Effect of Agricultural Management Intensity on the Organic Carbon Fractions and Biological Properties of a Volcanic-Ash-Derived Soil

Abstract

Intensive agricultural management affects the physical, chemical, and biological properties of soil, potentially contributing to a decrease in soil carbon storage. In this study, the effects of soil management intensity on soil organic carbon (SOC) content and its labile fractions, i.e., water-soluble organic carbon (OC-sol) and permanganate oxidizable carbon (POXC), were evaluated in a volcanic-ash-derived soil (Andisol) with a very high soil organic matter (SOM) content (>20%). These indicators were associated with water-stable aggregates (WSAs) and biological indicators, namely, earthworm density, cellulase activity, and autoclaved-citrate-extractable (ACE) proteins, related to the decomposition of SOM and its physical protection. The conditions evaluated were secondary native forest (SF), naturalized grassland (NG), no-till (NT), and conventional tillage (CT), considering the last item to be representative of a higher agriculture management intensity. Soil samples were collected by horizon. The SF and NG soil showed higher contents of SOC, OC-sol, and POXC. When comparing the evaluated annual cropping systems, NT showed higher values than CT (p < 0.05) in the first horizon (Hz1), while similar values were found at deeper horizons. The highest cellulase activity, ACE protein levels, and earthworm densities were found in NG and SF. NT also showed significantly higher levels of the aforementioned factors than CT (p < 0.05). A positive and significant relationship was found between the SOC content and WSA (R² = 0.76; p < 0.05) in the whole profile and between POXC and WSA for Hz1 (R² = 0.67; p < 0.05). Soil C storage was affected by the intensity of agricultural management, mainly because of the effect of tillage on structural stability, considering that biological activity synthesizes compounds such as enzymes and proteins that react and adhere to the mineral fraction affecting aggregate stability. The C content stored in the soil is consequently a key indicator with which to regulate SOM and protect SOC.

1. Introduction

Soils perform diverse ecosystem functions, including the storage of soil organic carbon (SOC), which is a key function for C sequestration [1,2]. However, soils are susceptible to degradation, which leads to SOC losses through carbon dioxide (CO₂) emissions, depending on the soil type, climate, management, and land use [3]. Inadequate management of production systems and unregulated changes in land use have resulted in a loss of stored C and thus a 20–25% increase in CO₂ emissions [4]. The latter is mainly due to the destruction of soil structures (e.g., over-tilling), which accelerates soil organic matter (SOM) mineralization rates and thus SOC, leading to an estimated overall loss of 133 Pg of C in the soil profile [5,6]. For this reason, it is essential to understand the factors that influence SOC storage, its persistence, and suitable soil management schemes [7,8].

SOC is considered a master property [9], and it is the most widely used soil quality/health indicator [10] because it is involved in almost all soil functions and ecosystem services [1]. SOC, which is part of SOM, is also differentiated by stable fractions [11]. The most stable fraction of C is associated with the mineral fraction, which is bound to plant compounds that have been solubilized and transformed by microorganisms (mineral-associated organic matter, MAOM). The less stable fraction (particulate organic matter, POM) is formed as a result of the partial decomposition of plant compounds and the physical fragmentation of plant structural components by macro-organisms such as earthworms, which are key organisms in SOM decomposition and enhance its protection [12,13], and beetles [5,14,15]. The physical stabilization of these soil fractions is mainly provided by the stability of soil aggregates, which blocks the access of soil organisms to organic compounds through physical occlusion, hydrophobicity, and adhesion to mineral surfaces [16,17,18], promoting the stability and storage of SOC at different hierarchical levels within the soil structure [19,20].

Soil management intensity (e.g., forest, grassland, and cropping systems) directly influences SOC dynamics [17,21]. Therefore, different degrees of intensity affect SOM inputs and SOC storage. Forests are considered reference systems within a soil management intensity gradient since they are not mechanically managed and usually store large amounts of C (25 Mg C ha⁻¹). Grassland systems are subject to a lower degree of intervention than cropping systems because they are not mechanically managed, plant and animal residues are constantly incorporated in them, and their vegetation cover is maintained, providing them with a high C sequestration potential [22]. In cropping systems, no tillage (NT) has been proposed as a conservation farming practice that decreases degradation and SOC losses [1,17,23,24]. It has been shown that reduced tillage or the addition of organic fertilizers can increase the POM fraction [25]. In this context, measuring soluble C indicators (associated with the POM fraction) has been used to evaluate the effect of land use management on SOC accumulation. This OC fraction can be measured using different methods, including soluble organic carbon (OC-sol) and permanganate oxidizable carbon (POXC), which are associated with dissolved organic matter (DOM), microbial biomass, and complex and lignin polysaccharides [26].

In volcanic-ash-derived soils, these fractions have been very scarcely assessed, and considering their potential for SOC accumulation and sequestration, it is important to evaluate how the biological properties that determine their structural stability are affected by the intensity of agricultural management, which alters the physical, chemical, and biological properties of the soil as well as the processes occurring in it [9,27]. Soils subject to volcanic influence in Southern Chile (Andisols, Ultisols and Inceptisols) are characterized by high SOC contents throughout their profiles, reaching surface values greater than 20% SOC [28,29,30]. This is associated with the presence of amorphous clays (such as allophane) and aluminum and iron oxides that react with SOM [30], forming organo–mineral complexes that contribute to the physicochemical protection of SOC [12,31]. Due to their great capacity to store SOC and their presence in highly productive areas of the world [32], it is essential to evaluate the management practices that foster this accumulation of SOC and minimize losses due to land use change (LUC). Thus, the objective of this work was to evaluate the effect of different management intensities on carbon labile fractions (OC-sol and POXC) and biological properties and their relationship with the structural stability of a soil derived from volcanic ash.

2. Materials and Methods

2.1. Experimental Site Description and Sampling

Sampling of all sites was carried out during the autumn of 2022 over two days. Soil samples were collected from farms (“Fundo Brasil”, “Chile Chico” and “Monte verde”) near the town of Máfil, Valdivia, Los Ríos Region, Chile. The soil corresponds to an Andisol, Los Ulmos Series (Udands), known as “red clayey,” with a volcanic influence and a loamy surface texture [33]. These soils tend to have high SOC content on the surface (4–12%), while drastic decreases have been observed at depth (0.4%, [34]). The average annual rainfall is approximately 1800 mm, and the average annual temperature ranges from 11 to 13 °C.

The sampled sites had different management intensities, considered from lower to higher intensities according to their degrees of anthropogenic intervention and management history (

Table 1. Management history for each land use zone.

Land Use	Physical Intervention	Vegetation	Residues Inputs	Time
CT	Plowing (0–20 cm)	Wheat monoculture	A portion of the residues is incorporated	20 years
NT	Without plowing (last 2 years, surface movement: 2 cm)	Wheat–vetch–rape rotations, sometimes incorporating peas	Residues are kept on the ground	>25 years
NG	Grazing (high loads in short periods)	Grasses (Agrostis capillaris L., Arrhenatherum elatius spp., Holcus lanatus L., Hypochaeris radicata). Legumes (Trifolium pratense and Locus pedunculatus)	Maintenance of vegetation cover and grazing	20 years
SF	No intervention	Maqui (Aristotelia chilensis), Arrayán (Luma apiculata), Roble (Nothofagus obliqua), Quila (Chusquea quila), Ulmo (Eucryphia cordifolia)	Maintenance of vegetation cover	>20 years

CT: conventional tillage, NT: no tillage, NG: naturalized grassland, SF: secondary native forest. All soils with different land uses were subjected to fertilizer and/or pesticide application, with the exception of NF.

): native forest (NF), grasslands for more than 20 years (NG), and crops under zero tillage (NT) and conventional tillage (CT), both cultivated under rotations of wheat (Triticum aestivum), oats (Avena sativa), rape (Brassica napus), and peas (Pisum sativum). For chemical and biological parameters, disturbed soil samples were taken (n = 5) at each soil horizon and land use zone.

2.2. Soil Measurements

2.2.1. Soil Organic Carbon (SOC), Soluble Carbon (OC-sol), and Active Carbon (POXC)

The SOC content was measured through the Walkley and Black wet-digestion method [35]. OC-sol was determined via a titration method, using the potassium sulfate (K₂SO₄) method [36] titrated with FeSO₄ [35]. POXC was evaluated via extraction with potassium permanganate [37]. A solution containing potassium permanganate (KMnO₄) dissolved in calcium chloride (CaCl₂) was prepared; this was used to create the standard curve (0.005, 0.01, 0.015, and 0.02 M) and the reaction solutions (2.5 g of soil, 2 mL of stock solution, and 18 mL of distilled H₂O). From the reaction solutions, 0.5 mL was removed and added to the dilution solution along with 49.5 mL of distilled H₂O. POXC is a method that oxidizes different aromatic compounds and lignin, so it is highly dependent on the input (amount and type) of organic matter and the effects of tillage [25]. POXC was calculated using the following equation:

$$POXC\left(\mathrm{mg}{\mathrm{kg}}^{-1} \mathrm{soil}\right)=\left(0.02 \mathrm{mol}{\mathrm{L}}^{-1}-\left(a+b\ast Abs\right)\right)\ast \left(9000 \mathrm{mgC}{\mathrm{mol}}^{-1}\right)\ast (0.02 \mathrm{L} {\mathrm{ss}}^{-1})$$

(1)

where a = standard curve intercept; b = standard curve slope; and Abs= sample absorbance.

2.2.2. Cellulase Activity and Autoclaved-Citrate-Extractable Protein

Cellulase activity (CM-cellulose) was determined according to the method employed by [38]. Enzymes are sensitive to changes in physical, chemical, and biological properties. Therefore, soils under management schemes that promote structural stability and SOM generally exhibit higher activity [39]. CM-cellulose was used as the substrate, and the reduction of sugars generated from the hydrolysis of the glycosidic bonds of the cellulose molecule was measured. The results were obtained as the glucose equivalent (GE) using the following equation:

$$Cellulaseactivity\left(\mathsf{\mu }\mathrm{m}\mathrm{GE}\ast {\mathrm{g}}^{-1}\mathrm{dm}\ast 24{\mathrm{h}}^{-1}\right)=\left({\displaystyle \frac{\left(S-C\right)\ast 30\ast 40}{10}}\right)\ast 100\%\mathrm{dm}$$

(2)

where S = mean value of the samples (μg GE); C = control (μg GE); 30 = volume of incubation mixture (mL); 40 = dilution factor of the filtrate; 10 = initial weight of the soil (g); and 100% dm = conversion factor for dry soil weight.

In this study, autoclaved-citrate-extractable protein (ACE) was used to measure protein-like substances found in SOM. The protocol modified by Wright and Upadhyaya [40] and Clune [41] was performed, in which 3 g of soil was placed in glass tubes along with 24 mL of a sodium citrate buffer (20 mM, pH 7.0). This was then shaken for 5 min at 180 rpm and autoclaved for 30 min at 121 °C. Two mL of the suspension was then removed, put into microcentrifuge tubes, and centrifuged at 10,000 rpm. A subsample of the extract was taken for the standard protein quantification assay (ACE), in which it was incubated at 60 °C for 30 min and then read using a spectrophotometer according to the color the sample developed [42]. The formula for calculating the extractable protein content is shown in the following equation:

$$ACEprotein={\displaystyle \frac{Proteinextractcontent\ast 24\mathrm{mL}}{drysoilweight}}$$

(3)

2.3. Earthworm Biomass and Density

Five samples were taken per soil management, considering a 20 cm × 20 cm × 20 cm soil block. This block was broken up to weigh and count the earthworms [43]. In turn, these earthworms were divided into two ecological categories, epigean and endogean, as they present different behaviors, thereby affecting the incorporation and fragmentation of SOM [44]. Then, the proportion of epigean and endogean earthworms with respect to the total biomass (g m⁻²) and earthworm density (number of individuals m⁻²) was calculated. Three density ranges were considered: low (< 100 m⁻²), medium (100–500 m⁻²), and high (500 m⁻²) [43,45].

2.4. Water Stability of Aggregates (WSA)

WSA was evaluated according to the method used by Kemper and Rosenau [46], sieving the wet sample and then saturating it via capillarity. WSA was then calculated with the following equation:

$$WSA\%={\displaystyle \frac{(retainedmass-sandmass)}{(totalmassofthesample-sandmass)}}$$

(4)

A sieve < 0.25 mm and NaOH were used as dispersing agents for the sand content.

2.5. Statistical Analysis

An ANOVA was performed to evaluate the effect of management intensity on SOC, the evaluated labile fractions (OC-sol and POXC), and biological indicators in each horizon separately. Means were separated using Tukey’s test (p < 0.05). A principal component analysis (PCA) and linear and nonlinear regression analyses were performed to determine the relationship between OC fractions and biological indicators of structural stability. The analyses were performed with Rstudio software version 4.3.0.3.

3. Results

3.1. Changes in SOC, Soluble OC, and POXC Content According to Management Intensity

The SOC content varied between 3 and 5% due to the agricultural management intensity (Figure 1A), mainly in Hz1, where SF > NG and NT > CT (F_3,12 = 14.53, p < 0.05). For OC-sol (Figure 1B), the same dynamics were observed (F_3,12 = 12.42, p < 0.05). For POXC (Figure 1C), it was found that NG > NT and SF > CT (F_3,12 = 28.56, p < 0.05). In the deeper horizons, a decrease in SOC, OC-sol, and POXC content was observed, where NT presented lower content compared to CT.

3.2. Changes in Biological Indicators According to Management Intensity

Cellulase activity (Figure 2A) and the ACE protein content (Figure 2B) showed differences between soil management types in each horizon but mainly in Hz1 (F_3,16 = 1017, p < 0.05; F_3,16 = 3.807, p < 0.05). In Hz1, NG had the highest values, with a cellulase activity two to three times higher than the other managements evaluated. CT had the lowest (p < 0.01) cellulase activity at Hz1. In contrast, in Hz2, this management scheme promoted twice as much activity compared to SF and NG (F_3,16 = 2173, p < 0.05). The ACE protein content was higher for both NT and SF (>25 mg g⁻¹), being similar (p > 0.05) between these management types, in contrast with CT, for which the value was significantly lower (<25 mg g⁻¹).

The highest earthworm biomass (Figure 3A) occurred in NG and NT (>1000 earthworms m⁻²), followed by SF and then CT, with a significantly lower biomass (p < 0.05). Under CT, endogean earthworms weighed approximately 50% more than epigean earthworms (Figure 3C). However, when analyzing earthworm density (Figure 3B) no significant differences between categories (p > 0.05) were observed, while significant differences between management strategies were found, where NG > NT = SF > CT. The CT-managed soil had low to no earthworm density (< 50 earthworms m⁻²) and a lower biomass since only epigean earthworms were found.

The PCA showed that PC1 (65.1%) most explained the variability in SOC, POXC, WSA, and proteins. The WSA and protein indicators mostly reflected the SOC and POXC content. In addition, a high correlation between these variables was found (Figure 4B). On the other hand, in PC2 (29.9%), the cellulase activity and earthworm variables were most represented and presented a high correlation (Figure 4B).

3.3. Relation Between Structural Stability, Organic Carbon Content, and Biological Indicators

A significant relation was observed between WSA and SOC (p < 0.001), both for the complete profile (Figure 5A) and the first horizon (Figure 5B). In addition, ACE protein was associated with WSA (p < 0.001; Figure 5C). Likewise, there was a positive relation between WSA and labile C fractions (p < 0.001), mainly with respect to POXC in the first horizon (Figure 5D).

4. Discussion

4.1. Changes in SOC Content and Labile Fractions According to Soil Management Intensity

At lower management intensities (SF and NG), higher SOC content was observed (Figure 1A). This shows that agricultural management intensity affects labile C content and fractions [1,9,47,48]. The forest system in this study (SF) that was considered as a reference (i.e., it was not subjected to mechanical or chemical intervention) presented the highest SOC content. In this context, C content can change according to the vegetation type of the forest, which determines the amount and type of SOM [49]. In this study, the SF consisted of mainly evergreen species and some native broadleaf deciduous species (Table 1); therefore, the litter renewal rate was slower, but the type of litter decomposed faster (lower C:N and lignin ratio), allowing SOC accumulation and greater labile fractions (Figure 1B,C) [50].

The naturalized grassland (NG) also presented high SOC content and labile fractions. These systems tend to have high SOM levels (approximately 350 Mg ha⁻¹) as a result of the decomposition of organic residues of both plant and animal origin [51], with a higher proportion of MAOM and a lower proportion of POM [52]. These productive systems store a third of the world’s terrestrial C reserves and have a high potential for C sequestration, especially if grazing is properly managed, for example, by increasing species diversity and incorporating legumes [22]. The NG in this study was under rotational grazing management with high stocking for short periods, involving leguminous and grass species (Table 1). This type of system has a high SOM input and allows for recovery from disturbance, in addition to maintaining vegetation cover throughout its cycle, which favors biological activity, soil structure and, therefore, SOC accumulation [1].

When comparing the two evaluated cropping systems, NT had higher SOC content and labile fractions (OC-sol and POXC) at Hz1 but not in the deeper horizons. This has been observed in several studies, where NT has proven to have a higher SOC content in the topsoil [17,47], while CT has been found to have a greater SOC content in the subsoil [18,53]. The latter may be due to tillage mixing the residues and redistributing SOC throughout the profile, thus permitting greater root growth at greater depth [47,53,54]. However, it has been proposed that adopting NT practices would increase the stock of SOC [55].

4.2. Influence of Biological Indicators on C Content Under Different Soil Management Intensities

Cellulase activity and labile C fractions proved to be sensitive to management intensity (Figure 2A) [56,57,58] and showed the highest content in the NG system at the first horizon (Hz1). Both of these indicators also showed higher activity in NT than in CT [59,60]. Cellulase activity measures the hydrolysis of cellulose, a complex structural component found in large proportions in plant residues that decompose mainly on the surface of soil [61,62,63]. Therefore, the action of this enzyme produces simpler and more soluble carbon compounds that could be associated with a higher content of labile C fractions (mainly with respect to POXC, as shown in Figure 4) that constantly enter and/or maintain plant and animal residues, given that the activity of this enzyme depends fundamentally on contact with SOM [64]. Enzymes are also sensitive to changes in physical, chemical, and biological properties introduced by management and/or changes in land use; therefore, systems with higher residue inputs generally have higher enzyme activity [39]. Thus, extensive grazing can stimulate both soil and plant biodiversity, increasing enzyme activity [65,66]. This results in greater ecological functionality of the soil [67] because these enzymes participate in the release of nutrients for plants and C for the metabolism of microorganisms, making them key indicators in terms of soil quality [68].

The ACE protein content was mainly affected by tillage (Figure 2B). This method is an indicator of substances like proteins (including glomalin and other proteins) that are synthesized by soil microorganisms (mainly in the hyphae of arbuscular mycorrhizal fungi), which have been positively associated with aggregate stability because they act as cementing agents of soil mineral fractions [40,42,69,70]. Therefore, tillage by physically disturbing the soil and fragmenting the aggregates causes a decrease in these proteins. Borie et al. [71] evaluated the concentrations of these proteins in different tillage systems in the first 20 cm of soil; NT presented higher values compared to CT for a soil derived from volcanic ash in Southern Chile and was related to higher SOC content. This was also observed in the results of this study, since the SOC content was positively associated with ACE protein at the first horizon (Figure 4B).

Earthworms perform important ecological functions in the soil [42]. They play a crucial role in the C cycle because they can protect SOM in soil aggregation and increase C mineralization as they metabolize SOM and promote soil microbial activity [72], which could explain the positive correlation observed between enzyme activity and earthworm indicators (Figure 4B). Earthworms’ functional traits can vary. Epigeans inhabit the upper layers of the soil profiles and have the ability to digest OM and fragment it, facilitating its decomposition [13]. However, Barthod et al. [73] suggested that the presence of these earthworms can slow down the mineralization of SOC by forming products that are less easily decomposed and are associated with the mineral fraction, suggesting that earthworms influence the persistence of SOC and therefore its accumulation. In contrast, endogeans are usually heavier than epigeans [45] because they are found at greater depths, although they can also digest organic matter from the surface, as well as soil mineral particles, a trait that has also been associated with the protection of SOM [12,74]. In accordance with previous studies, our results suggest that CT negatively affects the density and biomass of earthworms in the soil, both epigean and endogean, because CT negatively affects the structure of soil [13,75,76].

4.3. Influence of Aggregate Stability on SOC Persistence and Labile C Fractions

The persistence of SOM depends on several factors, including C use efficiency and microbial access, which are mainly determined by their association with minerals and occlusion in fine aggregates [5]. A close relation between SOC and microaggregate stability (<53 um) was observed in this study (Figure 5A), as noted in other studies on volcanic-influenced soils [30]. Likewise, a positive relationship with POXC was found, although only at Hz1, most likely because it is the horizon that presents the highest biological activity and is associated with an increase in labile C fractions [77]. These relationships with soil structural stability may principally exist because the occlusion of SOC and labile C fractions in microaggregates prevents the access of microorganisms, thus accelerating SOC mineralization [16,78]. An acceptable relation was also observed with ACE proteins (R² = 0.65, p < 0.05), which corroborates the notion that the presence of these proteins could have an impact on the overall structural stability in the soil due to their reactivity with the mineral surface [42].

4.4. Importance of SOC Storage and Protection in Volcanic-Ash-Derived Soils

Soils influenced by volcanoes are key in SOC storage. They are soils with unique and particular properties that make them highly reactive (due to the presence of Al/Fe oxides and allophane), allowing them to form organo–mineral complexes and store high amounts of SOM [28,29,79]. The results of this study show that forest (SF) and grassland (NG) systems are important C sinks, a fact that has been demonstrated by several authors [22,62,80]. These productive systems cover a third the world’s land surface [81], while in Chile, they cover approximately 95% of the useful area and are largely concentrated in the southern part of the country over volcanic-ash-derived soils [82]. In addition, it is important to consider that naturalized grasslands account for 47.8% of the grassland systems in the Los Ríos and Los Lagos Regions, being an important forage resource for farmers in the southern zone of Chile [83]. There are also important native forest and silvo-agricultural areas in this region [82]. Therefore, it is crucial for these systems to be properly managed because they can have a great impact on C sequestration and, therefore, climate change mitigation [84]. Furthermore, if the volcanic-ash-derived soils under these systems are not managed properly, they may exhibit weak structural stability and be susceptible to SOC losses [30]. It is essential to consider the impact of tillage on these soils and evaluate management practices that promote SOM and protect soil structures to maintain and/or increase SOC content [85].

5. Conclusions

In this work, tillage had a negative effect on the biological indicators measured. These indicators were associated more with structural stability than residue decomposition, which suggests that when there is greater biological activity in the soil, SOC is not necessarily mineralizing because the synthesis of compounds that react with the mineral fraction of the soil favors structural stability, thus protecting the SOC in the aggregates. The latter is a key element in stabilization and should be considered in future research related to SOC dynamics, ideally for different C fractions and aggregates. In addition, it is essential to advance the evaluation of labile C indicators, such as POXC, in different soil types, uses, and management schemes in Chile, since these indicators have been poorly studied throughout the country. It would also be interesting to establish the ranges in which these vary, mainly in volcanic-ash-derived soils that are characterized by their high SOM content, generally causing these indicators to be above the ranges established in other soil types.

Finally, we affirm that promoting the use of conservation tillage practices (reducing tillage to minimum or zero tillage) in cropping systems can benefit carbon sequestration. As observed in this study, no-tillage seems to be effective for storing SOC and producing the conditions necessary for structural stability and the maintenance of biological soil properties. Although no-tillage schemes favor soil properties, they still require agrochemicals, which can affect soil health in the long term, so it is essential to integrate crop diversity and/or rotations and reduce or change chemical fertilizers through the use of organic fertilizers to increase the resilience and sustainability of these systems by increasing carbon inputs.