Molecular Analysis of Genetic Diversity of Pectobacterium carotovorum Subsp Carotovorum Isolated in Morocco by PCR Amplification of the 16S-23S Intergenic Spacer Region

Abstract

Aims: Pectobacterium carotovorum is a ubiquitous bacterium that causes soft rot in different crop plants throughout the world. In Morocco, approximately 95% of the Strains isolates from potato plants with tuber soft rot are P. carotovorum. In this study, we test whether PCR ribotyping can be used to distinguish strains of Pectobacterium carotovorum isolated from soft rot potato and to differentiate among strains from different geographic regions. Place and Duration of Study: Laboratory of Virology, Microbiology and Quality / Ecotoxicology and Biodiversity, department Biology, Faculty of Sciences and Techniques, University Hassan-II Mohammedia Casablanca. Methodology: Eighty-three pectolytic enterobacteria were collected from potatoes rotten in Morocco, the strains were isolated in the Cristal Violet Pectate (CVP) medium and were purified in Original Research Article Amdan et al.; BBJ, 7(3): 102-110, 2015; Article no.BBJ.2015.051 103 LPGA agar (yeast extract, peptone, glucose and agar). After purification, strains were identified by physiological and biochemical tests. The confirmation of species was performed by PCR using primers Y1 and Y2. The genetic diversity of Pectobacterium carotovoum was investigated by PCR ribotyping using primers G1/L1, which are complementary to conserved regions of the rRNA operon. Furthermore, the profiles obtained were compared by the Unweighted Pair Group Method. Results: The biochemical and physiological analysis demonstrated that the predominant pectolytic enterobacterium present in Morocco is Pectobacterium carotovorum subsp carotovorum. The specific confirmation of species P. carotovorum by PCR has yielded a 434 bp DNA fragment of the pelY gene with all isolates. Further, PCR amplification of the 16S-23S Intergenic spacer Region (ITS-PCR) has presented a specific pattern made of 2-6 fragments ranging from 300 bp to 800 bp. The UPGMA tree has shown that there is considerable genetic diversity in P. carotovorum strains, which can be divided into four distinct groups.