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Ultrastructure of submerged non-alkaloid producing strains of Claviceps purpurea (Fr.) Tul.
Bioehern. Physiol. Pflanzen 175, 552 - 561 (1980)
Ultrastructure of Submerged Non-Alkaloid Producing Strains of Olaviceps purpurea (FR.) TUL. W. LÖSECKE, D. NEDMANK, H.-P. SCHMADDER and D. GRÖGER Institute of Plant Biochemistry, Academy of Sciences of the GDR, Halle (Saale) K e y Term In d e x: Non-alkaloid producing strains, ultrastructure, Lipid storage; Claviceps purpurea.
Summary Two non-alkaloid producing strains of Claviceps purpurea were investigated after cultivation in two different media. The ultrastructure of the hyphae depends on the composition of the eulture broth. The strain Pur 221 forms in the "production" medium NL 720 thin-walled hyphae with several nuclei numerous mitochondria and ER membran es. In older hyphae most of the cell volume is oceupied by polysaccharides. The strain Pur 218, grown in a rieh medium, shows relatively thin-walled hyphae with large nudei, short ER membranes and only a smallllllmber of lipid droplets. In ,L "produetion" medium hyphae of the same age eontain 50 % lipid related to the eell volume. It can be concluded from the electron micrographs that the composition of the lipids is ehanged during the cultivation. Morphometric analysis of the most important eell organelles allows a more precise description of the development of the fungus during cultivation and under different environmental conditions.
Iutroduction Many strains of Claviceps purpurea are unable to produce alkaloids under saprophytic conditions. Our knowledge about the reason of the lacking alkaloid synthesis is still obscure. Observations about a connection between lipid and alkaloid synthesis and special morphological properties in alkaloid producing strains(MANTLE and TONOLO 1968) cannot explain thc mechanism of the induction of synthesis of secondary mctabolite formation. Certain biochcmical and apparcntly morphological conditions are prerequisites for alkaloid synthcsis. We investigated the ultrastructure of nonproducing strains of Claviceps purpurea in order to describe the devclopment of the hyphae under different conditions by morphometrical methods. Material aud Methods Grawth media Medium NI, 720:
Medium NI, 821: Medium NI, M 10: Medium NI, M 10:
200.0g saecharose; 15.0g NH~-eitrat; 1.0 g Ca (N0 3)2; 0.25 g KH 2 P0 4 ; 0.3 g MgS0 4 • 7 H 2 0; 0.01 g FeS0 4 . 7 H 2 0; 0.0308 g ZnS0 4 • 7 H 2 0; 0.1 g KCl were made up with distilled w,Ltcr to 1,000 ml and sterilized ,Lt 110°C for 30 min. NI, 720 with 0.3 % of yeast extract. 5 % beer worth with O.B % of yeast extraet. "rieh" medium.
U1trastructure of Claviceps purpurea
553
Media NL 720 and NL 821 allow a good alkaloid production if suitable strains are used for cultivation. We designate them in this paper as " production" media.
Organisms The strains Pur 218 and Pur 221 were isolated from ergotamine-co ntaining sclerotia.
Light microscopy For light microscopic investigations spores, germinating spores and 6 days old mycelium of a submerged culture (strain Pur 218) were fixed with glutaraidehydejOs04 (see electron microscopy).
Electron microscopy KMn0 4-Fixation: Submerged cultivated cells were fixed 1, 3, 6, 9, 13 days after incubation with
1.2 % KMnOt jPalade buffer (1: 1, pH 7.4) for 30 min (LUFT 1956), dehydrated in g~aded series of aceton and embedded in Araldit. Sections were prepared with glass knives (U1tramicrotome Om U 2, Reichert, Austria), stained with Pb according to REYXOLDS (1963) and viewed with a transmission electron microscope (SEM 3j2, Fernsehelektronik DDR).
Glycol methacrylate embedding: 10 days old cells (strain Pur 218) gro wn in a rich medium (NLM 10) were prefixed with 5 % glutaraldehydejphosphate buffer (pR 7.4) for 2 hand subsequentlyfixed with 1 % Os04jPa.]ade buffer (pR 7.4) for 1-3 h. After staining in 0.5 % uranylace tate for 2 h the cells were dehydra,ted in glycol methacrylate und embedded in prepolymerized glyco l methacrylate (LE Duc and BERNHARD 1967). The polymerization was performed for 1-3 days with U.v. light. Detection or polysaccharides : Polysaccharides were detected b y per.iodic acid as described before (Lös ECKE et al. 1980).
Scanning electron microscopy: Ryphae of 3, Gand 13 days old submerged cultures (strain Pur 218j NL !VI 10 und NL 821) were prefixed in 5 % glutaraldehydejREPES-buffer (pH 8) for 2 h, subsequently fi xed in 1 % Os04jRE PES-buffer (pR 6.8) for 2 h, dehydrat ed in acetone and air dried. After coating with gold (RBA 120/2, Zeiss DDR) the objects were viewed in a scnnning electron microscope (J!VIS- U3, Jeol Japan). Morphometry: 30-50 cells of each sampie (strnin Pur 218PL M 10 und NL 821) were used for morphometri c nnaylsis. A r egular point lattice wns used to determine the proportion in volume of different cell organelles.
Results
Strain Pur 221 The strain Pur 221 forms under submerged conditions (medium NL 720) a nonsporulating, filamentous mycelium. It is not able to produce alkaloids (BÖHMER et a1. 1978). Young hyphae have a narrow cell wall, several nuclei, small vacuoles, numerou'l mitochondria and equally in thc cytoplasm distributcd ER membranes (Fig. 1). The hyphal cells contain a fluffy electron transparent material associated with a small number of lipid droplets. During a submcrged cultivation of the fungi the amount of this fluffy material increases permanently (Fig. 2), occupying most of the cell volume in old hyphae (Fig. 3). Thc lipid content remains constant, also old cells show only very little lipid droplets (Figs. 2, 3). The fluffy electron transparent material could be identified as polysaccharide (may be glycogen) due to its strong rcaction with perjodic acid anti. thiosemicarbazide (Fig. 10).
554
W. Lös ECKE et al.
Fig. 1 - 3. Ultrastructure of submerged non-alkaloid p1'Oducing cultures of Claviceps purpurea, strain Pur 221 grou'n 1;'11 medium N L 7:20 (Fixation IDln(\). 1. Ultrastructure of a hypha from a 3 days olll cnltnre with Bu merons mitorhondria (M), two nu clei (N), glycogen fG) ami droplets (L). x 24,000. 2, Part of a hyph
Ultrastrlll'ture of Claviceps purpurea
555
&train Pur 218 The cultivation of this strain was started with spores (Fig. 4). In both media NL 821 and NL M 10 the spores form (Fig. 5) after germination thin-waHed hyphae. Later they secluded conidiospores (Fig. 6, compare also Figs. 13 -16). The ultrastructure of the hyphal ceHs depends from the environmental conditions. While 6 days old ceHs grown in medium NL M 10 (rich medium) show large nuclei, a smaH number of mitochondria, short ER membranes and very smaH amounts of lipids (Fig. 7), ceHs in the same age, grown in NL 821 ("production" medium) have thick ceH walls and a very large number of lipid droplets (Fig. 8). In rich medium (NL M 10) cultivated mycelium shows only after 10-12 days larger amounts of lipid, phagocitozed by the vacuoles (Fig. 9). The same ceHs embedded in glycolmethacrylate possess partially extracted lipid droplets, some of them are incorporated in the vacuoles. The vacuoles give a very strong contrast with OS04 under these conditions (Figs. 11, 12). In the scanning microscope there are only little differences after cultivation in the two media. In both cases the mycelium consists of long, smaH and only little branched cells, closely interwoven and forming compact coils (Fig. 13). The thick-walled hyphae grown in medium NL 821 show irregular distributed pores (Figs. 14, 15). Older cells possess a higher number of pores which are enlarged in size (Fig.16).
M orphometry Fig. 17 shows the changes in the volume ratios of the most importallt cell organelles during the growth of the mycelium in rich (NL M 10) and "production" medium (NL 821). Thc proportion in volume of nuclei, mitocholldria, ER and vacuoles is diminished continuously during the cultivation in medium NL 821. To the same extend the lipid content is drastically increased during the third to sixth day of cultivation (3th day = 10 %; 6th day = 45 %; related to the cell volume). Together with the lipids the amount of polysaccharide is also increased (3th day = 7 %; 13th day = 18 %, related to the cell volume). In contrast thc average proportion in volume of nuclei, mitochondria and ER to the cell volumc remains constant up to the 9th day of fermentation in rich medium (nuclei: 20%; mitochondria: 10%; ER: 2,5%). The maximum of the vacuolization is reached on the 6th day of cultivation (23 % of the cell volume) and decreases up to 5 -7 % during the next days. Lipid droplets (maximal 35 % of the cell volume) and polysaccharides (maximal 8 % of th(' cell volume) are observed only in later stages of development (betwcen the 10th and 12th day) for thc strain Pur 218 cultivated in rich medium. Discussion
Non-alkaloid producing strains of Claviceps purpurea (FR.) TUL. have a pattern like to a sphacelial mycelium and form in general conidia (ToNoLO et al. 1961; MANTLE and TOKoLO 1968). The mycelium consists of long, small and rarely branched hyphae with a relatively low content of lipid (STRNADOVA and KYBAL 1976). 38
Biochclll. Physiol. Pflanzen, Bd. 175
556
W. Lös ECKE et al.
Ultrastructure of Clavieeps purpurea
557
The investigated strain Pur 221 does not form conidia under certain conditions. After cultivation in a "production" medium large amounts of polysaccharides are accumulated in older hyphae, but only very little lipid droplets were observed. It seems that the strain Pur 221 is not able to transform polysaccharides into lipids. This biochemical conversion, however, might be in some cases eorrelated to the formation of alkaloids as it was shown in parasitically growll sclerotic1 (LÖSECKE et al., 1980) and submerged cultivated alkaloid-producing strains (REH,\CEK and MALIK 1972; LÖSECKE et al., unpublished). In contrast strain Pur 218 is able to produce spores. With this relatively homogenous material a morphometric analysis gives good reproducible results. After eultivation in rieh medium the strain Pur 218 has filamentous hyphae, showing only in old hyphae lipid droplets, whieh are absorbcd into vacuolcs. Hydrolases in the vacuoles eould degradc the lipids. This assumption is supported by the observation that lipid droplct absorbing vaeuoles are very osmiophilie aftcr glyeol mcthacrylate embedding duc to thc increase of double bonds in course of the breakdown of lipids. In a "production" medium cultivated mycelium shows already between the third and 5th day of fermcntation large amounts of lipid droplcts (55 % lipid rclated to the eell volume). Regarding thc lipid content thc cells show a similar pattern as the mature sclerotia. Howevcr, the hyphae of strain Pur 218 posscss other morphological properties and do not form plectel1chymatic pellets. Despite a remarkable formation of lipids in this strain no alkaloid biosynthesis was recorded. This points to the fact, that lipid formation and alkaloid synthesis may occur very often concomitantly hut do not strictly depent on cach othcr. Bcsides lipid formation other differentiation steps are necessary to allow alkaloid formatiOll.
Fig.4-9. Light and eleetron mierographs of submerged non-alkaloid produeing eultures of Clavieeps purpurea: spores, germinating spores and hyphae of the strain Pur 218 grown in rieh (N L M 10) and produetion medium (NL 821). (Fixation: GlutaraldehydejOs04 for light microscopy; KMn0 4 for electron microscopy). 4. Spores of the strain Pur 218. x 320. 5. Germinating spores (NL .\1 10). x 320. 6. Hyphae of a 6 days old culture grown in "production" medium (NL 821). At the end the hyphae conidiospores (arrow). x 320. 7. Part of a hypha of a 6 days old culture grown in rieh medium (NL M 10). In the cytoplasm besides mitochondria (:VI), nuclei (~) and endoplasmic reticulum (ER) only very little lipid droplets (L) and glycogen. 8. Part of a hypha of a 6 days old culture grown in "production" medium (NL 821). Note the large number of lipid droplets and the glycogen deposits (G) in the cytoplasm; cell wall (W). x 24,000. 9. Part of a hypha of a 13 days old culture grown in rieh medium (NL M 10). Large lipid droplets (L) are phagoeytosed by the vacuoles (V). x 22,000. 3S*
Fig. 10 - 1:2. Polysaccluwili s Imd li:pids in submel'ged cultures of strains Pur '218 and Pur '221. (Fixatio n: KMn0 4 ). 10. Deteetion of polysaccharides in n hyph n of the strain Pur :2:21 grown in medium NL 720 (3 dnys old). The polysaccharide (glycogen = G) is associated with the lipid droplets (L). 11, 12. In glyeol metltalTylate cmbedcled hyphae of the strain Pur 218 (10 days grown in medium M 10). The strong osmi ophilir vaeuoles (Y) take up lipid dropl ets (L), which are partially extracted dming fixation und clllbcdding; tell wall (W). X 12,600.
559
Ultrastructure of Claviceps purpurea
Fig. 13 - 16. Scanning electron micrograplls of Claviceps purpurea (Fixation: Glutaraldehyde 0801)' 13. Scanning micrograph of hyphae of the strain Pur :218 (NI, JI 10).
X
:2,000.
14. Scanning micrograph of filamentolls hyphae of the strain Pur 218 (grown for 6 days in N L821).
x 4,000.
15. Scanning micrograph of a hypha (Pur 21SjNL 821) with round pores on the cell surface. x 7,000. 16. Scanni ng mierograph of an olr! hypha (PlIr 218jNK 821) with X 5,000.
11
strong articnlaed ('ell slIrface .
w. LÖSECKE et al. M/O
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~
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50 1,0
30 20
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~
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/3d
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Ultrastructnre of Claviceps purpurea
561
Fig. 17. Avarage proportiollin volUlne on the cell volume of hyphae of the straill Pur 218 in "produclion" (NL 821) and rich medium (NL 2'vl10) lrith standard error. a, nuclei; b, mitochondria; e, endoplasmie reticulum; d, vacuoles; e, lipids; f, glycogen.
References BÖHMER, S.-A., LEIS1'NER, H.- U., und SCHLEE, D.: Stamm- und milieuspezifische Aminosäuremuster in Claviceps purpurea bei saprophytischer Kultur. Z. Allg. Mikrobiol. 18, 233-241 (1978). LEDue, E. H., and BERNHARD, W.: Recent modifications of the glycol methaerylate embedding procedure. J. Ultmstruct. Res. 19, 19G-199 (19G7). LösEcKE, W., NEU1L\NX, D., GRÖGElt, D., ltnd SCHMAUDEIt, H.-P.: Ultrastructure of cytoplasmie ehanges during development of Selerotia in Claviceps purpurea (FR.) TUL. Arch. Microbiol. 125, 251- 257 (1980). LUFT, J. 11.: Permanganate - " new fix"tivc for electron microscopy. J. Biophys. Biochem. Cytol. 2, 799 (195G). MAN1'LE, P. G., and TONOLO, A.: Relationship between the morphology of Claviceps purpurea and the prodlletion of alkaloids. Trans. Br. mycol. Soc. 51, 499 -505 (19G8). REHACEK, Z., ,tnd MAL1K, K. A.: Physiological status of sllbmerged Claviceps during enzymatic assem bly of ergot "lkaloids. Folü, Microbiol. 17, 490 -499 (1972). REYNOLDS, E. S.: The use of lead citrate at high pli as an elecron-op"que stain in electron microscopy. J. Cell Biol. 1i, 208-212 (1963). STRNADOVA, E., und KYBAL, J.: Morphogenetische Veränderungen bei Claviceps purpurea infolge eines mutagenen Eingriffes salpetriger Säure. Plan ta medica 30,395-398 (1976). TONOLO, A., SC01'1'1, T., and VERO-RARCELLONA, L.: Morphological observations on different speeies of Claviceps TLL. grown in submerged culture. Sei. Repts. Ist. Super. Sanita 1,404-422 (1961).
Received February 20, 1980. Authors' address: W. LÖSECKE, Dr. D. NEUM.HN, Dr. H.-P. SCHMAUDER and Prof. Dr. D. GRÖGER, Institut für Biochemie der Pflanzen eier AdW, DDR - 4020 Halle (Saale), Weinberg 3.
Ultrastructure of Submerged Non-Alkaloid Producing Strains of Olaviceps purpurea (FR.) TUL. W. LÖSECKE, D. NEDMANK, H.-P. SCHMADDER and D. GRÖGER Institute of Plant Biochemistry, Academy of Sciences of the GDR, Halle (Saale) K e y Term In d e x: Non-alkaloid producing strains, ultrastructure, Lipid storage; Claviceps purpurea.
Summary Two non-alkaloid producing strains of Claviceps purpurea were investigated after cultivation in two different media. The ultrastructure of the hyphae depends on the composition of the eulture broth. The strain Pur 221 forms in the "production" medium NL 720 thin-walled hyphae with several nuclei numerous mitochondria and ER membran es. In older hyphae most of the cell volume is oceupied by polysaccharides. The strain Pur 218, grown in a rieh medium, shows relatively thin-walled hyphae with large nudei, short ER membranes and only a smallllllmber of lipid droplets. In ,L "produetion" medium hyphae of the same age eontain 50 % lipid related to the eell volume. It can be concluded from the electron micrographs that the composition of the lipids is ehanged during the cultivation. Morphometric analysis of the most important eell organelles allows a more precise description of the development of the fungus during cultivation and under different environmental conditions.
Iutroduction Many strains of Claviceps purpurea are unable to produce alkaloids under saprophytic conditions. Our knowledge about the reason of the lacking alkaloid synthesis is still obscure. Observations about a connection between lipid and alkaloid synthesis and special morphological properties in alkaloid producing strains(MANTLE and TONOLO 1968) cannot explain thc mechanism of the induction of synthesis of secondary mctabolite formation. Certain biochcmical and apparcntly morphological conditions are prerequisites for alkaloid synthcsis. We investigated the ultrastructure of nonproducing strains of Claviceps purpurea in order to describe the devclopment of the hyphae under different conditions by morphometrical methods. Material aud Methods Grawth media Medium NI, 720:
Medium NI, 821: Medium NI, M 10: Medium NI, M 10:
200.0g saecharose; 15.0g NH~-eitrat; 1.0 g Ca (N0 3)2; 0.25 g KH 2 P0 4 ; 0.3 g MgS0 4 • 7 H 2 0; 0.01 g FeS0 4 . 7 H 2 0; 0.0308 g ZnS0 4 • 7 H 2 0; 0.1 g KCl were made up with distilled w,Ltcr to 1,000 ml and sterilized ,Lt 110°C for 30 min. NI, 720 with 0.3 % of yeast extract. 5 % beer worth with O.B % of yeast extraet. "rieh" medium.
U1trastructure of Claviceps purpurea
553
Media NL 720 and NL 821 allow a good alkaloid production if suitable strains are used for cultivation. We designate them in this paper as " production" media.
Organisms The strains Pur 218 and Pur 221 were isolated from ergotamine-co ntaining sclerotia.
Light microscopy For light microscopic investigations spores, germinating spores and 6 days old mycelium of a submerged culture (strain Pur 218) were fixed with glutaraidehydejOs04 (see electron microscopy).
Electron microscopy KMn0 4-Fixation: Submerged cultivated cells were fixed 1, 3, 6, 9, 13 days after incubation with
1.2 % KMnOt jPalade buffer (1: 1, pH 7.4) for 30 min (LUFT 1956), dehydrated in g~aded series of aceton and embedded in Araldit. Sections were prepared with glass knives (U1tramicrotome Om U 2, Reichert, Austria), stained with Pb according to REYXOLDS (1963) and viewed with a transmission electron microscope (SEM 3j2, Fernsehelektronik DDR).
Glycol methacrylate embedding: 10 days old cells (strain Pur 218) gro wn in a rich medium (NLM 10) were prefixed with 5 % glutaraldehydejphosphate buffer (pR 7.4) for 2 hand subsequentlyfixed with 1 % Os04jPa.]ade buffer (pR 7.4) for 1-3 h. After staining in 0.5 % uranylace tate for 2 h the cells were dehydra,ted in glycol methacrylate und embedded in prepolymerized glyco l methacrylate (LE Duc and BERNHARD 1967). The polymerization was performed for 1-3 days with U.v. light. Detection or polysaccharides : Polysaccharides were detected b y per.iodic acid as described before (Lös ECKE et al. 1980).
Scanning electron microscopy: Ryphae of 3, Gand 13 days old submerged cultures (strain Pur 218j NL !VI 10 und NL 821) were prefixed in 5 % glutaraldehydejREPES-buffer (pH 8) for 2 h, subsequently fi xed in 1 % Os04jRE PES-buffer (pR 6.8) for 2 h, dehydrat ed in acetone and air dried. After coating with gold (RBA 120/2, Zeiss DDR) the objects were viewed in a scnnning electron microscope (J!VIS- U3, Jeol Japan). Morphometry: 30-50 cells of each sampie (strnin Pur 218PL M 10 und NL 821) were used for morphometri c nnaylsis. A r egular point lattice wns used to determine the proportion in volume of different cell organelles.
Results
Strain Pur 221 The strain Pur 221 forms under submerged conditions (medium NL 720) a nonsporulating, filamentous mycelium. It is not able to produce alkaloids (BÖHMER et a1. 1978). Young hyphae have a narrow cell wall, several nuclei, small vacuoles, numerou'l mitochondria and equally in thc cytoplasm distributcd ER membranes (Fig. 1). The hyphal cells contain a fluffy electron transparent material associated with a small number of lipid droplets. During a submcrged cultivation of the fungi the amount of this fluffy material increases permanently (Fig. 2), occupying most of the cell volume in old hyphae (Fig. 3). Thc lipid content remains constant, also old cells show only very little lipid droplets (Figs. 2, 3). The fluffy electron transparent material could be identified as polysaccharide (may be glycogen) due to its strong rcaction with perjodic acid anti. thiosemicarbazide (Fig. 10).
554
W. Lös ECKE et al.
Fig. 1 - 3. Ultrastructure of submerged non-alkaloid p1'Oducing cultures of Claviceps purpurea, strain Pur 221 grou'n 1;'11 medium N L 7:20 (Fixation IDln(\). 1. Ultrastructure of a hypha from a 3 days olll cnltnre with Bu merons mitorhondria (M), two nu clei (N), glycogen fG) ami droplets (L). x 24,000. 2, Part of a hyph
Ultrastrlll'ture of Claviceps purpurea
555
&train Pur 218 The cultivation of this strain was started with spores (Fig. 4). In both media NL 821 and NL M 10 the spores form (Fig. 5) after germination thin-waHed hyphae. Later they secluded conidiospores (Fig. 6, compare also Figs. 13 -16). The ultrastructure of the hyphal ceHs depends from the environmental conditions. While 6 days old ceHs grown in medium NL M 10 (rich medium) show large nuclei, a smaH number of mitochondria, short ER membranes and very smaH amounts of lipids (Fig. 7), ceHs in the same age, grown in NL 821 ("production" medium) have thick ceH walls and a very large number of lipid droplets (Fig. 8). In rich medium (NL M 10) cultivated mycelium shows only after 10-12 days larger amounts of lipid, phagocitozed by the vacuoles (Fig. 9). The same ceHs embedded in glycolmethacrylate possess partially extracted lipid droplets, some of them are incorporated in the vacuoles. The vacuoles give a very strong contrast with OS04 under these conditions (Figs. 11, 12). In the scanning microscope there are only little differences after cultivation in the two media. In both cases the mycelium consists of long, smaH and only little branched cells, closely interwoven and forming compact coils (Fig. 13). The thick-walled hyphae grown in medium NL 821 show irregular distributed pores (Figs. 14, 15). Older cells possess a higher number of pores which are enlarged in size (Fig.16).
M orphometry Fig. 17 shows the changes in the volume ratios of the most importallt cell organelles during the growth of the mycelium in rich (NL M 10) and "production" medium (NL 821). Thc proportion in volume of nuclei, mitocholldria, ER and vacuoles is diminished continuously during the cultivation in medium NL 821. To the same extend the lipid content is drastically increased during the third to sixth day of cultivation (3th day = 10 %; 6th day = 45 %; related to the cell volume). Together with the lipids the amount of polysaccharide is also increased (3th day = 7 %; 13th day = 18 %, related to the cell volume). In contrast thc average proportion in volume of nuclei, mitochondria and ER to the cell volumc remains constant up to the 9th day of fermentation in rich medium (nuclei: 20%; mitochondria: 10%; ER: 2,5%). The maximum of the vacuolization is reached on the 6th day of cultivation (23 % of the cell volume) and decreases up to 5 -7 % during the next days. Lipid droplets (maximal 35 % of the cell volume) and polysaccharides (maximal 8 % of th(' cell volume) are observed only in later stages of development (betwcen the 10th and 12th day) for thc strain Pur 218 cultivated in rich medium. Discussion
Non-alkaloid producing strains of Claviceps purpurea (FR.) TUL. have a pattern like to a sphacelial mycelium and form in general conidia (ToNoLO et al. 1961; MANTLE and TOKoLO 1968). The mycelium consists of long, small and rarely branched hyphae with a relatively low content of lipid (STRNADOVA and KYBAL 1976). 38
Biochclll. Physiol. Pflanzen, Bd. 175
556
W. Lös ECKE et al.
Ultrastructure of Clavieeps purpurea
557
The investigated strain Pur 221 does not form conidia under certain conditions. After cultivation in a "production" medium large amounts of polysaccharides are accumulated in older hyphae, but only very little lipid droplets were observed. It seems that the strain Pur 221 is not able to transform polysaccharides into lipids. This biochemical conversion, however, might be in some cases eorrelated to the formation of alkaloids as it was shown in parasitically growll sclerotic1 (LÖSECKE et al., 1980) and submerged cultivated alkaloid-producing strains (REH,\CEK and MALIK 1972; LÖSECKE et al., unpublished). In contrast strain Pur 218 is able to produce spores. With this relatively homogenous material a morphometric analysis gives good reproducible results. After eultivation in rieh medium the strain Pur 218 has filamentous hyphae, showing only in old hyphae lipid droplets, whieh are absorbcd into vacuolcs. Hydrolases in the vacuoles eould degradc the lipids. This assumption is supported by the observation that lipid droplct absorbing vaeuoles are very osmiophilie aftcr glyeol mcthacrylate embedding duc to thc increase of double bonds in course of the breakdown of lipids. In a "production" medium cultivated mycelium shows already between the third and 5th day of fermcntation large amounts of lipid droplcts (55 % lipid rclated to the eell volume). Regarding thc lipid content thc cells show a similar pattern as the mature sclerotia. Howevcr, the hyphae of strain Pur 218 posscss other morphological properties and do not form plectel1chymatic pellets. Despite a remarkable formation of lipids in this strain no alkaloid biosynthesis was recorded. This points to the fact, that lipid formation and alkaloid synthesis may occur very often concomitantly hut do not strictly depent on cach othcr. Bcsides lipid formation other differentiation steps are necessary to allow alkaloid formatiOll.
Fig.4-9. Light and eleetron mierographs of submerged non-alkaloid produeing eultures of Clavieeps purpurea: spores, germinating spores and hyphae of the strain Pur 218 grown in rieh (N L M 10) and produetion medium (NL 821). (Fixation: GlutaraldehydejOs04 for light microscopy; KMn0 4 for electron microscopy). 4. Spores of the strain Pur 218. x 320. 5. Germinating spores (NL .\1 10). x 320. 6. Hyphae of a 6 days old culture grown in "production" medium (NL 821). At the end the hyphae conidiospores (arrow). x 320. 7. Part of a hypha of a 6 days old culture grown in rieh medium (NL M 10). In the cytoplasm besides mitochondria (:VI), nuclei (~) and endoplasmic reticulum (ER) only very little lipid droplets (L) and glycogen. 8. Part of a hypha of a 6 days old culture grown in "production" medium (NL 821). Note the large number of lipid droplets and the glycogen deposits (G) in the cytoplasm; cell wall (W). x 24,000. 9. Part of a hypha of a 13 days old culture grown in rieh medium (NL M 10). Large lipid droplets (L) are phagoeytosed by the vacuoles (V). x 22,000. 3S*
Fig. 10 - 1:2. Polysaccluwili s Imd li:pids in submel'ged cultures of strains Pur '218 and Pur '221. (Fixatio n: KMn0 4 ). 10. Deteetion of polysaccharides in n hyph n of the strain Pur :2:21 grown in medium NL 720 (3 dnys old). The polysaccharide (glycogen = G) is associated with the lipid droplets (L). 11, 12. In glyeol metltalTylate cmbedcled hyphae of the strain Pur 218 (10 days grown in medium M 10). The strong osmi ophilir vaeuoles (Y) take up lipid dropl ets (L), which are partially extracted dming fixation und clllbcdding; tell wall (W). X 12,600.
559
Ultrastructure of Claviceps purpurea
Fig. 13 - 16. Scanning electron micrograplls of Claviceps purpurea (Fixation: Glutaraldehyde 0801)' 13. Scanning micrograph of hyphae of the strain Pur :218 (NI, JI 10).
X
:2,000.
14. Scanning micrograph of filamentolls hyphae of the strain Pur 218 (grown for 6 days in N L821).
x 4,000.
15. Scanning micrograph of a hypha (Pur 21SjNL 821) with round pores on the cell surface. x 7,000. 16. Scanni ng mierograph of an olr! hypha (PlIr 218jNK 821) with X 5,000.
11
strong articnlaed ('ell slIrface .
w. LÖSECKE et al. M/O
20 10
o~ I
~
5
~
50
9d
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50 1,0
30 20
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o~ r q"
~
5
/3d
MB2/
9d
Ultrastructnre of Claviceps purpurea
561
Fig. 17. Avarage proportiollin volUlne on the cell volume of hyphae of the straill Pur 218 in "produclion" (NL 821) and rich medium (NL 2'vl10) lrith standard error. a, nuclei; b, mitochondria; e, endoplasmie reticulum; d, vacuoles; e, lipids; f, glycogen.
References BÖHMER, S.-A., LEIS1'NER, H.- U., und SCHLEE, D.: Stamm- und milieuspezifische Aminosäuremuster in Claviceps purpurea bei saprophytischer Kultur. Z. Allg. Mikrobiol. 18, 233-241 (1978). LEDue, E. H., and BERNHARD, W.: Recent modifications of the glycol methaerylate embedding procedure. J. Ultmstruct. Res. 19, 19G-199 (19G7). LösEcKE, W., NEU1L\NX, D., GRÖGElt, D., ltnd SCHMAUDEIt, H.-P.: Ultrastructure of cytoplasmie ehanges during development of Selerotia in Claviceps purpurea (FR.) TUL. Arch. Microbiol. 125, 251- 257 (1980). LUFT, J. 11.: Permanganate - " new fix"tivc for electron microscopy. J. Biophys. Biochem. Cytol. 2, 799 (195G). MAN1'LE, P. G., and TONOLO, A.: Relationship between the morphology of Claviceps purpurea and the prodlletion of alkaloids. Trans. Br. mycol. Soc. 51, 499 -505 (19G8). REHACEK, Z., ,tnd MAL1K, K. A.: Physiological status of sllbmerged Claviceps during enzymatic assem bly of ergot "lkaloids. Folü, Microbiol. 17, 490 -499 (1972). REYNOLDS, E. S.: The use of lead citrate at high pli as an elecron-op"que stain in electron microscopy. J. Cell Biol. 1i, 208-212 (1963). STRNADOVA, E., und KYBAL, J.: Morphogenetische Veränderungen bei Claviceps purpurea infolge eines mutagenen Eingriffes salpetriger Säure. Plan ta medica 30,395-398 (1976). TONOLO, A., SC01'1'1, T., and VERO-RARCELLONA, L.: Morphological observations on different speeies of Claviceps TLL. grown in submerged culture. Sei. Repts. Ist. Super. Sanita 1,404-422 (1961).
Received February 20, 1980. Authors' address: W. LÖSECKE, Dr. D. NEUM.HN, Dr. H.-P. SCHMAUDER and Prof. Dr. D. GRÖGER, Institut für Biochemie der Pflanzen eier AdW, DDR - 4020 Halle (Saale), Weinberg 3.