1.6.10
PATHOGENICITY FACTORS OF CLAVIBACTER MICHIGANENSIS SUBSP. SEPEDONICUS

RM NISSINEN, TM LINDELL, MJ LAINE and MC METZLER

University of Turku, Department of Plant Physiology and Molecular Biology, BioCity A 6, 20520 Turku, Finland

Background and objectives
Clavibacter michiganensis subsp. sepedonicus (Cms) is a Gram-positive bacterium that causes potato ring rot. This disease is difficult to control because environment and cultivar affect symptom development in hosts, and Cms tends to cause latent infections. Although the causal organism was described in 1910, the pathogenicity factors and molecular biology of this bacterium are still very poorly understood. The majority of Cms strains from diverse geographical locations harbour a -50 kbp native plasmid, pCS1, either as free or integrated form. The highly conserved nature of the plasmid suggests that it contains genes essential for Cms, but none of these genes has been characterized, and their role in pathogenicity is unknown. The aim of this work was to study the significance of plasmid sequences for virulence of Cms.

Results and conclusions
Pathogenicity values of several Cms strains were confirmed by eggplant inoculation assay. The same strains were tested for elicitation of hypersensitive reaction (HR) in non-host plant tobacco. All pathogenic strains gave a strong HR within 36 h. Only one non-pathogenic strain, P45, gave an HR similar to pathogenic strains, whereas all other non-pathogenic strains failed to elicit any visible resoponse on tobacco. Strains were tested for production of cellulose by indicator plate assays. All strains were able to hydrolyse carboxymethyl cellulose, except the non-pathogenic strain P45, for which no activity was detected.

Strains were screened for the presence of native plasmid pCS1 by Southern hybridization using 22- and 29-kb fragments of pCS1 as probes. All strains except P45 gave identical hybridization patterns. DNA from strain P45 did not hybridize at all with the 22-kb fragment of pCS1, which contains the endoglucanase gene of Cms. Thus lack of cellulose activity of P45 seems to be due to absence of the cellulose gene in this strain. P45 was transformed with the cellulose gene and other fragments of pCS1 on transformation vectors and monitored for restoration of pathogenicity.

We conclude that strain P45 contains no sequences of native plasmid pCS1, in agreement with [1]. However, in conflict with earlier reports [2], no cellulose activity was detected in P45, which is confirmed by the absence of the cellulose gene from this strain.

These data support our belief that cellulose is an important pathogenicity factor for Cms. Because we find that cellulose-negative strains are able to elicit an HR on tobacco, and cellulose-positive strains unable to elicit an HR are both non-pathogenic, it would appear that both cellulose production and ability to elicit an HR are necessary for full virulence of Cms.

References
1. Mogen BD, Oleson AE, Sparks RB et al., 1988. Phytopathology 78, 1381-1386.
2. Baer D, Gudmestad NC, 1995. Canadian Journal of Microbiology 41, 877-888.