Stock plant growth conditions.

Stock plants of ‘Albion’, ‘Fronteras’, and ‘Monterey’ were grown in a glass-glazed greenhouse (Toledo Botanical Garden, Toledo, OH, USA) and replaced yearly with new plant material to maintain plant vigor and active stolon development. Daughter plants harvested from these stock plants were propagated and used as the starting material for the two research experiments. Stock plants were planted in 20.3-cm diameter pots filled with a peat-based substrate. Greenhouse conditions were maintained at 26/19 °C day/night air temperature and a 16-h photoperiod (0600 to 2200 HR). Light-emitting diode (LED) fixtures (Icarus Ti2; BIOS Lighting, Carlsbad, CA, USA) provided 200 µmol·m⁻²·s⁻¹ photosynthetic photon flux density (PPFD) when ambient light intensities were <300 µmol·m⁻²·s⁻¹ PPFD at canopy level. The plants were irrigated as needed. At every other irrigation, they were fertilized with a 15N–2.2P–12.5K water soluble fertilizer (Jack’s Professional 15-5-15 Ca-Mg; JR Peters, Inc., Allentown, PA, USA) at 100 mg·L⁻¹ N, supplemented with 306 mg·L⁻¹ magnesium sulfate heptahydrate (Magriculture; Giles Chemical, Waynesville, NC, USA). Substate solution pH and electrical conductivity (EC) were checked monthly (HANNA HI9814 GroPro; Hanna Instruments, Woonsocket, RI, USA). If EC drifted above 2.0 mS·cm⁻¹, the containers were leached to get rid of excess salts. If pH drifted below 5.5, 1.04 L·m⁻³ flowable lime (CalOx; BioSafe Systems, LLC, East Hartford, CT, USA) was applied to increase pH by approximately 0.5 units. Inflorescences were removed weekly, and stolons containing daughter plants were harvested as needed for research studies. About every 2 months, extra stolons containing at least one daughter plant were removed to maintain crown vigor but ensure a continuous supply of daughter plants that could be harvested and propagated for research studies.

Propagation of starting plant material.

Stolons of strawberry ‘Fronteras’ were harvested from stock plants on 27 May 2022 (Expt. 1) and from ‘Albion’, ‘Fronteras’, and ‘Monterey’ on 20 Dec 2022 (Expt. 2). The harvested stolons were stored in a cooler in the dark at 4 °C for 7 d before propagating. Daughter plants were cut from the stolon approximately 1 cm above and below the node and graded into size categories. Daughter plants with a crown diameter of 5 to 7 mm, visible root initials, and two to three unfolded leaves were selected; this crown diameter corresponded to the small to medium size categories described by Xu and Hernández (2020). Daughter plants were stuck in 50-cell count propagation trays (100-mL cell volume) filled with a commercial peat-based substrate (LM-111 All Purpose Mix; Lambert, Rivière-Ouelle, QC, Canada). Plants were propagated in a reach-in growth chamber (BDR16; Conviron, Winnipeg, MB, Canada) for 5 weeks (Expt. 1) or 4 weeks (Expt. 2). Environmental conditions were set to a constant 24 °C air temperature, a 16-h photoperiod, and a relative humidity (RH) of 80%. Humidity domes were placed over the trays for 7 d to maintain a high RH, and plants were hand misted twice daily. After 7 d, the humidity domes were removed, and the chamber RH setpoint was decreased to 70% for the remainder of the propagation interval. Initial PPFD was approximately 125 µmol·m⁻²·s⁻¹ for the first 2 weeks and then increased to approximately 225 µmol·m⁻²·s⁻¹ for the remaining 3 (Expt. 1) or 2 (Expt. 2) weeks of propagation. The plants were initially irrigated with tap water and then fertigated as needed with a diluted formulation of the 15N–2.2P–12.5K + magnesium sulfate fertilizer blend provided to the stock plants. The fertilizer was diluted to supply 30 mg·L⁻¹ N for the first 14 d, 50 mg·L⁻¹ N from days 15 to 28, and 75 mg·L⁻¹ N after 28 d. Stolons and inflorescences were removed as they became visible during the propagation phase. These rooted daughter plants were the starting plant material for the research studies.

Transplant and acclimation of research plants.

On 8 Jul 2022 (Expt. 1) and 26 Jan 2023 (Expt. 2), the rooted strawberry plants were transplanted into 20.3-cm diameter pots (2.90-L; mum pan; HC Companies, Middlefield, OH, USA), with one plant per pot. We refer to these rooted plants, after transplant, as the mother plants in our research studies. In Expt. 1, the pots were filled with a commercial peat: perlite substrate (85/15 v/v) (LM-111; Lambert). In Expt. 2, the commercial substrate (LM-111) was amended with additional coarse perlite (P.V.P. Industries, Inc., North Bloomfield, OH, USA) to increase porosity, and the final peat: perlite composition was 70/30 (v/v). The plants were grown in a glass-glazed greenhouse (Toledo Botanical Garden) for a 3-week acclimation period. The pots were irrigated as needed with 75 mg·L⁻¹ N from a 15N–2.2P–12.5K nutrient solution supplemented with magnesium sulfate. Visible stolons and inflorescences were removed twice weekly.

Greenhouse environmental conditions for Expts. 1 and 2.

Greenhouse setpoints were 26 °C day/23 °C night (25 °C average daily air temperature), ambient RH, and a 16-h photoperiod. LED fixtures (Icarus Ti2; BIOS Lighting) provided 200 µmol·m⁻²·s⁻¹ PPFD when ambient light intensities were <300 µmol·m⁻²·s⁻¹ PPFD at canopy level. Actual conditions were measured with an aspirated thermocouple, RH sensor, and quantum light sensor. The values were recorded every 10 min using a data logger (WatchDog 2475; Spectrum Technologies, Aurora, IL, USA), and mean values [± standard deviation (SD)] are reported in Table 1.

Table 1. Daily mean air temperature, relative humidity, and daily light integral of strawberry plants grown in the greenhouse (± standard deviation).

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Experimental design and treatments.

Six %${\text{NO}}_3^{-}$ treatments (0%, 20%, 50%, 60%, 80%, and 100% ${\text{NO}}_3^{-}$) were evaluated in both experiments. As mentioned previously, Expt. 1 evaluated only one strawberry cultivar (Fronteras), whereas Expt. 2 evaluated three strawberry cultivars (Albion, Fronteras, and Monterey) at each %${\text{NO}}_3^{-}$ treatment. Each pot (containing one plant) was an experimental unit. There were six pots per treatment combination, arranged in a randomized complete block design, for a total of 36 pots (Expt. 1) or 108 pots (Expt. 2). Treatments started on 4 Aug 2022 (Expt. 1) and 15 Feb 2023 (Expt. 2). The nutrient solution was a modified strawberry nutrient solution (Yamazaki 1982) comprised of 100 mg·L⁻¹ total N. The %${\text{NO}}_3^{-}$ treatments were 0%, 20%, 50%, 60%, 80%, or 100% ${\text{NO}}_3^{-}$, with the remaining N supplied by ammonium (${\text{NH}}_4^{+}$); this maintained the same total N across treatments (Table 2). The nutrient solutions were mixed using ultra-purified water (18 MΩ), then pH was checked (HANNA HI9814 GroPro; Hanna Instruments) and adjusted to a range of 5.6 to 5.9 ± 0.1 (Table 2) using 1 M potassium hydroxide (KOH). The target nutrient solution pH increased as the %${\text{NO}}_3^{-}$ supplied decreased (and %${\text{NH}}_4^{+}$ supplied increased) to help minimize pH drift in the pots (Table 2). A sample of each batch of nutrient solution was collected, stored at 4 °C until analysis, and analyzed using inductively coupled plasma-optical emission spectroscopy (ICP-OES) (iCAP 6300 Duo; Thermo Electron Corp., Waltham, MA, USA) (Supplemental Table 1).

Table 2. Modified strawberry nutrient solution (Yamazaki 1982), mixed to a final concentration of 100 mg·L⁻¹ N.

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Drip emitters (slimline EZ close plastic weights; Netafim, Fresno, CA, USA) delivered 77 ± 8 mL·min⁻¹ (± SD) to the pots every other day for the first 8 weeks of treatment and then daily for the second 8 weeks of the 16-week trial. The pots were irrigated for approximately 6 to 7 min to reach container capacity plus an approximately 10% leaching fraction. Beginning at 8 weeks of treatments, the pots were flushed with 18 MΩ water once every other week (approximately 20% leaching fraction). Additionally, flowable lime (CalOx; BioSafe Systems) was applied twice during Expt. 2 to increase the substate pH of the 0% to 60% ${\text{NO}}_3^{-}$ treatments. On 16 May 2023, flowable lime application rates were 3 mL per pot (0% and 20% ${\text{NO}}_3^{-}$ treatments), 1.5 mL per pot (50% ${\text{NO}}_3^{-}$), or 0.8 mL per pot (60% ${\text{NO}}_3^{-}$). On 25 May 2023, treatment applications were 3 mL per pot (0% and 20% ${\text{NO}}_3^{-}$) and 1.5 mL per pot (50% and 60% ${\text{NO}}_3^{-}$). The flowable lime was suspended in water, and a final volume of 250 mL was applied to each pot; nontreated plants received an equivalent volume of water. Plant canopies were sprayed weekly with 3.6 mM of calcium chloride (Sigma-Aldrich, St. Louis, MO, USA) (Expt. 2) to alleviate tip burn (Kroggel and Kubota 2017).

Data collection.

Leachates were collected every 4 weeks (Expt. 1) or at 8 and 16 weeks of treatment (Expt. 2) using the pour-through technique (LeBude and Bilderback 2009). Leachate pH and EC were measured (HANNA HI9814 GroPro; Hanna Instruments), and then the leachates were stored at 4 °C until they could be further analyzed using ICP-OES to quantify nutrient concentrations.

Inflorescences were counted and removed weekly. Stolon number, daughter plant number, and daughter plant morphology were assessed every 4 (Expt. 1) or 8 weeks (Expt. 2). Stolon number was counted at each interval. On nonfinal harvest intervals, stolons containing at least one daughter plant were harvested; stolons without a daughter plant remained on the plant until the next harvest interval. At the final harvest, all stolons (with and without daughter plants) were harvested. For Expt. 2 only, the presence of secondary branching was noted when stolons were harvested, as well as the number of daughter plants that formed on primary vs. secondary (or higher) branches. Daughter plant crown size (mm) was measured at the widest point using digital calipers (Fisher Traceable; Fisher Scientific, Pittsburgh, PA, USA) and later categorized using previously published size categories (Xu and Hernández, 2020), defined as extra small (XS; <3.5 mm), small (S; 3.5 to 6.0 mm), medium (M; 6.0 to 8.5 mm), large (L; 8.5 to 11.0 mm), and extra large (XL; >11.0 mm). Root number, daughter plant fresh mass, and stolon fresh mass (aggregated by pot) were also collected. Daughter plants and stolons, separately, were dipped into 0.1 M HCl-acidified water, rinsed in 18 MΩ water, placed in a paper bag, dried in a forced-air oven at 60 °C for at least 3 d, and weighed for dry mass.

At the end of the 16-week trial, mother plant morphology was also assessed. Canopy height was measured from the substrate surface to the top of the plant canopy. Canopy width (cm) was measured at the widest diameter of the canopy and perpendicular to the widest diameter and then divided by two. Total crown number was counted. Crown diameter was measured just above the substrate surface using digital calipers (Fisher Traceable; Fisher Scientific) at the widest point, perpendicular to the widest point, and divided by two. Fresh mass was weighed, mother plants were dipped into 0.1 M HCl-acidified water, rinsed in 18 MΩ water, placed in a paper bag, dried in a forced-air oven at 60 °C for at least 3 d, and weighed for dry mass. Total plant biomass was calculated as the mother plant plus the sum of the stolons and daughter plants at each harvest.

After drying, daughter plants (pooled by pot at each harvest interval) and mother plants (leaf tissue only) were finely ground for elemental analysis. To quantify %N, approximately 2.5 mg of dry tissue was measured into tin capsules (EA Consumables, Marlton, NJ, USA) and analyzed with a CN analyzer (vario MICRO cube; Elementar, Hanau, Germany). For other elements (except N), approximately 0.25 g of dry tissue was weighed, placed in a Teflon vessel, digested using a programmable microwave (MARS6; CEM Corp., Matthews, NC, USA), and analyzed with ICP-OES (iCAP 6300 Duo, Thermo Electron Corp.) using methods described by Boldt and Altland (2021).

Data analysis.

In Expt. 1, stolon and daughter plant number were analyzed at each harvest interval (4, 8, 12, and 16 weeks) and cumulatively. Each harvest interval was analyzed separately. Regression analysis was conducted using PROC REG (SAS version 9.4; SAS Institute, Cary, NC, USA) to evaluate linear or quadratic growth responses to %${\text{NO}}_3^{-}$.

The data collected in Expt. 2 were analyzed initially in SAS (version 9.4) as a two-way factorial (cultivar × %${\text{NO}}_3^{-}$) using PROC GLM to conduct an analysis of variance (ANOVA). When cultivar differences existed (P < 0.05), they were assessed using Tukey’s honestly significant difference post hoc test at α = 0.05. Next, regression analysis was conducted using PROC REG to evaluate linear or quadratic responses to %${\text{NO}}_3^{-}$. For variables with a significant ANOVA cultivar × %${\text{NO}}_3^{-}$ interaction effect, regression analysis was performed individually by cultivar. For variables without a significant cultivar × %${\text{NO}}_3^{-}$ interaction effect, the data for the three cultivars were pooled together for the regression analysis.

Stolon and daughter plant number.

In Expt. 1, %${\text{NO}}_3^{-}$, ranging from 0% to 100%, did not statistically affect ‘Fronteras’ stolon number or dry mass, daughter plant number, or daughter plant morphology (crown diameter, root number, or dry mass at any of the harvest intervals (P > 0.05 for all; Table 3). Cumulative stolon number averaged 22 ± 1 [mean ± standard error (SE)], and cumulative daughter plant number averaged 24 ± 2.

Table 3. Stolon, daughter plant, and mother plant attributes for strawberry (Fragaria ×ananassa ‘Fronteras’) grown in a soilless substrate for 16 weeks.

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Mother plant.

‘Fronteras’ mother plant growth (canopy height, canopy width, crown diameter, and crown dry mass) was unaffected by %${\text{NO}}_3^{-}$ (regression analysis P values were > 0.05), and therefore, the overall means are presented in Table 3. Mean canopy height was 20.9 ± 2.5 cm, canopy width was 39.6 ± 3.5 cm, crown diameter was 11.8 ± 1.8 mm, and crown dry mass was 14.5 ± 2.6 g.

Tissue %N.

Percent ${\text{NO}}_3^{-}$ did not affect foliar %N of daughter plants at any of the four harvests or in the mother plant (Table 3). Pooled daughter plant %N at the individual harvests ranged between 2.53% and 2.86% N. Mother plant foliar %N averaged 2.22% ± 0.06%.

Stolon number.

No significant linear or quadratic trends occurred in response to increasing %${\text{NO}}_3^{-}$ at 8 weeks (Fig. 1A). After 16 weeks of treatment, stolon number for ‘Albion’ and ‘Fronteras’ exhibited quadratic responses to increasing %${\text{NO}}_3^{-}$ (Fig. 1B). ‘Albion’ stolon number increased from 4 to 8 as %${\text{NO}}_3^{-}$ increased from 0% to 100% (P = 0.01; adjusted r² = 0.19). ‘Fronteras’ stolon number increased from 7 at 0% ${\text{NO}}_3^{-}$ to a calculated maximum of 11 at 56% ${\text{NO}}_3^{-}$ and then declined to 8 at 100% ${\text{NO}}_3^{-}$ (P = 0.03; adjusted r² = 0.15). ‘Monterey’ stolon number increased linearly as %${\text{NO}}_3^{-}$ increased, from 7 at 0% ${\text{NO}}_3^{-}$ to 16 at 100% ${\text{NO}}_3^{-}$ (P < 0.0001; r² = 0.49; Fig. 1B).

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Cumulative stolon number represents the sum of the two harvest dates (8 and 16 weeks of treatment). Like the 16-week harvest, stolon number for ‘Albion’ and ‘Fronteras’ exhibited quadratic responses to increasing %${\text{NO}}_3^{-}$, whereas ‘Monterey’ had a linear response to increasing %${\text{NO}}_3^{-}$ (Fig. 1C). ‘Albion’ stolon number decreased slightly from 0% to 20% ${\text{NO}}_3^{-}$ and then increased from 8 at 20% ${\text{NO}}_3^{-}$ to 13 at 100% ${\text{NO}}_3^{-}$ (P = 0.04; adjusted r² = 0.12). ‘Fronteras’ stolon number increased from 9 at 0% ${\text{NO}}_3^{-}$ to a calculated maximum of 14 at 64% ${\text{NO}}_3^{-}$ and then decreased to 13 at 100% ${\text{NO}}_3^{-}$ (P = 0.04; adjusted r² = 0.13). ‘Monterey’ stolon number increased from 14 at 0% ${\text{NO}}_3^{-}$ to 22 at 100% ${\text{NO}}_3^{-}$ (P = 0.0003; r² = 0.33).

In addition to stolon number, the percentage of stolons with secondary branching was assessed. No significant linear or quadratic regression responses occurred for ‘Albion’ or ‘Fronteras’. The percentage of stolons with only primary branching ranged between 20% and 39% in ‘Fronteras’ and between 49% and 74% in ‘Albion’ (Fig. 2). However, ‘Monterey’ had a quadratic response. The percentage of stolons with only primary branching increased from 35% of stolons at 0% ${\text{NO}}_3^{-}$ to a calculated maximum of 79% of stolons at 67% ${\text{NO}}_3^{-}$ and then decreased to 67% of stolons as %${\text{NO}}_3^{-}$ further increased to 100%.

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After 8 weeks of treatment, ‘Albion’ had the greatest stolon dry mass (2.94 ± 0.19 g), followed by ‘Monterey’ (2.42 ± 0.11 g), and ‘Fronteras’ had the least (1.63 ± 0.1 g) (P < 0.0001; Table 4). After 16 weeks of treatment, ‘Albion’ and ‘Monterey’ had similar stolon dry masses (2.40 ± 0.16 and 2.16 ± 0.14 g, respectively), and ‘Fronteras’ had a lower dry mass (0.87 ± 0.08 g) (P < 0.0001 for cultivar). Additionally, stolon dry mass at 16 weeks increased linearly from 1.26 to 2.11 g as %${\text{NO}}_3^{-}$ increased from 0% to 100% ${\text{NO}}_3^{-}$ (P = 0.001; r² = 0.10; data not shown).

Table 4. Cultivar differences for daughter plant and stolon morphology of strawberry (Fragaria ×ananassa) ‘Albion’, ‘Fronteras’, and ‘Monterey’ grown in a soilless substrate for 16 weeks.

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Daughter plant number.

After 8 weeks of treatment, ‘Monterey’ daughter plant number decreased linearly as %${\text{NO}}_3^{-}$ increased, but no significant linear or quadratic responses occurred for ‘Albion’ or ‘Fronteras’ in response to %${\text{NO}}_3^{-}$ (Fig. 1D). After 16 weeks of treatment, ‘Fronteras’ and ‘Monterey’ had quadratic responses as %${\text{NO}}_3^{-}$ increased, whereas ‘Albion’ did not exhibit a trend (Fig. 1E). ‘Fronteras’ daughter plant number increased to a calculated maximum of 28 at 61% ${\text{NO}}_3^{-}$ and then declined to 21 at 100% ${\text{NO}}_3^{-}$ (P = 0.03; adjusted r² = 0.15). ‘Monterey’ daughter plant number increased from 24 at 0% ${\text{NO}}_3^{-}$ to a calculated maximum of 69 at 85% ${\text{NO}}_3^{-}$ and then declined to 67 at 100% ${\text{NO}}_3^{-}$ (P < 0.0001; adjusted r² = 0.44). At the 16-week harvest, ‘Fronteras’ had a higher percentage of daughter plants borne on primary (vs. secondary) stolons (81% ± 2%) than ‘Albion’ (65% ± 3%) or ‘Monterey’ (55% ± 2%) (P < 0.0001 for cultivar; Table 4).

Cumulative daughter plant number exhibited responses similar to the 16-week data. ‘Albion’ daughter plant number increased linearly, from 27 at 0% ${\text{NO}}_3^{-}$ to 37 at 100% ${\text{NO}}_3^{-}$ (P = 0.04; r² = 0.11; Fig. 1F). ‘Fronteras’ and ‘Monterey’ exhibited quadratic responses to increasing %${\text{NO}}_3^{-}$. ‘Fronteras’ daughter plant number increased from 18 at 0% ${\text{NO}}_3^{-}$ to a calculated maximum of 34 at 66% ${\text{NO}}_3^{-}$ and then decreased to 30 at 100% ${\text{NO}}_3^{-}$ (P = 0.04; adjusted r² = 0.12). ‘Monterey’ daughter plant number increased from 49 at 0% ${\text{NO}}_3^{-}$ to a calculated maximum of 87 at 81% ${\text{NO}}_3^{-}$ and then decreased to 85 at 100% ${\text{NO}}_3^{-}$ (P < 0.0001; adjusted r² = 0.39).

Daughter plant morphology.

Daughter plant morphology (e.g., crown diameter, number of root initials, and dry mass) was primarily affected by cultivar (Table 4). After 8 weeks of treatment, ‘Fronteras’ had the largest daughter plant crown diameter (10.2 ± 0.3 mm), followed by ‘Albion’ (8.1 ± 0.2 mm), then ‘Monterey’ (7.1 ± 0.2 mm; P < 0.0001 for cultivar). After 16 weeks of treatment, ‘Fronteras’ and ‘Albion’ had larger but similar daughter plant crown diameters (7.3 ± 0.2 and 7.2 ± 0.1 mm, respectively), compared with ‘Monterey’ (6.5 ± 0.1 mm; P < 0.0001 for cultivar).

Based on the crown diameter size classifications defined by Xu and Hernández (2020), the percentage of daughter plants distributed into each size category was not affected by %${\text{NO}}_3^{-}$ (P > 0.05) at 8 or 16 weeks. When pooled across %${\text{NO}}_3^{-}$ treatments and harvest date, the size category with the greatest fraction of daughter plants was M for all cultivars (Fig. 3). ‘Monterey’ had a higher percentage of S- and M-sized daughter plants (71%) than ‘Albion’ (67%) and ‘Fronteras’ (49%). ‘Fronteras’ tended to have a higher amount of L and XL daughter plants (46%) compared with ‘Albion’ (31%) and ‘Monterey’ (23%).

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After 8 and 16 weeks of treatment, root number per daughter plant was affected by cultivar (P < 0.0001 for both; Table 4). ‘Fronteras’ had the highest root number per daughter plant (16 ± 1 at both harvests). ‘Monterey’ and ‘Albion’ had lower but similar root numbers per daughter plant at 8 weeks (10 ± 0 and 8 ± 0, respectively) and at 16 weeks (12 ± 1 and 11 ± 1, respectively). Daughter plant root number at 16 weeks decreased linearly as %${\text{NO}}_3^{-}$ increased from 18 at 0% ${\text{NO}}_3^{-}$ to 10 at 100% ${\text{NO}}_3^{-}$ (P < 0.0001; r² = 0.20; data not shown).

‘Fronteras’ daughter plants had a higher dry mass after 8 weeks of treatment (2.93 ± 0.23 g) than ‘Albion’ and ‘Monterey’ (1.18 ± 0.04 and 0.83 ± 0.02 g, respectively; P < 0.0001 for cultivar; Table 4). After 16 weeks, ‘Fronteras’ (1.28 ± 0.11 g) and ‘Albion’ (1.03 ± 0.08 g) daughter plants had higher dry masses than ‘Monterey’ (0.66 ± 0.02 g; P < 0.0001).

Mother plant morphology.

Inflorescence number, crown number, and crown diameter varied with cultivar but not %${\text{NO}}_3^{-}$ (P < 0.0001 for all; Table 5). Cumulative inflorescence number was highest in ‘Monterey’ (28 ± 2), followed by ‘Albion’ (8 ± 1) and then ‘Fronteras’ (0 ± 0). ‘Monterey’ produced the most branch crowns (7 ± 1), followed by ‘Albion’ (3 ± 0), and then ‘Fronteras’ (2 ± 0). ‘Monterey’ had the largest overall crown diameter (53.0 ± 6.5 mm), whereas ‘Albion’ and ‘Fronteras’ had smaller but similar crown diameters (39.0 ± 3.6 and 29.6 ± 2.4 mm, respectively).

Table 5. Cultivar differences for mother plant growth of strawberry (Fragaria ×ananassa) ‘Albion’, ‘Fronteras’, and ‘Monterey’ grown in a soilless substrate for 16 weeks.

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Canopy height increased linearly as %${\text{NO}}_3^{-}$ increased (P = 0.006; r² = 0.07; Fig. 4A). Canopy width increased linearly in ‘Monterey’ (P < 0.0001; r² = 0.41) and increased quadratically in ‘Albion’ (P = 0.02; adjusted r² = 0.13) as %${\text{NO}}_3^{-}$ increased from 0% to 100% (Fig. 4B); no significant linear or quadratic trends occurred in ‘Fronteras’ in response to %${\text{NO}}_3^{-}$. Mother plant dry mass increased linearly in ‘Albion’ from 21 to 36 g (P = 0.04; r² = 0.12) and in ‘Monterey’ from 37 to 98 g (P < 0.0001; r² = 0.45) as %${\text{NO}}_3^{-}$ increased from 0% to 100% (Fig. 4C); no significant linear or quadratic trends occurred in ‘Fronteras’ in response to %${\text{NO}}_3^{-}$.

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Total cumulative aboveground dry mass (stolons and daughter plants at weeks 8 and 16, plus mother plants at week 16) exhibited linear increases for ‘Albion’ and ‘Fronteras’ as %${\text{NO}}_3^{-}$ increased (Fig. 4D). ‘Albion’ increased from 63 to 99 g (P = 0.003; r² = 0.23), and ‘Fronteras’ increased from 62 to 92 g (P = 0.03; r² = 0.14) as %${\text{NO}}_3^{-}$ increased from 0% to 100%. ‘Monterey’ dry mass increased quadratically, from 96 g at 0% ${\text{NO}}_3^{-}$ to 210 g at 100% ${\text{NO}}_3^{-}$ (P < 0.0001; adjusted r² = 0.89).

Cultivar differences occurred for biomass allocation (P < 0.0001; Fig. 5). Within a cultivar, Albion and Fronteras allocated the greatest fraction of their biomass to daughter plants (43% and 61%, respectively), whereas ‘Monterey’ allocated the greatest fraction of its biomass to mother crowns (43%). Across cultivars, Fronteras had the highest percentage of dry mass allocated to daughter plants (61%), followed by ‘Albion’ (43%) and ‘Monterey’ (33%). ‘Monterey’ had the highest allocation to mother crowns (43%), followed by ‘Albion’ (29%) and ‘Fronteras’ (21%).

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Tissue nutrient composition for daughter and mother plants.

The primary focus of tissue nutrient analyses was on whether the N form supplied affected total %N in mother and daughter plants. After 8 weeks of treatment, %${\text{NO}}_3^{-}$ did not affect daughter plant foliar N (Fig. 6A), although cultivars differences did occur (P < 0.0001). ‘Albion’ had the highest %N (2.88% ± 0.03%). ‘Fronteras’ and ‘Monterey’ had lower but similar %N values (2.56% ± 0.06% and 2.64% ± 0.03%, respectively; Table 4). After 16 weeks of treatment, daughter plant %N had a quadratic response to increasing %${\text{NO}}_3^{-}$ (P = 0.004; adjusted r² = 0.07; Fig. 6B). The %N decreased to a calculated minimum at 41% ${\text{NO}}_3^{-}$ and then increased as %${\text{NO}}_3^{-}$ increased to 100%. ‘Fronteras’ had a higher %N (2.61% ± 0.05%) than ‘Albion’ and ‘Monterey’ (2.47% ± 0.04% and 2.48% ± 0.03%, respectively; P = 0.03 for cultivar; Table 4). In mother plants, foliar %N in all three cultivars exhibited quadratic responses to increasing %${\text{NO}}_3^{-}$ (Fig. 6C; adjusted r² values of 0.11 to 0.31). Foliar %N decreased to a calculated minimum at 30% ${\text{NO}}_3^{-}$ for ‘Fronteras’, 52% ${\text{NO}}_3^{-}$ for ‘Albion’, and 61% ${\text{NO}}_3^{-}$ for ‘Monterey’; then foliar %N increased as the %${\text{NO}}_3^{-}$ supplied increased to 100% ${\text{NO}}_3^{-}$.

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Daughter plant foliar macronutrient concentrations of P, K, Ca, Mg, and S were affected by %${\text{NO}}_3^{-}$ at one or both harvests (Supplemental Figs. 1 and 2). Despite these responses, all daughter plants appeared healthy, with no visual deficiency or toxicity symptoms. One response to highlight is that %Ca increased linearly as %${\text{NO}}_3^{-}$ increased in daughter plants at 16 weeks of treatment (r² values of 0.17 to 0.41) and in mother plants (r² = 0.23; Supplemental Fig. 2B and 2C). Also, %S decreased linearly as %${\text{NO}}_3^{-}$ increased in daughter plants after 8 weeks of treatment (r² = 0.33), and it generally decreased quadratically in daughter plants at 16 weeks of treatment (r² and adjusted r² values of 0.47 to 0.85) and in mother plants (adjusted r² values of 0.46 to 0.90; Supplemental Fig. 2H and 2I).

Foliar micronutrient concentrations of B, Cu, Fe, Mn, and Zn all decreased linearly or quadratically in response to increasing %${\text{NO}}_3^{-}$ in mother (Cu, Fe, Mn, and Zn) or daughter (B, Fe, Mn, and Zn) plants (Supplemental Figs. 3 and 4). Mo increased linearly or quadratically as %${\text{NO}}_3^{-}$ increased in mother and daughter plants (Supplemental Fig. 4D–F).

Substrate pH and EC.

Leachates collected at 8 and 16 weeks of treatment had quadratic increases in pH in response to increasing %${\text{NO}}_3^{-}$ (adjusted r² values of 0.86 to 0.97; Fig. 7A and 7B). At 8 weeks, substrate pH increased from 4.01 to 6.53 in ‘Albion’, from 4.29 to 6.21 in ‘Fronteras’ and from 3.81 to 6.80 in ‘Monterey’ as %${\text{NO}}_3^{-}$ increased from 0% to 100% (Fig. 7A). At 16 weeks, pH increased from 3.72 to 6.29 in ‘Albion’, from 3.65 to 6.13 in ‘Fronteras’, and from 3.77 to 6.25 in ‘Monterey as %${\text{NO}}_3^{-}$ increased from 0% to 100% (Fig. 7B). Conversely, EC at 8 and 16 weeks had quadratic responses and generally decreased as %${\text{NO}}_3^{-}$ increased (adjusted r² values of 0.50 to 0.88; Fig. 7C and 7D). At 8 weeks, EC peaked at calculated maximums of 5.31 mS·cm⁻¹ at 12% ${\text{NO}}_3^{-}$ in ‘Albion’, 5.66 mS·cm⁻¹ at 21% ${\text{NO}}_3^{-}$ in ‘Fronteras’, and 4.60 mS·cm⁻¹ at 29% ${\text{NO}}_3^{-}$ in ‘Monterey’ and then decreased to between 1.48 and 2.33 mS·cm⁻¹ at 100% ${\text{NO}}_3^{-}$ (Fig. 7C). At 16 weeks, substrate solution EC peaked at a calculated maximum of 41% ${\text{NO}}_3^{-}$ and then declined to 2.09 mS·cm⁻¹ at 100% ${\text{NO}}_3^{-}$ (Fig. 7D).

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