Biostimulation Effect of the Seaweed Extract (Ecklonia maxima Osbeck) and Plant Growth Promoting Bacteria (Bacillus subtilis Ehrenberg) on the Growth of the European Beech (Fagus sylvatica L.) Seedlings

Abstract

In forest tree nursery production, bioactive compounds are sought to promote plant growth in response to changing environmental conditions. This study evaluated the effects of the Ecklonia maxima (Osbeck) seaweed extract and the bacterium Bacillus subtilis (Ehrenberg) and their combination on the growth and development of beech (Fagus sylvatica L.) seedlings. A two-factor experiment was conducted in 2023 at the Didactic and Research Station, Department of Ecology and Silviculture, University of Agriculture in Krakow. The nursery experiment investigated the effects of foliar applications of varying doses: Ecklonia maxima at 960 and 1920 cm³·ha⁻¹ and Bacillus subtilis at 112 and 224 g·ha⁻¹ on European beech (Fagus sylvatica L.) seedlings. The application of E maxima seaweed extract a dose of 960 cm³·ha⁻¹ promoting beech seedlings increase in the area of the root system (101.0 ± 17.8 cm²). Seedlings sprayed with B. subtilis at a dose of 112 g·ha⁻¹ were characterized by the longest root system, the largest root collar diameter, and the highest DQI (Dickson Quality Index) values (22.9 ± 3.4 cm, 6.00 ± 0.4 mm, and 0.74 ± 0.2, respectively). Applying B. subtilis at 224 g·ha⁻¹ resulted in an increased SQ (Sturdiness Quotient) value (from 6.33 ± 0.7 for the control variant to 6.62 ± 0.7) and the lowest SHI (Seedling Health Index) value (5.59 ± 0.9). Applying higher doses of Ecklonia maxima (1920 cm³·ha⁻¹) and B. subtilis (224 g·ha⁻¹) increased the SQ index value but decreased the root system area. The best DQI and SHI values were observed with the highest dose combinations of E. maxima and B. subtilis. Selecting suitable biological substances promoting growth can support the sustainable production of beech seedlings while improving the adaptability of forest tree seedlings.

1. Introduction

European beech (Fagus sylvatica L.) is a dominant climax species characteristic of maritime and suboceanic climates and is the predominant deciduous tree species in the forests of Central Europe [1,2,3]. Its geographic distribution encompasses considerable environmental diversity, resulting in unique adaptations to regional conditions and varied strategies for managing environmental pressures [4,5,6,7,8]. Fagus sylvatica is a shade-tolerant species in the early stages of growth, making it suitable for shelterwood and selection forestry systems [9]. Its stands respond positively to thinning, even at an advanced age [10]. Besides its ecological importance, it is also a significant source of raw timber [11]. However, despite its resilience to management, Fagus sylvatica is sensitive to drought and heat waves. This sensitivity is particularly pronounced during the initial phases of ontogeny, when it exhibits remarkable susceptibility to various environmental pressures [12,13]. Consequently, as a key ecosystem engineer, it may be increasingly threatened by the more frequent droughts associated with ongoing climate change [14,15].

Climate change is impacting regions designated for forest management and resulting in adverse effects [16,17]. The exacerbation of climate change, particularly droughts, presents novel problems for forest management within the framework of forest ecosystem management [18,19,20]. The adaptation capacity of forest tree species is insufficient to mitigate the adverse impacts of the growing prevalence of water scarcity in forests [21].

The primary method for recovering forest ecosystems is the cultivation of forest crops. In European Union nations, about 30% of afforestation and artificial forest regeneration use forest tree seedlings predominantly sourced from state-operated nurseries [22,23]. Abiotic and biotic factors contribute to heightened mortality rates in forest tree seedlings, while the application of biostimulants may alleviate the impacts of environmental stressors.

Biostimulants are preparations containing bioactive compounds that influence plant growth [24,25]. In principle, their mechanism of action is aimed at alleviating [26] environmental stresses occurring during plant growth and development. The definition and idea of biostimulants are still developing [27,28]. The development of biostimulants 2.0 concept is underway, founded on a meticulous methodology informed by a comprehensive understanding of the molecular and metabolic mechanisms affecting specific biocompounds in plants and their microbiomes. These are no longer mere “supplements,” but rather sophisticated agricultural instruments [29,30].

Plant biostimulants based on extracts from seaweed species such as Ecklonia maxima (Osbeck), Kappaphycus alvarezii (Doty ex P.C. Silva), Ascophyllum nodosum (Le Jolis), Laminaria digitata (Hudson), Laminaria hyperborea (Gunnerus), Fucus vesiculosus (L.), Durvillea potatorum (Labillardière), and Fucus serratus (L.). Components of seaweed, including macro- and microelements, amino acids, vitamins, cytokinins, auxins, and abscisic acid (ABA)-like growth factors, influence cellular metabolism in treated plants, resulting in enhanced growth and yields. Seaweed extracts demonstrate biological action at low doses. Also seaweed and its derivatives are widely employed in agriculture due to the many active chemicals that enhance plant growth and development [31,32,33,34].

Another highly promising category of biostimulants are microorganisms [35]. Plant growth promoting bacteria (PGPB) are the group of microorganisms have positive effect on the growth and development, physiology of the plants. PGPB can enhance nitrogen fixation, phosphorus mobilization, phytohormone enhancement, and the induction of natural plant resistance through their interactions with plants [36,37]. Bacillus subtilis (Ehrenberg) bacteria promote plant growth by improving nutrient absorption and modifying phytohormonal molecular pathways [38]. In agricultural production, the application of appropriate seaweed extract and plant growth promoting bacteria (PGPB) necessitates various ways, and the correct concentration of the active ingredient in the formulation is essential [39]. The selection of various biochemical chemicals and microorganisms to enhance growth and development corresponds with the contemporary notion of biostimulants [40]. Although widely applied in agriculture and horticulture, the effects of biostimulants on forest tree species remain largely unexplored [41,42,43].

We hypothesize that treatments with preparations containing Ecklonia maxima seaweed extract and plant growth promoting bacteria Bacillus subtilis may alter the biometric characteristics of beech. Subsequently, this research aims to evaluate the efficacy of Ecklonia maxima extract and Bacillus subtilis in the nursery cultivation of Fagus sylvatica seedlings to determine their potential for enhancing artificial forest regeneration under climate change pressures.

2. Materials and Methods

2.1. Experiment and Field Studies

The nursery experiment was conducted under semi-controlled conditions in 2023 at the Didactic and Research Station, Department of Ecology and Silviculture, University of Agriculture in Krakow (49°27′3.043″ N; 20°57′17.083″ E), (

Table 1. Factors and their levels in the forest nursery experiment.

Factor	Levels of the Factors
	Low	Medium	High
Factor 1: Plant Growth Promoter— Biostimulant	0 cm3·ha−1—Ecklonia maxima (0 dm3·ha−1—Kelpak SL) K0	960 cm3·ha−1—Ecklonia maxima (3 dm3·ha−1—Kelpak SL) K1	1920 cm3·ha−1—Ecklonia maxima (6 dm3·ha−1—Kelpak SL) K2
Factor 2: Plant Growth Promoting Bacteria—Biofungicide	0 g·ha−1—Bacillus subtilis (0 dm3·ha−1—Serenade ASO) SR0	112 g·ha−1 —Bacillus subtilis (8 dm3·ha−1—Serenade ASO) SR1	224 g·ha−1—Bacillus subtilis (16 dm3·ha−1—Serenade ASO) SR2

). The two-factor nursery experiment was set up in a completely randomized design, in triplicate, in a plastic tunnel. The tunnel was 30.0 m long, 6.0 m wide, and 3.5 m high at the ridge and was constructed form steel. The covering was made of double polyethylene foil with a thermal layer, which helped reduce daily temperature fluctuations. The facility was equipped with a natural side ventilation system. The area of one experimental plot was 1.8 m² (1.2 × 1.5 m). The seed came from a selected seed stand located in the Lutowiska Forest District (MP/1/43275/05). Pre-sowing preparation, carried out at the Forest Seed Station Dukla, showed that the seeds were characterized by the following parameters: purity—94.5%, viability—94.5%, weight of one thousand seeds—246.3 g, and the number of seeds able to germinate in 1 kg of stock—3625. Seed sowing was carried out on 24 April 2023. A uniform sowing standard of 300 g was applied to each experimental plot (20 rows), which was calculated based on the seed sowing value. Immediately after sowing, a foil was placed over the tent structure. It was removed on 01 July 2023, so that seedling production was carried out under controlled conditions in May–June and under uncontrolled conditions in July–September.

Our study investigated the individual and combined effects of Ecklonia maxima (Osbeck) seaweed extract and Bacillus subtilis (Ehrenberg) on the Fagus sylvatica (L.) seedling growth. The first experimental factor was the Kelpak SL biostimulant. The levels of the first experimental factors included three doses of the preparation: Low—K0—0 dm³·ha⁻¹, Medium—K1—3 dm³·ha⁻¹, and High—K2—6 dm³·ha⁻¹. The biostimulant Kelpak SL (Kelp Products International (Pty) Ltd., Cape Town, South Africa) is a commercial preparation of an extract from the seaweed Ecklonia maxima (Osbeck), containing polysaccharides such as laminarin, alginates, and carrageenins; micro- and macronutrients; sterols; N-containing compounds, including betaines; and phytohormones. The doses of the preparations were administered in line with the manufacturer’s recommendations. The concentration of E. maxima extract in 1 dm³ of Kelpak SL is 320 cm³. The Kelpak SL biostimulant was applied in foliar spray on 2 June 2023 at the stage of three fully development leaves of beech (Table 1). All doses of the biostimulant Kelpak SL were applied as a volume of the commercial product in dm³·ha⁻¹, corresponding to a specific amount of active substance in cm³·ha⁻¹, according to the manufacturer’s information.

The second experimental factor was the microbial preparation Serenade ASO test. The levels of the second experimental factor included three doses: Low—SR0—0 dm³·ha⁻¹, Medium—SR1—8 dm³·ha⁻¹, and High—SR2—16 dm³·ha⁻¹. The microbial preparation Serenade ASO (Bayer AG, Kaiser-Wilhelm-Allee 1, 51373 Leverkusen, Germany), which contains the bacterium Bacillus subtilis (Ehrenberg), strain QST 713—13.96 g·dm⁻³. The doses of the preparations were administered in line with the manufacturer’s recommendations. Doses of Serenade ASO were applied in foliar spray on 2 June 2023, at the stage of three fully development leaves of beech. All dosages of the microbiological preparation Serenade ASO were administered as the volume of the commercial product in dm³·ha⁻¹, equating to a specified quantity of active ingredient in g·ha⁻¹, as per the manufacturer’s specifications.

2.2. Physico-Chemical Properties of Peat-Sawdust Substrate

The nursery experiment substrate was a 1-year peat and sawdust mixture combined at a 1:1 ratio (50% high peat, 50% fir, and spruce sawdust). Pre-sowing fertilization was applied at 2 kg per 1 m³ of substrate using Azofoska fertilizer (composition: N—13.6%, P—6.4%, K—19.1%, MgO—4.5%). The initial pH values of the substrate in H₂O and KCl were 5.22 and 4.68, respectively, while at the end of the experiment, they were 5.93 and 4.72, respectively (

Table 2. Physical and chemical properties of the substrate.

Substrate Parameters (TTf-1 #)		Date
		Before Sowing (24 April 2023)	After Seedlings Picking (11 October 2023)
pH	H2O	5.22	5.93
	KCl	4.68	4.72
Ncałk. [g·kg−1]		6.31	5.11
Ccałk. [g·kg−1]		350	258
Exchangeable cations	Ca2+ [cmol(+)·kg−1]	13.49	12.74
	Mg2+ [cmol(+)·kg−1]	4.06	1.21
	K+ [cmol(+)·kg−1]	7.19	5.84
	Na+ [cmol(+)·kg−1]	1.48	0.97
Sorption properties	Hydrolytic acidity Hh [cmol(+)·kg−1]	27.05	24.27
	Sum of exchangeable cation of the soil complex [cmol(+)·kg−1]	26.22	20.75
	Sorption capacity of the soil complex T [cmol(+)·kg−1]	53.26	45.02
	Degree of saturation of the sorption complex with bases V [%]	49.02	46.12
Bioavailable macronutrients	P [mg·kg−1]	92.27	52.58
	K [mg·kg−1]	2331.44	561.35
	Mg [mg·kg−1]	97.81	91.84
Volumetric density [Mg·m−3]		5.52
Solid phase density [Mg·m−3]		1.32
Total porosity [%]		76.0

# TTf-1—one-year-old sawdust and peat substrate mixed in a ratio of 1:1.

). The pH values in H₂O and KCl were determined according to ISO 10390 [44]. The total N and C concentrations in the prepared substrate were 6.31 and 350 g·kg⁻¹, respectively, while after the experiment, they were 5.11 and 258 g·kg⁻¹, respectively (Table 2). Total carbon and sulfur contents were determined using the high-temperature IR combustion method. Analyses of total N and total C were performed using a CNS-Leco TruMac elemental analyzer (LECO CNS TruMacAnalyzer, Leco, St. Joseph, MI, USA) by ISO 10694 and ISO 13878 [45,46].

The content of cations (Ca²⁺, Mg²⁺, K⁺, Na⁺) was determined after extraction in 1 M ammonium acetate (pH = 7.0) by inductively coupled plasma analysis (ICP-OES Thermo iCAP 6500 DUO, Thermo Fisher Scientific, Cambridge, UK). The content of exchangeable cations (Ca²⁺, Mg²⁺, K⁺, Na⁺) in the prepared substrate was 13.49 cmol₍₊₎·kg⁻¹, 4.06 cmol₍₊₎·kg⁻¹, 7.19 cmol₍₊₎·kg⁻¹, and 1.48 cmol₍₊₎·kg⁻¹, respectively, while at the end of the experiment, it was 12.74 cmol₍₊₎·kg⁻¹, 1.21 cmol₍₊₎·kg⁻¹, 5.84 cmol₍₊₎·kg⁻¹, and 0.97 cmol₍₊₎·kg⁻¹, respectively (Table 2). The hydrolytic acidity (Hh), the sum of exchangeable cations (S), the capacity of the sorption complex (T), and the degree of saturation of the alkali complex (V) of the substrate measured after harvesting beech seedlings decreased compared to the parameters measured before beech sowing. Hydrolytic acidity (Hh) and the sum of exchangeable bases (S) were determined using the Kappen method [47]. The capacity of the sorption complex was determined by summing the exchangeable bases and hydrolytic acidity. The saturation level of the sorption complex with bases (V) was determined by calculating the percentage of the total exchangeable bases in relation to the overall capacity of the sorption complex. The concentrations of bioavailable forms of P, K, and Mg in the prepared substrate were 92.27, 2331.44, and 97.81 mg·kg⁻¹, respectively. After the experiment, they were 52.58, 561.35, and 91.84 mg·kg⁻¹, respectively (Table 2). The contents of bioavailable P and K were determined using the Egner-Riehm method, while bioavailable Mg was determined using the Schachtschabel method (Table 2).

Physical properties were also determined for the peat-thinning substrate used in the experiment: bulk density, solid phase density, and total porosity (Table 2). Volumetric density and solid phase density were determined for five samples of the peat–sawdust substrate, taken using 100 cm³ measuring cylinders. The substrate’s volumetric density and solid phase density were 5.52 and 1.32 Mg·m⁻³, respectively. Total porosity was determined as the ratio of the volume of free spaces to the total volume. In the peat-thorn substrate used, total porosity was 76.0%.

2.3. Hydrothermal Conditions

The course of hydrothermal conditions during the growing period of the beech seedlings under controlled conditions is presented in

Table 3. Outdoor and sheltered climate conditions in 2023.

Month	Sum of the Monthly Temperatures [°C]	Average Monthly Temperature [°C]	Rainfall [mm]	Difference Between Average Temperature and Multi-Year Average Temperature [°C] #	Difference Between Precipitation and Multiyear Precipitation [mm] #	Sielianinov Hydrothermal Index * [K]	Classification of the Month	Temperature Undercover [°C]	Moisture Content Undercover [%]
May	495	10.3	34.80	−0.72	−75.8	0.70	Dry	19.7	45.7
June	689	14.5	66.80	0.41	−65.2	0.97	Dry	19.8	53.3
July	835	17.4	61.00	1.59	−64.9	0.73	Dry	Foil cover was removed from the tunnel
August	860	18.0	79.60	2.75	−21.3	0.93	Dry
September	730	15.2	62.00	4.26	−22.5	0.85	Dry

#—temperature and precipitation from the multiyear period 1971–2020 was developed based on data from the meteorological station located at Didactic and Research Station “Kopciowa”. * —K ≤ 0.4—extremely dry month; 0.4 < K ≤ 0.7—very dry; 0.7 < K ≤ 1.0—dry; 1.0 < K ≤ 1.3—fairly dry; 1.3 < K ≤ 1.6—optimal; 1.6 < K ≤ 2.0—moderately humid; 2.0 < K ≤ 2.5—humid; 2.5 < K ≤ 3.0—very humid; K > 3.0—extremely humid [48].

. On 1 July 2023, the foil was removed from the tunnels where the beech seedlings were growing. During the period from 24 April 2023, to 1 July 2023, in the growing season of beech seedlings under cover, irrigation was applied at a rate of 2 mm daily (before removing the cover on July 1). From 1 July 2023 (after removal of the cover), irrigation was applied at weekly intervals at a rate of 10 mm. The pluvial-thermal conditions in the different months of the growing season were characterized using the Sielianinov index (Table 3). The Sielianinov hydrothermal index (K) was calculated according to the formula

K = P/0.1∑t

where P—the monthly sum of precipitation in mm and Σt—the monthly sum of mean air temperatures > 0 °C [48].

The analysis of external hydrothermal conditions in 2023 was based on data from the meteorological station at the Didactic and Research Station of the Department of Ecology and Silviculture (Table 3). In 2023, the mean air temperature was higher than the values for the multiyear period (1971–2020). The growing season was characterized by a shortage of precipitation compared to the multiyear average. May had lower temperature (10.3 °C) and precipitation (34.8 mm) relative to the multiyear baseline. Nonetheless, these conditions did not impede the growth of beech seedlings owing to the presence of the foil cover. Under controlled conditions, the mean air temperature under the cover from May to June was similar, ranging from 19.7 to 19.8 °C. Humidity under controlled conditions in May and June was 45.7% and 53.3%, respectively (Table 3).

2.4. Laboratory Studies on Biometric Features

Biometric parameters were analyzed on 15 beech seedlings taken from each experimental plot on 11 October 2023. The mean of 15 seedlings for each experimental plot variable was computed and utilized as a singular observation in the statistical analysis (n = 3). The following biometric parameters were measured: root collar diameter, height, root system length, dry weight of the aboveground part, dry weight of the root system, and dry weight and number of the leaves. The dry weight of beech seedlings—separately for shoots, roots, and leaves—was determined using the drying method in a Memmert [Memmert GmbH + Co. KG, Schwabach, Germany] forced-air oven at 70 °C for 48 h, until a constant mass was achieved. The drying procedure was adapted from standard methods commonly used in plant physiological research [49,50]. An analysis of the root systems of beech seedlings was also conducted. For this purpose, an Epson Perfection V800 scanner (Seiko Epson Corporation, Suwa, Japan 2023) was used to acquire digital images of root systems, which were then analyzed using Win Rhizo™ Reg 20021 (Regent Instruments Inc., Quebec City, QC, Canada). Assimilation area of plant and leaf were analyzed using the Li-Cor 3200 (Li-Cor Biosciences, Tucson, AZ, USA).

An index analysis of beech seedling quality was carried out using four indices: seedling strength index or sturdiness quotient (SQ), shoot–to–root ratio (S/R), Dickson quality index (DQI), and seedling health index (SHI) [51,52,53,54,55,56,57].

The SQ index is the proportion of the seedling height to the diameter of the root collar, determined according to the formula

$$\mathrm{SQ}={\displaystyle \frac{\mathrm{S}\mathrm{H}}{\mathrm{R}\mathrm{C}\mathrm{D}}}$$

where SH—height of the seedlings [cm] and RCD—diameter of the root collar of the seedling [mm].

A low ratio indicates stronger (sturdy) seedlings that are more resistant to unfavorable abiotic conditions. On the other hand, a high value indicates less resilient seedlings, which may reduce their ability to adapt in forest cultivation [51,52,53].

The shoot-to-root ratio (S/R) denotes the correlation between the dry mass of the shoot and the dry mass of the root system of the cutting:

$$\mathrm{S}/\mathrm{R} ={\displaystyle \frac{SDW}{RDW}}$$

where SDW—dry weight of the shoot [g] and RDW—dry weight of the root system [g].

The S/R ratio expresses the proportions between the root system and the above-ground part [54]. For seedlings produced in nursery substrates, its value should not exceed 2:1 [51,55].

The Dickson Quality Index (DQI) is a tool used to assess seedling quality. A high value of this index indicates seedlings with better adaptability after planting in a forest crop [54]. The DQI was calculated according to the formula

$$\mathrm{DQI}={\displaystyle \frac{\mathrm{T}\mathrm{D}\mathrm{W}}{\frac{\mathrm{S}\mathrm{H}}{\mathrm{R}\mathrm{C}\mathrm{D}}+\frac{\mathrm{S}\mathrm{D}\mathrm{W}}{\mathrm{R}\mathrm{D}\mathrm{W}}}}$$

where TDW—total dry weight of seedling [g], SH—height of seedling [cm], RCD—root collar diameter [mm], SDW—shoot dry weight [g], and RDW—root system dry weight [g].

Another indicator used to assess seedling quality and suitability for forest cultivation is the seedling health index (SHI), which was calculated using the following formula [57]:

$$\mathrm{SHI} = ({\displaystyle \frac{\mathrm{R}\mathrm{C}\mathrm{D}}{\mathrm{S}\mathrm{H}}}+{\displaystyle \frac{\mathrm{R}\mathrm{D}\mathrm{W}}{\mathrm{S}\mathrm{D}\mathrm{W}}})\times \mathrm{T}\mathrm{D}\mathrm{W}$$

where variable explanations are as given in the DQI formula.

2.5. Statistical Analysis

Results from the two-factor nursery experiment in a completely randomized design were subjected to analysis of variance using the ANOVA module of the Statistica 13.3 software package (TIBCO Software Inc., Palo Alto, CA, USA) [58]. The Tukey test was used to compare object means. Before statistical analysis, the data distribution was tested for normality using the Shapiro–Wilk test and homogeneity of variance was assessed using Leven’s test. Significant levels for the tested features were determined for p = 0.05. Principal component analysis (PCA) was performed in the Multivariate and Exploratory Analysis module of the Statistica 13.1 software package (TIBCO Software Inc., Palo Alto, CA, USA) [58]. The graphs in Figures 1 and 2 represent the results at the mean ± 95% confidence interval.

3. Results

The use of Ecklonia maxima (Osbeck) algae extract significantly influenced specific morphological traits of beech seedlings. Alterations were noted in root system length, SQ index, assimilation area, leaf count, and root system area and volume. The control beech seedlings, which were not treated with the extract, exhibited the longest root development, suggesting that the application of the biostimulant does not consistently promote root elongation. The application of a dose of 960 cm³·ha⁻¹ resulted in a decrease in the SQ index, a reduction in the assimilation area, and a decrease in the number of leaves, while simultaneously promoting an increase in the area of the root system (101.0 ± 17.8 cm²). The lowest root volume was observed at a dose of 1920 cm³·ha⁻¹, indicating that increased concentrations of the extract may exert an inhibitory effect (1.16 ± 0.2 cm³).

Applying Bacillus subtilis (Ehrenberg) at a dose of 112 g·ha⁻¹ to beech seedlings resulted in the longest root system, the largest root collar diameter, and the highest DQI value (22.9 ± 3.4 cm, 6.00 ± 0.4 mm, and 0.74 ± 0.2, respectively). Compared to the untreated control, the application of B. subtilis (224 g·ha⁻¹) significantly altered the growth indices of beech seedlings. The SQ index was higher in the treated group (6.62 ± 0.7 vs. 6.33 ± 0.7), while the SHI index was lower (5.59 ± 0.9). Conversely, the S/R index was highest in the control group, with its value exceeding that of the B. subtilis-treated seedlings by nearly 0.1. In the control, beech seedlings were also characterized by the largest assimilation area of the leaf, root surface area, and root system volume—11.6 ± 0.8 cm², 109.4 ± 21.7 cm², and 1.39 ±0.3 cm³, respectively (

Table 4. Biometric parameters of beech seedlings.

Features	Ecklonia maxima				Bacillus subtilis
	K0 *	K1	K2	p-Value	SR0 *	SR1	SR2	p-Value
Plant height [cm]	37.2 ±2.9 a,**	35.6 ±4.2 a	35.7 ±3.6 a	0.063	36.6 ± 4.5 a	35.6 ± 3.7 a	36.3 ± 2.5 a	0.326
Root system length [cm]	24.6 ± 3.6 a	19.3 ± 4.3 c	21.2 ± 3.0 b	0.000	21.6 ± 4.5 b	22.9 ± 3.4 a	20.7 ± 4.6 b	0.002
Root collar diameter [mm]	5.78 ± 0.4 a	5.95 ± 0.3 a	5.73 ± 0.6 a	0.101	5.82 ± 0.4 b	6.00 ± 0.4 a	5.65 ± 0.6 b	0.006
Stem dry weight [g]	2.74 ± 0.4 a	2.81 ± 0.5 a	2.63 ± 0.5 a	0.318	2.73 ± 0.6 a	2.82 ± 0.5 a	2.63 ± 0.4 a	0.300
Root system dry weight [g]	2.29 ± 0.5 a	2.28 ± 0.5 a	2.26 ± 0.5 a	0.955	2.37 ± 0.5 a	2.36 ± 0.6 a	2.10 ± 0.3 b	0.027
Leaves dry weight [g]	1.01 ± 0.1 a	0.97 ± 0.1 a	0.99 ± 0.2 a	0.722	0.95 ± 0.1 a	1.04 ± 0.2 a	0.97 ±0.1 a	0.065
Plant dry weight [g]	6.04 ± 0.9 a	6.06 ± 1.0 a	5.87 ± 1.2 a	0.704	6.04 ± 1.1 a	6.22 ± 1.2 a	5.70 ± 0.8 a	0.104
SQ	6.51 ± 0.5 a	6.04 ± 0.5 b	6.40 ± 0.5 a	0.005	6.33 ± 0.7 b	6.00 ± 0.6 b	6.62 ± 0.7 a	0.000
S/R	1.28 ± 0.2 a	1.34 ± 0.2 a	1.26 ± 0.1 a	0.307	1.21 ± 0.2 b	1.33 ± 0.2 a	1.34 ± 0.1 a	0.040
DQI	0.67 ± 0.2 a	0.71 ± 0.2 a	0.67 ± 0.3 a	0.383	0.69 ± 0.2 b	0.74 ± 0.2 a	0.62 ± 0.2 b	0.004
SHI	6.19 ± 1.7 a	6.21 ± 1.7 a	6.21 ± 1.4 a	0.998	6.47 ± 1.0 a	6.56 ± 1.6 a	5.59 ± 0.9 b	0.008
Assimilative surface area of the plant [cm2]	214.6 ± 26.1 a	187.3 ± 24.5 b	206.0 ± 29.4 ab	0.014	209.6 ± 29.4 a	204.8 ± 33.0 a	193.5 ± 27.2 a	0.223
Assimilative area of leaf [cm2]	11.3 ± 0.9 a	11.2 ± 1.3	10.9 ± 1.1 a	0.594	11.6 ± 0.8 a	11.2 ± 1.0 ab	10.6 ± 0.6 b	0.025
Leaves number in the plant [pcs.]	20.4 ± 2.7 a	17.9 ± 2.4 b	19.7 ± 2.4 ab	0.044	19.2 ± 1.7 a	19.3 ± 2.0 a	19.5 ± 2.2 a	0.963
Root system area [cm2]	99.1 ± 15.2 ab	101.0 ± 17.8 a	87.4 ± 16.2 b	0.024	109.4 ± 21.7 a	92.4 ± 21.4 b	85.7 ± 7.34 b	0.000
Root system volume [cm3]	1.30 ± 0.3 a	1.30 ± 0.3 a	1.16 ± 0.2 b	0.049	1.39 ± 0.3	1.23 ± 0.3 b	1.14 ± 0.1 b	0.000

*—Comprehensive elucidation of shortcuts in Table 1; K0—0 cm³·ha⁻¹–Ecklonia maxima, K1—960 cm³·ha⁻¹—Ecklonia maxima, K2—1920 cm³·ha⁻¹—Ecklonia maxima, SR0—0 g·ha⁻¹—Bacillus subtilis, SR1—112 g·ha⁻¹—Bacillus subtilis, SR2—224 g·ha⁻¹—Bacillus subtilis.**—The means of the individuals were compared for the levels of the applied factors. Only means denoted by lowercase letters are compared, and means with distinct lowercase letters exhibited significant differences at p < 0.05.

).

The length of the root system of beech seedlings varied depending on the dose of Ecklonia maxima (Osbeck) algae extract and the amount of Bacillus subtilis (Ehrenberg) applied (biostimulant × biopreparation interaction, p = 0.006), (Figure 1A). Beech seedlings treated with B. subtilis at 112 g·ha⁻¹ had longer root systems than those treated at 224 g·ha⁻¹ or untreated, specifically on sites previously sprayed with E. maxima algae extract at 960 cm³·ha⁻¹.

The application of Bacillus subtilis bacteria in the presence of a previously applied Ecklonia maxima algae extract (biostimulant × biopreparation interaction, p = 0.001) modified the root collar diameter of seedlings (Figure 1B). An increase in root collar diameter was observed after the application of B. subtilis at a dose of 112 g·ha⁻¹ compared to other doses of the bacterium on sites previously not sprayed with E. maxima algae extract. Application of B. subtilis at 112 g·ha⁻¹ increased the root collar diameter of beech seedlings compared to seedlings treated with 224 g·ha⁻¹ of the bacterium on plots previously sprayed with 1920 cm³·ha⁻¹ of algal extract.

When analyzing the effect of applying Ecklonia maxima algae extract and Bacillus subtilis bacteria (biostimulant × biopreparation interaction, p = 0.001), differences were observed in the SQ index values of beech seedlings (Figure 2A). Spraying beech seedlings with B. subtilis at 224 g·ha⁻¹ increased the SQ index value relative to seedlings sprayed with the same bacterium at 112 g·ha⁻¹ on sites previously treated with algal extract at 1920 cm³·ha⁻¹.

The foliar application of B. subtilis in the form of a spray, in the presence of a previously applied Ecklonia maxima algae extract (biostimulant × biopreparation interaction, p = 0.002), affected the root system area of beech seedlings (Figure 2B). The reductions in the root system area were observed after applying B. subtilis at 112 and 224 g·ha⁻¹ to beech seedlings previously treated with algal extract at 960 cm³·ha⁻¹.

PCA showed a positive correlation between the SQ and S/R indices and the first component. The SQ index and plant height were strongly negatively correlated with the second component. Together, the two principal components explained 75.9% of the total variability (Figure 3). The DQI and SHI indices were negatively correlated with the S/R index, while the DQI and SHI indices were positively correlated with each other, as well as with root system area and root collar diameter (Figure 3A). Factor coordinate analysis indicated the presence of five clusters (Figure 3B). In the first quadrant, beech seedlings sprayed with the following combinations of biopreparations—E. maxima algae extract only at 960 cm³·ha⁻¹, B. subtilis only at 224 g·ha⁻¹, E. maxima + B. subtilis at 960 cm³·ha⁻¹ and 112 g·ha⁻¹, and E. maxima + B. subtilis at 1920 cm³·ha⁻¹ and 112 g·ha⁻¹—were characterized by higher root collar diameter, greater root system volume, higher DQI, and lower SHI. In the second quadrant, variation in dry weight, plant height, plant assimilative area, and root system length was observed in beech seedlings as a result of spraying by E. maxima at 960 cm³·ha⁻¹ or B. subtilis at 224 g·ha⁻¹. These parameters were negatively correlated with the first component. Beech seedlings in the third quadrant were characterized by the highest value of SQ and S/R index values, resulting from the combined application of E. maxima algae extract and B. subtilis bacteria at a dose of 1920 cm³·ha⁻¹ and 224 g·ha⁻¹, respectively (Figure 3B).

4. Discussion

The use of biologically active chemicals in biopreparations to enhance plant growth and development is a prevalent technique in plant production [59]. Biologically active chemicals serve multiple purposes, chiefly by promoting plant growth and development, safeguarding against phytopathogens, or activating the plant’s innate defense mechanisms [60]. The mechanisms of action of biostimulants are complex and multifaceted. These include direct interactions with plant signaling cascades [61], stimulation of overall metabolism [62], and facilitation of nutrient uptake and absorption [63]. Bioactive substances contained in biostimulants exert their effects, among other ways, by reducing the production of reactive oxygen species, stabilizing cell membranes, modulating enzyme activity, and improving photosynthetic processes [64]. Alongside established physiological responses, the mechanisms of Ecklonia maxima (Osbeck) and Bacillus subtilis (Ehrenberg) likely encompass hormonal regulation, which is increasingly acknowledged as a fundamental aspect of their biostimulatory capabilities. Extracts of E. maxima have demonstrated an impact on auxin, cytokinin, and gibberellin dynamics, facilitating root development, enhancing cell division, and improving stress tolerance [65]. B. subtilis can synthesize phytohormones like IAA and activate systemic resistance pathways linked to jasmonic acid and ethylene signaling. The interplay of hormonal and physiological processes enhances nutrient acquisition, promotes plant vigor, and improves responses to biotic and abiotic stresses [66].

The application of biostimulants from a category of growth-promoting biopreparations constitutes a component of sustainable nursery production, a method of plant cultivation [17]. Biostimulants are relevant to various sectors of plant production, including large-scale forestry operations [67]. Multiple research investigations have recorded the effects of bioactive chemicals that promote plant growth and development in agricultural production [68,69,70].

The findings of our study evaluating the effects of Ecklonia maxima algae extract and Bacillus subtilis bacteria on the biometric parameters of beech seedlings indicate diverse reactions. The administration of Ecklonia maxima extract at a dosage of 960 cm³·ha⁻¹ enhanced the root system surface area of beech seedlings. Biostimulants derived from seaweed extracts, such as E. maxima, are recognized for their beneficial impacts on plant growth and development [31,71]. In our investigation, we observed that the longest root systems, the assimilative surface area of plant, and the number of leaves per plant were found in beech seedlings that were not subjected to the E. maxima algal extract treatment. Administering double the manufacturer’s suggested dosage (1920 cm³·ha⁻¹) of E. maxima algal extract resulted in a decrease in the root system volume of beech seedlings. This outcome suggests that an initial increase in biostimulant dosage enhances growth and some biometric indicators; however, excessive concentrations may hinder plant development [72]. The response of forest tree seedlings is contingent upon intraspecific variability. The utilization of an aqueous extract of E. maxima, marketed as Kelpak SL, as a foliar spray led to enhanced tree height and needle length in ponderosa pine (Pinus ponderosa Dougl. ex C. Lawson) and an increased branch count in Colorado blue spruce (Picea pungens Engelm.). No morphological changes were found in eastern white cedar (Thuja occidentalis L.) in response to the applied algal extract [73]. The substantial body of data on edible and decorative plant species indicates that biostimulants enhance plant development [74,75]. The disparity in response between edible and woody plants arises from the particular efficacy of biostimulants contingent upon the growth type [76,77].

Bacillus subtilis bacteria significantly contribute to plant growth by colonizing roots, enhancing phytohormone synthesis, and augmenting the availability of macro- and micronutrients in the rhizosphere [38,78,79]. In our investigation, the application of this microbe at a dosage of 112 g·ha⁻¹ enhanced root system length and root collar diameter. B. subtilis enhanced the growth of Pinus taeda (L.) seedlings by 67.1% in root biomass and 33.1% in shoot biomass, establishing it as a suggested and advantageous microbe in forestry activities [80]. Liu et al. [81] utilized Bacillus subtilis as an inoculum for seedlings of Pinus bungeana (Zucc.), resulting in enhanced root system length and surface area, increased root and shoot biomass, as well as greater plant height and root collar diameter. Bacillus subtilis markedly enhances the growth of poplar seedlings, augmenting their height by 62.0% and biomass by 37.0% [82]. Nonetheless, the administration of an elevated dosage (224 g·ha⁻¹) of B. subtilis in our research diminished the dry weight of the root system in beech seedlings. From a nursery practice standpoint, the precise selection of the B. subtilis dosage is essential for altering the biometric parameters of beech seedlings. Administering an elevated dosage of B. subtilis (224 g·ha⁻¹) led to a significant enhancement in the SQ index (exceeding 6.5), signifying diminished seedling quality and underscoring the necessity of choosing the correct dosage of this bacterium. Our analysis demonstrated that the application of both a lower (112 g·ha⁻¹) and a higher (224 g·ha⁻¹) dose of B. subtilis caused an alteration in the ratio of above-ground to below-ground components of beech seedlings, as shown by the S/R index values. Liu et al. [81] indicated that inoculating Platycladus orientalis (L.) seedlings with a B. subtilis inoculum increased the dry weight of shoots in well-watered and drought-stressed seedlings by 34.85% and 19.23%, respectively, and elevated root weight by 15.45% and 13.99%. Consequently, the shoot-to-root ratio (S/R) significantly decreased, indicating that the beneficial effect of PGPR on aboveground growth exceeded that on root development. Notably, despite this shift, the 112 g·ha⁻¹ dosage considerably raised the Dickson Quality Index (DQI), indicating that increased total biomass and stem diameter offset the modified biomass partitioning. This aligns with the definition of DQI as a comprehensive metric of seedling quality that equilibrates vigor and robustness [82]. Similar results have been observed in other species, where B. subtilis inoculation enhanced seedling development and optimized resource-use efficiency [83,84]. These findings collectively affirm that B. subtilis functions as a plant growth-promoting rhizobacterium (PGPR); however, the extent and nature of its influence on shoot-root balance are contingent upon dosage, plant species, and environmental context.

The intricate interplay between E. maxima algal extract and B. subtilis bacteria altered root system length and root neck diameter. In our investigation, a dosage of 960 cm³·ha⁻¹ of E. maxima algal extract mixed with 224 g·ha⁻¹ of B. subtilis reduced root system length, potentially indicating an overactivation of physiological and metabolic processes in plants. The utilization of E. maxima algae extract and B. subtilis bacteria markedly influenced the root system length, root collar diameter, and SQ index of beech seedlings. The interaction between the specified doses of both preparations was significant for both parameters (p = 0.006 and p = 0.001, respectively). This finding is corroborated by the research of Zhang et al. [85], which shown that interactions between microbial biostimulants and algal extract-based biostimulants can, in certain instances, lead to plant growth suppression. The choice of the suitable biologically active compound, the accurate dosage at the correct concentration, and the formulation of the preparation employed are essential for attaining a synergistic effect, consistent with the notion of biostimulants 2.0 [29,30].

The findings align with prior research demonstrating the stimulatory impact of algal extracts on tree root development, attributed to phytohormones such auxins and cytokinins [32]. Bacillus subtilis bacteria boost root development by inhabiting the rhizosphere and synthesizing chemicals that enhance nutrient absorption [67]. The augmentation of root system length following the application of a lower bacterial dose (112 g·ha⁻¹) relative to a larger dose (224 g·ha⁻¹) in E. maxima-treated plants can be ascribed to elevated levels of bacterial metabolites in the rhizosphere. The SQ index values and root system surface area indicate a synergistic impact from the application of B. subtilis at a dosage of 224 g·ha⁻¹ and E. maxima extract at a dosage of 1920 cm³·ha⁻¹. An elevation in the SQ index, coupled with a reduction in the surface area of the root system, disrupts the equilibrium between the root system and the aerial portion of the seedling. Ensuring the proper ratio among these components relies on the accurate dosage of the biopreparation [76,86].

Principal component analysis (PCA), accounting for 75.9% of the variability, reveals the presence of five different clusters. The findings indicate that applying E. maxima and B. subtilis at rates of 1960 cm³·ha⁻¹ and 224 g·ha⁻¹, respectively, enhances the DQI and SHI indices. The inverse relationship between the DQI and SHI indices and the S/R ratio, along with findings from the third quadrant of the PCA (indicating optimal index values for beech seedlings at elevated biopreparation doses), underscores the necessity for meticulous dose determination based on the targeted seedling quality metrics [87].

The findings of this study align with contemporary research trends demonstrating the efficacy of extracts from algae and microorganisms in the cultivation of forest tree seedlings. Research indicates that extracts from the algae Ecklonia maxima and the bacterium Bacillus subtilis enhance the biometric parameters of seedlings, hence augmenting their resilience to environmental stress [26,88]. This study’s findings underscore the significance of the interaction between dosages of Ecklonia maxima algae extract and Bacillus subtilis bacteria in biopreparations, as well as their effects on the biometric parameters and quality of beech seedlings.

5. Conclusions

Ecklonia maxima (Osbeck) seaweed extract does not consistently enhance the growth of beech seedlings; in certain instances, it may exert an inhibitory effect, particularly at elevated doses (1920 cm³·ha⁻¹), resulting in a 10.7% decrease in root system volume relative to the control group. At lower doses (960 cm³·ha⁻¹), a 12.7% reduction in the plant’s assimilating surface and a 21.5% decrease in root system length were observed. The optimal biostimulation effect occurred at a Bacillus subtilis (Ehrenberg) dosage of 112 g·ha⁻¹, resulting in a 5.7% increase in root system length, a 3.0% increase in root collar diameter, and a 5.0% enhancement in the DQI index relative to the control, signifying an improvement in seedling quality. The elevated dosage of B. subtilis (224 g·ha⁻¹) influenced growth parameters, resulting in a 4.3% increase in SQ, while concurrently reducing leaf assimilation area and root system volume by 8.6% and 18.0%, respectively, in comparison to the control group.

The interactions between algae and bacteria exhibited both synergistic and antagonistic effects; for instance, there was an enhancement in root collar diameter when Ecklonia maxima at 1920 cm³·ha⁻¹ was combined with B. subtilis at 112 g·ha⁻¹, whereas a loss in root system area occurred when E. maxima (960 cm³·ha⁻¹) was paired with Bacillus subtilis (application at dose 112 g·ha⁻¹ and 224 g·ha⁻¹). Sprayed beech seedlings with B. subtilis at 224 g·ha⁻¹ combined with algal extract at 1920 cm³·ha⁻¹. elevated the SQ index value compared to seedlings treated with the identical bacterium at 112 g·ha⁻¹ in conjunction with algal extract at 1920 cm³·ha⁻¹. PCA analysis validated the presence of several variant groups, characterized by their predominant morphological traits—ranging from seedlings exhibiting enhanced stability (greater root collar width, elevated DQI) to those prioritizing above-ground growth (greater plant assimilation area and root length). The findings suggest that moderate doses of B. subtilis combined with Ecklonia maxima extract can enhance the quality of beech seedlings. Nonetheless, accurate dose selection is essential, as it can markedly enhance beech seedling survival and promote their growth and adaptation in natural environments.