3.3.36
IDENTIFiCATION, RESISTANCE TO ANTIBIOTICS AND UTILIZATION OF CARBON SOURCES BY XANTHOMONAS CAMPESTRIS PV. VITIANS STRAINS ISOLATED FROM DIFFERENT LETTUCE-GROWING AREAS

V TOUSSAINT1, CE MORRIS2 and O CARISSE1

1Agriculture and Agri-Food Canada, CRDH, Saint-Jean-sur-Richelieu, Quebec, J3B 3E6, Canada; 2INRA, Station de Pathologie Vegetale, Montfavet, 84143, France

Background and objectives
Bacterial leaf spot of lettuce, caused by X. c. pv. vitians, is endemic in many lettuce growing areas of the world [1]. In the last three years, for unknown reasons, it has become important in the lettuce growing areas of the province of Quebec, Canada. Experiments using different pesticides have not led to the control of the disease in the field. We are attempting to develop a biological control approach to reduce the severity of the disease, but more knowledge on the biology of the pathogen is needed to realize this kind of control. As the group X. c. pv. vitians has been recently reclassified into two species [2], it is important to determine which types of the pathogen are present in Quebec and to compare these strains to those isolated from other regions. The purposes of this study were to identify strains, with the Biolog GN microplate system, isolated from Quebec and different parts of the world, to characterize these strains for their resistance to antibiotics, for the utilization of different carbon sources, and finally to compare the results to those obtained from saprophytes isolated from lettuce leaves.

Materials and methods
Strains were identified with Biolog GN system. Stock culture were subcultured on nutrient agar for 72 hours at room temperature. One isolated colony was streaked onto tryptic soy agar and incubated for 24 hours at 28 C. To prepare the inoculum, colonies were transferred with sterile cotton swabs and suspended in sterile saline. The optical density was adjusted to 0.2 (590 nm). Biolog GN microplates were inoculated with 150 gl of this suspension per reaction well and incubated at 28C. The microplates were read with a microplate reader at 590 nm and the strains were identified with the Microlog software (version 3.7). The resistance to antibiotics was determined with the antibiograms ATB UR 14030 and ATB ANA 14260 (BioMerieux). Antibiograms ATB ANA 14260 were incubated under aerobic conditions. Visual observations were made after 24 and 48 hours. For the utilization of carbon sources by X. c. pv. vitians and saprophytes, 0.01 M of each carbon source was diluted in a minimal medium and wells were filled with 150 gi of these solutions. The microplates were inoculated with 150 gi of bacterial suspension adjusted at 0.2 (590nm). Bacterial growth was determined at 590 rim after 24, 48, and 72 hours of incubation at 28 C.

Results and conclusions
Variability of the strains was observed according to Biolog identifications. More than 67% of the strains were identified as X. c. pv. vitians type C, 2% as X. c. pv. vitians type A, and 13% as X. c. pv. carotae or X. c. pv. vitians type B. The last 18% were identified as X. campestris, but the similarity index was too low to identify the pathovar. All strains isolated in Quebec were identified as X. c. pv. vitians type C. The strains identified as X. c. pv. carotae were isolated in France, Greece, and USA. For resistance to antibiotics, strains were particularly resistant to P-lactams and glycopeptides, but they were sensitive to aminoglycosides and quinolones. Finally, the results for the different carbon sources showed that X. c. pv. vitians strains used L-phenylaianine, L-ornithine, Lpyroglutanic acid, cellobiose, dextrin, and putrescine, but few strains used thymidine and D-galactonic acid lactone which are used by most of the saprophytic bacteria of lettuce leaves.

References
1. Pernezny K, Raid, RN, Stall RE, Hodge NC, Collins J, 1995. Plant Disease 79, 359-60.
2. Vauterin L; Hoste B, Kersters K, Swings J, 1995. International Journal of Systematic Bacteriology 45, 472-89.