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Leaf-cutting ants rear clonal fungi for food and transmit the fungi from mother to daughter colonies so that symbiont mixing and conflict, which result from competition between genetically different clones, are avoided. Here we show that despite millions of years of predominantly vertical transmission, the domesticated fungi actively reject mycelial fragments from neighboring colonies, and that the strength of these reactions are in proportion to the overall genetic difference between these symbionts. Fungal incompatibility compounds remain intact during ant digestion, so that fecal droplets, which are used for manuring newly grown fungus, elicit similar hostile reactions when applied to symbionts from other colonies. Symbiont control over new mycelial growth by manurial imprinting prevents the rearing of multiple crops in fungus gardens belonging to the same colony.