Int. J. Pharm. Sci. Rev. Res.

, 20(2), May – Jun 2013; n° 35, 190-195 ISSN 0976 – 044X

Research Article

Individual and Combined Effect of Biofertilizer, Chemical Fertilizer and Vermicompost

on Amaranthus Tristis
Kavitha*, Sivagami Srinivasan, Ranjini
Avinashilingam Institute for Home Science and Higher Education for women, Coimbatore – 43, Tamilnadu, India.
*Corresponding author’s E-mail: kavitharaj111@gmail.com

Accepted on: 28-03-2013; Finalized on: 31-05-2013.

ABSTRACT
Fertilizer is a very essential input in crop production. Biofertilization technology has taken a part to minimize production costs and at
the same time avoid the environmental hazards. Organic agriculture is one of the ways that can produce high quality crops and
increase yield. Hence this study is to investigate that how different combinations of biofertilizer (Azospirillum), chemical fertilizers
and vermicompost would affect the growth of a green leafy vegetable, Amaranthus tristis. After the growth period of about 40 days
the biometric and biochemical analysis was done and it shows that there is significant change in biometric parameters and increase
in the biochemical constituents.
Keywords: Azospirillum, Amaranthus tristis, biofertilizer, chemical fertilizers, vermicompost.

INTRODUCTION human health and creating an eco-friendly, sustainable

and economical bio-system models.

I ndia is the second-largest producer and consumer of N

fertilizer in the world, after China, with close to a 15
per cent share of the global total 1. Fertilizer use has
been and will continue to be a major factor in the
increasing agricultural production and productivity.
Amaranth is widely cultivated in various regions of the
world as well as in India as a food and leafy vegetable.
The plant chosen for the study is Amaranthus tristis,
which is commonly known as “araikeerai” in Tamil. It is
Fertilization increases root growth, drought tolerance and used to treat stomach flu, diarrhea, gastroenteritis, stop
resistant to frost. It helps plants to resist attack of pests excessive menstruation and contraception. When applied
and diseases and stalk lodging. The application of externally, it can reduce tissue swelling from sprains and
chemical fertilizers over a period has resulted in poor soil tick bites. It is gluten-free and hence is a good alternative
health, reduction in produce and increase in incidences of to wheat. Amaranth can reduce total cholesterol and LDL
pest and disease and environmental pollution2. and can increase HDL.
Continuous imbalanced fertilization causes depletion in
This study is conducted by growing the Amaranthus tristis
soil fertility, drop in crop yields, poor crop quality and
plants in various treatments which include the individual
diminishing profits.
and combined applications of biofertilizer (Azospirillum),
Organic wastes and biofertilizers are the alternate chemical fertilizers and vermicompost, and finding the
sources to meet the nutrient requirement of crops and to changes in its biometric and biochemical parameters.
bridge the future gaps3. Biofertilizer, organic manuring Hence the main purpose of the study is to find that which
and bio-control have emerged as a promising component of the treatments would help in increasing the yield and
of integrating nutrient supply system in agriculture. nutritional content of the plant.
Microbiological fertilizers are important to environment
MATERIALS AND METHODS
friendly sustainable agricultural practices4. They can be
conveniently produced and partially substitute the The Biofertilizer (Azospirillum) and chemical fertilizer
chemical fertilizers to avoid environmental pollution. (urea and potash) were collected from Tamilnadu
Azospirillum spp. contribute to increased yields of Agricultural University, Coimbatore. The vermicompost
cereal and forage grasses by improving root and seeds of Amaranthus tristis were collected from
development in properly colonized roots, increasing Super seeds and nursery, Coimbatore. A pot culture study
the rate of water and mineral uptake from the soil and was laid out in completely randomized design with four
by biological nitrogen fixation. It is one of the important replications for each treatment. Each pot was uniformly
biofertilizer, which is found to fix nitrogen in association filled with 8 kg of soil and treated with different
with world’s most staple food crops like rice, maize, combinations of Azospirillum (10g per pot), chemical
5
sorghum, wheat and millets . fertilizer (0.2g urea and 0.08g potash per pot) and
vermicompost (0.6kg per pot). The treatments were as
Therefore organic farming helps to provide many
follows;
advantages such as; eliminate the use of chemicals in
the form of fertilizers/pesticides, recycle and regenerate T1- Control,
waste into wealth; improve soil, plant, animal and
T2- Chemical fertilizer,

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Int. J. Pharm. Sci. Rev. Res., 20(2), May – Jun 2013; n° 35, 190-195 ISSN 0976 – 044X
7
T3- Azospirillum, Carotenoid estimation
T4- Vermicompost, 5g of the sample was weighed and saponified for about
30 minutes in a shaking water bath at 37°C after
T5- Azospirillum + Vermicompost,
extracting the sample in 12% alcoholic KOH. The
T6- Azospirillum + Chemical fertilizer, saponified extract was transferred into a separating
funnel packed with glass wool and calcium carbonate
T7- Chemical fertilizer + Vermicompost,
containing 10 to 15 ml of petroleum ether layer. The
T8- Chemical fertilizer + Azospirillum + Vermicompost. lower aqueous phase was transferred to another
separating funnel, and the petroleum ether extract
About 50 seeds were sown in each pot and allowed to
containing the cartenoids pigments to amber colored
germinate. They were then thinned to 15 plants per pot.
bottle. The extraction of the aqueous phase was
The pots were watered daily and holes were made at the
repeated. A small quantity of anhydrous sodium sulphate
base of the pot to prevent water logging condition. The
was added to the petroleum ether extract to remove the
plants were uprooted on the 40th day without any
turbidity. The final volume of the petroleum ether extract
damage. The adhering soil particles were removed by
was noted and diluted by a known dilution factor. The
washing gently with water and the water droplets were
absorbance of the extracts at 450nm and 503 nm was
removed by blotting with the filter paper. Then these
noted in spectrophotometer.
plants were used for the biometric observation and
biochemical analysis.
Biometric Observations
Where,
The fresh plants were used to determine the fresh
P – Optical density of the sample
weight, shoot length (measured from the point of first
cotyledonary node to the tip of the longest leaves), root V – Volume of the sample
length (measured from the crown region of the plant to
W – Weight of the sample
the tip of the root) and number of leaves. For dry weight,
the plants were dried at 70°C overnight and weighed. Estimation of Total carbohydrates 8
Biochemical Analysis 100mg of the sample was weighed into a boiling tube,
6 was hydrolysed in a boiling water bath for 3 hours with
Chlorophyll estimation
5ml of 2.5N hydrochloric acid and cooled to room
1g of the sample of leaf tissue was weighed and ground temperature. This was neutralized with solid sodium
into a fine pulp with the addition of 20 ml of 80% carbonate until the effervescence ceases. The volume was
acetone. The supernatant was centrifuged (5000 rpm for made up to 100ml and centrifuged and the supernatant
5 minutes) and transferred to 100 ml volumetric flask. was collected. 0.5 to 1.0ml aliquots were used for
The residue was ground with 20 ml of 80% acetone; the analysis. The volume was made up to 1 ml in all tubes by
supernatant was centrifuged and transferred to the same adding distilled water. 4 ml of anthrone reagent was then
volumetric flask. This procedure was repeated until the added and heated for 8 minutes in a boiling water bath.
residue turned colorless. The mortar and pestle were Tubes were then cooled rapidly and read the absorbance
washed thoroughly with 80% acetone and the clear at 630 nm.
washings were collected in the volumetric flask. The
Protein estimation9
volume was made up to 100 ml with 80% acetone. The
absorbance value of the solution was read at 645 and 663 500mg of the sample was ground well with a pestle and
nm against the solvent (80% acetone) blank. mortar in 5-10ml of the phosphate buffer, centrifuged
and 0.1ml of the supernatant was used for the protein
The amount of chlorophyll present in the extract as mg
estimation.
chlorophyll per g tissue was calculated using the following
equations. The volume was made up to 1ml in all the test tubes. A
tube with 1ml of water served as a blank. 5ml of reagent
C was added to all the test tubes including the blank,
mixed well and allowed to stand for 10minutes. 0.5ml of
reagent D was then added, mixed well and was incubated
at room temperature in the dark for 30minutes. The blue
colour developed was read in a spectrophotometer at
Where, 660nm. A standard graph was drawn and the amount of
protein in the sample was calculated.
A – Absorbance at specific wavelengths,
Where, Reagent A is 2% sodium carbonate in 0.1N sodium
V – Final volume of chlorophyll extract in 80% acetone hydroxide.
W – Fresh weight of tissue extract

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Int. J. Pharm. Sci. Rev. Res., 20(2), May – Jun 2013; n° 35, 190-195 ISSN 0976 – 044X

Reagent B is 0.5% copper sulphate (CuSo4.5H2O) in 1% Statistical analysis like arithmetic mean and variance was
potassium sodium tartarate. employed to predict the results of the experiment.
Reagent C is alkaline copper sulphate solution: 50ml of RESULTS AND DISCUSSION
reagent A was mixed with 1ml of reagent B, prior to use.
Biometric parameters
Reagent D is Folin-Ciocalteau Reagent
Root length: The application of vermicompost alone (T4)
Vitamin A estimation10 or in combination with Azospirillum and chemical
fertilizer (T8) highly influenced the root length of
2µl of the sample in duplicates was used for this
Amaranthus tristis plants. This was followed by the
estimation. The volume in all the tubes was made upto
treatment T5 (Azospirillum + vermicompost). The rest of
0.1 ml with chloroform. Using a fast delivery pipette, 2 ml
the treatments recorded insignificant root length when
of TCA reagent was added rapidly mixing the contents of
compared to the control (shown in table 1). A study by
the tube. The absorbance was recorded immediately at
Islam et al showed similar results, where the root length
620nm in a spectrophotometer. A standard graph was
of the rice plants increased significantly on the combined
constructed and the concentration of vitamin A was
application of Azospirillum and chemical fertilizers13.
calculated.
Shoot length: All the treatments except T2 (chemical
Vitamin C estimation11
fertilizer) and T3 (Azospirillum) significantly (p<0.05)
1g of the sample was homogenized in 10ml of 4% TCA enhanced the shoot length. Among the treatments, T8
and centrifuged (2000rpm for 10 minutes). To the with the tripartite application of Azospirillum, chemical
supernatant obtained, a pinch of activated charcoal was fertilizer and vermicompost was found to be superior in
added, shaken well and kept for 10 minutes. This was registering the maximum shoot length, which was
centrifuged once again and the charcoal residue was followed by T7 (chemical fertilizer + vermicompost), T5
removed. The volume of the clear supernatants was then (Azospirillum + vermicompost) and T4 (vermicompost)
noted. 0.5 and 1.0 ml aliquots of this supernatant were respectively. The shoot length of the treatment, T6
taken for the assay. The assay volumes were made up (Azospirillum + chemical fertilizer) was on par with the
2.0ml with 4%TCA. 0.5ml of DNPH (2, 4-dinitrophenyl control. Thus the application of vermicompost along with
hydrazine) reagent was added to all the test tubes. 2 Azospirillum and chemical fertilizer has highly influenced
drops of 10% thiourea solution was then added and the growth of the shoot of Amaranthus tristis plants.
incubated at 37°C for 3 hours. The osazones formed were Figure 1 shows the appearance of plants as on 40th day
dissolved in 2.5ml of 85% sulpuric acid, in cold, drop by after sowing. The combined application of vermicompost
drop, with no appreciable rise in temperature. DNPH with chemical fertilizer recorded significantly higher vine
reagent and thiourea solution after the addition of H2SO4 length in Pisum sativum14.
served as the blank. The tubes were incubated for 30
Fresh and dry weights: It is clear from the table 1 that the
minutes at room temperature, and the absorbance was
fresh and dry weights of the treatment T8 (Azospirillum +
read spectrophtometrically at 540nm. The content of
chemical fertilizer + vermicompost) was found to be
ascorbic acid in the sample was calculated using the
maximum, which was followed by T4 (vermicompost), T5
standard graph.
(Azospirillum + vermicompost) and T7 (chemical fertilizer
Phosphorus estimation12 + vermicompost) respectively. Thus the application of
vermicompost along with Azospirillum and chemical
The ash of 2g of vegetable sample was dissolved in 2 to 3
fertilizer influenced the fresh and dry weights of the
drops of concentrated hydrochloric acid and the volume
Amaranthus tristis plants. A study by Zarei et al had also
was made up to 25 ml with distilled water. 0.1ml of the
shown that the inoculation of Azospirillum increased fresh
ash solution was taken, made up the volume to 4.7 ml 15
and dry weights of soybean . The dry weight of sweet
with water and added 0.5 ml of molybdate I solution and
potato increased with the combined application of
0.2 ml of ANSA (amino naphthol sulphonic acid). The color
organic and inorganic fertilizers was also reported by
developed was read after 20 minutes in a colorimeter
Saikia et al16.
using red filter against a reagent blank.
Number of leaves and moisture content: The maximum
Iron estimation
number of leaves was registered in T8 (Azospirillum +
Iron estimation was carried out using wong’s method. chemical fertilizer + vermicompost) which was followed
2ml of the ash solution (sample) was taken and the by T7 (chemical fertilizer + vermicompost) and T5
volume was made up to 6.5 ml with distilled water. A (Azospirillum + vermicompost) respectively. The
blank using 6.5 ml of distilled water was used. 1ml of 30% treatments T5 (Azospirillum + vermicompost) and T7
H2SO4, 1ml of 7% Potassium per sulphate and 1.5 ml of 40 (chemical fertilizer + vermicompost) registered a
% Potassium thiocyanate solution were added. The red comparable number of leaves. The rest of the treatments
colour developed was measured within 20 min at 540nm did not significantly influence the number of leaves of
in a spectrophotometer. The concentration of iron in the Amaranthus tristis plants. Similar results were reported
sample solution was calculated using standard graph. by Jala-abadi et al, where the application of organic

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manure and biofertilizer increased the grain yield and Amaranthus tristis plants. The rest of the treatments
leaves in wheat17. recorded comparable moisture content. Yadav et al.,
reported that the application of biofertilizers increased
The application of vermicompost alone (T4) or in 18
the moisture content in wheat crops .
combination with Azospirillum (T5) and chemical fertilizer
(T7 & T8) highly influenced the moisture content of the

Table 1: Biometric observations in Amaranthus tristis on the 40th day of growth

Root length Shoot length Fresh weight Dry weight Number of leaves Moisture
Treatments
(cm) (cm) (g) (g) (Number/plant) content (g)
T1 5.33 7.43 0.33 0.04 16.07 0.29
T2 4.93 6.93 0.37 0.03 7.06 0.33
T3 4.98 6.21 0.42 0.05 15.03 0.37
T4 8.08 11.45 1.75 0.19 24.01 1.56
T5 7.05 12.43 1.53 0.18 26.01 1.34
T6 3.43 7.42 0.36 0.06 16.04 0.30
T7 5.98 12.84 1.73 0.16 26.17 1.54
T8 8.25 13.13 1.76 0.25 30.02 1.51
CD(0.05) 0.009 0.095 0.009 0.031 0.154 0.006

Figure 1: Amaranthus tristis plants after uprooting, on the 40th day of growth.

Table 2: Biochemical parameters in Amaranthus tristis at the 40th day of growth

Total
Chlorophyll Carotenoids Protein Vitamin A Vitamin C Phosphorus Iron
Treatments carbohydrates
(mg/g) (mg/g) (mg/g) (mg/g) (mg/g) (mg/g) (mg/g)
(mg/g)
T1 2.18 0.84 29.10 28.03 1.31 112.10 172.10 1.42
T2 2.50 1.05 36.10 36.03 1.45 144.07 252.03 2.21
T3 1.70 1.06 32.00 34.00 1.55 149.10 214.13 2.41
T4 2.20 1.34 46.10 42.03 1.70 160.10 265.23 2.67
T5 2.80 0.79 52.23 43.03 1.90 154.10 283.26 3.01
T6 2.12 0.74 39.23 33.03 1.40 118.03 153.30 1.31
T7 1.80 1.88 50.73 45.00 2.20 170.10 248.90 2.85
T8 1.81 1.54 61.17 48.03 2.80 191.07 294.10 3.85
CD(0.05) 0.015 0.005 0.283 0.173 0.017 0.172 0.240 0.008

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Biochemical constituents Iron: The combined application of Azospirillum, chemical

fertilizer and vermicompost was found to be superior in
Chlorophyll: It is clear from table 2 that the chlorophyll
registering the maximum iron content, which was
content of the treatment T5 (Azospirillum +
followed by the treatment T5 (Azospirillum +
vermicompost) was found to be maximum when
vermicompost). The study by Densilin et al also revealed
compared to the other treatments. This was followed by
that the combined application of biofertilizer, inorganic
the treatments T2 (chemical fertilizer), T4 (vermicompost)
fertilizers and vermicompost increased the biochemical
and T6 (Azospirillum + chemical fertilizer) respectively.
constituents in chilli fruits21.
Thus the application of Azospirillum and vermicompost
highly influenced the chlorophyll content of the CONCLUSION
Amaranthus tristis plants, which in turn reflects the
Hence the conclusion of the study is that the combined
growth. The application of biofertilizers in combination
application of Azospirillum, chemical fertilizer and
with other fertilizers greatly influenced the amount of
19 vermicompost (T8) improved the biometric parameters
chlorophyll in dill (Anethum gragraveoles) leaves .
(shoot length, fresh and dry weight and number of
Carotenoids: Among the various treatments, T7 (chemical leaves). Also it increased the protein, total carbohydrates,
fertilizer + vermicompost) recorded the highest amount vitamin A, vitamin C, phosphorus and iron content of the
of carotenoids, which was followed by T8 (Azospirillum + Amaranthus tristis plants. The application of
chemical fertilizer + vermicompost) and T4 vermicompost (T4) increased the root length and the
(vermicompost) respectively. moisture content of the Amaranthus tristis. The dual
application of Azospirillum and vermicompost (T5)
Protein: The protein content of all the treatments was
enhanced the chlorophyll content, whereas the
found to be significantly higher when compared to the
application of chemical fertilizer and vermicompost (T7)
control. Among the treatments T8 (Azospirillum +
improved the carotenoids content.
chemical fertilizer + vermicompost) recorded the highest
protein content, which was followed by T5 (Azospirillum + The study clearly indicates that the combined application
vermicompost) and T7 (chemical fertilizer + of vermicompost, Azospirillum and chemical fertilizer is
vermicompost) respectively. Thus the application of superior in enhancing the growth and development of the
vermicompost along with the chemical fertilizer and green leafy vegetable, Amaranthus tristis. Thus the
Azospirillum highly influenced the protein content of increased growth and nutrient content of the Amaranthus
Amaranthus tristis. tritsis plants was proved by the application of
biofertilizers along with chemical fertilizers and
Total carbohydrates: The level of total carbohydrate
vermicompost. Hence it is recommended that the use of
content of the Amaranthus tristis was highly enhanced by
biofertilizer and vermicompost along with the chemical
the application of Azospirillum, chemical fertilizer and
fertilizer would be beneficial to the environment as it
vermicompost (T8). This was followed by the application
would reduce the use of inorganic fertilizers and promote
of vermicompost along with the chemical fertilizer (T7)
sustainable agriculture.
and Azospirillum (T5) respectively. These findings are
supported by the study led by Zaller et al, who had found Acknowledgement: We are extremely grateful to our
that the application of vermicompost highly enhanced the dean, Dr. R. Parvatham (Faculty of Science, Professor and
carbohydrate content in tomato plants20. Head, Department of Biochemistry, Biotechnology and
Bioinformatics, Avinashilingam Deemed University for
Vitamin A: As depicted in Table 2, the vitamin A level was
Women, Coimbatore) for the help rendered for the
found to be higher in the treatment T8 (Azospirillum +
completion of this study.
chemical fertilizer + vermicompost) which was followed
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Source of Support: Nil, Conflict of Interest: None.

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