Aims Dickeya species are high consequence plant pathogenic bacteria; associated with potato disease outbreaks and subsequent economic losses worldwide. Early, accurate and reliable detection of Dickeya spp. is needed to prevent establishment and further dissemination of this pathogen. Therefore, a multiplex TaqMan qPCR was developed for sensitive detection of Dickeya spp. and specifically, Dickeya dianthicola. Methods and Results A signature genomic region for the genus Dickeya (mglA/mglC) and unique genomic region for D. dianthicola (alcohol dehydrogenase) were identified using a whole genome‐based comparative genomics approach. The developed multiplex TaqMan qPCR was validated using extensive inclusivity and exclusivity panels, and naturally/artificially infected samples to confirm broad range detection capability and specificity. Both sensitivity and spiked assays showed a detection limit of 10 fg DNA. Conclusion The developed multiplex assay is sensitive and reliable to detect Dickeya spp. and D. dianthicola with no false positives or false negatives. It was able to detect mixed infection from naturally and artificially infected plant materials. Significance and Impact of the Study The developed assay will serve as a practical tool for screening of propagative material, monitoring the presence and distribution, and quantification of target pathogens in a breeding programme. The assay also has applications in routine diagnostics, biosecurity and microbial forensics.

AimsDickeya species are high consequence plant pathogenic bacteria listed among the quarantine pathogens of the European Union; associated with potato disease outbreaks and subsequent economic losses worldwide. Early, accurate, and reliable detection of Dickeya spp. is needed to prevent establishment and further dissemination of this pathogen. Therefore, a multiplex TaqMan qPCR was developed for sensitive detection of Dickeya spp. and specifically, D. dianthicola. Methods and ResultsA signature genomic region for the genus Dickeya (mglA/mglC) and unique genomic region for D. dianthicola (alcohol dehydrogenase) were identified using a whole genome based comparative genomics approach. The developed multiplex TaqMan qPCR was validated using extensive inclusivity and exclusivity panels, and naturally/artificially infected samples to confirm broad range detection capability and specificity. Both sensitivity and spiked assays showed detection limit of 10 fg DNA. ConclusionThe developed multiplex assay is sensitive and reliable to detect Dickeya spp. and D. dianthicola with no false positives or false negatives. It was able to detect mixed infection from naturally and artificially infected plant materials. Significance and ImpactThe developed assay will serve as a practical tool for screening of propagative material, monitoring the presence and distribution, and quantification of target pathogens in a breeding program. The assay also has applications in routine diagnostics, biosecurity and microbial forensics.

Aim:The newly defined species Pectobacterium parmentieri has emerged as an aggressive pathogen that causes soft rot and blackleg diseases on potato and has been widely disseminated across the globe, jeopardizing the productivity and potato food safety. The implementation of a fast and accurate detection tool is imperative to control, monitor and prevent further spread of these pathogens. The objective of this work was to develop a specific and sensitive multiplex TaqMan qPCR to detect P. parmentieri and distinguish it from all known Pectobacterium species. A universal internal control was included to enhance the reliability of the assay. Methods and Results:A comparative genomics approach was used to identify Oacetyltransferase and the XRE family transcriptional regulator as specific targets for primers/probe design for the detection of the Pectobacterium genus and P. parmentieri, respectively. Specificity was assessed with 35 and 25 strains included in the inclusivity and exclusivity panels, respectively, isolated from different geographical locations and sources. The assay specifically detected all 35 strains of Pectobacterium sp. and all 15 P. parmentieri strains. No cross-reactivity was detected during assay validation. Our assay detected up to 10 fg genomic DNA and 1 CFU ml −1 bacterial culture. No change in the detection threshold (1 CFU ml −1 ) was observed in spiked assays after adding host tissue to the reactions. The assay was validated with naturally and artificially infected host tissues and soil rhizosphere samples. All infected plant samples containing the target pathogens were accurately amplified. Conclusion:The presented multiplex TaqMan qPCR diagnostic assay is highly specific, sensitive, reliable for the detection of Pectobacterium species and P. parmentieri with no false positives or false negatives. Significance and Impact of the Study:The developed assay can be adopted for multiple purposes such as seed certification programmes, surveillance, biosecurity, microbial forensics, quarantine, border protection, inspections and epidemiology.

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Disease-free planting stocks and formal certification systems are critical for the profitable production of vegetatively propagated crops. Propagation through seed tubers is the standard and preferred process for potato production. Seed tuber certification systems are used in the United States and throughout the world to mitigate degeneration and promote the use of high-quality planting stocks. The seed certification uses molecular and serological diagnostic methods for stock testing. While the molecular assays available are well-validated and offer high sensitivity, the rapidly evolving pathogens they target routinely develop mutations that allow them to evade detection. An example of these limitations are the potato diseases soft rot and blackleg, caused by Pectobacterium and Dickeya species. These diseases are challenging for current diagnostics tools because of high pathogen genomic variability, identical symptom development, and co-infections. Given the rapidly changing genetics of soft rot and blackleg bacteria, metagenomic sequencing should be adopted as a tool for routine validation of currently accepted diagnostic methods, and for the development of new diagnostic assays. We propose metagenome sequencing usage to enhance the diagnostic capabilities and complement molecular epidemiology approaches. We envision that metagenomics will accelerate diagnosis, shorten response time to outbreaks caused by unknown microorganisms, and provide more accurate and sustainable management strategies for seed certification programs.