Locating enzyme activities and nutrients in the rhizosphere: Combining zymography and DET methods

Organic fertilization is a recognized sustainable practice in agriculture and represents a major nutrient source for microbes and plants in these systems. Microbes produce hydrolytic enzymes to mineralize nutrients from organic forms into mineral forms to satisfy their own needs, and thus can compete with plants for these mineralized nutrients. Thus, interactions between plants and microbes in the rhizosphere determine nutrient availability and flows. We investigated these relations, using a spatial approach that combined zymography with the method of diffusive equilibrium in thin films (DET) to localize enzyme activity and N and P availabilities simultaneously. Basil ( Ocimum basilicum L.) was grown in rhizoboxes filled with an organo-mineral crop soil (MS) or 100 % organic peat soil (OS) that was unfertilized or fertilized locally with horn meal for 20 days. In general, enzyme activities were higher in MS than in OS, but the stimulation of leucine aminopeptidase (LAP) activity and associated decrease in nutrient availability was 2 times as strong in OS as in MS. A rhizosphere effect, in which rhizodeposits stimulated enzyme activity, was clearly observed in OS. Fertilization increased LAP activity and nutrient availability near the location of fertilization, which increased basil growth in OS but not in MS. β-glucosidase, acid phosphatase and N-acetyl-glucosaminidase activities responded weakly to fertilization and the rhizosphere. By relating enzyme activities mapped by zymography to nutrient availability mapped by DET, we identified microbial hotspots in the rhizosphere where most nutrient mobilization processes and competition between plants and microbes occurred. © 2025 The Authors.