Discrimination of Ralstonia solanacearum isolates by genetic signatures produced by single-strand conformation polymorphism and low-stringency single specific primer PCR analysis

摘要

Development of molecular biology based techniques have led to reliable characterization and determination of the genetic diversity among phytopathogens. Single-strand conformation polymorphism (SSCP) and Low-stringency single specific primer (LSSP)-PCR were assessed for genetic typing of Ralstonia solanacearum isolates from suspicious bacterial wilt fields. R. solanacearum isolates obtained were amplified by colony PCR analysis with egl specific primers which amplified a PCR product of 237 bp. These amplified products were denatured and separated in a polyacrylamide gel to develop PCR-SSCP fingerprints, which confirms R. solanacearum by producing similar four banding patterns. The amplified product of colony-PCR was subsequently used as a template for LSSP-PCR analysis. The individual genotyping of each R. solanacearum obtained by LSSP-PCR were able to discriminate solanaceae and ginger isolates into two different clusters along with pathogenic and non-pathogenic. The LSSP-PCR profile of R. solanacearum isolates were closely related and evolved by the genome of host origin and diverge in genomic stability which was further confirmed by sequence analysis. In conclusion, SSCP and LSSP-PCR techniques were most effective compared to biochemical and physiological assays for identification and genetic variability in R. solanacearum, which has high genetic divergence. The rapid identification of R. solanacearum plays a crucial role in prevention of bacterial wilt.