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Chitinolytic actinobacteria isolated from an Algerian semi-arid soil: development of an antifungal chitinase-dependent assay and GH18 chitinase gene identification

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Abstract

The purpose of this study was to explore the microbial potential of a semi-arid sandy soil from south-central Algeria in order to isolate new chitinolytic actinobacteria. This soil is subjected to high temperatures (up to 43 °C) and has low nutrient content. Strains were isolated by plating soil suspensions on Bennett and Colloidal Chitin (CCM) medium. An initial clustering of isolates was made through BOX-PCR genetic profiling. Next, a 16S rRNA gene sequencing of representative isolates was realized. We also identified optimum physicochemical conditions for chitinolytic activity. A rapid in vitro assay based on glucose catabolic repression was developed to select isolates having a chitinase-dependent antifungal activity against two phytopathogenic fungi. Gene identification of glycosyl hydrolase family 18 (GH18) permitted us to assess the divergence of chitinase genes. Forty isolates were obtained from the semi-arid sandy soil. The molecular identification permitted us to assign them to Streptomyces or Micromonospora genera with seven possibly new bacterial species. For chitinolytic activity, 100% of isolates were able to grow and degrade colloidal chitin at pH 7 and at a temperature ranging from 30 to 40 °C. We also observed that Micromonospora strains had atypical activity patterns, with a strong chitinase activity maintained at high temperature. Finally, three strains presented an interesting chitinolytic potential to reduce fungal growth with new GH18 sequences. This study presents a new rapid method to detect antifungal chitinase-dependent activity that allowed to identify potentially new species of actinobacteria and new GH18 gene sequences.

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Acknowledgments

This work was supported by University Frères Mentouri Constantine1 (Algeria). LC thanks the Spanish government for a postdoctoral fellowship (Programa Nacional de Movilidad de Recursos Humanos del Plan Nacional de I-D+i 2008–2011). We thank Prof. A. Boulahrouf (Constantine University) for the help with this work. Thanks are expressed to France Chitin (www.france-chitine.com) for the gift of chitin. We thank Dr Feteh-el-Zahar Haichar (University of Lyon) for the technical advice and Dr Marc Bardin (INRA, Avignon) and Dr. Merouane Fateh (Constantine University) for the gift of Botrytis cinerea Bc21 and Fusarium oxysporum, respectively.

Funding

This work was funded by the Ministry of Higher Education and Scientific Research, Algeria.

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  1. Lorena Carro

    Present address: Departamento de Microbiología y Genética, Universidad de Salamanca, Salamanca, Spain

Authors and Affiliations

  1. Laboratoire de Génie microbiologique et applications, Université des Frères Mentouri Constantine 1, Campus Chaâbat Erssas, 25000, Constantine, Algeria

    Meriem Gasmi

  2. Laboratoire de Biologie appliquée et Santé, Université des Frères Mentouri Constantine 1, Route Ain El Bey, 25000, Constantine, Algeria

    Meriem Gasmi & Mahmoud Kitouni

  3. Ecologie Microbienne, Université Lyon 1, Université de Lyon, CNRS, UMR 5557, USC INRA 1193, Villeurbanne, F69622, Villeurbanne Cedex, France

    Lorena Carro, Petar Pujic, Philippe Normand & Hasna Boubakri

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Fig. A1 (a) Isolation of actinobacteria on MCC media. (b) Macroscopic aspect of isolates (C13, C28, C33, and C37) cultivated on ISP-2 medium. Fig. A2 Light microscopy observations on the representative strains of Streptomyces (C27) (a) and Micromonospora (C33) (b). Micromonospora is characterized by the presence of individual spores formed at the tip of branched mycelium, not found in the other strains close to Streptomyces. Fig. A3 Chitinolytic activity measured at different pH after 7 days of growth on CCM media. (#) significant difference was observed between two conditions (Wilcoxon rank sum tests, p-value < 0.05). Fig. A4 Chitinolytic activity measured at different temperatures after 7 days of growth in CCM media and sorted according their activity at 45°C. Strains on the left of the graph (C27, C39 and C40) did not grow at 45°C and strains on the right of the graph (from C1 to S. coelicolor) did not show chitinase activity at 45°C. Better score of activity was found for six strains at 30°C (C3-C4-C13-C19-C32-C34), and one strain at 45°C (C37). (#) significant difference was observed between two conditions yielding the highest activity (Wilcoxon rank sum tests, p-value < 0.05) (PPTX 1328 kb)

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Gasmi, M., Kitouni, M., Carro, L. et al. Chitinolytic actinobacteria isolated from an Algerian semi-arid soil: development of an antifungal chitinase-dependent assay and GH18 chitinase gene identification. Ann Microbiol 69, 395–405 (2019). https://doi.org/10.1007/s13213-018-1426-z

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