Influence of glyphosate on Rhizoctonia and Fusarium root rot in sugar beet

Transcription

1 Pest Mngement Siene Pest Mng Si 62: (26) Influene of glyphoste on Rhizotoni nd Fusrium root rot in sugr eet Ree L Lrson, 1, Amy L Hill, 1 Ann Fenwik, 1 Andrew R Kniss, 2 Lind E Hnson 1 nd Stephen D Miller 2 1 USDA-ARS, Sugreet Reserh Unit, Fort Collins, CO, USA 2 Deprtment of Plnt Sienes, University of Wyoming, Lrmie, WY, USA Astrt: This study tests the effet of glyphoste pplition on disese severity in glyphoste-resistnt sugr eet, nd exmines whether the inrese in disese is fungl or plnt medited. In greenhouse studies of glyphosteresistnt sugr eet, inresed disese severity ws oserved following glyphoste pplition nd inoultion with ertin isoltes of Rhizotoni solni Kuhn nd Fusrium oxysporum Shleht. f. sp. ete Snyd. & Hns. Signifint inreses in disese severity were noted for R. solni AG-2-2 isolte R-9 nd modertely virulent F. oxysporum isolte FOB13 on oth ultivrs tested, regrdless of the durtion etween glyphoste pplition nd pthogen hllenge, ut not with highly virulent F. oxysporum isolte F-19 or n isolte of R. solni AG-4. The inrese in disese does not pper to e fungl medited, sine in vitro studies showed no positive impt of glyphoste on fungl growth or overwintering struture prodution or germintion for either pthogen. Studies of glyphoste impt on sugr eet physiology showed tht shikimi id umultion is tissue speifi nd the rte of umultion is gretly redued in resistnt ultivrs when ompred with suseptile ultivr. The results indite tht preutions need to e tken when ertin soil-orne diseses re present if weed mngement for sugr eet is to inlude post-emergene glyphoste tretments. 26 Soiety of Chemil Industry Keywords: Bet vulgris; Rhizotoni solni; Fusrium oxysporum; glyphoste; shikimi id 1 INTRODUCTION Weed ontrol is ostly nd neessry prt of sugr eet prodution, relying hevily on severl postemergene heriide tretments with or without preemergene heriide pplied t plnting. Heriide tretment ost rnges from 171 to 319 $US h 1, 1 with dditionl osts for ultivtion nd hnd lor required to remove weeds left ehind fter heriide pplitions. Reent developments in weed ontrol inlude the use of trnsgeni plnts resistnt to heriides suh s glyphoste. 2 Glyphoste-resistnt (GR) sugr eet ws pproved for prodution in the United Sttes in Use of glyphoste ould provide sugr eet produers with rod-spetrum weed ontrol t frtion of the urrent ost for effiious weed ontrol in this rop. 1 Glyphoste trgets enolpyruvylshikimte-3- phosphte synthse (EPSPS), the enzyme responsile for onverting shikimte to horismte. 4 The inhiition of EPSPS loks the shikimi id (k shikimte) pthwy whih produes preursors neessry for the iosynthesis of romti ompounds, 5 inluding phenyllnine, tyrosine nd tryptophn. 6 The shikimi id pthwy lso gives rise to sliyli id 7 nd phytolexins, 8 oth of whih re importnt in plnt defense proesses. GR sugr eet ws developed through introdution of n Agroterium sp. strin CP4 EPSPS tht is resistnt to glyphoste. 9 Repeted glyphoste pplition impts soil miroil popultion dynmis, 1 nd miroorgnisms hve vrile response to glyphoste. Certin pseudomonds n onvert glyphoste into essentil mino ids, 11 nd some fungi re le to utilize glyphoste s nitrogen 12 or phosphorus 13 soure. Alterntively, glyphoste n hve negtive effets on ertin fungi, nd it inhiits growth of yest y preventing melniztion. 14 The effet of glyphoste use in GR rops on disese severity hs een exmined with vrile results. GR soyen ws more suseptile, following glyphoste pplition, to ertin isoltes of Fusrium in greenhouse 15 nd field 16 tests. Use of glyphoste on GR soyen lso inresed disese severity used y Slerotini slerotiorum (Li) de Bry in some soyen ultivrs ut not in others, 17 ut hd no impt on disese severity in single GR ultivr in nother study. 18 In yet nother study, GR soyen ws more suseptile to yst nemtode infetion thn the sme glyphoste-suseptile (GS) ultivr following glyphoste pplition. 19 Alterntively, use of glyphoste on GR otton redued generl seedling disese severity. 2 To dte, the potentil intertions Correspondene to: Ree L Lrson, USDA-ARS, Sugreet Reserh Unit, Fort Collins, CO, USA E-mil: ree.lrson@rs.usd.gov Both uthors ontriuted eqully. (Reeived 15 Deemer 25; revised version reeived 3 My 26; epted 2 June 26) Pulished online 2 Septemer 26; Soiety of Chemil Industry. Pest Mng Si X/26/$3.

2 Influene of glyphoste on disese inidene in sugr eet of GR plnts, glyphoste nd miroorgnisms hve not een exmined extensively. GR sugr eet is not urrently in ommeril prodution ut is expeted to e in the ner future. Therefore, it is importnt to understnd the potentil impt of glyphoste on sugr eet pthogen intertions efore ommeril prodution egins. The present study exmines the intertion of glyphoste tretment with two mjor soil-orne fungl diseses in sugr eet: Fusrium yellows, used y Fusrium oxysporum Shleht. f. sp. ete Snyd. & Hns, nd Rhizotoni root rot, used y Rhizotoni solni Kühn. Both fungl pthogens re widespred threts to sugr eet prodution in the United Sttes nd rod, using redued yield nd, in the se of Fusrium yellows, inresed impurity in the extrted syrup. 21 Additionl studies sought to determine the sis for the inrese in disese y exmining the impt of glyphoste on (1) fungl growth nd overwintering struture prodution nd viility nd (2) the effiy of EPSPS in GR nd GS sugr eet ultivrs. 2 EXPERIMENTAL METHODS 2.1 Plnt ulture Sugr eet (Bet vulgris ltissim Doell) vrieties B4RR (GR; Betseed In., Kimerly, ID), H16 (GR; Hilleshog, Longmont, CO) nd B4 (GS; Betseed In., Kimerly, ID) were seeded into 2 m dimeter pots ontining psteurized (3 h t 72 C nd.4 r) Metro-Mix 2 (The Sotts Compny, Mrysville, OH). For disese severity nlysis, t 1 week postplnting, seedlings were individully trnsplnted into 12 m pots or 6.7 m dimeter 25 m plnting ones for Rhizotoni root rot or Fusrium yellows tests respetively. For shikimi id nlysis, t 1 week post-plnting, seedlings were trnsplnted to 3.8 m dimeter 21 m deep plnting ones (Steuwe & Sons, In., Corvllis, OR) for plnts 2, 3 nd 4 weeks of ge nd to 6.7 m dimeter 25 m plnting ones when llowed to mture to 6 weeks of ge. Plnts were mintined in glsshouse t 22 ± 5 C, were wtered dily nd were kept under 16 h of dylight to mintin vigorous growth. B4RR hs resistne to Rhizotoni solni AG-2-2 (Stnder JR, privte ommunition). H16 is not reported to hve resistne to R. solni, nd neither ultivr hs resistne to Fusrium yellows. 2.2 Fungl ulture Two isoltes eh of Fusrium oxysporum nd Rhizotoni solni were used in this study. Rhizotoni solni isoltes R-9 (AG-2-2) nd R-1411 (AG- 4) re highly nd modertely virulent respetively, s determined y pthogeniity tests. 22 Fusrium oxysporum f. sp. ete isoltes were F-19 (highly virulent) nd FOB13 (virulent) (Hnson L nd Hill A, unpulished dt). Stok ultures of the isoltes were mintined on potto dextrose gr (PDA) (Beton, Dikinson nd Co., Sprks, MD) t 25 ± 2 C with 8 h of supplementl light per dy. For long-term storge, Fusrium isoltes were stored dried on sterile filter pper t 2 C s desried y Peever nd Milgroom, 23 exept tht isoltes were grown on wter gr (WA) (Beton, Dikinson nd Co., Sprks, MD), nd glss mirofier filter pper ws used. For long-term storge, R. solni isoltes were stored on olonized utolved rley grins t 2 C. 24 Rhizotoni inoulum ws prepred y growing eh R. solni isolte on moist utolved rley grins. Infested rley ws ir dried nd ground ording to the methods of Pierson nd Gskill. 25 Fusrium inoulum ws prepred y trnsferring 4 mm plug of fungl hyphe from the tively growing edge of fungl olony on PDA to hlf-strength V8 gr. 26 Pltes were inuted under 8:16 h light:drk photoperiod t C for 2 weeks. Sterile distilled wter (7.5 ml per plte) ws used to srpe hyphl mteril nd spores from the pltes with sterile ent glss rod. The ontents of 24 pltes were strined through sterile heeseloth, nd the spore onentrtion ws determined with hemytometer nd djusted to pproximtely onidi ml Plnt tretments Glyphoste-potssium 54 g AE L 1 SL (Roundup WetherMx; Monsnto Co., St Louis, MO) ws pplied to sugr eets t rte of.84 kg AE h 1 in n overll spry volume of 18 L h 1 using Reserh Trk Spryer (DeVries Mnufturing, Hollndle, MN) with TeeJet 82 nozzle (Sprying Systems Co., Wheton, IL). Ammonium sulfte (12 mg L 1 ) ws dded to the spry liquid ording to the mnufturer s reommendtion to give inresed glyphoste effiy. The surftnt ontrol tretment onsisted of 1 µl L 1 Tween 2 ontining 12 mg L 1 mmonium sulfte. Tween ws seleted for use s surftnt ontrol s it is non-toxi to fungi t this level nd the rrier from Monsnto ws not ville. Ammonium sulfte ws dded to the ontrol to remin onsistent with the glyphoste tretment. 2.4 Disese severity tests For Rhizotoni root rot tests, ground inoulum ws pplied to the rowns of six-week-old sugr eets [pproximtely.6 ml (2 4 fu) per plnt, 1 plnts per tretment]. Plnts were rrnged in ompletely rndomized design, wtered dily nd mintined t C. At 3 dys post-inoultion, roots were hrvested nd individully rted using sle of (no visile dmge) to 7 (plnt ded nd root ompletely rotted). 27 For Fusrium yellows tests, six-week-old plnts were removed from soil nd rinsed under running tp wter. Roots were soked in Fusrium spore suspension or sterile wter (ontrol) for 8 min with intermittent gittion. Following inoultion, eets were replnted into their originl plnting ones. Plnts were rrnged in ompletely rndomized design, wtered dily nd mintined t C. Plnt symptoms were rted Pest Mng Si 62: (26) 1183

3 RL Lrson et l. weekly for 6 weeks using modified rting sle 28 of to 5 ( = no visile disese; 1 = leves my e wilted, smll hloroti res on lower leves, ut most of leves still green; 2 = leves showing interveinl hlorosis, with entire leves hloroti; 3 = leves with neroti spots or eoming neroti nd dying, ut less thn hlf of the leves ffeted; 4 = hlf or more of the leves ded, plnts stunted, most living leves showing some symptoms; 5 = deth of the entire plnt). After 6 weeks, plnts were hrvested nd roots exmined for vsulr disolortion. The presene of the pproprite Fusrium isolte ws onfirmed through morphologil omprison of isoltes reovered from infeted plnts (t lest two roots per tretment), with originl isoltes used for inoultion. The re under the disese progress urve (AUDPC) ws determined for the 6 week period, llowing for exmintion of disese progress over time when nlyzed with non-destrutive smpling. The experiments were repeted 3 times. 2.5 Glyphoste effet on Fusrium oxysporum f. sp. ete in vitro The methods of Snogo et l. 15 were dpted for determintion of fungl growth, sporultion nd spore germintion in the presene of glyphoste. Briefly, 7 mm gr plugs (three per isolte per glyphoste onentrtion) from the mrgin of five-dy-old stok ultures of F-19 nd FOB13 isoltes were trnsferred to hlf-strength PDA (3 ml per 9 m dimeter petri plte) mended with filter-sterilized (.2 µm) glyphoste (pure tehnil grde; Supelo, Bellefont, PA) t, 1., 4., 8. or 4 µg ml 1, representing negtive ontrol, environmentl rekdown, stndrd pplition rte, identl duplite spry nd spillge respetively, s determined y onentrtion of tive ingredient per unit re. Pltes were inuted (25 ± 2 C; 8 h light) nd mesurements were tken dily until myeli rehed the edges of the pltes (pproximtely 7 dys fter plug trnsfer). Pltes were inuted until 21 dys fter plug trnsfer, t whih time spores were hrvested in 15 ml distilled wter nd onentrtions were determined using hemeytometer. The viility of spores in the presene of glyphoste ws determined s follows: 7 mm plugs of F-19 nd FOB13 were trnsferred to hlf-strength PDA (3 ml per 9 m dimeter petri dish) nd inuted (25 ± 2 C; 8 h light) until 14 dys fter plug trnsfer, t whih time spores were hrvested in 15 ml sterile distilled wter. For eh fungl isolte, 1 µl of spore suspension (three per isolte per glyphoste onentrtion) ws dded to one 9 µl glyphoste dilution (1., 4., 8. or 4 µg ml 1 ) or sterile distilled wter (negtive ontrol). Spores were inuted for 6 h in the drk t room temperture. The totl numer of germinting nd non-germinting onidi ws determined using hemeytometer. Perentge germintion ws lulted y dividing the numer of germinting onidi y the totl numer of onidi in the suspension. The experiments were replited 3 times. 2.6 Glyphoste effet on Rhizotoni solni in vitro The methods of Hrikrishnn nd Yng 29 were used to exmine the effet of glyphoste on R. solni in vitro. Briefly, 7 mm myelil plugs (three per isolte per glyphoste onentrtion) from the mrgin of twody-old stok ultures of R-9 nd R-1411 were trnsferred to the edge of PDA pltes mended with filter-sterilized (.2 µm) glyphoste (, 1., 4., 8. or 4 µg ml 1 ). Bsed on the rpidity of growth, R-1411 plugs were trnsferred to 15 m petri pltes (6 ml PDA) nd R-9 plugs were plted on 9 m pltes (3 ml PDA). Pltes were inuted (25 ± 2 C; 8 h light) nd mesurements of rdil growth were reorded dily until myeli rehed the edges of the pltes (pproximtely 7 dys fter plug trnsfer). R- 9 pltes were inuted for n dditionl 3 weeks to llow for sleroti prodution. R-1411 pltes were lso inuted for 3 weeks, ut no sleroti were produed y R At 4 weeks fter plug trnsfer, ll sleroti were mnully hrvested from pltes using foreps (three pltes per glyphoste onentrtion). Sleroti were dried overnight in lminr flow hood nd the totl weight ws then determined. The following dy, sleroti were ounted nd 5 individul sleroti were trnsferred to PDA (6 ml per 15 m petri plte). Pltes were inuted overnight (25 ± 2 C) nd the numer of germinting sleroti ws reorded. Perentge germintion ws lulted y dividing the numer of germinting sleroti y 5 (totl numer of sleroti plted). The experiments were repeted 3 times. 2.7 Shikimi id nlysis For sugr eet nlyses in experiments testing ge, ll leves were hrvested nd used in extrtions. For tissue-speifi response mesurements, otyledons, treted leves (leves present during glyphoste pplition), newly emergent untreted leves (leves tht emerged fter glyphoste ws pplied) nd roots were hrvested independently nd used in extrtions. For Fusrium studies, hyphe were hrvested from either non-glyphoste-supplemented PDA plte or plte ontining 4 µg ml 1 glyphoste. Shikimi id extrtions from sugr eet nd fungl mteril were rried out ording to Singh nd Shner. 3 Briefly, tissue ws olleted (three plnts per tretment per ultivr; three myeli olletions per tretment per isolte), weighed nd immeditely ground in liquid nitrogen. Following grinding, hydrohlori id (.25 M; 3 ml g 1 fresh weight) ws dded to eh smple. Smples were entrifuged for 3 min t 14 g. Superntnt ws used immeditely in the shikimi id onentrtion ssy. Extrtions were repeted on three independent osions for sugr eet nlyses nd two seprte osions for fungl nlyses. A spetrophotometri pproh for determining shikimi id onentrtions ws employed. 3 Briefly, 25 µl of superntnt olleted from the extrtion ws trnsferred to mirofuge tue ontining periodi 1184 Pest Mng Si 62: (26)

4 Influene of glyphoste on disese inidene in sugr eet id (1 mg L 1 ; 5 µl). Smples were llowed to oxidize t room temperture for 3 h. Following the inution, sodium hydroxide (1 M; 5 µl) nd glyine (.1 M; 3 µl) were dded to eh smple. The smple ws vortexed riefly nd the OD t 38 nm ws determined immeditely. A stndrd urve ws estlished using.5 6 µmol shikimi id (Sigm, St Louis, MO) dded to periodi id inuted under similr onditions. 2.8 Sttistil nlyses All sttistil nlyses for disese severity tests were performed using SAS (SAS Institute, Cry, NC). Dt from experiments were omined, nd liner model ws fit to these dt using PROC MIXED. Experiment ws treted s rndom effet. Beuse vrine in disese indies differed mong the isoltes, the group option in the repeted sttement ws used to fit models tht llowed heterogeneity of vrine mong ftor omintions. Sine the overll model fit indited tht there were signifint isolte effets nd signifint intertions etween isoltes nd other tretment ftors, nlyses were run on isoltes seprtely. Men seprtions for disese indies or AUDPC for eh isolte were onduted using the mro PDMIX8.SAS 31 with Bonferroni djustments to ontrol the type I error rte. All experiments testing diret effets of glyphoste on F. oxysporum nd R. solni were nlyzed with one-wy ANOVA. Mens seprtions were onduted with Tukey Krmer post ho test (α =.5) using PDMIX8.SAS. For shikimi id experiments, dt were nlyzed using nlysis of vrine in the generl liner models proedure of SAS. Sine no differene etween experiments ws deteted for shikimi id nlyses, experimentl dt were omined. Men seprtions etween tretments y tissue type for four-week-old sugr eet were onduted y Fisher s lest signifint differene (LSD) test (α =.5) using SAS. 3 RESULTS 3.1 Disese severity The GR plnts showed no visile dmge from the glyphoste tretments. There were no signifint differenes in folir yellowing or nerosis etween glyphoste-treted nd ontrol plnts in the sene of pthogens. There ws no signifint effet of experimentl replition on disese severity, nd therefore dt from the three experiments were pooled nd nlyzed together. For Rhizotoni root rot tests, nlysis of vrine showed two min effets (ultivr nd isolte) tht were highly signifint nd one min effet tht ws orderline signifint (spry, P =.519). Of the two-wy intertion effets, ultivr spry, spry isolte nd ultivr isolte were ll highly signifint. Additionlly, the three-wy intertion effet of ultivr spry isolte ws highly signifint. All other intertion effets were not sttistilly signifint (Tle 1). Therefore, no signifint effet of the mount of time etween glyphoste pplition nd pthogen inoultion ws deteted in this test. In the sene of glyphoste, sugr eet ultivr B4RR (Rhizotoni root rot resistnt) hd signifintly lower disese severity rtings thn the other sugr eet ultivr (H16) when the AG-2-2 isolte, R-9, ws used, ut B4RR ws not signifintly different from H16 in disese severity used y the AG-4 isolte, R-1411 (Fig. 1(A)). However, following tretment with glyphoste, oth sugr eet ultivrs showed sttistilly indistinguishle disese levels for eh of the two isoltes (P.5), nd the rting for ultivr B4RR ws signifintly higher thn the surftnt tretment when hllenged with isolte R-9 regrdless of the durtion etween glyphoste tretment nd pthogen hllenge. In Fusrium yellows tests, minimum evidene of disese ws oserved in non-inoulted ontrols, ut oth pthogen isoltes used detetle disese (Fig. 1(B) nd (C)). The presene of the pproprite Fusrium isoltes ws onfirmed nd the ontrol roots were free from Fusrium. As no sttistilly signifint effet of experiment ws oserved, dt for ll experiments were nlyzed together. All min effets (spry, ultivr, isolte nd dy) were signifint. The two-wy intertion effets of spry isolte, ultivr isolte, dy ultivr nd dy isolte were ll signifint. The two-wy intertion of dy spry ws orderline signifint (P =.674). The only three-wy intertion with signifine ws dy ultivr isolte (Tle 1). Isolte F-19 ws more virulent thn FOB13. F-19 killed 4% or more of the plnts within the 6 week rting period. FOB13 did not use ny plnt deth during the time period exmined with ny of the tretments. Disese severity used y isolte FOB13 ws signifintly higher following glyphoste tretment thn on surftnttreted plnts for oth ultivrs regrdless of the durtion etween tretment pplition nd pthogen inoultion (Fig. 1(B) nd (C)). There were no sttistilly signifint differenes etween plnts treted with isolte F-19 for either spry tretment t either time, lthough in the plnts treted t 1 dy fter sprying the two ultivrs differed signifintly in disese severity used y F-19 (Fig. 1(B) nd (C)). Cultivr H16 showed signifintly higher AUDPC thn ultivr B4RR for oth tretments with F-19 when inoulted 1 dy fter sprying, ut there ws no signifint differene etween the two ultivrs in verge AUDPC when they were inoulted t 9 dys fter sprying. 3.2 Fungl growth To determine the effet of glyphoste on fungl growth, the two isoltes of F. oxysporum (FOB13 nd F-19) nd R. solni (R-1411 nd R-9) were exmined in vitro. All isoltes showed similr rte of growth t glyphoste onentrtions etween 1 Pest Mng Si 62: (26) 1185

5 RL Lrson et l. Tle 1. Anlysis of vrine for min nd intertion effets of glyphoste on disese severity used y Rhizotoni solni nd Fusrium oxysporum in two glyphoste-resistnt sugr eet ultivrs Pthogen Effet F vlue P > F Rhizotoni Spry solni Cultivr Cultivr spry Isolte d <.1 Spry isolte 1.5 <.1 Cultivr isolte 17.9 <.1 Cultivr spry isolte Dy e Dy spry Dy ultivr Dy ultivr spry Dy isolte Dy spry isolte Dy ultivr isolte Dy ultivr spry isolte Fusrium Spry <.1 oxysporum f Cultivr Cultivr spry Isolte g 97.6 <.1 Spry isolte <.1 Cultivr isolte Cultivr spry isolte Dy Dy spry Dy ultivr Dy ultivr spry Dy isolte Dy spry isolte Dy ultivr isolte Dy ultivr spry isolte Results re from rtings tken t 4 weeks post-inoultion, using rting sle of to 7. Two spry tretments were used, either stndrd field pplition rte of glyphoste (.84 kg AE h 1 ) or surftnt ontrol. Two sugr eet ultivrs were used: B4RR nd H16. Cultivr B4RR hs resistne to Rhizotoni solni AG-2-2. Both re suseptile to Fusrium oxysporum. d Two isoltes of R. solni were used: R-1411 (AG-4) nd R-9 (AG-2-2). Control ws sterile ground rley. e Plnts were spryed either 1 dy or 9 dys efore inoultion with fungl isoltes. f Results re from verge re under the disese progress urve (AUDPC), representing rtings tken weekly for 6 weeks postinoultion using rting sle of to 5. g Two isoltes of F. oxysporum were used: FOB13, modertely virulent, nd F-19, highly virulent. Control ws sterile wter. nd 4 µg ml 1 when ompred with the negtive ontrol (P.5). At glyphoste onentrtion of 4 µg ml 1 there ws signifint inhiition of fungl growth for ll isoltes tested; however, the inhiition ws inomplete (17 nd 11% inhiition for F. oxysporum isoltes FOB13 nd F-19 respetively, nd 3.7% nd 25% inhiition for R. solni isoltes R-1411 nd R-9 respetively). 3.3 Prodution of overwintering strutures Spore prodution nd germintion were determined for the two isoltes of F. oxysporum in the presene of glyphoste. FOB13 showed signifint derese in spore prodution nd germintion t 8 µg ml 1 when ompred with the ontrol (Tle 2). However, spore prodution ws similr to the ontrol from the higher glyphoste onentrtions (12 µg ml 1, 2 µg ml 1 nd 4 µg ml 1 ). Spore prodution remined omprle with the ontrol levels t ll glyphoste onentrtions for F-19, ut spore germintion ws signifintly redued t glyphoste onentrtion of 4 µg ml 1. Spore prodution remined similr to the ontrol for FOB13 nd F-19 t glyphoste onentrtion of 4 µg ml 1 (dt not shown). Spore germintion showed sttistilly signifint redution t 1 mg ml 1 glyphoste for oth FOB13 nd F % nd 13.% respetively. Sleroti prodution, weight nd viility were nlyzed in the presene of glyphoste for R. solni R-9. Between nd 4 µg ml 1 of glyphoste there ws no signifint (α =.5) effet of glyphoste on sleroti prodution, with sleroti rnging from 96 to 14 per plte. However, t 4 µg ml 1 of glyphoste there ws signifint (α =.5) 6% redution in sleroti prodution ompred with the non-mended medi ontrol. There ws no signifint (α =.5) differene in sleroti weight or sleroti viility t ny of the glyphoste onentrtions tested. In tests exmining shikimi id levels in the presene of glyphoste, no differene ws noted etween F. oxysporum isoltes FOB13 nd F-19 when grown in 4 µg ml 1 glyphoste-supplemented medi or n unmended ontrol (dt not shown). Tle 2. Effet of glyphoste on spore prodution nd germintion of two isoltes of Fusrium oxysporum Glyphoste Spore prodution, Spore germintion (%), onentrtion (µg ml 1 ) FOB 13 d F-19 d FOB 13 F n.d. e n.d. n.d n.d. n.d. n.d d 16.9 Totl spore ount t 4 weeks fter myelil gr plug trnsfer. Numers followed y the sme letter re not sttistilly different s determined y Tukey Krmer post ho test (P <.5). Experiments were repeted 3 times. Perentge of totl spore ount following 6 h of exposure to glyphoste. d FOB13 = Fusrium oxysporum f. sp. ete isolte FOB13; F-19 = Fusrium oxysporum f. sp. ete isolte F-19. e n.d. = no dt Pest Mng Si 62: (26)

6 Influene of glyphoste on disese inidene in sugr eet Averge disese rting A B4RR surftnt B4RR glyphoste H16 surftnt H16 glyphoste Control/1 Control/9 R-1411/1 R-1411/9 R-9/1 R-9/9 Isolte/dy of hllenge Averge AUDPC B Control Fo13 F19 Averge AUDPC C Control FOB 13 F-19 Isolte Figure 1. Response of two glyphoste-tolernt sugr eet lines to different fungl isoltes in the presene or sene of the heriide glyphoste. All results re the verge rtings of 3 plnts. (A) Plnts inoulted 1 dy or 9 dys fter glyphoste or surftnt ontrol pplition with two different Rhizotoni solni isoltes, R-9 (AG-2-2) nd R-1411 (AG-4), in ground rley. The ontrol plnts were treted with sterile ground rley. Eh root ws rted for disese on (no disese) to 7 (ompletely rotted) rting sle. (B) Plnts inoulted with Fusrium oxysporum isoltes F-19 or FOB13 t 1 dy fter glyphoste or surftnt tretment. (C) Plnts inoulted with Fusrium oxysporum isoltes F-19 or FOB13 t 9 dys fter glyphoste or surftnt tretment. Vlues re verge re under the disese progress urve (AUDPC) of six weekly rtings ( 5 rting sle). Control tretment for Fusrium experiments ws inoultion with sterile wter. Sine nlysis of vrine showed sttistilly signifint effet of time of glyphoste pplition on disese severity for F. oxysporum, the dt were nlyzed seprtely. Brs on eh grph for given isolte with the sme letter re not signifintly different y men seprtion with Bonferroni djustments (P =.5). 3.4 Effets of glyphoste tretment on shikimi id levels in glyphoste-resistnt nd suseptile sugr eet ultivrs The impt of glyphoste pplition on sugr eet physiology in GS nd GR sugr eet ultivrs ws monitored y mesuring the umultion of shikimi id following glyphoste pplition. The nlysis of vrine showed tht ll min effets (ultivr, tretment, ge nd dy), two of the twowy intertions (ultivr tretment nd tretment dy) nd the three-wy intertion (ultivr tretment dy) were highly signifint (Tle 3). However, the two-wy intertion of plnt ultivr ge ws not signifint. Anlysis of the min effets y ultivr showed tht tretment, ge nd dy were ll highly signifint regrdless of glyphoste tolerne (Tle 4). There ws signifint inrese in shikimi id following oth surftnt nd glyphoste tretment (P <.1) for ll ultivrs, ut the rte of umultion ws gretest for the glyphoste-treted Tle 3. Anlysis of vrine ross ultivrs for min nd intertion effets of glyphoste on shikimi id levels in glyphoste-resistnt nd glyphoste-suseptile sugr eet ultivrs F P > F Cultivr 8.47 <.1 Tretment <.1 Age Dy <.1 Cultivr tretment 92.6 <.1 Tretment ge Tretment dy 4.49 <.1 Cultivr tretment dy GS vriety, B4 (dt not shown). The differene etween shikimi id levels in oth GR vrieties when treted with glyphoste versus surftnt ontrol ws only signifint in three- nd four-week-old eet smples (dt not shown). The growth rte of the GS vriety ws negtively ffeted with glyphoste Pest Mng Si 62: (26) 1187

7 RL Lrson et l. Tle 4. Anlysis of vrine for min effets y ultivr to exmine umultion of shikimi id following glyphoste pplition on glyphoste-suseptile nd glyphoste -resistnt sugr eet ultivrs Sugr eet ultivr B4 B4RR H16 F P > F F P > F F P > F Tretment < < <.1 Age < <.1 Dy d < < <.1 B4 is GS sugr eet ultivr nd B4RR nd H16 re oth GR sugr eet ultivrs. Eh ultivr ws treted with stndrd field pplition rte of glyphoste (.84 kg AE h 1 ) or surftnt ontrol. Shikimi id umultion ws monitored in ll plnt ultivrs t 2, 3, 4 nd 6 weeks of ge. d Leves were smpled t, 1, 2, 3, 4, 7, 9, 11, nd 14 dys post-pplition of glyphoste or the surftnt ontrol. The rte of shikimi id umultion (slope of line over 14 dys of smpling) etween tretments ws (B4), (B4RR) nd (H16) for surftnt ontrol nd glyphoste pplition respetively. Fresh weight (g) Fresh weight (g) Fresh weight (g) surftnt glyphoste m = m = m =.1288 m = A B C m =.912 m = Dys post-pplition Figure 2. Lef tissue umultion in glyphoste- or surftnt-treted GS, B4 (A), nd two GR ultivrs of sugr eet, B4RR (B) nd H16 (C). Dt re the men of three independent replitions, ontining three smples per replite. The slope of the line, representing tissue umultion, is noted s m. Differenes etween slopes for GS ultivr re signifint (P <.1), wheres the slopes for GR ultivrs re not signifintly different. tretment, nd the ffet on the GR vrieties ws negligile (Fig. 2). Shikimi id umultion ws monitored ross tissue types for oth the GR nd GS vrieties (Fig. 3). Levels of shikimi id were signifintly different etween glyphoste- nd surftnt-treted plnts for ll tissue types in the GS ultivr (P <.1) nd for ll tissue types exept the roots (P =.727 nd.117 for B4RR nd H16 respetively) for the GR vrieties. In ll instnes, the level of shikimi id umultion ws higher for the GS vriety thn for the GR vrieties following glyphoste pplition. In the GS vriety, shikimi id inresed rpidly nd rehed plteu over time. In omprison, the response to glyphoste tretment ws delyed nd trnsient in the GR ultivrs. The gretest nd most prolonged differene in response to glyphoste ppered in the newly emergent lef tissue for the GR ultivrs. The shikimi id level ws higher following glyphoste tretment t ll smpling time points. 4 DISCUSSION AND CONCLUSIONS There hve een severl reports of inresed disese inidene following glyphoste pplition in GR rops. 15,16,19 In the present study, the Rhizotoni root rot resistnt GR ultivr (B4RR) hd signifint inrese in disese severity when inoulted with n AG-2-2 isolte of R. solni, ut not with n AG- 4 isolte, inditing possile loss of resistne following glyphoste pplition. The ultivr B4RR hs resistne to AG-2-2 ut not to AG-4 whih typilly infets seedlings. 32 This inresed disese severity is proly not fungl-medited response. There ws no signifint differene in growth rte in the presene of glyphoste for either isolte tested. Additionlly, isolte R-9 produed equl numers of equivlently sized sleroti in the presene of glyphoste s ompred with wter-mended ontrol. This is in ontrst to other studies where, in the presene of glyphoste, other R. solni isoltes produed more, smller sleroti following exposure to glyphoste without ffeting viility. 29 Alterntively, the inresed disese severity used y R-9 ould reflet possile ultivr- or isolte-speifi response to glyphoste tretment. Other reserhers hve reported inresed disese severity in some soyen ultivrs with some pthogeni isoltes nd not in others. 17 The inrese in disese severity t oth times tested nd on oth ultivrs for one of the 1188 Pest Mng Si 62: (26)

8 Influene of glyphoste on disese inidene in sugr eet shikimi id (µmol) shikim id (µmol) shikimi id (µmol) A B4 surftnt B4 glyphoste B4RR surftnt B4RR glyphoste H16 surftnt H16 glyphoste B C n.d d Dys post-pplition Figure 3. Shikimi id umultion in GR (B4RR nd H16) nd GS (B4) ultivrs of sugr eet following glyphoste or surftnt pplition. Shikimi id onentrtions were monitored in (A) otyledons, (B) treted leves nd (C) newly emergent, untreted leves. Dt re the men of three independent replitions ontining three smples per time point per tretment. Anlysis of vrine for eh ultivr ross time showed sttistilly signifint differenes in shikimte levels etween tretments in severl tissue types (P <.1 for ll B4 tissue types; P <.1 for otyledons, treted leves nd newly emergent leves for B4RR nd P =.727 for roots; P <.1 for otyledons nd newly emergent leves for H16 nd P <.5 nd.177 for H16-treted leves nd roots respetively). Columns with the sme letter designtion re not sttistilly different s determined using Fisher s lest signifint differene test (P =.5). n.d. = no dt for those time points. F. oxysporum isoltes tested is onsistent with reports tht glyphoste tretment n inrese disese y Fusrium in oth GS 33 nd GR 15 rops. Synergism etween glyphoste nd some soil-orne pthogens, 34 inluding Fusrium, 35 hs een oserved nd n e expressed s inresed disese severity when glyphoste tretment is omined with pthogen exposure. 36 From in vitro studies it ws pprent tht the two isoltes of F. oxysporum were tolernt to glyphoste. Levels of glyphoste 1 times the reommended field pplition rte were neessry to hieve even slight growth inhiition. The slight derese in spore prodution with FOB13 in the presene of 8 µg ml 1 glyphoste ws most likely n experimentl rtift, sine further exmintion t 12 nd 2 µg ml 1 showed no differene from tht of the ontrol (dt not shown). Additionlly, the two isoltes ppered to hve similr physiologil response to glyphoste, sine shikimi id levels in the presene of 4 µg ml 1 glyphoste were similr for oth isoltes ompred with negtive ontrol (dt not shown). Finding sttistilly signifint inrese for only one of the two F. oxysporum isoltes is onsistent with reports for other pthogens tht isoltes n vry in their response to heriide tretments. 15,17 Furthermore, it my e tht, with the more virulent pthogen (F-19), the disese ws so severe tht no signifint differenes in disese ould e deteted with the methods used. The lk of evidene for fungl-medited mode of inresed disese severity led to the exmintion of sugr eet physiology following glyphoste pplition. Glyphoste inhiits the shikimi id pthwy whih gives rise to essentil romti mino ids nd plnt defense ompounds. 6 8 Inhiition of the shikimi id pthwy ould result in redued defensive pilities of sugr eet. This hs een oserved in GS tomto treted with sulethl dose of glyphoste. 36 Applition of the heriide leds to n inrese in shikimi id, 37 whih serves s n inditor of EPSPS effiy nd s mesure of the impt of glyphoste pplition on physiology. In preliminry studies using sugr eet seedlings, signifint differenes in shikimi id levels etween glyphoste Pest Mng Si 62: (26) 1189

9 RL Lrson et l. nd surftnt negtive ontrols were oserved (dt not shown). In ll ges of sugr eet (two, three, four nd six weeks old) tested, the rte of shikimi id umultion ws greter following glyphoste pplition thn following surftnt tretment in oth GR ultivrs. This suggests rndom insertionl event during trnsformtion, or the kground of the trnsformed mteril is not responsile for the sensitivity to glyphoste pplition nd the impt of glyphoste is not tied to prtiulr development stge. As expeted, shikimi id levels following glyphoste pplition were signifintly lower for the GR ultivrs thn for the GS ultivr. Glyphoste hd the gretest impt on tively growing tissue in sugr eet, whih is expeted sine it is phloem moile nd is trnsported to 38 4 metoli sinks suh s meristemti tissue. In tissue type studies, newly emergent, tively growing tissues hd the gretest, most prolonged shikimi id umultion. However, in every tissue type, umultion ws trnsient. Nevertheless, slight inhiition of EPSPS my inhiit plnt defenses in the sme mnner s tivtion of plnt defenses ffets plnt growth. 41 It is possile tht the inhiition of EPSPS is sustntil enough to limit sustrte vilility for onversion into sliyli id nd phytolexins without negtively ffeting growth, sine growth rte ws not ffeted y glyphoste in GR vrieties. Future studies need to ddress the impt of shikimi id umultion on the prodution of key plnt defense ompounds, inluding phytolexins. The indution of the phytolexin etvulgrin hs een reported in sugr eet roots inoulted with R solni, 42 ut the role of phytolexins in Fusrium yellows is not known. Alterntively, stress s result of heriide pplition hs een ssoited with inreses in disese. 39 Glyphoste is roken down into minomethylphosphoni id in GR soyen; this is phytotoxi ompound 43 tht lso my e soure of plnt stress in sugr eet. Thus, exmintion of glyphoste movement, onentrtion nd metoli yprodut prodution in sugr eet would provide lues out whether phytotoxiity reltes to disese severity. Additionlly, lthough the evidene from in vitro studies suggests tht the inrese in disese is not fungl medited, in plnt oservtions of lk of effet of glyphoste on fungl pthogens ould help onfirm tht inresed disese severity is result of hnge in plnt metolism. With the introdution of GR sugr eet into ommeril prodution on the horizon, the response of GR sugr eet to glyphoste needs further investigtion. The results of the present experiment suggest tht the response to glyphoste differs y ultivr nd pthogen isolte. The ultivrs used in this study re not the ultivrs tht re now urrently ville for ommeril prodution, nd therefore these new events should e inluded in these roder-spetrum studies. Nevertheless, ultivr y isolte disese severity study should e onduted for reeding lines eing used or onsidered for GR sugr eet prodution. For F. oxysporum there ws sttistilly signifint response to the mount of time etween glyphoste pplition nd pthogen inoultion (Tle 1). While no signifint diret effet on the intertion of spry tretment nd disese severity ws oserved, this indites tht timing of pplition my e n importnt onsidertion in pthogen intertions. Other reserhers hve reported tht the timing of glyphoste pplition n ffet disese severity. For exmple, glyphoste redued the severity of lef rust in whet if pplied efore infetion hd ourred, with effiy lsting for 21 dys fter pplition ut not more thn 35 dys fter pplition. 44 A etter understnding of the impt of the timing of glyphoste pplitions my llow the development of n optimized spry shedule for sugr eet to mnge potentil deleterious effets on disese severity. A future diretion inludes understnding the impt of GR rops on rottion. Inreses in Rhizotoni root rot ould inrese the soil pthogen popultion nd ffet other suseptile rops in rottion with GR sugr eet, suh s dry en, soyen nd orn. 45 Glyphoste pplition hs een reported to inrese propgules of Fusrium 33 in soil, hnging the inoulum level in the groeosystem. Furthermore, glyphoste pplition hs mde other rops suseptile to normlly non-pthogeni isoltes of Fusrium, 36 so rising onerns out new pthogeni threts to GR sugr eet, whih serves s symptomless host for ertin Fusrium isoltes. 46 Lstly, glyphoste n redue the numer of potentil ntgonists to pthogens in field soil 47 nd lter the intertion mong fungi on medi nd in soil. 48 Although this ws not the underlying mehnism for loss of resistne in the greenhouse studies, this is n re tht needs further investigtion under field onditions. ACKNOWLEDGEMENTS Speil thnks to Jquelyn Ani Akmkjin for her ssistne in tissue preprtion. Additionl thnks to Drs Jim Hunter, Leigh Towill nd Dn Blumenthl for onstrutive review of the mnusript. Thnks to Dr Mrk West for help with sttistil nlyses. REFERENCES 1 Kniss AR, Wilson RG, Mrtin AR, Burgener PA nd Fuez DM, Eonomi evlution of glyphoste-resistnt nd onventionl sugr eet. Weed Tehnol 18: (24). 2 Kishore GM, Pdgette SR nd Frley RT, History of heriidetolernt rops, methods of development nd urrent stte of the rt emphsis on glyphoste tolerne. Weed Tehnol 6: (1992). 3 Duke SO, Tking stok of heriide-resistnt rops ten yers fter introdution. Pest Mng Si 61: (25). 4 Steinruken J nd Amrhein N, The heriide glyphoste is potent inhiitor of 5-enolpyruvylshikimi id-2-phosphte 119 Pest Mng Si 62: (26)

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