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Non-lethal immobilization of zoospores of Phytophthora infestans by Li+
Mycol. Res. 95 (8): 970-972 (1991)
Printed in Great Britain
- - - - - -----------.-------- - -
970 -----
Non-lethal immobilization of zoospores of Phytophthora infestans by Li+
T. ERSEK Planl Prolection Instilule, Hungarian Academy of Sciences, P.G. Box 102, H-1525 Budapesl, Hungary
u. HOLKER AND M. HOFER BOlanisches Inslitute der Universitiil, Kirschallee 1, D-5300 Bonn, Germany
Li+ (100 mM) effected irreversible but non-lethal immobilization of Phytophthora infeslans zoospores in their pyriform shape and with attached flagella. The immobilization was immediate but the capacity of cells to encyst was dependent on the duration of the Li+ treatment and on the addition of Ca 2 +. It is suggested that the loss of zoospore motility and their ability to encyst is caused by a remarkable decrease of intracellular K+ and Ca 2+, respectively. The maintenance of zoospores of Phytophthora spp. in a healthy motile condition has been assumed to be due, at least partly, to the provision of essential cations in adequate amounts from sporangia (Gooding & Lucas, 1959; Cameron & Carlile, 1980). On the other hand, zoospores are able to form cell walls (cysts) either asynchronously during an extended stage of motility, or immediately upon various stimuli. Vigorous agitation, pectin or high concentration of cations, such as Na+, Ca 2 + or 5r2 +, cause synchronous encystment (Tokunaga & Bartnicki-Garcia, 1971; Paktitis, Grant & Lawrie, 1986; Grant, Griffith & Irving, 1986). Initial events that lead to encystment involve the loss of motility followed by a release of flagella. The newly formed cysts readily germinate. U+ (3-10 mM) also induces encystment but not cyst germination (Grant et al., 1986). In this communication a rather unique and unexpected effect of 100 mM U+ on the development of Phytophthora zoospores is reported. Phytophthora infestans (Mont.) de Bary, race 2.3.4, was cultivated on potato tuber slices. Both the zoospore release from sporangia and their viability were conSiderably higher when grown on potatoes than on any other common media. Zoospore suspensions (at cell density of 10 6 ml- 1 ) were obtained by allowing thoroughly washed sporangia to discharge zoospores into glass double-distilled water at 10-12 °C for 2-3 h. Zoospores were separated from sporangia by filtration through Whatman filter paper No. 4 CErsek, 1975). U+ was added to a final concentration of 100 mM. U+ and the other cations were supplied as chlorides (analytical reagent grade purchased from Merck). U+ immediately terminated the zoospores motility. However, the flagella remained attached and, as proven by the lack of cell fluorescence after staining with calcofluor, cell walls were not formed. Zoospores retained their pyriform shape for 6-8 h when kept at 10° (Figs 1 and 2) and eventually lysed. When U+ was added at room temperature, zoospores tended
to deteriorate rapidly (cytoplasmic aggregation) even though the flagella remained firmly attached (Fig. 3). In neither case could the encystment of zoospores be induced by vigorous agitation (I min in Vortex mixer). The same concentration of other cations, namely Na+, K+, Mg 2 + or Ca 2 +, had no effect comparable to that of U+. On the other hand, the above effect of U+ in hypotonic suspensions was also observed in isoosmotic conditions, Le. 200 mM sorbitol. Removal of U+ by pelleting zoospores at 1000 g for 30 s and resuspending the pellet in distilled water, even when containing essential cations such as Na+, K+, Mg 2 +or Ca 2 +, did not yield motile zoospores. Regardless of the duration of u+ treatment, zoospores rapidly lysed. However, cells capable of forming cell walls and germ-tubes were obtained by replacement of U+ by Ca 2 + and by Ca 2 + plus K+ (I mM each), respectively. This process was time dependent. Within the first 10 min of the U+ treatment more than 90% of cells formed cysts (in Ca 2 +) and germinated (in Ca 2 + plus K+), (Table 1). During the next 10 min the capacity of cells to encyst and germinate decreased proportionally with time. An extended treatment of cells with U+ (above 20 min) caused the majority of cells to lyse. No other combination of cations (including Na+ and Mg 2+) initiated encystment or prevented zoospores from lysing (Table 1). Measurements of essential cations, Na+, K+, Mg 2 +and Ca 2 +, by atomic absorption spectrometry after a 5 min exposure of zoospores to U+, revealed a remarkable drop in intracellular K+ and Ca 2+ concentrations (Table 2). This observation rationalizes the cation replacement experiments and is consistent with our earlier results, according to which the initiation of encystment by agitation is associated with efflux of K+ and influx of H+ due to a suddenly altered plasma membrane permeability for the two cations (Ersek & Hofer, 1990). In contrast, maturing cysts accumulate K+. Hence, the drop in intracellular K+ concentration upon the U+ treatment
T. Ersek,
u. Holker
971
and M. Hofer
Figs 1-3. Light micrographs of zoospores of P. infesfans immediately upon treatment with 100 mM-Li+, Bars 10 IJm. Figs 1-2. Li+ was added to zoospore suspension at 10° Fig. 3. Li+ was added to zoospore suspension at room temperature. Note that flagella are
attached to deteriorated zoospores.
or agitation may be a signal for termination of motility and/or encystment. Encystment, however, can take place only at an adequate intracellular level of Ca 2+. Contrary to these findings, the role of K+ in the signalling process has recently been questioned (Iser et al., 1989). Unlike agitation, Li+ treatment induces Ca 2 + leakage. A lowered intracellular level of Ca 2 + may be the reason for the observed inability of zoospores to encyst even though they had lost their motility in Li+. Thus, Ca 2 + seems to playa key role in maintaining the zoospore state and in the transition to the cyst stage (Paktitis et aI., 1986; Griffith, Iser & Grant, 1988). Because Li+ treatment under both isoosmotic and the above low osmolarity conditions brought about the same response, the efflux of K+ and Ca 2 + is due to
some direct (chaotropic) effect(s) rather than to an interference with the osmoregulatory system of the cells. In conclusion, the non-lethal, irreversible immobilization of P. infestans zoospores in their pyriform shape with flagella Table 2. Concentrations of essential cations in supematants of zoospore suspensions (10' cells ml- 1) and in cells of P. infestans before and after a 5 min treatment with 100 mM LiConcentration Supernatant (IJM)a
Intracellular (mM)b Efflux
Cation
-Li+
+Li-
-Li+
+Li-
(%)
Na+ K-
51'0±9'5 79'0±3'8 I2'0±I'2 7'3±0'7
51'0±5'5 92'7±5'9 15'0±2'1 II'7±2'6
53 109 86 17
53 63 76 7
0 43 II 59
Table 1. Response of P. infestans zoospores to a replacement of Li+ (l00 mM) by individual cations or by their combinations (I mM each).
Mg"+ Ca'+
Before the cation replacement, the zoospores were incubated for 5 min with Li+
a Values from 3 separate experiments denote mean (± S.E,) extracellular cation concentrations in supernatants ot live zoospore suspensions. Cation concentrations were measured directly. using a type 360 Perkin-Elmer atomic absorption spectrometer equipped with a combined Na-K, Mg or Ca cathode lamp as a light source. Each salt used for calibration was dissolved in glass double-distilled water. Conditions for measurements were set up according to the manual of the manufacturer. " Intracellular concentrations were calculated from the concentration differences between supernatants of boiled and live zoospore suspensions on the basis of an average cell diameter of 10 IJm.
Cation
Na+
K+
Mg'+
Ca'+
Na+ K+ Mg'+ Ca"+
lyse
lyse lyse
lyse lyse lyse
encyst encyst and germinate encyst/lyse encyst
+
See reverse combination.
Immobilization of zoospores of Phytophthora infestans attached and the loss of their ability to encyst, induced by U+, are effected by a decrease in the intracellular K+ and Ca 2 + concentrations. Encystment and germination can proceed only when the two essential cations are supplemented. This work has been supported by a DFG grant No. H0555/12 to M. H. T. E. was a recipient of a DAAD exchange fellowship.
REFERENCES Cameron. ]. N. Ii< Carlile, M. ]. (1980). Negative chemotaxis of zoospores of the fungus Phytophthora palmivora. Journal of Gmeml Microbiology !lO, 347-353. Ersek, T. (1975). The sensitivity of Phytophthora infestans to several antibiotics. Zeitschnft fur Pflanzenkrankheiten und PflanUn5chutz 82, 614--617. Ersek, T. Ii< HOfer, M. (1990). Some aspects of the metamorphosis of
(Received for publication 25 September 1990 and in revised form 3 November 1990)
972 Phylophthora infestans zoospores. Phytophthom Newslelter 16, 37-38. An extended version of Abstracts of papers of the International Phytophthom Symposium held in Dublin. Sept. 1989. pp. 31-32. Gooding, G. V. Ii< Lucas, G. B. (1959). Factors influencing sporangial formation and zoospore activity in Phytophthora parasitica vaT. nicotianae. Phytopathology 49, 277-281. Grant, B. R.• Griffith. ]. M. Ii< Irving. H. R. (1986). A model to explain ioninduced differentiation in zoospores of Phytophthora palmivora. Erperimmtal Mycology 10, 89-98. Griffith,]. M., Iser,]. R. Ii< Grant, B. R. (1988). Calcium control of differentiation in Phytophthora palmivora. Archives of Microbiology 149. 565-571. Iser. ]. R., Griffith. ]. M., Balson. A. Ii< Grant, B. R. (1989). Accelerated ion fluxes during differentiation in zoospores of Phytophthora palmivora. Cell Differentiatioll and Development 26, 29-38. Paktitis, S., Grant, B. Ii< Lawrie, A. (1986). Surface changes in Phytophthora palmivora zoospores follOWing differentiation. Protoplasma 135, 119-129. Tokunaga, ). Ii< Bartnicki-Garcia, S. (1971). Cyst wall formation and endogenous carbohydrate utilization during synchronous encystment of Phytophthora palmivora zoospores. Archiv fur Mikrobiologie 79, 283-292.
Printed in Great Britain
- - - - - -----------.-------- - -
970 -----
Non-lethal immobilization of zoospores of Phytophthora infestans by Li+
T. ERSEK Planl Prolection Instilule, Hungarian Academy of Sciences, P.G. Box 102, H-1525 Budapesl, Hungary
u. HOLKER AND M. HOFER BOlanisches Inslitute der Universitiil, Kirschallee 1, D-5300 Bonn, Germany
Li+ (100 mM) effected irreversible but non-lethal immobilization of Phytophthora infeslans zoospores in their pyriform shape and with attached flagella. The immobilization was immediate but the capacity of cells to encyst was dependent on the duration of the Li+ treatment and on the addition of Ca 2 +. It is suggested that the loss of zoospore motility and their ability to encyst is caused by a remarkable decrease of intracellular K+ and Ca 2+, respectively. The maintenance of zoospores of Phytophthora spp. in a healthy motile condition has been assumed to be due, at least partly, to the provision of essential cations in adequate amounts from sporangia (Gooding & Lucas, 1959; Cameron & Carlile, 1980). On the other hand, zoospores are able to form cell walls (cysts) either asynchronously during an extended stage of motility, or immediately upon various stimuli. Vigorous agitation, pectin or high concentration of cations, such as Na+, Ca 2 + or 5r2 +, cause synchronous encystment (Tokunaga & Bartnicki-Garcia, 1971; Paktitis, Grant & Lawrie, 1986; Grant, Griffith & Irving, 1986). Initial events that lead to encystment involve the loss of motility followed by a release of flagella. The newly formed cysts readily germinate. U+ (3-10 mM) also induces encystment but not cyst germination (Grant et al., 1986). In this communication a rather unique and unexpected effect of 100 mM U+ on the development of Phytophthora zoospores is reported. Phytophthora infestans (Mont.) de Bary, race 2.3.4, was cultivated on potato tuber slices. Both the zoospore release from sporangia and their viability were conSiderably higher when grown on potatoes than on any other common media. Zoospore suspensions (at cell density of 10 6 ml- 1 ) were obtained by allowing thoroughly washed sporangia to discharge zoospores into glass double-distilled water at 10-12 °C for 2-3 h. Zoospores were separated from sporangia by filtration through Whatman filter paper No. 4 CErsek, 1975). U+ was added to a final concentration of 100 mM. U+ and the other cations were supplied as chlorides (analytical reagent grade purchased from Merck). U+ immediately terminated the zoospores motility. However, the flagella remained attached and, as proven by the lack of cell fluorescence after staining with calcofluor, cell walls were not formed. Zoospores retained their pyriform shape for 6-8 h when kept at 10° (Figs 1 and 2) and eventually lysed. When U+ was added at room temperature, zoospores tended
to deteriorate rapidly (cytoplasmic aggregation) even though the flagella remained firmly attached (Fig. 3). In neither case could the encystment of zoospores be induced by vigorous agitation (I min in Vortex mixer). The same concentration of other cations, namely Na+, K+, Mg 2 + or Ca 2 +, had no effect comparable to that of U+. On the other hand, the above effect of U+ in hypotonic suspensions was also observed in isoosmotic conditions, Le. 200 mM sorbitol. Removal of U+ by pelleting zoospores at 1000 g for 30 s and resuspending the pellet in distilled water, even when containing essential cations such as Na+, K+, Mg 2 +or Ca 2 +, did not yield motile zoospores. Regardless of the duration of u+ treatment, zoospores rapidly lysed. However, cells capable of forming cell walls and germ-tubes were obtained by replacement of U+ by Ca 2 + and by Ca 2 + plus K+ (I mM each), respectively. This process was time dependent. Within the first 10 min of the U+ treatment more than 90% of cells formed cysts (in Ca 2 +) and germinated (in Ca 2 + plus K+), (Table 1). During the next 10 min the capacity of cells to encyst and germinate decreased proportionally with time. An extended treatment of cells with U+ (above 20 min) caused the majority of cells to lyse. No other combination of cations (including Na+ and Mg 2+) initiated encystment or prevented zoospores from lysing (Table 1). Measurements of essential cations, Na+, K+, Mg 2 +and Ca 2 +, by atomic absorption spectrometry after a 5 min exposure of zoospores to U+, revealed a remarkable drop in intracellular K+ and Ca 2+ concentrations (Table 2). This observation rationalizes the cation replacement experiments and is consistent with our earlier results, according to which the initiation of encystment by agitation is associated with efflux of K+ and influx of H+ due to a suddenly altered plasma membrane permeability for the two cations (Ersek & Hofer, 1990). In contrast, maturing cysts accumulate K+. Hence, the drop in intracellular K+ concentration upon the U+ treatment
T. Ersek,
u. Holker
971
and M. Hofer
Figs 1-3. Light micrographs of zoospores of P. infesfans immediately upon treatment with 100 mM-Li+, Bars 10 IJm. Figs 1-2. Li+ was added to zoospore suspension at 10° Fig. 3. Li+ was added to zoospore suspension at room temperature. Note that flagella are
attached to deteriorated zoospores.
or agitation may be a signal for termination of motility and/or encystment. Encystment, however, can take place only at an adequate intracellular level of Ca 2+. Contrary to these findings, the role of K+ in the signalling process has recently been questioned (Iser et al., 1989). Unlike agitation, Li+ treatment induces Ca 2 + leakage. A lowered intracellular level of Ca 2 + may be the reason for the observed inability of zoospores to encyst even though they had lost their motility in Li+. Thus, Ca 2 + seems to playa key role in maintaining the zoospore state and in the transition to the cyst stage (Paktitis et aI., 1986; Griffith, Iser & Grant, 1988). Because Li+ treatment under both isoosmotic and the above low osmolarity conditions brought about the same response, the efflux of K+ and Ca 2 + is due to
some direct (chaotropic) effect(s) rather than to an interference with the osmoregulatory system of the cells. In conclusion, the non-lethal, irreversible immobilization of P. infestans zoospores in their pyriform shape with flagella Table 2. Concentrations of essential cations in supematants of zoospore suspensions (10' cells ml- 1) and in cells of P. infestans before and after a 5 min treatment with 100 mM LiConcentration Supernatant (IJM)a
Intracellular (mM)b Efflux
Cation
-Li+
+Li-
-Li+
+Li-
(%)
Na+ K-
51'0±9'5 79'0±3'8 I2'0±I'2 7'3±0'7
51'0±5'5 92'7±5'9 15'0±2'1 II'7±2'6
53 109 86 17
53 63 76 7
0 43 II 59
Table 1. Response of P. infestans zoospores to a replacement of Li+ (l00 mM) by individual cations or by their combinations (I mM each).
Mg"+ Ca'+
Before the cation replacement, the zoospores were incubated for 5 min with Li+
a Values from 3 separate experiments denote mean (± S.E,) extracellular cation concentrations in supernatants ot live zoospore suspensions. Cation concentrations were measured directly. using a type 360 Perkin-Elmer atomic absorption spectrometer equipped with a combined Na-K, Mg or Ca cathode lamp as a light source. Each salt used for calibration was dissolved in glass double-distilled water. Conditions for measurements were set up according to the manual of the manufacturer. " Intracellular concentrations were calculated from the concentration differences between supernatants of boiled and live zoospore suspensions on the basis of an average cell diameter of 10 IJm.
Cation
Na+
K+
Mg'+
Ca'+
Na+ K+ Mg'+ Ca"+
lyse
lyse lyse
lyse lyse lyse
encyst encyst and germinate encyst/lyse encyst
+
See reverse combination.
Immobilization of zoospores of Phytophthora infestans attached and the loss of their ability to encyst, induced by U+, are effected by a decrease in the intracellular K+ and Ca 2 + concentrations. Encystment and germination can proceed only when the two essential cations are supplemented. This work has been supported by a DFG grant No. H0555/12 to M. H. T. E. was a recipient of a DAAD exchange fellowship.
REFERENCES Cameron. ]. N. Ii< Carlile, M. ]. (1980). Negative chemotaxis of zoospores of the fungus Phytophthora palmivora. Journal of Gmeml Microbiology !lO, 347-353. Ersek, T. (1975). The sensitivity of Phytophthora infestans to several antibiotics. Zeitschnft fur Pflanzenkrankheiten und PflanUn5chutz 82, 614--617. Ersek, T. Ii< HOfer, M. (1990). Some aspects of the metamorphosis of
(Received for publication 25 September 1990 and in revised form 3 November 1990)
972 Phylophthora infestans zoospores. Phytophthom Newslelter 16, 37-38. An extended version of Abstracts of papers of the International Phytophthom Symposium held in Dublin. Sept. 1989. pp. 31-32. Gooding, G. V. Ii< Lucas, G. B. (1959). Factors influencing sporangial formation and zoospore activity in Phytophthora parasitica vaT. nicotianae. Phytopathology 49, 277-281. Grant, B. R.• Griffith. ]. M. Ii< Irving. H. R. (1986). A model to explain ioninduced differentiation in zoospores of Phytophthora palmivora. Erperimmtal Mycology 10, 89-98. Griffith,]. M., Iser,]. R. Ii< Grant, B. R. (1988). Calcium control of differentiation in Phytophthora palmivora. Archives of Microbiology 149. 565-571. Iser. ]. R., Griffith. ]. M., Balson. A. Ii< Grant, B. R. (1989). Accelerated ion fluxes during differentiation in zoospores of Phytophthora palmivora. Cell Differentiatioll and Development 26, 29-38. Paktitis, S., Grant, B. Ii< Lawrie, A. (1986). Surface changes in Phytophthora palmivora zoospores follOWing differentiation. Protoplasma 135, 119-129. Tokunaga, ). Ii< Bartnicki-Garcia, S. (1971). Cyst wall formation and endogenous carbohydrate utilization during synchronous encystment of Phytophthora palmivora zoospores. Archiv fur Mikrobiologie 79, 283-292.