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Pycnidial development in Phoma betae
Short Communications
369
Pycnidial development in Phoma betae E. MONTE, P. M. MARTIN AND I. GARCiA-ACHA Departamento de Microbiolog{a, Genetica, Medicina Preventiva y Saluti Publica e Instituto de Microbiologla Bioqu{mica, Universidad de Salamanca/CSIC, 37071 Salamanca, Spain
Pycnidial development in Phoma betae. Mycological Research 92 (3): 369-372 (1989). Pycnidial ontogeny in Phoma betae is compared with developmental types already described in this genus and other Coelomycetes. Time lapse microscopic observation shows the development of pycnidia and 'holdfasts'. Key words: Phoma betae, Pycnidial ontogeny.
Studies on the origin and ontogeny of pycnidial conidiomata in Coelomycetes was initiated by De Bary (1884) but this field of study has not been developed by modem mycologists. This is probably due to the enormous morphological diversity at the genus level and even within species, which makes such characteristics of little value for diagnostic and classificatory purposes. De Bary (1884) described two ways in which pycnidia may develop based on origin from growth of one or alternatively of several hyphae. Kempton (1919) also suggested that a single cell could function as the origin of a conidioma. However, Kempton (1919), Punithalingam (1966) and Sutton (1977) admitted variations and combinations of both basic types of ontogeny and Maiello & Peterson (1976) included hyphal rings as an additional way in which pycnidial development could be initiated. In the present work several strains of Phoma betae Frank (teleomorph: Pleospora betae (Berl.) Nevodovsky) isolated from beet seedlings with 'black leg' in Salamanca and Sevilla, as well as isolate CECT 2348 (DSM 63181) from the Coleccion Espanola de Cultivos Tipo, Burjasot (Valencia), Spain, have been used. Pycnidia of P. betae were laid on agar blocks between a microscope slide and a coverslip and placed in a damp chamber at 28°C. The culture media used were CMA, PDA and Czapek-Dox agar. These media were also used to observe production of conidiomata in Petri dishes at 24 and 28°. A solid support is necessary for the initiation of a pycnidium which may be immersed in the substrate or produced on its surface. Pycnidia can also develop on the thin 6lm formed at the surface of static liquid cultures. The so-called thin hyphae (Monte & Garda-Acha, 1988) anastomose or they form bridges with adjacent hyphae (Figs 1-3). More frequently the thick hyphae become repeatedly septate, invaginated and aggregated (Figs 4, 5) according to the meristogenous developmental process if the pycnidium originates from a single hypha, or initially symphogenous if the pycnidium develops from a net of anastomosing hyphae. This process allows the formation of an irregular mass of
dark dematiaceous thick-walled very septate hyphae, around which will eventually rise the thin hyphae that form the external layer of the pycnidium from which, in tum, both types of hyphae will develop (Figs 6-9). Sometimes two (Fig. 10) or more (Fig. 11) hyphae lie adjacent as a ropy bundle, but not as thick as described by Maiello & Peterson (1976) in Phyllosticta antirrhini. They become septate and produce branches, giving rise to a pycnidium with compound meristogenous development. On PDA, CMA and Czapek-Dox agar modified with sodium acetate sole hyphae produce loops or coils (Fig. 12). This occurs when arbutin, cellulose, ethanol, glycerol and inosine are utilized as the sole carbon source for growth. The phenomenon is not restricted to P. betae for it also frequently happens in other Coelomycetes. This type of structure described by Maiello & Peterson (1976) as 'primary ringed primordia' in Phyllosticta antirrhini, is the immature stage and the formation of pycnidia from them in P. betae has not been observed. On CMA (Fig. 13), the presence of 'secondary ringed primordia' has also been observed, as recorded by Maiello & Peterson (1976) and Maiello (1978), but the loop formed by a string of hyphae does not seem able to give rise to pycnidia in P. betae. The presence of 'tertiary ringed primordia' (Maiello & Peterson, 1976) with a coil produced by the connexion of several hyphae close together, has also been observed in CMA cultures in a damp chamber, but further development of pycnidiil has never been observed. If pycnidia do develop from such primordia, development should be termed simple symphogenous ringed development (Fig. 14) in order to distinguish it from those cases in which the coil is due to the connexion of adjacent strings of hyphae, where the pycnidium so produced would be by a compound symphogenous ringed development. Pycnidia have never been observed arising from the apex of a major hypha. However, as described by Monte & GardaAcha (1988), it is easy to find swollen tips in hyphae belonging to the aerial mycelium in the colonies of P. betae. These structures are also present in hyphae in contact with the surface of the culture medium (Fig. 15). 26·2
Short Communications
370
Figs 1-9. Successive stages in the formation of a pycnidium of P. betae growing on CMA. Figs 1-3. At an early stage anastomosis and union bridges between hyphae are frequent. Figs 4, 5. A great number of transverse septa appear inside the thick hyphae, which become invaginated. Figs 6-8. Pycnidial development proceeds and a spherical structure is eventually formed. Fig. 9. Mature pycnidiurn from which numerous hypha emerge.
Short Communications
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Figs 10-16. Different theoretical modes of pycnidial development in P. betae. Fig. 10. Compound meristogenous (two hyphae). Fig. 11. Compound meristogenous (string of hyphae). Fig. 12. Primary ringed primordium. Fig. 13. Secondary ringed primordium. Fig. 14. Symphogenous ringed primordium. Fig. 15. Swollen hyphal tip in contact with surface of the culture medium. Fig. 16. Symphogenous.
The initiation of 'holdfasts' (Mangan, 1971) and that of pycnidia is very similar. However, pycnidia never develop in the rather anaerobic conditions in which 'holdfasts' are formed in great numbers. In preparations containing a small block of solid culture medium under the coverslip, pycnidia only develop at the external surfaces of the agar block where aerobiosis takes place. In this area pycnidia and 'holdfasts' are mixed together but as shown in Figs 17-23, 'holdfasts' and pycnidia follow different developmental paths. It is still difficult to define precisely pycnidial ontogeny and a revision of the terminology would be approximate as has happened for the concepts of conidiogenesis (Minter et al., 1983) and chlamydospores (Hughes, 1985). Pycnidial ontogeny in P. betae is much more diverse than was expected according to the observations by Bjorling (1945). Pycnidia in P. betae may be meristogenous (Figs
17-23), symphogenous (Fig. 16), or show intermediate stages as described by Boerema & Van Kesteren (1964) in other Phoma spp. The early steps in the pycnidial formation (Figs 1-5) resemble those described by Punithalingam (1966) for pycnidia of Septoria leucanthemi with simultaneous meristogenous and symphogenous development. The production of branched and multiseptate hyphae, with wide and short cells (Figs 6, 10) is similar to the 'loose type' of sclerotia formation in Rhizoctonia solani and to some degree resembles the initial development of 'terminal type' sclerotia in Botrytis allii (Townsend & Willetts, 1953). The development of a conidioma surrounded by a metabolically active thick hypha which is situated at the periphery of the future fruiting body (Figs 5, 7, 8), the pycnidial mass of which is formed by thin-walled narrower
372
Short Communications
Figs 17-23. Time lapse sequence of simultaneous development of a meristogenous pycnidium and a group of holdfasts. Fig. 17. Time = 0 h. Fig. 18. Time = 5 h. Fig. 19. Time = 8 h. Fig. 20. Time = 12'30 h. Fig. 21. Time = 26 h. Fig. 22. Time = 29 h. Fig. 23. Time = 34 h.
.20,urn \ 22
20,urn
_....-:._.....0.:........--"'_ _.....
cells, resembles the process observed by DiCosmo & Cole (1980) in Chaetomella acutiseta where a seta acts as the initial support for the conidioma. The most logical explanation for the formation of the pycnidial cavity is schizogenous and lysogenous activity (Punithalingam, 1966). However, this does not seem to be the case when pycnidia develop as ringed primordia from loops of one or more hypha(e). We are indebted to Drs B. C. Sutton and E. Punithalingam for their help in bibliographic work. REFERENCES BJORLING, K. (1945). Undersokningar rorande Phoma betae (Oud.) Fr. State Viixtskyddsastalt Meddelande 44, 1-96. BOEREMA, G. H. & VAN KESTEREN, H. A. (1964). The nomenclature of two fungi parasitizing Brassica. Persoonia 3, 17-28. DE BARY, A. (1884). Comparative Morphology and Biology of the Fungi, Mycetozoa and Bacteria. Oxford: Clarendon Press. DiCOSMO, F. & COLE, G. T. (1980). Morphogenesis of conidiomata in Chaetomella acutiseta (Coelomycetes). Canadian Journal of Botany 58, 1129-1137.
HUGHES, S.]. (1985). The term Chlamydospore. In Filamentous
23
Microorganisms. Biomedical Aspects (ed. T. Arai). Tokyo: Japan Scientific Societies Press. KEMPTON, F. E. (I919). Origin and development of the pycnidium. Botanic Gazette 68, 233-261. MAIELLO, ]. M. (1978). The origin of the pycnidium in Macrophomina phaseoli. Mycologia 70, 176-179. MAIELLO, ]. M. & PETERSON, ]. L. (1976). Pycnidium ontogeny in Phyllosticta antirrhini. Mycologia 68, 1121-1125. MANGAN, A. (1971). A new method for the detection of Pleospora bjoerlingii infection of sugar beet seed. Transactions of the British Mycological Society 57, 169-172. MINTER, D. W., SUTTON, B. C. & BRADY, B. L. (1983). What are phialides anyway? Transactions of the British Mycological Society 81, 109-120.
MONTE, E. & GARCfA-ACHA, 1. (1988). Vegetative and reproductive structures of Phoma betae in vitro. Transactions of the British Mycological Society 90, 233-245. PUNITHALINGAM, E. (1966) Development of the pycnidium in Septoria. Transactions of the British Mycological Society 49, 19-25. SUTTON, B. C. (I973). Coelomycetes. In The Fungi IV A (ed. G. C. Ainsworth, F. K. Sparrow & A. S. Sussman). New York: Academic Press. TOWNSEND, B. B. & WILLETTS, H. J. (1953). The development of sclerotia of certain fungi. Transactions of the British Mycological Society 33, 213-221.
369
Pycnidial development in Phoma betae E. MONTE, P. M. MARTIN AND I. GARCiA-ACHA Departamento de Microbiolog{a, Genetica, Medicina Preventiva y Saluti Publica e Instituto de Microbiologla Bioqu{mica, Universidad de Salamanca/CSIC, 37071 Salamanca, Spain
Pycnidial development in Phoma betae. Mycological Research 92 (3): 369-372 (1989). Pycnidial ontogeny in Phoma betae is compared with developmental types already described in this genus and other Coelomycetes. Time lapse microscopic observation shows the development of pycnidia and 'holdfasts'. Key words: Phoma betae, Pycnidial ontogeny.
Studies on the origin and ontogeny of pycnidial conidiomata in Coelomycetes was initiated by De Bary (1884) but this field of study has not been developed by modem mycologists. This is probably due to the enormous morphological diversity at the genus level and even within species, which makes such characteristics of little value for diagnostic and classificatory purposes. De Bary (1884) described two ways in which pycnidia may develop based on origin from growth of one or alternatively of several hyphae. Kempton (1919) also suggested that a single cell could function as the origin of a conidioma. However, Kempton (1919), Punithalingam (1966) and Sutton (1977) admitted variations and combinations of both basic types of ontogeny and Maiello & Peterson (1976) included hyphal rings as an additional way in which pycnidial development could be initiated. In the present work several strains of Phoma betae Frank (teleomorph: Pleospora betae (Berl.) Nevodovsky) isolated from beet seedlings with 'black leg' in Salamanca and Sevilla, as well as isolate CECT 2348 (DSM 63181) from the Coleccion Espanola de Cultivos Tipo, Burjasot (Valencia), Spain, have been used. Pycnidia of P. betae were laid on agar blocks between a microscope slide and a coverslip and placed in a damp chamber at 28°C. The culture media used were CMA, PDA and Czapek-Dox agar. These media were also used to observe production of conidiomata in Petri dishes at 24 and 28°. A solid support is necessary for the initiation of a pycnidium which may be immersed in the substrate or produced on its surface. Pycnidia can also develop on the thin 6lm formed at the surface of static liquid cultures. The so-called thin hyphae (Monte & Garda-Acha, 1988) anastomose or they form bridges with adjacent hyphae (Figs 1-3). More frequently the thick hyphae become repeatedly septate, invaginated and aggregated (Figs 4, 5) according to the meristogenous developmental process if the pycnidium originates from a single hypha, or initially symphogenous if the pycnidium develops from a net of anastomosing hyphae. This process allows the formation of an irregular mass of
dark dematiaceous thick-walled very septate hyphae, around which will eventually rise the thin hyphae that form the external layer of the pycnidium from which, in tum, both types of hyphae will develop (Figs 6-9). Sometimes two (Fig. 10) or more (Fig. 11) hyphae lie adjacent as a ropy bundle, but not as thick as described by Maiello & Peterson (1976) in Phyllosticta antirrhini. They become septate and produce branches, giving rise to a pycnidium with compound meristogenous development. On PDA, CMA and Czapek-Dox agar modified with sodium acetate sole hyphae produce loops or coils (Fig. 12). This occurs when arbutin, cellulose, ethanol, glycerol and inosine are utilized as the sole carbon source for growth. The phenomenon is not restricted to P. betae for it also frequently happens in other Coelomycetes. This type of structure described by Maiello & Peterson (1976) as 'primary ringed primordia' in Phyllosticta antirrhini, is the immature stage and the formation of pycnidia from them in P. betae has not been observed. On CMA (Fig. 13), the presence of 'secondary ringed primordia' has also been observed, as recorded by Maiello & Peterson (1976) and Maiello (1978), but the loop formed by a string of hyphae does not seem able to give rise to pycnidia in P. betae. The presence of 'tertiary ringed primordia' (Maiello & Peterson, 1976) with a coil produced by the connexion of several hyphae close together, has also been observed in CMA cultures in a damp chamber, but further development of pycnidiil has never been observed. If pycnidia do develop from such primordia, development should be termed simple symphogenous ringed development (Fig. 14) in order to distinguish it from those cases in which the coil is due to the connexion of adjacent strings of hyphae, where the pycnidium so produced would be by a compound symphogenous ringed development. Pycnidia have never been observed arising from the apex of a major hypha. However, as described by Monte & GardaAcha (1988), it is easy to find swollen tips in hyphae belonging to the aerial mycelium in the colonies of P. betae. These structures are also present in hyphae in contact with the surface of the culture medium (Fig. 15). 26·2
Short Communications
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Figs 1-9. Successive stages in the formation of a pycnidium of P. betae growing on CMA. Figs 1-3. At an early stage anastomosis and union bridges between hyphae are frequent. Figs 4, 5. A great number of transverse septa appear inside the thick hyphae, which become invaginated. Figs 6-8. Pycnidial development proceeds and a spherical structure is eventually formed. Fig. 9. Mature pycnidiurn from which numerous hypha emerge.
Short Communications
371
Figs 10-16. Different theoretical modes of pycnidial development in P. betae. Fig. 10. Compound meristogenous (two hyphae). Fig. 11. Compound meristogenous (string of hyphae). Fig. 12. Primary ringed primordium. Fig. 13. Secondary ringed primordium. Fig. 14. Symphogenous ringed primordium. Fig. 15. Swollen hyphal tip in contact with surface of the culture medium. Fig. 16. Symphogenous.
The initiation of 'holdfasts' (Mangan, 1971) and that of pycnidia is very similar. However, pycnidia never develop in the rather anaerobic conditions in which 'holdfasts' are formed in great numbers. In preparations containing a small block of solid culture medium under the coverslip, pycnidia only develop at the external surfaces of the agar block where aerobiosis takes place. In this area pycnidia and 'holdfasts' are mixed together but as shown in Figs 17-23, 'holdfasts' and pycnidia follow different developmental paths. It is still difficult to define precisely pycnidial ontogeny and a revision of the terminology would be approximate as has happened for the concepts of conidiogenesis (Minter et al., 1983) and chlamydospores (Hughes, 1985). Pycnidial ontogeny in P. betae is much more diverse than was expected according to the observations by Bjorling (1945). Pycnidia in P. betae may be meristogenous (Figs
17-23), symphogenous (Fig. 16), or show intermediate stages as described by Boerema & Van Kesteren (1964) in other Phoma spp. The early steps in the pycnidial formation (Figs 1-5) resemble those described by Punithalingam (1966) for pycnidia of Septoria leucanthemi with simultaneous meristogenous and symphogenous development. The production of branched and multiseptate hyphae, with wide and short cells (Figs 6, 10) is similar to the 'loose type' of sclerotia formation in Rhizoctonia solani and to some degree resembles the initial development of 'terminal type' sclerotia in Botrytis allii (Townsend & Willetts, 1953). The development of a conidioma surrounded by a metabolically active thick hypha which is situated at the periphery of the future fruiting body (Figs 5, 7, 8), the pycnidial mass of which is formed by thin-walled narrower
372
Short Communications
Figs 17-23. Time lapse sequence of simultaneous development of a meristogenous pycnidium and a group of holdfasts. Fig. 17. Time = 0 h. Fig. 18. Time = 5 h. Fig. 19. Time = 8 h. Fig. 20. Time = 12'30 h. Fig. 21. Time = 26 h. Fig. 22. Time = 29 h. Fig. 23. Time = 34 h.
.20,urn \ 22
20,urn
_....-:._.....0.:........--"'_ _.....
cells, resembles the process observed by DiCosmo & Cole (1980) in Chaetomella acutiseta where a seta acts as the initial support for the conidioma. The most logical explanation for the formation of the pycnidial cavity is schizogenous and lysogenous activity (Punithalingam, 1966). However, this does not seem to be the case when pycnidia develop as ringed primordia from loops of one or more hypha(e). We are indebted to Drs B. C. Sutton and E. Punithalingam for their help in bibliographic work. REFERENCES BJORLING, K. (1945). Undersokningar rorande Phoma betae (Oud.) Fr. State Viixtskyddsastalt Meddelande 44, 1-96. BOEREMA, G. H. & VAN KESTEREN, H. A. (1964). The nomenclature of two fungi parasitizing Brassica. Persoonia 3, 17-28. DE BARY, A. (1884). Comparative Morphology and Biology of the Fungi, Mycetozoa and Bacteria. Oxford: Clarendon Press. DiCOSMO, F. & COLE, G. T. (1980). Morphogenesis of conidiomata in Chaetomella acutiseta (Coelomycetes). Canadian Journal of Botany 58, 1129-1137.
HUGHES, S.]. (1985). The term Chlamydospore. In Filamentous
23
Microorganisms. Biomedical Aspects (ed. T. Arai). Tokyo: Japan Scientific Societies Press. KEMPTON, F. E. (I919). Origin and development of the pycnidium. Botanic Gazette 68, 233-261. MAIELLO, ]. M. (1978). The origin of the pycnidium in Macrophomina phaseoli. Mycologia 70, 176-179. MAIELLO, ]. M. & PETERSON, ]. L. (1976). Pycnidium ontogeny in Phyllosticta antirrhini. Mycologia 68, 1121-1125. MANGAN, A. (1971). A new method for the detection of Pleospora bjoerlingii infection of sugar beet seed. Transactions of the British Mycological Society 57, 169-172. MINTER, D. W., SUTTON, B. C. & BRADY, B. L. (1983). What are phialides anyway? Transactions of the British Mycological Society 81, 109-120.
MONTE, E. & GARCfA-ACHA, 1. (1988). Vegetative and reproductive structures of Phoma betae in vitro. Transactions of the British Mycological Society 90, 233-245. PUNITHALINGAM, E. (1966) Development of the pycnidium in Septoria. Transactions of the British Mycological Society 49, 19-25. SUTTON, B. C. (I973). Coelomycetes. In The Fungi IV A (ed. G. C. Ainsworth, F. K. Sparrow & A. S. Sussman). New York: Academic Press. TOWNSEND, B. B. & WILLETTS, H. J. (1953). The development of sclerotia of certain fungi. Transactions of the British Mycological Society 33, 213-221.