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 genus Dickeya belongs to the family Enterobacteriaceae and is comprised of pectinolytic Gram negative plant pathogenic bacteria, responsible for the soft rot diseases worldwide. Due to their association with catastrophic diseases, Dickeya species have been listed among the top ten most destructive bacterial plant pathogens (Mansfield et al. 2012) . Soft rot Enterobacteria are also associated with the most serious problems facing potato production worldwide. The impact of soft rot disease varies from country to country; in the Netherlands, the annual estimated losses are around €30 million per year, while in Israel, potato yield losses of 20-25% have been observed. Symptoms caused by Dickeya spp. are more severe in high moisture and temperature environments (Czajkowski et al. 2009 ). Infected plants show stunting, chlorosis, wilting, black discoloration and soft rot, which begins at the stem base and progresses upward. Once infection has occurred, the pathogen colonizes the vascular tissues and moves throughout the plant (Raoul des Essarts et al. 2016); severe infections lead to whole plant collapse. Latent infections can result in severe losses during storage, especially in warehouses that lack refrigeration facilities (Laurila et al. 2008 ). In recent years, D. dianthicola has emerged as one of the most important pathogens infecting potatoes worldwide, and symptoms resemble those of blackleg caused by Pectobacterium species (Boluk and Arif 2019). Due to high economic impact in the European potato growing countries, D. dianthicola (syn. Erwinia chrysanthemi pv. dianthicola) has been listed in the EPPO A2 List of pests recommended for regulation as quarantine pests A2/53 (Pg 8); http://archives.eppo.int/EPPOStandards. At present, neither resistant varieties nor effective physical, chemical or biological controls have been reported to manage D. dianthicola diseases under field conditions (Czajkowski et al. 2014) . Only preventive measures are recommended, which include planting certified seeds, eradicating contaminated plant materials, screening for latent infections, monitoring appropriate storage conditions and inspecting fields (Motyka et al. 2017) . Therefore, early, accurate and sensitive detection of Dickeya species is important to identify inoculum sources before they become a problem. TaqMan qPCR assay for detection of Dickeya spp. and described a number of primer pairs to detect different Dickeya sp. TaqMan qPCR with the probes labelled with different fluorogenic dyes enables simultaneous detection of multiple pathogens or pests in a single tube (Arif et al. 2015) . Multiplex TaqMan can be very useful for detection of different Dickeya species, which produces nearly indistinguishable disease symptoms without compromising the sensitivity and specificity of the assay.