| Rehmannia glutinosa is a perennial herbaceous medicinal plant of the Scrophulaceae family,and there are serious continuous cropping obstacles in its production.In this paper,the root zone soil of Rehmannia glutinosa was taken as the research object and the variety "wen 85-5" was used as the experimental material to analyze the accumulation and distribution of dry matter and effective components in the growth of Rehmannia glutinosa.Also,water soluble extracts from root zone soil and plant extracts of Rehmannia glutinosa at different growth stages were extracted to verify their allelopathic autotoxic effects.Four different roll-type membrane elements of 0.2 μm microfiltration membrane,0.004 μm ultrafiltration membrane（5000 D）,0.001 μm nanofiltration membrane（150 D）,and reverse osmosis membrane were used to enrich allelophenol in the soil water extract of Rehmannias root region,and we measured the enrichment efficiency.In addition HPLC chromatography and LC-MS chromatography were used to identify potential allelochemicals in the root zone soil of Rehmannia glutinosa.The allelopathic effects of different interception solution were verified by biological test of Rehmannia glutinosa seed germination.At last,seven phenolic acids enriched in the water extract of the root region of Rehmannia glutinosa were knockouted by HPLC to verify the contribution of phenolic acids to allelopathic autotoxicity effects of Rehmannia glutinosa.The results showed that:1.The accumulation of dry matter in Rehmannia glutinosa gradually increased,and the distribution center of dry matter in Rehmannia glutinosa was gradually shifted to the underground part;The contents of chlorophyll and soluble sugar increased first and then decreased,while the contents of polysaccharide gradually increased.The contents of water-soluble extract and alcohol-soluble extract all increased first,then decreased and then increased.The total contents of catalpol,amuoside,rehmannioside D and salidroside all showed an increasing trend,while the total contents of rehmannioside A and mullicoside showed an increasing trend at first and then decreased,the distribution of different active substances in different parts of the plant was different,and all of them moved to the underground part;Correlation analysis showed that the yield and quality of Rehmannia glutinosa was significantly positively correlated with the accumulation of asparoside,rehmannioside D,rehmannioside A,salidroside,cellinoside,chlorophyll and water-soluble extracts,but negatively correlated with the accumulation of alcohol-soluble extracts.2.Rehmannia glutinosa plants and root zone soil of extracts at different growth stages had inhibitory effects on growth.The effect of the extract was generally expressed as "low promotion and high inhibition".The aboveground extract of Rehmannia glutinosa had stronger inhibitory effect than the underground extract,and In the extracts of different growth periods,the expansion period had stronger inhibitory effect than that in the seedling period.Correlation analysis showed that the root length of three different extracts was positively correlated with the content of ferulic acid,which indicated that ferulic acid may promote the growth of Rehmannia glutinosa.Correlation analysis between the active component indexes of Rehmannia glutinosa at different growth stages and the root length of Rehmannia glutinosa seeds treated with three different extracts showedthat the root length of Rehmannia glutinosa seeds with root zone soil water extract was not highly correlated with the content of several substances.The root length of Rehmannia glutinosa seeds which added with the aboveground water extract of Rehmannia glutinosa was significantly positively correlated with the content of catalpol.The root length of Rehmannia glutinosa seeds with the water extract of the ground part of Rehmannia glutinosa was significantly positively correlated with the content of aucubin.3.Four membrane elements with different molecular diameters were used to enrich the root zone soil water extract,and the nanofiltration membrane had the highest enrichment efficiency,which reached 10.2785 mg·kg-1.HPLC showed that the contents and types of phenolic acid allelopathic substances in the intercepted solution of nanofiltration membrane were the most,and the types of iridoid glycosides and phenylethanol glycosides in the intercepted solution of reverse osmosis membrane were the most.The results of LC-MS also indicated that more iridoids were preserved in the nanofiltration membrane and reverse osmosis membrane intercepts.The seed germination experiment showed that the interception solution of different membrane elements had inhibitory effects on the germination potential and germination rate of Rehmannia glutinosa seeds,and the reverse osmosis membrane interception solution had the greatest inhibitory effect.The effects on root length and stem length were different,with promotion and inhibition.The inhibition effect of the reverse osmosis membrane intercepted solution reached the maximum when the concentration was 50 g·m L-1.Combined with the previous pot experiment,the allelopathic substances that were not beneficial to the growth of Rehmannia glutinosa were probably mainly retained in the intercepted solution of nanofiltration membrane and reverse osmosis membrane.4.Using HPLC to knock out the seven phenolic acids enriched in the soil water extract of Rehmannia glutinosa root area and then carried out pot experiment,the test results showed that there was no significant difference in the emergence rate of the knock-out group and the repeated cropping group,but with the growth of Rehmannia glutinosa,the mortality of each component showed significant difference,and the death rate of replanting Rehmannia glutinosa was the highest.While there was no death in the knockout group;According to the indexes of leaf length,leaf width,plant height,crown width and number of leaves,the growth of knockout group was not significantly different from that of normal stubble after 120 days.As can be seen from the results of root scanner,after 120 days of growth,there was no significant difference between the growth of the knockout group and the normal crop.It can be seen from the change of fresh and dry quality during the growth process,the fresh and dry weight of the underground part had no significant difference between the normal stubble and the knockout,but had significant difference with the repeat stubble at 120 days of growth.The activities of SOD,POD,CAT and APX in each group were increased continuously,among which the repeat crop group had the highest performance.The MDA content of the normal crop and the knockout group did not increase significantly,but the MDA content of the repeat crop group increased significantly,which also indicated the high degree of membrane lipid peroxidation and rapid aging in the repeat crop group.At the same time,the changes in the content of total iridoids and total phenylethanol glycosides in Rehmannia glutinosa were analyzed,with the growth of Rehmannia glutinosa,the content of total iridoid glycosides and total phenylethanol glycosides in leaves and tubers increased,At 120 days of growth,the total iridoid glycoside content in leaves differed significantly among the components.And the normal crop group had the highest content.Also the total phenethyl glycoside content differed significantly among the components,the knockout group had the highest content;There was no significant difference between the knockout group and the normal cropping group of iridoid glycosides,and the total phenethyl glycoside content of the knockout group was significantly different from the repeated group and normal group.The comprehensive analysis from three aspects of agronomic index,physiological index and quality index showed that the elimination of phenolic acids in soil greatly alleviated the continuous cropping obstacle of Rehmannia glutinosa,which had an important guiding significance for alleviating the continuous cropping obstacle of Rehmannia glutinosa. |
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