Evaluación de la expresión de los genes involucrados en la biosíntesis de lipopéptidos antimicrobianos en Bacillus megaterium mediante RT-qPCR.

Uno de los principales problemas en la agricultura es controlar fitopatógenos, para ello ha surgido la necesidad de aplicar agentes biológicos como controladores de plagas y evitar el uso de pesticidas. Por lo tanto, en la presente investigación se planteó evaluar la expresión de genes involucrados en la biosíntesis de lipopéptidos antimicrobianos en B. megaterium mediante RT-qPCR. La expresión génica relativa (EGR) se evaluó para los genes FEND e ITUDI que codifican fengicinas e iturinas respectivamente. En primera etapa se realizó pruebas de antagonismo de B. megaterium frente a Alternaria sp. y Botrytis sp., durante nueve días, evidenciándose que la bacteria puede controlar el crecimiento de ambos patógenos. Para corroborar este resultado, la segunda etapa consistió en pruebas moleculares; se evaluó la expresión en nueve días de los genes FEND e ITUDI de muestras pertenecientes a los cultivos duales anteriores, se obtuvo que la expresión del gen FEND de Bacillus megaterium frente a Alternaria sp. aumentó en 20,16 veces y frente a Botrytis sp. aumentó 3,20 veces, ambos al día 5. De igual manera se trató para la bacteria en fermentación, a diferentes tiempos, dando como resultados que la expresión de FEND e ITUDI aumentó en 95,14 veces y 18,70 veces, respectivamente, concluyendo así que existe un aumento en la expresión génica cuando Bacillus megaterium se encuentra frente al patógeno específico a ser controlado y cuando el crecimiento de Bacillus megaterium se da por fermentación líquida, la expresión de los genes es aún mayor.One of the main problems of agriculture is to control phytopathologies, due to this factor, there is a need to apply biological agents as pest controllers to prevent the use of chemicals. Therefore, in the present research was performed to evaluate the expression of genes involved in antimicrobials lipopeptides’ biosynthesis in B. megaterium by using RT-qPCR. The relative gene expressions (RGE) evaluated were the FEND and ITUDI genes which encode for fengicines and iturines respectively. The first stage consisted of antagonist tests of B. Megaterium vs. Alternaria sp. and Botrytis sp., performed for nine days, evidencing that the bacteria controlled growth of both pathogens. To corroborate this result, the second stage consisted of various molecular tests, evaluated the expression of FEND and ITUDI genes during nine days; which were taken from the dual microbial culture previously discussed, resulting in the expression of the FEND gene of Bacillus megaterium vs. Alternaria sp. increased by 20,16 times and against Botrytis sp. increased 3,20 times, both at day 5. In the same way the bacteria were put into a fermentation process, in different times, giving an increase of 95,14 and 18,70 times more than the normal expression of the FEND and ITUDI genes respectively, concluding that there is an actual increase in the genetic expression when Bacillus Megaterium grows next to a pathogen, as previously reported. Thus, the growth of Bacillus Megaterium in fermentation conditions increases the genetic expression even more

Quantification of FEND and ITUDI Anti-fungal Lipopeptide Gene Expression in Bacillus megaterium using RT-qPCR

Phytopathogenic diseases are a major concern in modern agriculture, and for decades, pesticides have been used to prevent potential damage. Bacillus megaterium is proposed as a biological controlling agent, and gene expression of the lipopeptide genes FEND and ITUDI was assessed using RT-qPCR. Inhibition effects of B. megaterium on Alternaria sp. and Botrytis sp. were examined over a period of nine days, which confirmed the potential use of this bacterium to counteract these two pathogens. In addition, expression of FEND and ITUDI genes was assessed over nine days in the aforementioned dual cultures and inhibition tests. FEND expression in B. megaterium increased 20.16-fold in response to Alternaria sp., and ITUDI expression increased 3.20-fold in response to Botrytis sp. on day five of incubation. These results were corroborated by gene expression data obtained from B. megaterium during fermentation, where FEND and ITUDI gene expression increased 95.14- and 18.70-fold, respectively. In conclusion, B. megaterium can increase lipopeptide synthesis when exposed to these particular phytopathogens and can significantly increase the respective expression during fermentation