Direct trade-off between cyanogenesis and resistance to a fungal pathogen in lima bean (Phaseolus lunatus L.)

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1 Journl of Eology 21, 9, oi: /j x Diret tre-off etween ynogenesis n resistne to fungl pthogen in lim en (Phseolus luntus L.) Dniel J. Bllhorn 1 *, Anre Pietrowski 2 n Reinhr Lieerei 3 1 Generl Botny n Plnt Eology, University of Duisurg-Essen, Universit tsstrsse 5, 5117 Essen, Germny; 2 Deprtment of Myology, University of Byreuth, Universit tsstrsse 3, 95 Byreuth, Germny; n 3 Bioenter Klein Flottek n Botnil Gren, University of Hmurg, Ohnhorststrsse 1, 2269 Hmurg, Germny Summry 1. Plnts re simultneously ttke y multiple herivores n pthogens. While some plnt efenes t synergistilly, others tre-off ginst eh other. Suh tre-offs mong resistnes to herivores n pthogens re usully expline y the osts of resistne, i.e. resoure limittions ompromising plnt s overll efene. 2. Here, we emonstrte tht tre-offs n lso result from iret negtive intertions mong efensive trits. We stuie ynogenesis (relese of HCN) of lim en (Fee: Phseolus luntus) n effets of this effiient nti-herivore efene on resistne to fungl pthogen (Melnoniee: Colletotrihum gloeosporioies). 3. Lef tissue estrution y fungl growth ws signifintly higher on high ynogeni (HC) lim en essions thn on low ynogeni (LC) plnts. The suseptiility of HC essions to the fungl pthogen ws strongly orrelte to reue tivity of resistne-ssoite polyphenol oxises (PPOs) in leves of these plnts. LC essions, in ontrst, showe high PPO tivity, whih ws orrelte with istint resistne to C. gloeosporioies.. Experimentlly pplie, gseous HCN reue PPO tivity n signifintly inrese the size of lesions use y C. gloeosporioies in LC leves. 5. Fiel oservtions of wil lim en popultion in Mexio revele higher infetion rte of HC ompre to LC plnt iniviuls. The types of lesions oserve on the ifferent ynogeni plnts in nture were similr to those oserve on HC n LC plnts in the lortory. 6. Synthesis. We suggest tht ynogenesis of lim en iretly tres off with plnt efene ginst fungl pthogens n tht the usl mehnism is the inhiition of PPOs y HCN. Our finings provie funtionl explntion for the oserve phenomenon of the low resistne of HC lim ens in nture. Key-wors: Colletotrihum gloeosporioies, iret efene, multiple efene synrome, plnt pthogen intertion, plnt resistne, polyphenol oxise, PPO, tre-off Introution *Corresponene uthor. E-mil: niel.llhorn@uni-ue.e In nturl systems, plnts efen themselves ginst ttk from multiple herivores n pthogens with vrious resistne strtegies. Some efenes ginst herivores re effetive lso ginst pthogens, while others re speifi to prtiulr ttkers (Mithell-Ols et l. 1995; Juenger & Bergelson 199; Stout et l. 199; Tiffin & Rusher 1999; Stinhome & Rusher 21). However, in mny ses the funtionl interply of ifferent efene mehnisms is little unerstoo n experimentl pprohes tht quntittively nlyse interting plnt efenes ginst herivores n pthogens re rre (Felton & Korth 2; Pul, Hther & Tylor 2). To fill this gp, we fouse on two iret efensive trits of lim en ynogenesis n tivity of polyphenol oxises (PPOs). Polyphenol oxises re enzymes tht re involve in ro rry of efene-ssoite plnt responses inluing responses to pthogen ttk (Cmpos et l. 2). Cynogenesis is the enzymtilly elerte relese of toxi hyrogen ynie (HCN) from pre-forme ynie-ontining preursors in response to ell mge (Møller & Seigler 1999). This trit is wiely istriute mong higher plnts n is highly effiient plnt efene ginst herivores (e.g. Bernys et l. 1977; Bernys 1991; Bllhorn, Lieerei & Gnzhorn Ó 29 The Authors. Journl ompiltion Ó 29 British Eologil Soiety

2 Cynogenesis in plnt pthogen intertions ; Bllhorn, Heil & Lieerei 26; Bllhorn & Lieerei 26; Bllhorn et l. 27, 2, 29). In ontrst to stuies on herivore plnt intertions, omprly little reserh hs een onute on the role of ynogenesis in plnt intertions with fungl pthogens (Lieerei et l. 199; Lieerei, Fok & Biehl 1996; Nielsen et l. 25). Hyrogen ynie is highly toxi to ll eukryotes ue to inhiition of the mitohonril respirtion pthwy y loking the ytohrome 3-epenent oxise (Solomonson 191). During infetion y pthogens, gseous HCN iffuses from estroye plnt ells through the tissue surrouning the infete re n, thus, oth the plnt itself n the pthogen re in iret ontt with the relese HCN (Lieerei, Fok & Biehl 1996). Strikingly, espite the strong n generl toxiity of ynie to eukryoti ells, no onsistent funtionl ssoition etween ynogenesis n resistne of plnts to pthogeni fungi hs een foun so fr (Rissler & Millr 1977; Fry & Myers 191; Lieerei 19; Lieerei et l. 199; Nielsen et l. 25). Prtly, this is ue to the effetive HCN etoxifition in some pthogeni fungi, e.g. y ynie hyrtses (Osourn 1996). However, severl stuies suggest tht highly ynogeni plnts re even more suseptile to fungl pthogens thn onspeifis with lower ynogeni properties (Dirzo & Hrper 192; Lieerei et l. 199; Lieerei, Fok & Biehl 1996; Osourn 1996). This pprent ontrition n e expline y negtive effets of ynie on plnt enzymes involve in nti-pthogen efene (Solomonson 191; Lieerei 27). Hyrogen ynie quntittively ffets mny enzymti tivities, most of ll those of metl-ontining enzymes. In ition to tlses n peroxises, PPOs represent n importnt group of plnt enzymes involve in plnt efenes ginst pthogens n herivores. Polyphenol oxises oxite phenoli ompouns to retive quinones (Felton et l. 199, 1992; Bi et l. 1995; Stout, Workmn & Duffey 1996; Thler et l. 21, 22; Krnthi, Krnthi & Wnjri 23). These enzymes hve een reporte to e involve in plnt efene ginst rthropo (e.g. Felton et l. 199, 1992; Stout, Workmn & Duffey 1996) n nemtoe herivores (e.g. Myer 26) s well s to provie resistne to teril n fungl pthogens (e.g. Lieerei et l. 199; Niholson 1992; Cmpos et l. 2; Thipypong, Stout & Attjrusit 27). In our present stuy, we use lim en (Fee: Phseolus luntus L.) s experimentl plnt. Lim en is well-stuie onerning inuile iniret efenes ginst herivores (e.g. Dike 1999; Dike & Vet 1999; Arimur, Kost & Boln 25; Mitho fer, Wnner & Boln 25; Choh, Kugimiy & Tkyshi 26, ; Heil & Silv Bueno 27; Kost & Heil 2). However, in ition to iniret efenes, some lim en genotypes re hrterize y strong ynogenesis n therefore re lso effiiently iretly efene ginst herivores (Bllhorn, Lieerei & Gnzhorn 25; Bllhorn, Heil & Lieerei 26, Bllhorn & Lieerei 26; Bllhorn et l. 27, 2, 29). Although lim en exhiits these two efene mehnisms, we reently emonstrte tht ynogenesis n totl relese of voltile orgni ompouns (VOC) in lim en re negtively orrelte to eh other, i.e. quntittive tre-off ours (Bllhorn et l. 2, ). Whether tre-offs lso exist etween ynogenesis n nti-pthogen efene of lim en remins elusive so fr. Thus, the im of our stuy ws to quntify puttive effets of ynogenesis on nti-pthogen efene. By this pproh, we intene to provie new insights into the funtionl interply of efenes ginst ifferent ttkers. We use six ultivte essions of lim en with highly ifferent onentrtions of ynogeni preursors in their leves n mesure their resistne to the fungl pthogen Colletotrihum gloeosporioies. To experimentlly onfirm the oserve tre-off etween ynogenesis n nti-pthogen efene, we pplie gseous HCN to leves inoulte with C. gloeosporioies. In the present stuy, our gol ws to emonstrte tht quntittive tre-offs etween nti-herivore n nti-pthogen efenes hve to e onsiere in funtionl nlyses of plnt eology in networks of interting speies. Mterils n methos PLANT MATERIAL For iossys with pthogens, we use three high ynogeni (HC) (CV_2357, CV_7, CV_1315) n three low ynogeni (LC) (CV_21, CV_73, CV_67) essions of lim en (Fee: Phseolus luntus L.). Selete essions represente the upper n lower rnge of ynie onentrtion in leves of wil-type plnts mesure uner nturl fiel onitions (Bllhorn et l. 29). Sees were provie y the Institute of Plnt Genetis n Crop Plnt Reserh (IPK) in Gtersleen, Germny. PATHOGEN MATERIAL In preliminry experiments, we sreene efine strins of the fungl en pthogen Colletotrihum linemuthinum (Sro & Mgnus) Briosi & Cvr [strins 1225 n 631 otine from the DSMZ (Deutshe Smmlung von Mikroorgnismen un Zellkulturen, Brunshweig, Germny)] n the losely relte generlist plnt pthogen Colletotrihum gloeosporioies (Penzig) Penzig & Sro (strins n 6216) for their ility to effiiently infet lim en leves uner lortory onitions. Inoultion of plnts with spore solutions showe tht the strin 6216 of C. gloeosporioies ws most omptile to lim en genotypes. In ition, this strin ws most relile in prouing spores, whih ws n oligtory preonition for onuting reprouile inoultion experiments. Thus, we selete C. gloeosporioies strin 6216 for iossys. The osmopolitn fungus C. gloeosporioies is fulttive hemiiotrophi plnt pthogen. Infetion of plnt tissue ours through wouns or stomt s well s vi penetrtion of intt plnt surfes (Wijesuner, Biley & Byre 19; Biley et l. 1992). At this stge, the fungus lives iotrophilly n evelops primry infetion hyphe (Niholson 1992; Bergstrom & Niholson 1999). When the host tissue is estroye in the ourse of n infetion, the fungus evelops seonry nerotrophi hyphe n proues new spores fter few ys. CULTIVATION OF PLANTS AND PATHOGENS Plnts were ultivte uner glsshouse onitions (16: L:D with photon flux ensity of 35 5 lmol m )2 s )1 t the plnt ontiner n 95 lmol m )2 s )1 t the top of the plnts, epening on Ó 29 The Authors. Journl ompiltion Ó 29 British Eologil Soiety, Journl of Eology, 9,

3 22 D. J. Bllhorn, A. Pietrowski & R. Lieerei nturl sun light). Aitionl light ws provie y W high-pressure soium lmps (Son-T Agro ; Philips Ò ; Hmurg, Germny). To voi hot spots uner the lmps, the position of the plnt ontiners ws hnge every 3 ys. Temperture [25 C (y): 2 C (night)] n mient reltive ir humiity (6 7%) were ontrolle y integro Ò (Surrey, Cn) softwre. Plnts were fertilize with nitrogen phosphte fertilizer (Blukorn Ò -Nitrophosk Ò -Perfekt; Compo GmH; Mu nster, Germny) twie week (3 mg per pot). A 1:1 mixture of stnr sustrte (TKS Ò -1-Instnt, Florgr Ò ;Olenurg, Germny) n sn (grin size.5 2. mm) serve for ultivtion in plnt ontiners (1 m in imeter). Twenty-eight plnts per ession were grown (full sis). Plnts use for the experiments were 5 weeks ol n h evelope five to six leves. Colletotrihum gloeosporioies ws ultivte in Petri ishes (9.5 m in imeter) on ot meium, ph 5. (% flour, 1.5% gr). Cultures were store in limti hmer t temperture of 25 C, reltive ir humiity of 5%, n uner light regime of 1 min light (15 lmol m )2 s )1 ) per 12 h to inue evelopment of spores. LEAF MATERIAL USED FOR BIOCHEMICAL ANALYSES AND BIOASSAYS Young leves of efine evelopmentl stge were use for ll iohemil nlyses n iossys to ssess effets of ynogeni fetures n PPO tivity on pthogen growth. From eh plnt the thir trifolite lef from the top ws selete (Fig. 1). These leves were hrterize y soft lef tissue, h een unfole for 5 6 ys n i not exee one-thir to one hlf of the finl lef size. Lef mss per re (LMA) ws onsiere s n inex of slerophylly to test for similr morphologil fetures of leves mong experimentl plnts (Tle 1). The im of this stuy ws to estlish iret quntittive reltionship of hemil lef hrteristis n their quntittive effets on pthogen performne. For this purpose, trifolite leves were prtitione for the respetive nlyses (Fig. 1). One leflet per lef ws nlyse for gseous HCN relese fter hemil ell isruption (HCN; ynogeni pity). The seon leflet ws ut long the miri. One hlf ws use for nlysis of ynogeni potentil (HCNp; onentrtion of ynogeni preursors) n for nlysis of Biossy C A HCNp+ + -gluosisetivity tivity HCN B PPO tivity Fig. 1. Smpling of lef mteril. Iniviul trifolite leves inserting three positions own the pex were use for quntittive nlyses of plnt trits n their effets on pthogen performne (n = 1plnts per ession with, n = 1 lef per plnt). Two intt leflets per lef were use for iossys with Colletotrihum gloeosporioies (A) n HCN nlysis (B), respetively. The thir leflet (C) ws prtitione n one hlf ws use for nlysis of HCNp n -gluosise tivity (using the sme lef extrt), while the other hlf ws use for nlysis of polyphenol oxise (PPO) tivity. The seletion of leflets (terminl or lterl) for the respetive nlysis or experiment ws set t rnom. -gluosise tivity. For oth nlyses, the sme lef extrt ws use. The other hlf serve for quntifition of PPO tivity. The thir leflet ws use for iossys with C. gloeosporioies (Fig. 1). Beuse the leflets of the sme lef were use to onut the iohemil n pthogen growth nlyses, it ws possile to relte the results on pthogen performne iretly to the iohemil hrteristis of tht sme lef. QUANTIFICATION OF CYANOGENIC FEATURES For nlysis of ynogeni potentil (HCNp), fresh leflets were weighe to the nerest.1 g n groun with liqui nitrogen n oole mortr n pestle ( C) uner ition of the fourfol volume (v fresh weight )1 )ofie-oln 2 HPO solution (67 mmol L )1 ). Smples were quntittively nlyse for their HCNp y omplete enzymti hyrolysis of ynogeni preursors with sustrte-speifi -gluosise isolte from ruer tree [Euphoriee: Heve rsiliensis (WILLD. ex JUSS) MUELL. ARG]. We use lose glss vessels (Thunerg vessels) for inution (2 min t 3 C in wter th) of lef extrts together with enzyme solution juste to n tivity of 2 nkt. Quntifition of relese HCN ws rrie out spetrophotometrilly t 55 nm using the Spetroqunt Ò ynie test (Merk KGA, Drmstt, Germny) oring to Bllhorn, Lieerei & Gnzhorn (25). Ativity of -gluosise in leflets ws nlyse oring to Bllhorn, Heil & Lieerei (26). Lef extrts were entrifuge for 15 min t 2 g n C (RC5C; Sorvll Instruments, DuPont Co. Bioreserh Systems, Wilmington, DE, USA). The protein-ontining superntnt ws onentrte y mmonium sulphte frtiontion n filtere through memrne ps with pore size <1 kd (Shleiher & Shuell BioSiene GmH, Dssel, Germny). We use p-np-gluosie (Merk KGA) s hromogeni rtifiil sustrte. Relese p-nitrophenol ws quntifie spetrophotometrilly ( nm, Ultrspe 3; Amershm Phrmi Bioteh, Freiurg, Germny) fter 1 min of inution t 3 C n fter the retion ws stoppe y ing 1 ml ie-ol soium ronte solution (1 mol L )1 ). Cynogeni pity (HCN; relese of gseous hyrogen ynie per unit time) ws mesure using n irflow system oring to Bllhorn, Lieerei & Gnzhorn (25). In this experimentl set-up, hloroform (35 ll per leflet) ws pipette onto intt leflets n the susequent relese of gseous HCN from the isintegrte ells ws etete quntittively (Fig. 2). The irflow in the system ws juste to 7. L h )1. In orer to test for ny remining non-leve ynogeni preursors in the lef mteril fter HCN nlysis, we prepre extrts from leflets, e speifi exogenous -gluosise n nlyse the ynogeni potentil (HCNp) of lef mteril in Thunerg vessels s esrie previously. QUANTIFICATION OF POLYPHENOLOXIDASE ACTIVITY We quntifie enzymti tivity of PPOs y mesuring the O 2 onsumption uring the oxition of polyphenols n their erivtes to quinones (Rihter, Lieerei & von Shwrtzenerg 25). The tivity ws etermine polrogrphilly, using the Clrk eletroe system (Yellow Springs Instruments, Yellow Springs, OH, USA), y mesuring the onomitnt oxygen epletion. The eletroe ws lirte to 1% O 2 sturtion using O 2 -sturte Sörensen uffer (phosphte itrte uffer, ph 5.6). We use -methylthehol (Fluk Chemie GmH, Deisenhofen, Germny) s stnr. The utoxition of -methylthehol ws mesure uner stirring efore eh PPO tivity nlysis. The solution for mesuring Ó 29 The Authors. Journl ompiltion Ó 29 British Eologil Soiety, Journl of Eology, 9,

4 Cynogenesis in plnt pthogen intertions 229 Tle 1. Cynogeni n morphologil trits of lim en leves. Young leves from ifferent iniviul plnts were sreene for their ynogeni fetures (n = 1 iniviul plnts per essions). Vlues shown for lef hrteristis re mens±sd. Signifint ifferenes etween essions were lulte y post ho nlysis (Tukey s HSD; P <.5) fter one-wy nov n re inite y ifferent letters in prentheses Cynogeni trits Morphologil trits* Aessions Cynogeni type [high (HC) n low (LC)] Cynogeni potentil HCNp (lmol HCN g )1 fresh wt.) Cynogeni pity HCN (lmol HCN g )1 fresh wt. min )1 ) -gluosise tivity (lkt g )1 ry wt.) Lef texture Lef mss per re LMA (mg m )2 ) CV_2357 HC 67.22±.62 ().63±.7 () 1.±.23 () Soft.±.7 () CV_7 HC 63.1±5. ().5±.9 () 1.91±.15 () Soft.6±.13 () CV_1315 HC 5.1±6.1 ().52±.9 () 1.67±.25 () Soft.5±.12 () CV_21 LC 27.7±2.7 ().12±.2 () 1.19±.11 () Soft.5±.11 () CV_73 LC 22.59±.3 ().2±.3 () 1.±.23 () Soft.7±.1 () CV_67 LC 19.61±2.51 ().1±. () 1.6±.16 () Soft.5±.12 () *We selete lim en essions with similr lef texture from lrger set of experimentl plnts, sine some genotypes re hrterize y hr-texture leves (Bllhorn, Heil & Lieerei 26). Aessions were ssigne to ifferent ynogeni types (HC n LC) oring to signifint ifferenes in their HCNp n HCN. Air flow HCN A B C D KCN solution Fig. 2. Applition of gseous HCN to inoulte leflets. The irflow in the vessel system ws provie y iphrgm pump with justle pity (A). The ir psses the HCN genertor (B) ontining 5 L of n unuffere, slightly lkline KCN solution (.1,.5 n 1. mmol KCN L )1, respetively), whih ws juste to ph 7. with n equte mount of phosphori i. Proue gseous HCN (.3, 1., n 3.61 lmol HCN L )1 ir) ws trnsporte into the inution hmer (C) ontining inoulte leflets of lim en. At the outlet of the system gseous HCN ws etete quntittively (D) to ontrol for onstnt mient ynie onentrtion in the inution hmer. utoxition onsiste of 2.9 ml Sörensen uffer n 1 ll -methyltehol (225 mmol L )1 ). For extrtion of PPOs, leflets were homogenize in the threefol volume (v fresh weight )1 ) Sörensen uffer. Extrtion ws rrie out in Eppenorf Ò tues (1.5 ml) t 25 C to llow enzymtilly elerte eomposition of ynogeni preursors in the plnt extrt. The onentrtion of free HCN in the smple ws nlyse y use of the Spetroqunt Ò ynie test (Merk). Thus, quntittive effets of relese HCN on PPO tivity oul e evlute. Susequently, lef extrts were entrifuge ( g, 1min, C) n the superntnt ws use for nlysis of PPO tivity. The solution in the O 2 eletroe onsiste of 1 ll superntnt n 2. ml O 2 -sturte uffer. After 1 min of stirring, 1 ll of -methyltehol (225 mmol L )1 ) ws e n O 2 epletion ws etermine polrogrphilly over time perio of 6 min. This proeure gives reltive vlues of O 2 epletion inluing enzymti O 2 epletion in the ourse of sustrte oxition, utoxition of the sustrte, n other O 2 onsuming proesses in the lef extrts. Thus, we sutrte oth vlues of utoxition of the sustrte n O 2 onsumption of the smples (mesure in the O 2 eletroe without smple or sustrte solution, respetively) from totl O 2 epletion. As prerequisite for speifi PPO ssy, we heke whether the system oul e inhiite y ynie. In ontrol experiments, potssium ynie (KCN) ws e to lef extrts prepre from LC plnts (showing high nturl PPO tivity) in onentrtion of 15, 3 n 6 lmol ynie g )1 fresh wt. (6 lmol ynie g )1 fresh wt. orrespons to ynie onentrtions nturlly ourring in HC plnts). INOCULATION OF LEAFLETS WITH COLLETOTRICHUM GLOEOSPORIOIDES Leflets were inoulte with spore suspension juste to onentrtion of 1 5 spores ml )1. Spore smples were tken from -y-ol fungl ultures, ilute with 5 ml qu est. n mixe three times for 1 min (Whirl Mix; Eppenorf AG, Hmurg, Germny). Mirosopi etermintion of spore onentrtion ws rrie out y use of n Improve-Doule-Neuuer ounting hmer. Seven smples of eh suspension were ounte uner the mirosope (igonl squres were ounte in row). Leflets were injure on the lower surfe (one injury per leflet) using.9-mm glss pin, Ó 29 The Authors. Journl ompiltion Ó 29 British Eologil Soiety, Journl of Eology, 9,

5 23 D. J. Bllhorn, A. Pietrowski & R. Lieerei n 5 ll of spore suspension were pipette onto the woun. Sterile wter inste of spore suspension serve s ontrol. Leflets were kept t room temperture until the spore suspension h evporte n or ws sore y the lef tissue. EVALUATION OF FUNGAL PERFORMANCE Eh inoulte leflet (n = 1 leflets per ession n per tretment) ws ple in Petri ish (9.5 m in imeter) line with moist filter pper to ensure wter supply. Seven Petri ishes were ple in the inution hmer of the irflow system for h simultneously (Fig. 2). We use four irflow systems in prllel, whih were ple in the limti hmer uner mient onitions s esrie ove. Positions of the Petri ishes were hnge twie y uring exhnge of the KCN solution in the HCN genertor. After the inution perio, the imeter of lesions (here efine s lef tissue estroye y fungl growth) ws evlute. For this purpose, leves were igitlly photogrphe on sle light ox (Fleishhker GmH & Co. KG, Shwerte, Germny) n lesion size ws quntifie using the nlysis softwre (Olympus Soft Imging Solutions GmH, Mu nster, Germny). APPLICATION OF HCN TO INOCULATED LEAVES We rrie out four experimentl series using ifferent HCN regimes (i.e. tretments with ifferent onentrtions of gseous HCN) with ll six essions of lim en. Applition of gseous HCN to inoulte leves ws onute y use of n irflow system s esrie in Bllhorn, Lieerei & Gnzhorn (25) with some moifitions. Here, we use two 1-L glss vessels, one s HCN genertor, the other s n inution hmer for inoulte leves (Fig. 2). The irflow ws juste to 3.6 L h )1. The HCN genertor ontine 5 L of slightly lkline non-uffere KCN solution of ifferent onentrtions (.1,.5 n 1. mmol ynie L )1 ) epening on the experimentl set-up. The relese of gseous HCN from these KCN solutions resulte in HCN tmospheres in the irflow system of.3, 1. n 3.61 lmol HCN L )1, respetively. These onentrtions overe the rnge of HCN tht nturlly n e relese from lim en leves (Bllhorn, Lieerei & Gnzhorn 25). For the ontrols, the HCN genertor ws fille with 5 L qu est. inste of KCN solution. For proution of gseous HCN, KCN solutions were juste to ph 7. y ing phosphori i in equimolr mounts to elerte relese of gseous HCN. The KCN solution ws exhnge twie y to gurntee onstnt onentrtion of tmospheri HCN. In the irflow system, gseous HCN relese from KCN solution ws ishrge into the inution vessel n trppe t the outlet of the irflow system in test tue ontining 1 ml of NOH solution (.1 mol L )1 ). The onentrtion of HCN in the tmosphere ws heke for onstny five times y y quntifying the HCN trppe per time perio (1 min). Quntifition ws rrie out using the Spetroqunt Ò ynie test (Merk) s esrie ove. FIELD OBSERVATIONS In nturl popultion of wil-type lim ens ner Puerto Esonio ( N, W, elevtion 12 m.s.l., Ox, southern Mexio) in Otoer 27, we sreene efine lef evelopmentl stges (fully unfole leves lote three insertion positions own the pex; n = 21 plnts) for the onentrtion of ynie-ontining preursors. Ourrene n type of lesions (smll n rk rown vs. lrge n light rown, see Fig. S1 in Supporting Informtion) on iniviul plnts ws note. STATISTICAL ANALYSES We pplie neste nov esign using Cynogeni Type (fixe ftor) n Aession (rnom ftor) to nlyse the t set for ifferenes in onstitutive PPO tivity, inue PPO tivity n pthogen mge etween HC n LC lim en essions. Aession ws neste within Cynogeni Type (i.e. HC or LC), sine we use six lim en essions of whih three were HC n three were LC essions. The effet of Cynogeni Type ws teste ginst the effet of Aession. To test for orreltions etween ynogeni fetures n onstitutive PPO tivity (i.e. to serh for geneti orreltions), we pplie Person s orreltions. To nlyse effets of exogenous ynie pplition on PPO tivity n size of eveloping lesions on lim en leves, we pplie neste generlize liner moel (GLM) with Cynogeni Type, Aession (neste within Cynogeni Type ) n Tretment s ftors. The effet of Cynogeni Type n its intertion with Tretment ws teste ginst Aession n its intertion, respetively. All sttistil nlyses were rrie out using spss 17 (SPSS for Winows; SPSS In., Chigo, IL, USA). Results CYANOGENIC FEATURES OF EXPERIMENTAL PLANTS Cynogeni fetures of lim en leves [ynogeni potentil (HCNp) n ynogeni pity (HCN)] were signifintly lower for LC thn for HC essions. Within groups of HC essions, HCNp showe no signifint ifferenes, while in the group of LC plnts the lest ynogeni ession CV_67 exhiite signifintly lower HCNp thn the two other LC essions [oring to post ho nlysis (Tukey s HSD, P <.5) fter nov; n = 1 plnts per ession; Tle 1]. The tivity of -gluosises, whih ruilly etermines the kinetis of hyrogen ynie relese from the ynogeni preursors in se of ell mge, ws higher for HC essions s ompre to LC essions. However, while the ifferenes were signifint for HC essions CV_2357 n CV_7, -gluosise tivity of the HC ession CV_1315 ws not signifintly ifferent from the LC essions CV_73 n CV_67 (Tle 1). Conerning HCN, no signifint ifferenes were oserve within the groups of HC n LC plnts, respetively. In ontrst to strong quntittive ifferenes of ynogeni fetures etween HC n LC plnts, leves showe istint homogeneity onerning their LMA. No signifint ifferenes were foun for this trit mong essions (Tle 1). CONSTITUTIVE POLYPHENOL OXIDASE ACTIVITY Polyphenol oxise tivity in untrete leves of HC essions ws lower s ompre to LC genotypes (Fig. 3). Cynogeni Type (HC or LC) n Aession(Cynogeni Type) h signifint effet on onstitutive PPO tivity (oring to neste nov esign; Tle 2). Among the three HC essions, ifferenes of onstitutive PPO tivity were not signifint [oring post ho nlysis (Tukey s HSD; P <.5) fter one-wy nov, Tle S1], wheres Ó 29 The Authors. Journl ompiltion Ó 29 British Eologil Soiety, Journl of Eology, 9,

6 Cynogenesis in plnt pthogen intertions 231 HC essions LC essions PPO tivity [µmol O 2 h 1 g 1 fresh wt.]. CV_ CV_ CV_7 16 CV_ CV_ CV_ Const. PPO tivity Const. PPO tivity Const. PPO tivity Applition of gseous ynie [µmol L 1 ] Const. PPO tivity Const. PPO tivity Const. PPO tivity Fig. 3. Polyphenol oxise tivity in leflets uner ifferent HCN-tmospheres. Ativity of polyphenole oxises (PPOs) in leflets of ifferent low ynogeni (LC) n high ynogeni (HC) lim en essions ws mesure uner ifferent experimentl onitions. PPO tivity ws mesure in inoulte leflets inute uner ifferent mient ynie onentrtions (,.3, 1. n 3.61 lmol HCN L )1 ir). Constitutive PPO tivity represents enzymti tivity in non-inoulte leves inute uner HCN-free onitions in the irflow system (lst olumn). Vlues shown for ifferent tretments represent mens + SD (n = 1 leves per tretment n ession; eh one lef ws erive from one iniviul plnt). Different letters inite signifint ifferenes mong tretments [oring to post ho nlysis (Tukey s HSD, P <.5) fter one-wy nov]. Tle 2. Effet of ynogeni type n ession of plnts on polyphenol oxise tivity n lesion size. We mesure onstitutive polyphenol oxise (PPO) tivity n PPO tivity fter inoultion with Colletotrihum gloeosporioies in leflets of ifferent ynogeni lim en essions ( Cynogeni Type = HC or LC essions). In ition, we mesure the size of lesions ourring on leves. Effets of Cynogeni Type n Aession(Cynogeni Type) were lulte using neste nov esign. The term Aession(Cynogeni Type) is neste euse eh mesure omes from only one omintion of the Aession n Cynogeni Type. The effet of Cynogeni Type ws teste ginst the men squres of Aession(Cynogeni Type) Soure Depenent vrile SS.f. F P-vlue Cynogeni Type Constitutive PPO tivity <.37 Aession(Cynogeni Type) <.1 Error Cynogeni Type Pthogen-inue PPO tivity <.1 Aession(Cynogeni Type) <.1 Error Cynogeni Type Lesion size <.1 Aession(Cynogeni Type) <.1 Error mong LC essions, CV_67 (the lest ynogeni essions mong LC plnts) showe signifintly higher PPO tivity thn the other two LC essions (Tle S1). Among ll six essions, onstitutive tivity of PPOs ws negtively orrelte to plnt ynogeni fetures [Person s orreltion: HCNp: r = ).659, P <.1; -gluosise tivity: r = ).9, P <.1; HCN: r = ).691, P <.1]. Within eh ession, we foun no signifint orreltions etween onstitutive PPO tivity n ynogeni trits (t not shown). This fining is ue to the high quntittive onsisteny of oth onstitutive PPO tivity n ynogeni trits mong plnts of given ession. In ontrol experiments onute to ssess the effet of ynie on PPO tivity in lef extrts uring preprtion, ing KCN t onentrtion of 6 lmol ynie g )1 fresh wt. (orresponing to the men HCNp of HC plnts) to liquots of extrts prepre from LC leves (n = 2 extrts, i.e. n = 1 extrts per ession) reue onstitutive PPO tivity y 9.7 ± 2.39% (men ± SD; Tle S2). Aing hlf the onentrtion (3 lmol ynie g )1 fresh wt.) reue PPO tivity in lef extrts y ± 3.52% (n =2),wheres Ó 29 The Authors. Journl ompiltion Ó 29 British Eologil Soiety, Journl of Eology, 9,

7 232 D. J. Bllhorn, A. Pietrowski & R. Lieerei ynie e t onentrtion of 15 lmol g )1 fresh wt. resulte in n inhiition y 27.5 ± 5.2% (n =2)ofthe onstitutive PPO tivity in LC lef extrts (Tle S2). POLYPHENOL OXIDASE ACTIVITY IN INOCULATED LEAFLETS Inoultion with C. gloeosporioies resulte in signifint inrese of PPO tivity in oth, HC n LC leves (oring to Wiloxon Signe Rnks test for two epenent vriles; HC leves: P =.2,Z = )5.19; LC leves: P <.1, Z = )5.65; n = 1 replitions per tretment n ession; Fig. 3). However, the inrese of PPO tivity in response to pthogen inoultion ws lower for HC thn for LC plnts. Differenes in inue PPO tivity epening on Cynogeni Type n Aession(Cynogeni Type) were signifint oring to neste nov esign (Tle 2). Within the group of HC plnts, we foun no signifint ifferenes in enzymti tivity [oring to post ho nlysis (Tukey s HSD, P <.5) fter one-wy nov; Tle S1]. Among LC plnts, inue PPO tivity ws signifintly higher for the lest ynogeni ession CV_67 s ompre to the other (higher ynogeni) LC essions CV_73 n CV_21 (Tle S1). PATHOGEN DAMAGE OF HC AND LC LEAFLETS After experimentl inoultion, men size of lesions use y C. gloeosporioies uner ontrol, i.e. HCN-free onitions, ws 6.6 times higher for HC thn for LC essions. Differenes in men lesion size epening on Cynogeni Type n Aession(Cynogeni Type) were signifint (oring to neste nov esign; Tle 2). Among HC essions (CV_2357, CV_7 n CV_1315), estrution of leflet tissue y the fungus ws signifintly lower for ession CV_1315 (the lest ynogeni essions mong HC plnts) ompre to CV_2357 (the highest ynogeni ession) while men lesion size on ession CV_7 took n intermeite position n ws not signifintly ifferent to oth other HC essions [oring to post ho nlysis (Tukey s HSD, P <.5) fter one-wy nov; Tle S3]. Among LC plnts we foun no signifint ifferenes in lesion size (Tle S3). EFFECT OF HCN TREATMENT ON POLYPHENOL OXIDASE ACTIVITY Aoring to GLM, HCN tretment of inoulte leves ws signifint soure of vrine for PPO tivity (Tle 3). In ition, the ftor Tretment Cynogeni Type h signifint effet on PPO tivity, initing quntittively ifferent responses in HC n LC essions to HCN tretment. Compre to HC essions, reution of PPO tivity in LC essions in response to HCN tretments ws stronger. Among HC essions, enzymti tivity of PPOs in inoulte leves trete with intermeite n high onentrtions of externl HCN ws slightly reue y the ftors.9 ±.15 (1. lmol HCN L )1 ) n.75 ±.1 (3.61 lmol HCN L )1 ;men±sd,n = 1 replitions per tretment n Tle 3. Tretment effets on polyphenol oxise tivity. Results otine using the GLM nlysis of vrine fter univrite esign using polyphenol oxise tivity s vrile. The term Aession(Cynogeni Type) is neste euse eh mesure omes from only one omintion of the Aession n Cynogeni Type. The effet of Cynogeni Type n its intertion with Tretment ws teste ginst the men squres of Aession(Cynogeni Type) n its intertion with Tretment, respetively Soure SS.f. F P-vlue Cynogeni Type <.1 Tretment <.1 Cynogeni Type Tretment <.1 Aession(Cynogeni Type) <.1 Aession(Cynogeni <.5 Type Tretment) Error ession), wheres PPO tivity in inoulte leves of HC essions uner the lowest HCN regime (.3 lmol HCN L )1 ) ws similr to PPO tivity mesure uner ontrol onitions, i.e. inoulte leves uner ynie-free tmosphere (ftor: 1.2 ±.19; n = 1 replitions per tretment n ession). For ll LC essions, we foun istint quntittive effets of gseous HCN pplition on pthogen-inue PPO tivity uner ll HCN regimes (Fig. 3). At the lowest mient HCN onentrtion of.3 lmol HCN L )1,PPOtivityws reue y ftor.5 ±.11 s ompre to ontrols. Higher onentrtions le to sustntil reution of enzymti tivity y the ftors.6 ±.6 (1. lmol HCN L )1 ) n.23 ±. (3.61 lmol HCN L )1 ). EFFECTS OF HCN APPLICATION ON LESION SIZE Experimentl pplition of gseous HCN to inoulte leflets of HC n LC essions inrese fungl growth, i.e. lesion size. Effets of HCN tretment on lesion size were signifint oring to GLM (Tle ). Corresponing to Tle. Tretment effets on lesion size. Results otine using the GLM nlysis of vrine fter univrite esign using lesion size s vrile. The term Aession(Cynogeni Type) is neste euse eh mesure omes from only one omintion of the Aession n Cynogeni Type. The effet of Cynogeni Type n its intertion with Tretment ws teste ginst the men squres of Aession(Cynogeni Type) n its intertion with Tretment, respetively Soure SS.f. F P-vlue Cynogeni Type <.1 Tretment <.1 Cynogeni Type Tretment <.1 Aession(Cynogeni Type) Aession(Cynogeni <.5 Type Tretment) Error Ó 29 The Authors. Journl ompiltion Ó 29 British Eologil Soiety, Journl of Eology, 9,

8 Cynogenesis in plnt pthogen intertions 233 PPO tivity, the ftor Tretment Cynogeni Type h signifint effet on lesion size, initing quntittively ifferent responses of the fungl pthogen epening on ynogeni type (HC or LC) of host plnts. Applition of gseous ynie in onentrtion of.3 lmol HCN L )1 inrese lesion size on HC leves y ftor of 1.16 ±., wheres lesion size of LC plnts ws inrese y ftor of 5.97 ± 1.. Higher HCN tmospheres (1. n 3.61 lmol HCN L )1 ) resulte in sustntilly inrese lesion sizes y ftors 1.2 ±.9 n 1.5 ±. for HC plnts n ftors.56 ± 1.26 n ± 1.59 for LC plnts (Fig. ). FIELD OBSERVATIONS Lesion imeter [mm 2 ] HC essions CV_2357 CV_7 CV_1315 LC essions CV_21 CV_73 CV_ Applition of gseous ynie [µmol L 1 ] Fig.. Lesion size on leflets uner ifferent HCN tmospheres. Lef mge of low ynogeni (LC) n high ynogeni (HC) essions of lim en y Colletotrihum gloeosporioies ws mesure s size of lesions fter h of inution uner ontrol ( HCN-free) n ifferent mient ynie onentrtions (.3, 1. n 3.61 lmol HCN L )1 ir). Vlues represent mens + SD (n =1 leves per tretment n ession; eh one lef ws erive from one iniviul plnt). Different letters inite signifint ifferenes mong tretments [oring to post ho nlysis (Tukey s HSD, P <.5) fter one-wy nov]. HCNp [µmol HCN g 1 fresh wt.] Plnt iniviuls Define lef evelopmentl stges of wil-type lim en plnts in nture showe istint vriility of ynogeni fetures. Cynogeni potentil (HCNp) of young leves rnge from to 7.6 lmol ynie g )1 lef fresh wt. n thus overe the rnge of HCNp expresse y lim en genotypes selete for lortory experiments. High ynogeni plnts t the nturl site were more ommonly olonize y pthogeni fungi thn lower ynogeni wil-type plnts. Cynogeni potentil n ourrene of lesions on leves were highly signifintly orrelte (oring to Person s orreltion: r =.71, P <.1). Lesions oserve on HC plnts were lrger n less rown thn the sporilly ourring lesions oserve on the lower ynogeni iniviuls (Figs 5 n S1). Disussion Lrge n light rown lesions Smll n rk rown lesions Fig. 5. Lesion type vs. ynogeni potentil of lim en plnts in nture. In nturl popultion of wil-type lim en plnts we nlyse efine lef stges of iniviul plnts (n = 21 plnts) for ynogeni potentil (HCNp). Leves were sreene for ourrene of lesions use y fungl pthogens n type of lesions (smll n rk rown lrge n ple to light rown) ws reore. Cynogenesis is n effiient efene ginst inset herivores, whih represent mjor group of plnt ntgonists (e.g. Bernys et l. 1977; Bllhorn, Lieerei & Gnzhorn 25; Agrwl 26; Bllhorn et l. 29). However, in ition to herivores, plnts hve to fe lmost uiquitously ourring pthogens (Osourn 1996). Thus, the generl question rises whether efenes ginst herivores re lso effetive ginst pthogens or vie vers, or if tre-offs mong suh efenes our (Thler et l. 1999; Thler, Owen & Higgins 2). We use set of lim en plnts with efine ynogeni fetures (Tle 1), whih llowe utilizing the nturl vriility of ynogenesis for funtionl nlyses in plnt pthogen intertions. Cynogeni potentil of essions orrespone to the HCNp of wil-type lim en plnts in nture (Fig. 5). In previous stuies, the HC lim en essions use here (CV_2357, CV_7 n CV_1315) were emonstrte to e effiiently efene ginst herivores, wheres LC essions (CV_21, CV_73 n CV_67) were only wekly efene (Bllhorn, Lieerei & Gnzhorn 25; Bllhorn & Lieerei 26; Bllhorn et l. 27). Here, s n itionl omponent of lim ens efene synrome, we onsiere polyphenol oxise (PPO) tivity. We foun low onstitutive PPO tivity in leves of HC essions, wheres LC plnts showe sustntil onstitutive PPO tivity (Fig. 3) suggesting geneti tre-off or onstrint for these two efene pthwys. Furthermore, HC essions showe only moest inrese in PPO tivity following infetion, wheres the nturlly high PPO tivity in LC essions ws strongly enhne in response to infetion with Colletotrihum gloeosporioies (Fig. 3). Although retive quinones provie y PPO tivity n generlly ffet oth herivores n pthogens (Krishik, Goth & Bros 1991; Constel 1999; Biere, Mrk & vn Dmme 2), in previous omprtive stuies on the sme LC n HC lim en essions use here, we emonstrte tht effiieny Ó 29 The Authors. Journl ompiltion Ó 29 British Eologil Soiety, Journl of Eology, 9,

9 23 D. J. Bllhorn, A. Pietrowski & R. Lieerei of extensive PPO tivity in LC plnts s n nti-herivore efene must e low (Bllhorn et l. 27). This is in orne to other reent stuies, in whih limite effets of PPOs on herivores hve een reporte (e.g. Brehenn et l. 27). In ontrst to the limite effiieny of PPO tivity s n nti-herivore efene of lim en, PPO tivity hs een esrie s trit ruilly ffeting the resistne of ens to the fungl pthogen Colletotrihum linemuthinum (Cmpos et l. 2). In our present stuy, the growth of the losely relte generlist plnt pthogeni fungus C. gloeosporioies in lef tissue ws signifintly orrelte to oth PPO tivity (negtive orreltion) n onentrtion of ynie in lef tissue (positive orreltion). Thus, in ontrst to potentilly limite effiieny of PPO tivity in nti-herivore efene, our iossys with C. gloeosporioies revele high effiieny of PPOs in nti-pthogen efene, onfirming the results of Cmpos et l. (2). However, sine orreltions etween PPO tivity, ynogenesis n suseptiility to pthogens o not neessrily reflet funtionl oherenes, we experimentlly pplie HCN t ifferent regimes to leves inoulte with C. gloeosporioies to support our finings. Gseous externl HCN quntittively inhiite PPO tivity (Fig. 3) n resulte in signifintly inrese lesion sizes (Fig. ). In ition, high PPO tivity in extrts of LC leves oul e inhiite y ing ynie (KCN) to lef extrts (Tle S2) in onentrtions resemling HCNp of HC leves. It urrently remins elusive whether other enzymes in ition to PPOs (e.g. tlses n peroxises) involve in plnt resistne to pthogens re lso inhiite y extensive ynogenesis (Solomonson 191). However, inhiition of other enzymes, in ition to PPOs, woul inrese the impt of efensive tre-offs on plnt fitness. In ition to iret inhiitory effets of ynie on PPO tivity, limiting effets of ynie on plnt resistne to pthogens might result(iniretly)from inhiiting ATP synthesis (Solomonson 191). Inue or tive resistne to pthogens is generlly n energy-requiring proess, whih thus n e onstrine y interrupte proution of energy equivlents. In ition, the tivtion of lterntive ynie-insensitive oxises (tht o not ontriute to effiient respirtory ATP proution) my entil metoli osts tht limit resoures for effiient resistne to pthogens (Vnlererghe & MIntosh 1997). However, sine ynie experimentlly e to lef extrts (Tle S2) immeitely inhiite PPO tivity quntittively, our results strongly suggest tht PPO tivity is iretly involve in limiting pthogen resistne of lim en (Fig. ). In ition, iret positive effets of ynie on fungl growth, s oserve for Miroylus ulei (somyetes) (Lieerei et l. 193), pthogen of the ynogeni ruer tree (Euphoriee: Heve rsiliensis), oul e lso exlue here, sine we foun no effets of ynie pplition on the evelopment of C. gloeosporioies ultures on pure mei (D.J.B., pers. os.). Our results inite sustntil quntittive tre-offs etween nti-herivore n nti-pthogen efene inferre y the funtionl interply of PPO tivity n plnt ynogenesis. Uner evolutionry spets, suh tre-offs etween plnts resistnes to multiple enemies might e of high importne. For some plnt speies genetilly se qulittive vrition of ynogeni fetures hs een esrie (Jones 1962, 1966) n popultions of some ynogeni speies my prtly or lmost entirely e ompose of ynogeni genotypes (Dy 195, ; Shppert & Shore 1995; Jones 199). Thus, some ountertive fores n selet ginst ynogenesis. The inrese suseptiility to fungl pthogens of HC iniviuls in popultion my e one of the ruil pressures seleting ginst ynogenesis uner nturl environmentl onitions speifilly fvouring pthogen evelopment (Dirzo & Hrper 192). So fr the question remins unnswere whether the tre-off we oserve here in the lortory hols true uner nturl fiel onitions. However, it seems very likely tht the results of our stuy n e trnsferre to plnt pthogen intertions in nturl systems sine mge y (unknown) pthogens on wil-type lim en plnts in nturl popultion in South Mexio were oserve minly on HC plnt iniviuls, wheres LC genotypes lthough mge strongly y herivores (Bllhorn et l. 29) showe signifintly less n smller lesions (Fig. 5). In ition, the lesions oserve on ifferently ynogeni plnts in nture were highly similr in olour n shpe to the type of lesions (smll n rk rown on LC plnt vs. lrge n light rown to olourless on HC plnts; Fig. S1) eveloping on lim en leves in response to inoultion with C. gloeosporioies uner lortory onitions (D.J.B., pers. os.). Furthermore, in preliminry experiments onute to ssess suitility of ifferent pthogens s experimentl orgnisms for this stuy, we foun tht lesions on HC n LC plnts use y C. linemuthinum ( ommonly ourring pthogenonlimen;cmposet l. 2) showe the sme hrteristis s lesions resulting from inoultion with C. gloeosporioies (D.J.B., pers. os.). Lst ut not lest, the onentrtions of ynogeni preursors in the HC n LC ultivrs we h selete for our stuy overe the minimum n mximum rnge of HCNp of wil-type lim en plnts in nture (Tle 1; Fig. 5) n the onentrtions of gseous HCN use for experimentl pplition to inoulte leves orrespone to onentrtions of HCN tht nturlly n e relese y lim en leves in response to mge (Bllhorn, Lieerei & Gnzhorn 25). Sine we refully selete experimentl orgnisms n juste experimentl onitions to reflet those in nture, n sine our results re supporte y fiel oservtions, the finings of our stuy re most likely trnsferle to nturl systems. However, in future stuies, knok-out plnt lking PPO tivity woul e useful in estlishing the finl proof of usl link etween PPO tivity n resistne to fungl pthogens uner lortory onitions. In prllel fiel stuies t nturl sites, the eologil relevne of lim ens intrspeifi vriility of investment in either ynogenesis or PPO tivity shoul e nlyse onsiering multi-speies networks. Ó 29 The Authors. Journl ompiltion Ó 29 British Eologil Soiety, Journl of Eology, 9,

10 Cynogenesis in plnt pthogen intertions 235 Aknowlegements The uthors re thnkful to the IPK for proviing see mteril for experimentl plnts n to the Universities of Hmurg n Duisurg-Essen for finnil support of this reserh projet. To Wihelm (Chigo) n Dr. Mrtin Shäler (Mrurg) re grtefully knowlege for omments on erlier versions of this mnusript. We further woul like to thnk Mrtin Heil (Irputo) for finnil support in Mexio (grnt He ). Referenes Agrwl, A.A. (26) Mroevolution of plnt efense strtegies. Trens in Eology n Evolution, 22, Arimur, G.I., Kost, C. & Boln, W. (25) Herivore-inue, iniret plnt efenes. Biohimi et Biophysi At Lipis n Lipi Metolism, 173, Biley, J.A., O Connell, R.J., Pring, R.J. & Nsh, C. (1992) Infetion strtegies of Colletotrihum speies. Colletotrihum: Biology, Pthology n Control (es J.A. Biley & M.J. Jeger), pp. 12. CAB Interntionl, Wllingfor. Bllhorn, D.J., Heil, M. & Lieerei, R. 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