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Ultrastructure of the zoosporangia of Albugo ipomoeae-panduratae as revealed by conventional chemical fixation and high pressure freezing followed by freeze substitution

     Department of Plant Pathology, University of Georgia, Athens, Georgia 30602

Elizabeth A. Richardson

     Department of Plant Biology, University of Georgia, Athens, Georgia 30602

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

Both conventional chemical fixation and high pressure freezing followed by freeze substitution (HPF/FS) were used to prepare zoosporangia of the oomycete Albugo ipomoeae-panduratae inside infected host leaves for study with transmission electron microscopy. Both fixations gave good preservation of ultrastructural details and data from the two sample types were highly complementary. However, HPF/FS gave better overall specimen contrast and superior preservation of microtubules, basal bodies and curved vacuoles closely associated with basal bodies. The basal body-associated vacuoles appear to represent cleavage vesicles involved in zoospore formation. Although HPF/FS did result in the rupture of some vacuoles and the extraction of lipid bodies, these problems did not interfere with our study. Overall zoosporangium morphology was similar to that reported previously for A. candida. Each zoosporangium was multinucleate and contained numerous mitochondria, lipid bodies, a variety of large and small vacoules/vesicles, and conspicuous arrays consisting of parallel strands of rough endoplasmic reticulum. Golgi cisternae and a pair of basal bodies were closely associated with each nucleus.

Key words: Cryofixation, sporangia, transmission electron microscopy

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Since the initial use of plunge freezing followed by freeze substitution (PF/FS) to prepare fungal hyphae for examination with transmission electron microscopy (Howard and Aist 1979), this fixation protocol has become the gold standard for ultrastructural studies of fungal germ tubes and hyphae (Howard 1981, Roberson and Fuller 1988). In addition, PF/FS has been shown to provide excellent preservation of ultrastructural details in various types of fungal spores including those of a number of plant pathogenic species. Spores of plant pathogenic fungi examined thus far include basidiospores of the rust Gymnosporangium juniperi-virginianae (Mims et al 1988), teliospores of the fungus Sporisorium sorghi (Mims and Snetselaar 1991), and conidia of a variety of species including Uncinuliella australiana (Mims et al 1995a), Blumeria graminis f. sp. hordei (Roberts et al 1996), Colletotrichum graminicola (Mims et al 1995b), C. truncatum (Van Dyke and Mims 1991), Alternaria cassiae (Mims et al 1997), Monilinia vaccinii-corymbosi (Mims and Richardson 1999), Entomosporium mespili (Mims et al 2000), Phyllostica ampelicida (Shaw et al 1998), Magnaporthe grisea (Hamer et al 1988), and Nectria haematococca (Caesar-TonThat and Epstein 1991). Good results also have been obtained for zoospores of the plant pathogenic oomycete Phytophthora palmivora (Cho and Fuller 1989).

In our opinion, the key to successful PF of spores involves rapid and direct exposure of spores to liquid propane, the cryogen employed in this procedure. However, while we have excellent results with spores resting on small pieces of dialysis membrane and those directly exposed on leaf surfaces, we have had little success with those inside host tissues. In an attempt to obtain better results with the latter types of spores, we have explored the use of high pressure freezing (HPF) followed by FS. Unlike PF, which is limited to very small samples measuring only a few micrometers in diameter, HPF permits the freezing of much larger samples without ice crystal damage (Gilkey and Staehelin 1986, Moor 1987). Here we report results we have obtained using HPF/FS to prepare zoosporangia of the oomycete Albugo ipomoeae-panduratae (Schwein) Swingle for study with transmission electron microscopy (TEM). Commonly known as white rust, this pathogen produces its zoosporangia in sori that develop beneath the epidermis of infected leaves of various species of Ipomoea. These propagules arise in chains from a layer of short, club-like sporangiophores that line the base of each sorus.

Because of concerns raised by Hyde et al (1991c) regarding damage done to zoosporangia of the oomycete Phytophthora during HPF/FS, we also prepared zoosporangia of Albugo ipomoeae-panduratae for comparison purposes using a conventional chemical fixation protocol. Currently, all information on the ultrastructure of zoosporangia of white rust fungi has come from the study of conventionally fixed samples of A. candida (Berlin and Bowen 1964, Kahn 1976, 1977).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Stems of Ipomoea hederacea (L.) Jacq. (ivy-leaf morning glory) bearing leaves infected by A. ipomoeae-panduratae were collected in the field near Athens, Georgia in August 2001 and placed in water for transport to the laboratory. A razor blade was used to excise 1-mm² pieces of leaf tissue bearing one or more sori that were prepared for study using either conventional chemical fixation or HPF/FS. For HPF, a single piece of tissue was placed in either a brass or an aluminum planchette (Technotrade International, Manchester, New Hampshire) along with a small amount of 15% (w/v) dextran in water, which served as a cryo-protectant, and frozen using a Balzers HPM 010 High Pressure Freezing Machine. Following freezing, samples were transferred rapidly to liquid nitrogen and then to a substitution fluid consisting of 2% OsO₄ in acetone containing 0.05% uranyl acetate. Samples were substituted as follows: -85 C for 4 d, -20 C for 2–3 h, 4 C for 2 h, room temperature for 45 min. After removal of substitution fluid, samples were rinsed in 3 changes of 100% HPLC acetone for 15 min each, infiltrated with a mixture of 50% Araldite/Embed 812 and 50% Spurr's resin and polymerized for 48 h at 60 C in Lux Contour Permanox disposal tissue culture dishes. For conventional fixation, the glutaraldehyde/OsO₄ protocol of Taylor and Mims (1991) was used. These samples were dehydrated in ethyl alcohol and infiltrated and embedded as described above. Thin sections of sori were cut using an ultramicrotome equipped with a diamond knife, picked up on slot grids and allowed to dry onto formvar-coated aluminum racks (Rowley and Moran 1975). Sections were post-stained for 3 min each with a saturated aqueous solution of uranyl acetate followed by lead citrate (Reynolds 1963) and examined using a Zeiss EM 902A transmission electron microscope operating at 80 kV.

A combination of light microscopy (LM) and scanning electron microscopy (SEM) was used to study overall sorus and zoosporangium morphology. For LM, 1 µm-thick sections were collected on a glass microscope slide and stained with toluidine blue O prior to examination. In the case of SEM, samples were fixed following the protocol of Enkerli et al (1997), dehydrated in a graded series of ethyl alcohol, critical point dried, sputter coated with gold, and examined with a JEOL 5800 scanning electron microscope operating at 15 kV.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Zoosporangia of A. ipomoeae-panduratae were produced in small to large and often confluent sori near the abaxial surfaces of infected leaves of I. hederacea and became exposed when the leaf epidermis ruptured (Fig. 1). Zoosporangia were borne in chains from short club-like sporangiophores that lined the sorus base (Fig. 3). As zoosporangia matured they separated from one another and accumulated in the sorus (Figs. 1, 2). Mature zoosporangia were short cylindric in shape and measured 14–18 x 15–20 µm. Except for a faint terminal secession scar at each end, the zoosporangium surface appeared smooth when viewed with SEM (Fig. 2).

View larger version (153K): [in this window] [in a new window]    FIGS. 1–3. Albugo ipomoeae-panduratae. 1. A scanning electron micrograph showing a sorus filled with zoosporangia. The ruptured leaf epidermis is shown at the arrowheads. Bar = 100 µm. 2. Higher magnification view of zoosporangia from Fig. 1. Faint secession scars (arrows) are visible on some zoosporangia. Bar = 10 µm. 3. A light micrograph of a 1 µm-thick section of a sorus stained with toluidine blue O. Sporangiophores (SP), chains of developing zoosporangia (arrows) and numerous disarticulated zoosporangia are visible in the sorus. Bar = 50 µm

View larger version (203K): [in this window] [in a new window]    FIGS. 4–8. Transmission electron micrographs of conventionally fixed zoosporangia of Albugo ipomoeae-panduratae. 4. Near median longitudinal section of a mature zoosporangium in which two nuclei (N), numerous mitochondria (M), lipid bodies (LB), and both electron-dense (single arrows) and electron-transparent (double arrows) vacuoles are visible. The zoosporangium wall is visible at W. Bar = 3 µm. 5. Section of a nucleus (N) illustrating excellent preservation of the nuclear envelope (NE). Strands of rough endoplasmic reticulum are visible at the arrowheads. Bar = 0.3 µm. 6. Elements of the Golgi apparatus (G) are evident near the surface the nucleus (N). A microtubule is visible (arrowhead) as well as a cross section of a basal body (BB). Bar = 0.3 µm. 7. A section showing two basal bodies (BB1,, BB2) visible in longitudinal section lying end to end near the surface of a nucleus (N). An electron-transparent vacuole (asterisk) is present near the end of basal body BB2. Microtubules are visible at the arrowheads. Bar = 0.2 µm. Fig. 8. An adjacent section of basal body BB1 from Fig. 7. A large electron-transparent vacuole (asterisk) is in contact with the end of the basal body. The nucleus is shown at N. Bars = 0.2 µm

View larger version (200K): [in this window] [in a new window]    FIGS. 9–12. Transmission electron micrographs illustrating the cytoplasmic contents of conventionally fixed zoosporangia of Albugo ipomoeae-panduratae. 9. Section showing an array of parallel strands of rough endoplasmic reticulum (arrowheads). Some electron-transparent vacuoles (double arrows) also are visible as well as a portion of an electron-dense vacuole (single arrow). Bar = 0.3 µm. Fig. 10. Section showing some large electron-dense vacuoles (arrow) and a few lipid bodies (LB). Bar = 0.3 µm. Fig. 11. Example of a large vacuole containing electron-dense membranous whorls (asterisks) surrounded by an electron-transparent area. Bar = 0.3 µm. Fig. 12. A section showing a cluster of small vacuoles (arrowheads) of moderate electron density. Note the striations visible in some of these vacuoles. Some electron-transparent vacuoles are visible at the double arrows. Bar = 0.15 µm

HPF/FS also yielded good preservation of ultrastructural details in many zoosporangia of A. pomoeiae-panduratae. All the various organelles and cytoplasmic inclusions present in conventionally fixed samples also could be identified in cryofixed samples (Figs. 13–21), although the overall appearance of some structures differed in the two samples. Large electron-dense vacuoles up to about 2 µm in diameter were evident in zoosporangia along with numerous electron-transparent vacuoles of various sizes (Fig. 13). However, large vacuoles containing membranous whorls similar to the one shown in Fig. 11 were not observed in cryofixed zoosporangia. Nuclei and associated Golgi cisternae were well preserved in cryofixed samples (Fig. 14) and the paired basal bodies found near each nucleus were particularly prominent (Figs. 15–18). Mitochondria were easy to identify (Figs. 13, 19), but appeared much more electron-transparent than in conventionally fixed zoosporangia. Microtubules commonly were observed near nuclei and basal bodies (Figs. 17, 18), and were more prominent in cryofixed samples. Lipid bodies were, however, extracted in cryofixed samples leaving small, electron-transparent, spherical areas in the cytoplasm (Figs. 13, 19). Arrays consisting of parallel strands of rough endoplasmic reticulum similar to those found in conventionally fixed zoosporangia also were present in cryofixed zoosporangia (Fig. 19) although the membranes comprising the individual strands were not as prominent as in conventionally fixed samples.

View larger version (201K): [in this window] [in a new window]    FIGS. 13–18. Transmission electron micrographs of high pressure frozen/freeze-substituted zoosporangia of Albugo ipomoeae-panduratae. 13. Low magnification view of a zoosporangium showing three nuclei (N), numerous mitochondria (M), the extracted remains of lipid bodies (LB), large electron-dense vacuoles (single arrows), electron-transparent vacuoles (double arrows) and the zoosporangium wall (W). Bar = 2 µm. 14. A section illustrating Golgi cisternae (G) near the surface of a nucleus (N). The nuclear envelope is visible at NE. Bar = 0.3 µm. 15. A pair of basal bodies (BB1, BB2) shown in longitudinal section oriented end to end near the surface of a nucleus (N). Note the curved vacuoles (arrows) associated with the ends of the basal bodies. Bar = 0.25 µm. 16. Slightly higher magnification view of basal body BB2 shown in Fig. 15 from an adjacent section. Note the vacuole at the arrow and profiles of similar vacuoles at the arrowheads. Bar = 0.15 µm. 17. An example of a curved vacuole (arrow) positioned at the end of a basal body (BB). Numerous profiles of other vacuoles are visible to the left of the one shown at the arrow. Microtubules are shown at the arrowheads. Bar = 0.3 µm. 18. A section showing paired basal bodies (BB1, BB2) and associated microtubules (arrowheads) near a nucleus. A portion of a curved vacuole is visible at the arrow. Bar = 0.3 µm

View larger version (191K): [in this window] [in a new window]    FIGS. 19–23. Transmission electron micrographs from high pressure frozen and freeze-substituted zoosporangia of Albugo ipomoeae-pandurartae. 19. A section showing mitochondria (M), an array of parallel strands of rough endoplasmic reticulum (arrowheads), the extracted remains of lipid bodies (LB) and two types of vacuoles (V1, V2). Bar = 0.3 µm. 20. A section showing a high magnification view of a V2 vacuole (arrow). Note the fine striations within the vacuole. Bar = 0.1 µm. 21. A section showing large electron-dense vacuoles (arrows) as well as V1 and V3 vacuoles. One of the electron-dense vacuoles appears to be ruptured (arrowhead). Bar = 0.3 µm. 22. Numerous profiles of curved vacuoles are evident at the arrows. The apparent rupture of one of the vacuoles is visible at the arrowhead. Remains of extracted lipid bodies are visible at LB. Bar = 0.3 µm. 23. Example of a ruptured electron-dense vacuole (arrow). The extruded vacuolar contents are visible at the asterisks. V1 and V3 vacuoles are also visible. Bar = 0.5 µm

In addition to the curved vacuoles associated with basal bodies and the electron-dense and electron-transparent vacuoles illustrated earlier, three additional types of vacuoles also were observed in cryofixed zoosporangia. One type reached a maximum diameter of slightly over 1µm and was filled with moderately electron-dense material (Fig. 19). A second type (Figs. 19, 20) with a maximum diameter of about 0.2 µm contained distinctly striated material. A third type (Fig. 21) possessed an electron-dense flocculent core surrounded by an electron-transparent margin, and exhibited a maximum diameter of about 0.5 µm.

Evidence of ruptured vacuoles was common in cryofixed samples (Figs. 21–23). In the curved vacuoles found in association with basal bodies, rupture of the tonoplast often was observed without extreme distortion of the remainder of the vacuole (Fig. 22). However, in the case of large electron-dense vacuoles, tonoplast rupture often was accompanied by the extrusion of vacuolar material into the surrounding cytoplasm (Fig. 23). The overall quality of ultrastructural details in the immediate vicinity of ruptured electron-dense vacuoles was poor.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
This study reports the successful use HPF/FS to prepare oomycete zoosporangia within infected host leaves for study with TEM. While the value of this fixation technique for the elucidation of precise ultrastructural details of the host-pathogen interface in plants tissues infected by fungal pathogens already has been demonstrated (Knauf et al 1989, Mims et al 2001), this appears to be the first report of its successful use on pathogen propagules inside host tissue. We believe that the information provided by our cryofixed samples contributes significantly to a better understanding of zooporangium ultrastructure in A. ipomoeae-panduratae. However, we still believe that direct comparisons of cryofixed and conventionally fixed samples are warranted and provide valuable information. In our opinion the two fixation protocols are highly complementary.

Due to the mechanically violent nature of HPF, it has been our experience that most fragile fungal structures exposed on host surfaces are lost during freezing. For this reason, PF probably will remain the best option for cryofixation of exposed propagules of plant pathogenic fungi despite limitations relating to specimen size. On the other hand, Roberson (1993) has clearly shown that HPF can yield good results for detached masses of spores. The overall quality of fixation he obtained for the thick-walled teliospores of the rust fungus Gymnosporangium clavipes compares very favorably to results we have obtained for a variety of different thick-walled spores prepared by PF. The most severe artifacts Roberson (1993) reported were breaks in the spore wall and disruptions in the nuclear envelope. In the case of the present study, rupture of vacuoles, a problem first reported in high pressure frozen zoosporangia of Phytophthora by Hyde et al (1991c), clearly was the most serious problem associated with our use of HPF. However, we believe that the advantages afforded by HPF greatly outweigh this negative aspect. While the extraction of lipid bodies in both high pressure and plunge frozen samples is also a problem, it is easily overcome by direct comparisons of cryofixed and conventionally fixed samples.

In comparing the advantages and disadvantages of cryofixation to conventional fixation in our study, we feel that cryofixation was superior for the preservation of vacuoles despite the rupture of vacuoles. For example, we believe that the electron-dense curved appearance of the vacuoles found in association with basal bodies in cryofixed samples (Figs. 15–18) is more life-like than the electron-transparent, swollen, spherical appearance of these structures in conventionally fixed samples (Figs. 7, 8). We believe that these vacuoles correspond to so-called cleavage vesicles reported in zoosporangia of Phytophthora cinnamomi by Hyde et al (1991a, b). In conventionally fixed zoosporangia of P. cinnamomi (Hyde et al 1991a), these vesicles possessed circular profiles and were filled with flocculent contents of low electron density. These workers also demonstrated a very large cleavage vesicle which they called an axonemal vesicle associated which the terminal plate of the basal body. This structure appears to be identical to the large vacuole visible in our Fig. 8. In plunge frozen zoosporangia of P. cinnamomi and P. palmivora (Hyde et al 1991b), cleavage vesicles were electron dense and much like the curved vacuoles that we observed near basal bodies of A. ipomoeae-panduratae. Interestingly, however, the cleavage vesicles these workers found in the high pressure frozen zoosporangia of Phytophthora were electron transparent rather than electron dense.

Hyde et al (1991b) reported that cleavage vesicles in Phytophthora disappeared with the formation of the first cleavage membranes involved in zoospore delimination. While it is unknown exactly when these vesicles first formed in Phytophthora, our study indicates that they were present in the zoosporangia of A. ipomoeae-panduratae before the process of zoospore formation began. In regard to zoosporogenesis in other fungi, it is interesting to note that Fisher et al (2000) have reported that cleavage elements in zoosporangia of the chytrid Allomyces macrogynus also were closely positioned to basal bodies.

As evident from this study, zoosporangia of A. ipomoeae-pandurae contained a variety of different types of vacuoles/vesicles. However, it was very difficult to correlate or match with a high degree of confidence certain types of vacuoles/vesicles observed in conventionally fixed samples with those observed in cryofixed samples. In extensively studied species of Phytophthora where vacuole/vesicle specific antibodies are available for immunolabeling, Hyde et al (1991a) were able to demonstrate that a number of different types of vacuoles/vesicles named for their eventual location in zoospores actually were present in zoosporangia before zoospore induction. These included so-called large peripheral vesicles, two types of smaller peripheral vesicles, and both dorsal and ventral vesicles.

Overall zoosporangium morphology in A. ipomoeae-panduratae was quite similar to that described for A. candida by Kahn (1976). However, Kahn (1976) reported only two types of vacuoles in what he considered to be mature zoosporangia of A. candida. These included smaller structures he termed multivesicular bodies and large electron-transparent vacuoles sometimes containing either dark globules or whorls of electron-dense material similar to those shown our Fig. 11. He made no mention of either electron-dense vacuoles similar to those present in our conventionally fixed samples or any type of vacuole associated with basal bodies. He reported that zoosporangia of A. candida were multinucleate and contained a complement of cytoplasmic organelles similar to those we found in A. ipomoeae-panduratae, including large arrays of parallel strands of rough endoplasmic reticulum. Similar arrays also have been reported in zoosporangia of Phytophthora palmivora by Hyde et al (1991a). Zoosporangium wall structure also appears similar in A. ipomoeae-panduratae and A. candida. However, we consistently found that the zoosporangium wall in A. ipomoeae-panduratae was thicker in conventionally fixed samples than in cryofixed samples. This agrees with previous observation of Mims and Richardson (1999) regarding conventionally fixed and cryofixed conidia of Monilinia vaccinni-corymbosi.

	FOOTNOTES

 
¹ Corresponding author, cwmims{at}arches.uga.edu

Accepted for publication May 24, 2002.

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This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Mims, C. W. Articles by Richardson, E. A. Search for Related Content PubMed Articles by Mims, C. W. Articles by Richardson, E. A. Agricola Articles by Mims, C. W. Articles by Richardson, E. A.