International Journal of Applied Research in Natural Products

Vol. 1(1), pp. 20-24, March/April 2008

Available online http://www.healthy-synergies.com
©2008 Healthy Synergies Publications

Original Article

Influence of bio-fertilizers on the availability of nutrients

(N, P and K) in soil in relation to growth and yield of
Stevia rebaudiana grown in South India
Das K1*, Dang R2, Shivananda TN3
1
St. John’s Pharmacy College, R.P.C. Layout, Vijayanagar, Bangalore-40, India. 2Al-Ameen College of
Pharmacy, Hosur Main Road, Bangalore- 27, India. 3IIHR, Medicinal and Aromatic Plant Division,
Hessaraghatta Lake Post, Bangalore-80, India.

Summary: A field experiment was conducted at Ripponpet (Dist. Karnataka), India in an acidic soil (pH 6.10) to study the response
of Stevia plant by the application of bio-fertilizers. The results showed the amount of available nitrogen (N), phosphorus (P) and
potassium (K) contents in soil have been found to increase significantly up to third month and thereafter, the amount of the same
decreased with the progress of the plant growth up to sixth month irrespective of treatments. However, the results further envisaged
that the total yield of fresh biomass has been recorded an increase up to sixth month, being highest in the combined application of bio-
fertilizers over that of their corresponding sole applications. The percent increase of bio-mass yield was recorded highest (22.14%) in
the treatment when all the bio-fertilizers were applied together.

Industrial relevance: The demand of Stevia is increasing widely due to its non caloric nature and usages as sugar supplement. The
leaves are having commercial importance due to presence of di-terpene sweet glycosides which are 300-400 times sweeter than sugar
without any side effects. The plant was domesticated in India in last 20th century from the wide source. So there is a need to set up
certain protocols for cultivation of Stevia in various soil conditions so that farmers can be benefited by selling, and industries also can
get healthy leaves throughout the year for isolate the active components and can formulate economical market products.

Keywords: acid soil, bio-fertilizers, nutrient availability, Stevia, yield

Introduction
Herbals are the oldest form of primary healthcare which had been used by all cultures throughout history. It was an
integral part of the development of modern civilization. Oflate Stevia is a plant which was originated from South America
(Paraguay and Brazil), belongs to the family Compositae (Bertoni, 1905). The Asian markets consume over 85% of the
global supply of the fluffy white crystalline Stevia extracts. The current extract market is 1.5 million kg, processed from
12 million kg of Stevia leaf (Stevia Facts, Web page). Stevioside is one of the active constituents which is available
abundantly in the leaf of Stevia (5-10% of dry weight basis) and is 300- 350 times sweeter than sucrose (Zhang et al.,
1999). Stevia acts as flavor enhancer and in medicinal field it has hypoglycemic, oral contraceptive, cardiovascular,
antimicrobial activity. It is also used to weight loss, digestive and skin problems (Mourey, 1992). Literature survey
revealed active constituents especially stevioside content in Stevia plant is greatly depends on the package of practices for
the cultivation of Stevia and adoption of modern agro- techniques (Nepovim et al., 1998 and Geuns, 2003) and water
management (Fronza and Folegatti, 2003) that has been proved by research trials conducted at New Delhi, India, indicated
attainable yield in the order of 2000 to 3000 kg/ha dry leaf and stevioside content ranged 15-20%, which was double than
that of other parts of the world where Stevia has been already been grown and processed commercially (Barathi, 2002). It
was also stated that cultivation of Stevia will allow a 7-fold increase in production of sugar equivalent (Akatov et al.,
2004). In view of the above fact, cultivation of Stevia is gradually coming into focus in Indian agric-field due to having no
optimum agronomic management practices. Since chemical fertilizers are made up of only few minerals and impede the
uptake of other minerals that results the imbalance the whole minerals pattern of the plants body and biofertilizers can
restore or increase the amount of deficient nutrients and enrich the soil nutrients where deficiency in the soil were
observed. In combination it helps biological nitrogen fixation for the benefit of plants. Along with the biofertilizers farm
yard manures (FYM) are also mixed to the soil to control the deficiency of phosphate content in soil and increase the water
holding capacity of the soil to stimulate the activity of beneficial microorganisms that makes the plant food elements in the
soil readily available to the plants. Not only that the chemical fertilizers are more expensive than biofertilizers and hence
to overcome these problems, the use of biofertilizers is being encouraged. Different pilot study were carried out with

*Corresponding Author:
Tel: +91(0) 9886146982
Fax: +91(0) 8023350035
E-mail: [email protected]
Accepted 11 February 2008

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 Biofertilizer study on Stevia

chemical and biofertilizers in green house for Stevia cultivation and seen higher yield obtained by applied biofertilizers
which would also reflected the simultaneous higher stevioside content in Stevia plant (Das, 2004). Application of
biofertilizers and their effects on Stevia plant in terms of yield and yield attributes in Green house conditioned were
reported (Das et al., 2007) but the same trial was not carried out under the field conditions to investigate the effects of
biofertilizers on the nutrient contents in soil and plants which further related to the biomass production of Stevia and
hence, the present investigation was undertaken.

Materials and Methods

Cutting of Stevia plants, collected from Ankur Nursery, Ripponpet (Shimoga, Karnataka), India, were used as a test
plant for the present study. A field experiment was conducted in the month of November 2005 at the Ripponpet, Shimoga
on acidic soil reaction. Before start of the experiment, initial soil pH, org carbon, Cationic Exchange Capacity (CEC),
available N, P and K were determined separately. Soil pH were determined by pH meter (Elico, India) by preparing
solution with ration of soil: water, 1: 2.5 w/v, organic carbon by wet digestion with mixing of potassium dichromate, CEC
by Kjeldahl method after extracting the soil with ammonium acetate. After extracting the soil samples, available N was
determined with the help of 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) (Piper, 1966 and
Jackson 1973). The relevant physicochemical properties of soils were: pH 6.10; organic carbon, 0.54%; CEC, 9.89 Cmol
(p+)/kg, available N, 55.89 mg/kg, available phosphorus in form of phosphorus pentaoxide (P2O5), 6.8 mg/kg, available K,
86 mg/kg, Diethyl triamine penta acetic acid (DTPA) - extractable Zinc, Copper, iron and Manganese were estimated of
0.54 mg/kg, 0.15 mg/kg, 5.2 mg/kg and 3.4 mg/kg respectively. Fields were divided into beds according to treatments. The
following treatments were used.
T1-- Control, only FYM @ 10 kg, without application of bio-fertilizers. T2 – soil application of FYM @ 10 kg mixed
with 250 g of Phosphorus Solubilizing Bacteria (PSB) (Bacillus megatheriam). T3 – soil application of FYM @ 10 kg
mixed with 250 g of Azospirillum (AZO). T4 – soil application of FYM @ 10 kg mixed with 500 g of Vesicular
Arbuscular Mycorrhiza (VAM). T5 – soil application of FYM @ 10 kg mixed with 250 g of PSB and 250 g of AZO. T6 –
soil application of FYM @ 10 kg mixed with 250 g of PSB and 500 g of VAM. T7 – soil application of FYM @ 10 kg
mixed with 250 g of AZO and 500 g of VAM. T8 – soil application of FYM @ 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 X 3) beds, with each bed size of 1 M X 1.25 M. Cutting of Stevia plants were planted at the same time in
all the beds. Each bed contains 6 rows and 6 columns that contain 36 plants (6X6), with total of 864 plants (36 X 24). The
moisture content was maintained to 60 % of water holding capacity (WHC) by irrigating three times in a week. The
periodic collection of soil samples were made and analysed for pH, available N, P and K in soil by following the method
described by Jackson (1973). Besides, different yield attributes and yields were also recorded periodically.

Results and Discussion

Changes in Ammoniated nitrogen (NH4-N) content in soil:

The results (Table-1a) shown that the content of NH4-N was found to be gradually increased with the progress of
crop growth irrespective of treatments and reaches a maximum within 3 months of crop growth and thereafter, the
amount decreased up to 6 months. The magnitude of such increase was recorded at the initial stage of the crop and
decrease at the later period. The highest increase (33.37 mg/kg) was found at 3 months of crop growth in the treatment
where recommended levels of combined bio-fertilizers were applied.
Changes in Nitrate-nitrogen (NO3-N) content in soil:
The result (Table-1b) for the changes in the NO3-N content in soil showed a similar trend to that of NH4-N content.
The mean content of NO3 –N in soil was also recorded highest (32.28 mg/kg) in T8 treatment when FYM + PSB +AZO +
VAM were applied simultaneously which was closely followed by the combined treatment with FYM + AZO + VAM.
Changes of available P content in soil:
The results (Table-2) showed that the available P content initially increased and thereafter, the amount of the same
gradually decreased with the progress of crop growth. However, the magnitude of such changes varied with treatments.
The highest available P content (12.23 mg/kg) was recorded at 3 months of crop growth in the treatment where
recommended level of combined bio-fertilizers was applied.
Changes of available K content in soil:
The result (Table-3) showed that the periodic changes in available K content varied with treatments, being highest
(90.79 mg/kg) in the treatment where bio-fertilizers were applied simultaneously, followed by the treatment (88.46
mg/kg) where combination of PSB and VAM were applied in the plot.
Biomass yield:
The results (Table-4) show that the amount of fresh biomass yield has been found to increase progressively
irrespective of treatments over control. However the magnitude of such changes varied with treatments, being recorded
highest (928 g) in the treatment T8 at 6 months of plant growth which might be due to combined application of bio-
fertilizers which caused maximum fixation of atmospheric nitrogen, increased uptake of soil P and K by the Stevia plant.
The percent response of microbial inoculant towards the total biomass yield was recorded highest (22.14%) in the
treatment where VAM+PSB+AZO was inoculated altogether which was closely followed by PSB+AZO (18.61%) and
VAM+AZO (17.12%).

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 Das et al

Table-1(a). Effect of modes of bio-fertilizer applications on the changes in NH4-N content (mg/kg) in soil (Mean of 3 replications).

Treatments 1st month 2nd month 3rd month 4th month 5th month 6th month
T1 27.85 27.42 30.94 30.48 28.48 27.12
T2 27.79 27.64 31.33 31.03 28.76 27.92
T3 28.01 28.07 30.52 30.37 27.63 27.11
T4 28.11 28.03 30.37 29.71 27.03 26.46
T5 28.70 28.94 31.94 30.47 27.93 27.82
T6 29.07 28.83 31.99 30.42 29.24 28.11
T7 28.80 29.02 30.43 30.38 27.59 27.17
T8 30.87 31.03 33.37 32.87 29.17 28.81
CD (P=0.05) 1.22 1.68 1.89 2.12 1.34 0.72

CD= Critical difference; T= Treatments

Table-1(b). Effect of modes of bio-fertilizer applications on the changes in N03N content (mg/kg) in soil (Mean of 3 replications)

Treatments 1st month 2nd month 3rd month 4th month 5th month 6th month
T1 26.05 26.31 30.64 30.40 26.72 24.98
T2 26.01 26.23 30.65 30.07 27.34 25.70
T3 27.16 26.46 60.46 29.40 27.17 25.35
T4 27.09 27.27 29.39 29.27 26.45 25.74
T5 28.07 27.49 30.44 29.81 25.85 24.58
T6 27.60 28.07 30.23 29.88 26.64 25.87
T7 27.97 28.06 30.46 29.28 27.13 26.93
T8 28.63 30.47 32.28 30.51 28.63 26.95
CD (P=0.05) 0.82 1.74 1.88 0.48 1.33 0.86

CD= Critical difference; T= Treatments

Table-2. Effect of modes of bio-fertilizer applications on the changes in available P content (mg/kg) in soil (Mean of 3 replications).

Treatments 1st month 2nd month 3rd month 4th month 5th month 6th month
T1 5.80 5.97 8.50 8.30 7.60 7.20
T2 6.43 7.07 8.06 7.90 7.80 7.60
T3 5.90 5.93 6.60 6.20 5.98 6.10
T4 7.63 7.65 8.18 8.00 7.38 7.00
T5 8.90 9.22 10.99 9.86 8.86 8.12
T6 8.43 8.60 9.85 8.87 7.98 7.28
T7 9.00 9.33 10.74 10.10 9.10 8.84
T8 10.17 11.03 12.23 11.69 9.89 9.46
CD (P=0.05) 1.16 1.88 1.74 2.10 1.38 1.79

CD= Critical difference; T= Treatments

Table-3. Effect of modes of bio-fertilizer applications on the changes in available K content (mg/kg) in soil (Mean of 3 replications)

Treatments 1st month 2nd month 3rd month 4th month 5th month 6th month
T1 80.27 80.47 82.77 81.79 77.86 72.64
T2 79.03 79.57 83.06 82.33 78.10 73.19
T3 82.23 82.90 84.35 83.66 79.20 74.50
T4 82.60 83.67 84.67 83.60 79.10 74.38
T5 85.40 86.90 88.06 84.92 80.45 75.20
T6 84.03 84.90 85.88 83.69 79.66 74.88
T7 85.60 87.27 88.46 85.20 80.48 75.38
T8 87.57 88.93 90.79 87.40 83.44 78.92
CD (P=0.05) 2.32 2.79 2.98 2.48 3.12 3.59

CD= Critical difference; T= Treatments

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Table-4. Effect of bio-fertilizer inoculation on fresh biomass yield (g) of Stevia plant (Mean of 3 replications).

Treatments 1st month 2nd month 3rd month 4th month 5th month 6th Total %
month (g) response
T1 400 560 648 694 720 740 3.762 ----
T2 460 610 720 756 798 810 4.154 10.42
T3 470 670 750 750 765 783 4.188 11.32
T4 380 612 690 798 822 859 4.161 10.61
T5 473 720 765 790 822 836 4.406 17.12
T6 495 690 770 816 836 855 4.462 18.61
T7 468 620 780 800 828 848 4.344 15.47
T8 350 735 815 865 902 928 4.595 22.14
CD (P=0.05) 54.78 39.45 37.32 41.68 59.44 65.27

CD= Critical difference; T= Treatments

Table- 5. Co-efficient of Correlation between bio-mass yield and nutrient contents in soil

Biomass yield NH4-N NO3-N P K

Biomass
yield 1
NH4-N 0.65* 1
NO3-N 0.51 0.30 1
P 0.58* 0.59* 0.62* 1
K 0.83** 0.61* 0.63* 0.71* 1

* = Significant at 5%
** = significant at 1%

Table- 6. Regression equations between bio-mass yield and nutrient contents in soil

R2
Biomass yield = -1.93 + 0.22 NH4-N 0.65
Biomass yield = 0.25 + 0.16 NO3-N 0.26
Biomass yield = 3.20 + 0.14 P 0.33
Biomass yield = -4.19 + 0.11 K 0.70

Discussion
The results reveal that the mean content of NH4- N and NO3-N in soil were found highest in T8 treatment when FYM
+ PSB +AZO + VAM were applied simultaneously up to three months, this may be due to positive interaction among the
bio-fertilizers and soil micronutrients and decrease at the later period may be explained by the dilution effect due to
higher biomass production. This result also confirm to the findings of Rakshit et al, 2002, in which they reported that the
interaction between rhizobia and VAM fungi has received considerable attention because of the relatively high
phosphorus demand of nitrogen fixation. The two symbioses typically act synergistically, resulting in greater nitrogen
and phosphorus content in combination than when each is inoculated on to the legume alone. It is observed that the
absolute values of NO3-N 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 soil also followed the same trend as earlier result, being higher with the treatment of
combined applications of biofertilizers, this is due to 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 phosphorus
sorbed at metal hydroxide surfaces through ligand exchange reactions and dissolve metal oxide surfaces that sorb
phosphorus (Fox et al., 1990). The decrease of P content at the later period of crop growth might be due to greater
uptake by crops. Where as with the combined application of bio-fertilizers suggest 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 + PSB + AZO + VAM were applied simultaneously.
In case of simultaneous increase biomass yield, it was reported with the application of N, P and K at 60, 30 and 45
kg/ha, respectively, produced higher dry leaf yield with the simultaneous higher nutrient uptake by Stevia plant
(Chalapathi et al., 1997 and 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 show that
the percent increase in the bio-mass yield of Stevia was recorded highest in the treatment receiving FYM + PSB + AZO
+ VAM simultaneously followed by the treatment T6 where FYM + PSB +VAM were applied together. The bio-mass
yield of Stevia (Table- 5) has been found to be significant positively correlated with available NH4-N (r = 0.65* ) , P (r
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 Das et al

=0.58* ) and K ( r = 0.83** ) contents in soil while non-significant positive correlation with NO3-N (r = 0.51 ) contents
in soil. From the regression equation (Table 6) it was observed that the amount of NH4-N, NO3-N , P and K contents in
soil accounted for 65 , 26, 33 and 70 % of the variability towards contributing the bio-mass yield of Stevia respectively.

Conclusion
Although the sole application of bio-fertilizers have been found to be increased the available N, P and K contents in
soils and their total contents in plant and biomass yield of plants over the control. The results suggested that the
combined applications of biofertilizers (PSB+VAM+AZO) has been found to be further increased the same significantly
over sole application suggesting a necessity for the balanced bio-fertilizer application in order to derive optimum
growth and nutrition of the Stevia plant.

Acknowledgements
The authors are grateful to the Mr. Anantha Murthi Javali, Ankur Nursry, Ripponpet, Shimoga, India, for providing
Stevia cuttings and field provided for Stevia cultivation.

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