Occurrence of blackleg disease of tobacco caused by Pectobacterium carotovorum subsp. carotovorum in China

The "floating tray system" is now the predominant tobacco seedling production system in Yunnan Province, China. An unusual disease of tobacco seedlings was observed in this system for the first time in 2001 in Dali, and was found in the rest of tobacco production areas in Yunnan Province from then on. The disease symptoms appeared under the high humidity and temperature conditions found in the polythene tunnels used for seedling production. Brownish-black necrotic spots developed on the lower part of the seedling stems (Fig. 1) and soft rot of the stems developed if the humidity was very high, resulting in death of the seedlings.

Five isolations were made from the disease spots onto nutrient agar plates. After incubation at 28°C for 48 hours, a bacterium with whitish-grey, opaque, round colony morphology was consistently isolated. This was identified as Pectobacterium carotovorum subsp. carotovorum (syn. Erwinia carotovora subsp. carotovora) on the basis of quantitative fatty acid methyl ester analysis and metabolic profiling (Biolog™). A 16S rDNA fragment of about 1.5 Kb was amplified by PCR using universal primers fD2 and rP1 and sequenced (Weissburg & Barns, 1991). The 1459 bp sequence (GenBank Acc. No. DQ333384) showed a very high (>99%) homology with the comparable 16S rDNA gene sequences of known P. carotovorum subsp. carotovorum strains (GenBank Acc. Nos. AJ233411, Z96089 and AF373189).

Pathogenicity was confirmed using three randomly selected isolates. These were suspended in distilled water (10⁷ CFU per ml) and needle-punctured to the leaves of healthy tobacco seedlings. Two days after inoculation, the inoculated leaves began to rot. Four days after inoculation, characteristic brownish-black spots appeared on the stem of all the inoculated plants. Bacteria with the same characteristics as those inoculated were successfully re-isolated from diseased tobacco seedlings. Control plants inoculated with water remained healthy.

P. carotovorum subsp. carotovorum is well known as the pathogen of hollow stalk disease in tobacco fields (McIntyre et al.,1978) and causes blackleg disease on seedlings in the floating tray system in the USA (Nesmith, 2003; Seebold, 2006). To our knowledge, this is the first report of the occurrence of blackleg disease of tobacco seedlings caused by P. carotovorum subsp. carotovorum in China.