Bradyrhizobium spp. aislados de nódulos de Lupinus mutabilis cultivadas en Mollebamba, La Libertad - Perú
No Thumbnail Available
Date
2025
Journal Title
Journal ISSN
Volume Title
Publisher
Universidad Nacional de Trujillo
Abstract
El presente trabajo tuvo como propósito aislar e identificar a especies de Bradyrhizobium a partir de nódulos radiculares de L. mutabilis cultivadas en el caserío de Miraflores, distrito de Mollebamba, La Libertad – Perú. Para ello se recolectó el sistema radicular con nódulos activos de 30 plantas de L. mutabilis. sin presencia de signos y/o síntomas de alguna enfermedad, de los cuales se aislaron colonias con características culturales y semejantes a las de Bradyrhizobium en agar levadura manitol (YMA) con rojo de congo, las colonias presuntivas de Bradyrhizobium se replicaron en medio agar levadura manitol (YMA), se incubó a 28°C por 5 a 10 días con la finalidad de obtener cultivos puros. Se realizo la caracterización fenotípica en medio agar levadura manitol (YMA) con azul de bromotimol, lo cual permitió distinguir al género Bradyrhizobium de otros géneros, dado que Bradyrhizobium produce alcalinidad en este medio. Los cultivos seleccionados fueron identificados genotípicamente mediante la técnica molecular de secuenciación del gen 16S ARNr para lo cual se solicitó los servicios brindados por el laboratorio Ecobiotechnology SAC. Se extrajo el ADN cromosómico, se realizó una PCR punto final, para lo cual se usaron los oligonucleótidos 518F: 5'-CCAGCAGCCGCGGTAATACG-3' y 800R: 5'-TACCAGGGTATCTAATCC-3'. Se realizó secuenciamiento SANGER del gen 16S ARNr, y las secuencias obtenidas se compararon a través de EZ BioCloud para determinar su similitud con otras cepas bacterianas tipo. La jerarquía taxonómica completa de los cultivos bacterianos identificados fue construida usando el sofware MEGA vr.11.0.13. Los cultivos puros BL-02-1 y BL-30-1 presentaron una similitud de 99.79% y 99.58% respectivamente con la cepa tipo Bradyrhizobium canariense BTA-1T según la base de datos de EZ BioCloud; mientras que el cultivo BL-13-2 presentó una similitud de 99.22% con la cepa tipo Bradyrhizobium subterraneum 58 2-1 según la base de datos de EZ BioCloud. Así pues, se logró aislar e identificar a Bradyrhizobium canariense y Bradyrhizobium subterraneum a partir de nódulos radiculares de Lupinus mutabilis cultivadas en Mollebamba, La Libertad – Perú.
The purpose of this work was to isolate and identify Bradyrhizobium species from root nodules of L. mutabilis grown in the hamlet of Miraflores, Mollebamba district, La Libertad - Peru. For this purpose, the root system with active nodules of 30 L. mutabilis plants was collected. These plants did not show signs and/or symptoms of any disease. From these colonies with cultural characteristics similar to those of Bradyrhizobium were isolated on yeast mannitol agar (YMA) with Congo red. Presumptive Bradyrhizobium colonies were replicated on yeast mannitol agar (YMA) medium and incubated at 28°C for 5 to 10 days in order to obtain pure cultures. Phenotypic characterization was performed on yeast mannitol agar (YMA) medium with bromothymol blue, which allowed distinguishing the genus Bradyrhizobium from other genera, since Bradyrhizobium produces alkalinity in this medium. The selected cultures were genotypically identified using the molecular technique of 16S rRNA gene sequencing, for which the services provided by the Ecobiotechnology SAC laboratory were requested. Chromosomal DNA was extracted, and end-point PCR was performed, for which oligonucleotides 518F: 5'-CCAGCAGCCGCGGTAATACG-3' and 800R: 5'-TACCAGGGTATCTAATCC-3' were used. SANGER sequencing of the 16S rRNA gene was performed, and the sequences obtained were compared through EZ BioCloud to determine their similarity with other type bacterial strains. The complete taxonomic hierarchy of the identified bacterial cultures was built using the MEGA vr.11.0.13 software. The pure cultures BL-02-1 and BL-30-1 presented a similarity of 99.79% and 99.58% respectively with the type strain Bradyrhizobium canariense BTA-1T according to the EZ BioCloud database; while the culture BL-13-2 presented a similarity of 99.22% with the type strain Bradyrhizobium subterraneum 58 2-1 according to the EZ BioCloud database. Thus, Bradyrhizobium canariense and Bradyrhizobium subterraneum were isolated and identified from root nodules of Lupinus mutabilis cultivated in Mollebamba, La Libertad - Peru.
The purpose of this work was to isolate and identify Bradyrhizobium species from root nodules of L. mutabilis grown in the hamlet of Miraflores, Mollebamba district, La Libertad - Peru. For this purpose, the root system with active nodules of 30 L. mutabilis plants was collected. These plants did not show signs and/or symptoms of any disease. From these colonies with cultural characteristics similar to those of Bradyrhizobium were isolated on yeast mannitol agar (YMA) with Congo red. Presumptive Bradyrhizobium colonies were replicated on yeast mannitol agar (YMA) medium and incubated at 28°C for 5 to 10 days in order to obtain pure cultures. Phenotypic characterization was performed on yeast mannitol agar (YMA) medium with bromothymol blue, which allowed distinguishing the genus Bradyrhizobium from other genera, since Bradyrhizobium produces alkalinity in this medium. The selected cultures were genotypically identified using the molecular technique of 16S rRNA gene sequencing, for which the services provided by the Ecobiotechnology SAC laboratory were requested. Chromosomal DNA was extracted, and end-point PCR was performed, for which oligonucleotides 518F: 5'-CCAGCAGCCGCGGTAATACG-3' and 800R: 5'-TACCAGGGTATCTAATCC-3' were used. SANGER sequencing of the 16S rRNA gene was performed, and the sequences obtained were compared through EZ BioCloud to determine their similarity with other type bacterial strains. The complete taxonomic hierarchy of the identified bacterial cultures was built using the MEGA vr.11.0.13 software. The pure cultures BL-02-1 and BL-30-1 presented a similarity of 99.79% and 99.58% respectively with the type strain Bradyrhizobium canariense BTA-1T according to the EZ BioCloud database; while the culture BL-13-2 presented a similarity of 99.22% with the type strain Bradyrhizobium subterraneum 58 2-1 according to the EZ BioCloud database. Thus, Bradyrhizobium canariense and Bradyrhizobium subterraneum were isolated and identified from root nodules of Lupinus mutabilis cultivated in Mollebamba, La Libertad - Peru.
Description
Keywords
Bradyrhizobium, Lupinus mutabilis, Bradyrhizobium canariense, Bradyrhizobium subterraneum, nódulos, gen 16S ARNr, oligonucleótidos, secuenciamiento SANGER, sofware MEGA vr.11.0.13.