Organic Fertilizer Optimization for Enhanced Growth and Nutrient Uptake in Bell Pepper Transplants (Capsicum annuum L.)

Abstract

Organic fertilization management for vegetable transplant production is challenging to growers due to the slow and unpredictable release nature of organic fertilizers. Nutrients in organic fertilizers, particularly nitrogen (N), often fail to meet the demands of rapidly growing transplants in soilless substrate. This study aimed to develop fertilization guidelines for organic bell pepper (Capsicum annuum L.) transplants by evaluating the performance of one conventional fertilizer, two organic fertilizers (Drammatic, Pre-Empt), and one naturally derived fertilizer (Bio-Matrix) at a range of N rates in supporting transplant growth. Bell pepper transplants were grown in an indoor growing chamber for 28 days with weekly fertilizer application. We found that the initial nitrate-N concentration in the fertilizer solution was the sole predictor of shoot dry weight (R² = 0.62), confirming that N availability was the primary limiting factor for transplant growth. The conventional fertilizer produced the largest transplants (370.9 mg/plant in shoot dry weight) while Drammatic resulted in the lowest maximum shoot growth (196.6 mg/plant), likely due to its high salinity and the accumulation of ammonium in the substrate. Bell pepper transplants exhibited low nutrient uptake capability and resulted in low N recovery efficiency, especially with the two organic fertilizers, Drammatic and Pre-Empt (15.6% and 23.8%, respectively). Furthermore, we found no carryover effects of the fertilizer treatments during the transplant stage on bell pepper growth after being transplanted to the greenhouse for 18 days. The final shoot dry weight only correlated with transplant shoot dry weight at the time of transplanting (R² = 0.87) but not with fertilizer type (p = 0.2849). Overall, Pre-Empt emerged as the most effective fertilizer for organic bell pepper transplant production. It is cost-effective, has low electrical conductivity, and is associated with low ammonium accumulation in the substrate. Therefore, it can be applied at high N rates to meet the N demand of bell pepper transplants. Based on our growing conditions, we recommend 23.1 g/L substrate of Pre-Empt for organic bell pepper transplant production.

1. Introduction

Bell pepper is a popular vegetable crop worldwide. According to the National Agricultural Statistic Service of the United States Department of Agriculture, the USA produced over 455 million kg (one billion pounds) of bell peppers in 2021, with a corresponding production value of USD 462 million. In bell pepper production, transplanting is a common practice which can produce uniform and resilient plants, enable earlier fruit production, and result in higher yield [1,2]. Obtaining vigorous and uniform bell pepper transplants is the crucial first step towards a high marketable yield.

Organic transplants are not widely available commercially, so organic vegetable growers often produce transplants themselves. One major challenge they face in transplant production, similar to organic vegetable production, is fertilization management [1]. Many materials can be used as organic fertilizers, including manure, green manure, compost, and byproducts of other agricultural activities such as slaughterhouse byproducts, fish scraps, and molasses from cane sugar production [3,4]. Using organic waste and byproducts promotes material recycling and improves the sustainability of agriculture practices [5]. However, two major challenges exist. First, the nutrient composition in these materials often does not match the crop demand, and the nutrient release relies on microbial activity [3,6,7,8,9]. Nutrients in organic forms must be converted by microbes into plant-available inorganic ions, such as nitrate (NO₃⁻), ammonium (NH₄⁺), and phosphate (PO₄³⁻). This process is called mineralization and is a slow process that is hard to predict and manipulate, which leads to a mismatch between nutrient supply and the high demands of rapidly growing transplants [6]. In contrast, conventional transplant production uses conventional, inorganic fertilizers that provide nutrients in predictable, readily available inorganic forms. Second, organic fertilizers often contain compounds other than nutrients, leading to salinity stress or phytotoxicity [6,10]. Nitrite (NO₂⁻) and ammonium accumulation during the mineralization process also cause phytotoxicity [11,12]. Attempts to increase the application rate of organic fertilizers to meet the high nutrient demands of crops can lead to salinity stress, phytotoxicity from non-nutrient compounds, or the accumulation of toxic levels of ammonium and nitrite [13].

For organic transplant production, the slow and unpredictable nutrient supply from organic fertilizers is a particular challenge, because the short growing cycles do not allow for sufficient mineralization, and the soilless substrate may not contain high enough microbial populations that mineralize organic nutrients effectively [14]. Within soilless substrates, both fertilizer and crop types have been shown to affect microbial populations. Organic fertilizers typically foster higher microbial activity than chemical fertilizers [15], and the microbial population and activity were influenced by fertilizer type and, in the long term, by the crop species growing in the substrate [16,17]. Growers need fertilization guidelines for organic vegetable transplant production. However, a significant knowledge gap in understanding and predicting nutrient dynamics in soilless substrate remains.

Previous research from our laboratory on organic watermelon transplants highlighted these complexities. A study comparing different organic fertilizers at different nitrogen (N) application rates found that while increasing N rates generally improved shoot growth, the type of fertilizer significantly affected root development [18]. Sustane 4-6-4 (Sustane), an organic fertilizer from composted turkey manure and feather meal, resulted in the largest root systems at a N rate of 0.56 g N/L substrate [18]. Conversely, Drammatic O Organic Fertilizer with Kelp 2-4-1 (Drammatic), a fish-based fertilizer, promoted the best shoot growth among all organic fertilizers but resulted in poor root growth, which was hypothesized to be a result of its high phosphorus (P) and low potassium (K) content [18]. A follow-up study tested the nutrient imbalance hypothesis in watermelon by blending Drammatic with N-rich and K-rich organic fertilizers to create more balanced N:P:K ratios [19]. The results demonstrated that adjusting the nutrient ratios did not significantly improve transplant growth. Instead, low N availability, specifically the nitrate concentration in the fertilizer solutions, was the primary factor limiting both shoot and root growth [19].

Furthermore, fertilizers derived from organic materials often contain other biologically active compounds. For example, Drammatic is made from fish protein hydrolysates, which are typically rich in amino acids such as glutamic acid, aspartic acid, glycine, alanine, and leucine [20,21]. These compounds serve as a readily available source of organic nitrogen for the soil microbiome. Hence, it is advertised to ‘boost their soil microbial activity’. Drammatic also contains kelp extracts that have been shown to improve the nutrient uptake and photosynthetic activity of crops, enhance crop growth and yield, and enhance crop nutritional value [22]. Pre-Empt organic fertilizer (Pre-Empt) is made from fermented molasses, the by-production of cane sugar production. A wide range of amino acids can be produced from molasses through bacterial fermentation [23]. In addition, it contains humic acid, fulvic acid, and arrays of vitamins, which can act as biostimulants to promote lateral root growth and chelate micronutrients to enhance their availability to plants [24,25]. Pre-Empt is also inoculated with a combination of aerobic and anaerobic microbes. Microbial inoculants, depending on the microbe population, offer a wide range of benefits to crops, including but not limited to the following: increasing resistance to abiotic and biotic stresses, improving nutrient availability and crop uptake, improving yield, enhancing plant development and enhancing overall crop health [26]. MicroLife Bio-Matrix 7-1-3 (Bio-Matrix) is derived from plant and fish amino acids and contains a ‘huge microbial inoculation’ with 100 thousand colony-forming units per ml (https://microlifeblank.wpenginepowered.com/wp-content/uploads/2025/04/ML-Bio-Matrix-Spec-Sheet.pdf, accessed on 30 June 2025). It also contains several plant hormones, growth stimulators and vitamins and is marketed as a dual liquid fertilizer and biostimulant. With the unique compositions and bio-active compounds in these fertilizers, it is likely that the different microorganisms and other biostimulant compounds would affect transplant growth and leave a carryover effect on the nutrient acquisition ability of transplants even after being transplanted to the field or larger containers.

This study aims to explore the effects of fertilizer types and N rates on the growth and nutrient uptake of bell pepper transplants and to establish relationships between fertilizer composition, nutrient dynamics in the substrate, and transplant performance. We hypothesized that (1) the growth and development of bell pepper transplants would be primarily determined by the N availability of fertilizers, similar to the findings from watermelon transplants [19], (2) the fertilization treatment and bioactive compounds from fertilizers would have a carryover effect on the performance of bell pepper plants after transplanting, (3) high salinity and ammonium toxicity from organic fertilizers would limit growth at high N rates, even when the nutrient supply was no longer a limiting factor, and (4) shoot size (dry weight) is the most reliable predictor of post-transplant performance because it is directly linked to the photosynthetic capacity and nutrient reserves of a plant.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

Pepper seeds (cv California Wonder; SEEDWAY, Hall, NY, USA) were sown on 9 October and 20 November 2024 for the 1st and the 2nd experiment. A peat-based organic germination substrate containing no starter fertilizer (customized OM2; Berger, Saint-Modeste, QC, Canada) was used. The seeds were sown in 72 -cell horticultural trays, with each tray containing about 4 L (380.54 g dry weight) of substrate. Two seeds were sown per cell. Immediately after sowing, the trays were misted with tap water and covered with humidity domes to maintain humidity. Humidity domes were removed nine days after sowing (DAS), when germination exceeded 50%. Bell pepper transplants were thinned to one per cell at 12 DAS, immediately before the fertilizer application. Transplants were illuminated by warm white LEDs (Ray44 PhysioSpec Indoor; Fluence, Austin, TX, USA) with a photosynthetic photon flux density of 191.0 ± 23.0 µmol·m⁻²·s⁻¹ (mean ± standard deviation) with a 16 h photoperiod. The average air temperature in the growth chamber was 27.5 ± 1.4 °C and 27.2 ± 1.3 °C during the 1st and 2nd experiment, respectively. The average vapor pressure deficit was 1.3 ± 0.5 kPa and 1.5 ± 0.7 kPa during the 1st and 2nd experiment, respectively.

2.2. Fertilizer Treatments

We tested four fertilizers of different N rates in this study: one conventional fertilizer (Peter’s Professional 20-20-20; ICL Group Ltd., Tel Aviv, Israel), one naturally derived fertilizer (Bio-Matrix), and two organic fertilizers (Pre-Empt and Drammatic). Both Pre-Empt and Drammatic are certified organic products listed in the Organic Materials Review Institute (OMRI). Drammatic (Drammatic O Organic Fertilizer with Kelp 2-4-1; Dramm Corporation, Manitowoc, WI, USA) is derived from fish scraps. Pre-Empt (Pre-Empt; Coastal Fertilizer & Supply Inc., Labelle, FL, USA) is fermented colloidal molasses and does not have guaranteed analysis for N, P, and K. The NPK label of Pre-Empt was estimated to be 6-1-1.7. The naturally derived fertilizer, Bio-Matrix (MicroLife Bio-Matrix 7-1-3; San Jacinto Environmental Supplies, Houston, TX, USA), is advertised to have organic origins but is not OMRI-certified.

Twenty and 18 trays were used in the 1st and 2nd experiment, respectively, with each tray considered an experimental unit. Seventy two plants were grown in each tray. Across the two experiments, the range of the N rate was 0.15–1.20 g N/L for the conventional fertilizer and Bio-Matrix, 0.15–0.80 g N/L for Drammatic, and 0.15–3.00 g N/L for Pre-Empt (Table S1). The corresponding P and K rates for each fertilizer type are also detailed in Table S1. Fertilizers were divided into 3 weekly applications: 25% of the total fertilizer rate (the 1st fertilizer application) at 12 DAS, an addition 25% was applied (the 2nd fertilizer application) at 19 DAS, and the remaining 50% was applied at 26 DAS (the 3rd and last fertilizer application). For each application, the fertilizers were dissolved in 1 L of tap water then applied as subirrigation.

2.3. Chemical Properties of Liquid Fertilizers

To characterize the nutrient content in each fertilizer, the initial ion concentration of each fertilizer was analyzed. Fertilizer solutions for each fertilizer were prepared with reverse osmosis (RO) water to a concentration of 400 mg N/L to quantify the concentrations of nitrate-nitrogen (nitrate-N), ammonium-nitrogen (ammonium-N), nitrite-nitrogen (nitrite-N) and phosphorus. The ion concentrations were measured by a spectrophotometer (HI 83200; Hanna Instruments, Woonsocket, RI, USA). The inorganic N concentration in fertilizer solutions was calculated as the sum of nitrate-N and ammonium-N concentrations. K concentration was not quantified due to the low accuracy of the testing protocol (HI 83299 K test; ± 60 mg/L ± 14% of reading). Additionally, before the 1st fertilizer application, fertilizer solutions made with tap water were measured for pH and electrical conductivity (EC) by a pH/EC tester (HI 98129; Hanna Instruments).

To monitor nutrient dynamics in substrate, leachate was collected from each tray by the PourThru method [27] at 13 and 27 DAS. The leachate collected on 13 DAS was immediately measured for pH and EC. The leachate from 27 DAS was stored at −20 °C. Prior to analysis, leachate samples were thawed and filtered through filter paper (Fisherbrand P8; Fisher Scientific, Hampton, NH, USA). Filtered leachate was then measured for nitrate-N, ammonium-N, nitrite-N and phosphorus concentrations, pH, and EC, similar to the fertilizer solutions.

2.4. Transplant Growth, Morphological, and Physiological Measurements

Top view photos of transplants were taken on 13, 20, and 27 DAS to monitor canopy expansion. Transplant height was measured at 27 DAS on six randomly selected representative transplants. On the same day, the Soil–Plant Analysis Development (SPAD) index (by SPAD 502; Minolta Co., Ltd., Osaka, Japan) was measured on the newest fully expanded leaf of six different transplants. Maximum quantum yield of photosystem II (F_v/F_m) was measured by a chlorophyll fluorometer (OS5p+; Opti-Sciences Inc., Hudson, NH, USA) after being dark-adapted for at least 30 min. Six transplants that were not subjected to SPAD measurements were selected for F_v/F_m from each tray, because of possible damage by the clip of the SPAD meter.

Transplants were harvested three times: 14, 21, and 28 DAS. Eighteen transplants per treatment were destructively harvested at each harvest. Both transplants and the associated substrate plugs were removed, and the amount of fertilizer to apply to each tray was re-calculated to account for the lower substrate volume. For the 18 transplants harvested on the same tray, total shoot fresh weight was recorded by a digital scale and the total leaf area was measured by a leaf area meter (LI-3100C; LI-COR Biosciences, Lincoln, NE, USA). Shoots were dried in an oven at 80 °C for at least 72 h, and their respective dry weights were recorded. Shoot compactness was calculated as the shoot dry weight divided by height (measured on 27 DAS). For the 3rd and final harvest at 28 DAS, on top of the above-mentioned measurements, shoot tissues from each tray were combined, ground into fine powder, and sent to Texas A&M AgriLife Extension Service Soil, Water, and Forage Testing Laboratory (College Station, TX, USA) for mineral analysis. After the N, P, and K concentrations in dried shoot samples were measured, the N, P, and K recovery efficiency was calculated. First, the total mineral (N, P, and K) content in the shoot tissues of each transplant was calculated as:

$$\mathrm{S}\mathrm{h}\mathrm{o}\mathrm{o}\mathrm{t} \mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{l} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}\left(\mathrm{g}\right)=\mathrm{s}\mathrm{h}\mathrm{o}\mathrm{o}\mathrm{t} \mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{l} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\mathrm{\%}\right)\times \mathrm{s}\mathrm{h}\mathrm{o}\mathrm{o}\mathrm{t} \mathrm{d}\mathrm{r}\mathrm{y} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h} \mathrm{p}\mathrm{e}\mathrm{r} \mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t}\left(\mathrm{g}\right)$$

Next, the mineral recovery efficiency was calculated for N, P, and K as:

$$\mathrm{M}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{l} \mathrm{r}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{y} \mathrm{e}\mathrm{f}\mathrm{f}\mathrm{i}\mathrm{c}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{y} (\mathrm{\%})={\displaystyle \frac{\mathrm{s}\mathrm{h}\mathrm{o}\mathrm{o}\mathrm{t} \mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{l} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}\left(\mathrm{g}\right)-\mathrm{s}\mathrm{h}\mathrm{o}\mathrm{o}\mathrm{t} \mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{l} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t} \mathrm{w}\mathrm{i}\mathrm{t}\mathrm{h}\mathrm{o}\mathrm{u}\mathrm{t} \mathrm{f}\mathrm{e}\mathrm{r}\mathrm{t}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{z}\mathrm{e}\mathrm{r}\left(\mathrm{g}\right)}{\mathrm{f}\mathrm{e}\mathrm{r}\mathrm{t}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{z}\mathrm{e}\mathrm{r} \mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{l} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\left(\frac{\mathrm{g}}{\mathrm{L}}\right)\times \frac{4}{72} \mathrm{L} \mathrm{s}\mathrm{u}\mathrm{s}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e} \mathrm{p}\mathrm{e}\mathrm{r} \mathrm{t}\mathrm{r}\mathrm{a}\mathrm{n}\mathrm{s}\mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t}}}$$

The shoot mineral content (g) was calculated from the previous equation. Shoot mineral content without fertilizer was the shoot mineral content of the two treatments without fertilizer (the first two treatments in Table S1). The applied mineral (N, P, and K) rates can be found in Table S1. Since one tray held a 4 L substrate and 72 transplants, the substrate volume per transplant was calculated as ${\displaystyle \frac{4}{72}} \mathrm{L} \mathrm{s}\mathrm{u}\mathrm{b}\mathrm{s}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e} \mathrm{p}\mathrm{e}\mathrm{r} \mathrm{t}\mathrm{r}\mathrm{a}\mathrm{n}\mathrm{s}\mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t}$ (L). Therefore, the denominator of this equation was the total amount of nutrients (N, P, and K) each transplant received.

Roots of six plants were selected in the 3rd harvest, washed in water, and scanned by a root scanner (Perfection V850; Epson America Inc., Long Beach, CA, USA) and were quantified by WinRHIZO software (version 2022a, Regent Instruments Inc., Québec City, QC, Canada). Root tissues were dried at 80 °C for 72 h and weighed for dry weight. The root–shoot ratio was calculated by dividing the root dry weight by the shoot dry weight for each treatment. Substrate samples were also collected from the 3rd harvest, air dried, and sent to the Soil, Water, and Forage Testing Laboratory for mineral content analysis and substrate pH and EC.

2.5. Post-Transplanting Greenhouse Study

To evaluate the post-transplant performance and the potential carryover effect of fertilizer treatments, a follow-up greenhouse study was conducted. In both replicates, 29 DAS (the day after the 3rd harvest), five pepper transplants per treatment were transplanted to 1.4 L round pots filled with a peat-based soilless substrate (BM6; Berger). One teaspoon (approximately 0.93 g) of Sustane 4-6-4 organic fertilizer (Sustane Corporate, Cannon Falls, MN, USA) was mixed in the substrate of each pot. This created a low nutrient condition (about 26.6 mg N/L substrate) to assess the nutrient acquisition ability of bell pepper plants. Pots were randomly arranged on greenhouse benches and re-randomized twice a week in a greenhouse in Dallas, Texas (32°59′ N, 96°46′ W). Plants were irrigated with tap water when the substrate surface was dry. The average air temperature in the greenhouse was 18.9 ± 2.9 °C and 26.1 ± 1.7 °C for the 1st and 2nd experiment, respectively. Daily light integral was 13.7 ± 5.3 mol·m⁻²·d⁻¹ and 7.7 ± 4.3 mol·m⁻²·d⁻¹ for the 1st and 2nd experiment, respectively. Performance index (PI) was measured by OS5p+ on a newly expanded leaf of all pepper plants 4 h after dark on 18 and 16 days after transplanting for the 1st and 2nd experiment, respectively. SPAD was measured on a fully expanded leaf for all plants. Eighteen days after transplanting, pepper plants were destructively harvested for both replicates and shoot dry weights were recorded after drying at 80 °C for 72 h.

2.6. Data Analysis

Regression analysis was performed to determine the relationships between N rates and chemical characteristics of fertilizers and transplant growth parameters with SigmaPlot 11.0 (Systat Software Inc., San Jose, CA, USA). Specifically, the following parameters were regressed against N rate for each fertilizer: pH, EC, ion concentrations of fertilizer solutions and leachates, transplant canopy size, height and compactness, SPAD, leaf number, total leaf area, shoot fresh and dry weight, shoot mineral concentration, nutrient use efficiency and root dry weight, and bell pepper plant SPAD, PI, and shoot dry weight 18 days after being transplanted to greenhouse. Other correlations were tested by Microsoft Excel (Microsoft, Seattle, WA, USA).

To identify the most influential chemical properties of the fertilizer and leachate solutions on transplant growth (shoot and root dry weight), stepwise regression analyses were performed using SAS OnDemand for Academics (SAS 9.4 M9, SAS Institute, Cary, NC, USA). The following independent variables were tested: pH and EC, nitrate-N, ammonium-N, total inorganic-N, nitrite-N, and phosphorus concentrations in freshly made fertilizer solutions, pH and EC of leachate solutions on 13 and 27 DAS, and nitrate-N, ammonium-N, nitrite-N and phosphorus concentrations in leachate solutions on 27 DAS. These independent variables were first assessed for multicollinearity. High multicollinearity indicates high correlations between the independent variables, which increases the instability of models constructed by stepwise regression. To reduce the multicollinearity, the variables with variance inflation >10 were removed before entering stepwise regression analysis. The remaining variables were then used to construct models for shoot dry weight and root dry weight with forward stepwise regression analyses. pH and EC of freshly made fertilizer solution, nitrate-N and ammonium-N in freshly made fertilizer solution, pH and EC of leachate solution of 13 and 27 DAS, nitrate-N and nitrite-N concentration in leachate solution were used to construct stepwise regression.

To test the carryover effects on the post-transplant growth from bio-active compounds in fertilizers during transplant stage, Analysis of Covariance (ANCOVA) was performed on the final shoot dry weight data by SAS OnDemand for Academics for bell pepper plants after being transplanted to the greenhouse. The fertilizer type during the transplant stage was the main factor, and the transplant shoot dry weight on 28 DAS was used as the covariate to account for the initial growth differences.

3. Results

3.1. Fertilizer Chemical Properties

Fertilizer solutions at a N concentration of 400 mg/L showed significant difference in their chemical compositions (

Table 1. Chemical properties of fertilizer solutions at a nitrogen (N) concentration of 400 mg/L. Nitrate-nitrogen (NO₃⁻-N), ammonium-nitrogen (NH₄⁺-N), nitrite-nitrogen (NO₂⁻-N), and phosphorus (P) concentration were measured using a spectrophotometer. Inorganic nitrogen concentrations were calculated as the sum of NO₃⁻-N and NH₄⁺-N concentrations. The inorganic N and measured P represented the N and P available to crops. The percentage of inorganic N and measured P represent relative to N and P content indicated by fertilizer labels (%N to label and %P to label) are also shown.

Fertilizers	Fertilizer Rate (g/L)	Label N Rate (mg/L)	Label P Rate (mg/L)	NO3− (mg/L)	NH4+ (mg/L)	NO2− (mg/L)	Inorganic N (mg/L)	Measured P (mg/L)	Available N	Measured P
Conventional	2.0	400	175	140	4	0	144	172	36.0%	99%
Drammatic	20.0	400	349	139	84	0	223	470	55.8%	135%
Pre-empt	6.7	400	36	66	25	0	92	57	23.0%	195%
Bio-Matrix	5.7	400	25	94	95	0.02	189	71	47.2%	285%

). The percentage of available N relative to fertilizer label was highest in Drammatic, followed by Bio-Matrix and Conventional fertilizer, while Pre-Empt had the lowest %N to label (Table 1). Conventional fertilizer had a low ammonium-N concentration, while Drammatic and Bio-Matrix had a higher ammonium-N concentration, with Pre-Empt being intermediate. All fertilizers had low nitrite concentration, with only Bio-Matric having only a detectable level of nitrite-N at 0.02 mg/L (Table 1). Drammatic had the highest P content, followed by conventional fertilizer, then by Bio-Matrix and Pre-Empt (Table 1). Bio-Matrix and both organic fertilizers provided more P than indicated by their fertilizer labels (Table 1). The potassium concentration in fertilizer solutions was not measured.

The pH of the Bio-Matrix solution was above 7 across all N rates, while the pH of conventional fertilizer, Drammatic, and Pre-Empt solutions decreased as N rates increased (Figure S1A). The pH of leachate collected the day after the 1st fertilizer application (13 DAS) showed a similar trend to the pH of fertilizer solutions (Figure S1C). At 27 DAS (after the 3rd fertilizer application), leachate pH from trays that received Drammatic and Bio-Matrix was no longer affected by N rate of fertilizers, while leachate pH with the conventional fertilizer and Pre-Empt continued to decrease with increasing fertilizer N rate (Figure 1A). At the same N rates, Drammatic solution had the highest EC while Pre-Empt had the lowest EC (Figure 1B). The EC of fertilizer solutions was positively correlated with the EC of leachate at both 13 and 27 DAS, as well as with the EC of substrate samples (Figure 1C).

On 27 DAS (one day after the 3rd fertilizer application), leachate collected from the conventional and Bio-Matrix treatments had higher nitrate-N concentrations than leachate from the two organic fertilizers at the same N rate (Figure 2A). Drammatic resulted in the highest ammonium-N in leachate and Pre-Empt left the least amount of ammonium-N, with the conventional fertilizer and Bio-Matrix being intermediate (Figure 2B). Consequently, the inorganic N concentration (sum of nitrate- and ammonium-N concentrations) was highest in leachate for Drammatic treatments and was the lowest for the Pre-Empt treatment at the same N rate (Figure 2D). In terms of nitrite-N, which is toxic to roots, N rates of conventional fertilizer and Bio-Matrix did not correlate with nitrite-N in leachate (Figure 2C), which indicated a minimal production of nitrite. In contrast, the nitrite-N concentration in leachate increased with the N rate for both Drammatic and Pre-Empt (Figure 2C). The nitrite-N concentration ranged between 0.0 and 9.2 and 0.4–54.0 mg/L for Drammatic and Pre-Empt, respectively (Figure 2C).

Phosphorus in leachate was also affected by fertilizer type and N rate (Figure 2E). The phosphorus concentration was highest with Drammatic treatment, likely due to its high phosphorus content (Figure 2E). Pre-Empt and Bio-Matrix fertilizers had lower P content and resulted in similarly low P concentration in leachate, while conventional fertilizer resulted in intermediate leachate P concentration (Figure 2E). Interestingly, both leachate and substrate P concentration correlated strongly with P content indicated by the fertilizer labels (Figure 3A), but did not correlate with available P as measured by the spectroradiometer (p = 0.37 and p = 0.69, respectively).

At harvest (28 DAS), nitrate-N in substrate increased with fertilizer N rates for all four fertilizers we tested (Figure 2F), which indicated nitrate-N accumulation in substrate. This increase was fastest with Bio-Matrix, followed by conventional fertilizer and Drammatic, then by Pre-Empt (Figure 2F). Substrate P and K concentrations increased with N rates of all four fertilizers (Figure 2G,H), which suggested P and K accumulation in the substrate rather than effective crop uptake. For conventional fertilizer and Drammatic, the increase in substrate P followed a quadratic pattern, suggesting stronger accumulations than Pre-Empt and Bio-Matrix (Figure 2G). The P concentration in the substrate was strongly affected by fertilizer P rates and aligns with this pattern (Figure 3A). The quadratic relationship between substrate K concentration and fertilizer N rate (Figure 2H) as well as the correlation between substrate K concentration and fertilizer K rate (Figure 3B) together indicated excessive K supply from all four fertilizers, similar to P.

3.2. Shoot Growth of Pepper Transplants

One day after the first fertilizer application (13 DAS), there was no clear effect of fertilizer N rate on transplant canopy size for any of the fertilizers (Figure 4A). At 20 and 27 DAS (one day after the 2nd and 3rd fertilizer application, respectively), canopy size exhibited a quadratic response to N rate (Figure 4B,C). The canopy size of bell pepper transplants at 27 DAS responded to the conventional fertilizer and Bio-Matrix similarly: both peaked at 0.83 g N/L substrate and reached a canopy area of 67.0 and 61.8 cm², respectively (Figure 4C,

Table 2. Maximum values of bell pepper transplant growth parameters, compactness, and SPAD index predicted from the regression analyses and corresponding N rates for each fertilizer tested in this study.

Fertilizer	Max Canopy Area (cm2/Plant) and N Rate (g/L)		Max Shoot Dry Weight (mg/Plant) and N Rate (g/L)		Max Shoot Compactness (g/m) and N Rate (g/L)		Max SPAD Index and N Rate (g/L)		Max Root Dry Weight (mg/Plant) and N Rate (g/L)
	Max Canopy Area	N Rate	Max Shoot Dry Weight	N Rate	Max Shoot Compactness	N Rate	Max SPAD Index	N Rate	Max Root Dry Weight	N Rate
Conventional	67.0	0.83	370.9	0.93	3.77	1.03	46.6	0.81	20.4	0.72
Drammatic	46.2	0.67	196.6	0.56	2.44	0.70	46.4	0.64	15.5	0.48
Pre-Empt	63.0	2.03	325.7	2.11	3.42	2.42	49.7	2.30	19.3	1.73
Bio-Matrix	61.8	0.83	337.3	0.82	3.77	0.94	50.2	0.90	21.4	0.70

). Drammatic achieved the lowest maximum canopy size of 46.2 cm² (Figure 4C, Table 2). Pre-Empt reached a maximum canopy size of 63.0 cm² at a much higher N rate of 2.03 g/L (Figure 4C, Table 2).

The leaf area and shoot fresh and dry weight followed similar patterns in response to the N rate (Figure 5 and Figure S2). At 14 DAS, shoot dry weight did not show a clear response to different N rates, except for Bio-Matrix (Figure 5A). By 21 DAS, shoot dry weight exhibited a quadratic pattern for all fertilizers: the shoot dry weight initially increased with N rates, reached a peak then decreased again (Figure 5B). This quadratic pattern persisted through the final harvest at 28 DAS (Figure 5C). For conventional fertilizer and Bio-Matrix, the N rates to achieve the highest shoot dry weight were similar (0.93 and 0.82 g/L, respectively, Figure 5C, Table 2). Drammatic resulted in the lowest maximum shoot dry weight (196.6 mg/plant) at the lowest N rate of 0.56 g/L (Figure 5C, Table 2). For Pre-Empt, shoot dry weight did not reach the maximum until a N rate of 2.11 g/L, and the maximum shoot dry weight was 325.7 mg/plant, which was comparable to the conventional fertilizer and Pre-Empt (Figure 5C, Table 2).

A forward stepwise regression showed that nitrate-N concentrations in fertilizer solutions were the only predictor of pepper transplant shoot dry weight (R² = 0.6220, p < 0.0001). The stepwise regression analysis also determined that pH (p = 0.4585) and EC (p = 0.4245) of freshly made fertilizer solution, ammonium-N concentration in freshly made fertilizer solution (p = 0.0786), pH (p = 0.3327) and EC (p = 0.2979) of leachate solution on 13 DAS, pH (p = 0.1685) and EC (p = 0.9460) of leachate solution on 27 DAS, and nitrate-N (p = 0.1334) and nitrite-N (p = 0.1283) concentrations in leachate solution on 27 DAS did not affect shoot dry weight.

Plant height at 27 DAS also exhibited a quadratic response to the N rate for different fertilizers (Figure S3). Shoot compactness similarly followed a quadratic pattern (Figure 6). This pattern indicates that the low N rates at insufficient range and the high N rates at toxic range both resulted in suboptimal compactness (Figure 6). The conventional fertilizer, Pre-Empt, and Bio-Matrix reached similar compactness (3.77, 3.42, and 3.77 g/m, respectively, Figure 6, Table 2). But Drammatic resulted in a lower maximum compactness of 2.44 g/m at 0.70 g N/L (Figure 6, Table 2).

The SPAD index of pepper transplants was also affected by fertilizer type and N rate (Figure 7). The maximum SPAD values achieved by all four fertilizers were similar: 46.6, 46.4, 49.7, and 50.2 for conventional fertilizer, Drammatic, Pre-Empt, and Bio-Matrix, respectively (Figure 7, Table 2).

Shoot mineral concentrations at the final harvest were also affected by fertilizer type and N rate (Figure 8). For conventional fertilizer and Bio-Matrix, the shoot N concentration initially increased steeply with N rates before plateauing at 1.28 and 1.31 g N/L, respectively (Figure 8A). For Drammatic, the shoot N concentration increased linearly within the N range we tested, up to 0.8 g N/L (Figure 8A). With Pre-Empt, the shoot N concentration of pepper transplants increased more gradually with N rate, and is predicted to reach a plateau at a much higher N rate of 3.97 g/L (Figure 8A). Nitrogen recovery efficiency also varied significantly depending on the fertilizer type and N rate (Figure 8B). For Drammatic and Pre-Empt, N recovery efficiency was unaffected by N rate and was around 15.6% and 23.8%, respectively (Figure 8B).

For shoot P concentration, Drammatic resulted in a linear increase in shoot P in response to N rate (Figure 8C). The conventional fertilizer and Bio-Matrix exhibited a quadratic response, with shoot P concentration reaching a plateau before declining with N rate (Figure 8C). On the other hand, Pre-Empt resulted in a much more gradual linear increase in shoot P concentration. The P recovery efficiency decreased with increasing N rates for all fertilizers (Figure 8D). The highest P recovery efficiency was observed with Bio-Matrix, followed by Pre-Empt, while the conventional fertilizer consistently showed the lowest P recovery efficiency (Figure 8D). The shoot potassium (K) concentration increased linearly with the N rate for all fertilizers, except for Drammatic (Figure 8E). This increase was faster with conventional fertilizer (Figure 8E). K recovery efficiency, much like P recovery efficiency, decreased as the N rate increased across all treatments (Figure 8F). The highest K recovery efficiency was observed with the Pre-Empt and Bio-Matrix (Figure 8F).

3.3. Pepper Transplant Root Growth

Similar to the trends observed for shoot growth (Figure 5C), root length and root dry weight were higher with conventional fertilizer and Bio-Matrix than with Drammatic across the tested N rates (Figure 9A,B). Peak root dry weight with Drammatic (15.5 mg/plant) was also achieved at a lower N rate of 0.48 g/L (Figure 9B, Table 2). In contrast, conventional fertilizer and Bio-Matrix resulted in a peak root dry weight (20.4 and 21.4 mg/plant, respectively) at N rates of 0.72 and 0.70 g/L, respectively (Figure 9B, Table 2). For Pre-Empt, maximum root dry weight (19.3 mg/plant) was lower than conventional fertilizer and Bio-Matrix but was achieved at a substantially higher N rate of 1.73 g/L (Figure 9B, Table 2). The peak root growth was achieved at lower nitrogen rates than shoot growth for all fertilizers we tested (Table 2).

The root–shoot ratio also varied significantly with N rate (Figure 9C). For conventional fertilizer, Drammatic and Bio-Matrix, the root–shoot ratio decreased linearly with increasing N rates (Figure 9C). Meanwhile, for Pre-Empt, the root–shoot ratio decreased rapidly when the N rate was low, then the decrease slowed down when the N rate reached 1.8 g/L (Figure 9C).

A forward stepwise regression analysis showed that root dry weight was positively correlated with nitrate-N concentrations of leachate solutions (R² = 0.1793, p = 0.0081). Regression analysis also showed that root dry weight was not correlated with pH (p = 0.5298) and EC (p = 0.8514) of freshly made fertilizer solution, nitrate-N (p = 0.5356), ammonium-N (p = 0.9898) and phosphorus (p = 0.2921) concentrations in freshly made fertilizer solutions, pH (p = 0.7815) and EC (p = 0.1291) of leachate solution on 13 DAS, pH (p = 0.6703) and EC (p = 0.7283) of leachate solution on 27 DAS, and nitrite-N (p = 0.2926) concentration in leachate solutions on 27 DAS.

3.4. Plant Growth After Transplanting

After being transplanted and grown in the greenhouse, neither SPAD nor PI of bell pepper plants were significantly affected by fertilizer type or N rate during the transplant stage (Figure S4). After 18 days in the greenhouse, shoot dry weight exhibited a very similar pattern as transplant shoot dry weight on 28 DAS (Figure 10A, compared to Figure 5C). The shoot dry weight at 18 DAT was strongly correlated with the transplant shoot dry weight at 28 DAS (Figure 10B). ANCOVA analysis indicated that, after accounting for the initial transplant size, the final plant dry weight was unaffected by fertilizer types during the transplant stage (p = 0.2849). It indicated that larger transplants resulted in larger mature plants, and their growth was not affected by fertilizer type during the transplant stage, despite our original hypothesis that different microorganisms and other biostimulant compounds would affect the plants’ ability to acquire nutrients.

4. Discussion

The production of high-quality bell pepper transplants is the foundational step for successful field establishment and, ultimately, high marketable yield and high profit. The fertilization strategy during this critical stage affects the vigor of transplants, thus affects the crop performance after transplanting and productivity [28]. Conventional, inorganic fertilizers offer nutrients in readily available forms and can be precisely tailored to meet the needs of specific crops. Nutrient release in organic fertilizers depends on microbial mineralization, which is a relatively slow process and is affected by many factors such as fertilizer type, substrate characteristics, and microbial community [29]. The mineral release from organic fertilizers often results in mismatch between nutrient supply and crop demand, especially in transplant production in soilless substrate under controlled environments [6,19]. Our study was designed to address this challenge in organic bell pepper transplant production.

4.1. Fertilizer and Rhizosphere pH and EC

The recommended EC range of pepper transplants is 0.6–0.9 mS/cm with the PourThru method and 0.2–0.3 mS/cm with 1:2 extraction method (https://urbanagnews.com/blog/news/e-gro-nutritional-factsheet-pepper-transplants, retrieved on 28 March 2025). High EC is known to reduce shoot and root growth in short term and cause salt accumulation in crops after prolonged exposure, which in turn inhibits the cell metabolism process and causes organelle damage [30]. In most of these treatments, especially with higher N rates, the leachate and substrate EC (Figure 1B and Figure S1) deviated significantly from these recommended values. At 28 DAS, the highest root dry weight using conventional fertilizer, Drammatic, Pre-Empt and Bio-Matrix were at N rates of 0.72, 0.48, 1.73, and 0.70 g/L (Figure 9A), which corresponded to a leachate EC of 1.7, 2.2, 1.5, and 1.6 mS/cm, respectively (Figure 1B). This discrepancy suggested that the ‘optimal’ EC level for maximum growth may not be a static value but determined by a trade-off between nutrient supply and salinity stress. Using EC as guidelines to direct organic fertilizer application for organic transplant production is unlikely to be reliable. Similarly, a study on bell pepper grafted to salt-tolerant rootstock showed a decrease in shoot and root biomass only at an EC above 6 mS/cm [31]. Unfortunately, since stepwise regression only seeks the best linear relationship, it did not identify any factor that led to the decline in shoot dry weight at high N rates of fertilizers. High EC likely remains an important factor in limiting shoot and root growth at high N rates.

4.2. Nitrogen and Growth of Bell Pepper Transplants

4.2.1. Nitrogen Availability as the Main Driver of Transplant Growth

Nitrogen availability varied widely among the four fertilizers tested in this study (Table 2). The conventional fertilizer had only 36.0% N in readily available forms (nitrate and ammonium). However, this value was likely underestimated. Its label suggested that 57.5% of its total N is in urea form. Although urea cannot be measured by our spectrophotometry, it is highly available to crops as it quickly hydrolyzes to ammonium in substrate [32]. Our previous research showed 87.1% of N from the conventional fertilizer was successfully assimilated into shoot tissues of watermelon transplants after 21 days of growth [19], which further supports the high N availability of the conventional fertilizer. Drammatic had the second highest available N in fertilizer solution (55.8%), followed by Bio-Matrix (47.2%, Table 2). Our previous study also showed that an additional 11.1% of N in Drammatic was mineralized from organic forms and became available on top of the 55.8% already available N after 21 days [19]. In contrast, Pre-Empt had the lowest initial inorganic N, with only 23.0% of N was in a form immediately available to bell pepper transplants (Table 1). The difference in N availability directly affected transplant growth.

The stepwise regression analysis indicated that N availability, especially nitrate-N concentration in fertilizer solution, was the sole predictor of shoot dry weight (R² = 0.62). This single factor explained 62.2% of the variation in shoot dry weight across all treatments. The stepwise regression analysis for root growth also showed that root dry weight was affected by nitrate-N concentrations of leachate solutions only (R² = 0.18), also suggesting that N is important for root growth. Interestingly, for root growth, the only predictor was the nitrate-N concentration in the leachate, and not the nitrate-N concentration in the fertilizer solution. This may imply that root growth was limited more by the roots’ ability to acquire available N from the substrate rather than by N supply. Nevertheless, results from both stepwise regression analyses highlighted the importance of nitrogen availability in fertilizers, especially nitrate-N, to crop growth, aligned with findings from our previous study [19].

4.2.2. Limited N Supply: Competition Between Transplants and Substrate Microbes

The N recovery efficiency of bell pepper transplants was surprisingly low, especially for the two organic fertilizers Drammatic and Pre-Empt (Figure 8B). From the two organic fertilizers, the N recovery efficiency after 28 days (23.8% ± 3.0% and 15.6% ± 2.0% for Drammatic and Pre-Empt, respectively, Figure 8B) was even lower than the available N in the fertilizer solution (55.8% and 23.0%, respectively, Table 1). This indicated that bell pepper transplants were not capable of absorbing even the readily available N from organic fertilizers. Watermelon transplants, on the other hand, were able to assimilate 66.9% of N from 0.4 g N/L Drammatic even with a shorter growing period of 21 days [19]. A study on pepper seedlings grown in vermiculite in a greenhouse similarly showed that N recovery efficiency ranged between 5 and 28% [33]. This is consistent with our observation of the low N recovery efficiency of bell pepper. Mass balance analysis revealed that ≥51% of plant-available N from organic fertilizers was unaccounted for (Table S2), likely due to microbial activity in the substrate.

Possible processes through which N was consumed were as follows: (1) it was immobilized by substrate microorganisms and (2) lost as gaseous N forms through microbial activity. Organic fertilizers were shown to promote microbial activity compared to inorganic fertilizers [15]. Pre-Empt was derived from fermented molasses, which were rich in labile carbon. It likely promoted N immobilization during the decomposing of carbon and consumed N in substrate. A meta-analysis found that, in soil, when using crop-derived residues used as soil amendment, it resulted in significantly higher immobilization rates than manure and synthetic amendments [34]. Crop-derived residue was rich in carbon and promoted carbon cycling by soil microbes [34]. This process can be beneficial in soil since it retained N in soil, reduced N loss, and could supply N throughout the growing season. For transplant production in substrate, which has short production cycles, N immobilization is undesirable. Also, the previous literature has shown that high carbon content enhanced the overall denitrification process and resulted in higher N loss through gaseous forms [29,35]. The slow growth of bell pepper transplants fertilized by Pre-Empt at low N rates could be a result of both the low nitrogen availability of Pre-Empt and more intense microbial competition of N supply.

4.2.3. Factors Beyond the Nitrogen Availability Limit Shoot and Root Growth at High Nitrogen Rates

Shoots and roots of bell pepper transplants grew to a maximum size at optimal N rates (Figure 5C and Figure 9B). Nitrate-N accumulated more rapidly in substrate as N rate increased, except for Pre-Empt (Figure 2F). These results indicated that, above the optimal N rates, transplant growth was no longer limited by N supply but was limited by rootzone stresses (ammonium toxicity and salinity stress) or planting density (light interception and propagation cell size).

Ammonium toxicity induces leaf chlorosis, inhibits both shoot and root growth, inhibits nutrient uptake by roots and ultimately reduces biomass accumulation and yield, although the mechanism of toxicity is not completely understood [11,36]. Pepper plants are known to be sensitive to ammonium [37,38]. In our preliminary study, hot pepper (cv. Corno di Toro) transplants were stunted by an organic fertilizer Nature Safe 7-7-7 that supplied nitrogen only in ammonium form and contained no nitrate-N (Figure S5). This further highlights pepper’s ammonium sensitivity. In this study, Drammatic resulted in the highest ammonium concentration in substrate leachate (Figure 2B). Subsequentially, Drammatic achieved much lower maximum shoot and root dry weight than the other fertilizers (Figure 5C and Figure 9A, Table 2). The initial Bio-Matrix solution was also high in ammonium concentration (Table 1). But unlike Drammatic, Bio-Matrix did not produce a similarly high ammonium concentration in substrate leachate (Figure 2B), possibly due to its microbial inoculants. Similarly, despite the high urea content in the conventional fertilizer, the conventional fertilizer did not result in as high ammonium accumulation in the substrate as Drammatic (Figure 2B) and resulted in the best shoot growth among all fertilizers (Figure 4 and Figure 5). Salinity stress leads to osmotic stress in the short-term and disturbs the ion balance of crops in the long term [39]. Salinity stress damages the photosynthetic machinery and reduces overall shoot and root growth [39]. As discussed in the Fertilizer and rhizosphere pH and EC Section, substrate EC with Drammatic was quite high. Subsequently, shoot and root dry weight with Drammatic started to decrease at low N rates compared to other fertilizers and also reached lower maximum values (Figure 5 and Figure 9).

For root growth, the R² of the root model (R² = 0.1793) was much lower than the shoot model, while leachate nitrate was shown to be the only predictor of root growth. The lower sensitivity of root growth to fertilization than shoot growth was similarly observed in Arabidopsis (Arabidopsis thaliana) and watermelon [19,40,41,42]. Other than ammonium and salinity stress, additional factors likely limited root growth, including propagation cell size [43,44,45] and the presence of bioactive compounds in substrate (e.g., kelp extract in Drammatic, microbial inoculants in Bio-Matrix, and humid acid in Pre-Empt). The effects of these factors on root growth were, unfortunately, not captured by regression analysis.

The negative effect of salinity stress and the ammonium toxicity of fertilizers, especially Drammatic, on bell pepper transplant growth represents an important area for future research and optimization in organic agriculture production. Utilizing biostimulants can potentially overcome these limitations in pepper transplant production. Biostimulants can increase the salinity and drought resistance of crops, enhance nutrient acquisition, improve crop growth and, eventually, increase crop yield and quality [46,47,48,49]. Also, microbe-based biostimulants can promote the conversion of ammonium and nitrate and thus alleviate ammonium toxicity in organic fertilizers, as discussed earlier with Bio-Matrix. Applying a biostimulant along with Drammatic can potentially take advantage of the high N availability of Drammatic and alleviate the salinity and high ammonium stress associated with Drammatic. This is one of our future research directions.

4.3. Phosphorus and Potassium Were Less Limited than Nitrogen to Pepper Transplant Growth

Despite lower P and K rates compared to N rates (Table S1, comparing columns 5 and 6 to column 4), both P and K accumulated in the substrate (Figure 2E,G,H) at much higher concentrations than N (Figure 2D,F). This accumulation indicated that P and K were not the main limiting factors of transplant growth in this study. This conclusion was further confirmed by the stepwise regression which identified nitrate-N concentration as the only predictor of shoot and root growth, with P and K concentration unrelated to shoot or root growth. This finding is consistent with previous studies showing that shoot growth parameters, such as fresh and dry weight, leaf number, total leaf area, and the canopy height of watermelon transplants grown in containers were sensitive to N but not P [41].

The strong correlation between both substrate P and K concentrations and fertilizer P and K rates is surprising (Figure 3). These indicate that, unlike N, the content of P and K as listed on the fertilizer labels are good indicators of the effective P and K availability over the 28-day production cycle.

4.4. Optimizing Transplant Production with Organic Fertilizer

It is desirable for transplants to have a well-developed root, compact shoot growth, sufficient leaf area, and green color for high photosynthetic capability without excessive water loss after transplanting [18,50,51]. In our study, the maximum root growth occurred at lower N rates than maximum shoot growth for all fertilizers (comparing Figure 5C and Figure 9B). However, there was no optimal N rate for the highest root–shoot ratio, as it decreased for all fertilizers as the N rate increased within the range tested in this study (Figure 9C). Pre-Empt tended to result in a high root–shoot ratio, especially at higher N rates, than other fertilizers (Figure 9C), possibly due to its lower EC and ammonium accumulation compared to other fertilizers at the same N rates (Figure 1B). Unfortunately, the effect of EC and concentrations of phytotoxic compounds were not identified by stepwise regression, as discussed earlier. For the above ground tissues, higher SPAD and compactness are desirable for transplants. High SPAD is often associated with higher photosynthesis capabilities and high compactness implies less leggy plants and higher resistance to wind and other mechanical stress. In our study, different fertilizers achieved similar highest SPAD (Figure 7). Maximum shoot compactness was highest with the conventional fertilizer and Bio-Matrix, followed by Pre-Empt, while Drammatic had the lowest compactness (Figure 6). Further, a well-developed shoot provides the necessary photosynthetic capacity to support future growth and pepper fruit production. But an excessively high leaf area might cause water stress after transplants, which is the hallmark of transplant shock [52].

Our results suggested that shoot growth is a good indicator for bell pepper transplant production, since bell pepper plant growth after transplanting strongly correlated with transplant shoot dry weight (Figure 10). This finding aligns with a previous study that larger sweet pepper transplants resulted in higher photosynthetic capability after transplanting and ultimately, higher yield [28]. Also, the root and shoot growth are interconnected: a large root system efficiently provides water and nutrients that supports vigorous shoot growth, and large shoots result in high photosynthesis to supply carbon to support root growth [53,54]. In our study, shoot and root growth showed similar quadratic patterns in response to N rates (Figure 5C and Figure 9B). Furthermore, shoot growth is a parameter that is easy to evaluate for growers, compared to root dry weight, root–shoot ratio, shoot compactness, or SPAD. Currently, fertilization guidelines for organic transplant production remain limited. Our findings showed that shoot size can be used as a practical and reliable reference to guide organic fertilization strategies for bell pepper transplants for growers.

Towards the end of 28 days of the propagation cycle, bell pepper transplants were cultivated with a density of 36 plants per tray in 72-cell insert, which was equivalent to 279 plant/m², and about 55 mL substrate per plant. The recommendation of fertilization was developed based on these conditions. Higher planting density and smaller root volume may impose additional limitations on transplant growth. The N rates that led to maximum shoot dry weight were 0.93, 0.56, 2.11, and 0.82 g/L for the conventional fertilizer, Drammatic, Pre-Empt, and Bio-Matrix, respectively, which corresponds to fertilizer rates of 4.6, 28.0, 35.2, and 11.7 g/L, respectively. Rates above these optimal values led to reduced growth, likely due to salinity and/or ammonium stress. Therefore, to achieve approximately 90% of the maximum growth while leaving a safety margin against salinity stress, the recommended fertilizer rates are 3.1 g/L (the conventional fertilizer), 18.3 g/L (Drammatic), 23.1 g/L (Pre-Empt), and 7.7 g/L (Bio-Matrix). Growers can modify the rates that best fit their production window and desirable transplant sizes.

4.5. Limitations in Statistical Analyses

While this study identified nitrogen availability as the primary driver of bell pepper transplant growth, our regression analysis focused on univariate relationships to test the hypothesis that N availability was the primary driver of transplant growth. This approach was unable to identify other limiting factors such as salinity and ammonium toxicity. Multivariate approaches such as Partial Least Squares Discriminant Analysis (PLS-DA) may further elucidate how fertilizer properties other than N availability (e.g., nutrient ratios, salinity, bioactive compounds) collectively affect transplant performance. Additionally, repeated measures ANOVA or mixed-effects models may better capture temporal changes in transplant growth, particularly for parameters measured across multiple growth stages (canopy size, shoot fresh, and dry weight). We plan to integrate these methods in our future studies.

5. Conclusions

Nitrate-N availability was shown as the primary limiting factor for organic bell pepper transplant shoot (R² = 0.62) and root (R² = 0.18) growth. Optimal N rates were fertilizer-specific: 3.1 g/L for conventional fertilizer, 18.3 g/L for Drammatic, 23.1 g/L for Pre-Empt, and 7.7 g/L for Bio-Matrix. Higher N rates beyond these values resulted in high salinity and/or ammonium stress which reduced growth, especially with Drammatic. Contrary to our second hypothesis, bioactive compounds in fertilizers (e.g., microbial inoculants) showed no carryover effects in this study. Instead, bell pepper transplant shoot size strongly correlated with post-transplanting performance (R² = 0.87), suggesting that shoot size was a more reliable indicator of future performance. Overall, Pre-Empt (23.1 g/L substrate) emerged as the best organic fertilizer, with low risks of salinity and ammonium stress that allowed for a higher application rate to supply sufficient nutrients.