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Evaluation of the Toxicity of Supernatant Cultures and Spore–Crystal Mixtures of Bacillus thuringiensis Strains Isolated from Algeria

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Abstract

Bacillus thuringiensis (Bt) is the most used technology for biological control of insect pathogens worldwide. In order to select new Bt candidates challenging the emergence of insect’s resistance, a mass bioassay and molecular screening was performed on an autochthonous collection. Toxicity assays against neonate larvae of three lepidopteran species (Mamestra brassicae, Grapholita molesta, and Spodoptera exigua) were conducted using spore–crystal mixtures and supernatant cultures of 49 Bt isolates harboring at least one gene coding for a lepidopteran-specific insecticidal protein. A threshold of 30% of “functional mortality” was used to discriminate between “nontoxic” and “toxic” isolates. The toxicity of many Bt isolates competed with that of Btk-HD1. However, only three of them (Bl4NA, Bl5NA, and Bl9NA) showed high toxicity in both spore–crystal mixtures and supernatant cultures against the three lepidopteran species. The Bt isolates Bl4NA and Bl9NA express a protein of 130 kDa whereas the Bt isolate Bl5NA expresses a protein of 65–70 kDa. The LC–MS/MS results indicate that the major peptides in the 130 kDa band of Bl9NA were Cry1Da, Cry1Ca, Cry1Ab, and Cry1Aa, and those in the 70 kDa band of Bl5NA were Cry1Aa and Cry1Ca. The evaluation of the protein content of the supernatants by comparison to Btk-HD1 indicates the overproduction of Vip3 proteins in these strains (most likely Vip3Aa in Bl4NA and Bl9NA and Vip3Ca in Bl5NA). In addition, these three Bt strains do not produce β-exotoxins. Based on our results, the three selected strains could be considered promising candidates to be used in insect pest control.

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Acknowledgements

We thank Dr. Yolanda Bel for her valuable comments related to toxicity bioassays, Dr. Joaquín Gomis-Cebolla for his help in the analysis of the proteomics data, and Dr. Patricia Hernández-Martínez and Daniel Pinos for their help in SDS-PAGE and Dot-Blot assays. We thank Rosa Maria González-Martínez for her support in laboratory assistance and Oscar Marín-Vázquez for his help with insect rearing. This work was supported by PNE research grant from the Algerian Ministry of Higher Education and Scientific Research (n° 100/PNE/ENS./ESPAGNE/2015–2016), by the Spanish Ministry of Science and Innovation (Grant Ref. AGL2015-70584-C2-1-R), by a Grant of the Generalitat Valenciana (GVPROMETEOII-2015-001) and by European FEDER funds. The proteomic analysis was performed in the proteomics facility of SCSIE University of Valencia that belongs to ProteoRed, PRB2-ISCIII, supported by Grant PT13/0001 of the PE I+D+i 2013–2016, funded by ISCIII and FEDERPT13/0001.

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Authors and Affiliations

  1. Microbiology Group, Laboratory of Cellular and Molecular Biology, Faculty of Biological Sciences, USTHB, El Alia, BP 32, 16111, Bab Ezzouar, Algiers, Algeria

    Zahia Djenane & Farida Nateche

  2. ERI de Biotecnología y Biomedicina (BIOTECMED), Department of Genetics, Universitat de València, 46100, Burjassot, Spain

    Zahia Djenane, Maria Lázaro-Berenguer & Juan Ferré

  3. Department of Biology, Faculty of Sciences, University Yahia Farès, 26000, Médéa, Algeria

    Zahia Djenane

Authors
  1. Zahia Djenane
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  2. Maria Lázaro-Berenguer
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  3. Farida Nateche
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  4. Juan Ferré
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Contributions

JF and FN conceived and designed the experiments; ZD and ML-B performed the insecticidal and molecular characterization of the strains; ZD, ML-B, and JF analyzed the results and wrote the manuscript.

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Correspondence to Juan Ferré.

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Animal ethics approval is not required for studies using Mamestra brassicae, Grapholita molesta, and Spodoptera exigua invertebrates.

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Djenane, Z., Lázaro-Berenguer, M., Nateche, F. et al. Evaluation of the Toxicity of Supernatant Cultures and Spore–Crystal Mixtures of Bacillus thuringiensis Strains Isolated from Algeria. Curr Microbiol 77, 2904–2914 (2020). https://doi.org/10.1007/s00284-020-02110-3

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