1.3.21
ENZYME ACTIVITY COMPARED IN CAPSICUM ANNUUM INFECTED WITH XANTHOMONAS AXONOPODIS PV. VESICATORIA (X. CAMPESTRIS PV. VESICATORIA)

C M WALKES and LW O'GARRO

Microbial Pathogenicity Research Group, Department of Biological and Chemical Sciences, University of the West Indies, PO Box 64, Bridgetown, Barbados

Background and objectives
The presence of a recognized invading pathogen will trigger an active defence in plants which results in the accumulation of pathogenesis-related (PR) proteins. Several PR proteins possess antifungal and antibacterial activity and some have been identified as chitinases or glucanases [1]. Phenylalanine ammonia-lyase (PAL) activity has been indicated in pathogen defence including the resistance of Capsicum annuum to Phythophthora capsici [2]. A previous study conducted to compare the response of pepper leaves and flowers to infection with a strain of Xanthomonas campestris pv. vesicatoria (XCV) found that chitinase and beta-1,3-glucanase were induced in infected leaf tissue when infiltrated with an inoculum containing 103 or 104 cells/ml [3]. The present study sought to compare the intercellular fluid (IF) activities of several enzymes in susceptible and resistant pepper cultivars. Enzymes assayed for were beta-glucanase, beta-glucosidase, chitinase and phenylalanine ammonia-lyase.

Materials and methods
Early Calwonder (ECW) and ECW20R cultivars were used in this study. The strain of XCV was a pepper race 1 designated S18. Fully expanded leaves were infiltrated with an inoculum containing 108 cells/ml. Sterile distilled water was used as a control. Until leaf abscission in the pathogen-infected plants, leaves were harvested, vacuum infiltrated with SDW and the IF collected by centrifugation at 1000 r.p.m. for 15 min at 4C. The IF was filter-sterilized and stored at -20C. Standard assays were used to determine enzyme activities in duplicate samples.

Results and conclusions
Beta-glucanase: in ECW the presence of the pathogen resulted in a decline in the level of activity until leaf abscission after 48 h. In contrast, the control showed a twofold increase in activity before returning to the initial level (in 72 h). In ECW20R, enzyme activity was similar for pathogen and control before leaf absicission after 30 h.

Beta-glucosidase: in both cultivars, activity was low in the control plants and 2-4 times higher in the presence of the pathogen.

Chitinase: activity showed a sevenfold increase in both cultivars in the presence of the pathogen when compared to the control over 30 or 48 h and before leaf absicission.

PAL: activity increased significantly in the ECW cultivar from zero level with the pathogen and in the control over time, however the level in the infected plants declined toward abscission in contrast to the control which peaked at that time period. No PAL activity was recorded for the ECW20R control, in contrast to the infected leaves where activity increased up to the 30 h before leaf abscission.

Both cultivars showed the presence of all the enzymes except PAL from zero hours, indicating they are normally found in the IF. Beta-glucosidase and chitinase were both found at higher levels in the presence of the pathogen than in the control over time in both cultivars, however levels in ECW20R were higher than in ECW. Beta-glucanase activity was suppressed in ECW in the presence of the pathogen. Beta-glucosidase and chitinase would appear to be general defence PR proteins whose activities are greatly enhanced in response to the presence of XCV in pepper plants.

References
1. Hammond-Kosack KE, Jones JDG, 1996. Plant Cell 8, 1773-1791.
2. Okey EN, Duncan EJ, Sirju-Charran G, Sreenivasan TN, 1997. Journal of Phytopathology (in press).
3. O'Garro LW, Charlemagne E, 1994. Physiological and Molecular Plant Pathology 45, 181-188.