Screening Of PGPR Strains Of Typical Legumes On Alpine Grassland And Construction Of Synthetic Bacteria

Alpine grasslands are a major component of the Qinghai-Tibet Plateau(QTP)and play an irreplaceable role in maintaining regional and global ecological security.Since the end of last century,QTP grassland ecosystem services have been severely degraded by a combination of endogenous and exogenous factors such as habitat fragility,climate change and human activities.The main constraints for the restoration of degraded alpine grasslands include plant species,soil microorganisms,soil nutrients and human factors,and restoration measures based on soil microorganisms and soil nutrients are a kind of near-natural restoration measures.By screening the strains of Plant growth-promoting rhizobacteria(PGPR)suitable for degraded grassland restoration and developing bacterial agents,the microbial constraints of degraded grassland restoration can be solved;the vegetation restoration technology based on soil nutrient regulation can solve the soil constraints of degraded grassland restoration.At present,there are few microbial agents developed and produced for grassland and forage at home and abroad,and the existing agents have a single function,mostly medium-temperature bacteria,which are not suitable for alpine grassland with low temperature all year round.In addition,according to the study of soil dynamics of degraded grassland in alpine meadows on Qinghai-Tibet Plateau,the organic carbon and total nitrogen content of surface soil(4-20 cm)decreased with the increase of grassland degradation.The number and species of legumes in degraded grasslands decreased,and the healthy growth of legumes is especially important to improve the productivity of grasslands,which shows that the establishment of legumes in degraded alpine grasslands is especially important.However,the severely reduced soil fertility in degraded alpine grasslands results in phosphorus deficiency that not only limits plant productivity,but also seriously affects the nitrogen fixation efficiency of legumes.In view of this,this study was conducted to isolate and identify the culturable bacteria in the inter-rhizosphere soil of typical legume forage grasses in degraded grasslands on the Tibetan Plateau,and to screen and identify the excellent PGPR strains with insoluble phosphate solubilizing ability,and to establish the corresponding strain resource library.Based on the different probiotic properties of the strains,we constructed a synthetic colony with Phosphate-solubilizing bacteria(PSB)as the core functional strain,and investigated the effects of inoculating different combinations of synthetic colony on plant growth to obtain the combination with the best probiotic effect.The main results obtained were as follows:1.A total of 191 culturable strains were isolated from the inter-rhizosphere soil samples of different legumes.At the phylum level,they were mainly distributed in Actinobacteria,Firmicutes,Proteobacteria and Bacteroidetes;at the genus level,they were mainly distributed in Arthrobacter,Lysobacter,Pseudomonas and Bacillus,and 60 other genera.Sixty-four PSB strains were obtained from the soil samples using selective media screening.They were mainly distributed in the phyla Proteobacteria,Actinobacteria and Firmicutes,and 14 genera including Pseudomonas,Streptomyces and Bacillus.2.10 bacterial strains were selected from the above strains by phylogenetic analysis and molecular biology identification as research materials.The results showed that these 10 strains had different growthpromoting characteristics(nitrogen fixation,phosphorus solubilization,and growth hormone secretion);the experimental results showed that the inoculation of these 10 strains promoted the growth of Arabidopsis thaliana and Medicago sativa L.to different degrees compared with the blank control.The growth of Arabidopsis thaliana and Medicago sativa L.were evaluated by principal component analysis.The effects of 10 strains on the growth of alfalfa were comprehensively evaluated by principal component analysis,and six candidate strains were obtained for the next study based on the evaluation results;firstly,the antagonistic effects between these six candidate strains were tested by plate standoff assay,and four candidate strains were identified for the construction of synthetic microbial communities,namely: Pseudomonas sp.GBXD-49,Bacillus sp.BHQ-R1,Serratia sp.XMKD-R6,and Pararhizobium sp.GBXD-R56.The strain GBXD-R49 with the highest overall score and high phosphate solubilization capacity was used as the main effective strain of the synthetic microbial communities.3.Based on the above experiments,the growth curves of the four candidate strains were measured,and the results showed that the OD600 values of the logarithmic growth period of the four candidate strains ranged from 0.2to 1.0;the API identification system was used to determine the substrate utilization ability of the four candidate strains,and the results showed that the strains GBXD-R49,BHQ-R1,XMKD-R6 and GBXD-R56 had The results showed that some combinations of synthetic colonies had higher phosphorus solubilization and growth hormone(Indole-3-acetic acid,IAA)production ability compared with the single strain GBXD-R49.The results showed that inoculation with different combinations of synthetic flora significantly increased the plant height,root length,biomass,chlorophyll content and root vigor of alfalfa,especially the synthetic flora combinations of PB(GBXDR49+BHQ-R1),PS(GBXD-R49+XMKD-R6)and PSP(GBXD-R49+XMKDR6+GBXD-R56)had significant effects.In this study,a large number of indigenous strains from alpine grasslands were obtained,and the synthetic bacterial flora constructed on this basis had good growth-promoting effects on alfalfa.The results of this study will provide habitat-applicable inter-rhizosphere growth-promoting strains for the restoration of degraded grasslands on the Tibetan Plateau,and provide some theoretical basis for solving the microbial constraints and soil constraints of degraded grassland restoration.