An improved bioassay for the detection of Bacillus thuringiensis β-exotoxin

Innes, Thomas Cameron Mac; Bouwer, Gustav

doi:10.1016/j.jip.2009.03.007

Cited by 15 publications

(14 citation statements)

References 12 publications

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“…The phospholipases, haemolysins, enhancin-like proteins and chitinases are synthesized in the insect midgut during vegetative growth, facilitating the dissemination of the bacteria through the peritrophic membrane and towards the hemocoel [1,17]. The β-exotoxins, also known as thuringiensins, are secreted, heat-stable, secondary metabolites analogues to the nucleotide adenine and with low molecular weight (~700 Da) [18]. …”

Section: Introductionmentioning

confidence: 99%

Draft Genome Sequences of Two Bacillus thuringiensis Strains and Characterization of a Putative 41.9-kDa Insecticidal Toxin

Palma

Muñoz

Berry

et al. 2014

Toxins

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In this work, we report the genome sequencing of two Bacillus thuringiensis strains using Illumina next-generation sequencing technology (NGS). Strain Hu4-2, toxic to many lepidopteran pest species and to some mosquitoes, encoded genes for two insecticidal crystal (Cry) proteins, cry1Ia and cry9Ea, and a vegetative insecticidal protein (Vip) gene, vip3Ca2. Strain Leapi01 contained genes coding for seven Cry proteins (cry1Aa, cry1Ca, cry1Da, cry2Ab, cry9Ea and two cry1Ia gene variants) and a vip3 gene (vip3Aa10). A putative novel insecticidal protein gene 1143 bp long was found in both strains, whose sequences exhibited 100% nucleotide identity. The predicted protein showed 57 and 100% pairwise identity to protein sequence 72 from a patented Bt strain (US8318900) and to a putative 41.9-kDa insecticidal toxin from Bacillus cereus, respectively. The 41.9-kDa protein, containing a C-terminal 6× HisTag fusion, was expressed in Escherichia coli and tested for the first time against four lepidopteran species (Mamestra brassicae, Ostrinia nubilalis, Spodoptera frugiperda and S. littoralis) and the green-peach aphid Myzus persicae at doses as high as 4.8 µg/cm2 and 1.5 mg/mL, respectively. At these protein concentrations, the recombinant 41.9-kDa protein caused no mortality or symptoms of impaired growth against any of the insects tested, suggesting that these species are outside the protein’s target range or that the protein may not, in fact, be toxic. While the use of the polymerase chain reaction has allowed a significant increase in the number of Bt insecticidal genes characterized to date, novel NGS technologies promise a much faster, cheaper and efficient screening of Bt pesticidal proteins.

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Section: Introductionmentioning

confidence: 99%

Draft Genome Sequences of Two Bacillus thuringiensis Strains and Characterization of a Putative 41.9-kDa Insecticidal Toxin

Palma

Muñoz

Berry

et al. 2014

Toxins

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“…B. thuringiensis CT-43 contains 140-kDa, 130-kDa, and 68-kDa ICPs and is highly toxic to lepidopterous and dipterous insects (9). More importantly, it can produce high levels of the nucleotide analogue thuringiensin (4), which has insecticidal activities against a wide range of insects (6).…”

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confidence: 99%

Complete Genome Sequence of Bacillus thuringiensis subsp. chinensis Strain CT-43

Wang

Yin

et al. 2011

J Bacteriol

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Bacillus thuringiensis has been widely used as an agricultural biopesticide for a long time. As a producing strain, B. thuringiensis subsp. chinensis strain CT-43 is highly toxic to lepidopterous and dipterous insects. It can form various parasporal crystals consisting of Cry1Aa3, Cry1Ba1, Cry1Ia14, Cry2Aa9, and Cry2Ab1. During fermentation, it simultaneously generates vegetative insecticidal protein Vip3Aa10 and the insecticidal nucleotide analogue thuringiensin. Here, we report the finished, annotated genome sequence of B. thuringiensis strain CT-43.

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“…Two other Btt strains, HD125 and T09, derived from temperate climates (USA and France, respectively), are known to produce β-exotoxins [34,35]. In order to confirm whether the Btt 01 strain from a tropical origin (Foz do Iguaçu-PR, Brazil) also produces this same type of toxin, an experiment was carried out to compare the toxic activities of these strains.…”

Section: Resultsmentioning

confidence: 99%

Requirement of Simultaneous Assessment of Crystal- and Supernatant-Related Entomotoxic Activities of Bacillus thuringiensis Strains for Biocontrol-Product Development

Argôlo-Filho

Costa

Pinheiro

et al. 2014

Toxins

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Bioinsecticides with lower concentrations of endospores/crystals and without loss of efficiency are economically advantageous for pest biocontrol. In addition to Cry proteins, other Bacillus thuringiensis (Bt) toxins in culture supernatants (SN) have biocontrol potential (e.g., Vip3A, Cry1I, Sip1), whereas others are unwanted (β-exotoxins), as they display widespread toxicity across taxa. A strain simultaneously providing distinct toxin activities in crystals and SN would be desirable for bioinsecticides development; however, strains secreting β-exotoxins should be discarded, independently of other useful entomotoxins. Entomotoxicity of crystals and SN from a Brazilian Bt tolworthi strain (Btt01) was tested against Spodoptera frugiperda to assess the potential for biocontrol-product development based on more than one type of toxin/activity. Tests showed that 107 endospores mL−1 caused >80% of larvae mortality, suggesting Btt01 may be used in similar concentrations as those of other Bt-based biopesticides. When it was applied to cornfields, a significant 60% reduction of larvae infestation was observed. However, bioassays with Btt01 SN revealed a thermostable toxic activity. Physicochemical characterization strongly suggests the presence of unwanted β-exotoxins, with isolate-specific temporal variation in its secretion. Knowledge of the temporal pattern of secretion/activity in culture for all forms of toxins produced by a single strain is required to both detect useful activities and avoid the potential lack of identification of undesirable toxins. These findings are discussed in the contexts of commercial Bt product development, advantages of multiple-activity strains, and care and handling recommended for large-scale fermentation systems.

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An improved bioassay for the detection of Bacillus thuringiensis β-exotoxin

Cited by 15 publications

References 12 publications

Draft Genome Sequences of Two Bacillus thuringiensis Strains and Characterization of a Putative 41.9-kDa Insecticidal Toxin

Draft Genome Sequences of Two Bacillus thuringiensis Strains and Characterization of a Putative 41.9-kDa Insecticidal Toxin

Complete Genome Sequence of Bacillus thuringiensis subsp. chinensis Strain CT-43

Requirement of Simultaneous Assessment of Crystal- and Supernatant-Related Entomotoxic Activities of Bacillus thuringiensis Strains for Biocontrol-Product Development

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