Azolla-Anabaena Symbiosis-From Traditional Agriculture to Biotechnology

Indian Journal of Biotechnology Vol 2, January 2003, pp 26-37

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 of the association. It has been Subramanian, 1995). The morphological differences estimated that nitrogen comprises 3-6% of the dry between cultured and freshly separated samples have weight of the association (Braun-Howland & also given use to the concept of major/minor or Nierzwicki-Bauer, 1990). Nitrogenase activity has been primary/secondary symbionts in Azolla (Gebhardt & shown to increase from negligible values at the shoot Nierzwicki-Bauer, 1991). apex to maximum level at approximately leaf number Azalia exhibits both sexual (involving a complex seven and then decline in older leaves. Photosynthesis is cycle) and asexual or vegetative modes of the ultimate source of all ATP and reductant (NADPH) reproduction (Fig. 1). Vegetative reproduction occurs required for nitrogenase activity. In dark, acetylene by fragmentation via an abscission layer that forms at reduction is known to proceed only until the supply of the base of each branch (Watanabe, 1982; Van Hove, photosynthate through photosynthesis and cyclic 1989). During sexual reproduction, Azalia has two photophosphorylation is available. This suggests a distinct life phases, the diploid sporophyte and strong interaction between photosynthesis and N2 haploid gametophyte or spores. The process of fixation in this association (Braun-Howland & sporulation is quite complex and a number of Nierzwicki-Bauer, 1990). The reported rates of nitrogen environmental factors are known to play an important fixation varies greatly and range from 20-200 umol role (Kar et al, 2001; Singh et al, 2001). C2~ g' dry wt m-I (Becking, 1976), 1.0-3.6 kg N2 ha' Physiology day" (Watanabe, 1982) to 116-695 nmol C2H4 g-I fresh Carbon and Nitrogen Metabolism wt hr' (Wagner, 1997). The outstanding attributes of Azalia-Anabaena Some genera of eubacteria are also known to association are directly related to its high possess nitrogenase, and may contribute to nitrogen productivity, ability to fix nitrogen at substantial rates, fixing potential of this association (Lindblad et al, and exhibit photosynthetic rates higher than most C4 1991) but their rate of nitrogen fixation and plants. contribution towards the association has not been Sporophyte investigated. The Azalia association is capable of

Vegetative fragmentation significant light dependent, nitrogenase - catalyzed H2 I evolution (Peters et ai, 1977). Observations from both Sporophyte ---+-r field and laboratory studies indicate unidirectional I hydrogenase activity in the symbiont. It presumably Ventral lobe initial functions in recycling of electrons and ATP by oxidizing H2 produced during nitrogen fixation 11 (Peters et al, 1977). Microporocarps Megasporocarps I I It is well established that freshly separated I I Oosphere Antherozoids Anabaena azollae releases about 40-50% of the Microsporangia Megasporangi dinitrogen fixed as ammonia into the immediate I environment (Meeks et al, 1987). Both the symbiont Archegonia Antheridia and Azalia exhibit GS/GOGAT and GDH activities (Ray et al, 1978). The ammonia-assimilating enzymes are primarily associated with Azolla rather than Female Anabaena, since Azalia accounted for almost 90% of Prothallus total GS activity and 80% total GDH activity of the Microspore association (Ray et al, 1978). Studies on the kinetics ! of incorporation of I3N into glutamine and glutamate '------1'------Male prothallus-----' and the use of GS inhibitors have clearly indicated that the fern assimilates both Ny-derived and exogenously supplied NH4 + by the glutamate synthase cycle with little or no contribution from biosynthetic Fig. I-Life cycle of Azalia GDH (Meeks et al, 1987). PABBY et al: AZOLLA-ANABAENA SYMBIOSIS 29

Cyanobacterial symbionts generally have low or machinery involving N metabolism (Pabby et al, undetectable levels of GS and only 10% of GS 2003). Distinct differences in cell size and shape activity of the association is attributed to the were observed not only between symbionts from endosymbiont (Ray et al, 1978). Such low levels of different species but also among. the cultured and GS have been suggested as a biochemical mechanism freshly separated cyanobionts of each species. Growth responsible for increased ammonia excretion of the attributes such as chlorophyll a, sugars, proteins were endosymbionts. It has been postulated that the host significantly higher in the freshly separated symbionts Azolla may produce effector molecules which modify while the cultured symbionts exhibited higher the ammonia-assimilating pathways of the activities of N-assimilation enzymes such as GS, NR endosymbiont by exhibiting or altering activity or and nitrogenase. synthesis (Rai et al, 1986). Appreciable levels of GDH has, however, been observed in the endophyte Utilization as Biofertilizers (Ray et al, 1978). But GDH, having a appreciably Nitrogen is the element most often limiting food lower affinity for ammonia than GS, could provide a production in the world. Agricultural production is regulatory role, enabling effective reassimilation of known to be directly dependent on nitrogen. In India, released ammonia at high intra-cavity ammonia rice contributes about 45% of the total cereal concentrations. production and is the staple food for majority of its One of the unique properties of Arolla-Anabaena population. Nitrogen can be supplied to rice crop symbiosis is that both the eukaryotic Azolla and the either through chemical fertilizers or through prokaryotic Anabaena have the ability to fix biological agents. Besides blue green algae, which are atmospheric CO2. Azolla contains chlorophyll a and b important biological inputs in rice cultivation, Azolla as well as carotenoids, while endophytic Anabaena forms another inexpensive, economical and contains chlorophyll a, phycobilins and carotenoids ecofriendly biofertilizer which provides unseen The phycocyanin content has always, in general, been benefits in terms of carbon and nitrogen enrichment observed to be higher than allophyocyanin and of soil and overall improvement in soil/crop phycocerythrin (Samal & Kannaiyan, 1994). The management practices and fertility status (Kaushik & photosynthetic pigments of the two organisms are Prasanna, 1989). complementary and, therefore, broader portion of The most suitable crop for application of Azalia is solar energy spectrum is harvested for photosynthesis. lowland rice, since both rice crop and fern require The formation of anthocyanins in Azolla is known to similar environmental conditions. The characteristics be triggered under high light intensity or low that make Azolla suitable as biofertilizer for rice temperature giving reddish appearance to Azolla include requirement of shallow freshwater habitat, fronds under these conditions (Lumpkin & Plucknett, rapid growth, high nitrogen fixing capacity, quick 1980). decomposition, and growth alongwith rice without

Both the partners in the symbiosis fix CO2 via competing for light and space (Vlek et al, 1995). In Calvin cycle and sucrose is the primary addition, a thick mat of Azolla suppresses weeds and photosynthetic end product in the association. reduces volatilization of ammonia in rice fields

However, when 14C02 was added to Anabaena (Singh, 2000). azollae isolated from leaf cavities, 14C-sucrose was Experiments have demonstrated the effectiveness not detected as the major product of cyanobacterial of Azolla as a biofertilizer on rice but the extent of photosynthesis. Hence, it has been suggested that A. benefit varies greatly because of different climatic azollae, in the mature leaf cavities, may be capable of conditions, methods of application, Azolla species, photoheterotrophic or mixotrophic metabolism and etc. Increase in grain yields of rice from 14% to 40% sucrose produced by the fern may be utilized as a have been reported with Azolla being used as dual reduced carbon source (Van Hove, 1989). crop. The use of Azolla as monocrop during fallow The cultured vs. freshly separated symbionts from season has shown to increase rice yield by 112% as six species of Azolla (belonging to the germplasm of against unfertilized controls by 23% when applied as National Centre for Conservation and Utilization of intercrop with rice, and by 216% when applied both Blue-Green Algae) were examined for their as monocrop and intercrop (Peters, 1975). Singh morphological attributes and growth parameters such (l977b) obtained a 6% increase in rice yield when A. as proteins, Chlorophyll and also enzymatic pinnata was grown with rice and an increase from 9% 30 INDIAN J BIOTECHNOL, JANUARY 2003 to 38% when Azolla was incorporated into the soil. (Marwaha et al, 1992). Sharma et al (1999) recorded Application of A. pinnata as intercrop twice, first 5 highest yield of wheat with application of 20 t of days after transplanting of rice and then after 27 days, Azalia and 60 kg N. resulted in 27% increase in grain yield while Although the agronomic potential of Azolla is well application of Azolla as monocrop and an intercrop documented, a number of environmental and incorporated after 40 days resulted in a 30.6% nutritional factors have restricted its widespread use. increase in rice grain yield. Studies have indicated that a single crop of Azolla can provide 20-40 kg N Role of Environmental Factors ha-', but remained insufficient to meet the total Environmental factors such as humidity, light nitrogen requirement of the target crop. Therefore, use intensity, photoperiod, salinity and temperature play of Azalia in combination with chemical nitrogen an important role in controlling growth and fertilizers affords a feasible alternative practice. physiology of the association. Water is a fundamental Singh and co-workers (1992) observed similar grain prerequisite for growth and multiplication of yield with Azalia alone and application of 150 kg N Azalia.The plant is made up of 90-95% water which is ha' as chemical fertilizer. An increase in yield of 0.9- required for the maintenance of structural integrity 2.0 t ha' and 0.8-1.0 t ha' was found when Azalia and major physiological processes. A water depth of was incorporated as compared to ammonium sulphate 3-5 em is recommended for optimal growth of AzalIa treated plots. Azalia incorporated with different doses in both nature and laboratory. Azalia requires relative of inorganic nitrogen (Subudhi & Singh, 1980) gave humidity between 85-90% for its normal growth. higher yield as compared to use of inorganic nitrogen Maximum nitrogenase activity has been observed in alone. Azalia incorporation along with 30 kg N ha' A. caroliniana at 88-95% moisture levels (Braun- and 50 kg N ha-' as ammonium sulphate (Singh, Howland & Nierzwicki-Bauer, 1990). 1977c) increased the yield to as much as obtained Light intensity and photoperiod greatly affect rates with the application of almost 60 and 80 kg N ha'. of growth and nitrogen fixation in Azalia. High light On comparing the effectiveness of Azalia with intensity was considered responsible for the other bio-fertilizers, rice grain yield was highest with senescence of the fern. Azalia attains maximum the application of Azolla + 120 kg N ha' (5.0 t ha-'), biomass at light intensities greater than 50% sunlight, followed by blue-green algae + Azalia + 60 kg N ha' while exposure to high light intensities usually (4.62 t ha') and lastly by 120 kg N ha" (4.61 t ha') resulted in decreased rates of nitrogen fixation (Vlek (Singh et al, 1992). Application of either Azalia or et al, 1995). Azalia can survive in a wide range of pH blue green algae along with phosphorus fertilizer (3.5-10.0) but optimum growth was observed in the (Singh & Singh, 1990) increased grain yield. The pH range from 4.5-7.0 (Kannaiyan, 1979). Growth application of phosphorus fertilizer either during was not supported in acidic (pH 3.0-3.8) and alkaline intercropping or as phosphorus enriched Azalia soil (pH 8.4) (Singh, 1977b). Azalia is capable of increased nitrogen uptake by rice grain (Singh & growing in a wide range of temperatures from 5-45"C. Singh, 1995). The growth rate of A. mexicana, A. microphylla and Azolla can be used as green manure in cultivation A. pinnata was highest at temperatures above 30°C of Water bamboo (Zizanica aquatica), arrowhead but A. filiculoides and A. caroliniana grew better at (Sagittaria sagiuifoliai, taro tColocasia esculantai, temperatures below 35°C (Peters et al, 1980). wheat (Triticum aestivumi and rice (Oryza sativa). The fern requires all macro and micro nutrients Utilization of Azolla as green manure in waterlogged which are essential for the normal growth and is soil resulted in rapid mineralization with the release sensitive to the presence of excess or absence of of 60-80% of the N within two weeks. Azalia applied suitable concentrations of nutrients. Phosphorus, as monocrop between the wheat and rice crop or as an potassium, calcium and magnesium are important intercrop with rice had significant beneficial effect on macronutrients, while micronutrients such as iron, subsequent wheat crop (Kolte & Misra, 1990). molybdenum, cobalt and zinc have shown to be Mahapatra & Sharma (1989) found beneficial effects essential for its growth and nitrogen fixation (Braun- on subsequent wheat crop with increase in grain yield Howland & Nierzwicki-Bauer, 1990). Among by 56% to 67% over control with application of macronutrients, phosphorus is the most common Azalia along with Sesbania. Incorporation of fresh element limiting growth of Azalia. As its content in fronds of Azalia also increased grain yield of wheat soil solution and in paddy water is generally too low PABBY et al: AZOLIA-ANABAENA SYMBIOSIS 31 to meet the requirement of Azolla, the addition of million hectares. An important biotic factor phosphorus is recommended for better growth and influencing biomass production of Azalia and its multiplication of Azolla (Kannaiyan et al, 1981). utilization is the prevalence of insect attacks, However, Azolla can grow without phosphorus in especially when temperatures rise above 28°C. It has soils having Olsen-Po Subudhi & Singh (1979) been estimated that insect population can reach up to observed reduction in growth and chlorophyll content 90,000 m-2 during peak summer and can completely and increase in soluble sugars and amino-nitrogen. damage Azolla crop within 3-5 days. More than 3 I Kannaiyan and coworkers (1981) reported that 6 kg insect pests belonging to Diptera, Coleoptera. ha phosphorus to be optimum for growth of Azolla. Lepidoptera, Homoptera and Orthoptera have been Higher heterocyst frequency of Anabaena azollae, reported. In India, Nymphula and Chironomids. nitrogen fixation and biomass production was which have been reported as important pests of Azalia observed with split application of phosphorus than (Singh, 1979b), cause rolling of the leaves and their with single application and with no phosphorus feeding results in brown patches. (Singh & Singh, 1988). The concentration of medium Carbofuran--An insecticide commonly used in rice phosphorus (0-0.5 flM) appeared to be critical for cultivation has been reported to stimulate the growth optimum growth of Azolla (Sah et al, 1989). A mutant and nitrogen fixation of Azolla at low dosages (Kar & strain with 0.75 rnM phosphorus requirement has been Singh, 1979). A strain from Bangladesh was reported developed (Vaishampayan et al, 1992). Subudhi & to be resistant to insecticides and could be used as Singh (1979) observed reduction in growth and biofertilizer during rainy season when incidence of nitrogen fixation when grown in Ca and P deficient pest control is high (Singh, 1979a). Herbicides such medium. A decrease in levels of nitrogen and as Butachlor, 2,4-DEE and 2,4-0 decreased biomass, phosphorus and increase in lipids and activity of acid chlorophyll, nitrogen content and nitrogen fixation, and alkaline phosphatase can act as physiological when used at recommended rates of 2.5, 1.5, 1.0 and indicators of phosphorus deficiency in Azolla (Pabby 0.4 kg a.i. ha-'. Monocrotophos, phorate, thiodan, et al, 2000a). Under phosphorus starvation, cells of quinalphos, carbosulfan, chloropyrifos and furadan endosymbionts Anabaena azollae show pale green were also effective in controlling pests, besides coloration and appear deformed. stimulating Azolla growth and nitrogenase activity. The symbiotic association of Azolla does not Neem cake @ 500kg/ha and carbofuran @ 75 kg/ha require any nitrogen in the growth medium as it were able to control attack of pests effectively (Singh houses the diazotrophic endosymbiont in its lobes. etaZ,1987). Different nitrogen sources significantly affect The incidence of fungal diseases in Azalia was nitrogen fixation but not fresh biomass (Manna & reported in India in 1979 «Sasi et al, 1979) which Singh, 1991), but a higher relative growth rate of A. was further aggravated due to snail attacks. A. nilotica caroliniana and A. pinnata was observed at 5 rnM was found to be most sensitive, while A. carliniana nitrate (Singh et al, 1992). Studies carried out both at was least. To control fungal diseases caused by laboratory and field level indicated inhibitory effect of Rhizoctonia solani, fungicides such as benomyl @ O. I nitrogenous fertilizers on the ammonia-assimilating % and carbondarin @ 0.2% were reported to be enzymes (Venkataramanan & Kannaiyan, 1986). In A. effective. Neemcake @ 500 kg ha" controlled black microphylla, ammonia assimilatory enzymes achieved root rot. Although genetic approaches such as highest activity at 24 hr and disappeared thereafter introduction of toxin genes into Azolla can be a whereas in A. pinnata GS/GOGAT system retained its feasible alternative, no breakthrough has been function up to 14th day of incubation in urea (Pabby obtained so far. et al. 2000b). It can surmised that this symbiotic association, Spore Production Technology involving a diazotrophic partner shows adaptation to The use of sporocarps of Azolla as primary source N-deficient and N-supplemented environment of inoculum in field can overcome several problems mediated mainly through the GS-GOGAT pathway of associated with the current biofertilizer technology the cyanobiont. which involves a bulky amount of initial inoculum Pests and Diseases of Azolla and their Control besides difficulties in its transportation. It has been estimated that Azolla is currently used But the factors triggering sporulation in nature are on only 2% of the world's rice fields i.e. around 3 poorly understood and are apparently species or strain 32 INDIAN J BIOTECHNOL, JANUARY 2003

specific. Also, the frequency of sporulation varies given geographical location or sporulate only during a widely among different species/strains of Azolla and certain part of the year. Often, strains with higher is stimulated by a combination of environmental biofertilizer potential have poor sporulation (Singh et factors including stress factors such as low al, 2001). Although the successful use of sporocarps temperature (Kannaiyan & Rains, 1985), low light for raising Azalia sporophytes under field conditions intensity (Becking, 1976), fluctuating photoperiod, was demonstrated in China, this practice has not less availability of phosphorus (Singh & Singh, 1988) become commercial because of poor growth of and overcrowding (Kannaiyan, 1979). These are sporophyte during initial stages of its development. thought to play interacting roles in induction of Information on use of sporocarps under field sporulation in natural habitats. conditions as primary inoculum is also scarce in India. In A. filiculoides, sporocarp formation is associated Kannaiyan (1994) suggested that the application of with mat formation, while in temperate regions this presoaked (soaked in water for 12 hrs) frond based phenomenon occurs during summer. A. pinnata sporocarp material @ 5 kg ha-1 a week after sporulate in winters in India and shown to be related transplanting of rice is as effective as vegetative with plant age and winter season (Singh, 1977b; inoculum. Kannaiyan, 1979). Incorporation of ferric chloride @ Uncertain and limited sporulation of Azalia strains 50 ppm, foliar spraying of growth regulators has been a major constraint and biotechnological particularly GA3@ 100 ppm and organic amendments interventions involving artificial induction are induce sporulation in Azalia (Kannaiyan et al, 1988). essential at this stage (Singh & Mahapatra, 2000). In A. microphylla, the sporulation frequency (number of sporulating plants/lOO plants) and number of sporocarps were highest in February and lowest in Genetic Improvement of Azolla June (Singh et al, 2001) while, in Philippines, this Although Azalla-Anabaena symbiosis has been species sporulates better during November- January at exploited for multiple uses, its poorly understood relatively low day/night temperature and shorter day symbiotic interactions has restricted its amenability to length. In A. mexicana, sporocarps/plant were genetic manuplation. Among the major types of maximum at 25115°C and a complete inhibition of abioticlbiotic stress that affect the growth and sporulation occurred at 38/25°C (Kannaiyan et al, performance of Azalia as a biofertilizer, its sensitivity 1988). The sporulation frequency and number of to temperature and water stress are most significant. micro, mega and total sporocarps/plant were observed to be negatively correlated with the average minimum Development of Stress Tolerant Azolla temperature and day length in A. microphylla (Kar et In recent years, soil salinity has become a serious al, 2001). problem in global agriculture, more in areas where An Azalia sporocarp technology involving water deficit prevails over a long period of time. production, collection, storage and germination of Azalia species are generally sensitive to NaCI sporocarps has been developed by Kannaiyan (1990) concentrations beyond 30 mM. Rai & Rai (1999, and referred as "Frond based dried spore inoculum" of 2000) were able to obtain Azalia plants tolerant to Azalia. The major limitation in sporocarp production 60mM NaCI concentrations through stepwise transfer is phosphorus management. The sporocarp yield in to higher concentrations. On analysis of such plants, it Azalia depends on total biomass and intensity of was observed that this is due to development of sporulation. Application of P (4.4-6.6 kg ha-') was capability by Azolla to regulate ion concentration, observed to be necessary for biomass production which may be related to modulation of genome (Singh & Singh, 1990) but this suppresses sporulation expression, as observed in higher plants. Salinity also significantly (Singh et al, 1987; Kar et al, 2001). This increased photosynthetic O2 evolution in both adapted problem can be tackled by either using Azalia strains and unadapted plants of A. pinnata. The response of with better sporulation under P fertilization or Azolla species to water stress (both salt and osmotic developing appropriate management practices to stress) showed that the Amazonian (RAR) specimens minimize the adverse effects of P on sporulation or of A. caroliniana are more tolerant to both types of through production of sexual hybrids. Uncertain and stress (Zimmerman, 1985). Similarly A. pinnata could limited sporulation in Azolla is another problem. tolerate 40mM NaCI (Rajarathinanam & Padhya, Many strains of Azalia either ?o not sporulate in a 1989). PABBY et al: AZOLLA-ANABAENA SYMBIOSIS 33

Although salinity tolerance was feasible in Azalia cyanobacterial microsymbiont from high temperature through physiological interventions, it has not been tolerant species A. microphylla to high temperature possible to develop temperature tolerant Azolla using sensitive species A. filiculoides resulting in increased such techniques. Among the approaches employed in temperature tolerance of A. filiculoides (Watanabe et this direction, development of hybrids has been al, 1989). Sarma & Deka (1987) produced callus successful to a certain extent. using leaf explant on Schenk and Hilderrandt medium supplemented with IAA, NAA, 2,4-D and benzyl 1 Production of Hybrids aminopurine at the rate 1,0.5, 1 and 0.5 ug L- • Callus Sexual hybridization can be an important method differentiation appeared 22 days after inoculation at for developing suitable hybrids with improved 25±I°C and 1000 lux light intensity. Frequent agronomic traits such as higher nitrogen fixing ability, subculturing further led to reduction in doubling time. relative growth rate, biomass production, sporulation, The callus lacked cells of the symbiont and therefore, temperature tolerance and resistance to pest and can be used to obtain Azolla protoplasts for fusion disease coupled with better adaptation under different with the strains of Anabaena having improved N agroecological conditions. fixing ability. A temperature tolerant mutant (up to Hybridization between A. microphylia (female) and 38°C) of A. pinnata has been obtained using ethyl A. filiculoides (male) improved annual biomass methane sulphonate (Dey, 1999). production. The hybrid produced biomass comparable to that of A. filiculoides in spring and of A. Fingerprinting of Azolla-Anabaena Symbiosis microphylla in summer and autumn, thus increasing In recent years, different techniques have been overall annual production. On the other hand, the employed to identify and group the cyanobacteria hybrid with A. filiculoides as female parent exhibited forming symbiosis with the different Azolla species. higher sensitivity to elevated temperatures than the Although ELISA, quantitative immunobinding assays hybrid with A. microphylla as female parent and other fluorescent labeling techniques revealed a (Watanabe et at, 1993). Hybrids between these high degree of similarity between freshly isolated species were intermediate to those of parents (DoVan endosymbionts from several Azolla species, distinct Cat et al, 1989), were not stressed (red colour) under differences were observed in the cultured symbionts. P or Ca deficient conditions and had higher nitrogen Fatty acid profiles of the symbionts also clearly content than that of parent, A. microphylia. Their brought out the distinction between cyanobionts from biomass production in field was also higher than A. Arolla plants belonging to section Euazolla and those microphylla, indicating positive heterosis. Attempts to from the section Rhizosperma (Caudales et al, 1995). hybridize A. mexicana or A. pinnata with other Azolla Franche and Cohen-Bazire (1987) studied the strains have been unsuccessful. This has been distribution of restriction sites around the nif HDK attributed to the absence of fertilization between a genes of endosymbionts extracted from four Azolla member of Old World Species (A. pinnata) and A. species and one free living Anabaena azollae and mexicana (New World Species). demonstrated that the arrangement of nif HDK genes In India, Azolla hybrids AHC-l, AHC-2 and AHC- was strongly conserved among Azolia species, 3 were derived from crosses within species of A. regardless of their geographical origin. . They also microphylla and AHA between A. pinnata imbricata demonstrated that the cyanobionts from Azolla plants x A. filiculoides and A. microphylla at Tamil Nadu within Euzolla and Rhizosperma belong to two Agricultural University, Coimbatore. All the hybrids different evolutionary lines. Moreover, even within exhibited higher biomass along with higher heterocyst Euazolla, two subgroups could be distinguished--one frequency and thereby N2 fixation. They also consisting of cyanobionts from A. caroliniana and A. exhibited higher chlorophyll content, nutrient content filiculoides and the other consisting of cyanobionts and activity of ammonia-assimilating enzymes from A. microphylla and A. mexicana. But (Gopalaswamy & Kannaiyan, 1997). Selection of cyanobionts of Rhizosperma could not be subdivided. promising strains with thermal tolerance was The Azolla accessions, fingerprinted and classified attempted by growing them in a phytotron (Do Van (Zimmerman et al, 1991) by enzyme electrophoresis Cat et al, 1989). Success has also been obtained in and leaf trichome morphology, provided a working transferring Anabaena from one species of Azolla to taxonomy for identification and utilization of Azolla another. Liu et al (1989) transferred the as a biofertilizer. Later in 1990, Plazinski et al 34 INDIAN J BIOTECHNOL, JANUARY 2003

(1990a) found that cyanobionts of A. caroliniana, A. identification of accessions of Azolla-Anabaena intact mexican a and A. microphylla need to be grouped symbioses. The advantage of DAF technique which together and differed from the cyanobiont of A. utilizes very short primers of ~ 5 nucleotides in filiculoides. On the other hand, Van Coppenolle et al length, is its utility for distinguishing among closely (1993) grouped the symbionts from seven Azolla related genotypes of both prokaryotic and eukaryotic species into three groups using nif gene probes and organisms. DAF was observed to be useful, therefore, chloroplast DNA probes and demonstrated the utility for not only defining the contribution of macro and of RFLP analysis of host plant or symbiotic micro symbionts in the fingerprints of Azolla but also cyanobacteria. DNA polymorphisms using RFLPs confirming sexual hybridization and maternal reinforced the conclusions derived from phenetic data transmission of Anabaena azollae strain (Eskew et al, and located additional polymorphisms among strains 1993). A rapid diagnostic system for Azolla and its which were enzymatically related (Zimmerman et al, cyanobionts using random primers, and primers for 1991). Cumulative RFLP analysis using heterologous the chloroplast-encoded introns of the tRNA-Leucine and random homologous gene probes reinforced the (UAA) gene was also developed (Kim et al, 1997). species relationships presented from isozyme analyses This was highly useful for rapid assessment of and trichome anatomies and members of A. similarities among Anabaena azollae and minor caroliniana- A. mexicana- A. microphylla cluster Anabaena isolates from Azolla. STRR-PCR showed distinct genetic similarity. DNA-DNA fingerprinting can be a valuable tool for analyzing hybridization techniques were also utilized for genetic diversity of the cyanobionts and phylogentic authentication of new Azolla-Anabaena symbiotic tree generated distinguished three clusters--one associations and strain and species specific probes containing the four species from Section Euazolla. a were generated which revealed that Azolla hosts can second, the isolate from A. filiculoides and the third harbour more than one Anabaena symbiont (Plazinski containing the three isolates from Section et al, 1990a). RFLP analysis using selected DNA Rhizosperma (Zheng et al, 1999). probes revealed genetic variation in cyanobacterial According to the knowledge and belief of authors, symbionts of Azolla and its closer relationship to free comparative molecular characterization of the living cyanobacterial strains (Plazinski et al, 1990a). endosymbionts, both freshly separated and cultured, Gebhardt and Nierzwicki-Bauer (1991) carried out has not been undertaken using PCR based primers. molecular and morphological characterization of free Future efforts of authors are, therefore, in this living and symbiotic counterparts from Azolla direction for a better understanding of the nature and mexicana and A. pinnata and suggested the ubiquitous identity of the symbionts and this unique association presence of a culturable minor cyanobacterial between a prokaryote and an eukaryote. symbiont in atleast three species of Azolla. Conclusions/Future Approaches Plasmid biology has been investigated in the From an agricultural point of view, the most symbionts from seven Azolla species and presence of important N-fixing prokaryotes form symbiotic 1-3 cryptic plasmids (35-100 MD) have been associations, especially with plants. However, the reported. Several heterologous DNA probes, high energy costs of biological nitrogen fixation often including Rhizobium symbiotic genes such as nod box sets a limit to the amount of fixation that a host can and nod MN and exopolysaccharide gene exoY support. Cyanobacterial associations are very valuable showed hybridization with plasmids belonging to the in this regard as the symbiont is itself a symbionts (Anabaena) and suggest that these photoautotroph. However, for Azotla-Anabaena plasmids may play a role in symbiotic interactions symbiosis, it has been calculated that 80% or more of with Azolla fern (Plazinski et al, 1990b). the photosynthate used for N-fixation is provided by Coppenolle et al (1995) utilized random primers for the host. It is, therefore, important to undertake identification and phylogenetic analysis of this fern research efforts towards critically evaluating the using accessions from all over the world. They were physiological capabilities of the symbiont vs. host and able to construct a phylogenetic classification of also elucidate the host signals/recognition processes Azolla using 10 random primers which was indicative between Azolla and Anabaena. These efforts also of the usefulness of RAPD techniques in evaluating need to include molecular dissection of the genetic diversity. A novel tool for DNA controls/regulatory elements involved in heterocyst fingerprinting-DAF has been utilized for positive and akinete differentiation processes. PABBY et al: AZaLIA-ANABAENA SYMBIOSIS 35

Another area of biotechnological research is Anabaena azollae based on RFLPs detected in Azolla increasing the usefulness of Azolla-Anabaena Anabaena DNA complexes using nif gene probes. Theor Appl Genet, 91,589-597. association as a biofertilizer. Hybridization studies Dey T, 1999. Induction and characterization of Arolla-Anabaena and reconstitution of symbiotic associations using symbiotic N2 fixing mutants and their assessment in rice altered genetically manipulated symbionts have iOryza sativa). Ph. D. Thesis, Banaras Hindu University. indicated that this can be an area of fruitful research. Varanasi, India. The benefits of Azolla is curbing ammonia Do Van Cat, Watanabe I, Zimmerman W J, Lumpkin T A & De Waha Baillonville T, 1989. Sexual hybridization among volatilization from flooded rice systems further Azolla species. Can J Bot, 67, 3482-3485. strengthens the promise of integrating Azolla in rice Eskew 0 J, Caetano A, Bassam B J & Gresshoff P M, 1993. management systems under field conditions (Singh, DNA amplification fingerprinting of the Arolla-Anabaena 2000). symbiosis. Plant Mol Bioi, 21, 363-373. Franche C & Cohen- Bazire G, 1987. Evolutionary divergence in In this age, when mankind is threatened by drastic the nif HDK gene region among nine symbiotic Anabaena changes in the global environment, triggered by man- azollae and between Anabaena azollae and some free living made activities, there is an urgent need to use heterocystous cyanobacteria. Symbiosis, 3, J 59-\78. sustainable and environmentally appropriate practices Gebhardt J S & Nierzwicki-Bauer S A, J 991. Identification of a in agriculture. 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