Characteristics Of Specific Microbial Community In The Rhizosphere Of Continuous Cropping Salvia Miltiorrhiza Bge.Based On ¹³C Stable Isotope Nucleic Acid Probe Technology

Objective:To clarify the morphological and physiological differences between continuous cropping and non-continuous cropping of S.miltiorrhiza.Identify the strains isolated and purified from the diseased strains of continuous cropping S.miltiorrhiza;Analyze the structural differences of specific microbial communities in the rhizosphere soil of S.miltiorrhiza under continuous cropping and non-continuous cropping conditions,and determine the composition of bacterial communities related to the growth and metabolism of S.miltiorrhiza by tracing the operation of ¹³C isotopes in the"plant-soil-soil microbial"system.Through the above research,further clarify the interaction between rhizosphere microorganisms and continuous cropping of S.miltiorrhiza and the relationship between them and the formation of continuous cropping obstacles,and provide new research ideas for clarifying the formation of continuous cropping obstacles of S.miltiorrhiza.Methods:The growth and metabolism of continuous cropping and non-continuous cropping S.miltiorrhiza were measured and analyzed by morphological observation and physiological index detection.Morphological and polygenic analysis was used to determine the fungus types isolated from various parts of the affected S.miltiorrhiza.Through PCR amplification,high-throughput sequencing technology and Miseq sequencing platform,the composition of bacterial and fungal communities in the rhizosphere soil of S.miltiorrhiza continuous cropping and non-continuous cropping were analyzed,and the changes of microbial community composition in the rhizosphere soil of S.miltiorrhiza before and after continuous cropping were determined.With the help of stable isotope nucleic acid probe（DNA-SIP）technology,S.miltiorrhiza plants were labeled with¹³C,and the¹³C-DNA and¹²C-DNA of rhizosphere microorganisms were separated by ultrahigh speed density gradient centrifugation technology,so as to determine the composition of rhizosphere bacterial community interacting with S.miltiorrhiza,and to explore the interaction mechanism between plants and rhizosphere microorganisms under continuous cropping conditions and the relationship between them and the occurrence of continuous cropping obstacles.Results:After continuous cropping,the biomass of S.miltiorrhiza decreased significantly,the root lesion shrank,and the chlorophyll content and sugar content in leaves decreased.The content of Pseudomonas,Lachnospirales and other bacteria in the rhizosphere soil of continuous cropping S.miltiorrhiza decreased,but the content of fungi such as Mortierella、Mortierella＿stylospora increased significantly.68 strains were isolated from the diseased strains of S.miltiorrhiza and rhizosphere soil,and were identified as 10kinds of fungi,among which the soil-borne pathogens Colletotrichum gloeosporioides、Alternaria tenuissima、Rhizoctonia solani and Fusarium oxysporum accounted for a large proportion.After S.miltiorrhiza marking experiment,the distribution ratio of ¹³C-photosynthetic carbon was root>shoot>rhizosphere soil,and the carbon assimilation ability of continuous cropping S.miltiorrhiza was lower than that of non-continuous cropping S.miltiorrhiza.DNA extracted from rhizosphere soil can be divided into DNA bands of different quality after ultrahigh speed centrifugation.Through the difference of DNA concentration in each layer of experimental group（group F,group L）and CK group and the change of the proportion of similar bacteria in each layer after high-throughput sequencing,the 1-6layers of group F,the 1-3 layers and 10-12 layers of group L,which have significantly increased mass concentration and a large number of fine bacteria,are selected as the target layers containing¹³C-DNA,and the composition of bacterial community at the level of phylum,class,order,family,genus and species of DNA components in each layer was analyzed.At present,specific bacteria in the rhizosphere soil of continuous cropping S.miltiorrhiza have been initially screened,including Bacteroidota、Cyanobacteria、Bacteroidia、Lachnospirales、Nitrososphaeraceae、Burkholderiaceae、Pseudomonas、Ralstonia、Bacillus＿aryabhattai、Romboutsia＿ilealis、Lachnospiraceae＿bacterium＿COE1、Corynebacterium＿imitans.Among them,the harmful bacteria account for a large proportion of ¹³C-DNA in the rhizosphere soil of continuous cropping S.miltiorrhiza,while the beneficial bacteria account for a small proportion.Conclusion:After continuous cropping,the growth and development of S.miltiorrhiza was affected,the photosynthetic capacity decreased,and the content of effective components decreased.There are structural differences in the composition of rhizosphere microbial communities between continuous cropping and non-continuous cropping S.miltiorrhiza,and the relative content of fungi is increased.Colletotrichum gloeosporioides、Rhizoctonia solani and Fusarium oxysporum in the infected S.miltiorrhiza plants and rhizosphere soil are relatively high,so it is speculated that the imbalance of rhizosphere microorganisms is likely to cause plant continuous cropping obstacles.Through ¹³C isotope tracing and DNA-SIP technology,the harmful bacteria Ralstonia、Pseudomonas、Corynebacterium＿imitans and the growth-promoting bacteria Bacillus＿aryabhattai that are related to the growth and metabolism of S.miltiorrhiza after continuous cropping were initially screened.