Screening,Optimization Of Culture Medium And Genome Sequencing Of PGPR From The Rhizosphere Of Peony

Peony is the national flower of China.It is not only an important ornamental plant,but also an important economic plant,mainly in Heze,Shandong province,Luoyang,Henan province and other places.In recent years,due to the long-term influence of peony planting,cropping cultivation,peony is affected by soil borne diseases increasingly.The spread of root rot badly affected the quantity and quality of peony.Rhizosphere soil samples were collected from the peony planting area in Heze,and the microbial separation of peony rhizosphere soil was carried out by series gradient dilution method.A total of 1304 isolates were obtained and 26 strains of antagonistic bacteria were screened by the method of plate confrontation.The antagonistic activity of MDJK10,MDJK30,MDYZ,MDJK11 and MDJK44 is better than others.Based on the analysis results of morphological,biochemical and 16S rRNA gene sequences,the 5 strains respectively belong to Bacillus siamensis,Bacillus paralicheniformis,Bacillus amyloliquefaciens or Bacillus siamensis,Streptomyces albireticuli and Streptomyces alboflavus.6 strains of phosphate-solubilizing bacteria,4 strains of organ ophosphate-degrading bacteria,11 strains of protein degrading bacteria and 10 strains of IAA producing bacteria were obtained by the selective medium.The medium of MDJK10,MDJK30 and MDYZ was optimized.The growth medium was optimized by dilution coating method,and the fermentation medium was optimized by oxford cup method.The suitable carbon source,nitrogen source and inorganic salt were selected by using a single factor experiment to add 3%sucrose to the culture medium.The single factor test for carbon source is glucose,sucrose,lactose,corn flour,corn starch and soluble starch;the nitrogen source is soybean meal,yeast,peptone,urea,?NH₄?₂SO₄ and NaNO₃;the inorganic salt is KH₂PO₄,K₂HPO₄,MgSO₄·7H₂O,CaCO₃,Cu SO₄·5H₂O and Fe SO₄·7H₂O.The optimum medium formula was determined by orthogonal test.The best growth medium of MDJK10 is:2%sucrose,1.5%soybean meal,0.003%Cu SO₄·5H₂O,and the best fermentation medium is:2%corn flour,1.5%peptone,0.003%Cu SO₄·5H₂O.The best growth medium of MDJK30 is:3%glucose,2%Na NO₃,0.002%FeSO₄·7H₂O,and the best fermentation medium is:1%sucrose,2%Na NO₃ 0.3%K₂HPO₄.The best growth medium of MDYZ is:1%sucrose,1%Na NO₃,0.1%MgSO₄·7H₂O,and the best fermentation medium is:1%corn flour,2%yeast extract,0.3%Mg SO₄·7H₂O.The genomes of MDJK30,MDJK11 and MDJK44 were analyzed by whole genome sequencing.The genome of Bacillus paralicheniformis MDJK30 strain is a circular chromosome with 4,352,468bp,and the content of GC is 45.94%.4,363 genes were annotated,including 4,134 encoding genes,110 RNA genes?including 24 r RNA genes,81 tRNA genes and 5 ncRNA genes?and 119 pseudo genes.The results of antismash3.0.5 analysis showed that the MDJK30 genome contains 11 secondary metabolic synthesis gene clusters,including4 known functional gene clusters and 7 unknown functional gene clusters.The genome of Streptomyces albireticuli MDJK11 is a linear chromosome with 8,144,417 bp,and the content of GC is 72.82%.6,971 genes were predicted,including 73 tRNA genes and 21 rRNA genes.The results of antismash3.0.5 analysis showed that the MDJK11 genome contains 46secondary metabolic synthesis gene clusters,including 34 known functional gene clusters and12 unknown functional gene clusters.The genome of Streptomyces alboflavus MDJK44 is composed of one linear chromosome and two linear plasmids p SJK1 and p SJK2.Chromosome size is 9,622,415bp and the GC content is 72.09%.8,885 genes were predicted,including 68 tRNA genes and 18 r RNA genes.The results of antismash3.0.5 analysis showed that the MDJK44 genome contains 31 secondary metabolic synthesis gene clusters,including25 known functional gene clusters and 6 unknown functional gene clusters;analyzed by antismash 3.0.5,MDJK44 contains 31 gene clusters.The size of pSJK1 is 88,700bp,and the content of GC is 67.46%,which contains 81 genes.The size of p SJK2 is 256,434bp,the content of GC is about 70.08%,which contains about 285 genes,and most of the genes on the plasmid are unknown.