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 (Cu
2+) 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:
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).