4. Discussion
4.1 Rhizosphere effects on soil C and N mineralization depend on plant phenology
For all levels of planting densities, the SOM-C and N mineralization showed similar unimodal patterns throughout maize growth stages, with the higher rates at flowering (Fig. 1c). This indicates that plant development stages exert an important control on the dynamic of soil C and N, as similarly reported by earlier studies on maize (Li et al. 2017; Kumar, Shahbaz, Blagodatskaya, Kuzyakov & Pausch 2018) and other crops (Cheng, Johnson & Fu 2003; Pausch et al. 2013; Zhuet al. 2018). The phenology-dependent effect on C and N fluxes from SOM is plausibly explained by plant-microbial interactions that are driven by (i) root growth and altered quality and quantity of rhizodeposits and (ii) changes in plant morphological traits with growth stages, and (ii) plant-associated changes in soil properties and nutrient status.
While root biomass remained almost constant from the heading to ripening stages (across all planting densities; Table 1), root-derived CO2 efflux strongly declined (Fig. 1b), even after normalization for root biomass (specific root-derived CO2, Fig. 2b). This indicates the decreased root respiration and indirectly a reduction of the amount of rhizodeposits with maize growth. A major reason for this could be that annual crops allocate more C belowground during early phases of vegetation stages, whereas the newly assimilated C remain in aboveground tissues for producing biomass and cobs at later growth stages, despite the increased shoots likely producing more photo-assimilated C (Gregory & Atwell 1991; Pausch & Kuzyakov 2018; Chen, Palta, Wu & Siddique 2019). This down-regulated С translocation from shoots to roots with maize growth was indicated by a negative relationship between root-derived CO2 efflux and shoot biomass (Fig. S4a).
At the maize heading stages, the higher root-mediated C release (as indicated by root-derived CO2) was coincident with the relatively abundant mineral N in soil (Fig. 1b and 2b; Table 2). The microbial growth and activity may be promoted with a better supply of C and nutrients sources (Hessen et al. 2004). This condition, however, favored microbial community to directly utilize easily degradable rhizodeposits over recalcitrant SOM for C and energy requirements (preferential substrate utilization, Blagodatskaya et al., 2011; Hagedorn et al., 2003). This is indicated by the much larger contributions of root-derived CO2 to total CO2 efflux (> 50% of total CO2) at the heading than at the other two stages, with consequent lower SOM mineralization (for all planting densities, Fig. 1a). Likewise, microorganisms might preferably assimilate the available N source and thus decreased N mineralization. Therefore, preferential substrates utilization by microorganisms results in the lower SOM decomposition and gross N mineralization at the earlier development stages, in contrast to that at the other growth stages.
The rates of SOM-C and N mineralization were highest at maize flowering, though root-derived C inputs decreased substantially (Fig. 1b and 2b) with a simultaneous decline of soil available N (DN and mineral N contents, Table 2). One mechanistic explanation for the enhanced SOM-C and N mineralization could be that the microbial community accelerates SOM mineralization to mine for N (Craine et al. 2007; Chen, Senbayram & Blagodatsky 2014; Sun et al. 2018). The accelerated gross N mineralization that released additional ammonium further resulted in higher gross nitrification at the flowering (Table 1). Moreover, SOM-C mineralization was accompanied by slight increases in MBC and a significant reduction in DOC contents (for all levels of planting density; Table 2). Hence, the SOM-derived CO2cannot be mostly originated from microbial overflow respiration and accelerated microbial turnover. Besides, microbial communities likely have switched to the consumption of available dissolved C in the soil to maintain their functionality to mineralize SOM regardless of the reduced root-derived C supply (Blagodatskaya et al. 2014). Another explanation could be that the maize at the flowering stage has adapted root morphology, i.e., longer root length per unit root biomass and larger root surface areas on average, which potentially improved microbial mineralization for N mining (see chapter 4.2. Fig 2a, Table 1).
At the maize ripening stage, the relative lower SOM-C and N mineralization are likely due to the biotic and abiotic factors that constrain microbial activity. Root-mediated microbial activation was possibly inhibited because of the very low inputs of root-derived C after maize maturity (Fig. 1b). This is supported by our studies showing that the stimulating effect of roots on the activities of C-, N- and P-acquiring enzymes were lower at maize maturity compared to the earlier stages (the presence vs. the absence of maize; Kumar et al.2018). Due to the depletion of soil available N (Table 2), the intensified plant–microbial competition for N could suppress microbial activity and hence SOM mineralization (Kuzyakov & Xu 2013). Furthermore, abiotic environmental conditions such as cooler temperature, have affected microbial activity and turnover directly (Price & Sowers 2004), but may also have altered plant-microbial interactions through changes in plant activity (e.g., photosynthetic activity, transpiration and nutrient uptake) (Nord & Lynch 2009), with respective feedbacks for microbial processes.
Taken together, SOM-C mineralization positively related to gross N mineralization throughout maize growth (across all planting densities; Fig. 3a), which agrees with previous studies on grassland and forest soils (Dijkstra et al. 2009; Phillips et al. 2011; Bengtson, Barker & Grayston 2012). This indicates that soil C and N cycles are tightly coupled in this arable soil. Furthermore, the C-to-N mineralization ratios of SOM tended to increase with soil DN contents (Fig. 3b), which suggests that the intensity of N-fluxes associated with SOM mineralization was dependent on microbial N availability. When soil mineral N was depleted by plant N uptake (as suggested by a negative relationship between plant biomass and soil mineral N, Fig. S4b), microorganisms likely acted much more on specific N-rich components of the heterogeneous SOM to mine N contained within (Murphy et al., 2015). We, therefore, conclude that microbial N mining hypothesis underlies the coupled turnover of C and N in this arable soil across plant growth stages.
4.2 Rhizosphere effects on soil C and N mineralization depending on intraspecific competition
In light of microbial activation by living roots (Cheng & Kuzyakov 2005; Blagodatskaya et al. 2014; Kumar et al. 2016), one would expect that, with higher plant density, the increased root biomass and on the other hand the rapid exhaustion of the available nutrients by root uptake may stimulate microorganisms to mine for nutrients from SOM. However, planting density did not affect the root-derived CO2 efflux, and the SOM-C and gross N mineralization (when comparing one growth stage; Fig. 1). Also, neither C nor N mineralization showed a clear relation with root biomass and root-derived CO2 efflux (across plant growth stages; Fig. S3). This is inconsistent with earlier studies suggesting that the stimulation of microbial decomposition is largely dependent on the root biomass and their associated rhizodeposits (e.g., Bengtson et al., 2012; Dijkstra et al., 2006; Shahzad et al., 2015; Wang et al., 2015; Yin et al., 2018). We suggest that a possible reason is that plants at higher densities expressed other traits such as root morphology in regulating microbial decomposition as a result of the intensified intraspecific competition.
Competition occurs when plant growth and nutrition are constrained by neighbors as a result of the reduction above- or belowground resources, such as light, water, and nutrients (Aerts 1999; Colom & Baucom 2019). Given the positive relative competition intensity (RCI) of shoots and roots under both double and triple planting densities (Table 1), it is clear that higher planting densities induced the intensive above- and belowground intraspecific competition throughout the growth stages. The competition belowground is more intensive at maize flowering (Table 1), as similarly shown by other maize fields (Li et al. 2019). Root plasticity in morphological traits could be decisive for competitive success for nutrients and water uptake (Kuzyakov & Xu 2013; Ahkami, Allen White, Handakumbura & Jansson 2017; Wen, Li, Shen & Rengel 2017). With increased intraspecific competition at maize flowering, plant altered morphological traits towards thinner and longer root per biomass unit, rather than producing more roots and exudates (as indicated by decreased specific root-derived CO2; Fig. 2a). Such root morphology adjustment is an efficient way to occupy a larger soil volume for exploring temporal and spatial available resources (Li et al. 2019). Accordingly, specific root length tended to increase with belowground intraspecific competition (RCI of roots) with increasing planting densities (Fig. S2b).
Furthermore, root morphologic traits were largely responsible for soil C and N turnover at higher planting densities, since both SOM-C and N mineralization increased with specific root length for the double and triple planting density, expect for single density (Fig. 4). Here, we suggest two possible effects of root morphology in regulating microbial decomposition of SOM. First, the longer root length and larger surface-area-to-volume ratios may largely extend the distribution of rhizodeposits for fueling microorganisms and simultaneously causes an evener nutrient depletion, facilitating microbial nutrient mining. Second, root morphology is a vital driver affecting soil properties such as aggregations (Dorodnikov et al. 2009). The increasing plant densities enhanced the proportion of smaller aggregates size classes (< 2000-250 μm), which favored microbial and enzymatic activities due to the better supply of water and substrates (Kumar et al. 2017).
In addition to intraspecific competition, higher planting density intensified the plant-microbial competition for nutrients and could retard microbial activity for SOM decomposition due to very strong nutrient limitations (nutrient competition hypothesis; Dormaar 1990; Kuzyakov 2002). This could be one mechanistic explanation for the unaffected SOM-C and N mineralization rates at higher planting densities as compared to those at low planting density (Dijkstra et al.2010; Pausch et al. 2013; Yin et al. 2018). However, given the stable microbial biomass and enzyme activities at increasing planting densities, we cannot explicitly confirm the negative effects of nutrient competition on SOM mineralization (Table 2, Kumar et al.2017). This might be attributed to the high initial nutrients contents in arable soils compared to those in natural ecosystems. Therefore, in situ tracer labeling (i.e., 15N) for reliably quantifying the plant-microbial competition for nutrients is needed (Kuzyakov & Xu 2013). Future work also needs to encompass other competitive and mutualistic interactions such as N fixation by rhizobia and mycorrhizal fungi. In summary, plants modify root morphology to obtain a greater capacity to forage nutrients in response to intraspecific competition. This, in turn, affected microbial mineralization for SOM.
In conclusion, our study provides in situ evidence for the predominant mechanisms of rhizosphere effects on soil C and N mineralization in an arable soil under field condition. Root-mediated acceleration of microbial activity and nutrient mining from SOM is a major mechanism driving C and N cycling as indicated by the coupled SOM-C and N mineralization throughout plant growth. The mineralization rates of C relative to N are regulated by microbial N availability and demand. Furthermore, owing to higher intraspecific competition at flowering, maize adjusted its root morphology for competing nutrients by the occupation of new soil volume, and strongly impact on SOM mineralization. Instead of root mass per se , SOM-C and N mineralization under higher planting densities were more related to root morphology (i.e., specific root length) (Fig. 5. Consequently, due to an elevated demand for nutrients under plant-plant and/or plant-microbial competition, root adaptation traits and soil nutrients availability plays an important role in modulating the activity and processes of microbial C and N cycling.