Biol Fertil Soils (2000) 32 :484–493 Q Springer-Verlag 2000

W.M.H.G. Engelaar (Y) 7 T. YoneyamaPlant Nutrition Diagnosis Laboratory,National Agriculture Research Center,Kannondai 3-1-1 Tsukuba, Ibaraki 305-8666, Japane-mail: willeme6narc.affrc.go.jpFax: c81-298-388951

T. MatsumaruSchool of Environmental Sciences, University of Tsukuba,Tsukuba, Ibaraki 305-8572, Japan

ORIGINAL PAPER

Willem M.H.G. Engelaar 7 Taigo MatsumaruTadakatsu Yoneyama

Combined effects of soil waterlogging and compaction

on rice (Oryza sativa L.) growth, soil aeration,

soil N transformations and 15N discrimination

Received: 4 February 2000

Abstract The combined effects of soil compaction andsoil waterlogging on the growth of two rice cultivars(Oryza sativa L., cultivars Kanto 168 and Koshihikari)and soil N transformations were studied in pots. Al-though waterlogging eliminated initial differences inmechanical resistance between compacted and loosesoils, Kanto 168 and Koshihikari roots had, respective-ly, less biomass and a lower porosity if soil was com-pacted prior to waterlogging. The cause for this wasprobably established before waterlogging. Redox val-ues showed that upland soils were well aerated. Loosewaterlogged soils contained oxic sites, but compactedwaterlogged soils did not. Potential denitrification wasstimulated by waterlogging and, to a larger extent, byplant presence. Waterlogging lowered potential nitrify-ing capacities, by competition between plants and mi-cro-organisms for NH4

crather than by oxygen shortage.Compaction prior to waterlogging benefited the poten-tial nitrifying capacity of soils with either cultivar andthe potential denitrifying capacity for soils with Koshi-hikari. Compaction had no effect on nitrification or de-nitrification in upland soils. N recoveries were low, es-pecially in pots without plants, as a result from sam-pling strategy and N loss. On day 42/43 after potting,total d15N values of waterlogged pots were positive,whereas after 22 days all pots had negative total d15Nvalues. Final d15N values of plant parts from waterlog-ged and upland soils were positive and negative, re-spectively. Although the d15N values generally ac-corded well with the other results, they did not support

higher N losses from compacted waterlogged soils thanfrom loose waterlogged soils with plants, as suggestedby potential denitrifying activities.

Keywords d15N 7 N transformations 7 Rice (Oryzasativa L.) 7 Soil aeration 7 Soil mechanical resistance

Introduction

The predominance of either ammonium or nitrate in asoil is the result of processes influencing their produc-tion, consumption and distribution. The compositionand size of the microbial population, the species andbiomass of plants and the presence or absence of oxy-gen in the soil are important parameters that influencethese processes. When the oxygen in a soil runs out as aresult of obstructed oxygen diffusion in combinationwith consumption by plant roots and soil organisms, ni-trification is replaced as most important N-transform-ing process by denitrification and/or nitrate ammonifi-cation (Laanbroek 1990). Many intermediates of thelatter two processes are volatile and may be lost fromthe soil (Conrad 1995), replacing nitrate leaching as themain cause of N loss from the soil.

Oxygen diffusion into the soil is inhibited when thecontinuity of gas-filled pores is limited, e.g. by a com-pacted soil layer (pincreased bulk density) or a highsoil moisture content, which can be caused by treadingby cattle or machinery and waterlogging, respectively.Some plant species can supply atmospheric oxygen totheir rhizosphere by radial oxygen loss from highly por-ous (aerenchymatous) roots (Both et al. 1992; Engelaaret al. 1995), thus relieving some of the stress of oxygendeficiency. Localised oxygen loss from roots increasesheterogeneity in oxygen status of the soil, and hencepossibly the occurrence of nitrification and denitrifica-tion. This capacity depends, among other parameters,on the fractional root porosity and continuity of theaerenchyma (Armstrong and Beckett 1987; Armstronget al. 1991).

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Soil compaction may not only inhibit oxygen diffu-sion into the soil, but also the formation of highly por-ous roots as a result of increased mechanical resistance(Engelaar et al. 1993). The actual compaction effectwill depend on soil particle size distribution and watercontent (Boone et al. 1986), with a high and low watercontent generally favouring hypoxia and increased soilpressures, respectively (Boone et al. 1986; Borchert andGraf 1988).

Rice (Oryza sativa L.) is very suitable for studyinginteractions between soil compaction and waterlogging,with respect to soil aeration and N cycling. The envi-ronmental importance of N transformations in ricefields has been recognised by studies on the possibleinfluence of rice on nitrification-denitrification relatedN losses from soil (Smith and Delaune 1984), and morerecently, on the effect of ammonium-based fertilizationon methane oxidation in the rice rhizosphere (Bodelieret al. 2000). Rice cultivars produce aerenchymatousroots to different extents (Das and Jat 1977), and oxy-gen is only released at the root tip, which results in highspatial and temporal variations in oxygen availability(Justin and Armstrong 1987; Flessa and Fischer 1992).Combined with the fact that rice is grown both underupland conditions and in waterlogged paddy fields, thiscreates highly diverse and dynamic plant-soil systems inwhich N is not only transformed but also continuouslyredistributed.

The measurement of d15N (natural abundance of15N), a comparatively new tool in ecological studies ofN cycling (Handley and Scrimgeour 1997; Högberg1997), may be very useful in studying such complex sys-tems with many interacting parameters and has beenused in a number of studies on the N cycling in paddysoils (Watanabe et al. 1981; Fillery and Vlek 1982; Ni-shio 1994; Nishio et al. 1994). d15N measurements notonly provide information about quantities of nitrate,ammonium and organic N, like conventional extractionand digestion methods do, but also reveal the possiblehistory of the N pool under study (Yoneyama et al.1990), such as the origin of N incorporated in plants(Tobita et al. 1994).

In this paper we describe the effects of combinedsoil waterlogging and increased bulk density on tworice cultivars, Kanto 168 (grown on relatively dryfields) and Koshihikari (grown in waterlogged pad-dies), soil aeration and nitrogen transformations anddistribution. We hypothesise that the presence of aer-enchymatous rice roots in waterlogged soils would in-crease the spatial variation in oxygen availability and,subsequently, in occurrence and rate of microbial Ntransformations. Such variations would not be expectedwhen the production of highly porous roots in hypoxicsoils is limited, as may be caused by the combination ofhigh soil strengths and small soil pore size in compactedsoils or insufficient adaptation to waterlogging of aplant species/cultivar that normally does not grow onwaterlogged soils, such as Kanto 168. The d15N valuesof several N pools in the rice plant-soil system were

measured, together with root and shoot development,soil aeration, potential nitrification and denitrificationactivities, and plant and soil N contents. These data arediscussed in view of the above hypothesis.

Materials and methods

Soil and plant preparations

An originally alluvial soil (pH-H2Op5.8) was collected from out-door plots previously grown with upland crops for 10 years at theNational Agriculture Research Center, Tsukuba, Japan. The soilwas passed through a 2 mm sieve and its water-holding capacity(WHC) measured. The sieved soil contained 6.3% coarse sand,18.3% fine sand, 45.0% silt and 30.5% clay, as determined withthe pipet method (Gee and Bauder 1986), which classifies it as alight clay to silty clay soil in the ISSS system. The organic C con-tent was 14.6 g C kg–1 dry soil and total N content was 1.68 g Nkg–1 dry soil (d15Npc6.07‰), with exchangeable NH4

c-N(d15Npc0.8‰) and NO3

–-N (d15Npc0.5‰) contents of 4.9 and7.7 mg kg–1 dry soil, respectively. In total, 80 polycarbonate potseach containing 1.7 l (inner height 18.7 cm, mean i.d. 10.8 cm)were prepared, 40 with loose soil (bulk density p 0.92 Mg dry soilm–3) and 40 with compacted soil (1.09 Mg m–3). For both groups1450 g fresh soil (p1197 g dry soil) was used per pot. To each pot1.5 g (NH4)2SO4 (d15Np0.0B0.1‰) and 0.5 g KH2PO4 were ad-ded. Pots with loose soil were filled in time and deionised waterwas added to a total of 0.35 kg water per kg dry soil (p60% ofWHC). Soil and water in compacted pots were added in threelayers. Twenty-four hours after applying a layer it was compactedwith a rubber stamper and water and the surface was scratchedbefore applying the next layer. To compacted soils only 0.29 kgwater per kg dry soil (p50% of WHC) was added, to preventexcessively wet conditions. Seeds of Kanto 168 and Koshihikariwere germinated on moistened cotton wool at 25 7C in the darkfor 4 days and then transferred to water-saturated quartz sand ina glasshouse. After another 9 days they were transferred, in pairs,to 12 pots with loose and 12 pots with compacted soil per cultivar.The remaining 32 pots contained only soil. Dying and dead seed-lings were replaced so that after 5 days all pots with plants con-tained two viable seedlings.

Treatments and harvests

The temperature in the glasshouse was kept at 25 7C. Light condi-tions were dominated by sunlight, but additional lighting was pro-vided by halogen lamps (National, MF400 L/BU-P) at a photo-synthetic photon flux density of 57 mmol s–1 m–2 (400–700 nm) atplant level, from 6.00 a.m. until 8.00 p.m. Daily watering withdeionised water to a predetermined pot weight compensated forevapotranspiration. After 22 days, five pots with loose and fivepots with compacted soil without plants were collected, as well astwo loose and two compacted pots with plants for both cultivars.Plants selected for harvest had clearly larger or smaller shootsthan the remaining plants. We assumed their roots also to be thelargest and smallest of all plants present and, because all har-vested plants had healthy roots, it was concluded that the remain-ing plants also had healthy roots. At day 22, half the number ofremaining pots was waterlogged to a level of 1–2 cm over the soilsurface. This level was maintained until the final harvest on day42 for Kanto 168 and pots with soil only, and on day 43 for Ko-shihikari. Non-waterlogged soils are referred to as upland soils,even though an originally alluvial soil was used. At harvest, theplants were removed from the soil and soil samples were taken asclose to the roots as possible. Shoots and roots were separatedand roots thoroughly washed. Samples from pots without plantswere taken from similar depths as the maximum rooting depth intheir planted equivalents. Soil samples and roots were stored at

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4 7C until further analysis. Shoots were weighted, dried at 70 7Cand weighed again. Pot sizes and harvest times were chosen toachieve a high root density in the soil without limiting plantgrowth. Plants were harvested before panicle onset or major die-back of shoots, which are both likely to induce major changes inN allocations and d15N values and thus complicate data interpre-tation.

Analysis

Root porosity

The washed roots were divided into thick and thin roots. Thickroots consisted mainly of non-branched roots without lateral rootgrowth. Thin roots included thin laterals and highly branchedthick roots that could not be removed without damaging the lat-erals. Root porosity was measured according to Jensen et al.(1969), with modifications. Pycnometers with an internal volumeof 10 ml were used and the amount of roots was generally be-tween 200 and 600 mg. In four cases an amount of roots rangingfrom 129 to 200 mg was used, due to insufficient biomass produc-tion. Cut root segments of 1 cm length were placed in a pycnom-eter and the air was extracted until no more bubbles escapedfrom the roots or water. Vacuum was applied once more, afterroots extruding from the water surface during the first air extrac-tion were re-submerged. Roots not used for the porosity measur-ements were dried at 70 7C and weighed. Total root dry weightsand porosities were calculated.

Soil strength

Directly after removal of the shoot, the mechanical resistance ofthe soil was measured with a penetrograph (Daiki, Tokyo, Japan)with a cone of 3 cm2 and a tip angle of 607.

Soil redox potential

Three days after the first harvest, Pt-electrodes were placed at5 cm depth in two out of five waterlogged pots from all series andin one out of five upland pots from every series with plants (18electrodes in total). From then on the redox potential was moni-tored using a mV-meter and an AgCl2/saturated KCl referenceelectrode (TOA Electronics, HS 205C). In upland pots, redox po-tentials were measured directly after daily watering, giving a morestable reading. One day before final harvesting, the Pt-electrodeswere taken out and re-inserted at three different depths: 5 cm,10 cm and the bottom, in some of the pots to be harvested thenext day. After several hours of stabilisation, redox potentialswere measured once more.

Potential nitrifying activity (PNA) was calculated, with somemodifications in the method of Schmidt and Belser (1994), fromthe increase in NO3

– plus NO2– content of a soil–medium mixture

with time. Fresh soil (20 g) was weighed into a 250-ml flask con-taining 150 mg CaCO3. A solution (50 ml) at pHp7.5, containing330 mg (NH4)2SO4, 140 mg K2HPO4 and 27 mg KH2PO4 l–1, wasadded. The flasks were covered with aluminium foil with smallholes and incubated on a rotary shaker at 150 rpm and 25 7C.Samples (1.5 ml) were taken after 1 and 7 h; the increase in NO3

–

plus NO2– contents was linear during this period. After centrifuga-

tion and filtering (0.45 mm), solutions were stored at 4 7C untiltheir NO3

– and NO2– contents were measured with a Dionex ion-

chromatograph, model 2010i.Potential denitrifying activity (PDA) was calculated from the

N2O production in a soil-medium mixture as described by Tiedje(1994), with some modifications. Fresh soil (20 g) was weighedinto a 225-ml serum bottle. A solution (50 ml) at pH 7.4, contain-ing 1.01 g KNO3, 140 mg K2HPO4, 27 mg KH2PO4, 100 mg chlo-ramphenicol and 1.8 g glucose l–1, was added. The bottle wascrimp-sealed and the internal atmosphere was replaced by argon

gas twice. Finally, 20 ml of the argon gas was extracted and re-placed by 20 ml acetylene. The bottles were incubated horizontal-ly on a rotary shaker at 150 rpm and 25 7C. After 1 and 7 h, gassamples (1 ml) were taken from the headspace of the bottles andtheir N2O concentration analysed with a gas chromatograph (Shi-madzu, model GC-14B); the increase in N2O concentration waslinear between 1 and 8 h.

N and d15N analyses

NH4c-N and NO3

–-N in soil extracts were separated by steam distil-lation (Mulvaney 1996). Fresh soil (approximately 50 g dry soil)was weighed into jars, 250 ml 1 M KCl solution was added andthe jars were shaken vigorously for 1 h. The extracts were de-canted over filter paper and 25 ml clear fluid was transferred to adistillation flask. After addition of 1 g MgO, steam was ledthrough the mixture and cooled in a condenser flask. The first25 ml of the distillate, containing the NH4

c from the soil extractwas collected in a flask that contained 5 ml of 3% boric acid withpH-indicator bromocresol green/methyl red solution (KantoChem). To the cooled remaining fluid in the distillation flask 0.8 gDevarda’s alloy was added, reducing the NO3

– in the soil extractto NH4

c. Steam distillation was repeated and this fraction was col-lected in another flask with pH-indicator. The coloured sampleswere titrated colourless with 2.5 mM H2SO4 and the NH4

c andNO3

– concentrations in the original extract were calculated.A portion (150 ml) of the filtered KCl extracts was transferred

to a new distillation flask and 2–3 g MgO was added. Distillationswere carried out as above, but this time 50 ml of the cooled steamfraction was collected in a flask containing sulphuric acid, inwhich the total amount of protons equalled twice the samples’calculated NH4

ccontent. The remaining fraction was divided overtwo distillation flasks to prevent overflow. From both parts, 50 mlof cooled steam was collected in sulphuric acid solution, after ad-dition of 1.5 g Devarda’s alloy, and combined. Subsequently, allcollected fractions were heated in beakers on a hotplate until thefluid had completely evaporated. The residue was redissolved indeionised water to an estimated final N concentration of 500 mgl–1, using the results of the acid titration for calculations. Of thesesolutions, 0.1 ml was transferred a tin capsule and freeze-dried.Because of low NH4

cconcentrations, samples had to be combined,reducing the number of observations to three per group of fiveidentically treated pots.

Dried root and shoot samples were ground with a mechanicalgrinder (Heiko, Japan). Approximately 4 mg of the powder wasweighed into tin capsules, with a precision of 1 mg. The tin cap-sules, with either plant or soil samples, were placed in an ANCAmass spectrometer (Europa Scientific, Crewe) and N content andd15N values were analysed by single N mode. d15N in a samplewas calculated relative to atmospheric N2, as ‰ deviation usingthe equation: (Rsample-Rair)/Rair!1000, where R was 15N/14N.

Devarda’s alloy does not always reduce NO3– only, resulting in

possible contamination of the NO3– fraction with other nitroge-

nous anions, and total recovery of NH4cin the distillation is essen-

tial because of the high fractionation that can occur in the NH4c/

NO3– equilibrium (Robinson and Conroy 1999). Reliability of

steam distillation was tested by incubating two series of five potswith fresh soil (150 g dry soil pot–1) from the fields, 1 g saccharoseand 0.5 g NH4NO3, for 12 days at 25 7C. Soils were either water-logged or moistened to 60% of WHC. The NO3

– recovery bysteam distillation as percentage (BSD) of the amount measuredwith ion-specific colorimetry, were 99.0B2.3 and 99.2B21.2 forupland and waterlogged soils, respectively. For NH4

c, recoverywas respectively 100.0B2.6 and 97.8B1.9. Thus, our proceduresfor determining exchangeable NH4

cand NO3– were reliable for this

soil.

Statistical analyses

Unless stated otherwise, biomass production, thin root:thick rootratios, root porosity, soil inorganic N contents and PNA were

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Table 1 Results of ANOVA and Kruskal-Wallis tests for effectsof soil moisture (Wp60% of WHC or waterlogged after 22 days),soil bulk density (Cp0.92 or 1.09 Mg dry soil m–3) and their inter-

action (C!W) on plant and soil parameters in pots with Kanto168, Koshihikari or soil only. For Kruskal-Wallis tests the signifi-cance of overall treatment effects is given under W. np5

Plantroot

Biomassshoot

Rootporosity

PNA N content d15N

Plant Soil NO3P Soil NH4

c Pot Plant Soil NO3P Soil NH4

c Pot

Kanto 168 W *** *** ** *** * *** * ** *** ** * **C *** ** * NS NSW!C * NS NS NS ***

Koshihikari W *** NS *** ** NS *** * ** ** *** * ***C NS NS NS NS ***W!C NS NS ** NS NS

Soil only W NS * * NS *** * NSC NS NSW!C NS ***

*pP^0.05, **pP^0.01, ***pP^0.001, NS non-significant

Fig. 1 Mean shoot and root biomass production (g DW pot–1c1SD) of Koshihikari and Kanto 168 grown on loose (L) and com-pacted (C) soils (0.92 and 1.09 Mg dry soil m–3, respectively) un-der upland (U) and waterlogged (W) conditions, 42/43 days afterpotting. Waterlogging started at day 22. Different letters indicatesignificant differences between treatments within one variety.P^0.05, np5

tested by two-way ANOVA (Sokal and Rohlf 1995) with soilmoisture regime and soil bulk density as main treatments. Addi-tionally, a Tukey test or Tamhane T2 test (both P^0.05) was per-formed to identify differences between individual groups (Sokaland Rohlf 1995). Because there was not always a normal distribu-tion or equal variances, PDA, plant-N, soil-N and total pot-Ncontents and soil, plant and total pot d15N values were tested withKruskal-Wallis tests. These were followed by a series of Mann-Whitney U-tests, with P^0.05 per test (Sokal and Rohlf 1995).Analyses were carried out with SPSS 9.0 software (SPSS, Chica-go).

Results

Soil mechanical resistance

At first harvest, penetrograph values for loose andcompacted soils were 0.15 and 1.0 MPa, respectively.Penetrograph values for upland soils without plants didnot change between the two harvests, but with cv. Ko-shihikari they increased to 0.35 and 1.4 MPa and withcv. Kanto 168 to 0.45 and 1.75 in loose and compactedsoils, respectively. Upon waterlogging, the mechanicalresistance decreased to almost 0 MPa, regardless of ini-tial soil bulk density. Final moisture content of uplandsoils with and without plants dropped to 71–77% and90–93% of their initial content, respectively.

Shoot and root development

Plants of both cultivars developed normally after22 days, with shoot dry weights ranging from 250 to550 mg per pot and root dry weights ranging from 17 to92 mg per pot. This large variation was the result ofchoosing the smallest and largest plants for the harvest.Final biomass production for the different treatments isshown in Fig. 1. Waterlogging had a significant effecton root biomass of both cultivars and shoot biomassproduction of Kanto 168 and compaction significantlyreduced both shoot and root biomass of Kanto 168 (Ta-ble 1; Fig. 1). Furthermore, for root biomass of Kanto

168 an interaction between waterlogging and compac-tion effects was found. Thin root : thick root ratios de-creased significantly for both cultivars as a result of wa-terlogging. For Kanto 168 they were (BSD) 3.02B0.69,

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Fig. 2 Mean fractional root porosities (%, c1 SD) of roots ofKoshihikari and Kanto 168 grown on loose (L) and compacted(C) soils (0.92 and 1.09 Mg dry soil m–3, respectively) underupland (U) and waterlogged (W) conditions, 42/43 days after pot-ting. Waterlogging started at day 22. Different letters indicate sig-nificant differences between treatments within each cultivar.P^0.05, np5

Table 2 Soil redox values (mV) measured at 5 cm depth fromday 23 to 41/42, those over 330 mV, those at different depths ofindividual pots with a value over 330 mV at final measurement

and number of observations at final harvest (day 42/43). Soilswere either continuously upland (U) or waterlogged from day 22(W) with a bulk density of 0.92 (L) or 1.09 (C) Mg dry soil m–3

Continuous measurements Final observations

Soil conditions Range Pots measured Observations Depth

Total 1330 mV 5 cm 10 cm Bottom

Kanto 168LU 331–422 1 3 3 423 351 386CU 334–423 1 3 3 418 391 411LW 29–225 2 14 2 71 446 176

112 393 100CW 28–177 2 14 0

KoshihikariLU 443–531 1 1 1 527a

CU 415–480 1 1 1 466LW 78–308 2 15 2 105 170 389

90 384 270CW 49–296 2 15 0

Soil onlyLW 87–328 2 10 3 299 297 398

344 167 341CW 62–178 2 11 0

a Pt electrodes inserted at 5 cm depth only, therefore no values for 10 cm and bottom

2.20B0.91, 0.32B0.15 and 0.38B0.06 in upland loose,upland compacted, waterlogged loose and waterloggedcompacted soils, respectively. These ratios were1.43B0.50, 0.89B0.18, 0.40B0.14 and 0.28B0.05 forthe same series with Koshihikari.

Treatment effects on total root porosity were differ-ent for the two cultivars (Fig. 2; Table 1). Kanto 168showed a significant increase upon waterlogging andcompaction caused a significant reduction. For Koshihi-kari a significant increase in root porosity after water-logging and a significant interaction between waterlog-ging and compaction effects was found. Additional t-tests showed significant differences in root porosity be-

tween loose waterlogged and compacted waterloggedsoils with Koshihikari (Pp0.043) and Kanto 168(Pp0.06).

Redox potentials

The redox values at 5 cm depth were higher than330 mV in upland soils and lower than 330 mV in wa-terlogged soils (Table 2). There was no clear differencebetween loose and compacted soils during the growthperiod and in most pots redox values remained fairlyconstant. After re-inserting the Pt-electrodes just be-fore harvest, values over 330 mV were measured at var-ious depths in some waterlogged loose soils, whichwere not found in compacted waterlogged soils.

Nitrification and denitrification

Waterlogging caused a shift from nitrification to deni-trification, which was much larger in the presence of aplant (Fig. 3). This was the result of a significant de-crease in PNA in soils with plants and an increase inPDA in all soils after waterlogging (Table 1). FinalPDA in soils without plants was low, when compared tosoils with plants, and the PNA of these soils was notaffected by waterlogging. Although compaction had nosignificant overall impact on PNA (Table 1), averagePNAs of loose waterlogged soils were only 48% and49% of those in compacted waterlogged soil for Kanto168 and Koshihikari, respectively. Similarly, PDA val-ues in loose waterlogged soil with Kanto 168 and Ko-

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Fig. 3 Mean potential nitrifying activities(nmol (NO3–cNO2

–) g–1

dry soil h–1, c1 SD ) and potential denitrifying activities (nmolN2O g–1 dry soil h–1, c1 SD) of upland (U) and waterlogged (W)soils, with either Koshihikari or Kanto 168 or soil only, at twobulk densities (0.92 (L) and 1.09 (C) Mg dry soil m–3), 42/43 daysafter potting. Different letters indicate significant differences be-tween treatments within the groups. P^0.05 for PNA andP^0.05 per test between two treatments for PDA, np5

shihikari were 68% and 35% of those in compacted wa-terlogged soils. Additional t-tests between loose andcompacted waterlogged soils revealed a significantlyhigher PNA (Pp0.039) and PDA (Pp0.041) in com-pacted pots with Koshihikari, and a significantly higherPNA (Pp0.017) in compacted waterlogged soil withKanto 168.

Nitrogen contents and recoveries

The amounts of N in soils and plants and their d15Nvalues are presented in Figs. 4 and 5 for the first andfinal harvest, respectively. Total recovery of N as plantN and soil inorganic N (NH4

c-NcNO3–-N) was low, es-

pecially in pots containing only soil (Fig. 6). In all soilsalmost all inorganic N was NO3

–, and shoots contained85–91% of the plant’s N (Fig. 5). Total recovery in potswith plants was highest in upland pots, due to a highercontent of soil inorganic N (Fig. 6) Plants had incorpo-rated more N in waterlogged pots than in upland pots.In pots without plants these trends were not so obvious,

Fig. 4 Mean exchangeable NH4c and NO3

– in soil and total-N inshoots and roots of rice (mg pot–1, c1 SD) and their respectived15N values (‰, c1 SD) in pots with loose (UL) or compacted(UC) soils, 22 days after potting. np2 and 5 for pots with andwithout plants respectively

although there too waterlogged compacted soil showedthe lowest recovery.

d15N values

At first sampling, d15N values of roots and shoots ofKanto 168 did not differ much and were higher thansoil NO3

– values (Fig. 4). The d15N value of exchange-able NH4

c was generally higher than that of plant N orsoil NO3

–. Koshihikari roots had higher d15N valuesthan the shoots in loose upland soils, but this was re-versed in compacted upland soils. Compacted soils withKoshihikari were the only ones with a lower d15N valuefor NH4

c than for NO3– (Fig. 4). At final harvest, d15N

values of NO3– in all soils with plants had increased and,

except for loose upland soil with Koshihikari, had be-come positive. The d15N values of NO3

– and total inor-ganic soil N in the waterlogged series were more posi-tive than their upland counterparts (Figs. 5, 6).

All parts of plants grown on upland soil showed ne-gative d15N values at final harvest (Fig. 5). In contrast,all parts of plants grown on waterlogged soil had posi-tive d15N values. In all individual plants from waterlog-

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Fig. 5 Mean exchangeable NH4c and NO3

– in soil and, total-N inthin roots, thick roots and shoots of rice (mg pot–1, c1 SD) withtheir respective d15N values (‰, c1 SD) in pots with looseupland (UL), loose waterlogged (WL), compacted upland (UC)or compacted waterlogged (WC) soils, 42/43 days after potting.Waterlogging started 22 days after potting. np5. *pNH4

camounttoo low for accurate d15N analyses

Fig. 6 Mean inorganic soil-N (NH4c-NcNO3

–-N), total plant-N,and total pot-N contents (mg pot–1, c1 SD), with their respectived15N values (‰, c1 SD) in pots with loose upland (UL), loosewaterlogged (WL), compacted upland (UC) and compacted wa-terlogged (WC) soils, 22 and 42/43 days after potting. Waterlog-ging started 22 days after potting. Different letters indicate signifi-cant differences between treatments within the series. Per test be-tween two treatments, P^0.05, np5ged soils, thick roots had a more positive d15N value

than thin roots with intermediate d15N values forshoots. In upland pots shoot d15N values were less ne-gative than either thin or thick roots (Fig. 5). Com-pared with first harvest, the total d15N values of all potsincreased, except for upland pots without plants(Fig. 6). This increase was much bigger for waterloggedpots than for upland pots. In upland pots without plantsthe d15N values became slightly more negative.

Discussion

The increase in mechanical resistance of upland soilswith plants could not depend on roots hindering theprobe in penetrating the soil. Indeed, root architecturewas almost the same in upland and waterlogged soils,but the resistance in waterlogged soils did not increaseas in upland soils. Probably the decrease in moisturecontent was the cause of the increased mechanical re-sistance in upland soil with plants. Consistent with thisidea, moisture levels were slightly lower in upland soilswith Kanto 168, which had a slightly higher resistance.In some additionally prepared pots without plants, the

resistance in compacted soil dropped rapidly after wa-terlogging. Therefore, the compaction effects on plantdevelopment in waterlogged pots (Table 1) were notlikely to have been caused by a higher resistance. Also,root systems at the start of waterlogging were too smallto account for significant differences in root porosityand growth between loose and compacted waterloggedsoils. When soils were waterlogged from the start, nocompaction effect on root porosity of Koshihikari wasfound (unpublished results), strongly suggesting thatthe cause for compaction effects in waterlogged soilswas established before waterlogging.

Redox values below 330 mV indicate the disappear-ance of free oxygen from the soil (Pearsall and Mortim-er 1939; Gambrell and Patrick 1978), and values above330 mV indicate soil sites where oxygen is probablypresent. Redox values below 220 mV, the boundary fornitrate disappearance (Laanbroek 1990) were found inseveral of the waterlogged soils in this experiment, butNO3

– was abundant in these soils (Table 2; Fig. 5). Thismeans that several N-transforming processes took placesimultaneously or, as supported by the results in Ta-

491

ble 2, the measured redox values can not be extrapo-lated to all sites because the soil was non-homogenous.Isolated oxic sites in otherwise reduced soils (Table 2)can result from oxygen loss from individual rice roots(Justin and Armstrong 1987; Flessa and Fischer 1992).A lower number of highly porous roots or less oxygenloss from such roots in compacted waterlogged soil,both consistent with the lower fractional root porosityin such soils (Fig. 2), would then explain why oxic spotswere not found in compacted waterlogged soil. Similar-ly, Rumex plants grown on compacted soils lost lessoxygen from their roots than when they were grown onloose soils (Engelaar et al. 1993). However, none of theabove explains the difference in oxygenation betweencompacted and loose waterlogged soils without plants(Table 2). The cause was probably related to physicalcharacteristics, such as a reduced gas diffusion rate incompacted soils, which may also have played a role inpots with plants.

Although their part in the oxygenation of waterlog-ged soil was not clear, plants obviously played a majorrole in determining size and composition of the nitrify-ing and denitrifying population (Fig. 4). PNA and PDAvalues do not represent actual counts, but they can beused to make comparisons between treatments whenexperimental conditions were similar, as here. Plantscan influence the nitrifying and denitrifying popula-tions through the supply of oxygen (Engelaar et al.1995), C compounds (Whipps 1984) and competitionfor N (Engelaar et al. 1991) or O2, and their impact de-pends on plant size and soil conditions.

The shift from nitrification to denitrification afterwaterlogging (Fig. 3) was expected, since the roots werenot able to keep most of their rhizosphere aerated(Laanbroek 1990). The difference in PDA betweenpots with and without plants was not determined by thepresence or absence of free oxygen, since none of thecompacted waterlogged pots showed signs of free oxy-gen presence (Table 2), but PDAs in pots withoutplants were lower (Fig. 4). Probably PDA in waterlog-ged soils with plants was higher because of the in-creased availability of C itself. Supply of C can increasePDA either by stimulating the aerobic growth of heter-otrophic bacteria, including those capable of denitrifi-cation under anoxic conditions, or by promotinggrowth of denitrifying bacteria under anoxic conditions(Bodelier et al. 1998). The slightly better aeration ofloose waterlogged soils when compared with com-pacted waterlogged soils (Table 2) may have led to thelower PDAs in the former soils (Fig. 3). However, thisdifference was not clear and only significant for cv. Ko-shihikari. There is also no clear reason why the PDA inupland soils with Koshihikari was higher than in soilswith Kanto 168, as oxygen and NO3

– availability weresimilar. Possibly differences in the composition orquantity of released exudates affected the compositionand activity of the soil microbiota.

Oxygen limitation was not the main cause for thesignificantly declining PNA after waterlogging, as indi-

cated by the higher PNA values in the compacted wa-terlogged soils than in the loose waterlogged soils(Fig. 3), the latter showing signs of local oxygen availa-bility (Table 2). Also, the PNA was not affected by wa-terlogging in pots without plants (Table 1). Higherplant biomass (Fig. 1) coincided with lower PNA values(Fig. 3) and a competition for NH4

cbetween plants andmicro-organisms may be hypothesised. Indeed, compe-tition for oxygen could be ruled out, as discussedabove. Since final exchangeable NH4

c contents wereequally low in all treatments (Fig. 5) and no correlationwas found between PNA and exchangeable NH4

c con-tents, the above hypothesis can only be valid if prior tothe final harvest larger differences occurred betweentreatments or if higher mineralisation rates ensured ahigher rate of NH4

c supply in soils with a high PNA.Differences in NH4

c content between soils were nothigher at first harvest (Fig. 4), but plant and soil d15Nvalues confirmed mineralisation in at least some treat-ments, as discussed below.

The recoveries of total N (plant N and soil mineralN) given in Fig. 6 are likely to reflect both the samplingprocedure and N disappearance due to denitrificationor ammonia volatilisation. In these soils (pH-H2Op5.8), the latter process was probably not signifi-cant. The water layer of waterlogged pots was not ana-lysed. Also, as a result of differences in rooting depths,different fractions of the soil were sampled for differenttreatment series. The highly mobile NO3

– possibly be-came unevenly distributed in the soil, due to daily wa-tering and subsequent evapotranspiration by soils andplants, and an average sample was not obtained. Thisshould be kept in mind in the interpretation of the Nrecoveries. However, since mainly soil surrounding theroots was sampled, the d15N values reflected the plant-available N.

As discussed in the Materials and methods section,the d15N values of soil nitrate and ammonium obtainedby steam distillation are vulnerable to contamination orfractionation and Robinson and Conroy (1999) omitteddata with a discrepancy of 10% or more between meas-urements by steam distillation and ion-specific colori-metry, thus excluding two-thirds of their data for ni-trate. Even with the near 100% correspondence withvalues obtained by ion-specific colorimetry, as foundfor the combination of soil and extraction procedureused in this experiment, one can not totally exclude in-fluences by contaminants with extraordinarily high orlow d15N values.

The increased d15N value of soil NO3– in waterlogged

pots without plants (compare Figs. 4 and 5) indicatesthat although these soils still had a reasonably high po-tential nitrifying activity (Fig. 3), the actual rate of deni-trification exceeded that of nitrification. Indeed deni-trification usually favours the lighter isotope, leading toan increase in d15N value of the remaining substrate(Yoneyama 1996). Although the net effect of mineralis-ation on d15NH4

c is not completely clear (Yoneyama1996), the decreased d15N values for NH4

c in pots with-

492

out plants (compare Figs. 1 and 2) implies that somemineralisation must have taken place between first andfinal harvest, generating NH4

c with a low d15N value asfresh substrate for nitrification.

All upland soils with plant showed an increase ind15NO3

–, that was always notably lower than in their wa-terlogged counterparts and, in the case of compacted,upland soils with Koshihikari, the d15NO3

– remained ne-gative. In contrast, for soils without plants an increasein d15NO3

– was found for waterlogged pots only andupland soils showed a slight decrease. Thus, in soilswith plants at least one plant-mediated process leadingto 15NO3

– enrichment must have taken place. In hydro-ponic cultures with rice, no significant 15N enrichmentof the medium was found when NO3

– was supplied atconcentrations of up to 2 mM, but a significant 15N en-richment was found for a NO3

– concentration of 8 mM(unpublished data). The soil NO3

– concentration was inmost cases much higher than 2 mM in this experiment(Fig. 5, maximum amount of water per pot approx. 1 l).Although uptake itself can not cause such isotopic frac-tionation, changes in soil d15NO3

– values can be causedby the excretion of 15N enriched NO3

–; thus excretiononly occurs when NO3

– concentrations are high and theexcreted NO3

– is that not reduced to NH4c, as confirmed

by the model of Robinson et al. (1998). The d15N val-ues of Kanto 168 plants on upland soil were negativeand stable in time, while those of Koshihikari becameeven more negative. With the increase in biomass takeninto account, that means that N uptake by plants wouldhave had a notable contribution to the increase in d15Nof the soil. As discussed above, the lower d15N value ofplants compared to soil NO3

– may be due to plant excre-tion of a part of the NO3

– taken up and this process oc-curs at soil NO3

– concentrations as found in these soils.The more positive d15NO3

– in waterlogged pots than inupland pots can not be explained totally by the abovementioned process, because the amount of plant-Ndoes not differ enough between the two treatments.Also, the more positive total d15N balance for all water-logged series (Fig. 6) implies that more N with a rela-tively low d15N value was lost from this plant-soil sys-tem, than from the upland system. Probably N losseswere due to denitrification, a hypothesis that is sup-ported by the increases in d15NO3

– and PDA in all se-ries, after waterlogging (Figs. 3, 5). The higher PDA incompacted waterlogged soils suggests that denitrifica-tion may have contributed more to the high d15NO3

–

values in these soils than in loose waterlogged soils(Figs. 3, 5). But total pot d15N values were almost equalin the two treatments (Fig. 6), which is not expected ifdifferent quantities of NO3

– were lost due to denitrifica-tion (Blackmer and Bremner 1977). An alternative ex-planation may be that the actual denitrifying activitiesduring the plant growth period were similar in the twotreatments and that they did not agree with the poten-tial activities measurements.

The higher d15N values for shoots than for roots ofupland plants are indicative of some discrimination

during N transport or metabolism in the plant. Shootnitrate reductase activity (NRA) may have discrimi-nated for 14N, leaving 15N-enriched NO3

– in the storagepool of the shoot (Yoneyama and Kaneko 1989). Alter-natively, root NRA may have discriminated for 14N,which means that NO3

– transported to the shoot wouldbe 15N-enriched.

A number of observations cannot be explainedunambiguously. For instance, the fact that thick rootsof individual plants of waterlogged soils had higherd15N values than the earlier produced thin roots. No Npool in the plant can account for higher d15N values inthick roots than in thin roots or shoots, unless N be-came enriched during storage, transport or incorpora-tion.

The main conclusions are that generally differencesbetween pots with Koshihikari and Kanto 168 weresmall. In waterlogged pots, bulk density effects on plantbiomass, root porosity and soil aeration were found,which were not related to soil mechanical resistance.Soil oxygenation by plants could not be proven. NH4

c

limitation was the most likely cause for decliningPNAs, after waterlogging. Exudation of C compoundsby plants and soil anoxia caused by waterlogging, pro-moted PDA. d15N analyses confirmed nitrification, de-nitrification and N incorporation by plants, and theirfractionation effects. It also confirmed higher denitrifi-cation rates in waterlogged than in upland soils. Thed15N analyses showed that although PNA and PDAprovide a tool for comparing active microbial popula-tions, they probably do not reflect actual N transforma-tion rates in soils. Finally, potentially important proc-esses, such as N immobilisation, could not be identifieddue to low N recoveries and, future experiments shouldinclude quantification and d15N measurements of vola-tile N compounds and mobile soil N.

Acknowledgements We thank Dr G. Ishioka of the National Ag-riculture Research Center for the analyses of the soil particle sizedistribution.

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