First Report of Dickeya fangzhongdai Causing Soft Rot of Phalaenopsis aphrodite in China

Phalaenopsis aphrodite is very popular in China. In the summer of 2017, a soft rot of P. aphrodite was found at the base of Phalaenopsis plants in Foshan city, Guangdong Province, with the incidence of about 10%. The infected leaves displayed water-soaked small round spots initially, and the spots expanded quickly with the increase of humidity and temperature, becoming translucent when facing a light source. Several days later, the inclusions exuded as the lesions decayed. The bacteria further infected the leaf sheath, resulting in the discoloring and perishing of leaf sheaths, falling off of leaves, and even the death of the whole plant. Finally, the leaves became as dry as paper. Five leaves with typical symptoms were randomly collected; after washing with 75% ethanol for 30 s, with 0.1% HgCl₂ for 2 min, and with sterile water three times, small pieces of tissues (5 × 5 mm) were removed from lesion borders, plated on LB solid medium, and incubated at 37°C for 24 to 48 h. Thereafter, 10 representative isolates were chosen for further identification. Generally, these strains were gram negative, cultivated at 37°C, and utilized D-arabinose, melibiose, raffinose, and mannitol but not 5-keto-D-gluconate, β-gentiobiose, or casein. A GEN III MicroPlate system (Biolog) was used for examination, and the 10 isolates were identified as Dickeya chrysanthemi (SIM 0.856). PCR was used to amplify the 16S rDNA gene with primers 27f and 1492r (Tillett et al. 2000), recA (Waleron et al. 2002), and dnaX (Sławiak et al. 2009). BLASTn was carried out online, and the underlying phylogeny trees were obtained based on these gene sequences using MEGA 5.05 through 100% bootstrap values. The results showed that representative tested strain FSPAD1, whose sequences were deposited in GenBank under accession numbers MK394174 (16S rDNA), MK405705 (recA), and MK405706 (dnaX), had the highest homology with D. fangzhongdai for 16S rDNA(JN940859), recA (KT992693), and dnaX (KT992713), with the identities of 100, 97, and 97%, respectively. Therefore, this isolate was identified as D. fangzhongdai strain FSPAD1. Pathogenicity tests were performed on healthy tender leaves of P. aphrodite. Sterile filter paper (1 cm in diameter) was used to absorb sterile water and the bacterial suspension (1 × 10⁸ CFU/ml). Then, the paper with sterile water (control) or the bacterial suspension were attached to tender leaves with three acupuncture points in each leaf. All tested plants were incubated at 28°C and kept moist with plastic bags. After 2 days, soft rot symptoms were observed in the pathogen-inoculated leaves, whereas sterile water (control) inoculated leaves exhibited no symptoms. The pathogenicity test was conducted three times. Koch’s postulates were achieved by reisolating D. fangzhongdai from the inoculated leaves. Meanwhile, 16S rDNA sequence alignment confirmed that the new reisolation was consistent with that of the inoculated plant. This pathogenic bacterium has been reported on Pyrus pyrifolia in China (Tian et al. 2016). So far, this is the first report of D. fangzhongdai causing soft rot disease on P. aphrodite.