scholarly journals Genetic Variability of Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) Populations from Latin America Is Associated with Variations in Susceptibility to Bacillus thuringiensis Cry Toxins

2006 ◽  
Vol 72 (11) ◽  
pp. 7029-7035 ◽  
Author(s):  
Rose Monnerat ◽  
Erica Martins ◽  
Paulo Queiroz ◽  
Sergio Ordúz ◽  
Gabriela Jaramillo ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Genetic Variability ◽  
Latin American ◽  
Spodoptera Frugiperda ◽  
Insect Pest ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cry Toxins ◽  
Geographical Regions ◽  
Insect Populations

ABSTRACT Bacillus thuringiensis strains isolated from Latin American soil samples that showed toxicity against three Spodoptera frugiperda populations from different geographical areas (Mexico, Colombia, and Brazil) were characterized on the basis of their insecticidal activity, crystal morphology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of parasporal crystals, plasmid profiles, and cry gene content. We found that the different S. frugiperda populations display different susceptibilities to the selected B. thuringiensis strains and also to pure preparations of Cry1B, Cry1C, and Cry1D toxins. Binding assays performed with pure toxin demonstrated that the differences in the toxin binding capacities of these insect populations correlated with the observed differences in susceptibility to the three Cry toxins analyzed. Finally, the genetic variability of the three insect populations was analyzed by random amplification of polymorphic DNA-PCR, which showed significant genetic diversity among the three S. frugiperda populations analyzed. The data presented here show that the genetic variability of S. frugiperda populations should be carefully considered in the development of insect pest control strategies, including the deployment of genetically modified maize in different geographical regions.

scholarly journals Insecticidal activity of culture supernatants from Bacillus thuringiensis Berliner strains against Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) larvae

1999 ◽  
Vol 28 (4) ◽  
pp. 675-685 ◽  
Author(s):  
Marliton R. Barreto ◽  
Leandro L. Loguercio ◽  
Fernando H. Valicente ◽  
Edilson Paiva
Keyword(s):  
Bacillus Thuringiensis ◽  
Protein Secretion ◽  
Spodoptera Frugiperda ◽  
Wide Spectrum ◽  
Insect Pest ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fall Armyworm ◽  
Tropical Maize ◽  
Potential Applicability ◽  
Culture Supernatants

Novel vegetative insecticidal proteins (Vips) identified in the supernatant of Bacillus thuringiensis (B.t.) cultures have shown to provide adequate control over a wide spectrum of economically important crop pests. To evaluate the potential applicability of these proteins against fall armyworm (Spodoptera frugiperda Smith) larvae, the most important insect pest for tropical maize, the characteristics and mortality effects of culture supernatants from five B.t. strains were investigated. Striking differences among strains were detected, not only in terms of efficiency in killing the insect, but also regarding to mortality effects of heated and non-heated supernatants, which were used to distinguish the heat-sensitive protein-derived insecticidal fraction from a thermostable one, with a non-protein nature (b-exotoxinas). The qualitative, quantitative and temporal patterns of total protein secretion in the medium (supernatant) were assessed through spectrophotometry and polyacrylamide gel electrophoresis. The strains showed remarkably distinct rates of growth and timing for protein secretion relative to cell density in culture. Moreover, the electrophoretic-banding patterns also varied in a strain-specific manner, both in denaturing and non denaturing conditions. Polypeptides displaying a molecular weight that is very close to the expected for previously identified Vip3A proteins were found for the strains with high supernatant-mortality ratios. The data suggest the feasibility and usefulness of searching for protein-derived (Vip-like) insecticidal fractions in B.t. supernatants as a mean of developing especific and efficient alternatives of biological control to be employed in integrated pest management programs of S. frugiperda in tropical maize.

scholarly journals Solubilization, Activation, and Insecticidal Activity of Bacillus thuringiensis Serovar thompsoni HD542 Crystal Proteins

2008 ◽  
Vol 74 (23) ◽  
pp. 7145-7151 ◽  
Author(s):  
Samir Naimov ◽  
Rumyana Boncheva ◽  
Rumyana Karlova ◽  
Stefan Dukiandjiev ◽  
Ivan Minkov ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Pieris Rapae ◽  
Insect Pest ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cydia Pomonella ◽  
Sodium Dodecyl ◽  
Crystal Proteins ◽  
Stable Product ◽  
Insect Gut

ABSTRACT Cry15Aa protein, produced by Bacillus thuringiensis serovar thompsoni HD542 in a crystal together with a 40-kDa accompanying protein, is one of a small group of nontypical, less well-studied members of the Cry family of insecticidal proteins and may provide an alternative for the more commonly used Cry proteins in insect pest management. In this paper, we describe the characterization of the Cry15Aa and 40-kDa protein's biochemical and insecticidal properties and the mode of action. Both proteins were solubilized above pH 10 in vitro. Incubation of solubilized crystal proteins with trypsin or insect midgut extracts rapidly processed the 40-kDa protein to fragments too small to be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas the Cry15 protein yielded a stable product of approximately 30 kDa. Protein N-terminal sequencing showed that Cry15 processing occurs exclusively at the C-terminal end. Cry15 protein showed in vitro hemolytic activity, which was greatly enhanced by preincubation with trypsin or insect gut extract. Larvae of the lepidopteran insects Manduca sexta, Cydia pomonella, and Pieris rapae were susceptible to crystals, and presolubilization of the crystals enhanced activity to P. rapae. Activity for all three species was enhanced by preincubation with trypsin. Larvae of Helicoverpa armigera and Spodoptera exigua were relatively insensitive to crystals, and activity against these insects was not enhanced by prior solubilization or trypsin treatment. The 40-kDa crystal protein showed no activity in the insects tested, nor did its addition or coexpression in Escherichia coli increase the activity of Cry15 in insecticidal and hemolytic assays.

scholarly journals Mitochondrial markers to distinguish Spodoptera frugiperda populations associated with corn and cotton crops

2016 ◽  
Vol 51 (5) ◽  
pp. 692-696 ◽  
Author(s):  
Paulo Roberto Queiroz ◽  
Carolina Almeida Ramiro ◽  
Érica Soares Martins ◽  
Mário Soberón ◽  
Alejandra Bravo ◽  
...  
Keyword(s):  
Genetic Variability ◽  
Feeding Behavior ◽  
Spodoptera Frugiperda ◽  
Dna Markers ◽  
Genetic Profile ◽  
Mitochondrial Markers ◽  
Geographical Regions ◽  
Insect Populations ◽  
Mitochondrial Nd1 ◽  
Lepidoptera Noctuidae

Abstract: The objective of this work was to analyze the genetic variability of Spodoptera frugiperda (Lepidoptera: Noctuidae) populations collected from corn and cotton crops in Brazil. The samples were analyzed by DNA markers. The dendrogram produced by the 20 RAPD primers evaluated showed a correlation between genetic profile and feeding behavior. The analysis of the mitochondrial ND1 gene allowed identifying the insect populations in both crops, and, in corn, in several geographical regions. The presented strategy allows the identification of Spodoptera frugiperda populations associated with corn and cotton crops.

scholarly journals Parasporal Body Formation via Overexpression of the Cry10Aa Toxin of Bacillus thuringiensis subsp. israelensis, and Cry10Aa-Cyt1Aa Synergism

2009 ◽  
Vol 75 (14) ◽  
pp. 4661-4667 ◽  
Author(s):  
Alejandro Hernández-Soto ◽  
M. Cristina Del Rincón-Castro ◽  
Ana M. Espinoza ◽  
Jorge E. Ibarra
Keyword(s):  
Bacillus Thuringiensis ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Microbial Control ◽  
Control Agent ◽  
Mass Spectrometry Analysis ◽  
Parasporal Crystal ◽  
Spectrometry Analysis ◽  
Crystal Complex

ABSTRACT Bacillus thuringiensis subsp. israelensis is the most widely used microbial control agent against mosquitoes and blackflies. Its insecticidal success is based on an arsenal of toxins, such as Cry4A, Cry4B, Cry11A, and Cyt1A, harbored in the parasporal crystal of the bacterium. A fifth toxin, Cry10Aa, is synthesized at very low levels; previous attempts to clone and express Cry10Aa were limited, and no parasporal body was formed. By using a new strategy, the whole Cry10A operon was cloned in the pSTAB vector, where both open reading frames ORF1 and ORF2 (and the gap between the two) were located, under the control of the cyt1A operon and the STAB-SD stabilizer sequence characteristic of this vector. Once the acrystalliferous mutant 4Q7 of B. thuringiensis subsp. israelensis was transformed with this construct, parasporal bodies were observed by phase-contrast microscopy and transmission electron microscopy. Discrete, ca. 0.9-μm amorphous parasporal bodies were observed in the mature sporangia, which were readily purified by gradient centrifugation once autolysis had occurred. Pure parasporal bodies showed two major bands of ca. 68 and 56 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These bands were further characterized by N-terminal sequencing of tryptic fragments using matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, which identified both bands as the products of ORF1 and ORF2, respectively. Bioassays against fourth-instar larvae of Aedes aegypti of spore-crystal complex and pure crystals of Cry10Aa gave estimated 50% lethal concentrations of 2,061 ng/ml and 239 ng/ml, respectively. Additionally, synergism was clearly detected between Cry10A and Cyt1A, as the synergistic levels (potentiation rates) were estimated at 13.3 for the mixture of Cyt1A crystals and Cry10Aa spore-crystal complex and 12.6 for the combination of Cyt1A and Cry10Aa pure crystals.

Characterization ofBacillus thuringiensismutants and natural isolates by molecular methods

1997 ◽  
Vol 43 (5) ◽  
pp. 403-410 ◽  
Author(s):  
Yong Chul Jung ◽  
Sung Uk Kim ◽  
Song Hae Bok ◽  
Ho Yong Park ◽  
Jean-Charles Côté ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Bacillus Thuringiensis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Rice Grain ◽  
Chain Reaction ◽  
Hyphantria Cunea ◽  
Natural Isolates ◽  
Grain Dust ◽  
Polymerase Chain

Two Bacillus thuringiensis var. kurstaki HD-1 mutants, two Bacillus thuringiensis var. israelensis HD-500 mutants, and four rice grain dust isolates were characterized using microscopic examination and protein profiles of purified crystals on sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Specific detection of cryI- and cryIV-type genes was performed in a polymerase chain reaction using cryI and cryIV-specific oligonucleotide primers. The cry-type genes under study consisted of cryIA(a), cryI(A)b, cryI(A)c, cryIB, and cryIV. Presence or absence of the cryI- and cryIV-type genes was further confirmed by Southern blotting followed by hybridization with specific cryI and cryIV gene fragments. A genetically modified strain of B. thuringiensis var. kurstaki HD-1, called OZK-13 and obtained following mutagenesis with ozone, was shown to contain cryIA(a), cryIA(b), and cryIA(c) genes. Another kurstaki HD-1 mutant, called NGK-13 and obtained following treatment with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), was shown to have lost the cryIA(b) gene while retaining the cryIA(a) and cryIA(c) genes. NGI-23-1, an oligosporogenous–multicrystalliferous mutant of B. thuringiensis var. israelensis (Bti) HD-500, obtained following treatment with MNNG contained cryIV-type genes. NGI-22, an oligosporogenous–acrystalliferous mutant of Bti HD-500, contained no cryI- nor cryIV-type genes. The rice grain dust isolate BT-285 contained the cryIA(a) and cryIA(c) genes. Isolate BT-14 contained only the cryIA(c) gene, whereas isolate BT-209 contained cryIA(a), cryIA(b), and cryIB genes. Isolate BT-205 contained no cryI- nor cryIV-type genes. Bacillus thuringiensis mutants and natural isolates shown to contain cryI-type genes were tested for their insecticidal activities in a series of bioassays against Hyphantria cunea Drury (Lepidoptera: Arctiidae). All cryI-carrying strains were toxic against the insect larvae. BT-205 was also tested and exhibited no toxicity against the insect larvae.Key words: Bacillus thuringiensis, δ-endotoxin crystal, cry-type genes, polymerase chain reaction.

scholarly journals Bacillus thuringiensis Cry1Ab Domain III β-22 Mutants with Enhanced Toxicity to Spodoptera frugiperda (J. E. Smith)

2020 ◽  
Vol 86 (22) ◽  
Author(s):  
Isabel Gómez ◽  
Josue Ocelotl ◽  
Jorge Sánchez ◽  
Sotero Aguilar-Medel ◽  
Guadalupe Peña-Chora ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Spodoptera Frugiperda ◽  
Insect Pest ◽  
Transgenic Maize ◽  
Crop Damage ◽  
Binding Interaction ◽  
Alanine Scanning Mutagenesis ◽  
Content Type ◽  
Lepidopteran Pests ◽  
Domain Iii

ABSTRACT The fall armyworm, Spodoptera frugiperda, is an invasive maize pest that has spread from the Americas into Africa and Asia and causes severe crop damage worldwide. Most populations of S. frugiperda show low susceptibility to Bacillus thuringiensis (Bt) Cry1Ab or Cry1Ac toxins, which have been proved to be effective against several other lepidopteran pests. In addition, S. frugiperda has evolved resistance to transgenic maize expressing Cry1Fa toxin. The specificity and toxicity of Cry toxins are determined by their binding to different larval midgut proteins, such as aminopeptidase N (APN), alkaline phosphatase (ALP), and cadherin (CAD), among other proteins, by means of exposed domain II loop regions and also by the domain III β-sheets β-16 and β-22. Here, we analyzed different Cry1Ab mutants with mutations in the domain III β-22 region. Alanine-scanning mutagenesis of this region revealed that all mutants showed increased toxicity against a nonsusceptible Cry1Ab S. frugiperda population. Further analysis of the mutant toxin Cry1AbS587A (bearing a mutation of S to A at position 587) revealed that, compared to Cry1Ab, it showed significantly increased toxicity to three other S. frugiperda populations from Mexico but retained similar toxicity to Manduca sexta larvae. Cry1AbS587A bound to brush border membrane vesicles (BBMV), and its higher toxicity correlated with higher binding affinities to APN, ALP, and CAD recombinant proteins. Furthermore, silencing the expression of APN1 and CAD receptors in S. frugiperda larvae by RNA interference (RNAi) showed that Cry1AbS587A toxicity relied on CAD expression, in contrast to Cry1Ab. These data support the idea that the increased toxicity of Cry1AbS587A to S. frugiperda is in part due to an improved binding interaction with the CAD receptor. IMPORTANCE Spodoptera frugiperda is an important worldwide pest of maize and rice crops that has evolved resistance to Cry1Fa-expressing maize in different countries. Therefore, identification of additional toxins with different modes of action is needed to provide alternative tools to control this insect pest. Bacillus thuringiensis (Bt) Cry1Ab and Cry1Ac toxins are highly active against several important lepidopteran pests but show varying and low levels of toxicity against different S. frugiperda populations. Thus, the identification of Cry1A mutants that gain toxicity to S. frugiperda and retain toxicity to other pests could be of great value to produce transgenic crops that resist a broader spectrum of lepidopteran pests. Here, we characterized Cry1Ab domain III β-22 mutants, and we found that a Cry1AbS587A mutant displayed increased toxicity against different S. frugiperda populations. Thus, Cry1AbS587A could be a good toxin candidate to produce transgenic maize with broader efficacy against this important insect pest in the field.

Insect Populations on Bacillus thuringiensis Transgenic Sweet Corn

2002 ◽  
Vol 37 (4) ◽  
pp. 285-292 ◽  
Author(s):  
Richard L. Hassell ◽  
B. Merle Shepard
Keyword(s):  
Bacillus Thuringiensis ◽  
Plant Height ◽  
Spodoptera Frugiperda ◽  
Transgenic Line ◽  
Sweet Corn ◽  
Transgenic Crop ◽  
Transgenic Corn ◽  
Late Season ◽  
65 H ◽  
Insect Populations

Insect populations and damage in three plantings (early, mid-, and late-season) of Bacillus thuringiensis (Bt) transgenic sweet corn were compared to a non-transgenic isoline at Charleston, SC, during 2000. The transgenic corn was expressing the crylA(b) gene from B. thuringiensis var. kurstaki. No fall armyworms, Spodoptera frugiperda (J. E. Smith), were present in the early-planted crop, and numbers of corn earworms, Heliocoverpa zea (Boddie), were significantly reduced in the Bt-transgenic corn compared to the non-Bt isoline. The non-Bt isoline had 65% H. zea infested ears compared to only 10% ears from the Bt-transgenic line. Damage and numbers of H. zea and S. frugiperda also were significantly higher in the non-Bt sweet corn planted in mid- and late-season plantings. Percent damaged ears by H. zea was 77% in the non-transgenic sweet corn and 21% in the Bt-sweet corn during the mid-season crop. In the late-planted crop, populations of H. zea averaged 15 per 120 ears in the non-BT isoline compared to less than 2 per 120 ears in the Bt-transgenic crop. Whorl damage by fall armyworms significantly reduced plant height in both mid-season and late-season non-transgenic crops but not in Bt-transgenic sweet corn. Numbers of other insects and spiders were low and not significantly different between the two treatments. Bt transformed sweet corn will play a major role in reducing populations of H. zea, S. frugiperda and limiting pesticides in this crop.

scholarly journals New Strategy for Isolating Novel Nematicidal Crystal Protein Genes from Bacillus thuringiensis Strain YBT-1518

2008 ◽  
Vol 74 (22) ◽  
pp. 6997-7001 ◽  
Author(s):  
Suxia Guo ◽  
Mei Liu ◽  
Donghai Peng ◽  
Sisi Ji ◽  
Pengxia Wang ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Meloidogyne Hapla ◽  
Microscopy Observation ◽  
Root Knot Nematode ◽  
Dna Library ◽  
Sds Page ◽  
New Strategy ◽  
Key Steps

ABSTRACT We have developed a strategy for isolating cry genes from Bacillus thuringiensis. The key steps are the construction of a DNA library in an acrystalliferous B. thuringiensis host strain and screening for the formation of crystal through optical microscopy observation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses. By this method, three cry genes—cry55Aa1, cry6Aa2, and cry5Ba2—were cloned from rice-shaped crystals, producing B. thuringiensis YBT-1518, which consists of 54- and 45-kDa crystal proteins. cry55Aa1 encoded a 45-kDa protein, cry6Aa2 encoded a 54-kDa protein, and cry5Ba2 remained cryptic in strain YBT-1518, as shown by SDS-PAGE or microscopic observation. Proteins encoded by these three genes are all toxic to the root knot nematode Meloidogyne hapla. The two genes cry55Aa1 and cry6Aa2 were found to be located on a plasmid with a rather small size of 17.7 kb, designated pBMB0228.

scholarly journals Activity of Free and Clay-Bound Insecticidal Proteins from Bacillus thuringiensis subsp. israelensis against the Mosquito Culex pipiens

2003 ◽  
Vol 69 (7) ◽  
pp. 4111-4115 ◽  
Author(s):  
LanNa Lee ◽  
Deepak Saxena ◽  
G. Stotzky
Keyword(s):  
Bacillus Thuringiensis ◽  
Molecular Mass ◽  
Culex Pipiens ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot ◽  
Enhanced Chemiluminescence ◽  
Adsorbed Proteins ◽  
Hydrolysis Of

ABSTRACT Bacillus thuringiensis subsp. israelensis produces parasporal insecticidal crystal proteins (ICPs) that have larvicidal activity against some members of the order Diptera, such as blackflies and mosquitoes. Hydrolysis of the ICPs in the larval gut results in four major proteins with a molecular mass of 27, 65, 128, and 135 kDa. Toxicity is caused by synergistic interaction between the 25-kDa protein (proteolytic product of the 27-kDa protein) and one or more of the higher-molecular-mass proteins. Equilibrium adsorption of the proteins on the clay minerals montmorillonite and kaolinite, which are homoionic to various cations, was rapid (<30 min for maximal adsorption), increased with protein concentration and then reached a plateau (68 to 96% of the proteins was adsorbed), was significantly lower on kaolinite than on montmorillonite, and was not significantly affected by the valence of the cation to which the clays were homoionic. Binding of the toxins decreased as the pH was increased from 6 to 11, and there was 35 to 66% more binding in phosphate buffer at pH 6 than in distilled water at pH 6 or 7.2. Only 2 to 12% of the adsorbed proteins was desorbed by two washes with water; additional washings desorbed no more toxins, indicating that they were tightly bound. Formation of clay-toxin complexes did not alter the structure of the proteins, as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the equilibrium supernatants and desorption washes and by dot blot enzyme-linked immunosorbent assay of the complexes, which was confirmed by enhanced chemiluminescence Western blot analysis. Free and clay-bound toxins resulted in 85 to 100% mortality of the mosquito Culex pipiens. Persistence of the bound toxins in nonsterile water after 45 days was significantly greater (mortality of 63% ± 12.7%) than that of the free toxins (mortality of 25% ± 12.5%).

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