Germination and parasitation of the resting sporangia ofSynchytrium endobioticum

Summary The cytoplasmic organization of the long-lived, thick walled resting stage of the sporangium ofSynchytrium endobioticum (Schilb.) Perc. is described. The cytoplasm of the resting sporangium contains a large number of closely packed lipid bodies and irregular electron dense bodies, which are interspaced with fine channels of cytoplasm. These ultrastructural observations are discussed in relation to the hypothesis ofBally (1912) andCurtis (1921) that zoospore primordia are already present during the resting stage. It is shown that the zoospore primordium is actually a lipid body and an osmiophilic body and the strands postulated to connect the individual zoospore primordia are actually the fine channels of cytoplasm.A new inner wall layer is laid down prior to the start of the germination. It is this wall layer which will protrude to form the vesicle in which sporogenesis takes place. The germination process observed, protrusion of a vesicle through a crack in the sporangial wall, the migration of the sporangial content into the vesicle, and the formation of a single, membrane-bound sporangium within this vesicle, is in full agreement with the recent light microscopic studies ofSharma andCammack (1976). These observations support the transfer ofS. endobioticum from the subgenusMesochytrium to the subgenusMicrosynchytrium (bothsensu Karling 1964).A major objective of the study, to obtain ultrastructural evidence for the location of the meiotic divisions in the life cycle, was not fulfilled.Three different fungi were observed to parasitize the resting sporangium ofS. endobioticum. These infections are discussed in relation to other mycoparasites of plant pathogenic fungi. The possibility of using a mycoparasite for the biological control of potato wart disease is considered to be without practical relevance.     

A stable mitomycin-induced LC-type albino mutant of Blastocladiella emersonii

In an effort to obtain orange mutants ofBlastocladiella emersonii Cantino &Hyatt, wild type zoospores were treated with mitomycin. From the variants produced, we obtained a stable, albino mutant (Ma-1) that differs significantly from another, previously described (Shaw &Cantino, 1969) UV-induced, albino variant. This report concerns the origin of Ma-1, its distinguishing features, and its apparent similarity to the few LC (late colorless) plants that normally appear in wild type populations. A preliminary note regarding mitomycin-induced variants ofB. emersonii has been published (Matsumae &Cantino, 1970).     

Development of the resting sporangia ofSynchytrium endobioticum,the causal agent of potato wart disease

Summary An ultrastructural study of the development of the resting sporangium ofSynchytrium endobioticum (Schilb.) Perc. infecting potato cells is presented. The resting sporangium is found to have a single large, centrally placed nucleus with a prominent nucleolus through its entirein situ development. The cytoplasmic organization of the resting sporangium is further characterized by numerous membrane-bound lipid bodies and osmiophilic bodies. The latter have a characteristic sieve-like appearance, probably because certain storage components have been extracted during preparation for electron microscopy. Because of the similar location and appearance of these osmiophilic bodies it is suggested that they are identical to what has earlier (based on light microscopy) been described as chromatin granules; and the ultrastructural studies presented here show that nucleolar discharge which was described from light microscopic observations as leading to chromatin granules in the cytoplasm, and finally forming the nuclei of the zoospores (bally 1912,curtis 1921,percival 1910) simply does not occur.The appearance of dense fibrillar-like structures on the sporangial surface at an early stage of resting sporangium development ultrastructurally distinguishes the resting sporangium from the zoosporangium. The development of the layered portion of the thick sporangial wall is shown to be due to the fusion of vacuoles containing pre-made wall fibrils with the cell membrane. It is suggested that the inner compact wall layer which is essentially substructureless is formed by the membrane itself.The characteristic wings of the matureS. endobioticum resting sporangium originate from the potato host cell wall. Remnants of host cell organelles in the outermost layer of the resting sporangium wall show that degradation of the host cell cytoplasm contributes to wall formation of the parasite.     

Hair morphology inPlantago sect.Coronopus (Plantaginaceae)

The presence of bottle-like hairs has been described in four hitherto uninvestigated taxa ofPlantago sect.Coronopus sensuDietrich. This hair type is a feature characteristic of sect.Coronopus.     

The endobiotic thallus ofPhysoderma maydis,the causal agent ofPhysoderma disease of maize

Summary The endobiotic thallus ofPhysoderma maydis is characterized by the presence of an extremely fine rhizomycelium which passes through the host cell wall, allowing the spread of the disease, and irregularly shaped turbinate cells, which may be septate or nonseptate and which are in close association with developing resting sporangia. The formation of the resting sporangium wall is first seen as localized depositions on the rounded surface of the sporangium and only later on the flattened surface of the sporangium which will form the operculum. The substructure of the resting sporangium wall is typical for members of theBlastocladiales. The resting sporangium is contiguous with the rhizomycelium during development and is finally sealed-off from the rhizomycelium by a further deposition of wall material. After the sealing-off of the resting sporangium from the rhizomycelium the content of the sporangium is compartmentalized and the two inner wall layers are deposited. The centre of the sporangium is filled with an electron dense accretion. At the periphery of the sporangium is a layer of lipid bodies. Between the lipid bodies and the central electron dense accretion is a thin layer of cytoplasm which contains the nuclei. The outer surface of the resting sporangium is smooth.     

Germination of the resting sporangia ofPhysoderma maydis,the causal agent ofPhysoderma disease of maize

Summary The structural and developmental characteristics of the resting sporangium in uniflagellate phycomycetes, together with the type of zoospore, are of high taxonomic value. Among these fungi, however, only a few electron microscopic investigations have been published on this topic, mainly due to technical problems. In the present study ofPhysoderma maydis (Blastocladiales) these problems were overcome as the resting sporangia in this species are formed synchronously, in large numbers, the germination is readily induced and the impermeability of the resting sporangium wall can be circumvented by shaking the prefixed sporangia with glass beads.The germination of the resting sporangia ofP. maydis is described by correlative light and electron microscopic studies and discussed in relation to related investigations on sporogenesis: The germination process starts by a breakdown of large electron-dense accretions found in the resting stage. Simultaneously, the peripheral location of the lipid bodies is lost. The large operculum is pushed open by a protrusion of the inner sporangial wall; an additional wall layer is formed during this process. Synaptonemal complexes are found in the nuclei at this stage, as are nuclear division figures which suggests anEuallomyces type of life cycle for this fungus. Cleavage vesicles, formed from dictyosomes or endoplasmic reticulum, ultimately separate the sporangial content into meiospores. The sequential assembly of organelles into the side body complex is described. Sequestering of the ribosomes into a nuclear cap is interpreted as taking place immediately prior to zoospore discharge.     

Polyamines and ornithine decarboxylase activity during growth and differentiation in Blastocladiella emersonii

The activity of ornithine decarboxylase (EC 4.1.1.17, L-ornithine carboxy-lyase) was determined during the life cycle of Blastocladiella emersonii. The specific activity of the enzyme was found to be low in the zoospores, to rise 20-fold during germination and early growth, to fall during growth and to rise again during sporulation. This rise in enzyme activity was shown to be dependent on protein synthesis. Putrescine levels, on a per mg of protein basis, paralleled the fluctuations found in ornithine decarboxylase activity. Putrescine and spermidine were the only polyamines found in extracts of B. emersonii.     

Mitochondrial alternative oxidase is determinant for growth and sporulation in the early diverging fungus Blastocladiella emersonii

Blastocladiella emersonii is an early diverging fungus of the phylum Blastocladiomycota. During the life cycle of the fungus, mitochondrial morphology changes significantly, from a fragmented form in sessile vegetative cells to a fused network in motile zoospores. In this study, we visualize these morphological changes using a mitochondrial fluorescent probe and show that the respiratory capacity in zoospores is much higher than in vegetative cells, suggesting that mitochondrial morphology could be related to the differences in oxygen consumption. While studying the respiratory chain of the fungus, we observed an antimycin A and cyanide-insensitive, salicylhydroxamic (SHAM)-sensitive respiratory activity, indicative of a mitochondrial alternative oxidase (AOX) activity. The presence of AOX was confirmed by the finding of a B. emersonii cDNA encoding a putative AOX, and by detection of AOX protein in immunoblots. Inhibition of AOX activity by SHAM was found to significantly alter the capacity of the fungus to grow and sporulate, indicating that AOX participates in life cycle control in B. emersonii.     

An improved (lysine enriched) synthetic medium for the growth of Blastocladiella emersonii on agar media

Numerous combinations of amino acids supported growth ofBlastocladiella emersonii populations on defined media solidified with agar. Lysine was a particularly influential ingredient in these media.     

Concanavalin A-induced lysis of zoospores of Blastocladiella emersonii

Concanavalin A (ConA) induces lysis of zoospores of the aquatic fungus Blastocladiella emersonii. The rapid lysis can be completely blocked by α-methyl- -mannoside. At lower temperatures the cells are more tolerant towards lysis compared with cells at room temperature. Dimeric ConA derivatives and Lens culinaris hemagglutinin A are less effective, whilst other lectins tested do not induce lysis at all. We suggest that cluster formation of adjacent ConA receptors precedes lysis.     

Observations on Phyllachora aravalliensis Dakshini et al. Affecting oropetium thomaceum (Linn.) Trin.

The paper presents results of observations on the type material ofPhyllachora aravalliensis Dakshini et al. (Ph. aravalliense original) affectingOropetium thomaceum (Linn.)Trin. Three distinct types of spore bodies were observed in addition to the normal perithecia. These are spermogonia, microconidial locules and sterile perithecia with beaded filaments, reminiscent of asci, and constitute addition to the original diagnostic characters of the species ofPhyllachora.     

Basic nuclear proteins in the Oömycete fungus Achlya bisexualis

Summary A comparison was made of the basic proteins extracted from the chromatin and nuclei of Achlya bisexualis, Blastocladiella emersonii, and Pisum sativum. Extraction of purified chromatin and nuclei of the Chytridiomycete, B. emersonii followed by gel electrophoresis produced no detectable protein bands. Extractions of purified nuclei and chromatin by either mineral acid or CaCl₂ from the Oömycete A. bisexualis resulted in several protein bands following separation by disc gel electrophoresis. Monitoring the nucleic acids during the nuclear isolation procedure as well as comparing electropherograms of basic nuclear proteins with basic ribosomal proteins suggests no significant contamination of the nuclear preparations with ribosomes. Carboxymethyl cellulose chromatography of the A. bisexualis nuclear basic proteins resolved three distinct fractions. Gel electrophoresis of the CM cellulose fractions indicated heterogeneity of each fraction. Amino acid analysis of the CM fractions showed that they were all lysine-rich and meet the basisity requirements of histones.     

Beitrag zur Cytologie und Entwicklung der siphonalen GrünalgeDerbesia marina

Zusammenfassung 1. Im Vordergrund vorliegender Untersuchungen steht die Klärung der Sporenentwicklung vonDerbesia marina.2. In die Sporangienanlagen treten aus dem Thallus zahlreiche Kerne ein, die sich noch teilen, schließlich aber die Anzahl von hundert nicht überschreiten.3. Wenn ein Sporangium etwa seine Endgröße erreicht hat, durchläuft ein Teil der Kerne (15–40) synchron die Meiosis, in der 2 n=16 Chromosomen zu erkennen sind.4. Erst nach der Meiosis wird das Sporangium durch eine Doppelwand vom Plasma des Thallus getrennt.5. Die zur Sporenbildung determinierten Kerne schwellen stark an und verteilen sich in gleichen Abständen an der Peripherie des Sporangiums. Ihre Zahl liegt in der Größenordnung der meiotischen Prophasekerne. Die übrigen Kerne sowie die vegetativen Kerne werden resorbiert.6. Noch vor Bildung der Sporenmembran entstehen die ringförmigen Blepharoplasten, und zwar unmittelbar am Rande einer napfförmigen Eindellung des Sporenkerns. Sie lösen sich vom Kern, nehmen an Umfang zu und erscheinen schließlich als Doppelringe, deren Struktur sich lichtoptisch noch weiter auflösen läßt.7. Der primäre Sporenkern teilt sich anschließend in einer großen Spindel. Nach weiteren Mitosen besitzt jede Spore schon innerhalb des Sporangiums etwa 10 bis 12 kleinere Kerne.8. Die Untersuchungen an derHalicystis-Generation bestätigen weitgehend die Angaben in der Literatur, insbesondere auch überHalicystis parvula.9. Die männlichen Gameten besitzen einen borstenförmigen Fortsatz, der nach unpublizierten elektronenoptischen Befunden wahrscheinlich aus einem überdauernden Spindelrest besteht.10. Die Lebendbeobachtung der Kopulation brachte ein von älteren Darstellungen abweichendes Ergebnis.11.Derbesia tenuissima zeigt cytologisch keine Abweichungen vonDerbesia marina und besitzt die gleiche Chromosomenzahl.12. Eine vonKornmann (1966) isolierte Variante vonDerbesia marina ohneHalicystis-Generation erwies sich als haploid. Bei ihrer Sporenentwicklung erfolgt keine Meiosis.13. Die Unterschiede der Sporenentwicklung beiDerbesia marina undDerbesia lamourouxii nach den Ergebnissen vonDavis (1908) werden diskutiert und die Entstehung des Basalringes der Geißeln mit der beiOedogonium verglichen.

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Contribution to cytology and development of the siphonaceous green algaDerbesia marina

This paper deals primarily with spore formation ofDerbesia marina. In an apical zone of the sporangium 15–40 rounded nuclei undergo meiosis. At this stage 8 pairs of chromosomes are observed. After meiosis the cytoplasm of the sporangium is separated from the thallus by the well-known double cell wall. Only one of the daughter nuclei, resulting from each meiotic division, proceeds towards final spore formation. These primary spore nuclei enlarge up to 3 times the diameters of the meiotic prophase nuclei. The remaining nuclei abort. Prior to formation of the spore membranes the ring of the flagellar bases (blepharoplasts) appear on the border of a depression formed on one side of the nucleus. This is followed by a mitotic division, in which long chromosomes are attached with a large spindle. Further mitotic divisions result in multinucleate spores, which are then delimited by the developing spore walls. The details of spore formation inDerbesia tenuissima are similar to those outlined forDerbesia marina. Investigations on cytology and development of the gametophyte produced results similar to those of previous authors studyingHalicystis ovalis andHalicystis parvula. Unpublished electron micrographes of the male gamete ofDerbesia marina suggest that the posterior bristle-like process contains remnants of the spindle fibres of the preceeding nuclear division. A further cytological investigation on an isolate ofDerbesia marina, which lacks theHalicystis generation (Kornmann 1966), has shown this sporophyte to be haploid. Spore formation in the isolate does not involve meiosis. The differences in spore formation betweenDerbesia marina andDerbesia lamourouxii (Davis 1908) are discussed and the formation of the ring of the flagellar bases compared with that ofOedogonium (Kretschmer 1930).

     

Study of the nuclear cycle of four species of strictly anaerobic rumen fungi by fluorescence microscopy

The nuclear cycles of four species of strictly anaerobic rumen fungi (Neocallimastix frontalis, Piromonas communis, Neocallimastix joyonii, andSphaeromonas communis) were compared by fluorescence microscopy. The existence of two types of sporangium development and thalli was confirmed in this study. The first type involved monocentric species fitted with an endogenously developing single sporangium and with rhizoids, such asN. frontalis, P. communis, andS. communis, characterized by the presence of rhizoid-like vesicles. The second type concerned polycentric species with an exogenous sporangium development such asN. joyonii. This species is characterized by a rhizomycelium and gamma particle-like bodies within the zoospore.     

Stimulation of the anaerobic growth ofSalmonella typhimurium by reduction ofl-carnitine,carnitine derivatives and structure-related trimethylammonium compounds

In view of the development of al-carnitine deficiency, the metabolism ofl-carnitine and structure-related trimethylammonium compounds was studied inSalmonella typhimurium LT₂ by means of thin-layer chromatography (TLC).l-Carnitine, crotonobetaine and acetyl-l-carnitine stimulated the anaerobic growth in a complex medium significantly. The stimulation depended on the formation of -butyrobetaine. The reduction ofl-carnitine proceeded in two steps: (1) Dehydration of thel-carnitine to crotonobetaine, (2) hydrogenation of crotonobetaine to -butyrobetaine. The reduction of crotonobetaine was responsible for the growth stimulation. Terminal electron acceptors of the anaerobic respiration such as nitrate and trimethylamine N-oxide, but not fumarate, suppressed the catabolism ofl-carnitine completely. Glucose fermentation, too, inhibited the reduction ofl-carnitine but optimal growth with a high carnitine catabolism was achieved byd-ribose. The esters of carnitine with medium- and long-chain fatty acids inhibited the growth considerably because of their detergent properties.Abbreviations TLC thin-layer chromatography     

Bicarbonate-induced synthesis of polysaccharide during morphogenesis by synchronous,single-generations of blastocladiella emersonii

Summary Relative to the formation of an ordinary colorless plant of Blastocladiella emersonii (which does not require bicarbonate for its formation), the bicarbonate-induced genesis of a resistant-sporangial plant (R.S.) is associated with: (a), a two-fold increase in the rate of exponential, net synthesis of an endogenous, apparently highly branched, glycogen-like polysaccharide; and (b), a three-fold increase in the total pool of this polysaccharide built up within the plant.The polysaccharide accumulates during the early development of an R. S. plant in synchronous, single-generation culture when glucose is being consumed rapidly from the medium without detectable production of lactic acid.The accumulated polysaccharide pool disappears again during the maturation period of a resistant-sporangial plant, when glucose is no longer being consumed from the medium. During this time, (a), approximately one mole of lactic acid is liberated per mole of polysaccharideglucose consumed; and (b), the specific activity of glucose-6-phosphate dehydrogenase rises sharply. These observations are consistent with the possibility that the final maturation of the resistant sporangium is associated with an increase in glucose metabolism via the hexose monophosphate shunt.     

Effects of 2-deoxy-d-glucose,amiloride, vasopressin,and ouabain on active conductance andE Na in the toad bladder

Summary The effects of various agents on active sodium transport were studied in the toad bladder in terms of the equivalent circuit comprising an active conductanceK ^a, an electromotive forceE _Na, and a parallel passive conductanceK ^p. For agents which affectK ^a, but notE _Na orK ^p, the inverse slope of the plot of total conductance against short-circuit currentI ₀ evaluatesE _Na, and the intercept representsK ^p. Studies employing 5×10^–7 m amiloride to depressK ^a indicate a changingE _Na, invalidating the use of the slope technique with this agent. An alternative suitable technique employs 10^–5 m amiloride, which reducesI ₀ reversibly to near zero without effect onK ^p. Despite curvilinearity of the -I₀ plot under these conditions,K ^p may therefore be estimated fairly precisely from the residual conductance. It then becomes possible to follow the dynamic behavior ofK ^a andE _Na (in the absence of 10^–5 m amiloride) by frequent measurements of andI ₀, utilizing the relationshipsK ^a=K-K ^p, andK _Na=I _O/(K-K ^p). 2-deoxy-d-glucose (7.5×10^–3 m) depressedK ^a without affectingE _Na. Amiloride (5×10^–7 m) depressedK ^a and enhancedE _Na. Vasopressin (100 mU/ml) enhancedK ^a markedly and depressedE _Na slightly. Ouabain (10^–4 m) depressed bothK ^a andE _Na. All of the above effects were noted promptly;K ^p was unaffected. The electromotive force of Na transportE _Na appears not to be a pure energetic parameter, but to reflect kinetic factors as well, in accordance with thermodynamic considerations.     

Effect of cotton dust and 2,4-D on Host-parasite relationship. (I) cotton and Fusarium-wilt

Summary The pathogenFusarium solani sensuSnyder &Hansen, as identified byW. L. Gordon was recorded in the present work as a new species causing cotton wilt in Egypt. Cotton dust in varying concentrations does not significally alter the normal infection of both Menoufi and Giza 26 cotton varieties towardsFusarium. Similarly the potency ofFusarium filtrate to induce wilting did not appreciably change with previous treatment of cotton plants with cotton dust. On the other hand leaves treated with 2,4-D showed the maximal water loss. Percentage infected Giza 26 cotton seedlings were found to be comparatively less in soil infected with 2,4-D treatedFusarium mycelium than in that infected with untreated mycelium.     

Decoupling of heart muscle cells: Correlation with increased cytoplasmic calcium activity and with changes of nexus ultrastructure

Summary Purkinje fibers of the sheep heart were exposed to (a) 0.1mm dihydro-ouabain (DHO), followed by (b) 0.1mm DHO in Na-free solution or to (c) 1mm dinitrophenol (DNP). The degree of electrical decoupling was characterized in terms of the inside longitudinal resistancer _i as measured with a 3-microelectrode voltage-clamp technique. Procedurea increasedr _i by a factor of 3.7±1.1 (mean±sd),b by a factor of 9.8±2.2, whereas inc incomplete voltage control indicated nearly complete uncoupling. Intracellular calcium activity (aCa _i ) was monitored with a microelectrode system. At control conditionsaCa _i was below 0.1 m. The procedures listed above increasedaCa _i to (a) 4±1.5 m, (b) 8±2 m, and (c) 36±12 m. The increase ofaCa _i was in good correlation with the changes in core resistance. Effects on nexus ultrastructure, investigated with freeze-fracture techniques, are shown in histograms. At control conditions, the particle diameter distributed around a single peak (8.3±0.5 nm). Proceduresb andc induced a second population at 10.8 nm; increased decoupling reduced the control population in favor of the 10.8 nm population. Decoupling enlarged the width of the nexus gap by a factor of 1.6; again, the control population decreased in favor of a new population. In the decoupled state the height of the particle was smaller. Pits on the E-face displayed a more regular array and a nearly unchanged center-to-center spacing. Separation into several peaks was not possible due to scatter of the data.We interpret the findings to mean that elevatedaCa _i induces a conformational change of the nexus subunits which corresponds to a transition from an open to a closed state. The conformational change can be formally described by a particle contraction which disrupts the continuity with the particle of the adjacent membrane. Purkinje fibers exposed to DNP for 1 hr showed thinned (7.7±0.5 mm) and elongated particles. We suggest that this is a secondary event and not a precursor of functional uncoupling.