Screening Of Plant Growth Promoting Rhizobacteria And Its Application In Promoting Alfalfa Growth By Coupling With Black Liquor

Long term application of chemical fertilizers leads to gradual degradation of arable land,soil ecology damage and the decrease of crop yield and quality,and even leads to a vicious cycle that crop yield doesn’t increase with increasing chemical fertilizer.Therefore,seeking environmentally friendly alternatives to fertilizers has become a hotpot.In this study,we tried to use Black Liquor produced by lignocellulose pretreatment,and plant growth promoting rhizobacteria（PGPR）to replace chemical fertilizers,while ensuring the yield of alfalfa.This is expected to be a good method to maintain soil health,and develop sustainable agricultural production.In this study,11 plant rhizosphere microorganisms were screened.And the mechanism of Enterobacter sp.PB promoting alfalfa growth was preliminarily revealed.The results indicated that PB promoted the growth of alfalfa by enhancing its photosynthesis.Firstly,PB enhances alfalfa photosynthesis by inducing upregulation of genes related to photosynthesis,such as LHCB1.Secondly,PB optimized the structure of soil microbial communities,resulting in 10times increase in the abundance of Rhizobiaceae.Correspondingly,the content of total nitrogen in soil and alfalfa,which provide nitrogen for chloroplast synthesis,increased 30%and 1 time,respectively.In addition,the organic acids secreted by PB converted insoluble phosphorus in the soil into available phosphorus.And then,the total phosphorus content in the soil and alfalfa increased.The increase of total phosphorus content is conducive to the enhancement of photosynthesis.Furthermore,some organic acids secreted by PB,such as citric acid and oxalic acid,enhanced photosynthesis in alfalfa.For the above reasons,the chlorophyll content of alfalfa increased by 45%and the net photosynthetic rate increased by 4.5 times.Then,the fresh weight of alfalfa increased by 6 times and the protein content increased by 2 times.Potassium alkali process Black Liquor was firstly proved that it not only had the potential to replace fertilizers,but also optimized the community composition of soil microorganisms and endophytic bacteria in alfalfa roots.Black Liquor was used as fertilizer to increase the content of nitrogen,phosphorus,and organic matter in soil.The content of available nitrogen and available phosphorus in the soil were increased by 1 time,and the organic matter content was increased by 24%.The increase in soil organic matter was beneficial for improving soil microbial communities.Black Liquor increased the abundance of soil nitrogen fixing Cyanobacteria,which became a new dominant microbial community.Black Liquor also transformed the dominant genus of endophytic bacteria in alfalfa roots from Novosphingobium（12.98%）to Ensifer（38.32%）,which was beneficial for improving the yield and quality of alfalfa.Compared with the control group,the treatment group treated with black liquor increased the yield of fresh alfalfa by 1.5 times and the protein content by one time.The combination of plant growth promoting rhizobacteria PB and Black Liquor improved the growth promoting effect of alfalfa compared to using them alone.Because PB and Black Liquor interact and promote each other in the soil.PB converted nitrogen from Black Liquor into NH₄⁺,making it more easily absorbed by plants.Black Liquor provided nutrients for PB,enhance its colonization in the rhizosphere of alfalfa,and enable PB to better optimize the soil microbial community structure.Compared to the control group,the abundance of Lysobacter,Flaviolibacter,Bdellovibrio,Pseudomonas,and Ensifer in the treatment group treated with Black Liquor combined with PB increased.In addition,the combination of PB and Black Liquor can also enhance the photosynthesis of alfalfa.The expression of genes related to carotenoid synthesis and photosynthesis has increased.Compared with the application of PB or Black Liquor alone,the combined treatment increased the total nitrogen content in soil by 40%and20%,and increased the total nitrogen content in alfalfa increased by 23%and 19%,respectively.The fresh weight of alfalfa were increased by 15%or 8%,and the protein content were increased by 1%or 18%,respectively.In summary,both plant growth promoting microorganisms and Black Liquor improved the yield and quality of alfalfa,and have the potential to replace fertilizers.Among them,the combination of Black Liquor and plant growth promoting microorganisms may have better application prospects.Replacing fertilizers by Black Liquor not only may solve industrial pollution problems,but also may reduce the costs of related industries such as potassium alkali papermaking and cellulose production.