Improving Soil Properties and Microbiomes by Mixed Eucalyptus–Cupressus Afforestation

Monoculture plantations of <i>Eucalyptus</i> in China have raised ecological concerns due to water depletion, soil degradation, and fire risk. Integrating <i>Eucalyptus</i> with <i>Cupressus</i> offers a sustainable approach to improving forest ecosystem health. In this study, we established five forest treatments, pure <i>Eucalyptus</i> (1:0), mixed <i>Eucalyptus</i>-<i>Cupressus</i> at three ratios (2:1, 1:1, and 1:2), and pure <i>Cupressus</i> (0:1), to assess their effects on soil properties, microbial diversity, and metabolomic profiles. Laboratory analyses revealed significant differences in physicochemical soil properties (such as water content (<i>p</i> < 0.05), pH (<i>p</i> < 0.001), organic carbon (<i>p</i> < 0.001), and nitrogen (<i>p</i> < 0.001)) among various groups within the mixed forests. Microbial community investigations highlighted a unique microbial signature in <i>Eucalyptus</i>-<i>Cupressus</i> mixed forests, especially when the tree ratio was 1:2, characterized by a rich (Chao1, <i>p</i> < 0.05) and diverse (Shannon, <i>p</i> < 0.05) array of bacterial taxa. The mixed <i>Eucalyptus</i>-<i>Cupressus</i> forest also exhibited an uplift in microbial communities, bacterial genera such as <i>RB41</i>, and fungal genera including <i>Penicillium</i>, <i>Talaromyces</i>, and <i>Mortierella</i>, which are associated with enhanced organic matter decomposition and nutrient cycling. Interactive networks within microbial communities were revealed through co-occurrence and Spearman correlation analyses, highlighting potential symbiotic relationships and ecological complexities. Metabolomic analysis, coupled with pathway analysis, further illuminated metabolic shifts in the mixed forests, emphasizing alterations in key metabolic pathways such as phenylpropanoid biosynthesis, tyrosine metabolism, arachidonic acid metabolism, and isoquinoline alkaloid biosynthesis. Collectively, these results show that moderately mixed <i>Eucalyptus</i>-<i>Cupressus</i> forests improve soil fertility and microbial multifunctionality, providing a practical model for sustainable and resilient forest management in subtropical regions.