Padronização de ensaio molecular por qPCR para a quantificação absoluta de Bacillus methylotrophicus em raízes de soja

Abstract

Soybean (Glycine max) belonging to the fabaceae family is the most widely cultivated species in the world and the fourth most important crop produced annually in terms of harvested area and production. Its successful cultivation depends on different factors, highlighting the efficient management of pests and diseases, which can be achieved with the use of biocontrol agents. The identification and quantification of the colonization of these beneficial microorganisms, including Bacillus methylotrophicus, has been carried out phenotypically. This study aimed to quantify, by qPCR, B. methylotrophicus in soybean roots using oligonucleotides designed by our research group. At first, qualitative analyzes were carried out to determine the presence/absence of B. methylotrophicus in the samples to simplify the tests. Amplification of the 16S reference gene was successful, confirming the presence of Bacillus in the collected material. We constructed an absolute standard curve and, due to the complexity of the rhizospheric environment, we evaluated the interactions of B. methylotrophicus with other biocontrol microorganisms commonly used by the partner company. We obtained, by qPCR, a higher concentration of B. methylotrophicus when applied together with B. subtilis. In contrast, the non-treated control and the treatment with the isolated microorganism showed no statistically significant difference in their recovery. Furthermore, we suggest the occurrence of a possible competition between B. methylotrophicus and Trichoderma asperellum. Therefore, the molecular marker developed for B. methylotrophicus proved to be effective in the molecular characterization of this species and can be applied in several studies related to the identification, interaction and concentration of B. methylotrophicus.