Pitfalls in screening for camalexin-deficient mutants: effects of kinetics, pathogen choice and inoculum concentration on apparent camalexin accumulation in wild-type (Columbia) and pad2 Arabidopsis

Putative camalexin-deficient mutants of Arabidopsis thaliana (Columbia ecot ype), identified through analysis of camalexin in drop diffusates after ino culation with Cochliobolus carbonum (incompatible fungal pathogen), were no t camalexin-deficient, in response to Pseudomonas syringae pv. maculicola ( Psm ES4326, compatible bacterial pathogen). One pad (phytoalexin-deficient) mutant, identified in a screen using Psm ES4326, appeared to produce wild- type amounts of camalexin in response to C. carbonum. Camalexin accumulatio n was compared in wild-type and pad2 Arabidopsis inoculated with C. carbonu m or Psm ES4326 to determine if the plants responded differently to the two pathogens. The pad2 mutant accumulated similar amounts of camalexin (10-25 % of wild-type), with similar kinetics of accumulation, in response to both pathogens. Wild-type plants tended to accumulate camalexin more rapidly, a lthough in slightly lower amounts, in response to C. carbonum than in respo nse to Psm ES4326, However, these trends were not invariable, and these res ults provided possible explanations for some of the initially-observed disc repancies in response to the two pathogens. The kinetics of accumulation in wild-type, and the amounts accumulated, could vary such that wild-type pro duced less camalexin than pad2 at 24 h postinoculation. The relative distri bution of camalexin between leaf tissue and drop diffusates was notably dif ferent at spore concentrations of 10(5) and 10(6) spores per ml, although t he total amount of camalexin produced was similar at both spore concentrati ons. Comparisons of camalexin accumulation in plants inoculated with C. car bonum and Pseudomonas syringae pv. syringae strain D20 (incompatible pathog en) revealed no camalexin in Pss-inoculated plants, although sample sizes w ere adequate for detecting camalexin in Psm-inoculated plants. These studie s demonstrate that identification of mutants deficient in a phytoalexin (a useful tool for studying the role of phytoalexins in disease resistance) re quires consideration of phytoalexin accumulation kinetics, inoculum concent ration, relative distribution of phytoalexin in leaves and spore droplet di ffusates. and type of pathogen. (C) 2001 Academic Press.