| 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. |
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