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First report of bacterial leaf spot disease on sunflower (Helianthus annuus) caused by Pseudomonas viridiflava in South Korea.

In September 2019, bacterial leaf spot symptoms were observed on sunflowers in an experimental field in Eumseong, South Korea. The leaves of infected plants initially showed irregular brown spots surrounded by haloes; as the disease progressed, the spots became enlarged and darkened (eXtra Fig. 1a). At the flowering stage, leaves became dry and showed signs of blight including defoliation; dark brown spots were also observed on sunflower stems and petioles but not on floral discs (eXtra Fig. 1b). Disease incidence ranged from 5% to 30% in three surveyed plots of the field. Symptomatic leaf tissue was surface-sterilized, macerated with sterile distilled water, and cultured on nutrient agar plates at 28°C for 48 h. After incubation, nine bacterial isolates, representing individually collected samples from each field, were selected for further study. All nine isolates were Gram-negative and fluorescent pigments produced under UV on King's medium B. With the LOPAT test, the isolates were levan negative, oxidase negative, positive for pectinolytic activity, arginine dihydrolase negative, and positive for tobacco hypersensitivity. Based on 16s rRNA sequences, all isolates shared 100% identity with Pseudomonas viridiflava strain KNOX209.1 (GenBank accession no. AY604847). The 16s rRNA sequences of nine isolates were deposited in GeneBank (accession nos. MT393747, MW446479 to MW446486). Based on the phylogenetic analysis of the 16s rRNA, all isolates were grouped with P. viridiflava strains isolated from globe artichoke, Chinese cabbage, and rape (Myung et al. 2010, Sanver et al. 2019, and Liu et al. 2019). The isolates CPB 19362, CPB 19366 and CPB 19372, which represent each plot were selected for further phylogenetic analysis and pathogenicity assays. The identity of these isolates was confirmed by sequences of housekeeping genes of the gyrase B subunit (gyrB) and RNA polymerase σ70 factor (rpoD) (Yamamoto et al. 2000) (GenBank accession nos. MT409400, MW446487 and MW446494 for gyrB and MT409401, MW446495 and MW446502 for rpoD). Based on the phylogenetic analyses of gyrB and rpoD, the three isolates belong to the same clade as the P. viridiflava pathotypes and were distinguished from P. syringae complex (eXtra Fig. 2). These results indicated that the bacteria isolated from the spots on the sunflower plants were P. viridiflava strains. To confirm the pathogenicity, bacterial suspensions (approximately 108 CFU/mL) of three representative isolates sprayed onto 4-week-old sunflower (cv. Common) seedlings separately until runoff occurred. Sterile distilled water was used as a control and inoculated in the same manner. After inoculation, plants were covered with transparent plastic bags at room temperature for 24 h. Plastic bags were then removed and plants were grown on a plant growth shelf at 25°C in 50% relative humidity. The leaves of plants inoculated with the bacterial suspensions developed small brown spots after 24 h. After 3 days, brown spots surrounded by chlorotic or necrotic areas were observed on infected leaves (eXtra Fig. 1c). These spots gradually increased in size and formed brown lesions with haloes similar to those of infected field-grown plants (eXtra Fig. 1d), but not on the controls treated with sterile water. The pathogenicity test was repeated three times. Isolates recovered from infected leaves showed the same morphological, biochemical, and molecular characteristics as the original isolates from field samples. To our knowledge, this is the first report of bacterial leaf spot on sunflower caused by P. viridiflava in South Korea.