Effects of substrate type on plant growth and nitrogen and nitrate concentration in spinach

The effects of three commercial substrates (a mixture of forest residues, composted grape husks, and white peat, black peat and coir) on plant growth and nitrogen (N) and nitrate (NO3) concentration and content were evaluated in spinach (Spinacia oleracea L. cv. Tapir). Spinach seedlings were transplanted at 45 days after emergence into Styrofoam boxes filled with the substrates and were grown during winter and early spring in an unheated greenhouse with no supplemental lighting. Each planting box was irrigated daily by drip and fertilized with a complete nutrient solution. The NO3 content of the drainage water was lower in coir than in the other substrates. However, shoot NO3 concentration was not affected by substrate type, while yield and total shoot N and NO3 content were greater when plants were grown in peat than in the mixed substrate or the coir. Leaf chlorophyll meter readings provided a good indication of the amount of N in the plants and increased linearly with total shoot N.


Introduction
The use of substrates and soilless culture systems for production of horticultural crops is increasing worldwide.2][3][4] Despite these many benefits, there is currently very little information available concerning the influence of substrate type on plant growth and nutrient uptake in many crops, including leafy vegetables.Physical and chemical properties such as bulk density, water holding capacity, pH, cation exchange capacity, and nutrient content vary considerably among substrates and, therefore, likely have a considerable influence on plant development and nutrition.Tissue nitrate (NO 3 ) concentrations tend to be higher when plants are grown in soilless culture systems, 5,6 and leafy vegetables such as spinach can accumulate levels that may be harmful to human health. 7,80][11][12][13] For example, a reduction of light from 800 to 200 mol×m -2 ×s -1 increased total shoot NO 3 concentration in spinach by more than 200%. 14][17] The objective of this study was to evaluate the influence of different substrate types on plant growth and shoot nitrogen (N) and NO 3 concentrations of spinach grown in an unheated greenhouse during the winter and early spring.

Growth conditions and substrates
The experiment was conducted in a greenhouse located at the Herdade Experimental da Mitra (38º31 52 N; 8º01 05 W), University of Évora, Portugal.The greenhouse was covered with thermal polyethylene and had no supplemental lighting.Air temperatures inside of the greenhouse ranged from 5 to 26ºC, and solar radiation ranged from 34 to 248 W•m -2 •d -1 .The experiment comprised three different commercial substrates: a mixture of forest residues, composted grape husks, and white peat (Substrato Universal Agriloja); a black peat blend (Super Terra Torfkultursubstrat 1; Hawita Flor, Germany); and a coir blend (Pelemix España S.L., Spain).Physical and chemical characteristics of the substrates, according manufacturer, are shown in Table 1.Mass wetness, moisture content, and bulk density were determined following the methods described by Fonteno and Harden (Table 2). 18pinach (Spinacia oleracea L. cv.Tapir) seedlings were transplanted at 45 days after emergence into to Styrofoam planting boxes (100-cm long × 25-cm wide × 10-cm high) filled with 16 L of substrate.The seedlings were spaced 8-cm apart in three rows per box and 10-cm apart between rows.Treatments were arranged in a randomized complete block design with five replicate boxes per substrate treatment.
Each planting box was irrigated using 4 L•h -1 pressure-compensating drip emitters.Irrigation was controlled by a timer and averaged 20 to 30% drainage (leaching fraction) at each application.Nutrient solution was applied daily by fertigation, from transplanting to the day before harvest.The solution was made from fresh tap water [electrical conductivity (EC) of 0.3 dS•m -1 ; pH 7; and 0.10-0.30mmol•L -1 NO 3 ] and initially contained 4.78 mmol and 7.64 µmol•L -1 Zn.The concentration was adjusted for plant growth at 21 days after transplanting (DAT) to 8.62 mmol Mo, and 7.64 µmol•L -1 Zn.The final pH of both solutions was 5.9.

Measurements
The pH, EC, and the concentration of NO 3 of the drainage water from each box was measured weekly using a potentiometer (pH Micro 2000 Crison), a conductivity meter (LF 330 WTW, Weilhein, Germany), and an ion-specific electrode and meter (Crison Instruments, Barcelona, Spain), respectively, following the procedures outlined by Prazeres. 19A portable chlorophyll meter (Minolta SPAD-502: Soil Plant Analysis Development, Minolta Co., Osaka, Japan) was used to measure leaf greenness at 24 and 36 DAT.Two recently expanded leaves were selected from three plants in each box for the chlorophyll readings, and three measurements were taken on each leaf on both dates.The plants were harvested at 36 DAT.The shoots of the plants were cut off at 1 cm above the substrate surface.Four representative plants (shoots) from each box were washed, oven-dried at 70ºC for 2-3 days, weighed, ground, and analyzed for total N using a combustion analyzer (Leco Corp., St. Joseph, MI, USA).Additional leaf samples were stored at −80°C for NO 3 determination. 20The samples were oven-dried at 65°C for 48 h, weighed (0.1000 g), macerated in a mortar, homogenized in a test tube with 10 mL of distilled water, agitated in a vortex, and incubated for 1 h at 45°C in a shaking water bath.Filtrated extract was then mixed with salicylic acid in 5% sulphuric acid (1:4), incubated for 20 min at room temperature, and mixed with 9.5 mL of 2 M sodium hydroxide.The concentration of NO 3 in the solution was then determined using UV-VIS spectrophotometer (Thermo Scientific, Genesys 10S) at 338 and 440 nm.

Data analysis
Data were analyzed by analysis of variance using SPSS Statistics 21 software (Chicago, IL, USA).Means were separated at the 5% level using Duncan's new multiple range test.

Drainage water
The pH in the drainage water was influenced by substrate type (Figure 1A).In general, pH was greater in the drainage water collected from the peat substrate than from the other two substrates.The pH also increased over time in each treatment, which was likely due to the differential uptake of ions from the nutrient solution.For instance, when N is supplied in the NO 3 form, there is an increase in hydroxide ion (OH -) concentration in the drainage water. 21The nutrient solution used in the present study provided ≈80% of the N as NO 3 .On average, the pH increased at a rate of 0.14 to 0.19 units per week in the three drainage solutions.
The EC and concentration of NO 3 in the drainage water were also affected by substrate type (Figure 1B,C).In the former case, EC was initially greater with coir than with the other two substrates or in the nutrient solution (1.5 dS.m -1 ).Coir often has high levels of Na and Cl. 1,2,22[25][26]     Plants grown in peat had greater shoot dry weight and more yield (fresh weight) than those grown in other two substrates (Table 3).The yields were similar to those obtained when spinach was grown in a floating system 8 and greater than those obtained in soil. 27,28af chlorophyll and shoot nitrogen and nitrate

Article
Plants grown in peat were greener and had higher chlorophyll meter readings at 24 and 36 DAT than those grown in the other substrates (Table 4).The readings increased linearly with shoot N content and were within the range reported by others (Figure 2). 8,29,30one of the plants in the treatments showed visual symptoms of N deficiency.However, plants grown in the mix of forest residues, husks, and peat had lower shoot N concentration than those grown in the other substrates (Table 4).In general, shoot N concentrations were higher than those reported in Florida (<30 g kg -1 ) but on the low end of the level considered to be sufficient for spinach at this stage of development (35-55 g kg -1 ). 31,32hoot NO 3 concentration was not affected by substrate type (Table 4).In each case, the values were higher than allowed by Regulation (EU) nº1258/2011 of the European Commission for fresh spinach (3.5 mg g -1 fresh weight).Therefore, these substrates do not appear to be a means of preventing high shoot NO 3 concentrations in spinach.Leaf NO 3 concentrations of spinach in a greenhouse, whether grown in soil or soilless culture systems, often exceed the value allowed by the EU.Siomos and colleagues found that plants from a soilless culture system had greater NO 3 and total N, P, and K content than plants harvested from soil. 33he high NO 3 concentrations in the present study were likely related to the environmental conditions in the greenhouse, nutritional factors, and the cultural techniques used.Light intensity was low in the greenhouse, not only due to the time of year (winter and early spring), but also due to the fact that the plastic film on the greenhouse was not totally transparent, and because a high planting density (64 plants/m 2 ) led to a considerable amount of leaf shading.][11][12] Peet and colleagues cited by Gruda found that the amount of daylight received was reduced by 30% or more by the glasshouse structure, while the other environmental factors, including the availability of water and nutrients, were usually at optimal levels. 34The high ratio of NO 3 :NH 4 (≈80) may have also led to high leaf NO 3 concentrations in our study.It has been reported that spinach accumulates more NO 3 when grown with solutions containing high NO 3 :NH 4 ratios. 8However, in that case, the total amount of N applied was 12 mmol L -1 and greater used in the present study (5.9 mmol L -1 from planting to 20 DAT and 10 mmol L -1 from 21 DAT until the day before harvest).

Conclusions
Black peat substrate produced more yield and a higher content of N and NO 3 in the shoots than the mix of forest residues, composted grape husks, and white peat or the coir

Article
after transplanting; DW, dry weight; FW, fresh weight.a,b Means followed by different letters within a column are significantly different at P≤0.05.

Figure 1 .
Figure 1.Effects of three commercial substrates (I, forest residues, husks, and peat; II, peat; III, coir) on pH (A), electrical conductivity (B) and concentration of nitrate (C) in the drainage water.Each symbol represents the mean of four replicates, and the error bars represent ±1 standard error.

Table 2 . Mass wetness, moisture content, and bulk density of three commercial sub- strates.
a,b,c Means followed by different letters within a column are significantly different at P<0.05.

Table 3 . Effects of three commercial substrates on shoot dry weight and fresh yield of spinach. Substrate Shoot dry weight (g/plant) Yield (kg•m -2 )
a,b Means followed by different letters within a column are significantly different at P≤0.05.