First report of Dickeya fangzhongdai causing soft rot of onion in New York State

In early July 2014, an outbreak of soft rot disease of onion (Allium cepa L.) occurred in Orange County, NY. Symptoms developed following rainy, hot, humid weather and included chlorosis, maceration of leaves, browning, and plant collapse within a few days. Bulbs from diseased plants appeared normal, but inner tissues were macerated. Disease was first apparent in a field of red onions (cv. Redwing); later yellow onions (cultivar unknown) in an adjacent field were symptomatic. Standard techniques were used to isolate bacteria from diseased red onion bulbs (Bonasera et al. 2017). Briefly, 14 onion bulbs were cut open with a disinfected knife, and diseased tissues were probed with sterile wooden applicators and streaked on onion extract medium agar (Zaid et al. 2012). Plates were incubated for 2 days at 28°C, and individual colonies were streaked to purity on Luria–Bertani (LB) agar. Small white bacterial colonies with irregular margins were obtained as the predominant type from three samples. A representative colony was identified as Dickeya sp. based on the sequence of a portion of its 16S rRNA gene amplified by fD1/rP2 (Weisburg et al. 1991). Several more colonies were assessed using ADE1 and ADE2 PCR primers specific to Dickeya genus (Nassar et al. 1996), which also indicated that these isolates belonged to the genus Dickeya. Genomic DNA of one of these colonies, AP6, was extracted and sequenced using an Illumina NextSeq500 by the Institute of Biotechnology at Cornell University. Raw reads were processed (Ma et al. 2019) and used for de novo assembly with SPAdes 1.12. The draft genome assembly resulted in 260 contigs, with an N50 size of 50,007 bp, totaling 5.11 Mbp, and it was deposited in GenBank as accession number VSRM00000000. We conducted average nucleotide identity and in silico DNA-DNA hybridization tests to determine the species of Dickeya strain AP6 using Python package pyANI and GGDC (https://ggdc.dsmz.de), respectively. These analyses indicated that AP6 belongs to species Dickeya fangzhongdai. Koch’s postulates were completed with D. fangzhongdai AP6. Two leaves each of three 80-day-old, healthy-appearing, controlled environment-grown onion plants (cv. Fortress for the first replicate, cv. Redwing for the second and third replicates) were inoculated with D. fangzhongdai AP6. The leaves were first punctured with sterile toothpicks, and then the wound was smeared with bacteria grown overnight on LB agar. One plant was punched with sterile toothpicks as a negative control. Elongated lesions with water-soaking at the margins were observed around the inoculation sites after incubating for 24 h at 30°C; no symptoms were observed on control plants. Disease symptoms progressed very rapidly; after 72 h, all AP6-inoculated plants had collapsed completely, and foliage was quite water-soaked. The experiment was conducted three times. We isolated three isolates from water-soaked lesions 24 h after inoculation and used dnaX sequencing on one of the reisolated colonies to confirm that bacteria in the diseased tissue were identical to the D. fangzhongdai inoculum. D. fangzhongdai was first identified in China as causing bleeding canker necrosis on pear (Tian et al. 2016). D. fangzhongdai was reported to cause soft rot in Welsh onion in Taiwan (Tsai et al. 2019), diseased Phalaenopsis orchids in Slovenia and Aglaonema in St. Lucia, and was found in water samples from Scotland and Malaysia (Alic et al. 2018; Pritchard et al. 2013). Soft rot disease of onion was reported in the United States (Ceponis et al. 1986), but its causal organism had not been investigated thoroughly. Here we report that D. fangzhongdai caused an outbreak of soft rot of onions in Orange County, NY.