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Effect of temperature on germination, viability and fine structure of Conidia of Bremia lactucae
[ 509 ] Trans. Br. mycol. Soc. 63 (3), 50g-5 18 (1974) Printed in Great Britain
EFFECT OF TEMPERATURE ON GERMINATION, VIABILITY AND FINE STRUCTURE OF CONIDIA OF BREMIA LACTUCAE By J. A. SARGENT
AND
H. L. PAYNE
Agricultural Research Council Unit of Developmental Botany, 181 A Huntingdon Road, Cambridge (With Plates 78 to 84.) Conidia of Bremia lactucae Regel germinated readily in water over the temperature range 0-20 °C. As the temperature was raised germination was progressively retarded and the capacity of spores to germinate on cooling declined. Early changes in the fine structure of conidia during germination at 15° included the proliferation of endoplasmic reticulum and the stimulation of dictyosomes to produce cytoplasmic vesicles which accumulated at the distal papilla from which the germ tube emerged. Concomitantly, peripheral lipid droplets moved inwards and their affinity for electron stain fell. At 28° conidia remained quiescent for at least 24 h. The only major change in their fine structure was the inward movement of the lipid droplets. However, the lipid continued to stain intensely. Most conidia maintained at 28° eventually became highly vacuolate and their membranes were disrupted. It is proposed that high temperatures prevent the conversion of lipid reserves to intermediates necessary for the supply of energy and the maintenance and synthesis of membrane systems required for germination.
Bremia lactucae is the cause of the downy mildew disease oflettuce. Under conditions of high relative humidity infected leaves produce abundant conidia which are readily dispersed by wind or splash droplets. These spores germinate readily provided that they are maintained in free water, and they will infect an available host within 3 h (Sargent, Tommerup & Ingram, 1973). Since conidia are the propagules by which the fungus normally spreads, a knowledge ofthe factors which influence their viability and germination and the changes which occur in their fine structure during this process is essential to an understanding of the biology of the disease. Temperature has long been recognized as an important factor controlling the germination of fungal spores. Among the earlier reports on its effect on the germination of conidia of B. lactucae, those by Powlesland (1954) and Verhoeff (1960) were the most detailed. Nevertheless their findings differed. Powlesland observed that conidia germinated readily in water over a 24 h period throughout the temperature range I to 10°. Germination decreased above 20° for young conidia and above 15° for older spores. Few conidia, whether young or old, germinated above 25°. Verhoeff, however, reported germination over the range - 3° to 31°. Maximal numbers germinated between 4° and 10° for conidia produced on host plants grown at 20 to 22° and between 2° and
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8° for those produced at 10° to IS°. (Neither of these workers reported the production of zoospores even at the lower temperatures and we have never observed their formation.) The earlier workers did not investigate the effects of temperature on germination beyond 24 h and no attempt was made to establish whether the low percentage of germination recorded at the high temperatures resulted from reversible suppression of germination or from death of the conidia. In an attempt to clarify this issue we first determined the sequence of changes which occur in the fine structure of the conidium of B. lactucae during germination and then reexamined the effect of a wide range of temperatures on germination. Particular attention was given to the effect of prolonged incubation at high temperatures on the potential viability and the fine structure of the conidium. MATERIALS AND METHODS
Conidia production andpreparation B. lactucae, race W5, was maintained as previously described (Sargent et al. 1973) on detached cotyledon pairs of the lettuce cultivar 'Trocadero Improved'. After inoculation with an aqueous suspension of B. lactucae conidia, the cotyledons were incubated in a saturated atmosphere at IS° in a Gallenkamp incubator (IH280) illuminated internally for 12 h each day by six 8 W daylight fluorescent tubes. The fungus began sporulating within 6 days and conidia were harvested on the 7th or 8th day. This was achieved by swirling the cotyledons in a flask with distilled water, decanting the water in which the conidia had become suspended and pelleting the spores by centrifugation at 1000 g for 2 min. To remove a water-soluble inhibitor of germination it was necessary to wash and pellet the spores 3 times over a 10 min period (Mason, 1973). No attempt was made to remove contaminating bacteria but cotyledons were discarded if, after incubation, they showed signs of rotting.
Preparation
ofconidia for electron microscopy
Conidia, whether freshly harvested or after incubation in water, were pelleted and fixed by resuspending them for 2 h in 6 % glutaraldehyde buffered to pH 7'1 with 0'1 M sodium cacodylate. They were then pelleted and resuspended in buffer four times over a period of 2 h before treatment for 2 h with a buffered solution of 2 % osmium tetroxide. After rinsing with water they were pelleted in a tapered plastic TAAB embedding capsule to which was subsequently added a few drops of warm I %water agar. The capsule was immediately centrifuged which sedimented the fixed conidia to the lower surface of the agar before it gelled. The capsule was chilled, cut open and the agar containing the spores cut into a number of small blocks. Each block was dehydrated through an ethanol series and finally embedded in Spurr's resin which was polymerized at 100° for I'5 h. Thin sections were cut, collected on uncoated grids and stained sequentially in a saturated solution of uranyl acetate in 50 % ethanol for 7 min and Reynold's lead citrate solution for 4 min.
Bremia lactucae conidia.], A. Sargent and H. L. Payne 511 Germination of conidia During preliminary attempts to devise a rapid assay of the potential viability offreshly collected conidia it was observed that the proportion of washed conidia which germinated duringi ncubation between 5° and 15° corresponded closely with the proportion which did not rapidly absorb acid fuchsin from dilute solution. Thus the viability of preparations of fresh conidia was routinely estimated by stirring a drop of 0'5 % acid fuchsin in I % acetic acid into a few drops of the spore suspension and determining immediately the percentage of conidia which remained unstained (normally 90-95 %). Determinations of actual percentage germination were made 24 h after plating a suspension of conidia (approx. 4 x 106 per ml) into welled glass slides and incubating them in darkness at a high relative humidity. Duplicate slides were prepared and approximately 1000 conidia on each slide were scored for germination. No difficulty was experienced in assessing whether germination had occurred after 24 h since even at 0° a germ-tube was clearly visible, its length exceeding that of the conidium. RESULTS
Quiescent conidium Structural details of the freshly harvested, ungerminated conidium are shown in PI. 78, 79, figs. 1-4. The ovoid spore measured on average 18 x 16 flm and retained at the proximal end part of the fractured sterigma. At the distal end was a papilla from which a single germ tube would arise during germination (PI. 78, fig. I). The wall of the conidium was approximately 0'24 flm thick except at the papilla where it narrowed to o- I 7 flm (PI. 78, fig. 2) and was uniformly fibrillar. However, at the proximal end of the spore were fragments of a cuticle-like layer overlying the wall and separated from it by a lightly staining layer approx. 0'05 flm thick (PI. 79, fig. 3). Sections through the conidium revealed profiles of nuclei, many of them with prominent nucleoli (PI. 78, fig. I; PI. 79, fig. 3). Mitochondria were abundant and more isodiametric than elongate (PI. 78, figs. I, 2; PI. 79, fig. 3). The dictyosomes were compact but the cisternae showed peripheral dilations (PI. 79, figs. 3, 4). Profiles of endoplasmic reticulum were sparse, relatively smooth, and apart from those associated with dictyosomes, short (PI. 79, figs. 3, 4). Densely packed ribosomes, few of which appeared associated into polyribosomes, gave a granular appearance to the ground plasm (PI. 78, fig. 2; PI. 79, figs. 3, 4). In addition to those organelles the conidium contained numerous vacuoles, many of which enclosed a strongly osmiophilic droplet (PI. 78, figs. 1,2; PI. 79, figs. 3,4), and darkly staining lipid droplets which were arranged predominantly around the periphery of the spore (PI. 78, fig. I; PI. 79, fig. 3). There was no cytoplasmic differentiation in the region of the papilla (PI. 78, fig. 2).
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Transactions British Mycological Society Germinating conidium
Sections through conidia 1'5 h after incubation at 15° are shown (PI. 80, figs. 5-7; PI. 8 I, fig. 8). The stage of germination represented by Fig. 8 is slightly in advance of that shown in Figs. 6 and 7. As might be expected the most conspicuous structural changes associated with germination occurred in the region of the papilla. By 1'5 h the papilla was densely packed with cytoplasmic vesicles which had displaced the larger organelles shown in PI. 78, fig. 2. Concomitant with this reorganization was a general orientation of organelles parallel to the long axis of the conidium: nuclei and mitochondria had elongated in the direction of the papilla (PI. 80, fig. 6; PI. 81, fig. 8) and microtubules were directed similarly (PI. 81, fig. 8). Dictyosomes had enlarged and, apparently, become activated. Many cytoplasmic vesicles, similar in appearance to those which aggregated at the papilla, surrounded the dictyosomes and seemed to have arisen from both the cisternae and the associated endoplasmic reticulum (PI. 80, fig. 5). Germination also involved a dramatic increase in the number and length of endoplasmic reticulum profiles each of which was characteristically lined by ribosomes (PI. 80, fig. 7; PI. 8 I, fig. 8). Those ribosomes which were not associated with membrane had, to a large extent, formed polysomal aggregates. The lipid droplets which in the quiescent spore stained deeply no longer had such an affinity for electron stains and had moved inwards from the cell periphery. Plate 81, fig. 8 illustrates a number of features associated with the emergence of the germ tube. Swelling of the papilla had resulted in rupture of the conidial wall and formation of a clearly distinguishable germ-tube wall. The profile of plasm~lemma was very irregular in this region and lomasomes were conspicuous, Plate 81, fig. 10 shows a section through a germ-tube which had developed within 2 h. It demonstrates both the dense nature of the cytoplasm within the germ-tube and the very rapid development which occurred at 15°. The tip of the germ-tube (PI. 81, fig. 9) was densely packed with vesicles which had differing affinities for the stains but resembled those which aggregated in the enlarging papilla (PI. 80, fig. 7; PI. 81, fig. 8).
EFFECT OF TEMPERATURE ON GERMINATION
To determine the effect of temperature on the germination of conidia, suspensions ofthe spores were maintained at various temperatures between 0° and 28° for 24 h. Table I shows the percentage of conidia which germinated. All the potentially viable spores germinated at the intermediate temperatures and even at 0°, but above 20° the number declined. At 28° no conidia germinated over 24 h but if those spores were subsequently cooled to 15°, germination then occurred. To investigate further the inhibition of germination at high temperatures, a series of experiments was set up to investigate the effects of prolonged exposure to these temperatures on both the germination of conidia and their potential to germinate at a lower temperature. The responses of
Bremia laetucae conidia.J. A. Sargent and H. L. Payne 513 Table
1
% Germination
Temperature (0C) o 7 9 15 21 25 28
90 ' 2 92 '7 9 1' 0 9 2 '3 87'4 27'5 o (8g'6)
The effect of temperature on the germination of washed conidia of B, lactucae over a 24 h period, The figure in brackets indicates the percentage of conidia which germinated on transfer to 15° for a further 24 h period, 9 I '2 % of conidia did not stain with acid fuchsin,
Table 2 Time (h) >
Expt. I, 25° Control = 94'7 Viability = 96'3 Expt. II, 28° Control = 94'2 Viabil ity = 92'9 Expt. III, 31° Control = 92'6 Viab ility = 90'S
24
48
72
26'8 (9 1'9)
76'2 (gl '7)
88'4
0 (87'0)
4,8 (70'8)
10'2 (14'2)
0 (2'2)
0 (0)
96
120
6'0 (5'2)
5'1 (5'5)
Percentage germination of conidia of B, lactucae incubated for varying periods at 25°, 28°, or 31°, The figures in brackets indicate the total percentage of conidia which germinated after the spores had been incubated for a further 24 h at 7°. Control = % germination over 24 h at 7°. Viability = % which failed to absorb acid fuchsin rapidly at the start of the experiment.
conidia held at 25°, 28° and 31° are shown in Table 2. During the first 24 h at 25° only 26·8 % of the spores germinated but continued incubation at this temperature resulted in 76'2 % germination after 48 h and 88'4 % after 72 h. Thus, although germination was retarded at 25° it was not permanently inhibited and, given time, almost all the initially viable spores germinated, Transfer of conidia at any time from 25° to 7 ° resulted in their germination within 24 h. At 28° the pattern of germination was more complex: conidia did not germinate during the first 24 h yet 87 % germinated if subsequently incubated at 7°. Prolonged incubation at 28° allowed some conidia to germinate but, unlike those held at 25°, only about 10 % had germinated after 72 h. Moreover, th e potential viability of these spores fell markedly after 24 h and few retained the capacity to germinate after 72 h. At 31° conidia were not only inhibited from germinating but their viability was reduced over 24 h to about 2 %.
Ultrastructural changes induced at 28° At 28° no conidia produced a germ-tube within 24 h but a swelling was conspicuous in the region of the papilla. The few conidia which
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germinated after 48 h at 28° produced only a narrow germ-tube which did not swell at its tip. Since the incubation of conidia at 2° not only delayed their germination but also resulted in a lowering of their viability it seemed pertinent to determine what changes occurred in their fine structure at that temperature. Electron micrographs of sections of conidia fixed after 3, 24 or 72 h at 28° are shown in Plates 82-84, figs. I 1-17. After 3 h (PI. 82, figs. I I, 12) the structure of the conidium resembled, in most respects, that of the freshly harvested spore (PI. 78, 79, figs. 1-4). Only the location of the lipid droplets differed: they were no longer confined to the periphery of the cell, many having moved inwards. By 24 h (PI. 83, figs. 13, 14) only small additional structural changes were observed in the majority of conidia. They included the continuation of the inward movement of the lipid droplets and the generation of additional small vacuoles. Many of the profiles of these new vacuoles revealed the inclusion of deeply staining droplets characteristic of those found in the vacuoles of fresh conidia. The smallest vacuoles, which usually contained electron dense granular aggregates, were continuous with profiles of double membrane. The dictyosomes were somewhat less compact than at 3 hand appeared to have given rise to some cytoplasmic vesicles. However, they did not resemble those characteristic of the germinating conidium. Little change in the structure of the papilla had occurred except for the loss of lipid droplets (PI. 81, fig. 10). The structure of a small proportion of the conidia fixed after 24 h was markedly different from that already described. Their contents had become disorganized and they were probably the spores which would have failed to germinate on cooling to a lower temperature. Throughout these cells (PI. 84, fig. 15) the integrity of the membranes appeared to have been lost: mitochondria were swollen and their cristae disorganized, the unit membranes of the nuclei had separated and the endoplasmic reticulum and dictyosome cisternae were dilated. Vacuolation had increased. Apparently many of the smaller vacuoles had coalesced and degenerate mitochondria appeared to have contributed to the overall vacuolation of the conidium. Few lipid droplets characteristic of viable spores remained but a number of large, more deeply stained drops were scattered throughout the cell. The condition of these disorganized conidia probably represents a stage through which all the spores pass if incubation at 28° is continued. By 72 h all conidia which had not previously germinated were severely disorganized (PI. 84, figs. 16, 17). Organelles were barely recognizable and membranes, with the exception of the tonoplast, had largely disappeared. Only fragments of the plasmalemma remained, the outer unit membrane of the mitochondrial envelope was occasionally distinguishable but the membranes of the nuclear envelope had completely disappeared. Some sites offormer endoplasmic reticulum profiles could be located by the rows of ribosomes which had lined the membrane, but there were no traces of dictyosomes. Most of the spore was occupied by a vacuole.
Bremia lactucae conidia.]. A. Sargent and H. L. Payne 515 DISCUSSION
These observations demonstrate the extensive and rapid changes which occur within the germinating conidium of B. lactucae even before the emergence of the germ-tube. Within 90 min the conidium is transformed from a state of quiescence to one in which organelles have become activated and orientated in preparation for the growth of the germ-tube. The assembly of ribosomes into polysomes and their association with the expanding endoplasmic reticulum is probably indicative of the activation of a protein synthesizing mechanism which is, without doubt, essential for germination. Additional membranous components arise (endoplasmic reticulum, dictyosome cysternae, cytoplasmic vesicles) and protein is also required for the synthesis of enzymes necessary for the utilization of storage material, alteration of the conidium wall to permit the emergence of the germ-tube, and the synthesis of germ-tube wall material. It seems that the dictyosomes are an important component of the mechanism for the synthesis of enzymes and wall-building components as well as the membranes with which those products are compartmentalized. Cytoplasmic vesicles appear to arise from the dictyosomes and to be directed, possibly under the influence of microtubules, towards the papilla. The contents of the vesicles stain to varying intensities reflecting, possibly, the separation of specific enzymes or wall precursors into distinct vesicles. The lomasomes associated with the ruptured conidium wall in the region of the papilla probably arise from the discharge of vesicles, containing wall-softening enzymes, through the plasmalemma and since the population of vesicles at the papilla closely resembles that found in the appressorium in the vicinity of the penetration peg (Sargent et al. 1973) it is likely that even during the early stages of germination, enzymes which aid the entry of the fungus through a host epidermal wall are being mobilized. Germination in water must rely upon an adequate supply of stored reserves. The lipid droplets originally lining the interior of the conidium are probably major contributors to this supply. Very early during germination they move inwards and the subsequent reduction in their affinity for the electron stains probably indicates alterations in their chemical structure as they become metabolized. The present study also focuses attention on the suppression of germination by high temperatures and the accompanying changes in the fine structure of the conidium. Our data demonstrate that at 25° germination can be delayed but not prevented; most conidia eventually germinate at this temperature. It is puzzling that some spores in a population should germinate readily while others remain quiescent for up to 72 h. Age differences might account in part for this differential response but when the spores were collected none could have been mature for as long as three days. Conidia differ somewhat in size and in this laboratory 1. Tommerup has shown that their complement of nuclei can vary between 9 and 22. Under conditions of stress, such variation in size and quantity of specific components might well have a profound effect on the attainment of levels of certain metabolites necessary for germination. That the majority of conidia held at 28° for 24 h remain in a state of
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quiescence is confirmed by their fine structure. Except for the dispersal of lipid from the periphery of the cytoplasm their structure changes very little over 3 h. At 15° a sporcling would have developed in that time and, if in contact with a host, have penetrated an epidermal cell (Sargent et at. 1973). The retention of a high affinity for electron stains by the lipid droplets at 28° suggests that high temperatures prevent the conversion of lipid to intermediates necessary to provide the energy required for germination and for the synthesis of membrane systems. Indeed, if utilization of the lipid reserves of the conidium is necessary for the maintenance of existing membrane systems and if high temperatures not only block the conversion of those reserves but, as is likely, promote the degradation of membranes it would not be surprising if those membranes soon lost their integrity at high temperatures. This is, in fact, what is observed to occur as incubation at 28° proceeds. With the exception of the tonoplast which, presumably, is continually being added to as degenerating organelles discharge their contents into the enlarging central vacuole, no membranes remain intact. King, Colhoun & Butler (1968) described similar changes in ageing sporangia of Phytophthora infestans (Mont.) de Bary. They too observed the formation of large osmiophilic inclusions in ageing spores. We attribute the formation of these to the condensation of phospholipid products of membrane dissolution. Presumably the few conidia which show similar symptoms by 24 h had, at the start of incubation, a smaller pool of intermediates available for incorporation into membranes. We propose, therefore, that a conidium maintained at 28° does not normally germinate because the mechanism for converting lipid to intermediates required for energy and the synthesis of new and repair of old membrane is inoperative. Provided that the mechanism is activated by lowering the temperature, before irreparable damage occurs to the existing membrane systems, death can be avoided and germination will occur. We wish to thank our colleagues, particularly D. S. Ingram, I. C. Tommerup and D.]. Maclean for many hours of helpful discussion. REFERENCES
KING, J. E., COLHOUN, J. & BUTLER, R. D. (1968). Changes in the ultrastructure of sporangia of Phytophthora infestans associated with indirect germination and ageing. Transactions ofthe British Mycological Society 51, 26g-281. MAsoN, P. A. (1973). Studies on the biology of Bremia lactucae Regel. Ph.D. Thesis, University of Cambridge. POWLESLAND, R. C. (1954). On the biology of Bremia lactucae. Transactions of the British Mycological Society 37,362-371. SARGENT, J. A., TOMMERUP, 1. C. & INGRAM, D. S. (1973). The penetration of a susceptible lettuce variety by the downy mildew fungus Bremia lactueae Regel. Physiological Plant Pathology 3, 231-239. VERHOEFF, K. (1960). On the parasitism of Bremia lactucae Regel on lettuce. Tijdschrift over Planteziekten 66, 133-204.
Bremia laetucae conidia.J. A. Sargent and H. L. Payne 517 EXPLANATION OF PLATES
78 TO 84
Abbreviations m = mitochondrion mt = microtubule n = nucleus pa = papilla st = sterigma v = vacuole
cw = conidial wall cv = cytoplasmic vesicle d = dictyosome e = endoplasmic reticulum gw = germ-tube wall I = lipid droplet to = lomasome Scale: bar =
I }tm.
PLATE 78 Fig. I. A median longitudinal section through a freshly collected conidium of B. lactucae. At the proximal end is a fragment of the sterigma and distally is the papilla. Several nuclei occur within the relatively dense cytoplasm. Mitochondria are rounded and evenly distributed amongst numerous small vacuoles, most of which contain an osmiophilic inclusion. Intensely staining lipid droplets are mostly confined to the periphery of the cytoplasm. Fig. 2. An enlargement of the papilla sectioned in Fig. I. The conidial wall is thinner in this region but the cytoplasm within the papilla is not distinguishable from that in other parts of the spore (cf, Fig. 3). PLATE 79 Fig. 3. A section through the proximal end of a freshly collected conidium. The fractured sterigma and adjacent area of the conidial wall are covered by a lightly staining layer and a cuticle-like layer. Between the larger organelles the ground plasm is densely packed with ribosomes. Dictyosomes are relatively quiescent and there is a paucity of endoplasmic reticulum profiles. Fig. 4. A section through two dietyosomes in a freshly collected conidium. Each is compact and relatively quiescent. The associated endoplasmic reticulum is smooth. PLATE 80
Fig. 5. A section through a dictyosome in a conidium incubated for 1'5 h at IS°. It is larger than those in the quiescent conidium and numerous cytoplasmic vesicles appear to arise from the margins of the cisternae and from the associated endoplasmic reticulum. Fig. 6. A section through a conidium incubated for 1'5 h at IS°. Nuclei and mitochondria are elongate and orientated towards the papilla from which the larger organelles have become displaced. The lipid droplets are less intensely stained. Fig. 7. A section through the distal end of a conidium incubated for 1'5 h at IS°. The papilla is filled almost exclusively with cytoplasmic vesicles. The remaining cytoplasm contains abundant profiles of rough endoplasmic reticulum. The lipid droplets are no longer confined to the periphery of the cytoplasm and stain only lightly. PLATE 81
Fig. 8. A section through the distal end of a germinating conidium incubated for 1'5 h at IS° but slightly more advanced than those shown in Figs. 6 and 7. The germ-tube is emerging through the ruptured conidial wall, adjacent to which are conspicuous lomasomes. Microtubules are directed towards the germ pore. Fig. 9. A longitudinal section through the tip of a germ-tube from a conidium incubated for 2 h at IS°. Cytoplasmic vesicles with contents which stain at different intensities are abundant. Fig. 10. A section through a germ-tube and germ pore of a conidium incubated for 2 h at IS 0. The germ tube is densely packed with cytoplasm and cytoplasmic vesicles are more abundant distally. (The section does not pass through the tip of the germ-tube.) PLATE 82
Fig. I I. A section through the cytoplasm of a conidium incubated for 3 h at 28°. Only the location of the lipid droplets distinguishes this cytoplasm from that of the freshly collected spore (cf. Fig. 3). Fig. 12. A section through the distal end of a conidium incubated for 3 h at 28°. No differentiation of the cytoplasm has occurred in the region of the papilla (cf. Fig. 2). 33
/dYC
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Transactions British Mycological Society PLATE 83
Fig. 13. A section through the cytoplasm of a conidium incubated for 24 h at 28°. Small vacuoles, many containing granules and continuous with profiles of double membrane, are present (cf. Fig. II). Fig. 14. A section through the distal end of a conidium incubated for 24 h at 28°. Except for the almost total loss of lipid droplets from the periphery of the cytoplasm, the region of the papilla has changed little since 3 h. PLATE 84 Fig. 15. A section through a conidium which is representative of a small proportion of the total after incubation for 24 h at 28°. Mitochondria are swollen and their cristae disorganized. The unit membranes of the nuclear envelope are separated and the endoplasmic reticulum and dictyosome cisternae are dilated. Vacuolation is extensive and large intensely staining drops are scattered throughout the spore. Figs. 16,17. Sections of conidia incubated for 72 h at 28°. The cell is highly vacuolate and the major organelles are extensively disorganized. Only the tonoplast appears intact. The plasmalemma is fragmented or absent. All membranes of the mitochondria are absent except for fragments of the outer membrane of the envelope, Nuclear and endoplasmic reticulum membranes have been lost leaving only lacunae to indicate their former sites.
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EFFECT OF TEMPERATURE ON GERMINATION, VIABILITY AND FINE STRUCTURE OF CONIDIA OF BREMIA LACTUCAE By J. A. SARGENT
AND
H. L. PAYNE
Agricultural Research Council Unit of Developmental Botany, 181 A Huntingdon Road, Cambridge (With Plates 78 to 84.) Conidia of Bremia lactucae Regel germinated readily in water over the temperature range 0-20 °C. As the temperature was raised germination was progressively retarded and the capacity of spores to germinate on cooling declined. Early changes in the fine structure of conidia during germination at 15° included the proliferation of endoplasmic reticulum and the stimulation of dictyosomes to produce cytoplasmic vesicles which accumulated at the distal papilla from which the germ tube emerged. Concomitantly, peripheral lipid droplets moved inwards and their affinity for electron stain fell. At 28° conidia remained quiescent for at least 24 h. The only major change in their fine structure was the inward movement of the lipid droplets. However, the lipid continued to stain intensely. Most conidia maintained at 28° eventually became highly vacuolate and their membranes were disrupted. It is proposed that high temperatures prevent the conversion of lipid reserves to intermediates necessary for the supply of energy and the maintenance and synthesis of membrane systems required for germination.
Bremia lactucae is the cause of the downy mildew disease oflettuce. Under conditions of high relative humidity infected leaves produce abundant conidia which are readily dispersed by wind or splash droplets. These spores germinate readily provided that they are maintained in free water, and they will infect an available host within 3 h (Sargent, Tommerup & Ingram, 1973). Since conidia are the propagules by which the fungus normally spreads, a knowledge ofthe factors which influence their viability and germination and the changes which occur in their fine structure during this process is essential to an understanding of the biology of the disease. Temperature has long been recognized as an important factor controlling the germination of fungal spores. Among the earlier reports on its effect on the germination of conidia of B. lactucae, those by Powlesland (1954) and Verhoeff (1960) were the most detailed. Nevertheless their findings differed. Powlesland observed that conidia germinated readily in water over a 24 h period throughout the temperature range I to 10°. Germination decreased above 20° for young conidia and above 15° for older spores. Few conidia, whether young or old, germinated above 25°. Verhoeff, however, reported germination over the range - 3° to 31°. Maximal numbers germinated between 4° and 10° for conidia produced on host plants grown at 20 to 22° and between 2° and
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8° for those produced at 10° to IS°. (Neither of these workers reported the production of zoospores even at the lower temperatures and we have never observed their formation.) The earlier workers did not investigate the effects of temperature on germination beyond 24 h and no attempt was made to establish whether the low percentage of germination recorded at the high temperatures resulted from reversible suppression of germination or from death of the conidia. In an attempt to clarify this issue we first determined the sequence of changes which occur in the fine structure of the conidium of B. lactucae during germination and then reexamined the effect of a wide range of temperatures on germination. Particular attention was given to the effect of prolonged incubation at high temperatures on the potential viability and the fine structure of the conidium. MATERIALS AND METHODS
Conidia production andpreparation B. lactucae, race W5, was maintained as previously described (Sargent et al. 1973) on detached cotyledon pairs of the lettuce cultivar 'Trocadero Improved'. After inoculation with an aqueous suspension of B. lactucae conidia, the cotyledons were incubated in a saturated atmosphere at IS° in a Gallenkamp incubator (IH280) illuminated internally for 12 h each day by six 8 W daylight fluorescent tubes. The fungus began sporulating within 6 days and conidia were harvested on the 7th or 8th day. This was achieved by swirling the cotyledons in a flask with distilled water, decanting the water in which the conidia had become suspended and pelleting the spores by centrifugation at 1000 g for 2 min. To remove a water-soluble inhibitor of germination it was necessary to wash and pellet the spores 3 times over a 10 min period (Mason, 1973). No attempt was made to remove contaminating bacteria but cotyledons were discarded if, after incubation, they showed signs of rotting.
Preparation
ofconidia for electron microscopy
Conidia, whether freshly harvested or after incubation in water, were pelleted and fixed by resuspending them for 2 h in 6 % glutaraldehyde buffered to pH 7'1 with 0'1 M sodium cacodylate. They were then pelleted and resuspended in buffer four times over a period of 2 h before treatment for 2 h with a buffered solution of 2 % osmium tetroxide. After rinsing with water they were pelleted in a tapered plastic TAAB embedding capsule to which was subsequently added a few drops of warm I %water agar. The capsule was immediately centrifuged which sedimented the fixed conidia to the lower surface of the agar before it gelled. The capsule was chilled, cut open and the agar containing the spores cut into a number of small blocks. Each block was dehydrated through an ethanol series and finally embedded in Spurr's resin which was polymerized at 100° for I'5 h. Thin sections were cut, collected on uncoated grids and stained sequentially in a saturated solution of uranyl acetate in 50 % ethanol for 7 min and Reynold's lead citrate solution for 4 min.
Bremia lactucae conidia.], A. Sargent and H. L. Payne 511 Germination of conidia During preliminary attempts to devise a rapid assay of the potential viability offreshly collected conidia it was observed that the proportion of washed conidia which germinated duringi ncubation between 5° and 15° corresponded closely with the proportion which did not rapidly absorb acid fuchsin from dilute solution. Thus the viability of preparations of fresh conidia was routinely estimated by stirring a drop of 0'5 % acid fuchsin in I % acetic acid into a few drops of the spore suspension and determining immediately the percentage of conidia which remained unstained (normally 90-95 %). Determinations of actual percentage germination were made 24 h after plating a suspension of conidia (approx. 4 x 106 per ml) into welled glass slides and incubating them in darkness at a high relative humidity. Duplicate slides were prepared and approximately 1000 conidia on each slide were scored for germination. No difficulty was experienced in assessing whether germination had occurred after 24 h since even at 0° a germ-tube was clearly visible, its length exceeding that of the conidium. RESULTS
Quiescent conidium Structural details of the freshly harvested, ungerminated conidium are shown in PI. 78, 79, figs. 1-4. The ovoid spore measured on average 18 x 16 flm and retained at the proximal end part of the fractured sterigma. At the distal end was a papilla from which a single germ tube would arise during germination (PI. 78, fig. I). The wall of the conidium was approximately 0'24 flm thick except at the papilla where it narrowed to o- I 7 flm (PI. 78, fig. 2) and was uniformly fibrillar. However, at the proximal end of the spore were fragments of a cuticle-like layer overlying the wall and separated from it by a lightly staining layer approx. 0'05 flm thick (PI. 79, fig. 3). Sections through the conidium revealed profiles of nuclei, many of them with prominent nucleoli (PI. 78, fig. I; PI. 79, fig. 3). Mitochondria were abundant and more isodiametric than elongate (PI. 78, figs. I, 2; PI. 79, fig. 3). The dictyosomes were compact but the cisternae showed peripheral dilations (PI. 79, figs. 3, 4). Profiles of endoplasmic reticulum were sparse, relatively smooth, and apart from those associated with dictyosomes, short (PI. 79, figs. 3, 4). Densely packed ribosomes, few of which appeared associated into polyribosomes, gave a granular appearance to the ground plasm (PI. 78, fig. 2; PI. 79, figs. 3, 4). In addition to those organelles the conidium contained numerous vacuoles, many of which enclosed a strongly osmiophilic droplet (PI. 78, figs. 1,2; PI. 79, figs. 3,4), and darkly staining lipid droplets which were arranged predominantly around the periphery of the spore (PI. 78, fig. I; PI. 79, fig. 3). There was no cytoplasmic differentiation in the region of the papilla (PI. 78, fig. 2).
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Sections through conidia 1'5 h after incubation at 15° are shown (PI. 80, figs. 5-7; PI. 8 I, fig. 8). The stage of germination represented by Fig. 8 is slightly in advance of that shown in Figs. 6 and 7. As might be expected the most conspicuous structural changes associated with germination occurred in the region of the papilla. By 1'5 h the papilla was densely packed with cytoplasmic vesicles which had displaced the larger organelles shown in PI. 78, fig. 2. Concomitant with this reorganization was a general orientation of organelles parallel to the long axis of the conidium: nuclei and mitochondria had elongated in the direction of the papilla (PI. 80, fig. 6; PI. 81, fig. 8) and microtubules were directed similarly (PI. 81, fig. 8). Dictyosomes had enlarged and, apparently, become activated. Many cytoplasmic vesicles, similar in appearance to those which aggregated at the papilla, surrounded the dictyosomes and seemed to have arisen from both the cisternae and the associated endoplasmic reticulum (PI. 80, fig. 5). Germination also involved a dramatic increase in the number and length of endoplasmic reticulum profiles each of which was characteristically lined by ribosomes (PI. 80, fig. 7; PI. 8 I, fig. 8). Those ribosomes which were not associated with membrane had, to a large extent, formed polysomal aggregates. The lipid droplets which in the quiescent spore stained deeply no longer had such an affinity for electron stains and had moved inwards from the cell periphery. Plate 81, fig. 8 illustrates a number of features associated with the emergence of the germ tube. Swelling of the papilla had resulted in rupture of the conidial wall and formation of a clearly distinguishable germ-tube wall. The profile of plasm~lemma was very irregular in this region and lomasomes were conspicuous, Plate 81, fig. 10 shows a section through a germ-tube which had developed within 2 h. It demonstrates both the dense nature of the cytoplasm within the germ-tube and the very rapid development which occurred at 15°. The tip of the germ-tube (PI. 81, fig. 9) was densely packed with vesicles which had differing affinities for the stains but resembled those which aggregated in the enlarging papilla (PI. 80, fig. 7; PI. 81, fig. 8).
EFFECT OF TEMPERATURE ON GERMINATION
To determine the effect of temperature on the germination of conidia, suspensions ofthe spores were maintained at various temperatures between 0° and 28° for 24 h. Table I shows the percentage of conidia which germinated. All the potentially viable spores germinated at the intermediate temperatures and even at 0°, but above 20° the number declined. At 28° no conidia germinated over 24 h but if those spores were subsequently cooled to 15°, germination then occurred. To investigate further the inhibition of germination at high temperatures, a series of experiments was set up to investigate the effects of prolonged exposure to these temperatures on both the germination of conidia and their potential to germinate at a lower temperature. The responses of
Bremia laetucae conidia.J. A. Sargent and H. L. Payne 513 Table
1
% Germination
Temperature (0C) o 7 9 15 21 25 28
90 ' 2 92 '7 9 1' 0 9 2 '3 87'4 27'5 o (8g'6)
The effect of temperature on the germination of washed conidia of B, lactucae over a 24 h period, The figure in brackets indicates the percentage of conidia which germinated on transfer to 15° for a further 24 h period, 9 I '2 % of conidia did not stain with acid fuchsin,
Table 2 Time (h) >
Expt. I, 25° Control = 94'7 Viability = 96'3 Expt. II, 28° Control = 94'2 Viabil ity = 92'9 Expt. III, 31° Control = 92'6 Viab ility = 90'S
24
48
72
26'8 (9 1'9)
76'2 (gl '7)
88'4
0 (87'0)
4,8 (70'8)
10'2 (14'2)
0 (2'2)
0 (0)
96
120
6'0 (5'2)
5'1 (5'5)
Percentage germination of conidia of B, lactucae incubated for varying periods at 25°, 28°, or 31°, The figures in brackets indicate the total percentage of conidia which germinated after the spores had been incubated for a further 24 h at 7°. Control = % germination over 24 h at 7°. Viability = % which failed to absorb acid fuchsin rapidly at the start of the experiment.
conidia held at 25°, 28° and 31° are shown in Table 2. During the first 24 h at 25° only 26·8 % of the spores germinated but continued incubation at this temperature resulted in 76'2 % germination after 48 h and 88'4 % after 72 h. Thus, although germination was retarded at 25° it was not permanently inhibited and, given time, almost all the initially viable spores germinated, Transfer of conidia at any time from 25° to 7 ° resulted in their germination within 24 h. At 28° the pattern of germination was more complex: conidia did not germinate during the first 24 h yet 87 % germinated if subsequently incubated at 7°. Prolonged incubation at 28° allowed some conidia to germinate but, unlike those held at 25°, only about 10 % had germinated after 72 h. Moreover, th e potential viability of these spores fell markedly after 24 h and few retained the capacity to germinate after 72 h. At 31° conidia were not only inhibited from germinating but their viability was reduced over 24 h to about 2 %.
Ultrastructural changes induced at 28° At 28° no conidia produced a germ-tube within 24 h but a swelling was conspicuous in the region of the papilla. The few conidia which
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germinated after 48 h at 28° produced only a narrow germ-tube which did not swell at its tip. Since the incubation of conidia at 2° not only delayed their germination but also resulted in a lowering of their viability it seemed pertinent to determine what changes occurred in their fine structure at that temperature. Electron micrographs of sections of conidia fixed after 3, 24 or 72 h at 28° are shown in Plates 82-84, figs. I 1-17. After 3 h (PI. 82, figs. I I, 12) the structure of the conidium resembled, in most respects, that of the freshly harvested spore (PI. 78, 79, figs. 1-4). Only the location of the lipid droplets differed: they were no longer confined to the periphery of the cell, many having moved inwards. By 24 h (PI. 83, figs. 13, 14) only small additional structural changes were observed in the majority of conidia. They included the continuation of the inward movement of the lipid droplets and the generation of additional small vacuoles. Many of the profiles of these new vacuoles revealed the inclusion of deeply staining droplets characteristic of those found in the vacuoles of fresh conidia. The smallest vacuoles, which usually contained electron dense granular aggregates, were continuous with profiles of double membrane. The dictyosomes were somewhat less compact than at 3 hand appeared to have given rise to some cytoplasmic vesicles. However, they did not resemble those characteristic of the germinating conidium. Little change in the structure of the papilla had occurred except for the loss of lipid droplets (PI. 81, fig. 10). The structure of a small proportion of the conidia fixed after 24 h was markedly different from that already described. Their contents had become disorganized and they were probably the spores which would have failed to germinate on cooling to a lower temperature. Throughout these cells (PI. 84, fig. 15) the integrity of the membranes appeared to have been lost: mitochondria were swollen and their cristae disorganized, the unit membranes of the nuclei had separated and the endoplasmic reticulum and dictyosome cisternae were dilated. Vacuolation had increased. Apparently many of the smaller vacuoles had coalesced and degenerate mitochondria appeared to have contributed to the overall vacuolation of the conidium. Few lipid droplets characteristic of viable spores remained but a number of large, more deeply stained drops were scattered throughout the cell. The condition of these disorganized conidia probably represents a stage through which all the spores pass if incubation at 28° is continued. By 72 h all conidia which had not previously germinated were severely disorganized (PI. 84, figs. 16, 17). Organelles were barely recognizable and membranes, with the exception of the tonoplast, had largely disappeared. Only fragments of the plasmalemma remained, the outer unit membrane of the mitochondrial envelope was occasionally distinguishable but the membranes of the nuclear envelope had completely disappeared. Some sites offormer endoplasmic reticulum profiles could be located by the rows of ribosomes which had lined the membrane, but there were no traces of dictyosomes. Most of the spore was occupied by a vacuole.
Bremia lactucae conidia.]. A. Sargent and H. L. Payne 515 DISCUSSION
These observations demonstrate the extensive and rapid changes which occur within the germinating conidium of B. lactucae even before the emergence of the germ-tube. Within 90 min the conidium is transformed from a state of quiescence to one in which organelles have become activated and orientated in preparation for the growth of the germ-tube. The assembly of ribosomes into polysomes and their association with the expanding endoplasmic reticulum is probably indicative of the activation of a protein synthesizing mechanism which is, without doubt, essential for germination. Additional membranous components arise (endoplasmic reticulum, dictyosome cysternae, cytoplasmic vesicles) and protein is also required for the synthesis of enzymes necessary for the utilization of storage material, alteration of the conidium wall to permit the emergence of the germ-tube, and the synthesis of germ-tube wall material. It seems that the dictyosomes are an important component of the mechanism for the synthesis of enzymes and wall-building components as well as the membranes with which those products are compartmentalized. Cytoplasmic vesicles appear to arise from the dictyosomes and to be directed, possibly under the influence of microtubules, towards the papilla. The contents of the vesicles stain to varying intensities reflecting, possibly, the separation of specific enzymes or wall precursors into distinct vesicles. The lomasomes associated with the ruptured conidium wall in the region of the papilla probably arise from the discharge of vesicles, containing wall-softening enzymes, through the plasmalemma and since the population of vesicles at the papilla closely resembles that found in the appressorium in the vicinity of the penetration peg (Sargent et al. 1973) it is likely that even during the early stages of germination, enzymes which aid the entry of the fungus through a host epidermal wall are being mobilized. Germination in water must rely upon an adequate supply of stored reserves. The lipid droplets originally lining the interior of the conidium are probably major contributors to this supply. Very early during germination they move inwards and the subsequent reduction in their affinity for the electron stains probably indicates alterations in their chemical structure as they become metabolized. The present study also focuses attention on the suppression of germination by high temperatures and the accompanying changes in the fine structure of the conidium. Our data demonstrate that at 25° germination can be delayed but not prevented; most conidia eventually germinate at this temperature. It is puzzling that some spores in a population should germinate readily while others remain quiescent for up to 72 h. Age differences might account in part for this differential response but when the spores were collected none could have been mature for as long as three days. Conidia differ somewhat in size and in this laboratory 1. Tommerup has shown that their complement of nuclei can vary between 9 and 22. Under conditions of stress, such variation in size and quantity of specific components might well have a profound effect on the attainment of levels of certain metabolites necessary for germination. That the majority of conidia held at 28° for 24 h remain in a state of
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quiescence is confirmed by their fine structure. Except for the dispersal of lipid from the periphery of the cytoplasm their structure changes very little over 3 h. At 15° a sporcling would have developed in that time and, if in contact with a host, have penetrated an epidermal cell (Sargent et at. 1973). The retention of a high affinity for electron stains by the lipid droplets at 28° suggests that high temperatures prevent the conversion of lipid to intermediates necessary to provide the energy required for germination and for the synthesis of membrane systems. Indeed, if utilization of the lipid reserves of the conidium is necessary for the maintenance of existing membrane systems and if high temperatures not only block the conversion of those reserves but, as is likely, promote the degradation of membranes it would not be surprising if those membranes soon lost their integrity at high temperatures. This is, in fact, what is observed to occur as incubation at 28° proceeds. With the exception of the tonoplast which, presumably, is continually being added to as degenerating organelles discharge their contents into the enlarging central vacuole, no membranes remain intact. King, Colhoun & Butler (1968) described similar changes in ageing sporangia of Phytophthora infestans (Mont.) de Bary. They too observed the formation of large osmiophilic inclusions in ageing spores. We attribute the formation of these to the condensation of phospholipid products of membrane dissolution. Presumably the few conidia which show similar symptoms by 24 h had, at the start of incubation, a smaller pool of intermediates available for incorporation into membranes. We propose, therefore, that a conidium maintained at 28° does not normally germinate because the mechanism for converting lipid to intermediates required for energy and the synthesis of new and repair of old membrane is inoperative. Provided that the mechanism is activated by lowering the temperature, before irreparable damage occurs to the existing membrane systems, death can be avoided and germination will occur. We wish to thank our colleagues, particularly D. S. Ingram, I. C. Tommerup and D.]. Maclean for many hours of helpful discussion. REFERENCES
KING, J. E., COLHOUN, J. & BUTLER, R. D. (1968). Changes in the ultrastructure of sporangia of Phytophthora infestans associated with indirect germination and ageing. Transactions ofthe British Mycological Society 51, 26g-281. MAsoN, P. A. (1973). Studies on the biology of Bremia lactucae Regel. Ph.D. Thesis, University of Cambridge. POWLESLAND, R. C. (1954). On the biology of Bremia lactucae. Transactions of the British Mycological Society 37,362-371. SARGENT, J. A., TOMMERUP, 1. C. & INGRAM, D. S. (1973). The penetration of a susceptible lettuce variety by the downy mildew fungus Bremia lactueae Regel. Physiological Plant Pathology 3, 231-239. VERHOEFF, K. (1960). On the parasitism of Bremia lactucae Regel on lettuce. Tijdschrift over Planteziekten 66, 133-204.
Bremia laetucae conidia.J. A. Sargent and H. L. Payne 517 EXPLANATION OF PLATES
78 TO 84
Abbreviations m = mitochondrion mt = microtubule n = nucleus pa = papilla st = sterigma v = vacuole
cw = conidial wall cv = cytoplasmic vesicle d = dictyosome e = endoplasmic reticulum gw = germ-tube wall I = lipid droplet to = lomasome Scale: bar =
I }tm.
PLATE 78 Fig. I. A median longitudinal section through a freshly collected conidium of B. lactucae. At the proximal end is a fragment of the sterigma and distally is the papilla. Several nuclei occur within the relatively dense cytoplasm. Mitochondria are rounded and evenly distributed amongst numerous small vacuoles, most of which contain an osmiophilic inclusion. Intensely staining lipid droplets are mostly confined to the periphery of the cytoplasm. Fig. 2. An enlargement of the papilla sectioned in Fig. I. The conidial wall is thinner in this region but the cytoplasm within the papilla is not distinguishable from that in other parts of the spore (cf, Fig. 3). PLATE 79 Fig. 3. A section through the proximal end of a freshly collected conidium. The fractured sterigma and adjacent area of the conidial wall are covered by a lightly staining layer and a cuticle-like layer. Between the larger organelles the ground plasm is densely packed with ribosomes. Dictyosomes are relatively quiescent and there is a paucity of endoplasmic reticulum profiles. Fig. 4. A section through two dietyosomes in a freshly collected conidium. Each is compact and relatively quiescent. The associated endoplasmic reticulum is smooth. PLATE 80
Fig. 5. A section through a dictyosome in a conidium incubated for 1'5 h at IS°. It is larger than those in the quiescent conidium and numerous cytoplasmic vesicles appear to arise from the margins of the cisternae and from the associated endoplasmic reticulum. Fig. 6. A section through a conidium incubated for 1'5 h at IS°. Nuclei and mitochondria are elongate and orientated towards the papilla from which the larger organelles have become displaced. The lipid droplets are less intensely stained. Fig. 7. A section through the distal end of a conidium incubated for 1'5 h at IS°. The papilla is filled almost exclusively with cytoplasmic vesicles. The remaining cytoplasm contains abundant profiles of rough endoplasmic reticulum. The lipid droplets are no longer confined to the periphery of the cytoplasm and stain only lightly. PLATE 81
Fig. 8. A section through the distal end of a germinating conidium incubated for 1'5 h at IS° but slightly more advanced than those shown in Figs. 6 and 7. The germ-tube is emerging through the ruptured conidial wall, adjacent to which are conspicuous lomasomes. Microtubules are directed towards the germ pore. Fig. 9. A longitudinal section through the tip of a germ-tube from a conidium incubated for 2 h at IS°. Cytoplasmic vesicles with contents which stain at different intensities are abundant. Fig. 10. A section through a germ-tube and germ pore of a conidium incubated for 2 h at IS 0. The germ tube is densely packed with cytoplasm and cytoplasmic vesicles are more abundant distally. (The section does not pass through the tip of the germ-tube.) PLATE 82
Fig. I I. A section through the cytoplasm of a conidium incubated for 3 h at 28°. Only the location of the lipid droplets distinguishes this cytoplasm from that of the freshly collected spore (cf. Fig. 3). Fig. 12. A section through the distal end of a conidium incubated for 3 h at 28°. No differentiation of the cytoplasm has occurred in the region of the papilla (cf. Fig. 2). 33
/dYC
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Fig. 13. A section through the cytoplasm of a conidium incubated for 24 h at 28°. Small vacuoles, many containing granules and continuous with profiles of double membrane, are present (cf. Fig. II). Fig. 14. A section through the distal end of a conidium incubated for 24 h at 28°. Except for the almost total loss of lipid droplets from the periphery of the cytoplasm, the region of the papilla has changed little since 3 h. PLATE 84 Fig. 15. A section through a conidium which is representative of a small proportion of the total after incubation for 24 h at 28°. Mitochondria are swollen and their cristae disorganized. The unit membranes of the nuclear envelope are separated and the endoplasmic reticulum and dictyosome cisternae are dilated. Vacuolation is extensive and large intensely staining drops are scattered throughout the spore. Figs. 16,17. Sections of conidia incubated for 72 h at 28°. The cell is highly vacuolate and the major organelles are extensively disorganized. Only the tonoplast appears intact. The plasmalemma is fragmented or absent. All membranes of the mitochondria are absent except for fragments of the outer membrane of the envelope, Nuclear and endoplasmic reticulum membranes have been lost leaving only lacunae to indicate their former sites.
(Accepted for publication
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