Mobilization and transfer of nutrients from litter to tree seedlings via the vegetative mycelium of ectomycorrhizal plants

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1 RESEARCH New Phytol. (2), 145, Mobiliztion nd trnsfer of nutrients from litter to tree seedlings vi the vegettive mycelium of ectomycorrhizl plnts J. PEREZ-MORENO AND D. J. READ* Deprtment of Animl & Plnt Sciences, University of Sheffield, Sheffield S1 2TN, UK Received 17 June 1999; ccepted 18 October 1999 SUMMARY The bility of the mycorrhizl fungus Pxillus involutus to mobilize nitrogen nd phosphorus from discrete ptches of beech (Fgus sylvtic), birch (Betul pendul) nd pine (Pinus sylvestris) litter collected from the fermenttion horizon of three forest soils, nd to trnsfer the nutrients to colonized B. pendul Roth seedlings, ws investigted in trnsprent observtion chmbers. The mycelium of P. involutus forged intensively in ll three types of litter, leding to significnt decline in their phosphorus contents fter 9 d. Over the sme period only one of the litter types, beech, showed more thn 1% loss of its N contents. Exploittion of the litter led to invigortion of the vegettive mycelium of the fungus throughout the chmbers s well s to significnt increses of biomss production nd lef re in seedlings grown in the plus litter ( L) reltive to those in minus litter ( L) systems. The yield increses were ssocited with gins in whole plnt tissue content nd concentrtion of P, but in content only in the cse of N. Clcultions suggest tht mjor proportion of the phosphorus lost from litter originted in its orgnic frction. The possible bsis of the discrepncy between vlues of N loss from litter nd gin by the plnt is discussed nd the extent to which the distinctive pttern of nutrient mobiliztion is feture peculir to this fungus-plnt combintion is considered. It is concluded tht nutrient mobiliztion from nturl orgnic substrtes in the fermenttion horizon of forest soils my be key function of the vegettive mycelium of mycorrhizl systems. The need for experimentl nlyses of greter rnge of fungus-plnt prtnerships is stressed. Key words: mycorrhiz, nutrient mobiliztion, vegettive mycelium, phosphorus, nitrogen, Pxillus, Betul. INTRODUCTION Melin nd co-workers (Melin & Nilsson, 195, 1953) were the first to demonstrte tht the vegettive myceli of ectomycorrhizl (ECM) plnts were ble to cpture nitrogen nd phosphorus ions nd fcilitte their trnsfer over distnces of severl centimetres to roots. Wheres these pioneering studies were crried out under septic conditions, using minerl solutions, ttention hs turned more recently to questions concerning the bility of ECM mycelil systems to mobilize nd trnsport nutrients from more complex substrtes, representtive of those in which ectomycorrhizl roots selectively proliferte in nture. These re predominntly orgnic residues of the trees, their fungl symbionts nd other components of the soil microflor, locted below the surfce litter lyer in the fermenttion horizon (FH). Bending & Red (1995) observed tht loclized res of FH mteril plced s discrete blocks within *Author for correspondence (tel 44 () ; fx 44 () ; e-mil D.J.Red Sheffield.c.uk). homogeneous pet becme intensively occupied by myceli of Suillus bovinus (Fr.) O. Kuntze nd Thelephor terrestris (Ehrh) s they grew from ectomycorrhizl roots of Pinus sylvestris L., the exploittion of the orgnic residues being ssocited with progressive reduction of their N nd P contents. Similrly, Entry et l. (1991) showed tht in Dougls fir forest, the N nd P contents of litter bgs, plced into soil occupied by dense mycelil mts of the ECM fungus Hysterngium setchellii Fisher, were lower thn those in bgs plced into soil with norml levels of hyphl occurrence. The mechnisms involved in nutrient mobiliztion from nturl substrtes hve not been exmined intensively but Bending & Red (1995b) observed significnt increses in the ctivities of proteinse, cid phosphtse nd polyphenol oxidse enzymes in FH residues being colonized by the mycelium of Pxillus involutus (Fr.) Fr. growing from mycorrhizl seedlings of Betul pendul Roth. Similrly, the ctivities of proteinse, phosphtse nd peroxidse enzymes were seen to be higher in soils supporting mycelil mts of rnge of hypogeous

2 32 RESEARCH J. Pe rez-moreno nd D. J. Red ECM fungi under Dougls fir, reltive to those in non-mt soils (Griffiths & Cldwell, 1992). While it is implicit in these studies tht nutrients re mobilized by, nd trnsferred from, the substrtes in myceli of ectomycorrhizl fungi, there pper to hve been no quntittive nlyses of coupling between such removl nd the cquisition of the resources by the mycorrhizl plnt. Here we describe experiments using birch seedlings grown in observtion chmbers with the ECM fungus P. involutus, in which mesurements of nutrient depletion, from rnge of different FH mterils colonized by the fungus, re coupled with nlysis of plnt response in terms of both growth nd nutrient content. MATERIALS AND METHODS Preprtion of mycorrhizl seedlings Seeds of B. pendul were collected from trees growing in Hmsterley Forest, Durhm, UK. They were stored dry t 5 C until required, then surfce sterilized with H O for 25 min nd rinsed with 5 ml of sterile distilled wter, before being plced onto wter gr in Petri dishes to germinte. Mycorrhizs were synthesized using modifiction of the procedure described by Brun et l. (1995). The fungus used ws fresh isolte of P. involutus (strin USPI97) obtined from fruit body growing under Betul in Cropton forest, North Yorkshire, UK. Plugs of mycelium were cut from stock culture nd were plced onto cellophne overlying gr supplemented with dilute nutrient solution. This ws modified from tht of Norkrns (1953) to contin only 16 mg P l,24mgnl nd 1 g glucose l. The medi were djusted to ph 5.7. When mycelium hd grown to cover pproximtely one third of the re of the cellophne disc, seedlings of B. pendul were trnsferred to the inoculum dishes, their roots being plced in direct contct with the fungus. Lterl roots were converted to mycorrhizs within 1 d, t which time seedlings were trnsferred to trnsprent observtion chmbers. Experimentl design Observtion chmbers of the kind described by Bending & Red (1995) were modified in the following wy. The trnsprent crylic pltes were reduced in size to cm. Insted of using pet lone s the bsl growth medium, thin lyer (c. 1 mm depth) of wter gr ws first poured onto the bse plte. To the gr surfce ws then dded, in ech chmber, 3 g d. wt of cid wshed 2-mm dimeter expnded cly (Lec) grnules. Finlly thin lyer consisting of 2.5 g d. wt of Sphgnum pet, ws spred evenly over the grnules, giving 12:1 (w:w) rtio of grnules to pet in ech chmber. This ws selected to minimize the potentil for the pet to influence seedling nutrient blnce. The pet, the cly grnules nd the gr collectively formed lyer c. 4 mm in thickness. These modifictions were bsed upon preliminry experiments showing tht the gr provided both n dhesive support nd nutrient free moisture supply for the grnule-pet mixture. The ddition of the grnules enhnced mycelil growth, probbly by fcilitting gs exchnge in the medium. Single mycorrhizl seedlings of B. pendul were trnsferred to ech of series of such observtion chmbers, their roots being plced in direct contct with the surfce pet lyer. After n upper plte hd been ttched, the chmbers were individully wrpped in sheets of luminium foil, through which the shoots emerged. The chmbers were stcked verticlly in plstic propgtor units with trnsprent, vented lids (Stewrt Plstics Ltd, Croydon, UK). The propgtors were incubted in controlled environment growth room with dy temperture of 15 C, night temperture of 1 C nd n irrdince of 15 µmol m s with photoperiod of 18 : 6 h light:drk. When the vegettive mycelium of P. involutus hd grown to occupy c. two thirds of the pet-grnule surfce the chmbers were removed from the growth room. Trys of litter previously collected from the FH of three forest soils were dded using modifiction of the pproch used by Bending & Red (1995). Selection, preprtion nd deployment of FH orgnic mtter Wheres Bending & Red (1995) used litter collected only from the FH of pine stnd, in the present study, in ddition to FH of P. sylvestris, these mterils were lso obtined from pure birch (B. pendul), nd beech (Fgus sylvtic L.) stnds. These orgnic residues were processed in the sme wy nd supplied in equivlent quntities s potentil nutrient sources for exploittion by the birch - Pxillus mycorrhizl system. Fresh FH mterils were ir-dried, pssed through 2 mm sieve nd their moisture content determined. The ph of the FH mterils from pine, birch nd beech stnds ws 3.8, 4.2 nd 4.1, respectively. Aliquots of.4 g f. wt of ech type were then dded to 3 cm weighing bots nd trnsferred to the observtion chmbers, where they were plced in res of uncolonized pet few millimetres in dvnce of the growing front of P. involutus mycelium. Pet ws scrped from the re so tht the bse of the litter bot could dhere directly to the gr with its outer rim fitting flush with the surrounding pet surfce. One try of ech of the three types of litter ws dded to ech of one series ( L) of observtion chmbers, while nother control series received no FH ddition ( L). There were three replicte chmbers with nd three

3 RESEARCH Nutrient trnsfer by mycorrhizl fungi 33 () F P B F P B (c) (d) F P B Fig. 1. ( c) Sequentil development of mycorrhizl seedlings of Betul pendul ssocited with Pxillus involutus in observtion chmbers contining trys of litter of beech (F), pine (P) nd birch (B). () 8 d fter litter plcement t stge of initil coloniztion of litter by P. involutus mycelium; fter 35 d showing intensive coloniztion of beech (F) litter nd erly plnt response; (c) fter 9 d showing further plnt growth nd externl mycelium development long mrgins of chmbers (rrows) s well s in trys of litter. (d) Mycorrhizl control plnt fter 9 d growth in chmber without litter ddition.

4 34 RESEARCH J. Pe rez-moreno nd D. J. Red without litter ddition. The ppernce of representtive L nd L chmbers is shown in Fig. 1. Initil mesurements were mde of plnt height nd stem dimeter t the time of litter ddition. All chmbers were then returned to the controlled environment room where they were incubted for further period of 9 d fter which time hrvests were tken nd the nutrient composition of the FH mteril nd of the plnt tissues were determined. In the course of the 9 d incubtion period, chmbers were periodiclly exmined to determine the progress of coloniztion of the FH trys by the mycelium of P. involutus. Nitrogen nd phosphorus nlyses of bsl pet nd litter Amounts of extrctble mmonium (NH ) nd phosphorus (PO ) in the bsl Sphgnum pet were determined in replicte subsmples tken t the time the observtion chmbers were set up. NH -N ws extrcted by shking the pet for 3 min with 2 M KCl nd PO -P ws obtined from prllel smples using.5 M NHCO djusted to ph 8.5 with.5 M NOH s extrctnt (Olsen et l., 1954). Extrcts were centrifuged t 25 rpm for 1 min nd sequentilly vcuum filtered through Whtmn No. 1 filter ppers nd.45 µm millipore filters. The extrcts were stored t 2 C before colorimetric nlysis using Tector 512 Anlyser (Foss Anlyticl, Didcot, UK). Totl mounts of N nd P in the Sphgnum pet were determined t time zero nd 9 d fter coloniztion in replicte sub-smples. Totl N nd P contents of the three types of litter were lso determined t the strt nd t the end of the experiment. Replicte weighed oven-dried subsmples of ech litter type nd of the pet were digested in concentrted H SO using the procedure of Allen et l. (1986). The N content of the digest ws determined by the indophenol blue method (Scheiner, 1976) nd its P contents by the mmonium molybdte ntimony trtrte blue ssy (John, 197). Extrctble N nd P of the litters were determined t the sme times using the extrctnts nd procedures employed for these mesurements in the bsl Sphgnum pet. Anlysis of response of Betul plnts At hrvest, birch plnts were prtitioned into roots nd shoots. The proportion of lterl roots converted to mycorrhiz ws clculted in ll plnts. Roots nd shoots were then oven-dried t 8 C nd their dry weights were mesured. N nd P concentrtions in the plnt shoot nd roots were then determined by the sme methods used for the FH mterils. In ddition, shoot heights nd folir res of ech seedling were mesured. RESULTS Growth responses of Betul pendul following litter coloniztion Visul observtions of the chmbers over the course of the 9 d growth period reveled progressive increse in shoot vigour in the L tretments, there being continuous production of new leves (Fig. 1 c). While occsionlly, expnsion of new leves ws ssocited with yellowing of the most mture lef (Fig. 1c) the overll pttern of folir development in the L chmber contrsted strongly with tht in the L tretment where shoots filed to develop (Fig. 1d) nd most of the leves present on the plnts t time zero were shed. Root growth in the presence of litter dditions ws lso more vigorous thn in the L tretment (Figs 1c,d, 3). Hevy mycorrhizl coloniztion ws retined on root systems of both nd L plnts throughout the experiment (Fig. 1b,c,d). At the finl hrvest 87% of root tips supported Pxillus mycorrhizs in L chmbers, the corresponding vlue in L systems being 9%. The stem lengths (Fig. 2) nd dimeters (Fig. 2b) of Betul plnts grown in the presence of litter diverged progressively from those of plnts without litter through the 9 d from initil coloniztion of the trys by P. involutus mycelium. The difference between the L nd L plnts were significnt (P.5) from dy 6 onwrds. By the finl hrvest both height nd stem dimeter of L plnts were round Stem dimeter (mm) Plnt height (mm) () Time (d) Fig. 2. Sequentil chnges in height () nd stem dimeter of Betul pendul plnts grown in the ectomycorrhizl condition with Pxillus involutus, in chmbers with (closed squres) or without (open circles) litter dditions. Mesurements were mde from the time t which litter ws dded ( d) to finl hrvest (9 d). Verticl brs indicte 95% confidence limits.

5 RESEARCH Nutrient trnsfer by mycorrhizl fungi 35 Dry weight (mg) () Folir re (cm 2 ) Root Shoot Whole plnt Fig. 3. Dry weights () nd folir res of Betul pendul grown in the ectomycorrhizl condition with Pxillus involutus, in chmbers with (closed brs) or without (open brs) litter dditions nd hrvested 9 d fter coloniztion of the litter. Verticl brs indicte 95% confidence limits. Totl P (µg) Totl N (µg) () Root Shoot Fig. 4. Totl nitrogen () nd phosphorus contents in Betul pendul plnts grown in the ectomycorrhizl condition with Pxillus involutus, in chmbers with (closed brs) or without (open brs) litter dditions nd hrvested 9 d fter ddition of the litter. Verticl brs indicte 95% confidence limits. double those of L systems. Whole plnt, shoot nd root dry weight of L plnts were significntly greter thn those of L plnts t hrvest (Fig. 3). Nutrient concentrtion in litter P (µg mg 1 d.wt) N (µg mg 1 d.wt) () c Betul litter b b b d Fgus litter b d Pinus litter Fig. 5. Nitrogen () nd phosphorus concentrtions in birch, beech nd pine litter before (open brs) nd 9 d fter (closed brs) coloniztion by P. involutus. Verticl brs indicte SE of the men. Brs with the sme letter re not significntly different with Tukey multiple comprison test, P.5. Yields in L chmbers were 3 times greter thn in those without litter. These chnges were ssocited with increse of lef re (Fig. 3b) so tht by dy 9 plnts in L chmbers hd lef res which gin were three times greter thn their L counterprts. Phosphorus nd nitrogen contents of the plnts Differences between P contents of L nd L plnts were significnt in both root nd shoots (Fig. 4b), there being 3 times more P in roots nd 4 times more in shoot of plnts grown with litter ddition. Totl nitrogen contents of shoots of L plnts were significntly greter thn in the L tretment (Fig. 4). There were lrge vritions in the mounts of N in the roots of individul plnts. As result, even though L plnts contined nerly double the totl quntities of N, the difference between their N contents nd those of L roots were not significnt. Nutrient depletion of litter colonized by P. involutus Only in the cse of the Fgus litter ws there significnt reduction of the N concentrtion of the litter s result of coloniztion by P. involutus (Fig. 5). In contrst, ll three types of litter showed significnt depletion of their P contents (Fig. 5b), the biggest reduction being seen in litter of Betul where the concentrtion ws lmost hlved. While differences between the litter types in terms of their initil nutrient contents were smll (Tble 1) the pttern of exploittion of these resources by P. involutus ws

6 36 RESEARCH J. Pe rez-moreno nd D. J. Red Tble 1. Phosphorus nd nitrogen depletion in beech, birch nd pine litters fter 9 d of coloniztion by Pxillus involutus grown in ssocition with Betul pendul. Amount (%) of P nd N reduction ttributble to loss from the orgnic frction Reduction of P nd N in reltion to initil contents, expressed s weight (µg) nd percentge Initil nutrient contents per try (µg) P N P N Totl P Extrctble P Totl N Extrctble N (µg) (%) (µg) (%) (%) (%) Tretment Birch litter Pine litter Beech litter Totl per chmber Vlues re mens SE, n 3 6. distinctive. Coloniztion of litter by the fungus led to men reduction of totl P content fter 9 d of 37%, there being little difference between litter types. A higher proportion, c. 1%, of totl P ws present in extrctble form thn in the cse of NH, where the vlue ws closer to 1% (Tble 1). Reltionships between the nutrient contents of litter nd pet nd the gins of P nd N by mycorrhizl plnts By compring the mounts of P nd N in plnts grown in nd L tretments t 9 d, the quntities gined from the litter cn be clculted (Tble 2). In the cse of P the difference in totl P contents ws 124 µg. However, P loss from the litter mounted to 369 µg (Tble 1) indicting tht only portion, c. 34%, of the P extrcted from the FH mteril ws trnsferred from the fungus to the plnt. Evidently, over the 9 d of the experiment the fungus itself ws the biggest sink for mobilized P, 66% of which ws retined by the mycelium. The 124 µg of P cquired by the plnts in the L systems represented only 13% of the 981 µg originlly present in the litter. This dditionl P supply ws, nonetheless, cpble of producing considerble increse of tissue P concentrtion (Tble 2). The sme clcultions for N trnsfer produce different outcome. The reduction in totl N in the litter of 995 µg (Tble 1), lmost ll of it from litter of Fgus, constituted only 5% of the originl litter N content. However, becuse of the high initil N contents nd N : P rtio of the litters this mount of N loss ws still three times greter thn tht of P. The min feture of the N budget, however, is the discrepncy between the 995 µg of N lost from the litter nd the 1589 µg gined by the plnts in the L tretments (Tble 2). The possible bsis of this pprent nomly is discussed lter. Despite such gins of totl N by plnts in the L chmbers, the tissue concentrtions chieved by the sme plnts were lower thn those of the L chmbers. The dditionl P gined by the L plnts produced N: P rtio of 17: 1 which ws lmost hlf tht observed in the L chmbers (Tble 2). Anlyses of the totl N nd P contents of the pet t time zero nd 9 dys fter its coloniztion by the fungl mycelium reveled tht wheres there were no significnt chnges (Tukey multiple comprison test, P.5) in N contents ( mg N g pet t time zero vs mg N g pet t 9 d, mens SE, n 4), the P content incresed significntly (Tukey multiple comprison test, P.5) from.2.8 mg P g pet t time zero to mg P g pet (mens SE, n 4) t 9 d. Men levels of extrctble NH -N in the pet were 6.25 µg g dry weight, equivlent to 151 µg per chmber nd of PO -P, 1 µg g dry weight, equivlent to 25 µg per chmber.

7 RESEARCH Nutrient trnsfer by mycorrhizl fungi 37 Tble 2. Totl N nd P contents, concentrtions nd N to P rtios in Betul pendul plnts grown in the ectomycorrhizl condition with Pxillus involutus in chmbers with nd without litter Whole plnt contents (µg) nd tissue concentrtions (%) of P nd N P N Tretment Totl Concentrtion Totl Concentrtion N: P Microcosms with litter :1 Microcosms without litter :1 Difference Vlues re mens SE, n 3. DISCUSSION The results cn be considered in terms of losses nd gins respectively of the mjor nutrients, nitrogen nd phosphorus, in the litter nd plnt comprtments, nd from the perspective of the overll N nd P budgets of the systems. Anlysis of the litter comprtments reveled tht coloniztion by Pxillus mycelium led to significnt losses of P from ll litter types but only to smll losses of N from single type, originting from F. sylvtic. There hs until recently been n unfortunte shortge of informtion concerning the bility of mycorrhizl fungi to mobilize the nutrient resources of nturl substrtes nd those studies tht hve been mde hve investigted different ectomycorrhizl systems. Entry et l. (1991) exmined field soils colonized by mt forming mycelil systems of unknown, but probbly hypogeous, mycorrhizl fungi nd compred their N nd P sttus with tht of djcent uncolonized soils. They reported reductions of both N nd P of round 33% from such mterils. Over the sme period losses of these elements from non-mt soils were round 17%. Using microcosm pproch similr to tht employed in the present study but with P. sylvestris grown in mycorrhizl ssocition with S. bovinus or T. terrestris, Bending & Red (1995) observed tht the extent of depletion of N nd P differed between the fungi. Suillus bovinus produced significnt reduction of c.23%of N nd P in pine litter while T. terrestris reduced N content by 13% while filing to provide significnt reduction of P. The min contrst between the results of erlier studies nd those of the present work is tht in the Betul-Pxillus system removl of P from the litter occurred to much greter extent thn N. The vlues of P reduction, rnging from 35% in the cse of pine litter to 39.9% in tht of birch re not dissimilr from those reported in mt soils by Entry et l. (1991). Over the sme period P. involutus ws ble to remove 1% or less of the N from birch nd pine litter while losses from tht of beech were under 14%. Clerly ny difference between results obtined in studies of this kind cn be ttributble to the different nutrient mobilizing cpbilities of the fungi employed nd to the distinctive chrcteristics of the substrtes on which they were grown, but the pprent bility of P. involutus to selectively remove P requires further exmintion. The effectiveness of the vegettive mycelium of P. involutus in scvenging for minerl P hs been demonstrted previously (Finly & Red, 1986; Andersson et l., 1996). In their study Andersson et l. (1996) compred the bilities of spruce nd birch seedlings grown in mycorrhizl ssocition with P. involutus nd those of non-mycorrhizl plnts, to recover phosphte ions spryed onto Sphgnum pet s H PO. The mycorrhizl seedlings cptured 22% of the pplied P by 8 d while only 7% ws recovered by uncolonized plnts. However, the pools of extrctble P in Sphgnum pet in the present study were smll nd were clerly not sufficient to support vigorous growth in the bsence of litter. Indeed, totl P contents of the pet in the L chmbers incresed over the 9 d incubtion period, presumbly s result of trnsfer in the mycelium from the litter. Mesurements of the difference between men totl combined mount of P lost from the litters in the microcosms (369 µg) nd their KCl extrctble minerl P contents (117 µg), indicte tht 252 µg of non-extrctble P ws recovered from the FH residues by P. involutus. This lrge pool probbly represents lbile orgnic frction. If we ssume tht the 252 µg is lrgely mde up of orgnic P it constitutes very lrge proportion, c. 69%, of the totl P removed. The bility of P. involutus to mobilize P from polymeric sources is not surprising since the fungus is known to produce the necessry phosphtse (Dighton, 1983; Ho, 1989; Antibus et l., 1992; Kieliszewsk-Rokick, 1992; Timonen & Sen, 1998) nd phytse (Liho, 197 ; McElhinney & Mitchell, 1993) enzymes. Further, when Bending & Red (1995b) compred cid phosphtse ctivities of litter being exploited by P. involutus s it grew from mycorrhizl roots of B. pendul, with those occurring in equivlent uncolonized mteril they found highly significnt increses in the presence of the fungus. The pprent filure of P. involutus to reduce the

8 38 RESEARCH J. Pe rez-moreno nd D. J. Red nitrogen sttus of the litter to significnt extent is, t first sight, surprising since the fungus is known to be n effective scvenger of mmonium (Abuzindh & Red, 1986b; Finly et l., 1989) nd mino-cid N (Abuzindh & Red, 1989) nd to be ble to express the cid proteinse enzymes required for the mobiliztion of orgnic N (Abuzindh & Red 1986, Bending & Red, 1995b). However while P. involutus ws ctegorised by Abuzindh & Red (1986b) on the bsis of its bility to use polypeptides s N sources, to be protein fungus, they lso observed in the sme study tht within this ctegory P. involutus retined unusully high mounts of the cquired N, which ccumulted to give concentrtion of c. 6.8% in its vegettive mycelium. If N immobilized in this wy ws retined in the hyphe colonizing the litter, ny mobiliztion chieved in the course of exploittion of the substrte would be msked. Sequentil hrvests over lrger time intervl would be required to determine whether litter N initilly immobilized in this wy in the mycelium ws subsequently trnsferred to the plnt. It is lso worthy of note tht of the three so clled protein fungi, employed s mycorrhizl symbionts by Abuzindh & Red (1989) in study of peptide ssimiltion nd N trnsfer to Betul, P. involutus generlly provided the smllest growth response nd lowest tissue N content in the plnts. It must lso be borne in mind tht the fungl prtners in the ectomycorrhizl symbiosis show very considerble intr-specific vribility in their bility to use nutrients. In the cse of P. involutus it hs been repetedly shown tht individul strins hve mrkedly different bilities to ssimilte defined N sources (Liho, 197; Finly et l., 1992; Keller, 1996). There is therefore the possibility tht the prticulr strin of P. involutus selected for use in the present study ws one of those which hve low bility to mobilize orgnic N. The discrepncy between the 995 µg N lost from the litter nd the 1589 µg gined by the plnts in the L tretment clerly requires further considertion. The possibility tht invigortion of the vegettive mycelium of P. involutus in the L chmbers (Fig. 1c) might hve led to enhncement of N cpture from the pet ws evluted. However, the smll chnge in the totl N contents of the pet over the 9 d of incubtion were insufficient to ccount for the dditionl 6 µg N gined by the plnts in this tretment. A more plusible explntion for the plnt gin ws the occurrence of N -fixing ctivity in the litter supplements. There is some evidence for the occurrence of N fixtion in the mycorrhizosphere of nturlly occurring plnts (Li & Hung, 1987; Chnwy & Holl, 1991; Li et l., 1992) but prerequisite for effective fixtion is reduction of oxygen prtil pressure in the immedite environment of the heterotrophs. It cn be hypothesised tht the extremely vigorous growth of ectomycorrhizl mycelium in the litter ptches will led, by enhncement of CO relese, both to decresed O vilbility nd to relese of crbon s substrte for ny N-fixing popultion. Nurmiho Lssil et l. (1997) provided evidence for the presence of bcteril colonies nd of individul bcteri in ssocition with the externl mycelium of P. involutus growing from P. sylvestris seedlings into nturl forest soil. Subsequent nlysis of these bcteril communities (Timonen et l., 1998) reveled lrge number of Bcillus species mny of which re puttive nitrogen-fixers. Clerly if significnt mounts of N fixtion did occur, it would, by compensting for N export, explin the pprent discrepncy between N loss of the litter nd N gin by the plnt. It will be priority of future studies to determine whether exploittion of litter by mycorrhizl hyphe cn led to enhncement of N fixing ctivity. The importnce to the plnts of n bility to cpture resources, prticulrly P, from the litter is evident from the strong growth responses seen in the L chmbers. A number of feed-bck loops re probbly involved. Up to the time of litter encounter, nd throughout the experiment in the cse of L chmbers, reltively smll investment of crbon in thinly but widely distributed mycelium is mintined. Access to the litter nutrient pool enbles growth, with lef expnsion, nd s result, lmost certinly, enhncement of ssimilte supplies to the fungus. It is not cler to wht extent the invigortion of the fungus seen in L chmbers is dependent upon crbon originting from the plnt or the litter, but other studies (e.g. So derstro m & Red, 1987) suggest tht current photosynthte is likely to be the most importnt source. In such circumstnces, feedbck to the mycelium would enble more vigorous explortion for new resource ptches. This perhps intermedite step in the feed-bck, my explin the reltively smll mount of N relesed to the plnt over the period of mesurement. The finl N:P rtio of 17:1 in tissues of B. pendul growing in L tretments is higher thn the optimum of 1:1 estblished by Ericsson & Ingestd (1988) for this species growing under hydroponic conditions. However it is not dissimilr from vlues found in nturlly occurring ectomycorrhizl trees of Africn (Ho gberg & Alexnder, 1995) nd North Americn (Comerford & Fisher, 1984) woodlnds which rnge from 9 17:1. Proportionlly higher N : P rtios such s the 31:1 found in the L chmbers re n indiction of very severe P deficiency. On this bsis lone it cn be deduced tht the response of Betul to litter ddition is lrgely determined by incresed ccess to P. Ericsson & Ingestd (1988) hypothesised tht under nutrient impoverished conditions, which re typicl of those previling in the present experiment nd frequently in the nturl environment of Betul seedlings, there will be

9 RESEARCH Nutrient trnsfer by mycorrhizl fungi 39 proportionlly higher demnd for P thn for N. This hypothesis is bsed on the expecttion tht the plnts will hve reltively smll requirement for N s ctivities nd turnover of the photosynthetic enzyme Rubisco will be low, while the need for P to sustin the ATP dependent nutrient forging ctivity will increse. Further studies using different fungl symbionts will be required to determine the extent to which phosphte priming is prticulr feture of mycorrhizs produced by P. involutus nd to evlute the extent to which mixed popultions of fungi confer distinctive litter mobiliztion qulities. ACKNOWLEDGEMENTS We thnk CONACYT, Colegio de Postgrdudos (Mexico) nd the British Chevening Scholrships Progrmme for provision of scholrship to J. P-M. We lso thnk Irene Johnson nd Glynn Woods for ssistnce. REFERENCES Abuzindh RA, Red DJ The role of proteins in the nitrogen nutrition of ectomycorrhizl plnts I. 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