Potassium homeostasis influences the locomotion and encystment of zoospores of plant pathogenic oomycetes




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Revised for Fungal Genetics and Biology: 3rd November, 2004

Potassium homeostasis influences the locomotion and encystment of zoospores of plant pathogenic oomycetes
Alex A. Appiah, Pieter van West, Meave C. Osborne, and Neil A.R. Gow
Aberdeen Oomycete Group,

College of Life Sciences and Medicine,

University of Aberdeen, Foresterhill,

Aberdeen, AB25 2ZD,

Scotland, UK.

Author for correspondence: Prof. Neil AR Gow

Tel. +44 1224 555879

Fax. +44 1224 555844

E-mail: n.gow@abdn.ac.uk


Present address: Environmental Entomology and Plant Pathology,

Central Science Laboratory (Defra), Sand Hutton, York, North Yorkshire, YO41 1LZ, England, UK.


Short title: Potassium ion regulation of zoospore swimming
Keywords: Oomycetes; ionophores; Phytophthora infestans; Phytophthora megakarya; Phytophthora palmivora; Pythium aphanidermatum; Pythium dissotocum; zoospore behavior;

Abstract
Zoospores of plant pathogenic oomycetes exhibit distinct swimming speeds and patterns under natural conditions. Zoospore swimming is influenced by ion homeostasis and changes in the ionic composition of media. Therefore, we used video microscopy to investigate swimming patterns of five oomycete species in response to changes in potassium homeostasis. In general, zoospore speed tended to be negatively correlated with zoospore size. Three Phytophthora species (Ph. palmivora, Ph. megakarya and Ph. infestans) swam in straight patterns with speeds ranging from 50-250 m/sec whereas two Pythium species (Py. aphanidermatum and Py. dissotocum) swam at similar speeds ranging from 180-225 m/sec with a pronounced helical trajectory and varying amplitudes. High external concentrations of potassium salts reduced the swimming speed of Ph. palmivora and induced encystment. This was not observed for Py. aphanidermatum. Application of the potassium ionophores gramicidin, nigericin and valinomycin resulted in reduced swimming speeds and changes in the swimming patterns of the Phytophthora species. Therefore potassium ions play a key role in regulating zoospore behavior.

1. Introduction
The group of Oomycetes (kingdom: Stramenopila) contains many economically important plant pathogens, particularly within the genera of Pythium, Phytophthora and Aphanomyces. Diseases caused by Oomycetes account for multibillion dollar annual losses of many important food and cash crops, ornamental plants and forest species worldwide (Erwin and Ribeiro, 1996; van West et al., 2003). Oomycetes produce free-swimming zoospores, which are agents for dispersal and infection of new hosts. Released zoospores swim in films of water in search of host tissues (seeds, roots, stems or leaves). Combinations of tactic signals help them to localize suitable infection sites (Gow et al., 1999; van West et al., 2003). In the rhizosphere, the tactic responses occur toward gradients of nutrient exudates that exist at root tips or around wound sites (Ho and Hickman, 1967; Hickman, 1970; Carlile, 1986; Morris & Ward, 1992; Deacon and Donaldson, 1993; Gow et al., 1999). In addition, electrotaxis towards electric gradients generated by plant roots in the rhizosphere has been shown to be an important influence in taxis towards and away from the root surface (Morris and Gow, 1993; Morris et al., 1995; van West et al., 2002).

There is accumulating evidence that calcium signaling is central to zoospore taxis and encystment (Byrt et al., 1982; Irving et al., 1984; Iser et al., 1989; Donaldson and Deacon, 1992; 1993a; Deacon and Donaldson, 1993; Reid et al., 1995; reviewed by van West et al., 2003). In many motile eukaryotes, calcium ion channels are involved in the regulation of motility and the conductance of the channel is in turn related to the membrane potential of the cell (Hinrichsen and Schultz, 1998; Nultsch and Dolle, 1987). Because the membrane potential is also regulated in most cells by potassium ion fluxes and so we wished to test the hypothesis that potassium ions regulate zoospore motility.

Donaldson and Deacon (1992; 1993b) reported that up to 1 mM Na+, K+ and Li+ had little effect on the swimming of zoospores and the germination of cyst of three Pythium species. However, Thomas and Butler (1989) reported that 1 mM-K+ caused zoospores of Achyla to slow down and swim in circular pattern. The role(s) of monovalent cations in zoospore biology therefore remains to be clarified. Here we examine the role of K+ ions in the locomotion of oomycete zoospores by video microscopy and image analysis. We conclude that modulation of cytoplasmic K+ strongly influences swimming and turning of zoospores, but that distinct genus-specific differences exist in the effects of alterations in K+ homeostasis on zoospore swimming.
2. Materials and methods
2.1. Culturing of isolates and zoospore production
Phytophthora palmivora and Phytophthora megakarya isolates were grown on 20% V8 agar media [200 ml Campbells V8 juice, 3 g CaCO3, 15 g micro agar (Duchefa, Haarlem, The Netherlands), 3 mg cholesterol and 800 ml de-ioinised water]. Isolates of Ph. palmivora (P6039) and Ph. megakarya (AKA24) were incubated in 9 cm Petri dishes at 26oC in the dark for 2 days, then under continuous light for a further 2-3 days (for Ph. palmivora) or 5-8 days (for Ph. megakarya). Motile zoospores of Ph. palmivora and Ph. megakarya were obtained by pouring 10 ml of 2 mM sodium phosphate buffer (pH 7.2) over mycelial cultures and gently scraping colonies with a sterile glass rod to release sporangia. The suspension of sporangia was placed in a beaker that was then placed in a –20ºC freezer for 4 min. After the cold-shock the suspension was left at room temperature (19-22 ºC) for 30 min, during which time zoospores were released. The average number of zoospores generated in such cultures was approximately 106 zoospores per ml. The zoospore densities were determined by an empirical relationship (Reid et al., 1995). The zoospores were filtered through Whatman No1 paper to remove sporangial and hyphal debris.

Isolates of Pythium aphanidermatum and Pythium dissotocum were grown on 10% V8 agar medium (100 ml Campbells V8 juice, 15 g micro agar (Duchefa, Haarlem, The Netherlands) and 900 ml de-ioinised water) and were incubated at 26oC in the dark for 4-5 days (for Py. aphanidermatum) and 7 days (for Py. dissotocum). Zoospores of Py. aphanidermatum and Py. dissotocum were obtained by placing cut strips of growing cultures on agar (15 mm squares) in Petri dishes. The strips were flooded with 20 ml of 2 mM sodium phosphate buffer (pH 7.2) and left at room temperature (19-22oC) for 4-6 h. The average number of zoospores released was approximately 104 zoospores per ml. Suspensions were filtered to remove debris and the zoospore densities were determined as described above.

An isolate of Phytophthora infestans (88069) was grown in the dark for 12 days on rye agar medium supplemented with 2% (w/v) sucrose (Caten and Jinks, 1968) at 18oC. Zoospores were released by flooding culture plates with 10 ml of cold 2 mM sodium phosphate buffer (pH 7.2) and the sporangia were gently dislodged at the surface of the agar using a sterile glass rod. The flooded plates were incubated for 3 h in a cold room at 4oC to release the zoospores. The suspension was filtered and the density adjusted to approximately 106 zoospores per ml.
2.2. Swimming characteristics of zoospores of different oomycete species
The swimming speed and trajectories of individual zoospores were measured using video-microscopy and coupled image analysis programs. Swimming speed has been reported to be influenced by temperature and an increase in temperature from 15C to 26C caused a corresponding increase in speed from 122 ± 32 to 232 ± 43 m/sec (Ho and Hickman, 1967). We therefore performed all experiments at a room temperature of 18ºC (range 19-22oC).

The characteristics of the different zoospores including dimensions (length and breadth) and their swimming behavior (speed and motility pattern) were measured while swimming in 2 mM sodium phosphate buffer (pH 7.2), which was the buffer used for zoospore discharge. The length and breadth of 10 randomly selected zoospores from untreated zoospore suspensions (control) of each species were measured using an image analysis program (below). The length/breadth (l/b) ratios were calculated. The natural average speed range for each species was obtained from approximately 30 individual zoospores that were tracked (10 from each of the 3 control treatments recorded for the 3 different ionophore experiments).


2.3. The role of different cations in zoospore motility
Initial experiments examined the effects of different salt concentrations (NaCl, KCl, CaCl2, K2SO4 and Na2SO4) on zoospore motility of Phytophthora palmivora (P6039) and Pythium aphanidermatum. The pH of solutions was checked to maintain a constant pH of 7.2. Control experiments showed that the buffer composition did not affect the behavior of Ph. palmivora or Py. aphanidermatum zoospores. For CaCl2, the zoospores were released into 2 mM Tris buffer pH 7.2 to prevent Ca-phosphate precipitation. Zoospore swimming behavior after treatments were recorded and analyzed using an imaging system comprising of a Zeiss (Axioplan2) upright microscope, mounted with a Hamamatsu digital camera (C4742-95) connected to a G3 Applemac computer housing the image analysis software package (Openlab, Improvision Ltd., Coventry, UK). Vaseline was smeared onto microscope slides in a square shape to create a central well into which the zoospore suspensions of treatments could be placed. A coverslip was then applied to form a well of zoospores in which movements could be observed microscopically. This arrangement ensured there was sufficient medium depth for the zoospores to swim freely without frequent collisions with the slide or cover slip. Zoospores were able to swim freely (for at least 1 h in control treatments), in about 100 l of liquid. The slides for each treatment were transferred quickly to the microscope stage and movements were recorded using an automated burst capture macro. A series of 50 consecutive photographic frames of the swimming zoospores were taken at high speed over an interval of approximately 5.5 sec. The effect of light and temperature on zoospore swimming was kept to the minimum by using the lowest possible brightness. Recordings were made a soon as possible over a recording time of 5.5 sec. The swimming patterns of individual zoospores were then tracked from recordings and their swimming speeds were calculated automatically over 15-20 frames.
2.4. Effect of potassium ionophores on zoospore behavior
To further examine the role of K+ on the motility of zoospores from Phytophthora and Pythium, the K+ ionophores gramicidin, nigericin and valinomycin were added to zoospore suspensions. Potassium ionophores were initially dissolved in 100% ethanol to a concentration of 1 mg/ml and then tested at working concentrations of 0, 0.5, 2.0, 4.0 and 10 g/ml. These concentrations were similar to those used in other studies investigating fungal and mammalian cells (Buchan et al., 1993; Donaldson and Deacon, 1993b) and were considered to cause non-specific changes in cell physiology. The effect on zoospore motility (speed and trajectory) was analyzed using the computer-based imaging system described above. The external concentration of K+ ions in buffered media was determined by flame photometry. Differences in the external K+ ion concentration in zoospore suspensions reflected the differences in potassium ion content of the culture media on which they were grown.
2.5. Statistical Analyses
The statistical analysis was conducted with GraphPad Prism Version 3.0 statistical software (GraphPad Software, San Diego, CA, USA). Means of samples with standard errors of the means ( SEM) were calculated. Data was subjected to one-way analysis of variance at 95% confidence level (P  0.05) or higher to determine significant differences between treatments. Experiments showing significant differences were further subjected to the Newman-Kuels tests. All experiments for the different treatments were repeated on different days using independent cultures.
3. Results

3.1. Characteristics of zoospores of different oomycete species
The natural dimensions and zoospore motility characteristics of the different species of oomycetes used in the studies were determined. The zoospores of the species differed in sizes, motility pattern and significantly in their speed of movement (Table. 1). Phytophthora infestans zoospores (Fig. 1A) were the largest in size (length 17.49 ± 3.47 (mean  s.d.) and breadth 11.39 ± 0.65 m) compared to the other species. Zoospores of Ph. megakarya zoospores (Fig. 1B) were the smallest (10.74 ± 0.97 and 7.74 ± 0.58 m). However, the zoospores of the different species were similar in their length/breadth ratios (1.4 - 1.6) with Py. aphanidermatum having the highest l/b ratio (1.6). The zoospore swimming speed of the species (except Ph. megakarya) correlated approximately with zoospore diameter. Ph. infestans zoospores had the largest zoospores and the slowest speed (ranging between 50 – 105 m/sec) while the smaller zoospores of Ph. palmivora (180 – 250 m/sec) and Py. dissotocum (198 – 225 m/sec) swam faster. The swimming pattern of the Phytophthora species predominantly followed straight trajectories while the Pythium species swam in helical trajectories with varying amplitudes. However, zoospores of Ph. infestans and Ph. megakarya, showed increased frequency of turning at very sharp angles when they approached or collided with other zoospores.
3.2. Effects of cations on zoospore motility
The effects of additions of different salts (0-10 mM) on zoospore motility of Py. aphanidermatum and Ph. palmivora was analyzed (Fig. 2). Both Na2SO4 and NaCl (10 mM) had little effect on the zoospore motility of Ph. palmivora (Fig. 2A) whereas CaCl2 reduced swimming speed from 172  6 m/sec to 128  11 m/sec. The swimming trajectory was not noticeably affected (data not shown). Addition of KCl reduced the swimming speed of Ph. palmivora, from 216  8.0 m/sec to 21  4.0 m/sec in a concentration-dependent manner. At 4 mM KCl zoospores of Ph. palmivora swam in perpetual circles (Fig. 3A). The addition of 10 mM KCl caused the zoospores to swim in a jerky fashion. K2SO4 also reduced swimming speed significantly at higher concentrations (Fig. 2A). The reduction in swimming speed of the Ph. palmivora zoospores was again accompanied by a change in their swimming pattern (Fig. 3A). It should be noted that high concentrations of potassium also increased zoospore encystment.

Addition of both KCl and K2SO4 did not cause significant changes in the swimming speed or pattern of Py. aphanidermatum zoospores, even at the highest potassium ion concentrations tested (Fig. 2B and 3B). The zoospores still swam in a helical or spiral motion.



3.3. Effect of potassium ionophores on zoospore behavior
The role of potassium on oomycete zoospores was further investigated by treating swimming zoospores with potassium ionophores. Gramicidin, nigericin and valinomycin solutions were applied to zoospore populations of five oomycete species, including Ph. palmivora, Ph. megakarya, Ph. infestans, Py. aphanidermatum and Py. dissotocum. The external concentrations of K+ ions in the 2 mM Na phosphate buffer was determined by flame photometry as 1.45  0.171 mM. The K+ ion concentration of media into which zoospores were discharged varied between 2.5 - 3.5 mM for media used for Phytophthora species and 5 - 6 mM for Pythium media. Eukaryotic microbes typically have an internal K+ concentration of 50-200 mM (Harold, 1987). Therefore the addition of ionophores to the bathing solution would result in net efflux of K+ from the cells.
3.4. Gramicidin

Gramicidin is a channel-forming ionophore that allows permeation of monovalent cations in a hydrated form down a concentration gradient (Rosenberg and Finkelstein, 1978). The ionophore is selective for K+ over Na+ and conductance is limited only by diffusion of the cation. This produced the most dramatic effect on the zoospore swimming speed and pattern in all species tested (Fig. 4). Even low concentrations of ionophore caused a significant reduction in speed in all species (P  0.05; at 0.5 g/ml). Gramicidin at 2.0 g/ml caused >80% reduction in speed of Ph. palmivora zoospores (Fig. 4) there was a concomitant change in swimming pattern from straight to a spiral motion (Fig. 5) and some zoospore lysis occurred. Further increases in gramicidin concentration caused zoospores to swim in more tight spiral motion and the swimming speed was further reduced. The most dramatic effect of gramicidin on zoospore swimming pattern was with Ph. megakarya where a strong circular and wave-like motion was observed even at the lowest tested concentration (0.5 g/ml) (Fig. 5). A significant (P  0.001) reduction in speed also occurred at this concentration, and approximately 50% of the zoospores encysted after 5 min of exposure without any associated cell lysis. Gramicidin had least effect on the swimming speed of Ph. infestans. The zoospores tended to swim more straight with little turning (Fig. 5). Little encystment of zoospores occurred even at higher gramicidin concentrations.

Gramicidin also caused a highly significant (P  0.0001) reduction in swimming speed of Py. aphanidermatum zoospores and significant (P  0.05) effects were observed on swimming speed and trajectory (Figs. 4 and 5). The effect of gramicidin on Py. dissotocum was similar to that seen for Py. aphanidermatum. A similar reduction of 85% in speed occurred at 2.0 g/ml but this was more abrupt in Py. dissotocum, and near instantaneous encystment of zoospores occurred at gramicidin concentrations above 2.0 g/ml. The swimming pattern of Py. dissotocum zoospores seemed unaffected at 0.5 g/ml gramicidin but swimming occurred in tight circles at 2.0 g/ml (Fig. 5).
3.5. Nigericin
Nigericin is a mobile carrier of ions in the plasma membrane that catalyses the electroneutral exchange of monovalent cations and protons. Efflux of potassium ions would therefore lead to concomitant acidification of the cytoplasm and toxicity due to perturbation of both K+ and H+ gradients across the zoospore membrane. The addition of nigericin to zoospore suspensions of Ph. palmivora caused a concentration-dependent decrease in swimming speed (Fig. 4). This decrease was statistically significant at all concentrations of nigericin investigated (P  0.0001). At  4 g/ml nigericin zoospores encysted more quickly (after less than 1 min exposure) and lysis was induced. Nigericin did not affect the straight swimming trajectory of Ph. palmivora, but induced a zig-zag motion with frequent sharp turning of straight-swimming zoospores (Fig. 6).

Nigericin did not have a marked effect on the speed and trajectory of Ph. megakarya zoospores at low concentrations (Fig. 4 and 6) but caused a significant decrease in the speed of swimming (P 0.001) at 4 and 10 g/ml. At these concentrations of nigericin, zoospores swam more erratically, turned more on contact with other zoospores and encystment was increased but no enhanced lysis occurred.

Nigericin treatments had less of an effect on the swimming speed of Ph. infestans zoospores, and significant differences (at least P  0.05) were only observed at highest ionophore concentrations. However, the swimming trajectory was affected markedly and zoospores swam in a rolling, twisting and turning motion, with frequent quick jerky or spinning movements and sharp turns when they collided with other zoospores. Encystment was not markedly effected - taking more than 10 min to induce 60% encystment at the highest concentrations.

The swimming speed and swimming pattern of Py. aphanidermatum zoospores were not immediately affected by nigericin (Figs. 4 and 6). It took more than 2 min even at relatively high concentrations before observable effects on swimming patterns could be observed. Likewise there was no dramatic effect of nigericin on the swimming speed of Py. dissotocum zoospores at lower concentrations of nigericin (Fig. 4). Large circular zoospore trajectories were observed in some zoospores when treated with 2 g/ml nigericin. Only 30% reduction in swimming speed was observed in both Py. aphanidermatum and Py. dissotocum at 2 g/ml nigericin.


3.6. Valinomycin
Valinomycin is a mobile carrier ionophore that catalyses the electrogenic uniport of monovalent cations and has a marked selectivity of K+ over Na+. Valinomycin treatment had a significant (P  0.0001) but mild effect on the swimming speed of Ph. palmivora zoospores up to 4 µg/ml treatment. It caused less than 30% reduction in speed at 2 µg/ml and there was no significant effect at 0.5 µg/ml (Fig. 4). The reduction in P. palmivora swimming speed was accompanied by an alteration in swimming pattern, with zoospores swimming in elliptical or circular patterns (Fig. 7). Higher zoospore encystment and significantly more lysis of zoospores were observed (within 5 min) with increasing concentrations of valinomycin when compared to gramicidin.

Similarly, valinomycin had a significant (P  0.0001) effect on Ph. megakarya zoospores. It caused 36% reduction in speed at 2 µg/ml and there was no significant difference between treatments above 2 µg/ml. A slight effect on swimming pattern was observed characterized by quick sharp turns on contact with other zoospores above 2 µg/ml treatment. Zoospore encystment and lysis increased in a concentration-dependent fashion.

Valinomycin significantly (P  0.0001) affected both swimming speed and pattern of Ph. infestans zoospores (Figs. 4 and 7). At 0.5 µg/ml an increase in the swimming speed occurred and the zoospores swam in a straight pattern (Fig. 7). The swimming speed was only slightly reduced with increasing concentration of ionophore concentration. Zoospores swam in a ‘dash-stop-dash’ manner and encysted early. There was also evidence of zoospore lysis induced by valinomycin.

Addition of valinomycin to Py. aphanidermatum zoospores produced a significant (P = 0.0015) reduction in the swimming speed. However, this effect was weak compared to those observed in the Phytophthora species. At 2.0 g/ml treatment less than 20% reduction in speed was obtained and significantly relevant differences between the control and other treatments were observed at 4 and 10 g/ml (Fig. 4 and 7). Zoospore swimming pattern was not affected greatly by valinomycin. Encystment of zoospores occurred slowly even at higher ionophore concentrations and lysis of zoospores was observed after longer exposures (within 10 min).

Similarly, valinomycin had little effect on the motility of Py. dissotocum zoospores. Significant (P  0.0001) effect of valinomycin on the swimming speed was only obtained at 10 g/ml. A slight effect on swimming pattern could be observed with some zoospores exhibiting circular motions at higher concentrations (Fig. 7), and there were little and slow effect on encystment and cell lysis.

4. Discussion
The five oomycete species studied are all important plant pathogens, whose life cycles can involve infection of foliar regions or roots of plants. Ph.  infestans mainly infects the aerial parts and also tubers of potatoes as well as tomatoes (van West et al., 2003). Ph. megakarya is the most aggressive of the Phytophthora species that cause black pod disease on cocoa (Appiah et al., 2003). It affects pods and causes stem and root cankers. Ph.  palmivora also causes fruits and root rot of many crop plants including cocoa and coconut (Erwin and Ribeiro, 1996). Py. aphanidermatum and Py. dissotocum are root pathogen that cause severe losses in many crops particularly those grown in closed soil less systems (Owen-Going et al., 2003, Bates and Stanghellini, 1984). These Pythium species cause root and crown rot in both seedlings (damping-off) and older plants (Moulin et al., 1994; Stanghellini and Rasmussen, 1994).

The zoospore dimensions of Ph.  palmivora (length 12.8 ± 1.2 m and breadth 8.5 ± 0.7 m) were similar to those previously reported (length 12.9 ± 0.8 m and breadth 8.9 ± 0.6 m) (Carlile, 1983). Zoospore dimensions have not been reported for any of the other four species. We found that swimming speed was correlated inversely with zoospore size. This suggests that swimming speed may be influenced by the fluid resistance opposing forward motion that in turn would be influenced by the cross-sectional area of the zoospore and the effect of its shape of streamlining.

The specific effects of K+ ions on behavior of zoospores from oomycetes were analyzed by addition of potassium salts or ionophores to the zoospore suspensions (Roquist and Waldenstrom, 2003; Harold, 1987). Addition of 5, 10 mM KCl and 10 mM of K2SO4 caused a significant reduction in swimming speed and a change in swimming pattern of Ph. palmivora zoospores but not Py. aphanidermatum (Fig. 3). These responses were similar to the responses observed in both species after the addition of nigericin to the zoospore suspensions (Fig. 6). These changes are not due to changes in the osmotic potential because additions of equimolar solutions of other salts had no effect. Therefore we conclude that it is a direct or indirect consequence of the change in the internal K+ concentration that is responsible for alteration of zoospore motility in these experiments.

Ionophores can facilitate ionic traffic across membranes either by acting as mobile ion-carriers or by forming pores that conduct ions through the pore complex (André, 1995; Harold, 1987; Rosenberg and Finkelstein, 1978). Gramicidin, a channel forming ionophore, induces cationic permeability by insertion into the membrane as a stationary ion-conducting channel with selectivity for monovalent cations. It effects the dissipation of K+ and H+ gradients (Schaller and Frasson, 2001; Harold, 1987; Pressman, 1976). Gramicidin has a high selectivity of K+ over Na+ of 3:1 (Wallace, 2000). Mobile carriers, such as valinomycin and nigericin, on the other hand concentrate within the lipid bilayer of the membrane, allowing cations to equilibrate with the aqueous phases on either side (Harold, 1987; Nicholls, 1982; Pressman, 1976). The molecular weights of valinomycin and nigericin, 1111 and 747 g/mol respectively, therefore nigericin would be predicted to carry more K+ ions at equivalent molar concentrations. We employed concentrations of the ionophores similar to those used in other studies investigating oomycetes and mammalian cells (Buchan et al., 1993; Donaldson and Deacon, 1993b). Experiments using these ionophores showed that altering potassium homeostasis during zoospore swimming significantly influenced zoospore speed, swimming pattern, and encystment.

The zoospores of different Pythium and Phytophthora species differed significantly in their response to the various ionophore treatments. Generally, the potassium ionophores, gramicidin, nigericin and valinomycin, affected the motility of the Phytophthora species zoospores more than those of Pythium species. For example, we observed that addition of potassium to zoospore suspensions of Ph. palmivora resulted in decreased motility, whereas swimming speed of zoospores from Py. aphanidermatum was not significantly affected (Fig. 2).

Gramicidin was found to cause the most dramatic changes in swimming with marked reductions in speed and swimming trajectories, especially for the Phytophthora species. The observed changes in the zoospore motility and swimming patterns in both Phytophthora and Pythium species may be the consequences of changes in potassium homeostasis on the cell membrane potential, which could impinge on flagellar movement. Changes in membrane potential influence ciliary beating flagellation of certain ciliates and algae (Nultsch and Dolle, 1987, Hinrichsen and Schultz, 1988). Treatments perturbing K+ homeostasis did not affect the different zoospore types equally. Modulation of swimming may be influenced by different threshold membrane potentials in different zoospores or by signaling systems that directly or indirectly respond to changes in cytoplasmic K+ concentration.

Both gramicidin and valinomycin cause increased K+ permeability of the cell membrane. However, the induced transport of K+ is much faster with gramicidin than valinomycin. This may also explain the toxic effect of this ionophore on the zoospores indicated by increases in cell lysis (Figs. 4 and 5). Valinomycin also caused a gradual increase in cell lysis in both Phytophthora and Pythium zoospore species over time. At high concentrations (10 g/ml) it caused a change in swimming pattern and a reduction in speed, which seemed to be species-dependent. Similar observations were made when valinomycin was added to spermatozoa of boar, whereby motility was progressively eliminated, without effecting plasma membrane integrity (Andersson et al., 1998). Therefore some of the effects observed on cell lysis are likely to be due to non-specific toxicity.

Valinomycin is a neutral uniport ionophore that dissipates the electrochemical gradient of potassium (Schaller and Frasson, 2001). Valinomycin therefore can be used to induce membrane permeability in order to estimate, or abolish membrane potentials depending on the conditions of the experiment (Nicholls, 1982). Cells normally have an internally negative membrane potential (Harold, 1977). Therefore, an efflux of K+ ions would cause hyperpolarization of the membrane potential. Alterations in the trans-membrane potential has already been proposed to explain changes in zoospore turning frequency due to alterations in external pH (Morris et al., 1995; Cameron and Carlile, 1980). Nigericin exchanges K+ for protons and will therefore also influence internal pH. Therefore it is possible that some of the effects seen on zoospore swimming may be due to alterations in cytoplasmic pH. Evidence has been published that implicated Ca2+ and surrogate divalent cations in the regulate zoospore swimming behavior (Deacon and Saxena, 1998; von Brombsen et al., 1997; Warburton and Deacon, 1998). It is therefore also possible that the treatments used here that lead to changes in membrane potential may also influence Ca2+ transport and hence swimming.

Therefore zoospore swimming of oomycetes is a highly regulated process, responding to changes in the ionic concentration of K+, Ca2+ and H+ by altering swimming speed, turning frequency, the trajectory of swimming and ultimately the docking encystment process that allows the zoospore to perpetuate its life cycle at the expense of the target organism that it seeks to infect.
Acknowledgements
The authors would like to acknowledge the Biotechnology and Biological Sciences Research Council (BBSRC) for financial support (BRE13669), The Royal Society for providing funding and a personal fellowship to P. van West, and the Natural and Environmental Research Council (NERC) for a studentship to M.C. Osborne.

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Table 1. Zoospore characteristics of phytopathogenic oomycete species used in the study.




Oomycete species

Phytophthora infestans

Phytophthora megakarya

Phytophthora palmivora

Pythium aphanidermatum

Pythium

dissotocum

Length (µm)

17.49 ± 3.47

10.74 ± 0.97

12.83 ± 1.22

13.82 ± 0.98

12.73 ± 0.54

Breadth (µm)

11.39 ± 0.65


7.74 ± 0.58

8.47 ± 0.72

8.82 ± 0.55

9.22 ± 0.92

L/B Ratio

1.5

1.4

1.5

1.6

1.4

Speed (µm/sec)

50 – 105

122 - 154

180 - 250

180 - 210

198 - 225

Swimming Pattern

Straight with sharp angled turning

Straight with more frequent sharp angled turning

Straight

Helical (sine wave)

Helical (sine wave with higher amplitude)

Length and breadth values are means  SEM of 10 randomly selected zoospores swimming in 2 mM Na phosphate buffer solution. Swimming speed values of each species represent the range from the 3 sets of recordings for 30 zoospores in the absence of ionophores.



Figure legends
Fig. 1. Zoospore morphology of five phytopathogenic oomycete species: Phytophthora (Ph. palmivora, Ph. megakarya and Ph. infestans) and Pythium (Py. aphanidermatum and Py. dissotocum) species. Scale bar represents 10 m.
Fig. 2. The effect of different salt concentrations on the motility (speed) of Ph. palmivora (A) and Py. aphanidermatum (B) zoospores. Effects on zoospores were assessed at 4 concentrations (0, 1, 5 and 10 mM) of salts. Zoospore speeds were calculated from recordings using the Openlab image analysis software (see Materials and Methods for details).
Fig. 3. Ph. palmivora (A) and Py. aphanidermatum (B) zoospore trajectories in 2 mM Na phosphate buffer plus KCl and K2SO4. Zoospores were photographed at high speed at approximately 0.11 sec per frame for a total of 5.5 sec per treatment. The movements of an average of 10 zoospores per salt concentration were tracked. Scale bar represents 100 m.
Fig. 4. Effect of K+ ionophores (gramicidin, nigericin and valinomycin) on the swimming behavior (speed) of zoospores of phytopathogenic oomycete species. (A) Ph. palmivora, (B) Ph. megakarya, (C) Ph. infestans, (D) Pythium aphanidermatum and (E) Py. dissotocum. Values are means  SEM and were obtained from tracing the speed (µm/sec) of an average 10 zoospores per treatment over 20 frames.
Fig. 5. Effect of gramicidin on the swimming pattern of different Oomycete species zoospores. Zoospores trajectories were tracked over a total of 50 frames (0.11 sec per frame for a total of 5.5 sec per treatment) when they remain in the field of view throughout treatment recording. Scale bar represents 100 m.
Fig. 6. Effect of nigericin on the swimming patterns of five oomycete species zoospores. Zoospores trajectories were tracked as described in Figure 5. Scale bar represents 100 m.
Fig. 7. Effect of valinomycin on the swimming patterns of five oomycete species zoospores. Zoospores trajectories were tracked as described in Figure 5. Scale bar represents 100 m.





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Figure 7



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