Short-term response of soil greenhouse gas fluxes to alfalfa termination methods in a Mediterranean cropping system

ABSTRACT

Soil acts as a natural source and sink for greenhouse gases (GHG) that are responsible for global warming and climate change. As the agricultural sector has an important impact on GHG (e.g., CO₂, CH₄, N₂O) emissions, the definition of mitigation strategies is needed, especially for the Mediterranean climate areas that appear most vulnerable to climate change. The introduction of perennial legumes, such as alfalfa (Medicago sativa L.), falls within this scope, but it requires the application of a termination method aimed at reduction of GHG emissions. With the aim of assessing the short-term effects of two different alfalfa termination methods (Tillage vs No tillage plus herbicide), we defined the hypothesis that alfalfa termination by Tillage will increase soil GHG emissions compared to its termination by No tillage plus herbicide. Soil CO₂, CH₄ and N₂O emissions were monitored over ~2 months (71 days; October–December, 2017) following alfalfa termination, using closed static chambers. The soil total GHG emissions after 71 days were significantly different for Tillage and No tillage plus herbicide: 311.90 ±21.21 versus 195.89 ±11.14 g CO₂ equivalent m⁻², respectively. For both termination methods, CO₂ (up to a maximum value of 1.24 ±0.18 and 0.65± 0.07 kg C-CO₂ ha⁻¹ h⁻¹, respectively) was the greatest contributor to the total soil GHG emissions (about 96%), compared to N₂O (up to a maximum value of 0.37 ±0.13 and 0.31± 0.10 g N-N₂O ha⁻¹ h⁻¹, respectively) and CH₄ (up to a maximum value of −0.01 ±0.03 and 0.07 ±0.03 g C-CH₄ ha⁻¹ h⁻¹, respectively). These data suggest that over the short term, legume perennial crop termination by No tillage plus herbicide better supports the purpose of climate regulations.

KEY WORDS:

• CO₂
• CH₄
• ecosystem services
• N₂O
• tillage
• herbicide

1. Introduction

Global warming and climate change are closely related to increased emissions of long-lived greenhouse gases (GHG) (Manabe 2019), which are due to energy, industry, transport, building sectors (76% of emissions), and to agriculture, forestry, and other land use (24% of emissions) (IPCC 2014). In particular, climate and land-use changes increase the vulnerability (e.g., declining soil fertility, reduction of water availability, and increasing risk of forest fire) of the Mediterranean regions (Schröter et al. 2005). About one fifth of the global anthropogenic GHG one fith derives mainly from agricultural production, land use, and land-use change activities (Smith et al. 2014). As soil is a natural source of GHG, the definition of mitigation strategies for reduction of agricultural soil GHG emissions is needed, especially under Mediterranean conditions (Trozzo et al. 2020).

From a climate change perspective in general, the use of cover crops can be an effective adaptation strategy (Kaye and Quemada 2017; Krstić et al. 18), but the introduction of perennial legumes into agricultural crop rotation can provide many other advantages. These advantages include, in particular: (i) lower GHG emissions through absence or reduction of nitrogen (N) fertilization (Sanz-Cobena et al. 2017); (ii) high-quality forage production; and according to Cosentino et al. (2015), (iii) positive effects on organic matter content and soil structure due to reduction in soil tillage frequency and depth, and to continuous release of organic residues; (iv) reduction of soil erosion; and (v) reduction of N leaching through enhanced soil structure and soil covering during heavy rain. In the evaluation of agro-ecosystems and their functions, these aspects are commonly defined as ecosystem services that are the benefits that humans obtain from ecosystems (D’Ottavio et al. 18).

Among the perennial legumes used at a global level, including the Mediterranean region, alfalfa (Medicago sativa L.) is one of the most widely cultivated forage crops (Suttie 2000). Stands of alfalfa last 3 years on average, although when there is no economic convenience to their termination, they can remain longer (e.g., for 6 or 7 years, or even longer). In these cases, alfalfa is commonly used for hay production and aftermath-grazed during the winter by transhumant or sedentary flocks (Caballero et al. 2009). Alfalfa is also included in crop rotations, where it is often alternated with winter cereals (Trozzo et al. 2020). Before sowing the cereals, the alfalfa stands are usually terminated by tillage or by herbicide application, with subsequent sod-seeding.

The method used for crop termination might have different effects on soil carbon (C) sequestration and on the magnitude of soil GHG emissions (Malhi, Lemke, and Schoenau 2010; Abido, Hadházy, and Henzsel 2020). Indeed, the crop termination method (e.g., tillage, no tillage, and minimum tillage), influences the soil conditions and therefore also the soil microbial activities and related processes (e.g., nitrification, denitrification, soil respiration, and methanogenesis) (Kyaw and Toyota 2007; Oertel et al. 2016). Soil disturbance by tillage is one of the main factors that affects soil carbon dioxide (CO₂) emissions and soil C storage. Abdalla et al. (2013) reported that conservation practices (i.e., conservation tillage and subsequent effects on crop residues) undertaken under appropriate climatic conditions can reduce soil CO₂ emissions. Also, the absence of tillage can lead to stabilization of and increases in soil organic C (Sanz-Cobena et al. 2017), which usually results in lower soil CO₂ fluxes compared to tillage (Abido, Hadházy, and Henzsel 2020; Francioni et al. 2020). Even if the absence of tillage can positively influence soil organic carbon and reduces soil CO₂ emissions, this effect might not be seen for methane (CH₄), as reported by Guardia et al. (2016) for a semi-arid Mediterranean climate, where there were no differences in soil CH₄ uptake for three different soil tillage systems (i.e., no tillage, minimum, and conventional tillage) under a long-term tillage system. Furthermore, in the immediate year after alfalfa termination, the method used can affect nitrous oxide (N₂O) emissions, as shown by (Malhi, Lemke, and Schoenau 2010) in a field experiment. They reported higher soil N₂O emissions following alfalfa termination by tillage compared to herbicide, due to the lower levels of soil nitrate under herbicide termination.

In addition to the crop termination method, the type of crop residues can affect the magnitude of soil GHG emissions, and in particular of N₂O (Shan and Yan 2013), which has a global warming potential of 265 times that of CO₂ over a 100-year timescale (IPCC 2014). The effects of crop residues on soil GHG emissions are closely related to the crop residue C:N ratio (Hütsch 2001; Toma and Hatano 2007). Indeed, previous studies have shown that soil N₂O emissions increase at low C:N ratios of crop residues (Toma and Hatano 2007; Shan and Yan 2013). This was associated with easier mineralization of the residues, with a high probability of nitrification and denitrification, and consequent higher N₂O emission. In contrast, in a semi-arid agroecosystem, Badía, Martí, and Aguirre (2013) reported that incorporation of high C:N ratio straw increased soil CO₂ emissions compared to soil without straw amendment. This was associated with the degradation of the added residues, but also of the native soil organic matter, corresponding to a positive priming effect. Indeed, the added labile C stimulates the decomposition of the recalcitrant soil organic matter fraction by providing energy required for its mineralization and to mobilize nutrients for microbial activity and growth. The C:N ratio of crop residues might also influence soil CH₄ fluxes. Indeed, according to Hütsch (2001), crop residues with a low C:N ratio can enhance mineralization processes, which might cause strong inhibition of CH₄ oxidation, with a reduction of the soil CH₄ uptake.

Despite the influence of crop termination methods, types of residues and management on the magnitude of soil GHG emissions, studies comparing crop termination methods in terms of soil GHG emissions are limited (Guardia et al. 2016; Trozzo et al. 2020), especially under Mediterranean conditions.

Based on the above analysis, we hypothesize that alfalfa termination by soil tillage will increase soil GHG emissions over the short term (i.e., ~2 months; 71 days), compared to alfalfa termination by no tillage plus herbicide. To investigate this hypothesis and to fill in the above-mentioned knowledge gaps, the aim of the present study was to evaluate the short-term effects of two different alfalfa termination methods (i.e., Tillage vs No tillage plus herbicide) on soil N₂O, CH₄ and CO₂ emissions in an alfalfa–wheat crop rotation system.

2. Materials and methods

2.1. Study area

The study was carried out in a hilly area of Ancona Province, Marche Region, central Italy (43° 33ʹ N, 13° 25ʹ E; 100 m a.s.l.; SW exposure; 23% slope). The climate was a temperate oceanic sub-Mediterranean variant, with a mean annual rainfall of 788 mm and a mean annual temperature of 14.6 °C. According to Trozzo et al. (2020), at the beginning of the study, the soil at 0.1 m depth was characterized as 254 g kg⁻¹ clay, 363 g kg⁻¹ sand, and 383 g kg⁻¹ silt, pH 8.11. The further soil parameters defined were: C:N ratio, 8.4; soil organic matter, 14.97 g kg⁻¹; total organic carbon, 8.6 g kg⁻¹; total nitrogen, 1.03 g kg⁻¹; humic and fulvic acids, 5.17 g kg⁻¹; cation exchange capacity, 22.53 meq. (100 g)⁻¹; field capacity, 24.6%; and wilting point 17.9%.

2.2. Experimental design

The field experiment began in October 2017, when a homogeneous area in a 6-year-old alfalfa meadow was fenced off, and it ended in late December 2017, 71 days following the alfalfa termination carried out on 11 October 2017 (as day 0 post alfalfa termination).

The two methods of alfalfa termination were Tillage and No tillage plus herbicide. The experimental design adopted was a complete randomized block design with three replicates. The effect of alfalfa termination methods was tested on two subplots, trenched with transparent polyvinyl chloride onduline (diameter, 1 m; height, 0.4 m; open at both ends), included in the main plots of 25 m² (2.5 m × 10.0 m). The type and timing of the soil tillage and herbicide application used for alfalfa termination are given in Table 1.

Table 1. Type and timing of the management practices applied for the two alfalfa termination methods

Alfalfa termination		Days after alfalfa	Management practice
Method	Date	Termination	Operation	Soil depth (m)
Tillage	11 October 2017	0	Spading	0.20
	16 October 2017	5	Harrowing 1	0.15
	21 November 2017	41	Harrowing 2	0.15
No tillage plus herbicide	11 October 2017	0	Herbicide	na

na, not applicable.

2.3. Assessment of soil temperature and water-filled pore space

Soil temperature and soil moisture were measured in each plot from mid-October 2017 to the end of December 2017, with a total of 16 recordings, from 2 days to 9 days apart. For each soil GHG sampling, a hand-held earth digital thermometer (620–0909; VWR, International, Italy) was used to record the soil temperature at a depth of 0.1 m. At the same time, to estimate soil moisture using the oven-drying method, a manual auger was used to collect soil samples at a depth of 0.1 m outside the delimited sub-plot area assuming comparable soil moisture condition outside and inside the sub-plot for this period. The soil moisture content was expressed as water-filled pore space (WFPS) (Schaufler et al. 2010) using Equation (1)(1) $WFPS=SWC/\left(1-BD/PD\right)\times 100$(1) (Linn and Doran 1984):

(1) $WFPS=SWC/\left(1-BD/PD\right)\times 100$(1)

where SWC is the soil water content (vol. %), BD is the soil bulk density (g cm⁻³), and PD is the soil particle density (2.65 g cm⁻³).

2.4. Sampling and analysis of soil greenhouse gas fluxes

The sampling of soil N₂O, CH₄ and CO₂ emissions was performed over ~2 months (71 days) starting on day 7 following the alfalfa termination, with roughly weekly sampling, and after any rain, for a total of 16 recordings. These soil GHG fluxes were measured using closed static chambers (Parkin and Venterea 2010) (height, 0.15 m; diameter, 0.25 m)  equipped with an internal thermometer. At the time of the gas sampling, the chambers (n = 1 per sub-plot; n = 6 per treatment) were fitted onto a polyvinyl chloride base that was permanently installed in the soil (depth, 0.1 m), as reported by Trozzo et al. (2020) who assessed, at the same experimental site, the magnitude of soil N₂O emissions in alfalfa-wheat crop rotation system. Gas sampling was carried out simultaneously for the two alfalfa termination methods, between 9.00 a.m. and 12.00 noon (Krauss et al. 2017). Four gas samples were taken for analysis using a syringe, within a deployment time of 45 min after closing the chamber, which were immediately injected into pre-evacuated vials (30 mL). The N₂O and CH₄ gas concentrations were determined by gas chromatography (GC8A; Shimadzu Corporation, Kyoto, Japan), while CO₂ was determined using an infrared gas analyzer (Li-7000; LI-COR, Lincoln, NE, USA). The fluxes were calculated by verifying the linear slope of the gas concentration over the sampling time (45 min) (Rowlings et al. 2012; Reinsch et al. 18; Vitale et al. 18; Trozzo et al. 2020), with only linear correlation with R² ≥ 0.8 accepted for N₂O and CH₄ and ≥ 0.9 for CO₂. The soil GHG fluxes were calculated according to the following equation:

$F=\frac{M}{V0}\frac{P}{P0}\frac{\left(273+T0\right)}{273+T}h\frac{dC}{dt}$

where T0, P0, and V0 are the absolute air temperature, atmospheric pressure, and molar volume under standard conditions, respectively; M is the molecular weight of gas X; P is the pressure outside the chamber; dC/dt is the slope of the curve of gas X concentration variation with time; h is the height of the chamber from the base ring to the top (Rolston 1986; Bellingrath-Kimura et al. 2015).

The mean emission rates were calculated as the average of the soil GHG fluxes over the study period and were expressed as g N-N₂O ha⁻¹ h⁻¹, g C-CH₄ ha⁻¹ h⁻¹ and kg C-CO₂ ha⁻¹ h⁻¹, respectively. The cumulative soil GHG fluxes over the study period were calculated by linear interpolation between the two sampling dates, and numerical integration using the trapezoidal rule (Rosenstock et al. 2016). The cumulative soil GHG fluxes were used to calculate the total GHG emissions of the soil and are expressed as t CO₂ equivalent (eq.) ha⁻¹, using the conversion factors of 265, 28, and 1 for N₂O, CH₄ and CO₂, respectively (IPCC 2014).

2.5. Statistical analysis

Statistical analysis was performed using SPSS Statistics, version 25.0 (SPSS Inc., IBM, Chicago, IL, USA). Repeated measures ANOVA was used to determine the effects of time (within factor), termination method (between-factor), and their interaction (time × termination method) for the soil temperature, WFPS, and GHG emissions. The normality of the data was monitored by visual inspection, and sphericity was tested using Mauchly’s tests. As the data for the soil GHG emissions violated the normality assumption, Box-Cox transformation was carried out. Differences within each sampling date and between soil total GHG emissions were analyzed with paired Student’s t-tests. Regression analysis was used to relate the flux data to the soil temperature (linear function relationship for N₂O and CO₂, quadratic function for CH₄) and WFPS (linear function for N₂O and CO₂, quadratic function for CH₄). Statistical significance was set at P < 0.05.

3. Results

3.1. Effects of alfalfa termination method on soil temperature and water-filled pore space

During the study, the soil temperature varied with a generally decreasing trend that coincided with an increasing trend for WFPS (Figure 1(a)). The soil temperature at 0.1 m depth ranged from 4.1 ±0.1 °C to 17.9 ±0.9 °C following alfalfa termination by Tillage, and from 4.2 ±0.3°C to 18.1 ±0.7°C following alfalfa termination by No tillage plus herbicide. Soil WFPS at 0.1 m depth ranged from 46.7% ±3.5% to 82.3% ±1.2% following alfalfa termination by Tillage, and from 56.8% ±0.9% to 88.2% ±1.2% following alfalfa termination by No tillage plus herbicide. For the days of measurements, the alfalfa termination method occasionally altered the soil temperature and the WFPS. However, no interactions emerged between the two termination methods and time for both soil temperature and WFPS (Table 2). Conversely, both time and termination method significantly affected the mean soil temperature and WFPS (Table 2). Lower soil temperature and WFPS were generally observed following alfalfa termination by Tillage, compared to that following termination by No tillage plus herbicide (Figure 1(a), Table 2).

Table 2. Mean data and results of repeated measures ANOVA for the soil parameters following the Tillage and No tillage plus herbicide alfalfa termination methods over the study period

Variable	Termination method (mean ±SE)		Source of variation	Statistic
	Tillage	No tillage plus herbicide		DF	F-value	P-value
Soil temperature	10.9 ±0.3	11.3 ±0.3	Time	1.58	1257.45	0.00
(°C)			Termination method	1	62.370	0.02
			Time × termination method	1.58	3.221	0.17
Water-filled pore	66.5 ±0.8	75.4 ±0.2	Time	1.38	21.93	0.02
space (%)			Termination method	1	146.77	0.01
			Time × termination method	1.38	2.30	0.25
Soil N2O flux	0.16 ±0.03	0.14 ±0.03	Time	1.73	3.71	0.14
(N g ha^−1 h^−1)			Termination method	1	1.51	0.34
			Time × termination method	3.46	1.12	0.41
Soil CH4 flux	−0.10 ±0.02	−0.03 ±0.01	Time	1.61	2.81	0.19
(C g ha^−1 h^−1)			Termination method	1	11.92	0.07
			Time × termination method	1.61	1.95	0.27
Soil CO2 flux	0.59 ±0.02	0.37 ±0.02	Time	1.89	34.98	0.00
(C kg ha^−1 h^−1)			Termination method	1	19.85	0.05
			Time × termination method	1.89	3.45	0.14

DF, degrees of freedom; SE, standard error.

Figure 1. Seasonal variations as days after alfalfa termination (day 0) for the soil temperature (ST) and water-filled pore space (WFPS) at 0.10 m depth (a), and of the soil nitrous oxide (N₂O, b), methane (CH₄,c) and carbon dioxide (CO₂, d) fluxes. Data are means ±standard error (n = 3). *, P < 0.05, alfalfa termination by Tillage versus No tillage plus herbicide.

3.2. Effects of alfalfa termination method on soil greenhouse gas dynamics and total emissions

The soil GHG fluxes varied markedly during the study period (Figure 1(b–d)). The soil N₂O fluxes ranged from 0.03 ±0.07 to 0.37 ±0.13 g N-N₂O ha⁻¹ h⁻¹ following alfalfa termination by Tillage, and from 0.03 ±0.04 to 0.31 ±0.10 g N-N₂O ha⁻¹ h⁻¹ following alfalfa termination by No tillage plus herbicide. Both termination methods showed fluctuations in the soil N₂O fluxes throughout the study period, with a general decrease in the emissions (Figure 1(b)). A soil N₂O emission peak was recorded for 37 days after alfalfa termination, when there was an evident increase in the soil N₂O emission flux following alfalfa Tillage (0.37 ±0.13 g N-N₂O ha⁻¹ h⁻¹). Within each sampling day, at day 16 after alfalfa termination, the soil N₂O emission following Tillage was significantly higher compared to No tillage plus herbicide, while at day 44 after alfalfa termination, it was significantly lower (Figure 1(b)). No interactions emerged between these two termination methods and time, and although Tillage generally showed higher soil N₂O emissions, these differences did not reach significance (Table 2).

The soil CH₄ fluxes ranged from −0.29 ±0.04 to −0.01 ±0.03 g C-CH₄ ha⁻¹ h⁻¹ following alfalfa termination by Tillage, and from −0.10 ±0.01 to 0.07 ±0.03 g C-CH₄ ha⁻¹ h⁻¹ following alfalfa termination by No tillage plus herbicide. Both termination methods showed fluctuations in the soil CH₄ fluxes during the study period, with a general uptake following alfalfa termination by Tillage, while No tillage plus herbicide also showed some emissions (Figure 1(c)). Within each sampling day, for days 29, 37 and 51 after alfalfa termination, there was significantly higher soil CH₄ uptake following alfalfa termination by Tillage compared to No tillage plus herbicide (Figure 1(c)). In general, alfalfa termination by Tillage showed higher levels of soil CH₄ uptake compared to alfalfa termination by No tillage plus herbicide, except for day 42, when a small opposite trend was briefly observed. No interaction emerged between the two termination methods and time, and despite Tillage showing higher soil CH₄ uptake, these differences did not reach significance (Table 2).

The soil CO₂ fluxes ranged from 0.15 ±0.05 to 18 kg C-CO₂ ha⁻¹ h⁻¹ following alfalfa termination by Tillage, and from 0.11 ±0.01 to 0.65 ±0.07 kg C-CO₂ ha⁻¹ h⁻¹ following alfalfa termination by No tillage plus herbicide. During the study period, the CO₂ fluxes of both of the alfalfa termination methods showed fluctuations, although with a general decrease, except for two CO₂ emissions peaks recorded for days 16 and 26 after alfalfa termination by Tillage (Figure 1(d)). Within the days of the measurements, alfalfa termination by Tillage showed significantly higher emissions compared to alfalfa termination by No tillage plus herbicide on days 9, 20 and 29 after alfalfa termination (Figure 1(d)). No interactions emerged between the two termination methods and time, although the alfalfa termination method did affect the soil CO₂ fluxes over time (Table 2). Overall, for the first month or so of the monitoring following alfalfa termination, soil CO₂ emissions for Tillage were higher compared to No tillage plus herbicide (Figure 1(d)).

Alfalfa termination by Tillage resulted in significantly higher soil total GHG emissions (as CO₂ eq.) compared to termination by No tillage plus herbicide (Figure 2). For both of these termination methods, CO₂ was the greatest contributor (~96%) to total soil GHG emissions, compared to the N₂O and CH₄ (Figure 2, box).

Figure 2. Soil total greenhouse gas (N₂O + CH₄ + CO₂) emissions over the 71-day study period following alfalfa termination by Tillage and by No tillage plus herbicide. Inset: The weight of each gas compared to the total soil greenhouse gas emissions following each alfalfa termination method. Positive values represent losses; negative values represent uptake. Data are means ±standard error (n = 3). *, P < 0.05, alfalfa termination by Tillage versus

3.3. Relation between soil greenhouse gas emissions and soil temperature and water-filled pore space

The seasonal variations of the soil temperature explained 46% of the soil N₂O emissions for the No tillage plus herbicide methods (Figure 3(a)), with 51% of the soil CH₄ emission for Tillage (Figure 3(b)), and 61% and 74% of the soil CO₂ emissions for Tillage and No tillage plus herbicide, respectively (Figure 3(c)). Conversely, no significant relationships emerged between soil temperatures and soil N₂O emissions for the tillage method (Figure 3(a); P = 0.08) nor between soil temperature and soil CH₄ emissions for the No tillage plus herbicide method (Figure 3(b)).

Figure 3. Relationships between the variations in soil GHG emissions (N₂O, CH₄, CO₂) and soil temperature at 0.1 m depth (a) and soil water-filled pore space (WFPS) at 0.1 m depth (b), following the alfalfa termination by Tillage (dashed line, open circles) and by No tillage plus herbicide (continuous line, closed circles). Data are means ±standard error (n = 3). *, P < 0.05.

The seasonal variations of WFPS explained 62% of the soil CH₄ emission for Tillage (Figure 3(e)), and 52% and 30% of the soil CO₂ emissions for Tillage and No tillage plus herbicide, respectively (Figure 3(f)). Conversely, there were again no significant relationships between WFPS and soil N₂O emissions for the Tillage (P = 0.05) and No tillage plus herbicide (P = 0.06) methods, respectively (Figure 3(d)) nor between WFPS and soil CH₄ emissions for the No tillage plus herbicide method (Figure 3(e)).

For both of the termination methods, the increases in soil temperature lead to increases in soil N₂O (Figure 3(a)) and CO₂ (Figure 3(c)) fluxes, and conversely, the increases in WFPS lead to decreases in these fluxes (Figure 3(d,f)). The dynamics for the soil CH₄ fluxes were less clear, which decreased with soil temperature only following the Tillage termination (Figure 3(b)), and increased with WFPS (Figure 3(e)).

4. Discussion

According to many studies (Abido, Hadházy, and Henzsel 2020; Francioni et al. 2020; Trozzo et al. 2020), soil GHG emissions depend on numerous factors that are related to crop management (e.g., crop rotation, tillage, and residues type) and pedo-climatic conditions (e.g., soil moisture and temperature). This study tested the hypothesis that over the short term, alfalfa termination by No tillage plus herbicide could reduce the soil GHG emissions compared to alfalfa termination by Tillage.

As also seen by other authors for other conservation practices, such as no tillage (Moraru and Rusu 2012), in the present study, the No tillage plus herbicide method showed a higher soil temperature than for Tillage over the monitored period. This might be linked to the generally higher soil moisture for the No tillage plus herbicide method over Tillage, which leads to slower cooling of the soil (Blanco-Canqui and Ruis 18). Moreover, this higher soil moisture for the No tillage plus herbicide method over Tillage might be linked to a lower soil evaporation rate (Schwartz, Baumhardt, and Evett 2010), which would occur mainly in the period immediately after the alfalfa termination and before major rain events. Similar data have been reported under other systems where neither tillage nor herbicides have been applied, with the residues left on top of the soil, as shown by Brunel-Saldias et al. (18).

In the present study, the soil temperature was one of the drivers of the soil N₂O (only for No tillage plus herbicide), CH₄ (only for Tillage) and CO₂ emissions, while the soil N₂O and CH₄ flux was not affected by soil temperature for the Tillage and No tillage plus herbicide method, respectively. Moreover, another important driver of the soil GHG emissions was the soil moisture, in particular for soil CH₄ (only for Tillage) and CO₂ emissions, while the soil N₂O and CH₄ (only for No tillage plus herbicide) flux was not affected by soil moisture.

Similar data were reported by Duan et al. (2019), who investigated the effects of soil addition with manure, biochar, and nitrification inhibitors on soil N₂O emissions. They defined a negative relationship between the fraction of residual unreduced N₂O and soil moisture. This might be related to the processes of complete denitrification (reduction to N₂) (Cayuela et al. 2017) and to reduced diffusion and transport of the gases (Chapuis-lardy et al. 2007) under less aerated soil conditions. In line with the data for the present study, Zhang, Lei, and Yang (2013) also reported decreased soil CO₂ emissions with high soil moisture due to the low effective porosity, with decreased soil temperature to be linked to soil microbes activity and C-depletion (Francioni et al. 2020).

The soil N₂O fluxes did not show any differences between the termination methods, although the mean N₂O flux following alfalfa termination by Tillage was slightly higher than that for No tillage plus herbicide. This was probably due to the soil aeration and the incorporation of residues with low C:N ratios (Trozzo et al. 2020). Contrary to the data here, when Malhi, Lemke, and Schoenau (2010), compared different termination times and methods, they reported that alfalfa termination by Tillage showed significantly higher soil N₂O emissions compared with Herbicide termination, which they attributed to lower levels of nitrate with the Herbicide method. Furthermore, the soil CH₄ fluxes also showed no significant differences between the termination methods here. The higher seasonal mean CH₄ uptake following alfalfa termination by Tillage was probably due to the lower soil moisture for the Tillage method, which might have promoted CH₄ consumption by methanotrophic bacteria (Oertel et al. 2016). It is possible that over the short term, such single tillage does not affect the methanotrophic bacteria, in contrast to the regularly tilled reported by Hütsch (2001).

The higher soil CO₂ fluxes observed following alfalfa termination by Tillage might be due to soil aggregate breakdown (Álvaro-Fuentes et al. 2008a), which leads to exposure of the soil organic matter to decomposition, with a consequent increase in soil CO₂ production. The data obtained in the present study are in line with Álvaro-Fuentes et al. (2008b), who carried out a field experiment in a Mediterranean environment and reported lower seasonal mean soil CO₂ emissions with no tillage than for conventional tillage. These soil CO₂ emission trends are also reflected in the soil total GHG emission for the study period.

Despite the specific trends over the study period, each of the three gases monitored had an impact on the soil total GHG emissions, which were significantly higher for the Tillage termination method compared to No tillage plus herbicide termination (Figure 2), with a greater contribution for CO₂ as observed also by Badagliacca et al. (2020). Following the alfalfa termination by Tillage, greater amounts of alfalfa residues were incorporated into the soil compared to alfalfa termination by No tillage plus herbicide. These residues are characterized by a low C:N ratio, which will probably have affected respiration of the soil microbes, which will lead to increases in soil CO₂ emissions (Nguyen and Marschner 2016).

5. Conclusions

Overall, the data from the present study indicate that these two different alfalfa termination methods influenced the soil GHG emissions over the short term. The termination by Tillage, which leads to the incorporation of crop residues with low C:N ratios and to facilitation of the decomposition of the soil organic matter, will have promoted greater soil CO₂ emission than termination by No tillage plus herbicide. Overall, the highest soil GHG emissions were recorded immediately after the alfalfa termination by Tillage, and were especially linked to higher soil CO₂ emissions. This confirms our hypothesis of reduction soil GHG emissions with the No tillage plus herbicide method. Some questions linked to these soil GHG emissions still remain open, particularly concerning the role of the degradation of the alfalfa roots, and to the effects of herbicide use on the microbial activities over a long-term perspective.

Based on the results of this study, alfalfa termination by No tillage plus herbicide appears to be the practice that better supports the purpose of climate regulation over the short term. At the same time, there is a need to be aware that this management practice can have effects on biodiversity and the associated ecosystem services. Therefore, the potential side effects of the use of herbicides on all the other soil agro-ecosystem components and functions also need to be taken into account for the introduction of such strategic management practices to reduce soil GHG emissions.

Contributions

MT and PD contributed equally; LT, MF, AWKM, RS, MT, PD, conceptualization; LT, MF, AWKM, methodology; LT, MF, AWKM, PD, MT, formal analysis; LT, MF, MT, PD, investigations; AWKM, RS, MT, PD, resources; LT, MF, PD, data curation; LT, MT, PD, MF, original draft preparation; LT, PD, MT, AWKM, RS, MF, review and editing; MT, PD, AWKM, supervision; MT, PD, project administration; MT, PD, AWKM, funding acquisition.

Acknowledgments

We would like to thank the anonymous reviewers for their valuable suggestions and corrections that helped to improve this manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.