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versión On-line ISSN 2521-9766versión impresa ISSN 1405-3195

Agrociencia vol.52 no.4 México may./jun. 2018


Crop Science

Effectiveness of biofertilizers and brassinosteroids in Stevia rebaudiana Bert.

Juan F. Aguirre-Medina1 

Francisco O. Mina-Briones1 

Jorge Cadena-Iñiguez2  * 

Ramón M. Soto-Hernández3 

1 Facultad de Ciencias Agrícolas. Universidad Autónoma de Chiapas. Entronque carretera costera y Estación Huehuetán. 30660. Huehuetán, Chiapas, México. (; (

2 Postgrado en Innovación en Manejo de Recursos Naturales, Campus San Luis Potosí, Colegio de Postgraduados. 78620. Iturbide No. 73, Col. Centro, Salinas Hidalgo, SLP. (

3 Postgrado en Botánica, Campus Montecillo, Colegio de Postgraduados. 56230. km 36.5 carretera México-Texcoco, Montecillo, Estado de México. México. (


The demand for low-calorie products has increased. One of them is a natural product extracted from stevia (Stevia rebaudiana Bert.) whose diterpene glucosides generate outstanding sweetening power. The organic cultivation of this species is increasing in the southern region of Chiapas, México, but its asexual establishment through cuttings presents agronomic limitations. This can be improved with the incorporation of biofertilizers. It is the case of the efficiency in N fixation by Azospirillum brasilense and the endomycorrhizae Rhizophagus intraradices, which improve the transport of nutrients and water to the host plant. These microorganisms have been used in biofertilizer induction programs in annual and perennial crops in México. The objective of this study was to evaluate the effect of R. intraradices (Schenck & Sm.) Walker & Schüßler and A. brasilense Tarrand, Krieg & Döbereiner in interaction with brassinosteroids in S. rebaudiana Bert. plants through morphological, physiological and biochemical changes. The cv Morita 2 was established in a greenhouse, in plastic bags with 6 kg of fluvisol-euthric soil plus washed river sand (1:1) and 4 g of each inoculant at the moment of transplant. The concentration of A. brasilense was 9X106 bacteria g-1 and that of R. intraradices 40 spores g-1 of soil with 95 % colonization of the root system. The brassinosteroid (CIDEF-4, Natura del Desierto, S. A. de C.V., steroid content 80 % with 10 % i.a.) (2 mg L-1 of water) was sprayed on the foliage after 28 d of sowing, plus three later applications with frequency of 14 d. The experimental design was completely random with seven repetitions; the treatments were the microorganisms and the brassinosteroid applied individually and combined, and the control without application or with inoculum. The experimental unit was one pot. The variables recorded were yield, percentage of mycorrhizae root colonization, content of sweetener in leaves, and of N and P in shoots 90 d after sowing. The inoculated plants and treated with brassinosteroid in different modality increased the leaf area (1728 cm2 per plant), content of stevioside (35.8 mg g-1), rebaudioside (29.5 mg g-1) and steviol (5 mg g-1) compared to the control (p≤0.05). The content of N and P increased with the application of R. intraradices alone and combined with brassinoesteroid.

Keywords: Rhizophagus intraradices; Azospirillum brasilense; brassinosteroid; steviosides; nitrogen and phosphorus


Stevia rebaudiana is a plant from the subtropical forest of high Paraná, native of the mountainous hills of Paraguay, where the inhabitants have used it as sweetener and in herbal medicine (Dacome et al., 2005; Prakash et al., 2008; Anton et al., 2010). The intense sweet taste in the leaves of this species is due to the presence of diterpene glucosides, of which stevioside and rebaudioside A, are in greater proportion. The sweetening power of these compounds is 150 to 300 times higher than sucrose (Midmore and Rank, 2002; Liu et al., 2010) and without caloric content (Anton et al., 2010). Therefore, its demand to sweeten foods and drinks has increased (Brandle and Telmer, 2007; Durán et al., 2012). In México there are environmental conditions for the cultivation of S. rebaudiana (Ramírez-Jaramillo et al., 2011) and it can be profitable.

Research about mineral nutrition of stevia is scarce (Das et al., 2007); the studies have evaluated chemical fertilization (Jaitak et al., 2008; Jarma et al., 2010). The substrates with high content of organic matter and inoculation by microorganisms increase secondary metabolites (Brandt and Møgaard, 2001) and improve plant growth. The increase in use of biofertilizers (mainly with microorganisms) has allowed decreasing the use of up to 69 589 t of chemical fertilizers, which allowed reducing 22.7 mil t of CO2 between 2014 and 2015 (SAGARPA, 2016).

The stevia leaf is the organ with highest concentration of steviosides (Gardana et al., 2010) and its content is influenced primarily by the light hours, phenological stage, and cultivar (Madan et al., 2010). Certain agricultural practices, such as fertilization (Das et al., 2007) and application of endomycorrhizae microorganisms (Portugal et al., 2006), increase nutrient absorption, mainly of P, improve the synthesis of secondary metabolites (Riipi et al., 2002), and can impact the concentration of flavonoids and isoflavonoids (Das et al., 2008; Hanan et al., 2008).

The environmental conditions of the coast of Chiapas, México, differ from those of the place of origin of Stevia and its persistence as a crop tends to decrease. Thus, the possibility of applying exogenous brassinosteroid emerges, which is a hormone that induces diverse responses in the plants, such as favoring the polarization of the cell membrane and increasing the resistance to biotic and abiotic stress (Singh and Shono, 2005; Reyes et al., 2008).

This study had the objective of identifying the influence of Rhizophagus intraradices and Azospirillum brasilense combined with brassinosteriod on the morphological, physiological and biochemical changes of Stevia rebaudiana Bert.

Materials and Methods

Experiment description

The study was carried out in a nursery during the spring of 2015, in the Experimental Field of the Agricultural Sciences School, Campus IV, Huehuetán, Chiapas, located in the junction of the coastal road and Estación Huehuetán, municipality of Huehuetán, Chiapas (15° 00’ and 15° 30’ N, 94° 30’ and 95° 00’ W, altitude of 44 m and minimum and maximum temperature of 15 and 38 °C). The soil used, from the group of the euthric fluvisols, was mixed with washed river sand (1:1). The texture of the substrate was sandy-loam (Bouyoucos, 1962), with 80.7 % sand, 13.3 % loam, 5.8 % clay, 2.6 % organic matter (Walkley-Black, 1934), electric conductivity of 0.05 ds m-1, 5 Meq 100g-1 of cationic exchange capacity, pH 5.7, 0.13 % of N (quantified with Micro-Kjeldahl; Irigoyen et al., 1992), P 14.2 mg kg-1 (evaluated by colorimetry), K++ 64.2 mg kg-1, Ca++ 474 mg kg-1 (determined through atomic spectrophotometry, Thermo Fisher Scientific, model 400 ¼), Mg++ 58.0 mg kg-1 and Na++ 102.5 mg kg-1.

Cuttings of S. rebaudiana Bert. of the Morita 2 variety, were obtained from a plantation, 10 km from the experimental site, from vigorous plants without apparent damage by insects or disease. The cuttings with longitude of 10±2 cm and 60 d of age were obtained from the upper third of the plant and once obtained they were transported in sterile water for their sowing. Six kg of the soil mixture, without sterilization, was placed in black plastic bags (caliber 700) with perforations on the base for drainage. The treatments were: 1) A. brasilense, 2) R. intraradices, 3) A. brasilense plus R. intraradices, 4) Brasinoesteroide, 5) R. intraradices plus brassinosteroid, 6) A. brasilense plus brassinosteroid, 7) A. brasilense, R. intraradices plus brassinosteroid and 8) control (only soil). Four g of the inoculum from each microorganism, depending on the treatment, were deposited in the cavity where the cutting would be placed at the time of transplant. The brassinosteroid (2 mg L-1) was applied 28 d after sowing and then every 14 d.

The host plant of R. intraradices was Brachiaria decumbens Stapf in sterile soil, the colonization reached 95 % in roots and the soil. This substrate was used as inoculum; when it was applied it contained 40 spores g-1 of soil plus propagules. The inoculum of A. brasilense was obtained from the Soil Microbiology Laboratory of the Benemérita Universidad Autónoma de Puebla (México), at the time of the inoculation presented 9X106 cells g-1, and this was impregnated in the peat as substrate. The soluble brassinosteroid CIDEF-4 (Natura del Desierto, S. A. de C. V. in México) had 80 % of the steroidal content and 10 % i.a., is not toxic and is compatible with fertilizers, insecticides and fungicides.

The experimental design was completely random with seven repetitions per treatment. The experimental unit was a pot with one plant. The analysis of N, P, rebaudioside A, and stevioside was determined in dehydrated leaves with four repetitions. The irrigation events were carried out with distilled water. Ninety days after sowing, the leaves were harvested and their biomass and content of N, P and sweeteners were determined.

Number of leaves, number of branches, biomass and leaf area

The total number of leaves and branches was counted; the dry biomass of the plant structures was obtained in an analytical scale (0.1 mg; Ohaus, NJ, USA), after being dehydrated in a stove, with circulating air, at 75-80 °C. The foliar area was recorded in cm2 with an integrator of foliar area (LI-COR, LI 3100, USA).

Content of sweeteners in leaf

The steviosides, rebaudioside A and steviol were extracted from dehydrated leaves at 60 °C and crushed in an electric mill. The compounds were determined by HPLC (Agilent Technologies 1200 Series, California, USA), in an analytical Zorbax CDB C-18 column of 4.6X150 mm and 5 (m of particle size, according to the method described by Hashimoto and Moriyasu (1978), in the Plant Chemistry Laboratory at Colegio de Postgraduados, Montecillo, México.

Content of N and P

The content of N and P was determined in the shoot, the N with the Microkjeldahl method and the P with the Olsen et al. (1954) method in a spectrophotometer (Thermo Fisher Scientific Model 400 ¼), at the Soil, Water and Plant Laboratory of the Agricultural Sciences School in Universidad Autónoma de Chiapas, Huehuetán Chiapas, México.

Mycorrhizae colonization in root

The colonization was quantified solely in the treatments with R. intraradices, with the Phillips and Hayman (1970) technique in 100 segments of root, with 1.5-1.6 cm length, and in optic microscope with immersion objective (100 X).

Statistical analysis

The experimental design was completely random and the data were analyzed through ANDEVA, with the SAS (version 9.0) GLM procedure, and the means were compared with the Tukey test (p≤0.05). The values in percentage were transformed to arcus-sine for the statistical analysis.

Results and Discussion

The highest number of leaves (p≤0.05) was found with the mixture of R. intraradices and brassinosteroid. The inoculation of the two microorganisms alone and co-inoculated plus brassinosteroid increased between 5 and 13 % the number of leaves compared to the control. The microorganisms and the brassinosteroid separated did not modify the number of leaves with regard to the control (Table 1).

Table 1 Development characteristics of Stevia rebaudiana Bert. plants inoculated with microorganisms plus brassinosteroid, cultivated in fluvisol-euthric soil from del Soconusco, Chiapas, México.  

Tratamiento Número Peso seco (g planta-1) Área foliar (cm2
Hojas Ramas Raíz Tallo y ramas Lámina foliar
R. intraradices 317±7.8 c 6.71±0.35 bc 1.57±0.08 bcd 4.43±0.05 e 4.76±0.14 cd 1173±47 cd
A. brasilense 310±10.7 c 5.86±0.26 bc 1.75±0.05 abc 4.98±0.18 cde 5.33±0.20 bc 1084±23 d
R. intraradices ×A. brasilense 373±9.9 b 6.00±0.30 bc 1.95±0.10 ab 6.22±0.15 b 5.97±0.17 ab 1390±48 b
Brasino-esteroide 339±9.9 bc 5.71±0.47 cd 1.94±0.10 abc 5.88±0.19 bc 4.70±0.19 cde 1179±30 cd
Brasino-esteroide ×R. intraradices 516±9.0 a 11.29±0.42 a 2.05±0.08 a 7.31±0.22 a 6.21±0.18 a 1728±58 a
Brasino-esteroide ×A. brasilense 246±6.1 d 3.86±0.26 d 1.52±0.12 cd 4.99±0.11cde 3.91±0.15 e 1151±20 d
Brasino-esteroide ×R. Intraradices ×A. Brasilense 344±8.1 bc 7.71±0.56 b 1.64±0.10 abc 5.57±0.30 bcd 5.00±0.25c 1356±50 bc
Testigo 328±10.5 c 6.57±0.57 bc 1.15±0.07 d 4.72±0.29 ed 4.11±0.17 de 1152±54 d
CV 6.9 16.5 14.6 9.8 9.9 9.1

Brassinosteroid CIDEF-4. CV: coefficient of variation (%). Means with different letter in a line are statistically different (Tukey, p≤0.05).

The increase in the number of leaves from inoculation with R. intraradices allows suggesting that the absorption of nutrients and water through the radical system increased, since when increasing the growth of the mycelium of these fungi it allows them to act as an extension of the root (Leigh et al., 2009) and to favor changes in their physiology (Barea et al., 2002). The increase in the number of leaves coincided with what was observed in perennial species like Theobroma cacao L. (Aguirre-Medina et al., 2007), Coffea arabica L. (Sánchez et al., 2005), Coffea canephora (Pierre) ex Froehner (Ibarra-Puón et al., 2014) and Tabebuia donnell-smithii Rose (Aguirre-Medina et al., 2014).

The brassinosteroid also favored the growth of the leaf and the number of branches. This seems due to some stress, according to Núñez and Mazorra (2001) who recorded this response in other species in tropical temperatures. In our study the stress could be due to the change in temperature of the austral place of origin of stevia (15 and 30 °C) and of the study site (up to 38 °C). The brassinoesteroid is a growth promotor through the increase in elongation and cell division (Salgado et al., 2008; Clouse, 2011), thermo-tolerance inductor in Bromus inermis (Wilen et al., 1995) and in tomato (Solanum lycopersicum Lam.) (Sing and Shono, 2005).

The mycorrhization of R. intraradices in combination with the brassinosteroid promoted (p≤0.05) the accumulation of biomass with regard to the other treatments; it was 44 and 24 % higher than in the treatment with R. intraradices and with brassinosteroid. The mycorrhization has been related to the transport of nutrients and water to places where the root cannot be explored (Sylvia, 2005). The inoculation with R. intraradices in association with brassinosteroid increased the foliar area (1728±58 cm2 per plant) more than the treatment with R. intraradices plus A. brasilense (1390±48 cm2 per plant) and with brassinosteroid plus R. intraradices plus A. brasilense (1356±50 cm2 per plant); these results contrasted with the values of the control (1152±54 cm2 plant-1). The number of branches and the root weight showed higher coefficients of variation than the other variables, although they did not reach the statistically permissible 20 % in field studies. This indicates that the use of microorganisms and the application of steroids are efficient to modify the physiology of stevia.

Plant growth is increased when combining associative bacteria, such as Azospirillum, and mycorrhizae fungi (Miller and Jastrow 2000). This effect was observed in validation plots of maize and bean in México (Aguirre-Medina, 2006; Trabelsi and Mhamdi, 2013). The control had 55 % lower biomass than that of the best treatment.

The initial increase of biomass in the leaf of S. rebaudiana with R. intraradices influenced the increase in total biomass during its whole development. The leaves are a principal source of photosynthates for other organs of the plant, which is why their increase and persistence favor the net rate of assimilation and relative rate of growth. When the mycorrhizae fungi favor the nutrition of plants, the photosynthetic rate improves (Wright et al., 2005). This fact, according to Milthorpe and Moorby (1982), establishes a positive relationship between the supply of mineral nutrients and rate of photosynthesis, which influences the whole photosynthetic complex.

Content of sweeteners

The S. rebaudiana plants inoculated with R. intraradices in presence of brassinosteroid presented significantly (p≤0.05) more stevioside (35.8±1.76) than the other treatments. The treatment with the two microorganisms plus the brassinosteroid (32.2±1.12), and with A. brasilense without combining (29.7±1.28) were in the same statistical group (Table 2). The arbuscular mycorrhizae fungi increase the absorption of nutrients, primarily P, which is an essential part of molecules like uridine diphosphate glucose which is a glucose donator in the synthesis of diterpene glucosides (Shibata et al., 1995; Richardson et al., 2009).

Table 2 Content of sweeteners in Stevia rebaudiana Bert. leaves inoculated with R. intraradices, A. brasilense and leaf spraying with brassinosteroid.  

Tratamientos Mg g de peso seco-1
Esteviósido Rebaudiósido A Esteviol Total edulcorantes
R. intraradices 21.5±0.76 d 17.7±0.90 a 1.6±0.12 a 40.9±1.47 b
A. brasilense 29.7±1.28 abc 26.3±6.66 a 1.1±0.66 a 57.2±7.78 ab
R. intraradices ×A. brasilense 25.8±1.56 cd 17.4±3.19 a 5.0±0.98 a 48.2±4.12 ab
Brasinoesteroide 27.2±1.14 bcd 21.9±5.72 a 1.6±0.96 a 50.8±5.32 ab
Brasinoesteroide ×R. intraradices 35.8±1.76 a 27.9±2.23 a 4.5±1.20 a 68.4±4.84 a
Brasinoesteroide ×A. brasilense 29.4±1.06 bc 29.5±3.61 a 4.2±1.18 a 63.2±5.10 ab
Brasinoesteroide ×R. intraradices ×A. brasilense 32.2±1.12 ab 35.1±7.84 a 1.5±0.88 a 68.8±9.41 a
Testigo 25.0±1.4 cd 17.8±0.60 a 1.5±0.90 a 44.9±2.00 b
CV 2.6 37.8 68.9 20.2

Brassinosteroid CIDEF-4. CV (%): coefficient of variation. Means with different letter in a line are statistically different (Tukey, p≤0.05).

Azospirillum increases the root development of the host plant through the hormones produced and N fixation (Bashan and De Bashan, 2010), something necessary for the normal growth of plants (Jarma et al., 2010).

The plants in presence of endomycorrhizae fungi can modify their content of steviosides and isoflavonoids (Hanan et al., 2008). Portugal et al. (2006) observed a higher concentration of stevioside (72 mg per plant) in stevia plants inoculated with R. intraradices, compared to the control plants (16 mg plant-1) without inoculating. In this case, the highest values of stevioside represented 50 % with R. intraradices plus brassinosteroid.

The coefficient of variation showed high variability of the content of rebaudioside A and steviol, and the difference between the treatments does not suggest a relation in their biosynthesis; that is, the correlation of the content of steviosides and of rebaudiosides is not significant, although the glucosides share a metabolic path with rebaudioside A (Madan et al., 2010). The low contents of steviol showed high CV, although the contents of the plants inoculated with microorganisms and brassinosteroid stood out (5.0±0.98, 4.5±1.2, 4.2±1.18).

Mycorrhizal plants generally absorb higher amounts of micronutrients, such as Mn (Pacovsky et al., 1985), which is a cofactor of enzymes that intervene in the synthesis of ent-kaurenoic acid. This is the precursor of the main sweeteners, such as steviosoid in stevia (Jarma et al., 2010); in addition, S, Cu, Zn and Fe (Habte and Aziz, 1985).

Jarma et al. (2010) pointed out that the stevioside can vary from 3 to 8 % in the dry tissue of stevia leaves. The statistical differences were present only with the stevioside and the highest value was found with the application of brassinosteroid plus R. intraradices (p≤0.05) with the inoculated microorganisms alone, plus the brassinosteroid.

The content of sweeteners, nutrients and biomass increased with the application of brassinosteroid. This indicates the possibility of stevia plants being stressed, possibly from the temperature of the study region. Sing and Shono (2005) treated tomato plants with 24 epibrassinolide and reported higher tolerance at 38 °C and higher photosynthetic efficiency in comparison to the plants treated at 25 °C.

Content of N and P

The plants inoculated with each microorganism or their combination presented a higher content of N compared to the control plants and when brassinosteroid was applied (Figure 1). The content of N increased and was statistically different from the control (p≤0.05) when inoculating A. brasilense plus the brassinosteroid. A. brasilense induces root growth in the host plant as a result of the production of plant hormones (Hungria et al., 2004), such as indole-acetic acid (Dobbelaere et al., 2003; Bashan and de Bashan, 2010), which modifies the morphology and increases the root biomass (Lalitha et al., 2011). Endomycorrhizae fungi transport N through the hyphae from sites of the soil that are inaccessible for the root (Hodge 2003). The inoculation with endomycorrhizae fungi in crops of Phaseolus sp. (Tajini and Drevon, 2012), Coffea arabica L. (Aguirre-Medina et al., 2011) and Cedrela odorata L. (Aguirre-Medina et al., 2014) showed similar results. The highest content of P in the plant tissue was found in the treatment with A. brasilense and when adding brassinosteroid (p≤0.05). Other studies have shown that mycorrhizal plants absorb the P from the soil efficiently in comparison to non-colonized plants (Andrade et al., 2009) from soil regions beyond the zone of exhaustion around the root (Wright et al., 2005).

Figure 1 Percentage of phosphorus (P) and nitrogen (N) in S. rebaudiana Bert. leaves biofertilized with R. intraradices, A. brasilense and foliar application of CIDEF-4 (brassinosteroid) under greenhouse conditions (n=4) ± standard error. Different letters in each treatment (N or P) are statistically different (p≤0.05). CV=2.6 % for N and CV=5.8 % for P. 

The root colonization of all plants inoculated with R. intraradices was high. In average, the colonization was 83 to 90 %. In the case of the control without application of the endomycorrhizae fungus, the root colonization was 52 %. The root colonization in the control treatment and in the treatment with brassinosteroid or A. brasilense is surely due to native mycorrhizae that are found in the substrate.


S. reabudiana plants inoculated and in presence of brassinosteroid, in different modes of association, showed certain increases in foliar area and in content of stevioside, rebaudioside and steviol. The content of N and P increased with R. intraradices inoculated alone and plus brassinosteroid.

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Received: March 2017; Accepted: February 2018

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