The glomalin-related soil protein content as influenced by crop rotation (spring barley monoculture/Norfolk crop rotation), straw application, and tillage

Long-term field experiments were established in 1969 at the gleyic fluvisol to assess the effects of soil management on soil organic matter and glomalin-related soil proteins. Two systems were studied: Hordeum vulgare L. monoculture (spring barley monoculture, SBM) and Norfolk crop rotation (NCR) consisting of the following crops: Trifolium or Medicago spp. (clover/alfalfa), Triticum aestivum L. (winter wheat), Zea mays L. (maize), and Hordeum vulgare L. (spring barley). Experimental factors included: i) tillage-ploughing (PL, 22 cm) vs. chiselling (CC, 12–14 cm); ii) straw management-straw removed (SR) vs. straw incorporated (SI; only in SBM); iii) soil sampling depths-0–10 cm, 10–20 cm, and 20–30 cm. Treatments were arranged as split-strip plots with four replications. Chisel cultivation enhanced total glomalin content and increased carbon in soil organic matter (C_SOM) content compared to ploughing, particularly in upper soil layers (0–20 cm) in the SBM. Straw incorporation promoted C_SOM accumulation more effectively under ploughing than under chisel cultivation but had no measurable effect on the easily extractable glomalin-related soil protein content. The Norfolk crop rotation system significantly improved both C_SOM levels and the glomalin-related soil protein content. These results highlight that adopting conservation-oriented soil management can improve soil structure and carbon retention, providing practical guidance for farming systems aiming to maintain long-term soil fertility

1 Introduction

Fertilisation and tillage practices significantly influence carbon sequestration in arable soils (1, 2). Compared to conventional tillage, no-tillage systems promote carbon in soil organic matter (C_SOM) accumulation in the topsoil (0–20 cm) (3) through the physical protection of macroaggregates (4) and the association of soil organic matter (SOM) with the mineral fraction (5).

A key factor influencing SOM is a group of glomalin-related soil proteins (GRSP) (6, 7). The GRSP promote long-term carbon sequestration primarily through strong physicochemical interactions with soil minerals. GRSP contains abundant carboxyl, hydroxyl, and hydrophobic functional groups that bind to Fe/Al oxides and clay surfaces, forming highly stable organo-mineral complexes that protect carbon from microbial decomposition (6–8). Its hydrophobic, thermally stable, and glycated molecular structure confers exceptional resistance to enzymatic degradation, making GRSP more persistent in soil than most microbial or plant-derived proteins (9, 10). In addition, GRSP acts as a key binding agent in soil aggregation, enhancing both microaggregate formation and macroaggregate stability, thereby physically occluding organic matter within aggregates and reducing its accessibility to decomposers (7, 11). By strengthening aggregate structure, GRSP indirectly promotes the accumulation and preservation of microbial necromass, which forms the dominant fraction of mineral-associated organic carbon (12). The continuous production of GRSP along arbuscular mycorrhizal hyphae further enhances spatial enmeshment of soil particles, contributing to both aggregate formation and the protection of soil carbon within hyphal networks (13).

The AMF are an important component of soil biota, playing a central role in supporting sustainable crop production and serving as indicators of soil health (14, 15). The AMF form symbiotic relationships with approximately 72% of all terrestrial plant species, receiving reduced organic compounds from the host in exchange for soil-derived nutrients and water (16, 17). According to Thomapoulos et al. (17), the AMF play a significant role in soil aggregate stability primarily through their hyphal networks, as evidenced by a strong correlation between phospholipid fatty acid (PLFA) biomarkers and water-stable aggregates (WSA). In contrast, the role of AMF in pore structure formation appears to be more closely linked to GRSP production than to the hyphal network itself.

The tillage disrupts fungal hyphae, which inhibits GRSP production. Although ploughing generally increases nutrient mobility and can stimulate hyphal growth, the physical disruption of hyphae has a dominant negative effect on GRSP production. Agnihotri et al. (18) found that a no-tillage approach significantly increased AMF biomass.

Compared to conventional tillage, no-tillage also increases AMF diversity, which has been positively correlated with GRSP abundance (19). Overall, the positive effect of no-tillage on GRSP accumulation appears to result primarily from the diversity and abundance of AMF species rather than from total AMF biomass.

AMF species vary greatly in functional traits, including colonisation strategies, organic compound uptake from plants, and nutrient utilisation (20, 21). It can therefore be concluded that a more diverse AMF community offers greater stability in the ecosystem. The results of Brito et al. (22), and Säle et al. (23) demonstrate a decline in AMF species diversity under conventional tillage compared to reduced tillage or no-tillage systems. On the other hand, in the experiments by Hu et al. (24), where maize and wheat were alternated, no negative effects of conventional tillage were observed.

It has been established that different AMF species have varying tolerance to soil structure disturbance. For example, representatives of the Scutellospora genus are highly sensitive to tillage, while representatives of the Glomus genus are commonly found in soils where ploughing is practised (25). Furthermore, the impact of soil compaction on AMF communities has been demonstrated due to changes in water content and oxygen availability (26). Therefore, deep ploughing can sometimes reduce compaction in the topsoil and provide better conditions for AMF growth, as well as alter the AMF community. Supporting host plant growth allows for better organic compound supply to the fungi, which can influence the AMF community (27).

It has been shown that autumn tillage can negatively affect the viability of hyphae for the subsequent spring crop (28). Since AMF are biotrophic, their viability gradually declines in the absence of host plants, such as during fallow periods, even in no-tillage systems. The introduction of mycotrophic cover crops and their sowing immediately after harvest of the previous crop (autumn tillage) can benefit the AMF development, even in ploughed systems. Therefore, crop rotations that include winter crops are more favourable for GRSP production than growing only spring crops. This effect can be further enhanced when soil tillage is performed in the autumn prior to the spring crop (28). McGonigle et al. (29) report that as the number of tillage operations increases, the amount of AMF hyphae decreases.

Several studies indicate that GRSP is stable and degrades slowly in the soil (30–33). Evidence for this is based on observations that when land use changes, such as the conversion of pastures to arable land, SOM decreases more rapidly than GRSP. The slower decline of GRSP leads to a relative increase in the GRSP/C_SOM ratio.

Other studies report a higher proportion of GRSP in C_SOM in soils with low C_SOM content due to their management, such as vineyard soils and intensively cultivated cereal fields (34–36). Wang et al. (37) found a decrease in GRSP content with increasing soil depth. However, the SOM content decreased more rapidly with depth (37), leading to an increased proportion of GRSP in C_SOM.

Some studies suggest that soil management practices may not always be the dominant factor influencing microorganism diversity and SOM levels (38). For example, crop rotation (39) can significantly limit the impact of tillage technology (40). As reported by Angers et al. (41), there was a higher C and N content in the 0–10 cm layer under no-tillage, but higher levels were found at depths >20 cm under ploughing. Although AMF do not have saprophytic capacity, some studies have shown that AMF can directly benefit from organic matter in the soil (42, 43). Yang et al. (44) report that the application of manure increased AMF growth and also spore production.

Alguacil et al. (45) emphasise that the application of straw combined with deep ploughing significantly affected the AMF community compared to ploughing alone. Herman et al. (46) report that applied organic matter influences AMF growth and community changes primarily by altering nutrient availability, as well as by changing the soil microbial community.

In our long-term maize monoculture experiments, a positive effect of incorporating wheat straw (5 t ha^-¹) combined with mineral N fertilisation on C_SOM content was observed (47). In long-term trials (22 years) on luvisols with a simple crop rotation (maize, winter wheat, spring barley), a positive effect of straw application (5 t ha^-¹ year^-¹) with N fertilisation on C_SOM content, humic acids, the ratio of humic acids to fulvic acids, and the content of EE-GRSP and T-GRSP was determined (48). The positive effect of straw application on GRSP content is also reported by Nie et al. (49) and Liang et al. (50). In our long-term experiments (27 years) with a simple crop rotation (potatoes, winter wheat, spring barley) on cambisols, a positive effect of the N + straw combination on C_SOM content, humic acids, EE-GRSP, and T-GRSP was observed compared to the unfertilized control variant. However, no significant differences were found between the N and N + straw variants (51). Similarly, in soil monitoring in the Czech Republic, no effect of straw application on C_SOM and GRSP content was observed (52). The presented findings clearly show that conclusions on the effect of straw application on GRSP content in soil are inconsistent or contradictory.

It is evident that carbon sequestration in arable soils is significantly influenced by: a) the crop species used in the rotation; b) the intensity of fertilisation, particularly the use of high-quality organic fertilisers (e.g. farmyard manure); c) the soil cultivation method (conventional tillage vs. no-tillage). This study aimed to determine: i) the effect of different soil tillage methods (ploughing vs. chiselling) on the content of carbon in soil organic matter (C_SOM) and glomalin-related soil proteins (EE-GRSP, T-GRSP); ii) the effect of long-term straw application on C_SOM and GRSP content (EE-GRSP, T-GRSP); iii) the effect of the Norfolk crop rotation (NCR) system compared to spring barley monoculture (SBM) on the content of C_SOM and GRSP (EE-GRSP, T-GRSP).

2 Materials and methods

2.1 Site and long-term field characteristics

The long-term field experiments were established in the autumn of 1969 in the Field Experimental Station of Mendel University in Žabčice. The site Žabčice is located in the South Moravian region. The first field experiment is focused on spring barley monoculture (SBM) (Hordeum vulgare L.) due to the tradition of growing and breeding in the Moravian region. A second experiment with Norfolk crop rotation (NCR) included four crops: clover or alfalfa (Trifolium or Medicago), winter wheat (Triticum aestivum L.), maize (Zea mays L.) for grain with 25 t ha^-1 of farmyard manure since 2002 - sugar beet (Beta vulgaris L.) in earlier years -, and spring barley (Hordeum vulgare L.). The initial crop was clover in 1972. Other crops followed as per Norfolk crop rotation convention. The NCR experiment was established on the same site as SBM in year 1972 (53). Both long-term experiments were conducted in parallel. As the experiments with spring barley monoculture and Norfolk crop rotation were going on, the cultivars were changing according to the demand of breweries and nowadays, only cv. Bojos is grown. The experimental factors described in Table 1 included in long-term field trials were: i) two soil management methods, ploughing (PL, 22 cm depth) and chiselling (CC, 12–14 cm depth); ii) two straw management methods, straw removed (SR), and straw chopping followed by incorporation (SI, only in monoculture); iii) three depths of soil sampling, 0–10 cm (D_0-10), 10–20 cm (D_10-20), and 20–30 cm (D_20-30). Stubble breaking was applied after the barley harvest (in July) with a disc cultivator in both SR and SI. Later, as a primary soil management methods, the soil was cultivated either by chisel cultivator (in CC treatments) or by ploughing (in PL treatments) in mid-October. The same process was also applied in NCR. The experiments are conducted as a small-plot stationary trial, arranged as a combination of split-strip plots (soil tillage strips) (54). The treatments are repeated 4 times in the experiment. For spring barley, nitrogen fertilisation was implemented at a dose of 60 kg N ha^-1 in ammonium sulphate before sowing. Phosphorus and potassium were applied in a dose of 39.6 kg P ha^-1 and 99.6 kg K ha^-1 (triple superphosphate, and potassium salt, respectively) for all fields in the autumn.

2.2 Soil properties

The studied soil was classified as Gleyic Fluvisol, heavily textured, with medium organic carbon content. The soil type was classified according to the Němeček et al. (55) and the IUSS Working Group WRB (56). The soil is heavily textured and weakly acidic, with high cation exchange capacity and medium C_SOM content. Basic site and soil properties are documented in Table 1, the experimental factors are described in Table 2.

Table 1

Table 1. Characteristics of experimental fields.

Table 2

Table 2. The experimental factors influencing the soil organic matter content and quality.

Table 3

Table 3. Significance of experimental factors and their interactions over the C_SOM content and SOM quality, spring barley monoculture (SBM).

2.3 Sampling and sample preparation

Five samples were collected from each plot and pooled. Soil samples were collected from D_0-10, D_10-20, and D_20–30 after the harvest of spring barley. For SBM, sampling was always conducted on the same plot. For NCR, the sampling was conducted on the same plot in the years 2017 and 2021. A different plot was sampled in the years 2019 and 2023 due to the influence of crop rotation. The soil sampling was conducted after the harvest of spring barley, that is when the crop rotation was over. This led to the shift of harvested plots. Collected soil samples were air-dried at 40°C until a constant weight was achieved. Dried soil samples were sieved through a mesh with 2 mm size holes for chemical analysis. Part of the 2 mm fine earth was further sieved through a mesh with 0.4 mm size holes for the CN analysis.

The harvested area of each plot was 9.84 m² (4.8 by 2.05 m). The harvest of ripe spring barley was carried out using a small plot combine harvester. The grain yield was calculated in tonnes per hectare from grain harvested at 14% moisture.

2.4 Chemical analysis

Carbon in soil organic matter (C_SOM), total soil nitrogen (N_T) content was determined via oxidation using the CN Analyser Elementar Vario Macro (Elementar Analysensysteme, Hanau, near Frankfurt am Main, Germany) (57) after the removal of carbonates from samples by HCl. This also allowed for the calculation of the C_SOM/N_T ratio.

The easily extractable glomalin-related soil protein (EE-GRSP) and total glomalin-related soil protein (T-GRSP) were analysed following the method described by Wright et Upadhyaya (6). Briefly, 1.00 g of air-dried soil (< 2 mm) was mixed with 8 mL of sodium citrate solution (20 mmol L^-¹ at pH 7.0 for EE-GRSP and 50 mmol L^-¹ at pH 8.0 for T-GRSP). The mixture was autoclaved at 121°C for 30 minutes for EE-GRSP and 60 minutes for T-GRSP, then allowed to cool and centrifuged at 5000 rpm for 10 minutes for EE-GRSP and 15 minutes for T-GRSP. For T-GRSP, the extraction was repeated five times until the characteristic red-brown colour, typical of glomalin, was no longer visible. Both EE-GRSP and T-GRSP were quantified colorimetrically using bovine serum albumin (BSA) as the standard and the Bradford protein assay (both provided by Bio-Rad, CA, Hercules, USA) to monitor the colour change. The glomalin content in the extracts was measured using the Tecan Infinite M Plex multimode microplate reader (Männedorf, Switzerland) at 595 nm. This allowed the calculation of the EE-GRSP/T-GRSP, EE-GRSP/C_SOM and T-GRSP/C_SOM ratios. Each sample for each analysis was analysed in two replicates.

2.5 Statistical analysis

The normality of all variables was tested using the Shapiro-Wilk test (P < 0.05), and no violations were detected. To evaluate the effects of experimental factors on soil organic matter indicators, we applied analysis of variance (ANOVA) with interactions. In this model, the categorical explanatory variables were soil management system (ploughing vs. chiselling), straw management (straw removed vs. straw incorporated; the latter only in SBM), soil sampling depth (0-10, 10-20, 20–30 cm), and sampling year. The dependent variables analysed in all models were C_SOM, N_T, EE-GRSP, and T-GRSP (Tables 3, 4). Interaction terms included year × soil management system and year × soil management system × straw management, as reported in the significance tables. To further examine individual factor effects, each categorical variable was additionally analysed using one-way ANOVA, where the same dependent variables (C_SOM, N_T, EE-GRSP, T-GRSP) were tested separately against single explanatory factors (soil management, straw management, depth; Tables 5–7). Post hoc comparisons were performed using Tukey’s HSD test (P < 0.05). Pearson correlation coefficients were calculated to characterise the relationships among SOM content and quality indicators at significance levels P < 0.05; P < 0.01; P < 0.001. All statistical analyses were conducted in TIBCO Statistica 12.3. (TIBCO Software Inc., Palo Alto, CA, USA),

Table 4

Table 4. Significance of experimental factors and their interactions over the C_SOM content and quality, Norfolk crop rotation (NCR).

Table 5

Table 5. The influence of soil management system (CC/PL) and depth on the C_SOM content and GRSP in the straw removed (SR) system in spring barley monoculture (SBM).

Table 6

Table 6. The influence of soil management system (PL/CC) on the C_SOM content and GRSP in the straw incorporation (SI) system in spring barley monoculture (SBM).

Table 7

Table 7. The influence of straw incorporation (SI) on the C_SOM content and quality in the spring barley monoculture (SBM).

3 Results

3.1 Spring barley monoculture

The grain of spring barley yields during the monitored period (2017-2023) are presented in Table 8 as calculated in Prudil et al. (53). The results show no statistically significant differences between the soil management systems (PL and CC) on the grain yields. A similar result was observed in the NCR system. The yields correspond to long-term average values for this region of Moravia under fertilisation intensity (60 kg N ha^-¹). It should be noted that yield data are not the focus of this study and are provided solely to characterise the long-term experiment. This study focuses primarily on monitoring changes in C_SOM and GRSP (EE-GRSP, T-GRSP) contents.

Table 8

Table 8. Spring barley grain yields, average for 2017–2023 period (t ha^-1).

The results in Table 3 show temporal changes only for EE-GRSP. Furthermore, significant interactions of year x management system, and year x soil management system x straw management were observed for EE-GRSP, suggesting that short-term responses of this GRSP fraction may be influenced by both management and interannual variability. The results confirm that EE-GRSP is the more labile component of GRSP content, and its changes are influenced more by the conditions of the individual year. The T-GRSP content was significantly influenced by the soil cultivation system, depth, and straw application, while no significant influence of sampling year was present. A similar pattern was found for C_SOM and N_T content. While noteworthy, a detailed interpretation of the interactions exceeds the intended scope of this study. To allow for a more objective evaluation, a combined dataset was created regardless of the year of sampling. More information regarding the results of individual factors on the EE-GRSP and T-GRSP content is presented in Tables 5, 6.

3.1.1 The influence of the soil management system (PL/CC) in spring barley monoculture

The results for both observed systems (PL/CC) in combination with the straw removed (SR) system are presented in Table 5. A positive effect of CC compared to PL on C_SOM content was observed across all monitored soil depth profiles. A similar pattern was found for N_T content. However, the soil management practices did not affect the C_SOM/N_T ratio. The content of EE-GRSP was not significantly affected by the soil management system. However, T-GRSP content was significantly higher under CC in the D_0–10 and D_10–20 profiles compared to PL. The lowest T-GRSP values for both systems were recorded in the D_20–30 profile. The results suggest a tendency towards higher proportions of EE-GRSP and T-GRSP in C_SOM under the PL system across all observed depth profiles. The EE-GRSP/C_SOM ratio was significantly affected by the soil management system only in the D_0–10 profile (9.36% in PL and 8.61% in CC). Similar significant effect was observed also for the T-GRSP/C_SOM ratio (PL – 37.4%; CC – 35.5%).

The total contents of C_SOM and GRSP in the entire topsoil profile are presented in Figure 1. For the SR combination, bulk density was measured during the monitoring years. In the PL system, the total C_SOM content was 50.35 t ha^-¹, while in the CC system it reached 57.89 t ha^-¹ across the entire topsoil profile. The CC system contributed to carbon sequestration in the topsoil, resulting in a 14.9% increase. In contrast, the increases in GRSP were relatively lower-8.8% for EE-GRSP and 10.9% for T-GRSP.

Figure 1

Figure 1. The carbon in soil organic matter (C_SOM) and glomalin-related soil protein (GRSP) content in the spring barley monoculture (SBM) topsoil as influenced by soil cultivation, straw removed (SR) treatments.

3.1.2 The influence of soil management system (PL/CC) in combination with straw incorporation in spring barley monoculture

The effect of soil management system (PL/CC) on the content and quality of SOM in combination with straw incorporation (SI) is presented in Table 6. Under the CC system, the C_SOM content is significantly higher in the D_0–10 and D_10–20 layers compared to PL. In the CC system, the C_SOM content is significantly lower in the D_20–30 profile compared to the D_0–10 and D_10–20 layers. In contrast, no difference was observed between the D_10–20 and D_20–30 layers under the PL system. Overall, the C_SOM content was higher under the CC system. Similar differences between the observed systems were also found for the N_T content. The C_SOM/N_T ratio was not affected by the soil management system (PL/CC) or by soil depth. The EE-GRSP content was not affected by the soil management system or soil depth. In contrast, the T-GRSP content was significantly higher under the CC system compared to the PL system in the D_0–10 profile. Furthermore, the T-GRSP content was significantly higher under the CC system compared to the PL system in the D_20–30 profile.

3.1.3 The influence of straw application (SI/SR) in spring barley monoculture

To compare the effects of straw incorporation (SI) and straw removal (SR), we used average contents of the observed parameters across the entire topsoil profile (Table 7). This approach was adopted due to the lack of bulk density data for the SI + CC/PL combinations, making it impossible to calculate values as in Figure 2. Although this evaluation is subject to some inaccuracy, it provides useful information and enables a comprehensive comparison between SI and SR. Straw incorporation contributed to an increase in C_SOM content, with a greater effect under the PL system (increase of 9.1%) than under CC (increase of 6.3%). A similar trend was observed for N_T content (increases of 9.2% and 4.3% for PL and CC, respectively). The smaller increase in N_T compared to C_SOM was likely due to the high C/N ratio of straw (79.1). Nevertheless, the C_SOM/N_T ratio remained nearly unchanged following straw application, indicating the strong stability of this parameter. Furthermore, the results indicate that the EE-GRSP content was not affected by SI in either soil management system (PL or CC). There was a tendency toward increased T-GRSP under SI, with a stronger effect in combination with CC than with PL (increases of 7.6% and 4.0%, respectively).

Figure 2

Figure 2. SR – straw removed; SI – straw incorporated. Ploughing and chiselling results were pooled. Different letters describe significant differences between soil management systems (Tukey's HSD test, P < 0.05, n = 96).

In order to investigate the overall effect of straw application in the SBM, data from chiselling and ploughing were pooled. Straw application significantly influenced the content of C_SOM and T-GRSP (p < 0.05) (Figure 3). The C_SOM content was significantly increased in the SI (1.36%) in comparison with the SR (1.27%). The T-GRSP content was likewise increased in the SI (4972 mg kg-1) in comparison with the SR (4699 mg kg-1).

Figure 3

Figure 3. PL – ploughing; CC – chisel cultivation. * the numbers underneath the horizontal axis represent the ratio of chisel cultivation and ploughing (CC/PL) (%). The values are calculated from the entire topsoil profile (0-30 cm). Only the treatments with straw removed were used for the calculation of this table.

3.2 Norfolk crop rotation

The influence of the sampling year is presented in Table 4. Similarly to the SBM, EE-GRSP content in the NCR was significantly affected by the sampling year. The T-GRSP content was unaffected. The T-GRSP content was significantly influenced by the sampling depth and an interaction between the soil management system and sampling depth. The same interaction was also statistically significant for C_SOM and N_T content, suggesting that the effects of soil management on SOM properties may vary with soil depth. More information related to the influence of individual factors is presented in Table 9.

Table 9

Table 9. The influence of soil management system and depth on the C_SOM and GRSP content in the straw removed (SR) system in Norfolk crop rotation (NCR).

In the NCR system, only the SR + PL/CC combinations were monitored over the course of the experiment (Table 9). Unlike in SBM, PL here contributed to an increase in C_SOM content, with statistically significant increases observed in the D_10-20 (1.72%) and D_20-30 (1.67%) profiles in comparison to the CC (1.62% and 1.41%, respectively). No significant differences in N_T content or the C_SOM/N_T ratio were found between the soil management systems (PL/CC). The effect of NCR was dominant and outweighed the influence of soil management system on GRSP content (EE-GRSP and T-GRSP), as well as on the EE-GRSP/C_SOM and T-GRSP/C_SOM ratios across the evaluated topsoil profiles (Table 9).

The measured contents in the topsoil profiles were recalculated to express the total amount of C_SOM, EE-GRSP, and T-GRSP per hectare of topsoil (Figure 2). Based on the relative CC/PL ratios, it is evident that the PL system had a positive effect on carbon sequestration. In the CC system, the topsoil contained 64.5 t C ha^-¹, representing 90.5% of the value observed under the PL system. Very similar proportions were found for EE-GRSP and T-GRSP, reaching 93.0% and 92.2%, respectively.

3.3 The influence of spring barley monoculture and Norfolk crop rotation on the carbon in soil organic matter content and glomalin-related soil protein content

The evaluation of results obtained from SBM and NCR confirms a positive impact of NCR on carbon sequestration in topsoil and GRSP content (Figure 4). In the PL system under SBM, the C_SOM content in the topsoil was 50.35 t ha^-¹, representing only 70.6% of the C_SOM content under NCR (71.3 t ha^-¹). In the case of CC, the difference was partially reduced (89.7%), due to the stronger effect of CC under SBM (57.9 t ha^-¹) and a weaker effect in NCR (64.5 t ha^-¹). The use of PL in NCR significantly increased the content of EE-GRSP (by 6.0%) and T-GRSP (by 16.8%) in comparison with the SBM. Inclusion of clover (average hay yield 7.62 t ha^-¹) and alfalfa (average hay yield 11–5 t ha^-¹) in NCR had a positive effect on C_SOM and GRSP. This is further supported by the positive effect of farmyard manure application (25 t ha^-¹ once every four years) to maize (previously to sugar beet).

Figure 4

Figure 4. SBM – spring barley monoculture; NCR – Norfolk crop rotation; PL – ploughing; CC – chisel cultivation. NCR = 100%. The numbers underneath the horizontal axis represent total content of carbon in soil organic matter (C_SOM), easily extractable glomalin-related soil protein (EE-GRSP), and total glomalin-related soil protein (T-GRSP) in the 30 cm death of the soil per hectare.

3.4 The overall influence of the chiselling and ploughing over the soil organic matter content and quality

In order to evaluate the overall influence of the soil management system, the data from SBM and NCR were pooled (Figure 5). No significant differences were observed for the C_SOM and EE-GRSP content. On the other hand, the T-GRSP content increased significantly in the CC (5077 mg kg^-1) in comparison to the PL (4878 mg kg^-1) (p < 0.05). Because SBM and NCR data were pooled, the observed differences reflect the overall effect of soil management irrespective of crop sequence. The significance of such small difference can be attributed to the large number of observations (n = 144).

Figure 5

Figure 5. PL – ploughing; CC – chiselling. Monoculture and Norfolk crop rotation results were pooled. Different letters describe significant differences between soil management systems (Tukey's HSD test, P < 0.05, n = 144).

4 Discussion

Long-term stationary experiments provide essential information about the potential for carbon sequestration in agricultural soils, as well as changes in SOM quality. The long-term SBM trials are a rare case on both the European and global scale. They were originally established in response to the fact that, during the 1960s, the South Moravian region (Czech Republic) was a dominant producer of spring barley used to obtain high-quality malt for beer production. In crop rotations, barley was grown repeatedly, and there was a practical need to determine the maximum feasible proportion of barley in cropping systems. This led to an extreme solution: long-term monoculture of spring barley. From this perspective, the trial has lost its original purpose. On the other hand, it remains a valuable source of information for studying soil organic matter (SOM), especially in comparison with the simultaneously conducted crop rotation experiment (NCR).

4.1 The influence of soil management system (PL/CC) in the spring barley monoculture

The impact of the reduced soil management system was particularly evident in the SBM. Although only a chiselling cultivator (CC) was used, the soil was cultivated to a depth of approximately 12–14 cm without turning of soil layers compared to the ploughing system (PL) with a depth of 22 cm. Significant changes in the content of C_SOM and other SOM quality indicators were observed.

The combination of SR + CC showed a positive effect on the C_SOM content compared to SR + PL across all observed soil profile depths. A similar trend was observed for N_T content. The applied soil management technologies did not affect the C_SOM/N_T ratio. A calculation revealed that the C_SOM content in the entire soil profile was 50.35 t ha^-1 (PL) and 57.87 t ha^-1 (CC), representing a 14.9% increase. As Mazzoncini et al. (1) and Smith (2) state, fertilisation and soil management significantly influence carbon sequestration in arable soils. Compared to conventional tillage, reduced tillage or no-tillage promotes SOM accumulation in the soil (3), primarily through physical protection of large aggregates (4) and binding SOM with the soil mineral fraction (5). Reduced tillage and no-tillage systems also maintain a stable environment for microbial activity (58), which impacts the distribution and stability of C_SOM in soil aggregates (5). Soil aggregates are considered one of the primary mechanisms controlling C_SOM sequestration. Reduced soil disturbance helps form large aggregates that physically trap free carbon (such as polysaccharides) and prevent rapid mineralisation, which facilitates C_SOM accumulation (59).

A key factor influencing soil organic matter is GRSP (6, 7). The content of EE-GRSP was not significantly affected by the soil management system in the individual profiles. However, when the contents in the individual profiles are recalculated to the amount of EE-GRSP in the entire topsoil, a noticeable increase of 8.8% is evident. The content of T-GRSP was significantly higher in the CC system in the D_0–10 and D_10–20 profiles compared to PL. Overall, there was 10.9% more T-GRSP in the CC system compared to PL. The tillage disrupts hyphae and reduces AMF biomass, which has a dominant and negative effect on production (18). Reducing the tillage and adopting no-tillage practices also increases AMF diversity, which positively correlates with GRSP content (19). It is important to note that some AMF genera like Glomus spp. may tolerate increased levels of soil disturbance due to tillage (24), or its effects may be reduced due to a shift in the AMF community toward more disturbance-tolerant species under repeated ploughing (45).

As Rillig et al. (9) and Nichols et Wright (60) state, GRSP is stable in soil and is considerably less prone to mineralisation than other SOM components. The GRSP/C_SOM ratio is a good indicator of changes in SOM content and quality Wang et al. (61).

For CC, the EE-GRSP/C_SOM ratio was 8.9%, while for PL it was 9.4%. Similarly, the T-GRSP/C_SOM ratio was 36.5% for CC and 37.8% for PL. It is clear that GRSP contributes to the stabilisation of SOM and carbon sequestration, even in systems that negatively affect SOM content (36). The conventional tillage system causes a higher rate of C_SOM mineralisation which in turn leads to increase in the GRSP/C_SOM ratio. The EE-GRSP/T-GRSP ratio was 24.9% for PL and 24.3% for CC. As Černý et al. (52) mention, higher proportions of EE-GRSP are typically found in sites with poorer SOM quality. In our case, the differences between the PL and CC systems are small enough to not consider them influencing the SOM quality.

4.2 The influence of straw application (SR/SI) in the spring barley monoculture

In comparing the influence of straw incorporation (SI) and removal (SR), we used the average contents of the monitored parameters for the entire arable soil profile. The applied straw contributed to an increase in SOM, with a more significant increase observed in the PL system (9.1%) compared to CC (6.3%). Although it seems that ploughing has a positive effect, this result comes from relatively smaller influence of chiselling (CC treatment) in the SI system in comparison to chiselling (CC) in the SR system. A positive influence of straw on C_SOM content was also observed in our long-term experiments with maize monoculture under the ploughing system (47, 48). A significant combined influence of the straw incorporation and reduced tillage on the C_SOM content is also mentioned in other works (62). The reduced tillage (in our case, the CC) can reduce the soil disturbance and promote root growth, enhancing the aggregate stability and increasing SOM in the soil profile (63). Straw incorporation increases the supply of organic matter and promotes the formation and stabilisation of macroaggregates that physically protect SOM from mineralisation (4, 64), which contributes to long-term increases in C_SOM under systems with repeated straw application (47–50).

The content of EE-GRSP was not affected by straw incorporation. However, there was a tendency toward increased T-GRSP levels by 7.6% in the CC system and 4.0% in the PL system. Our results are in accord with the conclusions of Nie et al. (49) and Liang et al. (50). On the other hand, these results are in contrast with Balík et al. (51), where no differences in GRSP content were detected between treatments with mineral nitrogen alone and those combining mineral nitrogen with straw application. Based on current results, the EE-GRSP is more influenced by a combination of the depth of the soil profile and soil management system.

The significant increase in T-GRSP under SI can be attributed to mechanisms linked to AMF activity and organic matter dynamics. The GRSP contributes to SOM stability through its aromatic carbon structures (9, 19) and through its role in promoting macroaggregate formation and stabilisation (6, 11). Since GRSP is also relatively stable and degrades slowly in soil (30–33), even moderate increases in its production associated with enhanced AMF activity may accumulate over time. Straw incorporation has been shown to influence AMF communities by altering nutrient availability and the broader microbial environment (46), and several studies have reported positive effects of straw application on both C_SOM and GRSP content (47–50). These mechanisms help explain the higher T-GRSP levels observed under SI compared to SR.

The higher C_SOM content in the SI is likewise consistent with previous findings. Straw incorporation provides an external carbon input and promotes the formation of macroaggregates that physically protect SOM from mineralisation (4, 64). Straw addition combined with adequate nutrient supply has repeatedly been associated with increased C_SOM, humic substances, and GRSP fractions in long-term field experiments (47, 48, 50). However, the reports on the effect of straw application on C_SOM and GRSP are inconsistent or contradictory (52), which may be related to differences in crop rotation, fertilisation intensity (specifically nitrogen), soil type, and AMF community composition in the individual studies (39–41). Nonetheless, the present results align with studies showing that straw incorporation supports SOM accumulation and enhances GRSP content under long-term management conditions.

4.3 The influence of soil management system (CC/PL) in the Norfolk crop rotation

The results from the NCR indicate that PL contributed to an increase in SOM content, with significant increases observed in the D_10–20 and D_20–30 profiles. No significant differences in between the PL and CC were found in the D_0–10 layer. These results contradict the findings of Naorem et al. (65), who reported significantly higher SOM content in the 0–10 cm depth for reduced tillage compared to conventional tillage. Additionally, Angers et al. (41) found higher C_SOM content in the 0–10 cm layer with no-tillage. With conventional tillage, Angers et al. (41) found higher C_SOM content in profiles deeper than 20 cm, which is in agreement with our results. Some studies, however, suggest that tillage methods may not always be dominant in influencing SOM content and microbial diversity (38). A significant role is also played by crop rotations (39).

When recalculating for the soil depth (30 cm), the C_SOM content was estimated as 71.32 t ha^-1 for PL and 64.5 t ha^-1 for CC, meaning that PL had 9.5% more C_SOM in the topsoil. The differences in the GRSP content were smaller (cca. 7%). That also shows the relatively higher stability of the GRSP content in comparison with the C_SOM content.

The results clearly demonstrate the effect of ploughing on GRSP content even within the NCR system. Under chiselling the CC variant, a higher amount of EE-GRSP (109.8%) or an equal amount of T-GRSP (100%) was observed in NCR compared to SBM (Figure 4). This supports the notion that physical disruption of hyphae has a dominant negative effect on GRSP production (18).

4.4 The influence of crop rotation (SBM/NCR) under ploughing system

The assessment of carbon sequestration efficiency in topsoil under the PL system showed significantly more favourable results in the NCR compared to the SBM. The amount of soil organic matter in the topsoil was substantially lower in SBM, representing only 70.6% of the C_SOM content observed in NCR. In SBM, 50.35 t C_SOM ha^-¹ was accumulated, while in NCR it was 71.322 t C_SOM ha^-¹. In NCR, the inclusion of clover (average hay yield of 7.62 t ha^-¹) and alfalfa (average hay yield of 11.5 t ha^-¹) had a very positive effect on C_SOM content. A global meta-analysis showed that crop rotation increased SOC by ~6.6% compared with continuous monoculture, highlighting that diversified cropping enhances long-term carbon storage in agricultural soils (66). The positive impact of incorporating clover and alfalfa into crop rotations on C_SOM levels has also been reported by several other authors (67, 68). Furthermore, the average grain yield of winter wheat in NCR (6.46 t ha^-¹) was substantially higher than the yield of spring barley in SBM, which also implies a greater root biomass and thus a larger input for SOM formation. In accordance with our previous results, the application of farmyard manure (25 t ha^-¹ every four years) also had a positive effect (69). Similarly, Börjesson et al. (70) observed a significant increase in SOM content in long-term experiments (35 years) under a crop rotation system-three years of grass-clover followed by one year of cereal-compared to a rotation with cereals only (spring barley, oats, spring wheat). A major contributing factor to the observed differences was the length of vegetation cover. In the NCR, crops such as winter wheat, alfalfa, and clover were already established in autumn, while in the SBM, fields remained bare from July to March. This led to more intensive SOM mineralisation in SBM.

The C_SOM/N_T ratio is considered a stable characteristic of SOM, relatively unaffected by soil management practices or fertilisation (47, 48). This is supported by our results, where no differences were found between PL and CC systems across soil depths. However, a higher C_SOM/N_T ratio was observed in NCR compared to SBM: 9.82-10.1 vs. 9.42-9.64, respectively, indicating more stable and higher-quality SOM in NCR (51). The PL in NCR also significantly increased GRSP levels-EE-GRSP by 6% and T-GRSP by 16.8%. This is attributed to the inclusion of crops such as maize and legumes in NCR, which have a strong affinity to AMF (71, 72). The increase in the GRSP content could have been caused by the increase in the C_SOM content. Although AMF do not have saprophytic capacity, some studies have shown that AMF can directly benefit from organic matter in the soil (42, 43). Yang et al. (44) report that the application of manure increased AMF growth and also spore production. The AMF persistence in ecosystems depends on the formation and survival of propagules (e.g., spores, hyphae, colonised roots) (28). Spores are considered resilient, long-term propagules, especially in the absence of host plants. However, autumn tillage has been shown to reduce hyphal viability for the following crop (28). Because AMF are biotrophic, their viability gradually declines without host plants, such as when land is left fallow-even under no-tillage systems. The inclusion of a mycotrophic cover crop, sown immediately after autumn soil cultivation, can support AMF development even in plough-based systems (28). In our case, the fallow period between barley harvest (July) and sowing (March) likely reduced AMF viability. AMF species differ significantly in functional traits, including colonisation strategy, uptake of organic compounds from plants, and nutrient utilisation (20, 21). Several long-term studies have observed reduced AMF species richness under conventional tillage (22, 23). However, Hu et al. (24) found no negative effect in systems rotating maize and wheat. This suggests that the crop diversity in NCR may have enhanced AMF species richness. Additionally, AMF species vary in their tolerance to soil disturbance. For example, members of the genus Scutellospora are highly sensitive to ploughing, while Glomus species are commonly found in ploughed soils (25).

Soil compaction has also been shown to influence AMF communities due to changes in water content and oxygen availability (25). In some cases, deep ploughing can reduce topsoil compaction and create better conditions for AMF development. Enhanced growth of host plants improves the supply of organic compounds to fungi and may affect the AMF community (27).

Several studies indicate that GRSP is stable in soil and mineralises slowly (30–33). When land use changes, soil organic matter declines more rapidly than GRSP. The slower dynamics of GRSP reduction lead to a relative increase in its proportion within SOM (GRSP/C_SOM ratio). Our results confirm this conclusion. In ploughing technology and monoculture, the EE-GRSP/C_SOM ratio was 9.4%, and the T-GRSP/C_SOM ratio was 37.9% in the entire 0–30 cm profile. On the other hand, the EE-GRSP/C_SOM and T-GRSP/C_SOM ratios in the NCR were 7.1% and 32.1% 0–30 cm profile. respectively. From this perspective, the SOM in the NCR has a higher proportion of easily mineralizable organic matter. Similarly, higher proportions of GRSP in C_SOM have been found in intensively farmed areas with cereals or vineyards, where the C_SOM content is low (34–36). The EE-GRSP and T-GRSP ratios in C_SOM observed in this study are higher than those reported by Černý et al. (52), who found EE-GRSP/C_SOM between 3.5-4.0% and T-GRSP/C_SOM between 16.9-27.0%, depending on soil type. Similarly, Balík et al. (47, 48) established average values for Luvisol 6.3%, 22.1%, and for Cambisol 5.0%, 18.6% for EE-GRSP/C_SOM and T-GRSP/C_SOM, respectively. It is clear that the GRSP/C_SOM ratio varies depending on soil conditions (soil type, soil texture) and the crops grown. Staunton et al. (73) report an EE-GRSP/C_SOM range of 0.6-9.6% and a T-GRSP/C_SOM range of 2.0-19.0%.

The EE-GRSP/T-GRSP ratio was 24.9% for SBM and 22.0% for NCR. As noted by Černý et al. (49), this ratio is significantly influenced by soil type. The authors report an EE-GRSP/T-GRSP ratio of 29.8% for sandy soils, 18.1% for clayey soils, 14.4% for Chernozem, and 15.4% for Cambisol. The relatively higher value of the EE-GRSP/T-GRSP ratio likely indicates poorer SOM quality at the site. However, whether this means poorer SOM quality at SBM compared to NCR cannot be confirmed. For comparison, Cissé et al. (74) report a value of 28.3%, and Staunton et al. (73) report a value of 40%.

4.5 The overall influence of the chiselling and ploughing over the soil organic matter content and quality

The general influence of the soil management system on the content and quality of the soil organic matter was investigated by pooling of the data from the SBM and NCR experiments. This would allow for uncovering the influence of CC and PL without the influence of the crop. There were no significant differences in the C_SOM and EE-GRSP content based on the soil management system. On the other hand, the T-GRSP content was significantly increased in the CC system (5077 mg kg^-1) in comparison to the PL (4878 mg kg^-1). The higher T-GRSP content observed under chiselling may be associated with the lower degree of disturbance of AMF hyphal networks compared to ploughing. As noted in the Introduction, tillage disrupts fungal hyphae and suppresses GRSP production (18), whereas reduced soil disturbance generally promotes AMF biomass (18) as well as AMF diversity, which has been positively correlated with GRSP abundance (19). Because AMF species differ in their tolerance to soil structure disturbance (25), chiselling is likely less detrimental to sensitive AMF taxa than ploughing, potentially supporting a community structure more conducive to GRSP production. Furthermore, GRSP is relatively stable and degrades slowly in soil (30–33); therefore, even minor annual differences in its production may accumulate over time. These mechanisms may explain why a statistically significant difference in T-GRSP was detected despite the small absolute difference between the two management systems.

This long-term study confirms that carbon sequestration and the accumulation of glomalin-related soil proteins in arable soils are significantly influenced by soil cultivation methods, straw management, and crop rotation design. Chisel cultivation enhanced total glomalin content and increased C_SOM compared to ploughing, particularly in upper soil layers (0–20 cm) in the SBM. Straw incorporation promoted C_SOM accumulation more effectively under ploughing than under chisel cultivation but had no measurable effect on EE-GRSP content. The Norfolk crop rotation system significantly improved both C_SOM levels and the C_SOM/N_T ratio relative to spring barley monoculture, highlighting its role in enhancing soil organic matter stability. These findings emphasise the importance of reduced soil management, diversified crop rotations, and organic residue management in improving soil carbon dynamics and soil quality in long-term arable systems.

Data availability statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Author contributions

JB: Writing – original draft, Conceptualization, Supervision. PR: Formal analysis, Writing – review & editing. PS: Data curation, Writing – original draft. TD: Methodology, Resources, Writing – review & editing. VS: Writing – review & editing. JČ: Methodology, Validation, Writing – original draft. MK: Writing – review & editing, Supervision. OS: Methodology, Writing – original draft, Validation. PM: Methodology, Writing – review & editing.

Funding

The author(s) declared financial support was received for this work and/or its publication. Research in this manuscript was funded by: the Ministry of Agriculture of the Czech Republic grant numbers QK21010124, QK23020056, and Project of the Technology Agency of the Czech Republic, Project No. SS02030018.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declare that Generative AI was not used in the creation of this manuscript.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fsoil.2025.1676426/full#supplementary-material

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