High-throughput cultivation and screening of plant-promoting bacteria with nitrogen fixation from mangrove sediments

Aims The present study was to isolate, cultivate, and screen plant growth-promoting bacteria (PGPB) from mangrove sediments at high throughput, and compare the differences in microbial diversity and PGPB screening efficiency between the high-throughput and traditional agar plate methods. Methods and Results A high-throughput method combining limiting dilution and two-sided barcode PCR was developed to effectively isolate and identify bacteria from mangrove sediments. Moreover, the metagenome and 16S rRNA amplicon sequencing were used for identification of potential PGPB genera and investigation of microbial diversity in different pooled cultures. The results showed that the microbial communities of the bacteria cultured by the high-throughput method had a significantly lower Simpson index (p < 0.05) and a higher proportion of rare species than that by the agar plate method. A diverse bacterial consortium encompassing 79 species of 39 genera and 6 phyla were successfully isolated by the high-throughput method. Furthermore, three species of Bacillus and four species of Pseudomonas exhibiting plant growth-promoting properties were isolated and purified from a series of Bacillus and Pseudomonas genera, including a previously uncultured Pseudomonas strain. In contrast, all the PGPB screened by the agar plate method belonged to the Vibrio genus, which has been reported to be pathogenic to humans. Conclusions The study demonstrated that the high-throughput method was superior to traditional plate methods for isolating a broader spectrum of microbial diversity from mangrove sediments, particularly rare species. When combined with metagenome sequencing, this approach enables a more efficient screening for PGPB potentially applicable for agriculture or environmental protection.