Plant and Soil221: 107–112, 2000.© 2000Kluwer Academic Publishers. Printed in the Netherlands.

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Contribution of N 2 fixing cyanobacteria to rice production: availability ofnitrogen from 15N-labelled cyanobacteria and ammonium sulphate to rice

E. Fernandez Valiente1,∗, A. Ucha1, A. Quesada1, F. Leganes1 and R. Carreres21Dpt. Biologıa, Universidad Aut´onoma de Madrid, 28049-Madrid, Spain and2Dpt. del Arroz, IVIA, Sueca,46410-Valencia, Spain

Received 30 March 1999. Accepted in revised form 22 September 1999

Key words:Cyanobacteria, ‘in situ’ N2 fixation, N fertilizer,15N balance, rice

Abstract

This study investigate the potential contribution of nitrogen fixation by indigenous cyanobacteria to rice productionin the rice fields of Valencia (Spain). N2-fixing cyanobacteria abundance and N2 fixation decreased with increasingamounts of fertilizers. Grain yield increased with increasing amounts of fertilizers up to 70 kg N ha−1. No furtherincrease was observed with 140 kg N ha−1. Soil N was the main source of N for rice, only 8–14% of the total Nincorporated by plants derived from15N fertilizer. Recovery of applied15N-ammonium sulphate by the soil–plantsystem was lower than 50%. Losses were attributed to ammonia volatilization, since only 0.3–1% of applied N waslost by denitrification. Recovery of15N from labeled cyanobacteria by the soil–plant system was higher than thatfrom chemical fertilizers. Cyanobacterial N was available to rice plant even at the tillering stage, 20 days after Napplication.

Introduction

Nitrogen is a key factor for lowland rice production.However, the efficiency of N fertilizer in rice crop isone of the lowest among the plant nutrients, due tolarge N losses from flooded soil (De Datta and Bur-esh, 1989). In fact, soil N is the principal source ofN for lowland rice. More than 50% of the N used byrice receiving N fertilizers derived from native soil N(Broadbent, 1984). Soil N pool is believed to be main-tained through biological nitrogen fixation (Kundu andLadha, 1995; Roger and Ladha, 1992) and fertilizerN. Among indigenous nitrogen-fixers in rice fields,cyanobacteria are the main contributors to N2 fixa-tion (Roger and Ladha, 1992). Direct evidences of thetransfer of cyanobacterial N to rice plant are, however,scarce (Roger, 1996).

Most studies on N utilization by rice and the con-tribution of N2 fixation to soil fertility in rice fieldswere conducted in Asia. Little work has been done inEuropean rice fields where agrochemical use markedly

∗ FAX No: +34 1 913978344.E-mail: eduardo.fernandez@uam.es

differs from that in Asia. Agrochemical use increasesyields, but involves numerous environmental problemsleading to a progressive loss of soil quality due to theirnegative effects on soil micro-organisms, particularlyon N2-fixing cyanobacteria (Roger and Ladha, 1992).The improvement of soil utilization, by preserving anddeveloping the natural ability of N2-fixing organismsfor biofertilization, would permit a reduction in N fer-tilizers use, with obvious ecological, economic andenvironmental benefits.

The occurence and distribution of N2-fixing cy-anobacteria and N2 fixation in the rice fields of Valen-cia (Spain) and their relationship with the physical andchemical characteristics of water and sediments weredescribed in previous papers (Quesada and FernándezValiente, 1996; Quesada et al., 1997). N2-fixing cy-anobacteria were present at similar densities than inAsian rice fields (Quesada and Fernández Valiente,1996). The estimate of the nitrogen fixed in placeswhere no cyanobacterial presence was visually appar-ent ranged from 0.23 to 75.5 kg N ha−1 year−1. Whencyanobacterial blooms were present, nitrogen fixationreached 2 kg N ha−1 day−1 (Quesada et al., 1997).

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This work evaluates the agronomic role of N2-fixing cyanobacteria and biological N2 fixation in therice fields of Valencia, Spain. The objectives were:(1) To investigate the effect of different amounts ofinorganic N fertilizers on N2 fixation and rice yieldand (2) to evaluate the recovery by rice of N fromcyanobacteria and ammonium sulphate, using15Nlabelled material.

Materials and methods

The rice fields studied are located in the EasternIberian Peninsula, surrounding the coastal lagoon of‘La Albufera’ near Valencia. Spain. A detailed de-scription of the site and rice crop cycle is givenelsewhere (Quesada et al., 1995a,b).

Agronomic study

Field experiments were conducted in three consecut-ive crop seasons (1990–92) in the experimental fieldsof the Rice Department (Valencian Institute for Agri-cultural Research at Sueca-Valencia). Field trials wereperformed in plots (5×20 m), laterally isolated byplastic sheets embedded into the soil to a depth of10 cm. Treatments consisted of five levels of nitrogen(0, 17.5, 35, 70 and 140 kg N ha−1) supplied as am-monium sulphate. A basal dose of 100 kg ha−1 P2O5was supplied, as superphosphate, in all plots. Plotswere laid out in a randomized complete block design,with four replicates per treatment. Basal N and P doseswere applied as a single broadcast application andcovered by about 3 cm of soil, 1–2 days before flood-ing. After the initial flood, around mid May, plots werehand-sown at 180 kg ha−1 seed (rice variety Senia),pre-soaked in tap water. At crop maturity grain andstraw yield and N content in plants were determined(Carreres et al., 1996).

Cyanobacterial population in the soil was meas-ured in cores collected from each plot, at 4–5-weeklyintervals throughout the growth cycles, as previouslydescribed (Quesada and Fernández Valiente, 1996).Likewise, ‘in situ’ assays of nitrogenase activity(ARA) (Quesada et al., 1989) were performed in foursampling periods, May, June, July and September,every year . At each sampling period, three assaychambers were placed in each plot.

15N labeling study

Availability of N from chemical15N-fertilizers to rice

was studied in three consecutive crop seasons (1995–97) in two experimental fields of the Rice Depart-ment (Valencian Institute for Agricultural Research atSueca-Valencia) located 20 km from each other. Tri-als were performed in microplots (1 m2), isolated byplastic sheets embedded into the soil to a depth of 10cm. Treatments consisted of three levels of nitrogen (0,70 and 140 kg N ha−1) supplied as15N-ammoniumsulphate (5–13.5 atom % excess15N, depending onthe year). Fertilizer was spread evenly on the soil sur-face and then mixed in the top 3 cm soil layer, 1–2days before flooding. Three days later rice seedlingswere transplanted at 20 cm spacing (24 rice hills permicroplot). At harvest all plants were collected, andgrain, straw, and root yields determined. Plant sampleswere analyzed separately for total N and15N using anautomatic nitrogen analyzer coupled to a mass spec-trometer. Soil samples were collected at 0–10, 10–20and 20–30 cm depth and analyzed separately for totalN and15N. N2 fixation (ARA) and denitrification weremeasured throughout the crop cycle in all microplots.Denitrification was measured by the acetylene inhibi-tion technique and by direct measurement of15N2 and15N2O evolved from soil (Buresh and De Datta, 1990),using the same assay chambers as for ARA.

Experiments to compare the recovery by plants ofnitrogen from15N-labelled cyanobacteria (25.7 atom% excess15N) and from15N-ammonium sulphate (10–13.5 atom % excess15N) were performed in shallowponds filled with soil from the rice fields (1996) or inthe field (1997). Cyanobacteria and ammonium sulph-ate were added in the same amounts (18–26 kg Nha−1, depending on the year). Trials in the ponds orin the field were performed as above except that mi-croplots were 0.25 m2and accommodated nine ricehills. Labelled cyanobacteria were obtained in thelaboratory by culturing cells ofNostocUAM 206 (isol-ated from the rice fields of Valencia) in BG-11mediumcontaining 15N-labelled KNO3 (75 atom % excess15N). Cells were collected by centrifugation, dried at60◦C and gently ground by pestle and mortar.

N recovery was calculated by the15N tracermethod and by the non-isotopic difference method(Diekmann et al., 1993).

15N tracer method:

15N recovery fraction =

atom % excess15N in the plant×N uptakein fertilizer treatmentatom % excess15N in the applied fertilizer× fertilizer N applied

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Table 1. Effect of ammonium fertilization on cyanobacterial abundance, N2 fixation, grain yield and N responses of rice: mean data ofthree consecutive crop seasons (1990, 1991, 1992)

N applied Cyanobacteria N2 fixation Grain yield N uptake N-use efficiency

(kg N ha−1) (CFU cm−2.105) (µmol ethylene m−2 h−1) (t ha−1) (kg ha−1) (kg grain kg N−1)

0 2.20 291 5.95 77.2 —

17.5 1.86 269 6.60 90.1 48.7

35 1.69 175 6.73 90.5 25.2

70 1.34 159 7.37 99.4 20.8

140 1.16 114 7.59 105.6 11.1

LSD (0.05) NS NS 0.51 11.5 8.4

Difference method:

Apparent recovery (AR) =N uptake in fertilized treatments−N up-take in control (no fertilizer) treatmentsFertilizer N applied

The so-called ‘priming effect’ or added N inter-action was equivalent to the difference between Nrecoveries calculated by the difference method and bythe15N tracer method (Diekmann et al., 1993).

Results and discussion

Agronomic study

A significant negative correlation (r= −0.53; P <

0.05;n= 35) between the abundance of N2-fixing cy-anobacteria and amount of N fertilizer applied wasobserved (Table 1). These results agree with previousecological data which indicated a negative correla-tion between the concentration of inorganic nitrogenin floodwater and cyanobacterial abundance (Quesadaand Fernández Valiente, 1996). However, differencesamong treatments were small and not significant byone-way analysis of variance.

In situ measurements of N2 fixation confirmedthe inhibitory effect of N fertilizers on N2-fixing cy-anobacteria. Nitrogenase activity decreased linearlyas the amount of N fertilizer increased (Table 1). Asignificant negative correlation (r= −0.90;P < 0.05;n= 60) between acetylene reducing activity and theamount of N fertilizer was observed. The inhibitionwas only partial and differences among treatmentswere only significant (one-way analysis of variance) intwo years (1990, 1991). Further studies in followingyears in the microplots used for15N-labeling exper-iments gave similar results (data not shown). Mean

values for the six years (1990–92 and 1995–97) were:240 ± 190 µmol ethylene m−2 h−1 in unfertilizedplots; 140± 154 µmol ethylene m−2 h−1 in plotsfertilized with 70 kg N ha−1 and 109± 153 µmolethylene m−2 h−1 in plots fertilized with 140 kg Nha−1. One-way analysis of variance of the mean datafor the 6 years indicated significant differences amongtreatments (P<0.012). Multiple comparisons by theBonferronit-test indicated that differences were onlysignificant between 0 and 140 kg N ha−1.

Assuming a ratio of acetylene reduced to N2 fixedof 5:1, a crop season of 137 days, and that nitrogenaseactivity during 1 h at noon is 8.3% of total fixationthroughout the day (Quesada et al., 1997), the dataof acetylene reducing activity may be used to make arough estimate of N fixed. The estimate range from 30kg N ha−1 crop−1 in unfertilized plots to 13 kg N ha−1

crop−1 in plots fertilized with 140 kg N ha−1. Thesevalues are in the range to those recorded in tropicalrice fields (Roger and Ladha, 1992).

Grain yield increased with increasing amounts ofN-fertilizers applied (Table 1). Grain yield in unfertil-ized plots was, during the 3 years, only 16–25% lowerthan in plots fertilized with 140 kg N ha−1. This meansthat the amount of N released in unfertilized plots wassufficient to assure an adequate supply of N to rice.Interestingly, there were no significant differences ingrain yield between plots fertilized with 70 or 140kg N ha−1 during the three consecutive crop seasons.These results indicated that the amount of N-fertilizersusually added by farmers in Valencian rice fields (140kg N ha−1) may be excessive and can be reduced by a50% without significant loss of productivity.

The amount of N utilized by plants increased withthe level of N-fertilizers (Table 1). The highest Nuptake was recorded in plots fertilized with 140 kgN ha−1, though there were no significant differences

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Table 2. N uptake, and recovery by plants and soil of15N-labeled fertilizer: mean of field experiments in three consecutive crop seasons(1995, 1996 and 1997)± standard deviation

15N recovery

N applied N uptake Plant (kg N ha−1) Soil (kg N ha−1) Total

(kg ha−1) (kg ha−1) Grain Straw Roots Total 0–10 cm 10–20 cm 20–30 cm Total (%)

0 117±26 – – – – – – – – –

70 137±44 7.0±1 3.4±1 0.7±0.1 11.1±2 15.3±4 4.5± 3 0.4±0.4 19.8±2 44.1

140 172±46 14.0±1 8.1±1 1.8±0.2 23.9±1 26.9±5 6.2±4 1.3±0.5 34.4±1 41.6

Table 3. Recovery of applied 15N-cyanobacteria and15N-ammonium sulphate: shallow pond experiment in the cropseason of 1996 (mean±SD of four replicates)

15N recovery (%)

Treatmenta Tillering Harvest

Plant Plant Soil Total

Cyanobacteria 12.3± 0.6 8.0± 0.5 47.8± 6 55.8

(NH4)2SO4 12.0± 0.4 7.2± 0.3 34.4± 5 41.6

a18 kg N ha−1.

when compared with plots fertilized with 70 kg Nha−1. In plots fertilized with 140 kg N ha−1, theamount of N utilized by plants was lower than thequantity applied as N fertilizers, indicating a pos-sible pollution of water bodies. In the other cases,the amount of N utilized by plants was higher thanthe applied as N fertilizers. The difference betweenN removed and N applied was higher at lower N in-put levels. A significant positive correlation (r=0.85;P<0.05) between N2 fixation and the differencebetween removed and applied N was found, suggest-ing that N2 fixation contributes to the maintenance ofgrain yield and fertility in rice fields.

Values of fertilizer N-use efficiency were signi-ficantly lower than that reported for other rice fields(Yanni, 1992), and decreased linearly as rate of N in-creased (Table 1). This low efficient use of fertilizersuggested high N losses in valencian rice fields.

15N labeling study

Nitrogen uptake and recovery from15N-ammoniumsulphate were studied in microplots experiments, toobtain a more direct information on the dynamics ofutilization of N by rice plants.

As in other studies (e.g., Sisworo et al., 1990),grain yield (not shown) and N uptake (Table 2) in the

microplots experiments were greater than those previ-ously observed in large plots (Table 1), although thedifferences among treatments were in the same range.Nitrogen recovery by plant was lower than in otherstudies (Sisworo et al., 1990; Tirol et al., 1982). Thepercentage of15N recovered by plants ranged from13.5 to 20%, depending on the year and treatment,with mean values of 15.8 and 17.0, respectively, forthe treatments of 70 and 140 kg N ha−1 (Table 2).Most of the fertilizer was recovered in the grain (about60%) since panicle is the part of the plant with higherN content (not shown).

From the data shown in Table 2 we can estimatethe origin of the N incorporated in the plants: 85% ofthe total N incorporated by plants fertilized with 70 kgN ha−1 derived from the soil N, 8% derived from fer-tilizer N and 7% derived from soil N by priming effectof fertilizer (increase in uptake of soil N in treatmentswhere N was applied). In plants fertilized with 140 kgN ha−1, 68% of the total N derived from soil N, 14%derived from fertilizer N and 18% derived from soil Nby priming effect.

At the end of the crop, the soil retained 28.2% ofapplied N in plots fertilized with 70 kg N ha−1, and24.5% in plots fertilized with 140 kg N ha−1 (Table2). About 78% of the total15N remaining in the soilwas found in the top 10 cm, suggesting that losses byleaching were not important.

The total15N recovered from the soil–plant systemat the end of the crop ranged from 40 to 47.2%, de-pending on the years and treatment, with mean valuesof 44.1 and 41.6, respectively, for the treatments of 70and 140 kg N ha−1 (Table 2). This means that morethan 50% of applied N was lost during the crop cycle.These losses cannot be due to denitrification since thisprocess took only place for a few days after floodingand with low rates (0.4–4 g N/ha h−1), in such a waythat only 0.3–1% of applied N was lost from denitri-fication. Therefore, since leaching and denitrification

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Table 4. Effect of N fertilization with cyanobacteria or ammonium sulphate on rice yield, N uptake, and recovery by plants and soil of15N-labeled fertilizers: data from field experiments in the crop season of 1997 (mean± SD of four replicates)

Yield (t ha−1) N uptake 15N recovery (%)

Treatmenta Grain Straw (kg ha−1) Plant Soil Total

Cyanobacteria 6.8± 1.0 4.6± 0.7 101± 15 10.2± 0.9 36.4± 2.4 46.6

(NH4)2SO4 7.2± 0.3 4.8± 0.5 100± 7 4.9± 0.6 21.1± 1.8 26.0

a26 kg N ha−1.

were not significant, losses have to be due to volatil-ization of ammonia, which should be favoured by thehigh values of pH found in water (from 7.5 to 10.5, de-pending on the time of day and on the crop phase) andsediments (from 7.4 to 7.6) (Quesada et al., 1995a,b).

The other objective of this study was to determ-ine the availability of cyanobacterial nitrogen to riceplant. Recovery of cyanobacterial nitrogen was com-pared with the recovery of the same amount of labeledammonium sulphate, in shallow ponds and field exper-iments.

The availability of cyanobacterial nitrogen to riceplant in shallow ponds experiments was similar to thatof chemical fertilizer, even at the tillering stage, in-dicating a fast mineralization of organic nitrogen inthe soil and a fast transfer of fixed nitrogen to riceplants (Table 3). The amount of cyanobacterial nitro-gen recovered in plants was lower than in other studies(Tirol et al., 1982), but in the same range than thatof ammonium sulphate. However, the amount of cy-anobacterial nitrogen recovered in soil was higher thanthat from chemical fertilizer, which was in the samerange than in the previous experiments.

No differences in grain and straw yields and inN uptake between plants fertilized with cyanobacteriaand ammonium sulphate were found in field experi-ments (Table 4). However, recovery of cyanobacterialnitrogen, both in plant and soil, was higher than thatof ammonium sulphate. The amount of ammoniumsulphate recovered in this experiment was significantlylower than that of the experiments commented aboveand could be underestimated.

In agreement with other reports (Norman et al.,1992; Wilson et al., 1989) the percentage of15N re-covered at maturity was lower than at tillering (Table3). These results suggest that the plant depends on thenative soil N to meet its N requirements during the lat-ter phases of its vegetative growth (Kundu and Ladha,1995; Wilson et al., 1989).

The results of this study show that valencian ricefields have favourable conditions for the development

of indigenous N2-fixing cyanobacteria and biologicalnitrogen fixation. Isotopic experiments reveal that Nfixed by cyanobacteria replenish soil N pool, which isthe main source of N for rice (67–85%), and is readilyavailable to rice plant in a short time. Thus, the abund-ance of these microorganisms improves soil fertilityallowing to obtain high grain yields (about 6 t/ha) inunfertilized plots. The input of N fertilizers up to 70kg N ha−1 give rise to a moderate increase in grainyield (about 20%) without severely affect N2 fixingcyanobacteria. No further increase can be obtained ingrain yield with higher levels of fertilizers, which, inturn, negatively affects N2 fixing cyanobacteria andbiological nitrogen fixation. The low efficiency ofN-fertilizers revealed by agronomic and isotopic ex-periments seems to be a consequence of the significantlosses by ammonia volatilization, a chemical processwhich is difficult to avoid due to the alkaline con-ditions of waters and sediments and to the climaticconditions of the geographic area with moderate butfrequent winds along the crop season.

In conclusion, our results indicate that a reasonableutilization of biological nitrogen fixation in combina-tion with inorganic nitrogen fertilization would allowa significant reduction in N fertilizer input without lossof productivity and with the ecological benefit that itmay represent for the ecosystem.

Acknowledgements

This work was supported by grants from ComisiónInterministerial de Ciencia y Tecnología (CICYTAGR89-0217-CO2-01,02; AGF93-0807-CO2-01,02;AGF97-0303-CO2-01,02).

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