First Report of Pseudomonas syringae pv. syringae Associated with Bacterial Blossom Blast on Apple (Malus pumila) in the United States

In May 2017, dead buds at pink and tight cluster growth stages were observed on young apple trees of cultivar SnapDragon at two orchard sites in Marlboro, NY. Disease symptoms included necrosis of lateral and terminal flower buds that were wilted and drooping. Bark below the dead buds on severely affected trees showed black streaking with or without interspersing white streaks. On the cross-section of branch wood, just below the buds, light brown xylem and glossy water-soaked sections resembling frost damage were observed. After bark shaving, dark xylem tissue was visible along the branch length, in contrast to the light or pale color of the healthy tissue. The incidence of the disease on trees was 3 to 5%, and 0.5% of trees died. There were five near-frost events recorded on 4, 8, 9, 10, and 11 May 2017, leading us to suspect that the observed symptoms were blossom blast caused by the ice-nucleating strains of Pseudomonas syringae. Isolation from the affected tree samples was performed on King’s B (KB) and nutrient sucrose agar (NSA) media. After 3 days at 25°C, most of bacterial isolates formed round, shiny, gray-white colonies producing green fluorescent pigment on KB medium and typical levan-positive colonies on NSA. Eight isolates, inducing a hypersensitive reaction on tobacco plants, were further characterized by conventional bacteriological and molecular tests (Braun-Kiewnick and Sands 2001). The analyzed bacterial isolates were Gram-negative, aerobic, hydrolyzed gelatin and aesculin, but not pectate, and were negative for oxidase and arginine-dihydrolase activity. All strains were ice-nucleation active and utilized sucrose, sorbitol, and inositol as carbon sources, but not tyrosine and tartaric acid. Results of levan, cytochrome c oxidase, potato soft rot, arginine dihydrolase, and tobacco hypersensitive reaction (LOPAT) tests indicated that the isolates belong to P. syringae group Ia, and gelatin hydrolysis, aesculin hydrolysis, tyrosinase activity, and tartrate utilization (GATTa) tests designated them as pathovar (pv.) syringae (Kaluzna et al. 2012). Polymerase chain reaction (PCR) with specific primers B1/B2 (Sorensen et al. 1998) and SyD1/SyD2 (Bultreys and Gheysen 1999) showed that isolates possessed genes responsible for synthesis (syrB) and secretion (syrD) of syringomycin, respectively. Partial sequencing of the 16S rDNA gene of three isolates (GenBank MG788015 to MG788017) showed 99 to 100% identity with sequences of P. syringae pv. syringae previously deposited in the NCBI GenBank database. To complete Koch’s postulates, pathogenicity of the four isolates was confirmed by inoculation of 1-month-old potted apple plantlets grown from seeds collected from mature apple fruit of cultivar SnapDragon. One plant per isolate was inoculated multiple times as follows: using a needleless syringe, two leaves per plant were spot-infiltrated with bacterial suspension (10⁶ CFU/ml); on the same plants, stem was also inoculated by inserting a sterile toothpick previously dipped into a bacterial colony. Sterile distilled water was used as a negative control. Inoculated plantlets were sealed within plastic bags for 48 h and further incubated at 22°C. Inoculation test was repeated in two independent experiments. Necrosis of the leaf and stem tissue developed at the point of inoculation, and its progression was observed daily for 2 weeks. No symptoms developed on the control plants. Bacteria were reisolated from inoculated tissues and confirmed as P. syringae pv. syringae by PCR for syrB and syrD genes. This is the first report of P. syringae pv. syringae causing blossom blast symptoms on developing apple buds in commercial orchards in the United States. Even though it rarely occurs on apples, with most recent frosts significantly lowering apple yields in Michigan (2012) and New York (2016), blossom blast might become a more common problem in U.S. orchards. Previously, P. syringae pv. syringae was isolated from dormant apple buds without and with internal necrosis in the United States (Burr and Katz 1984).