Electrical signaling is a fundamental mechanism for integrating environmental stimuli and coordinating responses in living organisms. While extensively studied in animals and plants, the role of electrical signaling in fungi remains a largely underexplored field. Early studies suggested that filamentous fungi generate action potential-like signals and electrical currents at hyphal tips, yet their function in intracellular communication remained unclear. Renewed interest in fungal electrical activity has fueled developments such as the hypothesis that mycorrhizal networks facilitate electrical communication between plants and the emerging field of fungal-based electronic materials. Given their continuous plasma membrane, specialized septal pores, and insulating cell wall structures, filamentous fungi possess architectural features that could support electrical signaling over long distances. However, studying electrical phenomena in fungal networks presents unique challenges due to the microscopic dimensions of hyphae, the structural complexity of highly modular mycelial networks, and the limitations of traditional electrophysiological methods. This review synthesizes current evidence for electrical signaling in filamentous fungi, evaluates methodological approaches, and highlights experimental challenges. By addressing these challenges and identifying best practices, we aim to advance research in this field and provide a foundation for future studies exploring the role of electrical signaling in fungal biology.

Bacteriophages play a crucial role in shaping bacterial communities, yet the mechanisms by which nonmotile bacteriophages interact with their hosts remain poorly understood. This knowledge gap is especially pronounced in structured environments like soil, where spatial constraints and air-filled zones hinder aqueous diffusion. In soil, hyphae of filamentous microorganisms form a network of 'fungal highways' (FHs) that facilitate the dispersal of other microorganisms. We propose that FHs also promote bacteriophage dissemination. Viral particles can diffuse in liquid films surrounding hyphae or be transported by infectable (host) or uninfectable (nonhost) bacterial carriers coexisting on FH networks. To test this, two bacteriophages that infect Pseudomonas putida DSM291 (host) but not KT2440 (nonhost) were used. In the absence of carriers, bacteriophages showed limited diffusion on 3D-printed abiotic networks, but diffusion was significantly improved in Pythium ultimum-formed FHs when the number of connecting hyphae exceeded 20. Transport by both host and nonhost carriers enhanced bacteriophage dissemination. Host carriers were five times more effective in transporting bacteriophages, particularly in FHs with over 30 connecting hyphae. This study enhances our understanding of bacteriophage dissemination in nonsaturated environments like soils, highlighting the importance of biotic networks and bacterial hosts in facilitating this process.

Filamentous fungi and fungal-like organisms contribute to a wide range of important ecosystem functions. Evidence has shown the movement of liquid across mycelial networks in unsaturated environments, such as soil. However, tools to investigate liquid movement along hyphae at the level of the single cell are still lacking. Microfluidic devices permit the study of fungal and fungal-like organisms with cellular resolution as they can confine hyphae to a single optical plane, which is compatible with microscopy imaging over longer timescales and allows for precise control of the microchannel environment. The aim of this study was to develop a method that enables the visualization and quantification of liquid movement on hyphae of fungal and fungal-like microorganisms. For this, the fungal-fungal interaction microfluidic device was modified to allow for the maintenance of unsaturated microchannel conditions. Fluorescein-containing growth medium solidified with agar was used to track liquid transported by hyphae via fluorescence microscopy. Our key findings highlight the suitability of this novel methodology for the visualization of liquid movement by hyphae over varying time scales and the ability to quantify the movement of liquid along hyphae. Furthermore, we showed that at the cellular level, extracellular movement of liquid along hyphae can be bidirectional and highly dynamic, uncovering a possible link between liquid movement and hyphal growth characteristics. We envisage that this method can be applied to facilitate future research probing the parameters contributing to hyphal liquid movement and is an essential step for studying the phenomenon of fungal highways. (c) 2024 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution-NonCommercial 4.0International (CC BY-NC) license (https://creativecommons.org/licenses/by-nc/4.0/).

Arbuscular mycorrhizal fungi (AMF) form symbiotic associations with the majority of land plants and deliver a wide range of soil-based ecosystem services. Due to their conspicuous belowground lifestyle in a dark environment surrounded by soil particles, much is still to be learned about the influence of environmental (i.e., physical) cues on spore germination, hyphal morphogenesis and anastomosis/hyphal healing mechanisms. To fill existing gaps in AMF knowledge, we developed a new microfluidic platform – the AMF-SporeChip – to visualise the foraging behaviour of germinating Rhizophagus and Gigaspora spores and confront asymbiotic hyphae with physical obstacles. In combination with timelapse microscopy, the fungi could be examined at the cellular level and in real-time. The AMF-SporeChip allowed us to acquire movies with unprecedented visual clarity and therefore identify various exploration strategies of AMF asymbiotic hyphae. We witnessed tip-to-tip and tip-to-side hyphal anastomosis formation. Anastomosis involved directed hyphal growth in a “stop-and-go” manner, yielding visual evidence of pre-anastomosis signalling and decision-making. Remarkably, we also revealed a so-far undescribed reversible cytoplasmic retraction, including the formation of up to 8 septa upon retraction, as part of a highly dynamic space navigation, probably evolved to optimise foraging efficiency. Our findings demonstrated how AMF employ an intricate mechanism of space searching, involving reversible cytoplasmic retraction, branching and directional changes. In turn, the AMF-SporeChip is expected to open many future frontiers for AMF research.

Spores are highly resistant dormant cells, adapted for survival and dispersal, that can withstand unfavourable environmental conditions for extended periods of time and later reactivate. Understanding the germination process of microbial spores is important in numerous areas including agriculture, food safety and health, and other sectors of biotechnology. Microfluidics combined with high-resolution microscopy allows to study spore germination at the single-cell level, revealing behaviours that would be hidden in standard population-level studies. Here, we present a microfluidic platform – the so-called four-conditions microfluidic chemostat (4CMC) – for germination studies where spores are confined to monolayers inside microchambers, allowing the testing of four growth conditions in parallel. This platform can be used with multiple species, including non-model organisms, and is compatible with existing image analysis software. In this study, we focused on three soil dwellers, two bacteria and one fungus, and revealed new insights into their germination. We studied endospores of the model bacterium Bacillus subtilis and demonstrated a correlation between spore density and germination in rich media. We then investigated the germination of the obligate-oxalotrophic environmental bacterium Ammoniphilus oxalaticus in a concentration gradient of potassium oxalate, showing that lower concentrations result in more spores germinating compared to higher concentrations. We also used this microfluidic platform to study the soil beneficial filamentous fungus Trichoderma rossicum, showing for the first time that the size of the spores and hyphae increase in response to increased nutrient availability, while germination times remain the same. Our platform allows to better understand microbial behaviour at the single-cell level, under a variety of controlled conditions. While we used it to decipher the responsiveness of soil dwellers’ spores, it would also be suitable for other spores from bacteria or filamentous fungi, but also vegetative cells and yeast, and even microbial communities.

Abstract This study presents an inexpensive approach for the macro- and microscopic observation of fungal mycelial growth. The ‘fungal drops’ method allows to investigate the development of a mycelial network in filamentous microorganisms at the colony and hyphal scales. A heterogeneous environment is created by depositing 15–20 µl drops on a hydrophobic surface at a fixed distance. This system is akin to a two-dimensional (2D) soil-like structure in which aqueous-pockets are intermixed with air-filled pores. The fungus (spores or mycelia) is inoculated into one of the drops, from which hyphal growth and exploration take place. Hyphal structures are assessed at different scales using stereoscopic and microscopic imaging. The former allows to evaluate the local response of regions within the colony (modular behaviour), while the latter can be used for fractal dimension analyses to describe the hyphal network architecture. The method was tested with several species to underpin the transferability to multiple species. In addition, two sets of experiments were carried out to demonstrate its use in fungal biology. First, mycelial reorganization of Fusarium oxysporum was assessed as a response to patches containing different nutrient concentrations. Second, the effect of interactions with the soil bacterium Pseudomonas putida on habitat colonization by the same fungus was assessed. This method appeared as fast and accessible, allowed for a high level of replication, and complements more complex experimental platforms. Coupled with image analysis, the fungal drops method provides new insights into the study of fungal modularity both macroscopically and at a single-hypha level.

Soil and plant roots are colonized by highly complex and diverse communities of microbes. It has been proposed that bacteria and fungi have synergistic effects on litter decomposition, but experimental evidence supporting this claim is weak. In this study, we manipulated the composition of two microbial kingdoms (Bacteria and Fungi) in experimental microcosms. In microcosms that were inoculated with fungi, litter loss was 47% higher than in microcosms that were not inoculated or only inoculated with bacteria. Combined inoculation with both bacteria and fungi did not significantly enhance decomposition compared with the fungi-only treatments, and, as such, we found no evidence for complementary effects using our experimental setup. Inoculation with fungi also had a positive impact on plant growth after 4 and 8 weeks (480% and 710% growth stimulation, respectively). After 16 weeks, plant biomass was highest in microcosms where both bacteria and fungi were present pointing to fungal-bacterial complementarity in stimulating plant growth. Overall, this study suggests that fungi are the main decomposers of plant litter and that the inoculated fungi contribute to plant growth in our experimental system.

As an endosymbiont of the ecologically and medically relevant fungus Rhizopus microsporus, the toxin-producing bacterium Mycetohabitans rhizoxinica faces myriad challenges, such as evading the host's defense mechanisms. However, the bacterial effector(s) that facilitate the remarkable ability of M. rhizoxinica to freely migrate within fungal hyphae have thus far remained unknown. Here, we show that a transcription activator-like (TAL) effector released by endobacteria is an essential symbiosis factor. By combining microfluidics with fluorescence microscopy, we observed enrichment of TAL-deficient M. rhizoxinica in side hyphae. High-resolution live imaging showed the formation of septa at the base of infected hyphae, leading to the entrapment of endobacteria. Using a LIVE/DEAD stain, we demonstrate that the intracellular survival of trapped TAL-deficient bacteria is significantly reduced compared with wild-type M. rhizoxinica, indicative of a protective host response in the absence of TAL proteins. Subversion of host defense in TAL-competent endobacteria represents an unprecedented function of TAL effectors. Our data illustrate an unusual survival strategy of endosymbionts in the host and provide deeper insights into the dynamic interactions between bacteria and eukaryotes.

This review highlights new advances in the emerging field of ‘Fungi-on-a-Chip’ microfluidics for single-cell studies on fungi and discusses several future frontiers, where we envisage microfluidic technology development to be instrumental in aiding our understanding of fungal biology. Fungi, with their enormous diversity, bear essential roles both in nature and our everyday lives. They inhabit a range of ecosystems, such as soil, where they are involved in organic matter degradation and bioremediation processes. More recently, fungi have been recognized as key components of the microbiome in other eukaryotes, such as humans, where they play a fundamental role not only in human pathogenesis, but also likely as commensals. In the food sector, fungi are used either directly or as fermenting agents and are often key players in the biotechnological industry, where they are responsible for the production of both bulk chemicals and antibiotics. Although the macroscopic fruiting bodies are immediately recognizable by most observers, the structure, function, and interactions of fungi with other microbes at the microscopic scale still remain largely hidden. Herein, we shed light on new advances in the emerging field of Fungi-on-a-Chip microfluidic technologies for single-cell studies on fungi. We discuss the development and application of microfluidic tools in the fields of medicine and biotechnology, as well as in-depth biological studies having significance for ecology and general natural processes. Finally, a future perspective is provided, highlighting new frontiers in which microfluidic technology can benefit this field.

BACKGROUND:To disperse in water-unsaturated environments, such as the soil, bacteria rely on the availability and structure of water films forming on biotic and abiotic surfaces, and, especially, along fungal mycelia. Dispersal along such "fungal highways" may be driven both by mycelial physical properties and by interactions between bacteria and fungi. However, we still do not have a way to disentangle the biotic and abiotic elements.RESULTS:We designed and 3D printed two devices establishing stable liquid films that support bacteria dispersal in the absence of biotic interactions. The thickness of the liquid film determined the presence of hydraulic flow capable of transporting non-motile cells. In the absence of flow, only motile cells can disperse in the presence of an energy source. Non-motile cells could not disperse autonomously without flow but dispersed as "hitchhikers" when co-inoculated with motile cells.CONCLUSIONS:The 3D printed devices can be used as an abiotic control to study bacterial dispersal on hydrated surfaces, such as plant roots and fungal hyphae networks in the soil. By teasing apart the abiotic and biotic dimensions, these 3D printed devices will stimulate further research on microbial dispersal in soil and other water-unsaturated environments.

Creating unique microenvironments, hyphal surfaces and their surroundings allow for spatially distinct microbial interactions and functions at the microscale. Using a microfluidic system and pH-sensitive whole-cell bioreporters (Synechocystis sp. PCC6803) attached to hyphae, we spatially resolved the pH along surfaces of growing hyphae of the basidiomycete Coprinopsis cinerea. Time-lapse microscopy analysis of ratiometric fluorescence signals of >2400 individual bioreporters revealed an overall pH drop from 6.3 ± 0.4 (n = 2441) to 5.0 ± 0.3 (n = 2497) within 7 h after pH bioreporter loading to hyphal surfaces. The pH along hyphal surfaces varied significantly (p < 0.05), with pH at hyphal tips being on average ~0.8 pH units lower than at more mature hyphal parts near the entrance of the microfluidic observation chamber. Our data represent the first dynamic in vitro analysis of surface pH along growing hyphae at the micrometre scale. Such knowledge may improve our understanding of spatial, pH-dependent hyphal processes, such as the degradation of organic matter or mineral weathering.

In recent years, microfluidic technologies have become widespread in biological science. However, the suitability of this technique for understanding different aspects of spore research has hardly been considered. Herein, we review recent developments in 'spores-on-a-chip' technologies, highlighting how they could be exploited to drive new frontiers in spore research.

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic matter, as well as regulation of nutrient levels. There they also find numerous opportunities to interact with a variety of other microbes such as bacteria or other fungi. Studying fungal interactions at the cellular level, however, can be challenging owing to the black box-like nature of soil. New microfluidic tools are being developed for the study of fungal interactions; two platforms designed to study bacterial-fungal and fungal-fungal interactions are highlighted. Within these microchannels, fungal-microbial interactions can be monitored in controlled physico-chemical environments at higher temporal and spatial resolution than previously possible. Application of these tools have yielded numerous novel biological insights, such as the observation of bacterial polar attachment to hyphae or revealing uncharacterised fungal-fungal antagonisms. A key feature of these methodologies regards the ease of use of this tool by non-experts, yielding highly translatable technologies for use in microbiology labs.

Routinely, fungal–fungal interactions (FFI) are studied on agar surfaces. However, this format restricts high-resolution dynamic imaging. To gain experimental access to FFI at the hyphal level in real-time, we developed a microfluidic platform, a FFI device. This device utilises microchannel geometry to enhance the visibility of hyphal growth and provides control channels to allow comparisons between localised and systemic effects. We demonstrate its function by investigating the FFI between the biological control agent (BCA) Clonostachys rosea and the plant pathogen Fusarium graminearum . Microscope image analyses confirm the inhibitory effect of the necrotrophic BCA and we show that a loss of fluorescence in parasitised hyphae of GFP-tagged F. graminearum coincides with the detection of GFP in mycelium of C. rosea . The versatility of our device to operate under both water-saturated and nutrient-rich as well as dry and nutrient-deficient conditions, coupled with its spatio-temporal output, opens new opportunities to study relationships between fungi.

Control over cell growth by mobile regulators underlies much of eukaryotic morphogenesis. In plant roots, cell division and elongation are separated into distinct longitudinal zones and both division and elongation are influenced by the growth regulatory hormone gibberellin (GA). Previously, a multicellular mathematical model predicted a GA maximum at the border of the meristematic and elongation zones. However, GA in roots was recently measured using a genetically encoded fluorescent biosensor, nlsGPS1, and found to be low in the meristematic zone grading to a maximum at the end of the elongation zone. Furthermore, the accumulation rate of exogenous GA was also found to be higher in the elongation zone. It was still unknown which biochemical activities were responsible for these mobile small molecule gradients and whether the spatiotemporal correlation between GA levels and cell length is important for root cell division and elongation patterns. Using a mathematical modeling approach in combination with high-resolution GA measurements in vivo, we now show how differentials in several biosynthetic enzyme steps contribute to the endogenous GA gradient and how differential cellular permeability contributes to an accumulation gradient of exogenous GA. We also analyzed the effects of altered GA distribution in roots and did not find significant phenotypes resulting from increased GA levels or signaling. We did find a substantial temporal delay between complementation of GA distribution and cell division and elongation phenotypes in a GA deficient mutant. Together, our results provide models of how GA gradients are directed and in turn direct root growth.

Phytopathogenic Verticillia cause Verticillium wilt on numerous economically important crops. Plant infection begins at the roots, where the fungus is confronted with rhizosphere inhabiting bacteria. The effects of different fluorescent pseudomonads, including some known biocontrol agents of other plant pathogens, on fungal growth of the haploid Verticillium dahliae and/or the amphidiploid Verticillium longisporum were compared on pectin-rich medium, in microfluidic interaction channels, allowing visualization of single hyphae, or on Arabidopsis thaliana roots. We found that the potential for formation of bacterial lipopeptide syringomycin resulted in stronger growth reduction effects on saprophytic Aspergillus nidulans compared to Verticillium spp. A more detailed analyses on bacterial-fungal co-cultivation in narrow interaction channels of microfluidic devices revealed that the strongest inhibitory potential was found for Pseudomonas protegens CHA0, with its inhibitory potential depending on the presence of the GacS/GacA system controlling several bacterial metabolites. Hyphal tip polarity was altered when V. longisporum was confronted with pseudomonads in narrow interaction channels, resulting in a curly morphology instead of straight hyphal tip growth. These results support the hypothesis that the fungus attempts to evade the bacterial confrontation. Alterations due to co-cultivation with bacteria could not only be observed in fungal morphology but also in fungal transcriptome. P. protegens CHA0 alters transcriptional profiles of V. longisporum during 2 h liquid media co-cultivation in pectin-rich medium. Genes required for degradation of and growth on the carbon source pectin were down-regulated, whereas transcripts involved in redox processes were up-regulated. Thus, the secondary metabolite mediated effect of Pseudomonas isolates on Verticillium species results in a complex transcriptional response, leading to decreased growth with precautions for self-protection combined with the initiation of a change in fungal growth direction. This interplay of bacterial effects on the pathogen can be beneficial to protect plants from infection, as shown with A. thaliana root experiments. Treatment of the roots with bacteria prior to infection with V. dahliae resulted in a significant reduction of fungal root colonization. Taken together we demonstrate how pseudomonads interfere with the growth of Verticillium spp. and show that these bacteria could serve in plant protection.

Plant roots adapt their development and metabolism to changing environmental conditions. In order to understand the response mechanisms of roots to the dynamic availability of water or nutrients, to biotic and abiotic stress conditions or to mechanical stimuli, microfluidic platforms have been developed that offer microscopic access and novel experimental means. Here, we describe the design, fabrication and use of microfluidic devices suitable for imaging growing Arabidopsis roots over several days under controlled perfusion. We present a detailed protocol for the use of our exemplar platform-the RootChip-8S-and offer a guide for troubleshooting, which is also largely applicable to related device designs. We further discuss considerations regarding the design of custom-made plant microdevices, the choice of suitable materials and technologies as well as the handling of the specimen.

The association of the agriculturally significant phytopathogenic fungus Rhizopus microsporus with the bacterial endosymbiont Burkholderia rhizoxinica is a remarkable example of bacteria controlling host physiology and reproduction. Here, we show that a group of transcription activator-like effectors (TALEs) called Burkholderia TALE-like proteins (BATs) from B. rhizoxinica are essential for the establishment of the symbiosis. Mutants lacking BAT proteins are unable to induce host sporulation. Utilising novel microfluidic devices in combination with fluorescence microscopy we observed the accumulation of BAT-deficient mutants in specific fungal side-hyphae with accompanying increased fungal re-infection. High-resolution live imaging revealed septa biogenesis at the base of infected hyphae leading to compartmental trapping of BATdeficient endobacteria. Trapped endosymbionts showed reduced intracellular survival, suggesting a protective response from the fungal host against bacteria lacking specific effectors. These findings underscore the involvement of BAT proteins in maintaining a balance between mutualism and antagonism in bacterial-fungal interactions and provide deeper insights into the dynamic interactions between bacteria and eukaryotes.