Review. Arbuscular mycorrhizal fungi (AMF) as bioprotector agents against wilt induced by Verticillium spp. in pepper

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1 Instituto Nionl de Investigión y Tenologí Agrri y Alimentri (INIA) Aville online t Spnish Journl of Agriulturl Reserh 21, 8(S1), S25-S42 ISSN: X Review. Arusulr myorrhizl fungi (AMF) s ioprotetor gents ginst wilt indued y Vertiillium spp. in pepper N. Goioehe 1 *, I. Grmendi 1,2, M. Sánhez-Díz 1 nd J. Aguirreole 1 1 Deprtmento de Biologí Vegetl. Seión Biologí Vegetl (Unidd Asoid l CSIC, EEAD, Zrgoz e ICVV, Logroño). Fultdes de Cienis y Frmi. Universidd de Nvrr. C/ Irunlrre, Pmplon. Spin 2 Present ddress: Deprtmento de Cienis de l Tierr y del Medio Amiente. Fultd de Cienis. Universidd de Alinte. Ctr. Sn Viente del Rspeig, s/n. Apdo. Correos Alinte. Spin Astrt Vertiillium dhlie Kle. is vsulr pthogen tht lters wter sttus nd growth of pepper plnts nd uses drsti redutions in yield. Its ontrol is diffiult euse it n survive in field soil for severl yers. The pplition of rusulr myorrhizl fungi (AMF) s ioprotetor gents ginst V. dhlie is n lterntive to the use of hemils whih, in ddition, is more respetful with the environment. The estlishment of the mutulisti ssoition of plnt roots nd AMF involves ontinuous ellulr nd moleulr dilogue etween oth symionts tht inludes the pretivtion of plnt defense responses tht my enhne the resistne or tolerne of myorrhizl plnts to soil-orne pthogens. Some AMF n improve the resistne of Cpsium nnuum L. ginst V. dhlie. This is espeilly relevnt for pepper ultivrs (i.e. v. Piquillo) tht exhiit high suseptiility to this pthogen. Compred with non-myorrhizl plnts, myorrhizl pepper n exhiit more lned ntioxidnt metolism in leves long the first month fter pthogen inoultion, whih my ontriute to dely oth the development of disese symptoms nd the derese of photosynthesis in Vertiillium-inoulted plnts with the susequent enefit for yield. In stems, myorrhizl pepper show erlier nd higher deposition of lignin in xylem vessels thn nonmyorrhizl plnts, even in sene of the pthogen. Moreover, AMF n indue new isoforms of idi hitinses nd superoxide dismutse in roots. Myorrhizl-speifi indution of these enzymti tivities together with enhned peroxidse nd phenyllnine mmoni-lyse in roots my lso e involved in the ioprotetion of Vertiillium-indued wilt in pepper y AMF. Additionl key words: iologil ontrol, Cpsium nnuum, myorrhizl symiosis, Vertiillium dhlie Kle. Resumen Revisión. Los hongos miorríio rusulres (MA) omo gentes ioprotetores del pimiento frente l se induid por Vertiillium spp. Vertiillium dhlie Kle. lter el estdo hídrio y el reimiento de ls plnts de pimiento y redue l produión del fruto. El empleo de hongos miorríio rusulres (MA) omo ioprotetores ontr V. dhlie es un lterntiv l uso de produtos químios más respetuos pr el medio miente. L soiión entre ls ríes de ls plnts y los hongos MA onllev un diálogo elulr y moleulr entre mos simiontes, que inluye l pretivión de respuests de defens en l plnt, lo ul puede inrementr l resisteni o tolerni de ls plnts miorrizds hi los ptógenos edáfios. Algunos hongos MA inrementn l resisteni del pimiento (Cpsium nnuum L.) ontr V. dhlie. Esto es espeilmente relevnte undo el ultivr (v. Piquillo, por ejemplo) es muy suseptile hi el ptógeno. En omprión on ls plnts no miorrizds, ls hojs de ls plnts de pimiento miorrizds muestrn un metolismo ntioxidnte más equilirdo lo lrgo del primer mes trs l inoulión del ptógeno, lo que puede retrsr el desenso de l fotosíntesis, on el onsiguiente enefiio pr l produión. L deposiión de lignin en el xilem es más temprn en los tllos de ls plnts miorrizds y los hongos MA induen nuevs isoforms de enzims quitins áid y superóxido dismuts en ls ríes. L indu- * Corresponding uthor: niegoi@unv.es Reeived: ; Aepted:

2 S26 N. Goioehe et l. / Spn J Agri Res (21) 8(S1), S25-S42 ión espeífi de ests isoenzims unid uns tividdes peroxids y fenillnin monio-lis más elevds en ls ríes de ls plnts miorrizds tmién pueden fvoreer l ioproteión del pimiento frente l se induid por Vertiillium. Plrs lve diionles: Cpsium nnuum, ontrol iológio, simiosis miorríi, Vertiillium dhlie Kle. Introdution Peppers elong to the fmily of Solnee nd the genus Cpsium. Although this genus inludes 25 speies, most of the peppers ultivted in temperte nd tropil res elong to the speies C. nnuum, thought to originte in Mexio nd Centrl Ameri (Andrews, 1995). The ultivted peppers re hereous, frost-sensitive plnts tht n e grown s n nnul or s perennil rop in field or greenhouses. This rop is widely onsumed s fresh vegetle or ondiment nd used for phrmeutil nd osmeti purposes (Boslnd, 23). Pepper plnts n suffer disorders, diseses nd pests tht redue fruit qulity nd yield. Diseses n e used y wide rnge of iologil gents, inluding teri, fungi, viruses, prsiti higher plnts, insets, nemtodes, irds nd mmmls (Boslnd, 23). The most importnt diseses used y fungi nd Oomyetes re Anthrnose, Cerospor, Lef Spot, Chrol Rot, Chonephor Blight, Dmping- Off nd Root Rot, Fusrium Stem Rot, Fusrium Wilt, Gry Lef Spot, Gry Mold, Phytophthor Blight, Powdery Mildew, Southern Blight, White Mold nd Vertiillium Wilt. Vertiillium Wilt onstitutes relevnt eonomi prolem euse it produes gret yield losses in Ameri (Evns nd MKeen, 1975) nd Mediterrnen ountries (Thnssoulopoulos nd Kitsos, 1972; Grí-Min et l., 1996; Pomr et l., 21) nd it n ffet pepper ultivted in oth field (Grí- Min et l., 1996) nd greenhouse (Gyoso et l., 27). Vertiillium Wilt of pepper n e used y oth Vertiillium lo-trum Reinke nd Berthlod nd V. dhlie Klehn, eing the ltter the most frequently reported s the usl orgnism (Golderg, 23). Cusl gents of Vertiillium Wilt in pepper Both Vertiillium lo-trum nd V. dhlie re virulent plnt pthogeni speies with low sprotrophi ilities. However, while V. dhlie form mirosleroti, whih re lk melnised lumps formed y udding of myelil ells, V. lo-trum produes only drk nd melnised resting myelium (Brr nd Clewes, 23). Those resting strutures re responsile for the gret diffiulties to ontrol Vertiillium wilt diseses. Moreover, the ontrol eomes extremely diffiult when the pthogen forms miroesleroti euse these resting strutures n survive 1 to 15 yers in the soil (Hele nd Krpp, 1999). Aording to Brr nd Clewes (23), V. dhlie should inlude ll isoltes whih produe only mirosleroti. The epidemiology of V. dhlie hs een widely studied. As explined y Zhou et l. (26), the infetion proess of V. dhlie n e divided into two phses. Phse I egins with the germintion of mirosleroti in response to plnt root exudtes (Mol nd Sholte, 1995). The fungus enters the root through epidermis or wounds, rossing ortil root to gin ess to immture xylem elements. Then phse II tkes ple, whih inludes hyphl prolifertion nd onidi prodution (Bekmn, 1987). The onidi re spred in the vsulr elements, where they germinte nd oloniztion ours. The new set of onidi ontinues the oloniztion of the upstrem vessels (Bekmn, 1987; Gold et l., 1996). In lter stge nd oiniding with senesene of leves, the fungus enters sprophyti stge. It goes out of the xylem elements nd olonizes the surrounding nonvsulr tissues in shoots nd roots. Afterwrds, new mirosleroti tht pper in the dying stems nd leves (Mol, 1995) will e inorported in the soil nd will onstitute new soure of inoulum. Vertiillium dhlie uses, therefore, monoyli disese, whih mens tht only one yle of disese nd inoulum prodution tke ple during growing seson (Frdin nd Thomm, 26). In ontrst to V. dhlie, V. lo-trum, during the sprophyti stge, my produe onidi on infeted plnt tissues tht n eome irorne. The dispersion of onidi fvors fungl spred so tht nother yle of disese n e initited Arevitions used: AMF (rusulr myorrhizl fungi), CAT (tlse), CER (CO 2 exhnge rte), GPX (guiol peroxidse), g w (lef ondutne), PAL (phenyllnine mmoni-lyse), RWC (reltive wter ontent), SOD (superoxide dismutse), T (trnspirtion rte), Ψ (wter potentil), Ψ p (pressure potentil).

3 Bioprotetion of pepper ginst Vertiillium-indued wilt y rusulr myorrhizl fungi S27 (Jiménez-Díz nd Millr, 1988). This is the reson why the diseses used y V. lo-trum my sometimes e polyyli (Frdin nd Thomm, 26). In the dormnt phse, V. lo-trum only produes drk nd melnised resting myelium whih exhiits shorter survivl potentil thn mirosleroti produed y V. dhlie. Growth, physiology, metolism nd fruit yield of pepper plnts infeted y Vertiillium spp. Symptom severity of Vertiillium Wilt in pepper widely vries with soil nd ir tempertures nd nutrient vilility (Golderg, 23). In ddition, the pthogeniity of V. dhlie to pepper n e determined y the previous ropping history nd the host of origin (Novo et l., 26). Moreover, when reserh studies re performed under ontrolled onditions, the effet of V. dhlie on pepper growth is lso dependent on plnt phenology when the inoultion with the pthogen is rried out (Goioehe et l., 2, 21; Grmendi et l., 24). In ft, while mrked redution in shoot growth ourred when V. dhlie infeted pepper plnts during their vegettive growth, the pthogen did not ffet plnt height when it ws inoulted t flowering. Furthermore, the negtive effet of V. dhlie on totl plnt iomss is lso dependent on the degree of tolerne exhiited y pepper ultivrs. For exmple, the derese in fresh weight ws 69% in the tolernt ultivrs Pdrón nd Yolo Wonder nd hieved 81% in the suseptile v. Luesi (Pomr et l., 24). In reent study, Novo et l. (26), working with Yolo Wonder nd Luesi pepper ultivrs, used the redution in plnt dry weight to lssify different V. dhlie isoltes ording to their ggressiveness. Differene in the degree of ggressiveness mong the different isoltes suggested high vriility mong smpling popultions independent of their originl lotion. When pplied rel-time PCR tehniques for deteting fungl myelium in infeted pepper, Gyoso et l. (27) found good orreltion etween the severity of the symptoms displyed y Vertiillium-infeted plnts nd the reltive mount of fungl DNA mesured s fungus/plnt DNA rtio. Vertiillium dhlie n lso tivte xillry uds in infeted pepper (Grmendi et l., 24), suggesting tht pil dominne of infeted plnts ould e ffeted y some hnges in hormone-like signls involved in the intertion etween host plnt nd pthogen. When the development of xillry rnhes is lose to the fruit set, the growth of seondry shoots my result in enhned ompetition for ssimiltes to the detriment of oth fruit prodution nd ripening. Goioehe et l. (2) oserved tht the lef wter potentil (ψ) in Vertiillium-infeted pepper delined efore ny hnge in the reltive wter ontent (RWC) ws still oserved. In ddition, the deline in lef ψ ourred efore the pperne of visile symptoms of the disese, whih indites tht V. dhlie relesed toxi sustnes tht ltered the physiology of the plnt host even when the fungus ws still lolized in the roots. In ft, oth V. dhlie nd V. lo-trum re reported to produe phytotoxins nd other moleules tht indue host ell deth (Pegg, 1965). One the infetion hd progressed, lef RWC shrply delined nd this redution ws onomitnt with drsti inreses in free proline nd totl solule sugrs (TSS), suggesting tht the umultion of proline nd TSS ould e sensor of the dmge used y the fungl infetion (Goioehe et l., 2). Vertiillium dhlie reinfores the redution in the rte of CO 2 ssimiltion nd lef ondutne experiened y pepper plnts s onsequene of the nturl senesene proess. However, the deline of photosynthesis in infeted pepper n e greter thn tht mesured in the lef ondutne even without ny signifint hnge in the totl horophyll ontent (Goioehe et l., 21), suggesting tht other ftors prt from stomtl losure nd hlorophyll degrdtion i.e. Ruiso tivity n e lso involved in the CO 2 exhnge rte redution. Defolition is nother ftor to tke into ount when tlking out gs exhnge. When infeted with defoliting V. dhlie strins, the redued gs exhnge of the entire plnt n e minly due to deresed lef re rther thn to redued photosyntheti effiieny (Gent et l., 1995). Pomr et l. (24) found tht, in the tolernt pepper v. Yolo Wonder, hnges in stem lignins (monomer omposition nd rosslinking) nd peroxidse tivity were relted with the mintenne of lef photosyntheti integrity during Vertiillium Wilt. Conversely, in the suseptile v. Luesi, photohemil proesses were strongly dmged s onsequene of the disese. The inresed lignin ontents in stems of tolernt pepper ultivr did not stop fungl hyphe penetrtion ut ould hve restrited the growth of the fungl hyphe in the xylem. Reently, Novo et l. (27), lso working with Yolo Wonder nd Luesi ultivrs, me-

4 S28 N. Goioehe et l. / Spn J Agri Res (21) 8(S1), S25-S42 sured inreses in elementl sulphur levels similr to the umultion of phenoli ompounds previously reported y Pomr et l. (24) in the mentioned ultivrs fter V. dhlie inoultion, whih suggests tht, in pepper plnts, sulphur though lerly not onferring full resistne my form prt of multifeted response tht retes physil nd hemil rriers to oloniztion y V. dhlie. Working with very suseptile ultivr of pepper (v. Piquillo), Grmendi et l. (26) oserved tht V. dhlie neither stimulted the phenylpropnoid pthwy nor eliited hydrolyti tivities in infeted pepper roots. However, this pprent mintenne of enzymti tivities ould e due to the time intervl (one week) etween plnt hrvests, whih my possily e too long to detet temporl hnges relted to the defense response. Vertiillium dhlie n elerte the reprodutive ility of pepper plnts, possily through some hnges in host hormonl lne. However, mny of the flowers my fll premturely in inoulted plnts (Goioehe et l., 21), whih produes signifint deline in fruit set nd, onsequently, in yield (Grmendi et l., 24). Totl yield per plnt my lso derese euse of the smll size hieved y individul fruits in Vertiillium-infeted plnts (Grmendi et l., 24). Fruit ripening n e ltered y the infetion with V. dhlie. However, in experiments rried out under ontrolled onditions, it is possile to find ontrsting results depending on plnt phenology when the pthogen is inoulted. If the pthogen ttk tkes ple during the vegettive growth of pepper plnts, fruit mturtion my e delyed. In ontrst, when V. dhlie is inoulted t flowering, fruit ripening my e elerted (Grmendi et l., 24). Suh different ehviors ould e due to differenes in ompetition etween vegettive nd reprodutive orgns in oth ses. As explined previously, when the tivtion of xillry uds in Vertiillium-infeted plnts ours lose to the fruit set, the growth of seondry shoots my result in enhned ompetition for photossimiltes to the detriment of oth fruit prodution nd ripening. Control of Vertiillium Wilt For intensively mnged rops, it hs een ommon prtie to fumigte with methyl romide (MB) prior to plnting in order to redue soil-orne pthogens (Jrvis, 1993; Chellemi, 2). However, MB uses soil nd wter ontmintion nd severely redues the diversity of the miroflor in the treted soil. These resons, together with the inlusion of MB mong the sustnes with high ozone-depleting potentil y the Montrel Protool, n interntionl trety sponsored y the United Ntions Environment Progrmme (UNEP), hve led to the serh for lterntives to the use of MB. Chloropirin (CP) (trihloronitromethne) hs trditionlly een used in formultions together with MB to roden the spetrum of tivity nd enhne the ontrol of soil-orne fungl pthogens (Wilhelm et l., 1961). In the Europen Union, however, the use of 1,3- dihloropropene nd CP my e foridden in the future. For this reson, mny governments re in urgent need of non-hemil lterntives to the use of MB, 1,3-dihloropropene nd CP to ontrol soil pests. Crop rottion hs een used to redue, t lest to some extent, propgules tht remin in the soil nd my infet suseptile plnts. However, the effiy of this mngement strtegy my e redued euse some propgules of Vertiillium spp. n persist y sprophyti oloniztion of plnt deris nd reprodution on nonhosts or weed speies (Golderg, 23). In ses where the profit otined from rop plnt is high, frmers prefer the use of hemils to rop rottion. In ddition, some «monoultures» hve little or no possiilities for rop rottions. This is the se of peppers in Spin. In this ountry, the Ntionl Projet on Methyl Bromide Alterntives ws foused on studying the mjor prolems tht the «phsed-out» use of MB ould rete in reltion to the exploittion of highly eonomil nd soilly importnt hortiulturl rops, suh s strwerries, peppers nd some «ut flower» ornmentls (i.e., the rntion). The Spnish Ministry of Agriulture requested tht INIA the Ntionl Institute for Agriulturl nd Food Reserh nd Tehnology developed Reserh Progrm imed t finding potentilly vile pprohes for «Alterntives to Methyl Bromide Conventionl Uses». The Reserh Progrm ws lunhed in June 1997, finished in 22 nd inluded severl projets foused on the serh for possile ulturl prties, other hemil produts or omintion of the two s lterntives to the use of MB. These reserh projets were developed in ollortion with frmers oopertives nd produers ssoitions nd field trils were rried out in the sme ropping frms nd geogrphil res of the ropping speies under study. After two yer trils, results showed tht hemil tretments sed on 1,3- dihloropropene + hloropirin (pprox. 65:35%) gve

5 Bioprotetion of pepper ginst Vertiillium-indued wilt y rusulr myorrhizl fungi S29 similr results to MB in terms of rop yield for pepper plnts. Physil methods for soil pest ontrol, suh s «io-fumigtion + solriztion» lso gve good results under pproprite onditions. However, dt on solriztion or io-fumigtion lone hve to dte shown poorer performne of these individul tretments, s ompred to MB, under the experimentlly pplied onditions. On 22 nd Novemer 26, the Spnish government pulished new Resolution to ontinue the serh for hemil, physil, ulturl nd iologil lterntives to the use of MB. There re severl lterntives to the pplition of MB nd other hemils to ontrol Vertiillium Wilts: the use of resistnt ultivrs, resistne indution, iologil methods nd the pplition of pproprite mngement strtegies. These lterntives n e pplied lone or my e inluded in wider progrm of integrted ontrol. Resistne is onsidered to e the most prudent prevention strtegy for the ontrol of diseses due to its effetiveness, ese of use nd lk of potentil negtive effets on the environment (Boslnd, 23). In ddition, mnipultion nd enhnement of plnt defense responses using geneti engineering tehnology hs the potentil to produe rop plnts with improved resistne to pthogens (MFdden, 2). Polygeni resistne to Vertiillium spp. hs een identified in severl plnt speies, inluding lflf, otton, potto nd strwerry while single dominnt resistne genes hve een identified in otton, sunflower, potto nd tomto speies (see review y Frdin nd Thomm, 26). In tomto, the Ve lous tht provides resistne ginst Vertiillium Wilt hs een used y plnt reeders for 6 yers nd is introdued in most ultivted tomtoes. Unfortuntely, lthough there re some tolernt pepper ultivrs (i.e., Pdrón nd Yolo Wonder), resistne genes hve not een identified for the C. nnuum- V. dhlie intertion (Pomr et l., 24). In ddition, s ll ultivted speies of Cpsium hve 2n = 24 hromosomes, the rossility mong the speies is limited nd reeders hve only een le to mke little differenes in disese resistne (Greenlef, 1986). In ft, no ommeril ultivrs resistnt to the Vertiillium Wilt re urrently ville (Golderg, 23). However, studies of Jung et l. (23, 25) onstitute n importnt dvne in the knowledge on genes involved in the response of pepper plnts when sujeted to pthogens, ioti nd environmentl stresses. These uthors reported the isoltion nd funtionl nlysis of pepper CALTP genes enoding three lipid trnsfer proteins, CALTPI nd CALTPII nd CALTPIII. While CALTPI nd CALTPIII genes were minly expressed in vrious pepper tissues infeted y pthogens, Phytophthor psii, pthogeni fungus tht uses wilt disese in pepper (Aguirreole et l., 1995), did not indue the trnsription of the CALTPII gene. The identifition of ntifungl proteins nd their expression in trnsgeni plnts provides new possiilities of introduing resistne to pthogens in suseptile ultivrs. Cpsium nnuum, however, is one of the most relitrnt diotyledonous speies for geneti engineering. In ft, there is not universl proedure suitle to trnsform different ultivrs within eh speies vi Agroterium tumefiens (Smith & Townsend) Conn. However, Pozuet-Romero et l. (21) developed tissue ulture regenertion protool tht opened new prospets for Agroterium-medited trnsformtion in ell pepper. Soil solriztion using ler plsti mulh for preplnt soil tretment is ommon prtie in modern griulturl systems s n lterntive to MB. Indeed, this pproh hs een reported to e n effetive tehnique to erdite mirosleroti of V. dhlie in the soil nd to ontrol Vertiillium Wilt in severl hereous nd woody hosts. However, the effiy of soil solriztion n e influened y prtiulr environmentl nd soil onditions suh s physil soil hrteristis nd irrigtion (López-Esudero nd Blno- López, 2). Biofumigtion is non-hemil pproh tht omines orgni mtter or green mnure with solriztion to ontrol diseses, nemtodes nd weeds (Kirkegrd et l., 2). In some ses, however, the joint pplition of solriztion nd iofumigtion to ontrol some pthogens led to mrked derese in soil myoflor (Mrtínez et l., 24). Another mngement strtegy tht n e useful for ontrolling V. dhlie is the pplition of soil mendments. The min soil mendments used to ontrol Vertiillium Wilts s well s the mjor prolems nd limittions ssoited with their use hve een reently disussed y Goioehe (29). As exposed in this review, the numerous ftors tht n influene the effiy of orgni mendments ginst Vertiilliumindued wilts hve led some reserhers to e pessimisti out the hnes of suess for the pplition of suh mterils in Agriulture. However, the orgni industry is rpidly eoming signifint plyer in the glol griulturl prodution sene. Aording to the New South Wles (NSW) Deprtment of Primry Industries of Austrli (28), the orgni industry is fst moving

6 S3 N. Goioehe et l. / Spn J Agri Res (21) 8(S1), S25-S42 wy from its «nihe» industry sttus nd into minstrem griulture, with n estimted nnul growth of up to 3% per yer. Mngement of soil-orne pthogens (inluding Vertiillium spp.) with orgni mendments would e, s expressed y Lzrovits (21), «disese ontrol strtegy slvged from the pst». The type nd level of orgni mendment would lerly vry depending on soil hrteristis, limte of the re, kind of rop nd even the Vertiillium isolte to e suppressed. However, ll of these prmeters must lso e tken into ount when pplying hemil produts to ontrol pests nd diseses nd some of the min limittions previously sried to orgni mendments (i.e. potentil risk for humn helth) my e even more pronouned for some syntheti hemil ompounds. Therefore, the use of orgni produts my represent very interesting tool for the suppression of Vertiillium-indued wilt, espeilly when they re inluded in wider progrm of integrted ontrol. Biologil ontrol gents for plnt diseses re urrently eing exmined s lterntives to syntheti pestiides due to their pereived higher level of sfety nd miniml environmentl impt. However, suh gents my hve potentil risks to other orgnisms present in the eosystem nd these should lso e onsidered (Brimmer nd Bolnd, 23). As mirosleroti re the most importnt strutures tht ensure the survivl of V. dhlie, severl ioontrol strtegies hve foused on () the inhiition of mirosleroti formtion on disesed plnt tissues fter plnt deth, () redution in survivl of mirosleroti, () prevention of their germintion nd/or (d) prevention of root infetion y germinting mirosleroti (Tjmos, 2). In the erly 197s, severl reserhers identified miroil popultions in the rhizosphere s onstituting the first rrier to pthogen infetion. Nowdys, it is well known tht some soils re nturlly suppressive to some soil-orne plnt pthogens inluding Fusrium, Geumnnomyes, Rhizotoni, Pythium nd Phytophthor. The groups of miro-orgnisms with ntgonisti properties towrds plnt pthogens re diverse nd inlude oth plnt-ssoited prokryotes nd eukryotes (Bre et l., 25). The use of ntgonisti miroorgnisms is widely pplied strtegy for ontrolling V. dhlie, with Tlromyes flvus (Klöker) Stolk & Smson eing the most widely studied iologil ontrol gent (Tjmos, 2). Other gener tht hve een reported s ioontrol gents ginst vsulr or soilorne fungl pthogens re Billus, Pseudomons, Chryseomons, Peniillium, Sphingomons, Stenotrophomons, Streptomyes nd Serrti. For exmple, Lren et l. (23) proposed tht Peniillium oxlium Thom. ould e used s ioontrol gent ginst Vertiillium Wilt of tomto under oth greenhouse nd field onditions. More reently, Berg et l. (26) onfirmed tht Pseudomons, Serrti, Billus nd Streptomyes were the min groups of Vertiillium teril ntgonist in the rhizosphere of strwerry nd oilseed rpe. Aprt from teri, ertin fungl root endophytes n e used to ontrol plnt diseses iologilly (Brimmer nd Bolnd, 23), inluding wilt indued y V. dhlie. A group of rhizospheri fungi tht re of inresing interest re the rusulr myorrhizl fungi (AMF) nd this is euse the mjority of rop nd hortiulturl plnts re ssoited with them to the mutul enefit of oth plnt host nd fungus. The use of AMF s n lterntive for ontrolling Vertiillium Wilt in suseptile pepper ultivrs In Nvrr, Northern Spin, wilt indued y V. dhlie is one of the most ommon diseses tht ffets pepper, nd drstilly redues yield. In ddition, the most ppreited pepper ultivr in this re, C. nnuum v. Piquillo, is very suseptile to V. dhlie. This ultivr, whih minly grows in the Southwest of Nvrr, hs Gurntee of Origin nd it is very ppreited for tering. In the ontext of Sustinle Agriulture, the lk of geneti resistne in pepper v. Piquillo together with restritions on hemil produts indue to look for other lterntives. As exposed previously, one possiility is the use of AMF. Sine AMF n enefit plnt growth nd helth there is n inresing interest in sertining their effetiveness in prtiulr plnt prodution prties. In ft, the mutulisti symiosis etween AMF nd plnt roots plys ruil role in nutrient yling in the eosystem nd n protet plnts ginst soil-orne pthogens (Azón-Aguilr nd Bre, 1997). Severl mehnisms n e involved in ioprotetion y AMF (Azón-Aguilr et l., 22): () the improvement of plnt nutrient sttus n help myorrhizl plnts to overome pthogen s ttk; () the inrese in root iomss in myorrhizl plnts n ompenste tissue dmge y the pthogen; () myorrhizl nd pthogeni fungi my ompete for host photosynthtes nd root oloniztion sites; (d) AMF n indue hnges in the ntomy nd rhite-

7 Bioprotetion of pepper ginst Vertiillium-indued wilt y rusulr myorrhizl fungi S31 ture of the root system nd in the rhizosphere; (e) AMF n tivte plnt defense mehnisms. Effetive ioprotetion is umultive result or ll these mehnisms working seprtely nd/or together (Hrrier nd Wtson, 24). Influene of AMF on physiology nd iohemistry of pepper plnts inoulted or not with V. dhlie Leves The development of visile symptoms of the disese in pepper inoulted with V. dhlie n differ depending on the presene or sene of AMF ssoited with plnt roots. In ft, Grmendi et l. (24) oserved tht, while 1% of non-myorrhizl plnts exhiited wilted, hloroti nd/or neroti leves 3 weeks fter pthogen inoultion, t tht time, folir disese symptoms hd ppered in only 15% of plnts ssoited with Glomus desertiol (Trppe, Bloss nd Menge) (Fig. 1). These sme uthors, working with C. nnuum v. Piquillo, lso found tht the disese index remined unhnged in the myorrhizl tretment etween the third nd the sixth weeks fter inoultion, oiniding with the fruit set period (Fig. 1). The study of wter sttus prmeters hs reveled tht the redution in lef Ψ in non-myorrhizl disesed plnts (Fig. 21) ours efore ny derese in RWC is still oserved (Fig. 21) (Grmendi et l., 24). As it hs een widely demonstrted, most wilt pthogens inrese the resistne to wter movement. The derese in lef Ψ ould e mehnism developed y infeted pepper plnts in order to hieve the required tension to let wter move from soil to shoot (Goioehe et l., 2). However, Grmendi et l. (24) demonstrted tht, in myorrhizl plnts inoulted with V. dhlie, lef RWC lwys remined unhnged (Fig. 22) despite the redution in lef Ψ (Fig. 22). The reltionship etween RWC nd Ψ hs sometimes een used to quntify the dehydrtion tolerne of tissues (Sánhez-Díz nd Krmer, 1971). It hs een suggested tht tissues le to retin high RWC s Ψ delines re more tolernt to dehydrtion. Also, mintenne of RWC t ny given Ψ my reflet greter rigidity of the ell wlls nd their ility to withstnd mehnil ollpse s wter is eing lost from the tissue (Bennet-Clrk, 1959). Grmendi et l. (24) lso oserved tht, two months fter pthogen inoultion, lef RWC in pepper ssoited with G. desertiol (Fig. 22) ws higher thn tht mesured in non-myorrhizl plnts (Fig. 21) despite their similr lef Ψ (Fig. 21, 22), suggesting n improved hydruli ondutne in myorrhizl plnts (Mushin nd Zwizek, 22). Moreover, Grmendi et l. (25) mesured higher pressure potentil (Ψ p ) in leves of myorrhizl pepper ompred to tht found in nonmyorrhizl plnts, whih my e due to different wll elstiity in the lef ells from the two types of plnts. Vertiillium dhlie n use signifint dereses of CO 2 exhnge rte (CER), lef ondutne (g w ) nd trnspirtion rte (T) in oth non-myorrhizl nd myorrhizl plnts (Fig. 3) ording to Grmendi et ) fr ) * * * * * * * * 4 3 * fl 2 * * Dys fter V. dhlie inoultion Dys fter V. dhlie inoultion NM+V M+V Vertiillium-inoulted plnts with symptoms (%) Disese index in Vertiillium-inoulted plnts (%) Figure 1. Perentge of plnts with visile symptoms of disese () nd disese index (%) () in non-myorrhizl (NM, ) nd myorrhizl (M, ) pepper inoulted with V. dhlie. Perentges of plnts showing disese symptoms (Fig. 1) were sujeted to r-sin trnsformtion efore pplying χ 2 test. Eh point in Figure 1 represents the men ± SD of 8-1 plnts. Mens were ompred with the Student s t-test. Within eh grph, sterisks indite signifint differenes (p.5). fl: flowering. fr: eginning of fruit set. This figure hs een extrted from the Europen Journl of Plnt Pthology 11, (24) with kind permission of Springer Siene nd Business Medi.

8 S32 N. Goioehe et l. / Spn J Agri Res (21) 8(S1), S25-S42 l. (24). However, these uthors found some differenes etween non-myorrhizl nd myorrhizl disesed plnts. First, pepper plnts olonized y G. desertiol exhiited greter CER (Fig. 32), g w (Fig. 32) nd T (Fig. 32) thn non-myorrhizl ones (Figs. 31, 31, 31). Seond, the delines in photosynthesis nd g w were firstly deteted in non-myorrhizl plnts (Figs. 31, 31). Third, while the redution in g w in non-myorrhizl plnts ws onomitnt with the development of the first visile symptoms of the disese (Fig. 31), the derese in g w in myorrhizl pepper ourred 15 dys fter symptoms were first oserved (Fig. 32). All these findings reinfore the ide tht AMF my e espeilly importnt for plnts sujeted to dverse onditions (Peñ et l., 1988; Sánhez-Díz et l., 199; Goioehe et l., 1997, 24). As fruit growth is minly sustined y the supply of urrent photossimilte (Ho, 1992), the lower derese in CER oserved in Vertiillium-inoulted myorrhizl plnts ompred to non-myorrhizl ones ould enefit the development of peppers in plnts ssoited with G. desertiol. 1).2 fl Ψ (MP).4 fr + s Dys fter V. dhlie inoultion 1) 2) RWC (%) RWC (%) Ψ (MP) 2).2 fl.4 fr + s Dys fter V. dhlie inoultion Dys fter V. dhlie inoultion Dys fter V. dhlie inoultion NM V NM+V M V M+V Figure 2. Wter potentil (Ψ) (MP) () nd reltive wter ontent (RWC) (%) () in leves of non-myorrhizl (NM) plnts inoulted (+V, ) or not ( V, ) with V. dhlie (1, 1) nd in myorrhizl (M) plnts inoulted (+V, ) or not ( V, ) with V. dhlie (2, 2). On eh dy, vlues were nlysed with one-wy ANOVA. Mens ± SD (n = 8-11 dt) were lulted nd, when the F-rtio ws signifint, lest signifint differenes were evluted y he Tukey- test. Within eh prmeter nd dy fter pthogen inoultion, vlues followed y the sme letter do not differ signifintly (p.5). s: first folir disese symptoms. fl: flowering. fr: eginning of fruit set. This figure hs een extrted from the Europen Journl of Plnt Pthology 11, (24) with kind permission of Springer Siene nd Business Medi.

9 Bioprotetion of pepper ginst Vertiillium-indued wilt y rusulr myorrhizl fungi S33 Studies foused on the ntioxidnt metolism in pepper infeted with V. dhlie nd ssoited or not with AMF (Grmendi et l., 24) demonstrted tht suh metolism ws modified in leves of Vertiilliuminoulted plnts efore symptoms of the disese were evident (Fig. 4), whih grees with the ide tht Vertiillium my relese toxi sustnes tht lter the metolism nd physiology of the host plnt even when the fungus is still lolized in the roots (Lorenzini et l., 1997). However, the study performed y Grmendi et l. (24) showed the existene of some differenes etween the ntioxidnt metolism of non-myorrhizl nd myorrhizl pepper. When pthogen-inoulted plnts were ompred with their respetive helthy ontrols on dy 13 fter the inoultion with V. dhlie, there ws enhned superoxide dismutse (SOD) tivity in leves of non-myorrhizl pepper infeted with the pthogen (Fig. 41) nd inresed tlse (CAT) tivity in leves from plnts olonized y G. desertiol nd inoulted with V. dhlie (Fig. 42). Grí- Limones et l. (22) oserved higher tivities of oth SOD nd CAT in stems of suseptile ultivr of hikpe infeted y Fusrium oxysporum, nd no hnges were found when the intertion etween hikpe nd F. oxysporum ws inomptile. The erly enhnement of SOD tivity in non-myorrhizl pepper (Fig. 41) whih ws not o-ordinted with inreses in CAT (Fig. 41) or other peroxidse tivities (i.e. guiol peroxidse, GPX, Fig. 41) ould hve resulted in n umultion of H 2 O 2 in leves nd susequent oxidtive dmge (Aro et l., 21). In ddition, deresed CAT tivity in Vertiillium-inoulted non- 1) 1) CER (µmol CO2 m 2 s 1 ) g w (mol H2O m 2 s 1 ) T (mmol H2O m 2 s 1 ) fl fr + s Dys fter V. dhlie inoultion Dys fter V. dhlie inoultion Figure 3. CO 2 exhnge rte (CER) (µmol CO 2 m 2 s 1 ) (), lef ondutne (g w ) (mol H 2 O m 2 s 1 ) () nd trnspirtion rtes (T) (mmol H 2 O m 2 s 1 ) () in non-myorrhizl (NM) plnt inoulted (+V, ) or not ( V, ) with V. dhlie (1, 1, 1) nd in myorrhizl (M) plnts inoulted (+V, ) or not ( V, ) with V. dhlie (2, 2, 2). Otherwise, s for Figure 2. This figure hs een extrted from the Europen Journl of Plnt Pthology 11, (24) with kind permission of Springer Siene nd Business Medi. CER (µmol CO2 m 2 s 1 ) gw (mol H2O m 2 s 1 ) T (mmol H2O m 2 s 1 ) fl fr + s Dys fter V. dhlie inoultion Dys fter V. dhlie inoultion 1) 2) Dys fter V. dhlie inoultion Dys fter V. dhlie inoultion NM V NM+V M V M+V 2) 2)

10 S34 N. Goioehe et l. / Spn J Agri Res (21) 8(S1), S25-S42 myorrhizl plnts on dy 23 (Fig. 41) suggests tht the mount of H 2 O 2 umulted in leves ould even hve inresed from dy 13. Ative oxygen speies n reinfore plnt ell-wlls, t s toxi gents ginst the pthogens nd/or prtiipte s seond messengers in routes leding to the tivtion of plnt defenserelted genes (Grí-Limones et l., 22). However, in C. nnuum v. Piquillo, the hypothetil umultion of H 2 O 2 ould e relted to the elerted senesene (Fig. 1) nd the deline in photosynthesis in plnts infeted with V. dhlie (Fig. 31), espeilly when pepper ws not ssoited with G. desertiol 1) SOD (U mg protein 1 min 1 ) y y x x x x SOD (U mg protein 1 min 1 ) y y x x x x 2) Dys fter V. dhlie inoultion Dys fter V. dhlie inoultion 1) 7 7 2) CAT (µmol H 2 O 2 mg protein 1 min 1 ) x x y x CAT (µmol H2O2 mg protein 1 min 1 ) x x xy y 1 y y y y Dys fter V. dhlie inoultion Dys fter V. dhlie inoultion 1) x 2) Guiol peroxidse (nmol oxidized guiol mg protein 1 min 1 ) x x x x x x Guiol peroxidse (nmol oxidized guiol mg protein 1 min 1 ) y y x y y Dys fter V. dhlie inoultion Dys fter V. dhlie inoultion NM V NM+V M V M+V Figure 4. Superoxide dismutse (SOD) (U mg protein 1 min 1 ) (1, 2), tlse (CAT) (µmol H 2 O 2 mg protein 1 min 1 ) (1, 2) nd guiol peroxidse (GPX) (oxidized nmol guiol mg protein 1 min 1 ) (1, 2) speifi tivities in non-myorrhizl (NM) (1, 1, 1) nd myorrhizl plnts (M) (2, 2, 2) inoulted (+V) or not ( V) with V. dhlie. Mens ± SD (n = 4 plnts) were nlyzed with one-wy nlysis of vrine (ANOVA) nd, when the F-rtio ws signifint, lest signifint differenes were ompred with the Tukey- test. Within eh prmeter nd dy fter pthogen inoultion, dt with the sme letter (, ) do not differ signifintly. Within eh prmeter nd tretment, dt with the sme letter (x, y) do not differ signifintly. Signifine levels were lwys set t 5% level of signifine.

11 Bioprotetion of pepper ginst Vertiillium-indued wilt y rusulr myorrhizl fungi S35 Tle 1. Totl phenoli ompounds (mg glli id g 1 DM) in stems nd roots from non-myorrhizl (NM) plnts nd plnts ssoited with Glomus desertiol (M), inoulted (+V) or not ( V) with Vertiillium dhlie. Determintions were performed t the moment of the pthogen inoultion (dy ) or 7 dys lter. Mens ± SD (n = 4-6 plnts) were ompred with the Tukey- test. Vlues followed y ommon letter within eh olumn (dy nd plnt orgn) (,, ) or line (tretment nd plnt orgn) (x, y, z) re not signifintly different (p.5) Dys fter pthogen inoultion Tretment 7 7 Stem Root NM V 4.12 ± 1.65 x 1.94 ±.45 y 2.7 ±.3 x 2.11 ±.3 x NM+V 4.12 ± 1.65 x 4.4 ±.55 x 2.44 ±.55 x 2.99 ±.57 x M V 4.88 ± 1.15 x 1.66 ±.47 y 2.7 ±.3 x 2.73 ±.68 x M+V 4.88 ± 1.15 x 2.55 ±.79 y 2.44 ±.55 y 3.46 ±.43 x (Grmendi et l., 24,). The inrese in CAT tivity in leves of pthogen-inoulted non-myorrhizl plnts etween dys 23 nd 3 (Fig. 41) oserved y Grmendi et l. (24) did not seem to e high nd/or erly enough to void the development of disese symptoms. In ontrst, GPX tivity inresed in leves of pepper olonized y G. desertiol one month fter pthogen inoultion (Fig. 42). This mehnism of H 2 O 2 detoxifition ould e relted to the slower development of the disese oserved in these plnts when ompred with non-myorrhizl ones (Fig. 1). The more lned SOD, CAT nd GPX tivities in myorrhizl plnts ould hve lso ontriuted to mintin ontrol photosyntheti rtes for longer (Fig. 32) nd, onsequently, would hve enefit fruit yield (Tle 2) in plnts ssoited with G. desertiol (Grmendi et l., 24,). Stem Aording to Pomr et l. (24), working with C. nnuum vs. Pdrón, Yolo Wonder nd Luesi, the phenoli metolism of pepper is ltered during the Vertiillium Wilt. In ft, the levels of totl free phenolis lerly inresed in stems of disesed plnts, lthough the mount of hlorogeni id, the min free phenoli in peppers (Díz et l., 1997), ws omprle in helthy nd disesed plnts. In ontrst, the mount of free phenoli ompounds in stems of C. nnuum v. Piquillo hd not inresed two months fter their inoultion with V. dhlie (Tle 1) (Grmendi et l., unpulished results). However, on dy 7 th, plnts inoulted with the pthogen exhiited higher ontent of phenolis thn helthy plnts, eing levels greter in non-myorrhizl thn in myorrhizl plnts. It is known tht AMF n indue the synthesis of lignin nd/or other ound phenolis ids (Morndi, 1996) tht n ply role in strengthening the ell-wlls nd preventing invsion y pthogens. Lignins re three-dimensionl phenoli heteropolymers ovlently ssoited with polyshrides in plnt ell wlls (Lewis et l., 1999) nd they re minly lolized in supporting tissues nd in the xylem. Lignin deposition provides mehnil resistne to the stems nd protets ellulose fiers from hemil nd iologil degrdtion in the fe of fungl ttk (Grer et l., 1998). Sine lignifition minly ours in the seondry xylem ell wll, whih is in potentil ontt with V. dhlie, it my e expeted tht xylem lignifition ts s physil rrier ginst V. dhlie (Smit nd Duery, 1997). Aording to mirosopi oservtions of ross-stem setions tken from pepper v. Piquillo nd stined with fst green-sfrnine (Fig. 5), there were not ler differenes etween non-myorrhizl (NM + V) nd plnts ssoited with G. desertiol (M + V) one month fter their inoultion with V. dhlie (Grmendi et l., unpulished results). However, when ompred helthy non-myorrhizl (NM V) nd myorrhizl (M V) plnts, lignin deposition ws higher in the xylem of stems elonging to plnts olonized y G. desertiol. The erly enhnement of peroxidse tivity in roots of plnts ssoited with G. desertiol (Grmendi et l., 26), ould e involved in higher stem lignifition of myorrhizl peppers. It is known tht peroxidses tlize the oxidtive polimeriztion of phenylpropnols to produe lignin nd the ross-linking of ell wll proteins, on-

12 S36 N. Goioehe et l. / Spn J Agri Res (21) 8(S1), S25-S42 Tle 2. Fruit set (%), fruit DM (g fruit 1 ), length (m) nd dimeter (m) of peppers, nd fruit yield (g plnt 1 ) in non-myorrhizl (NM) plnts nd plnts ssoited with Glomus desertiol (M), inoulted (+V) or not ( V) with Vertiillium dhlie. Mens (n = 4-6 plnts) were ompred with the Tukey- test. Within eh olumn vlues followed y the sme letter re not signifintly different (p.5) Tretment Fruit set Fruit DM Fruit length Fruit dimeter Pepper yield (%) (g fruit 1 ) (m) (m) (g DM plnt 1 ) NM V NM+V d M V M+V DM: dry mtter. This tle hs een extrted from the Europen Journl of Plnt Pthology 11, (24) with kind permission of Springer Siene nd Business Medi. triuting to enhne ell wll reinforement during fungl penetrtion (Mittler, 22). It is not ler if the enhned lignin deposition in myorrhizl pepper plnts v. Piquillo ws diret onsequene of plnt response to fungl oloniztion or it ws due to possile hnges in plnt phenology. In ny se, lignin deposition ould hve improved host plnt ility to restrit the fungl growth in the xylem. Root Grmendi et l. (26) found tht phenyllnine mmoni-lyse (PAL) tivity remined unhnged or NM V NM+V M V M+V Figure 5. Lignin deposition (red olour) on xylem vessels in ross-stem setions of non-myorrhizl (NM) nd myorrhizl (M) pepper plnts inoulted (+V) or not ( V) with V. dhlie one month fter pthogen inoultion. even slightly deresed in non-myorrhizl pepper plnts v. Piquillo when they were inoulted with V. dhlie (Fig. 6). Resistnt plnts re hrterized y rpid inrese in PAL tivity one they re ttked y Vertiillium spp. (Frdin nd Thomm, 26) ut C. nnuum v. Piquillo does not exhiit geneti resistne ginst V. dhlie (Plzón, 1985). However, there n e other resons to explin the lk of PAL enhnement in roots of Vertiillium-infeted pepper plnts oserved y Grmendi et l. (26). First, in this study, pepper inoulted with V. dhlie hd not still shown visile symptoms of the disese two weeks fter pthogen inoultion. Guillon et l. (22) oserved tht the highest PAL trnsript only ourred when lesions on the hypootyls were very evident in en plnts ttked y Rhizotoni solni. Seond, the possiility of trnsient indution of PAL t erly stges of the infetion of C. nnuum v. Piquillo with V. dhlie nnot e disrded. Moreover, the redution in PAL tivity n oinide with high expression of pl genes (Kervinen et l., 1998). Although oloniztion of roots y AMF n stimulte the phenylpropnoid pthwy (see Morndi, 1996 for review), the presene of G. desertiol never indued PAL tivity in roots of helthy pepper (Fig. 6) in the study rried out y Grmendi et l. (26), whih grees with oservtions of Guillon et l. (22). The determintion of totl solule phenolis in roots of four months old plnts orroorted tht helthy non-myorrhizl nd pepper plnts ssoited with G. desertiol hd similr levels of these ompounds (Tle 1) (Grmendi et l., unpulished results). However, when ompred PAL tivity in roots of V. dhlie-infeted pepper, plnts olonized y G. desertiol exhiited the highest vlue two weeks fter pthogen inoultion (Fig. 6) (Grmendi et l., 26). Similrly, Dehne nd Shönek (1979) oserved tht the simultneous

13 Bioprotetion of pepper ginst Vertiillium-indued wilt y rusulr myorrhizl fungi S37 ) 7 7 ) Dys fter V. dhlie inoultion Dys fter V. dhlie inoultion PAL (nmol trns-innmi id g 1 DM h 1 ) Figure 6. Phenyllnine mmoni-lyse (PAL) tivities (nmol trns-innmi id g 1 DM h 1 ) in roots from non-myorrhizl (NM) () nd myorrhizl (M) () plnts inoulted (+V) or not ( V) with V. dhlie. Mens (n = 3 plnts) ± SD just efore inoulting Vertiillium were ompred with Student s t-test nd dt on dys 7 nd 14 fter pthogen inoultion were nlysed y Tukey--test. Histogrms followed y ommon letter re not signifintly different (p.5). This figure hs een extrted from the journl BioControl 51, (26) with kind permission of Springer Siene nd Business Medi. PAL (nmol trns-innmi id g 1 DM h 1 ) V +V infetion of tomto with G. mossee nd F. oxysporum inresed PAL tivity of roots more thn the myorrhizl fungus lone or the pthogen lone. The inresed PAL tivity in roots of plnts olonized y G. desertiol nd infeted with V. dhlie (Grmendi et l., 26) ould explin the enhned mount of solule phenoli ompounds in myorrhizl roots two months fter pthogen inoultion (Tle 1) (Grmendi et l., unpulished results). The presene of G. desertiol indued three new idi hitinse (Fig. 7), two new SOD (Fig. 8) nd one new peroxidse (Fig. 9) isoforms in roots of C. nnuum v. Piquillo t erly stges of the symiosis in helthy plnts (Grmendi et l., 26). Assoition of roots with AMF n eliit plnt hitinse (Pozo et l., 1996), peroxidse (Gininzzi nd Gininzzi-Person, 1992) nd SOD (Lmis et l., 23) tivities. The estlishment of the mutulisti ssoition etween the roots of host plnts nd AMF involves ontinuous Dys fter pthogen inoultion 7 14 V +V V +V NM M NM M NM M NM M NM M Dys fter pthogen inoultion 7 14 V +V V +V NM M NM M NM M NM M NM M Figure 7. Aidi hitinse tivity fter seprtion of proteins under ntive onditions y Dvid system in 15% (w/v) polyrylmide gel ontining glyol hitin s sustrte. Roots extrts in MIlvine uffer (7 µg of protein per smple) from non-myorrhizl (NM) nd myorrhizl (M) plnt inoulted (+V) or not ( V) with V. dhlie were nlyzed just efore inoulting the pthogen s well s 1 nd 2 weeks lter. Brs indite onstitutive isoforms nd rrows mrk dditionl isoforms. This figure hs een extrted from the journl BioControl 51, (26) with kind permission of Springer Siene nd Business Medi. Figure 8. Superoxide dismutse (SOD) isoenzymes fter seprtion of proteins under ntive onditions using Dvid system in 15% (w/v) polyrylmide gels. Root protein extrts in MIlvine uffer (7 µg of protein per smple) from non-myorrhizl (NM) nd myorrhizl (M) plnts inoulted (+V) or not ( V) with V. dhlie were nlyzed just efore inoulting the pthogen s well s 1 nd 2 weeks lter. Brs indite onstitutive isoforms nd rrows mrk dditionl isoforms. This figure hs een extrted from the journl BioControl 51, (26) with kind permission of Springer Siene nd Business Medi.

14 S38 N. Goioehe et l. / Spn J Agri Res (21) 8(S1), S25-S42 Dys fter pthogen inoultion 7 14 V +V V +V NM M NM M NM M NM M NM M ) Fruit set (%) e d Figure 9. Peroxidse tivity fter seprtion of proteins under ntive onditions y Dvid system in 15% (w/v) polyrylmide gels. Root protein extrts (7 µg of protein per smple) from non-myorrhizl (NM) nd myorrhizl (M) plnts inoulted (+V) or not ( V) with V. dhlie were nlyzed just efore inoulting the pthogen s well s 1 nd 2 weeks lter. After protein seprtion, gels were inuted in.1 M Tris-HCl (ph 7.6) with 4-hloro-1-npthol nd H 2 O 2. Brs indite onstitutive isoforms nd rrows mrk dditionl isoforms. This figure hs een extrted from the journl BioControl 51, (26) with kind permission of Springer Siene nd Business Medi. ellulr nd moleulr dilogue etween oth symionts (Bonfnte-Fsolo, 1984) tht inludes the pretivtion of defense responses tht n ontriute to enhne the resistne of myorrhizl plnts to soilorne pthogens (Azón-Aguilr et l., 22). Influene of AMF on yield in pepper plnts inoulted or not with V. dhlie When studying fruit hrteristis nd yield in helthy plnts, Grmendi et l. (24) oserved, in some ses, dereses in fruit set, individul fruit dry mtter (DM) nd totl yield per plnt in myorrhizl pepper ompred to non-myorrhizl plnts (Tle 2). The lower fruit set, DM nd yield in plnts ssoited with G. desertiol ould e due to the prtil trnsport of photossimiltes from leves to AMF in roots (Bethlenflvy et l., 1982) in detriment to fruit development (Ho, 1992). However, the ssoition of pepper with AMF not lwys uses redution in the prodution of fruits (Fig. 1) (Grmendi et l., 24). In ontrst, the inoultion of C. nnuum v. Piquillo with V. dhlie lwys hd detrimentl effets on yield (Grmendi et l., 24,). It hs een found tht V. dhlie redues oth the quntity nd qulity of pepper fruits in plnts ultivted in oth greenhouse ) ) Pepper yield (g DM plnt 1 ) Fruit DM (g fruit 1 ) d NM Gd Gi Gm Tretments V +V Figure 1. Fruit set (%) (), pepper yield (g plnt 1 ) () nd fruit DM (g fruit 1 ) () in non-myorrhizl (NM) plnts nd plnts ssoited with Glomus desertiol (Gd), G. intrrdies (Gi) or G. mossee (Gm), inoulted (+V) or not ( V) with Vertiillium dhlie. Vlues re mens ± SD (n = 4-6 plnts). Mens were ompred with the Tukey- test. Within eh grph, vlues followed y the sme letter re not signifintly different (p.5). DM: dry mtter. This figure hs een extrted from the journl Biologil Control 31, (24) with kind permission of Elsevier (Liense Numer ). (Grmendi et l., 24) nd field (Grí-Min et l., 1996) due to the relevnt derese of photosynthesis omined with the premture fll of flowers (Goioehe et l., 21). The redution in the perentge of fruit set nd pepper yield s onsequene of pthogen inoultion ws more evident in non-myorrhizl plnts nd in plnts olonized y G. intrrdies (Shenk nd Smith) nd G. mossee (Niol. nd Gerd.) Gerd. nd Trppe thn in pepper ssoited with G. desertiol (Tle 2, Figs. 1, 1) (Grmendi et l., 24,). In ddition, only non-myorrhizl plnts showed redution in fruit length nd DM ompred to their respetive helthy ontrols (Tle 2) (Grmendi et l., 24). As suggested previously, the lower derese in CER oserved in plnts ssoited with G. desertiol (Fig. 32) fter their inoultion with the pthogen ompred to non-myorrhizl ones e d d

15 Bioprotetion of pepper ginst Vertiillium-indued wilt y rusulr myorrhizl fungi S39 (Fig. 31) would hve enefit the development of fruits in plnts ssoited with the mentioned AMF (Tle 2) (Grmendi et l., 24). In summry, slight ut signifint improvement of fruit yield per plnt hs een oserved fter the pplition of G. desertiol s ioprotetor gent of pepper v. Piquillo ginst Wilt indued y V. dhlie. Tking into ount the high numer of pepper plnts ultivted in greenhouses, this slight improvement of yield per plnt would imply relevnt profits for frmers. Finl onsidertions Despite the positive results when pplying G. desertiol to protet C. nnuum v. Piquillo ginst V. dhlie, the effetiveness of AMF in ontrolling soil orne pthogens n e ffeted y severl ioti nd ioti ftors (Singh et l., 2). In ft, different Glomus speies n exhiit different degree of suess s ioprotetor gents ginst prtiulr pthogen (Hte et l., 1999; Delerk et l., 22). In study foused on testing the ility of G. desertiol, G. mossee nd G. intrrdies s hypothetil ioprotetor gents ginst Vertiillium-indued wilt in pepper, Grmendi et l. (24) onluded tht only G. desertiol ws effetive to void gret yield redutions in disesed pepper plnts (Fig. 1). The enhned growth showed y plnts inoulted with G. mossee ws not onomitnt with n improvement of fruit prodution when they were infeted with V. dhlie. Only in helthy plnts, the ssoition of pepper with G. mossee enefited yield (Fig. 1). The deline in yield oserved in disesed non-myorrhizl plnts nd disesed plnts olonized y G. intrrdies or G. mossee ws due to oth redued fruit set (Fig. 1) nd fruit size (Fig. 1). In plnts ssoited with G. desertiol, the negtive effet of the pthogen on totl yield per plnt ws only due to deresed fruit size (Fig. 1). The ioprotetive effet of AMF on the development of pepper wilt is lso determined y the plnt phenology t the moment when V. dhlie is inoulted (Grmendi et l., 24). In ft, the highest effetiveness of G. desertiol ourred when the pthogen ws inoulted during the vegettive stge of pepper plnts. In ddition, the ggressiveness of V. dhlie n e ffeted y other ioti ftors, suh s soil moisture. Grmendi et l. (25) oserved tht the deleterious effet of this soil orne pthogen on fruit set nd yield n e mplified when pepper plnts undergo drought period efore their inoultion with V. dhlie. These uthors lso found tht the wter defiit did not improve the effetiveness of G. desertiol s ioprotetor gent ginst Vertiillium Wilt in pepper. The intertions etween AMF nd rhizosphere miro-orgnisms n lso influene the iologil ontrol of root pthogens (Azón-Aguilr nd Bre, 1997; Bre et l., 25). As exposed y Bre et l. (25), key point is to know to wht extent n ntifungl ioontrol gent will negtively ffet AMF. As severl studies (see Bre et l., 25 for review) hve demonstrted tht miroil ntgonists of fungl pthogens (i.e. PGPR, other fungi) do not exert ny ntimiroil effet ginst AMF, the dul inoultion of AMF nd PGPR would men new possiilities for the ioontrol of plnts ginst soil orne pthogens, inluding V. dhlie. In onlusion, pepper plnts v. Piquillo ssoited with G. desertiol exhiited more lned ntioxidnt metolism in leves fter pthogen inoultion thn non-myorrhizl plnts, whih ould ontriute to dely oth the development of disese symptoms nd the derese of photosynthesis in Vertiilliuminoulted plnts with the susequent enefit for yield. In ddition, the higher deposition of lignin in the xylem vessels of stems together with the indution of hydrolyti nd ntioxidnt enzymes in roots ould lso e involved in the ioprotetor effet of G. desertiol ginst Vertiillium Wilt in pepper v. Piquillo. However, the effetiveness of AMF in proteting pepper ginst Vertiillium Wilt n vry mong different Glomus speies. In ddition, high effetiveness in improving plnt nutrition nd growth is not neessry relted to gret effetiveness s plnt ioprotetor gents (Grmendi et l., 24). Moreover, ioprotetion ginst Vertiillium Wilt n e onditioned y plnt phenology t the moment of pthogen ttk (Grmendi et l., 24) nd other ioti ftors suh s drought n lso influene oth the ggressiveness of V. dhlie nd the effet of AMF s ioprotetor gents ginst Vertiillium-indued wilt in pepper (Grmendi et l., 25). Aknowledgements The uthors wish to thnk Fundión Universitri de Nvrr (FUNA, PIUNA) nd Goierno Vso for supporting the work. They re lso very grteful to Springer Siene nd Business Medi for giving per-

16 S4 N. Goioehe et l. / Spn J Agri Res (21) 8(S1), S25-S42 mission to reprodue Figures 1, 4 nd 6 nd Tle 3 from the Europen Journl of Plnt Pthology (24) 11, , nd Figures 1, 2, 4, 5 from BioControl (26) 51, The uthors re lso very grteful to Elsevier for llowing reproduing Figure 4 from Biologil Control (24) 31, Referenes AGUIRREOLEA J., IRIGOYEN J.J., SÁNCHEZ-DÍAZ M., SALAVERRI J., Physiologil ltertions in pepper during wilt indued y Phytophthor psii nd soil wter defiit. Plnt Pthol 44, ANDREWS J., Peppers, the domestited Cpsiums. University of Texs Press, Austin, Texs, USA. 186 pp. AROCA R., IRIGOYEN J.J., SÁNCHEZ-DÍAZ M., 21. Photosyntheti hrteristis nd protetive mehnisms ginst oxidtive stress during hilling nd susequent reovery in two mize vrieties differing in hilling sensitivity. Plnt Si 161, AZCÓN-AGUILAR C., BAREA J.M., Applying myorrhiz iotehnology to hortiulture: signifine nd potentils. Si Horti 68, AZCÓN-AGUILAR C., JAIZME-VEGA M.C., CALVET C., 22. The ontriution of rusulr myorrhizl fungi for ioremedition. In: Myorrhizl tehnology in griulture. From genes to ioproduts (Gininzzi S., Shüepp H., Bre J.M., Hselwndter K., eds). Birkhäuser Verlg, Berlin, Germny. pp BARBARA D.J., CLEWES E., 23. Plnt pthogeni Vertiillium speies: how mny of them re there? Mol Plnt Pthol 4, BAREA J.M., POZO M.J., AZCÓN R., AZCÓN-AGUILAR C., 25. Miroil o-opertion in the rhizosphere. J Exp Bot 56, BECKMAN C.H., The nture of wilt diseses of plnts. APS Press, St Pul, MN, USA. 182 pp. BENNET-CLARK I.A., Wter reltions of ells. In: Plnt physiology. Vol. 2 (Stewrd F.C., ed). Ademi Press, NY. pp BERG G., OPELT K., ZACHOW C., LOTTMANN J., GÖTZ M., COSTA R., SMALLA K., 26. The rhizosphere effet on teri ntgonisti towrds the pthogeni fungus Vertiillium differs depending on plnt speies nd site. FEMS Miroil Eol 56, BETHLENFALVAY G.J., BROWN M.S., PACOVSKY R.S., Prsiti nd mutulisti ssoitions etween myorrhizl fungus nd soyen: development of the host plnt. Phytopthology 72, BONFANTE-FASOLO P., Antomy nd morphology of VA myorrhize. In: VA myorrhiz (Powell C.L., Bgyrj D.J., eds). CRC Press, Bo Rton, Florid, USA. pp BOSLAND P.W., 23. Introdution. In: Compendium of pepper diseses (Pernezny K., Roerts P.D., Murphy J.F., Golderg N.P., eds). APS Press, St Pul, MN, USA. pp BRIMMER T., BOLAND G.J., 23. A review of the nontrget effets of fungi used to iologilly ontrol plnt diseses. Agri Eosyst Environ 1, CHELLEMI D.O., 2. Adpttion of pprohes to pest ontrol in low input griulture. Crop Prot 19, DECLERCK S., RISEDE J.M., RUFYIKIRI G., DELVAUX B., 22. Effets of rusulr myorrhizl fungi on severity of root rot of nns used y Cylindroldium spthiphylli. Plnt Pthol 51, DEHNE H.W., SCHÖNBECK F., Investigtions on the influene of endotrophi myorrhiz on plnt diseses. II. Phenol metolism nd lignifition. Phytopthol Z 95, DÍAZ J., ROS BARCELÓ A., MERINO F., Chnges in shikimte dehydrogense nd the end produts of the shikimte pthwy, hlorogeni id nd lignins, during the erly development of seedlings of Cpsium nnuum. New Phytol 136, EVANS G., MKEEN C.D., A strin of Vertiillium dhlie pthogeni to sweet pepper in southwestern Ontrio. Cn J Plnt Si 55, FRADIN E.F., THOMMA B.P.H.J., 26. Physiology nd moleulr spets of Vertiillium wilt diseses used y V. dhlie nd V. lo-trum. Mol Plnt Pthol 7, GARCÍA-LIMONES C., HERVÁS A., NAVAS-CORTÉS J.A., JIMÉNEZ-DÍAZ R.M., TENA M., 22. Indution of ntioxidnt enzyme system nd other oxidtive stress mrkers ssoited with omptile nd inomptile intertions etween hikpe (Cier rietinum L.) nd Fusrium oxysporum f. sp. ieris. Physiol Mol Plnt Pthol 61, GARCÍA-MINA J.M., JORDANA R., AGUIRREOLEA J., HERNÁNDEZ M.A., The effet of speil orgni mendment on the development of pepper plnts ultivted in soil infested with Vertiillium dhlie. In: Fertilizers nd environment (Rodríguez-Brrueo C., ed). Kluwer Ademi Pulishers, Netherlnds. pp GARMENDIA I., GOICOECHEA N., AGUIRREOLEA J., 24. Plnt phenology influenes the effet of myorrhizl fungi on the development of Vertiillium-indued wilt in pepper. Eur J Plnt Pthol 11, GARMENDIA I., GOICOECHEA N., AGUIRREOLEA J., 24. Antioxidnt metolism in symptomti leves of Vertiillium-infeted pepper ssoited with n rusulr myorrhizl fungus. J Phytopthol 152, GARMENDIA I., GOICOECHEA N., AGUIRREOLEA J., 24. Effetiveness of three Glomus speies in proteting pepper (Cpsium nnuum L.) ginst vertiillium wilt. Biol Control 31, GARMENDIA I., GOICOECHEA N., AGUIRREOLEA J., 25. Moderte drought influenes the effet of rusulr myorrhizl fungi s ioontrol gents ginst Vertiillium-indued wilt in pepper. Myorrhiz 15, GARMENDIA I., AGUIRREOLEA J., GOICOECHEA N., 26. Defene-relted enzymes in pepper roots during intertions with rusulr myorrhizl fungi nd/or Vertiillium dhlie. BioControl 51,

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18 S42 N. Goioehe et l. / Spn J Agri Res (21) 8(S1), S25-S42 D.R., eds). The APS Press, St Pul, Minnesot, USA. pp LORENZINI G., GUIDO L., NALI C., CIOMPI S., SOLDATINI G.F., Photosyntheti response of tomto plnts to vsulr wilt diseses. Plnt Si 124, MARTÍNEZ M.A., GUERRERO M.M., MARTÍNEZ M.C., BARCELÓ N., ROS C., LACASA A., TELLO J.C., 24. Efeto de l iofumigión on solrizión sore l miroflor del suelo en ultivos de pimiento en inverndero. Ats del XII Congreso de l Soiedd Espñol de Fitoptologí, Lloret de Mr, Giron, Spin, Sept 26-Ot 1. p. 89. [In Spnish]. MFADDEN H., 2. Prospets for ontrolling vsulr wilt diseses of otton nd other rops y geneti engineering. In: Advnes in Vertiillium. Reserh nd disese mngement (Tjmos E.C., Rowe R.C., Hele J.B., Frvel D.R., eds). APS Press, St Pul, MN, USA. pp MITTLER R., 22. Oxidtive stress, ntioxidnts nd stress tolerne. Trends Plnt Si 7, MOL L., Effet of plnt roots on the germintion of Vertiillium dhlie. II. Quntittive nlysis of the luring effet of rops. Eur J Plnt Pthol 11, MOL L., SCHOLTE K., Formtion of mirosleroti of Vertiillium dhlie Kle. on vrious plnt prts of two potto ultivrs. Potto Res 38, MORANDI D., Ourrene of phytolexins nd phenoli ompounds in endomyorrhizl intertions, nd their potentil role in iologil ontrol. Plnt Soil 185, MUSHIN T.M., ZWIAZEK J.J., 22. Etomyorrhizs inrese poplsti wter trnsport nd root hydruli ondutivity in Ulmus merin seedlings. New Phytol 153, NEW SOUTH WALES (NSW) DEPARTMENT OF PRIMARY INDUSTRIES, 28. [on line]. Aville in [15 Otoer, 28]. NOVO M., POMAR F., GAYOSO C., MERINO F., 26. Cellulse tivity in isoltes of Vertiillium dhlie differing in ggressiveness. Plnt Dis 9, NOVO M., GAYOSO C.M., POMAR F., LUCAS M.M., ROS BARCELÓ A., MERINO F., 27. Sulphur umultion fter Vertiillium dhlie infetion of two pepper ultivrs differing in degree of resistne. Plnt Pthol 56, PALAZÓN C., L «se» o «tristez» del pimiento. Nvrr Agrri 7, [In Spnish]. PEGG G.F., Phytotoxin prodution y Vertiillium lo-trum Reinke nd Berthold. Nture 28, PEÑA J.I., SÁNCHEZ-DÍAZ M., AGUIRREOLEA J., BECANA M., Inresed stress tolerne of nodule tivity in the Medigo-Rhizoium-Glomus symiosis under drought. J Plnt Physiol 133, POMAR F., BERNAL M.A., COLLAR J., CARAMELO C., GAYOSO C., NOVO M., PREGO C., SAAVEDRA A., SILVAR C., MERINO F., 21. A survey of «Tristez» of pepper in Glii nd the fungl pthogens using the disese. Cpsium News 2, POMAR F., NOVO M., BERNAL M.A., MERINO F., ROS BARCELÓ A., 24. Chnges in stem lignins (monomer omposition nd rosslinking) nd peroxidse re relted with the mintenne of lef photosyntheti integrity during Vertiillium wilt in Cpsium nnuum. New Phytol 163, POZO M.J., DUMAS-GAUDOT E., SLEZACK S., CORDIER C., ASSELIN A., GIANINAZZI S., GIANINAZZI- PEARSON V., AZCÓN-AGUILAR C., BAREA J.M., Indution of new hitinse isoforms in tomto roots during intertions with Glomus mossee nd/or Phytophthor niotine vr prsiti. Agronomie 16, POZUETA-ROMERO J., HOULNÉ G., CAÑAS L., SCHANTZ R., CHAMARRO J., 21. Enhned regenertion of tomto nd pepper seedling explnts for Agroterium-medited trnsformtion. Plnt Cell Tiss Org 67, SÁNCHEZ-DÍAZ M., KRAMER P.J., Behvior of orn nd sorghum under wter stress nd during reovery. Plnt Physiol 48, SÁNCHEZ-DÍAZ M., PARDO M., ANTOLÍN M., PEÑA J., AGUIRREOLEA J., 199. Effet of wter stress on photosyntheti tivity in the Medigo-Rhizoium-Glomus symiosis. Plnt Si 71, SINGH R., ADHOLEYA A., MUKERJI K.G., 2. Myorrhiz in ontrol of soil orne pthogens. In: Myorrhizl iology (Mukerji K.G., Chmol B.P., Singh J., eds). Kluwer Ademi Pul, NY. pp SMIT F., DUBERY I.A., Cell wll reinforement in otton hypootyls in response to Vertiillium dhlie eliitor. Phytohemistry 44, THANASSOULOPOULOS C.C., KITSOS G.T., Vertiillium wilt in Greee. Plnt Dis Rep 56, TJAMOS E.C., 2. Strtegies in developing methods nd pplying tehniques for the iologil ontrol of Vertiillium dhlie. In: Advnes in Vertiillium. Reserh nd disese mngement (Tjmos E.C., Rowe R.C., Hele J.B., Frvel D.R., eds). The APS Press, St Pul, MN, USA. pp WILHELM S., STORKAN R.C., SAGEN J.E., Vertiillium wilt of strwerry ontrolled y fumigtion of soil with hloropirin nd hloropirin-methyl romide mixtures. Phytopthology 51, ZHOU L., HU Q., JOHANSSON A., DIXELIUS C., 26. Vertiillium longisporum nd V. dhlie: infetion nd disese in Brssi npus. Plnt Pthol 55,