Centella asiatica (L.) Urb., a perennial herb of the Apiaceae family, is an important medicinal plant with diverse therapeutic applications. In this study, we integrated phenotypic evaluation, SSR markers, and HPLC fingerprinting to assess the diversity of 24 C. asiatica accessions collected from Yunnan Province, China. Considerable phenotypic, genetic, and biochemical diversity was observed. The Shannon–Wiener index ranged from 2.20 to 3.14 for qualitative traits. Principal component analysis identified five components accounting for 82.54% of the total phenotypic variance, with leaf morphology as the primary determinant. Q-type cluster analysis grouped the accessions into five phenotypic clusters. Transcriptome analysis revealed 16,799 SSR loci, with dinucleotide repeats as the most frequent motif (44.31%). UPGMA clustering based on SSR markers delineated five genetic groups that correlated with geographic origin, with accessions from the two northernmost sites forming a distinct cluster. HPLC fingerprints identified 16 common peaks, among which five major bioactive constituents—madecassoside, asiaticoside, kaempferol, madecassic acid, and asiatic acid—were quantified. The total content of these five compounds varied markedly among accessions (4.09–20.18 mg·g⁻¹), with the highest found in Zhaoyang (S5). Most accessions exhibited high chemical similarity (>0.9), except for Yiliang (S12), which displayed a distinct profile. The partial correlation between SSR-based classifications and geographic origin, together with the inconsistency between phenotypic and chemical groupings, indicates that morphological variation is shaped by both environmental factors and complex genetic regulatory mechanisms. These findings provide a scientific basis for germplasm conservation, selective breeding, and the sustainable utilization of C. asiatica.

Medicago sativa is one of the world’s most important forage plants, possessing strong nitrogen-fixing and regrowth capabilities. Promoting its growth not only enhances stress resistance but also reduces the use of chemical fertilizers. The value of Centella asiatica is primarily reflected in its medicinal properties. Currently, endophytic fungal resources of C. asiatica are scarce, and their potential to promote medicinal components and the underlying mechanisms remains unclear. This study employed DNA extraction techniques to isolate and identify endophytic fungi from different parts of C. asiatica. We systematically analyzed the plant growth-promoting traits of endophytic fungi. After screening for the optimal strain and inoculating it into Medicago sativa, we elucidated the mechanisms underlying its growth-promoting effect using metabolomic sequencing. Research findings: A total of 18 endophytic fungal strains were isolated, belonging to 12 genera. Among them, five indole-3-acetic acid (IAA) strains were identified, with strain J4 demonstrating the highest IAA production (17.157 mg·L⁻¹). The J4 strain has iron-transporting carrier activity, while 15 strains exhibit nitrogen-fixing activity. Inoculation with the Plectosphaerella plurivora strain significantly increases M. sativa’s germination rate, fresh weight, dry weight, and plant height. Metabolomic analysis indicates that P. plurivora may promote anthocyanin and jasmonic acid accumulation by regulating pathways such as flavonoid biosynthesis and pyrimidine metabolism, thereby promoting growth. This study reveals the mechanism by which endophytic fungi enhance M. sativa growth at the metabolomic level. This study reveals the growth-promoting mechanism of endophytic fungi in M. sativa from a metabolomic perspective, providing a theoretical basis for increasing forage yield and offering new insights into sustainable agricultural development.