Argentinean Bacillus thuringiensis strains exhibiting distinct morphology of their parasporal crystals

CECILIA PERALTA; DIEGO SAUKA; ANTONELLA MAROZZI; ELEODORO E. DEL VALLE; LEOPOLDO PALMA*

REVISTA ARGENTINA DE MICROBIOLOGíA

ASOCIACION ARGENTINA MICROBIOLOGIA

Lugar: Buenos Aires; Año: 2020

0325-7541

Bacillus thuringiensis is a gram-positive and sporulated bacterium exhibiting insecticidal activity against a wide range of insects3. During sporulation, this bacterium produces a number of different proteins forming crystalline inclusions adjacent to the spore (parasporal crystals). Among these insecticidal proteins, the most abundant are the commonly known as Cry (Crystal) proteins which are responsible to exert toxic activity (upon ingestion) against insects of different species5. This property has bestowed B. thuringiensis as the most efficient and used bioinsecticide to date2. However, Spodoptera cosmioides, Spodoptera eridania and Agrotis sp. (Lepidoptera) are species that are not yet controlled by some transgenic crops (e.g. Intacta RR2Pro soybean). Thus, in attempt to enlarge the host spectrum of this bacterium it is necessary to search for novel strains. In this work we show a sporulated B. thuringiensis Bt-UNVM_84 strain exhibiting a number of rare amorphous to spherical crystal combinations whereas sporulated B. thuringiensis strain Bt-UNVM-94 showed quasi symmetric bipyramidal parasporal crystals, by using Scanning Electron Microscopy (SEM) (Figure 1). Strains Bt-UNVM_84 and Bt-UNVM_94 were isolated from Oncativo (Córdoba, Argentina) and Cululú (Santa Fe, Argentina), respectively. The insecticidal activity of these different B. thuringiensis strains is currently under investigation. Each strain was grown in liquid CCY sporulation medium6 during 48 hs (150 rpm) until the absence of vegetative cells was found at a light microscope. The presence of parasporal crystals was first determined using Coomassie blue stained slides 1 (1000) using a Nikon E100 light microscope and confirmed later by using a Nikon Ti-Eclipse phase contrast microscope (1000) (data not shown). For SEM analysis, aliquots of 1 mL were centrifuged during 5 minutes (16,000 g) at room temperature. Each pellet was washed three times with sterile distilled water and fixed with 100 µl 4% formaldehyde. Each fixed preparation was then sent to Centro Integral de Microscopía Electrónica (CIME - CONICET - UNT) for SEM examination.