There are many examples of plants, bacteria
and algae that have formed intimate symbiotic associations or
"marriages" with each other. Divorce is practically nonexistent
in these marriages, and separations may result in the death of
one or both partners. In some cases the relationship is
decidedly one-sided, with only one partner actually benefiting.
These relationships are often termed parasitic, especially when
the non-benefiting partner is actually harmed by the
relationship. In other marriages the relationship is mutually
beneficial. Algae and fungi live together in an association
called lichen, and nitrogen-fixing bacteria live symbiotically
inside the root nodules of legumes. But one of the most
fascinating of all plant marriages involves a tiny aquatic water
fern (Azolla) and a microscopic filamentous blue-green alga
or cyanobacterium (Anabaena azollae). They grow together
at the surface of quiet streams and ponds throughout tropical and
temperate regions of the world.
Ponds along the San Dieguito River (San Diego County, California) are covered with a reddish carpet of Azolla filiculoides during the fall months. Photo also shows clump of cattails (Typha latifolia) and naturalized Australian Eucalyptus camaldulensis. The Eucalyptus trees were introduced into California near the turn of the century for fast-growing hardwood lumber for railroad ties, but proved inadequate because the spikes would not hold in the badly checked wood. Now these trees have literally taken over parts of San Diego County.
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A pond along the San Dieguito River (San Diego County, California) covered with a reddish carpet of Azolla filiculoides. The bright green areas are masses of the filamentous green algae, Mougeotia and Spirogyra.
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Virtually any sample of Azolla
examined under a microscope will have filaments of
Anabaena living within ovoid cavities inside the leaves.
Like nitrogen-fixing bacteria living inside the root nodules of
legumes, the relationship appears to be mutually beneficial.
Since Azolla is easy to maintain in aquarium cultures, it
is an excellent source of prokaryotic cells and heterocysts
for general biology laboratory exercises on cell structure and
function. It also has an interesting heterosporous life cycle and
can readily be adapted to laboratory exercises on symbiosis. In
addition, this little fern and its algal partner provide an
important contribution toward the production of rice for a hungry
world.
It has been estimated that there are at
least 10,000 different species of ferns in the world, from large
tree ferns of tropical rain forests to small rock ferns of desert
canyons and alpine crevices. Fossil evidence indicates that many
additional species of ferns flourished on earth during the
Carboniferous period, some 300 million years ago. But of all the
great diversity of ferns, relatively few kinds have colonized the
water. Azolla belongs to the Salvinia Family
(Salviniaceae), although some authorities now place it in the
monotypic family, Azollaceae. Six species are distributed
worldwide, three of which occur in the United States: A.
filiculoides, A. mexicana and A. caroliniana.
Individual Azolla plants have
slender, branched stems with minute, overlapping scalelike leaves
only one millimeter long. Each plant resembles a little floating
moss with slender, pendulous roots on its underside. The plants
tend to clump together and often form compact mats on the water
surface. Azolla is sometimes called "duckweed fern" and
commonly grows with one or more species of duckweeds (Lemnaceae),
including Lemna, Spirodela, Wolffia and Wolffiella. When growing
in full sunlight, particularly in late summer and fall,
Azolla may produce reddish anthocyanin in the leaves, in
contrast with the bright green carpets of duckweed and
filamentous green algae.
Several water fern plants (Azolla filiculoides) floating on the water surface. The plant in upper right with oval fronds is a duckweed (Lemna minuta). The minute plants which resemble tiny green bubbles are Wolffia borealis and another interesting species W. columbiana, two of the world's smallest flowering plants.
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The stems of Azolla pull apart
readily and the plants reproduce by fragmentation at an
astonishing rate. In fact, some species can double their biomass
in three days under optimal environmental conditions. Entire
ponds and muddy banks may be completely covered by a carpet of
velvety green or pink. In addition to providing food for various
water fowl, Azolla also provides the habitat and food for
numerous kinds of freshwater insects, worms, snails and
crustaceans.
A related African water fern (Salvinia rotundifolia), also listed as S. auriculata in some floras, is mentioned in the Guinness Book of World Records (1985
UK Edition) as the "most intransigent weed." This mat-forming
aquatic fern was detected on the filling of Kariba Lake in May
1959. Within 11 months it covered an area of 77 square
miles (199 km2), and by 1963 it covered 387 square
miles (1002 km2).
The water fern (Salvinia rotundifolia), a ubiquitous floating fern in quiet waters of streams and ponds throughout tropical America, Africa and Florida. The small duckweed in photo is Landoltia punctata.
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Sexual Reproduction In Azolla
Not only are Azolla species prolific
vegetative reproducers, but they also have a very interesting and
uniquely specialized sexual cycle. Like all ferns, Azolla
produces spores; however, unlike most ferns, Azolla
produces two kinds of spores. If you carefully examine Azolla
filiculoides during the summer months you can easily find
numerous spherical structures called sporocarps on the undersides
of the branches. The sporocarp of Azolla is homologous to
the sorus of other ferns, and the sporocarp wall represents a
modified indusium. The male sporocarp is a greenish or reddish
case about two millimeters in diameter, and inside are numerous
male sporangia which look like the egg mass of an insect or
spider inside a transparent case. Male spores (microspores) are
extremely small and are produced inside each microsporangium.
Close-up view of Azolla filiculoides showing scalelike, overlapping leaves and several globose reproductive structures called sporocarps. The male sporocarp (middle right) contains microsporangia that resemble eggs inside an egg sac. One sporocarp has broken open releasing many spore clusters or massulae. The minute ovoid plants in center are Wolffia borealis, a minute flowering seed plant.
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Closeup view of Azolla filiculoides showing a small female sporocarp flanked by two larger, globose male sporocarps. These tiny structures are so small that they could easily fit on the head of an ordinary straight pin.
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One very curious thing about microspores is that they tend to stick together in little clumps or masses called massulae. In the
American species (Subgenus Euazolla), each spore mass (massula)
is covered with minute hairs (barbed at the tips) called
glochidia. Under high magnification the massulae look like
strange space satellites with radiating antennae. The female
sporocarps are much smaller, and contain a single sporangium and
a single functional spore. Since an individual female spore is
considerably larger than a male spore, it is termed a
megaspore.
Highly magnified view (400X) of one spore mass (massula) of Azolla filiculoides with unique barbed projections called glochidia. In a related species of water fern (A. mexicana), the glochidia are septate with several distinct partitions.
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Although rarely seen by the casual
observer, the spores of most ferns develop into a fleshy, heart-
shaped structure called a prothallus or gametophyte, which
produces the actual sex organs (the female archegonium and male
antheridium). Azolla has microscopic male and female
gametophytes that develop inside the male and female spores. The
female gametophyte protrudes from the megaspore and bears one to
several archegonia, each containing a single egg. According to
Scagel, et al. (An Evolutionary Survey of the Plant Kingdom,
1966), the microspore forms a male gametophyte with a single
antheridium which produces eight swimming sperm. The barbed
glochidia on the male spore clusters presumably cause them to
cling to the female megaspores, thus facilitating
fertilization.
According to some references, the universal
occurrence of Anabaena azollae inside the leaves of
Azolla suggests that reproduction of this water fern may
be chiefly vegetative; however, it has been shown that this
cyanobacterium and fern may develop in synchrony. Short filaments
of Anabaena, called hormogonia, often survive under the
"indusium cap," on top of the germinating megaspore. Hormogonia
may be entrapped by the embryo Azolla plant during
differentiation of the shoot apex and dorsal lobe primordia of
the first leaves. It is fascinating to speculate on just how and
when these two diverse organisms formed such an intimate
association. Although filamentous cyanobacteria (with cells
resembling heterocysts) date back more than two billion years,
fossil Azolla plants are known only from late Cretaceous
deposits less than 80 million years ago.
Anabaena and Nitrogen Fixation
Close examination of an Azolla leaf
reveals that it consists of a thick, greenish (or reddish) dorsal
(upper) lobe and a thinner, translucent ventral (lower) lobe
emersed in the water. It is the upper lobe that has an ovoid
central cavity, the "living quarters" for filaments of
Anabaena. Probably the easiest way to observe
Anabaena is to remove a dorsal leaf lobe and place it on a
clean slide with a drop of water. Then apply a cover slip with
sufficient pressure to mash the leaf fragment. Under 400X
magnification the filaments of Anabaena with larger, oval
heterocysts should be visible around the crushed fern leaf. The
thick-walled heterocysts often appear more transparent and have
distinctive "polar nodules'' at each end of the cell. According
to Dr. C. P. Wolk at Michigan State University Plant Research
Laboratory (personal communication, 1984), the "polar nodules"
may be the same composition as cyanophycin granules (co-polymer
of arginine and aspartic acid). Cyanophycin granules occur in
many cyanobacteria and may serve as a nitrogen storage
product.
Modern-day filamentous cyanobacteria (Anabaena azollae) from cavities within the leaves of the ubiquitous water fern (Azolla filiculoides). The larger, oval cells are heterocysts (red arrow), the site of nitrogen-fixation where atmospheric nitrogen (N2) is converted into ammonia (NH3). Polar nodules are visible in some of the heterocysts. The water fern benefits from its bacterial partner by an "in house" supply of usable nitrogen. The cellular structure of these bacteria has changed very little in the past one billion years.
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Nitrogen fixation is a remarkable
prokaryotic skill in which inert atmospheric nitrogen gas
(N2) is combined with hydrogen to form ammonia
(NH3). This vital process along with nitrification
(formation of nitrites and nitrates) and ammonification
(formation of ammonia from protein decay) make nitrogen available
to autotrophic plants and ultimately to all members of the
ecosystem. Although Azolla can absorb nitrates from the
water, it can also absorb ammonia secreted by Anabaena
within the leaf cavities.
Recent studies have shown that the actual
site of nitrogen fixation occurs within the thick-walled
heterocysts. As the heterocyst matures, the photosynthetic
membranes (thylakoid membranes) become contorted or reticulate
compared to regular photosynthetic cells of Anabaena, and
they become non-photosynthetic (and do not produce oxygen). This
fact is especially noteworthy because nitrogen fixation requires
the essential enzyme nitrogenase, and the activity of
nitrogenase is greatly inhibited by the presence of oxygen.
Azolla and Rice Productivity
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 Wilson Clark (Science
80: Sept./Oct. 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.
In addition to nitrogen fixation, Azolla
has a number of other uses. Several California aquafarms grow
Azolla in large vats of circulating fresh water. Apparently fish
and shrimp relish the Azolla. In fact, Azolla was grown for fish
food and water purification at the Biospere II project in Arizona
(a 2.5 acre glass enclosure simulating an outer space
greenhouse). Fresh Azolla and duckweed (Wolffia) can also be
used in salads and sandwiches, just as alfalfa and bean sprouts
are used. Dried, powdered Wolffia and Azolla make a nutritious,
high protein powder similar to the popular alga (cyanobacterium)
Spirulina that is sold in natural food stores. Azolla has also
proved useful in the biological control of mosquitos. The
mosquito larvae are unable to come up for air because of the
dense layer of Azolla on the water surface. Azolla grows very
quickly in ponds and buckets, and in makes an excellent
fertilizer (green manure) and garden mulch.
A male tree frog (Hyla regilla) floating in a pond of Azolla filiculoides. On a warm summer night, the amazing chorus of dozens of these small frogs can be almost deafening.
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Azolla is certainly a valuable
laboratory plant that will thrive with very little care. In fact,
at the WAYNE'S WORD headquarters, the hippo staff has maintained
buckets of Azolla in ordinary tap water, although it may
exhibit seasonal fluctuations in density and vigor. Depending on
the sophistication of viewing equipment and level of study, this
plant can fascinate biology students from junior high school to
college. But its value goes far beyond the classroom. The use of
Azolla may be an important factor in the world's future
food needs and may play an important role in reducing the world's
reliance on fossil fuel-based fertilizers. The significance of
its symbiotic relationship with Anabaena is astounding
when one considers that millions of lives depend on these two
organisms.
References
- Armstrong, W.P. 1979. "A Marriage Between a Fern and an Alga." Environment Southwest No. 500: 20-24.
- Armstrong, W.P. 1985. "A Fern-Alga Symbiosis." Carolina Tips 48: 9-11.
- Clark, W. 1980. "China's Green Manure Revolution." Science 80 1: 69-73.
- Lumpkin, T.A and D.L. Plucknett. 1980. "Azolla: Botany, Physiology, and Use as a Green Manure." Economic Botany 34: 111-153.
- Scagel, R.F., R.J. Bandoni, G.E. Rouse, W.B. Schofield, J.R. Stein and T.M.C. Taylor. 1966. An Evolutionary Survey of the Plant Kingdom. Wadsworth Publishing Co., Inc., Belmont, California.
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