Taxonomic Marker Validation for Accurate Bacillus Species Identification

FIGUEROA, SOL A.; PETITTI, T.; TORRES MANNO, MARIANO A.; DAURELIO, LUCAS D.; ESPARIZ, MARTIN

Chapadmalal

Congreso; XVIII Congreso de la Sociedad Argentina de Microbiología General (SAMIGE); 2023

SAMIGE

Our laboratory focuses on the study of soil microbiome composition using high-resolution molecular markers and its correlation with crop condition in order to predict actions that enhance its yield.The Bacillus subtilis clade is usually classified in 5 groups, B. subtilis, B. amyloliquefaciens, B. gobiensis, B. pumilus, and B. licheniformis. It has been reported that some Bacillus species have PGPR activity, but there are multiple cases of incorrect classifications or incoherences between taxonomic classification and genomic or phenotypic characteristics. For the development of innovative inoculants -which could serve as an environmentally friendly substitute for conventional fertilizers-, it is crucial to accurately classify bacterial strains and establish a clear connection between their specific effects on crops.Given this background, a curated genome database was generated, 29,5% of analyzed sequences were reasigned considering Average Nucleotide Identity and multi-locus sequence analysis results, and 7,3% strains were classified as new species. After that, the performance of the 16S gene to classify groups was tested. An accuracy of 0.78 and a recall of 0.81 were observed. Hence, a new gene marker is needed to classify groups of Bacillus subtilis clade, and therefore, species within them. Considering these results, the performance of 14 genetic markers was evaluated. Two molecular markers out of them were selected to perform precise identification of species within the Bacillus genus. Subsequently, two sets of degenerate primers were designed, enabling PCR amplification of these markers for up to 40 different species.Afterward, efforts were dedicated to refining the metagenomic soil extraction process and marker amplification from soil, rhizosphere and root samples from wheat cultures under different conditions.