Bioencapsulation and delivery to mosquito larvae of Bacillus thuringiensis H14 toxicity by Tetrahymena pyriformis

Zaritsky, Arieh; Zalkinder, Valery; Ben‐Dov, Eitan; Barak, Ze’ev

doi:10.1016/0022-2011(91)90195-v

Cited by 8 publications

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“…Vacuoles excreted from B. thuringiensis subsp. israelensis-loaded cells should contain intact spores and crystals (34). Their fate inside the vacuoles was determined at various times after excretion by phase-contrast and electron microscopy as well as by viable-cell counting.…”

Section: Resultsmentioning

confidence: 99%

“…We have previously shown that toxicity of B. thuringiensis subsp. israelensis to larvae of Aedes aegypti and Anopheles stephensi was enhanced three-and eightfold, respectively, by bioencapsulation in food vacuoles of the protozoan Tetrahymena pyriformis, in which the toxins remain stable; each T. pyriformis cell concentrates in its food vacuoles between 180 and 240 spores and their associated ICP (5,20,21,34) and delivers them to the target organisms ( Fig. 1).…”

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confidence: 99%

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Germination, Growth, and Sporulation of Bacillus thuringiensis subsp. israelensis in Excreted Food Vacuoles of the Protozoan Tetrahymena pyriformis

Manasherob

Ben‐Dov

Zaritsky

et al. 1998

Appl Environ Microbiol

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Spores of Bacillus thuringiensis subsp.israelensis and their toxic crystals are bioencapsulated in the protozoan Tetrahymena pyriformis, in which the toxin remains stable. Each T. pyriformis cell concentrates the spores and crystals in its food vacuoles, thus delivering them to mosquito larvae, which rapidly die. Vacuoles containing undigested material are later excreted from the cells. The fate of spores and toxin inside the food vacuoles was determined at various times after excretion by phase-contrast and electron microscopy as well as by viable-cell counting. Excreted food vacuoles gradually aggregated, and vegetative growth of B. thuringiensis subsp.israelensis was observed after 7 h as filaments that stemmed from the aggregates. The outgrown cells sporulated between 27 and 42 h. The spore multiplication values in this system are low compared to those obtained in carcasses of B. thuringiensissubsp. israelensis-killed larvae and pupae, but this bioencapsulation represents a new possible mode of B. thuringiensis subsp. israelensis recycling in nontarget organisms.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Germination, Growth, and Sporulation of Bacillus thuringiensis subsp. israelensis in Excreted Food Vacuoles of the Protozoan Tetrahymena pyriformis

Manasherob

Ben‐Dov

Zaritsky

et al. 1998

Appl Environ Microbiol

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“…Approximately 5 x 109 CFU (50 ,ul of cells) were used for each transformation. To achieve the maximum field strength employing the Bio-Rad Gene Pulser (Bio-Rad, Richmond, Calif.), 0.2-cm cuvettes were used with settings of 100 fl, 25 ,uF, and 2.5 kV. Immediately after voltage application, the cells were diluted in 400 ,ul of cold S27 broth (23), spread onto a 0.2-p.m cellulose acetate membrane (Micro Filtration Systems, Dublin, Calif.), and placed on the agar surface of SC plates (23).…”

Section: Methodsmentioning

confidence: 99%

“…Introduction of genes coding for entomocidal proteins into other host microbes has resulted in expression of larvicidal activity for control of insect pests (14,20,25). Endophytic bacteria such as C. xyli subsp.…”

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Integrative Cloning, Expression, and Stability of the cryIA(c) Gene from Bacillus thuringiensis subsp. kurstaki in a Recombinant Strain of Clavibacter xyli subsp. cynodontis

Lampel¹,

Canter

Dimock

et al. 1994

Appl Environ Microbiol

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A bacterial endophyte was engineered for insecticidal activity against the European corn borer. The crylA(c) gene from Bacillus thuringiensis subsp. kurstaki was introduced into the chromosome of Clavibacter xyli subsp. cynodontis by using an integrative plasmid vector. The integration vectors pCG740 and pCG741 included the replicon pGEM5Zf(+), which is maintained in Escherichia coli but not in C. xyli subsp. cynodontis; tetM as a marker for selection in C. xyli subsp. cynodontis; and a chromosomal fragment of C. xyli subsp. cynodontis to allow for homologous recombination between the vector and the bacterial chromosome. Insertion of vector DNA into the chromosome was demonstrated by DNA hybridization. Recombinant strains MDR1.583 and MDR1.586 containing the cryL4(c) gene were shown to produce the 133,000-kDa protoxin and several smaller immunoreactive proteins. Both strains were equally toxic to insect larvae in bioassays. Significant insecticidal activity was demonstrated in planta. The cryL4(c) gene and the tetM gene introduced into strain MDR1.586 were shown to be deleted from some cells, thereby giving rise to a noninsecticidal segregant population. In DNA hybridization experiments and insect bioassays, these segregants were indistinguishable from the wild-type strain. Overall, these results demonstrate the plausibility of genetically engineered bacterial endophytes for insect control. Clavibacter xyli subsp. cynodontis is a fastidious gram-positive coryneform bacterium that naturally inhabits the xylem of Bermuda grass (Cynodon dactylon L.) (6). C. xyli subsp. cynodontis also colonizes the vascular system of corn (Zea mays L.) when artificially inoculated (16). High populations of C. xyli subsp. cynodontis are distributed in the xylem elements following the introduction of inoculum into the tissues of the whorl or stem of corn seedlings or after germination of corn seed infused with the bacterium. C. xyli subsp. cynodontis does not enter developing kernels and is therefore not present in the progeny seed of inoculated plants (5). Additionally, a DNA transformation system and the development of plasmid cloning vectors have been recently reported (13, 19).

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Formulation of Bacteria, Viruses and Protozoa to Control Insects

Burges¹,

Jones²

1998

Formulation of Microbial Biopesticides

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Formulation of bacteria, viruses and protozoa to control insects 3.6 Application to water 87 3.6.1 Problems of the aquatic environment and its target insects 87 3.6.2 Suspension concentrates and wettable powders 89 3.6.3 Penetration of foliage for mosquito control 90 3.6.4 Floating and slow-release products 91 3.6.5 Encapsulation 95 3.6.6 Monomolecular surface films 96 3.7 Future trends and research in formulation technology 97 3.7.1 Pathogen production 97 3.7.2 Trends in formulation for use on land 99 3.7.2a Products applied dry 99 3.7.2b Products applied as sprays 99 3.7.2c Additives 102 3.7.3 Products for use in water 104 3.7.3a Suspension concentrates and wettable powders 104 3.7.3b Granules, pellets and briqu.ettes 104 3.7.3c Monomolecular layers 106 3.7.3d Bacillus sphaericus 106 3.7.3e Products without live spores 106 3.7.4 Systemic expression of microbial factors 106 3.7.5 Novel strategies for control 108 3.7.6 Research priorities 108 References 109Table 3.2 Production of a Bacillus thuringiensis technical powder by centrifugation and spray-drying fermenter broth (adapted from Lisanky et aI., 1993) Ingredients (Appendix l) Centrifuged broth Aqueous 40% Bevaloid 211 Gum arabic Percentage (w/w) 97 2 1 Function Agent Dispersant Drying protectant Cost ($/kg product) 28.00 0.03 0.03 Preparation 1 Stabilize broth by adjusting pH to 4.1 with 5 M H2S04 2 Spin sample of broth in bench centrifuge at 2: 8000g; supernatant should be clear* 3 Sample for later tests on pH, solids, microbiological purity and cell count 4 Centrifuge at 2: 8000 g and < 35°C with continuous stirring/ agitation of the feed 5 Sample concentrate as for step 2 1 6 Add 2% dispersant and 1% gum arabic or 5% lactose slowly while stirring for 5 min, ensuring that no lumps remain 7 Spray-dry+, using a rotary atomizer, to 6-7% water content; lower levels may cause loss of potency; higher levels will allow caking and contaminants to grow 8 Sieve to < 50lIm and mill oversize material at < 35 DC 9 Bioassay potency of product. The relationship between potency of a 20 gil solids broth and that of the technical powder is about x5 for ssp. kurstaki and x3 for ssp. israelensis * If cloudy, cool sample to 5'C to clear possible interference by antifoam and check microscopically. If there are> 1-2 spores and crystals per field, add 4 mill Superfloc C 577 to bulk broth, but with ssp. israelensis dilute bulk broth to 1.5% with cold water, then add 6 mill of Superfloc to flocculate. t If the concentrate is not to be spray dried immediately, stabilize by adding 2 gIl potassium sorbate and adjust pH to 4.1 ± 0.1 with 5 M H 2 S0 4 , or cool to 5°C. t Spray driers vary. Check residence time of product in drier as over-exposure to heat will lower potency. Typical temperatures are inlet 180-210 °C and outlet 85°C. Experiment with temperatures and throughput rates.Preparation 1 Egg masses of a laboratory strain (New Jersey) of the gypsy moth are held for 150 days at 6 'C to complete diapause 2 Eggs are dehaired (Cosenza et al., 1963;Tardif and Secrest, 1970, modified by...

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Bioencapsulation and delivery to mosquito larvae of Bacillus thuringiensis H14 toxicity by Tetrahymena pyriformis

Cited by 8 publications

References 0 publications

Germination, Growth, and Sporulation of Bacillus thuringiensis subsp. israelensis in Excreted Food Vacuoles of the Protozoan Tetrahymena pyriformis

Germination, Growth, and Sporulation of Bacillus thuringiensis subsp. israelensis in Excreted Food Vacuoles of the Protozoan Tetrahymena pyriformis

Integrative Cloning, Expression, and Stability of the cryIA(c) Gene from Bacillus thuringiensis subsp. kurstaki in a Recombinant Strain of Clavibacter xyli subsp. cynodontis

Formulation of Bacteria, Viruses and Protozoa to Control Insects

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