First report of crown gall on sweet cherry trees caused by Agrobacterium larrymoorei in South Korea

In October 2024, crown gall was observed in 4-year-old cherry trees (Prunus avium) in an orchard at Sancheong, Gyeongsangnam-do, South Korea. The gall affected approximately 10% of all 200 trees in the orchard (0.4 ha). Cultivar information for field samples was unavailable. The diseased cherry trees had wilted leaves, dried branches, and galls of various sizes (2–20 cm) within the crown. To isolate the causative agent of this crown gall disease, which was suspected to be a tumorigenic agrobacterium (Kuzmanović et al. 2015), five young galls with a diameter of about 2 cm were washed with tap water, surface sterilized with 70% ethanol for 30 s, and dried at room temperature for 10 min. Three fragments (5 mm × 5 mm) from each gall were ground using a disposable polypropylene grinding rod fitted with a micro-grinding pestle in 1.5-ml e-tubes and suspended in sterile distilled water (SDW). After serial dilution, each suspension from five galls was spread on yeast mannitol (YM) agar and incubated at 28°C for 4 days. We isolated and purified 20 of the resulting dominant colonies from YM agar plates. These colonies were white to cream in color, smooth, convex, glistening, mucoid, and circular with entire edges, resembling the colony morphology of Agrobacterium species. Diagnostic polymerase chain reaction (PCR) was performed using virD2A/virD2C primers (Haas et al. 1995) and purified genomic DNA as template (Choi et al. 2019). Five isolates (C24a−e) produced a 224-bp PCR product. Their biochemical and physiological characteristics were similar to those of Agrobacterium species. All five isolates were positive for oxidase, growth at 35°C, and 2% NaCl, but negative for citrate utilization and acid production on potato dextrose agar supplemented with CaCO3. Pathogenicity was assessed by syringe inoculating 50 μl of overnight culture in YM liquid broth onto stems of 1-month-old tomato plants as surrogate hosts, as previously described (Choi et al. 2019). Sterile distilled water was used as a negative control. Inoculated tomatoes were placed in a growth chamber at 25℃/20℃ (light/darkness) for 5 weeks. Tomato plants inoculated with bacterial isolates developed tumors at the inoculation site, whereas negative controls showed no disease symptoms. The bacteria were re-isolated from tomatoes, and virD2 gene locus sequencing was performed on the re-isolated bacteria. For identity confirmation, three housekeeping genes (atpD, recA, and rpoB) from all five isolates were amplified with 800F/1350R, F7386/ArecAR, and 2040F/2718R, respectively (Aujoulat et al. 2011; Puławska et al. 2016; Shams et al. 2013). Additionally, sequences from the representative isolate C24a were deposited in GenBank (acc. nos. PV879807 to PV879809). Concatenated sequences of the three loci were used to conduct a maximum-likelihood phylogenetic analysis in MEGA11 (Mafakheri et al. 2019). All five isolates were grouped with A. larrymoorei. NCBI BLAST search of three loci with identical sequences in five isolates showed best matches of 97.86%, 99.78%, and 99.68% homology with Agrobacterium larrymoorei, respectively. Using biosensor strains, we found that all five isolates produce nopaline-type opines, similar to the type strain A. larrymoorei AF3.10T (Bouzar et al. 1995; Choi et al. 2019). This is the first report of crown gall caused by A. larrymoorei in cherry trees in South Korea. Our findings provide useful basic data for establishing strategies for managing and controlling cherry crown gall disease.