Diversity Of ACC Deaminase Activity Bacterium In Rhizosphere Soil And Inoculation Effects Of Codonopsis Pilosula With High Efficient Strain

Codonopsis pillosula,belonging to the genus Codonopsis of the Campanulaceae,which is mainly produced in the southeast Shanxi Province and is a medicinal herb.In recent years,with the increasing demand for C.pilosae and a large number of harvests have taken place,wild cockroach and codonopsis resources are gradually facing shortages.Plant growth promoting rhizobacteria?PGPR?plays an important role in the growth and development of plants due to its unique metabolic activities,including phosphate solubilizing bacterium,potassium-releasing bacterium,nitrogen-fixing bacterium,and ACC deaminase-producing bacterium and so on.The diversity ofbacterium ACC deaminase produced in rhizosphere soil of C.pilosula in the southeast of Shanxi and the inoculation effects of high-efficient strain was investigated.the rhizosphere soil of C.pilosula from the Pingshun County,Changzhi City,Lingchuan County,and Ze zhou County was collected.Strains were obtained fromrhizosphere soils using pure culture cultivation method,and morphological characteristics,physiological and biochemical experiments,Biolog GenIII system and16S rDNA identification techniques were used to identified and investigate the diversity of ACC deaminase-producing bacterium in the rhizosphere soil of C.pilosula.BIOLOG assay was used to study the diversity of microbial communities in the rhizosphere soil of C.pilosula,and the selected strain of LDS17 was used to study the production of IAA,siderophore,NH₃ production,HCN production,nitrogen fixation,and phosphorus solubilization,as well as its enzyme-producing conditions,salt tolerance,and broad-spectrum bacteriostatic properties.The high-producing ACC deaminase strain LDS17 was inoculated into the rhizosphere of C.pilosula.The seedling height,ground diameter,biomass,chlorophyll content,soil microbial metabolic intensity,amount of culturable microorganisms in the rhizosphere,enzyme activities and soil enzyme activities of the C.pilosula seedlings were measured.The study of functional diversity of soil microorganisms revealed the effect of high-yield ACC deaminase bacteria LDS17 on the growth of C.pilosula,which provided a theoretical basis for the high yield cultivation of C.pilosula as well as the development of high-efficiency compound microbial fertilizer.The main findings are as follows:1.A total of 33 strains with ACC deaminase activity were isolatedfrom the main producing areas of C.pilosula,which belonging to 5 genera and 10 species.twenty one strains were Klebsiella oxytoca,one strain was Bacillus siamensis,one strain was B.nakamurai,one strain was Exiguobacterium aurantiacum,three strains were B.aryabhattai,two strains were B.megaterium,one strain was B.idriensis,one strain was B.tequilensis,and one strain was Acinetobacter acetate,one strain was Acinetobacter calcoaceticus,one strain was Pseudomonas brassicacearum,which provides a rich strain resource for the cultivation,production,and germplasm innovation of C.pilosula.2.The strain LDS17 had the growth-promoting characteristics of IAA,siderophore,NH₃ production,HCN production,nitrogen fixation,phosphorus solubilization,and can tolerate a salt concentration of 6%.Through the research on the enzyme production conditions of highly productive ACC deaminase strain LDS17,it was determined that the optimum pH value for enzyme production was 6,the best incubation time was 24 hours,the optimum temperature was 30?,and the optimum rotation speed was 150 r/min,the specific activity of strain LDS17 was highest when fructose,peptone and sodium chloride were used as carbon sources,nitrogen sources and inorganic salts,respectively.Strain LDS17 was tested against the tested pathogens Rhizoctonia solani,Camellia oleifera anthracnose,Grape anthracnose,Dothiorella gregaria,Fusicoccum aesculi,Phomopsis macrospore,Rhizoctonia sp.,and Cytospora chrysosperma all have certain antibacterial activity.3.When the culture reached 168 h,the AWCD value of each sampling site was FT>DP>LDS>M.The Shannon index and McIntosh index of the soil rhizosphere of C.pilosula were significantly different in four plots,but there was no significant difference in Simpson index?P<0.05?.The order of Shannon index in four plots was ZZ>PS>CZ>LC;the order of Simpson index was PS>ZZ>LC>CZ;the order of McIntosh index was ZZ>PS>LC>CZ.The order of carbon source utilization from the highest to the lowest for the four sampling sites was:ZZ>PS>LC>CZ,and the highest degree of utilization of carbon sources at four sampling sites was L-asparagine,L-asparagine,D-malic acid and Tween 80 respectively.4.The GFP transformants with strong and stable genetic expression of LDS17strain were successfully obtained,which shows that the marker strain can be further used in the study of the colonization rules of C.pilosula.The recovery of GFP-labeled strains in the rhizosphere soil by dilution plate coating method showed that the number of LDS17 transformed bacteria in the rhizosphere soil of C.pilosula was5×10⁵ CFU/g.With the increase of inoculation time,the number of bacteria increased gradually,and the number of bacterial cells reached 1.07×10⁴ CFU/g at 25 days after inoculation.The roots of C.pilosula were observed after being inoculated for 40 days with fluorescence microscope,and a large number of green fluorescent GFP markers were found.The presence of cells further demonstrated that GFP-labeled strains could colonize the roots of C.pilosula seedlings for a long period of time.5.After inoculated with the strain LDS17,the seedling height,ground diameter,biomass,chlorophyll content,soil culturable microbial population?bacteria,actinomycetes,and fungi?and soil enzyme activity?invertase,urease and hydrogen peroxide?in the experimental group enzymes were higher than the control group;inoculation of strain LDS17 could increase the metabolic activity and diversity of microbial communities in the rhizosphere and the utilization of carbon sources by microorganisms,and increase the diversity of microbial structure and function.6.After the seedlings of C.pilosula were cultured for 60 days,each group was treated with different water treatments:drought treatment and normal water treatment.The height,MDA content,soluble protein content,SOD activity,POD activity,and CAT activity of the group were determined.After inoculation with strain LDS17 in drought conditions shows higher height and soluble protein content,higher catalase activity,peroxidase activity and superoxide dismutase activity increased,and lower malonaldehyde content compared with the control group.