Cyst wall formation and endogenous carbohydrate utilization during synchronous encystment of Phytophthora palmivora zoospores

Summary Vigorous agitation caused the zoospores of Phytophthora palmivora to undergo rapid synchronous encystment. The rate of encystment was determined by counting the number of cells with an alkali-resistant cyst wall. 50% of the zoospores formed an alkali-resistant cyst wall within 60 sec of agitation; after 120 sec, essentially all zoospores had encysted. The rate of spontaneous encystment in nonagitated suspensions was much slower. The flagella of nearly all zoospores disappeared within 30 sec of agitation, i.e. prior to the formation of an alkali-resistant cyst wall. Zoospores depend on internal reserves for synthesizing their cyst walls. Approximately 70% of the total carbohydrate in motile zoospores was extracted with water after treating the cells with 70% éthnol. During synchronous encystment, this carbohydrate fraction composed largely of glucans decreased markedly while the insoluble carbohydrate fraction (cyst wall glucan) increased correspondingly. Clearly, the conversion of cytoplasmic glucan into wall glucan plays a major role in zoospore encystment.     

Adhesion of Phytophthora palmivora zoospores: detection and ultrastructural visualization of concanavalin A-receptor sites appearing during encystment.

The binding of concanavalin A (Con A) to the cell surface of zoospores and cysts of Phytophthora palmivora was studied by radiometry (125I-Con A), ultraviolet microscopy (fluorescein-Con A) and electron microscopy peroxidase-diaminobenzidine technique). Zoospores were found to secrete during the early stages of encystment a Con A-binding material susceptible to trypsin digestion. This glycoprotein is contained in the so-called peripheral vesicles and is probably responsible for the adhesion of the encysting zoospores to solid surfaces.     

Adhesion of Phytophthora palmivora zoospores: electron microscopy of cell attachment and cyst wall fibril formation.

Zoospores of Phytophthora palmivora adhered to a plastic film surface were examined by electron microscopy. Three stages of adhesion were compared: (1) non-adhesive, unencysted zoospores, (2) adhered incipient cysts, and (3) adhered mature cysts. Thin sections of incipient cysts revealed cells attached to the film surface through the partially discharged contents of the so-called peripheral vesicles; this seems to be the first step in cell adhesion. In mature cysts, the adhesive appeared to have been compacted into an electron-dense deposit binding the cyst wall to the plastic surface. The adhesion zone was also examined in face view after lysing attached incipient cysts with sodium dodecyl sulphate. Cyst wall microfibrils were seen together with an amorphous substance (presumably the adhesive material). The microfibrils were in various stages of formation. Seemingly, adhesion and microfibril formation take place concurrently. The possibility was considered that the material contained in the peripheral vesicles serves in both cell adhesion and microfibril elaboration.     

The ultrastructure of cell wall formation and of gamma particles during encystment of Allomyces macrogynus zoospores

Structural changes during cell wall formation by populations of semisynchronously germinating zoospores were studied in the water mold Allomyces macrogynus. Fluorescence microscopy using Calcofluor white ST (which binds to -1,4-linked glycans) demonstrated that Calcofluor-specific material was deposited around most cells between 2–10 min after the induction of encystment (beginning when a wall-less zoospore retracts its flagellum and rounds up). During the first 15 min of encystment there was a progressive increase in fluorescence intensity. Ultrastructural analysis of encysting cells showed that within 2–10 min after the induction of encystment small vesicles 35–70 nm diameter were present near the spore surface, and some were in the process of fusing with the plasma membrane. The fusion of vesicles with the zoospore membrane was concomitant with the appearance of electron-opaque fibrillar material outside the plasma membrane. Vesicles similar to those near the spore surface were found within the gamma () particles of encysting cells. These particles had a crystalline inclusion within the electron-opaque matrix. During the period of initial cyst cell wall formation numerous vesicles appeared to arise at the crystal-matrix interface. Approximately 15–20 min was required for the cell wall to be formed. We suggest that the initial response of the zoospore to induction of encystment is the formation of a cell wall mediated by the fusion of cytoplasmic vesicles with the plasma membrane.Non-Standard Abbreviations GlcNac N-Acetylglucosamine - DS sterile dilute salts solution - PYG peptone-yeast extract-glucose broth     

Retraction of flagella by Phytophthora parasitica var. nicotiana zoospores

Summary Light and electron microscope evidence is presented to show that retraction of flagella may occur in Phytophthora parasitica zoospores during encystment.     

Scanning electron microscopic studies on the external morphology of Azotobacter vinelandii during encystment and germination.

Aspects of the external morphology of Azotobacter vinelandii cells during encystment and germination processes were observed with scanning electron microscopy. Most of the vegetative cells have a smooth surface but some have warty surfaces. The intact cysts have wrinkled surfaces and occasional small heaves. Mucoid materials are present on the surface of the cysts. During the encystment process, an extensive peeling-off of coat materials was noted, then the excretion and aggregation of new capsular materials was immediately followed. The germination process was initiated by an expansion of the central body (the cell), then the emerging of this cell from the cyst coats was observed.     

Specific induction of encystment ofLagenidium giganteum zoospores by concanavalin A and derivatives of chitin and chitosan

Summary Zoospores of the mosquito pathogenic fungusLagenidium giganteum preferentially attach to and encyst on the cuticular surface of the immature stages of many species of mosquitoes as the initial step in the infection process. Recognition by zoospores of specific chemical or physical signals on the cuticular surface triggers attachment. A number of compounds likely to be present on the surface of mosquito larvae were evaluated for efficacy in eliciting zoospore encystment. Free amino acids and oligomers, a number of phenolic and polyphenolic compounds and most carbohydrates did not induce encystment at concentrations less than 500 g/ml. Colloidal chitin and chitin films were also ineffective as was O-carboxy-methylchitin; however, glycol chitin and glycol chitosan induced rapid encystment at concentrations at or below 1 g/ml. Zoospores also attached to and encysted in great numbers on fibers of oxycellulose, but not on cellulose. Concanavalin A was the only lectin which induced encystment at concentrations less than 10 g/ml, which suggests that a glycoprotein with terminal mannose and/or glucose residues is involved in encystment. A number of phenols were metabolized by peroxidase on the zoospore surface. Addition of hydrogen peroxide to zoospore suspensions reduced the time needed to induce zoospore encystment by some phenols; however, there was no consistent relationship between the presence or absence of this synergistic effect and the ability ofL. giganteum peroxidase to metabolize a given substrate. The sterol-binding compound amphotericin B induced immediate encystment at 3.5 g/ml, suggesting that sterols, which are required for the induction of zoosporogenesis, were present on the zoospore membrane.     

Scanning electron microscope observation of the swarming phenomenon of Vibrio parahaemolyticus.

Scanning electron microscopy was used to study the production of lateral flagella and the swarming phenomenon in Vibrio parahaemolyticus. Differences in the size and diameter of the sheathed, polar flagellum and lateral flagella were apparent in these preparations. Swarming of V. parahaemolyticus was found to be similar to the swarming of Proteus spp. in that swarm cells which are heavily flagellated and elongated are formed.     

Zoospore encystment and pathogenicity of Phytophthora and Pythium species on plant roots

Seven plant species (lucerne, maize, oat, sugarbeet, sorghum, tomato, wheat) and 12 Pythium and Phytophthora species were used in a comparative study designed to investigate the effects of plant and oomycete inter-specific variation on zoospore encystment density and pathogenicity. Zoospores showed differential encystment behaviour and they encysted more on dicotyledonous than on monocotyledonous plants. Pythium aphanidermatum, P. deliense, and Phytophthora nicotianae were the most aggressive species. Sugarbeet was the most severely attacked plant species followed by tomato while oat plants were relatively unaffected. The relationship between zoospore encystment on roots and disease severity depended on the oomycete-plant combination. Correlation analysis between zoospore encystment density and disease severity indicated low and no significant levels (p.05) of association for most plant-oomycete combinations.     

Scanning electron microscope observations of Amoeba proteus during phagocytosis.

Phagocytosing Amoeba proteus at different stages of forming foodcups have been observed by scanning electron microscopy. A nonphagocytosing ameba is characterized by dorsal and lateral ridges running longitudinally over the posterior half of the cell and its attachment to the substrate over small areas. When stimulated by prey organisms, the ameba loses polarity and ridges, and adheres to the substrate more firmly over a wider area of contact. Then it forms broad pseudopods to surround its prey and this results in the formation of foodcups. The surface of all ameba is covered with small projections, and membranous blebs are often seen on the surface of phagocytosing organisms.     

康氏粉蚧蜡泌物的扫描电镜观察

采用制作玻片标本和扫描电镜技术研究了康氏粉蚧Pseudococcus comstocki(Kuwana)在不同发育阶段的显微形态特征及蜡泌物的超微形态。结果表明:三格腺是康氏粉蚧最主要的腺体,随着虫体的发育,数量增多,分布变广,每个腺孔分泌一根蜡丝,覆盖体表;管腺只在特定的时期分泌长的空心蜡管构成卵囊;多格腺分泌多棱形卷曲的小蜡丝粘附在卵粒上,防止其相互粘连。     

In vitro fusion of Acanthamoeba phagolysosomes. II Quantitative characterization of in vitro vacuole fusion by improved electron microscope and new light microscope techniques

To investigate the properties of phagolysosome (PL) fusion in Acanthamoeba homogenates, it was necessary to develop reliable methods for measuring in vitro PL fusion. The need to distinguish PL fusion from PL adhesion was met by the development of a quantitative electron microscope assay. Initial characterization of the fusion reaction by this method was followed by the development of a more rapid light microscope assay. Results obtained by the two methods were found to be in close agreement. By use of these new techniques, the in vitro PL fusion reaction was demonstrated to occur in a quantitatively reproducible manner. Under the present conditions employed, PL breakdown was not detected at any time during the in vitro incubation, while PL fusion was observed to proceed linearly for approximately 10 min, at which time the reaction ceased. Incubation of mixtures of two distinct PL types resulted in increases in hybrid PL types that were paralleled by decreases in nonhybrid PL types. The relative changes in PL concentrations observed were quantitatively consistent with PL fusion occurring randomly with respect to PL type. PL fusion was strongly inhibited by low concentrations of KF (50% inhibition at 2.7 mM), and by approximately tenfold higher concentrations of KCl, while KCN and 2,4-dinitrophenol (2,4-DNP) had little effect. In addition to further defining the nature of the PL fusion reaction in this system, these results demonstrate that, by use of the techniques described, quantitative study of the biochemical properties of this reaction is now possible.     

Protein synthesis during encystment of Azotobacter vinelandii.

Proteins synthesized during the encystment of Azotobacter vinelandii were radiolabeled with [35S]methionine and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Pulse labeling was used to demonstrate that early encystment-specific proteins were beginning to be synthesized at 2 h and reached peak levels about 12 h after initiation of encystment. One such protein was identified as a beta-ketoacyl acyl-carrier protein synthase. The concentration of early proteins began to decrease at 16 h, when intermediate proteins specific to the differentiation process began to be synthesized. The cessation of synthesis of intermediate proteins began at 20 h postinitiation, and the labeling pattern of proteins then remained constant throughout the remaining 4 days of encystment.     

Scanning electron microscope observation of the dog mesenteric lymph node

Summary The perfused large mesenteric lymph node of the dog was observed under the scanning electron microscope.The lymph sinus contains reticulum cells which mostly are two-dimensionally formed stellate plates oriented in a uniform direction. Large round macrophages are loosely fixed by the reticulum cell processes. No intermediate type between both cells has been observed. Macrophages having a few long tentacle-like projections are densely covered by clubbed cytoplasmic processes. Smaller round cells, probably plasma cells and lymphocytes also remained in the sinus.The pulp of the node is built up by reticulum cells, much smaller than those in the sinus, and by densely packed round cells including a few macrophages.The trabeculae and the reticulum of the nodal parenchyme form a continuous structure.Cordial thanks are expressed to Dr. Toshihiro Ishii, Professor of Anatomy of the Tohoku University Medical School, for his valuable advice. Thanks are also due to the kind cooperation of Mr. Akira Kubotsu of the Central Research Laboratories, Kuraray Co. Ltd.     

Release of highly elicitor-active glucans by germinating zoospores of Phytophthora megasperma f. sp. glycinea

An in-vitro culture system allowing the simultaneous germination of cysts was used to study the early host-independent release of phytoalexin elicitors by Phytophthora megasperma f. sp. glycinea, a soybean pathogen. Significant elicitor activity could be detected in the culture medium as early as 2 h after germination of P.m. f. sp. glycinea, race 1, cysts. The phytoalexin elicitor was heat-stable and heterogeneous in size. The apparent molecular mass ranged from 3 to 80 kDa. Anion exchange and lectin-affinity chromatography followed by sugar analysis confirmed that the elicitor activity resided primarily in glucans. The time course of elicitor release could then be accurately monitored by means of a competitive radioligand-displacement assay using the -glucan elicitor-binding sites of soybean (Glycine max (L.) Merr.) membranes. Linkage-composition analysis of the glucan elicitors showed that they were primarily (1 3)-linked with (1 6)--branches, a composition similar to that of glucans obtained by heat release from mature mycelium but different from that of elicitors obtained by acid hydrolysis or from spontaneous autohydrolytic release by senescent cultures. The naturally released elicitors displayed a biological activity in soybean cotyledon bioassays higher than purified acid-hydrolysed glucan elicitor or than the hepta-(1 3, 1 6)--glucoside, the smallest known carbohydrate elicitor for soybean. The present results demonstrate that elicitor release from the pathogen and perception by the potential host can take place in this system as early as during germ-tube formation and independent of the presence of host-produced endoglucanases.Abbreviations EC₅₀ effector concentration necessary for halfmaximal response - GC-MS gas chromatography-mass spectrometry - HG-APEA 1-[2-(4-aminophenyl)ethyl]amino-1-[hexagluco-syl]-deoxyglucitol - IC₅₀ inhibitor concentration necessary for half-maximal inhibition - P.m. f. sp. glycinea Phytophthora megasperma f. sp. glycinea - V₀ void volume Deceased on March 19, 1990This work was supported by the Deutsche Forschungsgemeinschaft (SFB 206). We thank Dr. H. Mayer, C. Warth and D. Borowiak (Max-Planck-Institut für Immunbiologie, Freiburg) for helpful discussions and experienced technical assistance (glucosyl-linkage analysis).     

A scanning electron microscope study of microvascular anastomoses.

The abdominal aortas of 30 rats were sutured under an operating microscope. The results were studied under a scanning electron microscope at 8 different periods after operation, ranging from 3 minutes to one month. The observations are presented.