The occurrence of fungicidal, elemental S is well documented in certain specialized prokaryotes, but has rarely been detected in eukaryotes. Elemental S was first identified in this laboratory as a novel phytoalexin in the xylem of resistant genotypes of Theobroma cacao, after infection by the vascular, fungal pathogen Verticillium dahliae. In the current work, this phenomenon is demonstrated in a resistant line of tomato, Lycopersicon esculentum, in response to V. dahliae. A novel gas chromatographymass spectroscopy method using isotope dilution analysis with 34 S internal standard was developed to identify unambiguously and quantify 32 S in samples of excised xylem. Accumulation of S in vascular tissue was more rapid and much greater in the disease-resistant than in the disease-susceptible line. Levels of S detected in the resistant variety (approximately 10 g g Ϫ1 fresh weight excised xylem) were fungitoxic to V. dahliae (spore germination was inhibited Ͼ90% at approximately 3 g mL Ϫ1 ). Scanning electron microscopy-energy dispersive x-ray microanalysis confirmed accumulation of S in vascular but not in pith cells and in greater amounts and frequency in the Verticillium spp.-resistant genotype. More intensive localizations of S were occasionally detected in xylem parenchyma cells, vessel walls, vascular gels, and tyloses, structures in potential contact with and linked with defense to V. dahliae. Transient increases in concentrations of sulfate, glutathione, and Cys of vascular tissues from resistant but not susceptible lines after infection may indicate a perturbation of S metabolism induced by elemental S formation; this is discussed in terms of possible S biogenesis.Phytoalexins are defined as low-M r , anti-microbial compounds that are both synthesized by and accumulate in plants after exposure to micro-organisms (Mansfield, 2000). A wide range of organic compounds such as phenolics and terpenoids has been identified as phytoalexins and they are synthesized from remote precursors. Although some phytoalexins are well known for their role in pathogen resistance in plants, the idea that elemental S (S 0 ), which has long been used by man as a protectant fungicide, may similarly function in defense, is relatively new Resende et al., 1996). In the typical, multiple phytoalexin response of resistant cultivars of Theobroma cacao to the vascular pathogen Verticillium dahliae, the most fungitoxic of four phytoalexins was S 0 . It accumulated to fungitoxic levels in xylem and not in other tissues and persisted for Ͼ60 d. This was the first report of S 0 as an induced antimicrobial substance and of any inorganic element (other than structural functions in cell walls of calcium or silicon) contributing directly to active defense . Elemental S formation is a property of many specialized prokaryotes Visser et al., 1997;Reinartz et al., 1998) but until recently had only been described in eukaryotes for a few algae (Ikawa et al., 1973;Izak et al., 1982;Kraus et al., 1984). It appears however, that this phenomenon may be ...
