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Section of Genetics, Cell Biology and Development, Department of Biology, University of Patras, 26100—Patra, Greece
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Certain phytopathogenic fungi differentiate by forming sclerotia by an unclear biochemical mechanism. We have proposed that sclerotial differentiation might be regulated by fungal antioxidant defense. Part of this defense might be ascorbic acid, which in its reduced form is a well-known antioxidant. This natural antioxidant was studied in Sclerotium rolfsii in relation to oxidative-growth conditions, developmental stages and strain-differentiating ability. The transition of a sclerotial strain from the undifferentiated to the differentiated stage was accompanied by a sharp shift in the ratio of reduced/oxidized ascorbate toward the oxidized form. Ascorbate profiles and lipid peroxidation levels were different between the sclerotial strain grown under high- and low-oxidative stress conditions, as well as between a nonsclerotial S. rolfsii strain grown under high-oxidative stress conditions. In addition, the ratio of reduced/oxidized ascorbate in the nonsclerotial strain remained unchanged throughout growth. Lipid peroxidation under high-oxidative stress conditions in sclerotial S. rolfsii colonies one day before differentiation was 3.6-fold higher than in same-day colonies of this strain grown under low-oxidative stress conditions and 2.5-fold higher than in similar-day colonies of the nonsclerotial strain grown under high-oxidative stress conditions. Exogenous ascorbate caused a concentration-dependent reduction of lipid peroxidation and a proportional inhibition of the degree of sclerotial differentiation in the sclerotial strain grown under high-oxidative stress conditions by lowering its lipid peroxidation before differentiation to levels similar to the strain grown under low-oxidative stress conditions and to the nonsclerotial strain. Ascorbic acid might be produced by the sclerotial strain to reduce oxidative stress, although less efficiently than the nondifferenting strain. The data of this study support our theory that oxidative stress might be the triggering factor of sclerotial differentiation in phytopathogenic fungi.
Key words: fungi, lipid peroxidation, oxidative stress, sclerotia
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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, Willetts 1971, Agrios 1988). Because of the biological and agricultural significance of the sclerotiogenic fungi, many efforts have been directed toward elucidating the mechanism of sclerotial biogenesis. Understanding sclerotial differentiation (viewed as a primitive form of biological differentiation) would help in understanding complex forms of differentiation and might provide information for the development of nontoxic control methods, as an alternative to traditional fungicides (Christias 1990).
In a previous study we have shown that biogenesis of sclerotia in Sclerotium rolfsii is characterized by a high degree of lipid peroxidation, providing for the first time strong indication of a relationship between sclerotial differentiation and oxidative stress (Georgiou 1997). This result prompted us to advance a theory suggesting that sclerotial differentiation is triggered by oxidative stress caused by free radicals. We have presented more evidence to support this theory by showing that certain hydroxyl radical scavengers (known to reduce oxidative stress) inhibit lateral sclerotial differentiation in S. rolfsii and in Sclerotinia minor (Georgiou et al 2000a).
In the context of our theory, we investigated whether the cytosolic antioxidant ascorbic acid (Bendich et al 1986, Halliwell and Gutteridge 1999) is related to differentiation of S. rolfsii. Ascorbate plays important biochemical roles (Englard and Seifter 1986), such as in cell growth and differentiation (Alcain and Buron 1994), in most organizisms. One of its many roles is as an antioxidant scavenging peroxyl radicals (Bendich et al 1986), superoxide radicals (Aver'yanov and Lapikova 1988), singlet oxygen (Kwon and Foote 1988) and hydrogen peroxide via ascorbate peroxidase (Yamasaki and Grace 1998). It also can act directly as a lipid antioxidant, as well as indirectly, by restoring the lipid antioxidant properties of vitamin E (Halliwell 1988, Hamre et al 1997, McCay 1985, Wright et al 1981). By acting as an antioxidant, ascorbate is converted to its oxidized form (dehydroascorbate), which readily can be converted by glutathione to its reduced, antioxidant form (Guaiquil et al 1997, May et al 1996, Vethanayagam et al 1999).
Ascorbate is produced by fungi (Petrescu et al 1992, Spickett et al 2000), but its role has been unclear, although it has been suggested that it is an antioxidant in the yeast Saccharomyces cerevisiae (Spickett et al 2000). Furthermore, exogenous ascorbate has been shown to inhibit differentiation (sporulation) in fungi (Hansberg and Aguirre 1990). It also has been tested as a fungicidal agent to control gray mold (Botrytis cinerea) and white mold (Sclerotinia sclerotiorum) in crops (Elad 1992).
We investigated how production of ascorbic acid in a sclerotial S. rolfsii strain, compared to a nonsclerotial strain, is related to development and lipid peroxidation under high- and low-oxidative stress conditions and how exogenous ascorbic acid affects lipid peroxidation and degree of differentiation (number of produced sclerotia) in the sclerotial strain.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). Both sclerotial strains showed no antagonism when paired with each other and with the nonsclerotial strain (group rating = 0).
Strains were grown in 9-cm Petri dishes on cellophane membrane disks (9-cm diam) placed on top of 25-mL agar medium, consisting of 15 mM NH4NO3, 2 mM NaCl, 0.5 mM MgSO4, ±10 µM FeCl3, 15 µM MnCl2, 70 µM ZnCl2, 1 µM thiamin, 0.1 M glucose, 0.1% (w/v) yeast extract and 1.5% (w/v) agar, in 10 mM potassium phosphate buffer, pH 7.0 (Georgiou et al 2001a). Basal growth medium and glucose (as 10x stock solution) were sterilized separately to avoid formation of oxidizing Maillard reaction products (Bridson and Brecker 1970). Cellophane membranes (type 300PT, British Cellophane Ltd., Bridgewater, Somerset, England) were boiled in 10 mM EDTA and washed four times in de-ionized-distilled water before sterilization. The medium constituents and the cellophane membranes were sterilized for 15 min at 120 C and 1 atm.
Fungal cultures were inoculated with agar disks (0.4-cm diam) cut from the margins of a 4-d-old colony and placed in the center of Petri dishes. Fungi were grown in single dish layers in an incubator at 23 C with 65% relative humidity and 12:12-h period of light:dark. Light intensity was 40 µE m-2 s-1, provided by Philips fluorescence lamps TLD 36W/965 (400–800 nm emission range). The sclerotial strain was grown under high- and low-oxidative stress conditions and the nonsclerotial strain under high-oxidative stress conditions. High-oxidative stress was established by growing the strains in light (12:12-h period of light:dark) with iron in the growth medium. Low-oxidative stress was established by the absence of light during growth with iron in the growth medium. The sclerotial strain grown under low-oxidative stress conditions was used as the control because this condition forms ca 200 sclerotia, compared to ca 1000 sclerotia under high-oxidative stress conditions. As an additional control, we used the nonsclerotial strain that does not produce sclerotia under high-oxidative stress conditions. Both controls were used to compare their ascorbate- and oxidative-stresses with those of the sclerotial strain.
Slerotial S. rolfsii initially covered the Petri dish with mycelia in 4 d. Thereafter, mycelia formed differentiated sclerotia that developed in three main stages: On Day 5, mycelia formed loosely bound, intertwined, white hyphal branches, called sclerotial initials (SI). Then, SI progressively developed to full-size, light brown, compact sclerotia, designated as early-, mid- and late-developed sclerotia (SD) at Days 6, 7 and 8, respectively. SD sclerotia matured on Day 9 to form dark sclerotia (SM, mature sclerotia) densely covered with melanin pigments. SD and SM sclerotia secreted droplets called exudate. The nonsclerotial S. rolfsii strain developed only hyphae throughout its growth.
Ascorbic acid assay –
Ascorbic acid (reduced and oxidized) in fungal samples was assayed by a colorimetric method we developed by modifying and combining previously reported methods (Galley et al 1996, Omaye et al 1979, Roe 1954). In principle, the method directly measures only the oxidized form of ascorbate (derivatized by 2,4-dinitrophenylhydrazine). Reduced ascorbate was determined indirectly by subtracting oxidized from total ascorbate (oxidized plus reduced; the latter was chemically oxidized during the assay) (see below). Ascorbic acid was quantified in whole colonies, mycelia (undifferentiated and differentiated), sclerotia and exudate. Mycelial tissue was sampled in 1-cm-wide strips cut from the center to the periphery of the colony. Undifferentiated mycelia were sampled from 2 to 4 d and 2 to 9 d from colonies of the sclerotial and the nonsclerotial strain, respectively. Differentiated mycelia (the mycelial portion of the colony that remained after removal of sclerotia) were sampled as well. Sclerotia (SI, SD and SM) were picked very gently from stems with stainless-steel forceps covered with Teflon ribbon. Exudate was collected from sclerotia with Pasteur pipette.
Fungal samples (0.4 g fresh wt of mycelia or sclerotia) were washed with 0.03% (w/v) metaphosphoric acid (MPA) and were ground in a porcelain mortar in liquid nitrogen to prevent artificial oxidation of endogenous ascorbate by possible traces of Fe(III) and Cu(II) during homogenization (Omaye et al 1979, Roe 1954). The resulting powder was mixed with 2.6 mL 0.03% MPA and was further homogenized on ice. At this stage, total sample dry wt was determined by drying 0.5-mL homogenate at 90 C for 3 h. The remaining homogenate was centrifuged at 25 000 g for 15 min, and the supernatant was adjusted to 4% (w/v) trichloroacetic acid (TCA) (from a 50% stock). Then it was incubated on ice for 10 min, and the precipitated proteins were removed by centrifugation at 15 000 g for 5 min. In assaying ascorbate in exudate, samples were adjusted to 4% (w/v) TCA from the same TCA stock. At this stage, the TCA-supernatant (or the TCA-exudate) could be stored at -70 C and assayed for oxidized and total ascorbate (reduced ascorbic acid is stable at acidic pH in cold for a week).
Reagent A (consisting of 20 mg thiourea, 2.5 mg CuSO45H2O and 0.15 g 2,4-dinitrophenylhydrazine, all dissolved in 9 N H2SO4 in final volume 5 mL) was used for assaying total ascorbate (oxidized plus reduced ascorbate). Here, the endogenous reduced ascorbate was oxidized by the copper ions of Reagent A (Omaye et al 1979, Roe 1954). Reagent B (same as Reagent A but without the copper reagent) was used for assaying endogenous oxidized ascorbate. Two 0.25-mL samples from the TCA-supernatant (or the TCA-exudate), one mixed with 0.05 mL Reagent A and the other with 0.05 mL Reagent B, were used. In the corresponding blanks, 0.25 mL 0.03% MPA in 4% TCA was used in the place of reagents A or B. The mixtures were incubated at 37 C for 3 h, 0.375-mL ice cold 75% (w/w) H2SO4 was added, and after incubation for 30 min at room temperature (for color development) their absorbance was measured at 520 nm with an LKB Ultrospec II 4050 UV-Visible spectrophotometer.
Total ascorbate was determined from a standard curve made with pure L-ascorbate (sodium salt) (0–60 µM, in 0.03% MPA and 4% TCA) treated with Reagent A (i.e., chemically oxidized by the Cu ions of Reagent A). Endogenous oxidized ascorbate was determined from another standard curve made with oxidized ascorbate treated with Reagent B. Oxidation was done in 3 mL 60 µM ascorbate stock solution (also dissolved in 0.03% MPA plus 4% TCA), mixed with 60 mg activated charcoal (HCl-washed) by 1 min vortexing 4 times. Charcoal was removed by passing ascorbate stock solution through a cellulose acetate syringe filter (25-mm diam, 0.2-µm pore, Nalgene Co, New York, USA). Ascorbate concentration was expressed in µmoles g-1 fungal dry wt or in µM (in sclerotium exudate).
Effect of ascorbate on sclerotial differentiation – Exogenous ascorbate was added to the growth medium in final concentrations 0–15 mg mL-1. Its effect on growth and differentiation of the sclerotial S. rolfsii strain was evaluated by measuring fungal generation time (G.T.), % sclerotial differentiation (%S.D.) and sclerotial differentiation delay (S.D.D.) (Georgiou et al 2000a). Sclerotial differentiation is defined as the portion of the mycelial colony that has been transformed to sclerotia. Transformation of SI to SD and SM sclerotia is another level of differentiation and is out of the scope of this study. Sclerotial differentiation is quantified by counting the number of sclerotia in a mycelial colony when they reach their final developmental stage (SM). At this stage, sclerotia are of uniform size (ca 1 mm diam). Briefly, sclerotial differentiation %S.D. is the percent number of SM sclerotia formed in a 9-d-old colony at various concentrations of ascorbate (100% S.D. is the number of SM sclerotia formed in the absence of the antioxidant). Sclerotial differentiation delay S.D.D. is the time of SI appearance minus the SI appearance of the control colony.
Lipid peroxidation assay – We tested whether the sclerotial and the nonsclerotial S. rolfsii strains differed in oxidative-stress levels during development. We used lipid peroxidation as an indicator of oxidative stress. Lipid peroxidation was measured at high-oxidative stress in a colony of the sclerotial S. rolfsii strain before it starts differentiating (4-d-old) and in a differentiated SD colony (7-d-old) of the same strain. As a control, we measured lipid peroxidation in same-day colonies of the sclerotial strain grown under low-oxidative stress conditions and of the nonsclerotial strain grown under high-oxidative stress conditions. We also studied the concentration-dependent lipid antioxidant effect of ascorbate on the sclerotial strain by measuring lipid peroxidation in the 4-d- and 7-d-old colonies, relative to the inhibition of differentiation caused by 2.5- and 5-mg mL-1 ascorbate added to the growth medium.
Fungal samples were frozen in liquid nitrogen and ground in a porcelain mortar. The resulting powder was suspended in 100 µM ethylenediaminetetraacetic acid (EDTA, disodium salt) solution (1:6 fresh w/v) and homogenized on ice. The homogenate was centrifuged at 25 000 g for 30 min and the EDTA-supernatant was assayed for lipid peroxidation by a modification of the TBA method (Castilho et al 1995). Specifically, 0.5-mL supernatant was mixed with 0.5-mL TBA reagent [0.67% w/v 2-thiobarbituric acid (TBA) dissolved in 5% w/v TCA] and with 5 µL 2% (w/v) butylated hydroxyanisol (BHA, dissolved in absolute ethanol). BHA was used as lipid antioxidant to prevent artificial lipid peroxidation during the assay. The mixture was incubated at 100 C for 20 min and was centrifuged at 15 000 g for 3 min. Absorbance of the supernatant was measured at 535 and 600 nm against a sample blank (using 0.5 mL 5% TCA in place of TBA reagent). The absorbance difference A (535–600 nm) was converted to nmoles of malondialdehyde (MDA) equivalents, using extinction coefficient for MDA 1.55 x 105 M-1 cm-1 (Nickander et al 1996). Lipid peroxidation was expressed in nmoles MDA mg-1 protein.
Protein concentration was assayed by a modification of a Coomassie Brilliant Blue (CBB)-based method (Sedmak and Grossberg 1977). Briefly, 0.1-mL supernatant was mixed with 0.9 mL 0.033% (w/v) CCB-G250 (dissolved in 0.5 N HCl), and after 5 min of incubation at room temperature its absorbance was measured at 620 nm and converted to mg protein from a bovine serum albumin (fraction V) standard curve (0–50 µg).
Statistical analysis of data –
Ascorbic acid and lipid peroxidation data (Figs. 1–3 and 5) were obtained from three independent experiments. In each experiment, three replicates were used for each sampling point. The replicate values in each experiment and among the three experiments were tested by Analysis of Variance (F-test). In both tests, the analysis did not show statistically significant differences (P 
0.05) and all values per sampling point were combined and used to determine the mean value and the standard error (SE). Mean values and SE for G.T., %S.D. and S.D.D. were measured in three independent experiments.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). Iron as well is an oxidative-stress generator because it can react with hydrogen peroxide to form hydroxyl radicals (Halliwell and Gutteridge 1999). Light and iron have been shown to induce sclerotial differentiation and hydrogen peroxide production in S. rolfsii (Sideri and Georgiou 2000). Figure 1 shows the concentration profiles of reduced and oxidized ascorbate in colonies of the sclerotial strain under high- and low-oxidative stress conditions and of the nonsclerotial strain under high-oxidative stress conditions. During the undifferentiated stage of the sclerotial strain, there was a progressive reduction in the concentration of reduced and oxidized ascorbate (up to two fold) irrespective of degree of oxidative stress. Furthermore, the undifferentiated stage started with reduced predominating to oxidized ascorbate 1.7 fold, with their ratio (reduced/oxidized) progressively decreasing to form a plateau around 1 at the end of that stage (4 d) (Fig. 1A). This reduction occurred one day earlier in the fungus that was grown under high- as compared to low-oxidative stress conditions. Furthermore, no reduced/oxidized ascorbate ratio plateau was formed under low-oxidative stress conditions (Fig. 1B). On the other hand, reduced and oxidized ascorbate concentrations in the nonsclerotial strain started from the same levels as those in the sclerotial strain (with the oxidized predominating to the reduced form 1.4 fold) and remained constant during corresponding days (Fig. 1A).
Sclerotial differentiation (generation of sclerotia) in the sclerotial strain was accompanied by a reversal of the reduced and oxidized ascorbate profiles. Under high-oxidative stress conditions, oxidized ascorbate increased six fold in entire mycelial colonies (mycelia and sclerotia) by the end of differentiation, while the reduced ascorbate remained at constant low levels (Fig. 1A). This resulted in a five-fold decline in their reduced/oxidized ratio. Under low-oxidative stress conditions (Fig. 1B), the ratio of reduced over oxidized ascorbate was 2.5-fold less than under high-oxidative stress conditions. Furthermore, oxidized ascorbate concentration at the end of differentiation was three-fold less under low- rather than high-oxidative stress conditions. This much lower conversion of reduced to oxidized ascorbate during differentiation under low-oxidative stress conditions, might be a reflection of the oxidative-stress status of the sclerotial strain, and might be related to the fact that under low-oxidative stress conditions the fungus produces five-fold fewer sclerotia than under high-oxidative stress conditions. In contrast, in the nonsclerotial strain, the reduced/oxidized ascorbate ratio remained constant throughout growth, although the amount of reduced and oxidized ascorbate increased ca two fold at the end of growth (Fig. 1B).
We examined how the reduced and oxidized ascorbate fractions in the differentiated whole mycelia (Fig. 1A) were distributed among their constituents, sclerotia and differentiated mycelia (Fig. 2), under high-oxidative stress conditions (i.e., with the maximum variation between oxidized and reduced ascorbate). Young sclerotia (SI), like young colonies, produced high concentrations of reduced and oxidized ascorbate (2.6-fold higher than in 2-d-old colonies) (Figs. 1A, 2A). This might be because SI sclerotia are composed of young, newly synthesized, intertwined hyphae. In addition, reduced and oxidized ascorbate in SI sclerotia (Fig. 2A) was 5.5- and 6.5-fold higher, respectively, than in the corresponding differentiated mycelia (Fig. 2B). As SI sclerotia further developed to SD and SM sclerotia, the concentration of reduced and oxidized ascorbate fell four and 2.5 fold, respectively. The reverse phenomenon was observed for oxidized ascorbate in the differentiated mycelia, where its concentration gradually increased six fold at the SM stage, while reduced ascorbate concentration remained low and constant during the SD and SM stage (Fig. 2B). This suggests that the observed increase of oxidized ascorbate concentration in whole mycelia during differentiation (Fig. 1A) was mainly due to the increase of the oxidized ascorbate in the differentiated mycelia.
Figure 3 shows the concentration of ascorbate in the exudate of sclerotia in various developmental stages. Reduced ascorbate was traced only in the exudate of early and mid-SD sclerotia. As sclerotia further developed to SM stage, their exudate accumulated concentrations of oxidized ascorbate (reaching almost 0.4 mM). Therefore, the actual production of oxidized ascorbate by S. rolfsii during differentiation might be much higher, because a very high fraction of it ends up in the exudate of sclerotia, possibly secreted or resulting from dead sclerotial cells. Such cells and cell-wall remnants have been observed in the outer layer of SM sclerotia (Chet et al 1969).
We suggest that ascorbate is produced by S. rolfsii in certain ratios and concentration gradients in response to formation of ROS during growth. It seems that high ROS might cause more accumulation of oxidized than reduced ascorbate during differentiation, despite attempts by the fungus to maintain a minimum supply of reduced ascorbate, possibly for antioxidant protection. This hypothesis is supported by the fact that, during differentiation, S. rolfsii shows a high increase of lipid peroxidation (Georgiou 1997). Furthermore, oxidized ascorbate accumulation in undifferentiated and differentiated mycelia (Figs. 1A, 2B) under high-oxidative stress conditions, correlate proportionally with their accumulated lipid peroxidation levels (Georgiou 1997). We have advanced a hypothesis proposing that some fungi survive in nature by differentiating (forming sclerotia) as a result of their inability to reduce oxidative stress (Georgiou 1997). According to our theory, if ascorbate acts as an antioxidant and reduces oxidative stress, it also is expected to reduce sclerotial differentiation (the number of formed sclerotia).
We have applied five criteria for establishing the ascorbate antioxidant role in relation to the oxidative-stress role on sclerotial differentiation in S. rolfsii: Exogenous ascorbate effect (i) on differentiation and (ii) on oxidative stress (lipid peroxidation) is expected to be concentration dependent; (iii) ascorbate must not affect growth and (iv) must not be used as the sole carbon source; (v) oxidative stress in the sclerotial S. rolfsii strain before it starts differentiating is expected to be higher than in the same strain grown under low-oxidative stress conditions (and possibly higher than in the nonsclerotial strain). These criteria were based on the facts that (a) ascorbate can be readily transported in eukaryotic cells (especially the oxidized form) (Guaiquil et al 1997, May et al 1995, Vera et al 1993, Welch et al 1995) and (b) its antioxidant action is concentration dependent (Wayner et al 1986).
To test these criteria, we added various concentrations of ascorbate (up to 15 mg mL-1) in the growth medium and we studied its effect on sclerotial differentiation under high-oxidative stress conditions (Fig. 4). Ascorbate at the tested concentrations did not support fungal growth as the sole carbon source, nor did it affect growth rate (generation time) with glucose as carbon source (Fig. 4A). Similar data have been obtained in another study (Maxwell and Bateman 1968). The effect of ascorbate on sclerotial differentiation of S. rolfsii was concentration dependent, showing a maximum 85% decline in the number of sclerotia per colony at 15 mg mL-1 in the growth medium (Fig. 4B). Ascorbate also delayed differentiation (i.e., the time appearance of SI sclerotia) in a concentration-dependent manner. Ascorbate at 10 mg mL-1 delayed differentiation 4 d (Fig. 4C). Ascorbate previously had been tested on S. rolfsii at much lower concentration (0.5 µg mL-1) and caused a 10% increase in sclerotial differentiation (Zoberi 1980). This might be explained by the fact that ascorbate can act as pro-oxidant (i.e., by redox cycling Fe) (Halliwell and Gutteridge 1999) at low concentrations and thus it might cause sclerotial differentiation, as our theory predicts.
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We propose that ascorbate is produced by S. rolfsii in concentration gradients and reduced/oxidized ratios in response to oxidative stress caused by ROS during growth. The sclerotial strain seems to handle oxidative stress less efficiently than the nonsclerotial strain, as is shown by their differences in the reduced/oxidized ascorbate ratios and the lipid peroxidation levels. Thus, it might be suggested that the sclerotial strain responds to its inability to reduce oxidative stress during early growth and differentiates by forming sclerotia. Exogenous ascorbic acid reduces oxidative stress in the sclerotial strain via its antioxidant action to levels near those of the nonsclerotial strain and inhibits differentiation, as our theory on sclerotial differentiation predicts (Georgiou 1997).
Our hypothesis on the differentiation-inhibiting antioxidant action of ascorbate is supported also by the similar effect this antioxidant had on other sclerotia-forming phytopathogens, such as Sclerotinia sclerotiorum, Rhizoctonia solani and Sclerotinia minor (Georgiou and Petropoulou, 2001a, b, 2002). Furthermore, similar differentiation-inhibiting effects by various antioxidants with hydroxyl-radical scavenging action have been documented in these fungi (Georgiou et al 2000a, b). In addition, ß-carotene, another natural antioxidant, has been identified and quantitated in S. rolfsii and S. sclerotiorum and has been shown to inhibit their differentiation (Georgiou et al 2001a, b). Our theory seems to hold true among sclerotiogenic fungi because the above mentioned fungi represent the four main types of sclerotial differentiation in phytopathogenic fungi—lateral-chained type (Sclerotium rolfsii), lateral-simple (Sclerotinia minor), loose (Rhizoctonia solani and terminal (Sclerotinia sclerotiorum) (Coley-Smith and Cooke 1971, Le Tourneau 1979, Willetts 1971, 1978).
Further support of our theory comes from studies by Hansberg and others showing the inducing action of hyperoxidant states caused by ROS on fungal differentiation, particularly on the conidiation process of Neurospora crassa (Hansberg and Aguirre 1990, Hansberg et al 1993, Lledias et al 1999, Toledo and Hansberg 1990, Toledo et al 1991). The appearance of ascorbate-concentration gradients during the undifferentiated and the differentiated stages of S. rolfsii also is in accordance with a complementary hypothesis proposing that differentiation at its initial stages depends on the establishment of redox and ROS generation gradients (Allen 1991, Allen and Balin 1989).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication June 9, 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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, •), oxidized (
, 
) and ratio of reduced/oxidized (
, 
) ascorbic acid. Standard errors are denoted as bars
