Characterization of lepidopteran-specific cry1 and cry2 gene harbouring native Bacillus thuringiensis isolates toxic against Helicoverpa armigera

Lone, Showkat Ahmad; Malik, Abdul; Padaria, Jasdeep Chatrath

doi:10.1016/j.btre.2017.05.001

Cited by 7 publications

(3 citation statements)

References 33 publications

(31 reference statements)

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“…The insecticidal activity of spore/crystal mixtures is an effective way to search for native Bt isolates having new entomocidal properties [ 7 , 32 , 34 , 35 , 36 ]. In our study, preliminary assays indicated that some of the 18 selected isolates in the collection had high toxicity against Lepidoptera.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Bacillus thuringiensis isolates by their insecticidal activity and their production of Cry and Vip3 proteins

et al. 2018

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Bacillus thuringiensis (Bt) constitutes the active ingredient of many successful bioinsecticides used in agriculture. In the present study, the genetic diversity and toxicity of Bt isolates was investigated by characterization of native isolates originating from soil, fig leaves and fruits from a Turkish collection. Among a total of 80 Bt isolates, 18 of them were found carrying a vip3 gene (in 23% of total), which were further selected. Insecticidal activity of spore/crystal mixtures and their supernatants showed that some of the Bt isolates had significantly more toxicity against some lepidopteran species than the HD1 reference strain. Five isolates were analyzed by LC-MS/MS to determine the Cry protein composition of their crystals. The results identified the Cry1Ac protein and a Cry2A-type protein in all isolates, Cry1Ea in 3 of them and Cry1Aa in one. The sequence analysis of the new vip3 genes showed that they had a high similarity to either vip3Aa, vip3Af or vip3Ag (94–100%). The vip3Aa gene of the 6A Bt isolate was cloned and sequenced. The protein was named Vip3Aa65 by the Bacillus thuringiensis Nomenclature Committee. The expressed and purified Vip3Aa65 protein was tested against five lepidopteran species and its toxicity compared to that of a reference protein (Vip3Aa16). Both proteins had similar toxicity against Grapholita molesta and Helicoverpa armigera, whereas Vip3Aa65 was less active than Vip3Aa16 against three species from the Spodoptera genus. A tetrameric structure of the Vip3Aa65 protein was detected by gel filtration chromatography. The study revealed some isolates with high insecticidal activity which can be considered promising candidates to be used in pest control.

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Section: Discussionmentioning

confidence: 99%

Characterization of Bacillus thuringiensis isolates by their insecticidal activity and their production of Cry and Vip3 proteins

et al. 2018

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“…In contrast to synthesis crystal proteins, it controls protein synthesis, including exotoxins, chitinases and phospholipases (Chelliah et al, 2019;Wei et al, 2019). The Cry2 gene encodes the Cry2A crystal protein, a parasporal crystal protein with insecticide activity (Lone et al, 2017;Palma et al, 2014). XRE and cry2 genes can be used as target genes to identify Bt (Wei et al, 2019).…”

Section: Bacterial Isolation and Identificationmentioning

confidence: 99%

Genotype and virulence gene analyses of Bacillus cereus group clinical isolates from the Chinese softshell turtle (Pelodiscus sinensis) in Taiwan

Cheng

Rao

Poudyal

et al. 2021

Journal of Fish Diseases

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Chinese softshell turtles (Pelodiscus sinensis) (CST) are susceptible to infections by bacteria belonging to the Bacillus cereus group (Bcg). Bcg includes several closely related species, two of which, B. cereus and B. thuringiensis, are pathogens of aquatic animals or insects. In the present study, we collected 57 Bcg isolates obtained from diseased CST from 2016 to 2019 in Kaohsiung and Pingtung, the areas with the most CST farms in Taiwan. All isolates were divided into four genotypes with two restriction enzymes, SmaI and NotI, by pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR).Representative isolates from each genotype were subjected to phylogenetic tree analysis using 16S rDNA and pyruvate carboxylase genes as phylogenetic markers, and these CST isolates appeared in different clades. PCR was performed targeting six selected virulence genes, four of which were detected in CST isolates, including cytotoxin K (1/57), hblC of the haemolysin BL complex (46/57), nheA of the nonhaemolytic enterotoxin complex (52/57) and enterotoxin FM (57/57), whereas cereulide synthetase and cereulide peptide synthase-like genes were not detected in any isolates.

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“…Phylogenetic trees were constructed using MEGA 5 following the multiple alignments of the sequences by Cluster W. The trees were constructed using the neighbor-joining (NJ) method and the maximum parsimony option model, with 1000 bootstrap replicates to estimate the support for each branch Cry-gene specific and competition-binding studies were carried out with lectins, and specific sugars confirmed that Cry1Ac and Cry1Fa share similar numbers of the midgut binding sites of both Anticarsia gemmatalis and Chrysodeixis includes (Bel et al 2017). Also, 5 pairs of universal primers have been used to detect the highly conserved Cry-gene sequences and differentiate 34 out of about 60 genes known in the following groups: 20 Cry1, 3 Cry2, 4 Cry3, 2 Cry4, 2 Cry7, and 3 Cry8 genes (Ben-Dov et al 1997); PCR screening using universal primers of two pairs to detect Cry1 and Cry2 gene families in their collection is performed by Lone et al (2017). Therefore, Cry1 protein and other Cry proteins found in the 12 Bt isolates could have similar binding sites on the midguts of S. littoralis and D. melanogaster.…”

Section: Discussionmentioning

confidence: 99%

Isolation and identification of Bacillus thuringiensis strains native of the Eastern Province of Saudi Arabia

Hassan

Ismail

Elashtokhy

et al. 2021

Egypt J Biol Pest Control

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Background Bacillus thuringiensis (Bt) produces a group of δ-endotoxin proteins designated as cry toxins. No doubt that Bt isolates are excellent potential candidates for biological control strategies. Results The present study showed that 12 Bt strains were isolated and characterized at morphological, biochemical, and molecular levels. All the tested 12 Bt strains were gram-positive, endospore-forming, and possessing typical Bt crystal structures under the scanning electron microscopy (SEM). Universal primers direct and reverse of five pairs were used to detect five Cry-type genes (Cry1, Cry2, Cry3, Cry4, Cry7, and Cry8) by the PCR sizes produced from the studied Bt strains. The 16S rRNA PCR technique, 16S gene primer, DNA template, dNTPs, and Taq polymerase produced unique and distinguishable restriction patterns used for the molecular characterization of the studied Bt strains. Based on the PCR products, the frequency of Cry-gene distribution among the tested strains was Cry1 100%, Cry4 85%, and Cry3 62%, and Cry2 and Cry7 frequency was 54%. Based on the activity of insecticidal of the tested Bt strains, Bt1, Bt9, Bt10, and Bt11 were extremely pathogenic; their pathogenicity ranged from 93 to 100% against dipteran and lepidopteran larvae, compared to the other Bt isolates. The nucleotide sequences of amplified 1500 bp conserved region of 16S rRNA genes of four strains blasted using NCBI database compared to NCBI database sequences, and they were reported as native strains of Bt showing high homology with the known Bt strains (99–100%). The nucleotide sequences of Bt1, Bt9, Bt10, and Bt11 were placed in the GenBank database under accession numbers MN860017, MN843958, MN843959, and MN843960, respectively. Conclusion The strategies of enhancing the sustainability of crops and vegetables that are targeted by a large number of pathogenic insects require a great effort of exploring novel species and strains of Bt. Herein, native strains of Bt were documented from the eastern province of Saudi Arabia that displayed bio-insecticidal action on larvae of Diptera and Lepidoptera.

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Characterization of lepidopteran-specific cry1 and cry2 gene harbouring native Bacillus thuringiensis isolates toxic against Helicoverpa armigera

Cited by 7 publications

References 33 publications

Characterization of Bacillus thuringiensis isolates by their insecticidal activity and their production of Cry and Vip3 proteins

Characterization of Bacillus thuringiensis isolates by their insecticidal activity and their production of Cry and Vip3 proteins

Genotype and virulence gene analyses of Bacillus cereus group clinical isolates from the Chinese softshell turtle (Pelodiscus sinensis) in Taiwan

Isolation and identification of Bacillus thuringiensis strains native of the Eastern Province of Saudi Arabia

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