DOI: 10.5281/zenodo.17201908
title: Dataset: A guide-assay for isolation of plant growth-promoting microorganisms using traditional and low-cost methodologies

description: The aim of this project was to sequence native isolates from the soil and rhizosphere microbiome of three Agave species planted in a semi-arid region of Brazil. The Agave species used were: i) Agave sisalana; ii) Agave hybrid H11648 (Agave amaniensis x Agave angustifolia) x Agave amaniensis; and iii) Agave hybrid H400f (Agave sp., with unknown parents).

Five soil samples and five rhizosphere samples were used for each Agave species analysed (five biological replicates per Agave species). In order to isolate plant growth-promoting microorganisms in these environments, ten different semi-selective culture media were used: i) Pseudomonas Isolation Agar (PIA); ii) JNFb; iii) NFb; iv) Verma; v) HP; vi) MacConkey Agar (MCK). Bacillus ChromoSelect Agar® (BCSA); viii) T3; ix) Dichloran Rose-Bengal Chloramphenicol Agar (DRBC), and; x) Glucose-Peptone-Yeast Extract Agar (GPYA).

Therefore, we have two environmental sources of isolation (soil and rhizosphere), three hosts for the microbiota (A. sisalana, H11648, and H400f), and ten semi-selective culture media used for isolation, totalling 60 distinct factors for the origin of the isolates obtained. These 60 factors were used to name the strains obtained after the isolation protocol, using the following nomenclature:



The 16S rRNA gene of the isolated prokaryotes was subjected to Sanger sequencing using didesoxynucleotides (primers 27F and 1492R), while the ITS region of filamentous fungi and yeasts was analysed using the same method (primers ITS1F and ITS4). Subsequent to the process of sequencing, the resulting .ab1 files were collected and processed via the Biopython (v.1.85) software. 

All files were converted to the. fasta format, resulting in the creation of the following compiled files:

i)                    16S_multifasta.fasta for prokaryotes;

ii)                  ITS_multifasta.fasta for eukaryotes.

For the 16S gene sequences, taxonomic identification was performed using the ACT tool from the SILVA rRNA database. The ITS sequences were aligned by similarity, with the NCBI/BLASTn database serving as a source of taxonomic data for the identification of the isolated strains. The identification of the isolates is described in the file entitled "16S_ITS_Taxonomy.xlsx".

Sequence alignment, both for 16S and ITS, was performed using MAFFT software (v.7.526) in automatic mode, with the TrimAI tool (v.1.5.0) also in automatic mode to remove sequences incompatible with the alignment. The pipeline generated two files: 

i)                    16S_multifasta_aligned.fasta for sequences from prokaryotes;

ii)                  ITS_multifasta_aligned.fasta  for the fungi and yeasts analyzed. 

Subsequently, phylogenetic clustering was performed using IQ-TREE 2 software (v.3.0.1). This step generated two tree files:

i)                    16S_IQtree.treefile  for prokaryotes sequences;

ii)                  ITS_IQtree.treefile  for the fungal and yeasts sequences.

These files were then uploaded to the iTOL platform for the visual exploration and annotation of the phylogenetic trees. Further details about the pipeline and the analyses can be found in the main article associated with this project.