Selected Ectomycorrhizal Fungi of Black Spruce (Picea mariana) can Detoxify Phenolic Compounds of Kalmia angustifolia

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1 J Chem Eol (2006) 32: DOI /s Seleted Etomyorrhizl Fungi of Blk Sprue (Pie mrin) n Detoxify Phenoli Compounds of Klmi ngustifoli Ren Sen Zeng & Azim U. Mllik Reeived: 17 Deemer 2004 / Revised: 15 Jnury 2006 / Aepted: 21 Jnury 2006 / Pulished online: 23 My 2006 # Springer Siene + Business Medi, In Astrt Allelopthy hs een implited s ftor ontriuting towrd filure of lk sprue (Pie mrin) regenertion in Klmi ngustifoli-dominted sites in estern Cnd. Severl phenoli ids of Klmi origin inhiit primry root growth of lk sprue. We tested the hypothesis tht some well-dpted onifer etomyorrhize n degrde nd detoxify wter-solule phenoli ompounds produed y Klmi nd use the degrded produts s ron soure to stimulte growth. We found tht hyphl growth of Pxillus involutus, ommon etomyorrhizl fungus of lk sprue, ws stimulted y wter lehtes of Klmi lef nd litter. An equimolr mixture of three phenoli ids (feruli, o-oumri, nd o- hydroxyphenyleti id), ommonly found in Klmi, hd no negtive effets on fungl growth t 1 mm onentrtion. The o-hydroxyphenyleti (o-hpa) id, whih is known to e toxi to lk sprue, ws found to stimulte the growth of Lri lt, L. iolor, nd P. involutus (isoltes nd ) y 38.4, 29.3, 25.0, nd 18.9%, respetively, t 1 mm. Pure feruli, o-oumri, nd o-hpa ids were degrded y 100, 98, nd 79.5%, respetively, within 10 d in the presene of P. involutus However, L. lt ould not tolerte high onentrtions of the Klmi lehtes. P. involutus nd L. iolor used o-hpa id s ron soure when ultured in nonron nutrient medium. The 0.5 nd 0.2 mm o-hpa id inhiited the root growth of lk sprue. However, fter solutions hd een exposed to ulture of P. involutus, they hd no signifint effet on seedling growth of lk sprue. We onluded tht some etomyorrhizl fungi, suh s P. involutus nd L. iolor, re le to degrde Klmi phenolis. Our findings point to R. S. Zeng: A. U. Mllik (*) Deprtment of Biology, Lkehed University, Thunder By, Ontrio, Cnd P7B 5E1 e-mil: zim.mllik@lkehedu. R. S. Zeng Institute of Tropil nd Su-Tropil Eology, South Chin Agriulturl University, Wushn, Gungzhou , Chin

2 1474 J Chem Eol (2006) 32: mehnism y whih etomyorrhizl speies n ontrol speies intertions in higher plnts y hnging the rhizosphere hemistry. Keywords Etomyorrhizl fungi. Phenoli ompounds. Detoxifition. Pie mrin. Klmi ngustifoli Introdution Nturlly regenerting nd plnted onifers in nutrient-poor orel forests of estern Cnd often suffer from growth inhiition due to erieous understory shrus (Mllik, 1987, 2001; Mllik nd Newton, 1988; Thiffult et l., 2004). Following ler utting nd fire, sheep lurel (Klmi ngustifoli L. vr. ngustifoli, herefter referred to s Klmi), ommon erieous understory plnt in estern Cnd, n seriously hinder onifer regenertion, espeilly lk sprue [(Pie mrin (Mill.)]. One use hs een ttriuted to llelopthi effets of Klmi phenolis (Mllik, 1987; Thompson nd Mllik, 1989; Zhu nd Mllik, 1994). Phenoli ids re relesed into the soil s lef nd litter lehtes, root exudtes, or s deying plnt residues (Siqueir et l., 1991; Gllet nd Pellissier, 1997). Severl re reported to t s signl moleules, nd some my influene signl trnsdution pthwys in symioti systems (Lynn nd Chng, 1990). Fries et l. (1997) reported tht ertin phenoli ids stimulte rusulr myorrhizl (AM) oloniztion, whih my promote growth of host plnts. The mehnism of onifer growth inhiition in the presene of erieous plnts is omplex nd the role of Klmi phenolis in lk sprue growth inhiition is unler (Wllstedt et l., 2002). Redued etomyorrhizl (ECM) oloniztion of Norwy sprue [Pie ies (L.) Krst] y Cenooum grniforme, L. lt (Pellissier 1993), nd Heelom rustuliniforme (Souto et l., 2000) in the presene of Vinium myrtillus L. (herefter referred to s Vinium) hs een suggested s use of its regenertion filure in sulpine forests in Europe. Hndley (1963) found few myorrhizl fungi ssoited with Norwy sprue root systems when grown in the presene of hether (Cllun vulgris L.). Ymski et l. (1998) reported signifintly lower stem height, root shoot iomss, nd redued myorrhiztion of lk sprue grown ner (<1 m) Klmi thn those grown wy (>1 m) from Klmi under field onditions. Klmi lef lehtes hve een found to e inhiitory to myorrhizl formtion nd myelil growth of some etomyorrhizl fungi of lk sprue (Mllik et l., 1998). Etomyorrhizl fungi often experiene hemil stress from forest floor phenolis (Bending nd Red, 1997). The role of phenoli ids in etomyorrhizl ssoitions is not well understood. Blk sprue Klmi forests of estern Cnd nd Norwy sprue Vinium forest of sulpine Frne nd Fennosndi produe lrge mounts of litter on the forest floor (Inderjit nd Mllik, 1999; Berg nd Dise, 2004). Erieous plnts, suh s Klmi nd Vinium, produe lrge mounts of phenoli ids, whih my umulte on the forest floor through lehing from leves (Zhu nd Mllik, 1994), litter, humus (Pellissier 1994), nd possily root exudtes. Mny of these phenoli ids inhiit primry root growth in lk sprue (Zhu nd Mllik, 1994), seed germintion nd seedling growth in

3 J Chem Eol (2006) 32: Norwy sprue, nd myorrhizl fungl growth (Pellissier 1993). Severl uthors hve reported inhiitory effets of erieous litter on growth nd respirtion of sprue myorrhiz (Bouflis et l., 1994; Bouflis nd Pellissier, 1994). Souto et l. (2000) showed tht humus phenolis of Vinium were more toxi to the sprue myorrhizl fungus, H. rustuliniforme, thn to the erioid (Vinium) myorrhizl fungus, Hymenosyphus erie. In etomyorrhizl symioses, plnt roots nd fungi funtion together s unit. Formtion of ECMs in plnts often llows them to estlish in hitts tht neither symiont my e le to oupy individully (Nehls et l., 2000). Etomyorrhize re le to llevite toxi effets of hevy metl tions in host plnts (Denny nd Wilkin, 1987; Colpert nd Vn Asshe, 1993). They my hve dditionl or relted mehnisms to del with phenoli llelohemils on the forest floor. From ontrolled experiment, Mllik et l. (1998) showed tht the growth inhiitory effets of Klmi lehte on lk sprue ould e overome y inoulting lk sprue with seleted myorrhizl fungi. Beuse myorrhizl fungi re loted t the interfe etween the soil-llelohemil reservoir nd roots of the host plnts, we hypothesized tht if ertin sprue ECM fungi n degrde Klmi phenolis, nd use the degrded produt(s) s ron soure, then these etomyorrhizl fungi would e le to protet lk sprue ginst Klmi phenolis nd enhne their growth. The ojetives of our study were (1) to identify ertin ECM fungi tht n withstnd the toxiity of Klmi lef, litter, nd humus lehtes nd their ssoited phenoli ompounds, nd (2) to test whether these ECM fungi n degrde nd detoxify the phenoli ids of Klmi. Methods nd Mterils Plnt nd Etomyorrhizl Fungl Mterils Blk sprue seeds were otined from Hills Nursery, Thunder By, Cnd. P. involutus (Btsh. Ex Fr.) Fr , , nd L. lt (Sop.:Fr.) Cooke were otined from the Cndin Colletion of Fungl Cultures, Ottw. Pisolithus tintorius Coker nd Couh ws otined from Prof. Mingqin Gong t the Reserh Institute of Tropil Forestry in Gungzhou, Forestry Ademy of Chin. Lri iolor (Mire) P.D. Orton ws olleted from Thunder By. P. tintorius ws used to ompre responses of ECM fungi from erieous-dominted nd nonerieous-dominted res. Chemils Feruli, o-oumri, nd o-hydroxyphenyleti (o-hpa) ids were purhsed from Sigm (St. Louis, MO, USA). Mlt extrt nd grnulted gr were ought from Beton Dikinson Co. All solvents used were nlytil or HPLC grde. Seed Surfe Steriliztion Blk sprue seeds were surfe-sterilized with 1% NClO 3 for 10 min nd then rinsed with sterile wter 5 to remove surfe orne pthogens. Sterilized seeds were dried with utolved filter pper nd kept t 4-C for susequent use.

4 1476 J Chem Eol (2006) 32: ECM Fungl Cultures Modified Melin Norkrns nutrient solution (MMN) ws used for growing the etomyorrhizl fungi (Mrx, 1969). Petri dishes ontining the MMN, Klmi lehtes, nd phenoli ids were inoulted with 4-mm-dim gr plugs ut from the edge of 2-wk-old etomyorrhizl fungl olonies. The inoulted pltes were inuted t room temperture (20 25-C). Dimeters of the fungl olonies were mesured every 5 d. All experiments onsisted of t lest three replites. Klmi Lehtes Klmi fresh leves (mture, 1-yr-old), litter (dry leves on the ground), nd humus (0 10 m) were olleted from 10 rndom lotions in Klmi-dominted heth originting from nturl fire 25 yr go ner the villge of Terr Nov just outside the oundry of Terr Nov Ntionl Prk, Newfoundlnd. Composite smples of fresh leves, litter, nd humus were mde y mixing the 10 smples. Corse roots nd rnhes were removed from the humus smples prior to mixing. Twenty g of fresh leves nd litter were soked seprtely in 100-ml distilled wter for 24 hr t room temperture (20 25-C). The onentrtions otined for lef nd litter lehtes were 0.2 g FW equivlent of leves or litter per ml of lehte. Klmi humus lehte ws otined y drining 200 ml distilled wter t slow rte for 24 hr through 100 g fresh humus in glss hromtogrphy olumn (30 mm) t room temperture. The filtrte of humus lehte ws djusted to 100 ml, nd its onentrtion ws 1 g FW equivlent of humus per ml of lehte. The wter lehtes of leves, litter, nd humus were pssed through Whtmn No. 42 filter pper nd then sterilized y pssing through two lyers of 0.45-mm Millipore filters. Experiment 1: Effets of Klmi Wter Lehtes on ECM Fungi We mesured the growth responses of two fungl isoltes, P. involutus nd L. lt , to three onentrtions of lef nd litter lehtes nd two onentrtions of humus lehte. The 0.1 g FW/ml lehte ws prepred y dding 50 ml originl lehte (0.2 g FW/ml) into 50 ml utolved MMN medium ontining 1.5 g gr t C. The 0.05 g FW/ml lehte ws prepred y dding 25 ml originl lehte nd 25 ml sterilized wter into 50 ml utolved MMN gr medium. The 0.01 g FW/ml lehte ws prepred y dding 5 ml originl lehte nd 45 ml sterilized wter into 50 ml utolved MMN medium. Conentrtions of 0.25 nd 0.05 g FW/ml of humus lehte were used. The originl lehtes of humus (25 ml, 1 g FW/ml) nd 25 ml sterilized wter were mixed with 50 ml utolved MMN medium ontining 1.5 g gr t C to otin 0.25 FW/ml of humus lehte. The 0.05 g FW/ml humus lehte ws prepred y dding 5 ml originl lehte nd 45 ml sterilized wter into 50 ml utolved MMN gr medium. The ontrol ws mde from hlf-strength MMN medium. The medium ws poured into 9-m-dim Petri dishes (20 ml medium in eh dish). A disk of inoulum (dim 4 mm) from eh of the five ECM fungi ws ut from the edge of 2- to 3-wk-old olony nd inoulted in Petri dish. After they were ultured for 28 d t C, dimeters of the fungl olonies were mesured.

5 J Chem Eol (2006) 32: Experiment 2: Effet of Pure Phenolis on ECM Fungi o-hpa, feruli, nd o-oumri ids were the mjor phenoli ids in Klmi lef lehtes (Zhu nd Mllik, 1994) nd humus (Mllik, unpulished dt). Therefore, we mesured the responses of five ECM fungl isoltes, P. involutus (211804, ), L. lt , P. tintorius 99132, nd L. iolor to these ompounds. o- HPA id ws diretly dissolved in sterile wter to otin 2-mM solution. Feruli nd o-oumri ids were dissolved in sterile hot wter (80-C). Phenoli solutions were sterilized y pssing through two lyers of 0.45-mm Millipore filters. Equivlent volumes of 2-mM phenoli solution nd doule onentrtion MMN gr were mixed to otin medium with 1 mm phenoli id nd full-strength MMN. The equimolr mixture of the three phenoli ids ws otined y mixing equivlent volumes of their respetive 1 mm medi for finl onentrtion of 0.33 mm for eh id. The ontrol onsisted of only MMN medium. Petri dishes ontining the ove-mentioned medi were inoulted with the etomyorrhizl fungi employed in Experiment 1, with four replites for eh tretment. The inoulted pltes were inuted t C nd the dimeters of the fungl olonies mesured fter 20 d. Experiment 3: Phenoli Aids s Cron Soure for ECM Fungi In this experiment, we investigted whether P. involutus nd L. iolor ould grow with o-hpa id s the only soure of ron. To prepre fungl inoul, P. involutus nd L. iolor were ultured on MMN gr for d. Myelil disks (dim 10 mm) were ut from the edges of fungl olonies nd trnsferred to dishes ontining wter gr. Lrge-dimeter inoul (10 mm s opposed to stndrd 4 mm) were used to provide nutrition for fungl growth. Wter gr ws used to exlude the possile nutrient effet of inoulted plugs on nonron nutrient experiment. These fungi were ultured in wter gr for 22 d. After 22 d, plug of inoulum (dim 4 mm) ut from the olony edge on wter gr ws inoulted on the nonron MMN medium (MMN gr medium without surose nd mlt extrts) without o-hpa id (ontrol) nd nonron MMN medium with 1 mm o-hpa id. There were four dishes for eh fungus nd tretment omintion. The experiment ws onduted t room temperture (20 25-C). Dimeters of the fungl olonies were mesured fter 25 d. Biomss Assy in Liquid Medi Beuse myelil iomss in the nonron medium ws very smll, hlf strength of MMN liquid medium ws used to ulture P. involutus nd L. iolor. Eh Petri dish (9 m) ontined 12 ml liquid medium with or without 1 mm o-hpa id, prepred s desried ove. There were 10 dishes for eh tretment. After utolving, U-shped glss rod (dim 2 mm) wrpped with piee of 4 4m unlehed pper towel ws dded to eh dish nd ws inoulted with two plugs (dim 4 mm) of P. involutus inoul on the surfe of the unlehed pper. The glss rod nd unlehed pper towel filitted the ulturing of the ECM fungi in liquid medi nd susequent olletion of myeli. Five ml of sterile wter were dded fter 15 d. After 30 d ulture, the myeli were hrvested from the unlehed pper. Ten dishes were divided into five groups, nd myeli from two dishes were omined, dried t 60-C for 24 hr, nd weighed to T0.1 mg.

6 1478 J Chem Eol (2006) 32: Experiment 4: Phenoli Redution y ECM Fungi in Pure Culture We tested whether P. involutus ould metolize phenolis. P. involutus ws ultured on MMN gr medium. The Petri dish ws inoulted t the enter with one piee of 4 mm myelil plug. Cultures were mintined t C for 20 d. Then two U-shped glss rods (dim 2 mm) were pled on the surfe of the medium. Aetone solutions of the phenoli ompounds (10 mm, 200 ml) were spotted on piee of Whtmn No. 3 filter pper (dim 12 mm). When the etone hd ompletely evported, 200 ml sterile wter were dded to the filter pper. Filter ppers were pled over eh rod. Eh tretment onsisted of three replites. The filter ppers were tken out 10 d lter nd they were soked in 5 ml methnol for 24 hr. The methnol extrts were onentrted to 2.5 ml t 40-C under redued pressure nd pssed through Whtmn No. 1 filter pper. Phenoli ompounds in the filtrtes were nlyzed y using Vrin Prostr HPLC equipped with Chrompk olumn ( mm) nd PDA detetor (Model 330) monitoring the sorne of the elution t 280 nm. The solvent system ws s follows: 0 9 min, 10% methnol nd 90% wter ontining 2.5% formi id; 9 20 min, 40% methnol nd 60% wter ontining 2.5% formi id; min, 60% methnol nd 40% wter ontining 2.5% formi id; 25 min, 100% methnol. Flow rte ws 1.5 ml/min, nd temperture ws 32-C. Pure ompounds were used s stndrds, nd phenoli ids were identified y omprison of retention times nd UV spetrum. Experiment 5: Detoxifition Biossy We investigted whether inoultion of sprue seedlings with ECM fungus redued the phytotoxiity of o-hpa id. Beuse o-hpa id ws the most toxi phenoli id in Klmi lef lehtes ffeting lk sprue seedlings (Zhu nd Mllik, 1994), it ws used to ondut further detoxifition studies. Sine full-strength MMN medium itself inhiits seedling growth of lk sprue, ll medi used were hlfstrength MMN. P. involutus ws ultured in MMN liquid medium (hlf strength) ontining 0.2 nd 0.5 mm o-hpa id. Eh Petri dish (10 m) ontined 15 ml liquid medium. P. involutus ws inoulted nd ultured s desried in Experiment 4. Noninoulted ulture ws used s the ontrol. Pltes were inuted t room temperture (20 23-C). Five ml sterile wter were dded fter 15 d. The ulture solution ws dented from the dishes fter 30 d nd pssed through Whtmn No. 42 filter pper. Filtrtes were djusted with sterile wter to initil volume nd used for the phytotoxiity test with lk sprue seedlings. Eh dish ontined 20 surfe-sterilized lk sprue seeds pled on piee of Whtmn 3 filter pper to whih 5 ml ulture solution were dded. The hlf-strength MMN medium ws used s negtive ontrol, nd hlf-strength MMN medium with 0.2 nd 0.5 mm o-hpa id were used s positive ontrols. The iossy ws onduted t C with 16-hr photoperiod. Two ml sterile wter were dded to eh Petri dish during inution. The lengths of lk sprue primry root nd shoot were mesured fter 15 d. Experiment 6: Phenoli Degrdtion y Inoulted Blk Sprue Blk sprue seedlings were inoulted with P. involutus following the method desried y Mrx nd Kenney (1982) with some modifitions to

7 J Chem Eol (2006) 32: determine whether the ECM inoulted seedlings re le to degrde o-oumri, feruli, nd o-hpa ids nd enhne the root shoot growth of lk sprue. ECM Inoulum Preprtion Vermiulite (pproximtely 600 ml) nd MMN liquid medium (300 ml) were dded to 1-l glss jrs. The jr lids hd holes tightly plugged with otton. After utolving for 30 min, eh jr ws inoulted with 10 myelil disks. After 45 d inution, the inoulum ws wrpped with four lyers of heeseloth nd irrigted with tp wter for 5 min to remove ny remining nutrient. This mteril ws used for lk sprue seedling inoultion for the following experiment. Seedling Inoultion Autolved (121-C for 30 min) vermiulite nd pet/moss mixture (1:1) ws dded to multipots (120 ml/ell) for seedling ulture. Pregerminted lk sprue seedlings (5 d) developed from surfe-sterilized seeds were sown in multipots nd kept in growth inutor mintined t 26-C with 16-hr photoperiod, 150 Md/m 2 /s PAR nd 60% reltive humidity. Seedlings were wtered dily nd fertilized one week with NPK. After 60 d inution, the seedlings were removed to new, lrger multipots (250 ml/ell) tht were filled with the myorrhizl fungl inoul nd vermiulite/pet moss mixture t the rtio 1:15. Eh ell ontined one seedling. Inoulted seedlings were grown in the sme inutor nd under the sme environmentl onditions s in the other experiments. The roots of inoulted seedlings were surrounded y dense myeli fter 135 d inoultion. Noninoulted (ontrol) seedlings were prepred nd grown in the sme mnner without inoultion. No myorrizl infetion ws found in ontrol seedlings. At this point, 30 ml of 1 mm phenoli id solution were dded to eh ell. There were six seedlings per tretment. Five d lter, the growing medi (pet/vermiulite mixture) of seedlings were extrted with 300 ml distilled wter 3. The wter extrts were pooled nd prtitioned ginst hexne 3 to remove lipids, then extrted with ethylette 3. Ethylette extrts were omined nd onentrted to dry form t 40-C under redued pressure, nd then dissolved with 5 ml methnol/ethyl ette (4:1). The phenoli ids were nlyzed with HPLC. Sttistil Anlysis All dt were normlly distriuted. One-wy nlysis of vrine (ANOVA) ws used, nd tretment differenes mong mens were tested t P = 0.01 with Dunn s multiple rnge test. Results Experiment 1: ECM Fungl Response to Klmi Lehtes Growth response of the ECM fungi to Klmi lehtes depended on the type of lehte (lef, litter, or humus), its onentrtion, nd the speies of ECM fungus. Higher onentrtions of lef lehtes nd lower onentrtions of litter lehtes

8 1480 J Chem Eol (2006) 32: hd stimultory effets on P. involutus. The 0.1 nd 0.05 g FW/ml lef lehtes signifintly stimulted the growth of P. involutus y inresing the olony dimeter of the fungus y nd 60.1%, respetively (Tle 1). However, these two onentrtions inhiited L. lt, whose olony dimeters were 12.6 nd 11.4% smller with 0.1 nd 0.05 g FW/ml lef lehtes, respetively, reltive to ontrols. The 0.1 g FW/ml litter lehte hd no effet on olony dimeter of P. involutus. However, the 0.05 nd 0.01 g FW/ml litter lehtes stimulted olony dimeter y 72.6 nd 49.0%, respetively. The 0.1 nd 0.05 g FW/ml litter lehte hd little effet on olony dimeter of L. lt, ut with 0.01 g FW/ml litter lehte, there ws inrese of 8% ompred to ontrol. The 0.25 nd 0.05 g FW/ml humus lehte hd no effets on olony dimeter of either P. involutus or L. lt (Tle 1). Experiment 2: ECM Response to Pure Phenoli Aids The response of fungl isoltes to pure phenoli ids lso differed depending on the type of phenoli id nd its onentrtion. P. involutus ws the most tolernt fungl isolte to the three phenoli ids nd their equimolr mixture (Fig. 1). Indeed, the olony dimeter of this isolte ws inresed y 25% in the presene of 1.0 mm o-hpa id, nd the mixture of the three phenoli ids did not inhiit growth t either onentrtion. However, P. involutus ws inhiited y feruli id t 1 mm. Both the 1.0 nd 0.1 mm phenoli mixtures inhiited olony dimeter of P. involutus nd L. lt Colony dimeter of L. iolor ws redued y 41.7% on 1 mm phenoli mixture, ut it ws inresed (y 17.8%) in the presene of the 0.1 mm mixture. Colony dimeter of oth L. iolor nd L. lt ws inresed y 1.0 mm o-hpa id. Colony dimeter of L. lt ws lso inresed y 0.1 mm o-hpa id. P. tintorius ws the most sensitive of the fungl isoltes to the phenoli mixture t 1.0 mm, nd its olony dimeter ws inhiited y 80.5% on the 1.0 mm mixture. With respet to the effet of individul phenoli ompounds on the myorrhizl fungi, feruli id ws the most toxi, strongly inhiiting growth of ll tested ECM fungi t 1 mm. The derese in olony dimeter of P. involutus , , nd Tle 1 Effet of Klmi lef, litter, nd humus lehtes on olony dimeter of P. involutus nd L. lt Lehtes Conentrtions (g FW/ml) P. involutus Colony dimeters [mm (men T SE)] L. lt Vlues re men T stndrd error (N = 4), mens followed y the sme letter(s) in the sme olumn re not signifintly different (P < 0.01) Control T T 1.0 Lef T T 1.2e Lef T T 1.5de Lef T T 1.7 Litter T T 0.6 Litter T T 1.5d Litter T T 1.0 Humus T T 1.7 Humus T T 0.6

9 J Chem Eol (2006) 32: Control Phenoli mixture o-hydroxyphenyleti id Feruli id Coumri id Colony dimeter (mm) d e 0 60 Control Phenoli mixture o-hydroxyphenyleti id Feruli id Courmri id Colony dimeter (mm) d d d d e 0 P. involutus P. involutus L. iolor L. lt P. tintorius ECM fungl isolte Fig. 1 Effet of three ommon Klmi phenoli ids nd their equimolr mixture on olony dimeter of five etomyorrhizl fungi t () 0.1 mm nd () 1 mm. Vlues re men T stndrd error (N = 4). Signifint differenes (P < 0.01) mong tretments in eh group re indited y different letters P. tintorius ws 95.0, 72.5, nd 92.5%, respetively (Fig. 1). The ompound lso inhiited growth of P. involutus nd P. tintorius t 0.1 mm, ut it stimulted the growth of L. lt t this onentrtion. The o-hpa id stimulted the olony dimeter of L. lt, L. iolor, nd the two isoltes of P. involutus y 38.4, 29.3, 25.0, nd 18.9%, respetively, t 1.0 mm; it lso stimu-

10 1482 J Chem Eol (2006) 32: Control o-hydroxyphenyleti id 14 Colony dimeter (mm) Myelil dry iomss (mg) P. involutus L. iolor ECM fungl isolte Fig. 2 Effets of 1 mm o-hydroxyphenyleti id on () olony dimeter nd () myelil dry iomss of P. involutus nd L. iolor in ron-free liquid MMN medium. Vlues re men T stndrd error (N = 5). Signifint differene (P < 0.01) mong tretments in eh group re indited y different letters ove rs

11 J Chem Eol (2006) 32: lted the olony growth of P. involutus nd L. lt t 0.1 mm. It did not stimulte olony growth of P. tintorius. Coumri id inhiited the olony growth of ECM fungi exept P. involutus t 1 mm. At 0.1 mm, oumri id did not inhiit ny fungus, nd stimulted the growth of P. involutus (Fig. 1). Experiment 3: Phenoli Aids s Cron Soure for ECM Fungi When P. involutus ws ultured on nonron MMN gr medium, its olony dimeter inresed y 41.7% in the presene of 1 mm o-hpa id (Fig. 2). The hyphl density of the fungus in the medium with o-hpa id ws muh higher thn tht of the ontrol. Control pltes hd little myeli. L. iolor did not grow t ll on the nonron MMN medium, ut did in the presene of 1 mm o-hpa id. L. lt did not grow either on ontrol or o-hpa id medium. The myelil iomss of P. involutus nd L. iolor ws inresed y 20.1 nd 33.9%, respetively when the two fungi were ultured seprtely in MMN liquid medium ontining 1 mm o-hpa id (Fig. 2). Experiment 4: Phenoli Degrdtion y ECM Fungus in Pure Culture The onentrtions of ll three phenoli ompounds, feruli, oumri, nd o-hpa ids were redued in the presene of P. involutus in pure ulture. While the reovery of oumri nd o-hpa ids ws redued y 97.9 nd 79.5%, respetively, we ould not detet ny feruli id following exposure to the fungl ulture on the medium (Fig. 3). 2.5 Control P. involutus Conentrtion (mm) o-coumri id o-hydroxyphenyleti ids Feruli id Phenoli ids Fig. 3 Conentrtions of o-oumri, o-hydroxyphenyleti, nd feruli ids in filter pper extrt without (ontrol) or with P. involutus ulture. Vlues re men T stndrd error (N = 4). Signifint differenes (P < 0.01) mong tretments in eh group re indited y different letters ove rs

12 1484 J Chem Eol (2006) 32: Control P. involutus Root length (mm) Shoot length (mg) Conentrtion (mm) Fig. 4 Phytotoxi effets of o-hydroxyphenyleti id t onentrtions of 0.2 nd 0.5 mm with nd without P. involutus ulture on () root length nd () shoot length of lk sprue. Vlues re men T stndrd error (N = 15). Signifint differenes (P < 0.01) mong tretments re indited y different letters ove rs

13 J Chem Eol (2006) 32: Uninoulted Inoulted Conentrtion (mg/l) o-coumri id o-hydroxyphenyleti ids Feruli id Phenoli ids Fig. 5 Conentrtions of o-oumri, o-hydroxyphenyleti, nd feruli ids in growing medi ontining uninoulted (ontrol) nd inoulted lk sprue seedling with P. involutus Vlues re men T stndrd error (N = 3). Signifint differenes (P < 0.01) mong tretments in eh group re indited y different letters ove rs Experiment 5: Detoxifition of o-hpa id y ECM Fungi Root growth of lk sprue seedlings ws inhiited y 82.0 nd 63.9%, respetively, in the presene of o-hpa id t 0.5 nd 0.2 mm (Fig. 4). However, no signifint effet of o-hpa id on root nd shoot length ws found when seedlings were grown with P. involutus ulture roths (Fig. 4,). Experiment 6: Phenoli Degrdtion y Inoulted Blk Sprue The onentrtions of phenoli ids in pet/vermiulite mixture with myorrhiz inoulted lk sprue were muh lower thn those ontining noninoulted lk sprue (Fig. 5). Five d fter dding o-hpa id to the growing medium ontining the ECM inoulted lk sprue, less thn 3% of the ompound ws reovered. Similrly, the reovery of o-oumri id nd feruli id ws only nd 10.4%, respetively, in the pet vermiulite mixture tht hd P. involutus inoulted lk sprue. Disussion Our results show tht the different ECM fungl isoltes hd differentil growth response to Klmi lehtes nd phenoli ompounds. L. lt ws less resistnt thn P. involutus. The growth of P. involutus ws signifintly stimulted y lef nd litter lehtes even in the presene of full-strength lef lehte (0.1 g FW/ ml). This growth enhnement my e used either y dditionl nutrients in the lef nd litter lehte nd/or the degrdtion produts of Klmi phenolis in the

14 1486 J Chem Eol (2006) 32: lehte tht the ECM fungi n use s ron soure. Erlier, Mllik nd Zhu (1995) reported tht growth of one isolte of P. involutus (NF4) ws stimulted in the presene of Klmi lef lehte. Coté nd Thiult (1988) found n isolte of P. involutus (0077) to e more sensitive to lef lehte toxiity of rsperry (Ruus ideus) ompred to other ECM fungi. In this study, wter lehte of Klmi humus showed no signifint effet on olony dimeter of P. involutus nd L. lt. Phenoli profiles of lef nd litter lehtes were different from tht of humus. It is possile tht y the time lef nd litter were deomposed into humus, mny of the influentil lleohemils were degrded y miroorgnisms. It is lso possile tht degrdtion nd use of humus phenolis is more diffiult for ECM fungi thn those of lef nd litter. Beuse different isoltes of the sme speies of ECM fungus responded differently to prtiulr phenoli id, our results suggest tht odpttion my influene ECM fungl tolernes to phenolis. P. tintorius ws the most sensitive fungus to the phenoli mixture; its olony dimeter ws inhiited y 80.5% in the presene of 1.0 mm mixture. This isolte ws olleted from the sutropil region of Gungdong, South Chin, wheres the other four fungl isoltes were olleted from orel forests of Cnd. In South Chin, we know of no report of high onentrtions of phenoli ids in soils, presumly euse of high preipittion, high litter qulity, rpid deomposition, nd little orgni mtter umultion. Therefore, this isolte my not hve mehnisms to del with high onentrtions of phenoli ompounds, wheres the fungi olleted from Cnd my possess this dpttion (Cllwy nd Ridenour, 2004; Vivno et l., 2004). In the nonron growing medium, oth P. involutus nd L. iolor grew signifintly etter with respet to olony dimeter nd myelil iomss in the presene of o-hpa id thn those without the phenoli id (Fig. 2). Sine o-hpa id ws the only ron soure in the medium, we onlude tht the fungus must hve used o-hpa id s ron soure to inrese its iomss. Our phenol redution experiment (Experiment 4) onduted with three pure phenoli ids onfirmed the ide tht P. involutus is le to redue or oxidize phenoli ids quite esily within short time (Fig. 3). However, high onentrtion (1 mm in the medi) of feruli id inhiits the growth of P. involutus (Fig. 1). After P. involutus ws ultured in 0.5 nd 0.2 mm o-hpa, its phytotoxiity disppered (Fig. 4). o-hpa id, t 0.2 mm, ws toxi to lk sprue seedlings in the sene of P. involutus, ut the 0.5 mm o-hpa id solution eme nontoxi in the presene of the fungl ulture. From this result, we onlude tht the onentrtion of 0.5 mm o-hpa id ws redued to less thn 0.2 mm, s result of fungl ulture. In other words, P. involutus detoxified the 0.5 mm o-hpa id to level lower thn 0.2 mm, nd llowed the seedlings to grow. Zhu nd Mllik (1994) hve shown tht lk sprue seedlings n grow in the presene of feruli nd oumri ids ut nnot tolerte high onentrtions of o-hpa id. From this study, we suggest tht the symioti ECM fungi of lk sprue n not only tolerte high onentrtions of o-hpa, ut n lso degrde the ompound nd use it s ron soure. Most of the dded phenoli ids in the pet/vermiulite mixture ontining P. involutus inoulted lk sprue disppered within 5 d, wheres sustntil mounts of the dded ids were reovered from the growing medium of the noninoulted (ontrol) seedlings. We suggest tht the P. involutus-inoulted

15 J Chem Eol (2006) 32: seedlings re le to degrde Klmi phenolis in the potting medium. However, the inomplete reovery of o-oumri nd feruli id in the uninoulted (ontrol) medium suggests tht preinoultion of lk sprue seedlings with suitle etomyorrhizl fungi my promote onifer regenertion in Klmi dominted sites. The prolem of Klmi-indued growth inhiition in lk sprue is omplex nd the reltionship etween Klmi phenolis nd myorrhize is still unler (Wllstedt et l., 2002). From field study tht showed poor growth, folir N nd P onentrtions nd myorrhiztion of lk sprue seedlings growing loser to Klmi thn those growing wy, Ymski et l. (1998) onluded tht Klmi limits the growth of lk sprue in wys other thn diret nutrient ompetition. They suggested tht sine myorrhize nd plnt nutrition re losely relted, fewer myorrizl short roots in lk sprue growing ner Klmi mens less nutrient quisition due to poor myorrhizl symiosis, thus resulting into poorer growth in these seedlings. Another use of poor growth ws ttriuted to the suseptiility of lk sprue ner Klmi to potentil pthogeni fungus, Philoephl dimorphospor. Ymski et l. (1998) suspet tht higher mount of myorrhizl short roots in seedlings growing wy from Klmi my hve proteted them from the pthogeni fungus nd onsequently llowed etter growth. One ould lso suspet long-term dominne of Klmi in the sene of n pproprite host (in this se, lk sprue) tht the enefiil ECM would e redued in soil inoulum. Our previous lortory studies hve shown tht out of 51 fungl isoltes tested, the growth of 41 isoltes ws redued in the presene of Klmi lef lehte in liquid MMN gr. Ymski et l. (1998) suspet tht Klmi ompounds ould ffet the survivl of fungl inoulum in soil or the fungl symionts in lk sprue roots. Roinson (1972) proposed similr hypothesis in explining the growth inhiition of Sitk sprue [Pie sithensis (Bong.) Crriere] seedlings in the presene of nother heth-forming erieous shru, C. vulgris. Response of ECM to erieous ompounds n e negtive, positive, or neutrl depending on the speies nd strins of the ECM (Mllik nd Zhu, 1995). In this study, we found tht ertin lk sprue ECM (prtiulrly P. involutus) n respond positively to Klmi phenolis with inresed iomss. This, in turn, enhnes seedling growth under lortory nd greenhouse onditions. Given the ft tht iophysil onditions in the field n e very different from those in the ontrolled lortory nd greenhouse, the logil next step of this reserh is to test if lk sprue seedlings preinoulted with P. involutus out plnted in Klmi dominted sites n overome the growth inhiition y mintining the symioti reltionship. We n drw severl onlusions from this study. First, our results prove tht ertin ECM fungi n not only offer protetion to host plnts ginst phenoli llelohemils relesed from neighoring plnts, ut they n lso use them s ron soure. This, in turn, n enefit the host plnt. Our findings hve importnt eologil signifine in the sense tht they point to mehnism y whih etomyorrhizl fungl speies n ontrol speies intertions in higher plnts y hnging the rhizosphere hemistry. Seond, the ility of ECM fungi to degrde nd detoxify phenoli llelohemils is not only speies-speifi, ut lso speifi to different strins of the sme fungl speies. Third, tree seedlings preinoulted with enefiil ECM fungi n degrde plnt phenolis in pet/vermiulite mixture. This offers some hope towrd overoming phenoli-indued onifer growth inhiition in

16 1488 J Chem Eol (2006) 32: erieous-dominted sites y outplnting tree seedlings preinoulted with refully seleted enefiil ECM fungi. Aknowledgments We knowledge the finnil support from the Nturl Siene nd Engineering Reserh Counil (NSERC) disovery grnt (to AUM) nd the Ntionl Nturl Siene Foundtion of Chin ( , ), Gungdong Nturl Siene Foundtion of Chin (039254, ), the Ntionl 973 projet of Chin (2006CB100200), Progrm for New Century Exellent Tlents in University (to RSZ). We thnk Dr. Christine Gottrdo, Deie Leh, nd Andre Aguirre of the Chemistry Deprtment, Lkehed University, nd Hihong Bi of the Deprtment of Eology, South Chin Agriulturl University for their help in HPLC nlyses, nd Dr. Ed Setliff of the Fulty of Forestry nd the Forest Environment, Lkehed University for his oopertion during the inoultion experiments. Comments of two nonymous reviewers, the journl editor, Drs. Roert D. Guy nd Jenelle Curtis (Fulty of Forestry, University of British Columi), Leonrd Huthison, nd Brin MLren (Fulty of Forestry nd the Forest Environment, Lkehed University) were helpful in revising the mnusript. Referenes BENDING, G. D. nd READ, D. J Lignin nd solule phenoli degrdtion y etomyorrhizl nd erioid myorrhizl fungi. Myol. Res. 101: BERG, B. nd DISE, N Clulting the long-term stle nitrogen sink in northern Europen forest. At Oeol. 26: BOUFALIS, A. nd PELLISSIER, F Allelopthi effets of phenoli mixtures on the respirtion of two sprue myorrhizl fungi. J. Chem. Eol. 20: BOUFALIS, A., PELLISSIER, F., nd TROSSET, L Responses of myorrhizl fungi to llelopthy: Cenooum geophilum nd Lri lt growth with phenoli ids. At Bot. Gll. 141: CALLAWAY, R. M. nd RIDENOUR, W. M Novel wepons: invsive suess nd the evolution of inresed ompetitive ility. Front. Eol. Environ. 2: COLPAERT, J. V. nd VAN ASSCHE, J. A The effets of dmium on etomyorrhizl Pinus sylvestris L. New Phytol. 123: COTÉ, J. F. nd THIBAULT, J. R Allelopthi potentil of rsperry folir lehtes on growth of etomyorrhizl fungi ssoited with lk sprue. Am. J. Bot. 75: DENNY, H. J. nd WILKIN, D. A Zin tolerne in Betul spp. IV. The mehnism of etomyorrhizl meliortion of zin toxiity. New Phytol. 106: FRIES, L. L. M., PACOVSKY, R. S., SAFIR, G. R., nd SIQUEIRA, J. O Plnt growth nd rusulr myorrhizl fungl oloniztion ffeted y exogenously pplied phenoli ompounds. J. Chem. Eol. 23: GALLET, C. nd PELLISSIER, F Phenoli ompounds in nturl solutions of oniferous forest. J. Chem. Eol. 23: HANDLEY, W. R. C Myorrhizl ssoition with Cllun hethlnd fforesttion. Bull. For. Commun. 36:1 70. INDERJIT nd MALLIK, A. U Nutrient sttus of lk sprue (Pie mrin (Mill) BSP forest soils dominted y Klmi ugustifoli L. At Oeol. 20: LYNN, D. G. nd CHANG, M Phenoli signls in ohittion: Implitions for plnt development. Annu. Rev. Plnt Physiol. Plnt Mol. Biol. 41: MALLIK, A. U Allelopthi potentils of Klmi ngustifoli to lk sprue (Pie mrin). For. Eol. Mnge. 20: MALLIK, A. U Blk sprue growth nd understory speies diversity in ontiguous plots with nd without sheep lurel (Klmi ngustifoli). Agron. J. 93: MALLIK, A. U. nd NEWTON, P. F Inhiition of lk sprue seedling growth on the forest floor sustrtes of entrl Newfoundlnd. For. Eol. Mnge 23: MALLIK, A. U. nd ZHU, H Overoming llelopthi growth inhiition y myorrhizl inoultion, pp , in Inderjit, K. M. M. Dkshini, nd F. A. Einhellig (eds.). Allelopthy: Orgnisms, Proesses, nd Applitions. Amerin Chemil Soiety, Wshington, DC. MALLIK, A. U., ZHU, H., nd PARK, Y. G Overoming Klmi indued growth inhiition of Pie mrin y myorrhizl inoultion. J. Koren For. So. 87:

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