Fungus Gnat Feeding and Mechanical Wounding Inhibit Pythium aphanidermatum Infection of Geranium Seedlings

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1 Eology n Epiemiology Fungus Gnt Feeing n Mehnil Wouning Inhiit Pythium phniermtum Infetion of Gernium Seelings S. E. Brun, J. P. Snerson, E. B. Nelson, M. L. Dughtrey, n S. P. Wright First n seon uthors: Deprtment of Entomology, n thir n fourth uthors: Deprtment of Plnt Pthology n Plnt-Miroe Biology, Cornell University, Ith, NY ; n fifth uthor: Unite Sttes Deprtment of Agriulture Agriulturl Reserh Servie, Roert H. Holley Center for Agriulture & Helth, Ith, NY Aepte for pulition 18 July ABSTRACT Brun, S. E., Snerson, J. P., Nelson, E. B., Dughtrey, M. L., n Wright, S. P Fungus gnt feeing n mehnil wouning inhiit Pythium phniermtum infetion of gernium seelings. Phytopthology 99: A series of lortory tests were onute to investigte potentil effets of fungus gnt (Brysi imptiens) feeing mge on suseptiility of gernium seelings (Pelrgonium hortorum) to infetion y the root rot pthogen Pythium phniermtum. Effets were ompre with those from similr tests in whih the seelings were mehnilly woune y severing the root tip with slpel. Assys of gernium seelings in petri ishes revele pronoune negtive fungus gnt Pythium intertion, with exposure to fungus gnt lrve 24 h prior to inoultion with P. phniermtum zoospores resulting in up to 47% fewer seeling eths thn woul hve een expete if the two gents h te inepenently. Similr results were oserve when seelings were sujete to mehnil wouning 24 h prior to zoospore inoultion. In ontrst, no intertion ourre when seelings were mehnilly woune immeitely prior to inoultion. The egree of plnt mge inflite y the feeing tivities of the lrvl fungus gnts h no signifint effet on the omine mge from fungus gnts n Pythium in petri ishes. Anillry stuies showe tht Pythium evelopment on V8 gr ws not inhiite y the presene of fungus gntssoite miroorgnisms, nor were seelings inoulte with these miroes less suseptile to Pythium infetion. The preise mehism or mehnisms unerlying the oserve intertions were not eluite; however, the results strongly suggest tht oth fungus gnt feeing n mehnil wouning tivte systemi efenses tht me the seelings more resistnt to Pythium infetion. Plnts re onstntly thretene y numer of ioti stresses. Plnt response to herivore or pthogen ttk is vrile in spe n time n epens on the ility of plnt to respon with inue resistne or to tolerte ttking orgnisms. Aioti n ontogeneti ftors influene plnt-ntgonist intertions s well. Plnts re often ttke y more thn one orgnism t time, foring them to mount n integrte system of efense ginst iverse enemies. The orer in whih ifferent ntgonists ssoite with plnt ffets the type n egree of response (5,24,35). Plnt pthogens vetore y insets n hve evstting effets on plnts. This ssoition of insets with plnt pthogens hs een oumente for more thn entury (39). In ommeril greenhouses, insets previously onsiere nuisnes hve een implite in the spre of soilorne pthogens. One key exmple of suh inset pests is rk-winge fungus gnt of the genus Brysi (Dipter: Sirie) (19). Ault fungus gnts trnsmit vriety of plnt pthogens tht proue eril ispersl stges, inluing spore-prouing fungi suh s Fusrium veneum (Fr.) S., F. foetens Shroers, O Donnell, Byen & Hooftmn, F. oxysporum f. sp. riis-lyopersii Jrvis & Shoemker, Thielviopsis siol (Berk. & Broome) Ferrris, n Vertiillium lo-trum Reinke & Berthol (8,9,12,18,23). Wouning y fungus gnts my lso influene pthogen infetion (19). Leth n Newton (25) foun tht lflf n re lover plnts injure y fungus gnt (Brysi sp.) lrve prior to inoultion with Fusrium spp. ie t muh higher rte thn uninjure plnts. A possile orreltion etween Brysi fungus Corresponing uthor: S. E. Brun; E-mil ress: se35@ornell.eu oi: / PHYTO The Amerin Phytopthologil Soiety gnt infesttions n root rot iseses use y Pythium hs lso een oserve. Plnts infete with Pythium re often lso infeste with fungus gnt lrve. Both of these orgnisms re importnt pests of greenhouse floriulture, n it is generlly thought tht fungus gnts vetor Pythium. However, intertions mong fungus gnts, Pythium, n florl rops hve not een thoroughly investigte experimentlly (11,21). The primry ojetive of this stuy ws to etermine if wouning mge inflite y fungus gnt lrve ffets suseptiility of gernium seelings (Pelrgonium hortorum L. H. Biley) to infetion y Pythium phniermtum (Eson) Fitzp. Aitionl ojetives inlue etermining whether P. phniermtum infetion is impte y the miroflorl ommunity ssoite with fungus gnts or y mehnil wouning. Preliminry stuies were lso onute to estimte the mein lethl onentrtion (LC 50 ) of P. phniermtum zoospores ginst gernium seelings in the iossy systems to e esrie. This informtion ws neee to etermine n pproprite trget zoospore ose for tests involving single-ose inoultions, whih were prt of the min ojetives. MATERIALS AND METHODS Inset rering. A lortory olony of Brysi imptiens (Johnnsen) ws estlishe with ults ollete from greenhouse on the Cornell University mpus. Aults n lrve were routinely rere in plsti ontiners (9.8 m in imeter y 6.2 m in epth; Pioneer Plstis, Dixon, KY) with lis ering holes (5.5 m in imeter) overe with nylon mesh (95 µm) for ventiltion. Fifty fungus gnt ults (preomintely femles) were ple in eh ontiner for egg proution n provie with 40 g of growing meium (Premier Pro-Mix BX, Qukertown, PA) sturte with tp wter n 10 g (ry weight) of Vol. 99, No. 12,

2 groun pinto ens (Goy Foos, In., Seuus, NJ). Colony ontiners were mintine t 27 ± 1 C with 14 h of light n 10 h of rkness. New ontiners were estlishe ily to provie onstnt supply of ll life stges. To otin n evenge ohort of fourth-instr fungus gnt lrve for use in iossys, 200 ult fungus gnts were ollete from olony ontiners n relese into 45-y-45-y-45.5-m ge in the lortory t mient temperture ( 24 C) n llowe to oviposit on 90-mm-imeter petri ishes (Beton Dikinson Lwre, Frnklin Lkes, NJ) tht ontine sturte otton overe with piee of lk filter pper spre with thin lyer ( 1.6 g) of groun pinto ens (Phseolus vulgris L.). After 24 h, petri ishes were remove from ges n the fungus gnt ultures mintine until the lrve omplete evelopment to the fourth instr. Aitionl wter n pinto ens were e to the ishes s neee to provie equte foo n wter. Fourthinstr lrve were reily ientifie on the surfe of the filter pper n trnsferre to iossy ontiners using fine rush. Plnt propgtion. Assys were onute in petri ishes for simpliity n reful oservtion; other ssys were onute with vermiulite growing meium in plug ells to simulte ommeril growing onitions. Gernium sees (Pelrgonium hortorum Orit White, Golsmith Sees) for petri ish iossys were surfe sterilize in 0.5% soium hypohlorite solution for 3 min, rinse in sterile istille wter (SDH 2 O), n germinte iniviully on top of one isk of filter pper (42.5 mm in imeter) sturte with 400 µl of SDH 2 O in tight (frition)-li petri ishes (47 mm in imeter). The see were inute in rkness for 3 ys t room temperture. Seelings were mintine t 27 ± 1 C with 14 h of light n 10 h of rkness n wtere ily (with 200 µl of SDH 2 O) until they were use in iossys. The seelings were oriente horizontlly in the petri ishes (lying on the filter pper). See use in vermiulite/plug try iossys were lso surfe sterilize (s esrie ove) n germinte on top of two isks of filter pper (90 mm in imeter) sturte with 2.2 ml of SDH 2 O in petri ishes (90 mm in imeter) sele with Prfilm. The sees were inute in rkness t room temperture until germintion (3 to 4 ys) n then plnte in wter-sturte, orse vermiulite within iniviul plug ells ( 26-ml volume) tht were ut from 128-well plug trys (Dillen Prouts/Myers Inustries In., Milefiel, OH). Plug ells were ple in 55-ml plsti vils n SDH 2 O ws e t rtes esrie elow for eh experiment. Vermiulite ws selete s the potting meium euse Pythium infetion ws not onsistent in vriety of petse plug mixes ommonly use y growers. At the initition of iossys, seelings were, in most ses, 7 to 8 ys ol (ys post see hyrtion), 4 m in length, n onsiste of short hypootyls terminting in two otyleons n n unrnhe root with ense root hirs. Pthogen mintenne. Pythium phniermtum (strin P58) ultures were grown in 60-mm-imeter petri ishes on V8 juie gr (ompose of 100 ml of V8 juie, 400 ml of H 2 O, 1.5 g of CCO 3, n 10 g of Bto Agr) n mintine in the rk t 27 ± 1 C. One-week-ol ultures of P. phniermtum were use in ll experiments. Every 2 weeks, the pthogen ws reisolte from 11-y-ol gernium seelings tht h een inoulte 3 ys erlier y pling 6-mm-imeter gr isk (from 1-week-ol ulture) of P. phniermtum on the root tip. Eh infete seeling ws ple in 90-mm-imeter petri ish n overe with 2% molten wter gr supplemente with the ntiiotis rifmpiin, peniillin G, n knmyin (t 1.0, 1.0, n 2.5 ml/liter of SDH 2 O, respetively) (WARP). After the gr soliifie, pltes were inute s esrie ove. P. phniermtum hyphe tht grew rpily through the gr were exise from the surfe of the meium n trnsferre to fresh WARP plte. In the following esriptions of methos, the term replite ssys refers to ssys onute on ifferent ys, using freshly prepre P. phniermtum inoul. Inoulum preprtion. Zoospore suspensions were proue y pling one 20-mm-imeter gr isk from 1-week-ol P. phniermtum ulture into 60-mm petri ish ontining 10 ml of SDH 2 O. Zoospore ulture pltes were strte t 1700 h on the y prior to the strt of n ssy. The ish ws sele with prfilm n inute s esrie ove. The sterile wter ws reple with 10 ml of fresh SDH 2 O the following morning n zoospores were ollete 6 h lter for use in the ssy. Approximte esire onentrtions of zoospores for iossys were otine y prepring seril ilutions in SDH 2 O. Conentrtions of zoospores in stok suspensions n initil ilutions in series were estimte y stnr hemytometer ounting. Four inepenent smples from eh ilution were ounte. Numers of zoospores in ilute suspensions were etermine y spreing 200-µl smple onto V8 gr in 90-mm-imeter petri ish (three replite pltes per ilution). CFU were ounte on eh plte fter 24 h of inution s esrie ove. P. phniermtum LC 50 estimtions. Four replite multiose petri ish iossys were onute to etermine the LC 50 of P. phniermtum zoospores. Four zoospore ose levels n sterile wter ontrol were use in eh ssy. For the first three ssys, the zoospore stok suspension ws serilly ilute 10, 10, 3, n 3. In the fourth ssy, the stok suspension ws serilly ilute 10, 5, 3, n 3. Iniviul seelings in tightli petri ishes (47 mm in imeter) were inoulte y pipetting 400 µl of one of the four zoospore ilutions onto the filter pper jent to the root tip of the plnt. Controls were inoulte with TABLE 1. Mein lethl onentrtion (LC 50 ) estimtes from replite four-ose iossys of Pythium phniermtum ginst gernium seelings in petri ishes n plug trys Assy Dose rnge (zoospores/ml) Mortlity rnge (%) LC 50 (zoospores/ml) Proit regression slope Heterogeneity χ 2 Filter pper Mens ± SE 49.7 ± ± Vermiulite , , , , , , , , , , Mens ± SE 125,128 ± 34, ± Seeling roote either on moist filter pper in petri ishes (Filter pper) or in vermiulite in plug trys (Vermiulite); SE = stnr error. Rnge of oses (expresse s vile zoospores per milliliter) pplie in eh iossy. Rnge of gernium seeling mortlity reore ross oses. Heterogeneity χ 2 (with 2 egrees of freeom) initing gooness-of-fit of the t to the proit-log ose liner regression moel (10) PHYTOPATHOLOGY

3 400 µl of SDH 2 O. In ll, 100 seelings were inoulte per ssy (20 per zoospore ose n ontrol). Mortlity of seelings ws reore 7 ys postinoultion. Five replite multiose iossys were onute to etermine the LC 50 of P. phniermtum zoospores ginst gernium seelings roote in vermiulite. For ll ssys, three 5 ilutions with SDH 2 O were me to the zoospore stok suspension. Iniviul 8-y-ol gernium seelings in vermiulite-fille plug ells were inoulte y pipetting 2 ml of either one of the four zoospore suspensions or sterile wter ner the rown of the plnt. In ll, 65 to 90 seelings were use per ssy (13 to 18 per zoospore ose n ontrol). The seelings were mintine t 27 ± 1 C with 14 h of light n 10 h of rkness n wtere ily with 1 ml of sterile wter. Mortlity of seelings ws reore 7 ys postinoultion. Effet of fungus gnt wouning on Pythium infetion. In totl, 24 ssys were onute to etermine the effets of plnt wouning on susequent P. phniermtum infetion of gernium seelings growing on wter-sturte filter pper in petri ishes. Twenty-one of these ssys inlue tretments esigne to etermine whether the time of seeling exposure to fungus gnt lrve influene infetion. Eh ssy ws esigne s two-y-two ftoril with ll omintions of fungus gnt lrvl presene or sene n P. phniermtum inoulum presene or sene s the four tretments (see sttistil nlysis setion). Gernium see were germinte s esrie ove. When the seelings were 7 ys ol, fourth-instr fungus gnt lrve were ple in hlf of the ishes (three lrve/ish) n llowe to fee on the seelings. The plnts were expose to the lrve for 24 h t 27 ± 1 C with 14 h of light n 10 h of rkness, fter whih time the lrve were remove. Preliminry experiments emonstrte tht fungus gnts relily use plnt mge tht ws visile y 24 h. Fungus gnt mge ws ssesse on eh plnt oring to the following sle: 1 = no visile mge or mge root hirs or minor rsions; 2 = lerly evient feeing mge srring the root, hypootyl, or otyleon surfe; n 3 = severe root, tunneling in stem or root olumn, hole in otyleon. Dishes to e trete with P. phniermtum were then inoulte y pipetting 400 µl of P. phniermtum zoospore suspension (150 ilution of stok suspension) onto the filter pper ner the root tip of the plnt. Doses (CFU) pplie in eh of the replite ssys were quntifie using the ove-esrie CFU plting metho. Eh tretment inlue 20 to 25 seelings. Mortlity of seelings ws reore 7 ys postinoultion. Four lortory iossys were onute to etermine the effets of exposure to fungus gnt lrve on susequent P. phniermtum infetion of gernium seelings roote in vermiulite-fille plug ells ontine in plsti vils (see plnt propgtion setion). Eh ssy ws esigne s two-y-two ftoril with ll omintions of fungus gnt lrvl presene or sene n P. phniermtum inoulum presene or sene s the four tretments. Fourth-instr fungus gnt lrve were then ple in hlf of the ells (three lrve/ell) n llowe to fee on the 7-y-ol seelings. At 24 h fter introution of the lrve, the ishes were inoulte with P. phniermtum y TABLE 2. Perent mortlity of gernium seelings in 28 mge-plnt ssys Assy Dose Control FGD Pythium FGD + Pythium e MD f MD + Pythium g 24-h MD h 24-h MD + Pythium i Filter pper Vermiulite 25 46, , , , , Seeling roote either on moist filter pper in petri ishes (Filter pper) or in vermiulite in plug trys (Vermiulite). Zoospores per milliliter use in eh iossy. FGD = fungus gnt mge; eh seeling expose to three fourth-instr fungus gnt lrve for 24 h. Eh seeling inoulte with Pythium zoospores. e Eh seeling expose to three fourth-instr fungus gnt lrve for 24 h n then inoulte with Pythium; lrve remove immeitely prior to Pythium pplition. f MD = mehnil mge; eh seeling woune y utting 1-m-long setion from istl portion of root. g Eh seeling woune y utting 1-m-long setion from istl portion of root n immeitely inoulte with Pythium zoospores. h Eh seeling woune y utting 1-m-long setion from istl portion of root 24 h prior to strt of ssy. i Eh seeling woune y utting 1-m-long setion from istl portion of root n inoulte with Pythium zoospores 24 h lter. Vol. 99, No. 12,

4 pipetting 2 ml of stok zoospore suspension onto the vermiulite jent to the rown of the plnt. Doses pplie in eh of the four replite ssys were quntifie y hemytometer. In ll, 68 seelings were use per ssy (17 per tretment). The seelings were mintine t 27 ± 1 C with 14 h of light n 10 h of rkness. At initition of the ssys, SDH 2 O ws e to eh vil to level 1 m ove the ottom of the plug ell, n wter ws susequently e one ily to restore this level. Mortlity of seelings ws reore 7 ys postinoultion. Effet of mehnil wouning on Pythium infetion. Of the ove-mentione 24 petri ish ssys, 14 inlue tretments to etermine the effets of mehnil wouning of plnts on susequent P. phniermtum infetion of gernium seelings. Eh ssy ws esigne s two-y-two ftoril with ll omintions of mehnil wouning presene or sene n P. phniermtum presene or sene s the four tretments. Mehnil wouning ws hieve y utting 1-m-long setion from the istl portion of the root of eh seeling using sterilize slpel. In eh of the 14 ssys, P. phniermtum zoospores were pplie to 8-y-ol seelings immeitely fter wouning. In 9 of the 14 ssys, zoospores were pplie to seelings tht h een woune 24 h previously (when 7 ys ol). Twenty seelings were use per tretment. Mortlity of seelings ws ssesse 7 ys postinoultion. Effet of fungus gnt-ssoite miroes on Pythium infetion. Four iossys were onute to etermine the possile effets of the fungus gnt lrvl miroil ommunity on P. phniermtum infetion of gernium seelings. Fungus gnt rinste ws generte y pling three fourth-instr fungus gnt lrve in sterile 2-ml entrifuge tue with 10 µl of sterile wter. The tue ws then vortexe for 30 s to remove miroorgnisms from the fungus gnts oies. Eighty 7-y-ol seelings per ssy were trete y pplying 10 µl of fungus gnt rinste vi pipette to either the rown or the root tip of seeling (20 seelings/tretment). In orresponing ontrol tretments, SDH 2 O ws pplie inste of rinste. Three replite 200-µl smples of eh rinste (ilute 2,000 ) were lso pipette onto V8 gr pltes for CFU ounts. One y fter pplition of the rinste tretments, the seelings were inoulte with P. phniermtum zoospores n monitore for mortlity s esrie ove. An itionl ssy ws onute in vitro to ssess possile ntgonisti effets of the fungus gnt-ssoite miroes on P. phniermtum infetion. One 6-mm-imeter gr isk from 1-week-ol ulture of P. phniermtum ws ple on the surfe of V8 meium in the enter of eh ish. In one tretment, three 10-µl roplets of fungus gnt rinste were pplie iniviully long the irumferene of the ish equiistnt from the P. phniermtum plug. In the seon tretment, three 10-µl TABLE 3. Effets of inoultion with Pythium phniermtum n exposure to fungus gnt lrve on mortlity (± stnr error) of gernium seelings in petri ish iossys Tretments Oserve plnt mortlity (%) Expete plnt mortlity (%) Synergism (+) or ntgonism ( ) ANOVA PL 12.7 ± 5.0 P: F [1,9] = 2.73; P = 0.13 FG 10.5 ± 4.0 FG: F [1,9] = 1.50; P = 0.25 FG + PL 9.7 ± % ( 55.9%) P FG: F [1,9] = 3.06; P = 0.11 IA: F [1,3] = 21.7; P = PM 43.3 ± 5.0 P: F [1,27] = 59.13; P < FG 9.3 ± 3.0 FG: F [1,27] = 0.11; P = 0.74 FG + PM 25.6 ± % ( 47.3%) P FG: F [1,27] = 11.99; P = IA: F [1,9] = 10.66; P = PH 88.6 ± 3.4 P: F [1,18] = ; P < FG 3.6 ± 3.1 FG: F [1,18] = 13.34; P = FG + PH 47.1 ± % ( 47.1%) P FG: F [1,18] = 25.69; P < IA: F [1,6] = 31.7; P = PA 52.6 ± 4.9 P: F [1,60] = 98.67; P < FG 7.6 ± 3.3 FG: F [1,60] = 1.50; P = 0.23 FG + PA 29.8 ± % ( 47.0%) P FG: F [1,60] = 17.02; P = IA: F [1,20] = 36.12; P < PL = low ose of Pythium, n = 4 ssys, 25 to 73 zoospores/ml; FG = fungus gnts, eh seeling expose to three fourth-instr fungus gnt lrve for 24 h; PM = meium ose of Pythium: n = 10 ssys, 100 to 194 zoospores/ml; PH = high ose of Pythium: n = 7 ssys, 320 to 800 zoospores/ml; PA = ll oses; FG + Pythium ose = seelings expose to fungus gnt lrve for 24 h n then inoulte with Pythium; lrve remove immeitely prior to Pythium pplition. Control preite if the gents exhiit inepenent tion; vlue lulte using the formul for omintion of inepenent proilities (34). Antgonism lulte s the ifferene etween oserve n expete perent plnt mortlity. Numers in prentheses inite the proportionl ifferenes etween the oserve n expete perent plnt mortlity. Ftoril nlysis of vrine (ANOVA) testing min effets n intertion; one-wy ANOVA testing hypothesis of inepenent tion (IA). TABLE 4. Effets of inoultion with Pythium phniermtum n exposure to fungus gnt lrve on mortlity (± stnr error) of gernium seelings roote in vermiulite Tretments Oserve plnt mortlity (%) Expete plnt mortlity (%) Synergism (+) or ntgonism ( ) ANOVA Pythium sp. (P) 35.4 ± 4.8 P: F [1,12] = 50.8, P < Fungus gnts (FG) 3.6 ± 4.6 FG: F [1,12] = 0.26, P = 0.62 P + FG 23.6 ± % ( 37.4%) P FG: F [1,12] = 3.40, P = 0.09 IA: F [1,4] = 27.4, P = Control preite if the gents exhiit inepenent tion; vlue lulte using the formul for omintion of inepenent proilities (34). Antgonism lulte s the ifferene etween oserve n expete perent plnt mortlity. Numer in prentheses inites the proportionl ifferene etween the oserve n expete perent plnt mortlity. Ftoril nlysis of vrine (ANOVA) testing min effets n intertion; one-wy ANOVA testing hypothesis of inepenent tion (IA). Test omprising n = 5 ssys, 2.2 to zoospores/ml PHYTOPATHOLOGY

5 roplets of rinste were pplie iniviully to 3-y-ol gernium plnts situte long the irumferene of 90-mm plte equiistnt from the P. phniermtum plug. Pltes were mintine t 27 ± 1 C with 14 h of light n 10 h of rkness, n growth of the Pythium sp. ws exmine 3 ys postinoultion. Twelve replites of eh tretment were use. In three replites, 200-µl smples of the fungus gnt rinste (ilute 8,000 ) were lso pipette onto iniviul V8 gr pltes (90-mm-imeter petri ish) for CFU ounts. Sttistil nlyses. LC 50 s of P. phniermtum zoospores were estimte y proit nlysis using the personl omputer progrm PoloPlus (26). All other sttistil tests were onute using the JMP softwre version 7.0 (36) using nlysis of vrine (ANOVA) n signifine levels of α = All perent mortlity t were rsine trnsforme prior to prmetri ANOVA (37) n ligne-rnk trnsforme for the lterntive nonprmetri test (27,33). It ws not possile to extly replite the zoospore ose ross ssys. Therefore, in orer to ssess the effet of P. phniermtum ose on infetion of gernium seelings, it ws neessry to omine oses into tegories. Three tegories were efine: <100 zoospores/ml, 100 to 299 zoospores/ml, or 300 zoospores/ml. Assy ws inlue s ftor (experimentl lok) in ll nlyses ut intertions with ssy were not exmine ue to restrition error (37). In ition, euse ssy ws onfoune with ose, it ws not possile to test ose tretment intertions. Of the 24 petri ish iossys, 14 omprise suexperiments investigting effets of exposure to oth fungus gnts n mehnil mge on mortlity of Pythium-inoulte gernium seelings. To simplify the intertion nlysis n presenttion of results, we onute seprte two-y-two ftoril nlyses of these ftors even though inepenent sets of ontrols n Pythium-only tretments were not onute for eh suexperiment. Though this pproh enles strightforwr presenttion of results, ution is lle for with ny srifie of inepenene. We o not onsier this signifint prolem in this se, euse (i) no ontrol mortlity ws oserve in ny of the iossys, initing no vrine ssoite with the estimte of 0% ontrol mortlity, n (ii) trens in the t were onsistent ross lrge numer of inepenently replite ssys, trnslting in mny ses to high levels of signifine of the teste intertions (espeilly the fungus gnt Pythium intertions) (P vlues 0.001). The lk of ontrol mortlity oes, however, rete prolem with respet to unequl vrine ross tretments. As onsequene, we orroorte the ANOVA results using the nonprmetri ligne rnk test reommene for ftoril esigns with intertion (27,33). Results in ll ses were similr to the stnr ANOVA (often prouing inrese levels of signifine), n we opte to present just the ANOVA results. In ition, we employe one-wy ANOVAs to test for inepenene of the Pythium n fungus gnt (or Pythium n mehnil mge) mortlity ftors. Mortlity preite y inepenent tion ws lulte using the formul for omintion of inepenent proilities (34), n this expete mortlity ws ompre iretly vi ANOVA with the oserve mortlity use y the gents ting in omintion. This omprison exlues the prolemti (zero-vrine) ontrol tretments from the nlysis n, euse ontrol mortlity ws zero, orretions for ontrol mortlity were lso unneessry. The sme prolem with inepenene exists with the tests exmining effets of vrile fungus gnt mge (seprte ontrol n Pythium-only tretments were not onute for eh mge level). In this se, there were fewer replites n smller smple sizes, n we opte not to onut the ftoril nlysis. Inste, the fungus gnt + Pythium tretments were nlyze y one-wy ANOVA (perent mortlity t rsine trnsforme n weighte y smple size) to etermine whether there ws signifint effet of fungus gnt mge level on mortlity of Pythium-inoulte seelings. RESULTS Estlishment of P. phniermtum LC 50 vlues. The men LC 50 ± stnr error (SE) etermine from four replite iossys in petri ishes equle 49.7 ± 15.2 zoospores/ml, with proit regression slope ± SE equl to ± The onentrtions of vile zoospores per milliliter use in the four ssys ws 0 to 897 n proue mximum of 100% mortlity (Tle 1). All seelings remine helthy in the ontrol tretments for the urtion of the experiment. Signs of isese in Pythiuminfete plnts inlue wtersoking tht me the tissue pper rker green in olor n hyphl growth on the surfe of the seeling roots n hypootyl within 7 ys fter inoultion. The men LC 50 ± SE etermine from five replite iossys with seelings roote in vermiulite equle ± zoospores/ml; men proit regression slope ± SE ws 1.04 ± The onentrtions of vile zoospores per milliliter use in the five ssys ws 70 to 193,000 n proue mximum of TABLE 5. Effets of fungus gnt (FG) mge level on perent mortlity (± stnr error) of gernium seelings expose to Pythium phniermtum FG mge level Tretment Anlysis of vrine Low ose FG 3.8 ± ± ± 5.5 F 2,6 = 4.2, P = FG + P 8.7 ± ± ± 0.6 F 2,6 = 0.5, P = 0.62 Ajuste FG + P 7.7 ± ± F 2,6 = 5.3, P = Meium ose FG 4.8 ± ± ± 1.8 F 2,18 = 2.3, P = 0.13 FG + P 13.6 ± ± ± 2.8 F 2,18 = 7.5, P = Ajuste FG + P 11.7 ± ± ± 2.6 F 2,18 = 1.9, P = 0.18 High ose FG ± 2.3 F 2,12 = 11.7, P = FG + P 39.6 ± ± ± 5.1 F 2,12 = 0.9, P = 0.44 Ajuste FG + P 39.6 ± ± ± 5.1 F 2,12 = 0.43, P = 0.66 All oses FG 2.9 ± ± ± 3.1 F 2,51 = 13.5, P < FG + P 22.5 ± ± ± 5.8 F 2,51 = 1.5, P = 0.24 Ajuste FG + P 21.5 ± ± ± 6.1 F 2,51 = 0.1, P = 0.87 FG: seelings expose to FGs lone; FG + P: seelings expose to FGs n then trete with Pythium sp.; juste FG + P: mortlity ttriutle to FGs lone eute from mortlity in the FG + P tretments using Aott s formul (1). Low ose = 25 to 73 zoospores/ml, 4 iossys; Meium ose = 100 to 194 zoospores/ml, 10 iossys; High ose = 320 to 800 zoospores/ml, 7 iossys; All oses = 21 iossys. FG mge ws ssesse on eh plnt oring to the sle 1 = no visile mge or mge root hirs or minor rsions; 2 = lerly evient feeing mge srring the root, hypootyl, or otyleon surfe; n 3 = severe root, tunneling in stem or root olumn, hole in otyleon. Vol. 99, No. 12,

6 80% mortlity (Tle 1). Control tretments (SDH 2 O) h miniml effet in most ses, with ll seelings in three ssys remining pprently helthy until the ssys were terminte. Signifint levels of ontrol mortlity were reore in two ssys (11 n 22%); however, none of the ffete seelings showe signs of Pythium infetion, n no Pythium sp. ws re-isolte from the e seelings. Intertion stuies. Results from the 28 ssys investigting effets of plnt wouning on Pythium infetion re presente in Tle 2. Assys 1 to 24 were onute with gernium seelings on wet filter pper in petri ishes, while ssys 25 to 28 were onute with seelings roote in vermiulite in plug ells. No seeling mortlity ws oserve in ny of the ontrol tretments ross ssys n, therefore, ll mortlity in the fungus gnt-lone n Pythium-lone tretments ws ttriutle to tretment effets (fungus gnt mge or Pythium infetion). Intertions were pprent etween the min effets of exposure to fungus gnt lrve n inoultion with Pythium s well s etween mehnil mge n Pythium inoultion. Evlution of the signifine of these intertions ws exmine vi ftoril ANOVA. Effets of fungus gnt wouning on Pythium infetion. Exposure to fungus gnt lrve lone i not use ny signifint plnt mortlity, wheres Pythium lone ws highly virulent when zoospores were pplie t the intermeite n high oses (Tle 3). There ws, however, n overll signifint intertion etween the fungus gnt n Pythium tretments. The exposure of seelings to fungus gnts 24 h prior to Pythium inoultion resulte in up to 47% fewer seeling eths n seeling mortlity up to 42 perentge points lower thn woul hve ourre h the two gents operte inepenently (Tle 3). Similr trens were oserve with seelings roote in vermiulite (Tle 4). A negtive intertion etween the fungus gnt n TABLE 6. Effets of mehnil wouning n Pythium phniermtum ose on gernium seeling mortlity (± stnr error) Tretments Oserve plnt mortlity (%) Expete plnt mortlity (%) Synergism (+) or ntgonism ( ) ANOVA Dmge t inoultion PL 25 ± 8.8 P: F [1,6] = 25.91; P = MD e 0 MD: F [1,6] = 0.003; P = 0.96 PL + MD f 25 ± % (0%) P MD: F [1,6] = 0.003; P = 0.96 IA: F [1,2] = 0.006, P = 0.94 PM 70 ± 5.9 P: F [1,12] = ; P < MD e 0 MD: F [1,12] = 0.005; P = 0.95 PM + MD f 69 ± % ( 1.4%) P MD: F [1,12] = 0.005; P = 0.95 IA: F [1,4] = 0.006, P = 0.94 PH 85 ± 4.3 P: F [1,15] = ; P < MD e 0 MD: F [1,15] = 0.51; P = 0.49 PH + MD f 90.8 ± % (+6.8%) P MD: F [1,15] = 0.51; P = 0.49 IA: F [1,5] = 0.48, P = 0.52 PA 66.8 ± 4.3 P: F [1,39] = ; P < MD e 0 MD: F [1,39] = 0.06; P = 0.80 PA + MD f 68.9 ± % (+3.1%) P MD: F [1,39] = 0.06; P = 0.80 IA: F [1,13] = 0.19, P = 0.67 Dmge 24 h prior to inoultion g PL 25 ± 7.9 P: F [1,6] = 14.55; P = MD h 0 MD: F [1,6] = 2.55; P = 0.16 PL + MD i 8.3 ± % ( 66.8%) P MD: F [1,6] = 2.55; P = 0.16 IA: F [1,2] = 29.74, P = 0.03 PM 82.5 ± 1.2 P: F [1,3] = ; P = MD h 0 MD: F [1,3] = 9.98; P = 0.05 PM + MD i 47.5 ± % ( 42.4%) P MD: F [1,3] = 9.98; P = 0.05 IA: F [1,1] = 16.55, P = 0.15 PH 88.7 ± 3.8 P: F [1,9] = ; P < MD h 0 MD: F [1,9] = 20.62; P = PH + MD i 47.5 ± % ( 46.4%) P MD: F [1,9] = 20.62; P = IA: F [1,3] = 49.31, P = PA 66.1 ± 6.7 P: F [1,24] = 98.48; P < MD h 0 MD: F [1,24] = 7.84; P = 0.01 PA + MD i 34.4 ± % ( 48.0%) P MD: F [1,24] = 7.84; P = 0.01 IA: F [1,8] = 53.33, P < Control preite if the gents exhiit inepenent tion; vlue lulte using the formul for omintion of inepenent proilities (34). Synergism n ntgonism lulte s the ifferene etween oserve n expete perent plnt mortlity. Numers in prentheses inite the proportionl ifferenes etween the oserve n expete perent plnt mortlity. Ftoril nlysis of vrine (ANOVA) testing min effets n intertion; one-wy ANOVA testing hypothesis of inepenent tion (IA). Mehnil mge (MD) t time of Pythium inoultion. PL = low ose: n = 3 ssys, 73 to 113 zoospores/ml; PM = meium ose: n = 5 ssys, 120 to 282 zoospores/ml; PH = high ose: n = 6 ssys, 320 to 440 zoospores/ml; PA = ll oses. e Eh seeling woune y utting 1-m-long setion from istl portion of root. f Eh seeling woune y utting 1-m-long setion from istl portion of root n immeitely inoulte with Pythium zoospores. g MD 24 h prior to Pythium inoultion. PL = low ose: n = 3 ssys, 73 to 113 zoospores/ml; PM = meium ose: n = 2 ssys, 187 to 282 zoospores/ml; PH = high ose: n = 4 ssys, 320 to 440 zoospores/ml; PA = ll oses. h Eh seeling woune y utting 1-m-long setion from istl portion of root 24 h prior to strt of ssy. i Eh seeling woune y utting 1-m-long setion from istl portion of root n inoulte with Pythium zoospores 24 h lter PHYTOPATHOLOGY

7 Pythium tretments ws pprent, with 37% fewer seeling eths n mortlity 14 perentge points lower thn preite y inepenent tion. There ws little eviene tht the egree of plnt mge inflite y the feeing tivities of the lrvl fungus gnts h ny signifint effet on the ove-esrie fungus gnt Pythium intertion (Tle 5). At the meium zoospore oses, seeling mortlity inrese with inresing mge (from 14 to 38%, P = 0.004); however, this tren ws pprent lso in the sene of Pythium, n signifine ws lost (P inrese to 0.18) when mortlity ttriutle to fungus gnts lone ws eute from mortlity in the fungus gnt + Pythium tretments using Aott s formul (1). In the ses of the low n high oses n when ll oses were omine, there were no ifferenes in seeling mortlity over the three mge levels (Tle 5). Effets of mehnil wouning on Pythium infetion. Mehnil wouning lone i not hve signifint effet on plnt mortlity, wheres Pythium sp. lone h highly signifint effet on plnt mortlity t ll oses (Tle 6). There ws no signifint intertion etween mehnil wouning n Pythium tretments (Tle 6). In ontrst, mehnil wouning of seelings 24 h prior to Pythium inoultion signifintly reue susequent Pythiuminue mortlity t the high ose ut not t the intermeite or low oses, n signifint intertion ws etete etween mehnil wouning n Pythium (Tle 6). The exposure of seelings to mehnil wouning 24 h prior to Pythium inoultion resulte in up to 46% fewer seeling eths n seeling mortlity up to 41 perentge points lower thn preite y inepenent tion (Tle 6). Effet of fungus gnt miroes on Pythium infetion. Treting seelings with miroes wshe from fungus gnts h no signifint effet on mortlity ue to Pythium infetion (F [3,9] = 1.1, P = 0.40). Perent mortlity of seelings in the rinste rown, sterile wter rown, rinste root tip, n sterile wter root tip tretments ws 46.2 ± 3.1, 51.2 ± 4.4, 52.5 ± 3.2, n 56.2 ± 2.6, respetively. The onentrtions of zoospores use in the four ssys ws 180 to 412 zoospores/ml. The fungus gnt rinstes ontine n verge of teri/ml. In the presene of fungus gnt rinstes on V8 gr plnts, no myelil inhiition ws oserve. Furthermore, ll of the plnts trete with rinstes showe symptoms of P. phniermtum infetion within 2 ys. The fungus gnt rinstes ontine n verge of teri/ml. DISCUSSION The primry gol of this stuy ws to etermine if wouning mge from fungus gnt lrve ffets the suseptiility of gernium seelings to infetion y P. phniermtum. The fining tht fungus gnt lrvl feeing reues infetion y P. phniermtum presents new n unexpete piture with respet to the role of fungus gnts in the evelopment of plnt isese. For exmple, some previous stuies with Brysi spp. hve shown tht feeing my promote plnt infetion y pthogeni fungi (15,25), wheres other stuies (12) hve emonstrte no enhne infetion from fungus gnt feeing (22). Our results revel reltionship etween fungus gnt feeing n inue resistne to pthogens tht hs not previously een emonstrte (19). Fungus gnt lrve hve een shown to inue plnt efense responses: the jsmonte pthwy (16,20) plys ritil role in proteting Ariopsis thlin from ttk y these insets (29). It is unler, though, whih trits tivte y the jsmonte pthwy re responsile for inrese resistne n whether ross-tlk my exist mong multiple plnt-efense-signling pthwys (20,29). Furthermore, it is possile tht efense responses inue y inset feeing my influene susequent ioti ttkers (5). For exmple, tomto plnts previously infeste with silverlef whitefly, Bemisi rgentifolii, re more resistnt to the fungl pthogen Erysiphe ihorerum thn re ontrol plnts (28). Beuse jsmoni i meites pthwys tht onfer prtil tolerne to nerotrophi pthogens suh s Pythium (13,31), it is possile tht fungus gnt feeing inhiite Pythium infetion vi the jsmonte response in the urrent stuy. Inresing fungus gnt feeing mge i not inrese the level of Pythium infetion. If inrese mge were iretly orrelte with the level of inue resistne, the expete result woul e eresing mortlity with inresing mge. This ws not the se (Tle 5). These finings gree with reent stuies initing tht plnts n initite rpi efense responses within minutes of n inset ontting plnt, n even the nonfeeing tivities (movements) of single inset my initite response (17). Furthermore, the proution of some plnt efensive ompouns suh s ertin terpenoi voltiles n methyl sliylte respon in nonliner mnner to mge level (30). Inset feeing n mehnil wouning n inue the sme efensive pthwys of plnt ut the plnt response levels n gene trnsript profiles my iffer (6,32,40). In the present stuy, however, oth exposure to fungus gnt lrve n mehnil mge 24 h prior to Pythium inoultion resulte in signifint reutions in seeling mortlity. Explntion for the lk of signifint reltionship etween plnt wouning n Pythium infetion when mehnil mge ws inflite immeitely prior to inoultion my relte to the ft tht zoospores re ephemerl, typilly enysting n germinting within n hour of loting host (14). Despite rpi plnt efense retions fter herivore ontt, other stuies with Niotin sylvestris revel tht systemi jsmoni i responses o not reh mximum until 180 min fter wouning (3). In the present stuy, when wouning ws one immeitely prior to zoospore inoultion, there my not hve een suffiient time for the gernium seelings to mount strong efensive response efore eoming infete. Consiering tht fungus gnt lrve re lmost lwys present in the greenhouse setting, plnt efense retions my our frequently in response to wouning n inset feeing. Plnts must regulte these responses, however, to lne efense n fitness osts. An itionl gol of this stuy ws to investigte potentil inhiitory hrteristis of the fungus gnt-ssoite miroil ommunity on P. phniermtum infetion. Miroorgnisms my ply importnt roles in intertions etween mroorgnisms n etween mroorgnisms n their pthogens. For exmple, miroorgnisms regurgitte y herivores hve een implite in the inution of plnt efense responses (4,7,38). Also, nonpthogeni rhizoteri hve een shown to inue systemi resistne in plnts similr to pthogen-inue systemi quire resistne. Rhizoteri-inue systemi resistne hs een shown with fungi, teri, n viruses on vrious plnts (4). Although miroorgnisms living in the soil my positively or negtively impt intertions mong Pythium spp., fungus gnts, n gernium plnts, there ws no signifint impt of the fungus gnts externl miroil ommunity on susequent P. phniermtum infetion of gernium seelings in this stuy. Our investigtions of fungus gnt Pythium gernium seeling intertions nnot e onsiere exhustive. For exmple, we i not investigte potentil inhiitory effets of miroes extrte from the guts of fungus gnts on P. phniermtum suseptiility. There is lso the possiility of fungus gnt lrve iretly interting with plnt pthogens n impting their ility to infet plnts. For exmple, slivry seretions from the fungus gnt, Brysi oprophil, hve een shown to inhiit germintion of sleroti from the plnt pthogen Slerotini slerotiorum (2). Also, lrve of the fungus gnt B. imptiens tht ingeste oospores n myelium of P. phniermtum n then fe on uumer roots growing in rokwool pprently introue the pthogen to these plnts (21). Vol. 99, No. 12,

8 Although our stuies emonstrte tht previous exposure of gernium seelings to fungus gnt lrve n result in reue levels of Pythium infetion, the evelopment of new mngement strtegies for fungus gnts is not wrrnte. Diret root feeing mge y fungus gnt lrve n trnsmission of plnt-pthogeni oniil fungi y ult fungus gnts is well oumente; thus, these insets shoul still e onsiere pests in most ontexts (12,19,23). Furthermore, the mehnisms of the oserve intertion my e omplex, with vrious itionl ftors ffeting expression in ommeril rop proution systems, n investigtion of these mehnisms is wrrnte. Overll, the finings of this stuy enhne our unerstning of the ssoition etween fungus gnts n Pythium in greenhouse floriulture n inite tht eologil stuies re neee to thoroughly explore soil pthosystems. ACKNOWLEDGMENTS This reserh ws fune in prt through Speifi Coopertive Agreement etween the Unite Sttes Deprtment of Agriulture Agriulturl Reserh Servie (USDA-ARS) Plnt Protetion Reserh Unit n the Cornell University Deprtment of Entomology, Ith, NY (Speifi Coopertive Agreement ) fune y the USDA- ARS, s prt of the Floriulture n Nursery Reserh Inititive. We thnk M. Rmos n K. C. Bennett for tehnil support n L. Cstrillo, A. Jk, n M. Rmos for proviing useful omments on the mnusript. LITERATURE CITED 1. Aott, W. S A metho of omputing the effetiveness of n insetiie. J. Eon. Entomol. 18: Ans, O., Alli, I., n Reeleer, R. D Inhiition of germintion of sleroti of Slerotini slerotiorum y slivry gln seretions of B. oprophil. Soil Biol. Biohem. 21: Blwin, I. T., Zhng, Z., Di, N., Ohnmeiss, T. E., MClou, E. S., Lyns, G. Y., n Shmelz, E. A Quntifition, orreltions n mnipultions of woun-inue hnges in jsmoni i n niotine in Niotin sylvestris. Plnt 201: Bros, P., Krishik, V. A., n Jones, C. 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