Bacillus thuringiensis serovar higo (flagellar serotype 44), a new serogroup with a larvicidal activity preferential for the anopheline mosquito

M. OHBA, H. SAITOH, K. MIYAMOTO, K. HIGUCHI AND E. MIZUKI. 1995. Eight strains of Bacillus thuringiensis , isolated in Japan, formed spherical parasporal inclusions and exhibited low to moderate larvicidal activities for two mosquito species, Anopheles stephensi and Culex pipiens molestus , but not for another dipteran, Telmatoscopus albipunctatus , or two lepidopterans, Bombyx mori and Hyphantria cunea . The anopheline toxicity (LC₅₀= 6.3 μg ml^-1) was > 10 times greater than the activity on the Culex mosquito. These strains were assigned to a previously undescribed flagellar (H) antigenic group. On the basis of the representative strain, 92–KU-137–4, a serogroup with H antigen 44, Bacillus thuringiensis serovar higo was established as new.     

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.     

Formulation of a flowable liquid concentrate of Bacillus thuringiensis serotype H-14 spores and crystals as mosquito larvicide

Bacillus thuringiensis serotype H-14 spores and crystals, produced in 51 fermenters, were centrifuged and resuspended in emulsified palm olein to give 3.2 x 10(11) colony forming units (cfu)/ml. The suspension was mixed with a cassava-molasses-palm olein-charcoal (CMPC-2) mixture which served as the carrier, adhesive, dispersant and protectant. The final concentration of the formulation was 3.2 x 10(9) cfu/ml. The lethal concentrations capable of killing 50% of the test population (LC50) of CMPC-2 during 0, 1 and 2 years of storage at 32 +/- 4 degrees C were 0.056, 0.058 and 0.058 mg/ml respectively as against 0.054, 0.051 and 0.054 mg/ml for the Institut Pasteur Standard-1978 (IPS-78) during the corresponding period. The chi 2 tests showed that the results were homogeneous at P = 0.05. The relative potencies of the preparations were 964.3, 879.3 and 931 International toxic units (ITU) Aedes aegypti as compared with the 1000 ITU assigned to IPS-78. At 95% confidence limits there was no significant difference between the potencies of CMPC-2 and IPS-78. Field tests showed that CMPC-2 provided between 87.5 and 100% control of natural populations of Aedes spp. and Cutex spp. Sedimentation tests showed that CMPC-2 settled markedly during storage. This, therefore, required that the product be thoroughly shaken before use.     

Formulation of a flowable liquid concentrate of Bacillus thuringiensis serotype H-14 spores and crystals as mosquito larvicide

A.O. EJIOFOR AND N. OKAFOR. 1991. Bacillus thuringiensis serotype H-14 spores and crystals, produced in 5 1 fermenters, were centrifuged and resuspended in emulsified palm olein to give 3.2 × 10¹¹ colony forming units (cfu)/ml. The suspension was mixed with a cassava—molasses—palm olein—charcoal (CMPC-2) mixture which served as the carrier, adhesive, dispersant and protectant. The final concentration of the formulation was 3.2 × 10⁹ cfu/ml. The lethal concentrations capable of killing 50% of the test population (LC₅₀) of CMPC-2 during 0, 1 and 2 years of storage at 32 ± 4.C were 0.056, 0.058 and 0.058 mg/ml respectively as against 0.054, 0.051 and 0.054 mg/ml for the Institut Pasteur Standard—1978 (IPS-78) during the corresponding period. The² tests showed that the results were homogeneous at P = 0.05. The relative potencies of the preparation were 964.3, 879.3 and 931 International toxic units (ITU) Aedes aegypti as compared with the 1000 ITU assigned to IPS-78. At 95% confidence limits there was no significant difference between the potencies of CMPC-2 and IPS-78. Field tests showed that CMPC-2 provided between 87.5 and 100% control of natural populations of Aedes spp. and Cutex spp. Sedimentation tests showed that CMPC-2 settled markedly during storage. This, therefore, required that the product be thoroughly shaken before use.     

Characterization of Bacillus thuringiensis ser. jordanica (serotype H71), a novel serovariety isolated in Jordan

The novel strain of Bacillus thuringiensis J112 isolated from a soil sample in Jordan was classified and characterized in terms of toxicity against dipteran and nematode larvae, crystal protein pattern, plasmid profile, and cry gene content. A new name, Bacillus thuringiensis serovariety jordanica (H serotype 71), is proposed for the reference strain J112. The parasporal crystal proteins were toxic to 3(rd) instar larvae of Drosophila melanogaster and to 2(nd) stage juveniles of root knot nematodes Meloidogyne javanica and M. incognita, but showed poor mosquitocidal activity towards Culex pipiens molestus and Culiseta longiareolata larvae. Solubilized and trypsin-digested crystal proteins possessed moderate hemolytic activity against sheep erythrocytes. SDS-polyacrylamide gel electrophoresis revealed that crystals are composed of several polypeptides ranging from 24 to 170 kDa, of which the 20-, 42-, 140-, and 170-kDa proteins were the major components. Analysis of the plasmid pattern of J112 revealed the presence of two large plasmidic bands of about 160 and 205 kbp. PCR with total DNA from strain J112 and specific primers for cry1, cry2, cry3, cry4, and cyt2A genes revealed that cry1, cry3A, cry4, cry5 and cyt2a genes are present.     

Tandem repeats in a new toxin gene from Bacillus thuringiensis and in other cry11-like genes

A new gene, cry11Bb2 from a field isolate of Bacillus thuringiensis, was cloned for expression in Escherichia coli. The encoded protein, with a deduced molecular mass of 89.5 kDa, exhibits 97 and 79% identities with the overlap regions of Cry11Bb1 from B. thuringiensis ssp. medellin and Cry11Ba1 from ssp. jegathesan, respectively. It is however longer than Cry11Bb1 by 42 amino acids in its carboxy-terminus, of which 32 comprise 2 tandem repeats additional to the 5 existing in the latter polypeptide. Possible roles for this recurrent motif among Cry toxins and their accessory proteins, and for their encoding genes are proposed.     

Cloning of cry2Aa and cry2Ab genes from new isolates of Bacillus thuringiensis and their expression in recombinant Bacillus thuringiensis and Escherichia coli strains

Bacillus thuringiensis (Bt) is the major source for transfer of genes to impart insect resistance in transgenic plants. Cry2A proteins of Bt are promising candidates for management of resistance development in insects due to their difference from the currently used Cry1A proteins, in structure and insecticidal mechanism. Two insecticidal crystal protein genes of Bt, viz. cry2Aa and cry2Ab were cloned from new isolates of Bt, 22-4 and 22-11, respectively. Expression of both the genes was studied in an acrystalliferous strain of Bt (4Q7) by fusing the cry2Aa and cry2Ab genes downstream of cry2Aa promoter and orf1 + orf2 sequences. Western blot analysis revealed a low level expression of the cloned cry2Aa and cry2Ab genes in the recombinant Bt strains. High-level expression of cry2Aa and cry2Ab genes was achieved in the recombinant E. coli by cloning the cry2A genes under the control of the T7 promoter.     

cry Genes profiling and the toxicity of isolates of Bacillus thuringiensis from soil samples against American bollworm,Helicoverpa armigera

Aims: The aim of this study was to search for Bacillus thuringiensis (Bt) harbouring cry1A gene which could effectively control cotton pest, American bollworm, Helicoverpa armigera. Methods and Results: cry gene profiling of 50 Bt isolates showed the presence of cry1, cry2, cry3, cry4, cry7, cry8 and cry9 genes. None of the isolates harboured cry1 gene alone. It was always found in combination with cry3. There was no isolate positive for cry10 gene. Considering isolates with single cry genes, the frequency of cry4 was predominant (22%) followed cry2 (6%), cry3 (4%) and cry8 (2%). Isolates having two cry genes in combination had 14% incidence for cry2 + cry4, 12% for cry3 + cry4 and 10% for cry1 + cry3. The most dominant three gene linkage was cry1 + cry3 + cry4. Further profiling of cry1 gene showed that cry1K gene was abundantly present in all combinations such as cry1A, cry1D, cry1F and cry1I. However, cry1C existed independent of other subtypes. Finally, the Bt isolates with cry1A were analyzed for 16S rRNA gene, which showed two distinct groups of isolates on the basis of sequence homology. Bioassays of spore–crystal mixtures of SBS‐Bt4, 8, 17, 21 and 26 harbouring cry1 against neonate larvae of H. armigera showed LC₅₀ 1288, 1202, 467·7, 524·8 and 108·5 μg ml^?1. The SBS‐Bt26 showed fourfold higher toxicity than the cry 1Ac harbouring positive control, HD‐73. Conclusions: None of the isolates harboured single cry 1 gene. They were always in combination of two or three genes. A Bt isolate (Bt26) had fourfold higher toxicity against H. armigera larvae compared with the positive control HD 73 and hence can be commercially exploited to control insect pest. Significance and Impact of the Study: The inter relationship between the cry genes content and the toxicity may allow better understanding of Bt ecology.     

Characterization and cloning of the cry2A gene from indigenous isolates of Bacillus thuringiensis

Molecular Biology - Discovery of novel cry genes of Bacillus thuringiensis (Bt) with higher toxicity is important for the development of transgenic Bt crops resistant to target pests. Two new...     

Bacillus subtilis soil isolates: plasmid replicon analysis and construction of a new theta-replicating vector

We have searched for plasmids in a collection of 55 Bacillus subtilis strains isolated from various natural sources of the territory of Belarus. Twenty percent of the strains contained one or two plasmids of either 6-8 or approximately 90 kb. Small plasmids were shown to carry a rolling circle replicon of the pC194 type. Four out of the eight large plasmids contained a related theta replicon that has no homolog in databases as shown by sequence determination. A B. subtilis/Escherichia coli shuttle vector based on this replicon was constructed. It has a low copy number (6 units per chromosome) and is stably inherited in B. subtilis. It might thus be a useful tool for DNA cloning. These data extend previous observations, indicating that most of the small plasmids of B. subtilis replicate as rolling circles and belong to the pC194 family. On the contrary, large plasmids appear to form a large pool of theta-replicating determinants, since three different replicons have already been isolated from them.     

Screening and characterization of Bacillus thuringiensis isolates from Brazil for the presence of coleoptera-specific cry genes

Forty-three Bacillus thuringiensis isolates from Brazil and 3 from Argentina were screened, using the polymerase chain reaction (PCR), for various coleoptera-specific cry genes. Seven isolates produced specific and/or nonspecific DNA fragments in a PCR reaction with primers specific for two coleopteran cry genes and 4 of these produced DNA fragments with primers specific for 7 known coleopteran cry genes. These isolates showed, by electron microscopy, the presence of spherical crystals. They also showed proteins of around 70 kDa which were immunologically similar to the Cry3Aa protein from B. thuringiensis subsp. tenebrionis. The 3 isolates from Argentina were toxic to T. molitor, and although no isolate from Brazil showed toxicity, they might show toxicity to another insect species.     

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.     

Isolation of a Bacillus thuringiensis strain (serotype 8a:8b) highly and selectively toxic against mosquito larvae

An isolate of Bacillus thuringiensis designated as PG-14 obtained from the Philippines was highly toxic to the mosquitoes Aedes aegypti and Culex molestus but nontoxic to the silkworm, Bombyx mori, and adults of a daphnid. The degree of toxicity to mosquito larvae was the same as that of the reference strain of Bacillus thuringiensis subsp. israelensis (serotype 14). Parasporal inclusion produced by the isolate PG-14 was spherical or irregular in shape and morphologically similar to that produced by the reference strain of subsp. israelensis. The H antigenic structure of the isolate was identical to that of the reference strain of B. thuringiensis subsp. morrisoni (serotype 8a:8b). Differences were shown in the O antigenic structures and in the production of lecithinase. Thermostable exotoxin was not produced by the isolate PG-14. The results indicate the isolation of a B. thuringiensis strain which shows the same toxicity as that of subsp. israelensis.     

Description of Culicoides lisicarruni (Diptera: Ceratopogonidae), a new species from Cundinamarca, Colombia

A new species of Culicoides of the subgenus Diphaomyia Vargas from high altitudes of the Andes in Colombia is described and photographied. The species is compared with its similar congener Culicoides marinkellei Wirth & Lee. Data on the collecting site and notes on the species daily activity are also provided.     

Persistence and biological activity in soil of the insecticidal proteins from Bacillus thuringiensis, especially from transgenic plants

The Dune System of Doñana National Park (SW Spain) exhibit a mosaic of environmental characteristics, with different plant communities, all under the same Mediterranean climate, creating an interesting field laboratory for the study of plant responses to stressing conditions. Fourteen woody plant populations were selected, belonging to either xerophytic or hygrophytic plant communities on stabilised dunes, where topography causes differences in soil water availability. Plants were tagged and morphological and ecophysiological measurements were recorded in winter and summer of 1999. Seasonal differences in ecophysiological measurements together with morphological variables were used as plant traits to identify the main adaptive responses of the species. Cluster analysis of traits separated three groups of plant strategies: spiny legume species; sclerophyll, and semideciduous species. In addition, another two kind of strategies have been found in the semideciduous group of species, those withstanding water shortages, attaining very negative water potentials, low photochemical efficiency, and leaf proline accumulation in summer, as opposed to those tolerating water deficit, with moderate seasonal differences in water potential, proline content and photochemical efficiency. The results of this study indicate that legume species behave as a different functional group and drought-semideciduous species present different adaptive responses under the same environmental stress. Ecophysiological measurements must be used as plant traits to detect functional groups under Mediterranean climate.     

Description of Lentistivalius philippinensis,a new species of flea (Siphonaptera,Pygiosyllomorpha, Stivaliidae), and new records of Ascodipterinae (Streblidae) on bats and other small mammals from Luzon,The Philippines

During May 2009 and July 2011, we collected 357 mammals and examined each for ectoparasites. Among the ectoparasites collected, a new species of flea was discovered. This new species, Lentistivalius philippinensis, is described from the male sex only. Two males were recovered from two specimens of the soricid Crocidura grayi Dobson in Municipality Maria Aurora, Aurora Province, Luzon, Philippines. Additional fleas included Thaumapsylla breviceps orientalis Smit, Thaumapsylla longiforceps Traub, and Ischnopsyllus indicus Jordan. Although the latter species is common in Japan and documented in Guam (as well as mainland Southeast Asia) also on Pipistrellus javanicus (Gray), Ischnopsyllus indicus represents a new record in the Philippine Islands. The ascodipterinae (Streblidae) Maabella stomalata and Ascodipteron speiserianum Muir collected from Rhinolophus inops K. Andersen and Rhinolophus subrufus K. Andersen, respectively, also represent new host records. A key to the species of the flea genus Lentistivalius Traub is provided.     

Purification and partial amino acid sequence of thuricin S, a new anti-Listeria bacteriocin from Bacillus thuringiensis

We report the isolation and characterization of a new bacteriocin, thuricin S, produced by the Bacillus thuringiensis subsp. entomocidus HD198 strain. This antibacterial activity is sensitive to proteinase K, is heat-stable, and is stable at a variety of pH values (3-10.5). The monoisotopic mass of thuricin S purified by high performance liquid chromatography, as determined with mass spectrometry ESI-TOF-MS, is 3137.61 Da. Edman sequencing and NanoESI-MS/MS experiments provided the sequence of the 18 N-terminal amino acids. Interestingly, thuricin S has the same N-terminal sequence (DWTXWSXL) as bacthuricin F4 and thuricin 17, produced by B. thuringiensis strains BUPM4 and NEB17, respectively, and could therefore be classified as a new subclass IId bacteriocin.