AGRONOMIC STUDIES ON THE POPULATION DYNAMICSOF VERTICILLIUM DAHLIAE

Transcription

1 AGRONOMIC STUDIES ON THE POPULATION DYNAMICSOF VERTICILLIUM DAHLIAE CENTRALE LAN D BO UWC AT A LOG US

2 Promotor: dr ir P.C. Struik, hooglerr in de kkerbouw vn de gemtigde klimtsgebieden Co-promotor: dr ing. K. Schölte, universitir docent bij de vkgroep Agronomie

3 /vj/oö^ol (<^s9 L.Mol AGRONOMIC STUDIES ON THE POPULATION DYNAMICS OF VERTICILLIUM DAHLIAE PROEFSCHRIFT ter verkrijging vn de grd vn doctor in de lndbouw- en milieuwetenschppen, op gezg vn de rector mgnificus, dr C.M. Krssen, in het openbr te verdedigen op vrijdg 23 juni 1995 des nmiddgs te vier uur in de ul vn de Lndbouwuniversiteit te Wgeningen (»Vi - i UIÖZ-

4 Omslg: Boeren n het werk op een veld. Vincent vn Gogh, Arles Vincent vn Gogh Stichting/ Vn Gogh Museum, Amsterdm. CIP-DATA KONINKLIJKE BIBLIOTHEEK, DEN HAAG Mol, L. Agronomie studies on the popultion dynmics of Verticillium dhlie I L. Mol. -[S.l. : s.n. Thesis Lndbouwuniversiteit Wgeningen. - With réf. - With summry in Dutch. ISBN Subject hedings: popultion dynmics / Verticillium dhlie I pthogens. The reserch described in this thesis ws prt of the reserch progrmme of the CT. de Wit Grdute School Production Ecology. BJBIJLOTÜtlFK

5 STELLINGEN 1. Tergdringing vn het gebruik vn grondontsmettingsmiddelen leidt tot een toenme vn de schde door Verticilliwn dhliein het gews rdppel. 2. Wortels vn cultuurgewssen stimuleren de kieming vn microscleroti vn V. dhlie in de bodem, mr dit proces heeft voor de beheersing vn de ziekte nuwelijks betekenis. Dit proefschrift 3. De virulentie vn een Verticilliwn dhliepopultie in de bodem hngt f vn de teeltgeschiedenis vn het perceel en het te telen gews. Dit proefschrift 4. De reproduktie vn V. dhlieis, onder gunstige weersomstndigheden, lger bij mechnische loofdodingstechnieken dn met chemische loofdoding. Dit proefschrift 5. Bij de veldkeuring vn pootrdppelen is visuele beoordeling op ntsting door Verticillium dhlie onmogelijk, onnodig en elk resultt misleidend. 6. Bij de Lndbouwuniversiteit hoort de boer de hoogste troef te zijn. 7. Regelgeving door de overheid met betrekking tot de lndbouw wordt beïnvloed door zoveel prtijen die een te geringe kennis vn de prktijk hebben dt regels vk tot te hoge kosten voor de ondernemers leiden en te weinig doeltreffend blijken. 8. Door een gebrek n specifieke kennis bij milieu- en ntuurbeschermingsorgnisties heeft veel kritiek op boeren en jgers een emotionele bsis gekregen. Door bij propgnd ook op de emoties vn het publiek in te spelen wordt bewust de kloof tussen opvttingen vn de prtijen vergroot.

6 9. Alleen grondgebonden produktie vn plntrdige en dierlijke produkten behoort lndbouw genoemd te worden. Als deze definitie wordt nvrd, wordt de lndbouw meteen een stuk milieuvriendelijker. 10. Flexibilisering vn de werktijden vn chuffeurs in het streekvervoer kn eenvoudig en zonder extr kosten worden ingevoerd door de onregelmtigheidstoeslgen n te pssen en elke chuffeur individueel deze toeslg uit te betlen. (Dit ltste in tegenstelling tot de bij sommige bedrijven nu gngbre gemiddelde onregelmtigheidstoeslg. ) 11. Een onmisbre vrdigheid vn de promovendus is dt hij het hndschrift vn de promotor goed en snel kn ontcijferen. 12. Door de ontwikkeling vn een goede methode om uien pnklr n te bieden kunnen veel trnen in de keuken worden voorkomen. 13. Mollen grven dieper dn menigeen denkt. 14. Vn het kunnen is slechts één bewijs: het doen! Stellingen behorende bij het proefschrift: "AGRONOMIC STUDIES ON THE POPULATION DYNAMICS OF VERTICILLIUM DAHLIAE" Leon Mol Wgeningen, 23juni 1995

7 ABSTRACT Mol, L., Agronomie studies on the popultion dynmics of Verticillium dhlie. Doctorl thesis, Wgeningen Agriculturl University, Wgeningen, The Netherlnds, X pp., English nd Dutch summries. Verticillium dhlie reduces crop yields by cusing erly senescence. The fungus survives in the soil s microscleroti formed on senescing tissue of colonised plnt prts. V. dhlie is endemic in mny soils becuse of the high potentil survivl of its microscleroti nd its wide host rnge. The primry im of the reserch ws to investigte the fesbility of reducing the inoculum density in the soil by stimulting the microscleroti to germinte. A second im ws to quntify the formtion of microscleroti on vrious crop species nd cultivrs, nd vrious prts of potto plnts. Hulm tretments to control the formtion of microscleroti on potto were tested nd finlly theoreticl model describing the long-term dynmics of the inoculum density in the soil ws developed nd tested. All crops investigted stimulted the germintion of microscleroti in the soil. Host plnts such s potto nd field ben induced more microscleroti to germinte thn non-host such s brley, but none of the crops ws ble to reduce the soil inoculum density effectively. The highest totl microsclerotil yield occurred in flx, followed by potto cultivrs; the other crops lgged fr behind. In potto, the mture eril prts hd the highest numbers of microscleroti. Potto cv. Element nd field ben proved to be most sensitive to their own isolte. In plots cropped with good hosts, soil inoculum density incresed very rpidly. Removing the debris of potto, field ben nd flx from the field ws effective in reducing the increse of the inoculum density. Mechnicl hulm tretments reduced the formtion of microscleroti more thn chemicl tretment. A theoreticl model built on the bsis of biologicl nd ecologicl principles gve very high correltion when it ws fitted to the dt obtined in long-term field experiment. This study provides quntittive bsis on the interctions between crops nd V. dhlie tht deserves to be expnded in further reserch. Keywords: crop rottion, crop species, crop debris, cultivrs, hulm killing, Hordeum vulgre, imge nlysis, inoculum density, Linum usittissimum, microscleroti, modelling, popultion dynmics, potto, removl, reproduction, resistnce, root density, root exudtes, root observtion, soil infesttion, Solnum tuberosum,tolernce, Verticillium dhlie, Vici fb, yield

8 Voor mijn vder

9 WOORD VOORAF De milieuvriendelijke beheersing vn ziekten en plgen in de lndbouw is niet nieuw. Al sinds meer dn 100 jr wordt getrcht door middel vn teeltmtregelen schde door pthogenen te beperken. Vruchtwisseling speelt hierbij een prominente rol. De vkgroep Agronomie vn de Lndbouwuniversiteit heeft l een lnge en "duurzme" trditie op het gebied vn vruchtwisselingsonderzoek. Sinds jren wordt hiern vormgegeven door dr ing. K. Schölte. Er mg mijns inziens dn ook gesproken worden vn de "School Schölte". Het is niet vreemd dt in het kder vn het dditioneel onderzoeksprogrmm vn het Meerjrenpln Gewsbescherming ook teeltkundig onderzoek is opgenomen. Immers, de gronomie is de discipline bij uitstek om bsiskennis uit verschillende deelgebieden op een zo hoog ggrtieniveu te integreren dt het verkregen eindprodukt een grote mtschppelijke relevntie krijgt. Het uitvoeren vn een promotieonderzoek is niet het werk vn de promovendus lleen. Zonder de hulp vn vele personen zou dit proefschrift nooit tot stnd zijn gekomen. Ik dnk hen llen hrtelijk hiervoor! Zonder nderen tekort te doen, wil ik een ntl mensen met nme noemen. In de eerste plts ben ik veel dnk verschuldigd n mijn begeleider en co-promotor dr ing. K. Schölte. Hij ws het die het onderzoeksvoorstel schreef en de ideeën hiervoor verzmelde. Kls, door jouw grote kennis, je nuchtere npk en je gerichte commentr ws het ltijd prettig om met jouw te werken en vn gedchte te wisselen. Een groot deel vn je schrse tijd heb je voor mij ingeruimd. Mijn promotor, professor dr ir P.C. Struik, dnk ik hrtelijk voor zijn stimulerende begeleiding. Nooit heb ik iemnd gezien die zo flexibel met een overvolle gend kn omgn. Mnuscripten wren ltijd binnen enkele dgen voorzien vn gerichte opbouwende kritiek. Pul, het is voor mij een eer om bij jou te mogen promoveren. Dr ir J. Vos bednk ik voor zijn kritische inbreng tijdens de tlrijke discussies en voor het becommentriëren vn de mnuscripten. Een specile plts bij de uitvoering vn de proeven werd ingenomen door de ssistent kkerbouw, dhr L. Hlstr. Lmmert, jouw grote prktische ervring, je interesse in en je kritische kijk op het onderzoek mkten het uitvoeren vn de proeven extr ngenm. De medewerkers vn onze proefccommodtie (nu onderdeel vn UNIFARM) onder leiding vn ing. L.A. Mol en zijn opvolger M. vn de Wrt dnk ik voor de verzorging vn de vele proeven. Specile dnk verdienen Jn vn der Pl die er voor heeft gezorgd dt mijn plnten nooit wtergebrek hdden en René Alles, Teus Bleijenberg, Henk vn Roekei, Wim vn der Slikke en Steven vn de Kleut voor het grondverzet en het verzorgen vn de veldproeven. Ton

10 Blokzijl en Herry Theunissen dnk ik voor het klren vn de vele technische klussen. Gerrit Besselink en Guido vn Hsselt dnk ik voor het tellen vn vele duizenden microscleroti. Medewerkers vn de vkgroep Fytopthologie dnk ik voor de prettige smenwerking en de nuttige commentren en dviezen. In het bijzonder geldt dit voor dr ir A.J. Termorshuizen. Ad, hoewel we n het zelfde onderwerp werkten, zijn we niet eikrs concurrent geworden, mr hebben we op een nuttige wijze smen kunnen werken. Bednkt hiervoor. Stgiir(e)s en studenten verlichtten het werk en zorgden voor bedrijvigheid om me heen. Mirnd Berendsen volgde een stge voor de Middelbre Lbortoriumopleiding te Arnhem. Henk vn Riessen werkte n het onderzoek mee in het kder vn een fstudeervk. Jochem vn Hlteren deed zowel zijn stge voor de Agrrische Hogeschool te Dronten ls een fstudeervk binnen mijn project. Ik heb vn het werk vn jullie veel profijt gehd en erg genoten. I thnk dr Mri Schütz for the prticiption in the experiments described in Chpter 2. I would like to express my deep grtitude to Dr O.C. Huismn from the Deprtment of Environmentl Science, Policy nd Mngement, University of Cliforni, Berkeley for giving me the opportunity to work for three months under his supervision in the United Sttes. Oen nd Mry Ann thnks for your hospitlity nd friendship! Het IPO-DLO dnk ik voor het beschikbr stellen vn de beeldnlyse-prtuur en ing. E.M.J. Meijer voor de ondersteuning bij het gebruik en de smenwerking bij het schrijven vn Hoofdstuk 3. Het ws zonder jullie hulp nooit mogelijk geweest om zoveel monsters te nlyseren. Dr C.H.J. Booij en dr W. vn der Werf dnk ik voor het becommentriëren vn de mnuscripten vn respectievelijk de Hoofdstukken 3 en 8. Mevr. J. Burrough-Boenish corrigeerde op nuwgezette wijze de Engelse tekst vn een ntl hoofdstukken. Ik bednk de VSB-bnk, het NWO en het Johnn Westerdijkfonds voor het subsidiëren vn buitenlndse reizen nr de Verenigde Stten en Isrël in 1992, 1993 en Het Hilbrnds Lbortorium voor Bodemziekten te Assen en het PAGV te Lelystd dnk ik voor het beschikbr stellen vn grond voor het verkrijgen vn gewspecifieke isolten vn V. dhlie. Ik drg dit proefschrift op n mijn vder. Hij heeft me betrokken bij de groei en de teelt vn gewssen en me geleerd ls boer nr gewssen te kijken. Ik heb dit de fgelopen jren ls onmisbr ervren. Leon

11 NOTE All ppers included in this thesis re submitted to or ccepted by vrious interntionl journls. The nme of the journl concerned is printed t the top of the first full pge of ech chpter or section. If possible, reference to the contents of the ppers should be mde by citing the originl publictions. As presented in this thesis they differ from the originl ppers in the following wys: 1. the 'keywords' of the individul ppers hve been combined into one list t the end of the 'Abstrct' ; 2. the cknowledgements re given in the 'Woord voorf'; 3. the 'References' of the generl introduction, the different ppers nd the generl discussion hve been combined into one list; 4. some minor chnges were mde to stndrdize presenttion. I thnk Kluwer Acdemic Publishers (Section 1.2, Chpters 2 & 3), Blckwell's (Chpters 5, 6 & 8) nd the editoril bords of Potto Reserch (Section 4.2; Chpter 7) nd the Netherlnds Journl of Agriculturl Science (Section 4.1) for their kind permission to include the ppers in this thesis.

12 CONTENTS 1 Generl introduction Introduction Life cycle nd ecology of Verticillium dhlie in potto Aim of the reserch Structure of the thesis 17 2 Effect of plnt roots on the germintion of microscleroti of Verticillium dhlie Use of root observtion boxes to ssess differences mong crops Quntittive nlysis of the luring effect of crops 31 3 Quntifiction of microscleroti of Verticillium dhlie in plnt mteril by 41 imge nlysis 4 Formtion of microscleroti on crops Formtion of microscleroti of Verticillium dhlieon vrious crops Formtion of microscleroti of Verticillium dhlieon vrious plnt prts of two 65 potto cultivrs 5 Effects of crop species, cultivrs, nd two isoltes of Verticillium dhlie on the 73 popultion of microscleroti in the soil, nd consequences for crop yield 6 Effects of crop rottion nd removl of crop debris on the soil popultion of 87 two isoltes of Verticillium dhlie 7 Effect of hulm tretments on the formtion of microscleroti of Verticillium 97 dhlie on potto 8 Theoreticl pproch to the dynmics of the inoculum density of Verticillium 105 dhlie in the soil: simple model nd its first test 9 Generl discussion nd conclusions 127 References 139 Summry 147 Smenvtting 153 Curriculum vite 159

13 CHAPTER 1 GENERAL INTRODUCTION

14 SECTION 1.1 INTRODUCTION Verticillium dhlie Kleb, is serious pthogen ffecting potto {Solnum tuberosum L.) in most countries where this crop is grown. The fungus survives in the soil by microscleroti tht persist for mny yers. The density of microscleroti of V. dhliein the soil minly depends on the cropping history, since the primry source of the inoculum is infested plnt debris. Plnt roots cn be colonised if microscleroti germinte in the vicinity of the root tip (Fitzell et l., 1980). Colonistion is followed by systemic infection of the vsculr system of the plnt, wherefter V. dhlie is dispersed within the host by conidi nd mycelil growth. Systemic infection by V. dhlie ffects plnt growth nd depresses plnt yield. Yield reduction is minly cused by closure of the stomt nd erly senescence of the cnopy fter blockge of the vsculr system of the hulm (Bowden et l., 1990; Hverkort et l., 1990). Microscleroti re formed bundntly in infested tissue upon deth of the host plnt. Since V. dhliehs very brod host rnge, the influence on soil infesttion of ll crops in the rottion must be considered. Verticillium dhlie shows synergistic interctions with other pthogens, such s endoprsitic nemtodes: Globoder spp., Prtylenchus spp. nd Meloidogyne spp., nd the fungi: Colletotrichum coccodes, Fusrium spp. nd Rhizoctoni solni (Riedel & Rowe, 1985; Evns, 1987; Schölte, 1989; Schölte & s'jcob, 1989). Thus, it my be expected tht reduction in the use of nemticides will indirectly increse the dmge cused by V. dhlie. A lower cropping frequency of potto my result in lower densities of V. dhlie in the soil, but is not n ttrctive control method to the frmer, becuse in The Netherlnds potto is mjor csh crop. Therefore, other methods to control the inoculum build-up in the soil deserve to be investigted. Since in Dutch crop rottions potto is the crop tht suffers most from V. dhlie nd since the pthogen cn form lrge number of microscleroti on potto tissue, the project described in this thesis is focused on this crop. A welth of literture concerning life cycle nd ecology of V. dhlie is vilble. A short review is given in Section 1.2. The im of the reserch is described in Section 1.3, fter which the structure of the thesis is given (Section 1.4).

15 In: Hverkort, A.J. & D.K.L. McKerron (eds). Ecology nd modelling of potto crops under 5 conditions limiting growth. Kluwer Scientific Publishers, Dordrecht, The Netherlnds: SECTION 1.2 LIFE CYCLE AND ECOLOGY OF VERTICILLIUM DAHLIAE IN POTATO L. Mol nd A.J. Termorshuizen Summry Verticillium dhlie is serious pthogen in most countries where potto is grown. The density of microscleroti of V. dhlie in soil minly depends on the cropping history. Plnt roots cn be colonised if microscleroti germinte in the vicinity of the root tip. Colonistion is followed by systemic infection of the vsculr system of the plnt. During colonistion of the root cortex nd systemic infection of the plnt there re interctions with mny soil orgnisms. Culturl prctices cn lessen the colonistion of the roots nd the severity of the disese. In the vsculr system of the plnt, V. dhlie is dispersed by conidi nd mycelil growth. Wilting symptoms pper fter the rections of the plnt to the presence of the pthogen. Yield reduction is minly cused by closure of the stomt nd erly senescence of the cnopy fter blockge of the vsculr system of the hulm. The fungus forms microscleroti on ded plnt tissue. Externl climtologicl fctors nd hulm killing prctices hve lrge influence on the number of microscleroti formed per unit hulm mteril. Since V. dhlie hs very brod host rnge, ttention should be pid to the control of this pthogen in ll crops in rottion. In some crops host specificity hs been found, but this is grdul property. Introduction Diseses cused by Verticillium dhliekleb, re wide-spred throughout the world, wherever susceptible crops re grown, nd re of economic importnce in most countries (Pegg, 1984). Verticillium dhlie is supposed to be the mjor component of the potto erly dying complex tht dmges crops by cusing erly mturity of the crop by wilting. Hosts include ll dicotylodonous plnts, with the most importnt crops ffected being potto, cotton, egg-plnt,

16 Section 1.2 tomto, mint, nd olive. Verticillium dhliesurvives by mens of microscleroti (MS). The high survivl potentil of MS nd the wide host rnge mke V. dhlieendemic to mny griculturl soils (Powelson, 1970). Verticillium dhlie is clssified s soil invding or root inhbiting fungus (Powelson, 1970). These fungi re chrcterised by prsitic phse on the living host plnt (Fig. 1: I), nd by sprophytic phse fter the deth of the host (Powelson, 1970) (Fig. 1: II). The two phses will be discussed seprtely. Dynmics of the soil popultion of V. dhlie A schemtic representtion of processes concerning the MS popultion in the soil is shown in Fig. 1: II. The popultion of MS in soil vries in composition nd density depending on the cropping history of the field. After germintion of microsclerotium, the hyph my infect plnt root. Due to their low competitive sprophytic bility, the mjority of the hyphe re not successful in reching plnt root nd they die. The next prgrphs will be focused on: survivl of MS (Fig. 1: ), germintion of MS (Fig. 1: b), nd colonistion of plnt roots (Fig. l:c). Survivl of MS (Fig.l: ) Ben-Yephet nd Szmulewich (1985) reported tht MS of V. dhlie survive longer in the field thn in the lbortory. After 5 yers of storge of ir-dried soil smples t C no V. dhliecould be detected, wheres 4% of the originl popultion density remined vible fter 7 yers of crop rottion. Wilhelm (1955) found tht V. dhlie persisted for 14 yers in field soil with no hosts present. The long persistence of the fungus in the field is probbly due to its bility to colonise nd produce new MS on the root systems of nerly ll plnt species including monocots (Mrtinson & Horner, 1962). The density of MS in soil ws monitored by Itoh et l. (1989) following Chinese cbbge by using infested soil. A first order rte eqution ws fitted to the observed decrese in the number of MS which ws more rpid t higher tempertures. A liner reltion ws observed between the logrithmic vlue of the hlf life nd the temperture in the rnge of 5-31 C. Survivl of MS of V. dhlieppers to be best under ir-dry conditions (Coley-Smith& Cooke, 1971). The mechnism for survivl hs not been elucidted. There is cler reltion between the presence of melnin in orgnisms nd their persistence (Bloomfield & Alexnder, 1967). Temperture my exert n indirect influence on survivl through direct effects on

17 Life cycle nd ecology of V. dhlie in potto f -reduced photosynthesis -reduced growth -oneven chlorosis nd uneven deth of leves -vessel coting -occlusion with gels -vsculr discolortion g prticulr physiologicl stge of the plnt -permetion of surrounding tissue -formtion of MS in both bove nd below ground prts -cultivr differences -dormncy -physiologicl heterogeneity -decomposition processes PLANT GROWTH breking of dormncy -multiple germintion -root exudtes -sporultion in the vsculr I system -germintion of conidi -dispersl in the I vsculr system s -colonistion of the cortex of ny plnt -interction with soil microorgnisms -interction with other orgnisms -penetrtion of the endodermis -formtion of lignitubers Fig. 1. Schemtic life cycle of V. dhlie showing prsitic phse on host (I), nd sprophytic phse fter plnt deth nd in soil (II). The scheme is explined in the text. MS= microscleroti. germintion of MS (Coley-Smith & Cooke, 1971) nd decomposition of the plnt mteril they re embedded in to chieve their relese into the soil (Hncock & Benhm, 1980). The influences of the temperture nd decomposition re entngled. Decomposition is the result of more thn one process, nd n increse in temperture, within certin rnge, will ccelerte most biologicl processes. The relese of MS from colonised plnt debris lsts t lest one yer following its incorportion into the soil nd consequently ffects the inoculum density in soil. In two cultivted cotton-field soils studied by Evns et l. (1967), the density of MS in soil declined throughout the growing seson, but incresed gin t hrvest time when mechnicl dmge to colonised cotton plnts cused the relese of fresh MS. There ws further increse in the soil inoculum density when the ded cotton stlks were returned to the soil in preprtion for the next crop. Huismn nd Ashworth (1976) observed the soil inoculum density of V. dhlie in eight commercil fields with different cropping histories t bout monthly intervls for

18 Section 1.2 yers. High inoculum densities in soil persisted under continuous cultivtion of cotton. The inoculum density usully incresed rpidly following one yer of cotton, with higher inoculum density occurring in the second yer regrdless of the susceptibility of the subsequent crop. The slow relese of MS from decomposing plnt debris probbly cuses dely in the build-up of the inoculum density in soil. It is not known how the survivl of MS embedded in plnt mteril compres to the survivl of relesed 'free' MS. Presumbly, the deth rte is higher in bound MS becuse of the higher microbil ctivity in nd round the decomposing plnt debris. Germintion of microscleroti (Fig. 1: b) Microscleroti of V. dhlie hve no constitutive dormncy where development is delyed becuse of n endogenous property of the propgule (Pegg, 1974), but they do hve requirement during germintion for exogenous nutrients (Emmtty & Green, 1969) from root exudtes of either hosts or non-hosts (Schreiber & Green, 1963). Concentrtions of exogenous nutrients cn be influenced by orgnic mendments to the soil, which influence generl soil microbil ctivity nd concentrtion of soluble compounds in the soil solution. Incresed soil microbil ctivity hs been reported to reduce the proportion of germintion of microscleroti nd growth of germ-tubes of V. dhlie in the rhizosphere (Jordn et l., 1972). In greenhouse nd field experiments, mendments with chopped brley or ot strw t severl rtes reduced the inoculum density of V. dhlie in soil nd disese incidence in potto plnts (Tolmsoff & Young, 1959; Hrrison, 1976). The effect of orgnic mendments hs not been elucidted, but it hs been suggested to be n increse in production of ntifungl substnces by the enriched microflor of the rhizosphere (Curl & Truelove, 1986). However, the interctions between the pthogen nd the microflor in the rhizosphere re probbly more importnt, since there is still doubt tht ntibiotic substnces produced in soil cn endure microbil degrdtion for long enough to ct s effective inhibitors (Curl & Truelove, 1986). Microscleroti hve the bility to germinte more thn once. Frley et l. (1971) showed tht MS germinted nd sporulted every time fter re-moistening soil with sucrose solution or wter up to nine times. Germintion percentge nd the number of germ-tubes decresed with succeeding germintions. Where susceptible crop is plnted in field in soil nturlly infested with V. dhlie, the behviour of the MS is likely to be different from tht in experiments where rtificil inoculum is pplied (Menzies& Griebel, 1967). Unless severl yers hve elpsed since the lst susceptible crop ws grown, the inoculum my be mostly in the form of MS embedded in prtly decomposed plnt residues. Some of these MS my lredy hve undergone cycle of germintion nd be depleted, while others my be freshly exposed during

19 Life cycle nd ecology of V. dhlie in potto cultivtion opertions nd my go through pek of germintion s the host seedling roots re growing in their vicinity (Menzies & Griebel, 1967). Colonistion of plnt roots by V. dhlie (Fig. 1: c) Germintion hyphe of MS my infect susceptible hosts by penetrtion of the root cortex followed by systemic invsion of the xylem vessels (Powelson, 1970). Infectious hyphe of V. dhlieemerging from MS penetrte roots primrily ner the root tip nd in the root-hir zone (Fitzell et l., 1980; Gerik & Huismn, 1988). The density of colonistion t distnce of more thn 1cm from the root pex ppered to be constnt (Gerik & Huismn, 1988). Most of the colonies re removed when the roots re surfce-sterilised, indicting tht V. dhlie is predominntly restricted to superficil sites in the root cortex (Evns& Gleeson, 1973). Host-pthogen interctions A schemtic representtion of the host-pthogen interctions is shown in Fig. 1:1. After colonistion of the plnt root, the fungus crosses physicl brriers in the plnt to rech the vsculr system. During systemic infection, colonistion of the plnt my result in resistnce rections, yield reduction, nd, finlly, production of MS on the plnt debris. The next prgrphs will be focused on systemic infection of plnt roots (Fig. 1: d). systemic colonistion of the plnt (Fig. 1: e), plnt rections nd yield reductions (Fig. 1: f), nd reproduction of V. dhlie (Fig.l: g). Systemic infection of plnt roots (Fig. 1: d) In response to the initil invsion by the fungus, the inner tngentil wll of the epidermis becomes swollen (Bell, 1973). Occsionlly gum-like encpsultion zone my be present in the outer corticl cells of the plnt roots round the hyphe of V. dhlie; these formtions re referred to s lignitubers (Griffiths, 1973). Lignitubers result from the extrusion of vesicles from the protoplst into the re between the cell wll nd plsmlemm. When the fungus strts to penetrte the cell wll, the vesicles ggregte nd lose their individul identity s they form the lignituber. Subsequently, the fungl cell wll becomes surrounded by the lignituber, while the host plsmlemm pprently remins intct. In most cses the invding hyphe lyse, but in few cses successful penetrtion occurs nd the fungus proceeds through the lyer of corticl cells (Bell, 1973).

20 10 Section 1.2 Fungi tht successfully penetrte the cortex encounter second brrier t the endodermis (Bell, 1973). Most of the penetrting hyphe tht were not stopped by lignitubers re so t the endodermis; the few hyphe tht penetrte this brrier progress into the vsculr tissue nd invde the vessels by penetrting through the pits. Alterntively, hyphe my rech the vsculr system by colonising the young root tip, where the endodermis still hs to be formed. The systemic infection of the plnt root is the property determining the suitbility of the plnt s host. For exmple, susceptible mint (Menthspp.) roots showed more extensive systemic invsion, while the number of root cortex infections ws similr to tht of resistnt mint species (Lcy & Horner, 1966). Infection of plnt roots by V. dhliemy be influenced by other orgnisms. In most cses, potto erly dying cn be ttributed to complex of soil microorgnisms including V. dhlie, Globoder spp., Prtylenchus spp., Meloidogyne spp., Colletotrichum coccodes, Fusrium spp. nd Rhizoctoni solni (Schölte, 1989; Schölte nd s'jcob, 1989). Other pthogenic gents tht hve been ssocited with potto erly dying include Erwini crotovornd Potto Virus X (Rouse, 1985). It hs been estblished tht potto erly dying is frequently cused by V. dhlie in combintion with endoprsitic nemtodes. In microplot studies on potto cv. Russet Burbnk, Kotcon et l. (1985) did not find significnt contribution by either C. coccodesnor R. solni to the disese syndrome either lone or in combintion with V. dhlieor P. penetrns. However, in potto cv. Amethyst yield loss by V. dhlie ws lmost doubled in the presence of C. coccodes (Schölte et l., 1985), nd Schölte nd s'jcob (1989) found three-fctor interction on yield loss between Meloidogyne spp. or P. neglectusnd R. solni nd V. dhlie with severl potto cultivrs. The mechnism of the interction between V. dhliend P. penetrns tht results in the synergistic expression of symptoms nd yield loss hs been subject of specultion. Green (1981) considered tht the mechnism could be wounding from nemtode feeding, which gives the fungus esier ccess to the vsculr system. Alterntively he proposed physiologicl mechnism where the plnt becomes more susceptible to the fungus becuse of trnslocted substnce. Wheeler et l. (1992) compred model in which yield loss of potto is proportionl to both V. dhlie nd P. penetrns with model in which yield loss is proportionl only to the popultion density of V. dhlie nd the presence of P. penetrns leds to more severe yield loss function thn in the bsence of the nemtode. However, their comprison ws regrded not conclusive considering the vribility in the expression of potto erly dying nd confounding effects of environmentl conditions. In evluting his mechnistic model, Johnson (1992) described potto crop losses cused by multiple biotic stress fctors, excluding nemtodes. He concluded tht crop loss by multiple

21 Life cycle nd ecology of V. dhlie in potto 11 stress fctors ws less thn the sum of losses from ech stress fctor cting lone. This ws illustrted by competitive defolition between V. dhlie nd Phytophthor infestons. Systemic colonistion of the plnt (Fig. 1: e) The process of production of conidi in the vsculr system is still uncler. A hypothesis put forwrd by severl uthors is tht directly fter V. dhliehs penetrted the vsculr system, it strts to produce conidi (Howell, 1973; Schnthorst, 1981). Conidi re produced by simple conidiophores or by budding (Tolmsoff, 1973) nd re pssively distributed through the vsculr system throughout the plnt. Conidi my germinte, nd penetrte vessel wlls (Grber, 1973; Tolmsoff, 1973). More reserch concerning the process of sporultion nd distribution in the vsculr system is needed. For exmple the influence of the nutrient concentrtion in the xylem nd the influence of the vitlity of the plnt on these two processes re indicted s origins of differences in the number of V. dhlieprticles in the stems of plnts, but the effect hs never been proven in experiments. In susceptible nd tolernt cotton cultivrs, colonistion ws equl up to the point where the pthogen pssed the endodermis nd reched the xylem (Grber, 1973). The number of vessels invded ppers to be good mesure of the severity of wilt disese, becuse the number of invded vessels ws relted to the number of systemic infections nd to the number of hyphe tht progressed through the cortex from the points of colonistion. As long s petioles of potto plnts re green, distribution of V. dhlie in the plnt is limited to the xylem, but in plnts with severe disese symptoms the pith, cmbium, nd cortex re invded (Grber, 1973). In the leves, infection my be confined to single pinn, or my involve the entire lef (Grber, 1973). Plnt rections nd yield reductions (Fig. 1: fi In potto, symptoms of V. dhliere difficult to distinguish from norml senescence nd my initilly involve only reduced growth (Street nd Cooper, 1984: Hverkort et l., 1990). Erly folir symptoms my pper s unilterl chlorosis of lower leves on few plnts. Lter some wilting of whole leflets or leves my occur, but the unilterl deth of lower leves is more typicl (Isc & Hrrison, 1968). The ctivities of the fungus stimulte the plnt to produce suberin-like coting, tyloses nd gels inside the vsculr elements. Tyloses re extensions from prenchym cells into the xylem (Newcombe & Robb, 1988). Gels rise from perfortion pltes, end-wlls nd pit membrnes by process of distension of primry wll nd middle lmell constituents (Molen et l ). This cn led to occlusion of the vessels in the vsculr system immeditely bove primry

22 12 Section 1.2 infection sites (Molen et l., 1977; Hrrison & Beekmn, 1982; Newcombe & Robb, 1988). A light brown vsculr discolourtion is often visible t the stem bse when sliced (Isc& Hrrison, 1968). The ccumultion of specific chemicls such s terpenoid ldehydes in the vessels ccompnies or follows the presence of the pthogen nd reduces the vibility of the fungl propgules (Hrrison& Beekmn, 1982). As colonistion of the xylem proceeds, the vessels my become plugged by hyphe (Grber, 1973). In potto plnts the plugging hs been trced from the root tip up to the top of the stem. Inoculted potto plnts produced no symptoms until tuberistion commenced (Busch& Edgington, 1967), suggesting tht before symptom expression of Verticillium wilt becomes evident, the host must be in n dvnced stge of development (Busch et l., 1978). By ltering the photoperiod to prevent tuberistion, few or no symptoms developed (Busch& Edgington, 1967). These observtions re consistent with other observtions of n ssocition between lteness of cultivr nd resistnce to V. dhlie (Busch et l., 1978). Koteon et l. (1985) ssocited disese incidence of V. dhliewith reduced root growth, folir weight, nd tuber yield. Infected plnts exhibited lower specific lef res (re produced/dry weight of lef tissue), higher lef weight rtios (dry weight of the lef system/dry weight of the whole plnt) nd higher lef re rtios (re of the leves produced/dry weight of the whole plnt), nd, under dry conditions, lower reltive growth rtes nd lower lef growth rtes (increse in dry weight/unit lef re/week) (Hrrison& Isc, 1969). Bowden et l. (1990) nd Hverkort et l. (1990) showed tht the initil decrese in photosynthesis cused by V. dhlie ws cused by stomtl closure. The low stomtl conductnce ws correlted with low lef wter potentil (Bowden et l., 1990). In potto, V. dhlie cused reduction in the light conversion efficiency, but stronger reduction of stomtl conductnce, resulting in decresed internl/externl C0 2 rtios nd in higher net photosynthesis t similr vlues of stomtl conductnce (Hverkort et l., 1990). The reduction of net photosynthesis does not seem to be responsible for more thn 10% of the reduction of dry mtter production. In res where the disese cuses n erly nd rpid senescence of the lef cnopy, reduction of intercepted rdition my be more importnt component of dmge thn reduction of photosynthesis (Hverkort et l., 1990). Although there re interconnections between the vsculr bundles in the stem, these re bsent in the petiole. As consequence blockge of petiole bundles cn be more dmging thn proportionl blockge of stem bundles (Grber, 1973). A lesser root length of potto plnts due to V. dhlie my decrese the wter supply nd cuse the development of folir symptoms (Kotcon et l., 1984). Also root surfce res nd volumes were reported to be ffected negtively by V. dhlie.

23 Life cycle nd ecology of V. dhlie in potto 13 Reproduction of V. dhlie (Fig. 1: g) As infected plnts become senescent, the fungus permetes the surrounding tissues nd forms MS within ded tissue (Powelson, 1970). There is no evidence of other thn trnsient increses in inoculum from sources other thn plnt debris. Potto stems colonised by V. dhliewill be filled with MS. A 1cm segment of stem my contin 8,000-20,000 vible MS nd popultions up to 1,000 MS per grm of soil hve been reported in fields repetedly cropped to potto, which roughly equls 50 million MS in the soil volume occupied by the roots of one plnt (Menzies, 1970). Differences in production of MS from 7,000-9,000 propgules per grm of stem tissue hve been reported in severl potto cultivrs (Slttery, 1981). In most plnt species infected with V. dhlie, wter is required for formtion of MS (Powelson, 1970). However, in the temperte zones, humidity is usully sufficiently high during the senescence of the potto hulms to ensure bundnt MS formtion without rin. Externl fctors cn hve lrge influence on the formtion of microscleroti. Autumngrown crops of potto in the Negev re of Isrel hd pproximtely 100 times higher microsclerotil production thn the spring-grown crops (Ben-Yephet & Szmulewich, 1985). Either the cool nd moist conditions in the utumn-winter seson enbled the plnts to dry slowly, fvouring formtion of MS, or the cooler wether llowed better survivl of the fungus in the plnt tissue. Ionnou et l. (1977b) exmined the formtion of MS in tomto debris in soil subjected to different irrigtion nd flooding regimes under field conditions. Few, if ny MS were produced during the flooding tretment. This inhibition ws ttributed to decresed O, nd incresed CO, concentrtions in the flooded soil. Upon dringe, the concentrtions of O, nd CO, returned rpidly to norml tmospheric levels nd formtion of MS ws resumed. The numbers of MS eventully produced following 10-, 20-, nd 40-dy flooding tretments were 90, 44, nd 46% respectively, of the verge numbers in the non-flooded tretments. The mjor prt of the new inoculum is produced in the eril prts of the potto plnt (Ben- Yephet & Szmulewich, 1985). A direct wy for control of V. dhlie is to interfere with the formtion nd dispersl of MS. In prctice this is ccomplished by vrious snittion mesures such s removl or destruction of disesed plnt mteril before it enters the soil or before it releses the inoculum otherwise. This process of field snittion is not widely prctised becuse of expense, lck of equipment, nd through frmers' version to destroying orgnic mtter. Production of MS hs been reported in plnt roots without systemic infection. Microscleroti were formed in lrge numbers in only few sections of whet roots (Krikun & Bernier, 1990). Tking into ccount totl root length of whet nd the number of MS found (up to 100 per root

24 14 Section 1.2 frgment of 1.3x0.4 mm), the contribution to the inoculum in soil my be significnt (Krikun& Bernier, 1990). Weed control is importnt to limit multipliction of V. dhlie (Woollims, 1966; Johnson et l., 1980). Host species include mny common weeds nd ntive plnts. As in crop plnts, symptoms re not lwys pprent in infected weed plnts. Host specificity Adpttion of V. dhlie, leding to host specificity, my confuse the serch for possible resistnce or tolernce mechnisms. Zilberstein et l. (1983b) reported tht germintion on gr nd pthogenicity of MS of V. dhlieto egg-plnt, potto nd tomto ws ffected by growth medium nd host origin. The virulence of n isolte of V. dhliedepends on the host species, nd geogrphicl origin, susceptibility of the host cultivr/genotype, nd the orgn on the plnt (Zilberstein et l., 1983b; Michil, 1989). Isoltes obtined from susceptible cultivrs ttcked these cultivrs only. Isoltes from resistnt cultivrs ttcked both susceptible nd modertely resistnt cultivrs (Michil, 1989). Production of MS vrints might permit the fungus to dpt to new host species nd vrieties or to new environmentl conditions (Tolmsoff, 1973). Mint isoltes were originlly not pthogenic to tomto. After one pssge through tomto, however, the isoltes becme more pthogenic to tomto nd lost pthogenicity to mint. The isoltes from region of continuous cropping to one crop tend to be similr nd ll disply high virulence ginst tht prticulr crop but generlly wek virulence ginst other species which they cn infect nevertheless (Vigoroux, 1971). Thus notion of "preferentil" nd "occsionl" hosts is formed. It is known tht gret vribility is possible in Verticillium species (Vigoroux, 1971). Puhll nd Hummel (1983) found 16different vegettive comptibility groups in V. dhlie. Using nother method, Joquim nd Rowe (1990, 1991) were ble to reduce the number of vegettive comptibility groups to four. Isoltes from two different vegettive comptibility groups showed significnt differences in pthogenicity towrds potto (Joquim & Rowe, 1991). Thus, in soils, popultions of individuls with different properties cn be built up. It ppers tht the kind of crop constitutes determinnt fctor for the quntittive nd qulittive composition of the popultion of V. dhlie in cultivted soils (Vigoroux, 1971; Tjmos, 1981). So, the choice of cultivr, crop rottion, nd culturl prctices will not be sufficient to keep the crop from infection, but they will still be key fctors in the controlling the severity of the disese.

25 15 SECTION 1.3 AIM OF THERESEARCH In this thesis, vrious processes in the life cycle of V. dhlie re nlysed quntittively to rrive t holistic nlysis of potentil control strtegy. Quntittive informtion bout formtion nd mortlity of microscleroti of V. dhlieis poor nd not consistent. Since both hosts nd non-hosts cn induce germintion of microscleroti (Schreiber & Green, 1963), possible strtegy to control V. dhliemight be to grow non-hosts. Indeed, inducing microscleroti to germinte, without producing bundnt propgules fter plnts hve been infected, my be mjor fctor in the control of Verticillium wilt when non-hosts re used in rottions (Schnthorst, 1981). The effect of the roots of commonly grown field crops on microscleroti is not known, but needs to be quntified before the serch for specil 'luring' crop cn be strted. Formtion of microscleroti occurs in lrge numbers on hosts. Also on non-host crops V. dhlie my form microscleroti on the roots without systemiclly infecting the plnt. This limits the possibilities to reduce the popultion by induction of the germintion of microscleroti. The most direct wy of controlling V. dhlie is to limit the formtion nd dispersion of new inoculum. Potto hulm tretments in the field or removl of crop debris from the field re potentilly effective mesures to prevent the formtion in plnt tissue or the ccumultion of microscleroti in the soil. The effect of those mesures depends on the numbers of microscleroti formed on both eril nd subterrnen plnt prts. When most of the production of microscleroti tkes plce on the eril plnt prts, then hulm tretment could ffect the increse of the inoculum potentil of V. dhlie. For n ccurte estimtion of the effect of tretments of potto hulm on the formtion of microscleroti, the density of microscleroti on the vrious plnt orgns, the dry weight rtio of the plnt orgns, nd the totl number of microscleroti per plnt orgn nd per plnt should be quntified. These dt nd the effects of hulm tretments should lso be known for different crop species nd cultivrs to study popultion dynmics. In prctice, the potto hulm is killed t vrious stges of mturity, depending on the purpose for which the crop is grown. Dvis et l. (1983) found shrp increse of the density of the microscleroti when the hrvest ws delyed. The hrvest time my interct with hulm tretments nd this interction should be investigted.

26 16 Section 1.3 Becuse of the very high numbers formed in plnt tissue, microsclerotil formtion is not esily quntified. Imge nlysis my offer possibility to improve the cpcity to count microscleroti. In crop sitution, the blnce between formtion nd mortlity of microscleroti determines the soil inoculum density. Moreover, the effect of the inoculum density on crop cn vry, depending on the provennce of the pthogen (Tjmos, 1981; Zilberstein et l., 1983b). It is hypothesized tht lso in Dutch crop rottions, host specificity of V. dhlie is importnt. The long persistence of microscleroti nd the long time tht microscleroti sty embedded in host debris obscure the effects of tretments in short-term experiments nd mke model n ttrctive nd necessry tool to nlyse dt sets over more yers. Modelling my lso provide tool to get n ide of the dynmics in soil inoculum nd cn predict the consequences of vrious crop rottions for the development of the soil inoculum density. In summry, the objectives of this project were: - to explore the effects of plnt roots on the germintion of microscleroti of V. dhlie; - to nlyse the formtion of microscleroti nd vrious mesures to reduce formtion of microscleroti; - to develop model tht describes the popultion dynmics in the soil under vrious crop rottions.

27 17 SECTION 1.4 STRUCTURE OF THE THESIS In Chpter 2 experiments re described in which the effects were ssessed of single plnt roots nd crops on microscleroti in the soil. Root observtion boxes were used to crry out quntittive study on the germintion of microscleroti. Root densities were mesured in field experiment to clculte the frction of the soil volume ffected by plnt roots in crop sitution. In Section 2.2 the influence of vrious crops on the popultion of microscleroti in the soil is clculted from results from the experiments reported in Section 2.1 nd from two dditionl experiments. In Chpter 3, method is described to count microscleroti in plnt mteril by imge nlysis. Chpter 4 dels with the formtion of microscleroti on vrious crop species nd cultivrs. In the experiments described in Section 4.1, plnts of vrious crop species nd potto cultivrs were inoculted or grown in infested soil to quntify the formtion of microscleroti. In Section 4.2, the results of two greenhouse experiments re described in which the production of microscleroti ws mesured in different prts of two potto cultivrs t two hrvest dtes. Chpter 5 reports on n experiment in which chnges in soil inoculum density were mesured s influenced by growing ten different crop species nd cultivrs during two subsequent yers; crop yields re reported s well. Soil ws initilly infested in two densities with two isoltes of V. dhlieobtined from soils with different cropping histories. In the third yer susceptible potto cultivr ws grown to nlyse the effects of the crop species nd cultivrs, grown in the previous two yers, nd of the initil infesttion level. In field experiment which is reported in Chpter 6, the effect of removl of crop debris on soil inoculum level of V. dhlie ws investigted for two isoltes. Chnges in inoculum density were recorded for period of three yers for different crop sequences including susceptible potto cultivr, field ben nd brley. In Chpter 7, the results of four pot experiments re described in which the formtion of microscleroti on potto hulm of vrious lengths, covered with soil or kept on the soil surfce, ws studied. Formtion of microscleroti with these tretments ws compred with formtion of microscleroti fter hulm killing with herbicide or by heting, on two hrvest dtes. In Chpter 8, the dynmics of the soil inoculum density re pproched theoreticlly with quntittive model. An eqution ws developed tht models the inoculum densities of V. dhlie

28 18 Section 1.4 over long time spns (yers) bsed on inputs of initil inoculum densities nd cropping sequences. The number of systemic infections of plnt roots during crop growth ws relted to soil inoculum density. In turn, formtion of microscleroti in debris nd reduction of the mount of the debris of different crops were relted to the number of systemic infections. Finlly, grdul relese nd mortlity of microscleroti in the soil were included to clculte subsequent inoculum densities in the soil. The formtion of microscleroti nd the production of crop debris were relted to series of observtions on soil inoculum densities, to correlte soil inoculum density to crop yield nd subsequent inoculum densities. The resulting eqution is bsed on biologiclly nd ecologiclly meningful principles nd mesurble prmeters. The thesis ends with generl discussion focusing on the potentil of gronomic mesures studied to control the pthogen.

29 CHAPTER 2 EFFECT OF PLANT ROOTS ON THE GERMINATION OF MICROSCLEROTIA OF VERTICILLIUM DAHLIAE

30 Europen Journl of Plnt Pthology 101, (in press) 21 SECTION 2.1 EFFECT OF PLANT ROOTS ON THE GERMINATION OF MICROSCLEROTIA OF VERTICILLIUM DAHLIAE I USE OF ROOT OBSERVATION BOXES TO ASSESS DIFFERENCES AMONG CROPS L. Mol nd H.W. vn Riessen Summry Root observtion boxes were used to study the effects of hosts nd non-hosts on the germintion of microscleroti of V. dhlie. The effects of roots on microscleroti were exmined within rdius of 1mm round the root tip. Host plnts such s potto nd field ben induced higher percentge of germintion of the microscleroti thn non-host such s brley. A susceptible potto cultivr stimulted germintion more thn resistnt cultivr. The germintion percentge nd the number of hyphe per microsclerotium decresed with distnce from the root surfce regrdless of the plnt species or cultivr. Introduction Microscleroti (MS) of Verticillium dhlie (Kleb.) remin dormnt in the soil but re stimulted to germinte by root exudtes. The infectious hyphe tht emerge from MS penetrte roots minly in the res of cell differentition nd in the root hir zone (Schnthorst, 1981). Only very smll proportion of the hyphe will successfully infect the root. If species or cultivr cn be systemiclly infected by V. dhlie it is clled host. However, lrge numbers of colonies per unit root length hve been found in non-host plnts s well in host plnts (Evns& Gleeson, 1973). Verticillium dhlieprimrily colonises the root cortex ner the root tip; the mximum density of penetrtion is 1cm from the root pex (Gerik& Huismn, 1988). Microscleroti of V. dhliecn germinte more thn once, but eventully become exhusted (Frley et l., 1971). Since both hosts nd non-hosts cn induce germintion of MS (Schreiber & Green, 1963), possible strtegy to control V. dhliemight be to grow non-hosts. Indeed, inducing MS to germinte without producing bundnt propgules fter plnts hve been

31 22 Section 2.1 infected, my be mjor fctor in the Verticillium wilt control, tht hs often been reported when non-hosts re used in rottions (Schnthorst, 1981). The resons for the root's greter stimulting effect on microscleroti round its tip compred with other sites long the root xis my be tht exudtes re excreted from the zone of root elongtion (Rovir & Dvey, 1974; Curl & Truelove, 1986) nd tht there re reltively low densities of orgnisms competing for nutrients here (Olsson et l., 1987). Fitzell et l. (1980) found tht the density of root colonistion by V. dhlie incresed up to 20 mm from growing root tip in whet (Triticum estivum)nd thornpple (Dtur strmonium), but Gerik nd Huismn (1988) showed tht in cotton the occurrence of colonies of V. dhlie did not increse beyond 5 mm from the root tip. Both these studies (Fitzell et l., 1980; Gerik & Huismn, 1988) give only n indiction of the number of colonies t the root surfce. Becuse of possible interctions between MS, root exudtes, nd rhizosphere microorgnisms, the colonistion of the root does not necessrily give good indiction of the effect of root on the germintion of MS in the soil. After germintion most of the hyphe of the MS stop growing nd die. To understnd the contribution of the induction of MS germintion by plnt roots to the control of the pthogen, quntittive informtion on the influence of crop roots on the germintion of MS in the soil is needed. This pper reports on reserch to ddress this by mesuring the effect of single root tips on MS in soil non-destructively. The results of two experiments in which the germintion of MS ws mesured s influenced by host plnts nd non-host plnts re presented. Mterils nd methods Production of microscleroti Green potto stems from the field were cut so they were long enough to be contined upright in n Erlenmeyer flsk. The stems were utoclved for 20 min. t 120 C. Two-week old sporulting V. dhlieculture on PDA slnts, isolted from potto, ws blended with sterile wter. The stems were dipped in the solution, nd were incubted under sterile conditions for four weeks t 22 C. By the end of this period the stems were completely covered with microscleroti. The stems were ir-dried, ground nd kept in dry plce t room temperture until used.