Screening Of γ-Polyglutamate Producing Strain With High-Efficiency And Its Influence On Soil Microecology Of Pakchoi

At present,it is low for the nutrient utilization efficiency of chemical fertilizer in China.It is important to explore effective and green agricultural inputs for nutrient utilization and accelerate the research and promotion of new technologies.γ-polyglutamic acid（γ-PGA）is a new multifunctional biological product,which has the excellent characteristics of easy-disperse,non-toxic,biodegradable.It can be used as the fertilizer synergist in agriculture to reduce fertilizer,improve quality and increase fertilizer efficiency.In this study,a high-efficiencyγ-PGA producing strain was screened and identified.The fermentation condition was optimized,and the whole genome sequencing was used to explore the metabolic pathways ofγ-PGA synthesis and degradation.The pot experiment was carried out by using the pureγ-PGA andγ-PGA fermentation broth to explore their growth promoting and efficiency increasing ability under the background of reducing nitrogen fertilizer by 30%.Finally,the effects on the rhizosphere soil microenvironment of green vegetables were analyzed.The main results are as follows:（1）.The strain A-5,a efficientγ-PGA producing,was screened from the microbial resource bank of our laboratory,and it was finally identified as Bacillus subtilis.The optimal amount of nitrogen source,carbon source and fermentation precursors in fermentation medium by Bacillus subtilis A-5 was determined by orthogonal test.The highestγ-PGA yield and Mw of Bacillus subtilis A-5 reached 34.21 g·L^-1and 4699 kDa,respectively.（2）.The complete genome of Bacillus subtilis A-5 was consisted of one chromosome（Chr）and two plasmids（p A,p B）with the length of 4,190,775 bp.A total of 4605 protein-encoding genes,3,664,629 bp in total length,accounted for 87.45%of the entire genome.Functional gene annotation shows the gene function of Bacillus subtilis A-5 is mainly in amino acid transport and metabolism.In addition,γ-PGA synthesis related gene cluster pgsBCA,key factor pgsE andγ-PGA degradation related genes pgdS,ggt and cwlO of Bacillus subtilis A-5 were predicted,which laid a foundation for improving itsγ-PGA production capacity.（3）.Under the background of reducing 30%nitrogen fertilizer,the effects of pureγ-PGA（PGA）,Bacillus subtilis A-5 bacterial solution（A5）,andγ-PGA fermentation solution（FJY）treatments on the growth and yield of Brassica rapa L.ssp.chinensis L.were studied with the pot experiment.The results show that,PGA,A5,and FJY treatments had a good growth promoting effect on Brassica rapa L.ssp.chinensis L..The yield of FJY treatment was significantly higher than other treatments（P<0.05）,which were 1.64,1.15,1.38,1.15 and 1.10 times higher than no fertilization（CK）,conventional fertilization（CF）,nitrogen reduction（N）,PGA,and A5 treatments,respectively.The leaf area of PGA treatment was also different from N treatment.In addition,FJY treatment also shows certain advantages in plant absorption of nutrients,activation of soil nutrients,and improvement of soil urease activity,and its utilization rates of nitrogen and phosphorus fertilizer were the highest among all treatments,reaching 52.15%and 8.03%respectively.It has the potential to improve soil environment,yield and quality.（4）.Compared with N treatment,A5 and FJY treatments decreased TN and TP in rhizosphere soil,but increased enzyme activity in rhizosphere soil,especially urease activity（UE）,which was significantly higher than other treatments（P<0.05）.A5 and FJY treatments decreased rhizosphere microbial diversity,and they were also significantly affected the structure and composition of indigenous rhizosphere bacterial community.The most dominant phyla were Actinobacteria and Proteobacteria,with relative abundances of 34.55～40.90%and 20.55-27.03%,respectively;ANOVA analysis showed,at the top 10 of phyla abundance,Chlorofloxi,Myxococcota,Cyanobacteria,Gemmatimonadota,and Methylmirabilota were the bacteria with significant difference among the treatments（P<0.05）.The top 10 genus level in abundance included unclassified＿f＿Micrococcaceae,Bacillus,norank＿f＿norank＿o＿Vicinamibacterales,norank-f＿Vicinamibacteraceae,Sphingomonas,Nocardioides,Lysobacter,Gaiella,unclassified＿f＿Intrasporangiaceae,and unclassified＿f＿Nocardioidaceae.ANOVA analysis showed,at the top 10 of genus level abundance,unclassified＿f＿Micrococcaceae,Bacillus,Nocardioides and Gaiellawere were the bacteria with significant difference（P<0.05）.The key environmental factors affecting the structure of soil microbial community structure in the rhizosphere are soil urease and pH.In addition,FAPROTAX prediction showed that the microbial agents significantly changed the bacterial abundance of rhizosphere soil in chemical energy heterotrophic,material decomposition,and nitrogen cycle.In conclusion,Bacillus subtilis A-5 is a functional strain with high-efficiently producing ofγ-PGA.It contains genes related to the synthesis and degradation ofγ-PGA.γ-PGA fermentation broth produced by Bacillus subtilis A-5 has good fertilizer synergistic effect.It not only improves plant nutrient absorption,yield and soil fertility environment,but also improves the composition and structure of rhizosphere soil microbial community,which has the strong application value in agriculture.