Contribution of glomalin-related soil proteins to soil organic carbon in trifoliate orange

Highlights

• GRSP contribution to SOC in trifoliate orange was studied.

• The C content of EE-GRSP and DE-GRSP was 1.01 ± 0.19 mg g^-1 and 1.70 ± 0.34 mg g.

• C contribution of total GRSPs achieved 23.26 ± 2.67% of total SOC.

• C content of GRSP and C contribution of GRSP to SOC were higher in DE-GRSP than in EE-GRSP.

Abstract

Arbuscular mycorrhizal fungi (AMF) produce glomalin-related soil protein (GRSP) that influences organic carbon (C) storage in soil; however, how much purified GRSP fractions contribute to soil organic carbon (SOC) is yet not known. The present study evaluated the contribution of GRSP towards changes in SOC in trifoliate orange grown in a rootbox divided into a roots + hyphae chamber (roots colonized by AMF hyphae and AMF extraradical hyphae) and a hyphae chamber (only the presence of AMF extraradical hyphae, without roots). Three AMF species (Diversispora epigaea, Paraglomus occultum and Rhizoglomus intraradices) were inoculated into the roots + hyphae chamber. Following four months of plant growth, P. occultum showed higher AMF hyphal growth in roots, soils and nylon mesh than D. epigaea or R. intraradices. Mycorrhizal inoculation improved the plant growth performance and increased easily extractable GRSP (EE-GRSP) and difficultly extractable GRSP (DE-GRSP) concentrations in both chambers, regardless of AMF species. The C content observed in total GRSP of the soil after purification was 2.71 ± 0.49 mg g⁻¹, while purified EE-GRSP and DE-GRSP showed the C content of 1.01 ± 0.19 mg g⁻¹ and 1.70 ± 010.34 mg g⁻¹, respectively. The C contribution by purified EE-GRSP and DE-GRSP accounted for 8.67 ± 0.95% and 14.59 ± 2.21%, respectively, of total SOC, with a total C contribution of purified GRSPs accounting for 23.26 ± 2.67% of total SOC. A significantly higher C content of GRSP and the C contribution of GRSP to SOC were observed in DE-GRSP than in EE-GRSP, as well as the soil of the roots + hyphae chamber than the soil of the hyphae chamber. The proportionate distribution of water-stable aggregate in 2–4 and 1–2 mm sizes and their stability were higher under AMF hyphae than under non-AMF hyphae. This study thus provided a database evidence of increased contribution of GRSP towards build-up of SOC in response to mycorrhizal symbiosis.

Introduction

Arbuscular mycorrhizal fungi (AMF) are one of the most predominant soil microbial communities and can establish symbiotic associations, namely, arbuscular mycorrhizas (AMs), with up to 80% of terrestrial plants (Keymer and Gutjahr, 2018). AMF are known to facilitate several important functions of host plants including improved plant performance, changed rhizosphere microbial diversity, and enhanced ability of plants to tolerate abiotic stress (Park et al., 2016; Jiang et al., 2017; Wu et al., 2019; Zhang et al., 2020). On the other hand, hyphae and spores of AMF secrete a kind of glycoprotein (glomalin), which is deposited into the soil named as glomalin-related soil protein (GRSP) (Wright and Upadhyaya, 1996; Rillig, 2004; Schindler et al., 2007). The GRSPs are broadly divided into three types: easily extractable GRSP (EE-GRSP), difficultly extractable GRSP (DE-GRSP), and total GRSP (T-GRSP) as the sum of EE-GRSP and DE-GRSP (Wu et al., 2014). The carbon (C) stored in GRSP is considered highly recalcitrant in nature, lasting for a minimum of 12–22 years (Zou et al., 2016), and plays an active role in the development of soil structure (Chi and Wu, 2017).

AMF are reported to consume 4–20% of the photosynthates synthesized by host plants. In return, C from mycorrhizas is deposited into the soil via extraradical hyphae as sink for C storage, where mycorrhizal contribution towards organic C accumulated into soil ecosystem involved approximately 54–900 kg hm⁻² (Zhu and Miller, 2003). Collectively, AMF hyphae and AMF-released GRSP are important components of soil organic carbon (SOC) pools (Tian et al., 2009), as evidenced from the positive correlation between GRSP and SOC (Wright et al., 1998; Rillig et al., 2003). GRSP acts as the most important source of C towards SOC build-up (Kumar et al., 2018). The C content of GRSP has been observed to be 2–24 times higher than that of soil humus, accounting for as high as 27% of SOC (Rillig et al., 2000). In the oldest soil (oxidized soil, >4 million years), C in purified T-GRSP accounted for 4% of total C, while the C contribution of T-GRSP was substantially higher than the microbial biomass C (Rillig et al., 2001). In the peat soil, purified T-GRSP accounted for as high as 52% of the total SOC, and a large amount of GRSP gradually transformed to an active soil C source (Rillig et al., 2000). Zhang et al. (2017a) reported that purified T-GRSP extracted from highly saline soils contained relatively high C content (43.41%). However, Wang et al. (2018) observed relatively low C content (17.6%) in T-GRSP from marine sediments in the Older Yellow River delta. These observations display the large variation in the C content of GRSP, and thereby more works need to be explored.

Based on the extraction method, GRSP is a mixture of mycorrhizal and non-mycorrhizal original compounds, including proteinaceous, humic, lipidic, and inorganic materials (Gillespie et al., 2011). To obtain purified GRSP, on the basis of the study by Wright et al. (1998), Rillig et al. (2001) suggested a protocol to purify the extracted T-GRSP. Wang et al. (2014) used 3D fluorescence spectroscopy to analyse purified T-GRSP, which consisted of mixture of substances similar to calcofluor white, fulvic acid, humic acid, nitrobenzoxadiazole, soluble microbial byproduct, tyrosine protein, and tryptophan protein. Most of these studies focused on the role of T-GRSP in soil SOC pool, while the contribution of purified EE-GRSP and DE-GRSP on SOC is still unknown. Earlier studies indicated that EE-GRSP is newly produced by AMF as an active substance to contribute an improvement in soil aggregate stability and soil moisture (Wu et al., 2014; Zou et al., 2014). Conversely, DE-GRSP derives from the turnover of EE-GRSP and is an old glomalin contributing towards soil C pools (Wu et al., 2014; Gao et al., 2020), which suggests that DE-GRSP plays a bigger role in C contribution than EE-GRSP, but the evidence is currently lacking.

The production and contribution of GRSP are affected by number of external factors, including roots, root exudates, and AMF species (Rillig, 2004; Bedini et al., 2009; Zou et al., 2016). Two-chambered rootboxes are widely used to evaluate the role of GRSP in soil aggregate formation and its stability (Wu et al., 2014; Ji et al., 2019). Such rootboxes allow the compartmentalisation of single AM fungal extraradical hyphae, free of roots, which aids in precise analysis on hyphal behaviour (Bedini et al., 2009), including the release and contribution of different GRSP fractions. AMF inoculations induced an increase in GRSP concentrations, dependent upon AMF species (Bedini et al., 2009; Wu et al., 2015). Therefore, more studies on variety of mycorrhizal fungal species would provide better insights in understanding the role of GRSP contribution towards SOC.

Citrus is a globally acclaimed fruit crop, mainly grown in mountainous areas. In China, citrus orchards have soil with SOC of 1–2%, a relatively low content (Wu et al., 2011). The management of SOC in citrus orchards, especially through mycorrhizal GRSP, can provide the guidance for organic citrus cultivation. Trifoliate orange (Poncirus trifoliata L. Raf.) is a popular citrus rootstock used in southeast Asia, and is a mycorrhiza-dependent plant, as reported by Wu et al. (2013). Here, we hypothesized that EE-GRSP and DE-GRSP could contribute to SOC, and the contribution of DE-GRSP is greater than EE-GRSP. To confirm the hypothesis, we used a two-chambered rootbox with a roots + hyphae chamber and a hyphae chamber to analyse and compare the contribution of purified GRSP fractions (EE-GRSP and DE-GRSP) to SOC pools in trifoliate orange colonized by three different AMF species.