【作者】 张树杰；

【导师】 胡锋；

【作者基本信息】 南京农业大学 ， 生态学， 2009， 博士

【摘要】 近年来,蚯蚓在重金属污染土壤植物修复中的应用得到了普遍的关注。许多研究表明,蚯蚓不仅能够提高污染土壤中重金属的生物有效性,还能促进植物生长、提高植物对重金属的吸收和向地上部的运输。这些结果说明,蚯蚓在螯合诱导植物修复重金属污染土壤中具有应用的潜力。但遗憾的是,蚯蚓提高土壤中重金属生物有效性、促进植物生长以及富集重金属的作用机理目前还不清楚。因此,本文设置了一系列的培养试验,主要研究：(1)威廉腔环蚓(Metaphire guillemi)粘液对Cd胁迫下番茄(Lycopersicon esculentum)合作903幼苗生长及Cd富集的影响；(2)蚯蚓粘液及其氨基酸模拟溶液对番茄幼苗生理指标及其体内Cd富集的影响；(3)蚯蚓粘液及其氨基酸模拟溶液对番茄幼苗中Cd的亚细胞分布及化学形态的影响；(4)蚯蚓对模拟污染土壤中Cd化学形态分布的影响,试图揭示蚯蚓提高土壤重金属生物有效性,促进植物对重金属吸收及向地上部运输的作用机理,为蚯蚓在植物修复重金属污染土壤中的应用提供理论依据。主要结果如下：(1)通过营养液培养试验研究了威廉腔环蚓(Metaphire guillemi)粘液对番茄(Lycopersicon esculentum)合作903幼苗生长及Cd富集的影响。结果发现,在Cd浓度为0和5mg L-1时,蚯蚓粘液显著提高了番茄幼苗干生物量,分别使根系及地上部干生物量增加了12.4-33.2%和13.8～44.5%；而在Cd浓度为10 mg L-1时,对番茄幼苗干生物量没有产生显著影响,甚至在添加高浓度蚯蚓粘液时使番茄幼苗地上部干生物量比对照降低4%。同时,本研究还发现,随着外源Cd浓度和蚯蚓粘液添加量的增加,番茄幼苗根系和地上部Cd浓度及富集量显著增加,且根系Cd浓度及富集量显著高于地上部。蚯蚓粘液中含有大量的可溶性有机碳(DOC)、NH4+-N、N03--N、P和K等植物可利用营养成分,促进了植物生长及对重金属的富集,这可能是蚯蚓促进植物生长、提高植物对重金属富集的作用机理之一(2)通过营养液培养试验发现,与对照相比,蚯蚓粘液显著增加了番茄幼苗根系和叶的干生物量,而其氨基酸模拟溶液仅使番茄根系幼苗干生物量较对照显著增加。同时,蚯蚓粘液及其氨基酸模拟溶液均使番茄幼苗平均根直径、根体积较对照显著增加。这些结果表明,蚯蚓粘液可以促进番茄幼苗生长,这可能与其提高番茄幼苗叶片中叶绿素含量,促进番茄幼苗对Fe、Mn、Zn和Cu等四种微量元素的吸收和运输有关。而蚯蚓粘液的主要成分氨基酸模拟溶液并未对番茄幼苗生长,叶绿素含量,以及Fe、Mn、Zn、Cu等微量元素的吸收和运输产生显著的影响。(3)在Cd胁迫下,蚯蚓粘液显著提高了番茄幼苗体内叶绿素含量和抗氧化酶活性,如超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT),显著促进了番茄幼苗对Fe、Mn、Zn、Cu等四种微量元素的吸收及运输。这可能是蚯蚓粘液促进番茄幼苗生长及对Cd富集的原因之一。但是,氨基酸模拟溶液虽然也能提高番茄幼苗体内叶绿素含量和抗氧化酶活性,促进番茄幼苗对Fe、Mn、Zn、Cu的吸收和运输,但其促进作用显著低于蚯蚓粘液。(4)在Cd胁迫下,蚯蚓粘液分别使番茄幼苗根系、茎和叶的鲜重增加123.9%、16.2%和32.0%,Cd浓度升高22.5%、14.4%和28.9%,Cd富集量增加173.2%、14.5%和75.4%。这可能与蚯蚓粘液提高了番茄幼苗亚细胞分布中可溶态Cd浓度、化学形态中无机及溶解形态Cd浓度有关。同时,氨基酸模拟溶液也具有同样的作用,即促进了番茄幼苗生长及对Cd的富集,并且提高了番茄幼苗亚细胞分布中可溶态Cd浓度、化学形态中无机及溶解态Cd浓度。但是,氨基酸模拟溶液的这种作用显著低于蚯蚓粘液。这些结果表明,蚯蚓粘液通过改变番茄幼苗体内Cd的亚细胞分布及化学形态,促进了番茄幼苗的生长及对Cd的富集。(5)蚯蚓(Metaphire guillemi)活动显著提高了模拟Cd污染土壤高沙土(10 mgCd kg-1, Orthic aquisols)中Cd的生物有效性,特别是蚯蚓活动频繁场所蚓穴及蚯蚓代谢产物蚓粪中Cd的生物有效性显著高于接种蚯蚓土壤土体。与对照相比,蚯蚓活动显著提高了土体中可交换态和碳酸盐结合态Cd浓度,而蚓穴壁及蚓粪中可交换态和碳酸盐结合态Cd浓度又显著高于接种蚯蚓土壤土体。同时,蚯蚓体内和体表可以分别富集和分泌Cd,且蚯蚓体内和粘液中Cd浓度均随培养时间的延长显著升高,二者之间存在显著的正相关关系。这些结果表明,蚯蚓可以提高土壤中Cd的生物有效性,其作用机理可能包括：1)蚯蚓活动显著降低了土壤pH,尤其是蚓穴壁及蚓粪pH；2)产生有机物质并使之与重金属Cd充分混合；3)体表分泌。以上这些试验结果表明,(1)蚯蚓粘液显著促进番茄幼苗生长及对Cd的富集,这可能是蚯蚓促进植物生长及对Cd富集的作用机理之一；(2)蚯蚓粘液促进植物对重金属的富集可能与其促进植物生长、加快植物代谢有关；(3)蚯蚓粘液中的氨基酸在蚯蚓粘液促进植物生长及对Cd富集中具有一定的作用,但其作用效率远低于蚯蚓粘液；(4)蚯蚓粘液促进植物生长及对重金属的富集可能与其含有的IAA激素及其它未知物质的关系更大；(5)蚯蚓活动可以提高土壤中Cd的生物有效性,其可能的机理包括三个方面：一是降低土壤pH；二是产生有机质,并使土壤中的有机质与Cd充分混合；三是通过体表分泌。更多还原

【Abstract】 Recently, many researches have been focused on the effects of earthworms on the phytoremediation of heavy metal contaminated soils which demonstrated that earthworms not only were able to increase heavy metal bioavailability in contaminated soils, but also enhance plant growth and heavy metal accumulations. These researches results indicated that earthworms had a potential role in modifying the efficiency of phytoremediation of heavy metal contaminated soils. However, the basic mechanisms of earthworms increase heavy metal bioavailability in contaminated soils, and enhance plant growth and heavy metal accumulations were still unclear. Therefore, we had designed a series of cultivated experiments to investigate the basic mechanisms of earthworms increase heavy metal bioavailability in contaminated soils, and enhance plant growth and heavy metal accumulation. These studies were mainly focused on:(1) Effects of earthworm (Metaphire guillemi) mucus on tomato(Lycopersicon esculentum, Hezuo 903) seedlings growth and cadmium (Cd) accumulations; (2) Influences of earthworm mucus and a solution of amino acids matching those in earthworm mucus on tomato seedlings physiological activities and Cd accumulations; (3) Effects of earthworm mucus and a solution of amino acids matching those of earthworm mucus on Cd subcellular distribution and chemical forms in tomato seedlings; (5) Influences of earthworm(Metaphire guillemi) on Cd species in spiked soils. The main results were shown as following:(1) A hydroponic study was conducted to investigate the effects of earthworm mucus (Metaphire guillemi) on tomato seedlings(Lycopersicon esculentum, Hezuo 903) growth and Cd accumulations. Results showed that, at 0 and 5 mg L-1 Cd concentrations, compared with control, earthworm mucus significantly increased dry weights of tomato seedlings roots and shoots by 12.4～33.2% and 13.8-44.5%, respectively. While, at 10 mg L-1 Cd concentration, earthworm mucus was no significant influence on tomato seedlings growth and even decreased dry weights of plant shoots by 4% at high earthworm mucus addition level treatment compared with control. Meanwhile, present study also showed, with Cd and earthworm mucus addition levels increasing, Cd concentrations and accumulations in tomato seedlings were significantly increased and the Cd concentrations and accumulations in tomato seedlings roots were much higher than those in corresponding shoots. Earthworm mucus contained abundant available nutrient materials, such as dissolved organic carbon (DOC), NH4+-N, NO3--N, P and K, which may be responsible for earthworm mucus increasing plants growth and heavy metals accumulation. This may be a mechanism of earthworm enhancing plant growth and heavy metal accumulations.(2) The results of a hydroponic study showed that, compared with CK, earthworm mucus significantly increased dry weights of tomato seedlings roots and stems; while a solution of amino acids matching those of earthworm mucus significantly increased dry weights of tomato seedlings roots only, but slightly decreased dry weights of plants stems and leaves. Meanwhile, both earthworm mucus and amino acids significantly increased the root average diameters and root volumes of tomato seedlings compared with CK, but the extents increased by earthworm mucus were much higher those than by amino acids. These results suggest that earthworm mucus could increase tomato seedlings growth through increasing chlorophyll contents of tomato seedlings leaves and enhancing microelements (including Fe, Mn, Zn and Cu) uptake and transport in tomato seedlings. While amino acids have slight influences on tomato seedlings growth, chlorophyll contents and microelements uptake and transport in tomato seedlings compared with earthworm mucus.(3) Earthworm mucus significantly increased chlorophyll contents and antioxidative enzymes activities, such as superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), and significantly enhanced four essential elements, such as Fe, Mn, Zn and Cu, uptake and transport in tomato seedlings under Cd stress. These may be explaination for earthworm mucus enhancing tomato seedlings growth and Cd accumulations. However, although a solution of amino acids matching those of earthworm mucus significantly increased chlorophyll contents, antioxidative enzymes activities and microelements uptake and transport in plants, these effects of amino acids were much lower than those of earthworm mucus.(4) Under Cd stress, compared with control, earthworm mucus significantly increased the fresh weights of tomato seedlings roots, stems and leaves by 123.9%,16.2% and 32.0%, the Cd concentrations in tomato seedlings roots, stems and leaves by 22.5%,14.4% and 28.9%, and the Cd accumulations in tomato seedlings roots, stems and leaves by 173.2%, 14.5% and 75.4%, respectively. These might be due to earthworm mucus significantly increased soluble fraction Cd concentrations of Cd subcellular distribution and significantly increased inorganic and soluble forms Cd concentrations of Cd chemical forms in tomato seedlings. Meanwhile, a solution of amino acids matching those of earthworm muus had same functions as earthworm mucus that significantly enhanced tomato seedlings growth and Cd accumulation, and significantly increased soluble fraction Cd concentrations of Cd subcellular distribution and inorganic and soluble forms Cd concentrations of Cd chemical forms in tomato seedlings, but these effects of amino acids solution were much lower than those of earthworm mucus. These results indicated earthworm mucus increased tomato seedlings growth and Cd accumulation through changing Cd subcellular distribution and chemical forms in plants.(5) Earthworm (Metaphire guillemi) activities significantly increased cadmium bioavailability in spiked soils (10 Cd mg kg-1, Orthic aquisols). Especially, the Cd bioavailabilities in burrow walls and casts, which were earthworm frequently moving place and metabolizing production, respectively, were significantly higher than those in bulk soils with earthworm. Compared with control, earthworm activities significantly increased the Cd concentrations of exchangeable and bound to soil carbonates fractions in bulk soil with earthworms, and the Cd concentrations of exchangeable and bound to soil carbonates fractions in burrow walls and casts were significant higher than those in bulk soils with earthworms. Meanwhile, earthworm tissue and epidermis could accumulate and excrete Cd, respectively. The Cd concentrations in earthworm tissue and mucus were significantly increased with incubation time. There was a significant positive correlation between the Cd concentrations of earthworm tissue and epidermal excretion. These results indicated that earthworm increased Cd bioavailability in contaminated soils, which might be due to earthworms 1) significantly decreased the pH of soils, especially in burrow walls and casts in which the pH significantly lower than the pH of bulk soil with earthworms,2) produce organic materials and fully mixed them with Cd, and 3) excete Cd through their epidermis.In conclusion, these results indicated:1) Earthworm mucus significantly increased tomato seedlings growth and Cd accumulation, which might be a mechanism of earthworm activities increasing plants growth and Cd accumulation; 2) Earthworm mucus increase Cd accumulation by tomato seedlings might be due to earthworm mucus enhancing plants growth and metabolism activities; 3) A solution of amino acids matching those of earthworm mucus play a lower role than earthworm mucus on increasing plants growth and Cd accumulation; 4) Earthworm mucus increasing plants growth and heavy metals accumulation might be due to IAA-like substances and other unknown materials; 5) Earthworm activities could increase Cd bioavailability in contaminated soils through decreasing soil pH, producing organic matters and mixing Cd thoroughly with organic matters, and excreting Cd through epidermis.更多还原