Induction of resistance to downy mildew (Peronospora parasitica) in cauliflower by DL-β-amino-n-butanoic acid (BABA)

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1 Plnt Pthology (), Blkwell Siene Ltd Indution of resistne to downy mildew (Peronospor prsiti) in uliflower y DL-β-mino-n-utnoi id (BABA) D. Silué *, E. Pjot nd Y. Cohen Bretgne Biotehnologie Végétle (BBV), Penn-r-prt, F- Sint Pol-de-Léon, Frne; nd Fulty of Life Sienes, Br-Iln University, Rmt Gn, Isrel Of the three isomers of minoutyri id, only the β isomer (BABA) ws effetive in induing resistne ginst Peronospor prsiti, the usl gent of downy mildew, in uliflower (Brssi olere vr. otrytis). A single folir spry pplied to -dy-old seedlings proteted the plnts ginst Peronospor prsiti for t lest dys. Of the enntiomers (R nd S), only the R ws effetive. Resistne ws ompnied y hypersensitive-like retion (neroti spots) whih ws evident efore inoultion. BABA ws systemilly effetive when pplied to the roots, ut filed to protet otyledons djent to treted ones. Unlike other hemil induers, BABA ws effetive when pplied severl hours postinoultion. It hd no effet on P. prsiti spore germintion. In uliflower seedlings, BABA did not indue the umultion of the pthogenesis-relted protein PR-, PR-, PR-, PR- nd PR-. Only treted nd hllenged seedlings umulted PR-. Keywords: AABA, BABA, GABA, indued lol nd systemi resistne, pthogenesis-relted (PR) proteins, priming Introdution Downy mildew, used y the oomyete Peronospor prsiti, is n importnt disese of rssis. Indution of disese resistne y,-dihloroisoniotini id (INA), potssium phosphte (K HPO ) or ienzolr-smethyl (ASM) hs een shown to provide effetive protetion of vrious plnt speies ginst virl, teril, fungl nd oomyete diseses (Kogel et l., ; Ryls et l., ; Vn Loon, ; Reuveni & Reuveni, ). Suh ompounds my serve s good lterntives to lssil pestiides if proven to e sfer nd effiient under field onditions. In uliflower, only limited informtion is ville on indued resistne. Thus, K HPO nd ASM were tested nd shown to e effetive in lortory onditions ginst downy mildew isoltes of different origins nd pthogeniity ptterns (Godrd et l., ; Béot et l., ; Zidi et l., ). DL-β-mino utyri id (BABA) ws shown to indue resistne in mny plnt speies ginst oomyete, fungl, teril, virl nd nemtode diseses (Ok et l., ; Jk et l., ). The mehnisms y whih BABA indues resistne in plnts re still poorly understood. It *To whom orrespondene should e ddressed. E-mil: silue@v.s-rosoff.fr Aepted August is not ler if BABA-indued resistne involves the sliyli id pthwy or nother pthwy. Siegrist et l. () indited tht resistne ginst TMV ws not operting in NhG too mutnt plnts (not umulting sliyli id), wheres Y. Cohen (unpulished results), working with Peronospor tin in too, nd Zimmerli et l. (), working with P. prsiti in Aridopsis thlin, showed tht BABA indued resistne in NhG mutnts nd lso in mutnts impired in the ethylene nd jsmoni id pthwys. Pthogenesis-relted (PR) proteins hve een shown to e good mrkers of indued resistne (IR) in severl plnt speies (Vn Loon, ). In the se of BABA, few reports hve shown tht PR proteins were indued in tomto nd too (Cohen et l., ; Cohen, ; Siegrist et l., ). The im of this study ws to test minoutyri ids for their ility to indue resistne in uliflower ginst different downy mildew isoltes, nd to nlyse whether the indued plnts umulted PR proteins. Mterils nd methods Plnt mterils Experiments onduted in Frne used the uliflower (Brssi olere vr. otrytis) v. Billong. This ession is known to e highly suseptile to downy mildew (Silué BSPP

2 D. Silué et l. et l., ; Godrd et l., ). Seedlings were produed in the greenhouse s previously desried (Godrd et l., ; Béot et l., ). In Isrel, the F hyrid uliflower v. Shugon ws used. Plnts were used t either the otyledon stge or when they rehed the third-lef stge. Chemils, fungl isoltes nd inoultion of seedlings Aqueous solutions of DL-α-(AABA), DL-β-(BABA) nd γ-minoutyri id (GABA) (Sigm, Sint Quentin Fllvier, Frne) were used t onentrtions of,,,, nd mm. Test solutions were spryed on the seedlings ( µl per seedling) nd the treted seedlings were then kept in seled propgtors in the growth inet ( h light, C dy nd C night) for dys. Aienzolr-S-methyl (CGA or BTH) ws lso used t mm s positive ontrol (Godrd et l., ). Seedlings were inoulted ( µl per seedling) with spore suspensions of P. prsiti ( spores ml ). After inoultion, seedlings were kept in the drk in moist hmer for h nd then t C/ C (dy/night) with h photoperiod for dys (Zidi et l., ). In other experiments, roots of -dy-old seedlings were dipped in mm BABA solution overnight, inoulted s desried ove nd then kept in sterile wter. To test whether the IR ws isolte-speifi, three isoltes (FP, FP nd FPA) of P. prsiti (Béot et l., ) were used to inoulte seedlings treted with mm BABA solution. FP is insensitive to metlxyl nd Remiltine (D. Silué, unpulished results). Experiments were onduted twie with seedlings in eh experiment. In Isrel, lol isolte of P. prsiti ws used. The two enntiomers, R-BABA nd S-BABA (kindly supplied y Syngent, Stein, Switzerlnd), were tested on seedlings treted with,, nd mm solutions. They were inoulted dy fter spry. Durtion of protetion nd urtive effiy To test the length of protetion onferred y BABA, - dy-old seedlings were spryed with mm solution nd inoulted either, or dys lter. Seven dys fter inoultion, plnts were sored for disese resistne. To test whether BABA hs urtive tion, seedlings were first inoulted with P. prsiti nd treted with mm BABA either, or dys lter. Three independent experiments were done with seedlings in eh experiment. Systemi tion of BABA To eluidte whether BABA indues systemi resistne, two types of experiment were done. In the first, one otyledon of seedling ( seedlings per tretment) ws spryed with BABA ( mm) nd, dys lter, the whole seedling ws hllenged with isolte FP. Treted nd nontreted otyledons were then sored seprtely for disese resistne. In the seond, roots of -dy-old seedlings were pled in mm BABA solution overnight, trnsferred into sterile wter nd then inoulted. Seven dys lter, seedlings were sored for disese resistne. Disese ssessment The severity of disese ws mesured using six-sore sle (,,,,, ) nd men perentge protetion provided y eh tretment lulted s previously desried (Zidi et l., ). Experiments onduted in Isrel exmined the effet of R-BABA nd S-BABA on sporultion. Sporultion of P. prsiti ws indued y pling the infeted plnts in dew-hmer ( C, drkness) for h. Cotyledons were removed, pled in % ethnol (one otyledon in ml), shken for h nd the numer of spores per otyledon determined with the id of hemoytometer. Assessment of AABA-, BABA- or GABA-indued resistne in three-lef plnts ws done similrly, exept tht oservtion of sporultion ws done on lef diss ( mm dimeter) removed from treted-hllenged leves prepred s desried ove. Pthogenesis-relted protein nlysis To nlyse PR proteins, seedlings were treted with either wter or BABA. Other plnts were treted with either wter or BABA nd then hllenged with P. prsiti (isolte FP). A positive ontrol onsisted of plnts spryed with ASM. Hrvest of seedlings nd their storge, s well s protein extrtion nd SDS-PAGE nlysis, were done s desried y Zidi et l. (). Five ntiodies rised ginst too idi PRs (provided y Dr B. Fritig, IBMP, Strsourg, Frne) were used: PR-(C), PR-(), PR-(Q), PR-(S), nd PR-. Results Level of indued resistne Resistne indued in -dy-old seedlings y AABA, GABA nd BABA is shown in Fig.. AABA indued prtil resistne t mm, ut disese indies (DIs) were not signifintly different from tht of the wter ontrol. GABA ws not effetive in induing resistne. In ontrst, BABA provided high levels of protetion nd t onentrtions of mm ws not signifintly different from tretment with ASM (DI =, no sporultion). Inoulted seedlings treted with mm showed hypersensitive-like neroti spots (not shown). Their growth ws redued y % dys fter tretment. Phytotoxiity ws lso oserved t mm of BABA (not shown), regrdless of the hllenge inoultion. Figure shows the results otined on three-lef plnts treted with mm of either AABA, BABA or GABA nd hllenged dy lter with P. prsiti. Sporultion ws lmost ompletely suppressed in plnts treted with BABA ut less so in plnts treted with AABA. No suppression of sporultion ws oserved with GABA. None BSPP Plnt Pthology (),

3 Resistne to P. prsiti indued y BABA AABA Disese index GABA Figure Dose response nlysis of (from top to ottom) AABA-, GABA- nd BABA-indued resistne in uliflower seedlings ginst downy mildew. seedlings were treted with vrious onentrtions ( mm) of the ompound nd hllenged dys lter with P. prsiti. ASM ( mm) ws inluded for omprison. Disese index ws ssessed dys lter. Vlves re mens of t lest independent experiments nd rs represent stndrd devitions. Tretments with the sme letter re not signifintly different t p =. Wter ASM BABA Conentrtion (mm ) of the three isoltes of P. prsiti differed signifintly in their response to mm BABA. BABA-treted nd nontreted otyledons were sored seprtely for disese expression. BABA-treted otyledons were proteted (DI = ) wheres untreted otyledons were s hevily disesed (DI = ) s in the wter ontrol seedlings (DI = ). BABA ( mm) solution redued disese (DI = ) when pplied to the roots, ompred with wter ontrol tretments with men DI =. Slight phytotoxiity, however, ws oserved on otyledons of the BABA-treted seedlings. Durtion of protetion Seedlings tht were first inoulted with P. prsiti nd treted with BABA few hours lter (dy ) showed signifint redution in disese (DI = ) ompred with wter ontrol (DI = ) (Fig. ). When BABA ws pplied or dys fter inoultion, disese severity ws redued ut less so (DI = ). Further dt otined showed tht myelium oloniztion ws lredy pprent in tissues of suseptile plnts dys fter inoultion (D. Silué unpulished results). When seedlings were inoulted, nd dys fter tretment, DI vlues were, nd, respetively, with only few onidiophores produed (Fig. ). The effet of the R-BABA nd S-BABA pplied t vrious onentrtions on sporultion of the pthogen in otyledons is illustrted in Fig.. R-BABA signifintly redued sporultion t ll onentrtions tested (,, mm), wheres S-BABA hd no signifint effet on sporultion ompred with untreted ontrol seedlings. BSPP Plnt Pthology (),

4 D. Silué et l. No. spores/lef dis Wter AABA BABA GABA Tretment Figure Effets of AABA, BABA nd GABA ( mm) pplied s folir spry to three-lef uliflower plnts on sporultion of Peronospor prsiti. Plnts were inoulted dy fter spry nd spore ounts in lef diss were tken dys fterwrds. Brs represent stndrd devitions. Tretments with the sme letter re not signifintly different t the % proility level. It should e noted tht neither enntiomer hd ny dverse effet on either sporngil germintion of P. prsiti in vitro or penetrtion of the pthogen into the lef tissue t onentrtions of up to mm in vivo (dt not shown). PR protein nlysis BABA-treted unhllenged seedlings hd only tres, or did not umulte PR-, PR-, PR-, PR- (dt not shown) or PR- (Fig. ). All these PRs were lso sent from the wter ontrol seedlings (dt not shown exept for PR- shown in Fig. ). Previous dt (Zidi et l., ) showed ross-retivity of these too ntiodies with uliflower PRs. In ontrst, BABA-treted seedlings, hllenged with isolte FP, showed mssive umultion of PR- t nd dys fter hllenge. At dys posthllenge, BABA nd ASM indued similr umultion of PR- (Fig. ). Wter-treted nd P. prsitiinfeted seedlings lso umulted PR- t dys, ut to lesser extent. Disese index d Dys efore/fter tretment Figure Durtion of protetion onferred y BABA ginst downy mildew (grey rs). Seven-dy-old uliflower seedlings were treted with mm BABA nd hllenged, nd dys lter with isolte FP. In seond experiment, seedlings were first inoulted with isolte FP, then spryed immeditely (), or dys lter with BABA. Disese soring ws done dys fter inoultion. Control seedlings were treted with wter (white rs). Dt re men disese indies otined from three independent experiments with seedlings tested in eh. Brs represent stndrd devitions. Tretments with the sme letter re not signifintly different t the % proility level. No. spores/otyledon Conentrtion (mm) S-BABA R-BABA Figure Effet of R nd S enntiomers of BABA on sporultion of Peronospor prsiti. Seedlings were spryed with mm of either enntiomer nd inoulted dy lter with P. prsiti. The numer of spores per otyledon ws ounted dys lter. Brs represent stndrd devitions. BSPP Plnt Pthology (),

Resistne to P. prsiti indued y BABA Figure Anlysis of pthogenesis-relted PR- protein umultion in uliflower seedlings hrvested nd dys fter tretment.

5 Resistne to P. prsiti indued y BABA Figure Anlysis of pthogenesis-relted PR- protein umultion in uliflower seedlings hrvested nd dys fter tretment. The seedlings were treted with mm BABA (lnes ) or treted with BABA nd inoulted with Peronospor prsiti isolte FP (lnes ). Control seedlings were treted with wter nd either uninoulted (lnes ) or inoulted with FP (lnes ). A positive ontrol onsisted of uninoulted seedlings treted with ienzolr-s-methyl (ASM) (lne ). Lne is the protein size mrker. The ntiser used were rised ginst too PR-. Disussion The present study shows tht of the three isomers of minoutyri id, the R enntiomer of BABA ws the est resistne-induing gent with onentrtions of nd mm, produing omplete protetion (no sporultion) nd without phytotoxiity of uliflower seedlings inoulted with P. prsiti. BABA indued hypersensitivelike retion (HR) in plnts efore hllenge s oserved y different uthors in other plnt speies (Cohen et l., ; Sunwoo et l., ; Siegrist et l., ). GABA filed to indue resistne in uliflower ginst P. prsiti t the onentrtions tested ( mm). Neither phytotoxiity nor hypersensitive nerosis ws oserved on the treted seedlings. Similr filures hve lso een reported in other pthosystems (Cohen et l., ; Siegrist et l., ). These results suggest tht, in generl, GABA nnot indue resistne in plnts. The third isomer of minoutyri id, AABA, hd some effet t the highest onentrtion ut the redution in disese ws not signifintly different from the wter ontrol. Cohen et l. () oserved tht AABA did not redue sporultion of vine downy mildew, Plsmopr vitiol, nd Siegrist et l. () notied tht the numer of Too mosi virus (TMV) lesions ws only slightly redued y AABA. Tretment with AABA on older threelef stge plnts signifintly redued sporultion. Previously, Cohen et l. () nd Cohen () reported similr results in the tomto/phytophthor infestns nd too/peronospor tin pthosystems. Other studies showed tht BABA indued resistne in different plnts ginst oomyete, fungl, teril, virl (for review, see Jk et l., ) nd nemtode diseses (Ok et l., ). BABA pplied up to dys fter inoultion showed some urtive effet. Zimmerli et l. () limed tht BABA hd no urtive effet ginst P. prsiti in Aridopsis thlin when BABA ws pplied s lte s dys fter inoultion nd dy efore sporultion ws ssessed. Suh lte pplition is ineffetive even for systemi fungiides suh s metlxyl (D. Silué, unpulished dt). In generl, urtive effet is not limed for systemi quired resistne or SAR-induing ompounds (Ryls et l., ). BABA ws found to move systemilly in tomto, too nd grpe plnts (Cohen & Gisi, ; Cohen et l., ). This my explin the systemi protetion ginst diseses oserved in tomto (Ok et l., ), pepper (Sunwoo et l., ), grpevine (Cohen et l., ) nd too (Siegrist et l., ). In the uliflower/ downy mildew system reported here, no resistne ws oserved in the untreted otyledon djent to treted one. Redued lterl moility of the ompound my e responsile for this lk of systemi resistne. In ontrst, root pplition did indue systemi resistne, s oserved in tomto (Cohen & Gisi, ), too (Cohen, ) nd grpe (Cohen et l., ). The ropetl trnsport of the ompound vi the xylem is proly responsile for the omplete protetion oserved. In lettue, oth folir nd root tretment with BABA indued systemi resistne ginst downy mildew used y Bremi ltue (D. Silué, unpulished results). BABA t mm proteted uliflower seedlings ginst downy mildew ut used phytotoxiity on most seedlings. At mm it provided omplete protetion without phytotoxiity ut plnt growth ws ffeted ( % redution). In nother study, Godrd et l. () showed tht uliflower plnt growth ws dversely ffeted y ASM. The mehnisms governing resistne indued y BABA re not ler. In tomto, PR proteins were strongly indued y folir or root pplition of BABA (Cohen et l., ), wheres in too they were indued y folir spry ut not y stem injetion or root pplition (Cohen, ). Siegrist et l. () showed tht PR- umulted in BABA-treted too plnts. Zimmerli et l. () showed tht Aridopsis thlin treted with BABA did not produe mrna of the PR- protein. This mrna ws, however, visile in treted seedlings tht were hllenged with P. syringe pv. tomto. The sme uthors stted tht llose deposition, seen fter inoultion of BABA-treted seedlings, ws mjor rrier BSPP Plnt Pthology (),

6 D. Silué et l. ginst P. prsiti. However, they did not look for the presene of PR- in suh seedlings. The present pper shows tht the idi PR-, PR-, PR-, PR- nd PR- proteins did not umulte in BABA-treted uliflower seedlings. However, BABA-treted seedlings hllenged with P. prsiti umulted PR-. Seedlings treted with BABA t lest dys prior to inoultion showed signifintly less disese. Similr results were otined in other pthosystems (Cohen, ; Cohen et l.,, ). Field trils onduted in Isrel indited tht BABA lone or in omintion with ASM nd/or K HPO -like ompound effetively ontrolled downy mildew in grpevine (Reuveni et l., ) nd similr trils re required for P. prsiti on uliflower. Aknowledgements Speil thnks to the CERAFEL (Morlix), ONIFHLOR (Pris), The Conseil Régionl de Bretgne, The Conseil Générl du Finistère (Quimper) nd The Ministère de l Agriulture (Pris) for finnil support. Dov Cohen (Tehnologil Shool of Br-Iln University) is lso knowledged for onduting the sporultion ssys. Referenes Béot S, Pjot E, Le Corre D, Monot C, Silué D,. Phytogrd (K HPO ) indues lolized resistne in uliflower to downy mildew of ruifers. Crop Protetion,. Cohen Y,. Lol nd systemi ontrol of Phytophthor infestns in tomto plnts y DL-β-mino-n-utyri ids. Phytopthology,. Cohen Y,. β-aminoutyri id indues systemi resistne in too ginst Peronospor tin. Physiologil nd Moleulr Plnt Pthology,. Cohen Y, Gisi U,. Systemi trnslotion of C-DL-βminoutyri id in tomto plnts in reltion to indued resistne ginst Phytophthor infestns. Physiologil nd Moleulr Plnt Pthology,. Cohen Y, Nidermn T, Mosinger E, Fluhr R,. β-minoutyri id indues the umultion of pthogenesis-relted proteins in tomto plnts nd resistne to lte light infetions used y Phytophthor infestns. Plnt Physiology,. Cohen Y, Reuveni M, Bider A,. Lol nd systemi tivity of BABA (DL--minoutyri id) ginst Plsmopr vitiol in grpevines. Europen Journl of Plnt Pthology,. Godrd JF, Zidi S, Monot C, Le Corre D, Silué D,. Benzothidizole (BTH) indues resistne in uliflower (Brssi olere vr. otrytis) to downy mildew of ruifers used y Peronospor prsiti. Crop Protetion,. Jk G, Cottier V, Toquin V, Rigoli G, Zimmerli L, Métrux J-P, Muh-Mni B,. β-minoutyri id-indued resistne in plnts. Europen Journl of Phytopthology,. Kogel KH, Bekhove U, Dreshers J, Munh S, Romme Y,. Aquired resistne in rley the resistne mehnism indued y,-dihloroisoniotini id is phenoopy of genetilly sed mehnism governing re-speifi powdery mildew resistne. Plnt Physiology,. Ok Y, Cohen Y, Speigel Y,. Lol nd systemi indued resistne to the root-knot nemtode in tomto y DLβ-mino-n-utnoi id. Phytopthology,. Reuveni R, Reuveni M,. Folir-fertilizer therpy onept in integrted pest mngement. Crop Protetion,. Reuveni M, Zhvi T, Cohen Y,. Controlling downy mildew (Plsmopr vitiol) in field-grown grpevine y β-minoutyri id (BABA). Phytoprsiti,. Ryls JA, Neuenshwnder UH, Willits MG, Molin A, Steiner H-Y, Hunt MD,. Systemi quired resistne. The Plnt Cell,. Siegrist J, Oroer M, Buhenuer H,. β-aminoutyri id-medited enhnement of resistne in too to too mosi virus depends on the umultion of sliyli id. Physiologil nd Moleulr Plnt Pthology,. Silué D, Nsht NI, Tirilly Y,. Differentil responses of Brssi olere nd B. rp essions to seven isoltes of Peronospor prsiti t the otyledon stge. Plnt Disese,. Sunwoo JY, Lee YK, Hwng BK,. Indued resistne ginst Phytophthor psii in pepper plnts in response to DL-β-mino-n-utyri id. Europen Journl of Plnt Pthology,. Vn Loon LC,. Indued resistne in plnts nd the role of pthogenesis-relted proteins. Europen Journl of Plnt Pthology,. Zidi S, Bredette S, Godrd JF, Monot C, Le Corre D, Silué D,. Prodution of pthogenesis-relted proteins in the uliflower (Brssi olere vr. otrytis)-downy mildew (Peronospor prsiti) pthosystem treted with ienzolr-s-methyl. Plnt Pthology,. Zimmerli L, Jk G, Métrux JP, Muh-Mni B,. Potentition of pthogenesis-speifi defense mehnisms in Aridopsis y β-minoutyri id. Proeedings of the Ntionl Ademy of Sienes (USA),. BSPP Plnt Pthology (),

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Archived at Control of Bremi ltue in Fiel-Grown Lettue y DL-3-Amino-n-Butnoi Ai () Y. Cohen 1, A. Bier 1, D. Gotlie 1, E. Ruin 1 Key wors: lettue, inue resistne, owny milew, eliitors, Bremi ltue Astrt DL-3-mino-n-utnoi