DETECTION AND IDENTIFICATION OF POTATO SOFT ROT PECTOBACTERIUM CAROTOVORUM SUBSP. CAROTOVORUM (DYE1969) BY PCR USING DIFFERENT SETS OF PRIMERS

Dr. Ibtihal Abu-Obeid, Hamed khlaif, Nida salem

Abstract


Various bacterial species are known to be agents causing soft rot of potatoes. In this study different detection methods were used to investigate these bacteria.
Two hundreds and four rotted potato samples were collected from different potato growing areas including: Amman, AR-Ramtha, Jordan Valley, Ma'an, Madaba, Mafraq, and commercial stores through different growing seasons in these areas. One hundred and thirty one bacterial isolates were identified as Pectobacterium carotovorum subspecies carotovorum (Pcc) by different biochemical and physiological tests. Results of reactions of the tested bacteria were found to be identical to the Pcc reference culture (NCPPB312) for the same tests.
Furthermore, the identity of the 131 Pcc Jordanian (Jo) isolates were identified by PCR analysis of total DNA extracted from isolates that biochemically identified as Pcc using different sets of primers. Primers used were; 16S rDNA universal primer Fd1/Rd1, species-specific primer ExpccF/ExpccR, recAF/recAR and pathogencity gene pmrAF/pmrAR. Sequencing of representative PCR products amplified with all sets of primers were done.
In addition to Pcc, different rots causal agents were detected by PCR amplification and further sequencing. The sequencing data revealed similarities to Pseudomonas fluorescence, Enterobacteriaceae genera such as Enterobacter spp., Serratia spp. and Klebsiella spp.,
The four sets of primers varied in their specificity in detection soft rot pathogens, where recA and pmrA sets of primer were found to be more efficient in detecting Pcc. On the other hand, Expcc sets of primer were not so specific in detecting Pcc isolates where it was able to detect only 51% of the isolates.
This study indicated that using molecular techniques such as amplification of different regions by using different sets of primers is sensitive and specific for detecting Pcc as potato soft rot causal agent. The results of this study showed that potato soft rot is widely spread in different potato planting areas in Jordan.

Keywords


Pectobacterium carotovorum subsp. carotovorum (Pcc), soft rot of potato, PCR, sequencing, detection

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