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Europ. J. Agronomy 26 (2007) 113–120

ffect of incorporation of Brassica napus L. residues in soils on mycorrhizalfungus colonisation of roots and phosphorus uptake by maize (Zea mays L.)

Sylvain Pellerin a,∗, Alain Mollier a, Christian Morel a, Christian Plenchette b

a INRA, Unite mixte de recherche 1220 INRA-ENITA Transfert sol-plante et cycle des elements mineraux dans les ecosystemes cultives,71 Avenue Edouard Bourlaux, B.P. 81, 33883 Villenave d’Ornon Cedex, France

b INRA, Unite mixte de recherche 1210 INRA-ENESAD-Universite de Bourgogne Biologie et gestion des adventices, 17 rue Sully,B.P. 86510, 21065 Dijon Cedex, France

Received 22 August 2005; received in revised form 5 July 2006; accepted 16 July 2006

bstract

Plants in the Brassicaceae family are known to contain thioglucoside compounds that produce isothiocyanates when tissues are disrupted. Thesehemicals have a negative effect on soil-borne fungal pathogens, and possibly on vesicular–arbuscular mycorrhizal (AM) fungi. We investigatedhe effect of incorporation of Brassica napus L. residues in a soil on mycorrhizal colonisation of roots, P uptake and growth of following maizeZea mays L.) crop. A pot experiment was carried out in a glasshouse with pre-inoculation with Glomus intraradices (+I and −I), incorporationf B. napus L. residues (+R and −R) and mineral P fertilization (+P and −P) as studied factors. The soil used was a neutral loamy soil with lowavailability. The pots were planted with maize (Z. mays L.). Phosphorus uptake, plant biomass, total leaf area per plant and area of individual

eaves (rank 4–7) were significantly larger in +P treatments than without P addition, thus confirming that the soil used for the experiment was Peficient. Incorporation of B. napus L. residues had also a positive effect on P uptake, plant biomass, total leaf area per plant and area of individual

eaves (rank 5–7). These effects were more pronounced in −P treatments than in +P treatments. There was no effect of pre-inoculation with G.ntraradices on P uptake and P dependant variables. The percentage of the root length that was colonised by mycorrhizae was lower in +P treatmentsut it was not significantly affected by other studied factors. Altogether the results showed that B. napus L. residues have mainly acted as a sourcef P. There was no evidence of a negative effect of the incorporation of B. napus L. residues on the colonisation of maize roots by mycorrhizae.

2006 Elsevier B.V. All rights reserved.

izae; P

foaeS

eywords: Biofumigation; Brassica napus L. residue; Glucosinolate; Mycorrh

. Introduction

Several Brassicaceae are known to contain thioglucosideompounds which produce isothiocyanates when tissues areisrupted. These chemicals have a negative effect on soil-borne

ungal pathogens, a process referred to as biofumigationSchreiner and Koide, 1993a; Angus et al., 1994; Brown and

orra, 1997; Sarwar et al., 1998). These compounds have also aegative effect on vesicular–arbuscular mycorrhizal (AM) fungind there is evidence of their involvement in the lack of AM

Abbreviations: Cp, phosphorus concentration (in mg P L−1) in the solutionf a soil suspension (1 g dry soil:10 mL deionised water); FC, field capacity (in gater per g dry soil); L, leaf length (in cm); LER, leaf elongation rate (in mm peregree-days); NEL, number of expanded leaves; NVL, number of visible leaves;L, root length (in m); TT, thermal time after emergence (in degree-days); AM,esicular–arbuscular mycorrhizal fungi; Wm, leaf width (in cm)∗ Corresponding author. Tel.: +33 5 57 12 25 12; fax: +33 5 57 12 25 15.

E-mail address: pellerin@bordeaux.inra.fr (S. Pellerin).

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161-0301/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.eja.2006.07.007

hosphorus; Zea mays L.

ungus colonisation of most Brassicaceae. Indeed, the releasef thioglucoside compounds in the rhizosphere of Brassicaceaend subsequent production of isothiocyanates has an inhibitoryffect on spore germination (Vierheilig and Ocampo, 1990;chreiner and Koide, 1993a,b). Since root colonisation byM is known to enhance uptake of immobile nutrients likehosphorus, the question arises whether previous croppingith Brassicaceae may have a negative effect on mycorrhizal

olonisation of roots and P uptake by the subsequent crop. Aegative effect of previous cropping with rapeseed (Brassicaapus L.) on root colonisation by mycorrhiza and P uptake ofhe following maize (Zea mays L.) has sometimes been reportedGavito and Miller, 1998a,b; Arihara and Karasawa, 2000). Thisegative effect may have two origins: (i) a reduction of the AM

opulations because rapeseed is a non-host species and (ii) andditional inhibitory effect on AM populations during and afterhe rapeseed culture because of antifungal compounds producedy roots and/or rapeseed residues. The objective of this study

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14 S. Pellerin et al. / Europ. J

as to investigate this latter effect by analysing the effect ofncorporation of rapeseed residues in a soil on mycorrhizalolonisation of roots, P uptake and growth of succeeding maize.

. Materials and methods

.1. Experimental design

A pot experiment was carried out in a glasshouse betweenarch and May 2002 at INRA, Bordeaux, France. The effects of

he three following experimental factors on mycorrhizal coloni-ation of roots, P uptake and growth of maize were studied:

pre-inoculation with the AM fungus Glomus intraradices(+I) or no pre-inoculation (−I). The objective of the pre-inoculation was to reinforce the initial AM infective potentialof the soil so as to highlight the possible negative effect ofrapeseed residues;incorporation of rapeseed residues (+R) or no incorporationof rapeseed residues (−R);fertilization with mineral P (+P) or no fertilization with min-eral P (−P).

On the whole, eight experimental treatments were com-ared. The experimental design was randomized with six blocksnd two replications per block and experimental treatmentombination.

The soil used for the experiment was a Haplic Luvisol (FAO-nesco, 1989) collected from a long-term P fertilization trial

ocated near Paris in the experimental domain of the Insti-ut National Agronomique Paris-Grignon (Thiverval-Grignon,rance). The soil was the topsoil of experimental plots hav-

ng received no P fertilization for several years. A soil with aow P availability was chosen so as to highlight the effects ofxperimental treatments on P uptake and maize growth. Its Pontent was 6 mg Olsen P per kg of dry soil. The P concentra-ion of the soil solution measured in a soil suspension (1 g dryoil:10 mL deionised water) was 0.02 mg P L−1. The numberf infective AM propagules, estimated with the most probableumber technique (Porter, 1979) was 8 propagules per 100 g soilrange 2–26). Other characteristics of the soil were: 281 g kg−1

lay (<2 �m); 597 g kg−1 silt (2–50 �m); 122 g kg−1 sand50–2000 �m); 15.4 g kg−1 organic carbon; pH in water 8.1. Theoil was sieved, homogenized and kept wet before filling pots.

Rapeseed residues (B. napus spp. oleifera cv Constant) wereollected on 11 March 2002 in an experimental field at Auzeville,outhwest France, when plants were at the 2.3 stage (pedicelslongating) (Berkencamp, 1973). A cultivar with a non-reducedontent of glucosinolates was chosen for the experiment so aso highlight the possible negative effects of rapeseed residuesn mycorrhizal colonisation and P uptake. Shoots and easilyxtractable roots were collected and transported to the laboratoryhere they were kept for 1 or 2 days at −4 ◦C before man-

al grinding and incorporation in soils. Immediately after plantampling, and just before grinding and incorporation in soils, aew plants were randomly sampled and immediately frozen at80 ◦C for measuring glucosinolate concentrations. Glucosino-

rtaw

nomy 26 (2007) 113–120

ate concentrations were measured at the CETIOM laboratoryArdon, France). The samples were freeze–dried and glucosi-olates were measured using high performance liquid chro-atography of desulphated derivatives (ISO 9167-1 method).

mmediately after plant sampling, the glucosinolate concentra-ion was 13.1 and 20.7 �mol g−1 fresh weight in shoots andoots, respectively. It was slightly lower just before grinding andncorporation in soils, probably because of losses during plantonservation (12.5 and 16.5 �mol g−1 fresh weight in shootsnd roots, respectively). These results are in the range of val-es reported by other authors for B. napus (Kirkegaard andarwar, 1998, 1999; Sarwar and Kirkegaard, 1998; Potter etl., 2000; Morra and Kirkegaard, 2002). Glucosinolate concen-rations were higher in roots than in aboveground parts of thelant, as already observed by other authors (Gardiner et al., 1999;irkegaard and Sarwar, 1999). Other plants were sampled for

nalysing their concentrations of N, P, K, Ca, Mg and Na. Phos-horus concentration, determined by an adaptation of Malachitereen colorimetric technique (Van Veldhoven and Mannaerts,987), was 5.49 and 3.37 mg g−1 dry matter in shoots and roots,espectively. Nitrogen concentration, determined by the Dumasethod, was 53.1 and 22.7 mg g−1 dry matter in shoots and

oots, respectively.The AM fungus inoculum was produced at INRA, Dijon,

rance following the method developed by Plenchette et al.1982). The inoculum was a mixture of leek roots colonised by. intraradices (Schenck and Smith, 1982) and calcined mont-orillonic clay (Terragreen, Oil Dry Company, Chicago, USA).

.2. Practical preparation

Opaque polypropylene pots (0.095 m basal diameter, 0.135 mop diameter, 0.27 m height and 0.0245 m3 internal volume) werelled with 14 kg of wet soil (11.8 kg of dry soil and 0.186 g ofater per g of dry soil). Depending on the experimental treat-ent, the soil was previously mixed with the inoculum of G.

ntraradices, ground rapeseed residues or a solution of mineral.

In pots inoculated with G. intraradices, 130 g of inoculumepresenting about 182,000 vesicles per pot (15,400 vesicles perg of dry soil) were mixed to the soil. The P content of the inocu-um was measured as the sum of the extractable P in distilledater (1 g inoculum in 10 mL deionised water) plus the mineralbound to the soil solid phase that was isotopically exchange-

ble in less than 1 month (Fardeau et al., 1991), plus organic. This determination was preferred to the measurement of theotal P content since this latter was very likely to overestimatehe bioavailable P. The amount of P added by incorporating thenoculum was 4.1 mg P per kg of dry soil.

In pots receiving rapeseed residues, 308 g of fresh biomass68 g of roots and 240 g of shoots) were manually grinded5 mm), incorporated and mixed to the soil, which represents6 g of fresh matter per kg of dry soil. The amount of rapeseed

esidues incorporated was calculated so as to represent about 1.5imes the amount incorporated in a field on an area basis. Themounts of N and P added by incorporating rapeseed residuesere 148 mg N and 16 mg P per kg of dry soil.

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In P fertilized pots, the soil was mixed with a solution ofaH2PO4·2H2O so that 50 mg P was added per kg of dry soil

200 kg P per ha). A P free nutrient solution was added to allots to avoid any limitation of other nutrients. The amount ofutrients added per kg of dry soil were: 50 mg N as NH4NO3,0 mg K as KSO4, 20 mg of Mg as MgSO4·7H2O, 2 mg Cu asuCl2·2H2O, 2 mg Mn as MnCl2, 1 mg Zn as ZnCl2, 1 mg B as3BO3 and 0.1 mg Mo as (NH4)6Mo7O24·4H2O.Maize (Z. mays L. cv Cecilia) was sown on 21 March 2002.

hree seeds were sown per pot but only one seedling was kepter pot after emergence. Pots were regularly irrigated with indi-idual drippers. A control pot was continuously weighed andrrigation started automatically when water content droppedelow 80% of field capacity (FC) (FC = 0.432 g water per g dryoil). Air and soil temperatures were measured with 0.2 mmiameter copper-constantan thermocouples. Air relative humid-ty was measured with a relative humidity probe (HMP35AC,aisala, Finland). All sensors were connected to a data logger

21×, Campbell Scientific, UK). Measurements were performedvery 15 min, but only hourly average values were recorded. Theverage air temperature during the experiment was 19.2 ◦C andhe average relative humidity was 62.7%. Thermal time aftermergence (TT in ◦C days) was calculated on a daily basis asollows:

T =∑ [(

TX + TN

2

)− Tb

]

here TX is the maximum daily air temperature (in ◦C), TNhe minimum daily air temperature (in ◦C) and Tb is the baseemperature (10 ◦C).

.3. Soil sampling and measurements

Soil cores (0.8 cm × 0.8 cm × 6 cm) were removed from threeots per experimental treatment at plant emergence (28 March),n 29 April (266 degree-days after emergence) and 13 May400 degree-days after emergence) with an auger sampler. Threeeplicates were removed per pot. Only the soil sampled between0 and 16 cm from the soil surface was kept for further analyses.he three replicates were mixed, air-dried and the P concentra-

ion (Cp, in mg P L−1) was measured in the solution of a soiluspension (1 g dry soil:10 mL deionised water) as proposed byardeau et al. (1991).

.4. Plant samplings and measurements

The number of visible leaves (NVL), the number of expandedeaves (NEL) and the length (L, in cm) and maximum widthWm, in cm) of individual leaves were recorded every 2 days onne plant per block and treatment. The area (A, in cm2) of anndividual leaf was calculated as follows:

= k × L × Wm

ith k = 0.75 for expanded leaves and k = 0.5 for visible, not fullyxpanded leaves (Bonhomme et al., 1982). The total leaf areaer plant was calculated by adding area of individual leaves.

wAUc

nomy 26 (2007) 113–120 115

Leaf elongation is known to be very responsive to P availabil-ty (Plenet et al., 2000; Assuero et al., 2004). Leaf elongationates (LER) were therefore recorded daily on leaves 4–7. Theeneral shape of the elongation curve of a maize leaf blade ver-us thermal time since its initiation is sigmoid (Arkebauer andorman, 1995). The first phase of the elongation curve occurshen the leaf is hidden by the leaf sheaths of the lower leaves.he elongation rate gradually increases, becomes about constant

quasi-linear elongation phase) and then gradually decreases.he leaf elongation rates were measured daily during this quasi-

inear elongation phase on six plants per treatment (one plant perlock). A fixed horizontal ruler was placed in contact with theeaf blade allowing the leaf to grow beside it. Marks were drawnn the lamina every day, which corresponded to the position ofhe ruler at time of marking. The LER (in mm per ◦C days)ere calculated as the ratio of the distance between two neigh-ouring marks to the duration of the period. To assess the effectf each experimental factor (pre-inoculation (I), incorporationf rapeseed residues (R) or fertilization with mineral P (P)),elative leaf elongation rates (LER for the −X treatment/LERor the +X treatment) and relative final leaf lengths (L for theX treatment/L for the +X treatment) were calculated for each

eaf rank (with X corresponding to I, R or P, depending on thereatment).

Six plants per experimental treatment (one plant per block)ere sampled between 29 April and 3 May (287 ◦C days after

mergence) and between 13 and 17 May (417 ◦C days aftermergence) for biomass determination, observations on rootsnd chemical analyses. Plants were removed from their pot andhe root systems were washed. A sample of fine root segments10 mm length) was carefully collected and placed in plasticags at 4 ◦C for later determination of AM colonisation. Shootsnd roots were dried at 50 ◦C for 96 h and weighed. Shoots andoots then were ground for chemical analyses. The P contentas determined by ICP (induced coupled plasma).

.5. Root colonisation by AM fungi

Root samples were washed in water and a sub-sample cor-esponding to about 2 g of fresh root was collected. Roots werehen plunged in a KOH solution (10%), heated at 90 ◦C for 1 h,nd rinsed in water. Roots were then plunged in a staining solu-ion (90% lactic acid, 98% glycerol, distilled water (1:1:1, v/v/v)ith 0.5% fuchsine), heated at 90 ◦C for 30 min, and rinsed inater. Roots then were cut in 5 mm segments and scattered onPetri dish with a 12.7 mm × 12.7 mm square pattern. The per-entage of the root length which was colonised by mycorrhizaeas estimated by the grid–line intersect method (Newman, 1965;ennant, 1975).

.6. Statistics

For most measured variables, analyses of variance (ANOVA)

ere carried out at harvest 1 and harvest 2 using the procedureNOVA of SYSTAT 10 for Windows (SPSS Inc. Chicago, IL,SA). Means were separated using the LSD at p = 0.05 signifi-

ance level.

116 S. Pellerin et al. / Europ. J. Agronomy 26 (2007) 113–120

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ig. 1. Total P uptake per plant at harvest 1 (287 degree-days after emergence)or all experimental treatments. Vertical bars indicate ±S.D. of the mean (n = 6lants per treatment).

. Results

.1. Phosphorus uptake, plant biomass and total leaf areaer plant

Phosphorus uptake and plant biomass at the first harvest wereignificantly higher in experimental treatments with mineral Pdded (p < 0.001) and in experimental treatments with incorpo-ation of rapeseed residues (p < 0.001) (Figs. 1 and 2). However,here was no significant effect of inoculation on both variables

p = 0.412 and 0.222 for P content and biomass, respectively).nteractions between factors were not significant. Very similaronclusions were obtained at the second sampling date (data nothown).

ig. 2. Total dry biomass per plant (shoots + roots) at harvest 1 (287 degree-daysfter emergence) for all experimental treatments. Vertical bars indicate ±S.D.f the mean (n = 6 plants per treatment).

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ig. 3. Number of visible leaves as a function of thermal time after emergenceor all experimental treatments. Vertical bars indicate ±S.D. of the mean (n = 6lants per treatment).

Leaf appearance was significantly delayed (α = 0.05) in −Preatments from 146 degree-days after emergence and in −Rreatments from 163 degree-days after emergence (Fig. 3). Totaleaf area per plant was significantly lower (α = 0.05) in −Preatments from 128 degree-days after emergence and in −Rreatments from 146 degree-days after emergence (Fig. 4). Foroth variables, the difference between −P and +P treatments wasess pronounced when rapeseed residues were incorporated. Noffect of inoculation was observed on any of these variables.

.2. Leaf elongation rates and final leaf sizes

A strong relationship was observed between the relative leaflongation rate (LER for the −X treatment/LER for the +X treat-ent, with X corresponding to I, R or P) and the relative final

ig. 4. Total leaf area per plant as a function of thermal time after emergenceor all experimental treatments. Vertical bars indicate ±S.D. of the mean (n = 6lants per treatment).

S. Pellerin et al. / Europ. J. Agronomy 26 (2007) 113–120 117

Table 1Final leaf length (in cm) per leaf rank and experimental treatment (n = 6 leaves per leaf rank and experimental treatment)

P Res. Inoc. Leaf rank

1 2 3 4 5 6 7

+P +R +I 5.4a 13.9a 27.8a 44.5ab 59.3a 71.4a 80.0a−I 5.5a 14.1a 27.1a 43.1abc 57.8a 70.5a 79.1ab

−R +I 5.7a 14.3a 27.6a 43.1abc 55.8ab 68.0ab 75.5ab−I 5.7a 14.6a 28.6a 45.3a 58.0a 68.2ab 75.3ab

−P +R +I 5.5a 14.0a 26.8a 40.7bcd 50.8bc 62.0bc 73.0ab−I 5.6a 14.0a 27.0a 39.0cd 47.7cd 59.6c 70.5bc

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or each leaf rank, means followed by the same letter are not significantly diffe

eaf length (L for the −X treatment/L for the +X treatment), forll leaf ranks between leaf 4 and leaf 8 and all experimental treat-ents (Fig. 5). Points were close to the bissectrix, which shows

hat lower final leaf lengths were mainly accounted for by lowerER during the quasi-linear elongation period. However, thenal leaf length was slightly less affected than the leaf elongationate for leaves whose elongation was heavily reduced, suggest-ng that the duration of leaf elongation was slightly increasedor these leaves. Moreover, plotting the relative final leaf lengthL for the −X treatment/L for the +X treatment) versus the rel-tive final leaf width (Wm for the −X treatment/Wm for the +Xreatment) shows that leaf length and leaf width were similarlyffected by experimental treatments (data not shown). Furthernvestigations about the effects of experimental treatments oneaf growth were therefore carried out on the final leaf length

ince this variable reflects and summarises other effects on leafrowth.

Addition of mineral P had a pronounced positive significantffect on final leaf length from leaf 4 to leaf 7 (Tables 1 and 2).

ig. 5. Relative leaf elongation rate during the quasi-linear elongation phaseLER for the −X treatment/LER for the +X treatment) vs. the relative final leafength (L for the −X treatment/L for the +X treatment) for leaves 4–8 (n = 6eaves per treatment and leaf rank). X corresponds to P, R or I, depending on thereatment.

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his positive effect of mineral P addition was more pronouncedn absence of rapeseed residue incorporation, thus resulting insignificant R × P interaction. Incorporation of residues had aositive significant effect on final leaf length from leaf 5 to leaf. This positive effect was more pronounced in the absence ofineral P addition. Inoculation had no effect on final leaf lengthshatever the other factors.

.3. Root infection by mycorrhizal fungi

The percentage of the root length that was colonised by myc-rrhizal fungi was quite low at the first sampling date (betweenand 12%) (Fig. 6). It was higher at the second sampling date

between 3 and 20%). The percentage of the root length that wasolonised was significantly reduced at both sampling dates whenineral P was added (p < 0.001). It was significantly higher in

he +I treatments at the second sampling date only (p < 0.01). Itas not significantly different between +R and −R treatments.

.4. P added, P concentration in the soil solution and Pptake

Phosphorus concentration in the soil solution (Cp) decreased

ith time for most experimental treatments, especially +P treat-ents, but this effect was not statistically significant due to the

igh variability within treatments (Fig. 7). This Cp decrease withime may be interpreted as the consequence of sorption of the

able 2nalysis of variance for the final leaf length for each leaf rank (p-values)

actor Leaf rank

1 2 3 4 5 6 7

NS NS NS NS NS NS NSNS NS NS NS *** *** ***NS NS * *** *** *** ***

× R NS NS NS NS NS NS NS× P NS NS NS NS NS NS NS× P NS NS NS NS * *** **

× R × P NS NS NS NS NS NS NS

, **, ***: significant at the 0.05, 0.01 and 0.001 probability level, respectively;S: not significant at the 0.05 probability level.

118 S. Pellerin et al. / Europ. J. Agronomy 26 (2007) 113–120

Fig. 6. Percentage of the root length which was colonised by mycorrhizae athao

asmit0Ntraar

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Fig. 8. Relationship between P uptake by plants and P added to the soil for allexperimental treatments. The P added was calculated as the sum of the P addedunder a mineral form (+P treatments), the P contained in rapeseed residues (+Rtreatments) and the P added by the inoculum (+I treatments). The P content ofthe inoculum was measured as the sum of the extractable P in distilled water(sa

4

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arvest 1 (287 degree-days after emergence) and harvest 2 (417 degree-daysfter emergence) for all experimental treatments. Vertical bars indicate ±S.D.f the mean (n = 6 plants per treatment).

dded P on the soil solid phase and P uptake by plants. At eachampling date, Cp values were significantly larger in +P treat-ents than in −P treatments. Cp values were also always larger

n +R treatments than in −R treatments, but this effect was sta-istically significant at the second date only (p = 0.19, 0.04 and.06 for the first, second and third sampling date, respectively).o significant differences were observed between +I and −I

reatments. Considering experimental treatments altogether, aelationship was observed at each sampling date between Cp

nd P added (data not shown). A positive linear relationship waslso observed between total P uptake and total P added (fertilizer,esidues and inoculum) (Fig. 8).

ig. 7. Phosphorus concentration in the soil solution (in mg P L−1) as a func-ion of thermal time. The +P−R+I treatment was discarded because of a verynexpected value at one sampling date probably due to an experimental error.

tspaptiamn(tsrhaateta

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1 g inoculum in 10 mL deionised water), plus the mineral P bound to the soilolid phase that was isotopically exchangeable in less than 1 month (Fardeau etl., 1991), plus organic P.

. Discussion

In our experiment, P uptake by plants and all P related plantariables like plant biomass, leaf appearance, leaf elongationnd final leaf size were higher in experimental treatments withineral P added (+P treatments) (Figs. 1–4). This confirms that

he soil used for the experiment was P deficient. This is con-istent with the fertilization regime applied on the experimentallot where it was collected (no mineral P added for several years)nd the low P Olsen value measured on the initial soil (6 mg Per kg of dry soil). The number of infective AM propagules ofhe initial soil (8 propagules per 100 g of soil) compares well buts in the lower range of values reported by Gianinazzi-Pearson etl. (1985) for 10 French agricultural soils. However, the roots ofaize plants grown on this soil without mineral P addition were

ormally colonised by mycorrhizae at the second harvest dateabout 20% of root length colonised; Fig. 6), even in experimen-al treatments without pre-inoculation, which confirms that theoil contained sufficient AM inoculum. This is consistent withesults of Anderson et al. (1987) and Amijee et al. (1989) whoave shown that the AM infective potential of soils was gener-lly high in soils receiving no mineral P fertilization. This maylso explain why additional inoculation with G. intraradices (+Ireatments) had only a slight effect on root colonisation and noffect on P uptake and subsequent P related variables. Anyhowhis show that this low P, naturally inoculated soil was appropri-te for answering the question.

Results obtained in our experiment show that rapeseedesidues were a significant source of P since P uptake, plantrowth and all P related plant variables were higher whenesidues were incorporated (Figs. 1–4; Tables 1 and 2). The

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S. Pellerin et al. / Europ. J

ystematic difference between +R and −R treatments strength-ns the idea that a significant part of the P contained in rapeseedesidues was released during the experiment. A rapid release ofhe P contained in plant residues was observed by other authorsnd was partly attributed to the presence of soluble inorganic Pn the residues (Friesen and Blair, 1988). Consistently, Cp val-es were higher in +R treatments than in −R treatment, althoughhe difference was statistically significant at one date only. Thismall difference between Cp values may be explained by theact that P released from rapeseed residues in this low P soil wasuickly sorbed by the soil solid phase and/or taken up by thelant.

Considering all experimental treatments, a linear relationshipas observed between total P uptake and total P added (fertiliz-

rs, residues and inoculum), which strongly suggests that effectsf experimental factors upon P uptake and plant growth werelmost entirely accounted for by their effect on P availabilityFig. 8). No negative effect of rapeseed residues incorporationn maize root colonisation by mycorrhizae was evidenced fromhis experiment (Fig. 6). This negative effect, if it exists, wasargely dominated by the positive effect of P addition by rapeseedesidues. Maize roots were normally colonised by mycorrhizalungi in +R treatments (Fig. 6), which suggests that a hypothet-cal negative effect of rapeseed residues on mycorrhizae, if itxists, is probably insignificant. A possible explanation is thatntifungal compounds are released by rapeseed residues imme-iately after incorporation and their concentration decreasesapidly due to volatilisation and microbial decomposition, sohat they have almost no further effects on spore germinationnd mycorrhization. This hypothesis is supported by results fromorra and Kirkegaard (2002) who have observed that maximum

sothiocyanate concentrations in a soil were observed immedi-tely after incorporation of rapeseed residues. In their study, aow concentration of isothiocyanates was observed after 4 days.imilar results were obtained by Gardiner et al. (1999). If so, thisuggests that the main effect of rapeseed on mycorrhizae occursuring the rapeseed culture, both because it is a non-host plantnd maybe because antifungal compounds are produced dur-ng plant growth in the rhizosphere (Rumberger and Marschner,003).

Our experiment was carried out with a rapeseed genotypeith a non-reduced content of glucosinolates. Moreover, rape-

eed residues were harvested at a vegetative stage, when thelucosinolate concentration in plant tissue was maximal (Sarwarnd Kirkegaard, 1998). Since no negative effect of rapeseedesidues incorporation was observed in our experiment, suchnegative effect is not expected to appear with genotypes withreduced content of glucosinolates, which are now widely used

n agriculture, or when mature residues are incorporated. Ourxperiment was carried out on a low P soil and the questionsemains whether similar results would be obtained on a high Poil, which is more common in agricultural soils. In that case,dditional P supplied by the residues is not expected to strongly

ffect plant P uptake. However, since no effect of incorporationf rapeseed residues was observed on maize root colonisationy mycorrhizae in our low P soil, it is not expected that such anffect could be observed in a high P soil. In that case, root coloni-

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ation by myccorhizae is expected to be lower. Moreover, theontribution of mycorrhizae to P uptake is expected to be lowern high P soils. Nevertheless, additional experiments would beecessary to test our results in an experimental situation wherehe side effect of residues incorporation on P availability isvoided or less pronounced. If such experiments confirm thatapeseed residues have no negative effect on AM populations,urther studies should focus on possible negative effects dur-ng the rapeseed culture itself, because rapeseed is a non-hostpecies and/or because of antifungal compounds produced byoots during rapeseed growth.

cknowledgements

Thanks are due to Aurelie Ganteil who carried out the experi-ental work and to Stephane Thunot, Christian Barbot and Ericartin for technical assistance. This research was founded by

he Association de Coordination Technique Agricole (France).he soil was collected in the experimental domain of the Insti-

ut National Agronomique Paris-Grignon (Thiverval-Grignon,rance).

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