1. bookVolume 29 (2021): Issue 2 (December 2021)
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2353-3978
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30 Jul 2013
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The Influence of Biostimulants on Tomato Plants Cultivated under Hydroponic Systems

Published Online: 28 Oct 2021
Volume & Issue: Volume 29 (2021) - Issue 2 (December 2021)
Page range: 107 - 116
Received: 01 Dec 2020
Accepted: 01 Jul 2021
Journal Details
License
Format
Journal
eISSN
2353-3978
First Published
30 Jul 2013
Publication timeframe
2 times per year
Languages
English
INTRODUCTION

Sustainable agriculture is a new style of farming that seeks to optimize production technology to obtain long-term stability of the agricultural sector, environmental protection, and human safety through the application of natural substances or microorganisms to enhance plant development, save soil fertility, and produce a healthy product (Robertson & Harwood 2013). Sustainable agriculture therefore aims to mitigate adverse impacts on farm environments while providing a sustained production and profit level (Fereres & Villalobos 2016; Corell et al. 2019).

According to the European Council of Biostimulants Industry (EBIC), biostimulants are natural substances or microorganisms that stimulate and enhance nutrient uptake, nutrient efficiency, tolerance to abiotic stresses, and natural processes to plants or the rhizosphere. The biostimulant concept has gained attention in recent years as a potential solution to some of the negative impacts of a changing climate on agriculture (Yakhin et al. 2017).

In line with the EBIC, Maxifol (Agromaster, Russia) is classified as a biostimulant. It contains, as the main active ingredient, seaweed extract. Seaweeds are multicellular, macroscopic organisms found in coastal and marine ecosystems that contain polysaccharides, polyunsaturated fatty acids, enzymes, and bioactive peptides (Shukla et al. 2016; Okolie et al. 2018). Seaweeds can live in extreme conditions, including those of temperature, salinity, and light. Compared to terrestrial organisms, seaweeds generate more stress-related substances, essential for their survival in hostile environment (Shukla et al. 2018). They are a source of plant biostimulants and can be applied to improve agricultural productivity (du Jardin 2015; Oosten et al. 2017). Extracts from Ascophyllum nodosum have been demonstrated to promote plant growth, reduce some biotic and abiotic plant stresses, and increase yield and quality (Shukla et al. 2019). Seaweed extracts are eco-friendly alternatives to the use of synthetic fertilizers and growth regulators (Carvalho et al. 2019). Radifarm (Valagro, Italy) is a plant biostimulant containing amino acids, glycosides, saponins, betaines, polysaccharides, organic acids, vitamins, and microelements (Tkalec et al. 2010). Radifarm, applied in the early stages of growth, promotes an extensive root system by speeding up the elongation of lateral roots and producing more adventitious roots (Parađiković et al. 2019; Bulgari et al. 2019).

Biostimulants can enhance plant stress tolerance by means of active components, such as polysaccharides, proteins, amino acids, and glycosides. Simultaneously, plants treated with biostimulants increasingly develop their roots, stems, and leaves, which is necessary for better plant adaptation after transplantation (Zeljković et al. 2010). This study aims to determine the impact of two biostimulants on the growth and yield of tomato plants cultivated in hydroponic system under greenhouse conditions.

MATERIALS AND METHODS
Growing conditions and plant material

A greenhouse experiment was conducted with the tomato hybrid cultivar ‘Merlice’, which is recommended for greenhouse cultivation on artificial substrates (De Ruiter, the Netherlands). The tomato seeds were sown on plastic trays filled with artificial wool substrates and fertilized with a nutrient solution (EC = 2.5 dS·m−1, pH 5.5). Through six weeks, tomato seedlings were kept in the greenhouse with temperatures from 18 °C to 22 °C during the day and from 16 °C to 18 °C at night at 60–65% relative air humidity. The greenhouse was continuously ventilated to avoid plant diseases. Seedlings with 4–5 true leaves were transplanted to the hydroponic system, using mineral wool as a substrate. After setting the seedlings on plastic boxes filled with wool substrate, they were fertilized with 100 ml per plant per day of a Hoagland nutrient solution (0.54 g·L−1 of KNO3, 0.84 g·L−1 of Ca(NO3)2, 0.14 g·L−1 of KH2PO4, 0.25 g·L−1 of MgSO4, and 0.2 g·L−1 of Fe). In the vegetative phase, the EC of the nutrient solution was 3.5 dS·m−1, and in the flowering phase, it was 5.0 dS·m−1; pH was adjusted weekly to pH 5.0–5.5. The light was set for 16 hours with an intensity of 500 μmol·m−2·s−1 and temperature at 25 °C day/night, with 60–65% relative humidity.

Biostimulant treatments

The experiment was conducted as a completely randomized process, with three replications per treatment and 50 plants per replication. Ten plants of the replicate were taken for analysis of growth parameters. The following concentrations of each biostimulant were studied: Maxifol (MF 2.5, SW 5.0, and 7.5 mg·L−1) and Radifarm (RF 2.5, 5.0, and 7.5 mg·L−1). They were applied three times: for soaking seeds before seeding and spraying in a volume of 65 ml per plant at the flowering and early fruit development stages. The control plants were sprayed with water. Tomato seeds were soaked in biostimulant solutions for 30 minutes before seeding, while control seeds were soaked in distilled water for the same time.

Biometric parameters

Biometric parameters of 30 plants per treatment were each analyzed three times: before transplanting seedlings, at fruit set, and at fruit formation phase (35, 70, and 105 days after seeding, respectively). The following parameters were scored: root length (only at transplanting), the plant height, number of the leaves per plant, total leaf area per plant, and fresh and dry weights calculated by multiple average leaf area and number of leaves per plant. The dry weight of whole plants was obtained after washing with deionized water and drying in an oven at 60 °C for more than 48 hours (10 samples per replicate). The average leaf area was measured using the Image J program as described by Abdelkader et al. (2019a). The relative growth rate (RGR) was calculated according to the formula: RGR = (ln W2 − ln W1)/(t2t1), and expressed as g·g−1·week−1. The crop growth rate (CGR) is the total dry matter produced per plant over a period of time. It was calculated by the following formula: CGR = (W2 − W1)/(t2 − t1), where W1 = dry weight of the plant at time t1, and W2 = dry weight of the plant at time t2 and expressed as g·week−1·plant−1. The net assimilation rate (NAR) was measured using the following equation: NAR = (W2 − W1)(lnL2 − lnL1)/(t2 − t1)(L2 − L1), and expressed as (g·m−2·week−1), where ln = natural log, W1 = dry weight of plant (g) at time t1, and W2 = dry weight of plant (g) at time t2 (Usuda 2004). Yield components were calculated by measuring the following parameters: number of fruits per plant, weight of individual fruit, and fruit yield per plant.

Biochemical parameters

Ten tomato fruits at the red-ripening stage were collected from each replicate to conduct biochemical analyses. Three replicates for each treatment and three separate analyses for each replication were conducted. Biochemical properties were evaluated to determine the following parameters: fresh and dry mass of fruit (after drying at 105 °C), the titratable acidity (TTA), expressed as a percentage of citric acid by the methods described by the Association of Analytical Communities (Considine 2005), the concentration of ascorbic acid, expressed as mg·100 g−1, determined by the 2,6-dichlorophenolindophenol method, carotenoids (expressed as mg·100 g−1), and nitrate (mg·kg−1). Sugar to acid ratio (maturity index) was calculated by dividing total soluble solid, measured as °Brix using a refractometer, by the titratable acidity of the given samples under analysis as described by Mohammed et al. (1999). The taste index was calculated using the equation of Navez et al. (1999) using the formula: Tasteindex=Brixlevel20(acidity)+acidity. {\rm{Taste}}\;{\rm{index}} = {{{\rm{Brix}}\;{\rm{level}}} \over {20\;\left( {{\rm{acidity}}} \right)}} + {\rm{acidity}}.

Statistical analysis

Data were analyzed using one-way analysis of variance (ANOVA) in a completely randomized design followed by Tukey’s HSD test ± SD (p = 0.05) with the help of the MINITAB (v. 19.0) program.

RESULTS
Biometric parameters

Soaking tomato seeds in a biostimulator solution before sowing influenced the root length of the seedlings (Fig. 1). Radifarm increased the root length by 4.3–7.8% and RutfarmMaxifol by 1.2–2.4% in comparison with the control. The seedlings soaked in Radifarm with a concentration of 7.5 mg·L−1 had the maximum root length (7.6 cm). RutfarmMaxifol increased the seedling root length only when it was used at a concentration of 7.5 mg·L−1, but to a lesser extent than Radifarm. Generally, the application of RutfarmMaxifol and Radifarm significantly enhanced growth and other biometric parameters if applied in concentrations of 5 and 7.5 g·L−1 compared with the control, and the effect increased with concentration (Table 1). RutfarmMaxifol was more effective than Radifarm in the increase of values of the studied parameters.

Figure 1

Root length at transplanting (35 days after seeding) of tomato seedlings hybrid cultivar ‘Merlice’, influenced by seeds soaking in biostimulants Radifarm and RutfarmMaxifol. The different letters indicate significant differences between the treatments at p < 0.05 established by Tukey’s multiple range test; values represent means ± SD

Biometric parameters of tomato plants under the influence of biostimulators (mg·L−1) under hydroponic system

Biometric parametersDays after plantingControlRF 2.5RF 5.0RF 7.5RM 2.5RM 5.0RM 7.5
Plant height (cm)3517.9 ± 2.0 d18.0 ± 1.7 d21.2 ± 2.1 ab21.5 ± 2.3 a20.1 ± 2.1 c20.9 ± 1.3 b21.0 ± 1.4 b
70143.5 ± 5.8 b146.5 ± 6.6 b171.5 ± 7.3 a178.0 ± 7.0 a159.2 ± 7.3 ab171.0 ± 5.6 a174.0 ± 6.1 a
105174.5 ± 7.9 d180.0 ± 8.6 d193.5 ± 9.0 c197.5 ± 10.7 bc198.5 ± 10.3 bc206.5 ± 9.4 b217.0 ± 9.3 a
Number of leaves356.5 ± 0.28 c6.8 ± 0.68 bc7.2 ± 0.74 ab7.2 ± 0.35 ab6.8 ± 0.67 bc7.3 ± 0.71 ab7.5 ± 0.80 a
7032.1 ± 2.1 d37.1 ± 1.9 bc39.1 ± 2.6 ab40.6 ± 2.4 a35.9 ± 1.9 c37.6 ± 1.6 bc39.0 ± 2.2 ab
10543.6 ± 2.8 c50.0 ± 2.2 bc52.4 ± 2.4 ab53.9 ± 2.2 a48.4 ± 2.2 c50.5 ± 1.8 bc52.0 ± 2.7 ab
Plant fresh weight (g)359.7 ± 1.0 d9.9 ± 0.9 d10.7 ± 1.1 c11.7 ± 1.2 b10.5 ± 1.0 c12.4 ± 1.2 ab13.0 ± 1.3 a
70150.7 ± 7.3 c151.1 ± 8.4 c152.8 ± 6.6 bc154.7 ± 7.9 b154.2 ± 7.0 bc158.9 ± 8.7 a161 ± 7.2 a
105201.4 ± 10.8 c216.1 ± 11.1 bc230.2 ± 12.8 b238.7 ± 12.2 ab204.0 ± 12.0 c249.7 ± 16.9 a251.0 ± 14.3 a
Plant dry weight (g)352.4 ± 0.1 e2.6 ± 0.3 de2.7 ± 0.2 cd2.8 ± 0.1 cd2.8 ± 0.2 bc3.1± 0.1 ab3.3 ± 0.2 a
7019.1 ± 2.1 d22.1 ± 2.2 c28.8 ± 2.6 b30.4 ± 2.7 ab24.2 ± 2.4 c30.9 ± 3.0 ab32.0 ± 1.8 a
10546.5 ± 2.7 d55.1 ± 3.0 c58.9 ± 2.3 b60.3 ± 3.5 b58.9 ± 3.3 b73.3 ± 4.0 a75.0 ± 4.2 a
Total leaf area per plant (dm2)350.47 ± 0.01 c0.55 ± 0.01 bc0.59 ± 0.1 b0.60 ± 0.02 b0.59 ± 0.01 b0.73± 0.02 a0.75 ± 0.02 a
7021.2 ± 0.68 b23.4 ± 0.67 a24.1 ± 0.89 a23.8 ± 0.80 a23.4 ± 0.75 a23.8 ± 0.73 a23.6 ± 0.80 a
10533.2 ± 1.6 ab33.6 ± 1.3 a34.0 ± 1.8 a33.9 ± 1.9 a32.8 ± 1.7 b33.4 ± 1.8 a34.1 ± 1.5 a

The different lower case letter(s) in each single row indicates significant differences between the treatments at p < 0.05 established by Tukey’s multiple range test ± SD

Figure 2 shows RGR, CGR, and NAR of tomato plants grown under the influence of biostimulants during two periods of growth expressed as weeks after planting (WAP) – the first (5–10 WAP) and second (10–15 WAP). The RGR ranged between 400 and 500 mg·g−1·week−1 at the first growth stage, while in the second growth stage, RGR values decreased to 120–180 mg·g−1·week−1. At the first growth stage, RF 5.0 and RF 7.5 treatments produced the highest RGR, but this effect differed at the second stage. There was a contradictory relationship between RGR values and the age of plants. Generally, RGR declines with increasing plant size (Fig. 2A). Concerning CGR, both biostimulants increased this parameter at the first stage, with a tendency to increase with concentration. At the second growth stage, only Radifarm had the same pattern; the effect of RutfarmMaxifol was different. The concentrations 5 and 7.5 mg·L−1 did not increase this parameter (Fig. 2B). Biostimulants in this experiment stimulated NAR only at the first stage of growth; this parameter in the older plants was not influenced by the biostimulants (Fig. 2C).

Figure 2

Relative growth rate (A1, A2), crop growth rate (B1, B2), and net assimilation rate (C1, C2) of tomato hybrid cultivar ‘Merlice’, influenced by biostimulators – Radifarm: RF 2.5, RF 5.0, and RF 7.5; RutfarmMaxifol: SW 2.5, SW 5.0, and SW 7.5 + control (nontreated) under hydroponic system

Yield components

For the determination of tomato yield components, the number of fruits per plant (pcs), fruit weight (g), and tomato fruit yield (kg per plant) were measured (Fig. 3). The values of all the above parameters increased with increasing concentration of biostimulators. RutfarmMaxifol was more effective than Radifarm at each concentration. The highest fruit number, fruit weight, and fruit yield gained the highest values at 7.5 g·L−1 of RutfarmMaxifol. The highest concentrations of biostimulants increased fruit number by 15 (Radifarm) and 20 (RutfarmMaxifol) over the control. The single fruit weight increased at the highest concentrations by 14% (Radifarm) and 18% (RutfarmMaxifol). The total yield of the plant increased by 20–70% when biostimulants were applied.

Figure 3

Yield components: number of fruits per plant (A), fruit weight (B), fruit yield (kg per plant) (C) of hybrid cultivar ‘Merlice’, influenced by biostimulants under hydroponic system. The different lower case letters indicate significant differences between the treatments at p < 0.05 established by Tukey’s multiple range test; values represent means ± SD

Biochemical analysis of tomato fruit

Radifarm enhanced dry matter of tomato fruits from 6.35% to 6.85% and RutfarmMaxifol by 6.25–6.45% (Fig. 4A). The value higher by 14% over control was obtained with Radifarm 7.5 g·L−1. The concentrations of total soluble solids (TSSs) and ascorbic acid increased with biostimulator concentration with the same pattern, more with the application of Radifarm (Fig. 4B, C).

Figure 4

Biochemical parameters: dry matter (A), total soluble solid (B), ascorbic acid (C), carotenoid contents (D), acidity (E), and nitrate contents in tomato fruits hybrid cultivar ‘Merlice’ (F), influenced by biostimulants under hydroponic system. The different lower case letters indicate significant differences between the treatments at p < 0.05 established by Tukey’s multiple range test; values represent means ± SD

In the present study, the concentration of carotenoids increased significantly only as an effect of the application of 5.0 and 7.5 g·L−1 of Radifarm (Fig. 4D). The acidity rate tended to decrease as an effect of RutfarmMaxifol treatment, less so in Radifarm treatment. Nitrate concentration in the tomato fruits increased with the concentration of Radifarm by 12% at 7.5 g·L−1 (Fig. 4F). Both biostimulants affected maturity degree and taste index in the same way (Fig. 5A, B). Significant increases were obtained at 5.0 and 7.5 g·L−1, more with the application of Radifarm.

Figure 5

Maturity degree (A) and taste index of tomato fruits hybrid cultivar ‘Merlice’ (B), applied with two biostimulants under hydroponic system. The different lower case letters indicate significant differences between the treatments at p < 0.05 established by Tukey’s multiple range test; values represent means ± SD

DISCUSSION

The application of biostimulants positively impacts plant growth by enhancing metabolism and modification of biochemical processes. Both biostimulants used in our experiment contain several compounds beneficial for the growth and yield of plants. RutfarmMaxifol contains polysaccharides, polyun-saturated fatty acids, enzymes, bioactive peptides, and free amino acids. Radifarm contains amino acids, glycosides, saponins, betaines, polysaccharides, organic acids, vitamins, and microelements. All of them are important for plants’ growth and fruiting. A wide range of beneficial effects of using seaweed extract on plants was reported, including improved germination of seeds, increased yield and yield quality, and improved postharvest shelf life of fruits (Norrie & Keathley 2006). Rayorath et al. (2008) reported that seaweed extracts enhanced root growth of Arabidopsis thaliana at very low concentrations (0.1 g·L−1). The positive results of the seaweed extract on tomato plants are attributed to its nutritional effects, parallel to their stimulative effects. Seaweed plays a positive role in seed germination and growth and development associated with hormonal effects (du Jardin 2015). In fact, known plant hormones and others compounds such as sterols and polyamines were identified in seaweed extracts (Craigie 2011). The increase in plant height, number of leaves, and fresh and dry biomass of tomato plants supplemented with biostimulants may be a synergy effect between its compounds and the resulting increase in endogenous levels of cytokinins and auxin, which can stimulate plant growth by affecting photosynthesis, respiration, and activation of plant defense systems, as was found in Kentucky bluegrass (Zhang et al. 2003).

Supplementation with biostimulants containing low-molecular-weight fractions exerts positive effects on plasma membrane functions (Nardi et al. 2002), which enables an increase in nutrient uptake (Chen & Aviad 2015) by improving the root cell membrane permeability (Valdrighi et al. 1996), as in the present study can indirectly confirm the increase in root length (Fig. 1). RGR, CGR, and NAR were derived from these equations by tacitly assuming that plants’ growth is a function of time. They represent the balance between metabolism and catabolism (Pereira & Machado 1987). According to Metcalf et al. (2006) and Rees et al. (2010), RGR and plant size are contradictory because of self-shading by the leaf canopy, increased allocation to structural components, and decreases in leaf area ratio (Philipson et al. 2012). Our experiment also showed that RGR declined with increasing plant size (Fig. 2A). As RGR is almost always a function of plant size, high values of RGR can occur either because plants are simply smaller or because they truly grow faster (Turnbull et al. 2008).

Biostimulant applications increased yield components compared to the control. This may be connected with the participation of these growth-stimulating hormones in different growth and development stages: pollen germination, fruit set, cell division, and elongation after pollination. Generally, the final yield (kg per plant) showed an increase of values in all the parameters in treatments with biostimulator applications. The yield was significantly affected by biostimulant concentrations (Abdelkader et al. 2019b; Francesca et al. 2020).

The effect of biostimulant on photosynthesis and secondary metabolism may also enhance the quality of tomato fruit (Colla et al. 2017) by increasing TSS and carotenoid contents. Overall, the main effect of biostimulants is to induce physiological responses in the treated plants, and most of these responses are able to affect plant metabolism, growth, and development (Wally et al. 2013a, b). The content of ascorbic acid in tomatoes is regulated by agriculture practices and environmental conditions (Zushi et al. 2020).

Radifarm treatments highly increased nitrate concentration compared to the other treatments (13% and 14% over control). The content of nitrates in tomato fruits can increase depending on various parameters associated with the plant and the environment, such as genotype, maturity, temperature, and nutrition (Abdelkader & Puchkov 2019). According to WHO, adults consume between 20 and 70 mg nitrate daily, and the maximum safe amount of nitrate daily entering the human body should be less than 3.65 mg·kg−1 (EFSA 2008).

CONCLUSIONS

Biostimulants RutfarmMaxifol and Radifarm applied at three phases – for seed soaking and twice foliarly on the plants of tomato hybrid cultivar ‘Merlice’ grown under a hydroponic system increased several growth and fruit the parameters. All concentrations: 2.5, 5.0, and 7.5 mg·L−1 of biostimulants, were beneficial and usually the highest concentration was the most effective. Radifarm had a more positive effect on tomato fruits’ quality and RutfarmMaxifol on the growth and yield parameters.

Figure 1

Root length at transplanting (35 days after seeding) of tomato seedlings hybrid cultivar ‘Merlice’, influenced by seeds soaking in biostimulants Radifarm and RutfarmMaxifol. The different letters indicate significant differences between the treatments at p < 0.05 established by Tukey’s multiple range test; values represent means ± SD
Root length at transplanting (35 days after seeding) of tomato seedlings hybrid cultivar ‘Merlice’, influenced by seeds soaking in biostimulants Radifarm and RutfarmMaxifol. The different letters indicate significant differences between the treatments at p < 0.05 established by Tukey’s multiple range test; values represent means ± SD

Figure 2

Relative growth rate (A1, A2), crop growth rate (B1, B2), and net assimilation rate (C1, C2) of tomato hybrid cultivar ‘Merlice’, influenced by biostimulators – Radifarm: RF 2.5, RF 5.0, and RF 7.5; RutfarmMaxifol: SW 2.5, SW 5.0, and SW 7.5 + control (nontreated) under hydroponic system
Relative growth rate (A1, A2), crop growth rate (B1, B2), and net assimilation rate (C1, C2) of tomato hybrid cultivar ‘Merlice’, influenced by biostimulators – Radifarm: RF 2.5, RF 5.0, and RF 7.5; RutfarmMaxifol: SW 2.5, SW 5.0, and SW 7.5 + control (nontreated) under hydroponic system

Figure 3

Yield components: number of fruits per plant (A), fruit weight (B), fruit yield (kg per plant) (C) of hybrid cultivar ‘Merlice’, influenced by biostimulants under hydroponic system. The different lower case letters indicate significant differences between the treatments at p < 0.05 established by Tukey’s multiple range test; values represent means ± SD
Yield components: number of fruits per plant (A), fruit weight (B), fruit yield (kg per plant) (C) of hybrid cultivar ‘Merlice’, influenced by biostimulants under hydroponic system. The different lower case letters indicate significant differences between the treatments at p < 0.05 established by Tukey’s multiple range test; values represent means ± SD

Figure 4

Biochemical parameters: dry matter (A), total soluble solid (B), ascorbic acid (C), carotenoid contents (D), acidity (E), and nitrate contents in tomato fruits hybrid cultivar ‘Merlice’ (F), influenced by biostimulants under hydroponic system. The different lower case letters indicate significant differences between the treatments at p < 0.05 established by Tukey’s multiple range test; values represent means ± SD
Biochemical parameters: dry matter (A), total soluble solid (B), ascorbic acid (C), carotenoid contents (D), acidity (E), and nitrate contents in tomato fruits hybrid cultivar ‘Merlice’ (F), influenced by biostimulants under hydroponic system. The different lower case letters indicate significant differences between the treatments at p < 0.05 established by Tukey’s multiple range test; values represent means ± SD

Figure 5

Maturity degree (A) and taste index of tomato fruits hybrid cultivar ‘Merlice’ (B), applied with two biostimulants under hydroponic system. The different lower case letters indicate significant differences between the treatments at p < 0.05 established by Tukey’s multiple range test; values represent means ± SD
Maturity degree (A) and taste index of tomato fruits hybrid cultivar ‘Merlice’ (B), applied with two biostimulants under hydroponic system. The different lower case letters indicate significant differences between the treatments at p < 0.05 established by Tukey’s multiple range test; values represent means ± SD

Biometric parameters of tomato plants under the influence of biostimulators (mg·L−1) under hydroponic system

Biometric parameters Days after planting Control RF 2.5 RF 5.0 RF 7.5 RM 2.5 RM 5.0 RM 7.5
Plant height (cm) 35 17.9 ± 2.0 d 18.0 ± 1.7 d 21.2 ± 2.1 ab 21.5 ± 2.3 a 20.1 ± 2.1 c 20.9 ± 1.3 b 21.0 ± 1.4 b
70 143.5 ± 5.8 b 146.5 ± 6.6 b 171.5 ± 7.3 a 178.0 ± 7.0 a 159.2 ± 7.3 ab 171.0 ± 5.6 a 174.0 ± 6.1 a
105 174.5 ± 7.9 d 180.0 ± 8.6 d 193.5 ± 9.0 c 197.5 ± 10.7 bc 198.5 ± 10.3 bc 206.5 ± 9.4 b 217.0 ± 9.3 a
Number of leaves 35 6.5 ± 0.28 c 6.8 ± 0.68 bc 7.2 ± 0.74 ab 7.2 ± 0.35 ab 6.8 ± 0.67 bc 7.3 ± 0.71 ab 7.5 ± 0.80 a
70 32.1 ± 2.1 d 37.1 ± 1.9 bc 39.1 ± 2.6 ab 40.6 ± 2.4 a 35.9 ± 1.9 c 37.6 ± 1.6 bc 39.0 ± 2.2 ab
105 43.6 ± 2.8 c 50.0 ± 2.2 bc 52.4 ± 2.4 ab 53.9 ± 2.2 a 48.4 ± 2.2 c 50.5 ± 1.8 bc 52.0 ± 2.7 ab
Plant fresh weight (g) 35 9.7 ± 1.0 d 9.9 ± 0.9 d 10.7 ± 1.1 c 11.7 ± 1.2 b 10.5 ± 1.0 c 12.4 ± 1.2 ab 13.0 ± 1.3 a
70 150.7 ± 7.3 c 151.1 ± 8.4 c 152.8 ± 6.6 bc 154.7 ± 7.9 b 154.2 ± 7.0 bc 158.9 ± 8.7 a 161 ± 7.2 a
105 201.4 ± 10.8 c 216.1 ± 11.1 bc 230.2 ± 12.8 b 238.7 ± 12.2 ab 204.0 ± 12.0 c 249.7 ± 16.9 a 251.0 ± 14.3 a
Plant dry weight (g) 35 2.4 ± 0.1 e 2.6 ± 0.3 de 2.7 ± 0.2 cd 2.8 ± 0.1 cd 2.8 ± 0.2 bc 3.1± 0.1 ab 3.3 ± 0.2 a
70 19.1 ± 2.1 d 22.1 ± 2.2 c 28.8 ± 2.6 b 30.4 ± 2.7 ab 24.2 ± 2.4 c 30.9 ± 3.0 ab 32.0 ± 1.8 a
105 46.5 ± 2.7 d 55.1 ± 3.0 c 58.9 ± 2.3 b 60.3 ± 3.5 b 58.9 ± 3.3 b 73.3 ± 4.0 a 75.0 ± 4.2 a
Total leaf area per plant (dm2) 35 0.47 ± 0.01 c 0.55 ± 0.01 bc 0.59 ± 0.1 b 0.60 ± 0.02 b 0.59 ± 0.01 b 0.73± 0.02 a 0.75 ± 0.02 a
70 21.2 ± 0.68 b 23.4 ± 0.67 a 24.1 ± 0.89 a 23.8 ± 0.80 a 23.4 ± 0.75 a 23.8 ± 0.73 a 23.6 ± 0.80 a
105 33.2 ± 1.6 ab 33.6 ± 1.3 a 34.0 ± 1.8 a 33.9 ± 1.9 a 32.8 ± 1.7 b 33.4 ± 1.8 a 34.1 ± 1.5 a

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