Changes in SOD isozyme in mycorrhizal asparagus inoculated with Fusarium oxysporum

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1 26 Originl reserh rtile Chnges in SOD isozyme in myorrhizl sprgus inoulted with Fusrium oxysporum Ji Liu 1 nd Yoh-ihi Mtsur 2 1 The United Grdute Shool of Agriulturl Siene, Gifu University, Gifu , Jpn 2 Fulty of Applied Biologil Sienes, Gifu University, Gifu , Jpn Corresponding uthor: Yoh-ihi Mtsur, E-mil: ymtsu@gifu-u..jp, Phone: , Fx: Reeived on My 1, 216; Aepted on Septemer 26, 216 Astrt: Symiosis-speifi hnges in ntioxidtive ility nd superoxide dismutse (SOD) isozyme in myorrhizl sprgus plnts through the inoultion with Fusrium oxysporum f. sp. sprgi (Fo, MAFF35556) were investigted. Dry weight of shoots nd roots inresed more in AMF (Gi, Glomus intrrdies; GM, Gigspor mrgrit)-inoulted plnts thn the ontrol. Eight weeks fter Fo inoultion, the inidene nd severity of Fusrium root rot were llevited in oth AMF-inoulted plnts, ompred to the ontrol, whih were not llevited. Superoxide dismutse (SOD) tivity, sorte peroxidse (APX) tivity, nd 1,1-diphenyl-2-pirylhydrzyl (DPPH) rdil svenging tivity inresed in oth AMF-inoulted plnts more thn in the ontrol efore nd fter Fo inoultion, nd inresed in ll sprgus roots due to Fo inoultion. As for SOD isozymes in roots efore Fo inoultion, the Rf =.37 (Cu/Zn-SOD) nd hd higher intensity in oth Gi nd GM ompred to the ontrol. Eight weeks fter Fo inoultion, Rf =.33 nd.37 (Cu/Zn-SODs) showed higher intensity in oth Gi nd GM ompred to the ontrol. Though AM fungl differene ppered in the intensity of Rf =.25 (Mn/Fe-SOD); GM hd higher intensity thn Gi, Cu/Zn nds hd lerly higher intensity thn Mn/Fe nd. These findings supposed tht tolerne to Fusrium root rot in myorrhizl sprgus plnts might e losely ssoited with the hnges in SOD tivity nd isozymes, espeilly in Cu/ Zn-SODs. Keywords: Arusulr myorrhizl fungi, Bioontrol, Fusrium oxysporum f. sp. sprgi, SOD isozyme Introdution Fusrium rown nd root rot, used minly y Fusrium oxysporum f. sp. sprgi (Fo), results in erly deline of yields in ultivted sprgus (He et l. 22, Elmer 215). It is diffiult to ontrol this disese suessfully with ulturl nd hemil methods due to the perennil nture of the rop, nd highly resistnt ultivrs hve not een developed (Pontroli et l. 2, He et l. 22, Elmer 215). Arusulr myorrhizl fungi (AMF) re uiquitous soil inhitnts, nd form symioti reltionship with the roots of most of the terrestril plnts. AMF promotes host plnt growth y enhning phosphorus uptke through symiosis (Mrshner nd Dell 1994), nd hene is n lterntive to high inputs of hemil fertilizers in sustinle rop prodution systems. Additionlly, severl investigtors erlier reported higher resistne of myorrhizl plnts to ioti nd ioti stresses (Grmendi et l. 26, Wu et l. 26). As for sprgus, Wker et l. (199) reported tht pplition of AMF in greenhouse trils ws diretly ssoited with the redued dmge from rown nd root rot, nd Liu et l. (216) demonstrted the tolerne to Fusrium root rot in myorrhizl sprgus plnts in deline fields. However, mny points remin unler out the mehnisms of disese tolerne in myorrhizl plnts. Liu J nd Mtsur Y 216 Chnges in SOD isozyme in myorrhizl sprgus inoulted with Fusrium oxysporum. Plnt Root 1: doi:1.3117/plntroot.1.26 Copyrights 216, Plnt Root (JSRR),

2 27 When plnts suffer pthogenesis infetion, prodution of higher onentrtion of retive oxygen speies (ROS) suh s H 2 O 2, superoxide nion (O 2 ), nd hydroxyl rdil hs een shown to rete ytotoxi onditions (Shoo et l. 27, Whid nd Close 27). To overome this negtive onsequene of ROS, plnts hve evolved vrious protetive mehnisms either to redue or ompletely eliminte ntioxidtive ilities of produing ntioxidnt enzymes nd sustnes (Moghddm et l. 26, Shoo et l. 27). The superoxide dismutse (SOD) plys primry role in defensive retions nd detoxifies superoxide (O 2 ) mong the ntioxidnt enzymes; thus, SOD tivity is onsidered the most importnt key enzyme in plnt s ntioxidnt ility (Fridovih 1986). In ddition, SODs re lssified into three different types in plnts, depending on their site nd metl ion: mitohondril mngnese (Mn-SODs), ytosoli nd hloroplsts opper/zin (CuZn-SODs), nd hloroplsti iron forms (Fe-SOD); these isoenzymes differ in their sensitivity to H 2 O 2 nd KCN nd n e esily distinguished y gel eletrophoresis (Fridovih 1986). In myorrhil plnts, in Pisum stivum-g. mossee symiosis, no qulittive differenes mong the SOD isozyme ptterns in oth myorrhizl nd non-myorrhizl roots were oserved, lthough higher SOD tivity ws deteted in myorrhizl pe roots (Arines et l. 1994). Pozo et l. (22) reported tht Mn-SOD s new isozyme ws deteted in myorrhizl (G. mossee) tomto plnts efore nd fter Phytophthor prsiti inoultion without SOD tivity nlysis. However, studies on the reltionship etween SOD tivity nd SOD isozyme hnges through AMF symiosis nd pthogeninoulted myorrhizl plnts re still rrely reported. In ddition, there re no reports on the hnges in SOD isozymes in myorrhizl plnts inoulted with Fusrium oxysporum s mjor pthogen, inluding AM fungl differene. We imed to lrify symiosis-speifi hnges in SOD tivity nd isozymes in myorrhizl sprgus plnts (Asprgus offiinlis L., v. Welome, suseptile to Fusrium root rot) y Fusrium oxysporum f. sp. sprgi (Fo) inoultion. Firstly, hnges in ntioxidtive enzymes [tivity of SOD nd sorte peroxidse (APX) tivity], 1,1-diphenyl-2-pirylhydrzyl (DPPH) rdil svenging tivity (frequently estimted s totl ntioxidtive sustne) y AMF nd Fo inoultion were determined. Then, SOD isozymes in myorrhizl sprgus plnts through the inoultion with Fo were investigted. Mterils nd Methods Inoultion of AMF Seeds of sprgus (Asprgus offiinlis L., v. Welome, suseptile to Fusrium root rot) were sown in ommeril soil (utolved t 1.2 kg m -2 nd 121 C for 1 hour) in plsti ontiner (19.W 33.5L 15.5H m). During the time of seed sowing, plnt holes were mde, nd eh hole ontins 3 g plnt -1 ommeril AMF of Glomus intrrdies (Gi, supplied y Idemitsukosn Co. Ltd. Tokyo, Jpn.), Gigspor mrgrit (GM, supplied y Centrlgrss Co. Ltd. Tokyo, Jpn.). After sowing, plnt holes were overed with soil nd dministered y mixed fertilizer (N: P: K = 13: 11: 13,.5 g plnt -1 ). Twenty plnts per plot with three replitions were irrigted regulrly nd grown in greenhouse. Eight weeks fter AMF inoultion, the dry weight of the shoots nd roots were investigted. The verge ws lulted from the vlues of 1 plnts in eh replition. Inoultion of Fusrium oxysporum f. sp. sprgi Fusrium oxysporum f. sp. sprgi (Fo; MAFF35556) were grown on potto dextrose gr medi. The onidi were hrvested in potto surose liquid medi nd inuted t 25 C in the drk ondition for 7 dys. The onidil suspension ws sieved nd the onentrtions djusted to 1 6 onidi ml -1. Eight weeks fter AMF inoultion, eh plnt ws inoulted y pouring 5 ml of the onidil suspension onto the soil. Eight weeks fter Fo inoultion, the symptoms were tegorized into five degrees: of perentge of storge roots with root lesions in plnt: 1, less thn 2%; 2, 2-4%; 3, 4-6%; 4, 6-8%; 5, 8-1%. In ddition, the disese index ws lulted y the following formul: Disese index = Σ(numer of plnts numer of degree in symptom) Totl numer of plnts 5 (mximum degree in symptom) 1

3 28 Evlution of AMF oloniztion level Eight weeks fter AMF-inoultion nd 8 weeks fter Fo inoultion, the roots of the sprgus were preserved with 7% ethnol nd stined ording to Phillips nd Hymn (197). The perentge of AM fungl oloniztion in 1-m segments of the lterl roots (revited PFCSL) ws lulted from pproximtely 3 smples of 1-m segments per plnt, for five plnts per plot nd time point. Anlysis of ntioxidtive ility Eight weeks fter AMF inoultion nd 8 weeks fter Fo inoultion, plnts were smpled nd prtitioned into shoots nd roots. Then, the smples were frozen in liquid nitrogen. Antioxidtive ilities were nlyzed y the following methods. As for SOD tivity, liquots (.1 g) of the roots nd shoots were homogenized in 4 ml of 5 mm phosphte-orte uffer (ph 7.8). The filtrte ws entrifuged (EF-13, Tomy Co., Ltd., Tokyo, Jpn) t 13, rpm for 1 min. The superntnt ws used for rude enzyme extrt. The tivity ws determined using the Nitro Blue Tetrzolium (NBT) redution method (Beuhmp nd Fridovih, 1971). APX ws extrted with 3 ml of n ie-old 5 mm phosphte uffer (ph 7.), nd the filtrte ws entrifuged t 13, rpm for 15 min nd determined using the method of Wu et l. (26) y mesuring the oxidtion of sorte t 29 nm using spetrophotometer (U-19, HITACHI). DPPH rdil svenging tivity ws mesured ording to the method of Burits nd Bur (2). A one grm smple ws ground in 4 ml of 9% methnol. The 1 μl of the extrt ws introdued into test tues, nd 2.7 ml of DPPH solution ws dded. The tues were mixed nd left to stnd for 3 min in the drk. Asorne ws red ginst lnk t 52 nm using spetrophotometer (U-19, HITACHI). SOD isozyme ws mesured ording to the method of Dvis (1964) nd Sohoo et l. (27). Roots (.5 g, fresh weight) were frozen in liquid nitrogen nd pulverized in mortr with 3 ml extrtion uffer ontining 75 mg L -1 polyoinylpolypyrrolidone (PVPP), 12.1 g/l Tris, 68 g L -1 surose, 17 mg L -1 EDTA,.31 ml L -1 Tween 8 nd 8 mg L -1 sodium-thioglyolte. The filtrte ws entrifuged t 13, rpm for 15 min in 5 C nd the superntnt (18 μl) ws mixed with 2 μl 4% surose nd 1 μl BPB. All extrts were nlyzed y 12.5% polyrylmide gel (e-pagel E-T 12.5L, ATTO Co., Ltd., Tokyo, Jpn). Eletrophoresis (PAGE) under ntive onditions (1 V, 2 ma, 13 min) using n eletrophoresis pprtus (AE-65, ATTO Co., Ltd., Tokyo, Jpn) ws rried out. SOD isoforms were deteted diretly on the gel fter eletrophoresis y the method desried y Beuhmp nd Fridovih (1971) sed on the inhiition to the stining uffer. The reltive distne (Rf vlue) of the nds ws derived from the men vlue of mny runs, lulted fter ssigning Rf = 1 to the fstest nd nd Rf = to the strt point of the runing gel (Mngris nd Alston 1992). Sttistil nlysis Men vlues were seprted y t test or Tukey s multiple rnge test t P <.5. All nlyses were performed using XLSTAT pro sttistil nlysis softwre (Addinsoft, New York, NY). Result Eight weeks fter AMF inoultion, the dry weight of the shoots nd roots of AMF plnts signifintly inresed ompred to the ontrol, espeilly in Gi (Fig. 1). AMF oloniztion ourred in ll the inoulted plnts suessfully. RFCSL rehed more thn 6% in oth AMF plnts, nd no signifint differene ourred mong the plots throughout the experimentl period (Fig. 2). In the nlysis of Dry weight of shoots (g) Dry weight of roots (g) Fig. 1. Dry weight of shoots nd roots in sprgus plnts 8 weeks fter AMF inoultion. C, ontrol; Gi, Glomus intrrdies; GM, Gigspor mrgrit. Brs represent stndrd errors (n = 1). Columns denoted y different letters indite signifint differene ording to Tukey s test (P <.5).

4 PFCSL (%) NS NS NS NS 2 Gi GM Gi GM Fo- Fo+ Fig. 2. AMF oloniztion level (PFCSL) in sprgus plnts efore nd fter Fusrium oxysporum f. sp. sprgi (Fo) inoultion. Gi, Glomus intrrdies; GM, Gigspor mrgrit. Fo-, efore Fusrium oxysporum f. sp. sprgi inoultion; Fo+, fter Fusrium oxysporum f. sp. sprgi inoultion. Brs represent stndrd errors (n = 5). NS, indite no signifint differene mong the tretments ording to Tukey s multiple rnge test (P <.5). SOD tivity in shoots (units g -1 FW) SOD tivity in roots (units g -1 FW) APX tivity in shoots (AsA μmol g -1 FW) APX tivity in roots (AsA μmol g -1 FW) DPPH rdil svenging tivity in shoots (mg g -1 FW) DPPH rdil svenging tivity in roots (mg g -1 FW) Fig. 3. Superoxide dismutse (SOD) tivity, sorte peroxidse (APX) tivity nd 1,1-diphenyl-2-pirylhydrzyl (DPPH) rdil svenging tivity in shoots nd roots 8 weeks fter AMF inoultion. C, ontrol; Gi, Glomus intrrdies; GM, Gigspor mrgrit. Brs represent stndrd errors (n = 1). Columns denoted y different letters indite signifint differene ording to Tukey s test (P <.5). ntioxidtive ility eight weeks fter AMF inoultion, SOD nd APX tivity were signifintly higher in the AMF plnts thn the ontrol in oth the shoots nd roots, espeilly in Gi (Fig. 3). On the other hnd, DPPH rdil svenging tivity inresed with signifine in shoots nd roots of the AMF plnts, espeilly in GM. Eight weeks fter Fo inoultion, inidene of Fusrium root rot in roots rehed up to 8% in the

5 3 ontrol plnts (Fig. 4), ut ws lower in Gi plnts (3%) nd GM plnts (5%). The severity of the symptoms in the roots eme lower in oth Gi nd GM ompred to ontrol. The disese indies of Fusrium root rot rehed more thn 4 in ontrol plnts, wheres it ws s low s 8 in Gi nd 14 in GM (Fig. 4). Hene, the disese indies nd inidene of Fusrium root rot for the AMF nd ontrol plnts followed similr pttern. As for ntioxidtive ility 8 weeks fter Fo inoultion, SOD nd APX tivity were signifintly higher in AMF roots, espeilly in Gi (Fig. 5). DPPH rdil svenging tivity inresed signifintly in AMF roots ompred to the ontrol; GM ws higher thn Gi. In ddition, SOD, APX nd DPPH rdil svenging tivity in roots inresed in ll the sprgus roots due to Fo inoultion (Figs. 3 nd 5). As for SOD isoenzymes in roots, Cu/Zn- (Rf =.33 nd.37) nd Mn/Fe-SOD (Rf =.25) were deteted in oth AMF nd ontrol roots efore nd fter Fo inoultion (Fig. 6). In this se, Cu/Zn nds hd higher intensity thn Mn/Fe in ll the plots, nd no differene ppered in the nd positions mong the plots. Before Fo inoultion (Fo-), SOD-1 (Rf =.37, Cu/Zn-SOD) nds eme higher in intensity in oth Gi nd GM ompred to the ontrol. Eight weeks fter Fo inoultion (Fo+), SOD-1 nd -2 (Rf =.37,.33, Cu/Zn-SODs) showed higher intensity in oth Gi nd GM thn the ontrol. The intensity of those Cu/ Zn nds deresed fter Fo inoultion in ontrol. In ddition, SOD-3 (Rf =.25, Mn/Fe-SOD) in GM inresed in intensity ompred to ontrol fter Fo inoultion. Compring sttes efore nd fter Fo inoultion, SOD-1,2 (Cu/Zn-SODs) deresed in the ontrol nd GM, ut inresed in Gi; SOD-3 (Mn/Fe-SOD) inresed in GM due to Fo inoultion. Disussion In this study, the dry weight of shoots nd roots inresed in AMF plnts ompred to the ontrol, suggesting tht growth-promoting effet through AMF symiosis ppered in sprgus plnts. As for tolerne to Fusrium root rot, the inidene nd severity of symptoms were llevited y AMF, with Gi hving etter results thn GM, though no signifint differene ppered in AMF oloniztion level. These results indited tht tolerne to Fusrium root rot ourred in sprgus plnts inoulted with AMF. In ddition, the ntioxidtive ility of myorrhizl plnts inresed efore nd fter Fo inoultion ompred to ontrol. In this se, the enhnement of SOD ntioxidnt pity in oth Gi nd GM plnts is onsidered s result of inresed isozymes. From these fts, it is suggested tht tolerne to Fusrium root rot in myorrhizl plnts is losely ssoited with the inrese in ntioxidtive ility. In the Pisum stivum-g. mossee symiosis, no qulittive differenes mong the isozyme ptterns in oth myorrhizl nd non-myorrhizl roots were oserved, lthough higher SOD tivity ws deteted in myorrhizl pe roots (Arines et l. 1994). In pthogen stress ondition, Pozo (22) reported tht Mn-SOD s new isozyme ws deteted in myorrhizl (G. mossee) tomto plnts without SOD tivity nlysis. In the report, no hnges ppered in the nd pttern efore nd fter Phytophthor inoultion, so tht only AMF oloniztion ffeted Mn-SOD isozymes. In this study, the intensity of the Cu/Zn isozyme hnged through AMF oloniztion, nd Fusrium inoultion indued dditionl hnges in the intensity of Cu/Zn- nd Fe/Zn-SOD isozymes inluding AM fungl differene. These re new findings ompred to those former reports. Some reports desried tht AMF oloniztion itself indued temporry inrese in ntioxidtive ilities suh s SOD, guiol peroxidse (G-POD), tlse (CAT), APX, DPPH rdil svenging tivity nd flvonoid ontent, suggesting tht oloniztion might e temporry stress for host plnts (Wu et l. 26, Zhu et l. 21). Antioxidnt levels in plnts nd tivities of ROS svenging enzymes hve een reported to e orrelted with tolerne to different environmentl stresses (Chitny et l. 22) inluding pthogen ttk in plnts (Miyzw et l. 1998). Our results showed similr ptterns to those findings s the inrese in SOD, APX nd DPPH rdil svenging tivity y AMF oloniztion efore nd fter Fo inoultion. On the other hnd, Moghddm et l. (26) mentioned tht higher SOD tivity nd newly formed SOD isozyme (Rf =.53) ws onfirmed in resistnt strwerry ultivr thn suseptile one with Myospherell frgrie infetion; the SOD isozyme ws supposed to e onerned with the disese resistne. In this experiment, Gi plnts showed higher dry weight nd lower disese index thn ontrol. In ddition, suppression of the Cu/Zn- SODs (Rf =.33,.37) ppered in ontrol roots, nd inresed intensity of those Cu/Zn-SODs ppered in Gi fter Fo inoultion. From these findings, espeilly, Cu/Zn isozymes might e losely ssoited with resistne to Fusrium root rot in myorrhizl sprgus. Antioxidtive sustnes, frequently estimted totlly s DPPH rdil svenging tivity, hve

6 31 Inidene of Fusrium root rot (%) Disese index of Fusrium root rot Fig. 4. Inidene nd disese indies of Fusrium root rot in sprgus plnts 8 weeks fter Fusrium oxysporum f. sp. sprgi inoultion. C, ontrol; Gi, Glomus intrrdies; GM, Gigspor mrgrit. Rtio of disesed storge roots;, -2 ;, 2-4 ;, 4-6;, 6-8;, 8-1 ( % ). Brs represent stndrd errors (n = 2). Columns denoted y different letters indite signifint differene ording to Tukey s test (P <.5). SOD tivity in roots (units g -1 FW) APX tivity in roots (AsA μmol g -1 FW) DPPH rdil svenging tivity in roots (mg g -1 FW) Fig. 5. Superoxide dismutse (SOD) tivity, sorte peroxidse (APX) tivity nd 1,1-diphenyl-2-pirylhydrzyl (DPPH) rdil svenging tivity in roots 8 weeks fter Fusrium oxysporum f. sp. sprgi inoultion. C, ontrol; Gi, Glomus intrrdies; GM, Gigspor mrgrit. Brs represent stndrd errors (n = 1). Columns denoted y different letters indite signifint differene ording to Tukey s test (P <.5). Fo- Fo+ C Gi GM SOD-3 (Rf =.25) Mn/Fe SOD-2 (Rf =.33) SOD-1 (Rf =.37) Cu/Zn Fig. 6. Chnges in superoxide dismutse (SOD) isozyme of myorrhizl sprgus roots efore nd fter Fusrium oxysporum f. sp. sprgi (Fo) inoultion. Gi, Glomus intrrdies; GM, Gigspor mrgrit; Fo-, efore Fusrium oxysporum f. sp. sprgi inoultion; Fo+, fter Fusrium oxysporum f. sp. sprgi inoultion. Rf = reltive moility (i.e., reltive eletrophoreti moility with respet to the romophenol lue nd).

7 32 lower eletron redution potentil thn the eletron redution potentil of oxygen rdils; s result, they diretly svenge retive oxygen intermedites without promoting further oxidtive retions (Ainsworth et l. 27). Vnith et l. (29) mentioned tht totl phenol ontent inresed teril wilt in tomto upon Rlstoni solnerum inoultion. Hihem et l. (29) reported tht slt stress indued DPPH free rdil svenging tivity nd polyphenoli ompounds in mize. In myorrhizl St. John s wort plnts inoulted with Colletotrihum gloeosporioides, sori id ontent nd disese tolerne inresed (Rihter et l. 211). In this experiment, disese index eme lower in oth Gi nd GM thn ontrol, nd the inrese in DPPH rdil svenging tivity lso ourred in oth AMF plnts. Hene, ntioxidtive sustnes lso hve n ssoition with tolerne to Fusrium root rot in myorrhizl sprgus plnts, the sme s ntioxidtive enzymes. Normn et l. (1996) reported tht the inidene of the symptom used y Phytophthor frgrie in strwerry plnts ws redued y the inoultion of AMF, though the effet differed with AM fungl speies. Ozgonen nd Erkili (27) reported tht growth promotion nd tolerne to Phytophthor psii hd no orreltion with the myorrhizl oloniztion levels in pepper. In this study, the AMF oloniztion level ws only heked twie, so it is diffiult to estimte when AMF rehed mximum oloniztion level during the experimentl period, nd how the oloniztion level ffets SOD isozymes in host plnts. Further study would e needed to lrify the reltionship etween AMF oloniztion level nd SOD isozyme inluding the reltion with other host nd pthogeni fungi. Conlusion AMF (Gi nd GM) plnts showed n inrese in the dry weight of shoots nd roots. Eight weeks fter Fo inoultion, the inidene nd severity of Fusrium root rot were oth llevited in AMFinoulted plnts to greter extent thn ontrol. SOD nd APX tivity nd DPPH rdil svenging tivity inresed in oth AMFinoulted plnts ompred with the ontrol efore nd fter inoultion. Eight weeks fter Fo inoultion, espeilly, Rf =.33 nd.37 nds (Cu/Zn-SODs) showed higher intensity in oth Gi nd GM ompred to the ontrol, nd AM fungl differene ppered in the Rf =.25 (Mn/Fe-SOD) intensity. These findings suggest tht tolerne to Fusrium root rot in myorrhizl sprgus plnts might e losely ssoited with the hnges in SOD tivity nd isozymes, espeilly in Cu/Zn- SODs. Aknowledgements This study ws supported y Grnts-in-Aid for Sientifi Reserh (No ), Jpn Soiety for the Promotion of Siene (JSPS). Referenes Ainsworth EA, Gillespie KM 27 Estimtion of totl phenoli ontent nd other oxidtion sustrtes in plnt tissues using Folin-Ciolteu regent. Nt. Prot. 2: Arines J, Quintel M, Vilriño A, Plm JM 1994 Protein ptterns nd superoxide dismutse tivity in nonmyorrhizl nd rusulr myorrhizl Pisum stivum L. plnts. Plnt Soil 166: Beuhmp C, Fridovih I 1971 Superoxide Dismutse: Improved ssys nd n ssy pplile to rylmide gels. Anl. Biohem. 44: Burits M, Bur F 2 Antioxidnt tivity of Nigell stiv essentil oil. Phytother. Res. 14: Chitny KV, Sundr D, Msilmni S, Reddy R 22 Vrition in het stress-indued ntioxidnt enzyme tivities mong three mulerry ultivrs. Plnt Growth Reg. 36: Dvis BJ 1964 Dis eletrophoresis. II. Method nd pplition to humn serum proteins. Ann. N. Y. Ad. Si. 121: Elmer WH 215 Mngement of Fusrium rown nd root rot of sprgus. Crop Prot. 73: 2-6. Fridovih I 1986 Biologil effets of the superoxide rdils. Arh. Biohem. Biophys. 247: Grmendi I, Aguirreole J, Goioehe N 26 Defenerlted enzymes in pepper roots during intertions with rusulr myorrhizl fungi nd/or Vertiillium dhlie. Bioontr. 51: He CY, Hsing T, Wolyn DJ 22 Indution of systemi disese resistne nd pthogen defene responses in Asprgus offiinlis inoulted with nonpthogeni strins of Fusrium oxysporum. Plnt Pthol. 51: Hihem H, Mounir D, Neur EA 29 Differentil responses of two mize (Ze mys L.) vrieties to slt stress: hnges on polyphenols omposition of folige nd oxidtive dmges. Ind. Crops Prod. 3: Liu J, Tniguhi A, Mtsur Y, Kojim T, Tkhshi K 216 PCR-SSCP nlysis nd disese ontrol y the pplition of iologil nd hemil gents in sprgus deline. At Hort. (In press) Mngnris AG, Alston FH 1992 Inheritne nd linkge reltionships of peroxidse isoenzymes in pple. Theor. Appl. Genet. 83: Mrshner H, Dell B 1994 Nutrient uptke in myorrhizl symiosis. Plnt Soil 159: Miyzw J, Kwt T, Ogswr N 1998 Indution of n idi isozyme of peroxidse nd quired resistne to

8 33 wilt disese in response to tretment of tomto roots with 2-furoi id, 4-hydroxyenzoi hydrzide or sliyli hydrzide. Physiol. Mol. Plnt Pthol. 52: Moghddm B, Chrles MT, Crisse O, Khnizdeh S 26 Superoxide dismutse responses of strwerry ultivrs to infetion y Myospherell frgrie. J. Plnt Physiol. 163: Normn JR, Atkinson D, Hooker JE Arusulr myorrhizl fungl-indued ltertion to root rhiteture in strwerry nd indued resistne to the root pthogen Phytophthor frgrie. Plnt Soil 185: Ozgonen H, Erkili A 27 Growth enhnement nd Phytophthor light (Phytophthor psii Leonin) ontrol y rusulr myorrhizl fungl inoultion in pepper. Crop Prot. 26: Phillips JM, Hymn DS 197 Improved proedures for lering roots nd stining prsiti nd vesiulrrusulr myorrhizl fungi for rpid ssessment of infetion.trns. Br. Myol. So. 55: Pontroli AC, Cmdro EL, Bine FJ, Rido A 2 Responese of Asprgus offiinlis pollen to the ulture filtrte of Fusrium oxysporum f. sp. sprgi. Si. Hort. 84: Pozo MJ, Cordier C, Dums-Gudot E, Gininzzi S, Bre JM, Azón-Aguilr C 22 Lolized vs systemi effet of rusulr myorrhizl fungi on defene response to Phytophthor infetion in tomto plnts. J. Exp. Bot. 53: Rihter J, Bltrusht H, Krodt K, Shellenerg I 211 Impt of rusulr myorrhiz on the St. John s wort (Hyperium perfortum) wilt disese indued Colletotriuhum f. gloeosporioides. J. Plnt Dis. Prot. 118: Shoo MR, Dsupt M, Kole PC, Bht JS, Mukherjee A 27 Antioxidtive enzymes nd isozymes nlysis of tro genotypes nd their implitions in Phytophthor light disese resistne. Myopthol. 163: Vnith SC, Nirnjn SR, Umesh S 29. Role of phenyllnine mmoni lyse nd polyphenol oxidse in host resistne to teril wilt of tomto. J. Phytopthol. 157: Wker TL, Sfir GR, Stephens CT 199 Effet of Glomus fsiultum growth of sprgus nd the inidene of Fusrium root rot. J. Amer. So. Hort. Si. 115: Whid A, Close TJ 27 Expression of dehydrins under het stress nd their reltionship with wter reltions of sugrne leves. Biol. Plnt. 51: Wu Q, Zou Y, Xi R 26. Effet of wter stress nd rusulr myorrhizl fungi on retive oxygen metolism nd ntioxidnt prodution y itrus (Citrus tngerine) roots. Eur. J. Soil Biol. 42: Zhu X, Song F, Xu H 21 Influene of rusulr myorrhiz on lipid peroxidtion nd ntioxidtive enzyme tivity of mize plnts under temperture stress. Myorrhiz 2: