Exploring the sensitivity of thermal imaging for Plasmopara viticola pathogen detection in grapevines under different water status

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1 CSIRO PUBLISHING Funtionl Plnt Biology, 28, 35, Exploring the sensitivity of therml imging for Plsmopr vitiol pthogen detetion in grpevines under different wter sttus Mnfred Stoll A,C, Hns R. Shultz A nd Bete Berkelmnn-Loehnertz B A Institut für Weinu und Reenzühtung, Fhgeiet Weinu, Forshungsnstlt Geisenheim, D Geisenheim, Germny. B Institut für Biologie, Fhgeiet Phytomedizin, Forshungsnstlt Geisenheim, D Geisenheim, Germny. C Corresponding uthor. Emil: m.stoll@f-gm.de Astrt. The effet on sptil vriility of lef temperture of inoulting grpevine leves (Vitis vinifer L. v. Riesling) with fungl pthogen (Plsmopr vitiol) ws studied in either well irrigted or non-irrigted plnts. The results from therml imgery were ompred with stomtl ondutne mesured y lef gs exhnge. The high sensitivity of lef temperture to stomtl ondutne mens tht infrred thermogrphy n e used to monitor irregulrities in temperture t n erly stge of development fter either infetion or other stress-relted hnges ffeting the mount of wter trnspired. Contrsting therml effets due to the pthogen ttk were found etween mesurements on well irrigted nd wter stressed plnts. With irrigted vines, pthogen development used n inrese in lef temperture t the point of infetion. In ontrst, under severe wter stress, the inoulted plnts showed lower temperture t the sites of inoultion ompred with the rest of the lef. Anlysis of the sptil nd temporl sensitivity of the temperture profile, otined from the devition of individul pixels from the men long stright line, suessfully distinguished etween helthy nd infeted positions on the lef irrespetive of the plnt wter sttus. Under greenhouse onditions nd for predefined res of the lef surfe, evidene ws lso quired for hrteristi therml responses to e pprent not lter thn 4 dys pst inoultion; tht is, t lest 3 dys efore visile symptoms ppered. Thus, erly nd remote detetion using therml imgery hs the potentil for pre-symptomti dignosis of ioti stress. Additionl keywords: ioti stress, infrred thermogrphy, pre-symptomti dignosis, stomtl ondutne. Introdution Most ultivrs of the world s finest nd widely plnted grpevine speies (Vitis vinifer L.) re suseptile to Plsmopr vitiol, the usl gent of downy mildew. The disese n use severe losses in yield nd qulity. Hene, the protetion of the vines generlly requires numerous fungiide tretments. The penetrtion of this pthogen ours vi stomt, suggesting reltionship etween P. vitiol nd trnspirtion. However, the stimuli nd moleulr mehnisms y whih the pthogen infets the leves re still unknown. Trnspirtion results in evportive ooling of the lef surfe (Gtes 1968) nd there hs een n inresing interest in using non-destrutive therml imgery to determine lef temperture. Thus, lef temperture n e surrogte for estimting stomtl ondutne (Jkson 1986; Jones 1999). Therml imgery revels the temperture distriution on surfes y deteting long wve infrred rdition. Lef temperture vries with evportion nd hene eomes funtion of stomtl perture. The theory is tht reduing trnspirtion y diminishing stomtl perture restrits het dissiption, using n inrese in lef temperture. It is noteworthy tht strong orreltion ws oserved etween stomtl ondutne lulted from lef temperture nd estimtes otined from diffusion porometry (Jones 1999; Grnt et l. 27) s well s estimtions of lef wter potentil y therml imgery (Cohen et l. 25). Lef temperture hs een proven s n inditor of plnt responses to vrious stressors (Cherle nd Vn Der Streten 2), in prtiulr, for grpevine wter vilility (Jones et l. 22; Möller et l. 27; Stoll nd Jones 27). Diminishing trnspirtion y reduing stomtl perture is the min response to drought stress. The reognition of vritions in lef temperture led to the omined use of infrred thermogrphy nd visile imgery to identify plnt stress (Leinonen nd Jones 24). Therml imgery ws pplied to non-destrutive monitoring of plnt s physiologil sttus in response to pthogen ttk (Cherle et l. 24). Furthermore, infrred thermogrphy n e pplied s useful tool for pre-symptomti detetion of downy mildew in uumer. In this ltter exmple, the mximum temperture differene (MTD) within lef turned out to e suitle method to differentite etween infeted nd noninfeted lef tissue (Lindenthl et l. 25; Oerke et l. 26). In grpevine leves, the myelium of the oomyete P. vitiol grows first in the interellulr spe of the spongy mesophyll. When onditions for sporultion re fulfilled, hyphe grow out of stomt on the xil side of the lef, forming speifilly rnhed sporngiophores. However, the symptoms on leves vry with growing onditions nd lef ge. On young leves nd Ó CSIRO /FP /8/4281

2 282 Funtionl Plnt Biology M. Stoll et l. under field onditions, the disese will first pper on the upper surfe s yellow spots referred to s oilspots. Under greenhouse onditions, oilspots hrdly emerge nd sporultion needs to e indued t the end of the inution period y supplying wet nd drk onditions. Allègre et l. (27) hve shown tht grpevine stomt remin open in the drk nd in response to wter defiit fter sprying the entire xil lef surfe with P. vitiol sporngi, therefore deregulting the trnspirtion rte. However, different lef pthogens my ffet trnspirtion in different wys, either using stomt to lose, i.e. the terium Pseudomons syringe pv. syringe (Di Giorgio et l. 1996) nd Too Mosi Virus (Cherle nd Vn Der Streten 21) or to open efore nerosis, i.e. Puini striiformis West. (stripe rust of whet, Smith et l. 1986) nd Cerospor etiol S. (plnt fungus in sugr eet; Cherle et l. 24). Lindenthl et l. (25) hve shown tht Pseudoperonospor uensis exhiited pre-symptomti derese in lef temperture up to.8 C lower thn non-infeted tissue due to norml stomtl opening. Inresed wter loss from dmged ells nd the inility of infeted plnt tissue to regulte stomtl perture used n inrese in trnspirtion rte, thus, ooling the lef surfe. Although oth, pthogen ttk nd wter reltion my show different responses on lef trnspirtion nd lef temperture, s yet there hs een little ttempt to nlyse the omined effets. The im of this study ws to implement therml imgery for ontemporneously monitoring lef temperture of onfined, infeted v. non-infeted res upon the ttk y P. vitiol nd of wter stressed v. well wtered grpevines under greenhouse onditions. Furthermore, experiments were rried out to ompre therml imging nd onventionl lef gs-exhnge mesurements s tools for distinguishing etween oth stressors. The opportunities of using different shpes nd sizes s tools for therml imge nlyses were lso investigted. Mterils nd methods Plnt mteril nd experimentl environment All plnts were grown in greenhouse t the Geisenheim Reserh Center (Geisenheim, Germny). Grpevines (Vitis vinifer L. v. Riesling) were propgted from two node uttings, grown with stndrd potting medi (Einheitserde ED73, Ptzer, Sinntl-Joss, Germny) using pots of 15-m dimeter nd 15-m height, until they hd developed eight leves. Thermogrphi s well s gs-exhnge mesurements were performed on three leves of the sme lef stge per plnt using five replites per tretment. The different irrigtion tretments were imposed one dy efore the inoultion of the pthogen. The tretments were: () ontrol: irrigted to field pity nd non-inoulted vines; () irrigted to field pity nd inoulted vines; () nonirrigted nd non-inoulted vines; (d) non-irrigted nd inoulted vines. Wtering ws pplied either every dy or withheld during the entire experiment. The temperture in the greenhouse ws set to 25 C for dytime nd 18 C for night time. Reltive humidity nd rdition were not ontrolled nd no dditionl lighting ws provided. Environmentl onditions (ir temperture nd RH) in the greenhouse during the experiment were olleted using DtHog (Eijkelkmp, Agriserh Equipment, Gieseek, The Netherlnds). The limti onditions during the experiment re summrised in Fig. 1C, D. Pthogen Sporultion of the pthogen ws indued y pling vines 7 dys fter inoultion with Plsmopr vitiol symptoms in drkened moist hmer overnight t 1% RH. Leves ering zoosporngi were used diretly for inoultion. For this purpose, tp wter ws used to disple the zoosporngi from the dxil side of the lef. The suspension ws djusted to sporngi ml --1 using Thom ell ounting hmer. To void ny run off, the droplets were positioned t three intersetions of min veins of eh lef of the three preseleted leves. Susequently, three drops of 3 ml of sporngi suspension were inoulted to the dxil lef surfe t the intersetions of min veins overing n re of ~.7 m 2 (.1 m 2 ). Control plnts were treted with 3 mlof tp wter in ple of the sporngi solution in the sme onfigurtion s for the inoulum on treted plnts. Immeditely fter inoultion, plnts were overed overnight with wet polyethylene gs t 1% RH in order to provide optimum infetion onditions. Both inoulted nd noninoulted plnts were kept in the greenhouse. Plnts were ssessed dily for downy mildew development y visul rting. On dy 8, plnts were kept in drkened moist hmer over night t 1% RH to indue the sporultion of P. vitiol nd to quntify the visul dmge. Therml imging Therml imges were otined using non-ooled fol plne rry infrred mer (TH712 MX, NEC, Tokyo, Jpn). The instrument opertes in the wvend etween 8 nd 14 mm. The detetor rry hs geometri resolution of 1.58 m rd (32 24 pixels fol plne rry nd field of view lens with minimum fous distne of.3 m). The therml resolution is.6 C nd ury of solute temperture mesurement lose to 2 C. Throughout the entire experiment, the emissivity ws set to.95, whih is within rnge quoted for plnt leves y Idso et l. (1969). The imger ws mounted on tripod nd held ~3 m from the leves, whih remined horizontl. Before ommening the mesurements the mer ws limtised to the greenhouse onditions for t lest 3 min. At the intersetions of min veins of the leves mesurements were onduted on three positions per lef nd on three leves per vine one dy etween 1 nd 123 hours. Imges were nlysed using the Pi-Win-IRIS softwre (es, Munih, Germny). The sptil sensitivity of lef temperture ws studied either long stright line rossing n inoultion re or from irulr re mesurements of inoulted nd non-inoulted lef tissue. For the nlysis of temperture profiles, line of 25 pixels length nd one pixel width over distne of 1.2 m ws used. The devition of eh individul pixel from the men ws lulted for eh intersetion of the min veins. To determine the mximum temperture differene, irle-shped res of.8 m 2 representing the re infeted were used. The mximum temperture differene (MTD) ws

3 Monitoring pthogen ttk under different soil wter sttus Funtionl Plnt Biology 283 Lef temperture ( C) (A) Control Irrigted/inoulted Non-irrigted/non-inoulted Non-irrigted/inoulted Air temperture ( C) (C) g s (mmol m 2 s 1 ) (B) Dys pst inoultion (DPI) VPD (kp) (D) Dys pst inoultion (DPI) Fig. 1. Lef temperture, stomtl ondutne nd limti onditions in the greenhouse fter inoultion with Plsmopr vitiol. (A) Lef temperture. (B) Stomtl ondutne (Vitis vinifer v. Riesling); ontrol, irrigted nd inoulted, non-irrigted nd non-inoulted nd non-irrigted nd inoulted plnts; different letters indite signifint differenes t the 5% level; n = 5 plnts per tretment with three mesurements on eh replite (s.e.). Averge, minimum nd mximum of (C) ir temperture nd (D) vpour pressure differene (VPD) during mesuring period (etween 1 nd 123 hours). identified y lulting the temperture differene of the men of inoulted nd non-inoulted, djent res. Physiologil mesurements Stomtl ondutne of speifi lef positions where inoultion hd tken ple ws mesured with n open photosynthesis system (GFS 3, Wlz, Effeltrih, Germny). The mesurements of stomtl ondutne (g s ) nd of lef temperture were performed on the sme leves, with lef gs-exhnge mesurements immeditely following therml imging. The leves were lmped in lef hmer of uvette re of 3 m 2. Mesurements were tken y seleting uvette temperture nd ron dioxide set to mient. CO 2 onentrtions rnged from 44 to 455 mmol mol --1. Airflow ws fed through the hmer t 75 ml min --1. Mesurements were mde in ll ses etween 1 nd 123 hours. A totl of three leves were mesured for eh plnt. For eh mesurement the instrument ws llowed to stilise s determined y the reltime monitoring within the system. Dt nlysis nd sttistis All experiments were onduted three times within one growing seson. The effets of tretments were nlysed y nlysis of vrine (two-wy ANOVA, with two levels of oth ftors) using R (R Foundtion; University of Auklnd, NZ). Where signifint tretment effet ws demonstrted y the ANOVA (P.5) signifint differenes etween mens were determined with Tukey s test. Results Reltionship etween lef temperture, stomtl ondutne nd wter defiit Using irulr re of the size positioned where the gsexhnge uvette ws pled, oth therml imgery nd lef gs-exhnge mesurements ould distinguish only for differenes of plnt wter sttus ut not for differenes due to pthogen ttk (Fig. 1). In this se, the re of investigtion ws pproximtely four times lrger thn the re of the size of the infetion. However, for ll irrigted plnts, either non-inoulted or inoulted, the lef temperture (Fig. 1A) ompred with the irtemperture (Fig.1C) ws lower. In ontrst, the temperture differene etween leves of non-irrigted plnts nd ir temperture ws less pronouned. In ft, for dys 5 nd 6, when VPD inresed to 1.7 nd 1.58 kp, respetively (Fig. 1D), lef nd ir temperture for non-irrigted vines were

4 284 Funtionl Plnt Biology M. Stoll et l. similr. When irrigted/inoulted nd irrigted/non-inoulted (ontrol) vines were ompred, smll ut minly no signifint differenes in lef temperture were oserved with inoulted leves showing higher temperture. Nevertheless, only 4 dys pst inoultion (DPI) when VPD ws lowest nd on 8 DPI efore the pperne of typil disese symptoms did these tretments differ signifintly (P.5). When inoulted nd noninoulted tretments of non-irrigted vines were ompred the lef temperture differenes were mrginl nd no signifint differenes were found. Stomtl ondutne, s mesured with the lefgsexhnge instrument, indited tht vines with high lef temperture showed lower stomtl ondutne nd vie vers (Fig. 1B). Even so, the lef temperture of irrigted/inoulted vines ws higher nd stomtl ondutne hd een redued ompred with ontrol, in most ses no sttistil differenes were found. Likewise, stomtl ondutne of non-irrigted vines, either non-inoulted or inoulted did not differ signifintly. As result, using lef gs exhnge mesurements or the solute lef temperture of the size of the uvette ould not determine differenes in the pthogen ttk. However, the vrition of lef temperture ompred with ir temperture in the greenhouse remined within 6 C. Under suh onditions, the lef-to-ir temperture differene (T lef T ir ) s funtion of stomtl ondutne ws relted for ll tretments to the extent tht higher lef tempertures were ssoited with lower stomtl ondutne (Fig. 2). There were sttistilly signifint differenes (P <.1) in the slopes of the regressions when ompring inoulted nd non-inoulted tretments inditing different response of the leves due to the pthogen ttk. There ws no sttistilly signifint differene (P =.132) etween the slopes of the regression when irrigted nd non-irrigted tretments were ompred. Reltionship etween lef temperture nd pthogen detetion The intertion etween P. vitiol nd the grpevine lef ws deteted thermogrphilly on 3 or 4 DPI efore ny visul symptoms ourred. Five dys fter inoultion, only 3% of the leves showed visul symptoms. Nonetheless, on dy 8, fter the plnts hd een kept in drkened moist hmer, visul symptoms lerly ppered t ll the inoulted lef positions. The lef temperture response differed etween irrigted nd wter stressed leves (Fig. 3). With irrigted vines inoultion nd pthogen development used n inrese in lef temperture t the point of infetion. In ontrst, under severe wter stress, when stomtl ondutne hd een redued to ~2% of the ontrols, ll of these inoulted plnts showed lower temperture with inresed trnspirtion t the sites of inoultion ompred with the rest of the lef. Mximum temperture differene nd temperture frequeny distriution The mximum temperture differenes (MTD) were derived from irle shped res of ~.8 m 2 ompring inoulted nd T lef T ir ( C) g s (mmol m 2 s 1 ) g s (mmol m 2 s 1 ) 1 (A) 1 (C) Control Irrigted/inoulted 1 y =.25x y =.325x R 2 =.49 P <.1 2 R 2 =.51 P < T lef T ir ( C) T lef T ir ( C) (B) g s (mmol m 2 s 1 ) (D) g s (mmol m 2 s 1 ) Non-irrigted/non-inoulted y =.243x R 2 =.25 P <.1 T lef T ir ( C) Non-irrigted/inoulted y =.354x.7339 R 2 =.53 P <.1 Fig. 2. Lef to ir temperture differene (T lef -- T ir ) s funtion of stomtl ondutne (g s ) of non-inoulted nd Plsmopr vitiol-inoulted vines under different wter sttus. The R 2, P-vlue nd eqution for the fitted liner regression re shown in eh grph (Vitis vinifer v. Riesling; n = 5 plnts per tretment; eh. Dt point shows men nd stndrd error for three replite leves; mesurements following 7 onseutive dys fter inoultion t 24 2 C nd 55 1% RH).

5 Monitoring pthogen ttk under different soil wter sttus Funtionl Plnt Biology 285 (A) ( C) (B) ( C) Fig. 3. Therml imge, presented in grey sle, of defined lolistions fter pthogen infetion on dy 4 fter inoultion. Leves (Vitis vinifer v. Riesling) inoulted with three drops of Plsmopr vitiol sporngi suspension on the lower lef surfe t three intersetions of the min veins of eh lef. (A) Irrigted/inoulted lef. (B) Non-irrigted/inoulted lef. non-inoulted lef tissue (Fig. 4). This size ws lmost equivlent to the size of the surfe of infetion.7 m 2 (.1 m 2 ). For oth non-inoulted tretments, either irrigted or non-irrigted, no signifint differenes were found in MTD etween djent tissue. During ll the experiments, oth tretments showed MTD smller thn.1 C. As result, efore the ourrene of ny visul symptoms the MTD of oth inoulted tretments ws higher thn irrigted or non-irrigted ontrols inditing hnges in lef temperture t the position of inoultion. For the irrigted/ inoulted tretment the MTD ws highest on dy 6 (.89 C) ut for non-irrigted/inoulted vines the MTD during the experiment ws lower with highest MTD on 5 DPI (.31 C). No signifint differenes were found etween inoulted/nonirrigted nd non-inoulted, either irrigted or non-irrigted on dy 7 nd 8 DPI. The generl redution in trnspirtion of nonirrigted vines ompred with irrigted vines nd, hene, the higher lef temperture of the former showed smller MTD ompred with irrigted inoulted vines. Furthermore, redued MTD ( C) Control Irrigted/inoulted Non-irrigted/non-inoulted Non-irrigted/inoulted Dys pst inoultion (DPI) Fig. 4. Mximum temperture differene (MTD) of djent res of inoulted or non-inoulted lef tissue of the intersetions of min veins (inoultion with Plsmopr vitiol). Vitis vinifer v. Riesling; n =5 plnts per tretment (s.e.). Signifint differenes etween tretments re indited y different letters following ANOVA. temperture of inoulted res of non-irrigted plnts ompred with non-inoulted nd djent setions of the lef (Fig. 3) lso result in negtive vlues of the MTD further delining the solute vlues. Thus, smll MTD under wter stress onditions my dissimulte the pperne of pthogen infetion. When nlyses of neighouring pixels rther thn lrger surfes, i.e. irulr shped res either of the size of the uvette or the size of the inoultion were ompred there were tretment effets distinguishing etter for ioti nd ioti stressors. The histogrms (Fig. 5) demonstrte the sptil nd temporl sensitivity of lef temperture profiles tken 2, 4 nd 7 dys fter P. vitiol inoultion. The line of 25 pixels (equivlent of 1.2 m) overed inoulted s well s non-inoulted re. The temperture frequeny distriution ws grouped in lsses of.1 C with eh vlue representing the devition of pixel from the men long the line. For noninoulted leves, 84 nd 92% of the temperture vlues were within rnge of.2 C devition from the men, respetively, for irrigted nd non-irrigted vines. Crossing only minor lef veins, the influene on the temperture profile ws smll. Hene, this temperture flutution my ount for temperture vritions or smll differene of trnspirtion within lef. The resulting urve during the entire experiment pproximtes Gussin distriution. With the ongoing pthogen development, oth irrigted/inoulted nd nonirrigted/inoulted plnts showed inreses in spred (vrine) of the distriution. The sptil nd temporl sensitivity of temperture lerly indites differenes due to the pthogen ttk. On 4 nd 7 DPI the irrigted/inoulted plnts hd etween 39.7 nd 46.1% of pixels outside the threshold of.2 C ompred with ontrol vines of 14.4 nd 14.1% for 4 nd 7 DPI, respetively. A similr sitution ourred for the non-irrigted tretment where 2.4 nd 35.2% of the temperture vlues were out of the threshold for 4 DPI nd 7 DPI, respetively, ompred with non-irrigted nd non-inoulted plnts with 8.7 nd 4.8% eyond the threshold. Disussion Prior to the pperne of visul symptoms, the lol infetion of the pthogen (P. vitiol) ws detetle y infrred thermogrphy. The hnges in lef temperture during downy

6 286 Funtionl Plnt Biology M. Stoll et l. (A) Frequeny (%) Control Non-irrigted/non-inoulted Irrigted/inoulted Non-irrigted/inoulted (B) Frequeny (%) (C) 4 Frequeny (%) m m m m m m m m m m T men T pix ( C) T men T pix ( C) T men T pix ( C) T men T pix ( C) m m Fig. 5. Histogrms showing the frequeny distriution of the temperture differene etween individul pixels nd the men temperture mesured long line (T men, 25 pixels; T pix, one pixel representing.4 m). The dtes of mesurements were (A) 2 dys pst inoultion (DPI), (B) 4 DPI nd (C) 7 DPI.The devition from the men (m) --.2 C, etween--.2 C nd.2 C nd.2 C is expressed (%). Vitis vinifer L. v. Riesling; n = 5 plnts per tretment with three mesurements on eh replite. mildew pthogenesis resulted in onsiderle heterogeneity in sptil nd temporl sensitivity. Under defined onditions in the greenhouse nd for smll, pre-determined positions on the leves, therml imging deteted the onset of n infetion. Under typil greenhouse onditions where rdition sored is less thn 1 W m --2 nd with miniml ir movement, the lef temperture differed from ir temperture y more thn 5 C. This is in greement with the theoretil pproh y Jones (1999). For detetion of wter stress, the omined use of solute lef temperture ompred with different referene surfes (Jones et l. 22; Grnt et l. 26) or the implementtion of different stress indies might e pplile (Möller et l. 26; Grnt et l. 27). However, ft tht needs to e onsidered in our stress monitoring experiments is the intertion of ioti nd ioti ftors, oth of whih influene lef temperture nd stomtl ondutne. The presented results indite tht under non-wter stressed onditions pre-symptomti inrese of temperture ours fter P. vitiol inoultion t the intersetions of min veins of grpevine leves; in ontrst, under severe wter stress the temperture t the inoulted res further delined. This is in greement with Allègre et l. (27), who found tht men temperture of totlly olonised leves delined during wter stress. In their experiments the stomtl deregultion ourred quite erly nd ws oserved within 3 DPI nd 6 DPI. In ddition, these uthors hve shown tht P. vitiol indues n inrese in stomtl perture in the drk s opposed to the expeted stomtl losure. Among the first host retions to fungl infetion re hnges in the semipermele properties of plsm memrnes (Lus 1998) using the umultion of metolites indued y norml phloem trnsport (Guimres nd Stotz 24; Rudelle et l. 25). In the pthogenesis of P. uens, suh shifts towrds strong sinks for nutrients hve een reported, using mssive trnsfer into the developing sporngi (Cohen et l. 1971). In the presene of wter stress due to enhned trnspirtion of infeted tissue nd the loss of memrne integrity, higher xylem trnsport towrds the infeted re my our using stomtl perture to inrese (Lindenthl et l. 25). Aprt from temperture differenes indued y the pthogen itself, the ntomy of the lef my lso e of mjor importne. The heterori hrter of grpevine leves, due to the ourrene of undle sheth extensions, n e seen s n dptive dvntge for this speies (Krourniotis et l. 2). Furthermore, the lerly defined lolistion fter pthogen infetion on grpevine leves monitored y lef temperture differenes within lef my further support this dptility nd hene limit the dmge to the entire lef. In ontrst, homori leves lk this omprtmenttion. Although heterori leves onstrin the effet to smller omprtments, homori leves my not e le to restrit the re infeted. In Cuumis stivus, fst spred of hyphe into djent mesophyll of this homori lef type hs een reported within dys nd visulised thermogrphilly (Lindenthl et l. 25). For this lef type nd under non-wter stress ondition, n

7 Monitoring pthogen ttk under different soil wter sttus Funtionl Plnt Biology 287 overll temperture inrese y lmost 2 C hs een reported s erly s 2 dys fter inoultion (Oerke et l. 26). On smller sle, temperture vritions within lef n give n indition of the sptil vriility of stomtl perture. A non-uniform stomtl losure indued y vrious environmentl effets hs een determined from hlorophyll fluoresene signls nd modelled from onventionl gs exhnge informtion (Downton et l. 1988). Furthermore, stomtl perture n e oordinted within ut not mong lol regions of lef; phenomenon whih hs een termed stomtl pthiness of leves (for reviews, see Weyers nd Lwson 1997; Bukley 25). The mehnism of pthiness is unknown. However, growing evidene suggests tht it my e used y two fetures: stomtl osilltion nd hydruli ondutivity. The norml response of stomt to drkness (Allègre et l. 27) nd wter stress (this study) indued y pthogen intertion etween grpevine leves nd P. vitiol further suggests the importne of hydruli mehnisms involved in pthy stomtl ehviour. Further, even though the re infeted overed pproximtely one fourth of the uvette re, we were not le to lerly detet the pthogen intertion y lef gs-exhnge mesurements in this greenhouse experiment. Compred with the density of pthogen ttk s well s the size of the uvette used in other studies where effets of downy mildew on lef gs-exhnge hve een reported (Moriondo et l. 25), for our own experiments the surfe of inoultion ws little nd the size of the uvette used ws rther lrge. These uthors hve shown tht for well ommodted plnts, P. vitiol infetion redues the funtionl lef re nd the ssimiltion rte nd tht the green tissues wy from the lesion were not ffeted y the pthogen. Hene, the heterori lef hrter n e seen s n importnt dpttion of grpevine leves to vrious environmentl onditions, e.g. pthogen ttk. It hs een suggested tht differenes etween the highest nd lowest temperture within lrge re is perhps useful for deteting pthogen ttk in uumer leves (Oerke et l. 26). In grpevines, when irrigted nd non-irrigted plnts, oth inoulted, were ompred, we found vritions in the MTD with non-irrigted plnts showing smller MTD ompred with irrigted plnts. The lower MTD oserved in non-irrigted/ inoulted plnts ws due to lower overll stomtl ondutne nd trnspirtion of the wter stressed leves, thus, giving higher lef temperture nd smller MTD. A smll MTD under wter stress onditions my therefore dissimulte the pperne of pthogen infetion. Hene, the wter sttus of the plnt needs to e inorported further, lso ffeting temperture differene within the leves. Using the temperture frequeny distriution nd the nlysis of smll distnes n further improve the therml nlyses. If the re infeted is smll, ny inrese in size of the mesuring re redues the sptil informtion of the tehnique used. Even though the therml resolution of the imger we used is stted s.6 C, the ury is quoted s 2 C or n error of 2%, whihever is lrger. In our pplition, however, we re only onerned with reltive vrition in temperture so the therml resolution is the more relevnt. The temperture profile shows irregulrities irrespetive of the wter sttus. Furthermore, suh nlyses do not require the inorportion of environmentl ftors or referene tempertures. Indeed our pproh hs shown potentil to revel lef temperture irregulrities independent of environmentl onditions nd the solute ury of the sensor. By defining temperture thresholds for lef sed on temperture profiles in nturlly ourring situtions, the imge nlysis hs the potentil to sreen for temperture irregulrities s result of smll size pthogen infetions. In onlusion, due to the high sptil nd temporl sensitivity, infrred thermogrphy proved to e suitle tool for sptil nd temporl nlysis on the ourrene s well s on the effets of P. vitiol under different plnt wter sttus nd under ontrolled onditions in the greenhouse. Sine the tehnique interferes s little s possile with the leves nd the environment, therml imging n e seen s n interesting tool in the reserh of plnt responses to stress. The tehniques ville for imge nlyses open up severl opportunities (Jones 24; Cherle et l. 27). Although we nnot onlusively stte wht is the min mehnism involved using the ontrsting effets on lef temperture under vrying plnt wter sttus the temperture frequeny distriution nlysis proposed n e used s n inditor for erly detetion of stress nd providing speifi informtion on the intertion of ioti nd ioti stressors. Therml imgery my eome onvenient pproh to monitor lef temperture nd my e useful for presymptomti forest models. Future studies should im t (i) verifying the imge nlysis tehnique upon pthogen ttk under field onditions nd (ii) utomting the proessing lgorithms of imge nlyses to detet sptil temperture irregulrities. Aknowledgements The expert tehnil support of Silke Klingeiel nd Winfried Shönh (oth Geisenheim Reserh Centre, Setion Phytomediine) s well s the ssistne of In Glogger (University of Applied Sienes, Wiesden), is grtefully knowledged. We thnk Prof. H.G. Jones (Dundee Division of Environmentl nd Applied Biology, U. of Dundee) nd Dr C.J. Sor (South Austrlin Reserh nd Development Institute) for their omments on the mnusript. Referenes Allègre M, Dire X, Héloir MC, Trouvelot S, Merier L, Adrin M, Pugin A (27) Stomtl deregultion in Plsmopr vitiol-infeted grpevine leves. New Phytologist 173, doi: / j x BukleyTN (25) Theontrol of stomt y wter lne. New Phytologist 168, doi: /j x Cherle L, Vn Der Streten D (2) Imging tehniques nd the erly detetion of plnt stress. 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