- Email: [email protected]
Rhodocybe stangliana, a parasite on other agarics?
Mycol. Res. 98 (I): 88-90 (1994)
88
Printed in Great Britain
Rhodocybe stangliana, a parasite on other agarics?
THOMAS LiESS0E Herbarium, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AE, u.K.
S0REN ROSENDAHL Department of Mycology, University of Copenhagen, 0ster Farimagsgade 2D, DK-1353, Copenhagen K., Denmark
Isozyme patterns in the 'protocarpic tubers' and in the pileus of Rhodocybe stangliana point to a parasitic lifestyle with the possible host being an unknown agaric species.
The taxonomy of Rhodocybe stangliana (Bresinsky & Pfaff) Riousset & Joss. is controversial. The taxon was originally described in the genus Squamanita (Bresinsky & Pfaff, 1968), but more detailed anatomical and ultrastructural studies by Riousset, Josserand & Capellano (1977) made the combination
Fig. 1. Rhodocybe stangliana, A. Basidiomes B. Section through base
of a basidiome.
in Rhodocybe Maire necessary. The unique character of the species, a 'protocarpic tuber' (sensu Bas, 1965), is the focal point of the controversy, having been variously described as a bulb or a sclerotium. Sandor (1957) suggested that the highly unusual basal structure in reality is a mixture of a host organism and a parasitic agaric. This hypothesis was subsequently rejected or ignored by later authors (Schwabel & Wandel, 1958; Bresinsky & Pfaff, 1968). Material (Fig. I) collected by LreSS0e (Lress0e, 1990) made it possible to study this rare species once more using a novel approach. Fungi parasitic on other fungi are well known, although a detailed study is lacking for most species (Hawksworth, 1981). Several species belonging to the Agaricales are known to parasitize other agarics, e.g. Psathyrella epimyces (Peck) A. H. Smith, Asterophora Ditm.: Fr. (2 species), and Volvariella surreeta (Knapp) Singer. It has also been hypothesised that the members of Squamanita, to which R. stangliana was originally aSSigned, have a parasitic relationship with agarics (Harmaja, 1988; Nagasawa, Hongo & Narita, 1990). The isozyme pattern and the banding pattern of denatured proteins from a fungus are phenotypic traits that reflect the genotype more directly than the morphological characteristics. For this reason isozyme analy~is has been used in taxonomic studies of controversial groups of fungi (Micales, Bonde & Peterson, 1986; Rosendahl, 1989). Specific isozymes have also been used for detection and quantification of arbuscular mycorrhizal fungi (Glomales) in plant roots even when multiple infections occur (Rosendahl & Sen, 1992). The aim of the present study was to compare the isozyme pattern and the pattern of denatured proteins from the stipe and pileus of the basidiome of R. stangliana in order to decide whether the basidiome consists of one or two distinct taxa.
T. Lcess0e and S. Rosendahl C
B
C
B
89 C
B
B
----~-
•
(a)
--
B
C
B
C
--
-
C
B
C
B
B
C
-----------
- -- -
II II II
. c=J.
C
---
--
c:::J _
c:::J _
c:::J _
c::::::J _
c::::::J _
c::::::J _
c::::::J c:::::::J
c=J
c::::::J
c=J c=J
+ + +
(e)
(b)
Fig. 2. Diagrams of the acrylamide gels showing the pattern of enzymes from the stipe (B) and the pileus (C) of three basidiomes of Rhodocybe stangliana. (a) Peptidase; (b) esterase; (c) malate dehydrogenase. (b)
(a)
./
(d)
(e)
----------------7) + Mobility
Fig. 3. Densitometric tracing of a gel stained for malate dehydrogenase. The solid line is extracts from the pileus, the dotted line represents extracts from the stipe of a Rhodocybe stangliana basidiome.
MATERIALS AND METHODS Three basidiomes of R. stangliana were examined. The material was taken from T. Lcess0e 2073, Denmark Jylland, Houskov, 8 Oct. 1989 (vouchers deposited at C and K). The pileus and stipe of the basidiomes of Agaricus bisporus (J. Lange) Imbach, Lepista nuda (Bull.: Fr.) Cooke and Flammulina ve1utipes (Curt.: Fr.) Karst. were used as controls. From the pileus and the stipe of three basidiomes, 1 g of material was sampled and frozen. The material was ground in an ice-chilled glass homogenizer adding 50 mg of insoluble polyvinylpoly-pyrroliodone (PVPP) and 1 ml standard extraction buffer (STEP) (Rosendahl & Sen, 1992), amended with 10% sucrose and 0'1 % Triton X-lOa. The homogenized material was transferred to Eppendorf tubes and centrifuged 30 min at 4 °C and 10 000 g.
-----------"71
Mobility
+
---------"') + Mobility
Fig. 4. Densitometric tracings of the banding pattern of denatured proteins after SD5-PAGE from (a) Rhodocybe stangliana; (b) Agaricus bisporus; (c) Lepista nuda; (d) Flammulina velutipes. The solid lines are protein bands from the pileus, the dotted lines are from the stipe of the basidiomes.
Electrophoresis was carried out in 80 x 80 x 0'8 mm, vertical, polyacrylamide gels using 24'8 mM-Tris 0'19 M glycine, pH 8'3, as an electrode buffer. The stacking gel contained 3'8% acrylamide and 125 mM-Tris-HCI, and the separation gel 7'5% acrylamide and 375 mM-Tris-HCI. Ten lJl of the protein extract were loaded into each well. Gels were stained for malate dehydrogenase (MDH, EC 1.1. 1,37), esterase (EST, EC 3, 1, 1, 1) and peptidase (pEP, EC 3.4, 1.1). The staining procedures were as described by Rosendahl & Sen (1992). Sodium dodecyl sulphate (SDS)-gel electrophoresis was
Rhodocybe stangliana made by extracting 100 mg of fungal material in 100 IJl buffer with 41 mM-Tris, 40 mM boric acid, pH 8'6, containing 1 % SDS, 10% sucrose and 0'1 M mercaptoethanol. The extracts were centrifuged 30 min at 10 000 g and the supernatant recovered and denatured by boiling for 5 min. Electrophoresis was carried out in a vertical discontinuous buffer system. The stacking gel contained 4'5 % acrylamide, 54 mM-Tris, 27 mMH 2 S0 4 and 0'1 % SDS and 20% sucrose. The separation gel contained 12% acrylamide 424 mM Tris, 31 mM-HCl and 0'1 % SDS. The upper tray was the same as the extraction buffer, but without rnercaptoethanol and sucrose. The lower tray buffer was the same as in the separation gel. The gels were stained in Coomassie blue after fixing in 50% methano!' 10% acetic acid and 40% H 2 0. The gels were measured on a GS 300 scanning densitometer (Hoeffer Scientific Instruments) at 585 nm wave length. Percentage similarity (5) in protein banding pattern between the pileus and the stipe of the basidiomes was calculated from the formula: 5 = (C x 100)/( C + T + B), where Tis the number of bands found only in the stipe, B is the number of bands found only in the pileus and C is the number of bands occurring in both the pileus and the stipe.
90
could pOSSibly be the host. Whether this is a possible host could be established by comparing protein patterns from the stipe of R. stangliana with patterns of non-parasitized fungi in the area, assuming that normal basidiome from non-parasitized mycelia occur on the same site. The banding pattern of proteins from different structures of a fungus may differ. This has been observed between different parts of the basidiome in Coprinus cinereus (Schaeff.: Fr.) S. F. Gray (Moore & Jjrjis, 1981) and Agaricus bisporus (Paranjpe, Chen & Jong, 1979). However, the difference in enzyme banding pattern detected in Coprinus pileus and stems, were quantitative rather than qualitative. The high degree of similarity between the pileus and the stipe of the three control species in the present study, therefore, does not necessarily contradict the results of Moore & Jirjis (1981) and Paranjpe et al. (1979). This study was supported by a grant to S. R. from the Danish Natural Science Research Council. We wish to thank Dr D. N. Pegler, Dr H. Knudsen and Mycological Research reviewers for their comments on the manuscript.
REFERENCES RESUL TS AND DISCUSSION The isozyme patterns of the stipe and the pileus of R. stangliana showed only limited similarity. The MDH bands that were found in the pileus were also found in the stipe (Fig. 2c). A similar result was obtained for peptidase (Fig. 2a). No esterase bands were found in extracts from the pileus, although several enzyme bands were detected in extracts of the stipe (Fig. 2 b). The pattern of denatured protein showed 11 % similarity between the pileus and the stipe, and only some similarity in the faster moving proteins could be detected (Fig. 3 a). In comparison, the banding pattern of proteins from the pileus and the stipe of A. bisporu5, L. nuda and F. velutipes, showed BB, 56 and 73 % similarity respectively, between the two parts (Fig. 3 b-d). The results further support the hypothesis that two taxa are involved in the 'basidiome' of R. starlgliana. This is in agreement with Sandor (1957), who found considerable differences between the bulb-like basal structure and the rest of the basidiome in their reaction to various chemical reagents. This suggested that the bulb was a parasitized fungus. The PEP and the MDH bands from the pileus found in the present study were also detected in the stipe (Fig. 2a, c). The high activity of these enzyme bands in the stipe imply that the 'pileus' is a biotrophic parasite, able to alter the gene expression of the host in order to prevent basidiome formation. This is known in Psathyrella epimyces which grows on species of Coprinus Pers. and frequently suppresses the host development to the extent that a positive identification is impossible (Smith, 1972). In the present study the potential host organism was not found. Several collection notes mention an associated species of Tephrocybe Donk and this (Accepted 8 July 1993)
Bas, C. (1965). The genus Squamanita. Persoonia 3, 331-359. Bresinsky, A & Pfaff, K. (1968). Ober eine bislang nicht benannte Art der Gattung Squamanita (Agaricales). Zeitschrift fur Pilzkunde 34, 169--174. Harmaja, H. (1988). Studies on the agaric genera Singerocybe n. gen. and Squamanita. Karstenia 27, 71-75.
Hawksworth, D. L. (1981). A survey of the fungicolous conidial fungi. In: The Biology of Conidial Fungi, vol. 1 (ed. G. T. Cole & B. Kendrick), pp. 171-244. Academic Press: New York, U.S.A Liessoe, T. (1990). Rhodocybe stangliana - ny for Danmark. Svampe 22,24-26. Micales, J. A, Bonde, M. R. & Peterson, G. L. (1986). The use of isozyme analysis in fungal taxonomy and genetics. Mycotaxon 27, 405-449. Moore, D. & Jirjis, R. I. (1981) Electrophoretic studies in carpophore development in the basidiomycete Coprinus cinereus. New Phytologist 87, 101-113.
Nagasawa, E., Hongo, T. & Narita, D. (1990). Squamanita odorata (Agaricales) from Japan. Reports of the Tottori Mycological Institute 28, 135-141. Paranjpe, M. S.. Chen, P. K. & Jong, S. C. (1979). Morphogenesis of Agaricus bisporus: changes in proteins and enzyme activity. Mycologia 71, 469-478. Reid, D. (1983). A second British collection of Squamanita paradoxa. Bulletin of the British Mycological Society 17, 111-113.
Riousset, L., Josserand, M. & Capellano, A (1977). Position systematique et description de 'Rhodocybe stangliana' (Bres. et Pfaff) Riousset et Joss. Basidiomycete Tricholomaceae (=' Squamanita stangliana' Bresinsky et Pfaff). Architecture de sa paroi sporique. Bulletin Mensuel de la Sociite Linneenne de Lyon 46, 119-130.
RosendahL S. (1989). Comparisons of spore-cluster forming Glomus species (Endogonaceae) based on morphological characteristics and isoenzyme banding patterns. Opera Botanica 100, 215-223. RosendahL S. & Sen, R. (1992). Isozyme analysis of mycorrhizal fungi and their mycorrhizas. In: Methods in microbiology vol. 24: Experiments with Mycorrhizae (ed. A K. Varma, D. J. Read & J. R. Norris), pp. 169-194. Academic Press. Sandor, R. (1957). Wenig bekannte Pilze aus der Miinchner Umgebung. Zeitschrift fur Pilzkunde 23, 48-52.
SchwabeL H. & WandeL J. (1958). Zur Kliirung einiger Pilze aus der Miinchner Umgebung. Zeiftschrift fur Pilzkunde 24, 52-53. Smith, A H. (1972). The North American species of Psathyrella. Memoirs of the New York Botanical Garden 24, 1-633.
88
Printed in Great Britain
Rhodocybe stangliana, a parasite on other agarics?
THOMAS LiESS0E Herbarium, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AE, u.K.
S0REN ROSENDAHL Department of Mycology, University of Copenhagen, 0ster Farimagsgade 2D, DK-1353, Copenhagen K., Denmark
Isozyme patterns in the 'protocarpic tubers' and in the pileus of Rhodocybe stangliana point to a parasitic lifestyle with the possible host being an unknown agaric species.
The taxonomy of Rhodocybe stangliana (Bresinsky & Pfaff) Riousset & Joss. is controversial. The taxon was originally described in the genus Squamanita (Bresinsky & Pfaff, 1968), but more detailed anatomical and ultrastructural studies by Riousset, Josserand & Capellano (1977) made the combination
Fig. 1. Rhodocybe stangliana, A. Basidiomes B. Section through base
of a basidiome.
in Rhodocybe Maire necessary. The unique character of the species, a 'protocarpic tuber' (sensu Bas, 1965), is the focal point of the controversy, having been variously described as a bulb or a sclerotium. Sandor (1957) suggested that the highly unusual basal structure in reality is a mixture of a host organism and a parasitic agaric. This hypothesis was subsequently rejected or ignored by later authors (Schwabel & Wandel, 1958; Bresinsky & Pfaff, 1968). Material (Fig. I) collected by LreSS0e (Lress0e, 1990) made it possible to study this rare species once more using a novel approach. Fungi parasitic on other fungi are well known, although a detailed study is lacking for most species (Hawksworth, 1981). Several species belonging to the Agaricales are known to parasitize other agarics, e.g. Psathyrella epimyces (Peck) A. H. Smith, Asterophora Ditm.: Fr. (2 species), and Volvariella surreeta (Knapp) Singer. It has also been hypothesised that the members of Squamanita, to which R. stangliana was originally aSSigned, have a parasitic relationship with agarics (Harmaja, 1988; Nagasawa, Hongo & Narita, 1990). The isozyme pattern and the banding pattern of denatured proteins from a fungus are phenotypic traits that reflect the genotype more directly than the morphological characteristics. For this reason isozyme analy~is has been used in taxonomic studies of controversial groups of fungi (Micales, Bonde & Peterson, 1986; Rosendahl, 1989). Specific isozymes have also been used for detection and quantification of arbuscular mycorrhizal fungi (Glomales) in plant roots even when multiple infections occur (Rosendahl & Sen, 1992). The aim of the present study was to compare the isozyme pattern and the pattern of denatured proteins from the stipe and pileus of the basidiome of R. stangliana in order to decide whether the basidiome consists of one or two distinct taxa.
T. Lcess0e and S. Rosendahl C
B
C
B
89 C
B
B
----~-
•
(a)
--
B
C
B
C
--
-
C
B
C
B
B
C
-----------
- -- -
II II II
. c=J.
C
---
--
c:::J _
c:::J _
c:::J _
c::::::J _
c::::::J _
c::::::J _
c::::::J c:::::::J
c=J
c::::::J
c=J c=J
+ + +
(e)
(b)
Fig. 2. Diagrams of the acrylamide gels showing the pattern of enzymes from the stipe (B) and the pileus (C) of three basidiomes of Rhodocybe stangliana. (a) Peptidase; (b) esterase; (c) malate dehydrogenase. (b)
(a)
./
(d)
(e)
----------------7) + Mobility
Fig. 3. Densitometric tracing of a gel stained for malate dehydrogenase. The solid line is extracts from the pileus, the dotted line represents extracts from the stipe of a Rhodocybe stangliana basidiome.
MATERIALS AND METHODS Three basidiomes of R. stangliana were examined. The material was taken from T. Lcess0e 2073, Denmark Jylland, Houskov, 8 Oct. 1989 (vouchers deposited at C and K). The pileus and stipe of the basidiomes of Agaricus bisporus (J. Lange) Imbach, Lepista nuda (Bull.: Fr.) Cooke and Flammulina ve1utipes (Curt.: Fr.) Karst. were used as controls. From the pileus and the stipe of three basidiomes, 1 g of material was sampled and frozen. The material was ground in an ice-chilled glass homogenizer adding 50 mg of insoluble polyvinylpoly-pyrroliodone (PVPP) and 1 ml standard extraction buffer (STEP) (Rosendahl & Sen, 1992), amended with 10% sucrose and 0'1 % Triton X-lOa. The homogenized material was transferred to Eppendorf tubes and centrifuged 30 min at 4 °C and 10 000 g.
-----------"71
Mobility
+
---------"') + Mobility
Fig. 4. Densitometric tracings of the banding pattern of denatured proteins after SD5-PAGE from (a) Rhodocybe stangliana; (b) Agaricus bisporus; (c) Lepista nuda; (d) Flammulina velutipes. The solid lines are protein bands from the pileus, the dotted lines are from the stipe of the basidiomes.
Electrophoresis was carried out in 80 x 80 x 0'8 mm, vertical, polyacrylamide gels using 24'8 mM-Tris 0'19 M glycine, pH 8'3, as an electrode buffer. The stacking gel contained 3'8% acrylamide and 125 mM-Tris-HCI, and the separation gel 7'5% acrylamide and 375 mM-Tris-HCI. Ten lJl of the protein extract were loaded into each well. Gels were stained for malate dehydrogenase (MDH, EC 1.1. 1,37), esterase (EST, EC 3, 1, 1, 1) and peptidase (pEP, EC 3.4, 1.1). The staining procedures were as described by Rosendahl & Sen (1992). Sodium dodecyl sulphate (SDS)-gel electrophoresis was
Rhodocybe stangliana made by extracting 100 mg of fungal material in 100 IJl buffer with 41 mM-Tris, 40 mM boric acid, pH 8'6, containing 1 % SDS, 10% sucrose and 0'1 M mercaptoethanol. The extracts were centrifuged 30 min at 10 000 g and the supernatant recovered and denatured by boiling for 5 min. Electrophoresis was carried out in a vertical discontinuous buffer system. The stacking gel contained 4'5 % acrylamide, 54 mM-Tris, 27 mMH 2 S0 4 and 0'1 % SDS and 20% sucrose. The separation gel contained 12% acrylamide 424 mM Tris, 31 mM-HCl and 0'1 % SDS. The upper tray was the same as the extraction buffer, but without rnercaptoethanol and sucrose. The lower tray buffer was the same as in the separation gel. The gels were stained in Coomassie blue after fixing in 50% methano!' 10% acetic acid and 40% H 2 0. The gels were measured on a GS 300 scanning densitometer (Hoeffer Scientific Instruments) at 585 nm wave length. Percentage similarity (5) in protein banding pattern between the pileus and the stipe of the basidiomes was calculated from the formula: 5 = (C x 100)/( C + T + B), where Tis the number of bands found only in the stipe, B is the number of bands found only in the pileus and C is the number of bands occurring in both the pileus and the stipe.
90
could pOSSibly be the host. Whether this is a possible host could be established by comparing protein patterns from the stipe of R. stangliana with patterns of non-parasitized fungi in the area, assuming that normal basidiome from non-parasitized mycelia occur on the same site. The banding pattern of proteins from different structures of a fungus may differ. This has been observed between different parts of the basidiome in Coprinus cinereus (Schaeff.: Fr.) S. F. Gray (Moore & Jjrjis, 1981) and Agaricus bisporus (Paranjpe, Chen & Jong, 1979). However, the difference in enzyme banding pattern detected in Coprinus pileus and stems, were quantitative rather than qualitative. The high degree of similarity between the pileus and the stipe of the three control species in the present study, therefore, does not necessarily contradict the results of Moore & Jirjis (1981) and Paranjpe et al. (1979). This study was supported by a grant to S. R. from the Danish Natural Science Research Council. We wish to thank Dr D. N. Pegler, Dr H. Knudsen and Mycological Research reviewers for their comments on the manuscript.
REFERENCES RESUL TS AND DISCUSSION The isozyme patterns of the stipe and the pileus of R. stangliana showed only limited similarity. The MDH bands that were found in the pileus were also found in the stipe (Fig. 2c). A similar result was obtained for peptidase (Fig. 2a). No esterase bands were found in extracts from the pileus, although several enzyme bands were detected in extracts of the stipe (Fig. 2 b). The pattern of denatured protein showed 11 % similarity between the pileus and the stipe, and only some similarity in the faster moving proteins could be detected (Fig. 3 a). In comparison, the banding pattern of proteins from the pileus and the stipe of A. bisporu5, L. nuda and F. velutipes, showed BB, 56 and 73 % similarity respectively, between the two parts (Fig. 3 b-d). The results further support the hypothesis that two taxa are involved in the 'basidiome' of R. starlgliana. This is in agreement with Sandor (1957), who found considerable differences between the bulb-like basal structure and the rest of the basidiome in their reaction to various chemical reagents. This suggested that the bulb was a parasitized fungus. The PEP and the MDH bands from the pileus found in the present study were also detected in the stipe (Fig. 2a, c). The high activity of these enzyme bands in the stipe imply that the 'pileus' is a biotrophic parasite, able to alter the gene expression of the host in order to prevent basidiome formation. This is known in Psathyrella epimyces which grows on species of Coprinus Pers. and frequently suppresses the host development to the extent that a positive identification is impossible (Smith, 1972). In the present study the potential host organism was not found. Several collection notes mention an associated species of Tephrocybe Donk and this (Accepted 8 July 1993)
Bas, C. (1965). The genus Squamanita. Persoonia 3, 331-359. Bresinsky, A & Pfaff, K. (1968). Ober eine bislang nicht benannte Art der Gattung Squamanita (Agaricales). Zeitschrift fur Pilzkunde 34, 169--174. Harmaja, H. (1988). Studies on the agaric genera Singerocybe n. gen. and Squamanita. Karstenia 27, 71-75.
Hawksworth, D. L. (1981). A survey of the fungicolous conidial fungi. In: The Biology of Conidial Fungi, vol. 1 (ed. G. T. Cole & B. Kendrick), pp. 171-244. Academic Press: New York, U.S.A Liessoe, T. (1990). Rhodocybe stangliana - ny for Danmark. Svampe 22,24-26. Micales, J. A, Bonde, M. R. & Peterson, G. L. (1986). The use of isozyme analysis in fungal taxonomy and genetics. Mycotaxon 27, 405-449. Moore, D. & Jirjis, R. I. (1981) Electrophoretic studies in carpophore development in the basidiomycete Coprinus cinereus. New Phytologist 87, 101-113.
Nagasawa, E., Hongo, T. & Narita, D. (1990). Squamanita odorata (Agaricales) from Japan. Reports of the Tottori Mycological Institute 28, 135-141. Paranjpe, M. S.. Chen, P. K. & Jong, S. C. (1979). Morphogenesis of Agaricus bisporus: changes in proteins and enzyme activity. Mycologia 71, 469-478. Reid, D. (1983). A second British collection of Squamanita paradoxa. Bulletin of the British Mycological Society 17, 111-113.
Riousset, L., Josserand, M. & Capellano, A (1977). Position systematique et description de 'Rhodocybe stangliana' (Bres. et Pfaff) Riousset et Joss. Basidiomycete Tricholomaceae (=' Squamanita stangliana' Bresinsky et Pfaff). Architecture de sa paroi sporique. Bulletin Mensuel de la Sociite Linneenne de Lyon 46, 119-130.
RosendahL S. (1989). Comparisons of spore-cluster forming Glomus species (Endogonaceae) based on morphological characteristics and isoenzyme banding patterns. Opera Botanica 100, 215-223. RosendahL S. & Sen, R. (1992). Isozyme analysis of mycorrhizal fungi and their mycorrhizas. In: Methods in microbiology vol. 24: Experiments with Mycorrhizae (ed. A K. Varma, D. J. Read & J. R. Norris), pp. 169-194. Academic Press. Sandor, R. (1957). Wenig bekannte Pilze aus der Miinchner Umgebung. Zeitschrift fur Pilzkunde 23, 48-52.
SchwabeL H. & WandeL J. (1958). Zur Kliirung einiger Pilze aus der Miinchner Umgebung. Zeiftschrift fur Pilzkunde 24, 52-53. Smith, A H. (1972). The North American species of Psathyrella. Memoirs of the New York Botanical Garden 24, 1-633.