Detection Of Pathogen Dickeya Dadantii Causing Bacterial Stem And Root Soft Rot Disease Of Sweet Potato

China is the largest sweet potato-producing country in the world.The stem and root rot disease of sweet potato caused by the bacterium Dickeya dadantii is a destructive disease for sweet potato and seriously threatens sweet potato production in China.The pathogen causing the stem and root rot disease of sweet potato has been listed on the Catalogue of Quarantine Pests for Import Plants to the People’s Republic of China whereas no detection method specific for D.dadantii has been available.This study aimed to design diagnostic primers for D.dadantii based on the whole genome sequences of Dickeya bacteria and develop a specific and sensitive method for the detection of the pathogen D.dadantii causing the stem and root rot disease of sweet potato.The taxonomic status of Dickyea bacteria whose whole genome sequences have been available was determined by phylogenomic analysis and comparison of overall genome relatedness.Specific primers for D.dadantii were designed using the high-throughput Find Differential Primers Pipeline based on bacterial whole genome sequences and the Pan-Genomes Analysis Pipeline to find species-specific signature genes.A pair of diagnostic primers(Dad1-F:5’-CATATCAACCAGACCAGCCGTT;Dadl-R:5’-CGGCCTGCTTTTAAACAACGTATTA)specific for D.dadantii was screened out by PCR amplification.A 167-bp amplicon was detected only from D.dadantii strains.Based on this pair of primers,conventional PCR,real-time fluorescence quantitative PCR(qPCR),and immuno-capture PCR were developed for specific detection of D.dadantii.The minimal detection limit by the conventional PCR was 50 pg·μL-1 for purified DNA and 2×105 CFU g-1 for DNA extracted from soils,sweet potato stems and root tubers inoculated with D.dadantii.The minimal detection limit by qPCR was 500 fg·μL-1 for purified DNA and 2×104 CFU g-1 for DNA extracted from soils,sweet potato stems and root tubers inoculated with D.dadantii.The standard curve of qPCR amplification for this primer pair was y=-1.9271x+36.607 with a correlation coefficient 0.9883.The detection limit of the immuno-capture PCR was 2×107 CFU g-1 for sweet potato stems and root tubers inoculated with D.dadantii and 2×106 CFU g-1 for inoculated soils.This study developed conventional PCR,qPCR and immuno-capture PCR methods for specific and sensitive detection of a D.dadantii-specific signature gene,providing effective methods for identification and detection of the pathogen causing the stem and root rot disease of sweet potato.These methods are important for the inspection and quarantine of D.dadantii,monitoring D.dadantii in fields,and controlling the stem and root rot disease of sweet potato.