Biological control of Verticillium wilt of olive by Paenibacillus alvei, strain K165

Transcription

1 Biologicl control of Verticillium wilt of olive y Penicillus lvei, strin K1 1

2 Astrct In the present study, the efficiency of the iocontrol gent Penicillus lvei (strin K1) to suppress Verticillium wilt of olive tree ws evluted in greenhouse nd field experiments. In plnt iossys were conducted under greenhouse conditions nd reveled tht K1 significntly decresed symptoms on the susceptile cultivr Amfissis y.% nd 1.% of the finl disese severity index nd reltive AUDPC (Are Under Disese Progress Curve), respectively. Therefter, the suppressive effect of K1 ginst Verticillium dhlie ws studied for two consecutive yers (00 nd 00) in newly estlished olive orchrd of the susceptile cv Amfissis nd the resistnt cv Klmon, nturlly infested with V. dhlie. The evlution of K1 ws crried out y recording symptoms, isoltions nd qpcr quntifiction of the pthogen in olive tissues. In oth yers, Amfissis trees treted with K1 showed significntly lower finl disese severity nd reltive AUDPC vlues compred to the non treted controls; wheres, in 00 decresed symptom severity ws ssocited with significntly lower V. dhlie DNA levels in plnt tissues, indicting the suppressive effect of the iocontrol gent. However, no significnt suppression ws oserved in Klmon. Pthogen isoltions long with qpcr quntifiction reveled sesonl fluctution of V. dhlie iomss in olive tissues with higher mounts occurring in My, nd lower mounts in Ferury, August nd Novemer. This is the first report of iologicl control of Verticillium wilt of olive tree under field conditions, ssocited with reduced pthogen levels inside the xylem tissues. Keywords: iologicl control, olive, suppression, Verticillium dhlie, Verticillium wilt

3 Introduction Verticillium wilt cused y the soilorne fungus Verticillium dhlie Kle. is one of the most serious olive (Ole europe L.) diseses, worldwide, cusing severe losses nd plnt deth (Jimenez-Diz et l. 01; Lopez-Escudero nd Mercdo-Blnco 0). Microscleroti (msc), the resting structures of V. dhlie, constitute the min potentil infective inoculum of the pthogen in the field nd persist in the soil for more thn 0 yers (Wilhelm 1). The use of fungicides, esides hving potentilly toxic consequences nd representing n environmentl thret, hve little effect on them (El- Zik 1). The infectious hyphe tht emerge from msc penetrte roots minly in the res of cell differentition nd in the root hir zone (Prieto et l. 00). Therefore, the use of iocontrol gents (BCAs) cple of growing in the rhizosphere could constitute potentil disese mngement strtegy for olive producers. Numerous studies hve shown the efficcy of vrious BCAs to suppress Verticillium wilt in different hosts (Berg et l. 001, 00; Mercdo-Blnco et l. 00; Tjmos et l. 00). A limited numer of those studies hve een dedicted to iologicl control of V. dhlie in olive trees (Lopez-Escudero nd Mercdo-Blnco 0). In one of those studies Mercdo-Blnco et l. (00) reported tht the ppliction of different strins of the Pseudomons fluorescens complex reduced Verticillium wilt symptom development on the susceptile olive cv Picul under controlled conditions. Lter, Prieto et l. (00) determined the in plnt interction of the defoliting V. dhlie pthotype, with the endophytic iocontrol strin P. fluorescens PICF. In different study, ppliction of P. fluorescens isoltes reduced significntly disese incidence nd severity on the susceptile olive cv Zrd under greenhouse conditions (Snei nd Rzni 0). However, the effect of BCAs ginst

4 Verticillium wilt on susceptile nd/or resistnt olive cultivrs hve not yet een investigted under field conditions. Furthermore, in ll the forementioned greenhouse experiments, olive plnt mteril ws rtificilly inoculted y using conidil suspension, insted of msc, tht constitutes the min nturl infective inoculum under field conditions. An lredy known BCA is the Penicillus lvei strin K1 tht hs een isolted from the root system of tomto plnts grown in Verticillium suppressive soil (Tjmos et l. 00). Previous studies hve shown the efficcy of strin K1 in reducing Verticillium wilt symptom development in eggplnts nd potto plnts under glsshouse nd field conditions (Mrkkis et l. 00; Tjmos et l. 00). The mode of ction of K1 ginst V. dhlie hs een minly ttriuted to induced systemic resistnce (ISR), in slicylic cid-dependent pthwy (Tjmos et l. 00). Also, Antonopoulos et l. (00) hve reported tht ppliction of K1 resulted in the reduction of msc germintion of V. dhlie, in the root tip nd the zone of elongtion of eggplnts. However, the efficcy of K1 ginst V. dhlie hs never een tested in olive trees, lthough the olive tree is one of the most susceptile hosts of the pthogen nd lso hs tremendous economicl impct on the economy of the min olive producing countries, Spin, Itly nd Greece (FAO 01). In view of ll the ove, the min ojectives of this study were to ssess the efficcy of strin K1 to protect olive plnt mteril infested with V. dhlie msc inoculum under greenhouse conditions, s well s to evlute the iocontrol potentil ginst V. dhlie on resistnt nd susceptile olive cultivrs under field conditions. It ws lso investigted whether symptom development is ssocited with fungl prolifertion in vsculr tissues, in different time periods in the field.

5 Mteril nd methods Plnt mteril Plnt mteril for greenhouse experiments, consisted of -month-old rooted cuttings of olive cv Amfissis; wheres for field experiments, -yer-old rooted cuttings of cvs Amfissis nd Klmon were used. Amfissis nd Klmon hve een chrcterized s susceptile nd resistnt to V. dhlie, respectively (Antoniou et l. 00; Mrkkis et l. 00, 0). Inoculum preprtion A highly-virulent non-defoliting V. dhlie isolte originted from disesed Amfissis olive tree ws used in the greenhouse experiments (Antoniou et l. 00). The isolte ws cryopreserved y freezing suspension of conidi ml -1 in % queous glycerol t -0 C. Before eing used, the fungus ws trnsferred to potto dextrose gr (PDA; Merck) t C for dys. Verticillium dhlie msc were prepred in sucrose sodium nitrte (SSN) liquid medium. The liquid cultures were shken in n oritl incutor t C for weeks. Msc were centrifuged t.000 g t 0 C for min to remove growth medium then ir dried. Msc were resuspended in sterile distilled wter nd filtered through 0 µm mesh to select lrge (>0 µm) msc, which germinte esily nd show high levels of pthogenicity (Hwke nd Lzrovits 1). Biocontrol gent preprtion

6 A spontneous rifmpicin-resistnt derivtive of strin of K1 (Tjmos et l. 00) ws used in the greenhouse experiments, while wild-type K1 strin ws pplied in the field experiments. K1 ws grown in nutrient roth plus glycerol (NG) liquid culture in Erlenmeyer flsks of l cpcity, contining 1 l of the medium, in n oritl incutor t 10 rpm t 0 C for h. A finl concentrtion of cfu ml -1 (mesured y dilution plting) ws otined y wter dilution. V. dhlie K1 iossys under greenhouse conditions Olive plnts (-month-old) cv Amfissis were grown in plstic pots contining soil (Potground, Klsmnn, Deilmnn, Germny) nd K1 ws pplied y root drenching (00 ml, cfu/ml), twenty dys efore trnsplnting the plnts to plstic pots contining l soil infested with 0 V. dhlie msc g -1 soil. The olive plnts were trnsplnted crefully without removing the soil of the rhisozphere nd plced in greenhouse, t ± C with 1-h light nd drk cycle nd neither hericides nor pesticides were pplied. Verticillium wilt symptoms were recorded every week for 0 dys fter plnt trnsplnting to infested soil. The experiment ws repeted three times with replictes (plnts) per tretment nd experiment. 0 1 Determintion of the K1 rhizosphere popultion The ility of K1 to colonize the rhizosphere of K1 treted olive plnts ws evluted t the end of the greenhouse experiment (0 dys post-inocultion, dpi), using the dilution plting technique (Hrris nd Sommers 1). Ten plnts per

7 experiment were smpled nd the experiment ws performed three times ( totl of 0 plnts). To estimte rhizosphere popultions, rhizosphere soil ws collected nd shken for min in 0 mm phosphte uffer (ph.0) contining Tween 0 (0.0%). The suspension ws plted onto PDA supplemented with rifmpicin (0 µg ml -1 ). After incution t 0 C for h, the numer of K1 colonies per g of rhizosphere soil ws determined. Experimentl field A nturlly infested field, previously cultivted with highly susceptile V. dhlie vegetle crops ws used to estlish the experimentl olive orchrd. The V. dhlie susceptile crops (potto, tomto nd eggplnt) grown in the field, exhiited severe Verticillium wilt symptoms in the pst. Verticillium dhlie msc levels were determined in the field y soil smpling from 0 ritrry sites, following Z pttern extending to the whole re of the field. Approximtely 0 gr of soil were collected from ech smpling point. Soil smples were dried in n oven t 0 C nd stored t room temperture. Susequently, the soil ws ssessed for the numer of msc using the modified Anderson smpler technique (Butterfield nd De Vy 1). In rief, 0. g of pulverized soil ws distriuted onto Petri pltes contining V. dhlie selective medium (Ausher et l. 1), using the modified Anderson smpler (Butterfield nd De Vy 1). Pltes were incuted in the drk t C nd V. dhlie msc were counted under the stereoscope fter weeks. V. dhlie K1 iossys under field conditions

8 Three-yer-old rooted cuttings of Amfissis nd Klmon grown in plstic pots (1 l cpcity) contining soil sustrte (Potground, Klsmnn, Deilmnn, Germny) were plnted in the experimentl field, in Octoer 00. Sixty trees of ech cultivr were plnted nd thirty of them were treted with K1 y root drenching pproximtely l of 1 X cfu ml -1 cteril suspension per tree. The remining thirty trees were root drenched with l of non sterilized wter nd served s controls. The BCA ws pplied in Jnury, Mrch, My nd Septemer of two consecutive yers (00 nd 00). Levin et l. (00) proposed tht V. dhlie infection cycle strts in utumn, with the reinvsion of the fungus proly cused y the germintion of msc nd the upwrd strem of conidi in the xylem during winter nd spring, when disese expression is t mximum; nd is completed when the fungl popultion decreses during summer, proly ecuse of high tempertures (Tossi nd Zzzerini 1). Therefore, the months of Jnury, Mrch, My nd Septemer were considered s the most pproprite to pply K1, in order to suppress the disese. The experiment ws performed with rndomized lock design with locks nd four experimentl units (K1-treted Amfissis, control- Amfissis, K1-treted Klmon, control- Klmon ) per lock. Ech experimentl unit consisted of ten olive trees. 1 1 Disese ssessment 0 1 Verticillium wilt symptoms in greenhouse experiments were recorded every week for 0 dys fter trnsplnttion to the V. dhlie msc infested soil; while in field experiments symptoms were ssessed in monthly intervls from Ferury to Novemer, for two consecutive yers. In Novemer 00, disesed rnches were removed y pruning nd symptom recording ws continued in Ferury 00, when new infections

9 1 1 were oserved. Disese incidence ws estimted s the percentge of infected plnts wheres mortlity ws estimted s the percentge of ded plnts. The disese severity index of ech rnch ws sed on n ritrry scle from 0- where 0= helthy rnch, 1= dull green leves, = internlly rolled leves, = necrotic leves, nd = defolited-ded rnch. The percentge of disese index ws clculted from the disese rting y the formul: Disese severity index (%) = [Σ(rting no. no. of rnches in the rting)/ Totl no. of rnches highest rting] 0% (Mrkkis et l. 00). Disese rtings were plotted over time to generte disese progress curves. Susequently the re under disese progress curve (AUDPC) ws clculted y the trpezoidl integrtion method (Cmpell nd Mdden 10). Disese ws expressed s percentge of the mximum possile re with reference to the mximum vlue potentil reched over the whole period of the experiment nd is referred to s reltive AUDPC. 1 1 Pthogen isoltion nd rel-time quntittive PCR quntifiction Two representtive disesed or symptomless rnches from ech of rndomly selected trees per tretment nd experimentl unit were collected in Ferury, My, August nd Novemer of 00. Ten wood chips from ech rnch, surfce-disinfested with % ethnol, were plced onto cidified potto dextrose gr (PDA, Merck). Severl representtive V. dhlie isoltes recovered from symptomtic tissues were identified s the non-defoliting pthotype y multiplex PCR ssy using the primer pirs D1/D nd NDr/NDf (Mercdo-Blnco et l. 001, 00), ccording to the procedure implemented in Mrkkis et l. (00). The remining rnch tissues were used for DNA extrction. Brnches were cut to form -1 mm long pieces, the rk ws

10 1 1 1 removed nd susequently the tissues were freeze-dried nd ground to fine powder y using n utoclved mortr nd pestle, in the presence of liquid nitrogen. Totl DNA ws isolted ccording to Dellport et l. (1), with the pproprite modifictions (incution time t C ws incresed to 1h), nd ws quntified y spectrophotometry nd grose gel electrophoresis (1% grose in 1X TAE uffer, stined with ethidium romide). Rel-time quntittive PCR (qpcr) ssys for quntifiction of V. dhlie DNA in olive tissues were conducted ccording to Mrkkis et l. (00, 0). In rief, polymerse chin rection (PCR) products of the primer pir Verticillium dhlie internl trnscried spcer (ITS)1-F/ITS-R cloned into the pgem-tesy vector. Stndrd curve (y=-0.1x+1., R =0.) ws generted from plotting the log of known DNA concentrtions ( ng to 1 fg of DNA), ginst the cycle threshold (Ct) vlues otined from the quntittive PCR nd served to clculte the reltive mount of V. dhlie DNA in totl genomic DNA smples extrcted from infected olive tissues. 1 1 Sttistics Anlysis of vrince (ANOVA) ws used to determine the effects of tretment (control or K1) on disese incidence, finl disese severity, mortlity nd reltive AUDPC in greenhouse experiments. ANOVA ws lso pplied to determine the effects of cultivr (Amfissis or Klmon), tretment (K1 or control), smpling time point (Ferury, My, August or Novemer 00) nd their interctions on disese incidence, finl disese severity, mortlity, reltive AUDPC, percentge of positive V. dhlie isoltions nd V. dhlie DNA level, in field experiments (Tle 1 nd ). Prior to ANOVA, homogeneity of vrince cross tretments ws evluted, nd n rcsin

11 trnsformtion ws pplied to normlize vrince. When significnt F-test ws otined for tretments (P 0.0), the dt were sujected to mens seprtion y Tukey s honestly significnt difference test. In greenhouse experiments, dt on disese incidence, mortlity, disese severity for ech dy of oservtion seprtely nd reltive AUDPC, were nlyzed y crrying out two-smple t-test (P 0.0). Results Suppression of Verticillium wilt symptoms in greenhouse experiments Verticillium wilt symptoms, minly chlorosis, strted on dy 0 in oth K1 treted nd control Amfissis plnts, wheres significnt difference in symptom severity ws oserved no sooner thn dpi (Fig. 1). Susequently, disese severity index in K1 treted plnts ws significntly lower compred to controls, for ech dy of oservtion. The reltive AUDPC nlysis long with the finl disese severity nd mortlity demonstrted tht symptom development in K1 treted trees ws significntly lower thn in controls, showing the suppressive effect of the iocontrol gent ginst V. dhlie, under greenhouse conditions (Tle ). The monitoring of the K1 popultion t the end of the experiment (0 dys fter K1 ppliction), reveled tht the BCA is n efficient olive root coloniser since its popultion numer ws. ± 1. cfu g -1 rhizosphere. Microscleroti density in soil nd suppression of Verticillium wilt symptoms in field experiments

12 Soil nlysis of inoculum densities reveled high inoculum levels in the experimentl field, since V. dhlie msc density ws. ± 0. msc g -1 soil. The onset of symptom development ws oserved four months (Ferury 00) fter trnsplnting (Octoer 00), minly in Amfissis trees (Fig. ). The ffected plnts exhiited typicl symptoms of the disese including chlorosis, wilting, defolition nd rnch dieck. One month lter, similr symptoms were oserved on Klmon trees, ut to lower extent compred to Amfissis. The disese severity index in Amfissis nd Klmon incresed stedily until July, when curing in disese progress ws noticed for two months (August-Septemer 00). A further increse of disese severity index ws then recorded reching 0. nd 1.%, respectively, in control nd K1 treted Amfissis cultivr trees, nd 1. nd 1.%, respectively, in control nd K1 treted Klmon cv trees, in Novemer 00 (Fig. ). The finl disese severity nd reltive AUDPC nlysis demonstrted tht K1 ppliction reduced significntly symptoms in Amfissis ut not in Klmon trees (Tle ). Furthermore, symptom development in Klmon ws significntly lower thn in Amfissis, suggesting its resistnce ginst V. dhlie under field conditions. In Novemer 00, ll symptomtic rnches of ffected trees were removed y pruning. In 00, the first symptoms were oserved in Mrch nd disese severity index ws shrply incresing until June, reching. nd 1.1%, respectively, in control nd K1 treted trees of Amfissis, nd 1 nd 1.0%, respectively, in control nd K1 treted trees of Klmon. Afterwrds, slow progress of symptoms ws oserved, until Novemer (Fig. ). Like 00, finl disese severity nd reltive AUDPC nlysis for 00 demonstrted tht K1 ppliction suppressed significntly symptom development in Amfissis. Interestingly, the mortlity of K1-treted Amfissis trees ws significntly lower compred to the non-treted control, during the 1

13 second yer in field experiment (Tle ). On the other hnd, the ppliction of K1 in the resistnt Klmon did not result in significnt reduction of symptoms compred to control trees. Effect of K1 on V. dhlie isoltion nd DNA level in field experiments The pthogen ws isolted nd quntified in olive tissues during ll sesons of 00. Representtive V. dhlie isoltes recovered from symptomtic tissues were identified s the non-defoliting pthotype y multiplex PCR ssy (dt not shown). Overll, the percentges of positive V. dhlie isoltions oserved in My (.%), where significntly higher compred to Ferury (.%), August (.%) nd Novemer (1.%). Furthermore, the percentge of positive V. dhlie isoltions ws significntly higher in rnches of Amfissis thn Klmon, in every month (Fig. ). These results coincide with the qpcr results, which demonstrted significntly higher mount of V. dhlie DNA in olive tissues in My compred to the other three months. Also, significntly higher V. dhlie DNA level ws oserved in Amfissis thn in Klmon tissues (Fig. ). Although, no significnt differences in the percentge of positive isoltions were oserved etween K1 treted nd non-treted trees within ech cultivr (Fig. ), qpcr results reveled significntly lower V. dhlie DNA levels in K1-treted Amfissis trees compred to the rest of the tretments, in My, August nd Novemer (Fig. ). In ccordnce to the previous dt of symptom development nd percentge of positive V. dhlie isoltions, no significnt differences in fungl DNA mounts were oserved etween K1 treted nd non-treted trees of Klmon. 1

14 Discussion Verticillium wilt hs een often reported s the min phytopthologicl prolem of olive trees in the orchrds of Mediterrnen countries nd in Cliforni (Lopez- Escudero et l. 0; Snyder et l. 10). Since Verticillium wilt cnnot e currently controlled y chemicls, the use of resistnt or tolernt olive cultivrs is considered s the most efficient method for disese mngement (Lopez-Escudero et l. 00). Mngement of Verticillium wilts of woody hosts such s olive tree is difficult nd should e sed on n integrted disese control strtegy (Lopez-Escudero nd Mercdo-Blnco 0; Tjmos 1; Tjmos nd Jimenez-Diz 1). Exploiting the potentil of microil ntgonists for the protection of olive plnting mteril hs een proposed s desirle pre-plnting mesure for the integrted mngement of Verticillium wilt (Mercdo-Blnco et l. 00). Therefore, severl Pseudomons spp. strins hve een descried s eneficil for olive tree (Mercdo-Blnco et l. 00; Prieto et l. 00; Snei nd Rzni 0). The mode of ction of the most efficcious of those Grm negtive Pseudomons strins, PICF, hs een suggested to e the induction of host defence (Gómez-Lm Cnás et l. 01), like in the cse of the Grm positive sporulting P. lvei strin K1 (Tjmos et l. 00). However, the efficiency of these cteril strins under field conditions or their suppressive effect on resistnt or tolernt cultivrs hs never een tested efore. In the present study, the efficcy of the iocontrol gent P. lvei strin K1 ws evluted to protect plnting mteril of the highly susceptile olive Amfissis nd the resistnt Klmon ginst V. dhlie, under greenhouse nd field conditions. In the greenhouse experiments, n ttempt ws mde to simulte the nturl infection process of Verticillium in olive trees in the field y using msc s the infective inoculum, in contrst to previous studies 1

15 where the root system of olive trees ws dipped into conidil suspension of the fungus (Mercdo-Blnco et l. 00; Prieto et l. 00; Snei nd Rzni 0). Dt on the highly virulent defoliting pthotype of V. dhlie suggest thn inoculum densities greter thn msc g -1 in nturlly infested soils re considered very high for olive trees, since disese incidence cn rech vlues greter thn 0% in susceptile cultivrs (Lopez-Escudero nd Blnco-Lopez 00; Trpero et l. 01). Furthermore, in rtificil inocultion experiments with the non-defoliting pthotype of V. dhlie it ws shown tht the use of nd 0 msc g -1 soil, did not result in significnt differences in symptom development in the susceptile Amfissis nd the resistnt Klmon (Antoniou et l. 00). In the present study, the verge inoculum density in the experimentl field ws. msc g -1 soil nd 0 msc g -1 soil in the greenhouse experiments. According to the forementioned literture, these msc densities constitute significnt infectious inoculum for olive trees. The present dt showed tht K1 suppressed significntly disese symptoms on the susceptile Amfissis, under controlled nd field conditions, nd the oserved decrese of disese severity index in the field ws ssocited with significnt reduction of fungl iomss in olive tissues. In previous study, the ppliction of K1 resulted in significnt reduction of V. dhlie msc germintion, in the root tips nd the zone of elongtion of eggplnts compred to the control (Antonopoulos et l. 00). Therefore, the oserved decrese of fungl iomss in the olive tissues cn e possily ttriuted to the reduction of msc germintion, which susequently resulted in decresed fungl invsion in olive tissues nd lower symptoms severity on Amfissis. In contrst, neither symptom development nor V. dhlie DNA reduction ws oserved in the K1 treted Klmon trees, indicting non significnt suppressive effect of this iocontrol gent on the resistnt cultivr. 1

16 Mercdo-Blnco (01) suggested the use of BCAs either s efore-plnting (preventive) or s post-plnting (pllitive) ction in estlished orchrds comined with other control tools. Even though K1 post-plnting ppliction did not result in significnt reduction of reltive AUDPC or V. dhlie iomss in the resistnt cv Klmon, considerle decrese y nerly 0% of the finl disese severity index, reltive AUDPC nd fungl DNA levels ws recorded in the K1 treted trees compred to the control trees, in Novemer 00. Thus, the use of resistnt cultivrs comined with K1 or other BCAs could potentilly e n efficient pproch in the effort to control Verticillium wilt of olive tree. The results of symptom development nd fungl isoltions long with qpcr suggest sesonl ctivity of Verticillium wilt. Disese symptoms progressed rpidly from the end of winter to the middle of summer nd the highest V. dhlie mounts were detected in the end of spring. Susequently, the oserved curing of disese severity index ws ssocited with the lower levels of the pthogen in olive tissues, s it ws reveled y pthogen isoltions nd qpcr; symptoms further progressed from the middle to the end of utumn. This filure to increse fter My, proly reflects reduced sporultion of the fungus nd coincides with the cyclicl periods of fungl elimintion tht chrcterize the lifestyle of V. dhlie in the vsculr system of trees nd plnts (Gold et l. 1; Heinz et l. 1; Mercdo-Blnco et l. 00; Mrkkis et l. 00, 0). Other reserches hve likewise reported the curing or decrese of disese severity (Wilhelm nd Tylor 1; Levin et l. 00). This hs een ttriuted to the nturl phenomenon of recovery ssocited with mechnisms tht llow trees to overcome injury nd decy, nd cn e ctivted fter infections cused y vsculr pthogens such s V. dhlie (Lopez-Escudero et l. 00; Mrkkis et l. 00, 0). However, nturl recovery in itself is unlikely to e exploited in control strtegy if 1

17 soil inoculum remins present (Buici nd Cirruli 01). Eventully, it might e used s control mesure fter drstic soil inoculum reduction or under moderte or low disese pressure. Similrly, Lopez-Escudero nd Blnco-Lopez (00) reported tht periods of incresed disese incidence in olive trees were minly in spring nd utumn; wheres Nvs-Cortes et l. (00) reported tht highest infection rte occurred in the winter to spring period nd decresed to minimum vlues in the summer to fll period. In ddition, sesonl chnges in the coloniztion of the trees y the fungus were oserved, since the highest percentge of positive isoltions nd DNA levels of the pthogen were detected in My; wheres significntly lower V. dhlie mounts were noticed in Ferury, August nd Novemer. These findings re prtilly in ccordnce with previous study (Levin et l. 00), where the highest isoltion rtes in disesed trees were in winter nd spring ( nd %, respectively), nd the lowest were in utumn nd summer (1-0%). The low pthogen mounts detected in the end of winter (Ferury 00) could possily e explined y the fct tht disesed rnches were removed y pruning in Novemer 00, nd the second infection cycle of vsculr coloniztion hd not yet tken plce y ctively growing V. dhlie, proly ecuse of the low tempertures in winter (Tosi nd Zzzerini 1). Therefore, the present dt support the hypothesis of Levin et l. (00) tht Verticillium wilt of olive tree follows n infection cycle tht strts in utumn with the ctivtion of the fungus y the germinting msc, conidi re produced in the xylem nd spred from roots through the trnspirtion strem to the upper prts of the trees during winter nd spring, reching mximum disese expression. The cycle is completed when the fungl popultion decreses during summer, proly ecuse of high tempertures (Tosi nd Zzzerini 1), exhiiting curing in disese symptoms nd recovery (Lopez- Escudero et l. 00). 1

18 The present study revels for the first time the efficiency of BCA to control Verticillium wilt of olive tree in nturlly infested soils. K1 successfully suppressed disese symptoms nd fungl iomss in tissues of the susceptile Amfissis, while remrkle ut no sttisticlly significnt decrese ws oserved in the resistnt Klmon, under high inoculum densities. The success of iologicl control requires n understnding of the mode of ction of the ntgonist, its interctions with the plnt nd the pthogen, nd lso the dose of ppliction (Alouvette et l. 00). It lso depends on the ecologicl fitness of the BCAs, especilly when they trget soilorne plnt pthogens. Further study is therefore needed to determine the mode of ction of the ntgonist, its ecologicl fitness nd n efficient nd economiclly fesile delivery system, dpted to olive trees. One step forwrd would e to investigte the efficiency of K1 to protect other susceptile, tolernt or resistnt cultivrs, in reltion to vrious soil types nd climtic conditions. The use of host resistnce comined with BCAs under n integrted disese mngement strtegy is the only plusile frmework for the effective control of one of the most serious olive tree diseses (Mercdo-Blnco 01). 1

19 References Alouvette C, Olivin C, Migheli Q, Steinerg C (00) Microiologicl control of soil-orne phytopthogenic fungi with specil emphsis on wilt-inducing Fusrium oxysporum. New Phytol 1:- Antoniou PP, Mrkkis EA, Tjmos SE, Pplomts EJ, Tjmos EC (00) Novel methodologies in screening nd selecting olive vrieties nd root-stocks for resistnce to Verticillium dhlie. Eur J Plnt Pthol 1:-0 Antonopoulos DF, Tjmos SE, Antoniou PP, Rfeletos P, Tjmos EC (00) Effect of Penicillus lvei, strin K1, on the germintion of Verticillium dhlie microscleroti in plnt. Biol Control :1-10 Ausher R, Ktn J, Ovdi S (1) An improved selective medium for the isoltion of Verticillium dhlie. Phytoprsitic :1-1 Berg G, Fritze A, Roskot N, Smll K (001) Evlution of potentil iocontrol rhizocteri from different host plnts of Verticillum dhlie Kle. J App Mycol 1:-1 Berg G, Zchow C, Lottmnn J, Gotz M, Cost R, Smll K (00) Impct of plnt species nd site on rhizosphere-ssocited fungi ntgonistic to Verticillium dhlie Kle. App Env Microiol 1:0-1 Buici G, Cirruli M (01) Nturl recovery from Verticillium wilt in olive: cn it e exploited in control strtegy? Plnt nd Soil 1:- Butterfield EJ, DeVy JE (1) Ressessment of soil ssys for Verticillium dhlie. Phytopthology :- Cmpell CL, Mdden LV (10) Introduction to Plnt Disese Epidemiology. Wiley, New York 1

20 Dellport SL, Wood J, Hicks JB (1) A plnt DNA minipreprtion: version. Plnt Mol Biol Rep 1:1-1 El-Zik KM (1) Integrted control of Verticillium wilt of cotton. Plnt Dis :- FAO, Food nd Agriculture Orgniztion of the United Ntions (01) Gómez-Lm Cnás C, Schiliro E, Vlverde-Corredor A, Mercdo-Blnco J (01) The iocontrol endophytic cterium Pseudomons fluorescens PICF induces systemic defense responses in eril tissues upon coloniztion of olive roots. Front Microiol :1-1 Hrris RF, Sommers LE (1) Plte-Dilution Frequency Technique for Assy of Microil Ecology Appl Microiol 1:0- Hwke MA, Lzrovits G (1) Production nd mnipultion of individul microscleroti of Verticillium dhlie for use in studies of survivl. Phytopthology :-0 Heinz R, Lee SW, Sprno A, Nzr RN, Ro J (1) Cyclicl systemic coloniztion in Verticillium-infected tomto. Physiol Mol Plnt Pthol :- Jimenez-Diz RM, Cirulli M, Buici G, Jimenez-Gsco MM, Antoniou PP, Tjmos EC, (01) Verticillium wilt, mjor thret to olive production: current sttus nd future prospects for its mngement. Plnt Dis :0- Levin AG, Lvee S, Tsror L (00) Epidemiology of Verticillium dhlie on olive (cv. Picul) nd its effect on yield under sline conditions. Plnt Pthol :1-1 Lopez-Escudero FJ, Blnco-Lopez MA (00) Recovery of young olive trees from Verticillium dhlie. Eur J Plnt Pthol :- 0

21 Lopez-Escudero FJ, Blnco-Lopez MA (00) Reltionship etween the inoculum density of Verticillium dhlie nd the progress of Verticillium wilt of olive. Plnt Dis 1:1-1 Lopez-Escudero FJ, Del Rio C, Cllero JM, Blnco-Lopez MA (00) Evlution of olive cultivrs for resistnce to Verticillium dhlie. Eur J Plnt Pthol 1:- Lopez-Escudero FJ, Mercdo-Blnco J (0) Verticillium wilt of olive: cse study to implement n integrted strtegy to control soil-orne pthogen. Plnt Soil :1-0 Lopez-Escudero FJ, Mercdo-Blnco J, Roc JM, Vlverde-Corredor A, Blnco-Lopez MA (0) Verticillium wilt of olive in the Gudlquivir vlley (southern Spin): Reltions with some gronomicl fctors nd spred of Verticillium dhlie. Phytopthol Mediterr :0-0 Mrkkis EA, Tjmos SE, Antoniou PP, Pplomts EJ, Tjmos EC (00) Symptom development, pthogen isoltion nd Rel-Time QPCR quntifiction s fctors for evluting the resistnce of olive cultivrs to Verticillium pthotypes. Eur J Plnt Pthol 1:0- Mrkkis EA, Tjmos SE, Antoniou PP, Pplomts EJ, Tjmos EC (0) Phenolic responses of resistnt nd susceptile olive cultivrs induced y defoliting nd non defoliting Verticillium dhlie pthotypes. Plnt Dis :- Mrkkis EA, Tjmos SE, Chtzipvlidis I, Antoniou PP, Pplomts EJ (00) Evlution of compost mendments for control of vsculr wilt diseses. J Phytopthol 1:- Mercdo-Blnco J (01) Biologicl control of Verticillium wilt of olive within n integrted disese mngement frmework. Bull IOBC/WPRC Bull :1-1 1

22 Mercdo-Blnco J, Colldo-Romero M, Prrill-Arujo S, Rodriguez-Jurdo D, Jimenez-Diz RM (00) Quntittive monitoring of coloniztion of olive genotypes y Verticillium dhlie pthotypes with rel-time polymerse chin rection. Physiol Mol Plnt Pthol :1- Mercdo-Blnco J, Rodriguez-Jurdo D, Hervs A, Jimenez-Diz RM (00) Suppression of Verticillium wilt in olive plnting stocks y root-ssocited fluorescent Pseudomons spp. Biol Contr 0:- Mercdo-Blnco J, Rodriguez-Jurdo D, Perez-Artes E, Jimenez-Diz RM (001) Detection of the nondefoliting pthotype of Verticillium dhlie in infected olive plnts y nested PCR. Plnt Pthol 0:0 1 Mercdo-Blnco J, Rodriguez-Jurdo D, Perez-Artes E, Jimenez-Diz RM (00) Detection of the defoliting pthotype of Verticillium dhlie in infected olive plnts y nested PCR. Eur J Plnt Pthol :1 1 Nvs-Cortés JA, Lnd BB, Mercdo-Blnco J, Trpero-Css JL, Rodríguez-Jurdo D, Jiménez-Díz RM (00) Sptiotemporl nlysis of spred of infections y Verticillium dhlie pthotypes within high tree density olive orchrd in southern Spin. Phytopthology :1-10 Prieto P, Nvrro-Ry C, Vlverde-Corredor A, Amyotte SG, Doinson KF, Mercdo- Blnco J (00) Coloniztion process of olive tissues y Verticillium dhlie nd its in plnt interction with the iocontrol root endophyte Pseudomons fluorescens PICF. Microil Biotechnol. :- Snei SJ, Rzni SE (0) Suppression of Verticillium wilt of olive y Pseudomons fluorescens. Amer J Exper Agric 1:-0 Snyder WC, Hnsen HN, Wilhelm S (10) New host of Verticillium lo-trum. Plnt Dis Rep :-

23 Tjmos EC (1) Prospects nd strtegies in controlling Verticillium wilt of olive. Bull OEPP/EPPO Bull :0-1 Tjmos EC, Jimenez-Diz RM (1) Mngement of disese. Pges - in: A Compendium of Verticillium wilt in Trees Species, Ponsen & Looijen, Wgeningen, the Netherlnds Tjmos SE, Flemetkis E, Pplomts EJ, Ktinkis P (00) Induction of resistnce to Verticillium dhlie in Aridopsis thlin y the iocontrol gent K-1 nd pthogenesis-relted proteins gene expression. Mol. Plnt Microe Inter 1:- 1 Tjmos EC, Tsitsiginnis DI, Tjmos SE, Antoniou P, Ktinkis P (00) Selection nd screening of endorhizosphere cteri from solrized soils s iocontrol gents ginst Verticillium dhlie of solnceous hosts. Eur J Plnt Pthol 1:- Tosi L, Zzzerini A (1) Investigtion on the epidemiology of Verticillium wilt in centrl Itly. Olive 1:0- Trpero C, Serrno N, Arquero O, Del Río C, Trpero A, López-Escudero FJ (01) Field resistnce to Verticillium wilt in selected olive cultivrs grown in two nturlly infested soils. Plnt Dis :- Wilhelm S (1) Longevity of the Verticillium wilt fungus in the lortory nd field. Phytopthology :10- Wilhelm S, Tylor JB (1) Control of Verticillium wilt of olive through nturl recovery nd resistnce. Phytopthology :-1

24 Tle 1 Anlysis of vrince for disese incidence (DI), finl disese severity (FDS), mortlity (M) nd reltive re under disese progress curve (RAUDPC), for Amfissis nd Klmon cultivrs, treted with K1 or not (control) under field conditions, in 00 nd 00 F vlues Source df DI FDS M RAUDPC DI FDS M RAUDPC Cultivr 1.1* 1.***.000.0*.0* 1.01***.*.** Tretment 1.0.*.000.0**.0 1.***.000** 1.0*** Cultivr Tretment * df c Symols: *, **, nd *** indicte significnce t P 0.0, 0.01, nd levels, respectively, ccording to the F test degrees of freedom etween groups c degrees of freedom within groups

25 Tle Anlysis of vrince for percentge of positive Verticillium dhlie isoltions (Vd I) nd DNA level (Vd DNA), for Amfissis nd Klmon cultivrs, treted with K1 or not (control), in four smpling time points, under field conditions, in 00 F vlues Source df Vd I Vd DNA Cultivr 1 10.***.0*** Tretment 1.*.1** Time.*** 1.*** Cultivr Tretment 1..** Cultivr Time.01*.00*** Tretment Time Cultivr Tretment Time df c Symols: *, **, nd *** indicte significnce t P 0.0, 0.01, nd levels, respectively, ccording to the F test degrees of freedom etween groups c degrees of freedom within groups

26 Tle Vlues (±stndrd errors) of disese prmeters of Amfissis nd Klmon cultivrs, treted with K1 or not (control) in greenhouse nd field experiments, in 00 nd 00 Experiment Tretment Disese prmeters DI (%) FDS (%) M (%) RAUDPC (%) 1 1 Greenhouse Field 00 Amfissis-Control.±. 1.±. 0.0±. 0.±. Amfissis-K1 0.0±1..0±. 1.±..1±. Amfissis-Control.±. 0.±..±. 1.0±1. Amfissis-K1.±. 1.±. 0.0±0.0.±0. Klmon-Control 0.0±. 1.±.1 0.0±0.0.0±1. Klmon-K1.±1.0 1.±.0 0.0±0.0.±1.0 Amfissis-Control.±..0±..±. 0.1±. Amfissis-K1.±..±..±. 1.±. Field 00 Klmon-Control 0.0±.0.±. 1.±. 1.±. Klmon-K1.±1.0 1.±. 0.0±0.0.±1. Disese prmeters were evluted periodiclly on the sis of externl symptoms during period of 0 dys fter trnsplnting to the Verticillium dhlie microscleroti-infested soil (0 msc g -1 soil), in greenhouse experiments. In field experiments, disese prmeters were recorded periodiclly for 1 months fter plnting, until Novemer 00. Then, disesed rnches were removed y pruning nd symptom recording ws continued for nother 1 months, until Novemer 00. DI = finl disese incidence; RAUDPC = reltive re under the disese progress curve with reference to the mximum vlue potentilly reched over ech ssessment period; FS = finl men severity of symptoms; M = mortlity. Within experiments, vlues in columns followed y the sme letter re not significntly different ccording to t-test nd Tukey s HSD test t P 0.0, in greenhouse nd field experiments, respectively 1 1 1

27 0 0 K1 Control ** Disese Severity Index (%) * * * ** ** Dys Post Inocultion Fig. 1 Verticillium wilt disese severity index on olive cultivr Amfissis treted with the iocontrol gent K1 (- -) or non treted controls (- -) t 0,,, 1,,, nd 0 dys fter trnsplnting (d.p.i.) to the Verticillium dhlie microsclerotiinfested soil. Asterisks (* nd **) indicte significnce (t P 0.0 nd 0.01 levels, respectively), t ech oservtion time-point, ccording to t-test. Verticl rs indicte stndrd errors

28 0 1 Disese Severity Index (%) Ferury 00 AMF C AMF+K1 KAL C KAL+K1 Mrch 00 April 00 My 00 June 00 July 00 August 00 Septemer 00 Octoer 00 Novemer 00 Fig. Verticillium wilt disese severity index on olive cultivrs Amfissis nd Klmon plnted in nturlly infested fields nd treted either with the iocontrol gent K1 or wter (control), in monthly intervls from Ferury to Novemer 00. Columns with different letters refer to differences etween tretments t ech oservtion time-point, ccording to the Tukey HSD test (P 0.0). Verticl rs indicte stndrd errors

29 Disese Severity Index (%) AMF C AMF+K1 KAL C KAL+K1 0 cc 1 Ferury 00 Mrch 00 April 00 My 00 June 00 July 00 August 00 Septemer 00 Octoer 00 Novemer 00 Fig. Verticillium wilt disese severity index on olive cultivrs Amfissis nd Klmon plnted in nturlly infested fields nd treted either with the iocontrol gent K1 or wter (control), in monthly intervls from Ferury to Novemer 00. Columns with different letters refer to differences etween tretments t ech oservtion time-point, ccording to the Tukey HSD test (P 0.0). Verticl rs indicte stndrd errors

30 0 ΑΜF C 1 Percentge of Positive V. dhlie Isoltions (%) ΑΜF+Κ1 ΚΑL C ΚΑL+K1 FEBRUARY 00 MAY 00 AUGUST 00 NOVEMBER 00 Fig. Percentge of positive Verticillium dhlie isoltions from rnches of olive cultivrs Amfissis nd Klmon, plnted in nturlly infested fields, treted either with the iocontrol gent K1 or wter (control), nd smpled in Ferury, My, August nd Novemer 00. Columns with different letters refer to differences etween tretments t ech smpling time point seprtely, ccording to the Tukey HSD test (P 0.0). Verticl rs indicte stndrd errors 0

31 0 1 Reltive V. dhlie DNA Quntity (moleculesx )/0 ng totl DNA ΑΜF C ΑΜF+Κ1 ΚΑL C ΚΑL+K1 c c FEBRUARY 00 MAY 00 AUGUST 00 NOVEMBER 00 Fig. Reltive Verticillium dhlie DNA mount in tissues of olive cultivrs Amfissis nd Klmon (molecules per 0 ng totl DNA), plnted in nturlly infested fields, treted either with the iocontrol gent K1 or wter (control), nd smpled in Ferury, My, August nd Novemer 00. Columns with different letters refer to differences etween tretments t ech smpling time point seprtely, ccording to the Tukey HSD test (P 0.0). Verticl rs indicte stndrd errors 1