Culture Media and Generations Influence Diversity Assessment of Soil Culturable Phosphate-Solubilizing Bacteria

High-throughput sequencing technology was used to study the changes in the diversity of culturable phosphorus-solubilizing bacteria (PSB) during serial enrichment, to evaluate the effects of different media and generations on diversity assessment of culturable PSB in a paddy soil. Through serial passages with inorganic phosphorus medium (IPM) and organic phosphorus medium (OPM) in both solid and liquid forms, culturable "potential" PSB enrichments were obtained from the first (1st-En), second (2nd-En) and third (3rd-En) generation with the media and then for DNA extraction together with the tested soil. Bacterial 16S rRNA genes were analyzed by high-throughput sequencing technology to explore the enrichment laws of soil culturable "potential" PSB and their proportions to soil background indigenous bacterial community. The results showed that a total of 58 phyla, 160 classes, 373 orders, 575 families and 979 genera were detected in soil background bacterial communities, and 20 phyla, 35 orders, 80 orders, 121 families and 223 genera of the "potential" PSB were cultured by four media after three generations. The diversity of culturable "potential" PSB is generally higher in OPM than IPM. The proportions of culturable "potential" PSB in soil background indigenous bacterial community were the highest at the phylum level (8.62%~25.9%), while only 3.22%~12.5% at other taxonomic levels. Compared with the database of "the known PSB" constructed in this study, 110 genera of "the known PSB" were existed in soil background indigenous bacterial community; 83 genera of "the known PSB" were existed in the enrichments of four media after three consecutive generations, accounting for 75.5% of "the known PSB" in the background soil, thus at least about 24.5% of the genera of "the known PSB" in the soil were not enriched or omitted. During the culture of three generations with different media, the culturable dominant PSB mainly included Proteobacteria, Actinobacteriota and Firmicutes, and their relative abundance reached a total of 97.20~99.97%; at the genus level, PSB with different physiological and metabolic characteristics were enriched by different media and generations. In addition, a large number of 140 genera were also enriched in which phosphorus-dissolving function or characteristic genes have not been demonstrated, accounting for 62.8% of soil culturable "potential" PSB communities. Most of the genera above were rare with relative low abundance, but a few have growth advantages, such as Chelatococcus. Combined the high-throughput sequencing technology with the traditional microbial culture technology, our study reveals the diversity of soil culturable PSB can be strongly affected by the components and status of culture medium and the generations, which provides a reference for the directional exploration and research of environmental PSB resources.