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Observations on extra-matrical vesicles of Gigaspora margarita in vitro
168
Notes and brief articles
OBSERVATIONS ON EXTRA-MATRICAL VESICLES OF GIGASPORA MARGARITA IN VITRO BY F. PONS AND V. GIANINAZZI-PEARSON
Station d'Amelioration des Plantes, INRA, BV 154021034 Dijon-Cedex, France Observations on the formation and development of vesicles by Gigaspora margarita suggest that they could be a potential source of inoculum for this VA fungus. During a study of factors affecting spore germination by the vesicular-arbuscular (VA) mycorrhizal fungus Gigaspora margarita in axenic culture, hyphae formed characteristic 'clustered warty vesicles' (Becker & Hall, 1976; Sward, Hallam & Holland, 1978). We have made further observations on these vesicles. Large, white spores of Gigaspora margarita Becker & Hall, collected from pure cultures on onions, were surface-sterilized for 15 min in a solution of 2 % chloramine T + 0 '04 'Yc, streptomycin and transferred to 0'5 % water agar (pH 6'8) alone or with additions of 25,50, 100 or 200 p.p .m. Pas K 2HP0 4 (pH 7'5-7'8 ). Spores were incubated at 24 °C in darkness and germination occurred after 3 days on all media. Hyphal growth from spores was measured microscopically using a micrometer eyepiece. On media containing high levels of P, numerous germ-tubes were produced by each spore in an area around the bulbous suspensor hypha (F ig. 1). Hyphal growth stopped after 35 days and the longest hyphae measured 7'8 and 1 ·8 mm on 100 and 200 p.p.m. P respectively. Hyphae rapidly became septate and void of their cytoplasmic contents and ' vesicles' were never observed. These observations indicate that conditions for spore germination are not necessarily those favourable for hyphal growth and differentiation. On the other media used, including water agar, spores formed only one or two germ-tubes close to the suspensor hyphae (Fig. 2). These grew rapidly (1 '7 lim min-I) and branched, both on the surface and deep into the agar medium . Hyphae continued growing for about 1 month and formed a sparse mycelium reaching distances of up to 16 '0 mm
from the spore. As previously reported (Sward et al. , 1978) rapid uni- or bidirectional protoplasmic streaming (2 '3- 3'2 pm S-I ) could be observed within the growing hyphae. Five days after spore germination, hyphae began to give rise to small branches (F ig. 3) which developed into the so-called 'clustered warty vesicles'(Fig. 6). One spore could produce as many as 14 clusters within 10 days after germination. The formation of these clusters was so rapid that it could be continuously followed microscopically ; soon after its formation each small hyphal branch formed a number of outgrowths (F ig. 3) which coiled, thus giving rise to a cluster of coils (F ig. 4). Each hypha I coil appeared to differentiate, forming a group of between 8 and 16 smooth-walled rounded structures 20 to 30 lim diam (F ig. 5). These developed small outgrowths on their walls and became papillate (F ig. 6); each had a small subtending hyphal stalk. Cytoplasmic streaming occurred in the initial hyphal branches and could be followed during early stages of development (F ig. 3); septa were not evident in the subtending hyphae of the clusters. When hyphae attached to these clusters degenerated and emptied or when they were broken by manipulation, many new hyphae developed from them and radiated into the surrounding medium (F igs 7, 8), with an initial growth rate of o-y nrn min -I . Growth slowed down considerably one week after their emergence. These new hyphae did not appear to emerge directly from the vesicle but from the subtending hyphae close to the point of attachment. The large number of vesicle clusters that can be produced by each azygospore of G. margarita together with their ability to form many new hyphae suggests that these structures may be acting
Figs. 1-8. Gigaspora margarita in axenic culture. Fig. 1. Azygo spore germination with numerous germ-tubes (arrows) on 0'5 % water agar plus 200 p .p.m. P. Fig. 2. Azygospore germination on 0 '5 % water agar . Figs 3-6. Formation of a cluster of vesicle s on o-5 % water agar; dev elopment from the stage in Fig . 3 to that in Fig. 4 occurred within 3'5 h and the stage in Fig. 6 was reached within 20 h . Figs 7- 8. Hyphae developing from clusters of vesicles in which the subtending hyphae have been broken by manipulation.
Trans . Br . mycol. Soc. 84 ( 1), (1985)
Primed in Great Britain
Notes and brief articles
SO lim
SO lim
L...J
L...J
2
,
3
10 lim L.....J
4
'" 6
5
SO li m
7 Trans. Br. mycol. Soc. 84 (1), (1985)
Printed in Great Britain
8
Notes and brief articles as propagules for mycorrhizal infection in soil. This hypothesis is being investigated using axenic mycorrhizal culture systems. We thank Dr D. Maizonnier for his advice with photography and acknowledge support for one of us (F.P.) from the D.G.R.S.T.
REFERENCES
W. N.&HALL, I. R.(1976). Gigasporamargarita, a new species in the Endogonaceae. Mycotaxon 4, 155-160. SWARD, R. J" HALLAM, N. D. & HOLLAND, A. A. (1978). Endogene spores in a heathland area of South-Eastern Australia. Australian Journal of Botany 26, 29-43. BECKER,
OCCURRENCE OF INFUNDIBURA ADHAERENS IN AQUATIC HABITATS IN BRITAIN BY R. D. GOOS
Department of Botany, University of Kingston, Rhode Island, U.S.A. AND E. DESCALS
Department of Biological Sciences, University of Exeter Infundibura adhaerens, a sporodochial hyphomycete with naviculate conidia and infundibuliform appendages at each end, is described from Quercus and Pinus litter in shallow ponds in Britain. Infundibura adhaerens Nag Raj & Kendrick (1981) is an unusual hyphomycete characterized by the production of naviculate conidia with unique, infundibuliform, apical appendages. Nag Raj & Kendrick cited four collections of the fungus, made from widely separated localities, as follows: on cone scales of Pinus from Austria, on cone scales of Picea from British Columbia, on leaves of Agathis australis from New Zealand, and on decomposing leaves of an undetermined broadleafed tree collected in a stream in New Zealand. In a footnote, they mention, without details, a collection of a similar fungus from England. During a survey of aero-aquatic hyphomycetes occurrring on decaying leaf litter, I. adhaerens has been observed several times on leaves of Quercus and Pinus. The leaf material used for these studies was collected on 9 February 1984, from shallow ponds at Bystock, near Woodbury, Devon, England. On return to the laboratory, it was washed in tap water to remove soil and debris, placed in moist chambers and incubated at 10-12 °C, under diurnal light from a north-facing window, supplemented by continuous near-u.v, illumination. The fungus was observed two to three weeks after the leaves were incubated, appearing as raised, clear to white, water-soaked, gelatinous masses. With increasing age, the sporodochia became more whitish and somewhat more firm in texture. The British material agrees well with the original
Trans. Br. mycol. Soc. 84 (1), (1985)
description producing non-septate, naviculate conidia measuring 20-26 x 3-4 pm in size, and bearing both the peculiar apical appendage and the terete, basal appendage. The conidiophores are produced on setose sporodochia. The fungus grew and sporulated readily on corn meal agar, forming the characteristic gelatinous sporulating structures. Recovery of I. adhaerens from decaying leaves in stagnant water suggests that the fungus may be a frequent colonizer of leaves in aquatic habitats, particularly in standing water. Such speculation is consistent with the original report of finding the fungus on a leaf in a New Zealand stream. Use of the techniques employed for the study of aeroaquatic hyphomycetes may be useful in providing additional information on the occurrence and distribution of I. adhaerens. Specimens of the fungus have been deposited in Herb. IMI, Kew. It is a pleasure to thank Professor J. Webster and P. J. Fisher for collecting the leaf material used for these studies. We also thank Professor Webster for providing the photographs.
REFERENCE
RAJ, T. R. & KENDRICK, B. (1981). Infundibura, a new hyphomycete with unique appendages. Canadian Journal of Botany 59, 542-546.
NAG
Primed in Great Britain
Notes and brief articles
OBSERVATIONS ON EXTRA-MATRICAL VESICLES OF GIGASPORA MARGARITA IN VITRO BY F. PONS AND V. GIANINAZZI-PEARSON
Station d'Amelioration des Plantes, INRA, BV 154021034 Dijon-Cedex, France Observations on the formation and development of vesicles by Gigaspora margarita suggest that they could be a potential source of inoculum for this VA fungus. During a study of factors affecting spore germination by the vesicular-arbuscular (VA) mycorrhizal fungus Gigaspora margarita in axenic culture, hyphae formed characteristic 'clustered warty vesicles' (Becker & Hall, 1976; Sward, Hallam & Holland, 1978). We have made further observations on these vesicles. Large, white spores of Gigaspora margarita Becker & Hall, collected from pure cultures on onions, were surface-sterilized for 15 min in a solution of 2 % chloramine T + 0 '04 'Yc, streptomycin and transferred to 0'5 % water agar (pH 6'8) alone or with additions of 25,50, 100 or 200 p.p .m. Pas K 2HP0 4 (pH 7'5-7'8 ). Spores were incubated at 24 °C in darkness and germination occurred after 3 days on all media. Hyphal growth from spores was measured microscopically using a micrometer eyepiece. On media containing high levels of P, numerous germ-tubes were produced by each spore in an area around the bulbous suspensor hypha (F ig. 1). Hyphal growth stopped after 35 days and the longest hyphae measured 7'8 and 1 ·8 mm on 100 and 200 p.p.m. P respectively. Hyphae rapidly became septate and void of their cytoplasmic contents and ' vesicles' were never observed. These observations indicate that conditions for spore germination are not necessarily those favourable for hyphal growth and differentiation. On the other media used, including water agar, spores formed only one or two germ-tubes close to the suspensor hyphae (Fig. 2). These grew rapidly (1 '7 lim min-I) and branched, both on the surface and deep into the agar medium . Hyphae continued growing for about 1 month and formed a sparse mycelium reaching distances of up to 16 '0 mm
from the spore. As previously reported (Sward et al. , 1978) rapid uni- or bidirectional protoplasmic streaming (2 '3- 3'2 pm S-I ) could be observed within the growing hyphae. Five days after spore germination, hyphae began to give rise to small branches (F ig. 3) which developed into the so-called 'clustered warty vesicles'(Fig. 6). One spore could produce as many as 14 clusters within 10 days after germination. The formation of these clusters was so rapid that it could be continuously followed microscopically ; soon after its formation each small hyphal branch formed a number of outgrowths (F ig. 3) which coiled, thus giving rise to a cluster of coils (F ig. 4). Each hypha I coil appeared to differentiate, forming a group of between 8 and 16 smooth-walled rounded structures 20 to 30 lim diam (F ig. 5). These developed small outgrowths on their walls and became papillate (F ig. 6); each had a small subtending hyphal stalk. Cytoplasmic streaming occurred in the initial hyphal branches and could be followed during early stages of development (F ig. 3); septa were not evident in the subtending hyphae of the clusters. When hyphae attached to these clusters degenerated and emptied or when they were broken by manipulation, many new hyphae developed from them and radiated into the surrounding medium (F igs 7, 8), with an initial growth rate of o-y nrn min -I . Growth slowed down considerably one week after their emergence. These new hyphae did not appear to emerge directly from the vesicle but from the subtending hyphae close to the point of attachment. The large number of vesicle clusters that can be produced by each azygospore of G. margarita together with their ability to form many new hyphae suggests that these structures may be acting
Figs. 1-8. Gigaspora margarita in axenic culture. Fig. 1. Azygo spore germination with numerous germ-tubes (arrows) on 0'5 % water agar plus 200 p .p.m. P. Fig. 2. Azygospore germination on 0 '5 % water agar . Figs 3-6. Formation of a cluster of vesicle s on o-5 % water agar; dev elopment from the stage in Fig . 3 to that in Fig. 4 occurred within 3'5 h and the stage in Fig. 6 was reached within 20 h . Figs 7- 8. Hyphae developing from clusters of vesicles in which the subtending hyphae have been broken by manipulation.
Trans . Br . mycol. Soc. 84 ( 1), (1985)
Primed in Great Britain
Notes and brief articles
SO lim
SO lim
L...J
L...J
2
,
3
10 lim L.....J
4
'" 6
5
SO li m
7 Trans. Br. mycol. Soc. 84 (1), (1985)
Printed in Great Britain
8
Notes and brief articles as propagules for mycorrhizal infection in soil. This hypothesis is being investigated using axenic mycorrhizal culture systems. We thank Dr D. Maizonnier for his advice with photography and acknowledge support for one of us (F.P.) from the D.G.R.S.T.
REFERENCES
W. N.&HALL, I. R.(1976). Gigasporamargarita, a new species in the Endogonaceae. Mycotaxon 4, 155-160. SWARD, R. J" HALLAM, N. D. & HOLLAND, A. A. (1978). Endogene spores in a heathland area of South-Eastern Australia. Australian Journal of Botany 26, 29-43. BECKER,
OCCURRENCE OF INFUNDIBURA ADHAERENS IN AQUATIC HABITATS IN BRITAIN BY R. D. GOOS
Department of Botany, University of Kingston, Rhode Island, U.S.A. AND E. DESCALS
Department of Biological Sciences, University of Exeter Infundibura adhaerens, a sporodochial hyphomycete with naviculate conidia and infundibuliform appendages at each end, is described from Quercus and Pinus litter in shallow ponds in Britain. Infundibura adhaerens Nag Raj & Kendrick (1981) is an unusual hyphomycete characterized by the production of naviculate conidia with unique, infundibuliform, apical appendages. Nag Raj & Kendrick cited four collections of the fungus, made from widely separated localities, as follows: on cone scales of Pinus from Austria, on cone scales of Picea from British Columbia, on leaves of Agathis australis from New Zealand, and on decomposing leaves of an undetermined broadleafed tree collected in a stream in New Zealand. In a footnote, they mention, without details, a collection of a similar fungus from England. During a survey of aero-aquatic hyphomycetes occurrring on decaying leaf litter, I. adhaerens has been observed several times on leaves of Quercus and Pinus. The leaf material used for these studies was collected on 9 February 1984, from shallow ponds at Bystock, near Woodbury, Devon, England. On return to the laboratory, it was washed in tap water to remove soil and debris, placed in moist chambers and incubated at 10-12 °C, under diurnal light from a north-facing window, supplemented by continuous near-u.v, illumination. The fungus was observed two to three weeks after the leaves were incubated, appearing as raised, clear to white, water-soaked, gelatinous masses. With increasing age, the sporodochia became more whitish and somewhat more firm in texture. The British material agrees well with the original
Trans. Br. mycol. Soc. 84 (1), (1985)
description producing non-septate, naviculate conidia measuring 20-26 x 3-4 pm in size, and bearing both the peculiar apical appendage and the terete, basal appendage. The conidiophores are produced on setose sporodochia. The fungus grew and sporulated readily on corn meal agar, forming the characteristic gelatinous sporulating structures. Recovery of I. adhaerens from decaying leaves in stagnant water suggests that the fungus may be a frequent colonizer of leaves in aquatic habitats, particularly in standing water. Such speculation is consistent with the original report of finding the fungus on a leaf in a New Zealand stream. Use of the techniques employed for the study of aeroaquatic hyphomycetes may be useful in providing additional information on the occurrence and distribution of I. adhaerens. Specimens of the fungus have been deposited in Herb. IMI, Kew. It is a pleasure to thank Professor J. Webster and P. J. Fisher for collecting the leaf material used for these studies. We also thank Professor Webster for providing the photographs.
REFERENCE
RAJ, T. R. & KENDRICK, B. (1981). Infundibura, a new hyphomycete with unique appendages. Canadian Journal of Botany 59, 542-546.
NAG
Primed in Great Britain