Fungal thermotolerance revisited and why climate change is unlikely to be supercharging pathogenic fungi (yet).

Thermotolerance has been viewed as an uncommon characteristic among the fungi and one of the reasons that less than 1% of the described species operate as opportunistic pathogens of humans. Growth at 37°C is certainly a requirement for a fungus that invades the body core, but tens of thousands of nonpathogenic species are also able to grow at this temperature. Ergo, body temperature does not serve as a thermal barrier to the development of infections by many harmless fungi. The absence of other virulence factors must be more demanding. This observation raises questions about the hypothetical links between climate change and the increasing number of life-threatening human mycoses. Given the widespread distribution of fungal thermotolerance and the 1°C (2°F) increase in global temperature over the last 140 years it seems unlikely that the warming climate has driven the evolution of more virulent strains of fungi. More compelling explanations for the changes in the behavior of fungi as disease agents include their adaptation to the widening use of azole antifungals in hospitals and the wholesale application of millions of tons of the same class of heterocyclic chemicals in agriculture. On the other hand, climate change is having a significant effect on the spread of human mycoses by extending the geographical range of pathogenic fungi. A related increase in fungal asthma caused by spore inhalation is another likely consequence of planetary change.

Ascus function: From squirt guns to ooze tubes.

Unlike the mechanism of ballistospore discharge, which was not solved until the 1980s, the operation of asci as pressurized squirt guns is relatively straightforward and was understood in the nineteenth century. Since then, mycologists have sought to understand how structural adaptations to asci have allowed the ascomycetes to expel spores of different shapes and sizes over distances ranging from a few millimeters to tens of centimeters. These modifications include the use of valves at the tips of asci that maintain ascus pressure and expel spores at the highest speeds, and gelatinous appendages that connect spores after release and create larger projectiles with greater momentum than single spores. Clever experiments in the twentieth century coupled with meticulous microscopic studies led investigators to understand how asci with complicated apical structures worked and mathematical models produced estimates of launch speeds. With the recent application of high-speed video microscopy, these inferences about ascus function have been tested by imaging the motion of spores on a microsecond timescale. These experiments have established that ascospore discharge is the fastest fungal movement and is among the fastest movements in biology. Beginning with the history of the study of asci, this review article explains how asci are pressurized, how spores are released, and how far spores travel after their release. We also consider the efficiency of ascospore discharge relative to the mechanism of ballistospore discharge and examine the way that the squirt gun mechanism has limited the morphological diversity of ascomycete fruit bodies.

Goldilocks mushrooms: How ballistospory has shaped basidiomycete evolution.

Ballistospory has been a governing factor in mushroom diversification. Modifications to fruit body morphology are subject to a series of fundamental constraints imposed by this uniquely fungal mechanism. Gill spacing in lamellate mushrooms, tube width in poroid species, and other configurations of the hymenium must comply with the distance that spores shoot themselves from their basidia. This reciprocal relationship between the development of fruit bodies and spores may have been maintained by a form of evolutionary seesaw proposed in this article. The necessity of the accurate gravitropic orientation of gills and tubes is another constraint on mushroom development and physiology, along with the importance of evaporative cooling of the hymenium for successful spore discharge and the aerodynamic shaping of the fruit body to aid dispersal. Ballistospory has been lost in secotioid and gasteroid basidiomycetes whose spores are dispersed by animal vectors and has been replaced by alterative mechanisms of active spore discharge in some species. Partnered with the conclusions drawn from molecular phylogenetic research, the biomechanical themes discussed in this review afford new ways to think about the evolution of basidiomycetes.

The fastest short jump in nature: Progress in understanding the mechanism of ballistospore discharge.

The coalescence of fluid droplets on the surface of ballistospores powers their launch into the air at a speed of up to one meter per second with an acceleration of thousands of g's. This mechanism has been studied for more than a century and its solution is an emblem of mycological progress. Because the spores move too fast for the launch to be watched with a light microscope, early advances were made by inferences about what must be happening when the spores disappeared rather than direct observations. These investigations were followed by ingenious experiments that led to a satisfying explanation of ballistospory by the 1990s. Ultra-high-speed video recordings of spore discharge verified this model in the 2000s and subsequent research has shown how the mechanism has been adapted to launch spores over different distances. The available evidence suggests that many of these adaptations have been achieved by changes in spore morphology. Understanding the cellular and genetic basis of these modifications is one of the principal challenges for understanding the evolution of the basidiomycetes.

Fungal ecology: Truffle-guzzling birds.

A new study of truffle fungi in Patagonia reveals the importance of native bird species in spore dispersal. This has wide implications for the study of animal-fungal symbioses in the Southern Hemisphere.

Hyphal and mycelial consciousness: the concept of the fungal mind.

Like other cells, fungal hyphae show exquisite sensitivity to their environment. This reactiveness is demonstrated at many levels, from changes in the form of the hypha resulting from alterations in patterns of exocytosis, to membrane excitation, and mechanisms of wound repair. Growing hyphae detect ridges on surfaces and respond to restrictions in their physical space. These are expressions of cellular consciousness. Fungal mycelia show decision-making and alter their developmental patterns in response to interactions with other organisms. Mycelia may even be capable of spatial recognition and learning coupled with a facility for short-term memory. Now is a fruitful time to recognize the study of fungal ethology as a distinctive discipline within mycology.

Are mushrooms medicinal?

Despite the longstanding use of dried mushrooms and mushroom extracts in traditional Chinese medicine, there is no scientific evidence to support the effectiveness of these preparations in the treatment of human disease. Consumers should evaluate assertions made by companies about the miraculous properties of medicinal mushrooms very critically. The potential harm caused by these natural products is another important consideration. In a more positive vein, the presence of potent toxins and neurotropic compounds in basidiomycete fruit bodies suggests that secondary metabolites with useful pharmacological properties are widespread in these fungi. Major investment in controlled experiments and objective clinical trials is necessary to develop this natural pharmacopeia.

Mushrooms as Rainmakers: How Spores Act as Nuclei for Raindrops.

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.

Short-range splash discharge of peridioles in Nidularia.

The distinctive shapes of basidiomata in the bird's nest fungi reflect differences in the mechanism of splash discharge. In the present study, peridiole discharge was examined in Nidularia pulvinata using high-speed video. Nidularia pulvinata produces globose basidiomata that split open at maturity to expose 100 or more peridioles within a gelatinous matrix. Each peridiole contains an estimated 7 million spores. The impact of water drops splashed the peridioles horizontally from the fruit body, along with globs of mucilage, at a mean velocity of 1.2 m s(-1). Discharged peridioles travelled for a maximum horizontal distance of 1.5 cm. This launch process contrasts with the faster vertical splashes of peridioles over distances of up to one metre from the flute-shaped fruit bodies of bird's nest fungi in the genera Crucibulum and Cyathus. Peridioles in these genera are equipped with a funicular cord that attaches them to vegetation, placing them in an ideal location for ingestion by browsing herbivores. The absence of cords in N. pulvinata and its use of a sloppy discharge mechanism suggest that it is more likely to be dispersed by animals feeding on the forest floor.

Splash and grab: biomechanics of peridiole ejection and function of the funicular cord in bird's nest fungi.

The bird's nest fungi (Basidiomycota, Agaricales) package millions of spores into peridioles that are splashed from their basidiomata by the impact of raindrops. In this study we report new information on the discharge mechanism in Crucibulum and Cyathus species revealed with high-speed video. Peridioles were ejected at speeds of 1-5 m per second utilizing less than 2 % of the kinetic energy in falling raindrops. Raindrops that hit the rim of the basidiome were most effective at ejecting peridioles. The mean angle of ejection varied from 67 to 73° and the peridioles travelled over an estimated maximum horizontal distance of 1 m. Each peridiole carried a cord or funiculus that remained in a condensed form during flight. The cord unravelled when its adhesive surface stuck to a surrounding obstacle and acted as a brake that quickly reduced the velocity of the projectile. In nature, this elaborate mechanism tethers peridioles to vegetation in a perfect location for browsing by herbivores.

Against the naming of fungi.

The use of molecular bar-coding and consensus on nomenclatural practices has encouraged optimism about the future of fungal taxonomy and systematics. There are, however, profound deficiencies in our understanding of fungal diversity and broader problems with the taxonomic enterprise that deserve greater attention. For 250 years mycologists have tried to reconcile fungal diversity with the Linnean fantasy of a divine order throughout nature that included unambiguous species. This effort has failed and today's taxonomy rests on an unstable philosophical foundation. Rather than persisting with the present endeavour, it may be more fruitful to abandon the notion of fungal species pending further basic research. In the meantime, mycologists should consider tagging collections with digital codes and assigning these operational taxonomic units to higher taxonomic ranks whose objective reality is corroborated by strong phylogenetic evidence.

Agni's fungi: heat-resistant spores from the Western Ghats, southern India.

This study concerns the thermotolerance of spores of mesophilic fungi isolated from a tropical semi-arid habitat subject to dry season fire in the Western Ghats, southern India. Among 25 species of Ascomycota isolated from leaf litter, nine were able to grow after incubation in a drying oven for 2h at 100°C; the spores of two of these species survived 2h incubation at 110°C, and one survived exposure to 115°C for 2h. The range of thermotolerance among mesophilic fungi isolated from the leaf litter was surprising: filamentous fungi from other habitats, including species that colonize scorched vegetation after fires and thermophilic forms occurring in self-heating plant composts, cannot survive even brief exposure to such high temperatures. It is possible that the exceptional heat resistance of the Indian fungi is related to adaptations to surviving fires. Genetic analysis of the physiological mechanisms of heat resistance in these fungi offers prospects for future biotechnological innovations. The discovery of extreme thermotolerance among common saprotrophs shows that this physiological trait may be more widespread than recognized previously, adding to concern about the evolution of opportunistic pathogens on a warmer planet. The fungi in this study are among the most heat-resistant eukaryotes on record and are referred to here as 'Agni's Fungi', after the Hindu God of Fire.

Solving the aerodynamics of fungal flight: how air viscosity slows spore motion.

Viscous drag causes the rapid deceleration of fungal spores after high-speed launches and limits discharge distance. Stokes' law posits a linear relationship between drag force and velocity. It provides an excellent fit to experimental measurements of the terminal velocity of free-falling spores and other instances of low Reynolds number motion (Re<1). More complex, non-linear drag models have been devised for movements characterized by higher Re, but their effectiveness for modeling the launch of fast-moving fungal spores has not been tested. In this paper, we use data on spore discharge processes obtained from ultra-high-speed video recordings to evaluate the effects of air viscosity predicted by Stokes' law and a commonly used non-linear drag model. We find that discharge distances predicted from launch speeds by Stokes' model provide a much better match to measured distances than estimates from the more complex drag model. Stokes' model works better over a wide range projectile sizes, launch speeds, and discharge distances, from microscopic mushroom ballistospores discharged at <1 m s(-1) over a distance of <0.1mm (Re<1.0), to macroscopic sporangia of Pilobolus that are launched at >10 m s(-1) and travel as far as 2.5m (Re>100).

Why mushrooms form gills: efficiency of the lamellate morphology.

Gilled mushrooms are produced by multiple orders within the Agaricomycetes. Some species form a single array of unbranched radial gills beneath their caps, many others produce multiple files of lamellulae between the primary gills, and branched gills are also common. In this largely theoretical study we modeled the effects of different gill arrangements on the total surface area for spore production. Relative to spore production over a flat surface, gills achieve a maximum 20-fold increase in surface area. The branching of gills produces the same increase in surface area as the formation of free-standing lamellulae (short gills). The addition of lamellulae between every second gill would offer a slightly greater increase in surface area in comparison to the addition of lamellulae between every pair of opposing gills, but this morphology does not appear in nature. Analysis of photographs of mushrooms demonstrates an excellent match between natural gill arrangements and configurations predicted by our model.

How far and how fast can mushroom spores fly? Physical limits on ballistospore size and discharge distance in the Basidiomycota.

Active discharge of basidiospores in most species of Basidiomycota is powered by the rapid movement of a droplet of fluid, called Buller's drop, over the spore surface. This paper is concerned with the operation of the launch mechanism in species with the largest and smallest ballistospores. Aleurodiscus gigasporus (Russulales) produces the largest basidiospores on record. The maximum dimensions of the spores, 34 × 28 µm, correspond to a volume of 14 pL and to an estimated mass of 17 ng. The smallest recorded basidiospores are produced by Hyphodontia latitans (Hymenochaetales). Minimum spore dimensions in this species, 3.5 × 0.5 µm, correspond to a volume of 0.5 fL and mass of 0.6 pg. Neither species has been studied using high-speed video microscopy, but this technique was used to examine ballistospore discharge in species with spores of similar sizes (slightly smaller than A. gigasporus and slightly larger than those of H. latitans). Extrapolation of velocity measurements from these fungi provided estimates of discharge distances ranging from a maximum of almost 2 mm in A. gigasporus to a minimum of 4 µm in H. latitans. These are, respectively, the longest and shortest predicted discharge distances for ballistospores. Limitations to the distances traveled by basidiospores are discussed in relation to the mechanics of the discharge process and the types of fruit-bodies from which the spores are released.

Biomechanics of invasive growth by Armillaria rhizomorphs.

Rhizomorphs of wood-decay basidiomycetes are root-like structures produced by the coordinated growth of thousands of hyphae. Very little is known about their development nor the way that they penetrate soils and rotting wood. In this study, we applied techniques used in previous studies on hyphae to explore the mechanics of the invasive growth process in Armillaria gallica. Growth rate measurements were made in media with different gel strengths. The osmolyte composition of rhizomorph sap was determined spectroscopically and the forces exerted by growing tips were measured using a force transducer. Cultured rhizomorphs extended at much faster rates than unbundled hyphae (3.5mmd(-1) versus 1.5mmd(-1)) and their growth accelerated in response to increased medium gel strength (to 7.4mmd(-1)). Measurements of rhizomorph osmolality provided a turgor pressure estimate of 760kPa (7.5atm.), and spectroscopic analysis showed that this pressure was generated by the accumulation of erythritol, mannitol, and KCl. Forces exerted by growing tips ranged from 1 to 6mN, corresponding to pressures of 40-300kPa (0.4-3.0atm.). Pressures exerted by extending rhizomorphs are comparable to those produced by individual vegetative hyphae. This suggests that the mechanical behavior of hyphae is similar whether they grow as unbundled cells or aggregate to form macroscopic rhizomorphs.

Adaptation of the spore discharge mechanism in the basidiomycota.

BACKGROUND: Spore discharge in the majority of the 30,000 described species of Basidiomycota is powered by the rapid motion of a fluid droplet, called Buller's drop, over the spore surface. In basidiomycete yeasts, and phytopathogenic rusts and smuts, spores are discharged directly into the airflow around the fungal colony. Maximum discharge distances of 1-2 mm have been reported for these fungi. In mushroom-forming species, however, spores are propelled over much shorter ranges. In gilled mushrooms, for example, discharge distances of <0.1 mm ensure that spores do not collide with opposing gill surfaces. The way in which the range of the mechanism is controlled has not been studied previously. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we report high-speed video analysis of spore discharge in selected basidiomycetes ranging from yeasts to wood-decay fungi with poroid fruiting bodies. Analysis of these video data and mathematical modeling show that discharge distance is determined by both spore size and the size of the Buller's drop. Furthermore, because the size of Buller's drop is controlled by spore shape, these experiments suggest that seemingly minor changes in spore morphology exert major effects upon discharge distance. CONCLUSIONS/SIGNIFICANCE: This biomechanical analysis of spore discharge mechanisms in mushroom-forming fungi and their relatives is the first of its kind and provides a novel view of the incredible variety of spore morphology that has been catalogued by traditional taxonomists for more than 200 years. Rather than representing non-selected variations in micromorphology, the new experiments show that changes in spore architecture have adaptive significance because they control the distance that the spores are shot through air. For this reason, evolutionary modifications to fruiting body architecture, including changes in gill separation and tube diameter in mushrooms, must be tightly linked to alterations in spore morphology.