SPORE MORTALITY AND ENVIRONMENTAL STOCHASTICITY: UNDERSTANDING THE LONG DISTANCE DISPERSAL OF PLANT PATHOGENIC FUNGI

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1013478
• Project Start Date: Oct 1, 2017
• Project End Date: Sep 30, 2020
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218

Performing Department
Bacteriology

Non Technical Summary
The dispersal of fungi drives the spread of disease. In recent years, dispersal has received a great deal of attention in fields ranging from mathematics and physics to ecology and molecular biology, nonetheless, only a patchy framework exists to explain fungal dispersal over very long distances. To date research has focused on the genetics of spatially disjunct populations, with dispersal inferred from measured levels of gene flow, or the trapping of spores at relatively short distances away from a source population. More poorly understood are the mechanics of spore movement over long distances. Fundamental, unanswered questions include 1) how long can spores survive in the environments typical of the atmosphere and are there consistent differences between short distance and long distance dispersers? Can these differences be used to predict dispersal strategies? 2) how far does a spore travel within its lifetime? Using an interdisciplinary approach grounded in both biology and physics, and a combination of experiments and modeling, I aim to 1) collect demographic data on spores of both short distance and long distance dispersers, quantifying mortality over time and identifying a typical lifespan for each species; and 2) use historical weather data available from NOAA to model the movement of spores in winds over landscapes, plotting distances reached by spores liberated at different times and in different seasons. Target species include two species of Alternaria (alternata and solani), pathogens of potato, and Puccinia graminis, a pathogen of wheat.

Animal Health Component

15%

Research Effort Categories

Basic

85%

Applied

15%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	4020	1070	30%
212	4020	1102	30%
212	4020	2010	30%
212	4020	1060	10%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
4020 - Fungi;

Field Of Science
2010 - Physics; 1060 - Biology (whole systems); 1070 - Ecology; 1102 - Mycology;

Keywords

fungi

dispersal

biomechanics

spore liberation

hysplit

Goals / Objectives
Fungi disperse over multiple temporal and spatial scales. Many live cryptically, hidden in the soil as decomposers of dead matter, or in symbiosis with plants, animals or other fungi. The diversity of the kingdom remains largely undocumented, encompassing millions of undescribed species. They are simultaneously among the worst threats and most fundamental components of many ecosystems. Although they lack legs or wings, fungi routinely move across vast distances, and even across oceans, using spores. But while spore ejection is controlled to the slimmest level of precision, after ejection spore fate is dramatically affected by a series of stochastic events that appear difficult to anticipate. I propose to test whether and how apparent stochasticity shapes dispersal and ultimately, to explore the evolution of strategies for spore liberation.Initial target species include two species of Alternaria (alternata and solani), pathogens of potato, and Puccina graminis, a pathogen of wheat.Objective 1) Once spores leave the cover of a crop canopy and enter large scale atmospheric circulation, they are exposed to harsh environments; UV light and other stresses are a major cause of mortality (Rotem & Aust 1991). Anecdotal evidence suggests physiological costs to maintaining e.g. UV tolerance; therefore, a short distance disperser may not be UV tolerant. To test the ability of long distance (A. alternata) and short distance (A. solani) dispersers to withstand various combinations of UV, air moisture (relative humidity) and temperature, we will expose spores of both species and collect demographic data, quantifying spore mortality over time. To provide data for Objective 2) (below), we will also include P. graminis in experiments. Once protocols are established, and based on initial results tied to this objective and Objective 2), we will seek to include additional species, including species known to have successfully traveled extreme distances.Hypothesis: Clear differences in the demographic trajectories of A. alternata and A. solani will reflect the different abilities of species' spores to survive in the environments typical of the troposphere; the long distance disperser A. alternata will better withstand UV exposure.Objective 2) Use insights gleaned from Objective 1), in combination with historical weather data available from the National Oceanic and Atmospheric Administration (NOAA), to model the movement of P. graminis and other species' spores in winds over landscapes (Stein et al. 2015).Hypothesis: Large scale numerical simulations of atmospheric transport will confirm that the timing of a spore's liberation (e.g. in the morning or evening; during a storm, or not) shapes the ultimate reach of the spore; simulations will inform the mechanics of the Puccinia "pathway" and reveal whether migration is achieved by a series of short "hops", or by fewer longer distance dispersal events.With these data and models, we will be able to explore the role apparent stochasticity may play in shaping spore dispersal, for example testing if spore liberation is cued to particular meteorological phenomena.

Project Methods
Objective 1)The variables most frequently identified as shaping spore survival in the atmosphere are light, moisture, and temperature (Gregory 1961; Isard & Gage 2001). But data on the physiological tolerances of spores to solar radiation (especially UV), air moisture (relative humidity), and temperature are scarce (Rotem & Aust 1991). The resilience of a spore to any of these variables will vary by species. For example, urediniospores of Puccinia striiformis var. tritici die quickly when exposed to high solar radiation, while ascospores of Giberrella zeae are more susceptible to low relative humidities, and urediniospores of Phakopsora pachyrhizi cannot tolerate cold temperatures (Maddison & Manners 1972; Fernando et al. 2000; Park et al. 2008).To test the ability of closely related, putative short and long distance dispersing species to survive an array of UV, RH, and temperature levels typical of the middle-upper troposphere; the layer of the atmosphere in which long distance dispersing spores are reported to travel (Griffin et al. 2001), and in collaboration with Pringle laboratory graduate students, I will ask:What combination of UV, RH and temperature most favors spore viability through time? And in an optimal environment, how many days are needed to cause 100% spore mortality?Experimental protocols. While unable to account for random fluctuations in the atmosphere, we can replicate homogenous levels of UV, RH, and temperature (T) in Biotron experiments, using combinations typical of the upper-middle troposphere (Gregory 1961). By recording viability at different time points in each combination of UV-RH-T, I will identify the UV-RH-T combinations that maximize spore survival. Preliminary trials suggest the following parameters will be most informative:Each experiment will involve a total of 84 glass slides, 12 per tier, each slide housing 6,000 spores (approximately 10 spores per µm2). The radiant flux of UV-A and UV-B light will be manipulated by placing spores at different distances from the light sources; RH will be either 90, 75, 50, 25, or 10% and T will be either 10, 15, 20, or 25ºC (a total of 20 experiments per species). Each experiment will run (with 12-hour light/dark rotations) for 12 days, or until 100% mortality is reached. If spores survive beyond the 12 days, which is unlikely, experiments will be run again for more days. Every 24 hours, a random slide will be removed from each tier. First germination will be induced (incubation periods will vary per species). The number of viable spores will then be recorded by quantifying the number of live spores. Spores will be stained with an esterase vitality stain (CFDA, Invitrogen™) and propidium iodide, which fluorescently label live and dead spores, respectively. Equipment housed at the Newcomb Imaging Center (www.botany.wisc.edu/nic/) will be used to image stained spores. The number of live and dead spores, as well as the sizes of germinating colonies, will be quantified using ImageJ and the Cell Counter plugin. Given the large number of spores on each slide (6,000) we expect to observe monotonically declining survivorship. Subsequent demographic analyses begin with the construction of a synthetic cohort, based on a time series of survivorships within the same treatment, but using different subsamples (the different slides). A Kaplan-Meier survivorship curve for each treatment will then be constructed. Survivorship analysis using Cox regression or GLMM (depending on the applicability of the proportional hazards assumption) will then be used to examine the importance of humidity, temperature, and UV on survivorship.Objective 2)Objective 2 will be completed in collaboration with a physicist and long standing collaborator at the Institut de Physique de Nice (Université Côte d'Azur, CNRS); Dr. Agnese Seminara.A tremendous amount of meteorological data, and data on the movement of air parcels, is made available by NOAA (www.arl.noaa.gov). Databases are continuously updated, and North American data are now available at a resolution of every 12 km2 every 6 hours, making it possible to numerically simulate particle dispersal in the atmospheric boundary layer (ABL).One of the most widely used approaches to simulating the movement of particles over the globe, but normally used to model the dispersal of smoke, dust, or pollutants, involves HYSPLIT (Hybrid Single Particle Lagrangian Integrated Trajectory) models (Stein et al. 2015). HYSPLIT integrates NOAA's extensive meteorological database, with its thousands of recorded wind speeds (every observation linked to both a specific time and place) to model the trajectory of particle diffusion in the atmosphere (Stein et al. 2015). We propose using HYSPLIT to model dispersal from known source populations. Our models will track the movement of particles (spores) from specific locations, e.g., in Mexico or Texas (the origin of the Puccinia pathway each year) across North America. Models will use real weather data to plot potential trajectories for any time on any day of any year available, allowing us to compute, for example, the trajectories of spores released in any combination of, for example, the early morning vs. late afternoon, or May vs. July, or Mexico vs. Iowa. Using data of Objective 1, we can set a limit to the number of days the model will run; if a spore's maximum potential survival in the ABL is estimated as 5 days we can limit the model to run for just 5 days. In combination with data from Objective 1, we will use the data to plot spore density gradients per meter (i.e. dispersal kernels) recorded from each simulated release time, date, and location. Simulated dispersal kernels will enable us to quantify the probability of e.g. P. graminis uredospores reaching discrete distances from single source populations with respect to time and distance, and test whether the Puccinia pathway is more likely to result from dispersal events spanning e.g. Mexico to Nebraska, or a series of smaller distances. Once techniques are in place, we can use them to model the dispersal kernels of many other species.With these models, we will also be able to explore the role apparent stochasticity may play in shaping spore dispersal, and eventually test if spore liberation is cued to particular meteorological phenomena. To the best of our knowledge, no one has ever systematically explored the timing of spore release; we know remarkably little about when spores are ejected in nature (but see Gilbert and Reynolds 2005). Many hypotheses are plausible. Spore liberation may be cued, for example, by 1) diurnal changes in near surface winds (e.g. updrafts caused by the sun rising and warming the earth) 2) meteorological events (e.g. approaching thunderstorms and associated updrafts), 3) unknown variables, or 4) random phenomena without pattern. To explore these hypotheses, we must first know what kinds of events would impact LDD; comparing the dispersal kernels of spores released, e.g., at dawn vs. noon would give us the needed support to move forward with targeted observations of spore liberation in nature.

Progress 10/01/17 to 09/30/20

Outputs
Target Audience:My current target audience now includes scientists interested in fungi, dispersal, movement ecology, plant pathology, disease ecology and epidemiology, demography, and ecology or evolution. My audience also includes growers and the companies involved in selling products to combat or manage fungal disease. To reach my target audiences, I wrote papers and gave numerous talks. An abbreviated list of the talks I have given while this grant was in progress follows: Invited Talks (Academic Audiences) 2017 Drexel University 2017 Uppsala University (Sweden) 2017 Chulalongkorn University (Thailand) 2017 Michigan State University 2017 Kellogg Biological Station 2018 University of Miami 2018 Max Planck Institute for Evolutionary Biology (Germany) 2018 University of Colorado, Boulder 2018 University of Dar Es Salaam (Tanzania) 2018 Mexican Mycological Congress Keynote (via teleconference) 2018 University of Michigan 2019 Fungal Genetics Conference "The Spore" 2019 International Conference on Mycorrhizas 10 "Emerging Model Systems" (Mexico) 2019 Mycological Society of America "Taylor Festschrift" 2019 Boston University 2020 University of Mississippi 2020 University of British Columbia 2020 Presidential Address, Mycological Society of America (Virtual: COVID-19) 2021 American Society of Microbiology/ Microbe 2020 "Microbes in the Anthropocene" (Cancelled: COVID-19, rescheduled for 2021) 2021 New Phytologist Symposium "Plant/Fungal Invasions" (Brazil) Invited Talks (Popular Audiences/Science Outreach) 2017 iBiology (www.ibiology.org) (three lectures) 2018 Inaugural Meeting of the Madison Mycological Society 2018 Wisconsin Mycological Society (twice) 2019 Milwaukee Public Museum 2019 University of Wisconsin-La Crosse Mycology Club 2019 Bogotá Botanical Garden, Colombia Changes/Problems:We did strategically change some aspects of our experiments, for example focusing on just two species of Alternaria in demographic experiments. However, we have more than met our original aims and on balance, the research has been a total success. What opportunities for training and professional development has the project provided?Two students received training: one graduated with a Ph.D. in May 2020 and is now in his first year of law school. Jacob Golan is attending NYU, one of our nation's premier schools for the study of intellectual property law and aims to be an expert in fungal patents and fungal intellectual property. Yen-Wen Wang will likely graduate in May 2022 (his progress has been somewhat slowed by the pandemic). His stellar record will enable him to join a top lab for a postdoc and I am quite sure he will settle into an academic career as the head of his own laboratory. How have the results been disseminated to communities of interest?Since the begininng of this project, I've given 24 invited talks to both academic and popular audiences, and I will continue to talk about my Hatch research in future (for the moment, all conferences and talks are virtual). We have also published a series of often very high-profile papers describing our data and I continue my efforts to change the narrative of fungal dispersal, seeking to move scientists to understand fungi as capable of manipulating their own dispersal in hitherto unsuspected ways. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? I am happy to report we have accomplished a great deal and more than met the stated goals of our Hatch grant proposal. In addition to the accomplishments and progress I described in previous reports, we have now proved: (Objectives 1 and 2): By measuring the germination of spores exposed to realistic simulations of atmospheric environments we sought to understand patterns of survival during travel and aimed to predict the potential distances travelled by spores of different species. We focused on two agricultural pathogens of great economic importance: Alternaria alternata and A. solani. Both species are major threats to Wisconsin potato crops, and Wisconsin is the third largest producer of potatoes in the USA. Potatoes are a $4 billion/year industry. The spores of the two species are very different sizes: conidia of A. solani are nearly 10x longer than conidia of A. alternata. We tested how long spores could survive over a time scale consistent with reports of continental or trans-oceanic dispersal: ~12 days. The vast majority of A. solani conidia failed to germinate after 24 hours' exposure to atmospheric environments, whereas over half of A. alternata conidia were viable even after 12 days. In order to evaluate how differences in size and spore longevity influence long-distance dispersal we next used HYSPLIT simulations of atmospheric transport to model the dispersal ability of each species. The smaller conidia of A. alternata remain airborne longer than the conidia of A. solani. The model predicts 99% of A. solani conidia will settle within 24 hours, at which time many are still viable, with potential dispersal distances of up to (only)100 km. By comparison, A. alternata conidia remain remain airborne for much longer periods of time. Because they also survive after days of exposure to the atmosphere, they appear capable of extremely long-distance dispersal, including from Wisconsin to Greenland (~4,000 km)! Our data and models of the survival and trajectory of these species' spores highlight the radically different dispersal patterns of these two closely related economically important pathogens. (Objective 2) In synergy with our work on Alternaria, we modelled the movement of spores released from fungi growing in different locations across North America over different seasons. Our research is now published in PNAS, a very high impact journal, and I have described it in previous reports. In brief: we discovered the timing of spore release will dictate how long spores remain in the atmosphere before settling. We hypothesize fungal pathogens will evolve to manipulate the timing of spore release, releasing spores at different times of day or in different months to enable spores to travel for shorter or longer times. This critical insight into the evolution of spore release may enable novel approaches to pathogen control by enabling scientists to develop strategies to disrupt dispersal. (Objective 1) In a novel addition to our stated goals, we have been using population genomics data to infer dispersal patterns of a deadly, invasive mushroom: the death cap. Using these data, we have developed novel bioinformatic pipelines to infer whether the death cap spreads aerially, using spores, or underground. While this manuscript is still being written, at the moment it seems clear spores are critical to the death cap's invasion. However, spores appear to travel fairly short distances, making the death cap analogous to A. solani (not A. alternata). Previous reports describe other publications related to fungal dispersal and fitting within the aims of my Hatch, for example work on the invasive fly agaric (Amanita muscaria) and on the dispersal of yeast fungi in nature (to our knowledge, the first paper ever to record the movements of fungi in real time in a natural habitat).

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Boynton, P.J., C.N. Peterson, A. Pringle. 2019. Superior dispersal ability can lead to persistent ecological dominance throughout succession. Applied and Environmental Microbiology 85:e02421-18.
• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Vargas- Estupi��n N., S. Gon�alves, A.E. Franco-Molano, S. Restrepo, A. Pringle. 2019. In Colombia the Eurasian fungus Amanita muscaria is expanding its range into native, tropical Quercus humboldtii forests. Mycologia 111:758-771.
• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Oneto, D.L., J. Golan, A. Mazzino, A. Pringle, A. Seminara. 2020. Timing of fungal spore release dictates survival during atmospheric transport. Proceedings of the National Academy of Sciences (PNAS) 117:5134-5143.
• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Iapichino, M., Y.-W. Wang, S. Gentry, A. Pringle, A. Seminara. In press. A precise relationship among Bullers drop, ballistospore and gill morphologies enables maximal packing of spores within gilled mushrooms. Mycologia.
• Type: Journal Articles Status: Other Year Published: 2021 Citation: Golan J., C.A. Adams, H. Cross, H. Elmore, M. Gardes, S.I. Glassman, S.C. Gon�alves, J. Hess, F. Richard, Y.-W. Wang, B. Wolfe, A. Pringle. Draft. Native and invasive populations of the ectomycorrhizal death cap Amanita phalloides are sexual but dispersal limited.
• Type: Journal Articles Status: Other Year Published: 2021 Citation: Golan, J., S. Ding, D.L. Oneto, R. Kessenich, A. Mazzino, M. Sandler, T.A. Rush, D. Levitis, A. Seminara, A. Gevens, A. Pringle. Draft. Survival in the atmosphere shapes the effective reach of Alternaria pathogens dispersing from Wisconsin.
• Type: Theses/Dissertations Status: Other Year Published: 2020 Citation: Golan, J. Biophysical constraints & genetic and genomic consequences of fungal dispersal. Ph.D. Dissertation: Department of Botany, University of Wisconsin-Madison

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:My target audience still includes my small group of collaborators: graduate students and colleagues here (at Wisconsin) and at the Institut de Physique in Nice. However, this year my target audience expanded to include readers of a set of publications now published/in press or in revision, including: Golan J., C.A. Adams, H. Cross, H. Elmore, M. Gardes, S.I. Glassman, S.C. Gonçalves, J. Hess, F. Richard, Y.-W. Wang, B. Wolfe, A. Pringle. Draft. Native and invasive populations of the ectomycorrhizal death cap Amanita phalloides are highly sexual but dispersal limited. Iapichino, M., Y.-W. Wang, S. Gentry, A. Pringle, A. Seminara. In revision. A precise relationship among Buller's drop, ballistospore and gill morphologies enables maximal packing of spores within gilled mushrooms. Mycologia. Oneto, D.L., J. Golan, A. Mazzino, A. Pringle, A. Seminara. In press. Timing of fungal spore release dictates survival during atmospheric transport. Proceedings of the National Academy of Sciences (PNAS). Vargas- Estupiñán N., S. Gonçalves, A.E. Franco-Molano, S. Restrepo, A. Pringle. 2019. In Colombia the Eurasian fungus Amanita muscaria is expanding its range into native, tropical Quercus humboldtii forests. Mycologia 111:758-771. Boynton, P.J., C.N. Peterson, A. Pringle. 2019. Superior dispersal ability can lead to persistent ecological dominance throughout succession. Applied and Environmental Microbiology 85:e02421-18. I also gave several talks to both Academic and Popular Audiences: Academic Audiences: 2018 University of Miami 2018 Max Planck Institute for Evolutionary Biology (Germany) 2018 University of Colorado, Boulder 2018 University of Dar Es Salaam (Tanzania) 2018 Mexican Mycological Congress Keynote (via teleconference) 2018 University of Michigan 2019 Fungal Genetics Conference "The Spore" 2019 International Conference on Mycorrhizas 10 "Emerging Model Systems" (Mexico) 2019 Mycological Society of America "Taylor Festschrift" 2019 Boston University 2020 University of Mississippi 2020 University of British Columbia 2020 American Society of Microbiology/ Microbe 2020 "Microbes in the Anthropocene" 2020 New Phytologist Symposium "Plant/Fungal Invasions" (Brazil) 2020 Presidential Address, Mycological Society of America Popular Audiences: 2018 Inaugural Meeting of the Madison Mycological Society 2018 Wisconsin Mycological Society (twice) 2019 Milwaukee Public Museum 2019 University of Wisconsin-La Crosse Mycology Club 2019 Bogotá Botanical Garden, Colombia Changes/Problems:We did settle on using just the two Alternaria species. I am confident we are providing the world with a unique dataset describing in detail the dynamics of spore longevity and its impacts on the dispersal of two devastating plant pathogens. What opportunities for training and professional development has the project provided?My entire laboratory of six graduate students benefits directly or indirectly from our Hatch support. The two students who've been supported directly will either graduate Spring 2020 (this semester) or in a year's time, because of Hatch support, both have been able to delve into experiments (with spores) or bioinformatics (of genomics data) deeply and thoroughly. Data and analyses are enabling not only an understanding of plant pathogen dispersal dynamics, but also an understanding of the dispersal and genetical system of an invasive ectomycorrhizal fungus. How have the results been disseminated to communities of interest?Results have been disseminated through several publications and talks described in "Target Audience" and last year's report. What do you plan to do during the next reporting period to accomplish the goals?Next year, we will focus on Objective 1 and target publication of the experimental data taken using Alternaria.

Impacts
What was accomplished under these goals? In fiscal year 2019, our proudest accomplishement was a paper now in press at the Proceedings of the National Academy of Sciences; PNAS is a high impact journal likely to facilitate the spread of our discoveries to a very broad audience. The paper describes our simulations of spore dispersal (see last year's report and Objective 2 above). The non-technical abstract of its content reads as follows: Fungi move between habitats by dispersing small spores through the atmosphere. We ask what causes some species to release spores at a specific time every day versus irregularly. We find that timing of spore release dictates how long spores remain in the atmosphere before returning to the ground: spores released at night are likely to travel for hours while spores released during the day may linger for days. Drivers are stronger in lower, warmer latitudes. Because spores in the open atmosphere are likely to die from prolonged exposure to light and air, the timing of spore release will impact survival. We have discovered a constraint likely to shape observed patterns of spore liberation. In essence, we have discovered that fungi can manipulate the timing of spore dispersal to manipulate the amount of time a spore travels in the atmosphere. We are hard at work completing the aims of Objective 1, this far, our findings confirm our hypothesis: there are clear differences in the demographic dynamics of Alternaria alternata and Alternaria solani and these differences reflect the typically short distances traveled by A. solani and the longer distances traveled by A. alternata. A brief summary of each of our other papers (listed in "Target Audience" and under "Products"): Vargas-Estupinan et al. 2019: Establishes the fungus Amanita muscaria as invasive in Colombia. Significance: most work on fungal dispersal ignores humans as vectors, but our work clearly demonstrates humans are moving fungi across the globe. Boynton et al. 2019: The dominant yeast within carniverous pitcher plants is dominant because it is an excellent disperser, dispersing constantly throughout the summer; it is a weak competitor and is not dominant because it can outcompete other fungi (it cannot). Significance: to our knowledge, the first paper ever to record the movements of fungi in real time in a natural habitat. Iapichino et al. in revision: A mathematical model of gill structure in mushrooms. Significance: mushrooms are "optimally packed" with spores, in other words, mushroom structure enables the maximum number of spores to fit within a minimal amount of biomass. Golan et al. draft: The invasive death cap fungus spreads using spores. Significance: we have discovered how an invasive fungus spreads across landscapes.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Oneto, D.L., J. Golan, A. Mazzino, A. Pringle, A. Seminara. In press. Timing of fungal spore release dictates survival during atmospheric transport. Proceedings of the National Academy of Sciences (PNAS).
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Vargas- Estupi��n N., S. Gon�alves, A.E. Franco-Molano, S. Restrepo, A. Pringle. 2019. In Colombia the Eurasian fungus Amanita muscaria is expanding its range into native, tropical Quercus humboldtii forests. Mycologia 111:758-771.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Boynton, P.J., C.N. Peterson, A. Pringle. 2019. Superior dispersal ability can lead to persistent ecological dominance throughout succession. Applied and Environmental Microbiology 85:e02421-18.
• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Iapichino, M., Y.-W. Wang, S. Gentry, A. Pringle, A. Seminara. In revision. A precise relationship among Buller's drop, ballistospore and gill morphologies enables maximal packing of spores within gilled mushrooms. Mycologia.
• Type: Journal Articles Status: Other Year Published: 2020 Citation: Golan J., C.A. Adams, H. Cross, H. Elmore, M. Gardes, S.I. Glassman, S.C. Gon�alves, J. Hess, F. Richard, Y.-W. Wang, B. Wolfe, A. Pringle. Draft. Native and invasive populations of the ectomycorrhizal death cap Amanita phalloides are highly sexual but dispersal limited.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:For the moment my target audience remains my small group of collaborators, encompassing my graduate students and colleagues at the Institut de Physique de Nice in France. We are actively generating theory and testing hypotheses, and until we are sure of our data and message, we are not disseminating results widely. However, in 2017 and 2018 I gave a series of talks and did touch on the themes of our research in these talks. A list of these talks, and talks planned for 2019, includes: Academic Audiences: 2017 Drexel University 2017 Uppsala University (Sweden) 2017 Chulalongkorn University (Thailand) 2017 Michigan State University 2017 Kellogg Biological Station 2018 University of Miami 2018 Max Planck Institute for Evolutionary Biology (Germany) 2018 University of Colorado, Boulder 2018 University of Dar Es Salaam (Tanzania) 2018 Mexican Mycological Congress Keynote (via teleconference) 2018 University of Michigan 2019 Fungal Genetics Conference Popular Audiences: 2017 iBiology (www.ibiology.org) (three lectures) 2018 Inaugural Meeting of the Madison Mycological Society 2018 Wisconsin Mycological Society (twice) 2019 Milwaukee Public Museum 2019 University of Wisconsin-La Crosse Mycology Club Changes/Problems:The only obstacle we are facing is handling more than two species in our demographic experiments. The experiments are trickier than we'd imagined, and if we can do a good job with the two Alternaria species, it may be all we can afford to get done. However, the data will be (to the best of our knowledge) the most detailed demographic data ever taken for a population of spores. What opportunities for training and professional development has the project provided?Three graduate students have benefitted directly from Hatch support. One is nearing graduation; a second recently completed preliminary or qualifying exams and is now a dissertator. He passed his exams with the highest obtainable marks. A third will complete her qualifying exams this semester. How have the results been disseminated to communities of interest?Please see the talks described under "Target Audience". By talking with both academic and popular audiences, I aim to disseminate my research to 1.) scientists who care about dispersal and disease, and to 2.) a broader public curious about fungi. Please note, I also teach an undergraduate/graduate class "Botany/Plant Pathology 332: Fungi". In this class, I routinely discuss spores and dispersal. What do you plan to do during the next reporting period to accomplish the goals?1. In a second experiment planned for the coming month, we are collecting detailed demographic data over a 14-day time course for spores in the optimal environment (see description of our first experiment above). Our time frame translates to a realistic span for continental dispersal. We aim to understand how long the "luckiest" spore can live in an optimal environment, testing whether spores of any species have the potential for long distance dispersal. 2. We aim to publish the manuscripts currently in draft. 3. We also plan to use the results from theory and experiments to plot next steps; the result on turbulence is especially exciting and worth pursuing further.

Impacts
What was accomplished under these goals? To date we have accomplished the following: 1. In collaboration with physicists at INPHYNI in France, we have generated nearly 10,000 simulations of spore dispersal using HYSPLIT models. Theory and simulated data are pointing to a startling result: turbulence, and turbulence alone, dictates the trajectories of fungal spores in the atmosphere. Horizontal wind, about which so much has been written, appears relatively less important. By manipulating the timing of spore release, fungi can manipulate access to turbulence and directly impact the ultimate reach of a spore. 2. In an experiment, we have measured the mortality of spores of Alternaria alternata (hypothesized to be a short distance disperser) and A. solani (hypothesized to be a long distance dispersere) when spores are exposed to a variety of realistic atmospheric environments (e.g. combinations of UV, humidity, temperature, etc.). We are right now evaluating the environment in which the greatest number of spores of each species survives; this will be considered the optimal environment for dispersal and will directly inform models described above, and a second experiment described below. We are currently writing or planning five manuscripts related to our research on dispersal: 1. Lagomarsino Oneto, Pringle, Mazzino, Golan, Seminara. Draft. Turbulence dictates the survival of fungal spores in the atmosphere, shaping the timing of spore liberation. Target: PNAS or APR 2. Iapichino, Wang, Gentry, Pringle, Seminara. Draft. Precise relationships among Buller's drop, ballistospores and gill morphology result in both maximum ejection velocity and optimal packing of spores within gilled mushrooms. Target: Journal of the Royal Society Interface 3. Golan, Seminara, Lagomarsino Oneto, Levitis, Gevens, Ding, Pringle. The luckiest spore: short and long distance dispersal as tested within a biophysical framework using two Alternaria species. Target: TBD 4. Golan, Adams, Cross, Elmore, Gardes, Glassman, Goncalves, Hesse, Richard, Wang, Wolfe, Pringle. Native and invasive populations of the ectomycorrhizal death cap Amanita phalloides are highly sexual but dispersal limited. Target: New Phytologist 5. Golan, Wang, Kriebel, Duncritts, Chapek, Pringle. Draft. Spore shapes among the Amanita are not different between decomposer and ectomycorrhizal species. Target: Fungal Ecology.

Publications