Azolla pinnata (PROSEA)

Plant Resources of South-East Asia
Introduction

Protologue: Prodr. fl. Nov. Holl.: 167 (1810).

Family: Azollaceae

Chromosome number: 2n= 44 (diploid), 66 (triploid), 88 (tetraploid)

Synonyms

Salvinia imbricata Roxb. ex Griff. (1844), Azolla decomposita Zoll. (1854), A. imbricata (Roxb. ex Griff.) Nakai (1925).

Vernacular names

Azolla, ferny azolla, mosquito fern (En)
Indonesia: mata lele (Javanese), kayu apu dadak, kakarewoan (Sundanese)
Malaysia: mãn chiang húng (Chinese)
Thailand: nae daeng (central)
Vietnam: beo dau.

Origin and geographic distribution

The exact origin of A. pinnata is unknown. It is found throughout tropical Asia (including South-East Asia), in southern and eastern China, in southern Japan, northern Australia and in tropical and southern Africa (Madagascar included). In maritime locations in China (Wenzhou district) and Vietnam (La Van village) A. pinnata is known to have been cultivated since the 7th Century.

Uses

A. pinnata has become important as an organic fertilizer in tropical lowland rice production in South-East Asia. It can be grown in dual culture with wet rice, or as a cover crop grown during the fallow season of rice and incorporated into the soil as a green manure. It maintains a symbiotic association with the blue-green alga Anabaena azollae Strasb. ex Wittr., Nordst. & Lagerh. which is able to fix atmospheric nitrogen. Other crops where A. pinnata is applied as mulch or as green manure include taro ( Colocasia esculenta (L.) Schott), Manchurian wild rice ( Zizania latifolia (Griseb.) Turcz. ex Stapf) and arrowhead ( Sagittaria trifolia L.). Commercial production of azolla compost has been realized; the quality is good and the decomposition time may be reduced by 50%. A. pinnata is also used as fodder crop for pigs, rabbits, chicken, ducks and fish, mainly in China. Another use in rice cultivation is to control weeds and good results have been obtained in controlling e.g. Cyperus difformis L., Echinochloa glabrescens Munro ex Hook.f., Monochoria vaginalis (Burm.f.) Presl and Paspalum sp. A dense cover of A. pinnata on water is also quite effective as a means of mosquito control. Waste material of A. pinnata is used as a substrate for cultivating edible mushrooms, e.g. Pleurotus sp. In China small-scale biogas production units are exploited using azolla as biomass. Philippine farmers have used azolla for human consumption in various dishes such as salads, pinangat, mungo and omelette. The meal can be added to flour to increase the fibre content.

Production and international trade

A. pinnata is only produced and traded locally. In the first half of the 20th Century in North Vietnam, where this fern was widely used as green manure for spring rice, villages specialized in propagating and selling it with good profit as a source of inoculum for rice paddy. In the United States A. pinnata cultivation is prohibited by federal law because of the risk of inadvertent break outs. In South Africa A. pinnata is not considered harmful.

Properties

Per 100 g (dry weight basis) A. pinnata contains approximately: protein 13-30 g, fat 4-6 g, carbohydrates 41-45 g, fibre 39 g, cellulose 5-15 g, hemicellulose 9-18 g, lignin 9-35 g, ash 10-24 g (ranges of the elements N 2-5%, P 0.1-1.6%, K 0.3-6%, Ca 0.4-1.7%, Mg 0.2-0.6%, S 0.2-0.7%, Si 0.1-3.5%, Na 0.1-1.3%, Cl 0.6-0.9%, Al 0.04-0.6%, Fe 0.04-0.6%, Mn 0.06-0.3%, Cu 0-0.03% and Zn 0.002-0.1%). The N, K and Si contents are reasonably high. Deficiencies in methionine, histidine and fibre make A. pinnata unfit to be the sole feed source for animals, but the amino acid composition compares well with reference protein sources and although the methionine content is low, the lysine content is more than twice that of maize. In the tropics 60-75% of the N will be released as ammonia within 6 weeks after the onset of mineralization in the soil in a flooded field without rice.

Description

A small, free-floating, aquatic fern, triangular to trapezoidal in outline, about 2-4 cm × 1 cm, with branched, floating stem (rhizome) bearing roots and leaves. Roots solitary, growing from stem branching points, hanging down in the water, plumose, 1-5 cm long, with obliquely arranged groups of 3-6 root hairs. Leaves small, alternately in two rows, variably imbricate, sessile, bilobed with a dorsal lobe held above the water surface and a floating (resting on water surface) ventral lobe; dorsal lobe subelliptical, up to 1.5 mm × 1 mm, fleshy, chlorophyllous and photosynthetic, margin hyaline and (2-)3(-4) cells wide, apex obtuse, with 1-2-celled trichomes and papillae, inside on lower surface with a cavity containing mucilage and filaments of the blue-green alga Anabaena azollae , green but turning red under adverse environmental conditions; ventral lobe thin (one cell thick), flat, translucent, without chlorophyll, glabrous. Sporocarps borne in pairs or fours at base of branches (reduced ventral leaf lobe), initially covered by involucre of dorsal leaf lobe, each containing one megasporangium (containing a single megaspore) or many microsporangia (each containing 64 microspores). Spores trilete, of two kinds; megaspore bearing on the proximal side a columella, hair-like filaments and 9 apical massulae (called floats); microspores of one sporangium released as one mass (called a massula) which floats, its inner surface bearing 2-4 small, simple, spiniform trichomes without hooked apices; microspores germinate within the massula.

Growth and development

The sporophyte of A. pinnata reproduces vegetatively through fragmentation, made easy by an abscission layer present at the base of each branch. Secondary branches extending from older lateral branches bend the lateral branch and put pressure on its abscission layer, contributing to its separation. Lateral branches then drift away independently from their parent. Exponential growth may continue under optimum conditions for fragmentation and dispersal and consequently A. pinnata can become a troublesome weed. Initiation of the gametophytic cycle in most Azolla species seems to be stimulated by environmental stress, which also affects the ratio of micro- to macro-sporocarps. When a plant becomes fertile, sporocarps are formed in pairs after a few divisions by a fertile ventral lobe initial of the first leaf of a branch. The dorsal lobe of the fertile leaf retains its normal shape and function. Leptosporangiate development of the sporangial initials eventually gives rise to two megasporangia, each containing 32 megaspore initials produced by meiotic division. If a megaspore is to develop within the megasporangium, all but one of the 32 megaspore initials abort. If all of the megaspore initials abort, microsporangia initials arise from basal outgrowths on the stalk of the megasporangium. The resulting pair of sporocarps may be all of one sex or any mix. Sporocarps mature on the plant in a week or more. Microsporocarps contain up to 130 stalked microsporangia with 64 microspores. The microspores are not released directly into the water, but as cluster held together by hardened mucilage (massula). The massulae bear trichomes that facilitate coupling to the hairs of the megasporangia. The microspores germinate and release antherozoids to fertilize an archegonium produced by the megaspore gametophyte. When after successful fertilization the new sporophyte ruptures the megasporangium wall, Anabaena strands located there become entrapped in the shoot apex. As the funnel-shaped first leaf emerges, it dislodges the Anabaena and establishes a colony around an apical trichome that is incorporated into the dorsal leaf cavity.

Other botanical information

Within A. pinnata three subspecies are distinguished of which only one is found in South-East Asia,subsp. asiatica R.M.K. Saunders & K. Fowler. This has lax to slightly imbricate dorsal leaf lobes with narrow (width about 1/10 of leaf width) hyaline margins, ventral leaf lobes shorter and wider than dorsal ones, surface of megaspore with prostrate or erect elongate outgrowths. The other 2 subspecies are found in Australia and New Caledonia (subsp. pinnata ) and in Africa (subsp. africana (Desv.) R.M.K. Saunders & K. Fowler). Although many species have been described in the genus Azolla Lamk, only 6 or 7 are currently recognized. A. pinnata , together with A. nilotica Decne. ex Mett. (leaves up to 40 cm long, never red, restricted to tropical Africa) are classified in section Rhizosperma of the genus Azolla . Several other Azolla species, e.g. A. filiculoides Lamk, A. mexicana Presl and A. microphylla Kaulfuss (all in section Azolla ), have been introduced into South-East Asia and are used experimentally and in breeding.

Ecology

The geographical distribution of Azolla indicates that it is adapted to a wide range of climatic conditions. The primary requirement of Azolla is an aquatic habitat, since it is very sensitive to dryness. It dies within a few hours under dry conditions. Most of the Azolla species are widely distributed in temperate regions as they are generally sensitive to the higher temperature of the tropics. The optimum temperature range in which most of them grow well is 20-25°C. The most favourable temperature for growth and nitrogen fixation of A. pinnata is 20-30°C, it perishes below 5°C and above 45°C. Light intensity interacts with the effect of temperature. The optimum temperature for A. pinnata grown under 15 klux artificial light is 30°C. The growth rate is reduced under light intensities that fall outside the range of 15-60 klux. The optimum relative humidity needed for normal growth of azolla is 85-90%; values below 60% cause the leaves to dry up. High relative humidity causes a longer dew period, resulting in susceptibility of the plant to disease and pest infestation. In the tropics direct sunlight at midday suppresses the growth of the fern, while light exposure on cloudy days is more favourable. Wind tends to push all the leaves together so they collect on the same part of the water surface. A slightly acidic to neutral pH (4.5-7) has been found to be suitable, the optimum being pH 5.5-6.0. Wilting occurs where salt concentrations surpass 1.5 g/l during the summer. Azolla may suffer from competition from other floating water plants such as algae, Pistia L., Lemna L., and Salvinia molesta D.S. Mitchell. Because of its rapid vegetative reproduction, however, azolla often also becomes a troublesome water weed, clogging waterways and drainage systems, forming dense surface mats which interfere with boating, fishing and recreational activities and degrade water quality by reducing oxygen levels.

Ferns of the genus Azolla share a mutually beneficial symbiotic relationship with the nitrogen-fixing blue-green alga Anabaena azollae . The fern provides nutrients and a protective leaf cavity for Anabaena , which in turn provides ammonia to the fern. The Anabaena symbiont occupies a specialized cavity in aerial dorsal leaf lobes. Specialized trichomes are involved in the metabolite exchange. More of the alga is found in the apical meristem and some filaments penetrate beneath the megasporangial indusium where they inoculate new leaves and young sporophytes, respectively. Natural Azolla populations are rarely free of Anabaena . Arthrobacter bacteria also constitute a third partner in the Azolla-Anabaena symbiosis, but their role is not yet understood. Nitrogen fixation occurs only in specialized Anabaena cells called heterocysts. Most of the energy is supplied by metabolites produced by the photosynthesis of Azolla . Characteristically low levels of ammonium-assimilating enzymes in Anabaena azollae make the nitrogen fixation process very efficient. The fixed nitrogen is transported to the host, which incorporates it into amino acids. Part of the amino acids probably then go back to Anabaena . Because Anabaena azollae is associated with Azolla throughout its life cycle, a free-living stage of the symbiont is not needed. The frequency of heterocysts inside the leaf cavity is 3-10 times that found on free-living blue-green algae. The taxonomy of Anabaena azollae and its relation to free-living Anabaena species is still subject to discussion. Also it has been questioned whether the laboratory grown isolates match the genuine endosymbiont.

Propagation and planting

At present vegetative material of A. pinnata is generally used as planting material. In order to maintain the fern throughout the year, special multiplication nurseries are prepared to produce sufficient quantities of planting material as inoculant for further propagation. These nurseries require shade, an ample supply of water, plant nutrients, disease and pest control and some measures to protect the fern from extreme weather conditions. The field for the fern nursery should be thoroughly prepared. Super phosphate is applied, usually in three gifts at 4 day intervals. About 7 days after inoculation a systemic insecticide is applied to provide protection against insect pests (e.g. carbofuran granules). By repeated inoculation and harvesting in the nursery a sufficient amount of azolla inoculants can be obtained for the main rice field. Vegetative reproduction can be promoted by fragmenting the plants, e.g. by tapping them with a broomstick. Storage of harvested azolla is difficult and hence special care is necessary for long distance transport. Desiccation should be prevented and fresh azolla can be shipped packed in sealed polythene bags or petri dishes, surviving at least 2-3 weeks when kept at 5-10°C.

Propagation can also be done by spores, which allows large quantities of inoculum to be produced and transported. To encourage the azolla to produce spores, the plants have to occupy the available space completely, making vegetative reproduction no longer possible. After about a week some 70% of the plants will have produced spores. Then, about two thirds of the azolla mat is harvested. Under favourable conditions the remaining plants will produce spores again in about three weeks. A production field of 10 m × 25 m can be inoculated with 100 g dry spores. Spores start germinating after 7 days. After two weeks the young plants can be transplanted.

Husbandry

Under favourable conditions, a layer of A. pinnata covering a rice field of one ha releases 20-30 kg organic N. The economic return from azolla adoption is more than 10% of the total non-land cost for rice production in areas where conditions favour azolla growth. Constraints for its use include the phosphorus content of the soil, insect and pest control and labour requirements. The need for additional phosphorus fertilizer may not be a problem in its adoption because azolla normally grows when only 0.06 ppm P is available, though 30 ppm or more is advised. However, if more than 200 g/ha of a systemic insecticide such as carbofuran is necessary the economic benefits are eliminated. Labour costs of azolla application can become critical where wages are high. In some areas Azolla species that lack heat tolerance are taken advantage of by letting the ferns die in summer, thus freeing the nutrients and allowing the light to penetrate through the water to the submerged rice plants. Sometimes azolla fertilization is used in addition to inorganic N-fertilizer. In general, however, the average nitrogen fixation rate of azolla amounts to 1-2 kg N per ha per day which is sufficient to meet the nitrogen demand of the rice crop. The azolla-rice culture can be augmented with fish production (e.g. Tilapia nilotica ) and with ducks.

Diseases and pests

A serious disease of A. pinnata in rice fields is caused by the fungus Myrothecium verrucaria , characterized by white spots of mycelium and a rapid rot and death of azolla. A. pinnata is also highly susceptible to rice sheath blight, caused by Sclerotinia sclerotiorum and to black rot disease caused by Rhizoctonia solani . Disease severity is higher when the leaves are also attacked by snails. Numerous other fungal genera have been isolated from diseased azolla. Crude garlic extract was found to be very effective against fungal pathogens.

In South-East Asia pests are considered the major limiting factor in azolla cultivation. In the Philippines, the most important pests are the webworm (causing yield losses of 6-74 %), the lepidopteran spinningworm ( Ephestiopsis vishnu ) and the caseworm complex ( Elophila ( Nymphula ) enixalis , E. nigralbalis , E. responsalis ). In Thailand, Chironomus glauciventris, Cryptoblabes sp ., Polypedilum johannseni and Elophila enixalis are the main pests. In Thailand, azolla is also damaged by high temperatures in April and by competition from blue-green algae. The best insect control and the highest yield was obtained by spraying 0.5 kg/ha monocrotophos, applied 1 and 10 days after inoculation. Foliar sprays of carbosulfan or chlorpyrifos applied after field inoculation also gave good results. Treatment with these or with monocrotophos resulted in 3-fold increases in yield. A preparation (Bactospeine) containing Bacillus thuringiensis , applied after inoculation, provided some insect control and significantly increased the yield. To reduce application costs, it is recommended that the azolla stock culture be sprayed 2-3 days before it is transferred to the propagation field. Another application is necessary 7 days later. Because azolla cultivation should begin after transplanting rice, insecticide applications also have some effect on rice pests. Destruction by snails is an obstacle to azolla-breeding in India. Snails primarily responsible are Lymnaea luteola , Bellamya bengalensis, and Pila globosa . The golden apple snail, Pomacea canaliculata , originating from South America, was introduced as a promising export industry in South-East Asia but it has also escaped to rice fields in Taiwan, Japan and the Philippines (here Pomacea cuprina and Pomacea gigas have also been introduced) and azolla and young rice seedlings are preferred hosts. Control is possible by manual removal, draining of rice fields, releasing carp (prey on young snails), application of pesticides such as triphenyltin-acetate ("brestan", effective but highly toxic to Nile tilapia), clonitralid ("bayluscid", effective but toxic to fish), metaldehyde ("namekil", effective with low fish toxicity).

In southern China, the azolla midge ( Polypedilum ivinoense ), which is aquatic at larval instar stage, can devastate an azolla stand in 3-5 days. It is light red, 2-3 mm long, and makes its nest on the underside of azolla, eating roots and young leaves. In summer it is able to complete its life cycle in 12 days, building in a short time populations of 90 000 per m². Methods to prevent damage by azolla midge include protecting the Dytiscidae which are natural enemies, applying maceration extract of cake of tea oil and insecticides such as deltamethrin, carbofuran, carbaryl, and temephos. Pesticides and herbicides can have effects on the growth and nitrogen assimilation of the Azolla-Anabaena symbiosis. Most rice herbicides (e.g. bipyridylium and phenolic herbicides, chloramben, dicamba, simazine, benzoic, triazine, dinitroaniline and urea herbicides) are deleterious to azolla but delaying inoculation with azolla until after herbicide application reduces or eliminates the problem. The symbiosis is also severely damaged by atmospheric SO₂even at low concentrations, with significant reductions in growth, assimilation, protein synthesis and heterocyst development. Propanil is highly toxic, sinking the floating fern in about five days after inoculation.

Harvesting

Since A. pinnata floats on the surface of the water, the easiest way of collection is by using nets and transport in big baskets. For green manuring of rice a full cover of azolla mat can be directly incorporated into the soil using simple tools or rotary tillers.

Yield

Under optimum conditions A. pinnata can double in weight every 3-5 days. In an open field under tropical conditions, a full cover of azolla can yield about 20 t/ha fresh weight. When intercropped with rice the growth rate decreases with the development of the rice canopy, but repeated inoculation and harvest of the fern can give an annual yield of up to 40-50 t/ha fresh weight. Rice yields may increase by 0.4-1.5 t/ ha when thus fertilized.

Handling after harvest

To mix dried azolla as supplement to commercial layer or broiler rations, the freshly harvested azolla should be washed thoroughly to remove soil and pesticide residues. Then it is dried in the sun for 3-7 days, until it crumbles when squeezed. Subsequently, it is ground, either using a commercial grinder, or by crushing the dried plants in bags with the feet. The azolla meal is mixed with the layer/broiler rations 1:10 by weight or 1:5 by volume. To harvest the spores, the harvested plants are placed in a 50 cm deep, well-drained pit. The plants are left to decay for two weeks during summer or up to one month in the rainy season. The decayed plants are collected and dried in the sun and open air for 24 hours. The dried mass is sieved through a coarse screen (1 mm mesh). The particles that pass through are immersed in water and left to soak overnight. The floating spores are finally scooped from the water.

Genetic resources

Living A. pinnata germplasm collections are maintained at the International Rice Research Institute (IRRI) in the Philippines, the Azolla Research Centre, Hanoi, Vietnam, the Azolla Research Center, Fujian Academy of Agricultural Science, Fuzhou, Fujian, China, in the United States at the Washington State University and the University of California, Davis, in Africa at research stations of the Office de la Recherche Scientifique et Technique Outre-Mer (ORSTOM) and the West Africa Rice Development Association (WARDA) in Ivory Coast, and at the Université Catholique de Louvain, Belgium (UCL). In 1997 about 550 accessions were maintained at IRRI, covering all extant species (about 150 accessions of A. pinnata ) collected worldwide in cooperation with many researchers, including also unique material which cannot be obtained from natural habitats, such as sexual hybrids (79 accessions), Anabaena -free azolla (20), azolla with heterologous symbiotic cyanobacteria (6) and putative mutants (16). The IRRI collection is now maintained as shoot-tip agar cultures, which are renewed every 3-6 months. Most cultures of the section Azolla are duplicated at the Azolla Research Center of the Fujian Academy of Agricultural Science (Fuzhou, Fujian, China).

Breeding

At several azolla research stations, selection and breeding programmes are in progress to obtain more efficient, more pest resistant and more shade- and salt-tolerant genotypes. Interspecific hybrids have been tried but so far they have all proved sterile. Given the isolated position of A. pinnata in Azolla , fertile interspecific hybrids are not to be expected. Other Azolla species hybridize more easily and several promising hybrids have been bred in the Philippines, Thailand and China.

Prospects

The great expectations of A. pinnata as a natural source of nitrogen and weed control peaked around 1980-1990, but have declined since. Nevertheless, many promising initiatives aimed at the utilization of azolla are being developed especially in China and South-East Asia, and also in other parts of the world. Unfortunately, in most environments azolla is not a cost-effective substitute for inorganic nitrogen. Limiting factors to the use of azolla include the need for water, high phosphorus requirements, susceptibility to pests and limited temperature tolerance. High labour costs (for maintenance, transport, inoculation and burying), high opportunity costs of land and poor water control are major constraints to the economic feasibility of green manure. Improvements in azolla technology that increase nitrogen yield and pest resistance or reduce the opportunity costs of labour and land could make azolla economically feasible in a wider range of environments. Integrated production systems with azolla are promising. In the laboratory the symbiotic Anabaena cyanobacterium can be induced to produce free hydrogen. So far this has not led to commercial-scale hydrogen production of plants. Azolla is capable of extracting phosphates from eutrophicated water, acting as a decontaminant in sewage treatment. In Israel, biological processes for the removal of heavy metals from effluents by means of azolla have been developed. The process is suitable for uranium, cadmium, nickel, copper and chromium and the recovery of silver. These processes are superior to traditional methods of metal removal from effluents when environmental and ecological constraints exist and the concentrations of metals are low (1-20 ppm). At such low concentrations, no effective chemical means of metal removal are presently available. In tests the heavy metals were concentrated 500-1000 fold in the azolla biomass within 2-7 days of growth, and the content of metal in azolla was about 1% Cu, Cd, Zn, U and Ag, and 0.3% hexavalent Cr and Ti. Some 40-60% of the heavy metal was removed from the water body. The total content of metal in the azolla ash was 5%. Filters made of dried azolla were found to bind a high percentage of the metal in solution.

Literature

Cagauan, A.G., Pullin, R.S.V., Muir, J.F. & Roberts, R.J. 1994. Azolla in aquaculture: past, present and future. Recent Advances in Aquaculture 5: 104-130.
Capaya, D.T., 1979. International bibliography on Azolla. International Rice Research Institute (IRRI), Los Baños, The Philippines. 66 pp.
Kikuchi, M., Watanabe, I. & Hows, L.D., 1984. Economic evaluation of Azolla use in rice production. In: Organic matter and rice, proceedings of a conference. International Rice Research Institute, Los Baños, The Philippines.pp. 569-592.
Lejeune, A., Cagauan, A. & van Hove, C., 1999. Azolla research and development: recent trends and priorities. Symbiosis 27(3-4):333-351.
Mogi, M.T., Okazawa, I., Miyagi, S., Sucharit, S., Tumrasvin, W., Deesin, T. & Khamboonruang, C., 1986. Development and survival of anopheline immatures (Diptera: Culicidae) in rice fields in northern Thailand. Journal of Medical Entomology (USA) 23(3): 244-250.
National Azolla Action Program, 1986. Field problems of Azolla. College of Agriculture, University of the Philippines, Los Baños, Laguna, The Philippines. 47 pp.
National Azolla Action Program, 1987. Growing and collecting azolla spores. College of Agriculture, University of the Philippines, Los Baños, Laguna, The Philippines. 12 pp.
Prud'homme van Reine, W.F., 2001. Anabaena (Bory) ex Bornet & Flahault. In: Prud'homme van Reine, W.F. & Trono, G.C. (Editors): Plant resources of South-East Asia No 15(1). Cryptogams: algae. Backhuys Publishers, Leiden, The Netherlands. pp. 83-88.
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Authors

Soetjipto Partohardjono & P. Swatdee