With a diverse “Fungi” kingdom having more than a million species, fungus is among the most abundant microorganisms we can find on earth. They are complex microbes that grow and move rapidly to populate neighbouring habitats where they create new colonies. Fungi are of great importance in our ecosystems. They are playing a central role in the nutrient cycle and are essential to the health of plants and animals. Some types of fungi are also pathogenic, causing serious illnesses such as respiratory diseases (e.g. coccidioidomycosis). Given the impact of fungi, it is essential to study them and understand how they grow and disperse in the environment. Most fungi release spores (single-celled units) as part of their reproductive process, and these spores are dispersed into the environment via air or water (e.g. raindrops) in search of a new habitat [1]. The dispersal of spores is at the heart of the entire fungal colonization process – their role is similar to that of seeds in the plant world.
Therefore, to understand the spread of fungi, a key element is to unveil the dynamics of spore dispersal. Studying and predicting the progression of spores helps to solve many important problems, such as ensuring the timely interception of fungal spread in food and crops [2]. However, the spore dispersal process is highly dynamic and sensitive to various environmental changes – for example, airborne fungal spores travel further if released during the day compared to the night [3]. These unique and relatively random properties of fungal spore dispersal make it extremely difficult to study and predict. These difficulties are further compounded by the fact that existing fungal culture techniques rely entirely on conventional agar- or plate-based protocols, which, due to their static nature, fail to reproduce the naturally dynamic habitat of fungi [4, 5]. Moreover, real-time monitoring is also limited by traditional methods, which is crucial to closely observe fungal spreading.
The study of spore elimination dynamics can benefit from existing microfluidics technologies. In fact, they can better mimic natural habitats by providing dynamic microenvironments that can adapt to different biological conditions in real time. For instance, we can precisely control flow rates on microfluidic chips or selectively add new chemicals into channels, making it easier to simulate microbiological processes such as fungal propagation. These advantages, along with its unparalleled precision in handling small sample volumes down to single spore, make microfluidic technology an ideal candidate to study fungi [6, 7].
In this article, we will stay at the intersection of microfluidics and spore research. We will first deconstruct the process of fungal spore dispersal and then provide an overview of how microfluidic technology can play a role in improving our understanding of spore dynamics.
Spores are single cells that fungi release during reproduction (sexually or asexually). When fungi release spores, they go through a series of steps before finding the right habitat. Once the habitat is found, they settle down and start the germination process to produce more fungi. This cycle repeats over time, enabling fungi to colonize more and more areas [1]. As a general rule, the fungal spore dispersal process involves the following steps.
Once the spores are mature in the fruiting body (an organ of fungus that produces spores), they are released into the environment. This process is called spore “release”. There are two types of release: active or passive. Active release means that the spores are forced out by the fungi and this release is influenced by various factors such as spore maturity and environmental conditions. Passive release is due to external forces such as wind or rain flow, and can release spores even before they are fully mature.
Once the spores are released from the fungus, they start to move around their environment, which can be an open atmosphere, the surface of a leaf or soil, etc. As fungi can grow in a wide variety of environments, their spores are also capable of dispersing in many different media. Dispersal of fungal spores is typically done by air or water. Airborne dispersal is more common, with wind being the main driving force responsible for transporting spores over a certain distance – for example airborne spore dispersal on a leaf surface. In contrast, water dispersal mainly moves spores between different surfaces or locations on a macro or micro-site – for example, the motion of fungal spores through soil by water.
Spore transport is a dynamic process. The efficiency of the process, as well as the distance traveled by a spore, depend significantly on environmental conditions such as air flow, temperature, humidity level and so on. In addition, spores come in many different types and sizes, which ultimately influence the way they disperse. This interaction of multiple factors makes the spore dispersal process highly random and difficult to simulate in a laboratory environment.
After traveling a certain distance in the environment, the spores are deposited on nearby surfaces. This process is called “Spore Deposition”. However, it is important to note that not all spores deposited on substrates succeed in germinating fungi. The chemical and physical properties of the substrate play a significant role in the success of spore germination. In addition, appropriate environmental conditions such as temperature, humidity levels, nutrient availability, etc. are also necessary to facilitate spore germination and colonization.
The importance of studying fungal spore dispersal is undeniable, as it enables us to understand the dynamics of fungal spread. Understanding these dynamics helps us to predict fungal activity and make proactive decisions for example when intercepting a fungal disease in crops. However, the complexity and heterogeneity of environments containing fungi (e.g., soil) are very difficult to replicate under typical laboratory conditions, which are by nature simplified and mostly homogeneous conditions.
In light of these challenges, the current trend is to use microfluidic technology to culture fungi and study their spore dispersal process. Microfluidics has emerged as a mainstream technology capable of providing miniaturized platforms for various biological experiments. It enables precise and dynamic control of the environment, mimicking the natural fungi habitat, but also high-resolution imaging, essential for close observation of fungal growth [6, 7]. The co-culture of fungi with other microorganisms is another advantage of microfluidic technology, allowing the study of the interactions between fungi and microorganisms.
Despite these advantages, the efforts on developing microfluidic methods to study the fungal spore dispersal process on a chip have only recently begun, as dedicated microchips for this purpose are not yet mature [6]. In the following subsection, we present how microfluidic technology can be used for on-chip simulation of fungal spread, in particular the spore dispersal process.
Before looking into more detail at how microfluidics can help reproduce various fungal spread mechanisms on a chip, let’s recall the fundamental process of fungal spore dispersal. Spores are micro-sized particles ejected by fungi. They move around in the surrounding environment. Here, the fluid dynamics of spore dispersal mainly relies on the principles of “Laminar” flow, as Reynold’s number is generally low at microscale where viscous forces tend to overcome inertial forces. Add to this the fundamental principles of microfluidic technology, known for exploiting laminar flows to achieve precise fluid control, and the study of spore dispersal fluid dynamics via microfluidics seems perfectly suited.
Nevertheless, beyond precise flow control, other requirements need to be met to effectively simulate spore dispersal on a chip. Highly controlled microenvironments, real-time spore tracking, and co-culture of microorganisms are among the factors that need to be considered and all of them can be addressed using existing microfluidic technologies. To elaborate further, we highlight below some of the key features of microfluidic devices, which make them a viable platform for on-chip spore studies.
Relying on these concepts, numerous studies of on-chip culture of fungi have been reported. For example, a fully integrated microfluidic device, called “AMF-SporeChip” was recently presented [10]. It aims to discover the growth dynamics of a certain fungi type, the arbuscular mycorrhizal fungus (Figure 2). The proposed microchip was coupled with a high-resolution timelapse microscope to obtain clear images, thus enabling real-time monitoring of fungal growth.
Considering the advantages offered by microfluidics for spore studies, a lot of recent research efforts have tried to simulate the whole lifecycle of fungal spores. These efforts go far beyond the simple dispersal mechanism, and point to an entire new research area, the“Spore-On-Chip”. The realization of spore-on-chip studies is fueled by the fact that spores, like many other microfluidics-compatible microorganisms, can be easily manipulated on chip using existing microfluidic techniques [6]. Studying key spore mechanisms, such as dispersal and germination, reveals valuable fungal information, for example on microbe pathogenesis, crucial in many applications such as crop health management, air quality monitoring, disease spread prediction etc.
Another important advancement in spore-on-chip research is the possibility to achieve single-spore level resolution, thanks to the invention of droplet microfluidics (DMF) technology. Droplet microfluidics, which allows single cells to be trapped in microdroplets and then be processed individually, enables high-throughput spore analysis [6]. Individual spore handling also reveals complex 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 improving our understanding of fungal diseases and developing antifungal strategies.
Fungal spores are key elements in our ecosystems, as they are responsible for fungal spread by germinating fungi over new locations. Understanding the mechanisms of fungal spread consists of studying the spatial and temporal dynamics of the spore dispersal process. The conventional methods used to cultivate fungi in laboratories are not adapted to these studies as they are not able to mimic natural conditions. For its part, microfluidic technology offers unique advantages by providing dynamic microenvironments that can match natural fungi habitats. Nevertheless, the integration of microfluidics with spore research is still in its early stages and many new studies emerge every day. Concepts such as “Fungi-On-Chip” and “Spore-On-Chip” are getting more attention, as they provide information on fungi that is impossible to obtain using traditional methods. In summary, microfluidics-based spore studies are an active area of research that has potential to grow in near-future, as it bridges many important knowledge gaps in fungal ecology.
Written and reviewed by Louise Fournier, PhD in Chemistry and Biology Interface. For more content about microfluidics, you can have a look here.
[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://www.annualreviews.org/content/journals/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://www.sciencedirect.com/science/article/pii/S1749461321000038?via%3Dihub.
[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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