Bacterial leaf spot of tobacco caused by Pseudomonas aeruginosa in China

A new disease of tobacco seedlings was observed during 2006 and 2007, in Xichou and Cangning counties, in Wenshan prefecture and Baoshan city respectively, in the Yunnan province of China. Symptoms were common in the float beds and transplants, and first appeared as small, pale or dark green lesions (Fig. 1), sometimes with curving of lower leaves, and brown lesions on the stems (Fig. 2). Sometimes a wet rot developed, or irregularly shaped brown areas with yellow haloes developed on older leaves, with some plants eventually dying. These symptoms were consistent with those of Philippine bacterial leaf spot described by Shew & Lucas (1991).

A fluorescent bacterium was isolated from diseased tissues onto King's medium B. Twelve strains were tested according to Schaad (1980). They produced positive reactions in tests for oxidase, arginine dihydrolase, nitrate reduction, growth at 41°C, and utilisation of mannitol, geraniol, and benzoate. Negative reactions were obtained for utilisation of cellobiose, sorbitol, trehalose, sucrose, m-tartrate, D-tartrate, D-arabinose, L-rhamnose. On this basis they were identified as Pseudomonas aeruginosa. Metabolic profiling (Biolog Station ELX808BLG, Biolog Micrologâ„¢ 4.20 database and software, Biolog, Hayward, California) indicated 0.51-0.72 similarity to P. aeruginosa.

Molecular identification was done according to Liu et al. (2002). PCR amplification was done with primers UFPL (5'-AGTTTGATCCTGGCTCAG-3') and URPL (5'-GGTTACCTTGTTACGACTT-3'). The sequence of the 1491 bp product (GenBank Accession Number AM930266) had 98% similarity to 16S rDNA sequences of P. aeruginosa (AY486350) by using BLAST. Pathogenicity of five strains was confirmed on four-week-old tobacco seedlings by either infiltrating leaves or injecting stems with a suspension containing approx. 6 × 10⁶ CFU/ml. Sterile water was used as a control. A wet rot, curved leaves, and severe necrotic symptoms developed on inoculated plants within 7-21 days (Fig.3). No symptoms developed on control plants. The bacterium was re-isolated from inoculated plants. The bacterium was confirmed as Pseudomonas aeruginosa on the basis of the above characteristics. Bacterial leaf spot caused by P. aeruginosa has been reported previously in the Philippines (Shew & Lucas 1991), but this is the first report of the disease from China.