- Email: [email protected]
Effect of nutrient omission and pH on the biomass and concentration and content of steviol glycosides in stevia (Stevia rebaudiana (Bertoni) Bertoni) under hydroponic conditions
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Journal of Applied Research on Medicinal and Aromatic Plants journal homepage: www.elsevier.com/locate/jarmap
Effect of nutrient omission and pH on the biomass and concentration and content of steviol glycosides in stevia (Stevia rebaudiana (Bertoni) Bertoni) under hydroponic conditions ⁎
Geeta Gautam Kafle, David J. Midmore , Resham Gautam School of Health, Medical and Applied Sciences, Central Queensland University, Rockhampton, Queensland, 4702, Australia
A R T I C L E I N F O
A B S T R A C T
Keywords: Hydroponics Screen-house Macronutrient Micronutrient Photosynthesis SPAD reading
Steviol glycosides (SGs), have recently been approved in western countries as sources of intense natural sweeteners. SGs are found in Stevia rebaudiana, mainly in the leaves, and industry prefers rebaudioside A (Reb-A) over other steviol glycosides for its superior flavour profile. Hence leaf biomass and concentration of SGs (and their product, SG content) are of primary agronomic interest. Experiments were conducted under controlled conditions in nutrient solution to assess the effects of nutrient deficiencies and pH on biomass production, and concentration and plant content of SGs. Total SG content was low in plants deficient in the macronutrients N, P, S, Mg or Ca because of reduced photosynthesis and because of the decreased leaf yield, even though lack of N resulted in greater concentration of stevioside in the leaves. Lack of N or P reduced the proportion of Reb-A to total SGs. Plants deficient in K had less yield than in the nutrient-complete control, but not significantly so and SG concentration in the leaves was similar to that of the control. Deficiency of the micronutrients Cu and Fe led to low SG yield, because of reduced SG concentration in leaves, and because of reduced leaf yield, respectively. Lack of other micronutrients did not influence SG content. Neutral to alkali conditions reduced plant growth and leaf yield, most likely due to deficit of P, but pH had no effect on SG concentration. Our results are indicative, but preliminary, and require confirmation in open field trials over several years.
1. Introduction Stevia (Stevia rebaudiana (Bertoni) Bertoni) is known for its sweet tasting compounds, steviol glycosides (SG), found in greatest concentration in the leaves (Kulasekaran et al., 2006). Production of high SG concentration in plentiful leaf biomass is an important goal in commercial stevia production. Interest in commercial production of stevia has intensified with approval, firstly in Australia and then in the USA and more recently in Europe, of steviol glycosides as intense sweeteners (FSANZ, 2008; Anon., 2011). In its natural habitat, stevia is found in infertile, acid sands or muck soils (Shock, 1982) although commercial production tends to take place on somewhat better soils. It is well known that plant growth in general is stunted when deprived of nutrients, whether due to insufficient quantity or to pH-conditioned non-availability in the growing medium, or to insufficient water for uptake. Each element plays one or more particular biochemical/physiological roles, so deficiency of that element will result in a set of predictable metabolic and physiological disturbances. Reports on the influence of nutrient deficiency (and toxicity) on stevia growth and SG yield are scarce. Low availability of soil N leads to ⁎
low leaf N concentration, reduction of photosynthesis (Sharma et al., 2016) and low leaf yield compared to adequate soil N, but it also leads to a higher concentration of SG in leaves than with adequate soil N (Barbet-Massin et al., 2015). In a similar manner, higher rates of NPK or farmyard manure led to higher leaf yield but lower SC concentration in trials in the western Himalayas (Kumar et al., 2012), and highest leaf SG concentration (16.7% w/w) was achieved by omission of N compared to that found in the N-supplied control (11.5% – Das et al., 2006). Utumi et al. (1999) found that total above ground biomass decreased with deficiencies of any macronutrient, however, the percentage of reduction was significantly higher in treatments without N, K, or Mg than with deficiencies of S, Ca or P. The concentration of SG decreased with the deficiency of all macronutrients except for P (by 27% for Ca, 24% for N, to 41% for S), causing a reduction in content of SG per plant. This contrasts with the earlier mentioned increased SG concentration in leaves of plants lacking N, and the reported lack of response of total leaf SG concentration to deficiency of any macro or micronutrient by Jarma et al. (2012), although concentration of Reb-A did decline with lack of P, S, K or Cu. Plant nutrient availability is highly dependent on the pH of the
Corresponding author. E-mail address: [email protected] (D.J. Midmore).
http://dx.doi.org/10.1016/j.jarmap.2017.08.001 Received 21 January 2017; Received in revised form 31 July 2017; Accepted 3 August 2017 2214-7861/ © 2017 Elsevier GmbH. All rights reserved.
Please cite this article as: Midmore, D.J., Journal of Applied Research on Medicinal and Aromatic Plants (2017), http://dx.doi.org/10.1016/j.jarmap.2017.08.001
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
G.G. Kafle et al.
growing medium. Nutrients such as phosphorous, magnesium and calcium are less available when the pH is below 5 (Jones, 2005). Similarly, at high pH iron, manganese, copper, zinc and boron are only sparingly available due to their low solubility (Jones, 2005). As a result, when the pH is outside of the optimal range, deficiency symptoms are seen on the above ground plant, although it is commonly difficult to ascribe the symptoms to deficiencies of individual elements. For stevia, Shock (1982) reports that even though native to low pH (4–5) soils, it grows well on less acid soils ranging from 6.5–7.5. Rank and Midmore (2006) reported that plants grown on neutral to alkali soils showed less plant yield compared to that on acidic soils. The specific effects of pH and of nutrient deficiency on biomass yield and SG content in stevia are largely unknown. This study therefore set out to determine the consequences of macro and micronutrient deficiency on morphology, biomass accumulation, and SG concentration and content in stevia plants. This was complemented by a study to identify the optimum pH required for the same. Visual symptoms of nutrient deficiency on stevia shoot and root colour and morphology from these experiments are reported by Midmore et al. (2012).
Table 1 Effect of different nutrient deficiencies on leaf chlorophyll concentration, photosynthetic rate, leaf dry biomass and shoot to root dry weight ratios of stevia at the time of harvest (at four weeks after treatments imposed). Values within a column followed by the same letter are not significantly different (P < 0.05).
2. Material and methods
Treatments
Chlorophyll concentration (SPAD units)
Photosynthetic rate (μmol m−2 s −1)
Leaf biomass (g/plant)
Shoot to root ratio
Complete No Mn No Zn No Mo No B No Cu No Cl No K No Ca No S No Mg No Fe No P No N No Micro No NPK
53.45 a 52.90 a 50.45 a 50.15 a 50.00 a 49.90 a 46.50 a 46.45 a 33.50 b 31.70 bc 28.00 bcd 25.75 bcd 22.70 cd 22.05 d 21.35 d 20.90 d
12.73 bc 11.28 bc 11.57 bc 13.03 c 15.51 c 13.12 c 15.89 c 10.43 bc 0.66 a 5.78 ab 1.45 a 2.98 a 1.49 a 2.60 a 0.87 a 0.28 a
3.96 d 3.12 cd 2.70 bcd 4.11 d 2.19 abcd 3.31 d 3.18 cd 3.27 d 1.09 abc 0.73 ab 1.12 abc 1.11 abc 0.38 a 0.38 a 0.43 a 0.30 a
12.6 ef 8.6 cde 7.6 bcde 10.2 de 9.6 de 9.5 de 8.9 cde 17.5 f 10.9 de 2.6 ab 9.8 de 3.6 abc 2.3 ab 1.7 a 6.5 abcd 2.8 ab
2.1. Location from 25 to 30 °C. The experiment consisted of 16 treatments (Table 1) each randomised in two blocks. Each treatment in each block, i.e., each box, comprised four plants, giving a total of 128 plants in the experiment. Data were collected from each plant to estimate sampling error.
Two experiments (one nutrient omission and one with differing pH) were conducted at CQUniversity, Rockhampton (23° 22′, 0.345”S, 150° 31′ 0.53”E), Australia, using a non-circulatory hydroponics system (Midmore, 1994) inside a screen-house with 67% full sunlight. 2.2. Plant material
2.4. Treatments and experimental design for the pH experiment
Seeds of Stevia rebaudiana variety ‘Shoutain-2′ were sown into 1:1 perlite:vermiculite media in speedling trays inside the screen-house on 17/08/09 for the nutrient omission and on 25/04/10 for the pH experiment. Following germination, seedlings were watered with half strength Manutec hydroponic solution (Manutec Pty. Ltd.) for three weeks in the nutrient experiment and for four weeks in the pH experiment. They were then transferred to 7 cm diameter poly-pots lined with mesh and filled with perlite, for a further 4 weeks and watered as before. At the 6–8 leaf stage, with plant height ranging from 6 to 8 cm, seedlings for the nutrient experiment were supplied with reverse osmosis (RO) water for two weeks, and then subjected to treatment. Seedlings for the pH treatments were not preconditioned with RO water.
In common practice for plants grown in soilless culture pH is raised by adding NaOH or KOH and lowered by adding H2SO4 or HNO3 to the solution (Jones, 2005). Half strength commercially available hydroponics fertilizer (Manutec Pty. Ltd) was used as a nutrient medium with pH of 6.7 and EC 1.45 dS/m. To bring the pH to the desired level 710, 580 or 355 ml of 0.25 M H2SO4 were added to achieve pH values of 4, 5, or 6, and likewise 35 or 125 ml of 1.0 M NaOH per 170 l were added to achieve pH values of 7 or 8, respectively. Styrofoam boxes and solution monitoring were as in the nutrient experiment, and each treatment was replicated six times, in a completely randomised design. Solution pH was maintained at 4, 5, 6, 7 and 8 daily by adding appropriate amounts of H2SO4 or NaOH. Mean maximum and minimum temperatures during the experimental period were 24.1 and 13.2 °C.
2.3. Treatments and experimental design for the nutrient omission experiment
2.5. Data collection
The chemical composition of the nutrient-deficient solutions was similar to that reported by Roberts and Whitehouse (1976). In essence, the following (in mmol) was the composition of the complete nutrient solution: N (10.054), P (1.17), K (3.36), Ca (3.35), Mg (1.5), S (1.5), Fe (0.51), Mn (0.01), Cu (0.0012), M0 (0.0002), B (0.323), Zn (0.0022), Cl (0.1767) and Na (0.0005). Omission treatments were so designed that, with exception of Na (which ranged from 0.119 to 7.065), S in the −Mg (0.68), and Cl in the −Fe (0.27), concentrations did not differ from the control. Styrofoam boxes (53 cm x 23 cm × 25 cm) were lined each with a black plastic bag to prevent leakage. Four holes were made on the lid of each box to hold the 7 cm diameter poly-pots. The plant to plant and row to row distances were maintained at 13 cm and 25 cm, respectively. Solution was filled to a depth of 15 cm, and height of the solution and pH and electrical conductivity (EC) were monitored daily between 9:00 and 10:00 am and adjusted accordingly. The pH for the treatments was c. 5.5–6.0 and EC varied between treatments. Mean maximum and minimum temperatures during the experimental period were 33.1 and 20.1 °C. The temperature of nutrient solutions ranged
Leaf chlorophyll concentration was estimated using a SPAD meter (Konica, Minolta Japan), with readings taken 3 and 4 weeks after the start of treatment in the nutrient experiment and at fortnightly intervals for the pH experiment. Youngest fully expanded leaves were used for the measurement. Leaf gas exchange (photosynthesis, transpiration and stomatal conductance) for all the treatments in both experiments was measured using an IRGA (Infrared Gas Analyser, model LCA-4 from ADC-UK). Measurements were made at 4 weeks following the start of treatments in the nutrient experiment and at fortnightly intervals for the pH experiment. The IRGA readings were taken between 11:00 a.m. and 2:00 p.m., on the same leaves used for chlorophyll determination. For the measurement of leaf steviol glycoside concentration (i.e., amount of SG per unit dry weight of leaf, expressed as %), two youngest fully expanded leaves from each plant of every treatment were removed three weeks after treatments began. The eight leaves from each box were combined and oven-dried at 60 °C for 48 h, ground to a fine powder with a mini bead-beater, and stored in air-tight containers. The 2
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
G.G. Kafle et al.
Fig. 1. View of pH experiment inside the screenhouse with stevia plants growing in hydroponics.
SG concentration of the samples was analysed with HPLC using a modification of the procedure described by Hearn and Subedi (2009). For the SG quantification through HPLC, pure stevioside (99.9% purity) and rebaudioside A (Reb-A, 97.4% purity) obtained from Wako Pty Ltd., Japan were used to make standard solutions of 0.25, 0.5 and 1.0 g/L for both stevioside and Reb-A in EtOH 70% (w/w) providing a three point calibration as suggested by Hearn and Subedi (2009). Plants were harvested four weeks after treatments began in the nutrient omission experiment and after eight weeks for the pH experiment. Plant height (measured from the base of the stem to the apical tip) and leaf and stem fresh and dry weights were measured for each plant. Root weights were measured per box since it was not possible to separate on a per plant basis. The product of leaf dry weight and concentration of leaf SG is presented as content of SG. A view of the pH experiment is given in Fig. 1.
3.1.2. Leaf gas exchange The photosynthetic rate of plants grown in the complete nutrient solution (12.73 μmol CO2 m−2 s −1, LSD = 7.1) differed significantly from the treatments without Fe, N, P, Mg, micro, Ca and NPK (ranging from 2.98 to 0.28 μmol m−2 s −1). However, photosynthetic rates in treatments lacking B, Cu, Mo, Zn, Mn, K and S were similar to that of the control (Table 1). The treatment without Cl had the highest photosynthetic rate (15.89 μmol m−2 s −1) while that without NPK had the lowest. The transpiration rate differed significantly between treatments (data not presented) and followed the same pattern as for photosynthetic rate. Transpiration rate in the treatment without Cl was highest (12.46 mmol H2O m−2 s −1, LSD = 4.5), slightly less in the control (10.01 mmol H2O m−2 s −1) and the lowest (2.75 mmol H2O m−2 s −1) was recorded in the treatment without micronutrients.
2.6. Statistical analysis
3.1.3. Growth parameters Total above ground (shoot) biomass of stevia grown in solution without the micronutrients Mo, Cu, Cl, Mn, and Zn and the macronutrient K, did not differ significantly from plants grown in the complete nutrient treatment (Fig. 2). Likewise, plant height did not differ between these treatments and the control (data not presented). Significant reductions in shoot biomass were found for the treatment lacking B, Fe, Mg, Ca, S, P, N, NPK or all micronutrients when compared to the nutrient-complete control (Fig. 2). Leaf (Table 1) and stem (data not presented) dry weights responded similarly to omission of the various nutrients as did above ground biomass while root dry weight, besides varying less between extremes of treatments, only differed from the control when Ca, all micronutrients or the combination of NPK were omitted (Fig. 2). The shoot to root ratio was close to the highest in the complete treatment, being only higher in the treatment lacking K (Table 1), and lowest in the treatments lacking N, P or NPK.
Data were analysed using the statistical package for ANOVA (analysis of variance) through Genstat version 11.1. Difference between means is reported as significant at P < 0.05. 3. Results 3.1. Nutrient omission 3.1.1. SPAD reading (surrogate for chlorophyll concentration) After four weeks of treatment, the SPAD reading for the complete treatment was 53.5 SPAD units, and was not affected by treatments lacking Mn, Zn, Mo, B, Cu, Cl or K (Table 1). The treatment without NPK had the lowest SPAD reading (20.9), 61% less than that of the complete treatment, and together with absence of Mg (28.0), Fe (25.8), P (22.7), N (22.1) and micronutrients (21.4), were all significantly less that for the complete treatment. The SPAD reading were higher by 2–3% in the control treatment and in most of those similar to the control at week 4 compared to readings at week 3 (data not presented). Those below the control in week 4 were generally with values lower than at week 3.
3.1.4. SG concentration and content in stevia leaves The total SG (stevioside + Reb-A) concentration varied from 3.0–12.1% dwt, but was only significantly less than in the control with lack of Cu or lack of all micronutrients (Table 2). Absence of Zn raised 3
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
G.G. Kafle et al.
Fig. 2. Average total above ground biomass (left hand side) and root dry weight (right hand side) of stevia (g/plant) grown with various nutrient deficiencies. Plants harvested after 4 weeks of treatment. Treatments with the same letter do not differ significantly at P < 0.05.
3.2. Solution pH
Table 2 Effect of different nutrient deficiencies on stevioside and Reb-A and total leaf concentration and total leaf SG content of stevia at the time of harvest (at four weeks after treatments imposed). Values within a column followed by the same letter are not significantly different (P < 0.05). Treatments
Stevioside (% dw)
Reb-A (% dw)
Total SG (stevioside + Reb-A) (% dw)
Total SG leaf content (mg/ plant)
no Zn no P no N no Cl no Mn no S no Fe complete no B no Mo no NPK no Ca no K no Mg no micro no Cu
7.8 7.2 6.4 6.1 6.0 6.0 5.6 5.2 5.1 5.1 4.7 4.3 4.0 3.8 2.8 1.9
4.2 3.5 2.4 3.0 3.5 3.1 3.9 3.6 3.4 3.7 1.3 3.7 2.4 2.1 1.1 1.1
12.1 a 10.7 a 9.0 ab 9.2 ab 9.6 ab 9.2 ab 9.6 ab 8.9 ab 8.6 ab 8.9 ab 6.1 bc 8.1 bc 6.5 bc 5.9 bc 4.0 c 3.0 c
350.0 ab 18.3 d 38.6 d 314.5 abc 323.0abc 78.2 cd 109.6 bcd 379.7 a 198.6 abcd 414.3 a 14.3 d 96.1 cd 209.3 abcd 125.0 bcd 20.8 d 116.8 bcd
a ab abc abcd abcd abcd abcd bcde bcde bcde bcde cdef cdef def ef f
a a a a a a a a a a a a a a a a
3.2.1. SPAD readings and leaf gas exchange At pH 6 and above SPAD readings show that the chlorophyll concentration was reduced compared to the values at pH 4 and 5. None of photosynthetic rate, transpiration rate or stomatal conductance differed between pH treatments (Table 3) although stevia grown at pH 6–8 had markedly lower values for all three parameters.
3.2.2. Growth parameters Above ground plant biomass was generally greater in plants grown between pH 4 and 6 than at pH 7 and 8. The plant biomass at pH 8 was one half that at pH 4–6 (Table 4) although plant height did not differ significantly between treatments (data not presented). Leaf dry weight did not differ between plants grown at pH 4, 5, 6, and 7, and all were greater than that at pH 8. Leaf weight at pH 8 was 62% that at pH 4 (Table 4). Stem dry weight was more sensitive to high pH than was leaf dry weight, and leaf dry weight (in relative terms) was more sensitive to high pH that was root dry weight (Table 4). Nevertheless, root weight was markedly and significantly lower in plants grown at pH 8 than those at pH 4–7 (Table 4). Branch number was highest at pH 6, that with the highest stem, leaf and total above ground biomass (Table 4), followed by pH 5, 4 and 7. Lowest branch number was recorded at pH 8 (Table 4).
total SG concentration by 36% above that of the control, and the absence of P by 20% but the differences between these and the control were not significant. There was significant difference in stevioside concentration between the treatments (P < 0.01). Plants grown without Zn had highest stevioside concentration, and this was significantly greater than that of the control (Table 2). There was no significant difference in Reb-A concentration between the treatments. However, the highest value of RebA was without Zn and the order of response of Reb-A to nutrient omission was similar to that of stevioside. The SG content on a per plant basis (i.e., the product of leaf weight per plant and concentration of SG in the leaf) was highest in plants grown without Mo (414.3 mg/plant) followed by the complete treatment and by those lacking Zn, Mn, Cl, K, or B. The SG content was related to variations in either the total leaf yield or concentration of SG in the leaf (Tables 1 and 2). For example, in treatments lacking S, P, N and Ca the SG concentration was similar to that of the control but due to a reduction in their leaf biomass the total leaf SG yield was low. For Cu deficient plants the total SG concentration was the lowest (3%) but because of the relatively high leaf yield per plant the total leaf SG content was higher in this treatment than that in a number of other treatments, particularly that lacking all micronutrients (Table 2).
3.2.3. SG concentration and content No significant difference between the pH treatments was found for leaf concentration of either stevioside or Reb-A, nor of total SG (Table 5). On average plant SG content at pH 4 was the highest and it was lowest at pH 8.
Table 3 Photosynthetic rate, transpiration rate, stomatal conductance and leaf chlorophyll concentration (SPAD units) of stevia grown at different pH (average of three dates of measurement). Values within a column followed by the same letter are not significantly different (P < 0.05).
4
Solution pH
Photosynthetic rate (μmol m−2 s −1 )
Transpiration rate (mmol m−2 s −1 )
Stomatal conductance (mmol m−2 s −1 )
Chlorophyll concentration (SPAD units)
4 5 6 7 8
11.17 11.33 11.45 10.69 10.34
3.06 3.02 3.46 2.86 2.72
0.16 0.17 0.23 0.15 0.13
46.6 b 46.5 b 44.3 a 43.8a 42.8 a
a a a a a
ab ab b a a
a ab b a a
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
G.G. Kafle et al.
Malavolta, 1997; Utumi et al., 1999). Shoot biomass decreased proportionally more than root biomass with macronutrient deficiencies (i.e., shoot to root ratios were least in these treatments, although again lack of K was an exception – Table 1). This is consistent with the interpretation that the plant assigns resources to the growth of the root as the main nutrient absorption organ (or rather does not reduce resource allocation to the roots to the same extent as the reduction to the shoots – Hermans et al., 2009). The additional resource allocation to roots is not only to satisfy the demand for additional root biomass per se, but also for a proportionately higher rate of respiration in roots than that of shoots under diminished nutrient supply (Lambers et al., 2008). It is interesting that lack of K did not increase investment in root relative to shoot biomass, a result not consistent with the literature on stevia, for Utumi et al. (1999) reported that only magnesium deficiency among the elements trialled reduced root relative to shoot dry weight. Greater investment in roots (or poorer growth of above ground organs) was evident in the treatments that lacked the macronutrients N, P or S (Table 1).
Table 4 Dry weight of stem, leaf, root and total biomass, shoot to root ratio, and number of branches per plant at harvest of stevia grown at different pH. Values within a column followed by the same letter are not significantly different (P < 0.05). Solution pH
4 5 6 7 8
Dry weight (g/plant) Stem
Leaf
Root
Total above ground biomass
12.2 c 10.8 bc 12.7 c 7.8 ab 4.9 a
14.1 b 14.5 b 15.2 b 12.8 b 8.9 a
8.8 7.7 7.4 8.5 6.5
26.3 25.2 27.9 20.6 13.8
d bc ab cd a
bc bc c b a
Shoot to root ratio
Branch no. (per plant)
2.99 3.14 2.57 2.41 2.12
8.4 b 9.4 bc 11.6 c 7.8 ab 4.9 a
bc c c ab a
Table 5 Percent dry weight of stevioside, Reb-A and total steviosides in leaves of stevia, and total content per plant grown at different pH. Values are means of six replicates with one missing value. Solution pH
Stevioside (% dry weight)
Reb-A (% dry weight)
Total SG (% dry weight)
Total SG (mg/plant)
4 5 6 7 8
2.68 2.69 2.47 2.97 3.00
0.67 0.74 0.56 0.85 0.98
3.36 3.43 3.03 3.82 3.99
478 477 436 505 354
4.1.2. Micronutrients and biomass Turning to the effects of lack of micronutrients on stevia, on which very little has been published, deficiency of iron leads to a decrease in chlorophyll content (Salisbury and Ross, 1992), as was very evident by the low SPAD reading for iron omission in our study. Iron deficiency symptoms in treatments lacking Fe (lack of Fe alone or lack of all micronutrients) appeared after two weeks as general chlorosis (Midmore et al., 2012), and were severe after four weeks, with necrosis of young leaves. This would be expected to contribute to the low rate of photosynthesis and above ground biomass in this treatment (Table 1 and Fig. 2). Relative investment in shoot biomass compared to that in roots was low in plants which lacked Fe, alone or in the treatment without any micronutrients (Table 1). Plants lacking Cu showed deficiency symptoms (Midmore et al., 2012), chlorosis in older leaves and brown necrotic spots in mature leaves, similar to that reported for tomato (Weir and Cresswell, 1993) but photosynthetic rate and SPAD readings did not differ from the control and plant biomass, stem yield, leaf yield, root weight were only slightly reduced, but not significantly so, compared to the control. Copper is somewhat more mobile in plants than is iron, resulting in the treatment lacking copper having a higher measured rate of photosynthesis and less suppression of above ground biomass than in the treatment lacking iron. Deficiency symptoms in plants lacking Mo, Cl, Zn or Mn were not prominent (Midmore et al., 2012) and above ground biomass, root weight, photosynthetic rates and SPAD readings did not differ significantly from the complete treatment. Of these three are reasonably mobile within the plant (not Mn) and may have been mobilised to younger leaves, Mo for redox reactions, Cl for water splitting in photosynthesis and Zn for chlorophyll production, all leading to sustained biomass production over the period of deficiency.
4. Discussion 4.1. The effect of nutrient deficiency on biomass, SG concentration and SG content 4.1.1. Macronutrients and biomass Reduced growth is a means by which plants concentrate minerals in plant tissue when supply is constrained. As expected therefore (Sharma et al., 2016), biomass yield was decreased relative to that of the control treatment for all the macronutrient deficiency treatments, although the effect of lack of K was not significant. Insensitivity of stevia biomass to lack of K has been reported earlier by Kawatani et al. (1978). Photosynthetic rate of plants lacking K also did not differ from that of the control, whereas omission of N, P, S, Mg or Ca led to notably lower photosynthetic rates (Table 1) and biomass (Fig. 2) than those of the control. Utumi et al. (1999) reported above ground biomass of stevia to be respectively 47% and 85% that of the control when N and S were omitted. In the current study reductions were even greater – biomass was respectively only 11% and 20% that of the complete control when N or S were omitted (Fig. 2). This was in line with our data showing (a) that the rate of photosynthesis in the treatment lacking N was only 20% that of the control (2.60 vs. 12.73 μmol m−2 s −1, LSD 5% = 7.07) and (b) the SPAD reading for the treatment lacking N was less than one half that of the control (Table 1). Positive correlations between leaf N concentration and CO2 assimilation rates reported by Barbet-Massin et al. (2015) for stevia, back this up further. While Kawatani et al. (1978) reported that biomass production in plants lacking P was less affected than by lack of N, our data show that lack of P or lack of S was as suppressive of above ground biomass production and rates of photosynthesis as was lack of N. Biomass and photosynthetic rates for plants lacking Ca or Mg were also well below those of the control plants receiving all necessary nutrients. The visual symptoms associated with macronutrient (N, P, Ca, Mg and S) deficiency of poor root development, stunted growth and chlorosis and necrosis of the leaves as reported by Midmore et al. (2012) for the same plants were consistent with that expected for plants in general (e.g. Weir and Cresswell, 1993), and also matched the symptoms reported by previous authors for macronutrient deficiency in stevia (Lima Filho and
4.1.3. Macronutrients and SG concentration and content In contrast to primary metabolites, concentration of secondary metabolites such as SGs exhibit high plasticity under selection pressure (Brandle and Rosa, 1992; Hartmann 2007) and are subject to environmental factors and agronomic management (Ceunen and Geuns, 2013; Pal et al., 2015). Within agronomic management, nutrient deficiencies can affect the accumulation of secondary metabolites. For example, when grown under deficit K (Ferreira, 2007) and P (Usha and Swamy, 2002) conditions, Artemisia annua produces high concentrations of the secondary metabolite, artemisinin. Freitas et al. (2008) also reported an increased concentration of vitexin in the leaves of Passiflora alata when grown under N deficiency conditions. Kawatani et al. (1978) reported that neither deficiency of N nor P affected leaf SG concentration, but in contrast Utumi et al. (1999) showed decreased SG concentration in P and N deficient plants (by 7.8% and 22.8%, respectively) and an even lower SG content per plant due to reduced 5
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
G.G. Kafle et al.
that of stevioside (Table 2). Exceptions to this were treatments lacking N, P, NPK or all the micronutrients where Reb-A was less than one half of the total SG. Diminished supply of N was recently (Barbet-Massin et al., 2015; Tavarini et al., 2015) shown to reduce the ratio of Reb-A to stevioside concentration: quite possibly the lack of N suppresses UDPdependent glycosyltransferases responsible for the transformation of stevioside into Reb-A, in a similar manner to what occurs in leaves of stevia plants transferred from long to short days, both linked most likely to an increased plant capacity for ROS scavenging (Mohamed et al., 2011).
biomass. In our study, lack of N did not reduce SG concentration (although it did not increase it, at least not for Reb-A, in the same manner as reported by Barbet-Massin et al. (2015) and Tavarini et al. (2015)) but, because of its marked negative effect on biomass, SG content per Ndeficient plant was 10 times less than that of the control. This implies that, consistent with the carbon-nutrient balance hypothesis (Herms and Mattson, 1992), as the availability of N becomes limited the higher C:N ratio would lead to a greater concentration of carbon-based compounds and the synthesis of chlorophyll is reduced in favour of sustained accumulation of SGs (Barbet-Massin et al., 2015), stevioside in our study. Positive correlations between leaf N concentration and CO2 assimilation rates and negative correlations between leaf N and leaf SG concentration reported by Barbet-Massin et al. (2015) for stevia further support these findings. SG content of P deficient plants was suppressed even more than that of N-deficient plants; it was 20 times less than that of the control (Table 2). Reasons for this are still to be determined. Conditions that favour biomass and especially leaf production favour SG production (Barbet-Massin et al., 2015). An optimal supply of N in the field should lead to a balance between production of leaf biomass and an economically extractable concentration of leaf SG. Indeed, Tavarini et al. (2016) advocate an intermediate rate of N (150 kg ha−1) to optimise Reb-A concentration, and the Reb-A:stevioside ratio. They also related the concentration of total SG to leaf chlorophyll concentration, suggesting a causative relationship, but our data, across deficiencies of all macro- and micronutrients showed no such relationship (r = 0.171 ns, between SPAD and total SG), although SPAD was much more closely correlated with total SG content per plant (r = 0.867***). In plants deficient in K neither the leaf SG concentration (in line with data of Kawatani et al., 1978) nor the overall leaf SG content differed significantly from that of the complete nutrient treatment, although in absolute terms plant SG content was only 55% that of the control. Indeed the reduction of any macronutrient, with the exception of K, led to a marked decrease in plant SG content. Utumi et al. (1999) also stated that deficiencies of any of N, P, K, Ca, Mg and S reduce the SG content whereas Lima Filho et al. (1997) reported that only deficiency of Ca caused the same.
4.2. The effect of pH on biomass, SG concentration and SG content The solubility and availability of nutrients, and therefore biomass production, are known to depend on the pH of the rooting medium. Above ground biomass at pH 8 was about one half that of the maximum between pH 4–6 (Table 4), and stem and leaf yields were lowest at pH 8. While reduced stem weight contributed most, fewer branches and less leaf weight also contributed but to a lesser degree. As with the noted greater negative effects of nutrient omission on the above ground than on root biomass, the negative effect of high pH was less evident on root than above ground biomass. Of the macronutrients, only solubility of P is markedly reduced by high pH, as P starts to form insoluble compounds with Ca and Mg. In our pH study we found in line with the treatment of P omission, that SPAD readings, photosynthetic rate and biomass were all reduced at higher pH (although the negative effect of high pH was not as great as with lack of P), and that there was not a pronounced effect in either study on leaf SG concentration. At high pH lack of P may therefore have been responsible for the noted effects. Of the micronutrients for which high pH reduces availability (Fe, Mn, Cu, Zn and B), only lack of Fe or B in the nutrient omission trial led to a marked decrease in biomass (Fig. 2). No cracking and lignification of stems, symptomatic of B deficiency in the omission trial (Midmore et al., 2012), was apparent in the high pH treatment hence it is unlikely that B deficiency was the major cause of low yield at high pH. However, deficiency of Fe may have been, for the visual deficiency symptoms at high pH characteristic of Fe deficiency (Midmore et al., 2012), the low SPAD reading indicating a low leaf chlorophyll concentration (44 at pH 8 vs. 45–50 for pH 4–7, LSD 5% = 4.2), and the low rate of leaf photosynthesis are typical of such a deficiency. The lack of any marked effects of high pH on SG concentration, as was also noted for lack of Fe in the nutrient omission trial, further supports this argument. Data presented are from studies conducted in hydroponics, and, although distinct trends are evident, further field-scale trials are necessary to confirm the tentative conclusions.
4.1.4. Micronutrients and SG concentration and content Deficiency of Fe did not affect concentration of SG when compared to the control, but because biomass was markedly reduced so too was SG content per plant (Table 2). In contrast, biomass of plants lacking Cu was not diminished compared to that of the control, but the concentration of SG in the leaves was, leading to less content per plant than the control (Table 2). The likely role of Cu in maintaining SG concentration in leaves may be through synthesis of isoprenoids in stevia chloroplasts which is then related to production of SGs in stevia chloroplasts (Jain et al., 2008) although lack of Cu had no noticeable effect on SPAD readings nor on the rate of photosynthesis. The zinc deficient treatment was the only one with a significantly higher stevioside leaf concentration than that of the control (Table 2). The high SG concentration was offset by a lesser biomass than that in the control, such that the SG content per plant lacking Zn did not differ from that of the control. The same was so for treatments lacking Cl, Mn and Mo. It is unlikely that stevia seed contributed sufficient micronutrients to satisfy early growth (100 seed weight is c. 0.3–1.0 g) although the half strength nutrient solution used during early growth could potentially supply some micronutrient. However, the strong effects of the treatment lacking Fe on biomass and lacking Cu on SG concentration would suggest that there were real deficiencies of micronutrients.
5. Conclusions Our preliminary results and those of Rank and Midmore (2006) who reported that plants grown on neutral to alkaline soil had poor growth during trials in northern Queensland, Australia, imply that stevia should be grown in soil with pH < 7 to achieve maximum leaf yield. Avoidance of micronutrient deficiencies (especially Cu and Fe) and provision of adequate N and P are also requirements for good SG yield. Confirmatory field trials over several years are called for. Acknowledgements We thank the Rural Industries Research and Development Corporation and Sanitarium Plc Ltd for financial support for these studies and Dr Ria Reyes-Guzman for advice on the HPLC analyses.
4.1.5. Ratio of Reb-A: steviosides Higher concentration of Reb-A over other steviosides is desirable by the sweetener industry due to its more acceptable flavour profile (Yadav et al., 2011). For most treatments in our experiment, including the complete nutrient control, concentration of Reb-A was at least 50%
References Anon, 2011. Commission Regulation (EU) No 1131/2011 of 11 November 2011 amending Annex II to Regulation (EC) No 1333/2008 of the European Parliament and
6
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
G.G. Kafle et al.
Springer-Verlag, New York. Lima Filho, O.F., Malavolta, E., 1997. Symptoms of nutritional disorders in stevia Stevia rebaudiana (Bert.) Bertoni. Scientia Agricola 54, 53–61 (In Portuguese). Lima Filho, O.F., Malavolta, E., Yabico, H.Y., 1997. Influence of nutritional stress on content and production of stevioside during Stevia rebaudiana development. Pesqisa Agropecuaria Brasileira, Brasilia 32, 489–494 (In Portuguese). Midmore, D.J., Rank, A.H., Walsh, K.B., Reyes, R., Kafle, G., Hopkins, K.C., 2012. Further Development of the Stevia Natural Sweetener Industry. RIRDC Publication No. 12/ 051, Canberra Australia. Midmore, D.J., 1994. Simple hydroponics for food security. ILEIA Newsletter 10, 11–12. Mohamed, A.A., Ceunen, S., Geuns, J.M., Van den Ende, W., de Ley, M., 2011. UDPdependent glycosyltransferases involved in the biosynthesis of steviol glycosides. Journal of Plant Physiology 168, 1136–1141. Pal, P.K., Kumar, R., Guleria, V., Mahajan, M., Prasad, R., Pathania, V., Gill, B.S., Singh, D., Chand, G., Singh, B., Singh, R.D., Ahuja, P.S., 2015. Crop-ecology and nutritional variability influence growth and secondary metabolites of Stevia rebaudiana Bertoni. BMC Plant Biology 15, 67. http://dx.doi.org/10.1186/s12870-015-0457-x. Rank, A.H., Midmore, D.J., 2006. Stevia: An Intense, Natural Sweetener – Laying the Groundwork for a New Rural Industry. RIRDC Publication No 06/020, Canberra Australia. Roberts, J., Whitehouse, D.J., 1976. Practical Plant Physiology. Longman Group Limited, London and New York. Salisbury, F.B., Ross, C.W. (Eds.), 1992. Plant Physiology. Wadsworth Publishing Company, Belmont, California USA. Sharma, S., Walia, S., Singh, B., Kumara, R., 2016. Comprehensive review on agro technologies of low-calorie natural sweetener stevia (Stevia rebaudiana Bertoni): a boon to diabetic patients. Journal of the Science of Food and Agriculture 96, 1867–1879. Shock, C.C., 1982. Rebaudi's stevia: natural noncaloric sweeteners. California Agriculture 36, 4–5. Tavarini, S., Sgherri, C., Ranieri, A.M., Angelini, L.G., 2015. Effect of nitrogen fertilization and harvest time on steviol glycosides flavonoid composition, and antioxidant properties in Stevia rebaudiana Bertoni. Journal of Agricultural and Food Chemistry 63, 7041–7050. Tavarini, S., Pagano, I., Guidi, L., Angelini, L.G., 2016. Impact of nitrogen supply on growth, steviol glycosides and photosynthesis in Stevia rebaudiana Bertoni. Plant Biosystems – An International Journal Dealing with all Aspects of Plant Biology 150, 953–962. Usha, R., Swamy, P.M., 2002. Influence of nutrient deficiency and sucrose concentrations on the production of artemisinin by cultured cells of sweet wormwood (Artemisia annua L.). Indian Journal of Plant Physiology 7, 159–162. Utumi, M.M., Monnerat, P.H., Pereira, P.R.G., Fontes, P.C.R., VDPC, Godinho, 1999. Macronutrient deficiencies in Stevia: visual symptoms and effects on growth, chemical composition, and stevioside production. Pesquisa Agropecuária Brasileira 34, 1039–1043 (In Portugese). Weir, R.G., Cresswell, G.C. (Eds.), 1993. Plant Nutrient Disorders 3: Vegetable Crops. Inkata Press, North Ryde, NSW Australia. Yadav, A.K., Singh, S., Dhyani, D., Ahuja, P.S., 2011. A review on the improvement of stevia [Stevia rebaudiana (Bertoni)]. Canadian Journal of Plant Science 91, 1–27.
of the Council with regard to steviol glycosides. Official Journal of the European Union L295, 205–206. Barbet-Massin, C., Giuliano, S., Alletto, L., Daydé, J., Berger, M., 2015. Nitrogen limitation alters biomass production but enhances steviol glycoside concentration in Stevia rebaudiana Bertoni. PLoS One 10 (7), e0133067. Brandle, J.E., Rosa, N., 1992. Heritability for yield, leaf: stem ratio and stevioside content estimated from a landrace cultivar of Stevia rebaudiana. Canadian Journal of Plant Science 72, 1263–1266. Ceunen, S., Geuns, J.M.C., 2013. Steviol glycosides: chemical diversity metabolism, and function. Journal of Natural Products 76, 1201–1228. Das, K., Dang, R., Shivananda, T.N., 2006. Effect of major nutrients on biomass production and stevioside content of Stevia rebaudiana – a pot culture study. Biomed 1, 22–25. FSANZ, 2008. Final Assessment Report: A540 Steviol Glycosides as Intense Sweeteners. Food Standards Australia New Zealand, Barton, ACT, Australia. Ferreira, J.F.S., 2007. Nutrient deficiency in the production of artemisinin dihydroartemisinic acid, and artemisinic acid in Artemisia annua L. Journal of Agricultural and Food Chemistry 55, 1686–1694. Freitas, M.S.M., Monnerat, P.H., Viera, I.J.C., 2008. Mineral deficiency in Passiflora alata Curtis: vitexin bioproduction. Journal of Plant Nutrition 31, 1844–1854. Hartmann, T., 2007. From waste products to ecochemicals: fifty years research of plant secondary metabolism. Phytochemistry 68, 2831–2846. Hearn, L.K., Subedi, P.P., 2009. Determining levels of steviol glycosides in the leaves of Stevia rebaudiana by near infrared reflectance spectroscopy. Journal of Food Composition and Analysis 22, 165–168. Hermans, C., Hammond, J.P., White, P.J., Verbruggen, N., 2009. How do plants respond to nutrient shortage by biomass allocation? Trends in Plant Science 11, 610–617. Herms, D.A., Mattson, W.J., 1992. The dilemma of plants: to grow or defend. Quartely Review of Biology 77, 283–335. Jain, P., Kachhwaha, S., Kothari, S.L., 2008. Improved micropropagation protocol and enhancement in biomass and chlorophyll content in Stevia rebaudiana (Bert.) Bertoni by using high copper levels in the culture medium. Scientia Horticulturae 119, 315–319. Jarma, O.A., Combatt, C.E., Polo, S.J., 2012. Glycoside contents depending on the nutrient deficiencies in Stevia rebaudiana Bert. Revista U.D.C.A Actualidad & Divulgación Científica 15, 107–116 (In Spanish). Jones, J.B. (Ed.), 2005. Hydroponics: a Practical Guide for the Soilless Grower. CRC Press, Boca Raton, USA. Kawatani, T., Kaneki, Y., Tanabe, T., Sakamoto, I., Murakami, K., Tanaka, O., 1978. On the cultivation of Kaa He-e (Stevia rebaudiana Bertoni). IV: Response of Kaa He-e to nitrogen fertilization rates and to the three major elements of fertilizer. Japanese Journal of Tropical Agriculture 21, 173–178. Kulasekaran, R., Singh, V., Megeji, N.W., 2006. Cultivation of stevia (Stevia rebaudiana): a comprehensive review. Advances in Agronomy 89, 137–177. Kumar, R., Sharma, S., Ramesh, K., Prasad, R., Pathania, V., Singh, B., Singh, R.D., 2012. Effect of agro-techniques on the performance of natural sweetener plant: stevia (Stevia rebaudiana) under western Himalayan conditions. Indian Journal of Agronomy 57, 74–81. Lambers, H., Chapin III, F., Stuart, P., Thijs, L., 2008. Plant Physiological Ecology.
7
Contents lists available at ScienceDirect
Journal of Applied Research on Medicinal and Aromatic Plants journal homepage: www.elsevier.com/locate/jarmap
Effect of nutrient omission and pH on the biomass and concentration and content of steviol glycosides in stevia (Stevia rebaudiana (Bertoni) Bertoni) under hydroponic conditions ⁎
Geeta Gautam Kafle, David J. Midmore , Resham Gautam School of Health, Medical and Applied Sciences, Central Queensland University, Rockhampton, Queensland, 4702, Australia
A R T I C L E I N F O
A B S T R A C T
Keywords: Hydroponics Screen-house Macronutrient Micronutrient Photosynthesis SPAD reading
Steviol glycosides (SGs), have recently been approved in western countries as sources of intense natural sweeteners. SGs are found in Stevia rebaudiana, mainly in the leaves, and industry prefers rebaudioside A (Reb-A) over other steviol glycosides for its superior flavour profile. Hence leaf biomass and concentration of SGs (and their product, SG content) are of primary agronomic interest. Experiments were conducted under controlled conditions in nutrient solution to assess the effects of nutrient deficiencies and pH on biomass production, and concentration and plant content of SGs. Total SG content was low in plants deficient in the macronutrients N, P, S, Mg or Ca because of reduced photosynthesis and because of the decreased leaf yield, even though lack of N resulted in greater concentration of stevioside in the leaves. Lack of N or P reduced the proportion of Reb-A to total SGs. Plants deficient in K had less yield than in the nutrient-complete control, but not significantly so and SG concentration in the leaves was similar to that of the control. Deficiency of the micronutrients Cu and Fe led to low SG yield, because of reduced SG concentration in leaves, and because of reduced leaf yield, respectively. Lack of other micronutrients did not influence SG content. Neutral to alkali conditions reduced plant growth and leaf yield, most likely due to deficit of P, but pH had no effect on SG concentration. Our results are indicative, but preliminary, and require confirmation in open field trials over several years.
1. Introduction Stevia (Stevia rebaudiana (Bertoni) Bertoni) is known for its sweet tasting compounds, steviol glycosides (SG), found in greatest concentration in the leaves (Kulasekaran et al., 2006). Production of high SG concentration in plentiful leaf biomass is an important goal in commercial stevia production. Interest in commercial production of stevia has intensified with approval, firstly in Australia and then in the USA and more recently in Europe, of steviol glycosides as intense sweeteners (FSANZ, 2008; Anon., 2011). In its natural habitat, stevia is found in infertile, acid sands or muck soils (Shock, 1982) although commercial production tends to take place on somewhat better soils. It is well known that plant growth in general is stunted when deprived of nutrients, whether due to insufficient quantity or to pH-conditioned non-availability in the growing medium, or to insufficient water for uptake. Each element plays one or more particular biochemical/physiological roles, so deficiency of that element will result in a set of predictable metabolic and physiological disturbances. Reports on the influence of nutrient deficiency (and toxicity) on stevia growth and SG yield are scarce. Low availability of soil N leads to ⁎
low leaf N concentration, reduction of photosynthesis (Sharma et al., 2016) and low leaf yield compared to adequate soil N, but it also leads to a higher concentration of SG in leaves than with adequate soil N (Barbet-Massin et al., 2015). In a similar manner, higher rates of NPK or farmyard manure led to higher leaf yield but lower SC concentration in trials in the western Himalayas (Kumar et al., 2012), and highest leaf SG concentration (16.7% w/w) was achieved by omission of N compared to that found in the N-supplied control (11.5% – Das et al., 2006). Utumi et al. (1999) found that total above ground biomass decreased with deficiencies of any macronutrient, however, the percentage of reduction was significantly higher in treatments without N, K, or Mg than with deficiencies of S, Ca or P. The concentration of SG decreased with the deficiency of all macronutrients except for P (by 27% for Ca, 24% for N, to 41% for S), causing a reduction in content of SG per plant. This contrasts with the earlier mentioned increased SG concentration in leaves of plants lacking N, and the reported lack of response of total leaf SG concentration to deficiency of any macro or micronutrient by Jarma et al. (2012), although concentration of Reb-A did decline with lack of P, S, K or Cu. Plant nutrient availability is highly dependent on the pH of the
Corresponding author. E-mail address: [email protected] (D.J. Midmore).
http://dx.doi.org/10.1016/j.jarmap.2017.08.001 Received 21 January 2017; Received in revised form 31 July 2017; Accepted 3 August 2017 2214-7861/ © 2017 Elsevier GmbH. All rights reserved.
Please cite this article as: Midmore, D.J., Journal of Applied Research on Medicinal and Aromatic Plants (2017), http://dx.doi.org/10.1016/j.jarmap.2017.08.001
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
G.G. Kafle et al.
growing medium. Nutrients such as phosphorous, magnesium and calcium are less available when the pH is below 5 (Jones, 2005). Similarly, at high pH iron, manganese, copper, zinc and boron are only sparingly available due to their low solubility (Jones, 2005). As a result, when the pH is outside of the optimal range, deficiency symptoms are seen on the above ground plant, although it is commonly difficult to ascribe the symptoms to deficiencies of individual elements. For stevia, Shock (1982) reports that even though native to low pH (4–5) soils, it grows well on less acid soils ranging from 6.5–7.5. Rank and Midmore (2006) reported that plants grown on neutral to alkali soils showed less plant yield compared to that on acidic soils. The specific effects of pH and of nutrient deficiency on biomass yield and SG content in stevia are largely unknown. This study therefore set out to determine the consequences of macro and micronutrient deficiency on morphology, biomass accumulation, and SG concentration and content in stevia plants. This was complemented by a study to identify the optimum pH required for the same. Visual symptoms of nutrient deficiency on stevia shoot and root colour and morphology from these experiments are reported by Midmore et al. (2012).
Table 1 Effect of different nutrient deficiencies on leaf chlorophyll concentration, photosynthetic rate, leaf dry biomass and shoot to root dry weight ratios of stevia at the time of harvest (at four weeks after treatments imposed). Values within a column followed by the same letter are not significantly different (P < 0.05).
2. Material and methods
Treatments
Chlorophyll concentration (SPAD units)
Photosynthetic rate (μmol m−2 s −1)
Leaf biomass (g/plant)
Shoot to root ratio
Complete No Mn No Zn No Mo No B No Cu No Cl No K No Ca No S No Mg No Fe No P No N No Micro No NPK
53.45 a 52.90 a 50.45 a 50.15 a 50.00 a 49.90 a 46.50 a 46.45 a 33.50 b 31.70 bc 28.00 bcd 25.75 bcd 22.70 cd 22.05 d 21.35 d 20.90 d
12.73 bc 11.28 bc 11.57 bc 13.03 c 15.51 c 13.12 c 15.89 c 10.43 bc 0.66 a 5.78 ab 1.45 a 2.98 a 1.49 a 2.60 a 0.87 a 0.28 a
3.96 d 3.12 cd 2.70 bcd 4.11 d 2.19 abcd 3.31 d 3.18 cd 3.27 d 1.09 abc 0.73 ab 1.12 abc 1.11 abc 0.38 a 0.38 a 0.43 a 0.30 a
12.6 ef 8.6 cde 7.6 bcde 10.2 de 9.6 de 9.5 de 8.9 cde 17.5 f 10.9 de 2.6 ab 9.8 de 3.6 abc 2.3 ab 1.7 a 6.5 abcd 2.8 ab
2.1. Location from 25 to 30 °C. The experiment consisted of 16 treatments (Table 1) each randomised in two blocks. Each treatment in each block, i.e., each box, comprised four plants, giving a total of 128 plants in the experiment. Data were collected from each plant to estimate sampling error.
Two experiments (one nutrient omission and one with differing pH) were conducted at CQUniversity, Rockhampton (23° 22′, 0.345”S, 150° 31′ 0.53”E), Australia, using a non-circulatory hydroponics system (Midmore, 1994) inside a screen-house with 67% full sunlight. 2.2. Plant material
2.4. Treatments and experimental design for the pH experiment
Seeds of Stevia rebaudiana variety ‘Shoutain-2′ were sown into 1:1 perlite:vermiculite media in speedling trays inside the screen-house on 17/08/09 for the nutrient omission and on 25/04/10 for the pH experiment. Following germination, seedlings were watered with half strength Manutec hydroponic solution (Manutec Pty. Ltd.) for three weeks in the nutrient experiment and for four weeks in the pH experiment. They were then transferred to 7 cm diameter poly-pots lined with mesh and filled with perlite, for a further 4 weeks and watered as before. At the 6–8 leaf stage, with plant height ranging from 6 to 8 cm, seedlings for the nutrient experiment were supplied with reverse osmosis (RO) water for two weeks, and then subjected to treatment. Seedlings for the pH treatments were not preconditioned with RO water.
In common practice for plants grown in soilless culture pH is raised by adding NaOH or KOH and lowered by adding H2SO4 or HNO3 to the solution (Jones, 2005). Half strength commercially available hydroponics fertilizer (Manutec Pty. Ltd) was used as a nutrient medium with pH of 6.7 and EC 1.45 dS/m. To bring the pH to the desired level 710, 580 or 355 ml of 0.25 M H2SO4 were added to achieve pH values of 4, 5, or 6, and likewise 35 or 125 ml of 1.0 M NaOH per 170 l were added to achieve pH values of 7 or 8, respectively. Styrofoam boxes and solution monitoring were as in the nutrient experiment, and each treatment was replicated six times, in a completely randomised design. Solution pH was maintained at 4, 5, 6, 7 and 8 daily by adding appropriate amounts of H2SO4 or NaOH. Mean maximum and minimum temperatures during the experimental period were 24.1 and 13.2 °C.
2.3. Treatments and experimental design for the nutrient omission experiment
2.5. Data collection
The chemical composition of the nutrient-deficient solutions was similar to that reported by Roberts and Whitehouse (1976). In essence, the following (in mmol) was the composition of the complete nutrient solution: N (10.054), P (1.17), K (3.36), Ca (3.35), Mg (1.5), S (1.5), Fe (0.51), Mn (0.01), Cu (0.0012), M0 (0.0002), B (0.323), Zn (0.0022), Cl (0.1767) and Na (0.0005). Omission treatments were so designed that, with exception of Na (which ranged from 0.119 to 7.065), S in the −Mg (0.68), and Cl in the −Fe (0.27), concentrations did not differ from the control. Styrofoam boxes (53 cm x 23 cm × 25 cm) were lined each with a black plastic bag to prevent leakage. Four holes were made on the lid of each box to hold the 7 cm diameter poly-pots. The plant to plant and row to row distances were maintained at 13 cm and 25 cm, respectively. Solution was filled to a depth of 15 cm, and height of the solution and pH and electrical conductivity (EC) were monitored daily between 9:00 and 10:00 am and adjusted accordingly. The pH for the treatments was c. 5.5–6.0 and EC varied between treatments. Mean maximum and minimum temperatures during the experimental period were 33.1 and 20.1 °C. The temperature of nutrient solutions ranged
Leaf chlorophyll concentration was estimated using a SPAD meter (Konica, Minolta Japan), with readings taken 3 and 4 weeks after the start of treatment in the nutrient experiment and at fortnightly intervals for the pH experiment. Youngest fully expanded leaves were used for the measurement. Leaf gas exchange (photosynthesis, transpiration and stomatal conductance) for all the treatments in both experiments was measured using an IRGA (Infrared Gas Analyser, model LCA-4 from ADC-UK). Measurements were made at 4 weeks following the start of treatments in the nutrient experiment and at fortnightly intervals for the pH experiment. The IRGA readings were taken between 11:00 a.m. and 2:00 p.m., on the same leaves used for chlorophyll determination. For the measurement of leaf steviol glycoside concentration (i.e., amount of SG per unit dry weight of leaf, expressed as %), two youngest fully expanded leaves from each plant of every treatment were removed three weeks after treatments began. The eight leaves from each box were combined and oven-dried at 60 °C for 48 h, ground to a fine powder with a mini bead-beater, and stored in air-tight containers. The 2
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
G.G. Kafle et al.
Fig. 1. View of pH experiment inside the screenhouse with stevia plants growing in hydroponics.
SG concentration of the samples was analysed with HPLC using a modification of the procedure described by Hearn and Subedi (2009). For the SG quantification through HPLC, pure stevioside (99.9% purity) and rebaudioside A (Reb-A, 97.4% purity) obtained from Wako Pty Ltd., Japan were used to make standard solutions of 0.25, 0.5 and 1.0 g/L for both stevioside and Reb-A in EtOH 70% (w/w) providing a three point calibration as suggested by Hearn and Subedi (2009). Plants were harvested four weeks after treatments began in the nutrient omission experiment and after eight weeks for the pH experiment. Plant height (measured from the base of the stem to the apical tip) and leaf and stem fresh and dry weights were measured for each plant. Root weights were measured per box since it was not possible to separate on a per plant basis. The product of leaf dry weight and concentration of leaf SG is presented as content of SG. A view of the pH experiment is given in Fig. 1.
3.1.2. Leaf gas exchange The photosynthetic rate of plants grown in the complete nutrient solution (12.73 μmol CO2 m−2 s −1, LSD = 7.1) differed significantly from the treatments without Fe, N, P, Mg, micro, Ca and NPK (ranging from 2.98 to 0.28 μmol m−2 s −1). However, photosynthetic rates in treatments lacking B, Cu, Mo, Zn, Mn, K and S were similar to that of the control (Table 1). The treatment without Cl had the highest photosynthetic rate (15.89 μmol m−2 s −1) while that without NPK had the lowest. The transpiration rate differed significantly between treatments (data not presented) and followed the same pattern as for photosynthetic rate. Transpiration rate in the treatment without Cl was highest (12.46 mmol H2O m−2 s −1, LSD = 4.5), slightly less in the control (10.01 mmol H2O m−2 s −1) and the lowest (2.75 mmol H2O m−2 s −1) was recorded in the treatment without micronutrients.
2.6. Statistical analysis
3.1.3. Growth parameters Total above ground (shoot) biomass of stevia grown in solution without the micronutrients Mo, Cu, Cl, Mn, and Zn and the macronutrient K, did not differ significantly from plants grown in the complete nutrient treatment (Fig. 2). Likewise, plant height did not differ between these treatments and the control (data not presented). Significant reductions in shoot biomass were found for the treatment lacking B, Fe, Mg, Ca, S, P, N, NPK or all micronutrients when compared to the nutrient-complete control (Fig. 2). Leaf (Table 1) and stem (data not presented) dry weights responded similarly to omission of the various nutrients as did above ground biomass while root dry weight, besides varying less between extremes of treatments, only differed from the control when Ca, all micronutrients or the combination of NPK were omitted (Fig. 2). The shoot to root ratio was close to the highest in the complete treatment, being only higher in the treatment lacking K (Table 1), and lowest in the treatments lacking N, P or NPK.
Data were analysed using the statistical package for ANOVA (analysis of variance) through Genstat version 11.1. Difference between means is reported as significant at P < 0.05. 3. Results 3.1. Nutrient omission 3.1.1. SPAD reading (surrogate for chlorophyll concentration) After four weeks of treatment, the SPAD reading for the complete treatment was 53.5 SPAD units, and was not affected by treatments lacking Mn, Zn, Mo, B, Cu, Cl or K (Table 1). The treatment without NPK had the lowest SPAD reading (20.9), 61% less than that of the complete treatment, and together with absence of Mg (28.0), Fe (25.8), P (22.7), N (22.1) and micronutrients (21.4), were all significantly less that for the complete treatment. The SPAD reading were higher by 2–3% in the control treatment and in most of those similar to the control at week 4 compared to readings at week 3 (data not presented). Those below the control in week 4 were generally with values lower than at week 3.
3.1.4. SG concentration and content in stevia leaves The total SG (stevioside + Reb-A) concentration varied from 3.0–12.1% dwt, but was only significantly less than in the control with lack of Cu or lack of all micronutrients (Table 2). Absence of Zn raised 3
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
G.G. Kafle et al.
Fig. 2. Average total above ground biomass (left hand side) and root dry weight (right hand side) of stevia (g/plant) grown with various nutrient deficiencies. Plants harvested after 4 weeks of treatment. Treatments with the same letter do not differ significantly at P < 0.05.
3.2. Solution pH
Table 2 Effect of different nutrient deficiencies on stevioside and Reb-A and total leaf concentration and total leaf SG content of stevia at the time of harvest (at four weeks after treatments imposed). Values within a column followed by the same letter are not significantly different (P < 0.05). Treatments
Stevioside (% dw)
Reb-A (% dw)
Total SG (stevioside + Reb-A) (% dw)
Total SG leaf content (mg/ plant)
no Zn no P no N no Cl no Mn no S no Fe complete no B no Mo no NPK no Ca no K no Mg no micro no Cu
7.8 7.2 6.4 6.1 6.0 6.0 5.6 5.2 5.1 5.1 4.7 4.3 4.0 3.8 2.8 1.9
4.2 3.5 2.4 3.0 3.5 3.1 3.9 3.6 3.4 3.7 1.3 3.7 2.4 2.1 1.1 1.1
12.1 a 10.7 a 9.0 ab 9.2 ab 9.6 ab 9.2 ab 9.6 ab 8.9 ab 8.6 ab 8.9 ab 6.1 bc 8.1 bc 6.5 bc 5.9 bc 4.0 c 3.0 c
350.0 ab 18.3 d 38.6 d 314.5 abc 323.0abc 78.2 cd 109.6 bcd 379.7 a 198.6 abcd 414.3 a 14.3 d 96.1 cd 209.3 abcd 125.0 bcd 20.8 d 116.8 bcd
a ab abc abcd abcd abcd abcd bcde bcde bcde bcde cdef cdef def ef f
a a a a a a a a a a a a a a a a
3.2.1. SPAD readings and leaf gas exchange At pH 6 and above SPAD readings show that the chlorophyll concentration was reduced compared to the values at pH 4 and 5. None of photosynthetic rate, transpiration rate or stomatal conductance differed between pH treatments (Table 3) although stevia grown at pH 6–8 had markedly lower values for all three parameters.
3.2.2. Growth parameters Above ground plant biomass was generally greater in plants grown between pH 4 and 6 than at pH 7 and 8. The plant biomass at pH 8 was one half that at pH 4–6 (Table 4) although plant height did not differ significantly between treatments (data not presented). Leaf dry weight did not differ between plants grown at pH 4, 5, 6, and 7, and all were greater than that at pH 8. Leaf weight at pH 8 was 62% that at pH 4 (Table 4). Stem dry weight was more sensitive to high pH than was leaf dry weight, and leaf dry weight (in relative terms) was more sensitive to high pH that was root dry weight (Table 4). Nevertheless, root weight was markedly and significantly lower in plants grown at pH 8 than those at pH 4–7 (Table 4). Branch number was highest at pH 6, that with the highest stem, leaf and total above ground biomass (Table 4), followed by pH 5, 4 and 7. Lowest branch number was recorded at pH 8 (Table 4).
total SG concentration by 36% above that of the control, and the absence of P by 20% but the differences between these and the control were not significant. There was significant difference in stevioside concentration between the treatments (P < 0.01). Plants grown without Zn had highest stevioside concentration, and this was significantly greater than that of the control (Table 2). There was no significant difference in Reb-A concentration between the treatments. However, the highest value of RebA was without Zn and the order of response of Reb-A to nutrient omission was similar to that of stevioside. The SG content on a per plant basis (i.e., the product of leaf weight per plant and concentration of SG in the leaf) was highest in plants grown without Mo (414.3 mg/plant) followed by the complete treatment and by those lacking Zn, Mn, Cl, K, or B. The SG content was related to variations in either the total leaf yield or concentration of SG in the leaf (Tables 1 and 2). For example, in treatments lacking S, P, N and Ca the SG concentration was similar to that of the control but due to a reduction in their leaf biomass the total leaf SG yield was low. For Cu deficient plants the total SG concentration was the lowest (3%) but because of the relatively high leaf yield per plant the total leaf SG content was higher in this treatment than that in a number of other treatments, particularly that lacking all micronutrients (Table 2).
3.2.3. SG concentration and content No significant difference between the pH treatments was found for leaf concentration of either stevioside or Reb-A, nor of total SG (Table 5). On average plant SG content at pH 4 was the highest and it was lowest at pH 8.
Table 3 Photosynthetic rate, transpiration rate, stomatal conductance and leaf chlorophyll concentration (SPAD units) of stevia grown at different pH (average of three dates of measurement). Values within a column followed by the same letter are not significantly different (P < 0.05).
4
Solution pH
Photosynthetic rate (μmol m−2 s −1 )
Transpiration rate (mmol m−2 s −1 )
Stomatal conductance (mmol m−2 s −1 )
Chlorophyll concentration (SPAD units)
4 5 6 7 8
11.17 11.33 11.45 10.69 10.34
3.06 3.02 3.46 2.86 2.72
0.16 0.17 0.23 0.15 0.13
46.6 b 46.5 b 44.3 a 43.8a 42.8 a
a a a a a
ab ab b a a
a ab b a a
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
G.G. Kafle et al.
Malavolta, 1997; Utumi et al., 1999). Shoot biomass decreased proportionally more than root biomass with macronutrient deficiencies (i.e., shoot to root ratios were least in these treatments, although again lack of K was an exception – Table 1). This is consistent with the interpretation that the plant assigns resources to the growth of the root as the main nutrient absorption organ (or rather does not reduce resource allocation to the roots to the same extent as the reduction to the shoots – Hermans et al., 2009). The additional resource allocation to roots is not only to satisfy the demand for additional root biomass per se, but also for a proportionately higher rate of respiration in roots than that of shoots under diminished nutrient supply (Lambers et al., 2008). It is interesting that lack of K did not increase investment in root relative to shoot biomass, a result not consistent with the literature on stevia, for Utumi et al. (1999) reported that only magnesium deficiency among the elements trialled reduced root relative to shoot dry weight. Greater investment in roots (or poorer growth of above ground organs) was evident in the treatments that lacked the macronutrients N, P or S (Table 1).
Table 4 Dry weight of stem, leaf, root and total biomass, shoot to root ratio, and number of branches per plant at harvest of stevia grown at different pH. Values within a column followed by the same letter are not significantly different (P < 0.05). Solution pH
4 5 6 7 8
Dry weight (g/plant) Stem
Leaf
Root
Total above ground biomass
12.2 c 10.8 bc 12.7 c 7.8 ab 4.9 a
14.1 b 14.5 b 15.2 b 12.8 b 8.9 a
8.8 7.7 7.4 8.5 6.5
26.3 25.2 27.9 20.6 13.8
d bc ab cd a
bc bc c b a
Shoot to root ratio
Branch no. (per plant)
2.99 3.14 2.57 2.41 2.12
8.4 b 9.4 bc 11.6 c 7.8 ab 4.9 a
bc c c ab a
Table 5 Percent dry weight of stevioside, Reb-A and total steviosides in leaves of stevia, and total content per plant grown at different pH. Values are means of six replicates with one missing value. Solution pH
Stevioside (% dry weight)
Reb-A (% dry weight)
Total SG (% dry weight)
Total SG (mg/plant)
4 5 6 7 8
2.68 2.69 2.47 2.97 3.00
0.67 0.74 0.56 0.85 0.98
3.36 3.43 3.03 3.82 3.99
478 477 436 505 354
4.1.2. Micronutrients and biomass Turning to the effects of lack of micronutrients on stevia, on which very little has been published, deficiency of iron leads to a decrease in chlorophyll content (Salisbury and Ross, 1992), as was very evident by the low SPAD reading for iron omission in our study. Iron deficiency symptoms in treatments lacking Fe (lack of Fe alone or lack of all micronutrients) appeared after two weeks as general chlorosis (Midmore et al., 2012), and were severe after four weeks, with necrosis of young leaves. This would be expected to contribute to the low rate of photosynthesis and above ground biomass in this treatment (Table 1 and Fig. 2). Relative investment in shoot biomass compared to that in roots was low in plants which lacked Fe, alone or in the treatment without any micronutrients (Table 1). Plants lacking Cu showed deficiency symptoms (Midmore et al., 2012), chlorosis in older leaves and brown necrotic spots in mature leaves, similar to that reported for tomato (Weir and Cresswell, 1993) but photosynthetic rate and SPAD readings did not differ from the control and plant biomass, stem yield, leaf yield, root weight were only slightly reduced, but not significantly so, compared to the control. Copper is somewhat more mobile in plants than is iron, resulting in the treatment lacking copper having a higher measured rate of photosynthesis and less suppression of above ground biomass than in the treatment lacking iron. Deficiency symptoms in plants lacking Mo, Cl, Zn or Mn were not prominent (Midmore et al., 2012) and above ground biomass, root weight, photosynthetic rates and SPAD readings did not differ significantly from the complete treatment. Of these three are reasonably mobile within the plant (not Mn) and may have been mobilised to younger leaves, Mo for redox reactions, Cl for water splitting in photosynthesis and Zn for chlorophyll production, all leading to sustained biomass production over the period of deficiency.
4. Discussion 4.1. The effect of nutrient deficiency on biomass, SG concentration and SG content 4.1.1. Macronutrients and biomass Reduced growth is a means by which plants concentrate minerals in plant tissue when supply is constrained. As expected therefore (Sharma et al., 2016), biomass yield was decreased relative to that of the control treatment for all the macronutrient deficiency treatments, although the effect of lack of K was not significant. Insensitivity of stevia biomass to lack of K has been reported earlier by Kawatani et al. (1978). Photosynthetic rate of plants lacking K also did not differ from that of the control, whereas omission of N, P, S, Mg or Ca led to notably lower photosynthetic rates (Table 1) and biomass (Fig. 2) than those of the control. Utumi et al. (1999) reported above ground biomass of stevia to be respectively 47% and 85% that of the control when N and S were omitted. In the current study reductions were even greater – biomass was respectively only 11% and 20% that of the complete control when N or S were omitted (Fig. 2). This was in line with our data showing (a) that the rate of photosynthesis in the treatment lacking N was only 20% that of the control (2.60 vs. 12.73 μmol m−2 s −1, LSD 5% = 7.07) and (b) the SPAD reading for the treatment lacking N was less than one half that of the control (Table 1). Positive correlations between leaf N concentration and CO2 assimilation rates reported by Barbet-Massin et al. (2015) for stevia, back this up further. While Kawatani et al. (1978) reported that biomass production in plants lacking P was less affected than by lack of N, our data show that lack of P or lack of S was as suppressive of above ground biomass production and rates of photosynthesis as was lack of N. Biomass and photosynthetic rates for plants lacking Ca or Mg were also well below those of the control plants receiving all necessary nutrients. The visual symptoms associated with macronutrient (N, P, Ca, Mg and S) deficiency of poor root development, stunted growth and chlorosis and necrosis of the leaves as reported by Midmore et al. (2012) for the same plants were consistent with that expected for plants in general (e.g. Weir and Cresswell, 1993), and also matched the symptoms reported by previous authors for macronutrient deficiency in stevia (Lima Filho and
4.1.3. Macronutrients and SG concentration and content In contrast to primary metabolites, concentration of secondary metabolites such as SGs exhibit high plasticity under selection pressure (Brandle and Rosa, 1992; Hartmann 2007) and are subject to environmental factors and agronomic management (Ceunen and Geuns, 2013; Pal et al., 2015). Within agronomic management, nutrient deficiencies can affect the accumulation of secondary metabolites. For example, when grown under deficit K (Ferreira, 2007) and P (Usha and Swamy, 2002) conditions, Artemisia annua produces high concentrations of the secondary metabolite, artemisinin. Freitas et al. (2008) also reported an increased concentration of vitexin in the leaves of Passiflora alata when grown under N deficiency conditions. Kawatani et al. (1978) reported that neither deficiency of N nor P affected leaf SG concentration, but in contrast Utumi et al. (1999) showed decreased SG concentration in P and N deficient plants (by 7.8% and 22.8%, respectively) and an even lower SG content per plant due to reduced 5
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
G.G. Kafle et al.
that of stevioside (Table 2). Exceptions to this were treatments lacking N, P, NPK or all the micronutrients where Reb-A was less than one half of the total SG. Diminished supply of N was recently (Barbet-Massin et al., 2015; Tavarini et al., 2015) shown to reduce the ratio of Reb-A to stevioside concentration: quite possibly the lack of N suppresses UDPdependent glycosyltransferases responsible for the transformation of stevioside into Reb-A, in a similar manner to what occurs in leaves of stevia plants transferred from long to short days, both linked most likely to an increased plant capacity for ROS scavenging (Mohamed et al., 2011).
biomass. In our study, lack of N did not reduce SG concentration (although it did not increase it, at least not for Reb-A, in the same manner as reported by Barbet-Massin et al. (2015) and Tavarini et al. (2015)) but, because of its marked negative effect on biomass, SG content per Ndeficient plant was 10 times less than that of the control. This implies that, consistent with the carbon-nutrient balance hypothesis (Herms and Mattson, 1992), as the availability of N becomes limited the higher C:N ratio would lead to a greater concentration of carbon-based compounds and the synthesis of chlorophyll is reduced in favour of sustained accumulation of SGs (Barbet-Massin et al., 2015), stevioside in our study. Positive correlations between leaf N concentration and CO2 assimilation rates and negative correlations between leaf N and leaf SG concentration reported by Barbet-Massin et al. (2015) for stevia further support these findings. SG content of P deficient plants was suppressed even more than that of N-deficient plants; it was 20 times less than that of the control (Table 2). Reasons for this are still to be determined. Conditions that favour biomass and especially leaf production favour SG production (Barbet-Massin et al., 2015). An optimal supply of N in the field should lead to a balance between production of leaf biomass and an economically extractable concentration of leaf SG. Indeed, Tavarini et al. (2016) advocate an intermediate rate of N (150 kg ha−1) to optimise Reb-A concentration, and the Reb-A:stevioside ratio. They also related the concentration of total SG to leaf chlorophyll concentration, suggesting a causative relationship, but our data, across deficiencies of all macro- and micronutrients showed no such relationship (r = 0.171 ns, between SPAD and total SG), although SPAD was much more closely correlated with total SG content per plant (r = 0.867***). In plants deficient in K neither the leaf SG concentration (in line with data of Kawatani et al., 1978) nor the overall leaf SG content differed significantly from that of the complete nutrient treatment, although in absolute terms plant SG content was only 55% that of the control. Indeed the reduction of any macronutrient, with the exception of K, led to a marked decrease in plant SG content. Utumi et al. (1999) also stated that deficiencies of any of N, P, K, Ca, Mg and S reduce the SG content whereas Lima Filho et al. (1997) reported that only deficiency of Ca caused the same.
4.2. The effect of pH on biomass, SG concentration and SG content The solubility and availability of nutrients, and therefore biomass production, are known to depend on the pH of the rooting medium. Above ground biomass at pH 8 was about one half that of the maximum between pH 4–6 (Table 4), and stem and leaf yields were lowest at pH 8. While reduced stem weight contributed most, fewer branches and less leaf weight also contributed but to a lesser degree. As with the noted greater negative effects of nutrient omission on the above ground than on root biomass, the negative effect of high pH was less evident on root than above ground biomass. Of the macronutrients, only solubility of P is markedly reduced by high pH, as P starts to form insoluble compounds with Ca and Mg. In our pH study we found in line with the treatment of P omission, that SPAD readings, photosynthetic rate and biomass were all reduced at higher pH (although the negative effect of high pH was not as great as with lack of P), and that there was not a pronounced effect in either study on leaf SG concentration. At high pH lack of P may therefore have been responsible for the noted effects. Of the micronutrients for which high pH reduces availability (Fe, Mn, Cu, Zn and B), only lack of Fe or B in the nutrient omission trial led to a marked decrease in biomass (Fig. 2). No cracking and lignification of stems, symptomatic of B deficiency in the omission trial (Midmore et al., 2012), was apparent in the high pH treatment hence it is unlikely that B deficiency was the major cause of low yield at high pH. However, deficiency of Fe may have been, for the visual deficiency symptoms at high pH characteristic of Fe deficiency (Midmore et al., 2012), the low SPAD reading indicating a low leaf chlorophyll concentration (44 at pH 8 vs. 45–50 for pH 4–7, LSD 5% = 4.2), and the low rate of leaf photosynthesis are typical of such a deficiency. The lack of any marked effects of high pH on SG concentration, as was also noted for lack of Fe in the nutrient omission trial, further supports this argument. Data presented are from studies conducted in hydroponics, and, although distinct trends are evident, further field-scale trials are necessary to confirm the tentative conclusions.
4.1.4. Micronutrients and SG concentration and content Deficiency of Fe did not affect concentration of SG when compared to the control, but because biomass was markedly reduced so too was SG content per plant (Table 2). In contrast, biomass of plants lacking Cu was not diminished compared to that of the control, but the concentration of SG in the leaves was, leading to less content per plant than the control (Table 2). The likely role of Cu in maintaining SG concentration in leaves may be through synthesis of isoprenoids in stevia chloroplasts which is then related to production of SGs in stevia chloroplasts (Jain et al., 2008) although lack of Cu had no noticeable effect on SPAD readings nor on the rate of photosynthesis. The zinc deficient treatment was the only one with a significantly higher stevioside leaf concentration than that of the control (Table 2). The high SG concentration was offset by a lesser biomass than that in the control, such that the SG content per plant lacking Zn did not differ from that of the control. The same was so for treatments lacking Cl, Mn and Mo. It is unlikely that stevia seed contributed sufficient micronutrients to satisfy early growth (100 seed weight is c. 0.3–1.0 g) although the half strength nutrient solution used during early growth could potentially supply some micronutrient. However, the strong effects of the treatment lacking Fe on biomass and lacking Cu on SG concentration would suggest that there were real deficiencies of micronutrients.
5. Conclusions Our preliminary results and those of Rank and Midmore (2006) who reported that plants grown on neutral to alkaline soil had poor growth during trials in northern Queensland, Australia, imply that stevia should be grown in soil with pH < 7 to achieve maximum leaf yield. Avoidance of micronutrient deficiencies (especially Cu and Fe) and provision of adequate N and P are also requirements for good SG yield. Confirmatory field trials over several years are called for. Acknowledgements We thank the Rural Industries Research and Development Corporation and Sanitarium Plc Ltd for financial support for these studies and Dr Ria Reyes-Guzman for advice on the HPLC analyses.
4.1.5. Ratio of Reb-A: steviosides Higher concentration of Reb-A over other steviosides is desirable by the sweetener industry due to its more acceptable flavour profile (Yadav et al., 2011). For most treatments in our experiment, including the complete nutrient control, concentration of Reb-A was at least 50%
References Anon, 2011. Commission Regulation (EU) No 1131/2011 of 11 November 2011 amending Annex II to Regulation (EC) No 1333/2008 of the European Parliament and
6
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
G.G. Kafle et al.
Springer-Verlag, New York. Lima Filho, O.F., Malavolta, E., 1997. Symptoms of nutritional disorders in stevia Stevia rebaudiana (Bert.) Bertoni. Scientia Agricola 54, 53–61 (In Portuguese). Lima Filho, O.F., Malavolta, E., Yabico, H.Y., 1997. Influence of nutritional stress on content and production of stevioside during Stevia rebaudiana development. Pesqisa Agropecuaria Brasileira, Brasilia 32, 489–494 (In Portuguese). Midmore, D.J., Rank, A.H., Walsh, K.B., Reyes, R., Kafle, G., Hopkins, K.C., 2012. Further Development of the Stevia Natural Sweetener Industry. RIRDC Publication No. 12/ 051, Canberra Australia. Midmore, D.J., 1994. Simple hydroponics for food security. ILEIA Newsletter 10, 11–12. Mohamed, A.A., Ceunen, S., Geuns, J.M., Van den Ende, W., de Ley, M., 2011. UDPdependent glycosyltransferases involved in the biosynthesis of steviol glycosides. Journal of Plant Physiology 168, 1136–1141. Pal, P.K., Kumar, R., Guleria, V., Mahajan, M., Prasad, R., Pathania, V., Gill, B.S., Singh, D., Chand, G., Singh, B., Singh, R.D., Ahuja, P.S., 2015. Crop-ecology and nutritional variability influence growth and secondary metabolites of Stevia rebaudiana Bertoni. BMC Plant Biology 15, 67. http://dx.doi.org/10.1186/s12870-015-0457-x. Rank, A.H., Midmore, D.J., 2006. Stevia: An Intense, Natural Sweetener – Laying the Groundwork for a New Rural Industry. RIRDC Publication No 06/020, Canberra Australia. Roberts, J., Whitehouse, D.J., 1976. Practical Plant Physiology. Longman Group Limited, London and New York. Salisbury, F.B., Ross, C.W. (Eds.), 1992. Plant Physiology. Wadsworth Publishing Company, Belmont, California USA. Sharma, S., Walia, S., Singh, B., Kumara, R., 2016. Comprehensive review on agro technologies of low-calorie natural sweetener stevia (Stevia rebaudiana Bertoni): a boon to diabetic patients. Journal of the Science of Food and Agriculture 96, 1867–1879. Shock, C.C., 1982. Rebaudi's stevia: natural noncaloric sweeteners. California Agriculture 36, 4–5. Tavarini, S., Sgherri, C., Ranieri, A.M., Angelini, L.G., 2015. Effect of nitrogen fertilization and harvest time on steviol glycosides flavonoid composition, and antioxidant properties in Stevia rebaudiana Bertoni. Journal of Agricultural and Food Chemistry 63, 7041–7050. Tavarini, S., Pagano, I., Guidi, L., Angelini, L.G., 2016. Impact of nitrogen supply on growth, steviol glycosides and photosynthesis in Stevia rebaudiana Bertoni. Plant Biosystems – An International Journal Dealing with all Aspects of Plant Biology 150, 953–962. Usha, R., Swamy, P.M., 2002. Influence of nutrient deficiency and sucrose concentrations on the production of artemisinin by cultured cells of sweet wormwood (Artemisia annua L.). Indian Journal of Plant Physiology 7, 159–162. Utumi, M.M., Monnerat, P.H., Pereira, P.R.G., Fontes, P.C.R., VDPC, Godinho, 1999. Macronutrient deficiencies in Stevia: visual symptoms and effects on growth, chemical composition, and stevioside production. Pesquisa Agropecuária Brasileira 34, 1039–1043 (In Portugese). Weir, R.G., Cresswell, G.C. (Eds.), 1993. Plant Nutrient Disorders 3: Vegetable Crops. Inkata Press, North Ryde, NSW Australia. Yadav, A.K., Singh, S., Dhyani, D., Ahuja, P.S., 2011. A review on the improvement of stevia [Stevia rebaudiana (Bertoni)]. Canadian Journal of Plant Science 91, 1–27.
of the Council with regard to steviol glycosides. Official Journal of the European Union L295, 205–206. Barbet-Massin, C., Giuliano, S., Alletto, L., Daydé, J., Berger, M., 2015. Nitrogen limitation alters biomass production but enhances steviol glycoside concentration in Stevia rebaudiana Bertoni. PLoS One 10 (7), e0133067. Brandle, J.E., Rosa, N., 1992. Heritability for yield, leaf: stem ratio and stevioside content estimated from a landrace cultivar of Stevia rebaudiana. Canadian Journal of Plant Science 72, 1263–1266. Ceunen, S., Geuns, J.M.C., 2013. Steviol glycosides: chemical diversity metabolism, and function. Journal of Natural Products 76, 1201–1228. Das, K., Dang, R., Shivananda, T.N., 2006. Effect of major nutrients on biomass production and stevioside content of Stevia rebaudiana – a pot culture study. Biomed 1, 22–25. FSANZ, 2008. Final Assessment Report: A540 Steviol Glycosides as Intense Sweeteners. Food Standards Australia New Zealand, Barton, ACT, Australia. Ferreira, J.F.S., 2007. Nutrient deficiency in the production of artemisinin dihydroartemisinic acid, and artemisinic acid in Artemisia annua L. Journal of Agricultural and Food Chemistry 55, 1686–1694. Freitas, M.S.M., Monnerat, P.H., Viera, I.J.C., 2008. Mineral deficiency in Passiflora alata Curtis: vitexin bioproduction. Journal of Plant Nutrition 31, 1844–1854. Hartmann, T., 2007. From waste products to ecochemicals: fifty years research of plant secondary metabolism. Phytochemistry 68, 2831–2846. Hearn, L.K., Subedi, P.P., 2009. Determining levels of steviol glycosides in the leaves of Stevia rebaudiana by near infrared reflectance spectroscopy. Journal of Food Composition and Analysis 22, 165–168. Hermans, C., Hammond, J.P., White, P.J., Verbruggen, N., 2009. How do plants respond to nutrient shortage by biomass allocation? Trends in Plant Science 11, 610–617. Herms, D.A., Mattson, W.J., 1992. The dilemma of plants: to grow or defend. Quartely Review of Biology 77, 283–335. Jain, P., Kachhwaha, S., Kothari, S.L., 2008. Improved micropropagation protocol and enhancement in biomass and chlorophyll content in Stevia rebaudiana (Bert.) Bertoni by using high copper levels in the culture medium. Scientia Horticulturae 119, 315–319. Jarma, O.A., Combatt, C.E., Polo, S.J., 2012. Glycoside contents depending on the nutrient deficiencies in Stevia rebaudiana Bert. Revista U.D.C.A Actualidad & Divulgación Científica 15, 107–116 (In Spanish). Jones, J.B. (Ed.), 2005. Hydroponics: a Practical Guide for the Soilless Grower. CRC Press, Boca Raton, USA. Kawatani, T., Kaneki, Y., Tanabe, T., Sakamoto, I., Murakami, K., Tanaka, O., 1978. On the cultivation of Kaa He-e (Stevia rebaudiana Bertoni). IV: Response of Kaa He-e to nitrogen fertilization rates and to the three major elements of fertilizer. Japanese Journal of Tropical Agriculture 21, 173–178. Kulasekaran, R., Singh, V., Megeji, N.W., 2006. Cultivation of stevia (Stevia rebaudiana): a comprehensive review. Advances in Agronomy 89, 137–177. Kumar, R., Sharma, S., Ramesh, K., Prasad, R., Pathania, V., Singh, B., Singh, R.D., 2012. Effect of agro-techniques on the performance of natural sweetener plant: stevia (Stevia rebaudiana) under western Himalayan conditions. Indian Journal of Agronomy 57, 74–81. Lambers, H., Chapin III, F., Stuart, P., Thijs, L., 2008. Plant Physiological Ecology.
7