Fire Blight of Apple, Caused by Erwinia amylovora, a New Disease in Korea

In June and July 2015, symptoms typical of fire blight were observed on shoots and leaves of apple (Malus pumila L.) in two commercial orchards located 67.38 km apart in Anseong (36°57′31.15″ N, 127°15′55.15″ E) and Jecheon (37°9′43.23″ N, 127°59′16.72″ E) cities of Korea. Symptoms including leaf and shoot blight were observed on 313 and eight trees in orchards of Anseong and Jecheon, respectively. Fifteen bacterial isolates were recovered on trypticase soy agar from leaf and shoot lesions that were surface-sterilized in 70% ethyl alcohol for 20 s. All isolates were gram-negative, rod-shaped, and mucoid. Phenotypic tests were performed based on diagnostic protocol for Erwinia amylovora (OEPP/EPPO 2013). The isolates were positive for levan formation, production of acetoin and reducing substance from sucrose, gelatin hydrolysis, requirement of growth factor (nicotinic acid), hypersensitive reaction in tobacco, both oxidation and fermentation, and acid production from L-arabinose. The isolates were negative for growth at 36 and 39°C, pectate degradation, H₂S from cysteine, urease production, indole, nitrate reduction, fluorescent pigment on King’s B medium, oxidase, and acid production from salicin, α-methyl glucoside, melibiose, and inositol. The isolates produced a 1-kb amplicon with the species-specific primers set A/B for E. amylovora (Bereswill et al. 1992), but did not produce any amplicon with primer set CPS1/CPS2c for E. pyrifoliae (Kim et al. 2001). Pathogenicity tests were performed on apple seedlings and immature pear fruits. Pathogenicity of the isolates to apple seedlings was confirmed by infiltrating bacterial suspensions (10⁸ CFU/ml) in sterile distilled water (DW) into the veins near the base of young leaves on the shoot tips of 2-year-old apple (cv. Fuji). The leaves and stem on the shoot tips turned brown, and the top of shoot bent over into a characteristic shape similar to the top of a shepherd’s crook within 5 days. Pathogenicity tests on immature pear fruit (Steinberger and Beer 1988) were confirmed by inoculation of 10 μl of bacterial suspensions (10⁷ CFU/ml) in sterile DW on cut surfaces of freshly cut immature pears (cv. Shingo). The inoculated fruits were placed in a humid plastic box at 28°C for 2 days. The fruits had severe necrosis and showed bacterial ooze at the inoculated sites. No symptoms were observed on the control plants and immature pear fruits inoculated with sterile DW. All tests were repeated two times. The identity of the bacterium reisolated from blighted apple shoots was confirmed by sequencing of the atpD gene (Young and Park 2007). The atpD (1,221 bp, KU500342 to KU500356) and carA (1,022 bp, KU500326 to KU500340) genes of all isolates were partially sequenced, and compared with the atpD (KU500341) and carA (KU500325) genes of the type strain (BC 228 = ATCC 15580^T) of E. amylovora to aid in the identification (Young and Park 2007). All isolates had the same atpD and carA gene sequences. All isolates and the type strain of E. amylovora shared 100% and 99.9% (one base difference) similarities in the sequences of atpD and carA genes, respectively. On the basis of pathogenicity, the sequences and phenotypic assays, the 15 isolates were identified as E. amylovora. To our knowledge, this is the first report of fire blight of apple caused by E. amylovora in Korea. The disease is expected to have a significant economic impact on apple in Korea. Programs for eradication of fire blight of apple have been executed to prevent further spread of the bacterium to new areas.