Arbuscular mycorrhizal fungal families and exploration-based guilds exhibit distinct responses to long-term N, P and K deficiencies and imbalances.

Many agroecosystems face nitrogen (N), phosphorus (P) or potassium (K) deficiencies due to imbalanced or insufficient nutrient replenishment after biomass harvest. How this affects the symbiosis between plants and arbuscular mycorrhizal fungi (AMF) and the abundance of exploration-based AMF guilds (rhizophilic, edaphophilic and ancestral) remains largely unknown. We studied a 70-yr nutrient deficiency experiment in a managed grassland in central Austria, where aboveground biomass was harvested three times annually. N, P and K were fully, partially or not replenished, causing long-term nutrient deficiencies and imbalances. We analysed AMF communities in soil and roots by DNA/RNA amplicon sequencing and fatty acid biomarkers, alongside soil and plant community properties. Soil AMF communities were affected by N and P deficiencies, while root AMF communities were most susceptible to K deficiency, showing up to 50% biomass reduction, particularly when N was abundant. We observed a shift from rhizophilic to ancestral guilds under P deficiency in soil, and under K deficiency in roots. Families within each guild, particularly ancestral, showed differential responses, indicating complementary nutrient specializations at the family level. Our findings underscore the previously unrecognized role of K deficiency in AMF symbiosis and suggest the existence of nutrient-related functional subgroups within exploration-based AMF guilds.