A search for elicitors of the hypersensitive reaction in lettuce downy mildew disease

Abstract

Attempts were made to isolate elicitors of the hypersensitive reaction (HR) from various infection structures produced by Bremia lactucae including isolated mycelium, primary and secondary vesicles and haustoria. Potential elicitors were assayed by injection into lettuce cotyledons and also following their addition to lettuce protoplasts. The protoplast assay was designed to ensure contact between elicitors and putative receptor sites in the plant cell membrane. Scanning electron microscopy showed that enzymic digestion of cotyledons for 12 h released protoplasts free of residual wall fragments and that the wall reformed slowly following protoplast isolation. Conidiosporangia germinated poorly in the protoplast osmoticum. Protoplast viability (assessed with fluorescein diacetate) was reduced by 20–45% in the presence of germinating spores but no cultivar or isolate specific effects were observed. Prior germination of conidiosporangia in water or use of protoplasts recovered following enzymic digestion for 3, 5, 7, or 9 h did not affect the response of protoplasts. Frozen, thawed and ultrasonicated suspensions of conidiosporangia did not kill protoplasts rapidly but caused non-specific agglutination. Intercellular fluids recovered from infected lettuce did not kill lettuce or cabbage protoplasts. Mycelium of B. lactucae was recovered following enzymic digestion of infected cotyledons. Intercellular hyphae and haustoria recovered appeared to be packed with cytoplasm but were dead as indicated by their failure to exclude Evans blue stain. Primary and secondary vesicles were recovered by homogenization of epidermal strips and purified by combinations of sieving and application to sucrose density gradients. Very few infection structures were viable following extraction and all were dead within 10 min of isolation. The presence of cultivar specific elicitors of the HR was indicated in certain assays of primary vesicles of isolate NW3 recovered 6 h after inoculation of the susceptible cv. Cobham Green. Repeated experiments showed that the occurrence of differential activity was very inconsistent and may have been associated with the presence of many viable vesicles in the active extracts. Difficulties associated with the detection of elicitors of the HR produced by obligate parasites are discussed.