【作者】 王艳芳；

【导师】 毛志泉；

【作者基本信息】 山东农业大学 ， 果树学， 2015， 博士

【摘要】 本研究分别在田间和盆栽条件下进行。于山东省蒙阴、栖霞以及莱州三地新建连作果园,新建园幼树按原树穴和原行间定植,连续两年在春、夏和秋季取老果园树穴、新植幼树树穴和行间三个取样位置采集土样,取样位置为0-30 cm土层土壤,利用ASE-HPLC法测定土壤中根皮苷及其他酚酸类物质含量并分析了年动态变化;盆栽(砂培)条件下研究了土壤实测浓度的根皮苷等酚酸类物质对平邑甜茶幼苗生物量、光合速率、根系线粒体、叶片和根系抗氧化酶活性、MDA含量及超氧阴离子自由基(O2?–)产生速率和H2O2含量等指标的影响;盆栽条件下研究了生物炭、海藻肥以及甲壳素三种土壤添加剂对连作条件下平邑甜茶幼苗的生理生化指标和连作土壤中根皮苷等酚酸类物质的含量、土壤酶活性、微生物数量和真菌群落结构的影响。主要结果如下:1.连作苹果园土壤中酚酸类物质的总含量在不同地区、不同土壤类型和不同时间明显不同。蒙阴新建园为砂土,其连作果园土壤中酚酸总量呈现先降低后升高的趋势,第二年比第一年土壤中酚酸类物质总含量有所增加;栖霞同样是砂土,但其土壤中酚酸总量则呈现先降低又升高再降低的趋势,第二年比第一年酚酸总量略有增加;莱州为壤土,其土壤中酚酸类物质总量则在缓慢增加。栖霞与蒙阴土壤中根皮苷的含量呈现“W”形,而莱州则呈现倒“V”形。三地老果园土壤中酚酸和根皮苷含量变化不大,基本维持稳定状态。2.土壤实测浓度的根皮苷、根皮素、肉桂酸、对羟基苯甲酸和间苯三酚均可抑制平邑甜茶幼苗的生长,降低了幼苗叶片光合速率、光合色素含量、保护酶活性,并且破坏根系线粒体结构,降低了根系保护酶活性,增加了细胞膜过氧化程度和活性氧含量。其中以根皮苷抑制最为明显:植株的鲜质量和干质量分别为对照的72.2%和61.4%;幼苗叶片光合速率、叶绿素含量、SOD、POD和CAT活性分别比对照降低了66.9%、57.1%、42.7%、60.2%和81.5%;根系受损程度大于地上部分,表现为根冠比降低,根系线粒体膜通透性转换孔开放程度增多,线粒体膜电位降低,细胞色素Cyt c/a比值降低;增加了H2O2和O2?–含量,分别是对照的6.0和1.9倍;叶片和根系中MDA含量大大增加,分别是对照的3.6和6.3倍。间苯三酚抑制效果最小。3.连作土中分别添加0、5、20和80 g·kg-1的生物炭,其中80 g·kg-1生物炭添加量对苹果连作障碍的缓解效果最好,主要表现为:2013年9月份,该处理的平邑甜茶幼苗株高、鲜重和干重分别是对照的1.54、2.03和2.07倍;幼苗叶片的光合速率和叶绿素总含量分别比对照提高了35%和31.5%;明显提高了幼苗叶片和根系内抗氧化酶活性,减轻了细胞膜脂化程度,降低了叶片和根系的MDA含量,其含量分别是对照的53%和58%。生物炭的添加,还降低了土壤酚酸类物质含量,提高了土壤酶活性和土壤微生物多样性。当连作土壤中添加80 g·kg-1生物炭时,土壤中根皮苷、没食子酸和对羟基苯甲酸分别为对照的24%、29%和17%,土壤中细菌/真菌比值为对照的2.5倍,真菌多样性、丰富度则分别是对照的1.5和2.2倍。生物炭可通过吸附土壤中酚酸类物质来改变微生物群落结构,从而减轻苹果连作障碍。4.海藻肥可缓解苹果连作障碍。连作土中分别添加0、5、20和40 g·kg-1的海藻肥,其中40 g·kg-1海藻肥添加量对苹果连作障碍的缓解效果最好,主要表现为:2013年9月份,该处理的平邑甜茶幼苗株高和干重分别是对照的1.38和1.82倍;幼苗叶片的光合速率和叶绿素总含量分别比对照提高了80%和17%;明显提高了幼苗叶片和根系内抗氧化酶活性,减轻了细胞膜脂化程度,显著降低了叶片和根系的MDA含量。连作土壤中添加海藻肥,对根皮苷等酚酸类物质含量影响不大,但土壤酶活和微生物变化明显。当连作土壤中添加40 g·kg-1海藻肥时,土壤中的脲酶、磷酸酶和蛋白酶活性分别是对照的1.47、1.46和2.13倍,土壤中细菌/真菌比值为对照的2.7倍,土壤真菌多样性和丰富度也有所提高。由此可见,海藻肥不能改变连作土壤中的酚酸类物质含量,其减轻苹果连作障碍的原因可能是与其有较高的有机质和营养元素含量,可提高土壤酶活性,改变微生物群落结构有关。5.添加适量甲壳素有减轻苹果连作障碍的效果。连作土壤中分别添加0,0.5,1.0和2.5 g·kg-1的甲壳素,与对照相比,1.0 g·kg-1的甲壳素处理可显著促进平邑甜茶幼苗株高和干鲜重,分别比对照增加了36.8%、82.1%和100.8%;甲壳素能增加幼苗根系保护酶活性,其中1.0 g·kg-1甲壳素处理SOD、POD和CAT活性最高,其次为0.5 g·kg-1,而2.5 g·kg-1甲壳素处理显著抑制了幼苗根系保护酶活性。1.0 g·kg-1甲壳素处理可提高土壤中细菌/真菌值,并提高了土壤中蔗糖酶、脲酶、蛋白酶、磷酸酶、过氧化氢酶和多酚氧化酶活性,分别比对照提高了8.6%、40.5%、81.1%、15.3%、18.7%和49.8%,2.5 g·kg-1甲壳素处理则降低土壤酶活性或者使土壤酶活性与对照相当。甲壳素的加入对根皮苷等酚酸类物质含量无显著影响。更多还原

【Abstract】 The research included field and pot experiment. We built three replanted orchard in Mengyin, Laizhou and Qixia city of Shandong Province, China. Soil samples were collected from the previous tree pit, sites between the previous rows(inter rows). The concentration of phenolic acid in the soils of the replanted apple orchards was determined and analyzed by an accelerated solvent extraction system and high performance liquid chromatography(ASE-HPLC). In the pot experiment, apple rootstock Malus hupehensis Rehd. seedlings were used as materials. To understand the phytotoxic mechanisms induced by phenolic acids involved in this phenomenon, Malus hupehensis Rehd. seedlings were planted in sand and treated with five phenolic acids(phloridzin, phloretin, cinnamic acid, p-hydroxybenzoic acid, and phloroglucinol) at the same concentrations as found in orchard soils. The effects of these phenolic acids on the growth and function of mitochondria and antioxidant systems of Malus hupehensis Rehd. seedlings were analyzed. At the same time, we studied the effects of biochar, seaweed fertilizer and chitin on the growth of Malus hupehensis Rehd. seedlings, soil enzymy activities and fungi community diversity under monoculturing, Results were as follows:1. The concentration of phenolic acids in continuous cropping apple orchard were obviously different in different areas, different soil types and different time. The soil total phenolic acids present first decreases and then rising, and the total phenolic acids content increased gradually with the extension of time in Mengyin area, which soil is sand, but water management was better; Qixia is also sand, but the total phenolic acids concentration in replant soil reduced first and then raised, at last declined, the total phenolic acids content of the second year increased slightly compared with the first year, but the growth rate was smaller; Laizhou is loam, and its total phenolic acids concentration in the soil was increased slowly. The content of phlorizin in Qixia and Mengyin appeared "W" shape, while that of Laizhou was inverted "V" shape. The total phenolic acids concentration in old orchard of the three area maintained a stable state. The characteristics of the changes may be due to the nature of the soil itself, measures of orchard management such as water and natural rainfall.2. The effects of these phenolic acids on the function of mitochondria and antioxidant systems of Malus hupehensis Rehd. seedlings were analyzed by measuring plant growth, activity of mitochondrial permeability transition pores(MPTPs), membrane electric potential and cytochrome c/a superoxidase(SOD), catalase(CAT) and peroxidase(POD) activity, along with malondialdehyde(MDA), hydrogen peroxide(H2O2), and superoxide radical(O2.-) content. All five kinds of phenolic acids inhibited the growth of replanted seedlings, and reduced total root length and average root diameter. Phloridzin had more significant inhibitory effects than other phenolic acids, reducing under-ground dry weight, aboveground dry weight, total root length, and average root diameter by 56.5%, 32.9%, 31%, and 27.9%, respectively. Root/shoot ratios were significantly decreased, which indicates that the impact on roots was more serious than on shoots. The phenolic acids increased the opening of MPTPs, decreased membrane electric potential and cytochrome c/a. Furthermore, POD, SOD, and CAT activity declined, which could be responsible for H2O2, O2- and MDA accumulation under the phenolic acid stress. Phloridzin was more toxic to seedlings than the other four phenolic acids; it reduced SOD, POD, and CAT activity by 29.6%, 16.4%, and 27.5%, respectively, and increased MDA, H2O2, and O2.- content by approximately 6.3, 6.0, and 1.9-fold compared to the control. Based on the above results, it could be concluded that phenolic acids induce ROS generation, and reduce antioxidant enzyme activity, thereby inducing mitochondrial permeability transition(MPT), and releasing cytochrome c to the cytosol. Therefore, phloridzin is the main phenolic acid occurring in apple continuous cropping orchard soils, and the degradation of phloridzin is the key to alleviating ARD.3. Before the Malus Hupehensis Rehd. seedlings were planted in pots, biochar was added to pots filled with replant soil at four rates: 0, 5, 20, 80 g kg-1. The results showed that the addition of biochar significantly decreased the contents of phenolic acids in replant soil through the sorption of biochar. In comparison with the control, biochar applied to replant soil at 80 g kg-1 enhanced the plant height, fresh weight, and photosynthetic parameters. Furthermore, seedlings in soil treated with biochar, particularly at 80 g kg-1, exhibited higher activity of antioxidant enzymes including superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase. With the addition of biochar, the contents of malondialdehyde, O2·- and H2O2 significantly decreased, and the osmotic substances accumulation in leaves also declined. These results suggested that the addition of biochar can alleviate apple replant disease by activating antioxidant enzymes, decreasing lipid peroxidation, and significantly reducing the phenolic acids content of replant soil through the sorption of biochar.4. In this experiment, Malus hupehensis Rehd. seedlings in pots containing replant soil were treated with four seaweed fertilizer application rates 0, 5, 20, 40 g kg-1. As a result of seaweed fertilizer addition, the plant height and dry weight were significantly increased. Seedlings grown in soil treated with seaweed fertilizer, particularly in 40 g kg-1 soil, exhibited higher activitiy of antioxidant enzymes including superoxide dismutase, peroxidase and catalase, which was accompanied by lower malondialdehyde accumulation. Compared with the control, the activities of invertase, urease, proteinase and phosphatase in soil increased with the addition of seaweed fertilizer. Remarkable differences in T-RFLP profiles were observed among control, 5, 20 and 40 g kg-1 treatments. A significant increase of fungi was observed in the seaweed fertilizer application soil. Forty g kg-1 application had the highest Shannon diversity index, evenness index and richness index, which showed the lowest level in the control. T-RFLP analysis indicated that fungal function and structure in 40 g kg-1 soil were different from control, 5 and 20 g kg-1 seaweed fertilizer treatment. These results suggested that the seaweed fertilizer application could improve the soil enzymes activities, change the soil fungal communities and improve soil quality, which could be responsible for seedlings growth promotion, higher activity of antioxidant enzymes, lower lipid peroxidation in root, as a result, the apple replant disease was alleviated.5. Chitins were added to the replant soils in pots to the final concentrations at 0,0.5,1.0,2.5 g · kg-1, respectively. The seedlings of Malus hupehensis Rehd. were planted in the pots. The effects of chitin on the photosynthesis, the contents of reactive oxygen species(ROS) and the activities of antioxidative enzymes in leaves of Malus hupehensis Rehd. seedlings under the replant conditions were studied. The results showed that the addition of chitin at 1.0 g · kg-1 obviously enhanced the plant height, ground diameter, up and under ground dry weight, root shoot ratio and photosynthetic parameters. The root shoot ratio of plants treated with 1.0 g · kg-1 chitins was 1.51 higher than that of the control. Treatment with 1.0 g · kg-1 chitins also increased the content of photosynthetic pigments, net photosynthetic rate(Pn), stomatal conductance(Gs) and transpiration rate(Tr) in leaves of seedlings. The Pn of plants treated with 1.0 g · kg-1 chitins was 1.30 higher than that of the control. The seedlings treated with 1.0 g · kg-1 chitin exhibited higher activities of antioxidant enzymes including superoxide dismutase(SOD), peroxidase(POD), catalase(CAT), and ascorbate peroxidase(APX), which were 1.10,1.85,1.77 and 1.43 times as high as that of the control, respectively. Treatment with 1.0 g · kg-1 chitin not only decreased the contents of MDA, H2O2, and O2.- to 73%, 62% and 34% of the control, but also reduced the contents of proline(Pro) and soluble sugars. Addition of chitin at 2.5 g·kg-1 significantly decreased the biomass, photosynthetic rate and the activities of antioxidant enzymes, promoted the accumulation of the MDA, O2.-, and proline. Those results suggested that appropriate concentrations of chitin could alleviate the replant disease of apple.更多还原