Isolasi dan Karakterisasi Bakteriofag sebagai Agen Biokontrol Agrobacterium tumefaciens

Abstract

Agrobacterium tumefaciens adalah bakteri penyebab crown gall disease pada tumbuhan namun berguna sebagai vehicle cloning dalam transformasi genetik tanaman. Akan tetapi, transformasi menggunakan Agrobacterium tumefaciens sering menyebabkan overgrowth yang berujung pada kematian kalus. Bakteriofag dapat menjadi agen biokontrol terhadap infeksi dan pengontrol Agrobacterium tumefaciens karena spesifisitasnya yang tinggi. Penelitian ini bertujuan mengisolasi dan mengarakterisasi bakteriofag litik spesifik Agrobacterium tumefaciens dari sampel limbah perairan. Isolasi dilakukan dengan pengambilan dan perbanyakan bakteriofag spesifik dari sampel limbah cair, diikuti dengan teknik double layer menggunakan Luria Agar hingga terdeteksi munculnya plak (28 °C, 72 jam). Karakterisasi bakteriofag meliputi pengamatan morfologi plak, penentuan nilai multiplicity of infection (MOI), uji kisaran inang, uji laju adsorpsi, uji stabilitas suhu dan pH, isolasi genom, digestion, serta analisis Random Amplified Polymorphic DNA (RAPD). Dua isolat bakteriofag litik (ΦWB dan ΦSBM) berhasil diisolasi dan spesifik terhadap Agrobacterium tumefaciens strain AGLI WT dan EHA105. Rasio MOI untuk kedua isolat adalah 10-5 dan 10-3. Isolat ΦWB memiliki stabilitas suhu yang lebih baik, dan stabilitas pH yang sama dengan ΦSBM pada rentang 5-11. Kedua isolat memiliki laju adsorpsi optimal yang berbeda. Kedua bakteriofag memiliki materi genetik dsDNA dan berbeda secara genetik berdasarkan analisis RAPD. Isolat ΦWB berpotensi sebagai agen biokontrol Agrobacterium tumefaciens.

Agrobacterium tumefaciens is a bacterium that causes crown gall disease and become a serious problem for agricultural plants. Bacteriophages can be potential biocontrol agents against Agrobacterium infection because of their high specificity and efficiency. This study aimed to isolate and characterize lytic bacteriophages specific to Agrobacterium tumefaciens from wastewater samples. Isolation processes involved samples enrichment, followed by double layer to detect the presence of bacteriophage plaques. Bacteriophage characterization included plaque morphology, multiplicity of infection (MOI), host range test, adsorption rate, temperature, and pH stability. Molecular characterization included genome isolation, digestion, and Random Amplified Polymorphic DNA (RAPD) analysis. Two bacteriophages (ΦWB and ΦSBM) were classified as lytic bacteriophages based on their transparent plaque morphology. Both isolates specific to Agrobacterium tumefaciens strains AGLI WT and EHA105. Both isolates had MOI ratios of 10-5 and 10-3. Our results showed that both bacteriophages had temperature and pH stability in the range of 28-30 °C and pH 5-11. ΦWB and ΦSBM isolates demonstrated optimal adsorption rate at 18 and 27 minutes respectively. Genome characterization showed that both bacteriophages possessed dsDNA as genetic material and RAPD analysis showed that both bacteriophages were genetically different. ΦWB was found promising as biocontrol agent of Agrobacterium tumefaciens.