Azolla-Anabaena Symbiosis-From Traditional Agriculture to ... · Azolla-Anabaena Symbiosis-From Traditional Agriculture to Biotechnology Anjuli Pabby, Radha Prasanna and P K Singh*

Indian Journal of BiotechnologyVol 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 EastAsia. But its utilization and genetic improvement has been limited mainly due to problems associated with theisolation and characterization of cyanobionts and the relative sensitivity of the fern to extremes of temperature andlight intensity. This paper reviews the historical background of Azolla, its metabolic capabilities and present dayutilization in agriculture. An outline of biotechnological interventions, carried out in India and abroad, is alsodiscussed for a better understanding of the symbiotic interactions, which can go a long way in further exploitation ofthis association in agriculture and environmental management.

Keywords: Azolla, Anabaena, biofertilizer, fingerprinting, symbiont

IntroductionAzolla is a small aquatic fern of demonstrated

agronomic significance in both developed anddeveloping countries (Singh, 1979a; Lumpkin &Plucknett, 1980; Watanabe, 1982; Giller, 2002). Theassociation between Azolla and Anabaena azollae is asymbiotic one, wherein the eukaryotic partner Azollahouses the prokaryotic endosymbiont in its leafcavities and provides carbon sources and in turnobtains its nitrogen requirements. This mutualexchange of activities helps in quick growth andmultiplication of the fern under optimalenvironmental conditions.

The agronomic potential of this association isrelated to its ability to grow successfully in habitatslacking or having low levels of nitrogen and underwaterlogged conditions. The Asians have recognizedbenefits of growing Azolla as biofertilizer, human

*Author for correspondence:Tel: 011-25788431; Fax: 91-011-25766420E-mail: [email protected]:ATP: Adenosine 5' triphosphate; 2,4-D, 2,4-dichlorophenoxyacetic acid; DNA: Deoxyribose nucleic acid; DAF: DNAamplification fingerprinting; ELISA: Enzyme linkedimmunosorbent assay; C2H4:: Ethylene; 2,4-DEE: Ethyl ester ofdichlorophenoxyacetic acid; GS: Glutamine synthetase; GOGAT:Glutamineoxo-glutrate amino transferase; GDH: Glutamatedehydrogenase; IAA: Indole acetic acid; NAA: Napthylene aceticacid; NADPH: Nictinamide adenine dinucleotide phosphate(reduced): NR: Nitrate reductase; RFLP: Restriction fragmentlength polymorphism; RAPD: Random amplified polymorphicDNA; STRR: Short tandem repetitive repeat.

food and medicine, besides its role in environmentalmanagement and as controlling agent for weeds andmosquitoes. It also improves water quality by removalof excess quantities of nitrate and phosphorus and isalso used as fodder, feed for fish, ducks and rabbits(Wagner, 1997). Besides its extensive use as a N-supplement in rice-based ecosystems, it has also beenused in other crops such as taro, wheat, tomato andbanana (Van Hove, 1989; Marwaha et al. 1992).

However, its most outstanding attribute, which isrelevant as a biofertilizer, is related to its high rate ofmultiplication, which helps in covering the entiresurface of water body in which it is growing within 2-3 days. The growing concern about the conservationof environment and the need for developingrenewable, sustainable resources has further enhancedthe value of Azolla, particularly in agriculture, eitheralone or in combination with chemical nitrogenousfertilizers.

The exploitation of this symbiotic system is limiteddue to its sensitivity to high/low temperatures andhigh phosphorus requirement. Also, the identity ofendosymbionts, variously identified asAnabaena/Nostoc/Trichormus (due to difficulties inculturing in free living state), has been one of themajor reasons for lack of in-depth classification andbiotechnological interventions of this green goldmine. Although immunological and nif probes havebeen utilized to analyze the genetic nature of thesymbionts, no clear picture has emerged so far(Franche & Cohen-Bazire, 1987; Meeks et al, 1988.

PABBY et al: AZOLIA-ANABAENA SYMBIOSIS

Plazinski et al, 1990a; Coppenolle et al, 1993).Therefore, at the present juncture, although there are avast number of research publications and extensivereviews available on different aspects of theassociation (Lumpkin & Plucknett, 1980; Braun-Howland & Nierzwicki-Bauer, 1990; Wagner, 1997;Giller, 2002), there is a definite need to revisit andsynthesize the salient research findings on thisassociation, including the improvement of its potentialas a biofertilizer through the use of modern moleculartools and genomics. This paper reviews and analyzesresearch findings concerning the biology andagronomic utilization of Azolla, for facilitating itsfuture exploitation not only as model systems forunderstanding symbiotic interactions but also its moreefficient utilization in agriculture.

Taxonomy and MorphologyClassification

The word Azolla is a combination of two Greekwords azo (to dry) and allyo (to kill), reflecting theinability of plants to survive dry conditions (Lumpkin& Plucknett, 1980). Lamarck established the genusAzolla in 1973 alongwith the description of A.filiculoides. Azolla belongs to: Phylum, -Pteridophyta;Class - Filicopsida; Order-Salviniales; Family -Azollaceae, and the recognized species of this genusare grouped in two Sections, Euazolla (New WorldSpecies: A. caroliniana, A. microphylla. A.filiculoides, A. mexicana, A. rubra) and Rhizosperma(Old World Species) : A. pinnata, A. nilotica.

Azolla has symbiotic associations withcyanobacteria and eubacteria that remain associatedwith it throughout its life-cycle. Taxonomically, theAzolla cyanobiont is placed in Phylum-Cyanophyta,Order-Nostocales, and Family-Nostocaceae. It wasfirst described as Nostoc (Strasburger, 1873) and laterrenamed as Anabaena azollae (Strasburger, 1984).The classification at generic level is questionable--whether to designate it as Nostoc (Meeks et al, 1988;Plazinski et al, 1990a; Kim et al, 1997) or entirely anew genera Trichormus azollae (Bergman et al, 1992;Grilli Caiola et al, 1993). Controversial reports existregarding the presence of more than one strain ofAnabaena within a single species of Azolla andwhether same or different Anabaena sp. is harbouredin different Azalia species (Ladha & Watanabe, 1982;Gebhardt & Nierzwicki-Bauer, 1991). The thirdpartner of the association-eubacteria have beendistinguished on the basis of cell shape, cell wallstructure and cytoplasmic organisation. Most studies

27

indicated that Arthrobacter species comprisedapproximately 90% of the bacterial coloniesregardless of the Azalia species used as inoculum(Braun-Howland & Nierzwicki-Bauer, 1990). Someother bacteria observed include Pseudomonas species,Arotobacter species, Alcaligenes faecalis andCauZobacter fusiformis (Plazinski et al, 1990b;Malliga & Subramanian, 1995).

Morphological and Reproductive CharacteristicsThe Azalia macrophyte, referred to as frond, ranges

from 1-2.5 em in A. pinnata to 15 em or more in thelargest species A. nilotica. It consists of amultibranched, prostrate, floating rhizome that bearssmall alternately arranged bilobed leaves consisting offloating dorsal lobe which is chlorophyllous, and acolourless ventral lobe which is partially submerged.Unbranched, adventitious roots arise from the nodeson the ventral surface of the rhizome. Theendosymbiont Anabaena azollae, is housed in thespecialised leaf cavity within dorsal leaf lobe(Lumpkin & Plucknett, 1980; Giller, 2002).

The cyanobacterium, Anabaena azollae, consists ofunbranched trichomes-+containing three types ofcells-vegetative cells which are 6-8 urn long and 10-12 urn broad and bead like and highly pigmented(Singh, 1979b; Van Hove, 1989); heterocysts-lightlypigmented, larger than vegetative cells with thickwalls and akinetes which are thick walled, restingspores are formed from the vegetative cells (Lumpkin& Plucknett, 1980). Akinetes are not commonlyobserved and the average heterocyst frequency of thecyanobiont has been reported to range between 15-20% (Becking, 1976),23.1% (Peters, 1975) and 20-30% (Singh, 1977a) in different species of Azolla.Studies on the homology among the species ofAnabaena within the same genus and existence ofsimilar/different Anabaena in different species ofAzolla is limited due to restricted growth ofcyanobionts and altered morphology ofendosymbionts when grown in artificial media (Tanget al, 1990; Gebhardt & Nierzwicki-Bauer, 1991).The symbiotic cyanobacteria associated with theseven Azolla species were earlier designated as asingle species Anabaena azollae (Lumpkin &Plucknett, 1980). But, difficulties involved in growingthe isolated cyanobacteria on artificial media haverepeatedly questioned the taxonomical status of A.azollae, its resemblances to Nostoc and the need fordesignation of a new genus for the endosymbiontspecifically. Tang and co-workers (1990) observed

28 INDIAN J BIOTECHNOL, JANUARY 2003

restricted growth and limited multiplication of theendosymbiont, but there are contrasting reportscommitting successful isolation and culturing ofendosymbionts (Newton & Herman, 1979; Malliga &Subramanian, 1995). The morphological differencesbetween cultured and freshly separated samples havealso given use to the concept of major/minor orprimary/secondary symbionts in Azolla (Gebhardt &Nierzwicki-Bauer, 1991).

Azalia exhibits both sexual (involving a complexcycle) and asexual or vegetative modes ofreproduction (Fig. 1). Vegetative reproduction occursby fragmentation via an abscission layer that forms atthe base of each branch (Watanabe, 1982; Van Hove,1989). During sexual reproduction, Azalia has twodistinct life phases, the diploid sporophyte andhaploid gametophyte or spores. The process ofsporulation is quite complex and a number ofenvironmental factors are known to play an importantrole (Kar et al, 2001; Singh et al, 2001).PhysiologyCarbon and Nitrogen Metabolism

The outstanding attributes of Azalia-Anabaenaassociation are directly related to its highproductivity, ability to fix nitrogen at substantial rates,and exhibit photosynthetic rates higher than most C4plants.

Sporophyte

ISporophyte ---+-r

Vegetative fragmentation

IVentral lobe initial

11Microporocarps Megasporocarps

I IMicrosporangia Megasporangi

IOosphere

I

IAntherozoids

Archegonia Antheridia

FemaleProthallus

Microspore

!'---------1'------- Male prothallus-----'

Fig. I-Life cycle of Azalia

The symbiotic Anabaena is able to reduceatmospheric nitrogen through the activity of enzymenitrogenase present in the heterocysts and fulfill the totalnitrogen requirement of the association. It has beenestimated that nitrogen comprises 3-6% of the dryweight of the association (Braun-Howland &Nierzwicki-Bauer, 1990). Nitrogenase activity has beenshown to increase from negligible values at the shootapex to maximum level at approximately leaf numberseven and then decline in older leaves. Photosynthesis isthe ultimate source of all ATP and reductant (NADPH)required for nitrogenase activity. In dark, acetylenereduction is known to proceed only until the supply ofphotosynthate through photosynthesis and cyclicphotophosphorylation is available. This suggests astrong interaction between photosynthesis and N2fixation in this association (Braun-Howland &Nierzwicki-Bauer, 1990). The reported rates of nitrogenfixation varies greatly and range from 20-200 umolC2~ g' dry wt m-I (Becking, 1976), 1.0-3.6 kg N2 ha'day" (Watanabe, 1982) to 116-695 nmol C2H4 g-I freshwt hr' (Wagner, 1997).

Some genera of eubacteria are also known topossess nitrogenase, and may contribute to nitrogenfixing potential of this association (Lindblad et al,1991) but their rate of nitrogen fixation andcontribution towards the association has not beeninvestigated. The Azalia association is capable ofsignificant light dependent, nitrogenase - catalyzed H2evolution (Peters et ai, 1977). Observations from bothfield and laboratory studies indicate unidirectionalhydrogenase activity in the symbiont. It presumablyfunctions in recycling of electrons and ATP byoxidizing H2 produced during nitrogen fixation(Peters et al, 1977).

It is well established that freshly separatedAnabaena azollae releases about 40-50% of thedinitrogen fixed as ammonia into the immediateenvironment (Meeks et al, 1987). Both the symbiontand Azalia exhibit GS/GOGAT and GDH activities(Ray et al, 1978). The ammonia-assimilating enzymesare primarily associated with Azolla rather thanAnabaena, since Azalia accounted for almost 90% oftotal GS activity and 80% total GDH activity of theassociation (Ray et al, 1978). Studies on the kineticsof incorporation of I3N into glutamine and glutamateand the use of GS inhibitors have clearly indicatedthat the fern assimilates both Ny-derived andexogenously supplied NH4 + by the glutamate synthasecycle with little or no contribution from biosyntheticGDH (Meeks et al, 1987).

PABBY et al: AZOLLA-ANABAENA SYMBIOSIS

Cyanobacterial symbionts generally have low orundetectable levels of GS and only 10% of GSactivity of the association is attributed to theendosymbiont (Ray et al, 1978). Such low levels ofGS have been suggested as a biochemical mechanismresponsible for increased ammonia excretion of theendosymbionts. It has been postulated that the hostAzolla may produce effector molecules which modifythe ammonia-assimilating pathways of theendosymbiont by exhibiting or altering activity orsynthesis (Rai et al, 1986). Appreciable levels ofGDH has, however, been observed in the endophyte(Ray et al, 1978). But GDH, having a appreciablylower affinity for ammonia than GS, could provide aregulatory role, enabling effective reassimilation ofreleased ammonia at high intra-cavity ammoniaconcentrations.

One of the unique properties of Arolla-Anabaenasymbiosis is that both the eukaryotic Azolla and theprokaryotic Anabaena have the ability to fixatmospheric CO2. Azolla contains chlorophyll a and bas well as carotenoids, while endophytic Anabaenacontains chlorophyll a, phycobilins and carotenoidsThe phycocyanin content has always, in general, beenobserved to be higher than allophyocyanin andphycocerythrin (Samal & Kannaiyan, 1994). Thephotosynthetic pigments of the two organisms arecomplementary and, therefore, broader portion ofsolar energy spectrum is harvested for photosynthesis.The formation of anthocyanins in Azolla is known tobe triggered under high light intensity or lowtemperature giving reddish appearance to Azollafronds under these conditions (Lumpkin & Plucknett,1980).

Both the partners in the symbiosis fix CO2 viaCalvin cycle and sucrose is the primaryphotosynthetic end product in the association.However, when 14C02 was added to Anabaenaazollae isolated from leaf cavities, 14C-sucrose wasnot detected as the major product of cyanobacterialphotosynthesis. Hence, it has been suggested that A.azollae, in the mature leaf cavities, may be capable ofphotoheterotrophic or mixotrophic metabolism andsucrose produced by the fern may be utilized as areduced carbon source (Van Hove, 1989).

The cultured vs. freshly separated symbionts fromsix species of Azolla (belonging to the germplasm ofNational Centre for Conservation and Utilization ofBlue-Green Algae) were examined for theirmorphological attributes and growth parameters suchas proteins, Chlorophyll and also enzymatic

29

machinery involving N metabolism (Pabby et al,2003). Distinct differences in cell size and shapewere observed not only between symbionts fromdifferent species but also among. the cultured andfreshly separated cyanobionts of each species. Growthattributes such as chlorophyll a, sugars, proteins weresignificantly higher in the freshly separated symbiontswhile the cultured symbionts exhibited higheractivities of N-assimilation enzymes such as GS, NRand nitrogenase.

Utilization as BiofertilizersNitrogen is the element most often limiting food

production in the world. Agricultural production isknown to be directly dependent on nitrogen. In India,rice contributes about 45% of the total cerealproduction and is the staple food for majority of itspopulation. Nitrogen can be supplied to rice cropeither through chemical fertilizers or throughbiological agents. Besides blue green algae, which areimportant biological inputs in rice cultivation, Azollaforms another inexpensive, economical andecofriendly biofertilizer which provides unseenbenefits in terms of carbon and nitrogen enrichmentof soil and overall improvement in soil/cropmanagement practices and fertility status (Kaushik &Prasanna, 1989).

The most suitable crop for application of Azalia islowland rice, since both rice crop and fern requiresimilar environmental conditions. The characteristicsthat make Azolla suitable as biofertilizer for riceinclude requirement of shallow freshwater habitat,rapid growth, high nitrogen fixing capacity, quickdecomposition, and growth alongwith rice withoutcompeting for light and space (Vlek et al, 1995). Inaddition, a thick mat of Azolla suppresses weeds andreduces volatilization of ammonia in rice fields(Singh, 2000).

Experiments have demonstrated the effectivenessof Azolla as a biofertilizer on rice but the extent ofbenefit varies greatly because of different climaticconditions, methods of application, Azolla species,etc. Increase in grain yields of rice from 14% to 40%have been reported with Azolla being used as dualcrop. The use of Azolla as monocrop during fallowseason has shown to increase rice yield by 112% asagainst unfertilized controls by 23% when applied asintercrop with rice, and by 216% when applied bothas monocrop and intercrop (Peters, 1975). Singh(l977b) obtained a 6% increase in rice yield when A.pinnata was grown with rice and an increase from 9%


to 38% when Azolla was incorporated into the soil.Application of A. pinnata as intercrop twice, first 5days after transplanting of rice and then after 27 days,resulted in 27% increase in grain yield whileapplication of Azolla as monocrop and an intercropincorporated after 40 days resulted in a 30.6%increase in rice grain yield. Studies have indicatedthat a single crop of Azolla can provide 20-40 kg Nha-', but remained insufficient to meet the totalnitrogen requirement of the target crop. Therefore, useof Azalia in combination with chemical nitrogenfertilizers affords a feasible alternative practice.Singh and co-workers (1992) observed similar grainyield with Azalia alone and application of 150 kg Nha' as chemical fertilizer. An increase in yield of 0.9-2.0 t ha' and 0.8-1.0 t ha' was found when Azaliawas incorporated as compared to ammonium sulphatetreated plots. Azalia incorporated with different dosesof inorganic nitrogen (Subudhi & Singh, 1980) gavehigher yield as compared to use of inorganic nitrogenalone. Azalia incorporation along with 30 kg N ha'and 50 kg N ha-' as ammonium sulphate (Singh,1977c) increased the yield to as much as obtainedwith the application of almost 60 and 80 kg N ha'.

On comparing the effectiveness of Azalia withother bio-fertilizers, rice grain yield was highest withthe application of Azolla + 120 kg N ha' (5.0 t ha-'),followed by blue-green algae + Azalia + 60 kg N ha'(4.62 t ha') and lastly by 120 kg N ha" (4.61 t ha')(Singh et al, 1992). Application of either Azalia orblue green algae along with phosphorus fertilizer(Singh & Singh, 1990) increased grain yield. Theapplication of phosphorus fertilizer either duringintercropping or as phosphorus enriched Azaliaincreased nitrogen uptake by rice grain (Singh &Singh, 1995).

Azolla can be used as green manure in cultivationof Water bamboo (Zizanica aquatica), arrowhead(Sagittaria sagiuifoliai, taro tColocasia esculantai,wheat (Triticum aestivumi and rice (Oryza sativa).Utilization of Azolla as green manure in waterloggedsoil resulted in rapid mineralization with the releaseof 60-80% of the N within two weeks. Azalia appliedas monocrop between the wheat and rice crop or as anintercrop with rice had significant beneficial effect onsubsequent wheat crop (Kolte & Misra, 1990).Mahapatra & Sharma (1989) found beneficial effectson subsequent wheat crop with increase in grain yieldby 56% to 67% over control with application ofAzalia along with Sesbania. Incorporation of freshfronds of Azalia also increased grain yield of wheat

(Marwaha et al, 1992). Sharma et al (1999) recordedhighest yield of wheat with application of 20 t ofAzalia and 60 kg N.

Although the agronomic potential of Azolla is welldocumented, a number of environmental andnutritional factors have restricted its widespread use.

Role of Environmental FactorsEnvironmental factors such as humidity, light

intensity, photoperiod, salinity and temperature playan important role in controlling growth andphysiology of the association. Water is a fundamentalprerequisite for growth and multiplication ofAzalia.The plant is made up of 90-95% water which isrequired for the maintenance of structural integrityand major physiological processes. A water depth of3-5 em is recommended for optimal growth of AzalIain both nature and laboratory. Azalia requires relativehumidity between 85-90% for its normal growth.Maximum nitrogenase activity has been observed inA. caroliniana at 88-95% moisture levels (Braun-Howland & Nierzwicki-Bauer, 1990).

Light intensity and photoperiod greatly affect ratesof growth and nitrogen fixation in Azalia. High lightintensity was considered responsible for thesenescence of the fern. Azalia attains maximumbiomass at light intensities greater than 50% sunlight,while exposure to high light intensities usuallyresulted in decreased rates of nitrogen fixation (Vleket al, 1995). Azalia can survive in a wide range of pH(3.5-10.0) but optimum growth was observed in thepH range from 4.5-7.0 (Kannaiyan, 1979). Growthwas not supported in acidic (pH 3.0-3.8) and alkalinesoil (pH 8.4) (Singh, 1977b). Azalia is capable ofgrowing in a wide range of temperatures from 5-45"C.The growth rate of A. mexicana, A. microphylla andA. pinnata was highest at temperatures above 30°Cbut A. filiculoides and A. caroliniana grew better attemperatures below 35°C (Peters et al, 1980).

The fern requires all macro and micro nutrientswhich are essential for the normal growth and issensitive to the presence of excess or absence ofsuitable concentrations of nutrients. Phosphorus,potassium, calcium and magnesium are importantmacronutrients, while micronutrients such as iron,molybdenum, cobalt and zinc have shown to beessential for its growth and nitrogen fixation (Braun-Howland & Nierzwicki-Bauer, 1990). Amongmacronutrients, phosphorus is the most commonelement limiting growth of Azalia. As its content insoil solution and in paddy water is generally too low

PABBY et al: AZOLIA-ANABAENA SYMBIOSIS

to meet the requirement of Azolla, the addition ofphosphorus is recommended for better growth andmultiplication of Azolla (Kannaiyan et al, 1981).However, Azolla can grow without phosphorus insoils having Olsen-Po Subudhi & Singh (1979)observed reduction in growth and chlorophyll contentand increase in soluble sugars and amino-nitrogen.Kannaiyan and coworkers (1981) reported that 6 kgha phosphorus to be optimum for growth of Azolla.Higher heterocyst frequency of Anabaena azollae,nitrogen fixation and biomass production wasobserved with split application of phosphorus thanwith single application and with no phosphorus(Singh & Singh, 1988). The concentration of mediumphosphorus (0-0.5 flM) appeared to be critical foroptimum growth of Azolla (Sah et al, 1989). A mutantstrain with 0.75 rnM phosphorus requirement has beendeveloped (Vaishampayan et al, 1992). Subudhi &Singh (1979) observed reduction in growth andnitrogen fixation when grown in Ca and P deficientmedium. A decrease in levels of nitrogen andphosphorus and increase in lipids and activity of acidand alkaline phosphatase can act as physiologicalindicators of phosphorus deficiency in Azolla (Pabbyet al, 2000a). Under phosphorus starvation, cells ofendosymbionts Anabaena azollae show pale greencoloration and appear deformed.

The symbiotic association of Azolla does notrequire any nitrogen in the growth medium as ithouses the diazotrophic endosymbiont in its lobes.Different nitrogen sources significantly affectnitrogen fixation but not fresh biomass (Manna &Singh, 1991), but a higher relative growth rate of A.caroliniana and A. pinnata was observed at 5 rnMnitrate (Singh et al, 1992). Studies carried out both atlaboratory and field level indicated inhibitory effect ofnitrogenous fertilizers on the ammonia-assimilatingenzymes (Venkataramanan & Kannaiyan, 1986). In A.microphylla, ammonia assimilatory enzymes achievedhighest activity at 24 hr and disappeared thereafterwhereas in A. pinnata GS/GOGAT system retained itsfunction up to 14th day of incubation in urea (Pabbyet al. 2000b).

It can surmised that this symbiotic association,involving a diazotrophic partner shows adaptation toN-deficient and N-supplemented environmentmediated mainly through the GS-GOGAT pathway ofthe cyanobiont.

Pests and Diseases of Azolla and their ControlIt has been estimated that Azolla is currently used

on only 2% of the world's rice fields i.e. around 3

31

million hectares. An important biotic factorinfluencing biomass production of Azalia and itsutilization is the prevalence of insect attacks,especially when temperatures rise above 28°C. It hasbeen estimated that insect population can reach up to90,000 m-2 during peak summer and can completelydamage Azolla crop within 3-5 days. More than 3 Iinsect pests belonging to Diptera, Coleoptera.Lepidoptera, Homoptera and Orthoptera have beenreported. In India, Nymphula and Chironomids.which have been reported as important pests of Azalia(Singh, 1979b), cause rolling of the leaves and theirfeeding results in brown patches.

Carbofuran--An insecticide commonly used in ricecultivation has been reported to stimulate the growthand nitrogen fixation of Azolla at low dosages (Kar &Singh, 1979). A strain from Bangladesh was reportedto be resistant to insecticides and could be used asbiofertilizer during rainy season when incidence ofpest control is high (Singh, 1979a). Herbicides suchas Butachlor, 2,4-DEE and 2,4-0 decreased biomass,chlorophyll, nitrogen content and nitrogen fixation,when used at recommended rates of 2.5, 1.5, 1.0 and0.4 kg a.i. ha-'. Monocrotophos, phorate, thiodan,quinalphos, carbosulfan, chloropyrifos and furadanwere also effective in controlling pests, besidesstimulating Azolla growth and nitrogenase activity.Neem cake @ 500kg/ha and carbofuran @ 75 kg/hawere able to control attack of pests effectively (SinghetaZ,1987).

The incidence of fungal diseases in Azalia wasreported in India in 1979 «Sasi et al, 1979) whichwas further aggravated due to snail attacks. A. niloticawas found to be most sensitive, while A. carlinianawas least. To control fungal diseases caused byRhizoctonia solani, fungicides such as benomyl @ O. I% and carbondarin @ 0.2% were reported to beeffective. Neemcake @ 500 kg ha" controlled blackroot rot. Although genetic approaches such asintroduction of toxin genes into Azolla can be afeasible alternative, no breakthrough has beenobtained so far.

Spore Production TechnologyThe use of sporocarps of Azolla as primary source

of inoculum in field can overcome several problemsassociated with the current biofertilizer technologywhich involves a bulky amount of initial inoculumbesides difficulties in its transportation.

But the factors triggering sporulation in nature arepoorly understood and are apparently species or strain


specific. Also, the frequency of sporulation varieswidely among different species/strains of Azolla andis stimulated by a combination of environmentalfactors including stress factors such as lowtemperature (Kannaiyan & Rains, 1985), low lightintensity (Becking, 1976), fluctuating photoperiod,less availability of phosphorus (Singh & Singh, 1988)and overcrowding (Kannaiyan, 1979). These arethought to play interacting roles in induction ofsporulation in natural habitats.

In A. filiculoides, sporocarp formation is associatedwith mat formation, while in temperate regions thisphenomenon occurs during summer. A. pinnatasporulate in winters in India and shown to be relatedwith plant age and winter season (Singh, 1977b;Kannaiyan, 1979). Incorporation of ferric chloride @50 ppm, foliar spraying of growth regulatorsparticularly GA3@ 100 ppm and organic amendmentsinduce sporulation in Azalia (Kannaiyan et al, 1988).

In A. microphylla, the sporulation frequency(number of sporulating plants/lOO plants) and numberof sporocarps were highest in February and lowest inJune (Singh et al, 2001) while, in Philippines, thisspecies sporulates better during November- January atrelatively low day/night temperature and shorter daylength. In A. mexicana, sporocarps/plant weremaximum at 25115°C and a complete inhibition ofsporulation occurred at 38/25°C (Kannaiyan et al,1988). The sporulation frequency and number ofmicro, mega and total sporocarps/plant were observedto be negatively correlated with the average minimumtemperature and day length in A. microphylla (Kar etal, 2001).

An Azalia sporocarp technology involvingproduction, collection, storage and germination ofsporocarps has been developed by Kannaiyan (1990)and referred as "Frond based dried spore inoculum" ofAzalia. The major limitation in sporocarp productionis phosphorus management. The sporocarp yield inAzalia depends on total biomass and intensity ofsporulation. Application of P (4.4-6.6 kg ha-') wasobserved to be necessary for biomass production(Singh & Singh, 1990) but this suppresses sporulationsignificantly (Singh et al, 1987; Kar et al, 2001). Thisproblem can be tackled by either using Azalia strainswith better sporulation under P fertilization ordeveloping appropriate management practices tominimize the adverse effects of P on sporulation orthrough production of sexual hybrids. Uncertain andlimited sporulation in Azolla is another problem.Many strains of Azalia either ?o not sporulate in a

given geographical location or sporulate only during acertain part of the year. Often, strains with higherbiofertilizer potential have poor sporulation (Singh etal, 2001). Although the successful use of sporocarpsfor raising Azalia sporophytes under field conditionswas demonstrated in China, this practice has notbecome commercial because of poor growth ofsporophyte during initial stages of its development.Information on use of sporocarps under fieldconditions as primary inoculum is also scarce in India.Kannaiyan (1994) suggested that the application ofpresoaked (soaked in water for 12 hrs) frond basedsporocarp material @ 5 kg ha-1 a week aftertransplanting of rice is as effective as vegetativeinoculum.

Uncertain and limited sporulation of Azalia strainshas been a major constraint and biotechnologicalinterventions involving artificial induction areessential at this stage (Singh & Mahapatra, 2000).

Genetic Improvement of AzollaAlthough Azalla-Anabaena symbiosis has been

exploited for multiple uses, its poorly understoodsymbiotic interactions has restricted its amenability togenetic manuplation. Among the major types ofabioticlbiotic stress that affect the growth andperformance of Azalia as a biofertilizer, its sensitivityto temperature and water stress are most significant.

Development of Stress Tolerant AzollaIn recent years, soil salinity has become a serious

problem in global agriculture, more in areas wherewater deficit prevails over a long period of time.Azalia species are generally sensitive to NaCIconcentrations beyond 30 mM. Rai & Rai (1999,2000) were able to obtain Azalia plants tolerant to60mM NaCI concentrations through stepwise transferto higher concentrations. On analysis of such plants, itwas observed that this is due to development ofcapability by Azolla to regulate ion concentration,which may be related to modulation of genomeexpression, as observed in higher plants. Salinity alsoincreased photosynthetic O2 evolution in both adaptedand unadapted plants of A. pinnata. The response ofAzolla species to water stress (both salt and osmoticstress) showed that the Amazonian (RAR) specimensof A. caroliniana are more tolerant to both types ofstress (Zimmerman, 1985). Similarly A. pinnata couldtolerate 40mM NaCI (Rajarathinanam & Padhya,1989).

PABBY et al: AZOLLA-ANABAENA SYMBIOSIS

Although salinity tolerance was feasible in Azaliathrough physiological interventions, it has not beenpossible to develop temperature tolerant Azolla usingsuch techniques. Among the approaches employed inthis direction, development of hybrids has beensuccessful to a certain extent.

Production of HybridsSexual hybridization can be an important method

for developing suitable hybrids with improvedagronomic traits such as higher nitrogen fixing ability,relative growth rate, biomass production, sporulation,temperature tolerance and resistance to pest anddisease coupled with better adaptation under differentagroecological conditions.

Hybridization between A. microphylia (female) andA. filiculoides (male) improved annual biomassproduction. The hybrid produced biomass comparableto that of A. filiculoides in spring and of A.microphylla in summer and autumn, thus increasingoverall annual production. On the other hand, thehybrid with A. filiculoides as female parent exhibitedhigher sensitivity to elevated temperatures than thehybrid with A. microphylla as female parent(Watanabe et at, 1993). Hybrids between thesespecies were intermediate to those of parents (DoVanCat et al, 1989), were not stressed (red colour) underP or Ca deficient conditions and had higher nitrogencontent than that of parent, A. microphylia. Theirbiomass production in field was also higher than A.microphylla, indicating positive heterosis. Attempts tohybridize A. mexicana or A. pinnata with other Azollastrains have been unsuccessful. This has beenattributed to the absence of fertilization between amember of Old World Species (A. pinnata) and A.mexicana (New World Species).

In India, Azolla hybrids AHC-l, AHC-2 and AHC-3 were derived from crosses within species of A.microphylla and AHA between A. pinnata imbricatax A. filiculoides and A. microphylla at Tamil NaduAgricultural University, Coimbatore. All the hybridsexhibited higher biomass along with higher heterocystfrequency and thereby N2 fixation. They alsoexhibited higher chlorophyll content, nutrient contentand activity of ammonia-assimilating enzymes(Gopalaswamy & Kannaiyan, 1997). Selection ofpromising strains with thermal tolerance wasattempted by growing them in a phytotron (Do VanCat et al, 1989). Success has also been obtained intransferring Anabaena from one species of Azolla toanother. Liu et al (1989) transferred the

33

cyanobacterial microsymbiont from high temperaturetolerant species A. microphylla to high temperaturesensitive species A. filiculoides resulting in increasedtemperature tolerance of A. filiculoides (Watanabe etal, 1989). Sarma & Deka (1987) produced callususing leaf explant on Schenk and Hilderrandt mediumsupplemented with IAA, NAA, 2,4-D and benzylaminopurine at the rate 1,0.5, 1 and 0.5 ug L-1

• Callusdifferentiation appeared 22 days after inoculation at25±I°C and 1000 lux light intensity. Frequentsubculturing further led to reduction in doubling time.The callus lacked cells of the symbiont and therefore,can be used to obtain Azolla protoplasts for fusionwith the strains of Anabaena having improved Nfixing ability. A temperature tolerant mutant (up to38°C) of A. pinnata has been obtained using ethylmethane sulphonate (Dey, 1999).

Fingerprinting of Azolla-Anabaena SymbiosisIn recent years, different techniques have been

employed to identify and group the cyanobacteriaforming symbiosis with the different Azolla species.Although ELISA, quantitative immunobinding assaysand other fluorescent labeling techniques revealed ahigh degree of similarity between freshly isolatedendosymbionts from several Azolla species, distinctdifferences were observed in the cultured symbionts.Fatty acid profiles of the symbionts also clearlybrought out the distinction between cyanobionts fromArolla plants belonging to section Euazolla and thosefrom the section Rhizosperma (Caudales et al, 1995).Franche and Cohen-Bazire (1987) studied thedistribution of restriction sites around the nif HDKgenes of endosymbionts extracted from four Azollaspecies and one free living Anabaena azollae anddemonstrated that the arrangement of nif HDK geneswas strongly conserved among Azolia species,regardless of their geographical origin. .They alsodemonstrated that the cyanobionts from Azolla plantswithin Euzolla and Rhizosperma belong to twodifferent evolutionary lines. Moreover, even withinEuazolla, two subgroups could be distinguished--oneconsisting of cyanobionts from A. caroliniana and A.

filiculoides and the other consisting of cyanobiontsfrom A. microphylla and A. mexicana. Butcyanobionts of Rhizosperma could not be subdivided.The Azolla accessions, fingerprinted and classified(Zimmerman et al, 1991) by enzyme electrophoresisand leaf trichome morphology, provided a workingtaxonomy for identification and utilization of Azollaas a biofertilizer. Later in 1990, Plazinski et al


(1990a) found that cyanobionts of A. caroliniana, A.mexican a and A. microphylla need to be groupedtogether and differed from the cyanobiont of A.filiculoides. On the other hand, Van Coppenolle et al(1993) grouped the symbionts from seven Azollaspecies into three groups using nif gene probes andchloroplast DNA probes and demonstrated the utilityof RFLP analysis of host plant or symbioticcyanobacteria. DNA polymorphisms using RFLPsreinforced the conclusions derived from phenetic dataand located additional polymorphisms among strainswhich were enzymatically related (Zimmerman et al,1991). Cumulative RFLP analysis using heterologousand random homologous gene probes reinforced thespecies relationships presented from isozyme analysesand trichome anatomies and members of A.caroliniana- A. mexicana- A. microphylla clustershowed distinct genetic similarity. DNA-DNAhybridization techniques were also utilized forauthentication of new Azolla-Anabaena symbioticassociations and strain and species specific probeswere generated which revealed that Azolla hosts canharbour more than one Anabaena symbiont (Plazinskiet al, 1990a). RFLP analysis using selected DNAprobes revealed genetic variation in cyanobacterialsymbionts of Azolla and its closer relationship to freeliving cyanobacterial strains (Plazinski et al, 1990a).Gebhardt and Nierzwicki-Bauer (1991) carried outmolecular and morphological characterization of freeliving and symbiotic counterparts from Azollamexicana and A. pinnata and suggested the ubiquitouspresence of a culturable minor cyanobacterialsymbiont in atleast three species of Azolla.

Plasmid biology has been investigated in thesymbionts from seven Azolla species and presence of1-3 cryptic plasmids (35-100 MD) have beenreported. Several heterologous DNA probes,including Rhizobium symbiotic genes such as nod boxand nod MN and exopolysaccharide gene exoYshowed hybridization with plasmids belonging to thesymbionts (Anabaena) and suggest that theseplasmids may play a role in symbiotic interactionswith Azolla fern (Plazinski et al, 1990b).

Coppenolle et al (1995) utilized random primers foridentification and phylogenetic analysis of this fernusing accessions from all over the world. They wereable to construct a phylogenetic classification ofAzolla using 10 random primers which was indicativeof the usefulness of RAPD techniques in evaluatinggenetic diversity. A novel tool for DNAfingerprinting-DAF has been utilized for positive

identification of accessions of Azolla-Anabaena intactsymbioses. The advantage of DAF technique whichutilizes very short primers of ~ 5 nucleotides inlength, is its utility for distinguishing among closelyrelated genotypes of both prokaryotic and eukaryoticorganisms. DAF was observed to be useful, therefore,for not only defining the contribution of macro andmicro symbionts in the fingerprints of Azolla but alsoconfirming sexual hybridization and maternaltransmission of Anabaena azollae strain (Eskew et al,1993). A rapid diagnostic system for Azolla and itscyanobionts using random primers, and primers forthe chloroplast-encoded introns of the tRNA-Leucine(UAA) gene was also developed (Kim et al, 1997).This was highly useful for rapid assessment ofsimilarities among Anabaena azollae and minorAnabaena isolates from Azolla. STRR-PCRfingerprinting can be a valuable tool for analyzinggenetic diversity of the cyanobionts and phylogentictree generated distinguished three clusters--onecontaining the four species from Section Euazolla. asecond, the isolate from A. filiculoides and the thirdcontaining the three isolates from SectionRhizosperma (Zheng et al, 1999).

According to the knowledge and belief of authors,comparative molecular characterization of theendosymbionts, both freshly separated and cultured,has not been undertaken using PCR based primers.Future efforts of authors are, therefore, in thisdirection for a better understanding of the nature andidentity of the symbionts and this unique associationbetween a prokaryote and an eukaryote.

Conclusions/Future ApproachesFrom an agricultural point of view, the most

important N-fixing prokaryotes form symbioticassociations, especially with plants. However, thehigh energy costs of biological nitrogen fixation oftensets a limit to the amount of fixation that a host cansupport. Cyanobacterial associations are very valuablein this regard as the symbiont is itself aphotoautotroph. However, for Azotla-Anabaenasymbiosis, it has been calculated that 80% or more ofthe photosynthate used for N-fixation is provided bythe host. It is, therefore, important to undertakeresearch efforts towards critically evaluating thephysiological capabilities of the symbiont vs. host andalso elucidate the host signals/recognition processesbetween Azolla and Anabaena. These efforts alsoneed to include molecular dissection of thecontrols/regulatory elements involved in heterocystand akinete differentiation processes.

PABBY et al: AZaLIA-ANABAENA SYMBIOSIS

Another area of biotechnological research isincreasing the usefulness of Azolla-Anabaenaassociation as a biofertilizer. Hybridization studiesand reconstitution of symbiotic associations usingaltered genetically manipulated symbionts haveindicated that this can be an area of fruitful research.The benefits of Azolla is curbing ammoniavolatilization from flooded rice systems furtherstrengthens the promise of integrating Azolla in ricemanagement systems under field conditions (Singh,2000).

In this age, when mankind is threatened by drasticchanges in the global environment, triggered by man-made activities, there is an urgent need to usesustainable and environmentally appropriate practicesin agriculture. In India, research efforts need to beintensified towards:

• Improvement of 'spore production technology'and its large scale development at field levelfor increasing mass awareness especially infarmers growing rice crop.

• Molecular characterization of the cyanobiontsfor their improved efficacy as free living orsymbiotic diazotrophs in agriculture.

• Development of hybrids with tolerance topests, extremes of temperature/light intensity.

Focused efforts in this direction can definitelywiden the scope of utilization of biofertilizers such asAzalia, not only in agriculture but also in industry aswater purifiers and nutritional supplements indeveloping countries such as India.

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Azolla-Anabaena Symbiosis-From Traditional Agriculture to ... · Azolla-Anabaena Symbiosis-From Traditional Agriculture to Biotechnology Anjuli Pabby, Radha Prasanna and P K Singh*