| The dried tuber of Pinellia ternate?Thunb.?Beri.,a perennial herb in Araceae,is one of the commonly used traditional Chinese medicines for remedying vomiting,relieving cough,and eliminating phlegm.The herb has detoxifying,anti-inflammatory,antitussive,and antitumor properties.However,the continuous cropping of Pinellia ternata has faced serious obstacles.For instance,replanting the herb can only be done after traditional fallow for more than 7-8 years.This situation seriously restricts the regional economy,sustainable use of land,and the sustainable development of related industries.An increasing number of studies have shown that allelochemicals from Pinellas spp.and the deterioration of rhizosphere microbial communities are the main causes of continuous cropping obstacles.Therefore,this study focused on the changes of allelotoxic substances and rhizosphere microbial communities in different root zones in P.ternata continuous cropping-rotation system,aiming to determine the dynamic changes of allelochemicals and microbial communities in the P.ternata continuous cropping field after the wheat rotation,and to explore the relationship between the potential allelopathic autotoxin,rhizosphere microorganisms and the obstacles of continuous cropping.The study also aims to provide a theoretical basis for the elimination of obstacles in P.ternata continuous cropping.The main research results include the following aspects:1.Physiological indicators,qualitative indicators and active compound content of P.ternata recovered after three years'crop rotation with wheat.The result showed that the plant height and leaf area of P.ternata planted in rotation with wheat for 3 years were all significantly higher than those grown continuously for 1 year with no significant difference compared with plant grown in control soil.The rate of reverse seedling was just the opposite.The quality fraction of the extractum,succinic acid,and alkaloid were all presented.By cultivating P.ternata after rotation of wheat for 3 year,the composition quality fraction of in P.ternata was significantly higher than that continuously grown for 1 year,which has reached the pharmacopoeia standard.However,no significant difference from the control group.The experimental results also showed that the growth index and effective component content of soil under P.ternata continuous cropping returned to normal levels after the soil was used for P.ternata-wheat rotation for 3 years or more.2.Through the NaOH extraction method,phenolic acid substances in the rhizosphere soil and control soil under continuous cropping for 3 years were extracted.LC-MS technique was used for analysis,isolation and identification,and allelopathic validation.A total of 17phenolic acid allelopathic substances were identified.Through contrast analysis and literature review,the following eight phenolic acid substances in the rhizosphere soil under P.ternata continuous cropping might have strong allelopathic activity on the growth and development of P.ternata:gallic acid,protocatechuic acid,vanilla acid,clove acid,ferulic acid and so on.The effects of these chemical on P.ternata germination were tested.Ferulic acid had a promoting effect on the germination rate of P.ternata,and the effect was enhanced by the increase in its concentration.The germination was suppressed by the other seven phenolic acids.3.Eight phenolic acids from rhizosphere and root soil in P.ternata continuous cropping-rotation system were analyzed by high performance liquid chromatography?HPLC?.The content of total phenolic acids increased with the extension of continuous cropping time and this content decreased significantly after crop rotation with wheat.The dynamic change of phenolic acids in the rhizosphere soil and root soil of P.ternata showed significant difference.Compared with continuous cropping,the contents of most phenolic acids decreased.However,the change of phenolic acid decreased in the first year,then gradually increased in the second year,and finally began to stabilize in the third year.The contents of the seven phenolic acids in the soil after rotation was 50%lower than the content in soil under continuous cropping soil.4.The alleviating effect and mechanism of eight phenolic acids and exogenously added antioxidants in P.ternata rhizosphere soil on the self-toxicity of P.ternata root system were clarified and used as bases for the alleviation of the self-toxicity of P.ternata.Through the FDA/PI double staining method,the effects of the eight phenolic acid mixture on the activity of P.ternata root cells and the accumulation of reactive oxygen species were studied.The POD superoxide dismutase?SOD?catalase?CAT?activity,ascorbate peroxidase?APX?,dehydroascorbate reductase?DHAR?,and semi-dehydroascorbate reductase activities were determined,and the mechanism of relieving the autotoxicity of eight phenolic acid compounds by exogenous antioxidants was discussed.The FDA/PI double staining results showed that mixed phenolic acid stress on P.ternata seedlings increased the accumulation of reactive oxygen species?ROS?.Four hours later,the root cells were completely apoptotic due to the large accumulation of reactive oxygen.Adding different concentrations of antioxidant ascorbic acid?ASC?or gentiobiose in the liquid medium containing phenolic acids can significantly alleviate the allelopathic effect caused by phenolic acids and reduce the accumulation of root tip ROS.The activity of antioxidant enzyme SOD was significantly enhanced after ASC treatment for 36 h and gentiobiose treatment for 72 h.The largely accumulated O2-in root system was converted into H2O2,which was completely decomposed into harmless H2O in the pinnata root system,so as to reduce the accumulation of reactive oxygen species in root system and reduce oxidative damage.Exogenous antioxidants?ASC and gentianoglycan?may enable root cells to clear the excessive accumulation of ROS due to phenolic acid stress by promoting SOD activity and increasing ASC-GSH circulating enzymes?APX DHAR and MDHAR?.5.This study focused on the deterioration of rhizosphere bacterial community structure caused by continuous P.ternata cropping and the restoration of deteriorated community system caused by P.ternate-wheat cropping rotation.In this study,the collected original soil was used to design potted experiment under greenhouse environment,and rhizosphere soil samples from continuous cropping to rotation cropping group were obtained consecutively.Each sample represented rhizosphere soil with different duration of continuous cropping and rotation cropping.An Illumina PE 300 platform was used for the sequencing.The result showed that the diversity of?-bacterial community had a decreasing tendency when continuous cropping is extended.After the rotation of continuous cropping soil,the diversity level of the?-bacteria began to increase,and the diversity level increased with the duration of the rotation.Continuous cropping was proven to lead to the simplification of rhizosphere bacterial community composition,whereas rotation led to the restoration and diversification of the unitary community composition.Pseudomonas,Rhizobium,and Streptophyta were mainly enriched in the continuous cropping system after categorizing the OTUs?operable taxon?at the genus level.Streptophyta dominated the OTUs identified from the rhizosphere soil under continuous cropping for a period of 1-1.5 years.This result indicates that most of growth-promoting bacteria were“recruited”to the rhizosphere after half a year's?1 crop?P.ternata cultivation.Moreover,root lesions began to rot and Streptophyta bacteria inside the root began to infiltrate into the rhizosphere,after continuous cultivation for a period from 1year?2 crops?to 1.5 years?3 crops?.Given that no typical pathogenic bacteria OTUs were enriched,the huge change in rhizosphere bacterial community structure may lead to the opportunistic entry of pathogenic fungi,which caused the roots rot.In the soil with crop rotation using wheat following continuous cropping for 1.5 years,the enriched OTUs mainly belonged to Rhizobium,Pseudomonas,Flavobacterium,Sphingomonas,Rhizobacter,and Arthrobacter,indicating that cropping rotation enabled a large number of plant growth-promoting bacteria in the soil to return to the rhizosphere of P.ternata and to restore the rhizosphere microbial community.All soil samples were classified into three types according to the characteristics of community composition:healthy soil,disease stress soil,and diseased soil.The three soil types significantly differ in physical and chemical characteristics and community diversity.The METAGENassist database was used to predict the active microbiome of the three types of soil.The distribution of bacterial groups with functions of degrading lignans and chitins,and nitrogen-fixing bacteria significantly differs among the three types of soil.6.Illumina PE 300 platform was used to sequence the ITS2 region of fungi.The results showed that the diversity of fungal community decreased with the extension of continuous cropping.After the crop rotation of continuous cropping soil,the fungal community?-diversity recovered,but the level of?-diversity did not increase significantly along with the extension of rotation time.Rotation can properly restore the diversity of fungal community but cannot further enhance community diversity by extending rotation time.The?-diversity analysis showed that the structure of rhizosphere fungi can be changed obviously by continuous cropping,but the change did not increase with the extension of continuous cropping time.The community structure of soil after continuous cropping for 1.5 years basically restored to the community structure of soil with continuous cropping for six months,after treated with crop rotation.Cluster analysis on all the soils suggested that the fungal community structures of continuous cropping soil and rotation cropping soil basically form a cluster group,and the clustering results were not significantly correlated with the duration of continuous cropping or rotation.Thus,the formation of fungal community structure was mainly highly correlated with crop patterns.CAP analysis established the correlation between the fungal community and the physical and chemical parameters of soils,including continuous cropping,rotation cropping,and all the soil samples.The results showed that sucrase,phenol oxidase,and total potassium were significantly correlated with the change of community structure in the continuous cropping system;total potassium and organic carbon were significantly correlated with the change in the community structure in soil under the crop rotation system;the contents of available nitrogen,total potassium,and available potassium were significantly correlated with the community structure differences among the soil samples.OTUs with significant difference in abundance between continuous cropping,rotation and control group was analyzed using the generalized linear model of binomial distribution.The results showed that the continuous cropping soil highly enriched OTUs belonging to the pathogenic fungi,and the fusarium OTUs in these OTUs were dominant.Moreover,OTUs of beneficial fungi that break down cellulose polysaccharides,are suppressed during continuous cropping.Once the continuous cropping soil was rotated,the pathogenic fungal OTUs were significantly inhibited,and the abundance of beneficial fungal OTUs were significantly increased.Overall,this study demonstrated that 8 phenolic acids presented the allelopathy on Pinellia ternate.These chemicals could migrate from root surface towards the rhizosphere after the crop rotation using wheat,and thus counteracted the adverse effects of phenolic acids on the growth and development of P.ternate.Additionally,exogenous antioxidants such as ASC and gentiobiose could further alleviate the allelopathy caused by rhizosphere phenolic acids and reduce the accumulation of ROS in the root tip.We also illustrated that crop rotation using wheat restored the microbial community structural deterioration caused by continuous cropping.Crop rotation recruited the plant growth promoting bacteria from soil to P.ternate rhizosphere,and the pathogenic fungal OTUs were significantly inhibited,while the beneficial ones dramatically enriched. |
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