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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.

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.

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).

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.

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.

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.

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.

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.

The fowing explanation is from Jim Deacon of the Institute of Cell and Molecular Biology, The University of Edinburgh.

Two molecules of ammonia are produced from one molecule of nitrogen gas. The reaction requires 16 molecules of ATP and a supply of electrons and protons (hydrogen ions) plus the enzyme nitrogenase. Nitrogenase consists of two proteins, an iron protein and a molybdenum-iron protein. The reaction occurs while N2 is bound to the nitogenase enzyme complex. The Fe protein is first reduced by electrons donated by ferredoxin. Then the reduced Fe protein binds ATP and reduces the molybdenum-iron protein, which donates electrons to N2, producing HN=NH. In two futher cycles of this process (each requiring electrons donated by ferredoxin) HN=NH is reduced to H2N-NH2, and this in turn is reduced to 2 NH3. Depending on the type of microorganism, the reduced ferredoxin which supplies electrons for this process in generated by photosynthesis, respiration or fermentation.

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.

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. 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.

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.

In the online newsletter Inverse (2018) Science writer Sarah Sloat has summarized the article about the economic importance of Azolla in Nature Plants (2018) by Fay-Wei Li, Paul Brouwer & Kathleen M. Pryer:

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. Li, Fay-Wei, Brouwer, P. and K. M. Pryer. 2018. "Fern Genomes Elucidate Land Plant Evolution and Cyanobacterial Symbioses." Nature Plants 4: 460-472. 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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