Actividad antifúngica e identificación molecular de cepas nativas de Bacillus subtilis (2016)

espanolLa bioprospeccion de bacteria antagonicas, como agentes de control biologico para el manejo de hongos fitopatogenos, es una actividad preponderante para reducir el uso de fungicidas quimicos. La bacteria Bacillus subtilis ha mostrado una excelente capacidad para inhibir el crecimiento y esporulacion de una gama amplia de hongos fitopatogenos. Las caracteristicas bioquimicas para diferenciar entre subespecies de B. subtilis varian a nivel regional. En esta investigacion se aislaron cepas de Bacillus de la Peninsula de Yucatan en Mexico y se evaluaron sus actividades antagonicas por confrontacion directa en un diseno completamente al azar. Para identificar las cepas mas activas se realizo una caracterizacion molecular. Para una identificacion precisa se secuenciaron parte de los genes 16S rRNA, gyrA y rpoB y se compararon con los depositados en el GenBank. Las cepas mas activas CBCC2, CBMT2, CBMN22, CBRF24, CBCK36, CBCK47 y CBMT51 causaron la inhibicion del crecimiento micelial de 32 a 78 % en Alternaria alternata, Helminthosporium rostratum y Curvularia lunata en pruebas de confrontacion directa; asimismo, los filtrados de cultivos libres de celulas de estas cepas Bacillus mostraron diferencias significativas (p≤0.05) en el grado de inhibicion de germinacion conidial de los hongos fitopatogenos. La amplificacion, secuenciacion y comparacion de los genes 16S rRNA, gyrA y rpoB mostraron que las cepas tenian una identidad de 95 a 100 % con Bacillus subtilis subsp. inaquosorum y B. subtilis subsp. subtilis. El gen gyrA mostro la tasa evolutiva mas alta al usar el analisis de la relacion filogenetica con las cepas estudiadas. EnglishThe bioprospection for antagonistic bacteria, as biological control agents to manage plant pathogenic fungi, is a preponderant activity to reduce the use of chemical fungicides. The bacterium Bacillus subtilis has shown an excellent capacity to inhibit the growth and sporulation of a wide variety of plant pathogenic fungi. The phenotypic and biochemical characteristics to differentiate among subspecies of B. subtilis vary at the regional level. In this research, Bacillus strains were isolated from the Yucatan Peninsula in Mexico and their antagonistic activities were assessed by direct confrontation in a completely randomized design. To identify the most active strains, molecular characterization was performed. For an accurate identification, partial 16S rRNA, gyrA and rpoB genes were sequenced and compared with those deposited in the GenBank. The most active strains CBCC2, CBMT2, CBMN22, CBRF24, CBCK36, CBCK47, and CBMT51 caused 32 to 78 % inhibition of mycelial growth in Alternaria alternata, Helminthosporium rostratum and Curvularia lunata in direct confrontation tests; likewise the cell-free culture filtrates of these Bacillus strains showed significant differences (p≤0.05) in the degree of inhibition of conidial germination of the plant pathogenic fungi. The amplification, sequencing and comparison of 16S rRNA, gyrA and rpoB genes showed that the strains had 95 to 100 % identity with Bacillus subtilis subsp. inaquosorum and B. subtilis subsp. subtilis. The gyrA gene showed the highest evolutionary rate using the analysis of phylogenetic relationship within the strains studied.