Azolla as a biofertilizer

1. INTRODUCTION:

One of the paradoxes in nature is the abundance of nitrogen in the atmosphere and its relative
non-availability to plants and animals. Organized crop production has, therefore, to depend
largely on synthetic nitrogen fertilizers the production of which is based on non-renewable,
fossil fuel resources, the escalating oil prices, the widening gap between supply and demand for
this nutrient and the prevailing low purchasing of alternate renewable organic resources to meet
at least a part of the nitrogen demand of crops. Any saving in the consumption of chemical
nitrogen without affecting the crop productivity will, therefore, be not only an economic
advantage but also a strategic necessity, the present day global interest in biological nitrogen
fixation a direct consequence of this necessity to provide some economic assistance to the small
and marginal farmers and to introduce a judicious combination of linear and cyclic fertilization
of soils (Venkataraman,1967).For this, we put more emphasis on biofertilizers, the term
“Biofertilizers”or which can be more appropriately called “Microbial inoculants” can be
generally defined as preparations containing live or latent cells of efficient strains of N2-fixing,
phosphate solubilizing or cellulolytic microorganisms used for application to seed, soil or
composting areas with the objective of increasing the numbers of such microorganisms and
accelerate certain microbial processes to augment the extent of the availability of nutrients in a
form which can be easily assimilated by plants (Subba Rao,1979).The great demand of nitrogen
in agriculture can be seen from an investment of over ten thousand million US dollars in
chemical fertilizer plants all over the world. It is estimated that the total energy required wide
ammonium fertilizer is equivalent to 213000 barrels of soil per day to meet the nitrogen fertilizer
needs of the united states of America alone. By 1985 the global projection for the energy metric
tons of which 25% is expected to be consumed in the developing regions(Venkataraman,1967).
Azolla is considered to be the major contributors of soil N. Although average consumption of
chemical fertilizers has increased in the country but still it is much lower than the required doses
for harvest. The researches carried out on Azolla at various institutions in India during the past
several, years provided interesting information on nitrozen economy on crop soils (Sing,1979).

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 Azolla as a biofertilizer

2. AZOLLA:

2.1 Possible environmental conditions:

Analysis of central Arctic drilling cores recently showed that ancestral Azolla spp. grew and
reproduced in situ in the Arctic Ocean some 48.5 million years ago during the mid Eocene
(Brinkhuis et al., 2006). Interestingly, around this same period a climatic transition occurred
from greenhouse to icehouse. Using extant Azolla species we want to assess the possible
environmental conditions during the mid Eocene Azolla interval.Considering the symbiotic
relation of the Azolla - Anabaena complex, growth is likely to be limited by phosphorus. Water
soluble phosphate is easily absorbed to soil and its release to floodwater is often to slow to meet
the requirements for massive Azolla growth.A water level which allows Azolla roots to penetrate
the soil may increase the phosphorus availability to the fern. Drilling cores dated to the early
middle Eocene show intervals of millimeter-scale laminated, pyritic black sediments indicative
of a water anoxic basin (Moran et al., 2006). Thick mats of floating Azolla may have hampered
the gas exchange between the atmosphere and surface waters causing anoxic conditions. It is
known that the availability of phosphorus under anaerobic conditions is enhanced by reduction
of iron complexes, especially when also sulphate is reduced. Sulphide enhances the iron-based
phosphorus mobilization because it has a higher affinity for iron (Lamers et al., 2002).
Phosphorus availability may also increase under conditions favouring dissimilative nitrate
reduction when large quantities of easily degradable organic substances are present in the system
(Wienk et al., 2000). When salt concentrations are gradually increased, extant Azolla species
may tolerate salinities up to 5.5 ‰, but without pre-incubation Azolla can stand salinities only up
to1 - 1.6% NaCl (Rai & Rai, 1998; Arora & Singh, 2003). Tolerance of Azolla to high salinity
levels maybe influenced by elevated atmospheric CO2at high concentrations. CO2 may stimulate
biomass production of Azolla species concentrations.The atmospheric CO2 concentration during
the mid Eocene is thought to be much higher than the extant concentration of approximately 370
ppm. The sea surface temperature at the Arctic during the Azolla interval is estimated 10ºC
(Brinkhuis et al., 2006). However, the relation between atmospheric CO2 concentrations and the
greenhouse climate of the early Eocene is uncertain since proxy measurements from paleosols,
marine boron isotopes, and leaf stomatal indices give estimated atmospheric CO2 concentrations
between 100 and 3500 ppm (Lowenstein & Demicco, 2006).

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 Azolla as a biofertilizer

Light intensity and photoperiod have a profound effect on growth of Azolla. The most favorable
photoperiod for present-day Azolla species is thought to be 20 hours (Wagner, 1997).

2.2 Ecology:
Azolla floats on the surface of water by means of numerous, small, closely-overlapping scale-like
leaves, with their roots hanging in the water. They form a symbiotic relationship with the
cyanobacterium Anabaena azollae, which fixes atmospheric nitrogen, giving the plant access to the
essential nutrient. This has led to the plant being dubbed a "super-plant", as it can readily colonise
areas of freshwater, and grow at great speed - doubling its biomass every two to three days.Their
nitrogen-fixing capability of Azolla has led to it being widely used as a biofertiliser, especially in
parts of southeast Asia. When rice paddies are flooded in the spring, they can be inoculated with
Azolla, which then quickly multiplies to cover the water, suppressing weeds. The rotting plant
material releases nitrogen to the rice plants, providing up to nine tones of protein per hectare per
year (Moren et al., 2006). Azolla are also serious weeds in many parts of the world, entirely
covering some bodies of water. The myth that no mosquito can penetrate the coating of fern to lay
its eggs in the water gives the plant its common name "mosquito fern"(Wienk et al., 2000).
Azolla cannot survive winters with prolonged freezing, so is often grown as an ornamental plant at
high latitudes where it cannot establish itself firmly enough to become a weed. It is not tolerant
to salinity; normal plants can't survive in greater than 1-1.6‰, and even conditioned organisms
die in over 5.5‰ salinity (Rai & Rai, 1998).

2.3 Figures of AZOLLA:

Some figures of Azolla are given below:

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 Azolla as a biofertilizer

Fig 1: General features of Azolla.

Source: Web 1

3. GEOGRAPHICAL DISTRIBUTION:

The native distribution of Azolla species has been confirmed through collection or observation of
herbarium specimens by Lumpkin (1987).According to him A. caroliniana is distributed in the
eastern half of the United States through central America and in South America. A. filiculoides is
found in Western United States and Canada Through Central America &most of South America
and may also be native to Japan.A. mexicana is found from the west coast of the United States to
Mexico and Central America. The occurrence of A. microphylla has been reported in South
America while of A. nilotica in Africa. A. pinnata is found in East and South Asia through
equatorial Asia to northern Ausrtolia and equatorial and South Africa including Madagascar. A.
rubra is found in Japan, Korea, Austrolia and New Zeland. A. filiculoides has also been
introduced in the South Africa and China and A. pinnata into New Zeland. Different strains of all
the species have recently been introduced to research stations of all continents and are been
evaluated for field use(Lumpkin, 1987).Some of the introduced species were sometimes superior
to the indigenous ones, as in Philippines A. microphylla, A. caroliana and A. mexicana replaced
the indigenous A.pinnata in different regions. Similarly in northern China A. filiculoides

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 Azolla as a biofertilizer

introduced from Germany and A. pinnata introduced in to Senegal grew much better than the
indigenous ones(Watanabe, 1994).

4. GROWTH AND REPRODUCTION:

4.1 Growth:

Azolla is a traditionally grown under cool, wet conditions. The plant prefers a placed water
surface; temperatures between 20 & 35oC, water pH of 4.7 & rich in all essential plant nutrients
except N, solution salt content <0.3%, exposure to >25% full sunlight, long day length
&freedom from competitors, insects & diseases.

4.1.1 Environmental factors:

Like other plants, Azolla-Anabaena symbiosis is also affected by environmental factors. The
production of anthocyanin, giving the Azolla carpet a reddish colour of varying insities often
signals unfavourable conditions such as excessive temperature or light intensity, or unbalanced
nutrition. So, due to diversity of these factors no precise diagnosis can be formulated, however
the experienced user may recognize the usual cause of this phenomenon in the local ecological
conditions and will react in time to apply appropriate fertilizer or shading to improve the
situation (Van Hove, 1989). The other important factors are nutrient availability, temperature,
light and aspects of water quality such as pH, salinity and turbulence (Lumpkin & plucknett,
1982).

Mineral nutrients:

The information concerning the mineral requirements of Azolla is essential for successful
propagation of the fern in the laboratory, green house and under field conditions. Azolla like
other green plants, requires all the micronutrients (except N) and micronutrients for its growth
and nitrogen fixation by its symbiont (Becking, 1979). For growing Azolla different
concentrations of nutrients have been used in 11 different culture media e.g. H2P04 0.5-60.0, Ca
1.4-12.0, Mg 1.6-18.8, K 1.3-10.0, Na 0-2.2, SO4 0.4-24, Cl 0-13, Fe 0.01-3.0 meq/L (Becking,
1979). In laboratory cultivation the threshold concentration of P for Azolla growth was reported

5
 Azolla as a biofertilizer

as 0.08 and 0.03, Ca 0.4 and 0.5, Mg 0.3 and 0.4, K 0.3 and 0.4 and S 0.08 mole by Yatazawa et
al., (1980).

Water and humidity:

Water is the most important single factor affecting Azolla cultivation. At relative humidity
<60%, Azolla becomes dry and while complete drying kills the plant (Becking, 1979). The
optimum relative humidity is reported to be 85-90%, while very high or low air moisture were
not suitable for Azolla growth as at very high humidity the transpiration is low and hence
nutrient uptake is hundred (ZAAS, 1975).

Temperature:

After water, temperature is probably the most important environmental factor limiting spread of
Azolla cultivation , and it is difficult to control also. There are two temperature factors that are
important for the growth of Azolla i.e. air and water temperature. Of these air temperature is
generally more important during winter, while water temperature during severe summer as at
mid-day, temperature of water surface is often 4-7 oC higher than the air temperature and may
reach 42-45oC at some places. For most widely grown Azolla species, the optimum temperature
is about 25oC and at this temperature doubling time is 3-5 days and nitrogen fixation capacity is
also high, & as the temperature rises to 30oC or above its growth slows down while exposure to
temperature much above 40oC for a few hours can kill this plant (Lumpkin & Pluckneet, 1982;
ZAAS, 1975).

Light:

Like other photoautotrophs, light is also essential for Azolla was grown at 25-30 oC and under
400-600 µE/m2/g light conditions, the doubling time of four Azolla species was found to
decrease from 12-12, to 16-8 & 24-0 (Peters & Ito, 1984). Longer days during rice season has
also been reported to encourage Azolla growth in India (Singh, 1992).

The growth of Azolla is said to be saturated at approximately 25-50 % of full sunlight being 25-
50 lux respectively, and for optimum growth full sunlight was not necessary (Ashton, 1974).

pH:

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 Azolla as a biofertilizer

As regards the quality of water, it should have a suitable pH for growth and nitrogen fixation.The
effect of pH on Azolla also occur indirectly as it affects the availability of nutrients in
floodwater. At very low pH the solubility of Al, Fe and Mn may reach to a toxic level in acid
soils and may interfere the absorption of Ca, Mg and other basic cations, (Lumpkin & Plucknett,
1982).In alkaline pH the availability of Ca, Mg and P decreases due to their decreased solubility
and most of the micronutrients (except Mo) like Fe, Mn, Zn, Cu and B become less available.
The availability of P depends on pH and H2PO4- ion is more available than HPO42- ions, thus
relatively more available phosphate ions are predominate between pH 4.5- 7.5 and P fixation
occurs with Fe, Al, Mn and Mg at lower pH while at higher pH mostly with Ca (Brady, 1984).

Salinity:

Amount total salts in water may affect Azolla growth, however healthy Azolla at 30 different
sites was observed in waters having 70-3080 µmho/cm (Lumpkin & Plucknett, 1982). The
optimum salt concentration for Azolla is reported to be 90-150 mg/L, while salinity and
alkalinity problems were found in coastal regions and poorly drained soils (Singh, 1992),Van
Hove et al., 1983 reported than 1g NaCl/L was toxic for Azolla growth. A decrease in growth
and nitrogen fixation in A. pinnata due to increase in EC of culture medium from 0.75 -5.0 ds/m
was observed in green house conditions(Ali et al., 1990). A decrease in nitrogen fixation in some
species of Azolla due to addition of NaCl was also observed by Kannaiyan (1992).

4.2. Reproduction:

Azolla reproduces sexually, and asexually by splitting. Like all ferns, sexual reproduction leads to
spore formation, but Azolla sets itself apart from other members of its group by producing two
kinds. During the summer months, numerous spherical structures called sporocarps form on the
undersides of the branches. The male sporocarp is greenish or reddish and looks like the egg
mass of an insect or spider. It is two millimeters in diameter, and inside are numerous male
sporangia. Male spores (microspores) are extremely small and are produced inside each
microsporangium. Curiously, microspores tend to adhere in clumps called massulae (Wagner,
1997).Azolla has microscopic male and female gametophytes that develop inside the male and
female spores. The female gametophyte protrudes from the megaspore and bears a small number

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 Azolla as a biofertilizer

of archegonia, each containing a single egg. The microspore forms a male gametophyte with a
single antheridium which produces eight swimming sperm. The barbed glochidia on the male
spore clusters are assumed to cause them to cling to the female megaspores, thus facilitating
fertilization.

5. NITROGEN ACCUMULATION IN AZOLLA :

5.1 Nitrogen fixation by Azolla:

The aquatic fern Azolla is widely distributed in tropical and temperate countries. It floats on the
surface of shallow ponds, canal, rivers and fixes atmospheric nitrogen due to its algal symbiont,
Anabeana azolle. Azolla has long been used rice culture in China and Vietnam due to its rapid
multiplication and nitrogen fixation during the last decade, the potential of Azolla as an efficient
nitrogen contributor to rice crop has been confirmed in different countries ( Talley and Rains,
1990; Watenable et al.,1987).A single group of Azolla grown with rice, contributes 20-40 kg
N/ha to the rice crop (Talley and Rains, 1990; Watenable et al.,1987).A recommended rates of
nitrogen for rice are 40-60 and 80-100 kg N/ha for wet dry seasons respectively, Azolla may not
net total N requirement of rice. The use of Azolla in combination with inorganic fertilizer
practice to optimise rice yield. Prior work on the feasibility of using Azolla as an organic
fertilizers for rice culture is based on the work of (Talley et al.,1987), (Singh, 1990), (Singh,
Talley and Rains, 1990)among others. Most of the earlier studies on N fixation of Azolla in rice
field in the presence of N fertilizer were done by measuring total N with the micro using
acetylene reduction activity (ARA) besides total N estimation under field conditions, a previous
study indicated variations in biomass and N yield of different species strains of Azolla which
affected the grain yield of rice. This study was undertaken to compare the performance of two
Azolla species at varying N levels.

Table 1. Fresh weight acetylene reduction activity and total N yield of Azolla species at
varying levels of urea in planted rice field (wet seasons 1986):

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 Azolla as a biofertilizer

Treatment Species Fresh weight (t/ha) ARA activity Total N yield (kg/ha)
(n moletylase/g fresh
(DAI) (DAI)
Azolla) (DAI)

7 14 21 7 14 21 7 14 21.

No A. pinnata 4.0 10.4 4.8 12.0 21.1 8.1 5.9 15.5 7.3

Nitrogen A. 6.0 12.6 6.0 18.2 37.6 19.1 7.3 18.7 9.0
caroinana

10 A. pinnata 3.6 9.7 4.2 2.1 16.8 4.7 5.3 14.3 6.3
kgN/ha
A. 4.3 11.2 5.4 13.6 30.3 17.0 6.7 16.5 8.1
caroinana

20 A. pinnata 3.3 9.2 4.0 2.4 12.5 1.3 4.8 13.7 5.9
kgN/ha
A. 3.6 9.7 5.0 9.2 20.3 10.0 5.3 14.3 7.5
caroinana

Azolla received the urea applied at transplanting which amounted to 50% of that provided to the
rice crop. DAI= Days After Inoculation.

Source: Manna and Sing (1989)

Table 2. Fresh weight, acetylene reduction activity & total N yield of Azolla species at
varying levels of Urea in planted rice field (Dry season 1987).

Treatment species Fresh weight (t/ha) ARA activity Total N yield (kg/ha)
(n moletylase/g fresh
(DAI) (DAI)

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 Azolla as a biofertilizer

Azolla) (DAI)

7 14 21 7 14 21 7 14 21.

No A. pinnata 5.1 11.4 4.4 13.5 28.7 11.1 7.6 16.9 6.6

Nitrogen A. 5.9 13.2 6.7 17.4 44.3 16.7 8.8 19.6 10.4
caroinana

10 A. pinnata 4.9 10.1 3.7 11.1 18.6 6.2 7.2 15.0 5.6
kgN/ha
A. 5.2 11.8 6.0 12.2 35.0 10.4 7.7 17.6 9.6
caroinana

20 A. pinnata 4.2 8.8 3.3 1.8 10.8 1.8 6.2 13.1 4.1
kgN/ha
A. 4.8 10.1 4.8 1.8 19.6 5.4 7.1 15.0 7.2
caroinana

Azolla received the urea applied at transplanting which amounted to 50% of that provided to the
rice crop . DAI = Days After Inoculation.

Source: Manna and Sing (1989).

5.2. Decomposition of Azolla in soil:

Pinnata had a high nitrogen content, nitrogen mineralization was faster than in other fern species.
The mineralization of nitrogen in A. menieana, which had the lowest nitrogen was the slowest.
Fresh Azolla released at maximum 2-5 times more ammonium nitrogen from its body than dry
Azolla did Watanabe et al., (1997) reported that ammonium was released more rapidly more

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 Azolla as a biofertilizer

from fresh Azolla than from dried Azolla. Mineralization in fresh azolla was active until 16 days
after the start of incubation and than it reached a platean rapid mineralization during the first 4
days of incubation in the case of dry Azolla was followed by a slow release of ammonium. The
organic forms of nitrogen in Azolla may become resistant to the mineralization, in other words,
these may be less easily decomposible types that slowly release ammonium mineralization
throughout the incubation with the soil. The initial rapid mineralization of nitrogen in dry Azolla
may have been due to the small amount of easily decomposible substances that remained after
drying.

Brotonegoro and Kadir (1998) reported that the mineralization of nitrogen in fresh Azolla
incorporated into flooded soil reached the maximum 24 days after the start of the incubation, and
that the release of ammonium during the period amounted to about 70 micro g/g dry soil.

5.3. Fate of Azolla- Nitrogen:

The nitrogen loss which was highest when Azolla was placed on the soil surface amounted to
more than 60% after 6 weeks of inoculation (Table 3). About 95% of the nitrogen remaining in
the soil was present in the upper layer after 2 weeks, and there after the nitrogen from Azolla
moved down wards. When Azolla was grown on water, three-fourths of the nitrogen settled as
sediments or decomposed for 6 weeks; of that amount one-thired remained in the soil and the rest
was lost.

The mineralization of Azolla- nitrogen was highest when Azolla was incorporated (Table 4).
Only a small amount of exchangeable ammonium organizating from Azolla inoculum was found
in the treatment where Azolla was grown in water.

The results suggest that the incorporation of Azolla into soil reduces the loss of nitrogen and
increase the availability of nitrogen to the plant.

Table 3. Distribution of nitrogen (%) from fresh Azolla applied in 3 cm layer of floded soil
(Pot experiment).

Treatment Weeks after Soil N

inoculation
Water Upper layer (0-3cm)

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 Azolla as a biofertilizer

Placed 2 0.77 52.7

on 4 1.0 32.2
soil surface 6 0.18 24.3

Grown 2 0.54 4.3

on 4 0.24 19.2
water 6 0.08 26.7

Source : Ito and Watanabe, (1995)

Table 4. Distribution of 15N-labeled exchangeable ammonium in each layer of soil.

15
Treatment Weeks after N-labeled exchangeable ammonium
inoculation Upper layer (0-3cm). lower layer (6-9cm).

Placed on soil 2 220 0

surface
4 247 12.6

6 106 83.4

Grown on 2 5.05 1.24

water
4 28.9 3.38

6 20.8 5.90

Source : Ito and Watanabe, (1995)

Culture of Azolla with the rice plant, which is comparable to the treatment in which Azolla is
grown on water, may supply some of the nitrogen required by the plant. The contribution is
likely to be lower than when Azolla is incorporated, because the loss of nitrogen will be greater.

6. NUTRIENTS OTHER THAN NITROGEN:

Azolla accumulate macro and micronutrients which are essential for growth of rice plant. After
decomposition of Azolla plant, its nutrients become available for the growth of rice plant. Work
done at the central Rice research institute, Cuttaek (Singh,1987) has revealed the following:

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 Azolla as a biofertilizer

(1) Standing water of 5-10 cm deep and the application to superphosphate at 4-8 kg P2O5/ha are
essential for rapid growth of Azolla.

(2) Azolla nurseries in small plots (50-100sqm) are preferable to large plots to avoid wind
erosion. Concrete tanks are also suitable for this purpose.

(3) Azolla inoculum at 0.1-0.4 kg/sqm is most desirable for the rapid multiplication of the fern
in nurseries for production of 8-1 t/ha green matter in so days.

(4) A PH of 8.0 is ideal and acidic soils (below pH 4.6) are not auitare unless lime is used to
correct pH.

(5) Water temperature in the range of 14-35oC is tolerable but the optimum range is 20-30oC.

(6) Use of carbofuran at 1-2 kg/ha prevents the rapid spread of insects-parasites and the
consequent destruction of Azolla nurseries.

(7) Harvested Azolla accumulated as heaps decomposes rapidly in 7-10 days.

(8) the composition of Azolla is approximately 94% water, 1% of P, K, Ca, Mn and Fe and
5%N.

The limitations in the use of Azolla of the areas where water is not available or in areas where
establishment is difficult due to other factors such as adverse temperature and pests.
Transportations of Azolla from distant part is risky since it is quickly perishable after removal
from water.

6.1 Nitrogen content:

Azolla used in rice field as nitrogenous fertilizer. It contains high amount of N2 then other
essential nutrients. The content of N2 in Azolla varies in different countries. Such as in India 5%
N2 ( Subba Rao, 1995) on the dry matter basis.

6.2 P content:

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 Azolla as a biofertilizer

On the average Azolla contained 0.284% of Phosphorous on a dry weight basis.(Watanabe,

1995).

6.3 K content: Azolla contains 2-3% K (Liu chung-chu, 1987) has a strong ability to
concentrate K from the water in which it grows. The K absorption of Azolla is around 0.85 ppm
K2O when cultivated in various concentrations of K medium, i.e. 1g Azolla biomass is able to
takeup about 70% of K from 800 ml of culture solution containing 0.85 ppm K2O in 1 day
( Watanabe, 1995).

7. EXPERIMENT CARRIED OUT USING AZOLLA:

7.1 Azolla use in rice soils:

Rice is the single most important source of food for people and Azolla plays a very important
role in rice production. For centuries Azolla and its nitrogen-fixing partner, Anabaena, have been
used as "green manure" in China and other Asian countries to fertilize rice paddies and increase
production. Some authorities believe the use of Azolla enabled the Vietnamese to survive the
effects of the American blockade when imported fertilizers did not reach North Vietnam during
the war. According to Clark (1980), the People's Republic of China has 3.2 million acres of rice
paddies planted with Azolla. This provides at least 100,000 tons of nitrogen fertilizer per year
worth more than $50 million annually. Extensive propagation research is being conducted in
China to produce new varieties of Azolla that will flourish under different climatic and seasonal
conditions. According to some reports, Azolla can increase rice yields as much as 158 percent
per year. Rice can be grown year after year, several crops a year, with little or no decline in
productivity; hence no rotation of crops is necessary.

7.1.1 Experiment 1: (Winter Low Temperature Resistance)

Biomass increase, rates of growth (RGR) and survival at winter temperatures of the five strains
from Dec. 2000 to May 2001 are shown in Figure 2:

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 Azolla as a biofertilizer

Figure 2. Fresh weight RGR (Relative Growth Rate) = (lnw2-lnw1)/(t2-t1) of the five strains.
Experiment started on Dec. 6th 2000. Every RGR is calculated on a two-week period, the end of
which is reported in x-axis. Bars represent standard deviation. A progressive decrease of leaf
dimensions was registered in all strains, associated with accumulation of anthocyanins.

Source : Ladha et al., 2000.

A progressive decrease of leaf dimensions was registered in all strains, associated with
accumulation of anthocyanins. As time passed, the Japan and Italy strains assumed a tawny
colour (an indicator of drying preceding death). After 126 days from the beginning of the
experiment, on the Apr. 11th 2001, these strains were eliminated from the experiment. By the
second half of April, the leaf dimensions of the remaining strains were comparable to those at the
outset of the experiment, with the leaves of the Milan strain bigger than those of the Sweden and
Germany strains.

Temperature and biomass increase were clearly correlated (r=0.6**–0.7**) for the Milan and
Germany strains whereas the correlation index for the Sweden strain is lower and not significant.

Table 5. Shows Carbon and Nitrogen content, and C/N ratios in Azolla strains.

Strains %C %N C/N

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 Azolla as a biofertilizer

Milan mean 38.854 2.581 15.582

std 1.6 0.48 2.58

Sweden mean 37.722 2.097 18.166

std 2.04 0.18 1.73

Germany mean 38.244 2.248 17.165

std 1.87 0.18 1.60

Italy mean 40.374 2.631 15.606

std 0.98 0.33 2.08

Japan mean 41.294 0.025 14.336

std 1.26 0.59 3 .66

Source : Ladha et al., 2000.

We observed high quantities of nitrogen in the leaf of the Japan and Italy strains, which appeared
to be related to the decrease of its biomass during our test. The Milan strain showed lower
nitrogen content, but a good c C/N ratio associated with a high RGR (Figure 2).

7.1.2 Azolla use in India:

Azolla use for wet land rice culture is increasing in several rice growing regions in India. The
increasing cost of fertilizers, particularly system for increasing rice yield under low-cost rice
production technology. Biological Nitrogen fixation though Azolla-Anabaena complex is
considered a potential biological system for increasing rice yield at comparatively low
cost.Nitrogen fertilizers & Azolla dual cropped at 200g/m2 with IR20 and incorporated once at
tillering and again at maximum tillering increased grain yield significantly.

Table 6. Effect of Azolla on rice yield.

Grain yield (t/ha)

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 Azolla as a biofertilizer

Nitrogen With Azolla Without Azolla

(Kg/ha)

0 4.4 3.9

25 4.7 4.6

50 4.9 4.8

75 5.2 5.4

Source : Kannaniyan (1987).

7.1.3 Azolla use in Srilanka:

The potential of Azolla as a biofertilizer for rice has been examined by field growth observations
at severel sites within the dry intermediate and wet zones of Srilanka. The effect of Azolla on
rice yield compared to that of urea was evaluated over three crop seasons.

Result presented in table 3 show that from the 53 Kg N/ha applied as fresh Azolla, nearly 30%
want into the panicles and 13%, in to the straw, giving a total Nitrogen recovery of 43%, in the
case of the 80 kg N/ha given as urea, only 28% was recovered in the panicles and 9% in the
straw, a total of 37%.The efficiency of N utilization from Azolla is higher than from urea (Table
7).

Table 7. The recovery of N from Azolla and urea by field grown rice.

Fertilizer Plant part Dry matter Yield N derived N recovery

source (Kg/ha) %
from fert.

Azolla Straw panicle 1293 9.57 73.63 13.21

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 Azolla as a biofertilizer

53.28 total 2060 24.31 66.50 29.87

Kg/ha
3353 33.88 42.98

Urea 79.63 Straw panicle 1358 13.14 54.75 8.98

Kg/ha
total 2639 4048 55.25 27.90

3997 53.62 36.88

Source : Kulasooriya (1987)

The effect of Azolla on rice yield under different planting patterns is given Table 8. Yield
increased 14% for broadcast seeded rice, 22% for transplanted, and 47% for avenue planted, the
yield values per hectre, however, are low, which reflects the low fertility of these atypical fields
at Peradeniya.

Table 8. Grain yield and Straw yield of rice grown in three planting patterns in the wet
zones of peradeniya.

Planting
pattern Grain yield (t/ha) Increase% Straw yield (t/ha) Increase%

Without With Without With

Azolla Azolla Azolla Azolla

Broadcast 1.4 1.6 14 2.77 3.45 25

18
 Azolla as a biofertilizer

Seeded 2.2 2.7 22 2.56 3.10 21

Planted in 2.1 3.0 47 2.47 3.41 39

avenues

Source : Kulasooriya (1987)

8. ECONOMIC IMPORTANCE OF AZOLLA:

Food:

In addition to its traditional cultivation as a bio-fertilizer for wetland paddy (due to its ability to
fix Nitrogen into the soil), Azolla is finding increasing use for sustainable production of
livestock feed. Azolla is rich in proteins, essential amino acids, vitamins and minerals. Studies
describe feeding azolla to dairy cattle, pigs, ducks, and chickens, with reported increases in milk
production, weight of broiler chickens and egg production of layers, as compared to conventional
feed. One FAO study describes how azolla integrates into a tropical biomass agricultural system,
reducing the need for inputs (Wagner, 1997).

Companion plant:

Azolla has been used, for at least one thousand years, in rice paddies as a companion plant,
because of its ability to both fix nitrogen, and block out light to prevent any competition from
other plants, aside from the rice, which is planted when tall enough to poke out of the water
through the azolla layer.

Larvicide:

As an additional benefit to its role as a paddy biofertilizer, Azolla spp. have been used to control
mosquito larvae in rice fields. The plant grows in a thick mat on the surface of the water,
reducing the rate at which oxygen dissolves into the water, effectively choking the larvae
(Wagner, 1997).

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 Azolla as a biofertilizer

Climatic paleontology:

A study of Arctic climatology reported that Azolla may have had a significant role in reversing
an increase in greenhouse effect that occurred 55 million years ago that caused the region around
the north pole to turn into a hot tropical environment. This research conducted by the Institute of
Environmental Biology at Utrecht University claims that large dense patches of Azolla growing
around freshwater lakes formed by the climate change eventually consumed enough carbon
dioxide for the greenhouse effect to reverse.

9. CONCLUSION:

As a general conclusion, beneficial role of Azolla in crop fields appears to be true. Recent
research has shown the feasibility of using these Azolla as a biological input in rice cultivation
and of obtaining yield for which the incremental input cost will be low. The use of these
regenerative biological sources may also minimize the environmental hazard and maximize the
ecological benefits. Unlike chemical fertilizers, Azolla do not bring about spectacular visual
changes in growth and production.Long term field experiments on an ecological basis-relating
the occurrence of N2 fixing strains, the qualitative and quantitive evolution of the Azolla, and the
variations in phototrophic N2 fixing activity to the environmental parameters-are needed to
determine.A good deal of extensive work requires to be organized to create a better awareness
among farmers of the benefits of Azolla fertilizers and a consumer demand for the product has to

20
 Azolla as a biofertilizer

be created.A real impact is possible only by mobilizing the available information and
propagating it through a sustained extension network among small and marginal farmers.

10. REFERENCES:

 Ali, S. A., M. Yasin & S.M. Aslam. 1990. Effect of salinity & nitrozen application on
the growth and nutrient concentration of Azolla. Intl. Conf. on Current Development in
Salinity and Drought Tolerance of Plants. pp.20.
 Ashton , P. J. 1974. The effect of some environmental factors in the growth filiculoides
lam. In: The Orange River, Progessive Report. Inst: Environmental Sci, Univ of the O. F.
S. Bioemfontein, South Africa: pp.12.

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 Azolla as a biofertilizer

 Becking J. H. 1989. Environmental requirements of Azolla for use in tropical rice. In:
Nitrogen & Rice. Int Rice Res Inst (IRRI), P O Box 933 Manila, Philippines. pp. 345-
350.
 Brady, N. C. 1984. The Nature & Properties of Soils. 9th ed. Mac Millan P Company.
New York Collier Mac Millan Publishers, London. 750 pp.
 Brinkhuis, H. , T. S. Ventura, U. Singh, W. Ventura and I. Watanabe. 2006. Episodic
fresh surface waters in the early Eocene Arctic ocean and adjacent seas. Nature, in
review. pp. 112-120.
 Brotonegoro, S. & A.S. Kadir. 1978. The decomposition of Azolla pinnata in moist and
flooded soil. Ann. Borgorieness 6: 169-175.
 Casanova, D. , J. Goudriaan, M. M. C. Forner and J. C. M. Withagen. 2002. Rice yield
prediction from yield components and limiting factors. European Journal of Agronomy
17 (1) : 41–50.
 Clark, W. 1980. China’s Green Manure Revolution. Science 80 (1) : 69-73.
 Ito, O. & I. Watanabe.1985. Availability to rice plant of nitrozen fixed by Azolla. Soil
Sci. Plant Nutr. 31: 91-104.
 Kannaiyan, S. 1987. Use of Azolla in India. In: Azolla Utilization. The Fujian Academy
of Agricultural Science, Fuzhou, Fujian, China and The International Rice Research
Institute Los B anos, Laguna, Philippines. pp. 109-117.
 Kannaiyan, S. 1992. Studies on the factors influencing growth and nitrozen fixation in
Azolla. In: Biofertilizer Technology Transfer, Gangawane, L. V.(ed). Associated
Publishing Company, New Delhi. pp.239-253.
 Kulasooriya, S. A. & W.K. Hirimburegama. 1987. Use of Azolla in India. In: Azolla
Utilization. The Fujian Academy of Agricultural Science, Fuzhou, Fujian, China and The
International Rice Research Institute Los B anos, Laguna, Philippines. pp. 131-139.
 Ladha, J. K. , D. Dawe, T. S. Ventura, U. Singh, W. Ventura and I. Watanabe. 2000.
Long-term effects of urea and green manure on rice yields and nitrogen balance. Soil
Science Society of America Journal 64 (6): 1993–2000.
 Lowenstein, T. K. & R. V. Demicco. 2006. Elevated Eocene Atomspheric Co2 and Its
Subsequent Decline. Science 313:1928.

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 Azolla as a biofertilizer

 Lumpkin, T. A. & Plucknett, D. L. 1982. AZolla As a Green Manure: Use &

Management in Production. Westview Press, Boulder, Colorado. pp. 230.
 Lumpkin, T. A. 1987. collection, maintenance and cultivation of Azolla. In: Symbiotic
Nitrozen Fixation Technology. Elken, G. H (ed). Marcel Dekker, Inc. New York. pp. 55-
94.
 Manna, A. B. & P.K. Singh.1989. Growth and nitrozen fixation of AZolla pinnata and
Azolla Caroliniana and their influence on rice yield. Nature, in review. pp. 207-212.
 Moran, K. , J. Backman, H. Brinkhuis & S. C. Clemens.2006. The Cenozoic
Palaeoenvironment of the Arctic Ocean. Nature 441: 601-605.
 Peters, G. A. & O. Ito. (1984).Determining nitrozen fixation and N input in Azolla grown
with without combined nitrozen sources: keeping the acetylene reduction assay in the
properspective. In: Practical Application of Azolla for Rice Production. Silver, W. S. &
Schro,E.C (eds). Martinus Nijhoff Pub. Dordrecht. pp. 29-44.
 Rai & Rai.1999.Growth behaviour of Azolla pinnata at various salinity levels and
induction of high salt tolerance plant & soil. 206: 84.
 Saunders & Fowler.1992. The supraspecific taxonomy and evolution of the fern genus
Azolla. Plant Systematics & Evolution 184: 175-193.
 Singh, P. K. 1977. Azolla plants as fertilizer and feed. Indian Fing. 27: 19-21.
 Singh, P. K. 1979. Use of Azolla in rice production in India. In nitrozen and rice. IRRI.
Manila, Philippines.pp. 407-418.
 Singh, P. K. 1992. Biofertilizers for flooded rice ecosystem. In: Fertilizers, Organic
Manures, Recyclable Wastes & Biofertilizers. Tandon, H. L. S. (ed). Fertilizer
Development & Consultation Organization, New Delhi, India. pp. 113-131.
 Subba Rao, N. S. , K. V. B. R. Tilak. , M. Lakshmikumari & C. S. Singh. 1979.
Azospirillum-a new bacterial fertilizer for tropical crops. Science Reporter C. S. I. R.
(India) 16: 609-622.
 Talley, S.N. , Talley, B. J. & Rains, D.W. 1977. Nirtozen fixation by Azolla in rice fields.
Martinus Nijhoff Pub. Dordrecht. pp.254-260.
 Talley, S. N. & D. W. Rains. 1980. Azolla filiculorides lam, as a fallow-season manure for rice in
a temperature climate. Agron. J. 72(1): 11-18.

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 Azolla as a biofertilizer

 Van Hove, C. 1989. Azolla and Its Multiple Uses with Emphasis on Africa, Rome (Italy).
pp. 61.
 Venkataraman, G. S. 1967. Azolla & soil fertility. Proc. Nat. Acad. Sci. India Sect. 37:
380.
 Wagner, G. M. 1997. Azolla: A review of its biology & utilization. The Botanical
Review 63: 1-26.
 Watanabe, I. , C. R. Espinas, N. S. Berja & B.V. Alimagno. 1977. Utilization of the
Azolla-Anabaena complex as a nitrozen fertilizer for rice. IRRI. Research Paper Series
11:: 12-28.
 Watanabe, I. 1994. Genetic enhancement and Azolla collection-problems in applying
Azolla-Anabaena symbiosis. In: Nirtozen Fixation in Non-legumes. The American
University in Cairo Press. pp. 437-445.
 Wienk, L. D. , J. T. A. Verhoeven, H. Coops & R. Portielje. 2000. Peilbeheeren nutrient
RIZA report. 2000. 45: 20-32.
 Yatazawa, M. , N. Tomomatsu, N. Hosoda & K. Nunome.1980. Nitrozen fixation in
Azolla-Anabaena nutrients status. Soil Sci plant Nutr. 26 (3): 415-426.
 ZAAS. 1975. Cultivation, Propagation and Utilization of Azolla. Zhejian Academy of
Agricultural Sciences (ZAAS). Institute Soil and Fertilizer (compilers). Agric Publishing
House Beijing, Chaina. pp.52-60.
 Website:
1. www. picture of Azolla. com

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