135 Effet of resoure supply on intertions etween folir endophyti nd root myorrhizl fungi in perennil ryegrss Q. LIU 1,. J. PRSONS 1, H. XUE 1, J.. NEWMN 2 nd S. RSMUSSEN 1 1 greserh, P.B. 118, Plmerston North 2 Dept. Environmentl Biology, University of Guelph, Ontrio, Cnd N1G2W1 Susnne.rsmussen@greserh.o.nz strt Both folir endophytes nd root rusulr myorrhizl fungi n infet perennil ryegrss nd improve its performne, minly y improving herivore resistne nd phosphorus (P) uptke, respetively. Here, we report study of how the endophyti fungus (Neotyphodium lolii) nd two myorrhizl fungi (Glomus spp.) re ffeted y P supply nd perennil ryegrss (Lolium perenne) ultivrs differing in wter solule rohydrte ontent. Endophyte nd lkloid onentrtions were redued t high P supply nd in the high sugr grss (HSG) erdrt, nd these effets were modulted y the presene of myorrhizl fungi. supply resulted lso in strong redution of myorrhizl olonistion, while the effet of the high sugr grss ws sutle nd modulted y endophyte infetion. Our results show tht the simultneous ssoition of perennil ryegrss with oth endophyti nd myorrhizl endosymionts n negtively ffet eh endosymiont, ut this depends on other ftors suh s resoure supply nd geneti kground of the prtners. Keywords: ryegrss, phosphte, high sugr grss, endophyte, myorrhize, lkloids Introdution ield grown perennil ryegrss plnts re often infeted with endophyti Neotyphodium spp. nd root speifi rusulr myorrhizl Glomus spp. fungi. Both endosymionts n onfer enefits to temperte grsses, minly y improving herivore resistne nd phosphorus (P) uptke, respetively. Studies on the intertions of host plnts with either one of these fungl symionts re numerous, ut studies onsidering triprtite intertions etween endophyte nd myorrhizl fungi nd host plnts re rre. It hs een shown tht endophyti fungi n suppress myorrhizl olonistion nd fungl spore germintion (Müller 23; Omini et l. 26; ntunes et l. 28). Effets of myorrhizl olonistion on endophyte infetion hve een muh less studied, lthough it hs een reported tht myorrhizl infetion n redue the level of endophyte-onferred herivore resistne (Viri et l. 22). lkloid onentrtions in the grss-endophyte symiosis vry onsiderly depending on seson, wether nd nutrient supply, nd there is onfliting evidene of the effets of nutrients on lkloid prodution (Mlinowski & Belesky 2; Rsmussen et l. 27). In previous study we hve shown tht oth endophyte nd lkloid onentrtions were redued in perennil ryegrss t high nitrogen (N) nd in the HSG erdove (Rsmussen et l. 27), demonstrting tht simple hnges in nutrient supply nd resoure (energy) relted plnt trits n ffet the symioti ssoition etween ryegrss nd its fungl endophytes. Here, we tested if P supply hd similr effet on endophytes nd if intertions etween myorrhizl Glomus fungi nd folir endophytes re ffeted y nutrient nd resoure supply in different HSG erdrt. Methods We used two Lolium perenne ultivrs, HSG erdrt nd ontrol ennem tht differ inherently in wter solule rohydrte ontents. Seedlings were rtifiilly inoulted y two N. lolii endophyte strins, Common Strin Lp19 () or R1; nd/or y two myorrhizl fungi, G. mossee () or G. intrrdies (). Non-inoulted endophytefree (E) nd/or myorrhizl-free () plnts were used s ontrols. totl of 36 plnts were grown in snd medi nd kept in ontrolled limte hmers for 12 weeks (14 h light, 2 o C/ 1 h drk, 1 o C). Hlfstrength Hoglnd nutrient solution ontining either.5 mm (low P) or 2 mm (high P) KH 2 PO 4 ws dded weekly. During the whole growth period plnts were wtered every seond dy to pot field pity. Plnts were trimmed every 4 weeks to 6 m ove-ground, trimmings were oven-dried nd dry weights reorded. t the finl hrvest lef ldes were frozen nd ground in liquid nitrogen nd then freeze-dried. Endophyte onentrtions were estimted sed on quntittive polymerse hin retion (qpcr) of genomi DN (gdn) isolted from infeted plnt tissues (Rsmussen et l. 27). Colonistion of roots with myorrhize ws mirosopilly ssessed. Permine nd wter solule rohydrtes were extrted nd quntified s desried previously (Rsmussen et l. 27). The overll experiment ws split-unit design, with
136 Proeedings of the New Zelnd Grsslnd ssoition 72: 135-14 (21) two ultivrs, three levels of folir endophytes, nd three levels of myorrhizl fungi s the whole unit tretment (2 x 3 x 3 x 1 replites) with two levels of P supply s the su-unit tretment (genotypes ross P tretments eing identil). ll nlyses were onduted using JMP sttistil softwre version 7.. Box-Cox trnsformtion ws used to homogenise the error vrines for sttistil nlysis nd report, uniformly, the untrnsformed mens nd stndrd error of the mens. Tukey s Honestly Signifint Differene (HSD) test ws used to ssist in the interprettion of signifint effets nd intertions. Results In lef ldes, endophyte onentrtions were signifintly redued t high P ompred with low P supply (P<.1); the redution ws stronger in the HSG igure 1 NlChi (opies/ng lde gdn) Permine (µg/g lde DW) 45 4 35 3 25 2 15 1 5 5 45 4 35 3 25 2 15 1 5 () Conentrtions of endophyte () nd () nd permine (), (d) nd (e) in lef ldes. Endophyte onentrtions were ssessed y qpcr of n endophyte speifi gene (N. lolii hitinse ; NlChi) in totl (plnt nd fungl) gdn isolted from the individul tissues. = ennem ultivr; = erdrt ultivr; = myorrhizl-free; = G. mossee infeted; = G. intrrdies infeted; = endophyte ommon strin infeted; R1 = endophyte R1 strin infeted. Different letters denote mens tht re signifintly different ording to Tukey s HSD test. Error rs represent stndrd error (SE). () R1 () (d) (e) erdrt thn in the ontrol grss ennem (ig. 1). Myorrhizl inoultion per se did not ffet endophyte onentrtion; however, it did modulte the response of endophyte to P in omplex three-wy intertion (P<.5), whih lso depended on the endophyti strin (ig. 1). In infeted plnts, high P supply redued endophyte onentrtions y more thn 5%, ut only if plnts hd een inoulted with G. intrrdies. In R1 infeted plnts, the effet of P ws strongest in myorrhizl-free plnts, lso resulting in more thn 5% redution of endophyte onentrtions t high P ompred to low P supply. Permine onentrtions in lef ldes were signifintly redued t high P (ig. 1; P<.1), in the HSG erdrt (ig. 1d; P<.1), nd in plnts olonised with oth myorrhizl speies (ig. 1e; P<.1); none of the intertions etween ove ftors were signifint. Myorrhizl olonistion rtes in plnts grown t high P supply were low (mens ± SE: low P = 9.9% ±.92; high P =.41% ±.14); we therefore sttistilly nlysed only the dt sets from plnts grown t low P. t low P supply, myorrhizl olonistion rtes were ffeted y three-wy ultivr x myorrhize x endophyte intertion (P<.5). Colonistion rtes of G. mossee were not signifintly ffeted y endophyte infetion or grss ultivr (ig. 2). However, olonistion rtes of G. intrrdies were signifintly redued in endophyte infeted ennem plnts, ut not in erdrt. High moleulr weight wter solule rohydrtes (HMW WSC) were signifintly higher in lef ldes of the HSG erdrt ompred with the ontrol ultivr ennem (ig. 3; P<.1). Even though the intertion etween P supply nd myorrhizl inoultion ws signifint (P<.5), Tukey s HSD test ws unle to differentite etween the different tretment omintions (ig. 3). Low moleulr weight wter solule rohydrtes (LMW WSC) were not different etween the two ultivrs, ut the oserved inrese of LMW WSC levels t high P ompred with low P ws signifint only in ennem (ig. 3; P<.1). HMW WSCs in roots were inresed t high P ompred with low P supply in oth ultivrs, nd this effet ws stronger in ennem, resulting in signifintly higher levels of HMW WSCs in roots of ennem ompred with the HSG erdrt t high P (ig. 3d; P<.1). P supply lso interted with myorrhizl inoultion (P<.1), ut the intertion ws too sutle for Tukey s HSD test to distinguish etween tretment omintions (ig. 3e). LMW WSC onentrtions in roots were higher t high P ompred to low P supply (ig. 3f; P<.1); none of the other min effets or intertions were signifint.
Effet of resoure supply on intertions etween folir... (Q. Liu,. J. Prsons, H. Xue, J.. Newmn nd S. Rsmussen) 137 igure 2 35 3 olonistion (%) 25 2 15 1 5 rusulr myorrhizl fungl () olonistion in roots of perennil ryegrss plnts grown t low P supply. E = endophyte-free; = endophyte ommon strin infeted; R1 = endophyte R1 strin infeted; = G. mossee infeted; = G. intrrdies infeted. Different letters denote mens tht re signifintly different ording to Tukey s HSD test. Error rs represent stndrd error (SE). igure 3 WSCs (mg/g lde DW) 3 () 25 2 15 1 Conentrtions of high moleulr weight (HMW) nd low moleulr weight (LMW) wter solule rohydrtes (WSCs) in lef ldes (), () nd () nd roots (d), (e) nd (f). = ennem ultivr; = erdrt ultivr; = myorrhizl-free; = G. mossee infeted; = G. intrrdies infeted; = endophyte ommon strin infeted; R1 = endophyte R1 strin infeted. Different letters denote mens tht re signifintly different ording to Tukey s HSD test. Error rs represent stndrd error (SE). HMW () HMW () LMW E R1 E R1 ennem erdrt ennem erdrt Disussion Effets of P supply on folir endophytes nd myorrhizl fungi In the present study high P supply hd strong negtive effet on folir endophyte nd lkloid onentrtions in infeted lef ldes of perennil ryegrss plnts. This effet of high P supply, in reduing endophyte nd lkloids onentrtions, is similr to wht hs een seen for high N supply in our previous study. There we hypothesised tht high resoure supply might stimulte plnt shoot growth more thn endophyte growth, therey diluting endophyte nd lkloid onentrtions (Rsmussen et l. 27). supply lso strongly depressed myorrhizl olonistion, onsistent with previous studies on other plnts (Smith & Red 1997), nd it hs een shown tht signlling moleules suh s strigoltones, whih re exuded y P strved plnts only, medite this myorrhizl response (Yoneym et l. 27). Effets of rohydrte ontent on folir endophytes nd myorrhizl fungi High sugr grsses (HSG) hve een red for umultion of high frutn levels in lef ldes, nd here we show tht erdrt did umulte higher levels of frutns ompred to ennem, onsistent with previous studies (Prsons et l. 24). Endophyte nd lkloid onentrtions were redued in the HSG erdrt ompred with the ontrol ultivr ennem, lso onsistent with previous study using the HSG erdove (Rsmussen et l. 27; 28). However, we lso note here tht the negtive effet of the HSG erdrt on endophyte onentrtions ws E R1 E R1 WSCs (mg/g root DW ) 5 3 25 2 15 1 5 (d) HMW (e) HMW (f) modulted y P supply nd myorrhizl inoultion; endophyte onentrtions were different etween the two ultivrs only t high P supply, lerly showing tht the intertion etween high sugr grsses nd endophytes n e ffeted y other ftors suh s nutrient supply or the presene of myorrhizl fungi. In roots, wter solule rohydrte (WSC) onentrtions were the sme in the two ultivrs t low P supply. t high P supply, high moleulr weight WSCs were lower in the HSG erdrt ompred with ennem, possily inditing differene in sugr trnslotion in HSG ompred with other ryegrss ultivrs. Erlier we reported tht endophyte infetion inresed WSC onentrtions in the ontrol sugr grss ennem, ut not in the HSG erdrt (Rsmussen et l. 27; 28). We did not see n effet of endophyte infetion on WSC levels in the present study, ut onentrtions were muh higher ompred with our previous study (pprox. 35 nd LMW
138 Proeedings of the New Zelnd Grsslnd ssoition 72: 135-14 (21) 15 mg/g lef lde DW, respetively), proly due to longer regrowth periods fter utting (4 nd 2 weeks, respetively). This demonstrtes tht ny effet endophyte infetion might hve on host plnt wter solule rohydrtes depends on other ftors ffeting wter solule rohydrte onentrtions, suh s hrvesting time nd ultivr. Myorrhizl olonistion ws not differentilly ffeted in the two ultivrs. Intertions etween myorrhizl fungi nd folir endophytes In this study, we hypothesised tht folir endophyte nd root myorrhizl fungi intert with eh other if simultneously present in perennil ryegrss plnts, nd tht the intertions etween them re ffeted y resoure supply. Our results show tht the effets of myorrhizl inoultion on endophyte nd permine onentrtions interted with P supply, ultivr, nd endophyti strin. In lef ldes, endophyte onentrtions were redued y myorrhizl inoultion, ut only t high P supply nd in infeted plnts, ut not t low P supply or in R1 infeted plnts. This indites tht studies on the effets of myorrhizl olonistion need to ontrol for ftors like myorrhizl speies, resoure supply, geneti kground of plnts nd even folir endophyti strins, if the outomes of myorrhizl infetion re to e ompred or generlised ross rnge of eologil studies. The effet of endophyte infetion on myorrhizl olonistion ws sutle nd lso depended on other ftors; speifilly myorrhizl olonistion in G. intrrdies inoulted ennem plnts. redution in myorrhizl olonistion hs previously een reported for severl endophyte-infeted Lolium speies (Müller 23; Omini et l. 26) nd it hs een suggested tht endophyti lkloids diretly trnsferred to the roots or vi lehtes from endophyte-infeted grss litter might inhiit myorrhizl fungi (ntunes et l. 28). We show here tht myorrhizl olonistion ws redued in oth (produes permine, lolitrem B, nd ergovline) nd R1 (does not produe lolitrem B or ergovline) N. lolii infeted ennem. This mkes it unlikely tht the ntgonisti effets on myorrhizl fungi re used y the endophyti lkloids lolitrem B nd ergovline, ut our results do not rule out tht permine negtively ffeted myorrhizl olonistion. However, we hve demonstrted in previous metolomis studies of the N. lolii perennil ryegrss ssoition (Rsmussen et l. 28, ) tht wide rnge of other, non-lkloid metolites re ffeted in endophyte-infeted tissues, whih ould lso e involved in the negtive effets of folir-sed endophyti fungi on the root-sed myorrhizl fungi seen in this nd other studies. Our results show tht the simultneous ssoition of temperte grsses with oth folir nd root symioti fungi n result in the redution of eh endosymiont, ut this depends on other ftors suh s resoure supply or grss ultivr. s this ould hve oth negtive (e.g. from redued endophyti nti-herivorous lkloids nd/or myorrhizl P supply) nd positive effets (e.g. from redued nutrient requirements of the endosymionts from the host plnt), triprtite ssoitions my shift the lne of mutulismprsitism etween host plnt nd its symionts, nd the degree of this shift depends on rnge of other ftors, like nutrient supply nd geneti kground of host plnts, myorrhizl nd endophyti fungi. CKNOWLEDGEMENTS This work hs een prtilly funded y the NZ oundtion of Reserh, Siene nd Tehnology (ontrts GRX51, C1X23, PROJ 1333-ECOS-GR) nd gmrdt NZ (postdotorl fellowship), nd ws onduted t greserh Grsslnds, New Zelnd. We thnk Dr. Geoffrey. Lne (greserh) for providing us with mss spetrometry method for the nlysis of permine. REERENCES ntunes, P.M.; Miller, J.; Crvlho, L.M.; Klironomos, J.N.; Newmn, J.. 28. Even fter deth the endophyti fungus of Shedonorus phoenix redues the rusulr myorrhizs of other plnts. untionl Eology 22: 912-918. Mlinowski, D.P.; Belesky, D.P. 2. dpttions of endophyte-infeted ool seson grsses to environmentl stresses: Mehnisms of drought nd minerl stress tolerne. Crop Siene 4: 923-94. Müller, J. 23. rtifiil infetion y endophytes ffets growth nd myorrhizl olonistion of Lolium perenne. untionl Plnt Biology 3: 419-424. Omini, M.; Eggers, T.; Bonkowski, M.; Gnge,.C.; Jones, T.H. 26. Lef endophytes ffet myorrhizl sttus nd growth of o-infeted nd neighouring plnts. untionl Eology 2: 226-232. Prsons,.J.; Rsmussen, S.; Xue, H.; Newmn, J..; nderson, C.B.; Cosgrove, G.P. 24. Some high sugr grsses don t like it hot. Proeedings of the New Zelnd Grsslnd ssoition 66: 265-272. Rsmussen, S.; Prsons,.J.; Bssett, S. 27. High nitrogen supply nd rohydrte ontent redue fungl endophyte nd lkloid onentrtion in Lolium perenne. New Phytologist 173: 787-797. Rsmussen, S.; Prsons,.J.; Xue, H.; rser, K.; Newmn, J.. 28. Metoli profiles of Lolium
Effet of resoure supply on intertions etween folir... (Q. Liu,. J. Prsons, H. Xue, J.. Newmn nd S. Rsmussen) 139 perenne re differentilly ffeted y nitrogen supply, rohydrte ontent, nd fungl endophyte infetion. Plnt Physiology 146: 144-1453. Rsmussen, S.; Prsons,.J.; Popy,.; Xue, H.; Newmn, J.. 28. Plnt-endophyte herivore intertions: more thn just lkloids? Plnt Signling nd Behvior 3: 1-4. Smith, S.E.; Red, D.J. 1997. Myorrhizl Symiosis. demi Press, Sn Diego, C, US. 243-246 pp. Viri, M.; Hther, P.E.; yres, P.G. 22. Comined effet of folir nd myorrhizl endophytes on n inset herivore. Eology 83: 2452-2464. Yoneym, K.; Xie, X.; Kusumoto, D.; Sekimoto, H.; Sugimoto, Y.; Tkeuhi, Y.; Yoneym, K. 27. Nitrogen defiieny s well s phosphorus defiieny in sorghum promotes the prodution nd exudtion of 5-deoxystrigol, the host reognition signl for rusulr myorrhizl fungi nd root prsites. Plnt 227: 125-132.