Optimising PCR Detection of Ralstonia solanacearum and Pseudomonas savastanoi pv. savastanoi: Two Models, Two Approaches

Abstract

Available protocols and primers for PCR detection of Ralstonia solanacearum on water samples and Pseudomonas savastanoi pv. savastanoi on asymptomatic olive trees have relatively low reliability because of sensitivity and specificity problems.

In order to improve PCR detection of R. solanacearum in water samples a recently described Co-operational amplification technique (Co-PCR) was applied. The simultaneous use of OLI-1, OLI-2 and JE2 primers improved the detection, giving an amplification product of 408 bp and a sensitivity of < 1 CFU m1⁻¹ of water. This method was used for analysis of water samples from several origins and it was the most efficient for R. solanacearum detection when comparing to isolation and to previously described PCR protocols.

The PCR detection of P. s. pv. savastanoi by using IAALF and IAALR primers was improved with an efficient DNA extraction protocol (after pre-enrichment of the target bacterial population in PVF-1 liquid medium), designing IAALN1 and IAALN2 internal primers for a nested PCR in a single closed tube and by the colorimetric detection of the amplicon of 338 bp. Moreover, the four primers were included in a multiplex nested RT-PCR protocol that allows the simultaneous detection of the bacterial target and four RNA viruses in olive plants. Sensitivity in spiked samples was up to 1 CFU m1⁻¹ of plant extract by either method and both were successfully applied to the detection of P. s. pv. savastanoi on asymptomatic shoots from olive plants grown in orchards and nurseries.