Screening and biodiversity analysis of cultivable inorganic phosphate–solubilizing bacteria in the rhizosphere of Hydrilla verticillata

concentration of dissolved inorganic phosphorus of the corresponding blank control was removed from all data. Molecular biology identification of strains The total bacterial DNA was extracted with the bacterial genomic DNA extraction kit from Biotech Bioengineering (Shanghai, China.) Co., Ltd, following the kit instructions, and poly- merase chain reaction (PCR) amplification of 16S rRNA was performed with universal primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT- 3’) [20]. The PCR reaction system (20 μL) was as follows: 10× Ex Taq buffer, 2.0 μL; 5U Ex Taq, 0.2 μL; 2.5mM dNTP mix, 1.6 μL; 27F, 1 μL; 1492R, 1 μL; DNA, 0.5 μL; and ddH2O, 13.7 μL. The PCR reaction conditions were as follows: denaturation at 95˚C for 30 s, annealing at 56˚C for 30 s, extension at 72˚C for 90 s, 25 cycles, pre-denaturation before reaction for 5 min (95˚C), and extension at the end of the cycle for 10 min (72˚C). The PCR amplification products were detected by 0.8% agarose gel electrophoresis. The PCR products were purified using the kit and then sequenced using a 3730XL sequencer [Biotech Bioengineering (Shang- hai, China.) Co., Ltd.]. The obtained 16S rRNA full sequences were submitted to the Basic Local Alignment Search Tool (BLAST) in the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov/) to identify the species classification of the strains. The neighbor-joining method was used to construct a phylogenetic tree [21]. Statistical analysis Statistical analysis was performed using SPSS statistical software (version 20.0, IBM, Armonk,

NY, USA). The one-way analysis of variance (ANOVA) was used to analyze the difference of DIP among different IPB strains. Significance levels were using P = 0.05 in all statistical analy- ses. Duncan method was used for POST-HOC test. Results and discussion Separation screening results and biodiversity analysis A total of 28 strains were obtained from the roots and rhizosphere sediments of Hydrilla verti- cillata with a good phosphate-solubilizing effect. These 28 strains were sequenced and the sequencing results were submitted to the NCBI database for the BLAST homology analysis, and the results are depicted in Table 1. The phylo- genetic evolutionary tree is shown in Fig 1. The results show that the isolated strains belong to two phyla: Proteobacteria and Firmi- cutes, involving four orders (Betaproteobacteria, Alphaproteobacteria, Gammaproteobacteria and Bacilli), seven classes (Burkholderiales, Enterobacteriales, Caulobacterales, Moraxellales, Hyphomicrobiales, Aeromonadales and Bacillales), nine families (Comamonadaceae, Entero- bacteriaceae, Moraxellaceae, Aeromonadaceae, Caulobacteraceae, Yersiniaceae, Bacillales Fam- ily XII. Incertae Sedis, Rhizobiaceae and Bacillaceae), and nine genera (Bacillus, Asticcacaulis, Acinetobacter, Aeromonas, Serratia, Exiguobacterium, Comamonas, Ciceribacter and Priestia). The genus Bacillus has the highest number of IPB, with nine strains, followed by the genus Aci- netobacter, with five strains. Among these, Bacillus and Acinetobacter are the dominant genera, accounting for 32.1% and 17.9%, respectively.

A research revealed [22] that strains belonging to the genera Bacillus, Acinetobacter and Ser- ratia are the more common strains of phosphate-solubilizing bacteria with phosphate-solubi- lizing functions. This research isolates Asticcacaulis, Aeromonas, Exiguobacterium, Comamonas, Ciceribacter and Priestia as IPB, which are rarely reported. PLOS ONE Screening and biodiversity analysis of cultivable inorganic phosphate–solubilizing bacteria PLOS ONE | https://doi.org/10.1371/journal.pone.0297047 January 19, 2024 4 / 11