Phytolectin Affinity Of Azotobacter Chroococcum And Its Mutagenesis Of Atmospheric Pressure Room Temperature Plasma

Azotobacter chroococcum is an abiogenous azotobacter.Because it does not need to cooperate with a certain plant to fix nitrogen independently,it has the characteristics of wide adaptability and plays an important role in the nitrogen cycle of natural ecosystems.It is a commonly used biological nitrogen fertilizer in agriculture.Reasonable use of biological nitrogen fertilizer can effectively increase the yield of crops,and at the same time can reduce the use of industrial nitrogen fertilizer to reduce the pollution caused by nitrogen surplus in agriculture.Therefore,effectively improving the nitrogen-fixing capacity of the Azotobacter chroococcum is of great significance for improving the biological nitrogen fertility and sustainable agricultural development.Atmospheric pressure room temperature plasma mutagenesis breeding technology is a new type of mutagenesis breeding technology,which can generate a large-capacity mutation library through plasma jet and is easy to screen strains.Compared with traditional physical and chemical mutagenesis methods,ARTP has the advantages of high mutation rate,safe operation and simple operation.It is an excellent and efficient mutagenesis breeding method.At present,the research on ARTP involves many fields such as bacteria and fungi,but there is no mutagenesis research on abiogenous azotobacter.In this paper,the method of plant seed lectin agglutination screening was used to rapidly screen out a Azotobacter chroococcum CICC 21686 which can combine with specific crops.For the first time,ARTP mutagenesis was selected to obtain mutant strains with high nitrogenase activity and good effect on plant growth,and its nitrogenase activity and growth promoting effect were verified.The research results are as follows:1. The lectin agglutination test was used to screen out a strain of Azotobacter chroococcum CICC21686 that could specifically bind to Brassica rapa var.glabra seed lectin.In this experiment,the lectins of three plant seeds were extracted,namely Brassica chinensis L.,Raphanus sativus L.var.radculus pers.and Brassica rapa var.glabra,and the agglutination tests of the three seed lectins with Azotobacter chroococcum CICC21686,Azospirillum brasilense CGMCC1.10379,and Paenibacillus sp.were performed.It was found that the Azotobacter chroococcum CICC21686 and the seed lectin of Brassica rapa var.glabra had obvious agglutination.Subsequent field test results showed that the Brassica rapa var.glabra applied with the suspension of Azotobacter chroococcum CICC21686 had a significant increase in various biological indicators compared with applied only with nitrogen-free medium.The growth rates of plant root length and plant height were 36.25%and 14.82%,respectively.The growth rates of underground biomass and aboveground biomass were 24.36%and 7.98%,respectively,and the growth rate of total biomass was 10.80%.The results showed that the Azotobacter chroococcum CICC21686 was effective in promoting the growth of the specific combination plants.The results of OTU cluster analysis and soil microbial diversity analysis showed that the treatment of nitrogen-fixing bacteria of Azotobacter Chroococcum had a certain effect on the bacterial diversity of rhizosphere soil of Brassica rapa var.glabra,resulting in a significant increase in the abundance of Proteobacteria,which may be one of the factors promoting the growth of Brassica rapa var.glabrae.2. ARTP was used to treat Azotobacter chroococcum CICC21686.The acetylene reduction method was used to determine the nitrogenase activity of the strain after mutagenesis.After two rounds of screening,an mutant 28s-20 was obtained,and its nitrogenase activity increased by 101.72%compared to the starting strain,which was（207.47±6.73）nmol·mg^-1·h^-1.After 5 generations of cultivation,the nitrogenase activity of the strain remained stable,which confirmed that ARTP has a good mutagenic effect on the Azotobacter chroococcum,and can obtain mutant strains with excellent characteristics and genetic stability.3. To explore the effect of mutagenesis on the biological characteristics of the strains,the growth curves of the wild strain Azotobacter chroococcum CICC21686 and the mutant strain 28s-20 were plotted,and the two strains were cultured with gradient temperature,pH,and inoculum.The results showed that the optimal growth temperature of the wild strain Azotobacter chroococcum CICC21686 and the mutant 28s-20 were28℃,and the optimal inoculation amount was 3%,indicating that temperature and inoculation amount were not the main factors affecting the maximum growth of the mutant.The optimal initial pH of the two strains was 7.5,but the change of pH had a more obvious effect on the growth of the mutant,that is,the mutant was more sensitive to the change of pH,indicating that the mutation increased the sensitivity of the strain to pH.The study of biological characteristics provides a theoretical basis for the high-density fermentation of 28s-20 mutant steain with high nitrogenase activity.A pot experiment was conducted to explore the effects of wild and mutant strains on the dry weight and total nitrogen of maize plants.The results showed that the dry weight and total nitrogen content of maize increased significantly in the test group with mutant strain,increasing by 34.95%and 77.22%,respectively,compared with the control group with nitrogen-free medium.The experimental group treated with mutant strain increased by 21.16%and 34.36%,respectively,compared with the experimental group treated with wild strain Azotobacter chroococcum CICC21686.The strain with high enzyme activity after mutation has provided technical support for the optimization of the species of Azotobacter chroococcum and nitrogen fixation of agricultural organisms.