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Nuclei in hyphae and conidia of Cladosporium herbarum
Notes and brief articles REFERENCES
BATKO, S., MURRAY, J. S. & PEACE, T. R. (1958). Sclerophoma pityophila associated with needle-cast and its connection with Pullularia pullulans, Transactions of the British Mycological Society 41,
126-128. BUTIN, H. (1963). Uber Sclerophoma pityophila (Corda) v. Hohn. als Blaupilz an verarbeitetemHolz. Phytopathologische Zeitschrift 48, 298-305. BUTIN, H. (1964). Uber zwei Nebenfruchtformen von Sydotoia polyspora (Bref, et v, Tav.) Muller, Sydouna 17,114-118. HADDOW, W. R. (1941). Needle blight and late fall browning of Red pine (Pinus resinosa Ait.) caused by gall midge (Cecidomyiidae) and the fungus Pullularia pullulans Berkhaut, Transactions of the Royal Canadian Institute 23, 161-189.
HUDSON, H. J. (1966). An ascomycete with Aureobasidium pullulans-type conidia. Nova Hedwigia 10, 319-328. LEHMANN, P. (1974). The biology of fungi decomposing pine leaf litter. Ph,D. Thesis, University of Cambridge. PuGH, G. F. J. & BUCKLEY, N. G. (1971). The leaf surface as a substrate for colonization by fungi. In Ecology of leaf-surface micro-organisms (ed. T. F. Preece and C. H. Dickinson),pp. 432-445. London: Academic Press. ROBAK, H. (1952). Dothichiza pithyophila (Cda) Petr., the pycnidial stage of a mycelium of the type Pullularia pullulans (de B.) Berkhout, Sydowia 6, 361-362. XENOPOULOS, S. (1974). Relationship and pathogenicity of Sclerophoma pityophila and Aureobasidium pullulans on Scots pine. Ph.D. Thesis, University of Aberdeen.
NUCLEI IN HYPHAE AND CONIDIA OF CLADOSPORIUM
HERBARUM JURGEN REISS
Institut fiir Spezielle Botanik der Unioersitdt, 6500 Mains, Germany Cladosporium herbarum is a common member of the Fungi Imperfecti and has received much attention because it can cause serious spoilage of many foodstuffs. However, there is little information on the cytology of this species and especially on nuclear behaviour in hyphae and conidia. De Vries (1967) shows drawings of nuclei in the mycelium of several species of Cladosporium but details are lacking. In his study on the nuclei in vegetative hyphaeof several phytopathogenic fungi, Hoffmann (1968) observed that the average number of nuclei per hyphal cell of C. fulvum was 1'2. There are, however, investigations on genera related to Cladosporium, e.g, Stemphylium (van Warmelo, 1971 a, b) and Alternaria (Stall, 1958; Hartmann, 1964, 1966; Brushaber, Wilson & Aist, 1967). A comparison of these results with the nuclear behaviour in C. herbarum was therefore of interest. Cladosporium herbarum (strain F4) was cultured in Petri dishes on malt extract agar (Difco). Test preparations for the cytological study were made by growing the fungus on cover slips in malt extract solution (Oxoid) in moist chambers. After an incubation period of 24 h the preparations were fixed and stained. The preparations were fixed for 30 min in freshly prepared ethanol-glacial acetic acid (3: 1, vjv), rinsed in water and hydrolysed in 1 N-HCI at 60°C for 8 min. The nuclei were stained for 60 min Trans. Br, mycol. Soc. 68 (1), (1977).
with HCl-Giemsa, prepared according to Hrushovetz (1956): two drops of Giemsa stain (Chroma, Stuttgart, Germany) were added to 1 ml phosphate buffer (pH 7'0). After careful rinsing the stained preparations were mounted in Euparal (Chroma). Most of the hyphal cells contain only one large nucleus of oval to ellipsoid shape. In interphase the darkly stained chromatin surrounds the nucleolus which appears as a lighter centre (Fig. 1). Dikaryotic cells were very rare. A network of strands appears in the prophase nucleus which still seems to be surrounded by an intact membrane (Fig. 2). Discrete chromosomes can be seen during metaphase because of their greater contraction (Fig. 3). Five chromosomes were tentatively counted. In anaphase the groups of chromosomes separate and move away from one another (Figs. 4, 5). A spindle is probably involved in the separation of the daughter chromosomes (Fig. 5). A new cross wall is subsequently formed (Fig. 5) and may even penetrate the spindle elements (Fig. 6). Branching begins with the formation of hyphal proliferations into which one of the daughter nuclei migrates (Figs. 7, 8). Anastomoses often occur and their development is similar (Fig. 9). A nucleus from one of the fused hyphae migrates into the hyphal bridge (Fig. 10). During conidiation short side-hyphae develop buds at their tips into which the nucleus migrates
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Notes and brief articles 5 pm
3
2
5 pm
5 pm
5 Fig . 1. Fig. 2. Fig. 3. Fig. 4. Fig . 5. seen.
6
Monokaryotic mycelium with nucleus in interphase. Prophase with chromatin strands. Metaphase chromosomes. Anaphase. Anaphase at further stage than Fig. 4. Probable spindle elements and a newly formed cross wall can be
Fig. 6. Late anaphase with new cross wall.
(F ig. 11). The newly formed conidium is separated from the hypha by a new cross wall. In most cases this primary conidium develops secondary conidia in the same manner. When a more or less Trans. Br. mycol. Soc. 68 (1), (1977) .
elongated chain of conidia has been formed the lowest conid ium develops a proliferation (Fig . 12) which elongates . After one nucleus has migrated into this bud its tip is transformed into a new
Printed in Great Britain 5- 2
Notes and briefarticles 5 tim
•
8
7
5 tim
5 tim
9 Fig. 7. Branchinghyphal cell with migrating nucleus. Fig. 8. Branching at further stage than Fig. 7. Fig. 9. Formation of a bridge during anastomosis. Fig. 10. Anastomosis with migrating nucleus. conidium after developing a new cross wall (Fig. 13). This process is continued resulting in a characteristically branched conidial head (Fig. 14). Most of the conidia contain one nucleus and only very rarely were cells with two or four nuclei observed. Trans. Br. mycol. Soc. 68 (1), (1977).
The behaviour of the nuclei of C. herbarum during conidiation, the branching of hyphae and during the formation of anastomoses resembles that in Alternaria tenuis (Hartmann, 1964, 1966)and Stemphylium botryosum (van Warmelo, 1971 a, b),
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Notes and brief articles
133
5 um
5 pm
11
12
13
5pm
14 Fig. 11. Primary conidium. Figs. 12, 13. Formation of the branched conidial head. Fig. 14. Branched conidial head with monokaryotic conidia.
Notes and brief articles
134
thus indicating a possible relationship between these fungi. This is confirmed by similar chromosome numbers. C. herbarum has a haploid chromosome number of five, Alternaria tenuis five (Hartmann, 1964), and Stemphylium botryosum six (van Warmelo, 1971 a). Distinct chromosomes, their alignment on the metaphase plate and the separation of chromatids to daughter nuclei are the most important criteria for mitosis in fungi (Ward & Ciurysek, 1962). It is considered that in C. herbarum these requirements are met . Therefore, it must be assumed that the somatic divisions in C. herbarum are mitotic. REFERENCES
BRUSHABER, J. A. WILSON, C. L. & AIST, ]. R. (1967). Asexual nuclear behavior of some plant-pathogenic fungi. Phytopathology 57,43-46. DE VRIES, G. A. (1967). Contribution to the knowledge of the genus Cladosporium Link ex Fr. Bibliotheca Mycologica, vol. 3. Lehre: ]. Cramer.
HARTMANN, G. C. (1964). Nuclear division in Alternaria tenuis. American fournal of Botany 51, 209-212.
HARTMANN, G. C. (1966). The cytology of Alternaria tenuis. Mycologia 58, 694-701.
HOFFMANN, G. M. (1968). Kernverhaltnisse bei pflanzenpathogenen imperfekten Pilzen, insbesondere Arten der Gattung Fusarium. Zentralblatt filr Bakteriologie, II. Abteilung, 122,405-419. HRUSHOVETZ, S. B, (1956). Cytological studies of Helminth osporium sativum. Canadian Journal of Botany 34, 321-3 27. STALL, R. E. (1958). An investigation of nuclear number in Alternaria solani, American Journal of Botany 45, 657-659 . VAN WARMELO, K. T. (1971a) . Somatic nuclear division in Stemphylium botryosum. Bothalia 10, 3.29-334. VAN WARMELO, K. T. (1971b). Conidial nucleation in Stemphylium botryosum. Bothalia 10, 335-339. WARD, E. W. B. & CIURYSEK, K. W. (1962). Somatic mitosis in Neurospora crassa. American Journal of Botany 49, 393-399·
MORPHOLOGY AND BIOLOGY OF ENTOMOPHTHORA MYRMECOPHAGA STANISl.AW BAl.AZY
Wielkopolski National Park, 62-°40 Puszczykowo, Poland AND ANDRZEJ SOKOI.OWSKI
Academy of Agriculture, Institute of Forest Protection, 60-627 Poznan, Poland
The species Entomophthora myrmecophaga (Turian & Wuest, 1969) has been insufficiently described owing to meagre collection of infected specimens of the ant Lasius sp. MacLeod & Muller-Kogler (1973) did not include this species in their key to Entomophthora owing to lack of differentiating features in the description, whereas Waterhouse (1975) placed it in group III (Entomophthora grylii-group) on the basis of information given by Turian & Wuest (1969). In 1974-1975 the authors collected information concerning a strain of entomorphthoraceous fungus from the ants Formica rufa L. and F. polyctena Forst. After written consultation with Professor G . Turian this material is now presented. Infected worker ants of F. rufa and F. polyctena were collected in sections 124-126 of the Pulawy forest district (poland) where field observations concerning the fungus were made . The area of collection is young mixed coniferous forest with compact Scotch pine stand 35-45 years old, with minimal birch and leafy shrubs. Trans . Br. mycol. Soc. 68 (1), (1977).
On 23 August 1974 two dead specimens of F. rufa were collected together with three live ones showing atypical behaviour. Carefulsearch between 16 August and 20 September 1975 produced almost 300 dead specimens of both ant species. Cultures were made from some of this material and the remainder was fixed immediately after collection and examined by standard methods. Stains used were cotton blue in lactophenol, Heidenhein's haematoxylinaceto-carmine, and, unsuccessfully, safranin O. Development of fungus and spores was accomplished only in damp chambers. Numerous attempts at isolation of the fungus on different nutritive media, including coagulated egg yolk, failed. Experimental infections in laboratory conditions were attempted by prolonged contact of living ant specimens with a sporulating mycelium, but no positive infections were obtained. The materials are preserved in the collection of the Wielkopolski National Park, Puszczykowo, Poland. Infected worker ants were always found singly or
Printed in Great Britain
BATKO, S., MURRAY, J. S. & PEACE, T. R. (1958). Sclerophoma pityophila associated with needle-cast and its connection with Pullularia pullulans, Transactions of the British Mycological Society 41,
126-128. BUTIN, H. (1963). Uber Sclerophoma pityophila (Corda) v. Hohn. als Blaupilz an verarbeitetemHolz. Phytopathologische Zeitschrift 48, 298-305. BUTIN, H. (1964). Uber zwei Nebenfruchtformen von Sydotoia polyspora (Bref, et v, Tav.) Muller, Sydouna 17,114-118. HADDOW, W. R. (1941). Needle blight and late fall browning of Red pine (Pinus resinosa Ait.) caused by gall midge (Cecidomyiidae) and the fungus Pullularia pullulans Berkhaut, Transactions of the Royal Canadian Institute 23, 161-189.
HUDSON, H. J. (1966). An ascomycete with Aureobasidium pullulans-type conidia. Nova Hedwigia 10, 319-328. LEHMANN, P. (1974). The biology of fungi decomposing pine leaf litter. Ph,D. Thesis, University of Cambridge. PuGH, G. F. J. & BUCKLEY, N. G. (1971). The leaf surface as a substrate for colonization by fungi. In Ecology of leaf-surface micro-organisms (ed. T. F. Preece and C. H. Dickinson),pp. 432-445. London: Academic Press. ROBAK, H. (1952). Dothichiza pithyophila (Cda) Petr., the pycnidial stage of a mycelium of the type Pullularia pullulans (de B.) Berkhout, Sydowia 6, 361-362. XENOPOULOS, S. (1974). Relationship and pathogenicity of Sclerophoma pityophila and Aureobasidium pullulans on Scots pine. Ph.D. Thesis, University of Aberdeen.
NUCLEI IN HYPHAE AND CONIDIA OF CLADOSPORIUM
HERBARUM JURGEN REISS
Institut fiir Spezielle Botanik der Unioersitdt, 6500 Mains, Germany Cladosporium herbarum is a common member of the Fungi Imperfecti and has received much attention because it can cause serious spoilage of many foodstuffs. However, there is little information on the cytology of this species and especially on nuclear behaviour in hyphae and conidia. De Vries (1967) shows drawings of nuclei in the mycelium of several species of Cladosporium but details are lacking. In his study on the nuclei in vegetative hyphaeof several phytopathogenic fungi, Hoffmann (1968) observed that the average number of nuclei per hyphal cell of C. fulvum was 1'2. There are, however, investigations on genera related to Cladosporium, e.g, Stemphylium (van Warmelo, 1971 a, b) and Alternaria (Stall, 1958; Hartmann, 1964, 1966; Brushaber, Wilson & Aist, 1967). A comparison of these results with the nuclear behaviour in C. herbarum was therefore of interest. Cladosporium herbarum (strain F4) was cultured in Petri dishes on malt extract agar (Difco). Test preparations for the cytological study were made by growing the fungus on cover slips in malt extract solution (Oxoid) in moist chambers. After an incubation period of 24 h the preparations were fixed and stained. The preparations were fixed for 30 min in freshly prepared ethanol-glacial acetic acid (3: 1, vjv), rinsed in water and hydrolysed in 1 N-HCI at 60°C for 8 min. The nuclei were stained for 60 min Trans. Br, mycol. Soc. 68 (1), (1977).
with HCl-Giemsa, prepared according to Hrushovetz (1956): two drops of Giemsa stain (Chroma, Stuttgart, Germany) were added to 1 ml phosphate buffer (pH 7'0). After careful rinsing the stained preparations were mounted in Euparal (Chroma). Most of the hyphal cells contain only one large nucleus of oval to ellipsoid shape. In interphase the darkly stained chromatin surrounds the nucleolus which appears as a lighter centre (Fig. 1). Dikaryotic cells were very rare. A network of strands appears in the prophase nucleus which still seems to be surrounded by an intact membrane (Fig. 2). Discrete chromosomes can be seen during metaphase because of their greater contraction (Fig. 3). Five chromosomes were tentatively counted. In anaphase the groups of chromosomes separate and move away from one another (Figs. 4, 5). A spindle is probably involved in the separation of the daughter chromosomes (Fig. 5). A new cross wall is subsequently formed (Fig. 5) and may even penetrate the spindle elements (Fig. 6). Branching begins with the formation of hyphal proliferations into which one of the daughter nuclei migrates (Figs. 7, 8). Anastomoses often occur and their development is similar (Fig. 9). A nucleus from one of the fused hyphae migrates into the hyphal bridge (Fig. 10). During conidiation short side-hyphae develop buds at their tips into which the nucleus migrates
Printed in Great Britain
Notes and brief articles 5 pm
3
2
5 pm
5 pm
5 Fig . 1. Fig. 2. Fig. 3. Fig. 4. Fig . 5. seen.
6
Monokaryotic mycelium with nucleus in interphase. Prophase with chromatin strands. Metaphase chromosomes. Anaphase. Anaphase at further stage than Fig. 4. Probable spindle elements and a newly formed cross wall can be
Fig. 6. Late anaphase with new cross wall.
(F ig. 11). The newly formed conidium is separated from the hypha by a new cross wall. In most cases this primary conidium develops secondary conidia in the same manner. When a more or less Trans. Br. mycol. Soc. 68 (1), (1977) .
elongated chain of conidia has been formed the lowest conid ium develops a proliferation (Fig . 12) which elongates . After one nucleus has migrated into this bud its tip is transformed into a new
Printed in Great Britain 5- 2
Notes and briefarticles 5 tim
•
8
7
5 tim
5 tim
9 Fig. 7. Branchinghyphal cell with migrating nucleus. Fig. 8. Branching at further stage than Fig. 7. Fig. 9. Formation of a bridge during anastomosis. Fig. 10. Anastomosis with migrating nucleus. conidium after developing a new cross wall (Fig. 13). This process is continued resulting in a characteristically branched conidial head (Fig. 14). Most of the conidia contain one nucleus and only very rarely were cells with two or four nuclei observed. Trans. Br. mycol. Soc. 68 (1), (1977).
The behaviour of the nuclei of C. herbarum during conidiation, the branching of hyphae and during the formation of anastomoses resembles that in Alternaria tenuis (Hartmann, 1964, 1966)and Stemphylium botryosum (van Warmelo, 1971 a, b),
Printed in Great Britain
Notes and brief articles
133
5 um
5 pm
11
12
13
5pm
14 Fig. 11. Primary conidium. Figs. 12, 13. Formation of the branched conidial head. Fig. 14. Branched conidial head with monokaryotic conidia.
Notes and brief articles
134
thus indicating a possible relationship between these fungi. This is confirmed by similar chromosome numbers. C. herbarum has a haploid chromosome number of five, Alternaria tenuis five (Hartmann, 1964), and Stemphylium botryosum six (van Warmelo, 1971 a). Distinct chromosomes, their alignment on the metaphase plate and the separation of chromatids to daughter nuclei are the most important criteria for mitosis in fungi (Ward & Ciurysek, 1962). It is considered that in C. herbarum these requirements are met . Therefore, it must be assumed that the somatic divisions in C. herbarum are mitotic. REFERENCES
BRUSHABER, J. A. WILSON, C. L. & AIST, ]. R. (1967). Asexual nuclear behavior of some plant-pathogenic fungi. Phytopathology 57,43-46. DE VRIES, G. A. (1967). Contribution to the knowledge of the genus Cladosporium Link ex Fr. Bibliotheca Mycologica, vol. 3. Lehre: ]. Cramer.
HARTMANN, G. C. (1964). Nuclear division in Alternaria tenuis. American fournal of Botany 51, 209-212.
HARTMANN, G. C. (1966). The cytology of Alternaria tenuis. Mycologia 58, 694-701.
HOFFMANN, G. M. (1968). Kernverhaltnisse bei pflanzenpathogenen imperfekten Pilzen, insbesondere Arten der Gattung Fusarium. Zentralblatt filr Bakteriologie, II. Abteilung, 122,405-419. HRUSHOVETZ, S. B, (1956). Cytological studies of Helminth osporium sativum. Canadian Journal of Botany 34, 321-3 27. STALL, R. E. (1958). An investigation of nuclear number in Alternaria solani, American Journal of Botany 45, 657-659 . VAN WARMELO, K. T. (1971a) . Somatic nuclear division in Stemphylium botryosum. Bothalia 10, 3.29-334. VAN WARMELO, K. T. (1971b). Conidial nucleation in Stemphylium botryosum. Bothalia 10, 335-339. WARD, E. W. B. & CIURYSEK, K. W. (1962). Somatic mitosis in Neurospora crassa. American Journal of Botany 49, 393-399·
MORPHOLOGY AND BIOLOGY OF ENTOMOPHTHORA MYRMECOPHAGA STANISl.AW BAl.AZY
Wielkopolski National Park, 62-°40 Puszczykowo, Poland AND ANDRZEJ SOKOI.OWSKI
Academy of Agriculture, Institute of Forest Protection, 60-627 Poznan, Poland
The species Entomophthora myrmecophaga (Turian & Wuest, 1969) has been insufficiently described owing to meagre collection of infected specimens of the ant Lasius sp. MacLeod & Muller-Kogler (1973) did not include this species in their key to Entomophthora owing to lack of differentiating features in the description, whereas Waterhouse (1975) placed it in group III (Entomophthora grylii-group) on the basis of information given by Turian & Wuest (1969). In 1974-1975 the authors collected information concerning a strain of entomorphthoraceous fungus from the ants Formica rufa L. and F. polyctena Forst. After written consultation with Professor G . Turian this material is now presented. Infected worker ants of F. rufa and F. polyctena were collected in sections 124-126 of the Pulawy forest district (poland) where field observations concerning the fungus were made . The area of collection is young mixed coniferous forest with compact Scotch pine stand 35-45 years old, with minimal birch and leafy shrubs. Trans . Br. mycol. Soc. 68 (1), (1977).
On 23 August 1974 two dead specimens of F. rufa were collected together with three live ones showing atypical behaviour. Carefulsearch between 16 August and 20 September 1975 produced almost 300 dead specimens of both ant species. Cultures were made from some of this material and the remainder was fixed immediately after collection and examined by standard methods. Stains used were cotton blue in lactophenol, Heidenhein's haematoxylinaceto-carmine, and, unsuccessfully, safranin O. Development of fungus and spores was accomplished only in damp chambers. Numerous attempts at isolation of the fungus on different nutritive media, including coagulated egg yolk, failed. Experimental infections in laboratory conditions were attempted by prolonged contact of living ant specimens with a sporulating mycelium, but no positive infections were obtained. The materials are preserved in the collection of the Wielkopolski National Park, Puszczykowo, Poland. Infected worker ants were always found singly or
Printed in Great Britain