Strategies to develop simple multi-species cover crop mixtures to enhance aboveground biomass and quality

Cover crops are an important tool for the sustainable intensification of crop production systems. Species mixtures can enhance their multifunctional benefits, but intra- and interspecific interactions will affect their performance. These interactions can be managed by selecting complementary species and optimizing seed rates. However, there is a lack of evidence-based recommendations for optimizing the design of multi-species cover crop mixtures. This study aimed to evaluate the performance of different species used as cover crops and their interaction with varying environmental conditions, investigate the potential advantages of species mixtures over pure stands, analyze the role of facilitation and competition in mixture performance, and validate the predictive accuracy of yield-density model interaction coefficients for three-species mixtures. The common and contrasting species oil radish ( Raphanus sativus var. oleiformis Pers .), Egyptian clover ( Trifolium alexandrinum L.), lacy phacelia ( Phacelia tanacetifolia Benth .), and bristle oat ( Avena strigosa Schreb .) were systematically tested in pure stands and mixtures of different compositions and configurations in four environments (2 sites x 2 years). Aboveground biomass, nitrogen accumulation, C/N ratio, and stand density were monitored to assess the cross-environmental performance using linear mixed models and to fit yield density models quantifying intra- and interspecific interactions. Oat, phacelia, and radish performed similarly on average but differed in their response to environments, while clover showed significantly lower performance. Two and three-species mixtures improved average performance by up to 53 % compared to pure stands without compromising litter quality. Optimal species combination and seed rate configuration increased cover crop performance by up to 52 %. Oat and radish were the most competitive species, followed by phacelia and clover. Biomass of three-species mixtures was successfully predicted (R 2 = 0.89) using competition coefficients derived from the yield density model. Our findings indicate that cover crop mixtures offer a way to mitigate the risks of environmental variability. Combining non-legume species with legume species, combining strong competitors with less competitive species, rather than pairing weak competitors or strong competitors, and reducing the seed rate of radish while increasing the seed rates of phacelia and oat in mixtures can optimize cover crop performance. Cross-environmental trends in species-specific intra- and interspecific interactions of cover crops are presented, providing evidence-based strategies to facilitate the development of multi-species cover crop mixtures for more sustainable agriculture. • Cover crops offer key benefits for sustainable crop production and should be further promoted. • Mixtures boosted performance by up to 53 % without reducing litter quality and helped buffer environmental variability. • Optimized species combinations and seed rates improved cover crop performance by up to 52 %. • Yield density models quantified interactions in mixtures, explained facilitation and competition, and predicted biomass.