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Effects of Septoglomus viscosum inoculation on biomass yield and steviol glycoside concentration of some Stevia rebaudiana chemotypes
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Effects of Septoglomus viscosum inoculation on biomass yield and steviol glycoside concentration of some Stevia rebaudiana chemotypes Luigi Tedone*, Claudia Ruta, Francesca De Cillis, Giuseppe De Mastro University of Bari - Department of Agricultural and Environmental Science, Via Amendola, 165/A - 70126, Italy
A R T I C LE I N FO
A B S T R A C T
Keywords: Stevia rebaudiana Chemotype Open field cultivation Mycorrhiza Plant regrowth Steviol glycosides
There is growing interest in the medicinal properties of Stevia rebaudiana Bertoni, due to its ability to produce zero-calorie sweeteners, steviol glycosides (SGs). Inoculation of the roots with arbuscular mycorrhizal fungi (AMF) is a method used to improve the production of secondary metabolites by many plant species, but its effects on S. rebaudiana have not been extensively studied, especially in open field conditions in Italy. Therefore, a field experiment was conducted to study the effects of the fungus Septoglomus viscosum on the leaves and secondary metabolites of three micropropagated stevia chemotypes (L1, L2 and L3) over three years. Our results indicate that the establishment of symbiotic relations between AMF and stevia plants of selected chemotypes improved leaf yield, because the mycorrhizal (MIC) treated plants had a better tendency to regrowth, especially L1 and L3, which had survival rates of 80 % and 90 %, respectively. MIC treatment also had positive effects in terms of SG production, which was not significant in terms of concentration, but was significant in terms of production.
1. Introduction Stevia (Stevia rebaudiana Bertoni) is a perennial shrub of sub-tropical origin. The plant is native to northeastern Paraguay, but is now cultivated in Mexico, Central America, Japan, China, Spain, Belgium, the UK, the USA (Williams and Burdock, 2009) and Italy (Tavarini and Angelini, 2013). It belongs to the Asteraceae family and has an extensive root system; its brittle stems produce small elliptical leaves (Yadav et al., 2011). Since ancient times, stevia has been used for various medicinal purposes (Goyal and Goyal, 2010). In Europe, stevia leaves are used in infusions, but stevia extract is now more important, since it is a promising renewable calorie-free sweetener that can be used as a sugar substitute, or as an alternative to artificial sweeteners (Anton et al., 2010). Stevia is the common name for the stevioside extracted from the leaves of stevia; it consists of ent-kaurene diterpene glycosides, commonly known as steviol glycosides, which are natural sweeteners that can be isolated from its leaves and are 250−300 times sweeter than saccharose. Almost thirty diterpenes have been identified in stevia leaves (Gupta et al., 2016), including stevioside, steviolbioside, rebaudioside A, B, C, D, E, F and dulcoside (Geuns, 2003). Stevioside is the most abundant glycoside in stevia leaves, (4–13 % w/w), followed by rebaudioside-A (2–4 % w/w), rebaudioside-C (1–2 % w/w), and dulcoside (0.4-0.7 % w/w) (Jackson et al., 2009; Gardana et al., 2010; Cacciola et al., 2011). Steviol-glycosides can be used not only as
⁎
sweeteners, but also for medicinal purposes (Gupta et al., 2013). Stevioside and the related compounds are reported to have anti-hyperglycaemic, anti-hypertensive, anti-inflammatory, anti-tumor, antidiarrheal, diuretic, and immunomodulatory effects (Chatsudthipong and Muanprasat, 2009; Bernal et al., 2011). The growing demand for natural alternatives to artificial sweeteners has generated considerable commercial interest in stevia. Stevia sales will be worth approximately $700 million in the next few years, according to Rabobank (Muth, 2015). Today, stevia is cultivated on approximately 32,000 ha worldwide, with the largest production area in China (approximately 75 % of the world total) (Singh and Verma, 2015). There is a great need for cultivation techniques (Mandal et al., 2013) to use in intensive farming in order to increase production of S. rebaudiana low-calorie glycosides, and to improve its biomass yield and glycoside composition. The quality of stevia leaves can vary widely, due to many factors, including chemotype, environmental and soil conditions, irrigation methods, sunlight, farming practices, sanitation, processing, and storage (Yadav et al., 2011; Ceunen and Geuns, 2013). Many factors limit the large-scale cultivation of stevia plants, including its very low germination percentage. In addition, seed propagation produces a nonhomogeneous population, with a consequent variability in glycoside composition (Miyagawa et al., 1986), while vegetative propagation produces only a low number of plants. In order to meet these challenges, and to achieve rapid mass
Corresponding author. E-mail address: [email protected] (L. Tedone).
https://doi.org/10.1016/j.scienta.2019.109026 Received 4 August 2017; Received in revised form 30 October 2019; Accepted 10 November 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Luigi Tedone, et al., Scientia Horticulturae, https://doi.org/10.1016/j.scienta.2019.109026
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line were inoculated with a crude AMF inoculum of Septoglomus viscosum (Basionym: Glomus viscosum T.H. Nicolson (1995)). The AMF inoculum consisted of sandy soil containing spores, external mycelium, and infected strawberry root fragments from strawberry pot cultures. Approximately 10 g (approximately 100–120 spores) of each AMF inoculum was placed immediately below the roots. Non-mycorrhizal plants were obtained by adding approximately 10 g of the same sterile sandy soil, without AMF inoculum, and were used as controls. Acclimatization took place in greenhouse conditions at 18−25 °C with mist, gradually reducing the humidity level from 85 to 90% to 50–60 % over 20 days. Subsequently, the plants were grown at 20 °C and 40 % RH until they were transplanted in the field. No fertiliser was added to the pots during greenhouse growth. Following plant acclimatization in the greenhouse, bio-morphological characteristics were identified for each chemotype on 10 representative samples of S. rebaudiana plants, and 130 healthy plants were then transplanted into an open field for each treatment and line. Bio-morphological determination was performed by separating the leaves from the stems, and the foliar area was measured using a leaf area meter (Model Li-Cor 3000). Shoots and roots were then weighed to determine their fresh weight, oven-dried (48 h, 60 °C), and then reweighed to determine their dry weight. The percentage of mycorrhizal colonization (Trouvelot et al., 1986) was measured to verify the establishment of symbiosis on the set of 10 randomly chosen representative root portions per treatment. The roots were stained following the Phillips and Hayman (1970) method. Ten 1-cm root pieces per plant were selected at random from the stained root fragments and placed on a microscope slide. Ten microscope slides were prepared for each treatment. The root fragments were mounted in a drop of glycerol and observed using an optical microscope (Leica DMLB100).
propagation of stevia, in vitro micropropagation from shoot tips and leaves is suggested as an extremely efficient method to obtain more uniform plants on an industrial scale than is possible with seed reproduction (Yadav et al., 2011; Luwańska et al., 2015; Röck-Okuyucu et al., 2016); the use of bioreactors is suggested in order to increase stevia plant production (Vives et al., 2017). Stevia can grow well in a wide range of soils if there are optimal soil moisture conditions and an adequate drainage. To ensure sustainable production of stevia, arbuscular mycorrhizal fungi (AMF) involved in symbioses with the plant roots provide the host plants with increased biomass, improving the growth rate of aromatic plants (Tarraf et al., 2015), together with their pathogen resistance, mineral nutrient uptake and rate of photosynthesis when compared with non-mycorrhizal symbioses. Cruz et al. (2004) reported that plants inoculated with AMF develop a more efficient root system, with increased survival ex vitro (Estrada-Luna et al., 2000; Moraes et al., 2004; Ruta et al., 2009) and better physiological activity due to their improved nutrition status. AMF have been shown to enhance the production of secondary metabolites in oregano, fennel, Artemisia annua, sweet basil and St John's wort (Khaosaad et al., 2006; Kapoor et al., 2004, 2007; Toussaint et al., 2007; Zubek et al., 2012). Their effects on S. rebaudiana have recently been studied by Mandal et al. (2013), who inoculated stevia plants with Rhizophagus fasciculatus and thus obtained increased biomass production, due to the greater number of shoots per plant, and higher concentrations of steviol glycosides. Tavarini et al. (2018) have also reported the positive effects of R. irregulare on stevia production and quality. To improve glycoside content and composition, several plantbreeding procedures have been used to increase leaf yield and also rebaudioside-A concentration in leaves (Brandle et al., 1998). Based on the studies reported in the literature, it appears that there is sufficient genetic variability (Thiyagarajan and Venkatachalam, 2015) to allow significant genetic improvements to the content of steviol glycosides in leaves. The present study assessed the effects of mycorrhizal inoculation on selected micropropagated stevia chemotypes. The aim was to define the best combination to improve biomass production and glycoside yield in order to facilitate industrial processing.
2.3. Experimental site The field experiment was carried out over three years (2011–2013) at the “Enrico Pantanelli” experimental farm of the University Bari “A. Moro”, at Policoro (southern Italy; 40°10′20″ N, 16°39′04″ E). The site is 15 m above sea level, and the climate is typically Mediterranean, according to the De Martonne classification; it has an average annual rainfall of 554 mm, distributed mainly during autumn and winter, a maximum mean temperature between 12.8 °C and 31 °C, and a minimum temperature between 3.95 °C and 11.26 °C. The soil is loamy and over 1.2 m deep, with the following physical characteristics: sand 398 g kg–1, silt 374 g kg–1, clay 228 g kg–1. Its chemical characteristics are as follows: pH 7.7; total N (Kjeldahl method) 1.7‰, available P2O5 (Olsen method) 27.6 mg kg–1, exchangeable K2O (ammonium acetate method) 227 mg kg–1, organic matter content (Walkley-Black method) 2.3 %, total carbonate 15.0 g kg–1, active carbonate 5.0 g kg−1, saturated paste extract electrical conductivity (ECe) 0.95 dS m–1, ESP 1.9 %; bulk density 1.25 kg dm–3; soil moisture at field capacity (measured in situ) of 0.32 m3 m−3 and at wilting point (-1.5 MPa) of 0.15 m3 m−3 of soil dry weight.
2. Materials and methods 2.1. Stevia chemotype selection A previous experiment selected three different chemotypes on the basis of their steviol glycoside content and composition, and their agronomic performance. Chemotype L1 has a high rebaudioside-A content (over 5 g 100 g dm−1) and medium leaf production, chemotype L2 has a high stevioside content (over 10 g 100 g dm−1) and medium leaf production, and chemotype L3 has a medium level of stevioside and extremely high leaf production. By referring to a previous experiment on the same site, we considered average leaf production per year to be 3.5 t ha−1, considering cultivation over three years.
2.4. Experimental design and field assessment 2.2. Plant material and mycorrhizal inoculation After plant acclimatization in the greenhouse, 130 micropropagated healthy plants for each treatment and line were transplanted into an open field on 6th May 2011, at a density of 5 plants m−2, with rows 0.6 m apart (Lavini, et al., 2008), and 0.35 cm between plants on the rows. Stevia microplants were arranged using a split plot design with four repetitions, with the two treatments as the first variable: mycorrhizal inoculated (MIC) and non-inoculated (control) plants, and chemotypes (L1, L2 and L3) as the second variable. The plot had an area of 20 m2. Before planting, the soil was prepared by deep ploughing, which was followed by disc-ploughing and harrowing. All plants received an application of 80, 60 and 140 kg ha−1 of N, P and K using urea, single
The three chemotypes were multiplied by micropropagation. Multiplication was induced on a basal medium (BM) (see, Morone Fortunato et al., 2005) enriched with 6-benzylaminopurine (BAP 0.5 mg l−1) and IAA (3.0 mg l−1), following the protocol described by Ruta et al. (2009). For rooting, the BM was enriched with a thin layer of the same liquid nutrient solution containing 4.5 mg l-1 Indol Acetic Acid (IAA) (Ruta et al., 2009). The rooted microplants (300 for each line) were transplanted into individual plastic pots with a volume of 0.2 dm3 containing a mixture of sterile peat (46 % organic carbon, 1.2 % organic nitrogen, 80 % organic matter, pH 6) and perlite (2:1, v/v ratio). At the time of transplantation, half of the 150 microplants of each 2
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2.08 g, fresh roots 2.22 g, dry roots 0.46 g) (Table 2). L1 and L3 appeared to show a greater affinity with S. viscosum than with L2, thus confirming that the potential of AMF to promote growth of the host plant also depends on the host genotype (Baum and El-Tohamy, 2015). Establishment of symbiosis was confirmed by an infection percentage of over 50 %, measured two months after inoculation. There is wide support in the literature for the use of AMF to improve growth of micropropagated plantlets (Estrada-Luna et al., 2000; Ruta et al., 2009; Yadav et al., 2013), mainly related to improved nutrient and water uptake.
Table 1 Harvesting schedule for stevia during the study period. Treatment
2012 6-Aug
Mycorrhiza Control
st
1 cut 1st cut
2013 25-Sep nd
2 cut 2nd cut
28-Jul st
1 cut 1st cut
2014 26-Sep nd
2 cut 2nd cut
7-Aug st
1 cut 1st cut
28-Sep 2nd cut 2nd cut
superphosphate, and potassium sulphate, respectively, as fertilisers. In the second and third years, the fertilisers were distributed partly at the start of the cycle (phosphorus and 1/3 of nitrogen as ammonium superphospate) and partly during the cycle (2/3 of nitrogen after the first harvest as ammonium nitrate 34 %). Each year, a total of 80 kg nitrogen and 20 kg P2O5 was applied during vegetative growth. All fertilisers were applied to the soil. The first irrigation treatment was performed immediately after transplanting and repeated three days later. Water was subsequently supplied on a regular basis using a drip-irrigation system to maintain optimal water conditions, especially in summer, throughout the whole study period. During all three years, the first harvest was performed in summer by cutting the aboveground biomass at a height of about 5−7 cm. Due to the variability in photoperiod sensitivity, which depends on the daylength sensitivity of this species, the cultivation of stevia as a perennial crop in this study allowed a two-harvest per year management system (Table 1). Before each harvest, the leaf area index (LAI) was measured at field level using AccuPAR (LAI-2000 Ceptometer (Wilhelm et al., 2000)), while the total biomass weight, number of stems, and leaf weight were measured at each harvesting time for each treatment.
3.2. Plant density The ability of stevia plants to survive until the end of the experiment was compared to the time of planting by measuring the total number of surviving plants m−2 at each harvest time, per year (Fig. 1). This enabled us to evaluate differences between stevia chemotypes and mycorrhizal-inoculated plants in the open field. Over the study period, stevia plant density decreased and differed significantly among chemotypes and treatments (Fig. 1). The mean survival rate for MIC plants was 80 % in 2012 (458 and 508 days after planting (DAT), compared with 53 % for control plants. In 2013 (824 and 874 DAT), the survival rate was 73 % for MIC plants and 40 % for control plants, and in 2014 (1189 and 1241 DAT), the survival rate was 63 % for MIC plants and 34 % for control plants. Plant survival differed among the chemotypes; L1 and L3 survival rates were higher under MIC treatments than for control plants. 3.3. Yield and yield components Tables 3 and 5 show dry leaf (t ha−1) and stem yields (t ha−1) at different harvest times during the three years. Variations were found among chemotypes, mycorrhizal treatments and years. The results showed a decrease in leaf and stem production for the three chemotypes in the third year. The maximum stem dry yield occurred in the second year (Table 5). Biomass production was always higher at the first harvest, because temperature conditions were better than at the second harvest. Biomass yield fell in the third year, with a 67 % and 16 % reduction in leaf and stem yield, respectively, compared to the first year.
2.5. Extraction and analysis of steviol glycosides (SGs) Sweet diterpene glycosides were extracted from the dried stevia leaves following Kolb et al. (2001). HPLC analysis was performed using an Agilent 1100 quaternary pump and a UV detector 1260. Chromatographic separation was performed on a column of polyamide Zorbax Carbohydrate 15 cm x 4.6 cm with particles of 5 μm, coupled by a "guard column" with cartridges for Zorbax NH2. The mobile phase consisted of acetonitrile/water HPLC grade at a ratio of 80:15 v/v. The elution programme used a constant flow of 2.5 ml min−1 and isocratic elution for 15 min at room temperature. Analysis was performed by monitoring the signal in absorbance at 210 nm. The peak identification and calibration were determined using pure stevioside and rebaudioside-A standards from Sigma-Aldrich.
3.3.1. Dry leaf yield Dry leaf yield was collected twice a year in a two-harvest management system. Pal et al. (2015) indicate that a single harvest during the growing season may lead to a lower leaf yield, and also to lower glycoside production (Moraes et al., 2013) than a multi-cut management system. Results from central Italy (Andolfi et al., 2006) further highlighted the importance of two cuts per year in achieving higher yields. Following the two-harvest management system per year, the annual dry leaf yield refers to the sum of the two harvests carried out each year. For a detailed analysis of dry leaf yield, dry leaf yield is reported in terms of grams of dry leaves per plant (Table 3) and tons per hectare under all the treatments (Table 4). The overall results showed a sharp decrease in dry leaf production (grams per plant and tons per hectare) in the last year. The average annual quantities of dry leaf production per plant were stable during 2012 (93.6 g plant−1) and 2013 (88.8 g plant−1), but a sharp decrease occurred in 2014 (35.7 g plant−1). Table 3 shows that the first cut gave a higher amount of dry leaves per plant than the second (46.8 vs. 44.4 g plant−1). This appears to be associated with the shorter vegetation period for the second cut than for the first cut (Andolfi et al., 2006). With regard to chemotypes, L3 yielded the highest dry leaf production in both harvest periods (85.8 g), followed by L1 (81.0 g), while L2 gave the lowest yield (51.4 g). AMF inoculated plants gave a higher yield (79.7 vs 65.7 g) than non-inoculated plants. The most significant effect of AMF inoculation was observed with L1
2.6. Statistical analysis The data obtained were subjected to variance analysis (ANOVA). In addition, treatment means were compared using the LSD test at a 0.05 probability level. CoStat software was used for statistical analysis. 3. Results and discussion 3.1. Plant material and mycorrhizal inoculation Table 2 shows the increases in the bio-morphological parameters of MIC plants of the three different chemotypes compared with the control plants. Mycorrhizal inoculation successfully influenced the epigeal and hypogeal growth of all three chemotypes during acclimatization in the greenhouse (Ruta et al., 2009). Mycorrhiza treatment had a significantly positive effect on the averages of all parameters measured for all lines (plant height 20.5 cm, leaf number 2.6, leaf area 35.5 cm2, fresh shoots 39.4 g, fresh shoots 3
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Table 2 Bio-morphological characteristics of three chemotypes of Stevia rebaudiana (Bert.) Bertoni grown in a greenhouse on a substrate enriched with mycorrhizal inoculum (S. viscosum fungus) (MIC) in comparison to the control plants two months after transplanting. Treatments
Mycorrhiza (M) MIC Control Chemotype (L) L1 L2 L3 Interaction MxL
Plant height
Leaves
Leaf area
Shoots
Roots
N plant−1
cm2 plant−1
Fresh (g plant−1)
Dry
cm
23.2 a 17.7 b ***
39.3 a 31.5 a *
42.8 a 36.0 a *
2.3 a 1.8 b **
0.50 a 0.42 b *
2.48 a 1.96 b **
0.36 0.32 **
56.3 a 0b ***
27.2 a 18.9 b 15.3 b **
27.0 a 42.4 a 37.0 b ns
45.9 a 40.2 ab 32.0 a ns
2.6 a 2.3 a 1.4 b *
0.6 a 0.45 ab 0.35 b ns
3.10 a 2.55 a 1.00 b *
0.48 a 0.33 a 0.20 b *
69.4 a 48.5 b 50.9 b *
ns
ns
ns
ns
ns
ns
ns
ns
Fresh
Root mycorrhizal colonization Dry (%)
ns,*,**, *** :Non significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple- range test (P = 0.05). Table 4 Dry leaf yield (t ha-1) of stevia crop as affected by mycorrhizal inoculum (M), chemotype (L) and growing year (Y). Dry leaf yield (t ha−1) Source of variance
Fig. 1. Plant density over the years. Data are means ± SE per treatment (n = 4).
Mycorrhiza (M)
MIC Control
Chemotype (L)
L1 L2 L3
Year (Y)
2012 2013 2014
Interaction MxL MxY LxY MxLxY
Second cut
Total
47.0a 31.3b ** 41.6a 26.4b 49.5a * 58.3a 43.5a 15.7b ***
32.6a 34.4a ns 39.3a 25.0b 36.3a * 35.3a 45.3a 20.0b ***
79.7a 65.7b * 80.9a 51.4b 85.8a * 93.6a 88.8a 35.7b ***
* ns ns ns
* ns ns ns
* ns ns ns
Chemotype (L)
L1 L2 L3
Years (Y)
2012 2013 2014
Second cut
Total
2.4a 1.6b * 2.1a 1.3b 2.5a * 3.0a 2.3a 0.8b ***
1.3a 0.8b * 1.2a 0.6b 1.4a ** 1.3a 1.4a 0.5b ***
3.7a 2.4b * 3.2a 1.9b 3.8a * 4.2a 3.7a 1.4b ***
* ns ns ns
* ns ns ns
** ns ns ns
ns,*,**, *** :Non-significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple- range test (P = 0.05).
Dry leaf yield (g plant−1) First cut
MIC Control
Interaction MxL MxY LxY MxLxY
Table 3 Dry leaf yield (g plant−1) of stevia crop as affected by mycorrhizal inoculum (M), chemotype (L) and growing year (Y).
Source of variance
Mycorrhiza (M)
First cut
The average dry leaf yield of 2.96 t ha−1 was similar to that reported by Brandle and Rosa (1992), and close to the average dry leaf yield of 2.67 t ha−1 reported by Megeji et al. (2005) in India. Higher yields of 3.6 and 3.9 t ha−1 were reported in central and southern Italy by Andolfi et al. (2006) and Lavini et al. (2008), respectively. However, in the USA, Moraes et al. (2013) reported a lower leaf yield of 2.0 t ha−1. Our analysis of annual dry leaf production (sum of the first and second harvests) indicates the first year as the most productive (4.2 t ha1 ), the second year as slightly less productive 3.6 t ha-1, and the third year as significantly (P ≤ 0.05) less productive (1.3 t ha-1). The effect of mycorrhizal inoculation was evident; MIC treatments produced a higher dry leaf yield than control treatments for all chemotypes and across the years. The effect of MIC treatment was more pronounced in the second and third years, when production decreased and the rate of decrease was lower under MIC treatment (4.6 t ha−1 vs. 3.8 t ha-1, 4.6 t ha−1 vs. 2.5 t ha-1, and 1.8 t ha−1 vs. 0.8 t ha-1, reported in 2012, 2013 and 2013, respectively). This agreed with other studies (Mandal et al., 2013, 2015), which found a significant increase in S. rebaudiana biomass after AMF inoculation. Mandal et al. (2013) found that increases in biomass were due to improved P-nutrition under MIC treatments. Smith and Read (2008) further highlighted the importance
ns,*,**, *** :Non significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple- range test (P = 0.05).
dry leaf production, which was 110.3 g plant-1 under MIC treatment, compared with 51.5 g plant−1 for control plants. These parameters, together with higher plant survival rates, gave better results for MIC treatments. 4
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Table 5 Stem number (n. plant −1) of stevia crop as affected by mycorrhizal inoculum (M), chemotype (L) and growing year (Y).
Table 6 Stem yield (t ha−1) of stevia crop as affected by mycorrhizal inoculum (M), chemotype (L) and growing year (Y).
Stem number (n plant−1) Source of variance Mycorrhiza (M)
MIC Control
Chemotype (L)
L1 L2 L3
Year (Y)
2012 2013 2014
Interaction MxL MxY LxY MxLxY
Stem yield (t ha−1)
First cut
Second cut
Total
Source of variance
18.1a 18.4a ns 16.3ab 12.5b 25.9a * 11.3b 26.4a 17.0ab **
33.7a 39.7b ns 32.2ab 24.7b 51.9a * 40.6a 39.0a 30.3a ns
51.8a 53.6b * 43.1b 37.3ab 77.9a ns 51.9a 65.4a 47.4a ns
Mycorrhiza (M)
MIC Control
Chemotype (L)
L1 L2 L3
Year (Y)
2012 2013 2014
ns ns ns ns
ns ns ns ns
ns ns ns ns
Interaction MXL MXY LXY MXLXY
First cut
Second cut
Total
1.92a 1.16b * 1.72b 1.09a 1.80b * 1.11b 2.18a 1.32ab *
1.39a 0.96a * 1.28a 0.55b 1.70a * 1.48a 1.19a 0.86a ns
3.31a 2.12b * 3.00a 1.64b 3.51a * 2.59ab 3.37a 2.18b *
ns ns ns ns
ns ns ns ns
ns ns ns ns
ns,*,**, *** :Non significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple- range test (P = 0.05).
ns,*,**, *** :Non significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple- range test (P = 0.05).
of a symbiotic relationship in providing host plants with a higher biomass yield than non-mycorrhizal symbioses through an increase in plant uptake of soil nutrients (Govindarajulu et al., 2005; Atul-Nayyar et al., 2009). The effect of the chemotype on dry leaf yield was significant and showed some effects of interaction with mycorrhizal inoculation (Table 3). This was clear when leaf yield varied among chemotypes in control treatments in the order L3 > L2 > L1, while the order was the opposite among MIC treatments (L1 > L2 > L3), indicating that mycorrhizal inoculation can change the productive response of some stevia chemotypes. Under MIC treatment, L1 gave a higher annual leaf production (5.3 t ha−1) than L2 and L3 (2.0 and 3.6 t ha−1), while L3 was the highest producer among the control treatments (3.9 t ha−1), followed by L2 (2.1 t ha−1) and L1 (1.2 t ha−1). Considering the interaction effect of MxL, MIC treatment had positive effects on L1 and L3. The highest significant difference was observed in L1 (4.3 for MIC treatment vs 1.2 t ha-1 for the control).
produced a significantly higher stem yield, while L1 gave the lowest stem yield.
3.3.2. Stem yield Stem production was measured each year in terms of number per plant and yield. Table 5 shows the effect of mycorrhiza and chemotype on the stem number per plant per year. The number of stems per plant was influenced by the chemotype and harvest time. As a general effect, a higher stem production per plant was observed at the second harvest (36.6 vs. 18.2), with average values ranging between 24.7 and 51.9, i.e. almost twice those of the first harvest (average between 11.3 and 26.4). (Table 5). L3 produced a greater number of stems per plant (77.9) than the other chemotypes (43.1 and 37.3 in L1 and L2, respectively). Average stem dry yields are reported in Table 6, showing that the average value was 2.71 t ha−1, as reported by Megeji et al. (2005) in India (2.72 t ha−1). Stevia dry stem yield varied significantly (P ≤ 0.05) among the years, among chemotypes and among treatments. Annual stem dry yield was highest in 2013 (3.37 t ha−1), unlike dry leaf yield. The higher and more evenly distributed rainfall could explain why production in 2013 was higher than in 2012 and 2014. In MIC plants, the effect of harvest time on dry stem yield showed a higher yield at the first cut in 2013 and 2014, but higher at the second cut in 2012. The same trend was found in control treatments (Table 6). MIC symbiosis produced a significantly higher stem yield than control treatments (3.31 vs. 2.12 t ha−1). With regard to the chemotypes, L3
Table 7 Leaf area index (LAI) of stevia crop as affected by mycorrhizal inoculum (M), chemotype (L) and growing year (Y).
3.3.3. Leaf Area Index (LAI) The leaf area index (LAI) is an important parameter, correlated directly to crop production. The results of this study showed an average LAI of 2.71, which varied between a maximum of 6.47 and a minimum of 0.6 among the years and treatments (Table 7). Our average value was significantly lower than the LAI value of 4 reported by Lavini et al. (2008) and the 4.83 reported by Fronza and Folegatti (2003). The average LAI was 3.53 during the first harvest and 1.89 during the second. (Table 7). The effect of the year was significant, with an average of 3.67 in the first year, 3.25 in the second year and 1.22 in the third year. This data confirmed the result of Pal et al. (2015), who reported that LAI was significantly (P ≤ 0.05) affected by the crop cycle and harvesting regime. The overall results showed a positive effect of mycorrhizal symbiosis
Source of variance
LAI
Mycorrhiza (M)
MIC Control
Chemotype (L)
L1 L2 L3
Year (Y)
2012 2013 2014
Interaction MxL MxY LxY MxLxY
First cut
Second cut
Total
4.20a 2.86b ** 3.76ab 2.39b 4.44a * 5.15a 3.97a 1.47b **
2.26a 1.52b * 2.13a 1.12b 2.43a * 2.18a 2.53a 0.96b **
3.23a 2.19b ** 2.94a 1.75b 3.43a ** 3.67a 3.25a 1.22b **
* ns ns ns
* ns ns ns
* ns ns ns
ns,*,**, *** :Non significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple- range test (P = 0.05). 5
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Table 8 Leaves/stem dry weight ratio of stevia crop as affected by mycorrhizal inoculum (M), chemotype (L) and growing year (Y).
Table 9 Stevioside content of stevia leaves as affected by mycorrhizal inoculum (M), chemotype (L) and growing year (Y).
Leaves/ stem ratio Source of variance Mycorrhiza (M)
Chemotype (L)
Year (Y)
Interaction MxL MxY LxY MxLxY
MIC Control L1 L2 L3 2012 2013 2014
Stevioside content (g 100 g dm−1)
Source of variance
First cut
Second cut
Total
1.4a 1.5a ns 1.4a 1.5a 1.6a ns 2.9 0.9 0.6 **
0.7a 0.7a ns 0.8a 0.8a 0.6a ns 0.5 1.2 0.6 **
1.1a 1.1a ns 1.1a 1.1a 1.1a ns 1.7a 1.0a 0.6a **
ns ns ns ns
ns ns ns ns
ns ns ns ns
Mycorrhiza (M)
MIC Control
Chemotype (L)
L1 L2 L3
Year (Y)
2012 2013 2014
Interaction MXL MXY LXY MXLXY
ns,*,**, *** :Non significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple- range test (P = 0.05).
First cut
Second cut
Total
10.3a 9.0b * 4.6b 12.5a 11.9a *** 9.5 9.4 10.1 ns
7.5a 7.5a ns 3.0b 10.1a 9.5a *** 7.7 7.8 7.0 ns
8.9a 8.3a ns 3.8b 11.3a 10.7a *** 8.6a 8.6a 8.5a ns
* ns ns ns
* ns ns ns
* ns ns ns
ns,*,**, *** :Non significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple- range test (P = 0.05).
on LAI: 3.23 for MIC treatments compared to 2.19 for the control treatments. On average, these values varied among the different chemotypes, with averages of 2.94, 1.75 and 3.43 for chemotypes L1, L2 and L3, respectively. With regard to M x L interaction, L1 presented the most significant differences between MIC and control treatments (4.77 vs 1.12); the maximum LAI (6.08) was measured in L1 MIC plants at the first harvest, while the minimum LAI (0.79) was recorded in L1 control plants at the second harvest.
%) compared with an average rebaudioside-A concentration of 2.9 % (ranging between 0.4 and 5.9 %). Other studies (i.e. Yadav and Guleria, 2012; Tavarini and Angelini, 2013) have reported stevioside concentrations in leaves ranging from 4 to 14 %, and rebaudioside-A concentrations ranging from 2 to 4 %. In southern Italy, Lavini et al. (2008) found a stevioside concentration of 8.4 % in dry leaves and a rebaudioside-A concentration of 5.7 %. These were close to the concentrations reported in this study. In terms of total SG content, an important aspect is the ratio between stevioside, with its slightly bitter aftertaste, and rebaudioside-A, which has a more acceptable flavour (Brandle et al., 1998). With regard to this aspect, agronomical traits can influence these compounds (Ren and Shi, 2018). The overall results indicated that stevioside content (Table 9) was significantly higher in L2 and L3, with an average of 11.4 % and 10.5 % in MIC treatments, and 11.1 % and 10.8 % in control treatments. Similarly to the total glycoside content, the summer cut produced higher average stevioside contents than the autumn cut for all three chemotypes. Across the years and treatments, the first cut gave a higher stevioside content than the second cut (9.7 vs. 7.5 %). MIC plants showed some positive effects on stevioside content (8.9 %) compared to nonmycorrhizal-inoculated plants (8.3 %), although the differences between them were not significant. The cropping year had no effect on stevioside concentrations, which remained stable at 8.6 %. Moraes et al. (2013) reported a higher stevioside content, which varied between 10.6 % and 11.1 % in the first and second growing seasons, respectively. The effect of M x L interaction was significant in L1 (4.7 % for MIC treatment vs 2.9 % for control treatments) and L2 (11.6 vs 11.0 % for control treatments). Rebaudioside-A contents are reported in Table 10. The average rebaudioside-A content was 2.9 %, which varied significantly between chemotypes and mycorrhizal inoculation, whereas the cultivation year had no significant effect. A study by Lavini et al. (2008) in southern Italy reported a higher concentration (5.7 %), while Moraes et al. (2013) reported a rebaudioside-A content in leaves harvested in Mississippi, USA, which ranged between 3.1 % in the first growing season and 4.5 % in the second growing season. Analysis of the results indicated a wide difference between chemotypes: L1 had the highest rebaudioside-A concentration (Table 10) under all treatments and across years (5.9 %). L3 produced the second highest concentration, with a
3.3.4. Leaf/stem ratio The average leaf production/stem production ratio was 1.1 (Table 8). This is in line with Brandle and Rosa (1992), who reported a value of 1.2 for stevia cultivated in Delhi, India. Brandle and Rosa also suggested that the high leaf/stem ratio might be due to cultivation under a long day photoperiod, considering that stevia is an obligate short-day plant. Therefore, enhanced vegetative growth under long days is not surprising. Megeji et al. (2005) reported a lower leaf/stem ratio (0.8) for stevia cultivated in Palampur, India. The overall results showed a decreasing trend in the leaf/stem ratio over the years (1.7, 1.0 and 0.6 respectively in the first, second and third years) (Table 8). Following the same trend of leaf production, large differences were found between the first and the second harvest: a higher leaf/stem ratio in the summer harvest, the highest dry leaf production in summer, and the highest stem production in autumn. The mycorrhizal inoculation had a variable impact on this index. A higher leaf/stem ratio was found in MIC treatments in the first and the third years. The chemotype had no clear effect on this index, especially in MIC treatments (1.1, 1.0 and 1.1 vs. 1.1, 1.2 and 1.1 for L1, L2 and L3, respectively). 3.4. Steviol glycoside (SG) concentrations in stevia leaves The main compounds of stevia glycosides, stevioside and rebaudioside-A, were measured. The content and distribution of these SG compounds varies considerably according to harvest time and chemotypes. Mandal et al. (2013, 2015), found a significant increase in SG concentrations in response to AMF colonization. Similar observations were reported later in another study (Tavarini et al., 2018). Under all treatments, stevioside was the predominant SG, present at higher levels than rebaudioside-A. An average stevioside concentration of 8.6 % was found in stevia dry leaves (ranging between 3.8 and 11.3 6
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mychorrizal treatment and year of harvesting. Considering the total data of yield of each individual glycoside, L3 gave better results in terms of total SG production (530.3 kg ha−1) than L1 (379.4 kg ha−1) and L2 (232.6 kg ha−1) under both treatments and across all years. The total SG yield was higher at the first harvest (276.1 kg ha−1) than the second harvest (104.7 kg ha−1). MIC treatment had a positive effect on total SG yield, resulting in 464.4 kg ha−1 total SGs vs. 297.1 kg ha−1 for control plants. Total SG yield was higher during the first and second years (respectively 539.5 and 448.7 kg ha−1), while the third year saw a sharp fall to 154.1 kg ha−1.
Table 10 Rebaudioside-A content of stevia leaves as affected by mycorrhizal inoculum (M), chemotype (L) and growing year (Y). Rebaudioside-A content (G 100 g dm−1) Source of variance Mycorrhiza (M)
MIC Control
Chemotype (L)
L1 L2 L3
Year (Y)
2012 2013 2014
Interaction MXL MXY LXY MXLXY
First cut
Second cut
Total
4.0a 3.6a ns 7.5a 0.6c 3.3b *** 3.6a 4.3a 3.5a ns
2.3a 1.9b * 4.2a 0.2c 1.8b *** 2.2a 2.0a 1.9a ns
3.1a 2.7b * 5.9a 0.4c 2.5b *** 2.9a 3.1a 2.7a ns
** ns ns ns
** ns ns ns
** ns ns ns
4. Conclusions The performance of three stevia chemotypes inoculated and noninoculated with AMF was evaluated in terms of total biomass yield in order to achieve improved concentration and production of the best steviol glycosides. The results showed that the production of stevia secondary metabolites is affected by harvest time, chemotype, and mycorrhizal symbiosis. Our studies suggests that mycorrhizal inoculation of selected stevia chemotypes can significantly influence the biomass yield of stevia. In fact, an effective mycorrhizal symbiosis relationship between AMF and micropropagated stevia plantlets was established in selected chemotypes and resulted in higher biomass and steviol glycoside yields. In general, the production of glycoside sweeteners increases proportionally to the increase in dry leaf yield. The effect of AMF differed according to chemotype, and was found to be more pronounced in chemotype L1. When chemotypes are compared, L3 generally gave the best results in terms of productive potential. On the other hand, in terms of specific SGs, L1 must be considered interesting due to its rebaudioside-A content, which is an important aspect for acceptability of the product. The overall results showed that the cultivation of Stevia rebaudiana as a perennial crop is possible and could be economically profitable. In
ns,*,**, *** :Non significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple- range test (P = 0.05).
total average of 2.5 %, while a rebaudioside-A concentration of only 0.4 % was measured in L2. Under MIC treatment, the average rebaudioside-A concentration was 3.1 % (ranging between 4.0 and 2.3 % in the first and second harvests, respectively) higher than the concentration reported under the control treatment (2.7 %). M x L was significant in L1: 6.8 % for MIC plants vs. 4.9 % for the control plants. Table 11 summarises the main results regarding stevioside, rebaudioside-A, and total SG yields as an average for the chemotype,
Table 11 Stevioside, rebaudioside-A and total steviol glycoside production of stevia leaves as affected by mycorrhizal inoculum (M), chemotype (L) and growing year (Y). treatment
Stevioside production (kg ha−1) first cut
MIC
Control
Mycorrhiza (M) MIC 241.6 Control 141.7 ** Chemotype (L) L1 95.9 L2 165.4 L3 293.5 ** Year (Y) 2012 277.7 2013 203.9 2014 79.2 *** Interaction (M x L) L1 202,3 L2 168,5 L3 282,5
Rebaudioside-A production (kg ha−1)
Total SGs yield (kg ha−1)
second cut
total
first cut
second cut
total
first cut
second cut
total
97.6a 59.8b *
339.2 201.5 *
93.0 56.7 ***
29.1 14.8 ***
122.0a 71.5b ***
334.6 198.4
126.6 74.6 *
461.2 273.0 **
a b *
34.9b 61.9b 129.0a **
130.8 227.2 422.4 **
156.1 7.6 81.1 ***
49.0 1.1 24.1 ***
205.1a 8.7c 105.2b ***
252.0 173.0 374.6
83.9 63.0 153.1 **
335.9 236.0 527.7 **
ab b a **
96.3a 107.9b 35.8c **
374.0 311.8 115.0 **
104.1 92.9 27.6
27.8 27.8 9.8 **
131.9a 120.7a 37.4b *
381.8 296.8 106.9 *
124.1 135.8 45.5 ***
505.9 432.6 152.4 *
a a b *
66,0ab 58,9ab 126,4a
268,4 227,4 408,9
290.6 4.0 70.1
94.5 0.2 24.4
385.1a 4.2d 94.5bc
492.9 172.5 352.6
160.5 59.1 150.8
653.4 231.6 503.4
a b a c b a
L1 L2 L3
25,1 162,2 304,2
10,6 64,9 131,4
35,7c 227,0ab 435,6a
49.8 11.3 92.8
14.4 2.1 23.7
64.2bc 13.4c 116.6c
74.9 173.4 397.0
24.9 67.0 155.1
99.8 240.4 552.1
Interaction MxL MxY LxY MxLxY
* ns ns ns
* ns ns ns
* ns ns ns
** ns ns ns
** ns ns ns
** ns ns ns
* ns ns ns
* ns ns ns
* ns ns ns
ns,*,**, *** :Non significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple- range test (P = 0.05). 7
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addition, two harvests per year can be achieved with improved management practices, including an optimal water supply in summer. However, the biomass yield between the two harvest times is expected to vary significantly due to differences in the photoperiod and in weather conditions between the two seasons. Our results indicate that stevia can be adapted to the agro-climatic condition of the study area, providing good biomass and steviol glycoside yields. We found that selection of a suitable chemotype of S. rebaudiana can make it possible to satisfy the needs of the food industry to use stevia as a substitute for sugar in new products.
Jackson, A., Tata, A., Wu, C., Perry, R., Haas, G., West, L., Cooks, R.G., 2009. Direct analysis of Stevia leaves for diterpene glycosides by desorption electrospray ionization mass spectrometry. Analyst 134 (5), 867–874. https://doi.org/10.1039/ B823511B. Kapoor, R., Giri, B., Mukerji, K.G., 2004. Improved growth and essential oil yield and quality in Foeniculum vulgare Mill. on mycorrhizal inoculation supplemented with Pfertilizer. Bioresour. Technol. 93, 307–311. https://doi.org/10.1016/j.biortech.2003. 10.028. Kapoor, R., Chaudhary, V., Bhatnagar, A.K., 2007. Effects of arbuscular mycorrhiza and phosphorus application on artemisinin concentration in Artemisia annua L. Mycorrhiza V. 17 (7), 581–587. https://doi.org/10.1007/s00572-007-0135-4. Khaosaad, T., Vierheilig, H., Nell, M., Zitterl-Eglseer, K., Novak, J., 2006. Arbuscular mycorrhiza alter the concentration of essential oils in oregano (Origanum sp., Lamiaceae). Mycorrhiza 16, 443–446. https://doi.org/10.1007/s00572-006-0062-9. Kolb, N., Herrera, J.L., Ferreyra, D.J., Uliana, R.F., 2001. Analysis of sweet diterpene glycosides from Stevia rebaudiana: improved HPLC method. J. Agric. Food Chem. 49, 4538–4541. https://doi.org/10.1021/jf010475p. Lavini, A., Riccardi, M., Pulvento, C., De Luca, S., Scamosci, M., D’Andria, R., 2008. Yield, quality and water consumption of Stevia rebaudiana Bertoni grown under different irrigation regimes in southern Italy. Ital. J. Agron. 3, 135–143. https://doi.org/10. 4081/ija.2008.135. Luwańska, A., Perz, A., Mańkowska, G., Wielgus, K., 2015. Application of in vitro stevia (Stevia rebaudiana Bertoni) cultures in obtaining steviol glycoside rich material. Herba Pol. 61 (1), 51–61. https://doi.org/10.1515/hepo-2015-0010. Mandal, S., Evelin, H., Giri, B., Singh, V.P., Kapoor, R., 2013. Arbuscular mycorrhiza enhances the production of stevioside and rebaudioside-A in Stevia rebaudiana via nutritional and non-nutritional mechanisms. Appl. Soil Ecol. 72, 187–194. https:// doi.org/10.1016/j.apsoil.2013.07.003. Mandal, S., Upadhyay, S., Singh, V.P., Kapoor, R., 2015. Enhanced production of steviol glycosides in mycorrhizal plants: a concerted effect of arbuscular mycorrhizal symbiosis on transcription of biosynthetic genes. Plant Physiol. Biochem. 89, 100–106. https://doi.org/10.1016/j.plaphy.2015.02.010. Megeji, N.W., Kumar, J.K., Singh, V., Kaul, V.K., Ahuja, P.S., 2005. Introducing Stevia rebaudiana, a natural zero-calorie sweetener. Curr. Sci. 88 iyagawa. Moraes, R.M., De Andrade, Z., Bedir, E., Dayan, F.E., Lata, H., Khan, I., Pereira, A.M.S., 2004. Arbuscular mycorrhiza improves acclimatization and increases lignan content of micropropagated mayapple (Podophyllum peltatum L.). Plant Sci. 166, 23–29. https://doi.org/10.1016/j.plantsci.2003.07.003. Moraes, R.M., Donegal, M.A., Cantrell, C.L., Mello, S.C., McChesney, J.D., 2013. Effect of harvest timing on leaf production and yield of diterpene glycosides in Stevia rebaudiana Bertoni: a specialty perennial crop for Mississippi. Ind. Crops Prod. 51, 385–389. https://doi.org/10.1016/j.indcrop.2013.09.025. Morone Fortunato, I., Ruta, C., Castrignano, A., Saccardo, F., 2005. The effect of mycorrhizal symbiosis on the development of micropropagated artichokes. Sci. Hortic. 106, 472–483. https://doi.org/10.1016/j.scienta.2005.05.006. Muth, N.D., 2015. From Food to Fuel: Carbohydrates. in: Sports Nutrition for Health Professionals, 1 edition. F.A. Davis Company, Philadelphia, USA, pp. 5–16. Pal, P.K., Mahajana, M., Prasad, R., Pathania, V., Singh, B., Ahujac, P.S., 2015. Harvesting regimes to optimize yield and quality in annual and perennial Stevia rebaudiana under sub-temperate conditions. Ind. Crops Prod. 65, 556–564. https://doi.org/10. 1016/j.indcrop.2014.09.060. Phillips, J.M., Hayman, D.S., 1970. Improved procedures for clearing roots and staining parasitic and vesicular arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Br. Mycol. Soc. 55, 158–161. Ren, G., Shi, Y., 2018. Correlation between glycosides in leaves and agronomic traits of Stevia rebaudina Bertoni. Bangladesh J. Bot. 47 (2), 337–342. Röck-Okuyucu, B., Bayraktar, M., Akgun, I.H., Gurel, A., 2016. Plant growth regulator effects on in vitro propagation and stevioside production in Stevia rebaudiana Bertoni. HortScience. 51 (12), 1573–1580. Ruta, C., Perrini, S., Tagarelli, A., Morone-Fortunato, I., 2009. Use of arbuscular mycorrhiza for acclimatization of micropropagated plantlets of melon, oregano artichoke and Spanish broom. Acta Hortic. 812, 473–477. https://10.17660/ActaHortic.2009. 812.68. Singh, A., Verma, P.P.S., 2015. Survival and growth performance of stevia cutting under different growing media. J. Med. Plants Stud. 3 (2), 111–113. Smith, S.E., Read, D., 2008. Mycorrhizal Symbiosis (Third Edition). Academic Press ISBN: 978-0-12-370526-6. Tarraf, W., Ruta, C., De Cillis, F., Tagarelli, A., Tedone, L., De Mastro, G., 2015. Effects of mycorrhiza on growth and essential oil production in selected aromatic plants. Ital. J. Agron. 10 (633), 160–162. https://doi.org/10.4081/ija.2015.633. Tavarini, S., Angelini, L.G., 2013. Stevia rebaudiana Bertoni as a source of bioactive compounds: the effect of harvest time, experimental site and crop age on steviol glycoside content and antioxidant properties. J. Sci. Food Agric. 93 (9), 2121–2129. https://doi.org/10.1002/jsfa.6016. Tavarini, S., Passera, B., Martini, A., Avio, L., Sbrana, C., Giovannetti, M., Angelini, L.G., 2018. Plant growth, steviol glycosides and nutrient uptake as affected by arbuscular mycorrhizal fungi and phosphorous fertilization in Stevia rebaudiana Bert. Ind. Crops Prod. 111, 899–907. Thiyagarajan, M., Venkatachalam, P., 2015. Assessment of genetic and biochemical diversity of Stevia rebaudiana Bertoni by DNA fingerprinting and HPLC analysis. Ann. Phytomed. 4 (1), 79–85. Toussaint, J.P., Smith, F.A., Smith, S.E., 2007. Arbuscular mycorrhizal fungi can induce the production of phytochemicals in sweet basil irrespective of phosphorus nutrition. Mycorrhiza. 17, 291–297. https://doi.org/10.1007/s00572-006-0104-3. Trouvelot, A., Kouch, J., Gianinazzi-Pearson, V., 1986. Mesure du taux de mycorhization VA d’un syste`me radiculaire: recherche of method d’estimation ayant une
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Andolfi, L., Macchia, M., Ceccarini, L., 2006. Agronomic productive characteristics of two genotypes of Stevia rebaudiana in central Italy. Ital. J. Agron. 2, 257–262. https://doi. org/10.4081/ija.2006.257. Anton, S.D., Martin, C.K., Han, H., Coulon, S., Cefalu, W.T., Geiselman, P., Williamson, D.A., 2010. Effects of Stevia, aspartame, and sucrose on food intake, satiety and postprandial glucose and insulin levels. Appetite 55, 37–43. https://doi.org/10. 1016/j.appet.2010.03.009]. Atul-Nayyar, A., Hamel, C., Hanson, K., Germida, J., 2009. The arbuscular mycorrhizal symbiosis links N mineralization to plant demand. Mycorrhiza 19 (4), 239–246. https://doi.org/10.1007/s00572-008-0215-0. Baum, C., El-Tohamy, W., Grudac, N., 2015. Increasing the productivity and product quality of vegetable crops using arbuscular mycorrhizal fungi: a review. Sci. Hortic. 187, 131–141. https://10.1016/j.scienta.2015.03.002. Bernal, J., Mendiola, J., Ibáñez, E., Cifuentes, A., 2011. Advanced analysis of nutraceuticals. Pharm. Biomed. Anal. 55, 758–774. https://doi.org/10.1016/j.jpba. 2010.11.033. Brandle, J.E., Starrat, A.N., Gijzen, M., 1998. Stevia rebaudiana: its agricultural, biological, and chemical properties. Can. J. Plant Sci. 78, 527–536. https://doi.org/10.4141/ P97-114. Brandle, J.E., Rosa, N., 1992. Heritability for yield, leaf:stem ratio and stevioside content estimated from a landrace cultivar of Stevia rebaudiana. Can. J. Plant Sci. 72, 1263–1266. https://doi.org/10.4141/cjps92-159. Cacciola, F., Delmontea, P., Jaworska, K., Dugo, P., Mondello, L., Rader, J., 2011. Employing ultrahigh pressure liquid chromatography as the second dimension in a comprehensive two dimensional system for analysis of Stevia rebaudiana extracts. J. Chromatogr. A 1218 (15), 2012–2018. https://doi.org/10.1016/j.chroma.2010.08. 081. Ceunen, S., Geuns, J.M.C., 2013. Influence of photoperiodism on the spatio-temporal accumulation of steviol glycosides in Stevia rebaudiana (Bertoni). Plant Sci. 198, 72–82. https://doi.org/10.1016/j.plantsci.2012.10.003. Chatsudthipong, V., Muanprasat, C., 2009. Stevioside and related compounds: therapeutic benefits beyond sweetness. Pharmacol. Ther. 121, 41–54. https://doi.org/10. 1016/j.pharmthera.2008.09.007. Cruz, C., Green, J.J., Watson, C.A., Wilson, F., Martins-Loução, M.A., 2004. Functional aspects of root architecture and mycorrhizal inoculation with respect to nutrient uptake capacity. Mycorrhiza 14, 177–184. https://doi.org/10.1007/s00572-0030254-5. Estrada-Luna, A.A., Davies Jr., F., Egilla, J.N., 2000. Mycorrhizal fungi enhancement of growth and gas exchange of micropropagated guava plantlets (Psidium guajava L.) during ex vitro acclimatization and plant establishment. Mycorrhiza 10 (1), 1–8. Fronza, D., Folegatti, M.V., 2003. Water consumption of the Estevia (Stevia rebaudiana (Bert.) Bertoni) crop estimated through microlysimeter. Sci. Agric. 60 (3), 595–599. https://doi.org/10.1590/S0103-90162003000300028. Gardana, C., Scaglianti, M., Simonetti, P., 2010. Evaluation of steviol and its glycosides in Stevia rebaudiana leaves and commercial sweetener by ultra-highperformance liquid chromatography–mass spectrometry. Chromatography A. 1217 (9), 1463–1470. https://doi.org/10.1016/j.chroma.2009.12.036. Geuns, J., 2003. Molecules of interest: stevioside. Phytochemistry 64, 913–921. Govindarajulu, M., Pfeffer, P.E., Jin, H.R., Abubaker, J., Douds, D.D., Allen, J.W., Bucking, H., Lammers, P.J., Shachar-Hill, Y., 2005. Nitrogen transfer in the arbuscular mycorrhizal symbiosis. Nature 435, 819–823. https://doi.org/10.1038/ nature03610. Goyal, S., Samsher, Goyal, R., 2010. Stevia (Stevia rebaudiana) a bio-sweetener: a review. Int. J. Food Sci. Nutr. 61, 1–10. https://doi.org/10.3109/09637480903193049. Gupta, E., Purwar, S., Sandaram, S., Gai, G.K., 2013. Nutritional and therapeutic values of Stevia rebaudiana: a review. J. Med. Plants Res. 7, 3343–3353. https://doi.org/10. 5897/JMPR2013.5276. Gupta, E., Purwar, S., Sundaram, S., Tripathi, P., Rai, G., 2016. Stevioside and rebaudioside a – predominant ent-kaurene diterpene glycosides of therapeutic potential: a review. Czech J. Food Sci. 34 (4), 281–299. https://doi.org/10.17221/335/ 2015-CJFS.
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Yadav, A.K., Singh, S., Dhyani, D., Ahuja Can, P.S., 2011. A review on the improvement of stevia Stevia rebaudiana (Bertoni). Can. J. Plant Sci. 91 (1), 1–27. Yadav, S.K., Guleria, P., 2012. Steviol glycosides from stevia: biosynthesis pathway review and their application in foods and medicine. Critical Rev. Food Sci. Nutr. Nutr. 52 (11), 988–998. https://doi.org/10.1080/10408398.2010.519447. Yadav, K., Aggarwal, A., Singh, N., 2013. Arbuscular mycorrhizal fungi induced acclimatization and growth enhancement of Glycyrrhiza glabra L.: a potential medicinal plant. Agric. Res. 2, 43–47. https://doi.org/10.1007/s40003-012-0047-1. Zubek, S., Mielcarek, S., Turnau, K., 2012. Hypericin and pseudohypericin concentrations of a valuable medicinal plant Hypericum perforatum L. Are enhanced by arbuscular mycorrhizal fungi. Mycorrhiza. 22, 149–156.
signification fonctionelle. In: Dijon, Ier Seminaire (Ed.), Les Mycorhizes: Phisiologie and Genetique. INRA, Paris, pp. 217–221. Wilhelm, W.W., Ruhe, K., Schlemmer, M.R., 2000. Comparison of three leaf area index meters in a corn canopy. Crop Sci. 40, 1179–1183. https://doi.org/10.2135/ cropsci2000.4041179x. Williams, L.D., Burdock, G.A., 2009. Genotoxicity studies on a high-purity rebaudioside A preparation. Food Chem. Toxicol. 47, 1831–1836. Vives, K., Andújar, I., Lorenzo, J.C., Concepción, O., Hernández, M., Escalona, M., 2017. Comparison of different in vitro micropropagation methods of Stevia rebaudiana B. Including temporary immersion bioreactor (BIT®). Plant Cell Tissue Organ Cult. 131 (1), 195–199.
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Effects of Septoglomus viscosum inoculation on biomass yield and steviol glycoside concentration of some Stevia rebaudiana chemotypes Luigi Tedone*, Claudia Ruta, Francesca De Cillis, Giuseppe De Mastro University of Bari - Department of Agricultural and Environmental Science, Via Amendola, 165/A - 70126, Italy
A R T I C LE I N FO
A B S T R A C T
Keywords: Stevia rebaudiana Chemotype Open field cultivation Mycorrhiza Plant regrowth Steviol glycosides
There is growing interest in the medicinal properties of Stevia rebaudiana Bertoni, due to its ability to produce zero-calorie sweeteners, steviol glycosides (SGs). Inoculation of the roots with arbuscular mycorrhizal fungi (AMF) is a method used to improve the production of secondary metabolites by many plant species, but its effects on S. rebaudiana have not been extensively studied, especially in open field conditions in Italy. Therefore, a field experiment was conducted to study the effects of the fungus Septoglomus viscosum on the leaves and secondary metabolites of three micropropagated stevia chemotypes (L1, L2 and L3) over three years. Our results indicate that the establishment of symbiotic relations between AMF and stevia plants of selected chemotypes improved leaf yield, because the mycorrhizal (MIC) treated plants had a better tendency to regrowth, especially L1 and L3, which had survival rates of 80 % and 90 %, respectively. MIC treatment also had positive effects in terms of SG production, which was not significant in terms of concentration, but was significant in terms of production.
1. Introduction Stevia (Stevia rebaudiana Bertoni) is a perennial shrub of sub-tropical origin. The plant is native to northeastern Paraguay, but is now cultivated in Mexico, Central America, Japan, China, Spain, Belgium, the UK, the USA (Williams and Burdock, 2009) and Italy (Tavarini and Angelini, 2013). It belongs to the Asteraceae family and has an extensive root system; its brittle stems produce small elliptical leaves (Yadav et al., 2011). Since ancient times, stevia has been used for various medicinal purposes (Goyal and Goyal, 2010). In Europe, stevia leaves are used in infusions, but stevia extract is now more important, since it is a promising renewable calorie-free sweetener that can be used as a sugar substitute, or as an alternative to artificial sweeteners (Anton et al., 2010). Stevia is the common name for the stevioside extracted from the leaves of stevia; it consists of ent-kaurene diterpene glycosides, commonly known as steviol glycosides, which are natural sweeteners that can be isolated from its leaves and are 250−300 times sweeter than saccharose. Almost thirty diterpenes have been identified in stevia leaves (Gupta et al., 2016), including stevioside, steviolbioside, rebaudioside A, B, C, D, E, F and dulcoside (Geuns, 2003). Stevioside is the most abundant glycoside in stevia leaves, (4–13 % w/w), followed by rebaudioside-A (2–4 % w/w), rebaudioside-C (1–2 % w/w), and dulcoside (0.4-0.7 % w/w) (Jackson et al., 2009; Gardana et al., 2010; Cacciola et al., 2011). Steviol-glycosides can be used not only as
⁎
sweeteners, but also for medicinal purposes (Gupta et al., 2013). Stevioside and the related compounds are reported to have anti-hyperglycaemic, anti-hypertensive, anti-inflammatory, anti-tumor, antidiarrheal, diuretic, and immunomodulatory effects (Chatsudthipong and Muanprasat, 2009; Bernal et al., 2011). The growing demand for natural alternatives to artificial sweeteners has generated considerable commercial interest in stevia. Stevia sales will be worth approximately $700 million in the next few years, according to Rabobank (Muth, 2015). Today, stevia is cultivated on approximately 32,000 ha worldwide, with the largest production area in China (approximately 75 % of the world total) (Singh and Verma, 2015). There is a great need for cultivation techniques (Mandal et al., 2013) to use in intensive farming in order to increase production of S. rebaudiana low-calorie glycosides, and to improve its biomass yield and glycoside composition. The quality of stevia leaves can vary widely, due to many factors, including chemotype, environmental and soil conditions, irrigation methods, sunlight, farming practices, sanitation, processing, and storage (Yadav et al., 2011; Ceunen and Geuns, 2013). Many factors limit the large-scale cultivation of stevia plants, including its very low germination percentage. In addition, seed propagation produces a nonhomogeneous population, with a consequent variability in glycoside composition (Miyagawa et al., 1986), while vegetative propagation produces only a low number of plants. In order to meet these challenges, and to achieve rapid mass
Corresponding author. E-mail address: [email protected] (L. Tedone).
https://doi.org/10.1016/j.scienta.2019.109026 Received 4 August 2017; Received in revised form 30 October 2019; Accepted 10 November 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Luigi Tedone, et al., Scientia Horticulturae, https://doi.org/10.1016/j.scienta.2019.109026
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line were inoculated with a crude AMF inoculum of Septoglomus viscosum (Basionym: Glomus viscosum T.H. Nicolson (1995)). The AMF inoculum consisted of sandy soil containing spores, external mycelium, and infected strawberry root fragments from strawberry pot cultures. Approximately 10 g (approximately 100–120 spores) of each AMF inoculum was placed immediately below the roots. Non-mycorrhizal plants were obtained by adding approximately 10 g of the same sterile sandy soil, without AMF inoculum, and were used as controls. Acclimatization took place in greenhouse conditions at 18−25 °C with mist, gradually reducing the humidity level from 85 to 90% to 50–60 % over 20 days. Subsequently, the plants were grown at 20 °C and 40 % RH until they were transplanted in the field. No fertiliser was added to the pots during greenhouse growth. Following plant acclimatization in the greenhouse, bio-morphological characteristics were identified for each chemotype on 10 representative samples of S. rebaudiana plants, and 130 healthy plants were then transplanted into an open field for each treatment and line. Bio-morphological determination was performed by separating the leaves from the stems, and the foliar area was measured using a leaf area meter (Model Li-Cor 3000). Shoots and roots were then weighed to determine their fresh weight, oven-dried (48 h, 60 °C), and then reweighed to determine their dry weight. The percentage of mycorrhizal colonization (Trouvelot et al., 1986) was measured to verify the establishment of symbiosis on the set of 10 randomly chosen representative root portions per treatment. The roots were stained following the Phillips and Hayman (1970) method. Ten 1-cm root pieces per plant were selected at random from the stained root fragments and placed on a microscope slide. Ten microscope slides were prepared for each treatment. The root fragments were mounted in a drop of glycerol and observed using an optical microscope (Leica DMLB100).
propagation of stevia, in vitro micropropagation from shoot tips and leaves is suggested as an extremely efficient method to obtain more uniform plants on an industrial scale than is possible with seed reproduction (Yadav et al., 2011; Luwańska et al., 2015; Röck-Okuyucu et al., 2016); the use of bioreactors is suggested in order to increase stevia plant production (Vives et al., 2017). Stevia can grow well in a wide range of soils if there are optimal soil moisture conditions and an adequate drainage. To ensure sustainable production of stevia, arbuscular mycorrhizal fungi (AMF) involved in symbioses with the plant roots provide the host plants with increased biomass, improving the growth rate of aromatic plants (Tarraf et al., 2015), together with their pathogen resistance, mineral nutrient uptake and rate of photosynthesis when compared with non-mycorrhizal symbioses. Cruz et al. (2004) reported that plants inoculated with AMF develop a more efficient root system, with increased survival ex vitro (Estrada-Luna et al., 2000; Moraes et al., 2004; Ruta et al., 2009) and better physiological activity due to their improved nutrition status. AMF have been shown to enhance the production of secondary metabolites in oregano, fennel, Artemisia annua, sweet basil and St John's wort (Khaosaad et al., 2006; Kapoor et al., 2004, 2007; Toussaint et al., 2007; Zubek et al., 2012). Their effects on S. rebaudiana have recently been studied by Mandal et al. (2013), who inoculated stevia plants with Rhizophagus fasciculatus and thus obtained increased biomass production, due to the greater number of shoots per plant, and higher concentrations of steviol glycosides. Tavarini et al. (2018) have also reported the positive effects of R. irregulare on stevia production and quality. To improve glycoside content and composition, several plantbreeding procedures have been used to increase leaf yield and also rebaudioside-A concentration in leaves (Brandle et al., 1998). Based on the studies reported in the literature, it appears that there is sufficient genetic variability (Thiyagarajan and Venkatachalam, 2015) to allow significant genetic improvements to the content of steviol glycosides in leaves. The present study assessed the effects of mycorrhizal inoculation on selected micropropagated stevia chemotypes. The aim was to define the best combination to improve biomass production and glycoside yield in order to facilitate industrial processing.
2.3. Experimental site The field experiment was carried out over three years (2011–2013) at the “Enrico Pantanelli” experimental farm of the University Bari “A. Moro”, at Policoro (southern Italy; 40°10′20″ N, 16°39′04″ E). The site is 15 m above sea level, and the climate is typically Mediterranean, according to the De Martonne classification; it has an average annual rainfall of 554 mm, distributed mainly during autumn and winter, a maximum mean temperature between 12.8 °C and 31 °C, and a minimum temperature between 3.95 °C and 11.26 °C. The soil is loamy and over 1.2 m deep, with the following physical characteristics: sand 398 g kg–1, silt 374 g kg–1, clay 228 g kg–1. Its chemical characteristics are as follows: pH 7.7; total N (Kjeldahl method) 1.7‰, available P2O5 (Olsen method) 27.6 mg kg–1, exchangeable K2O (ammonium acetate method) 227 mg kg–1, organic matter content (Walkley-Black method) 2.3 %, total carbonate 15.0 g kg–1, active carbonate 5.0 g kg−1, saturated paste extract electrical conductivity (ECe) 0.95 dS m–1, ESP 1.9 %; bulk density 1.25 kg dm–3; soil moisture at field capacity (measured in situ) of 0.32 m3 m−3 and at wilting point (-1.5 MPa) of 0.15 m3 m−3 of soil dry weight.
2. Materials and methods 2.1. Stevia chemotype selection A previous experiment selected three different chemotypes on the basis of their steviol glycoside content and composition, and their agronomic performance. Chemotype L1 has a high rebaudioside-A content (over 5 g 100 g dm−1) and medium leaf production, chemotype L2 has a high stevioside content (over 10 g 100 g dm−1) and medium leaf production, and chemotype L3 has a medium level of stevioside and extremely high leaf production. By referring to a previous experiment on the same site, we considered average leaf production per year to be 3.5 t ha−1, considering cultivation over three years.
2.4. Experimental design and field assessment 2.2. Plant material and mycorrhizal inoculation After plant acclimatization in the greenhouse, 130 micropropagated healthy plants for each treatment and line were transplanted into an open field on 6th May 2011, at a density of 5 plants m−2, with rows 0.6 m apart (Lavini, et al., 2008), and 0.35 cm between plants on the rows. Stevia microplants were arranged using a split plot design with four repetitions, with the two treatments as the first variable: mycorrhizal inoculated (MIC) and non-inoculated (control) plants, and chemotypes (L1, L2 and L3) as the second variable. The plot had an area of 20 m2. Before planting, the soil was prepared by deep ploughing, which was followed by disc-ploughing and harrowing. All plants received an application of 80, 60 and 140 kg ha−1 of N, P and K using urea, single
The three chemotypes were multiplied by micropropagation. Multiplication was induced on a basal medium (BM) (see, Morone Fortunato et al., 2005) enriched with 6-benzylaminopurine (BAP 0.5 mg l−1) and IAA (3.0 mg l−1), following the protocol described by Ruta et al. (2009). For rooting, the BM was enriched with a thin layer of the same liquid nutrient solution containing 4.5 mg l-1 Indol Acetic Acid (IAA) (Ruta et al., 2009). The rooted microplants (300 for each line) were transplanted into individual plastic pots with a volume of 0.2 dm3 containing a mixture of sterile peat (46 % organic carbon, 1.2 % organic nitrogen, 80 % organic matter, pH 6) and perlite (2:1, v/v ratio). At the time of transplantation, half of the 150 microplants of each 2
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2.08 g, fresh roots 2.22 g, dry roots 0.46 g) (Table 2). L1 and L3 appeared to show a greater affinity with S. viscosum than with L2, thus confirming that the potential of AMF to promote growth of the host plant also depends on the host genotype (Baum and El-Tohamy, 2015). Establishment of symbiosis was confirmed by an infection percentage of over 50 %, measured two months after inoculation. There is wide support in the literature for the use of AMF to improve growth of micropropagated plantlets (Estrada-Luna et al., 2000; Ruta et al., 2009; Yadav et al., 2013), mainly related to improved nutrient and water uptake.
Table 1 Harvesting schedule for stevia during the study period. Treatment
2012 6-Aug
Mycorrhiza Control
st
1 cut 1st cut
2013 25-Sep nd
2 cut 2nd cut
28-Jul st
1 cut 1st cut
2014 26-Sep nd
2 cut 2nd cut
7-Aug st
1 cut 1st cut
28-Sep 2nd cut 2nd cut
superphosphate, and potassium sulphate, respectively, as fertilisers. In the second and third years, the fertilisers were distributed partly at the start of the cycle (phosphorus and 1/3 of nitrogen as ammonium superphospate) and partly during the cycle (2/3 of nitrogen after the first harvest as ammonium nitrate 34 %). Each year, a total of 80 kg nitrogen and 20 kg P2O5 was applied during vegetative growth. All fertilisers were applied to the soil. The first irrigation treatment was performed immediately after transplanting and repeated three days later. Water was subsequently supplied on a regular basis using a drip-irrigation system to maintain optimal water conditions, especially in summer, throughout the whole study period. During all three years, the first harvest was performed in summer by cutting the aboveground biomass at a height of about 5−7 cm. Due to the variability in photoperiod sensitivity, which depends on the daylength sensitivity of this species, the cultivation of stevia as a perennial crop in this study allowed a two-harvest per year management system (Table 1). Before each harvest, the leaf area index (LAI) was measured at field level using AccuPAR (LAI-2000 Ceptometer (Wilhelm et al., 2000)), while the total biomass weight, number of stems, and leaf weight were measured at each harvesting time for each treatment.
3.2. Plant density The ability of stevia plants to survive until the end of the experiment was compared to the time of planting by measuring the total number of surviving plants m−2 at each harvest time, per year (Fig. 1). This enabled us to evaluate differences between stevia chemotypes and mycorrhizal-inoculated plants in the open field. Over the study period, stevia plant density decreased and differed significantly among chemotypes and treatments (Fig. 1). The mean survival rate for MIC plants was 80 % in 2012 (458 and 508 days after planting (DAT), compared with 53 % for control plants. In 2013 (824 and 874 DAT), the survival rate was 73 % for MIC plants and 40 % for control plants, and in 2014 (1189 and 1241 DAT), the survival rate was 63 % for MIC plants and 34 % for control plants. Plant survival differed among the chemotypes; L1 and L3 survival rates were higher under MIC treatments than for control plants. 3.3. Yield and yield components Tables 3 and 5 show dry leaf (t ha−1) and stem yields (t ha−1) at different harvest times during the three years. Variations were found among chemotypes, mycorrhizal treatments and years. The results showed a decrease in leaf and stem production for the three chemotypes in the third year. The maximum stem dry yield occurred in the second year (Table 5). Biomass production was always higher at the first harvest, because temperature conditions were better than at the second harvest. Biomass yield fell in the third year, with a 67 % and 16 % reduction in leaf and stem yield, respectively, compared to the first year.
2.5. Extraction and analysis of steviol glycosides (SGs) Sweet diterpene glycosides were extracted from the dried stevia leaves following Kolb et al. (2001). HPLC analysis was performed using an Agilent 1100 quaternary pump and a UV detector 1260. Chromatographic separation was performed on a column of polyamide Zorbax Carbohydrate 15 cm x 4.6 cm with particles of 5 μm, coupled by a "guard column" with cartridges for Zorbax NH2. The mobile phase consisted of acetonitrile/water HPLC grade at a ratio of 80:15 v/v. The elution programme used a constant flow of 2.5 ml min−1 and isocratic elution for 15 min at room temperature. Analysis was performed by monitoring the signal in absorbance at 210 nm. The peak identification and calibration were determined using pure stevioside and rebaudioside-A standards from Sigma-Aldrich.
3.3.1. Dry leaf yield Dry leaf yield was collected twice a year in a two-harvest management system. Pal et al. (2015) indicate that a single harvest during the growing season may lead to a lower leaf yield, and also to lower glycoside production (Moraes et al., 2013) than a multi-cut management system. Results from central Italy (Andolfi et al., 2006) further highlighted the importance of two cuts per year in achieving higher yields. Following the two-harvest management system per year, the annual dry leaf yield refers to the sum of the two harvests carried out each year. For a detailed analysis of dry leaf yield, dry leaf yield is reported in terms of grams of dry leaves per plant (Table 3) and tons per hectare under all the treatments (Table 4). The overall results showed a sharp decrease in dry leaf production (grams per plant and tons per hectare) in the last year. The average annual quantities of dry leaf production per plant were stable during 2012 (93.6 g plant−1) and 2013 (88.8 g plant−1), but a sharp decrease occurred in 2014 (35.7 g plant−1). Table 3 shows that the first cut gave a higher amount of dry leaves per plant than the second (46.8 vs. 44.4 g plant−1). This appears to be associated with the shorter vegetation period for the second cut than for the first cut (Andolfi et al., 2006). With regard to chemotypes, L3 yielded the highest dry leaf production in both harvest periods (85.8 g), followed by L1 (81.0 g), while L2 gave the lowest yield (51.4 g). AMF inoculated plants gave a higher yield (79.7 vs 65.7 g) than non-inoculated plants. The most significant effect of AMF inoculation was observed with L1
2.6. Statistical analysis The data obtained were subjected to variance analysis (ANOVA). In addition, treatment means were compared using the LSD test at a 0.05 probability level. CoStat software was used for statistical analysis. 3. Results and discussion 3.1. Plant material and mycorrhizal inoculation Table 2 shows the increases in the bio-morphological parameters of MIC plants of the three different chemotypes compared with the control plants. Mycorrhizal inoculation successfully influenced the epigeal and hypogeal growth of all three chemotypes during acclimatization in the greenhouse (Ruta et al., 2009). Mycorrhiza treatment had a significantly positive effect on the averages of all parameters measured for all lines (plant height 20.5 cm, leaf number 2.6, leaf area 35.5 cm2, fresh shoots 39.4 g, fresh shoots 3
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Table 2 Bio-morphological characteristics of three chemotypes of Stevia rebaudiana (Bert.) Bertoni grown in a greenhouse on a substrate enriched with mycorrhizal inoculum (S. viscosum fungus) (MIC) in comparison to the control plants two months after transplanting. Treatments
Mycorrhiza (M) MIC Control Chemotype (L) L1 L2 L3 Interaction MxL
Plant height
Leaves
Leaf area
Shoots
Roots
N plant−1
cm2 plant−1
Fresh (g plant−1)
Dry
cm
23.2 a 17.7 b ***
39.3 a 31.5 a *
42.8 a 36.0 a *
2.3 a 1.8 b **
0.50 a 0.42 b *
2.48 a 1.96 b **
0.36 0.32 **
56.3 a 0b ***
27.2 a 18.9 b 15.3 b **
27.0 a 42.4 a 37.0 b ns
45.9 a 40.2 ab 32.0 a ns
2.6 a 2.3 a 1.4 b *
0.6 a 0.45 ab 0.35 b ns
3.10 a 2.55 a 1.00 b *
0.48 a 0.33 a 0.20 b *
69.4 a 48.5 b 50.9 b *
ns
ns
ns
ns
ns
ns
ns
ns
Fresh
Root mycorrhizal colonization Dry (%)
ns,*,**, *** :Non significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple- range test (P = 0.05). Table 4 Dry leaf yield (t ha-1) of stevia crop as affected by mycorrhizal inoculum (M), chemotype (L) and growing year (Y). Dry leaf yield (t ha−1) Source of variance
Fig. 1. Plant density over the years. Data are means ± SE per treatment (n = 4).
Mycorrhiza (M)
MIC Control
Chemotype (L)
L1 L2 L3
Year (Y)
2012 2013 2014
Interaction MxL MxY LxY MxLxY
Second cut
Total
47.0a 31.3b ** 41.6a 26.4b 49.5a * 58.3a 43.5a 15.7b ***
32.6a 34.4a ns 39.3a 25.0b 36.3a * 35.3a 45.3a 20.0b ***
79.7a 65.7b * 80.9a 51.4b 85.8a * 93.6a 88.8a 35.7b ***
* ns ns ns
* ns ns ns
* ns ns ns
Chemotype (L)
L1 L2 L3
Years (Y)
2012 2013 2014
Second cut
Total
2.4a 1.6b * 2.1a 1.3b 2.5a * 3.0a 2.3a 0.8b ***
1.3a 0.8b * 1.2a 0.6b 1.4a ** 1.3a 1.4a 0.5b ***
3.7a 2.4b * 3.2a 1.9b 3.8a * 4.2a 3.7a 1.4b ***
* ns ns ns
* ns ns ns
** ns ns ns
ns,*,**, *** :Non-significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple- range test (P = 0.05).
Dry leaf yield (g plant−1) First cut
MIC Control
Interaction MxL MxY LxY MxLxY
Table 3 Dry leaf yield (g plant−1) of stevia crop as affected by mycorrhizal inoculum (M), chemotype (L) and growing year (Y).
Source of variance
Mycorrhiza (M)
First cut
The average dry leaf yield of 2.96 t ha−1 was similar to that reported by Brandle and Rosa (1992), and close to the average dry leaf yield of 2.67 t ha−1 reported by Megeji et al. (2005) in India. Higher yields of 3.6 and 3.9 t ha−1 were reported in central and southern Italy by Andolfi et al. (2006) and Lavini et al. (2008), respectively. However, in the USA, Moraes et al. (2013) reported a lower leaf yield of 2.0 t ha−1. Our analysis of annual dry leaf production (sum of the first and second harvests) indicates the first year as the most productive (4.2 t ha1 ), the second year as slightly less productive 3.6 t ha-1, and the third year as significantly (P ≤ 0.05) less productive (1.3 t ha-1). The effect of mycorrhizal inoculation was evident; MIC treatments produced a higher dry leaf yield than control treatments for all chemotypes and across the years. The effect of MIC treatment was more pronounced in the second and third years, when production decreased and the rate of decrease was lower under MIC treatment (4.6 t ha−1 vs. 3.8 t ha-1, 4.6 t ha−1 vs. 2.5 t ha-1, and 1.8 t ha−1 vs. 0.8 t ha-1, reported in 2012, 2013 and 2013, respectively). This agreed with other studies (Mandal et al., 2013, 2015), which found a significant increase in S. rebaudiana biomass after AMF inoculation. Mandal et al. (2013) found that increases in biomass were due to improved P-nutrition under MIC treatments. Smith and Read (2008) further highlighted the importance
ns,*,**, *** :Non significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple- range test (P = 0.05).
dry leaf production, which was 110.3 g plant-1 under MIC treatment, compared with 51.5 g plant−1 for control plants. These parameters, together with higher plant survival rates, gave better results for MIC treatments. 4
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Table 5 Stem number (n. plant −1) of stevia crop as affected by mycorrhizal inoculum (M), chemotype (L) and growing year (Y).
Table 6 Stem yield (t ha−1) of stevia crop as affected by mycorrhizal inoculum (M), chemotype (L) and growing year (Y).
Stem number (n plant−1) Source of variance Mycorrhiza (M)
MIC Control
Chemotype (L)
L1 L2 L3
Year (Y)
2012 2013 2014
Interaction MxL MxY LxY MxLxY
Stem yield (t ha−1)
First cut
Second cut
Total
Source of variance
18.1a 18.4a ns 16.3ab 12.5b 25.9a * 11.3b 26.4a 17.0ab **
33.7a 39.7b ns 32.2ab 24.7b 51.9a * 40.6a 39.0a 30.3a ns
51.8a 53.6b * 43.1b 37.3ab 77.9a ns 51.9a 65.4a 47.4a ns
Mycorrhiza (M)
MIC Control
Chemotype (L)
L1 L2 L3
Year (Y)
2012 2013 2014
ns ns ns ns
ns ns ns ns
ns ns ns ns
Interaction MXL MXY LXY MXLXY
First cut
Second cut
Total
1.92a 1.16b * 1.72b 1.09a 1.80b * 1.11b 2.18a 1.32ab *
1.39a 0.96a * 1.28a 0.55b 1.70a * 1.48a 1.19a 0.86a ns
3.31a 2.12b * 3.00a 1.64b 3.51a * 2.59ab 3.37a 2.18b *
ns ns ns ns
ns ns ns ns
ns ns ns ns
ns,*,**, *** :Non significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple- range test (P = 0.05).
ns,*,**, *** :Non significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple- range test (P = 0.05).
of a symbiotic relationship in providing host plants with a higher biomass yield than non-mycorrhizal symbioses through an increase in plant uptake of soil nutrients (Govindarajulu et al., 2005; Atul-Nayyar et al., 2009). The effect of the chemotype on dry leaf yield was significant and showed some effects of interaction with mycorrhizal inoculation (Table 3). This was clear when leaf yield varied among chemotypes in control treatments in the order L3 > L2 > L1, while the order was the opposite among MIC treatments (L1 > L2 > L3), indicating that mycorrhizal inoculation can change the productive response of some stevia chemotypes. Under MIC treatment, L1 gave a higher annual leaf production (5.3 t ha−1) than L2 and L3 (2.0 and 3.6 t ha−1), while L3 was the highest producer among the control treatments (3.9 t ha−1), followed by L2 (2.1 t ha−1) and L1 (1.2 t ha−1). Considering the interaction effect of MxL, MIC treatment had positive effects on L1 and L3. The highest significant difference was observed in L1 (4.3 for MIC treatment vs 1.2 t ha-1 for the control).
produced a significantly higher stem yield, while L1 gave the lowest stem yield.
3.3.2. Stem yield Stem production was measured each year in terms of number per plant and yield. Table 5 shows the effect of mycorrhiza and chemotype on the stem number per plant per year. The number of stems per plant was influenced by the chemotype and harvest time. As a general effect, a higher stem production per plant was observed at the second harvest (36.6 vs. 18.2), with average values ranging between 24.7 and 51.9, i.e. almost twice those of the first harvest (average between 11.3 and 26.4). (Table 5). L3 produced a greater number of stems per plant (77.9) than the other chemotypes (43.1 and 37.3 in L1 and L2, respectively). Average stem dry yields are reported in Table 6, showing that the average value was 2.71 t ha−1, as reported by Megeji et al. (2005) in India (2.72 t ha−1). Stevia dry stem yield varied significantly (P ≤ 0.05) among the years, among chemotypes and among treatments. Annual stem dry yield was highest in 2013 (3.37 t ha−1), unlike dry leaf yield. The higher and more evenly distributed rainfall could explain why production in 2013 was higher than in 2012 and 2014. In MIC plants, the effect of harvest time on dry stem yield showed a higher yield at the first cut in 2013 and 2014, but higher at the second cut in 2012. The same trend was found in control treatments (Table 6). MIC symbiosis produced a significantly higher stem yield than control treatments (3.31 vs. 2.12 t ha−1). With regard to the chemotypes, L3
Table 7 Leaf area index (LAI) of stevia crop as affected by mycorrhizal inoculum (M), chemotype (L) and growing year (Y).
3.3.3. Leaf Area Index (LAI) The leaf area index (LAI) is an important parameter, correlated directly to crop production. The results of this study showed an average LAI of 2.71, which varied between a maximum of 6.47 and a minimum of 0.6 among the years and treatments (Table 7). Our average value was significantly lower than the LAI value of 4 reported by Lavini et al. (2008) and the 4.83 reported by Fronza and Folegatti (2003). The average LAI was 3.53 during the first harvest and 1.89 during the second. (Table 7). The effect of the year was significant, with an average of 3.67 in the first year, 3.25 in the second year and 1.22 in the third year. This data confirmed the result of Pal et al. (2015), who reported that LAI was significantly (P ≤ 0.05) affected by the crop cycle and harvesting regime. The overall results showed a positive effect of mycorrhizal symbiosis
Source of variance
LAI
Mycorrhiza (M)
MIC Control
Chemotype (L)
L1 L2 L3
Year (Y)
2012 2013 2014
Interaction MxL MxY LxY MxLxY
First cut
Second cut
Total
4.20a 2.86b ** 3.76ab 2.39b 4.44a * 5.15a 3.97a 1.47b **
2.26a 1.52b * 2.13a 1.12b 2.43a * 2.18a 2.53a 0.96b **
3.23a 2.19b ** 2.94a 1.75b 3.43a ** 3.67a 3.25a 1.22b **
* ns ns ns
* ns ns ns
* ns ns ns
ns,*,**, *** :Non significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple- range test (P = 0.05). 5
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Table 8 Leaves/stem dry weight ratio of stevia crop as affected by mycorrhizal inoculum (M), chemotype (L) and growing year (Y).
Table 9 Stevioside content of stevia leaves as affected by mycorrhizal inoculum (M), chemotype (L) and growing year (Y).
Leaves/ stem ratio Source of variance Mycorrhiza (M)
Chemotype (L)
Year (Y)
Interaction MxL MxY LxY MxLxY
MIC Control L1 L2 L3 2012 2013 2014
Stevioside content (g 100 g dm−1)
Source of variance
First cut
Second cut
Total
1.4a 1.5a ns 1.4a 1.5a 1.6a ns 2.9 0.9 0.6 **
0.7a 0.7a ns 0.8a 0.8a 0.6a ns 0.5 1.2 0.6 **
1.1a 1.1a ns 1.1a 1.1a 1.1a ns 1.7a 1.0a 0.6a **
ns ns ns ns
ns ns ns ns
ns ns ns ns
Mycorrhiza (M)
MIC Control
Chemotype (L)
L1 L2 L3
Year (Y)
2012 2013 2014
Interaction MXL MXY LXY MXLXY
ns,*,**, *** :Non significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple- range test (P = 0.05).
First cut
Second cut
Total
10.3a 9.0b * 4.6b 12.5a 11.9a *** 9.5 9.4 10.1 ns
7.5a 7.5a ns 3.0b 10.1a 9.5a *** 7.7 7.8 7.0 ns
8.9a 8.3a ns 3.8b 11.3a 10.7a *** 8.6a 8.6a 8.5a ns
* ns ns ns
* ns ns ns
* ns ns ns
ns,*,**, *** :Non significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple- range test (P = 0.05).
on LAI: 3.23 for MIC treatments compared to 2.19 for the control treatments. On average, these values varied among the different chemotypes, with averages of 2.94, 1.75 and 3.43 for chemotypes L1, L2 and L3, respectively. With regard to M x L interaction, L1 presented the most significant differences between MIC and control treatments (4.77 vs 1.12); the maximum LAI (6.08) was measured in L1 MIC plants at the first harvest, while the minimum LAI (0.79) was recorded in L1 control plants at the second harvest.
%) compared with an average rebaudioside-A concentration of 2.9 % (ranging between 0.4 and 5.9 %). Other studies (i.e. Yadav and Guleria, 2012; Tavarini and Angelini, 2013) have reported stevioside concentrations in leaves ranging from 4 to 14 %, and rebaudioside-A concentrations ranging from 2 to 4 %. In southern Italy, Lavini et al. (2008) found a stevioside concentration of 8.4 % in dry leaves and a rebaudioside-A concentration of 5.7 %. These were close to the concentrations reported in this study. In terms of total SG content, an important aspect is the ratio between stevioside, with its slightly bitter aftertaste, and rebaudioside-A, which has a more acceptable flavour (Brandle et al., 1998). With regard to this aspect, agronomical traits can influence these compounds (Ren and Shi, 2018). The overall results indicated that stevioside content (Table 9) was significantly higher in L2 and L3, with an average of 11.4 % and 10.5 % in MIC treatments, and 11.1 % and 10.8 % in control treatments. Similarly to the total glycoside content, the summer cut produced higher average stevioside contents than the autumn cut for all three chemotypes. Across the years and treatments, the first cut gave a higher stevioside content than the second cut (9.7 vs. 7.5 %). MIC plants showed some positive effects on stevioside content (8.9 %) compared to nonmycorrhizal-inoculated plants (8.3 %), although the differences between them were not significant. The cropping year had no effect on stevioside concentrations, which remained stable at 8.6 %. Moraes et al. (2013) reported a higher stevioside content, which varied between 10.6 % and 11.1 % in the first and second growing seasons, respectively. The effect of M x L interaction was significant in L1 (4.7 % for MIC treatment vs 2.9 % for control treatments) and L2 (11.6 vs 11.0 % for control treatments). Rebaudioside-A contents are reported in Table 10. The average rebaudioside-A content was 2.9 %, which varied significantly between chemotypes and mycorrhizal inoculation, whereas the cultivation year had no significant effect. A study by Lavini et al. (2008) in southern Italy reported a higher concentration (5.7 %), while Moraes et al. (2013) reported a rebaudioside-A content in leaves harvested in Mississippi, USA, which ranged between 3.1 % in the first growing season and 4.5 % in the second growing season. Analysis of the results indicated a wide difference between chemotypes: L1 had the highest rebaudioside-A concentration (Table 10) under all treatments and across years (5.9 %). L3 produced the second highest concentration, with a
3.3.4. Leaf/stem ratio The average leaf production/stem production ratio was 1.1 (Table 8). This is in line with Brandle and Rosa (1992), who reported a value of 1.2 for stevia cultivated in Delhi, India. Brandle and Rosa also suggested that the high leaf/stem ratio might be due to cultivation under a long day photoperiod, considering that stevia is an obligate short-day plant. Therefore, enhanced vegetative growth under long days is not surprising. Megeji et al. (2005) reported a lower leaf/stem ratio (0.8) for stevia cultivated in Palampur, India. The overall results showed a decreasing trend in the leaf/stem ratio over the years (1.7, 1.0 and 0.6 respectively in the first, second and third years) (Table 8). Following the same trend of leaf production, large differences were found between the first and the second harvest: a higher leaf/stem ratio in the summer harvest, the highest dry leaf production in summer, and the highest stem production in autumn. The mycorrhizal inoculation had a variable impact on this index. A higher leaf/stem ratio was found in MIC treatments in the first and the third years. The chemotype had no clear effect on this index, especially in MIC treatments (1.1, 1.0 and 1.1 vs. 1.1, 1.2 and 1.1 for L1, L2 and L3, respectively). 3.4. Steviol glycoside (SG) concentrations in stevia leaves The main compounds of stevia glycosides, stevioside and rebaudioside-A, were measured. The content and distribution of these SG compounds varies considerably according to harvest time and chemotypes. Mandal et al. (2013, 2015), found a significant increase in SG concentrations in response to AMF colonization. Similar observations were reported later in another study (Tavarini et al., 2018). Under all treatments, stevioside was the predominant SG, present at higher levels than rebaudioside-A. An average stevioside concentration of 8.6 % was found in stevia dry leaves (ranging between 3.8 and 11.3 6
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mychorrizal treatment and year of harvesting. Considering the total data of yield of each individual glycoside, L3 gave better results in terms of total SG production (530.3 kg ha−1) than L1 (379.4 kg ha−1) and L2 (232.6 kg ha−1) under both treatments and across all years. The total SG yield was higher at the first harvest (276.1 kg ha−1) than the second harvest (104.7 kg ha−1). MIC treatment had a positive effect on total SG yield, resulting in 464.4 kg ha−1 total SGs vs. 297.1 kg ha−1 for control plants. Total SG yield was higher during the first and second years (respectively 539.5 and 448.7 kg ha−1), while the third year saw a sharp fall to 154.1 kg ha−1.
Table 10 Rebaudioside-A content of stevia leaves as affected by mycorrhizal inoculum (M), chemotype (L) and growing year (Y). Rebaudioside-A content (G 100 g dm−1) Source of variance Mycorrhiza (M)
MIC Control
Chemotype (L)
L1 L2 L3
Year (Y)
2012 2013 2014
Interaction MXL MXY LXY MXLXY
First cut
Second cut
Total
4.0a 3.6a ns 7.5a 0.6c 3.3b *** 3.6a 4.3a 3.5a ns
2.3a 1.9b * 4.2a 0.2c 1.8b *** 2.2a 2.0a 1.9a ns
3.1a 2.7b * 5.9a 0.4c 2.5b *** 2.9a 3.1a 2.7a ns
** ns ns ns
** ns ns ns
** ns ns ns
4. Conclusions The performance of three stevia chemotypes inoculated and noninoculated with AMF was evaluated in terms of total biomass yield in order to achieve improved concentration and production of the best steviol glycosides. The results showed that the production of stevia secondary metabolites is affected by harvest time, chemotype, and mycorrhizal symbiosis. Our studies suggests that mycorrhizal inoculation of selected stevia chemotypes can significantly influence the biomass yield of stevia. In fact, an effective mycorrhizal symbiosis relationship between AMF and micropropagated stevia plantlets was established in selected chemotypes and resulted in higher biomass and steviol glycoside yields. In general, the production of glycoside sweeteners increases proportionally to the increase in dry leaf yield. The effect of AMF differed according to chemotype, and was found to be more pronounced in chemotype L1. When chemotypes are compared, L3 generally gave the best results in terms of productive potential. On the other hand, in terms of specific SGs, L1 must be considered interesting due to its rebaudioside-A content, which is an important aspect for acceptability of the product. The overall results showed that the cultivation of Stevia rebaudiana as a perennial crop is possible and could be economically profitable. In
ns,*,**, *** :Non significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple- range test (P = 0.05).
total average of 2.5 %, while a rebaudioside-A concentration of only 0.4 % was measured in L2. Under MIC treatment, the average rebaudioside-A concentration was 3.1 % (ranging between 4.0 and 2.3 % in the first and second harvests, respectively) higher than the concentration reported under the control treatment (2.7 %). M x L was significant in L1: 6.8 % for MIC plants vs. 4.9 % for the control plants. Table 11 summarises the main results regarding stevioside, rebaudioside-A, and total SG yields as an average for the chemotype,
Table 11 Stevioside, rebaudioside-A and total steviol glycoside production of stevia leaves as affected by mycorrhizal inoculum (M), chemotype (L) and growing year (Y). treatment
Stevioside production (kg ha−1) first cut
MIC
Control
Mycorrhiza (M) MIC 241.6 Control 141.7 ** Chemotype (L) L1 95.9 L2 165.4 L3 293.5 ** Year (Y) 2012 277.7 2013 203.9 2014 79.2 *** Interaction (M x L) L1 202,3 L2 168,5 L3 282,5
Rebaudioside-A production (kg ha−1)
Total SGs yield (kg ha−1)
second cut
total
first cut
second cut
total
first cut
second cut
total
97.6a 59.8b *
339.2 201.5 *
93.0 56.7 ***
29.1 14.8 ***
122.0a 71.5b ***
334.6 198.4
126.6 74.6 *
461.2 273.0 **
a b *
34.9b 61.9b 129.0a **
130.8 227.2 422.4 **
156.1 7.6 81.1 ***
49.0 1.1 24.1 ***
205.1a 8.7c 105.2b ***
252.0 173.0 374.6
83.9 63.0 153.1 **
335.9 236.0 527.7 **
ab b a **
96.3a 107.9b 35.8c **
374.0 311.8 115.0 **
104.1 92.9 27.6
27.8 27.8 9.8 **
131.9a 120.7a 37.4b *
381.8 296.8 106.9 *
124.1 135.8 45.5 ***
505.9 432.6 152.4 *
a a b *
66,0ab 58,9ab 126,4a
268,4 227,4 408,9
290.6 4.0 70.1
94.5 0.2 24.4
385.1a 4.2d 94.5bc
492.9 172.5 352.6
160.5 59.1 150.8
653.4 231.6 503.4
a b a c b a
L1 L2 L3
25,1 162,2 304,2
10,6 64,9 131,4
35,7c 227,0ab 435,6a
49.8 11.3 92.8
14.4 2.1 23.7
64.2bc 13.4c 116.6c
74.9 173.4 397.0
24.9 67.0 155.1
99.8 240.4 552.1
Interaction MxL MxY LxY MxLxY
* ns ns ns
* ns ns ns
* ns ns ns
** ns ns ns
** ns ns ns
** ns ns ns
* ns ns ns
* ns ns ns
* ns ns ns
ns,*,**, *** :Non significant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple- range test (P = 0.05). 7
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addition, two harvests per year can be achieved with improved management practices, including an optimal water supply in summer. However, the biomass yield between the two harvest times is expected to vary significantly due to differences in the photoperiod and in weather conditions between the two seasons. Our results indicate that stevia can be adapted to the agro-climatic condition of the study area, providing good biomass and steviol glycoside yields. We found that selection of a suitable chemotype of S. rebaudiana can make it possible to satisfy the needs of the food industry to use stevia as a substitute for sugar in new products.
Jackson, A., Tata, A., Wu, C., Perry, R., Haas, G., West, L., Cooks, R.G., 2009. Direct analysis of Stevia leaves for diterpene glycosides by desorption electrospray ionization mass spectrometry. Analyst 134 (5), 867–874. https://doi.org/10.1039/ B823511B. Kapoor, R., Giri, B., Mukerji, K.G., 2004. Improved growth and essential oil yield and quality in Foeniculum vulgare Mill. on mycorrhizal inoculation supplemented with Pfertilizer. Bioresour. Technol. 93, 307–311. https://doi.org/10.1016/j.biortech.2003. 10.028. Kapoor, R., Chaudhary, V., Bhatnagar, A.K., 2007. Effects of arbuscular mycorrhiza and phosphorus application on artemisinin concentration in Artemisia annua L. Mycorrhiza V. 17 (7), 581–587. https://doi.org/10.1007/s00572-007-0135-4. Khaosaad, T., Vierheilig, H., Nell, M., Zitterl-Eglseer, K., Novak, J., 2006. Arbuscular mycorrhiza alter the concentration of essential oils in oregano (Origanum sp., Lamiaceae). Mycorrhiza 16, 443–446. https://doi.org/10.1007/s00572-006-0062-9. Kolb, N., Herrera, J.L., Ferreyra, D.J., Uliana, R.F., 2001. Analysis of sweet diterpene glycosides from Stevia rebaudiana: improved HPLC method. J. Agric. Food Chem. 49, 4538–4541. https://doi.org/10.1021/jf010475p. Lavini, A., Riccardi, M., Pulvento, C., De Luca, S., Scamosci, M., D’Andria, R., 2008. Yield, quality and water consumption of Stevia rebaudiana Bertoni grown under different irrigation regimes in southern Italy. Ital. J. Agron. 3, 135–143. https://doi.org/10. 4081/ija.2008.135. Luwańska, A., Perz, A., Mańkowska, G., Wielgus, K., 2015. Application of in vitro stevia (Stevia rebaudiana Bertoni) cultures in obtaining steviol glycoside rich material. Herba Pol. 61 (1), 51–61. https://doi.org/10.1515/hepo-2015-0010. Mandal, S., Evelin, H., Giri, B., Singh, V.P., Kapoor, R., 2013. Arbuscular mycorrhiza enhances the production of stevioside and rebaudioside-A in Stevia rebaudiana via nutritional and non-nutritional mechanisms. Appl. Soil Ecol. 72, 187–194. https:// doi.org/10.1016/j.apsoil.2013.07.003. Mandal, S., Upadhyay, S., Singh, V.P., Kapoor, R., 2015. Enhanced production of steviol glycosides in mycorrhizal plants: a concerted effect of arbuscular mycorrhizal symbiosis on transcription of biosynthetic genes. Plant Physiol. Biochem. 89, 100–106. https://doi.org/10.1016/j.plaphy.2015.02.010. Megeji, N.W., Kumar, J.K., Singh, V., Kaul, V.K., Ahuja, P.S., 2005. Introducing Stevia rebaudiana, a natural zero-calorie sweetener. Curr. Sci. 88 iyagawa. Moraes, R.M., De Andrade, Z., Bedir, E., Dayan, F.E., Lata, H., Khan, I., Pereira, A.M.S., 2004. Arbuscular mycorrhiza improves acclimatization and increases lignan content of micropropagated mayapple (Podophyllum peltatum L.). Plant Sci. 166, 23–29. https://doi.org/10.1016/j.plantsci.2003.07.003. Moraes, R.M., Donegal, M.A., Cantrell, C.L., Mello, S.C., McChesney, J.D., 2013. Effect of harvest timing on leaf production and yield of diterpene glycosides in Stevia rebaudiana Bertoni: a specialty perennial crop for Mississippi. Ind. Crops Prod. 51, 385–389. https://doi.org/10.1016/j.indcrop.2013.09.025. Morone Fortunato, I., Ruta, C., Castrignano, A., Saccardo, F., 2005. The effect of mycorrhizal symbiosis on the development of micropropagated artichokes. Sci. Hortic. 106, 472–483. https://doi.org/10.1016/j.scienta.2005.05.006. Muth, N.D., 2015. From Food to Fuel: Carbohydrates. in: Sports Nutrition for Health Professionals, 1 edition. F.A. Davis Company, Philadelphia, USA, pp. 5–16. Pal, P.K., Mahajana, M., Prasad, R., Pathania, V., Singh, B., Ahujac, P.S., 2015. Harvesting regimes to optimize yield and quality in annual and perennial Stevia rebaudiana under sub-temperate conditions. Ind. Crops Prod. 65, 556–564. https://doi.org/10. 1016/j.indcrop.2014.09.060. Phillips, J.M., Hayman, D.S., 1970. Improved procedures for clearing roots and staining parasitic and vesicular arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Br. Mycol. Soc. 55, 158–161. Ren, G., Shi, Y., 2018. Correlation between glycosides in leaves and agronomic traits of Stevia rebaudina Bertoni. Bangladesh J. Bot. 47 (2), 337–342. Röck-Okuyucu, B., Bayraktar, M., Akgun, I.H., Gurel, A., 2016. Plant growth regulator effects on in vitro propagation and stevioside production in Stevia rebaudiana Bertoni. HortScience. 51 (12), 1573–1580. Ruta, C., Perrini, S., Tagarelli, A., Morone-Fortunato, I., 2009. Use of arbuscular mycorrhiza for acclimatization of micropropagated plantlets of melon, oregano artichoke and Spanish broom. Acta Hortic. 812, 473–477. https://10.17660/ActaHortic.2009. 812.68. Singh, A., Verma, P.P.S., 2015. Survival and growth performance of stevia cutting under different growing media. J. Med. Plants Stud. 3 (2), 111–113. Smith, S.E., Read, D., 2008. Mycorrhizal Symbiosis (Third Edition). Academic Press ISBN: 978-0-12-370526-6. Tarraf, W., Ruta, C., De Cillis, F., Tagarelli, A., Tedone, L., De Mastro, G., 2015. Effects of mycorrhiza on growth and essential oil production in selected aromatic plants. Ital. J. Agron. 10 (633), 160–162. https://doi.org/10.4081/ija.2015.633. Tavarini, S., Angelini, L.G., 2013. Stevia rebaudiana Bertoni as a source of bioactive compounds: the effect of harvest time, experimental site and crop age on steviol glycoside content and antioxidant properties. J. Sci. Food Agric. 93 (9), 2121–2129. https://doi.org/10.1002/jsfa.6016. Tavarini, S., Passera, B., Martini, A., Avio, L., Sbrana, C., Giovannetti, M., Angelini, L.G., 2018. Plant growth, steviol glycosides and nutrient uptake as affected by arbuscular mycorrhizal fungi and phosphorous fertilization in Stevia rebaudiana Bert. Ind. Crops Prod. 111, 899–907. Thiyagarajan, M., Venkatachalam, P., 2015. Assessment of genetic and biochemical diversity of Stevia rebaudiana Bertoni by DNA fingerprinting and HPLC analysis. Ann. Phytomed. 4 (1), 79–85. Toussaint, J.P., Smith, F.A., Smith, S.E., 2007. Arbuscular mycorrhizal fungi can induce the production of phytochemicals in sweet basil irrespective of phosphorus nutrition. Mycorrhiza. 17, 291–297. https://doi.org/10.1007/s00572-006-0104-3. Trouvelot, A., Kouch, J., Gianinazzi-Pearson, V., 1986. Mesure du taux de mycorhization VA d’un syste`me radiculaire: recherche of method d’estimation ayant une
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Andolfi, L., Macchia, M., Ceccarini, L., 2006. Agronomic productive characteristics of two genotypes of Stevia rebaudiana in central Italy. Ital. J. Agron. 2, 257–262. https://doi. org/10.4081/ija.2006.257. Anton, S.D., Martin, C.K., Han, H., Coulon, S., Cefalu, W.T., Geiselman, P., Williamson, D.A., 2010. Effects of Stevia, aspartame, and sucrose on food intake, satiety and postprandial glucose and insulin levels. Appetite 55, 37–43. https://doi.org/10. 1016/j.appet.2010.03.009]. Atul-Nayyar, A., Hamel, C., Hanson, K., Germida, J., 2009. The arbuscular mycorrhizal symbiosis links N mineralization to plant demand. Mycorrhiza 19 (4), 239–246. https://doi.org/10.1007/s00572-008-0215-0. Baum, C., El-Tohamy, W., Grudac, N., 2015. Increasing the productivity and product quality of vegetable crops using arbuscular mycorrhizal fungi: a review. Sci. Hortic. 187, 131–141. https://10.1016/j.scienta.2015.03.002. Bernal, J., Mendiola, J., Ibáñez, E., Cifuentes, A., 2011. Advanced analysis of nutraceuticals. Pharm. Biomed. Anal. 55, 758–774. https://doi.org/10.1016/j.jpba. 2010.11.033. Brandle, J.E., Starrat, A.N., Gijzen, M., 1998. Stevia rebaudiana: its agricultural, biological, and chemical properties. Can. J. Plant Sci. 78, 527–536. https://doi.org/10.4141/ P97-114. Brandle, J.E., Rosa, N., 1992. Heritability for yield, leaf:stem ratio and stevioside content estimated from a landrace cultivar of Stevia rebaudiana. Can. J. Plant Sci. 72, 1263–1266. https://doi.org/10.4141/cjps92-159. Cacciola, F., Delmontea, P., Jaworska, K., Dugo, P., Mondello, L., Rader, J., 2011. Employing ultrahigh pressure liquid chromatography as the second dimension in a comprehensive two dimensional system for analysis of Stevia rebaudiana extracts. J. Chromatogr. A 1218 (15), 2012–2018. https://doi.org/10.1016/j.chroma.2010.08. 081. Ceunen, S., Geuns, J.M.C., 2013. Influence of photoperiodism on the spatio-temporal accumulation of steviol glycosides in Stevia rebaudiana (Bertoni). Plant Sci. 198, 72–82. https://doi.org/10.1016/j.plantsci.2012.10.003. Chatsudthipong, V., Muanprasat, C., 2009. Stevioside and related compounds: therapeutic benefits beyond sweetness. Pharmacol. Ther. 121, 41–54. https://doi.org/10. 1016/j.pharmthera.2008.09.007. Cruz, C., Green, J.J., Watson, C.A., Wilson, F., Martins-Loução, M.A., 2004. Functional aspects of root architecture and mycorrhizal inoculation with respect to nutrient uptake capacity. Mycorrhiza 14, 177–184. https://doi.org/10.1007/s00572-0030254-5. Estrada-Luna, A.A., Davies Jr., F., Egilla, J.N., 2000. Mycorrhizal fungi enhancement of growth and gas exchange of micropropagated guava plantlets (Psidium guajava L.) during ex vitro acclimatization and plant establishment. Mycorrhiza 10 (1), 1–8. Fronza, D., Folegatti, M.V., 2003. Water consumption of the Estevia (Stevia rebaudiana (Bert.) Bertoni) crop estimated through microlysimeter. Sci. Agric. 60 (3), 595–599. https://doi.org/10.1590/S0103-90162003000300028. Gardana, C., Scaglianti, M., Simonetti, P., 2010. Evaluation of steviol and its glycosides in Stevia rebaudiana leaves and commercial sweetener by ultra-highperformance liquid chromatography–mass spectrometry. Chromatography A. 1217 (9), 1463–1470. https://doi.org/10.1016/j.chroma.2009.12.036. Geuns, J., 2003. Molecules of interest: stevioside. Phytochemistry 64, 913–921. Govindarajulu, M., Pfeffer, P.E., Jin, H.R., Abubaker, J., Douds, D.D., Allen, J.W., Bucking, H., Lammers, P.J., Shachar-Hill, Y., 2005. Nitrogen transfer in the arbuscular mycorrhizal symbiosis. Nature 435, 819–823. https://doi.org/10.1038/ nature03610. Goyal, S., Samsher, Goyal, R., 2010. Stevia (Stevia rebaudiana) a bio-sweetener: a review. Int. J. Food Sci. Nutr. 61, 1–10. https://doi.org/10.3109/09637480903193049. Gupta, E., Purwar, S., Sandaram, S., Gai, G.K., 2013. Nutritional and therapeutic values of Stevia rebaudiana: a review. J. Med. Plants Res. 7, 3343–3353. https://doi.org/10. 5897/JMPR2013.5276. Gupta, E., Purwar, S., Sundaram, S., Tripathi, P., Rai, G., 2016. Stevioside and rebaudioside a – predominant ent-kaurene diterpene glycosides of therapeutic potential: a review. Czech J. Food Sci. 34 (4), 281–299. https://doi.org/10.17221/335/ 2015-CJFS.
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Scientia Horticulturae xxx (xxxx) xxxx
L. Tedone, et al.
Yadav, A.K., Singh, S., Dhyani, D., Ahuja Can, P.S., 2011. A review on the improvement of stevia Stevia rebaudiana (Bertoni). Can. J. Plant Sci. 91 (1), 1–27. Yadav, S.K., Guleria, P., 2012. Steviol glycosides from stevia: biosynthesis pathway review and their application in foods and medicine. Critical Rev. Food Sci. Nutr. Nutr. 52 (11), 988–998. https://doi.org/10.1080/10408398.2010.519447. Yadav, K., Aggarwal, A., Singh, N., 2013. Arbuscular mycorrhizal fungi induced acclimatization and growth enhancement of Glycyrrhiza glabra L.: a potential medicinal plant. Agric. Res. 2, 43–47. https://doi.org/10.1007/s40003-012-0047-1. Zubek, S., Mielcarek, S., Turnau, K., 2012. Hypericin and pseudohypericin concentrations of a valuable medicinal plant Hypericum perforatum L. Are enhanced by arbuscular mycorrhizal fungi. Mycorrhiza. 22, 149–156.
signification fonctionelle. In: Dijon, Ier Seminaire (Ed.), Les Mycorhizes: Phisiologie and Genetique. INRA, Paris, pp. 217–221. Wilhelm, W.W., Ruhe, K., Schlemmer, M.R., 2000. Comparison of three leaf area index meters in a corn canopy. Crop Sci. 40, 1179–1183. https://doi.org/10.2135/ cropsci2000.4041179x. Williams, L.D., Burdock, G.A., 2009. Genotoxicity studies on a high-purity rebaudioside A preparation. Food Chem. Toxicol. 47, 1831–1836. Vives, K., Andújar, I., Lorenzo, J.C., Concepción, O., Hernández, M., Escalona, M., 2017. Comparison of different in vitro micropropagation methods of Stevia rebaudiana B. Including temporary immersion bioreactor (BIT®). Plant Cell Tissue Organ Cult. 131 (1), 195–199.
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