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Scanning electron microscopy of the synnematal stage of Pycnostysanus azaleae
Notes and brief articles SCANNING ELECTRON MICROSCOPY OF THE SYNNEMATAL STAGE OF PYCNOSTYSANUSAZALEAE BY I. S. GBAJA AND S. R. CHANT
Biological Sciences Department, Chelsea College (University of London), Hortensia Road, London SWlO oQR, U.K. Investigations by scanning electron microscopy on the synnematal stage of Pycnostysanus azaleae have revealed the existence of single, multiple and branched forms. Mature synnemata bear conidia of variable shape including globose, globose-ovate and elongate.
Pycnostyanus azaleae (Peck) Mason (1941), previously ascribed to the genus Sporocybe (S. azaleae) by Saccardo (1886) is the causal agent of a disease of rhododendrons and azaleas known as bud and twig blight (Davis, 1939), or bud blast (Street, 1950; Howell & Wood, 1962). The fungus is imperfectly known but is generally recognized by synnemata that appear on the outer scales of the terminal flower buds about one year after infection. The purpose of this investigation is to amplify by scanning electron microscopy the information on synnematal structure reported by Davis (1940) and Viennot-Bourgin (1980). All leaves of Rhododendron ponticum L. Selon were removed, and the flower bud with subtending stem pre-cooled by immersion in liquid nitrogen before freeze-drying for 5 days. The outer scales of 1- and z-year-old infected buds were mounted on 10 mm wide double-sided adhesive tape on SEM stubs, coated with platinum in a Polaron SEM sputter-coating unit (E 5100) at 20 rnA for 2 min and then examined in a Philips SEM (501 B) at an accelerating voltage of 15 kV. Photographs were taken with Ilford HP3 220 film. The sclerotium appears first as a mass of intermingled hyphae forming a dense stroma approximately 40 p,m diam (Fig 1). Large mature sclerotia which may attain a diameter of 100 p,m produce synnemata from the centre, either singly (Fig. 2), in groups of 2-5 (Fig. 3), or in a multibranched assembly with a single origin (Fig. 4). Some synnemata show a more simple bi- or tri- furcate
branching near the base (Fig. 3). The stipe which is initially conical (Fig. 2) consists of densely packed hyphae (Fig. 5). The distal end of the synnema expands at first into a mass of chain-like structures bearing 2-8 unicellular, mainly globose spores (Figs 6, 7, 8) attached to each other by a short 'neck' (Fig. 9). Older synnemataalso bear penicillate conidiophores bearing elongate spores (Figs 10, 11) or globoseovate spores on short lateral sympodial branches (Figs 12, 13). The development of synnemata from the basal stroma appears more complex than that previously described by Davis (1940). Single, multiple and branched synnemata have been observed in the present study, although the factors which determine the type of structure produced are not known. Davis (1940) described and named five spore stages of P. azaleae in culture, namely chlamydosporic, cephalosporic,penicillioid, coremial ( = synnematal) and ascogenous. However, on synnemata he described only globose, ovate, ellipsoidal and oblong 'coremiospores' formed in chains of 1-9 at the tips of conidiophores. Similarly, Viennot-Bourgin (1980) mentioned only 'chain-like blastospores ', depicted in drawings to be of variable shape but mainly globose-ovate. From the present study it appears that synnemata are more complex than previously described, bearing spores of variable shape in at least three distinct forms of branching. Some of the forms described by Davis (1940) in cultures of this fungus are to be found associated in
Fig. 1. Sclerotium of Pycnostysanus azaleae on the bud scale of Rhododendron ponticum, Bar = 10 pm. Fig. 2. Immature synnema arising from the sclerotium on bud scale. Bar = 10 pm. Fig. 3. Developing on the bud scale of R. catawbiense arranged either singly or branched (arrowed). Bar = 100 pm. Fig. 4. 'Colonial' arrangement of highly branched synnemata of bud scale of R. ponticum. Bar = 100 pm. Fig. 5. T.S. Synnema showing arrangement of hyphal network with conidia mainly around the base of stipe. Bar = 10 pm. Fig. 6. Tip of developing synnema showing developing conidia. Bar = 10 pm. Fig. 7. Mature synnematal head and stipe showing chain-like arrangement of conidia; on R. catawbiense. (Bar = 10 pm.
Trans. Br. mycol. Soc. 83 (1), (1984)
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Notes and brief articles
Figs 1-7. For caption see opposite.
Trans. Br. mycol, Soc. 83 (1), (1984)
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179
180
Notes and brief articles
Fig. 8. Synnernata showing chain-like arrangement of conidia on R. ponticum. Bar = 10 I'm. Fig. 9. Synnematal head showing globose conidia connected by short necks (arrowed). Bar = 1 flm. Fig. 10. Penicillate conidiophore-bearing conidia in chains on synnema. Bar = 10 I'm. Fig. 11. Conidiophore showing arrangement of conidia and points of attachment (arrowed). Bar = 10 11m. Figs 12, 13. Conidiophores bearing globose to ovate conidia on short lateral sympodial branches on synnema. Bar = 10 I'm
Trans. Br. mycol. Soc. 83 (1), (1984)
Printed in Great Britain
Notes and brief articles the synnemata. It appears that the spore types described in this investigation could correctly be called conidia. REFERENCES
DAVIS, W. H. (1939). A bud and twig blight of azaleas caused by Sporocybe azaleae. Phytopathology 29, 5 17-5 19. DAVIS, W. H. (1940). New stages of Sporocybe azaleae. Phytopathology 30, 5°6--514. HOWELL, P. J. & WOOD, R. K. S. (1962). Some factors affecting rhododendron bud blast and its control. Annals of Applied Biology SO, 723-733.
181
MAsON, E. W.(1941).AnnotatedAccountofFungiReceived at the Imperial Mycological Institute, List 11, (Fasc. 3 - Special Part). Kew: Commonwealth Mycological Institute. SACCARDO,P.A. (1886). Syllogefungorumomnium hucusque cognitorum 20, 604-609. STREET, F. (1950). Some observations and notes on bud blast on Rhododendrons. Rhododendron Year Book S, 72-77· VIENNOT-BoURGIN, G. (1980). Observation simultanee en France du bud blast du Rhododendron et d'une cicadelle jouant Ie role de vecteur. Agronomie 1 (2), 87--9 2 •
CONCENTRIC BODIES IN CERCOSPORA BETICOLA (HYPHOMYCETES) BY NINOSKA PONS
Centro Nacional de Investigaciones Agropecuarias, Apdo. 4653, Maracay 2101, Venezuela J.L.GAY
Department of Pure and Applied Biology, Imperial College, London SW7 2BB AND B. C. SUTTON
Commonwealth Mycological Institute, Ferry Lane, Kew Transmission electron microscopy of stromatic cells and conidiophores of Cercospora beticola has demonstrated the presence of concentric bodies, typical of lichenized ascomycetes, and reported in a few free-living fungi. Ellipsoid bodies were first described by Brown & Wilson (1968) for certain inclusions in the mycobiont of the lichen Physcia aipolia. Griffiths & Greenwood (1972) showed that the structures were isodiametric and proposed the term 'concentric bodies 't;)describethem. Subsequently most reports of these microstructures concern their discovery in different species oflichens (Jacobs & Ahmadjian, 1969; Peveling, 1969; Griffiths & Greenwood, 1972) and Ahmadjian & Hale (1973) described them as characteristic of this group of organisms. Griffiths & Greenwood (1972) recorded concentric bodies in 43 lichenized fungi and also reported them in two non-lichenized fungi, Rhopographus jilicinus (Fr.) Fuckel and a fungal contaminant on cleistothecia of Sphaerotheca mors-uuae (Schw.) Berk. & Curt. (later reported by Martin, Gay & Jackson (1976) as unidentified hyphae with simplepored septa within cell lumina of mature cleistothecia). Knowledge of non-lichenized fungi with concentric bodies is comparatively limited. Bellemere (1973) reported them in Hysterographium fraxini (Pers.) de Not., Rhytisma acerinum (Pers.) Fr., Tryblidiopsispinastri (Pers.) Karst., Hypoderma rubi (Pers. ex Chev.) de Not., H eterosphaeria patella (Tode) Grev. and Ostropa barbara (Fr.) Nannf., and Granett (1974) described them for Venturia inaequalis (Cke) Wint. Similar inclusions found Trans. Br. mycol, Soc. 83 (1), (1984)
during the course of an ultrastructural investigation of conidiogenesis in Cercospora beticola Sacco are documented in this note. The isolate of C. beticola used was IMI 249002, obtained from seeds and leaves of Beta vulgaris cv. Renta, supplied by Dr F. Koch, KWS Kleinwanzlebener Saatzucht, AG-Einbeck, W. Germany. Cultures were grown on malt agar or potato dextrose agar at ca 25°C under white fluorescent lights (40 W), with alternate 12 h dark periods, or a combination of white fluorescent (20 W) and near u.v. (black) light (Philips, TL 20 W j08). Sporulation commenced after 9 days. Sporulation on leaves of B. vulgaris was obtained after 12 days by scratchinoculating, with drops of conidial suspension, r-rnonth-old plants kept in a humid chamber. Infected leaves were fixed by 2'5 % (v jv) glutaraldehyde in 0'1 M sodium cacodylate buffer at pH 7 for 6-14 days. Leaf pieces were transferred to fresh fixative and after 1 h given three zo-rnin washes in 0'1 M buffer, followed by post-fixation in 1 % (wjv) buffered OS04 for 2 h at ca 25°. The procedure was completed by dehydration in a 10 % graded series of ethanol and embedding in Epon 812. Epoxy propane was used as a link reagent. Sections were cut with a diamond knife on an LKB Ultratome 1 and stained with 2 % (w jv) aqueous uranyl acetate for 30 min at 60°. They were
Printed in Great Britain
Biological Sciences Department, Chelsea College (University of London), Hortensia Road, London SWlO oQR, U.K. Investigations by scanning electron microscopy on the synnematal stage of Pycnostysanus azaleae have revealed the existence of single, multiple and branched forms. Mature synnemata bear conidia of variable shape including globose, globose-ovate and elongate.
Pycnostyanus azaleae (Peck) Mason (1941), previously ascribed to the genus Sporocybe (S. azaleae) by Saccardo (1886) is the causal agent of a disease of rhododendrons and azaleas known as bud and twig blight (Davis, 1939), or bud blast (Street, 1950; Howell & Wood, 1962). The fungus is imperfectly known but is generally recognized by synnemata that appear on the outer scales of the terminal flower buds about one year after infection. The purpose of this investigation is to amplify by scanning electron microscopy the information on synnematal structure reported by Davis (1940) and Viennot-Bourgin (1980). All leaves of Rhododendron ponticum L. Selon were removed, and the flower bud with subtending stem pre-cooled by immersion in liquid nitrogen before freeze-drying for 5 days. The outer scales of 1- and z-year-old infected buds were mounted on 10 mm wide double-sided adhesive tape on SEM stubs, coated with platinum in a Polaron SEM sputter-coating unit (E 5100) at 20 rnA for 2 min and then examined in a Philips SEM (501 B) at an accelerating voltage of 15 kV. Photographs were taken with Ilford HP3 220 film. The sclerotium appears first as a mass of intermingled hyphae forming a dense stroma approximately 40 p,m diam (Fig 1). Large mature sclerotia which may attain a diameter of 100 p,m produce synnemata from the centre, either singly (Fig. 2), in groups of 2-5 (Fig. 3), or in a multibranched assembly with a single origin (Fig. 4). Some synnemata show a more simple bi- or tri- furcate
branching near the base (Fig. 3). The stipe which is initially conical (Fig. 2) consists of densely packed hyphae (Fig. 5). The distal end of the synnema expands at first into a mass of chain-like structures bearing 2-8 unicellular, mainly globose spores (Figs 6, 7, 8) attached to each other by a short 'neck' (Fig. 9). Older synnemataalso bear penicillate conidiophores bearing elongate spores (Figs 10, 11) or globoseovate spores on short lateral sympodial branches (Figs 12, 13). The development of synnemata from the basal stroma appears more complex than that previously described by Davis (1940). Single, multiple and branched synnemata have been observed in the present study, although the factors which determine the type of structure produced are not known. Davis (1940) described and named five spore stages of P. azaleae in culture, namely chlamydosporic, cephalosporic,penicillioid, coremial ( = synnematal) and ascogenous. However, on synnemata he described only globose, ovate, ellipsoidal and oblong 'coremiospores' formed in chains of 1-9 at the tips of conidiophores. Similarly, Viennot-Bourgin (1980) mentioned only 'chain-like blastospores ', depicted in drawings to be of variable shape but mainly globose-ovate. From the present study it appears that synnemata are more complex than previously described, bearing spores of variable shape in at least three distinct forms of branching. Some of the forms described by Davis (1940) in cultures of this fungus are to be found associated in
Fig. 1. Sclerotium of Pycnostysanus azaleae on the bud scale of Rhododendron ponticum, Bar = 10 pm. Fig. 2. Immature synnema arising from the sclerotium on bud scale. Bar = 10 pm. Fig. 3. Developing on the bud scale of R. catawbiense arranged either singly or branched (arrowed). Bar = 100 pm. Fig. 4. 'Colonial' arrangement of highly branched synnemata of bud scale of R. ponticum. Bar = 100 pm. Fig. 5. T.S. Synnema showing arrangement of hyphal network with conidia mainly around the base of stipe. Bar = 10 pm. Fig. 6. Tip of developing synnema showing developing conidia. Bar = 10 pm. Fig. 7. Mature synnematal head and stipe showing chain-like arrangement of conidia; on R. catawbiense. (Bar = 10 pm.
Trans. Br. mycol. Soc. 83 (1), (1984)
Printed in Great Britain
Notes and brief articles
Figs 1-7. For caption see opposite.
Trans. Br. mycol, Soc. 83 (1), (1984)
Printed in Great Britain
179
180
Notes and brief articles
Fig. 8. Synnernata showing chain-like arrangement of conidia on R. ponticum. Bar = 10 I'm. Fig. 9. Synnematal head showing globose conidia connected by short necks (arrowed). Bar = 1 flm. Fig. 10. Penicillate conidiophore-bearing conidia in chains on synnema. Bar = 10 I'm. Fig. 11. Conidiophore showing arrangement of conidia and points of attachment (arrowed). Bar = 10 11m. Figs 12, 13. Conidiophores bearing globose to ovate conidia on short lateral sympodial branches on synnema. Bar = 10 I'm
Trans. Br. mycol. Soc. 83 (1), (1984)
Printed in Great Britain
Notes and brief articles the synnemata. It appears that the spore types described in this investigation could correctly be called conidia. REFERENCES
DAVIS, W. H. (1939). A bud and twig blight of azaleas caused by Sporocybe azaleae. Phytopathology 29, 5 17-5 19. DAVIS, W. H. (1940). New stages of Sporocybe azaleae. Phytopathology 30, 5°6--514. HOWELL, P. J. & WOOD, R. K. S. (1962). Some factors affecting rhododendron bud blast and its control. Annals of Applied Biology SO, 723-733.
181
MAsON, E. W.(1941).AnnotatedAccountofFungiReceived at the Imperial Mycological Institute, List 11, (Fasc. 3 - Special Part). Kew: Commonwealth Mycological Institute. SACCARDO,P.A. (1886). Syllogefungorumomnium hucusque cognitorum 20, 604-609. STREET, F. (1950). Some observations and notes on bud blast on Rhododendrons. Rhododendron Year Book S, 72-77· VIENNOT-BoURGIN, G. (1980). Observation simultanee en France du bud blast du Rhododendron et d'une cicadelle jouant Ie role de vecteur. Agronomie 1 (2), 87--9 2 •
CONCENTRIC BODIES IN CERCOSPORA BETICOLA (HYPHOMYCETES) BY NINOSKA PONS
Centro Nacional de Investigaciones Agropecuarias, Apdo. 4653, Maracay 2101, Venezuela J.L.GAY
Department of Pure and Applied Biology, Imperial College, London SW7 2BB AND B. C. SUTTON
Commonwealth Mycological Institute, Ferry Lane, Kew Transmission electron microscopy of stromatic cells and conidiophores of Cercospora beticola has demonstrated the presence of concentric bodies, typical of lichenized ascomycetes, and reported in a few free-living fungi. Ellipsoid bodies were first described by Brown & Wilson (1968) for certain inclusions in the mycobiont of the lichen Physcia aipolia. Griffiths & Greenwood (1972) showed that the structures were isodiametric and proposed the term 'concentric bodies 't;)describethem. Subsequently most reports of these microstructures concern their discovery in different species oflichens (Jacobs & Ahmadjian, 1969; Peveling, 1969; Griffiths & Greenwood, 1972) and Ahmadjian & Hale (1973) described them as characteristic of this group of organisms. Griffiths & Greenwood (1972) recorded concentric bodies in 43 lichenized fungi and also reported them in two non-lichenized fungi, Rhopographus jilicinus (Fr.) Fuckel and a fungal contaminant on cleistothecia of Sphaerotheca mors-uuae (Schw.) Berk. & Curt. (later reported by Martin, Gay & Jackson (1976) as unidentified hyphae with simplepored septa within cell lumina of mature cleistothecia). Knowledge of non-lichenized fungi with concentric bodies is comparatively limited. Bellemere (1973) reported them in Hysterographium fraxini (Pers.) de Not., Rhytisma acerinum (Pers.) Fr., Tryblidiopsispinastri (Pers.) Karst., Hypoderma rubi (Pers. ex Chev.) de Not., H eterosphaeria patella (Tode) Grev. and Ostropa barbara (Fr.) Nannf., and Granett (1974) described them for Venturia inaequalis (Cke) Wint. Similar inclusions found Trans. Br. mycol, Soc. 83 (1), (1984)
during the course of an ultrastructural investigation of conidiogenesis in Cercospora beticola Sacco are documented in this note. The isolate of C. beticola used was IMI 249002, obtained from seeds and leaves of Beta vulgaris cv. Renta, supplied by Dr F. Koch, KWS Kleinwanzlebener Saatzucht, AG-Einbeck, W. Germany. Cultures were grown on malt agar or potato dextrose agar at ca 25°C under white fluorescent lights (40 W), with alternate 12 h dark periods, or a combination of white fluorescent (20 W) and near u.v. (black) light (Philips, TL 20 W j08). Sporulation commenced after 9 days. Sporulation on leaves of B. vulgaris was obtained after 12 days by scratchinoculating, with drops of conidial suspension, r-rnonth-old plants kept in a humid chamber. Infected leaves were fixed by 2'5 % (v jv) glutaraldehyde in 0'1 M sodium cacodylate buffer at pH 7 for 6-14 days. Leaf pieces were transferred to fresh fixative and after 1 h given three zo-rnin washes in 0'1 M buffer, followed by post-fixation in 1 % (wjv) buffered OS04 for 2 h at ca 25°. The procedure was completed by dehydration in a 10 % graded series of ethanol and embedding in Epon 812. Epoxy propane was used as a link reagent. Sections were cut with a diamond knife on an LKB Ultratome 1 and stained with 2 % (w jv) aqueous uranyl acetate for 30 min at 60°. They were
Printed in Great Britain