Isolation and Characterization of a Bacillus thuringiensis ssp. kurstaki Strain Toxic to Spodoptera exigua and Culex pipiens

A strain of Bacillus thuringiensis with dual toxicity was isolated from Korean soil samples and named K2. K2 was determined as ssp. kurstaki (H3a3b3c) by serological test and produced bipyramidal-shaped parasporal inclusions. The plasmid and protein profiles of B. thuringiensis K2 were different from those of the reference strain, ssp. kurstaki HD-1. To verify gene type of B. thuringiensis K2, PCR analysis with specific cry gene primers was performed. The result showed that B. thuringiensis K2 had cry1Aa, cry1Ab, cry1C, and cry1D type genes, whereas ssp. kurstaki HD-1 had cry1Aa, cry1Ab, cry1Ac, and cry2 type genes. In addition, B. thuringiensis K2 had high toxicity against Spodoptera exigua and Culex pipiens, whereas B. thuringiensis ssp. kurstaki HD-1 does not have high toxicity against these two insect species. Received: 19 January 2001 / Accepted: 21 February 2001     

Crystals of two mutants ofBacillus thuringiensis show increased toxicity against larvae ofSpodoptera littoralis

Crystals of two asporogenous mutants ofBacillus thuringiensis var.kurstaki strain HD-1 obtained following treatment with ozone and N-methyl-N′-nitro-N-nitrosoguanidine showed increased toxicity against larvae ofSpodoptera littoralis when compared to the wild-type crystal.      

Correlation between specific plasmids and δ-endotoxin production in Bacillus thuringiensis

Five strains of Bacillus thuringiensis that produce crystalline δ-endotoxin were used as parental strains in an effort to isolate acrystalliferous (Cry⁻) mutants: HD-2 (B. thuringiensis var. thuringiensis, flagellar serotype 1); HD-1 and HD-73 (both var. kurstaki, serotype 3ab); HD-4 (var. alesti, serotype 3a); and HD-8 (var. galleriae, serotype 5ab). The parental strains contain complex plasmid arrays that have been previously characterized (González and Carlton, 1980). The plasmid patterns of both Cry⁻ and Cry⁺ variants were analyzed and compared to the parental strains using a modified Eckhardt (1978) lysate-electrophoresis method. Most Cry⁻ mutants derived from strain HD-2 were found to exhibit a distinctive colony morphology which facilitated their isolation. Loss of crystal production was associated with loss of a 75-Md plasmid. A 50-Md plasmid of strain HD-73 was lost in the Cry⁻ mutants. Crystal production in strain HD-4 appears to be associated with a plasmid about 105 Md in size; in strain HD-1, a smaller plasmid (29 Md in size) seems to be involved. In strain HD-8, a large plasmid (˜130 Md in size) is implicated in crystal production. Direct bioassay of several of the mutant strains has confirmed the loss of δ-endotoxin activity in the acrystalliferous isolates. The evidence obtained supports the notion of a relationship between specific extrachromosomal DNA elements and δ-endotoxin production in B. thuringiensis, and suggests that in each strain only a single plasmid is involved, although the size of the implicated plasmid varies from one strain to another.     

Isolation and characterization of a strain of Bacillus thuringiensis ssp. kurstaki containing a new delta-endotoxin gene

A strain of Bacillus thuringiensis that showed significantly high toxicity to Plutella xylostella and Spodoptera exigua was isolated from a Korean soil sample and characterized. The isolate, named B. thuringiensis K1, was determined to belong to ssp. kurstaki (H3a3b3c) type by an H antisera agglutination test and produced bipyramidal inclusions. Plasmid pattern of K1 was different from that of the reference strain, ssp. kurstaki HD-1, but the parasporal inclusion protein profile of K1 had two major bands that were similar in size to those of ssp. kurstaki HD-1. To verify the δ-endotoxin gene types of K1, PCR analysis with specific cry gene primers was performed to show that K1 contained a new cry gene in addition to cry1Aa, cry1Ab, cry1Ac, cry1E and cry2 genes. PCR-amplified region of the new cry gene, cryX, showed 79% similarity to cry1Fa1 gene (GenBank Accession No. M63897). In an insect toxicity assay, K1 had higher toxicity against Plutella xylostella and S. exigua than ssp. kurstaki HD-1. Received: 21 December 2001 / Accepted: 28 January 2002     

Cloning and Expression of a Bacillus thuringiensis (L1-2) Gene Encoding a Crystal Protein Active Against Glossina morsitans morsitans and Chilo partellus

A local isolate of Bacillus thuringiensis, designated L1-2, that is toxic to Chilo partellus was found to be toxic to the adult tsetse fly, Glossina morsitans morsitans. The δ-endotoxin crystals derived from the isolate gave a major protein band with a molecular weight of M_r 130,000–140,000 on denaturing polyacrylamide gel electrophoresis. The sequence of the cloned gene was found to be similar to that of the B. thuringiensis subsp. kurstaki HD-73 cryIA(c) gene, having one amino acid difference at position 148 and four additional DNA differences. Received: 29 June 1996 / Accepted: 1 August 1996     

Analysis of <Emphasis Type="Italic">cry</Emphasis> Gene Profiles in <Emphasis Type="Italic">Bacillus Thuringiensis</Emphasis> Strains Isolated During Epizootics in <Emphasis Type="Italic">Cydia pomonella</Emphasis> L.

The crystal morphology and the profiles of genes encoding protein toxins (Cry and Cyt) were analyzed in 12 Bacillus thuringiensis strains isolated during epizootics in laboratory culture lines of Cydia pomonella, 2 isolates cultured from Leucoma salicis larvae, and 9 reference strains. Epizootic isolates produced crystals of the same bipyramidal shape; however, they revealed a variety of number and type of cry genes. Genes cry1I, cry2Ab, and cry9B were the most frequently observed in epizootic strains. Gene cry1I was noted in of 50% epizootic isolates. Eighty-three percent of them harbored gene cry2Ab. Gene cry9B was found for 42% of strains isolated during epizootics. Three isolates showed the largest number of cry genes and their variety; hence, they were chosen for the toxicity assay of their crystals and spores on C. pomonella larvae. One of them had approximately sixfold higher insecticidal activity than the reference strain B. thuringiensis subsp. kurstaki BTK STANDARD.     

Plasmid stability ofBacillus thuringiensis var.kurstaki (HD-1) during continuous phased cultivation

Summary Bacillus thuringiensis var.kurstaki (HD-1)_was grown as a continuous phased culture in a cyclone fermentor. During the time course of the continuous phased cultivation (CPC), the culture was sampled to determine the efficiency of sporulation and parasporal crystal formation. Concurrently, plasmid DNA was extracted and resolved on agarose gels. The plasmid profile remained constant throughout 328 h of cultivation. However, during the same time period, asporogenous, acrystalliferous variants increased from<1% to>90% of the cells harvested. Our data suggests that the disappearance of parasporal crystals inB. thuringiensis var.kurstaki (HD-1) during CPC occurs independent of plasmid copy but may be due to defective sporulation.     

Contribution of Bacillus thuringiensis Spores to Toxicity of Purified Cry Proteins Towards Indianmeal Moth Larvae

The influence of Bacillus thuringiensis subsp. kurstaki HD-1 spores upon the toxicity of purified Cry1Ab and Cry1C crystal proteins toward susceptible and BT-resistant Indianmeal moth (IMM, Plodia interpunctella) larvae was investigated. With susceptible larvae, HD-1 spores were toxic in the absence of crystal protein and highly synergistic (approximately 35- to 50-fold) with either Cry1Ab or Cry1C protein. With BT-resistant IMM larvae, HD-1 spores were synergistic with Cry1Ab and Cry1C protein in all three resistant strains examined. Synergism was highest (approximately 25- to 44-fold) in insects with primary resistance toward Cry1C (IMM larvae with resistance to B. thuringiensis subsp. aizawai or entomocidus). However, HD-1 spores also synergized either Cry1Ab or Cry1C toxicity toward larvae resistant to B. thuringiensis subsp. kurstaki at a lower level (approximately five- to sixfold). With susceptible larvae, the presence of spores reduced the time of death when combined with each of the purified Cry proteins. Without spores, the speed of intoxication and eventual death for larvae treated with Cry1C and Cry1Ab proteins was much slower than for the HD-1 preparation containing both spores and crystals together. Neither spores nor toxin dose affected the mean time of death of resistant larvae treated with either Cry1Ab or Cry1C toxins. Both Cry1Ab and Cry1C toxins appeared to reduce feeding and consequently toxin consumption. Received: 1 December 1995 / Accepted: 3 January 1996     

Biological characterization of two <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains toxic against <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis>

Two Bacillus thuringiensis strains isolated from diseased Spodoptera frugiperda larvae collected in the northwest of Argentina were molecularly and phenotypically characterized. Insecticidal activity against Spodoptera frugiperda larvae was also determined. Both strains were highly toxic against first instar larvae. One strain (Bacillus thuringiensis LSM) was found to be even more toxic than the reference strain Bacillus thuringiensis var. kurstaki 4D1. This strong biological effect was represented by both a higher mortality which reached 90%, and a shorter LT₅₀. Molecular characterization showed that Bacillus thuringiensis LSM carried a cry gene profile identical to that of Bacillus thuringiensis var. kurstaki 4D1. Evaluation of length polymorphism of the intergenic transcribed spacers between the 16S and 23S rDNA genes revealed an identical pattern between native strains and Bacillus thuringiensis var. kurstaki 4D1. In contrast, phenotypic characterization allowed differentiation among the isolates by means of their extracellular esterase profiles. Lytic activity that would contribute to Bacillus thuringiensis effectiveness was also studied in both strains. Analyses like those presented in the current study are essential to identify the most toxic strains and to allow the exploitation of local biodiversity for its application in biological control programmes.     

Expression of three genes coding for 135-kilodalton entomocidal proteins inBacillus thuringiensis kurstaki

The HD-1 strain ofBacillus thuringiensis (B.t.)kurstaki contains three homologous genes coding for 130–134-kilodalton entomocidal proteins [13]. In the present study, expression levels of these genes in strains of B.t.kurstaki were determined. In attempts to isolate a protein coded by a single gene, a number of variants were derived from strains of B.t.kurstaki, such as HD-263 and HD-1, by plasmid curing. The entomocidal proteins produced by the parental strains and their plasmid-cured variants were isolated by Sephacryl S-300 column chromatography and peptide-mapped by high performance liquid chromatography (HPLC). The results indicated that HD-263 produced two distinctive proteins, one identical with the protein of HD-73, which contains only a 6.6 kb Hind III class gene, and the other protein presumably coded by a 4.5 kb Hind III class gene. HPLC analysis revealed that 70% of the total protein in the HD-263 crystals consisted of the product of the 6.6 kb gene (6.6-kb protein), and the remaining 30% was the 4.5-kb protein. In the case of HD-1, the crystal consisted of at least two different proteins in equal amounts (50% each). The gene coding for one of these proteins was presumed to be a 5.3 kb Hind III class gene. The remaining 50% of the HD-1 crystal was accounted for by a protein similar to the 4.5-kb protein identified in HD-263. It appeared that the 6.6-kb protein was expressed poorly, if it was indeed expressed, in the HD-1 strain.     

Relationships between laboratory bioassay-derived potencies and field efficacies of Bacillus thuringiensis isolates with different spectral activities

Bacillus thuringiensis isolates with different spectral activities were not equally efficacious when applied to cabbage at the same number of IUs/ha for protection against larvae of the cabbage looper, Trichoplusia ni. Preparations of the isolates were standardized against T. ni larvae. Variety galleriae isolates (HD-196 and HD-153) were the most efficacious per applied IU, and a K-73 type variety kurstaki (HD-73) was the least efficacious per applied IU. A variety thuringiensis (HD-264) and a K-1 type variety kurstaki (HD-1) were intermediate in efficacy per applied IU. Speed of kill and, to some extent, differences in the amount of food consumed appear to be responsible for the differences in efficacy per applied IU. When more potent B. thuringiensis isolates are discovered and developed, the recommended field dosages for the new isolates must be determined by actual field experimentation rather than by extrapolation from existing HD-1 data.     

Activity spectra of Bacillus Thuringiensis δ-endotoxins against eight insect cell lines

Summary Eight continuous insect cell lines were tested for susceptibility to the δ-endotoxins of several lepidopteran-active strains and cloned-gene products of Bacillus thuringiensis. The assays were performed on cells suspended in agarose gel, which allowed the toxins activated at pH 10.5 to be applied directly in a high-pH buffer without causing solvent toxicity to the cells. The responses of the cell lines to the various toxins produced activity spectra that were used to identify functionally similar and dissimilar toxin proteins. IPRI-CF-1 and FPMI-MS-5, derived from neonate larvae of Choristoneura fumiferana and Manduca sexta, respectively, exhibited the greatest sensitivity to the toxins tested, whereas B. thuringiensis subsp. entomocidus had the broadest in vitro host range. Analysis of activity spectra led to the identification of the particular Cry protein that was responsible for the broad toxicity of this subspecies and demonstrated a distinct difference in toxin composition between two strains of subsp. sotto. The identical spectra observed for subsp. kurstaki HD-1 and NRD-12 is consistent with insect bioassay data obtained previously by other workers and supports the conclusion that there is virtually no difference in activity between these two strains. The in vitro assay system, referred to as the “lawn assay” and used to test B. thuringiensis activated toxins against insect cell lines, is particularly useful in mode-of-action studies and as a rapid, preliminary test for the presence of specific cytolytic proteins, rather than as a method for screening toxins of wild-type strains for insecticidal activity. The response of cells in vitro to B. thuringiensis toxins is often very different from that of the insect from which the cells were derived.     

Dual Insecticidal Activity of Spodoptera-Toxic Bacillus thuringiensis Strain Transformed with Lepidopteran-Specific Cry Toxin

The E. coli-B. thuringiensis shuttle vector for expression of cry1Ac, pHT1K-1Ac plasmid was introduced into acrystalliferous B. thuringiensis Cry⁻B and Spodoptera toxic STB-3 strain. The presence of a recombinant plasmid in transformants after electroporation was confirmed by PCR. The 1K-1Ac/Cry⁻B(Cry⁻B transformant) and 1K-1Ac/STB-3 (STB-3 transformant) produced bipyramidal-shaped parasporal inclusion that was 130 kDa in size as like B. thuringiensis subsp. kurstaki HD-73. In P. xylostella bioassay, these transformants showed significantly high toxicity than the wild-type recipients and further, in case of B. thuringiensis STB-3 transformant still had original Spodoptera toxicity. These results suggested that the pHT1K could be successfully applied for generating individual B. thuringiensis strains that produce various combinations of insecticidal proteins to expand their host spectrum and enhance insecticidal activity.     

Toxicity of a <Emphasis Type="Italic">Bacillus thuringiensis israelensis</Emphasis>-like strain against <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis>

Bacillus thuringiensis (Bt) Berliner is a promising agent for microbial control of agriculturally and medically important insects. This study aimed at searching for Bt strains encoding Cry proteins that act more efficiently against fall armyworm. Thirty Bt strains were isolated from soil samples in Pernambuco State and evaluated through bioassays. Among these, strain I4A7 was the most efficient against the fall armyworm, Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae), and thus it was characterized by biochemical sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and molecular (polymerase chain reaction (PCR) and sequencing reaction) methods. The protein pattern of this strain on a SDS–PAGE was similar to that of B. thuringiensis israelensis (Bti). Moreover, I4A7 cry DNA sequence showed high identity (99–100%) to genes cry4Aa, 4Ba, 10Aa, 11Aa, cyt1Aa and cyt2B from Bti. The toxicity of the newly isolated Bti-like strain upon S. frugiperda should be considered as this strain might be used in combination with other Bt strains, such as B. thuringiensis var. kurstaki (Btk). Handling Editor: Helen Roy.     

Host range and gene contents of Bacillus thuringiensis strains toxic towards Spodoptera exigua

Thirty-five strains of the entomopathogenic bacterium Bacillus thuringiensisactive on Spodoptera exigua, were characterized by means of serological identification and determination of crygene contents by PCR. The insecticidal activity of these 35 strains was further confirmed against S. exiguaand tested against two other species of the same genus: S. littoralisand S. frugiperda. The results indicate that serovars aizawai, thuringiensis, and kurstakiwere the most frequent within S. exigua-active strains and that serovar aizawaihad the highest number of strains exhibiting toxicity against the three species bioassayed. The presence in crygenes as determined by PCR suggests a non random distribution of some crygenes among serovars. Genes cry1C, cry1D, and cry1E, which are known to code for proteins toxic against Spodopteraspecies, were very common within S. exigua-active strains, specially in those belonging to serovar aizawai. However, some strains harbouring one or more of these genes were not toxic to S. littoralisor S. frugiperda; and some strains lacking all of the Spodoptera-active genes were found to be toxic to all three species. This suggests differences in the expression levels among strains bearing toxic genes and the involvement of other genes toxic to Spodopteraspecies. Since strains sharing the same crygenes exhibited different host ranges, the results indicate the need to perform toxicity bioassays in addition to other tests (serological identification and PCR) in order to determine the insecticidal activity of B. thuringiensisstrains.     

EFFECT OF TANNIC ACID ON THE EFFECTIVENESS OF BACILLUlS THURINGIENSIS VAR. KURSTAKI AGAINST HELICOVERPA ARMIGERA (HÜBNER)*

Abstract The role of tannic acid in increasing effectiveness of Bacillus thuringiensis var. kurstaki (HD-1) against Helicover pa armigera was examined in bioassays on semisynthetic diet. Concentrations of B. thuringiensis (0 %, 0.005 %, 0. 01 %, 0.015 %, 0.02 %, 0.025 % wet weight) were incorporated into the diet containing 0. 025% tannic acid and tannic acid-free diet. LD₅₀ of B. thuringiensis with tannic acid were 0.006% but that without tannic acid was 0.011%. Both B. thuringiensis and tannic acid retarded growth of H. armigera significantly, but there was no synergetic effect between them. Choice tests showed that B, thuringiensis deterred feeding of the fifth instar larvae of H. armigera but tannic acid had no such effect. Experiments on colony growth of B. thuringiensis on NBA media containing tannic acid (0, 1, 3, 6, 9, 12, 15, 18, 21 mg/100 ml) demonstrated that tannic acid reduced colony growth of B. thuringiensis, and inhibited sporulation above 15 mg/100 ml.     

Genetic and Biochemical Approach for Characterization of Resistance to Bacillus thuringiensis Toxin Cry1Ac in a Field Population of the Diamondback Moth, Plutella xylostella

Four subpopulations of a Plutella xylostella (L.) strain from Malaysia (F₄ to F₈) were selected with Bacillus thuringiensis subsp. kurstaki HD-1, Bacillus thuringiensis subsp. aizawai, Cry1Ab, and Cry1Ac, respectively, while a fifth subpopulation was left as unselected (UNSEL-MEL). Bioassays at F₉ found that selection with Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai gave resistance ratios of >95, 10, 7, and 3, respectively, compared with UNSEL-MEL (>10,500, 500, >100, and 26, respectively, compared with a susceptible population, ROTH). Resistance to Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai in UNSEL-MEL declined significantly by F₉. The Cry1Ac-selected population showed very little cross-resistance to Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai (5-, 1-, and 4-fold compared with UNSEL-MEL), whereas the Cry1Ab-, B. thuringiensis subsp. kurstaki-, and B. thuringiensis subsp. aizawai-selected populations showed high cross-resistance to Cry1Ac (60-, 100-, and 70-fold). The Cry1Ac-selected population was reselected (F₉ to F₁₃) to give a resistance ratio of >2,400 compared with UNSEL-MEL. Binding studies with ¹²⁵I-labeled Cry1Ab and Cry1Ac revealed complete lack of binding to brush border membrane vesicles prepared from Cry1Ac-selected larvae (F₁₅). Binding was also reduced, although less drastically, in the revertant population, which indicates that a modification in the common binding site of these two toxins was involved in the resistance mechanism in the original population. Reciprocal genetic crosses between Cry1Ac-reselected and ROTH insects indicated that resistance was autosomal and showed incomplete dominance. At the highest dose of Cry1Ac tested, resistance was recessive while at the lowest dose it was almost completely dominant. The F₂ progeny from a backcross of F₁ progeny with ROTH was tested with a concentration of Cry1Ac which would kill 100% of ROTH moths. Eight of the 12 families tested had 60 to 90% mortality, which indicated that more than one allele on separate loci was responsible for resistance to Cry1Ac.     

Efficient screening and breeding of Bacillus thuringiensis subsp. kurstaki for high toxicity against Spodoptera exigua and Heliothis armigera

Spodoptera exigua is one of the most renowned agricultural pest insects and relatively insensitive to Bacillus thuringiensis subsp. kurstaki strains which are widely used commercial products to control lepidopterans such as Heliothis armigera. In the current study, we have developed a new and efficient approach to screen and breed a B. thuringiensis subsp. kurstaki strain exhibiting high toxicity against S. exigua while retaining its high toxicity against H. armigera. UV and diethyl sulfate methods were used for mutagenesis, followed by an agar plug plate diffusion assay for preliminary screening of Zwittermicin A over-producing mutants, from which we obtained a mutant strain, designated here as B. thuringiensis subsp. kurstaki D1-23, with high toxicity against S. exigua. The toxicity of D1-23 against S. exigua and H. armigera was improved by 115.4 and 25.9%, respectively, compared to its parental commercial strain BMB005.     

Development of a recombinant strain of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">kurstaki</Emphasis> HD-73 that produces the endochitinase ChiA74

Bacillus thuringiensis subsp. kurstaki HD-73 was transformed with the homologous endochitinase gene chiA74 of B. thuringiensis subsp. kenyae LBIT-82 under the regulation of its own promoter and Shine–Dalgarno sequence. The plasmid, pEHchiA74, which harbors chiA74, was detected by southern blot analysis and showed high segregational stability when the recombinant strain was grown in a medium without antibiotic. The recombinant bacterium transformed with pEHchiA74 showed an improvement in chitinolytic activity three times that of the wild-type strain. Expression of ChiA74 did not have any deleterious effect on the crystal morphology and size, but sporulation and Cry1Ac production in rich medium (nutrient broth with glucose) was reduced by approximately 30%. No significant increase in the toxicity of the transformant bacterium toward Plutella xylostella was detected using the same amount of total protein. However, it is possible that ChiA74 synthesis compensated for the decrease in net Cry1Ac synthesis and toxicity observed with the recombinant strain.