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Ultrastructural studies of resting spore development in Polymyxa graminis
Mycol. Res. 102 (6) : 687–691 (1998)
687
Printed in the United Kingdom
Ultrastructural studies of resting spore development in Polymyxa graminis
J I A N P I N G C H E N1, Z H I Q I A N G W A N G1, J I A N H O N G2, C. R E B E C C A C O L L I E R3 A N D M I C H A E L J. A D A MS3* " Virology Department, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, People’s Republic of China # Electron Microscope Laboratory, Zhejiang Agricultural University, Hangzhou 311029, People’s Republic of China $ Crop and Disease Management Department, IACR-Rothamsted, Harpenden, Herts, AL5 2JQ, U.K.
Transmission electron microscopy was used to study the development and structure of resting spores and sporosori of Polymyxa graminis. Osmiophilic bodies generally distributed in the cytoplasm moved to the periphery as spores matured. The spore wall consisted of four or five layers, two of which extended into spines linking adjacent spores. An unstained plug in the wall, faced towards the outside of the sporosorus. Some spores germinated in situ by producing primary zoospores. Bundle-like structures in some spores may have been barley yellow mosaic virus particles.
Polymyxa graminis Ledingham (Ledingham, 1939) is a common obligate parasite of the roots of Poaceae. It is particularly important as the vector of several plant viruses, including barley yellow mosaic (BaYMV), barley mild mosaic (BaMMV), wheat spindle streak mosaic, wheat yellow mosaic and soilborne wheat mosaic viruses. Polymyxa is one of nine genera usually recognized in the Plasmodiophoromycetes (Karling, 1968), a class distinguished from other zoosporic fungi by the production of biflagellate, isokont zoospores. The genus contains two species, P. graminis and P. betae Keskin, which are distinguished by their host ranges, but there appears to be little or no consistent morphological distinction and their separate specific status is questionable (Barr, 1988). The life cycle is not completely understood but has two phases. In the sporangial phase, the plasmodium formed after infection by a zoospore develops into a lobed zoosporangium, which releases more zoospores either to the outside of the host, or to infect deeper layers of the root (Barr, 1988). Sporogenic plasmodia (using the terminology recommended by Braselton, 1995), initially indistinguishable from the sporangial ones, undergo what appears to be meiotic division (Braselton, 1988) and then develop into sporosori (often termed cystosori), each consisting of numerous, thick-walled resting spores which remain viable (together with any viruses they may be carrying) in soil for many years (Adams, 1990). Ultrastructural studies have been made of zoospores, their penetration mechanism and of sporangial plasmodia (Keskin & Fuchs, 1969 ; Barr & Allan, 1982 ; Chen & Adams, 1992), but less is known of sporogenic plasmodia and the development of sporosori, except for a scanning electron microscopy (SEM) study of P. betae by Ciafardini & Marotta (1988) and a report of cell wall ultra* Corresponding author.
structure of spores extracted from infected roots (Ciafardini et al., 1995). In this paper we report transmission electron microscopy (TEM) of different stages in sporosoral development and discuss the results with relation to published work using SEM and to observations on other genera of Plasmodiophoromycetes.
M A T E R I A L S A N D M E T H O DS Monofungal isolates of P. graminis were obtained by isolating sporosori from naturally infected barley roots and inoculating barley roots in irrigated sand culture (Adams, Swaby & Macfarlane, 1986 ; Adams & Jacquier, 1994). Roots from inoculated plants heavily infested with sporosori were dried and used as inoculum for further cultures. Pieces of root for electron microscopy were selected under a light microscope at different times after inoculation, fixed in 2±5 % glutaraldehyde, post-fixed with osmium tetroxide, dehydrated and embedded in L. R. White medium grade resin (London Resin Co.) containing 1 % (v}v) silicon fluid (Dow Corning 200) or in Agar 100 resin (TAAB Laboratories Ltd) using standard techniques. Ultramicrotome sections were stained in uranyl acetate and lead citrate before examination using a JEM1200EX transmission electron microscope at 60 kV.
RESULTS In sand cultures inoculated with sporosori and maintained at 12 °C, first infection of root cells was observed about 2 wk after inoculation and from 6–12 wk after inoculation numerous zoosporangia were seen easily in the fine roots. The first young, colourless, sporosori were detected at about 14 wk
TEM of resting spores of Polymyxa graminis
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Figs 1–6. Transmission electron micrographs of sections of resting spores of Polymyxa graminis. Fig. 1. Immature spores. Fig. 2. Whole sporosorus showing mature spores. Fig. 3. Part of a single spore showing association of osmiophilic bodies with membranes. Fig. 4. Detail of spore wall : 1–5 ¯ layers of spore wall, with edge of plug visible on extreme right. Fig. 5. Connection between adjacent spores. Fig. 6. Single spore with prominent plug. Bars, 1 µm. M, membrane ; N, nucleus ; O, osmiophilic bodies ; P, plug ; T, connection in transverse section.
J. Chen and others
689
1 100 nm
2 3 4 5 Plug Cytoplasm
Fig. 7. Diagram illustrating spore wall structure. The five layers are numbered and their thickness is approximately to the scale indicated by the bar.
and these became mature and dark brown over the following 5 wk. At the ultrastructural level, the first distinct stage in resting spore development was the development of transitional plasmodia containing nuclei with synaptonemal complexes. These were seen in the present study but are not described in detail because they have already been reported by Braselton (1984). This was followed by nuclear division and the division of the plasmodium into uninucleate cells by the formation of membranous sheets within the cytoplasm. A spore wall was then deposited between the cells and, while the walls remain fused in places, progressively larger spaces developed between the spores. In immature resting spores, osmiophilic bodies were distributed generally in the cytoplasm (Fig. 1) but these seemed to move to the periphery as the spores matured (Fig. 2) and sometimes appeared to be associated with a membrane system (Fig. 3). Mature resting spores were uninucleate and contained abundant mitochondria, strands of rough ER, Golgi bodies, well-distributed vacuoles, vesicles containing a granular matrix and numerous free ribosomes. Nuclei averaged 1±6 µm diam. and each contained a spherical acentric nucleolus (x` ¯ 0±4 µm). The mature spore wall consisted of four or perhaps five layers and adjacent spores had connecting links formed from the two outer layers (Figs 4–5). The outer layer was thin (around 25–30 nm) and extremely electron-dense, while the second layer was thicker (60–80 nm) and had the appearance of a matrix containing electron-dense granules. The third layer (usually about 25 nm) was the least osmiophilic and inside this there appeared to be one or perhaps two layers. The appearance varied between sections but, in some (e.g. Fig. 6), there was a rather fibrillar layer about 60–80 nm thick which was separated clearly from layer 3 and an innermost, darker, layer, with an appearance not dissimilar to that of the osmiophilic bodies in the cytoplasm. This inner layer may therefore represent an intermediate stage in the deposition of the inner spore wall. A prominent feature in many sections was an unstained plug within the fourth}fifth layer of the wall (Fig. 6). This was 100–120 nm thick, extended up to about one third of the circumference of the spore and in appropriate sections of the entire sporosorus it could be seen that these
plugs all faced towards the outside (Fig. 2). Layer 3 seemed to be thicker (up to about 70 nm) in the region of this plug. Our interpretation of the cell wall structure is summarised in Fig. 7. Some filamentous, virus-like bundles were observed inside a few resting spores (Fig. 8). They were always located near the plasmalemma and were seen only in the cytoplasm of an isolate of P. graminis transmitting barley yellow mosaic bymovirus (BaYMV), originating from Xiaoshan, Zhejiang, China. No such structures were ever seen within a virus-free isolate. In some spores, the cytoplasm contracted from the wall and the intervening space was filled with a granular matrix (Fig. 9). It is possible that the peripheral osmiophilic vesicles contributed to this process and that this was associated with primary zoospore differentiation, because flagella were seen in some other spores at this stage (Fig. 10). It was not unusual to find sporosori in which individual spores were at different stages of development (Fig. 11) and empty spores, usually associated with many bacteria, were sometimes found (Fig. 12) suggesting that germination had occurred in situ. DISCUSSION The general pattern of ultrastructural development of resting spores of P. graminis was similar to that of other species of Plasmodiophoromycetes (e.g. Plasmodiophora brassicae : Williams & McNabola, 1967). It began with the division of the plasmodium into uninucleate cells by the formation of membranous sheets within the cytoplasm, similar to that reported for P. betae (Barr & Asher, 1996) and Sorosphaera veronicae (Braselton & Miller, 1978). In a SEM study of the closely-related Polymyxa betae, Ciafardini & Marotta (1988) claimed that immature resting spore walls were folded with numerous empty spaces between them, while spores in older sporosori had an inflated appearance and adjacent spore walls were in contact without empty spaces. This seems to be the opposite of what we found by TEM and we suggest that artefacts introduced during specimen preparation for SEM (e.g. collapse of the thinner immature spore walls, but not of the thicker mature ones) may be responsible for the apparent difference. The cytoplasmic organelles within the resting spores had a similar appearance to those observed in zoospores (Barr & Allan, 1982 ; Chen & Adams, 1992). The osmiophilic bodies observed within the cytoplasm seemed to originate from a membrane system and move to the perimeter of the spore as it matured. It therefore seems more likely that these played a role in spore development and}or germination rather than being storage organelles. Spore walls of other species of Plasmodiophoromycetes have been described as having either one (Ligniera verrucosa : Miller, Martin & Dylewski, 1985), two (Polymyxa betae : D’Ambra & Mutto, 1975 ; Woronina pythii : Miller & Dylewski, 1983 ; Tetramyxa parasitica : Braselton, 1990), three (Spongospora subterranea : Lahert & Kavanagh, 1985 ; Plasmodiophora brassicae : Yukawa & Tanaka, 1979) or five (Polymyxa betae : Ciafardini et al., 1995) layers. It is possible that these reports partly reflect differences in specimen preparation, staining and
TEM of resting spores of Polymyxa graminis
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Figs 8–12. Fig. 8. Filamentous, virus-like bundles (V) in resting spore cytoplasm. Bar, 200 nm. Fig. 9. Contracting cytoplasm at start of primary zoospore differentiation. Bar, 500 nm. Fig. 10. Zoospore flagellum (F) inside spore. Bar, 1 µm. Fig. 11. Sporosorus with spores at different developmental stages. Bar, 2 µm. Fig. 12. Empty resting spores invaded by bacteria (B) after zoospore germination. Bar, 1 µm.
interpretation more than significant structural differences, although adjacent spores have been shown to associate in different ways. Those of Plasmodiophora are not aggregated, while in Woronina, spores are aggregated into a sporosorus but the walls of adjacent spores are not joined. Differing degrees of fusion between spores walls can be seen amongst
the other genera, with those of Sorosphaera veronicae showing the greatest degree of fusion and the most regular arrangement of spores (Braselton & Miller, 1978). In P. graminis, mature spores seem to be connected only by thin spines and it is relatively easy to fragment the sporosorus into individual spores (Adams & Collier, unpublished results).
J. Chen and others We assume that the plug in the wall is associated with germination, although it is not clear how it opens to release the primary zoospore. Similar structures can be seen in photographs of resting spores of P. betae (Ivanovic! , 1988) and Sorosphaera veronicae (Braselton & Miller, 1978) and also were described in Polymyxa betae by Ciafardini et al. (1995) who suggested that it disappeared before germination. Much smaller germination pores, with a clearly different morphology, have been described for S. subterranea (Lahert & Kavanagh, 1985), Plasmodiophora brassicae (Yukawa & Tanaka, 1979 ; Ikegami et al., 1982) and Woronina pythii (Miller & Dylewski, 1983). In their SEM studies of developing resting spores of Polymyxa betae, Ciafardini & Marotta (1988, 1989) suggested that holes developed at a point of weakness in the spore wall and then enlarged to permit zoospore germination, after which the spore walls folded back inside the empty spore. Our studies also show that at least some spores of a sporosorus may germinate once they are mature, although it is possible that some of the features shown by the scanning electron micrographs may represent specimen collapse during preparation. Primary zoospore formation within a spore has also been reported for Spongospora subterranea (Lahert & Kavanagh, 1985). The ability of Polymyxa species (and the viruses they transmit) to persist in soil for many years in the absence of a known host (Adams, 1990 ; Payne & Asher, 1990) suggests either that spores have a variable, inbuilt, dormancy, or that a host-specific stimulus is needed for their germination. Bundle-like structures (Fig. 8) within spores of an isolate transmitting BaYMV were similar to structures previously identified (by immunogold labelling) as barley mild mosaic bymovirus inside zoospores and zoosporangia (Chen, Swaby, Adams & Ruan, 1991). It therefore seems probable that these structures consist of BaYMV particles. Zhejiang Academy of Agricultural Sciences acknowledges grants from the European Commission (ISC CI1*CT93-0081), the National and Zhejiang Provincial Foundation for Natural Sciences and the Chinese Ministry of Agriculture. IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.
REFERENCES Adams, M. J. (1990). Epidemiology of fungally-transmitted viruses. Soil Use and Management 6, 184–189. Adams, M. J. & Jacquier, C. (1994). Infection of cereals and grasses by isolates of Polymyxa graminis (Plasmodiophorales). Annals of Applied Biology 125, 53–60. Adams, M. J., Swaby, A. G. & Macfarlane, I. (1986). The susceptibility of barley cultivars to barley yellow mosaic virus (BaYMV) and its fungal vector, Polymyxa graminis. Annals of Applied Biology 109, 561–572. Barr, D. J. S. (1988). Zoosporic plant parasites as fungal vectors of viruses : taxonomy and life cycles of species involved. In Developments in Applied (Accepted 21 July 1997)
691 Biology. 2. Viruses with Fungal Vectors (ed. J. I. Cooper & M. J. C. Asher), pp. 123–137. Association of Applied Biologists : Wellesbourne, Warwick. Barr, D. J. S. & Allan, P. M. E. (1982). Zoospore ultrastructure of Polymyxa graminis (Plasmodiophoromycetes). Canadian Journal of Botany 60, 2496– 2504. Barr, K. J. & Asher, M. J. C. (1996). Studies on the life-cycle of Polymyxa betae in sugar beet roots. Mycological Research 100, 203–208. Braselton, J. P. (1984). Karyotypic analysis of Polymyxa graminis (Plasmodiophoromycetes) based on serial thin sections of synaptonemal complexes. Canadian Journal of Botany 62, 2414–2416. Braselton, J. P. (1988). Karyology and systematics of Plasmodiophoromycetes. In Developments in Applied Biology. 2. Viruses with Fungal Vectors (ed. J. I. Cooper & M. J. C. Asher), pp. 139–152. Association of Applied Biologists : Wellesbourne, Warwick. Braselton, J. P. (1990). Ultrastructure and karyology of Tetramyxa parasitica (Plasmodiophoromycetes). Canadian Journal of Botany 68, 594–598. Braselton, J. P. (1995). Current status of the Plasmodiophorids. Critical Reviews in Microbiology 21, 263–275. Braselton, J. P. & Miller, C. E. (1978). Cyst formation in Sorosphaera veronicae (Plasmodiophoromycetes). Micron 9, 37–38. Chen, J. & Adams, M. J. (1992). Zoospore ultrastructure of Polymyxa graminis. Acta Mycologica Sinica 11, 216–220. Chen, J., Swaby, A. G., Adams, M. J. & Ruan, Y. (1991). Barley mild mosaic virus inside its fungal vector, Polymyxa graminis. Annals of Applied Biology 118, 615–621. Ciafardini, G., Arena, L. M. R., Mares, D. & Bruni, A. (1995). Cell wall ultrastructure in resting-spores of Polymyxa betae Keskin (Plasmodiophoraceae). Phyton (Horn) 35, 153–163. Ciafardini, G. & Marotta, B. (1988). Scanning electron microscopy of the sporosorus of Polymyxa betae (Plasmodiophoromycetes). Canadian Journal of Botany 66, 2518–2522. Ciafardini, G. & Marotta, B. (1989). Localization of fragile areas in walls of resting spores of Polymyxa betae. Canadian Journal of Botany 67, 3123–3126. D’Ambra, V. & Mutto, S. (1975). Ultrastruttura di Polymyxa betae Keskin. Plasmodio, sporangio e cistosoro. Rivista di Patologia Vegetale 11, 115–124. Ikegami, H., Naiki, T., Ito, T. & Imuro, Y. (1982). Ultrastructural growth process of Plasmodiophora brassicae in infected cells of Chinese cabbage and turnip (Studies on the clubroot of cruciferous plants IV). Research Bulletin of the Faculty of Agriculture, Gifu University 46, 9–19. Ivanovic! , M. (1988). Prouc) avanje biologije i ekologije Polymyxa betae Kesk. na s) ec0 ernoj repi u Jugoslaviji. Zas] tita bilja 39, 259–273. Karling, J. S. (1968). The Plasmodiophorales, 2nd Edition. Hafner : New York. Keskin, B. & Fuchs, W. H. (1969). Der Infektionsvorgang bei Polymyxa betae. Archiv fuX r Mikrobiologie 68, 218–226. Lahert, H. & Kavanagh, J. A. (1985). The fine structure of the cystosorus of Spongospora subterranea, the cause of powdery scab of potato. Canadian Journal of Botany 63, 2278–2282. Ledingham, G. A. (1939). Studies on Polymyxa n.gen. n.sp., a plasmodiophoraceous root parasite of wheat. Canadian Journal of Research C Botanical Sciences 17, 38–51. Miller, C. E. & Dylewski, D. P. (1983). Zoosporic fungal pathogens of lower plants. What can be learned from the likes of Woronina ? In Zoosporic Plant Pathogens. A Modern Perspective (ed. S. T. Buczacki), pp. 249–283. Academic Press : London. Miller, C. E., Martin, R. W. & Dylewski, D. P. (1985). The ultrastructure of plasmodia, sporangia and cystosori of Ligniera verrucosa (Plasmodiophoromycetes). Canadian Journal of Botany 63, 263–273. Payne, P. A. & Asher, M. J. C. (1990). The incidence of Polymyxa betae and other fungal root parasites of sugar-beet in Britain. Plant Pathology 39, 443–451. Williams, P. H. & McNabola, S. S. (1967). Fine structure of Plasmodiophora brassicae in sporogenesis. Canadian Journal of Botany 45, 1665–1669. Yukawa, Y. & Tanaka, S. (1979). Scanning electron microscope observations on resting sporangia of Plasmodiophora brassicae in clubroot tissues after alcohol cracking. Canadian Journal of Botany 57, 2528–2532.
687
Printed in the United Kingdom
Ultrastructural studies of resting spore development in Polymyxa graminis
J I A N P I N G C H E N1, Z H I Q I A N G W A N G1, J I A N H O N G2, C. R E B E C C A C O L L I E R3 A N D M I C H A E L J. A D A MS3* " Virology Department, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, People’s Republic of China # Electron Microscope Laboratory, Zhejiang Agricultural University, Hangzhou 311029, People’s Republic of China $ Crop and Disease Management Department, IACR-Rothamsted, Harpenden, Herts, AL5 2JQ, U.K.
Transmission electron microscopy was used to study the development and structure of resting spores and sporosori of Polymyxa graminis. Osmiophilic bodies generally distributed in the cytoplasm moved to the periphery as spores matured. The spore wall consisted of four or five layers, two of which extended into spines linking adjacent spores. An unstained plug in the wall, faced towards the outside of the sporosorus. Some spores germinated in situ by producing primary zoospores. Bundle-like structures in some spores may have been barley yellow mosaic virus particles.
Polymyxa graminis Ledingham (Ledingham, 1939) is a common obligate parasite of the roots of Poaceae. It is particularly important as the vector of several plant viruses, including barley yellow mosaic (BaYMV), barley mild mosaic (BaMMV), wheat spindle streak mosaic, wheat yellow mosaic and soilborne wheat mosaic viruses. Polymyxa is one of nine genera usually recognized in the Plasmodiophoromycetes (Karling, 1968), a class distinguished from other zoosporic fungi by the production of biflagellate, isokont zoospores. The genus contains two species, P. graminis and P. betae Keskin, which are distinguished by their host ranges, but there appears to be little or no consistent morphological distinction and their separate specific status is questionable (Barr, 1988). The life cycle is not completely understood but has two phases. In the sporangial phase, the plasmodium formed after infection by a zoospore develops into a lobed zoosporangium, which releases more zoospores either to the outside of the host, or to infect deeper layers of the root (Barr, 1988). Sporogenic plasmodia (using the terminology recommended by Braselton, 1995), initially indistinguishable from the sporangial ones, undergo what appears to be meiotic division (Braselton, 1988) and then develop into sporosori (often termed cystosori), each consisting of numerous, thick-walled resting spores which remain viable (together with any viruses they may be carrying) in soil for many years (Adams, 1990). Ultrastructural studies have been made of zoospores, their penetration mechanism and of sporangial plasmodia (Keskin & Fuchs, 1969 ; Barr & Allan, 1982 ; Chen & Adams, 1992), but less is known of sporogenic plasmodia and the development of sporosori, except for a scanning electron microscopy (SEM) study of P. betae by Ciafardini & Marotta (1988) and a report of cell wall ultra* Corresponding author.
structure of spores extracted from infected roots (Ciafardini et al., 1995). In this paper we report transmission electron microscopy (TEM) of different stages in sporosoral development and discuss the results with relation to published work using SEM and to observations on other genera of Plasmodiophoromycetes.
M A T E R I A L S A N D M E T H O DS Monofungal isolates of P. graminis were obtained by isolating sporosori from naturally infected barley roots and inoculating barley roots in irrigated sand culture (Adams, Swaby & Macfarlane, 1986 ; Adams & Jacquier, 1994). Roots from inoculated plants heavily infested with sporosori were dried and used as inoculum for further cultures. Pieces of root for electron microscopy were selected under a light microscope at different times after inoculation, fixed in 2±5 % glutaraldehyde, post-fixed with osmium tetroxide, dehydrated and embedded in L. R. White medium grade resin (London Resin Co.) containing 1 % (v}v) silicon fluid (Dow Corning 200) or in Agar 100 resin (TAAB Laboratories Ltd) using standard techniques. Ultramicrotome sections were stained in uranyl acetate and lead citrate before examination using a JEM1200EX transmission electron microscope at 60 kV.
RESULTS In sand cultures inoculated with sporosori and maintained at 12 °C, first infection of root cells was observed about 2 wk after inoculation and from 6–12 wk after inoculation numerous zoosporangia were seen easily in the fine roots. The first young, colourless, sporosori were detected at about 14 wk
TEM of resting spores of Polymyxa graminis
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Figs 1–6. Transmission electron micrographs of sections of resting spores of Polymyxa graminis. Fig. 1. Immature spores. Fig. 2. Whole sporosorus showing mature spores. Fig. 3. Part of a single spore showing association of osmiophilic bodies with membranes. Fig. 4. Detail of spore wall : 1–5 ¯ layers of spore wall, with edge of plug visible on extreme right. Fig. 5. Connection between adjacent spores. Fig. 6. Single spore with prominent plug. Bars, 1 µm. M, membrane ; N, nucleus ; O, osmiophilic bodies ; P, plug ; T, connection in transverse section.
J. Chen and others
689
1 100 nm
2 3 4 5 Plug Cytoplasm
Fig. 7. Diagram illustrating spore wall structure. The five layers are numbered and their thickness is approximately to the scale indicated by the bar.
and these became mature and dark brown over the following 5 wk. At the ultrastructural level, the first distinct stage in resting spore development was the development of transitional plasmodia containing nuclei with synaptonemal complexes. These were seen in the present study but are not described in detail because they have already been reported by Braselton (1984). This was followed by nuclear division and the division of the plasmodium into uninucleate cells by the formation of membranous sheets within the cytoplasm. A spore wall was then deposited between the cells and, while the walls remain fused in places, progressively larger spaces developed between the spores. In immature resting spores, osmiophilic bodies were distributed generally in the cytoplasm (Fig. 1) but these seemed to move to the periphery as the spores matured (Fig. 2) and sometimes appeared to be associated with a membrane system (Fig. 3). Mature resting spores were uninucleate and contained abundant mitochondria, strands of rough ER, Golgi bodies, well-distributed vacuoles, vesicles containing a granular matrix and numerous free ribosomes. Nuclei averaged 1±6 µm diam. and each contained a spherical acentric nucleolus (x` ¯ 0±4 µm). The mature spore wall consisted of four or perhaps five layers and adjacent spores had connecting links formed from the two outer layers (Figs 4–5). The outer layer was thin (around 25–30 nm) and extremely electron-dense, while the second layer was thicker (60–80 nm) and had the appearance of a matrix containing electron-dense granules. The third layer (usually about 25 nm) was the least osmiophilic and inside this there appeared to be one or perhaps two layers. The appearance varied between sections but, in some (e.g. Fig. 6), there was a rather fibrillar layer about 60–80 nm thick which was separated clearly from layer 3 and an innermost, darker, layer, with an appearance not dissimilar to that of the osmiophilic bodies in the cytoplasm. This inner layer may therefore represent an intermediate stage in the deposition of the inner spore wall. A prominent feature in many sections was an unstained plug within the fourth}fifth layer of the wall (Fig. 6). This was 100–120 nm thick, extended up to about one third of the circumference of the spore and in appropriate sections of the entire sporosorus it could be seen that these
plugs all faced towards the outside (Fig. 2). Layer 3 seemed to be thicker (up to about 70 nm) in the region of this plug. Our interpretation of the cell wall structure is summarised in Fig. 7. Some filamentous, virus-like bundles were observed inside a few resting spores (Fig. 8). They were always located near the plasmalemma and were seen only in the cytoplasm of an isolate of P. graminis transmitting barley yellow mosaic bymovirus (BaYMV), originating from Xiaoshan, Zhejiang, China. No such structures were ever seen within a virus-free isolate. In some spores, the cytoplasm contracted from the wall and the intervening space was filled with a granular matrix (Fig. 9). It is possible that the peripheral osmiophilic vesicles contributed to this process and that this was associated with primary zoospore differentiation, because flagella were seen in some other spores at this stage (Fig. 10). It was not unusual to find sporosori in which individual spores were at different stages of development (Fig. 11) and empty spores, usually associated with many bacteria, were sometimes found (Fig. 12) suggesting that germination had occurred in situ. DISCUSSION The general pattern of ultrastructural development of resting spores of P. graminis was similar to that of other species of Plasmodiophoromycetes (e.g. Plasmodiophora brassicae : Williams & McNabola, 1967). It began with the division of the plasmodium into uninucleate cells by the formation of membranous sheets within the cytoplasm, similar to that reported for P. betae (Barr & Asher, 1996) and Sorosphaera veronicae (Braselton & Miller, 1978). In a SEM study of the closely-related Polymyxa betae, Ciafardini & Marotta (1988) claimed that immature resting spore walls were folded with numerous empty spaces between them, while spores in older sporosori had an inflated appearance and adjacent spore walls were in contact without empty spaces. This seems to be the opposite of what we found by TEM and we suggest that artefacts introduced during specimen preparation for SEM (e.g. collapse of the thinner immature spore walls, but not of the thicker mature ones) may be responsible for the apparent difference. The cytoplasmic organelles within the resting spores had a similar appearance to those observed in zoospores (Barr & Allan, 1982 ; Chen & Adams, 1992). The osmiophilic bodies observed within the cytoplasm seemed to originate from a membrane system and move to the perimeter of the spore as it matured. It therefore seems more likely that these played a role in spore development and}or germination rather than being storage organelles. Spore walls of other species of Plasmodiophoromycetes have been described as having either one (Ligniera verrucosa : Miller, Martin & Dylewski, 1985), two (Polymyxa betae : D’Ambra & Mutto, 1975 ; Woronina pythii : Miller & Dylewski, 1983 ; Tetramyxa parasitica : Braselton, 1990), three (Spongospora subterranea : Lahert & Kavanagh, 1985 ; Plasmodiophora brassicae : Yukawa & Tanaka, 1979) or five (Polymyxa betae : Ciafardini et al., 1995) layers. It is possible that these reports partly reflect differences in specimen preparation, staining and
TEM of resting spores of Polymyxa graminis
690
Figs 8–12. Fig. 8. Filamentous, virus-like bundles (V) in resting spore cytoplasm. Bar, 200 nm. Fig. 9. Contracting cytoplasm at start of primary zoospore differentiation. Bar, 500 nm. Fig. 10. Zoospore flagellum (F) inside spore. Bar, 1 µm. Fig. 11. Sporosorus with spores at different developmental stages. Bar, 2 µm. Fig. 12. Empty resting spores invaded by bacteria (B) after zoospore germination. Bar, 1 µm.
interpretation more than significant structural differences, although adjacent spores have been shown to associate in different ways. Those of Plasmodiophora are not aggregated, while in Woronina, spores are aggregated into a sporosorus but the walls of adjacent spores are not joined. Differing degrees of fusion between spores walls can be seen amongst
the other genera, with those of Sorosphaera veronicae showing the greatest degree of fusion and the most regular arrangement of spores (Braselton & Miller, 1978). In P. graminis, mature spores seem to be connected only by thin spines and it is relatively easy to fragment the sporosorus into individual spores (Adams & Collier, unpublished results).
J. Chen and others We assume that the plug in the wall is associated with germination, although it is not clear how it opens to release the primary zoospore. Similar structures can be seen in photographs of resting spores of P. betae (Ivanovic! , 1988) and Sorosphaera veronicae (Braselton & Miller, 1978) and also were described in Polymyxa betae by Ciafardini et al. (1995) who suggested that it disappeared before germination. Much smaller germination pores, with a clearly different morphology, have been described for S. subterranea (Lahert & Kavanagh, 1985), Plasmodiophora brassicae (Yukawa & Tanaka, 1979 ; Ikegami et al., 1982) and Woronina pythii (Miller & Dylewski, 1983). In their SEM studies of developing resting spores of Polymyxa betae, Ciafardini & Marotta (1988, 1989) suggested that holes developed at a point of weakness in the spore wall and then enlarged to permit zoospore germination, after which the spore walls folded back inside the empty spore. Our studies also show that at least some spores of a sporosorus may germinate once they are mature, although it is possible that some of the features shown by the scanning electron micrographs may represent specimen collapse during preparation. Primary zoospore formation within a spore has also been reported for Spongospora subterranea (Lahert & Kavanagh, 1985). The ability of Polymyxa species (and the viruses they transmit) to persist in soil for many years in the absence of a known host (Adams, 1990 ; Payne & Asher, 1990) suggests either that spores have a variable, inbuilt, dormancy, or that a host-specific stimulus is needed for their germination. Bundle-like structures (Fig. 8) within spores of an isolate transmitting BaYMV were similar to structures previously identified (by immunogold labelling) as barley mild mosaic bymovirus inside zoospores and zoosporangia (Chen, Swaby, Adams & Ruan, 1991). It therefore seems probable that these structures consist of BaYMV particles. Zhejiang Academy of Agricultural Sciences acknowledges grants from the European Commission (ISC CI1*CT93-0081), the National and Zhejiang Provincial Foundation for Natural Sciences and the Chinese Ministry of Agriculture. IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.
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