Anabaena (PROSEA)
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Introduction |
Anabaena (Bory) ex Bornet & Flahault
- Protologue: Révis. Nostoc. hét.: 224 (1886) [1888].
- Family: Nostocaceae
- Chromosome number: Prokaryotic, thus no chromosomes
Major species and synonyms
Anabaena azollae Strasb. ex Wittr., Nordst. & Lagerh., Alg. aquae dulc. exs. 28: 1340 (1896), synonyms (Drouetian):
- Anabaena oscillarioides Bory ex Bornet & Flahault (1886) [1888],
- A. variabilis status azollae Fjerd. (1976).
- Accepted name is now: Trichormus azollae (Strasburger) Komárek & Anagnostidis (1989).
Anabaena flos-aquae (L.) Bory ex Bornet & Flahault, Révis. Nostoc. hét.: 241 (1886) [1888], synonym (Drouetian):
- Microcoleus vaginatus (Vaucher) Gomont ex Gomont (1892).
- Accepted name is now: Dolichospermum flosaquae (Brébisson ex Bornet & Flahault) P.Wacklin, L.Hoffmann & J.Komárek (2009).
Anabaena siamensis Antarik., Nova Hedw. 41: 343 (1985).
- Accepted name is now: Cylindrospermum siamense (Antarikanonda) J.R.Johansen & M.Bohunická (2014).
Origin and geographic distribution
Anabaena is a cosmopolitan genus and occurs primarily in freshwater, although a few species are found in marine environments. One taxon, occurring as a symbiont inside the leaves of water ferns of the genus Azolla Lamk, is usually considered to consist of one single species: Anabaena azollae. These water ferns are found in freshwater ecosystems of temperate and tropical regions throughout the world. In most of Asia the Azolla taxon is Azolla pinnata R. Br. subsp. asiatica Saunders & Fowler. Additional data on Azolla will be included in Prosea 15(2) on ferns and fern allies see Azolla pinnata (PROSEA).
Uses
The Azolla-Anabaena association is used as an alternative nitrogen source in agriculture, especially in wet-rice cultivation, and is regarded by many as a "green gold mine". The algal symbiont is capable of fixing atmospheric nitrogen at high rates. The enzyme necessary for nitrogen fixation, nitrogenase, is oxygen-labile and is assumed to be located in the heterocysts of Anabaena. Azolla is most effective when grown as a cover crop during the fallow season of rice and incorporated as green manure into the soil. However, nitrogen is also provided when Azolla is grown in dual culture with wet rice. Natural or cultured growths of Azolla may be harvested and applied to various crops including taro (Colocasia esculenta (L.) Schott) and wheat (Triticum spp.), either by incorporating them into the soil before planting the crop or by applying them as a mulch on top of the soil around the crop plants. Often, a combination of methods of application is used.
Although the use of Azolla is labour intensive, as much as 100 kg N/ha can be provided to a rice crop when planted in double rows. Azolla is cultivated on the paddy field and, after briefly draining the field, it is incorporated into the soil at intervals during the growing season. Azolla can also be used as a fodder for pigs, cattle, poultry and fish. In addition, Azolla (and thus also Anabaena azollae) can be used as human food, medicine and water purifier. When the Azolla-Anabaena association is grown in a nitrogen-free atmosphere and/or a water medium containing nitrate, the nitrogenase in the symbiont, instead of fixing nitrogen, evolves hydrogen from the water. Hydrogen is a non-polluting high-energy fuel.
Additional nitrogen supply can also be realized by growing blue-green algae alone on suitable soils. A. siamensis is very suitable as a -ricefield inoculum because of its good nitrogen-fixing capacity and because its akinetes are capable of surviving long dry periods. In general, using free-living blue-green algae does not increase the rice grain yield as much as applying the Azolla-Anabaena association. Under less favourable conditions the latter method is, however, profitable.
A. azollae isolates, when immobilized in polyurethane foam and added to rice seedlings, significantly increase root and shoot growth, chlorophyll content, and biomass production of these rice seedlings in laboratory cultures. Experimental inoculation of rice with A. azollae trichomes immobilized in polyurethane foam or sugar-cane waste has resulted in higher ammonia amounts in the flood water and increased rice grain and straw yields.
Production and international trade
Azolla (and thus also Anabaena azollae) is produced on a large scale in India, Bangladesh, Indonesia (Sulawesi), Thailand, Vietnam and China.
The Azolla-Anabaena associations have a long history as a green manure for wet rice in the Far East. Jia Si Xue published in 540 A.D. a book "Chih Min Tao Shu" (The art of feeding the people) describing the cultivation and use of Azolla in rice fields. Nevertheless, the application of Azolla in China did not expand markedly until 1962, since when it has been extended, first to about 1.34 million ha in 1978 then to 6.5 million ha in 1995. The use of Azolla in Vietnam can likewise be traced back many centuries, although in 1955 it was restricted to about 40 000 ha in the Red River delta, extending to 320 000 ha by 1965, covering about 40% of the area under rice. The association is mainly grown here during November-January in fallow, flooded fields to be used for "spring" rice.
Properties
All Anabaena are able to fix atmospheric nitrogen. A. azollae , as well as the non-symbiotic A. variabilis Kütz. ex Bornet & Flahault, when immobilized in polyurethane and polyvinyl foams or calcium alginate beads, release ammonia extracellularly, especially in the presence of L-methionine-D,L-sulphoximine. Different isolates differ in growth rate, biomass production and nitrogenase activity, and the immobilization usually further increases growth and ammonia excretion. Some soil Anabaena had positive plant growth regulator effects when extracts of these algae were administered to rice laboratory cultures.
Especially the planktonic A. flos-aquae is notorious for producing toxic blooms in freshwater. Anatoxins released by A. flos-aquae and A. circinalis (Kütz.) Rabenh. ex Bornet & Flahault can paralyze skeletal and respiratory muscles in fish, poultry, pets, cattle and humans, in some cases resulting in death. No antidotes or treatments are currently available. Different strains of A. flos-aquae produce different types of toxin: anatoxin-a is a neurotoxic secondary amine alkaloid and a potent neuromuscular blocking agent, while anatoxin-a(s) is a N-hydroxyguanidine methyl phosphate ester which is a potent cholinesterase inhibitor. The (s) is added because of the promotion of salivation in these intoxications. Some Anabaena strains are also able to produce other toxins, like lipopolysaccharide (LPS) endotoxins in their cell envelopes, causing fevers and inflammation after bathing or showering with water containing these cyanobacterial blooms. Microcystins (cyclic hepatopeptides) cause acute liver poisoning. The latter toxin is better known as being produced by the cyanobacterium Microcystis aeruginosa (Kütz.) Kütz.
Description
- Trichomes 2.5-14 μm in diameter, unbranched, straight or coiled, not tapering, made up of 8-18 cells, with extracellular polysaccharides not accumulating as a gelatinous matrix.
- Cells spherical to barrel-shaped, isodiametric, or slightly shorter or longer than diameter, with constrictions at the cross-walls.
- Heterocysts numerous, intercalary, basal.
- Spores (akinetes) usually larger and more elongate than vegetative cells, single or in series between heterocysts.
A. azollae.
- Trichomes solitary or in small groups inside cavity of leaves of Azolla water ferns, straight or curved, about 5 μm broad, without a visible sheath.
- Cells quadrangular, ellipsoidal or subglobose, 4-5.5 μm × 5-9.5 μm, greenish-blue, with granular contents.
- Heterocysts ellipsoidal, solitary, 6-10 μm × 7.5-11.5 μm, olive-green, occasionally lacking; usually heterocyst frequency 20-30% when actively fixing nitrogen.
- Spores lacking under natural conditions, observed in cultures: 6-7.5 μm × 9-13 μm, singly or in short series, usually not located near heterocyst; epispore smooth, yellowish.
A. flos-aquae.
- Trichomes circinate, planktonic, free-floating, 4-8 μm broad, without visible sheath.
- Cells ellipsoidal, rarely spherical, as long as broad or longer, 6-8 μm long, mostly with gas-vacuoles.
- Heterocysts ellipsoidal, 4-9 μm × 6-10 μm; heterocyst frequency usually 4-8%.
- Spores prominently bent, convex on outside, straight on inside, 7-13 μm × 20-35(-50) μm, singly, usually located near heterocyst; epispore smooth, colourless or yellowish, often surrounded by wide gelatinous sheath.
A. siamensis.
- Thalli mucilaginous, bright blue-green; trichomes short, single, straight, distinctly constricted at cross-walls, 30-50 μm long, without a visible sheath.
- Cells quadrangular to cylindrical, 2-3 μm × 2-5 μm, without gas-vacuoles.
- Heterocysts unipolar, spherical or subspherical, present at both ends of trichome, 2-4 μm in diameter, with a distinct yellowish smooth outer wall and hyaline yellow content.
- Spores (akinetes) formed in series, oval to subspherical, 5-10 μm in diameter, located away from heterocyst; epispore smooth, colourless.
Growth and development
The relationship between A. azollae and Azolla is one of permanent symbiosis, i.e. the two organisms are associated in all stages of the life cycle of the fern and the association persists from one generation to the other, regardless of whether reproduction of the fern is sexual or asexual. It appears that certain bacteria present in the leaf cavities of the fern, particularly an Arthrobacter sp., constitute a third partner in the Azolla-Anabaena symbiosis. The role of the bacterium in the partnership is not yet understood.
Akinetes of A. azollae are present in both microsporocarps and megasporocarps of Azolla, but are retained to maturity only in the latter. During the development of the Azolla zygote the Anabaena akinetes germinate to produce undifferentiated, generative filaments. These become associated with the shoot apex of the developing fern sporophyte, perpetuating the symbiosis through the reproductive cycle. Growth and development of the host and the symbiont are synchronous. This is a feature that does not occur in other plant-cyanobacteria symbioses. Each apical meristem of the fern has a small colony of Anabaena filaments associated with it and the growth rate of the endophyte is coordinated with that of the host. These Anabaena filaments associated with the plant apex lack heterocysts and do not exhibit nitrogenase activity. The establishment of algal filaments in each fern leaf begins in the young leaves contiguous with the apical Anabaena colony and is complete by the time each leaf emerges as a distinct mature entity. The development of the cavity in the dorsal lobes of the fern leaves begins in young leaves rapidly enlarging by cellular expansion. After that the cavity becomes inoculated with Anabaena and as the leaf matures, the Anabaena filaments in the leaf cavity multiply. Then also rapid differentiation of heterocysts occurs, accompanied by a rapid increase in nitrogenase activity. Attempts have often been used to isolate A. azollae from its host. In many cases isolates have been obtained that are still capable of fixing nitrogen, but in all cases these cultured isolates develop different characteristics from the fresh isolates. When immobilized in polyurethane foam, isolates enhance their heterocyst frequency as well as their nitrogenase activity. It has been hypothesized that the major cyanobacterial constituent of the association is an obligate endosymbiont incapable of in-vitro growth.
In mature trichomes of A. siamensis a vegetative cell in the mid-region of the trichome divides itself symmetrically, giving rise to two small daughter cells which will develop into heterocysts after the trichome breaks into two at the junction of the two newly formed incipient heterocysts. Akinetes in A. siamensis are usually also formed in the midregion of the trichome, where a vegetative cell becomes slightly enlarged and gradually transforms into a typical akinete by synthesizing an additional wall and accumulating inner granular contents. Successively, adjoining cells can also convert, resulting in a short string of akinetes.
In clonal cultures of A. siamensis, molecular nitrogen as well as ammonium or nitrate could be used as a nitrogen source, resulting in a doubling time of 3.4-3.7 days. The alga grows even faster (doubling time of 2 days) when the amino acid L-glutamine is used as the sole source of nitrogen.
The light-harvesting pigments of the two partners of the symbiotic Azolla-Anabaena association are complementary. The endophyte accounts for 10-20% of the association's total chlorophyll and about 16% of its total protein, with phycobiliproteins (phycocyanin and phycoerythrocyanin) accounting for 4-10% of the endophyte's protein. The Anabaena contributes 6-10% of the total photosynthetic capability of the association and its photosynthesis is not inhibited by atmospheric oxygen.
Other botanical information
Identification and classification of blue-green algae are usually heavily debated. Some morphological characters change in culture or are not expressed. The Anabaena species endophytic in Azolla might be growth forms of A. variabilis or some might be species of Nostoc Vaucher ex Bornet & Flahault or belong to Tricornus (Ralfs ex Bornet & Flahault) Komárek & Anagnost. Studies on fatty acid composition of the endophytes as well as immunological studies and lectin haemagglutination research suggest that these Azolla symbionts differ considerably from those of Anabaena and Nostoc. There has so far been no fully satisfactory demonstration of successful reestablishment of the symbiotic state with any isolate and an endophyte-free Azolla, though there are reports of success. Nevertheless, it will be difficult to acknowledge any of the isolates as the true endophyte until Koch's postulates have been fully demonstrated. Even the use of monoclonal antibodies has not yet solved the problem.
In the planktonic species the debate on variability is also far from reaching agreement, although database systems are being developed to help identifying the species. Because of the paired (young) intercalary heterocysts, A. siamensis might belong to Anabaenopsis Geitler.
Ecology
A. azollae occurs exclusively as an endophyte in the free-floating Azolla water ferns. The endophyte in mature leaf cavities of the Azolla fern releases newly fixed nitrogen as ammonia, which is rapidly assimilated by the fern. Since wind and wave action, as well as other turbulence, cause fragmentation of the fern and diminish growth, Azolla is not found on large lakes where the waters move swiftly. It is, however, capable of luxurious growth in ponds, marshes and canals, while paddy fields form an ideal environment for Azolla. The association can, however, survive only a few days in a paddy field once the field is drained. Although Azolla can colonize waters that are nitrogen deficient, its growth can be limited by the availability of other nutrients, such as phosphorus and iron. The most important factors influencing growth and distribution of the Azolla-Anabaena symbiosis are light, temperature, water, mineral nutrition, pH, salinity, physical disturbance and biotic interactions. Most populations of Azolla have an optimal temperature range of 20-25°C. The temperature regime is difficult for farmers to manage. Direct high temperature effects mainly decrease nitrogenase activity, but are not as serious as indirect effects, the latter resulting in higher losses due to pests. When conditions are favourable, Azolla will grow well under full sunlight.
A. flos-aquae is a planktonic freshwater alga which uses its gas vacuoles for buoyancy regulation. A. siamensis occurs on most soils and in paddy fields. Because of its prominent akinetes it can easily survive long dry periods. Both the free-living Anabaena filaments and the endophytic ones are able to reduce N2 under an air atmosphere and the nitrogenase is assumed to be localized in the heterocysts. A. siamensis is semihalotolerant and mesophilic. Its optimum growth temperature is 41°C.
Propagation and planting
Cultures of Azolla and its symbiont must be maintained vegetatively in nurseries throughout the year to be used as inoculum since the production of a large biomass of Azolla from spores is slow and difficult. Just prior to the rice-growing season the fern with the symbiont must be multiplied in large quantities to be ready for field cultivation. Inoculum transport presents obstacles in regions lacking means of transport. Dried Azolla is logistically preferable, but its N contribution is only half that of fresh plants.
During the multiplication phase of Azolla, prior to the rice-growing season, the fern mats must be continually subdivided to prevent competition for light, space and nutrients. In that way a high growth rate can be maintained. The level of inoculation of fresh Azolla into fields is (25-)30-200(-800) g/m2, depending on growth conditions. Fronds are often fragmented to aid growth and dispersal. Usually application of phosphorus is recommended to obtain significantly higher rice grain yields. However, Azolla pinnata is less affected by phosphorus-deficient conditions than most other Azolla species.
Aquaculture
Water is a fundamental requirement for successful cultivation and application of Azolla and its symbiont. Therefore, proper water control is essential. The Azolla-Anabaena association needs all the essential elements that are required by other plants as well as molybdenum and cobalt, both of which are required for atmospheric nitrogen fixation. Normally, all nutrients must be available in water throughout the period of growth of the fern. Threshold levels of P, K, Mg, and Ca required for Azolla growth are 0.03, 0.6, 0.5, and 0.5 mmol/l respectively; for the micronutrients Fe, Mn, Mo, and B 50, 20, 0.3, and 30 μmol/1, respectively. Phosphorus is the most important and often limiting nutrient for Azolla growth. Phosphorus deficiency is indicated by smaller, fragile, less vigorous plants with purple, bullet-shaped leaves, and developing very long roots. Maximum growth and atmospheric nitrogen fixation by the Azolla-Anabaena association requires greater P fertility than for rice alone, and this is a major barrier to applied use in many instances.
Azolla does not require any nitrogen in the culture medium, but the level of nitrogen in the water does positively affect growth and atmospheric nitrogen-fixing rates. Iron is a common limiting element, because it is an essential constituent of nitrogenase. In calcium-deficient Azolla the fronds become fragmented, while in potassium-deficient plants growth is stunted. Azolla is extremely sensitive to SO2 pollution in the atmosphere. Ozone pollution considerably reduces nitrogen-fixation rates and heterocyst frequency in the symbiont. Exposure to atmospheric NO2 pollution also decreases rates of growth, nitrogen fixation, heterocyst formation, and overall nitrogen cycling. The systemic fungicides Benlate (methyl-1-butyl carbamoyl-2 benzimidazole carbamate) and Vitavax (5,6-dihydro-2, methyl-1, 2-oxathiin-3-carboxanilide) stimulate ammonium production by immobilized A. azollae in a photobioreactor and inhibit the activity of the enzyme glutamine synthease.
Diseases and pests
Some cyanophages can infect Anabaena, but no studies are available regarding the importance of infection in natural populations. Pathogenic fungi and free-living algae can become damaging to Azolla, especially during hot, humid periods. Azolla is subject to a number of pests, in particular insects, some of which can be disastrous to successful propagation if they are not effectively controlled. Snails are also a common pest. One of these, Limnea natalensis, is a schistosomiasis vector and thus of concern for public health. The use of the insecticide Vophatox 0.005% is often fatal - very few Azolla plants survive a treatment. There are no pests known to attack the symbionts.
Harvesting
The Anabaena cyanobacteria are never harvested as an isolated crop. They are either included inside the Azolla plants or mixed with other cyanobacteria or plankton algae.
Yield
A dry weight of 4.8-7.7% of the fresh material has been found for Azolla, containing generally 3-6% nitrogen, but it is strongly influenced by growth conditions.
Handling after harvest
The harvested Azolla can be used immediately as green manure or can be air-dried for transport. Mixtures of soil cyanobacteria can be air-dried and used as new inoculum.
Genetic resources
Several Anabaena species and strains but not A. azollae are available from algal culture collections. No systematic collection of germplasm of Azolla has yet been recorded. The Azolla-Anabaena symbiotic nitrogen-fixing complex can be studied by genetic manipulation through tissue culture using excized frond meristems of the fern which carry the cyanobiont germplasm. There has been some success in transferring Anabaena from one species of Azolla to another.
Prospects
Because of the high costs of commercial N-fertilizer, the lasting concerns about environmental conservation, and the propagation of sustainable, renewable, non-polluting resources, it can be expected that both the application of Azolla-Anabaena associations and of suitable soil cyanobacteria will increase in the future. If no diseases and pests prevent future use of these organisms, they might become even more important as nitrogen sources for agriculture than they are at present. The popularity and the extent of their use will ultimately depend on the price of energy, which directly determines the availability and cost of inorganic N-fertilizer. Techniques to achieve a much lower phosphate requirement by the use of mutant Azolla plants might result in even greater popularity as a nitrogen fertilizer. However, the inability to establish free-living cultures of Anabaena azollae and the inability to control the induction of sporulation and germination in the field of the spores of the Azolla ferns are major limitations to research and applicability.
The toxic substances of some freshwater Anabaena might become the basis for future fine chemicals or pharmaceuticals.
Literature
- Antarikanonda, P., 1985. A new species of the genus Anabaena: Anabaena siamensis sp. nov. (Cyanophyceae) from Thailand. Nova Hedwigia 41: 343-352.
- Canini, A. & Grilli Caiola, M., 1995. Cyanobiont-host interactions in the Azolla association. In: Round, F.J. & Chapman, D.J. (Editors): Progress in phycological research 11. Biopress Limited, Bristol, United Kingdom. pp. 155-186.
- Codd, G.A., Edwards, C., Beattie, K.A., Lawton, L.A., Campbell, D.L. & Bell, S.G., 1995. Toxins from cyanobacteria (blue-green algae). In: Wiessner, W., Schnepf, E. & Starr, R.C. (Editors): Algae, environment and human affairs. Biopress Limited, Bristol, United Kingdom. pp. 1-17.
- Hiroki, M., Shimizu, A., Li, R., Watanabe, M. & Watanabe, M.M., 1998. Development of a database useful for identification of Anabaena spp. (Cyanobacteria). Phycological Research 46 (Supplement): 85-93.
- Kannaiyan, S., Aruna, S.J., Merina Prem Kumani, S. & Hall, D.O., 1997. Immobilized cyanobacteria as a biofertilizer for rice crops. Journal of Applied Phycology 9: 167-174.
- Metting, B., Zimmermann, W., Crouch, I. & Van Staden, J., 1990. Agronomic uses of seaweed and microalgae. In: Akatsuka, I. (Editor): Introduction to applied phycology. SPB Academic Publishing BV, The Hague, The Netherlands. pp. 589-627.
- Peters, G.A. & Calvert, H.E., 1983. The Azolla-Anabaena symbiosis. In: Goff, L.J. (Editor): Algal symbiosis. A continuum of interaction strategies. Cambridge University Press, Cambridge, Massachusetts, United States. pp. 7-145.
- Stulp, B.K. & Stam, W.T., 1984. Genotypic relationships between strains of Anabaena (Cyanophyceae) and their correlation with morphological affinities. British Phycological Journal 19: 287-301.
- Vaishampayan, A. & Kalayan Banerjee, 1995. Genetic approaches to accomplish reduced phosphate-dependence of Azolla-Anabaena symbiotic nitrogen fixing complex in wet agricultural fields. In: Kargupta, A.N. & Siddiqui, E.N. (Editors): Algal ecology: an overview. International Book Distribution, Dehra Dun, India. pp. 161-193.
- Wagner, G.M., 1997. Azolla: a review of its biology and utilization. The Botanical Review 63: 1-26.
Sources of illustration
Frémy, P., 1930. Les myxophycées de l'Afrique équatoriale française [The blue-green algae of French equatorial Africa]. Archives de Botanique 3, Mémoire 2. Fig. 308, p. 372 (habit A. azollae); Peters, G.A. & Calvert, H.E., 1983. The Azolla-Anabaena symbiosis. In: Goff, L.J. (Editor): Algal symbiosis. A continuum of interaction strategies. Cambridge University Press, Cambridge, Massachusetts, United States. Fig. 6, p. 119 (section of fernleaf with A. azollae). Van den Hoek, C., Mann, D.G. & Jahns, H.M., 1995. Algae, an introduction to phycology. Cambridge University Press, Cambridge, United Kingdom. Fig. 2.5.j, p. 23 (habit A. flos-aquae). Antarikanonda, P., 1985. A new species of the genus Anabaena: Anabaena siamensis sp. nov. (Cyanophyceae) from Thailand. Nova Hedwigia 41. Fig. 2 a & d, p. 352 (habit A. siamensis). Redrawn and adapted by P. Verheij-Hayes.
Authors
- W.F. Prud'homme van Reine
