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(American Journal of Botany. 1999;86:780-784.)
© 1999 Botanical Society of America, Inc.

The morphology of Cercophora palmicola (Lasiosphaeriaceae)1

Richard T. Hanlin

Department of Plant Pathology, University of Georgia, Athens, Georgia 30602-7274

Received for publication August 10, 1998. Accepted for publication December 8, 1998.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
A detailed study of ascomal morphology and development in Cercophora palmicola showed that ontogeny is ascohymeniaceous, giving rise to an ostiolate perithecium. Ascomal initials consist of a coiled ascogonium surrounded by several layers of hyphae whose cells become pseudoparenchymatous. The centrum of the young ascoma is composed of a few rows of large, thin-walled pseudoparenchymatous cells that line the ascomal wall, with the central region filled by tightly packed, filamentous paraphyses. The ascogenous system forms along the inside of the layer of pseudoparenchymatous cells at the base of the paraphyses and gives rise to unitunicate asci that grow up among the paraphyses. The wall of the mature perithecium is greatly thickened. It is composed of three regions: a thin outer region of darkly pigmented, angular cells with thickened walls; a broad central region of cells with gelatinized walls; and a thin inner region of flattened cells. Ascomal ontogeny in C. palmicola conforms well to the Sordaria type of development, as defined by Huang.

Key Words: ascomal development • Ascomycota • centrum ontogeny • Lasiosphaeriaceae • Sordariaceae • Sordariales


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Cercophora palmicola Hanlin & Tortolero is a sordariaceous perithecial ascomycete that occurs on palm stems in the tropics. It possesses several unusual morphological features that were noted in the original description (Hanlin and Tortolero, 1987 ), but which were not described in detail. The purpose of this paper is to provide additional information on the morphology of C. palmicola and to compare it with information published on related species.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
The material used in this study was collected on fallen, decaying stems of an unidentified palm in a wet forest at ~1500 m altitude near Sanare, Estado Lara, Venezuela. Some material was studied while fresh, then preserved as semipermanent mounts with lactophenol for future reference. Attempts to grow the fungus in culture were unsuccessful, thus the study was conducted on field-collected material. Most observations were made on sectioned material. Specimens to be sectioned were killed and fixed in the field in formalin-propionic acid-alcohol, and later dehydrated in a tertiary butyl alcohol series and embedded in Oxford Paraplast® Plus. Embedded material was sectioned at 6 or 8 µm on a rotary microtome, and the sections were mounted on standard microscope slides with Haupt's adhesive, then deparaffined and stained in iron hematoxylin. These procedures have been described in more detail previously (Hanlin and Tortolero, 1988 ). Paraffin sections to be examined under the scanning electron microscope (SEM) were cut at 10 µm and mounted on an 18-mm2 cover glass with Haupt's Adhesive (Gaudet and Kokko, 1984 ). After removal of the paraffin in xylene, the cover glass was affixed to a stub with silver paint, then coated with gold-paladium in a Hummer sputter coater. Prepared stubs were examined in a Phillips 505 SEM. Scanning electron micrographs were taken on Polaroid Type 55 P/N film, and light micrographs were taken either on a Zeiss Ultraphot with Polaroid Type 55 P/N film or on a Nikon Optiphot with Kodak technical pan film 2415. Both microscopes are equipped with Nomarski interference optics.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
The mycelium of C. palmicola grew internally in the host stem and became erumpent through the epidermis. The mycelium emerged through the epidermis as a broad layer of compact, parallel hyphae, which were oriented perpendicular to the long axis of the stem (Fig. 1). As this layer of hyphae elongated the epidermis broke and was pushed outward. When and how the fungus invaded the stem is not known, but this is presumed to have occurred after death of the host tissues. The hyphae in the inner cells of the cortex were hyaline and thin-walled, and they grew parallel to the long axis of the stem. In the outer rows of the cortex and in the epidermal cells, however, the hyphae became light brown and much-branched as they proliferated to completely fill the cells of these tissues (Figs. 2–3). Most of the host cell walls remained intact and the fungus mycelium was clearly visible inside them. After the hyphae emerged, they grew laterally along the surface of the stem epidermis, on the intact cuticle, where they formed a thin, compact, stromatic layer composed of dark-brown, interwoven hyphae. The ascomata formed directly on this external layer of hyphae.



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  		Figs. 1–10. Centrum morphology in Cercophora palmicola. 1. Compact parallel hyphae emerging through host epidermis and expanding laterally. Bar =100 µm. X138. 2. Knotted hyphae in host epidermal cells. Bar = 20 µm. X831. 3. Host epidermal and cortex cells filled with hyphae; several layers of hyphae are present on exterior of host tissues, outside the intact cuticle. Bar = 20 µm. X850. 4. Young ascoma with central ascogonial coil containing a binucleate cell. Bar = 30 µm. X577. 5. Coiled initials inside an incipient ascoma. Bar = 20 µm. X867. 6. Ascogonial cells (two binucleate) inside young ascoma. Bar = 15 µm. X1067. 7. Multinucleate hypha in center of young ascoma. Bar = 20 µm. X825. 8. Median section through young ascoma showing central paraphyses surrounded by a region of thin-walled pseudoparenchyma inside wall. Bar = 30 µm. X447. 9. Enlarged view of paraphyses in Fig. 8. Bar = 20 µm. X894. 10. Close-up of filamentous paraphyses and some large pseudoparenchyma cells. Bar = 20 µm. X920.

 
Ascomal development
Although some coiled hyphae were observed on the external mycelium, the earliest stages in ascomal development clearly recognizable as such were small, spherical, incipient ascomata (Fig. 4). These were composed of a central ascogonium surrounded by hyphae and they formed on the surface of the mycelium. In the smallest ascomata the ascogonium was in the form of a loose coil, which was surrounded by several layers of hyaline, thin-walled, isodiametric pseudoparenchyma cells (Fig. 4). In larger ascomata the cells of the ascogonium were larger and septate, with deeply staining nuclei (Figs. 5–7), and the outermost cells of the ascoma were thick-walled and pigmented, so that the exterior was dark brown. At least one cell in each coil was binucleate (Fig. 6).

The young ascoma increased greatly in size, forming a thick wall and a centrum filled with paraphyses and pseudoparenchyma.

Centrum structure
The inside of the perithecial wall was lined with a few rows of large, thin-walled pseudoparenchyma cells (Figs. 11, 12, 16, 18). This layer was thickest just beneath the ostiole, becoming thinner as it extended down the sides of the ascoma. Along the base of the centrum these cells were smaller, and they constituted a subhymenial pseudoparenchyma. Arising from the upper surface of the subhymenial pseudoparenchyma were numerous, filamentous, septate paraphyses (Fig. 14) that grew upward into, and filled, the interior of the ascoma as a compact mass (Figs. 8–10). These paraphyses could be readily dissected as a cluster from the ascomata at this stage of development. As they aged, the basal cells of the paraphyses swelled somewhat, so that the paraphyses tapered toward the apex. The ascogenous system lay at the interface of the paraphyses and the subhymenial pseudoparenchyma at the base of the centrum, where it was evident as a thin layer of deeply stained cells. As the asci developed, they grew up among the paraphyses, many of which subsequently disintegrated. The pressure of the developing asci also resulted in the crushing of the pseudoparenchyma cells inside the ascomal wall, so that they were not observed in fully mature ascomata.



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  		Figs. 11–20. Ascomal morphology in Cercophora palmicola. 11. Nonmedian section through developing ascoma with centrum filled with paraphyses surrounded by several rows of large, thin-walled pseudoparenchyma cells. Deeply staining ascogenous cells are visible at base of paraphyses, inside the layer of pseudoparenchyma. Bar = 50 µm. X267. 12. Oblique section through centrum pseudoparenchyma cells. Bar = 50 µm. X262. 13. Median section through ascoma with young asci growing up among the paraphyses. Note the thick ascomal wall. Bar = 150 µm. X96. 14. Septate, filiform paraphyses, tapered toward the tips. Bar = 12 µm. X1273. 15. Section through cluster of perithecia on basal hyphae. Bar = 40 µm. X417. 16. Section through developing ascoma showing the thick wall and centrum tissues. SEM. Bar = 30 µm. X523. 17. Close-up of ascomal wall composed of three regions: an outer layer of thick-walled, darkly pigmented, angular to flattened cells; a broad central region of cells with gelatinized walls and small lumina; and a thin, inner layer of flattened cells, inside of which are large pseudoparenchyma cells of the centrum. Bar = 50 µm. X357. 18. Section through ascomal wall as seen with SEM. Note the large, thin-walled pseudoparenchyma cells of the centrum. Bar = 40 µm. X404. 19. Close-up of papillate ostiole lined with periphyses. Bar = 50 µm. X350. 20. Angular cells comprising outermost layer of ascomal wall. Bar = 20 µm. X914.

 
Wall structure
The wall of the mature ascoma was thick and comprised nearly one-half the total width of the ascoma (Figs. 13, 16). It was composed of three distinct regions of cells (Figs. 17–18). The outermost wall region was 3–5 cells thick and consisted of angular pseudoparenchyma cells with a dark-brown wall (Fig. 20). The middle region consisted of 8–10 rows of cells that had greatly thickened, gelatinized walls and a small lumen; they were subhyaline to slightly yellowish in color. This region was also traversed by slender, dark-staining hyphae. The innermost wall region consisted of 3–4 rows of cells with lightly pigmented walls. Along the sides of the perithecium these cells were flattened, but at the base they became isodiametric and they extended through the middle wall region to the outer wall layer. The ascomal wall was thicker in the upper one-fourth of the ascoma than along the sides. The wall was perforated at the apex by an ostiole that was lined with slender periphyses (Fig. 19). On the exterior, the ostiole extended as a short papilla surrounded by parallel hyphae with dark-brown walls.

Ascospore development
When first delimited, ascospore initials were cylindrical and straight, with a long, tapering, gelatinous appendage at each end (Fig. 21). A septum formed in the upper one-third of the ascospore, and the upper cell enlarged until it was ~50% wider than the lower cell (Figs. 22, 23). A second septum occasionally formed in the lower cell, which often became curved. The upper cell then became dark brown (Fig. 24); the lower cell remained hyaline and usually disintegrated and fell off. When this occurred the remaining spore was one-celled, dark brown, and ventricose, with a truncate base (Fig. 25) and a rounded apex containing a germ pore (Fig. 27). Unpigmented ascospores were capable of germinating from both cells (Fig. 28). The narrowly clavate, unitunicate asci possessed an apical ring that did not stain in iodine (Fig. 26).



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  		Figs. 21–28. Ascospore development in Cercophora palmicola. 21. Newly delimited, cylindrical ascospore initial with terminal gelatinous appendages. Bar = 5 µm. X2000. 22. Older ascospore with enlarged upper portion. Bar = 8 µm. X1321. 23. Two-celled ascospore with terminal appendages. Bar = 8 µm. X1667. 24. Two- celled ascospore with pigmented upper cell and disintegrating, hyaline lower cell. Bar = 15 µm. X800. 25. Pigmented upper cell of ascospore, truncate at base where dehisced lower cell was attached. Bar = 15 µm. X686. 26. Structure of ascus of immature ascus. Bar = 4 µm. X2500. 27. Germ pore in end of pigmented upper cell of ascospore. Bar = 8 µm. X1600. 28. Germinated unpigmented, two-celled ascospore, with a phialide-like cell at end of uppermost germ tube. Bar = 8 µm. X1545.

 
Ascomata usually formed in a dense layer on the surface of the external mycelium, completely concealing it, but some isolated ascomata also occurred. Isolated ascomata were usually turbinate to subglobose in shape (Fig. 13), but when crowded together the ascomata may be oval and more elongated (Fig. 15).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Ascomal morphology and ontogeny in C. palmicola conform well to the Sordaria-type pattern of ascomal development, as defined by Huang (1976) in his study of Triangularia backusii L. H. Huang [{equiv} Apiosordaria backusii (L. H. Huang) Guarro]. This ontogenetic pattern is ascohymenial and is characterized by the formation of coiled ascomal intials that are enveloped by hyphae that will form the ascomal wall. The centrum of the developing ascoma is composed of several layers of large, thin-walled pseudoparenchyma cells that line the inside of the ascomal wall, with the central region being filled by a compact mass of slender paraphyses that arise from the basal region of the centrum. Asci subsequently form from a basal layer of ascogenous cells and grow up among the paraphyses.

Besides Apiosordaria and Cercophora, the Sordaria-type development has been described in Anixiella (Uecker, 1979 ), Gelasinospora (Ellis, 1960 ; Mai, 1977 ; Jensen, 1982 ; Sanni, 1982 ), Nemania (as Hypoxylon) (Jensen, 1981 ), Neurospora (Nelson and Backus, 1968 ), Podospora (Mai, 1976 ), Sordaria (Uecker, 1976 ; Mai, 1977 ; Sanni, 1984 ; Read and Beckett, 1985 ), and Triangularia (Moreau and Moreau, 1950 ). Of these genera, all are classified in the Sordariales, except Nemania, which is placed in the Xylariales (Eriksson and Hawksworth, 1993 ). Within the Sordariales, Apiosordaria, Cercophora, Podospora, and Triangularia are placed in the Lasiosphaeriaceae, whereas Anixiella, Gelasinospora, Neurospora, and Sordaria are placed in the Sordariaceae. This study supports the high degree of correlation between the families Lasiosphaeriaceae and Sordariaceae and the Sordaria type of ascomal ontogeny.

Luttrell (1951) used centrum morphology as the basis for distinguishing orders in his reclassification of the perithecioid ascomycetes. Subsequent studies, however, indicated that the correlation between centrum morphology and the presumed phylogenetic affinities of various genera and species is not absolute, causing more recent authors (Eriksson and Hawksworth, 1993 ) to abandon this character as a valid taxonomic criterion. There is, nonetheless, a high degree of correlation between the pattern of centrum morphology and particular families, as evidenced by the Sordariaceae.


   FOOTNOTES
 
1 The materials used in this study were collected under the auspices of NSF US/Venezuela Grant INT-8501713. The author thanks Jimmy O. Owens for histology and Alan H. Icard for photography. Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Ellis, J. J. 1960 Plasmogamy and ascocarp development in Gelasinospora calospora. Mycologia 52: 557–573.

Eriksson, O. E., and D. L. Hawksworth. 1993 Outline of the ascomycetes — 1993. Systema Ascomycetum 12: 51–257.

Gaudet, D. A., and E. G. Kokko. 1984 Application of scanning electron microscopy to paraffin-embedded plant tissues to study invasive processes of plant-pathogenic fungi. Phytopathology 74: 1078–1080.[ISI]

Hanlin, R. T., and O. Tortolero. 1987 A new species and a new combination in Cercophora. Mycotaxon 30: 407–416.

———, and ———. 1988 Morphology of Sclerotium coffeicola, a tropical foliar pathogen. Canadian Journal of Botany 67: 1852–1860.

Huang, L. H. 1976 Developmental morphology of Triangularia backusii (Sordariaceae). Canadian Journal of Botany 54: 250–267.

Jensen, J. D. 1981 The developmental morphology of Hypoxylon serpens in culture. Canadian Journal of Botany 59: 40–49.

———. 1982 The development of Gelasinospora reticulospora. Mycologia 74: 724–737.

Luttrell, E. S. 1951 Taxonomy of the pyrenomycetes. University of Missouri Studies 24: 1–120.

Mai, S. H. 1976 Morphological studies in Podospora anserina. American Journal of Botany 63: 821–825.

———. 1977 Morphological studies in Sordaria fimicola and Gelasinospora longispora. American Journal of Botany 64: 489–495.

Moreau, F., and Mme. Moreau. 1950 Étude du développement du Triangularia Bambusae (van Beyma) Boedijn. Revue de Mycologie 15: 146–158.

Nelson, A. C., and M. P. Backus. 1968 Ascocarp development in two homothallic Neurosporas. Mycologia 60: 16–28.

Read, N. D., and A. Beckett. 1985 The anatomy of the mature perithecium in Sordaria humana and its significance for fungal multicellular development. Canadian Journal of Botany 63: 281–296.

Sanni, M. O. 1982 Perithecium development of Gelasinospora longispora. Botanical Gazette 143: 542–545.

———. 1984 Perithecium development in Sordaria brevicollis. Nordic Journal of Botany 4: 65–69.

Uecker, F. A. 1976 Development and cytology of Sordaria humana. Mycologia 68: 30–46.

———. 1979 Development and cytology of Anixiella endodonta. Botanical Gazette 140: 310–317.





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