Molecular characterization, identification and detection of Xanthomonas axonopodis pv. manihotis, the causal agent of cassava bacterial blight disease in cassava (Manihot esculenta C.) by real - time pcr and pulsed-field gel electrophoresis

Cassava (Manihot escualanta Crantz) is one of the most important plant cultivar in Ghana. In this study, cassava samples showing symptoms of Cassava Bacterial Blight diseases were collected from eight different regions of Ghana. The putative pathogen was isolated and cultured on the Xanthomonas axonopodis pv. manihotis (Xam) semi-selective, Cefazolin Trehalose Agar (CTA) and Nutrient Agar (NA) media. The pathogenicity test of all the 32 isolates on the susceptible Esam cassava variety produced symptoms typical of Xam and was consistently re-isolated from the inoculated cassava plants and thereby satisfying the Koch's postulates. The classical PCR was conducted using Xanthomonas genus specific primer RST2/RST3 and Xam specific Variable Number Tandem Repeat (VNTRs) locus primer, XaG1_67F/R, which yielded 840 and 446 bp amplification products, respectively. The resulting PCR products were sequenced and analyzed using a BLASTn program, which revealed homology between 93 to 100% with several other Xam strains retrieved from the GenBank nucleotide database. Xanthomonas axonopodis pv. manihotis causing serious yield loss on cassava cultivation has been detected in a short time with a Real-Time PCR method, using the developed probe (LNA, Locked Nucleic Acid) and primer sets. The specificity of the developed probe and primer sets in this study were tested against different Xam strains, various other plant pathogenic bacteria from different genus and species and cassava genomic DNA. The Xam isolates were sensitively and selectively detected by Real-Time PCR using LNA probes. The results of the PCR analysis showed that Ashanti region had the highest incidence of CBB, which recorded 70%, followed by Volta region (60%); Brong Ahafo region (40%); Eastern region (40%) and Greater Accra region (20%). The developed method in this study was used for the detection of Xanthomonas axonopodis pv. manihotis from cell suspension, which showed sensitivity limit of 1 cells, whereas, the sensitivity of the detection limit of the genomic DNA in picogram (pg) of the bacterial was also found to be 13 pg. In conclusion, Real-Time PCR method using specific primer and probe sets detected Xanthomonas axonopodis pv. manihotis causing Cassava Bacterial Blight disease from both bacterial cell and diseased cassava plant tissue. The developed method was very quick and sensitive for the identification and detection of this pathogen within relatively short time (20-25 min). The genotypic differences between the Xam isolates isolated from the five most cassava producing regions of Ghana were examined by Pulsed-Field Gel Electrophoresis (PFGE) after digesting the genome with the rare-cutting restriction enzymes. The genetic differences between the 32 isolates collected from Ghana were determined by PFGE using SpeI enzyme. All the isolates were grouped into two different haplotypes by the PFGE.