Innovative Peat-Free Organic Substrates and Fertilizers Influence Growth Dynamics and Root Morphology of Fagus sylvatica L. and Quercus robur L. Seedlings One Year After Planting

Abstract

This study evaluated the effects of six innovative peat-free substrate formulations, combined with either a conventional solid fertilizer or a novel liquid fertilizer developed by the research team, on the early growth and root morphology of Fagus sylvatica L. and Quercus robur L. seedlings. Treatments were analyzed through two-way ANOVA and species-specific linear regression models. Following one year of field growth, survival rates remained high across all treatments. While R22 (a peat-free substrate with liquid fertilizer) exhibited the highest mean values for seedling height and diameter, only height showed statistically significant variation among treatments (p < 0.05), with no significant differences observed for diameter increment. It was further, revealed that seedlings treated with peat-free substrates and liquid fertilizers exhibited adequate survival, with several combinations especially R22 showing comparable performance to traditional peat-based media with solid fertilizer. Root morphological traits, particularly fine root length (≤0.50 mm) were strong predictors of above-ground growth in F. sylvatica, but less so in Q. robur, which relied more on total root length. The results highlight species-specific root–shoot coordination strategies, with beech exhibiting above-ground growth pattern and oak a gravitropic one. The findings concluded that R22 substrates confirmed exceptional performance with enhanced root growth comparable to peat after one year of forest planting, indicating strong potential for future development without the environmental concerns associated with peat use.

1. Introduction

Reforestation and afforestation initiatives are critical to combating climate change, enhancing biodiversity and stabilizing ecosystems [1,2]. Seedling survival after planting constitutes an essential component of the success of forest restoration programs [3,4,5,6]. This survival is strongly influenced by nursery cultural practices and silvicultural techniques, which play an important role in the performance of a seedling immediately after transplanting. [3]. Root system morphology is particularly important because its architecture directly affects water and nutrient uptake, thereby influencing tree seedling’s overall health and resilience [7]. The importance of root characteristics, in predicting seedling survival and adaptability to varying environments cannot be overemphasized [8].

Classifying root diameter is essential for understanding below-ground carbon dynamics [9]. Very fine roots, defined as those less than 0.5 mm in diameter, are more accurate indicators of root function than the broader traditional category of roots under 2 mm [10]. These very fine roots display species-specific traits and exhibit remarkable plasticity, adjusting their biomass and length across soil depths to optimize nutrient and water uptake [10,11,12]. Fine roots, ranging from 0.5 mm to 2 mm in diameter, are vital to nutrient cycling in terrestrial ecosystems [13,14]. Though they make up less than 5% of forest biomass, they are highly dynamic, functioning as both nutrient sources and sinks, and play a key role in carbon cycling and accumulation [10]. In contrast, coarse roots those greater than 2.0 mm in diameter differ significantly in morphology, nutrient content, and decomposition processes. Their size often correlates with aboveground biomass, and factors like tree size and age are commonly used to predict their development [9,15].

Known for its exceptional physical, chemical and biological properties, peat has long been a stable component in nursery substrates. Its superior water-holding capacity and consistent quality make it ideal for plant growth. However, the slow release of carbon from peat soils raises environmental concerns. Europe experienced a dramatic rise in peat excavation, with volumes soaring from 6000 tonnes in 2012 to 20 million tonnes by 2022 [16,17,18] This represents a staggering 333% increase over a decade, underscoring the expansion of peat extraction and its contribution to environmental degradation [19,20]. Consequently, EU Member States have been actively seeking to reduce peat consumption [21,22,23]. With impending restrictions on the availability of peat [24,25,26,27], the need to find alternative materials to replace peat demand an urgent attention, either partially or entirely. In response to this pressing issue, our team has developed and proposed a substrate designed to provide a sustainable solution [7,28].

Despite increasing efforts to promote peat-free substrates in forest nurseries, existing studies have notable limitations. Many studies evaluating peat-free substrates and fertilization strategies primarily assess above-ground parameters such as height, diameter, and biomass accumulation [28,29,30]. However, root system development, remains understudied. While solid fertilizers are widely used in forestry nurseries, recent advancements in liquid fertilizers suggest they may offer improved nutrient uptake efficiency [31,32,33,34,35]. These studies further examined fertilization effects in nurseries but rarely in the context of peat-free organic substrates. This study therefore, provides a direct comparison of solid and liquid fertilizers in combination with innovative organic substrates, assessing their effects on above-ground growth and below-ground root morphology one year after planting in a forest environment.

Therefore, this study aimed to evaluate the performance of innovative peat-free substrates in combination with two contrasting fertilization approaches: a conventional solid fertilizer and a novel liquid fertilizer formulation developed by our research team. The liquid fertilizer was designed specifically to complement the nutrient dynamics of organic peat-free substrates and optimize seedling uptake efficiency. The tested research hypotheses assumed that: (i) these innovative treatments would produce root and shoot traits comparable to conventional methods. (ii) Fagus sylvatica and Quercus robur would respond differently to the treatments due to their distinct growth strategies. (iii) specific root morphological traits may vary in their association with early shoot growth between the studied species.

2. Materials and Methods

2.1. Study Site

The study site was located in Barbarka, Miechow Forest District. The research area is situated at an altitude of approximately 370 m above sea level, in the Olkuska Upland, southern Poland (50°15′54.2″ N 19°53′36.5″ E). The experiment was situated in a forest complex managed by the National Forest Holding. The area was established in several gaps resulting from the clear-cutting of a Populus spp. plantation. The area of the Miechów Forest District is characterized by a diverse, upland landscape. The Olkuska Upland is a compact karst plateau made of limestone and marl. The climate is continental, characterized by significant temperature amplitude (21 °C) and a significant share of rainfall during the growing season. The average annual air temperature for the Forest District is 8.2 °C. The warmest month is July (19.6 °C), while the coldest is January (−3.0 °C).

2.2. Substrate Composition and Preparation

The peat rich in sphagnum used as the control variant (C) for growing the seedlings in this study was obtained from the nursery farm in Nędza (50.167964 N, 18.3138334 E). Its composition consisted of 93% peat and 7% perlite, with the addition of dolomite (3 kg per 1 m³ of substrate) to achieve a pH of 5.5. The elemental content (g/g of 100% dry weight of the growing medium at the beginning of the experiment) of 37.99 ± 0.69 (C), 0.74 ± 0.01 (N), 0.02 ± 0.01 (P) The peat-free substrates (R20, R21, and R22) were sourced from coniferous woody (mainly pine) they composed of a mixture of different components, including shavings, wood chips, straw, bark, perlite, core wood and mixed silage, with varying proportions as shown on

Table 1. Composition of the organic peat free substrate.

Substrate	Saw Dust	Wood Chips	Straw	Wood Bark	Perlite	Core Wood	Mixed Silage
					(%)
R20	73	10	-	10	4	2	1
R21	20	63	-	10	4	2	1
R22	50	-	10	33	4	2	1

. In total, four substrates (R20, R21, R22, and peat) were utilized, each subjected to two fertilization (S and U) variants. The first set received standard solid fertilization (SR20, SR21, and SR22 variants), while the second set was treated with a novel liquid fertilizer also developed by the University of Agriculture in Kraków (UR20, UR21, and UR22). The peat substrate served as the control in both fertilization scenarios, designated as SC and UC variants (

Table 2. Physicochemical properties of substrates used in seedling growth in the Nursery.

Substrate	Water Capacity (%)	Water Outflow Rate (L/min)	Bulk Density (g/cm3)	Solid Density (g/cm3)	Air Capacity (%)	Porosity (%)
R20	40.5 ± 2.9 b	0.595 ± 0.150 b	0.115 ± 0.009 a	0.64 ± 0.08 a	52.1 ± 3.19 c	92.6 ± 0.60 d
R21	33.1 ± 2.5 d	0.781 ± 0.114 a	0.098 ± 0.014 c	1.74 ± 0.07 a	60.8 ± 3.06 a	93.6 ±0.87 c
R22	37.8 ± 5.1 c	0.594 ± 0.150 b	0.104 ± 0.020 b	1.66 ± 0.11 a	55.8 ± 5.58 b	93.9 ± 0.98 b
Control	57.7 ± 5.4 a	0.417 ± 0.145 c	0.085 ± 0.007 d	1.69 ± 0.14 a	37.0 ± 5.72 d	94.7 ± 0.42 a
F	387.45	56.32	65.81	1.0717	295.79	76.48
p	0.0000	0.0000	0.0000	0.3870	0.0000	0.0000

Letters with different alphabet indicate statistically significant differences between means (p < 0.05).

).

Prior to filling, the substrate was pre-moistened using a line mixer with spray nozzles, and moisture levels were controlled organoleptically by the line staff to ensure the substrate reached the standard moisture level for container filling. The substrate’s moisture content was 75.9 ± 2.1%. The vibration intensity of the vibrating table was kept constant during the filling process, at 12.0 G maximum acceleration, as measured by the Voltcraft DL-131G device with ±0.5 G accuracy. Throughout the experiment, the line’s efficiency remained stable at 400 containers per hour, which is the standard rate at this nursery. All operating parameters line configuration, containers, and substrate types were consistent with those used in a previous experiment [35].

2.3. Seed Sowing and Germination

Using mechanical methods, the containers were filled with substrates and seeds immediately planted on 19 April 2022, at the Nursery Farm in Suków Papiernia (50.79613, 20.71011), Daleszyce Forest District. The experiment utilized V300 Styrofoam containers, which are commonly used in Poland for cultivating deciduous species such as beech and oak. To improve the germination process, oak seeds were scarified before sowing. After sowing, the containers were placed in a greenhouse for 4 weeks before being transferred to an external production field. During the seedling growth period in the nursery, manual weeding was carried out. The seedlings were cultivated for 5 months, following the standard procedure used in container nurseries [36]. Due to a total rainfall of only 78 mm during this period, irrigation was necessary to address the water deficit, and an automatic RATHMAKERS Gartenbautechnik sprinkler ramp was used for this purpose.

Osmocote fertilizer was incorporated into the substrate before sowing, with a total application rate of 3 kg per cubic meter of substrate. This was a mixture of 2 kg of Osmocote 3-4M and 1 kg of Osmocote 5-6M. The Osmocote 3-4M fertilizer had the following composition: 16% nitrogen (N), with 7.1% as N-NO₃- and 8.9% as N-NH₄+; 9% P₂O₅; 12% K₂O; 2.0% MgO; and included micronutrients (B, Fe, Cu, Mn, Zn, Mo). The Osmocote 5-6M fertilizer contained 15% nitrogen, with 6.6% as N-NO₃₋ and 8.4% as N-NH₄+; 9.0% P₂O₅; 12% K₂O; 2.0% MgO; and similar micronutrients. A new liquid fertilizer regimen was also employed, consisting of two different formulations. The first fertilizer contained 4.78% N, 1% P₂O₅, 2.64% K₂O, 2.65% CaO, 1.4% MgO, 0.71% SO₃, and 0.14% Na₂O. It was initially applied with a total volume of 3.14 dm³ (0.048 dm³ per square meter). The second fertilizer composition included 0.798% N, 0.166% P₂O₅, 0.440% K₂O, 0.441% CaO, 0.234% MgO, 0.118% SO₃, and 0.023% Na₂O. This was applied with a total volume of 15.09 dm³ (0.229 dm³ per square meter). During the period of seedling production, the first fertilizer variant was applied eight times at 10-day intervals, while the second variant was applied 15 times at 5-day intervals. This fertilization schedule was uniformly applied to both beech and oak seedlings throughout the nursery phase.

2.4. Plantation Establishment and Seedling Collection

After nursery production, the seedling was transported and planted into the forest on 5 September 2022. The field experiment was laid in a randomized complete block design with 8 treatments replicated 3 times. A total of 24 subplots were established for each species. In each subplot, 49 seedlings were planted with 1 × 1.7 m inter and intra-spacing making a total of 147 seedlings per treatment and species. For both species, therefore, were a total of 2352 seedlings were established. The plantation was established at the onset of the autumn season 2022. At the end of the growing season in 2023, 144 seedlings were selected (3 from each subplot) the seedlings were selected according to the average height of each subplot. The seedlings were carefully uprooted to obtain an intact root segment. The mean height of each subplot characterized the selected seedlings. They were carefully chosen from each of the eight treatment groups for onward laboratory analysis. This resulted in a total assessment of 144 seedlings for both species in the laboratory experiment. To reduce the impact of animals on the new forest, the area was fenced after the plantation was established. Above-ground data was collected on plant height, collar diameter, number of seedlings in perfect condition (SPC) total survived seedlings (TSS). The root morphological characteristics examined in this study include total root length (TRL), root surface area (RSA), average root diameter (ARD), and root volume (RV) to assess how different treatments impacted below-ground development. Root morphological diameters were further classified into very fine (≤0.5 mm), fine (0.5–2.0 mm) and coarse root (>2.0 mm).

2.5. Root Sample Preparation and Analyses

In the laboratory, all roots within each block were processed as follows: Root systems were carefully separated from soil and organic matter to keep the root segments intact and maintain attachment to the larger roots (>2 mm in diameter). The intact root segments were then gently rinsed with tap water followed by deionized water to remove residual soil while preserving delicate root tips. Morphological traits of roots in each diameter class were analyzed using WinRhizo™ Pro 2003b image analysis system (Regent Instruments Inc., Ville de Quebec, QC, Canada), an image analysis system specifically designed for root measurements. This analysis was conducted in the Laboratory of Biotechnology, Department of Ecology and Silviculture, University of Agriculture in Kraków.

2.6. Soil Sample Collection and Analysis

A 0.7-hectare research plot was established on a harvested Populus spp. site characterized by uniform parent material and soil type. Soil samples were collected from five different points within each subplot at two depth intervals: 0–10 cm and 10–20 cm, representing the top mineral horizons. Samples were placed in polyvinyl chloride (PVC) bags for transport and analysis. In total, 480 soil samples were collected. Each sample was air-dried, passed through a 2 mm sieve, and ground prior to physicochemical analysis. Soil pH was measured using the potentiometric method in both water and 1M KCl. Hydrolytic acidity was determined using the Kappen method, while exchangeable acidity and base content were assessed using the Sokołow method [37]. Total nitrogen and carbon contents were analyzed with a LECO CNS TruMac Analyzer (LECO Corporation, St. Joseph, MI, USA). The concentrations of alkaline cations (Ca²⁺, Mg²⁺, K⁺, Na⁺) were determined using 1M ammonium acetate extraction and quantified via inductively coupled plasma optical emission spectrometry (ICP-OES) with an iCAP 6500 DUO instrument (Thermo Fisher Scientific, Cambridge, UK). All analyses were conducted at the Laboratory of Forest Environment Geochemistry and Land Intended for Reclamation, Department of Ecology and Silviculture, Faculty of Forestry, University of Agriculture in Kraków, Poland.

2.7. Statistical Analyses

To analyze the effect of seedling growth on plantation survival, an overall effect of growth was assessed by fitting all sample units (2352 seedlings) in the population. Using destructive sampling method, nine root samples were selected per treatment, resulting in 72 data points analyzed per species for morphological evaluation. From WinRhizo™ Pro 2003b image analysis system (Regent Instruments Inc., Ville de Quebec, QC, Canada) the data were further separated into very fine, fine and coarse roots (≤0.5 mm, 0.5–2.0 mm, >2.0 mm respectively). To meet normality and homogeneity assumptions for further analyses, the Shapiro-Wilk test for compliance of variable distributions with normal distribution was used. The analyzed variables showed compliance with a normal distribution, and therefore, parametric tests were used. To evaluate treatment effects, on root morphological indices and diameter classification of very fine, fine, and coarse roots, two-way Analysis of Variance (ANOVA) was conducted separately for each species, using substrate type (peat-based vs. peat-free) and fertilizer type (solid vs. liquid) as fixed factors, and block as a random effect in a randomized complete block design. Duncan multiple range test (DMRT) was applied post hoc for pairwise comparisons and significance was set at p < 0.05. Multiple linear regression models were developed to explore the predictive relationships between root morphological traits and above-ground performance indicators. All statistical analyses were performed at a significance level of 95% confidence interval.

3. Results

3.1. Soil Properties

At both sites of beech and oak, the soils were slightly acidic, which is typical of forest environments. The pH values at 20 cm are slightly lower compared to those measured at 10 cm. Oak retains a uniform N concentration between both depths. Altogether, C contents were higher both at 10 cm in both sites and decreased at 20 cm as well P levels. Consistent trends in the C/N ratio across depths indicate that C and N cycling within the soil is balanced. Both sites also have more exchangeable cations at 10 cm than at 20 cm indicating that the upper soil layer plays a crucial role in seedling nutrient uptake, particularly in early growth stages. No significant differences were observed between the two sites based on statistical analysis of soil properties, although there were some small numerical differences among depths (

Table 3. Mean and SD of soil properties of sampled plot of oak and beech at Barbarka experimental site.

Soil Uptake Level (cm)						Exchangeable Cations ((cmol[+]/kg)
	pH (H20)	N (%)	C (%)	P2O5 (mg/100g)	C/N	Ca	K	Mg	Na
Fagus sylvatica site
0–10	5.21 ± 0.55	0.36 ± 0.10	2.41 ± 0.21	3.14 ± 0.21	13.78 ± 0.32	5.51 ± 0.52	0.18 ± 0.01	0.57 ± 0.06	0.70 ± 0.08
10–20	5.10 ± 0.57	0.34 ± 0.09	1.94 ± 0.15	2.87 ± 0.14	12.90 ± 0.32	4.81 ± 0.39	0.16 ± 0.01	0.42 ± 0.03	0.63 ± 0.06
Total	5.15 ± 0.56	0.35 ± 0.10	2.16 ± 0.12	2.99 ± 0.12	13.30 ± 0.23	4.22 ± 0.32	0.15 ± 0.01	0.49 ± 0.03	0.55 ± 0.07
p-value	0.187 ns	0.170 ns	0.061 ns	0.286 ns	0.065 ns	0.064 ns	0.062 ns	0.062 ns	0.062 ns
Quercus robur site
0–10	5.14 ± 0.59	0.45 ± 0.95	2.14 ± 0.22	4.70 ± 0.45	13.31 ± 0.33	5.11 ± 0.53	0.23 ± 0.02	0.55 ± 0.06	0.81 ± 0.26
10–20	5.03 ± 0.61	0.44 ± 0.09	1.83 ± 0.14	4.22 ± 0.40	12.23 ± 0.31	4.65 ± 0.36	0.21 ± 0.02	0.46 ± 0.03	0.82 ± 0.27
Total	5.08 ± 0.59	0.45 ± 0.10	1.55 ± 0.13	4.44 ± 0.30	12.73 ± 0.23	3.32 ± 0.32	0.22 ± 0.01	0.38 ± 0.03	0.82 ± 0.29
p-value	0.208 ns	0.945 ns	0.061 ns	0.427 ns	0.069 ns	0.061 ns	0.363 ns	0.061 ns	0.989 ns

ns = not significance.

). This implies a similar soil composition across the study area that can serve as a controlled background for comparison of treatment effects on seedling growth.

3.2. Growth Dynamics of F. sylvatica and Q. robur Seedlings One-Year After Forest Plantation

The result of above-ground growth parameters confirm that seedling establishment was successful across all treatments, with survival rates consistently exceeding 70%, the minimum benchmark for acceptable forest regeneration in Poland. The analysis of biometric increments following field transplanting revealed differences in species responses and treatment effectiveness. Notably, seedlings treated with liquid fertilizers exhibited reduced growth in both species, despite showing acceptable survival rates under both fertilization methods. After one year in the forest, treatment had a significant effect on height (p < 0.05), with R22 achieved height increment comparable to the peat-based control across both species. SC and SR22 (solid), UC and UR22 (liquid) were statistically grouped as the top performers for both height and diameter in both species (

Table 4. Biometric and increment rate of F. sylvatica and Q. robur seedlings after one year on crop.

	Fertilization Type				After 1 Year in the Forest		After Nursery Production Cycle		Absolute Increment (%)
Treatment		SPC	TSS	RSS (%)	Height (cm)	Collar Diameter (mm)	Height (cm)	Collar Diameter (mm)	Height	Collar Diameter
Fagus sylvatica
SR20	Solid	112	123	84	58.12 ± 9.89 b	9.11 ± 2.23 a	31.46 ± 3.56 a	5.63 ± 1.48 a	85	62
SR21		135	142	97	59.01 ± 10.42 ab	8.93 ± 2.01 a	30.25 ± 3.43 a	5.70 ± 1.62 a	95	55
SR22		138	143	97	61.92 ± 11.61 a	9.22 ± 1.96 a	31.64 ± 3.29 a	5.58 ± 1.15 a	96	58
SC		133	143	97	62.25 ± 12.02 a	9.61 ± 2.48 a	30.88 ± 3.23 a	5.72 ± 1.23 a	102	68
Total					60.32 ± 11.18	8.94 ± 2.44	31.06 ± 3.23	5.66 ± 1.26
p-value.					0.033 *	0.203 ns	0.473 ns	0.341 ns
UR20	Liquid	138	143	97	44.21 ± 7.77 f	7.33 ± 1.98 e	30.55 ± 3.23 e	5.22 ± 0.95 e	45	40
UR21		130	136	93	43.32 ± 7.18 f	7.41 ± 2.04 e	30.24 ± 2.98 e	5.36 ± 1.24 e	44	38
UR22		113	135	92	50.07 ± 8.49 e	7.68 ± 1.86 e	31.18 ± 3.19 e	5.21 ± 1.07 e	61	47
UC		118	135	92	54.04 ± 8.52 e	7.96 ± 1.49 e	31.07 ± 3.08 e	5.28 ± 0.99 e	56	51
Total					49.41 ± 3.19	7.60 ± 1.82	30.76 ± 3.17	5.27 ± 1.07
p-value.					0.024 *	0.176 ns	0.253 ns	0.316 ns
Quercus robur
SR20	Solid	129	134	91	56.76 ± 9.28 a	8.72 ± 1.89 a	31.15 ± 2.97 a	5.53 ± 1.56 a	82	58
SR21		123	130	88	55.74 ± 7.95 a	8.61 ± 1.78 a	31.73 ± 3.47 a	5.25 ± 1.43 a	76	64
SR22		132	140	95	56.79 ± 7.74 a	8.62 ± 2.03 a	30.95 ± 2.99 a	5.42 ± 1.39 a	77	59
SC		145	145	99	57.07 ± 6.89 a	8.93 ± 2.11 a	31.09 ± 2.99 a	5.66 ± 1.55 a	77	59
Total					55.58 ± 8.89	8.72 ± 1.85	31.23 ± 1.34	5.47 ± 1.67
p-value.					0.103 ns	0.473 ns	0.564 ns	0.271 ns
UR20	Liquid	139	142	97	41.72 ± 8.44 f	7.22 ± 1.10 e	31.51 ± 2.74 e	5.45 ± 0.89 e	32	32
UR21		134	140	95	41.03 ± 8.26 f	7.79 ± 1.22 e	30.43 ± 2.55 e	5.52 ± 0.92 e	35	41
UR22		139	144	98	42.69 ± 9.67 f	7.81 ± 1.54 e	31.49 ± 2.86 e	5.47 ± 0.95 e	36	43
UC		132	138	94	45.19 ± 9.93 e	7.94 ± 1.44 e	31.51 ± 3.02 e	5.66 ± 0.96 e	43	40
Total					42.66 ± 9.08	7.69 ± 1.32	31.23 ± 2.94	5.52 ± 0.94
p-value.					0.007 **	0.978 ns	0.305 ns	0.231 ns

SPC—Number Seedlings in perfect condition, TSS–Total survived seedlings, RSS—rate of seedling survival. S—State Forests solid fertilization, U—University novel liquid fertilization, R—novel substrates, C—control substrate (peat−perlite) (N = 147). Letters with different alphabet indicate statistically significant differences between means (p < 0.05). Alphabets ‘a’ and ‘b’ denote homogeneous groups under solid fertilization and ‘e’ and ‘f’ denote homogeneous groups under liquid fertilization. ns = not significance, * = p < 0.05, ** = p < 0.01.

).

3.3. Root Morphological Response of F. sylvatica and Q. robur Seedlings One Year After Forest Plantation Establishment

The result of root morphological parameters shows that the effect of solid fertilization was more pronounced on root morphological indices than the liquid once after one year of growth in the forest. The result of total root length revealed the R20 substrates recorded the higher mean length in beech regardless of fertilization method. The response of oak was different, showing different response to different fertilizer. Peat substrate recorded the highest mean under liquid fertilization while with significant variation while SR21 performed best with no Signiant variation (

Table 5. Morphological parameters of root system of F. sylvatica and Q. robur seedlings under different substrate fertilizer treatment after one year in the forest.

Treatment	Fertilization Type	TRL (cm)	RSA (cm2)	ARD (mm)	RV (cm3)
	F. sylvatica
SR20	Solid	1673.27 ± 252.94 a	171.66 ± 41.43 a	0.85 ± 0.15 a	3.59 ± 0.77 b
SR21		1265.52 ± 151.19 a	168.37 ± 39.55 a	0.82 ± 0.17 a	3.51 ± 1.32 b
SR22		1424.69 ± 265.10 a	182.79 ± 62.43 a	0.72 ± 0.16 a	3.37 ± 1.58 b
SC		1377.56 ± 104.39 a	226.28 ± 48.05 a	0.93 ± 0.19 a	4.10 ± 1.59 a
Total		1335.26 ± 207.75	187.28 ± 52.15 a	0.83 ± 0.18 a	3.89 ± 1.48
p-value.		0.285 ns	0.061 ns	0.087 ns	0.035 *
UR20	liquid	1132.69 ± 130.54 e	113.87 ± 19.61 e	0.83 ± 0.10 e	2.39 ± 0.89 e
UR21		1037.03 ± 92.90 e	88.98 ± 14.17 f	0.72 ± 0.08 f	1.80 ± 0.26 f
UR22		1029.58 ± 88.39 e	89.30 ± 6.50 f	0.72 ± 0.09 f	1.61 ± 0.56 f
UC		1056.09 ± 98.93 e	122.22 ± 10.22 e	0.74 ± 0.06 f	1.53 ± 0.42 f
Total		1063.85 ± 107.76	99.09 ± 16.65	0.75 ± 0.09	1.83 ± 0.65
p-value.		0.157 ns	0.001 **	0.024 *	0.015 **
	Q. robur
SR20	Solid	1396.90 ± 81.03 a	218.23 ± 38.22 a	0.97 ± 0.12 a	4.66 ± 0.56 b
SR21		1445.48 ± 83.89 a	219.83 ± 30.32 a	1.03 ± 0.13 a	6.01 ± 1.07 a
SR22		1443.91 ± 95.26 a	189.41 ± 18.21 b	0.79 ± 0.14 b	3.68 ± 0.48 c
SC		1386.20 ± 82.95 a	187.28 ± 20.75 b	1.03 ± 0.14 a	4.24 ± 0.76 bc
Total		1418.13 ± 86.59	203.69 ± 31.00	0.95 ± 0.16	4.65 ± 1.13
p-value.		0.336 ns	0.024 *	0.001 **	0.000 **
UR20	liquid	1329.14 ± 59.57 f	144.58 ± 19.36 f	0.99 ± 0.32 ef	3.41 ± 0.48 e
UR21		1345.08 ± 44.24 f	125.69 ± 25.66 g	0.81 ± 0.06 f	2.44 ± 0.49 e
UR22		1248.43 ± 68.06 g	109.37 ± 15.36 g	1.07 ± 0.19 e	2.99 ± 0.11 e
UC		1453.88 ± 65.88 e	164.48 ± 10.42 e	0.87 ± 0.18 ef	3.36 ± 1.19 e
Total		1344.13 ± 93.91	136.03 ± 27.42	0.94 ± 0.23	3.05 ± 1.43
p-value.		0.000 **	0.000 **	0.051 *	0.471 ns

TRL—Total root length, RSA—Root surface area, ARD—Average root diameter, RV—Root volume. Letters with different alphabet indicate statistically significant differences between means (p < 0.05). Alphabets ‘a, b’ and ‘c’ denote homogeneous groups under solid fertilization and ‘e, f’ and ‘g’ denote homogeneous groups under liquid fertilization. ns = not significance, * = p < 0.05, ** = p < 0.01.

). For F. sylvatica, RV was the only parameter to show statistically significant differences across treatments under solid fertilization. Although TRL, RSA, and ARD did not yield statistically significant differences (p > 0.05), numerical trends favored the SC treatment. Among the liquid fertilizer treatments, the UC and UR20 demonstrated marginally improved root architecture relative to other liquid combinations, although these differences were less pronounced. The treatment effects were more distinct among solid fertilizer treatments of Q. robur. The SC and SR21 treatments consistently produced superior results across these morphological parameters. TRL under solid fertilization did not differ significantly (p = 0.336), while the liquid fertilization result shows that TRL varied significantly (p = 0.000), with the control variant (UC) and UR20 recording the highest values (Table 5).

3.4. Root Diameter Classification of F. sylvatica and Q. robur Seedlings Under Different Treatments After One Year in the Forest

Root morphological responses across diameter classes showed distinct patterns between F. sylvatica and Q. robur, and were strongly influenced by fertilizer type and substrate combination (

Table 6. Root diameter classification of beech and oak seedlings under different treatments.

Treatment	Fertilization Type	Length < 0.5 mm	Length 0.5–2.0 mm	Length > 2.0 mm	Surface Area < 0.5 mm	Surface Area 0.5–2.0 mm	Surface Area > 2.0 mm	Volume < 0.5 mm	Volume 0.5–2.0 mm	Volume > 2.0 mm
Fagus sylvatica
SR20	Solid	465.22 ± 94.62 b	144.40 ± 30.07 b	36.04 ± 6.30 ab	23.72 ± 6.32 a	42.28 ± 9.08 b	58.19 ± 11.61 b	0.17 ± 0.02 a	1.13 ± 0.27 ab	10.59 ± 2.31 b
SR21		463.26 ± 99.38 b	118.89 ± 41.23 b	28.82 ± 8.58 b	21.27 ± 8.28 a	33.90 ± 11.14 b	57.00 ± 16.68 b	0.14 ± 0.06 a	0.90 ± 0.35 b	10.05 ± 3.55 b
SR22		472.41 ± 109.16 ab	133.92 ± 57.69 b	30.40 ± 7.53 b	24.48 ± 7.03 a	39.49 ± 19.08 b	59.43 ± 9.28 b	0.15 ± 0.05 a	0.97 ± 0.38 b	10.89 ± 1.73 b
SC		493.66 ± 109.71 a	194.83 ± 67.13 a	40.70 ± 9.64 a	26.32 ± 5.46 a	58.74 ± 20.40 a	72.39 ± 15.11 a	0.18 ± 0.04 a	1.52 ± 0.39 a	14.40 ± 4.54 a
Total		471.14 ± 111.71	148.01 ± 56.76	33.99 ± 9.11	23.19 ± 6.87	43.60 ± 17.71	60.75 ± 14.61	0.16 ± 0.04	1.13 ± 0.35	11.38 ± 3.03
p-value		0.048 *	0.021 *	0.015 *	0.376 ns	0.013 *	0.043 *	0.1236 ns	0.045 *	0.032 *
UR20	Liquid	325.34 ± 118.82 e	88.38 ± 25.80 ef	22.79 ± 7.43 e	21.22 ± 6.78 ef	23.21 ± 7.00 ef	42.16 ± 8.81 e	0.14 ± 0.04 e	0.56 ± 0.20 f	6.97 ± 1.40 f
UR21		302.71 ± 94.01 e	81.96 ± 30.18 f	23.84 ± 7.70 e	20.07 ± 5.66 f	21.24 ± 8.24 ef	40.79 ± 11.58 e	0.13 ± 0.04 e	0.50 ± 0.21 f	6.31 ± 2.02 f
UR22		360.41 ± 103.54 e	112.89 ± 45.33 ef	22.83 ± 3.78 e	24.60 ± 7.39 ef	29.68 ± 12.54 f	39.07 ± 4.52 e	0.17 ± 0.06 e	0.71 ± 0.33 e	7.13 ± 1.08 f
UC		382.04 ± 101.77 e	123.33 ± 41.95 e	24.88 ± 5.05 e	26.77 ± 5.66 e	34.31 ± 17.49 e	47.47 ± 8.63b e	0.18 ± 0.05 e	0.72 ± 0.35 e	8.32 ± 1.66 e
Total		342.62 ± 105.03	101.63 ± 39.12	23.58 ± 6.00	23.16 ± 6.70	27.11 ± 12.66	42.36 ± 8.96	0.16 ± 0.04	0.62 ± 0.27	6.89 ± 1.54
p-value		0.394 ns	0.070 ns	0.876 ns	0.124 ns	0.103 *	0.222 ns	0.285 ns	0.022 *	0.015 *
Quercus robur
SR20	Solid	597.88 ± 151.78 b	130.18 ± 19.66 a	37.64 ± 8.96 ab	33.85 ± 2.83 b	37.49 ± 4.85 a	47.87 ± 5.43 a	0.24 ± 0.07 a	0.99 ± 0.15 a	6.38 ± 0.48 a
SR21		664.72 ± 156.56 a	145.75 ± 27.53 a	34.94 ± 12.46 bc	39.58 ± 3.23 a	40.86 ± 3.84 a	56.42 ± 16.35 a	0.26 ± 0.06 a	1.09 ± 0.21 a	8.25 ± 1.81 a
SR22		670.63 ± 193.31 a	134.17 ± 48.93 a	46.29 ± 5.92 a	29.60 ± 6.76 b	38.72 ± 3.33 a	59.85 ± 13.16 a	0.22 ± 0.09 a	1.04 ± 0.36 a	7.62 ± 1.46 a
SC		672.41 ± 112.37 a	152.96 ± 17.73 a	36.57 ± 9.13 c	31.33 ± 6.61 b	41.46 ± 4.77 a	48.78 ± 15.77 a	0.22 ± 0.07 a	1.03 ± 0.19 a	8.69 ± 1.41 a
Total		641.41 ± 157.42	140.76 ± 31.07	36.36 ± 11.48	33.59 ± 6.27	39.63 ± 7.56	53.23 ± 13.81	0.23 ± 0.06	1.04 ± 0.22	7.74 ± 1.29
p-value		0.012 *	0.395 ns	0.001 *	0.002 *	0.674 ns	0.184 ns	0.191 ns	0.204 ns	0.152 ns
UR20	Liquid	443.93 ± 225.91 e	55.89 ± 29.77b ef	12.29 ± 2.96 e	25.54 ± 11.89 e	15.28 ± 7.80 ef	12.53 ± 3.73 f	0.16 ± 0.07 e	0.39 ± 0.21 f	0.95 ± 0.51 e
UR21		421.33 ± 202.95 e	56.70 ± 28.37b ef	13.08 ± 4.37 e	17.63 ± 10.41 e	13.71 ± 8.49 ef	13.25 ± 4.45 ef	0.11 ± 0.06 e	0.38 ± 0.24 f	1.12 ± 0.46 e
UR22		474.44 ± 91.74 e	71.06 ± 35.94 e	13.49 ± 5.74 e	24.22 ± 6.32 e	21.22 ± 9.63 e	11.91 ± 5.49 f	0.15 ± 0.07 e	0.59 ± 0.24 e	1.24 ± 0.43 e
UC		437.25 ± 94.64 e	54.60 ± 18.55 f	15.68 ± 2.94 e	21.92 ± 4.97 e	19.11 ± 5.25 f	17.42 ± 4.45 e	0.13 ± 0.06 e	0.23 ± 0.15 f	1.67 ± 0.58 e
Total		444.24 ± 171.88	52.06 ± 30.71	13.54 ± 4.19	22.33 ± 9.01	14.83 ± 8.78	13.78 ± 4.89	0.14 ± 0.06	0.40 ± 0.09	1.25 ± 0.49
p-value		0.147 ns	0.042 *	0.349 ns	0.267 ns	0.025 *	0.054 *	0.062 ns	0.042 *	0.097 ns

S—State Forests Solid fertilization, U—University novel liquid fertilization, R—novel substrates, C—control substrate (peat−perlite). Letters with different alphabet indicate statistically significant differences between means (p < 0.05). Alphabets ‘a, b’ and ‘c’ denote homogeneous groups under state fertilization and ‘e’ and ‘f’ denote homogeneous groups under novel liquid fertilization. ns = not significance, * = p < 0.05.

). In both species, seedlings treated with solid fertilizers, particularly under SC and SR22 treatments, exhibited significantly higher values across most diameter classes, especially in total length and surface area within the very fine root fraction (≤0.50 mm). Surface area and volume in this fine class were not significantly enhanced in these solid fertilizer treatments, but significantly enhanced in these liquid fertilizer treatments. Although, the differences were more pronounced in Q. robur of the same class, however, treatments R20 and R21 also demonstrated higher performance in few parameters, after peat-base substrate. Similar to solid once, treatment, UR22 again recorded higher output after UC in most of the accessed root parameters. Across both species, coarse volume (in the >2.00 mm) diameter class was less variable and showed fewer significant differences among treatments (Table 6).

The multiple linear regression models showed a strong association between below-ground root architecture and above-ground growth performance. Among the assessed variables, the abundance of very fine roots (≤0.50) classified by diameter emerged as the most influential factor driving both height and stem diameter development in beech. In oak, however, TRL was the only significant root trait influencing height, while none of the finer root classifications nor did other morphological parameters have a meaningful effect on diameter. This result brings a new perspective to forest nursery evaluations by demonstrating that root traits should not be universally applied as predictors across species. The differentiated role of root classifications especially the functional distinction of very fine, fine and coarse roots offers new practical implications. For beech, promoting fine root proliferation during nursery production may yield tangible benefits in early field performance. For oak, a broader view of establishment factors may be needed, extending beyond root architecture alone (

Table 7. Model estimates for above ground parameters of F. sylvatica and Q.robur one year after planting in the forest.

Species/Dependent Variable	Predictor	Coefficient	Std. Error	p-Value	CI Lower	CI Upper	Adjusted R2
F. sylvatica/Plant height	VFL	0.061	0.017	0.001	0.027	0.095	0.619
	VFSA	−3.195	1.213	0.011	−5.623	−0.766	0.619
F. sylvatica/collar diameter	VFL	0.011	0.002	0.000	0.006	0.016	0.644
	VFSA	−0.351	0.169	0.043	−0.689	−0.011	0.644
Q. robur/Plant height	TRL	−0.034	0.013	0.009	−0.059	−0.009	0.530

TRL—Total Root Length (cm), VFL (≤0.50)—Very-fine length (cm), VFSA (VFSA.≤0.50)—Very fine surface area (cm²).

).

4. Discussion

The findings of this study provide viability of innovative peat-free substrates and a novel liquid fertilizer in supporting early seedling development, in Fagus sylvatica and Quercus robur. Inline with the proposed hypotheses, several peat-free treatments most notably R22 demonstrated shoot growth and survival levels comparable to conventional peat-based controls. Species-specific differences reflected contrasting ecological foraging strategies in both species. F. sylvatica exhibited stronger correlations between shoot growth and very fine root development, while Q. robur’s growth aligned more closely with total root length (TRL). Therefore, root morphological traits predict early developmental dynamics under novel substrate and fertilizer regimes.

The analysis of soil properties showed slight numerical differences; however, these variations were not statistically significant (p-value < 0.05). The differences observed between soil depths and sampling locations did not significantly influence the growth or survival of seedlings [37,38,39,40]. The overall consistency in soil characteristics across both sites and depths suggests that soil conditions were not a major factor contributing to variations in seedling performance. Showing high survival rates across sites, beech and oak seedlings responded healthy to the applied treatments after one year of establishment. This agrees with previous research highlighting that the survival of seedlings is enhanced when individuals of the same species and age are planted together [41]. Moreover, successful root development remains a key factor in enabling seedlings to access soil moisture [41,42,43].

The high survival rates may also be due to the selection of superior seedlings from the nursery, a practice known to enhance field performance [44]. The consistently high survival rate observed across all treatments identified the potential of these innovative materials for successful seedling establishment. The ability of peat-free substrates to support similar survival outcomes to peat established their viability as sustainable alternatives in reforestation practices. This positive relationship between seedling size and survival has been observed in tropical species like Gmelina arborea and Khaya senegalensis [45,46] and in Mediterranean areas [44,47]. The study found a similar relationship in temperate species, likely due to the balance between root and foliar surface area and the ability to develop deep root systems before leaving the nursery [7,42,48]. However, some studies report no significant or even negative relationships between seedling size and survival [49,50].

The differences observed in seedling performance across treatments are in line with previous study [51] that report generally positive effects of peat-based substrates and solid fertilizers on early seeding development, although in this study, not all such trends reached statistical significance. This contradicts the preliminary report of Rotowa [7] on the same seedling after nursery production circle. Earlier investigation has shown that peat-based media provide favorable water retention, aeration, and structural consistency, which support both root elongation and nutrient uptake during the early growth phase [17]. The compositional analysis of the substrates used in this study confirms that peat substrates had more balanced organic matter and stable texture compared to the peat-free alternatives, which may have contributed to the improved morphological outcomes observed in the control treatments. Similarly, the physicochemical analysis revealed that the peat-based substrates maintained more favorable pH and EC ranges, which are known to influence nutrient availability and uptake efficiency, particularly in forest nursery system.

The limited performance of the novel liquid fertilizers, especially in Q. robur, aligns with previous findings that tree species differ in their tolerance to substrate variability and fertilization regimes [51,52,53]. While some studies report moderate success using composted or wood-based substrates, their performance often depends on precise control of nutrient formulation and substrate stabilization [54,55] These factors were optimized in the current experimental conditions. Furthermore, few studies have explored the role of root diameter classification as a predictor of shoot growth [9,10,11,12]. The strong positive association between very fine root length (≤0.50 mm) and shoot development in F. sylvatica provides new insight into the functional importance of absorptive root fractions in early seedling establishment. This suggests that simply measuring total root size may overlook key structural parameters that drive above-ground biomass accumulation. While Q. robur responded to total root length alone as a significant predictor. The effectiveness of these treatments in promoting increased growth reinforces the earlier recommendation by Rotowa et al., [7] to proceed with forest plantation using these seedlings once adequate root system formation was achieved after nursery production cycle. Though, the translation of those early gains into field performance was less consistent to earlier nursery performance. However, this evenness in results is consistent with the findings of previous studies by Kormanek et al., [56] on root growth of Quercus petraea seedlings, as well as studies on forest tree species grown in containers [57,58,59].

While the distinction between solid and liquid fertilizers was crucial to the experimental design, the observed differences in seedling growth are likely due not only to fertilizer form but also to variations in nutrient composition, availability, and release profiles. The solid fertilizer (Osmocote) used in this study is a controlled-release formulation that provides a balanced supply of essential micronutrients [60]. Its slow nutrient discharge over time ensured consistent availability during this crucial developmental stage, supporting stable root and shoot growth [61,62,63,64] In contrast, the liquid fertilizer although applied at regular intervals during nursery production period had lower overall nitrogen content and was more susceptible to leaching, especially in the more porous, peat-free substrates like UR22. Furthermore, previous work by Rotowa et al. [28] reported that this peat-based control substrate exhibited higher baseline nutrient concentrations prior to seed sowing compared to the innovative peat-free mixtures. This initial nutrient advantage, in combination with the solid fertilizer’s slow-release properties, likely created more favorable conditions for early seedling establishment in a new forest.

In this study, Fagus sylvatica and Quercus robur exhibited contrasting responses in below and above-ground coordination, particularly in how specific root traits influenced early shoot development. F. sylvatica showed a strong correlation between very fine root length (≤0.50 mm) and above-ground growth metrics, whereas Q. robur responded more strongly to TRL as a growth predictor. Although, these findings are consistent with prior studies that emphasize the functional importance of specific root traits in early seedling growth [8,10,11]. However, these variances reflect ecological and eco-physiological strategies of the two species. Beech has been reported to be a shade-tolerant, mesic-adapted species with a typically shallow, fibrous root system that facilitates efficient nutrient acquisition in surface soils [65,66,67,68]. Its early growth is often characterized by shoot elongation and crown development, strategies consistent with competitive light acquisition in closed-canopy environments. The observed association between shoot growth and very fine root traits may thus reflect this species’ reliance on dense absorptive roots for rapid resource uptake in the upper soil horizon. In contrast, oak has been reported to be more drought-tolerant, and characteristically develops a deep, vertically structured root system to access subsoil moisture, which is crucial for its establishment in open or water-limited sites [69,70,71,72]. The weaker correlation between very fine roots and shoot growth, and stronger association with total root length, likely reflects this more conservative growth strategy, which emphasizes rooting depth over fine root proliferation.

For effective reforestation, nursery practices should be tailored to meet species-specific ecological demands, particularly under evolving climate and soil conditions. The species-specific responses and root-shoot dynamics observed in this study offer valuable guidance for nursery management and reforestation planning. Fagus sylvatica, promoted fine root development through well aerated and nutrient-balanced substrates. In contrast, Quercus robur benefits from practices that support deep, vertically structured root systems, such as using containers and substrates that mimic natural soil conditions.

5. Conclusions

This study provides clear evidence that substrate and fertilizer choices significantly influence the early field performance of Fagus sylvatica and Quercus robur seedlings, with marked species-specific responses. Solid fertilizers consistently outperformed liquid formulations in promoting root development and shoot growth. The adoption of three root diameter classes identified that, very fine root traits (≤0.50 mm) offered novel predictive insights, especially for F. sylvatica, where strong root shoot harmonization was observed. Conversely, Q. robur showed a more gravitropic growth strategy, with total root length being the most informative predictor. The root systems of seedlings in the innovative substrates (R20, R21 and R22) were as well healthy, notable root growth was observed in R22, with values closely matching those of the peat-based control; however, these differences were not statistically in most cases after one year of planting into the forest, indicating a guaranteed prospect for future growth without environmental drawbacks like peat. While the study successfully captured early morphological responses under field conditions, limitations such as the absence of root: shoot biomass data and the inability to isolate nutrient form from their composition suggest directions for further research. Future studies should incorporate dry weight measurements, nutrient-matched fertilizer comparisons, and longer-term monitoring to strengthen conclusions on substrate and fertilizer efficacy. Overall, the adoption of these innovative substrates will not only reduce the environmental footprint but also aid biodiversity conservation, providing significant benefits in terms of seedling health and environmental sustainability during the early growth stages of forest seedlings in a new forest.