1.4.18
FOLIAR APPLICATION OF PHOSPHATE INDUCES LOCALIZED CELL DEATH, GENERATION OF REACTIVE OXYGEN SPECIES, SALICYLIC ACID, DEFENCE-RELATED PROTEINS AND SYSTEMIC ACQUIRED RESISTANCE IN CUCUMBER

M OROBER, J SIEGRIST and H BUCHENAUER

University of Hohenheim, Institute of Phytomedicine, D-70593 Stuttgart, Germany

Background and objectives
Foliar applied phosphates are known as inducers of systemic acquired resistance (SAR) in cucurbits. In cucumber, for example, a broad spectrum of protection was achieved against viral, bacterial and fungal pathogens [1]. However, little is known of the mechanisms which are involved in resistance induction by this chemical agent. Our aim is to detect early induced processes which lead to the expression of SAR. The mechanisms of phosphate-induced resistance were compared with those expressed by benzo[1,2,3]thiadiazole-7-carbothioic acid S-methyl ester (BionŽ) and tobacco necrosis virus (TNV).

Results and conclusions
Spray treatment of the lower leaves of cucumber plants with phosphate salts induced local and systemic resistance against anthracnose (Colletotrichum lagenarium) and powdery mildew (Sphaerotheca fuliginea). Concentrations between 20 and 80 mM of di- and tribasic sodium and potassium phosphates were most effective against both pathogens. SAR was already evident at 3 days and increased until 7 days after phosphate application. The maximum level of local and systemic protection to anthracnose induced by application of K2HPO4 (100 mM) 7 days before inoculation was 100 and 97%, respectively, and to powdery mildew 95 and 80%, respectively.

For inducing resistance, the formation of necrotic lesions on the inducer leaves appears to be necessary. The necrotic responses in the phosphate-treated tissue appeared within 24 h and were visible as small dots spread over the whole leaf surface. On leaves with SAR, no visible signs of necrosis were detected. Inoculation of cucumber leaves with TNV also caused localized necrotic symptoms.

Development of necrosis in phosphate-treated leaves was accompanied by the generation of reactive oxygen species. Accumulation of superoxide was detectable 4 h after application, whereas hydrogen peroxide was demonstrated when visible lesions appeared. In addition, localized cell death was identified microscopically using Trypan blue staining.

Spraying leaves of cucumber plants with phosphates or inoculating leaves with TNV resulted in accumulation of endogenous free and glucosylated salicylic acid (SA) which is a key molecule in the process of SAR-activation [2]. Glucosylated SA increased 50-fold in the phosphate-treated leaves and 230-fold in the TNV-inoculated leaves. The accumulation of SA was also detected in the untreated leaves expressing SAR. The increases in glucosylated SA were 6.7-fold and 14.9-fold, respectively. Treatment of leaves with BionŽ did not induce any SA accumulation in treated and untreated leaves.

Phosphate treatment resulted in a rapid activation of defence-related proteins such as peroxidase and polyphenoloxidase throughout the whole cucumber plant. Similar increases in activities of both enzymes were demonstrated in cucumber plants following TNV inoculation.

In conclusion, we assume that phosphates sprayed on leaves of cucumber plants induce resistance mechanisms corresponding with those activated by TNV. In this system a chemical inducer seems to elicit similar primary processes regarding expression of SAR as a biotic agent which are different from those induced by BionŽ.

References
1. Mucharromah E, Kuc J, 1991. Crop Protection 10, 265-270.
2. Molders W, Buchala A, Metraux J-P, 1996. Plant Physiology 112, 787-792.