Identification of Bacillus thuringiensis subsp. kurstaki Strain HD1-Like Bacteria from Environmental and Human Samples after Aerial Spraying of Victoria, British Columbia, Canada, with Foray 48B

Aerial applications of Foray 48B, which contains Bacillus thuringiensis strain HD1, were carried out on 9 to 10 May, 19 to 21 May, and 8 to 9 June 1999 to control European gypsy moth (Lymantria dispar) populations in Victoria, British Columbia, Canada. A major assessment of the health impact of B. thuringiensis subsp. kurstaki was conducted by the Office of the Medical Health Officer of the Capital Health Region during this period. Environmental (air and water) and human (nasal swab) samples, collected before and after aerial applications of Foray 48B, both in the spray zone and outside of the spray zone, were analyzed for the presence of strain HD1-like bacteria. Random amplified polymorphic DNA analysis, cry gene-specific PCR, and dot blot DNA hybridization techniques were used to screen over 11,000 isolates of bacteria. We identified bacteria with genetic patterns consistent with those of B. thuringiensis subsp. kurstaki HD1 in 9,102 of 10,659 (85.4%) isolates obtained from the air samples, 13 of 440 (2.9%) isolates obtained from the water samples, and 131 of 171 (76.6%) isolates from the nasal swab samples. These analyses suggest that B. thuringiensis subsp. kurstaki HD1-like bacteria were present both in the environment and in the human population of Victoria prior to aerial applications of Foray 48B. The presence of B. thuringiensis subsp. kurstaki HD1-like bacteria in human nasal passages increased significantly after the application of Foray 48B, both inside and outside the spray zone.     

Occurrence of natural Bacillus thuringiensis contaminants and residues of Bacillus thuringiensis-based insecticides on fresh fruits and vegetables

A total of 128 Bacillus cereus-like strains isolated from fresh fruits and vegetables for sale in retail shops in Denmark were characterized. Of these strains, 39% (50/128) were classified as Bacillus thuringiensis on the basis of their content of cry genes determined by PCR or crystal proteins visualized by microscopy. Random amplified polymorphic DNA analysis and plasmid profiling indicated that 23 of the 50 B. thuringiensis strains were of the same subtype as B. thuringiensis strains used as commercial bioinsecticides. Fourteen isolates were indistinguishable from B. thuringiensis subsp. kurstaki HD1 present in the products Dipel, Biobit, and Foray, and nine isolates grouped with B. thuringiensis subsp. aizawai present in Turex. The commercial strains were primarily isolated from samples of tomatoes, cucumbers, and peppers. A multiplex PCR method was developed to simultaneously detect all three genes in the enterotoxin hemolysin BL (HBL) and the nonhemolytic enterotoxin (NHE), respectively. This revealed that the frequency of these enterotoxin genes was higher among the strains indistinguishable from the commercial strains than among the other B. thuringiensis and B. cereus-like strains isolated from fruits and vegetables. The same was seen for a third enterotoxin, CytK. In conclusion, the present study strongly indicates that residues of B. thuringiensis-based insecticides can be found on fresh fruits and vegetables and that these are potentially enterotoxigenic.     

Detection of Bacillus thuringiensis kurstaki HD1 on cabbage for human consumption

The objectives of the study were to develop a specific procedure for quantification and identification of Bacillus thuringiensis kurstaki HD1, which is used as a biopesticide, and to quantify its presence in different kinds of cabbage for human consumption. We found that B. thuringiensis kurstaki HD1 can be distinguished from other B. thuringiensis strains by its unique random amplification of polymophic DNA-PCR pattern with the OPA9 primer and the presence of the flagellin genes, as detected by the primers FLAB1 and FLAB2. We detected from one to 100 Bacillus cereus-like bacteria in 10 batches of five different cabbage products for consumption. As many as 73 out of 134 isolates (53.7%) were identical with B. thuringiensis kurstaki HD1. The results show that B. thuringiensis kurstaki HD1 from biopesticides can be found in vegetables for human consumption.     

Transfer of chromosomal genes and plasmids in Bacillus thuringiensis.

A low frequency of chromosomal gene transfer from Bacillus thuringiensis to Bacillus cereus was detected by cell mating, with a tryptophan marker being the most frequently transferred gene among four that were tested. The process was resistant to DNase and was not mediated by cell filtrates. Among several B. thuringiensis subspecies tested, transfer was best with a derivative of B. thuringiensis subsp. kurstaki HD1, which lost several plasmids. All of the B. cereus recombinants contained at least one plasmid from the donor B. thuringiensis; frequently, it was a plasmid that encoded a protoxin gene. In matings with B. thuringiensis subsp. kurstaki HD1, a 29-megadalton plasmid that contained a ca. 2.5-kilobase region of homology with the chromosome was always transferred. No detectable transfer of chromosomal genes was found in B. thuringiensis subsp. kurstaki HD1 strains lacking this plasmid, suggesting that there may be chromosome mobilization.     

Transfer of chromosomal genes and plasmids in Bacillus thuringiensis

A low frequency of chromosomal gene transfer from Bacillus thuringiensis to Bacillus cereus was detected by cell mating, with a tryptophan marker being the most frequently transferred gene among four that were tested. The process was resistant to DNase and was not mediated by cell filtrates. Among several B. thuringiensis subspecies tested, transfer was best with a derivative of B. thuringiensis subsp. kurstaki HD1, which lost several plasmids. All of the B. cereus recombinants contained at least one plasmid from the donor B. thuringiensis; frequently, it was a plasmid that encoded a protoxin gene. In matings with B. thuringiensis subsp. kurstaki HD1, a 29-megadalton plasmid that contained a ca. 2.5-kilobase region of homology with the chromosome was always transferred. No detectable transfer of chromosomal genes was found in B. thuringiensis subsp. kurstaki HD1 strains lacking this plasmid, suggesting that there may be chromosome mobilization.     

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.     

Self-transfer and mobilisation capabilities of the pXO2-like plasmid pBT9727 from Bacillus thuringiensis subsp. konkukian 97-27

Recent characterisations of plasmids related to the anthrax virulence plasmids pXO1 and pXO2 in clinical isolates of Bacillus cereus and Bacillus thuringiensis have contributed to the emerging picture of a virulence-associated plasmid pool in the B. cereus sensu lato group. The family of pXO2-like plasmids includes the conjugative plasmid pAW63 from the biopesticide strain B. thuringiensis subsp. kurstaki HD73 and the heretofore cryptic plasmid pBT9727 from the clinical strain B. thuringiensis subsp. konkukian 97-27. Comparative sequence analysis of these three plasmids suggested that they were derived from an ancestral conjugative plasmid, with pAW63 retaining its self-transfer capabilities, and pXO2 having lost them through genetic drift. Such properties had not been investigated in pBT9727, but sequence homologies led us to predict that it may possess self-transfer capabilities. Here, we report that pBT9727 is indeed conjugative, and is able to promote its own transfer as well as that of small mobilisable plasmids.     

Expression of the crystal protein gene under the control of the alpha-amylase promoter in Bacillus thuringiensis strains.

The expression of an insecticidal crystal protein gene of Bacillus thuringiensis under the control of the alpha-amylase gene promoter was investigated. The cryIC gene, which encodes a protein known to have a unique activity against Spodoptera (armyworm) species, was used in this investigation. The cryIC gene was placed, along with the alpha-amylase promoter from B. subtilis, in a B. thuringiensis-derived cloning vector, generating a pair of recombinant plasmids, pSB744 and pSB745. The cloning vector that contains the minimal replicon of B. thuringiensis subsp. kurstaki HD73 is stably maintained in a variety of B. thuringiensis strains, as previously reported by Gamel and Piot (Gene 120:17-26, 1992). The present study confirmed that the recombinant plasmids are also stably maintained in B. thuringiensis subsp. kurstaki Cry-B and HD73 growing in media without selection pressure for at least 48 h. The cryIC gene on the recombinant plasmids were notably expressed at high levels in both recombinant strains. Expression of the introduced cryIC gene on the recombinant plasmid in B. thuringiensis subsp. kurstaki HD73 did not impair expression of the resident cryIA(c) gene. The CryIA(c) protein is known to have a high level of activity against loopers such as Trichoplusia ni (the cabbage looper). As a result of coexpression of the introduced cryIC gene and the resident cryIA(c) gene, recombinant strain HD73 acquired an additional insecticidal activity against Spodoptera exigua (the beet armyworm) whereas the original activity level against T. ni was maintained.     

Enhanced production of insecticidal proteins in Bacillus thuringiensis strains carrying an additional crystal protein gene in their chromosomes.

A two-step procedure was used to place a cryIC crystal protein gene from Bacillus thuringiensis subsp. aizawai into the chromosomes of two B. thuringiensis subsp. kurstaki strains containing multiple crystal protein genes. The B. thuringiensis aizawai cryIC gene, which encodes an insecticidal protein highly specific to Spodoptera exigua (beet armyworm), has not been found in any B. thuringiensis subsp. kurstaki strains. The cryIC gene was cloned into an integration vector which contained a B. thuringiensis chromosomal fragment encoding a phosphatidylinositol-specific phospholipase C, allowing the B. thuringiensis subsp. aizawai cryIC to be targeted to the homologous region of the B. thuringiensis subsp. kurstaki chromosome. First, to minimize the possibility of homologous recombination between cryIC and the resident crystal protein genes, B. thuringiensis subsp. kurstaki HD73, which contained only one crystal gene, was chosen as a recipient and transformed by electroporation. Second, a generalized transducing bacteriophage, CP-51, was used to transfer the integrated cryIC gene from HD73 to two other B. thuringiensis subsp. kurstaki stains. The integrated cryIC gene was expressed at a significant level in all three host strains, and the expression of cryIC did not appear to reduce the expression of the endogenous crystal protein genes. Because of the newly acquired ability to produce the CryIC protein, the recombinant strains showed a higher level of activity against S. exigua than did the parent strains. This two-step procedure should therefore be generally useful for the introduction of an additional crystal protein gene into B. thuringiensis strains which have multiple crystal protein genes and which show a low level of transformation efficiency.     

Unique regulation of crystal protein production in Bacillus thuringiensis subsp. yunnanensis is mediated by the cry protein-encoding 103-megadalton plasmid.

In sporulating cultures of Bacillus thuringiensis subsp. yunnanensis HD977, two cell types are observed: cells forming only spores and cells forming only crystals. Curing analysis suggested that the crystal proteins are plasmid encoded. Through plasmid transfer experiments, it was established that a 103-MDa plasmid is involved in the crystal production. Conjugal transfer of this plasmid to Cry- recipient cells of Bacillus thuringiensis subsp. kurstaki HD73-26 conferred the ability to produce crystals exclusively on asporogenous cells of the recipient, indicating that the 103-MDa plasmid mediates the unique regulation of Cry protein production. When the dipteran-specific cryIVB gene was introduced into wild-type (Cry+) and Cry- backgrounds of B. thuringiensis subsp. yunnanensis by phage CP51ts45-mediated transduction, similar to all other B. thuringiensis strains, irregular crystals of CryIVB protein were produced by spore-forming cells in both backgrounds. However, the synthesis of the bipyramidal inclusions of B. thuringiensis subsp. yunnanensis was still limited only to asporogenous cells of the transductant. Thus, it appears that the unique property of exclusive crystal formation in asporogenous cells of B. thuringiensis subsp. yunnanensis is associated with the crystal protein gene(s) per se or its cis acting elements. As the crystals in B. thuringiensis subsp. yunnanensis were formed only in asporogenous cells, attempts were made to find out whether crystal formation had any inhibitory effect on sporulation. It was observed that both Cry+ and Cry- strains of B. thuringiensis subsp. yunnanensis (HD977 and HD977-1, respectively) exhibited comparable sporulation efficiencies. In addition, the Cry- B. thuringiensis subsp. kurstaki host (HD73-26) and its Cry+ transconjugant (HD73-26-16), expressing the B. thuringiensis subsp. yunnanensis crystal protein, were also comparable in their sporulation efficiencies, indicating that production of the crystal proteins of B. thuringiensis subsp. yunnanensis does not affect the process of sporulation.     

The hypervariable region in the genes coding for entomopathogenic crystal proteins of Bacillus thuringiensis: nucleotide sequence of the kurhd1 gene of subsp. kurstaki HD1

One of the genes for the entomophatogenic crystal protein of Bacillus thuringiensis (subsp. kurstaki strain HD1) has been cloned in Escherichia coli, and its nucleotide sequence determined completely. The gene is contained within a 4360-bp-long HpaI-PstI DNA restriction fragment and codes for a polypeptide of 1,155 amino acid residues. The protoxin protein has a predicted Mr of 130,625. The E. coli-derived protoxin gene product is biologically active against Heliothis virescens larvae in a biotest assay. Extensive computer comparisons with other published B. thuringiensis subsp. kurstaki strains HD1, HD73, and B. thuringiensis subsp. sotto gene sequences reveal hypervariable regions in the first half of the protoxin coding sequence. These regions are responsible for the biological activity of the protein product of the cloned gene, and may explain the different biological activities of these different protoxins.     

A Change in a Single Midgut Receptor in the Diamondback Moth (Plutella xylostella) Is Only in Part Responsible for Field Resistance to Bacillus thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai

A population (SERD3) of the diamondback moth (Plutella xylostella L.) with field-evolved resistance to Bacillus thuringiensis subsp. kurstaki HD-1 (Dipel) and B. thuringiensis subsp. aizawai (Florbac) was collected. Laboratory-based selection of two subpopulations of SERD3 with B. thuringiensis subsp. kurstaki (Btk-Sel) or B. thuringiensis subsp. aizawai (Bta-Sel) increased resistance to the selecting agent with little apparent cross-resistance. This result suggested the presence of independent resistance mechanisms. Reversal of resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai was observed in the unselected SERD3 subpopulation. Binding to midgut brush border membrane vesicles was examined for insecticidal crystal proteins specific to B. thuringiensis subsp. kurstaki (Cry1Ac), B. thuringiensis subsp. aizawai (Cry1Ca), or both (Cry1Aa and Cry1Ab). In the unselected SERD3 subpopulation (ca. 50- and 30-fold resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai), specific binding of Cry1Aa, Cry1Ac, and Cry1Ca was similar to that for a susceptible population (ROTH), but binding of Cry1Ab was minimal. The Btk-Sel (ca. 600-and 60-fold resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai) and Bta-Sel (ca. 80-and 300-fold resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai) subpopulations also showed reduced binding to Cry1Ab. Binding of Cry1Ca was not affected in the Bta-Sel subpopulation. The results suggest that reduced binding of Cry1Ab can partly explain resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai. However, the binding of Cry1Aa, Cry1Ac, and Cry1Ca and the lack of cross-resistance between the Btk-Sel and Bta-Sel subpopulations also suggest that additional resistance mechanisms are present.     

The solubility of inclusion proteins from Bacillus thuringiensis is dependent upon protoxin composition and is a factor in toxicity to insects.

Bacillus thuringiensis subsp. aizawai HD133 is one of several strains particularly effective against Plodia interpunctella selected for resistance to B. thuringiensis subsp. kurstaki HD1 (Dipel). B. thuringiensis subsp. aizawai HD133 produces inclusions containing three protoxins, CryIA(b), CryIC, and CryID, and the CryIC protoxin has been shown to be active on resistant P. interpunctella as well as on Spodoptera larvae. The CryIA(b) protoxin is very similar to the major one in B. thuringiensis subsp. kurstaki HD1, and as expected, this protoxin was inactive on resistant P. interpunctella. A derivative of B. thuringiensis subsp. aizawai HD133 which had been cured of a 68-kb plasmid containing the cryIA(b) gene produced inclusions comprising only the CryIC and CryID protoxins. Surprisingly, these inclusions were much less toxic for resistant P. interpunctella and two other Lepidoptera than those produced by the parental strain, whereas the soluble protoxins from these strains were equally effective. In contrast, inclusions from the two strains were about as active as soluble protoxins for Spodoptera frugiperda larvae, so toxicity differences between inclusions may be due to the solubilizing conditions within particular larval guts. Consistent with this hypothesis, it was found that a higher pH was required to solubilize protoxins from inclusions from the plasmid-cured strain than from B. thuringiensis subsp. aizawai HD133, a difference which is probably attributable to the absence of the CryIA(b) protoxin in the former. The interactions of structurally related protoxins within an inclusion are probably important for solubility and are thus another factor in the effectiveness of B. thuringiensis isolates for particular insect larvae.     

Cloning and partial characterization of zwittermicin A resistance gene cluster from Bacillus thuringiensis subsp. kurstaki strain HD1

AIM: The study seeks to shed light on the aminopolyol, broad-spectrum antibiotic zwittermicin A gene cluster of Bacillus thuringiensis subsp. kurstaki HD1 and to identify any new uncharacterized genes with an eventual goal to establish a better understanding of the resistance gene cluster. METHODS AND RESULTS: We screened 51 serovars of B. thuringiensis by PCR and identified 12 zmaR-positive strains. The zmaR-positive B. thuringiensis subsp. kurstaki HD1 strain displayed inhibition zones against indicator fungal strain Phytophthora meadii and bacterial strain Erwinia herbicola as well as against Rhizopus sp., Xanthomonas campestris and B. thuringiensis subsp. finitimus. The zmaR gene cluster of strain HD1 was partially cloned using a lambda library and was extensively characterized based on the information available from a study performed on a similar group of genes in Bacillus cereus. CONCLUSIONS: Three of the five genes in the zwittermicin gene cluster, including the zmaR gene, had counterparts in B. cereus, and the other two were new members of the B. thuringiensis zmaR gene cluster. SIGNIFICANCE AND IMPACT OF THE STUDY: The two new genes were extensively analysed and the data is presented. Understanding antifungal activity of B. thuringiensis may help us to design suitable Cry toxin delivery agents with antifungal activity as well as enhanced insecticidal activity.     

Plasmid transfer between the Bacillus thuringiensis subspecies kurstaki and tenebrionis in laboratory culture and soil and in lepidopteran and coleopteran larvae

Plasmid transfer between Bacillus thuringiensis subsp. kurstaki HD1 and B. thuringiensis subsp. tenebrionis donor strains and a streptomycin-resistant B. thuringiensis subsp. kurstaki recipient was studied under environmentally relevant laboratory conditions in vitro, in soil, and in insects. Plasmid transfer was detected in vitro at temperatures of 5 to 37 degrees C, at pH 5.9 to 9.0, and at water activities of 0.965 to 0.995, and the highest transfer ratios (up to 10(-1) transconjugant/donor) were detected within 4 h. In contrast, no plasmid transfer was detected in nonsterile soil, and rapid formation of spores by the introduced strains probably contributed most to the lack of plasmid transfer observed. When a B. thuringiensis subsp. kurstaki strain was used as the donor strain, plasmid transfer was detected in killed susceptible lepidopteran insect (Lacanobia oleracea) larvae but not in the nonsusceptible coleopteran insect Phaedon chocleriae. When a B. thuringiensis subsp. tenerbrionis strain was used as the donor strain, no plasmid transfer was detected in either of these insects even when they were killed. These results show that in larger susceptible lepidopteran insects there is a greater opportunity for growth of B. thuringiensis strains, and this finding, combined with decreased competition due to a low initial background bacterial population, can provide suitable conditions for efficient plasmid transfer in the environment.     

Detection and phylogenic analysis of one anthrax virulence plasmid pXO1 conservative open reading frame ubiquitous presented within Bacillus cereus group strains

The presence of one of the anthrax virulence plasmid pXO1 conserved fragments was analyzed in 24 Bacillus cereus and B. thuringiensis strains, including 6 B. thuringiensis subspecies, by polymerase chain reactions. Twelve out of 24 strains showed PCR-positive for an ORF101 homologous sequence. Two pXO1-ORF101-like fragments from a B. cereus B-4ac and a commercial B. thuringiensis kurstaki HD1 were cloned, sequenced and expressed in Escherichia coli. Toxicity assays revealed that the product encoded by the pXO1-ORF101-like fragment had no impact on either Vero cells or Chinese Hamster Ovary cells, suggesting that this fragment probably not contribute to enterotoxic activity. Sequence alignment of the pXO1-ORF101 from three Bacillus anthracis and ORF101-like fragments from other 12 B. cereus group isolates indicated high identity (more than 90%) and the presence of subgroup- and strain-specific SNPs among these fragments.     

Efficacy tests and determination of optimal spray timing values to control nantucket pine tip moth (Lepidoptera: Tortricidae) infestations

The Nantucket pine tip moth, Rhyaciona frustrana (Comstock), a common regeneration pest of loblolly pine, Pinus taeda L., has been shown to reduce tree volume yields through larval feeding. Chemical applications can be effective in protecting trees from the growth losses associated with this feeding, and optimum spray timing values are commonly used to reduce the number of necessary applications and to increase insecticide efficacy. Optimal spray timing values for the Georgia Piedmont were obtained for the following four insecticides available for use in loblolly pine plantations: permethrin (Pounce), lambda-cyhalothrin (Warrior T), spinosad (SpinTor 2 SC), and Bacillus thuringiensis variety kurstaki Berliner (Foray 48B). Optimal timing values were similar between the first and second generations for each of these compounds. All of the insecticides used in this study significantly reduced tip moth damage below the control treatment levels. Lambda-cyhalothrin was the most efficacious and had the longest spray timing window. B. thuringiensis was the least effective and had the shortest timing window. Spinosad and permethrin were similar in efficacy and spray timing values. This information is applicable to regions where there are three tip moth generations per year, as found in the southern Piedmont region and the coastal plain of Virginia and most of North Carolina.     

Insecticidal toxins from Bacillus thuringiensis subsp. kenyae: gene cloning and characterization and comparison with B. thuringiensis subsp. kurstaki CryIA(c) toxins

Genes encoding insecticidal crystal proteins were cloned from three strains of Bacillus thuringiensis subsp. kenyae and two strains of B. thuringiensis subsp. kurstaki. Characterization of the B. thuringiensis subsp. kenyae toxin genes showed that they are most closely related to cryIA(c) from B. thuringiensis subsp. kurstaki. The cloned genes were introduced into Bacillus host strains, and the spectra of insecticidal activities of each Cry protein were determined for six pest lepidopteran insects. CryIA(c) proteins from B. thuringiensis subsp. kenyae are as active as CryIA(c) proteins from B. thuringiensis subsp. kurstaki against Trichoplusia ni, Lymantria dispar, Heliothis zea, and H. virescens but are significantly less active against Plutella xylostella and, in some cases, Ostrinia nubilalis. The sequence of a cryIA(c) gene from B. thuringiensis subsp. kenyae was determined (GenBank M35524) and compared with that of cryIA(c) from B. thuringiensis subsp. kurstaki. The two genes are more than 99% identical and show seven amino acid differences among the predicted sequences of 1,177 amino acids.     

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 (F(4) to F(8)) 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 F(9) 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 F(9). 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 (F(9) to F(13)) to give a resistance ratio of >2,400 compared with UNSEL-MEL. Binding studies with (125)I-labeled Cry1Ab and Cry1Ac revealed complete lack of binding to brush border membrane vesicles prepared from Cry1Ac-selected larvae (F(15)). 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 F(2) progeny from a backcross of F(1) 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.     

Persistence of Bacillus thuringiensis subsp. kurstaki in Urban Environments following Spraying

Bacillus thuringiensis subsp. kurstaki is applied extensively in North America to control the gypsy moth, Lymantria dispar. Since B. thuringiensis subsp. kurstaki shares many physical and biological properties with Bacillus anthracis, it is a reasonable surrogate for biodefense studies. A key question in biodefense is how long a biothreat agent will persist in the environment. There is some information in the literature on the persistence of Bacillus anthracis in laboratories and historical testing areas and for Bacillus thuringiensis in agricultural settings, but there is no information on the persistence of Bacillus spp. in the type of environment that would be encountered in a city or on a military installation. Since it is not feasible to release B. anthracis in a developed area, the controlled release of B. thuringiensis subsp. kurstaki for pest control was used to gain insight into the potential persistence of Bacillus spp. in outdoor urban environments. Persistence was evaluated in two locations: Fairfax County, VA, and Seattle, WA. Environmental samples were collected from multiple matrices and evaluated for the presence of viable B. thuringiensis subsp. kurstaki at times ranging from less than 1 day to 4 years after spraying. Real-time PCR and culture were used for analysis. B. thuringiensis subsp. kurstaki was found to persist in urban environments for at least 4 years. It was most frequently detected in soils and less frequently detected in wipes, grass, foliage, and water. The collective results indicate that certain species of Bacillus may persist for years following their dispersal in urban environments.