Components of Pink Snow Mould Resistance in Winter Wheat are Expressed Prior to Cold Hardening and in Detached Leaves

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1 J. Phytopthology 5, (6) Ó 6 The Authors Journl ompiltion Ó 6 Blkwell Verlg Norwegin Crop Reserh Institute nd Norwegin University of Life Sienes, A s, Norwy Components of Pink Snow Mould Resistne in Winter Whet re Expressed Prior to Cold Hrdening nd in Dethed Leves Å. A. Ergon nd A. M. Tronsmo AuthorsÕ ddress: Norwegin Crop Reserh Institute, Plnt Protetion Centre, Høgskolevn. 7, N- A s, Norwy nd Norwegin University of Life Sienes, Deprtment of Plnt nd Environmentl Sienes, P.O. Box 5, A s, Norwy (orrespondene to A. Ergon. E-mil: shild.ergon@um.no) Reeived August, 5; epted Novemer 7, 5 Keywords: dethed lef ssy, developmentl stge, Mirodohium nivle, prtil disese resistne, Tritium estivum Astrt Resistne to pink snow mould, used y Mirodohium nivle, ws investigted in four resistnt winter whet lines from the USDA World Cerel Colletion (CI9, CI6, PI7 nd PI868) nd three Nordi whet lines (, Rid nd V). Pink snow mould resistne ws tested in non-hrdened nd old-hrdened plnts inuted under rtifiil snow over nd in dethed lef segments mounted on wter gr nd inuted t either C in drkness or t room temperture with light during the dy. The whet lines CI9, CI6 nd PI868 were more resistnt thn the Nordi lines, oth efore nd fter old hrdening. Thus, lthough old hrdening strongly inreses the level of snow mould resistne in ll the whet lines, some resistne mehnisms re lso present prior to old hrdening in some of the resistnt lines. CI9, CI6 nd PI868 lso hd higher level of resistne thn the other lines in the dethed lef ssy, inditing tht these lines hve some resistne mehnisms ting in the leves. The resistne of PI7 ws expressed only in intt hrdened plnts nd not in non-hrdened plnts or in dethed leves. This indites tht this line relies on old hrdening-relted hnges in the rown for its resistne. In the dethed lef ssy the rte of lesion development vried gretly etween leves of different order. The highest orreltion with the whole plnt test ws otined when using seondry leves nd inution t C in the drk. Introdution Mirodohium nivle (pink snow mould) is one of severl fungi tht n use snow mould on erels nd grsses overed y snow. Mirodohium nivle n lso use other diseses, e.g. fusrium hed light, seedling light nd rown rot. Resistne to snow moulds in erels nd grsses inreses drmtilly during old hrdening (A rsvoll, 977; Tronsmo, 98; Gudet nd Chen, 987; Nkjim nd Ae, 996). In ddition, snow mould resistne is dependent on the developmentl stge of the plnts. In whet, young seedlings t the pretillering stge hve ertin level of snow mould resistne. This level drops trnsiently nd then inreses with the development of the plnts (Gudet nd Chen, 987; Gudet nd Kozu, 99; Nkjim nd Ae, 996). The inrese in snow mould resistne with oth developmentl stge nd old hrdening is thought to e relted to the umultion of rohydrte reserves (Bruehl nd Cunfer, 97; Kiyomoto, 987; Bengtsson, 989; Gudet et l., 999,, ) nd prtiulrly the inrese in the proportion of frutn (Ae nd Yoshid, 997; Gudet et l., ), whih is min rohydrte storge polymer in Poee speies in the temperte nd old zones (Meier nd Reid, 98). A lrge rohydrte storge inreses the ility of plnts to sustin longer under snow over, my enle plnts to tolerte the presene of snow mould fungi in their tissues nd/or my ensure the neessry resoures for defene retions ginst infetion t the end of the winter, the time when snow moulds usully develop. How frutn in prtiulr my ply role in snow mould resistne is unler, ut snow mould fungi might e less le to metolize frutn polymers ompred to mono- nd dishrides (Gudet et l., 999). Cold hrdening of plnts lso indues resistne to fungl pthogens tht infet during the growing seson, suggesting tht some generl defene-relted trit is present in old hrdened plnts (Tronsmo, 98; Tronsmo et l., 99). Aumultion of defene-relted proteins is thought to e involved in old hrdening-indued disese resistne (Tronsmo et l., 99; Hon et l., 995; Ergon et l., 998; Hiilovr-Teijo et l., 999; Kuwr et l., ; Gudet et l.,,). Redued ell wter potentil my lso ply role (Bruehl nd Cunfer, 97; Tronsmo, 986).

2 Components of Snow Mould Resistne in Whet In the 96s numer of snow mould resistnt winter whet lines in the USDA World Cerel Colletion were identified (Bruehl, 98). The resistne of these lines is thought to e relted to very rpid nd strong umultion of rohydrtes, prtiulrly frutn, during hrdening s well s low rte of onsumption during winter (Bruehl nd Cunfer, 97; Kiyomoto nd Bruehl, 977; Kiyomoto, 987; Bengtsson, 989; Ae nd Yoshid, 997; Yoshid et l., 998; Gudet et l.,, ). Resistne lso ppers to e somewht orrelted with higher degree of polymeriztion of frutn (Gudet et l., ). To our knowledge, resistne mehnisms of the resistnt lines hve only een studied in old hrdened plnts. In ddition to hrdening-dependent resistne mehnisms, resistnt mteril my lso depend on mehnisms present prior to hrdening. Suh mehnisms ould e the sme s, or different from, those indued y old hrdening. Whole plnt tests of snow mould resistne in ontrolled environments re prtiulrly time-onsuming nd ostly, sine the plnts hve to e inuted for mny weeks t low temperture. Although fster sreening t higher tempertures my e fesile (Nkjim nd Ae, 99) it is still diffiult to hit the pproprite inution length for the differentition etween genotypes. Severl inution periods re therefore ommonly used in prllel, inresing the size of the experiments. Dethed lef ssys for pthogen resistne re fster nd heper thn ssys using intt plnts. A dethed lef ssy for detetion of omponents of resistne to fusrium hed light in erels mkes use of M. nivle, euse this speies produes fr more distint nd redily oservle lesions on dethed leves thn other memers of the fusrium hed light omplex (Dimond nd Cooke, 999; Browne nd Cooke,, 5; Browne et l., 5). The im of the present study ws () to test if some of the snow mould resistnt winter whet lines identified in the USDA World Cerel Colletion prtly rely on resistne mehnisms present prior to old hrdening nd () to investigte whether hrdening-dependent, ge-dependent nd genotype-dependent pink snow mould resistne expressed in intt plnts re lso expressed in dethed leves. Informtion on this will lrify to wht extent the different forms of resistne re dependent on the rown, nd will indite whether there is potentil for the use of dethed lef ssys in prtil reeding of resistne to snow moulds. Mterils nd Methods Plnt mteril nd inoulum Seven winter whet (Tritium estivum L.) lines were used in this study; the Norwegin ultivr Rid nd reeding line V (Grminor AS, Ilseng, Norwy), the Swedish ultivr (Svlo f Weiull AB, Svlo v, Sweden), nd four snow mould resistnt lines seleted from the USDA World Cerel Colletion; CI9 (originting from Russi), PI7 (originting from Turkey), PI868 (originting from Afghnistn) nd CI6 (unknown origin) (Bruehl, 98). Seeds (three per pot) were sown t m depth in m pots ontining soil (ÔP-jordÕ; L.O.G., Oslo, Norwy). Four experiments were performed. In experiments nd, seeds were sown diretly, while in experiments nd, seeds were pregerminted for dys in petri dishes on the lortory enh prior to seeding. Three different growth regimes produed young non-hrdened plnts (Y), older non-hrdened plnts (O), nd old hrdened plnts (H). In experiments nd, plnts were grown under non-hrdening onditions in growth hmer for two (Y, H) or three (O) weeks t 8 C dy/ C night, 6 h photoperiod nd light intensity of 5 lmol/m /s (HPI uls, Philips, the Netherlnds). H plnts were susequently pled t C with 6 h photoperiod nd light intensity of 8 lmol/m /s (Philips HPI) for weeks. In experiments nd, plnts were grown in greenhouse t A s (59 5 N, E), Norwy, in June nd August, respetively, with 6 h dditionl light of pproximtely lmol/m /s (Powerstr HQI-BT, Osrm GmH, Mu nhen, Germny). The plnts grew fster in experiments nd thn in the first two experiments, nd they were therefore grown for only 8 (Y, H) or (O) dys. The dditionl old hrdening tretment of H plnts ws s in experiments nd. Prior to inoultion of whole plnts nd dethed leves, 5 rndom pots of eh ultivr nd growth regime were smpled for the mesurement of fresh nd dry weights of shoots, inluding the rown. The developmentl stge (numer of leves) ws lso reorded (Tle ). Inoulum of M. nivle (Fr.) Smuels nd Hllet [teleomorph Monogrphell nivlis (Shffnit) E. Mu ller] isolte 5/9 (Plnt Protetion Centre, A s, Norwy) ws prepred from myelil PDA disks stored t )8 C. A myelil suspension ontining geltine ( g/l) ws prepred s desried y Tronsmo (99). Whole plnt test A whole plnt test of resistne to pink snow mould ws performed in ll four experiments. The pots were orgnized into four inoulted nd four non-inoulted loks per inution period. In experiments nd the loks onsisted of one pot of eh whet line nd growth regime, giving totl of pots. This design llows for omprison of oth growth regimes nd whet lines. In experiments nd, plnts of different growth regimes were pled in seprte loks, giving four inoulted nd four non-inoulted loks per growth regime, eh with one pot per whet line. With this design, plnts from different growth regimes nnot e ompred. Inoulted loks were spryed with the myelil suspension (.5 ml per plnt), while the non-inoulted loks were spryed with geltine ( g/l) only. The pots were inuted under moist ellulose wdding nd plsti sheets t C in the drk, the pots elonging to the sme lok eing under one ommon Ôsnow overõ. In n ttempt to otin mximum differentition etween tretments, inution 5

3 6 Ergon nd Tronsmo Tle Size nd developmentl stge prior to inoultion Experiment Experiment Experiment Experiment Growth regime DW DW FW d NL e DW FW NL DW FW NL Y.6 f O H The vlues re verges of seven winter whet lines. Y, young non-hrdened plnts; O, older non-hrdened plnts; H, Y plnts old hrdened for n dditionl weeks. Dry weight (g). d Fresh weight (g). e Numer of leves. f Figures from the sme experiment not followed y the sme letter re signifintly different ording to the Student Newmn Keul test (P.5). periods were hosen ording to the disese progression oserved. In experiments nd, two inution periods were used; 5 nd 7 weeks in experiment, nd 6 nd 9 weeks in experiment. In experiments nd, only one inution period ws used, nd the length of this period differed etween plnts from the three growth regimes. The loks were inuted for 6 (Y), 6.5 (O) nd 5.5 (H) weeks in experiment nd for.5 (Y), 5 (O) nd (H) weeks in experiment. After inution, pots were trnsferred to the greenhouse, where the temperture ws pproximtely 6 C. Additionl light ( lmol/m /s) ws given 6 h dy. Plnts were ut t 7 m ove soil level nd llowed to regrow for week. Plnts were then ut t soil level, nd the fresh nd dry weight of shoot mteril in eh pot ws reorded. Resistne ws lulted s the reltive regrowth, tht is, the rtio etween the fresh weight of inoulted nd non-inoulted plnts. Dethed lef ssys Dethed lef ssys were performed in experiments nd only. Leves were smpled from three replite pots from eh growth regime nd whet line (other pots thn those used for the whole plnt test). The first, seond nd third (if present) lef lde were ut off t the se nd kept etween moist tissue pper. The term Ôlef lssõ will e used for the growth regime nd lef numer omintions. Four lef segments, one from eh of four leves, were pled on.5% wter gr in petri dishes with the dxil side fing up. Four petri dishes per lef lss nd whet line were inoulted with n 8 ll droplet of inoulum, pled on the middle of eh segment. Two petri dishes per lef lss nd whet line were mok inoulted with n 8 ll droplet of geltine ( g/l). Hlf of the petri dishes were inuted t room temperture (RT) on the lenh with light during the dy nd the other hlf were inuted t C in drkness. Oservtions of the length of the wter-soked lesion, the length of the myelil olony on the lef surfe (under inution t RT in light only) nd the presene of wter-soked kground ws mde t dys fter inoultion (d..i.) in petri dishes inuted t RT in light nd t 7, 9 nd nd d..i. in petri dishes inuted t C in drkness. Averge lesion nd olony length ws lulted for eh petri dish nd used in the sttistil nlysis. Dt nlysis Shoot weight prior to inoultion, reltive regrowth, length of wter-soked lesion nd length of myelil olony were sujeted to nlysis of vrine nd orreltion nlysis in the sttistil progrm pkge SAS 8. (SAS Institute In., Cry, NC, USA), using PROC GLM nd PROC CORR. Results Whole plnt test The effet of inoultion on regrowth of plnts fter inution depends on the length of the inution period. In experiment, inoulted plnts hd signifintly lower regrowth thn non-inoulted plnts fter the 7 weeks inution period, ut not fter the 5 weeks inution period. The effet of inoultion fter the long inution period ws present only in Y plnts nd not in O or H plnts (Tle ). In the other experiments the effet of inoultion ws signifint fter ll inution periods nd within ll growth regimes. H plnts hd signifintly etter reltive regrowth, nd thus higher degree of pink snow mould resistne, thn Y plnts while the reltive regrowth of O plnts differed etween experiments (Tle ). In experiment, in whih there ws low disese pressure, O plnts were similr to H plnts. In experiment, in whih there ws higher disese pressure, O plnts were similr to Y plnts or intermedite, depending on the length of inution nd the disese pressure otined. Vrition in resistne mong whet lines ws studied only in dt sets in whih there ws signifint effet of inoultion. This ws the se for Y plnts exposed to the long inution period in experiment, ll growth regimes nd inution periods in experiments. However, Y plnts in experiment ws exluded from nlysis euse ll inoulted plnts died nd therefore this dt set did not provide ny informtion out resistne. Thus, three experiments with interesting dt remined per growth regime

4 Components of Snow Mould Resistne in Whet 7 Tle Anlyses of vrine of plnt regrowth fter inoultion with Mirodohium nivle nd inution under rtifiil snow over Experiment Experiment Experiment Experiment Y (7 d ) O (7) H (7) Y (6) O (6) H (6) Y (9) O (9) H (9) Y (6) O (6.5) H (5.5) Y (.5) O (5) H () Soure d.f. I 69.6*** NS NS 98.***.9** 8.* 5.8*** 7.5** 5.9* 6.***.8*** 67. *** 6. *** 5.*** 5.*** B(I) 6 NS NS NS NS NS NS NS.8* NS.6* NS NS.* NS.* WL 6 5.8*** 7.7*** 6.5*** NS.** 5.*** NS NS 5.8*** 5.5*** NS 9.***.7*** NS NS I xwl 6.** NS NS.7***.* NS NS NS.** 5.5*** NS 5.*** 6.*** 5.95*** NS Fresh weight dt from eh growth regime nd inution period ws nlysed seprtely with the model Yijl ¼ I i + B(I) j +WL l + IxWL il + Error: I, inoultion; B, lok; WL, whet line. B(I) ws used s the error term when testing the effet of I. F-vlues re given. ***P.; **. < P <.; *. < P <.5. Degrees of freedom. Growth regime: Y, young non-hrdened plnts; O, older non-hrdened plnts; H, Y plnts old hrdened for n dditionl weeks. d Inution period (numer of weeks). Tle Resistne to Mirodohium nivle in winter whet plnts grown under different growth regimes Growth regime Experiment Experiment Seven weeks (. d ) Six weeks (.) Nine weeks (.6) Y.5 e.8.8 O.9.7. H Resistne ws mesured s the reltive regrowth; the rtio etween the fresh weight of inoulted plnts nd mok inoulted plnts fter inution nd regrowth. The verge vlues of seven winter whet lines re shown. Y, young non-hrdened plnts; O, older non-hrdened plnts; H, Y plnts old hrdened for n dditionl weeks. Inution period. d Disese pressure otined, lulted s ( ) verge reltive regrowth). e Numers not followed y the sme letter re signifintly different t the 5% level ording to the Student Newmn Keul test (within experiment nd inution period). (verge vlues of the two inution periods in experiment were used s one of the three dt sets). Sttistil nlysis ws performed on eh growth regime seprtely. There were signifint differenes etween whet lines in pink snow mould resistne (reltive regrowth) mong H plnts nd Y plnts. Among O plnts the effet of whet line ws not signifint (P ¼.7). Among the Y plnts, CI9, PI868 nd CI6 hd higher reltive regrowth nd thus higher levels of pink snow mould resistne thn, Rid, V nd PI7 (Fig. ). Among O plnts, CI9 hd the highest reltive regrowth. In experiment, where the verge reltive regrowth in O plnts ws s low s., only plnts of CI9, CI6 nd PI868 survived nd mong these CI6 hd the highest reltive regrowth. Among H plnts, CI9 hd etter reltive regrowth thn Rid nd, while the other lines were intermedite. In experiment, where the verge reltive regrowth of H plnts ws., very good seprtion etween lines ws otined. CI9, PI868, PI7 nd CI6 hd reltive regrowth vlues etween. nd.55, while the reltive regrowth of, Rid nd V were etween.5 nd.. Shoot weight of whet lines prior to inoultion ws not orrelted with reltive regrowth of whet lines in ny of the growth regimes (dt not shown). Dethed lef ssys Symptoms A distint wter-soked lesion developed from the inoultion point on inoulted lef segments, ut not on mok-inoulted lef segments, during inution t C in drkness. The myelium developing on the lef surfe ws very sprse nd diffiult to oserve with the nked eye. In some inoulted leves drk rown nerosis ppered t the inoultion point. At RT nd dylight the symptoms developed muh fster nd the wter-soked lesions were less distint nd often rownish. The myelium

5 8 Ergon nd Tronsmo Reltive regrowth (fresh weight) Y O H CI9 CI9 CI9 CI6 CI6 CI6 Fig. Resistne to Mirodohium nivle infetion in winter whet lines. Y, young non-hrdened plnts; O, older non-hrdened plnts; H, old-hrdened plnts (Y plnts old hrdened for n dditionl weeks). Resistne ws lulted s the rtio etween the fresh weight of the regrowth of M. nivle-inoulted plnts nd the fresh weight of the regrowth of non-inoulted plnts. Averge vlues ross three experiments in whih there were signifint effets of inoultion re shown. Note tht Y, O nd H plnts were not inuted for the sme numer of weeks. No ommon letters ove two olumns indite tht the whet lines re signifintly different ording to the Student Newmn Keul-test (P.5) PI7 PI7 PI7 PI868 PI868 PI868 Rid Rid Rid V V V ws not s sprse nd there ws higher frequeny of neroti retions. Under RT nd dylight, the verge lesion length t dys fter inoultion (d..i.) ws round mm, orresponding to the verge lesion length otined somewhere etween 9 nd d..i. under inution t C in drkness. Wter-soked kground ppered ross some lef segments in response to dethment nd/or inution (present on oth inoulted nd mok-inoulted lef segments). This ourred minly on primry leves of H plnts nd to some extent on seondry leves of H plnts nd primry leves of Y plnts. There ws no onsistent vrition etween whet lines in the frequeny of wter-soked kground. Wter-soked lesion At 7 nd 9 d..i. ( C nd drkness, experiment only) there were signifint differenes etween lef lsses in the length of the wtersoked lesion, ut there were no signifint differenes etween whet lines (Tle ). At d..i., however, there were effets of oth lef lss nd whet line in oth experiments. Under inution t RT nd light there ws lso n effet of lef lss. A signifint effet of whet line ws present only in experiment, presumly due to the higher numer of smples in this experiment. There were no signifint intertions etween lef lss nd whet line under ny of the inution onditions. The mjor vrition in lesion length ws due to differenes etween leves of different order. In generl, leves of higher order were more resistnt thn leves of lower order (Fig. ). When ompring lef lsses ross unhrdened plnts of different ges (Y nd O plnts), the older seondry leves from O plnts hd lrger lesions thn the younger seondry leves from Y plnts under oth inution onditions. However, mong primry leves this reltionship ws opposite, prtiulrly in the erly stges of inution (dt not shown). Thus it ppers tht old primry leves re more resistnt thn young primry leves, while old seondry leves re more suseptile thn young seondry leves. The primry lef of H plnts hd smller lesions thn the primry lef of Y plnts under oth inution onditions (Fig. ). The lesions developing on the seondry leves of H plnts were similr to those developing on the seondry leves of Y plnts. CI9, PI868 nd CI6 hd the smllest lesions under oth inution tretments (Fig. ). PI7, whih ws resistnt in the whole plnt experiments when old hrdened, did not exhiit ny resistne in the dethed lef ssys, not even when looking t hrdened leves only (dt not shown). Lesion length on the seondry lef inuted t C in drkness ws negtively orrelted with snow mould resistne of intt plnts in ll the growth regimes, the strongest orreltion eing mong H plnts (r ¼ ).9) (Tle 5). Under inution t RT with dylight signifint orreltion ws only otined in the primry lef of Y plnts, nd this orreltion ws weker thn tht of the seondry lef inuted t C in drkness. Myelil olony The length of the myelil olony ws reorded under inution t RT with dylight only. There were signifint differenes etween lef lsses (Tle 6). In oth experiments, the length of the myelil olony ws lrger in hrdened plnts thn in non-hrdened plnts (Fig. ). A signifint effet of

6 Components of Snow Mould Resistne in Whet 9 Tle Anlyses of vrine of wter-soked lesion length in dethed lef ssys of resistne to Mirodohium nivle Inution t C in drkness Inution t room temperture with dylight Experiment Experiment Experiment Experiment Soure d.f. d..i. d.f. 7 d..i. 9 d..i. d..i. d.f. d..i. d.f. d..i. LC 7.58*** 7 5.9***.*** 6.5*** 8.5** 7.*** WL 6.5** 6 NS NS.57* 6 NS 6.** LC WL NS NS NS NS NS NS The length of the wter-soked lesion ws nlysed using the model Y ij ¼ LC i +WL j +LC i WL j + Error, where Y ij, length of wtersoked lesion; LC i, lef lss; WL j, whet line, F-vlues re given; NS, not signifint, ***. < P <.; **. < P <.; *. < P <.5. Degrees of freedom. Dys fter inoultion. Fig. Differenes etween lef lsses in the length of the wtersoked lesion developing on lef segments inoulted with Mirodohium nivle in experiment ( nd ) nd experiment ( nd d), mesured fter dys of inution t C in drkness ( nd ) or dys of inution t room temperture ( nd d). Y, young non-hrdened plnts; O, older non-hrdened plnts; H, old-hrdened plnts (Y plnts old hrdened for n dditionl weeks). The numers fter Y, O nd H refer to the primry lef (), seondry lef () nd tertiry lef () on the plnt. Eh olumn represents the verge of seven winter whet lines. No ommon letters ove two olumns indite tht the lef lsses re signifintly different ording to the Student Newmn Keul-test (P.5). The numers elow the x-xis refer to the frequeny of mok-inoulted lef segments tht developed wter-soked kground Wtersoked lesion length (mm) 5 5 () Y Y O H H..75. () Y O H 5 5 () d d d d Y Y O O O H H H.7..8 (d) Y Y O O O H H H. whet line nd n intertion etween whet line nd lef lss ws present in experiment. CI9, PI868 nd CI6, whih hd the smllest lesions, lso hd the smllest myelil olonies. Disussion Whole plnt experiments The three Nordi whet lines were less resistnt to pink snow mould thn the four lines from the USDA World Cerel Colletion. Among Y plnts, CI9, PI868 nd prtilly CI6, were more resistnt thn the Nordi lines, ut PI7 were not. Among O plnts the only signifint differene reveled y the SNK-test ws the differene etween CI9 (resistnt) nd V (sensitive). However, in the experiment with the highest disese pressure only plnts of CI9, CI6 nd PI868 survived, inditing tht these hd higher resistne level thn the other lines. Among H plnts, CI9 ws more resistnt thn nd Rid, nd PI868, CI6 nd PI7 lso tended to e more resistnt thn the Nordi lines. The disese pressure otined for H plnts were low in two of the experiments, ut it ws high in one experiment nd in this experiment there ws very ler differene etween the Nordi lines (reltive regrowth ¼. to.55) nd the other four lines (reltive regrowth ¼.5 to.). The snow mould resistne of CI6, CI9, PI868 nd PI7 hve een demonstrted erlier (Bruehl, 967,,; Litshko et l., 988; Gudet nd Kozu, 99), ut these studies hve only involved old hrdened mteril. Our results demonstrte tht pink snow mould resistne is lso present t low level in some of the resistnt lines (CI9, PI868 nd CI6, ut not PI7) prior to hrdening nd tht hrdening-independent mehnisms

7 Ergon nd Tronsmo Wtersoked lesion length (mm) () () CI9 CI9 CI6 CI6 Fig. Differenes etween whet lines in the length of the wtersoked lesion developing on lef segments inoulted with Mirodohium nivle. Lesion length ws mesured fter dys of inution t C in drkness () or dys of inution t room temperture with dylight (). Eh olumn represents the verge of five lef lsses (Y, Y, O, H nd H) ross experiments nd () or the verge of ll lef lsses in experiment () (no signifint differenes etween whet lines in experiment ). No ommon letters ove two olumns indite tht the verge lesion size of the whet lines were signifintly different ording to the Student Newmn Keul-test (P.5) Tle 5 Correltion etween resistne sores otined with the whole plnt test nd with dethed lef ssys Growth regime First lef d..i. t C in drkness Seond lef of resistne therefore exist. It nnot e ruled out tht some mehnisms my e indued y low temperture during inution, ut suh mehnisms must then e independent of light nd will therefore not involve rohydrte umultion. Gudet nd Kozu (99) ompred frost resistne nd resistne to ottony snow mould in winter whet lines in study inluding some of the resistnt lines PI7 Third lef PI7 PI868 PI868 Rid Rid V V d..i. t room temperture with dylight First lef Seond lef Third lef Y NS ).89** ).78* d NS O NS d ).85* NS d NS d NS NS d H ).76* ).9** NS d NS d NS NS d Resistne of winter whet lines to Mirodohium nivle ws mesured s reltive regrowth in the whole plnt test nd s wter-soked lesion length in the dethed lef ssys. Correltions were tested within eh growth regime nd lef numer. NS, not signifint; **. < P <.; *. < P <.5. Dys fter inoultion. Y, young non-hrdened plnts; O, older non-hrdened plnts; H, Y plnts old hrdened for n dditionl weeks. d Dethed lef ssy performed in experiment only. Tle 6 Anlyses of vrine on myelil olony length in dethed lef ssys of resistne to Mirodohium nivle Soure Experiment Experiment d.f. d..i. d.f. d..i. LC 5.68*** 7 5.8*** WL 6 NS 6.77** LC WL NS.** The length of the myelil olony were nlysed using the model Y ij ¼ LC i +WL j +LC i WL j + Error, where Y ij, length of myelil olony; LC i, lef lss; WL j, whet line; F-vlues re given; NS, not signifint; ***. < P <.; **. < P <.; *. < P <.5. Degrees of freedom. Dys fter inoultion. from the USDA World Cerel Colletion. They oserved negtive orreltion etween these two types of resistne. In ontrst, Pulli et l. (996), found lose ssoition etween ultivr rnking on lotions with snow mould stress nd lotions with freezing stress. The study y Pulli et l. inluded minly Nordi ultivrs nd none of the resistnt lines. Norstr, whih is reltively suseptile ultivr ompred to some of the resistnt lines (Gudet nd Kozu, 99), ppered to e more resistnt thn mny of the Nordi ultivrs in snow mould hmer test performed y Pulli et l. (996). Together this indites tht the resistnt lines seleted from the world olletion hve some speifi snow mould resistne omponents not present in the Nordi mteril nd tht vrition in snow mould resistne in Nordi ultivrs my e lrgely due to vrition in the ility to old hrden. Dethed lef ssys Differenes etween lef lsses Lef lss hd mjor effet on the development of wter-soked lesions in response to inoultion. Most of the vrition ws due to lef position nd/or lef developmentl stge; mong plnts grown under the sme growth onditions the primry leves were more suseptile thn the seondry leves, nd the seondry leves more suseptile thn the tertiry leves. As wter-soked kground ws oserved mostly in primry leves, the oserved differenes etween leves of different order ould e due to lower level of tolerne to dethment nd inution in the primry leves. Unlike the sitution in the whole plnt experiments, growth regime hd less effet on the resistne expressed in the dethed lef ssys. This supports the ide tht the higher resistne of older nd/or hrdened plnts reltive to young non-hrdened plnts depends on rohydrte reserves in the rown, s this type of resistne will not show up in dethed lef ssys. However, s H leves ppered to e more suseptile to dethment nd inution, they my e more resistnt to pink snow mould thn wht is pprent in this study. The myelil olonies on the lef surfe developed fster on hrdened leves thn

8 Components of Snow Mould Resistne in Whet 6 () 6 () Fig. Effet of lef lss nd whet line on the length of the myelil olony developed dys fter inoultion with Mirodohium nivle nd inution t room temperture with dylight in experiment () nd ( nd ). Eh olumn represents the verge of seven whet lines ( nd ) or the verge of eight lef lsses (). No ommon letters ove two olumns indite tht the verge lesion size of the whet lines were signifintly different ording to the Student Newmn Keul-test (P.5) Myelil olony length (mm) () Y O H CI9 CI6 PI7 8 6 PI868 Y Y O O O H H H Rid V on non-hrdened leves, in spite of the ft tht the hrdened leves tended to hve smller wter-soked lesions. Cold hrdening is known to inrese the rigidity nd thikness of ell wlls (reviewed y Fujikw et l., 999). This my mke it hrder for fungl hyphe to digest the ells of hrdened plnts nd use the fungus to grow on the surfe of the leves rther thn inside the lef. This suggests tht hnges in the ell wll rigidity, thikness nd omposition during old hrdening ould e one of the mehnisms y whih old hrdened plnts eome more resistnt to pthogens. Differenes etween whet lines nd orreltion with whole plnt tests At time points where there ws signifint effet of whet line, PI868, CI9 nd CI6 lwys hd the smllest lesions. This shows tht these lines possess not only resistne mehnisms tht re independent of old hrdening s shown in the whole plnt experiments, ut lso mehnisms tht re expressed in dethed leves. The resistne of PI7, however, is dependent of old hrdening nd is lso not expressed in dethed leves. This indites tht PI7 relies on old hrdening-indued hnges in the stem nd rown nd does not hve dditionl hrdening-independent mehnisms or mehnisms expressed in leves. In the dethed lef ssy the length of the myelil olonies lso tended to e smller on leves of PI868, CI9 nd CI6 thn on the other lines. Together the results indite tht these lines re le to inhiit growth of M. nivle oth inside the lef s well s on the lef surfe, possily due to higher tivity of defene-relted proteins (Ergon, 999; Gudet et l.,,,). This mehnism ould represent some of the hrdening-independent resistne possessed y these lines. Results from the dethed lef ssy orrelted well with results from the whole plnt test, ut only for some lef lsses. Wter-soked lesion length on seondry leves inuted t C in drkness orrelted well with whole plnt resistne, prtiulrly mong hrdened plnts. The strong effet of lef order on lesion length hs implitions for sreening of resistne to fusrium hed light nd other diseses s well, nd my pose prolem if the mteril under testing vries in developmentl rte. However, sine the vrition etween leves of different order ould e due to vrition in tolerne to inution, it my e redued with the ddition of kinetin ( senesene retrder) to the wter gr, s done y Dimond nd Cooke (999) nd Browne nd Cooke (). A dethed lef ssy is not likely to pik up vrition in snow mould resistne relted to the rohydrte storge or other hrteristis speifi to the rown, ut detets resistne omponents present in leves. In the reeding of prtil disese resistne ginst snow mould the dethed lef ssy ould e useful in omintion with tests for rohydrte ontent in the rown. Aknowledgements This work ws funded y the Norwegin Reserh Counil. Experiments were performed in the Centre of plnt reserh in ontrolled limte (SKP), Ås, Norwy. Denis A. Gudet nd Helge Skinnes re knowledged for providing seeds of resistnt winter whet lines nd nordi lines, respetively. Gunhild Hgeskl, Grete Lund nd Elis Gusl re knowledged for their exellent tehnil ssistne.

9 Ergon nd Tronsmo Thnks to Ingerd S. Hofgrd for vlule disussions nd omments to the mnusript. Referenes Ae J, Yoshid M. (997) Influene of wter nd ir temperture on old hrdiness of whet. At Agri Hung 5: 9. Årsvoll K. (977) Effets of hrdening, plnt ge, nd development in Phleum prtense nd Festu prtensis on resistne to snow mould fungi. Si Rep Agri Univ Norwy 56:. Bengtsson B. (989) Solule sugr hnges during winter nd resistne to snow mould in winter whet. J Phytopthol :6 7. Browne RA, Cooke BM. () Development nd evlution of n in vitro dethed lef ssy for pre-sreening resistne to Fusrium hed light in whet. Eur J Plnt Pthol :9. Browne RA, Cooke BM. (5) A omprrtive ssessment of omponents of prtil disese resistne to Fusrium hed light using dethed lef ssy of whet, rley nd ots. Eur J Plnt Pthol :7 58. Browne RA, Murphy JP, Cooke BM, et l. (5) Evlution of omponents of fusrium hed light resistne in soft red winter whet germplsm using dethed lef ssy. Plnt Dis 89:. Bruehl GW. (967) Effet of plnt size on resistne to snow mold of winter whet. Plnt Dis Rep 5: Bruehl GW. (967) Lk of signifint pthogeni speiliztion within Fusrium nivle, Typhul idhoensis, nd T. inrnt nd orreltion of resistne in winter whet to these fungi. Plnt Dis Rep 5:8 8. Bruehl GW. (967) Correltion of resistne to Typhul idhoensis, T. inrnt nd Fusrium nivle in ertin vrieties of winter whet. Phytopthology 57:8. Bruehl GW. (98) Developing whets resistnt to snow molds in Wshington Stte. Plnt Dis 66:9 95. Bruehl GW, Cunfer B. (97) Physiologi nd environmentl ftors tht ffet the severity of snow mold of whet. Phytopthology 6: Dimond H, Cooke BM. (999) Towrds the development of novel in vitro strtegy for erly sreening of Fusrium hed light resistne in dult winter whet plnts. Eur J Plnt Pthol 5:6 7. Ergon Å, Klemsdl SS, Tronsmo AM. (998) Intertions etween old hrdening nd Mirodohium nivle infetion on expression of pthogenesis-relted genes in winter whet. Physiol Mol Plnt Pthol 5:. Ergon ÅG. Snow mould resistne in winter whet. Dotor Sientirum Theses 999:. Agriulturl Univiversity of Norwy, 999. Fujikw S, Jitsuym Y, Kurod K. (999) Determintion of the role of old limtion-indued diverse hnges in plnt ells from the viewpoint of voidne of freezing injury. J Plnt Reserh :7. Gudet DA, Chen THH. (987) Effets of hrdening nd plnt ge on development of resistne to ottony snow mold (Coprinus psyhromoridus) in winter whet under ontrolled onditions. Cn J Bot 65:5 56. Gudet DA, Kozu GC. (99) Sreening winter whet for resistne to ottony snow mold under ontrolled onditions. Cn J Plnt Si 7: Gudet DA, Lrohe A, Yoshid M. (999) Low temperturewhet-fungl intertions: A rohydrte onnetion. Physiol Plnt 6:7. Gudet DA, Lrohe A, Ergon Å, Mullin J. () Assoition etween plnt ge nd simple nd omplex rohydrte umultion mong winter whet ultivrs differing in resistne to snow moulds during limtion t low tempertures. At Agri Hung 8:. Gudet DA, Lrohe A, Puhlski B. () Seeding dte lters rohydrte umultion in winter whet. Crop Si : Gudet DA, Lrohe A, Frik M, Huel R, Puhlski B. () Cold indued expression of plnt defensin nd lipid trnsfer protein trnsripts in winter whet. Physiol Plnt 7:95 5. Gudet DA, Lrohe A, Frik M, Huel R, Puhlski B. () Plnt development ffets the old-indued expression of plnt defene-relted trnsripts in winter whet. Physiol Mol Plnt Pthol 6:75 8. Hiilovr-Teijo M, Hnnukkl A, Griffith M, Yu X-M, Pihkski-Munsh K. (999) Snow-mold-indued poplsti proteins in winter rye leves lk ntifreeze tivity. Plnt Physiol : Hon W-C, Griffith M, Mlynrz A, Kwok YC, Yng DSC. (995) Antifreeze proteins in winter rye re similr to pthogenesis-relted proteins. Plnt Physiol 9: Kuwr C, Tkezw D, Shimd T, Hmd T, Fujikw S, Arkw K. () Asisi id- nd old-indued thumtin-like protein in winter whet hs n ntifungl tivity ginst snow mould, Mirodohium nivle. Physiol Plnt 5:. Kiyomoto RK. (987) Cron dioxide exhnge nd totl nonstruturl rohydrte in soft white winter whet ultivrs nd snow mould resistnt introdutions. Crop Si 7: Kiyomoto RK, Bruehl GW. (977) Crohydrte umultion nd depletion y winter erels differing in resistne to Typhul idhoensis. Phytopthology 67:6. Litshko LD, Burpee LL, Goulty LG, Hunt LA, MKersie BD. (988) An evlution of winter whet for resistne to the snow mold fungi Mirodohium nivle (Fr.) Smu & Hll nd Typhul ishikriensis Imi. Cn Plnt Dis Surv 68:6 68. Meier H, Reid JSG. Reserve polyshrides other thn strh in higher plnts. In: Loewus FA, Tnner W (eds), Plnt Crohydrtes I. Intrellulr Crohydrtes. In: Pirson A, Zimmermnn MH (eds), Enylopedi of Plnt Physiology. New Series Vol A., Berlin Germny, Springer-Verlg, 98, pp Nkjim T, Ae J. (99) A method for ssessing resistne to the snow molds Typhul inrnt nd Mirodohium nivle in winter whet inuted t the optimum growth temperture rnges of the fungi. Cn J Bot 68: 6. Nkjim T, Ae J. (996) Environmentl ftors ffeting expression of resistne to pink snow mold used y Mirodohium nivle in winter whet. Cn J Bot 7: Pulli S, Hjortsholm K, Lrsen A, et l. (996) Development nd evlution of lortory testing methods for winterhrdiness reeding. Alnrp, Sweden, SLU, Nordi Gene Bnk, : 68. Tronsmo AM. (98) Predisposing effets of low temperture on resistne to winter stress ftors in grsses. At Agri Snd :. Tronsmo AM. (98) Resistne to the rust fungus Puini poenemorlis in Po prtensis indued y low temperture hrdening. Cn J Bot 6: Tronsmo AM. (986) Host wter potentils my restrit development of snow mould fungi in temperture-hrdened grsses. Physiol Plnt 68: Tronsmo AM. (99) Resistne to winter stress ftors in hlf-si fmilies of Dtylis glomert, tested in ontrolled environment. At Agri Snd, Set B, Soil Plnt Si : Tronsmo AM, Gregersen P, Hjeljord L, Sndl T, Bryngelsson T, Collinge DB. Cold-indued disese resistne. In: Fritig B, Legrnd M (eds), Mehnisms of Plnt Defene. Dordreht, the Netherlnds, Kluwer Ademi Pulishers, 99, pp. 69. Yoshid M, Ae J, Moriym M, Kuwr T. (998) Crohydrte levels mong winter whet ultivrs vrying in freezing tolerne nd snow mould resistne during utumn nd winter. Physiol Plnt :8 6.