Comparative genomics reveals signature regions used to develop a robust and sensitive multiplex TaqMan real-time qPCR assay to detect the genus Dickeya and Dickeya dianthicola

Shefali Dobhal; Gamze Boluk; Brooke Babler; Michael J. Stulberg; John Rascoe; Mark Nakhla; Toni A. Chapman; Alex B. Crockford; Michael Melzer; Anne M. Alvarez; Mohammad Arif

The effectiveness and robustness of any developed method is accessed by its ability to detect the pathogen from naturally infected samples. Therefore, the developed assay was tested with twentyfour asymptomatic and symptomatic, naturally infected plant samples. Thirteen samples showed positive amplification in both yellow and green channels, confirming the presence of D. dianthicola. Each infected sample that tested positive with TaqMan qPCR assay was further confirmed by sequencing the dnaA gene region followed by BLASTn (data not included). The BLASTn results confirmed 100% identity match with D. dianthicola. Furthermore, the developed method was also tested with 143 different plant and water samples received from various locations by the Wisconsin Potato certification laboratory. The method was able to detect 61 samples positive for Dickeya species and 60 for D. dianthicola. The developed method demonstrated the ability to be used for monitoring Dickeya species and D. dianthicola infections in naturally infected plant samples.