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Agricultural land abandonment is an emerging problem in European Union (EU), and about 11% of agricultural EU land is at high risk of abandonment in the coming 10 years. Land abandonment may have both positive and negative effects in ecosystems. Due to the potential for land abandonment to increase soil fertility, the study of vegetation succession effects on soil quality is of great importance. In this study, we investigated an abandoned vineyard where, after a period of 30 years, rows and alleys were characterized by two different forms of vegetation succession: natural recolonization by trees along the rows and by herbaceous vegetation in the alleys. No-tilled alleys covered by herbaceous vegetation of a neighboring conventionally cultivated vineyard were used as a comparison. Soil samples were chemically characterized (pH, extractable element, and available and total metals), and analyzed for the determination of carbon (C) and nitrogen (N) pools; hydrolytic and phenol oxidizing (PO) enzyme activities involved in C, N, and phosphorus (P) cycles; and the enzyme ratios. Results highlighted that natural recolonization by trees increased the organic C and N soil pools by 58% and 34%, respectively, compared to the natural recolonization by herbaceous vegetation. Moreover, natural recolonization by trees reduced β-glucosidase by 79%, urease by 100%, alkaline phosphastase by 98%, acid phosphatase specific hydrolytic activities by 50%, and catechol oxidase and laccase specific oxidative activities by 127% and 119%, respectively, compared to the renaturalization by herbaceous vegetation. In addition, the natural recolonization by trees reduced the C (βglu):C (PO) enzymes ratio by 16% compared to that of the conventional vineyard. Comparing the natural recolonization by herbaceous vegetation with that of the conventional vineyard revealed little significant difference (15% of the measured and calculated parameters); in particular, PO activities significantly decreased in the renaturalized vineyard with herbaceous vegetation by 49% (catechol oxidase) and 52% (laccase), and the C (βglu):C (PO) enzyme ratio showed a reduction (−11%) in the vineyard naturally recolonized by herbaceous vegetation compared to the conventional vineyard. This highlights that the type of vegetation succession that takes place after land abandonment may have a significant impact in terms of soil fertility and C accrual potential. These results help to focus attention on the practices used in agro-forestry that should be adopted in abandoned agro-ecosystems to increase their biodiversity, soil C stock, and soil quality, because these indicators are affected by the type of vegetative coverage. [ABSTRACT FROM AUTHOR]