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Biomechanical interaction between hyphae of two Pythium species (Oomycota) and host tissues
Fungal Genetics and Biology 37 (2002) 245–249 www.academicpress.com
Biomechanical interaction between hyphae of two Pythium species (Oomycota) and host tissues Erin MacDonald, Laurie Millward, J.P. Ravishankar, and Nicholas P. Money* Department of Botany, Miami University, Oxford, OH 45056, USA Received 21 February 2002; accepted 26 July 2002
Abstract Forces exerted by hyphae of the phytopathogen Pythium graminicola and mammalian pathogen Pythium insidiosum were compared with the mechanical resistance of their hostsÕ tissues. Hyphal apices of both species exerted a mean force of 2 lN, corresponding to mean pressures of 0:19 lN lm2 (or MPa) for P. graminicola, and 0:14 lN lm2 for P. insidiosum. Experiments with glass microprobes showed that the epidermis of grass roots resisted penetration until the pressure applied at the probe tip reached 1–12 lN lm2 . Previously published data show that mammalian skin offers even greater resistance (10–47 lN lm2 ). Clearly, tissue strength exceeds the pressures exerted by hyphae of these pathogens, verifying that secreted enzymes must play a critical role in reducing the resistance of plant and animal tissues. It is presumed that hyphae are sufficiently powerful to bore through any obstacles remaining after enzyme action. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Invasive hyphal growth; Kingdom Stramenopila; Pythium graminicola; Pythium insidiosum; Tissue penetration; Turgor pressure
1. Introduction Pathogenic fungi invade plant and animal tissues by secreting lytic enzymes and exerting force at their hyphal apices (Money, 2001). Many years of inconclusive gene disruptions failed to associate secreted enzymes with fungal virulence (Bastmeyer et al., 2002), until Tonukari et al. (2000) succeeded in demonstrating their importance by the targeted inactivation of SNF1 in Cochliobolus carbonum, a gene that controls the expression of several tissue-degrading enzymes. Even though the necessity for enzymes is now close to proven for C. carbonum (and for a handful of other fungi), gene inactivation studies do not discriminate between enzymes that are necessary for nutritional purposes and those that dissolve physical barriers. Many secreted enzymes probably serve both purposes, but it would be useful to identify those that have the greatest effect upon tissue strength. The relative importance of enzymes versus mechanics is not known for most interactions *
Corresponding author. Fax: 1-513-529-4243. E-mail address: [email protected] (N.P. Money).
between fungi and their plant or animal hosts, but is a crucial consideration whether we are interested primarily in understanding or in controlling a particular disease. It is certain that enzyme-catalyzed dissolution of tissues dominates some interactions, while mechanical force plays a greater role in others. Analysis of the secreted enzymes and the mechanical characteristics of a particular fungus offers the best prospects for ‘‘solving’’ its invasive mechanism. In a recent study, Ravishankar et al. (2001) demonstrated that the mammalian pathogen Pythium insidiosum (an oomycete fungus or stramenopile) must achieve a decisive reduction in tissue strength by proteinase secretion before it can penetrate cutaneous and sub-cutaneous tissues. This conclusion was reached by comparing the pressures exerted by single hyphae with the resistance of human and equine skin to needle insertion. In the present paper, we report the application of the same methodology to analyze the invasive mechanism utilized by Pythium graminicola. This oomycete is a widespread pathogen of graminaceous hosts that causes root rot and feeder root necrosis in a number of crop plants (Agrios, 1997; Deacon, 1997).
1087-1845/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 8 7 - 1 8 4 5 ( 0 2 ) 0 0 5 1 4 - 5
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E. MacDonald et al. / Fungal Genetics and Biology 37 (2002) 245–249
These data are compared with new measurements from a clinical isolate of P. insidiosum which was not included in the study by Ravishankar et al. (2001). Both Pythium species initiate infection with a biflagellate zoospore that attaches to the host surface and operates as a platform from which an infection hypha extends into the underlying tissue. The experiments indicate that the strength of intact host tissues exceeds the pressure exerted by vegetative hyphae of either Pythium species, verifying that tissue-degrading enzymes must effect a significant reduction in tissue strength to allow hyphal penetration of plant or animal tissues.
2. Materials and methods 2.1. Organisms and culture media Pythium graminicola Subramanian strain 96600 was obtained from the American Type Culture Collection (ATCC), Rockville, MD. It was originally isolated from an infected sugarcane root in Louisiana. P. insidiosum De Cock et al. ATCC strain 76049 was isolated from a human corneal infection in Haiti. The case history was described by Virgile et al. (1993) and a more recent report of P. insidiosum keratitis in Malaysia was published by Badenoch et al. (2001). Cultures were maintained on corn meal agar (CMA; Difco, Detroit, MI) and peptone–yeast extract–glucose (PYG) agar. Tissue strength measurements were made from the roots of seedlings of creeping bentgrass (Agrostis palustris Huds. ÔProvidenceÕ) and wheat (Triticum aestivum L.). Creeping bentgrass seedlings were 3 weeks old at the time of the experiments, while wheat seedlings were harvested 2 weeks after germination. 2.2. Measurement of hyphal force and applied pressure Overnight cultures were grown on PYG agar in customized culture dishes that clipped into a Delta TC3 microscope heating stage (Bioptechs, Butler, PA). When colony diameter reached ’ 5 mm, a rectangular block of agar was excised 1 mm in advance of the colony and the resulting well was filled with PYG broth. The match between the solid and liquid medium (PYG agar/PYG broth) in these experiments was considered superior to the previous use of CMA/distilled water (Ravishankar et al., 2001), because the turgor data (see next section) were also obtained from mycelia cultured in PYG broth. (Nevertheless, the estimates of applied pressure in Table 1 are very similar to those obtained from other strains of P. insidiosum growing in CMA/distilled water in the earlier study.) Hyphae were viewed through the glass base of the culture dishes with an inverted microscope (model IX70, fitted with long working distance objectives; Olympus,
Tokyo). Measurements of force were made as the hyphal apices penetrated the broth-filled well, using a miniature silicon bridge strain gauge (type AE-801; Capto a.s., Horten, Norway; marketed by SensorOne, Sausalito, CA) positioned with a motorized micromanipulator (MP-285; Sutter Instrument, Novato, California). Before use, the silicon beam was anchored to the base of the strain gauge with cyanoacrylate adhesive and painted with Sylgard (Dow Corning, Midland, MI) to reduce light sensitivity. Strain gauges were used in the single-ended or half-bridge mode. Signals were amplified and filtered through a 30 kHz low-pass filter (WPI, Sarasota, FL) and further amplified and filtered at 1 Hz before sampling. Calibration was performed with milligram and microgram weights. Instrument resolution was better than 0:1 lN. A Plexiglas microscope cage limited air movement around the strain gauge. For the same force applied per unit area of the hyphal apex, a larger hypha pushes against the strain gauge with greater total force than a smaller cell. To account for variations in hyphal size, the force measurements were converted to pressures by dividing force by the hyphal surface area in contact with the strain gauge. Contact areas (2pr2 ) were calculated from measurements of hyphal diameter (to the nearest 0:5 lm) from the region subtending the apex, using a 40 objective lens and eyepiece reticle (Ravishankar et al., 2001). In this way, each measurement of force was paired with an estimate of contact area from the same hypha. This allowed us to base mean values of applied pressure upon measurements of the pressures exerted by single hyphae. (In the earlier study, mean values of applied pressure were calculated by dividing mean force by mean contact area.) Experiments were performed on hyphae growing at room temperature (24 °C) and at elevated temperatures (34 °C for P. graminicola and 37 °C for P. insidiosum), to determine whether or not these microorganisms exhibited temperature-dependent changes in force and applied pressure. 2.3. Turgor measurement Cultures were grown in 100 mL PYG broth in Ehrlenmeyer flasks in Gyrotory water bath shakers (New Brunswick Scientific, Edison, NJ) at 70 r.p.m. Each flask was inoculated with ten 1-mm diameter plugs cut from an agar stock culture using a sterile Pasteur pipet. After 3–8 d incubation, each flask yielded ’100 mg fresh weight of mycelium. Mycelia and medium were separated by vacuum filtration and samples of each were transferred to microfuge tubes and frozen at )20 °C. After thawing, mycelia were disrupted using a Teflon plunger and centrifuged at 15,000g for 3 min to remove cell wall debris. Triplicate measurements of cytoplasmic and culture medium osmolality were made from each sample using a vapor pressure deficit osmometer (model
E. MacDonald et al. / Fungal Genetics and Biology 37 (2002) 245–249
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Table 1 Biomechanical data obtained from hyphae of P. graminicola and P. insidiosum Contact area (lm2 )
P a , applied pressure (lN lm2 or MPaÞb
P i , turgor pressure (lN lm2 or MPa)
Extension rate (lm min1 )
P. graminicola 96600 24 °C 1:7 0:3 ð19Þ 34 °C 2:0 0:4 ð15ÞNS
10 (19)a 10 (15)
0:17 0:03 ð19Þ 0:20 0:04 ð15ÞNS
0:84 0:04 ð16Þ 0:79 0:03 ð15ÞNS
8:1 0:4 ð23Þ 18:0 0:7 ð21Þ
P. Insidiosum 76049 24 °C 1:6 0:1 ð20Þ 37 °C 2:0 0:3 ð21ÞNS
15 2 ð20Þ 12 1 ð21ÞNS
0:13 0:01 ð20Þ 0:18 0:03 ð21ÞNS
0:39 0:02 ð30Þ 0:44 0:01 ð24Þ
4:4 0:1 ð20Þ 5:2 0:4 ð20ÞNS
Species, strain, temperature
Applied force (lN)
Note. Data reported as means standard error (sample size). NS/*: no significant difference/significant difference indicated by ANOVA at P >=< 0:05. a There was no measurable variation in contact area at 24 and 34 °C for P. graminicola. b Applied pressures cannot be calculated from mean values shown in table because they were derived from paired measurements (raw data) of force and contact area (see text).
5520; Wescor, Logan, Utah). Instrument resolution was 5 mmol kg1 and osmolality values were converted to osmotic pressures using the vanÕt Hoff equation. Turgor was calculated from the difference in osmotic pressure between the mycelium and culture medium. Turgor measurements are usually expressed in megapascals (MPa), but since these units are equivalent to lN lm2 , the latter are used throughout this paper to afford straightforward comparison with the micronewton forces exerted by hyphal apices, estimates of applied pressure, and data on tissue strength. 2.4. Tissue strength measurements Strength measurements from root epidermal cells were made with glass microprobes attached to the miniature strain gauges used in the hyphal force experiments (Fig. 1). Micropipets were pulled from glass microcapillaries on a micropipet puller (Sutter, Novato, CA) and then held close to a Bunsen flame to produce smooth semi-ellipsoidal tips with diameters of 5–8 lm. Straight-tipped micropipets were selected under a microscope and their wide ends were cut with a diamond knife to produce the microprobes. These were attached
Fig. 2. Micropenetration of root epidermal cell of creeping bentgrass seedling with glass microprobe (tip diameter 5 lm). Cell wall is pierced at position indicated by arrow.
to a plastic peg glued at 90° to the end of a calibrated strain gauge. Substrate strength measurements were made by pushing the tip of the microprobe into individual epidermal cells under the control of the motorized micromanipulator (Fig. 2). The root system of each seedling was secured in water agar to resist movement during penetration with the microprobe.
3. Results 3.1. Hyphal biomechanics I. P. graminicola
Fig. 1. Microprobe attachment to the strain gauge. The silicon beam is shown in profile: the cylindrical peg is glued to its flat surface. The strain gauge header is secured to a rod which is clamped onto the micromanipulator. Microprobes with different tip sizes are interchangeable.
The force exerted by hyphae of P. graminicola against the silicon beam of the strain gauge did not vary significantly between 24 and 34 °C (Table 1). The mean pooled from measurements at both temperatures was 1:9 0:2 lN ðn ¼ 34Þ. Hyphal size showed no measurable variation between the two temperatures, indicating a mean value for applied pressure of 0:19 0:02 lN lm2 (’2 atm). Turgor pressure also showed no significant variation between 24 and 34 °C, with a mean
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value of 0:81 0:03 lN lm2 (n ¼ 31 measurements from nine separate cultures). Reference to the applied pressure estimate above, shows that hyphal apices of P. graminicola exerted about 23% of this internal pressure against the strain gauge (0:19=0:81 lN lm2 ). At 34 °C, P. graminicola hyphae showed very fast extension rates, more than twice those measured at 24 °C. But when the temperature of the cultures was raised above 34 °C, hyphal bursting was a very frequent occurrence, consistent with extreme loosening of the apical cell wall. 3.2. Hyphal biomechanics II. P. insidiosum Unlike P. graminicola, P. insidiosum strain 76049 showed vibrant growth at 37 °C affording applied force and pressure measurements at this higher temperature. Small differences in force and hyphal size were indicated between 24 and 37 °C, but these apparent temperaturedependent trends were not statistically significant (Table 1). Based on data collected at both temperatures, hyphae of P. insidiosum produced a mean force of 1:8 0:2 lN ðn ¼ 41Þ, which corresponded to an applied pressure of 0:16 0:02 lN lm2 . Turgor pressure showed a significant increase from 0:39 lN lm2 at 24 °C to 0:44 lN lm2 at 37 °C. Calculations show that hyphae exerted 30–40% of their internal pressure against the strain gauge at these temperatures. 3.3. Tissue strengths Adjacent root epidermal cells showed quite different levels of resistance to pipet insertion (Fig. 2). Individual cells within wheat epidermis resisted penetration until pressures of 2:3–11:8 lN lm2 were applied at the tip of the glass microprobes. The mean strength value was 7:1 0:6 lN lm2 ðn ¼ 22Þ. The strength of root epidermis of creeping bentgrass seedlings varied from 1:1–9:6 lN lm2 with a mean value of 4:8 0:5 lN lm2 ðn ¼ 27Þ. Published experiments on the strength of human and equine skin—natural substrates for P. insidiosum—provided strength estimates of 10–47 lN lm2 (Ravishankar et al., 2001).
4. Discussion 4.1. Comparing the biomechanical characteristics of a phytopathogen and mammalian pathogen Beyond rather trivial differences in their biomechanical characteristics, the two pathogens exerted virtually the same level of force (’ 2 lN) and similar pressure (0:19 lN lm2 for P. graminicola and 0:14 lN lm2 for P. insidiosum). In the earlier study from our laboratory, small, temperature-dependent changes in force and applied pressure were measured in three strains of
P. insidiosum, but only under certain cultural conditions (Ravishankar et al., 2001). Similar trends were evident in P. graminicola and in the single strain of P. insidiosum examined in the present study, although these changes were not statistically significant (Table 1). The vitality of P. insidiosum hyphae at 37 °C is consistent with its appearance as a mammalian pathogen and contrasted with the instability of the apical hyphal wall of P. graminicola at temperatures above 34 °C. The turgor pressure in P. graminicola (0:81 lN lm2 ) was considerably higher than that measured in P. insidiosum (0:17–0:44 lN lm2 ; Table 1 and Ravishankar et al., 2001), but equivalent hydrostatic pressures have been reported for hyphae of other oomycetes (Money, 1994). Logic and experimental data suggest that hyphae exert pressure during invasive growth by loosening their apical walls, allowing some proportion of their internal turgor pressure to press upon the material in contact with the cell apex (Money, 2001). Any contribution to the pressure exerted by a hypha from its cytoskeleton is likely to be negligible (the argument is given in Money, 2001). According to this model, the upper limit on the invasive pressure exercised by the hypha is prescribed by the available pressure within the cell, and the ratio of applied pressure to turgor is a reflection of the degree of wall yielding at the extending hyphal tip. (This is supported by the observation that the magnitude of the applied pressure measurements have never exceeded estimates of turgor.) Despite differences in turgor, it appears that the degree of pressure transmission estimated for P. graminicola is comparable to the behavior of P. insidiosum: hyphae of P. graminicola (96600) and P. insidiosum (200269; Ravishankar et al., 2001), exerted 23–29% of their internal pressure against the silicon beam of the strain gauge. Other strains of P. insidiosum transmit 33–63% of their turgor against the strain gauge (Table 1 and Ravishankar et al., 2001). The applied pressure estimates for P. graminicola show that its hyphae could not breach epidermal cells that displayed the lowest level of resistance in our experiments (1:1 lN lm2 ) without the activity of wall-degrading enzymes, while the majority of healthy epidermal cells offered overwhelming resistance to penetration. Presumably, cells whose walls are weakened by abrasion against soil particles, or damaged by invertebrates, present superior sites for hyphal invasion by diminishing the demand on cell-wall-degrading enzymes. However, it has been demonstrated that Pythium zoospores target the intact roots of seedlings and can establish infections in healthy plants (Agrios, 1997; Deacon, 1997; Islam and Tahara, 2001). The reliance on secreted enzymes indicated for the invasive mechanism of P. graminicola also applies to P. insidiosum: maximum pressures are between 10- and 100-fold lower than the minimum resistance of mammalian skin (Ravishankar et al., 2001). We conclude
E. MacDonald et al. / Fungal Genetics and Biology 37 (2002) 245–249
that neither species of Pythium is strong enough to penetrate the tissues of its host without the concerted activity of secreted tissue-degrading enzymes. Once enzymes have reduced the resistance of host tissues, it is presumed that hyphae of both pathogens are sufficiently powerful to bore through any remaining obstacles. When pathogens that produce melanized appressoria invade cereal leaves, their penetration hyphae generate sufficient force to penetrate the cuticle and underlying cell wall by mechanics alone (Bechinger et al., 1999; Howard et al., 1991). There is no evidence that enzymes play any role in this process (Bastmeyer et al., 2002). Our experiments on vegetative hyphae of Pythium species suggest that leaf invasion by appressoria represents a very specialized mechanism, something that cannot be viewed as a model for understanding the majority of biomechanical interactions between fungi and their hosts.
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present conclusions upon other fungi (see discussion in Money, 2001). We are close to providing an authoritative answer to this question. Now that the necessary instrumentation for measuring the forces exerted by very small hyphae has been developed, the next phase of our research effort will involve a comprehensive survey of the biomechanical characteristics of species from Kingdom Fungi. When this is completed we will have a clear picture of the relative significance of force and enzymes during invasive growth by hyphal microorganisms. This will provide a firm empirical foundation for future research on invasive mechanisms.
Acknowledgments Support was furnished by National Science Foundation Grant IBN-9985546 to N.P.M.
4.2. Limitations of tissue strength data Zoospore cysts of P. graminicola adhere to the root epidermis and serve as infection platforms. (They are analogous to appressoria.) Although the measurements of root strength were made with microprobes matching the size of hyphae, this experimental design did not offer a perfect analog for the invading microorganism because the probe was not secured to the root surface before it was advanced into the host. One consequence of this experimental configuration is that the epidermal cell is free to buckle before the tip of the microprobe breaches its wall. This may lead to considerable overestimation of the invasive pressure required for a hypha which is firmly anchored to the host surface. Moreover, the plant cell wall may show somewhat greater resistance to penetration by a hydrophobic glass probe than it does to a hyphal surface that may be lubricated by hydrophilic components within its extracellular matrix. Our experiments on root epidermis provide the only data on the resistance of plant tissues to mechanical penetration since Manabu MiyoshiÕs (1895) measurements of cell wall strength in onion epidermis and Tradescantia leaves. Miyoshi estimated that these tissues resisted penetration until applied pressures reached 0:35–0:74 lN lm2 (i.e., close to the lower limit of our measurements from root epidermis). 4.3. Future research Biomechanical studies on the hyphae of stramenopile fungi have been facilitated by their relatively large size and fast growth rates. With the exception of the work on melanized appressoria, there has been very little research on hyphal mechanics on species within Kingdom Fungi. Therefore, it is important to question the bearing of the
References Agrios, G.N., 1997. Plant Pathology, fourth ed. Academic Press, San Diego, CA. Badenoch, P.R., Coster, D.J., Wetherall, B.L., Brettig, H.T., Rozenbilds, M.A., Drenth, A., Wagels, G., 2001. Pythium insidiosum keratitis confirmed by DNA sequence analysis. Br. J. Ophthalmol. 85, 502–503. Bastmeyer, M., Deising, H.B., Bechinger, C., 2002. Force exertion in fungal infection. Annu. Rev. Biophys. Biomol. Struct. 31, 321–341. Bechinger, C., Giebel, K.-F., Schnell, M., Leiderer, P., Deising, H.B., Bastmeyer, M., 1999. Optical measurements of invasive forces exerted by appressoria of a plant pathogenic fungus. Science 285, 1896–1899. Deacon, J.W., 1997. Modern Mycology, third ed. Blackwell Science, Oxford. Howard, R.J., Ferrari, M.A., Roach, D.H., Money, N.P., 1991. Penetration of hard substrates by a fungus employing enormous turgor pressures. Proc. Natl. Acad. Sci. USA 88, 11281–11284. Islam, M.T., Tahara, S., 2001. Chemotaxis of fungal zoospores, with special reference to Aphanomyces cochlioides. Biosci. Biotechnol. Biochem. 65, 1933–1948. Miyoshi, M., 1895. Die durchbohrung von membranen durch pilzf€aden. Jahrb. Wiss. Bot. 28, 269–289. Money, N.P., 1994. Osmotic adjustment and the role of turgor in mycelial fungi. In: Wessels, J.G.H., Meinhardt, F. (Eds.), The Mycota, vol. 1, Growth, Differentiation and Sexuality. Springer, Berlin, pp. 67–88. Money, N.P., 2001. Biomechanics of invasive hyphal growth. In: Howard, R.J., Gow, N.A.R. (Eds.), The Mycota, vol. 8, Biology of the Fungal Cell. Springer, Berlin, pp. 3–17. Ravishankar, J.P., Davis, C.M., Davis, D.J., MacDonald, E., Makselan, S.D., Millward, L., Money, N.P., 2001. Mechanics of solid tissue invasion by the mammalian pathogen Pythium insidiosum. Fungal Genet. Biol. 34, 161–175. Tonukari, N.J., Scott-Craig, J.S., Walton, J.D., 2000. The Cochliobolus carbonarum SNF1 gene is required for cell-wall-degrading enzyme expression and virulence on maize. Plant Cell 12, 237–247. Virgile, R., Perry, H.D., Pardanani, B., Szabo, K., Rahm, E.K., Stone, J., Salkin, I., Dixon, D.M., 1993. Human corneal ulcer caused by Pythium insidiosum. Cornea 12, 81–83.
Biomechanical interaction between hyphae of two Pythium species (Oomycota) and host tissues Erin MacDonald, Laurie Millward, J.P. Ravishankar, and Nicholas P. Money* Department of Botany, Miami University, Oxford, OH 45056, USA Received 21 February 2002; accepted 26 July 2002
Abstract Forces exerted by hyphae of the phytopathogen Pythium graminicola and mammalian pathogen Pythium insidiosum were compared with the mechanical resistance of their hostsÕ tissues. Hyphal apices of both species exerted a mean force of 2 lN, corresponding to mean pressures of 0:19 lN lm2 (or MPa) for P. graminicola, and 0:14 lN lm2 for P. insidiosum. Experiments with glass microprobes showed that the epidermis of grass roots resisted penetration until the pressure applied at the probe tip reached 1–12 lN lm2 . Previously published data show that mammalian skin offers even greater resistance (10–47 lN lm2 ). Clearly, tissue strength exceeds the pressures exerted by hyphae of these pathogens, verifying that secreted enzymes must play a critical role in reducing the resistance of plant and animal tissues. It is presumed that hyphae are sufficiently powerful to bore through any obstacles remaining after enzyme action. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Invasive hyphal growth; Kingdom Stramenopila; Pythium graminicola; Pythium insidiosum; Tissue penetration; Turgor pressure
1. Introduction Pathogenic fungi invade plant and animal tissues by secreting lytic enzymes and exerting force at their hyphal apices (Money, 2001). Many years of inconclusive gene disruptions failed to associate secreted enzymes with fungal virulence (Bastmeyer et al., 2002), until Tonukari et al. (2000) succeeded in demonstrating their importance by the targeted inactivation of SNF1 in Cochliobolus carbonum, a gene that controls the expression of several tissue-degrading enzymes. Even though the necessity for enzymes is now close to proven for C. carbonum (and for a handful of other fungi), gene inactivation studies do not discriminate between enzymes that are necessary for nutritional purposes and those that dissolve physical barriers. Many secreted enzymes probably serve both purposes, but it would be useful to identify those that have the greatest effect upon tissue strength. The relative importance of enzymes versus mechanics is not known for most interactions *
Corresponding author. Fax: 1-513-529-4243. E-mail address: [email protected] (N.P. Money).
between fungi and their plant or animal hosts, but is a crucial consideration whether we are interested primarily in understanding or in controlling a particular disease. It is certain that enzyme-catalyzed dissolution of tissues dominates some interactions, while mechanical force plays a greater role in others. Analysis of the secreted enzymes and the mechanical characteristics of a particular fungus offers the best prospects for ‘‘solving’’ its invasive mechanism. In a recent study, Ravishankar et al. (2001) demonstrated that the mammalian pathogen Pythium insidiosum (an oomycete fungus or stramenopile) must achieve a decisive reduction in tissue strength by proteinase secretion before it can penetrate cutaneous and sub-cutaneous tissues. This conclusion was reached by comparing the pressures exerted by single hyphae with the resistance of human and equine skin to needle insertion. In the present paper, we report the application of the same methodology to analyze the invasive mechanism utilized by Pythium graminicola. This oomycete is a widespread pathogen of graminaceous hosts that causes root rot and feeder root necrosis in a number of crop plants (Agrios, 1997; Deacon, 1997).
1087-1845/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 8 7 - 1 8 4 5 ( 0 2 ) 0 0 5 1 4 - 5
246
E. MacDonald et al. / Fungal Genetics and Biology 37 (2002) 245–249
These data are compared with new measurements from a clinical isolate of P. insidiosum which was not included in the study by Ravishankar et al. (2001). Both Pythium species initiate infection with a biflagellate zoospore that attaches to the host surface and operates as a platform from which an infection hypha extends into the underlying tissue. The experiments indicate that the strength of intact host tissues exceeds the pressure exerted by vegetative hyphae of either Pythium species, verifying that tissue-degrading enzymes must effect a significant reduction in tissue strength to allow hyphal penetration of plant or animal tissues.
2. Materials and methods 2.1. Organisms and culture media Pythium graminicola Subramanian strain 96600 was obtained from the American Type Culture Collection (ATCC), Rockville, MD. It was originally isolated from an infected sugarcane root in Louisiana. P. insidiosum De Cock et al. ATCC strain 76049 was isolated from a human corneal infection in Haiti. The case history was described by Virgile et al. (1993) and a more recent report of P. insidiosum keratitis in Malaysia was published by Badenoch et al. (2001). Cultures were maintained on corn meal agar (CMA; Difco, Detroit, MI) and peptone–yeast extract–glucose (PYG) agar. Tissue strength measurements were made from the roots of seedlings of creeping bentgrass (Agrostis palustris Huds. ÔProvidenceÕ) and wheat (Triticum aestivum L.). Creeping bentgrass seedlings were 3 weeks old at the time of the experiments, while wheat seedlings were harvested 2 weeks after germination. 2.2. Measurement of hyphal force and applied pressure Overnight cultures were grown on PYG agar in customized culture dishes that clipped into a Delta TC3 microscope heating stage (Bioptechs, Butler, PA). When colony diameter reached ’ 5 mm, a rectangular block of agar was excised 1 mm in advance of the colony and the resulting well was filled with PYG broth. The match between the solid and liquid medium (PYG agar/PYG broth) in these experiments was considered superior to the previous use of CMA/distilled water (Ravishankar et al., 2001), because the turgor data (see next section) were also obtained from mycelia cultured in PYG broth. (Nevertheless, the estimates of applied pressure in Table 1 are very similar to those obtained from other strains of P. insidiosum growing in CMA/distilled water in the earlier study.) Hyphae were viewed through the glass base of the culture dishes with an inverted microscope (model IX70, fitted with long working distance objectives; Olympus,
Tokyo). Measurements of force were made as the hyphal apices penetrated the broth-filled well, using a miniature silicon bridge strain gauge (type AE-801; Capto a.s., Horten, Norway; marketed by SensorOne, Sausalito, CA) positioned with a motorized micromanipulator (MP-285; Sutter Instrument, Novato, California). Before use, the silicon beam was anchored to the base of the strain gauge with cyanoacrylate adhesive and painted with Sylgard (Dow Corning, Midland, MI) to reduce light sensitivity. Strain gauges were used in the single-ended or half-bridge mode. Signals were amplified and filtered through a 30 kHz low-pass filter (WPI, Sarasota, FL) and further amplified and filtered at 1 Hz before sampling. Calibration was performed with milligram and microgram weights. Instrument resolution was better than 0:1 lN. A Plexiglas microscope cage limited air movement around the strain gauge. For the same force applied per unit area of the hyphal apex, a larger hypha pushes against the strain gauge with greater total force than a smaller cell. To account for variations in hyphal size, the force measurements were converted to pressures by dividing force by the hyphal surface area in contact with the strain gauge. Contact areas (2pr2 ) were calculated from measurements of hyphal diameter (to the nearest 0:5 lm) from the region subtending the apex, using a 40 objective lens and eyepiece reticle (Ravishankar et al., 2001). In this way, each measurement of force was paired with an estimate of contact area from the same hypha. This allowed us to base mean values of applied pressure upon measurements of the pressures exerted by single hyphae. (In the earlier study, mean values of applied pressure were calculated by dividing mean force by mean contact area.) Experiments were performed on hyphae growing at room temperature (24 °C) and at elevated temperatures (34 °C for P. graminicola and 37 °C for P. insidiosum), to determine whether or not these microorganisms exhibited temperature-dependent changes in force and applied pressure. 2.3. Turgor measurement Cultures were grown in 100 mL PYG broth in Ehrlenmeyer flasks in Gyrotory water bath shakers (New Brunswick Scientific, Edison, NJ) at 70 r.p.m. Each flask was inoculated with ten 1-mm diameter plugs cut from an agar stock culture using a sterile Pasteur pipet. After 3–8 d incubation, each flask yielded ’100 mg fresh weight of mycelium. Mycelia and medium were separated by vacuum filtration and samples of each were transferred to microfuge tubes and frozen at )20 °C. After thawing, mycelia were disrupted using a Teflon plunger and centrifuged at 15,000g for 3 min to remove cell wall debris. Triplicate measurements of cytoplasmic and culture medium osmolality were made from each sample using a vapor pressure deficit osmometer (model
E. MacDonald et al. / Fungal Genetics and Biology 37 (2002) 245–249
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Table 1 Biomechanical data obtained from hyphae of P. graminicola and P. insidiosum Contact area (lm2 )
P a , applied pressure (lN lm2 or MPaÞb
P i , turgor pressure (lN lm2 or MPa)
Extension rate (lm min1 )
P. graminicola 96600 24 °C 1:7 0:3 ð19Þ 34 °C 2:0 0:4 ð15ÞNS
10 (19)a 10 (15)
0:17 0:03 ð19Þ 0:20 0:04 ð15ÞNS
0:84 0:04 ð16Þ 0:79 0:03 ð15ÞNS
8:1 0:4 ð23Þ 18:0 0:7 ð21Þ
P. Insidiosum 76049 24 °C 1:6 0:1 ð20Þ 37 °C 2:0 0:3 ð21ÞNS
15 2 ð20Þ 12 1 ð21ÞNS
0:13 0:01 ð20Þ 0:18 0:03 ð21ÞNS
0:39 0:02 ð30Þ 0:44 0:01 ð24Þ
4:4 0:1 ð20Þ 5:2 0:4 ð20ÞNS
Species, strain, temperature
Applied force (lN)
Note. Data reported as means standard error (sample size). NS/*: no significant difference/significant difference indicated by ANOVA at P >=< 0:05. a There was no measurable variation in contact area at 24 and 34 °C for P. graminicola. b Applied pressures cannot be calculated from mean values shown in table because they were derived from paired measurements (raw data) of force and contact area (see text).
5520; Wescor, Logan, Utah). Instrument resolution was 5 mmol kg1 and osmolality values were converted to osmotic pressures using the vanÕt Hoff equation. Turgor was calculated from the difference in osmotic pressure between the mycelium and culture medium. Turgor measurements are usually expressed in megapascals (MPa), but since these units are equivalent to lN lm2 , the latter are used throughout this paper to afford straightforward comparison with the micronewton forces exerted by hyphal apices, estimates of applied pressure, and data on tissue strength. 2.4. Tissue strength measurements Strength measurements from root epidermal cells were made with glass microprobes attached to the miniature strain gauges used in the hyphal force experiments (Fig. 1). Micropipets were pulled from glass microcapillaries on a micropipet puller (Sutter, Novato, CA) and then held close to a Bunsen flame to produce smooth semi-ellipsoidal tips with diameters of 5–8 lm. Straight-tipped micropipets were selected under a microscope and their wide ends were cut with a diamond knife to produce the microprobes. These were attached
Fig. 2. Micropenetration of root epidermal cell of creeping bentgrass seedling with glass microprobe (tip diameter 5 lm). Cell wall is pierced at position indicated by arrow.
to a plastic peg glued at 90° to the end of a calibrated strain gauge. Substrate strength measurements were made by pushing the tip of the microprobe into individual epidermal cells under the control of the motorized micromanipulator (Fig. 2). The root system of each seedling was secured in water agar to resist movement during penetration with the microprobe.
3. Results 3.1. Hyphal biomechanics I. P. graminicola
Fig. 1. Microprobe attachment to the strain gauge. The silicon beam is shown in profile: the cylindrical peg is glued to its flat surface. The strain gauge header is secured to a rod which is clamped onto the micromanipulator. Microprobes with different tip sizes are interchangeable.
The force exerted by hyphae of P. graminicola against the silicon beam of the strain gauge did not vary significantly between 24 and 34 °C (Table 1). The mean pooled from measurements at both temperatures was 1:9 0:2 lN ðn ¼ 34Þ. Hyphal size showed no measurable variation between the two temperatures, indicating a mean value for applied pressure of 0:19 0:02 lN lm2 (’2 atm). Turgor pressure also showed no significant variation between 24 and 34 °C, with a mean
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value of 0:81 0:03 lN lm2 (n ¼ 31 measurements from nine separate cultures). Reference to the applied pressure estimate above, shows that hyphal apices of P. graminicola exerted about 23% of this internal pressure against the strain gauge (0:19=0:81 lN lm2 ). At 34 °C, P. graminicola hyphae showed very fast extension rates, more than twice those measured at 24 °C. But when the temperature of the cultures was raised above 34 °C, hyphal bursting was a very frequent occurrence, consistent with extreme loosening of the apical cell wall. 3.2. Hyphal biomechanics II. P. insidiosum Unlike P. graminicola, P. insidiosum strain 76049 showed vibrant growth at 37 °C affording applied force and pressure measurements at this higher temperature. Small differences in force and hyphal size were indicated between 24 and 37 °C, but these apparent temperaturedependent trends were not statistically significant (Table 1). Based on data collected at both temperatures, hyphae of P. insidiosum produced a mean force of 1:8 0:2 lN ðn ¼ 41Þ, which corresponded to an applied pressure of 0:16 0:02 lN lm2 . Turgor pressure showed a significant increase from 0:39 lN lm2 at 24 °C to 0:44 lN lm2 at 37 °C. Calculations show that hyphae exerted 30–40% of their internal pressure against the strain gauge at these temperatures. 3.3. Tissue strengths Adjacent root epidermal cells showed quite different levels of resistance to pipet insertion (Fig. 2). Individual cells within wheat epidermis resisted penetration until pressures of 2:3–11:8 lN lm2 were applied at the tip of the glass microprobes. The mean strength value was 7:1 0:6 lN lm2 ðn ¼ 22Þ. The strength of root epidermis of creeping bentgrass seedlings varied from 1:1–9:6 lN lm2 with a mean value of 4:8 0:5 lN lm2 ðn ¼ 27Þ. Published experiments on the strength of human and equine skin—natural substrates for P. insidiosum—provided strength estimates of 10–47 lN lm2 (Ravishankar et al., 2001).
4. Discussion 4.1. Comparing the biomechanical characteristics of a phytopathogen and mammalian pathogen Beyond rather trivial differences in their biomechanical characteristics, the two pathogens exerted virtually the same level of force (’ 2 lN) and similar pressure (0:19 lN lm2 for P. graminicola and 0:14 lN lm2 for P. insidiosum). In the earlier study from our laboratory, small, temperature-dependent changes in force and applied pressure were measured in three strains of
P. insidiosum, but only under certain cultural conditions (Ravishankar et al., 2001). Similar trends were evident in P. graminicola and in the single strain of P. insidiosum examined in the present study, although these changes were not statistically significant (Table 1). The vitality of P. insidiosum hyphae at 37 °C is consistent with its appearance as a mammalian pathogen and contrasted with the instability of the apical hyphal wall of P. graminicola at temperatures above 34 °C. The turgor pressure in P. graminicola (0:81 lN lm2 ) was considerably higher than that measured in P. insidiosum (0:17–0:44 lN lm2 ; Table 1 and Ravishankar et al., 2001), but equivalent hydrostatic pressures have been reported for hyphae of other oomycetes (Money, 1994). Logic and experimental data suggest that hyphae exert pressure during invasive growth by loosening their apical walls, allowing some proportion of their internal turgor pressure to press upon the material in contact with the cell apex (Money, 2001). Any contribution to the pressure exerted by a hypha from its cytoskeleton is likely to be negligible (the argument is given in Money, 2001). According to this model, the upper limit on the invasive pressure exercised by the hypha is prescribed by the available pressure within the cell, and the ratio of applied pressure to turgor is a reflection of the degree of wall yielding at the extending hyphal tip. (This is supported by the observation that the magnitude of the applied pressure measurements have never exceeded estimates of turgor.) Despite differences in turgor, it appears that the degree of pressure transmission estimated for P. graminicola is comparable to the behavior of P. insidiosum: hyphae of P. graminicola (96600) and P. insidiosum (200269; Ravishankar et al., 2001), exerted 23–29% of their internal pressure against the silicon beam of the strain gauge. Other strains of P. insidiosum transmit 33–63% of their turgor against the strain gauge (Table 1 and Ravishankar et al., 2001). The applied pressure estimates for P. graminicola show that its hyphae could not breach epidermal cells that displayed the lowest level of resistance in our experiments (1:1 lN lm2 ) without the activity of wall-degrading enzymes, while the majority of healthy epidermal cells offered overwhelming resistance to penetration. Presumably, cells whose walls are weakened by abrasion against soil particles, or damaged by invertebrates, present superior sites for hyphal invasion by diminishing the demand on cell-wall-degrading enzymes. However, it has been demonstrated that Pythium zoospores target the intact roots of seedlings and can establish infections in healthy plants (Agrios, 1997; Deacon, 1997; Islam and Tahara, 2001). The reliance on secreted enzymes indicated for the invasive mechanism of P. graminicola also applies to P. insidiosum: maximum pressures are between 10- and 100-fold lower than the minimum resistance of mammalian skin (Ravishankar et al., 2001). We conclude
E. MacDonald et al. / Fungal Genetics and Biology 37 (2002) 245–249
that neither species of Pythium is strong enough to penetrate the tissues of its host without the concerted activity of secreted tissue-degrading enzymes. Once enzymes have reduced the resistance of host tissues, it is presumed that hyphae of both pathogens are sufficiently powerful to bore through any remaining obstacles. When pathogens that produce melanized appressoria invade cereal leaves, their penetration hyphae generate sufficient force to penetrate the cuticle and underlying cell wall by mechanics alone (Bechinger et al., 1999; Howard et al., 1991). There is no evidence that enzymes play any role in this process (Bastmeyer et al., 2002). Our experiments on vegetative hyphae of Pythium species suggest that leaf invasion by appressoria represents a very specialized mechanism, something that cannot be viewed as a model for understanding the majority of biomechanical interactions between fungi and their hosts.
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present conclusions upon other fungi (see discussion in Money, 2001). We are close to providing an authoritative answer to this question. Now that the necessary instrumentation for measuring the forces exerted by very small hyphae has been developed, the next phase of our research effort will involve a comprehensive survey of the biomechanical characteristics of species from Kingdom Fungi. When this is completed we will have a clear picture of the relative significance of force and enzymes during invasive growth by hyphal microorganisms. This will provide a firm empirical foundation for future research on invasive mechanisms.
Acknowledgments Support was furnished by National Science Foundation Grant IBN-9985546 to N.P.M.
4.2. Limitations of tissue strength data Zoospore cysts of P. graminicola adhere to the root epidermis and serve as infection platforms. (They are analogous to appressoria.) Although the measurements of root strength were made with microprobes matching the size of hyphae, this experimental design did not offer a perfect analog for the invading microorganism because the probe was not secured to the root surface before it was advanced into the host. One consequence of this experimental configuration is that the epidermal cell is free to buckle before the tip of the microprobe breaches its wall. This may lead to considerable overestimation of the invasive pressure required for a hypha which is firmly anchored to the host surface. Moreover, the plant cell wall may show somewhat greater resistance to penetration by a hydrophobic glass probe than it does to a hyphal surface that may be lubricated by hydrophilic components within its extracellular matrix. Our experiments on root epidermis provide the only data on the resistance of plant tissues to mechanical penetration since Manabu MiyoshiÕs (1895) measurements of cell wall strength in onion epidermis and Tradescantia leaves. Miyoshi estimated that these tissues resisted penetration until applied pressures reached 0:35–0:74 lN lm2 (i.e., close to the lower limit of our measurements from root epidermis). 4.3. Future research Biomechanical studies on the hyphae of stramenopile fungi have been facilitated by their relatively large size and fast growth rates. With the exception of the work on melanized appressoria, there has been very little research on hyphal mechanics on species within Kingdom Fungi. Therefore, it is important to question the bearing of the
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