Research On Pyoverdine Extraction,Purification,Functional Analysis And Production Increase Of The Metabolite Of Pseudominas Chlorophyllas YL-1

Pseudomonads chlororaphis YL-1 is a biocontrol strain isolated from soybean rhizomes in our laboratory,which has certain efficacy against a variety of pathogenic fungi and bacteria.Preliminary research results show that: the whole genome of strain YL-1contains pyoverdine biosynthesis genes,this study used wild-type strain YL-1,mutantΔpvd S,Δpvd L,back-mutant Δpvd S::pvd S,and complemented strain Δpvd S-p UCP26 were the research objects.By measuring the content of viable bacteria and the production of pyoverdine in the above 5 strains,compare the indoor inhibitory effects of the 5 strains on the tested bacteria and the influence of the change of Fe³⁺concentration in the culture medium on the ability of the strain YL-1 to synthesize the iron and the antibacterial activity.The results show:（1）Knockout of the gene pvd S and pvd L which regulates the synthesis of pyoverdine has a greater impact on the production of pyoverdine,and the supplementation of the gene pvd S can make up for part of the degraded ability to produce pyoverdine;（2）Pyoverdine is the main antibacterial substance produced by strain YL-1 in an iron-deficient environment;（3）The Fe³⁺ in the environment will have a negative impact on the production of pyoverdine and the biocontrol effect of strain YL-1.In order to study the antibacterial effect of pure pyoverdine,the pyoverdine produced by the strain YL-1 in the SM culture broth was extracted by ion chromatography exchange method.After HPLC detection and purification,use pure pyoverdine for indoor antagonism test and outdoor pot experiment,and it was found that:（1）After liquid spectrum injection,the concentration of 2’20 "-3’00" pyoverdine sample is the highest,and the antibacterial effect is better.（2）The inhibitory effects of the pyoverdine samples on the iron-rich and iron-deficient media are similar to the tested bacteria;（3）With the increase of the dosage of pyoverdine,the inhibitory effect on the test bacteria is enhanced,and when the Fe³⁺in the environment is too high,the inhibitory effect of pyoverdine on the tested bacteria decreases.（4）Pyoverdine has a significant control effect on rice bacterial blight in a natural iron-deficient environment.The length of rice leaf disease spots after treatment with high and low concentration pyoverdine aqueous solutions were reduced by 61.8% and 36.3%.Preliminary research results show that: in indoor iron-deficient media and natural environment,the main bacteriostatic substance of Pseudomonas chlorophylla is the pyoverdine secreted by it.In order to increase the production of pyoverdine,use shake flask culture fermentation,through plackett-burman experimental design,central composite（CCD）experimental design and response surface method,optimizing the fermentation medium composition and fermentation conditions of YL-1 strain for high production of pyoverdine.Under the conditions,the best medium composition for obtaining YL-1 strain with high pyoverdine production is: succinic acid 1.52 g/L,sodium succinate 2.00 g/L,MgSO₄·7H₂O 0.88 g/L,（NH₄）₂SO₄ 0.50 g/L,sucrose 0.50 g/L,KH₂PO₄ 3.49 g/L,K₂HPO₄5.44 g/L,the best culture conditions are: temperature 26℃,p H value 7.0,fermentation time36 h,inoculation volume 2%,rotation speed 180 r/min,the liquid volume is 50 m L liquid in a 250 m L triangular flask.The results of the shake flask experiment showed that after optimizing the medium composition and culture conditions,the yield of strain YL-1pyoverdine was increased by 43.18%,and the ratio of OD405/OD600 was 2.36,and the optimization effect was obvious.