Microbial ecology of Bacillus thuringiensis: fecal populations recovered from wildlife in Korea

A total of 34 fecal samples, collected from 14 species of wild mammals in Korea, were examined for the occurrence of Bacillus thuringiensis. The organism was detected in 18 (53%) samples. Among the three food-habit groups, herbivorous animals yielded the highest frequency (69%) of samples positive for B. thuringiensis, followed by omnivorous animals (50%). Of the six fecal samples from carnivorous animals, only one sample contained B. thurin giensis. Among 527 isolates belonging to the Bacillus cereus - B. thuringiensis group, 43 (8%) were assigned to B. thurin giensis on the basis of the formation of parasporal inclusions. Of the 43 isolates, 13 were serologically allocated to the nine H-antigenic serotypes: H3ad (serovar sumiyoshiensis), H15 (dakota), H17/27 (tohokuensis/ mexicanensis), H19 (tochigiensis), H21 (colmeri), H29 (amagiensis), H31/49 (toguchini/muju), H42 (jinghongiensis), and H44 (higo). Other isolates were untestable or untypable by the 55 reference H antisera available. Insecticidal activity was associated with 23% of the fecal populations: three isolates killed larvae of the silkworm, Bombyx mori (Lepidoptera), and seven exhibited larvicidal activity against the mosquito, Aedes aegypti (Diptera). There was no larvicidal activity against the three lepidopterous insects: Plutella xylostella, Spodoptera exigua, and Spodoptera litura. The overall results suggest that wild animals in Korea are in contact with naturally occurring B. thuringiensis at high frequencies through the daily food intake of plants.     

Comparative study of the frequency and flagellar serotype flora of Bacillus thuringiensis in soils and silkworm-breeding environments

Two environments, soils and silkworm ( Bombyx mort )-breeding farms, in Kumamoto Prefecture, Japan, were explored for the comparative analysis of the frequency and the flagellar (H) serotype flora of Bacillus thuringiensis. This organism was found in 39.3% of the sericulture farms and 18.5% of the silkworm litter samples. The frequency of B. thuringiensis was 4.5% among the populations of the Bacillus cereus/B. thuringiensis group in silkworm-breeding insectaries. The predominant H serotype detected in silkworm litters was 4ac (35.6%), followed by serotypes 3abc (20.0%), 8ab (13.3%), 4ab (13.3%) and 7 (2.2%). The majority (87.1%) of sericulture-derived isolates exhibited larvicidal activity against the silkworm and/or the mosquito, Aedes aegypti. Of soil samples examined, 8.1% contained B. thuringiensis. The frequency of B. thuringiensis colonies was 0.6% among soil populations of the B. cereus/B. thuringiensis group. Of 22 soil isolates, 14 were assigned to five H serotypes: 10, 17, 18, 21 and 29. Most soil isolates showed no insecticidal activity against B. mori and A. aegypti , but two, belonging to H serotype 10 and an undescribed serotype, exhibited moderate larvicidal activity against A. aegypti.     

Recovery of Bacillus thuringiensis from Marine Sediments of Japan

Marine sediments from a Japanese bay were examined for the occurrence of Bacillus thuringiensis. Of 1313 colonies belonging to the Bacillus cereus/B. thuringiensis group, 22 (1.7%) were allocated to B. thuringiensis. Marine isolates of B. thuringiensis consisted of heterogeneous multiple H serogroups; 10 isolates were assigned to the eight serovars (kurstaki, sumiyoshiensis, sotto, aizawai, darmstadiensis, thompsoni, neoleonensis, and higo); two motile isolates failed to react with the reference antisera; and the others were serologically untestable. Insecticidal activities were associated with two kurstaki isolates (toxic to both Lepidoptera and Diptera) and a higo isolate (Diptera-specific). None of the parasporal inclusion proteins of the 22 isolates exhibited in vitro cytotoxic activity against two vertebrate cells, sheep erythrocytes and HeLa cells. All B. thuringiensis isolates had no halophilism, although seawater-based medium supported their growth, sporulation, and formation of parasporal inclusions. Received: 29 November 1999 / Accepted: 10 January 2000     

Bacillus thuringiensis soil populations naturally occurring in the Ryukyus, a subtropic region of Japan

Of 809 soil samples collected from the seven islands of the Ryukyus, Japan, 107 samples (13.2%) contained Bacillus thuringiensis. The frequency of B. thuringiensis among the B. cereus group was 1.1% (235/21842) on the average. The B. thuringiensis soil populations of the Ryukyus consisted of more than 22 H serogroups. The predominant H serotype was the H5ac/21 (serovar canadensis/colmeri), followed by the H3ad (serovar sumiyoshiensis) and H16 (serovar indiana). Geographically, most widely distributed H serogroups were the H16 and H10ac (serovar londrina); the former was recovered from five islands and the latter from three islands. Parasporal inclusions of the isolates were morphologically heterogeneous, roughly grouped into four categories: bipyramidal/cuboidal, spherical/ovoid, irregularly-pointed, and irregular-shaped. About 53% of the isolates formed spherical to ovoid parasporal inclusions. None of the isolates exhibited larvicidal activity against the silkworm, Bombyx mori. Only four isolates belonging to four different serotypes killed larvae of the mosquito, Aedes aegypti. These mosquito-specific isolates all produced spherical parasporal inclusions.     

Ubiquity of Bacillus thuringiensis on phylloplanes of arboreous and herbaceous plants in Japan

A total of 120 Bacillus thuringiensis strains was isolated from phylloplanes of 35 species of arboreous and herbaceous plants in an area of northern Kyushu, Japan. The isolates belonged to at least 17 serotypes and the group of H serotype 3 was predominant. Twenty strains were untypable by the existing reference H antisera and 47 were untestable due to autoagglutination or poor motility. Of the 120 isolates, 25 produced bipyramidal parasporal inclusions and the others, spherical or irregular-shaped. Insecticidal activity against mosquitoes ( Culex pipiens molestus and Anopheles stephensi ) and/or diamondback moth, Plutella xylostella , was associated with 28 isolates (23·3%). Overall results revealed that: B. thuringiensis is ubiquitous on a variety of plants; bacterial flora on phylloplanes consists of highly heterogeneous H serogroups; and there is little correlation between plant species and phenotypes of B. thuringiensis isolates.     

Occurrence of parasporin-producing Bacillus thuringiensis in Vietnam

A total of 63 Bacillus thuringiensis isolates were recovered from urban soils of Hanoi, Vietnam. Of these, 34 were identified to 12 H serogroups. None of the isolates showed larvicidal activities against three lepidopterous insects. Three isolates belonging to the two serovars, colmeri (H21) and konkukian (H34), were highly toxic to larvae of the mosquito Aedes aegypti. Parasporal inclusion proteins of four isolates exhibited cytocidal activities against HeLa cells. Immunologically, proteins of four isolates were closely related to parasporin-1 (Cry31Aa), a parasporal protein that preferentially kills human cancer cells. Haemolytic activities were associated with parasporal proteins of the three mosquitocidal isolates but not with those of the four cancer-cell-killing isolates. PCR experiments and nucleotide sequence analysis revealed that the genes of four anti-cancer isolates are closely related to the gene parasporin-1 (cry31Aa) but are dissimilar to those of the three other existing parasporins. Our results suggest that the soil of northern Vietnam is a good reservoir of parasporin-producing B. thuringiensis.     

Assessment of the efficacy of Japanese Bacillus thuringiensis isolates against the cigarette beetle,Lasioderma serricorne (Coleoptera: Anobiidae)

A total of 2,652 Japanese isolates of Bacillus thuringiensis, belonging to at least 54 H serogroups, were examined for assessment of the toxicity against the cigarette beetle, Lasioderma serricorne (Coleoptera: Anobiidae). When tested with spore/parasporal inclusion mixtures, strong larvicidal activities were associated with 28 isolates (1.1%). Serologically, these toxic isolates fell into 4 known H serovars: thuringiensis (9 isolates), kurstaki (2), kenyae (2), and darmstadiensis (15). Purified parasporal inclusions of the 10 selected isolates exhibited no larvicidal activity, while the supernatants of liquid cultures showed larvicidal and/or growth inhibitory effects. The activities were fully retained after heat treatment at 100 degrees C for 10 min. Overall results suggest that beta-exotoxin (or thuringiensin)-related substances are responsible for the toxicity of the present B. thuringiensis isolates against the cigarette beetle.     

Characterization and pathogenic evaluation of Bacillus thuringiensis and Bacillus sphaericus isolates from Argentinean soils

Eighty soil samples of different origin (from urban, agricultural, forested and horticultural areas) which had not previously been treated with bioinsecticides, were collected and examined to investigate the presence of Bacillus thuringiensis and B. sphaericus. From a total of 1473 bacterial isolates examined by differential staining techniques and growth on nutrient agar with the addition of penicillin and streptomycin, 31 (2.1%) strains of Bacillus sphaericus and 25 (1.6%) strains of Bacillus thuringiensis were isolated. These strains were tested for their pathogenicity against Diptera (Culex quinquefasciatus) and Lepidoptera (Anticarsia gemmatalis and Spodoptera frugiperda). Seven strains of Bacillus thuringiensis subspecies kurstaki were found to be pathogenic to Spodoptera frugiperda and twenty-two strains showed a pathological effect against Anticarsia gemmatalis. None of the strains of Bacillus thuringiensis nor the Bacillus sphaericus investigated, showed pathogenic activity against Culex quinquefasciatus. The strains of Bacillus thuringiensis were characterized serologically as belonging to six serotypes (darmstadiensis, entomocidus, kurstaki, muju, sotto and xianguangiensis). One strain seemed to be a new serotype. The electrophoretic profiles of the strains of Bacillus thruringiensis showed bands of 130 kDa similar to those found in strains pathogenic against Lepidoptera. Some physicochemical characteristics were also studied in the soil samples, in order to relate them to the presence or absence of these Bacillus species.     

Bacillus thuringiensis populations naturally occurring on mulberry leaves: a possible source of the populations associated with silkworm-rearing insectaries

Mulberry leaves were examined for the occurrence of Bacillus thuringiensis. This organism was recovered from both abaxial and adaxial surfaces: a total of 186 B. thuringiensis colonies were isolated from 24 (96·0%) out of 25 mulberry trees, and from 112 (11·2%) out of 1004 leaves from 25 trees. The frequency of B. thuringiensis colonies was 3·2% among 5900 colonies belonging to the Bacillus cereus/B. thuringiensis group. Single colonies were associated with 75·9% of the B. thuringiensis -positive leaves and 2–16 colonies were occasionally found on a single phylloplane. Flagellar (H) serotypying of the isolates revealed that, among the 19 H serotypes (serovars) detected, the H serotype 13 (serovar pakistani ) was the predominant, followed by the H serotypes 3abc ( kurstaki ), 6ac ( oyamensis ), 16 ( indiana ), 24 ( neoleonesis ), 4ac ( kenyae ), 7 ( aizawai ) and 10 ( darmstadiensis ). Larvicidal activity, against the silkworm ( Bombyx mori ) and/or the mosquito ( Aedes aegypti ), was exhibited by 18 isolates (9·7%) belonging to H serovars kurstaki, kenyae, canadensis and aizawai , and an unidentified H serogroup.     

Characterization of flagellar antigens and insecticidal activities of Bacillus thuringiensis populations in animal feces

In total, 287 Bacillus thuringiensis isolates, recovered from feces of 28 zoo-maintained animal species, were examined for flagellar (H) antigenicity and insecticidal activity. Serologically, 209 isolates (72.8%) were allocated to the 8 H serogroups, 4 were untypable, and 74 were untestable. Among the 8 H serotypes detected, H3abc (serovar kurstaki) predominated at a high frequency of 88.0%, followed by H6 (serovar entomocidus) with a frequency of 7.7%. Insecticidal activity was associated with 67.2% of the fecal populations: 188 isolates were toxic to both Bombyx mori (Lepidoptera: Bombycidae) and Aedes aegypti (Diptera: Culicidae), 2 isolates were specific for B. mori, and 3 isolates were toxic to A. aegypti only. Of the isolates with dual toxicity, 97.9% belonged to the serovar kurstaki, producing bipyramidal parasporal inclusions. All of the H7 (serovar aizawai) isolates were toxic to both insects.     

Haemolytic activity associated with parasporal inclusion proteins of mosquito-specific Bacillus thuringiensis soil isolates: A comparative neutralization study

Abstract Solubilized parasporal inclusions of the three mosquito-specific Bacillus thuringiensis isolates belonging to three different H serovars, co-isolated from a single soil microhabitat, showed haemolytic activity towards mammalian erythrocytes. Neutralization tests with antibodies against whole inclusion proteins resulted in crossed neutralization of haemolytic activity among the isolates and the type strain of B. thuringiensis serovar kyushuensis , indicating that the three soil isolates produce toxins related to the CytB toxin. No cross-neutralization occurred between the type strain of B. thuringiensis serovar israelensis and the three soil isolates.     

Isolation and Characterization of Bacillus thuringiensis Strains from Aquatic Environments in Spain

Samples collected from aquatic environments from Spain were analyzed for the occurrence and dipteran toxicity of Bacillus thuringiensis. From a total of 41 samples, 122 isolates were obtained, yielding a B. thuringiensis index of 0.22. Isolates were assigned to 13 different serovars, with serovar thuringiensis (serotype H1) the most frequently found. Toxicity tests carried out revealed that eight isolates (6.6% out of the total) were active against Tipula oleracea larvae. Serological tests assigned these toxic isolates to serovar thuringiensis. The toxicity found in these isolates against the tipulid was approximately seven times lower than that shown by the standard strain B. thuringiensis ser. israelensis IPS-82. Implication of Cry2A protein in toxic activity is hypothesized. Received: 3 December 1999 / Accepted: 5 January 2000     

Distribution, Frequency, and Diversity of Bacillus thuringiensis in an Animal Feed Mill

Bacillus thuringiensis was isolated from 36 of 50 residue samples obtained from an animal feed mill (a stored-product environment). Of 710 selected colonies having Bacillus cereus-B. thuringiensis morphology isolated from the samples, 477 were classified as B. thuringiensis because of production of parasporal delta-endotoxin crystals. There was a diverse population of B. thuringiensis, as revealed by differentiation of the isolates into 36 subgroups by using (i) their spectra of toxicity to the lepidopterans Heliothis virescens, Pieris brassicae, and Spodoptera littoralis and the dipteran Aedes aegypti and (ii) their parasporal crystal morphology. A total of 55% of the isolates were not toxic to any of these insects at the concentrations used in the bioassays; 40% of all isolates were toxic to one or more of the Lepidoptera; and 20, 1, and 1% of the isolates were toxic to only P. brassicae, H. virescens, and S. littoralis, respectively. The most frequent toxicity was toxicity to P. brassicae (36% of all isolates); 18% of the isolates were toxic to A. aegypti (5% exclusively), 10% were toxic to H. virescens, and 4% were toxic to S. littoralis. Toxicity to P. brassicae was more often linked with toxicity to H. virescens than with toxicity to S. littoralis. The frequency of toxicity was significantly greater in isolates that produced bipyramidal crystals than in isolates that produced irregular pointed, irregular spherical, rectangular, or spherical crystals.     

Unique activity associated with non-insecticidal Bacillus thuringiensis parasporal inclusions: in vitro cell-killing action on human cancer cells

Parasporal inclusion proteins from a total of 1744 Bacillus thuringiensis strains, consisting of 1700 Japanese isolates and 44 reference type strains of existing H serovars, were screened for cytocidal activity against human leukaemia T cells and haemolytic activity against sheep erythrocytes. Of 1684 B. thuringiensis strains having no haemolytic activity, 42 exhibited in vitro cytotoxicity against leukaemia T cells. These non-haemolytic but leukaemia cell-toxic strains belonged to several H-serovars including dakota, neoleonensis, shandongiensis, coreanensis and other unidentified serogroups. Purified parasporal inclusions of the three selected strains, designated 84-HS-1-11, 89-T-26-17 and 90-F-45-14, exhibited no haemolytic activity and no insecticidal activity against dipteran and lepidopteran insects, but were highly cytocidal against leukaemia T cells and other human cancer cells, showing different toxicity spectra and varied activity levels. Furthermore, the proteins from 84-HS-1-11 and 89-T-26-17 were able to discriminate between leukaemia and normal T cells, specifically killing the former cells. These findings may lead to the use of B. thuringiensis inclusion proteins for medical purposes.     

Isolation, geographical diversity and insecticidal activity of Bacillus thuringiensis from soils in Spain

Bacillus thuringiensis is a spore-forming bacterium showing the unusual ability to produce endogenous crystals during sporulation that are toxic for some pest insects. This work was performed to study the composition, ecological distribution and insecticidal activity of isolates of this entomopathogenic bacterium from the Spanish territory. Using a standard isolation method, B. thuringiensis was isolated from 115 out of 493 soil samples collected in the Iberian Peninsula and the Canary and Balearic Archipelagos. The percentages of samples with B. thuringiensis were 31.7, 27.6 and 18.5 and the B. thuringiensis index 0.065, 0.067 and 0.11 for the Iberian Peninsula, Canary and Balearic Archipelagos, respectively. The prairies were shown to be the worst source of B. thuringiensis while forests, urban and agricultural habitats showed similar percentages. Strain classification based on H-antigen agglutination showed a great diversity among the Spanish isolates, which were distributed among 24 subspecies, including three new ones andaluciensis, asturiensis and palmanyolensis. We differentiated 65 different protein profiles of spore-crystal mixtures by sulfate-polyacrylamide gel electrophoresis and we selected 109 isolates representative of these profiles to evaluate their insecticidal activity against insects from the Orders Orthoptera, Dictyoptera, Coleoptera, Lepidoptera and Diptera. We found variable percentages of isolates active against Coleoptera and Lepidoptera, one isolate highly active against mosquito larvae and for the first time, three isolates active against cockroaches and locusts.     

Natural occurrence of Bacillus thuringiensis on cabbage foliage and in insects associated with cabbage crops

Bacillus thuringiensis was isolated from the phylloplane of organically grown cabbage in one field during two growth seasons (1992-93). The frequency of B. thuringiensis varied between 0.02 and 0.67 of the total B. cereus/B. thuringiensis population, with an average of 0.11. Characterization of the B. thuringiensis isolates from foliage showed that the majority (64% of 150 isolates) belonged to serovar kurstaki, had bipyramidal crystals and toxicity towards Pieris brassicae and/or Trichoplusia ni. Other serovars were also found on the foliage but occurred at very low frequencies (one to three isolates of each serovar). Bacillus thuringiensis was also isolated from insects associated with the cabbage crop (Pieris rapae (Lep.), Delia radicum (Dip.), Syrphidae ribesii (Dip.) and Aleochara bilineata (Col.)), which were collected alive at different developmental stages in the same field. Serologically these isolates were assigned to the serovars kurstaki, aizawai, tochigiensis, colmeri and indiana/colmeri.     

Delta endotoxin of Bacillus thuringiensis subsp. israelensis.

From Bacillus thuringiensis subsp. israelensis, a proteinase-resistant protein was purified which exhibited toxicity to larval mosquitoes and cultured mosquito cells, lysed erythrocytes, and was lethal to mice. To extract the protein, a sporulating culture of B. thuringiensis subsp. israelensis was treated with alkali, neutralized, and incubated with trypsin and proteinase K. It was then purified by gel filtration and DEAE column chromatography. Up to 240 micrograms of toxic protein was purified from 1 g (wet weight) of culture pellet. Two closely related forms of toxic protein were obtained: the 25a and 25b proteins. The two forms comigrated near 25,000 daltons in a sodium dodecyl sulfate-polyacrylamide gel, were serologically related, and showed similar partial protease digestion profiles, but were distinguishable by DEAE chromatography and nondenaturing polyacrylamide gel electrophoresis. Protein sequencing data indicated the 25b protein lacked the two amino acids at the amino terminus of the 25a protein. A Western blot enzyme-linked immunosorbent assay of alkali-solubilized proteins that were not treated with proteases suggested the toxic 25a and 25b proteins were proteolytically derived from a larger molecule of about 28,000 daltons. Alkali-solubilized proteins from an acrystalliferous strain of B. thuringiensis subsp. israelensis and from B. thuringiensis subsp. kurstaki failed to cross-react with antibodies to the 25a protein.     

Cross-resistance spectra of Culex quinquefasciatus resistant to mosquitocidal toxins of Bacillus thuringiensis towards recombinant Escherichia coli expressing genes from B. thuringiensis ssp. israelensis

Sixteen Escherichia coli clones were assayed against susceptible and Bacillus thuringiensis-resistant Culex quinquefasciatus larvae. The clones expressed different combinations of four genes from Bacillus thuringiensis ssp. israelensis; three genes encoded mosquitocidal toxins (Cry11Aa, Cry4Aa and Cyt1Aa) and the fourth encoded an accessory protein (P20). The cross-resistance spectra of the mosquitoes were similar to the profiles for recombinant B. thuringiensis strains expressing B. thuringiensis toxin genes, but with varied toxicity levels. The toxicity of the recombinants towards resistant mosquito larvae was improved when p20 and cyt1Aa were expressed in combination with cry4Aa and/or cry11Aa. Recombinant pVE4-ADRC, expressing cry4Aa, cry11Aa, p20 and cyt1Aa, was the most active against the resistant Culex, and resistance levels did not exceed fourfold. These results indicate that B. thuringiensis ssp. israelensis genes expressed in a heterologous host such as E. coli can be effective against susceptible and B. thuringiensis-resistant larvae and suppress resistance.     

Isolation of Multiple Subspecies of Bacillus thuringiensis from a Population of the European Sunflower Moth, Homoeosoma nebulella

Five subspecies of Bacillus thuringiensis were isolated from dead and diseased larvae obtained from a laboratory colony of the European sunflower moth, Homoeosoma nebulella. The subspecies isolated were B. thuringiensis subspp. thuringiensis (H 1a), kurstaki (H 3a3b3c), aizawai (H 7), morrisoni (H 8a8b), and thompsoni (H 12). Most isolates produced typical bipyramidal crystals, but the B. thuringiensis subsp. thuringiensis isolate produced spherical crystals and the B. thuringiensis subsp. thompsoni isolate produced a pyramidal crystal. Analysis of the parasporal crystals by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the crystals from the B. thuringiensis subsp. kurstaki and aizawai isolates contained a protein of 138 kDa whereas those from B. thuringiensis subsp. morrisoni contained a protein of 145 kDa. The crystals from B. thuringiensis subsp. thuringiensis contained proteins of 125, 128, and 138 kDa, whereas those from B. thuringiensis subsp. thompsoni were the most unusual, containing proteins of 37 and 42 kDa. Bioassays of purified crystals conducted against second-instar larvae of H. nebulella showed that the isolates of B. thuringiensis subspp. aizawai, kurstaki, and thuringiensis were the most toxic, with 50% lethal concentrations (LC(inf50)s) of 0.15, 0.17, and 0.26 (mu)g/ml, respectively. The isolates of B. thuringiensis subspp. morrisoni and thompsoni had LC(inf50)s of 2.62 and 37.5 (mu)g/ml, respectively. These results show that a single insect species can simultaneously host and be affected by a variety of subspecies of B. thuringiensis producing different insecticidal proteins.     

Extracellular production of a heat-stable somatic antigen by Bacillus thuringiensis

Extracellular production of a heat-stable somatic antigen (HSSA) by Bacillus thuringiensis subsp. dendrolimus strain T84A1-A [flagellar (H) serotype 4a: 4b] was serologically detected. In Ouchterlony tests, the HSSA antiserum gave single precipitin lines against both untreated and heat-treated culture supernatants. These two precipitin lines fused completely. When colonies of strain T84A1-A were grown on nutrient agar plates containing the homologous HSSA antiserum, precipitin halos were formed around the colonies. Of 27 type strains of B. thuringiensis subspecies tested, only the type strains of B. thuringiensis subsp. sotto (H serotype 4a: 4b) and B. thuringiensis subsp. israelensis (H serotype 14) formed [precipitin halos on nutrient agar plates containing antiserum against the HSSA of strain T84A1-A.