Cropping systems including legume cover crops favour mineral–organic associations enriched with microbial metabolites in no-till soil Murilo G. Veloso A,C , Deborah Pinheiro Dick B , Janaina Berne da Costa B , and Cimélio Bayer A A Department of Soil Science, Federal University of Rio Grande do Sul, 7712 Bento Gon¸ calves Avenue, CEP 91540-000, Porto Alegre, RS, Brazil. B Institute of Chemistry, Federal University of Rio Grande do Sul, 9500 Bento Gon¸ calves Avenue, CEP 91501-970, Porto Alegre, RS, Brazil. C Corresponding author. Email: murilo.veloso@ufrgs.br Abstract. Long-term carbon (C) stabilisation in tropical and subtropical soils under no-tillage (NT) rests on the formation of mineral–organic associations (MOAs) that can be enriched with microbial metabolites. In this work, we assessed the role of long-term tillage and cropping systems and mineral N fertilisation in enriching MOAs with microbial metabolites in a subtropical soil. For this purpose, we sampled a sandy clay loam Acrisol up to 1 m depth involved in an ongoing 30-year-old experiment under two different tillage systems (conventional tillage and NT) in the presence and absence of legume cover crops and mineral nitrogen (N) fertilisation. The soil samples were subjected to particle size fractionation and n-alkane analysis. The NT and the presence of legume cover crops in the surface soil layer (05 cm) increased the abundance of plant-derived lipids (i.e. compounds with n-alkane chains of 2533 C atoms) in the whole soil. Microbial-derived lipids (i.e. compounds with shorter n-alkane chains (1524 C atoms)) were more abundant in the clay fraction of the surface (05 cm) and sub-surface soil layers (2030 and 75100 cm) in NT soil receiving high-quality residues of legume cover crops. However, N fertilisation decreased the abundance of microbial-derived lipids in the clay fraction of the 05 and 2030 cm soil layers. Our findings highlight the role of N-rich residues of legume cover crops, but not of mineral N fertilisation, in the long-term stabilisation of C in MOAs in NT soils through the action of microbial residues. Additional keywords: n-alkanes, no-tillage, soil carbon accumulation, soil lipids. Received 30 May 2019, accepted 13 August 2019, published online 19 September 2019 Introduction No-tillage (NT) is a widespread method of facilitating storage of organic carbon (C) in tropical and subtropical soils (Bayer et al. 2006; Veloso et al. 2018). Legume cover cropping is widely recognised as a key practice to favour storage of organic C in NT soils; however, the specific mechanisms behind C stabilisation in soils are incompletely understood. Veloso et al.(2019) recently found legume cover crops to play a major role in stabilising C by association with the silt–clay fraction in a kaolinitic Acrisol. This C stabilisation mechanism is especially important in tropical and subtropical soils, where mineral–organic associations (MOAs) account for 65–92% of total soil organic carbon (SOC; Amado et al. 2006). In addition to increasing soil nitrogen (N) input, legume cover crops may favour C storage by increasing the efficiency of microbial metabolism in stabilising C in plant residues enriched with N and biochemically labile structures. This hypothesis is supported by the fact that MOAs consist mainly of microbial residues (Puget et al. 1998; Kögel-Knabner et al. 2008; Rumpel et al. 2010). Also, labile residues are known to increase microbial biomass and hence to facilitate C accumulation through formation of MOAs (Rumpel et al. 2010; Cotrufo et al. 2013, 2015). The contribution of microbes to stabilising C in MOAs can be assessed with lipid analyses. Soil lipids represent the net result of processes such as the addition of plant residues or microbial synthesis and degradation (Dinel et al. 1990). Aliphatic compounds containing long chains of n-alkanes (2533 C atoms but especially C 27 ,C 29 ,C 31 and C 33 ) derive largely from plants, whereas those with shorter chains (usually 15–24 C atoms, but especially C 16 ,C 18 and C 20 ) are usually of microbial origin (van Bergen et al. 1998; Wiesenberg et al. 2004; Jandl et al. 2007). Not only the pattern distribution of extracted soil n-alkanes according to chain length, but also molecular proxies calculated from it, can be useful to elucidate soil organic matter (SOM) formation processes. Thus, the Journal compilation Ó CSIRO 2019 www.publish.csiro.au/journals/sr CSIRO PUBLISHING Soil Research https://doi.org/10.1071/SR19144