New Guinea impatiens (NGI), Impatiens hawkeri, has a $54-million wholesale market value in the United States (National Agricultural Statistics Service, 2019) and is highly resistant to Impatiens downy mildew (Plasmopara obducens) according to growers' experience (Warfield, 2011). In March 2019, NGI cv. Petticoat White in a New York greenhouse showed wilting, black stem streaks and vascular discoloration, with a 20% disease incidence. Symptomatic tissue pieces were added to sterile water in a test tube and streaks made on potato dextrose agar (PDA). After incubation at 26oC for two days, the most abundant colony type (mucoid, pale yellow) was transferred to PDA. One representative colony was selected and labeled as isolate 67-19. A single colony of isolate 67-19 was transferred to lysogeny broth (LB) (Bertani, 1951) and cultured at 28oC. Genomic DNA was extracted and polymerase chain reaction (PCR) performed using the 16S rRNA gene universal primers fD2 and rP1 resulting in a partial 16S rRNA amplicon (Weisburg et al., 1991). Basic Local Alignment Search Tool (BLASTn) analysis (Altschul et al., 1990) showed 99% identity with sequences of species belonging to Dickeya. Different primer sets have been developed to detect and identify the genus Dickeya and its various species (Pritchard et al., 2013). The primer sets used for genus identification, dnaX (Sławiak et al., 2009), Df/Dr (Laurila et al., 2010) and ADE1/ADE2 (Nassar et al., 1996), resulted in 500-bp, 133-bp, and 420-bp amplicons, respectively. Results suggested the bacterium was a Dickeya sp. To determine whether the species could be D. dianthicola, the specific primer set DIA-A was used (Pritchard et al., 2013) and the expected product of 150-bp was obtained. BLASTn results showed that the partial dnaX sequence (GenBank accession MT895847) of isolate 67-19 had 99% identity with the sequence of D. dianthicola strain RNS04.9 isolated in 2004 from potato (Solanum tuberosum) in France (GenBank accession CP017638.1). Therefore, this isolate 67-19 was designated as D. dianthicola. The complete genome of D. dianthicola strain 67-19 was generated using Nanopore and Illumina sequencing (GenBank accession CP051429) (Liu et al., 2020). Average nucleotide identity (ANI) determined by FastANI (v1.1) (Jain et al., 2018) showed 97.43% identity between the genome of D. dianthicola strain 67-19 and that of D. dianthicola strain NCPPB 453 (GenBank accession GCA_000365305.1), isolated in 1957 from carnation (Dianthus caryophyllus) in the UK. The pathogenicity of D. dianthicola strain 67-19 was shown on NGI cultivars Petticoat White and Tamarinda White. In July 2020, sterile toothpicks were used to make wounds and to transfer bacteria from a 48-hr PDA culture of D. dianthicola strain 67-19 to the stems of four plants of each cultivar. Four plants of each cultivar were mock inoculated similarly and all wound sites were wrapped with Parafilm before placing plants on a greenhouse bench. Ten days later, stems inoculated with D. dianthicola strain 67-19 showed necrotic lesions similar to the original symptoms, while control plants did not show symptoms. One month after inoculation, bacteria were re-isolated from all symptomatic stems. PCR was performed on the re-isolated bacteria as described. The dnaX sequence (GenBank accession MT895847) was confirmed to match that of D. dianthicola strain 67-19 (GenBank accession CP051429) 100% and fragments of the expected size were amplified (Liu et al., 2020). Stab inoculations of strain 67-19 into potato stems and tubers also resulted in blackleg and soft rot symptoms at the sites of inoculation, while mock-inoculated stem and tuber showed no symptoms. The sequence of the dnaX gene of the re-isolated bacterium from inoculated potatoes was confirmed to match that of D. dianthicola strain 67-19. To our knowledge, this is the first report of blackleg of New Guinea impatiens caused by D. dianthicola in the United States and worldwide. Since the disease caused by D. dianthicola poses a significant threat to the ornamentals and potato industries (Charkowski et al., 2020), further research on genome biology, epidemiology and management options is needed. LITERATURE CITED Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. 1990. Basic local alignment search tool. Journal of Molecular Biology 215:403-410. Bertani, G. 1951. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. Journal of Bacteriology 62:293-300. Charkowski, A., Sharma, K., Parker, M.L., Secor, G.A., and Elphinstone, J. 2020. Bacterial diseases of potato. Pages 351-388 in: The Potato Crop: Its Agricultural, Nutritional and Social Contribution to Humankind, H. Campos and O. Ortiz, eds. Springer International Publishing, Cham. Jain, C., Rodriguez-R, L.M., Phillippy, A.M., Konstantinidis, K.T., and Aluru, S. 2018. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nature Communications 9:5114. Laurila, J., Hannukkala, A., Nykyri, J., Pasanen, M., Hélias, V., Garlant, L., and Pirhonen, M. 2010. Symptoms and yield reduction caused by Dickeya spp. strains isolated from potato and river water in Finland. European Journal of Plant Pathology 126:249-262. Liu, Y., Helmann, T., Stodghill, P., and Filiatrault, M. 2020. Complete genome sequence resource for the necrotrophic plant-pathogenic bacterium Dickeya dianthicola 67-19 isolated from New Guinea Impatiens. Plant Disease. https://doi.org/10.1094/PDIS-09-20-1968-A. Nassar, A., Darrasse, A., Lemattre, M., Kotoujansky, A., Dervin, C., Vedel, R., and Bertheau, Y. 1996. Characterization of Erwinia chrysanthemi by pectinolytic isozyme polymorphism and restriction fragment length polymorphism analysis of PCR-amplified fragments of pel genes. Applied and Environmental Microbiology 62:2228-2235. National Agricultural Statistics Service. 2019. Floriculture crops 2018 summary. ISSN: 1949-0917. https://downloads.usda.library.cornell.edu/usda-esmis/files/0p0966899/rr1728124/76537c134/floran19.pdf Pritchard, L., Humphris, S., Saddler, G.S., Parkinson, N.M., Bertrand, V., Elphinstone, J.G., and Toth, I.K. 2013. Detection of phytopathogens of the genus Dickeya using a PCR primer prediction pipeline for draft bacterial genome sequences. Plant Pathology 62:587-596. Sławiak, M., van Beckhoven, J.R.C.M., Speksnijder, A.G.C.L., Czajkowski, R., Grabe, G., and van der Wolf, J.M. 2009. Biochemical and genetical analysis reveal a new clade of biovar 3 Dickeya spp. strains isolated from potato in Europe. European Journal of Plant Pathology 125:245-261. Warfield, C.Y. (2011). Downy Mildew of Impatiens. In GrowerTalks. https://www.growertalks.com/Article/?articleid=18921 Weisburg, W.G., Barns, S.M., Pelletier, D.A., and Lane, D.J. 1991. 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology 173:697-703.
Keywords: Causal Agent; Crop Type; Dickeya; Ornamentals; Prokaryotes; herbaceous/flowering plants.