Variation Among Some Fusarium oxysporum f. sp. melonis Isolates as Measured by Their Effect on Muskmelon Plant Growth and Wilt Severity

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1 The Africn Journl of Plnt Science nd Biotechnology 2007 Glol Science Books Vrition Among Some Fusrium oxysporum f. sp. melonis Isoltes s Mesured y Their Effect on Muskmelon Plnt Growth nd Wilt Severity Mejd Dmi-Remdi 1* Fkher Ayed 2 Hger Jeri 3 Hyf Jnoun-Khireddine 2 Mohmed El Mhjou 2 1 Regionl Centre of Reserch in Horticulture nd Orgnic Agriculture, Chott-Mriem, 4042 Sousse, Tunisi 2 Higher Agronomic Institute of Chott-Mriem, 4042 Sousse, Tunisi 3 Ntionl Institute of Agronomic Reserch of Tunisi, Street of Hèdi Krry, Arin, 2080 Tunisi Corresponding uthor: * dmi_rm@yhoo.fr ABSTRACT Fusrium wilt of muskmelon (Cucumis melo L.), cused y Fusrium oxysporum f. sp. melonis, is one of mjor constrints in melongrowing regions of Tunisi. Necrosis, gum exudtes, stunting, vine deth nd light to drk rown vsculr discolortion were recorded on disesed plnts lte in the seson. Eight isoltes of the vsculr wilt pthogen otined from different fields were identified on the sis of morphologic nd culturl criteri nd pthogenicity. All isoltes tested showed incidence of the disese on cv. Anns d Amérique plnts, estimted y the disese index, height nd fresh weight. A significnt negtive effect ws oserved on ll the mesured chrcteristics reveling the negtive influence of Fusrium wilt on the growth of muskmelon. Keywords: Cucumis melo L., Fusrium wilt, growth limittion, incidence, lef dmge INTRODUCTION Muskmelon (Cucumis melo L.) hs ecome the most importnt crop mong the cucurits grown in mny temperte nd su-tropicl countries. In Tunisi, over 10,000 h of muskmelon re grown nnully, nd nerly 116,000 tons were produced with yield of over 25 tons/h (Anonymous 2004). This cucurit is cultivted in glsshouses, minly in Tunisin Shel regions (Monstir, Sousse nd Mhdi), in low tunnels nd lso in the open in different regions of Tunisi (Arin, Bizerte, Sfx, Bèjà nd Jendou) (Jeri et l. 2004). The cultivted res hve grdully decresed during the lst decdes ecuse of the impct of severl fungl diseses, such s Verticillium nd Fusrium wilts (Mrtyn 1983; El Mhjou nd Ben Khedher 1987; El Mhjou et l. 1987; Ayed et l. 2007; Jnoun-Khireddine et l. 2007). Fusrium wilt of muskmelon, cused y Fusrium oxysporum f. sp. melonis, hs ecome one of the most criticl prolems in mny prts of the world (Netzer nd Weintll 1979; Zink nd Guler 1985; Jcoson nd Gordon 1990; Shreuder et l. 2000). This pthogen, including severl physiologicl rces (0, 1, 2, 1-2 y nd 1-2 w), cn cuse extensive losses of up to 90% (Mrtyn nd Amdor 1987; Chmpco et l. 1993; Ktn et l. 1994). It infects plnts through roots vi direct penetrtion, fter which the xylem vsculr tissue of the plnts is colonized, cusing stunting, vsculr wilting nd ultimtely plnt deth (El Mhjou nd le Picrd 1985). In Tunisi, during 2005 nd 2006 growing sesons, wilt nd sudden plnt collpse were oserved in some frms t the eginning of melon fructifiction nd progressing until hrvest. The pthogen ws susequently identified s Fusrium oxysporum. The ojectives of this study were to descrie the disese symptoms, identify the forme speciles nd to determinte the effect of F. oxysporum f. sp. melonis on seedling development. MATERIALS AND METHODS Pthogen isoltion During 2005, surveys for Fusrium wilt were crried out in the Tunisin muskmelon-growing res of Bizerte, Jendou, Bèjà nd Kiroun. Melon plnts showing symptoms of Fusrium wilt were collected from open fields. Isoltions were mde from root, crown nd stem tissues nd from fruits collected from infected muskmelon plnts. Stems nd roots were wshed under fine spry of tp wter nd cut into cm pieces. After surfce-disinfesting in sodium hypochlorite (10%) for 3 min, the plnt pieces were rinsed three times in sterile distilled wter nd ir-dried under lminr flow ench. Ech frgment ws plced on Potto Dextrose Agr (PDA) contining 300 mg/l of streptomycin sulfte nd incuted t 25 C for 4-5 dys in the drk. Single conidil isoltes of F. oxysporum were selected nd identified ccording to Messien nd Cssini (1968) nd Tivoli (1988). Eight isoltes otined from disesed muskmelon in 2005 were included in this study (Tle 1). For plnt inocultion, mycelium ws tken from the edge colony of ech isolte nd plced in Erlenmeyer flsks contining 150 ml of Potto Dextrose Broth (PDB) nd incuted t 25 C for 5 dys in rotry incutor (120 rpm). The liquid culture ws filtered nd the conidil suspension Tle 1 Origin of Fusrium oxysporum isoltes collected from fieldgrown muskmelon in Tunisi during Isolte Site of origin Cultivrs I 2 Bizerte Blnco I 4 Bizerte Blnco I 5 Bizerte Gold Myen I 7 Bizerte Anns d Amérique I 10 Jendou June Cnri I 12 Bèjà Lonni I 15 Kiroun Anns d Amérique I 20 Gès Lonni Received: 27 Ferury, Accepted: 10 August, Originl Reserch Pper

2 The Africn Journl of Plnt Science nd Biotechnology 1(1), Glol Science Books ws djusted to 10 7 conidi/ml y Mlssez cystometer (Nmiki et l. 1998). Plnt mteril Seeds of the muskmelon cultivr Anns d Amérique, susceptile to Fusrium wilt, were disinfected y immersion for 2 min in fresh 10% ethnol. They were then rinsed in sterile wter nd plced on moistened cotton wool in Petri dishes in the drk t 27 ± 2 C for 3 dys. The germinted seeds were plnted in pet, previously sterilized t 105 C for 1 h. The trys, fter inocultion t the expnsion of the first-true-lef stge, were plced in growth chmer t 26 ± 1 C during the dy nd 20 ± 1 C t night with 14 h photoperiod (64.75 μmol/m 2 /s). Pthogenicity tests After pproximtely 10 to 14 dys, seedlings were uprooted t the expnsion of the first-true-lef stge (Ltin nd Snell 1986). Roots were wshed in tp wter, pruned to ~2.5 cm nd dipped for 1 min in the inoculum suspension djusted t 10 7 conidi/ml (Jcoson nd Gordon 1988; Lorenzini et l. 1997). Ech isolte ws used to inoculte five seedlings. Controls were immersed in sterile distilled wter. Inoculted nd control seedlings were trnsplnted to plstic pots (0.7 l) filled with n utoclved mixture of pet nd perlite (3:1) nd plced in growth chmer t 26±1 C during the dy nd 20 ± 1 C t night with 14 h photoperiod. They were irrigted regulrly nd fertilized with the nutrient solution used y El Mhjou (1985) (per liter): g of KNO 3, 0.3 g of KH 2 PO 4, g of NH 4 Cl, 0.45 g of MgSO 4,7H2O, 0.3 g of (NH 4 ) 2 SO 4, 1.2 g of C(NO 3 ) 2, g of EDTA N, Fe nd other oligo-elements. Wilt symptoms, s descried y Jcoson nd Gordon (1988) (stunting, chlorosis, necrosis nd deth), were first oserved dys fter inocultion. If none of these symptoms were present, such isoltes were ctegorized s non-pthogenic to muskmelon. Reisoltions were mde, t the end of the iossy, from disesed plnts in order to confirm incrimintion of F. oxysporum in the induced symptoms. Disese incidence on muskmelon development The effect of F. oxysporum f. sp. melonis on seedling development ws ssessed through the index of disese severity using scle of 0-4 every 3-4 dys where: 0 = symptomtic lef, 1 = lef wilted, 2 = lef with hemiplegic yellowing, 3 = lef with necrosis, 4 = ded lef. Isolte virulence ws expressed s n index of lef dmge (I.L.D.) which is clculted for ech muskmelon plnt (Béye nd Lfy 1985): I.L.D. = notes/mx where notes = totl notes. Mx = 4 times the numer of developed leves. Plnt height nd fresh weight were recorded 43 dys fter inocultion. Pthogen isoltions were done, t the end of the iossy, to confirm its incrimintion in the disese development. Sttisticl nlyses Five plnts were inoculted for ech tretment (isoltes nd control). Anlyses of vrince were conducted for ech dependnt vrile to determine the effect of isoltes on plnt development. Dt re rrnged y completely rndomized design where tretments (seedlings inoculted y ech of eight Fusrium oxysporum isoltes nd control seedlings) represented the only fixed fctor. They were nlyzed using SPSS nd sujected to nlysis of vrince nd Fisher s lest significnt difference test LSD (t P 0.05). Fig. 1 Development of Fusrium wilt of field-grown muskmelon. (A) Plges hving Fusrium wilt symptoms in muskmelon field in Klt El Andlous in Bizerte, (B) Yellowing nd deth of the lower leves of muskmelon plnt, (C) Deth of some rnches, (D) Deth of entire muskmelon plnt, (E) Brown lesions with drk gum exudtes, (F) White mycelil growth on fruit surfce. A D B E C F 6

3 Muskmelon Fusrium wilt. Dmi-Remdi et l. Tle 2 Evolution of the Lef Dmge Index (I.L.D.) for muskmelon plnts, cv. Anns d Amérique, inoculted y F. oxysporum f. sp. melonis isoltes oserved 15 dys fter inocultion. Dys fter inocultion Index of Lef Dmge Control I 2 I 4 I 5 I 7 I 10 I 12 I 15 I c c c c c c d cd 0.54 cd c c c d cd cd cd 1.45 cd cd c c c c c c I n: F. o. f. sp. melonis Isoltes. z Within lines, mens followed y the sme letters re not significntly different (P 0.05) ccording to S.N.K.test. RESULTS Symptom development During 2005, muskmelon showing wilt symptoms were collected from field plntings in mjor Tunisin production res (Fig. 1A). The first symptoms of this disese were yellowing nd deth of some leves (Fig. 1B) followed y deth of one or more lterl vines, strting t the tip with runners dying ck towrds the crown (Fig. 1C) nd of ll plnt (Fig. 1D), respectively. Some runners my remin helthy. Reddish-rown lesions were oserved t the se of the plnt (Fig. 1E) developing long rnches. They could hve drk-rown gum exudtes (Fig. 1E). Furthermore, this soil-orne pthogen could lso colonize fruit infecting their tissues nd developing ove it white mycelium (Fig. 1F). Height (cm) Control I2 I4 I5 I7 I10 I12 I15 I20 F. oxysporum f. sp. melonis isoltes Fig. 3 Effect of inocultion y different F. oxysporum f. sp. melonis isoltes on muskmelon plnt height (cv. Anns d Amérique ), oserved 43 dys fter inocultion. c d Pthogenicity tests of F. oxysporum isoltes Typicl symptoms of Fusrium wilt disese were induced y ll F. oxysporum isoltes nd they ppered 15 to 25 dys fter inocultion. Inoculted muskmelon plnts showed hemiplegic yellowing t the lower leves, rowning in the vsculr region especilly in the stem nd scending wilt symptoms. These plnts wilted in the end of the iossy comprtively to the control plnts. Disese incidence on the development of muskmelon plnts Effect of Fusrium wilt on lef dmge All isoltes, used to inoculte seedlings y the root dip method, cused wilt on muskmelon cv. Anns d Amérique showing significnt difference etween helthy nd inoculted plnts (Tle 2). Inoculted Noninoculted Fig. 4 Effect of inocultion y F. oxysporum f. sp. melonis isolte on muskmelon plnt, cv. Anns d Amérique, comprtively to the noninoculted control oserved 25 dys fter inocultion. Noninoculted Inoculted Plnts developed disese symptoms, dys fter inocultion, which incresed differently during the iossy. The isolte I 15 is the most virulent showing higher disese severity noted t different evlution periods nd cusing the premture deth of plnts, 27 dys fter inocultion (Fig. 2). The other isoltes such s I 5, I 7 nd I 20 lso cused premture nd importnt wilt symptoms. Their lef dmge index reched , nd 5.175, respectively. However, I 2 nd I 10 were lesser wilt pthogens thn the other wilt isoltes showing nd in the end of the iossy respectively nd inducing lower disese incidence on plnts (Tle 2). Fig. 2 Comprison etween helthy muskmelon plnt, cv. Anns d Amérique (left), nd plnt inoculted with isolte I15 of F. oxysporum f. sp. melonis (right) 15 dys fter inocultion. Effect of Fusrium wilt on plnt height Regrdless of lef dmge, disese incidence ws evluted vi plnt growth. Indeed, these eight F. oxysporum f. 7

4 The Africn Journl of Plnt Science nd Biotechnology 1(1), Glol Science Books sp. melonis isoltes tested cused significnt reductions in muskmelon height (Fig. 3). The most importnt height reduction, 66.3%, ws induced y the isolte I 15 comprtively to non-inoculted plnts. In the sme wy, isoltes I 2, I 4, I 5 nd I 20 reduced clerly plnt length y 13.4 to 23.3% (Figs. 3, 4). Effect of Fusrium wilt on plnt fresh weight Fresh weight (g) cd cd cd c d c Fusrium wilt effect on plnt growth ws proved y reducing plnt fresh weight comprtively to non-inoculted plnts with significnt difference etween isoltes (Fig. 5). Isolte I 15 seemed to e the most ggressive, I 5 nd I 20 lso showed n importnt effect in reducing fresh weight y 76.3 nd 78.8%, respectively. DISCUSSION Fusrium wilt disese hs ecome serious thret for muskmelon crop in Tunisi in recent yers. It hs een reported in North Americ, Europe, Indi, Est Asi nd the Middle Est (Dutky et l. 1986; Jcoson nd Gordon 1988; Aou-Jwdh nd Al-Khoury 1996; de Cr et l. 2004). Fusrium symptoms, oserved under field conditions, such s wilting, necrosis, gum exudtes, stunting nd discolortion, differed from those oserved under greenhouse conditions, such s wilting, root rot nd yellowishornge discolourtion on the crown nd lower stem. Discolourtion ws first oserved in the vsculr tissues nd ws lter visile externlly s the pthogen spred into the corticl tissues. The sme symptoms were oserved y Punj et l. (2001) nd Kurt et l. (2002). On the sis of their ility to cuse disese on muskmelon, F. oxysporum isoltes re grouped into forme speciles melonis (Armstrong nd Armstrong 1981). These eight strins showed different levels of virulence on muskmelon plnts ffecting lef ltertion, height nd fresh weight. The negtive effects on melon growth were reported y Bletsos nd Thnssoulopoulos (2000). This soil-orne pthogen penetrtes nd invdes plnt tissue, overcomes host defenses nd optimizes growth in the plnt. To perform these tsks correctly, the fungus must perceive chemicl nd physicl signls from the host nd respond with the pproprite metolic nd morphogenetic chnges required for pthogenic development. Such chnges include direct hyphl growth, dhesion to the plnt surfce, occlusion of colonised vessels y gels, gums nd tyloses, differentition of specilized infection structures nd secretion of lytic enzymes nd phytotoxins (El Mhjou et l. 1984; El Mhjou 1985; di Pietro et l. 2001). In this respect, Mrtinez et l. (1991) reported tht the ility of the pthogen to cuse wilt symptom is correlted with high pectic ctivities such s exopolyglcturonse, endopolyglcturonse nd lyses. In the sme wy, Lorenzini et l. (1997) reported tht xylem colonistion y F. oxysporum increses resistnce to wter flow within the plnt, thus resulting in lef wter deficits tht might led to reductions in lef photosynthetic nd trnspirtion rtes, lef longevity nd consequently plnt fresh weight. Furthermore, some genes encode cell wlldegrding enzymes nd plnt sponin-detoxifying enzymes, which hve een implicted s possile key fctors in pthogenicity or virulence (di Pietro nd Roncero 1998; Inoue et l. 2001). Being one of the limiting fctors for melon production, the prevlence nd distriution of F. oxysporum f. sp. melonis rces in Tunisi reveled necessry in order to develop commercil melon cultivrs crrying resistnce to these rces. ACKNOWLEDGEMENTS Authors thnk The Higher Agronomic Institute of Chott-Mriem (ISAg-CM) for its finncil contriution. Mny thnks for Mohmed Youssef nd Aymen Youssef for their excellent technicl Control ssistnce. 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