Valorization of Underused Biomass of Acacia dealbata and Acacia melanoxylon Through Vermicomposting as an Alternative Substrate for Cucumber Production

Abstract

Invasive alien species are one of the main threats to global biodiversity, and pose significant management challenges in several areas outside their natural range. In southern Mediterranean Europe, the invasion of Acacia species is particularly severe and its control requires costly and often ineffective actions. The use of vermicompost derived from these species to replace peat-based substrates in horticulture offers a promising alternative to mitigate their economic and environmental impacts while enhancing the sustainability of their control. This study explored the potential of vermicompost produced from the fresh aboveground waste biomass (leaves + stems + flowers) of Acacia dealbata and Acacia melanoxylon (75:25 w/w), two of the most aggressive Acacia species in the Mediterranean, using Eisenia fetida over twelve weeks. In essence, this study aimed to evaluate the quality of the produced vermicompost and its suitability as a partial substitute for potting substrate in the production of cucumber (Cucumis sativus) seedlings for transplant. Four substrate mixtures containing 0%, 10%, 30%, and 50% of Acacia vermicompost (w/w), combined with commercial peat-based potting substrate and perlite (20%) were tested in polystyrene seedling trays. Seedling emergence, growth, and leaf biochemical parameters (photosynthetic pigments, phenolics, soluble sugars and starch, and total thiobarbituric acid-reactive substances—TBARSs) were evaluated. The results showed that the addition of Acacia vermicompost to the commercial substrate did not affect its germination but significantly enhanced seedling growth, particularly in mixtures containing 30% and 50% Acacia vermicompost. In addition, the absence of accumulation of TBARSs also reflected the superiority of these two treatments. These findings suggest that vermicompost derived from A. dealbata and A. melanoxylon biomass can be a viable peat-based substrate alternative for horticultural production, with the dual benefit of promoting sustainable agricultural practices and contributing to invasive species management.

1. Introduction

Invasive alien plant species (IAPS) are among the top five drivers of global environmental disruption, causing immeasurable losses in biodiversity, ecosystem function, and economy [1]. Acacia spp., mainly Acacia dealbata and A. melanoxylon, are two well established IAPS in Southern–Western and Mediterranean Europe, including Portugal, Spain, France, and Italy [2] presenting considerable management challenges. In Portugal, both species are listed as invasive (DL 92/2019) and their maintenance, breeding, reproduction, commercialization, introduction into the wild and repopulation is prohibited. Traditionally, management of these IAPS relies on manual and mechanical methods combined with herbicide application [3]. These methods usually require considerable human and economic resources and are rarely effective for long-term control [4]. Particularly challenging is their management in post-fire situations, due to high seed production, persistent soil seedbank [5,6], and vigorous resprouting from stumps and roots [7]. Both Acacia species also exhibit allelopathic/phytotoxic properties [8,9,10,11,12], which further complicate their management and impacts. Removing these Acacia species often generates large amounts of biomass waste and its improper disposal or abandonment on the ground can exacerbate economic and environmental issues [13,14]. The need for more sustainable, eco-friendly and even profitable ways to manage them is becoming increasingly urgent, and is recognized by the scientific community, decision-makers and land managers. Therefore, converting this abundant biomass waste into value-added products supports a zero-waste approach and aligns with circular economy principles [15]. Furthermore, such strategies can also ease management costs and generate revenue, supporting efforts to control IAPS spread [7], an ongoing priority of the EU Regulation no. 1143/20214. The EU Biodiversity Strategy 2030 (specifically commitment 9) and the European Green Deal also emphasize this priority and have the ambitious goal of reducing the IAPS spread by 50% by 2030.

In recent years, several attempts have been made to find potential ways of utilizing A. dealbata and A. melanoxylon residues for different agricultural purposes, as reviewed by Lorenzo & Morais [16]. Vermicompost, generated by the joint action of earthworms and microorganisms [17,18], is considered a promising value-added product, since, when added to the soil, it improves soil quality and health [19,20], promotes seed germination and plant growth [21], and reduces dependency on mineral fertilizers [22]. In this way, the use of vermicompost directly contributes to several Sustainable Development Goals (SDGs), particularly SDGs 2 (zero hunger), 6 (clean water and sanitation), and 15 (Life and land). In addition, the microbial and enzymatic activities promoted by the earthworms contribute to reduce or eliminate allelopathy and the toxicity of the substrates, making them more appropriate for land application [23,24]. In addition, earthworm activity may indirectly contribute to these species’ seed degradation [25], potentially helping to control IAPS spread.

Vermicomposting has been successfully tested in several terrestrial IAPS such as the annual herbs Parthenium hysterophorus L. [26,27,28,29,30,31] and Ageratum conyzoides L. [17,32], the herbaceous to woody perennial Chromolaena odorata (L.) R.M. King & H. Rob. [33,34], the perennial woody shrub Lantana camara L. [29,33,35], the climber Mikania micrantha Kunth [36,37] and aquatic IAS, namely, Eichhornia crassipes (Mart.) Solms [38,39,40,41,42], Pistia stratiotes L. [43], or Salvinia molesta D. S. Mitchell [44,45]. Most of the cited studies used the IAPS mixed with various proportions of animal manure, mainly cow dung, which does not provide a full understanding of the quality of IAPS vermicompost alone. Additionally, few studies have explored the agronomic value of the produced vermicompost. Hussain et al. [23,24] concluded that the allelopathic potential of L. camara and P. hysterophorus was totally eliminated through the vermicomposting and the resulting vermicompost improved germination and early growth of three common vegetables such as green gram (Vigna radiata), ladies finger (Abelmoschus esculentus), and cucumber (Cucumis sativus), when used at optimal concentrations. In a different study, Karthikeyan et al. [46] compared the vermicompost produced exclusively from L. camara with an inorganic fertilizer which had all the main macro- and micronutrients in concentrations equivalent to the ones present in the vermicompost, and concluded that L. camara vermicompost has the potential to support the germination, growth, and fruit yield of cluster bean (Cyamopsis tetragonoloba) better than equivalent quantities of inorganic fertilizers.

The production of high-quality seedlings is a key factor in crop production, and it is highly dependent on the substrate used [47]. Peat, and its derivatives, remains the most commonly used substrate in potting media, but due to environmental concerns regarding its extraction, it is crucial to find more environmentally friendly alternatives [48]. For example, Alami et al. [49] observed that the vermicompost produced from E. crassipes can be used as a peat substitute for lily cultivation. Regarding A. dealbata, Quintela-Sabaris et al. [50] highlighted its potential for producing vermicompost but did not explore its agricultural value. To the best of our knowledge, A. melanoxylon has not been previously studied for vermicompost production.

To bridge these knowledge gaps, a laboratory-scale experiment was conducted with the objective of evaluating the potential of Acacia vermicompost to partially replace a commercial peat-based pot plant substrate in the production of cucumber (Cucumis sativus) seedlings by analyzing seed emergence and seedling growth under outdoor conditions. This study is the first to investigate the combined use of A. dealbata and A. melanoxylon residues for vermicomposting and to explore the subsequent application of the obtained vermicompost in agricultural production, contributing to novel insights into sustainable agriculture and IAPS management.

2. Materials and Methods

2.1. Substrates

In the present study, two organic materials were considered to produce different substrates, one derived from the vermicomposting of a mixture of A. dealbata and A. melanoxylon, and a commercial peat-based pot plant substrate (BIO Siro, Leal & Soares, Lda., Mira, Portugal), composed of Sphagnum blonde peat, pine bark humus, coco peat, and biological organic fertilizer.

The aerial biomass (young branches, leaves, and flowers) of A. dealbata and A. melanoxylon used in the vermicomposting process was collected from the fields of the University of Trás-os-Montes and Alto Douro, Vila Real, Portugal. The fresh branches and leaves were shredded into 1–2 cm pieces before use, while the flowers were used whole. A mixture of A. dealbata and A. melanoxylon (75:25 w/w), based on the abundance of these invasive species in the area, was placed in 5 L plastic container, containing 10 g of the earthworm species, Eisenia fetida, obtained from our own production. The process was conducted with no pre-composting or manure supplementation under room temperature laboratory conditions and in darkness. During the process, the moisture level was maintained at around 70–80% and adjusted with water as needed. The life cycle of the earthworms was evaluated monthly by monitoring key indicators of their activity and reproduction, such as the number of individuals, their weight, and cocoon production. These parameters were used to assess the activity of the earthworms and the progress of the vermicomposting process. After twelve weeks, vermicomposting was completed, and a composite sample of the produced vermicompost was collected for analysis at UTAD’s Soil and Plant Analysis Laboratory, following established procedures [51].

The main characteristics of the Acacia vermicompost and of the commercial pot plant substrate are detailed in

Table 1. Characteristics of the Acacia vermicompost and the peat-based commercial SiroPlant substrate (Leal & Soares, Lda., Mira, Portugal) used in the pot experiment.

	Units	Acacia Vermicompost	Commercial Substrate
DM	%	22.5	41.5
pH (1:5)		7.3	5.8
EC (1:5)	dS m−1	1.61	0.77
OM	g kg−1 DM	798	749
C/N		14.70	56.52
N-NH4+	mg kg−1 FW	27.72	7.62
N-NO3−	mg kg−1 FW	423	145
N	g kg−1 DM	31.49	7.69
P	g kg−1 DM	1.38	1.37
K	g kg−1 DM	15.9	4.27
Ca	g kg−1 DM	18.8	17.3
Mg	g kg−1 DM	3.60	1.69
S	g kg−1 DM	2.02	1.69
B	mg kg−1 DM	16.5	10.7
Fe	mg kg−1 DM	985	5051
Cu	mg kg−1 DM	6.63	14.06
Zn	mg kg−1 DM	40.9	43.0
Mn	mg kg−1 DM	327	164
Ni	mg kg−1 DM	0.58	5.53
Cd	mg kg−1 DM	0.04	0.09
Pb	mg kg−1 DM	1.36	7.38
Cr	mg kg−1 DM	1.41	11.37
Hg	ug kg−1 DM	14.4	15.4

DM (dry matter); FW (fresh weight).

. The Acacia vermicompost had a higher organic matter (OM) content, electrical conductivity (EC), and pH values than the commercial substrate. It also exhibited higher macronutrients content, especially N, and lower amounts of micronutrients and metals. The vermicompost also had NH₄⁺-N content much lower than its NO₃⁻-N content, indicating a high level of maturity. The C/N ratio of the vermicompost was approximately 15, which indicates its maturity and is the preferable value for the agronomic use of compost [52]. Previous research work demonstrated the maturity and the disappearance of phytotoxicity in the Acacia vermicompost [53]. This was evidenced by a germination rate exceeding 85% and a germination index greater than 60%, compared to the control treatment involving distilled water. According to Portuguese legislation (Portaria n.o 185/2022 de 21 July 2022), this vermicompost is described as part of group 5 (organic correctives) and class I (regarding maximum admissible levels of metals), making it suitable for agricultural use.

2.2. Experimental Design

The experiment was conducted outdoors near the University of Trás-os-Montes and Alto Douro, Vila Real, Portugal (41°30′2′′ N, 7°38′55′′ W; 770 m altitude), during the summer season of 2024. The climate is Mediterranean, and during the experiment, from August 14 to September 7, the average minimum and maximum temperatures were 15.2 °C and 25.0 °C, respectively.

Cucumber (Cucumis sativus L.) cv. Market (Flora Lusitana Lds., Cantanhede, Portugal) was selected for the trial due to its popularity in the Mediterranean region and its fast growth [54]. In addition, cucumber is a moderate salt-tolerant plant, i.e., it can tolerate values of electrical conductivity up to 2.5 dS m⁻¹ [55], which is suitable for the vermicompost produced (Table 1). Cucumber also can grow in a wide range of pH values, but soils with pH between 6.0 and 6.8 are preferred [56]. The pH values of the vermicompost evaluated in this study were outside of the optimal range, and consequently it was mixed with the commercial peat-based substrate (BIO Siro, Leal & Soares, Lda., Mira, Portugal) to formulate three new substrates, with varying percentages of Acacia vermicompost (10%, 30% and 50% by weight, forming the substrates A10, A30, and A50, respectively). A control treatment (CT) with 0% vermicompost was included. All treatments contained the same proportion of perlite (20%) to enhance aeration. The pH values of the A10, A30, and A50 substrates were 6.44, 6.71, and 6.84, respectively.

Two cucumber seeds were placed in polystyrene trays with cells measuring 30 mm × 30 mm × 65 mm. After germination, only one seedling per cell was kept. Each treatment was replicated three times with 10 seedlings per replicate. The trays were watered daily with tap water and placed in natural sunlight to provide favorable conditions for germination and growth, with their positions re-randomized weekly. Seedlings were allowed to grow until they had developed two pairs of true leaves, which occurred ca. 3 weeks after sowing, the time at which seedlings are usually transplanted. No fertilizer was added throughout the experiment to ensure that any differences in seedling performance were solely due to the substrates tested.

2.3. Data Collection

Seed germination was monitored every 2 days during the first 10 days after sowing. Seeds were considered germinated when seedlings became visible above the soil (substrate) surface. Several germination-related parameters (

Table 2. Germination-related parameters determined 10 days after sowing.

Parameter	Formula
Germination percentage (G, %)	$G \left(\%\right)={\displaystyle \frac{{\sum }_{i=1}^{k}ni}{N}}\times 100$
Mean germination time (MGT, day)	$MGT={\displaystyle \frac{{\sum }_{i=1}^{k}ni\times ti}{{\sum }_{i=1}^{k}ti}}$
Coefficient of velocity of germination (CVG, %)	$CVG \left(\%\right)={\displaystyle \frac{{\sum }_{i=1}^{k}ni\times ti}{{\sum }_{i=1}^{k}ni}}\times 100$
Time of 50% of germination (T50, day)	$T50={\displaystyle \frac{ti+\left(\frac{{\sum }_{i=1}^{k}ni}{2}-ni\right)\times (tj-ti)}{nj-ni}}$
Synchrony of germination (Z)	$Z={\displaystyle \frac{{\sum }_{i=1}^{k}Cni, 2}{C\sum ni,2}}$, being C = ${\displaystyle \frac{ni (n1-1)}{2}}$

ni—number of seeds germinated in time “i”; N—number of seeds used; ti—time between the beginning of the germination and time “i”.

), including the germination percentage (G, %), mean germination time (MGT, day), coefficient of velocity of germination (CVG, %), time of 50% of germination (T50, day), and synchrony of germination (Z, without units) (Table 2) were determined [57,58] using the tool available at Agroinfo https://www.agroninfo.com/seed-germination-measurements/ (accessed on 9 April 2025).

Seedling growth parameters, including shoot height (cm), measured with a 30 cm ruler from the base to the tip of the seedling, and total number of leaves per plant, were recorded at 10, 16, and 24 days after sowing. At 24 days, plants were harvested for additional measurements, including root length (cm), root collar diameter (mm), and fresh shoot and root weight (g). Known quantities of plant material were oven-dried at 60 °C to a constant weight to calculate their dry weight. The root/shoot ratio was calculated for each seedling, by dividing the dry weight of the shoot by the dry weight of the root. The height/diameter ratio was also calculated for each seedling by dividing the shoot height by the root collar diameter. Leaf area (cm²) was determined using the Digimizer image analysis software (available at http://digimizer.com). Additionally, fresh leaf samples were collected after growth measurements, frozen in liquid nitrogen, and stored at −80 °C for biochemical analyses.

Leaf photosynthetic pigments (chlorophyll a, chlorophyll b, total chlorophyll, and total carotenoids) were extracted using 80% acetone (v/v), and quantified according to Sesták et al. [59] and Lichtenthaler [60]. Total phenols were determined by the Folin–Ciocalteu method [61]. Total soluble sugars and starch were quantified by using the method of Irigoyen et al. [62] and Osaki et al. [63], respectively. Lipid peroxidation, indicated by total thiobarbituric acid-reactive substances (TBARSs), was measured using the method of Heath & Packer [64].

2.4. Statistical Analysis

Germination, growth, and biochemical data were analyzed by one-way analysis of variance (ANOVA). Trends on growth parameters over time were subjected to a one-way repeated measures ANOVA, with day of observation as a within-factor and treatments as between-group factor. Mean differences were separated using the Tukey HSD test at a 5% level of significance. All data sets satisfied the ANOVA assumptions of homogeneity of variance and normality of errors. Statistical analyses were performed using IBM SPSS Statistics 29.0 software package (Statistical Package for the Social Sciences, IBM Corp., Armonk, NY, USA). In addition, a heatmap coupled with hierarchical clustering was created using the program Origin2025 to explore similarities among treatments. To avoid the influence of the unit of each parameter on the result, all data were standardized. Cluster analysis was performed based on Ward’s method and square Euclidean distance.

3. Results

3.1. Effect of Acacia Vermicompost on Cucumber Germination

The substitution of the commercial peat-based substrate with Acacia vermicompost did not negatively affect the germination of cucumber seeds (Figure 1) at any of the time points analyzed (p > 0.05). In general, cucumber seeds started to germinate 4 days after sowing, and the highest values were recorded 8 days after sowing (Figure 1). The A50 treatment reached its maximum value earlier, on day 6 after sowing.

At the end of the germination period, the percentage of germination (G) varied between 93.3% and 96.6% (

Table 3. Results of one-way ANOVA for germination percentage (G, %), mean germination time (MGT, day), coefficient of velocity of germination (CVG, %), synchrony of germination (Z), and time of 50% of germination (T50, day) determined 10 days after sowing. Data are present as mean ± SD (n = 3).

	G (%)	MGT (Day)	CVG (%)	Z	T50 (Day)
CT	93.3 ± 5.8	4.3 ± 0.2	23.4 ± 1.1	0.63 ± 0.14	3.6 ± 0.07
A10	96.7 ± 5.8	4.3 ± 0.2	23.4 ± 1.0	0.64 ± 0.14	3.6 ± 0.07
A30	93.3 ± 11.5	4.5 ± 0.2	22.3 ± 1.1	0.56 ± 0.06	3.7 ± 0.04
A50	96.7 ± 5.8	4.5 ± 0.2	22.1 ± 1.1	0.43 ± 0.13	3.9 ± 0.26
p-value	0.900	0.379	0.384	0.256	0.242

) with no statistically significant differences among treatments (p > 0.05). Other germination-related key parameters such as MGT, CVG, Z, and T50 (Table 3) followed the same trend, i.e., there were no significant differences among treatments. However, it was clear that the higher Acacia vermicompost proportion (A50 treatment) required a slightly longer germination time and exhibited lower synchrony, suggesting that this proportion could be the upper limit of Acacia vermicompost use.

3.2. Effect of Acacia Vermicompost on Early Growth of Cucumber

In contrast to the germination stage, seedling growth, especially shoot height, responded strongly to the Acacia vermicompost (Figure 2).

After 10 days, shoot height was similar among treatments, ranging from 2.84 ± 0.41 cm in CT to 3.26 ± 0.58 cm in A50. As time progressed, distinct differences emerged, with the A50 and the A30 treatments consistently showing superior shoot height compared to the other treatments at 16 days. The A10 treatment demonstrated an intermediate performance, with a mean shoot height of 4.45 ± 0.87 cm at 16 days, and the CT was the lowest one at the same time point (3.45 ± 0.65 cm). At the end of the observation period (24 days after sowing), the A50 and the A30 treatments yielded the maximum mean shoot height compared to the CT and A10 treatments, respectively (Figure 3A and

Table 4. Results of one-way ANOVA for morphological parameters determined at the end of the experimental period. Data are presented as mean ± SD (n = 3). For each parameter, different lowercase letters within the respective line indicate significant differences among the treatments (p < 0.05) according to Tukey’s test.

	CT	A10	A30	A50
Shoot height (cm)	4.54 ± 0.91 c	5.62 ± 1.05 b	6.81 ± 0.71 a	6.98 ± 1.18 a
Number of leaves	3.8 ± 0.5 a	3.9 ± 0.5 a	4.0 ± 0.5 a	4.0 ± 0.4 a
Root collar diameter (mm)	2.33 ± 0.34 d	2.69 ± 0.42 c	3.13 ± 0.47 b	3.41 ± 0.46 a
Height/root collar diameter	1.95 ± 0.33 a	2.13 ± 0.49 a	2.23 ± 0.41 a	2.06 ± 0.37 a
Root length (cm)	5.67 ± 1.18 a	5.35 ± 1.11 a	5.40 ± 0.85 a	5.61 ± 1.07 a
Shoot fresh weight (g)	0.62 ± 0.17 d	0.84 ± 0.24 c	1.28 ± 0.30 b	1.47 ± 0.38 a
Root fresh weight (g)	0.77 ± 0.52 a	0.55 ± 0.22 a	0.59 ± 0.23 a	0.65 ± 0.29 a
Shoot dry weight (g)	0.046± 0.016 b	0.051 ± 0.016 b	0.085 ± 0.018 a	0.092 ± 0.025 a
Root/shoot ratio	1.26 ± 0.28 a	0.96 ± 0.19 b	0.80 ± 0.17 c	0.81 ± 0.17 c
Leaf area (cm2)	8.92 ± 2.65 c	13.90 ± 3.95 b	20.26 ± 4.81 a	22.51 ± 6.53 a

).

The number of leaves also varied significantly with treatments over time (Figure 3B). At the first time point (10 days after sowing), the seedlings grown in all treatments had three leaves, and this number increased during the experiment; this occurred more rapidly in the A30 and A50 treatments.

At the end of the measurement period, most morphological parameters showed significant differences among treatments (Table 4). The Acacia vermicompost treatments (A10, A30 and A50) consistently presented higher values for shoot height, root collar diameter, shoot fresh weight and leaf area when compared to the CT. The A30 and A50 treatments exhibited the highest shoot heights, both approximately 50% higher than those observed in the CT. Plant diameter was also around 50% higher in the A50 treatment compared to the CT. The same pattern was found for shoot fresh weight, with the A50 treatment achieving values 138% higher than the CT. Shoot dry weight was significantly higher in the A30 and the A50 treatments, showing increases of 85–100% compared to the CT. Leaf area also followed the same trend, with the A30 and A50 treatments exhibiting 127–152% larger values than the CT. Conversely, the root/shoot ratio was higher in the CT, compared to the other treatments. The A30 and the A50 treatments demonstrated the largest decrease in this parameter, with a 35% reduction. The number of leaves, the diameter/root collar diameter, and the root fresh weight did not differ significantly among treatments (p > 0.05). Overall, the A30 treatment followed the trend of the A50 treatment, while the A10 treatment generally exhibited values closer to the CT.

The use of different proportions of Acacia vermicompost in the commercial peat-based substrate did not have a significant influence (p > 0.05) on the chlorophyll a, chlorophyll b, chlorophyll total, or carotenoids content (

Table 5. Results of one-way ANOVA for biochemical parameters determined at the end of the experimental period. Data are presented as mean ± SD (n = 3). For each parameter, different lowercase letters within the respective line indicate significant differences among the treatments (p < 0.05) according to Tukey’s test.

	CT	A10	A30	A50
Chlorophyll a (mg g−1 DW)	3.60 ± 0.90 a	4.31 ± 0.68 a	4.30 ± 1.33 a	4.76 ± 0.33 a
Chlorophyll b (mg g−1 DW)	1.91 ± 0.47 a	2.07 ± 0.46 a	1.99 ± 0.46 a	2.31 ± 0.19 a
Chlorophyll total (mg g−1 DW)	5.49 ± 1.36 a	6.35 ± 1.12 a	6.16 ± 1.77 a	7.03 ± 0.47 a
Carotenoids (mg g−1 DW)	0.73 ± 0.14 a	0.93 ± 0.07 a	0.97 ± 0.27 a	1.00 ± 0.06 a
Phenols (mg g−1 DW)	6.99 ± 0.25 a	6.12 ± 0.27 b	5.79 ± 0.40 bc	5.17 ± 0.17 c
Soluble sugars (mg g−1 DW)	5.29 ± 0.67 a	4.57 ± 0.54 ab	3.24 ± 0.49 b	3.45 ± 0.40 b
Starch (mg g−1 DW)	115.14 ± 4.11 a	85.96 ± 4.33 b	53.93 ± 1.72 c	58.46 ± 3.08 c
TBARS (mg g−1 DW)	0.033 ± 0.001 a	0.035 ± 0.002 a	0.038 ± 0.003 a	0.038 ± 0.003 a

). However, in general, photosynthetic pigment contents increased in the substrates with higher proportion of Acacia vermicompost. Conversely, phenolic content (Table 5) decreased progressively with increasing Acacia vermicompost proportions, showing reductions of about 12%, 17%, and 26% in the A10, A30, and A50 treatments, respectively, compared to the CT.

The soluble sugar content of the A30 and A50 treatments was significantly reduced by around 40% compared to the CT. On the other hand, the starch content decreased significantly under the A10, A30, and A50 treatments, with reductions of 25%, 53%, and 49%, respectively, compared with the CT. In contrast, none of the treatments influenced the TBARS content (p > 0.05).

3.3. Heatmap with Cluster Analysis

The cluster analysis performed on all data (Figure 4) revealed two groups, one formed by the CT and the A10 treatment, and another group distinct from this, composed by the A30 and the A50 treatments, showing a higher degree of similarity within these pairs. The cluster formed by the A30 and A50 treatments differed from the cluster composed of the CT and A10 treatments in terms of its higher values of morphological parameters and higher amount of photosynthetic pigments, reflecting the trends observed in the parameters analyzed.

4. Discussion

The results indicated that vermicomposting using the earthworm Eisenia fetida had a positive impact on the degradation of green biomass derived from two Acacia species, A. dealbata and A. melanoxylon. In addition, despite these IAPS being known for their allelopathic effects [8,9,10,11,12], the vermicompost did not exhibit these properties. To our knowledge, no previous studies on the specific mixture tested in the present study were performed, but the obtained results confirm the feasibility of vermicomposting residues of Acacia species. This finding aligns with the conclusions of Sabaris-Quintela et al. [50] that described the successful vermicomposting of A. dealbata using a different earthworm species (E. andrei). The physicochemical properties of the vermicompost produced in our study are comparable to those obtained in the aforementioned study [50], although the vermicomposting process lasted longer. These differences can be attributed to the earthworm species used, the initial composition of the mixture, and the earthworm density added to the container, as previously highlighted by Devi & Khwairakpam [17] and Bernal et al. [41] in studies with Parthenium hysterophorus and water hyacinth, respectively.

The end products of vermicomposting invasive plant species have been used in several applications [16], including their use in agriculture, for example, as organic material for potting substrate [32,49]. Selecting an appropriate potting substrate and optimizing its dosage is essential for providing mechanical support to the plant, as well as the nutrients and water required for its growth and development, all of which contribute to maximum transplantation success [47]. The present study shows that the addition of Acacia vermicompost at varying proportions (10%, 30%, and 50%) to the commercial substrate did not impair the germination of cucumber seeds. Germination occurred between the fourth and seventh day after sowing, with percentages ranging between 93 and 97% (Figure 1). Furthermore, no significant differences were observed in any of the parameters related to germination (Table 2), even for the higher proportion of Acacia vermicompost (50%). This indicates that, on the one hand, the EC and pH were favorable for germination, and, on the other hand, there was a well-balanced composition of nutrients in the substrate (Table 1) which upheld cucumber germination. The absence of negative effects of Acacia vermicompost on germination underscores its potential as a sustainable alternative to conventional peat-based substrates for cucumber germination. Alami et al. [49] reported similar results, observing that water hyacinth vermicompost supported successful growth of lily plants (Longiflorum × Asiatic cv. ‘Nashville’), indicating its potential for substituting peat in horticultural potting substrates.

The development of a robust aboveground part of the seedling is particularly important in trays where root growth is limited by the small volume of substrate in these containers [65]. This study indicated that while germination was not affected, increasing Acacia vermicompost up to 50% positively influenced shoot length, root collar diameter, shoot fresh and dry weight, and leaf area (Table 4), which is associated with its higher macronutrient availability, particularly with regard to N, P, K, Ca, and Mg (Table 1). These findings highlight the beneficial role of vermicompost in enhancing vegetative growth, and are consistent with the results reported on other vegetable seedlings, such as tomato [66,67], lettuce [66], pepper [66,67], and cornflower [67]. The similarity between the A30 and A50 treatments observed in the study suggests that increasing the vermicompost rate beyond a certain threshold does not result in further substantial benefits, potentially indicating a saturation point for plant growth response. This result aligns with findings by Blouin et al. [22], who observed maximum plant growth benefits with vermicompost proportions comprising between 30% and 50% of the soil volume. Conversely, other studies on cucumber plants have reported optimal vegetative growth at lower vermicompost proportions (20–40%) [68,69,70,71], which is undoubtedly associated with composition of the vermicompost used [65].

As an indicator of seedling strength [72], the height/root collar diameter found in the present study was similar in all treatments (Table 4), and within the values indicated by Adamczewska-Sowińska et al. [72]. The balance between the shoot height and its diameter indicates more robust seedlings, with a high probability of success when transplanted to the field.

Regarding the number of leaves, length of root, and root fresh weight (Table 4), the lack of significant differences among treatments suggests that these parameters are less sensitive to short-term changes in nutrient availability than other morphological parameters, such as shoot length or diameter. Similarly, chlorophyll content in cucumber leaves (Table 5) showed no significant differences among treatments, although there was a tendency toward increased chlorophyll content in vermicompost treatments (A10, A30, and A50) compared to the control (CT). This result suggests that Acacia vermicompost enhances photosynthetic capacity and growth. Jankauskiene et al. [71] also reported higher chlorophyll content in cucumber seedlings grown in vermicompost-enriched substrates compared to those grown only in peat. Furthermore, similar results were also reported in yellow mustard plants after substituting potting substrates with 5%, 10%, and 20% of vermicompost [73], and in radish and tomato, when vermicompost was used in proportions between 5 and 15% in the potting substrate [74].

There are very few reports on the impact of vermicompost use on the total phenolic content. A significant reduction in leaf phenolic content was observed in the current study with increasing vermicompost proportions, possibly reflecting the physicochemical attributes of the vermicompost used, which improved growing conditions. Calderon and Mortley [74] observed a similar result with increasing vermicompost proportions. Moreover, the lack of significant differences in TBARS content across treatments indicates that Acacia vermicompost provides favorable conditions for cucumber growth without inducing oxidative stress.

Interestingly, the soluble sugars and starch content decreased significantly with increasing vermicompost proportions but stabilized at higher levels (A30 and A50). This trend may indicate enhanced metabolic activity and nutrient bioavailability, supporting overall seedling growth. Higher sugar and starch allocation to biosynthetic processes likely contributed to increased biomass accumulation and shoot development, as reflected in the observed improvements in shoot height, foliar area, and shoot dry weight. These similarities between A30 and A50 treatments were further supported by cluster analysis (Figure 3), which grouped these treatments closely, suggesting comparable effects on plant growth parameters.

5. Conclusions

The results of this study indicate that Acacia vermicompost is free from toxic elements and possesses high nutritional value. It can partially replace peat-based commercial substrates, without the need for pre-composting and/or manure supplementation, and without negatively impacting the emergence or early growth of cucumber seedlings. While seed germination was unaffected, substituting peat-based substrates with Acacia vermicompost significantly enhanced seedling growth, as evidenced by increased shoot height, plant diameter, shoot fresh and dry weight, leaf area, and reductions in leaf phenolic content. These improvements were achieved without the use of additional fertilizers, which highlight the quality of the Acacia vermicompost.

The results also revealed that the highest proportion of Acacia vermicompost (50%) was equally as effective as a 30% proportion, suggesting that 50% may represent the upper limit for optimal use. To confirm this, future studies must explore a wide range of Acacia vermicompost proportions, evaluate its effects on other crops, and assess the behavior of seedlings after their transplantation under diverse growing conditions.

Based on the results, vermicomposting of Acacia species offers a promising eco-friendly and even profitable solution for producing horticultural substrates. It benefits from the high availability of Acacia biomass, which enhances the sustainability of IAPS management, promotes nutrient recycling, and reduces reliance on peat, thereby contributing to natural resource conservation and supporting circular economy approaches. In addition, this process can be adapted to different operational scales due to its simplicity and reduced resource requirements, enhancing its cost-effectiveness and sustainability. However, further studies should focus on conducting a cost–benefit analysis to assess the economic and environmental benefits of using Acacia vermicompost to produce horticultural substrates.