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Effect of biofertilizers on the nutrient

availability in soil in relation to growth,
yield and yield attributes of Stevia
rebaudiana
a b c
Kuntal Das , Raman Dang & T. N. Shivananda
a
Department of Pharmacognosy and Phytochemistry , St. John's
Pharmacy College , Vijayanagar, Bangalore
b
Department of Pharmacognosy and Phytochemistry , Al-Ameen
College of Pharmacy , Bangalore
c
Medicinal and Aromatic Plant Division, Indian Institute of
Horticultural and Research , Bangalore, India
Published online: 17 Aug 2009.

To cite this article: Kuntal Das , Raman Dang & T. N. Shivananda (2009) Effect of
biofertilizers on the nutrient availability in soil in relation to growth, yield and yield
attributes of Stevia rebaudiana , Archives of Agronomy and Soil Science, 55:4, 359-366, DOI:
10.1080/03650340802658432

To link to this article: http://dx.doi.org/10.1080/03650340802658432

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 Archives of Agronomy and Soil Science
Vol. 55, No. 4, August 2009, 359–366

Effect of biofertilizers on the nutrient availability in soil in relation

to growth, yield and yield attributes of Stevia rebaudiana
Kuntal Dasa*, Raman Dangb and T.N. Shivanandac
a
Department of Pharmacognosy and Phytochemistry, St. John’s Pharmacy College,
Vijayanagar, Bangalore; bDepartment of Pharmacognosy and Phytochemistry,
Downloaded by [Swinburne University of Technology] at 06:03 08 January 2015

Al-Ameen College of Pharmacy, Bangalore; cMedicinal and Aromatic Plant Division,

Indian Institute of Horticultural and Research, Bangalore, India
(Received 27 August 2008; final version received 25 November 2008)

A field experiment was conducted at Rani Chandramma Agricultural University

(Arabhavi, Belgaum), Karnataka, India, in basic soil to study the nutrient availability in
soil, yield and yield attributes of the medicinal plant stevia (Stevia rebaudiana). The
results showed that the availability of nutrients such as nitrogen (N), phosphorus (P)
and potassium (K) in the soil increased up to the fifth month; and thereafter, decreased
by the same amount with the progress of the plant growth up to the sixth month,
irrespective of treatments. Results also showed that the fresh and dry biomass yields
significantly increased up to the sixth month with different treatments, but the yield
attributes like plant height and the number of branches were observed to be varied with
various treatments, being highest in the combined application of biofertilizers over that
of their corresponding sole applications.
Keywords: basic soil; nutrient availability; stevia; yield

Introduction
The World Health Organization (WHO) estimates that 4.5 billion people around the world
use herbal medicines for their primary healthcare. It is estimated that the export earnings
from medicinal and aromatic plants would be raised to Rs 10,000 crores by 2010. The
demand for medicinal plant based on raw materials is increasing by 15–25%
internationally with alternative health delivery systems.
The leaves of stevia have commercial importance due to the presence of non-caloric
diterpenes and sweet glycosides, especially stevioside which is 300–400 times sweeter than
sugar without any side-effects. Certain protocols have been established for the cultivation
of stevia in various soil environments so that farmers can benefit from its sale and
industries can also obtain healthy leaves throughout the year for the isolation of the active
components to formulate commercial products.
Thus, the cultivation of stevia is gradually coming into focus in Indian agri-
culture due to having no optimum agronomic management practices. Several
literatures have reported that stevioside is the main active constituent of the stevia

*Corresponding author. Email: daskdphd@yahoo.co.in

ISSN 0365-0340 print/ISSN 1476-3567 online

Ó 2009 Taylor & Francis
DOI: 10.1080/03650340802658432
http://www.informaworld.com
 360 K. Das et al.

plant, is greatly dependable on the package of practices for the cultivation of stevia
as well as the adoption of modern agro-techniques (Nepovim et al. 1998; Geuns
2003). However, the influence of biofertilizers on the growth, yield and nutrient
content in stevia under field conditions has not been reported so far. The favourable
effects of sole and combined application of biofertilizers on the biomass production
as well as yield attributes are still lacking and hence, the present investigation was
undertaken.

Materials and methods

Cuttings of stevia plants, collected from Ankur Nursery, Ripponpet (Shimoga,
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Karnataka), India, were used as a test plant for the present study. A field experiment
was conducted in the month of February 2006 at the Aravavi, Belgaum, on basic soil
reaction. Before the start of the experiment, initial soil pH (soil:water, 1:2.5 w/v),
organic carbon (wet digestion with potassium dichromate), CEC (Kjeldahl method after
extracting the soil with ammonium acetate), available nitrogen (N), phosphorus (P) and
potassium (K) were determined by following the method as described by Piper (1966)
and Jackson (1973), respectively. After extracting the soil samples, available N was
determined by the Kjeldahl Method; available P was determined by spectrophotometer
(ECIL, made in India) using red filter at 660 nm; and available K was determined by
flame photometer (Elico, made in India). The relevant physicochemical properties of
soils were: pH 8.20; organic carbon 0.42%; CEC 14.66 cmol (pþ) kg71, available N
47.43 mg kg71, available P2O5 5.9 mg kg71, available K 79.8 mg kg71, DTPA
(Diethyltriaminepentaacetic acid) – extractable Zn, Cu, Fe and Mn were estimated at
0.41 mg kg71, 0.21 mg kg71, 3.6 mg kg71 and 2.1 mg kg71, respectively. Experimental
fields were divided into beds according to treatments. The following treatments were
used:

T1 – Control, only FYM (farmyard manure) at 10 kg, no application of biofertilizers;

T2 – Soil application of FYM at 10 kg mixed with 250 g of PSB (phosphorus solubilizing
bacteria).
T3 – Soil application of FYM at 10 kg mixed with 250 g of AZO (Azospirillum).
T4 – Soil application of FYM at 10 kg mixed with 500 g of VAM (vesicular arbuscular
mycorrhiza);
T5 – Soil application of FYM at 10 kg mixed with 250 g of VAM and 250 g of AZO;
T6 – Soil application of FYM at 10 kg mixed with 250 g of PSB and 500 g of AZO;
T7 – Soil application of FYM at 10 kg mixed with 250 g of PSB and 500 g of VAM;
T8 – Soil application of FYM at 10 kg mixed with 250 g of PSB, 250 g of AZO and 500 g of
VAM.

Each treatment was replicated thrice in a completely randomized design (CRD).

Altogether, there were 24 (8 6 3) beds, with each bed size of 1 6 1.25 m. Cuttings of
stevia plants were planted at the same time in all the beds. Each bed contains six rows and
six columns that contained 36 plants (6 6 6), with a total of 864 plants (36 6 24). The
moisture content was maintained to 60% of water holding capacity (WHC) by irrigating
three times a week. The periodic collection of soil samples were made and analyzed for pH,
available N, P and K in soil by following the method described above. Different yield
attributes, such as plant height, the number of branches and yields, were also recorded
periodically.
 Archives of Agronomy and Soil Science 361

Results
Changes in NH4-N content in soil
The results in Table 1(a) showed that the content of NH4-N was found to be gradually
increased with the progress in crop growth irrespective of the treatments and reached a
maximum within five months and thereafter decreased up to six months. The magnitude of
such an increase was recorded at the initial stage of the crop and the decrease at the later
period. The highest increase (35.08 mg kg71) was found at 5 months of crop growth in the
treatment where recommended levels of biofertilizers were applied in combination.
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Changes in N03-N content in soil

Changes in the N03-N content in soil showed a similar trend to that of NH4-N content
(Table 1(b)). The mean content of NO3-N in soil was also recorded the highest (34.80 mg
kg71) in the T8 treatment when FYM þ PSB þ AZO þ VAM were applied simultaneously
which was closely followed by the treatment receiving FYM þ PSB þ VAM (T7) together.

Changes of available P content in soil

Available P content in the soil initially increased and thereafter it gradually decreased with
the progress of crop growth (Table 2). However, the magnitude of such changes varied
with treatments. The highest available P content (14.40 mg kg71) was recorded at 3
months of crop growth in the treatment where the recommended level of biofertilizers (T8)
was applied in combination.

Changes of available K content in soil

Table 3 shows that the periodic changes in available K content varied with treatments,
being the highest (89.20 mg kg71) in the treatment where biofertilizers were applied
simultaneously (T8) and next followed by the treatment (88.10 mg kg71) where PSB and
VAM (T7) were applied in combination.

Table 1(a). Effect of modes of biofertilizer applications on the changes in NH4-N content
(mg kg71) in soil (mean of three replications).

Treatments 1st month 2nd month 3rd month 4th month 5th month 6th month
T1 24.34 24.66 24.87 25.32 25.63 25.62
T2 24.27 24.99 26.93 27.02 27.90 27.88
T3 25.11 26.86 27.63 28.17 28.69 28.62
T4 26.82 27.24 28.94 29.39 30.02 30.00
T5 26.97 28.01 29.87 30.09 30.76 30.64
T6 28.13 28.15 29.96 30.94 31.59 31.46
T7 27.07 27.83 30.10 31.43 31.98 31.83
T8 29.59 30.89 32.73 34.62 35.08 34.83
CD (p ¼ 0.05) 2.26 2.68 3.12 3.74 3.98 3.84

CD: Critical difference; T: Treatments; T1: FYM, T2: FYM þ PSB, T3: FYM þ AZO, T4: FYM þ VAM, T5:
FYM þ VAM þ AZO, T6: FYM þ PSB þ AZO, T7: FYM þ VAM þ PSB, T8: FYM þ VAM þ AZO þ PSB;
FYM: farmyard manure, PSB: P solubilizing bacteria, AZO: Azospirillum, VAM: vasicular arbuscular
mycorrhiza.
 362 K. Das et al.

Table 1(b). Effect of modes of biofertilizer applications on the changes in N03-N content
(mg kg71) in soil (mean of three replications).

Treatments 1st month 2nd month 3rd month 4th month 5th month 6th month
T1 23.21 24.32 24.43 24.96 25.44 25.40
T2 24.01 24.77 25.95 26.66 26.98 26.92
T3 24.81 25.58 27.09 27.77 28.41 28.36
T4 25.87 27.04 28.16 29.06 29.74 29.60
T5 26.77 27.36 28.46 29.74 30.51 30.38
T6 27.03 27.95 29.34 30.34 31.07 30.65
T7 26.81 27.57 29.78 31.67 31.50 31.21
T8 29.14 29.91 32.61 34.10 34.80 34.54
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CD (p ¼ 0.05) 2.24 2.56 2.89 3.15 3.58 3.46

CD: Critical difference; T: Treatments; T1: FYM, T2: FYM þ PSB, T3: FYM þ AZO, T4: FYM þ VAM, T5:
FYM þ VAM þ AZO, T6: FYM þ PSB þ AZO, T7: FYM þ VAM þ PSB, T8: FYM þ VAM þ AZO þ PSB;
FYM: farmyard manure, PSB: P solubilizing bacteria, AZO: Azospirillum, VAM: vasicular arbuscular
mycorrhiza.

Table 2. Effect of modes of biofertilizer applications on the changes in available P content

(mg kg71) in soil (mean of three replications).

Treatments 1st month 2nd month 3rd month 4th month 5th month 6th month
T1 6.30 6.60 7.10 7.80 7.90 7.80
T2 6.80 6.90 7.60 8.30 8.60 8.40
T3 6.60 7.00 7.90 8.80 9.20 9.10
T4 7.90 8.10 8.60 9.10 9.80 9.30
T5 8.40 8.80 9.30 9.90 10.60 9.90
T6 8.80 9.20 9.80 10.50 11.80 10.90
T7 9.10 9.50 10.40 11.20 12.40 11.80
T8 9.90 10.60 12.70 13.80 14.40 13.60
CD (p ¼ 0.05) 1.89 2.14 2.37 2.28 2.98 2.65

CD: Critical difference; T: Treatments; T1: FYM, T2: FYM þ PSB, T3: FYM þ AZO, T4: FYM þ VAM, T5:
FYM þ VAM þ AZO, T6: FYM þ PSB þ AZO, T7: FYM þ VAM þ PSB, T8: FYM þ VAM þ AZO þ PSB;
FYM: farmyard manure, PSB: P solubilizing bacteria, AZO: Azospirillum, VAM: vasicular arbuscular
mycorrhiza.

Biomass yield
Fresh biomass yield of stevia was increased progressively by irrespective of treatments over
control (Table 4). However, the magnitude of such changes varied with treatments; it was
recorded as the highest (887 g) in the treatment T8 at 6 months of plant growth which may
have been due to the combined application of biofertilizers which caused maximum
fixation of atmospheric nitrogen and increased uptake of soil P and K by the stevia plant.
The percentage response of the microbial inoculant towards the total biomass yield was
recorded as the highest (12.84%) in the treatment where VAM þ PSB þ AZO (T8) was
inoculated altogether, which was closely followed by VAM þ AZO (T5) (9.89%).

Plant height and the number of branches

The results in Tables 5 and 6 show that the plant height of the stevia was recorded highest
(27.30 cm) in the second month of growth while the number of branches was found to be
 Archives of Agronomy and Soil Science 363

Table 3. Effect of modes of biofertilizer applications on the changes in available K content

(mg kg71) in soil (mean of three replications).

Treatments 1st month 2nd month 3rd month 4th month 5th month 6th month
T1 80.40 80.80 81.20 81.90 82.00 81.90
T2 80.10 80.70 81.80 82.40 82.70 82.80
T3 80.60 80.90 82.20 82.90 83.30 83.20
T4 81.30 81.40 82.80 83.20 83.80 83.70
T5 81.70 81.90 83.10 83.80 84.90 84.80
T6 82.20 82.60 83.90 84.70 86.40 86.20
T7 81.90 82.20 84.70 85.90 88.10 87.90
T8 82.80 83.40 85.90 87.30 89.20 89.10
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CD (p ¼ 0.05) 1.24 1.46 2.06 2.12 2.96 2.84

CD: Critical difference; T: Treatments; T1: FYM, T2: FYM þ PSB, T3: FYM þ AZO, T4: FYM þ VAM, T5:
FYM þ VAM þ AZO, T6: FYM þ PSB þ AZO, T7: FYM þ VAM þ PSB, T8: FYM þ VAM þ AZO þ PSB;
FYM: farmyard manure, PSB: P solubilizing bacteria, AZO: Azospirillum, VAM: vasicular arbuscular
mycorrhiza.

Table 4. Effect of biofertilizer inoculation on fresh biomass yield (g) of stevia plant (mean of three
replications).

1st 2nd 3rd 4th 5th 6th Total %

Treatments month month month month month month (kg) response
T1 440 588 678 698 708 742 3.854 ––
T2 488 654 684 702 714 764 4.006 3.94
T3 496 692 698 708 721 774 4.089 6.10
T4 325 541 710 730 769 789 3.864 0.26
T5 516 708 717 731 742 821 4.235 9.89
T6 524 667 690 710 714 839 4.144 7.52
T7 508 648 694 703 744 856 4.153 7.76
T8 436 718 745 758 805 887 4.349 12.84
CD (p ¼ 0.05) 36.88 34.76 11.26 11.29 5.24 42.89 0.196

CD: Critical difference; T: Treatments; T1: FYM, T2: FYM þ PSB, T3: FYM þ AZO, T4: FYM þ VAM, T5:
FYM þ VAM þ AZO, T6: FYM þ PSB þ AZO, T7: FYM þ VAM þ PSB, T8: FYM þ VAM þ AZO þ PSB;
FYM: farmyard manure, PSB: P solubilizing bacteria, AZO: Azospirillum, VAM: vasicular arbuscular
mycorrhiza.

the highest (1612) at the sixth month of growth in the treatment T8 where VAM, PSB and
AZO were applied together. The number of branches progressively increased with the
treatment when compared to the control, whereas the plant height gradually decreased
with the progress of plant growth.

Statistical analysis
The results in Table 7 show that the biomass yield of the stevia plant was significant and
positively correlated with NH4-N (r ¼ 0.81**), NO3-N (r ¼ 0.80**), P content in soil
(r ¼ 0.81**), K content in soil (r ¼ 0.80**) and the number of branches (r ¼ 0.85**).
The results in Tables 8 and 9 show that the amount of NH4-N, NO3-N, P and K
content in the soil and the number of branches could be accounted for individually at
66%, 65%, 67%, 64% and 74% of the variability towards biomass yield of stevia,
 364 K. Das et al.

Table 5. Effect of biofertilizers on the plant height (cm) of stevia (mean of three replications).

Treatments 1st month 2nd month 3rd month 4th month 5th month 6th month
T1 25.66 24.47 22.84 21.23 21.53 21.01
T2 23.40 26.58 21.52 21.66 22.16 21.61
T3 22.03 26.82 21.61 21.62 21.97 21.31
T4 21.18 23.48 22.30 22.10 22.36 21.49
T5 23.02 26.03 20.77 21.76 22.30 21.56
T6 21.22 25.00 22.55 21.70 22.03 21.64
T7 16.53 24.43 22.39 22.06 21.78 21.63
T8 24.95 27.30 22.94 22.08 21.92 21.77
CD (p ¼ 0.05) 2.24 2.56 1.08 NS NS NS
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T1: FYM, T2: FYM þ PSB, T3: FYM þ AZO, T4: FYM þ VAM, T5: FYM þ VAM þ AZO, T6: FYM þ PSB
þ AZO, T7: FYM þ VAM þ PSB, T8: FYM þ VAM þ AZO þ PSB; FYM: farmyard manure, PSB: P
solubilizing bacteria, AZO: Azospirillum, VAM: vasicular arbuscular mycorrhiza.

Table 6. Effect of biofertilizers on the number of branches of (nos.) stevia plant (Mean of three
replications).

Treatments 1st month 2nd month 3rd month 4th month 5th month 6th month
T1 432 930 1278 1306 1412 1478
T2 456 997 1269 1360 1468 1548
T3 389 957 1295 1309 1414 1459
T4 183 940 1257 1348 1454 1560
T5 391 1126 1291 1403 1461 1578
T6 468 1071 1272 1414 1478 1590
T7 389 1002 1302 1406 1469 1568
T8 229 1139 1377 1503 1524 1612
CD (p ¼ 0.05) 21.58 32.89 41.46 43.39 46.25 47.58

T1: FYM, T2: FYM þ PSB, T3: FYM þ AZO, T4: FYM þ VAM, T5: FYM þ VAM þ AZO, T6: FYM þ PSB
þ AZO, T7: FYM þ VAM þ PSB, T8: FYM þ VAM þ AZO þ PSB; FYM: farmyard manure, PSB: P
solubilizing bacteria, AZO: Azospirillum, VAM: vasicular arbuscular mycorrhiza.

Table 7. Correlations among biomass yield, different soil and yield attributing parameters.

Yield Yield NH4-N NO3-N P mean K mean No. of branches

NH4-N 0.81 1
NO3-N 0.80 0.99 1
P mean 0.81 0.97 0.98 1
K mean 0.80 0.95 0.96 0.99 1
No. of branches 0.85 0.70 0.69 0.75 0.74 1

Table 8. Simple regression equations.

Regression equations R2
Biomass yield ¼ 404.51 þ 9.611 NH4-N 0.66
Biomass yield ¼ 413.58 þ 9.457 NO3-N 0.65
Biomass yield ¼ 555.26 þ 13.46 P 0.67
Biomass yield ¼ 7431.61 þ 13.34 K 0.64
Biomass yield ¼ 741.31 þ 1.84 No. of branches 0.74
 Archives of Agronomy and Soil Science 365

Table 9. Multiple regression equations.

Multiple regression equations R2

Biomass yield ¼ 7372.361 7 97.78 NH4-N þ 109.25 NO3-N 7 29.66 P 71.05 K 0.88
þ 2.17 No. of branches

respectively. From the multiple regression equation, it was found that the amount of NH4-
N, NO3-N, P and K content in soil and number of branches together accounted for 88%
of the variability towards producing biomass yield.
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Discussion
The results reveal that the mean content of NH4- N and NON in the soil were found to be
the highest in T8 treatment when FYM þ PSB þ AZO þ VAM were applied simulta-
neously up to three months; this may be due to the positive interaction among the
biofertilizers and soil micronutrients and the decrease in the later period may be explained
by the dilution effect due to higher biomass production. This result also confirms the
findings of Rakshit et al. (2002), who reported that the interaction between rhizobia and
VAM fungi has received considerable attention because of the relatively high phosphorus
demand for nitrogen fixation. The two symbioses typically act synergistically, resulting in
greater nitrogen and phosphorus content in combination than when each is inoculated onto
the legume alone. It is observed that the absolute values of NON content were recorded
lower than that of NH4-N content, which might be attributed to the anaerobic condition
prevailing in the soil due to maintenance of moisture regime to the level of field capacity.
The available P and K content in the soil also followed the same trend as an earlier
result, being higher with the treatment of combined applications of biofertilizers; this was
due to the physico-chemical release of inorganic and organic phosphorus by organic acids
through the action of low molecular weight organic anions such as oxalate which can
replace sorbed phosphorus at metal hydroxide surfaces through ligand exchange reactions
and dissolve metal oxide surfaces that sorbed phosphorus (Fox et al. 1990). The decrease
of P content at the later period of crop growth may have been due to greater uptake by
crops, whereas with the combined application of biofertilizers it suggests a greater role of
N in releasing K into the soil solution. Hence the mean potassium content in soil was
recorded highest in the treatment T8 treatment when FYM þ PSB þ AZO þ VAM were
applied simultaneously.
With the application of 60 kg N ha71, 30 kg P ha71 and 45 kg K ha71, stevia plants
achieved higher dry leaf yield and simultaneously higher nutrient uptake (Chalapathi et al.
1997, 1999). Sood and Kumar (1994) also reported that green and dry foliage yield
increased with increasing levels of N and P, which also confirmed the results obtained in
the present study. The results also showed that the percentage increase in the biomass yield
of stevia was recorded as the highest in the treatment receiving farmyard manure
(FYM) þ PSB þ AZO þ VAM (T8) simultaneously followed by the treatment T5, where
farmyard manure (FYM) þ AZO þ VAM were applied together.

Conclusions
The available N, P and K contents in soils and their total contents in the plant and its
biomass yield have been found to increase over the control by the application of
 366 K. Das et al.

biofertilizers. The results suggest that the combined application of biofertilizers (PSB þ
VAM þ AZO) have been found to further enhance the available N, P and K contents in
soil with the simultaneous increase in growth, yield and nutrition of stevia. Therefore, it
can be concluded from the present investigation that there is a need for the balanced and
integrated approach of fertilizer application including biofertilizers in order to derive
optimum growth and nutrition of the stevia.

Acknowledgements
The authors are grateful to Mr Anantha Murthi Javali of Ankur Nursery, Ripponpet, Shimoga,
India, for providing stevia cuttings and Dr Laxmi Narayan Hegde for providing a field for stevia
cultivation.
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