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Enhancement of Clover (Trifolium alexandrinum L.) Shade Tolerance and Nitrogen Fixation under Dense Stands-Based Cropping Systems

Improving legumes crops’ performance under dense stands shade environment (e.g., intercropped oats–clover) is needed to promote agroecological practices. Previous studies have revealed that ethylene produced by plants under dense standing conditions is among other factors that affect crops’ growth performance and reduce legumes’ ability to fix nitrogen (N). Here, we identified a Pseudomonas thivervalensis strain T124 as a high ACC deaminase-producing bacterium and evaluated its potential ability to alleviate the effects of reduced light (RL) and exogenous ethylene applied as ACC (ethylene precursor) on clover growth and development under controlled conditions and field conditions at dense stands of clover and oats intercrops. RL decreases clover root and shoots biomass, whereas the T124 strain counteracted RL effects, enhancing clover tolerance to shade. Exogenous ACC reduced clover growth and chlorophyll content while inducing overaccumulation of reactive oxygen species (H2O2 and O2•−). ACC-elicited cellular stress was suppressed by strain T124, suggesting the role of bacterial ACC deaminase activity. Combined with Rhizobium leguminosarum strain T618 (the strain identified as being able to fix N in symbiosis with clover), T124 prevents early nodule senescence by improving nodule leghemoglobin and reducing nodule nitric oxide levels. Co-inoculation with T124 + T618 increased shoot N content (+24%) more than T618 alone. Field experiments revealed that intercropping decreases Photosynthetic Active Radiation (PAR) at the top of clover due to oats, affecting clover photosynthesis assimilation. Interestingly, under T124 inoculation treatments, clover net photosynthetic rate (Anet) and stomatal conductance (Gs) were found to improve relative to the control and T618 inoculation treatments. Clover exhibits improved growth performance in terms of branching and nodulation after T124 inoculation. Most significant improvements occurred with the mixing of the two strains. Data suggest that co-inoculation with R. leguminosarum T618 and P. thivervalensis T124 potentially decreases the interspecific competition between clover and oats intercrops by reducing ACC (ethylene precursor) levels. Our study revealed that co-inoculation of legumes with competitive rhizobia and ACC deaminase-producing PGPRs is an eco-friendly approach to improving intercropping systems’ performance.