Showing papers on "Bacillus thuringiensis published in 1999"
TL;DR: In a laboratory assay, it is found that larvae of the monarch butterfly, Danaus plexippus, reared on milkweed leaves dusted with pollen from Bt corn, ate less, grew more slowly and suffered higher mortality than larvae rearing on leaves dusting with untransformed corn pollen or on leaves without pollen.
Abstract: Although plants transformed with genetic material from the bacterium Bacillus thuringiensis (Bt ) are generally thought to have negligible impact on non-target organisms1, Bt corn plants might represent a risk because most hybrids express the Bt toxin in pollen2, and corn pollen is dispersed over at least 60 metres by wind3. Corn pollen is deposited on other plants near corn fields and can be ingested by the non-target organisms that consume these plants. In a laboratory assay we found that larvae of the monarch butterfly, Danaus plexippus, reared on milkweed leaves dusted with pollen from Bt corn, ate less, grew more slowly and suffered higher mortality than larvae reared on leaves dusted with untransformed corn pollen or on leaves without pollen.
1,148 citations
TL;DR: It is found that a resistant strain of larvae on Bt cotton takes longer to develop than susceptible larvae on non-Bt cotton, and this developmental asynchrony favours non-random mating that could reduce the expected benefits of the refuge strategy.
Abstract: Crop plants genetically engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) are being grown on millions of hectares, but their success will be short-lived if pests adapt to them quickly1,2. The primary strategy for delaying insect resistance to transgenic Bt plants is to provide refuges of host plants that do not produce Bt toxins. This potentially delays the development of insect resistance to Bt crops by providing susceptible insects for mating with resistant insects. But our laboratory results with a worldwide pest of cotton, pink bollworm moths (Pectinophora gossypiella)3, contradict an important assumption of the refuge strategy. We find that a resistant strain of larvae on Bt cotton takes longer to develop than susceptible larvae on non-Bt cotton. This developmental asynchrony favours non-random mating that could reduce the expected benefits of the refuge strategy.
430 citations
TL;DR: It is shown that Bt toxin is released into the rhizosphere soil in root exudates from Bt corn.
Abstract: Bt corn is corn (Zea mays) that has been genetically modified to express insecticidal toxins derived from the bacterium Bacillus thuringiensis to kill lepidopteran pests feeding on these plants. Here we show that Bt toxin is released into the rhizosphere soil in root exudates from Bt corn.
407 citations
TL;DR: The results suggest that plants expressing high levels of a nonhomologous Bt protein should be able to overcome or at the very least, significantly delay, broad spectrum Bt-resistance development in the field.
Abstract: Evolving levels of resistance in insects to the bioinsecticide Bacillus thuringiensis (Bt) can be dramatically reduced through the genetic engineering of chloroplasts in plants. When transgenic tobacco leaves expressing Cry2Aa2 protoxin in chloroplasts were fed to susceptible, Cry1A-resistant (20,000- to 40,000-fold) and Cry2Aa2-resistant (330- to 393-fold) tobacco budworm Heliothis virescens, cotton bollworm Helicoverpa zea, and the beet armyworm Spodoptera exigua, 100% mortality was observed against all insect species and strains. Cry2Aa2 was chosen for this study because of its toxicity to many economically important insect pests, relatively low levels of cross-resistance against Cry1A-resistant insects, and its expression as a protoxin instead of a toxin because of its relatively small size (65 kDa). Southern blot analysis confirmed stable integration of cry2Aa2 into all of the chloroplast genomes (5, 000-10,000 copies per cell) of transgenic plants. Transformed tobacco leaves expressed Cry2Aa2 protoxin at levels between 2% and 3% of total soluble protein, 20- to 30-fold higher levels than current commercial nuclear transgenic plants. These results suggest that plants expressing high levels of a nonhomologous Bt protein should be able to overcome or at the very least, significantly delay, broad spectrum Bt-resistance development in the field.
336 citations
TL;DR: Several extracellular virulence factor genes are identified by the virtue of their PlcR‐regulated expression, which encode degradative enzymes, cell‐surface proteins and enterotoxins in pathogenic Bacillus cereus group.
Abstract: Members of the Bacillus cereus group (B. anthracis, B. cereus, B. mycoides and B. thuringiensis) are well-known pathogens of mammals (B. anthracis and B. cereus) and insects (B. thuringiensis). The specific diseases they cause depend on their capacity to produce specific virulence factors, such as the lethal toxin of B. anthracis and the Cry toxins of B. thuringiensis. However, these Bacillus spp. also produce a variety of proteins, such as phospholipases C, which are known to act as virulence factors in various pathogenic bacteria. Few genes encoding these virulence factors have been characterized in pathogenic Bacillus spp. and little is known about the regulation of their expression. We had previously reported that in B. thuringiensis expression of the phosphatidylinositol-specific phospholipase C gene is regulated by the transcriptional activator PlcR. Here we report the identification of several extracellular virulence factor genes by the virtue of their PlcR-regulated expression. These PlcR-regulated genes encode degradative enzymes, cell-surface proteins and enterotoxins. The PlcR-regulated genes are widely dispersed on the chromosome and therefore do not constitute a pathogenic island. Analysis of the promoter region of the PlcR-regulated genes revealed the presence of a highly conserved palindromic region (TATGNAN4TNCATA), which is presumably the specific recognition target for PlcR activation. We found that the plcR gene is also present in and probably restricted to all the members of the B. cereus group. However, although the polypeptide encoded by the B. cereus PlcR gene is functionally equivalent to the B. thuringiensis regulator, the polypeptide encoded by the B. anthracis gene is truncated and not active as a transcriptional activator. PlcR is the first example described of a pleiotropic regulator involved in the control of extracellular virulence factor expression in pathogenic Bacillus spp. These results have implications for the taxonomic relationships among members of the B. cereus group, the virulence properties of these bacteria and the safety of B. thuringiensis-based biopesticides.
334 citations
TL;DR: A crystal analysis was carried out in terms of morphology, δ‐endotoxin profiles and larvicidal activity for the newly identified serovars and it was found that atypical crystals, some with novel components, are becoming more common.
Abstract: The classification of Bacillus thuringiensis strains has been revised and updated based on flagellar antigens which have been in use for many years. Sixty-nine serotypes and 13 sub-antigenic groups have now been identified, giving 82 serovars among the 3500 B. thuringiensis isolates of the IEBC Collection. The number of serovars has gradually increased with the total number of strains. The biochemical characters used have also been investigated and their value assessed for identification of B. thuringiensis at the subspecies level. A crystal analysis was carried out in terms of morphology, δ-endotoxin profiles and larvicidal activity for the newly identified serovars. It was found that atypical crystals, some with novel components, are becoming more common. No insect susceptible to these serovars has been discovered among known target species. The number of cross-reacting H-antigens among B. cereus strains is increasing and may be of biological significance.
221 citations
TL;DR: Results indicated that field-collected colonies of H. virescens, H. zea, P. includens, and S. frugiperda were as susceptible as laboratory-reared colonies and those reported in the literature to the purified endotoxin proteins Cry1Ac and Cry1Ab.
Abstract: Susceptibility of Heliothis virescens (F.), Helicoverpa zea (Boddie), Pseudoplusia includens (Walker), Spodoptera exigua (Hubner), and Spodoptera frugiperda (J. E. Smith) to purified endotoxins and commercial formulations of Bacillus thuringiensis Berliner was measured in a wide range of colonies collected from 8 states in the U.S. Cotton Belt during 1992 and 1993. Results indicated that field-collected colonies of H. virescens, H. zea, P. includens, and S. frugiperda were as susceptible as laboratory-reared colonies and those reported in the literature to the purified endotoxin proteins Cry1Ac and Cry1Ab or the commercial formulations Javelin WG, Dipel ES, and Condor OF in diet-treated assays. Colonies of S. exigua collected from transgenic cotton expressing endotoxin protein had elevated median lethal concentrations (LC50s) compared with a colony collected from nontransgenic cotton or those from laboratory colonies. Ranges of LC50s for field-collected colonies of H. virescens generally were similar to the ranges observed for laboratory colonies and similar to those reported in the literature. Wider ranges of variation in LC50s were observed among populations of H. zea and S. exigua than among populations of the other species. However, the highest LC50 observed for H. zea was no higher than those reported in the literature. Only a few colonies of P. includens and S. exigua were tested. P. includens susceptibility was generally greater than that of H. virescens and less than that of H. zea. S. frugiperda was the least susceptible species studied. Variability in LC50s obtained with Cry1Ac ( r = 0.702) correlated with variability in LC50s for Cry1Ab across 13 colonies of H. virescens exposed to both proteins. Colonies of H. zea and H. virescens were pooled into single colonies and selected with the insecticidal proteins to produce endotoxin-resistant strains. Selection for resistance in H. virescens was not successful, but a strain of H. zea was selected that had elevated LC50s (10 times) after 2 generations and very high LC50s compared with a susceptible laboratory strain (100 times) after 8 generations, suggesting that H. zea has the genetic capacity to develop resistance to endotoxin proteins.
212 citations
TL;DR: The picture is not altogether positive - there is concern that the introduction of transgenic crops that are engineered to express a Bacillus thuringiensis toxin that confers resistance to insect predation was premature or should not have happened at all, and that the valuable insecticidal properties of Bacillian will be lost.
202 citations
TL;DR: The bacterium Bacillus thuringiensis is currently the source of insecticidal proteins in commercial insect-resistant transgenic plants and will remain the most important source during the next decade.
Abstract: : Insect-resistant transgenic plants have become an important tool for the protection of crops against insect pests. The acreage of insecticidal transgenic plants is expected to increase significantly in the near future. The bacterium Bacillus thuringiensis is currently the source of insecticidal proteins in commercial insect-resistant transgenic plants and will remain the most important source during the next decade. Insect resistance to B. thuringiensis Cry toxins is the main problem. Only one species, the diamondback moth, has evolved a resistance to B. thuringiensis-based formulations under field conditions. However, many other insect species were selected for resistance under laboratory conditions, indicating that there is a potential for evolution of resistance in most major pests. Many studies were conducted to elucidate the mode of action of the Cry toxins, the mechanisms and genetics of resistance, and the various factors influencing its development. This article reviews insect resistance...
199 citations
TL;DR: If field resistance turns out to be similar to this laboratory resistance, the usefulness of the high-dose/refuge strategy for resistance management in Bt maize may be diminished.
Abstract: Resistance in the European corn borer, Ostrinia nubilalis (Hubner), to a commercial formulation of Bacillus thuringiensis (Bt) Berliner toxin, Dipel ES, appears to be inherited as an incompletely dominant autosomal gene. This contrasts with the inheritance of resistance to Bt in other insects, where it has usually been characterized as a recessive trait. The proposed high-dose/refuge strategy for resistance management in Bt maize depends on resistance being recessive or partially recessive. If field resistance turns out to be similar to this laboratory resistance, the usefulness of the high-dose/refuge strategy for resistance management in Bt maize may be diminished.
198 citations
TL;DR: The hemolytic enterotoxin HBL seems to be broadly distributed among strains of the B. cereus group and relates neither to a certain species nor to a specific environment, and the consequences for food safety considerations need to be evaluated.
Abstract: The prevalence of the hemolytic enterotoxin complex HBL was determined in all species of the Bacillus cereus group with the exception of Bacillus anthracis. hblA, encoding the binding subunit B, was detected by PCR and Southern analysis and was confirmed by partial sequencing of 18 strains. The sequences formed two clusters, one including B. cereus and Bacillus thuringiensis strains and the other one consisting of Bacillus mycoides, Bacillus pseudomycoides, and Bacillus weihenstephanensis strains. From eight B. thuringiensis strains, the enterotoxin gene hblA could be amplified. Seven of them also expressed the complete HBL complex as determined with specific antibodies against the L(1), L(2), and B components. Eleven of 16 B. mycoides strains, all 3 B. pseudomyoides strains, 9 of 15 B. weihenstephanensis strains, and 10 of 23 B. cereus strains carried hblA. While HBL was not expressed in the B. pseudomycoides strains, the molecular assays were in accordance with the immunological assays for the majority of the remaining strains. In summary, the hemolytic enterotoxin HBL seems to be broadly distributed among strains of the B. cereus group and relates neither to a certain species nor to a specific environment. The consequences of this finding for food safety considerations need to be evaluated.
TL;DR: A procedure was developed and tested for the specific detection of the target organism in boiled rice that entailed 15 h of preenrichment followed by PCR amplification of the B. cereus-specific fragment and correlated well with results obtained with the 16S rDNA-based hybridization study but not with the results of their phenotypic characterization.
Abstract: As 16S rRNA sequence analysis has proven inadequate for the differentiation of Bacillus cereus from closely related species, we employed the gyrase B gene (gyrB) as a molecular diagnostic marker. The gyrB genes of B. cereus JCM 2152T, Bacillus thuringiensis IAM 12077T, Bacillus mycoides ATCC 6462T, and Bacillus anthracis Pasteur #2H were cloned and sequenced. Oligonucleotide PCR primer sets were designed from within gyrB sequences of the respective bacteria for the specific amplification and differentiation of B. cereus, B. thuringiensis, and B. anthracis. The results from the amplification of gyrB sequences correlated well with results obtained with the 16S rDNA-based hybridization study but not with the results of their phenotypic characterization. Some of the reference strains of both B. cereus (three serovars) and B. thuringiensis (two serovars) were not positive in PCR amplification assays with gyrB primers. However, complete sequencing of 1.2-kb gyrB fragments of these reference strains showed that these serovars had, in fact, lower homology than their originally designated species. We developed and tested a procedure for the specific detection of the target organism in boiled rice that entailed 15 h of preenrichment followed by PCR amplification of the B. cereus-specific fragment. This method enabled us to detect an initial inoculum of 0.24 CFU of B. cereus cells per g of boiled rice food homogenate without extracting DNA. However, a simple two-step filtration step is required to remove PCR inhibitory substances.
TL;DR: The proteins from 84‐HS‐1‐11 and 89‐T‐26‐17 were able to discriminate between leukaemia and normal T cells, specifically killing the former cells, and may lead to the use of B. thuringiensis inclusion proteins for medical purposes.
Abstract: Parasporal inclusion proteins from a total of 1744 Bacillus thuringiensis strains, consisting of 1700 Japanese isolates and 44 reference type strains of existing H serovars, were screened for cytocidal activity against human leukaemia T cells and haemolytic activity against sheep erythrocytes. Of 1684 B. thuringiensis strains having no haemolytic activity, 42 exhibited in vitro cytotoxicity against leukaemia T cells. These non-haemolytic but leukaemia cell-toxic strains belonged to several H-serovars including dakota, neoleonensis, shandongiensis, coreanensis and other unidentified serogroups. Purified parasporal inclusions of the three selected strains, designated 84-HS-1-11, 89-T-26-17 and 90-F-45-14, exhibited no haemolytic activity and no insecticidal activity against dipteran and lepidopteran insects, but were highly cytocidal against leukaemia T cells and other human cancer cells, showing different toxicity spectra and varied activity levels. Furthermore, the proteins from 84-HS-1-11 and 89-T-26-17 were able to discriminate between leukaemia and normal T cells, specifically killing the former cells. These findings may lead to the use of B. thuringiensis inclusion proteins for medical purposes.
TL;DR: The results suggest that the observed susceptibility differences reflect natural variation in B. thuringiemis susceptibility among corn borer populations rather than variation caused by prior exposure to selection pressures, and European corn borers are susceptible to B.Thuringiensis toxins among populations across most of their geographic range.
Abstract: Susceptibility to CrylAb and CrylAc toxins from Bacillus thuringiensis was deter- mined for 11 populations of neonate European corn borer,Ostrinia nubilalis (Hiibner), from the United States and 1 from northern Italy. Corn borer larvae were exposed to artificial diet treated with increasing B. thuringiensis concentrations, and mortality and growth inhibition were evaluated after 7 d. The range of variation in B. thuringiensis susceptibility indicated by growth inhibition was very similar to that indicated by mortality. Although interpopulation variation in susceptibility to both proteins was observed, the magnitude of the differences was small (54-fold) and comparable to the variability observed among generations within aparticular population (53-fold). Additionally, there was no indication that B. thuringiensis susceptibility was influenced by pheromone race, voltine ecotype, or geographic location. These results suggest that the observed susceptibility differences reflect natural variation in B. thuringiemis susceptibility among corn borer populations rather than variation caused by prior exposure to selection pressures. Therefore, European corn borers appar- ently are susceptible to B. thuringiensis toxins among populations across most of their geographic range.
TL;DR: It is shown here that the behaviour of non-target insects can also play a part in determining how their populations will be affected by Bt plants.
Abstract: Transgenic crops that express genes targeted against insect pests may also affect non-target insects. For example, lacewings1 and monarch butterflies2 have been reported to be susceptible to toxins from Bacillus thuringiensis (Bt) that are expressed in Bt transgenic plants, although these results were obtained in small-scale laboratory assays in which insects were exposed to high levels of transgenically expressed toxin in no-choice tests. We show here that the behaviour of non-target insects can also play a part in determining how their populations will be affected by Bt plants.
TL;DR: The biphasic nature of Lepidopteran genetic linkage is exploited to map this gene in diamondback moth with 207 amplified fragment length polymorphisms as DNA markers and provides a powerful tool for facilitating progress in understanding, monitoring, and managing resistance to Bt.
Abstract: Transgenic plants producing environmentally benign Bacillus thuringiensis (Bt) toxins are deployed increasingly for insect control, but their efficacy will be short-lived if pests adapt quickly. The diamondback moth (Plutella xylostella), a worldwide pest of vegetables, is the first insect to evolve resistance to Bt toxins in open-field populations. A recessive autosomal gene confers resistance to at least four Bt toxins and enables survival without adverse effects on transgenic plants. Allelic variants of this gene confer resistance in strains from Hawaii, Pennsylvania, and the Philippines. Here we exploited the biphasic nature of Lepidopteran genetic linkage to map this gene in diamondback moth with 207 amplified fragment length polymorphisms as DNA markers. We also cloned and sequenced an amplified fragment length polymorphism marker for the chromosome containing the Bt resistance gene. The results provide a powerful tool for facilitating progress in understanding, monitoring, and managing resistance to Bt.
TL;DR: This review describes the current knowledge of protease interactions with ICPs with special emphasis on the role of proteases in insect resistance to Bt toxins.
Abstract: The microbe Bacillus thuringiensis (Bt) produces crystals that contain insecticidal crystal proteins (ICPs) used to control many major pests. ICPs are degraded by proteases from a variety of sources, including those endogenous to the bacterium, those purified from animals and plants, or those found in insects. Proteases in the bacterium function in protein metabolism during sporulation; in some cases they hydrolyze ICPs. Insect proteases are implicated in Bt toxin specificity, mode of action and insect adaptation to Bt. This review describes the current knowledge of protease interactions with ICPs with special emphasis on the role of proteases in insect resistance to Bt toxins. Arch. Copyright 1999 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.
TL;DR: In vitro experiment mimicked the specific insecticidal action of the toxin in vivo well and introduced the BtR175 gene with a baculovirus, Spodoptera frugiperda Sf9 cells became susceptible to the CryIAa toxin.
TL;DR: It is demonstrated that high production ofCry1C protein can protect transgenic broccoli not only from susceptible or Cry1AR DBM larvae but also from DBM selected for moderate levels of resistance of Cry1C.
Abstract: A synthetic Bacillus thuringiensis (Bt) cry1C gene was introduced into broccoli (Brassica oleracea ssp. italica) by Agrobacterium-mediated transformation. Twenty-one Cry1C transgenic plants were regenerated from 400 hypocotyl and petiole explants. Variable amounts of stable steady- state cry1C mRNA accumulated in different transgenic plants. Cry1C protein (up to 0.4% of total soluble protein) was produced in correlation with the cry1C mRNA levels. Leaf section and whole-plant bioassays were done using diamondback moth (DBM) larvae from lines susceptible to Bt or resistant to Cry1A or Cry1C proteins (Cry1AR or Cry1CR, respectively). Plants with high levels of Cry1C protein caused rapid and complete mortality of all three types of DBM larvae with no defoliation. Plants with lower levels of Cry1C protein showed an increasing differential between control of susceptible of Cry1AR DBM. This study demonstrated that high production of Cry1C protein can protect transgenic broccoli not only from susceptible or Cry1AR DBM larvae but also from DBM selected for moderate levels of resistance of Cry1C. The Cry1C- transgenic broccoli were also resistant to two other lepidopteran pests of crucifers (cabbage looper and imported cabbage worm). These plants will be useful in studies of resistance management strategies involving multiple transgenes.
TL;DR: The imperfect correspondence between the model and observations suggests that reduced binding is not the only mechanism of resistance in the diamondback moth and that some, but not all, patterns of resistance and cross-resistance can be predicted correctly from the results of competitive binding analyses of susceptible strains.
Abstract: Insecticidal crystal proteins from Bacillus thuringiensis in sprays and transgenic crops are extremely useful for environmentally sound pest management, but their long-term efficacy is threatened by evolution of resistance by target pests. The diamondback moth (Plutella xylostella) is the first insect to evolve resistance to B. thuringiensis in open-field populations. The only known mechanism of resistance to B. thuringiensis in the diamondback moth is reduced binding of toxin to midgut binding sites. In the present work we analyzed competitive binding of B. thuringiensis toxins Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F to brush border membrane vesicles from larval midguts in a susceptible strain and in resistant strains from the Philippines, Hawaii, and Pennsylvania. Based on the results, we propose a model for binding of B. thuringiensis crystal proteins in susceptible larvae with two binding sites for Cry1Aa, one of which is shared with Cry1Ab, Cry1Ac, and Cry1F. Our results show that the common binding site is altered in each of the three resistant strains. In the strain from the Philippines, the alteration reduced binding of Cry1Ab but did not affect binding of the other crystal proteins. In the resistant strains from Hawaii and Pennsylvania, the alteration affected binding of Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F. Previously reported evidence that a single mutation can confer resistance to Cry1Ab, Cry1Ac, and Cry1F corresponds to expectations based on the binding model. However, the following two other observations do not: the mutation in the Philippines strain affected binding of only Cry1Ab, and one mutation was sufficient for resistance to Cry1Aa. The imperfect correspondence between the model and observations suggests that reduced binding is not the only mechanism of resistance in the diamondback moth and that some, but not all, patterns of resistance and cross-resistance can be predicted correctly from the results of competitive binding analyses of susceptible strains.
TL;DR: Relative fitness of resistant individuals is reduced in the absence of B. thuringiensis in the environment, and relaxation of selection pressure through refugia and insecticide rotation will favor decrease in the frequency of resistant alleles in beetle populations.
Abstract: Laboratory experiments were conducted to compare relative fitness of strains of Colorado potato beetle resistant and susceptible to Bacillus thuringiensis subsp. tenebrionis Cry3A toxin. Net replacement rates and intrinsic rates of population increase were calculated for resistant and susceptible populations. During the experiment, susceptible males on average copulated 13.3 6 1.5 times, whereas resistant males copulated only 8.0 6 1.0 times. Susceptible females produced an average of 824.2 6 68.1 eggs and 590.9 6 58.5 larvae, which was significantly .484.6 6 48.0 eggs and 334.9 6 39.7 larvae produced by an average resistant female. As a result, both net replacement rate and intrinsic rate of increase were reduced for the resistant population. Furthermore, twice as many susceptible beetles as resistant beetles survived overwintering diapause. Our results clearly indicate that relative fitness of resistant individuals is reduced in the absence of B. thuringiensis in the environment. Therefore, relaxation of selection pressure through refugia and insecticide rotation will favor decrease in the frequency of resistant alleles in beetle populations.
TL;DR: Btk sweet corn hybrids appear to be ideal candidates for use in integrated pest management (IPM) programs for both the fresh and processing sweet corn markets, and their use should drastically reduce the quantity of insecticides currently used to control these pests in sweet corn.
Abstract: Many of the lepidopterous insects which attack sweet corn, Zea mays L., are susceptible to insecticidal proteins produced by Bacillus thuringiensis ssp. kurstaki (Berliner) ( Btk ). Transgenic sweet corn expressing a synthetic cry gene for production of a Btk -insecticidal protein may provide a more environmentally acceptable means of sweet corn production. Eight transgenic sweet corn hybrids containing a synthetic gene for CryIA(b) protein production (BT11 event) were evaluated for resistance to the corn earworm, Helicoverpa zea (Boddie), and fall armyworm, Spodoptera frugiperda (J. E. Smith). Laboratory tests revealed that all Btk sweet corn hybrids were highly resistant to leaf and silk feeding by neonate 3 and 6 d old corn earworm larvae. Ear damage in the field to the Btk sweet corn hybrids caused by corn earworm was negligible. All Btk sweet corn hybrids, except Btk 95–0901, were moderately resistant to leaf and silk feeding by the fall armyworm. Survival and weight gain were reduced when neonates were fed excised whorl leaves of the Btk plants. Weight gain, but not survival, was reduced when 3- and 6-d-old fall armyworm larvae were fed excised whorl leaves of the Btk plants. Btk sweet corn hybrids appear to be ideal candidates for use in integrated pest management (IPM) programs for both the fresh and processing sweet corn markets, and their use should drastically reduce the quantity of insecticides currently used to control these pests in sweet corn. With appropriate cultural practices, it is highly unlikely that Btk sweet corn will contribute to the development of resistance to Btk proteins in these insects because of the high toxicity of the Cry proteins expressed in these sweet corn hybrids and the harvest of sweet corn ears from fields before larvae can complete development.
TL;DR: The selection and characterization of Bacillus thuringiensis strains, with ability to grow in a proteo-chitinaceous substrate (milled shrimp waste) as the sole ingredient, and the two most active proteolytic strains (Bt-103 and Bt-112) were characterized, which may show not only higher insecticidal activity, but also with the ability to produce extracellular enzymes with biotechnological applications.
Abstract: This paper reports the selection and characterization of Bacillus thuringiensis strains, with ability to grow in a proteo-chitinaceous substrate (milled shrimp waste) as the sole ingredient. Selected strains were able to produce crystal proteins, as well as proteases and chitinases as fermentation by-products. By a preliminary, qualitative screening of 152 B. thuringiensis strains, grown on media rich in protein and chitin, eight strains were selected. These strains were cultured in a liquid medium containing milled shrimp waste and their kinetics of protease production were followed. The two most active proteolytic strains (Bt-103 and Bt-112) were characterized by their crystal protein content, plasmid profiles, crystal ultrastructure, and toxicity towards Manduca sexta, Aedes aegypti and Leptinotarsa texana. The only activity recorded in these species was moderate toxicity of strain Bt-112 against Manduca sexta first instar larvae, as well as the highest proteolytic and chitinolytic activities. Its bipyramidal crystals were associated with semi-cuboidal inclusions and although its crystal proteins were similar to those of B. thuringiensis kurstaki (HD-1), its plasmid content was quite different. Serotyping of Bt-112 indicated that it belongs to serovar. tolworthi. Further studies with a similar strategy might render more strains with ability to grow in a rich waste by-product like the shrimp waste, which may show not only higher insecticidal activity, but also with the ability to produce extracellular enzymes with biotechnological applications.
TL;DR: No quantitative differences in total proteolytic activity were found between the strains, although qualitative differences related to the presence or absence of specific proteolytics activity bands using SDS-PAGE could be responsible for the observed resistance phenomenon.
Abstract: In a previous study, we demonstrated that resistance to Bacillus thuringiensis toxins in Heliothis virescens might be related to differences in the composition of the proteolytic extracts from insect midgut. There, we found specific proteolytic bands present in the gut extracts of the resistant strain and absent from the susceptible one. Here we report related facts using a new resistant strain (KCB) and a cross between the two strains used in our previous study. As would be expected, no quantitative differences in total proteolytic activity were found between the strains, although qualitative differences related to the presence or absence of specific proteolytic activity bands using SDS-PAGE could be responsible for the observed resistance phenomenon. Moreover, an SEM study made at different time intervals after intoxication shows that in the initial hours following intoxication, both the susceptible and the resistant strains show significant damage to the midgut epithelium. In the interval between 3 and 48 h, however, the resistant strain recovered such that by 48 h it had fully recovered whereas the susceptible strain did not. Arch. Copyright 1999 Wiley-Liss, Inc.
TL;DR: Geographic variation in the sensitivity of cotton bollworm to B. thuringiensis insecticide protein CryIA(c) was studied to establish a geographic baseline for comparing the future population responses to the increased use of B.Thurringiensis products in agriculture in China.
Abstract: Geographic variation in the sensitivity of cotton bollworm to B. thuringiensis insecticide protein CryIA(c) was studied to establish a geographic baseline for comparing the future population responses to the increased use of B. thuringiensis products in agriculture in China. The bollworm populations were collected from 5 ecological cotton areas of China, and the dose responses to CryIA(c) protein of mortality and growth inhibition were evaluated. The ranges of LC50s (50% lethal concentration) resulting in larval mortality and IC50s (50% inhibition concentration) of larvae grown into 3rd instars among different populations were 0.09–9.073 μg/ml and 0.011–0.057 μg/ml, respectively.
TL;DR: The results suggest that neem is a potential insect growth regulator on larvae of the Bt-S andBt-R strains of Colorado potato beetle and a possible B. thuringiensis resistance breaking compound.
Abstract: The toxicity of neem (Neemix, 0.25% azadirachtin [AI]) and combinations of neem and Bacillus thuringiensis was examined on 2nd instar of the B. thuringiensis susceptible (Bt-S) and resistant (Bt-R) strains of Colorado potato beetle, Leptinotarsa decemlineata (Say). Using a potato leaf-dipping method, the LC50 values of neem determined 2 d posttreatment to larvae of the Bt-S and Bt-R were 2.07 and 6.56 mg (AI)/liter, respectively. The LC50 values in both strains decreased significantly with increased exposure time. Cross-resistance between these toxins was not evident with a resistance ratio ranging from 1.3–3.2. Combinations of sublethal concentrations of neem (0.45 or 0.25 mg [AI]/liter) with a sublethal concentration of B. thuringiensis (0.74 mg [AI]/liter) to larvae of the Bt-S yielded an additive effect in larval mortality. In contrast, combinations of neem (0.78 or 0.43 mg [AI]/liter) and B. thuringiensis (319.8 mg [AI]/liter) resulted in a synergistic effect to larvae of the Bt-R strain. Sublethal concentrations of neem or B. thuringiensis applied separately or in combinations decreased the mean larval weight and retarded the larval growth of both strains. Our results suggest that neem is a potential insect growth regulator on larvae of the Bt-S and Bt-R and a possible B. thuringiensis resistance breaking compound. Field trials to determine the performance of neem in combination with B. thuringiensis on field populations of Colorado potato beetle are warranted.
TL;DR: Findings emphasize the need for careful deployment of B. thuringiensis -transgenic corn to preserve this effective pest management technology.
Abstract: Transgenic corn, expressing the insecticidal δ-endotoxin of Bacillus thuringiensis Berliner, provides high levels of control of some lepidopteran pests, particularly the European corn borer, Ostrinia nubilalis (Hubner). However, resistance to B. thuringiensis has been documented recently in laboratory colonies of agronomically important Lepidoptera, including O. nubilalis. For the past 4 yr, we have selected for Cry1Ac resistance in a population of O. nubilalis from southeastern Minnesota. Increasing resistance to B. thuringiensis was noted after only 8 generations of selection, with a peak at 162-fold resistance, based on comparisons of LC50s to a nonselected parental strain. This resistance was found to decrease at the same rate in the absence of B. thuringiensis selection, with one selected colony becoming nearly as sensitive to the Cry1Ac toxin as the nonselected colony after 9 generations without exposure to B. thuringiensis. The most resistant of the colonies, S-I, was only marginally cross-resistant to Cry1Ab, yet another selected colony, S-IV, did demonstrate a 16-fold cross-resistance. In addition, larvae from the S-IV colony had significantly greater weight gain when feeding on diet incorporated with B. thuringiensis -transgenic corn than did larvae from the nonselected parental colony. These findings emphasize the need for careful deployment of B. thuringiensis corn to preserve this effective pest management technology.
TL;DR: The data suggest that posttranslational modifications can have a significant effect on Cry1A toxin interactions with specific insect midgut proteins.
Abstract: Although extensively studied, the mechanism of action of insecticidal Bacillus thuringiensis Cry toxins remains elusive and requires further elucidation. Toxin receptors in the brush border membrane demand particular attention as they presumably initiate the cascade of events leading to insect mortality after toxin activation. The 170-kDa Cry1Ac toxin-binding aminopeptidase from the tobacco budworm (Heliothis virescens) was partially purified, and its corresponding cDNA was cloned. The cDNA encodes a protein with a putative glycosyl phosphatidylinositol anchor and a polythreonine stretch clustered near the C terminus with predicted O-glycosylation. Partial purification of the 170-kDa aminopeptidase also resulted in isolation of a 130-kDa protein that was immunologically identical to the 170-kDa protein, and the two proteins had identical N termini. These proteins were glycosylated, as suggested by soybean agglutinin lectin blot results. Cry1Ac toxin affinity data for the two proteins indicated that the 130-kDa protein had a higher affinity than the 170-kDa protein. The data suggest that posttranslational modifications can have a significant effect on Cry1A toxin interactions with specific insect midgut proteins.
TL;DR: The randomly amplified polymorphic DNA (RAPD) fingerprinting technique was applied to a collection of 101 strains of the genus Bacillus, including 61 strain of the B. cereus group and identified an 838-bp RAPD marker specific for Bacillus anthracis.
Abstract: Aiming to develop a DNA marker specific for Bacillus anthracis and able to discriminate this species from Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides, we applied the randomly amplified polymorphic DNA (RAPD) fingerprinting technique to a collection of 101 strains of the genus Bacillus, including 61 strains of the B. cereus group. An 838-bp RAPD marker (SG-850) specific for B. cereus, B. thuringiensis, B. anthracis, and B. mycoides was identified. This fragment included a putative (366-nucleotide) open reading frame highly homologous to the ypuA gene of Bacillus subtilis. The restriction analysis of the SG-850 fragment with AluI distinguished B. anthracis from the other species of the B. cereus group.
TL;DR: B. thuringiensis is ubiquitous on a variety of plants; bacterial flora on phylloplanes consists of highly heterogeneous H serogroups; and there is little correlation between plant species and phenotypes of B. thurringiensis isolates.
Abstract: A total of 120 Bacillus thuringiensis strains was isolated from phylloplanes of 35 species of arboreous and herbaceous plants in an area of northern Kyushu, Japan. The isolates belonged to at least 17 serotypes and the group of H serotype 3 was predominant. Twenty strains were untypable by the existing reference H antisera and 47 were untestable due to autoagglutination or poor motility. Of the 120 isolates, 25 produced bipyramidal parasporal inclusions and the others, spherical or irregular-shaped. Insecticidal activity against mosquitoes (Culex pipiens molestus and Anopheles stephensi) and/or diamondback moth, Plutella xylostella, was associated with 28 isolates (23·3%). Overall results revealed that: B. thuringiensis is ubiquitous on a variety of plants; bacterial flora on phylloplanes consists of highly heterogeneous H serogroups; and there is little correlation between plant species and phenotypes of B. thuringiensis isolates.