Intricate asymbiotic hyphal interactions of Rhizophagus irregularis revealed with single-plane observations in a new microfluidic device

Arbuscular mycorrhizal fungi (AMF) are essential symbionts to most land plants. There is great interest in using AMF inoculation treatments to enhance restoration or agricultural efforts. However, little is known about AMF asymbiotic interactions that could shape soil ecosystems and inoculation outcomes. Studying these early traits is challenging given the obligate nature of AMF. Microfluidic devices enable high-resolution, single-plane, time-resolved observation of AMF hyphal traits in controlled, plant-less environments. The AMF-AnastomosisChip, introduced in this work, additionally separates spores and hyphae into lanes for detailed observations prior to entering a shared interaction zone. Using this device with Rhizophagus irregularis, we phenotyped key hyphal traits from germination to anastomosis. We compared the effect of a fatty acid (FA) treatment to other environmental factors in shaping AMF growth and fusion (anastomosis). Among our results, we confirm known effects of the FA treatment in increasing hyphal branching and longevity but interestingly show that multiple hyphae in a lane can negate FA effects. We also newly reveal that anastomosis occurs for 100% of interactions among hyphae from different lanes, but only for 50% of in-lane interactions, potentially linked to spore origin. Our findings validate the AMF-AnastomosisChip as a versatile platform for increasing discovery in trait-based microbial ecology, including for asymbiotic AMF interactions with environmental factors. This work sets the foundation for future studies with the AMF-AnastomosisChip on the effects of nutrient content, plant-derived molecules, or microbial community members on the AMF traits characterized in this study.