Alfalfa is a leguminous plant with strong autotoxicity, which seriously affects regeneration and stability. In order to clarify the relationship between the key autotoxic substances and autotoxicity of alfalfa, this experiment determined the content of phenolic autotoxic substances in different varieties of alfalfa and the effect of different concentrations of alfalfa extracts on seed germination, seedling growth and physiology. The results showed that the content of single autotoxic substances in the eight alfalfa varieties was highest for total coumarin. The variety with the highest total coumarin content was “LZ”, and the lowest content was “656”. Principal component analysis of the autotoxicity of eight alfalfa varieties revealed that the variety with the strongest autotoxicity was “LZ” and the weakest was “656”. After treatment with extracts, the germination potential, germination rate, germination index and vigor index of 656 were higher than those of LZ, and the seeds of LZ and 656 did not germinate when the concentration was higher than C_0.025 and C_0.05, respectively. Compared with LZ, 656 had stronger osmotic regulation and antioxidant capacity, while the degree of membrane lipid peroxidation and ROS accumulation were lower. Further correlation analysis between the autotoxic substance content and autotoxicity observed that the content of total coumarin and autotoxic substances showed a significant positive association with autotoxicity (p < 0.01), and the total coumarin content showed a significant positive correlation with the content of autotoxic substances (p < 0.05). The total coumarin content is the major contributor to autotoxicity, and the higher the coumarin content, the higher the autotoxic substance content and the stronger the autotoxicity. Eight alfalfa varieties were systematically clustered on the basis of total coumarin content and autotoxicity, and the high-autotoxic alfalfa variety “LZ” and low-autotoxic alfalfa variety “656” were screened.

Endophytic rhizobia have the functions of dissolving organic phosphorus, secreting auxin, fixing nitrogen, and promoting growth. The proliferation of endophytic rhizobia in alfalfa and their symbiotic nodulation with alfalfa seedlings are regulated by various plant hormones. In this study, the alfalfa seeds (Medicago sativa L.) containing CFP-labeled rhizobium R.gn5f (isolated from the seeds of Gannong No.5 alfalfa) were used as materials, and the concentrations of 3-indoleacetic acid (3-IAA), 6-benzylaminopurine (6-BA) and homobrassinolide (HBR) suitable for the growth of R.gn5f were used for seed soaking treatment, and distilled water was used as the control. The proliferation of endophytic rhizobium, plant nodulation, nitrogen fixation performance and plant growth ability of alfalfa at different growth stages were determined. The effects of hormone types and concentrations on the proliferation and nitrogen fixation of endophytic rhizobia were analyzed to provide a theoretical basis for accurately promoting the nodulation, nitrogen fixation and growth-promoting ability of endophytic rhizobia in seeds. The results showed that the optimal concentrations of 3-IAA, 6-BA and HBR were 12 mg·L⁻¹, 16 mg·L⁻¹ and 2.47 mg·L⁻¹, respectively. The nitrogen fixation performance of endophytic rhizobium plants containing three hormones was higher at the branching stage and budding stage. The growth ability of the plant was better at the flowering stage. The hormone 2.47 mg·L⁻¹ of HBR was beneficial to the proliferation, nodulation, nitrogen fixation and plant growth of endophytic rhizobia in alfalfa at the vegetative and reproductive growth stages, and the number of R.gn5 f in the seeds of HBR plants at the mature stage was the largest (281.25 CFU·g⁻¹). Therefore, the hormone 2.47 mg·L⁻¹ of HBR was better for the proliferation of endophytic rhizobia R.gn5 f and plant growth in alfalfa. These findings provide a theoretical basis for precisely leveraging the nodulation and nitrogen-fixing capabilities of seed-borne endophytic rhizobia, thereby laying a foundation for the symbiotic breeding of alfalfa and rhizobia.

Although soil nematodes are central to belowground energy flow, how vegetation and soil aggregate characteristics interactively regulate the nematode community structure and energy dynamics remains poorly understood. We investigated 80 soil samples from five vegetation types—Prunus armeniaca L. (AV), Pinus tabuliformis Carrière (PT), Caragana korshinskii (CK), Medicago sativa L. (MS), and native grass Stipa bungeana (SB)—and four aggregate sizes (LMA > 2 mm, MMA 0.25–2 mm, SMA 0.053–0.25 mm, and MA < 0.053 mm) on the Loess Plateau. Vegetation types showed clear functional differentiation, in which AV dominated bacterivore diversity and energy flux in LMA, CK enhanced fungivore and herbivore energy flow, SB supported omnivore–carnivore energy flux, and PT exhibited suppressed communities. Fauna analysis of the EI (enrichment index)–SI (structural index) plot revealed aggregate-dependent food web structuring, where all vegetation types clustered in quadrant C (structured, low enrichment) in small aggregates, while PT and MS shifted to quadrant D (structured, enriched) in larger aggregates. SEM showed that energy flux and energy uniformity are driven by nematode abundance (p < 0.01) and diversity (p < 0.01), respectively, with soil aggregates promoting uniformity (p < 0.05) but suppressing total flux (p < 0.05), thus revealing a trade-off between energy throughput and distribution equity. CK maximizes total energy flux, while AV maintains high energy uniformity; as such, they could be keystone restoration species in the study area. This study provides mechanistic insights into soil food web energetics and offers an empirical foundation for optimizing vegetation restoration strategies on the Loess Plateau.