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Are fungal spores perfect projectiles? Evolution of ascospore shape

Final Report Summary - AFSPPEOAS (Are fungal spores perfect projectiles? Evolution of ascospore shape)

Project objectives

The project aims to investigate the successful process of fungal spore dispersal in the Ascomycota. In these species, the spores are forcibly discharged from an inflated cell called ascus. Although this pressure gun carries out accelerations that are unmatched in the plant and animal kingdoms, the ejected spores are decelerated to rest after traveling only few millimetres in air, due to their microscopic size. The probability of germination of the spore and the successful establishment of an independent fungus depend critically on the launch range. In fact, most species rely on air to disperse their spores to new potentially favourable environments. Spore discharge is therefore fundamental for the survival of the species and is expected to be under enormous evolutionary pressure. The broad objective of the proposal is the understanding of adaptations evolved by the fungi to maximise the range of spore discharge. The project lies at the interface between fluid dynamics and biology, and combines theory, numerical simulations and experiments. The objectives of the project are:
(i) understanding the physical limitations to spore dispersal;
(ii) understanding the physical constraints to the apparatus of spore launch;
(iii) explore the solutions evolved by fungi.

Description of the work

1) Puffing.
The cup fungi eject hundreds of thousands of spores synchronously in a fraction of a second in a process known as puffing. Synchronised ejection is widespread in the fungi, as it is thought to happen in about 9000 species of the 35 000 known species of ascomycete fungi. We have modelled the action of this collection of projectiles and simulated the air flow generated during the ejection. We have conducted experiments on two different species, Ascobolus f. furfuraceous and Sclerotinia sclerotiorum, to investigate the process, compare the theoretical expectations with the experimental realisation and explore possible coordination mechanisms. We collected high speed movies of the ejection filmed from the top of A. furfuraceous and from the side for S. sclerotiorum (illuminating with a vertical laser sheet); we performed particle image velocimetry (PIV) and measured the launch speed. In the attached document, puffing.avi I show an example of this mechanism of spore discharge in Sclerotinia sclerotiorum. We obtained this movie through laser sheet illumination and a fast camera at 3000 frames per second. The movie spans 200 ms, and it is slowed down 100 times.

2) Apical rings.
We have developed a complete model of the dynamics of spore ejection through the apical ring, an elastic aperture sealing the ascus and stretching to let the spore out during discharge. We included the full elastohydrodynamics of the apical ring to compute the dynamics of spore discharge. We identified the sources of energy dissipation in the system and performed numerical simulations to predict the optimal geometry of the apical ring. We performed a complete literature search to gather all the published data on the relevant morphological dimensions and verify the theoretical predictions.
In the attached document, spore_discharge.avi I show a high-speed movie of spore discharge in a cup fungus isolated from herbivore dung. We obtained this movie through a high speed camera connected to a microscope. The resolution is 128x128, the field of view is about 60 microns; the time resolution is 66 000 frames per second; it is slowed down 13 000 times, and it spans about 260 ms.

3) We reviewed the physical mechanism of spore ejection, summarising the constraints and organising the different morphological adaptations.

4) We have theoretically and experimentally investigated the growth of bacterial biofilms that are used as bio-control agents to prevent plant pathogen infection.

Main results

1) Cooperative spore ejection. We modelled the hydrodynamic interaction of spores with air as Stokes drag, numerically represented by a point force whose magnitude is proportional to the difference between the velocity of the spore and the local speed of air. Through asymptotic models and numerical simulations, we showed that the action of hundreds of thousands of spores launched in the same direction with extremely high velocity creates a jet of air that is able to carry the spores 10-20 times further than the range of an isolated spore. By comparing this prediction with PIV data obtained from high-speed movies of puffing, we showed that the model quantitatively predicts the structure of the jet of spores.
2) Moving around obstacles. As the spores mobilise the air, they transition from a purely ballistic dynamics to passive transport. This switch enables spores to travel around obstacles. We confirmed this expectation by filming puffing against a glass slide and observing the jet of spores moving around and pass the glass slide.
3) Cooperation among spores. High-speed movies of the fruit bodies during the puff show that the ejection is spatially coordinated. Few spores are initially discharged and initiate a wave of ejection that expands across the apothecium. Genetic conflicts might arise because the first spores to be ejected travel less far than the last spores in the puff, and because spores in a fruit body may not be genetically identical. We have shown theoretically and experimentally that spatial coordination enforces cooperation by mobilizing only a thin sheet of air above the asci that are triggered to eject their spores.
4) Apical ring. We have obtained a complete description of the dynamics of single spore ejection through the elastic ring at the apex of the cell that contains the spores. We have conducted numerical simulations of the equations of motion and found non-trivial conditions on the optimal design for the spore shooting apparatus. We have conducted a thorough literature search and showed data collapse on the theoretical prediction.
5) Bacterial biofilms. We have elaborated a quantitative model of biofilm growth and expansion that is relevant for the understanding of its effects as a bio-control agent against fungal spore invasion.

Conclusions and potential impact.

We expect our results to have potential impact both on fundamental scientific questions and on the control of fungal pathogens on plants and humans. Understanding how fungi disperse their spores is a key to understand how species move at all scales from millimetres to planetary scales, which is a fundamental requirement to predict range shifts in a time of global change. The ideas developed in this project provide:
(i) potentially new ways for the control of infections by Sclerotinia sclerotiorum, and other plant pathogens causing billions of dollars of damage per year;
(ii) a model for stable cooperation, with policing against cheating based on physical rather than genetic processes;
(iii) quantitatively accurate models to capture the biomechanics of spore discharge;
(iv) a novel framework to understand morphological diversity across phylogenies, based on the physical constraints on spore discharge. The results of this project strongly support the idea that spore range is a quantitatively accurate measure of individual fitness, providing a rare opportunity to probe how evolution operates in nature rather than in laboratory conditions.

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