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Effects of hormones on Entomophthora egressa morphogenesis
IOURN.4’.
OF INVERTEBRATE
Effects
33, 242-248 (1979)
PATHOLOGY
of Hormones
on Entomophthora
RICHARD Department
of Biology,
Memorial
A. NOLAN
University
egressa
Morphogenesis
AND GARY B. DUNPHY
of Newfoundland,
Sr. John’s,
Netifoundlund,
Canada
AlB
3X9
Received March 29, 1978 The morphological effects of a fungal sex hormone, trisporic acid (TA), and a synthetic insect juvenile hormone (JH) on mycelial cultures derived from protoplasts of the fungusEnromophthora egressa were determined. In comparison with the control treatment (no added hormone), only one treatment (JH and TA both added at 4Opg60 ml) produced all of the control cell types. Under both experimental conditions, normal hypha (without swollen tips), spherical hyphal bodies, irregularly shaped hyphal bodies, and thick-walled spheres were present. This JH plus TA treatment differed from the control in that normal rod-shaped hyphal bodies were also present and the thick-walled structures tended to be variable in shape. Hyphal tip swelling which did not lead to conidium production was common to all treatments in which only JH was added. The results of the addition of various concentrations and combinations of JH and TA with regard to the production of thickwalled cell types and three forms of hyphal bodies are given and discussed. KEY WORDS: Enromophrhora egressa; fungal morphogenesis; hormones, effects on fungal morphogenesis; insect juvenile hormone; fungal sex hormone; trisporic acid: fungal hyphal bodies.
INTRODUCTION
The eastern hemlock looper and the spruce budworm have been serious defoliators of balsam fir and spruce in Newfoundland (Otvos et al., 1971; Clark et al., 1973). Both of these forest insect pests are attacked by the fungus Entomophthora egressa (Otvos et al., 1973; Tyrrell, 1977). Tyrrell and MacLeod (1972) described the phenomenon in E. egressa of free protoplast release from germinating conidia and subsequent independent movement of protoplasts in modified Grace’s (1962, 1966) insect tissue culture medium. Subsequent published accounts have dealt with resting spore germination (Nolan et al., 1976), the development of the protoplasts on solid media with a liquid overlay, in complex liquid media, and in simplified liquid media (Dunphy and Nolan, 1977a,b), the comparison of growth of different isolates of E. egressa (Dunphy et al., 1978), and biochemical studies with one of the insect hosts (Dunphy et al., 1977a,b). The purpose of the present study was to examine the effects of (1) a naturally occurring fungal sex hormone, trisporic acid (TA), and (2) a synthetic insect juvenile hormone (JH) individually at different con-
centrations and together at two concentrations on the development of E. egressa. The ultimate goal of this and the previous series of studies is the mass fermentation production of dense resting spore (zygospore or azygospore) or other thick-walled cell suspensions for field application in biological control programs. MATERIALS
Stock cultures of E. egressa protoplasts (isolate 521) were maintained at 20°C in 15 ml of Grace’s insect tissue culture medium supplemented with 28 nil of heatinactivated fetal calf serum (Grand Island Biological Company, Grand Island, New Ybrk) added to 1 liter of medium. One milliliter of a 48-hr culture of cells was transferred to a 13-amino-acid medium without added vitamins (see Table 1; Dunphy and Nolan, 1977b); and, after 48 hr of growth, a l-ml aliquot was transferred to a Bellco flask (Bellco Glass, Inc., Vineland, New Jersey) containing 49 ml of the 13-aminoacid medium minus vitamins to further reduce carryover from the stock culture medium. This culture was incubated for 48 hr, and l-ml volumes were removed and used as the inoculum for each of the ex242
0022-201 l/79/020242-07$02.00/O Copyright 0 1979 by Academic Press. Inc. A11 rights of reproduction in any form reserved.
AND METHODS
HORMONES,
EFFECTS
ON
perimental flasks. The inoculum contained 2.6 x 10’ protoplasts/ml (SE = 2.5 x 106/ ml). After 19 days of incubation in darkness on a horizontal, rotary shaker (100 rpm, 20 + l”C), the cultures had been producing hyphal growth for 5 days and a mycelial ball had formed. Individual hyphae were protruding 2 to 4 mm beyond the main hyphal mass. At this time the hormones were added to the experimental flasks (three flasks per treatment). Two hormones were used. The fungal sex hormone, trisporic acid, was obtained as a mixture of >90% trisporic acid C and < 10% trisporic acid B from Dr. Richard Sutter (West Virginia University, Morgantown); and the synthetic juvenile hormone, AY-22, 342-3, consisting of a mixture in which the four most active isomers comprised approximately 72%, was obtained from Ayerst Laboratories (Division of Ayerst, McKenna and Harrison Ltd., Montreal). The trisporic acid was tested at concentrations of 0 (control), 20, 40, and 60pglml; the JH was tested at concentrations of 0 (control), 2, 4, 6, 20, 40, 60, 200, 400, and 6OOpg/50 ml. The two test compounds were used in combination, at both 40 and 60 ~8/50 ml. After the test chemicals had been added, the flasks were incubated for an additional 7 days: then the entire experimental flask yields were mounted in lactophenol either directly or after preliminary examination of a portion of the yield in the final incubation medium. All chemicals except the hormones were obtained from Sigma Chemical Company, St. Louis, Missouri, and were of the reagent grade. Grace’s medium was prepared according to Grace (1962) with the addition of fetal calf serum. All media were adjusted to pH 7.1 with 4 N KOH and tilter sterilized (0.2 pm, Nalgene filter unit, Sybron Corporation, Rochester, New York). Fifty-milliliter aliquots of media were added to 125-ml Bellco flasks. Osmolality, initially adjusted to 347 mM, was measured with an Advanced osmometer (model 3L: Advanced
FUNGAL
MORPHOGENESIS
243
Instruments, Needham Heights, Massachusetts). All glassware was washed in soapy water and rinsed with hot tap water before immersion in concentrated sulfuric acid saturated with potassium dichromate. The glassware was rinsed with hot tap water and finally with distilled water. RESULTS Control
The control flasks contained numerous germinating spherical hyphal bodies (Fig. 1). There was no rapid movement of the contents of the spherical hyphal bodies. A second prominent cell type was an irregularly shaped hyphal body (Fig. 2). The mycelial ball was stratified, with the spherical and irregularly shaped hyphal bodies being on the periphery of the ball. The older (central) areas of the ball contained a third cell type which was a thick-walled sphere (x = 27.46 pm diam, SE = 1.41 pm). These latter spheres had a highly globular interior and were in various stages of cellular lysis. The final medium pH was 7.0. Trisporic
Acid (TA)
For the 20-vg/50-ml treatment, the major difference between this treatment and all others was the presence of numerous spheres (Fig. 3) of which 2% had thick walls (i = 12.15 pm thick, SE = 0.00). These thick-walled spheres had a diameter of 30.62 pm (SE = 1.46 pm) and had a granulated cytoplasm with oil droplets. Spherical hyphal bodies composed 10% of the cell types and were most often found in the layer of the ball just below the previously mentioned cell type. The spherical hyphal bodies (25.5pm diam, SE = 2.6pm; wall thickness 9.6 pm, SE = 0.14 pm) were readily separated from the previous cell type because of their irregularly shaped vacuole. The 40+g/50-ml treatment possessed irregularly shaped hyphal bodies as the second most common cell type. The most common cell type was spherical hyphal bodies, many of which tended to be ellip-
FIG. FIG.
1. Germinating 2. Irregularly
spherical hyphal body (control). x 1300. shaped hyphal body (control). x 1100.
FIG. 3. Unidentified, thicker-walled, spherical cell (20 pg TN50 ml). FIG. 4. Ellipsoidal, spherical hyphal bodies, one with central septum 1300. FIG. 5. Bilobed structure with thin wall (40 /~g TA/50 ml). x 1300. FIG.
6. Hypha
with
swollen
tip (2 pg JH/SO ml). 244
x 1300.
x 1900. (arrow, 40 pg TAD0
ml).
x
FIG. FIG. FIG. 1600. FIG. JH/50 FIG. FIG.
7. Rod-shaped hyphal bodies (arrows, 4 pg JH/50 ml). x 400. 8. Spherical hyphal body with multiple germ tubes (200 pg JH/SO ml). 9. Thicker-walled, spherical cell with oil droplets in a granular cytoplasm 10. ml). 11. 12.
Irregularly shaped x 950. Irregularly shaped Fusing or dividing
hyphal
body
and two adhering
spherical
hyphal
x 900. (2OOpg bodies
JH/50
(arrows,
hyphal bodies (arrows, JH and TA at 40 pcLgI50 ml). x 600. spherical hyphal bodies (JH and TA at 60 /.~g/50 ml). x 1450. 245
ml).
x
600 pg
246
NOLAN
soidal, and displayed central septa (Fig. 4, arrow) between adjacent (daughter?) cells. An interesting bilobed structure (Fig. 5) was observed in this treatment at a 2% level; but it lacked a thick wall. The 20- and 40-pg/50-ml concentrations caused the most notable pH shifts in the final medium (6.5). The 6O+g treatment resulted in cell types that were either spherical or rod-shaped hyphal bodies. The final pH was 7.1. Juvenile Hormone (JH) The 2-pg treatment contained numerous germinating spherical hyphal bodies (29.2 + 1.1 pm diam); however, the most striking structures were the swollen hyphal tips (Fig. 6). The swollen hyphal tips were present at varying levels in all of the JH treatments, with the terminal swelling being 24.3 + 0.8 pm diam and having a vacuole (18.3 + 0.6Fm diam). The hyphae possessing the swellings were 18.2 t 0.7 pm in diameter and highly interwoven and penetrated into the center of the mycelial ball. The final p H was 6.9. The 4.0- and 6.0~pg treatment results were similar and resembled those of the 2-pg treatment except for the lack of spherical hyphal bodies, which were replaced by abundant rod-shaped hyphal bodies (Fig. 7, arrows). The final pH was 7.2 and 7.1, respectively. The 20-pg treatment produced primarily spherical hyphal bodies which developed differently depending upon their position in the hyphal ball. Those in the core failed to develop or only yielded poor germ-tube development. Those at or near the periphery developed long germ tubes. Occasionally a swelling developed at the end of the germ tube, but there was no evidence that this was a conidial initial. The firralpH was 6.9. The 40- and 60-pg levels produced only rod-shaped hyphal bodies. The final pH was 7.2 and 7.1, respectively. The 2OOqg treatment produced most notably spherical hyphal bodies with multiple germ tubes (Fig. 8). A spherical cell type (38.9 pm diam) with several oil drop-
AND
DUNPHY
lets in a granular cytoplasm was detected (Fig. 9). These cells were similar to those produced by the TA 20-pg treatment (Fig. 3). The final pH was 7.1. The 400~pg treatment produced primarily rod-shaped hyphal bodies and only a few spherical hyphal bodies and long hyphae as in all JH treatments. The firralpH was 7.1. The 6OOqg treatment produced a low incidence of germinating spherical hyphal bodies (27.19pm diam, SE = 1.2pm). Both rod-shaped hyphal bodies and irregularly shaped hyphal bodies (Fig. 10) were present. The most striking feature in these results was the presence of spherical hyphal bodies adhering to one another (Fig. 10, arrows). The linal pH was 7.0. Juvenile Hormone plus Trisporic Acid The 4O+g levels produced irregularly shaped hyphal bodies (Fig. 11, arrows) among normal hyphae. Germinating spherical hyphal bodies were also present and many thick-walled structures showing shape variation between spherical hyphal bodies and rod-shaped hyphal bodies. The final pH was 6.9. The 60-pg levels produced structures which were either dividing or fusing spherical hyphal bodies (Fig. 12), a few rodshaped hyphal bodies, and aberrant conidia (72-90 pm long, 41.31 t 1.73 pm at widest point, and 17.01 + 0.97 pm diam at the columella). There was a tendency toward wall thickening in some of the spherical hyphal bodies. The final pH was 7.1. DISCUSSION
In comparing the various experimental hormone treatment results with those of the control, only one produced all of the control cell types (JH and TA, 40 pg/50 ml). Under both conditions, normal hyphae (without swollen tips), spherical hyphal bodies, irregularly shaped hyphal bodies, and thick-walled spheres were present. The JH plus TA (4Opg/50 ml) treatment differed from the control in that normal rod-shaped hyphal bodies were also present and the thick-walled structures tended to be vari-
HORMONES,
EFFECTS
ON FUNGAL
able in shape. Thick-walled spheres were produced in the control; however, these were in the center of the mycelial ball and were lysing. The treatments which showed promise by the production of comparable, but nonlysing, thick-walled spheres were the TA 2O+g, JH 200~pg, and JH plus TA 40-pg treatments. It is difficult to speculate on the nature of the bilobed structure found in the TA 40-pg treatment and whether or not minor modifications of this experimental condition might produce more normal resting spores such as those investigated by Nolan et al. (1976), where similar but thicker-walled bilobed structures were encountered in conjunction with normal, germinating resting spores. All juvenile hormone treatments produced some degree of hyphal tip swelling, but this does not appear to be a possible forerunner of conidium formation in this instance. The hyphal swelling induced by JH was counteracted by 40 pg TA. The low JH concentrations of 2 and 20 pg appeared to favor spherical hyphal body formation, whereas concentrations of 200, 400, and 600 pg JH produced aberrant or suppressed germination and/or suppressed production of spherical hyphal bodies. Rod-shaped hyphal bodies were produced in a very irregular pattern: but TA at 60 pg both individually and in conjunction with JH favored their production. The aberrant conidia produced in the JH plus TA (60 pg/50 ml) treatment were approximately 2 to 3 times the mean length of normal conidia (28.0 to 49.5 pm,X = 36.5 + 4.8 pm; Otvos et al., 1973). Many of the thick-walled cells produced in the study fell within or near the size range of resting spores ofE. egressu (20.0 to 32.0pm diam, X = 25.0 k 0.8 pm; Otvos et., 1973). One problem with the study was the use of lactophenol for examining the cell types as this precluded the possibility of subsequent testing of the thick-walled spheres for germination to form primary conidia and, thus, positive classification of them as resting spores. It had been assumed that such ger-
247
MORPHOGENESIS
mination would occur in the liquid incubation medium as had been observed by Nolan et al. (1976). It is known that this fungus readily adapts to mass fermentation growth under the appropriate conditions (Nolan, unpubl.); however, the implications from this study are the possible problem of thick-walled-cell lysis within the mycelial ball and the advantageous use of JH and TA jointly for production of normal hyphae, abundant thick-walled cell types, and three forms of hyphal bodies. ACKNOWLEDGMENTS The authors would like to thank Dr. Richard Sutter and Ayerst Laboratories for the hormones. One of us (G.B.D.) held a National Research Council of Canada postgraduate scholarship. This study was supported by Canadian Forestry Service Contract OSU77-00048 and by NRCC Grant A-6665 to R.A.N.
REFERENCES I. S., AND PARDY, K. E. 1973. “Biological Agents Released in Newfoundland for the Control of Forest Insect Pests.” Canad. Dept. Envir., Canad. For. Serv., Inf. Rept. N-X-96,42 pp. DUNPHY, G. B., ANDNOLAN, R. A. 1977a. Regeneration of protoplasts of E~~ron@zthnrn egvessn. a fungal pathogen of the eastern hemlock looper. Canad. J. Bat.. 55, 107-113. DUNPHY, G. B., AND NOLAN, R. A. 1977b. Morphogenesis of protoplasts of Entomophthorrr egre~sa in simplified culture media. Canard. J. Bat.. 55, 3046-3053. DUNPHY, G. B., KEOUGH, K. M. W., ANDNOLAN, R. A. 1977a. Fatty acid composition and lipid content of late larval and pupal stages of the eastern hemlock looper. Lambdina fiscellariu fiscelluria (Lepidoptera: Geometridae). Canrrd. Entomol.. 109, 347-350. DUNPHY, G. B., NOLAN, R. A., AND OTVOS, I. S. 1977b. Ninhydrin-positive substance analysis of larval hemolymph of the eastern hemlock looper, Lumbdina fiscelluria fiscelluriu (Lepidoptera: Geometridae) and growth of Entomophthoru egresya protoplasts. Canad. Entomol.. 109, 341-346. CLARK,
DUNPHY,
R. C.,
OTVOS,
G. B.,
NOLAN,
R. A.,
AND
MACLEOD.
D.
M. 1978. Comparative growth and development of two protoplast isolates of Entomophthoro egresscr. J. Invertebr.
Pnthol..
31, 267-269.
GRACE, T. D. C. 1962. Establishment of four strains of cells from insect tissue grown in vitr0. Nature (London). 195, 788-789. GRACE, T. D. C. 1966. Establishment of a line of mosquito (Aedes aegypt; L.) cells grown in vitro. Nature (London), 211, 366367. NOLAN,
R. A.,
DUNPHY,
G.
B.,
AND
MACLEOD,
D.
248
NOLAN
AND DUNPHY
M. 1976. In vitro germination of Entomophthora egresting spores. Canad. J. Bat., 54, 1131-1134. OTVOS, I. S., CLARK, R. C., AND CLARKE, L. J. 1971. “The Hemlock Looper in Newfoundland: The Outbreak, 1966 to 1971; and Aerial Spraying, 1968 and 1969.” Canad. Dept. Envir., Canad. For. Serv., Inf. Rept. N-X-68, 62 pp. OTVOS, I. S., MACLEOD, D. M., AND TYRRELL, D. 1973. Two species of Entomophthora pathogenic to ressa
the eastern hemlock looper (Lepidoptera: Geometridae) in Newfoundland. Canad. Entomol., 105, 1435-1441. TYRRELL,
D. 1977. Occurrence of protoplasts in the natural life cycle of Entomophthora egressa. Exp. Mycol., 1, 259-263. TYRRELL, D., AND MACLEOD, D. M. 1972. Spontaneous formation of protoplasts by a species of Entomophthora.
J. Invertebr.
Pathol..
19, 354-360.
OF INVERTEBRATE
Effects
33, 242-248 (1979)
PATHOLOGY
of Hormones
on Entomophthora
RICHARD Department
of Biology,
Memorial
A. NOLAN
University
egressa
Morphogenesis
AND GARY B. DUNPHY
of Newfoundland,
Sr. John’s,
Netifoundlund,
Canada
AlB
3X9
Received March 29, 1978 The morphological effects of a fungal sex hormone, trisporic acid (TA), and a synthetic insect juvenile hormone (JH) on mycelial cultures derived from protoplasts of the fungusEnromophthora egressa were determined. In comparison with the control treatment (no added hormone), only one treatment (JH and TA both added at 4Opg60 ml) produced all of the control cell types. Under both experimental conditions, normal hypha (without swollen tips), spherical hyphal bodies, irregularly shaped hyphal bodies, and thick-walled spheres were present. This JH plus TA treatment differed from the control in that normal rod-shaped hyphal bodies were also present and the thick-walled structures tended to be variable in shape. Hyphal tip swelling which did not lead to conidium production was common to all treatments in which only JH was added. The results of the addition of various concentrations and combinations of JH and TA with regard to the production of thickwalled cell types and three forms of hyphal bodies are given and discussed. KEY WORDS: Enromophrhora egressa; fungal morphogenesis; hormones, effects on fungal morphogenesis; insect juvenile hormone; fungal sex hormone; trisporic acid: fungal hyphal bodies.
INTRODUCTION
The eastern hemlock looper and the spruce budworm have been serious defoliators of balsam fir and spruce in Newfoundland (Otvos et al., 1971; Clark et al., 1973). Both of these forest insect pests are attacked by the fungus Entomophthora egressa (Otvos et al., 1973; Tyrrell, 1977). Tyrrell and MacLeod (1972) described the phenomenon in E. egressa of free protoplast release from germinating conidia and subsequent independent movement of protoplasts in modified Grace’s (1962, 1966) insect tissue culture medium. Subsequent published accounts have dealt with resting spore germination (Nolan et al., 1976), the development of the protoplasts on solid media with a liquid overlay, in complex liquid media, and in simplified liquid media (Dunphy and Nolan, 1977a,b), the comparison of growth of different isolates of E. egressa (Dunphy et al., 1978), and biochemical studies with one of the insect hosts (Dunphy et al., 1977a,b). The purpose of the present study was to examine the effects of (1) a naturally occurring fungal sex hormone, trisporic acid (TA), and (2) a synthetic insect juvenile hormone (JH) individually at different con-
centrations and together at two concentrations on the development of E. egressa. The ultimate goal of this and the previous series of studies is the mass fermentation production of dense resting spore (zygospore or azygospore) or other thick-walled cell suspensions for field application in biological control programs. MATERIALS
Stock cultures of E. egressa protoplasts (isolate 521) were maintained at 20°C in 15 ml of Grace’s insect tissue culture medium supplemented with 28 nil of heatinactivated fetal calf serum (Grand Island Biological Company, Grand Island, New Ybrk) added to 1 liter of medium. One milliliter of a 48-hr culture of cells was transferred to a 13-amino-acid medium without added vitamins (see Table 1; Dunphy and Nolan, 1977b); and, after 48 hr of growth, a l-ml aliquot was transferred to a Bellco flask (Bellco Glass, Inc., Vineland, New Jersey) containing 49 ml of the 13-aminoacid medium minus vitamins to further reduce carryover from the stock culture medium. This culture was incubated for 48 hr, and l-ml volumes were removed and used as the inoculum for each of the ex242
0022-201 l/79/020242-07$02.00/O Copyright 0 1979 by Academic Press. Inc. A11 rights of reproduction in any form reserved.
AND METHODS
HORMONES,
EFFECTS
ON
perimental flasks. The inoculum contained 2.6 x 10’ protoplasts/ml (SE = 2.5 x 106/ ml). After 19 days of incubation in darkness on a horizontal, rotary shaker (100 rpm, 20 + l”C), the cultures had been producing hyphal growth for 5 days and a mycelial ball had formed. Individual hyphae were protruding 2 to 4 mm beyond the main hyphal mass. At this time the hormones were added to the experimental flasks (three flasks per treatment). Two hormones were used. The fungal sex hormone, trisporic acid, was obtained as a mixture of >90% trisporic acid C and < 10% trisporic acid B from Dr. Richard Sutter (West Virginia University, Morgantown); and the synthetic juvenile hormone, AY-22, 342-3, consisting of a mixture in which the four most active isomers comprised approximately 72%, was obtained from Ayerst Laboratories (Division of Ayerst, McKenna and Harrison Ltd., Montreal). The trisporic acid was tested at concentrations of 0 (control), 20, 40, and 60pglml; the JH was tested at concentrations of 0 (control), 2, 4, 6, 20, 40, 60, 200, 400, and 6OOpg/50 ml. The two test compounds were used in combination, at both 40 and 60 ~8/50 ml. After the test chemicals had been added, the flasks were incubated for an additional 7 days: then the entire experimental flask yields were mounted in lactophenol either directly or after preliminary examination of a portion of the yield in the final incubation medium. All chemicals except the hormones were obtained from Sigma Chemical Company, St. Louis, Missouri, and were of the reagent grade. Grace’s medium was prepared according to Grace (1962) with the addition of fetal calf serum. All media were adjusted to pH 7.1 with 4 N KOH and tilter sterilized (0.2 pm, Nalgene filter unit, Sybron Corporation, Rochester, New York). Fifty-milliliter aliquots of media were added to 125-ml Bellco flasks. Osmolality, initially adjusted to 347 mM, was measured with an Advanced osmometer (model 3L: Advanced
FUNGAL
MORPHOGENESIS
243
Instruments, Needham Heights, Massachusetts). All glassware was washed in soapy water and rinsed with hot tap water before immersion in concentrated sulfuric acid saturated with potassium dichromate. The glassware was rinsed with hot tap water and finally with distilled water. RESULTS Control
The control flasks contained numerous germinating spherical hyphal bodies (Fig. 1). There was no rapid movement of the contents of the spherical hyphal bodies. A second prominent cell type was an irregularly shaped hyphal body (Fig. 2). The mycelial ball was stratified, with the spherical and irregularly shaped hyphal bodies being on the periphery of the ball. The older (central) areas of the ball contained a third cell type which was a thick-walled sphere (x = 27.46 pm diam, SE = 1.41 pm). These latter spheres had a highly globular interior and were in various stages of cellular lysis. The final medium pH was 7.0. Trisporic
Acid (TA)
For the 20-vg/50-ml treatment, the major difference between this treatment and all others was the presence of numerous spheres (Fig. 3) of which 2% had thick walls (i = 12.15 pm thick, SE = 0.00). These thick-walled spheres had a diameter of 30.62 pm (SE = 1.46 pm) and had a granulated cytoplasm with oil droplets. Spherical hyphal bodies composed 10% of the cell types and were most often found in the layer of the ball just below the previously mentioned cell type. The spherical hyphal bodies (25.5pm diam, SE = 2.6pm; wall thickness 9.6 pm, SE = 0.14 pm) were readily separated from the previous cell type because of their irregularly shaped vacuole. The 40+g/50-ml treatment possessed irregularly shaped hyphal bodies as the second most common cell type. The most common cell type was spherical hyphal bodies, many of which tended to be ellip-
FIG. FIG.
1. Germinating 2. Irregularly
spherical hyphal body (control). x 1300. shaped hyphal body (control). x 1100.
FIG. 3. Unidentified, thicker-walled, spherical cell (20 pg TN50 ml). FIG. 4. Ellipsoidal, spherical hyphal bodies, one with central septum 1300. FIG. 5. Bilobed structure with thin wall (40 /~g TA/50 ml). x 1300. FIG.
6. Hypha
with
swollen
tip (2 pg JH/SO ml). 244
x 1300.
x 1900. (arrow, 40 pg TAD0
ml).
x
FIG. FIG. FIG. 1600. FIG. JH/50 FIG. FIG.
7. Rod-shaped hyphal bodies (arrows, 4 pg JH/50 ml). x 400. 8. Spherical hyphal body with multiple germ tubes (200 pg JH/SO ml). 9. Thicker-walled, spherical cell with oil droplets in a granular cytoplasm 10. ml). 11. 12.
Irregularly shaped x 950. Irregularly shaped Fusing or dividing
hyphal
body
and two adhering
spherical
hyphal
x 900. (2OOpg bodies
JH/50
(arrows,
hyphal bodies (arrows, JH and TA at 40 pcLgI50 ml). x 600. spherical hyphal bodies (JH and TA at 60 /.~g/50 ml). x 1450. 245
ml).
x
600 pg
246
NOLAN
soidal, and displayed central septa (Fig. 4, arrow) between adjacent (daughter?) cells. An interesting bilobed structure (Fig. 5) was observed in this treatment at a 2% level; but it lacked a thick wall. The 20- and 40-pg/50-ml concentrations caused the most notable pH shifts in the final medium (6.5). The 6O+g treatment resulted in cell types that were either spherical or rod-shaped hyphal bodies. The final pH was 7.1. Juvenile Hormone (JH) The 2-pg treatment contained numerous germinating spherical hyphal bodies (29.2 + 1.1 pm diam); however, the most striking structures were the swollen hyphal tips (Fig. 6). The swollen hyphal tips were present at varying levels in all of the JH treatments, with the terminal swelling being 24.3 + 0.8 pm diam and having a vacuole (18.3 + 0.6Fm diam). The hyphae possessing the swellings were 18.2 t 0.7 pm in diameter and highly interwoven and penetrated into the center of the mycelial ball. The final p H was 6.9. The 4.0- and 6.0~pg treatment results were similar and resembled those of the 2-pg treatment except for the lack of spherical hyphal bodies, which were replaced by abundant rod-shaped hyphal bodies (Fig. 7, arrows). The final pH was 7.2 and 7.1, respectively. The 20-pg treatment produced primarily spherical hyphal bodies which developed differently depending upon their position in the hyphal ball. Those in the core failed to develop or only yielded poor germ-tube development. Those at or near the periphery developed long germ tubes. Occasionally a swelling developed at the end of the germ tube, but there was no evidence that this was a conidial initial. The firralpH was 6.9. The 40- and 60-pg levels produced only rod-shaped hyphal bodies. The final pH was 7.2 and 7.1, respectively. The 2OOqg treatment produced most notably spherical hyphal bodies with multiple germ tubes (Fig. 8). A spherical cell type (38.9 pm diam) with several oil drop-
AND
DUNPHY
lets in a granular cytoplasm was detected (Fig. 9). These cells were similar to those produced by the TA 20-pg treatment (Fig. 3). The final pH was 7.1. The 400~pg treatment produced primarily rod-shaped hyphal bodies and only a few spherical hyphal bodies and long hyphae as in all JH treatments. The firralpH was 7.1. The 6OOqg treatment produced a low incidence of germinating spherical hyphal bodies (27.19pm diam, SE = 1.2pm). Both rod-shaped hyphal bodies and irregularly shaped hyphal bodies (Fig. 10) were present. The most striking feature in these results was the presence of spherical hyphal bodies adhering to one another (Fig. 10, arrows). The linal pH was 7.0. Juvenile Hormone plus Trisporic Acid The 4O+g levels produced irregularly shaped hyphal bodies (Fig. 11, arrows) among normal hyphae. Germinating spherical hyphal bodies were also present and many thick-walled structures showing shape variation between spherical hyphal bodies and rod-shaped hyphal bodies. The final pH was 6.9. The 60-pg levels produced structures which were either dividing or fusing spherical hyphal bodies (Fig. 12), a few rodshaped hyphal bodies, and aberrant conidia (72-90 pm long, 41.31 t 1.73 pm at widest point, and 17.01 + 0.97 pm diam at the columella). There was a tendency toward wall thickening in some of the spherical hyphal bodies. The final pH was 7.1. DISCUSSION
In comparing the various experimental hormone treatment results with those of the control, only one produced all of the control cell types (JH and TA, 40 pg/50 ml). Under both conditions, normal hyphae (without swollen tips), spherical hyphal bodies, irregularly shaped hyphal bodies, and thick-walled spheres were present. The JH plus TA (4Opg/50 ml) treatment differed from the control in that normal rod-shaped hyphal bodies were also present and the thick-walled structures tended to be vari-
HORMONES,
EFFECTS
ON FUNGAL
able in shape. Thick-walled spheres were produced in the control; however, these were in the center of the mycelial ball and were lysing. The treatments which showed promise by the production of comparable, but nonlysing, thick-walled spheres were the TA 2O+g, JH 200~pg, and JH plus TA 40-pg treatments. It is difficult to speculate on the nature of the bilobed structure found in the TA 40-pg treatment and whether or not minor modifications of this experimental condition might produce more normal resting spores such as those investigated by Nolan et al. (1976), where similar but thicker-walled bilobed structures were encountered in conjunction with normal, germinating resting spores. All juvenile hormone treatments produced some degree of hyphal tip swelling, but this does not appear to be a possible forerunner of conidium formation in this instance. The hyphal swelling induced by JH was counteracted by 40 pg TA. The low JH concentrations of 2 and 20 pg appeared to favor spherical hyphal body formation, whereas concentrations of 200, 400, and 600 pg JH produced aberrant or suppressed germination and/or suppressed production of spherical hyphal bodies. Rod-shaped hyphal bodies were produced in a very irregular pattern: but TA at 60 pg both individually and in conjunction with JH favored their production. The aberrant conidia produced in the JH plus TA (60 pg/50 ml) treatment were approximately 2 to 3 times the mean length of normal conidia (28.0 to 49.5 pm,X = 36.5 + 4.8 pm; Otvos et al., 1973). Many of the thick-walled cells produced in the study fell within or near the size range of resting spores ofE. egressu (20.0 to 32.0pm diam, X = 25.0 k 0.8 pm; Otvos et., 1973). One problem with the study was the use of lactophenol for examining the cell types as this precluded the possibility of subsequent testing of the thick-walled spheres for germination to form primary conidia and, thus, positive classification of them as resting spores. It had been assumed that such ger-
247
MORPHOGENESIS
mination would occur in the liquid incubation medium as had been observed by Nolan et al. (1976). It is known that this fungus readily adapts to mass fermentation growth under the appropriate conditions (Nolan, unpubl.); however, the implications from this study are the possible problem of thick-walled-cell lysis within the mycelial ball and the advantageous use of JH and TA jointly for production of normal hyphae, abundant thick-walled cell types, and three forms of hyphal bodies. ACKNOWLEDGMENTS The authors would like to thank Dr. Richard Sutter and Ayerst Laboratories for the hormones. One of us (G.B.D.) held a National Research Council of Canada postgraduate scholarship. This study was supported by Canadian Forestry Service Contract OSU77-00048 and by NRCC Grant A-6665 to R.A.N.
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