Screening Of Bupleurum Germplasm Resources And Study On Rhizosphere Soil Microorganisms

Chinese thorowax root is a genus of perennial herb in the umbelliferous family,which includes Bupleurum scorzonerifolium Wild.and Bupleurum chinense DC..The dried root is a common medicine for sweating.At present,the market demand of Bupleurum is large,the wild resources are increasingly exhausted,and the cultivation of Bupleurum has become the mainstream of the market.However,the cultivated Bupleurum was affected by many factors,such as multiple varieties and large variation of characters in different producing areas,resulting in uneven quality and difficult to achieve the expected curative effect.Therefore,it is urgent to select new bupleurum varieties with stable characters and good quality and regional characteristics to meet the market demand.In this paper,based on the previous research,the radix bupleuri in germplasm nursery,classified according to the phenotype screening of varieties,the identification of radix bupleuri provenance and the growth and photosynthetic characteristics,physiological and biochemical indexes,root dry weight,determination of effective component content and so on,to conduct a preliminary evaluation of radix bupleuri plants,at the same time of radix bupleuri rhizosphere soil microbial flora analysis,It provides theoretical basis for breeding new bupleurum species and field production.Objective:To establish the germplasm resource nursery of Bupleurum chinense.By observing the characters of the original plant of Bupleurum chinense and the seedlings of F1 and F2 generation in the resource nursery,different varieties were screened out,and the characters of different varities of Bupleurum chinense were determined and their DNA barcodes were identified.At the same time,the growth and photosynthetic characteristics,physiological and biochemical indexes,root weight and active component content of bupleurum were determined.To clarify the effects of different bacterial communities on the growth and occurrence of root rot of Bupleurum chinense,and to provide a basis for the breeding of superior varieties of Bupleurum chinense,and to provide a theoretical basis for solving root rot of Bupleurum chinense.Methods:1.Through wild collection and breeding,the germplasm resource nursery of Bupleurum chinense was established.According to the phenotype of Bupleurum chinense,3 varieties of Bupleurum chinense were preliminarily screened out.The plant height,leaf length,leaf width,node spacing,branch number,chlorophyll content and photosynthetic rate of the three variety were measured and analyzed.The activities of superoxide dismutase(SOD),peroxidase(POD),malondialdehyde(MDA)and catalase(CAT)in plants of the three varieties were determined.And the enzyme activities of soil sucrase(S-SC),soil neutral phosphatase(S-NP),soil alkaline phosphatase(S-AKP),soil urease(S-UE)and soil cellulase(S-Cl)in rhizosphere soil were also determined.The roots of each varieties were harvested in autumn to observe their characters and to preliminarily evaluate the three varieties of Bupleurum.2.Combined with phenotypes,DNA barcoding technology was used for molecular identification of the three selected varietiesvarieties to determine the provenances of Bupleurum chinense,which laid a foundation for subsequent breeding and selection of Bupleurum chinense.3.The contents of total saponins,total flavones,volatile oil and total polysaccharides of different selected varieties of Bupleurum were analyzed by spectrophotometry,and the contents of bupleurum saponins A,D and bupleurum saponins C in Bupleurum were detected by HPLC.4.High-throughput sequencing technology was used to analyze the community structure characteristics of rhizosphere soil microorganisms and rhizosphere soil bacteria and fungi of different varieties of Bupleurum chinense,and SPSS(Statistical Product and Service Solutions)was used to analyze the soil physical and chemical properties and enzyme activities.Results:1.One nursery of Bupleurum chinese germplasm resources was established,and 50 Bupleurum chinese germplasm resources from 25 different producing areas were preserved;According to the phenotypic classification of Bupleurum,three varieties were selected and named ZC01,ZC02 and ZC03 respectively.The results showed that the content of available N,available P and available K in the soil was 4.00 mg/kg,5.00 mg/kg,13.00 mg/kg,and p H value was 6.00.It was found that ZC01 had the highest plant height,ZC02 had the highest leaf length and photosynthetic rate,and the stem diameter was thicker.In the flowering stage of bupleurum in mid-July,varieties ZC01 and ZC02 had earlier flowering time,and variety ZC03 had later flowering time.Among them,ZC01 and ZC02 had no obvious difference in flower shape and dark yellow color,while ZC03 had longer calyx and light yellow color,which was obviously different from ZC01 and ZC02.In enzyme activity analysis,variety ZC02 had the highest SOD and POD enzyme activities,variety ZC01 had the highest CAT enzyme activity,and variety ZC03 had the lowest MDA content.The POD enzyme activity of variety ZC02 was significantly different from that of variety ZC01,but there was no significant difference in other enzyme activities.As for Bupleurum,ZC01 and ZC02 bupleurum had similar characters,the root was cylindrical,the root head was enlarged,most of the taproots were obvious,a few were not obvious.The variety ZC01 was0.25 g ～ 2.70 g in weight,6.22cm～ 21.91 cm in length and 0.10cm～0.36 cm in diameter,with 0 ～ 4 stem bases remaining at the top,most of which had fewer stem bases and most of which had more and finer branches.Surface yellow brown,with vertical wrinkles,less lenticular.The quality is brittle and easy to break,and the section is fibrous.Sweet smell,taste slightly bitter;Variety ZC02,0.30 g ～ 3.50 g in weight,2.21cm-12.64 cm in length,0.20cm-0.63 cm in diameter,with 0 ～4 stem bases remaining at the top,obvious branches,most of which are thick,with a yellowish-brown surface,fragrant and slightly bitter taste.Variety ZC03,the root is long conical,weight 0.10 g ～ 2.32 g,length2.70 cm ～ 17.82 cm,diameter 0.10 cm ～ 0.40 cm,no root,reddish brown surface,with vertical wrinkles,with oil septicosis taste.2.Provenances of three different varieties of Bupleurum chinese DC.were determined.ZC01 and ZC02 were Bupleurum chinese DC.And,a total of 25 loci of variation;Variety ZC03 is Bupleurum scorzonerifolium Wild.It is also known as Southern Bupleurum and has 19 mutation loci.Through the calculation of variation coefficient of Bupleurum,the order of variation index was as follows: stem diameter >Blade width& gt;Section spacing & gt;Leaves long & gt;Plant height.The variation coefficients of plant height,leaf length,leaf width and stem diameter were 14.28,20.89,32.90 and 46.19,respectively.The variation coefficient of nodal space of variety ZC02 was 22.51.3.The contents of total saponins,total flavonoids,volatile oil,total polysaccharides,saponins A,D and C of the three selected varieties were determined and analyzed.The results showed that the content of total saponins of the three varieties ranged from 2.20% to 2.60%,and ZC02 had the highest content of total saponins(3.10%).Variety ZC01 had the highest total polysaccharide content(5.60%).Variety ZC02 had the highest total flavonoid content(0.59%).In the content determination of saponins A,D and C,the content of ZC01,ZC02 and ZC03 were 0.30%,0.31% and 0.19%,respectively.The volatile oil content of ZC03 southern Bupleurum was higher than that of ZC01 and ZC02 northern Bupleurum,the content of ZC01,ZC02 and ZC03 was 0.44%,0.53% and 0.80%,respectively.4.The rhizosphere soil microorganisms of three different varieties of Bupleurum chinense showed high similarity in both bacteria and fungi,and no difference was found.In the level classification of bacteria,the dominant bacteria with high enrichment degree are Actinobacteriota,Proteobacteria,Chloroflexi,Gemmatimonas and so on.At the level of bacterial classification,the same bacterial communities are Arthrobacter,Bacillus,Blastococcus,Sphingomonas,Gaiellales and so on.In the level analysis of fungal phyla,the same bacterial communities were Ascomycota,Basidiomycota,Mortierellomycota and Glomomycota,respectively.The abundance of Ascomycota,Basidiomycota and Mortierellomycota was relatively high.At the level of fungi,Mortierella,Didymella,Cladosporium,Tausonia and Gibberella are the same flora.It was found that the proportion of the abundance of Chloroflexi,Acidobacteriota and Gemmatimonadota in the rhizosphere soil with root rot disease was higher than that in the healthy soil.The abundance of Actinobacteriota,Proteobacteria,Firmicutes,Myxococcota and Bacteroidota in bupleurum root rot soil was lower than that in healthy bupleurum root rot soil Soil.At the bacterial level,The abundance of Arthrobacter,Blastococcus,Sphingomonas,Microvirga and Bacillus in rhizosphere soil of P.bupleurum was lower than that of healthy p.bupleurum,the proportion of Norak-F-Gemmatimonadaceae and Norak-F-vicinamibacterales in rhizosphere soil was higher than that in healthy Bupleurum.At the fungal level,the content of Ascomycota decreased significantly,while that of Basidiomycota increased significantly.At the fungal level,the abundance of Didymella and Tausonia in the rhizosphere soil with root rot was higher than that in the healthy rhizosphere soil of P.bupleurum.The proportion of Gibberella,Mortierella,Fusarium,Cladosporium,Chaetomium and Alternaria in healthy soil was lower than that in healthy soil.In addition,two endemic species were found in the healthy rhizosphere soil microbes,namely Stachybotrys and Chordomyces.The results showed that both Melanophyllum and Cylindrocarpon were combined with Cylindrocarpon respectively.Conclusion:1.A Germplasm resource nursery of B.bupleurum was established,and 25 wild B.bupleurum germplasm resources in different regions of Chengde and 50 bred F1 and F2 seedlings of B.bupleurum were saved,from which 3 B.bupleurum varieties were screened out,and the morphological characteristics of each variety were greatly different.The variety ZC02 was evaluated in plant height,chlorophyll content,photosynthetic rate,enzyme activity in fresh leaves,soil enzyme activity and quality of medicinal materials.2.ZC01 and ZC02 were identified as Bupleurum chinense DC.The origin of ZC03 is Bupleurum scorzonerifolium Wild.The source.3.ZC01 and ZC02 had higher contents of total saponins,total flavonoids and total polysaccharides,and the content of Radix Bupleurum saponins A + D was higher than the pharmacopoeia standard.The volatile oil content of strain ZC03 was higher,and the saponin A + D content of Strain ZC03 did not meet the pharmacopoeia standard.The three strains of Bupleurum had advantages in different chemical composition content,and had potential breeding potential.4.There was no difference in the types of bacteria or fungi enriched in rhizosphere soil microorganisms of different strains of Bupleurum.The rhizosphere soil flora of the diseased and healthy Bupleurum were significantly different,with unique bacterial species and fungal species respectively.