The impact of different growth media and ammonium-nitrate ratio on yield and nitrate accumulation in lettuce (Lactuca sativa var. longifolia)

Ammonium (NH4) to nitrate (NO3) ratio and growth media significantly alter plant development and NO3 accumulation in lettuce. Nitrate accumulation is regarded harmful for environment and human health. The quality of lettuce is assessed by NO3concentration, size, and weight. This study determined the impact of five different growth media (cocopeat, peat, bark, perlite and rockwool) and four different NH4:NO3 ratios (0:100, 20:80, 40:60 and 60:40) on NO3 accumulation in lettuce, yield, and several growth attributes. The experimentation was conducted according to open feeding system of soilless agriculture. The ‘Cosmos’ variety of lettuce was used as experimental material in the study. Growth mediums and NH4:NO3 ratios significantly altered NO3 accumulation, head, leaf, stem and root traits. The results revealed that instead of growing lettuce with NO3 only in peat and rockwool, addition of NH4 (20:80 of NH4:NO3) into nutrient solution increased head weight. While head weight increased in perlite medium with the addition of NH4, it decreased in cocopeat and bark media. It is concluded that growth media and NH4:NO3 ratios pose significant impacts on NO3 accumulation in leaf and that the increase in NH4 ratio decreased NO3 accumulation in all growing media. Therefore, it is recommended that NH4 should be added in the nutrient solution to decrease NO3 accumulation, which will ultimately improve yield and quality of lettuce.


Introduction
Proper fertilization and provision of suitable ecological conditions is necessary to get higher yield best from crop plants. Nevertheless, production sustainability and safety of environment and human health must be considered while establishing the fertilization programs for getting higher yield and quality. Several other factors, including climate, developmental stage of plant, soil structure and nature of plants should also be considered while devising fertilization programs. It is commonly observed that high fertilizer input increases yield; however, leads to low quality and several other problems. Significant nitrate (NO3 -) accumulation, which contains high amount of nitrogen (N) is observed in below and aboveground parts of plants due fertilizers (Güneş, 1994;Ayaz and Yurttagül, 2006;Zandvakili et al., 2019). It is reported that the use of fertilizers containing high amount of NO3and nitrite (NO2 -) negatively affects human health and causes anemia. These also form carcinogenic nitrosamines by reacting with the secondary amines in human body (Oruç and Ceylan, 2001;Ayaz and Yurttagül, 2006;Özdestan and Üren, 2010). Nitrate and NO2are indigenous to soil, surface 2 and ground water, air, and plants (Aköz, 1981). Nitrate turns into NO2and become harmful since the later decrease's oxygen-carrying capacity of blood when reacts with hemoglobin and turns Fe +2 into Fe +3 . This situation known as Blue Baby Syndrome is very dangerous for children (Oruç and Ceylan, 2001;Özdestan and Üren, 2010). It is reported that acute toxicity of NO2is frequent and leads to death, whereas acute toxicity of NO3is rare (Özdestan and Üren, 2010). Approximately 87% of NO3intake to human body comes through vegetable consumption (Özdestan and Üren, 2010). It is suggested that NO3is a natural component of both vegetables and fruits and used as a preservative substance in meat and fish products (Ayaz and Yurttagül, 2006).
The NO3accumulation in plant tissues increases depending on nitrogenous fertilizers, low activity of nitrate reductase, low pH values, low light and soil moisture, high temperature, cloudy weather, and drought etc. (Rao and Rains, 1976;Ayaz and Yurttagül, 2006;Yardım, 2009;Çokuysal et al., 2016). The velocity of biochemical processes is slow and NO3level is high under low light availability. Moreover, cultivation season, storage and geographical conditions also affect NO3accumulation. Nitrate content in plants is related to their biological activities (Stepowska and Kowalczyk, 2001). Nitrate is digested in plant tissues by nitrate reductase enzyme. Nitrate accumulation increases when the activity of this enzyme is inhibited. Molybdenum (Mo) has a key role in the synthesis of nitrate reductase, and it is cofactor required for the enzyme (Ayaz and Yurttagül, 2006). The deficiency of Mo in growth medium inhibits the activity of nitrate reductase; thus, NO3accumulation may increase. Molybdenum deficiency leads to high NO3accumulation in many plant species (Moncada et al., 2018). It is suggested that NO3content in lettuce can be decreased through addition of Mo to the medium (Moncada et al., 2018). Zhao et al. (2016) reported that N has significant effect on plant growth, nutrient intake, and physiological processes of plants. Plants principally uptake N from soil in the form of NO3or NH4 + , and their ratio can easily change depending on pH of root zone, age and type of plant, quantity of light, and presence/absence of other ions. While NO3generally taken in acidic conditions, NH4 + is taken under alkaline conditions (Yardım, 2009;Çokuysal et al., 2016). The whole amount of N required by plants in soilless agriculture system can be given in NO3form. However, it is suggested to keep 15% NH4 + in nutrient solution due its positive effects (Gül, 2012). Growing vegetable in soilless agriculture is steadily around the world. Several growth media made up of organic or inorganic materials are used in soilless agriculture. Limited literature is available on the impact of growth media on NO3accumulation in plants. Furthermore, there are limited studies conducted on the impact of NH4 + :NO3ratio on plant growth and NO3accumulation in lettuce. Therefore, this study assessed the impacts of various growth media and different NH4 + :NO3ratios in nutrient solution on yield, plant growth and NO3accumulation in lettuce. This study aims to determine the appropriate NH4 + /NO3 − ratios according to the growing medium and thus to improve the quality of the crop by reducing nitrate accumulation in lettuce.

Experimental site
This study was conducted in polycarbonate greenhouse located at Faculty of Agriculture, Harran University, Şanlıurfa, Turkey during winter growing season of 2019.

Treatments
The 'Cosmos' lettuce (Lactuca sativa var. longifolia) variety was used as experimental material in the study. Many organic and inorganic media are used in the production of vegetables in greenhouses. In the study, 5 different organic and inorganic origin media, which are mostly used in production, such as cocopeat, perlite, peat, bark and rockwool were preferred. Four different NH4 + :NO3ratios each containing 150 ppm N, were applied to all growing media. The amount of N and other elements in solution was kept stable. The NH4 + :NO3ratios were adjusted by changing the amounts of ammonium sulphate, calcium sulphate and calcium nitrate.
The lettuce seedlings used in the experiments were bought from a local seedling firm. The seedlings were planted in 8-litre pots containing growth media on 1 st December 2018 and harvested on 20 th March 2019. All pots received same amount of fertilizer; however, different NH4 + :NO3ratios were applied 10 days after planting. The formulation of nutrient solution proposed by Gül (2012) for lettuce growing in winter months was used in the current study (Table 1). Nitrate analysis Nitrate contents in leaves were determined via salicylic acid method designed by Cataldo et al. (1975).
0.1 ml of the extracted leaf sample and 0.4 mL of 5% salicylic acid solution (dissolved in concentrated H2SO4) were mixed well. After 20 minutes at room temperature, 9.5 mL of 2N NaOH were added slowly to raise the pH above 12. Samples were cooled to room temperature and absorbance at 410 nm was determined in a spectrophotometer.

Head traits
The lettuces were harvested by cutting to growth media level. Afterwards, head diameter and length were measured by ruler. The heads were weighed in a precision scale for head fresh weight.

Leaf traits
The leaves in the harvested lettuce were separated from the stem and counted. The number of inner and outer leaves were counted separately. Total number of leaves were computed by adding inner and outer leaves. The leaves were weighed in a precision scale for fresh weight. Then, the leaves samples were dried in an oven at 65 o C until constant weight, and dry weight of leaves was recorded. Dry matter ratio was calculated by the equation 1 given below.

Stem and root traits
Leaves in the plants harvested were separated from stem and stem were weighed in a precision scale for fresh weight. The stems were dried in an oven at 65 o C until constant weight, and dry weight of stem was recorded. Roots were removed from growth media, washed and their length was measured. Afterwards roots were dried according to the above procedure and then root dry weight was recorded. However, roots of the plants grown in rockwool medium were not evaluated since they were not completely cleaned.

Statistical analysis
The experiment was laid out according to factorial design where growth media were main factor, while NH4 + :NO3ratios were regarded as sub-factor. All treatments had three replications. The collected data regarding plant attributes and NO3contents were tested for normality first through Shapiro-Wilk normality test. The normality test revealed that data had normal distribution; therefore, original data were used in the statistical analysis. Two-way Analysis of variance (ANOVA) was used to test the significance in the data. Least 4 significant difference test at 5% probability was used to determine the difference among treatment means. All statistical computations were done on MSTAT-C statistics software.

Results
Nitrate content and head traits Different growth media, NH4 + :NO3ratios, and the interaction significantly altered NO3accumulation ( Table 2). The highest NO3content was found in peat media, while the lowest was observed in perlite. Decreasing NH4 + :NO3ratio increased NO3accumulation. The highest NO3accumulation was recorded for 0:100 NH4 + :NO3ratio. However, the lowest NO3accumulation was noted for 60% ammonium and 40% nitrate. As to interaction between medium and NH4 + :NO3ratio, the highest nitrate accumulation was observed in cocopeat and peat media, in which the NH4 + :NO3ratio was 0:100. The lowest nitrate accumulation was seen in perlite with 60:40 or 40:60 NH4 + :NO3ratios in cocopeat, peat, bark and rockwool growth media.
Head weight was significantly influenced by growth media and interaction among NH4 + :NO3ratio and growth media; however, NH4 + :NO3remained non-significant. The highest head weight was noted for peat and cocopeat media, while rockwool resulted in the lowest head weight. Regarding interaction, the highest head weight was recorded for peat with 20:80 NH4 + :NO3ratio. The lowest head weights were obtained from all nitrate ratios of rockwool. Likewise, the impact of growing media, NH4 + :NO3ratios and their interaction on head diameter was significant. The plants grown in peat and perlite media recorded the largest head diameter. However, the lowest head diameter was recorded for the plants grown on rockwool. The 40:60 NH4 + :NO3ratio recorded the highest head diameter, while 0:100 and 20:80 ratios resulted in the lowest head diameter. Regarding interaction, the largest head diameter was noted for peat with 40:60 NH4 + :NO3ratio. The lowest head diameter was recorded for bark medium with 60:40 NH4 + :NO3ratio.
Head length was significantly altered by growth media, NH4 + :NO3ratio and their interaction. The plants grown in peat and bark media had the highest head length whereas rockwool had the lowest head length values. Peat medium with 20:80 NH4 + :NO3recorded the highest head length, whereas rockwool with 60:40, 40:60 and 20:80 NH4 + :NO3ratios resulted in the lowest head length.

Leaf traits
The values of inner, outer, and total leaf count, leaf fresh and dry weights, and dry weight percentage are given in Table 3 and Table 4. Growth media, NH4 + :NO3ratios, and their interaction significantly affected outer leaf count. The highest outer leaf count was noted for rockwool. The highest and the lowest outer leaf count was noted for 60:40 and 20:80 NH4 + :NO3ratios, respectively. Regarding interaction, cocopeat with 60:40 NH4 + :NO3ratio recorded the highest outer leaf count ( Figure 2A).
The NH4 + :NO3ratios, growth media and interaction between growth media and NH4 + :NO3ratio had significant effect on inner leaf count. The highest and the lowest inner leaf count was noted for cocopeat and rockwool, respectively. Generally, decreasing NO3and increasing NH4 + ratio in nutrient solution improved inner leaf count. The highest and the lowest inner leaf count was recorded for 60:40 and 0:100 NH4 + :NO3ratios, respectively. Regarding interaction, the highest and the lowest values for inner leaf count were recorded for cocopeat with 40:60 and rockwool with 40:60 NH4 + :NO3ratio, respectively ( Figure 2B).
Total leaf count was significantly affected by growth media and NH4 + :NO3ratios; however, interactive effect of growth media and NH4 + :NO3ratios was non-significant. Generally, decreased NO3in solution increased total leaf count. The highest total leaf count was recorded for 60:40 NH4 + :NO3ratio, 0:100 and 20:80 ratios resulted in the lowest total leaf count ( Figure 2C).  Different growth media and their interaction with NH4 + :NO3ratio had significant impact on leaf fresh weight; however, NH4 + :NO3ratios were non-significant in this regard. The highest and the lowest values for leaf fresh weight were noted for cocopeat and peat, and rockwool, respectively. Regarding interaction, peat media with 20:80 NH4 + :NO3ratio recorded the highest leaf fresh weight, whereas rockwool with 0:100, 40:60 and 60:40 ratios resulted in the lowest leaf fresh weight ( Figure 2D).
The individual and interactive effects of growth media and NH4 + :NO3ratios significantly altered leaf dry weight. The highest leaf dry weight was noted for peat medium, whereas rockwool and bark media resulted in the lowest dry weigh. The highest and the lowest values for leaf dry weight were obtained from 60:40 and 20:80 and 0:100 NH4 + :NO3ratios, respectively. Regarding interaction, peat with 60:40 and 20:80 NH4 + :NO3ratios recorded the highest leaf dry weight, rockwool with 0:100 NH4 + :NO3resulted in the lowest values ( Figure 2E). Dry matter ration was significantly affected by individual and interactive effects of growth media and NH4 + :NO3ratios. The highest dry matter ratio was recorded for rockwool medium. Similarly, 60:40 NH4 + :NO3ratios resulted in the highest dry matter ratio. Dry matter ratio decreased with increasing NO3ratio in the nutrient solution. Rockwool with 60:40 and 40:60 NH4 + :NO3ratios recorded the highest dry matter ratio, whereas cocopeat with 0:100 ration resulted in the lowest values of dry matter ratio ( Figure 2F).

Stem and root traits
The effects of growing media and NH4 + :NO3ratios on stem length were significant. Plants grown in peat medium had the longest stem length, while bark and rockwool medium gave the lowest stem length. It was observed that the application with the NH4 + :NO3ratio of 60:40 produced shorter stem length compared to the other applications. The longest stem plants were obtained from the 60:40 NH4 + :NO3application in peat medium, and the shortest stem from the 40:60 NH4 + :NO3ratio in bark medium ( Figure 3A).
Stem fresh weight was significantly affected by growth media. However, NH4 + :NO3ratio and interactive effect of growth media and NH4 + :NO3remained non-significant (Table 5). The highest values of stem fresh weight were noted for peat, cocopeat and perlite media, whereas bark and rockwool media resulted in the lowest values. Peat medium with 60:40 NH4 + :NO3ratio resulted in the highest stem fresh weight, while bark medium with 60:40 ratio resulted in the lowest stem fresh weight ( Figure 3B).  9 Different growth media, NH4 + :NO3ratios and their interaction had significant effect on root dry weight. The highest and the lowest dry weight was noted for cocopeat and perlite media, respectively. Increasing NH4 + and decreasing NO3ratio in nutrient solution increased root dry weight. The lowest root dry weight was recorded for 0:100 NH4 + :NO3ratio. Regarding interaction, cocopeat medium with 20:80 and 40:60 NH4 + :NO3ratio recorded the highest rot dry weight, while peat medium with 0:100 ratio resulted in the lowest root dry weight ( Figure 3D).
Growth media, NH4 + :NO3ratio and their interaction significantly altered root length. The bark medium resulted in the longest roots, whereas the shortest roots were recorded for peat and perlite media. Similarly, the longest and the shortest roots were noted for 60:40 and 0:100 NH4 + :NO3ratio, respectively ( Figure 3E).

Discussion
This study examined the impacts of various growth media and NH4 + :NO3ratios in nutrient solution on plant growth and NO3accumulation in lettuce. Growth media significantly affects plant morphology and biomass accumulation (Hahne and Schuch, 2006). Since NH4 + and NO3are two main sources of N absorbed by plant roots, it can be argued that N significantly impacts growth and chemical constituents of plants, including vegetables (Therios and Sakellariadis, 1988;Abu-Rayyan et al., 2004;Wang and Li, 2004). The impact of these two forms of N on plant growth depends on plant species and concentrations of these two (Assimakopoulou et al., 2019). Many crops, including vegetables, can use both NO3-N and NH4-N. Where they use less NH4-N, this is usually because there is less NH4-N in the soil (Vargas et al., 2020). The NH4 + :NO3ratio can be used as a strategy to eliminate abiotic stresses such as low light availability, salinity, drought, and heat. Numerous studies have suggested that growing plants with only NH4 + or NO3with various NH4 + :NO3ratios can affect tolerance of plants low light availability (Raza et al., 2021), water stress (Shang and Shen, 2018) or salinity (Meng et al., 2016;Ashraf et al., 2021).
In this study, NO3accumulation and head weight were higher in peat and cocopeat media than rest of the media. Head diameter was the highest in peat and perlite media, and head length was the highest in peat and bark media. However, the lowest values for head weight, diameter, and head length were noted on rockwool medium. The reason for a heavier head weight in cocopeat and peat media could be their higher water holding capacity, richer water content, and a lower dry matter rate.
Increased NH4 + level in nutrient solution decreased NO3accumulation. The lowest NO3accumulation was obtained for 60% NH4 + and 40% NO3 -. Although impact of NH4 + :NO3ratio was non-significant on head weight, decreasing NO3level in the medium caused a decrease in head weight. It was also observed that low NH4 + level improved plant growth. Moreover, decreased NO3level in nutrient solution increased head diameter; however, decreased head length.
Lettuce is one of the vegetables having high NO3level (Ayaz and Yurttagül, 2006). European Commission sets acceptable maximum NO3level as 4000-4500 mg kg -1 (fresh weight) for winter lettuce and 2500-3500 mg kg -1 (fresh weight) for summer lettuce (Anonimous, 2006). However, it difficult to keep NO3under maximum limit point in regions with low light availability. It is considered that proper NH4 + :NO3ratio in growth media could control NO3level in these regions. The NO3content ranged from 681.00 mg kg -1 (bark, 60:40 NH4 + :NO3 -) to 2995 mg kg -1 (cocopeat, 0:100 NH4 + :NO3 -) in the current study. It was recorded that if NH4 + :NO3ratio was merely changed, huge differences in NO3accumulation would be detected without much yield loss. However, all these levels were within acceptable range. Several studies suggested that a decrease in NO3levels in nutrient solution decreases NO3accumulation in leaves (Marsic and Osvald, 2002;Wang and Shen, 2011;Shang and Shen 2018). In studies on lettuce and iceberg, increasing N doses increased total N and NO3levels (Mordoğan et al., 2001). It is reported that decreased NO3amount in nutrient solution decreases head weight and head length in iceberg (Marsic and Osvald, 2002). Similarly, Assimakopoulou et al. (2019) reported that NH4 + :NO3ratios have no significant impact on total plant fresh weight of cabbage in the first 4 weeks of cultivation. However, they reported that 75:25 NH4 + :NO3ratio decreased many growth parameters eight weeks after cultivation. In another study, various NH4 + :NO3ratios applied on tomato plants under saline and non-saline conditions significantly affected plant length, yield, and fruit size (Ashraf et al., 2021).  noticed that the impacts of N forms on plant length vary depending on plant species. For instance, while a decrease in NH4 + :NO3ratio caused a shorter plant in pepper (Capsicum annuum) (Bar-Tal et al., 2001), it has non-significant impact on plant length in tomato (Lycopersicon esculentum) (Sandoval-Villa et al., 2001) and lily . On the other hand, another study examining the response of saccharum to changing NH4 + :NO3 − ratio reported that the lowest photosynthesis was noted where NH4 + rate was over 60% (Pissolato et al., 2019). It is suggested that if NH4 + is >30% in nutrient solution, photosynthesis and plant growth are disrupted (Pissolato et al., 2019). In another study Chen and Chen (2019) reported that invasive plants grown on only NH4 + had lower photosynthetic rate, pigment content and photo system II activity than the plants grown with NO3and NH4NO3. They also concluded that these plants prefer NO3form of N. The plants growing with aerobic respiration often prefer NO3over NH4 + . However, swapping partly NO3with NH4 + leads to better plant growth (Wang and Shen, 2011). In another study on Brassica pekinesis, NH4 + :NO3 -(10:90) rate increased N metabolism and nitric oxide levels in seedlings, leading to improved tolerance against low light density (Hu et al., 2021). The response of plant species to NH4 + :NO3ratio vary significantly.
Generally, dominant NH4 + content decreases plant growth as the plants prefer NO3 -. However, pH of the medium also decides whether plants prefer NO3or NH4 + . Moreover, the production of secondary metabolites in plants is affected by the NH4+:NO3-ratio in the nutrient solution. Ammonium or one of its assimilation products can serve as a stress signal, and high ammonium treatment can alter the metabolic pathways of plants, producing more reactive oxygen species (ROS) (Bybordi et al., 2012). Excessive accumulation of ROS can damage lipids, proteins, carbohydrates and nucleic acids in plants (Hatamzadeh et al., 2015) and inhibit the normal growth of plants (Du et al., 2021).
Plants grown in rockwool medium in the current study produced higher number of outer leaves than other growth media. Total leaf count in rockwool were like other media; however, it recorded the lowest leaf fresh weight. As the plants in this medium had a short head length, they had shorter leaves, leading to decreased leaf weight. The highest leaf dry weight was recorded on peat medium. Decreasing NO3rate in solution first slightly decreased outer leaf count, and a dramatic increase was recorded later. The highest inner, outer, and total leaf counts were recorded for 60:40 NH4 + :NO3ratio. However, increase in leaf count did not affect head weight. As the NH4 + ratio in nutrient solution increased, leaf dry weight and dry matter rates dramatically increased. The highest leaf dry weight and dry matter rate were obtained from the plants grown on 60:40 NH4 + :NO3ratio. The impact of NH4 + :NO3ratio in nutrient solution might vary depending on plant species.
For iceberg, it is reported that head fresh and dry weights decrease when NH4 + ratio increases (Marsic and Osvald, 2002). In a study on blueberry, it is illustrated that the highest shoot length, leaf count, and plant fresh and dry weights were noted for 5:1 NH4 + :NO3ratio (Yuan-Yuan et al., 2021). In another study on cabbage, it is reported that NH4 + :NO3ratio has no impact on leaf count (Assimakopoulou et al., 2019). Raza et al. (2021) demonstrate that only NH4 + or NO3applied to soybeans under normal or low light availability, leaf fresh and dry weights decreased. However, they also reported that if both N forms are applied together, these levels increase. In another study on the impact of NH4 + :NO3ratio on the growth of Brassica chinensis, it is reported that the highest biomass was obtained from 5:10 NH4 + :NO3ratio, while the lowest biomass value was recorded for 10:5 ratio (Shang and Shen, 2018).
In this study, stem fresh weights on cocopeat, peat and perlite media were higher than other media. The stem dry weights of the plants in cocopeat and stem length of the plant in peat were higher than the plants grown on other media. A decrease in NO3ratio of nutrient solution did not affect stem fresh weight, whereas increasing NH4 + ratio affected stem dry weight. The increase in stem dry weight can be explained with 11 decreasing NO3ratio or increasing dry matter rates in the growth medium. The highest stem length was obtained from 0:100 and 40:60 NH4 + :NO3ratios. Assimakopoulou et al. (2019) reported that increasing NH4 + in the growing medium leads to a decrease in stem fresh weight and stem length. These findings are in parallel with our study.
Root dry weights of the plants grown in cocopeat were considerably higher than the plants grown on other media. The plants grown on bark media recorded the longest roots. Adding NH4 + to nutrient solution increased root dry weight. The experiment in which N in the nutrient solution consisted of only NO3 -(100%) recorded the lowest root dry weights. Increasing NH4 + concentration in the growth medium increased root length as well. The plants grown on the lowest NO3ratio (40%) recorded the longest roots. Likewise, low NO3content increased root dry weight in iceberg (Marsic and Osvald, 2002) and cabbage (Assimakopoulou et al., 2019). Similarly, in another study on lettuce, the highest root dry weights were noted with 75:25 proportioned NH4 + :NO3ratio (Wang and Shen, 2011). In another study on pepper, the longest root and the highest root dry weights was recorded with 25:75 NH4 + :NO3ratio (Zhang et al., 2019). Du et al. (2021) obtained highest root dry weight in lettuce with NH4 + :NO3 -(50:50) or (75:25) ratios.

Conclusions
A proper fertilization is vital for increasing yield and quality of crop plants. The present study revealed that NH4 + :NO3ratios and growth media significantly affected plant growth and NO3deposition. Lettuce plants grow better under stable N availability if NH4 + is added as N source compared to NO3 --only applications. An increase in NH4 + ratio in the solution decreases NO3accumulation by suppressing it. This illustrates that a proper NH4 + :NO3ratio in the solution might support plant growth and decrease NO3accumulation in lettuce. It is hoped that findings of the current study may help in determining an appropriate NH4 + :NO3ratio and growth medium for decreasing NO3accumulation in soilless agriculture.

Authors' Contributions
The experimental design, experiments and data analysis of this article, preparation according to the journal writing rules, and editing works were carried out by SS. The author read and approved the final manuscript.