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Glutathione synthesis in response to captan: A possible mechanism for resistance of Botrytis cinerea to the fungicide
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
21, 412-416 (1984)
Glutathione Synthesis in Response to Captan: A Possible Mechanism for Resistance of Botrytis cinerea to the Fungicide’ ERANBARAK~ANDLLOYD Department of Environmental
V. EDGINGTON
Biology, University of Guelph, Guelph, Ontario NIG 2W1, Canada
Received August 8, 1983; accepted November 15. 1983 Two Botrytis cinerea isolates, field captan-resistant and normal type, were grown in liquid medium for 4 days prior to captan application. The glutathione (GSH) and oxidized glutathione (GSSG) of their mycelium and medium were analyzed at various times during the fifth day of their growth. Only slight differences in GSH levels between the isolates were observed, but marked differences in mycelial GSH were found in response to captan. Following the application of captan, less GSH was produced and after a longer lag period by the normal type of the fungus compared to the resistant isolate. This increase in the GSH level in the resistant isolate could make more nonvital thiol compound available for detoxifying captan and therefore prevent damage the fungicide could cause to the vital protein thiols of fungal cells. INTRODUCTION
Recently, the appearance of Botrytis cinerea Per. ex Fr. isolates resistant to captan under field conditions have been reported (1, 2). The ED,, values ranged from 2 to 13 times higher for resistant than for sensitive isolates. Whereas resistance to specific-site fungicides, such as benomyl or dicarboximide fungicides, is commonly observed under field conditions (3-5), resistance to captan was unexpected since its fungitoxicity arises from the reaction of its toxophores with numerous protein and nonprotein thiols vital to the normal function of fungal organisms (6, 7). Moreover, crossresistance to chlorothalonil and some other trichloromethylthio and dithiocarbamate fungicides was identified among captan-resistant isolates (8). It was suggested that this cross-resistance occurred due to the same mechanism of detoxification of these fungicides by low-molecular-weight thiols, such as GSH3 (8). GSH and GSSG play an important role in the regulation of many ’ Funded by a grant from the NSERC, Canada. ’ Fellow of Lady Davis Trust, Jerusalem, Israel. 3 Abbreviations used: GSH, reduced glutathione; GSSG, oxidized glutathione; OPT, o-phthalaldehyde; NEM, N-ethylmaleimide. 412 0048-3575184 $3.00 Copyright 0 1984 by Academic Press. Inc. All rights of reproduction in any form reserved.
physiological processes in the cell (9, lo), as well as representing a large pool of nonvital thiols which can readily react with the captan and its breakdown product-thiophosgene (11, 12, 13). The possibility that resistance to captan and some other alkylating agent fungicides in B. cinerea might be attributed to high levels of the nonprotein thiol GSH was studied in the present research. MATERIALS
AND METHODS
Materials. The isolates of B. cinerea used in this study, WT and 267, were found to be susceptible and resistant to captan under field conditions, with ED,, for spore germination of 0.12 and 1.12 PM, respectively. The maintenance, sources, and properties of these two isolates were described elsewhere (1). Captan (1,2,3,6-tetrahydro-N-(trichloromethylthio)phthalimide) was supplied by Chevron Chemical, Richmond, California, as a technical grade. The GSH, GSSG, EDTA, OPT and NEM were all obtained from Sigma Chemical Company, St. Louis, Missouri. Medium and tissue preparation. Twenty five mlliliters of sucrose-nitrate liquid me-
GLUTATHIONE
SYNTHESIS
dium, pH 5.7 (14), spore suspension (2 * lo3 spore ml-‘) was incubated in 9-cm-diameter Petri dishes at 20°C in the dark. After different periods of incubation, mycelia were filtered through preweighed Whatman 934 AH glass microfiber filters. Aliquots of 0.2 ml of the filtered medium were taken for GSH and GSSG assays. The mycelium, including the filter, was homogenized on ice with 6.0 ml of 0.1 M sodium phosphate, 5 mM EDTA buffer, pH 8.0, and 1.0 ml of 25% H,PO, solution. The total homogenate was centrifuged at 4°C at 45,OOOg for 30 min to obtain the supernatant for the assays. The dry weights of each of the extracted mycelial pellets were determined after heating at 100°C to constant weight and substracting the glass-fiber filter weights. Fungicide treatments. B. cinerea isolates WT and 267 were grown in liquid medium initially inoculated by their spores. After 4 days of incubation, captan dissolved in ethanol was incorporated to the medium to obtain final concentrations of 0. 3.3, or 19.8 FM (0.3% ethanol). Cultures were incubated at 25°C in still culture and measurements were made of growth GSH and GSSG over a time sequence. GSH assay. GSH content of mycelium and medium of the fungi was determined by a modification of the method used by Hissin and Hilf (15). For the mycelium assay, 3.0 ml of the phosphate-EDTA buffer was added to 2.0 ml of the supernatant. The final assay mixture (2.0 ml) contained 0.5 ml of diluted mycelium supernatant, 1.4 ml of phosphate-EDTA buffer, and 100 kg of OPT in 0.1 ml reagent-grade absolute methanol, which was prepared just prior to use. For the medium assay, 1.7 ml of the buffer and 0.1 ml of OPT solution were added to 0.2 ml of filtered medium of the fungus. After the final assay mixtures were prepared, they were thoroughly mixed. incubated for 15 min at room temperature, and transferred into quartz cuvettes. The fluorescence intensity at 440 nm was determined, with activation at 350 nm, by a Perkin-Elmer LS-5 fluorescence spectrophotometer.
IN
RESPONSE
TO CAPTAN
111
GSSG assay. For the mycelium assay. a 2.0-ml portion of the original 45,000g supernatant was incubated 30 min at room temperature with 0.1 ml of 40 mM NEM. The NEM complexes with GSH. preventing interference with the measurement of GSSG. To this mixture 2.9 ml of 0.1 N NaOH was added. A 0.5-ml portion of this mixture was taken for measurement of GSSG, using the procedure outlined above for GSH. except that 0.1 N NaOH was employed as diluent rather than phosphateEDTA buffer. For the medium assay, the final assay mixture (2.0 ml) contained 0.2 ml of the filtered medium and 50 t.~l of NEM solution, incubated at room temperature for 30 min. Following incubation, 1.65 ml ot NaOH and 0.1 ml of OPT solution were added. The solutions were transferred to quartz cuvettes and the fluorescence was determined as outlined above for GSH. Standards and recovery. GSH and GSSG standards were prepared simultaneously in phosphate-EDTA buffer or NaOH solution, respectively. The results for the analyses were corrected by the appropriate recovery value (always 88%), which was obtained by a similar procedure used by Hissim and Hilf ( 15). A standard curve wa> prepared plotting ug GSH or GSSG against fluorescence of the OPT complex. GSH and GSSG contents of the mycehum and the medium of the two isolates. in the presence of different concentrations of’ captan, were analyzed simultaneously, with four replicates of each. RESULTS
Mycelium dry weight (Fig. I) as well as GSH and GSSG contents of the mycelium (Fig. 3a) and the medium (Fig. 3b) were measured 0. 1, 6, 12, and 24 hr after the captan was applied. Captan was applied to the fungi at the log phase of their growth. when both the isolates had about the same dry weight (20-23 mg Petri dish-‘) (ca. Fig. 1). The growth rate of B. cinerea isolates during the experimental fifth day decreased after 12 hr. especially with the susceptible WT isolate. The mycelium growth of the
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glutathione levels either in the mycelium or the medium of the two isolates. Much more synthesis of glutathione by the resistant type was found in the first 12 hr after the captan was applied (Figs. 2b, c). On the i4 6 i2 6 i2 2.4 HR WT (S) 267 (R) other hand, there was no significant net FIG. 1. The growth of captan-susceptible (S, open production of glutathione by the WT during symbols) and -resistant (R, closed symbols) isolates of B. cinerea during the fifth day of incubation in su- this period. After 12 hr, the interpretation crose-nitrate liquid medium (20°C in the dark), in the of glutathione production becomes difficult presence of 0,3.3, or 19.8 PM captan (0, A, and 0, because the growth rate, especially of the respectively). Vertical bars denote the SE offour repWT isolate, was not in a log phase. Inlicates. creases in the glutathione content of the sensitive isolate were detected only 24 hr susceptible isolate, WT, after 24 hr of in- after the application of the higher dose of cubation in the presence of 3.3 and 19.8 ~.LM captan (Fig. 2~). captan, was inhibited more (16 and 23%, More details on the nature of the changes respectively), than the growth of the resis- in the glutathione level in the mycelium and tant isolate 267 (6 and 14%, respectively) the medium of the two isolates during the (Fig. 1). The total glutathione level detected fifth day of growth in response to captan in the absence of captan during the fifth day could be obtained from Fig. 3. In the abof the fungi growth was almost constant, sence of captan almost similar concentrawith little loss as a function of time (Fig. tions of GSSG were found in the mycelium 2a). Slightly higher levels of glutathione and in the medium of each of the isolates, were found for the resistant isolate (average but 11.5 and 3.5 times higher levels of GSH 12%) compared to the susceptible one were detected in the mycelium compared during this period of time. However, the to the medium of the 267 and WT isolates, presence of captan caused changes in the respectively (Fig. 3). In the presence of 3.3 FLM captan, rapid synthesis of GSH in the mycelium of the resistant fungus could be observed within 1 hr, accompanied by increases in the GSSG content (Fig. 3a). However, the response of the sensitive isolate to the lower concentration of captan was less intensive and slower. The GSH level of the mycelium of the sensitive isolate even decreased after the captan was applied, and it took about 6 hr for recovery (Fig. 3a), which suggests that adding the captan caused serious damage in the cells. Meanwhile, in the medium, adding the low levels of captan caused a similar decrease 2AOT 24 HR in GSH and a subsequent increase in GSSG FIG. 2. The total content of oxidized and reduced glutathione, expressed as kmol GSH g dry wt-‘, in with the susceptible isolate (Fig. 3b). the mycelium and the medium of captan-susceptible When a higher concentration of captan (open symbols) and -resistant (closed symbols) iso- (19.8 PM) was applied, the GSH levels in lates of B. cinerea (WT and 267, respectively) during the mycelium of both the isolates drastithe fifth day of incubation of sucrose-nitrate liquid medium, in the presence of (a) 0, (b) 3.3 or (c) 19.8 cally decreased, accompanied by rapid pro)LM captan. Vertical bars denote the SE offour rep- duction of GSSG (Fig. 3a). Simultaneously, licates. in the medium of both the isolates, very
GLUTATHIONE
SYNTHESIS
IN RESPONSE
TO CAPTAN
J1.i;
FIG. 3. GSH and GSSG contents of (a) the myceliam and (b) the medirrm of (.aptuI?-sIf.s(‘eptibic, (S, open symbols) and -resistant (R, closed symbols) isolates of B. cinerea during the fifth dais of incubation in sucrose-nitrate liquid medium (20°C in the dark), 24 hr after addition of 0. 3.3 or 19.8 (LM captan CC. A, and 0, respectively). Vertical bars denote the SE of.four replicates.
high concentrations of GSH and GSSG were detected (Fig. 3b). These levels of GSH and GSSG in the medium of the resistant isolate increased much faster, in response to the high concentration of captan, than in the medium of the susceptible isolate. DISCUSSION
Higher levels of glutathione were found in a field captan-resistant isolate of B. cinerea, during the fifth day of growth in liquid medium, compared to the normal susceptible isolate (12% difference). Likewise, GSH synthesis by the captan-resistant fungus, in response to the fungicide captan, was much faster and more intensive than by the susceptible isolate. The changes in glutathione levels in the medium during the fifth day of growth reflected those that occurred in the mycelium of the isolate in response to the lower concentration of captan. However, large amounts of glutathione were found in the medium in response to the higher concentration of captan. This was probably because membranes were damaged and both GSH and GSSG leaked into the medium. Captan and other trichloromethylthio fungicides are known as alkylating agents which react mostly with thiolic compounds
of the fungus (16). The nonvital pool of soluble thiolic compounds could react with these fungicides, detoxifying them and preventing the damage to cellular vital protein thiols (7. 17). GSH was found to be the main low-molecular-weight thiol involved in such a detoxification of captan in fungal cells (6, 1 I, 12). For this reason, in the present study, specific fluorimetric analysis of GSH and GSSG (15) was used. instead of another analysis for general thiol compounds. Captan reacts with thiols very fast (12). In addition, 80% of it is normally hvdrolyzed during 24 hr in aqueous solution under conditions similar to those used in this study (18). As a consequence of this, fast regeneration of GSH in response to captan could eliminate much of the damage of this fungicide to the fungal cells. In plants, it was suggested that some herbicides, which produce nontoxic conjugates with GSH, may elevate the level of GSH used in their own detoxification (19. 20). In fungi, it was suggested that a pool of thiol compounds within the cells was responsible for their resistance to mercury (21), although no difference in thiol concentration could be established between the resistant and the sensitive isolates. The present study showed little difference between glutathione levels in captan-
416
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AND EDGINGTON
resistant and -sensitive isolates. However, following the captan application, less GSH was produced and after a longer lag period by the normal type of B. cinerea compared to the field captan-resistant isolate. In addition to the practical solutions suggested to reduce the appearance of captanresistant B. cinerea isolates (l), the use of GSH synthesis inhibitor, together with captan, could be considered for improving the efficacy of captan as a fungicide. The loss of resistance of B. cinerea isolates to captan, as a result of their aging, as was previously shown (l), might be explained by the loss of activity of the enzymatic system responsible for the GSH synthesis due to the aging of the fungal inoculum. ACKNOWLEDGMENTS We thank Mrs. Karen D. Robinson for expert technical assistance. We appreciate useful discussions on various aspects of this research with Dr. G. Ezra and Dr. G. R. Stephenson. REFERENCES 1. E. Barak and L. V. Edgington 1984. Botryris cinerea resistant to captan; the effect of inoculum age and type on response to captan. Cnn. J. Plant Pathol. 6 (in press). 2. H. S. Pepin and E. A. MacPherson, Strains of Bofrytis cinerea resistant to benomyl and captan in the field, Plant Dis. 66, 404 (1982). 3. J. Dekker, Resistance, in “Systemic Fungicides” (R. W. Marsh, Ed.), p. 176, Longman, London, 1977. 4. T. Katan, Resistance to 3,5-dichlorophenyl - N cyclicimide (“dicarboximid”) fungicides in the grey mould pathogen Botrytis cinerea on protected crops, Plant Puthol. 31, 133 (1982). 5. M. Panayotakou and N. E. Malathrakis, Resistance of Botrytis cinereu to dicarboximide fungicides in protected crops, Ann. Appl. Biol. 102, 293 (1983). 6. M. R. Siegel, Reactions of certain trichloromethyl sulfenyl fungicides with low molecular weight thiols. In vivo studies with cells of Succhuromyces pusforiunus, .I. Agric. Food Chem. 18, 823 (1970). 7. M. R. Siegel, Reaction of the fungicide folpet (N(trichloromethylthio)phthalimide) with a thiol protein, Pestic. Biochem. Physiol. 1, 225 (1971).
8. E. Barak and L. V. Edgington 1983. The role of glutathione in the resistance of Botrytis cinerea to captan (Abstr) Can. J. Plant Parhol. 5, 200. 9. N. S. Kosower and E. M. Kosower, The glutathione status of cells, In?. Rev. Cytol. 54, 109 (1978). 10. H. Rennenberg, Glutathione metabolism and possible biological role in higher plants, Phytochemistry 21, 2771 (1982). 11. D. V. Richmond and E. Somers, Studies on the fungitoxicity of captan. VI. Decomposition of 3SS-labeled captan by Neurospora crassu conidia, Ann. Appl. Biol. 62, 35 (1968). 12. M. R. Siegel, Reactions of certain trichloromethyl sulfenyl fungicides with low molecular-weight thiols. In vitro studies wth glutathione, J. Agric. Food Chem. 18, 819 (1970). 13. A. Kaars Sijpesteijn, H. M. Dekhuijzen, and J. W. Vonk, Biological conversion of fungicides in plants and microorganisms, in “Antifungal Compounds” (M. R. Siegel and H. D. Sisler, Eds.) Vol. 2, p. 91, Dekker, New York, 1977. 14. K. E. Parry and P. K. S. Wood, The adaptation of fungi to fungicides: Adaptation to captan, Ann. Appl. Biol. 47, 1 (1959). 15. P. J. Hissim and R. Hilf, A fluorimetric method for determination of oxidized and reduced glutathione in tissues, Anal. Biochem. 74, 214 (1976). 16. R. J. Lukens, Heterocyclic nitrogen compounds, in “Fungicides” (D. C. Torgeson, Ed.), Vol. 2, p. 395, Academic Press, New York, 1969. 17. M. R. Siegel and H. D. Sisler, Reactions of folpet with purified enzymes, nucleic acids and subcellular components of Sacchuromyces pastoriunus, Phytopathology 58, 1129 (1968). 18. R. Frank, H. E. Braun, and J. Stanek, Removal of captan from treated apples, Arch. Environ. Contam. Toxicol. 12, 265 (1983). 19. G. R. Stephenson, A. Ali, and E M. Ashton, Influence of herbicides and antidotes on the glutathione levels of maize seedlings, in “Pesticide Chemistry: Human Welfare and the Environment” (J. Miyamoto and P C. Keamey, Eds), Vol. 3, p. 219, Pergamon, Oxford. 20. G. Ezra and J. Gressel, Rapid effects of a thiocarbamate herbicide and its dichloroacetamide protectant on macromolecular syntheses and glutathione levels in maize cell cultures, Pestic. Biochem. Physiol. 17, 48 (1982). 21. I. S. Ross and K. M. Old, Thiol compounds and resistance of Pyrenophora avenue to mercury, Trans. Brit. Mycol. Sot. 60, 301 (1973).
BIOCHEMISTRY
AND
PHYSIOLOGY
21, 412-416 (1984)
Glutathione Synthesis in Response to Captan: A Possible Mechanism for Resistance of Botrytis cinerea to the Fungicide’ ERANBARAK~ANDLLOYD Department of Environmental
V. EDGINGTON
Biology, University of Guelph, Guelph, Ontario NIG 2W1, Canada
Received August 8, 1983; accepted November 15. 1983 Two Botrytis cinerea isolates, field captan-resistant and normal type, were grown in liquid medium for 4 days prior to captan application. The glutathione (GSH) and oxidized glutathione (GSSG) of their mycelium and medium were analyzed at various times during the fifth day of their growth. Only slight differences in GSH levels between the isolates were observed, but marked differences in mycelial GSH were found in response to captan. Following the application of captan, less GSH was produced and after a longer lag period by the normal type of the fungus compared to the resistant isolate. This increase in the GSH level in the resistant isolate could make more nonvital thiol compound available for detoxifying captan and therefore prevent damage the fungicide could cause to the vital protein thiols of fungal cells. INTRODUCTION
Recently, the appearance of Botrytis cinerea Per. ex Fr. isolates resistant to captan under field conditions have been reported (1, 2). The ED,, values ranged from 2 to 13 times higher for resistant than for sensitive isolates. Whereas resistance to specific-site fungicides, such as benomyl or dicarboximide fungicides, is commonly observed under field conditions (3-5), resistance to captan was unexpected since its fungitoxicity arises from the reaction of its toxophores with numerous protein and nonprotein thiols vital to the normal function of fungal organisms (6, 7). Moreover, crossresistance to chlorothalonil and some other trichloromethylthio and dithiocarbamate fungicides was identified among captan-resistant isolates (8). It was suggested that this cross-resistance occurred due to the same mechanism of detoxification of these fungicides by low-molecular-weight thiols, such as GSH3 (8). GSH and GSSG play an important role in the regulation of many ’ Funded by a grant from the NSERC, Canada. ’ Fellow of Lady Davis Trust, Jerusalem, Israel. 3 Abbreviations used: GSH, reduced glutathione; GSSG, oxidized glutathione; OPT, o-phthalaldehyde; NEM, N-ethylmaleimide. 412 0048-3575184 $3.00 Copyright 0 1984 by Academic Press. Inc. All rights of reproduction in any form reserved.
physiological processes in the cell (9, lo), as well as representing a large pool of nonvital thiols which can readily react with the captan and its breakdown product-thiophosgene (11, 12, 13). The possibility that resistance to captan and some other alkylating agent fungicides in B. cinerea might be attributed to high levels of the nonprotein thiol GSH was studied in the present research. MATERIALS
AND METHODS
Materials. The isolates of B. cinerea used in this study, WT and 267, were found to be susceptible and resistant to captan under field conditions, with ED,, for spore germination of 0.12 and 1.12 PM, respectively. The maintenance, sources, and properties of these two isolates were described elsewhere (1). Captan (1,2,3,6-tetrahydro-N-(trichloromethylthio)phthalimide) was supplied by Chevron Chemical, Richmond, California, as a technical grade. The GSH, GSSG, EDTA, OPT and NEM were all obtained from Sigma Chemical Company, St. Louis, Missouri. Medium and tissue preparation. Twenty five mlliliters of sucrose-nitrate liquid me-
GLUTATHIONE
SYNTHESIS
dium, pH 5.7 (14), spore suspension (2 * lo3 spore ml-‘) was incubated in 9-cm-diameter Petri dishes at 20°C in the dark. After different periods of incubation, mycelia were filtered through preweighed Whatman 934 AH glass microfiber filters. Aliquots of 0.2 ml of the filtered medium were taken for GSH and GSSG assays. The mycelium, including the filter, was homogenized on ice with 6.0 ml of 0.1 M sodium phosphate, 5 mM EDTA buffer, pH 8.0, and 1.0 ml of 25% H,PO, solution. The total homogenate was centrifuged at 4°C at 45,OOOg for 30 min to obtain the supernatant for the assays. The dry weights of each of the extracted mycelial pellets were determined after heating at 100°C to constant weight and substracting the glass-fiber filter weights. Fungicide treatments. B. cinerea isolates WT and 267 were grown in liquid medium initially inoculated by their spores. After 4 days of incubation, captan dissolved in ethanol was incorporated to the medium to obtain final concentrations of 0. 3.3, or 19.8 FM (0.3% ethanol). Cultures were incubated at 25°C in still culture and measurements were made of growth GSH and GSSG over a time sequence. GSH assay. GSH content of mycelium and medium of the fungi was determined by a modification of the method used by Hissin and Hilf (15). For the mycelium assay, 3.0 ml of the phosphate-EDTA buffer was added to 2.0 ml of the supernatant. The final assay mixture (2.0 ml) contained 0.5 ml of diluted mycelium supernatant, 1.4 ml of phosphate-EDTA buffer, and 100 kg of OPT in 0.1 ml reagent-grade absolute methanol, which was prepared just prior to use. For the medium assay, 1.7 ml of the buffer and 0.1 ml of OPT solution were added to 0.2 ml of filtered medium of the fungus. After the final assay mixtures were prepared, they were thoroughly mixed. incubated for 15 min at room temperature, and transferred into quartz cuvettes. The fluorescence intensity at 440 nm was determined, with activation at 350 nm, by a Perkin-Elmer LS-5 fluorescence spectrophotometer.
IN
RESPONSE
TO CAPTAN
111
GSSG assay. For the mycelium assay. a 2.0-ml portion of the original 45,000g supernatant was incubated 30 min at room temperature with 0.1 ml of 40 mM NEM. The NEM complexes with GSH. preventing interference with the measurement of GSSG. To this mixture 2.9 ml of 0.1 N NaOH was added. A 0.5-ml portion of this mixture was taken for measurement of GSSG, using the procedure outlined above for GSH. except that 0.1 N NaOH was employed as diluent rather than phosphateEDTA buffer. For the medium assay, the final assay mixture (2.0 ml) contained 0.2 ml of the filtered medium and 50 t.~l of NEM solution, incubated at room temperature for 30 min. Following incubation, 1.65 ml ot NaOH and 0.1 ml of OPT solution were added. The solutions were transferred to quartz cuvettes and the fluorescence was determined as outlined above for GSH. Standards and recovery. GSH and GSSG standards were prepared simultaneously in phosphate-EDTA buffer or NaOH solution, respectively. The results for the analyses were corrected by the appropriate recovery value (always 88%), which was obtained by a similar procedure used by Hissim and Hilf ( 15). A standard curve wa> prepared plotting ug GSH or GSSG against fluorescence of the OPT complex. GSH and GSSG contents of the mycehum and the medium of the two isolates. in the presence of different concentrations of’ captan, were analyzed simultaneously, with four replicates of each. RESULTS
Mycelium dry weight (Fig. I) as well as GSH and GSSG contents of the mycelium (Fig. 3a) and the medium (Fig. 3b) were measured 0. 1, 6, 12, and 24 hr after the captan was applied. Captan was applied to the fungi at the log phase of their growth. when both the isolates had about the same dry weight (20-23 mg Petri dish-‘) (ca. Fig. 1). The growth rate of B. cinerea isolates during the experimental fifth day decreased after 12 hr. especially with the susceptible WT isolate. The mycelium growth of the
414
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AND
EDGINGTON
glutathione levels either in the mycelium or the medium of the two isolates. Much more synthesis of glutathione by the resistant type was found in the first 12 hr after the captan was applied (Figs. 2b, c). On the i4 6 i2 6 i2 2.4 HR WT (S) 267 (R) other hand, there was no significant net FIG. 1. The growth of captan-susceptible (S, open production of glutathione by the WT during symbols) and -resistant (R, closed symbols) isolates of B. cinerea during the fifth day of incubation in su- this period. After 12 hr, the interpretation crose-nitrate liquid medium (20°C in the dark), in the of glutathione production becomes difficult presence of 0,3.3, or 19.8 PM captan (0, A, and 0, because the growth rate, especially of the respectively). Vertical bars denote the SE offour repWT isolate, was not in a log phase. Inlicates. creases in the glutathione content of the sensitive isolate were detected only 24 hr susceptible isolate, WT, after 24 hr of in- after the application of the higher dose of cubation in the presence of 3.3 and 19.8 ~.LM captan (Fig. 2~). captan, was inhibited more (16 and 23%, More details on the nature of the changes respectively), than the growth of the resis- in the glutathione level in the mycelium and tant isolate 267 (6 and 14%, respectively) the medium of the two isolates during the (Fig. 1). The total glutathione level detected fifth day of growth in response to captan in the absence of captan during the fifth day could be obtained from Fig. 3. In the abof the fungi growth was almost constant, sence of captan almost similar concentrawith little loss as a function of time (Fig. tions of GSSG were found in the mycelium 2a). Slightly higher levels of glutathione and in the medium of each of the isolates, were found for the resistant isolate (average but 11.5 and 3.5 times higher levels of GSH 12%) compared to the susceptible one were detected in the mycelium compared during this period of time. However, the to the medium of the 267 and WT isolates, presence of captan caused changes in the respectively (Fig. 3). In the presence of 3.3 FLM captan, rapid synthesis of GSH in the mycelium of the resistant fungus could be observed within 1 hr, accompanied by increases in the GSSG content (Fig. 3a). However, the response of the sensitive isolate to the lower concentration of captan was less intensive and slower. The GSH level of the mycelium of the sensitive isolate even decreased after the captan was applied, and it took about 6 hr for recovery (Fig. 3a), which suggests that adding the captan caused serious damage in the cells. Meanwhile, in the medium, adding the low levels of captan caused a similar decrease 2AOT 24 HR in GSH and a subsequent increase in GSSG FIG. 2. The total content of oxidized and reduced glutathione, expressed as kmol GSH g dry wt-‘, in with the susceptible isolate (Fig. 3b). the mycelium and the medium of captan-susceptible When a higher concentration of captan (open symbols) and -resistant (closed symbols) iso- (19.8 PM) was applied, the GSH levels in lates of B. cinerea (WT and 267, respectively) during the mycelium of both the isolates drastithe fifth day of incubation of sucrose-nitrate liquid medium, in the presence of (a) 0, (b) 3.3 or (c) 19.8 cally decreased, accompanied by rapid pro)LM captan. Vertical bars denote the SE offour rep- duction of GSSG (Fig. 3a). Simultaneously, licates. in the medium of both the isolates, very
GLUTATHIONE
SYNTHESIS
IN RESPONSE
TO CAPTAN
J1.i;
FIG. 3. GSH and GSSG contents of (a) the myceliam and (b) the medirrm of (.aptuI?-sIf.s(‘eptibic, (S, open symbols) and -resistant (R, closed symbols) isolates of B. cinerea during the fifth dais of incubation in sucrose-nitrate liquid medium (20°C in the dark), 24 hr after addition of 0. 3.3 or 19.8 (LM captan CC. A, and 0, respectively). Vertical bars denote the SE of.four replicates.
high concentrations of GSH and GSSG were detected (Fig. 3b). These levels of GSH and GSSG in the medium of the resistant isolate increased much faster, in response to the high concentration of captan, than in the medium of the susceptible isolate. DISCUSSION
Higher levels of glutathione were found in a field captan-resistant isolate of B. cinerea, during the fifth day of growth in liquid medium, compared to the normal susceptible isolate (12% difference). Likewise, GSH synthesis by the captan-resistant fungus, in response to the fungicide captan, was much faster and more intensive than by the susceptible isolate. The changes in glutathione levels in the medium during the fifth day of growth reflected those that occurred in the mycelium of the isolate in response to the lower concentration of captan. However, large amounts of glutathione were found in the medium in response to the higher concentration of captan. This was probably because membranes were damaged and both GSH and GSSG leaked into the medium. Captan and other trichloromethylthio fungicides are known as alkylating agents which react mostly with thiolic compounds
of the fungus (16). The nonvital pool of soluble thiolic compounds could react with these fungicides, detoxifying them and preventing the damage to cellular vital protein thiols (7. 17). GSH was found to be the main low-molecular-weight thiol involved in such a detoxification of captan in fungal cells (6, 1 I, 12). For this reason, in the present study, specific fluorimetric analysis of GSH and GSSG (15) was used. instead of another analysis for general thiol compounds. Captan reacts with thiols very fast (12). In addition, 80% of it is normally hvdrolyzed during 24 hr in aqueous solution under conditions similar to those used in this study (18). As a consequence of this, fast regeneration of GSH in response to captan could eliminate much of the damage of this fungicide to the fungal cells. In plants, it was suggested that some herbicides, which produce nontoxic conjugates with GSH, may elevate the level of GSH used in their own detoxification (19. 20). In fungi, it was suggested that a pool of thiol compounds within the cells was responsible for their resistance to mercury (21), although no difference in thiol concentration could be established between the resistant and the sensitive isolates. The present study showed little difference between glutathione levels in captan-
416
BARAK
AND EDGINGTON
resistant and -sensitive isolates. However, following the captan application, less GSH was produced and after a longer lag period by the normal type of B. cinerea compared to the field captan-resistant isolate. In addition to the practical solutions suggested to reduce the appearance of captanresistant B. cinerea isolates (l), the use of GSH synthesis inhibitor, together with captan, could be considered for improving the efficacy of captan as a fungicide. The loss of resistance of B. cinerea isolates to captan, as a result of their aging, as was previously shown (l), might be explained by the loss of activity of the enzymatic system responsible for the GSH synthesis due to the aging of the fungal inoculum. ACKNOWLEDGMENTS We thank Mrs. Karen D. Robinson for expert technical assistance. We appreciate useful discussions on various aspects of this research with Dr. G. Ezra and Dr. G. R. Stephenson. REFERENCES 1. E. Barak and L. V. Edgington 1984. Botryris cinerea resistant to captan; the effect of inoculum age and type on response to captan. Cnn. J. Plant Pathol. 6 (in press). 2. H. S. Pepin and E. A. MacPherson, Strains of Bofrytis cinerea resistant to benomyl and captan in the field, Plant Dis. 66, 404 (1982). 3. J. Dekker, Resistance, in “Systemic Fungicides” (R. W. Marsh, Ed.), p. 176, Longman, London, 1977. 4. T. Katan, Resistance to 3,5-dichlorophenyl - N cyclicimide (“dicarboximid”) fungicides in the grey mould pathogen Botrytis cinerea on protected crops, Plant Puthol. 31, 133 (1982). 5. M. Panayotakou and N. E. Malathrakis, Resistance of Botrytis cinereu to dicarboximide fungicides in protected crops, Ann. Appl. Biol. 102, 293 (1983). 6. M. R. Siegel, Reactions of certain trichloromethyl sulfenyl fungicides with low molecular weight thiols. In vivo studies with cells of Succhuromyces pusforiunus, .I. Agric. Food Chem. 18, 823 (1970). 7. M. R. Siegel, Reaction of the fungicide folpet (N(trichloromethylthio)phthalimide) with a thiol protein, Pestic. Biochem. Physiol. 1, 225 (1971).
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