The aim of this study was to investigate the effects on nutrient uptake, photosynthetic pigment content, stomatal aperture and antioxidant system of alfalfa leaves after three consecutive years of phosphorus fertilizer application, so as to provide scientific fertilization methods for efficient production of alfalfa.

Field trials were conducted at the forage experiment station of Shihezi University in Xinjiang from 2019 to 2021. ‘WL366HQ’ alfalfa was used as the test material, and four fertilizer treatments in the experiment included 0 (CK), 50 (Low P, LP), 100 (Middle P, MP), and 150 (High P, HP) kg·hm^-2. Samples were taken at the first flowering of alfalfa to determine the hay yield, nitrogen and phosphorus content, pigment content, stomatal opening, antioxidant enzyme activity, and oxidizing substances of leaves.

The nitrogen and phosphorus content, photosynthetic pigment content and stomatal aperture of leaves of alfalfa increased significantly (P<0.05) after 3 years of continuous phosphorus fertilization, with the highest nitrogen and phosphorus content under MP treatment (54.74 and 2.99 g·kg^-1, respectively), the highest chlorophyll a and chlorophyll b content under MP, and the highest carotenoid content under LP or MP, and the lowest in CK. The stomatal aperture was the highest under MP and significantly higher than that under CK (P<0.05). Therefore, phosphorus fertilizer had different effects on the morphological and physiological characteristics of alfalfa leaves, and MP treatment significantly affected the photosynthetic and physiological characteristics of alfalfa leaves (P<0.05). Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities were the highest under MP, with the value of 162.55, 406.40 and 147.13 U·g^-1, respectively, and were significantly higher than those under CK (P<0.05). The malondialdehyde (MDA), hydrogen peroxide (H₂O₂) and proline (Pro) content were the lowest under MP, at 2.38 and 1.04 μmol·g^-1 and 56.85 μg·g^-1, respectively. According to the Pearson correlation analysis, the total N and P contents of alfalfa leaves showed significant positive correlation (P<0.05) with chlorophyll content, carotenoid content, stomatal aperture, SOD activity and POD activity, and significant negative correlation (P<0.05) with MDA and H₂O₂ content. The overall evaluation showed that the principal component scores of phosphorus application treatments were ranked as MP>HP>LP>CK.

The reasonable application of phosphorus fertilizer increased the nutritional characteristics and photosynthetic physiological characteristics of alfalfa leaves, thus avoiding the stressful effects brought by other environmental factors, enhancing the adaptive capacity of alfalfa, and making it better adapted to external environmental changes. Considering the leaf morphology, photosynthetic physiological characteristics and antioxidant enzymes and oxidizing substances of alfalfa, the phosphorus application rate of 100 kg·hm^-2 was suitable under this experiment.