INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

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 (S⁰), which has long been used by man as a protectant fungicide, may similarly function in defense, is relatively new (Cooper et al., 1996; 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 S⁰. It accumulated to fungitoxic levels in xylem and not in other tissues and persisted for >60 d. This was the first report of S⁰ 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 (Cooper et al., 1996). Elemental S formation is a property of many specialized prokaryotes (Schmidt et al., 1987; 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 more widespread and elemental S could have a frequent role in pathogen resistance. S could function in preformed defenses as suggested by its occurrence in the cuticular wax of several gymnosperms and angiosperms (Kylin et al., 1994). It may also be associated with hypersensitivity, a rapid, localized apoptotic response, and the phenotypic expression of many major genes for disease resistance (Jabs and Slusarenko, 2000).

S afforded the unusual opportunity of cellular localization of an antimicrobial substance by coupled scanning electron microscopy-energy dispersive x-ray microanalysis (SEM-EDX). This revealed high concentrations of S in scattered xylem parenchyma (XP) cells, within vessel walls and in gels occluding vessels, areas in direct contact with the xylem-invading pathogen (Cooper et al., 1996). It is thought that the presence of elemental S in XP cells could reflect accumulation in hypersensitive cells, which lack metabolic capabilities. The death of scattered XP cells is typical for vascular diseases (Mace et al., 1976; Cooper, 1981), and other phytoalexins such as phaseollin and wyerone accumulate to high levels in necrotic cells after production by adjacent living cells (Mansfield, 2000). The persistence of S⁰ in xylem tissues suggests unavailability to living cells, because wheat and spinach cells can metabolize elemental S in their chloroplasts (Legris-Delaporte et al., 1987; Joyard et al., 1988; Jolivet et al., 1995). This persistence also implies that localized accumulations were fungitoxic as many fungi can metabolize sublethal levels of S⁰ (Beffa, 1993). Therefore, it is emerging that diverse plant species can produce and accumulate elemental S in structures that may play a key role in defense.

The major source of S for plants is sulfate, which is reduced in a multistep pathway, predominantly in the chloroplasts, to sulfide. It then combines to form Cys, some of which is subsequently converted to Met or glutathione; the latter is the major store and transportable form of non-protein reduced S (Schmidt and Jäger, 1992; Hell, 1997; Leustek and Saito, 1999; Hawkesford and Wray, 2000). Production of elemental S in eukaryotes is by an uncharacterized pathway, which may involve oxidation of sulfide. It has been postulated that a sulfide oxidase may be responsible for elemental S production in spinach chloroplasts (Joyard et al., 1988) and oxidation by cytochromes has been suggested in the green alga Chlorella fusca (Kraus et al., 1984). Both of these enzymes have been implicated in bacterial production of S⁰ (Moriarty and Nicholas, 1970; Gray and Knaff, 1982; Cusanovich et al., 1991; Sasahira et al., 1992; Bang et al., 1995; Pattaragulwanit et al., 1998). The origin of the elemental S production in plants may be from glutathione or Cys degradation, possibly via the action of an, as yet uncharacterized, Cys desulfhydrase (Rennenberg et al., 1987; Schmidt, 1987). It is possible that sulfide is a by-product of the degradation of these thiols and it is this sulfide that is oxidized to form elemental S in a non-enzymic reaction (Steudel et al., 1986).

In the current work we demonstrate that elemental S is formed in tomato plants (Lycopersicon esculentum) in response to infection with V. dahliae. A comparison of a compatible and an incompatible interaction was made in isogenic lines lacking or containing the Ve gene for resistance to Verticillium spp. (Cooper and Wood, 1980; Diwan et al., 1999). This elemental S was extracted and quantified by gas chromatography-mass spectroscopy (GC-MS) as ³²S₈, the most abundant isotope and common form of S⁰. Tissue and cellular localization of S was similar to that in T. cacao (Cooper et al., 1996). S⁰ accumulation in xylem of inoculated, disease-resistant tomatoes was coincident with or followed an increase in sulfate, Cys, and glutathione.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Colonization of Tomato Plants by V. dahliae and Resulting Disease Symptoms

Symptoms became apparent in infected GCR 26 (disease-susceptible) tomato plants at approximately 10 to 13 d postinoculation (dpi). Plants expressed symptoms of water stress (flaccidity of petioles and leaves, data not shown) around midday but recovered by evening through to early morning. Epinasty of lower petioles was also apparent at this time. In the next week, wilt symptoms became irreversible and severe. Flaccidity, chlorosis, and necrosis of the lower leaves progressed to successive leaves up the plant, adventitious roots were produced, and by 21 dpi plants were severely wilted and stunted. Resistant (GCR 218) plants had chlorotic areas on the lowest leaves, whereas other parts of the plant appeared healthy and they were a similar height to control plants. Removal of the stem epidermis of susceptible infected plants revealed brown discoloration of underlying vascular bundles in contrast to the cream-colored xylem tissues of healthy and resistant plants.

Rapid, acropetal hyphal colonization occurred in infected GCR 26 stems progressing from 5% of vessels infected at internode 1 at 13 dpi (when initial symptoms were evident) to 57% at 28 dpi (Fig. 1). Colonization of internode 8 was slow initially and none was evident at internode 15 up to 20 dpi, however invasion then progressed rapidly at both internodes to reach around 30% at 28 dpi. In GCR 218 plants, hyphal colonization by V. dahliae was sparse. Only approximately 0.3% of vessels contained hyphae in internode 1 at 13 dpi and hyphae were not detected in this or in higher internodes 20dpi. Control plants showed no colonization in any sections.

View larger version (11K): [in this window] [in a new window]   Figure 1.   Colonization of susceptible (GCR 26) and resistant (GCR 218) tomato plants inoculated with V. dahliae. Analyses were performed at internodes 1 (), 8 (), and 15 () in susceptible plants and internode 1 (x) in resistant plants at 13, 20, and 28 dpi. Transverse sections of individual vascular bundles were cut from each internode of three replicate plants and percentage of vessels colonized was calculated. Points represent percentage of xylem vessels infected with fungal hyphae. Colonization for GCR 218 was only detectable at 0.3% in internode 1 at 13 dpi and so further data points for other internodes from the resistant variety are omitted for clarity. Chi-square tests at the 95% confidence level revealed significantly higher colonization of susceptible plants compared with resistant plants at internodes 1 and 8 at 13 and 20 dpi and in all internodes at 28 dpi.

Percentage of vessels containing vascular occlusions (tyloses and gels) was also significantly higher (as determined by chi-square) in vascular tissues of V. dahliae-inoculated resistant plants than of susceptible plants until 28 dpi when a similar number of tyloses was present only at internode 1 of both treatments (data not shown). Control plants showed no vascular occlusion.

GC-MS Analysis of S₈ in Xylem from Susceptible and Resistant Tomato Lines

Xylem from control plants did not accumulate elemental S and none was detected in inoculated plants at 7 dpi (before stem colonization). Subsequently, inoculated susceptible plants showed a slow increase in S₈ reaching 1.88 ± 0.71 µg g¹ at 21 dpi. In comparison, inoculated resistant plants showed a rapid and more substantial increase in elemental S and contained 10.4 ± 1.7 µg g¹ at 21 dpi (Fig. 2).

View larger version (10K): [in this window] [in a new window]   Figure 2.   GC-MS analysis for S8 of xylem tissue from resistant and susceptible tomato plants inoculated with V. dahliae. Xylem was harvested from three replicate control () and inoculated () susceptible plants and control () and inoculated (x) resistant plants at 7, 14, and 21 dpi for extraction and analysis by GC-MS. Values represent the mean with SE. Similar data were obtained in a repeated experiment.

SEM-EDX Localization of S in Vascular Tissue of Tomato Plants Inoculated with V. dahliae

Based on extent of colonization and the GC-MS analysis above, cryofixed and lyophilized, transverse and longitudinal sections were analyzed from the first internode of the tomato stems between 12 and 16 dpi and at 28 dpi to compare relative S levels and tissue and cellular distribution in resistant and susceptible, inoculated, and control plants. Cryofixed samples were coated in aluminum and lyophilized samples were coated in carbon before viewing by SEM. At 12 to 16 dpi general area analyses covering 25 vascular areas were made for each cryofixed treatment by x-ray analysis for the detection of S. S levels were recorded as "high" when the S peak was greater than 50% of the height of the potassium peak, which was always the predominant, endogenous element. Wherever a high level of S was detected, x-ray mapping was performed to enable visualization of any localized accumulations in the form of a dot map. These were compared with a secondary electron image to determine in which structure the S accumulation had occurred. Further localization studies were also attempted on lyophilized sections at 14 dpi and cryofixed and lyophilized samples at 28 dpi.

At 12 to 16 dpi only very low levels of S were detected in all areas analyzed from control plants (Fig. 3a, i and ii). In inoculated, resistant plants 18 of 25 vascular areas examined showed high S (Fig. 3d, i and ii), but in the pith cells only very low levels, equivalent to that in control plants, were present (Fig. 3b, i and ii). In inoculated susceptible plants, S was low in the majority of vascular areas (17 of 25; Fig. 3c, i and ii).

View larger version (76K): [in this window] [in a new window]   Figure 3.   Relative S levels in resistant and susceptible V. dahliae-inoculated and control stems of tomato plants. Transverse and longitudinal sections from the base of the stem were analyzed at 12 to 16 dpi for the detection of "high" (>50% of K peak) or "low" (<50% of K peak) S. Twenty-five area x-ray analyses were made for each treatment and a representative scanning electron image (i) and spectrum from that image (ii) is shown (a-d). Very low levels of S were detected in all areas of control stems analyzed (a, i and ii) and in the central pith cells from resistant inoculated plants (b, i and ii). In susceptible, inoculated plants most vascular areas contained low S (c, i and ii) in comparison to resistant, inoculated plants where the majority of vascular areas showed high S (d, i and ii). There were more vascular occlusions (gels and tyloses [d, i]) evident in the resistant vascular tissues at this time than in the susceptible line in which many vessels contained fungal hyphae (c, i). Note the aluminum peak derives from the coating evaporated onto the sample. V, Vessel lumen; VW, vessel wall; F, fungal hypha; T, tylose; P, stem pith cell; XP, xylem parenchyma cell; VG, vascular gel; TS, transverse section; and LS, longitudinal section.

For localization studies, cryofixed and lyophilized samples gave similar results. Because cryofixed samples could not be stored, lyophilized samples were used for subsequent analyses. In the vascular areas tested, from both resistant and susceptible stems at 12 to 16 dpi and at 28 dpi, those that had shown "low" S had no accumulations of S above background signal (Fig. 4a, i-iii) but in those that had shown "high" S, S had accumulated over much of the vascular tissue as evident from comparison with the background signal (Fig. 4b, i-iii). Occasionally, more intense localizations of S were detected in distinct XP cells (Fig. 4c, i and ii), gels (Fig. 4d, i and ii), tyloses, and vessel walls, in comparison with lower but still "high" levels detected in surrounding vascular structures.

View larger version (147K): [in this window] [in a new window]   Figure 4.   Distribution of S in vascular tissues of V. dahliae-inoculated and control stems of tomato plants. Transverse and longitudinal sections from the base of the stem were analyzed at 12 to 16 and 28 dpi for the detection of "high" (> 50% of K peak) or "low" (<50% of K peak) S. Wherever "high" S was detected an x-ray dot map was produced for localization of S. Representative SEM images are shown (i) with corresponding dot maps for S (ii) and background noise from the analyzer (iii; a-d). No accumulations of S were found in control samples above background (a, i-iii). In most samples that had high S, S was present over most of the vascular tissue in comparison to background signal (b, i-iii). Note accumulation of S in the tylose (b, ii). Occasionally, there were more intense spots of S in certain structures such as XP cells (c, i and ii) and gels (d, i and ii). For abbreviations see Figure 3 legend.

Temporal Effect of Infection on Sulfate, Glutathione, and Cysteine Levels in Tomato Tissues

Roots, stems (both extracted vascular tissue and total stem samples), and leaves from nodes 4, 8, and 15 from control and inoculated resistant and susceptible plants were analyzed for sulfate, glutathione, and Cys levels by HPLC.

Sulfate levels were higher in pathogen-inoculated roots (Fig. 5a), stem vascular (Fig. 5b), and total stem tissue (data not shown) from the lower half of the plant than in corresponding control material at 7 dpi. This increase also occurred in leaves 4 and 8, but later at 14 dpi (Fig. 5c) and later still in leaf 15 at both 14 and 21 dpi (Fig. 5d).

View larger version (18K): [in this window] [in a new window]   Figure 5.   Temporal effects of V. dahliae infection on sulfate levels in resistant tomato plants. Values represent the means of three replicates with SE. Plants were inoculated with either sterile water () or V. dahliae (x). At 7, 14, and 21 dpi, tissue samples were taken from root (a), total stem (up to node 8; data not shown), stem vascular tissue (up to node 8; b), leaf 4 (data not shown), leaf 8 (c), and leaf 15 (d) of plants and sulfate was estimated by HPLC.

In infected plants glutathione content of the stem vascular tissues and of leaves from the resistant but not susceptible line increased approximately 2- to 3-fold at 14 dpi (Fig. 6). No significant increase was detected in extracted entire stem or root samples (data not shown).

View larger version (18K): [in this window] [in a new window]   Figure 6.   Temporal effect of V. dahliae infection on glutathione levels in resistant and susceptible tomato plants. Values represent the means of three replicates with SE. Plants were inoculated with either sterile water () or V. dahliae (x). At 7, 14, and 21 dpi tissue samples were taken from root (data not shown), total stem tissue (data not shown) stem vascular tissue (a), leaf 4 (b), leaf 8 (c), and leaf 15 (d) of plants and glutathione content was estimated by HPLC. Glutathione levels in inoculated, susceptible plants were not significantly different from those in control plants and are omitted for clarity. A repeated experiment with stem vascular tissue produced similar data.

Cys levels followed a similar pattern and increased approximately 2- to 3-fold at 14 dpi but only in the vascular tissue of stems and only from the resistant genotype (Fig. 7).

View larger version (12K): [in this window] [in a new window]   Figure 7.   Temporal effects of V. dahliae infection on Cys in xylem tissue of resistant and susceptible tomato plants. Values represent the means of three replicates with SE. Plants were inoculated with either sterile water () or V. dahliae (x). At 7, 14, and 21 dpi tissue samples were taken from root, total stem, stem vascular tissue, leaf 4, leaf 8, and leaf 15 of plants, and Cys was estimated by HPLC. Only stem vascular tissue (above) showed a significant change in Cys levels on pathogen infection. Cys levels in inoculated susceptible plants were not significantly different from those in control plants and are omitted for clarity. The data are representative of two comparable experiments.

Toxicity of Elemental S to V. dahliae

Elemental S was found to be highly toxic to V. dahliae spores and mycelium by the two assays used. The slide assay designed to investigate the inhibition of spore germination by S⁰ revealed >90% inhibition of spore germination at 3.125 µg mL¹ (Fig. 8). The thin layer chromatography (TLC) bioassay was designed to investigate toxicity of elemental S to spore germination initially and then to mycelial growth resulting from germination of spores in areas surrounding the zones of S application. Some inhibition of spore germination was evident at 4 d at concentrations of 3.125 and 6.25 µg mL¹ as fungal growth was less dense than in surrounding areas and control spots. Between 12.5 and 50 µg mL¹, there was clear inhibition. From 100 µg mL¹ to 8,000 µg mL¹ inhibition also extended beyond the area of application suggesting that S may also act at a distance (Fig. 9). The patterns of inhibition did not change even after 40 d suggesting that mycelial growth (from surrounding germinated spores) into S treated zones was also inhibited at 3.125 µg mL¹ S and above.

View larger version (11K): [in this window] [in a new window]   Figure 8.   Inhibition of V. dahliae spore germination by elemental S. Values represent the percentage germination of three replicate analyses of 100 spores with SE. Probit analysis (the method commonly used to determine the potency of a toxin in a bioassay) was attempted but revealed that the data did not fit a typical dose response and therefore could not be analyzed to give an exact ED50 value. Therefore the ED50 value is expressed as between 1.56 and 3.125 µg mL1.

View larger version (125K): [in this window] [in a new window]   Figure 9.   TLC bioassay showing toxicity of elemental S to V. dahliae spores and mycelium. Fifty microliters of each S solution (concentration in micrograms per milliliter is shown below each application spot) was applied to the TLC plate and a suspension of V. dahliae spores (1 × 106 spores mL1) sprayed on the surface. The fungus began to show as pigmented black microsclerotia at 3 d against the white background of the plate and inhibition zones were obvious at 4 d (above). The plate was analyzed for 40 d but the growth pattern did not change significantly after 4 d.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

To date there are few examples of elemental S production by eukaryotes. The discovery of S in resistant lines of tomato and previously of T. cacao, in sufficient quantities, in the right place, and at the right time potentially to inhibit V. dahliae are the first to implicate the element in induced disease resistance (Cooper et al., 1996; Resende et al., 1996). Clearly, tomato offers a more tractable model for genetical and biochemical dissection.

Related plant families generally make use of chemically related compounds for defense. S is perhaps the only known phytoalexin that is produced by different taxa, but this may reflect that it is the only known inorganic antimicrobial agent produced by plants (Dixon, 2001).

Inoculation of near isogenic disease-resistant and -susceptible tomato lines resulted in rapid, acropetal, systemic spread of V. dahliae in susceptible GCR 26, whereas colonization of the resistant variety (GCR 218) was restricted to basal internodes and was very sparse (approximately 0.3% vessels infected at the first internode). This expression of the Ve gene for resistance concurs with previous data of Cooper and Wood (1980). The overall restriction and eventual visual disappearance of hyphae from the stem base of GCR 218 implies the production of antifungal compounds. Previously linked with disease resistance of tomato is the phytoalexin rishitin and chitinases (some of which can degrade fungal cell walls) (Bell and Mace, 1981). Elemental S may contribute to this antimicrobial environment, which is thought to be maintained by physical occlusion of vessels with tyloses and gels (Cooper, 2000). In this study, tyloses were abundant in the incompatible interaction but significantly less in the infected xylem of the susceptible genotype.

Kinetics and levels of S accumulation were revealed accurately for the first time as a result of the development of a method for accurate quantification of ³²S₈. S accumulation resembled that of various organic phytoalexins, with a more rapid and intensive production in the resistant than in the susceptible host; a pattern also suggested by SEM-EDX. This differential response is especially remarkable as it was inversely related to the amount of pathogen; fungal biomass in resistant xylem was negligible in contrast to the extensive colonization of xylem in the susceptible line.

Analogous patterns of phytoalexin accumulation in incompatible host-pathogen interactions, which result in rapid highly localized toxic levels coincident with inhibition of fungal growth, include the isoflavanoids phaseollin in bean (Phaseolus vulgaris) and glyceollin in soybean (Glycine max), the furanoacetylene wyerone in broad bean (Vicia faba), and the sesquiterpene rishitin in potato (Solanum tuberosum) (Mansfield, 2000).

SEM-EDX revealed that accumulation of S as the element and/or as organically bound S was widespread in tomato vascular tissue undergoing an incompatible interaction with V. dahliae. Occasional localizations were detected in scattered XP cells, vascular gels, and in xylem vessel walls in pathogen-inoculated plants and were detected in both cryofixed and lyophilized sections. These zones corresponded closely with the results of SEM-EDX on vascular tissue from resistant T. cacao plants inoculated with V. dahliae (Cooper et al., 1996). Terpenoid aldehyde phytoalexins of cotton are similarly formed in XP cells and these along with phytoalexins of some other species are exuded into xylem vessels to impregnate vascular occlusions (Bell and Mace, 1981). Impregnation of these structures with elemental S would be of direct relevance to resistance against vascular fungi by providing an effective barrier to vertical and lateral spread, which characterizes their mode of invasion (Cooper, 2000). Even based on the amounts of S⁰ detected in entire tissue extracts (probably containing only a small proportion of S-containing XP cells), which after 14 and 21 dpi were approximately 6 and 10 µg g¹, respectively, levels were greater than that required for inhibition of V. dahliae spore germination and hyphal growth; both structural forms are produced in infected xylem. Calculations of amounts of other phytoalexins such as phaseollin, have revealed that whole tissue extracts give a gross underestimate, because phytoalexins become concentrated in hypersensitive cells where they can be in considerable excess above that required for pathogen inhibition (Mansfield, 2000). SEM-EDX analysis indicated that S may also be concentrated in this way.

The origin and biosynthetic pathway of elemental S formation remains unresolved. The increased sulfate levels may reflect the over-expression of sulfate transporters in response to the burden on metabolism to produce elemental S. The observed transient peaks of Cys and glutathione, particularly as these occur in the vascular tissue samples, implicate these S-containing compounds in the phenomenon. The major site of S reduction is in the leaf tissues, and glutathione would be involved in transport of this S in the vascular tissues. Reduced glutathione can also have a protective role in anti-oxidative pathogen defense reactions, and pool size can be elevated in response to these demands (Kömives et al., 1998). Notably, marked accumulation of glutathione occurred in tomato cells carrying resistance genes in response to specific fungal elicitors (May et al., 1996). Localized accumulation of glutathione and subsequent degradation together with chemical oxidative processes (Steudel et al., 1986) could result in the observed depositions. Future work will focus on the identification of Cys degradation pathways, for example the identification of a Cys desulfhydrase (Schmidt, 1987). Novel components of a pathway leading to elemental S accumulation, induced by pathogen infection, may be identified by employing gene screening procedures relying on differential expression, such as differential display. We are currently applying these approaches to this tomato model system.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Plant Growth and Cultivation

Tomato (Lycopersicon esculentum) GCR 26 and GCR 218 (isogenic lines, which are susceptible and resistant, respectively, to V. dahliae) were either grown in Levingtons compost (fine grade 2 followed by medium grade 2) or a 1:1 sand:perlite mix in a greenhouse. Plants were fed three times a week with a full nutrient solution that contained KNO₃ (7 mM), MgCl₂.6H₂O (0.9 mM), KH₂PO₄ (1.0 mM), Mg(NO₃)₂·6H₂O (1.7 mM), NaCl (0.1 mM), EDTA FeNa (0.05 mM), Ca(NO₃)₂·4H₂O (5 mM), CaCl₂·6H₂O (2 mM), CH₃COOZn·2H₂O (1.3 µM), H₃BO₃ (24.5 µM), Cu(NO₃)₂·3H₂O (0.6 µM), (NH₄)₂ Mo·4H₂O (0.8 µM), FeNa EDTA (50.1 µM), Mn(NO₃)₂·4H20 (9.3 µM), and 1.0 mM SO₄² supplied as MgSO₄·7H₂O. The temperature was maintained at 25°C ± 3°C and supplementary illumination was supplied by Phillips 400-W high-pressure sodium lamps (Eindhoven, The Netherlands) for a 16-h daylength.

Pathogen Growth and Inoculation of Plants

V. dahliae, isolate Dvd-T5 race 1, was provided by K. Dobinson (Agriculture and Agri-food, Ontario, Canada). Spores were stored long term in 25% (v/v) glycerol at 70°C and when required were subcultured onto Czapek dox agar (Oxoid Ltd., Basingstoke, UK) and incubated at 25°C. To produce inoculum, a shake culture of V. dahliae was made in Czapek dox liquid medium at 25°C, 150 rpm. The resulting suspension was filtered through two layers of sterile muslin and centrifuged at 1,000g for 10 min. The pellet was resuspended twice in sterile milli-Q water (pH 7). Spore concentration was determined with a hemocytometer and diluted to give 1 × 10⁷ spores mL¹ with milli-Q water (pH 7).

Eight-week-old plants from each cultivar were root-inoculated by pouring 50 mL per plant of the V. dahliae spore suspension onto the soil or sand. Control plants were sham-inoculated with 50 mL of sterile milli-Q water (pH 7). After inoculation, all plants were watered to wash the inoculum into the soil.

Quantification of V. dahliae from Plant Stem Tissue

Quantitative colonization analysis was performed at different stem positions (internodes 1, 8, and 15) of GCR 26 and GCR 218 soil grown, inoculated, and control plants. This was done at three time points (13, 20, and 28 d) after inoculation. Thin transverse sections of individual vascular bundles were cut with a razor blade from each of the stated internodes of three replicate inoculated and control plants and examined by light microscopy at 400×. Percentage of xylem vessels infected with fungal hyphae and percentage of vessels occluded with gels or tyloses was calculated (Cooper and Wood, 1980).

Dissection and Preparation of Xylem for Elemental S, Sulfate, and Thiol Analyses

After root-inoculation, GCR 26 and GCR 218 V. dahliae-inoculated and control plants were left for one of three time intervals corresponding with those used for assessment of colonization (13, 20, and 28 dpi) for preliminary quantitative analysis of elemental S. S was present in both resistant and susceptible xylem at all time points but not in controls. Therefore the experiment was repeated but using plants grown in sand culture to ensure a defined sulfate regime and the plants were harvested at 7, 14, and 21 dpi for elemental S, sulfate, and thiol analyses. At each time point, three plants were harvested individually and the lower half of the stem up to node 8 was removed from each plant. The xylem tissue was excised by scraping away the epidermis with a scalpel, and then the vascular bundles were dissected out with forceps. All xylem tissue was frozen at 70°C and when required was comminuted in liquid nitrogen and subdivided for each analysis. For sulfate and thiol analyses roots, whole stem sections (up to node 8), leaf 4 (numbered from the base), leaf 8, and leaf 15 were additionally harvested.

Elemental S Detection by GC-MS

Dichloromethane (HPLC grade, Fisher Scientific UK. Ltd.) was added to the comminuted xylem sample (10 mL g¹) and left for 10 min to extract non polar compounds. At this point a defined amount of ³⁴S standard dissolved in dichloromethane was added to quantify the ³²S in the sample. An attempt was made to match the level of ³⁴S added to the level of natural ³²S predicted from previous experiments. Extracts were then filtered through 1PS filter paper (Whatman, Clifton, NJ) into round bottom flasks and the dichloromethane removed by rotary evaporation. The remaining residue was further purified by redissolving in 20 mL hexane (HPLC grade, Fisher Scientific UK. Ltd., Loughborough, Leicestershire, UK) and running through an 8-mL silica (60A) column (Extract Clean, Alltech, Deerfield, IL). The column was further eluted with 10 mL of hexane, and the effluent containing the elemental S was collected. The hexane was removed by rotary evaporation, and the residue was resuspended in 2 mL of dichloromethane and transferred to a 5-mL glass vial from which the dichloromethane was evaporated in a stream of nitrogen gas. The final residue was then resuspended in 250 µL of dichloromethane and analyzed by GC-MS.

Elemental S was quantified (as S₈) by GC-MS using a model 5791 GC-MS (Hewlett-Packard, Palo Alto, CA), with an SGE BPX5, 25-m × 0.25-mm column and helium as carrier gas at a pressure of 80 kPa. The internal standard (³⁴S₈) co-elutes with ³²S₈ but has a defined mass of 272 compared with 256. Single ion monitoring was used for detection and the 256 and 272 ions were analyzed in turn for 15 cycles of 50 ms to build up a peak. The column temperature regime adopted was: 35°C for 2 min, which was raised by 25°C per minute to 200°C, and then by 5°C per minute to 250°C, and finally by 15°C per minute to 320°C. The MS source temperature was 150°C and the transfer line temperature was 250°C. The ionization mode used was electron impact (+ve ion) at 70 eV. S₈ is thermally unstable at temperatures >119°C and was found to break down to S₂ during analysis. Although there was some recombination to S₈ in the cooler parts of the process, this was not complete and fragments of S₂, S₄, and S₆ were produced. Recombination of ³²S and ³⁴S ions also occurred giving mixed S compounds.

A cool, on-column injector with a 0.53-mm i.d. precolumn (retention gap) was used to prevent depolymerization at the start of the GC-MS process. Calibration curves were constructed with defined ratios of ³²S₈ and ³⁴S₈ to determine thermal splitting and recombination due to temperatures of the column, the MS, and the transfer line. The amount of ³²S₈ was then estimated by integration of the ³²S₈ and ³⁴S₈ peaks with reference to calibration curves. Calculations were performed by an HP Pascal Chemstation computer.

Localization of S by Coupled SEM-EDX

Transverse and longitudinal sections of thickness approximately 2 mm of individual vascular bundles were excised with a razor blade (dichloromethane washed) from the first internode of V. dahliae-inoculated and control, susceptible and resistant plants at 12 to 16 dpi (analysis of all treatments could not be completed in 1 d and samples could not be stored) and at 28 dpi for cryofixation. A 20-nm coating of aluminum was evaporated onto tissues (gold obscured the S peak during x-ray analysis), and the samples were viewed in a JSM-6310 SEM (JEOL, Tokyo). X-ray analysis was by an AN10000 energy dispersive x-ray analyzer (Oxford Instruments Ltd, Marlow, UK).

Lyophilized samples were also prepared at 14 and 28 dpi from all treatments as cryofixed samples could not be stored. Samples were plunged into liquid nitrogen for 5 min and then transferred under liquid nitrogen to aluminum carriers for lyophilization for 12 h. Samples were then mounted on carbon adhesive discs, which were adhered to aluminum planchettes (Agar Scientific Ltd, Stansted, UK). Mounted samples were carbon coated in an E12E Vacuum Coating Unit (Edwards High Vacuum Ltd, Crawley, UK). SEM and x-ray analysis was performed as above.

Two replicate plants were used for cryofixation and a further two for lyophilization for each treatment at each time point. For analysis of relative S levels present at 12 to 16 dpi, cryofixed material was used and 25 vascular areas were randomly chosen from two randomly selected sections from each plant. Localization of S in the form of dot maps was also performed on these areas as well as in lyophilized sections at 14 dpi and with both cryofixed and lyophilized sections at 28 dpi.

Analysis of Sulfate Ions by HPLC

Sulfate was measured after the protocol of Blake-Kalff et al. (1998) by extracting 0.1 g of lyophilized plant material in 1 mL of deionized water at 90°C for 1 h, after which the extract was filtered through filter paper (no. 42, Whatman). SO₄² concentrations in the extracts were determined by ion chromatography (Dionex 2000i/sp) using an AS9SC separation column fitted with an AS9G guard column (Dionex, Sunnyvale, CA). The eluent solution consisted of 1.8 mM Na₂CO₃, 1.7 mM NaHCO₃, and the column was regenerated with 0.025 N H₂SO₄.

Analysis of Thiols by HPLC

Comminuted tissue (0.1 g) was extracted in 1.5 mL of 0.1 N HCl, containing 0.1 g of acid-washed polyvinylpolypyrrolidone. This was mixed and left at room temperature for 1 h. Samples were then centrifuged at 10,000g for 5 min. Aliquots of 0.5 mL were filtered through 0.2-µm spin filters (Anachem, Luton, UK). One-hundred microliters of 0.25 M Ches (2-[N-cyclohexylamino] ethanesulfonic acid) was added to 100 µL of filtered sample to adjust to pH 8.0. Seventy microliters of 10 mM dithiothreitol was added before a 1-h incubation at room temperature followed by 10 µL of 25 mM monobromobimane. The components were rapidly mixed, and derivatization occurred at room temperature in the dark for 15 min. The reaction was terminated by the addition of 220 µL of 100 mM methylsulphonic acid.

Monobromobimane derivatives were then separated by HPLC using a Zorbax ODS 5-µ column (Jones Chromatography, Hergoed, UK). A gradient of 10% to 90% (v/v) methanol in 0.25% (v/v) acetic acid (pH 4.9) was used to elute the derivatives, which were detected fluorimetrically (excitation 380 nm, emission 480 nm) and compared with known standards for quantification.

Toxicity of Elemental S to V. dahliae

Two bioassays were devised to test toxicity of elemental S to V. dahliae, spore germination and mycelial growth.

Slide Bioassay of Spore Germination

Two-day-old liquid cultures of V. dahliae were grown in 100 mL of Czapek Dox (Oxoid) in 250-mL flasks at 150 rpm, in darkness at 25°C. Cultures were filtered through muslin and centrifuged at 3,000g for 10 min, and conidia were resuspended in sterile distilled water (pH 6.5) and diluted to 3 × 10⁵ spores mL¹. A 2-fold dilution series of ³²S (Aldrich, Milwaukee, WI) ranging from 1.6 to 100 µg mL¹ as well as 500, 1,000, and 8,000 µg mL¹ solutions were made up in dichloromethane. Fifty microliters of one of these solutions or pure solvent was pipetted into each of three 10-mm wells of a teflon-lined diagnostic slide (Merck, Darmstadt, Germany). The contents of each well was evaporated and 40 µL of the V. dahliae spore suspension added. Each slide was transferred to an individual Petri dish containing moistened filter paper and incubated for 15 h at 25°C. Immediately after incubation, 10 µL of 0.1% (w/v) aniline blue in lactophenol was pipetted into each well to stain conidia and arrest further growth. Conidia were considered to have germinated when germ tube length was longer than the spore diameter. Percentage germination was calculated from 100 spores from each of the three replicate wells. Data were subjected to probit analysis (Finney, 1964), the method commonly used to determine an ED₅₀ or potency value from a toxicity bioassay (see Fig. 8 legend).

TLC Bioassay

A V. dahliae conidial suspension (1 × 10⁶ spores mL¹) was made up in Czapek Dox medium. A 20- × 20-cm TLC aluminum sheet (silica gel 60 F₂₅₄, Merck) was pre run in dichloromethane and allowed to dry. Fifty microliters of each of the ³²S solutions quoted above and pure solvent as a control was pipetted slowly onto the TLC plate and the dichloromethane allowed to evaporate resulting in approximately 25-mm diameter zones. The V. dahliae spore suspension was then sprayed evenly onto the plate and incubated in darkness at 25°C and 100% relative humidity. Plates were analyzed daily for 7 d and then weekly until 40 d to detect inhibition of spore germination initially and then any mycelial growth, which would eventually colonize the majority of the silica gel and could subsequently invade initial zones of inhibition. The production of pigmented, melanized microsclerotia clearly revealed fungal growth and provided a sufficiently dark background to visualize white areas where growth did not occur.