- Email: [email protected]
Nuclear behaviour in mycelia, conidiophores and conidia of Erysiphe pisi
[ 481 ] Printed in Great Britain
NUCLEAR BEHAVIOUR IN MYCELIA, CONIDIOPHORES AND CONIDIA OF ERYSIPHE PISI By U. P. SINGH, H. B. SINGH Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras H indu University, Varanasi-221005, India AND AIKO SAKAI Biological Laboratories, National Women's University, 630 Nara , Japan
Light and electron microscope observations on resting and dividing nuclei in mycelia, conidiophores and ungerminated and germinated conidia of Erysiphe pisi, after fixation and staining with aceto-orcein and lead citrate, are described. The observations revealed that mitosis is similar to that in other fungi. During early stages of division the nucleolus was clearly visible. The chromosome number was n = 6 but there was little difference in morphology of the chromosomes. The nucleus and nucleolus were clearly visible under the electron microscope. Harper (1905) observed the resting nucleus, nuclear fusion and mitosis in powdery mildews. Colson (1938) obtained similar results while studying nuclear behaviour in asci of Phyllactinia corylea (Pers.) Karst. Allen (1936) studied the nuclear cytology in Erysiphe polygoni DC., a parasite on Delphinium species. Olive ( 1953) in his classical review suggested that nuclear structure and behaviour in fungi are essentially the same as those of higher organisms. Kimber & Wolfe (1966) studied the chromosome number in E. graminis DC. and Clare (1964) observed n=4 for eight species belonging to five genera of the family Erysiphaceae. McKeen (1971) observed Woronin bodies in E. graminis. McKeen (1972 a, b) also studied nuclear movement and mitosis in E. gramin is f.sp. hordei Lev. We report here studies on the mitotic divisions in mycelia, conidiophores and conidia of Erysiphe pisi DC. which causes powdery mildew of the pea (Pisum satiuum L. ).
for 30 min at 60°C and stored in stain for 2-24 h. The colonies were gently scraped from the disks and mounted on glass slides in a mixture of glycerine and water in equal quantities or in the staining solution and examined under the light microscope. Conidia of E. pisi were fixed on glass slides in the mucilage of Tradescantia tricolor (Thirumalachar & Pavgi, 1950) and incubated in moist chambers for germination at 20 o . Germinated conidia were fixed in Camoy's fluid and stained in 0 '1 % synthetic orcein prepared in 45 % glacial acetic acid (Johansen, 1940). The material was then rinsed several times in phosphate buffer (pH 7'0) for 3 h. It was rinsed again in 1'0 % osmium tetroxide (p H 7'0 ) for 3 h with the pH being adjusted with phosphate buffer. The materials were dehydrated in an ascending series of acetone and embedded in epoxy resin. Sections were made and stained with lead citrate and examined under a HU-12 electron microscope.
MATERIALS AND METHODS
RESULTS
Cultures of E . pisi were maintained in a glasshouse on 15-day-old susceptible pea seedlings by dusting conidia from infected plants. Seven days after inoculation leaves with mildew were cut into disks (3 mm diam) with a sharp cork borer. The disks were fixed in Carnoy's flu id (3 parts 95 % ethyl alcohol :2 parts chloroform: 1 part glacial acetic acid) for 15 min. The disks were then mordanted with 0.1 % iron alum and then transferred to 45 % glacial acetic acid containing 0.1 % synthetic orcein
The hyphal cells have one nucleus which is oval to round and 3 '0-4'5 p,m diam. It contains a darkstained nucleolus of about 0'5-0'75 p,m diam. The nucleolus is always attached to the nuclear membrane (Fig. 1). The nucleus divides by mitosis and a single nucleus migrates into the conidiophore initial (F ig . 2). As the nucleus moves further into the young conidiophore, the nucleus divides mitotically and a septum is formed thus giving two uninucleate cells (Fig. 3). The nucleus is round but
Nuclear behaviour in Erysiphe pisi
3
o
Figs 1-17. Erysiphe pisi stained with aceto-orcein and observed by light microscopy. Fig. 1. Resting nucleus showing deeply stained nucleolus in a mycelial cell . x 1700. Fig. 2. Resting nucleus at the base of conidiophore initial. x 1250. Figs 3, 4. Septum formation and migration of the nucleus towards the upper cell. x 1000. Fig. 5. A resting nucleus in the terminal cell of the conidiophore. x 2000. Fig. 6. Early prophase. x 2000. Figs 7, 8. Late prophase. x 2000. Fig. 9. Early metaphase. x 2000.
U. P. Singh, H. B. Singh and A. Sakai
•
II
13 Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig.
10. 11. 12 . 13. 14. 15 . 16. 17.
Early metaphase. x 2000. Metaphase showing six bivalents. x 2200. Two-celled conidiophore, each cell containing a single nucleus. x 750. Three-celled conidiophore. x 1000. Four-celled conidiophore. x 1000. A mature conidium containing a single nucleus. x 1500. A germinating conidium showing a single nucleus moving towards a germ-tube. x Nuclear migration into the conidial germ-tube. x 1200.
1500.
Nuclear behaviour in Erysiphe pisi
~\
,-
\ _ .......
nucleus divides mitotically and enters interphase. The chromatin material enlarges and becomes thread-like (F ig. 6) and it gradually condenses before metaphase. During prometaphase the chromosomes become very short but the identity of individual chromosomes is not clear (F igs 7-10). Well-defined bivalents are formed during metaphase and the haploid chromosome number would appear to be (n=6) (Fig. 11 ). In Fig. 11 chromosome a is the smallest and measures o- 5 x o· 5 p,m while chromosome b is slightly bigger (1 '0 x 0'75 p,m). There is little difference between the size of chromosomes c and d(l'O x 0'75 p,mand 1'0 x 1 '0 um, respectively) and chromosomes e andfare more or less the same size. Anaphase was not observed but a septum was formed between the daughter nuclei to give two uninucleate conidiophores (Fig. 12 ). The process is repeated and 3-4 conidia are formed, each of which is uninucleate (Figs 13-14). In some nuclei the nucleolus is distinct (Fig. 14). Each cell of the conidiophore becomes a conidium which is capable of dissemination. The nucleus in the mature conidium is almost round and is often located towards one end (F ig . 15). A mature conidium germinates to produce a germ-tube and the nucleus migrates to the base of the germ-tube (F ig. 16 ) and eventually enters the conidium (F ig. 17). Under the electron microscope the resting nucleus was seen near the tip of the apical cell of the conidiophore and it was faintly stained, but the nucleolus was densely stained and attached to the nuclear membrane (Fig. 18). The resting nucleus in a mature conidium is large, faintly stained and situated at one end of the conidium. The nucleolus is clearly visible as a small, oval, dark-stained body attached to the nuclear membrane (F ig. 19). DISCUSSION
19 Figs 18-19. E. pisi stained with lead acetate and observed by electron microscopy. Fig. 18 . Apical cell of conidiophore showing a single nucleus and a dark-stained nucleolus. x 2000. Fig. 19. Cross-section of a mature conidium showing the nucleus (N) surrounded by nuclear membrane and a dark-stained nucleolus (arrow). x 2500.
the nucleolus is not visible and the cells elongate to become cylindrical (F ig. 4). The resting nucleus in each conidiophore is round and is usually present near the centre of the cell but the nucleolus is not visible (F ig . 5). The
As E. pisi is an obligate parasite it cannot be grown in artificial media, so that it is difficult to study the nuclear divisions in the mycelia, conidiophores and conidia. However, the technique used in this study shows that it is possible to study the nuclear behaviour during mitosis. The nucleolus was clearly, but the nucleoplasm only faintly visible. Some of the stages, especially metaphase, were clearly visible. The haploid chromosome number is six (n = 6). The nucleolus was also deeply stained and attached to the nuclear membrane and the nucleoplasm was faintly stained. The central bodies, spindle microtubules and astral rays were not seen in any of the preparations. However, Harper (1905), Crackower & Bauer (19 7 1) and McKeen ( 19 72 a, b) have reported these organelles in several powdery mildews. The
U. P. Singh, H. B. Singh and A. Sakai nucleolus, which was clearly visible in resting nuclei during aceto-orcein staining, disappeared during mitosis. The nucleolus persists during most of mitosis in E. graminis f.sp. hordei (McKeen, 197 2 b). He observed its disappearance as the daughter nuclei developed. Westergaard & Wettstein (1970) reported the expulsion of nucleoli from the nucleus during interphase in the ascomycete Neottiella. There is no such evidence in E. pisi, and this was not observed by McKeen (1972b) in E. graminis f.sp. hordei. There seems to be a range of chromosome numbers in the powdery mildews. The ascomycetes, in general, have four chromosomes. Dangeard (1904) observed 8 chromosomes (n = 8) in Sphaerotheca castagnei, Harper (1905) observed 8 chromosomes (n = 8) in Phyllactinia species and Clare (1964) observed n=4 for Ph. corylea. McKeen (1972b) observed n=6-8 chromosomes in E. graminis f.sp. hordei. Based on genetic studies, Moseman (1963) suggested the possibility of at least n = 5 in this fungus. Two chromosomes (n = 2) are reported for E. graminis f.sp. tritici by Kimber & Wolfe (1966). We report here for the first time that in E. pisi n=6.
H. B. Singh is grateful to the Council of Scientific and Industrial Research, New Delhi for financial assistance. REFERENCES
ALLEN, RUTH F. (1936). A cytological study of Erysiphe polygoni on Delphinium. Journal ofAgricultural Research 53, 801-818.
CLARE, B. G. (1964). Erysiphaceae of Southern Eastern
Queensland. Queensland University. Papers of the Department of Botany 4, 11
McKEEN, W. E. (1971). Woronin bodies in Erysiphe graminis DC. Canadian Journal of Microbiology 17, 1557-1560.
McKEEN, W. E. (1972a). Nuclear movement in Erysiphe graminis DC. Canadian Journal of Microbiology 18, 1333-1336.
McKEEN, W. E. (197 2b). Somatic mitosis in Erysiphe
graminis hordei. Canadian Journal of Microbiology 18, 19 15-1922. MOSEMAN, J. G. (1963). Relationship of genes conditioning pathogenicity of Erysiphe graminis f.sp. hordei on barley. Phytopathology 53, 1326--133°. OLIVE, L. s. (1953). The structure and behaviour of fungus nuclei. Botanical Reoieu: 19, 439-586. THIRUMALACHAR, M. J. & PAVGI, M. S. (1950). Notes on spore germination and mounting techniques. Indian Phytopathology 3, 177-178. WESTERGAARD, M. & WETTSTEIN, D. (1970). The nucleolar cycle in an ascomycete. Compte rendu des travaux de Laboratoire de Carlsberg 37, 195-237.
(Received for publication 27 January 1984)
NUCLEAR BEHAVIOUR IN MYCELIA, CONIDIOPHORES AND CONIDIA OF ERYSIPHE PISI By U. P. SINGH, H. B. SINGH Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras H indu University, Varanasi-221005, India AND AIKO SAKAI Biological Laboratories, National Women's University, 630 Nara , Japan
Light and electron microscope observations on resting and dividing nuclei in mycelia, conidiophores and ungerminated and germinated conidia of Erysiphe pisi, after fixation and staining with aceto-orcein and lead citrate, are described. The observations revealed that mitosis is similar to that in other fungi. During early stages of division the nucleolus was clearly visible. The chromosome number was n = 6 but there was little difference in morphology of the chromosomes. The nucleus and nucleolus were clearly visible under the electron microscope. Harper (1905) observed the resting nucleus, nuclear fusion and mitosis in powdery mildews. Colson (1938) obtained similar results while studying nuclear behaviour in asci of Phyllactinia corylea (Pers.) Karst. Allen (1936) studied the nuclear cytology in Erysiphe polygoni DC., a parasite on Delphinium species. Olive ( 1953) in his classical review suggested that nuclear structure and behaviour in fungi are essentially the same as those of higher organisms. Kimber & Wolfe (1966) studied the chromosome number in E. graminis DC. and Clare (1964) observed n=4 for eight species belonging to five genera of the family Erysiphaceae. McKeen (1971) observed Woronin bodies in E. graminis. McKeen (1972 a, b) also studied nuclear movement and mitosis in E. gramin is f.sp. hordei Lev. We report here studies on the mitotic divisions in mycelia, conidiophores and conidia of Erysiphe pisi DC. which causes powdery mildew of the pea (Pisum satiuum L. ).
for 30 min at 60°C and stored in stain for 2-24 h. The colonies were gently scraped from the disks and mounted on glass slides in a mixture of glycerine and water in equal quantities or in the staining solution and examined under the light microscope. Conidia of E. pisi were fixed on glass slides in the mucilage of Tradescantia tricolor (Thirumalachar & Pavgi, 1950) and incubated in moist chambers for germination at 20 o . Germinated conidia were fixed in Camoy's fluid and stained in 0 '1 % synthetic orcein prepared in 45 % glacial acetic acid (Johansen, 1940). The material was then rinsed several times in phosphate buffer (pH 7'0) for 3 h. It was rinsed again in 1'0 % osmium tetroxide (p H 7'0 ) for 3 h with the pH being adjusted with phosphate buffer. The materials were dehydrated in an ascending series of acetone and embedded in epoxy resin. Sections were made and stained with lead citrate and examined under a HU-12 electron microscope.
MATERIALS AND METHODS
RESULTS
Cultures of E . pisi were maintained in a glasshouse on 15-day-old susceptible pea seedlings by dusting conidia from infected plants. Seven days after inoculation leaves with mildew were cut into disks (3 mm diam) with a sharp cork borer. The disks were fixed in Carnoy's flu id (3 parts 95 % ethyl alcohol :2 parts chloroform: 1 part glacial acetic acid) for 15 min. The disks were then mordanted with 0.1 % iron alum and then transferred to 45 % glacial acetic acid containing 0.1 % synthetic orcein
The hyphal cells have one nucleus which is oval to round and 3 '0-4'5 p,m diam. It contains a darkstained nucleolus of about 0'5-0'75 p,m diam. The nucleolus is always attached to the nuclear membrane (Fig. 1). The nucleus divides by mitosis and a single nucleus migrates into the conidiophore initial (F ig . 2). As the nucleus moves further into the young conidiophore, the nucleus divides mitotically and a septum is formed thus giving two uninucleate cells (Fig. 3). The nucleus is round but
Nuclear behaviour in Erysiphe pisi
3
o
Figs 1-17. Erysiphe pisi stained with aceto-orcein and observed by light microscopy. Fig. 1. Resting nucleus showing deeply stained nucleolus in a mycelial cell . x 1700. Fig. 2. Resting nucleus at the base of conidiophore initial. x 1250. Figs 3, 4. Septum formation and migration of the nucleus towards the upper cell. x 1000. Fig. 5. A resting nucleus in the terminal cell of the conidiophore. x 2000. Fig. 6. Early prophase. x 2000. Figs 7, 8. Late prophase. x 2000. Fig. 9. Early metaphase. x 2000.
U. P. Singh, H. B. Singh and A. Sakai
•
II
13 Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig.
10. 11. 12 . 13. 14. 15 . 16. 17.
Early metaphase. x 2000. Metaphase showing six bivalents. x 2200. Two-celled conidiophore, each cell containing a single nucleus. x 750. Three-celled conidiophore. x 1000. Four-celled conidiophore. x 1000. A mature conidium containing a single nucleus. x 1500. A germinating conidium showing a single nucleus moving towards a germ-tube. x Nuclear migration into the conidial germ-tube. x 1200.
1500.
Nuclear behaviour in Erysiphe pisi
~\
,-
\ _ .......
nucleus divides mitotically and enters interphase. The chromatin material enlarges and becomes thread-like (F ig. 6) and it gradually condenses before metaphase. During prometaphase the chromosomes become very short but the identity of individual chromosomes is not clear (F igs 7-10). Well-defined bivalents are formed during metaphase and the haploid chromosome number would appear to be (n=6) (Fig. 11 ). In Fig. 11 chromosome a is the smallest and measures o- 5 x o· 5 p,m while chromosome b is slightly bigger (1 '0 x 0'75 p,m). There is little difference between the size of chromosomes c and d(l'O x 0'75 p,mand 1'0 x 1 '0 um, respectively) and chromosomes e andfare more or less the same size. Anaphase was not observed but a septum was formed between the daughter nuclei to give two uninucleate conidiophores (Fig. 12 ). The process is repeated and 3-4 conidia are formed, each of which is uninucleate (Figs 13-14). In some nuclei the nucleolus is distinct (Fig. 14). Each cell of the conidiophore becomes a conidium which is capable of dissemination. The nucleus in the mature conidium is almost round and is often located towards one end (F ig . 15). A mature conidium germinates to produce a germ-tube and the nucleus migrates to the base of the germ-tube (F ig. 16 ) and eventually enters the conidium (F ig. 17). Under the electron microscope the resting nucleus was seen near the tip of the apical cell of the conidiophore and it was faintly stained, but the nucleolus was densely stained and attached to the nuclear membrane (Fig. 18). The resting nucleus in a mature conidium is large, faintly stained and situated at one end of the conidium. The nucleolus is clearly visible as a small, oval, dark-stained body attached to the nuclear membrane (F ig. 19). DISCUSSION
19 Figs 18-19. E. pisi stained with lead acetate and observed by electron microscopy. Fig. 18 . Apical cell of conidiophore showing a single nucleus and a dark-stained nucleolus. x 2000. Fig. 19. Cross-section of a mature conidium showing the nucleus (N) surrounded by nuclear membrane and a dark-stained nucleolus (arrow). x 2500.
the nucleolus is not visible and the cells elongate to become cylindrical (F ig. 4). The resting nucleus in each conidiophore is round and is usually present near the centre of the cell but the nucleolus is not visible (F ig . 5). The
As E. pisi is an obligate parasite it cannot be grown in artificial media, so that it is difficult to study the nuclear divisions in the mycelia, conidiophores and conidia. However, the technique used in this study shows that it is possible to study the nuclear behaviour during mitosis. The nucleolus was clearly, but the nucleoplasm only faintly visible. Some of the stages, especially metaphase, were clearly visible. The haploid chromosome number is six (n = 6). The nucleolus was also deeply stained and attached to the nuclear membrane and the nucleoplasm was faintly stained. The central bodies, spindle microtubules and astral rays were not seen in any of the preparations. However, Harper (1905), Crackower & Bauer (19 7 1) and McKeen ( 19 72 a, b) have reported these organelles in several powdery mildews. The
U. P. Singh, H. B. Singh and A. Sakai nucleolus, which was clearly visible in resting nuclei during aceto-orcein staining, disappeared during mitosis. The nucleolus persists during most of mitosis in E. graminis f.sp. hordei (McKeen, 197 2 b). He observed its disappearance as the daughter nuclei developed. Westergaard & Wettstein (1970) reported the expulsion of nucleoli from the nucleus during interphase in the ascomycete Neottiella. There is no such evidence in E. pisi, and this was not observed by McKeen (1972b) in E. graminis f.sp. hordei. There seems to be a range of chromosome numbers in the powdery mildews. The ascomycetes, in general, have four chromosomes. Dangeard (1904) observed 8 chromosomes (n = 8) in Sphaerotheca castagnei, Harper (1905) observed 8 chromosomes (n = 8) in Phyllactinia species and Clare (1964) observed n=4 for Ph. corylea. McKeen (1972b) observed n=6-8 chromosomes in E. graminis f.sp. hordei. Based on genetic studies, Moseman (1963) suggested the possibility of at least n = 5 in this fungus. Two chromosomes (n = 2) are reported for E. graminis f.sp. tritici by Kimber & Wolfe (1966). We report here for the first time that in E. pisi n=6.
H. B. Singh is grateful to the Council of Scientific and Industrial Research, New Delhi for financial assistance. REFERENCES
ALLEN, RUTH F. (1936). A cytological study of Erysiphe polygoni on Delphinium. Journal ofAgricultural Research 53, 801-818.
CLARE, B. G. (1964). Erysiphaceae of Southern Eastern
Queensland. Queensland University. Papers of the Department of Botany 4, 11
McKEEN, W. E. (1971). Woronin bodies in Erysiphe graminis DC. Canadian Journal of Microbiology 17, 1557-1560.
McKEEN, W. E. (1972a). Nuclear movement in Erysiphe graminis DC. Canadian Journal of Microbiology 18, 1333-1336.
McKEEN, W. E. (197 2b). Somatic mitosis in Erysiphe
graminis hordei. Canadian Journal of Microbiology 18, 19 15-1922. MOSEMAN, J. G. (1963). Relationship of genes conditioning pathogenicity of Erysiphe graminis f.sp. hordei on barley. Phytopathology 53, 1326--133°. OLIVE, L. s. (1953). The structure and behaviour of fungus nuclei. Botanical Reoieu: 19, 439-586. THIRUMALACHAR, M. J. & PAVGI, M. S. (1950). Notes on spore germination and mounting techniques. Indian Phytopathology 3, 177-178. WESTERGAARD, M. & WETTSTEIN, D. (1970). The nucleolar cycle in an ascomycete. Compte rendu des travaux de Laboratoire de Carlsberg 37, 195-237.
(Received for publication 27 January 1984)