Fertilization treatments.

All fertilizers were custom blends of the following individual technical-grade salts (Fisher Scientific, Pittsburgh, PA): calcium nitrate [Ca(NO₃)₂⋅4H₂O]; potassium nitrate (KNO₃); monopotassium phosphate (KH₂PO₄); potassium sulfate (K₂SO₄); magnesium nitrate [Mg(NO₃)₂]; iron chelate (Fe-DTPA); manganese chloride (MnCl₂⋅4H₂O); zinc chloride (ZnCl₂⋅7H₂O); copper chloride (CuCl₂⋅2H₂O); boric acid (H₃BO₃); and sodium molybdate (Na₂MoO₄⋅2H₂O).

Fertilization treatments began on the day of transplant. Three micronutrient fertilizer rates of 1×, 2×, and 4× (referred to M_1X, M_2X, and M_4X, respectively) of a modified Hoagland’s solution concentration (Barnes et al. 2012) were mixed using the previously described fertilizer salts (Table 1). The macronutrient fertility rate was altered during weeks 5 through 8, during which nitrogen (N), potassium (K), and calcium (Ca) were increased in all of the examined micronutrient fertility treatments (Table 1). The fertility treatments were mixed in 100-L barrels and applied through drip irrigation as needed at every irrigation with an estimated 10% leaching fraction. The solution was delivered via pumps (model 1A; Little Giant Pump Co., Oklahoma City, OK, USA) connected to 1.9-cm-diameter irrigation tubing fitted with circular drip emitters (Dramm USA, Manitowoc, WI, USA).

Table 1. The applied nutrient fertilizer concentration × micronutrient fertility concentration treatments.

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Data collection.

Twenty single plant replicates were transplanted for each treatment (Si rate × micronutrient fertility rate). At weeks 1, 3, 6, 9, and 12, substrate pH and electrical conductivity (EC) were evaluated using the pour-through method for the same six replicates for each treatment (Cavins et al. 2004). Plants were initially irrigated with 250 mL of the fertilizer solution to reach container capacity 30 min before each data collection, and 75 mL of deionized water (DI) was poured over the pots to displace 50 mL of leachate. The leachate was analyzed to determine the pH and EC using a Hanna portable pH/EC meter (HI9813–6; Hanna Instruments, Smithfield, RI, USA).

At weeks 6 and 12, six plants were sampled to determine plant height and measured from the substrate level to the apical meristem [(widest diameter + perpendicular axis) ÷ 2]; the diameter was also measured. After 6 weeks of growth, six plants were destructively harvested, and the most recently matured leaves and a sample of the roots were collected to evaluate the micronutrient and macronutrient tissue concentrations of each treatment. The root samples were washed to remove the substrate before drying. The most recently matured leaves and root samples were initially rinsed with DI; then, they were washed in a solution of 0.5 M HCl for 1 min and rinsed again with DI water (Henry et al. 2018). The remaining shoot tissue was harvested separately and dried to calculate the total plant biomass. At week 12, six plants were destructively harvested, during which floral material was collected for the cannabinoids, heavy metal, and micronutrient analysis; the remaining aboveground portion of the plant was collected to determine biomass production.

Upon sampling, the plant tissues and remaining aboveground plant biomass were dried at 70 °C for 96 h, and the dry mass was weighed and recorded. After drying, leaf tissue was ground in a Foss Tecator Cyclotec™ 1093 sample mill (≤0.5-mm sieve; Analytical Instruments, LLC, Golden Valley, MN, USA). Then, the ground tissue was placed in vials containing ∼3 g of tissue and analyzed at the Waters Laboratory (Warsaw, NC, USA). Plant material (0.5 g) was first rinsed in nitric acid (10 mL of HNO₃ at 15.6 N) and digested in a microwave digestion system for 30 min (MARS 6 Microwaves; CEM Corp., Matthews, NC, USA). After microwave digestion, the solution was diluted with 50 mL of DI; then, it was vacuum-filtered through acid-washed paper (Laboratory Filtration Group, Houston, TX, USA). After dilution, the plant mineral tissue concentration was determined using an inductively coupled plasma-optical emission spectrometry (ICP-OES) machine (Spectro Arcos EOP; Mahwah, NJ, USA).

Floral chemical analysis.

During the flowering harvest (8 weeks into floral development), the main apical meristem and four terminal axillary buds were harvested, creating a composite floral sample; half of this sample (∼4 g) was used for the heavy metal analysis by Waters Laboratory. The metals were analyzed using Environmental Protection Agency methods 3050B for digestion and 6010S for analyses by the ICAP-OES. Plant material (0.5 g) was rinsed using 70:30 (volume:volume) nitric acid:hydrogen peroxide and digested using an Environmental Express HotBlock metals digestion system (Environmental Express, Charleston, SC, USA). After digestion, plant samples were analyzed using a Spectro Arcos ICP-OES (Spectro Arcos EOP).

The remainder of the composite floral sample was used for the cannabinoid analysis. The composite sample was freeze-dried (Harvest Right, North Salt Lake, UT, USA) for 30 h. Dry mass was weighed, recorded, and submitted for the cannabinoid and terpene analysis (Delta 9 Analytics, Raleigh, NC, USA). On arrival, samples were lyophilized and ground; a 2-g (1.98–2.02 g) sub-sample from the composite buds was obtained. The analysis of cannabinoids was accomplished through high-pressure liquid chromatography (SHIMADZU 8050 and 8040 Triple Quadrupole UHPLC/MS/MS analysis; Austin, TX, USA). Exact details regarding the cannabinoid analysis cannot be provided because Delta 9 Analytics uses a proprietary protocol.

The cannabinoid analysis included both the active (decarboxylated) and acid forms of CBG, THC, CBD, and cannabichromene. Additional cannabinoids and forms exist, but they are not reported here (e.g., cannabidivarin and tetrahydrocannabivarin) because their concentrations were too low to detect, were not evaluated, or were present in similar concentrations regardless of treatment. The total CBD and THC were calculated by the following equations:$${\mathrm{\Delta }}^{\text{9}}\text{THC }+[0.877\times \text{tetrahydrocannabinol\hspace{0.17em}acid\hspace{0.17em}}(\text{THCA})]=\text{ Total THC}$$ [1] $$\text{CBD}\hspace{0.17em}+\hspace{0.17em}\left[0.877\times \text{cannabidiol\hspace{0.17em}acid\hspace{0.17em}}\left(\text{CBDA}\right)\right]=\text{ Total CBD}$$ [2]

Statistical analysis. The statistical analysis was conducted using SAS (version 9.4; SAS Institute, Cary, NC, USA). Plant growth metrics and leaf and flower nutrient values were analyzed for differences within each data collection (n = 6) as a 3 × 3 factorial of micronutrient fertility rate × Si amendment rate with substrate PROC GLM. Means were separated with Tukey’s honest significant difference (P < 0.05). Deviations in plant metrics, total plant dry weights, and leaf tissue nutrient values were calculated based on the percentage of controls (M_1X × Si_0X).

Substrate pH and EC.

Regarding the interaction of micronutrient fertility × Si substrate amendment at sampling intervals of 6 and 12 weeks, differences were observed among the substrate pH of the various treatments in which a general trend of plants that received the Si_1X or Si_0.5X amendment exhibited a lower substrate pH than those that received the Si_0X amendment (P = 0.043 and P < 0.001, respectively) (Tables 2 and 3). When examining the simple effects of the Si rate, plants that did not receive a Si amendment exhibited significantly greater substrate pH than those that received a Si amendment of Si_0.5X or Si_1X (P < 0.001) at both sampling intervals (Tables 2 and 3). These differences can be attributed to the lower amount of dolomitic limestone added to the Si_0.5X or Si_1X treatments to account for the basic properties of Si (Table 1). However, the difference was less than 0.4 units at both sampling dates and likely had no impact on plant growth (Tables 2 and 3).

Table 2. Growth metrics of Cannabis sativa ‘Auto CBG’ grown in soilless substrate amended with Si_0X, Si_0.5X, or Si_1X and supplied with micronutrient concentrations (M_1X, M_2X, or M_4X) for 6 weeks from transplant.

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Table 3. Growth metrics of Cannabis sativa ‘Auto CBG’ grown in soilless substrate amended with Si_0X, Si_0.5X, or Si_1X and supplied with micronutrient concentrations (M_1X, M_2X, or M_4X) for 12 weeks from transplant.

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After 6 weeks of growth, there were no observed differences in substrate EC for the interaction of micronutrient fertility × Si substrate amendment or any of the examined simple effects (Table 2). However, at week 12, although there were no significant differences in the interaction, when examining the simple effect of the micronutrient fertility rate, plants that received a fertility rate of M_1X exhibited significantly greater EC than all other micronutrient fertility treatments (P = 0.002) (Table 3). Previous studies reported an increase in substrate EC for sunflowers grown using substrates that received Si from a 20% rice hull amendment; however, petunias grown under the same conditions did not exhibit significant differences in EC (Boldt et al. 2018). This variability in differences regarding Si resulting in increased EC values can likely be related to the uptake patterns of the crop and the fact that excess nutrients were not available in the container.

Plant growth metrics.

After 6 weeks of growth, plant height, diameter, and shoot dry weight were not significantly different when comparing the interaction of micronutrient fertility × Si substrate amendment (Table 2). However, plant dry weight was significant when examining the simple effect of the micronutrient fertility rate; plants that received a M_1X fertility rate had significantly less biomass when compared with those that received a fertility rate of M_2X or M_4X (P < 0.001) (Table 2). Additionally, no phytotoxicity was observed after 6 weeks of growth on any of the treatments, and similar growth occurred (Fig. 1).

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After 12 weeks of growth, plants were screened for phytotoxicity caused by micronutrient accumulation. Plants that received a Si_0X rate and a M_4X fertility treatment exhibited the most severe phytotoxicity (Fig. 2). Additionally, less severe phytotoxicity was observed on plants that received Si_0.5X and a M_4X fertility rate. Phytotoxicity was not observed on plants that were grown with a M_4X micronutrient fertility rate and received a Si_1X substrate amendment or any other treatment. Additionally, plant height, diameter, and shoot dry weight were not significantly different when comparing the interaction or simple effects of micronutrient fertility rate × Si substrate amendment after 12 weeks of growth (Table 3).

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Currently, limited published research has examined the use of calcium silicate as a substrate amendment; however, researchers have examined other forms of Si supplements in soilless substrates, with rice hulls being one of them. The results of this study vary compared with those reported by Boldt et al. (2018), who reported decreased sunflower and petunia total aboveground dry weight when amending peat:perlite substrates with 20% rice hull incorporation. However, this could likely be due to the change in the physical properties of the substrate and not as a result of Si.

Root tissue analysis.

After 6 weeks of growth, when examining the interaction of the micronutrient fertility rate × Si rate, significant differences were observed for B, Zn, Mn, Fe, and Cu, and there was a general trend of plants that received a silicon substrate amendment of Si_1X and M_4X fertility rate exhibiting the lowest elemental concentrations (Table 4). Plants that received a M_1X fertility rate and Si_0.5X or Si_1X rate exhibited 48% and 52% lower Fe concentrations, respectively, when compared with plants grown without Si (Table 4). When the highest micronutrient rate was applied (M_4X) with the Si_1X rate, the root tissue of the plant exhibited B, Zn, Fe, Mn, and Cu concentrations that were lower by 64.5%, 67.3%, 74%, 67.2%, and 77.4%, respectively, when compared with plants that received the Si_0.5X or Si_0X rate (Table 4). These trends show that under excessive micronutrient conditions, the addition of Si helps limit micronutrient accumulation in the roots.

Table 4. Root tissue nutrient concentrations of Cannabis sativa ‘Auto CBG’ grown in soilless substrate amended with Si_0X, Si_0.5X, or Si_1X and supplied with micronutrient concentrations (M_1X, M_2X, or M_4X) for 6 weeks from transplant.

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Foliar tissue analysis.

The B leaf tissue concentrations exhibited significant differences in the interaction of the micronutrient fertility rate × Si rate (P < 0.001) (Table 5). When examining the simple effects of the micronutrient fertility rates for B, Zn, Fe, and Cu, significant differences were observed (Table 5). The Si concentration in the foliar tissue was significant when examining the interaction of the micronutrient fertility rate × Si rate (P = 0.039). However, when examining the simple effect of the Si amendment rate, plants that received a Si concentration of 1× exhibited a significantly greater Si concentration compared with those that did not receive Si (Si_0X) (P < 0.001) (Table 5). It has been reported that Si alleviates Mn toxicity in cucumber (Cucumbis sativus L.) (Rogalla and Römheld 2002); however, during this study, we did not observe a decrease in foliar Mn concentrations with the increasing Si rate. It has been reported that Mn toxicity can occur with concentrations as low as 47.88 mg⋅kg⁻¹ in cannabis plants (Cockson et al. 2019); however, even plants that did not receive Si (Si_0X) and a micronutrient fertility rate of 1× exhibited an Mn foliar concentration of 193.80 mg⋅kg⁻¹ without visual Mn foliar toxicity symptoms.

Table 5. Leaf tissue micronutrient concentration of Cannabis sativa ‘Auto CBG’ grown in soilless substrate amended with (Si_0X, Si_0.5X, or Si_1X) and supplied with micronutrient concentrations (M_1X, M_2X, or M_4X) for 6 weeks from transplant.

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The interaction of Si soil amendments × Fe nutrition has been widely examined among many different species. The results suggested that Si amendments strongly regulate the transport of Fe and Fe availability in the soil and root apoplast, thus lowering the Fe concentration and distribution within the plant (Becker et al. 2020; Gonzalo et al. 2013). Additionally, the interaction of Si × Cu toxicity has been examined in wheat (Nowakowski and Nowakowska. 1997) and Arabidopsis (Khandekar and Leisner 2011). The alleviation of Cu toxicity by Si resulted from the increased binding sites in the cell wall; although there was no decrease in the Cu concentration in the shoot, the Si deposits formed Cu-binding sites that prevented the high Cu concentrations from negatively impacting the plant (Pavlovic et al. 2021). This could potentially explain why, in our study, there were no differences in Cu concentrations of the foliage after 6 weeks of growth for plants that were grown using a Si substrate amendment.

Floral tissue analysis.

After 12 weeks of growth, B, Zn, Mn, Fe, Cu, and Si floral tissue concentrations were significantly different when the interaction of the micronutrient fertility rate × Si rate was examined (Table 6). As a general trend, plants that received a greater micronutrient fertility rate exhibited a greater micronutrient concentration in the floral tissue, excluding that of Fe (Table 6). Inversely, plants that received an Si amendment generally exhibited a greater Si concentration compared with those that did not receive an Si amendment (Table 6). In most cases, Si concentrations in floral material yielded a lower Si concentration when compared with the foliar material; this trend is similar to that reported by Boldt et al. (2018), who found that sunflower leaves exhibited the greatest concentration, followed by roots, stems, and flowers. It has been reported that Si reduces the infection of gray mold (Botrytis cinerea) in lettuce, tomato, and pepper when it is supplied in a hydroponic nutrient solution (Pozo et al. 2015). Gray mold is one of the most important diseases in cannabis production because it results in the greatest losses in yield (McPartland et al. 2000). Thus, regarding floral material accumulating Si without disease pressure, further research is needed to determine if the increased Si concentration can prevent yield losses caused by botrytis.

Table 6. Floral tissue nutrient concentrations of Cannabis sativa ‘Auto CBG’ grown in soilless substrate amended with Si_0X, Si_0.5X, or Si_1X and supplied with micronutrient concentrations (M_1X, M_2X, or M_4X) for 12 weeks from transplant.

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After 12 weeks of growth, CBG, CBGA, total CBG, total THC, and total cannabinoid concentrations were similar across all examined Si rates or micronutrient fertility treatments (Table 7). Therefore, the inclusion of Si in the substrate offers the advantage of not detrimentally impacting cannabinoid concentrations in cannabis.

Table 7. Cannabinoid concentrations of Cannabis sativa ‘Auto CBG’ grown in soilless substrate amended with Si_0X, Si_0.5X, or Si_1X and supplied with micronutrient concentrations (M_1X, M_2X, or M_4X) for 12 weeks from transplant.

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