Evaluation of Several Indigenous Microorganisms and Some Bio-Fungicides for Biocontrol of Potato Verticillium Wilt

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1 Pest Technology 2010 Glol Science Books Evlution of Severl Indigenous Microorgnisms nd Some Bio-Fungicides for Biocontrol of Potto Verticillium Wilt Hyf Jnoun-Khireddine 1 Mejd Dmi-Remdi 2* Fkher Ayed 3 Mohmed El Mhjou 1 1 Institut Supérieur Agronomique de Chott-Mriem, 4042, Chott-Mriem, Sousse, Tunisi 2 Centre Régionl des Recherches en Horticulture et Agriculture Biologique, 4042, Chott-Mriem, Sousse, Tunisi 3 Centre Technique de l Agriculture Biologique de Chott-Mriem, 4042, Chott-Mriem, Sousse, Tunisi Corresponding uthor: * dmi_rm@yhoo.fr ABSTRACT Verticillium wilt development ws completely suppressed with tretments sed on indigenous Trichoderm hrzinum, T. virens, Gliocldium ctenultum, Penicillium sp. nd rhizocteri incorported into the culture sustrte 15 dys prior inocultion with pthogen. The vsculr discolortion extent noted on ll potto (Solnum tuerosum L.) Spunt plnts treted iologiclly ws sttisticlly comprle to tht recorded on non-inoculted nd untreted plnts which were symptomless. Potto plnts treted with G. roseum, G. ctenultum, T. hrzinum, T. virens, Penicillium sp. nd indigenous rhizocteri showed significnt increse, y more thn 55%, of their height in comprison to the untreted nd inoculted control. Roots nd stem fresh weights increse, compred to the untreted nd inoculted control, with some indigenous ntgonistic tretments rnging etween nd %, respectively. Plnts treted with Penicillium sp. nd indigenous rhizocteri showed n increse of 44-46% of their tuer weight compred to the untreted nd inoculted control. The LDI recorded with io-fungicides sed tretments ws reduced y more thn 65% in comprison to the inoculted nd untreted plnts. Wilt development ws completely suppressed with Biofolr TM, sed on citric cid nd mint oil, which ws incorported into infested culture sustrte. All plnts treted with io-fungicides showed significntly incresed height of out 12 to 28% compred to the untreted nd inoculted control. With T. hrzinum (Biocont-T TM ) nd Pythium oligndrum (Polyversum ) sed io-fungicides, the roots, stem nd tuer fresh weights recorded were 28-86, nd 28-57% higher thn tht noted on untreted nd inoculted control plnts, respectively. Keywords: ntgonistic tretment, disese severity, inocultion, plnt growth, Solnum tuerosum L., yield INTRODUCTION Potto (Solnum tuerosum L.) is the third most importnt food crop in the world fter whet nd rice (Wng et l. 2008; Schieer nd Arnd Sldñ 2009; Visser et l. 2009). In Tunisi, Verticillium wilt (VW) is widespred in the mjor potto producing regions from the North-West to the Centre-Est of the country, nd fvoured y the mild Mediterrnen climte. Verticillium spp. cuse premture senescence resulting into 30-50% yield losses nd low qulity tuers (Johnson 1988; Rowe nd Powelson 2002). V. dhlie is oth soil- nd seed-orne Deuteromycete fungus (Alström 2001; Rowe nd Powelson 2002). Potto tuers represent the more efficient mens of disese spred ecuse initil infection is often unnoticed due to pthogen locliztion in the vsculr ring nd symptoms developed reltively lte during culture. Thus, VW represents serious constrint to locl seed production progrms. Furthermore, in recent study, Dmi-Remdi et l. (2011) hve shown tht three Verticillium species re involved in potto vsculr wilt symptoms nd contriuting, with vrile degrees, to the potto Verticillium wilt complex in Tunisi. In ddition, synergistic interctions etween V. dhlie nd certin phytoprsitic nemtodes such s Prtylenchus, Glooder, Meloidogyne species led to incresed wilt incidence nd severity (Tjmos 1989; Rotenerg et l. 2004; Dmi- Remdi et l. 2009). V. dhlie hs wide host rnge, including vegetle nd field crops, herceous ornmentls, ground cover, shrus nd trees (Bht nd Suro 1999; Frdin nd Thomm 2006). In Tunisi, V. dhlie ws isolted from tomto, pepper, eggplnt, potto, melon, cucumer, sqush, rtichoke nd olive tree (Jnoun-Khireddine et l. 2005, 2006; Triki et l. 2006; Jnoun-Khireddine et l. 2007, 2007, 2008). VW is difficult to control due not only to the endogenous growth of the pthogen, nd to its ility to infect multiple hosts, ut lso to the multi-yers longevity of its resting structures in the soil nd to the inefficcy of fungicides on pthogens once they rech the xylem (Hwke nd Lzrovits 1994; Alström 2001; Frdin nd Thomm 2006). Thus, VW control is limited to crop rottion (Scholte 1989), fumigtion with methyl romide nd other fumignts (Rowe et l. 1987), nd soil solriztion (Tjmos nd Pplomts 1988; Ktn nd DeVy 1991). However, these strtegies re either inefficient in reducing inoculum levels in the soil or hrmful to the environment nd humn helth (Uppl et l. 2007). The deploying of resistnt cultivrs is efficient, economiclly profitle nd environmentlly sfe for potto VW control. However, the most commercilized potto cultivrs re susceptile nd reeding lines for Verticillium resistnce ws undertken for stisfying disese control (Jdri et l. 1992; Wheeler et l. 1994; Cristinzio et l. 1995; Omer et l. 2000; Be et l. 2008). In fct, in Tunisi, most grown potto cultivrs exhiit vrying degrees of susceptiility to VW, rnging from moderte to high resulting in significnt reductions in yield (Dmi-Remdi et l. 2010). Other lterntive strtegies to control Verticillium wilt rely currently on the use of fungl (Dyf et l. 2003; El Hssni et l. 2004; Jnoun-Khireddine et l. 2009, 2009) or rhizocteril ntgonists (Ongen et l. 1999; Nrisw et l. 2002; Berg et l. 2005; Tjmos et l. 2005; Received: 28 Septemer, Accepted: 25 Ferury, Originl Reserch Pper

2 Pest Technology 4 (1), Glol Science Books Uppl et l. 2007; Li et l. 2008; Zhu et l. 2009), endophytes (Alström 2001; Nrisw et l. 2004; Shittu et l. 2009), mycorrhiz (Krginnidis et l. 2002; Grmendi 2004), nitrogenous nd orgnic mendments including composts (Tenut nd Lzrovits 2002; Biley nd Lzrovits 2003; Termorshuizen et l. 2006; Ochii et l. 2008; Giotis et l. 2009), iologicl soil desinfesttion (Blok et l. 2000; Gould et l. 2004) nd plnt extrcts (Uppl et l. 2008). In fct, the suppression of Verticillium ws chieved y use of rhizosphere-colonizing fungi of the gener Tlromyces, Trichoderm, Penicillium, Fusrium nd Gliocldium in severl studies in different countries (Mtt nd Grildi 1977; Hll nd Scheier 1984; Millr et l. 1984; Henni 1987; Kim et l. 1988; Berg et l. 1999; El Aissmi nd Lhlou 1999; D Ercole et l. 2000; Lren et l. 2003; Aghighi et l. 2004). Gliocldium species were lrgely used s ntgonists of Verticillium spp. nd prticulrly G. roseum ws successfully used for the control of V. dhlie nd reduction of microscleroti viility (Mrois et l. 1984; Keinth et l. 1991; Frvel 1996). Lren et l. (2003) used Penicillium oxlicum for reduction of tomto vsculr wilts. In previous study, the tomto VW severity ws significntly reduced, under growth chmer nd greenhouse conditions, y the use of endogenous Trichoderm spp., Gliocldium spp. nd Penicillium sp., colonizing nturlly vegetle roots (Jnoun-Khireddine et l. 2009, 2009). Rhizocteri were studied for their plnt growth-promoting effects nd lso for their ntgonistic potentil ginst plnt diseses (Kloepper nd Beuchmp 1992; Liu et l. 1995; Silv et l. 2004). Endophytic cteri were defined y Hllmnn et l. (1997) s cteri which cn e isolted from the surfce of disinfected root tissues or extrcted from the internl plnt tissues visily undmged. This root coloniztion cn reduce pthogen ction directly vi production of ntimicroil sustnces or competition for spce, nutrients or ecologicl niches or indirectly, vi induction of systemic resistnce (Kloepper nd Beuchmp 1992; Liu et l. 1995). Severl rhizosphere or endophytic cteri hve een reported s iocontrol gents ginst V. dhlie (Berg et l. 2001; Mercdo-Blnco et l. 2004; Berg et l. 2005; Mlndrki et l. 2008; Uppl et l. 2008). Bcillus sutilis ws shown to e efficient for control of the strwerry VW (Jordn nd Trr 1978). Similrly, rhizospheric strins of Bcillus, Erwini, Flvocterium, Pntoe, Pseudomons, Serrti, Sphingomons, Stenotrophomons nd Streptomyces isolted from Brssic spp., Cpsell spp. nd Frgri spp. produced sustnces with inhiitory ctivities ginst V. dhlie nd which suppressed strwerry nd colz VW (Berg et l. 1994; Berg 1996; Berg et l. 2000). The im of the present work ws to investigte the in vivo ntgonistic potentil of some endophytic fungi nd cteri, isolted from vegetle crops nd compred with chemicl fungicide nd known iocontrol reference gent, for their ility to control the potto VW. Some io-fungicides sed on plnt extrcts or ntgonistic microorgnisms re lso tested in seprte ssy for comprison. MATERIALS AND METHODS Plnt mteril Potto seed tuers (Solnum tuerosum L. cv. Spunt ), the most cultivted in Tunisi, were used. They were superficilly disinfected with 10% sodium hypochlorite solution for 5 min, rinsed with tp wter nd ir dried. They were plced under environmentl conditions fvourle for pre-germintion (15-20 C, 60-80% reltive humidity nd nturl room light). Pthogen inoculum V. dhlie isoltes used in the present study were otined from different hosts including solnceous nd cucurits plnts (Tle 1) showing wilt symptoms nd vsculr discolortion. They were Tle 1 Verticillium dhlie isoltes tested nd their origins. Isoltes Originl plnt hosts Origins Vd13 Tomto Chott Mriem Vd66 Tomto Téoul Vd95 Tomto Chott Mriem Vd14 Potto Neul Vd20 Potto Chott Mriem Vd28, Vd29 Potto Sidi Bou Ali Vd82 Potto Neul Vd64 Melon Chott Mriem Vd8 Eggplnt Téoul cultured t 20 C on PDA (potto dextrose gr) medium dded with 300 mg/l of streptomycin sulphte (Phrmdrug production Gmh-Hmourg, Germny). Liquid cultures used for sustrte inocultion were prepred on PDB (potto dextrose roth) nd incuted t 20 C under continuous gittion t 150 rpm during 4-5 dys. The spore suspension concentrtion used ws djusted to 10 7 spores/ml y Mlssez cytometer. For their long-term preservtion, pthogen isoltes were stored up to 12 months t -20 C in 25% glycerol solution. Fungl ntgonists Isoltes of Trichoderm hrzinum, T. viride, T. virens, Gliocldium ctenultum (three isoltes per species), G. roseum nd Penicillium sp. (two isoltes ech), used in the present study, were lredy screened for their ility to control tomto VW vi in vitro, in vivo nd in situ experiments (Jnoun-Khireddine et l. 2009, 2009). They were isolted from roots of severl vegetles (tomto, eggplnt, potto nd melon) nd the monoconidil cultures were identified ccording to the Kuicek nd Hrmn (1998) key. They were cultured t 25 C on PDA nd stored t -20 C in 10% glycerol solution until use. Liquid cultures were prepred on PDB nd the spore concentrtion used ws djusted to 10 7 spores/ml. Bcteril ntgonists Rhizocteri (Bcillus sp.) used were isolted from roots nd rhizosphere of helthy nd vigorous tomto nd pepper plnts, collected from regions of Chott Mriem nd Beml (Costl regions of Tunisi, Governortes of Sousse nd Monstir, respectively). Isoltion of cteri ws mde ccording to the soil dilution method of Wksmn (1922). In fct, 100 g of soil, collected with roots, were dded to 100 ml of sterile distilled wter (SDW) nd shken t 200 rpm during 30 min. Three to four successive dilutions were mde nd the otined soil suspensions were cultured on NA (nutrient gr) nd KB (King s B gr) medi (King et l. 1954; Rijmkers et l. 1995) dded with chlormphenicol (13 μg/l, 98%, Agros, Orgnics, New Jersey, USA) nd mpicillin (100 μg/l, 100%.i., Sigm, St. Louis, MO). A reference strin of Pseudomons fluorescens p 417, with known ntgonistic ctivity nd grtefully provided y Dr. Bkker (Deprtment of Plnt Ecology nd Evolutionry Biology, Section of Plnt Pthology, Utrecht University, Utrecht, the Netherlnds), ws included in the present study for comprison with Tunisin stins. Liquid cteril cultures were prepred on KB liquid medi nd stored t 6 C until use. For their long term preservtion, cteril isoltes were stored t 20 C in NA dded with 40% of glycerol following the Kim et l. (1997) technique. Chemicl nd iologicl fungicides Three iologicl products (io-fungicides) nd fungicide were tested in vivo ginst V. dhlie. Active ntgonistic gents or chemicl ingredients nd rtes used re presented in Tle 2. The chemicl fungicide ws included in the experiment s positive control. 36

3 Biocontrol of potto Verticillium wilt. Jnoun-Khireddine et l. Tle 2 Bio-fungicides tested nd doses used ginst V. dhlie in vivo. Active ingredient/gent Commercil nme Dose Trichoderm hrzinum Biocont-T WP TM 1 g/l Pythium oligndrum Polyversum 1 g/l Citric cid + mint oil Biofolr TM 200 cc/hl Effect of ntgonists tested on potto VW development At the multi-germ stge, tuers were plnted in plstic pots (25 cm dimeter) contining pet nd perlite mixture (2: 1), previously sterilized t 110 C for 1 h, nd humidified with 200 ml of liquid culture of ech mixture of fungl or cteril isoltes tested (5 tuers per ech fungl or cteril tretment). Five tuers plnted in pet nd perlite mixture humidified with similr volume of SDW only served s untreted controls. Five tuers plnted in uninfected culture sustrte wtered with 100 ml of oxyquinolin suspension (Cryptonol, Novrtis AG, Bsel, Switzerlnd) used t 3.5 l/hl) were included s positive control (control treted chemiclly). After emergence, plnts were wtered every 2 to 3 dys depending on environmentl conditions nd plnts needs. Potto plnts were inoculted with V. dhlie, 14 dys postemergence, y wtering ech pot with 100 ml of pthogen spore suspension (djusted to 10 7 spores/ml). Tuers plnted in sterile culture sustrte nd wtered with 100 ml of SDW or 100 ml of pthogen spore suspension served s untreted nd non-inoculted (NIC) nd untreted nd inoculted (IC) controls, respectively. Effect of io-fungicides on potto VW development Germinting tuers (5 tuers/elementry tretment) were individully dipped in different io-fungicides, except for Biofolr TM, prepred ccording to doses mentioned in Tle 2 then plnted in colonized pet nd perlite prepred s descried ove. However, Biofolr TM, which ws fertilizing, ws mixed with the culture sustrte just efore plnttion. Tuers dipped in SDW nd plnted in sterile or infested culture sustrte served s untreted nd non-inoculted (NIC) nd untreted nd inoculted (IC) controls, respectively. Plnts were plced under greenhouse conditions for 60 dys where tempertures rnged etween 11 nd 35 C (minim nd mxim, respectively). They were regulrly wtered nd fertilized with nutritious solution (Phrnd et l. 2002). Evlution criteri VW severity ws estimted, 60 dys post inocultion (DPI), vi the lef dmge index (LDI) nd ccording to 0-4 scle depending on symptom severity on leves s previously descried y Béye nd Lfy (1985) nd used in Jnoun-Khireddine et l. (2007). In ddition, the vsculr discolortion extent ws determined fter longitudinl sectioning of stems. Effects of iologicl nd chemicl tretments tested were lso evluted vi plnt growth nd production prmeters: height nd roots, stems nd tuers fresh weights. The presence of pthogen in the stem ws lso verified y reisoltion on PDA nd percentge of pthogen re-isoltion from treted plnts ws clculted for ech tretment tested. In fct, per elementry tretment, segments otined from the se, the medium nd the top of the stems were plted on PDA nd incuted t 20 C for 7 dys. The percentge of pthogen re-isoltion ws clculted vi the numer of segments colonized with V. dhlie in comprison to the totl numer of segments plced. Sttisticl nlyses Sttisticl nlyses were performed, for ll prmeters mesured, following completely rndomised design where tretments (fungl nd cteril ntgonists (or io-fungicides tested), IC nd NIC controls, nd/or the chemicl tretment) were the sole fixed fctor. The chemicl fungicide ws included in the ntgonist s ssy only. Five replictes were used per elementry tretment nd mens were seprted using Fisher s protected LSD test (t P 0.05). RESULTS Suppressive effects of fungl nd cteril ntgonists tested The efficcy of the ntgonists tested ws evluted, 60 DPI, vi severl prmeters expressing disese severity (LDI nd vsculr discolortion extent) nd plnt growth (height nd roots, stems nd tuer weights). Lef dmge index Wilt severity depended significntly on tretments tested. In fct, the LDI recorded on potto Spunt plnts treted iologiclly or chemiclly ws sttisticlly comprle to tht noted on non-inoculted nd untreted control plnts (NIC) Lef dmge index NIC IC Gr Gc Th Tv Tvs P Rh p 417 Oxy Tretment tested Fig. 1 Effect of ntgonists tested on the lef dmge index noted on potto Spunt plnts, 60 dys post-inocultion with V. dhlie in comprison to the different controls. Brs with the sme letter re not significntly different ccording to Fisher's protected lest significnt difference LSD test (P 0.05). NIC: untreted nd non-inoculted control, IC: untreted nd inoculted control, Gr: inoculted nd treted with G. roseum; Gc: inoculted nd treted with G. ctenultum, Th: inoculted nd treted with T. hrzinum, Tv: inoculted nd treted with T. viride, Tvs: inoculted nd treted with T. virens, P: inoculted nd treted with Penicillium sp., Rh: inoculted nd treted with indigenous rhizocteri; p 417: inoculted nd treted with the reference strin of Pseudomons fluorescens p 417; Oxy: inoculted nd treted with oxyquinolin, 11 < T < 35 C. 37

4 Pest Technology 4 (1), Glol Science Books NIC IC Tvs Th Tv Fig. 2 Potto Spunt plnts treted with 3 Trichoderm species in comprison to the untreted ut inoculted or non controls oserved 60 dys fter their incorportion into the culture sustrte nd their susequent inocultion with V. dhlie. NIC: untreted nd non-inoculted control, IC: untreted nd inoculted control, Th: inoculted nd treted with T. hrzinum, Tv: inoculted nd treted with T. viride, Tvs: inoculted nd treted with T. virens, 11 < T < 35 C. 25 Vsculr discolortion extent (cm) NIC IC Gr Gc Th Tv Tvs P Rh p 417 Oxy Tretment tested Fig. 3 Effect of ntgonists tested on the vsculr discolortion extent noted on potto Spunt plnts, 60 dys post-inocultion with V. dhlie in comprison to the different controls. Brs with the sme letter re not significntly different ccording to Fisher's protected lest significnt difference LSD test (P 0.05). NIC: untreted nd non-inoculted control, IC: untreted nd inoculted control, Gr: inoculted nd treted with G. roseum; Gc: inoculted nd treted with G. ctenultum, Th: inoculted nd treted with T. hrzinum, Tv: inoculted nd treted with T. viride, Tvs: inoculted nd treted with T. virens, P: inoculted nd treted with Penicillium sp., Rh: inoculted nd treted with indigenous rhizocteri; p 417: inoculted nd treted with the reference strin of Pseudomons fluorescens p 417; Oxy: inoculted nd treted with oxyquinolin, 11 < T < 35 C. which were symptomless nd it ws reduced y more thn 80% in comprison to the inoculted nd untreted plnts (IC). Incorportion of fungl (T. hrzinum, T. virens, G. ctenultum, Penicillium sp.) nd cteril (indigenous rhizocteri) ntgonists into the culture sustrte, dys prior inocultion y wtering potto plnts with mixture of V. dhlie isoltes, completely prevented the expression of VW symptoms, s lso oserved with the fungicidesed tretment (oxyquinolin), in comprison to the untreted nd inoculted control (Figs. 1, 2). Vsculr discolortion extent Wilt severity, lso estimted vi the vsculr discolortion extent (or discolortion height) depended significntly on tretments tested. In fct, this prmeter noted on ll potto Spunt plnts treted iologiclly or chemiclly ws sttisticlly comprle to tht recorded on non-inoculted nd untreted (NIC) plnts (Fig. 3) s lso concluded vi the LDI prmeter. The most importnt vsculr discolortion extent of out 18.7 cm, representing 78% of plnt height, ws registered on the untreted nd inoculted potto plnts. Plnt height Potto plnt height, noted 60 DPI (Fig. 4), depended significntly on tretments tested. In fct, plnts treted with G. roseum, G. ctenultum, T. hrzinum, T. virens, Penicillium sp. nd indigenous rhizocteri showed significnt increse, y more thn 55%, of their height in comprison to the inoculted nd untreted control (IC). However, plnts treted iologiclly with T. viride nd the P. fluorescens reference strin p 417 nd chemiclly with oxyquinolin showed sttisticlly comprle height s untreted nd inoculted plnts. Plnt weight Potto roots fresh weight, noted 60 DPI (Tle 3), did not significntly depend on tretments tested. However, plnts treted with G. roseum, T. hrzinum nd Penicillium sp. showed n increse of out %, even sttisticlly insignificnt, of their root fresh weight in comprison to the untreted nd inoculted control (IC). Plnts treted with indigenous rhizocteri showed incresed roots weight, of out 55%, which ws nerly three times higher thn tht 38

5 Biocontrol of potto Verticillium wilt. Jnoun-Khireddine et l Plnt height (cm) NIC IC Gr Gc Th Tv Tvs P Rh p 417 Oxy Tretment tested Fig. 4 Effect of ntgonists tested on potto Spunt plnts height noted, 60 dys post-inocultion with V. dhlie, in comprison to the different controls. Brs with the sme letter re not significntly different ccording to Fisher's protected lest significnt difference LSD test (P 0.05). NIC: untreted nd non-inoculted control, IC: untreted nd inoculted control, Gr: inoculted nd treted with G. roseum; Gc: inoculted nd treted with G. ctenultum, Th: inoculted nd treted with T. hrzinum, Tv: inoculted nd treted with T. viride, Tvs: inoculted nd treted with T. virens, P: inoculted nd treted with Penicillium sp., Rh: inoculted nd treted with indigenous rhizocteri; p 417: inoculted nd treted with the reference strin of Pseudomons fluorescens p 417; Oxy: inoculted nd treted with oxyquinolin, 11 < T < 35 C. Tle 3 Effects of different fungl nd cteril ntgonists on roots, stems nd tuers weight of potto plnts inoculted t plnting with V. dhlie in comprison to chemicl tretment nd to the untreted ut inoculted or non controls. Tretment Root weight (g)* Stem weight (g) Tuer weight (g) NIC cd IC d Gr cd Gc Th Tv cd Tvs c P Rh cd p cd Oxy cd * Different letters within column indicte significnt differences ccording to Fisher's protected lest significnt difference LSD test (P 0.05). NIC: untreted nd non-inoculted control, IC: untreted nd inoculted control, Gr: inoculted nd treted with G. roseum; Gc: inoculted nd treted with G. ctenultum, Th: inoculted nd treted with T. hrzinum, Tv: inoculted nd treted with T. viride, Tvs: inoculted nd treted with T. virens, P: inoculted nd treted with Penicillium sp., Rh: inoculted nd treted with indigenous rhizocteri; p 417: inoculted nd treted with the reference strin of Pseudomons fluorescens p 417, Oxy: inoculted nd treted with oxyquinolin, 11 < T < 35 C. otined with the reference strin of P. fluorescens p 417, compred to the untreted nd inoculted control. Tle 3 shows tht ll tretments tested on potto plnts ffected significntly the stems fresh weight noted 60 DPI. In fct, the most importnt stem weight ws recorded on plnts inoculted nd treted with Penicillium sp. which ws two times higher thn tht noted on untreted nd inoculted plnts. Plnts treted t plnting with Gliocldium nd Trichoderm species nd indigenous rhizocteri showed significntly comprle stem weight s the untreted nd noninoculted control plnts; moreover, their weight ws incresed y 28 to 62% compred to the inoculted nd untreted control. All tretments tested did not ffect significntly the tuers fresh weight noted 60 DPI. However, plnts treted with Penicillium sp. nd indigenous rhizocteri showed n increse of 44-46% of their tuer weight compred to the untreted nd inoculted control; this increse ws lso nerly two times higher, even sttisticlly insignificnt, thn tht recorded with oxyquinolin nd the reference strin P. fluorescens p 417. Pthogen re-isoltion Fig. 5 showed tht pthogen re-isoltion from potto plnts, 60 dys fter their inocultion vried depending on tretments tested (dt not nlyzed sttisticlly). In fct, V. dhlie ws isolted from 66% of segments plted on PDA issued from untreted nd inoculted plnts. However, pthogen ws not recovered from segments removed untreted nd non-inoculted plnts nd those inoculted nd treted with T. hrzinum, T. virens, Penicillium sp. nd the reference stin of P. fluorescens p 417. With the other iologicl tretments tested, the percentge of pthogen re-isoltion rnged etween 12 nd 33%, which ws 50 to 81% less thn tht noted on untreted nd inoculted control plnts, compred to 25% recorded with the oxyquinolin-sed tretment. Suppressive effects of io-fungicides tested The efficcy of the io-fungicides tested ws evluted, 60 DPI, vi severl prmeters expressing disese severity (lef dmge index only) nd plnt growth. Lef dmge index The LDI depended significntly on tretments tested on potto Spunt plnts (Fig. 6). In fct, the LDI recorded with io-fungicides sed tretments ws sttisticlly comprle to tht noted on non-inoculted nd untreted control plnts (NIC) nd it ws reduced y more thn 65% in comprison to the inoculted nd untreted plnts (IC). Moreover, wilt development ws completely suppressed, where LDI reduction reched 100% compred to IC tretment, with the io-fungicide Biofolr TM, sed on citric cid nd mint oil, incorported into culture sustrte. VW severity ws reduced y 65 nd 69% with Pythium oligndrum nd T. hrzinum sed io-fungicides (Polyversum nd Biocont-T TM, respectively). 39

6 Pest Technology 4 (1), Glol Science Books Pthogen re-isoltion (%) NIC IC Gr Gc Th Tv Tvs P Rh p 417 Oxy Tretment tested Fig. 5 Percentge of V. dhlie re-isoltion on PDA from potto Spunt plnts depending on tretments tested. NIC: untreted nd non-inoculted control, IC: untreted nd inoculted control, Gr: inoculted nd treted with G. roseum; Gc: inoculted nd treted with G. ctenultum, Th: inoculted nd treted with T. hrzinum, Tv: inoculted nd treted with T. viride, Tvs: inoculted nd treted with T. virens, P: inoculted nd treted with Penicillium sp., Rh: inoculted nd treted with indigenous rhizocteri; p 417: inoculted nd treted with the reference strin of Pseudomons fluorescens p 417; Oxy: inoculted nd treted with oxyquinolin, 11 < T < 35 C Lef dmge index NIC IC Biof Bioc Polyv Tretment Tretement tested Fig. 6 Effect of some io-fungicides on the lef dmge index noted on potto Spunt plnts, 60 dys post-inocultion with V. dhlie in comprison to the untreted ut inoculted or non controls. Brs with the sme letter re not significntly different ccording to Fisher s protected lest significnt difference LSD test (P 0.05). NIC: untreted nd non-inoculted control, IC: untreted nd inoculted control Polyv: inoculted nd treted with Polyversum sed on P. oligndrum; Bioc: inoculted nd treted with Biocont-T TM sed on T. hrzinum, Biof: inoculted nd treted with Biofolr TM sed on citric cid nd mint oil, 11 < T < 35 C. Plnt height Potto plnt height, noted 60 DPI (Fig. 7), depended significntly on io-fungicides nd controls used. In fct, ll plnts treted with io-fungicides showed significntly incresed height of out 12 to 28% compred to the untreted nd inoculted control. Moreover, plnts treted with Biocont-T TM, sed on T. hrzinum, showed significntly comprle height s untreted nd non-inoculted controls. Plnt weight Potto roots fresh weights, noted 60 DPI (Tle 4), did not significntly depend on io-fungicides nd tretments tested. However, plnts treted with Biocont-T TM, sed on T. hrzinum, nd Polyversum, composed of Pythium oligndrum, showed n increse of out 86 nd 28% respectively, even sttisticlly insignificnt, of their root fresh weight in comprison to the untreted control (IC). Tle 4 showed tht ll tretments tested on potto plnts did not ffect significntly the stems fresh weight noted 60 DPI. However, the most importnt stem weights were recorded on plnts inoculted nd treted with Biocont-T TM nd Polyversum which were 81 nd 48% higher, respectively, thn tht noted for untreted nd inoculted plnts. All io-fungicides nd tretments used ffected significntly the tuers fresh weight. In fct, the lest tuers fresh weight ws noted on potto plnts treted with Biofolr TM composed of citric cid nd mint oil. However, plnts treted with Biocont-T TM nd Polyversum produced more tuers for which the registered weight ws 57 nd 28% higher, respectively thn tht recorded on untreted nd inoculted plnts. Moreover, this tuer weight increse exceeded y 49 nd 22% tht noted on untreted nd noninoculted control (NIC) plnts for tretments sed on Biofolr TM nd Polyversum, respectively. DISCUSSION Biocontrol of soil-orne fungi such s V. dhlie hs een ctively explored worldwide (Berg et l. 2001; Pegg nd 40

7 Biocontrol of potto Verticillium wilt. Jnoun-Khireddine et l Plnt height (cm) c NIC IC Biof Bioc Polyv Tretment tested Fig. 7 Effect of some io-fungicides on potto Spunt plnts height noted 60 dys post-inocultion with V. dhlie in comprison to the untreted ut inoculted or non controls. Brs with the sme letter re not significntly different ccording to Fisher s protected lest significnt difference LSD test (P 0.05). NIC: untreted nd non-inoculted control; IC: untreted nd inoculted control; Polyv: inoculted nd treted with Polyversum sed on P. oligndrum; Bioc: inoculted nd treted with Biocont-T TM sed on T. hrzinum, Biof: inoculted nd treted with Biofolr TM sed on citric cid nd mint oil, 11 < T < 35 C. Tle 4 Effects of different io-fungicides on roots, stems nd tuers weight of potto plnts inoculted t plnting with V. dhlie in comprison to the untreted ut inoculted or non controls. Tretment Root weight (g)* Stem weight (g) Tuer weight (g) NIC IC Biof TM Bioc TM Polyv * Different letters within column indicte significnt differences ccording to Fisher's protected lest significnt difference LSD test (P 0.05). NIC: untreted nd non-inoculted control, IC: untreted nd inoculted control Polyv: inoculted nd treted with Polyversum sed on P. oligndrum; Bioc: inoculted nd treted with Biocont-T TM sed on T. hrzinum, Biof: inoculted nd treted with Biofolr TM sed on citric cid nd mint oil, 11 < T < 35 C. Brdy 2002) ut the present study is the first one deling with potto VW iocontrol in Tunisi. Potentil iocontrol of VW y indigenous ntgonists nd some io-fungicides with regrd to pthogen nd ntgonist inoculum nd ppliction conditions Given tht the key element for mximizing chnces of selection of iocontrol gents efficient ginst VW gents is firstly the ssessment of genetic vriility within Verticillium popultion, the inoculum used is composed of mixture of virulent isoltes of V. dhlie, the predominnt species in Tunisi, issued from different plnts nd regions nd for which ggressiveness hs een previously determined (Jnoun-Khireddine et l. 2007, 2007). As the rhizosphere is the site of interctions etween plnts, microil pthogens, nd ntgonistic fungi nd rhizocteri (Hndelsmn nd St 1996; Sörensen 1997; Berg et l. 2000, 2005), it constitutes n interesting setting for the interctions tht led to disese nd iocontrol of disese (Hndelsmn nd St 1996). In fct, ll iocontrol gents tested in this study elonged to the indigenous microflor. In fct, Trichoderm spp., Gliocldium spp. nd Penicillium sp. isoltes s well s rhizocteri of the genus Bcillus, were otined from soil nd roots of different Verticillium host plnts. These fungl isoltes were lso lredy tested for their inhiitory ctivity ginst three Verticillium species in vitro nd for their cpcity to reduce wilt severity nd to promote growth of tomto plnts in in vivo nd in situ previous experiments (Jnoun-Khireddine et l. 2009, 2009). Moreover, s the mjority of nturlly occurring iologicl control results likely from mixtures of microorgnisms in suppressive soils (Lemnceu nd Alouvette 1991), we used in the present work mixture of isoltes elonging to the different ntgonist species tested. Furthermore, rhizocteril isoltes were lredy evluted for their in vitro inhiitory ctivity ginst Verticillium spp. nd were shown to e ntgonistic to this pthogen (Jnoun-Khireddine, unpulished dt). In fct, ccording to Spdro nd Gullino (2005), y comining specific strins of microorgnisms, multiple trits ntgonizing the pthogen cn e comined nd this my result in higher level of protection. For exmple, when Pseudomons putid WCS358 strin, siderophore-producing cteri, ws comined with P. putid RE8 strin, inducing systemic resistnce, ginst F. oxysporum f. sp. rphni, Fusrium wilt suppression ws significntly enhnced (de Boer et l. 2003). The present results reveled tht potto ioprotection ginst VW is possile with some indigenous ntgonists nd io-fungicides tested nd this independently of their method of ppliction i.e. incorportion into culture sustrte or seed tuer dipping, respectively. Efficcy of VW iocontrol depends on the method of ppliction of ntgonists in the ecosystem, the numer of tretments nd the cpcity of the selected strin to dpt to different environments (Minuto et l. 2006). In fct, in the present study, the ntgonists tested were incorported into culture sustrte 15 dys prior inocultion with V. dhlie. Thus, this period seems to e lrgely sufficient for their estlishment nd dpttion ecuse normlly, s reported y D Ercole et l. (2000), iocontrol gents require short period for colonizing the environment. Consequently, the well estlished ntgonistic gents my hve doule ction: preventing pthogen progress, within host tissues, nd reducing its spreding vi its sprophytic ctivity nd its importnt cpcity of colonizing the rhizosphere outside tissues. A dely is necessry etween protective tretment nd occurring of susequent infection. The durtion of this period depended on the nture of Verticillium-host plnt pthosystem nd the type of the protective gent. This conclusion my explin the registered vriility of ntgonistic potentil of iocontrol gents used in the present study nd lso their successful estlishment. In fct, this dely is very short for protection of lucerne with Gliocldium roseum nd of cotton with Tlromyces flvus (Millr et l. 1984; Murry et l. 1997). In contrst, mple protection with Bcillus sutilis requires dely of three dys (Hll et 41

8 Pest Technology 4 (1), Glol Science Books l. 1986) wheres ppliction of Trichoderm needs 7 dys efore ggressive infection of eggplnts (Henni 1987). As prcticl consequence, ppliction of conidil suspension of P. oxlicum y wtering tomto plntlets, 7 dys prior trnsplnting, resulted in significnt reduction of Verticillium nd Fusrium wilts of tomto (Suquillo et l. 2005). Similrly, ppliction of ntgonists into culture sustrte, 15 to 20 dys prior inocultion with pthogen, controlled successfully VW of potto cused y V. dhlie. Ordentlich et l. (1990) lso showed tht ppliction of T. hrzinum in sowing rows reduced disese incidence nd incresed the totl yield y 38.5%. Moreover, sustrte ppliction of T. hrzinum, T. viride nd T. virens (10 8 spores/ ml) efore inocultion with F. oxysporum f. sp. tuerosi ws found to reduce Fusrium wilt severity on potto plnts compred to inoculted nd untreted plnts (Ayed et l. 2006). Effects of ntgonists nd io-fungicides on VW development nd the susequent effects on plnt growth According to culture nd incution conditions used in the present study, ppliction of ntgonists to potto plnts efore inocultion did not totlly prevent pthogen penetrtion ut hd contriuted in the dely of its prolifertion within tissues. In fct, the reltive vsculr discolortion extent compred to plnt height rnged etween 0 nd 11% for ll ntgonists tested wheres for untreted nd control plnts, it reched 78%. The positive protection chieved y these ntgonists ws proly due to ntgonism of V. dhlie microscleroti leding to inoculum reduction (Jnounkhireddine et l. 2009, 2009) in contct of roots nd/or stimultion of defense mechnisms of plnt hosts. Indigenous rhizocteril isoltes used in mixture in the present study hd reduced potto wilt severity nd enhnced plnt growth nd production. It seems tht these locl Bcillus sp. isoltes cted vi ntgonism nd plnt growth promotion s reduced symptom intensity ws ccompnied with incresed plnt height nd weight compred to the untreted nd inoculted control. Similrly, Rupch nd Kloepper (1998) found tht mixtures of different plnt growth promoting rhizocteri, pplied s seed tretment, showed n importnt enhncement of plnt growth nd reduction of severl cucumer diseses. Hs nd Défgo (2005) mentioned tht mixtures of plnt growth promoting rhizocteri, comining ntiiosis nd induced systemic resistnce my e very efficient in prctice. In fct, Hll et l. (1986) reported tht Bcillus sutilis strins, which colonized nturlly mple stem tissues, cn suppress VW. Furthermore, Tjmos et l. (2004) found tht three cteril isoltes, mong 18 tested under greenhouse conditions nd identified s Bcillus sp., showed significnt iocontrol ctivity ginst V. dhlie. The most efficient cteril isoltes, designed s K-165 nd colonizing nturlly the rhizosphere, inhiited the mycelil growth V. dhlie in dul-culture experiments nd successfully controlled VW of severl Solnceous species. Egmerdiyev (2006) lso found tht species of Pseudomons, Bcillus nd Arthrocter were identified s cteril ntgonists of V. dhlie. Berg et l. (2001; 2005) showed tht vrious species of the Pseudomons, Bcillus, Trichoderm gener, isolted from suppressive soils, were effective ginst V. dhlie. In recent study, Li et l. (2008) showed tht, in greenhouse experiments, suspension of the cells of the CH2 strin of Bcillus cereus, otined from the rhizosphere of eggplnt, reduced the severity of VW on eggplnt y 69.69%, nd tht this strin nd its chitinse hve good commercil potentil in controlling VW. The reference strin of P. fluorescens p 417 hd reduced VW severity ut its effect on plnt height nd weight ws significntly comprle to tht noted on untreted nd inoculted control plnts. Similr results were reported y Mercdo-Blnco et l. (2004) who found tht ppliction of different strins of the P. fluorescens complex reduced VW symptom development in olive trees. Similrly, Berg et l. (2000) recorded significnt reduction of VW fter dip tretments in cteril cultures ths, in greenhouse nd in fields nturlly infested with V. dhlie. However, nd contrrily to our results, Berg et l. (2000) noted n increse of the reltive hrvest which rnged etween 117 (Streptomyces lidoflvus S1) nd 344% (P. fluorescens P6) under greenhouse conditions, nd etween 113 (Streptomyces lidoflvus S1) nd 247% (P. fluorescens P6) in experimentl fields. The tretment of potto seeds with io-fungicides sed on T. hrzinum (Biocont-T TM ) nd Pythium oligndrum (Polyversum ) hd successfully controlled VW nd incresed tuer weight y 28 nd 57%, respectively, compred to the untreted nd inoculted control. Similr results were reported y Reknovic et l. (2007) who found tht the iofungicide Polyversum hd reduced y 66.6% the severity of VW cused y V. dhlie, when pplied prior inocultion of pepper plnts. However, in recent study, Giotis et l. (2009) mentioned tht the iologicl control product Polyversum, sed on Pythium oligndrum, hd no positive effect on VW incidence nd fruit yield, numer nd size. These io-fungicides tested ginst Fusrium oxysporum f. sp. tuerosi hd lso reduced potto Fusrium wilt incidence compred to the untreted control when incorported into culture sustrte 15 dys prior inocultion. Fung stop (sed on citric cid nd mint oil s Biofolr TM ) nd Biocont-T TM were shown to e the most ctive during the iossy wheres Polyversum hd lesser effect in controlling Fusrium oxysporum f. sp. tuerosi in vivo (Ayed et l. 2007). The incresed plnt growth nd production oserved with some iologicl tretments (ntgonists nd io-fungicides) in the present study my e ttriuted to n induction of systemic resistnce y protective gents involved. According to Vn Loon et l. (1998), the induced systemic resistnce ws qulified s n incresed plnt defence cpcity vi ctivtion of ltent resistnce mechnisms induced y vrious gents including rhizocteri. In fct, esides its ntifungl ctivities, T. hrzinum is le to induce, even t distnce of trget pthogen, resistnce of plnts to diseses. In recent studies, ppliction of this ntgonist round roots or into soil ws shown to induce resistnce in plnts ginst their specific pthogens (De Meyer et l. 1998; Howell et l. 2000; Hir et l. 2007). Certin Trichoderm strins re le to estlish durle coloniztion of roots nd to penetrte the epiderm tissue where they produced nd relesed compounds tht induced responses of loclised or systemic plnt resistnce. Plnts rected ginst the fungl invsion vi the synthesis nd ccumultion of phytolexins, flvonoïds, terpenoids nd other ntimicroil compounds. Trichoderm strins re generlly more resistnt to these compounds tht the mjority of the other fungi. Similrly, Tjmos et l. (2005) reported n induced systemic resistnce mechnism in rhizocteri s is the cse of the K165 strin of Penicillus which induced resistnce ginst V. dhlie in eggplnt. Moreover, susequent induction of systemic resistnce, K-165 delyed expression of VW symptoms in cucumer nd reduced the finl development of symptoms y 40 to 50% compred to the untreted control (Tjmos et l. 2001). In the sme wy, Uppl et l. (2008) explined the efficcy of the strins DF37 of P. fluorescens nd M1 of B. pumilus in the reduction of VW in potto cultivrs Kenneec nd Russet Burnk, in spite of the wek inhiitory effect ginst V. dhlie in vitro, y the involvement of these cteri in ctivting resistnce mechnisms in potto. In the light of the present promising results, further studies will e focused on the ssessment of the in situ efficcy of these indigenous microorgnisms, lone or in comintion with other control mesures, for reduction of VW severity in res destinted for seed tuers production. 42

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