Effects of salinity stress induced by hot spring water on tomato growth, yield and fruit quality under hydroponic cultivation in Japan

Summary: The objective of this research was to test hot spring water as a source of salt to improve tomato quality under the hydroponic system. This research was conducted at Yamagata University, in Japan from February to July 2017. Salt stress was induced using salts of hot spring wastewater collected from Yupoka Onsen (Tsuruoka, Japan). The treatments were EC 2, 4, 8 12, and 16 mS/cm which were arranged in a Randomized Complete Block Design (RCBD) with five replications. Tomato plants were grown at EC 2 until flowering and then subjected to different EC concentrations until harvesting. The data were collected on plant growth parameters and fruit quality. Fruits were harvested at the red stage until the 5 th truss. The results showed that fruits' Soluble Solids Content, organic acid, Nitrate contents and Sugar: Acid ratio increased significantly at EC 16 mS/cm and in the upper trusses compared to EC 2 and in the lower trusses. In contrast, fruit weight significantly decreased at EC 16 mS/cm and in upper trusses. Leaf thickness, size and SPAD, and specific leaf weight significantly declined at EC 16 mS/cm and upper leaves compared to EC 2 and in lower leaves. Plant height started to decline significantly after three weeks of treatment at EC 16 compared to EC 2.


Introduction
Tomato (Solanum lycopersicum) is economically among the most important vegetables grown worldwide (Agius et al., 2022).Tomato fruits are key ingredients in the preparation of meals in several countries.Tomato is also a vital source of vitamins, minerals and antioxidants essential for human health.Recently, more attention has been concentrated on the bioactive ingredients and health aspects of tomato fruits.The production of tomatoes in terms of quantity and quality aspects remains the priority for the satisfaction of consumers (Lu et al., 2019).The most significant quality aspects of any vegetable product are taste and aroma.Tomatoes are produced conventionally in soils or using soilless media.One of the soilless culture methods used to produce tomatoes is the hydroponic system.The hydroponic system uses a nutrient mineral solution for plant development.It improves the growth environment while limiting moisture and nutrient uncertainty.It contributes greatly to saving water and fertilizer thereby improving the water and nutrient use efficiency by crops (Zhang et al., 2016).Connections between the hydroponic environment and the salinity of the tomato plant are extremely complicated.Salt stress's destructive effects on tomatoes include slow plant growth (Kamrani et al., 2013) and the reduction of fruit yield and size (Rosadi et al., 2014).
Nevertheless, the high EC enhance quality aspects such as nutritional value and flavour.This results in an increased value in terms of market prices thereby balancing the yield losses (Ghoname et al., 2019).
There is no generalised level of salinity that could be recommended in tomato production, rather it varies according to quality traits and interaction between cultivars, climate factors, nutrient solution concentration as well as crop management (Safi et al., 2018;Zhang et al., 2016).Although tomato is moderately sensitive to salinity, yield reduction at higher salinity levels (> 2.5 dS m -1 EC) has been observed (Singh et al., 2012).This reduction of yield in tomatoes due to salinity is mainly a result of reduced biomass of roots, leaves and fruits.Nevertheless, salinity increases the content of tomato fruits in total soluble solids like sugars, amino acids and organic acids which are essential to human health (Ghoname et al., 2019;Parvin et al., 2015).To take advantage of this, many tomato growers use seawater for improving tomato fruit quality.However, it is difficult to use seawater for tomato production at high altitudes.Some "hot spring" water found at high altitudes has high salt concentrations.The present study aimed to investigate the response of tomato 'Reika' to salts collected from spring water under hydroponic cultivation.

Materials and methods
Reika tomato seeds were sown on 7 February 2017 on moist papers into Petri dishes in a culture room at 24 °C with a 14/10 h light/dark photoperiod and relative humidity of 45-50%.After three days, seeds were transferred into a cell tray (100 mL) filled with growing media (Baido 300, Pro-Bokash-KantoNosan, Tochigi, Japan) containing 0.22 g L -1 of nitrogen and grown for two weeks in the same culture room.Thereafter, seedlings were transplanted into plastic pots (500 mL) filled with growing media in a heated glasshouse (over 10 °C) for six weeks.
The hot spring water was naturally evaporated in the sheeting dam into the plastic greenhouse during the summer season, and the collected salts were used to increase solution EC.At the flowering initiation, 25 seedlings were transplanted into hydroponic established in the greenhouse.Styrofoam boxes (28 L) which were wrapped with aluminium foil to avoid excessively high temperatures were used for the cultivation.The boxes were filled with nutrient solutions of Otsuka 1 (macronutrient), Otsuka 2 (micronutrient), and Otsuka 5 (Oligo-elements) made by Otsuka Chemical in Osaka, Japan.The 75 g of Otsuka 1, 50 g of Otsuka 2, and 3 g of Otsuka 3 were diluted in 1 L of hot water without mixing the solutions, Then, both solutions were diluted with 97 L of tap water to make 100 L of the total solution, these solutions had EC 1.34 mS/cm and a pH of 6.5.Six weeks after planting, EC was enhanced up to EC 2 mS/cm by adding nutrient solution.The enhancement of ECs was continued every two days by adding 0.6 g of hot spring salts for 1L of water to get EC 4, EC8, EC 12 and EC 16 mS/cm treatments.Thus, five different salt stress treatments (EC 2 ± 0.2 mS/cm, 4 ± 0.2 mS/cm, 8 ± 0.2 mS/cm, 12 ± 0.2 mS/cm, and 16 ± 0.2 mS/cm) were provided, and the nutrient solution was renewed every week.These EC treatments to nutrient solutions were arranged in a Randomized Complete Block Design (RCBD) and replicated five times.Nitenpirum insecticide (Sumitomo Chemicals Co. Insecticide Best.Guard Grain) and fungicides (Tebuconazole, Agro China Pty Ltd, 188 Xinjunhuan Rd.Shanghai, P.R. China) were applied for controlling insect pests and powdery mildew.
The average temperature and relative humidity (RH) inside the greenhouse were 20.3 o C and 68.9% respectively and the average temperature of the nutrient solution was 18.2 o C during the growing season.The trellising was done with ropes and plants were kept as a single stem with seven trusses.The daily average temperature, RH and the average temperature of the medium solution in the greenhouse were 22.6 o C, 70.9% and 20.1 o C respectively during this experiment.

Data collection
Data for plant height were collected weekly during plant growth starting from salt stress induction until the 7 th truss developed.Leaf size, thickness and SPAD data were measured on the first leaf under the truss until the fifth truss.Leaf size and thickness were measured at fruit green maturity of each truss until the 5 th truss while SPAD was measured every week during the breaker stage.SPAD index was taken four times on each leave.Harvesting was done on all fruits on five trusses at full ripening.For the laboratory analyses of SCC, organic acids and NO 3 -ion, harvested fruits of the same colour were used.They were selected using the colour Reader (CR-10, Konica Minolta, Tokyo).These fruits were then cut into half longitudinally and samples were taken using the cork borer.Obtained samples were blended in a mortar and filtrated using a tissue filter.The extract was centrifuged at 6000 rpm for one minute.The SSC was measured using a Digital refractometer (PR-101, Atago, Tokyo), organic acid content was determined using the organic acid meter (PAL-BXIACID F5, Atago, Tokyo) and NO 3 -was measured using a nitrate ion meter (LAQUAtwin NO 3 -B-341, Horiba, Tokyo ATAGO).The use of materials consisted of opening and calibrating the glass electrode with distilled water/or standard solution and rinsing it with distilled water or cleaning it with tissue paper.Then after rinsing or cleaning, fill the juice sample slowly on the glass electrode without bubbling and measure the sample.
The juice for measuring organic acid was diluted with desalted water on a ratio of 1:50 (0.1 ml of juice into 4.9 ml of desalted water).The SSC was expressed in % Brix, organic acid in %, and NO 3 -in ppm.The sugar: acid ratio was calculated by dividing fruit SSC with an organic acid.

Data analysis
Data were analysed by GenStat 20 th Edition.The analysis of variance (ANOVA) was used to test the difference among treatments and the Least Significant Differences (LSD) test at 5% was used to separate treatment means.

Results
Fruit SSC, NO 3 -and Sugar Acid ratio significantly increased by high EC and by truss level while organic acid increased by higher EC but decreased by the level of the truss.Fruit weight and size significantly decreased by high EC and by the level of the truss.Fruit SSC, NO 3 -, Organic acid and Sugar Acid ratio were significantly increased by the interaction of salt stress and level of the truss while the results showed no significant difference in fruit weight and size (Table 1).
The highest average on fruit weight, size, SSC, NO 3 -, Organic Acid and Sugar Acid ratio was 240.3 g at EC 4 on truss 5, 70.8 mm at EC 4 on truss 2, 8.7 brix % EC 16 on truss 3, 1779 ppm at EC 12 on truss 4, 2.85% at EC 16 on truss 5, and 4.032 at EC 16 on truss 4 respectively.About 75 of the fruits obtained in low EC were larger fruit sizes (67-82 mm) while 80 of the fruits obtained in high EC were smaller than 57 mm (Table 1).
The plant height curve showed a difference significant (p<.001) among salt stress treatments after three weeks of salt stress.The highest was EC 8 with 173.8 Cm while the lowest was EC 16 with 145.4 Cm (Figure 1).Significant differences among salt stress and level of leaf were recorded on leaf size, thickness, SPAD and Leaf Specific Weight.Leaf size was increased in leaves at the higher level but decreased by higher EC.Leaf thickness, leaf specific weight (LSW) and SPAD were increased by high EC while declining by the higher level of leaves (Table 2).There was no significant interaction between salt treatment and leaf level on the leaf size, LSW and SPAD while a significant difference was observed in leaf thickness (Table 2).The highest leaf thickness was recorded at EC 12 on the leaf under the 2 nd truss (0.858 mm) while the lowest was at EC 2 on the leaf under the 5 th truss (0.548 mm).

Discussion
In the present study, salinity effects were assessed concerning tomato fruits SSC, OA, Sugar Acid ratio, NO 3 -, weight and size.On plant growth, the assessment was done plant height, leaf size, thickness, SPAD and LSW.The reasonable response may be an alternative to tomato growers along distances from the Seashore.The results showed that fruit SSC, NO3 -and Sugar Acid ratio increased by high increased EC and level of the truss while fruit weight and size decreased by high EC and level of truss (Table 1-2) (Figure 2).Similar results were reported by (Al Hassan et al. (2015) who showed that the total amount of soluble sugars slightly decreased in salt-stressed plants, but significant differences were registered only starting with 300 mM NaCl in both samplings.Under water stress conditions the decrease was also significant and more accentuated after a longer drought treatment.This indicates that fruit water reduction in high EC affected weight loss and size reduction.It can be produced by inhibition of water uptake by the root resulting in the reduction of water transport to the fruit and increased concentration of soluble solids (Oztekin & Tuzel, 2011;Shabala et al., 2012).
For instance, the fruit weight of tomato was reported to show a significant variation at p<.001 among different germplasms of tomato, and in general, there was a significant decrease in fruit yield compared to controls in plants treated with high concentrations of salt (Siddiky et al., 2015(Siddiky et al., , 2012)).However, fruit organic acid was increased by higher EC but decreased by the level of the truss.Thus, the increase in acidity of the fruit juice could be due to the higher Na content in the fruit juice since this is the only ion that has a higher concentration in hot spring water used as a source of salinity.The decrease of fruit acidity at the high level of truss can be explained by high evapotranspiration due to high temperature and RH in the greenhouse during tomato cultivation.The higher fruit acidity of fruit tomatoes was also reported by Agius et al. (2022) who stated that the concentration of citric acid and malic acid remained unaffected, the pH dropped by approximately 0.1 unit and the titratable acidity increased slightly at higher salinity levels tested (17 and 34 mM).
Most plant growth parameters were negatively affected by salt stress, although leaf thickness, leaf specific weight (LSW) and SPAD were increased (Table 2).The reduction of the vessels' diameter is a common phenomenon among plants that decreases the incidence of cavitation.The increased thickening of the xylem vessels improves stability by enhancing the mechanical properties of the secondary cell walls (Eckert et al., 2019).Hoffmann et al. (2021) reported that the bottom leaflets showed stronger stress signs and response, while the top leaflets were less impacted by the abiotic stressor and had an increased expression of cell wall-related genes involved in the expansion.According to Oztekin and Tuzel (2011), plant height showed a 29.03% reduction in 200 mM NaCl treatment compared to 50 mM.According to Azarmi et al. (2010), the total leaf area decreased with increasing salinity (EC2.5-6mS/cm).This overall decrease in plant growth may be associated with the reduction of plant water uptake in salinity conditions.

Conclusions
Results from this study showed that high EC induced by salinity obtained from hot spring water improves tomato fruit quality but yield and plant biomass are reduced.The hot spring water could be an alternative to salt stress at 8 mS/cm under hydroponic cultivation.It should create a balance between yield loss and fruit quality.

Figure 1 :
Figure 1: Effect of high salt treatment on plant height.

Figure 2 .
Figure 2. Relationship of tomato fruit SSC and weight.

Table 1 .
Chemical composition of hot spring water.

Table 2 .
Chemical composition of nutrients applied.

Table 3 .
Effect of interaction of salt stress and long exposure on fruit SSC, NO 3 -, organic acid, sugar: acid ratio, weight and size.

Table 4 .
Effect of interaction of salt stress and level of leaf on leaf size, specific weight, SPAD and thickness.