Adaptation of fine roots to annual fertilization and irrigation in a 13-year-old Pinus pinaster stand

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1 Tree Physiology 29, doi:1.193/treephys/tpn2 Adpttion of fine roots to nnul fertiliztion nd irrigtion in 13-yer-old Pinus pinster stnd M.R. BAKKER, 1,2 E. JOLICOEUR, 3 P. TRICHET, 4 L. AUGUSTO, 5 C. PLASSARD, 6 J. GUINBERTEAU 7 nd D. LOUSTAU ENITA de Bordeux, UMR 122 TCEM, F Villenve d Ornon, Frnce Corresponding uthor (mrk.bkker@bordeux.inr.fr) Fculte de foresterie, Universite de Moncton, Cmpus d Edmundston, 165 boulevrd He bert, Edmundston, Nouveu-Brunswick, Cnd INRA, UR 1263 EPHYSE, F Cests, Frnce INRA, UMR 122 TCEM, F Villenve d Ornon, Frnce INRA, UMR 1222 BSR, F-346 Montpellier, Frnce INRA, UPR 1264 MYCSA, F Villenve d Ornon, Frnce Received June 26, 28; ccepted September 3, 28; published online December 5, 28 Summry Effects of fertiliztion nd irrigtion on fine roots nd fungl hyphe were studied in 13-yer-old mritime pine (Pinus pinster Aït. in Solnd), 7 yers fter the initition of the tretments. The fertiliztion trils consisted of phosphorus tretment, complete fertilizer tretment (N, P, K, C nd Mg), nd n unfertilized tretment (control). Fertilizers were pplied nnully nd were djusted ccording to folir trget vlues. Two irrigtion regimes (no irrigtion nd irrigtion of set mount ech dy) were pplied from My to October. Root smples to depths of 12 cm were collected in summer of 25, nd the biomss of smll roots (dimeter 2 2 mm) nd fine roots (dimeter 2 mm) nd fine root morphology were ssessed. Biomss nd length of hyphe were studied by mesh ingrowth bg technique. Totl fine root biomss in the litter nd in the 12 cm soil profile rnged between 111 nd 296 g m 2. Results derived from the mesurements of biomss nd root length, or root re, showed tht both fertilizer tretments reduced the size of the fine root system, especilly in the top soil lyers, but did not ffect smll roots. Compred with control tretments, fine root morphology ws ffected by both fertilizer tretments with the fine roots hving incresed specific root length/ re, nd irrigtion tended to reinforce this finer morphology. The mount of hyphe in the mesh ingrowth bgs ws higher in the fertiliztion nd irrigtion tretments thn in the controls, suggesting further extension of the root system (ectomycorrhizl infection) nd thus of the uptke system. Irrigtion hd no significnt effect on the size of the fine root system, but resulted in shllower rooting system. Totl root to shoot rtios were unffected by the tretments, but fine root mss:needle mss nd fine root re index:lef re index rtios decresed with incresing nutrient supply. Overll, compred with the control fine roots, incresed nutrient supply resulted in lower fine root biomss but the dynmic frction of the finest roots ws greter. Irrigtion hd only limited effects on fine root size, distribution nd morphology. Keywords: nnul resource optimiztion, fine root density, fine root morphology, hyphe, root to shoot rtios. Introduction Although elevted tmospheric CO 2 concentrtions cn potentilly stimulte tree growth (DeLuci et l. 1999), this effect my be limited by low nutrient nd wter supplies (Oren et l. 21, Loustu et l. 25) nd ultimtely by the dptbility of the root system in fcilitting dequte nutrient uptke. To provide sufficient nutrient uptke from low fertility soils, trees cn disply morphologic plsticity by selectively llocting biomss for growth within nutrientrich ptches (Hutchings nd de Kroon 1994, George et l. 1997, Hodge 24), or physiologic plsticity by incresing uptke rte per unit of root length (Go rnsson et l. 26). The optimum morphology nd physiology of fine roots depend on how plnt cn best invest crbon to optimize benefits under given environmentl conditions (Chpin et l. 1987, Eissenstt 1992, Ryser 26). In ddition, trees in forest ecosystems cn form ssocitions with mycorrhizl fungi (Smith nd Red 1997). The growth of ectomycorrhizl hyphe cn gretly enhnce the bsorption cpcity of the root system, constituting up to 75% of the uptke potentil of young pine systems (Rousseu et l. 1994). Therefore, Ó The Author 28. Published by Oxford University Press. All rights reserved. For Permissions, plese emil: journls.permissions@oxfordjournls.org

2 23 BAKKER ET AL. crbon used for soil explortion by the hyphe of ectomycorrhizl fungi cn hve mjor effect on the nutrient uptke efficiency of forest trees. Trils on Pice bies ((L.)H.Krst)nd Pinus sylvestris (L.) in Sweden (Axelsson nd Axelsson 1986), Pinus ted (L.) in the southestern USA (Albugh et l. 1998), Pinus rdit (D. Don) in Austrli (Wterworth et l. 27), nd Pinus pinster (Aït. in Solnd) in Frnce t the site where our study ws crried out (Trichet et l. 28) hve shown tht increses in tree productivity cn be much lrger when nutrients or wter, or both, re optimized continuously or dded nnully insted of only immeditely fter plnting. These experiments highlighted the high responsiveness of trees to the ddition of either nutrients or wter, with some interctive effects of nutrients nd wter, depending on the locl conditions. Trichet et l. (28) showed tht nnul optimiztion of nutrients, but not irrigtion, resulted in significnt increses in boveground productivity of mritime pine in the first five growing sesons fter the beginning of the tretments. As soils in the region of our study site re poor in phosphorus nd wter deficits in summer hve occurred more frequently in recent yers (Trichet et l. 28), we investigted dpttions in the size nd morphology of the mritime pine fine root system, long with ssocited hyphe, to nnul fertiliztion nd irrigtion tretments. Previous studies on coniferous species showed decline in fine root biomss in response to nnul fertiliztion (Albugh et l. 1998, Mier nd Kress 2), nd overll fine root biomss of P. pinster ws lower t the most fertile sites in our study region compred with the lest fertile sites (Acht et l. 28). Consequently, we hypothesized tht trees tht re fertilized nnully cn meet their nutrient demnds with smller fine root uptke system thn unfertilized trees. As rooting depth is greter t drier sites thn t more humid sites (Bkker et l. 26) nd s most nutrients re concentrted in the top 2 cm of the soil profile, we predicted tht fine roots would be more concentrted in the upper lyers of the soil (shllower rooting) in irrigted plots thn in unirrigted plots. Mterils nd methods Site description The study site is locted in the Domine de l Hermitge experimentl forest in Pierroton, 2 km southwest of Bordeux, Frnce (44 42 Nnd 46 W). Site elevtion is 58 m.s.l. nd the topogrphy is flt. Men nnul ir temperture is 12.5 C nd nnul precipittion verges 95 mm, with frequent prolonged droughts in summer. The soils re sndy podzols developed on n eolin sndy deposit from the Quternry er. Folir nutrient concentrtions in the control tretment indicted tht the site is poor in phosphorus (Trichet et l. 28). Wter-tble depth rnges from bout.1 m in winter in wet yers to 1.6 m in summer in dry yers. In 1993, the site ws ploughed to depth of.3 m nd plnted with 1-yer-old mritime pine (P. pinster Aı t. in Solnd) seedlings t spcing of m. In 1998, when the stnd ws 6 yers old, rndomized block design ws estblished for the fctoril combintion of two irrigtion regimes nd three fertiliztion regimes: phosphorus only (P tretment), complete fertiliztion with N, P, K, C nd Mg (F tretment), nd no fertiliztion (C). Irrigtion (I) ws pplied dily t rte equivlent to the men potentil evpotrnspirtion (bout 6mmdy 1 ) during the summer (My October). Fertiliztion ws pplied ccording to folir nlyses nd trget vlues for optiml folir nutrition (13 mg g 1 for N nd N/P, N/K, N/C nd N/Mg rtios of.1,.37,.12 nd.6, respectively; Trichet et l. 28). Ech of the six tretment combintions hd four replictes, yielding 24 plots ech mesuring 6 36 m (.216 h). Externl border zones reduced the plot dimensions to 5 28 m (.14 h with 17 trees in ech of the 24 plots). The fertiliztion nd irrigtion tretments hve been pplied nnully since 1998, with the exception of 2 when the trees were recovering from storm event t the end of 1999 (see Trichet et l. 28 for further informtion). In 1997, the understory ws removed in the inter-tree lines with disking device ( 5 6 cm soil depth) resulting in some disturbnce of the superficil soil. In 1998 nd 1999, the understory ws controlled with chemicls. From 21 onwrd, mowing mchine ws used nd understory plnt mteril s well s ded tree brnches were crushed nd left on the soil surfce. Tble 1 shows some stnd chrcteristics for ech tretment for the yer 25, when our study ws conducted. Dimeter t brest height (DBH t 13 cm) rnged from 18.9 to 24. cm, bsl re from 25.5 to 3.5 m 2 h 1 nd lef re index (LAI) from 2.6 to 3.1. Additionl informtion on the site nd tretments hs been reported by Trichet et l. (28). Field dt cquisition Mesurements (DBH, tree height, folir nutrient concentrtions nd LAI) were performed every yer, on ll trees, or subsmple of trees, or in individul mesurement plots (e.g., for LAI). Allometric reltionships developed t the site were used to compute totl tree needle biomss, totl boveground tree biomss (cf. Trichet et l. 28), nd the sum of totl tproot + corse root biomss (Bert nd Dnjon 26). The llometric equtions were: needle biomss = (DBH )(AGE ), totl boveground biomss =.6575(DBH )(AGE.322 ), nd tproot + corse root biomss = 84.7(C ), where C13 is tree circumference t 13 cm nd biomss is expressed in kg tree 1,DBHincm,AGEinyersnd C13 in m. The three yers preceding our study (22 24) were dry yers (17 21% less rin thn the 3-yer men). Reltive to the men precipittion sums per seson TREE PHYSIOLOGY VOLUME 29, 29

3 EFFECTS OF FERTILIZER AND WATER ON FINE ROOTS OF P. PINASTER 231 Tble 1. Tretment nd stnd chrcteristics in 25 (stnd ge = 13 yers). Tretment code C P F I IP IF Irrigtion None None None Irrigtion Irrigtion Irrigtion Fertilizer Nil P b Complete F c None P b Complete F c Stocking (number h 1 ) DBH t 13 cm height d,e,g (cm) 18.9 (.1) 2.8 (.2) 21.9 (.2) 2.5 (.2) 22.3 (.2) 24. (.3) Totl eril biomss d,e (kg tree 1 ) 84.4 (1.4) 14.5 (2.2) (2.7) 1.7 (1.9) 12.6 (2.8) (4.) Bsl re d,f (BA, m 2 h 1 ) 25.5 (.2) 28.3 (.) 3.1 (.1) 28.5 (.3) 3.5 (.2) 3.5 (.2) Bsl re increment d,f (BAI, m 2 h 1 yer 1 ) 2.6 (.) 2.7 (.) 2.9 (.) 3.3 (.) 3.3 (.) 3.4 (.) LAI d,f (m 2 m 2 ) 2.6 (.) 3. (.) 3. (.) 2.8 (.) 3.1 (.) 3. (.) Dily irrigtion from My to October equivlent to bout 6 mm dy 1. b Men rte in of 32 kg P h 1 yer 1. c Men rte (in kg h 1 yer 1 ) for of 84 N, 34 P, 56 K, 22 C, 7 Mg, 1.3 B, 2.9 Cu, 2.1 Mn nd.6 Zn. d Vlues refer to men vlues nd SE (between brckets). e Men vlues bsed on ll trees present in the two blocks considered. f Men vlues bsed on sums for two blocks (n = 2). g Using qudrtic mens for men DBH. for , utumn 24 ws dry ( 22%), winter 25 ws very dry ( 43%), spring 25 ws verge ( 9%), nd summer 25 ws very dry ( 38%). Root smples were collected in erly July 25 in soil cores to depth of 12 cm in two blocks (two plots per tretment). In ech plot (.14 h), two smple zones (re round trget trees; i.e., m for m spcing) were selected from which four root smples were collected (yielding totl of 16 cores per tretment). The smplezoneswerechoseninreltiontotwosubsequenttrget trees within the sme tree line to enble comprisons between boveground tree dimensions nd belowground root dt bsed on the dimensions of the trees nd the root dt corresponding to ech zone. The trget trees hd to hve dimeters comprble to the men of the tretment nd hd to be surrounded by ll four neighboring trees in subsequent tree lines. The positions of the four root smple cores in ech zone were ligned perpendiculrly to the tree line t equl distnces from both trget trees. The distnce between smple points ws 1 m. The two centrl points were thus in the tree lines nd the two outer points in interlines. For ech core smple point, litter ws smpled with root corer device (internl dimeter of core hed 8 cm; mximum length 15 cm). Soil smples were tken with percussion drill (internl dimeter of 4 cm; mximum length 1 m) in two stges ( 6 nd 6 12 cm) nd were divided into 15-cm deep lyers. One yer lter, in My 26, 6 mesh bgs (3 lm mesh size) were instlled in the site s described by Wllnder et l. (24) to evlute length nd mss of hyphe. The mesh bgs were filled with 12 g cid-wshed qurtz snd (pssed through 2-mm sieve) nd buried in the C, P nd IP tretments in the upper 5 cm of the minerl soil (2 bgs per tretment). Bg positions were in tree lines t 1 m from the rndomly selected trees. Hlf of the bgs were left in the soil for 6 months (retrieved in November 26) nd the reminder for 12 months (retrieved in My 27). Both yers were slightly drier thn verge ( 22% precipittion for 26, 16% for 27). Root nd mesh bg smple processing Core smples were plced in plstic bgs, trnsported to the lbortory the sme dy, nd stored t 4 Cuntiltheywere processed (within 2 months). The moist smples were sieved through 4- nd 2-mm sieves without wter nd the root frgments were trnsferred from the sieves to floting bsin, from which live fine roots (dimeter < 2 mm) nd live smll roots (dimeter 2 2 mm) of P. pinster were collected mnully using forceps. Despite the nnul removl of understory vegettion, roots of understory shrubs nd herbs were present in the core smples. Roots of these understory species nd ded roots of P. pinster were not retrieved. Species nd vitlity were distinguished by visible chrcteristics nd root odor (Bkker et l. 26). The lengths of the smll roots were determined with mesuring rod. Root lengths, dimeters nd morphology of fine roots (specific root length, specific root re nd number of root tips per unit of root length) were obtined by imge nlysis. The clened fine roots were submerged in 1% ethnol t 4 C before being scnned. The imges were nlyzed using WinRHIZO Version 25 (Regent Instruments Inc., Nepen, ON, Cnd). The number of root tips counted during the nlysis of these imges (16 smples per tretment nd soil lyer) ws compred with mnul counts mde with the id of stereo microscope (n =4)while exmining ectomycorrhizl morphotypes. Becuse the Win- Rhizo nd mnul counts were similr, we subsequently used the output vlue from WinRHIZO to estimte the number of ectomycorrhizl root tips per unit of root length (ECM-RL). Root dry mss ws ssessed by drying the root mteril t 15 C to constnt mss. Mesh bgs contining qurtz snd nd the hyphl structures tht hd grown into them were kept t 4 C until processed. They were then TREE PHYSIOLOGY ONLINE t

4 232 BAKKER ET AL. opened nd the snd ws plced in trnsprent bsin to fcilitte visul inspection of the smple with the id of binoculr microscope. Hyphl length nd hyphl mss were ssessed s described by Wllnder et l. (24). Briefly, hyphl length ws mesured by plcing 15 2 g liquot in vil with 8 ml of deionized wter, shking for 1 min, nd then trnsferring 5 ml of the solution to filter pper. The filter pper ws spryed with methylene blue to stin the hyphe. The qudrnts of the filter pper were then observed with the id of stereomicroscope (1 2 mgnifiction) nd n opticl monoculr glss with regulr grid ws plced on the prts of the filter pper where hyphl structures were detected. Intersections of the grid (interline distnce of.625 mm) nd the hyphl structures were used to clculte hyphl length s described by Tennnt (1975). For hyphl mss (ssessed fter 12 months only), severl g smples were ech plced in smple vil contining 8 ml of deionized wter nd shken for 1 min. The entire solution ws trnsferred to filter pper in 5-ml steps. Observed hyphl structures were retrieved using forceps nd trnsferred to petri dishes. Remining smll hyphl frgments tht represented < 5% of the totl mount of hyphe were evluted visully (s percentge of the recovered hyphl structures in the petri dishes). The retrieved hyphl structures in the petri dishes were observed with the id of binoculr microscope nd clened by removing dhering snd grins using forceps. Hyphl dry mss of the smples ws expressed on n sh-free bsis (the difference between the dry mss fter 48 h t 6 C nd the sh content obtined by grdul ignition up to 55 C for 5 h). To determine whether the hyphe were ectomycorrhizl or sprophytic species, 1 subsmples of hyphe (dried t 6 C) s well s dried smples of crpophores collected in utumn 26 (six ectomycorrhizl species, five sprophytic species) were nlyzed for 13 C isotopic composition by mss spectrometry (Trcer Mss; Europ Scientific, Crewe, UK). Hyphl length nd biomss were first expressed on the bsisof12gsndndthenconvertedtonrebsis (m 2 ) by using 1.56 g cm 3 (cf. Wllnder et l. 24) s the men density of both soil nd snd nd ssuming tht the bgs were representtive of the 1 cm soil lyer. This vlue seems relistic s it is within the rnge of 1.58 ±.2 g cm 3 (men ± SE) for the sndy soils of the study region tht hve low C content (< 1 mg g 1 ; n = 19, Augusto nd Bkker unpublished dt). Specific hyphl length ws expressed s the rtio of hyphl length to hyphl mss. Sttisticl nlyses Effects of tretments were evluted seprtely for root distribution, root morphology, summed profile vlues of the roots, nd for boveground to belowground rtios. We used the SAS softwre Version 8.1 (SAS Institute, Cry, NC) to compute men vlues nd stndrd errors, nd to test differences between tretments nd soil depths. Two-wy ANOVA ws used to test the effects of phosphorus (P), irrigtion (I) nd their interction on root nd eril prmeters. Two-wy ANOVA ws lso used to test the effects of complete fertiliztion (F), I nd their interction. One-wy ANOVA ws used to ssess the generl effects of the stnd on hyphl prmeters, followed by the Bonferroni t test to distinguish between the men vlues per tretment. To ssess the effects of both tretment nd soil depth, mixed liner models were used with tretment, depth nd tretment deptheffectssfixedeffectsndtherootsmple point s rndom effect. The Bonferroni djustment in the lest squres mens differences procedure ws used to ssess differences between tretments s function of soil depth s well s differences between depth lyers within tretments. Arc sine trnsformtions were used for percentge vlues to meet model ssumptions. Results The size of the fine root system ws strongly dependent on soil depth (P <.1). Fine root length densities were low in the shllow litter lyer ( cm cm 3 ) nd highest in the top 15 cm of soil ( cm cm 3 ). Fine root length densities decresed with soil depth in similr mnner in ll tretments (Figure 1A nd B), nd no fine roots were recorded below 1 cm in cores of ny of the tretments. Fine root re density, fine root biomss density nd smll root biomss density showed similr ptterns with soil depth (dt not shown seprtely). The effect of P on fine root length density ws significnt only in the 15 3 cm soil lyer (P =.18), nd I nd the P I interction hd no significnt effects on fine root length density (Tble 2) t ny soil depth. The F tretment resulted in significnt decrese in fine root length density in the top 3 cm of the soil (P <.1), but I nd F Ihdnosignificnt effects (Tble 2). Fine root re density ws ffected by the tretments in similr wy s fine root length density (dt not shown in detil). The morphologicl prmeters of the fine root system, fine root dimeter, specific root length, specific root re nd number of ECM-RL, were ll significntly ffected by soil depth (P <.1 in ll cses). Overll, fine root dimeter incresed with soil depth (from mm in litter to.76.9 mm t 45 6 cm) nd specific root length (Figure 1C nd D; Tble 2); specific root re (from cm 2 g 1 in litter to cm 2 g 1 t 45 6 cm) nd number of ECM-RL (Tble 2) decresed with soil depth. Phosphorus ppliction significntly incresed specific root length nd decresed the number of ECM-RL in severl soil lyers (Tble 2; P <.5). In contrst, I hd only one significnt effect it decresed the number of ECM-RL (P <.46) in the 15 3 cm soil lyer nd the P I interction ws never significnt (Tble 2). Complete fertilizer ppliction resulted in significnt increse in specific root length (P <.5) nd TREE PHYSIOLOGY VOLUME 29, 29

5 EFFECTS OF FERTILIZER AND WATER ON FINE ROOTS OF P. PINASTER 233 A B cm cm C P Depth (cm) Depth (cm) F cm cm 3 I IP IF C D Depth (cm) Depth (cm) m g C P F m g I IP IF Figure 1. Fine root length density (cm cm 3 ) in nonirrigted plots (A) nd irrigted plots (B), nd specific root length (m g 1 ) in the nonirrigted (C) nd irrigted plots (D). Men vlues nd SE re shown. Abbrevitions: C, control tretment; P, phosphorus fertiliztion tretment; F, complete fertiliztion tretment; I, irrigtion tretment; IP, irrigtion + phosphorus fertiliztion tretment; nd IF, irrigtion + complete fertiliztion tretment. Tble 2. Tretment men vlues (rnge of six tretment men vlues) nd sttisticl significnce (P levels) for fine root length density (FRLD), specific root length (SRL), nd rmifictions (ECM-RL) t stnd ge of 13 yers, 7 yers fter tretment initition for soil depths of 6 cm (n = 16 root cores per tretment). Tretment men vlues P vlues P vlues P I P I F I F I FRLD cm cm 3 Litter cm ** cm * *** cm cm SRL mg 1 Litter * ** cm * ** cm cm * cm *.14 ** ECM-RL Number m 1 Litter cm * cm *.74 ** ** cm * cm *P <.5; **P <.1; ***P <.1. significnt decrese in the number of ECM-RL (Tble 2; P <.5) in severl soil lyers, wheres I significntly reduced the number of ECM-RL in the 15 3 cm soil lyer (P =.3), but hd no effect on specific root length. The F I interction ws only significnt for specific root length t depth of 45 6 cm (P =.7). The tretments ffected specific root re in similr wy s specific root length, lthough specific root re showed greter response to irrigtion. Significnt decreses in fine root dimeter in response to the F nd I tretments were recorded only in the litter lyer (dt not shown seprtely). Hyphl growth ws mesured in ingrowth mesh bgs 6 12 months fter incubtion in the C, P nd IP tretments only (Figure 2). Hyphl length in ingrowth bgs TREE PHYSIOLOGY ONLINE t

6 234 BAKKER ET AL. ws significntly greter in the IP tretment thn in the P nd C tretments (Figure 2A; P <.1, Bonferroni t test), but vlues did not differ significntly between 6 nd 12 months of incubtion (P =.12). Hyphl lengths rnged between.6 nd km m 2 in the C nd IP tretments fter 6 months of incubtion nd tended to increse to 3. nd km m 2, respectively, fter 12 months of incubtion (Figure 2A). After 12 months of incubtion, hyphl mss ws significntly higher in the IP tretment thn in the C tretment (P =.6), nd vlues in the P tretment were intermedite between those in the IP nd C tretments (Figure 2B). Totl hyphl mss rnged between 6 nd 17 g m 2 (Figure 2B). Specific hyphl length ws not significntly ffected by the tretments (P =.6) nd rnged between 44 nd 89 km g 1 in the C nd IP tretments (Figure 2C). Hyphe from the mesh ingrowth bgs hd d 13 C vlue of ±.15& (n = 1) tht ws comprble with the vlue of ±.32& (n = 6) mesured in the crpophores of species we identified s ectomycorrhizl species (dt not shown in detil). Both vlues differed significntly from the d 13 Cvlueof ±.61& (n = 5) obtined for crpophores of sprophytic fungl species (P <.1). Dt in Tble 3 summrize the tretment effects on the verticl depth distribution of fine roots. Bsed on biomss, length, or surfce re, between 52% nd 81% of ll fine roots occurred in the top 3 cm of the soil. The P nd P I tretments did not ffect this distribution significntly (Tble 3). In contrst, I resulted in significntly shllower A 1 2 km m months 12 months b Hyphl length b C P IP B g m months b verticl root distribution. The F tretment significntly ffected the percentge of fine root biomss nd fine root re in the top 3 cm (Tble 3), wheres I hd no significnt effects on these mesures but there were significnt F I interctions on the percentge of fine root length nd fine root re in the top 3 cm of soil. Aboveground growth ws ffected by the P, F nd I tretments, but not their interctions (Tble 4). Dimeter t brest height, totl needle mss nd totl eril tree mss were ll significntly incresed by P, F or I (Tble 4). Smll root biomss in the entire 12-cm soil profile, including the litter lyer, rnged between 112 nd 562 g m 2 nd ws not significntly ffected by the tretments (coefficient of vrition 9 24%). Summed fine root biomss for the entire 12-cm soil profile, including the litter lyer, rnged between 111 nd 296 g m 2. It decresed significntly in the P nd F tretments, but ws unffected by I nd the interctions between P nd I nd between F nd I (Tble 4). Fine root length vried from 1162 to 2486 m m 2 nd the F tretment significntly decresed fine root length, wheres P, I nd the interctions hd no significnt effects on fine root length (Tble 4). Summed fine root re rnged between 2.4 nd 4.79 m 2 m 2 nd only the F tretment significntly reduced this vlue (Tble 4). Totl number of ectomycorrhizl tips rnged between 62 nd tips m 2 (Tble 4). The F tretment significntly reduced this number wheres P, I, P IndF I did not significntly ffect the totl number of ectomycorrhizl tips (Tble 4). Rtios of root biomss:shoot biomss were significntly ffected only by Hyphl mss b C P IP C km g Specific Hyphl Length 12 months C P IP Figure 2. Hyphl length (1 2 km m 2 ) fter 6 nd 12 months of incubtion (A), hyphl mss (g m 2 ) (B), nd specific hyphl length (km g 1 ) (C). Men vlues for 1 ingrowth bgs +1 SE re shown. Different letters indicte significnt differences between tretments t P <.5 (Bonferroni t test). Differences between the 6- nd 12-month incubtion times were not significnt (P =.119; Bonferroni t test). Tble 3. Summry of tretment men vlues, SE, nd sttisticl significnce (P levels) on verticl root distribution t stnd ge of 13 yers, 7 yers fter tretment initition (n = 16 root cores per tretment). Vrible (%) Tretment men vlues (SE) P vlues P vlues C P F I IP IF P I P I F I F I FRB3 6 (4) 6 (4) 53 (4) 72 (5) 71 (5) 52 (6).99 **.92 * FRL3 68 (3) 67 (4) 7 (3) 79 (4) 81 (3) 62 (5).77 ** * FRA3 63 (3) 64 (4) 62 (4) 75 (5) 77 (4) 58 (5).56 **.78 *.22 * FRB, FRL or FRA3 = % of totl fine root biomss, length, or surfce in 12 cm confined to top 3 cm. *P <.5; **P <.1. TREE PHYSIOLOGY VOLUME 29, 29

7 EFFECTS OF FERTILIZER AND WATER ON FINE ROOTS OF P. PINASTER 235 Tble 4. Summry of tretment men vlues, SE nd sttisticl significnce (P levels) for eril vribles, summed root vlues for 12 cm, nd root:eril rtios t ges 13, 7 yers fter tretment initition. Vrible (units) Tretment men vlues nd SE P vlues P vlues C P F I IP IF P I P I F I F I Aeril vribles Number of trees DBH (cm) 18.9 (.1) 2.8 (.2) 21.9 (.2) 2.5 (.2) 22.3 (.2) 24. (.3) *** ***.77 *** ***.29 NB (kg tree 1 ) 8.2 (.1) 1. (.2) 11. (.2) 9.7 (.2) 11.4 (.2) 13.1 (.3) *** ***.92 *** ***.8 TAB (kg tree 1 ) 84 (1) 15 (2) 116 (3) 11 (2) 121 (3) 141 (4) *** ***.98 *** ***.6 Root vribles Number of cores SRB (g m 2 ) 379 (161) 359 (74) 562 (336) 214 (48) 193 (48) 112 (27) FRB (g m 2 ) 296 (44) 183 (14) 159 (17) 294 (53) 186 (17) 111 (2) ** *** FRL (km m 2 ) 2.5 (.4) 2. (.2) 1.4 (.1) 2.4 (.3) 2.2 (.2) 1.2 (.2) *** FRA (m 2 m 2 ) 4.8 (.7) 3.8 (.3) 2.8 (.2) 4.7 (.6) 4. (.4) 2.4 (.4) *** T-ECM (N in 1 m 2 ) 38 (51) 236 (41) 136 (19) 32 (52) 26 (41) 62 (1) *** Root:eril Number of rtios R:S.32 (.1).31 (.).31 (.).32 (.1).31 (.).31 (.) * FR:N.32 (.6).15 (.1).12 (.1).28 (.6).14 (.1).7 (.1) ***.5.72 *** RAI:LAI 1.88 (.3) 1.24 (.1).93 (.8) 1.68 (.22) 1.31 (.11).81 (.13) * *** This is the sum of ll trees in both replicted blocks tht were in zones tht hd not been ffected by the 1999 storm nd could be included in the clcultions. DBH, tree dimeter t brest height (cm); NB, needle biomss per tree; TAB, totl eril biomss per tree; SRB, summed smll root biomss in 12 cm soil nd litter; FRB, summed fine root biomss in 12 cm nd litter; FRL, summed fine root length in 12 cm nd litter; FRA, summed fine root surfce re in 12 cm nd litter; T-ECM, totl ectomycorrhizl root tips in 12 cm nd litter in 1 per m 2 ; R:S, totl boveground:totl belowground biomss (fine roots + sum of tp nd corse roots); FR:N, summed fine root biomss in 12 cm nd litter:men needle biomss of the four closest trees; RAI:LAI, fine root re index s summed fine root re for 12 cm nd litter in m 2 m 2 :LAI in m 2 m 2 for the corresponding tretment. Smll roots, roots with dimeter between 2 nd 2 mm; fine roots, roots with dimeter < 2 mm. *P <.5; **P <.1; ***P <.1. RAI:LAI b b C P F I IP IF F (Tble 4). The fine root mss:needle mss rtio nd the root re index:lef re index (RAI:LAI) rtio showed similr ptterns to those of fine root size with both rtios decresing significntly in response to the P nd F pplictions but not to I or the interctions including I (Tble 4; Figure 3). b Figure 3. Root re index: lef re index (RAI:LAI) rtios. Men vlues (n = 16 per tretment) +1 SE re shown. Different letters indicte significnt differences between tretments (P <.5; djusted Bonferroni t test). Abbrevitions: C, control tretment; P, phosphorus fertiliztion tretment; F, complete fertiliztion tretment; I, irrigtion tretment; IP, irrigtion + phosphorus fertiliztion tretment; nd IF, irrigtion + complete fertiliztion tretment. b Discussion Fine root size nd morphology Although the roots were smpled firly close to the stems of the trees (< 2 m wy), which is where highest rooting density should occur (Sudmeyer et l. 24), our summed fine root biomss for the 12 cm soil nd litter profile ws lower ( g m 2 ) thn the g m 2 rnge previously recorded for P. pinster in the region (Bkker et l. 26, Acht et l. 28). Smpling ws performed in summer in the cited studies nd should thus be comprble with our study. Vlues for the 15 cm soil lyers pper to be lower in erly spring nd somewht higher in utumn (n = 27 study sites; Bkker unpublished dt) thn vlues in midsummer, but this pttern my vry from yer to yer depending on climtic conditions. The proportion of fine root biomss or fine root length in the top 3 cm (reltive to the totl 12 cm soil profile) ws 52 81% which is similr to the 52 8% reported by Bkker et l. (26). Annul ppliction of P or F significntly decresed the size of the fine root system, wheres irrigtion resulted in shllower rooting profile. Thus, under our experimentl conditions, nutrient vilbility ws more importnt in explining differences in the size of the fine root system thn wter vilbility. Similr results were TREE PHYSIOLOGY ONLINE t

8 236 BAKKER ET AL. obtined with soil cores in nutrient nd wter resource optimiztion experiments on P. ted in USA (Albugh et l. 1998, Mier nd Kress 2), nd the reltive but not bsolute lloction to fine root biomss decresed in fertiliztion nd irrigtion experiment on P. sylvestris in Sweden (Axelsson nd Axelsson 1986). The bsence of strong effect of irrigtion on the size of the fine root system in our study is likely becuse the soil wter content of the unirrigted plots ws often sufficient to meet the wter demnd of the trees, even though nnul precipittion ws 17 21% lower thn norml in the 3 yers preceding our smpling. The site is on humid moorlnd with firly high wter tbles nd men yerly precipittion of 95 mm, but prolonged summer droughts cn led to significnt wter deficits becuse rooting depth is limited to bout 1 m by the high winter wter tbles nd the presence of discontinuous hrd pn (Dnjon et l. 1999, Bkker et l. 26, Acht et l. 28). Given the dry climtic conditions in recent yers, the fine root system my exhibit greter response to the combined effect of irrigtion nd fertilizers in the future becuse irrigtion hd significnt effect on the eril dimensions of the trees (Tble 4). The specific root lengths tht we hd mesured (6 35 mg 1 ) were greter thn those mesured in 55-yer-old P. pinster stnds in the region (4 12 m g 1 ), but the ectomycorrhizl root tips per meter of fine root length (68 255) ws comprble with the 9 35 ectomycorrhizl root tips per meter of fine root length mesured in the 55-yer-old P. pinster stnds (Bkker et l. 26). The P nd F tretments incresed specific root lengths nd specific root res in both the irrigted nd nonirrigted plots. The pek vlues of m g 1 in the P nd F tretments (Figure 1) ccord with the vlues for the finest root dimeter frctions in other studies: m g 1 for 1 mm dimeter roots mesured in 38-yer-old P. sylvestris in Sweden (Clemensson-Lindell nd Persson 1993), 22 3 m g 1 in newly formed P. sylvestris roots in greenhouse experiment (George et l. 1997), nd 2 52 m g 1 for the finest order roots of P. sylvestris nd Pinus nigr (Arnold) in Polnd, Finlnd nd Estoni (Withington et l. 26, Ostonen et l. 27, 27b). The high specific root lengths in the P nd F tretments indicte tht lrger proportion of the < 2 mm frction commonly considered s fine roots comprises the finer root frction (< 1 or <.5 mm dimeter) in these tretments. This will hve consequences on root uptke, respirtion nd turnover, becuse these processes differ mong root dimeter size frctions (Eissenstt nd Yni 1997, Pregitzer et l. 1998, King et l. 22, Pierret et l. 25). In ddition, hyphl lengths nd hyphl mss significntly incresed when irrigtion nd phosphorus were pplied simultneously. These hyphe belonged to ectomycorrhizl species bsed on their d 13 C vlues which were comprble with those collected from ectomycorrhizl mushrooms (cf. Wllnder et l. 24). Overll, under our experimentl conditions, incresing nutrient vilbility led to nutrient uptke system comprising n incresed proportion of fine roots of < 1 or even <.5 mm in dimeter s well s incresed extension of the ectomycorrhizl hyphl network. A finer root system for incresed nutrient vilbility is in ccordnce with the ssumption tht specific root lengths should be high t our productive study site (Delzon nd Loustu 25) becuse fst growth requires fst nd efficient cquisition of resources (Ryser 26). Our results re similr to studies in Estoni nd Finlnd including 11 Pice bies stnds, three Betul pendul (Roth) stnds nd three P. sylvestris stnds (Ostonen et l. 1999, 27) where, irrespective of tree species, specific root res incresed with incresing site fertility. Assuming tht fertiliztion ffects the different dimeter clsses (mycorrhizl short roots, < 1, 1 2 nd < 2 mm roots) differently (Ostonen et l. 27b), this my explin the pprent discrepncies between core nd minirhizotron methods (Albugh et l. 1998, King et l. 22). These two studies were crried out t the sme experimentl P. ted site nd showed less fine root stnding biomss in soil cores fter nnul nutrient optimiztion (Albugh et l. 1998) but more < 1 mm roots in minirhizotron imges (King et l. 22). Bsed on these results, we suggest tht fine root production nd turnover of the dynmic frction re positively correlted with nutrient vilbility (Ndelhoffer et l. 1985, King et l. 22, Mier et l. 24), wheres this is not the cse for the entire < 2 mm dimeter frction (Tble 4). Incresed root turnover is reported to be n efficient mechnism to increse phosphorus nd potssium uptke (Steingrobe 25). Under our conditions, the optiml crbon investment (Chpin et l. 1987, Eissenstt 1992, Ryser 26) in response to incresed nutrient vilbility is higher specific root length nd more ectomycorrhizl hyphe, nd perhps higher fine root turnover. Root:shoot reltionships Totl root:shoot rtios were not clerly ffected by the tretments but the fine root mss:needle mss rtios, tht rnged from.7 to.32, were significntly decresed by pplictions of P or F. Lower fine root mss:needle mss rtios with incresing P vilbility were lso recorded by Zerihun nd Montgu (24) nd by Helmisri et l. (27) t 16 sites where Pice bies nd P. sylvestris grew on nturl fertility grdient. In our study, irrigtion did not significntly ffect fine root mss:needle mss rtios, lthough t sites with lower soil wter content, higher root to shoot biomss rtios hve been reported (Cirns et l. 1997). The fine RAI:LAI rtios in our study rnged from.81 to 1.88 nd were lowest in the F tretment nd highest in the control tretments; irrigtion did not ffect these rtios. Overll, fertiliztion tretments ffected both fine RAI nd LAI. For the fine RAI mesurements, only fine root surfce dt were vilble from smples tken in 25, wheres LAI ws monitored on n nnul bsis. At our study site, LAI peked in 21 nd then plteued from TREE PHYSIOLOGY VOLUME 29, 29

9 EFFECTS OF FERTILIZER AND WATER ON FINE ROOTS OF P. PINASTER onwrd, reching 3. in the control tretment nd up to 3.9 in the fertilized plots (Trichet et l. 28). Vlues of 3. re higher thn those typiclly found in mture stnds of the highest fertility clss in the region (Delzon nd Loustu 25). The LAI declined to in 24 nd 25 s result of thinning nd wind throw. Hence, both tree dimensions nd LAI were lower in the control tretments thn in the P nd F tretments, wheres fine RAI ws higher. This my be the direct result of incresed nutrient vilbility (i.e., higher lloction to boveground growth nd crown biomss nd lower lloction to fine root stnding biomss), or my be relted to ontogeny- or size-relted differences mong the tretments (Ovington 1957, Helmisri et l. 22, Ritson nd Sochcki 23, Zerihun nd Montgu 24, Coyle nd Colemn 25, Coyle et l. 28). The root:shoot rtios decresed from.87 t the ge of 7 yers to.29 t 55 yers (Ovington 1957) in P. sylvestris in the UK, nd from.33 t the ge of 15 yers to.15 t 1 yers in P. sylvestris in Finlnd (Helmisri et l. 22). We obtined no cler indictions tht size-relted differences explined ny of the tretment effects. Overll root:shoot rtios did not differ significntly between tretments, but they decresed in the first decdes fter stnd estblishment in P. pinster in Austrli (Ritson nd Sochcki 23). Chnges in P. pinster tree or stnd physiology re reported to occur somewhere between the ges of 1 nd 32 yers in the study region (Delzon nd Loustu 25), but LAI vlues were stble in ll of our tretments. Given the ge of our trees nd the limited time spn of the tretments, we believe tht the observed shifts in lloction (towrd more needle biomss reltive to fine root biomss) together with n increse in the finest prt of the fine root system (including ectomycorrhizl hyphe) re more likely to be the result of nutrient vilbility thn of overll developmentl effects. We conclude tht nnul fertiliztion with either P or F for seven growing sesons led to reduction in the size of the fine root system in the P. pinster stnds by the ge of 13 yers. This supports our hypothesis tht lloction to fine roots decreses with incresing nutrient vilbility. So fr, irrigtion hs hd no significnt effect on the size of the fine root system, but hs resulted in shllower rooting system. This finding supports our second hypothesis tht roots re more concentrted in the top soil in irrigted stnds. However, the P nd F pplictions resulted in n increse in finer root morphology (higher specific root length/re) nd irrigtion tended to enhnce this finer root morphology, which we hd not predicted. The quntity of ectomycorrhizl hyphe tht grew into the mesh bgs ws lso higher in the P nd IP tretments thn in controls, suggesting further extension of the finest prt of the fine root system. Fine root to needle biomss or surfce re rtios were highest in control tretments nd decresed with incresing resources, which my be either direct consequence of resource vilbility on fine roots or the result of size-dependent lloction differences, i.e., less lloction to fine root systems s trees increse in ge or size. Intensive monitoring over longer time spn is needed to investigte whether, in the longer term, it is nutrient vilbility or tree size tht best explins the differences in tree growth (Coyle et l. 28). Our study suggests tht the fine root clss limit of 2 mm dimeter my well be inpproprite for studying nutrient nd fine root reltionships nd tht fine root production or turnover in the finest nd dynmic frction should receive more ttention (Withington et l. 26, Ostonen et l. 27b). Acknowledgments The uthors thnk Christin Brbot nd Sylvie Niollet for their help in collecting the field smples nd Cthe rine Pernot for prepring the mesh ingrowth bgs. Finncil support ws obtined from JPE (Jeunes Professionels à l Etrnger), enbling E. Jolicoeur s sty in Frnce. The PN-ACI ECCO project De terminnts nturels et nthropiques de l biodiversite rhizosphe rique et de l biodisponibilité du phosphore en foreˆ t tlntique funded the mesh bg study. The uthors thnk the INRA-Pierroton Experimentl Unit, under the supervision of Ptrick Pstuzsk nd Fre de ric Bernier,for the instlltion nd mintennce of the experimentl site. References Acht, D.L., M.R. Bkker nd P. Trichet. 28. Rooting ptterns nd fine root biomss of Pinus pinster ssessed by trench wll nd core methods. J. For. Res. 13: Albugh, T.J., H.L. Allen, P.M. Dougherty, L.W. Kress nd J.S. King Lef re nd bove- nd below-ground growth responses of loblolly pine to nutrient nd wter dditions. For. Sci. 44: Axelsson, B. nd E. Axelsson Chnges in crbon lloction ptterns of spruce nd pine trees following irrigtion nd fertilistion. Tree Physiol. 2: Bkker, M.R., L. Augusto nd D.L. Acht. 26. Fine root distribution of trees nd understory in mture stnds of mritime pine (Pinus pinster) on dry nd humid sites. Plnt Soil 286: Bert, D. nd F. Dnjon. 26. Crbon concentrtion vritions in the roots, stem nd crown of mture Pinus pinster (Ait.). For. Ecol. Mng. 222: Cirns, M.A., S. Brown, E.H. Helmer nd G.A. Bumgrdner Root biomss lloction in the world s uplnd forests. Oecologi 111:1 11. Chpin, F.S., A.J. Bloom, C.B. Field nd R.H. Wring Plnt responses to multiple environmentl fctors. Bioscience 37: Clemensson-Lindell, A. nd H. Persson Long-term effects of liming on the fine-root stnding crop of Pice bies nd Pinus sylvestris in reltion to chemicl chnges in the soil. Scnd. J. For. Res. 8: Coyle, D.R. nd M.D. Colemn. 25. Forest production responses to irrigtion nd fertiliztion re not explined by shifts in lloction. For. Ecol. Mng. 28: Coyle, D.R., M.D. Colemn nd D.P. Aubrey. 28. Abovend below-ground biomss ccumultion, production, nd distribution of sweetgum nd loblolly pine grown with irrigtion nd fertiliztion. Cn. J. For. Res. 38: TREE PHYSIOLOGY ONLINE t

10 238 BAKKER ET AL. Dnjon, F., D. Bert, C. Godin nd P. Trichet Structurl root rchitecture of 5-yer-old Pinus pinster mesured by 3D digitising nd nlysed with AMAPmod. Plnt Soil 217: DeLuci, E.H., J.G Hmilton, S.L. Nidu, et l Net primry production of forest ecosystem with experimentl CO 2 enrichment. Science 284: Delzon, S. nd D. Loustu. 25. Age-relted decline in stnd wter use: sp flow nd trnspirtion in pine forest chronosequence. Agric. For. Meteorol. 129: Eissenstt, D.M Costs nd benefits of constructing roots of smll dimeter. J. Plnt Nutr. 15: Eissenstt, D.M. nd R.D. Yni The ecology of root lifespn. Adv. Ecol. Res. 27:1 63. George, E., B. Seith, C. Scheffer nd H. Mrschner Responses of Pice, Pinus nd Pseudotsug roots to heterogeneous nutrient distribution in soil. Tree Physiol. 17: Go rnsson, H., H. Wllnder, M. Ingerslev nd U. Rosengren. 26. Estimting potentil nutrient uptke from different soil depths of Quercus robur, Fgus sylvtic, nd Pice bies. Plnt Soil 286: Helmisri, H.-S., K. 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Soil CO 2 evolution nd root respirtion in 11 yer-old loblolly pine (Pinus ted L.) plnttions s ffected by moisture nd nutrient vilbility. Cn. J. For. Res. 3: Mier, C.A., T.J. Albugh, H.L. Allen nd P.M. Dougherty. 24. Respirtory crbon use nd crbon storge in midrottion loblolly pine (Pinus ted L.) plnttions: the effect of site resources on the stnd crbon blnce. Globl Chnge Biol. 1: Ndelhoffer, K.J., J.D. Aber nd J.M. Melillo Fine root production in reltion to net primry production long nitrogen vilbility grdient in temperte forests: new hypothesis. Ecology 66: Oren, R., D.S. Ellsworth, K.H. Johnsen, et l. 21. Soil fertility limits crbon sequestrtion by forest ecosystems in CO 2 - enriched tmosphere. Nture 411: Ostonen, I., K. Lo hmus nd R. Lsn The role of soil conditions in fine root ecomorphology in Norwy spruce (Pice bies (L.) Krst.). Plnt Soil 28: Ostonen, I., K. Lo hmus, H.-S. Helmisri, J. Truu nd S. Meel. 27. 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