correlated with available Cd in soil.Cadmium and root activity increased the amount of AMF.Both microbial functional diversity and PLFAs profiles differed between RYS and SLS soil,and between the rhizosphere and nonrhizosphere environment.Microbial community in rhizospheric soil suffered less Cd stress,and was more stable.7.Evaluation indices system for soil Cd pollution was established based on soil-plant system and soil-microbe system.The values of Cd thresholds based on soil-microbe system were much higher than those accoding to soil-plant system,and thus the microbial indices didn’t fit to assess Cd pollution in soil.Cadmium thresholds for potential dietary toxicity should be considered in establishing evaluation indices system for soil Cd pollution.The species of vegetables,soil types and Cd availability should be all taken into consideration for establishing Cd thresholds in soil.Ammonium acetate extractable Cd was suggested to assess Cd thresholds for potential dietary toxicity in pakchoi,tomato and radish.The corresponding values were 0.066 and 0.166 mg kg^-1,0.089 and 0.092 mg kg^-1,0.051 and 0.045 mg kg^-1in RYS and SLS,respectively in pakchoi,tomato and radish-growing soil.更多还原