The grass-legume intercropping improved soil quality through soil biological and biochemical properties and increased soybean yields. The Cowpea intercropping yielded more soybeans than Pigeon pea intercropping and single-cropped. According to Laroca et al. (2018), the grass-legume intercropping with Cowpea and Pigeon pea resulted in higher soybean yields (416 and 338 kg ha− 1, respectively), compared to single cropping of grasses. The higher soybean yields were associated with higher microbial biomass C and N contents, qMIC and lower basal respiration and qCO2 (Table 2).
The basal respiration rate and the qCO2 are variables used to measure the metabolic activity of microbial biomass in the soil. Higher qCO2 values indicate C losses by the soil, showing stress conditions for microbiota according to the large amount of energy for its maintenance (Nunes et al. 2011). Low-qCO2 values are related to environments that are more stable or closer to their equilibrium state, as they indicate energy savings (Silva et al. 2010; Wardle and Ghani 1995). In addition, microbial biomass and qMIC are commonly used for soil quality assessment (Bastida et al. 2008; Mader et al. 2002; Salinas-Garcı́a et al. 2002). Therefore, the lower basal respiration and qCO2, and the greater qMIC and microbial biomass C may indicate higher soil microbial C utilization efficiency (Andersen et al. 2013; Anderson and Domsch 1978; Pleisner et al. 2016) under grass-legume intercropping (Table 2). This may be due to the higher contribution of labile C and N (with easy oxidation) from treatments under grass-legume intercropping (Crème et al. 2016) with residue inputs with low C/N ratio.
The residue inputs with lower C/N ratios under grass-legume intercropping contributes to an increase in microbial biomass and activity (Leite et al. 2013; Moraes et al. 2019). This indicates a higher conversion efficiency of the C and N in plant residues into C and N in microbial biomass (Balota and Chaves 2011; Spohn 2015; Veloso et al. 2019). Therefore, the grass-legume intercropping in ICLS improve soil microbial properties, which may contribute in the long run to achieve the equilibrium in the production system, since it showed low-qCO2 values, but maintained high qMIC values (Table 2), highlighting that the microbial biomass is not under stress and is able to use organic C efficiently (Fang et al. 2018; Hartman and Richardson 2013; Silva et al. 2010).
The microbial biomass C and N improvements under grass-legume intercropping (Table 2) were also reported by Hurisso et al. (2013) and Almeida et al. (2016). The microbial biomass N increments may be due to leguminous plants insertion, which favors soil N availability through the biological N fixation (Crème et al. 2016). On the other hand, in single grasses the soil microbiota consumes more C to maintain a lower microbial population. The greater the abundance and diversity of roots, the greater the exudation of organic compounds that will serve as a source of C and energy for soil microorganisms (Chávez et al. 2011; Dhakal and Islam 2018).
The total C and N stocks also increased in the soil, mainly under Pigeon Pea intercropping, compared to single grasses (Table 1). These results demonstrate that grasses, when intercropped with legumes, promote the C and N improvements in the soil. Similar results were found by Frasier et al. (2016) and Laroca et al. (2018), who observed increases of C and N stocks in an experiment using grass-legume intercropping. Frasier et al. (2016) assigned the increase of total N stock to the better residue quality (lower C/N ratio) provided by leguminous plants. Almeida et al. (2016) also documented improvements in microbial biomass C and N under grass-legume intercropping systems, which may result in higher soybean yields.
Soybean under N-fixing bacteria inoculation does not rely to N fertilizer because it reduces restricts carbohydrates to nodule metabolism (Denison and Harter 1995). However, some studies have shown that N addition via leguminous crops may positively influence the soybean yields (Nascente and Stone 2018; Tanaka et al. 1992). The higher-N input to the soil-plant system, derived from the biological N fixation, may contribute to improvements of soybean yield due to the N source, that is gradually released through mineralization (Cicek et al. 2014; Pacheco et al. 2017). The biological N fixation can contribute a considerable N fraction in the soil, releasing to the soybean in succession, reflecting in increases in grain yield (Pacheco et al. 2017). Likewise, soybean is positively influenced by N fertilization in pasture grasses in advance (Costa et al. 2021).
The β-glucosidase activity was also higher under grass-legume intercropping (Fig. 1A). This enzyme acts on the hydrolysis of organic compounds and it is important in the C life cycle (Bowles et al. 2014). It has a direct relationship with the N stock in the soil (Stieven et al. 2014), which was also higher under grass-legume intercropping (Table 2). The higher FDA activity found in grass-legume intercropping treatments (Fig. 1B) corroborate the results of Ferreira et al. (2017), which found higher FDA activity in agroecological production systems with legumes compared to conventional systems using single grasses.
The acid phosphatase and urease activity were not affected by treatments (Figs. 1D and 1E). Laroca et al. (2018) found similar results where the acid phosphatase activity was not influenced by legumes or grasses, because the activity of this enzyme is more related to phosphate fertilization, which was the same in all treatments. Ye et al. (2017) reported a reduction of the acid phosphatase activity, and associated this result with the excess available phosphorous in the soil. Lanna et al. (2010) documented greater urease activity related to higher N content in the soil. Despite not differing significantly (Fig. 1E), same effect was found in this study, where the urease activity was correlated (r = 0.51*) to total N stocks (Table 3).
Regarding the arylsulfatase activity, studies conducted in Brazil indicate that the quality of residues added to the soil are more determinant in the activity of this enzyme than the quantity of residues (Balota et al. 2011), and the input of more labile organic residues helps the activity of this enzyme (Lisboa et al. 2012). In addition, the higher contents of total organic C stocks (Table 1) can provide high levels of sulfur in the form of sulfate esters which, in turn, are substrates for this enzyme (Balota et al. 2014).
In summary, legume intercrops increased microbial activity and biomass and total C and N stocks, and qMIC, and decreased basal respiration and qCO2, compared to single grasses in pasture phase. However, the adoption of ICLS under no-till using single grasses in pasture phase is already an important way to improve soil quality (Sarto et al. 2020) and grain yield, even in the short-term (Carneiro et al. 2009). In the Brazilian Cerrado region, soil C improvements can reach 26 g kg− 1 under ICLS compared to U. decumbens as cover crop (Gazolla et al. 2015). Alves et al. (2011), evaluating microbial properties, documented a microbial biomass C 60% higher under ICLS compared to the native vegetation of Cerrado region. Therefore, grass-legume intercropping in the pasture phase of ICLS is an additional tool to maximize soil quality improvements and soybean yields even in the short-term.