6.

Azolla -Anabaena symbiosis - its physiology and

use in tropical agriculture

1. WATANABE

1. Introduction

Azolla is a water fem widely distributed in aquatic habitats like ponds, canals, and
paddies in temperate and tropical regions. This plant has been of interest to
botanists and Asian agronoTIÙsts because of its symbiotic association with a N 2 -
fIxing blue-green alga and rapid growth in nitrogen-defIcient habitats.
Recently, the interest in this plant-alga association has been renewed by the
demand for less fossil energy·dependent agricultural technology.
Reviews on updatinginformation were made by Moore [20], Watanabe [42] ,and
Lumpkin and Plucknett [19]. A bibliographic list was published by the Inter-
national Rice Research Institute [15] .

2. Biology and physiology of Azolla-alga relation

Azolla belongs to the Azollaceae, a heterosporous free-floating fem, and is close to

the family Salviniaceae. There are six extant species of Azolla (Table 1) and 25
fossil species are recorded [14]. These are divided into two subgenera: El{azolla,
a New World azolla, and Rhizosperma. Species differentiation is based on the
morphology of the sexual organ.
The number of septa in the glochidia was used as a taxonomic tool to
differentiate Euazolla. This criterion was questioned by taxonomists because of
variations within a given species [10] . In the subgenus Rhizosperma, the glochidia
are replaced by a root-like structure emerging from the massulae in the micro-
sporangium. In A. nilotica, neither the glochidia nor the root-lïke structure is
present on the massulae (Fig. 1).
Because the sporocarps are usually absent in naturally grown azolla, it is difft-
cult to identify species.
Four species of Euazolla originated from the New World, but currently these are
widely spread in the temperate regions of Europe and Asia. A. pinnata is widely
distributed in the subtropical and tropical regions of Asia and has been used for
agricultural purposes in Asia. A. nilotica is a giant water fem, about 10 cm long, and
is distributed in central Aftica.
The azolla plant has a branched floating stem that bears altemately arranged
overlapping leaves and true roots. Each leaf has two lobes - the ventral or lower

Y.R. Dommergues and H. G. Diem (eds.j, Microbiology of Tropical Soils

and Plant Productivity. ISBN 902472624 7.
© 1982 Martinus Niihoff/Dr W. Junk Publishers, The Hague/Boston/London.
170

Mes
2.

YI
Ind
'I[;~:U::\::'" Ma

Fig. J. Sexual organs of Azol/a.

1. Megasporocarp and Microsporocarp - Me: megasporocarp; Mi: microsporocarp; Mir: micro-
sporangium
2. Microsporangium - Ma: massulae
3. Massulae of A. pinnata - Tr: trichome; Mis: microspore
4. Massulae of A. [ilieuloides - GI: glochidium
5. Megasporocarp - Ind: Indisum; FI: float; Mes: megaspore
6. Germinating megasporocarp - YI: young leaf; Ma: massulae
Table J. Species of Azol/a
Subgenus No. of floats Species Major distribution before
on megasporocarps dispersal by men
Euazol/a 3 A. jilieuloides, Lamarck Southern South America
Western North America
A, earoliniana, Willd. Eastern North America
Central America
A. mexieana, Pres!. Northern South America
Western North America
A. mierophyl/a, Kaulfuss Tropical and subtropical
America
Rhizosperma 9 A. pinnata, R. Brown Tropical-subtopical
Asia and coastal Africa
A. lIilotiea, De Laisne Upper NUe and Sudan
Central Africa
 171

lobe and the dorsal or upper lobe. The dorsal lobes are chlorophyllous and aerial,
the ventral lobes are partly submerged, thin, and achlorophyllous.The frond is
about 1 to 3 cm long. In optimum condition, the lateral branch of stems of A.
filiculoides and A. nilotica and sometimes, of A. caroliniana, partly becomes aerial
and new shoots grow upward, thus giving a higher biomass than the flatly growing
ones. The roots occur at branch nodes on the ventral surface of the stem. They are
about 2 to 10 cm long, depending on species, have hairs and a sheathing root cap
that falls off with age. In shallow water, the roots adhere to the soil surface and
absorb nutrients from the soil.
In natural conditions, azolla multiply by vegetative reproduction. Under certain
circumstances, the formation of sexual organs is observed. A new generation is
formed from the fertilized embryo. Although a sporophytic life cycle is described
(Fig. 2), little is known about the conditions for spore formation and its ecological
significance. High temperature (early summer) in temperate regions and low
temperature in tropical and subtropical regions (A. pinnata) have been reported to
induce sporocarps. In southern China, sorne local strains of A. pinnata form spores
abundantly in June and July, and to a lesser extent in September and October. In
northern Vietnam, spores are formed in March-April. Formation of sexual organs
seems to be associated with high density of azolla population.

/_sp_orop_hYt_" I<------~
Ventral lobe initial Embryo

/ ~ ?~
~licrosporangium HegasPlrangium oos';;:'re Antherozoid

1 f
Microspore Megaspore

~
~--~
1
Female prothallus

Male prothallus

Fig. 2. Heterosporous life cycle of Azolla.

When sporocarps are formed, the vegetative growth rate is retarded [2, 3].
Sporocarps are borne by shoot stalks on the first ventral lobe initial of a lateral
branch and occur in pairs (Fig. 1).
Microsporocarps, the male organs, are larger than the mega(macro)-sporocarps
and are brown yellow or brownish red. They contain many microsporangia and
within the periplasmodium of a microsporangium, 32 or 64 microspores develop
and aggregate into massulae. From the massulae, glochidia develop. Microspores
germinate and release antherozoids which fertilize the oospore in the megaspore.
172

Megasporocarps, the female organ, are smaller and produce only one megaspore. In
mature megasporocarps, the megaspore is covered by 3 or 9 floats together with a
columella. One oospore is formed from a megaspore. The germination of embryo
and the subsequent growth of young seedIing is slow. It takes 1 or 2 months from
germination for an azolla to grow as big as a vegetatively growing fem with
branches. The germinating megasporocarps are attached by many massulae. The
fertilized megaspores can withstand desiccation and can survive more than 1 year
in dry condition [46]. Therefore, the fertilized megasporocarps can be used for
storage of azolla germplasm collection. Because of the slow growth of a new
azolla seedIing, the megasporocarps are unlikely to be used as seeding material for
agricultural use. Light is necessary for the germination of sporocarps.
The symbiont alga is found in the cavity formed in the proximal portion of the
dorsal lobes. The cavity has a mouth which opens toward the ventral side of the
dorsal lobes. This mouth probably acts as the site of gas exchange between the
atmosphere and the symbiont.
The symbiont alga was named Anabaena azollae Strasburger, but Fjerdingstad
[7] recently claimed that the alga is actually an ecoform of Anabaena variabilis.
The isolation of Anabaena azollae from azolla has been frequently reported. But
none has succeeded in the re-inoculation of the isolated symbiont to alga-free
azolla and nothing is known about the free-living state of this symbiont. Anabaena
azollae was claimed to be associated with the microsporocarps and megasporo-
carps. It is possible that Anabaena in young azolla seedlings originated from the
algal cells in megasporocarps.
During the differentiation of dorsal lobe primordia, the cavity occupied by the
symbiont is created by epidermal cell growth. Several algal cells sheltered in the
shoot apex are entrapped by the enclosing epidermal cells and colonized the cavity
[17]. The hair-like cell of plant origin is seen in the youngest cavity. Algal cells in
the youngest lobes do not have heterocysts. The frequency of heterocysts increases
to about 30% in the 15th leaf as the lobes are traced back from the shoot apex to
the basal parts. After the 20th leaf, the heterocysts begin to be senescent [12, 13].
The N2 -fIxing ability of each leaf is proportional to the frequency of heterocysts in
each leaf.
The frequency of heterocysts is higher in symbiotic alga in azolla than in free-
living heterocystous blue-green algae. Hair-like cells are seen in all cavities, which
are believed to be the site material exchange between the host plant and the alga
[26] . The interaction between both partners is still poorly understood. The growth
of azolla and alga may be synchronized. The alga-free azolla has also the cavity and
hair-like structure.
The N2 -fIxing ability of the symbiont is demonstrated in acetylene reduction
assay and \SN2 • The endophyte algae which are mechanically isolated from the fem
have the ability to fIx \SN2 or to reduce acetylene, although the rate is lower than
in association with the fem [23,24, 25, 27] .
The isolated alga excretes about half of the fIxed N2 as ammonium. Because
most of the ammonia-assimilating enzyme glutamine synthetase are found in the
fem parts of the association, the algae in the association provide fIxed N2 to the
 173

host plant mainly as ammonia and the plant converts it to amino acids [25]. N2
fIxation is associated with photosynthesis. Evidences show that the blue-green
algae in the association catch solar energy and use this for nitrogenase reaction
[28] . It is not known if the energy and reductant necessary for nitrogenase reaction
are partly provided by the fem.
The N2 -fIxing system in the fern-alga association is not strongly inhibited by
ammonium, nitrate, and urea. Azolla growing in 2.5 mM ammonium can maintain
its growth rate similarly in its absence and N2 fIxation is inhibited by approxi-
mately 30%. N2 -fIxing activity is quickly recovered upon the transfer to N-free
medium (Ito and Peters, unpublished). The N2 ·fIxing system in the endophyte
may be, by sorne unknown mechanisms, protected from the inhibitory action of
combined nitrogen. This is a unique nature of the Azolla-Anabaena symbiosis
unlike the legume-rhizobium symbiosis which is more sensitive to combined
nitrogen.

3. Environmental factors affecting growth and N 2 -fixing activities

The growth rate, maximum biomass, and N 2 -fIxing activity in optimum conditions
provide the estimate of the potential of Azolla-Anabaena symbiosis for agricultural
use.
In optimum light and temperature conditions in the laboratory, Peters et al.
[29] obtained about 2.0 days doubling time or less for A. [ilieuloides, A. earo-
liniana, A. mexieana, and A. pinnata. Doubling time of 2 days corresponds to 0.34
g-1 day-l. Growth in the liquid medium by Talley and Rains [39] with Azolla
filieuloides and Watanabe et al. [42] with Azolla pinnata showed that the maxi·
mum growth rate was about 2.5 days doubling time.
N2 ·fIxing rate is estimated by the relative growth rate and nitrogen content of
the fem. Assuming 0.277 daily relative growth rate (2.5 days doubling time) and
4% N content in dry matter, daily N2 -fIxing rate is calculated as 11.0 mg N g-l dwt.
Assuming 4: 1 electron ratio of N2 fIxation to acetylene reduction and 12-hr light
period a day, this N2 -fIxing rate corresponds to 130 ~mol C2 H4 g-1 dwt h-1. The
reported value of ARA (acetylene reduction activity) fluctuated from 20 to 200
[4] .
.The growth curve of azolla approximate1y follows a logistic curve until the bio-
mass reaches the maximum. The growth is characterized by the initial growth rate
and the maximum biomass. The growth rate is retarded as the plant density
increases [2] .
The maximum biomass or nitrogen accumulation reported by researchers is
summarized in Table 2. Because Azolla [ilieuloides grows upward from the water
surface and forms a thick mat, the maximum biomass is higher than that of other
azolla species. No data on the maximum biomass of A. nilotica, which forms
10-15 cm long stem, are available. The maximum daily N2 ·fIxing activity of A.
filieuloides per unit area was about 2.8 kg N ha-1 , whereas the maximum daily N2 •
fIxing rate of A. pinnata grown in open paddy fIelds in the Philippines was 3.1 kg
N ha- 1 (Watanabe, unpublished). Average daily N2 -fIxing rates, measured from
Table 2. Maximum biomass and average N, -fixing rate
Species Condition Maximum biomass Average N,- References
fixing rate
Dry matter N content Days
(kg ha- I day-I)
(kg ha -1) (kg ha- I )
A. filiculoides Fallow paddy, USA 1700 52 35 1.5 Talley and Rains [38]
Shallow pond, USA 1820 105 ? Talley et al. [37]
Paddy soi! in pots 5200 128 50 2.6 Tuzimura etai. [41]
Fallow paddy, USA 2300 93 46 2.0 Talley and Rains [39]
A. mexicana Ponds, USA 830 39 39 1.0 Talley et al. [37]
Paddy field, USA 1100 38 ? Talley and Rains [38]
A. pinnata Fallow paddy, Philippines 900-1200 48 30-25 1.9-1.6 Watanabe (unpublished)
Phytotron
26°(d)/18° C(n) 2170 96 37 2.6 Watanabe (unpublished)
33° (d)/25° C(n) 1500 33 22 1.5 Watanabe (unpublished)
37° (d)/29° C(n) 1120 30 23 1.3 Watanabe (unpublished)
(var africana) 640 26 15 1.8 Roger Reynaud [31]
greenhouse
A. filiculoides 26° (d)!l8° C(n) 3200 126 51 2.5 Watanabe (unpublished)
A. caroliniana 26° (d)/18° C(n) 3190 146 41 3.6 Watanabe (unpublished)

d = day; n = night
 175

inoculation to harvest, are presented in Table 2. The values fluctuate from 1.0 to
2.6 kg N ha- 1 . Watanabe et al. [44] reported that 26 crops of azolla yielded 450
kg N ha- 1 for 330 days in an open paddy field. Singh [34] reported an annual
production of 333 ton fresh weight ha by weekly harvest and estimated annual
nitrogen production at 840 kg N ha- 1 • Shen et al. [33] reported 93-152 kg N ha- 1
for 45 days.
From these figures, the high potential of azolla as a N2 -fixing crop is easily
realized. The fixing rate is almost comparable to the figure offorage legumes [22] .
Among environmental factors affecting the growth and N2 -fixing activity of
azolla, temperature, light, humidity, and mineral elements are described.

3.1. Temperature

The optimum temperature of A. pinnata, A. mexicana, and A. caroliniana, when

grown in constant temperature under 15 klux artificiallight [29] is about 30 oC.
A. filiculoides requires 25 oC. The response of nitrogenase activity to temperatures
ranging from 10 ° to 42 Oc also shows that A. filiculoides likes lower temperature
than A. pinnata [4]. Although A. pinnata is widely distributed in the tropics, it
grows better in cooler seasons. In northern Vietnam, the growth of A. pinnata is
best in January when the average air temperature is 17 oC. In Varanasi, India,
A. pinnata grew from July to December but was absent from the ponds [Il] in
hot summer (April to June). In southern China, azolla grows most abundantly from
February to May. In the Philippines, the growth is poorest in April and May when
monthly average temperature exceeds 32 Oc [44]. Watanabe et al. [43] reported
that in controlled temperature with 8 Oc differences between day and night, the
growth of A. pinnata in culture solution was about the same at 26 Oc (day)/18 Oc
(night), 29 oC/21°C, and 32 °C/24 oC, but was reduced by about 50% at 35 °/27 Oc
(average 31°C). The maximum biomass is more adversely affected by higher
temperature than the growth rate at low plant density [42]. A pinnata dies pro·
gressively at temperatures higher than 40 Oc and lower than 5 oC. Most of the
experiments to examine the response of the fern to temperature were conducted at
constant day temperature or without the shift of temperature from day to night.
A. filiculoides could not grow at 40 Oc (day)/30 Oc (night) temperature. However, if
the fems are grown at lower temperatures and then subjected to a step-wise increase
in temperature simulating dawn to midday of the diurnal cycle, the nitrogenase
activity increases with temperature up to 40°C and remains high at 45°C.
SiInilarly, A. filiculoides continues to fix N2 in the field during hot (40-45 OC)
afternoons [39]. Temperature response varies with light intensity [1]. The lower
the temperature, the lower the optimum light intensity for the growth of nitro-
genase [2, 39]. The temperature response is also dependent on the source of
nitrogen [2]. In the tropics, the poor growth of A. pinnata in hot summers (average
monthly temperature exceeding 30 oC) is a problem to be overcome for the agri-
cultural use of the fern-alga association.
176

3.2. Light

Short periods of exposure experiments on various light intensities showed that light
saturation to nitrogenase is about 250,uE m-2 sec- l (20 klux) by Talley and Rains
[39], 5 klux by Peters [23] and 10 to 5 klux by Watanabe [42]. For long-term
experiments, the fem requires higher light intensity than in the short-term exposure
experiments, because the growing fronds overlap each other. Ashton [2] observed
that the growth rate of A. filiculoides increased with increasing light intensity to a
maximum in 50% sunlight (49 klux), but further increase oflight intensity retarded
the growth rate. Talley and Rains [39] however, did not observe retardation of
growth of A. filiculoides under artificial light when light intensity was increased
from 500,uEm-2 sec- l to 1000 (ca. 80klux) when the temperature during illumi-
nation was higher than 25 oC. The apparent discrepancy may be due to the plant
density, temperature, and light source. Although shading reduces not only light
intensity but also temperature of water and air during sunny rnidday, experiences
tell that shading is beneficial for the growth of A. pinnata during hot summer. A.
pinnata, A. mexicana, and A. caroliniana have been observed to tum red in strong
sunlight and remain green in shading.

3.3. Humidity

Optimum relative hurnidity is reported to be 85-90% [47] . At a relative humidity

lower than 60%, azolla becomes dry and fragile and more susceptible to adverse
condition.

3.4. Mineral requirements

The mineral composition of azolla is summarized in Table 3. Although reported

figures vary greatly due to excess uptake, it would be reasonable to assume that
macronutrient contents in their sufficiently supplied levels are as follows (in
percent to dry weight): N: 4-5, P: 0.5, K: 1.0-2.0, Ca: 0.5, Mg: 0.5, Fe: 0.1.
In batch cultures of azolla in nutrient solution the levels of nutrients to induce
mineral deficiency symptoms are reported [41]. Yatazawa et al. [45] carefully
examined the threshold concentration of macroelements in nutrient solution by
using the inoculum that was precultured in the nutrient-deficient solution. Thres-
hold concentrations for A. pinnata growth were 0.03, 0.04, 0.4 and 0.5 rnmol
liter-l for P, K, Mg and Ca, respectively.
Phosphorus-deficient azolla turns reddish brown, fronds become fragile, and
roots are elongated and easily detached. Anabaena cells become pale green and
deformation of vegetative cells and heterocysts occur. The browning of dorsal
lobes starts from the newer leaves in A. mexicana, but from the older leaves at the
basal parts of stems in other species. Reddening of dorsal lobes is more intense in
Table 3. Mineral composition of Azolla
Species Conditions Percent in dry matter References
N P K Ca Mg Fe
A. filiculoides Water culture 0.79 6.5 0.25 0.30 Tuzimura et al. [41J
A. filiculoides Soil culture 0.95 1.6 1.0 0.36 Tuzimura et al. [41J
A. filiculoides Naturally grown 45 05 2.0 0.97 0.1 Buckingham etai. [5]
A. pinnata Naturally grown 45 05 245 0.4 0.5 0.06 Singh [34]
-0.9 1.0 -0.6 0.26
A. filiculoides Naturally grown 2 0.1 2.0 0.6 0.4 0.3 Lumpkinand
-3.4 -0.4 2.6 -0.8 -0.6 05 Plucknett [19]
A. pinnata Water culture 5.0 1.0 3.2 0.2 0.7 0.08 Watanabe (unpublished)
(deficient) (0.08) (0.4) (0.05) (0.016)

-
-...l
-...l
178

calcium-deficient azolla, than in phosphorus-deficient plants. The fronds turn

fragmented and algal cells are lost from the cavity in the extreme Ca-deficient plant.
In potassium-deficient plants, yellowish browning also occurs. In iron-deficient
azolla, cWorophyll content decreases and the plant turns yellowish [43] .
Effect of microelements are also reported by Yatazawa et al. [45] and Johnson
et al. [16] .
Azolla grows in aquatic habitats and absorbs nutrients mainly from the water. In
shallow water the plant root attaches to the soil and plant absorbs nutrients from
the soil. Because phosphorus content in soil solution or in paddy water is generally
too low to meet the requirement by azolla, the addition of phosphorus is necessary
for better growth of azolla [44] .
To determine the minimum level of phosphorus in water medium, continuous
flow culture was used. At 2 Ilmol P 1-1 , Azolla pinnata from Bangkok grew
normally, but at 11lmol P 1-1 , the azolla suffered phosphorus-deficiency symptoms
[35] . The growth of various species and strains of azolla and the difference in sup-
porting growth at 1 Ilmol 1-1 were observed and compared (Subudhi and
Watanabe, unpublished).

4. Agricultural use of AzolIa

A book on Chïnese agricultural techniques - the essence of feeding people, written

in 540 A.D. - describes the cultivation and use of azolla in rice fields. At the
beginning of the 17th century (end of Ming dynasty), many local records reported
the use of azolla as manure [18] .
Azolla used in China originated in Fujiang and Guangdong and spread to rice
fields in other provinces, south of the Yantze River. Since the foundation of the
People's Republic of China, azolla techniques as green manure for rice and as
animal feed have been greatly encouraged and have spread north of the Yantze
River. In double cropping of rice in central and southern China, azolla is grown
only before the early rice (March-May).
In northern Vietnam, A. pinnata has been used as a green manure crop for
centuries. It is believed that a peasant in La Van village of Thai Binh province
discovered and domesticated the azolla. Before the revolution, sorne families in the
village knew the techniques of rearing azolla starter from April to November. The
villagers began selling starter stocks of azolla to regional propagators at high prices
[21] . Nowaday~, the maintenance and multiplication of starters, and the further
propagation from the starters, are systematically conducted and distributed from
regional azolla multiplication centers to farmers' cooperatives. Azolla is grown in
January and February in paddy fields before spring rice and then incorporated
[40] .
 179

S. Green manure for rice

Because of its high N2 -fixing ability and nitrogen content, azolla has high potential
as green manure crop for wetland rice. It is grown either before or after trans-
p1anting rice.
One crop cff A. pinnata contains about 20-40 kg N ha- I . A Vietnamese study
showed that incorporation of 1 ton fresh azolla increased the average rough rice
yield by 28 kg in 1958-1967 [6]. If 20 t fresh weight of azolla ha -1 is produced
before transplanting (this figure is reasonable) about 0.5 t ha- I rice yield increase
will be obtained.
In 1975, a conference on azolla for southern China summarized findings from
1,500 experiments in 7 provinces (Chiangxu, Guandong, Fujien, etc). Azolla as
manure increased rice yield by 600-750 kg ha- I . Ninety percent of 422 field
experiments in Chekiang Province reported an average increase in rice grain yield of
700kgha- 1 or 18.6% [18].
Lately, interest in azolla as green manure for rice was resumed in other south
Asian countries. The International Rice Research Institute organized INSFFER
(International Network of Soil Fertility and Fertilization Evaluation for Rice)
collaborative network activity to test the effect of azolla as green manure. Scientists
from 5 countries joined the network and field experiments were conducted at 12
sites in 1979. The results are summarized in Table 4. Positive responses of azolla
incorporation either before or after transplanting rice over no nitrogen control were
obtained in 10 sites. Growing of azolla before or after the rice produced a rice
yield increase equivalent to that obtained from 30 kg N ha -1 as urea or ammonium
sulphate. Growing of azolla and its incorporation before and after rice increased

Table 4. Effects of azolla and nitrogen fertilizer on rice yield, International Network of Soil
Fertility and Fertilizer Evaluation for Rice (INSFFER) trialsa
Treatment Average Index
grain yield (t ha -1 )
1 No nitrogen 2.6 100
2 30 kg N ha -, chemical fertilizer 3.2 122
3 60 kg N ha- I 3.7 141
4 Azolla grown before transplanting, 3.2 122
incorporated
5 Azolla grown after transplanting, 3.1 118
incorporated
6 Azolla grown after transplanting 3.1 119
7 30 kg N ha-' + Azolla before transplanting, 3.7 143
incorporated
8 30 kg N ha- 1 + Azolla after transplanting, 3.5 134
incorporated
9 Azolla grown before and after transplanting, 3.6 139
incorporated

a Conducted in 8 sites in Thailand, 2 in India, and 1 each in China and Nepal

180

rice yield equivalent to that obtained from 60 kg N ha- 1 as chemical N fertilizer.

Whether azolla was incorporated or not after it covers fully the paddy surface in the
rice canopy did not affect rice yield.
An average N2 -fIxing activity of 1-2 kg N ha- 1 day-l, which is shown by the
Azolla-Anabaena complex, is suffIcient to meet the nitrogen requirement of rice if
azolla is grown for the period of one rice cropping.
By widening the distance between rice rows, azolla was grown continuously in
the rice canopy [18]. This technique was examined at the International Rice
Research Institute. Wide rows (53 cm) were alternated with narrow rows (13 cm).
Distance between hi1ls was 6.6 cm. Azolla was grown six or four times and
incorporated into the soil after water was drained. A total of 100-70 kg N ha- 1
were contained in the incorporated azolla. Grain yield was almost equivalent to that
obtained from 70-100 kg N ha- 1 chemical nitrogen fertilizer (Watanabe,
unpublished).
The decomposition of azolla is rapid and nitrogen efflciency of azolla is almost
comparable or slightly inferior to that of urea or ammonium sulphate [34, 43] .
Principally, azolla is grown in the fIelds, where rice is grown after the azolla harvest
or together with azolla as described above. Alternately, azolla is grown con-
tinuously year round in paddy fIelds or in the adjacent ponds. Excess of azolla,
after it has been used for rice, can be composted for dryland or vegetable crops.

6. Management practices

The inoculum of azolla must be healthy and fresh. It is continuously multiplied in

the inoculum preparation plots or ponds. The inoculum density is an important
factor in the effIcient production of azolla. Singh [34] recommends 2 tons fresh
weight ha -1 as the inoculum size. In Vietnam, 5 tons ha -1 or more is recommended.
When inoculum density is low, azolla is overgrown by algae and weeds.
In Vietnam, the half-saturation method is recommended. The saturated density
of A. pinnata is about 10-20 t fresh weight ha- 1 • First, the available inoculum is
spread in an area to keep the density at 0.5 kg fresh weight m-2 • After one week,
the surface is fully covered. Then, half of the azolla is transferred to the open area
which has about the size as the area where the azolla was taken (see Fig. 3). After
one week or so, both areas are fully covered by azolla. Again, half of the azolla is
taken from both areas and transferred to the fIeld about the size of the area where
the azolla was taken. By repeating this procedure, the area covered by azolla is
exponentially expanded. Azolla is notably responsive to phosphorus fertilizer and
requires a continuous supply of water-soluble phosphorus for rapid propagation.
Split application of superphosphate is more effIcient in promoting azolla growth
than basal application and 1 kg P2 05 ha- 1 every 4 days is recommended [44]. The
Vietnamese recommended 5-10 kg superphosphate (1-2 kg P2 0 S ha- 1 ) every
5 days. Singh [34] recommended 4-6 kg P2 0 S ha-1 every week. Superphosphate
 181

10 -15 kg fresh wt azalla

20m
2
~
t,.".
full caver

"'w

full caver
5 -7 dal after

160 m 2

t
>-.
transfer
1

full caver
5-7 days
after full caver
transfer ~
- - - - ;...

80 m2

Fig. 3. Schematic of the half-saturation method for A. pinnata.

must be applied on the surface of azolla. In China, 3% (w/w) suspension of super-

phosphate with 0.21 m- 2 is recommended.
In a suitable condition each kg of P2 05 results in 2 kg additional nitrogen in the
azolIa biomass [44] .
The application of potassium is recommended in light mineraI soils. Talley et
al. [37] obtained significant azolla growth response from a single application of 0.8
kg Fe ha- 1 as ferric chelate. When soil is flooded before azolla inoculations, iron
from the reduced layer becomes available to azolla growth on floodwater.
Water control is critical. Water depth should be kept at about 3-5 cm. When
water depth is less than 3 cm, the roots attach to the soil and mineral deficiency is
recovered. In hot summer, keeping the water shallow to allow azolla roots to attach
to the soil is effective in maintaining healthy azolla. When azolla is incorporated,
water is drained and azolla is easily turned under by rake.
0
Insect damage is serious, particularly in hot summer (higher than 30 C), because
generation time of insects decreases at higher temperature.
Tran and Dao [40] identified the main insect pests of azolla in Vietnam as larvae
182

of Pyralis, Nymphula, and Chironomus species. In China these genera are also the
major and most destructive insect pests [18]. Two species of Nymphula, brown
Nymphula (N. tarbata) and black Nymphula (N. swindol), are important. Adults of
brown Nymphula have yellow brown wings and those of black Nymphula have
black wings. Their eggs are laid on the back side of ventral lobes and are hatched
after 4 or 6 days at 30°C. The larvae attack azolla and form burrows embedded by
the silk and fragmented azolla leaves and roots. They come out of the sack while
eating azolla. They also eat other aquatic plants.
The gray pyralid·Pyralis sp. lays whitish oval eggs on the periphery of the space
between the dorsal and ventral lobes. The newly hatched larva is grayish white,
but becomes grayish blue or green in later stage. The duration of the Ist and 2nd
instar stage at about 30 Oc is 3 days. At the end of the 2nd instar, they construct
burrows and eat the shoot of the fem. Pupae live for 4-5 days and so do the
adults. Total development takes 20 days (Lin Shi·he, Guangxi Province, personal
communication). In southern China, this pyralis is the most destructive one. There
are 8-10 generations a year, the generations overlap. The damage is easily visible
by the lines of burrows. In addition to Pyralis and Nymphula, Chironornid (Poly·
pedilium, Mierospeetor, Tendipes, smout beetle (Bagous sp.), and snail (Radix
swinhoer) are recorded as pests in southern China.
In a hot climate, the damage by fungus attack is also serious, but probably the
fungus attack follows the damage by insects or desiccation. Sclerotium and
Rhizoetonia are recorded [32] .
Because insect damage is serious in a hot climate (> 30 oC), it is a major con-
straint in growing azolla in the tropics. A close watch is needed to predict the
sudden outbreak of insect pests. The surface of azolla mats should be carefully
examined for the presence or traces of insect burrows. The collecting lamp is also
usefuI for the prediction of lepidopterous insects.
The application of 3-4 kg a.i. carbofuran or ferithion per ha is effective, par-
ticularly when the application is made with phosphorus fertilizer. Dipping the
inoculum in 1000-3000 times diluted insecticide solution is also effective.
In China, bacterial insecticides have also been tried to control azolla pests [18] .

7. Other uses of azolla

Azolla can act as a weed suppressor, because the azolla mat covering the surface
depresses the growth of weeds.
Rains and Talley [30] reported that early development of A. filieuloides eH·
minated Cyperus difformus and Polygonum species from the paddy, but not
Eehinoehloa erusgalli which is taller. Total biomass was reduced by the azolla cover,
although Eehinoehloa predominated. In the International Rice Research Institute's
fields where azolla has been continuously grown, sporadic growth of Monoehoria
vaginalis (non-submerged weed) is noticed.
In Hawaii, taro growers used A. [ilieuloides as weed suppressor [8]. But there
 183

are opinions that azolla is harmful to nce. Fujiwara et al. [9] mentioned that a
Japanese farmer who used azolla as green manure observed that a thick mass of
azolla covered the young shoot of nce when water was flooded, resulting in the
death of rice seedlings. The azolla mat at the early stage of nce, when temperature
is still cool, lowers water temperature, resulting in the slight depression of tiller-
ing. In direct-seeded nce, quick growth of azolla may be harmful for young rice
seedlings.
Azolla has also been used as food to fish and domestic animals in Asia and
Africa, because it is rich in protein [36]. It is fed to cattle, pigs, ducks, and fish.
Buckingham et al. [5] analyzed the nutritional value of A. filiculoides and con-
cluded that protein in azolla had a low nutritive value for growing rat. Addition of
lysine, methionine, and histidine was effective in improving quality. The high (39%)
neutral fibre content of azolla was a major limiting factor in the efficient use of
azolla as protein source for monogastric animaIs. In vitro digestability data showed
it would be a useful source for ruminants. However, the azolla samples were taken
from the creek in an urban area and nothing was described about the age of the
fem. Fibre content of their samples was much higher than that reported by
Fujiwara et al. [9] . Change of nutritive value according to the age of azolla needs to
be examined. Singh [34] also suggested the possibility of azolla as human food.

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DEVELOPMENTS IN PLANT AND SOIL SCIENCES
VOLUME 5

MICROBIOLOGY
OF TROPICAL SOILS
AND PLANT PRODUCTIVITY
Y.R. DÜMMERGUES/H.G. DIEM
(EDITüRS)

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I.ihrar.\ ur Cougn·.... ('atalnJ..::il\~ in Pllhli(';lliulI n,lia

Main entry under title:

Microbiology of tropical soils and plan~

productivity.

(Developments in plant and soil sciences

v. 5)
Includes index.
1. Soil microbiology--Tropics. 2. Micro-
organi~ms, Nitr9gen-fixing:-Tropics: 3. Crops
and so~ls--Trop~cs. 4. So~ls--Trop~cs. 5. Optimal
soil fertility--Tropics. I. Dommerguès.,
Y. R. (Yvon R.) II. Diem, H. G. III. Series.
QRlll.M4 631.4'6 81-22536
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