Distribution, Serological Identification, and PCR Analysis of Bacillus thuringiensis Isolated from Soils of Korea

A total, 58 strains of Bacillus thuringiensis were isolated from soils of various regions in Korea. Serological tests showed that B. thuringiensis isolates represented 10 H serotypes, indicating a varied flora of B. thuringiensis. But the H serotypes did not have a significantly uneven distribution, ranging from 1 to 11 isolates. In toxicity tests, 35% of all isolates were toxic to lepidoptera, 20% were toxic to diptera, and 9% were non-toxic isolates. Especially, a large number of lepidopteran/dipteran-active isolates (36%) were found. Forty all lepidopteran-active isolates produced typical rhomboidial inclusions, and the remainder, which belong to dipteran-active and non-toxic isolates, were spherical in shape. In addition, lepidopteran/dipteran-active isolates produced rhomboidal or spherical inclusions. PCR analysis using cryI, II, III, IV, and V gene-specific primers showed that the frequency of the cryIC gene (57%) predominated, followed by the cryIA(b) (45%) and cryIIA genes (34%). But, the cryIE, cryIF, cryIII, cryIVC and cryV genes were not reactive. Several isolates had unusual PCR products and multiple insecticidal crystal protein genes. PCR results showed varied distribution of the cry-type gene. Seven isolates were selected for evaluation of novel activity according to the following criteria: flagellar serotypes, parasporal inclusion morphology, SDS-PAGE, plasmid DNA patterns, toxicity, and the cry-type gene in PCR analysis. Two isolates, named S333 (H7) and S225 (H7), among them synthesized PCR products of the cryIC gene, but the S333 isolate producing rhomboidal inclusion was toxic to both Plutella xylostella and Culex pipiens, whereas the S225 isolate having toxicity to only C. pipiens produced spherical inclusion. Received: 13 March 1998 / Accepted: 14 April 1998     

Secondary Structural and Phylogenetic Implications of Nuclear Large Subunit Ribosomal RNA in the Ectomycorrhizal Fungus Tricholoma matsutake

A number of Bacillus thuringiensis isolates from sericultural farm, soil, and granary samples in Korea were found. B. thuringiensis isolates were predominant in granary (40%), followed by sericultural farm (33% and 25% in the spring and fall isolation), and soil (10%). In toxicity tests for three areas, lepidopteran-active isolates were rich in the spring of sericultural farm and granary, but the fall isolation of sericultural farm displayed that a large number of B. thuringiensis isolates having dual specificity against both lepidopteran and dipteran larvae were found. The soil showed even distribution against lepidoptera and/or diptera. Most of B. thuringiensis isolates showed strong toxicity against tested insects. PCR analysis using cryI, cryII, cryIII, cryIV, and cryV gene-specific primers for determination of the cry gene contents of B. thuringiensis isolates indicated that the frequency of the cryIA, cryIC, cryID, and cryII among cry genes predominated, and the cryIB, cryIE, cryIF, cryIG, and cryIV were not popular. In contrast, no PCR products were detected for the cryIII and cryV templates. Several B. thuringiensis isolates produced unusual PCR products and complicated combinations consisting of multiple cry genes. Seven out of 11 B. thuringiensis isolates undetected by specific primers from sericultural farm, all out of 9 isolates from soil, and 19 out of 25 isolates from granary were toxic to lepidoptera and/or diptera. In addition, five nontoxic isolates of sericultural farm, all of five nontoxic isolates of soil, and 13 nontoxic isolates of granary produced the expected PCR products. PCR results showed varied distribution of cry genes for three areas, respectively. An evaluation of this novel activity demands that several criteria be measured: the frequency, flagellar serotype, crystal morphology, toxicity, and combination of the cry genes. Received: 28 March 2000 / Accepted: 26 April 2000     

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.     

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     

Two new subspecies of Bacillus thuringiensis isolated in Japan: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) and Bacillus thuringiensis subsp. tochigiensis (serotype 19)

Bacteriological and serological characteristics of three Bacillus thuringiensis isolates obtained in Japan were investigated. They formed typical rhomboidal parasporal inclusions but flagellar (H) antigens of these isolates were different from those of the known 17 H serotypes of B. thuringiensis. The three isolates were divided into two new serotypes (serotypes 18 and 19). The serotype 18 isolate (3–71) produced thermostable exotoxin and the inclusions of this isolate were toxic to larvae of the silkworm, Bombyx mori, but nontoxic to larvae of the mosquito, Aedes aegypti. The other isolate (119-72) belonging to serotype 18 produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. However, other bacteriological properties of the isolate 119-72 were similar to those of the isolate 3–71. The serotype 19 isolate (117-72) produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. Acid production from saccharose and the production of brownish purple pigment were observed in the two serotype 18 isolates, while neither of them was observed in the serotype 19 isolate. In other 29 biochemical properties tested, there was no difference among the three isolates. Based on these characteristics, the following two subspecies names are proposed: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) for the type strain 3–71 and Bacillus thuringiensis subsp. tochigiensis (serotype 19) for the type strain 117-72.     

Molecular Characterization of Novel Serovars of Bacillus thuringiensis Isolates from India

Novel Bacillus thuringiensis isolates GS4, GN24 and UP1 were isolated and characterized by determination of serotyping, insecticidal protein by SDS-PAGE, plasmid composition, cry gene content and insect toxicity. Serologically two isolates GS4 and UP1 were allocated to the H3abce which is a new serovar while isolate GN24 was of H3ab type. Isolate GS4 produced flat crystal inclusions while UP1 produced cuboidal crystals. PCR analysis found that both isolates contained cry1 and cry1Ac genes. The major protein bands found of isolate GS4 were of molecular weights 175, 135, 97, 88, 66, 54 and 27 kDa, isolate UP1 were of 85, 60 and 40 kDa and isolate GN24 were of 130, 90, 66 and 45 kDa. Though isolates GS4 and UP1 belonged to a new serovar H3abce, they showed different crystal inclusions and cry gene content. Isolate GS4 was toxic to lepidopteran insect larvae of Helicoverpa armigera but UP1 did not showed any toxicity.     

Bacillus thuringiensis: A Common Member of Microflora in Activated Sludges of a Sewage Treatment Plant

Bacillus thuringiensis was recovered at a high frequency from activated-sludge system environments in an urban sewage-digestive plant. All of the test materials, sampled at several digesting steps, contained the organism. Of 515 colonies belonging to the B. cereus/B. thuringiensis group, 45 (8.7%) were assigned to B. thuringiensis. The highest density of this bacterium was 1.6 × 10³ cfu/ml in a scum sample of the first aeration basin. Among the 45 isolates, 7 were assigned to the known H serovars. Two isolates of the serovar kenyae isolates exhibited Lepidoptera-specific toxicity. Diptera-specific toxicity was shown by an isolate of serovar israelensis and a serologically undefined isolate. Lectin activity was associated with 12 isolates. Received: 10 October 2000 / Accepted: 17 November 2001     

Bacillus thuringiensis isolates from northeastern Brazil and their activities against Plutella xylostella (Lepidoptera: Plutellidae) and Spodoptera frugiperda (Lepidoptera: Noctuidae)

Northeastern Brazil has been little explored in the search for Bacillus thuringiensis (Berliner) variants for the control of agricultural pests such as Plutella xylostella (L.) and Spodoptera frugiperda (J.E. Smith). The aim of this study was to isolate B. thuringiensis from soil and insect samples collected from the northeastern states and to evaluate their lethal and sublethal activities against the neonate larvae of P. xylostella and S. frugiperda. One hundred and four isolates were bioassayed and visualised for the presence of crystals. Bipyramidal crystals were present in 31% of the isolates. In pathogenicity tests, 13 and 19 isolates caused >30% mortality in P. xylostella and S. frugiperda, respectively. The Laboratory of Insect-Toxic Interactions (LIIT)-4311 isolate was the most toxic for P. xylostella, with toxicity similar to B. thuringiensis var. kurstaki (Dipel® WP) and B. thuringiensis var. aizawai (Xentari® WDG). For S. frugiperda, the LIIT-4311, LIIT-4306 and LIIT-4406 isolates were more active than B. thuringiensis var. aizawai. The LIIT-4311 and LIIT-4306 isolates caused high rates of larval growth inhibition in both P. xylostella and S. frugiperda. These results suggest a broad distribution of B. thuringiensis variants in areas of northeastern Brazil. Because LIIT-4306 and LIIT-4311 provided the highest levels of toxicity and larval growth inhibition for both P. xylostella and S. frugiperda, these isolates can be exploited to develop new technologies for pest management.     

Investigation of the cyt gene in Bacillus thuringiensis and the biological activities of Bt isolates from the soil of China

The presence of cyt genes was investigated in 80 type strains of Bacillus thuringiensis and 143 isolates obtained from soil samples of China by PCR amplification using two pairs of primers for the cyt1 and cyt2 genes. Three type strains of serotypes H11ac, H14 and H36, eight isolates belonging to H3, H14, H18 and H21, and one isolate of unknown serotype harbored cyt genes. We also tested the cytolytic activity for mammal cells, the hemolytic activity for sheep erythrocytes and insecticidal activity against mosquitoes of five isolates that contained cyt genes but did not belong to B. thuringiensis serovar israelensis. The protein profiles of the five isolates were different from those of the type strains of B. thuringiensis serovar israelensis, and among the five isolates, only Y-5 showed mosquitocidal activity against larvae of Culex quinquefasciatus. All five of the isolates exhibited hemolytic activity, but only three could cause the cell death of A549 cells. The cytopathological changes induced by NX-4 in some A549 cells were characterized with cell-ballooning.     

Discrimination among <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> H serotypes,serovars and strains based on 16S rRNA, <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">aroE</Emphasis> gene sequence analyses

Our aim was to investigate the capability of each of three genes, 16S rRNA, gyrB and aroE, to discriminate, first, among Bacillus thuringiensis H serotypes; second, among B. thuringiensis serovars from the same H serotype; and third, among B. thuringiensis strains from the same serovar. The 16S rRNA, gyrB and aroE genes were amplified from 21 B. thuringiensis H serotypes and their nucleotide sequences determined. Additional strains from four B. cereus sensu lato species were included for comparison purposes. These sequences were pair-wise compared and phylogenetic relationships were revealed. Each of the three genes under study could discriminate among B. thuringiensis H serotypes. The gyrB and aroE genes showed a discriminatory power among B. thuringiensis H serotypes up to nine fold greater than that of the 16S rRNA gene. The gyrB gene was retained for subsequent analyses to discriminate B. thuringiensis serovars from the same H serotype and to discriminate strains from same serovar. A total of 42 B. thuringiensis strains, which encompassed 25 serovars from 12 H serotypes, were analyzed. The gyrB gene nucleotide sequences were different enough as to be sufficient to discriminate among B. thuringiensis serovars from the same H serotype and among B. thuringiensis strains from the same serovar. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The isolates of Bacillus thuringiensis serotype 10 with a highly preferential toxicity to mosquito larvae

Comparative bacteriological and serological studies of three isolates and the reference strain of Bacillus thuringiensis subsp. darmstadiensis (serotype 10) were conducted. No difference was shown in the flagellar antigenic structure between the three isolates and the reference strain. Differences were observed in the O antigenic structures and in the following biochemical properties: lecithinase production, DNase production, arginine decarboxylase production, acid production from inulin, and malonate utilization. β-Exotoxin production was not detected in these three isolates. The reference strain produced parasporal inclusions toxic to the lepidopterous larvae but nontoxic to mosquito larvae. On the contrary, two among the three isolates, which produced spherical parasporal inclusions, were not toxic to the lepidopterous larvae but highly toxic to larvae of the mosquitoes, Culex tritaenlorhynchus, Culex molestus, and Aedes aegypti. Another isolate produced large irregular-shaped inclusions nontoxic to the insects of both orders. Accordingly, B. thuringiensis serotype 10 was divided into three groups from the viewpoint of toxicity against lepidopterous and mosquito larvae.     

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.     

Interaction between crystalline proteins of two Bacillus thuringiensis strains against Spodoptera exigua

The aim of the present article was to evaluate potential synergism between crystalline proteins produced by two Bacillus thuringiensis Berliner strains, MPU B6 and MPU B9, against beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae). Protein inclusions of bacterial strains were isolated from a spore‐crystal mixture. We estimated the 50% lethal concentration (LC₅₀) of crystals for S. exigua larvae. Insecticidal activity of MPU B6 and MPU B9 individual crystal preparations against caterpillars were determined and compared with the commercial pesticide Foray. Protein crystals of MPU B9 had the highest toxicity against S. exigua. The proteins were approximately 25× more toxic than Foray. Insecticidal activity of protein crystals of MPU B6 isolate was approximately 2.5× higher than that of Foray. A mixture of crystals suspensions of both isolates MPU B6/MPU B9 had an additive effect on S. exigua caterpillars. The high insecticidal potency of B. thuringiensis MPU B9 crystals against S. exigua predisposes the strain for additional studies on production of a new effective preparation against pest insects.     

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.     

Characterization of Bacillus thuringiensis isolates from Argentina that are potentially useful in insect pest control

In order to find novel strains of Bacillus thuringiensis that are toxic to some of the major pests that impact economically important crops in Argentina, we initiated a search for B. thuringiensis isolates native to Argentina. We succeeded in assembling a collection of 41 isolates, some of which show a high potential to be used in biological control programs against lepidopteran and coleopteran pests. About 90% of the strains showed toxicity against Spodoptera frugiperda and Anticarsia gemmatalis, two important lepidopteran pests in Argentina. It is noteworthy that only one of these strains contained a cry1-type gene, while another isolate showed a dual toxicity against the lepidopteran and coleopteran insects assayed. Genetic characterization of the strains suggests that the collection likely harbors novel Cry proteins that may be of potential use in biological insect pest control.     

Genetic Diversity of Bacillus cereus/B. thuringiensis Isolates from Natural Sources

The genetic diversity and relationships among 154 Bacillus cereus/B. thuringiensis isolates recovered from soil samples from five geographic areas in Norway were investigated with multilocus enzyme electrophoresis (MEE). Cluster analysis revealed two major groups (designated cluster I and cluster II) separated at genetic distance greater than 0.55. Cluster I included 62 electrophoretic types (ETs) originating from all five locations, whereas, in cluster II, all but one isolate were from the same location. The isolates were also serotyped with B. thuringiensis flagellar antisera, and 28 distinct serotypes were identified. In general, serotyping did not show correlation to the genetic diversity of the isolates. The presence of IS231- and IS240-like transposable elements was detected in 14% of the strains of cluster II only. Parasporal crystals were observed in three strains; ten other strains were toxic to Trichoplusia ni. We conclude that B. cereus/B. thuringiensis from soil exhibit a high degree of recombination. Received: 15 December 1997 / Accepted: 26 January 1998     

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     

Investigation of the cyt gene in Bacillus thuringiensis and the biological activities of Bt isolates from the soil of China

The presence of cyt genes was investigated in 80 type strains of Bacillus thuringiensis and 143 isolates obtained from soil samples of China by PCR amplification using two pairs of primers for the cyt1 and cyt2 genes. Three type strains of serotypes H11ac, H14 and H36, eight isolates belonging to H3, H14, H18 and H21, and one isolate of unknown serotype harbored cyt genes. We also tested the cytolytic activity for mammal cells, the hemolytic activity for sheep erythrocytes and insecticidal activity against mosquitoes of five isolates that contained cyt genes but did not belong to B. thuringiensis serovar israelensis. The protein profiles of the five isolates were different from those of the type strains of B. thuringiensis serovar israelensis, and among the five isolates, only Y-5 showed mosquitocidal activity against larvae of Culex quinquefasciatus. All five of the isolates exhibited hemolytic activity, but only three could cause the cell death of A549 cells. The cytopathological changes induced by NX-4 in some A549 cells were characterized with cell-ballooning.     

Characterization of Flagellar Antigens and Insecticidal Activities of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> 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. Received: 4 June 2002 / Accepted: 5 July 2002     

Serotyping of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> isolates,their distribution in different Jordanian habitats and pathogenicity in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The investigation of Bacillus thuringiensisin 40 different samples collected from 12 different Jordanian habitats involved the isolation of 80 Bacillus thuringiensis isolates. Out of these isolates, 47 were pathogenic to the third instar larvae of Drosophila melanogaster. The highest viable count of Bacillus thuringiensis was estimated among soil samples contaminated with decomposed animal bodies (14.25 × 10⁷ c.f.u./g), and the lowest viable count was obtained from soils contaminated with engine oil (0.17 × 10⁷c.f.u./g). Serotyping of the 80 isolates against 55 antisera indicated the presence of 13 serotypes, 12 were identical or cross-reacted withaizawai, higo,israelensis, kenyae, kumamotoensis, kurstaki, malaysiensis, morrisoni, pakistani,sooncheon,tohokuensis, andthuringiensis, whereas the remaining one reacted negatively with the 55 tested antisera indicating the presence of an unknown serotype. Israelensis was the dominant serotype among all the samples except those from decomposed animal and olive-cultivated soils. The pathogenic isolates were found to be in 11 of the 13 serotypes. Spherical parasporal crystals were the most common and toxic crystal types.