Persistance and growth of an egg pathogenic fungus applied in alginate granules to field soi! and its pathogenicity toward Globodera pallida

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1 Fundm. ppl. NemLOI., 1992, 15 (5), Persistnce nd grwth f n egg pthgenic fungus pplied in lginte grnules t field si! nd its pthgenicity twrd Glbder pllid Rlf-Peter SCHUSTER nd Richrd A. SIKORA Rheinische-Friedrich- Wilhelms- Universitiit zu BOrln, Institut!ür Pjlnzenkrnkheiten, Nussllee 9, 5300 Bnn, GemlrlY. Accepted fr publictin 4 December Summry - The effect f fungl pthgen incrprted int lginte grnules n Glbder pllid ppultin dynmics ws investigted nd the effects fund were due t reduced rt weight up t 76.4 % fter 2 weeks, which led t similr reductins in htching nd in rt penetrtin. Cmprisn between the number f newly frmed cysts nd level f infected G. pllid eggs frm different wn experiments shwed c1er negtive crreltin. A clser exmintin prved, tht nly inculted cysts were infected nd nt newly frmed nes. Even s the fungus ws ble t infect newly frmed femles; n experiment cncerning the durtin f infectivity in field si! prved decrese in the level f infectin f eggs 28 dys fter pplictin f the fungus. Durtin f fungl infectivity ws exmined 2 nd 8 weeks fter grnule pplictin. There were n differences in degrec f infectin but reductin in the number f eggs per cyst. Fungl grwth thrugh field sil ws mesured with templte system using the degree f infectin f G. pllid eggs s prmeter which shwed high levels f infectin when cysts were directly ttched t the fungl inculum. Perthphytic mde-f-ctin f the islte used is discussed. Résumé - Persistnce et crissnce d'un chmpignn prsite des œufs ppliqué en chmp sus frme de grnulés d'lginte; s pthgénie envers Glbder pliid - L'influence d'un chmpignn pthgène incrpré à des grnules d'lginte sur l dynmique des ppultins de Glbder pllid été étudiée. Les effets bservés cnsistent en une diminutin du pids des rcines puvnt ller jusqu'à 76,4 % prés deux semines, ce qui induit une diminutin de même rdre pur l'éclsin et l pénétrtin du némtde. Lrs de ces expériences, l cmprisn entre le nmbre de kystes néfrmés et le tux d'infesttin des œufs de G. pllid reléve d'une crréltin négtive nette. Un exmen plus ttentif mntre que seuls les kystes inculés snt infestés et nn les kystes néfrmés. Méme dns ces cnditins, le chmpignn peut infester les femelles néfrmées; une expérience en chmp sur l persistnce du puvir infestnt du chmpignn mntre une diminutin du tux d'infesttin des œufs, vingt-huit jurs près l'pplictin du chmpignn. L persistnce du puvir infestnt du chmpignn été testée deux et huit semines près l'pplictin des grnulés; il n'y ps différence dns le degré d'infesttin, mis une diminutin du nmbre des œufs pr kyste. L crissnce du chmpignn en chmp été mesurée grâce à un système clibré utilisnt le degré d'infesttin de G. pllid cmme prmètre, lequel démntre de huts niveux d'infesttin lrsque les kystes snt directement u cntct de l'inculum fngique. Le mde d'ctin perthphytique de l'islt utilisé est discuté. Key-wrds : Nemtdes, Glbder, pthgenic fungus, ppultin dynmics, lginte. A brd spectrum f fungi hve been shwn t hve the bility t infect the eggs f cyst nemtdes (Kerry, 1974; Gswmi & Rumpenhrst, 1978, Nigh et l., 1980; Ressner, 1987). Hwever nly few studies (Dckmnn, 1990) hve been cnducted with Glbder pllid, these fungi re ptentil bilgicl cntrl gents especil1y in thse situtins where resistnt cultivrs re lcking r in wter cnservtin znes, where nemticide use is prhibited. The lck f cmmercil1y cceptble pplictin technlgy fr fungl bsed bilgicl cntrl systems hs limited their intrductin int cmmercil griculture. Fr exmple, the pplictin f fungl egg pthgens fr G. pllid s; spre r mycelium suspensins, n clnized strw r brn prved t be unsuccessful (Sikr el l., 1990). Successful frmultin f fungi fr the bilgicl cntrl f fungl plnt pthgens hs been ccmplished by incrprting the fungl gent int lginte grnules; technique first used by Wlker nd Cnnick (1983) fr mycherbicides. Schuster nd Sikr (1992) demnstrted tht perthphytic fungl egg pthgens f G. pllid culd be incrprted successfully int field sil when frmulted in lginte grnules. The technique incresed bilgicl cntrl efficcy significntly under field cnditins. T mximize efficcy f the lginte frmultin system hwever, mre detiled studies re needed n the interreltinship between the nemtde, fungi nd lginte in the sil ecsystem. The fl1wing gls were set t gin better understnding f this interreltinship : i)effect ffungl pthgens incrpr- ISSN / /92/05/ $ 2.70/ GUlhier-Villrs - ORSTOM 449

2 R. P. SChUSler & R. A. Sikr ted int lginte grnules n nemtde ppultin dynmics; ii) influence f lginte frmultin n survivl nd grwth f fungl pthgens in field sil, nd iii) determintin f ptimum cnditins fr fungl infectin f eggs. Mteril nd methds GENERAL METHODS Is lte nd cullivtin f lhe fungus A nn-sprulting fungus (Fs), riginlly islted frm G. pllid eggs nd initilly shwn r cuse high levels f infectin f G. pllid eggs in in vitr tests (Sikr el l., 1990), ws used in ll experiments. The islte cused high levels f infectin when pplied in lginte grnules r field sil in pt experimenrs. The islte ws cnsidered r be perthphytic pthgen killing eggs befre clniztin by the ctin f txins (Schuster & Sikr, 1992). Inculum ws prduced by : 1) grwing the islte n 1 % ptr dextrse gr (PDA) in Petri dishes t 25 C fr 5 dys, 2) inculting individul 1 litre screw cp culturing flsks cntining 500 ml f glycerine-peptne nutrient brth with the fungl bimss nd gr f ne Petri dish, nd 3) incubting the cultures n rtry shker fr 7 dys t 21 C. Fermenttin ws terminted when pprximtely 40 g fresh weight (0.85 g dry weight) f fungl bimss ws present in the flsk. Prduin f lginte grnules The fungus ws incrprted int lginte grnules s described by Schuster nd Sikr (1992). In ll but ne experiment, the grnules cnrined the culture medi glycerine-peptne brth plus 2 % brn. Actul fungl bimss in the grnules ws pprximtely g fresh weight/grnule. An pplictin rte f 1 % (w/w) cnsisted f 330 grnules/0 g sil equivlent t 1.7 g fungl fresh weight/0 g sil. The fllwing nutrient cmbintins were used in the lginte grnules in the test t determine the effect f nutrient level n bilgicl cntrl: 1) ABNF = lginte + culture medi + whet brn + Fs; 2) ABN lginte + culture medi + whet brn; 3) AF = lginte + Fs; 4) A = lginte. The fungus ws seprted frm the brth in the grnules withut nutrients (AF), using 0!lm sieve nd the fungus resuspended in distilled wter. Fungl density per unit sil fter tretment ws nt mesured, becuse fungl grwth ut f the grnules s mycelium is dynmic prcess vrying quickly within shrt perid f time. This mkes quntifictin f specific fungus in nn-sterile field sil, withut specific mrking techniques r selective, nerly impssible. Evlutin Cysts were seprted frm the sil using the wet screen-decnttin methd (mdified frm Ayub, ) nd the cysts seprted frm the remmmg rgnic mtter by flttin in MgSO~ t density f g/ml. Bilgicl cntrl ws determined by estimting fungl infectin levels f eggs fllwing crushing f the cysts in hnd-held tissue hmgenizer. The eggs frm the crushed cysts were cllected n 25!lm sieve nd the resulting egg suspensin plted nt gr pltes. The degree f infectin ws determined using the technique described by Schuster nd Sikr (1992). The sil used ws mixture f cyst free field sil nd snd CI: 1). The experiments were cnducted in either the greenhuse r in climtic chmbers t men temperture f 21 C with 16 h illumintin. EXPERIMENTAL DESIGN Effe f fungl frmuli in in lginte grnules n pllo rl weighl, nemlode emergence nd penelrlin G. pllid sil frm greenhuse stck cultures ws dded t field sil : snd-mixture CI: 1) until pi f 1500 eggs nd juveniles in cysts per 0 g f sil ws btined. Greenhuse plstic pts Il cm in dimeter were then filled with 600 g f this sil mixture. Ech tretmenr ws replicted eight times. Alginte grnules (ABNF; ABN; AF; A) were incrprted inr the sil t 1 % (w/w) prir t plnting by thrughly mixing the ttl munt f sil required fr ech tretment with the grnules befre ptting (4.8 kg sil : 48 g lginte grnules). The nemrde infested cntrls were nt treted with grnules. One certified seed tuber f the cultivr" Hns ", 3.5 cm in dimeter, ws plnted int ech pt. Fresh rt weight ws determined 2 r 8 weeks fter plnting. The effect f the Fs-lginte frmultin n htch ws determined tw weeks fter plnting. The pts were flded tw times within 3 h with 150 ml distilled wter nd the slutin cllected frm belw the dringe hle in the pts. The slutin ws then pre-filtered n cttnwl filter (Schleicher & Schuell Nr ) nd pssed thrugh 0.2!lm filter (Srtrius SM 11307) in sterile filtrtin unit (Srtrius SM 165). The sterile slutin cllected frm ech plnt ws stred seprtely in the icebx t 5 C in plstic jrs. The exudtes frm ech replicte were tested individully fr their htch stimulus ctivity t 25 % dilutin with 25 cysts in fur replictes in glss vils. The number f emerged juveniles s well s the number f eggs nd juveniles remining in the cysts ws determined fter 2 weeks nd the percent emergence clculted. Penetrtin ws determined by stining the nemrdes in rts in n cid fuchsin - lcric cid slutin in micrwve yen (Hper, 1990). The rts were blended 2 x s t rpm in Wring Blendr (Bender & Hbein) nd the number f juveniles tht hd penetrted the rt tissue determined using stere micrscpe (0 x). Fundm. ppl. Nemwl.

3 Egg plhgenic fungus LOwrd Glbder pllid Fungl egg clniztin s ffeeted by cyst ge nd time Results frm ur studies (Schuster & Sikr, 1992) demnstrted, tht the level f infectin f G. pllid eggs frm the greenhuse stck culture ws cnsistently belw 2 %. The level f infectin in cntrls with pplictin f grnules withut fungus ws eqully lw. Cntrls with fungus free grnules were nt used in the fllwing experiments. The number f newly frmed G. pl/id cysts nd the level f infectin with the sme lginte tretment, pplictin time nd pplictin rte frm different wn experiments were cmpred sttisticlly using nnliner regressin. This prcedure ws used, becuse f the lrge degree fvritin in level f egg infectin existing between the experiments. The Fs-lginte frmulted grnules were pplied t 1 % w/w t plnting t sil inculted with 9 mnth-ld cysts (1500 eggs nd J2/ 0 g f sil). Eight Il cm plstic pts were filled with 600 g f the treted sil nd plnted with ne ptt tuber. After eight weeks in the greenhuse, the femles nd cysts were extrcted nd then divided int three grups: 1) 9 mnth ld cysts - thse used initilly s inculum, 2) beige cysts, nd 3) white femles - the ltter tw grups newly frrned during the curse f the 8 weeks test perid. The white femles, beige cysts nd 9 mnth ld cysts frm ech pt were then exmined seprtely fr level f Fs egg infectin s described befre. T study the bility f Fs in lginte grnules t infect eggs f different ges, infectin f eggs f 9 mnth ld cysts nd thse in white femles were cmpred. Hundred grms f sil per replicte were treted with 1 % Fs-lginte grnules nd inculted with fifteen newly frrned white femles cntining eggs r 9 mnth ld cysts. The tretments were replicted fur times nd the level f infectin determined fter 2 weeks. Fungl grwth thrugh field sil The bility f the fungus t vercme nturl fungistsis, grw specific distnce frm the incultin pint f the grnule nd still infect nemtde eggs ws mesured using templte system designed by Sikr et l. (1990). Using the templte, the cysts were plced t predeterminted distnce f 0, 0.5 nd 1 cm frm the Fs-lginte grnules. The level f infectin ws determined fter 3, 7, r 14 dys s lredy described. Ech distnce ws tested with 15 cysts nd grnules in ne Petri-dish nd ech distnce replicted fur times. Results EFFECT OF FS-ALGINATE GRANULE FORMULATION ON ROOT WEIGHT, EMERGENCE AND PENETRATION OF PO TATO The rt weight f ptt ws drsticlly reduced 76.4 % (ABNF 82.5 %; ABN 77.4 %; AF 69.9 %; A 75.6 %) ver the untreted cntrl 2 weeks fter pplictin fllwing the pplictin f lginte grnules with r withut Fs (Tble 1). There were n significnt differences between the individul lginte tretments. Hwever, the negtive effect n rt weight ws nt evident fter 8 weeks in thse tretments cntining dditinl nutrients (ABNF nd ABN) when cmpred t the cntrl. Cnversely the rt weight in the tretments withut nutrient (AF nd A) were still significntly lwer fter 8 weeks. Similr results were btined in htching tests cnducted with exudtes cllected 2 weeks fter plnting frm the sme experimentl pts (Tble 1). Emergence ws reduced 89.2 % (ABNF 94 %; ABN 89.2 %; AF 86.5 %; A 87.1 %) in the lginte tretments vs the cntrl. There were n differences in emergence between the lginte tretments. Durtin f infeetivity Durtin f ctivity f the sterile islte (Fs) in field sil ws deterrnined by d ding 1 % (w/w) lginte grnules cntining Fs per replicte t 0 g f field sil. Fifteen cyst/replicte were inculted 0, l, 2, 3, 4, 6 r 8 weeks fter pplictin. Ech tretment ws replicted fur times nd fungl egg infectin ws deterrnined 2 weeks fter cyst incultin. The durtin f fungl infectivity ws exmined by tw pprches : 1) 0 g f field sil cntining 1 % Fs-lginte grnules ws inculted with fifteen nine-mnth-ld cysts nd the cysts remved fter 2 r 8 week intervls nd the level f infectin nd number f eggs/cyst deterrnined r 2) the cysts were extreted frm the sil fter 2 weeks expsure te the fungus, reinculted int field sil withut Fs-lginte inculum nd infectin nd number f eggs/cyst deterrnined 8 weeks fter the first expsure t the fungus. The tretments were replicted ten times. Tble 1. Effect f different nutrients nd the sterile fungus (Fs) in lginte grnules n rt weight 2 nd 8 weeks fter pplictin, n htch nd n penetrtin f G. pllid. Rt weight (g). Cntrl Tretments 2 weeks 8 weeks hlch (%) penetrtin ABNF 0.70 b b 181 b ABN 0.87 b b 140 b AF 0.95 b 2.89 b 13.1 b 198 b A 0.64 b 3.66 b 12.9 b 330 b Within clumn, mens fllwed by the sme letter re nt significntly (p = 0.05) different ccrding t Duncn's Multiple Rnge Test. Vl. 15, no

4 R. P. Schusler & R. A. Sikr 40 Ihfectin (%) 1 >1( >1( 30 >1(>1( '" >1( n = 60 r = Infectin (Of) / / / - / c- V / c Number f cysts Fig. 1. Crreltin between the number f newly frmed G. pllid cysts nd the degree f egg infectin in different pt experiments with the sterile fungus (Fs) t the sme munt f lginte grnules pplied ( r = 0.71; n = 60). Cmprble results were btined in the penetrtin tests (Tble 1). A distinct reductin f 82.2 % (ABNF 89.2 %; ABN 91.5 %; AF 85 %; A 62.9 %) ws mesured in the lginte grnule tretments when cmpred t the cntrl. DYNAMICS Of COLONIZATION The results frm different wn experiments with the sme lginte tretments, pplictin time nd pplictin rte were cmpred sttisticlly using nnliner regressin. There ws c1er negtive crreltin between the number f newly frrned cysts nd level f infectin. Infectin f mre thn % ws nly detected when less thn 500 cysts per replicte were present in the pts (Fig. 1). When the Fs islte in lginte grnules ws pplied t plnting, nd the cysts in the tests were divided int three grups s t ge, distinct trend ws detected. There ws definite difference between the infectin leve1s with the Fs islte in eggs in newly frrned cysts nd white femles vs 9 mnth-ld cysts (Fig. 2). Fungl infectin, deterrnined 8 weeks fter pplictin, shwed tht the newly frmed beige clred cysts nd white femles hd infectin rtes f 3 % nd 0.38 % s cmpred t 45 % detected in the 9 mnth-ld cysts initilly used s inculum. The hypthesis generted in the previus test, tht the fungus ws unble t infect the eggs in femles r newly frmed cysts ws nt verified in the study with 9 mnth ld cysts nd newly frmed white femles with eggs. The results demnstrted tht pprximte1y the sme per- c- V b C / /1 v'- / V / /7 ld cysts new cysts white femles Fig. 2. Effect f the sterile fungus (Fs) pplicted t plnting in lginte grnules n the degree f egg infectin f inculted 9 mnth ld cysts, n the newly frmed brwn cysts nd n white femles f G. pllid 8 weeks fter pplictin. Mens with the sme letter re nt significntly different (p = 0.05) ccrding t Duncn's Multiple Rnge Test. centge 28.8 nd 29.5 % f eggs in the yung nd ld cysts were infected, respectively (Fig. 3). The incrprtin f cysts int Fs-lginte treted sil t different time intervls fter fungl pplictin demnstrted c1erly tht the level f infectin f the eggs ws nt due t difference in the ge f eggs in the inculum. The dt shwed tht the Fs islte is nly ble t infect eggs effective1y fr 3 weeks in field sil fter incultin in lginte t 1 % w/w. There is definite decrese in the level f infectin f eggs fter 28 dys in field sil (Fig. 4) Infectin (Of) V n.s. V V V / V V- V IV / white femles cysts Fig. 3. Effect f the sterile fungus (Fs) in lginte grnules n the egg infectin f 9 mnth ld cysts nd white femles f G. p/lid 2 weeks fter pplictin (n.s. = nt significnt). 452 Fundm. ppl. NemlOl.

5 Egg plhgenic fungus lwrd Glbder pllid Infectin (Of) v : <---- ~I... ~~ r-- f---- ~<---- C f---- C ~ Weeks fter pplictin f grnules where the distnce between the cysts nd the Fs-lginte grnules were exctly cntrlled. Fs infectin levels f up t 67.5 % were detected fter 14 dys when the cyst were in direct cntct with the Fs-lginte grnule (Tble 2). The mximum level f infectin t distnce f 0.5 nd 1.0 cm between the grnule nd the cyst f 30 % ccurred fter 7 dys. Infectin levels did nt increse with time therefter. Tble 2. Effect f time nd distnce f grnule frm cyst n infectin f G. pllid eggs by the sterile fungus (Fs) pplied in lginte grnules. (Degree f infectin (%).) Time fter pplictin f lginte grnules Fig. 4. Effect f time between pplictin f the sterile fungus (Fs) in lginte grnules nd the incultin f G. pllid cysts n the degree f egg infectin. Mens fllwed by the sme letter re nt significntly different (p = 0.05) ccrding t Bnferni - Hlm Test. Distnce (cm) 3 dys 7 dys dys 14 dys d 47.5 b e 36.8 cd 36.0 cd 39.8 c e 37.0 cd 35.8 cd 33.5 cd The results frm the experiments invlving different durtins f fungl expsure verified this dynmic prcess. In Figure 5, il tretments shw equl levels f infectin ( %). Of imprtnce is the tretment 2-8, becuse the cysts were seprted frm the fungl inculum fter 2 weeks f expsure. The number f eggs/cyst in the tretments exmined fter 8 weeks ws pprximtely 30 % lwer thn the cunts fter 2 weeks. Htching ws nt bserved during the experiment. The imprtnce f nutrient bse fr Fs survivl nd spred thrugh the sil ws demnstrted in the studies Infectin (Of) 0-' '-- 2 weeks 8 weeks tretments Inlectin _ eggb/cyst n. f eggs/cyst 36 -'---~----':'----':' ==----=---T '''' weeks Fig. 5. Egg infectin by the sterile fungus (Fs) nd number f eggs!cyst f G. pllid dependent n the time f exmintin nd the durtin f fungl presence in sil. Mens, seprtely fr egg infectin nd number f eggs!cyst, fllwed by the sme letter re nt significntly (p = 0.05) different ccrding t Duncn's Multiple Rnge Test. Mens fllwed by the s me lelter re net signifïcntly CP 0.05) different ccrding te Bnferni - Hlm Test. Discussin The results shwed direct influence f lginte used in the frmultin f egg pthgeruc fungi n initil rt grwth ptterns. Similr results were btined by Lhmnn (1989) with endprsitic fungi in lginte grnules fr cntrl f G. pllid. The reductin in rt grwth des nt seem t be due t direct txic effects n rt tissue but t retrdtin in germintin r spruting f the tuber. Rt grwth my be reduced by extensive fungl nd bcteril grwth rund the grnule, which cuid lter the COz/Oz rti leding t initil suppressin f germintin nd rt extensin. The negtive effects f lginte n rt grwth is nt detectble fter 8 weeks. In this time intervl the nutrients in the grnule re metblized by the etivity f the fungl egg pthgen nd sscited sil micrflr. This lss in nutrient vilbility frm the lginte grnule with time is ls reflected in the lss f fungl infectivity f eggs fter 4 weeks s weil s in the visible reductin in the size f the grnule fter 8 weeks. The cmbined reductin in rt weight, rt exudte levels nd therefre htch prbbly is respnsible fr the decrese in penetrtin in the lginte tretments. Rwsthme nd Brdie (1987) shwed reduced htching with smller rt systems. Furthermre Perry nd Clrke (1981) demnstrted tht penetrtin is direcùy ffected by fctrs ltering htching rte. The reductin in newly frmed cysts detected by Schuster nd Sikr CI 992) when fungl egg pthgens were pplied t field Vl. 15, no

6 R. P. Schuster & R. A. Sikr sil in lginte were prbbly ls due t the negtive effects f lginte n rt grwrh. Our hypthesis tht lginte bsed fungl frmultins pplied t the time f plnting culd be effective in bilgicl cntrl f the eggs in newly fnned cysts prved t be invlid. Neither regressin f infectin rte t number f cysts nr dditinl experimenttin shwed significnt levels f infectin f eggs in newly frmed cysts. The lck f significnt fungl grwrh in the sil fter 4 weeks in the cntrl tests clrifies why cntrl fter cmpletin f the life cycle is impssible. The fungus hs the bility t grw quickly ut f the lginte grnule int the sil when supplied with nutrients, but is then suppressed with time by ther cmpetitrs. The sensitivity f the islte t fungistsis my lie in its sterile nn-sprulting chrcteristics, in cntrst fr exmple t Verticillium chlmydsprium tht prduces chlmydspres. The results f test cmpring infectin between 2 nd 8 weeks fter fungl pplictin in lginte, demnstrted, tht n in-cyst infectin dynmic leding t incresing rtes f egg infectin des nt tke plce. The level f infectin within the cyst in this time intervl is nt dependent n the presence f the fungus in the sil, s demnstrted by the fct tht the sme levels f infectin were detected when cysts were cnstntly expsed t the fungus fr 8 weeks r were seprted frm the fungl inculum fter 2 weeks expsure. Hwever, there ws significnt reductin in the number f eggs nd juvenile per cyst fter 8 weeks when cmpred t 2 weeks expsure, becuse the eggs infected fter 2 weeks re degrded nd n lnger detectble. Knuth (1986) shwed tht V. chlmydsprium cmpletely clnized the eggs f Heterder vene fter 30 dys t 22 c. The thereticl in-cyst dynmics f fungl develpment in H. schchtii eggs ws prpsed by Nicly (1989) nd is cnsidered slw prcess. The reductin in number f eggs per cyst in ur experiment cn nt be due t htch, becuse G. pllid htches nly in the presence f hst rt exsudtes (Perry, 1986). The use f the templet fr precise mesurement f fungl grwrh thrugh sil nd detennintin n f infectin s influenced by time f expsure nd distnce between fungl inculum nd hst, demnstrted tht the mximum infectin levels were reched ver lng distnces fter 7 dys, regrdless f initil inculum density. The results ls shwed tht infectin levels incresed drsticlly when the cyst were plced directly djcent t the fungl inculted lginte grnule. The fct tht infectin levels did nt increse prgressively indictes n ther mechnism f infectin nd nt true prsitism. In dditin, infectin levels incresed prgressively with incresing inculum levels. The results demnstrte the presence f fungl bsed txic metblites tht initilly cuse egg mrtlity, with the fungus mst prbbly entering the egg fter deth. Nicly nd Sikr (1989) suggested tht mny fungi detected in 454 eggs re grwing sprphyticlly n ded tissue which my hve been initilly killed by perthtrphic behviur. Sikr et l. (1990) discussed the different definitins f egg clniztin nd discussed the perthphytic mde f infectin sscited with fculttive pthgens. The infectin rtes in ur tests were cnsistently nel' 30 %. The nly exceptin beeing thse tretments where the cysts were plced directly djcent t the lginte grnule. This indictes tht nly certin prtin f the eggs in the cyst re highly suspectible t infectin t ny ne time, nd prbbly indictes the clniztin f nn-vible eggs present in the cyst. Our results demnstrte srne f the dvntges nd limittins f using fungi fr the bilgicl cntrl f G. pllid n ptt. The dt cnfinned erlier findings, tht infectin f G. pllid eggs by the fungus ws bsed n perthphytic mde-f-ctin. Alginte s test fnnultin, lthugh cusing srne initil germintin nd rt grwrh inhibitin, prved t be n elegnt technique fr studying bilgicl cntrl nd fungl behviur twrd cyst nemtdes in field sil. Furthennre, the results shwed tht pplictin f perthphytic pthgen in lginte 8 weeks prir t plnting cn increse bilgicl cntrl by reducing the number f vible eggs in dnnnt cysts, thereby reducing nemtde erly rt infectin. The presence f txic metblites prduced by fungl pthgens is ften verlked in stndrd bissy tests fr bilgicl cntrl gents. Bissy systems used fr detectin f fungl egg pthgens r prsites shuld be exmined fr their suitbility t detect rgnisms with predetermined mde-f-ctin. The mechnism by which prsitism r pthgenicity ccurs beeing imprtnt fr ptimizing bicntrl. References AYOUB, S. M. (1977). Plnt NemLOlgy. An griculturltrining id. Scrment, USDA, 158 p. DACKMAN, C. (1990). Fungl prsites f the ptt cyst nemtde Glbder rslochiensis : Isltin nd reinfectin. J. NemLOI., 22 : GOSWAMI, B. K. & RUMPENHORST, H.]. (1978). Asscitin f n unknwn fungus with ptt cyst nemtdes, Glbder rslochiensis nd Glbder pl/id. NemLOlgic, 24 : HOOPER, D. ]. (1990). Extrcting nd prcessing f plnt nd sil nemtdes. In : Luc, M., Sikr, R. A. & Bridge, ]. (Eds). Plnt prsitic nemlodes in subtrpicl nd trpicl griculture. Willingfrd, UI<. CABI : KERRY, B. R. (1974). A fungus sscited with yung femles f the cerl cyst nemtde, Helerder vene. Nemllgic, : 259. KNUTH, P. (1986). Untersuchung vn Verbreitung und ppultinsdynmischer Bedelllung pi/zlicher Anlgnisten vn Fundm. ppl. NemLOI.

7 Egg plhgenic fungus IOwrd Glbder pllid Heterder vene Wllenw. in Bden-Würtemberg. Disserttin Hhenheim. NICOLAY, R. (1989). Einj7uss vn rgnischer Düngung und UmweltfklOren uf die Prsitierung vn Eiern des RübenzystennemlOden Heterder schchtii (Schmidt) durch Pilze. Disserttin, Univ. Bnn: 139 p. NICOLAY, R. & SIKORA, R. A (1989). Wirkung vn Gründüngung und nderen rgnischen Düngern uf die Ppu 1tinsdynmik des Rübenzystennemtden Heterder schchlii (Schmidt). Anz. SchiidlK., PjlSchUlz, UmwSchulZ, 62 (6) : NIGH, E. A, THMAsN, 1. J. & VAN GUNDY, S. D. (1980). Acremnium slriclum nd Fusrium xysprum, rw fungl prsites f eggs f Helerder schchlil~ the sugr beet cyst nemtde. Nemlrpic, : 4. PERRY, R. N. (1986). Physilgy f htching. In : Lmberti, F. & Tylr, C. E. (Eds). CYSI nemlodes : PERRY, R. N. & CLARKE, A J. (1981). Htching mechnism f nemtdes. Prsitlgy, 83 : RAWSTHORNE, D. & BRODlE, B. B. (1987). Mvement f ptt rt diffuste thrugh sil. J. NemlOl., 19 : ROESSNER, J. (1987). Pilze is Antgnisten vn Glbder rslochiensis. NemlOlgic, SCHUSTER, R.-P. & SIKORA, R. A. (1992). Influence f different frmultins f fungl egg pthgens in lginte grnules n bilgicl cntrl f Glbder pllid. Fund. ppl. NemlOl., 15 : SlKORA, R. A, HIEMER, M. & SCHUSTER, R.-P. (1990). Reflectins n the cmplexity f fungl infectin f nemtde eggs nd the imprtnce f fculttive perthphytic fungl pthgens in bilgic! cntrl f Glbder pllid. Medded. Fc. Lndbuww. Rijksuniv. Gent, 55 : WALKER, H. L. & CONNICK, W. L. Jr. (1983). Sdium lginte fr prductin nd frmultin f mycherbicides. Weed Sci., 31 : Vl. 15, no