| To explore the effects of the depth of descending phosphate fertilizer on physiological characteristics of the overwintering organs of alfalfa, the ‘Gibraltar’ alfalfa variety was sown on July 1, 2020. Five treatments of phosphate fertilizer depth included the ground surface, 7.5, 15, 22.5, 30 cm from the ground (respectively represented by S0, S1, S2, S3, S4), and no fertilization as the control (CK). Simulated low temperature treatment on the root crown of alfalfa during the overwintering period (4,-10,-20℃ and -30℃) was used to determine the relative conductivity, the content of nitrogen\|containing protective substances (soluble protein, proline, free amino acid content). The results showed that with the decrease of low temperature stress, the relative conductivity of alfalfa root crown displayed a continuous increase. The soluble protein and free amino acid had a trend of first increasing and then decreasing. The contents of soluble protein, proline and free amino acid were all reached the maximum value at -10℃, which increased by 65.18%～144.67%,46.24%～133.33% and 27.13%～57.01%, respectively, compared with 4℃ treatment. The relative conductivity of alfalfa root crown was the smallest under the treatment of S2 phosphorus application depth of 15 cm, which was reduced by 24.87%～57.21% compared with CK. The value of the soluble protein content of alfalfa root crown was the largest under the treatment of S2 phosphorus application depth of 15 cm, which was higher than that of CK and other treatments. The advanced phosphorus application treatment increased by 8.18%～79.81%. The proline and free amino acid content of the alfalfa root crown was significantly higher than other advanced phosphorus application treatments under the S0, S1 and S2 treatments, indicating that the application of phosphate fertilizer 15 cm (S2 treatment) from the soil surface was the most beneficial for increasing the nitrogen content of alfalfa, reducing the relative conductivity, and improving the cold resistance of alfalfa. |