- Email: [email protected]
External ultrastructure of fruit body initiation in Morchella
Mycol. Res. 109 (4): 508–512 (April 2005). f The British Mycological Society
508
doi:10.1017/S0953756204002126 Printed in the United Kingdom.
External ultrastructure of fruit body initiation in Morchella
Segula MASAPHY MIGAL, Galilee Technology Centre, POB 831, Kiryat Shmona, 11016, Israel. E-mail : [email protected] Received 27 May 2004; accepted 26 November 2004.
The external morphological changes occurring during initiation and early stages of fruit body development of a Morchella sp., before the development of asci, were examined by scanning electron microscopy. Four stages of primordial development were distinguished. First, disk-shaped knots of 0.5–1.5 mm were observed on the surface of the substrate. Next, the knot inflated and a primordial stem emerged from its centre. Afterward, the stem lengthened, oriented upward, and two types of hyphal elements developed : long, straight and smooth basal hairy hyphae and short stem hyphae, some of which were inflated and projected out of a cohesive layer of tightly packed hyphal elements. Finally, when the stem was 2–3 mm long, pre-apothecia emerged in the apical end, with ridges and pits having distinguished types of paraphyses. Extracelluar mucilage covered the ridge layer and helped give the tissue its shape and rigidity.
INTRODUCTION The ascomycete genus Morchella (morels) is an economically important edible mushroom that enjoys much interest. Many researchers have attempted to raise morels in controlled conditions. The first to successfully grow morel fruit bodies under controlled conditions was Ower (1982). However, over 20 years later, it is still difficult to cultivate them despite many efforts by researchers and growers. The development of morel ascomata, therefore, is usually studied on specimens found in nature. Since morels appear in a wide range of habitats and only for a short period of time, fundamental studies concerning ascocarp formation are limited and there is a lack of information regarding its life cycle and controversy regarding fruit initiation. Morchella fruit bodies have a complex morphology. At maturity, the fruit body consists of a hollow stalk with a conical sponge-like head and many apothecia on the surface. Little is known on morphology of the early stages of morel fruit bodies, and the dynamics of the development process. Ower (1982), the first to describe the developmental stages of ascomata grown in a controlled chamber, used simple photography. His note on Morchella ascoma ontogeny was followed by the in-depth cytological studies of Volk & Leonard (1989, 1990). To study the morel life-cycle they followed the development of ascoma fruiting in association with tuberous begonias (Begonia tuberhybrida) in semicontrolled conditions, from very small primordia to
fully developed fruit bodies. However, in this system they could only follow developing ascomata and not the early stages of initiation. They also studied morphological changes after the germination of sclerotia on agar and found that hyphae aggregate in a pin-like shape, which they suggested to be the first stage of primordium formation although primordia formation from the aggregate was not observed. In the MIGAL (Galilee Technology Centre, Israel) research facilities, we were able to grow ascomata of morels (Morchella sp., local strain) in a controlled room. In this system, the initiation of fruit bodies could be observed with the naked eye as knots 0.5–1.5 mm diam. The growing system enabled us to follow initiation and primordial development from the first changes in morphology. To increase our fundamental understanding of Morchella biology and the processes leading to morel ascoma development, external morphological changes during the early developmental stages of primordia formation were followed with a scanning electron microscope (SEM) and are described for the first time in the present paper.
MATERIALS AND METHODS Fungus and cultural conditions Fruit bodies of a local strain of Morchella sp. (MIGAL 730) were grown in controlled conditions in the
S. Masaphy MIGAL laboratory, after re-germination of the fungal sclerotia, at temperature of 16–20 xC and relative humidity of 90–98 %. Voucher material is preserved in the MI6AL collection. Sample preparation for SEM observations Samples for SEM observation were prepared according to a modification of the protocol of Masaphy et al. (1987). Morel primordia, in different developmental stages from the first observed knot (0.5–1.5 mm diam) up to 5 mm long (2 d later), were cut out of the growing substrate and transferred to a solution of 4 % glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 2 h at 4 x. They were dehydrated with a series of increasing ethanol concentrations, for different lengths of time (0 ethanol, 1 h ; 25 %, 15 min ; 50 %, 15 min ; 75%, 15 min ; 95%, 15 min ; 100 %, 10–60 min ; 100%, 10–60 min). After dehydration, the samples were critical point dried through liquid CO2, adhered to stubs, coated with a gold-palladium layer, and observed under an SEM (JEOL JSM 35C, Tokyo).
RESULTS The dynamic morphological changes in the hyphal configurations of the Morchella primordium observed by SEM corresponded to four development stages. Many initial Morchella primordia were first observed by the naked eye as tiny hyphal knots on the surface of the substrate. When viewed with the SEM, this early stage of primordium was seen as a flat disk-like knot (Fig. 1), consisting of a sheaf of hyphal elements, starting from the centre of the knot. Within 24 h, the knot had inflated to produce a globular structure, and a stem began to emerge from the centre (Fig. 2). While the first knot appeared to form anywhere on the surface of the substrate, the emerged stem was oriented upward and away from its substrate. This orientation became more prominent as the stem elongated (Fig. 3). With the emergence and growth of the stem, the outer layer of the primordium (the enveloping hyphae) began to differentiate into two types of hyphae, distinguishing the stem from the basal region (Figs 3–4). The stem hyphae consisted of a dense layer of short hyphal elements that produced a cohesive layer oriented vertically and outward from the stem centre. Thick, inflated, wrinkled cells projected out between short hyphal elements composing the stem enveloping tissue (Fig. 5). The cells were closely packed and joined together by a mucilagenous matrix, giving the stem its shape and rigidity. The basal hyphae were hair-like, long, smooth, straight, loosely packed and oriented upward from its substrate (Fig. 6). When the stem was 2–3 mm long, many preapothecia began to differentiate on its apical region (Figs 7–8), producing two distinguished zones on the stem : the stalk and the head. At this stage, the stalk
509 became bulbous at its lower end. This inflation was clearly visible to the naked eye in all the primordia produced on the substrate, giving them a wider base for holding onto the substrate. Occasionally, while inflating, the lower part of the stalk broke apart naturally, producing cracks in the tissue (Fig. 7) that possibly indicate the beginning of the hollow stalk formation. The pre-apothecia (pits) appeared in the apical end of the stem represent the head zone. The head had several preapothecia in different stages of development that varied in size. The cells building the pre-apothecia pits, which lined the bottom of the pits, were parallel and vertical, forming a very dense cohesive layer, seen with the SEM (Fig. 9). At this stage, all the hyphal tips looked similar, and probably all corresponded to the paraphyses sterile cells layer that, on maturation, will also contain acsi that bear ascospores (Ower 1982, Volk & Leonard 1990, Janex-Favre et al. 1998). Few projected cells were located within the lining layer inside the pits, but the margins of the pre-apothecia pits were rich in inflated projected cells. The outer ridges consisted of a dense layer of short thick hyphal elements, combined with longer projected, wrinkled cells. An extracellular mucilagenous matrix layer covered the surfaces of the ridge tissue and the hyphal layer inside the pits, joining the individual cells into a cohesive layer (Fig. 10).
DISCUSSION The external morphological changes and cell type differentiation, which were observed by SEM, demonstrated the developmental dynamic process of early stages of initial fruit bodies of Morchella. The process included the first hyphal unified knot, and then the emergence of the stem that differentiated into two zones : the globular base and the elongated stem. Next, the stem continued differentiating into the stalk and head while pre-apothecia were produced at its apical end. The organization of the hyphal elements in the base, stalk and head of the Morchella primordia, was tightly ordered. Unlike the description of Ower (1982), no interwoven mycelium was observed on the base or the stem of the early primordium. The organization of the hyphal elements of the Morchella was different from the interwoven aggregation of the enveloping hyphae of other carpophoral primordia such as the basidiomycete Agaricus bisporus (Masaphy et al. 1987), Pleurotus pulmonarius (Moore 1998a) or other ascomycetes such as Sordaria humana (Read 1983). However, the arrangement of the enveloping hyphal in the early stages of the stem of the Morchella primordium resembles the arrangement of the hyphae in the neck of the primordial perithecium of S. humana (Read 1983). Interestingly, it also resembles the organization and cell types that build the cohesive layer of the gill surface of the fruit body of some agarics such as
Morchella primordium development
1
510
4
s
2
b
5 s
b 3 6
Figs 1–6. Fruit body initiation in Morchella. Fig. 1. Stage I of primordium formation : radial knot of 1 mm diam. Note the long, loose, hairy hyphal elements and the beginning of inflation at the centre of the knot. Bar=500 mm. Fig. 2. Stage II of primordium formation : emergence of the primordial stem. The stem is about 0.5 mm long. Note the beginning of differentiation of two types of hyphae, basal (b) and stem (s). Bar=1 mm. Fig. 3. Stage III of primordium formation : upward elongation of the stem. Bar=1 mm. Fig. 4. The interface tissue between the base (b) and the stem (s), showing the distinct difference between the two types of hyphae. Bar=200 mm. Fig. 5. Closely packed stem hyphal elements with both short and thick wrinkled projecting hyphal elements (paraphyses ; arrows). Note the extracellular mucilage layer on and between the hyphal elements. Bar=50 mm. Fig. 6. Long, straight and loosely packed basal hyphae. Bar=50 mm.
S. Masaphy
511
7 sk
Coprinopsis cinereus and Volvariella bombycina (Chiu & Moore 1990a, b, Moore 1998b). The similarity between the Morchella primordial surface and the gill surface includes the external tightly ordered arrangement of hyphal elements as well as the similarly shaped projected inflated cells that are present within the cohesive layer (Horner & Moore 1987, Chiu & Moore 1990a, b). In agarics, these inflated cells are termed cystidia, while in ascomycetes they are called paraphyses. Both cystidia and paraphyses are sterile cells that can vary from abundant to absent, depending on the species (Smith 1966, Kendrick 2000). In Pezizales, they can differ morphologically from cylinder to inflated cells. Paraphyses are usually located in the hymenial layer interspersed among the asci. However, in Morchella, beside being present in the hymenial layer lining the apothecia, they also were found on the surfaces of the young stem, the stalk and the sterile ridges in the head. They varied morphologically depending on their location. In the present study, three types of paraphyses were observed : short compact cells, projected cells and cells lining the pits. Similar observations were reported by Janex-Favre et al. (1998). In their study they distinguished between two types of paraphyses in M. deliciosa : primitive paraphyses which covered the whole smooth surface of the young cap and then were restricted to the ridges in the growing primordium, and hymenial paraphyses which lined the pits. The role of the projected inflated cells on the surface of the primordium is unknown. They are present in different tissues of the developing primordium and their role might differ in each tissue. They probably have a role similar to the inflated cystidia found on the gill surface. Moore (1998a) suggested that cystidia are highly differentiated cells for their particular function. He suggested that their role in C. cinereus is mechanical, giving the gill its shape in the stretched-skin construction. In V. bombycina, cystidia have been observed with aqueous droplets adhering to them, suggesting they are secretory cells (Chiu & Moore 1990b). In
c
b
8
9 a
10
a p
Figs 7–10. Fruit body initiation in Morchella. Fig. 7. Stage IV of primordium formation : early differentiation of the stem to stalk (sk), above the base (b), and the head (c) containing the pre-apothecia on the apical end. The large pit in the bottom of the stalk was occasionally observed in growing primordia. Bar=2 mm. Fig. 8. Close-up of pre-apothecial layer on the stem. Bar=200 mm. Fig. 9. Close-up of pre-apothecium. Note the densely-packed parallel hyphal paraphysal elements in the bottom of the pre-apothecia (a), and the projected cells on the edge of the pre-apothecium and ridge tissue. Bar=100 mm. Fig. 10. Close-up of the ridge and pit interface. Note the extracellular polysaccharide cover of the hyphal layer on the ridge (p), the parallel hyphal paraphysal elements on the bottom of the pre-apothecium (a), and the projecting paraphysal cells on the edge of the pre-apothecium. Bar=20 mm.
Morchella primordium development primordia of Morchella, the projected cells are suggested to have both roles. They may be the source of the mucilagenous matrix observed on and between the projected inflated cells, which bounds the hyphae together to produce the head tissue. The chemical nature of the mucilagenous layer was not determined in the present study. The mucilaginous coat on the asci of Morchella was reported by Samuelson (1978). Mucilaginous sheaths are common in primordia of other genera of fungi (Chaubal et al. 1991, Connolly et al. 1995), and are composed of polysaccharides and proteinaceous materials (Palmer, Murmanis & Highly 1983). It was also seen on the surface of S. humana by Read (1983), who suggested it is a polysaccharide. The role of the mucilage on the surface of the enveloping layer of Morchella primordia is probably to bind the hyphae together and give the tissue shape and rigidity. It could also protect the tissue against dehydration and an adverse environment. This might help Morchella initials survive changes in climatic conditions or humidity and temperature of the soil (Schmidt 1983, Buscot 1989, Goldway et al. 2000). The morphological changes shown here in early Morchella primordia seem to be a continuation of the developmental process observed by Volk & Leonard (1990) on an agar medium. They showed the aggregation and cohesion of aerial hyphae into a pin-shaped strand from sclerotial germination, and believed it to be the fruit body initial. In the present paper, and as shown by Ower (1982), the first 1 mm knot seems to originate from one central zone. Ower (1982) believed the knot originated from a single cell, but it could have originated from a strand, as observed by Volk & Leonard (1990). In their tentative summary of the life cycle of morel, Volk & Leonard (1990) suggested that the fruit body could arise from a primary mycelium or a secondary heterokaryotic mycelium. In their work on an agar medium, the pin-shaped strand observed by Volk & Leonard (1990) did not continue to develop and become primordia and fruit bodies. Further studies on the subsurface behaviour of the fungal mycelium on the growing substrate now need to be carried out to determine the morphological changes that occur between sclerotia germination and fruit body formation and to learn the complete life cycle of Morchella.
512 REFERENCES
ACKNOWLEDGEMENT
Buscot, F. (1989) Field observations on growth and development of Morchella rotunda and Mitrophora semilibra in relation to forest soil temperature. Canadian Journal of Botany 67: 589–593. Chaubal, R., Wilmot, V. A. & Wynn, W. K. (1991) Visualization, adhesiveness and cytochemistry of the extracellular matrix produced by urediniospore germ tube of Puccinia sorghi. Canadian Journal of Botany 69 : 2044–2054. Chiu, S. W. & Moore, D. (1990a) A mechanism for gill pattern formation in Coprinus cinereus. Mycological Research 94: 320–326. Chiu, S. W. & Moore, D. (1990b) Development of the basidiome of Volvariella bombycina. Mycological Research 94: 327–337. Connolly, J. H., Chen, Y. & Jellison, J. (1995) Environmental SEM observation of the hyphal sheath and mycofibrils in Postia placenta. Canadian Journal of Microbiology 41: 433–437. Goldway, M., Amir, R., Goldberg, D., Hadar, Y. & Levanon, D. (2000) Morchella conica exhibiting a long fruiting season. Mycological Research 104: 1000–1004. Horner, J. & Moore, D. (1987) Cystidial morphogenetic field in the hymenium of Coprinus cinereus. Transactions of the British Mycological Society 88: 479–488. Janex-Favre, M.-C., Parguey-Leduc, A. & Bruxelles, G. (1998) L’hymenium de Morchella deliciosa Fr. (Ascomycetes, Discomycetes). Crytogamie, Bryologie Lichenologie 19: 293–304. Kendrick, B. (2000) The Fifth Kingdom. 3rd edn. Mycologue Publications, Newburyport, MA. Masaphy, S., Levanon, D., Tchelet, R. & Henis, Y. (1987) Scanning electron microscope studies of interactions between Agaricus bisporus (Lang.) Sing. hyphae and casing soil bacteria. Applied Environmental Microbiology 53: 1132–1137. Moore, D. (1998a) Initiation of structures. In Fungal Morphology (D. Moore, ed.) : 246–281. Cambridge University Press, Cambridge, UK. Moore, D. (1998b) Tissue domains. In Fungal Morphology (D. Moore, ed.) : 317–358. Cambridge University Press, Cambridge, UK. Ower, R. (1982) Notes on the development of the morel ascocarp. Mycologia 74 : 142–144. Palmer, J. G., Murmanis, L. & Highley, T. L. (1983) Visualization of hyphal sheath in wood-decay hymenomycetes. I. Brown-rotter. Mycologia 75 : 995–1004. Read, N. D. (1983) A scanning electron microscopic study of the external features of perithecium development in Sordaria humana. Canadian Journal of Botany 61: 3217–3229. Samuelson, D. E. (1978) Asci of the Pezizales. VI. The apical apparatus of Morchella esculenta, Helvella crispa, and Rhizina undulate. General discussion. Canadian Journal of Botany 56: 3069–3082. Schmidt, E. (1983) Spore germination of and carbohydrate colonization by Morchella esculenta at different soil temperatures. Mycologia 75 : 870–875. Smith, A. H. (1966) The hyphal structure of the basidiocarp. In The Fungi: an advanced Treatise (G. C. Ainsworth & A. S. Sussman, eds) 2: 151–177. Academic Press, New York. Volk, T. J. & Leonard, T. J. (1989) Experimental studies on the morel. I. Hetrokaryon formation between mono ascosporous strains of Morchella. Mycologia 81 : 523–531. Volk, T. & Leonard, T. (1990) Cytology of the life-cycle of Morchella. Mycological Research 94: 399–406.
This work was supported by the Office of the Chief Scientist, Ministry of Agriculture, Israel.
Corresponding Editor: J. K. Stone
508
doi:10.1017/S0953756204002126 Printed in the United Kingdom.
External ultrastructure of fruit body initiation in Morchella
Segula MASAPHY MIGAL, Galilee Technology Centre, POB 831, Kiryat Shmona, 11016, Israel. E-mail : [email protected] Received 27 May 2004; accepted 26 November 2004.
The external morphological changes occurring during initiation and early stages of fruit body development of a Morchella sp., before the development of asci, were examined by scanning electron microscopy. Four stages of primordial development were distinguished. First, disk-shaped knots of 0.5–1.5 mm were observed on the surface of the substrate. Next, the knot inflated and a primordial stem emerged from its centre. Afterward, the stem lengthened, oriented upward, and two types of hyphal elements developed : long, straight and smooth basal hairy hyphae and short stem hyphae, some of which were inflated and projected out of a cohesive layer of tightly packed hyphal elements. Finally, when the stem was 2–3 mm long, pre-apothecia emerged in the apical end, with ridges and pits having distinguished types of paraphyses. Extracelluar mucilage covered the ridge layer and helped give the tissue its shape and rigidity.
INTRODUCTION The ascomycete genus Morchella (morels) is an economically important edible mushroom that enjoys much interest. Many researchers have attempted to raise morels in controlled conditions. The first to successfully grow morel fruit bodies under controlled conditions was Ower (1982). However, over 20 years later, it is still difficult to cultivate them despite many efforts by researchers and growers. The development of morel ascomata, therefore, is usually studied on specimens found in nature. Since morels appear in a wide range of habitats and only for a short period of time, fundamental studies concerning ascocarp formation are limited and there is a lack of information regarding its life cycle and controversy regarding fruit initiation. Morchella fruit bodies have a complex morphology. At maturity, the fruit body consists of a hollow stalk with a conical sponge-like head and many apothecia on the surface. Little is known on morphology of the early stages of morel fruit bodies, and the dynamics of the development process. Ower (1982), the first to describe the developmental stages of ascomata grown in a controlled chamber, used simple photography. His note on Morchella ascoma ontogeny was followed by the in-depth cytological studies of Volk & Leonard (1989, 1990). To study the morel life-cycle they followed the development of ascoma fruiting in association with tuberous begonias (Begonia tuberhybrida) in semicontrolled conditions, from very small primordia to
fully developed fruit bodies. However, in this system they could only follow developing ascomata and not the early stages of initiation. They also studied morphological changes after the germination of sclerotia on agar and found that hyphae aggregate in a pin-like shape, which they suggested to be the first stage of primordium formation although primordia formation from the aggregate was not observed. In the MIGAL (Galilee Technology Centre, Israel) research facilities, we were able to grow ascomata of morels (Morchella sp., local strain) in a controlled room. In this system, the initiation of fruit bodies could be observed with the naked eye as knots 0.5–1.5 mm diam. The growing system enabled us to follow initiation and primordial development from the first changes in morphology. To increase our fundamental understanding of Morchella biology and the processes leading to morel ascoma development, external morphological changes during the early developmental stages of primordia formation were followed with a scanning electron microscope (SEM) and are described for the first time in the present paper.
MATERIALS AND METHODS Fungus and cultural conditions Fruit bodies of a local strain of Morchella sp. (MIGAL 730) were grown in controlled conditions in the
S. Masaphy MIGAL laboratory, after re-germination of the fungal sclerotia, at temperature of 16–20 xC and relative humidity of 90–98 %. Voucher material is preserved in the MI6AL collection. Sample preparation for SEM observations Samples for SEM observation were prepared according to a modification of the protocol of Masaphy et al. (1987). Morel primordia, in different developmental stages from the first observed knot (0.5–1.5 mm diam) up to 5 mm long (2 d later), were cut out of the growing substrate and transferred to a solution of 4 % glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 2 h at 4 x. They were dehydrated with a series of increasing ethanol concentrations, for different lengths of time (0 ethanol, 1 h ; 25 %, 15 min ; 50 %, 15 min ; 75%, 15 min ; 95%, 15 min ; 100 %, 10–60 min ; 100%, 10–60 min). After dehydration, the samples were critical point dried through liquid CO2, adhered to stubs, coated with a gold-palladium layer, and observed under an SEM (JEOL JSM 35C, Tokyo).
RESULTS The dynamic morphological changes in the hyphal configurations of the Morchella primordium observed by SEM corresponded to four development stages. Many initial Morchella primordia were first observed by the naked eye as tiny hyphal knots on the surface of the substrate. When viewed with the SEM, this early stage of primordium was seen as a flat disk-like knot (Fig. 1), consisting of a sheaf of hyphal elements, starting from the centre of the knot. Within 24 h, the knot had inflated to produce a globular structure, and a stem began to emerge from the centre (Fig. 2). While the first knot appeared to form anywhere on the surface of the substrate, the emerged stem was oriented upward and away from its substrate. This orientation became more prominent as the stem elongated (Fig. 3). With the emergence and growth of the stem, the outer layer of the primordium (the enveloping hyphae) began to differentiate into two types of hyphae, distinguishing the stem from the basal region (Figs 3–4). The stem hyphae consisted of a dense layer of short hyphal elements that produced a cohesive layer oriented vertically and outward from the stem centre. Thick, inflated, wrinkled cells projected out between short hyphal elements composing the stem enveloping tissue (Fig. 5). The cells were closely packed and joined together by a mucilagenous matrix, giving the stem its shape and rigidity. The basal hyphae were hair-like, long, smooth, straight, loosely packed and oriented upward from its substrate (Fig. 6). When the stem was 2–3 mm long, many preapothecia began to differentiate on its apical region (Figs 7–8), producing two distinguished zones on the stem : the stalk and the head. At this stage, the stalk
509 became bulbous at its lower end. This inflation was clearly visible to the naked eye in all the primordia produced on the substrate, giving them a wider base for holding onto the substrate. Occasionally, while inflating, the lower part of the stalk broke apart naturally, producing cracks in the tissue (Fig. 7) that possibly indicate the beginning of the hollow stalk formation. The pre-apothecia (pits) appeared in the apical end of the stem represent the head zone. The head had several preapothecia in different stages of development that varied in size. The cells building the pre-apothecia pits, which lined the bottom of the pits, were parallel and vertical, forming a very dense cohesive layer, seen with the SEM (Fig. 9). At this stage, all the hyphal tips looked similar, and probably all corresponded to the paraphyses sterile cells layer that, on maturation, will also contain acsi that bear ascospores (Ower 1982, Volk & Leonard 1990, Janex-Favre et al. 1998). Few projected cells were located within the lining layer inside the pits, but the margins of the pre-apothecia pits were rich in inflated projected cells. The outer ridges consisted of a dense layer of short thick hyphal elements, combined with longer projected, wrinkled cells. An extracellular mucilagenous matrix layer covered the surfaces of the ridge tissue and the hyphal layer inside the pits, joining the individual cells into a cohesive layer (Fig. 10).
DISCUSSION The external morphological changes and cell type differentiation, which were observed by SEM, demonstrated the developmental dynamic process of early stages of initial fruit bodies of Morchella. The process included the first hyphal unified knot, and then the emergence of the stem that differentiated into two zones : the globular base and the elongated stem. Next, the stem continued differentiating into the stalk and head while pre-apothecia were produced at its apical end. The organization of the hyphal elements in the base, stalk and head of the Morchella primordia, was tightly ordered. Unlike the description of Ower (1982), no interwoven mycelium was observed on the base or the stem of the early primordium. The organization of the hyphal elements of the Morchella was different from the interwoven aggregation of the enveloping hyphae of other carpophoral primordia such as the basidiomycete Agaricus bisporus (Masaphy et al. 1987), Pleurotus pulmonarius (Moore 1998a) or other ascomycetes such as Sordaria humana (Read 1983). However, the arrangement of the enveloping hyphal in the early stages of the stem of the Morchella primordium resembles the arrangement of the hyphae in the neck of the primordial perithecium of S. humana (Read 1983). Interestingly, it also resembles the organization and cell types that build the cohesive layer of the gill surface of the fruit body of some agarics such as
Morchella primordium development
1
510
4
s
2
b
5 s
b 3 6
Figs 1–6. Fruit body initiation in Morchella. Fig. 1. Stage I of primordium formation : radial knot of 1 mm diam. Note the long, loose, hairy hyphal elements and the beginning of inflation at the centre of the knot. Bar=500 mm. Fig. 2. Stage II of primordium formation : emergence of the primordial stem. The stem is about 0.5 mm long. Note the beginning of differentiation of two types of hyphae, basal (b) and stem (s). Bar=1 mm. Fig. 3. Stage III of primordium formation : upward elongation of the stem. Bar=1 mm. Fig. 4. The interface tissue between the base (b) and the stem (s), showing the distinct difference between the two types of hyphae. Bar=200 mm. Fig. 5. Closely packed stem hyphal elements with both short and thick wrinkled projecting hyphal elements (paraphyses ; arrows). Note the extracellular mucilage layer on and between the hyphal elements. Bar=50 mm. Fig. 6. Long, straight and loosely packed basal hyphae. Bar=50 mm.
S. Masaphy
511
7 sk
Coprinopsis cinereus and Volvariella bombycina (Chiu & Moore 1990a, b, Moore 1998b). The similarity between the Morchella primordial surface and the gill surface includes the external tightly ordered arrangement of hyphal elements as well as the similarly shaped projected inflated cells that are present within the cohesive layer (Horner & Moore 1987, Chiu & Moore 1990a, b). In agarics, these inflated cells are termed cystidia, while in ascomycetes they are called paraphyses. Both cystidia and paraphyses are sterile cells that can vary from abundant to absent, depending on the species (Smith 1966, Kendrick 2000). In Pezizales, they can differ morphologically from cylinder to inflated cells. Paraphyses are usually located in the hymenial layer interspersed among the asci. However, in Morchella, beside being present in the hymenial layer lining the apothecia, they also were found on the surfaces of the young stem, the stalk and the sterile ridges in the head. They varied morphologically depending on their location. In the present study, three types of paraphyses were observed : short compact cells, projected cells and cells lining the pits. Similar observations were reported by Janex-Favre et al. (1998). In their study they distinguished between two types of paraphyses in M. deliciosa : primitive paraphyses which covered the whole smooth surface of the young cap and then were restricted to the ridges in the growing primordium, and hymenial paraphyses which lined the pits. The role of the projected inflated cells on the surface of the primordium is unknown. They are present in different tissues of the developing primordium and their role might differ in each tissue. They probably have a role similar to the inflated cystidia found on the gill surface. Moore (1998a) suggested that cystidia are highly differentiated cells for their particular function. He suggested that their role in C. cinereus is mechanical, giving the gill its shape in the stretched-skin construction. In V. bombycina, cystidia have been observed with aqueous droplets adhering to them, suggesting they are secretory cells (Chiu & Moore 1990b). In
c
b
8
9 a
10
a p
Figs 7–10. Fruit body initiation in Morchella. Fig. 7. Stage IV of primordium formation : early differentiation of the stem to stalk (sk), above the base (b), and the head (c) containing the pre-apothecia on the apical end. The large pit in the bottom of the stalk was occasionally observed in growing primordia. Bar=2 mm. Fig. 8. Close-up of pre-apothecial layer on the stem. Bar=200 mm. Fig. 9. Close-up of pre-apothecium. Note the densely-packed parallel hyphal paraphysal elements in the bottom of the pre-apothecia (a), and the projected cells on the edge of the pre-apothecium and ridge tissue. Bar=100 mm. Fig. 10. Close-up of the ridge and pit interface. Note the extracellular polysaccharide cover of the hyphal layer on the ridge (p), the parallel hyphal paraphysal elements on the bottom of the pre-apothecium (a), and the projecting paraphysal cells on the edge of the pre-apothecium. Bar=20 mm.
Morchella primordium development primordia of Morchella, the projected cells are suggested to have both roles. They may be the source of the mucilagenous matrix observed on and between the projected inflated cells, which bounds the hyphae together to produce the head tissue. The chemical nature of the mucilagenous layer was not determined in the present study. The mucilaginous coat on the asci of Morchella was reported by Samuelson (1978). Mucilaginous sheaths are common in primordia of other genera of fungi (Chaubal et al. 1991, Connolly et al. 1995), and are composed of polysaccharides and proteinaceous materials (Palmer, Murmanis & Highly 1983). It was also seen on the surface of S. humana by Read (1983), who suggested it is a polysaccharide. The role of the mucilage on the surface of the enveloping layer of Morchella primordia is probably to bind the hyphae together and give the tissue shape and rigidity. It could also protect the tissue against dehydration and an adverse environment. This might help Morchella initials survive changes in climatic conditions or humidity and temperature of the soil (Schmidt 1983, Buscot 1989, Goldway et al. 2000). The morphological changes shown here in early Morchella primordia seem to be a continuation of the developmental process observed by Volk & Leonard (1990) on an agar medium. They showed the aggregation and cohesion of aerial hyphae into a pin-shaped strand from sclerotial germination, and believed it to be the fruit body initial. In the present paper, and as shown by Ower (1982), the first 1 mm knot seems to originate from one central zone. Ower (1982) believed the knot originated from a single cell, but it could have originated from a strand, as observed by Volk & Leonard (1990). In their tentative summary of the life cycle of morel, Volk & Leonard (1990) suggested that the fruit body could arise from a primary mycelium or a secondary heterokaryotic mycelium. In their work on an agar medium, the pin-shaped strand observed by Volk & Leonard (1990) did not continue to develop and become primordia and fruit bodies. Further studies on the subsurface behaviour of the fungal mycelium on the growing substrate now need to be carried out to determine the morphological changes that occur between sclerotia germination and fruit body formation and to learn the complete life cycle of Morchella.
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This work was supported by the Office of the Chief Scientist, Ministry of Agriculture, Israel.
Corresponding Editor: J. K. Stone