Modulation of Grapevine Physiological Performance by Compost and Vermicompost Obtained from Vine Pruning Residues

Abstract

Recycling vineyard pruning residues into compost and vermicompost represents a sustainable strategy to reduce viticulture’s reliance on chemical fertilizers. Nonetheless, their effects on plant performance remain poorly understood. This study evaluated the effect of vine pruning residues compost and vermicompost on the physiological, biochemical, and growth performance of Vitis vinifera L. cv. Touriga Franca, in comparison with mineral fertilization and an unfertilized control. A pot experiment was conducted from April to September 2024 in northern Portugal under Mediterranean climate conditions, using one-year-old grapevines and subjected to four fertilization treatments. Leaf gas exchange, chlorophyll a fluorescence, photosynthetic pigments, antioxidant and osmoprotective metabolites, and shoot and root development were assessed at three sampling dates during the growing season. Organic amendments enhanced photosynthetic performance and root growth relative to the unfertilized control. Vermicompost promoted higher CO₂ assimilation, stomatal conductance, and shoot and root elongation, whereas compost increased intrinsic water use efficiency, photochemical regulation, and root biomass. Biochemical analyses indicated that compost favored protein and carotenoid accumulation, while vermicompost increased proline and later protein levels, alongside reduced phenolic and flavonoid contents. Despite their similar chemical composition, compost and vermicompost induced distinct physiological responses driven by differences in biological activity and nutrient dynamics. These findings demonstrate that pruning-derived organic amendments can match mineral fertilization in supporting grapevine performance while offering additional benefits for stress regulation and sustainable vineyard management.

1. Introduction

With the global population projected to exceed 9 billion by 2050, food production will need to increase by 70% compared to the past two decades [1]. This increase will inevitably generate larger volumes of agricultural waste, whose disposal, use and management, are becoming a growing concern [2,3]. Numerous studies have highlighted the problem of the waste generation associated with intensified agricultural and industrial practices [4,5,6]. Farming intensification is also associated with increased use of synthetic fertilizers, which raises serious risks to natural resources, threatens human health, compromises environmental integrity, degrades soil quality, and contributes to the emergence of pathogen resistance [7,8]. However, due to synthetic fertilizers reliance on non-renewable energy sources and limited materials for their production, an imminent shortage is expected [9].

Viticulture is a major agricultural sector worldwide and involves a sequence of activities, including grape cultivation, pruning, vine training, pest and disease control, ripening, and harvesting [10,11]. These activities generate considerable amounts of organic residues, namely pruning wood, leaves, and grape stems [11,12]. In most regions, these residues are commonly left in the field to decompose or are burned [3], leading to considerable environmental consequences, such as the release of greenhouse gases and nutrient losses. Beyond these environmental drawbacks, leaving untreated woody material in the vineyard can also contribute to the spread of grapevine diseases, since such residues may act as reservoirs for pathogenic agents [13,14]. Therefore, the valorization of vineyard residues has become a strategic priority to improve soil fertility, reduce external inputs, and enhance system sustainability. Their management should align with the "waste management hierarchy," prioritizing prevention, reuse, recycling (including composting) and promoting energy recovery, with disposal being the last resort [15]. Preferably, management strategies should minimize energy consumption, reduce carbon emissions and nutrient loss, and focus on adding value to the residues [15].

In this context, composting and vermicomposting are promising alternatives for managing agricultural waste and for converting organic wastes into nutrient-rich soil stable amendments, reducing the dependence on synthetic fertilizers [16] and simultaneously promoting soil health and quality [9,17]. Composting is an aerobic biological process that transforms organic residues into a stabilized, sanitized, nutrient-rich humus-like product [18,19]. This process involves microorganisms that facilitate the breakdown and transformation of organic materials into a possible high quality product [20]. Similarly, vermicomposting relies on the combined action of earthworms and microorganisms to convert organic waste into valuable products, such as organic fertilizers and soil amendments that enhance the availability of essential nutrients [21]. Vermicompost has been described as an exceptional soil enhancer, that significantly boosts plant growth by improving the availability of vital nutrients such as nitrogen (N), phosphorus (P), potassium (K), and zinc (Zn) [22,23,24]. Several studies have shown that vermicompost can enhance germination rates, yield, plant height, stem thickness, leaf size, dry weight, leaf count, and grain weight, while lowering nitrate accumulation in crops and improving fruit quality with higher levels of vitamin C, carbohydrate, and protein [25,26]. Although the impacts of both compost and vermicompost have been studied in different crops, information regarding the effects of these organic amendments on grapevine physiology remains limited, namely on those obtained from vine pruning residues.

Organic amendments are increasingly advocated as sustainable alternatives to conventional fertilizers due to their ability to improve soil physical, chemical, and biological properties, including enhanced organic carbon content, nutrient availability, and microbial activity that support plant growth and soil health [27]. However, despite extensive work on soil quality outcomes, there is limited mechanical understanding of how amendments with similar chemical composition can produce distinct physiological responses in plants over time. In particular, long-term studies are needed to capture dynamic nutrient release, microbial community shifts, and plant response trajectories that are not evident in short-term trials [28]. Organic amendments influence soil processes on multiple temporal scales, from initial nutrient mineralization, through gradual formation of stable organic matter, to sustained microbial and enzymatic activity, all of which can alter plant nutrient uptake, stress responses, and growth patterns [29].

Although numerous studies have demonstrated the benefits of compost and vermicompost application in horticultural and annual crops, research addressing their effects on perennial crops remains limited, particularly in grapevine systems. Most available studies focus on short-cycle species, while long-lived crops such as grapevines present distinct physiological dynamics, nutrient demands, and soil–plant interactions. Therefore, the responses of grapevines to organic amendments derived from pruning residues are still poorly understood, highlighting the need for targeted research in perennial cropping systems. By conducting a full 1-year evaluation under defined soil conditions, this study provides new insight into soil–plant interactions, integrating physiological, biochemical, and growth responses of grapevines to compost and vermicompost derived from vine pruning residues throughout one growing cycle.

In this sense, this study aimed to evaluate the effect of compost and vermicompost, derived from vine pruning residues, on grapevine physiological, biochemical, and growth traits, in comparison with conventional fertilization. It was hypothesized that soil enrichment with compost and vermicompost derived from vine pruning residues will significantly enhance grapevine growth and lead to measurable improvements in key physiological and biochemical performance indicators compared to conventional fertilization. Specifically, this work aimed to (i) assess the impact of these organic amendments on leaf gas exchange, photosynthetic efficiency, and photosynthetic pigment composition; (ii) analyze their effects on key biochemical indicators related to stress regulation and metabolism, including antioxidant compounds, osmoprotective metabolites, soluble sugars, and proteins; and (iii) determine their influence on shoot and root growth and biomass allocation. Additionally, the study sought to identify potential differences in plant responses between compost and vermicompost, despite their similar origin, and to evaluate the potential of pruning-derived organic amendments as sustainable alternatives to synthetic fertilizers in viticulture.

2. Results and Discussion

2.1. Leaf Gas Exchange Parameters

Leaf gas exchange parameters under the different soil treatments are presented in

Table 1. Transpiration rate (E), stomatal conductance (g_s), net photosynthesis (A), intrinsic water use efficiency (A/g_s) and the ratio of intercellular to atmospheric CO₂ concentration (C_i/C_a) under different soil treatments C−, C+, CP, and VCP) at 43, 81 and 147 days after treatment (DAT). The results are expressed as mean ± SD (n = 6).

DAT	Treatment	E (mmol·m−2 s−1)	gs (mmol·m−2 s−1)	A (µmol·m−2 s−1)	A/gs (µmol·mol−1)	Ci/Ca
43	C−	1.37 ± 0.110 a	47.2 ± 5.09 ab	6.09 ± 0.422 a	135.9 ± 2.42 bc	0.432 ± 0.009 ab
	C+	2.36 ± 0.448 b	55.1 ± 10.9 b	6.95 ± 0.230 ab	129.7 ± 26.9 ab	0.442 ± 0.101 b
	CP	1.81 ± 0.046 ab	35.4 ± 0.887 a	6.16 ± 0.224 a	174.3 ± 10.6 c	0.279 ± 0.037 a
	VCP	4.53 ± 0.351 c	89.1 ± 9.01 c	7.75 ± 0.936 b	87.6 ± 14.16 a	0.585 ± 0.057 b
	p value	<0.001	<0.001	0.016	0.001	0.002
81	C−	0.348 ± 0.024 a	4.55 ± 0.431 a	1.20 ± 0.001 a	261.3 ± 18.8 b	0.254 ± 0.032 a
	C+	5.83 ± 0.771 c	87.9 ± 11.7 c	4.82 ± 0.003 c	78.4 ± 12.85 a	0.685 ± 0.024 b
	CP	1.38 ±0.356 a	16.9 ± 4.49 a	3.61 ± 0.001 b	85.9 ± 16.33 a	0.668 ± 0.094 b
	VCP	3.88 ± 0.490 b	47.9 ± 6.37 b	3.34 ± 0.223 b	76.0 ± 2.56 a	0.615 ± 0.010 b
	p value	<0.001	<0.001	<0.001	<0.001	<0.001
147	C−	1.95 ± 0.903 a	105.7 ± 68.4	4.549 ± 2.213 a	44.7 ± 13.9 a	0.786 ± 0.064 b
	C+	5.70 ± 0.239 b	163.0 ± 6.60	8.431 ± 0.398 b	48.6 ± 4.76 ab	0.745 ± 0.025 ab
	CP	4.94 ± 0.912 bc	131.6 ± 14.9	9.093 ± 0.233 b	68.8 ± 9.92 b	0.654 ± 0.040 a
	VCP	7.11 ± 0.540 c	175.3 ± 16.9	9.234 ± 0.008 b	57.7 ± 3.52 ab	0.694 ± 0.020 ab
	p value	<0.001	ns	0.015	0.003	0.045

Different lowercase letters indicate significant differences among treatments within each sampling date (p < 0.05) and non-significant differences are represented by ns (p > 0.05).

. At 43 days after treatment (DAT), vermicompost-treated (VCP) plants exhibited the highest net photosynthesis (A), stomatal conductance (g_s) and transpiration rate (E). VCP plants also showed the lowest intrinsic water use efficiency (A/g_s) indicating greater water consumption to sustain photosynthesis. In contrast, negative control (C−) and compost (CP) plants generally showed lower values overall but significantly higher A/g_s relative to the other treatments. The ratio of intercellular to atmospheric CO₂ concentration (C_i/C_a) was significantly lower in CP-treated plants in comparison with the positive control (C+) and VCP. At 81 DAT, C+ plants displayed significantly higher A, g_s and E than the remaining treatments, whereas C consistently presented the lowest values. Both CP and VCP treatments led to significantly higher A and C_i/C_a in comparison to the C−, with increases of 163% and 142%, respectively, suggesting better carbon assimilation. VCP showed intermediate g_s and E, lower than C+, but higher than C− and CP. The highest A/g_s values were found in the untreated plants (C−), while all fertilized treatments showed similarly lower values. The C_i/C_a values were higher in all treated plants compared to the C−. At 147 DAT, VCP-treated plants showed the highest E, while CP and C+ showed similar intermediate values, with all being significantly higher than C−, with increases of 266%, 154% and 193%, respectively. Relatively to A, all treated plants exhibited significantly higher values than C−, with increases of 100%, 103% and 85%, respectively. CP plants also showed significantly higher A/g_s than C− plants (54% of increase), but with no statistical differences with VCP and C+ vines. On the other hand, plants with CP application resulted in significantly lower C_i/C_a values compared with C−. Regarding the g_s values, there was a tendency of treated plants exhibited higher values than C−, but differences were not statistically significant.

Over the experimental period (43–147 DAT), gas-exchange responses showed clear temporal trends. A, g_s, and E declined at 81 DAT and recovered at 147 DAT, suggesting a mid-season reduction in stomatal activity. The highest values at 81 DAT were recorded in C+, reflecting the rapid response to mineral fertilization. A/g_s tended to decrease toward the end of the period, with VCP showing the lowest values in all dates, whereas CP consistently promoted higher values. The C_i/C_a ratio tended to increase slightly over time, but being highest in VCP at 43 and 81 DAT.

Overall, both CP and VCP treatments improved gas-exchange performance in grapevines when compared to the untreated plants. The findings of Mairata et al., corroborates the results of the present study, as they reported that compost applications enhanced photosynthesis and stomatal conductance in grapevines [30]. VCP consistently led to better photosynthetic activity, particularly at early stages (43 DAT), while CP favored greater water-use efficiency throughout the experiment. Although compost and vermicompost exhibit similar macronutrient content and organic matter characteristics, previous studies have shown that vermicompost contains higher levels of plant growth regulators such as kinetin, suggesting that bioactive compounds rather than overall nutrient composition may drive the distinct physiological responses observed in plants [31]. Organic amendments have been shown to significantly reshape soil microbial communities, carbon and nitrogen pools, enhancing nutrient cycling and enzymatic activity in vineyard soils, which may contribute to improved plant physiological performance [32]. The improved performance of VCP at the initial stage, through increased A, g_s and E suggests rapid physiological stimulation. These findings are consistent with previous studies reporting improved photosynthetic performance under several vermicompost amendments [33,34,35], possibly due to greater nutrient availability, such as N, P, Mg and Fe, which are essential for the development of the photosynthetic apparatus [36], and microbial activity [37]. Similarly, Hosseinzadeh et al. also reported an increase in E in chickpea (Cicer arietinum L.) with vermicompost use, which may be linked to the microbial activity, and is known to facilitate root water uptake [37]. Consequently, improved water retention helps maintain stomatal opening, thereby increasing transpiration rates [38,39]. The relatively higher g_s values observed in VCP reflects stomatal opening and greater CO₂ influx, supporting higher photosynthetic and transpiration rates [40,41], which is consistent with the key role of stomatal regulation in influencing E and, consequently, the overall water balance in plants [42]. Furthermore, the lower A/g_s values in VCP plants also indicate that more water is used to achieve higher CO₂ assimilation, which is a typical physiological strategy when plants prioritize carbon gain under favorable soil nutrient and moisture conditions [33,35]. On the other hand, the improved performance of CP, particularly regarding A/g_s, suggests a more efficient carbon gain per unit water loss, which can be associated with a gradual and efficient nutrient release and improved soil structure [43]. These improvements may increase soil moisture retention, supporting more conservative stomatal behavior and enhanced water-use efficiency over time.

2.2. Photochemical Traits

Chlorophyll fluorescence a is an important indicator of photosynthetic efficiency and plant responses to environmental factors [44]. The effective quantum yield of photosystem II (Φ_PSII), photochemical quenching (qP), and the maximum quantum efficiency of PSII (F_v/F_m) are parameters used to evaluate the photochemical efficiency in plants [45], while non-photochemical quenching (NPQ) quantifies the dissipation of excess energy absorbed as heat in the PSII antenna complex [46]. In general, an increase in NPQ may result either from protective mechanisms that prevent light-induced damage or from the occurrence of photodamage itself [46].

The photochemical performance of plants under different soil treatments is presented in

Table 2. Photochemical efficiency of photosystem II (Φ_PSII). Photochemical quenching (qP). Maximal quantum efficiency of photosystem II (F_v/F_m), and non-photochemical quenching (NPQ) under different soil treatments (C−, C+, CP, and VCP) at 81 and 147 days after treatment. (DAT). Results expressed as mean ± SD (n = 6).

DAT	Treatment	ΦPSII	qP	Fv/Fm	NPQ
81	C−	0.146 ± 0.028 a	0.569 ± 0.077 a	0.849 ± 0.041 b	7.98 ± 0.348 b
	C+	0.278 ± 0.012 c	0.722 ± 0.048 b	0.847 ± 0.044 b	7.43 ± 0.873 b
	CP	0.149± 0.014 a	0.643 ± 0.044 ab	0.723 ± 0.048 a	7.45 ± 0.603 b
	VCP	0.209 ± 0.024 b	0.597 ± 0.054 a	0.807 ± 0.066 b	6.36 ± 0.259 a
	p value	<0.001	0.001	0.001	0.001
147	C−	0.130 ± 0.008 c	0.386 ± 0.051 ab	0.756 ± 0.035 ab	7.87 ± 0.582 b
	C+	0.069 ± 0.003 a	0.517 ± 0.029 c	0.697 ± 0.047 a	4.65 ± 0.315 a
	CP	0.068 ± 0.009 a	0.431 ± 0.061 b	0.795 ± 0.047 b	11.2 ± 0.974 c
	VCP	0.112 ± 0.008 b	0.313 ± 0.056 a	0.806 ± 0.065 b	8.70 ± 0.774 b
	p value	<0.001	<0.001	0.014	<0.001

Different lowercase letters indicate significant differences among treatments within each sampling date (p < 0.05).

. At 81 DAT, C+ and VCP-treated plants exhibited significantly higher Φ_PSII than the C− plants (90% and 43% of increase, respectively). Furthermore, C+ plants also displayed the highest qP, with an increase of 27% compared to the C− plants, while both VCP and CP presented no statistically significant differences. F_v/F_m, values were similar among C−, C+ and VCP plants, being significantly higher than CP (increases of 17%, 17% and 11%, respectively). Finally, at 81 DAT, VCP-treated plants showed the lowest NPQ values, suggesting a reduced dissipation of excess light energy as heat, although no significant differences were observed. At 147 DAT, all treated plants exhibited lower Φ_PSII than C−, although VCP plants maintained higher Φ_PSII, when compared to the C+ and CP-treated plants (increase of 64%). CP and C+ plants showed a significantly higher qP than VCP-treated plants, with increases of 37% and 65%, respectively, with both composting treatments having no differences in comparison to C−. The F_v/F_m was higher in CP and VCP-treated plants than in C+, with increases of 14% and 15%, respectively. CP and VCP plants also presented significantly higher NPQ values compared to the C+ plants, increases of 141% and 87%, respectively.

Between both timepoints (81 and 147 DAT), our results revel that both Φ_PSII and qP decreased in all treatments. Furthermore, F_v/F_m values declined in C− and C+, with slight increases for CP and VCP plants, but remaining within the range typically associated with healthy PSII performance [46]. NPQ also decreased in C− and C+, but increased in CP and VCP, suggesting an enhancement of non-photochemical energy dissipation mechanisms that protect the photosynthetic apparatus, namely to the high temperatures and radiation conditions [47]. Overall, CP and VCP maintained higher photochemical stability compared to C− and C+, reflecting better physiological resilience throughout the experimental period. While VCP tended to promote higher photochemistry efficiency at 81 DAT, CP enhanced photoprotection at 147 DAT.

The higher Φ_PSII observed in C+ and VCP plants at 81 DAT indicates improved photosystem efficiency [48], which aligns with the higher gas-exchange values recorded for these treatments (

Table 3. Phenols, flavonoids, protein, ABTS, proline and soluble sugars content (mg g⁻¹) under different soil treatments (C−, C+, CP and VCP) at 43, 81 and 147 days after treatments (DAT). Results expressed as mean ± SD (n = 6).

DAT	Treatment	Phenols (mg·g−1)	Flavonoids (mg·g−1)	Protein (mg·g−1)	ABTS (mg·g−1)	Proline (mg·g−1)	Soluble Sugars (mg·g−1)
43	C−	164.3 ± 4.04	44.8 ± 11.9	12.5 ± 1.34 a	4.05 ± 0.166	0.24 ± 0.04 a	10.15 ± 2.11 b
	C+	175.7 ± 17.9	57.6 ± 6.08	11.2 ± 1.04 a	3.77 ± 0.201	0.55 ± 0.04 b	9.91 ± 0.98 b
	CP	147.8 ± 0.997	27.4 ± 15.6	15.6 ± 1.05 b	3.99 ± 0.250	1.46 ± 0.02 d	11.86 ± 0.82 b
	VCP	171.4 ± 13.8	56.7 ± 17.4	11.9 ± 1.08 a	3.89 ± 0.227	0.66 ± 0.11 c	5.82 ± 0.60 a
	p value	ns	ns	<0.001	ns	<0.001	<0.001
81	C−	29.0 ± 0.658 b	15.1 ± 2.08 b	10.1 ± 0.647 a	4.51 ± 0.095 c	0.28 ± 0.06 b	19.88 ± 0.92
	C+	27.5 ± 1.99 ab	5.59 ± 1.77 a	14.3 ± 0.707 c	3.74 ± 0.098 a	0.18 ± 0.03 a	20.81 ± 1.00
	CP	27.3 ± 1.44 ab	28.3 ± 4.55 c	11.1 ± 0.925 ab	4.62 ± 0.071 c	0.54 ± 0.08 c	19.46 ± 0.45
	VCP	26.6 ± 1.21 a	14.0 ± 1.11 b	12.1 ± 0.514 b	3.91 ± 0.111 b	0.53 ± 0.04 c	19.71 ± 0.18
	p value	0.041	<0.001	<0.001	<0.001	<0.001	ns
147	C−	26.2 ± 0.476 b	5.77 ± 0.633 a	15.9 ± 0.678	3.71 ± 0.008	0.60 ± 0.11 b	15.28 ± 0.84 c
	C+	26.3 ± 0.075 b	6.29 ± 0.881 a	15.2 ± 0.462	3.71 ± 0.022	0.06 ± 0.01 a	12.83 ± 0.19 a
	CP	26.1 ± 0.350 b	9.41 ± 0.767 b	14.7 ± 0.700	3.73 ± 0.029	0.03 ± 0.01 a	14.03 ± 0.70 b
	VCP	23.2 ± 0.768 a	8.89 ± 0.690 b	14.8 ± 1.444	3.72 ± 0.031	0.07 ± 0.01 a	13.72 ± 0.52 ab
	p value	<0.001	<0.001	ns	ns	<0.001	<0.001

Different lowercase letters indicate significant differences among treatments within each sampling date (p < 0.05) and non-significant differences are represented by ns (p > 0.05).

). Similarly, the higher qP values in C+ and CP plants, compared to the C−, suggest a greater proportion of open reaction centers and proper PSII functioning [35,49]. The F_v/F_m values near 0.8 in C−, C+ and VCP plants indicate lower photoinhibitory damage, whereas CP plants exhibited signs of stress [35]. Furthermore, the lower NPQ observed in VCP plants reflects a reduced need for thermal dissipation, suggesting that absorbed light was effectively utilized for photochemistry rather than being lost as heat [46].

At 147 DAT, VCP plants maintained higher F_v/F_m levels, suggesting better photochemical performance. Liu et al. reported similar results.in tomato, where vermicompost significantly increased the F_v/F_m ratio, a response that indicates better light energy absorption and utilization [50]. On the other hand, the higher NPQ values observed in CP-treated plants at 147 DAT reflect the activation of stronger photoprotective mechanisms [51]. The authors [50] also linked these enhancements to a better nutrient balance and improved F_v/F_m. In fact, Ortuño et al. demonstrated that compost, especially when combined with mycorrhizal inoculation, improved photosynthetic efficiency in Cistus albidus plants subjected to water stress [52].

Collectively, these findings highlight the distinct roles of CP and VCP in modulating photosynthetic efficiency and stress resilience. This supports the premise that organic amendments can significantly optimize plant physiological status, particularly during key phenological stages.

2.3. Biochemical Responses

2.3.1. Photosynthetic Pigments

The photosynthetic pigment content of vine leaves under different soil conditions is summarized in Figure 1. At 43 DAT, unfertilized plants (C−) showed the highest content of chlorophyll a, chlorophyll b, and total chlorophyll and carotenoids, whereas C+, CP and VCP plants exhibited similarly lower amounts, with no differences among fertilized treatments. By 81 DAT, no statistically significant differences were detected among treatments for any analyzed parameter. However, at 147 DAT, VCP-treated plants showed 47% lower chlorophyll b compared to C+, while CP-treated plants exhibited a sharp accumulation of carotenoids, exceeding C+ and VCP levels by 886% and 671%, respectively. A consistent decline in chlorophyll levels was observed across all treatments from 43 to 147 DAT, aligning with the onset of leaf senescence. In contrast, carotenoid concentrations showed a different pattern, remaining relatively stable between 43 and 81 DAT and increasing at 147 DAT, particularly in the CP treatment. Overall, the temporal trends observed in the present study highlight a shift from chlorophyll-driven photosynthetic activity toward enhanced photoprotection at later stages of plant development [53]. Vermicompost stimulated physiological activity early in the season without altering photosynthetic pigment contents, whereas compost enhanced photoprotection in the later stages, indicating distinct effects of organic amendments on grapevine physiology.

Photosynthetic pigments are essential components of the photosynthetic system, varying in type and concentrations [54]. Chlorophylls are responsible for light absorption at different wavelengths, and tend to decrease as leaf senescence settle, which was consistent with the results observed in the present study.

Although organic amendments often enhance pigment content [55,56,57,58,59], this effect was not observed in the present study, particularly at 43 DAT. The lower pigment concentration in fertilized plants may be associated with a reduction in their biosynthesis during early growth stages [60], although with no effect on the photosynthetic function as shown by gas-exchange parameters (Table 3). On the other hand, the increase in carotenoid content in CP plants at 147 DAT indicates enhanced photoprotective capacity due to their antioxidant properties. Carotenoids are essential for dissipating excess excitation energy and preventing oxidative damage, thereby protecting the photosynthetic apparatus from photodamage and photoinhibition [54]. This is consistent with the findings of Hussein et al. [61], who observed that higher fertilization rates promoted plant growth and were associated with increased carotenoid accumulation. Thus, while vermicompost can provide numerous agronomic benefits, stimulating early physiological activity, its exclusive use can lead to reduced chlorophyll and carotenoid contents in leaves as observed at 43 DAT in this study. On the other hand, compost application appeared to prime the vines for photoprotection during late-season senescence.

2.3.2. Antioxidant and Osmoprotective Responses

The results of leaf biochemical parameters related to antioxidant capacity and osmoprotection are summarized in Table 3.

At 43 DAT, significant differences among treatments were observed for protein, proline and soluble sugar content. The CP treatment led to the highest leaf protein and proline contents, suggesting enhanced nitrogen assimilation and osmoprotective metabolism. CP, C− and C+ plants also showed significantly higher accumulation of soluble sugars compared to VCP. No significant differences were observed among treatments for total leaf phenolics, flavonoids, or ABTS antioxidant activity.

At 81 DAT, the CP- and VCP-treated plants presented similar total phenolic and protein contents; however, CP promoted the highest flavonoid accumulation, nearly double that of the VCP treatment, indicating stronger stimulation of secondary metabolism. Regarding antioxidant activity, both CP and C− plants showed significantly higher ABTS radical scavenging activity compared to C+ (increases of 24% and 20%, respectively) and VCP-treated plants (increases of 15 and 18%, respectively). CP- and VCP-treated plants also exhibited the highest proline values, suggesting an active influence of these treatments on the plant’s osmoprotection system.

At 147 DAT, VCP plants exhibited the lowest leaf phenolic content, whereas both CP- and VCP-treated plants showed significantly higher flavonoid accumulation than C− and C+, suggesting enhanced photoprotection and antioxidant activity during late development. Conversely, proline and sugar contents declined in fertilized plants, indicating reduced osmotic stress and improved physiological status, in comparison to the C−.

Throughout the experimental period (43–147 DAT), phenols and flavonoids generally declined across all treatments, reflecting a progressive reduction in secondary metabolite accumulation as plants matured. Protein content remained relatively stable across time, with differences within natural variability among treatments. ABTS antioxidant activity also showed a tendency to decrease over time, consistent with the reduction in phenolic compounds. Conversely, proline exhibited a marked decrease from 43 to 147 DAT, particularly when soil was fertilized, contrasting with the C− plants, suggesting lower stress levels in these three groups. Soluble sugar content presented a different trend between dates: a slight increase from 43 to 81 DAT, followed by a decline toward the end of the season. Overall, these temporal trends indicate a metabolic shift from antioxidant defense towards osmotic regulation and energy storage as the phenological cycle progressed.

Observing these results, beyond their impact on photosynthetic performance, organic amendments also influenced leaf biochemical traits associated with the antioxidant metabolism and osmoprotection, highlighting their role in modulating plant physiological responses [32,62]. Phenolic compounds and flavonoids function as key non-enzymatic defense agents in plants, acting as biochemical markers that are highly responsive to environmental changes and stress conditions [63]. In the present study, the concentration of phenols and flavonoids decreased over time, which may indicate better physiological performance throughout the cycle. Although organic amendments typically stimulate phenol and flavonoid production via the shikimate pathway, through the upregulation of phenylalanine ammonia-lyase [64], this effect was not observed in the early stages of this study. This finding aligns with Yusof et al. [65] who found that in Clinacanthus nutans Lindau vermicompost did not affect total phenol content and actually reduced flavonoid content compared to unfertilized plants. Similar reductions in phenolic compounds and antioxidant capacity have been reported in lettuce [66], pak choi [67], and chincuya [68] plants, fertilized with vermicompost. These observations suggest that certain factors present in vermicompost could downregulate the synthesis of phenolic compounds [65]. The accumulation of secondary metabolites such as phenolics and flavonoids is influenced by interactions between plant genotype and environmental conditions, such as cultivation methods, seasonal variation, abiotic and biotic stresses, and the plant’s nutrient status [69,70]. In particular, the nutritional status is essential in supporting plant growth, with previous studies indicating that vermicompost supplementation enhances nutrient availability (particularly N) in readily absorbable forms, thereby improving nutrient uptake by plants [71]. As a result, as plants are not nutrient-limited, the synthesis of phenols and flavonoids tends to be lower [65]. Furthermore, the increased availability of N, a crucial component of amino acids, can lead to higher protein content in plant tissues [72]. This is in accordance with our protein results, as at 43 DAT, the CP treatment tended to increase the protein content in plants, while the same was verified at 81 DAT in the VCP plants.

Regarding the osmoprotectants (proline and soluble sugars), the application of compost and vermicompost resulted in higher proline accumulation at 43 and 81 DAT, likely due to nutrient availability rather than stress. Since proline is a nitrogenous-compound, increased N absorption could have enhanced proline synthesis and accumulation [73,74], and a greater stress resistance in the early dates [75]. These results coincide with previous finding in mango ginger [76] and in tomato [77]. Crucially, increased proline synthesis suppresses glutamate, which plays a key role in the biosynthesis of chlorophyll a, potentially explaining the reduction in chlorophyll content in the treated plants [37,78]. Although CP and VCP plants showed higher proline content than C− at 43 DAT, this dynamic diminished over time. In fact, by the end of the assay, all fertilized plants showed lower proline content than C−, with decreases ranging from 88% to 95%. This lower proline accumulation in CP and VCP at the end of the season suggests reduced stress and improved physiological status, which is in accordance with studies that highlight the stress-mitigating effects of vermicompost [74,79,80,81,82]. A similar trend was observed for soluble sugars, which declined by 8% to 16% relative to C− at 147 DAT. The absence of sugar accumulation in treated plants may be attributed to the positive influence of the organic amendments on leaf growth, leading to a stronger assimilation of sugar in sink-organs, thereby reducing soluble sugar accumulation [83]. Taken together, these results indicate that both organic amendments seemingly alleviated metabolic stress particularly at the end of the season.

2.3.3. Shoot and Root Length and Biomass

The length and weight of shoots and roots at 190 DAT are presented in Figure 2. Regarding shoot development, only VCP-treated plants exhibited significantly greater stem length compared to C−, showing an increase of 26%. However, in terms of stem biomass, neither CP nor VCP resulted in significant gains compared to C−, while C+ led to a significant increase in this parameter. For root development, all fertilization treatments (C+, CP, and VCP) significantly improved root length compared to the C−, with increases ranging from 59 to 70%. No significant differences in root length were found among the fertilized treatments themselves. However, for root biomass chemical fertilization (C+) and compost (CP) significantly increased root weight in comparison to C−, whereas VCP did not affect this parameter. This suggests that while vermicompost strongly stimulated root elongation, it did not translate into proportional biomass accumulation. Conversely, compost appeared to favor biomass accumulation, particularly in the root system.

Despite the limited number of growth traits evaluated in the present study, similar responses have been described in other studies assessing the effects of organic fertilization on grapevine growth. For instance, Gaiotti et al. [84] reported that compost derived from pruning residues significantly increased root growth, while other authors observed general improvements in vegetative development under organic fertilization [85,86]. Increased pruning wood biomass is often indicative of improved plant vigor and assimilating production, which could have also contributed to the greater root development observed here [87,88]. A key finding of this study was the differential effect of the amendments: CP favored biomass, while VCP favored elongation. The biomass accumulation in CP plants could be attributed to the slightly slower and sustained nutrient release characteristic of composts. Furthermore, vermicompost has also been previously associated with leaf area expansion and increases in photosynthetic capacity, leading to optimal plant growth and biomass accumulation [89,90]. In the present study, VCP was mainly associated with shoot and root length rather than to shoot and root biomass, probably due to improved nutrient availability, stimulation of microbial activity, and production of growth-regulating compounds such as humic substances and auxin-like molecules [37,67,74]. Organic soil amendments applied to vineyards influence microbial activity and nutrient availability, which in turn support increased plant vigor and biomass development [32]. In fact, these organic amendments can harbor a variety of plant growth-promoting bacteria (PGPB) that enhance plant productivity through processes such as nitrogen fixation, nutrient solubilization, and the production of growth hormones [91]. This growth-promoting capacity of composts and vermicompost has been widely demonstrated across diverse plant species, including quinoa [92], pigeon pea [93], corn [36], tomato [94], olive [95], pine [96,97], banana [98], papaya [99] and peach [100].

3. Materials and Methods

3.1. Plant Material and Experimental Design

The experiment was carried out from April to September of 2024 at the University of Trás-os-Montes e Alto Douro (41°17′14.8″ N 7°44′14.8″ W, 500 m above sea level), Baixo Corgo sub-region of the Douro Demarcated Region, Vila Real, northern Portugal. This region exhibits a Mediterranean climate, with hot, arid summers and cooler winters accompanied by significantly higher precipitation levels. Rainfall is markedly seasonal, with minimal precipitation during the summer months. Monthly total precipitation and minimum, average and maximum mean temperature recorded during the experimental period at a weather station in Vila Real are presented in Figure 3. Briefly, during the experimental period, the average temperature varied between 13.7 °C in April and 23.6 °C in August, while precipitation values ranged from 0 mm in August to 67.4 mm in April.

The experimental trial was conducted using one-year-old grapevines (Vitis vinifera cv. Touriga Franca, grafted onto 1103P rootstock) grown outdoors in 15 L pots filled with silty-loam soil (characterized in Section 3.1.2). The experimental design consisted of four treatments with six replicates each, totaling twenty-four plants. The treatments included (C−) and positive (C+) controls, and compost (CP), and vermicompost (VCP) soil applications. The plants were monitored daily, with weed control performed manually when necessary and irrigation adjusted at 90% of field capacity. Field capacity was determined by irrigating the pots until water drained freely from the bottom, indicating saturation. The pots were then allowed to drain, and this condition was considered as field capacity. The C− treatment consisted of soil without any type of fertilization or amendment, to which no mineral or organic inputs were applied during the experiment. The C+ treatment was supplied with 1.25 g NPK 12:11:18, 7.5 g single superphosphate 18% P₂O₅, and 0.25 g KCl 60% K₂O, per pot. Compost and vermicompost, produced under the conditions described in Section 3.1.1. were applied once at the beginning of the experiment, at 375 g fresh weight (FW) per pot. Application rates were defined according to the Portuguese legislation (Portaria no. 185/2022, 21st of July) based on metal concentrations (Section 3.1.1) and soil pH (Section 3.1.2).

Physiological and biochemical measurements were conducted on three dates: May 28th (43 DAT), July 5th (81 DAT), and September 9th (147 DAT). The initial sampling date (43 DAT) was selected to ensure plants had developed mature leaves suitable for the assessments. Chlorophyll a fluorescence data was collected only at 81 and 147 DAT due to technical constraints with the equipment during the first time point.

For the biochemical analyses, leaves used for the gas-exchange and chlorophyll a fluorescence measurement were flash-frozen in liquid nitrogen, ground to a fine powder and stored at −80 °C. Leaves were collected from the middle third of the plant, selecting fully expanded leaves with similar sun exposure across treatments and sampling dates. Additionally, at 190 DAT, half of the plants from each treatment were randomly selected for destructive growth analysis.

3.1.1. Compost and Vermicompost Production

Compost and vermicompost were produced from a mixture of vineyard pruning and sewage sludge, with the solid fraction of cattle slurry as inoculum as described in Morais et al. [101]. Composting was carried out in insulated containers (135 L) with aeration occurring 10 times during a 140-day thermophilic phase. Following this phase, part of the compost was transferred to wooden boxes (56 L) containing 150 g of Eisenia fetida earthworms, clitellated and non-clitellated, for vermicomposting, which lasted 100 days. The remaining compost continued its maturation phase in the same insulated containers and for the same duration as the vermicomposting process. Both final products, compost and vermicompost, were dried, sieved, and then analyzed for their chemical and microbiological characteristics (

Table 4. Chemical and microbiological properties of compost and vermicompost used as soil amendment.

Variables	Units	Compost	Vermicompost
Moisture	(%)	61.0	61.1
pH		6.4	6.5
Organic matter	g kg−1	879.5	846.7
Electrical conductivity	dS m−1	1.6	1.7
N	mg kg−1	28.6	28.4
P		4.6	4.3
K		16.4	16.5
Ca		17.1	19.0
Mg		4.4	4.3
S		3.1	3.1
B		25.0	26.2
Fe		2406.9	2665.3
Cu		109.6	103.7
Zn		319.5	313.5
Mn		225.7	228.9
Ni		5.7	6.0
Cd		0.3	0.3
Pb		9.5	11.9
Cr		6.6	7.9
Hg	µg kg−1	38.2	59.0
C/N		17.8	17.3
N-NH4+/N-NO3−		0.13	0.06
E. coli		Not present
Salmonella spp.		Not present

).

3.1.2. Soil Characterization

Soil samples were collected from the surface Ap horizon (0–20 cm), air dried and sieved (Ø < 5 mm). Chemical and physical properties were determined in the <2 mm fraction (

Table 5. Physico-chemical properties of the soil sampled at a depth of 0-20 cm and used in the trial.

Soil Properties	Units	Value
pH (H2O) (1:5)		5.4
pH (KCl 1M)		4.2
Electrical conductivity (1:5)	dS m−1	0.11
Organic matter	g kg−1	16.4
N		0.96
Extractable P2O5	mg kg−1	23.0
Extractable K2O		115.0
Exchangeable Ca	cmol(+) kg−1	6.98
Exchangeable Mg		2.03
Exchangeable K		0.27
Exchangeable Na		0.04
Exchangeable acidity		0.30
Effective cation exchange capacity		9.63
Extractable calcium	mg kg−1	1397.0
Extractable magnesium		244.0
Copper EDTA		4.07
Zinc EDTA		10.1
Iron EDTA		87.8
Manganese EDTA		43.6
Copper		26.2
Zinc		97.9
Lead		17.9
Cadmium		0.23
Chromium		43.8
Nickel		22.6
Mercury	µg kg−1	26.0
Ca:Mg		6.7
K:Mg		0.42
C:N		9.9
Coarse sand (0.2–2.0 mm)	(%)	16.6%
Fine sand (0.02–0.2 mm)		41.3%
Silt (0.002–0.2 mm)		31.5%
Clay (<0.002 mm)		10.6%
Texture classification		Silty loam

). The pH was measured both in a 1:5 soil-to-solution ratio using H₂O and 1 mol L⁻¹ KCl [102], and electrical conductivity (EC) in a 1:5 soil-to-water suspension [103]. Effective cation exchange capacity, calculated as the sum of Ca, Mg, K, and Na plus exchangeable acidity, was extracted with barium chloride and analyzed by ICP-OES [104]. P₂O₅ and K₂O were extracted using a 2:40 soil-to-ammonium lactate solution ratio [105]; P₂O₅ was quantified by the modified molybdenum–ascorbic acid blue method in a segmented flow autoanalyzer SanPlus (Skalar Analytical B.V., Breda, The Netherlands) [106], and K₂O was determined by atomic absorption spectroscopy. Micronutrients (Fe, Cu, Zn, Mn) were extracted in a 1:10 soil-to-AAAc-EDTA solution at pH 4.65 [107] and analyzed by atomic absorption spectroscopy. Particle size distribution (sand, silt, clay) was determined using a Skalar Robotic Analyzer SP2000 (Skalar Analytical B.V., Breda, The Netherlands) after removing organic matter with H₂O₂ and carbonates with HCl, with pyrophosphate added during analysis. Total carbon was measured by dry combustion with near-infrared detection using an elemental TOC analyzer, and boron was extracted in hot water (1:2 soil-to-water ratio) and determined by ICP-OES [108]. Total nitrogen was quantified using the Kjeldahl method [109].

3.2. Leaf Gas-Exchange and Chlorophyll a Fluorescence

Leaf gas-exchange measurements were performed on six leaves per treatment, during the morning period (9:00–10:30 a.m.), at 43, 81, and 147 DAT, using a portable InfraRed Gas Analyzer (LCpro+, ADC BioScentific Ltd., Hoddesdon, UK) equipped with a 6.25 cm² leaf chamber. Net CO₂ assimilation rate (A, µmol·m⁻² s⁻¹), stomatal conductance (g_s, mmol·m⁻² s⁻¹), transpiration rate (E, mmol·m⁻² s⁻¹), and ratio of intercellular to atmospheric CO₂ concentration (C_i/C_a) were calculated using the equations of von Caemmerer and Farquhar [110]. The A/g_s (µmol·mol⁻¹) ratio was used to estimate the intrinsic water use efficiency.

Chlorophyll a fluorescence parameters were measured at 81 and 147 DAT on the same leaves used for gas exchange measurements, using a Pulse Amplitude Modulated Fluorometer (Mini-PAM, Photosynthesis Yield Analyzer; Walz, Effeltrich, Germany). Prior to the following measurements, leaves were dark-adapted for 30 min using leaf clips. Maximum quantum efficiency of photosystem II (PSII) was calculated as F_v/F_m = (F_m − F₀)/F_m by measuring the fluorescence signal from a dark-adapted leaf when all reaction centers are open using a low-intensity pulsed measuring light source (F₀) and during a pulse saturating light (0.7 s pulse of 15,000 mol photons m⁻² s⁻¹ of white light) when all reactions centers are closed (F_m). Following F_v/F_m estimation, after a 20 s exposure to actinic light (1500 mol m⁻² s⁻¹), light-adapted steady-state fluorescence yield (F_s) was averaged over 2.5 s, followed by exposure to saturating light (15,000 µmol m⁻² s⁻¹) for 0.7 s to establish F_m′. The sample was then shaded for 5 s with a far-red light source to determine F₀′. Fluorescence parameters (photochemical quenching, qP; non-photochemical quenching, NPQ; and photochemical efficiency of photosystem II, Φ_PSII, were calculated according to [45,111].

3.3. Biochemical Analysis

The absorbance values of the following biochemical analyses were acquired using a SPECTRUM star Nano spectrophotometer (BMG Labtech GmbH, Munich, Germany).

3.3.1. Photosynthetic Pigments

To quantify the photosynthetic pigments (chlorophyll a, chlorophyll b, total chlorophyll and carotenoids), 10 mg of leaf samples were extracted in 4 mL of 80% acetone at 4 °C. The solution was then centrifuged at 1753× g at 4 °C for 10 min. After centrifugation, the absorbance was read at wavelengths of 663 nm, 645 nm, and 470 nm. As the pigments are photo and thermo-sensitive, the entire extraction and quantification process was carried out in the dark and on ice. The content of photosynthetic pigments was calculated according to Arnon [112] and Lichtenthaler [113], and results expressed as mg g⁻¹ FW.

3.3.2. Total Soluble Sugars

Total soluble sugars content was determined according to Irigoyen [114]. Briefly, soluble sugars were extracted by heating 10 mg of fresh sample in 5.0 mL of 80% ethanol for 1 h at 80 °C, following an anthrone sulfuric acid method. The determination of total soluble sugars was made in triplicate by reading the absorbance at 625 nm. A standard curve was prepared with glucose, and the results expressed as mg g⁻¹ FW.

3.3.3. Total Soluble Protein Quantification

For total soluble protein quantification, 10 mg of fresh leaf sample were extracted using a potassium phosphate buffer (pH 7.5) with ethylenediaminetetraacetic acid (EDTA) and quantified according to Bradford [115]. Summarily, after 30 min of centrifugation (12,000× g rpm) at 4 °C, the extract was added to the Coomassie Brilliant Blue reagent and the absorbance values were recorded at 595 nm. A standard curve was prepared with bovine serum albumin and the results were expressed as mg g⁻¹ FW.

3.3.4. Phenolic Compounds and Antioxidant Activity

For determining the total phenolics and flavonoids content as well as the ABTS, the extracts obtained with MeOH/H₂O (70:30, v/v) at the concentration of 4 mg FW mL⁻¹. The total phenolic compounds were determined by the Folin–Ciocalteu method [116,117] at 725 nm and expressed as mg gallic acid equivalents per gram of extract (mg GAE g⁻¹ FW). The aluminum chloride (AlCl₃) complex method, at 510 nm, was used for the quantification of the total flavonoids content and results were expressed as mg of catechin equivalents per gram of extract (mg CAE g⁻¹ FW) [116]. The antioxidant activity was determined using ABTS (2.2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging following the method of Trolox equivalent antioxidant capacity (TEAC) assay at 734 nm, and was expressed as mg of Trolox equivalents per gram of extract (mg TE g⁻¹ FW).

3.3.5. Proline Determination

Proline concentration was measured following the method of Bates et al. [118], with minor adjustments. Briefly, 200 mg of fresh leaf samples were homogenized in 3% (w/v) sulfosalicylic acid. Following centrifugation at 3500× g rpm for 20 min at 4 °C, 200 µL of the extract was mixed with 200 µL of ninhydrin acid and 200 µL of glacial acetic acid. The mixture was incubated at 100 °C for 1 h, and then cooled on ice. Subsequently, 1 mL of toluene was added to the mixture and the absorbance of the toluene phase was read at 520 nm. A standard curve prepared with proline was performed and the results were expressed as mg of proline per gram of FW (mg g⁻¹ FW).

3.4. Growth Parameters

To assess the cumulative effects of the compost and vermicompost treatments over the entire growth period, at the end of the experiment (190 DAT), three randomly selected plants from each treatment were harvested, and the length of both stems and roots (cm) were measured. The dry weight (DW) of aboveground and belowground organs (g) was determined after drying them in a force-draft oven at 70 °C until a constant weight. Total dry matter included both oven-dried stems and roots. These assessments were performed when plants were considered fully developed, allowing for a comprehensive evaluation of growth performance and the influence of the treatments on plant development and biomass allocation.

3.5. Statistical Analysis

Statistical analyses were performed with SPSS 20.0 software (Statistical Package for the Social Sciences, Chicago, IL, USA). After testing for ANOVA assumptions (homogeneity of variances with the Levene’s mean test, and normality with the Kolmogorov–Smirnov test), statistical differences between treatments (C−, C+, CP and VCP) within each sampling date were evaluated by one-way factorial ANOVA, followed by the post hoc Tukey’s test (p < 0.05).

4. Conclusions

This study demonstrates that compost and vermicompost derived from vine pruning residues, despite having similar chemical compositions, exert distinct influences on grapevine physiology and growth. Vermicompost enhanced gas exchange, photosynthetic rate, and shoot elongation, albeit at the cost of lower water-use efficiency. In contrast, compost promoted a more conservative growth strategy, characterized by moderate increases in photosynthetic activity while improving water-use efficiency, root biomass accumulation, and photoprotective capacity. These differences suggest that biological activity and nutrient release dynamics, rather than total chemical composition alone, drive the differential physiological responses.

From a practical perspective, vermicompost is recommended for enhancing early-season vigor and photosynthetic capacity, while compost may be preferable under water-limited conditions where improved water-use efficiency and root development are critical. Both amendments substantially improved plant performance compared with unfertilized controls and achieved physiological outcomes comparable to conventional fertilization, while offering additional benefits related to stress tolerance and sustainability. These findings emphasize that the choice between organic amendment should be guided by specific production goals and plant physiological targets

Although these pot-based results are promising, long-term field trials are essential to validate amendment performance under realistic vineyard conditions and across multiple growing seasons. Future research should also assess amendment responses under typical Mediterranean stress factors, including drought, heat, and nutrient-limited soils, to optimize organic residue valorization strategies for sustainable viticulture.