| Anoectochilus roxburghii（Wall.）LindI is a perennial herbaceous plant in the orchid family.It can be used as a whole herb,and has the titles of“King of Medicine”.Due to the exhaustion of wild resources,its source is mainly artificial planting.However,A.rhizosphere grows slowly in the process of artificial planting,the active ingredients are insufficiently accumulated,and it is susceptible to diseases and insect pests,which leads to a lower artificial planting efficiency.Plant growth promoting rhizobacteria（PGPR）has a growth-promoting and quality-improving biocontrol effect and has been developed as a microbial fertilizer for crops and Chinese medicinal plants.This study intends to screen out the probiotics that can promote the growth of the A.rhizosphere and resist pathogenic bacteria from the A.rhizobacteria flora,and explore its rhizosphere colonization ability and characteristics by using fluorescent labeling,and study the growth-promoting and quality-improving characteristics of growth promoting rhizobacteria on A.roxburghii through pot experiments.It provides guidance for the artificial and efficient cultivation of PGPR.The main results obtained are as follows:（1）Screening and identification of growth-promoting rhizobacteria in A.roxburghii.Ten strains with antibacterial activity against JXL-2 of beneficial rhizosphere microorganisms isolated from the rhizosphere soil of A.roxburghii in Lincang,Yunnan province.Among them,3 strains showed significant abilities of nitrogen fixation,phosphorus dissolving and IAA secretion.2#was identified as Paenibacillus terrae,9#was identified as Bacillus velezensis,10#was identified as Bacillus subtilis.The antagonism and biofilm experiments showed that there was no antagonism,but synergism between the 2#,9#and 10#strains,and they can be used as a combined strain.This research provided high-quality strain resources for subsequent growth-promoting of A.roxburghii,and further enriched the PGPR strain resource library.（2）The construction and colonization characteristics of fluorescent strains of growth-promoting rhizobacteria in A.roxburghii.Through the electroporation method,the fluorescent plasmids me Cherry and GFP were successfully transferred into the 9#and 10#strains,and the fluorescent strains 9#-me Cheery and 10#-GFP were obtained.Moreover,the plasmid can be maintained in a short period of time without selection pressure,which met the experimental requirements.In addition,it had no obvious effect on the growth status of the affected strains.Hydroponic experiments showed that 9#-me Cheery y and10#-GFP strains can successfully colonize the rhizosphere of A.roxburghii and form biofilm in the root hair area of A.roxburghii.The successful colonization of growth-promoting rhizobacteria provided a prerequisite for the subsequent establishment of A.roxburghii-PGPR co-culture.（3）Screening of chemotactic agents of growth-promoting rhizobacteria in A.roxburghii.The exogenously added PGPR strain competed with indigenous microorganisms for nutrient space and living space to occupy a dominant niche,which was the prerequisite for its function.The rapid response of PGPR to organic acids（amino acids）in root exudates and the ability to form biofilms were important factors to measure its colonization ability.In order to promote PGPR to play a better role in the dominant niche in the rhizosphere and apply it to agricultural production,the chemotactic agents citric acid and threonine were screened out through chemotaxis experiments and biofilm formation experiments,and their best application concentration were 40μmol/L and 60μmol/L,which provided the basis for the subsequent PGPR strain and chemotactic agents for promoting the growth of A.roxburghii.（4）The growth-promoting effect of strains and chemotactic agents on A.roxburghii.The effects of PGPR strains and different treatments with chemotactic agents on the growth and active ingredient accumulation of A.roxburghii were compared and studied under the PGPR-A.roxburghii artificial cultivation system.The results of the study showed that compared with the control group,the growth indicators such as plant height,fresh weight and root weight of A.roxburghii were significantly higher than those of the control group under the different treatments of PGPR;The active components of flavonoids of A.roxburghii were significantly higher than the control group,and soil enzymes activity also showed that PGPR strain promoted enzyme activity,which was consistent with its efficient nitrogen fixation and phosphorus dissolution.To sum up,we paper screened 3 beneficial microbial strains with growth promoting effect from the rhizosphere microorganisms of A.roxburghii,and studied their colonization characteristics in the root of A.roxburghii.Finally,a system of co-culture of beneficial bacteria and A.roxburghii was established to promote growth,providing guidance for the artificial and efficient planting of A.roxburghii. |
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