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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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.
KeywordsActinobacteria Chitinase BOX-PCR Catabolic repression Antifungal activity Gene capture GH18 Streptomyces Micromonospora
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.
This work was funded by the Ministry of Higher Education and Scientific Research, Algeria.
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Conflicts of interest
The authors declare that they have no conflict of interest.
Research involving human participants and/or animals
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