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Ascosporogenesis in Tuber magnatum
[ 201
Trans. Br. mycol. Soc. 80 (2), 201-207 (1983)
]
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ASCOSPOROGENESIS IN TUBER MAGNATUM By GRAZIELLA BERTA AND ANNA FUSCONI Istituto Botanico and Centro di Microscopia Elettronica dell' Unioersita, Turin, Italy Ascosporogenesis in Tuber magnatum is intermediate between the general pattern of Euascomycetes and that of Hemiascomycetes. T . magnatum ascospores are delimited individually by two membranes which originate from the ascus plasmalemma; wall material is deposited between these membranes. The epispore is deposited first . The outer perispore is initially expanded but then contracts and fills with increasingly compact electron-dense material to form the surface ornamentations. The electron-transparent endospore is deposited inside the epispore. The general features of ascosporogenesis are well known and may be divided into three stages: (1) two membranes delimit uninucleate regions of cytoplasm; (2) spore primary wall material is deposited between these membranes; (3) the inner membrane becomes the spore plasma membrane and the outer one the investing membrane. The ascal cytoplasm outside the ascospores is termed the epiplasm. Subsequent phases of wall development vary according to the species (Beckett , 1981). The mechanisms of spore delimitation in Hemiascomycetes and Euascomycetes are very different. In the former, spores are delimited individually in a variety of ways (Ashton & Moens, 1979). In Euascomycetes, the nuclei and cytoplasm are enveloped by a common involucre, a double membrane or ascus vesicle at the periphery of the ascus . This then invaginates around individual nucle i to delimit the ascospores (Beckett & Crawford, 1970; Bracker, 1967; Campbell, 1973; Carroll, 1967, 1969; Greenhagh & Griffiths, 1970; Reeves , 1967; Schrantz, 1970; Stiers, 1974; Wells, 1972 ; Wilsenach & Kessel, 1965). However in T. aestivum Vitt., T. rufum Pico and T . mesentericum Vitt., the ascus vesicle is absent and their spores are delimited singly as in Hemiascomycetes (janexFavre & Parguey-Leduc, 1976, 1980; PargueyLeduc & Janex-Favre, 1977a). In this paper, ascosporogenesis in Tuber magnatum Pico, the most commonly eaten truffle in Italy, is described. MATERIALS AND METHODS Small pieces of T. magnatum ascocarps were fixed for 4 h with 2 % glutaraldehyde in 0'1 M-sodium cacodylate buffer at pH 7'2, postfixed for 2 h in 1 % OsOJ in the same buffer , deh ydrated through a graded alcohols series, and embedded in Durcupan ACM.
Thick (1- 2 pm) and thin sections were cut with glass knives on a Reichert Ultracut ultramicrotome. Sections were stained with 1 % toluidine blue in 1 % borax for examination with a Wild M20 light microscope (L M) or with uranyl acetate and lead citrate for examination with a Philips EM 300 transmission electron microscope (T EM) . Some fixed pieces were dehydrated in absolute ethanol followed by a graded ethanol-amyl acetate series, and then dried in a DCP-1 (Denton Vacuum Inc. ) critical point dryer. Dried specimens were coated with gold in a Sputtering Hummer II (T echn ics) and examined using a Siemens Autoscan scanning electron microscope (SEM ). RESUL TS The T . magnatum ascus has a hemispherical apex and an elongated narrow base . Prior to ascospore delimitation, the ascus shows pronounced polarization (F ig. 1). The basal zone is filled with glycogen, whereas in the apex, which is the site of spore formation, nuclei, lipid droplets, numerous mitochondria and vacuoles, many of which contain electron-dense drop-like inclusions, are present (Fig. 2). Throughout this and following stages, the apical zone often contains vesicles with double membranes formed from invaginations of the ascus plasma membrane. At times , double membranes parallel to the ascus plasmalemma were also observed (F ig. 2). In some cases, they were extensions of vesicles and in others they originated directly from the ascus plasma membrane. Ascospores are initially irregular (Fig. 3) and later spherical (Fig. 6). They are delimited by two membranes (Figs 5, 7) and contain a nucleus, man y mitochondria, small vacuoles , endoplasmic reticulum and, occasionally, small lipid droplets (Figs 3, 7). The space between the delimiting membranes
202
Ascosporogenesis in Tuber magnatum
Graziella Berta and Anna Fusconi gradually increases and is filled by wall material which eventually forms the epispore. The outer membrane becomes the investing membrane and the inner becomes the plasma membrane of the developing ascospores. Following epispore formation the investing membrane extends and stretches irregularly. At first, the region between it and the epispore is virtually electron-transparent (Fig. 8) but later, fills with a floccular slightly electron-dense material (Figs 9, 10) to form the perispore. The epiplasm degenerates while the ascospores develop. The many, small epiplasmic vesicles which often contain spherical electron-dense inclusions, coalesce to form a single vacuole surrounding the ascospores. This vacuole contains membrane fragments, lipids and electron-dense clusters formed by fusion of the inclusions within the original vesicles (Fig. 8). The tonoplast becomes attached to the investing membranes, giving the impression that the spores are delimited by two outer membranes (Fig. 10). Deposition of electron-dense material into the peri spore begins when the investing membrane is most extended and is concurrent with deposition of the endospore between the epispore and the spore plasma membrane (Fig. 11). Theperisporecontracts during deposition and the electron-dense material becomes filamentous (Fig. 12). This material is probably derived from the epiplasmic vacuole and grows to form a network that is increasingly compressed outwards, to become dense and uniform in the mature spore. The endospore appears slightly stratified (Figs 14, 15) and is synthesized by the ascospore cytoplasm. During endospore deposition, the plasma membrane becomes convoluted, and lornasomes occur frequently between the plasma membrane and the forming wall (Figs 11-13).
203
SEM pictures of a mature spore show the characteristic honeycomb shaped ornamentations described by Malencon (1938). However, the ridges of the peri spore which form the honeycomb shapes are not straight, as Malencon thought, but slightly undulated (Fig. 16). The cytoplasm of mature ascospores is almost entirely filled with large lipid droplets (Fig. 15). DISCUSSION
Our results indicate that ascosporogenesis in T. magnatum resembles that found in practically all Euascomycetes (Beckett, 1981). The differences which occur during delimitation are related to ascus shape and organization: the Tuber ascus is pear-shaped and the sporogenous region is limited to the apex. Nuclei, mitochondria and other organelles form only a small part of the apex and are mostly found in the periphery. The remaining part of the apex is filled with vacuoles which probably force the nuclei and cytoplasm to the ascus periphery where the ascospores develop (Wells, 1972). The absence of an ascus vesicle in Tuber may result from this type of ascus organization. Although the double-membrane system adjacent to the ascus wall resembles the ascus vesicle it does not invaginate to delimit the ascospores. In agreement with Parguey-Leduc & janex-Favre (1977 a), it is suggested that Tuber ascospores are delimited individually by vesicles formed from invaginations of the ascus plasma membrane (Fig. 5). This type of development is normal in Hemiascomycetes but in the Euascomycetes it has been reported only in Ceratocystis fimbriata Ellis & Haist. (Stiers, 1976). However, in C. fimbriata delimitation may also arise from normal ascus vesicle development.
ABBREVIATIONS USED IN LEGENDS
AW, ascus wall; AZ, apical sporogenous zone; BZ, basal zone; EN, endospore; EP, epispore; EPL, epiplasm; ER, endoplasmic reticulum; G, glycogen; 1M, investing membrane; L, lipid droplets; M, mitochondria; PE, perispore; PM, spore plasma membrane; SC, spore cytoplasm; V, vacuoles with electron-dense inclusions. Figs 1-7. Tuber magnatum. Fig. 1. LM longitudinal section of ascus before ascospore differentiation. The apical sporogenous and basal zones are visible x 480. Fig. 2. TEM apical sporogenous zone of a young ascus. Note the double membrane parallel to the ascus plasma membrane (arrows) x 6900. Fig. 3. TEM recently formed ascospores with irregular shapes and bounded by the double delimiting membrane (arrows) x 8100. Fig. 4. TEM serial section of the spore of Fig. 3 showing vesicles forming from the ascus plasmalemma and which may be involved in the formation of the ascospore double delimiting membrane x 1200. Fig. 5. TEM part of the double delimiting membrane (arrow) of the ascospore in Fig. 3 x 37000. Fig. 6. TEM ascospore with rounded shape x 6200. Fig. 7. TEM double delimiting membrane of the ascospore in Fig. 6 x ~ 37000.
Ascosporogenesis in Tuber magnatum
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Graziella Berta and Anna Fusconi Delimitation is followed by progressive deterioration of the epiplasm. Epiplasmic vacuoles coalesce to form a single vacuole containing ascospores with the tonoplast often adjacent to the investing membrane. Increased vacuolization has been noted in Pyronema domesticum (Sowerby) Saccardo (Reeves, 1967), Ascobolus stercorarius (Bulliard) Schroeter (Wells, 1972), and in Saccobolus kervernii (Crouan) Boudier (Carroll, 1969). The tonoplast is frequently adpressed to the spore investing membrane, for example, in P. domesticum and T. magnatum and even more so in A . stercorarius. As in all Euascomycetes, wall formation in T. magnatum begins with pr imary wall material deposition between the two delimiting membranes. The subsequent phases of development depend on the species . The endospore is usually primary in origin, and material deposited subsequently is located between the endospore and the investing membrane (Delay, 1966; Mainwaring, 1972; Reeves, 1967; Wells, 1972). However, in T. magnatum the endospore is deposited in a second phase between the epispore and the spore plasma membrane. Similar centripetal sequences are not common in ascomycetes, although they have been observed in some yeasts, such as in Han senula anomala (Hansen) H . & P. Sydow (Bandoni, Bisalputra & Bisalputra, 1967) and in some Euascomycetes (Janex-Favre, 1978; Lutley & Wilson, 1972a , b; Schrantz, 1970 ; Stiers, 1974). Development in T. magnatum is the same as in T . mesentericum (Janex-Favre & Parguey-Leduc, 1980) but differs from T. rufum (j anex- Favre, 1977), where all the layers differentiate in th e region previously occupied by the primary wall. In T. magnatum and other Tuber species ascospore wall development is characterized by considerable expansion of the perispore. The stretched T. magnatum perispore greatly resembles the perispore sac in A. stercorarius (Wells, 1972), though the origin and development of the latter are different. In A. stercorarius the perispore sac is deposited immediately after the endospore and turns into a mucilaginous layer when mature. After stretching, the T. magnatum peri spore withdraws and fills with electron-dense material, which becomes more and more compact until it forms the
205
ornamental layer . The perispore in both T . rufum (Janex-Favre, 1977) and T. mesentericum (JanexFavre & Parguey-Leduc, 1980) forms the ornamental layer , whilst it disappears after stretching in T. aestivum , where the ornamental layer is formed by a layer which corresponds to the epispore in T. magnatum (Parguey-L educ & Janex-Favre, 1977b ). Although the ultrastructure of the ornamental layers are very similar, in species of Tuber, the view of [anex-Favre and Parguey-Leduc that ascospore development varies and heterogeneity exists in the genus Tuber, would seem to be correct. We thank Prof. S. Scannerini for his suggestions and critical reading of the manuscript and Dr Giovannetti for providing sporocarps. This work was the 265th by the Centro di Micologia del Terreno del Consiglio Nazionale delle Ricerche (C.N.R. ) of Turin. It was funded by C.N.R. and the University of Turin.
REFERENCES
ASHTON, M . L. & MOENS, P. B. (1979). Ultrastructure of sporulation in the Hemiascomycetes A scoidea corymbo sa, A . rubescens, Cephaloascus frag rans and Saccharomy copsts capsularts. Canadian Journal of Botany 57, 1191-1297. BANDONI, K . J., BISALPUTRA, A. A. & BISALPUTRA, T . (1967) . Ascospore development in Han senula anomala. Canadian J ournal of B otany 45, 361-366. BECKETT, A. (1981). Ascospore formation. In Th e Fungal Spor e: M orphogenetic Control s (ed . G. Turian & H. R . H ohl), pp . 107-129. New York : Academic Pre ss. BECKETT, A. & CRAWFORD, R. M . (1970). Nuclear behaviour and ascospore delimitation in X ylosphaera polymorpha. J ournal of General M icrobiology 63, 269-280 . BRACKER, C. E . (1967). Ultrastructure of fung i. A nnual Review of Phytopathology 5, 343-374. CAMPBELL, R. (1973). Ultrastructure of asci, ascospores, and spore release in Lophodermella sulcigena (Rostr .) v. Hohn, Protoplasma 78, 69-80. CARROLL, G . C. (1967). The ultrastructure of ascospore delimi tati on in Saccobolus keruerni, Journal of Cell Biology 33, 321-339· CARROLL, G. C. (1969) . A study of the fine structure of ascosporogenesis in Saccobolus kerverni. Archiv fur Mikrobiologie 66, 321-339.
Fig s 8-13. Tuber magnatum, TEM. Fig. 8. Dilation of the perispore. Note the coalescing vacuoles in the epiplasma x 4200. Fig s 9-10. The perispore fills with scanty electron-dense mate rial. The den se layer close to the plasma membrane forms the epispore x 7000. Fig . 11. Perispore at maximum dilation. Endospore synthesis has begun inside the pre -exi sting wall. Convoluted plasma membrane and some lomasomes (arrows) are visible x - 7000. Figs 12, 13. Developing ascospore wall showing the increased thickness of the endospore. The perispore shrinks and fills with electron-dense filamentous material x - 7000.
Ascosporogenesis in Tuber magnatum
206
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Graziella Berta and Anna Fusconi DELAY, C. (1966) . Etude de l'infrastructure de l'asque d'Ascobolus immersus Pers ., pendant la maturation des spores. Annales des Scien ces Naturelles Botanique et Biologie Vegetale, 8, 361-420. GREENHALGH, G . N . & GRIFFITHS, H. B. ( 1970). The ascus vesicle. Transactions of the British M y cologIcal Society 54, 489-492. JANEX-FAVRE, M. C. (1977 ). La paroi des ascospores du Tuber rufum Pico, Bulletin de la Societe mycologique de France 93, 407-424. JANEx-FAvRE, M . C. (1978). Formation et evolution des ascospores chez Ie Diatrype disciformis (H offin.) Fries, etude ultrastructurale. R evue de Mycologie 42, 265-275 . JANEX-FAVRE, M. C. & PARGUEY-LEDUC, A. (1976). La formation des ascospores chez deux Truffes: Tuber rufum Pico et Tuber aestiuum Vitt . (T uberacees). Comptes Rendus hebdomadaire des Seances de I' A cademie des sciences 283, 1173-1175. JANEX-FAVRE, M . C. & PARGUEY-LEDUC, A. (1980). Formation et evolution des ascospore du Tuber mesentericum Vitt. Bulletin de la Societe Mycologique de France 96 , 225-237· LUTLEY, M. & WILSON, I. M . ( 1972 a). Development and fine structure of ascospores in the marine fungus Ceriosporopsis halima. Transactions of the British M y cological Society 58, 393-402. LUTLEY, M. & WILSON, I. M . ( 1972 b). Ob servations on the fine structure of ascospores on the marine fungi: Halosphaeria appendiculata , Torpedospora radiata and Corollospora maritima. Transactions of the British My cological Socie ty 59, 219-227. MAINWARING, H . R. (1972). The fine structure of ascospore wall formation in Sordaria fim icola. Archie fur Mikrobtologie 81, 126--135.
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MALEN/yON, M . G . ( 1938). Les Truffes eur opeennes. Historique-Morphogenie-OrganographieClassification-Culture. R evue de Mycologie 3, 1--92. PARGUEY-LEDUC, A. & JANEx-FAVRE, M . C. (1977 a). L'organisarion des asques de deux truffes: Tuber rufum Pico et Tuber aestivum Vitt. Revue de Mycologie 41,1-32. PARGUEY-LEDUC, A. & JANEx-FAvRE, M . C. (1977b ). L'ornamentation des ascospores chez Ie Tuber aestivum Vitt. Extrait des trauaux dedies au Professeur Vienn ot-Bourgin. REEvEs, F. (1967). The fine structure of ascospore formation in Pyronema domesticum. Mycologia 59, 1018-1033. SCHRANTZ, J-P. (1970). Etude cytologique en microscopie optique et electronique, de quelques Ascomycetes. II. La paroi . Revue de Cytologie et de Biologie Vegeta le 33, 111-168. STIERS, D. L. ( 1974) . Fine structure of ascospore formation in Poroma punctata. Canadian Journal of Botany 52, 999-1°°3. STIERS, D. L. (1976 ). The fine structure of ascospore formation in Ceratocystis fimbriata. Canadian Journal of B otany 54,1714-1723. WELLS, K . (1972). Light and electron microscopic studies of Ascobolus stercorartus, II. Ascus and ascospore ontogeny. University of California Publications in B otany 62, 1--93. WILSENACH, R. & KESSEL, M. (1965). The role of lomasomes in wall formation in Peni cillium uermiculatum . J ournal of General MIcr obiology 4°,4°1-4°4.
(R eceiv ed for publication 10 S eptember 1981 )
Figs 14-16. Tuber magnatum. Fig. 14. TEM developing ascospore showing wall layers x 5800 . Fig. 15. TEM mature ascospore wall showing the slightly stratified endospore, the thin epispore and the uniform compact perispore x 7900. Fig . 16. SEM of a mature ascospore. The characteristic honeycomb ornamental layer is visible x 2000.
Trans. Br. mycol. Soc. 80 (2), 201-207 (1983)
]
Printed in Great Brita in
ASCOSPOROGENESIS IN TUBER MAGNATUM By GRAZIELLA BERTA AND ANNA FUSCONI Istituto Botanico and Centro di Microscopia Elettronica dell' Unioersita, Turin, Italy Ascosporogenesis in Tuber magnatum is intermediate between the general pattern of Euascomycetes and that of Hemiascomycetes. T . magnatum ascospores are delimited individually by two membranes which originate from the ascus plasmalemma; wall material is deposited between these membranes. The epispore is deposited first . The outer perispore is initially expanded but then contracts and fills with increasingly compact electron-dense material to form the surface ornamentations. The electron-transparent endospore is deposited inside the epispore. The general features of ascosporogenesis are well known and may be divided into three stages: (1) two membranes delimit uninucleate regions of cytoplasm; (2) spore primary wall material is deposited between these membranes; (3) the inner membrane becomes the spore plasma membrane and the outer one the investing membrane. The ascal cytoplasm outside the ascospores is termed the epiplasm. Subsequent phases of wall development vary according to the species (Beckett , 1981). The mechanisms of spore delimitation in Hemiascomycetes and Euascomycetes are very different. In the former, spores are delimited individually in a variety of ways (Ashton & Moens, 1979). In Euascomycetes, the nuclei and cytoplasm are enveloped by a common involucre, a double membrane or ascus vesicle at the periphery of the ascus . This then invaginates around individual nucle i to delimit the ascospores (Beckett & Crawford, 1970; Bracker, 1967; Campbell, 1973; Carroll, 1967, 1969; Greenhagh & Griffiths, 1970; Reeves , 1967; Schrantz, 1970; Stiers, 1974; Wells, 1972 ; Wilsenach & Kessel, 1965). However in T. aestivum Vitt., T. rufum Pico and T . mesentericum Vitt., the ascus vesicle is absent and their spores are delimited singly as in Hemiascomycetes (janexFavre & Parguey-Leduc, 1976, 1980; PargueyLeduc & Janex-Favre, 1977a). In this paper, ascosporogenesis in Tuber magnatum Pico, the most commonly eaten truffle in Italy, is described. MATERIALS AND METHODS Small pieces of T. magnatum ascocarps were fixed for 4 h with 2 % glutaraldehyde in 0'1 M-sodium cacodylate buffer at pH 7'2, postfixed for 2 h in 1 % OsOJ in the same buffer , deh ydrated through a graded alcohols series, and embedded in Durcupan ACM.
Thick (1- 2 pm) and thin sections were cut with glass knives on a Reichert Ultracut ultramicrotome. Sections were stained with 1 % toluidine blue in 1 % borax for examination with a Wild M20 light microscope (L M) or with uranyl acetate and lead citrate for examination with a Philips EM 300 transmission electron microscope (T EM) . Some fixed pieces were dehydrated in absolute ethanol followed by a graded ethanol-amyl acetate series, and then dried in a DCP-1 (Denton Vacuum Inc. ) critical point dryer. Dried specimens were coated with gold in a Sputtering Hummer II (T echn ics) and examined using a Siemens Autoscan scanning electron microscope (SEM ). RESUL TS The T . magnatum ascus has a hemispherical apex and an elongated narrow base . Prior to ascospore delimitation, the ascus shows pronounced polarization (F ig. 1). The basal zone is filled with glycogen, whereas in the apex, which is the site of spore formation, nuclei, lipid droplets, numerous mitochondria and vacuoles, many of which contain electron-dense drop-like inclusions, are present (Fig. 2). Throughout this and following stages, the apical zone often contains vesicles with double membranes formed from invaginations of the ascus plasma membrane. At times , double membranes parallel to the ascus plasmalemma were also observed (F ig. 2). In some cases, they were extensions of vesicles and in others they originated directly from the ascus plasma membrane. Ascospores are initially irregular (Fig. 3) and later spherical (Fig. 6). They are delimited by two membranes (Figs 5, 7) and contain a nucleus, man y mitochondria, small vacuoles , endoplasmic reticulum and, occasionally, small lipid droplets (Figs 3, 7). The space between the delimiting membranes
202
Ascosporogenesis in Tuber magnatum
Graziella Berta and Anna Fusconi gradually increases and is filled by wall material which eventually forms the epispore. The outer membrane becomes the investing membrane and the inner becomes the plasma membrane of the developing ascospores. Following epispore formation the investing membrane extends and stretches irregularly. At first, the region between it and the epispore is virtually electron-transparent (Fig. 8) but later, fills with a floccular slightly electron-dense material (Figs 9, 10) to form the perispore. The epiplasm degenerates while the ascospores develop. The many, small epiplasmic vesicles which often contain spherical electron-dense inclusions, coalesce to form a single vacuole surrounding the ascospores. This vacuole contains membrane fragments, lipids and electron-dense clusters formed by fusion of the inclusions within the original vesicles (Fig. 8). The tonoplast becomes attached to the investing membranes, giving the impression that the spores are delimited by two outer membranes (Fig. 10). Deposition of electron-dense material into the peri spore begins when the investing membrane is most extended and is concurrent with deposition of the endospore between the epispore and the spore plasma membrane (Fig. 11). Theperisporecontracts during deposition and the electron-dense material becomes filamentous (Fig. 12). This material is probably derived from the epiplasmic vacuole and grows to form a network that is increasingly compressed outwards, to become dense and uniform in the mature spore. The endospore appears slightly stratified (Figs 14, 15) and is synthesized by the ascospore cytoplasm. During endospore deposition, the plasma membrane becomes convoluted, and lornasomes occur frequently between the plasma membrane and the forming wall (Figs 11-13).
203
SEM pictures of a mature spore show the characteristic honeycomb shaped ornamentations described by Malencon (1938). However, the ridges of the peri spore which form the honeycomb shapes are not straight, as Malencon thought, but slightly undulated (Fig. 16). The cytoplasm of mature ascospores is almost entirely filled with large lipid droplets (Fig. 15). DISCUSSION
Our results indicate that ascosporogenesis in T. magnatum resembles that found in practically all Euascomycetes (Beckett, 1981). The differences which occur during delimitation are related to ascus shape and organization: the Tuber ascus is pear-shaped and the sporogenous region is limited to the apex. Nuclei, mitochondria and other organelles form only a small part of the apex and are mostly found in the periphery. The remaining part of the apex is filled with vacuoles which probably force the nuclei and cytoplasm to the ascus periphery where the ascospores develop (Wells, 1972). The absence of an ascus vesicle in Tuber may result from this type of ascus organization. Although the double-membrane system adjacent to the ascus wall resembles the ascus vesicle it does not invaginate to delimit the ascospores. In agreement with Parguey-Leduc & janex-Favre (1977 a), it is suggested that Tuber ascospores are delimited individually by vesicles formed from invaginations of the ascus plasma membrane (Fig. 5). This type of development is normal in Hemiascomycetes but in the Euascomycetes it has been reported only in Ceratocystis fimbriata Ellis & Haist. (Stiers, 1976). However, in C. fimbriata delimitation may also arise from normal ascus vesicle development.
ABBREVIATIONS USED IN LEGENDS
AW, ascus wall; AZ, apical sporogenous zone; BZ, basal zone; EN, endospore; EP, epispore; EPL, epiplasm; ER, endoplasmic reticulum; G, glycogen; 1M, investing membrane; L, lipid droplets; M, mitochondria; PE, perispore; PM, spore plasma membrane; SC, spore cytoplasm; V, vacuoles with electron-dense inclusions. Figs 1-7. Tuber magnatum. Fig. 1. LM longitudinal section of ascus before ascospore differentiation. The apical sporogenous and basal zones are visible x 480. Fig. 2. TEM apical sporogenous zone of a young ascus. Note the double membrane parallel to the ascus plasma membrane (arrows) x 6900. Fig. 3. TEM recently formed ascospores with irregular shapes and bounded by the double delimiting membrane (arrows) x 8100. Fig. 4. TEM serial section of the spore of Fig. 3 showing vesicles forming from the ascus plasmalemma and which may be involved in the formation of the ascospore double delimiting membrane x 1200. Fig. 5. TEM part of the double delimiting membrane (arrow) of the ascospore in Fig. 3 x 37000. Fig. 6. TEM ascospore with rounded shape x 6200. Fig. 7. TEM double delimiting membrane of the ascospore in Fig. 6 x ~ 37000.
Ascosporogenesis in Tuber magnatum
2°4
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-,
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Graziella Berta and Anna Fusconi Delimitation is followed by progressive deterioration of the epiplasm. Epiplasmic vacuoles coalesce to form a single vacuole containing ascospores with the tonoplast often adjacent to the investing membrane. Increased vacuolization has been noted in Pyronema domesticum (Sowerby) Saccardo (Reeves, 1967), Ascobolus stercorarius (Bulliard) Schroeter (Wells, 1972), and in Saccobolus kervernii (Crouan) Boudier (Carroll, 1969). The tonoplast is frequently adpressed to the spore investing membrane, for example, in P. domesticum and T. magnatum and even more so in A . stercorarius. As in all Euascomycetes, wall formation in T. magnatum begins with pr imary wall material deposition between the two delimiting membranes. The subsequent phases of development depend on the species . The endospore is usually primary in origin, and material deposited subsequently is located between the endospore and the investing membrane (Delay, 1966; Mainwaring, 1972; Reeves, 1967; Wells, 1972). However, in T. magnatum the endospore is deposited in a second phase between the epispore and the spore plasma membrane. Similar centripetal sequences are not common in ascomycetes, although they have been observed in some yeasts, such as in Han senula anomala (Hansen) H . & P. Sydow (Bandoni, Bisalputra & Bisalputra, 1967) and in some Euascomycetes (Janex-Favre, 1978; Lutley & Wilson, 1972a , b; Schrantz, 1970 ; Stiers, 1974). Development in T. magnatum is the same as in T . mesentericum (Janex-Favre & Parguey-Leduc, 1980) but differs from T. rufum (j anex- Favre, 1977), where all the layers differentiate in th e region previously occupied by the primary wall. In T. magnatum and other Tuber species ascospore wall development is characterized by considerable expansion of the perispore. The stretched T. magnatum perispore greatly resembles the perispore sac in A. stercorarius (Wells, 1972), though the origin and development of the latter are different. In A. stercorarius the perispore sac is deposited immediately after the endospore and turns into a mucilaginous layer when mature. After stretching, the T. magnatum peri spore withdraws and fills with electron-dense material, which becomes more and more compact until it forms the
205
ornamental layer . The perispore in both T . rufum (Janex-Favre, 1977) and T. mesentericum (JanexFavre & Parguey-Leduc, 1980) forms the ornamental layer , whilst it disappears after stretching in T. aestivum , where the ornamental layer is formed by a layer which corresponds to the epispore in T. magnatum (Parguey-L educ & Janex-Favre, 1977b ). Although the ultrastructure of the ornamental layers are very similar, in species of Tuber, the view of [anex-Favre and Parguey-Leduc that ascospore development varies and heterogeneity exists in the genus Tuber, would seem to be correct. We thank Prof. S. Scannerini for his suggestions and critical reading of the manuscript and Dr Giovannetti for providing sporocarps. This work was the 265th by the Centro di Micologia del Terreno del Consiglio Nazionale delle Ricerche (C.N.R. ) of Turin. It was funded by C.N.R. and the University of Turin.
REFERENCES
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Fig s 8-13. Tuber magnatum, TEM. Fig. 8. Dilation of the perispore. Note the coalescing vacuoles in the epiplasma x 4200. Fig s 9-10. The perispore fills with scanty electron-dense mate rial. The den se layer close to the plasma membrane forms the epispore x 7000. Fig . 11. Perispore at maximum dilation. Endospore synthesis has begun inside the pre -exi sting wall. Convoluted plasma membrane and some lomasomes (arrows) are visible x - 7000. Figs 12, 13. Developing ascospore wall showing the increased thickness of the endospore. The perispore shrinks and fills with electron-dense filamentous material x - 7000.
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(R eceiv ed for publication 10 S eptember 1981 )
Figs 14-16. Tuber magnatum. Fig. 14. TEM developing ascospore showing wall layers x 5800 . Fig. 15. TEM mature ascospore wall showing the slightly stratified endospore, the thin epispore and the uniform compact perispore x 7900. Fig . 16. SEM of a mature ascospore. The characteristic honeycomb ornamental layer is visible x 2000.