Metal Micronutrient and Silicon Concentration Effects on Growth and Susceptibility to Pythium Root Rot for Hydroponic Lettuce (Lactuca sativa)

Abstract

The objectives were to evaluate the effects of increasing metal micronutrient concentrations and silicon (Si) concentrations on plant growth and susceptibility to Pythium root rot with hydroponically grown lettuce (Lactuca sativa). In the first experiment, lettuce was grown in hydroponic solutions with metal micronutrients iron (Fe), manganese (Mn), copper (Cu), and zinc (Zn) supplied at either 0, 2.5, 5, or 10 mg∙L⁻¹. A standard commercial hydroponic solution was also included as a control, with metal micronutrients supplied at 2 Fe, 1 Mn, 0.5 Cu, and 0.5 Zn mg∙L⁻¹. In the second experiment, hydroponic lettuce was grown with Si at 0, 7, 14, 28, and 56 mg∙L⁻¹. Hydroponic treatment solutions for both experiments were either dosed with Pythium myriotylum (Pythium treatment) at 1.80 × 10⁴ oospores per L or deionized water as a non-Pythium control. Data were collected on leaf SPAD chlorophyll content, shoot height and width, total plant fresh mass, and root disease severity. Increasing the Cu concentration in solution decreased Pythium disease severity but reduced lettuce growth and yield. Increasing the concentration of the other metal micronutrients also tended to reduce lettuce growth but had no significant influence on root disease. Supplementing the hydroponic solution with Si had no effect on Pythium root disease severity and slightly decreased lettuce growth at 56 mg∙L⁻¹ Si. Results of this study suggest that the management of micronutrients and Si nutrition is not an effective strategy and, at best, a risky strategy for controlling Pythium in hydroponic lettuce. Growers would likely benefit from maintaining metal micronutrient and Si concentrations within the ranges of (in mg∙L⁻¹) 0.5 to 5.5 for Fe, 0.1 to 2.0 for Mn, 0.1 to 0.6 for Cu, 0.1 to 0.6 for Zn, and 0 to 28 for Si for many hydroponic crops. Supplementing Si has the potential to negatively influence plant growth and quality for certain plant species, and testing is necessary to evaluate phytotoxicity risks prior to implementing in commercial practice. Overall, successful mitigation of root rot pathogens in commercial hydroponic production requires the combination of proper sanitation, best management and cultural practices, appropriate hydroponic system design, and the implementation of a water treatment system with proper design and a multi-barrier approach.

1. Introduction

Waterborne pathogens such as Pythium spp. are major causes of root rot disease and crop loss in hydroponic production [1,2,3]. Pythium spp. are oomycete pathogens producing highly resilient oospores as well as motile zoospores [2,3], which persist in and spread rapidly through recirculating solutions. Commercial growers aim to mitigate pathogen entry into hydroponic systems using various preventative measures and proper sanitation [4] and limit pathogen distribution using a range of multi-barrier water treatment approaches and technologies [5]. Although critical to reducing pathogen risk in commercial horticulture, prevention and water treatment strategies alone do not guarantee disease-free production, and periodic Pythium outbreaks remain a challenging aspect of hydroponic production.

Plant nutrition has a role in mitigating pathogen risks in hydroponics [1,6,7,8]. For example, certain macronutrients such as nitrogen, phosphorus, and calcium are known to influence disease susceptibility and can be managed in soilless culture systems to reduce disease risks [6,8]. Increasing the concentration of copper (Cu) has also been suggested to control Pythium in hydroponics [7]. Copper salts have been widely used for centuries as a fungicide [5], and free copper (Cu²⁺) in solution at 0.5 to 1.0 mg∙L⁻¹ has been shown to significantly reduce Pythium, Phytophthora, Xanthomonas, and other pathogens distributed in irrigation water [9]. Other metal micronutrients such as manganese (Mn) and zinc (Zn) may also have a role in preventing disease in hydroponic culture [8,10], but the influence of these nutrients is less understood.

Several past studies have reported good control of Pythium ultimum and Pythium aphanidermatum by supplementing the hydroponic solution with up to 1.7 mM Si (47.6 mg∙L⁻¹ Si) for cucumber (Cucumis sativa, [11,12,13]). Supplementing with silicon (Si) has also been shown to suppress foliar diseases such as powdery mildews and leaf spot in a range of vegetable crop species [7,14]. Root zone Si is known to alleviate the toxicity of metals (metal micronutrients and “heavy metals”) as well as various biotic and abiotic plant stresses [15]. Few studies, however, have investigated the effects of increasing metal micronutrient and Si concentrations on plant growth and the suppression of root rot pathogens such as Pythium in hydroponic leafy green crops.

The objectives of this study were to evaluate the effects of increasing metal micronutrient and Si concentrations in the supplied hydroponic solution on plant growth and susceptibility to Pythium root rot in lettuce (Lactuca sativa). Metal micronutrients and Si were supplied at concentrations exceeding those typically used in commercial practice (see Results and Discussion, Section 3.4) to evaluate whether increasing concentrations above standard levels would be an effective and relatively low-risk disease management strategy. Metal micronutrient concentrations were also standardized (at 0, 2.5, 5, and 10 mg∙L⁻¹, see Materials and Methods, Section 2.3) across micronutrients in this study to allow for straightforward treatment comparisons. Emphasis was placed on comparing the effects of increased metal micronutrient/Si concentrations on plant performance when grown in a standard hydroponic control solution used in commercial production. The authors hypothesized that increasing the concentrations of certain micronutrients, particularly Cu, may increase Pythium suppression but also result in decreased plant growth.

2. Materials and Methods

Two experiments were conducted to evaluate metal micronutrient and silicon (Si) concentration effects on plant growth and resistance to Pythium root rot disease in lettuce (Lactuca sativa L.). The metal micronutrients evaluated included iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu). Both experiments were conducted concurrently in a controlled-environment polycarbonate greenhouse at the University of Arkansas in Fayetteville, AR, USA (36.0687° N, 94.1748° W). Average daily temperature (ADT) and daily light integral (DLI) during the experiments were (mean ± standard deviation) 27.0 ± 4.7 °C and 12.2 ± 8.6 mol∙m⁻²∙d⁻¹ of photosynthetically active radiation, respectively, and were measured using portable weather station loggers (WatchDog 2475 Plant Growth Station; Spectrum Technologies, Aurora, IL, USA). Hydroponic solution temperature was 26.8 ± 3.0 °C and was measured using portable battery-powered HOBO loggers (Onset Computer Corporation, Bourne, MA, USA).

2.1. Pythium Culture and Inoculum Preparation

Pythium myriotylum “PM1” isolates, originally isolated from soilless substrates growing hemp [16], were shown to cause disease in hydroponically grown “Rex” lettuce [17]. Cultures were plated on potato dextrose agar (PDA) in a sterile Petri dish and sealed with parafilm. Plates were incubated at 25 °C and allowed to develop mycelium for 3 d. The mycelium-dense Petri dishes served as stock plates used to further propagate Pythium for experimentation.

Plugs (4 × 4 cm) of mycelium grown on the Pythium stock plates were transferred to sterile Petri dishes at six plugs per dish. Liquid 10% V8 (Campbell’s, Camden, NJ, USA) media solution was dispensed into Petri dishes at 10 mL per dish, after which the Petri dishes were sealed with parafilm and incubated for 14 d at 25 °C under light-emitting diode (LED) lights to allow mats of mycelial tissue to develop. Mycelial tissue mats were then triple rinsed with 10 mL deionized water to remove the remaining liquid V8 media and induce sporulation. Plates were re-sealed and incubated for another 7 d for spore production prior to the preparation of the inoculum used in experimentation.

Pythium spore solution was prepared on the same day it was used to inoculate the hydroponic culture vessels during experimentation. Sporulating mycelial mats of Pythium were transferred to 50 mL centrifuge tubes and macerated for 2–3 min using a vortex and 3 mm sterilized glass beads, forming a well-blended spore solution. Mycelia were filtered from the spore solution, and the solution was dispensed into a sterilized glass beaker and stirred. The oospore concentration was measured and adjusted to 9.0 × 10⁶ oospores/L (9.00 × 10⁶ oospores/mL) using a hemocytometer. Each hemocytometer measurement consisted of 20 10 μL samples of spore solution, where spores were counted in each sample using a 20× microscope lens and averaged across samples.

2.2. Plant Culture

Seeds of “Rex” lettuce (Johnny’s Selected Seeds, Fairfield, ME, USA) were sown into 2.5 cm diameter rockwool cubes at one seed per cube (AO plugs 200 counts, 2.5 cm height; Grodan, Roermond, The Netherlands), and irrigated with a commercial 13N-0.9P-10.8K (Jack’s Professional LX, JR Peters, Inc., Allentown, PA, USA) water-soluble fertilizer mixed at 150 ppm-N with tap water. The tap water had an electrical conductivity (EC) of <0.3 mS∙cm⁻¹ and <60 ppm bicarbonate alkalinity. Seeded rockwool cubes were transferred to the greenhouse and sub-irrigated as needed with the same fertilizer solution previously mentioned. At approximately 14 d after sowing, when plants displayed at least two true leaves, seedlings were transferred into hydroponic culture vessels.

Hydroponic culture vessels consisted of 20 L black plastic containers (45.5 × 34.0 × 17.5 cm). A 2.6 cm thick polystyrene foam board (Styrofoam Utilityfit R-10; Dow, Midland, MI, USA) cut to 37.5 × 26.0 cm was used as a raft on top of the nutrient solution for each culture vessel. Each raft was covered with white-black plastic (Black and White Panda Film; Vivosun, Ontario, CA, USA), with the plastic extending over and down the culture vessel sides, preventing light from directly entering the nutrient solution. Each culture vessel contained a submersible fountain pump (Low Water Shut-off 80-GPH Submersible Fountain Pump; Smartpond, Niedersachsen, Germany) used to continually circulate the hydroponic solution. A clear plastic tube fitted with an aquarium air stone was inserted between the raft and side of the culture vessel, underneath the white-black plastic, and provided continuous aeration of the hydroponic solution. Plastic containers, rafts, plastic films, pumps, and aeration tubes were washed with a phosphate-free detergent and rinsed with deionized water before use in this experiment.

Seedlings were transplanted into the hydroponic culture vessels at four plants per vessel, where the rockwool cubes of each seedling were inserted into 2.5 cm square holes cut in the floating rafts, allowing roots to grow down into the nutrient solution. Plant spacing consisted of a 2 × 2 configuration for each vessel, where each lettuce plant was 15.5 cm from the adjacent plant.

A modified Hoagland’s solution was used as a standard hydroponic solution and was adjusted to supply the specific micronutrient and Si concentrations needed for treatments in both experiments. Macronutrient concentrations in the standard hydroponic solution were supplied at (in mg∙L⁻¹): 210 nitrate nitrogen (NO₃-N), 32 phosphorus (P), 234 potassium (K), 200 calcium (Ca), 48 magnesium (Mg), and 71 sulfate sulfur (SO₄-S). Micronutrient concentrations were (in mg∙L⁻¹): 2 iron (Fe), 1 manganese (Mn), 0.5 boron (B), 0.5 copper (Cu), 0.5 zinc (Zn), and 0.1 molybdenum (Mo). Macronutrients were derived from reagent-grade calcium nitrate, potassium nitrate, potassium phosphate, potassium chloride, and magnesium sulfate. Micronutrients were derived from commercial grade Fe-EDDHA, Mn-EDTA, Zn-EDTA, Cu-EDTA, boric acid, and sodium molybdate (JR Peters, Inc., Allentown, PA, USA). Fertilizer salts were mixed in tap water dechlorinated with 2.5 mg·L⁻¹ of sodium thiosulfate. Individual micronutrients and silicon were either added to or omitted from this standard hydroponic solution formulation for the experiment treatments described below.

2.3. Experiment #1: Effects of Metal Micronutrient Concentration

An augmented (4 × 4 + 1) × 2 factorial experiment was conducted using a randomized split-plot design. The first factor consisted of the four metal micronutrients (Fe, Mn, Cu, and Zn), and the second factor consisted of micronutrient concentrations at 0.0, 2.5, 5.0, and 10.0 mg∙L⁻¹ in the hydroponic solution. The experimental design was augmented with the addition of a control treatment consisting of the standard hydroponic formulation described earlier with Fe, Mn, Cu, and Zn supplied at 2.0, 1.0, 0.5, and 0.5 mg∙L⁻¹, respectively, as indicated by the +1 in the factorial experiment design. The third factor consisted of Pythium dose, where hydroponic systems were either dosed with Pythium spore solution or de-ionized water (non-Pythium control).

Data were analyzed separately by metal micronutrient, and for each analysis, the Pythium dose treatment was the whole-plot factor and micronutrient concentration plus the augmented control solution was the split-plot factor. Each hydroponic culture system was considered one observational unit and treatment replicate. There was one replicate culture vessel per micronutrient concentration treatment and two replicates per standard hydroponic control solution treatment for each Pythium and non-Pythium plot. Replication was achieved by repeating the experiment twice for a total of three experimental runs starting on 27 April 2022, 22 June 2022, and 10 August 2022.

Each experimental run started with the transfer of lettuce seedlings into the hydroponic culture vessels. At this time, each culture vessel was filled to 20.00 ± 0.05 L of the respective treatment solution, with pH adjusted to 6.00 ± 0.05 and EC averaging 1.33 mS∙cm⁻¹ across treatment solutions. Each experimental run was conducted using two adjacent and identical benches within the same greenhouse, on which the hydroponic culture vessels were placed. Solution pH in each vessel was monitored every 2–3 d and maintained within a pH of 5.5 to 6.0 by titrating with H₂SO₄ and KOH at 1 N.

The Pythium spore solution (previously described) was dosed into the respective hydroponic solution treatments 5 d after seedlings were transferred to the hydroponic culture vessels. Prior to dosing, the prepared spore solution was covered, placed in the greenhouse, and allowed to equilibrate to ambient temperature for 1 h. Pythium spore solution was dosed into culture vessels to supply 9.0 × 10⁴ oospores per plant (1.80 × 10⁴ oospores per L of hydroponic solution). Equivalent volumes (200 mL) of deionized water were dosed into non-Pythium control vessels.

At the end of each experimental run, final data were collected 14 d after hydroponic vessels were inoculated with Pythium (approximately 33 d after sowing seed) and consisted of measuring leaf SPAD chlorophyll content, shoot height and width, shoot and root fresh and dry mass, and severity of Pythium root lesions for each treatment replicate.

Leaf SPAD chlorophyll content was measured using a Minolta SPAD-502 Plus chlorophyll meter (Konica Minolta, Tokyo, Japan), which measures the ratio of light transmitted through leaves at 650 and 940 nm wavelengths [18]. Each SPAD value per treatment replicate consisted of an average of 12 measurements taken on three randomly selected leaves for each of the four plants per culture vessel.

Shoot height was measured from the top of the floating raft to the highest leaf point for each lettuce plant and averaged across the four plants per treatment replicate. Shoot width measurements (in cm) consisted of averaging two perpendicular width measurements taken on each lettuce plant and averaging all plants per treatment replicate.

Root disease severity was measured for each treatment replicate using a modified mid-point method and an ordinal scale emphasizing root disease severities of ≤50% [19]. The percentage of roots showing brown discoloration, a symptom of Pythium root rot, was assessed by one rater for each plant per replicate using a visual six-point index and ordinal scale where values of 0, 1, 2, 3, 4, and 5 corresponded to no root damage (0% damaged), 1 to 10% damaged roots, 11 to 25% damaged roots, 26 to 50% damaged roots, 51 to 75% damaged roots, and 76 to 100% damaged roots, respectively. A percent disease severity score for each treatment replicate was determined using the following equation:

DS = [Σ(CF × R) ÷ (TP) × (R_M)] × 100

(1)

where DS is the disease severity score as a percentage of roots showing discoloration, R is the mid-point percentage value for the 0 through 5 ordinal scale values mentioned above, CF is the frequency at which each rating was assigned per replicate, TP is the total plant number per replicate (e.g., four plants), and R_M is the maximum rating score (e.g., 100% disease severity).

For each treatment replicate, root tips measuring 5 cm in length (approximately 3 mg fresh mass) were collected from each lettuce plant and placed in Petri dishes with fresh PDA media to re-isolate Pythium. Petri dishes were incubated in the laboratory at 25 °C and monitored daily for mycelial growth, with data collected on whether Pythium was re-isolated or not re-isolated from the root samples.

Total fresh and dry mass per plant was measured for each treatment replicate by destructively harvesting shoot and root tissues. Total fresh mass was determined by combining harvested shoots and roots, after which tissues were oven-dried at 60 °C for 72 h for total dry mass determination.

2.4. Experiment #2: Effects of Silicon Concentration

A 5 × 2 factorial experiment was conducted using a randomized split-plot design. The first factor consisted of Si concentrations of 0, 7, 14, 28, and 56 mg∙L⁻¹ in the hydroponic solution. The second factor consisted of hydroponic systems dosed either with Pythium or deionized water (non-Pythium control). The Pythium dose treatment was the whole-plot factor, and Si concentration was the split-plot factor. Silicon was added to hydroponic solutions as potassium silicate, which supplied 0, 25.4, 50.8, 101.5, and 203.0 mg∙L⁻¹ of additional K, respectively. Each hydroponic culture system with four lettuce plants was considered one experimental unit and treatment replicate. There was one replicate culture vessel per Si concentration for each Pythium and non-Pythium plot, except for the 0 mg∙L⁻¹ Si treatment (same as the standard hydroponic control solution in Experiment 1), for which there were two replicate vessels per plot. Treatment replication, dosing with Pythium, and data collection were identical to the methods described in the first experiment.

2.5. Statistical Analysis

Analysis of variance (ANOVA) with PROC GLIMMIX from SAS 9.4 (SAS Institute, Cary, NC, USA) was used to evaluate the fixed effects of metal micronutrient/Si concentration and Pythium dose and interaction effects on leaf SPAD chlorophyll content, shoot height and width, total plant fresh and dry mass, and root disease severity. Random effects included the replication or block. The selection of mixed-model statistics using PROC GLIMMIX was made partially because the factorial design in the first experiment was augmented with the addition of a standard hydroponic control solution, and because of the additional replication for the hydroponic control solutions in both studies. Means separation used Tukey’s honestly significant difference (HSD) at α = 0.05, except for root disease severity, where the percentage of diseased roots at each metal micronutrient/Si concentration was compared to values observed with the standard hydroponic control solution using Dunnett’s test (α = 0.05).

3. Results and Discussion

3.1. Metal Micronutrient Concentration Effects on Leaf SPAD, Canopy Dimensions and Plant Growth

Concentration and Pythium dose treatments had either main or interaction effects on leaf SPAD chlorophyll content for each metal micronutrient (

Table 1. Solution copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn) concentrations and Pythium dose effects on leaf SPAD chlorophyll content in hydroponic lettuce at the end of the experiment. Data were collected approximately 33 d after sowing seed and 14 d after hydroponic solutions were dosed with a Pythium or no-Pythium treatment.

Leaf SPAD Chlorophyll Content
	Cu		Fe		Mn		Zn
	No Pythium	Pythium	No Pythium	Pythium	No Pythium	Pythium	No Pythium	Pythium
0.0 mg·L−1	27.4 a	28.9 a	25.3 b	25.2 b	27.1 a	25.8 b	30.0 a	29.0 a
Control x	27.2 a	28.2 a	27.2 a	28.2 a	27.2 a	28.2 a	27.2 a	28.2 a
2.5 mg·L−1	27.0 a	27.6 a	26.6 a	29.2 a	26.8 a	27.2 a	26.5 a	26.2 a
5.0 mg·L−1	26.4 a	27.1 a	28.5 a	28.3 a	28.1 a	25.7 a	27.3 a	28.6 a
10.0 mg·L−1	28.2 a	28.0 a	27.6 a	28.1 a	28.2 a	27.0 a	27.2 a	28.3 a
No Pythium	27.2 a		27.0 a		27.5 a		27.6 a
Pythium	28.0 a		27.8 a		26.8 a		28.1 a
Concentration	0.0634		0.0281		0.0322		0.2266
Pythium	0.0472		<0.0001		0.0637		<0.0001
Conc. * Pythium	0.6034		0.2593		0.0024		0.1723

Data are least-square means of 6, 3, and 18 replicates for control solution effects with no Pythium and Pythium, concentration effects with no Pythium and Pythium, and Pythium treatment effects, respectively. Mean separation used Tukey’s honestly significant difference (HSD) at the α = 0.05 significance level. In some scenarios, there was a significant treatment effect (p-value less than 0.05) but no difference between individual treatments according to Tukey’s mean separation. This is an artifact from the use of two statistical analyses (ANOVA and Tukey’s HSD) differing in statistical power on experimental data where the treatment effects were minimal. ^x Control solution metal micronutrient concentrations were (in mg·L⁻¹) 2.0 for Fe, 1.0 for Mn, 0.5 for Cu, and 0.5 for Zn. “Conc. * Pythium” refers to the interactive effect of Pythium dose and micronutrient concentration.

); however, leaf SPAD values were relatively similar overall, and there were no symptoms of chlorosis or visual differences between treatments. Iron supplied at 0 mg∙L⁻¹ as well as 0 mg∙L⁻¹ Mn with Pythium resulted in a decreased leaf SPAD compared to the control solution (Table 1), and symptoms of leaf chlorosis and micronutrient deficiency may have progressed further if the experiment continued for a longer period.

Micronutrient concentration and Pythium dose treatments influenced lettuce canopy width as shown in

Table 2. Solution copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn) concentrations and Pythium dose effects on shoot canopy width in hydroponic lettuce at the end of the experiment. Data were collected approximately 33 d after sowing seed and 14 d after hydroponic solutions were dosed with a Pythium or no-Pythium treatment.

Canopy Width (cm)
	Cu		Fe		Mn		Zn
	No Pythium	Pythium	No Pythium	Pythium	No Pythium	Pythium	No Pythium	Pythium
0.0 mg·L−1	22.4 a	22.1 ab	21.7 a	19.8 b	22.3 a	21.4 a	18.2 b	18.3 b
Control x	22.5 a	22.2 ab	22.5 a	22.2 a	22.5 a	22.2 a	22.5 a	22.2 a
2.5 mg·L−1	22.7 a	22.7 ab	22.4 a	21.0 ab	22.7 a	20.5 a	20.8 a	24.3 a
5.0 mg·L−1	22.6 a	23.6 a	22.9 a	21.3 ab	23.3 a	21.6 a	22.0 a	21.3 b
10.0 mg·L−1	20.4 b	21.5 b	20.6 b	21.7 ab	22.0 a	21.2 a	21.7 a	21.7 b
No Pythium	22.1 a		22.0 a		22.6 a		21.0 a
Pythium	22.4 a		21.2 b		21.4 b		21.6 a
Concentration	0.0011		0.0275		0.2507		<0.0001
Pythium	0.8215		0.0113		0.0002		0.2614
Conc. * Pythium	0.3059		0.0275		0.2447		0.0562

Data are least-square means of 6, 3, and 18 replicates for control solution effects with no Pythium and Pythium, concentration effects with no Pythium and Pythium, and Pythium treatment effects, respectively. Mean separation used Tukey’s honestly significant difference (HSD) at the α = 0.05 significance level. ^x Control solution metal micronutrient concentrations were (in mg·L⁻¹) 2.0 for Fe, 1.0 for Mn, 0.5 for Cu, and 0.5 for Zn. “Conc. * Pythium” refers to the interactive effect of Pythium dose and micronutrient concentration.

, although, like trends observed for leaf SPAD, canopy width measurements were overall similar and differed only by a couple of centimeters between treatments. Increasing Cu to 10 mg∙L⁻¹ caused a reduction in canopy width regardless of Pythium treatment (Table 2). Iron supplied at 10 mg∙L⁻¹ without Pythium or 0 mg∙L⁻¹ with Pythium resulted in decreased canopy width compared to the control solution (Table 2). Zinc supplied at ≥5 mg∙L⁻¹ with Pythium or 0 mg∙L⁻¹ also resulted in decreased canopy width (Table 2). Similar treatment effects on canopy height were observed; however, the relative differences in height between treatments were even smaller compared to canopy width, and therefore data were not shown.

Hydroponic culture vessels dosed with Pythium resulted in a decreased total fresh mass per lettuce plant across metal micronutrient treatments (p < 0.0001,

Table 3. Solution copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn) concentrations and Pythium dose effects on total fresh mass per hydroponic lettuce plant at the end of the experiment. Data were collected approximately 33 d after sowing seed and 14 d after hydroponic solutions were dosed with a Pythium or no-Pythium treatment.

Total Fresh Mass per Plant (g)
	Cu		Fe		Mn		Zn
	No Pythium	Pythium	No Pythium	Pythium	No Pythium	Pythium	No Pythium	Pythium
0.0 mg·L−1	90.9 a	68.6 ab	66.6 b	52.3 b	86.6 a	79.5 a	59.8 b	54.4 b
Control x	87.7 a	78.2 a	87.7 a	78.2 a	87.7 a	78.2 a	87.7 a	78.2 a
2.5 mg·L−1	81.3 ab	72.0 ab	76.1 ab	66.8 ab	81.1 a	68.7 ab	82.2 a	82.1 a
5.0 mg·L−1	73 b	71.5 ab	89.1 a	67.6 ab	84.7 a	69.9 ab	88.7 a	68.0 ab
10.0 mg·L−1	72.1 b	66.1 b	76.6 ab	72.0 ab	73.2 b	57.2 b	88.7 a	61.9 b
No Pythium	81.0 a		79.2 a		82.7 a		81.4 a
Pythium	71.3 b		67.4 b		69.1 b		68.9 b
Concentration	<0.0001		<0.0001		<0.0001		<0.0001
Pythium	<0.0001		<0.0001		<0.0001		<0.0001
Conc. * Pythium	0.0325		0.1661		0.3544		0.0118

Data are least-square means of 6, 3, and 18 replicates for control solution effects with no Pythium and Pythium, concentration effects with no Pythium and Pythium, and Pythium treatment effects, respectively. Mean separation used Tukey’s honestly significant difference (HSD) at the α = 0.05 significance level. ^x Control solution metal micronutrient concentrations were (in mg·L⁻¹) 2.0 for Fe, 1.0 for Mn, 0.5 for Cu, and 0.5 for Zn. “Conc. * Pythium” refers to the interactive effect of Pythium dose and micronutrient concentration.

). Increasing metal micronutrients to concentrations above those supplied by the control solution also decreased total fresh mass per plant for Cu, Fe, and Mn regardless of Pythium treatment (Table 3). Increasing Zn concentration up to 10 mg∙L⁻¹ decreased the fresh mass for Pythium-treated lettuce, but not for lettuce without Pythium inoculation, suggesting plants were tolerant of high Zn but not when combined with the stress of root infection by Pythium. Supplying 0 mg∙L⁻¹ Fe and Zn resulted in decreased total fresh mass compared to the control solution regardless of Pythium treatment (Table 3), whereas supplying 0 mg∙L⁻¹ Mn had no effect on fresh mass per plant. Supplying 0 mg∙L⁻¹ Cu appeared to result in a slight but non-statistical decrease in plant fresh mass for Pythium-treated lettuce but had no apparent effect on fresh mass for lettuce without Pythium inoculation.

Micronutrient concentration and Pythium dose treatment effects on total dry mass per plant, shown in

Table 4. Solution copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn) concentrations and Pythium dose effects on total dry mass per hydroponic lettuce plant at the end of the experiment. Data were collected approximately 33 d after sowing seed and 14 d after hydroponic solutions were dosed with a Pythium or no-Pythium treatment.

Total Dry Mass per Plant (g)
	Cu		Fe		Mn		Zn
	No Pythium	Pythium	No Pythium	Pythium	No Pythium	Pythium	No Pythium	Pythium
0.0 mg·L−1	4.94 a	4.45 ab	4.14 b	3.87 b	4.63 a	4.62 a	3.88 b	3.80 b
Control x	4.76 a	4.70 a	4.76 a	4.70 a	4.76 a	4.70 a	4.76 a	4.70 a
2.5 mg·L−1	4.31 b	4.21 b	4.23 ab	4.42 ab	4.58 a	4.51 a	4.52 a	4.51 ab
5.0 mg·L−1	4.16 b	4.24 b	4.29 ab	4.39 ab	4.49 a	4.42 ab	4.68 a	4.31 ab
10.0 mg·L−1	4.28 b	4.23 b	4.35 ab	4.46 ab	4.46 a	4.03 b	4.90 a	4.26 ab
No Pythium	4.49 a		4.52 a		4.81 a		4.67 a
Pythium	4.37 b		4.31 b		4.34 b		4.32 b
Concentration	0.2277		0.1004		0.0014		0.0064
Pythium	0.0001		0.0020		0.0150		<0.0001
Conc. * Pythium	0.5163		0.0336		0.0382		0.0658

Data are least-square means of 6, 3, and 18 replicates for control solution effects with no Pythium and Pythium, concentration effects with no Pythium and Pythium, and Pythium treatment effects, respectively. Mean separation used Tukey’s honestly significant difference (HSD) at the α = 0.05 significance level. ^x Control solution metal micronutrient concentrations were (in mg·L⁻¹) 2.0 for Fe, 1.0 for Mn, 0.5 for Cu, and 0.5 for Zn. “Conc. * Pythium” refers to the interactive effect of Pythium dose and micronutrient concentration.

, followed trends like those observed for total fresh mass in Table 3. Overall, Pythium-treated lettuce had a lower dry mass per plant across metal micronutrient treatments compared to the no-Pythium treatments (Table 3). Compared to the control solution, increasing solution Cu to ≥2.5 mg∙L⁻¹ and supplying 0 mg∙L⁻¹ Fe and Zn resulted in decreased total dry mass regardless of Pythium treatment (Table 4).

3.2. Silicon Concentration Effects on Leaf SPAD, Canopy Dimensions and Plant Growth

Leaf SPAD chlorophyll content was not influenced by Si concentration or Pythium treatment (

Table 5. Solution silicon (Si) concentration and Pythium dose effects on leaf SPAD chlorophyll content, shoot canopy width, total fresh mass, and dry mass per plant for hydroponic lettuce at the end of the experiment. Data were collected approximately 33 d after sowing seed and 14 d after hydroponic solutions were dosed with a Pythium or no-Pythium treatment.

	Leaf SPAD Chlorophyll Content		Canopy Width (cm)		Total Fresh Mass per Plant (g)		Total Dry Mass per Plant (g)
	No Pythium	Pythium	No Pythium	Pythium	No Pythium	Pythium	No Pythium	Pythium
Control (0 mg·L−1)	27.2 a	28.2 a	22.5 a	22.2 a	87.7 a	78.2 a	4.77 a	4.70 a
7 mg·L−1	26.9 a	27.7 a	21.6 a	23.3 a	86.3 a	75.5 a	4.73 a	5.08 a
14 mg·L−1	26.2 a	28.6 a	22.2 a	21.7 a	82.1 b	70.6 ab	4.60 a	4.48 a
28 mg·L−1	27.1 a	27.4 a	22.3 a	22.9 a	82.3 b	67.5 b	4.81 a	4.30 a
56 mg·L−1	27.4 a	30.0 a	22.4 a	20.8 b	83.8 b	67.6 b	4.95 a	5.04 a
No Pythium	27.0 a		22.2 a		84.4 a		4.77 a
Pythium	28.4 a		22.2 a		71.9 b		4.72 a
Concentration	0.1015		0.0211		<0.0001		0.6872
Pythium	0.2561		0.2520		0.0396		0.1279
Conc. * Pythium	0.4094		0.3965		0.8232		0.3477

Data are least-square means of 12, 6, and 18 replicates for the control solution and silicon concentration (0, 7, 14, 28, and 56 mg·L⁻¹) main effects and Pythium treatment main effects, respectively. Mean separation used Tukey’s honestly significant difference (HSD) at the α = 0.05 significance level. “Conc. * Pythium” refers to the interactive effect of Pythium dose and micronutrient concentration.

). Shoot canopy width was slightly reduced at 56 mg∙L⁻¹ compared to the control solution, particularly with Pythium-treated lettuce, but overall was not influenced by Pythium treatment or the interaction (Table 5). Similar to the results shown in Table 3 for metal micronutrients, Pythium-treated lettuce resulted in a lower total fresh mass per plant compared to lettuce without Pythium inoculation (Table 5). Increasing Si concentration to >14 mg∙L⁻¹ also decreased plant fresh mass. Silicon concentration and Pythium treatment had no effect on total dry mass per plant (Table 5).

3.3. Metal Micronutrient and Silicon Concentration Effects on Percent Root Disease

Across all experimental runs for both experiments, Pythium was re-isolated from 94.9% of lettuce plants grown in solution inoculated with Pythium. Pythium was not re-isolated from the roots of lettuce plants grown in solution without Pythium inoculation (no-Pythium control solutions), indicating Pythium was either not present or was below the detection and re-isolation limits for these samples.

Overall, lettuce plants without the Pythium dose treatment appeared visually healthy, with green foliage color and a primarily white root system. In contrast, most lettuce plants dosed with Pythium developed visual symptoms associated with Pythium root infection, including reduced shoot growth, brown discolored roots, root lesions, sloughing of root cortexes, and root tissue necrosis [20,21]. The combination of these visual symptoms and the high pathogen re-isolation rate from inoculated lettuce roots suggest the growth of Pythium-treated plants was negatively affected by Pythium root infection.

For lettuce grown without Pythium inoculation, the percentages of individual plant root systems showing symptoms of brown discoloration (likely symptoms of Pythium lesions and root rot) were relatively low and ≤6% across the replicates for the different metal micronutrient and Si concentration treatments (data not shown). Therefore, root disease percentage data analyzed and presented in

Table 6. Solution copper (Cu), iron (Fe), manganese (Mn), zinc (Zn), and silicon (Si) concentrations effects on percent disease severity of roots for hydroponic lettuce plants grown in solution dosed with Pythium. Data were collected approximately 33 d after sowing seed and 14 d after hydroponic solutions were dosed with a Pythium treatment.

	Percent Disease Severity
Concentration	Cu	Fe	Mn	Zn
0.0 mg·L−1	36.5%	33.8%	13.1%	16.3%
Control x	24.2%	24.2%	24.2%	24.2%
2.5 mg·L−1	12.6%	27.7%	29.0%	10.1%
5.0 mg·L−1	12.5%	24.2%	27.4%	29.2%
10.0 mg·L−1	6.3%	23.4%	18.8%	34.4%
p-value	0.0321	0.8702	0.3877	0.1804
±Std. Error	7.1%	11.8%	7.2%	7.8%
Concentration	Si
Control (0 mg·L−1)	24.2%
7 mg·L−1	25.4%
14 mg·L−1	28.0%
28 mg·L−1	26.4%
56 mg·L−1	20.2%
p-value	0.9647
±Std. Error	11.8%

Data are least-square means of 6 and 3 replicates for the control solution and micronutrient/Si treatments, respectively. Mean separation used Dunnett’s T at the α = 0.05 significance level, where micronutrient/Si concentration treatments were each compared to the control solution. The value that is bolded, italicized, and underlined indicates the treatment effect is statistically different from the control solution at α = 0.05. ^x Control solution metal micronutrient concentrations were (in mg·L⁻¹) 2.0 for Fe, 1.0 for Mn, 0.5 for Cu, and 0.5 for Zn.

only included treatments where Pythium was dosed into the hydroponic solution. Lettuce grown in the hydroponic control solution resulted in a root disease percentage of 24.2% (Table 6). Compared to the control solution, decreasing solution Cu concentration (0 mg∙L⁻¹ Cu) increased the root disease percentage, whereas increasing Cu resulted in a decreased root disease percentage (Table 6), which was only statistically lower at 10 mg∙L⁻¹ Cu (Table 6). The remaining metal micronutrients and Si treatments had no statistical influence on root disease percentage compared to the control solution.

3.4. General Discussion of Major Results

Overall, the results from this study indicate that increasing the Cu concentration in the hydroponic solution to ≥2.5 mg∙L⁻¹ has the potential to reduce root disease caused by Pythium (Table 6), but with the consequence of reducing plant growth and yield (Table 3 and Table 4). Increasing the concentrations of the remaining metal micronutrients (Fe, Mn, and Zn) did not influence the severity of root disease but tended to also reduce plant growth (Table 3, Table 4 and Table 6). As shown with Zn in Table 3, excessively high metal micronutrient concentrations at ≥5 mg∙L⁻¹ may cause additional root stress and increase plant susceptibility to root disease.

Reduced lettuce growth at Cu concentrations of ≥2.5 mg∙L⁻¹ was likely a result of Cu toxicity, which is supported by reports from Langenfeld et al. [7] suggesting phytotoxicity occurs with hydroponic lettuce at >1.3 mg∙L⁻¹ Cu. Raudales et al. [5] reported that Cu supplied up to 5.0 mg∙L⁻¹ in the fertilizer solution may be tolerated by certain crops grown in substrate; however, this is highly dependent on plant species and culture system, and thresholds for Cu toxicity are lower for plants grown in hydroponic solutions versus organic substrate or soil [8,22]. Increasing supplied Cu can also reduce plant growth by inducing a deficiency for one of the other metal micronutrients, particularly Fe [23], since metal micronutrients exist as divalent cations in solution and compete for root uptake [15]. In general, the antagonistic or synergistic effects that changing one individual nutrient concentration has on plant uptake of other nutrients is a common challenge when designing experiments for plant nutrient research and interpreting results.

Withholding plant essential micronutrients from the hydroponic solution may be expected to reduce plant growth and increase susceptibility to Pythium; however, these results did not occur for lettuce grown with 0 mg∙L⁻¹ Cu and Mn (Table 3 and Table 4). Trace amounts of metal micronutrients are known to leach into solution from the various plastics and metal equipment used in hydroponic systems [4,7,10], sometimes enough to satisfy plant demand. It was beyond the scope of this study to track nutrient levels in solution and plant tissues over time. However, aside from the differences in plant fresh and dry mass between treatments (Table 3 and Table 4), no visual symptoms of nutrient toxicity or deficiency were observed.

Supplementing the hydroponic solution with Si had no influence on lettuce susceptibility to Pythium but did reduce growth at the highest Si concentration of 56 mg∙L⁻¹ compared to the control. The cause of reduced growth is unclear; to the authors’ knowledge, there has not yet been a published scenario of Si toxicity in horticultural or agronomic crop species, and Si toxicity seems unlikely in hydroponic solutions because of the relatively low solubility and availability of Si for plant uptake compared to typical field soil conditions.

With container-grown floriculture and edible crops, weekly potassium silicate (KSiO₃) drench applications have resulted in changes in plant morphology and growth for certain species [24,25,26], sometimes with detrimental effects on plant quality. However, in these experiments, the use of KSiO₃ also increased root zone K, and in this study, the 56 mg∙L⁻¹ Si supplied from KSiO₃ nearly doubled the solution K to 437 mg∙L⁻¹ K compared to the 234 mg∙L⁻¹ K supplied in the control solution with no Si (see Section 2). Marschner [15] suggests an abundant supply and “luxury consumption” of K may interfere with the uptake and physiological availability of other nutrient cations such as Ca and Mg and therefore result in K-induced deficiencies. Although the results are inconclusive, it is possible the additional K, either in combination with or separate from Si, influenced nutrient uptake and reduced lettuce growth at the highest Si concentrations in this study.

Past research investigating the role of Si in plant biology has led to the classification of plant species into two main categories: Si “accumulator” and “non-accumulator” (sometimes called Si “excluder”) species, where Si concentrations in dried shoot tissues tend to be greater than 1000 mg∙kg⁻¹ for Si “accumulators” and less than 1000 mg∙kg⁻¹ for Si “non-accumulators” [27]. Voogt and Sonneveld [14] found providing supplemental Si increased yields and resistance to powdery mildew for the Si “accumulators” cucumber (Cucumis sativus), rose (Rosa sp.), and courgette (Solanum melongena). It is a generally accepted hypothesis that Si “accumulators” benefit the most from supplemental Si fertilizer applications in soilless or hydroponic production [14,25,28,29].

Lettuce has been classified as a Si “non-accumulator” by Voogt and Sonneveld [14], which may help explain why supplemental Si had little effect on growth and no influence on lettuce resistance to Pythium infection in this study. Heine et al. [28] found supplemental Si limited the infection and spread of Pythium aphanidermatum in the roots of the Si “accumulator” bitter gourd (Mormodica charantia) but not in the Si “non-accumulator” tomato (Solanum lycopersicum), even though tomato had the greater total Si content in root tissues. These authors attributed the greater resistance of bitter gourd to Pythium root rot to a higher proportion of Si taken up into the root symplast as opposed to the apoplast. Past research showing the positive effects of root zone Si on root resistance to Pythium infection has mostly been with plant species of the family Cucurbitaceae and other known Si “accumulators” [11,12,13]. Overall, the underlying physiological and molecular factors contributing to Si-mediated resistance in certain plant species are still not well understood [15,30] and deserve further investigation.

Several studies have begun to cast doubt on the view that Si must be accumulated in large amounts to provide a plant benefit [22], and supplementing the hydroponic solution with Si may still be advantageous for lettuce and other Si “non-accumulator” crops. For example, Voogt and Sonneveld [14] found that supplying 1.5 mmol∙L⁻¹ Si (42 mg∙L⁻¹ Si) alleviated Mn toxicity in hydroponic lettuce. Frantz et al. [22] showed Si helped alleviate Cu toxicity in the Si “non-accumulator” snapdragon (Antirrhinum majus) grown hydroponically. Other examples of supplemental Si benefits for Si “non-accumulator” species include documented reductions in viral symptoms in tobacco (Nicotiana tabacum) [31], decreased sensitivity to salinity and blossom-end rot with tomato [32], a longer shelf life and faster recovery from wilt with poinsettia (Euphorbia pulcherrima, N.S. Mattson, unpublished data), and increased resistance to Ralstonia solanacearum with tomato [33].

Increasing metal micronutrient and Si concentrations in the hydroponic solution has been suggested as a strategy to promote lettuce growth [7], and concentrations in this study were increased above those generally recommended to commercial growers to evaluate potential benefits. A review of >100 fertilizer solution formulations for various crops grown hydroponically and in soilless culture by several authors indicated recommended concentrations ranging from (in mg∙L⁻¹) 0.5 to 5.5 for Fe, 0.1 to 2.0 for Mn, 0.1 to 0.6 for Cu, 0.1 to 0.6 for Zn, and from 0 to 28 for Si [4,34,35]. Based on the results of this study, growers would likely benefit the most and avoid losses in yield by maintaining the supplied metal micronutrient and Si concentrations within these ranges. Several researchers have recommended adding Si to the hydroponic solution for all plants [7,10,36]; however, small-scale testing of supplemental Si applications is necessary to evaluate the potential for phytotoxicity prior to implementing in commercial practice [26]. Overall, managing micronutrients and Si nutrition seems to be a relatively ineffective and, at best, risky strategy for mitigating losses caused by Pythium in hydroponics.

4. Conclusions

Increasing the concentration of Cu in the hydroponic solution decreased the severity of root disease from Pythium, but at the cost of reduced lettuce growth and yield. Increasing the concentration of other metal micronutrients also tended to reduce lettuce growth, with no significant influence on Pythium root disease severity. Supplementing the hydroponic solution with Si had no effect on Pythium root disease severity and slightly decreased lettuce growth at the highest concentration of 56 mg∙L⁻¹. Lettuce is characterized as a Si “non-accumulator” species and does not take up large amounts of Si, which may be a reason why supplemental Si applications had no effect on Pythium resistance. Lettuce and other Si “non-accumulator” species may still benefit from Si added to the hydroponic solution in terms of alleviating metal micronutrient toxicity risks.

Results of this study suggest that managing micronutrient and Si nutrition is likely an ineffective strategy for preventing crop losses caused by Pythium in hydroponics. This study also emphasized the relatively low and narrow concentration ranges for which micronutrients and Si must be maintained in the root zone, where small increases above these ranges can potentially result in significant decreases in yield. Recommended concentrations range from (in mg∙L⁻¹) 0.5 to 5.5 for Fe, 0.1 to 2.0 for Mn, 0.1 to 0.6 for Cu, 0.1 to 0.6 for Zn, and 0 to 28 for Si. Supplementing the hydroponic solution with Si has the potential to negatively affect plant growth and quality for certain plant species, and small-scale testing is necessary to evaluate phytotoxicity risks prior to implementing in commercial practice. Overall, successful mitigation of root rot pathogens in commercial hydroponic production requires the combination of proper sanitation, best management and cultural practices, appropriate hydroponic system design, and the implementation of a water treatment system with proper design and a multi-barrier approach.