Elicitation of a flavonoid phytoalexin accumulation in Cassia obtusifolia by a mycoherbicide: Estimation by AlCl 3-spectrofluorimetry

Abstract

The attack of weed plants by mycoherbicidal organisms can be inhibited by the antifungal phytoalexins accumulated upon infection. The measurement of phytoalexin levels in inoculated leaves is problematic due to interference by pigments and other plant metabolites. A rapid and simple procedure is presented for measuring flavonoid phytoalexins after TLC in many samples of small amounts of tissue, based on the reaction of flavonoids with AlCl 3, yielding a fluorescent complex. A single flavonoid phytoalexin was isolated from 12- to 14-day-old leaves of Cassia obtusifolia inoculated with the mycoherbicidal pathogen Alternaria cassiae. The phytoalexin reacted with AlCl 3, with a fluorescence emission peak at 472 nm when excited at 340 nm (max), with a linear response between 1 to 200 nmol phytoalexin. The method was qualitatively as sensitive as detection by staining with AlCl 3 on a silica gel thin layer chromatography plate, yet enabled quantification of the phytoalexin from a few milligrams of leaf tissue at varying times after elicitation. An initial small increase in accumulation of the phytoalexin 4 hr after inoculation was detected using this spectrofluorimetric method, but not by measurement of ultraviolet absorbance, which is far less specific and less sensitive.