1.4.26
TREATMENT OF LETTUCE SEEDLINGS WITH DICHLOROISONICOTINIC ACID INDUCES SUSCEPTIBILITY TO A NORMALLY AVIRULENT ISOLATE OF BREMIA LACTUCAE

M BENNETT, A SOARES, O BONNAMOUR, R SESSA and J MANSFIELD

Department of Biological Sciences, Wye College, Ashford, Kent TN25 5AH, UK

Background and objectives
2,6-Dichloroisonicotinic acid (DCINA) has been shown to act as an inducer of both local and systemic acquired resistance (SAR) in a wide range of plant species. Treatment of lettuce seedlings cv. Diana with DCINA followed by inoculation with a virulent isolate (TV) of Bremia lactucae resulted in a slight decrease in sporulation. Surprisingly, inoculation with an avirulent isolate (CL9W), in which resistance mediated by the Dm7/A7 interaction is usually characterized by a late hypersensitive response (HR) [1], resulted in greatly increased levels of sporulation. The objective of the research was to investigate these seemingly contradictory results.

Materials and methods
Plants, isolates, inoculations and microscopy were as described in [1]. DCINA was applied by uptake through cut hypocotyls or spraying 7-day-old seedlings. Inoculation was usually 24 h after treatment with DCINA. A range of DCINA analogues which included isonicotinic acid, 2-chloroisonicotinic acid, 2-chloronicotinic acid, 2,6-dichloropyridine and 3,5-dichlorobenzoic acid were tested for activity. Quantification of the guaianolide sesquiterpene phytoalexin Lettucenin A following infection was as described in [2]. The timing of irreversible membrane damage (IMD), measured by the ability of infected epidermal cells to plasmolyse in a hypertonic solution, was determined [1].

Results and conclusions
Microscopical observations of fixed and cleared cotyledons indicated that fungal colonization by the avirulent isolate was more extensive and associated with a marked reduction in the yellow autofluorescent deposits typical of cells showing the HR in lettuce. Structure-activity analysis of analogues of DCINA indicated that only 2-chloroisonicotinic acid was as effective as DCINA, with other modifications being either less or totally ineffective, and that increased sporulation was always associated with a reduction in autofluorescent deposits. Lettucenin A accumulation in infected cotyledons was almost completely inhibited although more cells were infected following DCINA treatment. The reduction in autofluorescent deposits and phytoalexin accumulation introduced the possibility that DCINA may have been acting by inhibiting cell death and thereby delaying activation of resistance. Whilst analysis demonstrated a small but significant delay in the timing of IMD following treatment, all infected epidermal cells did eventually die. Although it was not possible to extend this technique to infected mesophyll cells, microscopy of fixed cotyledons indicated that these cells also died.

DCINA thus appears to act in two ways in lettuce seedlings. Whilst it is capable of inducing a low level of resistance to normally compatible isolates, it also delays or inhibits at least some of the defence responses typically associated with the HR, permitting increased growth of avirulent isolates.

References
1. Bennett M, Gallagher M, Bestwick C et al., 1994. Physiological and Molecular Plant Pathology 44, 321-333.
2. Bennett M, Gailagher M, Fagg J et al., 1996. Plant Journal 9, 851-865.