Occurrence of Bacillus thuringiensis on Cured Tobacco Leaves

A worldwide survey was conducted to evaluate the frequency and distribution of Bacillus thuringiensis populations on cured tobacco leaves during post-harvest storage. In total, 133 tobacco samples of different types and origins were analyzed. Nine percent of the samples showed the presence of B. thuringiensis, and 24 B. thuringiensis strains were isolated and characterized. The majority of the isolates produced bipyramidal crystals, and three fourths of them showed a second type of crystal protein (cuboidal or heterogeneous crystals). Only three isolates showed the rhomboidal crystal morphology characteristic of the anti-coleopteran B. thuringiensis subsp. tenebrionis. PCR analysis with primers specific for cry1 and cry3 genes revealed eight distinct cry gene profiles. The results of this study indicate that B. thuringiensis is naturally present at low frequency on the phylloplane of cured tobacco leaves and that its distribution is worldwide. Received: 26 August 1999 / Accepted: 5 October 1999     

Diversity analysis and characterization of Coleoptera-, Hemiptera- and Nematode-active cry genes in native isolates of Bacillus thuringiensis

Applications to combat non-lepidopteran insects are not as common as applications against lepidopteran insects. The aim of the present work was to isolate and identify Bacillus thuringiensis isolates from soil samples using five approaches, viz., analysis of crystal protein production by microscopy; detection of cry gene content by PCR, SDS-PAGE profiling; cloning and sequencing; phylogenetic analysis; and toxicity testing. Two hundred soil samples were used for isolation of B. thuringiensis and a total of 69 putative isolates of B. thuringiensis that produce parasporal crystalline inclusions were isolated from 5,267 Bacillus-like colonies. A bipyramidal inclusion was predominant in 32.2 % of the B. thuringiensis isolates compared to other shapes. Crystal protein profiling of B. thuringiensis isolates by SDS-PAGE analysis showed the presence of bands of 130, 73, 34, 25 and 13 kDa, among which 50–60 kDa bands were present abundantly. PCR analysis revealed the predominance of Coleopteran-active cry genes in these isolates. Variation in nucleotide sequences, crystal morphology and mass of crystal protein(s) purified from the isolates of B. thuringiensis revealed genetic and molecular diversity. Four strains containing Coleopteran-active cry genes showed higher toxicity against Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) adults when compared with B. thuringiensis subsp. morrisoni pathovar tenebrionis. These results are useful in emphasizing the distribution of cry genes and for prognostication of toxicity, and may contribute to the identification of novel candidate genes for bioengineered crop protection.     

Fluorescent Amplified Fragment Length Polymorphism Analysis of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis Isolates

DNA from over 300 Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis isolates was analyzed by fluorescent amplified fragment length polymorphism (AFLP). B. thuringiensis and B. cereus isolates were from diverse sources and locations, including soil, clinical isolates and food products causing diarrheal and emetic outbreaks, and type strains from the American Type Culture Collection, and over 200 B. thuringiensis isolates representing 36 serovars or subspecies were from the U.S. Department of Agriculture collection. Twenty-four diverse B. anthracis isolates were also included. Phylogenetic analysis of AFLP data revealed extensive diversity within B. thuringiensis and B. cereus compared to the monomorphic nature of B. anthracis. All of the B. anthracis strains were more closely related to each other than to any other Bacillus isolate, while B. cereus and B. thuringiensis strains populated the entire tree. Ten distinct branches were defined, with many branches containing both B. cereus and B. thuringiensis isolates. A single branch contained all the B. anthracis isolates plus an unusual B. thuringiensis isolate that is pathogenic in mice. In contrast, B. thuringiensis subsp. kurstaki (ATCC 33679) and other isolates used to prepare insecticides mapped distal to the B. anthracis isolates. The interspersion of B. cereus and B. thuringiensis isolates within the phylogenetic tree suggests that phenotypic traits used to distinguish between these two species do not reflect the genomic content of the different isolates and that horizontal gene transfer plays an important role in establishing the phenotype of each of these microbes. B. thuringiensis isolates of a particular subspecies tended to cluster together.     

Production of heat-stable exotoxin by Bacillus thuringiensis and related bacteria

Heat-stable exotoxin production by 740 strains of Bacillus thuringiensis and related bacteria was investigated using the housefly, Musca domestica, from the following viewpoints: (1) the relation-ship between B. thuringiensis flagellar (H) serotypes and exotoxin production and (2) the exotoxin production by Bacillus species other than B. thuringiensis. Of 437 isolates belonging to 11 serotypes of B. thuringiensis which had been confirmed to produce parasporal inclusions, 35 isolates belonging to serotypes 1, 3a:3b, 4a:4c, and 10 produced heat-stable exotoxin. Exotoxin was not detected in the isolates of serotypes 3a, 4a:4b, 5a:5b, 5a:5c, 6, 7, and 8a:8b. No heat-stable exotoxin was demonstrated in 28 acrystalliferous isolates which possessed H antigens of B. thuringiensis serotypes 1, 3a, 4a:4b, 4a:4c, 5a:5c, 6, 7, 10, 11a:11c, and 12. A total of 270 B. cereus isolates which did not possess B. thuringiensis H antigen were examined and three isolates were found to produce heat-stable exotoxin. No heat-stable exotoxin was produced by B. subtilis (two strains), B. natto (one strain), and B. megaterium (two strains). These results indicate that the heat-stable exotoxin production in B. thuringiensis is a strain-specific property rather than a serotype(subspecies)-specific property.     

Characterization of Tunisian Bacillus thuringiensis Strains with Abundance of kurstaki Subspecies Harbouring Insecticidal Activities Against the Lepidopteran Insect Ephestia kuehniella

The study of 257 crystal-producing Bacillus thuringiensis isolates from bioinsecticide free soil samples collected from different sites in Tunisia, was performed by PCR amplification, using six primer pairs specific for cry1, cry2, cry3, cry4, and vip3A genes, by the investigation of strain plasmid pattern, crystal morphology and delta-endotoxin content and by the assessment of insecticidal activities against the lepidopteran insect Ephestia kuehniella. Based on plasmid pattern study, 11 representative strains of the different classes were subjected to morphological and molecular analyses. The comparison of the PFGE fingerprints confirmed the heterogeneity of these strains. B. thuringiensis kurstaki strains, harbouring at the same time the genes cry1A, cry2, cry1Ia, and vip3A, were the most abundant (65.4%). 33.34% of the new isolates showed particular delta-endotoxin profiles but no PCR products with the used primer sets. B. thuringiensis israelensis was shown to be also very rare among the Tunisian B. thuringiensis isolates diversity. These findings could have considerable impacts for the set up of new pest control biological agents.     

Bacillus thuringiensis isolates from Korean forest environments

We investigated the distribution, toxicity, morphology, and protein profiles of Bacillus thuringiensis isolates from forests in Korea to isolate naturally occurring novel B. thuringiensis. A total of 170 B. thuringiensis isolates were obtained from 832 samples producing spore and parasporal inclusion bodies. In toxicity tests for lepidopteran, dipteran, and coleopteran insects, 57.6% isolates were toxic only to Lepidoptera, 5.3% were toxic only to Diptera, and 24.1% were toxic to both Diptera and Lepidoptera. The remaining collections (13.0%) were not toxic to the tested insects. The shapes of the parasporal crystals produced in B. thuringiensis isolates were bipyramidal, spherical, ovoid, or irregular. As their toxicities varied with parasporal crystal shape, B. thuringiensis isolates possessing bipyramidal or irregular parasporal crystals were largely toxic to lepidopteran species whereas those producing spherical parasporal crystals were mainly toxic to dipteran species. B. thuringiensis toxic to both dipteran and lepidopteran insects contained 130- and 70-kDa parasporal crystals, whereas B. thuringiensis toxic to lepidopteran insects expressed 130-kDa parasporal crystals. The results suggest that forest areas in Korea are a rich source of B. thuringiensis and need to be further explored to discover novel B. thuringiensis isolates.     

Genetic diversity and functional characterization of endophytic <Emphasis Type="BoldItalic">Bacillus thuringiensis</Emphasis> isolates from the North Western Indian Himalayas

A total of 15 endophytic Bacillus thuringiensis isolates were obtained from root nodules of six legumes (soybean, ricebean, gahat, frenchbean, lentil and pea). All of these isolates were characterized by the presence of one of two different types of crystalline inclusions (spherical and bipyramidal) and tolerance to a wide pH range (4–10; optimum 7.0) and NaCl concentrations up to 8%. Genetic diversity among the B. thuringiensis isolates was determined by repetitive extragenic palindromic PCR assays (rep-PCR) using the Bacillus cereus-repetitive extragenic palindromic, BOX, enterobacterial repetitive intergenic consensus sequence and (GTG)₅ primers. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis proteogram of the B. thuringiensis isolates revealed the presence of two major polypeptides (24.4 and 131.0 kDa). Maximum crystal protein profile was observed in the B. thuringiensis isolates producing the spherical crystal, while those isolates producing the bipyramidal crystal protein showed four four major polypeptides (24.4, 33.8, 81.2 and 131.0 kDa). The purified crystal protein profile of the B. thuringiensis isolates revealed the presence of only one major protein of 130 kDa mass. Isolates VRB1 and VLG15 possessing the cry1 and cry2 family genes demonstrated 100% mortality against first-instar larvae of the Bihar hairy caterpillar (lepidopteran pest). Our study of the ecological and molecular diversity among newly identified B. thuringiensis isolates suggests that these could be useful in planning new strategies for integrated pest management in sustainable agricultural systems.     

Immunological properties of entomocidal protein of Bacillus thuringiensis and its insecticidal activity

The immunological properties of the proteinaceous component of the parasporal crystal (δ-endotoxin) of Bacillus thuringiensis var. kurstaki were analyzed by rocket immunoelectrophoresis. Two antisera, one against the k-l-type crystal containing two components, and the other against the k-73-type crystal containing one component, were made in rabbits. The antigens consisting of purified and dissociated crystals were run in electrophoresis with these two antisera. The ratio between the two peak heights of precipitin lines, which were formed by the dissociated crystal of one B. thuringiensis isolate in two antisera, was compared with the ratios of other isolates under identical conditions. The difference in the ratio reflected a difference in the structure of the crystal component and correlated closely with the insecticidal activity spectrum. This method can be used to evaluate a newly isolated B. thuringiensis, and it can further differentiate the isolates which have been classified as one serotype.     

Isolation and characterization of Bacillus thuringiensis strains from olive-related habitats in Turkey

Aims: To isolate Bacillus thuringiensis strains from different olive‐related habitats (olive groves and olive oil factories) in Turkey and to characterize these strains by molecular methods. Methods and Results: A total of 150 samples, consisting of olive grove soil, green olive leaves, olive leaf residues, animal faeces, olive pomace and dust, were examined for the presence of B. thuringiensis. One hundred B. thuringiensis strains were isolated from 54 environmental samples (36%) and characterized in terms of crystal morphology, cry and cyt gene content by polymerase chain reaction, plasmid profiles and 16S‐internal transcribed spacer ribosomal DNA restriction fragment length polymorphism (16S‐ITS rDNA RFLP). The highest percentage of samples containing B. thuringiensis was found in 38 out of 54 total soil samples (70%). Of the 100 B. thuringiensis isolates, the most frequent crystal shapes were irregularly shaped (24%), spherical‐irregular pointed (19%), cuboidal (17%) and spherical (16%). The cry1 plus cry4 genotype was the most abundant genotype in our collection (21%). RFLP analysis of the amplified 16S‐ITS rDNA revealed 11 distinct patterns for the isolates and 10 reference strains. Conclusions: Bacillus thuringiensis isolates showed a great genetic diversity and crystal shape heterogeneity. Significance and Impact of the Study: This is the first study on the isolation and characterization of B. thuringiensis from olive‐related habitats in Turkey. No correlation was observed between the cry genotypes and insecticidal crystal shapes of the isolates. Restriction profiles of 23% of the isolates were found to be different from those of the 10 reference strains used.     

Pathogenomic sequence analysis of Bacillus cereus and Bacillus thuringiensis isolates closely related to Bacillus anthracis

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are closely related gram-positive, spore-forming bacteria of the B. cereus sensu lato group. While independently derived strains of B. anthracis reveal conspicuous sequence homogeneity, environmental isolates of B. cereus and B. thuringiensis exhibit extensive genetic diversity. Here we report the sequencing and comparative analysis of the genomes of two members of the B. cereus group, B. thuringiensis 97-27 subsp. konkukian serotype H34, isolated from a necrotic human wound, and B. cereus E33L, which was isolated from a swab of a zebra carcass in Namibia. These two strains, when analyzed by amplified fragment length polymorphism within a collection of over 300 of B. cereus, B. thuringiensis, and B. anthracis isolates, appear closely related to B. anthracis. The B. cereus E33L isolate appears to be the nearest relative to B. anthracis identified thus far. Whole-genome sequencing of B. thuringiensis 97-27and B. cereus E33L was undertaken to identify shared and unique genes among these isolates in comparison to the genomes of pathogenic strains B. anthracis Ames and B. cereus G9241 and nonpathogenic strains B. cereus ATCC 10987 and B. cereus ATCC 14579. Comparison of these genomes revealed differences in terms of virulence, metabolic competence, structural components, and regulatory mechanisms.     

Fluorescent Amplified Fragment Length Polymorphism Analysis of Norwegian Bacillus cereus and Bacillus thuringiensis Soil Isolates

We examined 154 Norwegian B. cereus and B. thuringiensis soil isolates (collected from five different locations), 8 B. cereus and 2 B. thuringiensis reference strains, and 2 Bacillus anthracis strains by using fluorescent amplified fragment length polymorphism (AFLP). We employed a novel fragment identification approach based on a hierarchical agglomerative clustering routine that identifies fragments in an automated fashion. No method is free of error, and we identified the major sources so that experiments can be designed to minimize its effect. Phylogenetic analysis of the fluorescent AFLP results reveals five genetic groups in these group 1 bacilli. The ATCC reference strains were restricted to two of the genetic groups, clearly not representative of the diversity in these bacteria. Both B. anthracis strains analyzed were closely related and affiliated with a B. cereus milk isolate (ATCC 4342) and a B. cereus human pathogenic strain (periodontitis). Across the entire study, pathogenic strains, including B. anthracis, were more closely related to one another than to the environmental isolates. Eight strains representing the five distinct phylogenetic clusters were further analyzed by comparison of their 16S rRNA gene sequences to confirm the phylogenetic status of these groups. This analysis was consistent with the AFLP analysis, although of much lower resolution. The innovation of automated genotype analysis by using a replicated and statistical approach to fragment identification will allow very large sample analyses in the future.     

Distribution of phenotypes among Bacillus thuringiensis strains

An extensive collection of Bacillus thuringiensis isolates from around the world were phenotypically profiled using standard biochemical tests. Six phenotypic traits occurred in 20–86% of the isolates and were useful in distinguishing isolates: production of urease (U; 20.5% of isolates), hydrolysis of esculin (E; 32.3% of isolates), acid production from salicin (A; 37.4% of isolates), acid production from sucrose (S; 34.0% of isolates), production of phospholipase C or lecithinase (L; 79.7% of isolates), and hydrolysis of starch (T; 85.8% of isolates). With the exception of acid production from salicin and hydrolysis of esculin, which were associated, the traits assorted independently. Of the 64 possible combinations of these six phenotypic characteristics, 15 combinations accounted for ca. 80% of all isolates, with the most common phenotype being TL (23.6% of isolates). Surprisingly, while the biochemical traits generally assorted independently, certain phenotypic traits associated with the parasporal crystal were correlated with certain combinations of biochemical traits. Crystals that remained attached to spores (which tended to be non-toxic to insects) were highly correlated with the phenotypes that included both L and S. Among the 15 most abundant phenotypes characterizing B. thuringiensis strains, amorphous crystals were associated with TLE, TL, T, and Ø (the absence of positive tested biochemical traits). Amorphous crystal types displayed a distinct bias toward toxicity to dipteran insects. Although all common phenotypes included B. thuringiensis isolates producing bipyramidal crystals toxic to lepidopteran insects, those with the highest abundance of these toxic crystals displayed phenotypes TLU, TLUA, TLUAE, and TLAE.     

Virulence of some native Bacillus thuringiensis isolates against Ephestia kuehniella (Zeller) (Lep., Pyralidae) and Pieris brassicae (Lep., Pieridae) larvae isolated from stored products of Urmia city

Bacillus thuringiensis isolates were recovered from numerous sources including soil, grain dust, plant leaves, diseased insect larvae from insectariums and sericulture environments. B. thuringiensis strains were isolated using acetate selection method with 0.025?M. concentration. The morphology of crystals was studied using light microscopy. Bioassay tests were conducted on Ephestia kuehniella (Zeller) (L.) as well as Pieris brassicae (L.). Based on the results, 35 B. thuringiensis strains were isolated from 140 samples. Majority of strains (%31.42) had bipyramidal crystals. There was a significant difference in toxicity to insects among B. thuringiensis isolates; 28.57 and 14.28% of the isolates were toxic to the larvae of P. brassicae and E. kuehniella, respectively, causing more than 50% mortality. Results indicated that B. thuringiensis isolates with insecticidal activity could be used in integrated pest management to control farm and stored product pests.     

Molecular Characterization and Genetic Diversity of Insecticidal Crystal Protein Genes in Native Bacillus thuringiensis Isolates

The Western Ghats of Karnataka natural ecosystem are among the most diverse and is one of the eight hottest hotspots of biological diversity in the world, that runs along the western part of India through four states including Karnataka. Bacillus thuringiensis (Bt) strains were isolated from soils of Western Ghats of Karnataka and characterized by molecular and analytical methods as a result of which 28 new Bt-like isolates were identified. Bt strains were isolated from soil samples using sodium acetate selection method. The morphology of crystals was studied using light and phase contrast microscopy. Isolates were further characterized for insecticidal cry gene by PCR, composition of toxins in bacterial crystals by SDS-PAGE cloning, sequencing and evaluation of toxicity was done. As a result 28 new Bt-like isolates were identified. Majority of the isolates showed the presence of a 55 kDa protein bands on SDS-PAGE while the rest showed 130, 73, 34, and 25 kDa bands. PCR analysis revealed predominance of Coleopteran-active cry genes in these isolates. The variations in the nucleotide sequences, crystal morphology, and mass of crystal protein(s) purified from the Bt isolates revealed genetic and molecular diversity. Three strains containing Coleopteran-active cry genes showed higher activity against larvae Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) than B. thuringiensis subsp. Morrisoni. Results indicated that Bt isolates could be utilized for bioinsecticide production, aiming to reduce the use of chemical insecticide which could be useful to use in integrated pest management to control agriculturally important pests for sustainable crop production.     

Phenotypic and genotypic features of new autoagglutinating Bacillus thuringiensis strains

A total of 28 autoagglutinating strains of Bacillus thuringiensis were isolated from different ecologic niches and distinct sites. Twenty-six strains demonstrated toxicity to mosquito larvae of Aedes aegypti and Culex quinquefasciatus. The electrophoretic protein profiles of the crystal components were studied. Twenty-three out of the 28 strains showed the same larvicidal activity and the same protein profiles as B. thuringiensis serovar israelensis. Using isoenzyme analysis (MLEE), it was observed the presence of three electrophoretic types (ETs). The mosquitocidal strains grouped into one ET. The random amplified polymorphic DNA analysis (RAPD) was evaluated using six primers, which demonstrated three different patterns for the 28 autoagglutinating strains, allowing correlation of the profiles obtained with the toxicity observed in the bioassays. The RAPD patterns for mosquitocidal strains were identical to the one of serovar israelensis. However, to strains of low toxicity, each primer generated distinctive RAPD patterns, which demonstrated that these strains belong to different serovars. Although the antigenic classification the 26 autoagglutinating strains of B. thuringiensis could not be determined by classical flagellar serotyping, MLEE and RAPD profiles proved these strains to be compatible with B. thuringiensis serovar israelensis.     

Phenotypic characterization of Bacillus thuringiensis and Bacillus cereus

One hundred and thirty-seven strains of Bacillus thuringiensis and 35 strains of Bacillus cereus were tested for the presence or absence of 99 traits. An analysis of these data indicated that strains of B. thuringiensis were indistinguishable from B. cereus, except for their ability to produce parasporal crystals. This conclusion was based on a comparison of the phenotypic properties of B. thuringiensis and B. cereus, as well as on the results of numerical analyses of the data which grouped strains into clusters on the basis of phenotypic similarity. In the resulting dendrograms, strains of B. thuringiensis and B. cereus were interspersed, exhibiting no tendency to segregate. In addition, with the exception of serovar israelensis, strains on B. thuringiensis belonging to the same flagellar serovar showed little or no tendency to group in different clusters. A comparison of the phenotypic differences between serovars indicated that the greater the number of strains in the serovars, the fewer, if any, phenotypic traits separating them. This suggests that the properties reported to differentiate serovars can be attributed to the internal phenotypic diversity of the species. Characterization of 10 mosquitocidal strains of Bacillus sphaericus indicated that the traits employed in this study readily distinguished these highly related organisms from strains of B. thuringiensis and B. cereus.     

Bacillus thuringiensis Isolates from Great Nicobar Islands

Bacillus thuringiensis (Bt) strains were isolated from soil samples of Great Nicobar Islands, one of the “hottest biodiversity hotspots,” where no collection has been characterized previously. The 36 new Bt isolates were obtained from 153 samples analyzed by crystal protein production with light/phase-contrast microscopy, determination of cry gene profile by SDS-PAGE, evaluation of toxicity against Coleopteran, and Lepidopteran insect pests, finally cloning and sequencing. Majority of the isolates showed the presence of 66–35 kDa protein bands on SDS-PAGE while the rest showed >130, 130, 73, and 18 kDa bands. The variations in crystal morphology and mass of crystal protein(s) purified from the isolates of Bt revealed genetic and molecular diversity. Based on the toxicity test, 50 % of isolates were toxic to Ash weevils, 16 % isolates were toxic to cotton bollworm, 38 % isolates were toxic both to ash weevil as well as cotton bollworm, while 11 % of the isolates did not exhibit any toxicity. PCR analysis unveiled prepotency of cry1B- and cry8b-like genes in these isolates. This study appoints the first isolation and characterization of local B. thuringiensis isolates in Great Nicobar Islands. Some of these isolates display toxic potential and, therefore, could be adopted for future applications to control some agriculturally important insect pests in the area of integrated pest management for sustainable agriculture.     

Fingerprinting of Bacillus thuringiensis Type Strains and Isolates by Using Bacillus cereus Group-Specific Repetitive Extragenic Palindromic Sequence-Based PCR Analysis

A total of 119 Bacillus thuringiensis strains (83 type strains and 26 native isolates), as well as five B. cereus group species, were analyzed by repetitive extragenic palindromic sequence-based PCR analysis (Rep-PCR) fingerprinting. Primers Bc-REP-1 and Bc-REP-2 were specifically designed according to an extragenic 26-bp repeated sequence found in the six B. cereus group genomes reported. A total of 47 polymorphic bands were detected, and the patterns varied from 5 to 13 bands in number and from 0.2 to 3.8 kb in size. Virtually each type strain showed a distinctive B. cereus (Bc)-Rep-PCR pattern, except for B. thuringiensis serovars dakota (H serotype 15 [H15]) and sotto (H4a,4b), as well as serovars amagiensis (H29) and seoulensis (H35), which shared the same patterns. As expected, serovar entomocidus (H6) and its biovar subtoxicus showed an identical pattern; similarly, serovars sumiyoshiensis (H3a,3d) and fukuokaensis (H3a,3d,3e), which share two antigenic determinants, also showed identical Bc-Rep-PCR patterns. Interestingly, serovars israelensis (H14) and malaysiensis (H36), which share several phenotypic attributes, also showed identical Bc-Rep-PCR patterns. Native, coleopteran-active strains, including the self-agglutinated LBIT-74 strain, showed Bc-Rep-PCR patterns identical or very similar to that of the tenebrionis strain. Likewise, native mosquitocidal strains (including some self-agglutinated strains) also showed patterns identical or very similar to that of the serovar israelensis IPS-82 strain. Additionally, native β-exotoxin-producing strains from serovar thuringiensis showed patterns identical to that of the B. thuringiensis type strain. The B. cereus group-specific Bc-Rep-PCR fingerprinting technique was shown to be highly discriminative, fast, easy, and able to identify B. thuringiensis serotypes, including nonflagellar and self-agglutinated strains.     

Conjugal transfer between Bacillus thuringiensis and Bacillus cereus strains is not directly correlated with growth of recipient strains

Bacillus thuringiensis and Bacillus cereus belong to the B. cereus species group. The two species share substantial chromosomal similarity and differ mostly in their plasmid content. The phylogenetic relationship between these species remains a matter of debate. There is genetic exchange both within and between these species, and current evidence indicates that insects are a particularly suitable environment for the growth of and genetic exchange between these species. We investigated the conjugation efficiency of B. thuringiensis var. kurstaki KT0 (pHT73-Em^R) as a donor and a B. thuringiensis and several B. cereus strains as recipients; we used one-recipient and two-recipient conjugal transfer systems in vitro (broth and filter) and in Bombyx mori larvae, and assessed multiplication following conjugation between Bacillus strains. The B. thuringiensis KT0 strain did not show preference for genetic exchange with the B. thuringiensis recipient strain over that with the B. cereus recipient strains. However, B. thuringiensis strains germinated and multiplied more efficiently than B. cereus strains in insect larvae and only B. thuringiensis maintained complete spore germination for at least 24 h in B. mori larvae. These findings show that there is no positive association between bacterial multiplication efficiency and conjugation ability in infected insects for the used strains.     

Isolation and molecular characterization of Bacillus thuringiensis found in soils of the Cerrado region of Brazil,and their toxicity to Aedes aegypti larvae

This study investigated the potential of Bacillus thuringiensis isolates obtained in the Cerrado region of the Brazilian state of Maranhão for the biological control of Aedes aegypti larvae. The isolates were obtained from soil samples and the identification of the B. thuringiensis colonies was based on morphological characteristics. Bioassays were run to assess the pathogenicity and toxicity of the different strains of the B. thuringiensis against third-instar larvae of A. aegypti. Protein profiles were obtained by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polymerase chain reaction assays were used to detect the toxin genes found in the bacterial isolates. Overall, 12 (4.0%) of the 300 isolates obtained from 45 soil samples were found to present larvicidal activity, with the BtMA-104, BtMA-401 and BtMA-560 isolates causing 100% of mortality. The BtMA-401 isolate was the most virulent, with the lowest median lethal concentration (LC₅₀) (0.004 × 10⁷ spores/mL), followed by the Bacillus thuringiensis var. israelensis standard (0.32 × 10⁷ spores/mL). The protein profiles of BtMA-25 and BtMA-401 isolates indicated the presence of molecular mass consistent with the presence of the proteins Cry4Aa, Cry11Aa and Cyt1, similar to the profile of Bacillus thuringiensis var. israelensis IPS-82. Surprisingly, however, none of the cry and cyt genes analyzed were amplified in the isolate BtMA-401. The results of the present study revealed the larvicidal potential of B. thuringiensis isolates found in the soils of the Cerrado region from Maranhão, although further research will be necessary to better elucidate and describe other genes associated with the production of insecticidal toxins in these isolates.