Rice rhizodeposition promotes the build-up of organic carbon in soil via fungal necromass

Highlights

• Contribution of rhizodeposits to soil organic C (SOC) under high CO2 is quantified .

• 13C was used to trace components in the plant-soil-microbe continuum .

• 13C-amino sugars were accumulated in mineral SOC fraction at the booting stage .

• Rice rhizodeposits build up SOC via fungal necromass under elevated CO2 .

• Microbial necromass inclusion in climate models helps SOC-sequestration prediction .

Abstract

Rice rhizodeposition plays an important role in carbon sequestration in paddy soils. However, the pathways through which rice rhizodeposits contribute to soil organic C (SOC) formation are poorly understood because of specific paddy soil conditions. Furthermore, microbial necromass has been largely ignored in studies examining the contribution of rhizodeposits to C sequestration during plant growth. To evaluate the contribution of microbial necromass to SOC formation via rhizodeposition, rice (Oryza sativa L.) plants were continuously labeled with ¹³CO₂ for 38 days under ambient (aCO₂, 400 μL L⁻¹) or elevated CO₂ (eCO₂, 800 μL L⁻¹) in a paddy field at two levels of N fertilization. The distributions of photosynthetic-¹³C in the shoots and roots, microbial communities, and SOC fractions were quantified. eCO₂ increased plant growth and, consequently, the total ¹³C incorporated into the shoots, roots, and SOC compared to aCO₂, while N fertilization (100 kg N ha⁻¹) decreased root biomass and rhizodeposits in the soil and microbial pools, including living biomass (phospholipid fatty acids, PLFA) and microbial necromass (amino sugars). Rhizodeposits were initially immobilized mainly by bacteria and preferentially recovered in fungal necromass (glucosamine). While ¹³C incorporation into PLFAs was slightly increased during plant growth, ¹³C in microbial necromass increased greatly between the tillering and booting stages. Fungal necromass, which is less decomposable compared to bacterial residues, was the largest contributor to C sequestration with rhizodeposits via the mineral-associated SOC fraction, particularly under elevated CO₂ without N fertilization. This study reveals the significance of the C pathways from rhizodeposits through fungal necromass and organo-mineral associations for the build up of SOC in paddy fields.

Introduction

Plant root-derived carbon (C) dynamics in the rhizosphere are critical for assessing soil C sequestration and mitigating the greenhouse effect (Lehmann and Kleber, 2015; Zhu et al., 2018). In general, C is transferred from the atmosphere to the soil in the form of water-soluble sugars and organic acids, as well as large-molecular-weight compounds, such as mucilage and sloughed root cells, through plant photosynthesis and rhizodeposition (Kuzyakov and Schneckenberger, 2004; Jones et al., 2009). However, the input of C into soil through rhizodeposition is difficult to observe, with previous measurements focusing on the C pool size, which remains comparatively stable (Paterson et al., 2009). On the other hand, C isotope labeling techniques allow for the tracing of the photosynthesized-C flows and fate in the soil-plant-microbe continuum (Jones et al., 2009; Jeewani et al., 2020; Radajewski et al., 2000). Approximately 20–35% of the photoassimilates are allocated below ground, 10–16% are incorporated into roots, 3–5% are released as rhizodeposits, and 8–12% are lost as root and microbial respiration (Pausch and Kuzyakov, 2018). Variations in the quantity of photosynthesized-C flows depend on plant species, growth stages, soil physico-chemical properties, and nutrient availability, as well as the size, activity, and composition of microbial communities (Ge et al., 2012; Liu et al., 2019).

In contrast to soil microbial biomass, which is small, the C fluxes through this pool are large (Jones et al., 2009). Understanding the microbial community and utilization of rhizodeposits in soil is crucial to accurately estimate the dynamics and sequestration of photosynthesized C. Root exudates shape the assembly of the rhizosphere bacterial community through substrate preferences, which in turn drives the decomposition of root-derived C cycling (Zhalnina et al., 2018). In addition, saprotrophic and mycorrhizal fungi are the main microbial groups that metabolize organic substrates released from the roots (Hannula et al., 2012). While dead microbial biomass (i.e., necromass) has been identified as a significant source of soil organic matter (Miltner et al., 2012), microbial pathways via necromass have been largely ignored and remain poorly reflected in current models of SOC sequestration (Wieder et al., 2013; Liang et al., 2017). However, recent studies have revealed that the role of microbial necromass in SOC stabilization is far greater than previously believed. It is estimated that living microbial biomass contributes to 2% of the SOC, while microbial necromass accounts for up to 80%, mainly because of the recalcitrance of cell wall materials (Liang and Balser, 2011) and allocation in micropores smaller than 150 μm (Kravchenko et al., 2019), resulting in a long residence time in soil. The inclusion of microbial necromass in SOC sequestration research and the understanding of the driving factors, such as land use and fertilization, and the edaphic and environmental variables that determine necromass accumulation, will improve predictions of soil C storage in plant-soil systems (Liang et al., 2017).

Numerous laboratory incubation and modeling studies have demonstrated that land use, climatic variables, plant characteristics (biomass, diversity, and growth stage), substrate chemical composition, soil types and properties (mineral content, C:N ratio), and soil management (e.g., fertilization) determine the contribution of microbial residue compounds to stabilized SOM (Zhu et al., 2016, 2017a; Cui et al., 2020; Liu et al., 2020; Li et al., 2021, Xiao et al., 2021). A recent review estimated the contribution of microbial necromass to agricultural (56%), forest (30%), and grassland (62%) soils by collecting data published between 1996 and 2018 (Liang et al., 2019). Similarly, Ni et al. (2020) found that the amino sugar content in soils, which reflects microbial necromass, from 131 forest, grassland, and cropland sites declined with depth (litter layer, O horizon soil, and mineral soil up to >100 cm) on a global scale. Angst et al. reported a larger contribution of microbial necromass to SOC in agro-ecosystems (up to 50%) compared to forests, mainly via aggregates and mineral associations. Apart from mineral-organic sorption, organic-organic interactions contribute to the abiotic adsorption of necromass. For example, yeast necromass promotes bacterial necromass accumulation (Buckeridge et al., 2020). In addition, soil oligotrophic or copiotrophic conditions also affect the microbial utilization of rhizodeposits and, subsequently, microbial residue production. For example, mollisol with larger C stocks allows for the maintenance of larger necromass production rates compared to C-poor Ultisol (Wang et al., 2020). In addition, perennial plant diversity also determines the accumulation efficiency of microbial necromass (Zhu et al., 2020; Kravchenko et al., 2019). However, shifts during plant growth have rarely been investigated. A recent study revealed that climate and plant productivity are the main drivers of necromass accumulation at regional scales (Zhang et al., 2021). Thus, theoretical frameworks that address the accumulation of microbial necromass affected by climate variables (i.e., increasing temperature and elevated CO₂) and crop management during plant growth are needed.

Indeed, numerous studies have reported on rhizodeposition-driven C sequestration under climate change and agricultural management. Generally, soil C sequestration can benefit from elevated CO₂ (eCO₂) conditions (Haase et al., 2007; Kuzyakov et al., 2019). Increasing atmospheric CO₂ concentration increases plant rhizodeposits, as it stimulates net primary production and shoot and root biomass, and increases the root/shoot ratio and rhizodeposition (Pendall et al., 2004). For example, a study found that 5 years of elevated CO₂ resulted in higher root biomass (123%) compared with ambient CO₂ levels, resulting in doubled rhizodeposition (83 vs. 35 g C m² year⁻¹) (Pendall et al., 2004). In addition to directly increasing C input, the major consequences on C cycling by high CO₂ occur through effects on soil microbial communities. Arbuscular mycorrhizal fungi (AMF), which act as a major conduit in the transfer of C from roots to soil, are upregulated by eCO₂ (Drigo et al., 2010). The dominant microbial groups in the rhizosphere are determined by the plant growth stage due to changes in the quantity and quality of rhizodeposits. This includes the succession of opportunistic microorganisms (such as bacteria) that specialize in the decomposition of readily available resources to slow-growing but efficient microorganisms (such as fungi) that feed on polymerized substances (de Graaff et al., 2010).

Nitrogen (N) availability is another factor that determines the quantity, quality, and accumulation of rhizodeposits in soil (Bowsher et al., 2017; Xiao et al., 2019a, 2019b). For instance, N fertilization increased C stabilization in N-deficient soil via increased rice rhizodeposits (Hill et al., 2007) and shifted microbial communities (Ge et al., 2016; Luo et al., 2018). Nitrogen addition is frequently toxic to fungi, whereby a high supply of N favors bacterial PLFA assimilation of root C over fungal growth. In addition, changes in soil C:N ratios by mineral fertilizers assemble the microbiome via stoichiometric control. Additionally, N shifts the composition (and availability) of rhizodeposited C by affecting plant growth, thus modulating the composition of microbial community and the life strategies in the rhizosphere (Fu et al., 2021). Previous studies have investigated the combined effects of CO₂ and N on the distribution of rhizodeposited C in soil pools (Haase et al., 2007; Hu et al., 2017; Xu et al., 2018; Li et al., 2020). As a result, the observed differences in rhizodeposition fate and C sequestration can be attributed to: (i) the amount of rhizodeposition depending on plant type and growth stage; (ii) changes in the structure of the microbial community. The manner by which rhizodeposits, especially in arable crops, alter the microbial biomass, activity, community, and functions during plant growth is important for deciphering the mechanisms underlying changes in SOC under environmental factors, such as eCO₂ and N.

Few studies have considered the contribution of both living and dead microorganisms to C sequestration via rhizodeposits during plant growth. Thus, the contribution of rhizodeposits to the SOC pool via microbial necromass has rarely been estimated (Peixoto et al., 2020). Tracing and quantifying the photosynthetic-C flow through the plant‒soil-microbe continuum, including both microbial proliferation and mortality, will improve our understanding of the mechanisms underlying rhizodeposit-C sequestration in soil. The development of biomarkers able to distinguish C sources and separate living microorganisms from microbial necromasses remains a significant challenge (Liang et al., 2019). One possibility is phospholipid fatty acid analysis in combination with stable isotope probing, which can be used to trace plant-derived C flows in living microbial communities (Treonis et al., 2004). The accumulation of amino sugars in soil is a good indicator of the contribution of dead microorganisms, considering that plants do not produce amino sugars (Glaser et al., 2004). Although more than 20 amino sugars have been identified in microorganisms, glucosamine (GlcN), galactosamine (GalN), and muramic acid (MurN) are the most commonly used biomarkers. Glucosamine in soil is mainly derived from the chitins of fungal cell walls, whereas MurN exclusively originates from bacterial cell walls, providing indications of the fungal (GlcN) and bacterial quantities (MurN), respectively (Joergensen, 2018; Liang et al., 2020).

Considering the large C sequestration potential of paddy soils, it is crucial to understand the contribution and path of rhizodeposits to stable C pools (Zhu et al., 2017b; Atere et al., 2020; Wei et al., 2021). In this study, we traced photosynthate flow within rice plants and belowground, with a special focus on pools of microbial necromass in paddy soils. Rice (Oryza sativa L.) received 0 or 100 mg N kg⁻¹ soil and was grown continuously in ¹³CO₂ atmosphere for 38 days under ambient (400 μL L⁻¹) or elevated CO₂ concentrations (800 μL L⁻¹). Stable isotope probing was combined with PLFA biomarkers and amino sugars, including GlcN and MurN, to determine the contribution of living and dead microbial biomass to SOC accumulation in pools of mineral-associated organic matter (MAOM) and particulate organic matter (POM) (Koyama et al., 2018). We hypothesized that: (i) compared to bacteria, a more stable fungal necromass would contribute more to SOC because of its recalcitrance, especially in the late booting stage, because of the slower adaption and growth of fungal hyphae during plant development; (ii) both elevated CO₂ and N fertilization would increase the total plant biomass and belowground C allocation, with the subsequent incorporation of rhizodeposits into SOC fractions, particularly the mineral-associated C pool.