| The research was conducted from March2011to March2014in the fruit roots laboratory of college of horticultural science and engineering, shandong agricultural university, National laboratory center for apple engineering and state key laboratory of crop biology, The field experiment were carried out in a piece of new built replanted orchards in Ciyao Town of Ningyang County, Daolang town of Daiyue district in Tai’an city and Jincheng town of Laizhou city in Yantai city of Shandong Province, China, respectively. Soil samples were collected from the previous tree hole (tree hole), sites between the previous rows (inter-rows) and sites between the previous trees (inter-trees) at2depths:0-30cm and30-60cm. The phenolic acid concentration in the soils of the replanted apple orchards was determined and analyzed using an accelerated solvent extraction system with high performance liquid chromatography (ASE-HPLC). In the sand experiment, apple rootstock Malus hupehensis Rehd. seedlings were used as materials. Effects of soil concentration of five phenolic acids and their mixtures on the biomass were investigated, such as root mitochondria-related indicators, leaf antioxidant enzyme activity, TCA cycle enzymes on seedlings of Malus hupehensis Rehd. At the same time, effects of soil concentration of five phenolic acids and their mixtures on the diversity, evenness, richness index of soil fungi in the pot experiment under the repant conditions were also determined. Results were as follows:1. To establish a new protocol based on the ASE-HPLC method, which could determine the content of phenolic acids in orchard soil rapidly and efficiently. Phlorizin was set as a sample for analysis, which is one of the characteristic phenolic acids in apple orchard soil. Accelerated solvent extraction (ASE) combined with high performance liquid chromatography (HPLC) was used to assess it. Specifically, the parameters of extraction solvent, extraction temperature, pressure and number of cycles were optimized, aiming at exploring the optimal conditions of ASE extraction for phenolic acids. The optimal conditions of ASE extraction for phenolic acids were as follows:Ethanol as extraction solvent initially, and methanol was the subsequent extraction solvent, extraction temperature at120℃, a pressure of10.300MPa,2cycles, every5min static time,60%purge volume, and purge time for90s. The process was simpler, and with good reproducibility and linearity, correlation coefficient up to0.990, recovery efficiency between83%and98%, detection limits to the amount of1.300×10-4-2.500×10-2βg/mL. ASE-HPLC is an accurate and rapid new protocol to mensurate phenolic acids, which is an improved and practical method.2. The composition and content of phenolic acids in the soils of replanted apple orchards significantly differed among different seasons, different sampling positions and different soil layers. The phenolic acid content tended to increase from the spring to fall. The soil phenolic acid content was the highest in the previous planting pit for all3of the orchards, especially in the fall. The soil phenolic acid content in the previous tree hole was5.080,5.010and1.700times that in the soils inter-row. In the spring, the soil phenolic acids were most abundant in shallow soils, and in the summer and fall, the soil phenolic acids were primarily distributed in the deep soils. Phlorizin, benzoic acid, salicylic acid, phloretin and vanillin were the main phenolic acid species in the replanted apple orchards. Comparison of different sampling locations, the growth of replanted trees in Daolang between rows is best. Comparison of different sampling locations, plant height, dry perimeter, numbers of branches of the replanted trees in original tree holes are lower than between rows.3. Soil concentration of phloridzin, phloretin, benzoic acid, vanillic aldehyde, salicylic acid and five kinds of phenolic acids mixture had an inhibited affect on the biomass, root vigor, root mitochondria related index, leaf protective enzyme, root TCA cycle enzymes of the Malus hupehensis Rehd seedlings, and the mixture of five kinds of phenolic acid had the most inhibitory effect. Treatments that caused damage to the biomass of Malus hupehensis Rehd. seedlings were sorted as:Mixed phenolic acids> Phlorizin> Phloretin> Vanillic aldehyde> Salicylic acid> Benzoic acid. Compared with the control, root protein spots were significantly different after treated by phlorizin, phloretin and benzoic acid,16(10up-regulated,6down-regulated),13(5up-regulated,8down-regulated),9(3up-regulated,6down-regulated), respectively; According to their functions, these proteins were classified as8functional groups:signal transduction, pathogen associated proteins, metabolism related proteins, free radical scavenging related protein, stress related protein and so on.4. Soil fungal diversity differences were presented among the samples, which were collected in Ciyao, Daolang and Jincheng towns. Hereinto, Shannon diversity index, Pielou evenness index and Dominance index in all samples were between0.430-2.470,0.170-0.850and0.120-0.810, respectively. Shannon diversity index, Pielou evenness index and Simpson index in all samples were between0.430-2.470,0.170-0.850and0.120-0.810, respectively. The highest Margalef richness index (R=4.550) was observed at0-30cm soil layer of tree hole in Jincheng and the lowest value (R=0.770) was obtained at30-60cm soil layer of tree inter-row in Ciyao. In all investigated sites and soil layers, original tree hole showed the highest diversity index, evenness index, richness index and the lowest simpson index. Soil fungal diversity index, evenness index, richness index of0-30cm soil layer was higher than those of30-60cm soil layer; however, Simpson index expressed a reverse trend. PC A and cluster analysis indicated that soil fungi of Ciyao, Daolang and Jincheng formed an independent community structure, respectively. And ITS clone library analysis the tree hole of Ciyao, Jincheng, Daolang apple replant orchard, the study found that apple replant orchards have some pathogenic fungi, including Cylindrocarpon, Fusarium and other pathogenic fungal species.5. Live colonies of F. moniliforme could be labeled with SiC quantum dots and that the fluorescence in the mycelium could be observed for at least40days. The images from a fluorescence microscope during the labeling process showed that F. moniliforme growth and division were slower without phlorizin. While growth was fast and division was rapidly with phlorizin, and with0.500mmol/L and1.000mmol/L of phlorizin, F. moniliforme growth was greater with increased phlorizin. The plate culture experiments showed that different soil concentrations of phlorizin, phloretin, benzoic acid, vanillic aldehyde, salicylic acid, and mixtures of five phenolic acids could promote the growth of F. moniliforme and F. oxysporum. Potting experiments showed that different soil concentrations of phlorizin, phloretin, benzoic acid, vanillic aldehyde, salicylic acid, and mixtures of five phenolic acids could increase the diversity of soil fungi, and make significant changes in the soil fungi community structure. |
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