A Biostimulant Containing Humic and Fulvic Acids Promotes Growth and Health of Tomato ‘Bush Beefsteak’ Plants

Abstract

Humic substances are a type of biostimulant used in greenhouse production to promote plant growth and health. Our goal was to evaluate the effect of three commercially available biostimulants containing humic and/or fulvic acids (HumaPro, FulviPro, and Micromate) on the performance and tissue nutrient concentration of Solanum lycopersicum L. ‘Bush Beefsteak’ grown in a peat-based substrate. We conducted four experiments testing application rate and fertility level (50 and 100 mg⋅L^–1 N) (Exp. 1), application rate and frequency (Exp. 2), direct Micromate incorporation into the substrate (Exp. 3), and FulviPro application method (drench vs. foliar spray) (Exp. 4). Plants were fertilized with 20N-1.3P-15.7K. Experiments 2, 3, and 4 were conducted under low fertility (50 mg⋅L^–1 N). Micromate promoted growth when applied as a weekly drench at 40 g·L⁻¹ or when incorporated into the substrate (20 g in 1 L of the substrate). Micromate-treated plants showed high P, S, and Si in the shoot and root tissues. FulviPro showed a negative effect when applied as a drench at higher rates, but foliar application increased greenness (Green Leaf Index). The negative effects of FulviPro might be due to the over-accumulation of Fe and Mn when applied as a drench.

1. Introduction

Humic substances originate from the decomposition of organic matter and include fulvic acid, humic acid, and humin. They can be extracted from various sources, including soil, compost, peat, and leonardite [1,2]. Fulvic and humic acids are commonly used in the formulation of plant biostimulants because they can increase substrate nutrient storage capacity and nutrient uptake by the plant, promote plant growth, and enhance plant stress tolerance [3,4]. Humic substances benefit plants by altering root morphology, enhancing nutrient uptake [5], modulating primary and secondary plant metabolism [6], enhancing abiotic stress tolerance [7], and stimulating beneficial plant–microbe interactions [8,9,10]. However, the efficacy of humic substances and derived commercial products depends on several factors, such as the application rate and frequency, application method, plant species, type of humic substance, product formulation, growing substrate, and environmental conditions [3,4,11,12].

Tomato (Solanum lycopersicum) was the third most produced vegetable in the United States (U.S.) in 2022 [13]. Although most of the U.S. tomato production is open-field, greenhouse tomato production has expanded in recent years. Under this management system, tomato is grown in soilless substrates, and nutrients are provided by a nutrient solution [14]. The tomato transplants used by commercial growers and home gardeners are also produced in greenhouses in containers of soilless substrate. Humic substances have been highlighted as a potential tool to improve fertilizer use efficiency in soilless production because they can increase the nutrient storage capacity of soilless substrates [4]. In tomato, humic substances have been reported to increase germination, growth, yield, and fruit quality [15,16,17,18]. Soil application of humic acids increased yield in tomato ‘Target NF1’ by up to 48%, and the foliar application resulted in a yield increase of up to 26% [15]. Soil applications of humic acids to tomato ‘Kero F1’ increased macro (N, P, and K) and micronutrient (Fe, Zn, and Cu) concentrations in leaves [17]. Alenazi and Khandaker reported that drench application of humic acid promoted growth and improved the physicochemical fruit quality of tomato ‘Luanova’, ‘Savarona’, and ‘Tessera’ under greenhouse conditions [18].

Despite the positive results reported in the literature, the efficacy of humic substances and derived commercial products is not consistent and limits their usage in improving crop performance [11,19,20]. Micromate, HumaPro, and FulviPro are commercial biostimulants that contain humic substances, but only Micromate has shown a positive effect on plant growth in our previous experiments [21]. The goal of this research was to determine whether Micromate, HumaPro, or FulviPro applications could increase growth, quality, and tissue nutrient concentrations of young tomato plants grown in soilless substrate.

2. Materials and Methods

2.1. Germination and Seedling Growth

For all experiments, tomato (Solanum lycopersicum) ‘Bush Beefsteak’ (Ivy Garth Seeds & Plants, Chesterland, OH, USA) seeds were germinated in a 128-plug tray filled with Pro-Mix PGX (Premier Tech Horticulture, Quakertown, PA, USA). Plug trays were covered with a plastic dome to maintain humidity. Seeds were germinated and grown under fluorescent lights with a 12 h photoperiod for three weeks and then moved to the greenhouse for acclimation. Seedlings were fertilized with water-soluble 20N-1.3P-15.7K Petunia FeED (Jack’s Professional; J.R. Peters, Allentown, PA, USA) at a rate of 50 mg·L⁻¹ N.

Four weeks after sowing, the seedlings were transplanted into 11.4 cm (6 in) azalea pots. These pots were filled with a peat-based substrate prepared in-house. The substrate included 80% peat (Pro-Moss, Premier Tech Horticulture) and 20% coarse perlite (PVP Industries Inc., North Bloomfield, OH, USA) by volume. The substrate was amended with a wetting agent (AquaGrowL; Aquatrols, Paulsboro, NJ, USA) at 7.7 mL per 100 L of the substrate, and dolomitic limestone (Oldcastle Lawn & Garden, Atlanta, GA, USA) to adjust pH to around 5.4.

2.2. Greenhouse Environmental Conditions

All experiments were conducted in a controlled environment glass greenhouse on The Ohio State University Wooster Campus. The day and night temperatures were set at 21.1–24.4 °C and 15.5–18.3 °C, respectively, and the target humidity was ~70%. A 14 h photoperiod was maintained with metal halide and high-pressure sodium lamps. Supplemental light was provided when the photosynthetically active radiation (PAR) was below 250 μmol·m⁻²·s⁻¹, and shade cloth was deployed when the PAR was above 400 μmol·m⁻²·s⁻¹.

2.3. Humic/Fulvic Acid-Based Biostimulants Evaluated

Three commercial biostimulants containing humic substances derived from leonardite were included in these experiments. HumaPro is a liquid biostimulant containing humic acid (16%) as the main active ingredient (Huma Inc., Gilbert, AZ, USA), FulviPro is a liquid biostimulant containing fulvic acid (20%) (Huma Inc.), and Micromate is a soluble powder composed of both humic and fulvic acids (24%) (Huma Inc.). The mineral nutrient concentrations of HumaPro, FulviPro, and Micromate were previously reported by Martins et al. [21].

2.3.1. Experiment 1—Evaluating Different Biostimulant Rates at Low and Optimal Fertility

Experiment 1 was organized in the greenhouse in a complete randomized block design with 12 replicates (n = 12). The experiment included 20 treatments within each block. The three biostimulants, HumaPro, FulviPro, and Micromate, were evaluated at three different rates, and control plants received no biostimulant treatment. Rates for HumaPro and FulviPro included 5, 10, and 20 mL·L⁻¹, and Micromate rates were 5, 10 and 20 g·L⁻¹. All biostimulants and rates were evaluated at a low (50 mg·L⁻¹ N) and an optimal (100 mg·L⁻¹ N) fertilizer rate from the water-soluble 20N-1.3P-15.7K Petunia FeED (Jack’s Professional, J.R. Peters). Micronutrients in the fertilizer included 1.34% Mg, 0.01% Cu, 0.2% Fe, 0.05% Mn, and 0.05% Zn. Plants were fertilized at each irrigation, and the biostimulant treatments were applied weekly as a 150 mL drench starting at the transplant. Fertilizer solutions and biostimulants were prepared with city water. Control plants grown at both low and optimal fertility were drenched with water on treatment day. The experiment was conducted from 30 June to 21 July 2022. Plants were harvested three weeks after transplant to evaluate the effects of the treatments on growth and plant health (details below in the following section: Parameters evaluated to determine the impact of biostimulant treatments on growth promotion and plant health).

2.3.2. Experiment 2—Evaluating Application Frequency

Experiment 2 was organized in the greenhouse in a complete randomized block design with 12 replicates (n = 12) and 28 treatments. Treatments were a combination of application frequency (once, biweekly, and weekly), biostimulant (HumaPro, FulviPro, and Micromate), and rate. HumaPro and FulviPro rates were 1.25, 2.5, 5, and 10 mL·L⁻¹, and Micromate rates were 5, 10, 20, and 40 mL·L⁻¹. These rates were determined based on results from experiment 1. Plants were grown under low fertility (50 mg·L⁻¹ N), and the fertilizer was 20N-1.3P-15.7K Petunia FeED (Jack’s Professional, J.R. Peters). Fertilizer and biostimulant treatments were prepared with city water. The biostimulants were applied as a 150 mL drench to each container starting at transplant. Control plants were drenched with 150 mL water on treatment day. The experiment was conducted from 12 September to 13 October 2022. Plants were harvested four weeks after transplant.

2.3.3. Experiment 3—Effect of Micromate Incorporated into the Growing Substrate

Experiment 3 was organized in the greenhouse in a complete randomized block design with 12 replicates (n = 12). The experiment tested two Micromate rates and a non-treated control. Micromate rates were 20 or 40 g per liter of the substrate (g·L⁻¹), and it was incorporated into the substrate before transplant. Plants were grown under low fertility (50 mg·L⁻¹ N), using 20N-1.3P-15.7K Petunia FeED water-soluble fertilizer (Jack’s Professional, J.R. Peters). The experiment was conducted from 16 November to 15 December 2022. Plants were harvested three weeks after transplant.

2.3.4. Experiment 4—Evaluating Drench versus Spray Applications of FulviPro

Experiment 4 was organized in the greenhouse in a complete randomized block design with 12 replicates (n = 12) and a 2 × 5 factorial arrangement. The application method had two levels: drench and spray. This experiment included one biostimulant product, FulviPro, at five rates (0, 1.25, 2.5, 5, 10 mL·L⁻¹). Plants were grown under low fertility (50 mg·L⁻¹ N) using 20N-1.3P-15.7K Petunia FeED water-soluble fertilizer (Jack’s Professional, J.R. Peters) prepared in city water. Treatments were prepared with reverse osmosis (RO) water and applied weekly as a drench (200 mL) or spray (10 mL using a spray bottle; plants also received 200 mL RO water drench to equal the amount of water applied to the drench-treated pots) starting at transplant. The experiment was conducted from 27 June to 26 July 2023. Plants were harvested four weeks after transplant.

2.4. Parameters Evaluated to Determine the Impact of Biostimulant Treatments on Growth Promotion and Plant Health

At the end of the experiments, root and shoot tissues were collected for further analysis. Shoot tissues (stems and leaves) were harvested and placed in paper bags. The substrate was washed from the roots, and the roots were placed in paper bags. The collected tissues were air-dried in an oven at 60 °C for one week, and the dry biomass of shoots and roots was recorded. For experiments 1 and 3 only, pooled samples were prepared from each treatment (n = 3) and sent for nutrient analysis (see details below). A pooled sample consisted of blended dry tissue from two experimental units.

In experiment 3, the growth index was used as a non-destructive measurement of plant size before harvest. Growth index (GI) is calculated using plant height and two perpendicular widths as follows: [(width 1 + width 2)/2 + height]/2 [22]. SPAD measurements were taken on the youngest fully expanded leaf using a SPAD chlorophyll meter (Minolta Camera Co., Osaka, Japan) and were used as an indicator of leaf greenness and plant health [23]. In experiments 3 and 4, digital plant phenotyping was carried out using the TraitFinder greenhouse phenotyping system (Phenospex, Heerlen, The Netherlands). The TraitFinder has two PlantEye F600 scanners, which acquire morphological and spectral reflectance data from the scanned plants. Of the parameters obtained from the TraitFinder, digital biomass was used to quantify plant growth promotion over time, and green leaf index (GLI), normalized difference vegetation index (NDVI), normalized pigment chlorophyll ratio index (NPCI), and plant senescence reflectance index (PSRI) were used to identify differences in plant health between the control and treated plants. GLI is a vegetation index used to evaluate vegetation greenness, and it is calculated from the amount of light reflectance in the green (G), red (R), and blue (B) wavelengths [GLI = (2 × G − R − B)/(2 × G + R + B)]. NDVI is calculated from the amount of light reflected in the R and near-infrared (NIR) wavelengths [NDVI = (NIR − R)/(NIR + R)]. The TraitFinder uses light reflectance from R and B wavelengths to calculate NPCI [NPCI = (R − B)/(R + B)], and PSRI is calculated from the amount of light reflected in the R, G, and NIR wavelengths [PSRI = (R − G)/NIR] [24,25].

2.5. Tissue Nutrient Analysis

Tissue nutrient analysis was conducted at the Service Testing and Research Laboratory (STAR Laboratory, The Ohio State University, Wooster, OH, USA). Shoot and root tissue were ground and digested using the Discover SP-D microwave digestion system (CEM Corporation, Matthews, NC, USA). The total concentration of phosphorus (P), potassium (K), aluminum (Al), boron (B), calcium (Ca), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), molybdenum (Mo), sodium (Na), sulfur (S), and zinc (Zn) was obtained from the Agilent 5110 ICP-OES (Inductively Coupled Plasma Optical Emission Spectroscopy) system (Agilent Technologies, Santa Clara, CA, USA). Total nitrogen (N) and carbon (C) were measured using the Vario Max Cube Carbon–Nitrogen Analyzer (Elementar Americas, Mt. Laurel, NJ, USA) [26].

2.6. Statistical Analysis

All statistical analyses and graphs were performed in R statistical software (version 4.3.1) [27]. Experiments 1, 2, and 3 were analyzed using a one-way analysis of variance (ANOVA) using the model Y = block + treatment. If ANOVA identified significant differences, the mean comparisons were conducted according to Tukey’s Honestly Significant Difference (HSD) test at α = 0.05 (p < 0.05). Experiment 4 was analyzed using a two-way ANOVA using the model Y = block + method + treatment + method × treatment. When the interaction was significant, treatment mean comparisons were conducted for each level of the method. Mean comparisons were conducted according to Tukey’s HSD test at α = 0.05 (p < 0.05). The normality of the residuals was checked visually using Shapiro–Wilk’s test. The homogeneity of variance of residuals was checked visually and using Levene’s test. Tissue nutrient data were subjected to a principal component analysis (PCA) using the package stats, and biplots were prepared using the package ggplot2.

3. Results and Discussion

3.1. Experiment 1—Humic Substances (Micromate) Promoted Growth of Tomato Plants in Soilless Substrate under Limited Fertilization

Under low fertility conditions (50 mg·L⁻¹ N), Micromate application promoted an increase in tomato shoot dry weight (DW) (Figure 1A,B). Plants treated with Micromate at 20 g·L⁻¹ were 61% larger than control plants (Figure 1B). FulviPro application reduced tomato shoot DW. The effect of FulviPro was dose-dependent, and the most significant reduction in shoot DW (73.7%) was observed at the 20 mL·L⁻¹ rate (Figure 1A,B). HumaPro did not influence shoot DW.

At optimal fertility (100 mg·L⁻¹), neither Micromate nor HumaPro influenced shoot DW (Figure 1B). In contrast, the effect of FulviPro application was consistent with the results observed under low fertility. Plants treated with FulviPro at 10 and 20 mL·L⁻¹ were significantly smaller than the control shoots (Figure 1B). Similarly, Martins et al. reported that Micromate promotes growth in petunias grown under low and optimal fertility and that FulviPro reduces petunia growth under both low and optimal fertility [21]. Also, no effect on shoot dry weight was observed in petunias or hydroponic lettuce treated with HumaPro [21,28].

Humic substances are reported to increase N, P, Fe, Zn, Mn, and Cu uptake by the plant [5]. Principal component analysis (PCA) was performed to compare the shoot nutrient concentration profiles of each treatment. Principal component (PC) 1 and PC2 accounted for about 53.7% of the total variance (Figure 2). Control samples from the low and optimal fertility conditions separated along PC1. Samples clustered according to product treatment. All samples were separated from the non-treated control in low fertility conditions. The shoot nutrient concentration profile of plants grown under low fertility and treated with Micromate at 20 g·L⁻¹ (green circles) was similar to the nutrient profile of plants grown at the optimal fertility rate (red triangles). As observed in the PCA biplot (Figure 2), shoot DW was positively correlated with P tissue concentration (Figure 2). Micromate treatment has been reported to increase the concentration of P in shoots of petunias grown under low fertility [21]. However, P shoot concentration in Micromate-treated tomatoes at 20 g·L⁻¹ (1.8 mg·g⁻¹) or at 40 g·L⁻¹ (1.8 mg·g⁻¹) was similar to that observed in untreated controls (1.8 mg·g⁻¹) under low fertility (

Table 1. Mineral macronutrient concentrations in tomato ‘Bush Beefsteak’ shoot dry tissue (experiment 1).

Fertility Level (mg·L−1 N)	Biostimulant	Rate (mL·L−1 or g·L−1)				Macronutrient (mg·g−1)
			N (%)	C (%)	P	K	Mg	Ca	S
100	Control	0	3.7 ± 0.1 BCD	38.4 ± 0.2 AB	1.9 ± 0.1 ABC	35.9 ± 0.3 CD	12.9 ± 0.1 C	14.4 ± 0.2 CD	4.0 ± 0.3
100	FulviPro	20	4.5 ± 0.2 A	36.0 ± 1 BCD	2.1 ± 0.1 A	47.9 ± 1.4 AB	16.9 ± 0.5 AB	17.7 ± 0.5 B	4.2 ± 0.1
100	HumaPro	20	4.0 ± 0.1 ABC	39.4 ± 0.3 A	1.8 ± 0.1 ABC	47.2 ± 2.8 ABC	13.8 ± 0.2 C	14.0 ± 0.9 CD	3.7 ± 0.2
100	Micromate	5	3.6 ± 0.1 BCD	35.9 ± 0.3 BCD	2.1 ± 0.04 AB	39.7 ± 2.1 ABCD	14.3 ± 0.6 C	15.4 ± 0.4 BCD	4.1 ± 0.1
100	Micromate	10	3.3 ± 0.4 CDE	34.6 ± 1.4 D	2.0 ± 0.1 AB	36.6 ± 3.1 BCD	13.3 ± 0.1 C	13.6 ± 0.5 D	4.0 ± 0.1
100	Micromate	20	3.5 ± 0.1 BCDE	38.4 ± 0.4 AB	2.2 ± 0.1 A	35.0 ± 1.3 D	12.6 ± 0.4 C	13.2 ± 0.4 D	4.2 ± 0.2
50	Control	0	4.3 ± 0.3 AB	37.9 ± 0.3 ABC	1.8 ± 0.1 ABC	40.6 ± 2.6 ABCD	14.8 ± 0.5 BC	16.3 ± 0.3 BC	4.4 ± 0.2
50	FulviPro	20	3.9 ± 0.1 ABCD	36.8 ± 1 ABCD	1.5 ± 0.1 C	39.9 ± 0.4 ABCD	18.9 ± 0.7 A	21.0 ± 0.5 A	4.0 ± 0.1
50	HumaPro	20	4.6 ± 0.2 A	39.6 ± 0.7 A	1.7 ± 0.1 BC	48.6 ± 2.3 A	14.1 ± 0.6 C	15.6 ± 0.3 BCD	4.1 ± 0.3
50	Micromate	5	3.6 ± 0.2 BCD	36.2 ± 0.3 BCD	1.8 ± 0.1 ABC	39.0 ± 3.4 ABCD	14.7 ± 0.2 BC	17.4 ± 0.5 B	4.3 ± 0.2
50	Micromate	10	3.1 ± 0.2 DE	35.0 ± 0.4 CD	1.8 ± 0.1 ABC	35.6 ± 2.5 D	15.0 ± 0.5 BC	17.5 ± 0.5 B	4.2 ± 0.1
50	Micromate	20	2.8 ± 0.1 E	38.8 ± 0.3 AB	1.8 ± 0.1 ABC	32.6 ± 2.1 D	12.8 ± 0.7 C	13.9 ± 0.4 CD	4.3 ± 0.3

The table shows the means ± standard error (n = 3). For each tissue, treatment means with different letters are significantly different according to the Tukey’s honestly significant difference test (α = 0.05).

).

Micromate- and FulviPro-treated plants separated along PC1 (Figure 2). The variables contributing the most to PC1 were DW, P, Mg, Fe, Mn, EC, and Ca. DW and P showed a negative correlation with Mg, Fe, Mn, EC, and Ca. Shoot tissue from FulviPro-treated plants contained higher levels of Ca, Mg, Fe, and Mn than control plants at both optimal and low fertility (Table 1 and

Table 2. Mineral micronutrient concentrations in tomato ‘Bush Beefsteak’ shoot dry tissue (experiment 1).

							Micronutrient (µg·g−1)
Fertility Level (mg·L−1 N)	Biostimulant	Rate (mL·L−1 or g·L−1)	Al	B	Cu	Fe	Mn	Mo	Na	Zn
100	Control	0	13.6 ± 1.9 A	43.1 ± 1.8 A	3.1 ± 0.2	114.4 ± 5.6 BC	78.9 ± 1.5 DEF	0.8 ± 0.1 BC	1539.3 ± 111.7 BC	95.1 ± 2 E
100	FulviPro	20	24.5 ± 6.7 AB	36.8 ± 1.5 AB	3.7 ± 0.4	197.7 ± 10.2 A	162.2 ± 11.4 B	1.0 ± 0.1 ABC	1842.0 ± 178.5 ABC	137.7 ± 1.8 AB
100	HumaPro	20	9.8 ± 0.3 A	39.9 ± 2.2 AB	7.2 ± 3.7	109.7 ± 12.8 BC	83.6 ± 10.5 DEF	0.7 ± 0.1 BC	1242.3 ± 50 C	134.0 ± 8.8 ABC
100	Micromate	5	17.6 ± 5.9 AB	44.6 ± 2.1 A	3.0 ± 0.2	102.5 ± 5.4 BC	78.6 ± 5.6 EF	0.8 ± 0.1 BC	1757.7 ± 226.5 ABC	108.5 ± 6.9 CDE
100	Micromate	10	16.3 ± 0.5 AB	39.1 ± 0.6 AB	2.8 ± 0.1	89.8 ± 6.8 C	71.5 ± 1.4 EF	0.8 ± 0.1 BC	1831.7 ± 155.7 ABC	102.8 ± 9.2 DE
100	Micromate	20	16.8 ± 2.8 AB	41.0 ± 1.9 AB	3.0 ± 0.2	87.1 ± 9.9 C	63.7 ± 1.7 F	0.6 ± 0.1 C	2230.7 ± 99.3 AB	98.8 ± 6.1 DE
50	Control	0	14.8 ± 0.8 AB	38.5 ± 0.7 AB	3.7 ± 0.2	133.1 ± 0.9 B	108.8 ± 5.6 CDEF	1.2 ± 0.1 AB	1758.3 ± 128.8 ABC	122.9 ± 4.3 ABCD
50	FulviPro	20	32.7 ± 4.3 A	40.5 ± 1.6 AB	5.8 ± 0.7	198.7 ± 4.4 A	218.4 ± 19.6 A	1.0 ± 0.1 ABC	2351.0 ± 176.9 A	122.9 ± 4.5 ABCD
50	HumaPro	20	14.9 ± 7.7 AB	33.4 ± 0.9 B	5.2 ± 0.4	118.9 ± 10.6 BC	116.4 ± 0.7 BCDE	1.2 ± 0.2 AB	1666.3 ± 61 ABC	148.1 ± 3.5 A
50	Micromate	5	20.5 ± 3.5 AB	41.7 ± 1.5 A	4.1 ± 0.1	117.6 ± 9.4 BC	124.8 ± 7.4 BCD	1.4 ± 0.1 A	1634.3 ± 117.5 ABC	107.3 ± 4 DE
50	Micromate	10	20.6 ± 3.6 AB	44.0 ± 1 A	3.8 ± 0.2	91.6 ± 6.5 BC	134.1 ± 16 BC	1.2 ± 0 AB	1621.0 ± 209.3 ABC	114.7 ± 5.1 BCDE
50	Micromate	20	19.8 ± 3.1 AB	38.7 ± 1.9 AB	3.3 ± 0	78.7 ± 5.7 C	83.0 ± 1.7 DEF	0.8 ± 0.1 BC	1708.7 ± 179.4 ABC	94.8 ± 1.3 E

The table shows the means ± standard error (n = 3). For each tissue, treatment means with different letters are significantly different according to the Tukey’s honestly significant difference test (α = 0.05).

). Shoot dry tissue from healthy tomato plants contains around 21 mg Ca, 5.4 mg Mg, 70–94.57 µg Fe, and 16.8–66.3 µg Mn per gram [29,30,31]. The Ca concentration of the shoot tissue from FulviPro (20 mL·L⁻¹) treated tomatoes under low (21 ± 0.5 mg·g⁻¹) fertility was similar to the Ca concentration reported in healthy tomato plants (21 mg·g⁻¹) [31]. In contrast, we observed higher shoot Mg concentration than previously reported [31], and FulviPro-treated plants showed the highest levels (Table 2). Increasing Fe and Mn in the nutrient solution reduces plant growth and increases nutrient accumulation in the tissue [29,30]. Growth reduction is observed in tomato plants when shoot Fe and Mn concentrations are equal to or higher than 80 µg·g⁻¹ and 207.4 µg·g⁻¹, respectively [29,30]. The Fe tissue concentration of FulviPro (20 mL·L⁻¹) treated plants was about 198 µg·g⁻¹, and the Mn tissue concentration was 162.23 µg·g⁻¹ under optimal fertility and 218.43 µg·g⁻¹ under low fertility. Both Fe and Mn concentrations in tissue are much higher than previously reported for tomato under Fe or Mn toxicity. Thus, micronutrient toxicity could be the reason for the observed reductions in growth when plants were treated with FulviPro drenches at 5, 10, and 20 mL·L⁻¹. Micromate and FulviPro separated from HumaPro along PC2. The variables contributing the most to PC2 were Al, B, S, Zn, K, Cu, and C. The nutrient profile of HumaPro-treated plants showed high Zn, K, Cu, and C but low Al, B, and S (Table 2). The formulation of HumaPro has higher Fe and Mn concentrations than FulviPro [21], but HumaPro did not reduce plant growth. Moreover, Fe and Mn tissue concentrations of HumaPro-treated plants were similar to control plants. Thus, it seems that FulviPro mainly increases Fe and Mn uptake, and at higher doses, it leads to toxicity.

3.2. Experiment 2—Humic Substances (Micromate) Increased Shoot Dry Weight but Not Root Dry Weight in Tomato Grown in Soilless Substrate under Limited Fertilization

Based on the results from experiment 1, experiment 2 tested lower concentrations of HumaPro and FulviPro and higher concentrations of Micromate. As observed previously (experiment 1), tomatoes treated weekly with Micromate at 20 g·L⁻¹ showed higher shoot DW than control plants, but the difference was not statistically significant. Only weekly and biweekly Micromate applications at 40 g·L⁻¹ significantly increased shoot DW (Figure 3A). Plants treated biweekly at 40 g·L⁻¹ received a total of 12 g Micromate, and the ones treated weekly at 20 g·L⁻¹ or weekly at 40 g·L⁻¹ received a total of 12 g or 24 g Micromate, respectively. Therefore, to observe a significant response in plant growth, at least 12 g Micromate should be provided to tomatoes, and this total product amount can be divided into weekly or biweekly applications. In contrast, Martins et al. observed growth promotion in petunia treated with at least 3 g Micromate per pot, while the best results were observed with 24 g per pot [21]. Thus, the Micromate dosage is species-dependent, and to observe a better response in tomato plants, higher Micromate rates (>24 g per plant) should be evaluated.

Similar to experiment 1, FulviPro showed a negative effect on shoot DW. Biweekly FulviPro applications at 10 mL·L⁻¹ and weekly FulviPro applications at 5 and 10 mL·L⁻¹ significantly reduced shoot DW (Figure 3A). In experiment 1, the negative effect of FulviPro applications was associated with Fe and Mn toxicity due to the high Fe and Mn concentration in FulviPro. Plants treated biweekly at 10 mL·L⁻¹ received a total of 3 mL FulviPro, and the ones treated weekly at 5 mL·L⁻¹ and 10 mL·L⁻¹ received a total of 3 and 6 mL FulviPro, respectively. Similarly, biweekly (10 mL·L⁻¹) and weekly (5 or 10 mL·L⁻¹) FulviPro drenches reduce growth in petunia ‘Picobella Blue’ [21]. Lower rates of FulviPro did not influence tomato shoot DW (Figure 3A). Under these experimental conditions, the application of HumaPro did not result in a significant increase in dry biomass (Figure 3A). Adverse or null effects have been previously reported in lupin growth and chlorophyll content when applying humic substances in combination with Fe sources [32].

We also assessed the impact of our treatments on the root dry weight of tomato plants. Tomatoes treated with Micromate and HumaPro did not exhibit a statistically significant difference in root dry weight when compared to the control group (Figure 3B). In contrast, Micromate application in petunia increased root dry weight [21]. The one-time, biweekly, and weekly FulviPro applications at a concentration of 10 mL·L⁻¹ showed a lower mean root dry weight than the control. However, the reduction in root dry weight was statistically significant only for the weekly applications (Figure 3B). A similar trend was observed in petunia, where increasing the FulviPro rate decreased root dry weight, but the differences were not statistically significant [21]. Similarly, applying humic substances to lupin plants grown in a vivianite (as Fe-source) amended substrate decreased root dry weight [32].

3.3. Experiment 3—Incorporating Humic Substances (Micromate) into Soilless Substrate Promoted Tomato Growth, Increased Leaf Greenness, and Increased the Mineral Nutrient Concentration of the Shoots and Roots

We learned from experiment 1 and 2 that the minimum amount of Micromate needed to observe plant growth promotion in tomato was 12 g per plant. Thus, here we tested if higher rates would provide additional benefits when Micromate was incorporated into the substrate. Incorporating Micromate into the growing substrate promoted growth in plants under low fertility (Figure 4). Shoot and root dry weight, digital biomass, and growth index measured at the end of the experiment were significantly higher in Micromate-treated plants (Figure 5). However, there was no difference between the 20 and 40 g·L⁻¹ rates. Thus, incorporating more than 20 g Micromate per liter of substrate does not further promote the growth of tomato plants grown under these low fertility conditions.

We evaluated the health of the canopy by measuring greenness and spectral reflectance. SPAD measurements were used as an indicator of greenness or leaf chlorophyll content [23]. SPAD did not capture differences in leaf greenness between control and Micromate-treated tomatoes (Figure 6A). Green leaf index (GLI), normalized difference vegetation index (NDVI), normalized pigment chlorophyll ratio index (NPCI), and plant senescence reflectance index (PSRI) are vegetation indexes calculated from the amount of light reflected at certain wavelengths, and they are used for vegetation health assessment [24,25,33]. Values for these indices were obtained from the TraitFinder Digital Greenhouse phenotyping system (Phenospex). The GLI of healthy tomato ‘Money maker’ was 0.25 [24]. The GLI of tomatoes treated with Micromate at 40 g·L⁻¹ was higher (0.21) than in untreated (0.19) tomatoes (Figure 6B).

NDVI is positively correlated with leaf chlorophyll content or crop N content [34]. Micromate (20 g·L⁻¹)-treated tomatoes showed slightly higher NDVI (0.62) than the untreated control (0.61) (Figure 6C). Similar NDVI values (~0.6) were reported in healthy tomato ‘Money maker’ [24]. The mean NDVI of Micromate-treated and untreated plants was lower than the previously reported threshold values for optimal tomato N content (0.724–0.787) [34]. NDVI has been shown to be species-dependent [35]. This makes it challenging to compare our observed values and those reported in the literature.

NPCI is a vegetation index negatively correlated with chlorophyll and nitrogen content [33,36]. The NPCI of healthy tomato ‘Money maker’ was 0.05 [24]. The NPCI of tomatoes treated with Micromate at 40 g·L⁻¹ was higher (0.035) than in untreated (0.023) tomatoes (Figure 6D). NPCI values in plants under nutrient deficiency [37,38], heat stress [39], drought [40], and biotic stress [41,42] are higher than in healthy plants grown under optimal conditions. The high NPCI values observed in Micromate-treated tomatoes at 40 g·L⁻¹ can suggest a potentially negative response of the plant to high doses.

In this experiment, we collected growth promotion and plant health data through conventional (shoot dry weight, growth index (GI), and SPAD) and digital phenotyping (digital biomass, GLI, NPCI, NDVI, and PSRI). The growth index is a non-destructive approach to evaluating plant growth, and the SPAD meter evaluates plant greenness. Both measurements are manual and time-consuming. In contrast, the TraitFinder phenotyping system allows for rapid evaluation of plant growth and provides various spectral vegetation indices used to assess plant health [43,44]. The TraitFinder digital biomass has been shown to correlate with growth index and shoot dry weight [43,44]. In addition, the TraitFinder captured small differences in canopy health that were missed by the SPAD measurements. It is important to consider that the TraitFinder covers a larger proportion of the canopy compared to the SPAD meter (2 × 3 mm measuring area). Moreover, SPAD only measures leaf absorbance of light in the red and near-infrared wavelengths [45].

Principal component analysis was performed to compare the shoot and root nutrient concentration profiles of each treatment. For the shoot samples, principal component PC1 and PC2 accounted for about 59.51% of the total variance (Figure 7A). Samples of Micromate-treated plants and control plants separated along PC1. The variables contributing the most to PC1 were Si, B, S, and P, and they positively correlated with shoot and root DW (Figure 7A). Moreover, Micromate-treated plants showed higher Si, S, and P concentrations in root and shoots, but B only increased in the root (

Table 3. Mineral macronutrient concentrations in tomato ‘Bush Beefsteak’ shoot and root dry tissue (experiment 3).

Tissue Type	Micromate Rate (g·L−1)					Macronutrient	(mg·g−1)
		N (%)	C (%)	P	K	Mg	Ca	S
Root	0	ND	ND	1.9 ± 0 B	20.1 ± 0.4	6.7 ± 0.2 B	5.1 ± 0.1 B	2.7 ± 0.1 B
Root	20	ND	ND	2.3 ± 0.1 A	18.9 ± 0.7	7.7 ± 0.2 A	5.8 ± 0.2 A	3.4 ± 0.1 A
Root	40	ND	ND	2.5 ± 0.1 A	18.1 ± 1.4	6.7 ± 0.2 AB	5.4 ± 0.2 AB	3.4 ± 0.1 A
Shoot	0	3.9 ± 0.1	37.9 ± 0.6	1.9 ± 0 B	36.3 ± 2.1	11.2 ± 0.3	14.0 ± 0.1	4.4 ± 0 B
Shoot	20	3.5 ± 0.1	38.8 ± 0.2	1.9 ± 0.1 B	34.2 ± 1.3	11.0 ± 0.2	14.3 ± 0.3	4.9 ± 0.3 AB
Shoot	40	3.6 ± 0.1	37.8 ± 0.2	2.4 ± 0.1 A	32.8 ± 2.2	10.8 ± 0.5	14.6 ± 0.4	5.5 ± 0.1 A

ND = Not determined. The table shows the means ± standard error (n = 3). For each tissue, treatment means with different letters are significantly different according to Tukey’s honestly significant difference test (α = 0.05).

,

Table 4. Mineral micronutrient concentrations in tomato ‘Bush Beefsteak’ shoot and root dry tissue (experiment 3).

					Micronutrient	(µg·g−1)
Tissue Type	Micromate Rate (g·L−1)	Al	B	Cu	Fe	Mn	Na	Zn
Root	0	122.8 ± 1.6 B	26.8 ± 0.8 A	6.4 ± 1.1	165.7 ± 6.9	51.7 ± 2.4 B	6829.0 ± 464.7 B	91.6 ± 5.3
Root	20	179.9 ± 1.6 A	23.3 ± 0.7 AB	6.0 ± 0.2	199.2 ± 10.9	68.3 ± 1.1 A	9966.0 ± 341.9 A	102.1 ± 2
Root	40	200.8 ± 19.5 A	21.5 ± 0.9 B	5.7 ± 0.1	211.5 ± 25.3	71.6 ± 4.6 A	9220.3 ± 634.5 A	107.8 ± 5.7
Shoot	0	26.3 ± 3.2	45.8 ± 0.3 B	2.9 ± 0.1	158.2 ± 6.5 A	91.5 ± 0.5	1178.9 ± 128	89.3 ± 4.5
Shoot	20	39.1 ± 7.2	46.7 ± 2.2 AB	3.0 ± 0.2	146.7 ± 10.5 AB	92.7 ± 4.9	1244.0 ± 41.4	83.6 ± 2.2
Shoot	40	34.9 ± 5.2	53.3 ± 1 A	3.0 ± 0.3	117.1 ± 7.3 B	82.8 ± 5	1346.3 ± 162.5	80.8 ± 4.7

The table shows the means ± standard error (n = 3). For each tissue, treatment means with different letters are significantly different according to Tukey’s honestly significant difference test (α = 0.05).

and

Table 5. Mineral concentrations in tomato ‘Bush Beefsteak’ shoot and root dry tissue (experiment 3).

				Micronutrient	(µg·g−1)
Tissue Type	Micromate Rate (g·L−1)	Si	Se	Sr	V	Li	Ba
Root	0	217.53 ± 16.47 A	2.54 ± 0.36	26.55 ± 0.09 B	0.37 ± 0.01	0.62 ± 0.04	16.87 ± 0.1 B
Root	20	406.90 ± 30.09 A	2.05 ± 0.07	29.38 ± 0.64 A	0.44 ± 0.05	0.72 ± 0.07	19.43 ± 0.54 A
Root	40	387.13 ± 21.01 B	3.23 ± 1.4	28.4 ± 0.12 A	0.49 ± 0.05	0.60 ± 0.05	17.43 ± 0.38 B
Shoot	0	516.23 ± 18.32	3.26 ± 0.58	28.52 ± 0.8 B	0.20 ± 0.01	0.76 ± 0.04	11.98 ± 0.47
Shoot	20	608.23 ± 64.23	3.22 ± 0.52	31.21 ± 0.05 AB	0.25 ± 0.03	0.72 ± 0.07	13.55 ± 0.05
Shoot	40	714.77 ± 56.06	3.97 ± 0.9	33.71 ± 0.93 A	0.22 ± 0.02	0.85 ± 0.06	12.60 ± 0.68

The table shows the means ± standard error (n = 3). For each tissue, treatment means with different letters are significantly different according to Tukey’s honestly significant difference test (α = 0.05).

). Martins et al. showed that Micromate drenches increase P concentration in petunias grown under low and optimal fertilization [21]. However, when Micromate was applied as a drench in tomato, we only observed an increase in P concentration under optimal fertility (Table 1). Thus, Micromate’s effect on tissue nutrient concentration was enhanced when applied as a substrate amendment. Accordingly, humic acid application was reported to increase N, P, Ca, Mg, S, Cu, Fe, Mn, and Zn in tomato shoots [46].

For the root samples, principal component PC1 and PC2 accounted for about 74.8% of the total variance (Figure 7B). The variables contributing the most to PC1 were Al, P, Mn, S, Si, and B. Root and shoot DW showed a negative correlation with B and a positive correlation with P, S, Si, Ca, and Na concentration in the roots (Figure 7B). Overall, Micromate treatment increased the average concentration of all nutrients analyzed in the root except B, Cu, and K. Similarly, Türkmen et al. reported that humic acids increased the nutrient concentrations in tomato roots [46]. However, they also observed that high rates led to a reduction in the nutrient concentration. Therefore, careful evaluation of application rates is needed to obtain the best plant outcomes from humic substance applications. The average nutrient concentration in shoots and roots per treatment is presented in Table 3, Table 4 and Table 5. Si is not considered an essential nutrient but has been reported to benefit plants under abiotic and biotic stress [47]. Micromate treatment increased Si in the root and shoot.

3.4. Experiment 4—Drench Application of FulviPro Negatively Impacted Plant Growth and Health, but Spray Application Increased Green Leaf Index (GLI) Values

The application method is an important factor in modulating the efficacy of humic substances [11]. Foliar and soil application of humic acids increased yield in tomato, but the spray application showed the highest increase [15]. Tomato growth and yield are positively influenced by the foliar application of fulvic acid, yet excessive application rates result in adverse effects [48]. We observed a negative effect when FulviPro was applied as a drench (experiments 1 and 2), so in this experiment, we also evaluated foliar spray applications at various rates.

We found a significant interaction between our treatments and application methods, so we analyzed simple effects for each level of the application method factor (drench vs. spray). When applied as a drench, the FulviPro application reduced the final shoot and root dry weight and digital biomass (Figure 8). The effect of FulviPro was dose-dependent, and the highest reduction in shoot DW was observed at the 5 and 10 mL·L⁻¹ rates (Figure 8A). According to our results, FulviPro was also reported to reduce growth in petunia ‘Picobella Blue’ when applied as a drench [21]. No significant differences in growth were observed when FulviPro was applied as a foliar spray (Figure 8A–C). In contrast, foliar application of fulvic acid promoted growth and increased chlorophyll content in tomato ‘Seogwang’, except at the highest dose of 1.6 g·L⁻¹, which caused toxicity [48].

The vegetation indices provided by the TraitFinder were used as indicators of plant health. GLI, NDVI, NPCI, and PSRI are vegetation indices calculated from the amount of light reflected at certain spectral bands, and their values are correlated to leaf chlorophyll content [24,25,33]. Overall, we observed a detrimental effect of the FulviPro drench applications and little effect of the foliar spray applications on plant health parameters. As observed with plant growth, the FulviPro effect was dose-dependent, and the strongest effects were observed on plants treated with a concentration of 10 mL·L⁻¹. Sudiro et al. reported that vegetation index values for healthy tomato ‘Money maker’ were GLI~0.25, NPCI~0.05, NDVI~0.6, and PSRI~0.04 [24]. At the end of the experiment (week 5), GLI values were significantly lower when plants were drenched with 10 mL·L⁻¹ FulviPro (0.12) compared to the control (0.18) (Figure 9). These differences in GLI were initially detected at three weeks after transplant. In contrast, the GLI values for FulviPro sprayed plants were higher than control plants on week 4 (rates 5 and 10 mL·L⁻¹) and week 5 (rates 2.5, 5, and 10 mL·L⁻¹) (Figure 9). Similar results were seen with foliar applications of fulvic acid that increased the leaf chlorophyll content in tomato ‘Seogwang’ [48]. The NDVI values of FulviPro-drenched plants at 10 mL·L⁻¹ were lower (0.60 on week three; 0.57 on week four) than the NDVI of controls (0.65 on week three; 0.64 on week four). There was no significant difference in NDVI at week five (Figure 10). Our tomatoes were grown under limited fertilization, which explains why the GLI and NDVI values of our control plants were lower than previously reported values in healthy tomato [24].

In contrast to GLI and NDVI, higher values for NPCI and PSRI indicate a less healthy plant [24,33,43,49]. At week two, the NPCI values of FulviPro-drenched plants at 5 (0.086) and 10 mL·L⁻¹ (0.103) were higher than the NPCI of controls (0.060), confirming a negative impact on plant health (Figure 11). By week four, the NPCI values of FulviPro-drenched plants at 2.5 (0.074), 5 (0.070), and 10 mL·L⁻¹ (0.093) were higher than the NPCI of controls (0.039) (Figure 11). At week four, the mean PSRI value of FulviPro-drenched plants at 10 mL·L⁻¹ (0.041) was higher than the mean PSRI value for the control treatment (0.016) (Figure 12). Similar results were observed at weeks two and three. NPCI, PSRI, and NDVI values were not significantly different among the drench treatments by week five (Figure 10, Figure 11 and Figure 12). Plants were grown under low fertility; therefore, NPCI and PSRI of control plants were similar or lower than previously reported for healthy tomato [24]. There were no significant differences in NPCI, NDVI, or PSRI when comparing control plants to those sprayed with different rates of FulviPro (Figure 10, Figure 11 and Figure 12).

4. Conclusions

In summary, Micromate promoted growth and health when applied via drench or incorporation into the substrate for tomatoes grown under low fertility. The lowest Micromate rate required to observe an effect was 12 g per plant, and applying more than 20 g per plant did not further increase its effects. The growth-promoting effect was associated with higher shoot and root nutrient concentrations, especially P, S, and Si. HumaPro did not affect tomato plant growth or health under the experimental conditions, and FulviPro showed a consistently negative effect on plants when applied as a drench, especially at high rates (5 and 10 mL·L⁻¹). Shoot DW was negatively associated with the tissue concentration of Fe, Mg, and Mn, and Ca, Fe, and Mn toxicity seems to be the main factor leading to a reduction in growth. Lower doses of FulviPro (<2.5 mL·L⁻¹) did not affect plant growth or health. Spraying FulviPro at 10 mL·L⁻¹ improved GLI, but the increase was very small. The rate of Micromate needed to observe a beneficial effect is species-dependent and justifies future research to optimize the application of humic substances on other greenhouse crops. Humic substances could be a potential alternative to reduce fertilization rates in tomato production while maintaining growth and proper nutrition. However, it is important to conduct trials to ensure the efficacy of the products before introducing them to the management programs.