Effect of Environmentally Friendly Nutrition Supply on Stevia ( Stevia rebaudiana B . ) Production

The experiment was carried out on raised bed (3 rows) with 33 cm row distance and 25 cm between the plants and 24 plant m density. The plantation was prepared on 28 May, 2015 with about 10 cm high transplants. The soil surface was mulched with wooden chips (5 cm layer). The aim was to evaluate the environmentally friendly fertilizers (Dudarit 150 g m, Sprintalga – 0.04% concentration four times 200 mL per plant). During the vegetation period pruning was applied at 30 cm plant height at the 4 double leaf from the soil surface and two cuttings (13 Aug and 29 Sept). By evaluating the effect of treatment on the leaf surface and chlorophyll content, statistically significant results were not found. Higher SPAD (relative chlorophyll content) value was measured in the middle of the growing period compared to the end of the vegetation when the plants were older and the temperature started to decrease. The positive effect of Dudarit and Sprintalga was clear on the plant height and biomass production. The dried herb yield was higher (4 kg m) on treated plots comparing to control (3 kg m). Although increased photosynthetic intensity was not detected on treated plots, the plantation showed greater vitality and side shoot improvement to control plots. Under our circumstances, the environmentally friendly fertilization (Dudarit and Sprintalga) has increased the dried leaves yield with 20% by similar stevioside and rebaudioside-A content.


Introduction
Stevia provides a good alternative for the sweet taste in the diet of people with diabetes besides of protecting against obesity.The dried leaves of stevia are 30 times sweeter than sugar due to the presence of bioactive compounds which bring about its sweet taste (Geuns et al., 2003).
The origin of this species is South America.In the 19 th century, Moises Santiago Bertoni extracted and discovered the favourable effects of bioactive compounds (steviolglycosides) which are 300 times sweeter than sugar.
The steviol glycosides are known as natural sweeteners and they consist of several compounds such as: stevioside, steviol bioside, rebaudioside A, B, C, D, E, F and dulcoside-A.Among them, the most important glycosides are stevioside and rebaudioside-A.The stevioside has a little bitter aftertaste whereas the rebaudioside-A determines the sweeter taste (Pal et al., 2014).
Today several food and drink processing firms use stevia in their products because of the thermal stability of its bioactive compounds.For instance, Kroyer (2010) reported that stevioside showed good stability up to 120 °C.
Firstly, Japan took the stevioside into market as a sweetener.Nowadays the largest production is located in China and 30% of their product is exported.Besides of China, there is successful production in the United States, Canada and Europe as well (Brandle et al., 1998;Amzad-Hossain et al., 2010).
Stevia is famous not only as a sweetener, but it is also known due to its several healing effects such as: anti-caries, lowers blood glucose level, calorie-free (Glycemic Index = 0), regulator of insulin level (Chatsudthipong-Muanprasat, 2009;Goyal et al., 2010;Lemus-Mondaca et al., 2012).For instance, Elnaga et al. (2016) have proved the slimming, blood sugar and cholesterol lowering effects in rat experiments.Furthermore, Khodjaeva et al. (2013) reported that the leaf extract of stevia has influences on hypertension and hyperglycemia.
On the other hand, shortness of calcium supply can cause decreased glycoside concentration (Filho et al., 1997).
During the first part of vegetation the growing of stevia is rather slow.In the middle of summer it can increase when the temperature is higher.During this period side-dressing the leaves and soil is recommended.
To increase the bunching of stevia, the 25-30 cm high plants should have the cutting at the 4 th double leaves; therefore, the side shoots will be increased.After cutting (first harvest) the proper water supply and side dressing is important for renewing the plantation.With this growing method 3-4 cuttings can be realized in a year (Takácsné Hájos et al., 2016).The cutting determines the renewing of the plant, therefore the depths should be taken care of.
The harvest method is mechanical with movable cutting machine which can collect the green biomass in one process.The yield depends on the growing area (number of cuttings), variety and soil conditions.For instance, in Canada 3000 kg ha -1 leaf yield was measured by 105 mg g -1 stevioside concentration (Brandle-Rosa, 1992).Furthermore, in Paraguay the yield by unirrigated condition is 1500-2500 kg ha -1 , and 4300 kg ha -1 by irrigation (Jordan Molero, 1984).
The fresh biomass needs to be spread and dried on aerated and protected area from direct sunshine immediately after cutting.The drying process is important to reduce the water content until 10-14%.For artificial drying the optimal air temperature is 50-60 °C.The binder drying machine is good for this process.After that, the dried leaves have to be removed from the stem.For storing, multilayer paper bags can be used to protect the leaves from humidity.
The aim of the experiment was to evaluate the effect of environmental friendly nutrition supply on the yield and bioactive compounds.

Materials and Methods
The experiment was carried out in the Horticultural Demonstration Garden of the Faculty of Agricultural and Food Science and Environmental Management, University of Debrecen in 2015.The transplanting was done on 28 of May, 2015 on raised bed where two rows have been prepared.The row distance was 33 cm and there was 25 cm between the plants.The experimental plots were mulched with composted food chips.For the treatment Dudarit (Table 1) and Sprintalga products were applied.
Dudarit was used for soil preparation (150 g m -2 ) and for side dressing with irrigation 5 times during the vegetation period (29 of July, 4 of August, 11 of August, 19 of August, 3 of Sept) in 0.04% concentration.
Sprintalga consist of peptides, alga extracts and amino acids.It can stimulate the root development and the intensity of mineral uptake (Biolchim Hungary Ltd.).For the treatment 0.04% solution was applied four times (30 of June, 14 of July, 4 of August, 11 of August) during the vegetation period with 200 mL per plant dose.The soil analysis before the treatment is presented in Table 2.
The data of temperature during the vegetation period is shown in Fig. 1.
Stevia rebaudiana B. belongs to the Stevia genus (Asteraceae).The plant is about 40-60 cm high, but in its origin it can reach even 100 cm (Mishra et al., 2010).Stevia is a perennial species, however, in Europe it is grown as one year production.The stevia's feeder root is extensive and is located near to the soil surface.Besides, it is sensitive to excessive moisture which is the main reason why irrigation must be strictly monitored.The stem and the leaves are green and lightly covered with gland cell.The white tubular flowers are on the end of seed stalk.Regarding to the seeds (2-3 mm), their ability to germinate is extremely low, therefore, vegetative propagation is often applied which can assure the constant stevioside level in the progeny plants (Singh and Rao, 2005).
Stevia demands high temperature, the optimal is between 20 to 24 °C.It cannot resist temperature below 9 °C, but in autumn it can tolerate even 0 °C (Singh and Rao, 2005).
For the temperature change it is rather sensitive, because its influence on stevioside synthesis.In India the development of stevia is good even on 30 -40 °C (Chalapthi et al., 1997).According to Barathi (2003) and Takácsné Hájos et al. (2016), the optimum condition for stevia is when the temperature does not increase above 40 °C during the day and does not decrease below 10 °C during the night.Besides, the length of sunlight hours and the intensity of sunshine have favourable effects on the vegetative improvement and on stevioside content (Valio-Rocha, 1966;Metivier-Viana, 1979;Zaidan et al., 1980;Viana, 1981;Mizukami et al., 1983).
The water need is medium; however, on temperate zone it should be irrigated.Drip irrigation is suggested with additional water supply (25-30 mm) occasionally.Therefore, high humidity can be avoided which is favourable for fungi diseases such as Septoria sp. and Sclerotinia sp.(Chang et al., 1997;Jain et al., 2014).During the vegetation it is necessary to ensure balanced water supply which can be carried out by mulching besides of protecting the plants against the weeds.Stevia has higher water needs after transplanting and cutting (Andolfi et al., 2002).
For the proper development of stevia, the soil needs to have pH between 5 to 7, low salt content and high water capacity.The optimal NPK supply is 50:25:25 kg per hectare which is given 2-3 times during the vegetation (Rank-Midmore, 2006).
Positive correlation was observed between the amounts of bioactive compounds such as: stevioside, rebaudioside-A and the proper nutrient supply of the soil.For instance, Sheu and co-workers (1987) increased the stevioside and rebaudioside-A content by boron treatment (5 ppm concentration).Besides, Acuna et al. (1997) detected that Humiforte (synthetic amino acid, N, P, K and microelements) and Aminol (amino acid and N) combination is one of the most efficient methods to increase the stevioside content.Furthermore, proper potassium supply can increase resistance, yield (Ma and Shi, 2011) and stevioside content (with 5.08%) in form of potassium-nitrate (Pal et al., 2013).
In the experimental year, the natural precipitation was not sufficient (200 mm) during the growing period; therefore, the plantation was irrigated regularly two times a week with 30 mm water.• dry weight (g m -2 and g per plant)

Measurements on open field
• leaf and stem ratio by different cutting times

Analytical measurements
• stevioside (g per 100 g dry weight) • rebaudioside-A (g per 100 g dry weight) with HPLC method (Kolb et al., 2001) Results and Discussion

Plant height
The evaluation of plant height depends on control and treated plots and the time of pinching and cutting (Fig. 2).
Slow development was detected until the 4 th measurement.After the pruning (between the 4 th and 5 th measurement) the side shoot development was increased.The pruning followed by increased temperature caused favourable effect on plant height.Thereby, it seems that the warm condition improved the regeneration of the plants after cutting.
The favourable effect of treatment on plant height was proved not only after pinching, but also at the final period of vegetation.Similar effect was observed on fresh biomass production (Fig. 3).
The treatment was efficient on the stevia plants, which can be explained by the improved root system and better ability of water and mineral uptake.
The marketable product of stevia is the dried leaf.Therefore, it is important to follow the leaf and stem ratio which is higher at the first cutting (Fig. 4).
The 1 st cutting showed higher leaf stem ratio against the 2 nd cutting.Between the treated and control plots no statistical differences were found.
Favourable effect of treatment was determined on the leaf weight per plant during the second cutting.The leaf yield was higher (10.23 g per plant) on the treated plots than on the control ones (6.33 g per plant) which is statistically significant.

SPAD measurements
The relative chlorophyll content of leaves was evaluated during the vegetation.The treated plants have showed higher SPAD values (30.08-41.63),however these values were not statistically significant (Fig. 5).Furthermore, great differences were found among the different measurement times.Lower values were detected at the end of the growing period which can be explained by elder leaves.LAI measurements Previously, positive correlation was observed between the weight of biomass and the leaf area.Thus, it was measured (leaf area m -2 ) in the experiment (Fig. 6).
The increasing effect was detected till the cutting time.The highest LAI value (2.23-2.39m 2 per m 2 ) was measured before the second cutting.

Correlation by Pearson
The results of correlation analysis by Pearson are shown in Table 3.
The correlation between the treatment and plant height (r = 0.508) was proved only by the second cutting.It can be explained by the favourable effect of Dudarit and Sprintalga which was expressed rather at the end of the growing period.Similar tendency was detected between the treatment and the biomass production, where the value for the first cutting was r = 0.635 and for second cutting r = 0.931.
The correlation between plant height and SPAD was medium strong negative (r = -0.509)at first cutting, while between the plant height and the biomass this value was r = 0.477.

Bioactive compounds
Stevioside and rebaudioside-A content were measured as the most important bioactive compounds of stevia (Fig. 7).Statistical differences were not found between the control and treated plots.
Stevioside concentration was measured between 161.5-168.7 mg g -1 on treated and control plots.These values are 204 higher than the ones detected by Brandle-Rosa (1992) in Canada (105 mg g -1 ).Vafadar et al. (2014) and Gautam-Kafle (2011) measured much lower amounts on non-field experiments, 48.05 mg g -1 and 57-76 mg g -1 respectively.The rebaudioside-A content was between 11.7-13.3mg g -1 which is similar to the amount measured by Gautam-Kafle (2011); 17-21 mg g -1 respectively.The lower amount can be explained with the negative correlation between stevioside and rebaudioside-A due to their biosynthetic relationship (Shibata et al., 1991).
The experiment proves that the Hungarian climatic condition is favourable for stevia production with good inner quality due to the influence of daily solar radiation during the summer period.

Conclusions
Our results proved that stevia production can be successful in Hungary propagated by transplants at the end of May.With the two cuttings the total biomass was about 4 kg m -2 which is more than 700 g dried material.The bioactive compounds of the dried material are the following: stevioside (161.5 mg g -1 ) and rebaudioside-A (11.7 mg g -1 ) which were measured on treated plots.The Dudarit and Sprintalga treatment could not show statistically favourable effect on these parameters to control plots.
In our experiment, the environmentally friendly fertilisers produced good effect on leaf yield (10.23 g per plant) comparing to control (6.33 g per plant) by second cutting.

Fig. 2 .
Fig. 2. The plant height (cm) of treated and control plots (Debrecen, 2015) Fig. 3. Yield of raw and dry weight (g m -2 ) (Debrecen, 2015) is valid with 95% confidence.**The result can be used with 99% confidence.Where is no sign (*) there is no statistically significant difference between the values.