With a diverse “Fungi” kingdom having more than a million species, fungus is among the most abundant microorganisms we can find on earth. These are complex microbes that grow and travel fast to nearby habitats, where they create new colonies. Fungi hold a great significance in our ecosystems, as they play a pivotal role in nutrient cycling and are crucial for plant and animal health. Some fungus types also function as pathogens and cause serious diseases such as respiratory illness (e.g., coccidioidomycosis). Considering such an impact of fungi, it gets critical to study fungus and understand the way it grows and disperses in the environment. Most fungi release spores (single cell units) as a part of their reproduction process, and these spores get dispersed in the environment via airflow or water (e.g., raindrops) in search for a new habitat [1]. Spore dispersal lies at the heart of the whole fungal colonization process – their role is similar to that of the seeds in the plant world.
Therefore, in understanding the fungal spread, one key element is to unveil the dynamics of spore dispersal. Studying and predicting spore progression solves many important issues e.g., ensuring timely interception of fungal spread in food and crops [2]. However, spore dispersal process is highly dynamic and is also sensitive to various environmental changes – e.g., airborne fungal spores travel more if released in the daytime compared to the night [3]. Such unique, fairly random properties of fungal spore dispersal make its study and prediction extremely challenging. These challenges get further pronounced by the fact that existing fungi culturing techniques rely entirely on conventional agar-based or well-plate-based protocols, which due to their static nature fail to replicate the naturally dynamic habitat of fungi [4, 5]. In addition, real-time monitoring is also limited with traditional ways, which is crucial to closely observe the fungal spread.
As an alternative, the study of spore disposal dynamics can benefit greatly from the existing microfluidics technologies, as they can better mimic the natural habitats by providing dynamic microenvironments that can adapt to different biological conditions in real time. For instance, we can accurately control flow rates on chip or selectively add new chemicals into the channels etc., essentially making it easier to simulate microbiological processes such as fungal spread. These benefits, along with its unprecedented precision in handling small sample volumes down to single spore, makes the microfluidics technology an ideal candidate to study fungi [6, 7].
Staying at the intersection of microfluidics and spore research, in this article, we will first deconstruct the fungal spore dispersal process and then provide insights on how microfluidics technology can play its part in improving our understanding of the spore dynamics.
Spores are single-cell units that fungus ejects during reproduction (either sexually or asexually). Starting from their initial release from the fungus, spores go through a series of steps before they can find a suitable habitat, where they settle down and start the germination process to produce more fungi. This cycle continues over time, allowing fungi to colonize more and more areas [1]. Typically, the dispersal process of fungal spores has the following steps involved.
Once the spores are mature enough in the fruiting body (an organ of fungus that produces spores), they are liberated or released into the environment – referring to the spore “Liberation” process. Liberation can be of two types i.e., either active or passive. Among these, active liberation indicates a forceful discharge of spores by the fungi and is influenced by various factors such as spore maturity level and environmental conditions. Passive release, on the other hand, takes place under the action of external forces such as wind or rain flow, and can liberate spores even before they are fully matured.
Once the spores are liberated from the fungus, they start traveling in the environment they are confined in, which can be open atmosphere, surface of a leaf or soil etc. As fungi can grow in a diverse variety of environments, their spores are also capable of dispersing through many different mediums such as air or water. However, airborne dispersal is more common where wind acts as the main driver responsible for transporting spores over a certain distance – e.g., airborne spore dispersal on a leaf surface. In contrast, dispersal in water medium mainly moves the spores between different surfaces or locations within a macro or micro-site – for example, waterborne movement of fungal spores within the pores located in soil.
Spore transport is a dynamic process, where the spore movement efficiency as well as the amount of distance a spore will travel depend significantly on the environmental conditions e.g., air flow, temperature, and humidity level etc. In addition, spores themselves have numerous types and come in various different sizes, which eventually influence the way they disperse. This interplay of multiple factors makes the spore dispersal process highly random and difficult to simulate in a lab setting.
After traveling a certain distance in the environment, the spores settle down on nearby surfaces known as the “Spore Deposition” process. However, it is worthwhile to note that not all spores that deposit on substrates go on to carry out a successful fungi germination. Chemical and physical properties of substrate play a significant role in defining the success of spore germination. In addition, appropriate environmental conditions such as temperature, humidity levels, availability of nutrients etc., are also necessary to facilitate spore germination and colonization.
The critical importance of studying fungal dispersal is undeniable, as it allows us to understand the dynamics of fungal spread. Comprehending these dynamics essentially helps us foresee fungal activities and make proactive decisions e.g., when intercepting fungal infestation in crops. However, the complexity and heterogeneity of environments containing fungi (e.g., soil) are very challenging to replicate using typical laboratory conditions that are simplified and mostly homogeneous in nature.
In light of these issues, there is an ongoing trend towards using microfluidics technology for cultivating fungi and studying their spore dispersal process. Over recent years, microfluidics has emerged as a mainstream technology capable of providing miniaturized platforms for various biological experiments. Microfluidics not only provides a precise and dynamic control over the environment thus mimicking the natural fungi habitat, but also enables high-resolution imaging that is critical for closely observing fungal growth [6, 7]. Co-cultivation of fungi with other microorganisms is yet another added benefit of microfluidics technology, allowing us to reveal fungal interactions that are important for understanding overall fungal ecology.
Despite these benefits, the efforts on developing microfluidic ways of studying fungal spore dispersal process on chip have begun only recently, with microchips dedicated for this purpose yet to be matured [6]. In the following subsection, we provide a perspective on how microfluidics technology can be leveraged for on-chip simulation of fungal spread, particularly the spore dispersal process.
Before delving deeper into how microfluidics can help replicate various fungal spread mechanisms on a chip, let’s recall the fundamental spore dispersal process. Spores are indeed micro-sized particles that get ejected from the fungi and then travel the surrounding environment. Here, the fluid dynamics of spore dispersal mainly relies on the “Laminar” flow principles, since Reynold’s number is usually low at microscale where viscous forces tend to overcome inertial forces. Coupling this with the fundamentals of microfluidics technology that is known for intelligently exploiting laminar flows to achieve precise control over fluids, studying the fluid dynamics of spore dispersal via microfluidics seems a perfect match.
Nevertheless, beyond precise flow control, there are also other requirements that need to be met for us to effectively simulate spore dispersal on chip. Highly controlled microenvironments, real-time spore tracking, and co-cultivation of microorganisms are among the factors to consider, all of which can be addressed using existing microfluidic technologies. To elaborate further, below we highlight some key features of the microfluidic devices, which make them a viable platform for on-chip spore studies.
Relying on these concepts, many on-chip fungi culturing studies have been reported. For instance, a fully integrated microfluidics device was recently reported “AMF-SporeChip” [10], which aims to uncover the growth dynamics of a certain fungi type i.e., arbuscular mycorrhizal (Figure 2). The proposed microchip was coupled with a high-resolution timelapse microscope to obtain clear visuals, thus allowing for real-time monitoring of fungal growth.
Considering that microfluidics technology provides an unmatched platform for spore studies, many research efforts have recently surfaced that attempt to simulate the whole lifecycle of fungal spores. These go well beyond just the dispersal mechanism, indicating a whole new research direction i.e., “Spore-On-Chip”. The realization of spore-on-chip concepts is fueled by the fact that spores, like many other microfluidics-compatible microorganisms, can be easily handled on chip using existing microfluidic techniques [6]. Studying key spore mechanisms, such as dispersal and germination, unveils valuable fungal insights, e.g., about microbe pathogenesis, that are crucial in many applications such as crop health management, air quality monitoring, disease spread prediction etc.
Another notable advancement spore-on-chip research has seen recently is the possibility to achieve single-spore level resolution, thanks to the invention of droplet microfluidics (DMF) technology. Droplet microfluidics, with its abilities to trap single cells inside microdroplets and then process them individually, enables high-throughput spore analyses [6]. Individual spore handling also reveals intricate spore-to-spore variations, which are impossible to extract when processing cells in bulk. One example of single-cell spore study is the microfluidic chip shown in Figure 3, which performs statistically rich spore germination analyses of pathogenic fungi [9]. Such studies are essential in enhancing our understanding of fungal diseases and developing antifungal strategies.
Fungal spores are key elements in our ecosystems, as they drive fungal spread by germinating fungi over new places. Hence, gaining an in-depth understanding of fungal spread mechanisms essentially boils down to revealing the spatial and temporal dynamics of the spore dispersal process. However, conventional methods to cultivate fungi in lab fall short due to their inherent inability to mimic natural conditions. In contrast, microfluidics technology offers unique benefits by providing dynamic microenvironments that can match natural fungi habitats. Nevertheless, the integration of microfluidics with spore research is still in its infancy, with many new studies emerging each day. Concepts such as “Fungi-On-Chip” and “Spore-On-Chip” are gaining plenty of attention, as they promise fungal insights that are otherwise impossible to capture with traditional ways. To sum up, microfluidics-based spore studies indicate an active area of research that has potential to grow substantially in near-future, as it bridges many important knowledge gaps in fungal ecology.
[1] M. Roper and A. Seminara, “Mycofluidics: The Fluid Mechanics of Fungal Adaptation,” Annual Review of Fluid Mechanics, vol. 51, no. Volume 51, 2019, pp. 511-538, 2019, doi: https://doi.org/10.1146/annurev-fluid-122316-045308.
[2] C. R. Davies et al., “Evolving challenges and strategies for fungal control in the food supply chain,” Fungal Biology Reviews, vol. 36, pp. 15-26, 2021/06/01/ 2021, doi: https://doi.org/10.1016/j.fbr.2021.01.003.
[3] D. Lagomarsino Oneto, J. Golan, A. Mazzino, A. Pringle, and A. Seminara, “Timing of fungal spore release dictates survival during atmospheric transport,” Proceedings of the National Academy of Sciences, vol. 117, no. 10, pp. 5134-5143, 2020/03/10 2020, doi: 10.1073/pnas.1913752117.
[4] A. Podwin, T. Janisz, K. Patejuk, P. Szyszka, R. Walczak, and J. Dziuban, “Towards microfluidics for mycology – material and technological studies on LOCs as new tools ensuring investigation of microscopic fungi and soil organisms,” Bulletin of the Polish Academy of Sciences Technical Sciences, vol. 69, no. No. 1, pp. e136212-e136212, 9.02.2021 2021, doi: 10.24425/bpasts.2020.136212.
[5] C. E. Stanley, G. Grossmann, X. Casadevall i Solvas, and A. J. deMello, “Soil-on-a-Chip: microfluidic platforms for environmental organismal studies,” Lab on a Chip, 10.1039/C5LC01285F vol. 16, no. 2, pp. 228-241, 2016, doi: 10.1039/C5LC01285F.
[6] L. S. Bernier, P. Junier, G.-B. Stan, and C. E. Stanley, “Spores-on-a-chip: new frontiers for spore research,” Trends in Microbiology, vol. 30, no. 6, pp. 515-518, 2022/06/01/ 2022, doi: https://doi.org/10.1016/j.tim.2022.03.003.
[7] F. Richter, S. Bindschedler, M. Calonne-Salmon, S. Declerck, P. Junier, and C. E. Stanley, “Fungi-on-a-Chip: microfluidic platforms for single-cell studies on fungi,” FEMS Microbiology Reviews, vol. 46, no. 6, p. fuac039, 2022, doi: 10.1093/femsre/fuac039.
[8] N.-T. Nguyen, X. Huang, and T. K. Chuan, “MEMS-Micropumps: A Review,” Journal of Fluids Engineering, vol. 124, no. 2, pp. 384-392, 2002, doi: 10.1115/1.1459075.
[9] L. J. Barkal, N. M. Walsh, M. R. Botts, D. J. Beebe, and C. M. Hull, “Leveraging a high resolution microfluidic assay reveals insights into pathogenic fungal spore germination,” Integrative Biology, vol. 8, no. 5, pp. 603-615, 2016, doi: 10.1039/c6ib00012f.
[10] F. Richter, M. Calonne-Salmon, M. G. A. van der Heijden, S. Declerck, and C. E. Stanley, “AMF-SporeChip provides new insights into arbuscular mycorrhizal fungal asymbiotic hyphal growth dynamics at the cellular level,” Lab on a Chip, 10.1039/D3LC00859B vol. 24, no. 7, pp. 1930-1946, 2024, doi: 10.1039/D3LC00859B.
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