AN ULTRASTRUCTURAL STUDY OF SEXUAL REPRODUCTION IN PYTHIUM ULTfMUM

Transcription

1 New Phytol. (1968) 67, AN ULTRASTRUCTURAL STUDY OF SEXUAL REPRODUCTION IN PYTHIUM ULTfMUM BY R. MARCHANT Department of Botany, University College, London (Received 12 May 1967) SUMMARY The original observations of sexual reproduction in Pythium ultimum have been re-interpreted in the light of electron microscope observations. The antheridium of P. ultimum penetrates the oogonium with only a slight constriction of the antheridium and not through a tube as described previously. During the development of the oospore the amount of storage material increases and the wall thickens. In the mature oospore there is a single centrally placed nucleus and most of the rest of the oospore is filled with storage material. The few remaining organelles are found in small pockets between the storage organelles. The lenticular body, originally described as the nucleus, appears to be an artifact produced from the periplasm. INTRODUCTION Descriptions of sexual reproduction in species of Pythium have been based solely on observations with the light microscope up to the present time. These observations have resulted in certain misconceptions about the structures and events leading to sexual reproduction in Pythium. Trow (1901) described a new saprophytic species, P. ttltimum which produces a single oospore in each oogonium, contained loosely within the oogonium wall (aplerotic). Fertilization is achieved by the penetration of the oogonium by a single antheridium, followed by subsequent nuclear fusion. The antheridium is produced from the branch immediately below the oogonium, and Trow (1901) figured the penetration of the oogonium wall by means of a constricted tube. The changes taking place hefore and after fertilization were described by Trow. He stated that storage material produced during the development of the oospore finally coalesced to form a large central storage globule in the mature oospore. A lenticular nucleus was also reported at one side of the storage globule in the mature oospore. Studies with the electron microscope, confirmed by light microscopy, have shown some of these observations to be erroneous. On the basis of the results presented in this paper, a re-interpretation of the sexual structures of P. ultimum will be made. MATERIALS AND METHODS Two strains of Pythium ultimum Trow were used in this study; a sexual one obtained from Queen Elizabeth College, London, and a parthenogenetic one obtained from Birkbeck College, London. Apart from the absence of antheridia and sexual fusion in the parthenogenetic strain, the structure and development of the oospores appeared identical and no further distinction will be made between the strains. The organism was grown at 25' C on oatmeal agar; prepared from the extract of 40 g/1 oatmeal, solidified with 2 o (w/v) 167

2 i68 R. MARCHANT Bacto Difco agar. Material for examination in the electron microscope was fixed in one of the following ways: 1, 2",, potassium permanganate in veronal acetate buffer (ph 6.5) for i hour at room temperature. 2, 6",, glutaraldehyde in veronal acetate buffer (ph 6,5) for 16 hours at 4 C, followed by thorough washing and post-fixation in 2 /,, osmium tetroxide (unbuffered) for 2 hours at room temperature, 3, 6/y acrolein in o.i M-cacodylate buffer (ph 7.2-7,4) for 2 hours at room temperature, followed by thorough washing and post-fixation in i "^'^ osmium tetroxide (Hess, 1966), After fixation and subsequent washing the material was dehydrated in tertiary butanol mixtures (Johansen, 1940). The material was then embedded in Araldite and sections cut with glass knives on an LKB ultratome. Sections were examined with a Zeiss EM9 or Siemens Elmiskop i electron microscope. Preparations were stained on the grids with lead citrate (Reynolds, 1963), Feulgen-stained material for observation in the light microscope was prepared in the following manner: 1. Material was fixed to a coverslip with Haupt's adhesive. 2. Hydrolysis carried out for io-i:; minutes at 60" C in i-n-hcl. 3. Preparations stained for 2 hours in feulgen stain, 4. Mounted in 45 (v/v) glacial acetic acid in water. Material prepared in this way was observed and photographed through a Zeiss photomicroscope. RESULTS Electron tnicroscopy Examination of sections of young unfertilized oogonia reveals the structure shown in Plate 2, No. 3, The oogonium wall is relatively thin and contained within this wall are large numbers of organelles. The mitochondria are nearly spherical in shape and contain large numbers of tubuli; there are also numerous small vacuoles and membrane-bounded organelles containing storage material (Plate 3, No. 5). There is an extensive system of discontinuous endoplasmic reticulum and vesicles, w hich may be important in the synthesis of the wall of the developing oogonium (Marchant, Peat and Banbury, 1967), Several nuclei can be seen in this section, which is in agreement with Trow's observ'ations on young oogonia. After fertilization has occurred and the oospore wall has started to thicken, the number of mitochondria seems to increase and the vacuoles disappear (Plate 2, No, 4). There is also synthesis of storage material which becomes orientated around the perimeter of the oospore (Plate 3, No. 6). At this stage, several nuclei are still visible despite the fact that the oospore is well developed towards maturity. The oogonium is separated from the hypha bearing it by a loose plug of material forming a type of septum across the supporting hypha (Plate 4, No. 8). This wall is poorly organized and does not resemble the more highly organized septa found in other organisms (Marchant, 1966). The function of this plug of material seems to be simply to separate the oogonium from the supporting hyphal system, that it does this effectively can be seen from the fact that the organelles in the supporting hypha become degenerate. In the fully mature oospore, wall thickening produces an irregular outer surface to the oospore (Plate 4, No. 9). The oogonium wall is still intact at this stage and can be seen

3 Ultrastructitre of sex organs in Pythium 169 some distance beyond the oospore. In none of the sections observed was there indication of the presence of any periplasm, resulting from the elimination of oogonial cytoplasm from the developing oospore. The synthesis of storage material has by this time produced a more or less continuous system of membrane-bounded storage organelles in the oospore (Plate 4, No. 9). Other organelles have become relegated to small areas between the storage organelles. The only prominent structure in the oospore, apart from the storage material, is a large centrally placed nucleus (Plate 4, No. 10). This nucleus is 8-12 /< in diameter and occupies a large proportion of the volume of the oospore. There is no evidence of any other large organelle in the mature oospore. 1 he reactions of the storage material with potassium permanganate and osmium suggest that it is probably composed of lipids. Normally only a single antheridium is produced for each oogonium, although occasionally two are produced. In Plate i. No. i, an antheridium which has not penetrated a fertilized oogonium can be seen pressed against the oogonium wall. The antheridia contain numerous mitochondria, and often several nuclei can be seen in one section of an antheridium. The antheridia are highly vacuolated and also have a certain amount of storage material. The penetration of the oogonium by the antheridium is achieved with only a slight constriction of the antheridium at the point of entry (Plate i. No. 2). The antheridium has an extremely thin wall and from the nature of the oogonium wall at the point of entry, it seems that penetration may be brought about not by the physical or chemical activity of the antheridium alone, but by some function of the oogonium itself. Marchant et al. (1967) have already reported lomasome-like structures from the hyphae of Pythium idtimum; these structures are seemingly produced by elaboration of the plasmalemma. These organelles are almost invariably seen in the tips of antheridia which have penetrated an oogonium. Marchant et al. (1967) suggested that these structures were not involved in wall synthesis in this organism. Girbardt (1961) thought that structures of this type might serve to increase the surface area of the plasmalemma, although it is difficult to envisage the function of such a system in the antheridium. Light microscopy To confirm the results of the electron microscopy, developing oogonia and oospores were examined with the light microscope. Staining with feulgen stain revealed that there is nuclear material throughout the young oospore (Plate 5, Nos. 11 and 12), which agrees with the observations in the electron microscope. In such fixed material a lenticular body can often be seen in the oospore. This structure is not stained with feulgen stain and on closer examination can be seen to be between the oospore and the oogonium wall (Plate 5, No. 11). This structure may have been the one which Trow (1901) described as the lenticular nucleus. It seems to be an artifact produced by the fixing and staining procedures. It is probably derived from the periplasm eliminated from the developing oospore. In the fully mature oospore (Plate 5, Nos. 13 and 14) a single central structure is visible, which stains with feulgen stain. This organelle corresponds to the one described by Trow as a central storage globule, but on the evidence of electron and light microscopy is undoubtedly a single central nucleus. DISCUSSION Trow (1901) based his description oi Pythium ultimum on light-microscope observations. The basic structure was observed in unstained preparations, while the cytology was

4 170 R. MARCHANT deduced from stained sections, using relatively non-specific nuclear stains. Trow's observations of many nuclei within the antheridia and oogonia have been confirmed, and many of his observations on zonation of the cytoplasm within the oogonium and oospore can be related to the fine structure described here. Observations of antheridia penetrating oogonia have shown that there is only slight constriction at the point of entry and not a tubular extension into the oogonial cytoplasm. The difficulty of observing the antheridium through the oogonium wall, in light microscopy, may account for this discrepancy. The major point of disagreement with Trow's original description is over the nucleus in the oospore, there seems little doubt, however, that the large central structure in the mature oospore is a nucleus and not a storage globule. The fine structure of the oogonia, oospores and antheridia shows no really unusual features. In the hyphae examined previously (Marchant et al., 1967) and in the reproductive structures of this organism no dictyosomes have been found. Hawker and Abbott (1963) reported dictyosomes in vegetative hyphae of P. debaryanum, but only one structure resembling a dictyosome has been observed in many sections of P. ultimum examined and this was probably only a chance configuration of endoplasmic reticulum. Hawker and Abbott (1963) also reported lomasomes in the vegetative hyphae of P. debaryanum. Marchant et al, (1967) have, however, pointed out that similar structures in P. ultimum seem to be formed from the plasmalemma and suggested that the name lomasome be reserved for structures produced from the endoplasmic reticulum. The function of this plasmalemmal structure is hard to determine; structures of this type appear to be completely absent from certain fungi, e.g. Fusarium culmorum (Marchant, 1966; Marchant et al., 1967) and Phycomyces blakesleeanus (Peat and Banbury, 1967). Marchant et al (1967) showed that the tubules and vesicles in tbese structures had electron-opaque contents and they may be sites of extracellular enzyme synthesis. ACKNOWLEDGMENT I would like to thank the Science Research Council for a research grant held during the completion of this work. REFERENCES GiRBARDT, M. (1961). Licht und elektronenmikroskopische L^ntersuchungen an Polystictus versicohr. W. Die Eeinstrukture von Grundplasma und Mitochondrien, Arch. MikrobioL, 39, 351. HAWKER, L, E, & ABBOT, P, MGV, (1963), Fine structure of the young vegetative hyphae of Pythium debaryanum. J. gen. MicrobioL, 31, 491. HESS, W. M, (1966), Eixation and staining of fungus hyphae and host plant root tissues for electron microscopy. Stain TechnoL, 41, 27, JOHANSEN, D. A, (1940), Plant Microtechnique. New York, MARCHANT, R, (1966), Fine structure and spore germination in Fusarium culmorum. Ann. Bot. N.S,, 30,441, M,ARCH,WT, R.,PE,AT, A, & BANBURY, G. H, (1967). The ultrastructural basis of hyphal growth, AfOTPM"'-. 66, 623. PE,\T, A. & BANBURY, G, H, (1967). L'ltrastructure, protoplasmic streaming, growth and tropisms of Phycomyces sporangiophores. New PhytoL, 66, 475. REYNOLDS, E. S, (1963), The use of lead citrate at high ph as an electron-opaque stain in electron microscopy, J. Cell Biol., 17, 208, TROW, A. H, (1901), Observations on the biology and cytology of Pythium ultimum, n. sp. Ann. Bot., ,

5 THE NEW PHYTOLOGIST, 67, Pi..\Ti-: I R. MAHCHA'ST ULTRASTRUCTURE OF SEX ORGANS JN PYTHIL'IVI (faeiitf;ftage 170)

6 THE NEW l'iiy'l'()l()(;is'l", 67, I PLATE 2 R. MARCHA:<iT ULTRASTRUCTURE OF SEX ORGANS IN PYTHIUM

7 THE NEW PHYTOLOGIST, 67, i OS K. yi\rchanr~ultrastructure OF SEX ORGANS IN PYTHIUM

8 Till-: \i':\v piiy'ix)l()(;ist, 67, PLATE 4 R. M.\RCH\NT ULTRASTRUCTURE OF SEX ORGANS IN PYTHIUM

9 THE NEW PHYTOLOGIST, 67, i PI ATK K. M.\RCH.\ST ULTRASTRUCTrRE OF SEX ORGANS IN PYTHILM

10 Ultrastructure of sex organs in Pythium 171 EXPLANATION OF PLATES The symbols used for all plates are as follows: a, antheridium wall; an, antheridial nucleus; ev, vesicular endoplasmic reticulum; 1, lipid storage material; lo, vesicular plasmalemma body; m, mitochondrion; n, nucleus; o, oogonium wall; op, oogonium septal plug; os, oospore wall; p, periplasm; v, vacuole. Scale marks represent i //, unless otherwise stated. PLATE I No. I. Section through an antheridium pressed against the wall of a fertilized oogonium. Acrolein, x 13,300. No. 2. Section through an antheridium penetrating an oogonium, the oogonium is at the left of the micrograph. Acrolein, x 39,600. PLATE 2 No. 3. Section of a young oogonium, showing vacuolation and lipid storage material. KMnO4, X No. 4. Section through a young oospore which has become separated from the oogonium. KMnO4, X PLATE 3 No. 5. Part of a section through a young oogonium, showing the organelles. KMnOj, X 16,800. No. 6. Part of a section through a young oospore, showing the organelles. KMnO4, x 16,800. PLATE 4 No. 7. Section through an antheridium within a fertilized oogonium, illustrating a vesicular plasmalemma body. Acrolein, x 84,000. No. 8. Section showing the loose plug of material isolating the oogonium from the supporting hypha. Acrolein, x 23,000. No. 9. Section of a mature oospore in the oogonium, showing the thick irregular oospore wall and the storage material. Glutaraldehyde, x 14,000. No. 10. Part of a section of a mature oospore, showing the central nucleus and storage material. Glutaraldehyde, x 37,800. PLATE 5 No. II. Light micrograph of a young oospore within an oogonium, stained with feulgen stain. The lenticular periplasm can be seen at one side, and there is no obvious nuclear material, x No. 12. Light micrograph of a young oospore within an oogonium, stained with feulgen stain. X No. 13. Light micrograph of a mature oospore in an oogonium, stained with feulgen stain. The large central nucleus is obvious, x No. 14. Light micrograph of mature oospores, stained with feulgen stain, showing the central nucleus in each, x 2000.

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