AMF-SporeChip provides new insights into arbuscular mycorrhizal fungal asymbiotic hyphal growth dynamics at the cellular level.

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.

Anastomosis within and between networks of Rhizophagus irregularis is differentially influenced by fungicides.

Arbuscular mycorrhizal (AM) fungi play key roles in soil fertility of agroecosystems. They develop dense extraradical mycelial (ERM) networks via mechanisms such as hyphal anastomosis. These connections between hyphae can be affected by agricultural practices such as the use of fungicides, but how these compounds affect anastomosis formation within and more importantly between networks of the same AM fungal strain remains poorly unexplored. Here, the impact of azoxystrobin, pencycuron, flutolanil, and fenpropimorph at 0.02 and 2 mg L-1 were tested in vitro on the anastomosis formation within and between networks of Rhizophagus irregularis MUCL 41833. Azoxystrobin and fenpropimorph had a particularly detrimental impact, at the highest concentration (2 mg L-1), on the number of anastomoses within and between networks, and for fenpropimorph in particular at both concentrations (0.02 and 2 mg L-1) on the number of anastomoses per length of hyphae. Curiously fenpropimorph at 0.02 mg L-1 significantly stimulated spore production, while with azoxystrobin, the reverse was observed at 2 mg L-1. The two other fungicides, pencycuron and flutolanil, had no detrimental effects on spore production or anastomosis formation within and between networks. These results suggest that fungicides with different modes of action and concentrations differentially affect anastomosis possibly by altering the hyphal tips of AM fungi and may thus affect the capacity of AM fungi to develop large hyphal networks exploring and exploiting the soil at the service of plants.

Fungi-on-a-Chip: microfluidic platforms for single-cell studies on fungi.

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.

Direct transfer of zinc between plants is channelled by common mycorrhizal network of arbuscular mycorrhizal fungi and evidenced by changes in expression of zinc transporter genes in fungus and plant.

The role that common mycorrhizal networks (CMNs) play in plant-to-plant transfer of zinc (Zn) has not yet been investigated, despite the proved functions of arbuscular mycorrhizal fungi (AMF) in crop Zn acquisition. Here, two autotrophic Medicago truncatula plants were linked by a CMN formed by Rhizophagus irregularis. Plants were grown in vitro in physically separated compartments (Donor-C and Receiver-C) and their connection ensured only by CMN. A symbiosis-defective mutant of M. truncatula was used as control in Receiver-C. Plants in both compartments were grown on Zn-free medium, and only the leaves of the donor plants were Zn fertilized. A direct transfer of Zn was demonstrated from donor leaves to receiver shoots mediated by CMN. Direct transfer of Zn was supported by changes in the expression of fungal genes, RiZRT1 and RiZnT1, and plant gene MtZIP2 in roots and MtNAS1 in roots and shoots of the receiver plants. Moreover, Zn transfer was supported by the change in expression of MtZIP14 gene in AM fungal colonized roots. This work is the first evidence of a direct Zn transfer from a donor to a receiver plant via CMN, and of a triggering of transcriptional regulation of fungal-plant genes involved in Zn transport-related processes.

Fungicides With Contrasting Mode of Action Differentially Affect Hyphal Healing Mechanism in Gigaspora sp. and Rhizophagus irregularis.

Fungicides are widely used in conventional agriculture to control fungal diseases, but may also affect non-target microorganisms such as arbuscular mycorrhizal (AM) fungi. These root symbionts develop extended mycelial networks within the soil via mechanisms such as anastomosis that indistinctly concerns intact and damaged hyphae, the latter being named hyphal healing mechanism (HHM). The HHM differs between Glomeraceae and Gigasporaceae. However, the effects of fungicides on this mechanism in unknown. Here, the impact of azoxystrobin, pencycuron, flutolanil, and fenpropimorph at 0.02 and 2 mg L-1 were tested in vitro on the HHM of Gigaspora sp. MUCL 52331 and Rhizophagus irregularis MUCL 41833, and repair events visualized carefully under a dissecting bright-field light microscope. Azoxystrobin was the more detrimental for both AM fungi at 2 mg L-1, while fenpropimorph impacted only R. irregularis (stimulating at low and inhibiting at high concentration). Conversely, flutolanil and pencycuron did not impact any of the two AM fungi. The mechanisms involved remains to be elucidated, but perturbation in the still-to-be firmly demonstrated spitzenkörper or in sterols content as well as a process of hormesis are possible avenues that deserve to be explored in view of a rationale management of chemicals to control fungal pathogens without harming the beneficial AM fungi.

Diesel fuel differentially affects hyphal healing in Gigaspora sp. and Rhizophagus irregularis.

Hydrocarbon pollution is an increasing problem affecting soil ecosystems. However, some microorganisms can cope with these pollutants and even facilitate plant establishment and thus phytoremediation. Within soil, arbuscular mycorrhizal fungi (AMF) have developed several strategies to survive and flourish under adverse conditions. Among these is the hyphal healing mechanism (HHM), a process allowing hyphae to re-establish integrity after physical injury. This mechanism differs among species and genera of AMF. However, whether and to what extent hydrocarbon pollution impacts the HHM is unknown. Here, the HHM was monitored in vitro on two AMF strains, Rhizophagus irregularis MUCL 41833 and Gigaspora sp. MUCL 52331, under increasing concentrations of diesel (1, 2, and 5% v:v). The addition of diesel slowed-down the HHM in both fungi. On Gigaspora sp., this effect was limited and most hyphae were able to heal after injury. Conversely, all steps of healing were severely impaired in R. irregularis. That fungus reconnected the injured hyphae at a much lower frequency than the Gigaspora sp., instead investing its energy to link neighboring hyphae or roots, or developing new branches from uninjured hyphae.

Rhizophagus irregularis MUCL 41833 Improves Phosphorus Uptake and Water Use Efficiency in Maize Plants During Recovery From Drought Stress.

Irregular precipitations are likely to affect maize production in the future. Arbuscular mycorrhizal fungi (AMF) have been reported to increase maize resistance to drought, but their role on the short-term inorganic phosphorus (Pi) uptake, leaf gas exchange parameters and water content during recovery after drought remains poorly understood. Here, we investigated these parameters in maize plants colonized or not by Rhizophagus irregularis MUCL 41833. The mycorrhizal (M) and non-mycorrhizal (NM) plants were grown for a 3-week period in a circulatory semi-hydroponic cultivation system and were submitted to well-, moderately-, or poorly-watered conditions (WW, MW, and PW, respectively), the two latter conditions corresponding to moderate and severe droughts. The plants were then watered at field capacity for 42 h with a Pi impoverished Hoagland nutrient solution and the dynamic of Pi depletion in the nutrient solution, corresponding to Pi uptake/immobilization by the maize-AMF associates, was evaluated at 0, 9, 21, and 42 h. The CO2 assimilation rate (A), stomatal conductance (gs), transpiration (E), and instantaneous water use efficiency (WUEi) were also assessed at 0 and 42 h of circulation. Plant biomass, plant water content, phosphorus concentration and content, and leaf relative water content were evaluated at harvest. During recovery, Pi uptake was significantly higher in M versus NM plants whatever the water regime (WR) applied before recovery. AMF did not affect leaf gas exchange parameters before recovery but modulated gs and E, and improved WUEi after 42 h of recovery. At harvest, no significant difference in dry biomass was found between M and NM plants but shoot fresh weight was significantly higher in M plants. This resulted in an increased shoot water content in M plants grown in the MW and PW treatments. Surprisingly, leaf relative water content was significantly lower in M plants when compared with NM plants. Finally, P content and concentration were significantly higher in roots but not in shoots of M plants. Our results suggested that AMF can play a role in drought resistance of maize plants by increasing the Pi uptake and WUEi during recovery after drought stress.

The arbuscular mycorrhizal fungus Rhizophagus irregularis MUCL 41833 increases the phosphorus uptake and biomass of Medicago truncatula, a benzo[a]pyrene-tolerant plant species.

The accumulation of phosphorus (P) in plants increases their biomass and resistance/tolerance to organic pollutants. Both characteristics are mandatory for the utilization of plants in phytoremediation. Arbuscular mycorrhizal (AM) fungi improve plant P nutrition, and thus growth. However, only a few studies have focused on the dynamics of inorganic P (Pi) uptake in AM fungal-colonized plants in the presence of organic pollutants. Indeed, most of the results so far were obtained after harvesting the plants, thus by evaluating P concentration and content at a single time point. Here, we investigated the effects of the AM fungus Rhizophagus irregularis MUCL 41833 on the short-term Pi uptake dynamics of Medicago truncatula plants grown in the presence of benzo[a]pyrene (B[a]P), a polyaromatic hydrocarbon (PAH) frequently found in polluted soils. The study was conducted using a non-destructive circulatory semi-hydroponic cultivation system to investigate the short-term Pi depletion from a nutrient solution and as a corollary, the Pi uptake by the AM fungal-colonized and non-colonized plants. The growth, P concentration, and content of plants were also evaluated at harvest. The presence of B[a]P neither impacted the development of the AM fungus in the roots nor the plant growth and Pi uptake, suggesting a marked tolerance of both organisms to B[a]P pollution. A generally higher Pi uptake coupled with a higher accumulation of P in shoots and roots was noticed in AM fungal-colonized plants as compared to the non-colonized controls, irrespective of the presence or absence of B[a]P. Therefore, fungal-colonized plants showed the best growth. Furthermore, the beneficial effect provided by the presence of the AM fungus in roots was similar in presence or absence of B[a]P, thus opening the door for potential utilization in phytomanagement of PAH-polluted soils.

Dynamics of Short-Term Phosphorus Uptake by Intact Mycorrhizal and Non-mycorrhizal Maize Plants Grown in a Circulatory Semi-Hydroponic Cultivation System.

A non-destructive cultivation system was developed to study the dynamics of phosphorus (Pi) uptake by mycorrhizal and non-mycorrhizal maize plantlets. The system consisted of a plant container connected via silicon tubes to a glass bottle containing a nutrient solution supplemented with Pi. The nutrient solution is pumped with a peristaltic pump to the upper part of the container via the silicon tubes and the solution percolate through the plantlet container back into the glass bottle. Pi is sampled from the glass bottle at regular intervals and concentration evaluated. Maize plantlets were colonized by the AMF Rhizophagus irregularis MUCL 41833 and Pi uptake quantified at fixed intervals (9, 21, and 42 h) from the depletion of the Pi in the nutrient solution flowing through the plantlets containers. Plants and fungus grew well in the perlite substrate. The concentration of Pi in the bottles followed an almost linear decrease over time, demonstrating a depletion of Pi in the circulating solution and a concomitant uptake/immobilization by the plantlet-AMF associates in the containers. The Pi uptake rate was significantly increased in the AMF-colonized plantlets (at 9 and 21 h) as compared to non-colonized plantlets, although no correlation was noticed with plant growth or P accumulation in shoots. The circulatory semi-hydroponic cultivation system developed was adequate for measuring Pi depletion in a nutrient solution and by corollary Pi uptake/immobilization by the plant-AMF associates. The measurements were non-destructive so that the time course of Pi uptake could be monitored without disturbing the growth of the plant and its fungal associate. The system further opens the door to study the dynamics of other micro and macro-nutrients as well as their uptake under stressed growth conditions such as salinity, pollution by hydrocarbon contaminants or potential toxic elements.

Short-term chromium (VI) exposure increases phosphorus uptake by the extraradical mycelium of the arbuscular mycorrhizal fungus Rhizophagus irregularis MUCL 41833.

Hexavalent chromium is a potent carcinogen, while phosphorus is an essential nutrient. The role of arbuscular mycorrhizal fungi (AMF) in the uptake of P is well known and was also reported, at low levels, for Cr. However, it is unclear whether the uptake of Cr can impact the short-term uptake dynamics of P since both elements have a similar chemical structure and may thus potentially compete with each other during the uptake process. This study investigated the impact of Cr(VI) on short-term P uptake by the AMF Rhizophagus irregularis MUCL 41833 in Medicago truncatula. Bi-compartmented Petri plates were used to spatially separate a root compartment (RC) from a hyphal compartment (HC) using a whole plant in vitro culture system. The HC was supplemented with Cr(VI). Chromium(VI) as well as total Cr and P were monitored during 16 h within the HC and their concentrations determined by the end of the experiment within roots and shoots. Our results indicated that the uptake and translocation of Cr from hyphae to roots was a fast process: roots in which the extraradical mycelium (ERM) was exposed to Cr(VI) accumulated more Cr than roots of which the ERM was not exposed to Cr(VI) or was dead. Our results further confirmed that dead ERM immobilized more Cr than alive ERM. Finally our results demonstrated that the short exposure to Cr(VI) was sufficient to stimulate P uptake by the ERM and that the stimulation process began within the first 4 h of exposure.