Abstract
Millions of tons of fungal spores are dispersed in the atmosphere every year. These living cells, along with plant spores and pollen grains, may act as nuclei for condensation of water in clouds. Basidiospores released by mushrooms form a significant proportion of these aerosols, particularly above tropical forests. Mushroom spores are discharged from gills by the rapid displacement of a droplet of fluid on the cell surface. This droplet is formed by the condensation of water on the spore surface stimulated by the secretion of mannitol and other hygroscopic sugars. This fluid is carried with the spore during discharge, but evaporates once the spore is airborne. Using environmental electron microscopy, we have demonstrated that droplets reform on spores in humid air. The kinetics of this process suggest that basidiospores are especially effective as nuclei for the formation of large water drops in clouds. Through this mechanism, mushroom spores may promote rainfall in ecosystems that support large populations of ectomycorrhizal and saprotrophic basidiomycetes. Our research heightens interest in the global significance of the fungi and raises additional concerns about the sustainability of forests that depend on heavy precipitation.
Million of tons of fungal spores are dispersed in the atmosphere every year [1]. An individual gilled mushroom can release 30,000 basidiospores every second, corresponding to a daily output of billions of microscopic particles [2]. Basidiospores are discharged from the gill surfaces by a catapult mechanism powered by the rapid movement of a drop of fluid over the spore surface (Fig 1). This fluid is called Buller’s drop in tribute to “the Einstein of Mycology,” A. H. R. Buller (1874–1944). Buller’s drop is formed by the condensation of water on the spore surface that is stimulated by the secretion of mannitol and other hygroscopic sugars [3, 4]. Water also condenses on a spot on the adjacent spore surface. The merger of Buller’s drop with this second volume of fluid (the adaxial drop) causes a rapid displacement of the center of mass of the spore. This fluid motion, driven by surface tension, imparts momentum to the spore and it is launched at an initial velocity of up to 1.8 m s-1 [5]. This mechanism was proposed in some detail in the 1980s [6], and verified later by high-speed video recordings [7].
Buller’s drop and the adaxial drop form via condensation of water on the spore surface and their coalescence causes a rapid shift in the center of mass of the spore that is responsible for the launch.
Discharged spores fall in the narrow spaces between the gills and are dispersed in airflow around the mushroom cap. The same discharge mechanism operates in poroid mushrooms, with fertile tubes rather than gills, in mushrooms with spines, and from the great diversity of fruit bodies that form spores on exposed surfaces. Fluid is carried with basidiospores after discharge, but evaporates once the spore is airborne. Mushroom spores are a primary source of mannitol detected in air samples above tropical forests [1]. Using mannitol as a biotracer, Elbert et al. (2007) estimated that 50 million tonnes of spores are dispersed in the atmosphere every year. This biomass is carried by an Avogadroian number of spores, corresponding to an average of 1,000 spores for every square millimeter of Earth’s surface. Other biogenic aerosols include plant spores and pollen grains. These “primary biological aerosol particles” appear to be particularly important in the Amazon Basin, and other densely forested locations, where they may serve as nuclei for cloud formation and precipitation [8]. Details of the hygroscopic behavior of mushroom spores have not been studied previously.
Environmental scanning electron microscopy (ESEM) has been used in a number of experiments to study the condensation of water on pollen and other particles that become aerosolized [9–13]. This technique allows investigators to study the properties of untreated biological samples to preserve natural surface chemistry and to visualize the condensation of water in real time. In the present study, we report ESEM experiments showing that the extraordinary mechanism of spore discharge in mushrooms has a specific effect on water condensation after spores are dispersed in the atmosphere. This suggests that mushroom spores are particularly powerful catalysts for raindrop formation in clouds and contribute to rainfall. This may be an important phenomenon in ecosystems that support large populations of ectomycorrhizal and saprotrophic basidiomycetes. Our research heightens interest in the global significance of the fungi and raises additional concerns about the sustainability of forests that depend on heavy rainfall.
Materials and Methods
Fungal specimens
Fresh specimens of basidiomycete fruit bodies were collected from the Miami University Natural Areas in Oxford, Ohio (Fig 2). Permits are not required for collecting specimens from these woodlands and none of the fungi are listed as protected or endangered species. These species included gilled and poroid mushrooms whose spores are discharged via the drop mechanism (species of Lactarius, Russula, and Suillus), and a puffball (Lycoperdon pyriforme) and earth-star (Geastrum saccatum) whose spores are expelled from the fruit body by the impact of raindrops. Gilled and poroid mushrooms were positioned above ESEM aluminum stubs to allow a fine deposit of spores to accumulate for 1–4 h. The puffball and earth-star spores were deposited on the ESEM stubs by gentle squeezing of the fruit bodies to cause puffing. The spores were not treated with any chemicals prior to imaging.
Environmental Scanning Electron Microscopy (ESEM)
Samples were analyzed using an environmental scanning electron microscope (FEI Quanta 200 ESEM, Hillsboro, OR). Non-coated stubs carrying the spore deposits were mounted on a Peltier cooling stage inside the microscope and the temperature was stabilized at 3 C. The relative humidity inside the sample chamber was controlled by maintaining a constant temperature and altering the vapor pressure (Hiranuma et al. 2008). This provides conditions of supersaturation inside the specimen chamber according to the Clausius-Clapyron equation [14]
RH = relative humidity
P = pressure in torr
PS = saturated vapor pressure at 273.15°C = 4.58 torr
LV = latent heat of vaporization of water = 2.26 x 106 J kg-1
RV = Gas constant for water vapor = 461.5 J/(kg K)
TC = temperature in °C
For the analysis of hygroscopic behavior, each spore deposit of each species was subjected to 2–4 repetitions of identical changes relative humidity.
Results
Dynamics of drop formation
Spores with prominent hilar appendices from gilled and poroid mushrooms were studied under conditions of increasing relative humidity (RH) using the ESEM. Water droplets developed on the hilar appendix (Fig 3) and on the adaxial surface (Fig 4) of these spores between relative humidities of 101% and 102%. Image sequences were captured showing the spore surface prior to droplet initiation, formation of the initial droplets, and subsequent expansion of droplets (Figs 3 and 4, S1 Video and S2 Video). These experiments show that Buller’s drop and the adaxial drop can reform on the spore after discharge.
(A, D) Spore prior to droplet formation with hilar appendix appearing as rounded protruberance from the base of the spore. (B, E) Droplet beginning to form, and (C, F) continuing to expand. Scale = 2 μm.
(A-C) Lactarius hygrophoroides, (D-F) Russula pulchra. The spores in this figure are viewed in profile with the hilar appendix protruding from the base and the adaxial surface above (arrows). (A-C) 103% RH; (D) 98% RH; (E,F) 102% RH. Scale = 5 μm.
https://doi.org/10.1371/journal.pone.0140407.g004
The sensitivity to drop expansion on the hilar appendix and adaxial surface of the spores was demonstrated by controlling the RH inside the sample chamber by increasing or decreasing the water vapor pressure within the sample chamber of the microscope (Fig 5). Water condensing on the spore forms large droplets when RH is increased to 102% RH, and shrinks when it is dropped below 100% RH, appearing as a thin film on the spore surface before complete evaporation. New drops were generated on the same spores after multiple rounds of dehydration and rehydration.
Several factors dictate the maximum size of Buller’s drop and the adaxial drop during spore discharge. Buller’s drop is constrained by spore size and geometry, hydrophobicity of spore surface, and the growth of the adaxial drop with which it fuses [5,15]. In the ESEM experiments, droplets were observed growing from the hilar appendix, and from the adaxial surface, but not from both parts of the same spore surface simultaneously. The maximum size of droplets growing from the hilar appendix of spores is comparable to those predicted from spore dimensions [5,15] and associated with normal spore discharge (Fig 3). Droplets growing from the adaxial surface of spores became much larger, with the diameter of droplets often exceeding the diameter of the spore (Fig 6). The largest drops associated with single spores expanded to a diameter of 13 μm and 27 μm drops surrounded clusters of three spores.
Source – Journals
