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Frozen-etched spores of Trichoderma viride
Transactions British Mycological Society mode of germination and nature and structure of promycelium and sporidia. Complete suppression of sporidia occurred in submerged conditions. The non-formation of normal promycelia and sporidia reported earlier by Duggar (1892) in Ravenelia cassiaecola may probably be explained on the basis of the submerged conditions under which the teliospores were germinated. The author is grateful to Professor M. N. Kamat, Head, Department of Mycology and Plant Pathology, for his deep interest and guidance to Dr G. B. Deodikar, Director, M.A.C.S. and Dr S. R. Sarvotam, Superintendent of Quality Control, Hindustan Antibiotics Ltd., Pimpri, Poona for laboratory facilities and encouragement. He is also thankful to Dr A. V. Sathe for his helpful suggestions and to Shri D. N. Nagpure for the drawings. REFERENCES
DUGGAR, B. M. (1892). Germination of the teliospores of Raoenelia cassiaecola. Botanical Gazette 17, 144-148.
FROZEN-ETCHED SPORES OF TRICHODERMA VIRIDE D. JONES
Department of Microbiology, Macaulay Institute for Soil Research, Craigiebuckler, Aberdeen, U.K. The freeze-etching technique for preparing biological samples for the electron microscope was introduced by Steere in 1957 and improved by Moor & Miihlethaler in 1963. The method enables the cell and its components to be examined in the transmission electron microscope without the complications of possible artifacts that might appear in chemically fixed material. It has a further advantage over the thin-section technique that surface features of organelles, in addition to cross-sectional views, are revealed. This paper presents some observations by freezeetching of Trichoderma viride Pers. ex Fr. spores, not hitherto examined by this technique. The organism is a very common soil species and the strain chosen was isolated from sclerotia of Sclerotinia sclerotiorum, a pathogen of many agricultural and horticultural crops. The spores when applied to the surface of sclerotia in sand or soil germinate and penetrate the rind cells to bring about lysis of the internal tissue, finally rendering the sclerotia non-viable (Jones & Watson, 1969). A previous paper (Jones & Johnson, 1970) has described freeze-etched spores of Coniothyrium minitans a fungus also found to parasitize the sclerotia. In this investigation a Model V 4-750 Polaron freeze-fracture and etching module (Polaron Equipment Ltd, Finchley, London, N'3, U.K.) Trans. Br. mycol. Soc. 57 (2), (1971). Printed in Great Britain
Notes and Brief Articles
349
has been used. This is an advanced design of the apparatus developed by Steere (1957). To the author's knowledge there have been no published accounts of fungal spor e replicas prepared by the Polaron module, although the freeze-etching technique has been increasingly used for the examination of fungi (Sassen, Remsen & Hess, 1967; Hess, 1968; Campbell, 1969; Sleytr, Adam & Klaushofer, 1969; Gool, M eyer & Lambert, 1970). Spores of T. viride were harvested from malt-extract agar cultures and immersed in 20 % glycerol for up to 5 h. A proportion of the spores was incubated in a liquid nutrient medium for 24 h at 25 °C before being transferred to glycerol. After centrifuging, samples of each of the spore pellets were transferred to a copper support stub which was then plunged into Freon 22 in a small vessel cooled in liquid nitrogen. The frozen specimen was transferred to the pre-cooled peg (- 160 °) of the freezeetching module placed on a pyrex glass spacer ring fitted on to the vacuum base plate of an AEI Type 12 evaporating unit; the chamber was evacuated to a pressure of 5 x 10- 5 bar. The temperature was raised to - 100° and the spore sample fractured manually with a precooled knife which consists of a highly manoeuverable poker-like rod at the end of which is fixed a portion of a scalpel blade. This device, together with the rotational ability of the specimen mount, provides excellent flexibility of movement throughout the preparation procedure. The cut surface of the frozen spore sample was etched by vacuum sublimation for 2 min at - 93 ° after which it was shadowed with platinum-carbon and coated with carbon. The shadowing gun of the module has been replaced by one made in the Institute workshop; as a result, the quality of the replicas has been improved. After coating, the sample was removed from the module, allowed to thaw at room temperature and the replica floated off in distilled water. Any remaining spores and debris were removed from the replica by floating it in 15 % sodium hypochlorite (I h) followed by 75 % sulphuric acid (2 h or overnight). The washed replica was transferred to a formvar-coated copp er grid (200 grade) and dried at 40 ° for 15 min before examining in an electron microscope (AEI EM6). The entire procedure takes at the most up to 24 h to complete, compared with up to 5 days to prepare ultrathin sections. Photographs reproduced in this paper were printed so that deposited platinum-carbon appears dark and shadow is light. Most of the spores which had been incubated for 24 h in the nutrient solution had not germinated to produce hyphal tubes but considerable swelling had occurred in many. Other features not found in the spores taken directly from the agar plates were also observed and are referred to below. Cross-fractured spores are generally spherical to oval in shape (PI. 2 I , figs. I, 2). Within the plasmalemma, or cytoplasmic membrane as it is sometimes called, the most striking features are the many spherical vacuoles of various diameters, fractured in different planes to reveal either the outer (OV) or the inner (I V) surface of the vacuolar membrane. Trans. Br. mycol. Soc. 57 (2), (1971). Printed in Great Britain
350 Transactions British Mycological Society It was difficult to identify lipid granules with certainty; one of the few fungal species previously examined by freeze-etching, Coniothrium minitans, differs from T. viride in that a lipid globule is generally present in the spores and this can occupy a large volume of the cell displacing the ovalshaped nucleus to one pole . No mitochondria could be identified in T. viride spores. Each spore generally has one quite large nucleus (N ) which is identified by the presence of nuclear pores (NP) in the nuclear membrane (PI. 21, fig. I). The cell illustrated in this figure had been fractured to reveal the outer surface of the nuclear membrane; some cells, not illustrated, had been fractured to reveal the inner surface of the nuclear membrane and this also showed nuclear pores. In the same way some cells had been fractured to reveal the inner surface of the plasmalemma (IP, PI. 21, fig. 3) with characteristic folds (F), which appear as invaginations when the outer surface (OP) of the plasmalemma was exposed as in PI. 21, fig. 5. Randomly-scattered particles (PT) appear on both surfaces of the plasmalemma and these are shown in detail on the inner surface in PI. 21, fig. 4. Microfibrils (FL) are seen to bridge some of the invaginations in the plasmalemma (PI. 21, fig. 6). Another interesting feature of the plasmalemma of some of the spores incubated in the liquid nutrient medium is the presence of almost elliptical depressions (D); these appear as ridges when the inner surface of the plasmalemma is exposed and often lie side by side in groups of two or three (PI. 2 I, figs. 3-5 ). They are less distinct in spores not incubated in the nutrient medium and have not been previously reported in freeze-etched fungal spores. The cell wall appears to be two-layered, the outer layer (0 W) bearing small protuberances (WP, PI. 21, fig. 3). Of the spores transferred immediately from the agar plates to the Freon, none revealed the surface of the spore wall on fracturing. However, many of the swollen spores from the liquid nutrient medium had fractured to reveal this surface which possessed wart-like protuberances (WP), some of which had their tips removed (PI. 21, fig. 7). Although the surface views of the plasmalemma, nucleus and vacuoles in T. viride spores present a similar picture to those of other fungal spores examined by the freeze-etching technique, certain features have not been previously reported. A systematic study by this technique of the ultrastructural features of fungal spores and hyphae from the different taxonomic groups might reveal further features not observed using other methods. I am indebted to Dr W. J. McHardy and Mr R. Swaffield for their help in preparing the replicas , in particular in the carbon coating and shadowing of the specimens. I also thank Dr R. P. C. Johnson of the Department of Botany, University of Aberdeen, for good advice on the technical aspects. REFERENCES
CAMPBELL, R. (1969). Further electron microscope studies of the conidium of Alternaria brassicicola. Archives Mikrobiologia 69, 60-68. GOOL, A. VAN, MEYER, J. & LAMBERT, R. (1970). The fine structure of frozen etched Fusarium conidiospores. Journal de Microscopic 9, 653-660. Trans. Br. mycol. Soc. 57 (2), (1971). Printedin Great Britain
Trans. Br. mycol. Soc.
Vol. 57.
Plate
21
(Facing p. 351)
Notes and Brief Articles
35 1
HESS, W. M. (1968). Ultrastructural comparisons of fungus hyphal cells using frozenetched replicas and thin sections of the fungus Pyrenochaeta terrestris. Canadian Joumal
of Microbiology 14, 205- 210. JONES, D. & JOHNSON, R. P. C. (1970). Ultrastructure of frozen, fractured and etched pycnidiospores of Coniothrium minitans. Transactions of the British Mycological Society
55,83--87·
JONES, D. & WATSON, D. (1969). Parasitism and lysis by soil fungi of Sclerotinia sclerotiorum (Lib.) de Bary, a phytopathogenic fungus. Nature 224 , 287-288 . MOOR, H. & MUHLETHALER., K. (1963). Fine structure in frozen-etched yeast cells.
Journal of Cell Biology 17, 609-628. SASSEN, M . M. A., REMSEN, C . C. & HEss, W. M. (1967). Fine structure of Penicillium megasporum conidiospores. Protoplasma 64, 75--88. SLEYTR, U., ADAM:, H. & KLAUSHOFER, H. (1969). Die Feinstruktur der Konidien von Aspergillus niger, v, Tiegh., dargestellt mit Hilfe der Gefrieratztechnik. Mikroskopie 25, 320-331. STEERE, R. L. (1957). Electron microscopy of structural detail in frozen biological specimens. Journal of Biophysical and Biochemical Cytology 3, 45-60. EXPLANATION OF PLATE 21
Shadowed replicas of frozen, fractured and etched spores of Trichoderma viride from agar plate cultures (spores in figs. 2-7 had been incubated for 24 h in liquid nutrient medium). Fig. I. Cross fractured spore showing various cell organelles including nucleus and vacuoles. Fig. 2 . Two fractured spores, the smaller revealing various cell structures, and the larger the outer surface of plasmalemma. Fig. 3. Spore fractured to reveal inner surface of plasmalemma. Fig. 4. Part of fig. 3 enlarged to show particles and depressions. Fig. 5. Spore fractured to reveal outer surface of plasmalemma. Fig. 6. Part of fig. 5 enlarged to show fibrils. Fig. 7. Spore fractured to reveal outer surface of wall. Abbreviations: N, nucleus; NP, nuclear pore; OP, outer surface of plasmalemma; IP, inner surface of plasmalemma; PT, particles; F, folds; IV, inner surface of vacuolar membrane; OV, outer surface of vacuolar membrane; FL, fibrils; IW, inner wall layer; OW, outer wa11 layer; WP, wart-like protuberances; D , depressions ; X, ice crystals and solutes. Arrow in circle indicates direction of shadow.
EFFECT OF PARAQUAT ON GROWTH AND SPORULATION OF SEPTORIA NODORUM AND SEPTORIA TRITICI D. GARETH JONES AND JILL R. WILLIAMS
Department ofAgricultural Botany, University College Aberystwyth
of Wales,
Paraquat, formulated as 'Gramoxone' W, is used in agricultural practice to kill weeds in cereal stubble prior to ploughing. Any fungus present on the stubble might, therefore, be exposed to this chemical. The effect of paraquat on the growth of various soil fungi has been investigated by Wilkinson & Lucas (Ig6ga). On agar plates incorporating 'Gramoxone' W, there was a reduction in the rate oflinear extension of mycelia, abnormalities in growth habit and in patterns of spore production, and suppression of spore germination. Consistent differences were Trans. Br, mycol. Soc. 57 (2), (1971). Printed in Great Britain
DUGGAR, B. M. (1892). Germination of the teliospores of Raoenelia cassiaecola. Botanical Gazette 17, 144-148.
FROZEN-ETCHED SPORES OF TRICHODERMA VIRIDE D. JONES
Department of Microbiology, Macaulay Institute for Soil Research, Craigiebuckler, Aberdeen, U.K. The freeze-etching technique for preparing biological samples for the electron microscope was introduced by Steere in 1957 and improved by Moor & Miihlethaler in 1963. The method enables the cell and its components to be examined in the transmission electron microscope without the complications of possible artifacts that might appear in chemically fixed material. It has a further advantage over the thin-section technique that surface features of organelles, in addition to cross-sectional views, are revealed. This paper presents some observations by freezeetching of Trichoderma viride Pers. ex Fr. spores, not hitherto examined by this technique. The organism is a very common soil species and the strain chosen was isolated from sclerotia of Sclerotinia sclerotiorum, a pathogen of many agricultural and horticultural crops. The spores when applied to the surface of sclerotia in sand or soil germinate and penetrate the rind cells to bring about lysis of the internal tissue, finally rendering the sclerotia non-viable (Jones & Watson, 1969). A previous paper (Jones & Johnson, 1970) has described freeze-etched spores of Coniothyrium minitans a fungus also found to parasitize the sclerotia. In this investigation a Model V 4-750 Polaron freeze-fracture and etching module (Polaron Equipment Ltd, Finchley, London, N'3, U.K.) Trans. Br. mycol. Soc. 57 (2), (1971). Printed in Great Britain
Notes and Brief Articles
349
has been used. This is an advanced design of the apparatus developed by Steere (1957). To the author's knowledge there have been no published accounts of fungal spor e replicas prepared by the Polaron module, although the freeze-etching technique has been increasingly used for the examination of fungi (Sassen, Remsen & Hess, 1967; Hess, 1968; Campbell, 1969; Sleytr, Adam & Klaushofer, 1969; Gool, M eyer & Lambert, 1970). Spores of T. viride were harvested from malt-extract agar cultures and immersed in 20 % glycerol for up to 5 h. A proportion of the spores was incubated in a liquid nutrient medium for 24 h at 25 °C before being transferred to glycerol. After centrifuging, samples of each of the spore pellets were transferred to a copper support stub which was then plunged into Freon 22 in a small vessel cooled in liquid nitrogen. The frozen specimen was transferred to the pre-cooled peg (- 160 °) of the freezeetching module placed on a pyrex glass spacer ring fitted on to the vacuum base plate of an AEI Type 12 evaporating unit; the chamber was evacuated to a pressure of 5 x 10- 5 bar. The temperature was raised to - 100° and the spore sample fractured manually with a precooled knife which consists of a highly manoeuverable poker-like rod at the end of which is fixed a portion of a scalpel blade. This device, together with the rotational ability of the specimen mount, provides excellent flexibility of movement throughout the preparation procedure. The cut surface of the frozen spore sample was etched by vacuum sublimation for 2 min at - 93 ° after which it was shadowed with platinum-carbon and coated with carbon. The shadowing gun of the module has been replaced by one made in the Institute workshop; as a result, the quality of the replicas has been improved. After coating, the sample was removed from the module, allowed to thaw at room temperature and the replica floated off in distilled water. Any remaining spores and debris were removed from the replica by floating it in 15 % sodium hypochlorite (I h) followed by 75 % sulphuric acid (2 h or overnight). The washed replica was transferred to a formvar-coated copp er grid (200 grade) and dried at 40 ° for 15 min before examining in an electron microscope (AEI EM6). The entire procedure takes at the most up to 24 h to complete, compared with up to 5 days to prepare ultrathin sections. Photographs reproduced in this paper were printed so that deposited platinum-carbon appears dark and shadow is light. Most of the spores which had been incubated for 24 h in the nutrient solution had not germinated to produce hyphal tubes but considerable swelling had occurred in many. Other features not found in the spores taken directly from the agar plates were also observed and are referred to below. Cross-fractured spores are generally spherical to oval in shape (PI. 2 I , figs. I, 2). Within the plasmalemma, or cytoplasmic membrane as it is sometimes called, the most striking features are the many spherical vacuoles of various diameters, fractured in different planes to reveal either the outer (OV) or the inner (I V) surface of the vacuolar membrane. Trans. Br. mycol. Soc. 57 (2), (1971). Printed in Great Britain
350 Transactions British Mycological Society It was difficult to identify lipid granules with certainty; one of the few fungal species previously examined by freeze-etching, Coniothrium minitans, differs from T. viride in that a lipid globule is generally present in the spores and this can occupy a large volume of the cell displacing the ovalshaped nucleus to one pole . No mitochondria could be identified in T. viride spores. Each spore generally has one quite large nucleus (N ) which is identified by the presence of nuclear pores (NP) in the nuclear membrane (PI. 21, fig. I). The cell illustrated in this figure had been fractured to reveal the outer surface of the nuclear membrane; some cells, not illustrated, had been fractured to reveal the inner surface of the nuclear membrane and this also showed nuclear pores. In the same way some cells had been fractured to reveal the inner surface of the plasmalemma (IP, PI. 21, fig. 3) with characteristic folds (F), which appear as invaginations when the outer surface (OP) of the plasmalemma was exposed as in PI. 21, fig. 5. Randomly-scattered particles (PT) appear on both surfaces of the plasmalemma and these are shown in detail on the inner surface in PI. 21, fig. 4. Microfibrils (FL) are seen to bridge some of the invaginations in the plasmalemma (PI. 21, fig. 6). Another interesting feature of the plasmalemma of some of the spores incubated in the liquid nutrient medium is the presence of almost elliptical depressions (D); these appear as ridges when the inner surface of the plasmalemma is exposed and often lie side by side in groups of two or three (PI. 2 I, figs. 3-5 ). They are less distinct in spores not incubated in the nutrient medium and have not been previously reported in freeze-etched fungal spores. The cell wall appears to be two-layered, the outer layer (0 W) bearing small protuberances (WP, PI. 21, fig. 3). Of the spores transferred immediately from the agar plates to the Freon, none revealed the surface of the spore wall on fracturing. However, many of the swollen spores from the liquid nutrient medium had fractured to reveal this surface which possessed wart-like protuberances (WP), some of which had their tips removed (PI. 21, fig. 7). Although the surface views of the plasmalemma, nucleus and vacuoles in T. viride spores present a similar picture to those of other fungal spores examined by the freeze-etching technique, certain features have not been previously reported. A systematic study by this technique of the ultrastructural features of fungal spores and hyphae from the different taxonomic groups might reveal further features not observed using other methods. I am indebted to Dr W. J. McHardy and Mr R. Swaffield for their help in preparing the replicas , in particular in the carbon coating and shadowing of the specimens. I also thank Dr R. P. C. Johnson of the Department of Botany, University of Aberdeen, for good advice on the technical aspects. REFERENCES
CAMPBELL, R. (1969). Further electron microscope studies of the conidium of Alternaria brassicicola. Archives Mikrobiologia 69, 60-68. GOOL, A. VAN, MEYER, J. & LAMBERT, R. (1970). The fine structure of frozen etched Fusarium conidiospores. Journal de Microscopic 9, 653-660. Trans. Br. mycol. Soc. 57 (2), (1971). Printedin Great Britain
Trans. Br. mycol. Soc.
Vol. 57.
Plate
21
(Facing p. 351)
Notes and Brief Articles
35 1
HESS, W. M. (1968). Ultrastructural comparisons of fungus hyphal cells using frozenetched replicas and thin sections of the fungus Pyrenochaeta terrestris. Canadian Joumal
of Microbiology 14, 205- 210. JONES, D. & JOHNSON, R. P. C. (1970). Ultrastructure of frozen, fractured and etched pycnidiospores of Coniothrium minitans. Transactions of the British Mycological Society
55,83--87·
JONES, D. & WATSON, D. (1969). Parasitism and lysis by soil fungi of Sclerotinia sclerotiorum (Lib.) de Bary, a phytopathogenic fungus. Nature 224 , 287-288 . MOOR, H. & MUHLETHALER., K. (1963). Fine structure in frozen-etched yeast cells.
Journal of Cell Biology 17, 609-628. SASSEN, M . M. A., REMSEN, C . C. & HEss, W. M. (1967). Fine structure of Penicillium megasporum conidiospores. Protoplasma 64, 75--88. SLEYTR, U., ADAM:, H. & KLAUSHOFER, H. (1969). Die Feinstruktur der Konidien von Aspergillus niger, v, Tiegh., dargestellt mit Hilfe der Gefrieratztechnik. Mikroskopie 25, 320-331. STEERE, R. L. (1957). Electron microscopy of structural detail in frozen biological specimens. Journal of Biophysical and Biochemical Cytology 3, 45-60. EXPLANATION OF PLATE 21
Shadowed replicas of frozen, fractured and etched spores of Trichoderma viride from agar plate cultures (spores in figs. 2-7 had been incubated for 24 h in liquid nutrient medium). Fig. I. Cross fractured spore showing various cell organelles including nucleus and vacuoles. Fig. 2 . Two fractured spores, the smaller revealing various cell structures, and the larger the outer surface of plasmalemma. Fig. 3. Spore fractured to reveal inner surface of plasmalemma. Fig. 4. Part of fig. 3 enlarged to show particles and depressions. Fig. 5. Spore fractured to reveal outer surface of plasmalemma. Fig. 6. Part of fig. 5 enlarged to show fibrils. Fig. 7. Spore fractured to reveal outer surface of wall. Abbreviations: N, nucleus; NP, nuclear pore; OP, outer surface of plasmalemma; IP, inner surface of plasmalemma; PT, particles; F, folds; IV, inner surface of vacuolar membrane; OV, outer surface of vacuolar membrane; FL, fibrils; IW, inner wall layer; OW, outer wa11 layer; WP, wart-like protuberances; D , depressions ; X, ice crystals and solutes. Arrow in circle indicates direction of shadow.
EFFECT OF PARAQUAT ON GROWTH AND SPORULATION OF SEPTORIA NODORUM AND SEPTORIA TRITICI D. GARETH JONES AND JILL R. WILLIAMS
Department ofAgricultural Botany, University College Aberystwyth
of Wales,
Paraquat, formulated as 'Gramoxone' W, is used in agricultural practice to kill weeds in cereal stubble prior to ploughing. Any fungus present on the stubble might, therefore, be exposed to this chemical. The effect of paraquat on the growth of various soil fungi has been investigated by Wilkinson & Lucas (Ig6ga). On agar plates incorporating 'Gramoxone' W, there was a reduction in the rate oflinear extension of mycelia, abnormalities in growth habit and in patterns of spore production, and suppression of spore germination. Consistent differences were Trans. Br, mycol. Soc. 57 (2), (1971). Printed in Great Britain