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Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois 60115-2861
Gordon W. Beakes
Department of Biology, University of Newcastle, Newcastle upon Tyne, NE1 7RU, UK
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ABSTRACT |
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An isolate of Myzocytiopsis vermicola, a holocarpic parasite of Rhabditis nematodes, was studied with transmission electron microscopy (TEM) to follow development during infection, asexual and sexual reproduction. Nematodes became infected after attachment of apical cystospore buds to the nematode cuticle. Apical buds were packed with vesicles with dense fibrillar contents, which were absent from the thallus. Some thalli developed into sporangia while others became paired gametangial cells. Zoospore cleavage was often intrasporangial, although during the early stages of an epidemic partially differentiated zoospores usually were released via an exit tube into a fine vesicle. Packets of tripartite tubular hairs (TTH) were not observed in the cytoplasm of either developing or mature sporangia. TEM of sectioned material and whole mounts of zoospores revealed biflagellate zoospores, some without hairs and others with a proximal row of very short hairs on the anterior flagellum. Gametangial contact was via a short, walled fertilization tube and surplus antheridial and oogonial nuclei remained in their respective gametangial cells until disintegration of the periplasm. The mature oospores had a scalloped, electron opaque, epispore wall layer. These observations will be discussed in relation to the likely phylogenetic position of the Myzocytiopsidales within the oomycetes.
Key words: infection, nematode parasite, oomycete, phylogeny, ultrastructure, zoosporogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSOBSERVATIONDISCUSSIONLITERATURE CITED |
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) but later was given its own binomial, Myzocytium vermicolum by Fischer (1892). The zoospores were reported to be oval with a shorter anterior flagellum and a longer posterior flagellum (Karling 1981). An isolate from littoral, marine, Rhabditis nematodes resulted in a comprehensive account of the species that included observations with video microscopy to record zoospore release into a vesicle and permitted the recognition of two morphologically distinct types of zoospore (Newell et al 1977). These authors also described a short septal papillum involved in the fertilization process, which supported Dangeard’s (1903) observation of a narrow fertilization tube in this species. Dick (1997) amalgamated most nematophagous species of Myzocytium and Lagenidium into a new genus, Myzocytiopsis (family Myzocytiopsidaceae), making M. lenticularis (Barron) Dick the type species.
Due to their host-dependent nature these biflagellate parasites of nematodes have been little studied, and until recently only nominal fine structural accounts of their reproductive development have been available (Saikawa and Anazawa 1985, Glockling 1994, Dick 1995, Glockling and Beakes 2000). The genus Myzocytiopsis displays much variation in spore type and infection methods (Glockling and Beakes 2000). Myzocytiopsis vermicola is one of three species that produces biflagellate zoospores that develop a succession of apical adhesive buds after zoospore encystment. These cystospores adhere to the nematode cuticle and initiate a new infection. The other two adhesive-spored species are M. humicola (Barron and Percy) M.W. Dick and M. glutinospora (Barron) M.W. Dick (Barron and Percy 1975, Barron 1976). M. glutinospora morphologically is comparable to M. vermicola but is reported to have pyriform zoospores with subapical flagellar insertion and zoospore release in the absence of a retaining vesicle (Barron 1976). The other species, M. humicola, is characterized by its smooth oospore wall and pyriform zoospores (Barron and Percy 1975). M. subuliformis (P.A.Dang.) M.W. Dick also has infection spores with an adhesive tip, but this species has no motile stage (aplanosporic) and produces elongate tapered aplanospores (Glockling and Beakes 2000).
Although it is possible to grow some species of holocarpic nematode parasites in pure culture (Glockling and Dick 1997), we were unable to grow this particular isolate of M. vermicola. This study will concentrate on details that are considered to be of phylogenetic significance such as general morphology and structure, zoospore base and rootlet arrangement, sporangial cleavage and the fertilization process. This first detailed account of thallus development in this little studied family within the oomycetes (Peronosporomycetes, Dick 2001) will enable structural features and differentiation to be compared with that previously documented in saprolegnialean and peronosporalean species.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSOBSERVATIONDISCUSSIONLITERATURE CITED |
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Light microscopy/zoospore release sequence.— – An infected nematode containing mature sporangia was mounted on a slide and covered with a cover slip. Still images of a mature sporangium exit tube were captured with a Nikon E600 microscope with phase contrast (40x) optics and DXM1200 digital camera. Images were captured from before zoospore release until after zoospore release and encystment.
Preparation of samples for transmission electron microscopy.— –
Specimens were prepared for TEM using techniques previously described (Beakes and Glockling 1998). Infected nematodes on the agar surface were covered with a 2 mm2 plastic sheet, cut out and encased in molten agar that was allowed to set before immersion in 2.5% glutaraldehyde. After a buffering step in 0.1 M cacodylate buffer, specimens were postfixed in 1% osmium tetroxide before being dehydrated in a graduated acetone series. Specimens were embedded in Epon resin and polymerized at 65 C for 24 h. Blocks were sectioned with a Reichert-Jung Super Nova ultramicrotome and collected on Formvar-coated copper slot grids. Sections were viewed and photographed on a Hitachi H-600 TEM. Negatives were developed and scanned into Adobe Photoshop with an Epson 2450 photo scanner.
Zoospore whole mounts.— – Sporulating specimens from the first and second isolates were washed and placed in a cavity slide in sterile water. Some water containing zoospores was collected with a tapered glass tube and placed on Formvar and carbon-coated copper slot grids. The grids were exposed briefly to osmium vapor and allowed to dry. Grids were taped to a glass slide and shadowed with gold palladium using an evaporator (Kinney Vacuum Co., Boston, Massachusetts).
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OBSERVATION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSOBSERVATIONDISCUSSIONLITERATURE CITED |
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). In Petri dish cultures asexual reproduction (FIG. 1a–h) occurred almost exclusively at the onset of infection and continued while nematode populations were high. A typical infected nematode containing a chain of ovoid thalli with lateral discharge tubes is illustrated (FIG. 2). Although zoospores were released into vesicles early in infections, later zoospores were released directly through the discharge tube without evidence of a vesicle (FIG. 2a–c). Sexual reproduction, which resulted in thick-walled oospores (FIGS. 1i–k, 47), occurred mainly in older cultures when the epidemic was in decline.
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, arrows). Internalized axonemes were observed in this cyst and along the side of the nucleus in another encysting zoospore (FIG. 4) indicating that flagellar retraction, rather than shedding of flagella had occurred. Pregermination cystospores were thicker walled, ovoid, and had a distinct polarity in the distribution of organelles around the central nucleus (FIG. 7). Mitochondria were located toward the bud-forming side of the spore whereas dense body vesicles (DBV) were concentrated on the opposite side (FIG. 7), an arrangement that becomes more pronounced in elongate budded spores (FIG. 6). During early stages in cystospore formation the amorphous bodies in the DBV were associated with the vesicle membrane (FIG. 7) and lacked the fingerprint lamellations seen in zoospore DBV (FIG. 39). A single Golgi body, which was associated with the cystospore nucleus (FIG. 5) and oriented toward the bud side of the spore, produced distinctive apical vesicles (av), which ultimately accumulated in the tips of developing buds (FIGS. 8–10).
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). The bud wall was continuous with the anterior cyst wall (FIG. 8) and each bud expanded to ca. 0.5 µm diam. Buds were filled with av, almost to the exclusion of every other organelle, except for a single, centrally located, mitochondrion; the nucleus remained in the main cystospore body (FIG. 6). The bud wall had a distinctive, ca. 50 nm thick, outer layer (FIG. 9, arrow), which we presumed to be adhesive material that enables these spores to stick to the nematode cuticle. Infection by M. vermicola relies on a passing nematode making contact with one of these sticky cystospore buds (FIG. 10). A collar of fibrillar material was present around the contact point between the apical bud and pitted nematode cuticle (FIG. 10, arrow).
The host cuticle was breached by a relatively narrow penetration hypha (FIG. 11, arrows), followed by the migration of the cystospore cytoplasm through the chain of buds and into the expanding "protothallus", which probably is enveloped by the invaginated host plasma membrane rather than puncturing it (FIG. 11). This uninucleate "protothallus" (FIG. 12) had characteristically dense cytoplasm containing mitochondria and small vacuoles but completely lacking av (FIGS. 11, 12). These relatively thick-walled "protothalli" became typically constricted with the oldest compartment adjacent to the point of penetration quickly becoming vacuolate (FIGS. 12, 16). At this early stage of infection the host protoplast appeared to have retracted from around the protothallus but still remained connected to it by fine protoplasmic strands (FIGS. 16, 17).
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). These rapidly enlarge to form the typically beaded thallus, which is characteristic of this species and which eventually may fill almost the entire body of the moribund host (FIGS. 1d, 2). Young septa were characteristically double-layered with a zone of amorphous material sandwiched between the two layers of wall (FIG. 25a, b, asterisk). As the thalli enlarge and become increasingly rounded these layers also become more obviously separated (FIG. 25b) although individual sporangia remain bound by the original outer thallus wall layer (FIG. 25, arrow). Young thalli were multinucleate (FIGS. 13, 14, 18, 19) but mitotic stages were never observed. Paired centrioles were present at the apex of each nucleus, which also had an associated Golgi body.
The cytoplasm contained scattered lipid globules, small DBV and mitochondria throughout (FIG. 13). The peripheral cytoplasm in young thalli contained localized regions packed with abundant tubular cisternae (FIG. 14, arrows) with uniformly electron opaque contents (FIG. 15). Some of these cisternae appeared to be oriented perpendicularly to the plasma membrane (FIG. 13, arrows), while others were organized in parallel stacks reminiscent of a Golgi-endoplasmic reticulum complex (FIG. 15a). Some cisternae expanded slightly at their ends, and the whole complex was interspersed with more expanded vesicles of similar density (FIG. 15b).
As thalli enlarged and matured a conspicuous central vacuole system developed, derived from the expansion and coalescence of some of the DBV fraction (FIGS. 18–21). This expanding vacuolar system contained variable amounts of electron opaque, globular material. The vacuolar inclusions initially formed conspicuous hemispherical aggregates associated with the membrane (FIG. 18) but at more advanced stages appeared to disperse into discrete globules within the vacuole (FIGS. 19, 20). The formation of this central vacuole system eventually resulted in the peripheral concentration of the nucleate cytoplasm (FIG. 20), which is rich in scattered DBV and mitochondria. Also during the period of thallus expansion, distinctive encystment vesicles first appeared in the cytoplasm. These were small (ca. 150–200 nm diam) spherical vesicles with a moderately opaque outer matrix and an irregular more electron-lucent core containing granular material (FIGS. 28, 38b). These vesicles eventually became aligned close to expanding cleavage and axonemal vesicle systems (FIGS. 26, 28).
Fully expanded thalli were recognized by the presence of a single lateral discharge tube (FIGS. 1e, 2, 21), which breached the nematode cuticle. At maturity the tips of some of these discharge tubes were filled with a plug of moderately electronopaque, fibrillar material (FIG. 22). At this stage the peripheral cytoplasm was organized into uneven masses delimited by the expanded central vacuole system (FIG. 21).
Zoospore cleavage and release.— –
During the final stages of thallus maturation the concentrated peripheral cytoplasm is converted into fully differentiated zoospores. This process, although usually occurring intrasporangially during later stages in an infection, also can occur in a transient external vesicle derived from the expanded apical plug material. Observations of living thalli revealed that the large central vacuole collapsed suddenly, either during intrasporangial cleavage or shortly before the partially differentiated mass of cytoplasm was released into a vesicle. Ultra-structural observations of differentiating thalli also confirmed that the relative timing of cleavage furrow and flagellum development varied vis a vis the collapse and assimilation of the central vacuole system. In most thalli observed zoospore differentiation was completed inside the sporangium (FIG. 27) and the fully differentiated zoospores escaped via the opened discharge tube without the restraint of a transient vesicle (FIG. 2a–c). Zoospores that remained inside the sporangium encysted and developed adhesive buds in situ (FIG. 6).
During the early stages of intrasporangial cleavage the large central vacuole system usually was maintained (FIG. 21) as additional cleavage cisternae became aligned around the developing uninucleate spore initials (FIGS. 23, 24, 26). These cleavage cisternae appeared to arise from the alignment and coalescence of vesicles, which have a thin electron-lucent outer region with a moderately opaque core (FIG. 23). Individual vesicle components fused and expanded (FIG. 24) to form a system of electron-lucent cleavage furrows delimiting individual zoospore initials (FIG. 27). Ultimately this coalescence process appeared to result in the assimilation of the plasmalemma and the vacuole tonoplast into the zoospore initial membranes in the fully cleaved sporangium (FIG. 27). Concurrently cisternae also delineated the differentiating flagella axonemes (FIGS. 28, 29). Flagellum differentiation was initiated when an axonemal cleavage cisterna capped the transitional plate of each of the paired kinetosomes before the generation of the axonemal tubules. As the axonemes were assembled they continued to be enveloped by the expanding cleavage cisternum (FIG. 29, asterisk), which eventually is assimilated into the cleavage vesicle system during the final stages of zoospore differentiation (FIG. 27). Each zoospore initial contained a nucleus surrounded by a cluster of mitochondria and a peripheral zone rich in DBV (FIGS. 26, 27). At this stage the DBV inclusions are reticulate and have characteristic fingerprint lamellations (FIG. 39).
The differentiated zoospores.— –
Zoospores that were released into an external vesicle before their escape had a fairly short swimming phase after the rupture of the vesicle. In contrast zoospores that were directly released from the sporangium (FIG. 2a–c) remained motile for a much longer period (ca. 30 min), as did the zoospores that remained trapped inside the open sporangium. Zoospores were ovoid (FIG. 33a) with a shallow ventral grove from which the flagella emerged. Shadow-cast preparations of zoospores from the first isolation revealed a zoospore with laterally inserted flagella (FIG. 33b). The posterior flagellum tapered to an acroneme at its tip, while the anterior flagellum was blunter (FIG. 33b). Whole mounts of several zoospores from the second isolation showed an anterior flagellum decorated with a single row of short proximal hairs (FIG. 34, arrows). These corresponded to a single row of short (50 nm) hairs decorating one side of the anterior flagellum that were seen in longitudinal TEM sections (FIG. 35, arrow).
As with other "secondary type" zoospores a water expulsion vacuole discharged into a ventral groove. The flagella originated from a more or less central boss (FIG. 36) on the ventral side of the spore, with the kinetosomes angled at about 170 degrees to each other (FIG. 37). The transitional helix (TH), which lay immediately above the transitional plate, appeared to be of a double helical type (although individual subunits were not well resolved) of about 8–12 gyres (FIG. 35). The kinetosomes were associated with electron-opaque plaques of material that surround and interconnect their bases (FIG. 37, asterisk). A posterior rootlet strap of six or seven microtubules was seen in TS in differentiating zoospores (FIGS. 30, 31, arrows). Fibers linked the microtubules in this strap to a striate, wedge-shaped fan between the kinetosomes (FIG. 30, arrow). These features were never resolved in postrelease zoospores. In differentiated zoospores (FIGS. 32, 36, 38) the spherical ev accumulated adjacent to the plasma membrane on the ventral side of the spore, particularly along the shoulder of the ventral groove (FIG. 38a, arrows). The DBV in zoospores now had prominent fingerprinting (FIG. 39) and were mainly in the dorsal region of the spore with the mitochondria mainly concentrated around the dorsal side of the nucleus (FIGS. 36, 38).
Gametangial copulation and oospore formation.— –
In M. vermicola gametangia arise from alternate thallus segments in mature infections (FIG. 40). In this species male and female gametangia were usually morphologically distinct, with ovoid antheridia and more or less spherical oogonia (FIGS. 1i, 40, 46). The mature male thallus produces an antheridial tube that penetrates into the oogonium until it contacts and penetrates the differentiated oosphere (FIG. 41). The male thallus at this stage (FIG. 42) was packed with mitochondria, large DBV and gametic nuclei. A thin wall delimited the young fertilization tube and its tip was packed with small vesicles with opaque granular contents. As the antheridial contents migrated into the oogonium, the antheridial cell became increasingly empty and vacuolated, except for a few residual peripheral nuclei. At later stages the fertilization tube became thicker walled (FIG. 44, arrows) and the antheridial contents began to disintegrate so that by the time the oospores are mature the male thallus is an empty shell (FIGS. 40, 45, 46).
At the time of fertilization the contents of the oogonium had differentiated into a uninucleate oosphere surrounded by vacuolated periplasm containing excluded oogonial nuclei (FIGS. 41, 43). The oosphere is defined by a thin, electron-opaque membrane, which is underlain in localized regions by clusters of electron-lucent vesicles (FIG. 43, arrows). The oosphere cytoplasm contained a large number of what appear to be expanding DBV (FIG. 41), which, as described in other oomycetes, coalesce to form the refactile ooplast visible in mature oospores (FIG. 47, asterisks). As with the formation of the somatic vacuole in differentiating thalli, the electron-opaque globules disintegrate and disperse as the vacuole enlarges. However, by the time the thick oospore wall had formed, the cytoplasm did not fix well and appeared opaque and packed with large lipid globules (FIGS. 44, 46). The surrounding periplasm does not persist and disappears by the time the oospore is mature (FIGS. 46, 48).
As the oospore matures a thick, multilayered, wall develops that gives the mature oospores their distinctive reticulate appearance (FIG. 1k). In light microscopy maturing oospores appear to have a thick, regularly scalloped wall (FIG. 47). In TEM view the scalloping is an undulating, electron-opaque "epispore wall" layer (FIG. 48). The depressions between the scallops were filled with an electron-lucent, loosely fibrillar, material, so that the outer margin of the oospore is actually smooth (FIG. 48, arrows) and not uneven as it appears in living spores (FIG 47). Inside of the electron-opaque epispore layer is a relatively thin electron-lucent endospore layer, which was delimited by the plasma membrane (FIG. 48).
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DISCUSSION |
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, 2001). Members of the Myzocytiopsidales are parasites of nematodes that traditionally were considered to be part of the holocarpic Lagenidiales (Sparrow 1973, 1976). Sparrow recognized that oomycete fungi could be divided into two main "galaxies", which Dick (2001) has renamed the Saprolegniomycetidae and the Peronosporomycetidae. Recent molecular analyses show that some terrestrial and marine holocarpic Lagenidium species cluster in the Pythiales with most species forming a sister clade to the main, plant parasitic peronosporaceous genera such as Phytophthora and Pythium (Cook et al 2001). 18S sequence data for two culturable Myzocytiopsis species, indicate this genus is a polyphyletic assemblage, with species clustering in both the major oomycete clades (Glockling and Embley unpublished data). Myzocytiopsis vermicola has morphological and structural traits in common with the Saprolegniomycetidae and in particular the Peronosporomycetidae, although it also possesses a number of unique features.
Zoospore discharge into transient restraining vesicles has been reported for other members of the Myzocytiopsidaceae (Glockling and Beakes 2000, Dick 2001), Lagenidium (renamed Salilagenidium by Dick, 2001) callinectes (Gotelli 1974) and Pythium species (Lunney and Bland 1976) and is a characteristic feature of the Peronosporomycetidae (Beakes 1987, 1994). However in some thalli of M. vermicola the zoospores differentiate completely before being discharged from the sporangium in a pattern commonly seen in saprolegniaceous oomycetes such as Saprolegnia (Beckett et al 1974, Beakes 1994) and Achlya (Money et al 1988) and marine genera such as Halipththoros (Overton et al 1983) and Eurychasma (Kuepper and Muller 1999).
On encystment the zoospores of M. vermicola retract their flagella rather than shedding them. This is characteristic of primary zoospores as opposed to secondary zoospores, which usually shed their flagella (Holloway and Heath 1977). Newell et al (1977) reported that their marine isolate of M. vermicola showed two morphologically distinct types of zoospore. The first-formed zoospores in a number of marine genera such as L. callinectes, Lagenisma (Schnepf et al 1978), Petersenia (Pueschel and van der Meer 1985) and Haliphthoros (Overton et al 1983) all appear to retract flagella. Of interest, zoospores in all these species are more pyriform than reniform. Flagellar retraction has been reported in a typical secondary zoospore of Phytophthora palmivora var nicotiana (Reichle 1969) so this behavior may not be exclusively associated with saprolegniaceous species.
The rather ill defined electron-opaque outer coat of the cystospore in M. vermicola appears to be derived from peripheral encystment vesicles. There is however no evidence of either a tripartite outer cyst coat decorated with spines or the large ventral K bodies that are associated with cystospores of Saprolegnia species (Beakes 1987, Lehnen and Powell 1989). However the encystment vesicles of M. vermicola do appear more structured than those typically found in most peronosporalean species (Beakes 1987, Gubler and Hardam 1988). In most saprotrophic and plant pathogenic oomycetes the zoospores are the main infective spores, which on making contact with the host discharge a ventral pad of adhesive material derived from a K-body or ventral vesicle systems (Lehnen and Powell 1989, Gubler and Hardman 1988). One of the unusual features of many Myzocytiopsis species is that they produce encysted zoospores that undergo further differentiation to produce budded infection cystospores (Glockling and Beakes 2000). In the M. vermicola spore the surface of the terminal bud has a more electron-opaque fibrillar outer coating, which appears to be the material that forms the localized adhesive pad when the bud makes contact with the host cuticle. This is reminiscent of the situation in plant pathogens such as Blumeria, where material coating the conidium flows to the site of contact with the leaf surface (Carver et al 1999). The cystospore buds were densely filled with vesicles with dense fibrillar contents closely resembling those reported in M. glutinospora and M. humicola (Glockling and Beakes 2000). The cystospores of M. vermicola and M. humicola previously have been compared to those of another holocarpic nematode parasite, Gonimochaete pyriforme G.L.Barron, but their development and structure appears different although they probably have the same function (Barron 1973, Saikawa and Anazawa 1985, Glockling and Beakes 2000). These vesicles probably contain the necessary enzymes to aid penetration of the tough nematode cuticle. The presence of a single mitochondrion in each adhesive bud either may provide energy for these processes or could serve as a reservoir for the calcium ions needed to trigger the exocytosis of these lytic vesicles.
The newly formed protothalli always were walled indicating infection does not involve the injection of protoplast as had been described in some other nematophagous pathogens such as Haptoglossa (Glockling and Beakes 2000). Although the quality of host cytoplasm fixation in the immediate vicinity of the invading pathogen was not good, it is possible that the host plasma membrane invaginates rather than being ruptured indicating infection may be more analogous to haustorium formation than to protoplast injection across the host membrane as described in Olpidium (Lesemann and Fuchs 1970). The protothalli have a distinctive host-pathogen interface, in which the walled pathogen is surrounded by a space bridged by narrow strands of membrane. This is reminiscent of plasma membrane of cells pulling away from tight junctions such as plasmodesmata. Although the effect may be a consequence of fixation induced shrinkage (of either the thallus or surrounding host cytoplasm) it does suggest that tight connections have been established between host and parasite. This may be important in establishing nutrient transfer in such a specialized obligate pathogen.
The septa, which divide the thallus into segments, were similar to those reported in other Myzocytiopsis species, including M. lenticularis. Although a double structure is produced like that observed in many saprolegnialean species (Beckett et al 1974), no vesicular material is trapped between the layers. Furthermore the septa differ from the callose-like plugs described in most peronosporalean species (Hemmes 1983).
As in other oomycete fungi the dense body vesicles (DBV) with electron-opaque contents are part of the cell’s vacuolar storage system (Beakes 1980, 1994). In M. vermicola, the provacuoles in young thalli are associated with rather large amorphous electron-opaque granules that form hemispherical aggregates attached to the tonoplast membrane. A similar organization of vacuolar granules has been illustrated in Phytophthora chlamydospores (Hemmes 1983). These granules eventually seem to disperse within the vacuole in a similar fashion to those described in germinating Saprolegnia oospores (Beakes 1980). The nonvacuolar, cytoplasmic DBV fraction eventually becomes incorporated into the zoospore initials, where the inclusions acquire their characteristic fingerprint substructure as described in many peronosporalean species (Hemmes 1983). In Phytophthora at this lamellate stage these vesicles contain phosphorylated mycolaminarins (Bartnicki-Garcia and Wang 1983).
Our study suggests that zoospore formation in M. vermicola can be considered a biphasic process. It begins with the concentration of the nucleate cytoplasm in a peripheral mass because of the formation of the large central vacuole. This is similar to the situation in the Saprolegniaceae where the large central vacuolar system, derived from the expansion and coalescence of dense body vesicles, cleaves out a peripheral array of zoospore initials (Gay and Greenwood 1966, Gay et al 1971, Money et al 1987). Partially differentiated zoospores form as a result of the fusion of the tonoplast with the plasma membrane, followed by the separate differentiation of the flagella. In Achlya, high molecular weight mycolaminarins released from the central vacuole are responsible for maintaining internal osmotic pressure within the sporangium because of the semipermeable nature of the sporangial wall (Money et al 1988). In Myzocytiopsis the central vacuole collapsed, presumably as a result of the centrifugal fusion of the tonoplast with the plasma membrane. Rather than individual zoospore initials, this results in the formation of a somewhat irregular mass of multinucleate cytoplasm that pulls away from the thallus wall. This release of mycolaminarins however would serve to provide the necessary internal pressure to drive the flow of partially differentiated cytoplasm into the extrasporangial vesicle or the flow of differentiated zoospores from the open exit tube (Money et al 1988).
The second phase of the process in Myzocytiopsis involves the cleavage of the cytoplasmic mass into individual zoospore initials and is similar to that reported in a number of Phytophthora species (Hohl and Hamamoto 1967, King and Butler 1968, Hyde et al 1991a). Individual zoospore initials are delimited by a series of cleavage cisternae, although the precise ontogeny of these vesicles is still uncertain and the exact timing of their disposition appears to be variable. In most accounts the Golgi system has been implicated in the synthesis of cleavage vesicles or cisternae (Hohl and Hamamoto 1967, Lunney and Bland 1974, Hyde et al 1991a, b). In Phytophthora parasitica a central vacuole has been reported to collapse before cleavage furrows are formed (Hohl and Hamamoto 1967), whereas in P. cinnamomi the mass of cytoplasm becomes delimited by cleavage cisternae without any apparent involvement of a central vacuolar system (Hyde et al 1991a). In M. vermicola the cleavage vesicles concurrently delimit the developing flagella as has been reported in Phytophthora species (Hohl and Hamamoto 1967, King and Butler 1968, Hyde et al 1991a, b). In many Pythium species, zoospore differentiation occurs within the extrasporangial vesicle (Lunney and Bland 1974). Because of the obligately parasitic nature of Myzocytiopsis and the scarcity of specimens, only chemical fixation could be used in this study. In Phytophthora this is known to result in a markedly different preservation of the cleavage vesicle system compared with cryofixation techniques (Hyde et al 1991b). In conventionally fixed material developing cleavage furrows seem to be defined by the coalescence of arrays of somewhat inflated vesicles as described here (Hyde et al 1991a). In contrast, in freeze substituted sporangia, cleavage planes are defined by a narrow cisternal system with electron-opaque contents (Hyde et al 1991b), so the system described here could be an artifact of chemical fixation.
The absence of packages of tripartite tubular hairs (TTH) in the differentiating cytoplasm of the mature sporangia is a notable feature of zoospore differentiation in this species. In most oomycetes packets of TTH form in cisternae of rough endoplasmic reticulum; these packets often are associated with mitochondria early in sporangium differentiation, typically preceding septum formation (Beakes 1994, Gay and Greenwood 1966, Heintz, 1971). TTH packets have been seen in developing sporangia of M. intermedia (Barron) M.W.Dick (Glockling and Beakes 2000) and M. bolata (Glockling 1994, Dick 1995). Zoospores of M. vermicola appear either bald or with a single row of short hairs, which is reminiscent of the arrangement described in the insect pathogen Crypticola clavulifera (Frances et al 1989). A marine isolate of M. vermicola produces dimorphic zoospores (Newell et al 1977), but it is not known whether dimorphism was linked to differences in flagellar ornamentation. However the lack of mastigoneme hairs on zoospores may be a feature of nematode parasites because Haptoglossa dickii also lacks such hairs (Beakes and Glockling 1998).
Sexual reproduction in this isolate of M. vermicola was not observed frequently in our cultures and always occurred when infection was in decline, as reported in M. glutinospora (Barron 1976). During sexual reproduction the antheridial protoplasm migrates into the oogonium by means of a short fertilization tube. The tube does not appear to be fully walled at first and is filled with small vesicles that either may be secreting enzymes or depositing wall materials. Ultrastructural studies of fertilization tubes in Phytophthora capsici showed a somewhat similar distribution of small vesicles (Hemmes and Bartnicki-Garcia 1975, Hemmes 1983). Although we observed only a few thalli at the oosphere stage it is nevertheless clear that oosphere differentiation in M. vermicola is periplasmic, a key characteristic of the Peronosporomycetidae. This contradicts previous conclusions based solely on light microscopic observations that species in this genus lack differentiation into an oosphere and periplasmic layer (Karling 1981). In the maturing oosphere the DBV appear to be coalescing into larger units (the ooplast) as described in other developing oospores (Beakes 1980, 1981, Hemmes 1983). The mature oospore wall of M. vermicola appears to have an uneven, scalloped, outline and is similar to the oospore in M. glutinospora, which has been described as "echinate" (Barron 1976). This contrasts with the usually smooth oospore walls in most peronosporaceous oomycetes (Beakes 1981, Hemmes 1983, Hemmes and Stasz 1984). The only species with somewhat similar uneven oospore walls that has been described at the ultrastructural level is Albugo candida (Beakes 1981). In M. vermicola the shallow spines seem to be the result of an undulating electron-opaque layer. However it is clear from our ultrastructural observations that the depressions between the spines are filled with an external matrix material. This could represent material laid down from the periplasm and thus be a true exospore layer as has been described in Albugo (Beakes 1981).
Overall this structural study of M. vermicola indicates that this species has many more features in common with the Peronosporomycetidae than with the Saprolegniomycetidae. This suggests that many members of the holocarpic Myzocytiopsidales are likely to be members of the Peronosporomycetidae as suggested by recent molecular studies (Cook et al 2001). However several features are similar to those in the Saprolegniomycetidae, which might indicate that this species may be relatively primitive. In this respect the similarity in the rather complex oospore wall with those of Albugo is of interest because this genus represents the most basal member of the peronosporalean lineage so far sequenced (Petersen and Rosendahl 2001). This study also suggests that the family Myzocytiopsidaceae as defined by Dick (2001) is unlikely to represent a natural grouping within the oomycetes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Current address: Genome Damage and Stability Centre, University of Sussex, Science Park Road, Falmer, Brighton, East Sussex BN1 9RQ, UK.
1 Corresponding author. E-mail: S.Glockling{at}sussex.ac.uk
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LITERATURE CITED |
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