Microbiological and molecular determination by real-time PCR of two bacteria of the genus Bacillus of agro biotechnological interest

Abstract

The comparison of growth curves was carried out at different ranges of pH, salinity, and temperature, and the size, shape, and position of the endospore of two different strains of the genus Bacillus were also determined. For molecular identification, nucleic acid extraction was performed using the Roche® High Pure PCR Template Preparation Kit and the amplification curves resulting from Real-Time PCR were subsequently analyzed. Growth kinetics were performed on each of the bacteria cultures for 72 hours and the curves were statistically analyzed using the statistical methods ANOVA and Tukey to determine significance (p<0.001). The factorial analysis for the variable "temperature" did not show a significant difference between strains and neither with the fixed values (28, 32, 37 °C), the variable "pH" did show an interaction with values (p<0.0001) between strains; the variable "salinity" also presented a significant difference between strains and with their respective interactions. In the analysis of the area under the curve, the variable "temperature" did not show a significant difference, while the variables "pH and salinity" presented values corresponding to (p <0.0001). For molecular determination, the amplification curves were analyzed, confirming the biochemical presumption of the BM strain as B. megaterium through the analysis of the phaC gene, as well as it was possible to corroborate that the BL strain corresponds to B. licheniformis when analyzing the gene. lchAA. Bacterial kinetics allowed establishing the key hours for growth analysis, confirming that the characteristics analyzed correspond to species belonging to the Bacillaceae family, with the application as biological controllers in agriculture.