Alfalfa (Medicago sativa) is a highly valuable perennial forage legume that suffers from autotoxicity, which decreases plant resistance, reduces soil fertility, causes serious soil-borne diseases, and promotes ecological imbalance. We evaluated the effects of autotoxicity on the seed germination of 22 alfalfa varieties, and then elucidated the oxidative damage and lipid peroxidation in two alfalfa varieties with contrasting autotoxicity tolerances. The technique for order of preference by similarity to ideal solution (TOPSIS) method was used to rank the germination of the 22 alfalfa varieties when exposed to six autotoxic concentrations (0, 0.025, 0.075, 0.125, 0.175, and 0.225 g∙mL⁻¹). We found WL656HQ and 3105C to be autotoxicity-tolerant and autotoxicity-sensitive varieties, respectively. The germination index mainly affects the comprehensive allelopathic index of WL656HQ and 3105C, which were the simple vigor index and radicle length according to the random forest model, respectively. 3105C eliminates reactive oxygen species (ROS) via antioxidant enzymes and antioxidants under T1 (0.025 g∙mL⁻¹), but the oxidative stress system and the oxidative scavenging system cannot maintain the balance under T2 (0.125 g∙mL⁻¹), causing oxidative bursts. In comparison, WL656HQ used its oxidative scavenging system (peroxidase (POD), ascorbate peroxidase (APX), and glutathione reductase (GR)) to maintain its redox dynamic balance by removing excess ROS at all concentrations. In conclusion, the positive and negative indicators of autotoxicity for the two varieties were ascorbate (ASA) and hydroxyl free radicals (OH^•), and proline (Pro) and dehydroascrobate (DHA), respectively. The most sensitive autotoxic concentrations of 3105C and WL656HQ were T2 (0.125 g∙mL⁻¹) and T1 (0.025 g∙mL⁻¹), respectively.

Allelopathy plays a major role in agricultural production, influencing plant protection, crop yield, and crop rotation systems. This study investigated the effects of root exudates on 3105c alfalfa (Medicago sativa) seeds and seedlings to identify crops with strong and weak allelopathic potential. The results revealed that corn (Zea mays L.) (T1) exhibited the strongest allelopathic effects, whereas soybean (Glycine max (Linn.) Merr.) (T10) exhibited the weakest effects. T1 promoted seed germination by increasing radicle length and the simple vitality index. Both T1 and T10 promoted 3105c seedling growth and enhanced antioxidant capacity, albeit through different mechanisms. T1 primarily increased antioxidant capacity by elevating ascorbate and dehydroascorbate levels while reducing malondialdehyde content. In contrast, T10 enhanced antioxidant capacity by increasing soluble sugar and protein levels via hydroxyl free radical inhibition. These findings demonstrate that the allelopathic properties of corn effectively promote alfalfa growth by enhancing seed germination and improving physiological stress resistance.

The allelopathic theory has garnered considerable attention in the field of agricultural production for its efficient plant protection, rapid crop yield increase, and scientific establishment of the crop rotation system. To study the effects of the main maize allelochemical DIMBOA (2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one) on the growth and development of alfalfa under autotoxic coumarin stress, we treated alfalfa seedlings with DIMBOA under coumarin stress and non-stress conditions in this study. Results show that 0.0342 mM coumarin significantly inhibited alfalfa seed germination percentage(Gp), germination potential(GP), radicle length, germ length, seeding height, and simple viability index (SVI), with decreases of 37.29%, 59.91%, 7.60%, 30.90%, 13.27%, and 45.70%, respectively. An amount of 0.6 mM DIMBOA could promote alfalfa seed Gp, GP, radicle length, germ length, seeding height, dry fresh ratio, and SVI, with increases of 12.38%, 23.91%, 48.69%, 48.65%, 48.68%, 295.12%, and 67.17%, respectively. However, the addition of DIMBOA under conditions of coumarin stress could effectively alleviate coumarin effects on alfalfa seedlings. Coumarin + DIMBOA treatment for 24 h mainly decreased reactive oxygen species (ROSs) and malondialdehyde (MDA) as well as soluble protein and soluble sugar, increasing some antioxidant enzyme activities and antioxidant content to alleviate the oxidative damage of alfalfa caused by coumarin stress. Administration of treatment for 72 h significantly promoted the morphological development of alfalfa seeding roots. Administration of treatment for 96 h significantly enhanced the photosynthetic capacity of alfalfa seedlings. The results of principal component analysis demonstrated that chlorophyll b(Chl b)and net photosynthetic rate(Pn) were the key indicators for coumarin + DIMBOA treatment to promote photosynthesis in alfalfa seedlings. Additionally, root length, mean root diameter, and root volume were the key indicators of root growth and development. Coumarin + DIMBOA treatment primarily increased catalase(CAT), peroxidase (POD), and ascorbate peroxidase (APX) activity and antioxidants(ASA) while reducing MDA and superoxide anion radical(O₂^•−). This study strongly suggested that DIMBOA can effectively improve the tolerance of alfalfa seedlings to coumarin stress through a combination of effects on root morphology, photosynthesis, and physiological indicators.