Larvae of Culex quinquefasciatus were exposed to infection by Lagenidium giganteum and various co... more Larvae of Culex quinquefasciatus were exposed to infection by Lagenidium giganteum and various concentrations of B.t.i. or B. sphaericus. The resulting larval mortalities, percentages of infected dead larvae and percentages of larval body regions containing the fungus were compared. Overall, the effectiveness of Lagenidum giganteum against the larvae was not significantly affected by the presence of B.t.i. or B. sphaericus, and the fungal and bacterial agents were compatible. In experiments using 3-day-old larvae, the extent of growth of the fungus in the infected larvae and the percentage of the larvae infected were related to the concentration of B.t.i. in the range of 0.057-0.456 ITU/ml tested but were not related to the concentration of B. sphaericus in the range of 0.6-4.8 x 10(4) spores/ml. With larvae of various ages treated with a low concentration of B.t.i. (0.114 ITU/ml), exposure to the fungus increased the mortality rate in early but not late instars. After single and multiple applications of B.t.i. and B. sphaericus in the presence of the fungus, followed by drying and reflooding, the fungus persisted and reinfected larvae while the B. sphaericus persisted but the B.t.i. did not.
ABSTRACT Bacillus thuringiensis produce d1 endotoxinas que requieren activación proteolítica para... more ABSTRACT Bacillus thuringiensis produce d1 endotoxinas que requieren activación proteolítica para ser activas. La activación de la toxina citolítica de 28 kDa (Cyt1Ab1) de B. thuringiensis subesp. Medellín con tripsina, quimotripsina y extractos intestinales de larvas del mosquito Culex quinquefasciatus fue analizada. La toxina Cyt1Ab1 de B. thuringiensis subsp. medellin fue procesada por todas las proteasa evaluadas a fragmentes entre 23 y 25 kDa, mientras que el procesamiento de la toxina Cyt1Aa1 produjo fragmentos entre 22.5 y 24.5 kDa. La toxina Cyt1Ab1 fue procesada preferencialmente in vitro en un pH de 12, produciendo un fragmento de 25 kDa cuando se trató con extractos intestinales de larvas de mosquito, y resultados similares se obtuvieron cuando la toxina fue activada in vivo por larvas de C. quinquefasciatus. La toxina solubilizada y los fragmentos resistentes a la acción de las proteasas generados en los experimentos in vitro, demostraron actividad hemolítica, pero no mosquitocida. La secuencia amino terminal del fragmento tratado con extractos intestinales de C. quinquefasciatus indica que el sitio de corte está localizado entre Lys31 y Asp32, generando una proteína con secuencia amino Terminal de DDPNEKNNHNS; mientras que la toxina tratada con tripsina mostró un sitio de corte entre Leu29 y Arg30, y la tratada con quimotripsina entre Arg30 and Lys31.
Bioprocess and Biosystems Engineering, Jul 22, 2007
An extended dynamical model for growth and sporulation of Bacillus thuringiensis subsp. kurstaki ... more An extended dynamical model for growth and sporulation of Bacillus thuringiensis subsp. kurstaki in an intermittent fed-batch culture with total cell retention is proposed. This model differs from reported models, by including dynamics for natural death of cells and substrate consumption for cell maintenance. The proposed model uses sigmoid functions to describe these kinetic parameters. Equations for time evolution of substrate, vegetative, sporulated and total cell concentration were taken from previous works. Model parameters were determined from batch experimental data obtained in pilot plant. Parameter identification was developed in two stages: (1) coarse identification using a multivariable optimization with constraints algorithm, (2) fine identification by heuristic fit of model parameters looking for a minimal model error. The proposed model estimates adequate time evolution of the process variables with a mean error of 2.6% on substrate concentration and 6.7% on biomass concentration.
To identify and characterize Bacillus thuringiensis strains highly toxic to Spodoptera frugiperda... more To identify and characterize Bacillus thuringiensis strains highly toxic to Spodoptera frugiperda, and to explore the genetic diversity of such strains. The insecticidal activity of 1100 strains of B. thuringiensis from Colombian soil samples was assayed against first instar S. frugiperda larvae, and 32 active strains were found. After a second bioassay evaluation, the eight most potent strains were selected for further characterization, which included crystal protein profiles determined by polyacrylamide gel electrophoresis, plasmid profile, plasmid restriction patterns, cry gene composition, qualitative determination of beta-exotoxin production, random amplified polymorphic DNA, serotyping, and toxicity to S. frugiperda. All Colombian strains contained cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C and cry1D genes. However, PCR profiles of the Colombian strains suggested the presence of variants of the cry1 genes. Serotyping indicated that these strains belong to the kurstaki, thuringiensis, canadiensis and indiana subspecies. Interestingly, three strains belonging to different serotypes and subspecies were found in the same soil sample, and toxicity ranged between 11 and 976 ng cm(-2) of diet. It has been shown that B. thuringiensis strains belonging to different serotypes and displaying variable potency to S. frugiperda larvae can be found in the same soil sample. The results obtained indicate that some of the B. thuringiensis strains studied could be of interest for further development for S. frugiperda control programmes.
Larvae of Culex quinquefasciatus were exposed to infection by Lagenidium giganteum and various co... more Larvae of Culex quinquefasciatus were exposed to infection by Lagenidium giganteum and various concentrations of B.t.i. or B. sphaericus. The resulting larval mortalities, percentages of infected dead larvae and percentages of larval body regions containing the fungus were compared. Overall, the effectiveness of Lagenidum giganteum against the larvae was not significantly affected by the presence of B.t.i. or B. sphaericus, and the fungal and bacterial agents were compatible. In experiments using 3-day-old larvae, the extent of growth of the fungus in the infected larvae and the percentage of the larvae infected were related to the concentration of B.t.i. in the range of 0.057-0.456 ITU/ml tested but were not related to the concentration of B. sphaericus in the range of 0.6-4.8 x 10(4) spores/ml. With larvae of various ages treated with a low concentration of B.t.i. (0.114 ITU/ml), exposure to the fungus increased the mortality rate in early but not late instars. After single and multiple applications of B.t.i. and B. sphaericus in the presence of the fungus, followed by drying and reflooding, the fungus persisted and reinfected larvae while the B. sphaericus persisted but the B.t.i. did not.
ABSTRACT Bacillus thuringiensis produce d1 endotoxinas que requieren activación proteolítica para... more ABSTRACT Bacillus thuringiensis produce d1 endotoxinas que requieren activación proteolítica para ser activas. La activación de la toxina citolítica de 28 kDa (Cyt1Ab1) de B. thuringiensis subesp. Medellín con tripsina, quimotripsina y extractos intestinales de larvas del mosquito Culex quinquefasciatus fue analizada. La toxina Cyt1Ab1 de B. thuringiensis subsp. medellin fue procesada por todas las proteasa evaluadas a fragmentes entre 23 y 25 kDa, mientras que el procesamiento de la toxina Cyt1Aa1 produjo fragmentos entre 22.5 y 24.5 kDa. La toxina Cyt1Ab1 fue procesada preferencialmente in vitro en un pH de 12, produciendo un fragmento de 25 kDa cuando se trató con extractos intestinales de larvas de mosquito, y resultados similares se obtuvieron cuando la toxina fue activada in vivo por larvas de C. quinquefasciatus. La toxina solubilizada y los fragmentos resistentes a la acción de las proteasas generados en los experimentos in vitro, demostraron actividad hemolítica, pero no mosquitocida. La secuencia amino terminal del fragmento tratado con extractos intestinales de C. quinquefasciatus indica que el sitio de corte está localizado entre Lys31 y Asp32, generando una proteína con secuencia amino Terminal de DDPNEKNNHNS; mientras que la toxina tratada con tripsina mostró un sitio de corte entre Leu29 y Arg30, y la tratada con quimotripsina entre Arg30 and Lys31.
Bioprocess and Biosystems Engineering, Jul 22, 2007
An extended dynamical model for growth and sporulation of Bacillus thuringiensis subsp. kurstaki ... more An extended dynamical model for growth and sporulation of Bacillus thuringiensis subsp. kurstaki in an intermittent fed-batch culture with total cell retention is proposed. This model differs from reported models, by including dynamics for natural death of cells and substrate consumption for cell maintenance. The proposed model uses sigmoid functions to describe these kinetic parameters. Equations for time evolution of substrate, vegetative, sporulated and total cell concentration were taken from previous works. Model parameters were determined from batch experimental data obtained in pilot plant. Parameter identification was developed in two stages: (1) coarse identification using a multivariable optimization with constraints algorithm, (2) fine identification by heuristic fit of model parameters looking for a minimal model error. The proposed model estimates adequate time evolution of the process variables with a mean error of 2.6% on substrate concentration and 6.7% on biomass concentration.
To identify and characterize Bacillus thuringiensis strains highly toxic to Spodoptera frugiperda... more To identify and characterize Bacillus thuringiensis strains highly toxic to Spodoptera frugiperda, and to explore the genetic diversity of such strains. The insecticidal activity of 1100 strains of B. thuringiensis from Colombian soil samples was assayed against first instar S. frugiperda larvae, and 32 active strains were found. After a second bioassay evaluation, the eight most potent strains were selected for further characterization, which included crystal protein profiles determined by polyacrylamide gel electrophoresis, plasmid profile, plasmid restriction patterns, cry gene composition, qualitative determination of beta-exotoxin production, random amplified polymorphic DNA, serotyping, and toxicity to S. frugiperda. All Colombian strains contained cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C and cry1D genes. However, PCR profiles of the Colombian strains suggested the presence of variants of the cry1 genes. Serotyping indicated that these strains belong to the kurstaki, thuringiensis, canadiensis and indiana subspecies. Interestingly, three strains belonging to different serotypes and subspecies were found in the same soil sample, and toxicity ranged between 11 and 976 ng cm(-2) of diet. It has been shown that B. thuringiensis strains belonging to different serotypes and displaying variable potency to S. frugiperda larvae can be found in the same soil sample. The results obtained indicate that some of the B. thuringiensis strains studied could be of interest for further development for S. frugiperda control programmes.
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