Vol 2, Issue
3 ,
2014
ISSN: 2321-6832
Original Article
USE OF AZOLLA BIOFERTILIZER
IN POT CULTURE STUDIES WITH PADDY CROP ORYZA SATIVA
MAHALINGAM.P.U, MUNIAPPAN.K*, ARUMUGAM.N AND SENTHIL MURUGAN.G
Department of Biology, Gandhigram Rural Institute – Deemed University, Gandhigram, Dindigul District, Tamil Nadu
Received:
24 July 2014,
Revised and
Accepted: 14 August 2014
ABSTRACT
The pure cultures of Azolla species were mass multiplied and used as potential biofertilizer to study the growth performance of paddy (BPT paddy variety) in pot culture. Pots were prepared with different compositions of Red soil, Vermicompost,
NPK
and Azolla cultures. Healthy seeds of BPT paddy variety were collected from Raj Agro Form, Dindigul
and showed in pots with thirteen treatments and watered regularly. The various growth
parameters such as percentage of Seed germination, Shoot length, Root length, No of leaflets, No of root branches, Wet and Dr y weight of whole
crops of different treatment were
observed periodically for 21 d. The total chlorophyll and carotenoid contents were estimated in all treatments including control. The rapid growth rate of paddy crop was recorded between 15 to 21 d of pot cultures due to the influence of Azolla biofertilizers. This study will attract the farmers to use Azolla biofertilizers for paddy cultivation.
Keywords: Azolla, BPT Paddy,
Oryza sativa, Biofertilizer. Nitrogen
fixation
INTRODUCTION
The quick multiplication rate and rapid decomposing capacity of
Azolla has become an important factor to used as green manure cum biofertilizer in paddy field [1]. This free-floating freshwater fern, fixes atmospheric nitrogen through the symbiotic association
with
Anabaena azollae that lives inside
the
dorsal lobes of Azolla leaves, potentially supplying a substantial amount of N2 to the paddy crop [2]. The
genus of Azolla species is very sensitive to lack of
water in aquatic ecosystems such as stagnant waters, ponds, ditches, canals
or
paddy fields. These areas may be seasonally covered
by
a mat of Azolla
associated
with other
free-floating
crops
species such as
Lemna minor [3]. Application of Azolla not only increases the growth and yield of paddy crops but also improves the soil fertility [4]. The
mass cultivation of Azolla
species is achieved in fresh
water under
strictly controlled conditions. The fresh water system is wildly
available in populated
countries like India; large scale production of Azolla
is feasible
in
tropical conditions in developing countries,
where land
costs and labor are comparatively cheaper. The mass
cultivation of Azolla in
dairy effluent with various combinations of
cow dung, whey water and tap water as low cost medium was reported [5]. Hence, utilization of
waste water from dairy, industry
also recommendable for
mass cultivation of Azolla.
This aquatic fern can be exploited as a potential source of bioferitilizer to increase the
production
of
paddy variety [6]. Hence; Preliminary studies were carried out to evaluate the growth
performance of paddy crop using Azolla biofertilizer.
MATERIALS AND METHOD
Mass multiplication of Azolla species
The large cement tub was filled with 2 Kg
of
fine red soil. Slurry was
made
by mixing one Kg of
cow dung, 15 gm of super phosphate in 5
liters of water. Only live and healthy culture of
half
kg Azolla was inoculated in the prepared tub bed. The optimum temperature 270 C and pH 5.0
to
5.7 is maintained for mass multiplication
[7].
Population estimation of Azolla species
Pure cultures of Azolla
species were procured from
TNAU, Coimbatore. The Population of Azolla was estimated by direct count method. Such as 50
gms wet wt of the culture was taken and spread
over the marked 1 sq. cm area of the clean grid petriplate. Then
it
was observed under 50 lux
white light source. The number of
individual Azolla were counted per grid field and the populations
were estimated by using Conn’s direct observation
method
[8].
Pot culture studies with paddy crop
using Azolla biofertilizer
Pots were prepared
with thirteen different treatment using red soil,
vermicompost NPK
and Azolla cultures. Healthy seeds of BPT paddy variety were collected from Raj Agro Farm dindigul and sowed in pots and watered regularly. The various growth parameters such
as percentage of seed germination,
Shoot length, Root length, No of leaflets, No of branches, Wet and
Dry weight of whole crops of different treatment were observed periodically for 21 d.
Analysis of Seed Germination Percentage of paddy crop
The germination percentage of paddy seed in various
treatments
were calculated using the following Formula [9].
Germination (%) =
Number
of seeds germination / Total number of
seeds X 100
Analysis of Shoot Length and Root Length of the paddy crop
The shoot length
of Azolla species treated paddy crops were measured
in all 30 d treated
crops. The average shoot length is expressed in centimeter for
each treatment and in control. Simultaneously the length of
root was measured from a fixed point
below the surface of
the
soil to the end of
the
root [10].
Analysis of Fresh and Dry Weight of the paddy Crop
The paddy crop from pots of
each treatment were carefully uprooted on 15d & 21d, washed with water and weighted the fresh weight using weighing balance. Followed
by the whole Crops were dried
in hot air oven at 50oC by gradually increasing the temperature up to
120o C for every two hrs for 24hrs. The dry weight of whole crop
was weighed accurately and the results were expressed in grams [11].
Estimation of Chlorophyll and
Carotenoid
Contents
in
Paddy
Crop
The chlorophyll &
carotenoid contents of paddy crop grown in
various
treatments
were
estimated
by standard
method
(Arnon,
1949). One gram of leaf from paddy crop was homogenized
in 20 ml acetone (80%)
and centrifuged at 10,000
rpm
for 5
min. The
contents of total chlorophyll (T-Chl), chlorophyll a
(Chl-a), chlorophyll
b (Chl-b) and carotenoid in
the
supernatant were
determined by spectrophotometrically at OD of 645, 662 and 470
nm. The chlorophyll and
carotenoid contents were calculated using the following
formulae [12]:
Chlorophyll
a (mg/g fw) = 11.75*A662-2.35*A645
Chlorophyll
b (mg/g fw) = 18.61*A645-3.96*A662
Total chlorophyll (mg/g fw) = Chlorophyll a + b
Carotenoids (µmole g
fw)
=1000 X A470 - 2.29*chlorophyll (a) –
81.4* chlorophyll (b) / 227.
Statistical Analysis of Selected Parameters
Statistical analysis
of
the data was carried out by using Prism
Software (PV6). Mean standard deviation
(SD) and percent variation was calculated. 'One Way Analysis of Variance' (ANOVA) was tested
in order to check the statistical significance in
parameters like Root
and Shoot length, No of leaflets, No
of
root branches, Fresh and Dry wt of treated paddy crops. The data was analyzed for three different levels of significance based on the ‘p’ values
RESULT AND DISCUSSION
Azolla is a free floating aquatic fern, which can be widely found
in freshwater
environments in temperate and tropical regions all over the world. It ranks among the fastest growing crops on earth and
due to its association with the nitrogen fixing Cyanobacteria Anabaena azollae, it is independent of external organic nitrogen. In that way Azolla species is not only known to fix
huge amounts of
carbon, but as well
to produce vast amounts of organic nitrogen. The nutrient, which mainly limits the growth
of Azolla, is phosphorous.
Azolla comes under
the
family of Salviniaceae [13].In the present
study the procured strains of Azolla were mass multiplied under controlled condition (FIG 1) without Lemna species contamination. The dead Azolla cultures were removed periodically in order to
enhance
the oxygen
content for a time.
The well grown Azolla cultures have been
treated
with the selected paddy cultivars for 21 days.
Fig. 1: Mass multiplied Azolla Species
Seed germination is the growth of an embryonic crop contained within a seed;
it results in the formation of the seedling, in the
present study the pots were prepared in different combinations of
red soil,
vermicompost, NPK and Azolla.
The
sowed
paddy seeds germination percentage was observed with in 7
to
10 d under controlled conditions.
Approximately 95%
seeds are germinated well compared with control pot (TABLE 1). The growth of shoot length
on 15d & 21d
was
observed as 8.9±1.05 & 16.83±0.351 cm in treatment 12, this drastic growth may be the influence of Azolla. The
graphical
comparison of shoot length on
15th and 21st d given in
(FIG 2)
Fig.
2: Root and shoot length of paddy
cultivars grown
in various treatments on 15d and 21d .
Well development of leaflets in (Treatment 12) and root branches T12 were observed in the Azolla treated paddy crops during the same period of course. The more number of leaflets may enhance the
rate of photosynthesis
which
helps to produce
more number of
carbohydrate productions in the form of reserved food
materials in
seeds due to presence of increased
level of chlorophyll contents [14] The enhanced level of chlorophyll
content in the treated
crops was higher compared to control (FIG 2 and
TABLE
4),
which was
observed in the treatment pots 1, 7 and 12 has high level of
chlorophyll content. The mean
value of
total
chlorophyll content was
expressed in
mg/g in TABLE: 4. the enormous development of
roots
in the crops
plays
a
major
role
in
absorption of minerals
and available
nutrients in the soil under controlled condition [15] (TABLE 1). Comparatively increased level
of wet and dry weight was
observed in 15th and 21st d of paddy cultivars of Azolla treated crops.
(TABLE 3) This indirectly indicates the
enhancement of
Biomass level [16].The statistical
analysis of selected parameter (TABLE 5)
showed statistically significant result due to the impact of Azolla in the present study.
|
|
Treatment
|
Treatment Condition
|
No of seeds Germination
|
% of germination
|
|
T0
|
Sand
+ Red sail
(1:1) -
Control
|
17 1
|
85.00
|
|
T1
|
Sand + Red soil (1:1) +
Recommended dose NPK (100%)
|
14.66
1.52
|
73.33
|
|
T2
|
Sand + Red soil(1:1) +
Vermicompost (100%)
|
17 1
|
85.00
|
|
T3
|
Sand + Red soil (1:1) +
Azolla microphylla (100%)
|
18.33
0.57
|
91.67
|
|
T4
|
Sand + Red soil(1:1) +
Azolla filiculoids
C4 hybrids (100%)
|
17.66
|
88.33
|
|
T5
|
Sand + Red soil (1:1) +
Azolla microphylla
(50%) +
Azolla filiculoids
C4 hybrids (50%)
|
18.66
0.57
|
93.33
|
|
T6
|
Sand + Red soil (1:1) +
|
17 1
|
85.00
|
|
Values are mean of triplicate ± standard error
Table 2: Number of leaf lets and Root branches in paddy crop on 15 d and 21 d
Treatment Treatment Condition
Number of leaf lets Number of root branches
(in cm)
15 d 21 d 15 d 21 d
T0 Sand + Red sail
(1:1) -
Control
1 2 4.66 0.57 5.0 1.0
T1 Sand
+ Red soil (1:1) +
Recommended dose NPK (100%)
T2 Sand
+ Red soil(1:1) + Vermicompost (100%)
T3 Sand + Red soil (1:1) +
Azolla
microphylla (100%) T4 Sand + Red soil(1:1) +
Azolla filiculoids C4 hybrids (100%)
T5 Sand + Red soil (1:1) +
Azolla microphylla
(50%) +
Azolla filiculoids
C4 hybrids (50%)
T6 Sand + Red soil (1:1) +
Azolla
microphylla (50%) + NPK (50%) T7 Sand + Red soil (1:1) +
Azolla filiculoides C4 hybrids (50%) + NPK
(50%)
T8 Sand + Red soil(1:1) +
Azolla filiculoids
C4 hybrids (25%)
+ Azolla microphylla (25%) + NPK (50%)
T9 Sand + Red soil(1:1) +
Azolla microphylla (50%) +
vermicompost
(50%)
T10 Sand+ Red
soil(1:1) +
Azolla
filiculoides
(C4 hybrids (50%) +
vermin compost (50%)
T11 Sand+ Red
soil (1:1) +
Azolla microphylla (25%) +
Azolla filiculoids C4 hybrids (25%) + Vermicompost (50%)
T12 Sand + Red soil (1:1) +
Azolla microphylla (25%)
Azolla filiculoids
C4 hybrids (25%)
+vermicompost
(25% ) + NPK
(25% )
2 2 4.66 1.52 8.0 1.73
2 2 4.0 1.73 6.33 1.52
2 2 5.0 1.0 6.66
0.57
2 2 3.33 0.57 6.0 2.0
3 4 5.0 1.0 6.0
1.0
2 2 3.66 2.08 10.0 1.0
3 4 3.0 1.0 8.66 1.52
2 3 4.33
1.15 7.66 1.52
1.15 7.66 1.52
2 3 5.33
0.57 6.33 0.57
0.57 6.33 0.57
2 3 4.66
1.52 5.33 1.15
1.52 5.33 1.15
2 3 4.66
1.52 6.66 0.57
1.52 6.66 0.57
|
|
Value are mean
of triplicate ± standard error
Table 3: Fresh weight and Dry
weight of whole paddy crop on 15 d and 21 d
Treatment Treatment Condition
Fresh weight in
whole paddy crop(in mg)
Dry weight
in whole paddy crop(in mg)
15 d 21 d 15 d 21 d
T0
Sand + Red sail (1:1) - Control 0.02 0.01 0.022 0.04 0.008 ± 0.002 0.011±0.0017
T1 Sand
+ Red soil (1:1) +
Recommended dose NPK (100%)
T2 Sand + Red soil(1:1) + Vermicompost (100%)
T3
Sand + Red soil (1:1) +
Azolla
microphylla (100%) T4 Sand + Red soil(1:1) +
Azolla filiculoids C4 hybrids (100%)
T5 Sand + Red soil (1:1) +
Azolla microphylla
(50%) +
Azolla filiculoids
C4 hybrids (50%)
T6
Sand + Red soil (1:1) +
Azolla
microphylla (50%) + NPK (50%) T7 Sand + Red soil (1:1) +
Azolla filiculoides C4 hybrids (50%) + NPK
(50%)
T8 Sand + Red soil(1:1) +
Azolla filiculoids
C4 hybrids (25%) +
Azolla
microphylla (25%) + NPK (50%) T9 Sand + Red soil(1:1) +
Azolla microphylla (50%) + vermicompost
(50%)
T10 Sand+ Red
soil(1:1) +
Azolla
filiculoides
(C4 hybrids (50%) +
vermin compost (50%)
T11 Sand+ Red
soil (1:1) +
Azolla microphylla (25%) +
Azolla filiculoids C4 hybrids (25%) + Vermicompost (50%)
0.02 0.04 0.043 0.001 0.0093 ±0.0032 0.027 ±0.002
0.04
0.01 0.0500.031 0.014
±0.0025 0.029 ± 0.021
0.01 0.0500.031 0.014
±0.0025 0.029 ± 0.021
0.03 0.008 0.044 0.012 0.028
±0.005 0.038 ±0.044
0.033 0.006 0.038 0.003 0.008
±0.0028 0.024 ±0.004
0.0330.005
0.0410.0005 0.009 ±0.0046 0.082 ±0.010
0.005
0.0410.0005 0.009 ±0.0046 0.082 ±0.010
0.0450.013 0.0410.014 0.008
±0.0026 0.023 ±0.005
0.013 0.0410.014 0.008
±0.0026 0.023 ±0.005
0.030.02 0.0450.0050 0.009 ±0.0020 0.027 ±0.005
0.02 0.0450.0050 0.009 ±0.0020 0.027 ±0.005
0.030.09 0.0350.008 0.007
±0.0045 0.017 ±0.009
0.09 0.0350.008 0.007
±0.0045 0.017 ±0.009
0.040.04 0.0370.005 0.008
±0.001 0.030 ±0.004
0.04 0.0370.005 0.008
±0.001 0.030 ±0.004
0.040.02 0.0450.005
0.011
±0.015 0.016 ±0.004
0.02 0.0450.005
0.011
±0.015 0.016 ±0.004
|
|
|
|
|
T12
|
Sand
+ Red soil (1:1) +
|
0.03
|
0.007
|
0.051
|
0.008
|
0.006 ±0.0015
|
0.01 ±0.007
|
|
|
Azolla microphylla (25%)
Azolla filiculoids
C4 hybrids (25%)
+vermicompost
(25% ) + NPK
(25% )
|
|
|
|
|
|
|
Value of mean triplicate ± standard error
Table 4: Chlorophyll and Carotenoid contents of paddy crop grown in various
treatmentson 21d
Chlorophyll a
Total
Treatment Treatment condition
(mg/g fw)
Chlorophyll
b
Chlorophyll
(mg/g fw)
Carotinoids
(mg/g fw)
(μ mole g
/fw)|
T0
|
Sand
+ Red sail
(1:1) – Control
Sand + Red soil (1:1) +
|
4.50±0.636
|
2.242±0.253
|
6.74
|
1.68±0.044
|
|
T1
|
Recommended dose NPK (100%) Sand + Red soil(1:1) +
|
10.08±1.732
|
1.210±0.590
|
11.29
|
3.89±0.015
|
|
T2
|
Vermicompost (100%) Sand + Red soil (1:1) +
|
5.06±0.999
|
2.820±1.04
|
7.88
|
1.46±0.04
|
|
T3
|
Azolla microphylla (100%)
Sand
+ Red soil(1:1) +
|
5.17±0.612
|
2.305±0.932
|
7.475
|
1.19±0.125
|
|
T4
|
Azolla filiculoids C4 hybrids (100%) Sand
+ Red soil (1:1) +
|
4.48±0.670
|
2.89±0.60
|
7.37
|
1.55±0.020
|
|
T5
|
Azolla microphylla
(50%) +
|
5.15±1.558
|
2.905±1.254
|
8.05
|
2.18±0.025
|
Azolla filiculoids
C4 hybrids (50%)
Sand
+ Red soil (1:1) +
T6 Azolla microphylla (50%) + NPK (50%)
Sand
+ Red soil (1:1) +
T7 Azolla filiculoides
C4 hybrids (50%) + NPK
(50%)
Sand
+ Red soil(1:1) +
T8 Azolla filiculoids C4 hybrids (25%) +
Azolla microphylla (25%) + NPK (50%) Sand + Red soil(1:1) +
7.57±1.515 2.762±1.172 10.332 2.75±0.043
6.91±2.035 3.265±0.70 10.175 2.93±0.02
8.34±0.57 2.19±1.28 10.53 2.946±0.037
T9 Azolla microphylla (50%) +
vermicompost
(50%)
Sand+ Red soil(1:1) + T10 Azolla
filiculoides
(C4 hybrids (50%) +
vermin compost
(50%) Sand+ Red soil (1:1) +
T11 Azolla microphylla (25%) +
Azolla
filiculoids C4 hybrids (25%) +
Vermicompost (50%)
Sand + Red soil (1:1) + T12 Azolla microphylla (25%)
Azolla filiculoids
C4 hybrids (25%)
+vermicompost
(25% ) + NPK (25% )
7.55±0.023 2.976±0.96 10.526 2.56±0.01
8.63±0.23 1.218±0.245 9.848 3.106±0.032
7.15±0.23 3.601±0.93 10.751 2.033±0.028
8.32±0.576 3.042±0.894 11.362 2.166±0.015
Value of mean triplicate ± standard error
Table 5: Statistical analysis on different growth parameters of paddy cultivars growth in various treatments
|
Parameters
|
15th d treatment
p value
|
Level of significance
With mean
|
21st d treatment
p value
|
Level of significance
With mean
|
|
Root Length in
cm
|
0.0269
|
*4.573 ± 0.4818
|
< 0.0001
|
****6.372 ± 0.3524
|
|
Shoot Length in cm
|
< 0.0001
|
****9.517 ± 0.2485
|
< 0.0001
|
****14.75 ± 0.3582
|
|
No of Leaflets
|
< 0.0001
|
****2.552 ± 0.08535
|
<0.0004
|
***2.083 ± 0.05921
|
|
No of Root branches in cm
|
< 0.0001
|
****7.024 ± 0.4968
|
< 0.0001
|
****7.303 ± 0.4524
|
|
Fresh wt of whole crop in mg
|
< 0.0001
|
****0.0340 ± 0.002026
|
< 0.0001
|
****0.0425 ± 0.001406
|
|
Dry wt of
whole crop in mg
|
0.1653
|
Not significant
|
0.0051
|
**0.02792 ± 0.005442
|
Significant (p = 0.01 to
0.05), ** Very Significant (p = 0.001 to 0.01) and *** Extremely Significant (p < 0.001) ****
strongly significant
(p<0.0001)
CONCLUSION
In the present preliminarily study,
the
procured Azolla species were
mass multiplied in controlled condition and the same was utilized for the growth
of paddy crops for 30 d. During the course of studies
for 30 d
the
fundamental parameters were investigated. It was
observed that, Azolla species started to show their impact from the
15thd and was completely degraded in the prepared tub bed on the
21std. Logarithmic growth rate of paddy crop was observed between
15th to 21st d of treatment. Hence further supplementation is recommended
to
enhance the growth and yield of paddy crops for longer
time
till
harvesting. The
functional role and interaction
between nitrogen fixation and utilization by paddy crop networks per square feet area namely measurement of Azolla
response, grain yield, Crop Nitrogen uptake, excretion of NH3 by Azolla species in free state conditions, rate of reproduction
through sporulation,
preparation of dried inoculum along with long shelf life of different Azolla species are yet to be analyzed
well. An extensive research
work has to be carried out to produce more quintals of paddy yield using organic fertilizers, which will help to increase the economic
status of our rural parts of India.
ACKNOWLEDGMENT
Authors are grateful to TNAU for
providing the various cultures of
Azolla species and
Raj
agro farms – Dindigul, for the supply of BPT
paddy seeds.
AUTHOR’S CONTRIBUTION
MPU, MK and
SMG designed the
experimental setup.
MK, SMG carried out the implementation
of
various methods. MPU, AN
interpreted the results and AN prepared
the
manuscript. All authors
read
and approved the manuscript.
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