First Report of Pseudomonas syringae pv. syringae Causing Bacterial Leaf Spots of Oil Pumpkin (Cucurbita pepo) in Serbia

Oil pumpkin (Cucurbita pepo L.) is commonly used for oil production, mainly in central and eastern Europe (1). In Serbia, it grows only in the north (Vojvodina Province), up to 1,500 ha. In June 2008, typical bacterial spot symptoms (dark green, water-soaked, transparent and greasy spots with yellow margins) were observed for the first time, cultivated at the experimental fields near Backi Petrovac. Since then, bacterial spots were regularly observed on oil pumpkin in the beginning of the growing seasons and during rainy weather, with disease incidence ranging from 5 to 20%. Bacteria isolated from 40 diseased leaves formed white, round, convex, and mucoid colonies on nutrient sucrose agar (NSA). Eight representative strains were aerobic, gram-negative, non-spore-forming rods. All strains produced fluorescent pigment and catalase. In levan-oxidase-potato rot-arginine dihydrolase-tobacco hypersensitivity (LOPAT) tests (3), they induced a hypersensitive reaction in tobacco leaves, did not cause soft rot of potato tubers, and were positive for levan and negative for oxidase and arginine dihydrolase. According to the LOPAT profile, they were classified in the Ia subgroup of pseudomonads (3). Strains hydrolyzed aesculin, but were unable to hydrolyze starch or reduce nitrates to nitrites. Negative reactions were obtained with hydrogen sulfide and indole. Reactions were identical to those of reference strain Pseudomonas syringae pv. syringae CFBP 1582, which was included in all biochemical, physiological, and molecular tests for comparison. To identify the pathogen, PCR and DNA sequencing were employed. Fragments of 752 bp for the syrB gene and 1,040 bp for the syrD gene were amplified from all strains, using B1/B2 and SyD1/SyD2 primer sets, respectively (2). The pathogenicity was tested on seeds and seedlings of oil pumpkin cv. Olinka. Strains were grown for 48 h on nutrient broth (NB) at 28°C and bacterial suspensions of ~108 CFU ml−1 were used for inoculations. Sterile water was used as negative control. Seeds (at the BBCH-1-0 stage) allowed to imbibe water were wounded by needle, immersed in the bacterial suspensions, and maintained in humid petri dishes to allow symptom development. The cotyledons of seedlings at the BBCH-10 stage were inoculated by hypodermic needle and potted plants were maintained at 25 ± 1°C and 75% relative humidity. Symptoms, including dark green, water-soaked spots, appeared 5 to 7 days after inoculation of both seeds and seedlings. The bacterium was re-isolated from spots of all seeds and seedlings tested, fulfilling Koch's postulates (the identity of re-isolated strains was confirmed by pathogenicity, morphology, and biochemical features). No symptoms were observed on controls. 16S rDNA amplicons obtained from representative strain Tk21 and re-isolated strain Tk21R with fD1/rD1 primers (4) were sequenced and deposited in GenBank under accession nos. KF305578 and KF735064, respectively. The sequences showed 100% similarity to each other and P. syringae pv. syringae from pepper (KC816630.1) (China), Ficus carica (JQ071937) (Serbia), and culture-collection ICMP:3023 (HM190217). On the basis of the symptoms, biochemical tests, and 16S rDNA sequence homology, the pathogen was identified as P. syringae pv. syringae. To our knowledge, this is the first report of P. syringae pv. syringae causing bacterial leaf spot on oil pumpkin in Serbia