Azolla - Anabaena: Symbiosis - Its Physiology and Use Tropical Agriculture
Azolla - Anabaena: Symbiosis - Its Physiology and Use Tropical Agriculture
Azolla - Anabaena: Symbiosis - Its Physiology and Use 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] .
Mes
2.
YI
Ind
'I[;~:U::\::'" Ma
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
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.
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
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
-
-...l
-...l
178
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
6. Management practices
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
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] .
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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Anabaena symbiosis. Planta 122, 179-184.
13. Hill, D.J. 1977 The ro1e of Anabaena in the Azolla-Anabaena symbiosis.
New Phytol. 78,611-616.
14. Hills, L.V. and Gopal, B. 1967 Azolla primaeva and its phy10genetic signifi-
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15. International Rice Research Institute. 1979 International Bibliography on
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16. Johnson, G.V., Mayeux, P.A. and Evans, H.J. 1966 A cobalt requirement for
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185
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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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