Mycologia, 95(3), 2003, pp. 506-512.
© 2003 by The Mycological Society of America
Conidioma development in Ophiodothella vaccinii
Richard T. Hanlin 1
Department of Plant Pathology, University of Georgia, Athens, Georgia 30602-7274
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ABSTRACT
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The perithecial ascomycete Ophiodothella vaccinii causes a leafspot disease of sparkleberry (Vaccinium arboreum), in which an anamorph is produced early in the life cycle of the fungus. The anamorph forms shiny, black, pulvinate conidiomata that contain a single central pore. After initial infection, fungal hyphae permeate the interior tissues of the leaf, creating lesions. Conidiomata are initiated by the formation of a small layer of intertwined, thicker-walled hyphae beneath the epidermis of the lesion. Near the center of this hyphal layer a subglobose collection of thick-walled hyphae is formed. This hyphal collection grows upward, becoming conical and pressing against the epidermis. Elongation of a columnar apex of the hyphal collection ruptures the epidermis, creating a pore. Subsequent expansion and development of conidiophores and conidia push the epidermis upward, lifting it away from the column, opening the pore and allowing conidia to emerge. The conidioma is regarded as a modified acervulus.
Key words: Acerviclypeatus poriformans, acervulus development, anamorph, Ascomycetes, conidioma morphology, sparkleberry, Vaccinium arboreum
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INTRODUCTION
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Ophiodothella vaccinii Boyd is the cause of flyspeck leafspot disease of Vaccinium arboreum Marshall (sparkleberry), a common understory shrub in the southeastern United States. The disease is characterized by the formation of circular, reddish-yellow lesions bearing the anamorph of the fungus (Hanlin 1990
) that appear in early summer. In her original description of this fungus, Boyd (1934) described the anamorph as an acervulus with scolecospores, but she did not assign it to a particular genus. She also noted the presence of an "umbonate structure" in some of the acervuli, but she did not characterize it further. Hanlin (1990) reexamined the fungus and erected the genus Acerviclypeatus, with the single species A. poriformans, to accommodate it because of the unusual structure of the conidioma. A subsequent study was undertaken into the development of the conidioma, the results of which are presented here.
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MATERIALS AND METHODS
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All studies were conducted on field-collected material. Both paraffin and plastic embedments were used to supplement observations of fresh material. Specimens for paraffin embedment were killed and fixed in formalin-propionic acid-alcohol (FPA), then dehydrated through a tertiary butyl alcohol series before infiltration and embedment in paraplast. Sections were cut on a rotary microtome at 6–8 µm thickness, mounted on standard microscope slides and stained in iron hematoxylin. For plastic embedment, material was fixed in 2.5% glutaraldehyde buffered in Na-cacodylate, post-fixed in osmium tetroxide and embedded in Spurr's resin. Sections were cut 1–2 µm thick on a Sorvall MT2B ultramicrotome, stained in toluidine blue and mounted on glass slides. Material for examination under the scanning electron microscope (SEM) was prepared similarly, then critical-point dried, mounted on stubs and coated with gold palladium before observation. These procedures have been described in greater detail (Hanlin and Tortolero 1989).
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RESULTS
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Lesion development –
The first sign of infection was the appearance of small chlorotic spots on the surface of bright green leaves. As these lesions spread outward, they became orange to reddish due to the deposition of red pigment granules in the infected cells; the source of these granules is unknown. Soon after pigmentation began, a single incipient conidioma became visible in the center of the lesion. As the lesion grew, other conidiomatal initials appeared in a roughly circular pattern nearer the periphery of the lesion (Fig. 1). Lesions averaged 5–6 mm in diameter but they varied greatly in size, from 2 mm-diameter lesions containing a single conidioma up to 10 mm-diameter lesions containing numerous conidiomata. Lesions often coalesced, covering large areas of the leaf. As conidioma grew within the leaf, the epidermis was pushed outward and a black pigment was secreted in the epidermal cells. The mature conidioma was circular to irregular in outline, ca 0.5 mm diameter, with a raised, shield-like, shiny black covering containing a single central pore. Several conidiomata formed in most lesions, and each contained numerous filamentous, (Fig. 2) one-celled, hyaline conidia.
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FIGS. 1–6. Surface characteristics of Ophiodothella vaccinii lesions. 1. Lesion with four mature conidiomata surrounded by a ring of incipient conidiomata (black dots). 2. Older lesion with mature conidiomata. Note central pores. 3. Young conidioma with a raised ring around the depressed center containing the dome with a slit-like rupture. 4. Close-up of slit-like rupture in epidermis. 5. Close-up of a triangular rupture in epidermis. 6. Close-up of apex of the column rupturing the epidermis. 3–6. SEM micrographs. Scale bars: 1 = 1 mm, 2 = 300 µm, 3 = 100 µm, 4–5 = 20 µm, 6 = 2 µm
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External conidioma structure –
The first stage of conidioma development was the appearance of a small dome-shaped protrusion in the host epidermis. As this protrusion rose, the epidermis was pushed outward, resulting in a broadly conical raised area. As the young conidioma increased in diameter, development of the conidiophores beneath the epidermis lifted it, creating a doughnut-shaped ring that was depressed toward the center, around the central protruding dome (Fig. 3). By now fungal cells beneath the dome had ruptured the epidermis by either a short, linear or triangular break (Figs. 4, 5), creating the opening that would become the pore. The rounded apical cells of the column that ruptured the epidermis were visible in the opening (Fig. 6). In mature conidioma, the pressure of the developing conidia beneath the epidermis pushed it upward, away from the central dome, opening the pore and giving the conidioma its raised, pulvinate shape (Fig. 2).
Conidioma ontogeny –
The tissue of the leaves was permeated by thin-walled hyphae that grew intra- and intercellularly (Figs. 7, 8); these hyphae proliferated outward from a central infection site, forming the lesions and increasing their diameter. Soon after a young lesion became evident, the internal mycelium formed a small, thin layer of hyphae with slightly thickened walls just beneath the epidermis in the center of the lesion; this marked the beginning of conidioma formation. The hyphae in this layer were much branched, with short, intertwined cells that formed an irregular, compact layer. On the lower side of the leaf, these hyphae aggregated in the spaces in the mesophyll, as well as in the adjacent host cells, some of which appeared disrupted (Fig. 8). On the upper side of the leaf, however, the appearance was somewhat different, due to the crowded layer of palisade cells. The hyphae filled the palisade cells, but they often were difficult to discern due to a brownish pigment in these cells, especially in the vacuole. Hyphae also formed a layer between the epidermis and the palisade cells (Fig. 9); it is on this layer that conidiogenous cells formed.
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FIGS. 7–12. Stages in conidioma development in Ophiodothella vaccinii. 7. Aggregation of hyphae in cells of mesophyll tissue on lower side of leaf, marking initiation of conidioma development. 8. Aggregation of hyphae in spaces and cells of mesophyll tissue on lower side of leaf, marking initiation of conidioma development. Note rupture of some cell walls. 9. Aggregation of hyphae beneath epidermis and in palisade cells on upper side of leaf. 10. Young globose collection of cells pressed against upper epidermis. 11. Further development of cell collection, which has crushed the epidermis but not broken the cuticle. 12. Vertical extension of cell collection extending through cuticle, creating rupture that will become the pore. Paraffin embedment. Scale bars: 7 = 50 µm, 8 = 40 µm, 9 = 10 µm, 10–12 = 20 µm
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Regardless of the position on the leaf, subsequent conidioma development was the same. In the center of this layer of thicker-walled cells, a compact collection of similar, enlarged hyphal cells formed. This collection of cells was globose roughly and extended slightly below the layer of thick-walled cells (Figs. 10, 13). On the upper side of the leaf, most of this collection of cells formed between the epidermis and the palisade, but the base extended into the upper portions of palisade cells, which were disrupted. The globose collection of cells then began to elongate vertically, forming a broadly conical structure that grew upward, pressing against the overlying epidermis (Fig. 11). This resulted in the dome-shaped protuberance on the surface of the leaf (Figs. 11, 12). Along with this upward growth, the cells in the basal layer spread laterally, increasing the diameter of the developing conidioma (Figs. 13, 15). The uppermost cells in this layer gave rise to a crowded layer of slender, erect, thin-walled hyphae that elongated upward, perpendicular to the long axis of the leaf. These hyphae were incipient conidiophores, which formed a ring around the central hyphal collection (Figs. 14, 15).
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FIGS. 13–19. Additional stages in conidioma development in Ophiodothella vaccinii. 13. Globose collection of thick-walled cells pressing against lower epidermis. Note lateral extension and convex shape of the young conidioma. 14. Vertical section through leaf with older conidioma on lower surface. The vertical column has raised but not broken the epidermis, and the development of conidiphores around the central collection of cells has raised the overlying epidermis, so that the center of the conidioma is depressed. 15. Later stage in which the central vertical column has breached the epidermis and conidiophore development has progressed. 16. Fully mature conidioma filled with conidia, with open pore and dark central column and layer of conidiophores. Note dark clypeus in epidermis on upper side of leaf. 17. Close-up of central collection of cells in Fig. 14, with vertical column raising epidermis. 18. Close-up of central collection in 15, with vertical column extending through epidermis. 19. Close-up of a cell collection and vertical column showing cell structure and arrangement. 16. Paraffin embedment; all others plastic embedment. Scale bars: 13 = 20 µm, 14–15 = 30 µm, 16 = 40 µm, 17, 19 = 10 µm, 18 = 15 µm
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As expansion of the basal layer and development of the incipient conidiophores occurred, the central hyphal collection also continued to develop. Continued upward growth resulted in a terete, a vertical column at the apex that was composed of tightly compact, thick-walled cells that pressed against the epidermis (Figs. 14, 17); as the column continued to elongate, it ruptured the epidermis, creating a pore (Figs. 15, 18, 19). This pore, however, remained plugged as conidioma development continued. By this time the elongating conidiophores had raised the epidermis, forming a ring around the depressed center that contained the dome with the protruding central column (Fig. 15).
The basal layer of the conidioma continued to expand laterally, increasing its diameter. As this occurred, the epidermis above the basal layer was lifted. The cells of the epidermis by now were filled with hyphae that secreted a dark brown to black substance, forming the clypeus and giving the surface of the conidioma its characteristic shiny, black appearance at maturity. The clypeus did not extend beyond the margins of the conidioma (Figs. 16, 22), and the host cuticle remained intact throughout conidioma development, except for the area over the pore. The conidioma now appeared pulvinate on the leaf surface.
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FIGS. 20–23. Structure of pore, column, and conidia in Ophiodothella vaccinii. 20. Cross-section through open pore surrounded by epidermal cells filled with pigmented hyphae, forming the clypeus. 21. Cross-section through vertical column showing cell structure and surrounding conidia. This section is beneath that in 20. 22. Cross-section through edge of conidioma showing uninvaded epidermal cells and conidia. 23. Close-up of cross-section through crowded conidia in conidioma. Plastic embedment. Scale bars: 20 = 20 µm, 21–23 = 10 µm
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The conidiophores matured and began to produce scolecosporous conidia; as the conidia elongated, they pushed against the overlying epidermis, lifting it away from the apex of the column and opening the central pore (Figs. 16, 20). The column of thick-walled hyphae that created the pore now was surrounded by conidia (Fig. 21), which appeared round in cross section (Fig. 23). The conidia were inclined toward the center of the conidioma and were surrounded by a gelatinous matrix that was visible when moist, freshly collected conidiomata were broken open. Under conditions of ample moisture, mature conidia emerged through the pore and floated away. When drier conditions prevailed, however, they were extruded through the pore in a yellowish cirrhus. The pore-forming column remained persistent throughout the life of the conidioma.
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DISCUSSION
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On the basis of developmental morphology, the conidioma in Ophiodothella vaccinii (Acerviclypeatus poriformans) must be regarded as an acervulus but one in which spore release is through a small pore rather than by a general rupturing of the overlying epidermis, as typically occurs in an acervulus (Sutton 1980). Relatively few ontogenetic studies have been conducted on acervular fungi. Sutton (1966) studied conidioma development in species of Colletotrichum, in which he distinguished two types of ontogeny, the pulvinate and the hypostromatic. In pulvinate development, the fungus hyphae penetrate the host tissues and cause the simultaneous rupture of the outer epidermal wall and the cuticle by the developing conidiophores, setae and stromatic tissue. This type of development is characteristic of C. lindemuthianum and the majority of other Colletotrichum species that have been studied. In hypostromatic development, fine hyphae arising from the epidermal stroma penetrate the outer epidermal wall, where they form a thin layer beneath the cuticle. The cuticle then is ruptured by the developing conidiophores and setae. Hypostromatic development is typical of C. graminicola and C. falcatum. These two ontogenetic types are not exclusive, however, because both may occur in the same species, apparently depending upon the particular strain of the fungus and/or the nature of the host tissues. Similarly, Watanabe et al (1998) reported that conidiomata in Truncatella sp. produced pycnidium-like structures that inverted at maturity to form a pulvinate acervulus. The development of the conidioma in O. vaccinii differs in that conidiophores form beneath the epidermis, which remains intact except for the pore.
Conidioma development in O. vaccinii (A. poriformans) is unique in that spore release occurs through a small pore in the epidermis formed by the fungus, which also is a characteristic of some pycnothyria. It differs, however, in the absence of a distinct fungal wall. As cited by Sutton (1973), the primary distinguishing characteristic between an acervulus and a pycnidium is the presence or absence of a wall composed of fungal tissue that encloses the conidiogenous system. Such a wall is lacking in an acervulus, whereas it is present in a pycnidium. It is clear from the early developmental stages of the conidioma in O. vaccinii that a wall is lacking; the conidiogenous cells that arise directly on the subepidermal layer of hyphae are not enclosed by any fungal tissue; this is typical of acervulus development. The dark clypeus that results from the growth of fungal hyphae in the host epidermis cannot be regarded as a wall, because it is not part of the spore-producing tissues. In this sense, conidia development in O. vaccinii can be considered similar to other acervulate species. The pore initially is formed by a narrow rupture of the overlying epidermis, suggesting that it is formed by mechanical, rather than by enzymatic, means. This appears to be the only species reported thus far with such a spore-release mechanism. Why such a unique structure would evolve in this particular fungus is unclear.
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ACKNOWLEDGMENTS
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I thank Jimmy Owens for assistance with histology, and Ikuho Amano for translation of the Japanese paper. The helpful suggestions of the reviewers also are appreciated.
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FOOTNOTES
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1 E-mail: rhanlin{at}uga.edu 
Accepted for publication September 9, 2002.
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LITERATURE CITED
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Boyd ES., 1934 A developmental study of a new species of Ophiodothella. Mycologia 26:456-469
Hanlin RT., 1990 Acerviclypeatus, a new genus for the anamorph of Ophiodothella vaccinii. Mycotaxon 37:379-384
———, Tortolero O., 1989 Morphology of Sclerotium coffeicola, a tropical foliar pathogen. Can J Bot 67:1852-1860
Sutton BC., 1966 Development of fructifications in Colletotrichum graminicola (Ces.) Wils. and related species. Can J Bot 44:887-897
———. 1973 Coelomycetes. In: Ainsworth GC, Sparrow FK, Sussman AS, eds. The fungi—an advanced treatise. Vol. IVA. A taxonomic review with keys: ascomycetes and fungi imperfecti. New York: Academic Press. p 513–582 .
———. 1980 The Coelomycetes. Commonwealth Mycol. Inst., Kew, England. 696 p
Watanabe K, Kobayashi T, Doi Y., 1998 Conidiomata of Truncatella sp. on different media. Nippon Kingakukai Kaiho 39:21-25. [In Japanese]
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