Microbial biobank-based strain phenotype identification of plant growth-promoting rhizobacteria in citrus

Plant growth-promoting rhizobacteria (PGPR) can increase nutrient availability and protect plants against diseases, thus benefiting host plants. Despite advances in the study of PGPR, the mechanism by which rhizobacteria promote plant growth remains largely unclear due to the lack of the microbial biobank for PGPR isolation especially in perennial woody crops such as citrus.By 16S rRNA amplicon sequencing of microbiota, we first investigated the rhizobacterial composition from five citrus-producing regions in China and identified 9887 amplicon sequence variants (ASVs) belonging to 28 rhizobacterial phyla, among which Proteobacteria phylum possessed a higher relative abundance in the perennial woody (citrus) plants than in annual herbaceous plants. Additionally, we established a citrus rhizosphere microbial biobank (containing 3142 bacteria strains) by a large-scale isolation of the citrus rhizobacteria, and screened 269 potential PGPR from 435 bacterial taxa in the biobank by examining the production of indole-3-acetic acid (IAA) and siderophores, the dissolution of phosphate, and their effect on Arabidopsis root growth. We verified the plant growth-promoting functions of 22 representative potential PGPR via a pot-culture inoculation of microbes onto the seedlings of citrus, tomato, and Arabidopsis, among which, PGPR strain 2H3 belonging to Proteobacteria phylum extensively distributed in different citrus habitats significantly promoted plant growth, probably by secreting active substrates (VHLD, RLLPA, 3-oxo-C8-HSL, and N-Ac-5-ASA) to the rhizosphere and increasing root nutrient uptake.In summary, we revealed a significant role of Proteobacteria in citrus, established a comprehensive citrus rhizobacterial biobank, characterized potential PGPR, and identified active substrates secreted by PGPR strain. Our findings provide theoretical basis and valuable reference for further development and utilization of beneficial rhizobacteria in citrus cultivation.