This is one of two Profiles on fungal zoospores (motile, flagellate spores). It introduces the subject, describes the structure and behaviour of zoospores of the economically important oomycota (e.g. potato blight), and links to a page on natural toxins produced by oat roots, which lyse zoospores (see Zoospores and saponins).

A further Profile deals with zoospores of chytridiomycota and plasmodiophorids, some of which are vectors of important plant viruses.

Introduction to zoospores: cells specialized for dispersal and site-selection

Several groups of fungi and fungus-like organisms produce motile, flagellate spores. These zoospores differ in number and types of flagella and in ultrastructural details in the different groups of organisms. But all zoospores share some common features:

• they are naked, wall-less cells, specialized for dispersal because they cannot divide nor absorb organic nutrients;
• they swim for many hours, using endogenous food reserves, then encyst by retracting or shedding their flagella and secreting a wall;
• they can respond to a range of environmental signals, which they use to locate the sites where they will encyst.

This precise site-selection or "homing" behaviour (Figure A) accounts for much of the interest in zoosporic fungi. In practical terms, it gives zoospores an important role in infection of plant and animal hosts, and sometimes as vectors of major plant viruses (Table 1).

Click the image for higher resolution (52KB)

Figure A. Homing sequence of zoospores of the oomycota, e.g. Pythium or Phytophthora species, Courtesy of Mel Fuller. Zoospores released from the 'Multiple Birth Center' are barred access to Food and Fuel, Cyber Sex (no minors or zoospores admitted!) and the Reproduction Center. At the end of their quest, they drop their flagella into the bin and encyst at their new home! [Reviewed by Deacon & Donaldson, 1993]

Structure and ultrastructure

The zoospore body (soma) of the oomycota is typically kidney-shaped, with a ventral groove (Figure B). Two flagella emerge about one-third of the distance along this groove. The posteriorly directed flagellum is long and smooth - a "whiplash-type" flagellum. The anterior flagellum is a shorter "tinsel-type" which bears tripartite hairs (mastigonemes) along its length.

Figure B. Scanning electron micrograph of a zoospore of Phytophthora [Image supplied by MS Fuller]

Only a small part of each flagellum is seen in Figure C. The flagella have a central core termed the axoneme, composed of microtubules and associated proteins. Over most of its length, the axoneme is surrounded by a membrane which is continuous with the cell membrane. The mastigonemes of the tinsel flagellum are glycoprotein extensions which project through the flagellar membrane.

Figure C. Transmission electron micrograph of a Phytophthora zoospore (about 15 micrometres diameter) prepared by freeze-substitution and sectioned through the region where the nucleus (N) extends towards the flagellar basal apparatus. The spores contain several mitochondria (M) and fingerprint vacuoles (FV). Three types of peripheral vesicle are shown, and flattened peripheral cisternae lie beneath most of the zoospore plasma membrane. The regions marked with an asterisk are thought to be where lipids were removed by solvents during fixation. [Image supplied by MS Fuller; from Cho & Fuller 1989]

Zoospores can swim for many hours, at 150 micrometres or more per second, so in theory they could disperse several metres from their point of release by their own activities. But in practice this seldom happens because they change direction frequently when they meet obstacles or by spontaneous, random turns. They are best suited for local dispersal but can be carried longer distances in moving water. In addition, some plant-pathogenic species (e.g. Phytophthora infestans, which causes potato blight) have wind-dispersed sporangia and these release zoospores when they land on a host surface.

The swimming mechanism is only partly understood. The flagellate bacteria have a rotor which causes the flagella to rotate, and spontaneous turns are achieved by a reversal of the rotor direction. In zoospores there is no flagellar rotor. Instead, the flagella beat in a sine wave which moves from the base to tip of each flagellum, and the zoospore swims in a straight helix, rotating about its axis (see "control" in Figure E). Hydrodynamic studies suggest that the posterior flagellum serves mainly as a rudder, while the anterior flagellum contributes up to 90% of the swimming speed because the mastigonemes act like oars, converting the forwards-directed sine wave to a backwards thrust [Reviewed by Carlile, 1983].

The corkscrew-like pattern of zoospore swimming, interspersed with random changes of direction, is shown in Figure D where the tracks of Pythium zoospores were photographed with a 5-second exposure, using darkfield microscopy. However, in this case many of the abrupt turns were caused when zoospores hit the surface of a glass coverslip.

The regulation of swimming pattern is strongly influenced by calcium, which regulates the behaviour of flagella or cilia in other eukaryotic cells (see Bloodgood, 1989). For example (Figure E), when external calcium is removed by a chelator (EGTA) then the zoospores swim in almost straight lines. They swim in circles when treated with substances (e.g. lanthanum or verapamil) that block calcium channels in the cell membrane. They swim in a skidding fashion when treated with compounds (e.g. trifluoperazine or dibucaine) that interfere with the calcium-binding protein calmodulin. And they swim in an erratic, jerky manner when treated with an ionophore (which equilibrates the calcium concentration across the cell membrane) or with amiloride (which disrupts ion exchange across the membrane).

Figure E. Zoospore tracks of Pythium aphanidermatum treated with various calcium-interfering substances (Control = no treatment). Each track shows the position of a single zoospore at intervals of 0.1 sec (control, channel blocker), 0.2 sec (EGTA, ionophore) or 0.3 sec (trifluoperazine) traced from a video screen; arrows show the initial direction of movement. [Donaldson & Deacon, 1993a].

It is still unclear exactly how these substances exert their effects on motility, but two general points are of interest. First, zoospores treated with any of these substances do not show the normal, periodic changes of direction and also cannot respond to attractants. So the normal swimming pattern is essential for the role of zoospores in site-selection, discussed below. Second, these findings illustrate one of the many unifying themes of biology, because calcium plays a central role in all eukaryotic organisms. In fact, some of these substances that affect zoospores by interfering with calcium-mediated processes are used as pharmacological agents to regulate heartbeat, kidney function or psychological disturbances. A nice demonstration of this was reported recently by NASA scientists, who tested the effects of various substances on the web-building activities of spiders. This serious piece of research showed that caffeine was one of the most disruptive agents. Caffeine exerts its effects by depleting the calcium levels in intracellular stores.

"Fortean Times" (The Journal of Strange Phenomena) picked up on this with a wonderful joke (or was it an advert?):

Based on a page in "Fortean Times", Number 84

Caffeine strongly disrupts normal zoospore function, and ethanol is a strong chemoattractant of some zoospores!

Having laid the foundations of zoospore structure and function, we now consider the homing sequence leading to infection of a host from a zoospore. The following diagram gives a composite view of this sequence, starting from a sporangium, such as one of the wind-dispersed sporangia of Phytophthora infestans (the cause of potato blight). However, it should be noted that this composite view has been built up from studies on various Phytophthora, Pythium and Aphanomyces species. Not all of these stages may apply to any one fungus.

Stage a (top left) in this diagram shows a sporangium of a Phytophthora infestans. In this fungus the sporangium can germinate in two ways: by hyphal outgrowth (b₁) or by undergoing protoplasmic cleavage (b₂) to release zoospores (c) by dissolution of the sporangial papillum. Zoospores (inset at bottom left) have an anterior tinsel flagellum and a posterior whiplash flagellum, inserted in a ventral groove close to the position of the nucleus (n) and water-expulsion vacuole (wev). Small dorsal vesicles (dv) and small ventral vesicles (vv) lie just beneath the plasma membrane. The sequence d₁ to g₁ shows the events when a zoospore is induced to encyst by a host surface component. The spore typically orientates, settles and adheres with the future, pre-determined germination site against the host. (e₁). Cyst coat glycoprotein is then released by exocytosis of the small dorsal vesicles, and proteinaceous adhesive is released from the ventral vesicles. Over the next few minutes a microfibrillar cyst wall is synthesised beneath the cyst coat (f₁). Then after 20-30 min the cyst germinates by a hyphal outgrowth towards the host (g₁); the hypha either infects directly or (not shown) produces a swollen appressorium before infection. An alternative sequence (d₂-g₂) occurs if a zoospores does not find a host. It encysts eventually and, after some hours, typically releases a further zoospore.

Figure F.

• 1. accumulation of zoospores at specific sites by sensing gradients of attractants or repellents (zoospore taxis);
• 2. settling and orientation on the host surface, perhaps by recognition of specific host surface components;
• 3. adhesion and encystment, involving the release of adhesins and production of a cyst wall;
• 4. germination with a fixed orientation, from an apparently predetermined point.

These four stages are remarkable for many reasons:

• they occur so rapidly that they can lead to infection from an originally motile spore within 30-40 minutes;

• during his short time the cells undergo major changes of organisation - the originally wall-less, motile cell is transformed into a walled cyst which then gives rise to a new hyphal apex (see apical growth);

• the cells can respond to many exogenous cues, including those that change the direction or speed of swimming, others that trigger encystment and yet others that can trigger cyst germination;

• throughout this time the cells do not take up external organic nutrients - this occurs only after the cyst has germinated and the germ-tube has grown to some length.

Thus, we can think of zoospores as being largely pre-programmed cells, which require only the appropriate signals to progress through a complex sequence of events leading to infection.

The term taxis can be used generally to describe a change in swimming orientation of a motile cell in response to an external stimulus. The zoospores of various fungi have been shown to respond to chemical gradients (chemotaxis), oxygen (aerotaxis), electrical or ionic fields (galvanotaxis, electrotaxis) or light (phototaxis). Some also can swim against a water current (rheotaxis) and some swim upwards in suspension (negative geotaxis) but probably not by sensing gravity as such. In addition to all these factors, when zoospore suspensions are sufficiently dense then the cells can autoaggregate - a phenomenon termed adelphotaxis and perhaps caused by the release of an attractant substance.

One or more of these factors can cause zoospores to accumulate at specific sites. For example, the zoospores of plant pathogenic fungi commonly accumulate at wound sites, or near the stomata of leaves, or near the root tips (Figure G). Similarly, the zoosporic parasites of small animals often accumulate at the body orifices (see Catenaria).

Zoospore taxis can also serve to bring zoospores to sites where they will be destroyed by toxins. A natural example of this is seen in the responses of zoospores to oat roots: see zoospores and saponins.

Figure G. Videotape images (shown as negatives) of Pythium zoospores accumulating on wheat roots: at 1 min 43 sec (left) and 8 min 25 sec (right) after roots were immersed in zoospore suspension. The left image shows a root tip surrounded by a ball of root tip mucilage containing shed root cap cells (rc). A mass of zoospores (z) has accumulated over the surface of the root tip mucilage. The right image shows a region just behind the tip of a root coated with two layers of alginate gel. Zoospores (z) have accumulated locally on the alginate in the zone where root hairs start to emerge. [From Jones et al., 1991]

Capillary model systems can be used to investigate the individual attractants of zoospores (Figure H) and thus give clues to the factors involved in host-location. Typically, the zoospores of root-infecting Pythium and Phytophthora species are attracted strongly to a few individual amino acids (e.g. aspartic or glutamic acid), sugars (e.g. glucose) or volatile compounds (e.g. ethanol, aldehydes), all of which are common components of root exudates. But the spores respond better to mixtures than to individual compounds. In addition, a few examples of host-specific attractants have been reported from capillary studies. For example, Phytophthora sojae, a host-specialised pathogen of soybean roots, is strongly attracted to the soybean flavonoids daidzein and genistein, and Aphanomyces cochlioides (from spinach) is strongly attracted to the host flavonoid, cochliophilin A. These reports are interesting because they parallel the behaviour of host-specific Rhizobium species, which also are attracted to the flavonoids of their hosts and can be repelled by non-host flavonoids.

It is assumed that the responses to different attractants are mediated by chemoreceptors in the zoospore or flagellar membrane. But no receptor has yet been purified and characterised from a zoosporic fungus.

Figure H. Left: zoospores of Phytophthora palmivora accumulating at the mouth of a capillary filled with a fluorescently tagged amino acid (dansyl-asparagine) [supplied by MJ Carlile, from JN Cameron & MJ Carlile, unpubl.]. Right: zoospores of Pythium aphanidermatum accumulated and encysted at the mouth of a capillary containing malt-extract agar. Germ-tubes have grown from the cysts into the agar in the capillary.

Zoospores can be induced to encyst by artificial agents such as high concentrations of chemicals or mechanical agitation in laboratory conditions. But in nature they probably respond to recognition of a host surface component. For example, the zoospores of many Pythium and Phytophthora species are induced to encyst by pectin or by root surface mucilage (which often has a similar composition to pectin). There may also be a degree of specific recognition. For example (see Table 2) three species of Pythium responded differently to a range of plant-derived polymers in laboratory assays, and the Pythium species that characteristically infect the roots of grasses and cereals show only a weak ability to encyst on non-grass roots (Table 3).

Data from Donaldson & Deacon (1993b); RT Mitchell & Deacon (unpubl.)

1. The flagella are shed, the zoospore rounds off, and the nucleus migrates to a central position.

2. Some of the peripheral vesicles (see Figure C) fuse with the cell membrane to release their contents onto the cell surface. These contents include a cyst coat glycoprotein from the small dorsal vesicles and an adhesive protein or glycoprotein from the small ventral vesicles.

3. The flat peripheral cisternae start to bud vesicles which will deposit a cyst wall underneath the cyst coat.

4. The production of the cyst wall is usually completed in about 5-6 minutes. Then the water-expulsion vacuole disappears, and a germ-tube emerges about 20-30 minutes later, to produce either a hypha or an appressorium (pre-infection swelling).

The disruption of any of these events could potentially lead to succesful control of disease caused by zoosporic fungi. An interesting example of this is the role of oat roots in disrupting the normal behaviour of zoospores - see Zoospores and saponins

When zoospores settle on a host surface, the cysts almost invariably germinate towards the host (Figure I). This raises the possibility that zoospores might pre-align during encystment. Several lines of evidence support this idea and also suggest that the site of germination is predetermined. In other words, a zoospore settles and encysts on a host with a precise orientation (docking) so that the emerging hypha can rapidly penetrate the host.

Figure I. Zoospore cysts of Pythium aphanidermatum germinating towards a wheat root after the zoospores settled and encysted just behind the root tip.

The first evidence for pre-alignment came from simple laboratory experiments with Pythium zoospores, shown diagrammatically in Figure J. Motile spores were placed in a chamber with a block of nutrient-rich agar (malt-peptone agar) at one end and a block of nutrient-free water agar at the other end. Most of the zoospores swam towards the nutrient-rich agar, then encysted and germinated towards the agar block. Fewer zoospores settled and encysted near the water agar, and these cysts germinated with random orientation.

Figure J. Simple experiment to show that zoospores of Pythium have a fixed (predetermined) point of germination. (see Mitchell & Deacon, 1986b)

These findings could be explained in two ways:

• either the zoospore has a fixed (predetermined) site of germination which is located next to the attractant source (e.g. host) by pre-alignment of the zoospore when it encysts
• or the zoospore cyst has the potential to germinate from any point, and the actual site of germination is influenced by the attractant source (e.g. host).

To distinguish between these possibilities, zoospores were encysted artificially (by agitation) then placed in a chamber. The pre-encysted cells always germinated with random orientation, even when they were positioned next to the nutrient-rich agar. So they seem to have a fixed site of germination which cannot be influenced by external factors such as nutrients. Nevertheless, the germ-tubes can change direction after they have emerged from a fixed position, and then grow towards a nutrient source (see hyphal tropisms).

This raises the possibility that the flagella might be involved in recognising host surface components, and again evidence has come from the use of MAbs. Of all the antibodies raised against zoospores of P. cinnamomi, only one caused rapid encystment when added to motile spores. This MAb binds specifically to the surface of both flagella, and it also induced rapid encystment of other Phytophthora and Pythium species. In contrast, MAbs that bind to the whole zoospore surface or to components of only the anterior flagellum did not cause rapid encystment.

We noted earlier that calcium has an important role in zoospore motility. It also is required for cyst adhesion and subsequent cyst germination. This is not surprising because calcium is an important second messenger in all eukaryotic cells: it is intimately involved in the signal transduction pathways that link the perception of an external stimulus to a cellular response. For example, the binding of a ligand to the external face of a membrane receptor needs to be translated into a cellular response, such as the activation of a gene or other change of cell behaviour. Calcium ions are one of the links in this chain. The binding of the receptor might open a calcium-specific membrane channel, allowing a localised influx of Ca²⁺. Any change in the intracellular free Ca²⁺ content can then have numerous effects, via interaction with cytoskeletal proteins or with calcium-binding proteins such as calmodulin.

All the substances that interfere with calcium-mediated processes can disrupt cyst germination when applied to pre-encysted cells. They include calcium channel-blockers, calcium ionophores, calcium chelators, calmodulin inhibitors, and compounds such as caffeine that deplete the calcium levels in intracellular stores, or other compounds (e.g. TMB-8) which block the release of calcium from intracellular stores. Equally important, zoospore cysts have an absolute requirement for external calcium, because the removal of this with calcium chelators inhibits germination.

These findings have spurred attempts to measure calcium changes in encysting zoospores. One approach to this is the use of calcium-sensing dyes, which change their fluorescent properties when they bind to calcium. For example, some of the "ratiometric dyes" change the excitation wavelength at which they fluoresce when exposed to calcium, so the ratio of fluorescence at different excitation wavelengths can be used to estimate intracellular free calcium levels in a cell. Figure K shows examples of living zoospores which absorbed these dyes and were observed by confocal microscopy. The calcium levels in different parts of the cells are then translated into false colour images. But in these examples the dyes were rapidly sequestered into cellular organelles - a common problem in fungi.

Figure K. False colour images obtained by confocal microscopy of Phytophthora zoospores treated with the calcium-sensing dyes fluo-3 (left) and calcium green (right). The left-hand image shows a recently encysted zoospore in two optical sections (towards the top of the cell - left; in near median plane of focus - right). The blue region, of lowest free calcium concentration, corresponds to the position of the nucleus and water-expulsion vacuole. The higher calcium concentrations (red or white) seem to be in a zone where many fingerprint vacuoles and mitochondria occur, suggesting that the dye was sequestered in these organelles. The right-hand image shows an optical section through a motile zoospore. The blue zone along the centre of the cell represents the ventral groove. [Adrian Warburton & JW Deacon, unpubl.]

The problem of dye sequestration in organelles can be overcome to some degree by using cell-impermeable (free acid) forms of the dyes, which remain outside of the cells so that fluorimetry can be used to measure changes in the calcium in the bathing medium of a population of zoospores. The dyes then measure the fluxes of calcium ions into and out of the cells during the stages of zoospore encystment. Recent studies of this sort (Figure L) show that the external calcium concentration drops markedly when zoospores are induced to encyst by agitation, indicating that the spores take up a large amount of calcium at this stage. Then they release calcium progressively during the next 20-30 minutes, representing an enormous net efflux of the cellular calcium reserves. When the experiment is repeated in the presence of a calcium channel blocker (lanthanum or verapamil) there is no net influx or efflux of calcium (Figure M), and the zoospores do not encyst. If TMB-8 is used instead of a channel blocker, the spores show an initial net influx of calcium, but no subsequent efflux (Figure N). These cells encysted but showed only low germination. [In mammalian systems, TMB-8 is known to block the release of calcium from intracellular calcium stores]

Figure L. Estimates of the free calcium concentration in the medium surrounding a population of Phytophthora zoospores, as measured by fluoriemtry of the calcium-sensing dye fura-2. Calcium levels in the medium were measured for an initial 200 seconds, then the cells were vortexed to induce encystment (70 seconds break in the diagram) before the recordings were continued. [A Warburton & JW Deacon, 1998]

Figure M. Estimates of the free calcium concentration in the medium surrounding a population of Phytophthora zoospores. Details as in Figure L except that lanthanum or TMB-8 was added immediately before the zoospores were vortex-encysted. [A Warburton & JW Deacon, 1998]

Studies such as these could form the basis for novel approaches to control zoosporic plant pathogens. The manipulation of external calcium levels can have profound effects on several stages of the zoospore homing sequence, of possible practical benefit in glasshouse irrigation systems (von Broembsen & Deacon, 1997).

Recent work has extended some of the above findings to the airborne sporangia of Phytophthora infestans, which causes potato late blight and was responsible for the Great Irish Potato Famine in the 1840s. It is still a significant pathogen of potato crops. In laboratory conditions these sporangia will release zoospores when incubated in water at 12^oC, or germinate by hyphal outgrowth at 20^oC. In either case, the germination of sporangia is suppressed by a wide range of treatments that would be expected to combine with any external calcium ions (e.g. calcium chelators such as EGTA or BAPTA, or even phosphate or pectin). Moreover, as shown in the diagram below, these treatments lead rapidly to death of the sporangia. This raises the prospect that we might be able to use mild, environmentally safe chemicals to control potato blight. But the effects have yet to be tested in realistic field conditions.

Death of Phytophthora infestans sporangia when incubated in the presence of 0.1% or 5 mM concentrations of various chemicals at 12^oC in laboratory conditions. From Hill et al., 1998.

Reviews

RA Bloodgood (1991) Transmembrane signalling in cilia and flagella. Protoplasma 164, 12-22.

MJ Carlile (1983) Motility, taxis and tropism in Phytophthora. In Phytophthora: Its Biology, Taxonomy, Ecology and Pathology (ed. DC Erwin, S Bartnicki-Garcia & PH Tsao) pp. 95-107. American Phytopathological Society, St Paul.

JW Deacon & SP Donaldson (1993) Molecular recognition in the homing responses of zoosporic fungi, with special reference to Pythium and Phytophthora. Mycological Research 97, 1153-1171.

AR Hardham (1995) Polarity of vesicle distribution in oomycete zoospores: development of polarity and importance for infection. Canadian Journal of Botany 73 (Supplement) S400-S407.

Research articles (hyperlinks go to Abstracts of recent papers)

CW Cho & MS Fuller (1989) Ultrastructural organization of freeze-substituted zoospores of Phytophthora palmivora. Canadian Journal of Botany 67, 1493-1499.

SP Donaldson & JW Deacon (1993a) Changes in motility of Pythium zoospores induced by calcium and calcium-modulating drugs. Mycological Research 97, 877-883.

SP Donaldson & JW Deacon (1993b) Differential encystment of zoospores of Pythium species by saccharides in relation to establishment on roots. Physiological and Molecular Plant Pathology 42, 177-184.

SW Jones, SP Donaldson & JW Deacon (1991) Behaviour of zoospores and zoospore cysts in relation to root infection by Pythium aphanidermatum. New Phytologist 117, 289-301.

RT Mitchell & JW Deacon (1986a) Differential (host-specific) accumulation of zoospores of Pythium on roots of graminaceous and non-graminaceous plants. New Phytologist 102, 113-122.

RT Mitchell & JW Deacon (1986b) Chemotropism of germ-tubes from zoospore cysts of Pythium spp. Transactions of the British Mycological Society 86, 233-237.

SL von Broembsen & JW Deacon (1997) Calcium interference with zoospore biology and infectivity of Phytophthora parasitica in nutrient irrigation solutions. Phytopathology 87, 522-528.

A Warburton & JW Deacon (1998) Transmembrane Ca²⁺ fluxes associated with zoospore encystment and cyst germination by the phytopathogen Phytophthora parasitica. Fungal Genetics and Biology 25, 54-62.

AE Hill, DE Grayson & JW Deacon (1998) Suppressed germination and early death of Phytophthora infestans sporangia caused by pectin, inorganic phosphate, ion chelators and calcium-modulating treatments. European Journal of Plant Pathology 104, 367-376.

Websites

Plasmodiophorid Home Page

Zoosporic Fungi Online

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