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Program for the Biology of Filamentous Fungi, Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas 77843
G.C. Carroll
Department of Biology, University of Oregon, Eugene, Oregon 97403
H.C. Hoch 1
Department of Plant Pathology, Cornell University, New York State Agricultural Experiment Station, Geneva, New York 14456
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ABSTRACT |
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It has been shown that conidia of Phyllosticta ampelicida require attachment to a substratum to initiate germination. Furthermore this attachment occurs only on hydrophobic surfaces. This study was initiated to ascertain the breadth of this phenomenon among other species of the genus Phyllosticta. We tested 23 isolates of Phyllosticta representing at least 14 named species. These isolates were collected from North America, Asia and Africa. For 22 of the 23 isolates tested spore attachment occurred at a rate of 60–100% on hydrophobic polystyrene but at 0–5% on hydrophilic polystyrene. The one exception to the preference for a hydrophobic substratum for attachment was an unnamed species of Phyllosticta from Rhus glauca that attached less than 10% on either surface. A similar response was observed when assaying germination and appressorium formation for 17 isolates. Germination and appressorium formation for these isolates proceeded on hydrophobic polystyrene but not on nutrient agar, which is hydrophilic. In five of the tested isolates germination was high on both hydrophobic polystyrene and hydrophilic nutrient media. The isolate from Rhus glauca did not germinate appreciably on either surface. Taken together these results suggest that the requirement for conidium contact/attachment to trigger germination is pervasive to the genus Phyllosticta.
Key words: adhesion, appresoria, Guignardia bidwellii, hydrophilic, hydrophobic
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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said that "the length of time for germination varies considerably and for no known reason", and that "often [Reddick] has not observed germination in less than twenty-four or even thirty-six hours". Viala and Ravaz (1888) indicated that similar spores germinated in 3 or 4 h. Until recently little was known regarding requirements for germination of P. ampelicida conidia on host surfaces other than such general parameters as temperature, moisture and pH regimes. Using better microscopes and more refined observational methods, initial morphological signs of germination have been consistently noted at 40–60 min (>90% germination) when the spores were deposited on "green" grape tissues, with mature appresoria present by 6 h (Kuo and Hoch 1996b). Germination in vitro on artificial media has been even more difficult to achieve. Rates of conidium germination were reported to be low, even during extended periods (24–36 h). For example, on nutrient media such as yeast-extract agar, germination was about 7% after 24 h at 25 C, a rate significantly less than on plant tissues (Caltrider 1961). Recent studies elucidated an important and overriding factor that promotes spore germination in P. ampelicida, whether on host or artificial substrata. That factor is contact and attachment of the spore with a substratum (Kuo and Hoch 1995, 1996, 1996b; Shaw and Hoch 1999, 2000; Shaw et al 1998). Such an interaction somehow signals initiation of the germination process. Furthermore studies by Hoch and colleagues (Kuo and Hoch 1995, 1996a, b; Shaw and Hoch 1999, 2000; Shaw et al 1998) have demonstrated that contact, and thus germination, is best achieved on hydrophobic substrata such as a grape leaf or an inert surface (e.g. polystyrene). Conidia do not readily attach to hydrophilic substrata such as agar media, clean glass or sulfonated polystyrene and thus do not germinate. These studies have lead to the conclusion that conidia of P. ampelicida will not germinate unless they contact and attach to a surface. Furthermore there is a strong preference for a hydrophobic surface for attachment to occur.
The observation that contact/attachment to a substratum triggers germination of spores is possibly more common than has been documented. For a few fungal species it has been noted that conidia germinated poorly if they were not in contact with a substratum. For example Colletotrichum trifolii and C. graminicola conidia apparently failed to germinate unless they were in contact with a substratum or unless they were in the presence of a carbon source (Dickman et al 1995, Warwar and Dickman 1996, Chaky et al 2001). For Magnaporthe grisea it was noted that conidia did not germinate in water if they were maintained in suspension or unless nutrients were added. If spores were allowed to settle onto a substratum, germination appeared to be enhanced regardless of the nutritional status of the medium (Lee and Dean 1993) and germination is generally perceived to occur very quickly after the conidium adheres to a surface (Hamer et al 1988, Talbot 1995). Similarly Bipolaris sorokiniana spores germinated at significantly higher rates when they were attached to a substratum (Apoga et al 2001). Conidia of Phyllosticta sphaeropsoidea, the causal agent of leaf blotch of horse chestnut, also have been documented with low germinated rates (Stewart 1916, Hudson, 1987). The clear documentation for the contact-germination phenomenon in Phyllosticta ampelicida and the casual notations for a few other pathogenic fungi indicate that such prerequisites may be more general and important than had been thought.
Experimental studies have been narrowly focused on P. ampelicida, thus we were interested in knowing whether we were working with a species that was somewhat unique (excepting the few reports as noted above) in its germination requirements or whether this phenomenon was characteristic of other Phyllosticta species as well. Thus our goal was to ascertain the breadth of the contact/attachment-germination phenomenon among other Phyllosticta species.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Isolates were maintained on one-half strength potato-dextrose agar (Difco, Detroit, Michigan) with the agar content readjusted to 1.5% at 25 C under continuous fluorescent light as reported by Kuo and Hoch (1995). For some isolates enhancement of conidium production was achieved by growing the isolates on microfiber glass filters (Whatman, Clifton, New Jersey) overlaid on the culturing media (G. Carroll unpubl). For routine use isolates were transferred weekly to fresh media, otherwise they were stored (after growth) on microfiber glass filters at –80 C.
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Attachment assays.— –
A total of 10 µL of the spore suspensions were placed on either hydrophobic (polystyrene) or hydrophilic (sulfonated polystyrene) substrata (Holboke and Pinnell 1989, Kuo and Hoch 1996a, Rubin 1966). The spores were allowed to settle onto these surfaces for 15 min at which time the total number of spores in five image fields (with a 40x objective, Olympus IMT-2 microscope) were determined. This equated 50–100 spores/image field. The membranes held by forceps were washed immediately with a strong stream from a faucet (flow ca. 2.5 L/min) for 3 s to remove any nonattached spores. Conidia remaining attached to the membranes after washing were counted as described above. The attachment assay was replicated with three aliquots of spore suspension (three separate drops) on each of three membranes with each species being tested on three separate occasions. Data are expressed as the percentage of adherent spores for each assay.
Germination and appressorium formation assays.— –
Germination and appressorium formation was assessed on both hydrophobic and hydrophilic surfaces. Polystyrene served as a hydrophobic surface and has been shown to support germination rates indistinguishable from that which occurs on host surfaces (Kuo and Hoch 1995). Potato-dextrose agar (PDA) (1.5%, Difco) coated glass cover slips served as a hydrophilic surface (with a nutrient base) and was used to assess conditions previously shown to support minimal germination from P. ampelicida conidia (Kuo and Hoch 1996a). Three replicate drops (10 µL of spore suspension) were applied to each of three replicated test surfaces. All test materials were placed in a humidified chamber and incubated at room temperature. One hundred conidia were counted within each drop after 6 and 24 h incubation with either a Zeiss Axiophot or an inverted IMT-2 Olympus microscope. Six h as the minimum incubation time was chosen because it was determined previously that P. ampelicida conidia readily germinate and form mature appresoria on both host leaf and polystyrene surfaces within this time period.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The one exception to the preference for a hydrophobic substratum was Phyllosticta sp. from Rhus glauca; that attachment rate was less than 9% on either surface.
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) were assayed for germination and appressorium formation on both polystyrene and a thin coating of nutrient agar on microscope slides (FIGS. 2, 3). Seventeen isolates exhibited germination rates of 30–100% on hydrophobic polystyrene but exhibited germination less than 10% on hydrophilic nutrient agar. Germination percentages were generally not significantly higher at 24 h than at 6 h, the exception being Phyllosticta sp. from Viola sempervirens in which the germination rate increased from 17–92% at 24 h. Five of the isolates, however, germinated at high rates on both hydrophobic and hydrophilic substratum (FIG. 3). Similar to the other 17 isolates, Phyllostica from Sarcostemma viminale does germinate at significantly higher rates on the hydrophobic substratum at 6 h, but by 24 h germination is ca. 90% on both substratum types. The other four isolates germinate at appreciable levels on the hydrophilic substratum even at 6 h. The isolate from Rhus glauca did not germinate appreciably on either surface. This was not surprising given that conidia of this isolate did not attach to the substratum effectively during adhesion assays (FIG. 1).
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, 3). Phyllosticta from Rhus glauca did not germinate at rates high enough to assess appressorium formation on either substratum. In all of the cultures tested we noticed conidia that did germinate on agar produced a single unbranched germ tube that ceased growth after about 20 h and died without establishing a mycelial colony. Therefore mycelial colonies are propagated only by serial transfer of mycelia initially derived from diseased plant tissue. We believe that Phyllosticta sp. conidia require appressorium formation to switch their developmental program to vegetative mycelial growth.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). None of these events occurred on hydrophilic surfaces, including sulfonated polystyrene, glass cover slips or agar (nutrient or water). In addition conidium germination also did not occur when spores were held in a hanging drop (i. e. were not allowed to settle onto a surface). We have described three developmental events for conidia from each isolate: (i) conidium attachment, (ii) conidium germination and (iii) appressorium formation. For each of these events we assessed each stage on both a hydrophobic and a hydrophilic surface. We conducted preliminary experiments to determine the most suitable substrata on which to conduct each assay. We found that for attachment assays polystyrene and sulfonated polystyrene were the most suitable substrata because (i) these substrata were easily manipulated for microscopic analysis, (ii) these substrata were readily placed under the water stream to actuate spore detachment and (iii) this was the substratum on which P. ampelicida strain PY3 spore attachment assays were performed and our results with P. ampelicida strains closely matched previous results. For our assays of conidium germination and appressorium formation we initially tested the phenomenon on numerous surfaces including hydrophobic polystyrene as well as hydrophilic surfaces such as sulfonated polystyrene, oven-muffled cover glasses and nutrient agar. On all hydrophilic surfaces germination and appressorium formation percentages were similar (data not shown); however we quickly determined that over long periods (approaching 24 h) it was impossible to keep the conidia in liquid suspension because the water droplet readily spread across the entire surface, creeping to and even over the edge of the surface due to the acute advancing angle of the water on each hydrophilic surface. This droplet then quickly dried even in humidified chambers. Such surfaces were adequate for the short term attachment assays but proved impossible to use for germination and appressorium formation. We experimented with hydrophobic barriers such as a wax ring, silicone rubber masks and lanoline, but in all cases germination was affected (usually inhibited) when the same material was placed on inductive hydrophobic surfaces as a control. We therefore decided to use nutrient agar as our experimental hydrophilic surface. We reasoned that (i) this was the most appropriate surface because the spores readily were retained in liquid suspension for the duration of the experiment and (ii) the presence of nutrients in the media would provide ample opportunity for initiation of germination if it was a factor in germination. Finally it should be noted that in our visual assessment of germination and appressorium formation we also could assess conidium attachment, although not quantitatively. For all isolates where germination did not occur the conidia were observed to be nonattached, moving about in a random Brownian-like motion within the water on the medium surface. Conversely in all isolates for which germination was observed the germlings were not moving as has been reported by Kuo and Hoch (1996a). Unpublished work (Hoch and Kuo) using the PY3 isolate established that nonattached and thus nongerminated conidia remained viable and capable of germination for extended periods. Using the double-sided chamber (Kuo and Hoch 1996a) conidia that did not attach (or germinate) after 10 h, germinated within 2 h after the holder was flipped over to let the same conidia settle and attach to the hydrophobic surface.
The isolates we chose are geographically diverse, originating from throughout the United States, Asia and Africa and isolated from a wide range of host plants (TABLE I). We also choose three isolates identified as P. ampelicida, the species studied with the most frequency for spore germination triggers (Kuo and Hoch 1995, 1996a, Kuo and Hoch b; Shaw and Hoch 1999, 2000; Shaw et al 1998). In addition we choose five isolates from unique hosts identified as P. capitalensis because this species is a ubiquitous endophyte with a broad host range (Baayen 2002, Okane et al 2003, Pandey et al 2003). Of these isolates six are identified as Phyllosticta sp., followed by the host from which they were isolated. Each has a unique ITS sequence (G. Carroll unpubl) and therefore is likely to represent a unique species yet to be described. A final determinate for inclusion in this study was our ability to maintain the conidiation capacity of the isolate. Phyllosticta species can be exceptionally difficult to maintain in a state primed for conidiation in the laboratory. Many, including several P. citricarpa isolates, were dropped from our study because of this difficulty.
All species of Phyllosticta, with the exception of Phyllosticta from Rhus glauca, exhibited a similar preference for hydrophobic substrata and for the induction of conidial germination as reported for P. ampelicida (Kuo and Hoch 1996, Shaw and Hoch 1999). This may not be surprising because all of the conidia assayed displayed the preformed gelatinous sheath that characterizes the genus Phyllosticta. While it has not been shown that the gelatinous sheath is directly involved in adhesion, the fact that it surrounds each conidium and is thus the first layer to contact the substratum, deduction would infer this role. Such an adhesive material with a role similar to that of the spore tip mucilage of M. grisea (Hamer et al 1988) is preformed and competent for use at spore maturity. Active metabolism is not required for attachment. Unlike M. grisea however the adhesive sheath of Phyllosticta covers the spore surface and is exposed to the environment at maturity (Shaw and Hoch 1999). The adhesive mucilage of Phyllosticta is unlike many other attachment systems that require active metabolism (e.g. Nectria haematococca [Kwon and Epstein 1997], Cochliobolus heterostrophus [Braun and Howard 1994] Bipolaris sorokiniana [Apoga et al 2001] and Blumeria graminis [Carver et al 1999]).
All isolates that germinated developed appresoria only on hydrophobic surfaces, similar to that of P. ampelicida as shown in this study and by Shaw et al (1998). Seventeen of the 23 isolates germinated and formed appresoria at appreciable levels only on hydrophobic substrata. The link between hydrophobic surfaces and appressorium formation is well established in many species (Staples and Hoch 1995).
For germination, however, this study represents the most comprehensive one to date demonstrating a preference for hydrophobic substrata to initiate germination of conidia. Why evolve a requirement for contact/attachment to a hydrophobic surface to trigger germination? Phyllosticta species are typically found growing on green tissue of plants that is hydrophobic due to the waxy, cuticle coating.
A spore that does not germinate until it encounters a hydrophobic surface increases its chances of germinating on a host that can support the organism. We have shown that both spore germination and appressorium formation are dependant on free external calcium ions and that these processes could be inhibited by pharmacological antagonists of calcium channels (Shaw and Hoch 2000). We have hypothesized that the act of attachment to the substratum deforms the spore plasma membrane, therefore opening calcium channels. The influx then sets in motion calcium-mediated signaling pathways leading to germination. In Aspergillus nidulans both RAS and cAMP signaling pathways have been implicated in spore germination control (Osherov and May 2001, Filinger et al 2002). Future work with P. ampelicida is necessary to determine precisely how signaling for germination proceeds. It has been suggested that the lipophilic, germination self-inhibitor pyriculol from M. grisea diffuses across the hydrophobic substratum therefore releasing the conidium to germinate (Hedge and Kolattukudy 1997). It is possible that a similar mechanism is operative in Phyllosticta species. Adhesion as a prerequisite for germination also has been documented for two aquatic fungi, Anguillospora longissima and Lunulospora curvula (Webster and Davey 1984). The conidia of Colletotrichum graminicola also appear to be stimulated to germinate on attachment to a hydrophobic surface, although this can be bypassed by the addition of a carbon source (Chaky et al 2001). Clearly more work is necessary to identify the precise means by which spore germination is triggered in numerous fungal systems.
In contrast to the 17 isolates mentioned above (FIG. 2) five isolates germinated readily on hydrophilic surfaces (FIG. 3), several of which exhibited high levels of germination by 24 h. The mechanism that allows for this is not understood. It is possible that these isolates might have evolved this ability because their hosts have fewer hydrophobic surfaces or they might belong to an outgroup of Phyllosticta that has lost a negative germination regulator. Unpublished ITS data from G. Carroll’s lab does not support this latter theory, however. While we have not resolved this question we are certain that all isolates are indeed Phyllosticta species due to morphological characteristics including spore shape, the presence of characteristic lipid bodies and the presence of the gelatinous sheath and appendage distinctive of the genus (van der Aa 1973). Following current species concepts no delineation can be drawn between different Phyllosticta species based on their teleomorphic connection because all described Phyllosticta species that have a known sexual stage have Guignardia teleomorphs.
Another characteristic of Phyllosticta conidia is that germination is "terminal" on agar media. Unlike most other plant pathogenic fungi, conidia of Phyllosticta develop unbranched germ tubes on agar, cease growth and within 20 h die. It appears that members of the genus Phyllosticta require appressorium formation to switch their developmental program to vegetative mycelial development. Mycelial cultures, to our knowledge, are not derived from Phyllosticta conidia in any of the tested species.
Previous work with P. ampelicida established that within 6 h germination and appressorium differentiation approached 100% (Kuo and Hoch 1996a). For this reason we assayed all isolates at 6 h. It became apparent for some species that the germination potential was not fully realized by 6 h, therefore we extended the assay for all isolates to 24 h. For the 17 isolates that have a substratum preference similar to P. ampelicida there was no appreciable difference in substratum response between 6 h and 24 h. For a few (especially Phyllosticta sp. from Sarcostemma viminale and P. encephalarti) germination increased markedly on the hydrophilic surface with an extended time. It is possible that the film of water that normally separates the spore from a hydrophilic surface was diminished, either by evaporation or absorption into the agar medium, therefore letting the conidium make contact with the normally noninductive surface, triggering spore germination. However, because these isolates consistently demonstrated this enhanced germination rate with time, it is likely that other mechanisms allowing germination are involved. Similar trends in elevated germination in vitro at 24 h, and even 72 h, have been noted for P. ampelicida (Caltrider 1961, Reddick 1911) and P. sphaeropsoidea (Hudson 1987, Stewart 1916), although at rates that are low compared to those reported by others (Kuo and Hoch 1995, a, 1996b; Shaw and Hoch 1999, 2000; Shaw et al 1998).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: hch1{at}cornell.edu
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LITERATURE CITED |
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