MOLECULAR DETECTION OF BACILLUS ALTITUDINIS 19RS3 AND T5S-T4 ISOLATED FROM YERBA MATE (ILEX PARAGUARIENSIS ST. HIL.) USING STRAIN-SPECIFIC PRIMERS

CORTESE, ILIANA JULIETA; 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: The genus Bacillus presents a great diversity of species that are widely distributed in the environment. It is one of the most studied genera and was shown to improve plant growth through acombination of mechanisms. It is used as a biofertilizer that holds promise to make sustainable agricultural practices and ecologically safe. In our previous studies, two endophytic endospore-formingbacteria coded as Bacillus altitudinis 19RS3 and T5S-T4 were isolated from Ilex paraguariensis St. Hil roots and selected for their plant growth-promoting (PGP) properties in vitro and in vivo. Strain-specificprimers to detect B. altitudinis 19RS3 and T5S-T4 strains were designed by whole-genome analysis.Objective: This study aimed to test the pre-designed strain-specific primers with DNA isolated from B. altitudinis 19RS3 and T5S-T4.Methods: Genomic DNA from liquid cultures incubated at 30°C for 24 h was extracted by using Sambrook work protocol modified. For the molecular detection, the primers designed for 19RS3: 873F 3´-ATTggCAAAgATAgCAggg-5´, 873R 3´-AgCATCAATCggCTgTggA-5´, 884F 3´-ggTCAgCCTgTAAAAACACCg-5´ and 884R 3´-gTCCCATCCATTAACCTTCA-5´; and for T5S-T4: 2341F 3´-ACACCACATCATTCACTggAgA-5´, 2341R 3´-gCCTTCTAACATCCTgCA-5´, 3296F 3´-gCTACATATCCAACTCCTCAgA-5´ and 3296R 3´-AgCAATAgTAACCgACTTCTCAg-5´ were used. Strain-specificprimers were tested in 20 μL standard PCR reactions. The reaction mixture contained 1X Taq DNA polymerase buffer (10X: 500 mM KCl, 100 mM Tris-HCl, pH 9.0 at 25°C, 1% Triton®X-100), 200 µM ofeach dNTP, 10 pmol of each primer, and 0.5 U of the enzyme Taq DNA polymerase (Inbio Highway, Argentina). Amplifications were performed in a thermal cycler multigene TM II (Labnet International Inc.,USA). During the first cycle, DNA was denatured at 94 °C for 5 min. For the subsequent 30 cycles, the tubes were kept at 94 °C for 40 s, at 57 °C for 70 s and at 72 °C for 65 s, followed by an extension at72 °C for 10 min. The annealing temperatures of 55; 57, and 60 °C were tested. The PCR products were visualized in 2% (w/v) agarose gel stained with GelRed® (Sigma-Aldrich, Germany). Theelectrophoretic run was performed in an electrophoretic vessel (Electrophoresis Subsystem 70 Labnet International) at 110 V for 30 min and bands were visualized using a UV transilluminator (Model MUV21-312-220).Results: The primers were specific for each B. altitudinis strain, as no amplification products were obtained for negative controls. Amplification products were produced using an annealing temperature of 57°C. Amplicons of approximately 500 and 300 bp were generated for B. altitudinis 19RS3 and T5S-T4, respectively.Conclusions: B. altitudinis 19RS3 and B. altitudinis T5S-T4 were successfully detected. The use of strain-specific primers are one of the cheapest and quickest methods to monitoring the colonization ofbacterial strains in nursery and field experiments. The strain-specific primers will be applied in future traceability experiments.