ABSTRACT: Plant-soil feedback (PSF) can be a major driver of plant performance in communities, and this concept can be used in selecting crop rotation sequences to maximize agricultural yields. Potential benefits of using PSF in this context include nutrient use optimization, pathogen reduction, and enhancement of mutualisms between crops and microbes. Yet the contributions of these combined mechanisms are poorly understood. Here we investigated the relative contributions of these mechanisms using five major crops commonly cultivated in rotation (alfalfa, canola, maize, soybean, and wheat) under controlled conditions. We trained soil by growing each of the five crops in a "training phase," and then reciprocally planted the five crops in the trained soils in a "feedback phase." To tease out soil biota from nutrient effects, we established three treatments: "control" (trained unsterilized soil used in the feedback phases), "biota" (sterilized soil in the feedback phase inoculated with soil biota from the control treatment after the training phase), and "nutrient" (sterilized soils in both phases). Plant-soil feedback for each crop was calculated by comparing the total biomass of each crop grown in soils trained by each of the four other crops (i.e., in rotation) against total biomass in self-trained soil (i.e., monocropping). We found that PSF values varied among crop combinations in all the treatments, but such variation was the greatest in the nutrient treatment. Overall, soil biota feedback tended to be lower, whereas nutrient feedback tended to be greater compared to the unsterilized control soil, suggesting that effects of antagonistic biota outweighed those of beneficial microbes in the biota treatment, and that plants optimized nutrient uptake when the soil microbiome was absent in the nutrient treatment. Furthermore, soils in the nutrient treatment trained by the legume crops (alfalfa and soybean) tended to provide the greatest positive feedback, emphasizing the important legacy of N2 fixers in crop rotation. Taken together, our data demonstrate how nutrients and soil biota can be integral to PSFs among crops, and that assessing PSFs under controlled conditions can serve as a basis to determine the most productive crop rotation sequences prior to field testing.