Rhizosphere Microecological Characteristics And Responses Of Gene Expression In Polygonatum Odoratum Under Continuous Cropping

Consecutive monoculture problem(CMP)has been seriously restricted the scale production of Polygonatum odoratum(Mill.)Druce and has become one of the important obstacle factors in the industrialization of P.odoratum.The differences of growth,soil nutrient,rhizosphere microbial community,transcriptome and miRNAs in roots and leaves were systematically investigated at morphological,physiological and molecular levels using the P.odoratum plants treated with continuous cropping(CC)and first cropping(FC)systems.The results preliminary revealed the characteristics of microecological changes in rhizosphere,and the molecular responding mechanisms in the plants,which is of great theoretical and practical significance for the analysis of CMP of P.odoratum.The main results were as follows:1.The growth and development of P.odoratum were significantly inhibited,the activities of antioxidant enzymes and other physiological indexes were affected,and the diseases were increased under CC.The plant height decreased by 19.1%,the average number of leaves per plant decreased by 1.67,the leaf area index decreased by 26.98%,the yield decreased by 77.48%,and the polysaccharide content decreased by 58.81 of P.odoratum under CC compared with FC.The content of chlorophyll a and total chlorophyll under CC significantly decreased by 57.45% and 50.72%,respectively,compared with FC at rhizome enlargement stage.At the flowering and rhizome enlargement stages,Peroxidase(POD)activity in leaves of P.odoratum under CC increased by 12.61% and 65.98%,whereas Catalase(CAT)activity decreased by 55.60% and 35.71%,and the content of Malondialdehyde(MDA)increased by 46.45% and 16.76%,respectively.Superoxide Dismutase(SOD)activity in leaves under CC increased at the seedling stage,but decreased at the flowering and rhizome enlargement stages,whereas the difference was not significantly displayed.The rate of root rot reached 46.42% and 71.53% at the flowering and rhizome enlargement stages,respectively,and the disease index was 21.51% and 39.86%,respectively,suggesting that pathogenic fungi of root rot were continuously accumulated under CC.2.The ecological balance of the available nutrients,enzymatic activity,bacteria and fungi in rhizosphere soils were damaged under CC.The alkali-hydrolyzed nitrogen(AN)content under CC was significantly lower than that of FC at the flowering and the rhizome enlargement stages,and decreased by 8.55% and 7.48%,whereas the available phosphorus(AP)was significantly higher,increasing by 60.57% and 30.18% respectively,and the available potassium(AK)increased by 30.59% at the rhizome enlargement stage compared with FC.At the rhizome enlargement stage,the activities of urease(UE),polyphenol oxidase catalase(PPO),catalase(CAT),sucrose(SU),acid phosphatase(ACP)in rhizosphere soil under CC were significantly lower than that of FC,and decreased by 39.53%,5.51%,12.95%,38.93%,and 17.71%,respectively.The abundance of bacterial community significantly decreased at the rhizome enlargement stage,and the abundance of fungal community was significantly decreased at the wilting stage under CC,whereas the Shannon and Simpson indexes of bacteria or fungi were not significantly different between CC and FC.The dominant population structure of rhizospheric microorganisms was significantly affected by CC,including beneficial bacteria such as Haliangium,Trichoderma and Paraglomus decreased significantly,whereas pathogens such as Fusarium,Neocosmospora,and Volutella increased significantly,and F.solani and F.oxysporum significantly increased especially.The changes of microbial diversity and dominant population structure in rhizosphere were significantly correlated with the environmental factors such as soil nutrient and enzyme activity.AK had the greatest effect on the Shannon index of bacteria,and PPO had a significant effect on the bacterial community richness,whereas AN had the most obvious effect on the fungal community abundance and diversity.AP was significantly positively correlated with Fusarium,Neocosmospora,and Podospora,and negatively correlated with Actinobacteria.The prediction of microbial functioning showed that CC might mainly affect the metabolism and information transmission process of bacterial community,and obviously increased pathogen-saprotroph fungi abundances,resulting in the rhizosphere microecological dysfunction and then inducing consecutive monoculture problem.3.The effects of CC on the expression of genes related to energy and secondary metabolism in roots and leaves of P.odoratum were revealed through transcriptome analysis.Four RNA-seq libraries were constructed from roots of P.odoratum under CC(CCR),leaves of P.odoratum under CC(CCL),roots of P.odoratum under FC(FCR),and leaves of P.odoratum under FC(FCL),and 615,786,632 clean reads were obtained.Due to lack of the reference genome sequences,the sequencing data were used to generate de novo assembly.After removing the low-quality sequences,the further assembly was performed using Trinity,and generated 842,213 transcripts and 510,970 unigenes.A total of 7,932 and 5,866 differentially expressed genes(DEGs)were up-regulated and down-regulated in CCL vs FCL,respectively,while 4,843 up-regulated and 10,945 down-regulated genes were identified in CCR vs FCR.Further screening of DEGs showed that transcription factors,including MYB,WRKY,and ERF,were responded positively to the stress of CC,and the expression of protein kinase genes such as LRR-RLK,LECRK,and MAPK were significantly affected,and the DEGs related defense response,such as PR1,thaumatin-like protein,and chitinase,peroxidase,ribosome-inactivating protein,plant thionin,and mannose-specific lectin,played important roles in response to CMP.In this study,161 candidated genes responding to CMP were selected,including 73 genes in leaves and 88 in roots.4.Continuous cropping responsive miRNAs in leaves and roots were identified by small RNA-seq.Four small RNA libraries were constructed from CCR,CCL,FCR,and FCL for high-throughput sequencing,and 284 conserved miRNAs and 81 new miRNAs were identified,including 142 miRNAs differentially expressed in CCR vs FCR and 126 miRNAs in CCL vs FCL.Using mi RNA-m RNA association analysis,we found 12 mi RNA-m RNA regulated modules might play an important role in the molecular response to rhizosphere microecological changes under CC,including regulated modules associated with disease resistance and defense response such as mi R156-SPL17,mi R156-LRR-RLKs,mi R319-LECRKs,mi R396-RIPs,mi R528-Peroxidase 5,mi R172-NPC2,and novel?182-ERD15,and regulated modules involved in regulation of growth,lipid metabolism,and isoflavone metabolism such as mi R156-SPL17,mi R156-CYP94B1,mi R156-SULTR4;1,mi R172-NPC2,and novel?182-CYP81E9,and regulated modules mediated the regulation of root development such as mi R156-SPL17,mi R156-CYP94B1,mi R477-WDL1,and novel?117-LOX1.5.Overall,this study analyzed the important characteristics of the imbalance of rhizosphere microecosystem,and the RNA-seq libraries and s RNA libraries from roots and leaves of P.odoratum were first constructed,and the differential gene expression profiles and miRNAs expression profiles were obtained,and the representative genes and miRNAs responding to CMP were screened.It was found that 12 mi RNA-m RNA regulatory modules may play an important regulatory role in the molecular response of plants to rhizosphere microecological changes under continuous cropping,resulting in abnormal growth and development,disorder of secondary metabolism,and decreased resistance to disease,and then inducing consecutive monoculture problem.The results provided the basic data for further understanding on the molecular response mechanism of P.odoratum under CC,and for the research of new technology to control CMP.