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Environmental signalling during induction of appressorium formation in Phytophthora
Mycol. Res. 101 (4) : 395–402 (1997)
395
Printed in Great Britain
Environmental signalling during induction of appressorium formation in Phytophthora
U R S B I R C H E R A N D H A N S R. H O H L Institute of Plant Biology, University of ZuX rich, Zollikerstr. 107, CH-8008 ZuX rich, Switzerland
Appressorium formation in vitro by Phytophthora palmivora started after about 1 h of incubation of zoospores in an aqueous solution and was influenced by topographical signals, substrate hydrophobicity and available nutrients. Free floating, non-adhering germlings did not form appressoria. On smooth substrates, no appressoria were formed under high nutrient conditions (20 % pea broth) independent of the hydrophobicity of the contact surface, while under low nutrient conditions (5 % pea broth) appressoria formed on smooth substrates with hydrophobic, but not on smooth substrates with hydrophilic, surfaces. If the same substrates were scratched, appressoria formed over these scratches independent of levels of exogenous nutrients and substrate hydrophobicity. These findings are summarized in a model of signalling for appressorium induction.
Phytophthora palmivora is an important plant pathogen attacking a broad range of economically important crops in warmer regions of the world (Chee, 1974). It infects mainly aerial parts of its hosts, while root invasions have been described in only a few cases (Chee, 1974). As with other oomycetes, zoospores are involved in dispersal in the soil or on aerial parts of host plants. After contacting a solid surface, the motile zoospore is transformed into a non-motile, spherical cyst. Zoospores of P. palmivora become adhesive very early during encystment due to rapid secretion of adhesive material (Sing & Bartnicki-Garcia, 1972, 1975 a, b). On the leaf surface of black pepper (Piper nigrum) most of the encysted zoospores of P. palmivora strain P113 and P. capsici form single germ-tubes (Feuerstein & Hohl, 1986). Occasionally these germ-tubes grow into the narrow furrows around trichomes. The invading germ-tubes are wedged in the furrows and penetrate the epidermis without forming appressoria (Feuerstein & Hohl, 1986). In most cases, however, germlings form appressoria which are morphologically similar to appressoria formed by other Phytophthora species (Pristou & Gallegly, 1954 ; Sto$ ssel, Lazarovits & Ward, 1980 ; Gees & Hohl, 1988), over periclinal or anticlinal walls of epidermal cells, while access via stomata has not been described (Feuerstein & Hohl, 1986). Unlike the situation with some other pathogenic fungi (see Hoch & Staples, 1991 ; Hardham, 1992 ; Read et al., 1992 ; Mendgen & Deising, 1993) information about exogenous and endogenous signals regulating the induction of appressorium formation in Phytophthora is lacking. In the present study we present data obtained with an in vitro system which indicate that appressorium formation in Phytophthora is influenced by contact with a solid substrate, the surface topography, the substrate hydrophobicity and available nutrients.
MATERIALS AND METHODS Growth of the pathogen and production of zoospores Phytophthora palmivora (E. J. Butler) E. J. Butler strain P113 was used throughout this study. The fungus was grown in 9 cm plastic Petri dishes on Borlotti bean agar (Ward et al., 1979 ; Hohl & Balsiger, 1986). After 5 d at 24 °C in darkness the cultures were transferred to room temperature (approx. 21°) and daylight for 2 d, which induced sporangium formation. The sporangia from three plates were harvested with an inoculation loop and transferred into 50 ml sterile double distilled water and centrifuged at 10 g (rav 13 cm) for 2 min. The supernatant was discarded and the pellet containing sporangia was resuspended in 5 ml of sterile double distilled water. The suspension was incubated at 4° for 10 min and for an additional 15–20 min at 25° to allow emergence of zoospores. After filtration through a sterile 20 µm nylon mesh (Nybolt P20, Schweizerische Gazefabrik AG), to separate the zoospores from the sporangial cases, the concentration of zoospores was determined using a haemocytometer. The zoospore suspension was diluted with pea broth and double distilled water to a final concentration of 5¬10% zoospores ml−", and used immediately. Addition of pea broth did not induce rapid encystment of zoospores. For the preparation of 1 l of pea broth, 75 g of frozen garden peas were boiled in 250 ml of distilled water for 20 min, filtered, and 10 g sucrose, 1 g asparagine, 0±25 g MgSO \7H O and 0±5 g KH PO were added. The medium % # # % was made up to 1 l with distilled water (Hohl & Balsiger, 1986). Test substances and measurements of osmolality To assess the effects of exogenous nutrients on appressorium
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396
Table 1. Time course of germling differentiation of P. palmivora in tissue culture plates* Germ-tubes (%)
Hours of incubation at 25° 1 2 3 4 5 6
Germination (%) 95±5³0±9 98±3³0±9 99±6³0±6 99±7³0±3 99±9³0±2 100³0
Non-differentiated
With slightly swollen apex
With appressorium
With appressorium, hyphae and} or vesicles
70±2³1±8 8±0³1±6 4±2³0±9 1±7³0±7 0±8³0±8 0±8³0±5
29±8³1±8 6±9³1±2 1±8³0±7 0±9³0±7 0±1³0±2 0±6³0±5
0 84±9³1±7 77±6³1±8 69±7³2±3 53±2³2±1 30±3³1±8
0 0±3³0±3 16±5³1±8 27±7³2±5 45±8³2±0 68±4³2±2
* Germlings were incubated in 5 % (v}v) pea broth. At least 200 germlings were analysed per incubation time in each of three replicates. Three independent experiments were performed. Values represent mean and .. (n ¯ 9).
formation, the following substances were used : NaCl, KCl, CaCl , Ca(NO ) \4H O, MgSO \7H O purchased from # $# # % # Merck (Dietikon, Switzerland), -glucose, -sucrose, -maltose and -arabinose from Fluka (Buchs, Switzerland) and asparagine and thiamine-HCl from Sigma (Buchs, Switzerland). The osmolality of the different test substances was determined with a Roebling Osmometer (Vogel, Giessen, Germany).
Measurements of contact angles The contact angle of droplets of 5 % (v}v) and 20 % (v}v) pea broth on each of the test substrates was determined using a NRL Model 100.00 Contact Angle Goniometer (Rame! -Hart Inc., Mountain Lakes, U.S.A.). The mean value for each substrate was estimated by measuring contact angles of ten 0±5 µl drops (Clement et al., 1993). Germ-tube growth assay
Appressorium formation assay To study appressorium formation in vitro the following substrates were chosen : cellophane (jam jar-seals from Migros Genossenschaftsbund, Switzerland), 18¬18 mm glass coverslips (Knittelgla$ ser or Menzelgla$ ser, Merck, Dietikon, Switzerland), 35 mm diam. polystyrene Petri dishes (Greiner GmbH, Nu$ rtingen, Germany) and Teflon PFA 1000 LP film (Du Pont de Nemours International SA, Geneva). The time course analysis was performed using polystyrene 24-well flat-bottom tissue culture plates (Corning, New York). To provide topographically structured surfaces cellophane, polystyrene and Teflon were scratched with a brass brush and glass surfaces were scratched with a diamond pencil. Scratched substrates as well as non-scratched control substrates were cleaned with 70 % ethanol followed by extensive rinsing with sterile distilled water. The cleaned substrates were airdried in a laminar-flow cabinet prior to use. Single pieces (1±5¬2±0 cm or 2±0¬2±5 cm) of each of the different test substrates (except polystyrene) were placed in 35 mm diam. polystyrene Petri dishes. For polystyrene, the Petri dishes themselves served as test substrates. Three samples (100–150 µl) of 5¬10% zoospores ml−" suspended in 0–20 % (v}v) pea broth were incubated on each of the different test substrates at 25° in darkness. After incubation, appressorium formation was immediately checked. The experiments were performed in triplicate. In each of three independent experiments at least 200 germlings from each of the three samples were analysed (n ¯ 9). To check the position of appressoria in respect to scratches in test substrates, in each of three independent experiments, the position of at least 100 appressoria (whether over scratches or not) was determined after incubation.
Circular glass coverslips (12 mm diam.) were cleaned with 70 % ethanol, extensively rinsed with distilled water and autoclaved. Sterile coverslips were placed in 35 mm diam. polystyrene Petri dishes. Zoospores (5¬10% ml−") suspended in 100 µl of 20 % (v}v) or 5 % (v}v) pea broth were placed onto the coverslips. After incubation for 3 h at 25° germlings were fixed in a freshly prepared aqueous solution of 3±7 % (w}v) p-formaldehyde. Germ-tube lengths were determined with a graphic program (Metris, written by Nils Antonsen, Hauptstr. 44, CH-5200 Brugg) after digitalization from photomicrographs on a graphic tablet. The length of 100 germ-tubes derived from two independent experiments was determined. Light microscopy Appressorium formation was checked with an inverted microscope (Nikon, TMS-F) equipped with a 20¬Ph 2 DL objective (Nikon). Interference contrast observations were made on a Zeiss Axiophot microscope equipped with a PlanNEOFLUAR 40 (Ph 2) objective. Scanning electron microscopy To examine the geometry of grooves in dry scratched substrates, the latter were sputter-coated with gold before examination. Specimens were examined with a Hitachi S-4000 field emission scanning electron microscope. RESULTS Time course of infection structure formation Table 1 shows the results of a quantitative analysis of the formation of appressoria on tissue culture plates. Zoospores
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Figs 1–8. Sequence of morphological changes of P. palmivora during appressorium formation on polystyrene. Fig. 1. Adhering cyst. A detached flagellum is visible (arrow head). Fig. 2. Germling with a single germ-tube. Fig. 3. Early appressorium development as indicated by a slight broadening of the hyphal apex. Figs 4, 5. The length of the germ-tube between the cyst and the appressorium is highly variable. Figs 6, 7. After a period of maturation, finger-like projections and}or one or several vesicles (V) emerge from the mature appressoria (A). Fig. 8. Emerging hyphae often form additional appressoria. Bar represents 10 µm for all figures.
which adhered to the substrate rounded up, discarded their flagella, formed a cyst wall (Fig. 1) and germinated, mostly by producing single germ-tubes (Fig. 2). The germination rate of adhering cysts was over 95 % after 1 h of incubation. At this
time, 29±8 % of the germ-tubes showed a slight broadening of the hyphal apex (Fig. 3) as a first sign of appressorium formation. The length of the germ-tube between the cyst and the appressorium was highly variable (Figs 4, 5). After 2 h of
Induction of appressorium formation in Phytophthora
398 Table 2. Influence of different substances on the frequency of appressorium formation of P. palmivora on polystyrene
100
Germlings with appressoria (%)†
Germination rate (%)
80 Test substances* 60 40 20
0 0
5
10
15
20
Appressorium formation (%)
100
NaCl KCl CaCl # Ca(NO ) \4H O $# # MgSO \7H O % # KH PO }Na HPO \2H O # % # % # KH PO }Na HPO \2H O # % # % # KH PO }Na HPO \2H O # % # % # KH PO }Na HPO \2H O # % # % # -glucose -sucrose -maltose -arabinose -asparagine -asparagine thiamine-HCl (3 µ)
(pH 5±0) (pH 6±0) (pH 7±0) (pH 8±0)
Control 5 % (v}v) pea broth in distilled water 20 % (v}v) pea broth in distilled water
80
84±3³4±2 89±5³3±5 84±7³4±9 84±4³1±7 65±8³7±4 82±1³3±8 85±0³1±9 81±4³3±6 78±7³3±9 83±6³3±8 90±5³1±2 87±7³1±5 79±7³5±6 79±3³4±1 81±9³3±5
90±4³2±9 4±5³2±2
* Osmolality for all test substances 11±5 m except for the 5 % (v}v) pea broth control (2±5 m). † Germlings were incubated for 3 h at 25°. At least 200 germlings were analysed per test substance in each of three replicates. Three independent experiments were performed. Values represent mean and .. (n ¯ 9).
60 40 20
of germlings forming appressoria) nor in 5% (v}v) pea broth (0±6³0±7 % of germlings forming appressoria).
0 0
5 10 15 Pea broth concentration (%)
20
Fig. 9. Influence of pea broth concentration on germination rate and frequency of appressorium formation in P. palmivora on polystyrene. Germlings were incubated for 3 h at 25°. At least 200 germlings were analysed per treatment in each of three replicates. Three independent experiments were performed. Values represent mean and .. (n ¯ 9).
incubation more than 90 % of the germlings formed spherical or ovoid appressoria, morphologically similar to those formed on host leaves (Feuerstein & Hohl, 1986 ; Gees & Hohl, 1988). Subsequently, finger-like projections (Fig. 6) and}or one or several vesicles (Fig. 7) emerged from the mature appressoria. Emerging hyphae often formed additional appressoria (Fig. 8). After 6 h of incubation, more than 98 % of the adhering germlings had developed appressoria. Up to this stage no pseudoseptum (callosity) (Gooday & Hunsley, 1971 ; Hohl & Suter, 1976) separating the germ-tube from the appressorium was visible by light microscopy. Effects of contact with substrate on appressorium formation Zoospores were incubated in 5 % (v}v) or 20 % (v}v) pea broth in Petri dishes rotating at 150 rpm on a shaker to prevent adhesion of the growing germlings to the substrate. After 3 h of incubation no appressoria were formed by free floating germlings, neither in 20 % (v}v) pea broth (0±1³0±2 %
Effects of exogenous nutrients on appressorium formation on smooth substrates The data shown in Fig. 9 indicate that in germlings of P. palmivora, exogenous nutrients influenced the germination rate and the frequency of appressorium formation on polystyrene after 3 h of incubation. High nutrient concentrations suppressed, while low concentrations were conducive to, appressorium formation. P. palmivora was able to produce appressoria using endogenous reserves exclusively, since appressoria were formed under nutrient-free conditions (Fig. 9). Under these conditions, formation of secondary zoospores from cysts (Hemmes & Hohl, 1971) was also observed frequently. From the results presented in Fig. 9 two pea broth concentrations were chosen for subsequent experiments : 5 % (v}v) pea broth (‘ low nutrient conditions ’) leading to high germination rates as well as high frequencies of appressorium formation on polystyrene, and 20 % (v}v) pea broth (‘ high nutrient conditions ’) leading to high germination rates but very low frequencies of appressorium formation on polystyrene. In the following experiments we tested the influence of different ions, amino acids and sugars on appressorium formation on polystyrene (Table 2). The test substances were applied at the same osmolality as a 20 % (v}v) pea broth solution (11±5 m), which inhibited appressorium formation (Fig. 9). The test substances were added after 20 min of incubation in 20 % (v}v) pea broth to allow adhesion and cyst formation in the pea broth solution. Of the tested substances,
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Table 3. Influence of substrate hydrophobicity and exogenous nutrients on the frequency of appressorium formation of P. palmivora in vitro Germlings with appressoria (%)* Smooth substrate
5 % pea broth
Φ† "
20 % pea broth
Φ† #
Teflon 87±3³5±1 89³3 0±6³0±4 89³3 Polystyrene 84±3³3±6 62³4 1±7³1±3 62³3 Glass 0±9³0±5 37³3 0±6³0±5 36³2 Cellophane 2±3³1±3 17³2 1±2³0±4 15³3 * Germlings were incubated for 3 h at 25°. At least 200 germlings were analysed per test substrate in each of three replicates. Three independent experiments were performed. Values represent mean and .. (n ¯ 9). † Contact angles measured with 5 % (v}v) pea broth (¯ Φ ) or 20 % (v}v) " pea broth (¯ Φ ). #
only MgSO \7H O showed a slight inhibitory effect on % # appressorium formation on polystyrene after 3 h of incubation. Appressorium formation on this substrate was not affected by the pH within the range tested (pH 5±0–8±0). Effects of substrate hydrophobicity on appressorium formation on smooth substrates The effect of the hydrophobicity of a contact surface on appressorium formation in vitro was determined using a variety of substrates (Table 3). It cannot be excluded,
however, that physical factors other than hydrophobicity also differed among these substrates. Under high nutrient conditions, very few appressoria were formed. This was true for hydrophilic as well as for hydrophobic substrates. Under low nutrient conditions appressoria were formed almost exclusively on substrates with hydrophobic surfaces. On hydrophilic substrates appressorium formation occurred only at very low levels. Germ-tube growth was somewhat affected by the pea broth concentration. After incubation for 3 h in 5 % (v}v) or 20 % (v}v) pea broth on glass, germlings formed germ-tubes of 64±2³24±6 µm and 74±9³26±6 µm in length, respectively (n ¯ 100). Appressorium formation on scratched surfaces Figures 10–17 show examples of grooves on the different test substrates. The morphology and the distribution of the grooves varied considerably between different substrates and also for each substrate. On each of the substrates tested some germ-tubes grew into and along a groove and left it again without any apparent morphological differentiation. However, on different parts of the same ridge or elsewhere on the same substrate, germtubes were stimulated to differentiate appressoria (Figs 18, 19).
Figs 10–17. Scanning electron micrographs of grooves on scratched test substrates. Figs 10, 14. Cellophane. Figs 11, 15. Glass. Figs 12, 16. Polystyrene. Figs 13, 17. Teflon. Bars represent 10 µm in Figs 10–13 and 50 µm in Figs 14–17.
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400
Figs 18, 19. Appressoria formed on scratched substrates. Fig. 18. Polystyrene. Fig. 19. Glass. Germlings were incubated in 20 % (v}v) pea broth which inhibits appressorium formation on smooth substrates (see Table 3). Bar represents 10 µm for both figures.
Table 4. Influence of scratches in substrates with different surface hydrophobicities on appressorium formation of P. palmivora in vitro*
Substrate
Germlings forming appressoria (%)†
Appressoria positioned over scratches (%)‡
Teflon Polystyrene Glass Cellophane
50±5³13±5 47±8³22±5 9±7³3±9 52±5³14±4
92±7³0±9 94±4³3±1 96±0³2±0 94±4³1±6
* Germlings were incubated in 20 % (v}v) pea broth for 3 h at 25°, which inhibits appressorium formation on smooth substrates (see Table 3). † At least 200 germlings were analysed per test substrate in each of three replicates. Six independent experiments were performed. Values represent mean and .. (n ¯ 18). ‡ At least 100 germlings were analysed in each of three independent experiments. Values represent mean and .. (n ¯ 3).
In Table 4 the results of a quantitative analysis of the influence of scratches in the test surfaces on appressorium formation are shown. The experiments were performed with 20 % (v}v) pea broth, which inhibited appressorium formation on the corresponding smooth substrates (Table 3). The results demonstrate that appressoria formed on scratched hydrophilic as well as on scratched hydrophobic surfaces after 3 h of incubation. The frequencies of appressorium formation ranged between 2±9 and 91±5 % with the lowest values on glass. The ..s of the mean were very high compared to those obtained on smooth substrates (Table 3). On each of the test substrates more than 92 % of appressoria were situated over grooves (Table 4).
Effects of changes in environmental conditions on appressorium formation The following experiments were performed to determine whether or not germlings growing under non-inducive conditions were able to form appressoria after conditions had been changed to become inducive. In the first experiment, germlings incubated on polystyrene in 20 % (v}v) pea broth for 3 h (non-inducive conditions) did not form appressoria. After the 20 % (v}v) pea broth solution was diluted to 5 % (v}v) with sterile distilled water (inducive conditions), 95±0³2±1 % of the germlings developed appressoria within 3 h, in some cases with hyphae and}or vesicles. In the second experiment, germlings were incubated in Petri dishes rotating at 150 rpm on a shaker to prevent adhesion of the germlings to the surface of the substrate (noninducive conditions). After incubating these germlings for an additional 3 h in still conditions on polystyrene, thereby allowing them to settle and adhere to the substrate, 83±3³3±6 % of the germlings incubated in 5 % (v}v) pea broth (inducive conditions) produced appressoria while only 2±5³1±2 % of those incubated in 20 % (v}v) pea broth (noninducive conditions) produced appressoria.
DISCUSSION Appressorium formation in phytopathogenic fungi may be influenced by a variety of physical and chemical signals (Hoch
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401
Contact with surface ? yes
Contact with inducive topographical signal ? * yes Appressorium formation
Undifferentiated mycelial growth
no Contact surface hydrophobic ? yes
Low nutrient conditions ? † yes
Appressorium formation
no
no Undifferentiated mycelial growth
no
Undifferentiated mycelial growth
Fig. 20. Model for the induction of appressorium formation in P. palmivora in vitro. * Inducive topographical signal provided by artificially scratched surface (see Materials and Methods). † ‘ low nutrient conditions ’ ¯ 5 % (v}v) pea broth, ‘ high nutrient conditions ’ ¯ 20 % (v}v) pea broth.
& Staples, 1991 ; Read et al., 1992 ; Mendgen & Deising, 1993). In most species, appressorium formation is affected by several factors, not all of which are known. In a general sense, they contribute to a conducive environment (Emmett & Parbery, 1975 ; Jelitto, Page & Read, 1994) for infection structure formation. In some, rather rare, cases appressoria appear to be induced by a single specific primary signal (Hoch & Staples, 1991 ; Read et al., 1992). In our study on P. palmivora, we found evidence for both of these possibilities. There apparently is a primary topographical signal on scratched substrates created by abrupt changes in the surface topography. In contrast to rust species (Hoch et al., 1987 ; Allen et al., 1991 a), in P. palmivora this primary signal does not appear to be narrowly defined since induction of appressorium formation was not affected by the strong variation in morphology of the grooves. A different situation was encountered on smooth surfaces. Here, both surface hydrophobicity and nutrient levels provided the main signals for appressorium induction. However, these factors were obviously interrelated and appressorium induction, or the lack of it, was the result of specific combinations of the two parameters, i.e. high or low surface hydrophobicity, high or low nutrient level. The experiments testing different components contained in the nutrient medium, which made use of a limited range of salts, sugars, amino acids and pH conditions, did not disclose a single factor to be responsible for the inhibitory activity of high nutrient levels. However, the results indicate that the effect of high nutrient levels on appressorium formation was
not simply due to an increased osmolality of the medium since none of the components tested at concentrations equal to that of the complete nutrient medium had any inhibitory activity. The aspects described above for P. palmivora are summarized in a model of signalling for appressorium induction (Fig. 20). It combines the data obtained in this study and also provides some speculation concerning interactions and possible ‘ hierarchies ’ among the inducive parameters. A major point included concerns the observation that a factor may overrule any positive or negative effect of another factor and, therefore, maintain a higher rank in the ‘ hierarchy ’. In this sense, ‘ contact with a surface ’ is located at the top, since it was a prerequisite for the induction of appressorium formation by either an inducive topographical signal or a hydrophobic contact surface under low nutrient conditions. ‘ Contact with an inducive topographical signal ’ is located below ‘ contact with a surface ’. The former factor induced appressorium formation independent of substrate hydrophobicity or nutrient conditions, factors which strongly affected appressorium formation on smooth substrates. ‘ Hydrophobic surface ’ and ‘ low nutrient conditions ’ which induced appressorium formation only in combination are located at the bottom. ‘ Hierarchy ’ in the sense of our model implies that two independent signal receptor mechanisms and}or signal transduction pathways are involved in appressorium induction in P. palmivora. One presumably becomes active after attachment and perception of an inducive topographical signal, while the other would be activated, also following attachment, through a combined action of low nutrient conditions and a hydrophobic contact surface. The various treatments given to the germlings in this study may have significance for the biology of the natural infection process. Thus, surface irregularities providing topographical signals appear, e.g. on edges of stoma guard cells, or in grooves over anticlinal walls, where the fungus may penetrate without entering the cytoplasm of host epidermal cells. Many pathogenic fungi (Hoch & Staples, 1991 ; Allen et al., 1991 b ; Read et al., 1992) including Phytophthora species (Sto$ ssel et al., 1980 ; Gees & Hohl, 1988) are known to form appressoria preferentially over these irregularities. Low nutrient levels and a hydrophobic surface also provide useful stimuli for appressorium formation, as aerial host surfaces are hydrophobic and lack high levels of nutrients. Furthermore, in P. palmivora, the level of nutrients influences the development of cysts. The presence of nutrients leads to the formation of a germ-tube. The absence of nutrients, suggesting that the encysted zoospore is not near a host, induces formation of a secondary zoospore (Hemmes & Hohl, 1971). Our results also show that germ-tubes may grow for several hours under noninducive conditions and then form appressoria upon encountering an appropriate signal, a property clearly of advantage for a pathogen growing on a host surface and attempting to gain access to the host tissue at an appropriate point of entry. Our in vitro system allows the induction or inhibition of appressorium formation by changing a single parameter such as the nutrient level or the surface topography. This offers the potential for unravelling the mechanisms and identifying
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402 Hoch, H. C. & Staples, R. C. (1991). Signaling for infection structure formation in fungi. In The Fungal Spore and Disease Initiation in Plants and Animals (ed. G. T. Cole & H. C. Hoch), pp. 25–46. Plenum Press : New York. Hoch, H. C., Staples, R. C. Whitehead, B., Comeau, J. & Wolf, E. D. (1987). Signaling for growth orientation and cell differentiation by surface topography in Uromyces. Science 235, 1659–1662. Hohl, H. R. & Balsiger, S. (1986). A model system for the study of fungus–host surface interactions : adhesion of Phytophthora megasperma to protoplasts and mesophyll cells of soybean. In NATO ASI Series, Vol. H4. Recognition in Microbe-Plant Symbiotic and Pathogenic Interactions (ed. B. Lugtenberg), pp. 259–272. Springer-Verlag : Berlin. Hohl, H. R. & Suter, E. (1976). Host–parasite interfaces in a resistant and a susceptible cultivar of Solanum tuberosum inoculated with Phytophthora infestans : leaf tissue. Canadian Journal of Botany 54, 1956–1970. Jelitto, T. C., Page, H. A. & Read, N. D. (1994). Role of external signals in regulating the pre-penetration phase of infection by the rice blast fungus, Magnaporthe grisea. Planta 194, 471–477. Mendgen, K. & Deising, H. (1993). Infection structures of fungal plant pathogens – a cytological and physiological evaluation. New Phytologist 124, 193–213. Pristou, R. & Gallegly, M. E. (1954). Leaf penetration by Phytophthora infestans. Phytopathology 44, 81–86. Read, N. D., Kellock, L. J., Knight, H. & Trewavas, A. J. (1992). Contact sensing during infection by fungal pathogens. In Perspectives in Plant Cell Recognition (ed. J. A. Callow & J. R. Green), pp. 137–172. Cambridge University Press : Cambridge, U.K. Sing, V. O. & Bartnicki-Garcia, S. (1972). Adhesion of zoospores of Phytophthora palmivora to solid surfaces. Phytopathology 62, 790. Sing, V. O. & Bartnicki-Garcia, S. (1975 a). Adhesion of Phytophthora palmivora zoospores : detection and ultrastructural visualization of concanavalin Areceptor sites appearing during encystment. Journal of Cell Science 19, 11–20. Sing, V. O. & Bartnicki-Garcia, S. (1975 b). Adhesion of Phytophthora palmivora zoospores : electron microscopy of cell attachment and cyst wall fibril formation. Journal of Cell Science 18, 123–132. Sto$ ssel, P., Lazarovits, G. & Ward, E. W. B. (1980). Penetration and growth of compatible and incompatible races of Phytophthora megasperma var. sojae in soybean hypocotyl tissues differing in age. Canadian Journal of Botany 58, 2594–2601. Ward, E. W. B., Lazarovits, G., Unwin, C. H. & Buzzell, R. I. (1979). Hypocotyl reactions and glyceollin in soybeans inoculated with zoospores of Phytophthora megasperma var. sojae. Phytopathology 69, 951–955.
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Environmental signalling during induction of appressorium formation in Phytophthora
U R S B I R C H E R A N D H A N S R. H O H L Institute of Plant Biology, University of ZuX rich, Zollikerstr. 107, CH-8008 ZuX rich, Switzerland
Appressorium formation in vitro by Phytophthora palmivora started after about 1 h of incubation of zoospores in an aqueous solution and was influenced by topographical signals, substrate hydrophobicity and available nutrients. Free floating, non-adhering germlings did not form appressoria. On smooth substrates, no appressoria were formed under high nutrient conditions (20 % pea broth) independent of the hydrophobicity of the contact surface, while under low nutrient conditions (5 % pea broth) appressoria formed on smooth substrates with hydrophobic, but not on smooth substrates with hydrophilic, surfaces. If the same substrates were scratched, appressoria formed over these scratches independent of levels of exogenous nutrients and substrate hydrophobicity. These findings are summarized in a model of signalling for appressorium induction.
Phytophthora palmivora is an important plant pathogen attacking a broad range of economically important crops in warmer regions of the world (Chee, 1974). It infects mainly aerial parts of its hosts, while root invasions have been described in only a few cases (Chee, 1974). As with other oomycetes, zoospores are involved in dispersal in the soil or on aerial parts of host plants. After contacting a solid surface, the motile zoospore is transformed into a non-motile, spherical cyst. Zoospores of P. palmivora become adhesive very early during encystment due to rapid secretion of adhesive material (Sing & Bartnicki-Garcia, 1972, 1975 a, b). On the leaf surface of black pepper (Piper nigrum) most of the encysted zoospores of P. palmivora strain P113 and P. capsici form single germ-tubes (Feuerstein & Hohl, 1986). Occasionally these germ-tubes grow into the narrow furrows around trichomes. The invading germ-tubes are wedged in the furrows and penetrate the epidermis without forming appressoria (Feuerstein & Hohl, 1986). In most cases, however, germlings form appressoria which are morphologically similar to appressoria formed by other Phytophthora species (Pristou & Gallegly, 1954 ; Sto$ ssel, Lazarovits & Ward, 1980 ; Gees & Hohl, 1988), over periclinal or anticlinal walls of epidermal cells, while access via stomata has not been described (Feuerstein & Hohl, 1986). Unlike the situation with some other pathogenic fungi (see Hoch & Staples, 1991 ; Hardham, 1992 ; Read et al., 1992 ; Mendgen & Deising, 1993) information about exogenous and endogenous signals regulating the induction of appressorium formation in Phytophthora is lacking. In the present study we present data obtained with an in vitro system which indicate that appressorium formation in Phytophthora is influenced by contact with a solid substrate, the surface topography, the substrate hydrophobicity and available nutrients.
MATERIALS AND METHODS Growth of the pathogen and production of zoospores Phytophthora palmivora (E. J. Butler) E. J. Butler strain P113 was used throughout this study. The fungus was grown in 9 cm plastic Petri dishes on Borlotti bean agar (Ward et al., 1979 ; Hohl & Balsiger, 1986). After 5 d at 24 °C in darkness the cultures were transferred to room temperature (approx. 21°) and daylight for 2 d, which induced sporangium formation. The sporangia from three plates were harvested with an inoculation loop and transferred into 50 ml sterile double distilled water and centrifuged at 10 g (rav 13 cm) for 2 min. The supernatant was discarded and the pellet containing sporangia was resuspended in 5 ml of sterile double distilled water. The suspension was incubated at 4° for 10 min and for an additional 15–20 min at 25° to allow emergence of zoospores. After filtration through a sterile 20 µm nylon mesh (Nybolt P20, Schweizerische Gazefabrik AG), to separate the zoospores from the sporangial cases, the concentration of zoospores was determined using a haemocytometer. The zoospore suspension was diluted with pea broth and double distilled water to a final concentration of 5¬10% zoospores ml−", and used immediately. Addition of pea broth did not induce rapid encystment of zoospores. For the preparation of 1 l of pea broth, 75 g of frozen garden peas were boiled in 250 ml of distilled water for 20 min, filtered, and 10 g sucrose, 1 g asparagine, 0±25 g MgSO \7H O and 0±5 g KH PO were added. The medium % # # % was made up to 1 l with distilled water (Hohl & Balsiger, 1986). Test substances and measurements of osmolality To assess the effects of exogenous nutrients on appressorium
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Table 1. Time course of germling differentiation of P. palmivora in tissue culture plates* Germ-tubes (%)
Hours of incubation at 25° 1 2 3 4 5 6
Germination (%) 95±5³0±9 98±3³0±9 99±6³0±6 99±7³0±3 99±9³0±2 100³0
Non-differentiated
With slightly swollen apex
With appressorium
With appressorium, hyphae and} or vesicles
70±2³1±8 8±0³1±6 4±2³0±9 1±7³0±7 0±8³0±8 0±8³0±5
29±8³1±8 6±9³1±2 1±8³0±7 0±9³0±7 0±1³0±2 0±6³0±5
0 84±9³1±7 77±6³1±8 69±7³2±3 53±2³2±1 30±3³1±8
0 0±3³0±3 16±5³1±8 27±7³2±5 45±8³2±0 68±4³2±2
* Germlings were incubated in 5 % (v}v) pea broth. At least 200 germlings were analysed per incubation time in each of three replicates. Three independent experiments were performed. Values represent mean and .. (n ¯ 9).
formation, the following substances were used : NaCl, KCl, CaCl , Ca(NO ) \4H O, MgSO \7H O purchased from # $# # % # Merck (Dietikon, Switzerland), -glucose, -sucrose, -maltose and -arabinose from Fluka (Buchs, Switzerland) and asparagine and thiamine-HCl from Sigma (Buchs, Switzerland). The osmolality of the different test substances was determined with a Roebling Osmometer (Vogel, Giessen, Germany).
Measurements of contact angles The contact angle of droplets of 5 % (v}v) and 20 % (v}v) pea broth on each of the test substrates was determined using a NRL Model 100.00 Contact Angle Goniometer (Rame! -Hart Inc., Mountain Lakes, U.S.A.). The mean value for each substrate was estimated by measuring contact angles of ten 0±5 µl drops (Clement et al., 1993). Germ-tube growth assay
Appressorium formation assay To study appressorium formation in vitro the following substrates were chosen : cellophane (jam jar-seals from Migros Genossenschaftsbund, Switzerland), 18¬18 mm glass coverslips (Knittelgla$ ser or Menzelgla$ ser, Merck, Dietikon, Switzerland), 35 mm diam. polystyrene Petri dishes (Greiner GmbH, Nu$ rtingen, Germany) and Teflon PFA 1000 LP film (Du Pont de Nemours International SA, Geneva). The time course analysis was performed using polystyrene 24-well flat-bottom tissue culture plates (Corning, New York). To provide topographically structured surfaces cellophane, polystyrene and Teflon were scratched with a brass brush and glass surfaces were scratched with a diamond pencil. Scratched substrates as well as non-scratched control substrates were cleaned with 70 % ethanol followed by extensive rinsing with sterile distilled water. The cleaned substrates were airdried in a laminar-flow cabinet prior to use. Single pieces (1±5¬2±0 cm or 2±0¬2±5 cm) of each of the different test substrates (except polystyrene) were placed in 35 mm diam. polystyrene Petri dishes. For polystyrene, the Petri dishes themselves served as test substrates. Three samples (100–150 µl) of 5¬10% zoospores ml−" suspended in 0–20 % (v}v) pea broth were incubated on each of the different test substrates at 25° in darkness. After incubation, appressorium formation was immediately checked. The experiments were performed in triplicate. In each of three independent experiments at least 200 germlings from each of the three samples were analysed (n ¯ 9). To check the position of appressoria in respect to scratches in test substrates, in each of three independent experiments, the position of at least 100 appressoria (whether over scratches or not) was determined after incubation.
Circular glass coverslips (12 mm diam.) were cleaned with 70 % ethanol, extensively rinsed with distilled water and autoclaved. Sterile coverslips were placed in 35 mm diam. polystyrene Petri dishes. Zoospores (5¬10% ml−") suspended in 100 µl of 20 % (v}v) or 5 % (v}v) pea broth were placed onto the coverslips. After incubation for 3 h at 25° germlings were fixed in a freshly prepared aqueous solution of 3±7 % (w}v) p-formaldehyde. Germ-tube lengths were determined with a graphic program (Metris, written by Nils Antonsen, Hauptstr. 44, CH-5200 Brugg) after digitalization from photomicrographs on a graphic tablet. The length of 100 germ-tubes derived from two independent experiments was determined. Light microscopy Appressorium formation was checked with an inverted microscope (Nikon, TMS-F) equipped with a 20¬Ph 2 DL objective (Nikon). Interference contrast observations were made on a Zeiss Axiophot microscope equipped with a PlanNEOFLUAR 40 (Ph 2) objective. Scanning electron microscopy To examine the geometry of grooves in dry scratched substrates, the latter were sputter-coated with gold before examination. Specimens were examined with a Hitachi S-4000 field emission scanning electron microscope. RESULTS Time course of infection structure formation Table 1 shows the results of a quantitative analysis of the formation of appressoria on tissue culture plates. Zoospores
U. Bircher and H. R. Hohl
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Figs 1–8. Sequence of morphological changes of P. palmivora during appressorium formation on polystyrene. Fig. 1. Adhering cyst. A detached flagellum is visible (arrow head). Fig. 2. Germling with a single germ-tube. Fig. 3. Early appressorium development as indicated by a slight broadening of the hyphal apex. Figs 4, 5. The length of the germ-tube between the cyst and the appressorium is highly variable. Figs 6, 7. After a period of maturation, finger-like projections and}or one or several vesicles (V) emerge from the mature appressoria (A). Fig. 8. Emerging hyphae often form additional appressoria. Bar represents 10 µm for all figures.
which adhered to the substrate rounded up, discarded their flagella, formed a cyst wall (Fig. 1) and germinated, mostly by producing single germ-tubes (Fig. 2). The germination rate of adhering cysts was over 95 % after 1 h of incubation. At this
time, 29±8 % of the germ-tubes showed a slight broadening of the hyphal apex (Fig. 3) as a first sign of appressorium formation. The length of the germ-tube between the cyst and the appressorium was highly variable (Figs 4, 5). After 2 h of
Induction of appressorium formation in Phytophthora
398 Table 2. Influence of different substances on the frequency of appressorium formation of P. palmivora on polystyrene
100
Germlings with appressoria (%)†
Germination rate (%)
80 Test substances* 60 40 20
0 0
5
10
15
20
Appressorium formation (%)
100
NaCl KCl CaCl # Ca(NO ) \4H O $# # MgSO \7H O % # KH PO }Na HPO \2H O # % # % # KH PO }Na HPO \2H O # % # % # KH PO }Na HPO \2H O # % # % # KH PO }Na HPO \2H O # % # % # -glucose -sucrose -maltose -arabinose -asparagine -asparagine thiamine-HCl (3 µ)
(pH 5±0) (pH 6±0) (pH 7±0) (pH 8±0)
Control 5 % (v}v) pea broth in distilled water 20 % (v}v) pea broth in distilled water
80
84±3³4±2 89±5³3±5 84±7³4±9 84±4³1±7 65±8³7±4 82±1³3±8 85±0³1±9 81±4³3±6 78±7³3±9 83±6³3±8 90±5³1±2 87±7³1±5 79±7³5±6 79±3³4±1 81±9³3±5
90±4³2±9 4±5³2±2
* Osmolality for all test substances 11±5 m except for the 5 % (v}v) pea broth control (2±5 m). † Germlings were incubated for 3 h at 25°. At least 200 germlings were analysed per test substance in each of three replicates. Three independent experiments were performed. Values represent mean and .. (n ¯ 9).
60 40 20
of germlings forming appressoria) nor in 5% (v}v) pea broth (0±6³0±7 % of germlings forming appressoria).
0 0
5 10 15 Pea broth concentration (%)
20
Fig. 9. Influence of pea broth concentration on germination rate and frequency of appressorium formation in P. palmivora on polystyrene. Germlings were incubated for 3 h at 25°. At least 200 germlings were analysed per treatment in each of three replicates. Three independent experiments were performed. Values represent mean and .. (n ¯ 9).
incubation more than 90 % of the germlings formed spherical or ovoid appressoria, morphologically similar to those formed on host leaves (Feuerstein & Hohl, 1986 ; Gees & Hohl, 1988). Subsequently, finger-like projections (Fig. 6) and}or one or several vesicles (Fig. 7) emerged from the mature appressoria. Emerging hyphae often formed additional appressoria (Fig. 8). After 6 h of incubation, more than 98 % of the adhering germlings had developed appressoria. Up to this stage no pseudoseptum (callosity) (Gooday & Hunsley, 1971 ; Hohl & Suter, 1976) separating the germ-tube from the appressorium was visible by light microscopy. Effects of contact with substrate on appressorium formation Zoospores were incubated in 5 % (v}v) or 20 % (v}v) pea broth in Petri dishes rotating at 150 rpm on a shaker to prevent adhesion of the growing germlings to the substrate. After 3 h of incubation no appressoria were formed by free floating germlings, neither in 20 % (v}v) pea broth (0±1³0±2 %
Effects of exogenous nutrients on appressorium formation on smooth substrates The data shown in Fig. 9 indicate that in germlings of P. palmivora, exogenous nutrients influenced the germination rate and the frequency of appressorium formation on polystyrene after 3 h of incubation. High nutrient concentrations suppressed, while low concentrations were conducive to, appressorium formation. P. palmivora was able to produce appressoria using endogenous reserves exclusively, since appressoria were formed under nutrient-free conditions (Fig. 9). Under these conditions, formation of secondary zoospores from cysts (Hemmes & Hohl, 1971) was also observed frequently. From the results presented in Fig. 9 two pea broth concentrations were chosen for subsequent experiments : 5 % (v}v) pea broth (‘ low nutrient conditions ’) leading to high germination rates as well as high frequencies of appressorium formation on polystyrene, and 20 % (v}v) pea broth (‘ high nutrient conditions ’) leading to high germination rates but very low frequencies of appressorium formation on polystyrene. In the following experiments we tested the influence of different ions, amino acids and sugars on appressorium formation on polystyrene (Table 2). The test substances were applied at the same osmolality as a 20 % (v}v) pea broth solution (11±5 m), which inhibited appressorium formation (Fig. 9). The test substances were added after 20 min of incubation in 20 % (v}v) pea broth to allow adhesion and cyst formation in the pea broth solution. Of the tested substances,
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399
Table 3. Influence of substrate hydrophobicity and exogenous nutrients on the frequency of appressorium formation of P. palmivora in vitro Germlings with appressoria (%)* Smooth substrate
5 % pea broth
Φ† "
20 % pea broth
Φ† #
Teflon 87±3³5±1 89³3 0±6³0±4 89³3 Polystyrene 84±3³3±6 62³4 1±7³1±3 62³3 Glass 0±9³0±5 37³3 0±6³0±5 36³2 Cellophane 2±3³1±3 17³2 1±2³0±4 15³3 * Germlings were incubated for 3 h at 25°. At least 200 germlings were analysed per test substrate in each of three replicates. Three independent experiments were performed. Values represent mean and .. (n ¯ 9). † Contact angles measured with 5 % (v}v) pea broth (¯ Φ ) or 20 % (v}v) " pea broth (¯ Φ ). #
only MgSO \7H O showed a slight inhibitory effect on % # appressorium formation on polystyrene after 3 h of incubation. Appressorium formation on this substrate was not affected by the pH within the range tested (pH 5±0–8±0). Effects of substrate hydrophobicity on appressorium formation on smooth substrates The effect of the hydrophobicity of a contact surface on appressorium formation in vitro was determined using a variety of substrates (Table 3). It cannot be excluded,
however, that physical factors other than hydrophobicity also differed among these substrates. Under high nutrient conditions, very few appressoria were formed. This was true for hydrophilic as well as for hydrophobic substrates. Under low nutrient conditions appressoria were formed almost exclusively on substrates with hydrophobic surfaces. On hydrophilic substrates appressorium formation occurred only at very low levels. Germ-tube growth was somewhat affected by the pea broth concentration. After incubation for 3 h in 5 % (v}v) or 20 % (v}v) pea broth on glass, germlings formed germ-tubes of 64±2³24±6 µm and 74±9³26±6 µm in length, respectively (n ¯ 100). Appressorium formation on scratched surfaces Figures 10–17 show examples of grooves on the different test substrates. The morphology and the distribution of the grooves varied considerably between different substrates and also for each substrate. On each of the substrates tested some germ-tubes grew into and along a groove and left it again without any apparent morphological differentiation. However, on different parts of the same ridge or elsewhere on the same substrate, germtubes were stimulated to differentiate appressoria (Figs 18, 19).
Figs 10–17. Scanning electron micrographs of grooves on scratched test substrates. Figs 10, 14. Cellophane. Figs 11, 15. Glass. Figs 12, 16. Polystyrene. Figs 13, 17. Teflon. Bars represent 10 µm in Figs 10–13 and 50 µm in Figs 14–17.
Induction of appressorium formation in Phytophthora
400
Figs 18, 19. Appressoria formed on scratched substrates. Fig. 18. Polystyrene. Fig. 19. Glass. Germlings were incubated in 20 % (v}v) pea broth which inhibits appressorium formation on smooth substrates (see Table 3). Bar represents 10 µm for both figures.
Table 4. Influence of scratches in substrates with different surface hydrophobicities on appressorium formation of P. palmivora in vitro*
Substrate
Germlings forming appressoria (%)†
Appressoria positioned over scratches (%)‡
Teflon Polystyrene Glass Cellophane
50±5³13±5 47±8³22±5 9±7³3±9 52±5³14±4
92±7³0±9 94±4³3±1 96±0³2±0 94±4³1±6
* Germlings were incubated in 20 % (v}v) pea broth for 3 h at 25°, which inhibits appressorium formation on smooth substrates (see Table 3). † At least 200 germlings were analysed per test substrate in each of three replicates. Six independent experiments were performed. Values represent mean and .. (n ¯ 18). ‡ At least 100 germlings were analysed in each of three independent experiments. Values represent mean and .. (n ¯ 3).
In Table 4 the results of a quantitative analysis of the influence of scratches in the test surfaces on appressorium formation are shown. The experiments were performed with 20 % (v}v) pea broth, which inhibited appressorium formation on the corresponding smooth substrates (Table 3). The results demonstrate that appressoria formed on scratched hydrophilic as well as on scratched hydrophobic surfaces after 3 h of incubation. The frequencies of appressorium formation ranged between 2±9 and 91±5 % with the lowest values on glass. The ..s of the mean were very high compared to those obtained on smooth substrates (Table 3). On each of the test substrates more than 92 % of appressoria were situated over grooves (Table 4).
Effects of changes in environmental conditions on appressorium formation The following experiments were performed to determine whether or not germlings growing under non-inducive conditions were able to form appressoria after conditions had been changed to become inducive. In the first experiment, germlings incubated on polystyrene in 20 % (v}v) pea broth for 3 h (non-inducive conditions) did not form appressoria. After the 20 % (v}v) pea broth solution was diluted to 5 % (v}v) with sterile distilled water (inducive conditions), 95±0³2±1 % of the germlings developed appressoria within 3 h, in some cases with hyphae and}or vesicles. In the second experiment, germlings were incubated in Petri dishes rotating at 150 rpm on a shaker to prevent adhesion of the germlings to the surface of the substrate (noninducive conditions). After incubating these germlings for an additional 3 h in still conditions on polystyrene, thereby allowing them to settle and adhere to the substrate, 83±3³3±6 % of the germlings incubated in 5 % (v}v) pea broth (inducive conditions) produced appressoria while only 2±5³1±2 % of those incubated in 20 % (v}v) pea broth (noninducive conditions) produced appressoria.
DISCUSSION Appressorium formation in phytopathogenic fungi may be influenced by a variety of physical and chemical signals (Hoch
U. Bircher and H. R. Hohl
401
Contact with surface ? yes
Contact with inducive topographical signal ? * yes Appressorium formation
Undifferentiated mycelial growth
no Contact surface hydrophobic ? yes
Low nutrient conditions ? † yes
Appressorium formation
no
no Undifferentiated mycelial growth
no
Undifferentiated mycelial growth
Fig. 20. Model for the induction of appressorium formation in P. palmivora in vitro. * Inducive topographical signal provided by artificially scratched surface (see Materials and Methods). † ‘ low nutrient conditions ’ ¯ 5 % (v}v) pea broth, ‘ high nutrient conditions ’ ¯ 20 % (v}v) pea broth.
& Staples, 1991 ; Read et al., 1992 ; Mendgen & Deising, 1993). In most species, appressorium formation is affected by several factors, not all of which are known. In a general sense, they contribute to a conducive environment (Emmett & Parbery, 1975 ; Jelitto, Page & Read, 1994) for infection structure formation. In some, rather rare, cases appressoria appear to be induced by a single specific primary signal (Hoch & Staples, 1991 ; Read et al., 1992). In our study on P. palmivora, we found evidence for both of these possibilities. There apparently is a primary topographical signal on scratched substrates created by abrupt changes in the surface topography. In contrast to rust species (Hoch et al., 1987 ; Allen et al., 1991 a), in P. palmivora this primary signal does not appear to be narrowly defined since induction of appressorium formation was not affected by the strong variation in morphology of the grooves. A different situation was encountered on smooth surfaces. Here, both surface hydrophobicity and nutrient levels provided the main signals for appressorium induction. However, these factors were obviously interrelated and appressorium induction, or the lack of it, was the result of specific combinations of the two parameters, i.e. high or low surface hydrophobicity, high or low nutrient level. The experiments testing different components contained in the nutrient medium, which made use of a limited range of salts, sugars, amino acids and pH conditions, did not disclose a single factor to be responsible for the inhibitory activity of high nutrient levels. However, the results indicate that the effect of high nutrient levels on appressorium formation was
not simply due to an increased osmolality of the medium since none of the components tested at concentrations equal to that of the complete nutrient medium had any inhibitory activity. The aspects described above for P. palmivora are summarized in a model of signalling for appressorium induction (Fig. 20). It combines the data obtained in this study and also provides some speculation concerning interactions and possible ‘ hierarchies ’ among the inducive parameters. A major point included concerns the observation that a factor may overrule any positive or negative effect of another factor and, therefore, maintain a higher rank in the ‘ hierarchy ’. In this sense, ‘ contact with a surface ’ is located at the top, since it was a prerequisite for the induction of appressorium formation by either an inducive topographical signal or a hydrophobic contact surface under low nutrient conditions. ‘ Contact with an inducive topographical signal ’ is located below ‘ contact with a surface ’. The former factor induced appressorium formation independent of substrate hydrophobicity or nutrient conditions, factors which strongly affected appressorium formation on smooth substrates. ‘ Hydrophobic surface ’ and ‘ low nutrient conditions ’ which induced appressorium formation only in combination are located at the bottom. ‘ Hierarchy ’ in the sense of our model implies that two independent signal receptor mechanisms and}or signal transduction pathways are involved in appressorium induction in P. palmivora. One presumably becomes active after attachment and perception of an inducive topographical signal, while the other would be activated, also following attachment, through a combined action of low nutrient conditions and a hydrophobic contact surface. The various treatments given to the germlings in this study may have significance for the biology of the natural infection process. Thus, surface irregularities providing topographical signals appear, e.g. on edges of stoma guard cells, or in grooves over anticlinal walls, where the fungus may penetrate without entering the cytoplasm of host epidermal cells. Many pathogenic fungi (Hoch & Staples, 1991 ; Allen et al., 1991 b ; Read et al., 1992) including Phytophthora species (Sto$ ssel et al., 1980 ; Gees & Hohl, 1988) are known to form appressoria preferentially over these irregularities. Low nutrient levels and a hydrophobic surface also provide useful stimuli for appressorium formation, as aerial host surfaces are hydrophobic and lack high levels of nutrients. Furthermore, in P. palmivora, the level of nutrients influences the development of cysts. The presence of nutrients leads to the formation of a germ-tube. The absence of nutrients, suggesting that the encysted zoospore is not near a host, induces formation of a secondary zoospore (Hemmes & Hohl, 1971). Our results also show that germ-tubes may grow for several hours under noninducive conditions and then form appressoria upon encountering an appropriate signal, a property clearly of advantage for a pathogen growing on a host surface and attempting to gain access to the host tissue at an appropriate point of entry. Our in vitro system allows the induction or inhibition of appressorium formation by changing a single parameter such as the nutrient level or the surface topography. This offers the potential for unravelling the mechanisms and identifying
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