Showing papers on "Bacillus thuringiensis published in 1997"

Initial frequency of alleles for resistance to Bacillus thuringiensis toxins in field populations of Heliothis virescens.

Abstract: The risk of rapid pest adaptation to an insecticide is highly dependent on the initial frequency of resistance alleles in field populations. Because we have lacked empirical estimates of these frequencies, population–genetic models of resistance evolution have relied on a wide range of theoretical estimates. The recent commercialization of genetically engineered cotton that constitutively produces an insecticidal protein derived from the biocontrol agent, Bacillus thuringiensis (Bt) has raised concern that we lack data needed to quantify the risk of insect pests such as Heliothis virescens rapidly adapting to this ecologically valuable class of toxins. By individually mating over 2,000 male H. virescens moths collected in four states to females of a Bt toxin-resistant laboratory strain, and screening F1 and F2 offspring for tolerance of the toxic protein, we were able to directly estimate the field frequency of alleles for resistance as 1.5 × 10−3. This high initial frequency underscores the need for caution in deploying transgenic cotton to control insect pests. Our single-pair mating technique greatly increases the efficiency of detecting recessive resistance alleles. Because alleles that decrease target site sensitivity to Bt toxins and other insecticides are often recessive, this technique could be useful in estimating resistance allele frequencies in other insects exposed to transgenic insecticidal crops or conventional insecticides.

Transgenic plants: An emerging approach to pest control

Abstract: Insect pests are a major cause of damage to the world's commercially important agricultural crops. Current strategies aimed at reducing crop losses rely primarily on chemical pesticides. Alternatively transgenic crops with intrinsic pest resistance offer a promising alternative and continue to be developed. The first generation of insect-resistant transgenic plants are based on insecticidal proteins from Bacillus thuringiensis (Bt). A second generation of insect-resistant plants under development include both Bt and non-Bt proteins with novel modes of action and different spectra of activity against insect pests.

Initial frequency of alleles for resistance to Bacillus thuringiensis toxins in field populations of Heliothis virescens (transgenic cropsyinsecticidesyevolution)

Abstract: The risk ofrapid pest adaptation to an insec- ticide is highly dependent on the initial frequency of resistance alleles in field populations. Because we have lacked empirical estimates of these frequencies, population-genetic models of resistance evolution have relied on a wide range of theoretical estimates. The recent commercialization of genetically engi- neeredcottonthatconstitutivelyproducesaninsecticidalprotein derivedfromthebiocontrolagent,Bacillusthuringiensis(Bt)has raised concern that we lack data needed to quantify the risk of insect pests such as Heliothis virescens rapidly adapting to this ecologicallyvaluableclassoftoxins.Byindividuallymatingover 2,000 maleH. virescensmoths collected in four states to females ofaBttoxin-resistantlaboratorystrain,andscreeningF1andF2 offspring for tolerance of the toxic protein, we were able to directly estimate the field frequency of alleles for resistance as 1.5 310 23 .Thishighinitialfrequencyunderscorestheneedfor caution in deploying transgenic cotton to control insect pests. Oursingle-pairmatingtechniquegreatlyincreasestheefficiency of detecting recessive resistance alleles. Because alleles that decreasetargetsitesensitivitytoBttoxinsandotherinsecticides are often recessive, this technique could be useful in estimating resistance allele frequencies in other insects exposed to trans- genic insecticidal crops or conventional insecticides.

Extended Screening by PCR for Seven cry-Group Genes from Field-Collected Strains of Bacillus thuringiensis

Abstract: An extended multiplex PCR method was established to rapidly identify and classify Bacillus thuringiensis strains containing cry (crystal protein) genes toxic to species of Lepidoptera, Coleoptera, and Diptera. The technique enriches current strategies and simplifies the initial stages of large-scale screening of cry genes by pinpointing isolates that contain specific genes or unique combinations of interest with potential insecticidal activities, thus facilitating subsequent toxicity assays. Five pairs of universal primers were designed to probe the highly conserved sequences and classify most (34 of about 60) genes known in the following groups: 20 cry1, 3 cry2, 4 cry3, 2 cry4, 2 cry7, and 3 cry8 genes. The DNA of each positive strain was probed with a set of specific primers designed for 20 of these genes and for cry11A. Twenty-two distinct cry-type profiles were identified from 126 field-collected B. thuringiensis strains. Several of them were found to be different from all published profiles. Some of the field-collected strains, but none of the 16 standard strains, were positive for cry2Ac. Three standard and 38 field-collected strains were positive by universal primers but negative by specific primers for all five known genes of cry7 and cry8. These field-collected strains seem to contain a new gene or genes that seem promising for biological control of insects and management of resistance.

Preimaginal Development, Survival, and Field Abundance of Insect Predators on Transgenic Bacillus thuringiensis Corn

Abstract: Laboratory studies determined the effects of feeding corn, Zea mays L., pollen expressing a Cry1Ab protein derived from Bacillus thuringiensis (Berliner) subsp. kurstaki on 3 predatory species: Coleomegilla maculata DeGeer (Coleoptera: Coccinellidae), Orius insidiosus Say (Heteroptera: Anthocoridae), and Chrysoperla carnea Stephens (Neuroptera: Chrysopidae). No acute detrimental effects of the transgenic B. thuringiensis pollen (Cry1Ab protein) on preimaginal development and survival were observed among these predators. The following percentage survival values (±SE) were observed: C. maculata , 89 ± 2.2% ( B. thuringiensis corn pollen), 69 ± 5.9% (non- B. thuringiensis corn pollen); O. insidiosus , 63 ± 12% ( B. thuringiensis corn pollen), 44 ± 10.2% (non- B. thuringiensis corn pollen); and C. carnea , 49 ± 3.5% ( B. thuringiensis and non- B. thuringiensis corn pollen). No detrimental effects were observed in the abundance of Ostrinia nubilalis (Hubner) (Lepidoptera: Pyralidae) predators (coccinellids, anthocorids, chrysopids) in B. thuringiensis corn compared with non- B. thuringiensis corn during 2 yr of field evaluations. Predator numbers observed before, during, and after pollen shed suggest that B. thuringiensis corn pollen will not affect natural enemy movement in corn. Additional studies are needed to test for chronic and reproductive effects over several generations before concluding that transgenic B. thuringiensis corn pollen has no effect on insect predators.

The Bacillus thuringiensis vegetative insecticidal protein Vip3A lyses midgut epithelium cells of susceptible insects.

Abstract: The Vip3A protein is a member of a newly discovered class of vegetative insecticidal proteins with activity against a broad spectrum of lepidopteran insects. Histopathological observations indicate that Vip3A ingestion by susceptible insects such as the black cutworm (Agrotis ipsilon) and fall armyworm (Spodoptera frugiperda) causes gut paralysis at concentrations as low as 4 ng/cm2 of diet and complete lysis of gut epithelium cells resulting in larval death at concentrations above 40 ng/cm2. The European corn borer (Ostrinia nubilalis), a nonsusceptible insect, does not develop any pathology upon ingesting Vip3A. While proteolytic processing of the Vip3A protein by midgut fluids obtained from susceptible and nonsusceptible insects is comparable, in vivo immunolocalization studies show that Vip3a binding is restricted to gut cells of susceptible insects. Therefore, the insect host range for Vip3A seems to be determined by its ability to bind gut cells. These results indicate that midgut epithelium cells of susceptible insects are the primary target for the Vip3A insecticidal protein and that their subsequent lysis is the primary mechanism of lethality. Disruption of gut cells appears to be the strategy adopted by the most effective insecticidal proteins.

Transgenic elite indica rice plants expressing CryIAc delta-endotoxin of Bacillus thuringiensis are resistant against yellow stem borer (Scirpophaga incertulas).

Abstract: Generation of insect-resistant, transgenic crop plants by expression of the insecticidal crystal protein (ICP) gene of Bacillus thuringiensis (Bt) is a standard crop improvement approach. In such cases, adequate expression of the most appropriate ICP against the target insect pest of the crop species is desirable. It is also considered advantageous to generate Bt-transgenics with multiple toxin systems to control rapid development of pest resistance to the ICP. Larvae of yellow stem borer (YSB), Scirpophaga incertulas, a major lepidopteran insect pest of rice, cause massive losses of rice yield. Studies on insect feeding and on the binding properties of ICP to brush border membrane receptors in the midgut of YSB larvae revealed that cryIAb and cryIAc are two individually suitable candidate genes for developing YSB-resistant rice. Programs were undertaken to develop Bt-transgenic rice with these ICP genes independently in a single cultivar. A cryIAc gene was reconstructed and placed under control of the maize ubiquitin 1 promoter, along with the first intron of the maize ubiquitin 1 gene, and the nos terminator. The gene construct was delivered to embryogenic calli of IR64, an elite indica rice cultivar, using the particle bombardment method. Six highly expressive independent transgenic ICP lines were identified. Molecular analyses and insect-feeding assays of two such lines revealed that the transferred synthetic cryIAc gene was expressed stably in the T2 generation of these lines and that the transgenic rice plants were highly toxic to YSB larvae and lessened the damage caused by their feeding.

One gene in diamondback moth confers resistance to four Bacillus thuringiensis toxins.

Abstract: Environmentally benign insecticides derived from the soil bacterium Bacillus thuringiensis (Bt) are the most widely used biopesticides, but their success will be short-lived if pests quickly adapt to them. The risk of evolution of resistance by pests has increased, because transgenic crops producing insecticidal proteins from Bt are being grown commercially. Efforts to delay resistance with two or more Bt toxins assume that independent mutations are required to counter each toxin. Moreover, it generally is assumed that resistance alleles are rare in susceptible populations. We tested these assumptions by conducting single-pair crosses with diamondback moth (Plutella xylostella), the first insect known to have evolved resistance to Bt in open field populations. An autosomal recessive gene conferred extremely high resistance to four Bt toxins (Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F). The finding that 21% of the individuals from a susceptible strain were heterozygous for the multiple-toxin resistance gene implies that the resistance allele frequency was 10 times higher than the most widely cited estimate of the upper limit for the initial frequency of resistance alleles in susceptible populations. These findings suggest that pests may evolve resistance to some groups of toxins much faster than previously expected.

Global variation in the genetic and biochemical basis of diamondback moth resistance to Bacillus thuringiensis

Abstract: Insecticidal proteins from the soil bacterium Bacillus thuringiensis (Bt) are becoming a cornerstone of ecologically sound pest management. However, if pests quickly adapt, the benefits of environmentally benign Bt toxins in sprays and genetically engineered crops will be short-lived. The diamondback moth (Plutella xylostella) is the first insect to evolve resistance to Bt in open-field populations. Here we report that populations from Hawaii and Pennsylvania share a genetic locus at which a recessive mutation associated with reduced toxin binding confers extremely high resistance to four Bt toxins. In contrast, resistance in a population from the Philippines shows multilocus control, a narrower spectrum, and for some Bt toxins, inheritance that is not recessive and not associated with reduced binding. The observed variation in the genetic and biochemical basis of resistance to Bt, which is unlike patterns documented for some synthetic insecticides, profoundly affects the choice of strategies for combating resistance.

Influence of Exposure to Single versus Multiple Toxins of Bacillus thuringiensis subsp. israelensis on Development of Resistance in the Mosquito Culex quinquefasciatus (Diptera: Culicidae).

Abstract: The impending widespread use of transgenic crop plants encoding a single insecticidal toxin protein of Bacillus thuringiensis has focused attention on the perceived risk of rapid selection of resistance in target insects. We have used Bacillus thuringiensis subsp. israelensis toxins as a model system and determined the speed and magnitude of evolution of resistance in colonies of the mosquito Culex quinquefasciatus during selection for 28 consecutive generations with single or multiple toxins. The parental strain was synthesized by combining approximately 500 larvae from each of 19 field collections obtained from the states of California, Oregon, Louisiana, and Tennessee. At least 10,000 larvae were selected in each generation of each line at an average mortality level of 84%. The susceptibilities of the parental and selected lines were compared in parallel tests in every third generation by using fresh suspensions of toxin powders. The normal toxin complement of B. thuringiensis subsp. israelensis consists of four toxins, CryIVA, CryIVB, CryIVD, and CytA. Resistance became evident first in the line that was selected with a single toxin (CryIVD), attaining the highest level (resistance ratio [RR], >913 at 95% lethal concentration) by generation F(inf28) when the study was completed. Resistance evolved more slowly and to a lower level (RR, >122 by F(inf25)) in the line selected with two toxins (CryIVA+CryIVB) and lower still (RR, 91 by F(inf28)) in the line selected with three toxins (CryIVA+CryIVB+ CryIVD). Resistance was remarkably low (RR, 3.2) in the line selected with all four toxins. The results reveal the importance of the full complement of toxins found in natural populations of B. thuringiensis subsp. israelensis as an effective approach to resistance management.

CytA enables CryIV endotoxins of Bacillus thuringiensis to overcome high levels of CryIV resistance in the mosquito, Culex quinquefasciatus

Abstract: Cry proteins produced by Bacillus thuringiensis are selective biodegradable insecticides used increasingly in bacterial insecticides and transgenic plants as alternatives to synthetic chemical insecticides. However, the potential for development of resistance and cross-resistance in target insect populations to Cry proteins used alone or in combination threatens the more widespread use of this novel pest control technology. Here we show that high levels of resistance to CryIV proteins in larvae of the mosquito, Culex quinquefasciatus, can be suppressed or reduced markedly by combining these proteins with sublethal quantities of CytA, a cytolytic endotoxin of B. thuringiensis. Resistance at the LC95 level of 127-fold for a combination of three CryIV toxins (CryIVA, B, and D), resulting from 60 generations of continuous selection, was completely suppressed by combining sporulated powders of CytA in a 1:3 ratio with sporulated powders of a CryIVA, CryIVB, and CryIVD strain. Combining the CytA strain with a CryIVA and CryIVB strain also completely suppressed mosquito resistance of 217-fold to the latter toxins at the LC95 level, whereas combination of CytA with CryIVD reduced resistance in a CryIVD-selected mosquito strain from greater than 1,000-fold to less than 8-fold. The CytA/CryIV model provides a potential molecular genetic strategy for engineering resistance management for Cry proteins directly into bacterial insecticides and transgenic plants.

Inheritance of Resistance to the Bacillus thuringiensis Toxin Cry1C in the Diamondback Moth

Abstract: Laboratory selection increased resistance to the Bacillus thuringiensis toxin Cry1C in a strain of diamondback moth (Plutella xylostella). The selected strain was derived from a field population that had evolved high levels of resistance to Bacillus thuringiensis subsp. kurstaki and moderate resistance to Cry1C. Relative to the responses of a susceptible strain of diamondback moth, the resistance to Cry1C of the selected strain increased to 62-fold after six generations of selection. The realized heritability of resistance was 0.10. Analysis of F(inf1) hybrid progeny from reciprocal crosses between the selected strain and a susceptible strain showed that resistance to Cry1C was autosomally inherited. The dominance of resistance to Cry1C depended on the concentration; inheritance was increasingly dominant as the concentration decreased. Responses of progeny from single-pair families showed that resistance to Cry1C and resistance to Cry1Ab were inherited independently, which enhances opportunities for managing resistance. However, compared with projections based on previously reported recessive inheritance of resistance to Cry1A toxins, the potentially dominant inheritance of resistance to Cry1C observed here could accelerate evolution of resistance.

Microbial Utilization of Free and Clay-Bound Insecticidal Toxins from Bacillus thuringiensis and Their Retention of Insecticidal Activity after Incubation with Microbes.

Abstract: The insecticidal toxins produced by Bacillus thuringiensis subspp. kurstaki and tenebrionis were resistant when bound on clays, but not when free, to utilization by pure and mixed cultures of microbes as sources of carbon and carbon plus nitrogen, and their availability as a nitrogen source was reduced. The bound toxins retained insecticidal activity both before and after exposure to microbes or pronase. The insecticidal activity of the toxins persisted for 40 days (the longest time evaluated) in nonsterile soil continuously maintained at the -33-kPa water tension and room temperature, alternately air dried and rewetted to the -33-kPa water tension, or alternately frozen and thawed, although alternate drying and wetting reduced the activity.

Transgenic sugarcane plants resistant to stem borer attack

Abstract: A truncated cryIA(b) gene encoding the active region of the Bacillus thuringiensis δ-endotoxin was expressed in transgenic sugarcane plants (Saccharum officinarum L.) under the control of the CaMV 35S promoter. Genetic transformation was accomplished by electroporation of intact cells. The levels of recombinant toxin were established and biological activity tests were performed against neonate sugarcane borer (Diatraea saccharalis F.) larvae. Transgenic sugarcane plants showed significant larvicidal activity despite the low expression of CryIA(b).

Inheritance, Stability, and Lack-of-Fitness Costs of Field-Selected Resistance to Bacillus thuringiensis in Diamondback Moth (Lepidoptera: Plutellidae) from Florida

Abstract: A colony of Plutella xylostella (L.), established from crucifer fields in Florida, was used to investigate resistance to Bacillus thuringiensis Berliner subsp. kurstaki . From an initial level of >1,500-fold, resistance fell within 3 generations in the absence of selection to ≈300-fold compared with susceptible larvae. Unlike previous cases of resistance to B. thuringiensis in P. xylostella , resistance in our Florida colony was stable at ≈300-fold without additional selection in the laboratory. High levels of resistance (>1,000-fold) recurred after a single exposure to B. thuringiensis subsp. kurstaki in the 4th generation. High levels of resistance did not recur after a 2nd selection in the 8th generation. Cage studies and genetic analysis of F1 larvae and backcross progeny. where the resistant parents were characterized by stable levels of resistance, showed that resistance was an incompletely recessive, autosomal trait probably controlled by a single allele that did not confer detectable levels of reduced fitness in the absence of exposure to B. thuringiensis . As one of the few shldies to demonstrate stable resistance to B. thuringiensis subsp. kurstaki from insects that were collected from the field and not subject to further selection in the laboratory, our results clearly emphasize the need to develop specific resistance management strategies for B. thuringiensis before there is widespread evolution of resistance.

Cloning of novel enterotoxin genes from Bacillus cereus and Bacillus thuringiensis.

Abstract: A novel enterotoxin gene was cloned from Bacillus cereus FM1, and its nucleotide sequence was determined. Previously, a 45-kDa protein causing characteristic enterotoxin symptoms in higher animals had been isolated (K. Shinagawa, p. 181-193, in A. E. Pohland et al., ed., Microbial Toxins in Foods and Feeds, 1990) from the same B. cereus strain, but no report of cloning of the enterotoxin gene has been published. In the present study, a specific antibody to the purified enterotoxin was produced and used to screen the genomic library of B. cereus FM1 made with the lambda gt11 vector. An immunologically positive clone was found to contain the full protein-coding region and some 5' and 3' flanking regions. The deduced amino acid sequence of the cloned gene indicated that the protein is rich in beta structures and contains some unusual sequences, such as consecutive Asn residues. In order to clone enterotoxin genes from Bacillus thuringiensis, two PCR primers were synthesized based on the nucleotide sequence of the B. cereus gene. These primers were designed to amplify the full protein-coding region. PCR conducted with DNA preparations from the B. thuringiensis subsp. sotto and B. thuringiensis subsp. israelensis strains successfully amplified a segment of DNA with a size almost identical to that of the protein-coding region of the B. cereus enterotoxin. Nucleotide sequences of the amplified DNA segments showed that these B. thuringiensis strains contain an enterotoxin gene very similar to that of B. cereus. Further PCR screening of additional B. thuringiensis strains with four primer pairs in one reaction revealed that some additional B. thuringiensis strains contain enterotoxin-like genes.

Microorganism and insecticide

Abstract: Bacillus thuringiensis serovar japonensis strain Buibui (FERM BP-3465) belonging to Bacillus thuringiensis serovar japonensis and capable of producing insecticidal toxin proteins to kill coleopterous larvae, and an insecticide containing, as an effective ingredient, the toxin proteins produced.

Phylogenetic relationships of Bacillus thuringiensis delta-endotoxin family proteins and their functional domains.

Abstract: Insecticidal crystal proteins (ICPs) from Bacillus thuringiensis have been used as biopesticides for the last 35 years. B. thuringiensis is a gram-positive bacterium which produces proteinaceous inclusions during sporulation; these inclusions can be distinguished as distinctively shaped crystals by phase-contrast microscopy. The inclusions are composed of proteins known as ICPs, Cry proteins, or d-endotoxins, which are highly toxic to a wide variety of important agricultural and healthrelated insect pests as well as other invertebrates. Due to their high specificity and their safety for the environment, ICPs are a valuable alternative to chemical pesticides for control of insect pests in agriculture and forestry and in the home. It has been proposed that the rational use of B. thuringiensis toxins will provide a variety of alternatives for insect control and for coping with the problem of insect resistance to pesticides. Intensive screening programs have identified strains of B. thuringiensis from soil samples, plant surfaces, dead insects, and stored grains from all over the world. The isolated strains show a wide range of specificity against different insect orders (Lepidoptera, Diptera, Coleoptera, Hymenoptera, Homoptera, Phthiraptera or Mallophaga, and Acari) and other invertebrates (Nemathelminthes, Platyhelminthes, and Sarcomastigorphora) (13). Currently 45 different serotypes have been catalogued, representing a total number of 58 serovars (28). Many of the ICP genes have been cloned, sequenced, and classified as cry and cty genes. The first classification was based on insecticidal activity (23), with the different Cry proteins denoting ICPs toxic to various insect and invertebrate groups as follows: CryI toxic to lepidopterans, CryII toxic to lepidopterans and dipterans, CryIII toxic to coleopterans, CryIV toxic to dipterans, and CryV and CryVI toxic to nematodes (14). Novel cry genes isolated recently have created some problems for this classification scheme, especially genes that were homologous to known genes but displayed different specificities and genes that had dual specificity. Recently, a novel nomenclature has been proposed based exclusively on amino acid identity (7). To date, over 50 cry gene sequences have been determined and classified into 15 families (7). The cry genes code for proteins with a range of molecular masses from 50 to 140 kDa. Upon ingestion by the susceptible target, the protoxins are solubilized and proteolytically processed to release the toxic fragment (23). During proteolytic activation, peptides are removed from both aminoand carboxyl-terminal ends of the protoxin. For the 130to 140-kDa protoxins, the carboxyl-terminal proteolytic activation removes half of the molecule, resulting in an active toxin fragment of 60 to 70 kDa. A generally accepted model for Cry toxin action is that it is a multistage process. First, the activated toxin binds to receptors located on the apical microvillus membrane of epithelial midgut cells (6, 22, 49). After the toxin binds the receptor, it is thought that there is a change in the toxin conformation allowing toxin insertion into the membrane. Oligomerization of the toxin follows, and this oligomer then forms a pore that leads to osmotic cell lysis (26a, 30, 32, 40). Receptor binding is a key factor in specificity. Two different insect proteins have been identified as receptors for Cry toxins, the 120-kDa aminopeptidase N Cry1Ac toxin-binding protein purified from brush border vesicles of Manduca sexta, Heliothis virescens, and Lymantria dispar (20, 25, 41, 48) and the 210-kDa cadherin-like glycoprotein Cry1Ab toxin-binding protein purified from M. sexta membranes (47). Specific binding involves two steps, one that is reversible and one that is irreversible. Recent data suggest that toxicity correlates with irreversible binding (31). Irreversible binding might be related to insertion of the toxin into the membrane but could also reflect a tighter interaction of the toxin with the receptor. The crystal structures of Cry3A (coleopteran-specific) and Cry1Aa (lepidopteran-specific) toxins have been reported (21, 30). The Cry3A protoxin has a molecular mass of 70 kDa and does not contain the large carboxyl-terminal extension contained in the Cry1Aa toxin. The crystal structure of Cry1Aa toxin was determined from the activated toxin fragment. Both toxins share 36% amino acid identity, and the two structures show high overall similarity (21). Both are globular molecules containing three distinct domains connected by single linkers. Domain I extends from residues 33 to 253 in Cry1Aa and from residues 58 to 290 in Cry3A; it is a seven a-helix bundle in which a central helix (helix a-5) is completely surrounded by six outer helices (Fig. 1A). This domain has been implicated in the channel formation in the membrane. The six a-helices are amphipathic and are long enough to span the 30-A-thick hydrophobic region of a membrane bilayer. Point mutations in the region encoding the central a-5 helix of the Cry1Ac toxin (residues 163 to 170) drastically affect toxicity without affecting binding to larval midgut vesicles (52). Residues 265 to 461 in Cry1Aa and 291 to 500 in Cry3A form domain II (Fig. 1B). Domain II consists of three antiparallel b-sheets with similar topologies packed around a hydrophobic core. This domain represents the most divergent part in structure between the two toxin molecules (21). This domain has been described as the specificity-determining domain, since reciprocal hybrid genes between closely related toxins * Mailing address: Department of Microbiology, Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, Apdo. Postal 510-3, Cuernavaca, Morelos, Mexico. Phone: (52) (5) 622-7635. Fax: (52) (73) 17-2388. E-mail: bravo@ibt.unam.mx.

Expression of a Bacillus thuringiensis cryIA(c) gene in transgenic peanut plants and its efficacy against lesser cornstalk borer.

Abstract: The invasion of peanut (Arachis hypogaea L.) pods and seeds by aflatoxin-forming species of Aspergillus is linked to injury by the lesser cornstalk borer and frequently causes a severe reduction in crop quality. The lesser cornstalk borer is susceptible to the lepidopteran-active Bacillus thuringiensis insecticidal crystal protein. We have introduced a codon-modified Bacillus thuringiensis cryIA(c) gene into peanut using microprojectile bombardment. The toxin-coding region of a Bt cryIA(c) gene was reconstructed for expression in plants and the resulting 3.4 kb gene cassette (promoter: 1.8 kb coding: 3') was directly cloned into the BglII site of plant transformation vectors. The vectors contained the hph gene, conferring resistance to the antibiotic hygromycin. Somatic embryos initiated from immature peanut cotyledons of two cultivars were used as the target for bombardment. DNA from hygromycin-resistant embryogenic cell lines, regenerated plants, and a progeny plant showed the presence and integration of hph and Bt genes by PCR and/or Southern blot analyses. ELISA immunoassay of the CryIA(c) protein from the hygromycin-selected plants showed the expression of CryIA(c) protein up to 0.18% of total soluble protein. Insect feeding bioassay of transformed plants indicated various levels of resistance to the lesser cornstalk borer, from complete larval mortality to a 66% reduction in larval weight. A negative correlation between percent survival or larval weight and the amount of Bt CryIA(c) protein was recorded indicating in general that the higher the protein level the lower the survival or larval weight of the insect. Based on leaf bioassay, transformation of peanut with vectors containing the Bt cryIA(c) gene may be effective in protecting the peanut plants from damage by lepidopteran insect larvae of lesser cornstalk borer.

Increased fitness of transgenic insecticidal rapeseed under insect selection pressure

Abstract: Rapeseed Brassica napus L. transgenic for a Bacillus thuringiensis (Bt) transgene was developed and was shown to be insecticidal towards certain caterpillars including the diamondback moth Plutella xylostella L. and the corn earworm Helicoverpa zea Boddie. To simulate an escape of the transgenics from cultivation, a field experiment was performed in which transgenic and nontransgenic rapeseed plants were planted in natural vegetation and cultivated plots and subjected to various selection pressures in the form of herbivory from insects. Only two plants, both transgenic, survived the winter to reproduce in the natural-vegetation plots which were dominated by grasses such as crabgrass. However, in plots that were initially cultivated then allowed to naturalize, medium to high levels of defoliation decreased survivorship of nontransgenic plants relative to Bt-transgenic plants and increased differential reproduction in favour of Bt plants. Thus, where suitable habitat is readily available, there is a likelihood of enhanced ecological risk associated with the release of certain transgene/crop combinations such as insecticidal rapeseed. This is the first report of a field study demonstrating the effect of a fitness-increasing transgene in plants.

Effects of Bacillus thuringiensis toxins in transgenic cotton and potato on Folsomia candida (Collembola: Isotomidae) and Oppia nitens (Acari: Orbatidae)

Abstract: Methods are reported for testing the effects of toxins or small molecules [ BaCillus thuringiensis ssp. kurstaki delta-endotoxins HD-1 CryIA (b) and HD-73 CryIA (c)] in plants (cotton and potato) on 2 nontarget soil arthropods, a collembolan, Folsomia candida Willem, and an orbatid mite, Oppia nitens Koch. Time to oviposition. egg production, and final body length were unaffected when F. candida were fed residues of transgenic cotton lines #81 or #249. Total production of O. nitens adults and nymphs was unaffected by feeding on leaves of both transgenic cotton lines. With B. thuringiensis ssp. tenebrionis CryIIIA in potato, no differences were seen in the 3 indices for F. candida . Overall, transgenic toxin sources had no negative effects, but similar tests with cadmium as a positive control showed a concentration-rate response of decreased reproduction.

Soil inactivation of the Bacillus thuringiensis subsp. kurstaki CryIIA insecticidal protein within transgenic cotton tissue : Laboratory microcosm and field studies

Abstract: The environmental fate of Bacillus thuringiensis subsp. kurstaki CryIIA insecticidal protein expressed within transgenic cotton plant tissue (= Bt-cotton) was evaluated by determining reduction in the biological activity of the protein incubated in soil for 120 days. Studies were conducted in a laboratory microcosm and under field conditions during the fall and winter of 1995−1996 in St. Louis, MO. An insect bioassay, based on growth inhibition of larval Heliothis virescens (F.), was used to estimate DT50 values (50% dissipation time = “half-life” of bioactivity) of the CryIIA protein. DT50 values were 15.5 and 31.7 days for the laboratory and field, respectively. The percentages of initial CryIIA protein bioactivity remaining after 40 days of incubation were similar for the laboratory and field samples. In both environments, <25% of the initial bioactivity remained after 120 days. These results indicate that the biological activity of CryIIA protein, as a component of postharvest Bt-cotton plants, readil...

Characterization of Bacillus thuringiensis isolated from infections in burn wounds

Abstract: Four strains of Bacillus thuringiensis were isolated from infections in burn wounds and from water used in the treatment of burn wounds. The strains produced large parasporal inclusion bodies composed of 141, 83, and 81 kDa protoxins. The four strains were tested for insecticidal activity against larvae of Pieris brassicae and Aedes aegypti but showed no activity; Vero cell assays for the production of enterotoxins were also negative. Attempts to classify the strains according to flagellar H-serotype showed them all to be non-flagellated. Apart from two occupational health accidents that occurred during the handling of highly concentrated B. thuringiensis fluids, this is the first report of B. thuringiensis causing non-gastrointestinal clinical infection in immunosuppressed patients.

Experimental evidence that refuges delay insect adaptation to Bacillus thuringiensis

Abstract: Theoretical projections suggest that refuges from exposure can delay insect adaptation to environmentally benign insecticides derived from Bacillus thuringiensis, but experimental tests of this approach have been limited. We tested the refuge tactic by selecting two sets of two colonies of diamondback moth (Plutella xylostella) for resistance to B. thuringiensis subsp. aizawai in the laboratory. In each set, one colony was selected with no refuge and the other with a 10 per cent refuge from exposure to B. thuringiensis subsp. aizawai. Bioassays conducted after nine selections were completed show that mortality caused by B. thuringiensis subsp. aizawai was significantly greater in the refuge colonies than in the no-refuge colonies. These results demonstrate that the refuges delayed the evolution of resistance. Relative to a susceptible colony, final resistance ratios were 19 and 8 for the two no-refuge colonies compared to 6 and 5 for the refuge colonies. The mean realized heritability of resistance to B. thuringiensis subsp. aizawai was 0.046 for colonies without refuges, and -0.002 for colonies with refuges. Selection with B. thuringiensis subsp. aizawai decreased susceptibility to B. thuringiensis toxin Cry1Ab, but not to Cry1C or B. thuringiensis subsp. kurstaki. Although the ultimate test of refuges will occur in the field, the experimental evidence reported here confirms modelling results indicating that refuges can slow the evolution of insect resistance to B. thuringiensis.

Resistance of Plutella xylostella (Lepidoptera: Plutellidae) to Bacillus thuringiensis Berliner in Central America

Abstract: Eighteen field populations of Plutella xylostella (L.) from Central America were tested for susceptibility to Bacillus thuringiensis subsp. kurstaki Berliner, and 7 of the 18 field populations were tested for susceptibility to B. thuringiensis subsp. aizawai. Tests with B. thuringiensis subsp. kurstaki were done with a leaf-dip bioassay and a single concentration (20.5 mg [Al]/liter) incorporated in diet. Only leaf-dip bioassays were used in tests with B. thuringiensis subsp. aizawai. The LC 50 s of B. thuringiensis subsp. kurstaki in the field populations from Guatemala, Honduras, and Costa Rica ranged from 4.3- to 18.3-, 9.3- to 77.2-, and 13.3- to 19.5-fold higher, respectively, than the LC 50 of a susceptible population from New York (Geneva 88). LC 50 s of B. thuringienis subsp. aizawai in the 7 field populations were 1.9- to 3.3-fold higher than the LC 50 of Geneva 88. Mortality in the single concentration test varied significantly among populations within countries, and 14 field populations displayed significantly lower mortality than Geneva 88. Results from both bioassay techniques suggest that populations of P. xylostella from Central America have evolved resistance to B. thuringiensis subsp. kurstaki. Monitoring populations of P. xylostella for baseline and changing levels of susceptibility to B. thuringiensis subspecies and the implementation of pesticide resistance management strategies are urgently needed in Central America.

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

Abstract: 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.

Production of transgenic eggplant (Solanum melongena L.) resistant to Colorado Potato Beetle (Leptinotarsa decemlineata Say)

Abstract: A modified gene of Bacillus thuringiensis var. Tolworthi (Bt), encoding a coleopteran insect-specific CryIIIB toxin, was transferred via Agrobacterium tumefaciens to the female parent of the eggplant commercial F1 hybrid ‘Rimina’. One-hundred and fifty eight transgenic plants were regenerated and tested by PCR and NPTII expression assays. The presence of the CryIIIB toxin in leaf extracts was demonstrated in 57 out of 93 transgenic plants tested by DAS-ELISA assay. High Bt-expressing plants contained a 74-kDa protein cross-reacting with serum anti-CryIIIB toxin. Twenty three out of 44 S. melongena plants tested by insect bioassay showed significant insecticidial activity on neonate larvae of Colorado Potato Beetle (CPB). The Bt transgene and the toxic effect on CPB larvae were transmitted to progenies derived by selfing. Thus, transgenic Bt eggplants represent a very effective means of CPB pest control.

Identification, isolation, culture and preservation of entomopathogenic bacteria

Abstract: Publisher Summary This chapter discusses identification, isolation, culture and preservation of entomopathogenic bacteria. Entomopathogenic bacteria are found among the Gracilicutes, and Firmicutes divisions within the kingdom Procaryotae. It is found that although several bacterial genera are able to produce endospores, the genus Bacillus is recognized by being rod-shaped, usually Gram-stain positive, producing catalase and being aerobic or facultatively anaerobic. Bacillus cells produce an endospore on completion of growth. The shape of the spores and the bacterial cells may be modified if the bacteria are grown under less than optimum growth conditions. Heat treatment of environmental samples and aerobic incubation will allow selection of Bacillus from global bacterial flora. Urea can be degraded by bacteria in alkaline ammonium carbonate which changes the indicator color. Sensitivity of bacteria to certain bacterial viruses called phages allows classification of Bacillus thuringiensis and Bacillus sphaericus according to this susceptibility. B. thuringiensis and B. sphaericus are soil bacteria, but they are also abundant in insects and, being cosmopolitan, can be found in any biotope. It is found that freeze drying is considered the most efficient for long-term storage and conservation of strain characteristics.