Isolation And Identification Of An Alfalfa Endophytic Actinomycete MtRt-6 And Evaluation Of Its Plant Growth-promoting Effects

Endophytes are an important biological resource,which can promote the stable and efficient production of plants.When used as biological fertilizer,it can reduce a series of environmental and health problems caused by excessive application of chemical fertilizers.In this thesis,an endophytic actinomycete MtRt-6 was isolated from alfalfa,and its growth-promoting effect on plants was explored to provide a theoretical basis for further understanding of its growth-promoting mechanism and its development and utilization in agriculture.The research content and results are as follows:(1)Isolation and identification of the endophytic actinomycete:Through surface-sterilization combined with four kinds of isolation medium(TWEY?YMA?MS?HV),the endophytic actinomycete MtRt-6 was isolated from roots of alfalfa.Combined with physiological and biochemical characteristics,16S r RNA sequence homology analysis and Non-Redundant Protein Database annotations,it was identified as the rare actinomycete Amycolatopsis tolypomycina.(2)Analysis of physiological and biochemical characteristics of endophytic actinomycete MtRt-6:Through culture experiments with different conditions,it is found that MtRt-6 has a wide range of adaptations to the types of media,carbon sources,temperature and p H.By using detection media,it was found that MtRt-6 can produce protease,chitinase,cellulase,tyrosinase,amylase and other hydrolase enzymes.In addition,MtRt-6 also has the growth-promoting properties of nitrogen fixation,phosphorus dissolution,potassium dissolution,and ACC enzyme production.The headspace-gas chromatography-mass spectrometry method was used to further identify the volatile substances produced by MtRt-6,and it was found that the components were ocimene,2-methyl-2-bornene and 2-methylisoborneol.(3)Whole genome sequencing and bioinformatics analysis of MtRt-6:The whole genome size of MtRt-6 is 10,575,343 bp and the G+C content is 71.63%.It is predicted that there are 9,777 protein-coding genes,7 r RNA operons,53 t RNA genes and 29 s RNA genes.In addition,through a comprehensive analysis of the annotation results of the KEGG database and the Swiss-Pro database,19 genes encoding enzymes related to nitrogen metabolism pathways such as nitrogen fixation,ammonia assimilation,nitric acid and nitrous acid reduction were found in the genome of MtRt-6,including isc U,glt BD,gln AE,nar UGHIK,gdh,acd S and other genes.At the same time,a total of 35 secondary metabolite biosynthetic gene clusters in genome of MtRt-6 were predicted and analyzed using anti SMASH.(4)Analysis of MtRt-6's characteristics of promoting host growth:MtRt-6 spore suspension with different concentrations(106,107,108 spores·m L^-1)were inoculated on Medicago truncatula and rice by root irrigation and embedding.It was found that the biomass of the inoculated plants was significantly higher than that of the uninoculated plants and the above-mentioned effect is dose-dependent.The concentration of MtRt-6spore suspension with the best effect on the growth of Medicago truncatula and rice is 10⁶spores·m L^-1and 10⁷ spores·m L^-1,respectively.The results of real-time fluorescent quantitative PCR showed that the relative expression levels of the housekeeping gene atp D of MtRt-6 and the key nitrogen assimilation gene GS of Medicago truncatula and rice were significantly up-regulated after inoculation,indicating that MtRt-6 can colonize stably in both Medicago truncatula and rice,and regulate the nitrogen metabolism in the host to promote the growth of the host plant.In conclusion,this study has isolated and identified a strain of alfalfa endophytic actinomycete Amycolatopsis tolypomycina MtRt-6,which has the potential to be developed as a biological fertilizer and inoculant.