Computational protocol: A novel plant-based-sea water culture media for in vitro cultivation and in situ recovery of the halophyte microbiome

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Protocol publication

[…] Throughout the microbiological analyses of tested halophytes, one hundred forty-six isolates were selected. Based on their cultural and morpho-physiological characteristics, forty-four representatives of various plants, spheres and culture media were selected for further characterisation. They were tested for PGP functions: nitrogen fixation, phosphate solubilization, indole acetic acid production, and salt tolerance. Based on results obtained, they were clustered (PAST3 software; https://folk.uio.no/ohammer/past), using Unweighted Pair Group Method with Arithmetic Mean (UPGMA). The resulting distance matrix was visualized in dendrogram, and reformatted using FigTree software (http://tree.bio.ed.ac.uk/software/figtree), and annotated using the online tool of Interactive Tree of Life (iTOL) (http://itol.embl.de). [...] Selected isolates were grown in liquid cultures of the corresponding culture media, then bacterial broth cultures were centrifuged at 9500g for 15 min., and DNA was extracted from bacterial pellets using the QIAGEN DNeasy plant mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The extracted DNA was used as a template to amplify the whole 16S rRNA gene using the primers 9bfm (GAGTTTGATYHTGGCTCAG) and 1512r (ACGGHTACCTTGTTACGACTT) . The reaction was performed in a total volume of 25 µL with 2 µL template DNA (ca. 2–18 ng µL−1), 12.5 µL of QIAGEN TopTaq master mix (Qiagen, Hilden, Germany), 5.5 µL PCR water, and 2.5 µL of 3.3 pmol of both primers, using the Bio-Rad C1000 Thermal Cycler (Bio-Rad, CA, USA). The thermal cycling program was adjusted as follows: 4 min of initial denaturation at 95 °C, 30 thermal cycles of 1 min denaturation at 95 °C, 1 min annealing at 56 °C, and 1 min of extension at 74 °C; PCR was finished by a final extension step at 74 °C for 10 min. QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) was used to purify the PCR product according to the manufacturers’ instructions.16S rRNA gene sequencing was performed according to Sanger enzymatic sequencing (Eurofins MWG Operon, Ebersberg, Germany). 16S rRNA gene sequences were compared with their closest matches in GenBank (www.ncbi.nlm.nih.gov/BLAST/) and GreenGenes (http://greengenes.lbl.gov/cgi-bin/nph-index.cgi) databases to determine the taxonomy of the bacterial strains. Together with 429 sequences representing all species of Bacillus spp. (280), Halomonas spp. (134), and Kocuria spp. (15), we constructed the phylogenetic tree using MUSCLE and the Neighbours-Joining methods based on the maximum composite likelihood model implemented in MEGA 6.0 . The bootstrap values were calculated after 1000 replicates and indicated at each node. The 16S rRNA gene sequences identified in this study have been deposited in the GenBank database under the accession numbers: KU836856–KU836865 [...] Analysis of Variance (ANOVA) and Fisher’s Least Significance Difference (LSD) were carried out using STATISTICA v10 (Statsoft, OK, USA). […]

Pipeline specifications

Software tools FigTree, iTOL, MEGA, Statistica
Applications Miscellaneous, Phylogenetics, 16S rRNA-seq analysis
Organisms Mesembryanthemum crystallinum, Hemisus marmoratus, Hordeum vulgare
Chemicals Sodium Chloride