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Control of phototactic migration in Dictyostelium discoideum
Printed in Sweden Copyright Q 1973 by Academic Press, Inc. All rights of reproduction in my form reserued
Experimental Cell Research 82 (1973) 236-240
CONTROL
OF PHOTOTACTIC
DICTYOSTELIUM
MIGRATION
IN
DISCOIDEUA4
K. L. POFF and W. F. LOOMIS,
JR
Department of Biology, University of California, San Diego, La Jolla, Calif. 92037, USA
SUMMARY Phototactic migration of pseudoplasmodia of the cellular slime mold, Dictyostelium discoideum, is directed by a response at the anterior tip. Horizontal light appears to be focused by refraction at the surface of the pseudoplasmodium such that it acts preferentially on the distal cells. We have been able to show that light stimulates the rate of pseudoplasmodial movement up to 80 %. This increase is dependent on the intensity of the incident light. Thus it appears that light can control the direction of migration by increasing the rate of movement on the distal side. The anterior cells are then turned toward the light by cohesion to the more slowly moving proximal side. Migration rate in the dark may be limited by the rate of synthesis or deposition of the surface sheath surrounding the pseudoplasmodium. It is suggested that light increases the rate of migration by stimulating the formation of the surface sheath. Localized stimulation would then result in a turning response.
During
development
of Dictyostelium discontaining up to IO5 cells are formed, and migrate under suitable conditions as individual units over distances of many cm before forming fruiting bodies [9, 111. Migration of pesudoplasmodia is positively phototactic with maximal response to light at ca 430 and 560 nm [5, lo]. We have been interested in the sequence of events whereby light energy is absorbed and translated into biochemical alterations which ultimately control the direction of migration. We have recently described a light-induced absorbance change observed in intact cells, in cell-free preparations, and in a cell-free fraction enriched in mitochondria [IO]. This in vitro response was shown to be sensitive to green light. The similarity of the spectral sensitivity for the in vitro response coideum, pseudoplasmodia
Exptl Cell Res 82 (1973)
and for phototactic migration suggests that this photoresponse is involved in phototaxis. Thus, light may control the direction of migration by directly modifying mitochondrial functions. The purpose of this study is to examine the immediate mechanisms whereby the direction of phototactic migration of the pseudoplasmodium may be controlled.
MATERIALS
AND METHODS
Conditions of growth and development Dictyostelium discoideum NC-4 was grown in association with Klebsiella aerogenes on SM agar plates [13]. The amoebae were collected from the plates, sedimented by centrifugation and washed 3 times in cold, distilled water. Washed amoebae were incubated for 15 h on filter supuorts to elicit svnchronous development of pseudoplasmodia [i3]. The pseudoplasmodia were then dissociated and the cells allowed to reaggregate on fresh 2% agar for 4 h in darkness before use.
Control of phototactic
00 60 40 20
ANTERIOR
POSTERIOR
1. Abscissa: distance from anterior tip of pseudoplasmodium (mm); ordinate: angle of turn (degrees). Localization of the phototactic turning response of a pseudoplasmodium of Dictyostelium discoideum. The pseudoplasmodium was irradiated on one side for 5 min and permitted to complete its turn in darkness for 15 min following the irradiation period. The angle of turn induced by light was plotted vs. the portion of the pseudoplasmodium irradiated. The diameter of the irradiating microbeam is represented by the diameter of the data points. Each data point value is the average of two separate determinations. Fig.
RESULTS Axial sensitivity to light Phototaxis in D. discoideum is known to involve a lens effect first described in Phycomyces [l]. In an aqueous environment, light appears to be focused by the convex surface of the pseudoplasmodium onto the distal cells which then respond preferentially to the light. If pseudoplasmodiaare immersed in a medium of higher refractive index than the cells, the phototactic responseis reversed, i.e. migration becomes photophobic [3]. Likewise, illumination of only one side of a pseudoplasmodium by a vertical light beam results in migration away from the illuminated side [5]. We have confirmed the results obtained with vertical microbeam illumination and
migration
in Dictyostelium
discoideum
137
have extended the technique to show that only cells in the anterior 5-lo?:, of a pseudoplasmodium are responsiblefor phototactic control of migration. For this expt. a 30 pm diameter microbeam was produced by inserting a small iris in the condenser of a conventional light microscope. Pseudoplasmodia of D. discoideumNC-4 were irradiated on one side at various distances from the anterior tip for 5 min periods. After a subsequent 15 min in darkness the angle of turn induced by the light was measured. Irradiation of one side of the tip of the pseudoplasmodium elicited a rapid advance of the irradiated cells resulting in a change in the direction of migration such that the pseudoplasmodium migrated away from the illuminated side. Irradiation of cells further removed from the anterior tip resulted in a gradually diminished response until about 150 pm back from the tip, no effect was elicited (fig. 1). Control of phototactic migration thus appears to be restricted to cells in the anterior 5-10 ‘%of the pseudoplasmodium. These data confirm the suggestion of Raper [l l] that both the receptive and directive centers must be in the apex of the pseudoplasmodium since it is the apex which veers toward the light. However, when the anterior third was removed from a series of pseudoplasmodia by microsurgery and the cut surfaces freed of surface sheath, new tips formed and the posterior portions contmued migration, orienting normally to light. It appears that the posterior cells either regained sensitivity to light upon removal of tip cells or that the posterior cells were previously sensitive to light but unable to affect the direction of migration due to their position in the pseudoplasmodium. Effects of light on migration rate A change in direction of migration requires that the distal surface of the pseudoExptl
Cell
Res 82 (1973)
238
K. L. Poff & W. F. Loomis, Jr
Table 1. Pseudoplasmodial Expt no. 1
2 ii 2 7 8 9 10 II 12 13 14 15 16 17 18
migration
rate
Strain
Lighting condition
NC-4 NC-4 NC-4 NC-4 NC-4 NC-4 NC-4 NC-4 NC-4 NC-4 NC-4 NC-4 NC-4 NC-4 NC-4 NC-4 L-25 L-25
Darkness Bi-lateral Bi-lateral Uni-lateral Uni-lateral Bi-lateral Bi-lateral Bi-lateral Bi-lateral Bi-lateral B&lateral Bi-lateral Bi-lateral Bi-lateral Bi-lateral Bi-lateral B&lateral Bi-lateral
sources sources source source sources sources sources sources sources sources sources sources sources sources sources sources sources
Light intensity (pW/cm”)
Migration rate
0
100
2 2 10 10 20 20 20 20 20 20 20 20 20 20 20 20 20
122 123 139 139 145 159 163 159 163 182 133 165 172 152 176 103 100
Relative to the migration rate in parallel control cultures incubated Average of 10 pseudoplasmodia.
plasmodium cover a longer path than the proximal surface in the same period of time. Since the lens effect experiments show that light is preferentially active on the distal cells, we would expect light to stimulate the rate of cell migration of distal cells rather than retard migration of proximal cells. However, Raper [l l] has reported that he was unable to observe any effect of light on the rate of migration. His experiments were done on a heterogeneous population of pseudoplasmodia of various sizes migrating on growth plates. Since the rate of migration is sensitive to the ionic environment and the size of the pseudoplasmodium [2], the system had considerable ‘noise’. We sought to re-examine the situation using a more defined system. Amoebae dissociated from pseudoplasmodia were permitted to reaggregate on fresh agar in the dark so as to form a population of pseudoplasmodia with a relatively homogeneous size distribution and rate of Exptl
Cd
Res 82 (1973)
in darkness which is taken as 100.
migration. This permitted small changes in migration rate to be more easily observed. To reduce wander of the pseudoplasmodia, a pair of light beams of equal intensity at right angles to each other were used to irradiate the plates. Under these conditions, the pseudoplasmodia migrated directly between such paired light beams, suggesting that phototaxis resulted from a balance in response to each light source. After incubation at 22°C for 24 h in the presence or absence of light, the plates were photographed and the length of slime track left by the 10 most rapidly migrating pseudoplasmodia was measured directly from the photographs. The effect of illumination from a single source was also determined. In all cases, irradiation increased the rate of migration, in some cases up to 80%; the increase was linearly dependent on the intensity of incident light over a ten-fold range (table 1). As a control, the rate of migration of a phototaxis-negative mutant,
Control of phototactic migration
in Dictyostelium discoideum 239
L-25 [7] was measured in a similar experiment. There was no significant stimulation of migration by irradiation of this strain (table 1). The average turning radius for a series of 1.Ox 0.1 mm pseudoplasmodia turning through a right angle in response to a unilateral light beam showed that during the turn, the distal surface of the pseudoplasmodia covered a 60 % greater distance than the proximal surface. Thus, the observed light-induced increase in migration rate is sufficient to account for directional control if localized to the distal side.
One component which occurs only during the development phase of the life cycle is the surface sheath which surrounds the cells late in aggregation and provides integrity during migration and culmination 18, I I]. As the cells migrate, new sheath is produced while previously formed sheath is left behind as a collapsed tube. The cells gain traction on the inside of the sheath and not directly on the surrounding medium. Thus. a light effect on traction could provide the ratelimiting control of migration. Although there is no direct evidence excluding this possibility, the cells are tightly packed within the elastic sheath in pseudoplasmodia and DISCUSSION do not appear to be limited by traction. There are several possiblemechanismswhereThe surface sheath limits lateral and by light might increase the rate of migration posterior movement and thus determines and thereby control migrational direction: the polarity of migration [6, 81. It also ex(a) by directly stimulating amoeboid move- tends over the apical anterior cells. Anterior ment of the individual cells; (b) by a secondary movement, therefore, would depend on the stimulation of amoeboid movement through production of new sheath material. tight an effect of light on a component present only may control the rate of migration by ultiin pseudoplasmodia. Direct stimulation by mately affecting the surface sheath. Francis light of amoeboid movement appears to be has suggested that light speeds stiffening ruled out by the observations that individual of surface sheath on the distal side such amoebae isolated before aggregation or that amoebae push through the weaker side dissociated from migrating pseudoplasmodia toward the light [5]. This model is not show no alteration in direction or rate of consistent with our present observations movement when exposed to light [3, 5, 121. since it predicts that bilateral light should It would appear therefore that the migration retard the rate of migration. rate is limited and controlled at the pseudoWe suggest that phototaxis results when plasmodium level. unilateral light stimulates the rate of sheath It has been suggested that pseudoplas- production by the distal cells. The increased modial migration is elicited by a chemotactic sheath production would permit those cells response to CAMP which is produced in to move more rapidly. Since there is little pulses entrained by cells at the anterior tip cell mixing in pseudoplasmodia and since [4]. However, no direct evidence for such the overall shape of the pseudoplasmodium pulses of CAMP has been found during is held by the forces of cell cohesion and migration and we were unable to observe surface sheath elasticity, the increased rate any effect of gradients of exogenous CAMP of movement by the distal side would turn on pseudoplasmodial migration. We feel, the direction of migration toward the light. therefore, that a direct effect of light on the After the pseudoplasmodium is directed toward the light, the cells on both sidesof the production of CAMP is unlikely. Exptl
Cell
Res 82 ( 19731
240
K. L. Poff & W. F. Loomis,
Jr
pseudoplasmodium would be exposed to equal intensities of light, and the rate of sheath production would be equal on both sides. The pseudoplasmodium would then continue to migrate directly toward the light source. This model is consistent with our present observations that light stimulates the rate of migration. We thank Dr James D. McElroy and Dr Warren L. Butler for their interest and support, and Kent Homnick, Robert Parker, and Sandra Swarbrick for technical assistance. This work was supported by grants from NSF.
3. Bonner, J T & Whitfield, F E, Biol bull 128 (1965) 51. 4. Cohen, M & Robertson, A, Dev biol 27 (1972) 589.
5. 6. 7. 8. 9. 10. 11. 12. 13.
Francis, D W, J cell camp physiol 64 (1964) 131. Garrod, D, J cell xi 4 (1969) 781. Loomis, W F Jr, Nature 227 (1970) 745. - Ibid 240 (1972) 6. Newell, P, Telser, A & Sussman, M, J bacterial 100 (1969) 763. Poff, K L, Butler, W L & Loomis, W F Jr, Proc natl acad sci US 70 (1973) 813. Raper, K B, J Elisha Mitchell sci sot 56 (1940) 241. Samuel, E D, Dev biol 3 (1961) 317. Sussman, M, Methods in cell physiology (ed D Prescott) vol. 2, p 397. Academic Press, New York (1966).
REFERENCES 1. Blaauw, A H, Ret trav bot neerl 5 (1909) 209. 2. Bormer, J T, Koontz, P G & Paton, D, Mycologia 45 (1953) 235.
Exptl
Cell
Res 82 (1973)
Received April 26, 1973
Experimental Cell Research 82 (1973) 236-240
CONTROL
OF PHOTOTACTIC
DICTYOSTELIUM
MIGRATION
IN
DISCOIDEUA4
K. L. POFF and W. F. LOOMIS,
JR
Department of Biology, University of California, San Diego, La Jolla, Calif. 92037, USA
SUMMARY Phototactic migration of pseudoplasmodia of the cellular slime mold, Dictyostelium discoideum, is directed by a response at the anterior tip. Horizontal light appears to be focused by refraction at the surface of the pseudoplasmodium such that it acts preferentially on the distal cells. We have been able to show that light stimulates the rate of pseudoplasmodial movement up to 80 %. This increase is dependent on the intensity of the incident light. Thus it appears that light can control the direction of migration by increasing the rate of movement on the distal side. The anterior cells are then turned toward the light by cohesion to the more slowly moving proximal side. Migration rate in the dark may be limited by the rate of synthesis or deposition of the surface sheath surrounding the pseudoplasmodium. It is suggested that light increases the rate of migration by stimulating the formation of the surface sheath. Localized stimulation would then result in a turning response.
During
development
of Dictyostelium discontaining up to IO5 cells are formed, and migrate under suitable conditions as individual units over distances of many cm before forming fruiting bodies [9, 111. Migration of pesudoplasmodia is positively phototactic with maximal response to light at ca 430 and 560 nm [5, lo]. We have been interested in the sequence of events whereby light energy is absorbed and translated into biochemical alterations which ultimately control the direction of migration. We have recently described a light-induced absorbance change observed in intact cells, in cell-free preparations, and in a cell-free fraction enriched in mitochondria [IO]. This in vitro response was shown to be sensitive to green light. The similarity of the spectral sensitivity for the in vitro response coideum, pseudoplasmodia
Exptl Cell Res 82 (1973)
and for phototactic migration suggests that this photoresponse is involved in phototaxis. Thus, light may control the direction of migration by directly modifying mitochondrial functions. The purpose of this study is to examine the immediate mechanisms whereby the direction of phototactic migration of the pseudoplasmodium may be controlled.
MATERIALS
AND METHODS
Conditions of growth and development Dictyostelium discoideum NC-4 was grown in association with Klebsiella aerogenes on SM agar plates [13]. The amoebae were collected from the plates, sedimented by centrifugation and washed 3 times in cold, distilled water. Washed amoebae were incubated for 15 h on filter supuorts to elicit svnchronous development of pseudoplasmodia [i3]. The pseudoplasmodia were then dissociated and the cells allowed to reaggregate on fresh 2% agar for 4 h in darkness before use.
Control of phototactic
00 60 40 20
ANTERIOR
POSTERIOR
1. Abscissa: distance from anterior tip of pseudoplasmodium (mm); ordinate: angle of turn (degrees). Localization of the phototactic turning response of a pseudoplasmodium of Dictyostelium discoideum. The pseudoplasmodium was irradiated on one side for 5 min and permitted to complete its turn in darkness for 15 min following the irradiation period. The angle of turn induced by light was plotted vs. the portion of the pseudoplasmodium irradiated. The diameter of the irradiating microbeam is represented by the diameter of the data points. Each data point value is the average of two separate determinations. Fig.
RESULTS Axial sensitivity to light Phototaxis in D. discoideum is known to involve a lens effect first described in Phycomyces [l]. In an aqueous environment, light appears to be focused by the convex surface of the pseudoplasmodium onto the distal cells which then respond preferentially to the light. If pseudoplasmodiaare immersed in a medium of higher refractive index than the cells, the phototactic responseis reversed, i.e. migration becomes photophobic [3]. Likewise, illumination of only one side of a pseudoplasmodium by a vertical light beam results in migration away from the illuminated side [5]. We have confirmed the results obtained with vertical microbeam illumination and
migration
in Dictyostelium
discoideum
137
have extended the technique to show that only cells in the anterior 5-lo?:, of a pseudoplasmodium are responsiblefor phototactic control of migration. For this expt. a 30 pm diameter microbeam was produced by inserting a small iris in the condenser of a conventional light microscope. Pseudoplasmodia of D. discoideumNC-4 were irradiated on one side at various distances from the anterior tip for 5 min periods. After a subsequent 15 min in darkness the angle of turn induced by the light was measured. Irradiation of one side of the tip of the pseudoplasmodium elicited a rapid advance of the irradiated cells resulting in a change in the direction of migration such that the pseudoplasmodium migrated away from the illuminated side. Irradiation of cells further removed from the anterior tip resulted in a gradually diminished response until about 150 pm back from the tip, no effect was elicited (fig. 1). Control of phototactic migration thus appears to be restricted to cells in the anterior 5-10 ‘%of the pseudoplasmodium. These data confirm the suggestion of Raper [l l] that both the receptive and directive centers must be in the apex of the pseudoplasmodium since it is the apex which veers toward the light. However, when the anterior third was removed from a series of pseudoplasmodia by microsurgery and the cut surfaces freed of surface sheath, new tips formed and the posterior portions contmued migration, orienting normally to light. It appears that the posterior cells either regained sensitivity to light upon removal of tip cells or that the posterior cells were previously sensitive to light but unable to affect the direction of migration due to their position in the pseudoplasmodium. Effects of light on migration rate A change in direction of migration requires that the distal surface of the pseudoExptl
Cell
Res 82 (1973)
238
K. L. Poff & W. F. Loomis, Jr
Table 1. Pseudoplasmodial Expt no. 1
2 ii 2 7 8 9 10 II 12 13 14 15 16 17 18
migration
rate
Strain
Lighting condition
NC-4 NC-4 NC-4 NC-4 NC-4 NC-4 NC-4 NC-4 NC-4 NC-4 NC-4 NC-4 NC-4 NC-4 NC-4 NC-4 L-25 L-25
Darkness Bi-lateral Bi-lateral Uni-lateral Uni-lateral Bi-lateral Bi-lateral Bi-lateral Bi-lateral Bi-lateral B&lateral Bi-lateral Bi-lateral Bi-lateral Bi-lateral Bi-lateral B&lateral Bi-lateral
sources sources source source sources sources sources sources sources sources sources sources sources sources sources sources sources
Light intensity (pW/cm”)
Migration rate
0
100
2 2 10 10 20 20 20 20 20 20 20 20 20 20 20 20 20
122 123 139 139 145 159 163 159 163 182 133 165 172 152 176 103 100
Relative to the migration rate in parallel control cultures incubated Average of 10 pseudoplasmodia.
plasmodium cover a longer path than the proximal surface in the same period of time. Since the lens effect experiments show that light is preferentially active on the distal cells, we would expect light to stimulate the rate of cell migration of distal cells rather than retard migration of proximal cells. However, Raper [l l] has reported that he was unable to observe any effect of light on the rate of migration. His experiments were done on a heterogeneous population of pseudoplasmodia of various sizes migrating on growth plates. Since the rate of migration is sensitive to the ionic environment and the size of the pseudoplasmodium [2], the system had considerable ‘noise’. We sought to re-examine the situation using a more defined system. Amoebae dissociated from pseudoplasmodia were permitted to reaggregate on fresh agar in the dark so as to form a population of pseudoplasmodia with a relatively homogeneous size distribution and rate of Exptl
Cd
Res 82 (1973)
in darkness which is taken as 100.
migration. This permitted small changes in migration rate to be more easily observed. To reduce wander of the pseudoplasmodia, a pair of light beams of equal intensity at right angles to each other were used to irradiate the plates. Under these conditions, the pseudoplasmodia migrated directly between such paired light beams, suggesting that phototaxis resulted from a balance in response to each light source. After incubation at 22°C for 24 h in the presence or absence of light, the plates were photographed and the length of slime track left by the 10 most rapidly migrating pseudoplasmodia was measured directly from the photographs. The effect of illumination from a single source was also determined. In all cases, irradiation increased the rate of migration, in some cases up to 80%; the increase was linearly dependent on the intensity of incident light over a ten-fold range (table 1). As a control, the rate of migration of a phototaxis-negative mutant,
Control of phototactic migration
in Dictyostelium discoideum 239
L-25 [7] was measured in a similar experiment. There was no significant stimulation of migration by irradiation of this strain (table 1). The average turning radius for a series of 1.Ox 0.1 mm pseudoplasmodia turning through a right angle in response to a unilateral light beam showed that during the turn, the distal surface of the pseudoplasmodia covered a 60 % greater distance than the proximal surface. Thus, the observed light-induced increase in migration rate is sufficient to account for directional control if localized to the distal side.
One component which occurs only during the development phase of the life cycle is the surface sheath which surrounds the cells late in aggregation and provides integrity during migration and culmination 18, I I]. As the cells migrate, new sheath is produced while previously formed sheath is left behind as a collapsed tube. The cells gain traction on the inside of the sheath and not directly on the surrounding medium. Thus. a light effect on traction could provide the ratelimiting control of migration. Although there is no direct evidence excluding this possibility, the cells are tightly packed within the elastic sheath in pseudoplasmodia and DISCUSSION do not appear to be limited by traction. There are several possiblemechanismswhereThe surface sheath limits lateral and by light might increase the rate of migration posterior movement and thus determines and thereby control migrational direction: the polarity of migration [6, 81. It also ex(a) by directly stimulating amoeboid move- tends over the apical anterior cells. Anterior ment of the individual cells; (b) by a secondary movement, therefore, would depend on the stimulation of amoeboid movement through production of new sheath material. tight an effect of light on a component present only may control the rate of migration by ultiin pseudoplasmodia. Direct stimulation by mately affecting the surface sheath. Francis light of amoeboid movement appears to be has suggested that light speeds stiffening ruled out by the observations that individual of surface sheath on the distal side such amoebae isolated before aggregation or that amoebae push through the weaker side dissociated from migrating pseudoplasmodia toward the light [5]. This model is not show no alteration in direction or rate of consistent with our present observations movement when exposed to light [3, 5, 121. since it predicts that bilateral light should It would appear therefore that the migration retard the rate of migration. rate is limited and controlled at the pseudoWe suggest that phototaxis results when plasmodium level. unilateral light stimulates the rate of sheath It has been suggested that pseudoplas- production by the distal cells. The increased modial migration is elicited by a chemotactic sheath production would permit those cells response to CAMP which is produced in to move more rapidly. Since there is little pulses entrained by cells at the anterior tip cell mixing in pseudoplasmodia and since [4]. However, no direct evidence for such the overall shape of the pseudoplasmodium pulses of CAMP has been found during is held by the forces of cell cohesion and migration and we were unable to observe surface sheath elasticity, the increased rate any effect of gradients of exogenous CAMP of movement by the distal side would turn on pseudoplasmodial migration. We feel, the direction of migration toward the light. therefore, that a direct effect of light on the After the pseudoplasmodium is directed toward the light, the cells on both sidesof the production of CAMP is unlikely. Exptl
Cell
Res 82 ( 19731
240
K. L. Poff & W. F. Loomis,
Jr
pseudoplasmodium would be exposed to equal intensities of light, and the rate of sheath production would be equal on both sides. The pseudoplasmodium would then continue to migrate directly toward the light source. This model is consistent with our present observations that light stimulates the rate of migration. We thank Dr James D. McElroy and Dr Warren L. Butler for their interest and support, and Kent Homnick, Robert Parker, and Sandra Swarbrick for technical assistance. This work was supported by grants from NSF.
3. Bonner, J T & Whitfield, F E, Biol bull 128 (1965) 51. 4. Cohen, M & Robertson, A, Dev biol 27 (1972) 589.
5. 6. 7. 8. 9. 10. 11. 12. 13.
Francis, D W, J cell camp physiol 64 (1964) 131. Garrod, D, J cell xi 4 (1969) 781. Loomis, W F Jr, Nature 227 (1970) 745. - Ibid 240 (1972) 6. Newell, P, Telser, A & Sussman, M, J bacterial 100 (1969) 763. Poff, K L, Butler, W L & Loomis, W F Jr, Proc natl acad sci US 70 (1973) 813. Raper, K B, J Elisha Mitchell sci sot 56 (1940) 241. Samuel, E D, Dev biol 3 (1961) 317. Sussman, M, Methods in cell physiology (ed D Prescott) vol. 2, p 397. Academic Press, New York (1966).
REFERENCES 1. Blaauw, A H, Ret trav bot neerl 5 (1909) 209. 2. Bormer, J T, Koontz, P G & Paton, D, Mycologia 45 (1953) 235.
Exptl
Cell
Res 82 (1973)
Received April 26, 1973