Effects Of AER314-4 Strain Inoculation On Endophytic Diversity During Seed Germination Of Ammodendron Bifolium

Ammodendron bifolium is a sandy deciduous shrub of the genus Ammodendron of Leguminosae,and it is a rare sand-fixing plant endemic to desert areas in our country.The germination rate of Ammodendron bifolium seeds is low under drought conditions,studies have confirmed that the difficulty of seed germination under drought conditions is one of the endangered factors.In order to solve the problem of seed germination of Ammodendron bifolium,in the early stage,isolated the endophytic bacterium AER314-4（Bacillus axarquiensis）from the root of Ammodendron bifolium,and it was confirmed that inoculating AER314-4 strain under drought conditions could increase the seed germination rate from 6% to 53%.Transcriptome analysis showed that inoculation with AER314-4 strain could restore the expression of drought inhibited genes in silver sand locust seeds and activate silenced genes.Metabolome analysis showed that the main factors that promoted the germination of Ammodendron bifolium seeds were small molecules,and their increase could alleviate drought stress and promote seed germination.In order to reveal the role of endophytes in symbiotic drought resistance,this study took the seeds of Ammodendron bifolium as the research object,using high-throughput sequencing technology,the composition and evolution of endophytic bacteria in three treatment groups（control group（CK）,drought stress group（DS）,drought stress plus bacteria group（DE））,different germination media（filter paper,sterilized in situ soil and in situ soil）conditions and early,middle and late stages of germination were studied,to understand the effects of germination medium,drought stress and inoculation of AER314-4 strain on the composition and diversity of endophytic bacterial community.The main findings are as follows:The high-throughput sequencing data of Ammodendron bifolium seed samples and soil samples in situ under sufficient water conditions were compared and analyzed.The results showed that at the phylum level,when filter paper was used as the medium,the dominant phylum of endophytic bacteria in the samples was Proteobacteria;when sterilized soil and in situ soil were used as media,the endophytic bacterial groups in the samples changed from Firmicutes in the early and middle stages of germination to Proteobacteria in the late germination stage.At the genus level,when filter paper was used as the medium,the endophytic bacterial groups in the samples changed from Ralstonia in the early and middle stages of germination to Bacillus and Massilia in the later stage.When the sterilized soil was used as the medium,the endophytic bacterial groups in the samples changed from Bacillus in the early stage of germination to Bacillus,Massilia,Delftia and Tumebacillus in the middle stage;in the later stage,Massilia,Pantoea and Pseudomonas are the main species.When the in situ soil was used as the medium,the dominant groups of endophytic bacteria in the samples evolved from Bacillus in the early and middle stages of germination to Massilia and Pseudomonas in the later stage.The results showed that the endophytic bacteria in the samples were mainly derived from the inherent endophytic bacteria of the seeds of Ammodendron bifolium;the dominant phyla and genera of endophytic bacteria in different germination media samples were different,indicating that the germination medium was one of the main factors affecting the dominant bacterial flora of Ammodendron bifolium seed.Under drought stress conditions,through the comparative analysis of high-throughput sequencing data of Ammodendron bifolium seeds and in situ soil samples,it was found that endophytic bacteria in the drought stress group were distributed in 24 phyla,59 classes,151 orders,270 families and 512 genera.At the phylum level,when filter paper was used as the medium,the endophytes of Proteobacteria in the samples were inhibited by drought stress.When the sterilized soil was used as the medium,the drought stress inhibited the endophytes of Proteobacteria in the early stage of germination and the endophytes of Firmicutes in the middle stage.When the in situ soil was used as the medium,the endophytes of Firmicutes in the samples were inhibited by drought stress.At the genus level,under drought stress conditions,the endophytic bacteria of Ammodendron bifolium seeds were mainly composed of Bacillus,Lysinibacillus,Pseudomonas,Klebsiella and Paenibacillus is the main genera that can resist drought stress in existing reports.Diversity and sharing analysis showed that drought stress hindered the survival of some strains in the seeds of Ammodendron bifolium,reducing the number of endophytic bacterial species in the samples by about 20%.Inoculation of exogenous strains under drought stress conditions,the results showed that the strain did enter the seeds and had an absolute dominance,the sequence of this strain accounted for more than 90% of the high-throughput sequencing data.After removed AER314-4（ie OTU1314）from the data,when filter paper is used as the medium,the dominant phylum in the sample is Firmicutes;when sterilized soil was used as the medium,inoculation with exogenous strains gradually recovered the endophytes of Proteobacteria that were suppressed under drought conditions,and the relative abundance of Firmicutes gradually decreased;when the in situ soil was used as the medium,the dominant phylum in the samples was Proteobacteria.At the genus level,inoculation with exogenous strains resulted in changes in the relative abundances of Bacillus,Massilia,Lysinibacillus,Pseudomonas,Klebsiella and Paenibacillus in the sample,It is speculated that the evolution of these microbial taxa regulates seed metabolism,thereby relieving drought and promoting seed germination.The results showed that Ammodendron bifolium seeds contained abundant and diverse endophytic bacteria,the endophytic bacteria in the samples were mainly derived from the inherent endophytic bacteria of Ammodendron bifolium seeds;drought stress hindered the survival of some strains of the seed itself and reduced the diversity of endophytic bacteria in the sample;the inoculation of AER314-4 strain can regulate the endophytic bacterial groups in the seeds of Ammodendron bifolium to alleviate drought stress and promote seed germination.The results of this study are of great significance to enrich the research on the symbiotic bacteria of Ammodendron bifolium,analyze the effects of drought stress and exogenous strain inoculation on the diversity of endophytic bacteria in the seeds of Ammodendron bifolium,and understand the symbiotic stress resistance mechanism of AER314-4 strain from the perspective of endophytic bacteria.The experiment and theoretical basis were provided for the application of endophytic bacteria in the stress resistance,population protection and breeding of rare plants.