Isolation And Identification Of Streptomyces Roseoflavus NKZ-259 And The Analysis Of Secondary Metabolites

Plant growth regulators(PGRs)are substances that regulate growth and development of the crop.They play an important role in increasing yield,improving quality,enhancing stress resistance,prolonging the storage period and preservation of agricultural products.At present,most of the plant growth regulators used in China are synthesized chemically,which easily caused food safety and environmental pollution problems.It seriously restricts the development of plant growth regulators in China.Therefore,it is necessary to develop a new,safe and efficient microbial plant growth regulators.An actinomycete NKZ-259 which can produce indole acetic acid was isolated in this study,which showed that the fermentation broth of actinomycete NKZ-259 had a significant promoting effect on the growth of tomatoes and peppers.On this basis,the whole genome of the strain was comparatively analyzed,and the plant growth regulators produced by the strain were detected by liquid chromatography-mass spectrometry(LC-MS).The content of IAA,the secondary metabolites of the NKZ-259 were increased by optimizing the fermentation medium.The results are as follows:1.The strain which can produce indole acetic acid was isolated from the soil of Qilian Mountain in Qinghai Province.It was identified as Streptomyces roseoflavus NKZ-259 by 16 S rDNA identification.The fermentation broth of NKZ-259 was prepared to irrigate the roots of tomatoes and peppers in greenhouse and field.The results of the experiment in greenhouse showed that fresh weight and dry weight were also increased compared with control,and the root length of tomatoes also increased by 73%.The field experiment results showed that the plant height,stem diameter,crown width and maximum diameter of tomato fruits increased by 11.29%,21.05%,29.68% and14.86% respectively compared with the control,and the plant height,stem diameter,crown width and maximum diameter of pepper fruits increased by 13.58%,14.57%,16.04% and 14.07% respectively.The results of the experiment showed that the fermentation broth of NKZ-259 had a significant growth-promoting effect on tomatoes and peppers.2.The whole genome of NKZ-259 was sequenced by PacBio third generation sequencing platform.The genome length of NKZ-259 was 8081455 bp and the G-C content was 72.13%.Secondary metabolic gene clusters was predicted by antiSMASH,it showed that there were 35 secondary metabolic gene clusters including: non-ribosomal peptides,nucleosides,butyrolactones,bacteriocins,polyketones,terpenes and ectoines.KEGG gene annotation and comparative genomic analysis of the strain were carried on.It was predicted that zeatin(ZA),salicylic acid(SA)and abscisic acid(ABA)might be produced during secondary metabolism of NKZ-259.3.The fermentation broth of NKZ-259 was analysed by organic solvent extraction,silica gel column chromatography,thin layer chromatography and liquid chromatography-mass spectrometry.IAA,ABA and SA were found in the fermentation broth of NKZ-259.All the results showed that NKZ-259 could produce the plant growth regulators IAA,ABA and SA,might produce ZA.4.The medium for IAA production by NKZ-259 was optimized by single factor test,Plackett-Burman design,central composite design and response surface analysis.The optimum medium formulation was obtained as follows: L-tryptophan 2.24 g/L,glucose 20.7 g/L,potassium diphosphate 0.5 g/L,soluble starch 10 g/L,peptone 3 g/L,potassium nitrate 4.5 g/L.The content of IAA produced by medium fermentation was 45.3774 ?g/mL,and that produced by original fermentation medium was 11.64 ?g/mL.The content of IAA was increased three times by optimized fermentation medium.In summary,Streptomyces roseoflavus NKZ-259 was isolated in this study.The fermentation broth of the strain had significant promoting effect on the growth of tomatoes and peppers.According to genome annotation,comparative analysis and LC-MS detection,it showed that IAA,ABA and SA can be produced in the secondary metabolic process of Streptomyces roseoflavus NKZ-259.The optimization of fermentation medium significantly increased the IAA content produced by NKZ-259,which laid a foundation for the development of new,green and efficient microbial plant growth regulators.