Calcium control of differentiation in Phytophthora palmivora

A method has been developed for the preparation of zoospores from Phytophthora palmivora which allows the ionic composition of the suspension medium to be closely controlled. Sub-micromolar concentrations of calcium ions have been shown to play a key role in maintaining the zoospore state and in the transition to the cyst stage. Restriction of free Ca²⁺ to between 0.2 and 1 M resulted in zoospores which could be maintained for several hours before they finally encysted and germinated. When exposed to citrus-pectin, or 3 mM SrCl₂, or to vigorous shaking, these zoospores underwent rapid synchronous encystment. At free Ca²⁺ concentrations below 0.1 M, zoospores lysed slowly. If exposed to inducers of encystment before lysis had occurred, the zoospores failed to respond to pectin or to vigorous shaking. However, they did differentiate in response to SrCl₂ addition. Provided the free Ca²⁺ was maintained between 0.02 and 0.2 M, zoospores survived gentle centrifugation, a procedure which previously had resulted in encystment.Abbreviations IM (ion-mix) release medium containing 100 M KCl, 10 M CaCl₂, and 10 M MgCl₂     

Formation of multinucleate zoospores inPhytophthora infestans (Mont.) De Bary (Oomycetes,Pythiaceae)

Summary Zoospore formation in sporangia ofPhytophthora infestans, the causal agent of potatoes downy mildew disease, was influenced by reduction of oxygen tension, by application of a respiratory poison (KCN), and by an inhibitor of cytokinesis (cytochalasine B). As demonstrated by different methods of light microscopy, in all three cases the percentage of morphologically abnormal, multinucleate, multiflagellate cells increased. High concentrations of KCN caused a complete inhibition of zoospore formation. These effects are interpreted as disorders of the process of cytokinesis during zoosporogenesis.     

Changes in the lysosome structure during the formation of zoospores inTrebouxia potteri

Summary Changes in the lysosome structures were examined by electron microscopy during the formation of zoospores inTrebouxia potteri. Lysosomes in vegetative cells were homogeneously filled with electron-dense material. At the beginning of zoospore formation, lysosomes invaginated or evaginated to take up mitochondria, ER, or cytoplasmic ground plasma. The ingested organelles became disorganized within the lysosomes. During this disruption of these organelles, the lysosomal contents became heterogeneous, suggesting a decrease in the amount of enzymes within the lysosomes. Golgi bodies and ER seemed to be involved with the disruption of the organelles, probably supplying some substances necessary for the functioning of the lysosomes. Amount of electron-dense materials decreased and, finally, only one to three small spherical aggregates remained in the lysosomes. Then the lysosomes appeared to shrink via loss of watery substances or cutting off of electron-transparent regions. After these changes in lysosome structure, nuclei started to divide successively for formation of the zoospores. The possibility is proposed that the drastic cytoplasmic changes operated by lysosomes trigger the following morphogenetic events in the formation of zoospores.Abbreviations ER endoplasmic reticulum - TGN trans Golgi network     

Characterisation of the water expulsion vacuole inPhytophthora nicotianae zoospores

Summary The water expulsion vacuole (WEV) in zoospores ofPhytophthora nicotianae and other members of the Oomycetes is believed to function in cell osmoregulation. We have used videomicroscopy to analyse the behaviour of the WEV during zoospore development, motility and encystment inP. nicotianae. After cleavage of multinucleate sporangia, the WEV begins to pulse slowly but soon attains a rate similar to that seen in motile zoospores. In zoospores, the WEV has a mean cycle time of 5.7 ± 0.71 s. The WEV continues to pulse at this rate until approximately 4 min after the onset of encystment. At this stage, pulsing slows progressively until it becomes undetectable. The commencement of WEV operation in sporangia coincides with the reduction of zoospore volume prior to release from the sporangium. Disappearance of the WEV during encystment occurs as formation of a cell wall allows the generation of turgor pressure in the cyst. As in other organisms, the WEV inP. nicotianae zoospores consists of a central bladder surrounded by a vesicular and tubular spongiome. Immunolabelling with a monoclonal antibody directed towards vacuolar H⁺-ATPase reveals that this enzyme is confined to membranes of the spongiome and is absent from the bladder membrane or zoospore plasma membrane. An antibody directed towards plasma membrane H⁺-ATPase shows the presence of this ATPase in both the bladder membrane and the plasma membrane over the cell body but not the flagella. Analysis of ATPase activity in microsomal fractions fromP. nicotianae zoospores has provided information on the biochemical properties of the ATPases in these cells and has shown that they are similar to those in true fungi. Inhibition of the vacuolar H⁺-ATPase by potassium nitrate causes a reduction in the pulse rate of the WEV in zoospores and leads to premature encystment. These results give support to the idea that the vacuolar H⁺-ATPase plays an important role in water accumulation by the spongiome in oomycete zoospores, as it does in other protists.Abbreviations BMM butyl methylmethacrylate - F fix 4% formaldehyde fixation - GF fix 4% formaldehyde and 0.2% glutaraldehyde fixation - V-ATPase vacuolar H⁺-ATPase - WEV water expulsion vacuole     

Effect of phosphate on the toxicity of phosphite in Phytophthora palmivora

The concentration of P_i in the growth medium had a marked effect on the amount of the fungicide phosphite accumulated by Phytophthora palmivora. The mass of mycelium, produced after 7 days growth in a medium containing excess P_i, was not markedly inhibited until phosphite concentrations of 10 mM or greater were supplied. In contrast, in a P_i-deficient medium, growth was inhibited by 0.1 mM phosphite. In the latter medium, the fungus took up significantly more phosphite once P_i had been depleted from the medium (from day 4 to 6). When 7 day old mycelium was presented with either 1 mM P_i or phosphite, the P_i-deficient mycelium transported either anion at almost eight times the rate found in the P_i-excess mycelium. The level of alkaline phosphatase present in the P_i-deficient mycelium was also significantly elevated.     

Elemental analysis of particles in fungal zoospores

The elemental composition of gamma particles in zoospores ofAllomyces macrogynus andBlastocladiella emersonii was determined by use of energy dispersive X-ray analysis of glutaraldehyde fixed, thin section zoospores. Isolated preparations of gamma particles were also examined. Gamma particles contained no detectable elements. Similar sized, globular, electron dense cytoplasmic inclusions contained phosphorus and calcium. We suggest that previous studies assigning calcium and phosphorus to gamma particles may have been based on confusion of these two types of cytoplasmic inclusions.     

Characterising adhesiveness ofPhytophthora cinnamomi zoospores during encystment

During encystment,Phytophthora cinnamomi zoospores bind firmly to the host surface. We have developed a microassay to study adhesion of the zoospores to solid surfaces, both biological and non-biological. The results show that timing of the acquisition of adhesiveness during encystment correlates closely with the secretion of high molecular weight glycoproteins. The adhesive phase is short lived, occurring between 1 and 4 min after induction of encystment. During this period, cells that come into contact with a variety of surfaces (glass, plastic, and onion epidermis) become firmly attached, while cells that come into contact with one of these substrata after this period are unable to bind. Our results also show that EGTA inhibits cyst adhesion, while addition of calcium promotes cyst adhesion, especially of cysts more than 4 min old. To help identify the cyst surface component involved in adhesion we tested a number of lectins for their ability to block cyst adhesion. Soybean agglutinin andHelix pomatia agglutinin, lectins which bind to the secreted high molecular weight glycoproteins, both inhibit adhesion in the presence and absence of the hapten sugar, indicating that inhibition was non-specific. Wheatgerm agglutinin, a lectin which does not bind to the cyst surface, also blocked adhesion non-specifically.     

Ultrastructure of chytridiomycete and oomycete zoospores using spray-freeze fixation

Summary The ultrastructure of zoospores of several zoosporic fungi was examined using a modified cryofixation technique. An atomizer was used to spray a zoospore suspension into the cold propane reservoir of a conventional plunge freeze-substitution apparatus. Spray-freeze fixation and freeze-substitution of zoospores porvided better fixation of vacuolar structures, membranes and the extracellular coat than that obtained with chemical fixation. The overall shape of cryofixed spores was closer to that seen in living zoospores. Two types of vacuoles were seen in cryofixed zoospores ofMonoblepharella andChytridium. One type of vacuole contained electron-opaque material within the lumen while the other type had no visible internal material in the lumen and appeared to be part of the water expulsion vacuole complex. Coated pits and coated vesicles were observed associated with both the water expulsion vacuoles and the plasma membrane inMonoblepharella andPhytophthora, suggesting that endocytosis of the plasma membrane and expulsion vacuoles is part of membrane recycling during osmoregulatory events. An extracellular coat was seen on the outer surface of cryofixed zoospores ofMonoblepharella sp.,Chytridium confervae andPhytophthora palmivora without the use of carbohydrate-specific stains. The spray-freeze method gave good and reproducible fixation of the wall-less spores in quantities greater than those obtained in previously described zoospore cryofixation studies. The technique is potentially useful for cell suspensions in that freeze damage from excess water is limited.Abbreviations ddH₂O deionized distilled water - PME Pipes/MgCl2/EGTA buffer - WEV water expulsion vacuole     

Localization of calmodulin in flagella of zoospores ofPhytophthora cinnamomi

Summary Calmodulin distribution in the tinsel and whiplash flagella of zoospores ofPhytophthora cinnamomi has been studied by immunofluorescence microscopy and immunogold labelling. In whole zoospores labelled with a monoclonal antibody raised against pea calmodulin, followed by a second antibody-FITC, both flagella appear to be weakly stained except for a region at the base of the tinsel flagellum which was stained intensely. A similar staining pattern was also detected in isolated flagella labelled with anti-calmodulin. To identify the calmodulin rich region of the tinsel flagellum, we labelled sections of zoospores embedded in Lowicryl K4M with anti-calmodulin followed by a second antibody gold probe. In the tinsel flagellum, the gold labelling was restricted to a paraxonemal swelling close to the base. Very little gold labelling was detected elsewhere. The swelling extends for 1.5–2.0 n from the base of the tinsel flagellum and is hook shaped in cross section. Immunoblot analysis confirmed that the staining was specific for calmodulin.     

Observations of the water expulsion vacoule ofPhytophthora palmivora

Summary Characteristic changes in the ultrastructure of the green algaScenedesmus armatus, grown in batch culture in the presence of aqueous fuel-oil extract (AFOE) have been observed. The changes affected mainly chloroplasts and mitochondria. The regular arrangement of the thylakoid stacks became distorted and the whole chloroplast lobed. Plastoglobules were more numerous in the treated cells than in the controls, especially after long-term exposure to AFOE.The mitochondrial matrix cells exposed to AFOE were more electron-translucent. An increase in the number of small mitochondrial profiles was observed after prolonged treatment with AFOE.The number and size of osmophilic bodies increased markedly in the cytoplasm of the treated cells. The cytochemical reaction of these bodies with Sudan black B indicated their lipid composition.Plasmalemma invagination into the cytoplasm and vacuoles, cytoplasmic layers, and an increase in size of the vacuolar compartment were observed in cells exposed to AFOE for a long time.The possibility that detoxification, involving microbody activity, may have occurred inScenedesmus is suggested.Abbreviation AFOE aqueous fuel-oil extract     

Polyphosphate granules in encysted zoospores of Rozella allomycis

Methods of ultracytochemistry and of X-ray energy dispersive analysis have been used to demonstrate that the gamma-like granules in encysted zoospores of the chytrid Rozella allomycis contain polyphosphate. The possibility that cysts contain two classes of polyphosphate granules which differ in structure, in function, and in origin is discussed.     

Sporangial structure inPhytophthora is disrupted after high pressure freezing

Summary The effects of high pressure on the ultrastructure of sporangia ofPhytophthora cinnamomi andP. palmivora have been examined by comparing sporangia frozen in a Balzers hyperbaric freezer or pressurized in a French pressure cell with sporangia plunge frozen at ambient pressure. Both freeze fixation methods provided excellent preservation of most cell structures, but one organelle type seen in plunge frozen material, the large peripheral vesicle (LPV), was not observed in high pressure frozen sporangia. Instead, these sporangia contained large irregularly shaped structures which exhibit the patterns of spatial distribution and, forP. cinnamomi, the monoclonal antibody binding characteristic of LPVs. These findings suggest that some factor of the hyperbaric freezing process causes LPVs to be degraded. Sporangia ofP. cinnamomi that had been pressurized in a French pressure cell also exhibited large structures with the spatial distribution and monoclonal antibody binding characteristic of LPVs. The apparent expansion of LPVs that follows from both pressurizing treatments causes considerable passive disruption of sporangial structure. This is the first report of a major disturbance of cell structure from use of the Balzers hyperbaric freezer, and reflects the lability, noted in previous work, of LPVs inPhytophthora.     

Action spectra for phototaxis in zoospores of the brown algaPseudochorda gracilis

Summary Action spectra for phototaxis in zoospores of brown alga,Pseudochorda gracilis (Laminariales), were examined in the wavelength range between 300 and 600 nm using the Okazaki Large Spectrograph and a video tracking system. The direction of swimming (both in percent cells swimming in parallel with the stimulating light, and in mean angle of cell movement) was dependent on the wavelength. The action spectra had two peaks at 420 and 460 nm, while light above 500 nm was not effective in changing the swimming direction of the cells.Abbreviations TCMA tracker-cell movement analyzer system - CMA cell movement analyzer program     

Properties of the phosphate and phosphite transport systems of Phytophthora palmivora

Germlings of Phytophthora palmivora possess at least two systems for the uptake of inorganic phosphate (P_i). The first is synthesized on germination in medium containing 50 M P_i and has a K_m of approx. 30 M (V_max=7–9 nmol P_i/h·10⁶ cells). The second is synthesized under conditions of P_i-deprivation and has a higher affinity for P_i (K_m=1–2 M), but a lower V_max (0.5–2 nmol P_i/h·10⁶ cells). The fungicide phosphite likewise enters the germlings via two different transport systems, the synthesis of which also depends on the concentration of P_i in the medium. The K_m of the lower affinity system is 3 mM (V_max=20 nmol phosphite/h·10⁶ cells) and that of the higher affinity system is 0.6 mM (V_max=12 nmol/h·10⁶ cells). P_i and phosphite are competitive inhibitors for each other's transport in both systems. However, whereas mM concentrations of phosphite are necessary to inhibit P_i transport, only M concentrations of P_i are required to inhibit phosphite transport. A third system of uptake for P_i also exists, since when phosphate-deprived cells are presented with mM concentrations of P_i, they transport the anion at a very high rate (around 100 nmol/h·10⁶ cells). High rates of transport of phosphite are also observed when these cells are presented with mM concentrations of this anion.     

Cell wall formation in zoospores of Allomyces arbuscula

Carbohydrate composition was determined in isolated cell walls of meiospores of Allomyces arbuscula after incubation for 15 min (encysted meiospores: cysts), 150 min (germlings: cysts + rhizoids) and 24 h (cysts + rhizoids + hyphae). The principal constituent in all cell wall samples is chitin, accounting for about 75% of the recovered carbohydrates. In addition, cell walls of all stages examined contain polysaccharides which release galactose, glucose, mannose, arabinose, xylose, fucose, and rhamnose on acid hydrolysis. While different developmental stages show minor quantitative changes in chitin, the ratio of galactose to glucose decreases sharply during differentiation of ungerminated cysts into germlings with rhizoids and hyphae. The increase in glucose is accompanied by a decrease in the amount of xylose and/or fucose and of galactose.List of Abbreviation TFA trifluoroacetic acid     

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).     

Encystment of zoospores of the fungus,Phytophthora cinnamomi,is induced by specific lectin and monoclonal antibody binding to the cell surface

Summary Only two of a number of macromolecules that bind to the surface of zoospores of the dieback fungus,Phytophthora cinnamomi, induce encystment when added to a suspension of actively swimming zoospores. One, the lectin Concanavalin A (ConA), binds to the entire surface of the zoospores including the surface of both flagella. Within 10 minutes more than 70% of the cells have encysted in the presence of 5 g/ml ConA. This encystment is inhibited by preincubation of the lectin with its hapten sugar, -methyl-D-mannoside. The other effective molecule, a monoclonal antibody designated Zf-1, is one of 35 that have been raised to components on the surface of zoospores and cysts ofP. cinnamomi. The antigen for Zf-1 occurs only on the surface of the two flagella. Purified Zf-1 at 15 g/ml causes encystment of 75% of the zoospores in 13minutes. To show that the induction of encystment by these two probes is not due simply to the presence of protein either in solution or bound to the zoospore a number of other proteins were tested, including other antibodies that bind to the zoospore surface. None of these other molecules caused encystment even at concentrations greater than 200 g/ml. The results are consistent with the surface components that bind ConA and Zf-1 being involved in the critical step of triggering encystment at the surface of a potential host during infection.     

Additive inheritance of resistance to pod rot caused by Phytophthora palmivora in cocoa

Summary Quantitative inheritance of resistance to Phytophthora pod rot (Ppr) was studied in cocoa hybrid progeny from 12 Trinitario x Amazonian crosses and their reciprocal crosses. The crossing scheme was similar to a factorial design. Disease was assessed by the number and percentage of infected pods on each tree. Highly significant differences due to general combining abilities (GCA) were obtained for all characters, except for the GCA of Trinitario on total pod production. Differences for specific combining ability (SCA) were not significant for all characters. There were no significant differences between reciprocal crosses. The Trinitario clone K82 provided the only source for the hybrid progenies of strong Ppr resistance to the hybrid progenies, while K20 provided moderate resistance. Other parental clones — KA2-101, KA5-201, KEE 2, KEE 5, and KEE 52 — produced progenies which were susceptible to Ppr. It is evident that resistance to Ppr in cocoa is inherited additively. Maternal and cytoplasmic effects were assumed to have no influence on inheritance of resistance. It is also concluded that resistance to Ppr of the kind shown by K82 is likely to be horizontal resistance. Breeding for high-yielding cultivars combined with Ppr resistance is the most effective way of controlling Ppr of cocoa on the crops of growers with small holdings in Papua New Guinea.     

Production of monoclonal antibodies against peripheral-vesicle proteins in zoospores of Phytophthora nicotianae

Summary. A coimmunisation protocol using microsomal fractions from Phytophthora nicotianae cells has enhanced the production of monoclonal antibodies directed towards proteins produced during asexual sporulation. Over 40% of the antibodies targeted three categories of zoospore peripheral vesicles. Five antibodies label the contents of dorsal vesicles, with three of these reacting with two P. nicotianae polypeptides with a relative molecular mass of approximately 100 kDa. Two antibodies label the contents of large peripheral vesicles and react with two very high-molecular-weight polypeptides in extracts of P. nicotianae cells. These antibodies cross-react with the contents of large peripheral vesicles in P. cinnamomi zoospores. Ten antibodies label the contents of P. nicotianae zoospore ventral vesicles and react with a single polypeptide with a relative molecular mass of 230 kDa. A number of these antibodies against the contents of ventral vesicles in P. nicotianae zoospores cross-react with ventral-vesicle proteins in P. cinnamomi cells in immunofluorescence and immunoblot assays. The study illustrates the value of the coimmunisation protocol and has produced antibodies that could be instrumental in the cloning of genes encoding peripheral-vesicle proteins.Correspondence and reprints: Plant Cell Biology Group, Research School of Biological Sciences, Australian National University, GPO Box 475, Canberra, ACT 2601, Australia.     

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.