Identification and characterization of the new bacillus thuringiensis serovars pirenaica (serotype H57) and iberica (serotype H59)

Two new Bacillus thuringiensis strains have been classified by the H antigen of the cells and differentiated by their morphological, biochemical and molecular characteristics. The flagellar agglutination showed that both strains bore specific H antigens which allowed their classification as the new serotypes H57 and H59. The serovar names proposed for the type strains characterized in this work are B. thuringiensis ser. pirenaica, for the H serotype 57, and B. thuringiensis ser. iberica, for the H serotype 59. Further characterization of these strains, by means of SDS-PAGE, Western inmunodetection, plasmid profile and cry-gene identification by polymerase chain reaction, confirmed the originality of the two novel serotypes. Toxicity tests carried out against several insect species, belonging to the orders Lepidoptera, Diptera and Coleoptera, showed no detectable insecticidal activity for either of the B. thuringiensis strains.

Characterization of Bacillus thuringiensis serovar bolivia (serotype H63), a novel serovar isolated from the Bolivian high valleys.

The type strain Bacillus thuringiensis var. bolivia (serotype H63), isolated from the Bolivian high valleys, has been characterized at different levels. Its parasporal crystal has an unusual shape and it is composed of a protein of 155 kDa which shows two bands of 75 and 80 kDa after activation. Analysis by PCR shows the presence of cry1 genes, and amplification with specific primers gave products for cry1 E, cry1 D, cry4 A and cry4 B with sizes different to those expected. Immunoblotting tests showed positive reaction for Cry1 E, Cry3 A, Cry4 A and Cry11 A crystal proteins. The plasmid pattern revealed two large and two small plasmids. Toxicity tests were performed against 14 insects and a slight toxicity was found against Plutella xylotella and Trichoplusia ni.

[Characterization of entomopathogenic Bacillus samples isolated in Senegal and study of their toxicity for malaria vectors]. / Caractérisation de souches de Bacillus entomopathogènes isolées au Sénégal et étude de leur toxicité pour les vecteurs du paludisme.

A screening program developed in Senegal to isolate new strains of entomopathogenic Bacillus has led to the isolation of 194 strains of Bacillus thuringiensis and 9 strains of Bacillus sphaericus from various sites and insect samples. The characterization of these strains regarding their H serotype, their crystal composition and their toxicity against mosquitoes (Culex pipiens, Aedes aegypti and Anopheles stephensi) has led to the isolation of 27 mosquitocidal strains. As malaria is an important public health problem in Senegal, these strains were more completely characterized looking for their toxicity against the two major malaria vectors in Senegal: Anopheles gambiae and Anopheles arabiensis.

Another Bacillus sphaericus serotype harbouring strains very toxic to mosquito larvae: serotype H6.

Ten isolates of Bacillus sphaericus from Ghana, very toxic to mosquito larvae, have been identified as belonging to serotype H6. These isolates can be represented by the head-group strain IAB59. They form crystals at the sporulation stage. Their larvicidal effect on Culex pipiens and Anopheles stephensi larvae is as high as that of the most toxic strains already known, e.g. 1593 and 2362 (serotype H5a,5b) and 2297 (serotype H25). Spore-crystal extracts of all these strains contain a 43-Kd polypeptide immunologically related to the 43-Kd polypeptide from strain 2362 described by other authors.

Comparison between bacteriophage typing and serotyping for the differentiation of Bacillus sphaericus strains.

Eleven bacteriophages were isolated using mosquito pathogenic strains of B. sphaericus as hosts. The phage morphology and host range on five DNA homology groups including 16 mosquito pathogenic and 32 non-pathogenic strains were determined. the same bacterial strains were studied serologically for their flagellar agglutination by antisera representing eight serotypes: six antisera prepared against the reference strains for the DNA homology groups and two others against selected mosquito pathogenic strains. Two new serotypes have been determined among the non-pathogenic strains. Both phage typing and serotyping identified subgroups within the IIA homology group which contains the mosquito pathogens. The same bacterial strains were found in the subgroups established by both methods. These subgroups often paralleled difference in the level of toxicity of the strains for mosquito larvae.

[Biochemical and serological characterization of "Bacillus sphaericus" strains, pathogenic or non-pathogenic for mosquitoes (author's transl)]. / Caractérisation biochimique et sérologique de souches de Bacillus sphaericus pathogènes ou non pour les moustiques.

A biochemical and serological study of 35 Bacillus sphaericus strains including some pathogenic for mosquito larvae, is reported. A classification of these, and a differentiation of the pathogenic strains, cannot be made on the basis of the 78 phenotypic characters which have been determined by conventional methods. Numerical analysis of the results given by the auxanograms on 160 substrates, gives good results. An even more valuable approach is the flagellar agglutination technique, which clearly distinguishes the pathogenic from the non-pathogenic strains and furthermore can specifically differentiate strains among the pathogenic isolates.

[Interest of some additional biochemical tests for the classification of "Bacillus" (author's transl)]. / Intérét de certains critères biochimiques supplémentaires pour la classification des souches de Bacillus

In addition to previous taxonomic work, eight biochemical tests have been applied to the study of 580 Bacillus strains belonging to 22 species. The attack of chitin, melibiose and amygdalin can be used for the differentiation of some species. It is the same with most of the enzymes studied here, although Tween-esterase and DNase are often present in the majority of the isolates. RNase is still more frequent, being found in nearly all the strains. On the other hand, arginin-dihydrolase appears to be very rare and ornithin-decarboxylase seems always absent.