Native bacteriophages infecting Bacillus thuringiensis: Morphology and thermostability

Abstract

The production of bioinsecticides based on Bacillus thuringiensis (Bt) is sensitive to bacteriophage infection. As lytic bacteriophages, they could act directly on the bacteria cells and/or bacteria could have prophages integrated to the genome or as a plasmid. Consequently, the isolation, characterization andidentification of the viruses infecting entomopathogenic strains are crucial for improvement the industrial process. Bt RT (EF638795.1) is a native strain isolated from an indigenous Spodoptera frugiperda (Sf) larva, and its efficacy against different pests including Sf has been proven. In this work, we proposedthe characterization of two environmental bacteriophages (M3 and M4) active against Bt RT. Those viruses were previously isolated from soil samples from Tucumán. Thermostability of M3 and M4 was evaluated as follow: Single lysis plaques of M3 and M4 phages were propagated in LB cultures of Bt RT. Suspensions were filtered (0,22 μm) and titrated by the Double Agar Overlay Plaque Assay. Thermal assays were carried out at 45, 50, 55, 60, 65, 70 and 75°C during 30 min. Samples were taken every 10 min. Later, 3 μl of each suspension were dropped onto a Bt RT lawn and incubated at 30°C ON. As a result, after 30 min of treatment at 60°C both bacteriophage suspensions were completely inactivated. For Transmission Electron Microscopy, a drop of purified bacteriophage was negatively stained with1% uranyl acetate on formvar-coated copper grids and photographed on a Zaiss EM109 microscope (Oberkochen, Germany). This study showed tailed bacteriophages. So, according to the morphological analysis, both bacteriophages belonging to the order Caudovirales. In this order, M3 belong to the Siphoviridaefamily based on the observed non-contractile and flexible tail (length= 293 nm; width= 9nm) with a capsid width= 58.6 nm and a capsid height= 55 nm). M4 showed a morphology similar to the Myoviridae family with the peculiar contractile tail (length= 66 nm; width= 18 nm) and with an isometric head with a diameter of ~66 nm. In general, both phages exhibited similar behavior regarding temperature. The results obtained will allow us to design more effective control procedures to avoid contamination in fermentations with loss of the product which may be partial or total depending on the type of bacteriophage. In additionthe isolated bacteriophages could be also used for the phagotyping of this bacterial species.