Surface tension propulsion of fungal spores

Most basidiomycetes, including many edible mushrooms, actively disperse their spores through a mechanism known as ballistospory(Buller, 1909-1950; Ingold, 1939). The spores, or ballistospores, are borne on the gills of mushroom caps or equivalent reproductive structures (Fig. 1A). Each spore develops on an outgrowth known as the sterigma to which it is attached via the hilum – a constriction of the sterigma that works as an abscission zone(Fig. 1B,C). Spore ejection is preceded by the condensation of Buller's drop at the hilar appendix located on the proximal end of the spore (Fig. 1D,E). Buller's drop is nucleated by the secretion of hygroscopic substances (such as mannitol) that decrease the vapor pressure of the incipient droplet (Webster et al.,1995). In the meantime, a film of water develops on the spore probably following a similar process. When the drop reaches a critical size,it touches the water film on the spore surface. At this point, surface tension quickly pulls the drop onto the spore thus creating the necessary momentum to detach the spore from the sporogenic surface. The spore can then fall freely under the action of gravity. Upon emerging from the cap, the spore is carried away by air currents to a distant location where it can germinate to produce a new mycelium and, ultimately, new mushrooms.

Surface tension is almost imperceptible at length scales at which humans operate. However, at microscopic length scales, surface tension forces dominate over the force of gravity. This fact can be understood from a simple scaling argument. The force of gravity on an object such as a spore scales as F_g ∼ ρgR^–3, where ρis the density of the object, g=9.8 m s^–2 is the gravitational acceleration and R is the characteristic length of the object. By contrast, the surface tension force is F_γ∼γR, where γ is the liquid's surface tension (γ=72×10^–3Nm^–1 for water at room temperature). Considering the ratio of these forces: F_γ/F_g ∼γ/ρgR²; it can be seen that as R gets small, the surface tension force becomes increasingly important and dominates the force of gravity for R smaller than 1 mm. This simple phenomenon has profound consequences on the release of spores. The dispersal of most fungal spores by wind requires that the spores be small thus making the force of gravity inconsequential compared with adhesion forces. As a result, spores tend to cling to each other and to the gills of mushroom caps. Active spore ejection provides a solution to this problem, which explains the great diversity of mechanisms for spore release in fungi and nonvascular plants(Straka, 1962). However,unlike other active dispersal mechanisms, which involve mass release of spores from specialized launching structures, ballistospores are self-propelled by water.

Given that a large mushroom can shed spores at the astonishing rate of 40 million spores per hour (Buller,1909-1950); the release of ballistospores has rightfully attracted some attention (Buller,1909-1950; Ingold,1939; Money,1998). As early as 1939, Ingold determined that the surface energy in Buller's drop is sufficient to account for the kinetic energy of the spore(Ingold, 1939). He, however,concluded his discussion of the topic remarking that `although there appears to be sufficient surface energy to discharge the spore it is not too easy to see how this energy could be mobilized to bring about discharge'(Ingold, 1939). More recently,Turner and Webster (Turner and Webster,1991) were able to predict the initial spore velocity with respectable accuracy based on a few judicious assumptions. The development of high-speed video cameras and their recent application to visualize ballistospore ejection (Pringle et al.,2005) provide, for the first time, a way to address Ingold's question with direct measurement of all key parameters in the problem.

Here, we present a detailed analysis of how surface tension is used for spore ejection in Auricularia auricula (`tree ears') and Sporobolomyces yeasts. In particular, we quantify the forces and energies of the three stages of the ejection process: the transfer of surface energy from the drop to the spore, the work of fracture required to release the spore from the sterigma and the kinetic energy of the spore after ejection. Our analysis reveals an exquisite fine-tuning of the different stages that yields a surprisingly high efficiency for the transfer of energy from Buller's drop to the spore.

To initiate spore development and spore discharge, dehydrated Auricularia auricula (Fr.) J. Schrot fragments were first imbibed on a wet towel and then kept under humid conditions with the fertile surface facing downward. After a few hours, spore ejection had begun as indicated by the presence of white spores on the bottom of the dish. We cut thin vertical sections (0.5 mm) of the fungus and laid them flat on a microscope slide covered with a thin (100–200 μm) layer of 2% agar. Sterigmas were now oriented horizontally so that spores were ejected perpendicular to the optical axis of the microscope. Spores from yeast-like species were isolated from leaves. Although the yeasts were not identified to the species level, they are members of the Urediniomycetes (the rust fungi), likely to be of the genus Sporobolomyces. The yeasts were plated from a primary culture onto a thin layer of a 2% nutrient agar. After a few days, the spores germinated to form hyphae, sterigmas and new spores. Our yeast cultures may have included more than one species but we found little quantitative differences between the different cultures. Therefore, for simplicity, we are treating all samples as a single taxon. All experiments were performed on A. auricula and the yeast species, except for the work of fracture of the hilum, which was performed on A. auricula only.

All imaging was done in transmitted light with ×20 and ×40 objectives. Images were captured with a Phantom V7.0 (Wayne, NJ, USA) or a Photron Ultima APX-RS (San Diego, CA, USA) high-speed camera at a frame rate of up to 250,000 frames s^–1 and exposure times as short as 1μs. The high acquisition rate necessary to capture spore ejection can be achieved only when image resolution is low (typically 32×128 pixels). Although our analyses were performed on these raw images, the frames from the time-lapse sequences are presented in the figures at higher resolution to improve clarity. We include as supplementary material three movies (AVI format) for A. auricula and one for the Sporobolomycesyeasts (see Movies 1–4 in supplementary material). The frame rates for Movies 1–4 are, respectively 90,000 frames s^–1; 80,000 frames s^–1; 250,000 frames s^–1; 90,000 frames s^–1.

We developed image analysis routines in Matlab (The MathWorks, Natick, MA,USA) to track the centroid of the spore and the rotation of the spore's major axis over the entire trajectory. Although spore translation in A. auricula and Sporobolomyces yeasts could be tracked reliably in all time-lapse sequences, only A. auricula offered two spores with rotation confined to the imaging plane that could thus be analyzed for their angular velocity. The Sporobolomyces yeasts could not be positioned such that the spore trajectory was confined to the focal plane of the microscope; the spores thus moved quickly out of focus. To compute the spore velocity, we used a 3-D tracking algorithm that relies on the size of the out-of-focus spore to infer its vertical position. The calibration for the vertical position was obtained by imaging particles at known vertical displacements above or below the focal plane and recording the size of the out-of-focus particles.

The rupture force of the hilum in A. auricula was measured with custom-made micropipettes calibrated on an analytical balance (0.1 μN precision). Using a micromanipulator, a micropipette was brought into contact with the top of the spore, perpendicular to the sterigma. A water film provided adhesion between the spore and the glass micropipette. In some experiments, we also used poly-l-lysine-coated micropipettes to enhance adhesion. The micropipette was then displaced slowly until the spore detached from the sterigma or until the adhesion between the spore and pipette failed. The force was calculated from the deflection of the micropipette with an error of ±5%. To infer the spring constant of the sterigma, we measured its elongation δ just prior rupture (error of 10%).

Spore ejection is best described by first analyzing the spore ballistics to infer the spore initial velocity and kinetic energy. We then proceed to a mechanical analysis of the stages that precede ejection.

According to Stokes' law, the decay times areτ _T=m_SD/6πμR andτ _R=m_SD/20πμR,where R and m_SD are, respectively, the mean radius and mass of the spore–drop complex (see Materials and methods)(Happel and Brenner, 1983). We can therefore compare the decay times inferred from the fitted spore displacement in Fig. 3 with those predicted by the theory. For the decay time associated with the translational velocity (τ_T), the mean ratio of the measured over the predicted decay times is 0.91 (standard deviation: σ=0.08, for N=4 measurements). For the decay time associated with the angular velocity (τ_R), the average ratio is 1.08 (σ=0.19, N=2). The measurements are therefore in surprising close agreement with the theory.

Finally, we can look at the kinetic energy of the spore. The translational energy is

We now address the most fundamental question of the ejection mechanism– how the surface energy stored in Buller's drop is transformed into kinetic energy. To answer this question, we need a proper understanding of the fusion process. Fusion takes place over a time interval of less than 4 μs and is therefore just below the temporal resolution of most high-speed cameras currently available. Using a frame rate of 250,000 images per second and a shutter speed of 1 μs, we obtained some new and critical information about the early stages of spore ejection (Fig. 5). The first frame in Fig. 5A shows the drop that has condensed at the base of the spore. On the second frame, the drop has touched the spore and coalesced. The drop has not spread over the spore completely because its outline can still be discerned. In the third frame, the spore has been ejected while the top border of the drop is still visible on the spore. Finally, the last frame shows the spore rotation in and out of the image plane. This sequence of images establishes that the drop travels only a short distance on the spore and does not spread over the entire surface. Fig. 5B,C offers additional evidence of the partial fusion of the drop,which, as we will show, has some important implications for the amount of surface energy available to release the spore.

Consideration of the forces acting during the coalescence of Buller's drop reveals that ballistospore ejection is the fungal equivalent of jumping(Fig. 6). The same three ingredients are present – a lowering of the center of mass, a quick release of energy and an interaction with a rigid support. Growth of Buller's drop at the proximal end of the spore lowers the spore's center of mass (i.e. it brings it closer to the sterigma) as well as provides the energy to be used during ejection. This step is the ballistospore's way of bending its `legs' in preparation for jumping. As soon as fusion begins, the drop exerts on the spore a surface tension force directed towards itself and the spore exerts on the drop a force of the same magnitude but of opposite direction(Fig. 6A). With no external interaction (isolated system), the drop and spore would move towards each other, and the global center of mass would remain immobile. Thus, there would be no ejection. In the case of ballistospores, the sterigma plays the role of the rigid support. Its presence prevents the spore from moving towards the drop by exerting a reaction force opposing the surface tension force applied by the drop. The sterigma force is the external force acting on the spore–drop complex that leads to the motion of the center of mass. The same requirement for interaction with a rigid support is found in jumping. There, the moments applied at the leg joints must be resisted by the ground to generate the impulse that will accelerate the center of mass. Ballistospore ejection, however, differs from jumping in one important way. The spore is not resting on the sterigma but is attached to it. Therefore, as the spore launches forward, it will put the sterigma under tension. The latter must break easily to release the spore.

This scenario emphasizes the critical role played by Buller's drop and the sterigma during spore ejection. We can subdivide the ejection process into four stages (Fig. 6). During the first stage, Buller's drop grows thus lowering the center of mass of the spore and storing the energy that will be used during ejection. The second stage encompasses the early coalescence during which the sterigma is under compression and provides the counter-acting force necessary to move the global center of mass of the spore–drop complex. It is this force that allows Buller's drop to be accelerated up to a characteristic speed V_D. In the third phase, the drop decelerates as it transfers its momentum to the spore. The sterigma is now under tension and needs to break easily to release the spore without dissipating its kinetic energy. Finally, the fourth stage is the release of the spore.

The simplest model for energy transfer suggests that the kinetic energy of the drop is equal to the difference in surface energy,Δ E_p, between the initial state just before coalescence and the final state just after coalescence (energy loss will be considered in the last section). The validity of this assumption can be ascertained by estimating the Reynolds number for the drop motion. We find Re=V_DR_D/ν≈50. The relatively large value for the Reynolds number confirms that viscous effects are small compared with inertial effects, leading to an efficient transfer of surface energy into kinetic energy. Therefore, we can write

The strength of the chitinous wall of the sterigma could easily exceed the force created by the fusion of Buller's drop. Therefore, to predict the initial velocity of the spore at ejection, the energy required to break the hilum must be known. We measured the rupture force (F_B) by pulling on spores with calibrated glass microcantilevers. The microcantilever was brought in contact with the distal end of the spore and gradually pulled away (Fig. 7, insets). Surface tension between the cantilever and the spore allowed us to put the spore and sterigma under tension. Our measurements reveal two spore classes(Fig. 7). Some spores are weakly attached to the sterigma and are removed with a force between 0.08 and 0.3 μN (mean F_B=0.15 μN). Other spores are strongly attached to the sterigma and cannot be removed with forces up to 1.2 μN(the maximal force that could be applied with the experimental set-up). For these spores, the force required to fracture the hilum is higher than the adhesion force between the cantilever and the spore. Most attempts to increase the adhesion between the spore and the cantilever, and thus apply higher forces on the hilum, failed; probably because the wet spore surface does not allow strong bonding. However, numerous trials with cantilevers coated with poly-l-lysine yielded a few strongly bonded cantilevers. For these experiments, the hilum either ruptures for forces in the low range observed before or for large forces above 1 μN and up to 4.8 μN(Fig. 7). The two spore classes provide direct evidence for the development of an abscission zone at spore maturity to allow easy release of the spore(van Neil et al., 1972; McLaughlin et al., 1985). The upper force range gives an estimate of the force required to rupture the hilum before the abscission zone has fully developed.

For a finite rupture force, the spore velocity is reduced by an amountΔ V that depends on the work done to fracture the hilum. During the late phase of the coalescence process, the sterigma is stretched until the hilar region is fractured. Given a stiffness k and an elongationδ for the sterigma, the elastic force acting on the sterigma is F_E=kδ. When F_E reaches F_B, the hilum breaks. The energy needed to sever the attachment is equal to the work done by the elastic deformation:

It is also possible to predict the angular velocity of the spore. A torque is exerted on the spore because the surface tension force is applied at some distance from the point of contact between the spore and the sterigma(Fig. 6A). This torque explains the rotation of the ejected spore. Using the conservation of angular momentum(Happel and Brenner, 1983), we have

The key assumption of our model is that the drop reaches a characteristic velocity V_D that can be predicted from the change in surface energy of the system. To test the validity of this assumption, we performed experiments on an artificial system that mimics the fusion of Buller's drop. A drop was placed on a highly hydrophobic plate while another plate, this one wettable, was approached slowly from above until it touched the drop. Contact with the wettable plate induced a fast upward motion of the drop (Fig. 8). The contrast of wettability between the two substrates was such that the drop moved in its entirety from the lower surface to the upper one. This coalescence process is very similar to what happens when a drop wets the spore. Biance et al. performed a similar experiment but with plates of similar wettability, leading to a final state where the drop is split between the two surfaces(Biance et al., 2004). They provided a scaling relationship for the horizontal growth dynamics of the neck. Here, we complement their analysis with a study of the vertical motion of the center of mass.

We found that after a brief acceleration, the drop's center of mass moves upward at a constant speed (Fig. 8H). Therefore, the fusion process is associated with a characteristic velocity of the center of mass. We measured this characteristic velocity in a series of experiments and plotted it as a function of the theoretical velocity predicted from Eqn 13 (Fig. 8I). The observed drop velocity is proportional to the predicted velocity, with a proportionality constant β=0.28. The parameter β is a measure of the efficiency of the transfer of surface energy to kinetic energy. The value ofβ below one indicates that a fraction of the surface energy is lost in the coalescence process and therefore not available to accelerate the drop. By the same token, we can interpret the velocity ratios listed in Table 2 as a measure of the efficiency of the energy transfer in the ballistospores. Auricularia auricula and the Sporobolomyces yeasts show a similar efficiency with more than two third of the surface energy liberated contributing to the kinetic energy of the spore.

The use of surface tension by ballistosporic fungi offers a new paradigm for performing work at the micron scale. One clear advantage of this mechanism is that work, being performed by the fusion of a water droplet, comes virtually for free. It is only under this condition that the innumerable spores contained in a mushroom cap can all be equipped with their own discharge apparatus. The ballistosporic mode of dispersal is in sharp contrast with the mass release of spores or propagules by specialized launching structures found in other taxa (Straka,1962). It is therefore of great interest to uncover the design principles that make surface tension an effective source of energy.

As first stated by Ingold (Ingold,1939), the key step for ballistospore release is the transfer of surface energy stored in Buller's drop to the spore. Our analysis emphasizes the critical role played by the sterigma. First, during the early phase of the coalescence process, the sterigma provides the external force that prevents the spore from moving toward the drop. The global center of mass of the spore–drop complex is thus projected forward leading to ejection. In the late phase of the coalescence process, the sterigma is now put under tension and should fracture easily to prevent dissipation of the spore energy. Our measurements of the force required to release the spore from the sterigma show that an active weakening of the hilum takes place before ejection. The characteristic rupture force of 0.15 μN (N=15) recorded for a weakened hilum is comparable with the rupture force of 0.1 μN reported for wind-dispersed fungal conidiospores (Aylor,1975). This value is large compared with the gravitational force acting on the spore (F_g<2×10^–6μN) but small compared with the surface tension force that can be exerted by a drop at this scale(F_γ=γ2πR_D<1.4 μN,where R_D=2.25 μm is the drop radius). However, a force of up to 4.8 μN is necessary to detach an unweakened spore(Fig. 7), i.e. three times the surface tension force. Therefore, without an active weakening mechanism, spore ejection would be impossible.

To predict the initial velocity of the spore, we developed a model that focuses on the surface energy freed during the coalescence process. This model predicts with surprising accuracy the initial translational and angular velocity of the spore, particularly if one makes allowance for energy dissipation during fusion. A prediction of the model is that the geometry of the fused drop affects the amount of energy available to eject the spore. Consequently, spore morphology and the wetting properties of the spore surface can play an important role in the transfer of surface energy to kinetic energy. The low efficiency of energy transfer in our artificial system when compared with ballistospores (Table 2) also emphasizes the challenges associated with the fine-tuning of such a mechanism. It is likely that the difference in scale between the two systems explains the higher efficiency for ballistospore ejection. It is also noteworthy that our model predicts similar efficiency of energy transfer for the two species studied despite differences in spore geometry and a threefold difference in the initial velocity between the Sporobolomyces and A. auricula spores.

A way to evaluate viscous loss is to calculate the energy loss in volume during the fusion process. This energy is E_V=T_Dμ∫ξ²dV,where T_D ∼ R_D/V_D is the characteristic time for the drop merging process and ξ is the shear rate. By taking a characteristic shear rate ξ ∼ V_D/R_D due to the small deformation of the drop, the integration gives

We have found it useful to compare ballistospore release with jumping in animals. We first note that the take-off velocity of the spore (1–2 m s^–1) falls precisely within the narrow range of take-off velocities (1–4 m s^–1) reported for good jumpers from insects to mammals (Vogel,2005a; Vogel,2005b). This striking observation suggests that a take-off velocity on the order of 1 m s^–1 is a fundamental limit for jumpers whether they achieve this velocity through muscle work or surface tension. Vogel (Vogel, 2005b)posited that the strength of biomaterials may impose limits on the stress that can be applied to accelerate jumpers and thus may set the maximal take-off velocity. However, it is doubtful that the same argument would apply to ballistospores. As we have shown, the surface tension force exerted by Buller's drop is F_γ=1.4 μN and is applied on a cross-section of 5 μm², which is a level of stress that most biomaterials can sustain.

In both insects and vertebrates, the velocity of the center of mass is known to increase monotonically during the active part of the jump up to the take-off velocity (Burrows,2006; Burrows,2008; Marsh and Johnalder,1994). The evolution of the center of mass velocity can be accounted for if a finite force is applied during the entire hind limb deployment. By analogy, it would be tempting to assume that the velocity of Buller's drop in ballistospores follows a similar evolution with surface tension, instead of muscle work, providing a roughly constant force over the entire distance traveled by the drop. However, as can be seen in our artificial system (Fig. 8H),this approach would lead to a gross overestimate of the drop velocity. The drop is in fact accelerated over a very short distance and then displaced at a constant characteristic velocity. We have argued that understanding what sets this characteristic velocity is the key to predicting the spore velocity at ejection. Our results show that the characteristic velocity scales with the surface energy freed during the coalescence(Fig. 8I) and thus highlight the importance, in ballistospores, of the final geometry of Buller's drop in determining the energy available for discharge and the take-off velocity of the spore.

LIST OF SYMBOLS

 
• A_D

  drop surface area after fusion

 
• A_S

  area of the spore covered by the drop after fusion

 
• a

  acceleration of the spore

 
• a, b

  minor and major semi-axes of the spheroid

 
• Ca

  capillary number

 
• D

  drag force

 
• E_B

  energy of fracture

 
• E_K

  translational kinetic energy

 
• E_R

  rotational kinetic energy

 
• E_V

  energy loss in volume

 
• F_B

  rupture force of the hilum

 
• F_D

  force applied by the drop

 
• F_E

  elastic force

 
• F_g

  force of gravity

 
• F_γ

  surface tension force

 
• F_R

  ground reaction force

 
• F_S

  force applied by the spore

 
• F_SD

  force applied by the spore–drop complex

 
• F_St

  force applied by the sterigma

 
• g

  gravitational acceleration

 
• k

  stiffness

 
• L_S

  length of the spore

 
• l

  distance between the global center of mass and the point of drop fusion

 
• M

  moment at the joints

 
• m

  mass

 
• m_D

  mass of the drop

 
• m_SD

  mass of the spore–drop complex

 
• R

  mean radius

 
• R_D

  radius of the drop before fusion

 
• \(R_{\mathrm{D}}^{{^\prime}}\)

  radius of the drop after fusion

 
• Re

  Reynolds number

 
• r_g

  spore's radius of gyration

 
• R_S

  spherical spore radius

 
• t

  time

 
• T_D

  characteristic time for drop coalescence

 
• V₀

  initial spore velocity

 
• V_D

  velocity of the drop

 
• W_S

  width of the spore

 
• v

  velocity

 
• x

  axis of discharge

 
• γ

  surface tension at liquid–vapor interface

 
• γSL

  surface tension at spore–liquid interface

 
• γSV

  surface tension at spore–vapor interface

 
• ΔE_p

  surface energy freed during the fusion process

 
• δ

  elongation

 
• θ

  angular position of the spore

 
• θ

  contact angle

 
• μ

  dynamic viscosity of air

 
• ν

  kinematic viscosity of air

 
• ξ

  shear rate

 
• ρ

  density

 
• τR

  characteristic decay time associated with angular velocity

 
• τT

  characteristic decay time associated with the translational velocity

 
• Ω0

  initial angular velocity