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Microscopic observations of germination and septum formation in pycnidiospores of Botryodiplodia theobromae
DEVELOPMENTAL
BIOLOGY
32, 1-14 (1973)
Microscopic
Observations
Formation
of Germination
in Pycnidiospores
and Septum
of Botryodiplodia
theobromae’ WILLIAM P. WERGIN,* LARRY D. DUNKLE, 3 JAMES L. VAN ETTEN, 3 GRANT ST. JULIAN,~ AND LEE A. BULLA, JR.~ Accepted
October 30, 1972
A population of aseptate pycnidiospores of the fungus Bottyodiplodia theobromae can be induced to germinate or to form septa delimiting two cells; this developmental process is dependent upon nutritional and environmental factors. Transmission electron microscope investigations indicate that during germination of the aseptate spore, a new inner wall layer is synthesized de nova at the site of germ tube emergence. Formation of the septum also involves the de nouo synthesis of an inner wall layer which comprises the majority of the septum and completely surrounds the spore. The wall of the germ tube emerging from the septate spore is a direct extension of this inner layer deposited during the formation of the septum. Although the early stages of spore germination may involve localized enzymatic degradation of the internal layers of the spore wall, transmission and scanning electron micrographs of germinating spores show that the outer wall layers are physically fractured by the emerging germ tube. It is suggested that spore germination and septum formation are initially similar processes regarding cell wall genesis but that some mechanism responsive to environmental and nutritional conditions determines the course of development.
ing, and both are pathogenic (Brown, 1968, 1971). The isolate of B. theobromae used in our studies on the synthesis of macromolecules during fungal spore germination typically produces 90-95% aseptate spores in culture. Unlike other isolates of this fungus (Ekundayo and Haskins, 1969), the aseptate spores are not converted to septate spores if they are stored dry at room temperature or at 4°C. However, a population of aseptate spores incubated in water can be converted almost quantitatively to a population of septate spores; both spore types, when incubated in a nutrient medium, will germinate. Thus, as outlined in Fig. lA, it is possible to regulate the developmental processes of the aseptate spore by environmental and nutritional factors. Since the sites of germ tube emergence in the aseptate spore and septum formation usually occur in the equatorial region of the spore, electron microscopic investigations were conducted to
INTRODUCTION
Pycnidiospores of Botryodiplodia theobromae Pat. (syn. Diplodia natalensis Pole Evans) occur as two distinct morphological types: single-celled (aseptate) hyaline spores and two-celled (septate) darkly pigmented spores. Generally, it is considered that the aseptate spores are immature and that they are converted to mature septate spores within the pycnidium or shortly after their release from it (Alasoadura, 1970; Brown, 1968; Ekundayo, 1970; Ekundayo and Haskins, 1969). Both spore types are capable of germinat’ Published as Paper Number 3473, Journal Series, Nebraska Agricultural Experiment Station. Research reported was conducted under Project No. 21-17. 2 Southern Weed Science Laboratory, U.S. Delta States Agricultural Research Center, Agricultural Research Service, Stoneville, Mississippi 38776. 3Department of Plant Pathology, University of Nebraska, Lincoln, Nebraska 68503. 1 Northern Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois 61604. 1 1973 by Academic Press, Copyrieht All rights o9reproduction in any form
Inc. reserved.
2
DEVELOPMENTALBIOLOGY
obtain comparative information on cell wall genesis during the early stages of germination and during septum formation. Changes in the structure of the spore surface and overall anatomy which accompany spore germination were also observed by scanning electron microscopy. MATERIALS
AND
METHODS
Cultural conditions. The isolate and the techniques for producing and harvesting B, theobromae spores have been described previously (Van Etten, 1968). The spores were stored at -80°C for periods up to 6 weeks without affecting the rate or capacI ty for germination or septum formation. The conversion of aseptate spores to septate spores was accomplished by incubating 500 mg of spores (90-95% aseptate) in baffled 500-ml Erlenmeyer flasks containing 100 ml of sterile distilled water. The flasks were incubated at 28°C on a rotary shaker for 20-22 hr. Both spore types were germinated by incubating 100 mg of spores in 100 ml of glucose-yeast extract medium [GYE; 1% (w/v) glucose, 0.2% (w/v) Difco yeast extract] in baffled 500 ml Erlenmeyer flasks. Phase-contrast and scanning electron microscopy. The techniques for observing specimens by phase-contrast and scanning electron microscopy were modified from those described earher (Bulla et al., 1969). For phase-contrast microscopy, mounting slides were prepared by spreading a thin film of 1% Noble agar evenly over the surface of glass microscope slides. A small amount (about 0.05 ml) of a cell suspension was placed on the solidified agar surface and covered with a cover slip. Cells were photographed on Panatomic-X film through Neofluar phase optics of a Zeiss WL microscope. Squares (10 by 10 mm) cut from glass microscope slides were placed on aluminum specimen stubs for scanning electron microscopy. The glass squares were adhered to the stubs with double-coated Scotch tape. About 0.1 ml of a diluted cell suspen-
VOLUME 32, 1973
sion was spread over the mounting surface, dried, and coated with gold-palladium (60/40 alloy) to a thickness of 15 nm. Specimens were examined in a Cambridge stereoscan Mark II scanning electron microscope at an accelerating voltage of 20 kV; the final aperture was 200 pm, and the beam specimen angle was 45”. Transmission electron microscopy. Spores incubated on GYE agar medium were fixed on the agar surface by adding 2 to 3 ml of 3% glutaraldehyde in 0.05 M phosphate buffer, pH 6.8. Sections (2-3 mma) of the agar medium containing the fixed spores were transferred to glass vials and incubated at room temperature for 2 hr in phosphate buffer containing 3% glutaraldehyde. This fixation was followed by washing the spores with six changes of the phosphate buffer over a period of 60 min. Next, the spores were postfixed in 2% osmium tetroxide for 2 hr, dehydrated in an acetone series, and embedded in Spurr’s (1969) medium. Silvergray sections of the embedded cells were cut on a Sorvall MT-2 ultramicrotome with a diamond knife and mounted on uncoated 300 x 75 copper grids. After mounting, the sections were stained with 2% aqueous uranyl acetate for 10 min followed by lead citrate for 5 min. The preparations were viewed and photographed in a Hitachi HU-11C electron microscope at an accelerating voltage of 75 kV; the objective aperture was 30 pm. RESULTS
Germination
and Septum Formation
The rate of septate spore formation by a population of aseptate spores is shown in Fig. 1B. The appearance of the septum was first observed by phase contrast microscopy after 5-7 hr of incubation, and the process was completed by approximately 16 hr. Neither the aseptate nor the septate spores germinated significantly (< 5%) in water at the spore concentration used (5 mg/ml). Following their formation by incubation in water, septate spores incu-
WERGIN et al.
0
6Vt Medium [21.34
Spore Germination
Cl
keptateSpore
I Cl Distilled
Water
128
6VE Medium 128.34 Cl
septate
Spora
FIG. 1. (A) Diagrammatic representation of Botryodiplodia theobromae spore septation and of aseptate and septate spore germination. (B) Time course of septation. (C) Time course of germination of a population of aseptate spores (0) or septate spores (0) incubated at 1 mg of spores/ml of GYE medium at 34°C.
bated in a nutrient medium germinated at a rate comparable to that of aseptate spores (Fig. lc). Furthermore, the spores were capable of germinating after being transferred to GYE medium at any stage of septum formation. For example, if aseptate spores were incubated in water for 10 hr (approximately 50% septate spores) and then transferred to GYE medium, the rate of germination was not reduced. If the spores were incubated in GYE medium for a short period of time (e.g., 1 hr) and then transferred to water, they would complete germination. These observations suggest that the commitment to germination is stronger than that to septum formation. Observations with the Scanning Microscope
Electron
Figure 2 presents a series of scanning electron micrographs with phase-contrast
and Septum Formation
3
photographs inserted and shows the morphological changes in the cell surface of B. theobromae from the ungerminated spore through germ tube formation. An ellipsoid ungerminated spore is shown in Fig. 2A. Prior to germ tube formation, the spores apparently become modified along one side of their longitudinal axis where a slightly collapsed area appears (Fig. 2B and C). The collapse in the spore wall is possibly the result of localized enzymatic activity which weakens the wall at the point of germ tube emergence. Phase-contrast photographs reveal a slight lengthening of the spore but show no such surface irregularity. Figure 2D shows an early stage in the formation of the germ tube; the depressed area of the spore has become highly differentiated. In phase-contrast optics (see insets, Fig. 2D and El, germ tube formation appears as a bulge on the spore longitudinal axis. Emergence of the germ tube is shown in Fig. 2E and F. The tip of the tube appears smooth when contrasted to the slightly irregular surface of the spore. Figure 2G shows a fully developed germ tube with misshapen spore attached. A noteworthy feature of germ tube growth is shown in Fig. 2F. The appearance of the germ tube wall at its tip, the region which remains extensible during germ tube elongation, is distinctly different from the wall behind this apical dome. Ultrastructural Germination
Aspects of Aseptate Spore
Ultrastructural aspects of aseptate spore germination and germ tube development are shown in transmission electron micrographs of transverse sections (Fig. 3). The spore wall consists of two layers (Fig. 3A and B): a thin, electron-dense outer layer and a broad, less electron-dense inner layer (designated primary wall). The outer layer (labeled a in Fig. 3B) is about 25 nm thick and appears as a compact layer of amorphous material that envelopes the en-
4
DEVELOPMENTALBIOLOGY
VOLUME 32, 1973
FIG. 2. Scanning electron micrographs with phase-contrast photographs inserted, of Botryod$odia theobromae spores at various times during germination. (A) Ungerminated pycnidiospore, 0 time; (B) 1 hr; (C) 2 hr; (D-E) 2.5-3.5 hr; (F) 3.5-4 hr; (G) 5 hr (x 2000-3040).
tire spore. Beneath this layer, and frequently dissociated from it by an electrontransparent zone (see arrow, Fig. 3B), is the primary wall. This wall is composed of concentrically aligned fibrils that lie within the transverse plane. In the outer region of
the primary wall the fibrils are compact, whereas in the inner region they are not compressed as tightly. Such structural differences probably account for the areas of variable electron density throughout the primary wall.
WERGINet al.
Spore Germination
and Septum Formation
5
FIG. 2. E and G.
Upon germination the primary wall differentiates into two distinct layers (Fig. 3C-F). The outer region (designated layer
b) becomes a compact layer of amorphous material that is barely distinguishable from layer a, and the inner region (desig-
FIG. 3. Ultrastructural aspects of aseptate spore germination and germ tube development in Botryodiplodia theobromae. (A) Transverse section of ungerminated spore (x 15,000). (B) Enlargement of spore wall showing outer layer (a), primary wall (PW), and an electron transparent zone (arrow) beneath layer a (x 40,000). (C) Transverse section of an early stage of germination (x 12,000). (D) Enlargement of the spore wall and develop6
FIG. 3. C and D. ing germ tube (arrow) layers o, b, and c, and of germ tube showing spore wall: outer layer
(x 40,000). (E and F) Germ tube emergence showing fracture of cell wall components: newly synthesized germ tube wall (d) (x 26,000 and 36,000, respectively). (G) Elongation new germ tube wall (d) and fractured spore wall layers ( x 15,000). (H) Enlargement of (a); primary wall, outer zone (b), and inner zone (cl: new germ tube wall (d) (x 62,000). 7
8
DEVELOPMENTAL BIOLOGY
VOLUME 32, 1973
FIG. 3. E and F.
FIG. 3. G and H. 9
10
DEVELOPMENTAL
BIOLOGY
nated layer c) becomes more heterogeneous than it was in the ungerminated spore. The most striking morphological change is the formation of a germ tube (see arrows, Fig. 3C and D). Although the germ tube almost always emerges from the same region of the spore, the spores of B. theobromae have no germ pore. Therefore, the spore wall must either be digested or physically ruptured during germination. The early stages might consist of at least a partial enzymatic digestion of layer c as suggested in Fig. 3D. As the germ tube emerges, a new wall layer (designated d, Fig. 3E and F) is formed at its tip. This layer is thickest at the extreme tip of the germ tube. The outer dense region (b) is clearly defined from the inner region (c), which has become reticular. Layers a, 6, and c appear frayed and fractured at the point of germ tube emergence (Fig. 3E-H). Only the newly synthesized wall (d) covers the tip of the germ tube. Ultrastructural aspects of septum formation and septate spore germination Figures 4A-C are representative micrographs of septum formation. The septum forms progressively with the production of wall material from the spore periphery to its center (arrows, Fig. 4A). Septation is complete when the spore is partitioned into two cells (Fig. 4B). The lateral walls of the septum seem to be continuous with an inner zone of the spore wall (layer e in Fig. 4C) that is deposited during septation. Subsequently, the septum thickens and a band of electron dense material, similar to that comprising the wall layer b, accumulates and surrounds the spore in the spetal region (denoted by the arrow in Fig. 4C). The final stages of septum formation consist of the deposition of a secondary wall (designated fin Fig. 4B) in the middle of the septum. Thus, the wall of the septate spore is composed of an outer layer (a), a primary wall with an outer electron-opaque re-
VOLUME
32. 1973
gion (b) and an electron-translucent region (c), and a newly synthesized layer (e) that is continuous with the septum. This new wall material, layer e, surrounds the emerging germ tube (Fig. 4D) of the septate spore. No additional wall material is laid down at the tip of the germ tube as is the case for the aseptate spore. Emergence of the germ tube disrupts the outer layer (a) and the two layers (b and c) of the primary wall (Fig. 4E). DISCUSSION
Bartnicki-Garcia (1968) has outlined three mechanisms of wall formation during fungal spore germination. The first, designated type I, involves formation of the germ tube wall by direct extension of the spore wall or one of its innermost layers. Type I is more common to the higher fungi and probably does not involve a change in the composition of the cell wall. Type II consists of the de novo synthesis of cell wall on protoplasts and is characteristic of zoospore encystment in aquatic Phycomycetes. Type III is de novo formation of vegetative (or germ tube) wall under the spore wall. Characteristic of lower fungi, particularly Mucorales, type III may involve changes in wall composition. However, recent studies on spore germination of Botrytis cinerea by Gull and Trinci (1971) indicate that the type III pattern may not be restricted to the Mucorales. Pycnidiospores of B. theobromae seem to occupy a unique position with regard to Bartnicki-Garcia’s (1968) categories. Aseptate spores apparently germinate by a type III mechanism, whereas septate spores germinate by a type I mechanism. New germ tube wall material is synthesized beneath the aseptate spore wall during germination. A new inner layer of wall material is synthesized upon septum formation, and it is the extension of this inner layer that constitutes the new germ tube wall of the septate spore. A similar situation has been described for a di-
WERGINet al.
Spore Germination
and Septum Formation
FIG. 4. Ultrastructural aspects of septum formation and subsequent germination of a septate spore. (A) Beginning septum formation (arrows denote septum formation) (X 7,000). (B) Fully developed septum (arrow denotes an intermediate layer f) (x 9,000). (C) Enlargement of septum (e) showing its continuation with the inner spore wall: layers a, b, and c are the same as in Fig. 3E (arrow denotes a band of electron dense material which surrounds the spore in the septal region) (x 25,000). (D) Tangential section of a germinated septate spore showing the respective spore wall layers and the newly formed germ tube wall as an extension of the septum wall (e) (x 8000) (the cross wall which appears to separate the germ tube from the spore is a result of the plane of section). (E) Enlargement of the wall region where the germ tube emerges (X 11,500).
FIG. 4. B and C. 12
WERGINet al.
Spore Germination
and Septum Formation
FIG. 4. D and E.
14
DEVELOPMENTALBIOLOGY
morphic organism, Mucor rouxii (BartnickiGarcia et al., 1968); cell wall genesis proceeds by a type III pattern during sporangiospore germination and by a type I pattern during the formation of a hypha from the yeast cell form. Depending upon the environmental and nutritional conditions, aseptate spores of B. theobromae are capable of forming either a germ tube or a septum. Both processes occur in the equatorial region of the aseptate spore; however, septum formation is initiated at the periphery of the equatorial plane, whereas germ tube formation is localized at a spherical segment of the spore. Therefore, septum formation in B. theobromae may merely be an alternative expression of the earliest events initiating germination of aseptate spores. We thank Gordon Adams and Frederick L. Baker of the Northern Laboratory for their able technical assistance. Mention of firm names or trade products does not imply that they are endorsed or recommended by the Department of Agriculture over other firms or similar products not mentioned. This investigation was supported in part by Public Health Service grant A108057 from the National Institute of Allergy and Infectious Diseases. REFERENCES ALASOADURA, S. 0. (1970). Culture
studies
on Bo-
VOLUME 32, 1973
tryodiplodia theobromae Pat. Mycopathol. Mycol. Appl. 42, 153-160. BARTNICKI-GARCIA, S. (1968). Cell wall chemistry, morphogenesis, and taxonomy of fungi. Annu. Reu. Microbial. 22, 87-108. BARTNICKI~ARCIA, S., NELSON, N., and COTA-ROBLES, E. (1968). Electron microscopy of spore germination and cell wall formation in Mucor rouxii. Arch. Mikrobiol. 63, 242-255. BROWN, G. E. (1968). Germination of immature and mature spores of Diplodiu natalensis. Phytopathology 58, 1044 (Abstract). BROWN, G. E. (1971). Pycnidial release and survival of Diplodia natalensis spores. Phytopathology 61, 559-561. BULLA, L. A., ST. JULIAN, G., RHODES, R. A., and HESSELTINE, C. W. (1969). Scanning electron and phase-contrast microscopy of bacterial spores. Appl. Microbial. 18, 490-495. EKUNDAYO, J. A. (1970). Pycnidium production by Botryodiploidia theobromae. III. Germination of the pycnidiospores. Can. J. Bat. 48, 67-70. EKUNDAYO, J. A., and HASKINS, R. H. (1969). Pycnidium production by Botryodiploida theobromae. II. Development of the pycnidium and fine structure of the maturing pycnospore. Can. J. Bot. 47, 1423-1424. GULL, K., and TRINCI, A. P. J. (1971). Fine structure of spore germination in Botrytis cinerea. J. Gen. Microbial. 68, 207-220. SPURR, A. R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-43. VAN ETTEN, J. L. (1968). Protein synthesis during fungal spore germination. I. Characteristics of an in vitro phenylalanine incorporating system prepared from germinated spores of Botryodiplodia theobromae. Arch. Biochem. Biophys. 125, 13-21.
BIOLOGY
32, 1-14 (1973)
Microscopic
Observations
Formation
of Germination
in Pycnidiospores
and Septum
of Botryodiplodia
theobromae’ WILLIAM P. WERGIN,* LARRY D. DUNKLE, 3 JAMES L. VAN ETTEN, 3 GRANT ST. JULIAN,~ AND LEE A. BULLA, JR.~ Accepted
October 30, 1972
A population of aseptate pycnidiospores of the fungus Bottyodiplodia theobromae can be induced to germinate or to form septa delimiting two cells; this developmental process is dependent upon nutritional and environmental factors. Transmission electron microscope investigations indicate that during germination of the aseptate spore, a new inner wall layer is synthesized de nova at the site of germ tube emergence. Formation of the septum also involves the de nouo synthesis of an inner wall layer which comprises the majority of the septum and completely surrounds the spore. The wall of the germ tube emerging from the septate spore is a direct extension of this inner layer deposited during the formation of the septum. Although the early stages of spore germination may involve localized enzymatic degradation of the internal layers of the spore wall, transmission and scanning electron micrographs of germinating spores show that the outer wall layers are physically fractured by the emerging germ tube. It is suggested that spore germination and septum formation are initially similar processes regarding cell wall genesis but that some mechanism responsive to environmental and nutritional conditions determines the course of development.
ing, and both are pathogenic (Brown, 1968, 1971). The isolate of B. theobromae used in our studies on the synthesis of macromolecules during fungal spore germination typically produces 90-95% aseptate spores in culture. Unlike other isolates of this fungus (Ekundayo and Haskins, 1969), the aseptate spores are not converted to septate spores if they are stored dry at room temperature or at 4°C. However, a population of aseptate spores incubated in water can be converted almost quantitatively to a population of septate spores; both spore types, when incubated in a nutrient medium, will germinate. Thus, as outlined in Fig. lA, it is possible to regulate the developmental processes of the aseptate spore by environmental and nutritional factors. Since the sites of germ tube emergence in the aseptate spore and septum formation usually occur in the equatorial region of the spore, electron microscopic investigations were conducted to
INTRODUCTION
Pycnidiospores of Botryodiplodia theobromae Pat. (syn. Diplodia natalensis Pole Evans) occur as two distinct morphological types: single-celled (aseptate) hyaline spores and two-celled (septate) darkly pigmented spores. Generally, it is considered that the aseptate spores are immature and that they are converted to mature septate spores within the pycnidium or shortly after their release from it (Alasoadura, 1970; Brown, 1968; Ekundayo, 1970; Ekundayo and Haskins, 1969). Both spore types are capable of germinat’ Published as Paper Number 3473, Journal Series, Nebraska Agricultural Experiment Station. Research reported was conducted under Project No. 21-17. 2 Southern Weed Science Laboratory, U.S. Delta States Agricultural Research Center, Agricultural Research Service, Stoneville, Mississippi 38776. 3Department of Plant Pathology, University of Nebraska, Lincoln, Nebraska 68503. 1 Northern Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois 61604. 1 1973 by Academic Press, Copyrieht All rights o9reproduction in any form
Inc. reserved.
2
DEVELOPMENTALBIOLOGY
obtain comparative information on cell wall genesis during the early stages of germination and during septum formation. Changes in the structure of the spore surface and overall anatomy which accompany spore germination were also observed by scanning electron microscopy. MATERIALS
AND
METHODS
Cultural conditions. The isolate and the techniques for producing and harvesting B, theobromae spores have been described previously (Van Etten, 1968). The spores were stored at -80°C for periods up to 6 weeks without affecting the rate or capacI ty for germination or septum formation. The conversion of aseptate spores to septate spores was accomplished by incubating 500 mg of spores (90-95% aseptate) in baffled 500-ml Erlenmeyer flasks containing 100 ml of sterile distilled water. The flasks were incubated at 28°C on a rotary shaker for 20-22 hr. Both spore types were germinated by incubating 100 mg of spores in 100 ml of glucose-yeast extract medium [GYE; 1% (w/v) glucose, 0.2% (w/v) Difco yeast extract] in baffled 500 ml Erlenmeyer flasks. Phase-contrast and scanning electron microscopy. The techniques for observing specimens by phase-contrast and scanning electron microscopy were modified from those described earher (Bulla et al., 1969). For phase-contrast microscopy, mounting slides were prepared by spreading a thin film of 1% Noble agar evenly over the surface of glass microscope slides. A small amount (about 0.05 ml) of a cell suspension was placed on the solidified agar surface and covered with a cover slip. Cells were photographed on Panatomic-X film through Neofluar phase optics of a Zeiss WL microscope. Squares (10 by 10 mm) cut from glass microscope slides were placed on aluminum specimen stubs for scanning electron microscopy. The glass squares were adhered to the stubs with double-coated Scotch tape. About 0.1 ml of a diluted cell suspen-
VOLUME 32, 1973
sion was spread over the mounting surface, dried, and coated with gold-palladium (60/40 alloy) to a thickness of 15 nm. Specimens were examined in a Cambridge stereoscan Mark II scanning electron microscope at an accelerating voltage of 20 kV; the final aperture was 200 pm, and the beam specimen angle was 45”. Transmission electron microscopy. Spores incubated on GYE agar medium were fixed on the agar surface by adding 2 to 3 ml of 3% glutaraldehyde in 0.05 M phosphate buffer, pH 6.8. Sections (2-3 mma) of the agar medium containing the fixed spores were transferred to glass vials and incubated at room temperature for 2 hr in phosphate buffer containing 3% glutaraldehyde. This fixation was followed by washing the spores with six changes of the phosphate buffer over a period of 60 min. Next, the spores were postfixed in 2% osmium tetroxide for 2 hr, dehydrated in an acetone series, and embedded in Spurr’s (1969) medium. Silvergray sections of the embedded cells were cut on a Sorvall MT-2 ultramicrotome with a diamond knife and mounted on uncoated 300 x 75 copper grids. After mounting, the sections were stained with 2% aqueous uranyl acetate for 10 min followed by lead citrate for 5 min. The preparations were viewed and photographed in a Hitachi HU-11C electron microscope at an accelerating voltage of 75 kV; the objective aperture was 30 pm. RESULTS
Germination
and Septum Formation
The rate of septate spore formation by a population of aseptate spores is shown in Fig. 1B. The appearance of the septum was first observed by phase contrast microscopy after 5-7 hr of incubation, and the process was completed by approximately 16 hr. Neither the aseptate nor the septate spores germinated significantly (< 5%) in water at the spore concentration used (5 mg/ml). Following their formation by incubation in water, septate spores incu-
WERGIN et al.
0
6Vt Medium [21.34
Spore Germination
Cl
keptateSpore
I Cl Distilled
Water
128
6VE Medium 128.34 Cl
septate
Spora
FIG. 1. (A) Diagrammatic representation of Botryodiplodia theobromae spore septation and of aseptate and septate spore germination. (B) Time course of septation. (C) Time course of germination of a population of aseptate spores (0) or septate spores (0) incubated at 1 mg of spores/ml of GYE medium at 34°C.
bated in a nutrient medium germinated at a rate comparable to that of aseptate spores (Fig. lc). Furthermore, the spores were capable of germinating after being transferred to GYE medium at any stage of septum formation. For example, if aseptate spores were incubated in water for 10 hr (approximately 50% septate spores) and then transferred to GYE medium, the rate of germination was not reduced. If the spores were incubated in GYE medium for a short period of time (e.g., 1 hr) and then transferred to water, they would complete germination. These observations suggest that the commitment to germination is stronger than that to septum formation. Observations with the Scanning Microscope
Electron
Figure 2 presents a series of scanning electron micrographs with phase-contrast
and Septum Formation
3
photographs inserted and shows the morphological changes in the cell surface of B. theobromae from the ungerminated spore through germ tube formation. An ellipsoid ungerminated spore is shown in Fig. 2A. Prior to germ tube formation, the spores apparently become modified along one side of their longitudinal axis where a slightly collapsed area appears (Fig. 2B and C). The collapse in the spore wall is possibly the result of localized enzymatic activity which weakens the wall at the point of germ tube emergence. Phase-contrast photographs reveal a slight lengthening of the spore but show no such surface irregularity. Figure 2D shows an early stage in the formation of the germ tube; the depressed area of the spore has become highly differentiated. In phase-contrast optics (see insets, Fig. 2D and El, germ tube formation appears as a bulge on the spore longitudinal axis. Emergence of the germ tube is shown in Fig. 2E and F. The tip of the tube appears smooth when contrasted to the slightly irregular surface of the spore. Figure 2G shows a fully developed germ tube with misshapen spore attached. A noteworthy feature of germ tube growth is shown in Fig. 2F. The appearance of the germ tube wall at its tip, the region which remains extensible during germ tube elongation, is distinctly different from the wall behind this apical dome. Ultrastructural Germination
Aspects of Aseptate Spore
Ultrastructural aspects of aseptate spore germination and germ tube development are shown in transmission electron micrographs of transverse sections (Fig. 3). The spore wall consists of two layers (Fig. 3A and B): a thin, electron-dense outer layer and a broad, less electron-dense inner layer (designated primary wall). The outer layer (labeled a in Fig. 3B) is about 25 nm thick and appears as a compact layer of amorphous material that envelopes the en-
4
DEVELOPMENTALBIOLOGY
VOLUME 32, 1973
FIG. 2. Scanning electron micrographs with phase-contrast photographs inserted, of Botryod$odia theobromae spores at various times during germination. (A) Ungerminated pycnidiospore, 0 time; (B) 1 hr; (C) 2 hr; (D-E) 2.5-3.5 hr; (F) 3.5-4 hr; (G) 5 hr (x 2000-3040).
tire spore. Beneath this layer, and frequently dissociated from it by an electrontransparent zone (see arrow, Fig. 3B), is the primary wall. This wall is composed of concentrically aligned fibrils that lie within the transverse plane. In the outer region of
the primary wall the fibrils are compact, whereas in the inner region they are not compressed as tightly. Such structural differences probably account for the areas of variable electron density throughout the primary wall.
WERGINet al.
Spore Germination
and Septum Formation
5
FIG. 2. E and G.
Upon germination the primary wall differentiates into two distinct layers (Fig. 3C-F). The outer region (designated layer
b) becomes a compact layer of amorphous material that is barely distinguishable from layer a, and the inner region (desig-
FIG. 3. Ultrastructural aspects of aseptate spore germination and germ tube development in Botryodiplodia theobromae. (A) Transverse section of ungerminated spore (x 15,000). (B) Enlargement of spore wall showing outer layer (a), primary wall (PW), and an electron transparent zone (arrow) beneath layer a (x 40,000). (C) Transverse section of an early stage of germination (x 12,000). (D) Enlargement of the spore wall and develop6
FIG. 3. C and D. ing germ tube (arrow) layers o, b, and c, and of germ tube showing spore wall: outer layer
(x 40,000). (E and F) Germ tube emergence showing fracture of cell wall components: newly synthesized germ tube wall (d) (x 26,000 and 36,000, respectively). (G) Elongation new germ tube wall (d) and fractured spore wall layers ( x 15,000). (H) Enlargement of (a); primary wall, outer zone (b), and inner zone (cl: new germ tube wall (d) (x 62,000). 7
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DEVELOPMENTAL BIOLOGY
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FIG. 3. E and F.
FIG. 3. G and H. 9
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DEVELOPMENTAL
BIOLOGY
nated layer c) becomes more heterogeneous than it was in the ungerminated spore. The most striking morphological change is the formation of a germ tube (see arrows, Fig. 3C and D). Although the germ tube almost always emerges from the same region of the spore, the spores of B. theobromae have no germ pore. Therefore, the spore wall must either be digested or physically ruptured during germination. The early stages might consist of at least a partial enzymatic digestion of layer c as suggested in Fig. 3D. As the germ tube emerges, a new wall layer (designated d, Fig. 3E and F) is formed at its tip. This layer is thickest at the extreme tip of the germ tube. The outer dense region (b) is clearly defined from the inner region (c), which has become reticular. Layers a, 6, and c appear frayed and fractured at the point of germ tube emergence (Fig. 3E-H). Only the newly synthesized wall (d) covers the tip of the germ tube. Ultrastructural aspects of septum formation and septate spore germination Figures 4A-C are representative micrographs of septum formation. The septum forms progressively with the production of wall material from the spore periphery to its center (arrows, Fig. 4A). Septation is complete when the spore is partitioned into two cells (Fig. 4B). The lateral walls of the septum seem to be continuous with an inner zone of the spore wall (layer e in Fig. 4C) that is deposited during septation. Subsequently, the septum thickens and a band of electron dense material, similar to that comprising the wall layer b, accumulates and surrounds the spore in the spetal region (denoted by the arrow in Fig. 4C). The final stages of septum formation consist of the deposition of a secondary wall (designated fin Fig. 4B) in the middle of the septum. Thus, the wall of the septate spore is composed of an outer layer (a), a primary wall with an outer electron-opaque re-
VOLUME
32. 1973
gion (b) and an electron-translucent region (c), and a newly synthesized layer (e) that is continuous with the septum. This new wall material, layer e, surrounds the emerging germ tube (Fig. 4D) of the septate spore. No additional wall material is laid down at the tip of the germ tube as is the case for the aseptate spore. Emergence of the germ tube disrupts the outer layer (a) and the two layers (b and c) of the primary wall (Fig. 4E). DISCUSSION
Bartnicki-Garcia (1968) has outlined three mechanisms of wall formation during fungal spore germination. The first, designated type I, involves formation of the germ tube wall by direct extension of the spore wall or one of its innermost layers. Type I is more common to the higher fungi and probably does not involve a change in the composition of the cell wall. Type II consists of the de novo synthesis of cell wall on protoplasts and is characteristic of zoospore encystment in aquatic Phycomycetes. Type III is de novo formation of vegetative (or germ tube) wall under the spore wall. Characteristic of lower fungi, particularly Mucorales, type III may involve changes in wall composition. However, recent studies on spore germination of Botrytis cinerea by Gull and Trinci (1971) indicate that the type III pattern may not be restricted to the Mucorales. Pycnidiospores of B. theobromae seem to occupy a unique position with regard to Bartnicki-Garcia’s (1968) categories. Aseptate spores apparently germinate by a type III mechanism, whereas septate spores germinate by a type I mechanism. New germ tube wall material is synthesized beneath the aseptate spore wall during germination. A new inner layer of wall material is synthesized upon septum formation, and it is the extension of this inner layer that constitutes the new germ tube wall of the septate spore. A similar situation has been described for a di-
WERGINet al.
Spore Germination
and Septum Formation
FIG. 4. Ultrastructural aspects of septum formation and subsequent germination of a septate spore. (A) Beginning septum formation (arrows denote septum formation) (X 7,000). (B) Fully developed septum (arrow denotes an intermediate layer f) (x 9,000). (C) Enlargement of septum (e) showing its continuation with the inner spore wall: layers a, b, and c are the same as in Fig. 3E (arrow denotes a band of electron dense material which surrounds the spore in the septal region) (x 25,000). (D) Tangential section of a germinated septate spore showing the respective spore wall layers and the newly formed germ tube wall as an extension of the septum wall (e) (x 8000) (the cross wall which appears to separate the germ tube from the spore is a result of the plane of section). (E) Enlargement of the wall region where the germ tube emerges (X 11,500).
FIG. 4. B and C. 12
WERGINet al.
Spore Germination
and Septum Formation
FIG. 4. D and E.
14
DEVELOPMENTALBIOLOGY
morphic organism, Mucor rouxii (BartnickiGarcia et al., 1968); cell wall genesis proceeds by a type III pattern during sporangiospore germination and by a type I pattern during the formation of a hypha from the yeast cell form. Depending upon the environmental and nutritional conditions, aseptate spores of B. theobromae are capable of forming either a germ tube or a septum. Both processes occur in the equatorial region of the aseptate spore; however, septum formation is initiated at the periphery of the equatorial plane, whereas germ tube formation is localized at a spherical segment of the spore. Therefore, septum formation in B. theobromae may merely be an alternative expression of the earliest events initiating germination of aseptate spores. We thank Gordon Adams and Frederick L. Baker of the Northern Laboratory for their able technical assistance. Mention of firm names or trade products does not imply that they are endorsed or recommended by the Department of Agriculture over other firms or similar products not mentioned. This investigation was supported in part by Public Health Service grant A108057 from the National Institute of Allergy and Infectious Diseases. REFERENCES ALASOADURA, S. 0. (1970). Culture
studies
on Bo-
VOLUME 32, 1973
tryodiplodia theobromae Pat. Mycopathol. Mycol. Appl. 42, 153-160. BARTNICKI-GARCIA, S. (1968). Cell wall chemistry, morphogenesis, and taxonomy of fungi. Annu. Reu. Microbial. 22, 87-108. BARTNICKI~ARCIA, S., NELSON, N., and COTA-ROBLES, E. (1968). Electron microscopy of spore germination and cell wall formation in Mucor rouxii. Arch. Mikrobiol. 63, 242-255. BROWN, G. E. (1968). Germination of immature and mature spores of Diplodiu natalensis. Phytopathology 58, 1044 (Abstract). BROWN, G. E. (1971). Pycnidial release and survival of Diplodia natalensis spores. Phytopathology 61, 559-561. BULLA, L. A., ST. JULIAN, G., RHODES, R. A., and HESSELTINE, C. W. (1969). Scanning electron and phase-contrast microscopy of bacterial spores. Appl. Microbial. 18, 490-495. EKUNDAYO, J. A. (1970). Pycnidium production by Botryodiploidia theobromae. III. Germination of the pycnidiospores. Can. J. Bat. 48, 67-70. EKUNDAYO, J. A., and HASKINS, R. H. (1969). Pycnidium production by Botryodiploida theobromae. II. Development of the pycnidium and fine structure of the maturing pycnospore. Can. J. Bot. 47, 1423-1424. GULL, K., and TRINCI, A. P. J. (1971). Fine structure of spore germination in Botrytis cinerea. J. Gen. Microbial. 68, 207-220. SPURR, A. R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-43. VAN ETTEN, J. L. (1968). Protein synthesis during fungal spore germination. I. Characteristics of an in vitro phenylalanine incorporating system prepared from germinated spores of Botryodiplodia theobromae. Arch. Biochem. Biophys. 125, 13-21.