NITROGEN FIXATION by the Azolla-Anabaena Azollae SYMBIOSIS

IAEA-TECDOC-325

ROLE ISOTOPETH F EO S IN STUDIES ON NITROGEN FIXATION AND NITROGEN CYCLING BY BLUE-GREEN ALGAE AND THE AZOLLA-ANABAENA AZOLLAE ASSOCIATION

PROCEEDING CONSULTANTA F SO S MEETING ORGANIZEE TH Y DB JOINT FAO/IAEA DIVISIO ISOTOPF NO RADIATIOD EAN N APPLICATIONS OF ATOMIC ENERGY FOR FOOD AND AGRICULTURAL DEVELOPMENT AND HELD IN VIENNA, 11-15 OCTOBER 1982

A TECHNICAL DOCUMENT ISSUEE TH Y DB INTERNATIONAL ATOMIC ENERGY AGENCY, VIENNA, 1985 THE ROLE OF ISOTOPES IN STUDIES ON NITROGEN FIXATION AND NITROGEN CYCLING BY BLUE-GREEN ALGAE AND TUEAZOLLA-ANABAENA AZOLLAE ASSOCIATION, IAEA, VIENNA, 1985 IAEA-TECDOC-325

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The International Atomic Energy Agency (IAEA) and the Food and Agriculture Organization e Uniteoth f d Nations (FAO) support research contracts, seminars, symposi varioud aan s other meetings concerned wit hwida e rang f nucleaeo r techniques. These activitie carriee y b sar t dou thier Joint FAO/IAEA Divisio f Isotopno Radiatiod ean n Application f Atomiso c Energ Foon yi d and Agricultural Development. This Consultants Meeting on the Role of Isotopes in Studies on Nitrogen Fixation and Nitrogen Cycling by Blue-green Algae and their Associations was held at the Vienna International Centr Viennaen i , Austria, Octobe , 1982—1 15 1 rparticipante Th . s from develope developind dan g countries attended at the invitation of the sponsoring organizations. The objective of the meeting was to evaluate the current status of research on nitrogen inputs to lowland rice fields by blue- green alga theid ean r associations, particularly Azolla, recommeno t d an d appropriate research problems which coul solvee db isotope-aidey db d studies. This document contains reports presented by several of the participants, and the recommendations developed durin meetine gth r isotope-aidegfo d research consultante Th . s group recommended that the FAO/IAEA Joint Division should initiate a coordinated research programme in this field and that initial emphasis should be placed on the Azolla-Anabaena symbiosis. Dr. M. Fried, Director of the FAO/IAEA Joint Division, served as Chairman of the scientific sessions and Dr. S.K.A. Danso as the Scientific Secretary. EDITORIAL NOTE

In preparing this material for the press, staff of the International Atomic Energy Agency have mounted paginatedand originalthe manuscripts submittedas authorsthe givenby and some attention to the presentation. The views expressed in the papers, the statements made and the general style adopted are the responsibility of the named authors. The views do not necessarily reflect those of the govern- ments of the Member States or organizations under whose auspices the manuscripts were produced. The use in this book of particular designations of countries or territories does not imply any judgement by the publisher, the IAEA, as to the legal status of such countries or territories, of their authorities and institutions of of the delimitation of their boundaries. The mention specificof companies theirof or products brandor names does implynot any endorsement recommendationor IAEA. partthe the of on Authors are themselves responsible for obtaining the necessary permission to reproduce copyright material from other sources. CONTENTS

Opening address ...... 7 M. Zifferero Nitrogen fixatio Azolla-Anabaenae th y nb azollae symbiosis ...... 9 . J.H. Becking Establishment and management of Azolla in rice fields ...... 21 S.A. Kulasooriya Growt nitrogehand n fixatio Azollanof pinnata var. africana: Environmental conditions and field plot inoculation assays ...... 39 P.A. Reynaud Sexual reproduction of Azolla species ...... 3 5 .

J.H. Becking The use oN-labellef 15 d dinitrogen in the study of nitrogen fixation by blue-green algae .... 63

K. Jones Use oNf 15 in the study of biological nitrogen fixation in paddy soils at the International Rice Research Institute ...... 81 . Watanabe,/ P.A. Roger Blue-green algae in rice fields. Their ecology and their use as inoculant ...... 99 P.A. Roger

Applicatio seriae th f lno dilution techniqu estimato et biomase eth -fixin2 N f so g blue- green algae under field conditions ...... 119 P.A. Reynaud Effect of inoculating blue-green algae and Azolla on rice yield ...... 129 S.A. Kulasooriya

Recommendations ...... 145 List of Participants ...... 163 OPENING ADDRESS

M. ZIFFERERO International Atomic Energy Agency, Vienna

Directore n behalO th f o f s Genera Fooe Agriculturd th an d f lo e Organization and the International Atomic Energy Agency of the United Nations, I have the pleasure to extend a warm welcome to all the participants in this Meeting. I hope you will find the available facilities in the Vienna International Centre or VIC, as our building is called, satisfactory and conduciv a successfu r efo l meeting.

At the current rate of population growth, there is an urgent need for increased food production. Ric graid ean n legumes, bein o vertw gy important food crops, will pla even a y r increasing rol avertinn ei g severe hunged ran malnutrition. All efforts should therefore be directed at maximizing the yield f thesso e cropslease th tt , a possible cost, especiall pooe th r r fo y peasant farmers, who incidentally produce the greatest bulk of human food in the developing countries.

Examining the constraints on achieving high level of production even with high yielding varieties of crops, one cannot but agree that soil nutrient deficiencie cruciale sar . Traditionally, expensive fertilizers have been used to correct soil nutrient deficiencies. Indeed ,a revie s showwha f no tha% 50 t the increase in rice yield after World War II can be attributed to increased fertilize userN . Nitroge d phosphorunan s have indeed been demonstraten i d moso mantw t y e importanth studie e b o t st elements which limit crop yields. Althoug n increaseha s beedha nrate advocatef fertilizeus o e P d o an t d rN mee growine th t g deman r foodfo d e shoul,w t losno de abilite sighth f f to o y peasant farmer o affort s purchase th d f fertilizerseo , whose prices aref projecte increaso t dyeare th sn i eahead attendane th wels , a s la t waste associated with uhe fertilizer applied, as plants are capable of taking up only a portion of the applied fertilizer in its year of application, a lot being lost through various processes.

The exploitation of cheaper alternatives or supplements to fertilizers have therefore been advocated e IAEs commiteTh .ha A d itself strongl usino t y g nuclear techniques to answer some of the pressing questions with many of the alternatives which have been identified, and for which there are prospects. f theso e e On areas whic s receiveha h d considerable whicr supporfo hd an t isotopes have proved very valuable is the area of N2 fixation by grain legumes A Join. t FAO/IAEA coordinated research programme, wholy devoteo t d this problem is in its fifth year, and many questions for which definitive answers weravailablet no e o poot re ,du techniques , have been answered clearly by the use of ^-'N. Last year, an FAO/IAEA consultants meeting on the mycorrhizae was convened to investigate the potential role of these mycosymbiont makinn i s g existing soil nutrient naturallr o s y occurrind an g cheap fertilizers more availabl r planfo e t growth A positiv. e recommendation was made, and it is our hope that a programme would be initiated in the near future.

r increaseOu d interes biologican i t l nitrogen fixatio a supplemen s na t or alternativ n certaie(i n situationsr ou fertilizerN o o t )t d le s sha convening this meeting. Evidence being assembled daily face pointth t o t s tha N tfixatio blue th e y greeb n n alga r theio e r associations, sucs ha Azolla-Anabacna is very important. Available figures from e.g. the Azolla symbiosis demonstrates that this sourc N coul f eo d rival legum eN fixation . For instance, the figures of 500 and 600 kg N/ha/yr of N fixed by Azolla in paddies quoted by the International Rice Research Institute (IRRI) and OSTROM n Senegai greatee lar r than woul expectee b d d from mostr legumesou s i t I . sincere feeling that the experience gained by using *% for field studies of N fixatio legumen nextendee i b n sca estimatino t d g accuratel fixeyN bluy b d e green algae in rice paddies, and how various management practices act on the amount of N fixed by these algae and their associations.

Attempts to develop microorganisms capable of fixing N directly into rice plant drawin e stile th sar n lo g board only. These blue-green algad ean Azolla plants which fix N in rice paddies therefore have to decompose before making the fixed N available for uptake by rice. This then is a significant difference betwee N fixe e n rici th dn e paddien legumes i here ar d e san e W . again happy to inform you of the IAEA's interest and previous involvements in programmes designe investigato t d efficience eth nutrienf yo t use. Isotopes provepowerfue b o t d l tool r sucsfo h studies e woulw mose d b d,an t interested to initiate programmes of nutrient availability from the blue-green algae based on your recommendations.

The Joint FAO/IAEA has invited you to this meeting, as a result of its commitmen findino t g way meand san f alleviatinso hungrye plighe th th g f to , especially in the developing countries. We therefore seek your assistance in identifying those areas of N fixation and N cycling by the blue-green algae and their associations, which could be tackled most effectively by isotope technique d recommendinsan g future research activities aime increasint a d gN fixation by these organisms leading to a less dependence on fertilizer N use for rice production. It would be the most ideal thought, to have the yield of the most important cereal crop, rice being highly independent of expensive fertilizer N application. lookinl al e g ar surm u forwara excitinn eyo I a o t dsuccessfud gan l meeting, and I would like to wish you all an enjoyable stay in Vienna. It is pleasurr ou youe b ro t ehost r thisfo s week. NITROGEN FIXATION BY THE Azolla-anabaena azollae SYMBIOSIS

J.H. BECKING Research Institute I.T.A.L., Wageningen, Netherlands

Abstract

A concise outlin presentes i e maie th n n do characteristic e th f o s AzpJLla association in relation to tropical wetland rice cultivation and the nitrogen econom f paddo ypresence th y nitrogea o t soilsf o e Du .n fixing cyanobiont occurrin speciaa n i g l leaf Azolle cavitth f ao y leaf, the water fern Azolla can grow in a nitrogen-deficient environment and is able to contribute considerably to the nitrogen status of the soil. Under favourable field conditiony dr s5.0-9. x g Azollfi N. n g 0m aca weight plant whic, tissue hy correspond,da doublina o t s g timf eo the biomass of 2.5-5.5 days in Azolla pinnata var. pinnata. For unlimited growt Azollf o h nitrogea n input intecosystee th o f mo 300-60 N.hag 0k ~ .year estimateds i " .

Environmental conditions suc s ligha h t intensity, temperature, humidit d othean y r weather conditions suc wins wated a h an d r agitation (waves y affec -fixinN )ma e th t g capacit ferne n generalth I .f o y ,a turbulent water surface is deleterious for a free-floating water fern like Azolla. Further watee ,th r managemen rice th e f f o tcroo s i p influenc n N-inpuo e t produce e benefiAzolle y th b dth d f aan o t accumulated N for the rice crop. Chemical factors such as inorganic nutrients may affect the growth of Azolla. In this respect in particular the availability of phosphates is important.

n experimentaA l set-u nitrogen-fixine th presentes i pw ho r fo d g capacity of Azolla plants can be measured in the field by means of the acetylene reduction assay using a rather simple glass vessel. A 15 compariso s madnwa e betwee fixatio_ N n Azolly b n d acetylenaan e reductio f ftzollno a plants under identical conditions A conversio. n rati C.H.:I\f o = 5.6-7.s observedL wa 1 : 9 , being therefore different frotheoreticae th m l valu f 3:1o e e availabilit.Th e th f o y nitrogen fixe y ftzolle ricb dth eo t acro discusseds i p . Some physiologica d biochemicaan l l characteristic e ftzollth f so a symbiosis are presented such as the inhibition of N fixation by CO and the observed difference pigmenn i s t compositio e ftnabaenth f o n a azollae symbionts in the various ftzolla species, which seems to be correlated -fixinN wite th h g capacit f theso y e associations.

Introduction

e nitrogen-fixinTh g capacit associatioe th f o y n ftzolle th a sppd an . blue-green algae ftnabaena azolla y contributema e considerable th o t y nitrogen economy of tropical wetland rice. In the present short outline some requirements and characteristics of ftzo 1la and its internal symbiont are discussed in relation to tropical wetland rice cultivation, ftzolla is a small water fern which has a world-wide distribution occurrin tropicaln i g , sub-tropica d temperatlan e regionst ,bu particularl S.En i y . Asia f agronomio , s wheri t i ec importance because of its very rapid growth and its large combined nitrogen inputs into the soil. Since 1971 we have (Becking 1972 a) studied the environmental requirements of Azolla in natural ecosystems in Indonesia (Java) and also in laboratory experiments. Together with work from other sources (see review "Nitrogen and Rice", I.R.R.I. 1979) there is now a good insigh d evidenc an tavailabilite th n o e d potentialan y f ftzoHso s a green manure in tropical wet rice cultivation.

Growt d Nitrogehan n Fixation

presence blue-greee th Duth o t ef eo n algal symbiont (ftnabaena azollae), ftzolla specie e abl gronitrogen-deficienar a so t en i w t medium. Usually, ftzolla ver s y ha arapi d growth. Under optimal conditions in the field its growth rate is 5.0-9.0 mg N per g dry weight per day,producing a dry matter increase of 0.135-0.290 g per g ftzolla dr.wt. per day. This value corresponds to a doubling time of 2.5-5.5 days (see review Becking 1979). Similar doubling times have been found by other workers (Brotonegoro & Abdulkadir 1976; Watanabe 1977, 1978 and others).

e maximaTh exponentia e lN inputh n i t l phas growtf eo r hfo unlimited growth of ftzo1la as estimated by the acetylene reduction assay is about 335-670 kg W per ha per year (Becking 1972 a, b). Values of

10 e samth e order haue been obtaine othey b d r workers growtn I . h experiments in open containers, Saubert (1949) estimated, on the basis f biomaso s productio N-contents it d nan ,a valu r nitrogefo e n fixation annumr pe a .h r Moreoverpe N g k ,0 Watanab31 f o e (1977)e founth n i d Philippines in field plots an accumulation of 330 kg N per ha in 220 days d wit,an h continuous growt f Azollaho , wit 2 crop2 h s harvesten i d . ha r days0 pe 33 N ,totaa g k l0 yiel46 f o d

e above-mentioneTh d estimate refel al so unlimite rt d continuouan d s growt f Azollho open i a n water areas with optimal mineral nutritionn I . agricultural practice, however growte th , f Azollt continuousho no s i a . e falloth n wI period between rice crops e field,th e generallar s y dr y and devoi f Azollo d a growth. Moreover, befor flowerine th e g stagf o e the rice plants, the fields are normally drained in order to stimulate tillering of the rice plants. Hence, after flooding of the newly established rice field, ftzolla usually starts from practically zerof i , the field is not artificially inoculated with Azolla. Also, because of space competition with the rice plants, complete cover of Azolla. is normally never obtained. For this reason, the optimal values of Azolla as obtained by the acetylene reduction assay should be corrected down to 25 - 50% Azolla cover, giving a yield of 62-125 kg N per ha per year (Becking 1975). Taking into account the observed dark I\L fixation of about 20-30% nitrogen fixation during night (see later), a figure for nitrogen fixation, under field condition, of 103-106 kg W per ha per estimatee b annu n ca m d (Becking 1976).

Factors Affecting Mitrogen fixation

Apart from agricultural practice ,numbea environmentaf ro l factor y affecsma e yiel d nitrogeth t an d n fixation potentiaf o l Azolla. These factors can be of a physical, chemical or biological nature, The effect of these limiting factors may also be different either for the blue-green algal symbiont or for the total plant-algal association.

Of the physical factors light intensity, temperature, humidity and weather (wind, waves, etc.) are particularly important for Azolla growth. With respect to light it can be stated that it affects the photosynthetic activitgrowte th d nitroged han an y n fixatioe th f o n plant arid the symbiont. High light intensities (above 90 k lux) show

11 11.25 11.55 12.25 1255 13.25 13.514.255 14.55 y da tim f o e

. 1 Fig. Acetylene reduction rat f flzolleo a pinnata intact plants in Indonesia as affected by light intensity during time of day (o full of sunlight, A dark). After 30 minutes' exposur e darth e k perio s startedi d .

n inhibitora y effec n nitrogeto n fixation (see Fig.l), whil w lighlo e t intensitie r shadino s g (particularl e ricth ey b plantsy ) seem o havt s e a favourable effect on the growth and multiplication of the fern. Day temperatures of 25-30 C are favourable for the growth of Azolla. However, higher temperatures e distinctl,ar e.gC 5 3 . y inhibitorr o y even detrimental for its growth. These responses may be, however, differen varioun i t s Azolla species, strain r varietiesso A certai. n humidity is further essential for the growth and multiplication of Azolla as a water fern. Hence, irrigation and water management of the rice field are important for the growth of a free-floating fern like Azolla. Wind wave an e generalld ar s t favourabl no ygrowte th f r ho fo e the fern. Agitation of the water tends to break the fronds, and this fragmentation usuall a seriou s ha y s limitinL gI\ effece th n to fixation capacit frondse th f o y. Large wave r typhons(o s e ver)ar y detrimenta Azollr fo l a growth.

12 chemicae th f O l mentionee factorsb n ca t di , that ftzolla needsa complete inorganic nutrient solution like most higher plants. However, with regard to nitrogen it can rely on atmospheric nitrogen (N ) for its total N-requirement. The presence of combined nitrogen (e.g. nitrate) in the substrate has remarkably only a small reducing effect on the nitrogen fixation capacity of the association. This is primarily due to the fact that the upper lobe of the bilobed leaf, which contain direcn e symbionti th s t tno contacs i , t wite wateth h r layer, but projects freely into the air. Of the main mineral requirements particularly phosphate, iron arid some trace elements (especially Mo for nitrogen fixation) are important. The pH of the medium usually affects the availability of these elements. In general Azolla prefer mediusa m clos neutralito t e r slightlo y y acidic, e facth probabl to t tha e tfavourabldu s y i thi e H p sth r fo e availabilit f somo y f theseo e mineral plantse th e supplo Th t s. f o y oxygen or nitrogen gas is usually not limiting, because of the free e fern th o thorougN .o t accesr hai w f investigatioso no o t p u s ha n bee nwhetheo t mad s a e r flzoll resistans i a o salint r semi-salino e e conditions.

biologicae th f O l factors, insects suc, Lepidopteras h a (caterpillars), Diptera (mosquito larvae), Cephalapoda, Crustaceae and snails may influence the growth of flzolla by their grazing on the Azolla biomass. However, the use of pesticides or herbicides are usually also detrimenta o ft.zpJL.lt l a (Becking 1979) o probabl,s e th y application of biological pest control methods should be explored. Microbiological pathogens (bacteria, fungi and virus) are another biological factor affectin growte gth ftzo1laf ho . Thes affecy ema t the growth and reproduction of both systems and also may show antagonisms, i.e. substances released by bacteria or other agents, which may reduce the growth and nitrogen fixation of the association.

Measurements and Assessment of Nitrogen Fixation

For the assessment of nitrogen fixation in the field a small glass vessel (Fig 2) of c. 70 ml capacity supplied with two rubber septa was used. The acetylene reduction assay was employed by introducing acetylene gas through the rubber septa with a

13 Fig. 2. Glass vessel (70 ml) used for the acetylene reduction assayo rubbee vessetw Th .s r ha l sept r monitorinafo g s thatmospherega e vesseth n ei l after acetylene introduction.

gas-tight syringe. Gas samples of the atmosphere of the vessel were also taken through the rubber septa. The experiments were performed with c. 10% acetylene in air and the ethylene production from acetylene was determined by means of a gas chromotograph fitted with a hydrogen-flame ionisation detector and a Porapak T (80/100 mesh) column operating at 60 C. A typical experiment of such an assay is give Tabln i n . 1 e

14 Table 1. Acetylene reduction by Azolla pinnata plants wi thAnabanea azollae

symbiosis in the field. Muara Experimental Station, Bogor, Indonesia.

C2H2 i ncubation n moles n moles C2Hi, n moles C2H/, n moles CoH/, time mg N g proteim n in air C2Hi» mg N mi n n mi 0 3 n mi 0 3 h min 10% acetylene 0-30 88 15.1 30.2 3.1 30- 60 86 l*. 3 29.6 3.1 0 9 - 60 6i< 11.0 22.0 2.3 0 12 - 90 103 17.7 35-^ 3-7 120- 150 113 . -119 * 38.8 it.O

Plants: dry weight 0.165 g 5.82 mg N (3.53% N)

Table 2.

Effec f £-2^2o t e nîtrogenasth ° n «° e (C2H2) activit f Azollao y c nc

£2^2 in air 5* 10* 20* 60* 80* ARA % 96 100 92 *»3 38 Inhibition % ^ - 8 57 62

To use the acetylene reduction assay, the optimum acetylene concentration for the particular organism (Azolla) must be used. As show n Tabli n , optimae2 l acetylene reduction activity. c (ARA t a s )i 10% C_H_ in air. conversioe Furtheth r rfo acetylenf o n e reduction values int oN fixation values e conversioth , n ratif o C„H.: N_ should be experimentally determined. The conversion 24 2 factor depends on environmental conditions and plant material. Tests under similar condition r acetylenfo s . N e reductio r fo d nan should be made in order to obtain a reliable value for the conversion ratio.

Under identical laboratory conditions (flat-bottomed Roux bottles with a thin layer of medium) we tested the nitrogen fixation of Azolla presencfronde % th (v/v 10 n i sf )eo N wit ato0 4 h mexces% N s 15 witr % o (v/v10 h ) acetylene (Becking, unpublished) botn I .h setf so

5 Tabl . Exposur3 e f AzoVLao e pinnata atofrond0 4 1 m% o t s N2

Exposure N Ato15 m% N-t con nte dr. wt. yg N fixe fixeN d g y d fixeN g y d time sample bi omass bi omass per per per h mg g b i ornas s mg . N wt . dr g

0 0.3688 1.78 0.0499 - - - 1 0.4471 A. 82 0.1407 9.8 2.04 69.8 2 0.5232 4.67 0.1478 18.5 3.96 125.1 3 0.5570 4.38 0.1421 21.1 4.81 148.3 It 0.5957 5.10 0.1615 29.5 5.79 182.8

15 % N10 Atmospher 2, ^ ( 0.3 1 2 218 e C0 , v/vA % :12

15 Tabl Compariso. 4 e f o nN 2 fixatio ^2^2d an n reductio Azollan i n

Method Time 1 h 2 h 3 h 4 h

yg N fixed 70 125 148 t w r d g . p 183

1SN2 yMol N fixed 10.56 p. g dr wt 4.99 8.93 13.05

yMol N2 2.49 4.47 p. g dr wt 5.30 6.53

Acetylene yMol C2Hj, 14 28 42 50 Reduction p. g dr wt

Ratio C2H^/N2 5-6 6.3 7.9 7-7

experiments same th ,e basi s mixturga c s use, wa e0- d % [7218 , %A 15 0.3% C02, (v/v)] and only the 10% N (v/v) in the N experiment was replaced by 10% C H (v/v) in the acetylene reduction experiments evidens A .e firs th tn t i fro , 4 m Tabld an 3 e four consecutive hours the ratio C H :W changes from 5.6 - 6.3 e conversioTh o 7.7-7. t . 4 , i 2 nd hourn 9i nd houran an facto3 s 1 s r N -C-hL is therefore in this case not the theoretical value 1:3, but on the average 1:6.

ây°LÎlability of Azolla nitrogen to the rice plant

The availability of Azolla nitrogen to the rice plant depends on the soil type, the ecosystem of the soil and the number and abilities e micro-organismth f o s present, sinc e combineth e d organic nitrogen

16 accumulate y Azollb d a mus e converteb t o minerat d l nitrogen, befort i e becomes available to the rice plant. The availability of mineralized nitrogen to the rice crop depends to a large extent also on the agricultural system practiced, i.e. whether a fallow period after the rice harvest with low humidity and oxygen access into soil is applied alss i ot I influence. e systeth f drainagey o mb d , tillage, ploughing, etc. d whethean , e ricrth e stubbl workes i e d inte soith o l or not.

e conversioTh n rat organif eo c nitroge minerao t n l nitrogen depends largely on the bacteria and fungus flora and these are in turn determined by the agricutural system practiced and the soil type involved.

The conversion of organic nitrogen in Azolla to mineral nitrogen d uptakan f thieo s nitroge e ricth ey e followeb nb plan n ca ty mean b d s of the tracer technique using nitrogen labelled with N. Here again several methods are possible. For instance the Azolla plants 15 d wit W labellefe he b e I\IO , b n e soiN_ dr n I\IH_ca th o ca lr .o <£ o T labelled previously and by "dilution" of the labelled soil l\l with nitrogen fixed by Azolla the N-turner can be estimated. If Azolla d witplantfe h e isotopisar c mineral nitrogen care shoul e takeb d n that all nitrogen is converted into organic nitrogen and that there is no residual pool of I\IO_ maintained in the plant.

From discussions of the FAO/IAEA consultants meeting it was evident that more emphasi futurn i s e e th wor n o k t shoulpu e b d turn—ove e fixerL th I\ raty Azolle soi b dd f th eo an l n i a /. availability and uptake of this N by the rice plant. Therefore one of the main recommendations is the application of N-15 and other isotopes in the nitrogen cycling of the agricultural systems using Azolla.

Some phy s iolog i cal characteristic s of the as soc iat ion

Nitrogen fixation of the Azolla symbiosis is sensitive to CO. As show concentratioe Tabln th i n , e5 0.1f no r produce%ai (v/v n i )O sC alread % reductio71 y l\L-asn i n e activit s measurea ye th y b d acetylene reduction test. With the concentration of 1.0% (v/v) CO the inhibitio nearls i n y complete (96%) e effeTh .probabls i t y causey b d

17 Tables. Effect of CO on the nitrogenase activity of Azolla

r ai Cn i O Q% 0.01* 0.1* 1.0*

ARA % 100 100 29 k

Inhibi tion 0 0 71- 96 %

a poisoning of the CO-sensitive system of cytochrornes of the flnabaena azollae symbiont.

Nitrogen fixatio e symbiosith f no s varies wite Azollth h a species or clone involved. For a scheme of classification and species differentiation of Azolla, see in the contribution "Sexual reproductio f Azollo n a species" e sectioth , n "Taxonom f Azolla"o y y B . means of biophysical methods such as pigment absorption and epiflourescence tests, it was shown that the algal symbiont of Azolla pinnata var. pinnat a somewha d aha t different pigment composition than algae th l symbion f otheo t r form d speciean s s like Azplla caroliana Filiculodes. A d an . Acetylene reduction tests performed concomitantly with the biophysical tests showed a higher nitrogen-fixing capacity in the Azolla pinnata var. pinnata association as compared to the other Azolla species e higTh .h nitrogenase activit e Azqllth f ao y pinnata var. pinnata associatio agreemenn i s i n t wite observatioth h n that although the chlorophyll content of the heterocysts was high (chlorophyll absorption tests) e fluorescenc,th e yiel f chlorophylo d l a in the heterocysts was low. Further, in heterocysts of the cyanobion f thio t s specie e phycocyanith s n content (phycocyan absorptio d alsphycocyanie an n th o w testslo s )wa n fluorescencen I . addition, a distinct gradient in phycocyanin concentration was observed in the vegetative cells decreasing gradually from the cells adjacen heterocystse th o t e highesTh . t phycocyanin concentration was always found in the vegetative cell next to the heterocyst of the cyanobion f Azollto a pinnata var. pinnat s distinctlawa y higher than in the heterocysts of free-living species such as Anafaaena cylindrica as shown in parallel chlorophyll absorption tests with the latter species.

Thus, apart from morphological differences between the various Azolla species, the algal symbionts of the individual species demonstrate differences in physiological properties probably as a

18 resul differena f to t genetic backgroun d presumablan d co-evolutioa y n of both partners.

REFERENCES

Becking, J.H. 1972 a. Ecological—hydrobiological study on irrigated rice fields in relation to the fixation of atmospheric nitrogen n Dutch(i , English summary). Report Netherlands Foundatior fo n the Advancemen Tropicaf to l Research (WOTRO) Haguee ,Th e ,Th Netherlands, 42 pp.

Becking, J.H. 197 . 2b Symbiosen : Stickstoff-Bindung. Fortschr. Bot. 14: 459-467.

Becking, J.H. 1975. Nitrogen fixatio somn i n e natural ecosystemn i s Indonesia. In "Symbiotic nitrogen fixation in Plants" (ed. P.S. Nutman), International Biological Programme I.B.P. Synthesis meeting, Edinburgh, Sept. 1973, vol. 7, Cambridge Univ. Cambridge-London 539-550. pp , .

Becking, J.H. 1976. Contributio f plant-algano l associationsn I . Proceedings of the 1 International Symposium on Nitrogen Fixation, vol. 2, June 1974. Washington State University Press, Pullman, U.S.A. 556-580. pp , .

Becking, J.H. 1979. Environmental requirements of ftzolla for use in tropical rice production n "NitrogeI . d Rice"an n , I.R.R.I., Manila 345-373. pp , .

Becking, J.H. and Donze, M. 1981. Pigment distribution and nitrogen fixatio Anabaenn i n a azollae. Plan d Soi, 203-226tan 61 l .

Brotonegoro, S. and Abdulkadir, S. 1976. Growth and nitrogen-fixing activit f Azollo y a pinnata. Ann. Bogorensi : 69-776 s .

International Rice Research Institute (ed. I.R.R.I.) 1979. "Nitrogen and Rice", Symposium, International Rice Research Institute, Los Banos, Laguna, Philippines, September 18-21, 1978, 499 pp.

19 Saubert, G.G.P. 1949. Provisional communication of the fixation of elementary nitroge floatina y nb g fern. Ann. Roy. Bot. Gard. Buitenzorg 51: 177-197.

Watanabe, I., Espinas, C.R., Berja, M.S., Alimagno, B.V. 1977. Utilization of the Azolla-Anabaena complex as a nitrogen fertilizer for rice. International Rice Research Institute, I.R.R.I. Res. Pap. Ser. No. 11, 15 pp.

Watanabe lowlann . i 1978,I e .us d s ricAzollit ed cultureaan . Tsuchi to Biseibutsu (Soi Microbes)d lan , Tokyo : 1-10,20 .

20 ESTABLISHMEN MANAGEMEND TAN F TO Azolla IN RICE FIELDS

S.A. KULASOORIYA Departmen f Botanyo t , University of Peradeniya, Sri Lanka

Abstract

This paper summarizes available informatio growte th d n an hno maintenance of Azolla in rice fields, and is based primarily upon reports from rice producing countrie Asian i s . Since methodr fo s large scale production of sporocarps are not available, Azolla inoculum must be produced vegetatively. For experimental purposes this can be accomplished in galvanized iron trays with a 2 cm layer of waterf o e onlm c Th .y5 soinecessard an l y additio concentrates i n d 2 super phosphate at a rate of 1.5g/m every 5 days. Phosphorous is the major nutrient limiting Azolla growth under field conditions, and P deficient Azoll mors i a e susceptibl damago t e higy b e h light intensity. Heavier inoculums favour establishment and growth of Azolla. To favour initial growth, the inoculum can be limited to a smaller area by floating bamboo or twisted rice straw ropes. Azolla is susceptible to several pests. Common pesticides used in rice culture also control most Azolla pests biologicat ,bu l control methods may also be effective. Suggestions are made on problems which could be profitably investigate. P 2 d 3 an N d usin5 , 1 C g 4 1

INTRODUCTION

The importance of Azolla. with its nitrogen fixing endophyte Anabaena azollae. especiall biofertilizea s a y ricer fo rwels i , l documented (Moore 1969, Tran & Tuan 1973, Talley et al 1977, Watanabe et al 1977, Liu Chung Chu 1979, Tuan & Thuyet 1979, Singh 1979, Rains Talle& y 197 Lumpkid 9an Plucknetn& t 1980). Informatios it n no morphology, development, physiology and biochemistry has been reviewed recently (Peters et al 1979). Such data are invaluable not only in the designin f isotopio g c studie fiela t a sd level t als,bu o usefun i l the formulatio technologa f o n whicy b y h thi s bettee b plan y rtma exploite n agriculturei d .

21 widespreae Th Azollf o e ricy us adb e farmer stils i s l morr eo less confine Chino t dVietnamd an a , whil othen i e r countries like Bangladesh, India, Indonesia, Philippines, Sri Lanka, Thailand and U.S.A. (California), this technolog staga t f laboratora eo s i y d an y field experimentation.

Rice farmer Chinn i sVietnad an a m have used Azolla s a fertilizer for rice for centuries, but even in these countries, the production and utilization of Azolla as an integral part of organized rice productio relativela s i n y recent developmen (Li. t u Chunu Ch g 1979, Tuan & Thuyet 1979).

In this presentatio shalI n l attemp o summarizt e th som f o e available information on the growth and maintenance of Azolla in rice fields. The information contained in this paper is primarily based upon reports from rice producing countries in Asia.

Introductio Azollf no rico at e fields

One of the foremost requisites for Azolla in rice cultivation is the production of sufficient material for field inoculation. Since factors cotrolling the formation and germination of sporocarps are still poorly understood, productio done f inoculub no n i eo t s mha situ, through vegetative multiplication, because the storage and transport of fresh, bulky material is not only cumbersome, but also impracticable, as Azolla undergoes rapid decomposition during storage under tropical conditions.

Azolla need gooa s d amoun f attentioto card an ne especially during the initial stages of its introduction to a new environment and bess ii t t attempte severaln i d , gradual stages. Suc excercisn ha e should be started preferably with a collection of different species, or at least different strains of the same species, found in a particular country n "AzollA . a Bank f thi"o s typ bess i e t maintained in the laboratory under controlled conditions, using chemically defined media. A period of adaptive multiplication could then be attempted preferabl e are e th whicb o n sit t r a yn e o plan i ut th hs i t introduced. Initially, such multiplications coul e carrien b di t ou d pits, ditche r shalloo s w tank f smalso l surface area, using soid an l growte wateth s a hr medium.

22 For this purpose, we have used galvanized-iron trays x 240cm x 60 cm x 10 cm (high) with a 2 cm layer of soil covered with tap water, made up to a level of 5 cm (Figure 1). A bent glass tube inserted at one end of the tray ensures drainage of water above a desired level and prevent e spillinth s g ove Azollf o r a when left outdoors during rainy weather. The only chemical nutrient regularly added during this growt phosphoruss i h , whic usualls i h y provide s concentratea d d super 2 phosphat ever, e m fertilizeryg/ fiv5 1. rate th f days eo t ,a e .W have found thaoutdooe th t r cultivatio Azollf no a from laboratory grown materia moss i l t successful whe outdooe nth r culturee ar s maintained under a partial shade during the first 3 to 5 days. This is especially so in the dry zones of Sri Lanka where the light intensity goes daytime abov th 5 klud 12 e an xe temperatur arouns i e d 36°C during a major part of the year.

o principaThertw e ar e l method growinf so g Azoll ricn i a e fields (Liu Chung Chu 1979, Tuan & Thuyet 1979): monocultura s A . 1 open i e n fields. 2. In dual culture with rice plants.

The former method can be used either when there is a long fallow period during which standing water is available in the fields, or where rapid multiplication of Azolla is desired so as to obtain a heavy cover within a short period. This heavy cover of Azolla when incorporated e capabilit inte soilth th os ,ha f providino y gooa g d basal dressing of nitrogen without much interference with the total duration of the rice production period. In this method, Azolla plants initially adapted unde partiaa r l shade fiel e addeth ar edo t d plots and allowed to grow under exposed conditions. During the next few day e fronds th man y turf sma o y n maroon-red w ten remaio fe t da t n,bu green e extenTh . f reddeninto populatioa n i g n varies with different strain Azollaf o se hav w d e ,an foun d that this featur d someha e relationshi originae th o t p l habitae strainth r instancef Fo to . ,a strain originally collected from a warm area had a lesser number of red fronds compare straia o t d n collected fro coolema r locality. However n invariabla t ,no this eswa character. Neverthelesse ,th fronds that remained green under exposed conditions adapted faster and multiplied rapidly, especially under frequent phosphate additions.

23 Field plot e inoculatear s d with fresh Azolla preferably during their active growth. The inoculum densities used, have varied from 50 filiculoide. A r g/mfo 2 s (Rain g/m0 40 2 Talles& o t 0 y10 1979o t ) 100o (Singt 0 0 g/mh25 d 19792 an (Tua) Thuyen& . tA r 1979fo ) pinnata. Our experience, as well as that of the others, is that the heavier the inoculum, the faster the growth and establishment. Use of heavy inocula has been also suggested as a measure to control pests, smotheree whicb rapie n th ca h dy b dgrowt Azollf ho a (Liu Chunu Ch g 1979). Howeve applicatioe th r heavf no y inocula become practicaa s l limitation when large area f fieldo s se inoculatedb hav o t e .

The dual culture method of growing Azolla with rice is perhaps of more widespread applicability because standing wate availabls i r n i e the field during the growth of rice from seedling to panicle initiation mosn i , t wetland rice fields. Azolla grows harmoniously with rice plant ofted an s n remains gree healthd an n y during such growth, being shaded from high light intensities, by the rice canopy. However, the biomass of Azolla under dual culture is obviously less, sinc e ricth ee plants occup larga y e field th are f .ao Chinese scientists have adopte e plantinchanga d th n i e g patter f ricno e leaving wider avenue between i s n row f ricso e hill permio t s t better growt Azollf ho a (Liu Chun 1979)u Ch g . This method, . testeDr y b d Watanabe and his group at the International Rice Research Institute, Peradeniyt a Philippine s u y b Centran i ad an si Lanka lSr s give,ha n very encouraging results. The results of our experiment show that an incorporatio Azollf o n a grown under "avenue planted" rice gav graia e n yiel % increased14 increasd % comparean 47 2 s 2 f o obtaineeo t d d with Azolla grown under transplanted and broadcast seeded rice respectively (Kulasooriya 1983). Another factoconsideree b o t r d durine th g initial field establishment of Azolla is dispersion of the fronds by wind. Turbulenc d fragmentatioan e n have been e detrimentashowb o t n l growte foth r Azollf o h a (Ashton 1974). Whe smalna l inoculuf mo Azolla is added to an open field, the fronds get dispersed by wind and very often such fronds either peris r tako h lona e g tim o initiatet e rapid growth. Such spreading of inoculum material can be minimised by dividin e fielth g d plots into smaller sub-plots using bamboy an r o other suitable floating material. This type of partitioning successfull diagramaticalls i ys u use y b d y show Figurn i n . 2 eThi s show stakee b tha directioe o nt th win e s intth dha o f o naccoun n i t

24 the subdivisio Azoll e plotse th th s af A . o n cove r increasese ,th bamboo removee ar s wind an dd itself assiste rapith n di se spreath f o d growing Azolla mat have .W e found that rice straw twisted into loose ropes can also be used for this purpose. Chinese farmers use rice rows as barriers to prevent the drifting of Azolla (Lumpkin & Plucknett 1980).

Growt nitroged an h n fixation

Under favourable conditions, Azolla multiplies very rapidly by vegetative means d suc,an h fragmentatio facilitates i n e th y b d abscission layers pointe formeth f branchingt o sa d . Reportf o s growth and nitrogen fixation of different species of Azolla from different localities, under various conditions have been well summarize Beckiny b d g (1979) e doublinTh . g times give Beckiny b n g (1979) rang 19.o et fro90 daysm2. . While these rate growtf o s h appear to be related to species and the growth conditions, laboratory growth measurements have generally given shorter doubling times than field measurements examplr Fo . e Tun She& g n (1981) have recordea d doublin . pinnat A 8 g day r 2. tim fo sf ao e under laboratory growta n i h defined medium, whereas the rate under field conditions, in soil-water was 5 days. We have recorded an 8-fold increase in biomass in 2 weeks (Figur50-fold an ) 3 ed increas days2 2 n i ,e giving doubling timef o s 4.8 and 3.9 days respectively for the same species of Azolla grown during the Yala 1980 (dry season) and Maha 1981/82 (wet season) at AmbalantotaLankai Sr y zon dr f eo .e th n i ,

Nitrogenase activity of Azolla measured by the acetylene reducing activity (ARA) ranges from 1.2. to 13.1 umoles/g (f.w.)/h. In the case of ARA, laboratory measurements have given values higher than the theoretica 1 conversio3: l bees nha nt ratioi suggeste d an , d thae th t conversion factor (C H :N ) for laboratory measurements be 4:1 (Becking 1979). For field grown strains of A. pinnata in monoculture, we have recorded ARA values ranging from 1.82 to 2.59 umol/g (f.w.)/h equivalent to 3.1 to 4.64 kg N/ha/day, when converted on a 3:1 ratio and extrapolate relation i de correspondin th o t n g Azolla biomassesn ,o the basis of a 12 h light period per day, (Table 1).

Measuremen strain 3 diurnaf . pinnatan to A i f A o s lÄR . grown under dual culture with rice, showe positiva d e correlation with light

25 with some variations among the different strains (Figure 4). These values when converted to the nitrogen fixed, gave rates of 0.37 to 1.14 kgN/ha/day (Tabl . Comparee2) monocultureo t d lowee ,th r rates of nitrogen fixation recorded in this case can be ascribed to the lesser biomasse Azollf o s a produced under dual culture.

Requirements for field growth

In defined media, under laboratory conditions, the nutrient requirement growte th Azollf r ho fo s a have been e founb o t d essentially similar to those of other green plants, the principal difference being that molecula e Azollus n aca r dinitroge meeo s t n it t entire nitrogen requirements. However, the major nutrient critical for growt nitroged an h n fixatio fiele th phosphorus i dn i n s (Thuyet& Tuan 1973, Watanab 1977l a t e , Singh 1977, 1979, Talle1977l a t ye , Becking 1979, Liu Chung Chu 1979, Lumpkin & Plucknett 1980). Watanabe l e(1977ta ) also demonstrated tha coule criticaa tF e b d l nutrient especially with s availabilitregarit o t d t alkalina y . epH

A common symptom of P deficiency is the reddening of the Azolla fronds due to anthocyanin formation, and the lengthening of roots which become brittle and are easily detached (Watanabe et al 1977). Besides P deficiency, anthocyanin formation in Azolla has been ascribe o generat d l stress factors suc higs a h h light botd ,an h high temperaturw anlo d e (Becking 1979). Vietnamese scientists recognise two varieties of Azolla pinnata. referred to as "green Azolla" and "red Azolla" which respond differently to stress factors (Thuyet & Tuan 1973 and Tuan &. Thuyet 1979). As the names imply the "green Azolla" remain green unde deficiencyrP higd ,an h light, while eth "red Azolla" turns red under such conditions. However, the "red Azolla" is more tolerant to low temperature and salinity. Watanabe et al (1981) have reported that, although the growth of A. pinnata (green) from Vietnam did not turn red under P deficiency culture, its growth was retarded. They suggest that it is a mutant strain lacking the ability to synthesise anthocyanins. We have found that the reddenin Azollf o g a pinnat resule n interactioa th s f i tao n between factors deficiencyP suc s a h , ligh temperaturd an t e (Kulasooriyt ae al. 1980) and similar results were reported from Malaysia by Tung & Shen same (1981th e r specie)fo Azollaf o s thein I . r work Tun Sheg& n

26 (1981) have demonstrated that the growth of field grown Azolla (doubling time adversels )i y affecte y increasb d lighn i e t undeP r deficiency e highesth , P t f ,o rat wherea m e epresencpp th 0 n 2 i s f o e of growth was achieved under full sunlight. In the absence of P, ÄRA is negatively related to light intensity, but in its presence, ARA at 50,75 and 100% sunlight was higher than at 25% sunlight. The light intensities recorde 0 k.lux12 d o t wer, 0 comparable3 r ou o t e intensitie Ambalantotat a d muct ,bu h higher than those reportee b o t d advers filiculoide. A o t e Ashtoy b s n (1974) recenA . t report from India indicated that Azolla (probably pinnata) multiplied well under high temperature (29-39 C) in the field and that there was not much differenc rate d multiplicatioth f an eo levelP n 0 i e5 o f tw o s e th t a n 2 100 g/m of superphosphate (Sindha Mathar et al 1981).

r experienceIou n , large scale monocultur Azollf e fieleo th n dai needs frequent P additions, in the form of powdered concentrated super 2 phosphate or triple super phosphate, broadcast at the rate of 58/m every five days. Such additions ten o keet d p Azolla gree d healthan n y and increases resistance to pest attakcs. Although such P additions e impracticablb y ma mase th sn i ecultivatio f Azollo n y ricab e farmers necessare ,b the maintenance y th yma n i y f healtho e y nurseries to be used as inoculum.

We have also observed that in a thick red cover of Azolla. a number of fronds in the subsurface remain green and healthy, probably due to the shading by the fronds above them. This may be one reason y Azollawh . which shows light saturatio r photosynthesifo n d an s nitrogen fixation at around 8000 to 5000 lux respectively in the laboratory (Peters 1976) nitrogex ,fi gro d wan n under much higher light intensities in the field.

Besides their direct effects, high light and temperature in exposed field conditions may have an indirect, adverse effect on Azolla. Under such conditions the rapid evapotranspiration of water results in an increase in salt concentration and Azolla growth is inhibite y thib d s high salinity. Chinese farmers overcome this problem by having a flow of fresh water into the field, which not only keeps the temperature down, but also compensates for the evaporation losses (Liu Chun 1979)u Ch g . These factors clearly show that Azolla

27 technology wil completele lb y successful onl arean i y s where good water supply and control is possible. The widespread use of Azolla in rice cultivatio s invariabli n y limites absolutit y b d e requirements for standing water and appropriate changes have to be adopted in areas like the dry zones of Sri Lanka, where water is available for rice cultivation only during certain periodyeare th .f so

Another constraint for Azolla use is its susceptibility to several pests (Liu Chung Chu 1979 and Lumpkin & Plucknett 1980). So far it is reassuring to find that common pesticides used in agriculture concentrationt a , s applie r rice fo de als ,ar o effective against pest Azollaf o s face aware th b .t f o eo t However s ha e ,on that resistance pest type y e evolvfutursma th n i e especially under extensive field growt Azollf ho ricn i a e growing countries. Recently a fungal disease of Azolla has been reported from Thailand (Parkpian Arunyanar 1982)l a t te . Certain insect pests have been effectively controlled by biolgical control methods notably by the use of Bacillus thurigiensis. while the protection of insect predators such as frogs and spider alss i s o advocated Tetramoriut an n A . m Ruineense bees .ha n found to destroy 67% of these insect pests (Liu Chung Chu 1979). Insect pests of Azolla develop into alarming proportions in Vietnam and China durin warmere th g , summer months; therefor e tropicth n i es where temperatures remain high throughou yeare tth e threa ,th f to thes ewidespreae pestth Azollf n o o s e aus d coulserioua e b d s problem. s alreadha t I y been pointe t thadearte ou dth t f informatioo h n no sporocarp formatio d theian n r germination under defined conditionss i , a major limitation for the large scale utilization of Azolla. Due to this deficienc knowledgen i y , Azolla productio e don sitb i o it eut s nha jus tutilizatios prioit o t r n because storagt possibleno s i e d ,an transport of bulky, fresh material is impracticable. Furthermore, without adequate knowledg e sexuath n lo eAzolle phasth f ao e life cycle o profitabl,n e programm e initiate b e artificia n th ca er fo d l breeding of Azolla strains more tolerant to high light and temperature and low levels of phophorus and more résistent to pest attacks. What is done at present is to pick up such strains produced randomly by natural selection and propogate them by vegatative means. This of cours vera s yi e unsatisfactory situation e puritth f s suc,o a y h strains cannot be sustained. A preliminary experiment on sporocarp germination under artificial conditions s reporte,wa Gurunathay b d n

28 and Sreerangasami (1980). They foun presencde th tha n i tf combine eo d nitrogen, dissected sporocarp dormanca d ha s y perio f abouo d t three months, while in a nitrogen free medium the sporocarps did not germinate at all. Watanabe et al (1981) indicated that sporocarp formation by A. mexicana was stimulated by low (26/18 C: day/night) temperature deficiencyP d an s . They observed that supernatant water from decomposing A. mexicana cultures or water extracts of Azolla also stimulated sporulatio d founan n d thae activtth e fractios nwa dialysable.

Possible area r isotopifo s c studes

bees ha n t pointeI t thaeffecte ou dth t Azollf so n soiao l fertility is related not only to its quantity, but also to its quality, (Liu Chun Chinese u 1979th gCh d )ean attribute this quality Azoll e N ratith primarilC/ f ae o th biomass bees o ha t yn t I foun. d that nitroge supplies i ncroe th p o mort d e readily when Azolla wita h C/N ratio of 10 is incorporated into soil, whereas if this ratio was t availablno e standins th wa o t eN ge th crop 7 1 .

It is therefore important that both CO fixation and N fixatio Azolly nb a under different environmental parametere ar s 14 15 thoroughly investigated, and isotopes such as C and N can profitabl e use b ysucn i d h studies. These studies coul e initiateb d d in laboratories, but should be extended to field conditions if more meaningful results are to be obtained.

e rol Th f phosphoru o e y nutrien fiele ke th a d r s growta fo ts f ho Azolla has already been emphasised. Unfortunately, however, no critical studies have been reporte phosphorue th n o d s metabolisf mo 32 Azolla. Studies using P would be useful to understand:

- The absolute necessity of P for Azolla. e necessitTh - r frequentfo y , small dose thif o s s nutrient. The interactions between P metabolism and environmental factors such as light, temperature and pH.

While such studies should provide information that woule b d useful to improve the field growth of Azolla both as a monoculture or

29 in dual culture with rice, further studie needee ar s o improvt d e th e availabilit Azollf o y a fixed nitroge e associateth o t n ds i crop t I . common practice eithe o incorporatt r e Azolla intgreea e soi s th oa l n manure prior to transplanting of rice, or to allow Azolla to grow side by side with rice, with periodic incorporations alon growte th g h cycle of the crop. It is apparent that both these methods combined, i.e. a basal incorporation followe duay b d l culture, with periodic incorporations should provide a maximum N-input. However, such a technology coul limitee b d numbea y b df othe ro r factorse b o T . successful, such a technology should have an all-year-round, assured supply of controllable water. But this situation is rather rare in most rice producing third world countries. Of the rice growing areas in South Eas trainfes i Asia % 70 ,d (Frene 1981)l a t ye . Eve watef i n r t suppllimitingno s i y , intensive rice cultur f raisineo t leasa g o ttw crop year spe r would leave hardl y falloan y w n intercroperioa r fo d p of Azolla. It is therefore more likely that only one of the above method practicalls i s y adaptable, rather tha combinatioa n f bothno .

In most rice growing Lanki areaSr an i s limitatio watef o n r suppl Azollf y o basala woulconstrain a e s us a e b d,e greeth n i tn manure fertilizer, while the prospects are better for its use as a dual culture with rice. In our field experiments we have therefore laid more emphasis on the latter method. The results of these experiment e give ar sanothen i n r paper presented here.

Another area that warrants investigatio fertilite th s i n y effect of Azolla under natural decomposition, without incorporation. At first sight, ludicroua thi y appeae sb ma o t r s suggestion whet ,bu n one becomes aware of certain widespread methods of rice cultivation practice developinn i s g countries liki LankaSr e , such studies become relevant. Under limitations of regulated water supply (rainfed) and due to the high cost of labour, rice is not grown under row planting in many parts of Sri Lanka. This type of broadcast seeded rice produce rather crowded growth, without any regular pattern of spacing. This would not only limit the space available for Azolla growth, but also impede its periodic incorporation without damage to crop plants. Under these circumstance necessare b y ma o allot t yi s w natural decompositio Azollf no o increasat e soil fertility evet a n minimal levels. Sirinivasan (1981) reported that manuring with

30 Azolla. without incorporation did not add any appreciable N-input to the soil, but in our experiments we obtained a 14% increase in grain yield ove controa r l without Azolla which receive N-fertilizero n d .

Chinese scientists have conducted some experiment methode th n o ss of incorporation of Azolla into soil, and their results have been summarized by Liu Chung Chu (1979). These results though interpreted in term f graiso n yiel t shof ricey qualitativo dno wan o ,d r eo quantitative data regarding nutrient availability to the crop under the different method f incorporationo s .

Azollf o e aUs prelabelled with isotope f carboso nitroged an n n y reveama l invaluable informatio mineralizatioe th n no n processes under these different systems, such data may enable the development of Azolla technologies that could benefi croe th tp more effectively, like differene th t fertilizer application schedules recommended with respec soio t l type, season, rice variety etc. Such studies would also enable the understanding of nitrogen losses from organically bound nitrogen under different environmental conditions.

Studies on Sporocarps

While attempting to break sporocarp dormancy by physico-chemical procedures, it may be worthwhile to examine the possibility of using ionizing radiations as mutagenic agents for the breeding of robust Azolla strains.

Acknowledgements

My sincer. W.KMr .o t eHirimburegame thankdu e s ar s hi r fo a assistance in conducting the experiments and preparation of figures and Tables alsm a oI .gratefu . S.WMr .o lt Abeyseker stafs hi fd an a for the cooperation extended during the field trials at the Ambalantota Rice Research Station. Experiments done in Sri Lanka, include thin i d s paper were supporte granty b d s froNaturae mth l Resources, Energ Sciencd an y e Authorite th i Lank Sr d aan f o y International Foundation for Science, Sweden.

31 REFERENCES

. 1 Ashton, P.J. (1974 effece Th )somf - t o e environmental factorn so the growth of Azolla f.iliculoides Lam. In: The Orange River Progress Report. (Institute for Environmental Sciences), Universit Bloemfonteinf yo , South 138 - Africa 3 ,12 p ,p 2. Becking, J.H. (1979 )Environmenta- l requirement Azollf so r afo use in tropical rice production. In: NITROGEN & RICE, The International Rice Research Institute s Banos,Lo , Philippinesp ,p 345 - 374. 3. Freney ,(1981. J.Ral t ).e Nitroge- n balanc irrigaten i e d wetland rice Wetselaar: .In Simpson, ,R. , J.R Rosswall.& . ,T (eds) Nitrogen cyclin Soutn i g h East Asia Monsoonat nWe l Ecosystems, Australian Acad. Sei., Canberra202- 9 .19 p ,p 4. Gurunathan, M. & Sreer Rangasami, S.R. (1980) - Studies on the biology of Azolla-Anabaena system. In: Azolla a Biofertilizer, Tamilnadu Agricultural University, Coimbatore. ,14 India- 7 p ,p 5. Kulasooriya, S.A., Hirimburegama, W.K. & de Suva R.S.Y. (1980) Effec f lightto , temperatur phosphorud ean growte th d n hsan o nitrogen fixation in Azolla pinnata native to Sri Lanka. Oecol. Plant. 1, (4), 355 - 365. Chunu Li g Chu. 6 Azoll.f o (1979 e ricn ai Us )e- production i n China NITROGE: .In RICEN& Internationae ,Th l Rice Research Institute, Los Banos, Philippines, pp 375 - 394. 7. Lumpkin, T.A Plucknett.& , D.L. (1980 )Azolla- ; Botany, grees Physiologa e n us manure d an y . Econ Bot. 153- . (2) 1 34 ,, ,11 . 8 Moore, A.M. (1969 )Azolla- ; Biolog agronomid an y c significance. Bot. Rev. , 35_, 17-35. 9. Parkpian Arunyanart, Arunee Surin, Wanchai Rochanahasadin & Somikd Disthaporn (1982 )Rotte- n diseas Azollaf eo . Int. Rice Res. Newslett.. ,10 T_'., 1 . Peters10 , G.A. (1976) Studie Azolla-Anabaenn so a azollae symbiosis. In: W.E.Newton & C.J.Nyman (eds) Proc 1st Int. Sytnp. on Nitrogen Fixation, Vol 2, Washington State Univ. Press, pp 592 - 610. 11- Peters, G.A., Mayne, B.C., Ray, T.B Toi.& a Jr., R.A. (1979) Physiolog biochemistrd an y Azolla-Anabaene th f yo a symbiosis. In: NITROGE RICEN& Internationae ,Th l Rice Research Institute, Los Banos, Philippines, pp 325 - 344.

32 . Rains12 , D.W Talley.& , S.N. (1979 )Use- Azollf so Nortn i a h America NITROGE: .In RICEN& Internationae .Th l Rice Research Institute Banoss ,Lo , Philippines 434- 9 .41 p ,p 13. Sindhamathar Krishnamoorthy, ,A. Anavaradhan& . ,S ,(1981L ) Azolla influence on rice yield. Int. Rice Res. Newslett., £:5, 23 - 24. . Singh14 , P.K Azoll.f o (1979 e ricn ai Us )e- productio Indian i n . In:NITROGEN & RICE, The International Rice Research Institute, Los Banos Philippines, pp 407 - 418. 15. Sirinivasan, S. (1981) - Effect of Azolla manuring without incorporation. Int. Rice Res. Newsl;ett., 6_:4, 22 - 23. 16. Talley, S.U., Talley, B.J. & Rains, D.W. (1977) - Nitrogen fixatio Azolly b n ricn i a e fields : Alexande.In r Hollaende. ed r Genetic Engineerin Nitroger fo g n Fixation., Plenum Press, N.Y. and Lond., pp 259 - 281. 17. Tran Q.T .Tuan& , D.T. (1973 )Azolla- gree;A n compost. Vietnamese Studies Agricultura, ,38 l Problems, Agronomic Datap ,p 119 - 127. 18. Tuan, D.T. & Tran, Q.T. (1979) - Use of Azolla in rice production in Vietnam NITROGE: In . RICEN& Internationae ,Th l Rice Research Insitute, Los Banos, Philippines, pp 395 - 406. 19. Tung, H.F. & Shen, T.C. (1981) - Studies on the Azolla pinnata-Anabaena azollae symbiosis: Growth and nitrogen fixation. New Phytol., 8_7_, 743 - 749. . Watanab20 Espinas, I. e , C.R., Berja, N.S Alimagno.& , V.B. (1977) Utilizatio Azolla-Anabaene th f no a comple nitrogea s a x n fertilizer for rice. IRRI Res. Paper Series., No.11, 10pp. 21. Watanabe, I., Khe-Zhi, Bai., Berja, N.S., Espinas, C.R., Ito, 0. and Subudhi, B.P.R. 1981) - The Azolla-Anabaena complex and its usricn i e e culture. IRRI. Res. Paper . 10ppSerie69 . .sNo

33 TABL Biomas- 1 E d acetylensan e reducing activity J^ARA.f o ) 15-day old monocultures of Azolla pinnata strains grown in 2 5 m field plots at Ambalantota, in the low-country, dry zone*o i LankafSr . Growth condition Fig.3n i same s th a e . e sar

b Azolla Fresh weight ARA N Fixation strain of Azollaa (I0~6mol (KgKAa/day) (g/plot) g (f.w.)A)

Debokkawa 8000 + 54 2.59 + 1.50 4.64 Bangkok 7892 7 2+ O £L/Ii 1.36 4.30 India 76004 ++12 1.82 + 1.18 3.10 Peradeniya 7125 + 712 2.41 + 1.36 3.84

) t-.eaa n valu f fouo e r replicates. Mea) b n valu eighf o e t samples incubated acetylen% wit 0 2 h e from 133 0o 143t 0 CST, unde 0 K.lu9 r t J> xa 37°Co k t . * Terrain: flat to undulating; rice soils: low humic gley expectanc' ^yp/ 5 e;7 y valu annuaf o e l rainfall: less than 500 mm.

TABLE 2 Nitrogen fixation by Azolla in dual culture with rice transplanted

Ambalantotat a m c 0 2 x .m c 0 2

* mean specific activity N- fixation Azolla strain Biomass during the day g. m pmol CjH« day" g~ (i'.w' ) g day" k ha "N (f. w)

Debokkawa 820 14.96 1.14

Bangkok 700 10.87 0.71

peradeniya 610 6.64 0.37

* used the theoritical 3:1 conversion factor.

34 galvanized iron tray Azolla

thick rubber tube

(a)

Figure 1 - Diagram of a p-alvanized-iron tray used for the outdoor, soil-water cultur Azollaf o e . a) Cross-sectional view of the draining system. b) View of tray shewing drain tube attachment.

35 wind direction water inlet water, outlet J—»

-2m- • 4m- • 6m-

(b)

Figur Diagra- . nurser2 ea f o m y cultur zolA lf a o e pinnata under field conditions. a) 1st stage - Bamboo poles (1), (2) £.• (3) placed at 1 m intervals. b) 2nd stage - Bamboo poles moved to double the area of each sub-plot. d stag3r Bambo- e) c o (30& pole ) > (1 s remove Azolls a d a form sa complet e cover.

36 Debokkawa strain Bangkok strain Indian strain Peradeniya strain

6 9 12 15 days after inoculation

Figure 3 - Growth patterns of the different strains of Azolla pinnata in 5 m field plots at Arabalantota, Sri Lanka, lïacb plot was initially inoculated with 900 g (f.l/t.) of Azolla togethe f freso g hr /K Azoll witg 6 hf o a triple-----uper-phosphate (T3P) and 1 g/Kg of fresh Azolla of Carbofuran (~5 % a.i.). Subsequently ÏS'P powde s broadcasrwa ) (1. m 5E/ t oveAzolle th r a cover ever day5 yd Carbofura san n (0«5 initiaG/ e s addeth rwa a ) t a dy l an sig f o n pest attcks. (Diurnal light intensity: 5 to 125 K.lux; daily temperature 37°C)o t 5 2 :.

37 Light 70-8x 5kl 100-125 klx x kl 7 5-

Temp. 28-JO °C 35-3C 4° 32-33 °C

2.0 . Debokkawa strain Bangkok strain PeradeniyT a strain 1.5 •

' 0 "1. 0

o I 0.5

0 0 0700 0900 1100 1300 1500 1700 1900

Centra] Standard Tiny eda (CSTe th f )o

Figure

Acetylene reduction activity during the day time of three Azolla isolates under field condition duan si l culture with rice. (Ambalantota)•

38 GROWTH AND NITROGEN FIXATION OF Azolla pinnata var. africana: ENVIRONMENTAL CONDITIONS AND FIELD PLOT INOCULATION ASSAYS

P.A. REYNAUD Offic rechercha l e d e e scientifiqut e technique Outre-Mer (ORSTOM), Dakar, Senegal

Abstract The growth and the nitrogen fixation of Azolla pinnata var. africana is discussed in regard to climatic conditions occuring in a dry tropical area. Optimum acetylene reducing activity (ÄRA) was observed at a light intensity of 60 klux, and both higher and lower light intensities reduced ARA. Optimum ARA is in the range of 25-35 C. The harmful effec f desiccatioto slowes i n y addindb g alginat d storinan e t a g . ConcentrationC 6 nitrate-f o s ammoniom-d an N N have different effects on Azolla. growth, and ARA. Plot assays show the importance of a residual effect on a second rice cycle.

Introduction

In order to measure the inoculation effect of Azolla on rice and on soil, one has first to determine the best conditions of growth and the best agronomical practices in a given area.

West Africa n possesstrainow s it s: Azolla pinnata var. africana which is recognized to occur in almost all the countries of the region. This region can be separated into two climatic areas (Charreau, 1974).

a semi-humid tropical area with a rainy season ranging from fiv o seveet n month d conditionan s s simila Asiatio t r c zones where Azolla grows; a dry tropical area with a rainy season ranging from two to five humid months, near 15 N latitude.

lattee Ith n r area solar radiatio highs i n thao s , t potential Azolla productivity shoul highe db . Unfortunately, water requiremenr fo t Azolla growth is also high and rainfall is often inadequate and varies widely from year to year, so that effective productivity is very low.

39 In this zone, adaptation of Azolla is a major problem. As Azolla could be very beneficial for the expected future nitrogen budget of these less developed countries, we have to consider its use under these extreme climatic conditions.

. EcologicaA l factors influencing N.-fixatio Growtd . nA an f ho africana.

I.LIGHT INTENSITY.

Experimental procedure: A tank was divided into five areas of 25 x y floatinb m c 0 g7 partition thao culturs e tth e medium addes dwa identica eacn i l h area. Each ares shade screea wa y b d n allowing transmissio eithef o n r 100%, inciden f 60%o % ,7 r t36%o sunlight% ,22 , whic reacn ca h h easil maximua y p.m1 0 klu 9 t a xDakarf n .m o i .

Each are s inoculateawa d witg (fres40 h h weight . africanaA f )o . day5 1 sd Afte fresan 8 r h weigh determines twa eacr fo dh area. Afte8 r dayAzolle th s a morpholog s observe ywa e affecte b o t d higy b d h light intensities: leave srootd becaman sd werere e stunted. Unde lowese rth t light intensity (Emax =6.3 klux) Azolla morphology was not apparently affecteproductivite th t w (Tablbu dlo s ey wa 1.) ratie .Th o chlorophyll/carotenoid decreased with increased incident sunlight.

After 15 days the ratio chlor./car. did not differ and productivity was proportional to incident sunlight intensity.

The Acetylene Reducing Activity (ARA) measured at 27 C in different sunlight intensities maximus i , m when Emax0 klu6 s xi . (300 2 n.mole /h/cH sC highe; m) lowed an r r light intensities (1.7 klux) both reduce ARA (Roger and Reynaud, 1979), showing that an optimum energy inpu requires i t o obtait d higa n h leveN -fixation f lo .

. TEMPERATUREII .

In optimum light intensity, experiments on ARA were carried out in the range of 25 C-40 C; temperatures higher than 35 C increased ARA during the first hour of incubation but then inhibited it, at 40 C totalls i A AR y inhibited withi hours6 n . . africanA Optimu f o s A i am AR in the range of 25-35 C.

40 In the dry area of West Africa the growth of A. africana is very slow almosd ,an t stops between Decembe Aprid an r l whetemperature th n e range is between 15-28 C. In April, when the temperature increases, the doubling tim reduces i e days3 o t d. This fac markedls i t y important s impossibli t ai s o product e e Azolla inocula between Decembed an r April, under these latitudes with the indigenous strain. However, we have teste . caroliniandA a during January 1981, wit temperatura h e range betwee ngreea 15-2n i n , hous8C havd an ee noticed thadoublins it t g fol3 tim s di e less tha f thin o . africana thaA se f us to e Th . introduced strain coul e indicatedb d .

III. EFFECT

y seasoDurin dr relative e th nth g e humidity varie hour6 n i ss time th e, coursfer e up 35%o allowes th t y i n fro f % dr e.I m95 o t d A fallAR zero f st o o withi 4 hour 2 nremoistenind an s propea n i g r growth medium doet restorno s emoro n ARA ed an growt, observes i h d (Reynaud, 1980). This harmful effect of desiccation can be slowed methodo tw dow y nb s summarize Tabln i d . 2 e

. 1 Adding alginate (0.05% growte w/vth )o ht medium resultn i s an ARA decrease of only 60% within 24 h during desiccation, and when the fern is remoistened, ARA is restored to its initial level.

. 2 Storing undeAzollC humiditw 6 lo rt a y conditions preserved ARA morr .fo e tha 8 hours4 n .

e havW e successfully used thes methodo etw protectinr fo s g Azolla durin e shipmenth g f inoculuo t e fieldth o m.t

IV. EFFECT OF NATURE AND CONCENTRATION OF COMBINED NITROGEN.

In the Fleuve region, north of Senegal, amounts of nitrogen as .require0 kg/he 15 higar s a a hr ric fo d e cultivatio thao s n t AzoTla has to be used along with chemical nitrogen fertilizer. Thus s importanii t o assest e effecth s f differeno t t formd an s concentrations of chemical N on growth and nitrogen fixation of Azolla.

41 Azoll s growa wa nutrien n i n tm medipp 8 8 a d containinan 8 8. , 0 g of ammonium-N or nitrate-N. In comparison to N-free medium, nitrate-N concentration had no effect on Azolla growth, but 88 ppm of NH -N had a negative effect and 8.8 ppm NH -N, a markedly positive effect (Table 3).

High nitrogen concentrations quickly depressed dinitrogen seemt i , s -N tha fixationO N e cast th f thio e n I s. fixatios i n replaced by nitrate-N assimilation. On the other hand 88 ppm of NH -N stopped dinitrogen fixation of Azolla. With 8.8 ppm of combined-N, the acetylene reduction of the symbiotic system is depresse abouo t d% afte40 t day2 1 r s exposure.

A further experiment was conducted to examine the effects of a rang ammonium-f o e N concentration Azolln o s a (Tabl . Growte4) f ho e fer positivth s i n comparison i e e growtth N-fren o i t nh e medium s depressewitwa A h ÄR concentration e dNH.-Nm th pp t 4 ,4 bu o t p u s concentrationl aal t NH.-Nf o s . s decrease Wit8 ppmwa 1. hA ,ÄR d to abou hale on tf after 6-12 dayrecoveret activits bu s it l al dy after 19 days of incubation.

These data show the limiting effect of low concentrations of e facth t d combined-thaan tA dinitrogeAR n o N n fixatiod an n combined-N assimilation takes place simultaneouslm pp y4 4 eve t a n concentratio f combineo n d nitrogen. Utilizatio nitratf o n e seems preferable to the use of ammonium (Liu Chung Chu, 1979) as ammonium competes with dinitroge younn i n g fronds. (Watanabe et.al. 1981).

r resultgoon i Ou de agreemenar s t with thos Yatazawf o e. al t ae (1980. pinnatA r )fo a var. imbricata whic consideres e i h th e b o t d Asiatic form of A. pinnata.

B. Plot Assays

Azolla p_innata var. africana trials were carried out in Senegal according to the recommendations of the International Network on Soil Fertility and Fertilizer Efficiency in Rice (INSFFER) for 1979. Four randomized plotsquar2 f so e metres, eaccontainine on h g

42 950 kg (dry weight) of sandy soil (N% = 0.140) covered in a plastic film to avoid N diffusion, were used for each treatment. The trials wer ORSTOe eth carriet Ma Statiot ou d Dakan i n r (Senegal). Experiment teso effecte t s th t Azollf so a inoculation were conducted during the wet season 1980 (August to November). Subsequently, the residual effec Azollf o t s investigateawa d durin e followinth g y dr g season 1981 (Februar Juneo t y ) (Tabl. 5) e

During the first assay the climatic conditions were best for growth of AzoJLla i.e.: maximum light intensity: 70 klux, maximum relative humidity: 98%, temperature range: 22-37 C.

A tenth treatmen nine s addeth te wa o t treatmentd s use othen i d r 1NSFFER trial Azolla: s . previousl groundd an s C y ,wa 0 drie 6 t a d incorporate e soi 0 dayth 1 ln i sd before transplanting e ratth et a , of 60 kg/ha. In all treatments with N addition, urea was the sole nitrogen source s appliewa t ;i thren i d e split applications: before transplanting, 20 days after transplanting and 40 days after transplanting. During the second cultivation cycle, only treatments receive3 d an 2 d urea-treatmente th l N Al (Tabl s. were6) e always provided witwaterp ta h .

I. RESULTS ON THE FIRST TRIALS.

Inoculation with Azolla increased the grain and straw yield in all treatments (Table 5). Inoculation with Azolla without N fertilizer (treatments 4, 5, 6) increased the grain 38-40%, which is similar to the increase resulting from the addition of 30 kg urea-N/ha e increas Th e stra. th n wi e yiel s highewa d r when Azolla was incorporated (37%) than when it was not (28%). When no N fertilizer was added, the highest yield increase (54%) was obtained with two Azolla inoculations in succession, the first one before, and the second after transplanting (treatment 9). The combination of Azqlla. inoculatio nN/hg k wit a0 3 happlicatio n (treatment 7,8) increased the yield more than did 60 kg urea-N/ha. The yield increases resulting from Azolla inoculation reported here highear e r than the average yield increases observed in INSFFER (1980) trials conducted in Asia, due to the small plot area.

43 Incorporatio previouslf no y dried Azolla (treat. 10)s ,wa significantly less favourable than other types of Azolla incorporation comparisoA . n between*treatmen urea-N/hag k 0 (6 3 t ) and N/hg treatmenk fore organi0 s aAzollth f a mo (6 n i 0 t1 acN powder) shows that the second form of N is less available to rice than the first one.

The growth of Azolla. expressed as total N from Azolla per ha (Table 6) shows that the development of Azolla was significantly better before (treatment 4,7) than after transplanting (treatment 5,6,8). Incorporation was always done 15 days after inoculation Azollg k (0.15 a fresh weight/square metre) growte Th . Azollf ho a was significantly increased with application of urea (treatment 7,8) at the rate of 30 kg N/ha.

II. RESIDUAL EFFECT.

During the dry season 1981, rice IR 1529 was replaced by KN1H300. This variety is assumed to grow better than IR 1529 with col climatiy ddr d (15-3an c ) 0conditionsC , althoug averags it h e yield y seasodurindr lowee s i nth g r tha nR 1529 I thas f It to . straw/grain rati alss i o o significantly highed R 1529an (I r8 :0. KN1H300: 1.3). Witexceptioe th h treatmenf no whos, t9 yielw elo d remains unexplicable graie ,th e soi th n e totaln yieldi th ld N an s after two cultivation cycles were higher in treatments with Azolla than in treatments with urea. Thus it follows than an Azolla inoculation-incorporation treatment brings to the rice, during two cultivation cycles equivalenn ,a t valu 90-12f eo urea-g 0k r Npe hectare e totahighes th i ls N Azolln A i r. a treatmen seemt i t s possible that the residual effect of Azolla inoculation could continue during 2nd rice cultivation cycle.

mose Th t significant event durin e seconth g e dgoo th cycld s ewa growth of rice in treatment 10. The yield was about the same as in treatments 3,7 and 8, which means that, during the first cultivation cycle, the majority of N in dried Azolla was not available for n explaica rice e W n.e fac th thi ty b stha t ther afte, is e r incorporation of dried Azolla in the wet soil, an aerobic mineralisatio onlf o n smala y l fractio Azolle th f ano material (20% f totao l nitroge mineralize s e i firsn th 5 1 s t5 days)a 1 t n i dBu .

44 centimeter f suiso l became quickly anaerobic 1982). (Loyeal t ,re denitrification occurred and this mineralized Azolla-N was lost and not assimilable for rice. Anaerobic fermentation transforms Azolla-N into a form that is easily mineralisable under the aerobic conditions which occurred during ploughin e seconth r d fo gric e culture. The poor availability of Azolla-N. during the first rice cycle, can be enhanced if Azolla is composted and incorporated just before transplanting.

CONCLUSIONS.

In regar o thest d e data:

e besTh t africangrowt. A f o h a occurs durin e shor th gt season twe .

Viabilit f freso y h Azolla inoculum during transpore b n ca t improve y soakinb d fronde th g s previousl alginatn i y e (0,05%) and. C the 6 nt a drainin h 8 4 d storin o an gt p u g

Nitrate has no effect on Azolla growth in nutrient media, but in the fieldN/hag k positiva 0 s 3 ,e rat, ha ureth f eo et a effect.

Inoculatio Azollf no a alwaypositiva d sha e effec n ricto e yield.

A residual effect of Azolla was found on a second rice culture.

The degradation of the fern in the soil is not elucidated and suppositions mus confirmee tb N assay y .b d

45 REFERENCES

CHARREAU, C., 1974. Soils of tropical dry and dry-wet climatic areas of West Africmanagementd an d thei an ae us r . Agronomy Mimeo 74-26, Cornell University, Ithaca.

International Rice Research Institute, 1980. firse Reporth tn o t trials of Azolla use to rice, INSFFER (1979), Los Banos, Laguna, Philippines.

U CHUNLI G CHU, 1979Azollf o e ricn .i aUs e productio Chinan i n . In: "Nitrogen and Rice". IRRI, Los Banos, Laguna, pp 375-394.

LOYER, J-Y., JACQ, V.AREYNAUDd an . , P.A., 1982. Variations physico- chimique rizière d l s so dane n inondésu t évolutione a l e d s biomasse algale et des populations microbiennes du cycle du soufre. Cahier ORSTOM. Serie Biologie in press.

REYNAUD, P.A., 1982. Fixation d'Azote che Cyanobacteries zle s libres ou en symbiose (Azolla); possibilities d'utilisation agronomique en Afrique Tropicale. Bull. Fed. FAO. 47, 63-80.

ROGER, P.A REYNAUDd .an , P.A., 1979. Premières donnéer su s l'écologie d*Azolla africana en zone sahelienne (Senegal). Oecol. Plant75-84, 1 , ..14

WATANABE, I., KE-ZHI BAI, BERJA, N.S., ESPINAS, C.R...ITO, 0. and SUBUDHI, B.P.R., 1981 Azolle .Th a Anabaene aus s compleit d an x in rice culture. I.R.P.S 1-11: pp 9 .6

YATAZAWA M., TOMOMATSU, N., HOSODA, N. and NUNOME, K., 1980. Nitrogen fixation in Azolla-Anabaena symbiosis as affected by mineral nutrient status. Soil. Sei. Plant Nutr 6 (3)2 . , 415-426.

46 Table 1 : Effect of shading on A. Africana biomass production and morphology

sunlight 100 60 36 22

Fresh weight afte day8 r s in g from 40 g of inoculum 115 126 125 148 91

Leaves* coloration green border green border green border light green bright green red yellow yellow

Chlorophyly dr o % la: weight 2.71 3.65 6.57

Fresh weight after 15 days of g from 40 g of inoculum 264 250 189 215 132

Leaves' coloration pink pink light green light green bright green

Chlorophyll a:%y odr weigth 5.83 7.1 7.8 Table 2 : Effect of temperature and alginate (0.05 %, 10 ) on fresh weight and Acetylene Reducing Activity of A/olla pinnata var. africana. Each e meaualuth ns i e of triplicates.

24 h 48 h Treatments on fresh Azolla 25°C : % fresh weight : % ARA : % fresh weight : % ARA

R. H .25°% 98 :C 94 95 92 90 R. H. : 30% 25°C 75 0 47 0 R. H. : 30% 0.05 alginat% e 25°C 92 45 76 0 C R6° . H% . 98 r 97 100 96 88 R. H. : 39%C ,6° 97 90 94 65

R.H. relative humidity obtained with K.SO. (98%) and 5(30-40%)0 H .

Table 3 : Effect of chemical form and concentration of N-nitrogen on the growth and the Acetylene Reducing Activity of Azpl'la pinnata var. af ricana, each value is the mean of triplicates.

Fresh weigh. tg % ARA Treatment day2 1 ss s y da day4 8 s day4 2 day8 1 ss

W-free medium 15 18 20 100 100 100 + H W m 8.pp 8 16 19 25 65 110 60 f H W m 8pp 8 14 15 15 60 35 10 O W m pp 8 8. 15 17 19 85 90 60 O W m pp 8 8 15 18 20 55 60 15

48 Tabl: Effec e4 combine f to d e nitrogeth n o ) n O ((NS ) H growt d Acetylenan h e Reducing Activit Azolln i y a pinnata var. africana.

fresh weight ARA

Culture solution 6 days 12 days 19 days 6 day day2 day9 1 1 ss s

Control - N 100 100 100 100 100 100 NH.-N : 1 . 8 ppm 110 108 100 50 45 95 4 : N - H 8.8 ppm 112 118 99 45 25 55 4 NH -m Npp :4 4 105 115 50 35 20 5 4 NH.-m N pp :8 8 90 80 35 25 12 3 4 : N - H 175 ppm 85 60 35 20 10 0 4

49 Table 5 : Effect of Azolla and urea on rice yield and straw conduct as proposed by INSFFER t seasowe d residuaa an nn o l nex e effecy seasoth tdr n n o ti n Dakar (Senegal) (rice variety a: IR1529, b: KN1H300)

Yields : (a) 1980 wet season, (b): residual effect dry season 1981 Grain Straw a T/ha Contro T/hb al% Control % a T/ha Contro T/hb al% Control%

1. Control 4 2. 3.7 0 10 100 3.2 100 3.3 100 2.N/hg k a0 3 8 2. 5.1 8 13 117 3.9 122 4.6 139 3N/h.g k a0 6 5.5 149 2.7 115 4.4 137 4 121 4. Azolla inc. before transplanting 5.1 138 3.8 161 4.4 137 4.4 133 5. Azolla grown after transp. then inc. 5.2 140 3.5 146 4.6 144 4 121 6. Azolla grown after transp. no inc7 .2. 5.1 8 13 112 4.1 128 3.8 115 7N/h.g k Azoll+ a 0 3 a inc. before transp. 5.9 159 3.3 140 4.4 137 3.7 112 8. 30 kg N/ha -i- Azolla after transp. inc. 5.7 154 3 125 4.5 141 3.6 109 9. Azoria grown before and after transp d incan .. 5.7 154 2.4 100 4.3 134 3.3 100 10. 60°C dried Azolla, incs a . N/hg k a0 6 1 3. 4.7 7 12 130 3.5 109 4.2 127 inc = incorporatio. ; transpn = .transplantin g N imported N from Azolla N exported with N%0 a" s ha ure . akg kg. ha"1 the yield kg. ha"1 soie inth l after Treatments a b Total a a b total the two cultures 1 Control 0 O O O 45 34 79 0,135 2 30 kg N/ha 30 30 60 0 60 42 102 0,145 N/hg k 3a0 6 60 60 120 O 66 40 1O6 0,149 4 Azolla inc. before transplanting O O O 20 62 51 113 0,16O 5 Azolla grown after transp. then inc. 0 0 0 7,5 64 46 110 0,140 6 Azolla grown after transp inco .n . 0 0 0 12,4 61 44 105 0,153 N/hg k 0 Azolla+ 3 7 a inc. before transp. 30 30 34 69 44 113 0,133 8 3O kg N/ha + Azolla after transp. inc. 30 30 15 68 4O 108 O,164 9 Azolla grown before and after transp. then inc. 35,8 1 10 0,14 4 3 679 1O 6O°C dried Azolla, inc. as N/h6g Ok a 60 55 43 98 0,156

inc. : incorporation, transp. : transplanting a : rice culture during 198O wet season, b: residual effect on a second rice culture during 1981 dry season.

Tabl Nitroge. e6 n budgerico tw en to cultivatio effecs n ure f tAzollao d aan . SEXUAL REPRODUCTION OF Azolla SPECIES

J.H. BECKING Research Institute I.T.A.L., Wageningen, Netherlands

Abstract

Azolla is a heterosporous fern and has a typical fern life cycle. Little is known of the factors inducing sporocarp formation or ecologicas it f o l significanc Azollan ei Anabaene Th . a azollae cyanobion transmittes i t e nexth t o t generatiod n througe th h macrosporocarp. The six existing species of Azolla are usually divided into two sub-genera Euazolla (4 species) and Rhizosperma (2 species) dependin whethen o g r the y9 outside float r havth o n e3 so e of their macrospores.

Introduction

A brief outline sexuath f o el reproduction syste Azollf mo s i a presented on request of the committee of the consultants meeting.

Azolla is a heterosporous fern. This means that two types of sporangia (sporocarps) are formed, producing two types of spores, i.e. macro- and micro-spores. Like in all ferns, in its life cycle Azolla forms firs gametophyta t e (prothallium), which produce n archegoniua s m and antheridiu d thesan m e structures giv eovu n a risr mo o et spermatozoids (or antherozoids) which form, after fertilization, the zygot embryo)r (o e .

Life cycl Azpliaf o e .

At the apex of the stem of the Azolla plant, the algal component Anabaena azollae occurs in a free-living, non-nitrogen fixing state subsequene th n I (Fig . t 1) .developmen t e leavestageth e f th so Anabaena cell e incorporatear s d intleae th of cavities (Becking 1978, 1979).

53 In natural conditions, Azolla usually multiplie vegetativy b s e reproduction, i.e .y fragmentatiob e frondsth f o n. However, under certain conditions the formation of sexual organs is observed.

Little is known of the factors inducing sporocarp formation or of its ecological significanc n Azollai e n temperatI . e regions, high temperature and high light intensity followed by low temperature may induce sporocarp formation n tropicaI . d subtropicaan l l regionw lo s temperature has been reported to induce sporocarps in A. pinnata. In south China, local A.pinnata strains form spores in June/July and to a lesser exten September/Octobern i t n nortI . h Vietnam, sporee ar s formed mainly in the months March/April and seem to be associated with high surface density. When sporocarp formede ar s , vegetative growth is usually retarded, either by an intrinsic factor or by overcrowding.

In general, sporocarps are formed on the shoot stalks near the base of the fronds. They are situated between the ventral and dorsal lobes nea lateraa r l branc thed an hy always occu pairsn i r , i.e. either two macro- or two micro-sporocarps or the combination of one macro- and one micro-sporocarp (Fig 2).

Micro-sporocarps, the male organs, are much larger than the macro mega-r (o - ) sporocarps. Thebrownise ar y h yello coloun i w d an r contain many microsporangia. Withi e periplasmodiunth f suco m a h microsporangium, 32 or 64 microspores develop and in a later stage these aggregate into massulae. Ripe massulae sho alveolan wa r structure with microspores inside and anchor-like (or arrow-like) projection outside th t a se lattee Th (Fi r. g3) structure s pla rola y e in the attachment of the microsporic massulae to the outer wall of the macrospore (or macro-sporocarp).

Macro-sporocarps, the female organs, are also born on the stalks e ofteanar d n associate e samth en i lead f axis witmuce th hh larger micro-sporocarp. They sho dark-colourewa d upper portioe th f o n indusium due to the lignified nature of the tip of the macro-sporocarp. In the development of the macro-sporocarp, algal cells of the symbiont are included below the upperpart of the indusium (Fig. 4). In a young macro-sporocarp the Anabaena cells completely surround the columella stalk within the macro-sporocarp down to the

54 ripe th bottocavitye en th i macro-sporocar f t mo ,bu e algapth l cells are pressed to the tip of the macro-sporocarp by the developing alveolar bodies withi sporocare th n p (Becking 1978) e ripTh .e macro-sporocarp, which is about 0.75 x 0.42 mm in diameter is only diametee aboue thirmucth e on e halth f h to on dr f o ro large r globular micro-sporocarp.

The algal cells within the macro-sporocarp are present in rows or fillaments. Theprobable ar y y resting cell r "akineteso s " since they posses thick cell wall contrasn (i s vegetativo t e cellso n d )an heterocysts (Becking 1978). Althoug ontogenetie th h c formatioe th f no micro-sporocar o somt s ei p extent paralle o thalt f to maero-sporocarps, the micro-sporocarps never contained algal cells (Becking 1978,1979).

A complete lif evegetative cyclth f eo d sexuaan e l reproduction Ajd.u3.lf o representes i a n thi I Tabln i ds . efigur1 e solieth d arrows indicate the continuation of the association (i.e. incorporation of the algal endophyt macro-sporocarps)e th n i e , wherea dottee sth d arrows indicate that the endophyte is not transmitted.

Taxonomy of Azolla

The morphology of the sexual organs of Azolla is closely connected to the taxonomic division of Azolla species, as they are morphologically distinct. The six existing forms of Azolla are usually divided int section2 o s depending whether the9 y r havo 3 e floats on the outside of the macrospore (Florschütz 1938; Hills and Gopal 1967).

e fouTh r speciee sectioth f o sn Euazolll originallal e ar a f yo Nort Soutd an h h American origi e section Th (Tabl . n e2) Rhizosperm a has only 2 species and is widespread in the Eastern Hemisphere, i.e. tropical Africa, southern Africa, S.E. Asia, Japa Australiad an n e .Th species Azolla pinnata has two morphologically distinct forms, i.e. A.pjnnata var. pinnat Brow. aR A.pinnatd an n a var. imbricata (Roxb.) o formtw Bonap se havTh . e basically different distribution patterns, which may be somewhat disturbed by human interference. The highly

55 imbricate forms sho clearlwa y disjoined distribution occurrine th n o g S.W. coast of Africa and Madagascar, and in .Australia (and to some extent in Papua/New Guinea); whereas, the pinnate forms occur in a conjoined area in S.E. Asia from India to Japan and from Indonesia to Papua (Swee Hilld tan s 1971).

REFERENCES

Becking, J.H. 1978. Ecolog physiologicad an y l adaptation Anabaenf o s a in the Azolla-Anabaena azollae symbiosis. In: "Environmental role of nitrogen-fixing blue-green algae and Asymbiotic Bacteria", Ecol. Bull. (Stockholm) 26: 226-281.

Becking, J.H. 1979. Environmental requirementn i e Azollf so us r afo tropical rice production. In: "Nitrogen and Rice", Symposium International Rice Research Institute, Publ. I.R.R.I., Manila: 345-373.

Florschütz, F. 1938. Die beiden Azolla-arten des Niederländischen Pleistozäns. Reel. Trav. Bot. Neerl. 3_5: 932-9457

Hills, L.V Gopald .an 1967. ,B . Azolla primaevphylogenetis it d an a c significance. Can Bot. J : .1179-119145 .

Sweet, A.R Hillsd .an , L.V. 1971 A stud. Azollf o y a pinnat Brow. aR n Am. Fern J. 71: 1-14.

56 vegetative reproduction< f SPOROPHYTE -*• SPOROPHYTE X vegetative reproduction

macrosporocarp micros porocarp I Y macrospore microspore i

macrogametophyte microgametophyte i l \ f archegonium antheridium l I $ ovum spermatozoid (antherozoid) s

EMBRYO

Tabl . Diagram-oI e e lifth f e cycl f Azollao e spp. Solid arrows represent the continuatio e associatioth f o n n with Anabaena azollae. Broken

arrows indicate no continuation of the association.

57 Table 2. Original distribution of the six existing Azolla species

Number of floats Subgenus on mega-sporocarps Species Main distribution

Euazolla . filieuloideA s Southern South America, Lamarck Western North America to Alaska

A. earoliniana Eastern North America, Wild Central America, North South America, West Indies

A. mexicana Northern South America, Presl. Western North America to British Columbia and eastward to Illinois

microphyll. A a Wester Northerd an n n Kaulfuss South America to Southern North America and the West Indies

Rhizosperma . pinnatA a Eastern Hemisphere: R. Brown Tropical Africd an a Southern Africa, S.E., Asia, Japand ,an Australia

. niloticA a Upper Nild Sudanan e , De Laisne Central Africa

58 Fig. l Section throug e ape Azollf th hxo a filiculoides stem showing the presence of free-living Anabaena azollae near the leaf e incorporatioth primordid an e e algastep th th n ti m i et f a o n an initial stage of leaf formation of the dorsal lobe. Scale . urn 0 3 r ba

59 . 2 Fig. Macro-sporocarp micro-sporocarpd an ) axie (a s th f o ln i ) (b s the upper leave e lobeth Azollf f so o a filiculoides. Scale bar 10 mm.

. Fig3 . Ripe massula of the micro-sporocarp showing its alveolar structure with microspores inside and the anchor-like projections (glochidia), which plaattachmene rolth ya n ei f to the microsporic massula outee th macrosporere o et walth f lo . . mm 1 Scal r eba

60 Fig. 4. Longitudinal section through the tip of a nearly ripe macro-sporocar Azollf po a filiculoides showin endophytie th g c algal cells pressed to the upper portion of the sporocarp by the development of some accessory structures. Scale bar 30 urn.

61 THE USE OF 15N-LABELLED DINITROGEN IN THE STUDY NITROGEF O N FIXATIO BLUE-GREEY NB N ALGAE

K. JONES Department of Biological Sciences, University,of Lancaster, Lancaster, United Kingdom

Abstract

Prior to the development of the acetylene reduction technique maie th s usen s wa Na qualitativ d quantitativd an e e measurf eo nitrogen fixatio y free-livinb n g cyanobacteri varieta n i a f aquatiyo c and terrestrial habitats. Despite its expense and the technical difficulty, N is a major tool in the study of cyanobacteria, for example, incorporation of H is the definitive test for nitrogen fixation; it is used in the determination of the correct ratio of acetylene reductio o nitroget n n fixation sitn i un i ,nitroge n fixation assays tracinn i , e formatioth g d fat an nf extra-cellulaeo r nitrogen and in measuring the turnover and grazing rates of cyanobacterial intra-cellular nitrogen. These latter studies show that N-labelled extra-cellular nitrogen can serve as nitrogen sources for a variety of bacteria, fungi, algae and higher plants, and that cyanobacteria are graced and digested by a variety of animals. The turn ver rates of cyanobacterial N-labelled cells are dependent on the type of cell, species, environmental condition e availabilitth d an s f degradino y g organisms e breakdowTh . n product rapidle ar s y mineralise d uses an da d nitrogen source y highesb r plants.

INTRODUCTION

The heavy isotope of nitrogenN ha15 s, bee n used to study nitrogen f fixatio blue-greey nb n algae (cyanobacteria followine th n }i g ways: In situ measurement of nitrogen fixation; In proving unequivocably that particular isolatex sfi nitrogen;

63 In determining the correct ratio to be used in calculating nitrogen fixation from acetylene reduction data; In tracin fate gnitrogeth f e o n fixed. In this.presentation these topic discussede sar .

IN SITU MEASUREMEN NITROGEF TO N FIXATIO HABITAIN NI S DOMINATED

BLUE-GREEN ALGAE developrcne Prioth o acetylenrt e th f to e reduction technique (Stewart _et_.al_., 1967 usuae th ) l metho measurinf do g nitrogen fixation ni the field was by estimât ng the uptake of 15N-labelled dinitrogen ( ^2). The appropriate methodology is described Cor terrestrial habitats by Stewart (1966 & 1967a) and for aquatic systems by Nsess et al. (1962). Tabl citee1 referencese th sorn literature f eo th n si e where has been employed to measure nitrogen fixation by blue-green algae in a variet habitatsf yo .

TABLE 1 In situ assays of nitrogen fixation by blue-green algae usin^ - g

Habitat Reference Tropical ocean Dugdale, Goering and Ryther (1964) Temperate lakes Borne and Fogg (1970) Tropical lake Hom Vined ean r (1971); GanHomd fan e (1975) Temperate rocky shore Stewart (1967) and dune slack Antarctic algal mats P^gg and Stewart (1968); Home (1972)

64 over The advantages of using «2 acetylene reduction include. certainte Th y that nitrogen fixatio beins ni g measured. The possibilit pinpointinf yo exace gth t locatio nitrogef no n fixation withi habitate nth . The avoidance cL the problems inherent in the acetylene reduction technique (see discussio y Fle al_nb t t,e? Watanabed 1975an e ;Le , 1977; Patriquin and Denicke, 1978; Lethbridge et a±, 1982). Bière are, however, disadvatages in using N for in situ measurements of nitrogen fixation and these include: Expense. Time consuming. Technically difficult Requirement for a mass spectrometer to measure relatively small ratiosN increaseo t . w n si The impracticability of carrying out large numbers of replicates. The difficulty in assaying undisturbed samples in truly in situ studies as, for example, those on acetylene reduction by sediments (ferbert, 1975; Jones, 1982). On balanc seemt ei s that acetylene reductio bese th t s methoni r dfo

15 s u measuring nitrogen fixation in the field and that N2 h° l<3 be used to check that nitrogen fixatio procese th s ni s being measure obtaio t d ndan the correct rati acetylenr ofo e reduction relativ nitrogeo et n fixation.

15 N2 FIXATIO DEFINATIVE TH S NA E TES NITROGER TFO N FIXATION To be absolutely certain that presumed nitrogen-fixing organisms are nitrogen fixers and not scavengers of combined nitrogen from the atmospher medir eo a they shoul testee db uptakr dfo *%„f eo . Good examples of this are found in the early papers of Stewart (1962; 1964; 1965) where he confirmed that marine strains of Calothrix scopulorum and

65 5 ———Itosto—c ————————entophyticur—o were nitroge- n fixers. More recentlyj * beeNs 2-ha n checa use datr s da kfo a obtained from acetylene reduction assaysn I .

tabl havI e2 e presente 2 duptak ^N datr speciey aefo b Nof so s toc isolated fro varietma habitatsf yo . This dat bees aha n useo t d

corroborate acetylene rr Auction results published in the literature. ^^tioo xa variouy i^ "b TABL Es2 Nostoc species

•"•TJ enrichment (atom % •LSN Duratiof no excess) assay Algae Atmosphere Sub-tropical grasslan) (i d 3 d 0.414 7.117 Salt marsh creek bank (ii) 1 d 0.129 8.022 Estuarine sediments (iii) 1 d 0.113 5.863 Freshwater canal 1 d 0.105 10.449 Moorland stream in association with moss (iv) 7 d 0.965 13.758

(i). Jones, K. (1977); (ii). Jones, K. (1974); (iii). Jones, K.

(1982); (iv). Jones and Wilson (1978).

N uptak15 dea f o f s e ïha inative eus e tesnitroger tfo n fixatios ni

2 particularly important when 'new nitroge1 n fixer isolatee sar wherd dan e acetylene reduction seem least ,a first ta t sight, improbabler Fo . example, while working on a salt marsh in 1972 I found that sediments colonised by the non-he terocystous blue-green alga, Lyngbya aestuarii,

N n consistently reduced acetylene. When tested for utilisation of 2 ^ an atmosphere containing ^ labelled to 45 atom % ^N excess for three days sedimente )th s showed significant enrichmen 0.03t ta 0 ato^ m% excess. Although low, compare thao dt nearb f to y creek ban intentd kse s

66 colonise Nostoy db c (enriche 0.53o dt 7 excess)N ato m% labellin e ,th g was consistent with fixation by a non-heterocystous blue-green alga capable only of fixation in microaerophylic environments. It is ironic

that nitrogen fixation chodesmlum^Sscillatorii b_Tr - a blooms detected using

N (Dugdal15 e et^al^. 19C4 / onls wa )y fully accepted after corroboratioy nb 2 extensive acetylene reduction experimentation.

DETERMINATION OF THE RATIO OP ACETYLENE REDUCTION TO NITROGEN FIXATION The pioneer acetylene th f so e reduction assa fielr yfo d measurements of nitrogen fixation (Stewart jet cd., 1967; Hardy cît _aJL , 1968) suggested that the ratio of acetylene reduced to nitrogen fixed should be checked for each system as the theoretical ratio of 3:1 may not always hold good. This need to check the ratio has become increasingly important with the almost universal adoption of acetylene reduction for measuring nitrogen fixation in the environment; with the extrapolation of the data over large areas; and their incorporation into mathematical models which have implication environmentar sfo l management. The main method employe separatelo t s di simultaneouslt ybu y measure nitrogen fixation in a series of replicates using both acetylene reduction

and nN2* Table 3 shows the results of just such an experiments recently carried out with Azolla filiculoides and A. caroliniana. The ratios varied from 2.4:1 to 6:1 for an assay which lasted 12 hours. This compares with ratios of 3.4:1 after 14 days, 1.6:1 after 19 days and 2.4:1 after 22 days for A. pinnata (Watanabe et ail, 1977).

67 TABL E3 Determinatio ratie th acetylen f oo f no e reductio nitrogeo nt n fixation for 2 Azolla symbioses

filiculoide. A carolinian. A s a

atmosphern i N e (atom -eN excess) 0.397 1.133

15 15

N in Azolla (atom N% excess) 0.035 0.031

15 15 6 26 3 24 Total nitrogen (ug) Nitrogen fixed (from 15N) (ug) 21 7 Ethylen 0 5 e production (ug42.) 5 Ratio of ethylene production to N fixe15 d 2.4:1 6: 1

(8 Azolla plants were placed in bijou bottles (vol. 7 cm) and the atmospher mixturs replaces ga ewa a ey db containin oxygen% g10 , 0.02% carbon dioxid removees witwa res e s hth dtga witargon m c syringh a 1 . e •3 ê

l5 and replaced with N-labelled N2 (3 bottles) or acetylene (6 bottles). Bottles were place ligha n do t rac root ka m temperatur hour2 1 r s efo afte r which they were teste ethylenr dfo e production Chromatograpusins ga ga h r an^-enrichmendfo t wit masha s spectrometer enrichmene Th . f to unexposed Azolla control 0.36s swa 8 ato m% ^j) . In a similar experiment carried out on Npstoc cultures isolated from the Lancaster area the ratio varied from 2.3:1 to 2.7:1 (Table 4).

68 TABLE 4 Extermination of the ratio of acetylene reduction to nitrogen fixatio Nostor nfo c spps. Canal NosLoc Estuarine Creek bank ttostoc Nostoc atmosphern i N 15 e (atoN excess15 m% ) 9.506 7.459 7.743 algn i a N (ato15 N excess15 m% ) 0.125 0.122 0.125 8 5 2 9 3 13 ïbtal nitrogen (ug) nitrogen fixed (from 15N) (ug) 1.75 1.51 0.94 Ethylene production (ug) 4.4 0 4. 52.1 6 Rati ethylenf oo e productioo nt 15N fixed 2.5:1 2.7:1 2.3:1

(3 cm algal suspension were transferred to 6 bijou bottles. 3 were tested for 2 fixation and 3 for acetylene reduction over a 22 hour period at room temperatur ligha n eo t rack enrichmene Th . unexposef to d controls swa 0.370 atom % 15N excess). It goes almost without saying tha acetylene tth e reduction techniqus ei

1 ) u take

much easier and quicker than ^ 2 P assays. The possible errors in the

e ar s roorYe numerou^assa 2 s and much care is needed. Ihe two major sources of error (in my experience) are: the estimations of total nitrogen on small samples and ensuring that the exact enrichment of all of the assay bottles is measured. One cannot assume that the enrichment of the nitrogen ^ generatio e inth n flas identicas ki assae thath o ylt n ti bottles n I . practice it rarely is. If , as an examination of the literature shows, the rati variabls oi lefs i te witeon usefulnesss querha it f yo . Perhape sw should re-emphasize that acetylene reduction data shoul transformee db o dt nitrogen fixation only after extensive investigatio thaf no t particular system. This is not to detact from the acetylene reduction method, which has been suc booha nitrogeo nt n fixation studies mort ,bu ereflectioa f no the difficulty in obtaining the correct ratios.

69 TRACIN 2 FIXEN FATE BLUE-GREEY DB Gp TH E O N ALGAE Nitrogen-fixing blue-green algae ten predominato t d environmentn ei s where there is little cornpetition, where their prokaryotic cell structure enables the withstano mt d extrem changeablr eo e condition wherd san e there levelw lo e combif so ar r .-d nitroge thao ns abilit e nitrogex tth fi o yt s ni an advantage, for example, rocky shores, salt marshes/ shallow marine sediments, desert crusts, lake tropicad san l oceans. Becaus levele eth f so combine dthesn i nitroge w e lo environment e nar might si expectee tb d that the nitrogen fixed by blue-green algae is of importance to the development of the ecosystem. ^N can be used as a tracer to see if this is indeed the case.

Transfer Experiments In these experiments -*N has been used to trace the nitrogen fixed by blue-green algae into other components of the ecosystem. A surface section particulae th f o r syste removeds mi , place containea n di exposed ran o dt 1 ^2* After a period of incubation the various components are dissected out and analysed for % content. Environments examined in this way include: desert crusts (Mayland et al. , 1966), sand dune slacks (Stewart, 1967), algal mats on a rocky shore (Jones and Stewart. 1969b), associated with Nostoc in the antarctic (Harne, 1971) and moss hummocks in a freshwater stream (Jones and Wilson, 1978). Table 5 contains data for a similar type experimenf o t carriesedimentn o t dou s dominate blue-greey db n algaa n ei

15 fixe d bv e salt marsh. The data show that the N2 ^ blue-green algae is transferred to higher plants and that in Salicomia dolichostachya the TJ is translocated to the shoots. These results are in agreement with those cited abov other efo r environments.

70 TABL E5 Transfe f ro fixe saly b d t marsh blue-gree higheo nt r plants

Componen systef to m Enrichment excess N (ato-* m% ) Anabaen sedimenta+ s 0.221 sediments only 0.050 Puccinellia maritima 0.058 Salicornia dolichostachya 0.025 Npsto sedimentc+ s 0.537 Salicornia dolichostachya roots 0.537 Salicornia dolichostachya shoots 0.048

(Surface portion sedimente th f so s together withighee hth r plants were place Erlenmyen di r flask whicn si atmosphere hth s replaces ega wa a y db mixture composed of 10% nitrogen labelled to 54 atom % *^J excess, 20% oxygen, 0.02% carbon dioxid argond ean . Afte day r3 varioue sth s components were separated out and analysed for "^ enrichment using a mass spectrometer. The labelling of unexposed controls was 0.367 atom % ^N.) uncleaIs ti r from these result releases si e whetheN th ^ y db e rth blue-green algae as extracellular products or by autolysis. It is possible that some of the enrichment of the higher plants, especially in the case of P. maritima, could be the result of nitrogen fixation by bacteria as it has been demonstrated in the rhizosphere (Jones, 1974).

Productio Extracellulaf no r Nitrogen Most of the blue-green algae liberate extracellular nitrogen during growth. Thi potentialls si importann ya t nitrogen source whe bluee nth - green alga is a nitrogen fixing species as it represents new combined nitrogen enterin ecosysteme gth release Bi . extracellulaf eo r nitroges nha

71 been most extensively investigated for the nitrogen-fxing marine blue-green alga, Calothrix scopulorum (Jones and Stewart, 1969a). We found that the highest quantities of nitrogen were released during growth under optimal environmental condition that highese sbu tth t proportional release (extracellular nitroge proportioa 3 nintracellula;i e th f no r nitrogen) occurred during lag phase growth and in unfavourable conditions. On average 40% of the nitrogen f ixeci by C. scopulorum is released extracellularly although this highefigure b n changinn erca i g conditions liberatee Th . d nitroge that no t s whicni juss hha t been fixed. Tabl containe6 s data which show that although after 16 days growth extracellular nitrogen comprised 20% "/ of the total nitrogen it contained only 6.1 bo 7.5^of the newly-fixed

TftBL E6 intracellulae Distributioth n i 3 T extracellulad f rnan o r nitroge Calothrif no x scopulorum day 6 grow1 sr undenfo r

Experimen tA ExperimentB Intracellular N (uglT) 4,847 4,559

Intracellular labellin1 g (atoN excess15 m% ) 13.580 11.800 Extracellular N (uglT1) 926 914 Extracellular labelling (atoN excess15 m% ) 4.055 4.510 % 15N intracellular 93.9 92.4 % 15N extracellular 6.1 7.6

was grown in an atmosphere containing 10% nitrogen labelled to 60 atom % J excess, 2% carbon dioxide and argon. J3 was grown similarly but with a nitrogen enrichment of 35.4 atom % ^N excess). Similar 15N data was obtained for a tfostoc culture isolated from moss hummocks thin I .s cas enrichmene eth intracellulae th f to r fractios nwa

72 19 628 atom % 15N excess and that of the extracellular fraction 1.937 atom % N excess. The nitrogen released in this way should not be confused with that transferred to other plants in 'the blue-green algal symbiosis (newly fixed inorganic nitrogen) , but it may account for sane of the leakage of organic nitrogen found in lichens exposed to alternate wetting and drying (Millbank, 1978). The extracellular nitrogen release nitrogen-fixiny db g blue-green algae is comprised mainly of small peptides and amino acids (Stewart, 1963; Jones and Stewart, 1969a; Walsby, 1974a and b; Jones and Wilson, 1978).

Uptak 15f N-Iabelleeo d Extracellular Nitrogen It has been demonstrated experimentally that extracellular nitrogen liberate blue-greey db n sourcusea e algab s da n combinef eo eca d nitrogen for the growth of other organisms. Generally this has involved growing the algcombined-nitrogen-frea n ai e inorganic medium under atmosphere containing ^9* ^e al9ae were then filtered from the medium leaving the extracellular nitrogen labelled with "N. The labelled medium was used as a nutrient source for growing test organisms. For example, extracellular nitrogen from Calothrix scopulorum was shown to serve as a nitrogen source for marine bacteria, fungi, unicellular alga macro-algad ean e (Joned san Stewart, als s 1969 wa 1969bo d t ashowan I ). n thanitrogee tth s nwa assimilated by an active process and was not the result of passive diffusion calculateds wa t I . ,^ results froe mth , that bacteria used 65%, fung i98% o frot ,9 m6 unicellula macro-algad r an alga % 99 e o frot e8 m3 fro m 11 to 82% of the available extracellular nitrogen. Fractionation of the test plants showed that the taken up became incorporated into the *r«, structural protein of the cells (partictt^ fraction) (table 7).

73 15 5 TADI.E 7 Distribution of N in algal cells grown j.n * N-labelled extracellular nitrogen fran Calothrix scopulorum

Organism % uptake of the Distribution of 15N within availably b N e the cell fractions whole plants (thaf %o t take) nup Particulate Soluble Cell wall

Enteromorpha 82.4 58.0 19.7 22.3 intestestinalis Porphyra 59.3 68.5 14.9 16.6 umbilicalis

Scytosiphon 34.7 54.6 19.4 26.0 lomentarius

(The algae, growing in the Calothrix zone on a rocky shore, were immersed in 10 on aliquots of extracellular products for 18 h in the light at 10°C. Shey were then removed, washed, smashed in a French press and separated into the three fractions for nitrogen analysis and mass spectrometry) absence Inth organismsf eo , which remove extracellulae dth r nitrogen almost immediately, the nitrogen became adsorbed onto both inorganic and organic surfaces where it could serve as a supply of nitrogen for epiphytes. Extracellular nitrogen liberate Nostoa y db c sp., isolated from moss hummock moorlana n si d stream, were show functioo nt nitroges na n sources for the following higher plants, Carex lepidocarpa, C. capillaris, Minuartia verna, the moss, Gymnostomun recurvirostrum, 'and the green alga, Hjrmidium subtile '(Jones and Wilson, 1978). In the case of G. recurvirostrum it was shown that new shoots and rhizoids were much more highly labelled than older one thid san s implies thaextracellulauptake e tth th f eo r nitrogen a s nwa active process and that the nitrogen was useful to the plant.

Turnove Intracollulaf ro r Blue-Green Algal Nitrogen Blue-green algae are the first colonizers of bare areas where they are majoa r sourc combinef eo d nitrogen (Webble Jonesd yan , 1971). Iherefore there is considerable interest in the rate at which the nitrogen fixed, and

74 thereby immobilise algaee th releasedn s ,di i . However, relatively little worbees kha n don thin ei s area usin. JoneT$ g s (1974) incorporated Nastoc and Anabaena spps. labelled wit intJ T ho sand whic uses groho wa dt w salt marsh plants. *TSf was taken up by the plants but it was not clear whether it came from autolysi bacteriar so l breakdow labellee th f no d algae. There is widespread evidence for the degradation of blue-green algae by bacteria. Watanab Kiyohard ean a (1960) showed decompositio blue-greef no n algae by a variety of bacteria and isolated a strain of Bacillus subtilis capable of converting 40% of blue-green cell-nitrogen to ammonia in 10 days. Verma and Martin (1976) investigated the rate of decomposition of 6 blue-green algae labelled wit. îhe^C hy found that cytoplas degrades mwa d more rapidly than cell walls, that up to 80% of the C was recovered within 22 weeks, and that kinetes were the most résistent of the blue-green cells to bacterial breakdown. There are also environmental differences, for example, Calothrix anomol mors ai e rapidly decompose pondn di s tha algan ni l mats (Gunnison and Alexander, 1975). It has been suggested that specialised • lytfec bacteria important in regulating algal populations. (Shilo, 1970; Daft and Stewart, 1971). lysis of blue-green algae by viruses and fungi has also been demonstrated (Safferman, 1973; Whitton, 1973). * Nitrogen fixe blue-greey db n algae]reventually converte bacteriy db o at ammonium susceptibl s whei t ni volatilisationo et , plant uptake, adsorption, nitrificatio denitrificatiod nan n (Jone Wilsond san , 1978; Skujtnd san Klubek, 1978).

Grazin Animaly gb s It had been generally accepted that blue-green algae were not a suitable source of nitrogen for animals (Arnold, 1971; Barter, 1973; Gunnison and Alexander, 1975). However, more recently evidence has been presented which suggest that this may not always be the case. Brenner £t . (1976_al ) showed that Talorchestia longicomis ,gammarideaa n amphipod, grazed nocturnall srln yo t marsh algal mats containin blue-greee gth n algae

75 Calothrix contarenii, J^JiË/ZË. aestuarii and Microcoleus chthonoplastes. An examination of gut contents together with grazing experiments with C labelled algae showed thaassimilatioe th t n efficienc aiiphipoe th f yo d feeding on this diet was 67%. This is in contrast to results for another amphipod, Hyalella axteca ,efficienc n whica d hha f onlo y y 5-15% whed fe n blue-green algae (Hargrave, 1970). Rxnan (1978) has shown that a marine copepod, Macrosetella gracillis, is,with other marine animal plankton/ dependen r nitroge fo tnitrogen-fixine th n no g blue-green alga Oscillatoria (Trichodesmium) grazind an , microcrustaceany gb s beesha n show o limit n e th t productivit f nitrogen-fixinyo g blue-green alga flooden ei d soils (Wilsot ne al., 1980) and rice paddies (Osa-Afiana and Alexander, 1981). Data for *^N experiments are scarce. Jones and Wilson (1978) describe experiments in which ^N-labelled Nostoe was fed to earthworms, slugs and organisme millepedesth f o l sAl becam. e labelled wit case ^ h• andth e n i , of the earthworm, it was possible to show that the % was carried throughou e bodth t y and, presumably, sourca use s da f nitrogeneo .

REFERENCES Arnold,D.E. (1971).. Ingestion,, assimilation, survivad an l reproduction by Daphnia pulex fed 7 snecies of blue- green algae.. Iiimnol.uceanogr.lj6,, 906-920.. Brenner, J):.., Valiela,!.,. V.onRaalte, £•,!>.:-and Car p enter, E „J.. (1976). Grazin 'Jalorchestiy gb a longicorni n i algan t a n ma o ls

a New England salt marsh.; J.:Exp.Mar,3iol.i:col.22tl6l-l69. Daft,M..J. Stewart,,Y/.D..P.d an . . (1971).. Bacterial pathogenf o s freshwater blue-green algae.' New Phytol..JO,.819-829. DugdalejR-G;.., Goering,J..J.. and Ryther,J.H. (1964). High nitrogen fixation in the Sargasso sea and the Arabian sea.. limnol.Oceanogr.9 »507-510.' Elet,R.x7.', Rudd,J^W.JM^ and Hamilton^RiB.! (1975).. Acetylene reduction assay r nitrogefo s n fixatio freshwatersn ni t a note of caution.: Appl.Microbiol.2$,580-583..

76 Ebgg,GJ2. S.tewart,WJ)JPd .an * (1968). sit.n I u determinatio biolf no - ogical nitrogen fixation in Antarctica.; Brit.Antarct.l Survey Bull.3J5,39-46. Ganf,G.&.' Horne,A.J.d .an : (1975).1 Diurnal stratification,,photosynthesis and nitrogen fixation in a shallow equatorial lake (Lake George,Uganda)." Freshwater Biol.5,13-39.-' Gunnison,;). and Alexander,M.i (1975). Resistance and susceptibility of alga decompositioo et naturay n"b l microbial

communities.I.imnol .Pceanogr,20,64-70 »i Hardy,R^W..,1 , Holsten,RJD.iv Jackson,^.K Burnsd »an jC;,.R (1968). ïïhe acetylene-ethylene fixation« assaN r yfo : laboratory and field evaluation. Physiol.43. .PI „ 1185-1207. s' Hargrav£,3..T* (1970). Utilization of benthic microflora by Hyalella azteca(Amphipoda). J.Anim.Ecol.3_2,357-437.. Herbert,H.A.: (1975). Heterotrophic nitrogen fixatio shallon ni w estuarine sediments.. J.JExp.Mar.Biol.08, 215-225. Horne,A.J.. (1972).. The ecology of nitrogen fixation in Signy Island,, South Orkney Islands. Brit.Antarct.Survey Bull.27,1-18. Horne,A.-J. Fogg,G.Ed .an „ (1970).. Nitrogen fixatio somn ni e English lakes.. Proc.3îoy.Soc.J3.175, 351-366. Hörne,A.J Viner,A.3.d .an . (1971).. Nitrogen fixatios it d nan significance in Uropical lake George.. Nature 232,417-418.

Jones,K. (1974).. Nitrogen fixation in a salt marsh. J.Ecol.'62t, 553-565.. Jones,JC.: (1977). Acetylene reductio blue-greey nb n alga subn ei - tropical grassland.. New.Phytol.78, 421-426.. Jones,K.. (1972).' Nitrogen fixatio temperate th n ni e estuarine intertidal sediment Rivee th rf so Lune . Limnol.. Oceanogr. 27,: 455-460.

Jones,K. and Stev;art,:V/JD.JP». (1969b;).. Nitrogen turnover in marine and brackish habitats.iv. Uptakextracellulae th f eo r products of the nitrogen-fixing alga Calothrix scopulorum. J.Mar.3iol.Ass.DJC .49., 701-716.

77 Jones,K. and Stewart,Y/.D..P. (I969a). Nitrogen turnover in marine and brackish habitats.iii productioe .Th extracellulaf no r nitrogen by Calothrix scopulorum.49,475-488.. Jones,K. and Wilson,R.E. (1978). The fate of nitrogen fixed by a free-livin e-greeu gbl n alga»; Ecol.Bull.(Stockholm). 26,, 158-163. Iee,KJC. and Watanabe,J. (1977).. Problems of the acetylene reduction technique applied to water-saturated paddy soils.. Appl.: and Environ. Microbiol.-34_, 654-660. Lethbridge,G.., Davids on, M.S.. and Sparling,G ^P.. (1982).. Critical evaluatio acetylene th f no e reduction tesestimatinr tfo g the activity of nitrogen-fixing bacteria associated with the roots of wheat and barley. Soil Biol.Biochcm.14,27-35. Mayland,H.P.., HcIntosh,T.H. and Fuller,V/.H. (1966). Fixation of isotopic nitroge semi-arià n no d soi algay lb l crust organisms. Proc.Amer.Soc.Soil Sci.jJO, 56-60. Millbank,J..V/* (1978). T3 :-> contribution of nitrogen-fixing lichens to the nitrogen status of their environment. Ecol'.Bull. (Stockholm).:2j6, 260-265. Keess,J.:, Dugdale,R., Dußdale,V/.. and Goering,J. (1962). Nitrogen metabolis lakes,!n mi . Measuremen nitrogef o t n fixation with %2»- Limnol.Oceanogr.7, 163-169. Osa-Afiana,,L.Oe and Alexander,M.I (1981). Factors affecting prédation bmicrocrustaceaya n (Cypris sp. nitrogen-fixinn )o g blue-green algae. Soil Biol.Biochem.l3_,27-32. Patrio.uin,.DjG.. and Déni eke ,,Di (1978). In situ acetylene reduction assay nitrogenasf so e activity associated wite hth emergent halophyte Spartina alterniflora loisel"..: Methodological problems. Aqu.Botan 211-226. y4, . Ebman,M.B^ (1978). Ingestio blue-greee th f no n alga Trichodesmiuy mb the harpactacoid copepod, Macrostella gracilis. Limnol. Oceanogr.23_, 1245-1248.

Porter,K.JG* (1973). Selective gra zindifferentiad gan l digestiof no alga Zooplanktony eb . Nature 244, 179-180.

78 Safferraan,R.5.. (1973). Phycoviruses. In The Biology of the Blue-Green JELgae. Eds.N.G..Car Whi214-237,., . A on t. t B d -ran Blackv/ell Scientific Publisher, Oxford. Shilo,M. (1970). Lysi blue-greef so n algamyxobactera y eb . s J.Bacteriol.l_04t 453-461. Sku;jcns,J Klubek,B.d .an . (1978). Nitrogen fixatio cyclind nan y gb blue-green algae-lichen-crusts in arid rangeland soils. Ecol.Bull.(Stockholm) 2j6, 164-171. Stev.-art,W.JD.I>.. (1963). liberation of extracellular nitrogen by two nitrogen-fixing blue-green algae.. Rature 200, 1020-1021. Stev/art,W.D.JP» (1964).. Nitrogen fixatio Myxophyceay nb e from marine environments. J.Gen.Microbiol.3J>, 415-422. Stev/art,V/.D.JP.. (1965).. Nitrogen turnove marinn ri brackisd ean h habitats.!...,Nitrogen fixation. Ann.Bot.29, 229-239. Stev/art,V/.JD.J?.. (1966). Nitrogen Fixation in Plants. 168pp. Athlone Press, London. Stewart,W.J)..P.. (I967a). Nitrogen turnover in marine and brackish habitat s.u. Use of % in measuring nitrogen fixation in the field.. Ann.Bot.3JL, 385-407. Stev/art,VJ.D.,P. (I967b). Transfer of biologically fixed nitrogen in a sand dune slack region.. Nature 214, 603-604. Stewart,W.D..P., Fitzgerald,G.J?.. and Burris,R.H* (1967). In situ

studies on N fixation using the acetylene reduction 2 technique. Proc.natn.Acad.Sci.U.S.A.58_, 2071-2078. V.erma,L Martin,J.J?d .an . (1976).. Decompositio algaf no l celld san component theid san r stabilisation through complexing with model humic acid-type phenolic polymers.. Soil Biol. Biochem.£, 85-90. Walsby,A.JE. (I974a). The extracellular products of Anabaena cylindrica Lemm.i. Isolation of a macromolecular pigment-peptide and other components. Br.Phyc.J.9_, 371-381. Walsby,A.E.. (1974b). The extracellular products of Anabaena cylindric& Lemm.ii. Fluorescent substances containing serine and

79 threonine and their role in extracellular pigment formation. Br.Phyc.J.9, 383-391.

Watanabe,A Kiyohara,Td .an . (I960). Decompositio blue-greef no n algne as effected by the action of soil bacteria. J.Gen.Appl. Microbiol.,Tokyo £, 175-179. Webbley,D.M. and Jones,D. (1971). Biological transformations of microbial residues in soil. In Soil Biochemistry,Eds. McLaren and Skujens, 446-485. Marcel Dekker,Inc. New York. Whitton,B.A'.. (1973). Interractions v.-ith other organismse Th n .I Biology of the Blue-Green Algae. Eds. N.JG.jCarr and 3.A.. Whitton, 415-433i Blackwell Scientific Publications, Oxford. Wilson,J.2.., Greene,S. and Alexander,M. (1980). Effect of microcrustaceans on blue-green algae in flooded soil. Soil Biol.Biochem.12, 237-240.

80 STUDE TH BIOLOGICA F YN O I N 1S F O E L US NITROGEN FIXATION IN PADDY SOILS AT THE INTERNATIONAL RICE RESEACH INSTITUTE

A . WT NI A ABE, P.A. ROGER* Soil Microbiology Department, The International Rice Research Institute, Los Banos, Laguna, Philippines

Abstract Nitrogen fixation studies for importann ma t researcaspece th f to h programme in the Soil Microbiology Department at the International Rice

Research Institute, particularly as dinitrogen fixation (Np-fixation) y factoke ideterminina sn ri g nitrogen suppl wetlann yi d rice soilf so developing areas. The N technique has been used - (i) to detect dinitroge incorporation_ nN fixatio e th y nb , (ii folloo )t behavioue wth r of nitrogen fixe dinitrogen-fixiny db g organism d (iiisan asseso )t e sth contribution of N -fixation by the N dilution method. Since the introduction of emission spectroscopic apparatu 1972n si volume ,th studief eo s using s increasedNha . Nitrogen fixation studies for importann ma t researce aspecth f to h progra Soin mi l Microbiology Departmen Internationae th t ta l Rice

Research Institute, particularly as dinitrogen fixation (N„-fixation) is a key factor in determining nitrogen supply in wetland rice soils of developin gtechniquN areas e detec o bees t Th . eha ) n t (i use d—

dinitrogen fixation by N? incorporation (1, 2, 3, 5, 6, 7, 10); (ii) to follow the behavior of nitrogen fixed by N_-fixing organisms (8, 9, 11, 12); and (iii) to assess the contribution of N -fixation by the 5N dilution method (10, 13). Sinc introductioe eth emissiof no n spectroscopic apparatu 1972n si ,

the volum studief eo increaseds usinha N g .

DETECTIO) 1 -FIXATION F NO INCORPORATION 5 Y NB N

Before the routine use of acetylene reduction technique, the N„

feeding techniqu onle th y s sensitivewa e assay techniqu dinitroger fo e n

fixation.

Office de la recherche scientifique et technique Outre-Mer (ORSTOM), France. 81 Soi) 11 l Samples

The effects of light and organic matter application to N -fixation

in flooded soils were firsA studielaboratore th . t 2) n triai d , (1 yl

to estimate phototrophic nitrogen fixation was conducted in 1968 using

soil from pot experiments incubated in test tubes under an atmosphere

enriched with N. The data (Table 1) shows that phototrophic NFA was

dominant in this soil and that the addition of N fertilizer remarkably

depressed the amount of nitrogen fixed. In soils exposed to the light,

without nitrogen fertilizer, NFA corresponding to 30 kg N.ha .month

estimates wa d (IRRI, 1968). However bees ha n t ,reportei d that small

scale experiments favo growte rth blue-greef ho n algae (Roged ran

Kulasooriya, largely 1980ma d )yan overestimate photodependent N_-fixation. The effect of various fertilizers on N_-fixation can be seen in Table 2. Inhibition was observed on both phototrophic and heterotrophi -fixatiocN n with almost complete inhibitio 0 ppr. 16 N a t na

Luxuriant growth of algae occurred in pots receiving ammonium N but the

amount of fixed N was not appreciable. This indicated that besides

an inhibitory effect on nitrogenase activity, a stimulation of the

growth of non-fixing algae by mineral nitrogen can limit N -fixing

blue-green algae growt competitioy hb antagonistid nan c effects (IRRI,

1968).

Comparisio) 12 n with acetylene reduction method

The relation between acetylene reduction . activitN d yan (3) uptake activit examines ywa soir fo dl sample . s Witisotope hth e

method, nitrogen fixatio 2.61s nwa , 2.5 2.5d 4an 9 yg/10 g/day after

incubating the soil under 0.2 atm 0 + 0.25 atm N for 1, 3, and 5 days,

respectively. Wit acetylene hth e reduction method, evaluated nitrogeu

fixatio 2.6s nwa 4 yg/day afte incubatioh r6 n (3:1 conversion ratio). methodo tw e sTh gave similar results despit difference eth incubatiof eo n period (3).

82 A similar comparison was made with Azolla. Azolla pinnata fixed 0.65 ymol N_/hr/g fresh weight for a 24 h exposure to N under

lighx kl t0 1 intensity a . Wit acetylene hth e reduction method, activity was 2.18 iimol C„H /hr/g fresh weight. Therefore, the ratio of C„H

reduced to N„ fixed was 3.3 (close to theoretical value) (0. Ito,

unpublished) .

13) N--fixation associated with rice

131) Dinitrogen fixation associated with wetland rics ewa

confirmed by exposing rice plant to an atmosphere containing N? gas shows wa fo ndayt r7 I tha s. t) (Tabl b bot , he3a roobasad tan l part of shoot are sites for N.-fixation. N -fixing activity measured by

N« incorporation was close to the values estimated by acetylene reduction assays (7).

132) Dinitrogen fixation associated with deepwater rice. It was observed that submerged parts of deepwater rice are colonized by blue-green algae. Photodependent acetylene reduction activities were

found to be associated with submerged roots, leaf sheaths, and to a lesser extent; culms confiro T . m N.-fixation associated with deepwater rice and to study the availability of the fixed nitrogen to the plant,

submergerged part deepwatef so of deepwater ric headint ea r ricge stagat headine wereg exposestageo t d wer e exposed to N- for 9 days and harvested after grain maturation (Table 4) . It s founwa d thasubmergee th t d root lead san f sheaths colonize bluey db - green alga highed e ha enrichmenN r t thaothee nth r plants e partth f so . plane th s recovere tn wa i N Approximatel de froaeriath e f mth o l% y40

parts which wer s estimatedirectlt ewa no t I yd. exposeN- tha to t d werN g em fixe8 planr dpe periody t da ove9 .ra This activits i y much higher than that reporte heterotrophir fo d c N„-fixation associated

with wetland rice. These results demonstrate that photodependent N - fixatio associates ni d with deepwater rice nitrogee ,th n fixed being utilized directl rice th e y planyb t (10) ,11 83 AVAILABILIT) 2 FATD BIOLOGICALLYF AN E O Y FIXED NITROGEN

welw no ls Ii testablishe d that N„-fixatio Azollay nb , blue-green alga heterotrophid ean c bacteria play vitasa soile th roll n ei

fertility and studies were undertaken to establish how much fixed nitroge availabls ni rice th e o eplantt .

21) Azolla The availability to the rice plant of Azolla N was examined in pots and in field experiments using A., pinna t a labeled with N-nitrate. The Azolla plants were either floated on the water, or incorporated in the soil. Over a 17 week period the rice plant in pot took up 53% of N from the incorporated Azolla and only 10% from the floating Azolla. When Azoll soiplacee s ath wa l n surfacdo e highest loss, amounting 60% for 6 weeks were observed; losses were 50% when it was floated on

the wate onld whe% ran y33 n incorporated fiele labeleN th dn I . d Azolla (41 kg N/ha) was added at 30 days after transplanting. The first rice crop was absorbed 26% and 13% of N from incorporated and floating Azolla, respectively. The availability to the second crop was only 5%

in both cases (9). As a consequence of multiplication of floating

Azolla the N content was diluted and the availability of N was much

lower than the availability of Azolla N per se.

22) Blue-green algae Ipreliminarna y experiment Gloeotrichi grows a wa n n. i asp

small pond with N-nitrate added. Fresh algal material (1.1% N in 2 dry matter, 8.89 ato excessm% incorporates )wa m plotl a . n di

Recovery of N in the grain, straw and root of the first crop was applieN e algas a dth f seconle o 14.7 massth % n dI .crop e ,th

recover onls ywa y 2.25%. Althoug remaininN h t no soi n gs i lwa

analyzed, this preliminary experiment suggeste availabilitw lo a d y of algal N to rice crop. Because the algal mass used in this

84 experiment had an unusual low N content and a high C/N ratio the results were of limited value (12).

Iseconna d experiment s growNosto. ,wa A . n sp cunde r laboratory

conditions, labeled with N-Nharvesteds wa O, kepd ,an t drier o d

fresh (7.3weighy dr %, tN basis). UptakfroN m f eo thi s material

b yd fiel ric an s studiedt ewa po experiment y db s (12, 13). Availability

of N from dried EGA incorporated in the soil was 23-28% for the first

crop and 27-36% for two consecutive crops (Table 5). Surface application

of algal material reduce availabilitN d firse 14-23o th t y tr %crofo p

and 21-27% for two crops. The availability of algal N was less than

that of ammonium sulfate, but for two crops its availability was very similar. Afte cropo tw r s mor eremaineN soin i d l from algae than from

ammonium sulfate (11).

Availability of N from the incorporated fresh algae was higher

than that from dried algae. It can be concluded that blue-green algal

N is less susceptible to N losses than inorganic fertilizers, its low

C/N ratio imparts a better N availability than such organic fertilizers

as farmyard manure.

Heterotrophi) 23 c bacteria

Experiments were designed to study the availability to the rice planfixeN f to d heterotrophicall paddn yi subsequens y it soid lan t transformations in soil. Soil samples enriched with glucose were incubated under N«. N-ammonium sulfate was added in other samples to compare transformation nitrogef so n derived from heterotrophic dinitrogen fixatio immobilized nan d ammonium (8). Results showed that biologically fixed-N undergoes similar transformation thao st f o t immobilized ammoniu mN (8) . Afte day2 r4 s rice plant absorbed sha d 34% of the fixed-N and 8% of the soil-N. This result showed that heterotrophically fixe remainedN readils a d y decomposabl. eN

85 DILUTION E TH N ) TECHNIQU3 E

31) Experiments with deepwater rice

A N dilution experiment was conducted simultaneously with the

N„ feeding experimen deepwaten to r rice described above. Deepwater

ric grows e wa pot n i n s wit labeleN h d ammonium sulfate (60 N/pot)g 0m .

After harvest, plants were split into various portions, as in the

feeding experimentcontenN e th t determinedd ,an contentN . f so various portions of the plants in N. feeding experiments were

negatively correlated to N contents of corresponding portions of the

plants grown in pots with N-ammonium sulfate (r = - 0.73) (Fig. 1),

This negative correlation suggests thadilutionN t , whic founs hwa d

to be more intense in submerged roots and leaf sheaths than other parts, photodependeno t e du s wa t nitrogen fixatio thesn no e parts (10).

Submerged root lead san f sheath deepwatef so r rice covered with black cloth

had highe contentN r s (less dilution) thasame nth e parts exposed

to light. This suggests that photodependent N -fixation diluted N

in the tissues of deepwater rice.

Rice was grown in deep and shallow water conditions, in the

Philippine Thailand san d (10). Rice grow deepwaten ni r condition

showe loweda contentN r , suggesting that nitroge watere th n ,ni

occurrin combines ga dinitrogen, dN bothr ,o , contributeo st

nitrogen nutritio deepwatef no r rice (Tabl. e6)

Experiments on deepwater rice suggests the potential of the

N dilution technique in identifying active sites of dinitrogen

fixation, providing that control experiments are carried out to

prove that the observed difference in N contents is due to solely

N -fixation.

Relationship) 32 s betwee dilutiobalancnN N d ean n

Nitrogen balance studies by total nitrogen analysis provide

86 approximation a nitroget ne f no nsysteme inputh n i t. Althougo hn

sophisticated technique incorporatio„ N s sa acetylenr no e reduction

requirede ar , this metho labos i d r intensiv timd ean e consuming. Principles of isotope dilution may be applied to assess the amount of

plant nitrogen derived from dinitrogen by comparison of a N -fixing

system fixing^bun witno ha t otherwise identical^system.

N-ammonium sulfat sucrosd ean e were adde moiso t d t soin i l

orde labeo t rnitrogee lth n fractio biomasse th f soie no s Th .lwa

then air-dried, remoistened and planted with two consecutive crops of

dryland ric wetlanr eo d rice (14). Tota lbalanccontentN N d ean s were measured in grains, straws and roots (Table 7). Dryland rice

had a lower positive nitrogen balance and a much higher N contents

than wetland rice. When pot surfaces were covered with black cloth, to suppress photodependen -fixationtN statisticallo ,n y significant

nitrogen gain was observed. N content of plants was higher than when photodependent N„-fixation by blue-green algae or Azolla was

allowe negativa d dan e correlation between nitrogen balancN d ean content in the plant was observed (Fig. 2). Therefore, it is

reasonabl assumo et e thalowee th N tcontent r ricn si e grown ni nitrogey b dilutioe unshadeN th o t nf o ne derive ddu pot s sdwa

from photodependent (free-livin symbioticd gan ) dinitrogen fixation. When wetland rice growing in pots with suppression of photodependent

N -fixation was taken as a control system, the calculated contribution

of nitrogen derived from photodependent N„-fixatio nitrogeo nt n ni the plant was 20-30%.

-FIXATION N I N 15 NFUTUR) F 4 O STUDIE E EUS S Undoubtedly N„-feeding experiments are necessary for direct evidenc N„-fixationf eo . expensivo Unfortunatelyto s i s ega _ N ,

for general field application. Growing one rice plant in an N.

atmosphere costs several thousand US dollars! 87 N dilution experiments are quite suitable for application to

wider aspects of dinitrogen fixation studies. Providing proper

control of the non N?-fixing system is selected, the N dilution technique could be useful in evaluating the effect of inoculation

by blue-green algae, Azolla or bacteria, identifying the sites of

N_-fixation and screening highly active N -fixing systems (blue-green algae, Âzolla, or rice in association with bacteria).

REFERENCES

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nitrogen fixatio photosynthetiy nb c microorganism submergea n si d

Philippine soil, Soil Sei. Plant Nutr., 19:117-123.

Ancajas. R ) . Yoshida3 R d . an 1973. ,T . Nitrogen fixing activitn yi

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4) Yoshida, T. and F. E. Broadbent. 1975. Movement of atmospheric

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Barraquio. L ) Watanabe5 . W d an .. ,I 1979 levelw Lo . fixef so d nitrogen

required for isolation of free-living ^-fixing organisms from rice roots, Nature 277:565-566.

Watanabe. I d ) Durbin6 an . .J . ,1980K . Sulfate reducing bacteria

and nitrogen fixatio flooden ni d rice soil, Soil Biol. Biochem.,

12:11-14.

Cabrera. ) D Ito7 Watanabe . I , ,d 0. ,an . 1980. Fixatio dinitrogen-1f no 5

associated with rice plants, Appl. Environ. Microbiol., 39:554-558.

88 8) Ito, 0. and I. Watanabe. 1981. Immobilization, mineralization and availability to rice plants of nitrogen derived from heterotrophic nitrogen fixation in flooded soil, Soil Sei. Plant Nutr., 27:169-176.

9) Watanabe, I., Bai K. Z., N. S. Berja, C. R. Espinas, 0. Ito, and

B.P.R. Subudhi. 1981 Azolla-Anabaene Th . n i e aus comples it d xan

rice culture, IRRI Res. Paper Series #69.

10) Watanabe, I., W. Ventura, W. Cholitkul, P. A. Roger, and S. A.

Kulasooriya. Potentia biologicaf lo l nitrogen fixation ni deepwater rice. Proceedin Internationaf go l Deepwater Workshop,

Nov. 1981, Bangkok (in press at IRRI).

) WatanabeVenturao 11 W d an .. , I 1982. Nitrogen fixatio bluey nb - green algae associated with deepwater rice. Curr. Sei. 51:462-464.

12) Tirol, A. C., P. A. Roger, and I. Watanabe. Fate of nitrogen fro bluma e green alga flooden ei d rice soil. Soil Sei. Plant Nutr press)n (i . .

13) Roger, P. A. and I. Watanabe. 1982. Research on algae, blue-green alga phototrophid ean c nitrogen fixatio IRRt na I (1961-1981),

summarization, problems and prospects. IRRI Res. Paper Series #78.

Watanabe) . VenturaI 14 d an . ,.W Relationship between nitrogen balance

dilutioN and ricn ni e soil system (submitted).

Kulasooriya. A . S d an ) Roger. 15 .A . 1980,P . Blue green algad ean

rice. The International Rice Research Institute, Los Banos,

Laguna, Philippines. 112 p.

89 Table 1. Nitrogen fixation under various soil treatments (IRRI, Annual Report for 1968).

Incubated darn i k Pretreatment* Incubated n lighi t Estimated amount 15** Estimated amount of fixed N Atom % excess N15** of fixed N Ato excesm% sN /month(ka g/h ) /monta /h g h(k plano N PK tLt .395 32.0 .038 3.2 PKD tt No plant .493 32.8 .020 1.2 PKS L1R .459 32.6 .026 1.9 PKD IR8 .514 36.0 .006 0.5 PKL Peta .517 38.1 .047 3.4 PetD PK a .410 27.2 .027 1.8 NPK plano LN t .043 5.7 .048 4.6 NPKD No plant .020 2.0 .000 0.0 NPKL IR8 .075 6.4 .017 1.5 NPKD 1RS .070 5.1 .024 1.8 NPKL Peta .151 13.3 .031 2.5 NPKD Peta .089 5.6 .000 0.0 *Treatment of Maahas clay soil in greenhouse pots from which soil samples were obtained 2 months after transplanting IR8 and Peta. ** Determined after incubatio soif n o glas n li sgreenhousee monttube1 th r n hsi fo . Ligh- L t (good growt indigenouf ho s algae). "D - Dark (no algal growth observed). Tabl . eEffec2 ammoniuf to mfertilizeN nitrogen ro n fixation (IRRI, Annual Repor 1968)r fo t .

„ , , _ Ato excesm% sN Level of N Fertilizer application Incubation Incubation (ppm) in light in dark

No fertilizer 0 0.150 0.100

Ammonium sulfate 80 0.042 0.036

160 0.009 0.000

Ammonium chloride 80 0.005 0.015 160 0.000 0.000

0 8 0.024 urea 0.043

0 16 0.000 0.000

91 N)

Table 3a. 15„ incorporation into rice plant (IR26 growta n )i h chambe days7 r .fo r

Apparen „ fixatiotN n Sampl. eno ) (g Drt yw % of N N content Amount of N (yg) rate/ (ymoN f lo (atom % excess) day)b 2

Root 14.2 0.495 0.714 503 5.40 Outer leaf sheath 5.14 0.569 1.57 461 4.95 Inner leaf sheath 17.3 1.03 0.052 92.0 0.98 Leaf blade 12.2 1.77 0.003 6.51 0.069 Young panicle 1.08 2.63 0/005 1.42 0.015

Root 10.89 0.554 0.818 493 5.29 Outer leaf sheath 3.20 0.510 2.57 420 4.51 Inner leaf sheath 21.37 0.922 0.091 179 1.92 Leaf blade 12.80 1.60 0.001 2.40 0.026 Young panicle 1.98 1.94 0.002 0.769 0.008

Basal node includede sar . 44.3%s wa s .ga Ato„ N exces% m f so . incorporatioN Tabl . e3b n into15 .rice plant growta n si h chambe days7 r .fo r

Apparent N. fixation Plant part Dry wt (g)N f o % N content Amount of N fixed rate (pmo N./f lo 1 (atom % excess) (yg) day)3

Uncovered

Root 2.33 0.765 0.501 89.3 1.77 Outer leaf sheath 2.29 0.727 0.947 158.0 3.11 Basal node 1.66 0.515 0.245 20.9 0.413 Inner leaf sheath 9.34 1.06 0.028 27.6 0.545 Leaf blade 4.02 1.97 0.031 24.6 0.486 Young panicle 3.58 1.16 0.017 7.07 0.139 Covered

Root 2.25 0.769 0.424 73.3 1.45 Outer leaf sheath 3.22 0.544 1.86 324.0 6.40 Basal node 1.57 0.549 0.385 32.2 0.656 Inner leaf sheath 9.25 0.929 0.059 50.7 1.00 Leaf blade 5.31 1.51 0.040 32.1 0.034 Young panicle 2.72 1.33 0.039 14.1 0.279

Atom % excess of N_ gas was 24.2. Ito et al (7).

vo Table 4. Nj-fixation by deepwater rice exposed to N„ for 9 days, 1980 wet season, IRRI.

Plant parts Total N Atom % Fixed N mg N/pot excess ug N/pota

Aerial parts

Grain 184 + 33 0.04 + 0.02 149

Leaf blade 75 + 17 1.77 + 0.88 2780

Leaf sheath 28 + 3 0.30 + 0.21 208

Culm 26 + 4 0.11 + 0.07 57

Floating parts

Leaf sheath 36 + 3 3.12 + 1.14 2310

Culm 27 + 1 0.48 + 0.15 264

Root 6 + 3 2.01 + 0.77 267

Submerged parts

Leaf sheath 29 + 3 1.37 + 0.46 831 Culm 28 + 4 0.27 + 0.07 153

Root 35 + 3 0.66 + 0.28 625

Root in soil 68 + 6 0.23 + 0.11 329

Whole plant 542 + 51 7973

Submerged weed 41 + 12 0.97 + 0.48 823

aAssuming the average of 48.1 atom % excess of N, during 9 days exposure. Watanabe et al (1980).

94 algan i Tabl ) e. (% e5 Recoverafte N cropo tw r ric f f s o yo soin plantei d lan s (IRRI Annual Report for 1981).

EGA incorporated Surface applied Experimental 1st d crocro2n pp Soil unaccounted 1st crop 2nd crop Soil Unaccounted

Fresh algae pot experiment 38 5 53 4 13 8 73 6

Dried algae pot experiment 28 8.5 58 7 5 5.5 7 14 22

Dried algae field experimen* 33 t 5 3. 23.5 * 32 4 23

0-1 e soile th layem 5 th c n .I f ro Roge Watanabd ran e (12).

in 15 14 Table 6. N/ N ratio in deep water rice plants growing under

shallow and deep water conditions (IRRI, Annual report for 1980).

15 15 14 Water Li ht Total N N N/ N ratio Site ** a/ mg/pot mg/pot B/A x 100 depth exposure* 6 — A B

Bangkhen Shallow + 718 111 15.4

Hantra Semi- deep + 979 86.2 8.8

Bangkok Deep + 952 97.6 10.2

IRRI Shallow + 502 194 38.6 Shallow 504 230 45.6

Deep + 737 201 27.2

Deep 530 114 21.5

— + : covered with sack cloth - : not covered with black cloth.

96 Tabl . e7 Balanc labelef eo unlabeled dan d nitroge contenN grainsn d i ntan , straw rootd san f so rice.

Balancf eo nitrogen (mg/pot) Atom % excess of N Treatment in plant* 15N* 14N

Dryland , unplanted - 58 125 -

Dryland, planted - 34 216 7.14 Flooded, unplanted - 49 - 658 - Flooded, planted - 27 814 3.93 Flooded, black cloth - 42 4ns 4.35 Flooded, Azolla + P ~ 41 938 3.62

Flooded, algae + P + Ca - 28 1013 3.80 Flooded , black cloth + P + Ca - 35 145ns 4.08

Standard error 10 115 0.07

15 Initial amount of N was 196 mg. Atom % excess of ammonium sulphate was 31.06.

**E Pot surface was covered by black cloth (Ventura and Watanabe, in press).

97 Ato «xcesm% s feedin12 5N g 4

I _L I 456 Atom % excess 15 N dilution

Fig. 1. Relationship between 15.N contents of various parts of deepwater rice, determined by N feeding and N dilution techniques.

Nitrogen botanc N/potg e(m )

1000

500

100

,T 4.5 Ato excesm% plann si t

Fig. 2. Relationship between nitrogen balance in flooded soil-rice ecosyste contenN d ricn i tman e plants.

98 BLUE-GREEN ALGA RICEN I E FIELDS Their ecology inoculant theiras and use

P.A. ROGER* Soil Microbiology Department, The International Rice Research Institute, Bafloss Lo , Laguna, Philippines

Abstract

This paper is a short review on blue-green algae in rice fields, their ecology and their use as inoculants. Some emphasis has been given to the recent studies of the relations between blue-green algae and rice which include the availability of algal nitrogen to the rice plant and epiphytic relationships.

1 INTRODUCTION

One fertilizee resulth f to r price increase durin lase th gt decad renewea s i e d interes biologican i t _ fixatiolN meana s f a nso fertilizerN f o reducine us e . th g However, biologica lN fixation requires energy generally obtained by the catabolism of photosynthetically fixed carbon (photosynthate). Among the N--fixing microorganisms, only blue-green algae (BGAable o ar )et generate their own photosynthate from CO and water. This trophic independence make especiallA sBG y attractiv biofertilizera s ea e Th . agronomic potential of BGA was recognized in 1939 by De, who attribute naturae th d l fertilit f tropicayo l paddy fieldo st N -fixing BGA. Since then, many trials have been conducted which attempted to increase rice yield by algal inoculation or by cultural practices favourin growte th g f indigenouho s BGA. The paddy field ecosystem provides a favourable environment for the growth of BGA with respect to their requirements for light, water, high temperature and nutrient availability. This could be the reason A gro higheBG n wi r abundanc paddn i e y soils tha uplann i n d soils (Watanab Yamamotod ean , 1971 reportes a widel)e th n yi d different climatic condition Indif o s a (Mitra, 1951 Japad )an n (Okudd aan Yamaguchi, 1952) pooliny B . g data obtaine Senegan i d appeart li s that

Offic rechercha l e ed e scientifiqu t techniquee e Outre-Mer (ORSTOM), France.

99 N -fixing BGA were recorded in 86 out of 89 paddy soils (Reynaud, 1980). However, Venkataraman (1975) pointe that ou dt "contraro t y general belief, N -fixing BGA are not invariably present in tropical rice soils, and that an all India survey showed that out of 2213 soil samples from rice fields, only about 33% harboured N -fixing forms" e heterogenouTh . d sometimesan s limited distributio- in f o n stils fixin i welt A lno l BG g understood becaus o systematien c analysis has correlate presence th d r absencwiteA o BG h f eo environmenta l factors (Lowendorf, 1980).

2 METHODOLOGICAL LIMITATIONS FOR THE STUDY OF BGA IN RICE FIELDS

Quantitativ1 2. e evaluations

The lack of completely satisfactory methods for estimating algal abundance and biomasses of the different algal groups (Fogg et al., 1973) is certainly a limiting factor for ecological studies with BGA. Plating techniques mose , th whict e frequentlar h y used methods, are advantageous in providing qualitative and quantitative results simultaneously. However accurace count,e th th f syo e depenth n do reliability of the particular dilution method. Filamentous forms are difficult to separate into individual cells. Plating techniques can be improve determininy b d meae th gn volum eacf eo h "count unit" (cell, filament, akinet colonyr eo , accordin e speciesth o t g directlyy )b , microscopically examining the first dilution and multiplying the results of enumerations by the corresponding "volume unit". This permits the expression of the results of enumerations in term of biomasses (Roger and Reynaud, 1976). Algal enumeration oftee sar n limite n inadequata y db e methodology in sampling. resulte Mosth expressee f to sar numbes a d f algaro r epe gram of dry soil which does not take into account algae in the floodwater of submerged soils and does not permit any extrapolation at the field level (What is the dry weight of soil colonized by algae in one hectar padda f eo y field?) expreso t s betteA i . sy rwa 2 enumeration usiny b numbes a s g m c algaf corr o r epe samples wita h well defined diameter, each core sample including the first centimetre e correspondin th f soio d lan g floodwater e soi th submerges i lf ,i d (Roge Reynaudd ran , 1976). This will p.emit comparisond san extrapolation fiele datth e dt th a leve f correca so f i l t densitf yo samplin bees gha n used.

100 The problem of the choice of a density of sampling in relation to distribitionae th l ecolog f algay o beeß eha n studie Rogey b dd ran Reynaud (1978). Results indicate that soil algae have log-normal distribution (logarithm numbef so normalle ar r y distributed thad )an t vera y high densit f samplinyo requires i g obtaio t d significana n t evaluation exampler Fo . meae ,th n valu Anabeanf eo biomassep as s based upo 0 sample 4 ncore0 1 f so each, take0.2a n i n5padda h y field, had a confidence interval of +32% and -27% of the mean. Such an evaluation clearly demonstrates tha composita t e sample obtainey b d mixing a large number of core subsamples is at least required to enumerate algae; of course replicate measurements on composite samples will give better results. Currently, result enumerationA BG f so o to soiln i se sar fragmentary and the methodology used too frequently open to criticism, to allo developmene wth generaf o t l concepts.

2.2 Nitrogen fixing activity.

N -fixation by BGA has been most frequently studied using the acetylene reducing activity method which may lead to erroneous results (Lowendorf, 1980). ARA variations during both the day and the growing rapie b cycld important n an d eca ; moreove log-normaa alsA s rÄR o ha l distribution (Roge al.t re , 1977). Therefore, large numbef ro replicates and very frequent measurements are needed to ensure a satisfactory measur totaf eo l ARA. However, this tedious work will stil ln imprecisa lea o t d e evaluatio nitrogee th f o n n fixing activity (NFA) as the conversion factor acetylene-nitrogen is not constant and needs to be determined experimentally for each set of experimental conditions (Peterso Burrisd vera nan s yi ,convenienA 1976)AR t Bu . t and reliable method for qualitative studies when the measurements are brief (David and Fay, 1977), when the problems of gas diffusion and greenhouse effect minimizee ar s whed an dn statistically valid sampling method adoptee sar d (Roge Kulasooriyad ran , 1980). Few reliable estimations of ARA have been hitherto published. The number of measurements and replicates have been generally too low. Moreove importance rth anaerobif eo c non-heterocystou- sN. fixing BGA was not appreciated until recently and field measurements nitrogenasf o e activity were carrie t unde ou daerobin ra phass ga c e only, therefore it is difficult to evaluate the N-input due to N -fixatio (StewartA BG y b n , 1978).

101 3 ECOLOG BLUE-GREEF YO N ALGAE

paddn I y field algad an s lA growt successionBG f ho governee ar s d by climatic, physiochemica biotid lan c factors.

Climati1 3. c factors

Among climatic factors, light intensit certainls i y mose th yt important. Light availability for soil algae depends upon the season and latitude plane ,th t canopy verticae ,th l locatio algae th n ef i n o photie th turbidite c th watere zon d th ean f .o y Light intensity reachin soiexcessiv e o vart y th g lma w y lo froo e mto o level t 0 (1 s 110,000 lux). In cultivated soils the screening effect of a growing crop canopy can cause a rapid decrease in the light reaching the algae. Thus the canopy of transplanted rice decreased light by 50% when plants wer day5 e1 s old % % afte ,mont85 95 o e d rtw on an h months (Kurasawa, 1956). e generallar A BG y sensitiv higo t e h light intensities. They develop various protective mechanisms like vertical migratione th n i s wate f submergero d soils; preferential growt morn i h e shaded zones like enbankements, unde r insidro e decaying plant materialw fe a r ,o millimeters belo e soiwth l surface; photophobotaxis; photokinesisd ;an stratificatio e strainth f algan o ni s l mats, where N.-fixing strains grow under a layer of eukaryotic algae, more resistant to high light intensities (Roger and Reynaud, 1982). In areas with high incident light intensities, BGA develop later in the crop cycle when the plant cover is dense enough to protect them from excessive light (Roge Reynaudd ran ,othe e 1977)th rn O .hand , light deficiency may also be a limiting factor. In Japan, available light under the canopy was lower than the compensation point of the phytoplankton during the second part of the growth cycle (Ichimura, 1954). In the Philippines, during the wet season, when light was moderate s highewa barA n i r,ÄR e soil tha planten i n d soil (Watanabe et al., 1977). The optimal temperature for BGA is about 30-35 C, Temperature is rarel limitina y paddn i g A yfacto BG field r rfo s becaus range eth e of temperature permitting their growt larges i h r than that requirey b d rice; however, temperature influence compositioe th s algae th lf no productivitye biomasth d an s w temperatureLo . s decrease productivity

102 and favour eukaryotic algae. High temperatures favour BGA and increas algae th e l productivity (Roge Kulasooriyad ran , 1980).

3.2 Soil properties

Among the soil properties, pH is the most important factor determining the algal flora composition. Under natural conditions BGA grow preferentially in environments that are neutral to alkaline. This explains why, positive correlations occur in the rice fields between sporeA numbeA BG BG d :sois- d an rsoi- wateH an lp H lp H rp and the N-fixing algal biomass in samples homogenous for stage of rice development, fertilization and plant cover density (Roger and Reynaud, 1982) beneficiae Th . l influenc growtA BG s n i highf o eo H hp demonstrated by the fact that the addition of lime increases BGA growt «„-fixatiod an h n (Roge Kulasooriyad ran , 1980). Howevere ,th presenc certaif eo soiln i strain A s BG wit f valueH so hp s between5 bees ha ann 6 dreporte d (Durrel, 1964; Aiyer, 1965). Besides pH, phosphorus availability is an important factor determining growt BGAf ho . Okud Yamaguchd aan i (1952) incubate7 d11 submerged soil noted an s d tha growtA tBG s closelhwa y relatee th o dt availabl conteneP soile th .f to e growt -fixinN Th ricn i f ho eA fieldBG g moss i s t commonly limited by low pH and P deficient, and application of P together with lim frequentls eha y produced positive results (Roge Kulasooriyad ran , 1980).

3.3 Biotic factors

Organisms that limit BGA growth are: pathogens, antagonistic organism grazersd san thesef O . , only grazers have been documented. The developmen Zooplanktof to n populations, especially cladocerans, copepods, ostracods, and mosquito larvae prevented the establishment of algal blooms within one or two weeks (Venkataraman, 1961). Grazing rates and algal diet preferences of ostracods were studied by Grant and Alexander (1981) who estimated the potential consumption of BGA at _2 an average field density of 10,000 ostracods m to be about 120 kg (fw) ha day . An economical alternative for controlling ostracod populations is the application of crushed seeds of the neem tree (Azadirachta indica) (Gran al.t te , 1982). Snails form another

103 grou f algapo l grazer submergen i s d paddy fields biomase ,th f so snails can be as high as 1.6 t/ha in certain rice fields in the Philippines (Roger and Kulasooriya, 1980). Commercial pesticides capabl controllinf eo g grazer expensive sar thud an es uneconomicao lt use (Grant et al.. 1982).

4 RELATIONS BETWEEN BLUE-GREEN ALGAE AND THE RICE PLANT

4.1 Availability of algal nitrogen to rice

uptake Th ricy eb nitrogef eo s demonstratewa nA fixeBG a y db n do qualitative basis by Renaut et al. (1975) and Venkataraman (1977), using N tracer technique. In a quantitative experiment Wilson et al (1980) recovered from a rice crop 37% of the nitrogen from N-labelled Aulosira sp spread on the soil and 51% of the nitrogen from the same material incorporated into the soil. This study was conducted on a laborataory scale and did not include analysis of N remaining in the soil. d fielan dt experimentPo s conducte Internationae th t a d l Rice Research Institute, using N labelled Nostoc sp, showed that availabilit f nitrogeo y n from drie A incorporateBG d h soit s n lwa i d 2 e th r firse fo th % tr 36 crofo betweed d % pan an 28 betwee7 d n2 an 3 n2 crops. Surface application of the algal material reduced the availability to 14-23% for the first crop and 21-27% for 2 crops (Tiro al.t le , 1982). Availabilit nitrogef o y n from fresh algal material was similar to that of dried material when surface applied (14%muct bu )h higher (38%) when incorporated (Roge Watanabed ran , 1982). The pot experiment was demonstrated that for the first crop algal nitroge s lesnwa s available than ammoniuo tw m r sulfatfo t ebu crop avaibilits it s vers ywa y simila thao t rf ammoniu to m sulfate (Tirol et al., 1982). That indicates the slow release nature of BGA nitrogen, which agrees with the cumulative effects of algal inoculation (Roge Kulasooriyar& balanc,N 1980) e plantn i eTh . s and soil after two crops (pot experiment, dried algae) showed that losses froanmoniuN m m sulfate were more than twice than froA mBG regardles mode applicationf th eo f so . From these result authore sth s concluded that, due to its organic nature, BGA material is less susceptible to nitrogen losses than inorganic fertilizer and that its N ratiC/ ow lo (5-7) givebettea t i s r nitrogen availability than those

104 of an organic fertilizer like farmyard manure. Relative availability f algao l nitroge rico nt e depende susceptibilitth n so o t y decomposition of the algal materai which varies with the strains (Gunnison and Alexander, 1975) but also with their physiological state as demonstrate discrepance th y db y betwee valuee th n s reportey db Wilson et al. (1980) and those reported by Tirol et al. (1982). The former authors use algan a d l material collected directly froe mth flask culture and blended after resuspension indistilled water, wherea lattee sth r authors use algan a d l material drie root a d m temperature, comprising mainly vegetative cells in dormancy and akinetes, and therefore less susceptible to decomposition.

4.2 Growth promoting effect

Besides increasing N fertility, BGA have been assumed to benefit higher plants by producing growth-promoting substances. This hypothesi e additiv bases th i s n o d e effectA inoculatioBG f so e th n i n presenc nitrogenouf eo s fertilizers. Mos thesf to e results have been obtained with ric similat ebu r results were observed also with vegatables such as radishes and tomatoes (Roger et al., 1979). More direct evidenc hormonaf eo l effect coms sha e primarily from treatment f ricso e seedlings with algal culture their so r extracts. Presoaking rice seeds wit cultureA hBG extractr so s enhances germination, promotes the growth of roots and shoots, and increases weighe th proteid an t n contengraie th nf t o (Roge d Kulasooriyaran , alss 1980)ha o t beeI . n established that algal growth-promoting substances are beneficial to other crops besides rice and that the productio f sucno h substance t confineno s i sBGAo t d . Whether these substances are hormones, vitamins, amino-acids or any other components is still unknown.

4.3 Epiphytism

Epiphyti havA BG ce been observe wetlann do d rice (Roge al.t re , 1981), deepwater rice (Kulasooriy al.t ae , 1981, Martine Catlingd zan , 1982), and on weeds growing in rice fields (Kulasooriya et al., 1981). Comparing these three different hosts (Kulasooriy al.t ae , s founwa 1980t di )tha t epiphytis associatee th wetlann d o an mA dAR d ric seedlingt a e , tillerin headind an g e submergeth g n stageo d dan s weed Char s predominantlawa colonieo t e ydu Gloeotrichif so p as

105 visible to the naked eye. The epiphytic algae on wetland rice at maturity e submergeth n ,o d weed Najasdeepwaten o d ,an r rice coule db observed only under the microscope and the dominant algae were Nostoc. Calothrix. and Anabaena. A unique finding was that B6A also exist •insid cavitiee th e f senescenso t rice leaf sheaths; this "endophytism", however, in addition to being not confined to rice, was not presen livinn i t g healthy tissues A frequen. t observatios nwa that older hoste partplantd th an sf so s with rough surfaces supported more numerous epiphytic BGA. From these facts, it was concluded that epiphytism is possibly related to an abiotic effect, of which a mechanical effect in relation to the roughness of the host surface f importanceo appear e b o t s . Rate AHAf so wetlann .o d rice gradually diminished from seedling to maturity mainly due to the concomitant decrease of Gloeotrichia epiphytis reductioe th d an mf availabl no e light deepwaten I . r rice also there was a decrease in specific ARA (activity per gram of host) from headin maturito t g t thi s compensateybu s wa n increasa y db n i e the host biomass so that a constant activity per plant was observed at both stages e resultmeasurementA Th .AR f so s indicated N thae th t contribution by N -fixing microorganisms epiphytic on wetland rice epihpytit bu w iA plalo s BG n cimportana y t rol inoculun i e m conservation because floating alga d soian el alga frequentle ear y washe t frofiele dou mth d during heavy othe e rainsth rn O hand. , epiphytic nitrogen fixation on deepwater rice makes a substantial N contributio thio t n s ecosystem (10-2 N/hag 0k e th ) mainlo t e ydu greater biomass available for colonization by epiphytic BGA. The importance of epiphytic N -fixation and the availability of epiphiytically evaluates fixewa N d Uatanaby db . (1981 al Watanabd t )e an Venturd ean a (1982) usin techniquesN g fiela n I d. experiment, ric s growewa n in pots containing N labelled ammonium sulfate, in shallow and deep (110 cm) water in the Philippines and Thailand. Rice plants in deepwater had lower N enrichment suggesting that nitrogen in floodwater as molecular nitrogen or combined or both, contributes to nutrition of deepwater rice (Watanabe et al., 1981). Direct evidence of N.-fixation associated with deepwater rice was obtained by exposing submerged parts of a plant to 15N_ for 9 days. The highest enrichment werN e f sfouno submergen i d 15 d nodal rootd san leaf sheaths wher groA eBG w epiphytically. Durinday9 e s th gperio 8 d maturityt a mg-plane s fixed th N wa fixee tan y db th ,dN f o abou % t40

106 s foun plante wa partn th i ddirectlt f sno o y _ exposeN o t d (Watanab Venturad ean , 1982). In shallow water-rice, epiphytic BGA make only a small contributio nitrogee th o t n n input, whereas deepwaten ,i r rice they produc substantiaea l nitrogen input whic especialls i h y important becaus thin i e s cropping system nitrogen fertilize seldos i r m applied.

5 ALGAL INOCULATIO RICN NI E FIELDS

BGA were amon firse gth t-fixinN g agents recognizee b o dt activ flooden i e d rice soils. Many trials have been conducteo t d increase rice yield by inoculating the soil with BGA. This practice, also called algalization, a terminology introduced by Venkataraman (1966), has been reported to have a beneficial effect on grain yield in different agroclimatic conditions. However, some reports also indicate failure of algalization. The conclusions of the review of Roger and Kulasooriya (1980) on algalization are summarized hereafter.

5.1. Methodology of the experiments

Most experiment algalization so n have "blacbeea n no k box" basis, where onllase th yt indirect effect (grain yieldf )o algalization was observed and the intermediate effects were not studied. Ther littls i e e informatio qualitative th n no d an e quantitative variation -fixinN e th gf so algaN le th flor d aan balance in inoculated paddy soils. Pot and field experiments have been conducted, usuall singla n yo e crop e relativTh . e increasn i e grain yield over the control was on average 28% in pot experiments and 15% in field experiments. The better growth of BGA in pot experiments is probably attributabl reductioe th o et f climatino c disturbancd an e to the mechanical effect of the pot walls, where BGA frequently seem to grow preferentially and profusely. Pot experiments may therefore only be suitable for qualitative studies, since they overestimate the effectA inoculationBG f so .fiele th Mos df to experiment e th n so other hand, were conducted in only one growing season and may underestimate the effects of algalization since the advantages of a slo wreleasN apparene b firse et th no fron t i ty m ma deaA dBG algalized crop.

107 5.2. Effec algalizatiof to rice th e n planno t

Algalization may affect plant size, nitrogen content, and the number of tillers, ears, spikelets, and filled grains per panicle. The most frequently used criterion for assessing the effects of algalization'has been better grain yield. Results of field experiments conducted mainly in India report an average yield increase of about 14% over the control, corresponding to about 450 kg grain cropr pe h,a where algal incoulatio s effectivenwa higheA . r grain yield increase was observed when algalization was in combination with limesometimed an ,P s molybdenum application. Unfortunatelt i y is not possible to separate the direct effect of PK fertilizers on rice from its indirect effect upon the growth of indigenous or introduced algae. The effects of algalization used with N fertilizers are controversial. Since biological N fixation is known to be inhibited by inorganic N the beneficial effect of algalization in the presenc N fertilizer f eo moss swa t frequently interprete resultins a d g from growth-promoting substances produce y algadb a alsr eo y ob temporary immobilization of added N followed by a slow release through subsequent algal decomposition permitting a more efficient utilization crope th .y b N f o

5.3 Effect of algalization on soil properties and microflora.

Grain-yield measurements suggest that algalization produces both a cumulative and residual effect. This was attributed to a build up of both the organic N content and the number of.BGA propagules in the soil, facilitatin reestablishmene th g biomassA BG e th . f to Severa l reports indicat n increasa e organin i e c matte organid an r . cN Algalizatio s alsnwa o reporte o increaset d : aggregation e statuth f so soil (Shiel Durreld an d , 1964), water-holding capacity (Singh, 1961), available P, and total microflora, Azotobacter, Clostridia, and nitrifiers (Ibrahi al.t me , 1971).

5.4. Limiting factors for algalization

Amon possible th g e limiting factors responsibl failure th r eefo f algalization-onlo d availablan H p y e P,conten e soith l f havto e been studied. Since in some soils, algalization is inefficient in spite of the addition of lime and phosphate (Okuda and Yamaguchi, 1952),

108 availabl conteneP probabls onle i t th y t factoyno r limitine th g effect of algalization. On the other hand, texture, organic matter content, CEC of saturated extracts, and total N are probably not important limiting factors (Subrahmanya al.t ne , 1965). Amone th g biotic factors thapossibln tca y limi A inoculuEG t m growth, graziny gb the Zooplankton has been already mentioned. Other possible mechanisms involved suc antagonismn a h , competition, etc. have been citedt ,bu their role is not clear. Low temperatures, heavy rains, and cloudy weather have also been reporte limio t destablishmene th t e th f to inoculum.

S.S. Algalization technology

The methodology of EGA production has been reviewed by Watanabe and Yamamoto (1970 Venkataramad )an n (1972) methode Th . fielf so d application have been reviewed by Venkataraman (1981). Methods of inoculum production in artificially controlled conditions have been developed mainl Japan i y n where algalizatio t usedno s .i n Producing the inoculum in artificially controlled conditions is well defined but relatively expensive. On the contrary, the open air soil culture, used in India, is simpler, less expensive and easily adoptable by the startea f o e r us cultur farmerse bases th i n t eo dI .a tha s i t multistrain inoculum of Aulosira. Tolypothrix. Scytonema. Nostoc. Anabaena and Plectonema, provided by the "All India Coordinated Project on Algae" (1979). The inoculum is multiplied by the farmer in _2 shallow trays or tanks with 5-15 cm water, about 4 kg soil m , 100 _2 g triple superphosphate m and insecticide. If necessary, lime is added to correct the soil pH to about 7.0-7.5. In 1 to 3 weeks, a thic t develop ksoima e th l n surfaco s d sometimeean s floats. Watering is stopped and water in the trays is allowed to dry up in the sun. Algal flakee e theth sar n n i d scrapestoree an us bagn f r i ddof fo s fields. With such a method, the ultimate proportion of individual straine algath n li s flake unpredictables i s assumes i t I .d that, becaus e inoculueth produces i m soin d i climati an l c conditions simila fielde thoso th t r n i e, dominant strain mosse wilth t e lb adaptelocae th lo t dconditions e recordeTh . d rate productiof so f no algal flakes in the open air soil culture range from 0.4 to 1.0 kg m -2 in IS days, indicating that a 2 m 2 tray can produce in 2-3 months enough algal materia inoculato lt ricf o ea h fielde1 r Fo .

109 transplanted rice the algal inoculum is generally applied 1 week after the transplanting. When rice is sown, seeds can be coated by mixing the algal calciug suspensiok 3 2- m d carbonatan n 10-2r epe seeg 0k d and air-dried in the shade. Recommendation r fielsfo d applicatio drief no d algal inoculum (algal flakes) give "Aly nb l Coordinated Projec Algaen to " (1979) indicate thaalgay tdr lf 8-1 o flake g 0k s applie wee1 d k after transplanting is sufficient to inoculate 1 .ha; although a larger amount will accelerate multiplicatio d establishmenan n fielde th n i .t Algalization can be used with high levels of commercial nitrogen fertilizer, but reduction of the N dose by one third is recommended. o benefiT t frocumulative mth e effec algalizationf to algae ,th e shoul appliee least db a r t fo dthre e consecutive seasons. Recommended pest-control measures and other management practices do not interfere witestablishmene th h d activitan t f thesyo e e fieldsalgath n i e.

6 RESEARCH NEEDS ON BGA IN RICE FIELDS

Looking at the literature on BGA, it is surprising to observe the disequilibrium betwee differene th n t topics. Taxonomy, morphology, micromorphology, physiolog enzymologd an y highle ar y y documented whereas ecolog stils i y l very poorly understood, most probably because methodologicaf o l problems. Test tube grow havA nBG e been extensively studied d severa,an l desirable physiological characteristicA BG f o s strains for field inoculation have been summarized by Stewart et al. (1979 followss )a : "Strain fiele th dn s i shoul selectee us e db r fo d fast-growing and capable of fixing N under aerobic, microaerobic and anaerobic conditions. They should also be able to grow photoautotrophically and chemoheterotrophically. and store endogenous carbohydrate reserves. They should evolve little H_ and liberate nitrogen in excess of their requirements for optimum growth. Cyanobacteria diffe whethe n thet i r no y r ro liberat e extracellular n inhibitioo . NH f glutamino n e synthetase e way whicn Th .i s h 4.— glutamine synthetas Cyanobacterif eo regulates i a d f coulo e db importanc strain i e n selection". Howeve selectioe th r "supef no r N_-fixing strains meanino n s "ha g unless such strain e ablo ar set survive, develop and fix nitrogen as programmed in the rice fields. Information is available on inoculum production under laboratory and outdoor conditions, but the successful field establishment of these

110 availabl econtenP probabls onle i t th y t factoyno r limitine th g effec f algalizationto othee th rn O hand. , texture, organic matter content, CEC of saturated extracts, and total N are probably not important limiting factors (Subrahmahya al.t ne , 1965). Amone th g biotic factors than possibltca y limi A inoculutBG m growth, graziny gb the Zooplankton has been already mentioned. Other possible mechanisms involved suc antagonismn a h , competition, etc. have been citedt ,bu thei rcleart no rolw temperatures s Lo i e. , heavy rains cloudd ,an y weather have also been reported to limit the establishment of the inoculum.

5.5. Algalization technology

methodologe Th productioA BG f yo bees nha n reviewe Watanaby db e and Yamamoto (1970) and Venkataraman (1972). The methods of field application have been reviewed by Venkataraman (1981). Methods of inoculum production in artificially controlled conditions have been developed mainly in Japan where algalization is not used. Producing the inoculu artificialln i m y controlled condition wels i s l definet bu d relatively expensive. On the contrary, the open air soil culture, use Indian i d simplers ,i , less expensiv d easilan e y adoptable th y eb startea f o e r us cultur farmerse bases th i n et o d I a tha. s i t multistrain inoculu Aulosiraf mo . Tolypothrix. Scytonema. Nostoc. Anabaena and Plectonema. provided by the "All India Coordinated Project on Algae" (1979). The inoculum is multiplied by the farmer in _2 shallow tray tankr so s wit waterm hc 5-10 5 ,10 abou, soig k lm 4 t _2 g triple superphosphat d insecticidean m e f necessaryI . , lims i e added to correct the soil pH to about 7.0-7.5. In 1 to 3 weeks, a thick mat develops on the soil surface and sometimes floats. Watering is stopped and water in the trays is allowed to dry up in the sun. Algal flakes are then scraped off and stored in bags for use in the fields. With such a method, the ultimate proportion of individual strains in the algal flakes is unpredictable. It is assumed that, because the inoculum is produced in soil and climatic conditions simila e fieldthoso th t r n i e, dominant strain smose wilth t e lb adapted to the local conditions. The recorded rates of production of algal flakes in the open air soil culture range from 0.4 to 1.0 kg 5 days1 n i , indicatin m -2 g m thatraproduc2 n 3 a tca y2- n i e 2 months enough algal material to inoculate 1 ha of rice field. For

109 transplanted rice the algal inoculum is generally applied 1 week after e transplantingth . When ric sowns i e ,coatee b seed n mixiny db ca s g the algal calciug suspensiok 3 2- m d carbonatan n seeg 10-2r k epe 0d and air-dried in the shade. Recommendations for field application of dried algal inoculum (algal flakes) give "Aly b nl Coordinated Projec Algaen to " (1979) indicate that 8-10 kg of dry algal flakes applied 1 week after transplantin sufficiens i g inoculato t althoug; ha e1 largea h r amount will accelerate multiplication and establishment in the field. Algalization can be used with high levels of commercial nitrogen fertilizer, but reduction of the N dose by one third is recommended. o benefiT t frocumulative mth e effec f algalizationto algae ,th e should be applied for at least three consecutive seasons. Recommended pest-control measures and other management practices do not interfere with the establishment and activity of these algae in the fields.

6 RESEARC RICN I HE A NEEDFIELDBG N SO S

Looking at the literature on B6A, it is surprising to observe the disequilibrium betwee differene nth t topics. Taxonomy, morphology, micromorphology, physiolog enzymologd an y highle ar y y documented whereas ecology is still very poorly understood, most probably because of methodological problems. Test tube grown BGA have been extensively studied, and several desirable physiological characteristics of BGA strain r fielfo s d inoculation have been summarize Stewary . db al t te (1979) as follows: "Strains selected for use in the field should be fast-growing and capable of fixing N_ under aerobic, microaerobic and anaerobic conditions. They should also be able to grow photoautotrophicall chemoheterotrophicallyd an y d stor.an e endogenous

carbohydrate reserves. They should evolve little H and liberate ? nitroge excesn i n theif o s r requirement r optimufo s m growth. Cyanobacteria differ in whether or not they liberate extracellular n inhibitioo . NH f glutamino n e synthetase e way whicTh n .i s h 4^ glutamine synthetas Cyanobacterif eo regulates i a d f coulo e db importance in strain selection". However the selection of "super N -fixing strains" has no meaning unless such strains are able to survive, develonitrogex fi d pan programmes na e ricth en i dfields . Information is available on inoculum production under laboratory and outdoor conditions, but the successful field establishment of these

110 inocula sporadice seemb o st pointes Gibsoy A .b t nou d (1981) virtually nothin e attribute knows th i g f no s permitting introduced strains to colonize the various hostile environments to which they will be exposed. Recent studies have shown the importance of grazers as a limiting factor for BGA growth. Neem cake or neem oil application permits cheap contro microcrustaceansf lo similaa t ,bu r technology is needed for snails that graze on BGA. Another important gap, is the mode of action of inoculated BGA. In field experiments average yield increase in absence of nitrogen fertilizers (14.6%) does not significantly differ from that in presence of nitrogen fertilizers (14.3%). Since biological N -fixatio e knowinhibites b i n o nt y inorganib d beneficiae th , cN l effec f algalizatioto presence th n fertilizerN i nf eo bees sha n most frequently interpreted as resulting from growth-promoting substances produced by BGA. Such a hypothesis still needs to be proved as algalization experiments have been conducted on a "black box" basis, where onllase th yt indirect effect (yieldn a f )o agronomic practice (algalization) was observed. No data area availabl nitrogen eo n fixatio algad nan l biomass measurementn a n i s inoculated paddy field. Therefore the relative importance of nitrogen fixatio y ihculatenb n increasini A BG d e ricth ge yield, compared with other possible effects like auxinic effects, effect n soiso l properties, increas availabilitP f eo y etc stils .i l unknown. Although it is claimed that BGA are widely used for rice production in certain countries (India, Burma) appeart ,i s that algal technologs i y still at a research level in most of the rice growing countries. This is most proably due to insufficient knowledge, which does not permit algalizatio recommendee b o nt d with confidenc farmerse th o et . Singh (1979) reported that the experiments conducted at CRRI have shown very clearly that fresh algae inoculation is always better than dried algae inoculation. hige Howeverth h o watet e ,rdu conten BGAf to e ,th weight of a fresh inoculum is very high: a value of 750 kg/ha was reported in a FAO Soils Bulletin on organic manure in China (1977). Using fresh inocula for algalization reduces one of the main advantage f thiso s technology ,e utilizatio th tha s i t stablea f o n , eas handleo t y , non-bulky y inoculum, dr essentia s i t I . achievo lt e f reliableo m thai e y inoculu,dr establiso t m h competitivele th n i y field. For such a purpose we need a better knowledge of the factors

111 affectin formatioe germinatioe th th ) i gd an n f algano l spored san propagules, ii) the establishment of algal inocula in situ.

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117 APPLICATION OF THE SERIAL DILUTION TECHNIQUE

TO ESTIMAT BIOMASE ETH NF 2S-FIXINO G BLUE-GREEN ALGAE UNDER FIELD CONDITIONS

P.A. REYNAUD Offic rechercha l e d e e scientifiqu t techniquee e Outre-Mer (ORSTOM), Dakar, Senegal

Abstract The serial-dilution method developed to estimate algal biomass in field sample describeds i s . This metho illustrates i d transeca y b d t experimen ricy dr ea fieldn i t t showI . log-normasa l distributiow la n for algal material e effecTh . taxof to n volume unit, enumeratiod an n sampling on the accuracy of the method is determined.

INTRODUCTION

The main problem in the measurement of the effect of inoculation with blue-green alga ricn i e e fielddetermino t s i s e principaeth l factors which might be involved in the inoculation effect. The review by Roger and Kulasooriya (1980) indicates that most work on algalization has been performe comparo t d graie eth n yiel treatmentn i d s inoculated or non-inoculated with algae. Experiments conducte thin o d s "black box" basis give no additional information on the qualitative and quantitative development of the algal inoculum and of the phototrophic nitrogen— fixing activity although these parameters are important and may have explaine algalizatioe th y dwh n effec s positivetwa , negativr eo residual. To have a better understanding of the evolution of blue-green algae during the rice cultivation cycle, it is necessary to estimate the total algal biomass along the development of the crop from seedling stag harvesto et .

Algal abundance has been estimated by three principal methods: direct observation, measuremen pigmentsf to platind ,an g techniques. e direcTh t microscopic examinatio generalls i n y user qualitativfo d e determinations, whereas the pigment analysis does not indicate the compositio algae th lf no flora . Plating techniques, whic more ar he frequently used, are advantageous in providing both qualitative and

119 quantitative information simultaneously, but the accuracy of such a method dependdilutioe th n so n procedure.

The purpose of the present paper, is to describe an improved serial-dilution method developed in our lab, to estimate algal biomass in field samples.

I. METHOD

e experimenTh s conductericy twa dr ea fieln i d d just after cropping sample7 ;5 s consistin thref go e soidiametem c l e coreon rf so and aboudeptm c t1 h each were collected alon transeca g 18.f to . 5m They were store bottley closen dr i d d san d unde temperatureb rla .

Each sample was weighed, crushed and resuspended in 30 ml deionised wate thoroughld an r y stirred. Total nitrogen, protein concentration, eacf o anhH dp suspensio measureds nwa e suspensioTh . dilutes nwa d from eacf o glas n hl i m dilutiose 0 on tube1 d s spreao t an snwa d0 1 onto a petri dish containing 30 ml of algal medium solidified with 1% —4 mediu1 1 6 agarB m usee e W .(Alieth d Stanied , nan 0 1 re 1968th r )fo 0 1 dilutiond an enumerato 0 t s1 totae eth l algal constituants 5 - 4 - —3 and the BG 11 medium minus nitrogen with 10 ,10 , 10 dilutions to enumerate specifically nitrogen fixing blue-green algae. For each dilution three petri dishes were used. Incubations were conducte ligha n i td chambe rdays1 2 (50 r . 0fo Count) luxC 0 3 ,s were performed with a stereomicroscope WILD M5. The petri dish was divided by a frame into 1 cm squares, and each square was examined under magnification2 1 x a . Enumeratio f algano l colonie s performeswa n o d entire th e surfac f eaceo h petri dish.

RESULT. II S

. VolumA e unit

In this method of enumeration each algal colony is presumed to have arisen from a propagule which could either be a short piece of filament or an akinete. To convert the algal numbers to biomass, it is necessary to calculate the mean volume of this propagule unit and the following method was adopted for this purpose. From these enumerations, 14 principal taxons were identified (Table 1).

120 Several colonies belonging to each taxon were picked from the petri dish and vigourously stirred for 10 minutes. With this suspension we measured, under microscope hundrea size ,f th e o d piecef o s filamen diametee th r to celf ro l aggregates. From these measurements volum,a e uni s calculatetwa eacr fo dh taxoy nb assuming that the form of the broken filaments corresponded roughly to either a cylinder, sphere or cone. The value of each taxon volume uni thus i t s specifi e algath eo t ccollecte thin i d s usee ecosyster otheb fo dn rca ecosystemt mbu e taxo th wel s f i ni s l identified.

The relative error determined here for r=5% is closely related abilite tth o o fragment y e dispersio th coloniee tth o t d f san no broken filaments during the stirring and diluting steps. For instanc size th ef filamenteo Nostof so c punctiforme (relative error mors i e) homogenou9% = s than thos filamentf eo Anabaenf so a .s_P_heri_ca (relative error = 20%) and the relative error of the estimation of this taxon biomass would be higher than for N. punctiforme.

B. Enumeration of taxons

Enumeration of taxons was normally conducted using two succesive dilutions and the result was expressed as the mean of the six petri dishes.

On the medium BG 11 minus nitrogen, eucaryotic algae and non nitrogen fixing blue-green algae grew normally durin firse th g t week, then their growth stopped and colonies became yellow confirming their inability to fix N (Reynaud and Roger 1977).

For N2~fixing blue-green algae (N -fix B6A) there was a good correlation between the number of colonies which developed, either on medium with or without nitrogen source.

The use of the two media and three dilutions allowed us to:

- distinguish slow growing taxons 'from fast growing ones - count eac hoptimas taxoit s na l dilution

121 - reduce possible competition between algal groups - easily isolate each taxon.

Petersen (1932) pointed out that the dilution method was not reliabl filamentour efo s typthad ean t some spreading types also od not form individual colonies in the agar medium. To have an estimatio thesf no e biomasse have sw e settlee scala th n f do eo 2 densit separatiof o y filamentf no thesr f o sfo em c taxon 1 n so agar plate; we have compared it with a direct enumeration of filaments under microscope for a suspension of Lyngbva sp. Means of triplicates estimations were identica methodso ltw wite th h.

C. Biomass determination

The accuracy of algal estimation and in situ ARA measurements depend upo densite nth distributioe th f samplin o yf o f o d an gw nla the variable. Earlier studies on the correlation between means and variance f enumeratioso f soino l microorganism sitn A i uAR d an s measurements, indicates that these variables have approximately a log-normal distribution (Roger et al., 1977, Roger and Reynaud, 1978) e firsTh . t implicatio s thaconfidence wa tth thif w n o la s e interval and parametric statistical variable (i.e.: t variable of Student Fisher) mus calculatee tb d usin logarithme th g algaf so l enumeration measurementsA AR r so confidence Th . e intervas lwa dissymmetrical inferios ,it r limi s generalltwa y slightly lower than that incorrectly calculated using the t variable of Student Fisher; the upper limit was generally higher. The validity of the logarithmic transformation mus checkee tb methoa y db de baseth n o d ratio between two correction coefficients established by Neyman and Scott (1960 programmed )an (1978). Rogey al db t re ; transformation is considere valis a d d whe ratie nth o c~/ includes ci d between 0.61.33d an 6 .

e transecTh t experimen gooa s di t demonstratio loge th —f no normal distribution law for algal material. When variables such as weigh, pH f sample to concentratioN d an s soilsn i n distributee ,ar d along the transect as a normal distribution, the log-normal transformation is justifiable for 10 taxons out of 11 (Table 2). In fact, Anabaena sphaerica has a c /c ratio of 3.465 depending of

122 two very high values, but this ratio diminished to 0.988 with their supression.

Most result expressee sar numbes a d algaf ro grar epe f soil mo ; these data don't take into account algae presen flooe th dn i twate r of submerged soils and do not permit extrapolation to the field level. A more satisfactory way to evaluate algal population is to 2 determine the number of algae per cm , each core sample includes the first centimetre of soil and the corresponding flood water column. As the soil has been sampled in a dry area we compared the o expressionstw precisioe samee :th th confidence ;th s nwa e interval on the weight of 57 samples was 5%, r=5%.

On the transect the total algal biomass was estimated at 2970 kg/ha wit confidenca h e interva 0 (lowe1 f lo r limit9 (uppe1 d )an r limit) for r = 5%. As we have determined that there is no autocorrelation function between algal material of two next samples e havw e calculate confidence th d e interval with leslesd san s samples within 18.5 meters.

lower limit upper limit 57 samples 10 19 29 samples 34 25 19 samples 53 32 15 samples 69 35 sample2 1 s 83 39

The accuracy was very high with 57 soil samples (that is every 30 cm intervals), and since biological evaluations could tolerate an accurac f abouyo t 50% ,seemt i s convenien thin i t s transeco t collect a sample every meter.

The serial-dilution method, is currently used as a standard procedure in our lab. This allows us to compare the algal biomasses studied here with those of other areas in Senegal: on 97 soils estimated coveree ar 1 lesy :2 algaef db o s g betweek 7 tha,5 0 n1 0 n1 tons2 1 n y A .b e on ton0 1 d sd an an betwee8 g 1 k , 0 kg n10 0 an10 d averag weighy edr equas i t 3.85o lt fresf %o h weight average th n ,o e weighy dr e otth f 5.5 %nitroges i n (Roger, Tiro Watanabed lan ,

123 unpublished); thus algae ,th e supplie rice th de fielf o dg k wit 2 h6. nitrogen.

CONCLUSIONS

The serial-dilution method is not an uni-versial technique for the quantitative evaluation of the algal biomass: when there is a thick algal bloom it is easier and more accurate to collect the algae and measure the pigment concentration.

The method described in this paper is time consuming and could lead to errors due to spore germination. However, it is useful in its application to large scale studies of non-bloom forming algae after their introductio e identificatioth r o fieldnfo t d an s f no dominant taxons. To compensate for the log normal distribution of these organims, algal enumeration bese sar t carrie compositn o t dou e samples prepared by mixing several surface soil cores removed from a field.

REFERENCES

H.H. ALLE R.Yd Nan . STANIER, 1968. Selective isolatio blue-greef no n algae from wate soild Gen. ran J .. Hicrobiol 203-209: 1 .5 .

J. NEYMASCOTT. E d N,an 1960. Correctio biar nfo s introducea y db transformation of variables. Ann. Math. Stat. 31 : 643-655.

J.B. PETERSEN, 1932 algae .Th l vegetatio Hammef o n r Bakkar. Bot. Tidsskr 42 : 1-48.

P.A. REYNAUD and P.A. ROGER, 1977. Milieux sélectifs pour la numera- tion des algues eucaryotes, procaryotes et fixatrices d'azote. Rev. Ecol 421-428: .) Sol(3 .4 .1

P. ROGER and P. REYNAUD, 1976. Dynamique de la population algale au cours d'un cycle de culture dans une rizière sahelienne. Rev. Ecol. Biol 545-560: . ) Sol(4 .3 .1

124 P.A. ROGER, P.A. REYNAUD, G.E. RINAUDO, P.E. DUCER T.Md .Fan TRAORE, 1977. Mis evidencn e distributioa l e d e n log-normalle ed l'activité réductrice d'acétylèn situn i e . Cah. ORSTOM. Ser. Biol. 12 : 133-140.

P.A. ROGER and P.A. REYNAUD, 1978. La numération des algues en sol distributioe submergd i lo : e organismes nde densitt se é d'echantillonage. Rev. Ecol. Biol. Sol., 1978, 15. (2) : 219-234.

P.A. ROGER, P.A. REYNAUMONNIAUX. G d an D , 1978. Normalisatios nde donnée calcut précisioe sa l e ld mesures nde microbiologin se e du sol. Cah. ORSTOM. Ser. 171-180: Biol) <2 . , .Vol13 .

P.A. ROGE S.Ad an R. KULASOORIYA, 1980. Blue-green alga riced an e . I.R.R.I., Los Banos, Philippines

125 K) TABLE I

Taxons Shape and size measured Numbe f elementro s Volume unit measured M 3 Pseudanabaen. asp Lengt diameted han f ro 112 1.5010 ± 11 X Narrow L.P.P. species cylindrical 132 1.45103 + 17 X Lyngby. asp trichomes 245 X 1.35101 1 + 4 Anabaena ambigua 103 X 3.20100 2 + 3 Anabaena vaginicola 105 8.13103 + 13 X Scytonema millei 100 1.30104 22 X Spirogyra spp. 138 6.5010 + 24 X

Nostoc microscopicum Trichomes length and 134 X 7 5.1011 + 0 Nostoc punctiforme diameter of hemispherical 134 4.9010 9 X Anabaena sphaerica cells 105 7.9010 + 20 X

Calothrix spp. Heigh diameted tan conicaf ro l 72 1.0010 + 18 X trichomes Gloeothece samoensis Diameter of cells aggregates 98 X 3 1.3412 ± 0 Unicellular green algae 20 X 3.80105 1 + 1

Navicul. sp a Double cone: length and diameter frustulf o e 20 X 0 3.5011 ± 0

Calculated volume unit and relative accurency to 95X confidence interval of principal taxons determined in a Senegal rice-field. Table 2

% Confidence interval Mean value Taxons kg/ha Lower Limit Upper Limit c'/c

Pseudanabaena sp. 193 6 21 1.032 Narrow L. P. P. strains 615 22 27 1.099 Lyngby. sp a 678 3 32 0.880 Total homocystous 1664 7 21 1.034 Nostoc microscopium 4.8 4 30 0.857 Nostoc punctiforme 23.2 3 32 0.807 Total Calothrix sp. 198.9 9 30 0.792 Gloeothece samoensis 94 21 32 0.891 Anabaena ambigua 35.7 13 33 0.823 Anabaena vaginicola 100.3 118 42 0.840 Anabaena spherica 104.4 960 39 3.465 Scytonema mi Ile i 43.2 14 44 0.970

Total N2-fixing forms 632.5 6 24 0.987 Total algal biomass 2970 10 19 1.029

Biomass estimations, confidence interval with a log-normal distribution, valid when 0.66.< C'/C <1.33. Mean e obtainear s d fro 7 5 msampling a padd n o sy field transece th n i t Fleuve region Senegal.

127 EFFECT OF INOCULATING BLUE-GREEN ALGAE AND Azolla RICN O E YIELD

S.A. KULASOORIYA Department of Botany, Universit f Peradeniyayo , Peradeniya, Sri-Lanka

Abstract Nitrogen fixing blue-green algae (BGA) and the Azolla-Anaebaena symbiosi e potentiasar l alternative source f nitrogeso r lowlannfo d rice production e literaturA surve.th f o y e showse th tha n to average, whe A inoculationBG effectives i n ,a ric e yield increasf o e 14% (450 kg grain ha ) has been observed. However, in Sri Lanka no significant increase grain i s n yield have beeA n BG observe o t e du d inoculation. Azolla inoculation in broadcast, transplanted, and avenue transplanted rice gave yiel d 48%an dd ,2 an increase2 , 12 f so was equivalent to 55 to 80 kg N ha as urea. Azolla was observed to reduce weed growth by 53%. Azolla is easier to establish in rice field e easilb sn sincyca t recognizei e d wit e naketh h d eye, however, BGA are better able to withstand periods of desication which occur in rain-fed rice production. Most algalization experiments have been performe a "blac n o d k box" basis where onlfinae th y l grain yiels ha d been measured. Isotope experiment n pla ca svitaa y l roln i e understanding the processes by which BGA and Azolla increase rice yields.

INTRODUCTION

Nitrogen along with water management, has been referred to as the realizatioe th keo t y yielf no d potentia f moderlo n rice varieties (Brady 1979). To supply this demand, synthetic nitrogen fertilizers were liberally used in the 1960's due to their low cost, availability and ease of application. With the energy crisis of the 1970's, the potential dange f completro e dependenc n suceo h fertilizers, manufactured primarily from fossil fuel by-products, dawned upoe nth scientific community dealing with crop production t thereforI . e became apparent that alternative sources of nitrogen have to be sought and a world-wide enthusiasm developed for biological nitrogen fixation.

129 Whether synthetic, natural or biological, the conversion of the inert dinitrogen molecule into a combined form initially requires energy, although the overall process is exothermic. In this respect, amon nitrogee th g n fixing organism d theisan r associationse ,th photosynthetic organisms, which utilize the almost inexhaustible resource of solar energy, are more attractive than their heterotrophic counterparts photosynthetie th f O . c nitrogen fixers activ a ric n i e field ecosystem, the blue-green algae (BGA) and the Azo1la-Anabaena symbiosis are more important than the photosynthetic bacteria, whose nitrogen fixatio limites o theii nt e rdu dabsolut e requiremenn a r fo t anaerobic environment for this process.

With the realisation of the importance of the nitrogen fixing BGA in maintaining soil fertility in rice fields (De 1939, Singh 1961, Watanabe 1959), attempts have been mad o inoculatt e e rice fields with selected strains of these organisms, and this process has been termed algalization (Venkataraman 1972). On the other hand, the use of flzoll ricn s beei a eha ntraditionaa l practic Chinn i e d Vietnaaan m r centuriesfo e scientifith t ,bu c interpretation beneficiae th f so l effect f thiso s practic vera s yi e recent development.

This presentation is confined to experiments done on the inoculation of BGA and Azolia into rice field, since other aspects of algal and flzolla use in rice cultivation are discussed by the other participants.

USE OF ALGAE

The effect of algal inoculation on rice has been studied extensivel e result th Indin i yd asan have been comprehensively presented in several review articles (Venkataraman 1972 & 1979). A e productiomanuath r fo l d utilizatior ricnan alsfo s w i eA ono BG f no available (Venkataraman 1981). Roger and Kulasooriya (1980) summarized the literature on the effects of algalization on the grain yield of rice in the presence and absence of additional treatments. From this literature surveys possiblwa t i , drao t e w certain, general inferences. It was observed that pot experiments gave better results than field experiments (relative increase*of grain yield over treatment is an average of 28% in pot experiments, compared to 15% in

130 field experiments). This was possibly due to less climatic and mechanical disturbances in the pot experiments. It may also be due to preferential growth of algae in pots, which provide a moist, solid substratum around a small area on to which algal filaments adhere and grow profusely. Results from pot experiments should only be considered as indicators of a potential and should not be extrapolated directl o fielt y d conditions. Froe resultth m f publisheo s d field experiments, Roger and Kulasooriya (1980) concluded that on an auerage, algal inoculation where effective, cause a relativd e increase in grai n% ove e comparablyiel14 rth f o d e non-algalized treatmentd san ove% controlse 16 rth d thi,an s increase corresponde abouo t d0 t45 kg/per hectar graif eo crop r npe .

Venkataraman (1979) reported result extensivf so e field experiments conducte Indin i d a wher effece th e f algalizatioto n have been investigate e presencth n i df differeneo t level f chemicao s l fertilizer. From these result concludede h s :

- In areas where chemical nitrogen fertilizers are not used, algal inoculatio givn farmere nca th e benefie th s f to applying 25-30 kg ftl/ha, - Where N-fertilizer usede amoune sar ,th N-fertilizef to n rca be reduced by about l/3rd through algal supplementation. - Even at high levels of nitrogen, algal inoculation has a beneficial effect.

The positive effect of algalization on grain yield at high N-level bees ha sproductioe n th interprete o t e growtf no du s a dh promoting substance algae th ey b s (Venkataraman e 1972)th t ,bu validity of this interpretation is open to question. Growth promoting substances are produced even by non-nitrogen fixing BGA and field inoculation with such algae in the presence of chemical N-fertilizers should permit the assessment of the effect of growth promoters produced by BGA, but so far such experiments have not been reported (Roge rKulasooriy& a 1980). Eve sucf i n h experiments give positive results woult i , difficule b d attributo t e beneficiath e l effects entirely to the production of biologically potent substances, unless e showb n ni ca tconlcusivel y that those substances came froaddee th m d algae. For instance, algal inoculation together with chemical

131 IM-fertilizer y promotma s e rapid algal growth, utilizind an g immobilizin addee th g d nitroge organin i n c matter goo,a d parf to which would have been otherwise lost due to leaching, ammonia volatilization and denitrification. Subsequent death of these algae and the slow release of the nitrogen in them may be compatible with a more effective utilization by the crop, resulting in better yields.

While ther e severaear l report f algaso e giving beneficial effects, there are also reports indicating a failure of algalizatipn. Even our own experiences from the field experiments with BGA in Sri Lanka, are not very encouraging. We have examined the effects of soil amendments such as addition of phosphorous, lime and molybdenum, which enhanc growte th e f nitrogeho n fixin resulte e gth th BGA t f so bu , qualitative and the quantitative improvement of algal growth were not positive experimentr ou n I . were sw e also unabl o recort e y an d significant increases in grain yield. These experiments were initially conducte t Peradeniya d Centran i a i LankalSr , wher e riceth e soils generallH havp ea y below neutrality d suc,an h soils seldom promote algal growth (Watanabe 1973, Yamaguchi 1976) alse W .o conducted a few algalization experiments at Maha Illuppallema (North Central Province) and Ambalantota (Southern Province), in the dry zones of Sri Lanka, where the soil reaction is near neutral. In these localities, initial algal growth in the fields was encouraging, but they produced only marginal increase grain i s n yield (Figur . Fro1) e m this figure some beneficial noticee effecb n tca d especially undew lo r level f fertilizeo s r nitrogen t thi clearls ,bu i s y depressed under higher fertilizer treatments e AmbalantotTh . a experiments also revealed another interesting effect of algalization, which resulted in higher numbers of filled grains per panicle, at all the fertilizer levels examined (Table 1). Such results have been reported earlier from India (Jalapath l 1977)have a w beet t t eno e n, bu o ablRa io t e repeat this experimen r interpreto t this result.

On the whole our experiments with BGA have given only low yield increases in rice, especially in comparison to our experiments with Azolla.

132 field experiments). Thi s possibl swa o les t se du climatiy d an c mechanical disturbances in the pot experiments. It may also be due to preferential growth of algae in pots, which provide a moist, solid substratum aroun a smald l whic o aret n aho algal filaments adherd ean grow profusely. Results from pot experiments should only be considere s indicatora d potentiaa f so extrapolatee d shoulb lan t no d d directly to field conditions. From the results of published field experiments, Roger and Kulasooriya (1980) concluded that on an auerage, algal inoculation where effective, cause relativa d e increase in grai n% ove comparable yiel14 rth f o d e non-algalized treatmentd san 16% over the controls, and this increase corresponded to about 450 kg/per hectare of grain per crop.

Venkataraman (1979) reported result extensivf so e field experiments conducted in India where the effect of algalization have been investigate e presencth n i d f differeneo t level f chemicao s l fertilizer. From these results he concluded:

arean I s- where chemical nitrogen fertilizer usedt no ,e sar algal inoculation can give the farmers the benefit of applying 25-30 kg N/ha, - Whore N-fertilizer usede amoune sar th , N-fertilizef to n rca be reduced by about l/3rd through algal supplementation. - Even at high levels of nitrogen, algal inoculation has a beneficial effect.

e positivTh e effec f algalizatioto grain no n yiel t higa d h N-levels has been interpreted as due to the production of growth promoting substances by the algae (Venkataraman 1972), but the validity of this interpretation is open to question. Growth promoting substance e producesar d eve y non-nitrogeb n n d fielfixinan A d BG g inoculation with such algae in the presence of chemical N-fertilizers should permi assessmene th t effece th growtf f to o h promoters produce r sucfa h o BGAy s experimentb d t ,bu s hav t beeeno n reported (Roge rKulasooriy& a 1980). Eve sucf i n h experiments give positive results, it would be difficult to attribute the beneficial effects entirel e productioth o t y f biologicallno y potent substances, unless it can be shown conlcusively that those substances came from the added algae. For instance, algal inoculation together with chemical

131 N-fertilizers may promote rapid algal growth, utilizing and immobilizin addee th g d nitroge organin i n c matter goo,a d parf to which would have been otherwis eo leaching t lose tdu , ammonia volatilization and denitrification. Subsequent death of these algae and the slow release of the nitrogen in them may be compatible with a more effective utilization by the crop, resulting in better yields.

While there are several reports of algae giving beneficial effects, there are also reports indicating a failure of algalizatipn. Even our own experiences from the field experiments with BGA in Sri t verLankano y e encouraging,ar have M .e examine effecte th d f soiso l amendments such as addition of phosphorous, lime and molybdenum, which enhance the growth of nitrogen fixing BGA, but the results of the qualitative and the quantitative improvement of algal growth were not positive r experimentou n I . were sw e also unabl o recort e y an d significant increase grain i s n yield. These experiments were initially conducte t Peradeniya d Centran i a i LankaSr l , wher e ricth ee scils have a pH generally below neutrality, and such soils seldom promote algal growth (Watanabe 1973, Yamaguchi 1976). We also conducte algalizatiow fe a d n experiment Maht a s a Illuppallema (North Central Province d Ambalantot)an a (Southern Province)y dr e th n i , i LankazoneSr f so , wher e soieth l reactio neas i n r neutral n thesI . e localities, initial algal growte fieldth s encouragingn i hswa t ,bu they produced only marginal increases in grain yield (Figure 1). From this figure some beneficial noticee effecb n tca d especially undew lo r levels of fertilizer nitrogen, but this is clearly depressed under higher fertilizer treatments. The Ambalantota experiments also revealed another interesting effect of algalization, which resulted in higher number f filleo s d graine fertilize th r panicle l pe s al t ,a r levels examined (Table 1). Such results have been reported earlier from India (Jalapathi Rao et al 1977), but we have not been able to repeat this experimen r interpreto t this result.

On the whole our experiments with BGA have given only low yield increases in rice, especially in comparison to our experiments with Azolla.

132 USE OF AZOLLA

We examined the effect of Azolla grown under dual culture with broadcast seeded 20cmx 0 ,2 s transplante IScmsx 0 3 d avenu)x an d 5 (1 e planted ricd theean n manually incorporated. This resulte grain i d n yield increases of 14, 22 and 48% over the .control plots with the corresponding planting patterns whico ,t h neither y Azollan r ano fertilizer nitrogen was added (Table 2). Field experiments at Ambalantota were designe examino t d effece th e f Azollto n ricao n i e comparison to different levels of chemical N-fertilizers. These experiments conducted during three consecutive rice growing seasons have given positive results (Table 3 and Fig 2). Yala and Maha, are the traditional rice growing seasoni Lanka Sr e Yal n Th i s.a season extends from Apri relativels o Augusi t l d an t y comparee dr y th o t d Maha season which extends from Octobe yeae on Februaro r t e f ro th f o y next year.

It can be seen from these results that the effect of two Azolla incorporations durin e ricth ge cultivation cycle have given yield responses equivalen comparablo t e plots thag k t 0 receive8 o t 5 5 d l\l/h f fertilizeo a treatmenA BG givt s urea ra no e a eTh d .t di significant yield increase.

We also evaluated the weedicidal effects of Azolla in the field, and found tha coult i t d suppress weed growt % (Tabl 53 mucs ha s ha e 4). So far we have been successful in the establishment of Azolla in rice fields at Undugoda & Peradeniya (midcountry wet zone), Ambalantota (low country dry zone) and Pannala (low country intermediate Lanki zoneSr af ) o (Figur. e3)

AZOLLA Vs BLUE GREEM ALGAE

From our present field experiments, Azolla therefore appears to have a greater potential than BGA as a biofertilizer for rice in Sri Lanka. Azolla also has a clear advantage over BGA in that the plant can be seen with the naked eye and with an initial familiarity, can be easily recognized by rice farmers. BGA on the other hand, are difficult to distinguish from other field algae and need microscopic observation for positive identification as species capable or

133 incapabl f nitrogeeo n fixation n thiI .s context woult i , easiee b d r to transfer ftzolla technology to the farmers in developing countries.

While ftzolla has these advantages over BGA, its widespread use is limited by the availability of water and its susceptibility to pest attacks. The requirement for year round availability of controllable water, which is necessary for ftzolla could be a serious limitation for the spread of ftzolla technology in South East Asia where nearly 70% of ric stils i e l cultivated under rainfed conditions (Frene l 1981)a t e y. In this regard, BGA is a better choice because they can withstand periodic drying of fields by producing resistant akinetes and hormogonia which germinate and initiate rapid growth immediately after rewetting alsA BG o. grow better under neutra alkalino t l e conditions withstann ca d an d salinity better than ftzolla. i LankaEveSr e n i nth , e higheNorte b th y potentian hi ma r wherA BG ef lo soil e calcareouar s s and alkaline and where profuse growth of N- fixing BGA occur naturally in rice fields during the rainy seasons (Thirukkanasan et al 1977). However we have still not performed any algalization experiments in this locality to investigate this potential. The dramatic successes of improving the fertility of the alkaline "Usar" soils in India by Singh (1961) is largely attributed to the ability of BGA to colonize and grow on such adverse soils.

AVAILABILIT F FIXEYO D NITROGEN

Although there are reports of release of fixed nitrogen into the medi algay ab e growing under laboratory conditions l 1973)(Foga t ge , most of the organic nitrogen in the algal cells becomes available to the crop afte deate rth d mineralizatio han algae th lf no s cells i t I . therefore important to realize that algalization often results in a cummulative improvement of soil fertility and the effects of algal inoculation increases year after year (Roger & Kulasooriya 1980). Singl (1981a t he ) have reporte n studieo d t spo carrien i t ou d experiments, with flooded rice soil o examint s availabilite th e N f o y and P, after incorporation of BGA, ftzolla, organic, and farmyard manur comparison i e ammoniuo nt m fertilizer e chemicaTh . l fertilizer release mosN e tth drapidl y days0 (871 n %i ) while ftzolla came second. Amon e specieth g f ftzollso a tested pinnat. ,ft a release£ N d more rapidly than ft.filiculoides and ft.mexicana. ft.pinnata, (the

134 Indian isolate) released 88% of its N as ammonium in 40 days whereas the Vietnamese isolate release % durin77 de sam th ge period. Compared to these releaseA ,BG nitroges it 0 days 4 f d o n onli n.% 38 yThi s evidence also supports the concept that BGA would have a delayed effect on a crop compared to other forms of inorganic and organic fertilizers.

POSSIBLE AREAS FOR ISOTOPIC STUDIES

It has been pointed out that BGA added to rice fields are subjecte variouo t d s environmenta biotid lan c factors (Roger& Kulasooriya 1980), whic y limima h t their growth e importancTh . f eo thoroughly investigating the ecology of BGA in rice fields has been emphasised (Roger & Reynaud 1979 and Roger & Kulasooriya 1980) primarily wit ha vie o properlt w y understand their exact roln i e benefittin associatee th g d crops. Unfortunately most algalization experiments have been carried out on a "black box" basis, where only the grain yield has been measured as a final index. This is primarily due to difficulties in estimating algal growth and nitrogen fixation quantitively under field conditions. Under such circumstancesa failur algalizatiof eo n canno explainede tb d evee successe,an th n s difficule ar o interpret t wit degreha accuracf eo y sufficieno t assign the positive effects only to algal activity. The use of isotope n pla ca svitaa y understandinle th rol n i e f boto g h photosynthesi d nitrogesan nd Azoll an fixatio A aBG undey b n r field conditions. Data obtained from such studies would be invaluable in improving biofertilizer technologies with the prospect of exploiting these organisms more profitabl ricn i y e cultivation.

Another are r isotopiafo c studie squalitative woulth e b d d ean quantitative analysi Azolle th d algaf aso an l nitrogen during mineralizatio ricn i n e soils. These woul t onlno dy enablo havt es eu a clear view of the intermediate degradatory products, but would also pave the way for innovations of new procedures for biofertilizer use.

s beeha n t I pointe t thae ricou dtth e crop seldom recovers more than 30-40% of the fertilizer-N added (Freney et al 1981), and this has been largely attributed to the various losses of field applied nitrogen (Watanabe et al 1981). It is generally believed that losse's during mineralizatio organif no c nitroge e mucnar h less, especially

135 when such organic matter is incorporated in the soil. However, losses through ammonia volatilizatio drastio t e ndu c a increases a H p n i s resul algaf to l activity have been reported (Watanab 1979p d Ap )an & e N-losses durin mineralizatioe th g f surfacno e bloom algaf so alsy ema o be high, but no data are available on these processes.

e potentiaTh d Azolll an N-inpu A aBG inty tb o rice field s beeha s n generally calculate o methodstw y b d :

1. Measuring the biomasses of these organisms and calculating n themi N . e e basith th e totaf n so th o U lI 2. Measuring nitrogenase activity (frequently by ARA) and extrapolatin e resultth g o fielt s d levels.

The inherent limitations in the ARA method and the inaccuracies extrapolatioe ith n f suco n h data have been pointeWatanaby b t ou d e& Cholitkul (1979) who stressed the need for N studies in rice fields.

All these factors warrant e launchinth s coordinatea f o g d programme of isotopic studies on nitrogen fixation in rice producing countries e thir,th mandn i f whicworldo y e har , where unfortunately facilitie r sucfo s h studie e limitear s d unlikel an de forthcomin b o t y g 15 in the near future. These studies should include the use of l\l and 14,C to understand the basic metabolic processes in the nitrogen fixing organisms, autotrophic, heterotrophic and symbiotic, that play a vital rol maintaininn i e g soil fertilit ricn f i yo ee fieldsus e Th . 32P may unravel several poorly understood aspects of P metabolism in these organisms whose widespread use is limited to a large extent by knowledgen i thip ga s .

In this manner new innovations may be developed, leading to better exploitatio f thesno e organisms, resultin lesn i g s dependence on fossil-fuel based chemical fertilizers, which are fast becoming a luxury beyond the reach of poor peasant farmers in many developing countries.

136 REFERENCES

1. Brady, N.C. (1979) - Opening Address. In: Nitrogen & Rice, The International Rice Research Institute s Banos,Lo , Philippinesp p , 1-4. P.K, De ,2 .(1939 e rol blue-greef Th eo ) - n alga nitrogen i e n fixatio ricn i n e fields, Proc. Roy. SOG. Lond. ,- 139 127B1 . 12 , 3. Fogg, G.E., Stewart, W.D.P., Fay, P. & Walsby, A.E. (1973) - 'THE BLUE-GREEN ALGAE1, Acad, Press Lond & N.Y., 459pp. 4. Freney et al (1981) - Nitrogen balance in irrigated wetland rice. In: Wetselaar Simpson. ,R , J.R. Rosswall& , . (eds),T , Nitrogen cycling in South-East Asian wet monsoonal ecosystems, Australian Acad. Sei., 202Canberra- .9 19 . ,pp 5. Jalapathi Rao, L., Venkatachari, A., Sundara Rao, W.V.B. and Reddy, K. Raj. (1977) - Individual and combined effect of bacteria d algalan l inoculatio yiele th f rice o dn no . Curr. Sei. 46, (2), 50 - 51. 6. Roger, P.A Kulasooriya& . , S.A. (1980 )- 'BLUE-GREE N ALGAD EAN RICE e Internationa1,Th l Rice Research Institute Banoss ,Lo , Philippines. 112pp. 7. Roger, P.A Reynaud& . , P.A. (1979 )Ecolog- blue-greef o y n algae in rice fields. In: Nitrogen & Rice, The International Rice Research Institute, Los Banos, Philippines, pp 287 - 310. 8. Singh, P.K., Panigrahi, B.C. and Satapathy, K.B. (1981) Comparative efficiency of Azolla, blue-green algae and other organic manure availabilitP d relation i san N floodea o t n i y d rice soil. Plan Soilt& 35-44, ,62 . 9. Singh, R.N. (1961) - Role of blue-green algae in nitrogen economy Indiaf o n agriculture. Indian Counci f Agriculturalo l Research. New Delhi., 175 pp. . Thirukkanasan10 Kulasooriya, ,A. , S.A Theivendirarajah£ . . ,K (1977 )periodiA c blue-greene surveth f o y s found abundantly during the monsoon period in a paddy field near Jaffna Campus at Vaddukoddai and a general survey of blue-greens in the Jaffna Peninsul e samth en i aperiod . Proc. 33rSessionsn An d'Sre ith f ,o Lanka Assoc. Avdmt. of Science, pp 50-51. 11. Venkataraman, G.S. (1972 )Alga- l Biofertilizer Rics& e Cultivation. Today & Tomorrow's Printers and Publishers, Faridabad (Haryana. pp 5 )7

137 12. Venkataraman, G.S. (1979 )Aga- l inoculatio ricn i n e fields: in , Nitroge nRice& Internationae Th . l Rice Research Institutes ,Lo Banos, Philippines 324- 1 .31 p p , 13. Venkataraman, G.S. (1981 )Blue-gree- n alga Ricr efo e Production FAO Soils Bulletin 46, Food and Agricultural Organization of the United Nations, Rome 102p p , . 14. Watanabe. A. (1959) Distribution of nitrogen fixing blue-green alga varioun i e s area f Soutso h East n APPLAsiaGe . J . Microbiol. 21-29, ,5 . 15. Watanabe, A. (1973) On the inoculation of paddy fields in the Pacific area with nitrogen fixing blue-green lagae. Soil Biol. Biochem 162- (1)5 . 1 .,16 16. Watanabe, I. & App, A. (1979) - Research needs for management of nitrogen fixatio flooden i n d rice crop systems : NitrogeIn . n& Rice e Internationa.Th l Rice Research Institute Banoss ,Lo , Philippines 490- 5 .48 p p , 17. Watanabe, I. & Cholitkul, W. (1979) - Field studies on nitrogen fixatio paddn i n y soils Nitroge: In . n& Rice e InternationaTh . l Rice Research Institute, Los Banos, Philippines, pp 223 — 240. 18. Watanabe Craswell. ,I , E.T App.& (1981. ,A )Nitroge- n cycling in wetland rice fields in south east and east Asia. In: Wetselaar Simpson. ,R , J.R .Rosswall& . (eds,T ) Nitrogen Cycling Monsoonat inWe l Ecosystems. Australian Acad. Scie., Canberrap ,p 4 - 17. 19. Yamaguchi, M. (1976) - Nitrogen fixation by microorganisms in paddy soil relation i s o theint r fertility e fertilitTh : In .f o y paddy soils and fertilizer applications for rice. Taipei, Taiwan, Food & Fertilizer Tech. Centre for Asia & Pacific, pp 60-75.

138 Table 1. Numbe f fillero d grain r paniclpe s n algalizei e non-algalized dan d treatments. (1980-81 liana Season. Arabalantûta, Sri Lanka) .

Treatment non-algalized algalized

Rice only 88. 4 95. 9 P. ON 9 . 99 94 .0 P. 20N 105.7 107.3 P. 40N 106. 6 108. 2 i P. 60N 108. 1 110. 3 N 80 . P 109:%9 112. 9

Table-2 Grain yield and straw yield oi rice grown in three planting pattern t a Peradeniyas *

«* Treatment Grain yield Straw yield

Planting Azolla t ha" */• increase t ha V» increase pattern

Broadcast seeded 1-44 2-77 seeded t- 1-64 14V3-45 . 25V.

Transplantedat at 2-25 2-56

m c 20c 0 2 m x + 2-74 22V3. 10 21V.

Transplanted in 2-07 2-47 avenues -t- 3-04 47V.3-41 39V.

2.86V= cv . cv = 13-5V.

# mean oi 4 replicates t wet zone

139 ble-o T 3 Elfect of Azollo and Blue Green Algae (BGA) on rice (dual culture and two incorporations ol Azolla after transplanting of rice) Grain yield 3 consecutivdat r fo a e seasons (1981) -82 monthsRic2 1/ e3 ( variet6 )1 t y-A

Grain yield (t ha"1 ) percentage increase over F. ON

Season 1981 19812 8 - 1982 1981 1981-82 1982 Treatment Yala Maha Yala Yala Maha Yala *•* Rice only 3 -74 a a 9 3-8 3-98a - - -

F^ON 3-99b 4-16b 4-3lb 0 0 0

F-20N 4-55cd 4-39b 454bc 14V. 5V. 5V.

F.40N 4-79d 4 59b 463bcd 20V. 10V. 7V.

F-6ÖN 509e 538c 4£7cde 27V. 29V. 13V. F.80N 527f 550C 5O4ef 32V. 32V. 17V.

F-ÎOON 5-56g 59lc 5-29f 39V. 42V. 23V. F.10N BGA 4-11b 3-94ab - 3V. - - F. ION Azolla- Pd 5-39fg 5-81C e d 2 A9 35V. 39V. UV. F ION Azolla- Dk 5-46 f g - - 37V. - - F ION Azolla -Bk 5-26ef - - 32V. - - cv=6-2V.) (cv= 9-2V.) (cv=^V.) 1 Yala season-April to Aug. . Maha season- Oct Febo t . . * F-Conc. super phosphate(60kgP CLhd Muriatd 1ah ) f Potash(75kgKOhaeo ) fertilizers. b * mean Mea replicateo * 4 f tw n o s y followe an same s- th ey db lette e ar r not significantly different at 5V. level

Pd - Peradeniya isolate. Dk-Deb6kkawa isolate. Bk-Bangkok isolate

140 Effec f Azollo t ween ao d growth

Planting Azolla Peradeniva expt. Ambalantota expt. -1* -1* pattern g.plot '/»suppression g.plot '/.suppression

Broadcast 35-0 n.d. seeded • 23-3 34V.

Random n.d. 384 transplanting • 0 18 53V.

Avenue 1006 510 transplanting * 55-6 45V. 297 42V. m c 0 2 x 2m 0c 74-3 699 transplanting » 48-6 35V. 331 52V.

n.d.-not determined * Dry weight ol weeds at 30 days after transplanting of rice

141 6 -

0.29$ t algalizeno 1 d 3.22$

-i K*j algalized 5-62$ ^^H *v* '« 5 ' > - A ••VV H 8.27$ | 1 •*• .

•0 3$ 0.41$ 1'6 S •*H 4. , • .•\ "X 1 *!•*' mM -**V *".•' •/»•" X- ! c ':'•} !-*.*' '.X ',x ''< •H ''**• 0) **•\ **r** *•"•• :v '•i %; *.•"• •V' o !*""•• ^"*" ***** ****** •'•'•'. •*".•' •*x .**, £*? •*•*' **•*' 3 • '& '*.***' X* 1 1 1 V.' \»j' ^•"' *«*v •*^"' t**»; X- :"•> "**•' •"•V <-i •A' ..•' •*0* ** * »**«• fe ;.';' '.";> '*£ v>: ^ •?*•' * ,•", • ** -•'x; A' •• ,* '*»*E •*.•*. **•** ^§ 2 ;•;*; " * " • *»•„• ^t" •^

N 80 F N 60 P N 40 P N 20 P N O * F Rice only kg N ha" &a Urea fertilizer

Cones a . a Supe h - 75k o s r2 * a g P P Phosphat a h 45k d 0 2 an egK Muriat potashf eo algalizel Al . à plots received Sodium molybdate

at the rate of 250g ha-1.

Figure 1. Grain yield of an algalization experiment. (1980-81 Maha Season,

Ambalantota, Sri Lanka).

(percentage increase valueth e yielf sar eo algalizatioy db n ovefertilizee rth r nitrogen controls)

142 6-0 1981 (April-A uç) Y A-138« 0-014X 1981-82(Ocl-Mar) Y 4.069«0-018X 1982 ( April-Auç) Y 4-305«0-009X

5-5

CHEMICAL FERTILIZER (UREA) ho LEVE N ) g k N L(

FIGURE 2- Estimated linear relationship between gram yield chemicadan l fertilizer (Ure levelN r ) threfo s e consecutive seasons

Figure n i sparenthesi s indicat e performanceth Azofle th af eo incorporated e fortreatmentth f mo n i N g termk m s f o s fertilizer

( Treatments are given m table 3)

143 DRY ZONE

-*Maha Illuppall

INlERMEDlj£ ZONE |

Col ombo

m k 0 25 5 Ambatantota

Figur . Experimenta e3 lLanki siteSr an si wher e Azolla pinnats awa grow ricn ni e field différenf so t climatic zones.

144 RECOMMENDATIONS

The Meeting stressed on the importance of blue-green algae and Azolla in increasing rice yields* These organisms are known to fix atmospheric N, which can then be used by rice plants, thereby cutting dow expensivn no e fertilize rrequireN produco t d e high yields of this crop potentiae Th . Azollf lo d bluaan e green alga bees eha n demonstrated mainly through yield responses obtaine fieln i d d trials, with and without Azolla and blue-green algae.

Valid estimate ? fixeN f blue-greey so b d n alga Azolld ean n ai the field ove growinra g season are, however, scarce. Thi, sis because method havr s fa usee o s beed n unreliable. Isotopic techniques, which will be of great value for estimating N. fixed in the field oveentire th r e growing perio rica f eo d crop, hav t beeeno n exploited Meetine Th . g therefore strongly recommendef o e us e th d isotope quantifo st blue-greee fixe. th yN y b d n alga Azolld ean n ai rice fields. In addition, several physical, chemical, biological factors and agronomic practices have been shown to affect algal and

Azolla growth in the field. Their possible effects on N_ fixed need to be studied fully, and N in particular, offers a unique tool for such investigations.

The benefit derived from these organisms comes through their use as biofertilizers. They therefore have to undergo decomposition so as to release the fixed N. for the growth of the rice plant. It is thus essential to study how much of the N in these blue-green algae and Azolla is made available for the rice crop, and how much of the

145 residual N in the biofertilizer becomes available for subsequent rice crops additionn I . , several factor managemend san t practices will affec rate d quantitth tean releaseN f yo d from these biofertilizers. Isotope providN f so onle th ey direc mosd tan t sensitive methor fo d estimating how much N from mineralized biofertilizer under these different environmental conditions becomes availabl ricr efo e growth.

The Meeting documented the need for the isolation and selection of highly efficien fixin« tN g strain blue-greef so n alga Azolld ean a efforin na maximiso t e N.fixed.n i AgainN role ,f th eo assessin fixe« gN eacy b df thes o h e strain greate s founb swa o t d, particularly under field conditions.

The participants recommende d IAEd an AthaO througtFÀ h their Joint FAO/IAEA Division coordinat Researcea f o h e Programmus e th n eo nuclear techniques in studies of N cycling by blue-green algae and Azolla in rice fields. As a beginning, however, it was recommended that isotopic aided field studies should place more emphasi Azolln so a sinc presentt ea , yield responses with Azolla have been more positive than with blue-green algae. advocates Alsowa t ,i d that untie lth coordinated Research Programme could be initiated, the Seibersdorf Laboratory shoul encouragee b d conduco t d t preliminary studies, which could forbasie th experimentr m fo s conductee b o st participanty b d s in such a coordinated research programme.

s recommendeIwa t d that there shoul greatea e b d r cooperation betwee Joine th n t FAO/IAEA Divisio advanced nan d Laboratories elsewhere, actively engaged in studies especially on the blue-green algae and Azolla, so as to provide more information which would serve as back-up for isotope-aided field studies.

146 The Meeting further recommended that the reports presented by participants recommendatione welth s , a s la s developed durine th g

meeting shoul compilee b d d int non-priceoa d document d thu,an s facilitat distributios eit scientifie th o nt c communit developef o y d and developing countries.

INOCULATION AND ESTABLISHMENT OF BLUE-GREEN ALGAE IN THE FIELD

The importance of N_-fixing blue-green algae in rice soils was highlighted in 1939. Since then many trials have been conducted to utilize these organisms to improve rice yields.

Informatio blue—green no n alga ricd recentls ean ewa y summarized by Roge Kulasooriyd ran a (1980) manuaa d r thei,an lfo r practicae lus was published by FAO (Venkataraman, 1981).

In paddy fields, blue-green algae can develop standing crops as high as 500 kg ha" (dw) corresponding to 30 kg N ha~ . Estimates for nitrogen fixation, mainly derived from acetylene reducing activity measurements range from 0 to 80 kg N ha~ and average 30 kg N ha . From reports on field experiments, conducted mainly in India, it appears that algalization could on an average, provide a 14 % increase in rice yield, corresponding to 450 kg ha~ of rice grain per crop. Suc increasen a h , which could stil highere lb , would havea significant impac ricn to e productio f thini s technology coule b d improved to have a widespread applicability in other rice growing countries.

Althoug claimes i t hi d that blue-green alga widele ear y usen i d countries such as India, Burma and Egypt for rice production, it

147 appears that this technology is still at a research level in many rice growing countries. This is due to an insufficient knowledge of the systems which therefore does not permit algalization to be recommended with confidence to the farmers. Information is available on inoculum production under laborator d outdooyan r conditions. However successfue ,th l field establishment of these inocula has been sporadic and little is known abou factore th t s influencing colonisatio developmend nan f to blue-green algae under natural conditions. Furthermore, little is known about the mechanisms of rice yield increase due to inoculation; most probably, the beneficial effect of blue-green algae is not only due to their N^-fixing activity.

Ther e severaar e lknowledgr ou gap n i s thesf eo e organisms under field condition d isotopisan c studie y profitablsma usee yb o t d unravel some of these mysteries. These gaps are listed as follows:

Factors affectin establishmene th g N«-fixinf to g blue-green algae inoculated in the field:

1) Climatic: light intensity, temperature ) Physico-chemical2 phosphoru, :pH changed san s inducey b d

the growth of rice. 3) Biotic: grazing by aquatic fauna; competition with indigenous flora. ) Agronomi4 c practices: inoculatio relation ni lano nt d preparation, water management, agrochemicals.

148 Inoculum Production: i) Production of a low cost, active, viable and durable inoculum in a form easy to handle, ) Selectioii strainf no s having good potentiar fo l

establishment and a high N2 fixing ability.

Isotopic studieusefue b understandinr y fo lsma g biotic factors, mainly grazin fixatio« N n cyclingN go isotopef d o nan e us ,s s a mutagenic agents for producing improved algal strains may also be studied.

INOCULATIO ESTABLISHMEND NAN AZOLLFIELE F TO TH DN AI

Azolla The potentia Azollf lo nitrogea s aa n fixing green manurr efo rice has been "rediscovered" recently by scientists. Rice farmers in Chin Vietnad aan m have used Azoll centuriesr afo evet ,bu thesn ni e countries productioe ,th utilizatiod nan Azollf integran o a s a l par organizef o t d rice productio relativels ni recenya t development.

Information on Azolla was summarized by Lumpkin (1982) and in an earlie SoiO rFA l Bulletin (1978) coverin studa g y tou Chinan i r . Field trials on Azolla have been conducted since 1979 in 8 countries Internationae bth y l Networ Soin ko l Fertilit d Fertilizeyan r Efficiency for Rice (INSFFER) which is coordinated by IRRI. One crop of Azolla grow ricn ni e fields increase graie th d nsame yielth e o t d ^•j extenf o s thata ta h obtaine applicatio. e N th g k y b d0 3 f no urea. The high potential of Azolla is shown in Vietnam, where grain e obtainear a dh t durin yield6 sprine 5. th g f so g crop with Azolla as the sole source of N. However, such high yields warrant labor intensive technology economice th d ,an sevaluatee b nee o t d d under different agro-economic conditions.

149 Widesprea Azollf o e limites i aus d environmentay b d d agronomicaan l l factors, mainly water management, temperature, pH, phosphorus availability and pests.

Main research needs are:

) Presently1 , inoculum productio confines i n vegetativo t d e multiplication which result problemn si storagf so d ean transport and also lead to a decrease in the productivity of the strains. Factors inducing sporocarp formation and spore germination are poorly understood. Such knowledge is essentia producinr lfo inoculun ga m whic easies hi o rt

handle and store, and having a constant potential productivity.

) Screenin2 temperaturf go e tolerant, pest resitantw ,lo phosphorus requiring strain specier so s would increase the chanc adoptinf eo g Azolla technolog tropicse th n yi .

3) Comparison of the efficiency of Azolla application

through green manuring and dual cropping. Timing and method of application under different cropping systems. Isotopic methodvere b yn helpfuca s thir lfo s purpose.

ESTIMATING BIOLOGICAL NITROGEN FIXATIO AZOLLY NB D AAN

BLUE GREEN ALGAE

15 f No 2e gasUs .) a

The earliest fixatio N application i . N n studiey f b no s swa

Burris and Miller (1941). This method has been used to provide direct however, N_ evidencf , o fixation„ involveN e r us efo e Th s.

150 the enclosur f planteo shorr sfo t duration n chambersi s supplied with

N enriche (Witts ga Dayd Z yan N d , 1978). Results obtained from such studies, therefore ,instantaneoue b ten o t d d subjecsan o t errors associated with extrapolating dat shorn i a t term studiea o st growing season which involves diurnal, daily and seasonal variations (Knowles, 1980). Due to these disadvantages this methodology seems

not to be appropriate for the evaluation of biological N fixation 2 by Azolla and/or blue green algae, under field conditions.

b) The Acetylene Reduction Technique.

The acetylene (CJEO reduction technique was developed as a

result of the finding by Schollhorn and Burris (1967) that N» fixatio inhibites ni acetyleney b d d furthermore,an , that acetylens ei

converted to ethylene (C2H.) by nitrogenase (Dilworth, 1966). The technique has been widely used as a measure of N. fixing activity. maie Th n disadvantage, however , thamethoe ,is th t d measures onlya short term enzyme activity, whose extrapolatio whola o nt e growing season involves many assumptions whic e ofte validhar t nno e Th . acetylene reduction technique cannot therefor recommendee eb r fo d

accurate estimatio fixatio- N blue-gree e f nth o y nb n algad ean fielde Azollth n .ai

In some simple experiments involving relative estimates, such as screenin largga e numbe N fixin f ro g blue-green algal strainse ,th acetylene reduction technique, however, becomes handy easied ,an r than most available methods. enricheN f o de Us substrate ) c .

151 15 14 Fried and Broeshart (1975) have demonstrated that the N/ N

ratio of N« fixing and appropriate non-N fixing controls grown on 2 N enriched substrates are different. This difference in the isotopic composition is used as a quantitative measure of N. fixed in variou 2 fixinN s g systems validite resulte Th .th f o ys strongly depends on the selection of a suitable non-fixing plant, called contro standarr o l d plant, which essentiall absoro t same s bth ha ye ratiN / o froe substrat absence th m fixe e th th n f i rs o e ea

N2 fixed (Fried et al, 1983). In the case of Azolla the possible standards coul: be d

1) Genotypes of Azolla, naturally free of Anabaena

2) Azolla treated with antibiotics to eliminate Anabaena

) Othe3 r specie aquatif o s c plants e.g. Pistia, Lemna

In the case of blue green algae, possible standards could be:

1. N2 fixing blue-green algae which have been prevented from fixing N„ by the application of high doses of inorganic N fertilizers 2. Genotypes of blue green algae unable to fix N- 3. Green algae, such as Spirogyra, which do not fix N-

The methodology for substrate labelling for these organisms has to be investigated. There are several possibilities for applying enriche materialN dfixatio. N r sfo n studies witblue-greee th h n algae and Azolla

These are:

(i) use of N enriched organic matter, N enriche15 f o e d(iiUs fertilizers) , (iii) use of N enriched fertilizers together with a high energy source, suc sucross ha e (Zapat Wagnerd aan , 1982)

152 RECOMMENDATIONS:

1. Preliminary field studies should be conducted to evaluate the suitabilit availablf yo e method substratf so e labelling.. 2. The suitability of different standard or control plants

should be studied.

FACTORS AFFECTING h\ FIXATION BY BLUE-GREEN ALGAE AND AZOLLA

Introduction

e factorTh s affectin fixatio_ gN Azolln ni d bluaan e green dividee algab n eca d int maio4 n groups. They physica) are(a : l factors chemica) (b , l factors biologica) (c , l ) factor(d d san agronomic factors effecte Th . f thesso fixatioe« N factore th nn so by the blue green algae and Azolla are often different, and not well understood.

Physica) (a l factors. physicae Oth f l factors, climatic conditions cannoe tb changed in the case of field experiments, although water management ansomo t d e degree light intensity (dependin growte th n ho g stagf eo the rice plan culturad tan l practice manipulatede b n )ca N . tracer woulvaluabla e b d e tooassessinr fo l fixatio~ gN n under different light conditions during the growth cycle of the rice plant. Light affects photosynthesis, a process which supplies energy and carbon skeletons for N« fixation. It is therefore important that 14CO. studies be conducted to evaluate the efficiency of different strain blue-greef so n alga Azolld ean photosynthesizo at e th d ean contribution of this to N. fixation.

153 (b) Biological factors.

e effectTh pathogensf so , antagonistic activitief so other organism e ric d 'grazersth e san n cycline i th N n f 'o go ecosyste assayee b n N studiesmeany ca mb d f so »

) Agronomi(c c practices. studieN shon sca w what effect different agronomic practice quantitse e ric th havfixej th N n e eo n f i e dsysteyo th y mb blue-green alga Azollad ean w thes wels ho , a es l a different practices affec release th tfroN f meo incorporated organic materials.

CONCLUSIONS AND RECOMMENDATIONS:

(1) Of the factors affecting N« fixation, chemical and biological factors wels , a agronomis la c practice manipulatee b n sca d to maximize atmospheric N. fixation and N transfer from blue green 15 32 algae and Azolla into the rice plant. (2) N and also P studies shoul employee b d evaluato t d e these processe orden si o t r increas potentiae th e f theslo e system biofertilizerss sa n I ) (3 . addition, 14C studies should be undertaken to assess the contribution mad y photosynthesieb fixatio_ N o st y thesnb e systems.

TURNOVER OF NITROGEN AND ITS AVAILABILITY

INTRODUCTION

The nitrogen fixe cyanobacteriy b d convertes ai d mainly into cellular protein. In order for this nitrogen to be made available for the growt ricef o h must ,i t firs degradede b t e rat Th whict .ea h this happen dependens si cyanobacteriae th n to l species microbiae ,th l

154 compositio padde th yf no soi environmentad lan l conditions. With Azolla, whic particularls hi y important rate ,releasf th eo slowes ei r when the carbon to nitrogen ratio and the silica content are high.

In order to better understand the release of the fixed nitrogen, essentias ii t l that these processe e investigatesar d further under field conditions. The use of N as a tracer will be an invaluable tool in demonstrating the pathway of N from fixer to rice plants and in manipulating the ecosystem to speed up the processes.

RESEARCH APPROACHES

1. Degradation Processes

Blue-green alga Azolld ean a cell decomposee sar y b d

proteolytic enzymes release bacteriy b d d fungaan i (organic N is also released during autolysis and attack by viruses,

myxobacteria and fungi). As the C:N ratio of blue-green alga s realativelei y low proteolytie th , c organisms releasea significant proportion of the nitrogen as ammonia.

Different species and strains of both Azolla and blue-green

algae differ in their susceptibility to decomposition. The speed at which decomposition takes place determines the rate at which the nitrogen is made available to the crop, and also how much is available for various losses of N to occur.

Ther littls ei e information availabl thin eo s under field conditions propose W . e that both blue-green alga Azolld ean a shoul labellee b dfate th e d dtrace an wit N relation i dh n followinge toth : Differen) (a t strain specied san s

155 cellratioe N th C: sf e so Th ) (b (c) Environmental-factors: soil chemistry, temperature pH and water regime (d) The interrelationships between N labelled cells and proteolytic bacteria

effecte concentrationi Th S ) f so (e Azolln si a productioe Th ) (f extracellulaf no r nitrogen.

2. Fat degradatiof eo n products

Ammonia is the main end product of the proteolytic and deaminating activit degradinf o y g bacteria. Thi availabls si o t e rice, but there is competition from other processes. These include

volatilization, uptake by microbes, algae and weeds, adsorption onto colloidal particles and nitrification.

. 3 Losses frosystee th m m (a) Volatilization: such losses are increased by high temperatures, wind speed higd san H (cause hp y b d photosynthesis of algal blooms). Although difficult, it is possibl measuro et e short term volatilization rates with N. This could be done at a later stage of the programme.

(b) Microbial growth: although the ammonia removed through microbial and plant growth will ultimately be released and become available for the growth of rice, there is no guarantee tha wilt ti mad e lb e availabl time th t ei t ea

is needed.

156 Uncontrolled growth of micro-organisms could have an effect on the establishment of blue-green algae, Azolla and rice, could promote anaerobiosis and cause the proliferation of fungal pathogens.

system because:

) nitrati subjecs ei denitrificationo t . ii) there is gaseous loss of NO during nitrification; iii) nitrate is more readily leached than ammonia;

usee b studo t dn ca y N these processe d shoulsan e b d employe thin i d s project.

(d) Leaching: this is mainly a problem after nitrification has occurred. Ammonium is less susceptible to leaching because,it is adsorbed onto colloidal surfaces and remains in the vicinity of rice roots. Tracer studies with N should be able to quantify losses by this process*

RECOMMENDATIONS:

An understandin processee th f go s involve turnovee th n i df ro fixed nitrogen is essential if the potential of biofertilizer is to be realised traceN . r studies shoul employee b d thir fo ds purpose.

The main objective shoulstudo t releas e fixee e yth b dth N df eo from the Azolla or blue-green algae (biofertilizer) and measure its availabilit rice th e o plantyt .

157 The processes involved in this release should also be studied.

The following major factors shoul takee b d n into account during such studies:

1. Existing agronomic practices 2. Incorporation versus surface application of the biofertilizer 3. The timing of the release of ammonia. 4. The residual effect of biofertilizer N on subsequent crops . 5 Susceptibilit degradatioo yt n shoul traia e b dt taken into account during strain selection . 6 Searc naturar hfo mutand lan t strains which liberate ammoni extracellulad aan r nitrogen.

7. The role played by the soil fauna. e effectTh pesticide. f 8 so biodégradationn so .

possible Th . 9 e effect growtf so h promoting substances

(there is premature release of nitrogen when the inoculum response th failplane th t s greatetsi bu f e o r than that due to nitrogen alone).

REFEBENCES:

1. Becking, J.H. (1978). Ecology and physiological adaptations of Anabaen Azolla-Anabaene th n ai a azollae symbiosis. Ecol. Bul. (Stockholm) j26:266-281. 2. Becking, J.H. (1978). Environmental requirements of Azolla for tropican i e us l rice production. Nitroge Ricd n. an e p 345-373, IRRI, Los Banos, Philippines. 3. Becking, J.H. and Donze, M. (1981). Pigment distribution i and nitrogen fixatio Anabaenn ni a azollae. Pland tan Soil 6:203-226. 158 4. Burrls, MillerR.Hd an . , C.E. (1941) Applicatioo t N 15 f no the stud biologicaf o y l nitrogen fixation* Science 93:114-15. 5. Dilworth, M.J. (1966) Acetylene reduction by nitrogen-fixing preparations from Clostrldium pasteurianum. Biochem. Biophys. Acta 127:285-294. 6. Fried, M. and Broeshart, H. (1975) An independent measure of the amount of nitrogen fixed by a legume crop. Plant and Soil 43:707-711. 7. Fried Danso, ,M. , S.R.À Zapatd .an methodologe 1983. aF Th . y

of measuremen fixatio- N f to nonlegumey nb inferres sa d from field experiments with legumes. Can. J. Microbiol. In press. SoilO FA s . Bulletin8 (19791 4 . )No . China : Azolla propogation

and small-scale biogas technology.

9. Gunnison, D. and Alexander, M. (1975). Resistance and susceptibilit algaf yo decompositioo et naturay nb l microbial communities. Liranol. Oceanogr. 20, 64-70. 10. Jones, K. (1982). The Use of N-labelled dinitrogen in the study of nitrogen fixation by blue-green algae. FAO/IAEA Consultants MeetinRole f Isotopeth eo n go n Studiesi n so Nitrogen Fixation and Nitrogen Cycling by blue-green Alga theid ean r Associations. OctoberVienna5 1 - 1 ,1 , 1982. Knowle. 11 (1981). sR measuremene Th . nitrogef to n fixationn I . Current Perspectives in Nitrogen Fixation. A.B. Gibson and W.E. Newton eds. Australian Acad. Sei., Canberra, Australia pp. 327-333.

159 12. Lumpkin T.A. Plucknett, D.L. (1982) Azolla as a Green Manure, use

and Management in Crop Production« Westview Press,

Boulder, Colorado, U.S.A. 13. Nitrogen and Rice. Int. Rice Research Institute. Manila, Philippines. (1979). . Reynaud14 (1982). .P . Growt Azollf o h a african Nitroged aan n fixation. Environmental conditions and plots inoculation assay. Consultant Meeting. FAO/IAEA Consultants Meeting Role of th Isotopeneo n Studiesi Nitrogen so n Fixation and Nitrogen Cycling by blue-green Algae and their Associations, October5 Vienna1 - 1 ,1 , 1982. 15. Roger P., and S.A. Kulasorriya (1980). Blue-green Algae and

Rice. IRRI Banoss ,Lo , Philippines.

16. Roger P.A., Watanabe I. 1982. Research on algae, blue-green algae, and photo tropic nitrogen fixation at the IRRI (1963 - 81), Summarization, Problems and Prospects. IRPS No. 78.

17. Schöllhorn, R. and Burris, R.H. (1967) Acetylene as a competitive inhibitor of N_ fixation. Proc. Nat. Acad. Sei. U.S.A. 38:213-216. . Venkataraman18 , G.S. (1981) Blue-green Alga Ricr efo e Production.

FAO Soils Bulleti. 46 . nNo 19. Verma, L. and Martin, J.P. (1976). Decomposition of algal cells and components and their stabilisation through complexing with model humic acid-type phenolic polymers. Soil Biol. Biochem. S_, 85-90. . Wagne21 r G.H Zapatad .an (1982). ,F . Field evaluatio referencf no e stude cropth nitroge f yo n si n fixatio legumey nb s using isotopic techniques. Agrom 74:607-612. .J .

160 21. Watanabe I. and Roger P.A* (1982). use of N in the study of biological nitrogen fixation in paddy soils at the IRRI

Consultants Meeting, FAO/IAEA Joint Project. Vienna.

22. Watanabe, I., Rai Ke-Zhi, Berger, N.S. Espinas, C.R. Ito, 0., B.P.R. Subudhi, 1981. The Azolla-Anabaena complex and

its use in rice culture. IRPS. No. 69

15 . Witty23 J.M,y J.PDa . d (1978)f .an o Nevaluatin n 2e i us . g asymbiotic N- fixation in Isotopes in Biological

Dinitrogen Fixation. IAEA, Vienna, Austria.

161 LIS PARTICIPANTF TO S

Danso, S.K.F., Scientific Secretary Soil Fertility, Irrigation and Fried, M., Chairman Crop Production Section Joint FAO/IAEA Division P.O. Box 100, A-1400 Vienna, Austria

Invited Speakers

Becking, J.H. Institut Atomir efo c Sciences in Agriculture 6 Keyenbergseweg, Postbus 48 Wageningen Netherlande ,Th s

Roger, P.A. The International Rice Research Institute 3 93 P.Ox Bo . Manila, Philippines

Reynaud, P.A. Office de la Recherche Scientifique et Technique Outre-Mer Centre Orstor Dakae nd r B.P. 1386, Dakar Senegal

Kulasooriya, S.A. Departmen Botanf to y University of Peradeniya Peradeniya, Sri Lanka

Junes. ,K Departmen Biologicaf to l Sciences University of Lancaster Lancaster LAI 4YO LancashireK ,U

Stewart, W.O.P. Department of Biological Sciences Universite Th DUNDEf yo E DUNDE 4HN1 EDD , Scotland

Dujardin. ,E Université de Liege Department of Botanique Sart Tilman, Liege Belgium

Ofori, O.S. FAO, AGL, Rome, Italy

163 Staff Members

Zifferero. ,M Deputy Director General Department of Research and Isotopes

Lamm. ,C Deputy Director Joint FAO/IAEA Division

Reichardt, K. Head, Soil Fertility, Irrigation and Crop Production Section

Zapata, F. Agricultural Section Seibersdorf Laboratory

Hardarson, G. Agricultural Section Seibersdorf Laboratory

Kawai, T. Plant Breedin Geneticd an g s Section

Pascuali. ,J Agricultural Section Seibersdorf Laboratory

in no

164