First Report of Bacterial Leaf Spot on Watermelon Caused by Pseudomonas syringae pv. syringae in Georgia
- B. Dutta
- R. D. Gitaitis
- J. E. Driver
- S. Smith , Department of Plant Pathology, University of Georgia, Tifton 31793.
In May 2015, watermelon leaves (Citrullus lanatus (Thunb.) Matsum. & Nakai) of cultivar Melody were observed with circular to irregularly shaped spots, each with a tan center and dark margin in a field in Cook Co., GA. The field exhibited 20 to 25% disease incidence with no symptoms on fruits. Symptomatic leaves with leaf spot were collected, and bacterial isolations were made on King’s medium B agar. After 48 h of incubation at 25°C, cream-colored, fluorescent blue (under UV light), round colonies with smooth margins were isolated. The isolates were Gram negative, oxidase negative, nonpectolytic on potato, arginine dihydrolase negative, and produced levan. Bacterial isolates also produced hypersensitive reactions on tobacco when inoculated with a bacterial suspension of 1 × 108 colony forming units (CFU)/ml. These characteristics indicated that the isolates belonged to Pseudomonas syringae van Hall LOPAT group Ia of Lelliott et al. (Lelliot et al. 1966). The 16S rRNA from four isolates (W-1, W-2, W-3, and W-4) was amplified (Leu et al. 2010), and the resultant PCR products were sequenced and BLAST searched in GenBank. The 16S rRNA sequences matched those of Pseudomonas syringae pv. syingae (Accession No. JQ071937) with 99 to 100% sequence identity. The GenBank Accession Nos. of 16S rRNA sequences for the four watermelon isolates (W-1, W-2, W-3, and W-4) are KT008095, KT008096, KT008097, and KT321517, respectively. The four test isolates also had ≥97% similarity with P. syringae pv. syringae when tested with BIOLOG (Biolog, Hayward, CA). Additionally, polymerase chain reaction (PCR) with HrpZ group III-specific primers (Inoue and Takikawa 2006) resulted in the amplification of the watermelon isolates along with a type strain of P. syringae pv. syringae (NCPPB 1770). These results indicate that the watermelon isolates belonged to the same HrpZ group III as that of P. syringae pv. syringae. Two-week-old watermelon seedlings cv. crimson sweet were inoculated by spraying with a suspension of 1 × 108 CFU/ml. Three experiments were conducted under greenhouse conditions using four test isolates (W-1, W-2, W-3, and W-4) with four replications each. Seedlings (n = 4) sprayed with sterile water were used as a control. Seedlings were maintained at ∼25°C and ∼70% RH with a 12-h photoperiod. Seedlings were evaluated after 14 days post inoculation for bacterial leaf spot. Disease incidence was recorded for each seedling per treatment. One hundred percent of the inoculated seedlings with four test isolates developed symptoms as above. The disease incidence was observed to be 100% each for the four isolates. Four control plants inoculated with sterile water remained asymptomatic. Bacterial colonies were reisolated from symptomatic seedlings that showed characteristics similar to those described above. The identity of isolated colonies was confirmed by amplifying and sequencing the 16S rRNA gene, which showed 99 to 100% similarity to P. syringae pv. syringae accessions in GenBank. In addition, reisolated colonies showed similar characteristics based on physiological (BIOLOG profile), and molecular assay (HrpZ group III), indicating that these strains are P. syringae pv. syringae. In addition, for further confirmation, watermelon isolates (n = 4) along with a type strain P. syringae pv. syringae (NCPPB 1770) were also spray inoculated onto two-months-old lilac (Syringa vulgaris L.) (n = 3/isolate/experiment), a natural host of P. syringae pv. syringae, as described above. All 12 lilac plants inoculated with any of the four watermelon isolates or with P. syringae pv. syringae (type strain) developed symptoms (irregularly-shaped spots, each with a tan center and dark margin) after 10 days post inoculation. Symptoms were not observed from the sterile water treated leaves. Reisolation and characterization of colonies using above-mentioned approach confirmed the colonies to be P. syringae pv. syringae. To our knowledge, this is the first report of P. syringae pv. syringae in watermelon in Georgia. Being a known seedborne bacterium of watermelon, it is possible that the pathogen might have been introduced through contaminated seeds. Knowledge of the presence of P. syringae pv. syringae in the agro-ecosystem of Georgia may encourage scientists to implement integrated management practices for this pathogen.