Isolation Of Enterobacter Cancerogenus JY65 And Its Molecular Mechanism Of Alleviating NaCl Stress In Rice

At present,soil salinity is one of the main problems affecting global crop yield reduction.Researchers have attempted to alleviate the effects of salt stress on plant growth using a variety of approaches,including genetic modification of salt-tolerant plants,screening higher salt-tolerant genotypes,and the inoculation of beneficial plant microbiome,such as plant growth-promoting bacteria(PGPB).PGPB mainly exists in the rhizosphere soil,plant tissues and the surfaces of leaves or stems,and can promote plant growth and increase plant tolerance to abiotic stress.Many halophytes recruit salt-resistant microorganisms,therefore,endophytic bacteria isolated from halophytes will help enhance plant stress responses.Beneficial plant-microbe interactions are widespread in nature,and microbial communities provide an opportunity to understand these beneficial interactions.In this study,we analyzed the structural differences of bacterial communities in Suaeda and checked whether they recruited PGPB,determined the plant growth promoting characteristics and saline-alkali tolerance of culturable bacteria,predicted the functional genes involved in plant growth promotion and stress response in Enterobacter cancerogenus JY65 by whole genome sequencing,and demonstrated its growth promoting mechanism against salt stress,which lays a theoretical foundation for utilization of PGPB.(1)After collecting stem,leaf,root,rhizosphere and bulk soil samples of two Suaeda species from Northeast Forestry University,Illumina MiSeq platform was used to conduct 16S rRNA gene sequencing on the bacterial community in these samples,and to compare the influence degree of different plant species and compartments and check whether they recruit PGPB.The results showed that the bacterial diversity of Suaeda species decreased gradually from soil to root system.The two Suaeda species established very similar plant-related microbiota in the rhizosphere and tissue environment.The dominant phyla of all the samples were Proteobacteria,Actinobacteria,Gemmatimonadetes,Planctomycetes,Bacteroidetes and Acidobacteria,of which Proteobacteria was extremely abundant in the tissue samples.Proteobacteria and Actinobacteria were the core microbiome in this species,indicating that their wide distribution may play an important role in the stability and function of the microbial community.There were abundant beneficial bacteria in different samples,and significant differences in the bacterial composition related to plant species and sample types.The difference of bacterial community mainly existed among different plant compartments but not plant species.The ecological networks of different plant compartments were non-random and have the topological characteristics of complex systems,such as being scale free,small world and modularity,giving the ecological networks stability and resilience,and the topological characteristics of different networks were significantly different.(2)Culturable bacteria were isolated and identified from the bulk soil,rhizosphere soil,root,stem and leaf samples of S.salsa,and their plant growth-promoting traits and salt-alkali tolerance were evaluated in vitro.The results showed that these culturable bacteria mainly belong to Proteobacteria,Firmicutes and Actinobacteria,and are mainly halobacteria.All strains have high saline-alkali tolerance,but their growth-promoting abilities vary greatly according to saline-alkali tolerance,IAA secretion and plant-bacteria interaction test.Among them,the JY65 strain has a strong saline-alkali resistance and a significant plant-promoting effect on the plant height,root length and fresh weight of rice against NaCl stress,suggesting that this strain is a PGPB.(3)The whole genome sequence of JY65 was obtained through the combination of third-generation single-molecule sequencing technology and second-generation high-throughput sequencing technology,and its related genes and metabolic pathways involved in plant growth-promoting traits and salinity tolerance were analyzed.The results showed that the genome of the JY65 strain has 4 916 634 bp with a GC content of 55%,4 497 coding sequences and 82 tRNA.Based on the 16S rRNA gene and average nucleotide identity,JY65 belongs to Enterobacter cancerogenus.We identified the genes encoding plant growth-promoting traits(phosphate dissolution,siderophore and IAA secretion),as well as the genes involved in host colonization(motility,chemotaxis,adhesion and secretion system).In terms of adaptation to the adverse environment,many enzymes and regulatory factors are found in response to oxidative stress,including antioxidant enzymes,Na+/H+antiporter,K+transporter,glycine-betaine and trehalose.The comparison of plant growth-promoting genes among different strains showed that the general classification pattern of the genes was similar,among which the motility,chemotaxis and secretion systems were the most significantly different.(4)We evaluated the effects of JY65 and different mutant strains in the protection of rice seedlings against NaCl stress using hydroponics.The result confirmed that the potential to JY65 strain has the potential to effectively alleviate the damage caused by NaCl stress to rice seedlings by secreting Na+/H+antiporter,biofilm,betaine,trehalose and T6SS.Inoculating rice plants subjected to salt stress with PGPB JY65 significantly increased the non-enzymatic(AsA and GSH)and enzymatic oxidation system activities(SOD,POD,APX and CAT)to remove the accumulation of reactive oxygen species,and promoted the expression of salt-related genes(OsSOS1,OsHKT1;1,OsNHX1,OsNHX2,OsNHX3,OsNHX5)to maintain the balance of cell osmotic stress,which effectively alleviate the damage caused by NaCl stress.