Isolation and characterization of Bacillus sphaericus strains obtained from larvae of Culex pipiens in La Plata city, Argentina.

TRANCHIDA M. C.; RICCILLO P.M.; GARCIA J.J.; MICIELI M. V.

Villa Carlos Paz, Cordoba, Argentina

Congreso; VI Congreso Argentino de Microbiología General; 2009

Sociedad Argentina de Microbiologia General

Bacillus sphaericus is an aerobic, spore–forming bacterium, isolated from the soil and mosquito larvae. Mosquitocida B. sphaericus strains have toxicity against mosquito larvae due to binary toxin encoded by genes bin. Thus are used in insect control program. The aims of this work were: 1- Isolation, 2- Phenotypic and molecular characterization of native Bacillus, with toxicity to immature stages of Cx. pipiens. 3- Determination of the toxicity and host range. 4-Identification of genes bin and mtx. Cx. pipiens larvae were collected from natural breeding sites. At laboratory, dead larvae were observed under microscope where the presence of bacteria with spores was detected. The native isolates were grown in NYSM agar media, which showed the presence of three different types of colonies. These isolates were named C107, C207 and C307. Gram and malachite green stain was performed. We evaluated its growth in different carbon sources, in gelatin, ability to utilize casein, acetoin, tyrosine, and lecithin. Resistance to antibiotic (streptomycin, erythromycin, tetracycline and chloramphenicol), growth in different concentrations of sodium chloride and boric acid were evaluated. We studied the catalase activity, the ability to hydrolyze urea and to produce hemolysis. Also sequencing of 16S rRNA genes and the comparation with sequences available in database BLAST were performed. The sequences from native and references (SPH88 and K7865) strains were aligned using CLUSTAL. The presence of genes encoding toxins in B. sphaericus were analyzed by PCR, using primers BSN1/BSN2, BS1/BSN2 to amplify genes bin B and BSN3/BSN4, BS3/BS4 to bin A.  The primers 100.1 and 100.2 were used to detect genes mtx. The host range was evaluated according to Institute Pasteur standard protocols. Were tested: Cx. pipiens, Ae. aegypti, Cx. dolosus, Cx. apicinus, Oc. albifasciatus and An. albitarsis. The LC50 was obtained through the Probit Analysis. The strains B. sphaericus 2362 and SPH88, and strain K7865 of B. fusiformis were used as reference strains. The isolates C107 and C207 showed mosquitocida activity. Cx. pipiens was the species more susceptible to C107 (LC 50 4x104 spore/ml) meanwhile Oc. albifasciatus showed more susceptibility to C207 (LC 50 3.4x106 spore/ ml). Ae. aegypti have not been susceptible to these strains. The presence of genes bin A and B, and mtx were recorded in both native strains. Analysis of the sequence of 16S rDNA genes, the cytomorphology and biochemical characterization showed that the C107 strain belongs to the group of B. sphaericus meanwhile the native strain C207 is more close to the B. fusiformisgroup. The C307 strain was identified as B. licheniformis and it did not have mosquitocida activity.