Indian Journal of Biotechnology Vol 2, January 2003, pp 26-37 Azolla-Anabaena Symbiosis-From Traditional Agriculture to Biotechnology Anjuli Pabby, Radha Prasanna and P K Singh* National Centre for Conservation and Utilization of Blue-Green Algae, Indian Agricultural Research Institute, New Delhi 110 012, India The Azolla - Anabaena symbiosis has attracted attention as a biofertilizer worldwide, especially in South East Asia. But its utilization and genetic improvement has been limited mainly due to problems associated with the isolation and characterization of cyanobionts and the relative sensitivity of the fern to extremes of temperature and light intensity. This paper reviews the historical background of Azolla, its metabolic capabilities and present day utilization in agriculture. An outline of biotechnological interventions, carried out in India and abroad, is also discussed for a better understanding of the symbiotic interactions, which can go a long way in further exploitation of this association in agriculture and environmental management. Keywords: Azolla, Anabaena, biofertilizer, fingerprinting, symbiont Introduction food and medicine, besides its role in environmental Azolla is a small aquatic fern of demonstrated management and as controlling agent for weeds and agronomic significance in both developed and mosquitoes. It also improves water quality by removal developing countries (Singh, 1979a; Lumpkin & of excess quantities of nitrate and phosphorus and is Plucknett, 1980; Watanabe, 1982; Giller, 2002). The also used as fodder, feed for fish, ducks and rabbits association between Azolla and Anabaena azollae is a (Wagner, 1997). Besides its extensive use as a N- symbiotic one, wherein the eukaryotic partner Azolla supplement in rice-based ecosystems, it has also been houses the prokaryotic endosymbiont in its leaf used in other crops such as taro, wheat, tomato and cavities and provides carbon sources and in turn banana (Van Hove, 1989; Marwaha et al. 1992). obtains its nitrogen requirements. This mutual However, its most outstanding attribute, which is exchange of activities helps in quick growth and relevant as a biofertilizer, is related to its high rate of multiplication of the fern under optimal multiplication, which helps in covering the entire environmental conditions. surface of water body in which it is growing within 2- The agronomic potential of this association is 3 days. The growing concern about the conservation related to its ability to grow successfully in habitats of environment and the need for developing lacking or having low levels of nitrogen and under renewable, sustainable resources has further enhanced waterlogged conditions. The Asians have recognized the value of Azolla, particularly in agriculture, either benefits of growing Azolla as biofertilizer, human alone or in combination with chemical nitrogenous fertilizers. *Author for correspondence: The exploitation of this symbiotic system is limited Tel: 011-25788431; Fax: 91-011-25766420 E-mail: [email protected] due to its sensitivity to high/low temperatures and Abbreviations: high phosphorus requirement. Also, the identity of ATP: Adenosine 5' triphosphate; 2,4-D, 2,4-dichlorophenoxy endosymbionts, variously identified as acetic acid; DNA: Deoxyribose nucleic acid; DAF: DNA Anabaena/Nostoc/Trichormus (due to difficulties in amplification fingerprinting; ELISA: Enzyme linked culturing in free living state), has been one of the immunosorbent assay; C2H4:: Ethylene; 2,4-DEE: Ethyl ester of dichlorophenoxyacetic acid; GS: Glutamine synthetase; GOGAT: major reasons for lack of in-depth classification and Glutamineoxo-glutrate amino transferase; GDH: Glutamate biotechnological interventions of this green gold dehydrogenase; IAA: Indole acetic acid; NAA: Napthylene acetic mine. Although immunological and nif probes have acid; NADPH: Nictinamide adenine dinucleotide phosphate been utilized to analyze the genetic nature of the (reduced): NR: Nitrate reductase; RFLP: Restriction fragment length polymorphism; RAPD: Random amplified polymorphic symbionts, no clear picture has emerged so far DNA; STRR: Short tandem repetitive repeat. (Franche & Cohen-Bazire, 1987; Meeks et al, 1988. PABBY et al: AZOLIA-ANABAENA SYMBIOSIS 27 Plazinski et al, 1990a; Coppenolle et al, 1993). indicated that Arthrobacter species comprised Therefore, at the present juncture, although there are a approximately 90% of the bacterial colonies vast number of research publications and extensive regardless of the Azalia species used as inoculum reviews available on different aspects of the (Braun-Howland & Nierzwicki-Bauer, 1990). Some association (Lumpkin & Plucknett, 1980; Braun- other bacteria observed include Pseudomonas species, Howland & Nierzwicki-Bauer, 1990; Wagner, 1997; Arotobacter species, Alcaligenes faecalis and Giller, 2002), there is a definite need to revisit and CauZobacter fusiformis (Plazinski et al, 1990b; synthesize the salient research findings on this Malliga & Subramanian, 1995). association, including the improvement of its potential as a biofertilizer through the use of modern molecular Morphological and Reproductive Characteristics tools and genomics. This paper reviews and analyzes The Azalia macrophyte, referred to as frond, ranges research findings concerning the biology and from 1-2.5 em in A. pinnata to 15 em or more in the agronomic utilization of Azolla, for facilitating its largest species A. nilotica. It consists of a future exploitation not only as model systems for multibranched, prostrate, floating rhizome that bears understanding symbiotic interactions but also its more small alternately arranged bilobed leaves consisting of efficient utilization in agriculture. floating dorsal lobe which is chlorophyllous, and a colourless ventral lobe which is partially submerged. Taxonomy and Morphology Unbranched, adventitious roots arise from the nodes Classification on the ventral surface of the rhizome. The The word Azolla is a combination of two Greek endosymbiont Anabaena azollae, is housed in the words azo (to dry) and allyo (to kill), reflecting the specialised leaf cavity within dorsal leaf lobe inability of plants to survive dry conditions (Lumpkin (Lumpkin & Plucknett, 1980; Giller, 2002). & Plucknett, 1980). Lamarck established the genus The cyanobacterium, Anabaena azollae, consists of Azolla in 1973 alongwith the description of A. unbranched trichomes-+containing three types of filiculoides. Azolla belongs to: Phylum, -Pteridophyta; cells-vegetative cells which are 6-8 urn long and 10- Class - Filicopsida; Order-Salviniales; Family - 12 urn broad and bead like and highly pigmented Azollaceae, and the recognized species of this genus (Singh, 1979b; Van Hove, 1989); heterocysts-lightly are grouped in two Sections, Euazolla (New World pigmented, larger than vegetative cells with thick Species: A. caroliniana, A. microphylla. A. walls and akinetes which are thick walled, resting filiculoides, A. mexicana, A. rubra) and Rhizosperma spores are formed from the vegetative cells (Lumpkin (Old World Species) : A. pinnata, A. nilotica. & Plucknett, 1980). Akinetes are not commonly Azolla has symbiotic associations with observed and the average heterocyst frequency of the cyanobacteria and eubacteria that remain associated cyanobiont has been reported to range between 15- with it throughout its life-cycle. Taxonomically, the 20% (Becking, 1976),23.1% (Peters, 1975) and 20- Azolla cyanobiont is placed in Phylum-Cyanophyta, 30% (Singh, 1977a) in different species of Azolla. Order-Nostocales, and Family-Nostocaceae. It was Studies on the homology among the species of first described as Nostoc (Strasburger, 1873) and later Anabaena within the same genus and existence of renamed as Anabaena azollae (Strasburger, 1984). similar/different Anabaena in different species of The classification at generic level is questionable-- Azolla is limited due to restricted growth of whether to designate it as Nostoc (Meeks et al, 1988; cyanobionts and altered morphology of Plazinski et al, 1990a; Kim et al, 1997) or entirely a endosymbionts when grown in artificial media (Tang new genera Trichormus azollae (Bergman et al, 1992; et al, 1990; Gebhardt & Nierzwicki-Bauer, 1991). Grilli Caiola et al, 1993). Controversial reports exist The symbiotic cyanobacteria associated with the regarding the presence of more than one strain of seven Azolla species were earlier designated as a Anabaena within a single species of Azolla and single species Anabaena azollae (Lumpkin & whether same or different Anabaena sp. is harboured Plucknett, 1980). But, difficulties involved in growing in different Azalia species (Ladha & Watanabe, 1982; the isolated cyanobacteria on artificial media have Gebhardt & Nierzwicki-Bauer, 1991). The third repeatedly questioned the taxonomical status of A. partner of the association-eubacteria have been azollae, its resemblances to Nostoc and the need for distinguished on the basis of cell shape, cell wall designation of a new genus for the endosymbiont structure and cytoplasmic organisation. Most studies specifically. Tang and co-workers (1990) observed 28 INDIAN J BIOTECHNOL, JANUARY 2003 restricted growth and limited multiplication of the The symbiotic Anabaena is able to reduce endosymbiont, but there are contrasting reports atmospheric nitrogen through the activity of enzyme committing successful isolation and culturing of nitrogenase present in the heterocysts and fulfill the total endosymbionts (Newton & Herman, 1979; Malliga & nitrogen requirement