MOLECULAR IDENTIFICATION AND PHYLOGENY OF TWO ENDOPHYTIC PLANT GROWTH-PROMOTING SPOREFORMING BACTERIA ISOLATED FROM YERBA MATE (ILEX PARAGUARIENSIS ST. HIL.)

CORTESE, ILIANA JULIETA; OLIVETTI, ORIEL AYRTON SVIERCZ; CASTRILLO, MARIA LORENA; ONETTO, ANDREA LILIANA; ZAPATA, PEDRO DARIO; LACZESKI, MARGARITA ESTER

Rondônia

Simposio; I Simpósio de Microbiologia de Rondônia: Saúde, Ambiente e Inovação; 2021

Fiocruz Rondônia

Introduction: Ilex paraguariensis St. Hil., also called yerba mate, is one ofthe most economically important crops in Argentina. It is widely marketedin South America; but also, it is consumed worldwide. It is emphasizedthat despite this overall consumption, yerba mate can only grow in certainregions of Argentina, Paraguay, and Brazil due to unique soilcharacteristics, such as lateritic soils, and warm and moist weather.Currently, there are plantations of very good performance in the region;however, there are concerns about the increase of yerba mate degradedcrops, as a result of the monoculture system, erosion and compaction ofsoil, and nutrient loss combined with little or no soil fertilization. In ourgroup, this motivated the research and development of a biofertilizer fromnative bacteria to recover crop performance. For this reason, twoendophytic bacteria coded as 19RS3 and T5S-T4 were isolated from I.paraguariensis St. Hil. roots and selected for their plant growth-promoting(PGP) properties showed in vitro. The strains were morphologicallyidentified as Bacillus. However, bacteria closely related cannot bedistinguished from other species simply by 1 6 S rRNA gene sequence.Objective: The aim of the present work was to molecularly identify twoendophytic plant growth-promoting spore-forming bacteria isolated fromyerba mate by the analysis of the 16S rRNA, 23S rRNA, and gyrBconcatenated gene sequences. Methods: Bacillus sp. 19RS3 and T5S-T4genomes were sequenced. The reported genome of B. altitudinis strainSGAir0031 (GenBank accession number: CP022319) was used as areference for the detection of 16S rRNA, 23S rRNA, and gyrB genes. Genesearch and analysis were performed with Geneious 11.0.1 software. Thesequences obtained were compared with the nucleotide and proteindatabases of the National Center for Biotechnology Information (NCBI),using the BLASTn and BLASTx platforms, respectively. Sequences of the16S rRNA, 23S rRNA, and gyrB genes from different species belonging tothe genus Bacillus were selected and manually concatenated using theMega 6.06 software. The sequences were analyzed by the NeighborJoining, Maximum Likelihood, and Maximum Parsimony methods using theBootstrap test with 1000 replicates. Results: Neighbor-Joining, MaximumLikelihood and Maximum Parsimony trees with high bootstrap values wereconstructed. Monophyletic clades were generated for each Bacillusspecies. Bacillus sp. 19RS3 and T5S-T4 were grouped in a monophyleticclade supported by a bootstrap of 100% and were identified as B.altitudinis. Conclusions: The use of 16S rRNA, 23S rRNA, and gyrBconcatenated genes sequences provides an accurate identification forbacterial isolates that involve a complex and extensive biochemicalidentification procedure. It is an efficient method for the identification andtaxonomic analysis of closely related isolates such as those of Bacillusgenus that are involved in plant growth promotion and have a potentialapplication as biofertilizer.