Long-term impacts of no-till and organic material applications on soil biological indicators in organic vineyards

Abstract

Background

The integration of conservation tillage and organic material applications presents a promising, sustainable approach to improving soil biological health in organic viticulture systems without relying on synthetic inputs. However, long-term studies evaluating their combined effects on soil microbial biomass and enzyme activities under organic management remain limited.

Methods

This ten-year field study (2012–2021) investigated three tillage systems (conventional, reduced, and no-tillage) combined with four organic material treatments (control, broccoli, Antep radish, and olive mill wastewater) in an organic vineyard. Basal soil respiration, organic carbon content, microbial biomass carbon (MBC) and nitrogen (MBN), and the activities of key soil enzymes (β-glucosidase, alkaline phosphatase, and urease) were measured annually.

Results

Basal soil respiration significantly increased (p <0.05) by 50.0%, from 23.8 mg CO₂-C 100 g⁻¹ in 2015 to 35.7 mg CO₂-C 100 g⁻¹ in 2021, with no-tillage systems showing the highest values (34.1 mg CO₂-C 100 g⁻¹). Soil organic carbon content significantly rose from 1.4% in 2015 to 1.7% in 2021 (p < 0.05), reaching 2.0% under olive mill wastewater treatment. Broccoli application resulted in the highest microbial biomass carbon (246.6 µg g⁻¹) and nitrogen (206.9 µg g⁻¹), both significantly higher than the control (p< 0.05). Enzyme activities, particularly β-glucosidase and alkaline phosphatase, were markedly enhanced under no-tillage and organic material treatments (p < 0.05), with alkaline phosphatase activity peaking at 1113.2 µg pNP g⁻¹ 2h⁻¹ under olive mill wastewater treatment. Correlation analysis revealed strong positive relationships between basal soil respiration and urease activity (r = 0.86), and between β-glucosidase and alkaline phosphatase activity (r = 0.95), indicating synergistic improvements in soil biochemical functioning. In contrast, microbial biomass parameters showed weak negative correlations with enzyme activities, indicating potential trade-offs between microbial growth and enzymatic turnover.

Conclusions

No-tillage systems consistently promoted higher microbial activity and organic carbon levels, confirming their long-term benefits for soil health. Combining no-tillage with organic material amendments, particularly broccoli and Antep radish, represents an effective strategy to enhance microbial and enzymatic functions critical for nutrient cycling. This integrated approach supports the sustainability of organic vineyards by improving soil biological properties and ecosystem resilience.

Introduction

Weed management in organic agriculture represents one of its most challenging aspects, primarily relying on preventive preventative methods such as cover crops, mulches, green manures, and intercropping, where allelopathy can play a significant role. Allelopathy refers to biological interactions between plants in which the growth of one species is suppressed by chemical compounds released by another [1]. These interactions occur through chemical compounds (allelochemicals) released via leaching, evaporation, decomposition, or root exudations. For allelopathy to influence plant growth under field conditions, allelochemicals must accumulate to phytotoxic levels, persist in the soil, and come into contact with target plants [2]. However, once in the soil, their persistence and efficacy are determined by processes such as sorption, transport, and transformation [3]. which are influenced by soil texture, chemical properties, and microbial activity [4, 5].

Numerous metabolites, including carbohydrates, proteins, vitamins, amino acids, and other organic compounds, are released into the rhizosphere by plant roots, serving as important nutritional sources for rhizosphere microorganisms. Low-molecular-weight root exudates can act as allelochemicals and mediate interactions between plants and other organisms [6, 7]. Soil microorganisms can utilize these organic molecules as a carbon source and decompose them, thereby preventing allelochemicals from accumulating to phytotoxic levels. The phytotoxicity stems from reactive oxygen species (ROS) generated during redox cycling between oxidized and reduced states of allelochemicals. Some soil organisms possess enzymes that detoxify ROS [8]. The efficacy of new chemicals emerging after allelochemicals undergo such enzymatic processes may either increase or decrease [9].

Weed suppression utilizing the allelopathic effects of plants belonging to the Brassicaceae family is common in organic systems [10]. These plants, including broccoli and radish, release Glucosinolates (GSL) into the soil as volatile substances to suppress target species such as Verticillium and Fusarium spp [11]. Isothiocyanates (ITCs), formed from the breakdown of GSLs by the myrosinase enzyme, function as biofumigation compounds [12]. However, since the present study did not directly measure GSLs or ITCs, the emphasis is placed on soil microbial and biochemical parameters that reflect the broader impacts of Brassicaceae residue incorporation. Soil microorganisms play a central role in mediating the effects of organic amendments through various biochemical transformations. Microorganisms perform reversible reactions such as oxidation/reduction and acetylation/hydrolysis [13]. Schmidt and Ley [14] suggested that carbon-limited soil organisms rapidly mineralize phenolic compounds due to their higher energy content by weight compared to simple sugars. Phenolic acids can be readily transformed by soil microorganisms from one compound to another with varying degrees of phytotoxicity (e.g., from ferulic acid to vanillic acid) [15]. Such biotransformations performed by soil microorganisms can result in compounds with different biological properties that directly affect the plant’s allelopathic capacity [16]. These microbial-mediated transformations are accompanied by changes in soil enzyme activities, which serve as sensitive indicators of soil biological functioning and organic matter turnover [16].

Olive mill wastewater produced during olive oil production contains readily degradable proteins, sugars, organic acids, polyalcohols, oils, and polyphenols [17]. Its application to soil can stimulate microbial populations (bacteria, yeasts, and fungi) and alter microbial community composition [18]. High-energy organic materials like olive mill wastewater cause a rapid but short-term increase in the number of copiotrophic bacteria colonizing plant roots, while reducing the growth rate of oligotrophic bacteria [19, 20]. The use of such organic materials has been reported to suppress soil-borne pathogens through both general suppression (enhanced overall microbial activity) and specific suppression (increased populations of antagonistic microbes) [21–23]. Moreover, the application of organic amendments influences soil respiration rates and microbial biomass, which are key indicators of microbial activity and nutrient cycling in agricultural soils [21–23]. In organic farming systems, practices such as green/organic fertilization and minimum tillage provide favorable conditions for soil biota. Additionally, the extent to which plants utilize nutrients in these systems depends largely on the decomposition and transformation activities of microorganisms. Tillage practices, ranging from conventional to no-tillage, influence soil structure, aeration, water retention, and the dynamics of soil organic matter decomposition, all of which affect microbial activity and enzyme functions. Understanding how these management practices interact with organic amendments to influence soil biological properties is essential for optimizing sustainable crop production systems.

Organic vineyards, characterized by perennial woody crops with specific soil management requirements, provide an appropriate research model to evaluate the long-term effects of conservation practices on soil biological functioning. Despite the growing body of research on allelopathy and organic soil amendments, long-term field studies assessing how conservation tillage combined with organic material applications affect soil respiration, microbial biomass, and enzyme activity in perennial systems such as organic vineyards, remain scarce. Most existing studies are short-term, focus on annual crops, or consider only a single management factor.

This study is novel in that it simultaneously examines the ten-year interactive effects of tillage intensity and organic material applications (broccoli, Antep radish, olive mill wastewater) on multiple soil biological indicators in organically managed vineyards. By addressing this gap, it provides new insights into sustainable soil management practices that enhance microbial functions and nutrient cycling in perennial horticultural systems. The specific objective of this study was to investigate the combined effects of different organic material applications and tillage practices on soil microbial and biochemical parameters associated with soil quality in organically managed vineyards.

Materials and methods

Experimental site and design

The experimental field at the Manisa Viticulture Research Institute (38°38′0.9″N, 27°23′59.4″E) was established in 2012 to evaluate new approaches to weed control and conservation tillage in organic grape production. Royal grape grafted onto 110 R rootstock was planted as seedlings in 2012 at a spacing of 2 × 3 m. Although the trial was maintained from 2012 to 2022, soil sampling for the present study was carried out in three specific years: 2015, 2018, and 2021. Please note this clarification to resolve the inconsistency between the stated experimental period and the actual soil-sampling years. Before planting, vesicular arbuscular mycorrhiza (Glomus spp.) was applied to the root zone at a rate of 25 propagules per seedling using a syringe to promote root development. The mycorrhizal inoculum (Glomus spp.) was obtained from the Culture Collection of the Department of Soil Science and Plant Nutrition, Faculty of Agriculture, Ege University (İzmir, Türkiye), maintained under sterile sand–soil culture with host plants until application. After inoculation, colonization success was confirmed microscopically using the grid-line intersect method in root samples, and seedlings showed improved root colonization rates compared to non-inoculated controls. The experiment was established using a split-split plot design with three different tillage methods as main plots and four different organic material applications as sub-plots, with three replications (12 vines per replication) for a total of 36 experimental plots. The experimental soil was characterized as having a loam texture, slightly alkaline reaction (pH 7.67), calcium carbonate (CaCO₃) content 4.88%, non-saline condition (0.0069%), and low organic matter content (1.61%). Each main plot measured approximately 20 m × 30 m, and each subplot covered an area of about 10 m × 15 m. One vineyard row (3 m) was left as a border between each main plot, while two vines were used as guard vines between adjacent subplots to prevent edge effects. In total, the experiment included 432 vines, with 12 vines per treatment replication, and an additional 504 isolation vines distributed throughout the vineyard to minimize treatment interference. Conservation agriculture methods constituted the main plots: shallow tillage with a chisel plow (breaking the soil from below at a depth of approximately 20–25 cm), soil cultivation with a disk harrow (10–15 cm), and no-tillage. Different organic material applications were arranged as the sub-plots. Soil sampling was conducted annually after harvest, using an auger to collect samples from the 0–20 cm soil layer at four random points within each subplot. These subsamples were then composited into one representative sample per subplot. In total, three composite samples were collected per treatment replication, corresponding to the three blocks, and stored at 4 °C until laboratory analyses.

Organic material applications

Four organic material treatments were applied in the study: broccoli (Brassica oleracea var. italica, BR), Antep radish (Raphanus sativus L., AT), olive mill wastewater (ZK), and a control (K) with no organic material application. Following soil tillage operations (Ç and GD) in autumn, 6 kg of shredded broccoli, 6 kg of shredded Antep radish, or 6 L of olive mill wastewater were applied per vine and incorporated into the soil in May. In zero-tillage plots, weeds were not incorporated into the soil but were only cut superficially once at the beginning of flowering using a mower. The organic materials used in this study were sourced locally. Fresh broccoli (Brassica oleracea var. italica) and Antep radish (Raphanus sativus L.) residues were obtained from commercial vegetable production fields in Manisa province, Türkiye, harvested at the end of their respective growing seasons. Olive mill wastewater was collected from a local olive oil production facility (olive mill) operating in Manisa province during the olive processing season (November-December). All materials were applied immediately after collection to ensure freshness and biological activity. Also, total phenol concentration was determined spectrophotometrically using Merck/WTW 14,551 phenol reagent kits with a Photometer Nova 60/Spectroquant. Fresh broccoli residues contained 46.8% total carbon and 2.6% total nitrogen, resulting in a C/N ratio of 18. Antep radish residues had a total carbon content of 44.2% and total nitrogen content of 1.6%, with a C/N ratio of 27.6. Olive mill wastewater was characterized by a total organic carbon content of 27.0 g L⁻¹, total nitrogen content of 0.7 g L⁻¹ (C/N ratio of 38.5), and total phenol content of 0.66 g L⁻¹.

Agronomic practices

To meet the nutritional requirements of the plants, organic manure (15 t ha⁻¹) and a green manure mixture consisting of barley (25 kg ha⁻¹), vetch (35 kg ha⁻¹), and broad bean (75 kg ha⁻¹) were applied annually. Throughout the experimental period, no synthetic fertilizers or pesticides were used.

Soil characteristics and soil sampling and analysis

Composite soil samples were collected from each plot at a depth of 0–20 cm in 2015, 2018, and 2021. For each plot, five subsamples were randomly collected and homogenized to form one composite sample, resulting in a total of 27 composite soil samples (9 treatments × 3 years × 1 composite sample per treatment) analyzed over the three sampling years. Before analysis, all soil samples were air-dried and sieved through a 2 mm mesh. For physical and chemical analyses, air-dried samples were used to determine soil texture, pH, water-soluble total salt, lime, organic matter, and total organic carbon (TOC). Subsequently, biochemical analyses were carried out, including soil respiration (TS), microbial biomass carbon (MBC), microbial biomass nitrogen (MBN), and enzyme activities such as dehydrogenase (DHG), alkaline phosphatase (APA), urease (UA), and β-glucosidase (GA). For microbiological analyses, soil samples were stored at field moisture at + 4 °C until processing. MBC and MBN were determined by the chloroform fumigation–extraction method using 0.5 M K₂SO₄ as the extractant. Soil respiration was measured as CO₂ evolution during incubation. Dehydrogenase activity was assessed by the reduction of triphenyltetrazolium chloride (TTC) to triphenyl formazan (TPF). Urease activity was quantified colorimetrically after incubation with urea, using sodium phenolate and sodium hypochlorite reagents to detect released NH₄⁺. Alkaline phosphatase activity was determined with p-nitrophenyl phosphate (pNPP) as the substrate, and β-glucosidase activity with p-nitrophenyl-β-D-glucopyranoside, both producing p-nitrophenol measurable at 400 nm.

Soil texture was determined according to Richards [24], while soil pH in saturated soil was measured following the method of Jackson [25]. Water-soluble total salt content was analyzed according to Soil Survey Staff [26], and lime content was determined based on the method of Çağlar [27]. Organic matter content was assessed using the Walkley and Black [28] method. The chemical characterization of organic materials (total carbon, total nitrogen, and polyphenol content) was determined using standard analytical procedures: total carbon and nitrogen by dry combustion method (LECO analyzer), and total polyphenols in olive mill wastewater by the Folin-Ciocalteu colorimetric method. Soil respiration was evaluated following the methods of Isermeyer [29] and Jäggi [30]. Microbial biomass carbon was determined using the procedures described by Kalembasa and Jenkinson [31] and Vance et al. [32], while mi-crobial biomass nitrogen was analyzed according to Hart and Brookes [33]. Enzyme ac-tivity measurements included dehydrogenase activity following Thalmann [34], alkaline phosphatase activity based on the methods of Tabatabai and Bremner [35] and Eivazi and Tabatabai [36], urease activity according to Kandeler and Gerber [37], and β-glucosidase activity following the method of Hoffman and Dedekan [38].

Data analysis

The study employed a Randomized Complete Block Design (RCBD) with three replications for all experiments. Quantitative data were analyzed to assess the effects of three main factors, years (2015, 2018, 2021), soil management practices (chisel plowing, disk harrowing, and no-tillage), and organic applications (broccoli, Antep radish, olive mill wastewater, and control), as well as their interactions on soil biochemical parameters. Prior to ANOVA, residual normality was tested using the Shapiro–Wilk test, and variance homogeneity was checked with Levene’s test. A comprehensive three-way analysis of variance (ANOVA) was performed using IBM SPSS Statistics V22.0. To identify statistically significant differences among treatment means, Duncan’s multiple range test was applied at a significance level of p < 0.05. Results are expressed as mean values with their corresponding standard errors (SE), while detailed data, including means and standard deviations (SD) for all treatments, are provided in the supplementary material. To further examine the relationships between variables, principal component analysis (PCA) was conducted using GraphPad Prism version 9.3.1 (GraphPad Software, LLC, San Diego, CA, USA). The PCA results were visualized through a biplot, following the approach outlined by Evgenidis et al. [39]. Additionally, a hierarchical clustering heat map was generated to illustrate the associations and intensity of interactions among the studied factors and parameters. This visualization was created using the SRPLOT online platform (https://www.bioinformatics.com.cn/en), accessed on February 10, 2025.

Results

Our findings revealed that Year (Y), Soil Management Treatment (SMT), and Organic Material Application (OMA) had significant effects on most soil biochemical parameters. Interaction effects varied; Y × OMA significantly influenced soil respiration (p = 0.022). Y × SMT had significant effects on soil organic carbon (p = 0.002), microbial biomass C (p = 0.001), dehydrogenase activity (p = 0.015), alkaline phosphatase activity (p = 0.011), and β-Glucosidase activity (p = 0.002). SMT × OMA showed no significant effects, while Y × SMT × OMA was significant only for microbial biomass C (p = 0.011) (Table 1).

Table 1.

ANOVA results for the effects of year (Y), soil management treatment (SMT), and organic material application (OMA) on soil parameters

Source	Y (F, p)	SMT (F, p)	OAA (F, p)	Y × SMT (F, p)	Y × OAA (F, p)	SMT×OAA (F,p)	Y × SMT×OAA (F, p)
Soil respiration	51.424, p < 0.001*	9.791, p < 0.001*	4.678, p = 0.005*	1.826, p = 0.133 (ns)	2.659, p = 0.022*	1.106, p = 0.368 (ns)	0.820, p = 0.630 (ns)
Soil organic carbon	68.009, p < 0.001*	13.351, p < 0.001*	20.291, p < 0.001*	4.546, p = 0.002*	0.848, p = 0.538 (ns)	2.106, p = 0.063 (ns)	1.201, p = 0.299 (ns)
Microbial biomass C	9.008, p < 0.001*	19.153, p < 0.001*	24.052, p < 0.001*	5.014, p = 0.001*	5.039, p < 0.001*	0.977, p = 0.447 (ns)	2.423, p = 0.011*
Microbial biomass N	3.031, p = 0.054 (ns)	13.443, p < 0.001*	6.532, p = 0.001*	0.716, p = 0.583 (ns)	0.448, p = 0.844 (ns)	0.833, p = 0.548 (ns)	0.462, p = 0.930 (ns)
Dehydrogenase activity	82.250, p < 0.001*	9.025, p < 0.001*	2.952, p = 0.038*	3.319, p = 0.015*	0.397, p = 0.879 (ns)	0.704, p = 0.647 (ns)	0.975, p = 0.480 (ns)
Urease enzyme activity	56.285, p < 0.001*	3.347, p = 0.041*	1.617, p = 0.193 (ns)	0.883, p = 0.479 (ns)	0.368, p = 0.897 (ns)	0.325, p = 0.922 (ns)	0.553, p = 0.872 (ns)
Alkaline phosphatase activity	197.303, p < 0.001*	22.882, p < 0.001*	2.577, p = 0.060 (ns)	3.503, p = 0.011*	1.484, p = 0.196 (ns)	0.309, p = 0.930 (ns)	1.136, p = 0.346 (ns)
β-Glucosidase activity	490.770, p < 0.001*	24.752, p < 0.001*	3.428, p = 0.021*	4.683, p = 0.002*	1.381, p = 0.234 (ns)	0.832, p = 0.549 (ns)	1.498, p = 0.145 (ns)

ns not significant; *p < 0.05. Values represent the results of ANOVA (F and p values) for the effects of year (Y), soil management treatment (SMT), and organic amendment application (OAA) on soil biochemical properties

Soil respiration and soil organic carbon

For soil respiration, values ranged from 19.5 mg CO₂-C 100 g^− 1 to 44.4 mg CO₂-C 100 g^− 1 across treatments. The average soil respiration increased over time, from 23.8 mg CO₂-C 100 g^− 1 in 2015 to 35.7 mg CO₂-C 100 g^− 1 in 2021, representing a 50.0% increase. No-tillage (NT) had the highest respiration (34.1 mg CO₂-C 100 g^− 1), while disc harrow had the lowest (30.4 mg CO₂-C 100 g^− 1). Among organic material applications, broccoli-treated plots showed the highest soil respiration (33.4 mg CO₂-C 100 g^− 1), while olive mill wastewater-treated plots had the lowest (29.8 mg CO₂-C 100 g^− 1). Soil organic carbon ranged from 1.2 ± 0.0 to 2.4% across treatments. The overall average increased from 1.4% in 2015 to 1.7% in 2021, showing a 21.4% rise. No-tillage maintained the highest organic carbon content (1.8%), while disc harrow had the lowest (1.5%). Among organic material treatments, olive mill wastewater application resulted in the highest organic carbon (2.0%), whereas the control treatment showed the lowest (1.4%). Among soil management techniques, no-tillage resulted in the highest average soil respiration (34.1 mg CO₂-C 100 g^− 1) and soil organic carbon (1.8%), while disc harrow had the lowest values. Organic material applications showed that broccoli and olive mill wastewater had the highest impact on soil respiration and organic carbon, respectively. When averaging the effects of disc harrow, chisel, and no-tillage, no-tillage exhibited superior results in both parameters. The overall average values for soil respiration and soil organic carbon were 31.1 mg CO₂-C 100 g^− 1 and 1.7%, respectively (Table 2).

Table 2.

Effect of soil management techniques and organic material applications on soil respiration and soil organic carbon over time

Soil Management Techniques	Organic Material Application	Soil respiration				Soil organic carbon
		2015	2018	2021	Average of Years	2015	2018	2021	Average of Years
Disc Harrow	Control	25.1 ± 1.6ns	31.7 ± 8.8ns	29.7 ± 0.8ns	28.8 ± 5.3ns	1.1 ± 0.1ns	1.3 ± 0.0ns	1.5 ± 0.3ns		1.3 ± 0.3ns
	Broccoli	22.3 ± 2.3	31.2 ± 2.7	46.5 ± 9.8	33.3 ± 11.8	1.3 ± 0.1	1.7 ± 0.1	1.9 ± 0.1		1.6 ± 0.3
	Antep radish	21.7 ± 1.6	34.7 ± 8.8	31.8 ± 3.6	29.4 ± 7.6	1.4 ± 0.2	1.5 ± 0.2	1.9 ± 0.3		1.6 ± 0.3
	Olive mill wastewater	22.4 ± 1.9	31.0 ± 7.7	37.0 ± 3.6	30.1 ± 7.7	1.5 ± 0.1	1.5 ± 0.3	2.0 ± 0.1		1.6 ± 0.3
Chisel	Control	20.3 ± 2.1	31.9 ± 6.9	27.1 ± 5.6	26.5 ± 6.8	1.2 ± 0.0	1.3 ± 0.1	1.2 ± 0.1		1.2 ± 0.1
	Broccoli	19.6 ± 5.0	32.0 ± 7.0	34.9 ± 1.3	28.9 ± 8.3	1.7 ± 0.0	1.8 ± 0.1	1.9 ± 0.0		1.8 ± 0.1
	Antep radish	27.0 ± 2.4	36.1 ± 10.6	33.2 ± 3.8	32.1 ± 7.0	1.4 ± 0.1	1.8 ± 0.1	2.2 ± 0.1		1.8 ± 0.3
	Olive mill wastewater	19.5 ± 3.5	34.4 ± 6.5	28.1 ± 3.0	27.4 ± 7.6	1.4 ± 0.2	2.1 ± 0.6	2.1 ± 0.4		1.8 ± 0.5
No-Tillage	Control	27.1 ± 2.2	31.6 ± 5.2	33.2 ± 2.3	30.6 ± 4.1	1.2 ± 0.0	1.6 ± 0.1	2.1 ± 0.1		1.6 ± 0.4
	Broccoli	31.9 ± 2.4	37.8 ± 9.3	44.4 ± 6.9	38.0 ± 8.0	1.6 ± 0.4	1.8 ± 0.2	2.4 ± 0.3		2.0 ± 0.4
	Antep radish	27.0 ± 5.9	39.3 ± 5.2	41.0 ± 1.3	35.8 ± 7.7	1.5 ± 0.3	1.7 ± 0.2	2.4 ± 0.3		1.9 ± 0.5
	Olive mill wastewater	21.8 ± 3.1	32.8 ± 4.7	41.4 ± 6.3	32.0 ± 9.5	1.4 ± 0.2	1.7 ± 0.1	2.2 ± 0.3		1.8 ± 0.4
Average of organic material application	Control	24.2 ± 3.5 de	31.7 ± 6.2 bc	30.0 ± 4.0 cd	28.6 ± 5.6 C	1.2 ± 0.1ns	1.4 ± 0.2	1.6 ± 0.4		1.4 ± 0.3ns
	Broccoli	24.6 ± 6.3 de	33.7 ± 6.7 bc	41.9 ± 8.0 a	33.4 ± 9.9 A	1.5 ± 0.3	1.7 ± 0.1	2.1 ± 0.3		1.8 ± 0.3
	Antep radish	25.2 ± 4.2 de	36.7 ± 7.6 ab	35.3 ± 5.1 bc	32.4 ± 7.6 AB	1.4 ± 0.2	1.7 ± 0.2	2.2 ± 0.3		1.8 ± 0.4
	Olive mill wastewater	21.2 ± 2.9 e	32.8 ± 5.8 bc	35.5 ± 7.1 bc	29.8 ± 8.2 BC	1.4 ± 0.1	1.7 ± 0.4	2.1 ± 0.3		1.7 ± 0.4
Average of soil management techniques	Average of disc harrow	22.9 ± 2.1ns	32.1 ± 6.5ns	36.2 ± 8.3ns	30.4 ± 8.3 B	1.3 ± 0.2 d	1.5 ± 0.2 cd	1.8 ± 0.3 b		1.5 ± 0.3 C
	Average of chisel	21.6 ± 4.4	33.6 ± 7.0	30.8 ± 4.7	28.7 ± 7.4 B	1.4 ± 0.2 d	1.7 ± 0.4 b	1.9 ± 0.4 b		1.7 ± 0.4 B
	Average of no-tillage	27.0 ± 4.9	35.4 ± 6.4	40.0 ± 6.0	34.1 ± 7.9 A	1.5 ± 0.3 cd	1.7 ± 0.1 bc	2.3 ± 0.3 a		1.8 ± 0.4 A

Means were separated by Duncan’s multiple range test at the p < 0.05 level. Within each column, means followed by different lowercase letters indicate significant differences among treatments or years, while means followed by different uppercase letters indicate significant differences among averages of organic material applications or soil management techniques. Values marked with ‘ns’ denote non-significant differences

Microbial biomass C and microbial biomass N

For microbial biomass C, values varied across years and treatments. In 2015, values ranged from 144.9 µg g^− 1 (Chisel-Control) to 276.8 µg g⁻¹ (No-Tillage-Broccoli). In 2018, the highest value was observed under Chisel-Antep Radish (230.0 ± 38.4 µg g^− 1), while the lowest was under Disc Harrow-Olive Mill Wastewater (129.3 µg g^− 1). By 2021, microbial biomass C ranged from 132.1 µg g^− 1 (Disc Harrow-Control) to 233.7 µg g^− 1 (Chisel-Broccoli). The average across years for microbial biomass C was highest in broccoli-applied soils (246.6 µg g^− 1) and lowest in control treatments (153.0 µg g^− 1). Among soil management techniques, No-Tillage had the highest average (213.5 µg g^− 1), while Disc Harrow had the lowest (195.1 µg g^− 1). For microbial biomass N, values also showed temporal and treatment-dependent variations. In 2015, the lowest value was 35.3 µg g^− 1 (Disc Harrow-Olive Mill Wastewater), while the highest was 75.9 µg g^− 1 (Chisel-Olive Mill Wastewater). In 2018, values ranged from 20.9 µg g^− 1 (Disc Harrow-Antep Radish) to 68.8 µg g^− 1 (Chisel-Broccoli). By 2021, microbial biomass N ranged between 22.5 µg g^− 1 (Disc Harrow-Olive Mill Wastewater) and 81.5 µg g^− 1 (Chisel-Broccoli). The highest average across years was observed in Broccoli-applied soils (206.9 µg g^− 1), while the lowest was in Control treatments (160.6 µg g^− 1). Among soil management techniques, Chisel had the highest average (206.6 µg g^− 1), whereas No-Tillage had the lowest (181.6 µg g^− 1). The overall average of microbial biomass C across all treatments and years was 200.7 µg g-1, while for microbial biomass N, it was 186.57 µg g^− 1 (Table 3).

Table 3.

Effect of soil management techniques and organic material applications on microbial biomass C and microbial biomass N over time

Soil Management Techniques	Organic Material Application	Microbial biomass C				Microbial biomass N
		2015	2018	2021	Average of Years	2015	2018	2021	Average of Years
Disc Harrow	Control	160.8±5.7 f-j	155.9±49.9 g-j	132.1±12.0 j	149.6±29.0ns	53.7±10.4ns	34.8±1.7ns	33.3±3.5ns	40.6±11.3ns
	Broccoli	208.2±33.8 b-f	169.2±18.4 f-j	161.3±8.3 f-j	179.6±29.3	62.5±8.5	52.0±6.3	50.0±6.4	54.9±8.5
	Antep radish	167.6±13.1 f-j	207.7±7.1 b-f	204.6±3.9 c-g	193.3±20.8	38.2±39.7	40.4±8.0	29.0±13.7	35.9±22.0
	Olive mill wastewater	207.1±7.3 b-f	129.3±22.0 j	151.4±19.0 h-j	162.6±37.8	35.3±25.8	29.0±5.1	22.5±8.1	28.9±14.8
Chisel	Control	144.9±28.8 ij	183.1±9.3 d-i	195.6±19.4 c-h	174.6±29.1	52.3±8.0	47.5±20.9	64.9±9.5	54.9±14.4
	Broccoli	254.7±20.2 ab	219.4±17.7 b-e	233.7±9.6 a-c	235.9±21.0	62.5±44.5	68.8±19.3	81.5±3.9	70.9±25.7
	Antep radish	223.4±35.0 b-e	230.0±38.4 b-d	233.1±28.9 a-c	228.8±30.0	65.3±14.4	51.1±1.2	57.7±10.3	58.0±10.8
	Olive mill wastewater	188.1±17.5 c-i	190.2±22.4 c-i	182.6±6.2 d-i	186.9±14.9	75.9±20.2	53.9±1.1	53.4±3.7	61.1±15.2
No-Tillage	Control	153.2±27.6 h-j	152.4±16.6 h-j	167.7±16.4 f-j	157.7±19.6	43.0±6.2	36.5±5.3	37.9±5.1	39.2±5.7
	Broccoli	276.8±44.5 a	193.3±55.0 c-i	145.6±26.0 ij	205.2±68.8	66.3±26.6	62.7±6.0	63.0±6.1	64.0±14.1
	Antep radish	233.7±31.5 a-c	175.9±20.6 e-j	176.6±18.8 e-j	195.4±35.6	65.4±39.2	42.3±3.8	45.1±0.8	50.9±22.5
	Olive mill wastewater	190.2±9.6 c-i	165.0±39.1 f-j	148.5±9.2 h-j	167.9±27.5	55.8±42.0	43.9±4.2	46.0±6.1	48.6±22.0
Average of organic material application	Control	153.0±21.3 d	163.8±30.4 cd	165.1±30.9 cd	160.6±27.4 B	49.7±8.9ns	39.6±12.3	45.3±15.8	44.9±12.9 B
	Broccoli	246.6±42.5 a	194.0±37.3 bc	180.2±43.2 b-d	206.9±49.1 A	63.8±26.3	61.2±12.9	64.8±14.5	63.2±18.2 A
	Antep radish	208.2±39.4 b	204.5±32.3 b	204.7±30.0 b	205.8±32.8 A	56.3±31.8	44.6±6.7	43.9±15.1	48.3±20.7 B
	Olive mill wastewater	195.1±13.9 bc	161.5±36.5 cd	160.8±19.7 cd	172.5±29.2 B	55.7±31.9	42.2±11.3	40.6±15.0	46.2±21.7 B
Average of soil management techniques	Average of disc harrow	185.9±27.9 ab	165.5±38.5 b	162.4±29.6 b	171.3±33.1 B	47.4±24.0ns	39.0±10.2	33.7±13.0	40.1±17.3 C
	Average of chisel	202.8±48.2 a	205.7±29.2 a	211.3±28.3 a	206.6±35.5 A	64.0±23.7	55.3±14.8	64.4±12.9	61.2±17.7 A
	Average of no-tillage	213.5±55.2 a	171.6±34.7 b	159.6±20.9 b	181.6±44.9 B	57.6±28.8	46.4±11.1	48.0±10.5	50.7±19.0 B

Means were separated by Duncan’s multiple range test at the p < 0.05 level. Within each column, means followed by different lowercase letters indicate significant differences among treatments or years, while means followed by different uppercase letters indicate significant differences among averages of organic material applications or soil management techniques. Values marked with ‘ns’ denote non-significant differences

Soil dehydrogenase activity and urease enzyme activity

Our results demonstrated significant variations in dehydrogenase and urease enzyme activities depending on soil management techniques, organic material applications, and years. Under the Disc Harrow method, dehydrogenase activity in the Control showed fluctuations, starting at 348.2 µg TPF g^− 1 in 2015, decreasing to 104.9 µg TPF g^− 1 in 2018, and rising again to 321.4 µg TPF g^− 1 in 2021. A similar trend was observed in the Broccoli treatment, where activity values were 337.1 µg TPF g^− 1 in 2015, 135.3 µg TPF g^− 1 in 2018, and 347.4 µg TPF g^− 1 in 2021. The Chisel system exhibited a different pattern, with the Control group decreasing from 212.1 µg TPF g^− 1 in 2015 to 114.4 µg TPF g^− 1 in 2018, followed by an increase to 257.8 µg TPF g^− 1 in 2021. The Broccoli treatment under Chisel showed a steady increase across years, with values of 269.0 µg TPF g^− 1 in 2015, 160.9 µg TPF g^− 1 in 2018, and 281.9 µg TPF g^− 1 in 2021. Under No-Tillage, dehydrogenase activity in the Control increased significantly over time, from 213.8 µg TPF g^− 1 in 2015 to 411.1 µg TPF g^− 1 in 2021. The Broccoli treatment exhibited a similar pattern, with activity levels increasing from 331.9 µg TPF g^− 1 in 2015 to 458.5 µg TPF g-1 in 2021. For urease enzyme activity, results varied across treatments and years. Under the Disc Harrow method, the Control had values of 90.6 µg N g^− 1 2 h^− 1 in 2015, increasing to 159.8 µg N g-1 2 h^− 1 in 2021. The Broccoli treatment followed a similar pattern, with values rising from 74.9 µg N g^− 1 2 h^− 1 in 2015 to 170.1 µg N g^− 1 2 h^− 1 in 2021. In the Chisel system, urease enzyme activity in the Control increased from 42.1 µg N g^− 1 2 h^− 1 in 2015 to 165.4 µg N g^− 1 2 h^− 1 in 2018, then slightly decreased to 143.8 µg N g^− 1 2 h^− 1 in 2021. The Broccoli treatment showed a more stable increase, reaching 162.3 µg N g-1 2 h-1 in 2021. Under No-Tillage, urease enzyme activity in the Control increased from 76.9 µg N g-1 2 h-1 in 2015 to 178.4 µg N g^− 1 2 h^− 1 in 2018, followed by a slight decline in 2021. The Broccoli treatment under No-Tillage exhibited an increasing trend, peaking at 232.4 µg N g^− 1 2 h^− 1 in 2021. When comparing organic material applications, dehydrogenase activity ranged from 235.6 µg TPF g^− 1 in the Control group to 289.0 µg TPF g^− 1 in the Antep radish treatment. The highest urease enzyme activity was recorded in the Antep radish treatment (160.6 µg N g^− 1 2 h^− 1). The overall average dehydrogenase activity across all treatments and years was 264.1 µg TPF g^− 1 while the overall urease enzyme activity was 144.9 µg N g^− 1 2 h^− 1 (Table 4).

Table 4.

Effect of soil management techniques and organic material applications on dehydrogenase activity and urease enzyme activity N over time. Means were separated by duncan’s multiple range test at the p < 0.05 level. Within each column, means followed by different lowercase letters indicate significant differences among treatments or years, while means followed by different uppercase letters indicate significant differences among averages of organic material applications or soil management techniques. Values marked with ‘ns’ denote non-significant differences

Soil Management Techniques	Organic Material Application	Dehydrogenase activity (µg TPF g− 1)				Urease enzyme activity (µg TPF g− 1)
		2015	2018	2021	Average of Years	2015	2018	2021	Average of Years
Disc Harrow	Control	348.2 ± 42.9ns	104.9 ± 31.8ns	321.4 ± 42.3ns	258.2 ± 120.4ns	90.6 ± 15.0ns	146.1 ± 61.4ns	159.8 ± 22.5ns	132.2 ± 46.2ns
	Broccoli	337.1 ± 48.1	135.3 ± 62.0	347.4 ± 28.7	273.3 ± 111.7	74.9 ± 8.5	196.8 ± 73.5	170.1 ± 13.2	147.3 ± 67.0
	Antep radish	210.2 ± 20.2	159.4 ± 52.9	403.2 ± 61.3	257.6 ± 119.0	57.9 ± 11.0	155.8 ± 68.7	210.2 ± 61.7	141.3 ± 81.5
	Olive mill wastewater	261.9 ± 55.9	146.4 ± 38.4	368.4 ± 6.7	258.9 ± 102.0	53.3 ± 4.0	170.9 ± 78.2	198.5 ± 59.1	140.9 ± 82.9
Chisel	Control	212.1 ± 88.1	114.4 ± 23.6	257.8 ± 42.1	194.8 ± 80.9	42.1 ± 5.9	165.4 ± 38.6	143.8 ± 16.8	117.1 ± 60.9
	Broccoli	269.0 ± 102.6	160.9 ± 22.5	281.9 ± 41.3	237.3 ± 80.6	81.3 ± 13.4	168.9 ± 53.9	162.3 ± 13.4	137.5 ± 51.0
	Antep radish	320.8 ± 262.7	147.0 ± 29.5	334.8 ± 106.5	267.5 ± 168.9	92.6 ± 22.6	186.4 ± 71.5	163.1 ± 20.1	147.4 ± 57.4
	Olive mill wastewater	243.3 ± 70.4	129.6 ± 51.6	267.1 ± 45.3	213.3 ± 80.4	68.0 ± 9.8	189.0 ± 95.5	141.0 ± 16.3	132.6 ± 71.8
No-Tillage	Control	218.5 ± 32.9	132.0 ± 14.3	411.1 ± 59.3	253.9 ± 128.5	76.9 ± 12.1	178.4 ± 68.8	168.2 ± 28.0	141.2 ± 61.3
	Broccoli	331.9 ± 49.2	191.5 ± 26.8	458.5 ± 36.6	327.3 ± 120.4	134.6 ± 24.0	188.6 ± 68.3	232.4 ± 52.4	185.2 ± 61.6
	Antep radish	381.8 ± 82.4	152.2 ± 31.5	491.2 ± 183.4	341.7 ± 181.1	100.0 ± 6.5	181.2 ± 76.7	198.2 ± 12.2	159.8 ± 59.9
	Olive mill wastewater	278.9 ± 80.9	149.9 ± 20.2	428.2 ± 45.6	285.7 ± 129.7	70.6 ± 2.3	194.1 ± 65.0	203.8 ± 55.3	156.2 ± 77.2
Average of organic material application	Control	259.6 ± 84.2ns	117.1 ± 24.2	330.1 ± 78.9	235.6 ± 111.5 B	69.8 ± 23.9ns	163.3 ± 51.9	157.3 ± 22.5	130.2 ± 55.3ns
	Broccoli	312.7 ± 70.0	162.6 ± 43.1	362.6 ± 83.3	279.3 ± 108.3 A	96.9 ± 31.8	184.8 ± 58.3	188.3 ± 43.4	156.7 ± 61.6
	Antep radish	304.2 ± 157.3	152.9 ± 34.6	409.8 ± 129.6	289.0 ± 157.1 A	83.5 ± 23.4	174.5 ± 64.3	190.5 ± 39.3	149.5 ± 65.0
	Olive mill wastewater	261.4 ± 62.4	141.9 ± 35.0	354.6 ± 77.6	252.6 ± 106.3 AB	64.0 ± 9.7	184.6 ± 70.5	181.1 ± 51.1	143.2 ± 75.0
Average of soil management techniques	Average of disc harrow	289.3 ± 69.8 c	136.5 ± 45.8 d	360.1 ± 46.3 b	262.0 ± 108.7 B	69.2 ± 17.8ns	167.4 ± 63.5	184.6 ± 43.7	140.4 ± 68.0 AB
	Average of chisel	261.3 ± 136.0 c	138.0 ± 34.2 d	285.4 ± 63.5 c	228.2 ± 108.4 B	71.0 ± 23.1	177.4 ± 59.2	152.5 ± 17.9	133.7 ± 59.1 B
	Average of no-tillage	302.8 ± 84.2 bc	156.4 ± 30.6 d	447.3 ± 91.6 a	302.2 ± 140.2 A	95.5 ± 28.7	185.6 ± 59.9	200.6 ± 42.3	160.6 ± 64.5 A

Means were separated by Duncan’s multiple range test at the p < 0.05 level. Within each column, means followed by different lowercase letters indicate significant differences among treatments or years, while means followed by different uppercase letters indicate significant differences among averages of organic material applications or soil management techniques. Values marked with ‘ns’ denote non-significant differences

Soil alkaline phosphatases activity and β-glucosidase

In our findings, Disc Harrow treatments showed alkaline phosphatase activity increasing from 481.6 µg p-NP g^− 1 h^− 1 in 2015 to 758.6 µg p-NP g^− 1 h^− 1 in 2021 for the control, and from 464.2 µg p-NP g^− 1 h^− 1 to 816.0 µg p-NP g^− 1 h^− 1 for broccoli, 364.2 µg p-NP g^− 1 h^− 1 to 964.3 µg p-NP g^− 1 h^− 1 for Antep radish, and 329.6 µg p-NP g^− 1 h^− 1 to 861.2 µg p-NP g^− 1 h^− 1 for olive mill wastewater. The β-glucosidase activity in disc harrow treatments initially decreased from 133.2 µg Saligenin g^− 1 3 h^− 1 in 2015 to 72.6 µg Saligenin g^− 1 3 h^− 1 in 2018 for control before increasing to 239.4 µg Saligenin g^− 1 3 h^− 1 in 2021. Chisel treatments displayed alkaline phosphatase values of 290.5 µg p-NP g^− 1 h^− 1 (2015), 464.7 µg p-NP g^− 1 h^− 1 (2018), and 729.8 µg p-NP g^− 1 h^− 1 (2021) for control plots. With organic material applications, chisel treatments reached 759.5 µg p-NP g^− 1 h^− 1 (broccoli), 816.1 µg p-NP g^− 1 h^− 1 (Antep radish), and 735.3 µg p-NP g^− 1 h^− 1 (olive mill wastewater) in 2021. For β-glucosidase, chisel treatments showed values of 61.0 µg Saligenin g^− 1 3 h^− 1 (2015), 69.8 µg Saligenin g^− 1 3 h^− 1 (2018), and 212.5 µg Saligenin g^− 1 3 h^− 1 (2021) for control plots. No-Tillage showed the highest enzyme activities with alkaline phosphatase values of 475.8 µg p-NP g^− 1 h^− 1 (2015), 538.0 µg p-NP g^− 1 h^− 1 (2018), and 909.2 µg p-NP g^− 1 h^− 1 (2021) for control, reaching as high as 1113.2 µg p-NP g^− 1 h^− 1 with olive mill wastewater in 2021. β-glucosidase activity in no-tillage reached 304.4 µg Saligenin g^− 1 3 h^− 1 with broccoli application in 2021, compared to 188.8 µg Saligenin g^− 1 3 h^− 1 in 2015. Among organic material applications, Antep radish produced the highest average alkaline phosphatase activity (632.3 µg p-NP g^− 1 h^− 1), followed by broccoli (623.2 µg p-NP g^− 1 h^− 1), olive mill wastewater (586.8 µg p-NP g^− 1 h^− 1), and control (563.8 µg p-NP g^− 1 h^− 1). For β-glucosidase, broccoli showed the highest average at 161.7 µg Saligenin g^− 1 3 h^− 1, followed by Antep radish (157.2 µg Saligenin g^− 1 3 h^− 1), control (142.9 µg Saligenin g^− 1 3 h^− 1), and olive mill wastewater (142.7 µg Saligenin g^− 1 3 h^− 1). The overall average alkaline phosphatase activity across all treatments increased from 429.5 µg p-NP g^− 1 h^− 1 in 2015 to 878.1 µg p-NP g^− 1 h^− 1 in 2021, while the overall average β-glucosidase activity increased from 104.8 µg Saligenin g^− 1 3 h^− 1 in 2015 to 267.6 µg Saligenin g^− 1 3 h^− 1 in 2021 (Table 5).

Table 5.

Effect of soil management techniques and organic material applications on alkaline phosphatases activity and β-Glucosidase activity over time

Soil Management Techniques	Organic Material Application	Alkaline phosphatases activity				β-Glucosidase activity
		2015	2018	2021	Average of Years	2015	2018	2021	Average of Years
Disc Harrow	Control	481.6 ± 29.7ns	425.8 ± 81.1ns	758.6 ± 95.4ns	555.4 ± 167.2ns	133.2 ± 18.4ns	72.6 ± 35.0ns	239.4 ± 10.1ns	148.4 ± 75.9ns
	Broccoli	464.2 ± 1.0	450.7 ± 82.8	816.0 ± 84.5	576.9 ± 188.9	113.0 ± 2.6	80.6 ± 33.8	280.4 ± 35.5	158.0 ± 96.1
	Antep radish	364.2 ± 11.0	491.9 ± 12.7	964.3 ± 85.9	606.8 ± 290.0	89.0 ± 39.1	78.5 ± 32.9	285.3 ± 21.0	150.9 ± 104.6
	Olive mill wastewater	329.6 ± 3.4	508.6 ± 18.7	861.2 ± 76.6	566.5 ± 254.5	67.2 ± 6.4	84.2 ± 43.8	288.3 ± 42.5	146.6 ± 110.9
Chisel	Control	290.5 ± 32.8	464.7 ± 85.1	729.8 ± 23.4	495.0 ± 197.3	61.0 ± 21.3	69.8 ± 23.2	212.5 ± 11.3	114.4 ± 75.5
	Broccoli	367.2 ± 29.2	517.0 ± 89.6	759.5 ± 99.5	547.9 ± 184.7	80.9 ± 13.5	82.9 ± 25.2	235.2 ± 7.3	133.0 ± 78.1
	Antep radish	397.4 ± 89.4	518.3 ± 10.1	816.1 ± 10.7	577.3 ± 206.8	80.1 ± 11.0	84.9 ± 21.4	257.1 ± 17.3	140.7 ± 88.6
	Olive mill wastewater	360.5 ± 43.6	459.0 ± 18.6	735.3 ± 84.0	518.3 ± 197.0	84.9 ± 14.0	75.0 ± 26.9	216.9 ± 14.5	125.6 ± 70.6
No-Tillage	Control	475.8 ± 61.5	538.0 ± 13.9	909.2 ± 57.4	641.0 ± 217.5	117.9 ± 29.3	81.4 ± 28.5	298.1 ± 18.6	165.8 ± 103.0
	Broccoli	619.7 ± 60.0	587.8 ± 14.8	1026.6 ± 84.2	744.7 ± 230.4	188.8 ± 11.0	89.6 ± 37.5	304.4 ± 62.5	194.3 ± 100.1
	Antep radish	594.8 ± 64.9	496.4 ± 21.6	1047.1 ± 193.5	712.8 ± 294.3	139.4 ± 21.6	98.1 ± 35.4	302.7 ± 30.0	180.1 ± 97.1
	Olive mill wastewater	408.1 ± 24.9	505.3 ± 13.2	1113.2 ± 109.7	675.6 ± 342.5	102.7 ± 30.5	74.0 ± 10.6	291.6 ± 35.5	156.1 ± 105.1
Average of organic material application	Control	416.0 ± 101.5ns	476.2 ± 101.2	799.2 ± 101.0	563.8 ± 197.1ns	104.0 ± 38.7ns	74.6 ± 25.9	250.0 ± 39.8	142.9 ± 85.2 B
	Broccoli	483.7 ± 115.3	518.5 ± 113.0	867.4 ± 144.5	623.2 ± 213.6	127.6 ± 48.8	84.3 ± 28.5	273.3 ± 47.2	161.7 ± 92.0 A
	Antep radish	452.1 ± 134.4	502.2 ± 136.3	942.5 ± 156.3	632.3 ± 263.1	102.9 ± 36.0	87.1 ± 27.8	281.7 ± 28.4	157.2 ± 94.7 AB
	Olive mill wastewater	366.1 ± 42.5	491.0 ± 148.0	903.2 ± 184.4	586.8 ± 269.2	85.0 ± 23.0	77.7 ± 26.7	265.6 ± 46.4	142.7 ± 94.3 B
Average of soil management techniques	Average of disc harrow	409.9 ± 84.5 ef	469.3 ± 112.3 de	850.0 ± 107.5b	576.4 ± 221.2B	100.6 ± 31.9 e	79.0 ± 31.6 e	273.3 ± 32.9 b	151.0 ± 93.6 B
	Average of chisel	353.9 ± 61.8 f	489.7 ± 108.6 de	760.2 ± 81.3c	534.6 ± 190.6B	76.7 ± 16.3 e	78.1 ± 21.6 e	230.4 ± 21.5 c	128.4 ± 75.7 C
	Average of no-tillage	524.6 ± 101.9 d	531.9 ± 142.6 d	1024.0 ± 129.6a	693.5 ± 266.7 A	137.2 ± 39.8 d	85.8 ± 27.2 e	299.2 ± 34.5 a	174.1 ± 98.0 A

Means were separated by Duncan’s multiple range test at the p < 0.05 level. Within each column, means followed by different lowercase letters indicate significant differences among treatments or years, while means followed by different uppercase letters indicate significant differences among averages of organic material applications or soil management techniques. Values marked with ‘ns’ denote non-significant differences

General evaluation

Principal Component Analysis (PCA) of soil microbial properties revealed distinct relationships between various soil parameters and sampling times. PC1 explained 51.92% of the total variance, while PC2 accounted for 21.48%. The analysis showed clear clustering patterns based on treatment conditions and sampling times. Microbial biomass C and N were strongly associated and positioned opposite to enzyme activities (dehydrogenase, β-glucosidase, alkaline phosphatases, and urease). Soil organic carbon and soil respiration demonstrated moderate positive correlation with PC1. Samples with prefixes 15- and 18- predominantly clustered on the left side of the plot, while those with 21- prefixes appeared on the right side, indicating temporal changes in soil microbial community structure and function. The first quadrant contained mainly 21-series samples with positive loadings on both PC1 and PC2, showing strong associations with dehydrogenase activity, β-glucosidase activity, and soil organic carbon. The second quadrant was primarily populated by 15-series samples with positive PC2 but negative PC1 values, correlating strongly with microbial biomass C and N parameters. The third quadrant contained a mixture of 15- and 18-series samples with negative loadings on both PC1 and PC2, lacking strong associations with any specific measured soil properties. The fourth quadrant featured primarily 18- and 21-series samples with positive PC1 but negative PC2 values, showing associations with soil respiration and urease enzyme activity (Fig. 1).

Fig. 1.

Principal component analysis (PCA) of soil biological parameters under different tillage systems and organic amendments across three years. 2015 (15), 2018 (18), 2021 (21), Disc Harrow (D), Chisel (C), No-Tillage (NT), Control (C), Broccoli (B), Antep radish (AR), Olive mill wastewater (OMW)

According to the correlation matrix results, strong positive correlations were observed between soil respiration and urease enzyme activity (r = 0.86), β-glucosidase and alkaline phosphatases activity (r = 0.95), β-glucosidase and dehydrogenase activity (r = 0.81), and alkaline phosphatases activity and soil organic carbon (r = 0.74). Moderate positive correlations were found between soil respiration and β-glucosidase (r = 0.53), dehydrogenase activity and soil organic carbon (r = 0.44), and urease enzyme activity and β-glucosidase (r = 0.46). Weak negative correlations were detected between microbial biomass parameters and several enzyme activities, including urease enzyme activity and microbial biomass N (r = −0.23), alkaline phosphatases activity and microbial biomass C (r = −0.15), and β-glucosidase and microbial biomass C (r = −0.12) (Fig. 2). In our study, the heatmap revealed distinct patterns in soil biological parameters across different tillage systems and organic material treatments over the three-year period (2015, 2018, and 2021). In 2015, treatments showed generally lower enzyme activities (alkaline phosphatase, β-glucosidase, and dehydrogenase) compared to 2021 treatments. The 2021 no-tillage treatments (21-NT-B, 21-NT-OBW, 21-NT-C, and 21-NT-AR) exhibited higher enzyme activities and soil organic carbon, while demonstrating lower microbial biomass C and N. The 2018 treatments formed a separate cluster with moderate values for most parameters. Treatments with olive mill wastewater amendment (OMW) in 2021 showed enhanced soil respiration and urease enzyme activity. The chisel tillage treatments with broccoli amendment (21–C–B) demonstrated elevated enzyme activities. Overall, a temporal shift was observed from 2015 to 2021, with no-tillage systems in 2021 showing the most pronounced enhancement of soil enzymatic activities and organic carbon content (Fig. 3).

Fig. 2.

Correlations analysis of soil biological parameters under different tillage systems and organic amendments across three years

Fig. 3.

Hierarchical clustering analysis of soil biological parameters under different tillage systems and organic amendments across three years

Discussion

Soil respiration and soil organic carbon

In our findings, No-tillage systems demonstrated superior performance in enhancing both soil respiration (34.1 mg CO₂-C 100 g^− 1) and soil organic carbon (SOC) (1.8%), which aligns with numerous studies in the literature. Indeed, Abdalla et al. [40] conducted a meta-analysis demonstrating that no-tillage practices significantly increase SOC sequestration compared to conventional tillage, particularly in the upper soil layers. This improvement is attributed to minimal soil disturbance, which preserves soil structure and reduces organic matter decomposition rates. The increased soil respiration under no-tillage, despite higher SOC content, represents a complex equilibrium between carbon inputs and outputs. Palm et al. [41] explain that no-tillage systems often develop more active and diverse microbial communities, which can lead to increased respiration rates while still maintaining higher overall carbon stocks. In contrast, the lower soil respiration (30.4 mg CO₂-C 100 g^− 1) and SOC (1.5%) values observed with disc harrow tillage corroborate findings by Zuber et al. [42], who reported that intensive tillage practices accelerate organic matter decomposition and reduce carbon sequestration potential. The intermediate values observed with chisel tillage suggest a gradient effect of tillage intensity on soil parameters, supporting Zhang et al. [43]’s demonstration that tillage intensity affects both the quantity and quality of SOC, with more intensive tillage leading to preferential depletion of labile carbon fractions crucial for microbial activity.

Our organic material applications revealed intriguing patterns related to allelopathic effects. Broccoli-treated plots exhibited the highest soil respiration (33.4 mg CO₂-C 100 g^− 1), attributed to the biochemical composition of brassica residues. Some authors reported that brassica crops produce glucosinolates and other bioactive compounds that stimulate microbial activity when incorporated into the soil [44, 45]. Haramoto and Gallandt [46] explained that broccoli tissues contain glucosinolates that hydrolyze into bioactive isothiocyanates upon decomposition, initially exhibiting antimicrobial properties followed by compensatory microbial growth as described by Rumberger and Marschner [47]. In contrast, olive mill wastewater-treated plots showed significant enhancement of SOC (2.0%) but the lowest soil respiration (29.8 mg CO₂-C 100 g^− 1), aligning with findings by Mechri et al. [48] and Komnitsas and Zaharaki [49]. This inverse relationship can be attributed to the strong allelopathic effects of olive mill wastewater as Chaari et al. [50] found that polyphenolic compounds in olive mill wastes can temporarily inhibit certain soil microbial groups. Saadi et al. [51] identified high concentrations of polyphenols in olive mill wastewater, including hydroxytyrosol and tyrosol, which possess significant antimicrobial properties that can persist in soil for extended periods, as demonstrated by Mekki et al. [52]. The Antep radish treatments showed intermediate performance in both soil respiration and SOC, suggesting a balanced effect on soil parameters. Poeplau and Don [53] noted that cover crops like radish contribute to soil carbon inputs through their extensive root systems, while Jabran et al. [54] explained that radish species contain various glucosinolates with allelopathic potential, though generally less pronounced than other Brassica oleracea.

Although no statistically significant interaction between no-tillage and allelopathic organic amendments was observed for any of the measured variables in this study, the combined use of these practices is discussed in the literature as a potentially complementary approach to enhancing soil health. Maillard et al. [55] reported that integrating no-tillage with organic inputs can provide additive benefits: no-tillage reduces physical disturbance and protects soil structure, while organic amendments supply additional carbon and nutrients. Furthermore, under no-tillage conditions, allelopathic compounds may persist longer in the surface soil layers due to reduced mixing and exposure to degradative processes, as shown by Kruidhof et al. [55]. The temporal dynamics observed in our study, with soil respiration increasing from 23.8 mg CO₂-C 100 g^− 1 in 2015 to 35.7 mg CO₂-C 100 g^− 1 in 2021 (50.0% increase) and SOC increasing from 1.4% to 1.7% (21.4% increase), further support the role of organic material in soil biological processes. Bonanomi et al. [56] demonstrated that soil microbial communities can develop adaptive tolerance to allelopathic compounds over time, potentially explaining the progressive increases in soil respiration across treatments despite initial inhibitory effects. These findings have significant implications for agricultural sustainability and ecosystem services, supporting Lal’s [57] growing consensus that conservation tillage practices are essential for sustainable soil management, while highlighting the importance of selecting appropriate organic inputs based on specific soil health objectives, particularly when considering their potential allelopathic properties and long-term effects on soil microbial communities and carbon dynamics. On the other hand, soil respiration showed moderate positive correlation with PC1 (51.92% of variance) in our principal component analysis. Strong positive correlation (0.86) was observed between soil respiration and urease enzyme activity according to the correlation matrix. Treatments with olive mill wastewater amendment in 2021 demonstrated enhanced soil respiration, indicating increased microbial activity in these plots. This finding aligns with Alef and Nannipieri [58], who suggested that organic amendments rich in phenolic compounds can stimulate heterotrophic respiration in agricultural soils.

Microbial biomass C and microbial biomass N

The findings of our study indicated significant variations in microbial biomass carbon (MBC) and microbial biomass nitrogen (MBN) across different soil management techniques and allelopathy applications, highlighting the complex interactions between management practices and soil microbial communities. The highest average MBC observed under no-tillage systems (213.5 µg g^− 1) aligns with findings by Six et al. [59], who reported that conservation tillage practices promote fungal-dominated microbial communities with higher carbon-use efficiency. This enhanced microbial biomass under no-tillage reflects increased substrate availability and improved habitat conditions for soil microorganisms. Li et al. [60] demonstrated that reduced soil disturbance preserves fungal hyphae networks and promotes microbial community development, explaining our observed higher MBC values under no-tillage compared to disc harrow tillage (195.1 µg g^− 1). The remarkable response of MBC to broccoli application (average 246.6 µg g^− 1) compared to control treatments (153.0 µg g^− 1) can be attributed to the biochemical composition of Brassica oleracea residues. Feng et al. [61] found that glucosinolate-rich brassica residues stimulate specific microbial groups after the initial biofumigation effect dissipates. The temporal variations in MBC following broccoli application, with particularly high values in 2015 under No-Tillage-Broccoli treatment (276.8 µg g^− 1), support findings by Kerner et al. [62] that brassica-derived compounds initially cause a shift in microbial community composition followed by enhanced microbial proliferation. The decline in some MBC values by 2021 suggests a stabilization of microbial communities over time, as described by Lupwayi et al. [63] who observed similar temporal patterns in long-term studies of organic amendments.

For MBN, the highest average observed under chisel tillage (206.6 µg g^− 1) rather than no-tillage (181.6) presents an interesting contrast to MBC patterns. This aligns with research by Balota et al. [64] suggesting that moderate tillage can temporarily increase nitrogen mineralization, making more nitrogen available for microbial immobilization. The strong response of MBN to broccoli application (206.9 µg g^− 1) compared to control treatments (160.6 µg g^− 1) corroborates findings by O’Farrell [65] that brassica green manures enhance nitrogen cycling through their relatively low C: N ratio and stimulation of proteolytic bacterial populations. The pronounced temporal variability in MBN values, particularly the fluctuations observed in Chisel–Olive Mill Wastewater treatments (from 75.9 µg g^− 1 in 2015 to lower values in subsequent years), is consistent with observations by Montemurro et al. [66] that olive mill waste applications can cause initial increases in soil nitrogen availability followed by immobilization as phenolic compounds are degraded. The differential response of MBC and MBN to management practices demonstrates the complex nature of soil microbial dynamics. The contrasting patterns between tillage effects on MBC versus MBN support the concept proposed by Mbuthia et al. [67] that different microbial functional groups respond uniquely to soil management. The overall average values of MBC (200.7 µg g^− 1) and MBN (186.6 µg g^− 1) across all treatments and years indicate a relatively balanced microbial stoichiometry, suggesting that despite treatment-induced fluctuations, the soil microbial communities maintained functional equilibrium over the six-year study period, as described by Mooshammer et al. [68] in their comprehensive review of microbial resource stoichiometry.

On the other hand, the allelopathic effects of different C-sources and soil management techniques demonstrated significant influence on soil microbial parameters through complex biochemical interactions. The consistently higher microbial biomass C observed in broccoli-treated plots, particularly under no-tillage (276.8 µg g^− 1 in 2015), can be explained through the allelopathic mechanisms described by Brown and Morra [69], who demonstrated that glucosinolate hydrolysis products from brassica tissues act as selective biofumigants that initially suppress certain microbial populations but subsequently stimulate proliferation of adapted microbial communities. This selective pressure contributes to the reshaping of microbial community structure and function, as evidenced by our observed temporal fluctuations in microbial biomass. The contrasting response pattern seen with olive mill wastewater applications, which yielded the lowest microbial biomass C values under disc harrow tillage (129.3 µg g^− 1 in 2018), aligns with findings by Karpouzas et al. [70] who identified that the high polyphenolic content of olive mill wastes exerts persistent antimicrobial effects, particularly when incorporated deeper into the soil profile through intensive tillage. The interaction between tillage intensity and allelopathic compounds is further elucidated by Tabaglio et al. [71], who found that allelopathic metabolites accumulate primarily in surface soil layers under no-tillage systems, creating stratified microbial habitats with distinct functional communities. Our observed differences in microbial biomass N distribution across treatments further support the concept that soil management techniques modify the persistence and bioactivity of allelopathic compounds, with moderate tillage (chisel) potentially optimizing the balance between allelopathic suppression and stimulation effects, as indicated by the highest average MBN values (206.6 µg g^− 1) under this treatment. In this regard, our findings demonstrate that the combined selection of appropriate organic inputs and tillage intensity can strategically harness allelopathic phenomena to enhance beneficial soil microbial properties while minimizing detrimental effects on soil quality. On the other side, microbial biomass C and N were strongly associated and predominantly clustered in the second quadrant with 15-series samples. Weak negative correlations were detected between microbial biomass parameters and several enzyme activities, including alkaline phosphatases (−0.15) and β-glucosidase (−0.12). The 2021 no-tillage treatments exhibited lower microbial biomass C and N despite showing higher enzyme activities, suggesting a shift toward more efficient microbial communities with enhanced enzymatic capabilities. Similar patterns of microbial efficiency were observed by Balota et al. [72] in their long-term conservation tillage studies. This apparent negative correlation may be explained by microbial community adaptation under long-term conservation practices. In nutrient-rich and stable environments, microorganisms tend to allocate resources to extracellular enzyme production rather than increasing their total biomass. Higher enzymatic activities with relatively lower microbial biomass indicate a functional shift toward more efficient decomposition and nutrient cycling, where fewer microbes maintain higher activity levels. In contrast, higher microbial biomass does not always translate into proportional enzyme production or respiration, as larger populations may include dormant or less active cells. Therefore, the observed pattern reflects functional efficiency rather than absolute microbial abundance.

Soil dehydrogenase activity and urease enzyme activity

In our results, soil dehydrogenase and urease enzyme activities exhibited distinct patterns across different soil management techniques and allelopathy applications, indicating their sensitivity to agricultural practices and temporal dynamics. The substantial increase in dehydrogenase activity under No-Tillage systems, particularly in Broccoli treatments (from 331.9 µg TPF g^− 1 in 2015 to 458.5 µg TPF g^− 1 in 2021), aligns with findings by Melero et al. [73], who reported that conservation tillage practices enhance soil enzyme activities due to increased organic matter accumulation and improved soil structure. The temporal fluctuations observed under Disc Harrow treatments, with dehydrogenase activity in Control plots decreasing from 348.2 µg TPF g^− 1 in 2015 to 104.9 µg TPF g^− 1 in 2018 before rising to 321.4 µg TPF g^− 1 in 2021, support the observations of Mbuthia et al. [67] that conventional tillage can cause immediate disruption of microbial habitats, leading to temporary decreases in enzyme activities followed by recovery periods. The differential response of dehydrogenase activity to organic material applications, with the highest average value observed in Antep radish treatments (289.0 µg TPF g^− 1) compared to Control treatments (235.6 µg TPF g^− 1), corresponds with research by Waliszewska et al. [74] demonstrating that brassica-derived biofumigants can stimulate specific microbial groups that contribute to enhanced oxidative enzyme activities. This stimulatory effect is attributed to the release of bioactive compounds during decomposition, as explained by Yim et al. [75], who found that isothiocyanates from Brassica tissues can modify soil microbial community structure and function. The positive response of dehydrogenase activity to organic amendments compared to control treatments is consistent with the findings of García-Ruiz et al. [76], who demonstrated that organic inputs provide carbon substrates that fuel microbial metabolic processes and associated enzyme production.

For urease enzyme activity, the progressive increase observed across most treatments over time, particularly under No-Tillage with Broccoli application (peaking at 232.4 µg N g^− 1 in 2021), aligns with findings by Nannipieri et al. [77] that stable soil environments promote the accumulation of persistent extracellular enzymes. The significantly higher urease activity in Antep radish treatments (160.6 µg N g^− 1) compared to other organic material applications supports research by Moeskops et al. [78] showing that cover crops with moderate C: N ratios can enhance nitrogen cycling enzyme activities. The temporal variations in urease activity under Chisel tillage, with the Control group increasing from 42.1 µg N g^− 1 in 2015 to 165.4 µg N g^− 1 in 2018 before declining slightly to 143.8 µg N g^− 1 in 2021, suggest complex interactions between soil disturbance and enzyme stabilization processes, as described by Burns et al. [79] in their review of soil enzyme dynamics under varying management practices. The overall average dehydrogenase activity (264.1 µg TPF g^− 1) and urease enzyme activity (144.9 µg N g^− 1) across all treatments and years reflect the general responsiveness of these enzymes to management practices. The higher values observed in the final year (2021) compared to the initial year (2015) across most treatments indicate a positive trajectory in soil biological quality, as suggested by Acosta-Martínez et al. [80], who proposed that enzyme activities serve as early indicators of improvements in soil health under sustainable management practices. The distinct patterns observed between dehydrogenase (an intracellular enzyme reflecting overall microbial activity) and urease (an extracellular enzyme involved in nitrogen cycling) highlight the importance of considering multiple enzyme classes when assessing soil biological responses to management, as emphasized by Trasar-Cepeda et al. [81] in their comprehensive analysis of soil enzyme activities as indicators of soil quality.

The allelopathic effects of different C-sources on soil enzyme activities reveal complex biochemical interactions that influence soil biological properties. The consistently higher dehydrogenase activity observed in Antep radish treatments (289.0 µg TPF g^− 1) compared to control (235.6 µg TPF g^− 1) demonstrates the stimulatory allelopathic potential of brassica-derived compounds, as described by Haramoto and Gallandt [46], who found that glucosinolate hydrolysis products initially suppress certain microbial groups but subsequently enhance the activity of adapted populations. This biphasic response pattern explains the temporal fluctuations in dehydrogenase activity, particularly under Disc Harrow treatments where activities decreased substantially by 2018 before recovering in 2021. The differential response of urease activity to Broccoli applications under No-Tillage (peaking at 232.4 µg N g^− 1 in 2021) compared to more moderate increases under Disc Harrow can be attributed to the persistence of allelopathic compounds in undisturbed soil profiles, as demonstrated by Petersen et al. [82], who found that isothiocyanates from brassica tissues have extended half-lives in no-till systems compared to conventionally tilled soils. The interaction between soil management techniques and allelopathic effects is further elucidated by Bonanomi et al. [56], who showed that soil disturbance alters the distribution and bioavailability of plant-derived allelochemicals, affecting their impact on enzyme-producing microorganisms. Our findings align with research by Zibilske and Makus [83], who demonstrated that allelopathic compounds can selectively inhibit certain microbial functional groups while stimulating others, leading to shifts in enzyme production patterns over time as soil microbial communities adapt to these chemical stressors. This explains the observed convergence of enzyme activities by 2021 across most treatments, suggesting the development of microbial tolerance mechanisms to allelopathic compounds as described by Mazzola et al. [84] in their long-term studies of brassica green manure effects on soil biological properties. In addition, dehydrogenase activity showed strong association with samples in the first quadrant, particularly with 21-series samples. Urease enzyme activity demonstrated strong positive correlation with soil respiration (0.86) and moderate correlation with β-glucosidase (0.46). The fourth quadrant of our PCA plot, featuring primarily 18- and 21-series samples, showed associations with urease enzyme activity, indicating temporal changes in nitrogen cycling potential. As noted by Kandeler et al. [85], these enzyme dynamics reflect the adaptation of microbial communities to changing substrate availability under different management practices.

Soil alkaline phosphatases activity and β-glucosidase

In our study, alkaline phosphatase and β-glucosidase activities exhibited remarkable temporal and treatment-dependent responses, reflecting their sensitivity to soil management techniques and organic material applications. The substantial increase in alkaline phosphatase activity under No-Tillage treatments, particularly with olive mill wastewater application reaching 1113.2 µg g^− 1 2 h^− 1 in 2021 compared to 475.8 µg g^− 1 2 h^− 1 in 2015 for control plots, aligns with findings by Dick et al. [86], who demonstrated that conservation tillage practices enhance enzyme stability through reduced physical disruption of soil aggregates and increased organic matter accumulation. This progressive increase across years supports the concept proposed by Sardans and Peñuelas [87] that soil enzyme activities can serve as early indicators of shifts in soil quality under different management regimes. The differential response of alkaline phosphatase to different C-sources, with Antep radish producing the highest average activity (632.3 µg g^− 1 2 h^− 1) followed closely by broccoli (623.2 µg g^− 1 2 h^− 1), corresponds with research by Reddy et al. [88] showing that brassica crops can enhance phosphatase activity through root exudation of organic acids that solubilize soil phosphorus. This stimulatory effect is particularly pronounced in combination with No-Tillage, as observed in our results, supporting the findings of Nannipieri et al. [89] that undisturbed soil profiles facilitate the accumulation of enzyme-stabilizing organic matter and the development of fungal hyphal networks that contribute significantly to phosphatase production.

For β-glucosidase activity, the marked increase across all treatments from 2015 to 2021, with particularly high values under No-Tillage with broccoli application (304.4 µg Saligenin g^− 1 3 h^− 1 in 2021 compared to 188.8 µg Saligenin g^− 1 3 h^− 1 in 2015), aligns with research by Stott et al. [90] demonstrating that this enzyme is highly responsive to management practices that enhance carbon substrate availability. The initial decrease observed under Disc Harrow treatments from 133.2 µg Saligenin g^− 1 3 h^− 1 in 2015 to 72.6 µg Saligenin g^− 1 3 h^− 1 in 2018 for control plots, followed by a substantial increase to 239.4 µg Saligenin g^− 1 3 h^− 1 in 2021, suggests a complex adaptation process of soil microbial communities to tillage disturbance, as described by Kabir [91] in studies of soil enzyme dynamics under varying tillage intensities. The consistently higher enzyme activities observed under No-Tillage compared to Chisel and Disc Harrow treatments across most fertilization applications support the findings of Balota et al. [72], who reported that reduced soil disturbance promotes the development of stratified microbial communities with enhanced enzyme production capacity. The significant response of both enzymes to brassica applications (broccoli and Antep radish) demonstrates the beneficial effects of these crops on soil biological activity, as documented by Njeru et al. [92] in their studies of cover crop effects on soil enzyme dynamics. The overall increases in average alkaline phosphatase activity from 429.5 µg g^− 12h^− 1 in 2015 to 878.1 µg g^− 12h^− 1 in 2021 and β-glucosidase activity from 104.8 µg Saligenin g^− 1 3 h^− 1 in 2015 to 267.6 µg Saligenin g^− 1 3 h^− 1 in 2021 across all treatments indicate a positive trajectory in soil biological quality over the six-year study period. This temporal pattern supports the concept proposed by Acosta-Martínez et al. [80] that sustainable management practices can progressively enhance soil enzyme activities through cumulative improvements in soil organic matter quality and microbial community structure. The concurrent increases in both phosphatase (involved in phosphorus cycling) and β-glucosidase (involved in carbon cycling) activities suggest a balanced enhancement of multiple nutrient cycling processes, as emphasized by Sinsabaugh et al. [93] in their ecological stoichiometry framework for soil enzyme activities.

The results of our study on soil enzyme activities revealed significant insights into the allelopathic effects of different treatments on soil biological properties. The data demonstrates clear trends in both alkaline phosphatase and β-glucosidase activities across various tillage methods and organic amendment applications over the six years from 2015 to 2021. The no-tillage system consistently demonstrated superior enzyme activities compared to disc harrow and chisel treatments. By 2021, no-tillage plots reached the highest alkaline phosphatase values (909.2 µg g^− 12h^− 1 for control, up to µg g^− 12h^− 1 1113.2 with olive mill wastewater). This aligns with Roldan et al. [94], who reported that conservation tillage practices preserve soil aggregates and organic matter, creating favorable conditions for microbial proliferation and enzyme production. We find it particularly interesting that no-tillage amplified the allelopathic effects of the organic amendments. This synergistic interaction between tillage systems and allelopathic substances suggests that minimal soil disturbance preserves bioactive compounds released by the amendments, allowing them to exert more pronounced effects on soil biological processes. Among the organic amendments, Antep radish produced the highest average alkaline phosphatase activity (632.3 µg g^− 12h^− 1), followed closely by broccoli (623.2 µg g^− 12h^− 1). These Brassicaceae species are known to contain glucosinolates, which, upon hydrolysis, release isothiocyanates with significant allelopathic properties [46]. In our experience, these compounds not only suppress certain pathogenic microorganisms but can also stimulate beneficial soil microbiota, potentially explaining the enhanced enzyme activities. For β-glucosidase, broccoli amendments resulted in the highest average activity (161.7 µg Saligenin g^− 1 3 h^− 1), followed by Antep radish (157.2 µg Saligenin g^− 1 3 h^− 1). This suggests that different allelopathic compounds may have specific effects on particular enzyme systems. The allelopathic effects of Brassicaceae residues on soil enzymes have been previously documented by Nannipieri et al. [77], who noted their ability to alter soil microbial community composition and function. Olive mill wastewater, despite showing considerable enhancement of enzyme activities compared to control, generally resulted in lower values than the Brassicaceae amendments. This differential response could be attributed to the phenolic compounds present in olive mill waste, which may have both stimulatory and inhibitory effects on soil microorganisms depending on concentration and soil conditions [95]. On the other hand, strong positive correlations were observed between β-glucosidase and alkaline phosphatases activity (0.95) and between alkaline phosphatases activity and soil organic carbon (r = 0.74). The 2021 no-tillage treatments exhibited higher enzyme activities compared to other treatments, with pronounced enhancement in enzymatic activities related to carbon and phosphorus cycling. The consistent increase in these enzyme activities from 2015 to 2021 across treatments suggests a progressive improvement in soil biological quality under sustainable management practices. This temporal pattern supports the findings of Sinsabaugh et al. [93], who proposed that coordinated enzyme activities reflect balanced stoichiometric relationships in nutrient cycling processes.

Conclusions

This study was the first to comprehensively evaluate the long-term effects of three tillage systems combined with four organic material treatments on soil enzyme activities and microbial properties in an organic vineyard. Our findings demonstrated that no-tillage practices significantly enhanced soil microbial biomass, enzymatic activities, and organic carbon accumulation compared to the other tillage methods. Among the organic material treatments, broccoli and Antep radish amendments consistently improved microbial activity, while olive mill wastewater application contributed to higher soil organic carbon levels. The strong correlations observed between soil respiration, enzymatic activities, and microbial biomass suggested that integrating specific organic amendments with reduced tillage optimized soil health in vineyard ecosystems. Based on these findings, we recommended adopting no-tillage systems alongside targeted organic amendments to enhance soil biological functions and sustainability in viticulture. Future studies should investigate the long-term stability of these microbial and enzymatic responses under different climatic conditions and soil types. To sum up, these results provide valuable guidance for vineyard management strategies, contributing to improved soil health, enhanced nutrient cycling, and sustainable agricultural practices.

Abbreviations

MBC

Microbial biomass carbon

MBN

Microbial biomass nitrogen

SOC

Soil organic carbon

GSL

Glucosinolates

ITC

Isothiocyanates

PCA

Principal component analysis

ANOVA

Analysis of variance

TPF

Triphenyl formazan

TTC

Triphenyltetrazolium chloride

pNPP

p-nitrophenyl phosphate

RCBD

Randomized Complete Block Design

ROS

Reactive oxygen species

Authors’ contributions

Conceptualization: N.O., F.A., O.K., H.H.K., F.K., H.H.V., and Ö.M.; Methodology: N.O., F.A., O.K., H.H.K., F.K., H.H.V., and Ö.M.; Investigation: N.O., F.A., O.K., H.H.K., F.K., H.H.V., and Ö.M.; Software: N.O. and O.K.; Formal analysis: N.O. and O.K.; Writing—original draft preparation: N.O. and O.K.; Writing—review and editing: O.K.; Visualization: N.O., F.A., and O.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by the General Directorate of Agricultural Research and Policies, Republic of Türkiye Ministry of Agriculture and Forestry (Project number: TAGEM/BBAD/12/A08/P08/02). The funding body had no role in the design of the study, collection, analysis, and interpretation of data, or in writing the manuscript.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Requests for materials should be directed to N.O. (nur.okur@ege.edu.tr), O.K. (kayaozkan25@hotmail.com or ozkan.kaya@ndsu.edu), and F.A. (fadimeates2@yahoo.com).

Declarations

Ethics approval and consent to participate

Not applicable, as this study involved soil and plant materials only, with no human or animal subjects.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.