A New Disease, Bacterial Leaf Spot of Rape, Caused by Atypical Pseudomonas viridiflava in South Korea

In March 2007, a bacterial leaf spot of rape (Brassica napus var. oleifera) was observed in fields near Seogwipo City, Jeju Province, South Korea. Symptoms on leaves included white and corky-brown spots and sometimes water-soaked spots on the lower leaf surface. Seven bacterial isolates (BC2495–BC2497 and BC2506–BC2509) were recovered on trypticase soy agar (TSA) from leaf spot lesions surface sterilized in 70% ethyl alcohol for 1 min. Isolates were gram-negative, aerobic rods with one to three flagella. Pathogenicity was evaluated on 2-week-old rape plants by spot and spray inoculation. Bacteria were grown on TSA for 48 h at 25°C. Five microliters of bacterial suspension in sterile distilled water (1 × 10⁵ CFU/ml) were spot inoculated on pinpricked positions of five detached leaves for each isolate. The detached leaves were incubated in a plastic box with high humidity at 20°C. Spot-inoculated surfaces turned white 48 h after inoculation followed by a brownish discoloration. A bacterial suspension in sterile distilled water (100 ml at 1 × 10⁵ CFU/ml) was sprayed onto three plants for each isolate. Plants were maintained in a growth chamber at 20°C and 90% relative humidity. Isolates induced identical symptoms 2 weeks after spray inoculation as those originally observed on rape in the fields. Bacteria were reisolated 18 days after inoculation from diseased lesions surface sterilized in 70% ethyl alcohol for 1 min. Pathogenicity of the reisolated bacteria was confirmed by spot inoculation as described above. No symptoms were noted on detached leaves and intact plants inoculated with sterilized distilled water. Using the Biolog Microbial Identification System, Version 4.2 (Biolog Inc., Hayward, CA), the isolates were identified as Pseudomonas viridiflava with a Biolog similarity index range of 0.52 to 0.72 after 24 h. Results of LOPAT tests (2) of isolates were identical to that of atypical P. viridiflava reported by Gonzalez et al. (1). Levan production and pectolytic activity of the isolates were variable. All isolates were positive for tobacco hypersensitivity and negative for oxidase reaction and arginine dihydrolase production. The 16S rDNA region (1,442 bp) of the isolates (GenBank Accession Nos. HM190218-HM190224; P. viridiflava CFBP2107(T) = HM190229), amplified by using universal PCR primers, shared 100% sequence identity with atypical P. viridiflava (GenBank Accession No. AM182934) (1). The gyrB sequence (638 bp) from the isolates (GenBank Accession Nos. HM190232-HM190238; P. viridiflava CFBP2107(T) = HM190239), amplified by using previously reported PCR primers (3), had a distance index value range of 0.029 to 0.031 with that of the P. viridiflava CFBP2107(T) (=BC2597) as determined by Jukes-Cantor model using MEGA Version 4.1 (4). On the basis of phenotypic characteristics and the sequences, the seven isolates were identified as atypical P. viridiflava. The disease is named “bacterial leaf spot”. To our knowledge, this is the first report of bacterial leaf spot of rape caused by atypical P. viridiflava.