A new diagnostic tool for simultaneous detection of Erwinia amylovora and Ralstonia solanacearum

Erwinia amylovora and Ralstonia solanacearum are polyphagous plant pathogenic bacteria included in the EPPO A2 list. To prevent their introduction and spread in new areas, their monitoring is crucial. This study aimed to develop a rapid and sensitive diagnostic assay for the simultaneous quantification of the two bacteria. Two primers/probe sets reported in the EPPO guidelines and targeting the amsC gene of E. amylovora and the 16S rRNA region of R. solanacearum were marked with two different fluorophores (FAM and HEX) and assessed in duplex qPCR and ddPCR assays. The specificity of both qPCR and ddPCR was confirmed against a panel of six bacteria and fifteen fungi mainly responsible for common diseases in nurseries. The detection limit for the duplex qPCR assay was 100 fg of DNA for both the bacterial species, while ddPCR resulted 10-fold more sensitive, quantifying < 1 copy μL–1 of each E. amylovora and R. solanacearum. Three different protocols for DNA extraction from wood of Lantana camara were tested in qPCR assays for in-field diagnostic tests, using A) Sample Homogenization and M1 Sample Prep Cartridge kit for DNA (Biomeme); B) QuickPick SML Plant DNA Kit (BN Products & Services); and C) REDExtract-N-Amp Plant PCR Kit (Sigma-Aldrich). Detection limits of five and ten cells for reaction were obtained with protocols C and B, respectively, while protocol A was less sensitive (> 20 cells per reaction). Protocols A and C were more accessible and more rapid (5 min and 15 min, respectively) than protocol B (50-60 min).