Quantification of Field Resistance to Verticillium dahliae in Eight Russet-Skinned Potato Cultivars Using Real-Time PCR

Transcription

1 Am. J. Potto Res. (2013) 90: DOI /s Quntifition of Fiel Resistne to Vertiillium hlie in Eight Russet-Skinne Potto Cultivrs Using Rel-Time PCR J. S. Pshe & A. L. Thompson & Neil C. Gumest Pulishe online: 14 Deemer 2012 # Potto Assoition of Ameri 2012 Astrt Chnges in potto proution over the pst 10 to 20 yers, hve resulte in inrese emphsis eing ple on reeing for resistne to Vertiillium wilt, use y Vertiillium hlie Kle. While mny russet-skinne ultivrs reently hve een relese with reporte resistne to Vertiillium wilt, informtion is lking on the level of pthogen oloniztion, n therefore, the level of true geneti resistne is not known. Eight russet-skinne ultivrs were grown in fiel trils with low n high levels of V. hlie in the soil, n evlute for wilt, stem oloniztion, yiel, n tuer vsulr isolortion. A reently evelope QPCR ssy ws vlite, with strong reltionships to ulture plting ssys over three stem smpling tes. Aitionlly, stem oloniztion levels, s etermine y QPCR, were relte to wilt n tuer vsulr isolortion. However, totl yiel i not exhiit strong reltionship to ny other prmeter evlute in this stuy. Results from these stuies inite tht vrying levels of true resistne re present in the russet-skinne ultivrs evlute, n tht the QPCR ssy n e relile in rpily evluting resistne to V. hlie uner fiel onitions. Bse on pthogen quntifition using stem oloniztion erive from tritionl plting ssys n QPCR, the resistne level of severl ultivrs is more lerly efine n isusse. Resumen Los mios en l prouión e pp en los psos ños, hn resulto en un umento en el énfsis uio en el mejormiento pr l resisteni l J. S. Pshe : N. C. Gumest (*) Deprtment of Plnt Pthology, North Dkot Stte University, Frgo, ND 58108, USA e-mil: Neil.Gumest@nsu.eu A. L. Thompson Deprtment of Plnt Sienes, North Dkot Stte University, Frgo, ND 58108, USA mrhitmiento por Vertiillium, uso por Vertiillium hlie Kle. Mientrs que muhos ultivres e piel tipo russet se hn liero reientemente on resisteni report l mrhitmiento por Vertiillium, l informión ree el nivel e olonizión el ptógeno, y e quí que no se onoz el nivel e resisteni genéti verer. Se semrron oho ultivres on piel tipo russet en ensyos e mpo on niveles jo y lto e V. hlie en el suelo, y evluos pr mrhitez, olonizión el tllo, renimiento, y eolorión vsulr el tuérulo. Se evluó reientemente un ensyo QPCR, on reliones fuertes ensyos e ultivos en pls sore tres fehs e muestreo el tllo. Aiionlmente, los niveles e olonizión el tllo, omo se eterminron por QPCR, se relionron l mrhitmiento y l eolorión vsulr el tuérulo. No ostnte, el renimiento totl no exhiió un relión fuerte ningún otro prámetro evluo en este estuio. Los resultos e estos estuios inin que están presentes iversos niveles e resisteni verer en ls vriees e piel tipo russet evlus, y que el ensyo e QPCR puee ser onfile en l evluión rápi e resisteni V. hlie jo oniiones e mpo. Con se en l untifiión el ptógeno meinte el uso e l olonizión el tllo eriv e los ensyos triionles e pls y QPCR, el nivel e resisteni e vrios ultivres está más lrmente efinio y isutio. Keywors Solnum tuerosum. Vertiillium hlie. Disese resistne. Polymerse hin retion. Vertiillium wilt. Cultivr resistne Introution Vertiillium wilt in Unite Sttes potto (Solnum tuerosum L.) proution is use y Vertiillium hlie Kle. n V. lo-trum Reinke n Berthol. V. hlie is often

2 Am. J. Potto Res. (2013) 90: the most ommon n evstting of these two pthogens, minly euse mirosleroti persist in the soil for s mny s 14 yers (Powelson n Rowe 1993; Wilhelm 1955). Due to the ese of introution of V. hlie into non-infeste fiels, the wie host rnge, n the longevity of miroslerotil survivl in the soil, most griulturl soils re infeste with the pthogen to some extent (Rowe 1985; Powelson et l. 1993). Effets on yiel n e more sustntil when suseptile rops re grown in short rottions. Aitionlly, the soil miro-environment hs eome inresingly fvorle for the uil-up n survivl of plnt pthogens in the surfe lyers of the soil, inluing V. hlie, with the implementtion of onservtion tillge n ropping prties (Bokus n Shroyer 1998; Tylor et l. 2005). The symptoms of suessful infetion y V. hlie inlue overll wilting of the host plnt, premture vine eth, folir hlorosis n nerosis. Infete vsulr unles pper light rown n plnts remin eret fter senesene, whih istinguishes symptoms of Vertiillium wilt from symptoms of other wilt iseses use y pthogens inluing Colletotrihum ooes (Wllr.) Hughes n Fusrium spp. (Rih 1983; Rowe1985). Symptoms of Vertiillium wilt my not e evient until plnts reh mturity n my not e istinguishle from nturl senesene t ny growth stge (Dvis 1985). Therefore, wilt symptoms shoul not e relie upon solely for evluting isese mngement strtegies inluing soil fumignts, ulturl prties, or geneti resistne. While wilting eventully les to premture eth of the host, it my not e iret inition of yiel loss. Aitionlly, vsulr isolortion in tuers n result in rowning of proesse prouts n my rener symptomti potto tuers unmrketle (Rowe 1985). While V. hlie resistne mehnisms in potto re not well unerstoo, host resistne n the level of pthogen in the soil often re onsiere the min prmeters ffeting the evelopment of Vertiillium wilt. In moertely suseptile v. Russet Burnk, stem oloniztion inrese with inresing V. hlie soil infesttion levels (Niot n Rouse 1987). However, mny ftors inluing temperture, soil-wter ontent, soil type, orgni mtter ontent, n the vilility of nutrients, lso ffet the level of Vertiillium wilt in potto rop (Ben-Yephet n Szmulewih 1985; Cppert et l. 1992; Dvis et l. 2001; Nnou n Hrrison 1979; Powelson n Rowe 1993). The ontrol of these ftors, when possile, n i in lowering isese severity y improving plnt helth n limiting the infetivity of inoulum n spre of the pthogen in the plnt. However, mngement of Vertiillium wilt hs proven extremely iffiult to hieve without the use of soil fumignts (Powelson et l. 1993; Rowe n Powelson 2002). Soil fumigtion with metm soium n result in n up to 80 % reution in soil infesttion levels n orresponing reutions in isese (Tylor et l. 2005). Unfortuntely, this metho of ontrol is expensive n hs etrimentl effets on the environment (Dvis 1985; Powelson et l. 1993). Aitionlly, regultions outline y the EPA will mke the pplition of this hemil muh more limite in the future (MRe n Noling 2010). Russet-skinne potto ultivrs for use in frozen proessing, s well s tle stok, omprise greter thn 70 % of ll potto rege in the Unite Sttes (Ntionl Potto Counil 2012). Of the top seven ultivrs proue for see-tuers in North Ameri in 2000, v. Russet Burnk ws the most ommonly grown omprising 30 % of totl res, n ws tegorize s moertely suseptile to Vertiillium wilt (Rowe n Powelson 2002). Two other russet-skinne ultivrs rnke in the top seven se on rege, Russet Norkoth, t 11 %, lssifie s very suseptile, n Rnger Russet t 4 %, lssifie s moertely resistnt (Novy, et l. 2003). This represents generl lk of resistne to Vertiillium wilt mong russet-skinne ultivrs (Rowe n Powelson 2002). However, over the pst two ees, numerous russet-skinne ultivrs hve een relese with reporte resistne to Vertiillium wilt (Johnsen et l. 1994; Love et l. 2002, 2005, 2006; Mosleyetl. 1999, 2000, 2001; Novyet l. 2002, 2003, 2006, 2008, 2010; Strk et l. 2009). The iffiulty in reommening the utiliztion of mny of these ultivrs lies in the ft tht mny hve not een evlute for true resistne, ut merely hve een evlute for symptoms of wilt. In the ltter se, growing these ultivrs n result in the uil-up of high levels of inoulum without losses in the urrent seson (Rowe 1985). Unfortuntely, eonomi losses n result when suseptile ultivr is plnte in tht soil. Aitionlly, when soil inoulum rehes high levels, it my require severl rottions wy from suseptile host, even if ontrol mesures suh s soil fumigtion re use. Reent reserh to quntify V. hlie in potto ws performe using tritionl plnt tissue plting ssys (Be et l. 2008; Jnsky 2009; Jnsky n Miller 2010). While results from this reserh provie vlule informtion onerning the evlution of Vertiillium wilt resistne in potto, stuies of this type re iffiult to perform ue to the time n lor intensive nture of plting ssys. A uplex QPCR metho reently ws evelope utilizing host normlizing gene for solute quntifition of the potto:v. hlie intertion (Pshe et l. 2013). Quntifition results from this QPCR ssy orrelte very well to tritionl plting ssys for pthogen quntifition, n to wilt ross eight russet-skinne potto ultivrs grown uner greenhouse onitions. Aitionlly, this ssy n e performe in frtion of the time require for tritionl plting ssys. One ojetive of this reserh ws to further vlite the reently evelope QPCR ssy (Pshe et l. 2012) for the

3 160 Am. J. Potto Res. (2013) 90: quntifition of V. hlie in potto stem tissue grown uner fiel onitions. Another ojetive ws to seprte true resistne from tolerne y evluting pthogen oloniztion, symptom evelopment n yiel, for russetskinne potto ultivrs with vrying levels of purporte resistne to Vertiillium wilt. The ojetives were omplishe y growing these russet-skinne potto ultivrs, long with ontrol ultivrs with estlishe resistne levels, in V. hlie infeste fiel reserh plots. Rel-time QPCR methos were ompre to tritionl plting methos to etermine the mount of oloniztion in stem tissue to quntify the host:pthogen intertion. Mterils n Methos Cultivr Seletion Eight russet-skinne potto ultivrs were selete for evlution of oloniztion y V. hlie (Tle 1). Among these were very suseptile or suseptile ontrol v. Russet Norkoth n moertely resistnt or resistnt ontrol v. Rnger Russet, inlue for omprison purposes. Cultivr Russet Norkoth is known to e hevily olonize y V. hlie uner fiel onitions n ommonly is use s suseptile ontrol (Be et l. 2007; Frost et l. 2007; Hoyos et l. 1991; Jnsky 2009). Cultivr Rnger Russet hs een emonstrte to e resistnt, lthough not ompletely, to oloniztion y V. hlie uner fiel onitions n hs een inlue in previous stuiessresistntontrol ultivr (Be et l. 2007, 2008; Jnsky2009). Cultivrs Russet Burnk n Dkot Trillzer (AOND Russ) lso hve een evlute previously for oloniztion n were lssifie s suseptile to resistnt, n very resistnt to pthogen oloniztion, respetively (Hoyos et l. 1991; Jnsky 2009). The other ultivrs evlute h een reporte previously to rnge from moertely suseptile/moertely resistnt to very resistnt, ut stem oloniztion h not een evlute. Previous lims of resistne were se solely on the presene or sene of wilt symptoms in ultivrs Umtill Russet (moertely suseptile/ moertely resistnt) (Mosley et l. 1999), Bnnok Russet (very resistnt) (Novy et l. 2002), Alturs (very resistnt) (Novy et l. 2003) n Premier Russet (moertely resistnt) (Novy et l. 2008) (Tle 1). Fiel Tril A omintion of four V. hlie isoltes from potto tissue proue in Minnesot ws use to inoulte potto plnts in fiel trils (Pshe et l. 2012). Two of these isoltes were kinly provie y Dr. Jim Breen, Deprtment of Plnt Tle 1 Totl yiel, evlute in 2009 n 2010, n potto tuer vulr islortion iniene n severity, evlute in 2010, ross eight russet-skinne potto ultivrs with vrying levels of reporte suseptiility to Vertillium wilt. Evlutions were performe on tuers grown in fiel soils infeste t low n high Vertiillium hlie levels Cultivr Reporte suseptiility Totl yiel (mt/h) Tuer vsulr isolortion f Mil Severe Totl Russet Norkoth very suseptile Rnger Russet resistnt e Russet Burnk moertely resistnt/moertely suseptile Umtill Russet moertely resistnt/moertely suseptile Dkot Trillzer very resistnt e Bnnok Russet very resistnt e Alturs very resistnt Premier Russet moertely resistnt e e P vlue < < Vlues within olumns with the sme letter re not sttistilly ifferent se on Fisher s protete lest signifint ifferene (α=0.05) Jnsky 2009 Mosley et l Novy et l Novy et l e Novy et l f Mil tuer vsulr isolortion: light isolortion not ontinuouse roun the vsulr ring. Severe tuer vsulr isolortion: rker in olor n ontinuous roun the vsulr ring, or nerly so

4 Am. J. Potto Res. (2013) 90: Pthology, University of Minnesot. The remining two originte from potto tissue grown in entrl Minnesot, n were isolte n store s esrie previously (Pshe et l. 2012). All four isoltes of V. hlie were sent to Northwest Myologil Consultnts, Corvllis, OR to e use to infest grin. Briefly, sterilize grin see ws infeste with eh of the four isoltes in iniviul gs eh ontining pproximtely 3.2 kg of fully hyrte grin. These mushroom spwn gs, with numer seven vents to llow gs exhnge, were susequently inute in the rk t 22 C for 3 weeks. Grin ws sent to North Dkot Stte University where it ws rie on greenhouse enhes for 7 to 14 ys efore milling in In 2010, grin remine in the spwn gs until soil infesttion. Certifie see tuers of eh ultivr were ut into see piees weighing pproximtely 70 g n suerize t 13 C for pproximtely 48 h prior to plnting. Sixteen tretments, onsisting of eight ultivrs y two infesttion levels, were plnte in rnomize omplete lok esign (RCBD) with four replites. Eh experimentl unit ontine two rows of ten plnts per replite. Soil smples were sent to Pest Pros, Plinfiel, WI for nlysis of V. hlie levels. Low infesttion onsiste of resient soil V. hlie popultions of 6.0 Vertiillium propgules per grm of soil (Vppg) in 2009, n 4.5 Vppg in High infesttion ws hieve y ing pproximtely 19 kg of rie n groun V. hlie-infeste grin pplie in-furrow t plnting, t rte of 96.7 g/row m in In 2010, 33 kg of hyrte grin ws pplie t g/row m. The inrese weight of grin ws e to ount for the ifferene etween ry n hyrte grin weight. The sme four V. hlie isoltes were plte to soli CV8 gr n grown in the rk t 25±2 C for 3 weeks, to e use s itionl inoulum inorporte uring hilling opertions. Contents of the ulture pltes (gr n fungus) were mixe with istille wter t rte of 25 ml/plte, puree with n eletri lener n juste to finl onentrtion of mirosleroti/ ml using hemoytometer. The gr n fungl slurry ws pplie to the soil of eh high infesttion plot t rte of 30 ml/row m with hn hel CO 2 pressure operte spryer n inorporte uring hilling opertions on June 11, 2009 n June 2, Trils were mintine following typil ommeril growing prties for northestern North Dkot, inluing overhe irrigtion n ultivtion, s well s fungiie, insetiie n heriie pplitions. Disese Evlution Visul ssessments for signs of Vertiillium wilt were performe four times from 83 to 110 ys fter plnting (DAP). Symptom evelopment ws evlute on perentge wilt severity sis for eh tretment/replite omintion in 2009 n 2010 n totl yiel ws otine t the en of eh growing seson. In 2010, tuers were evlute for stem en iniene of mil, severe, n totl vsulr isolortion from 9 to 18 ys post-hrvest. Tuers were ut long the short xis just elow the stolon tthment. Mil isolortion ws efine s light isolortion, whih i not follow the entire vsulr ring, while severe isolortion ws rker in olor n extene roun the vsulr ring, or nerly so. Soil smples were otine t hrvest to evlute the pthogen infesttion level in eh fiel replite. Stem Coloniztion Anlysis V. hlie oloniztion ws quntifie in potto stem smples using tritionl ulture plting ssys, n y QPCR. Fresh stem setions from the sl region of ll true stems (originting from the see-tuer) of five plnts in eh ultivr/replite were ollete t 82 n 104 DAP in 2009 n 84 n 104 DAP in Smples were proesse following previously esrie protools with some moifitions (Jnsky 2009). Stem setions were surfe sterilize n isk weighing pproximtely 1 g ws exise from eh stem n ple into selle plsti g n sterile istille wter ws e t 1:1 weight to volume rtio. Setions were rushe n 50 μl spre onto soli NP mei (Pshe et l. 2012). At hrvest, 139 n 132 DAP in 2009 n 2010, respetively, when plnts were senesent or nerly so, stem setions were rie, groun in Wiley Mill using 40 mesh sreen n 50 mg were plte onto soli NP mei. Pltes from oth rushe fresh n groun rie stem setions were inute in the rk for 4 to 5 weeks. Pltes from fresh stems were re iretly, while stem eris from rie stems ws wshe from pltes uner running tp wter n pltes were rie overnight efore exmintion uner 60 mgnifition using stereomirosope. The numer of olony forming units (CFU)/g stem tissue then ws lulte (Pshe et l. 2012). A uplex QPCR ssy ws performe using primers n proe evelope from the trypsin protese gene (VTP1)of V. hlie (Doinson et l. 2004) n internl ontrol tin gene (t) of Solnum tuerosum (Atllh n Stevenson 2006). Pthogen quntifition in potto stem tissue using the QPCR ssy ws ompre to tritionl plting methos esrie ove (Pshe et l. 2012). Stem tissue use in PCR quntifition ws otine iretly jent to, n simultneously with, tht use in tritionl ulture plting methos. Totl genomi DNA ws extrte from fresh n rie stems using the FstDNA Spin Kit (MPBiomeils, Solon, OH) following mnufturer s instrutions. Prior to DNA extrtion, tissue ws pulverize for 45 s using n MP FstPrep-24. In fresh n rie stems, 150 n 50 mg tissue ws use for DNA extrtion, respetively. This PCR ssy ws optimize for use in quntifition of DNA extrte from stem tissue, s esrie previously (Pshe et l.

5 162 Am. J. Potto Res. (2013) 90: ). Cyling onitions were initite with 2 min t 95 C followe y 40 yles of 30 s t 95 C, 60 s t 58 C n 30 s t 72 C with t pture fter the nneling step performe in Strtgene Mx3005P using polypropylene QPCR 96-well tue pltes, non-skirte (Agilent Tehnologies, In., Snt Clr, CA). Amplifition n quntifition of the VTP1 gene ws hieve using 0.5 μm VTP1-2 forwr n VTP1-2 reverse primers, 0.4 μm VTP1-2 Tqmn proe, 0.6 μm PotAt forwr n PotAt reverse primers, 0.1 μm PotAt Tqmn proe 1.5 mm MgCl 2, 0.2 mm NTP, 1 polymerse uffer, 1 unit GoTq DNA polymerse n 2.0 μl templte DNA in 25 μl retion. All QPCR retions performe on stems ollete from fiel trils were run in uplite. Sttistil Anlyses Coloniztion t from fiel trils onute in 2009 n 2010 were trnsforme using ue-root trnsformtions to meet normlity ssumptions for the nlysis of vrine (ANOVA). Dt then were omine se on the intertion of tril yer with the min effets of ultivr n infesttion level for wilt, oloniztion, yiel, n tuer vsulr isolortion. Two-wy ANOVA were onute on omine t from the 2009 n 2010 fiel trils using ultivr n soil infesttion level s min ffets in PROC GLM of SAS. Men wilt severity, ue-root trnsforme stem oloniztion, tuer yiel n vsulr isolortion were ifferentite using Fisher s protete lest signifint ifferene test (α=0.05). Reltionships mong ll prmeters were evlute using Person s orreltion. Results No signifint intertion ws oserve etween the min effets of ultivr n infesttion level for wilt severity t 83 (P=0.5855), 98 (P=0.0549), 104 (P=0.7263), or 110 (P= ) DAP. However, signifint intertion ws oserve for ue root trnsforme CFU/g erive from plting ssys t 82/84 (P=0.0312) n 104 (P=0.0064) DAP, s well s for V. hlie mg/g of stem tissue (P=0.0135) erive from QPCR nlyses of fresh stem tissue ollete 104 DAP. The intertion etween ultivr n infesttion level erive from ulture plting quntifition on the first smpling te t 82/84 DAP ws, in gret prt, ue to slight erese in stem oloniztion from low to high soil infesttion levels in v. Bnnok Russet, ompre to n inrese in ll other ultivrs. To emonstrte this, when the ANOVA ws performe without v. Bnnok Russet, the intertion etween min effets ws no longer present (P= ). At 104 DAP the sitution ws ifferent. Stem tissue oloniztion inrese ross ll ultivrs etween soil infesttion levels, ut ifferenes in oloniztion for Russet Norkoth, the suseptile ontrol ultivr, were greter thn tht oserve in other ultivrs. Therefore, when v. Russet Norkoth ws exlue from the nlysis, no intertions etween ultivr n infesttion level were oserve mong the other seven ultivrs for plting (P= ) or QPCR (P=0.0753) ssys t 104 DAP. No signifint ifferenes in oloniztion were oserve etween high n low soil infesttion levels of V. hlie for ny ultivr with some level of resistne. No signifint intertion of these min effets ws oserve in stems ollete t 82/84 DAP n evlute for V. hlie oloniztion with QPCR (P=0.0886). Aitionlly, no signifint intertions were oserve mong min effets of V. hlie stem oloniztion ollete t hrvest n evlute using tritionl ulture plting (P=0.2833) n QPCR (P=0.8754) ssys, totl yiel (P=0.4521), n for mil (P=0.2910), severe (P=0.1253), or totl (P=0.6296) vsulr isolortion in tuers. Beuse intertions of the min effets were not signifint in the mjority of prmeters, ll t re presente within the min effets of ultivr n soil infesttion level for eh prmeter. Development of Vertiillium Wilt Symptoms Signifint ifferenes in wilt severity mong ultivrs were oserve t ll four t olletions tes (Fig. 1). Wilt severity t the first t olletion te, 83 DAP, ws less thn 10 % in ll ultivrs. Highly suseptile ontrol v. Russet Norkoth isplye signifintly higher level of wilt ompre to vs. Umtill Russet, Bnnok Russet, Alturs, Dkot Trillzer, n Premier Russet. Wilt in vs. Rnger Russet n Russet Burnk ws not ifferent thn ny other ultivr. At the seon wilt evlution 15 ys lter, wilt severity inrese sustntilly, espeilly in ultivrs with little, or moerte, resistne to the pthogen. Wilt t 98 DAP in v. Russet Norkoth ws signifintly higher thn ll other ultivrs, wilt in v. Russet Burnk ws higher thn ll the remining ultivrs, n vs. Dkot Trillzer n Premier Russet were signifintly less wilte, ompre to vs. Russet Norkoth, Russet Burnk n Rnger Russet. Wilt symptoms ontinue to inrese throughout the reminer of the growing seson, ut not s rmtilly s ws oserve etween the first n seon evlutions. At 104 DAP, wilt in v. Russet Norkoth ws gin signifintly higher thn other ultivrs, followe y v. Russet Burnk. Vertiillium wilt ws lest severe in vs. Dkot Trillzer n Premier Russet, ut not signifintly lower thn tht oserve in Alturs n Bnnok Russet. At the finl t olletion te 110 DAP, wilt in suseptile ontrol v. Russet Norkoth rehe over 90 %, n remine signifintly higher thn ll other ultivrs. This gin ws followe y v. Russet Burnk, s well s Umtill Russet.

6 Am. J. Potto Res. (2013) 90: Wilt severity (%) Russet Rnger Russet Norkoth (HS) (R) Russet Burnk (MS) Umtill Russet (MS) e Dkot Bnnok Russet Trillzer (HR) (R) Alturs (R) 83 DAP 98 DAP 104 DAP 110 DAP e Premier Russet (HR) Fig. 1 Perentge Vertiillium wilt severity evlute t 83, 98, 104 n 110 ys fter plnting (DAP) in 2009 n Evlutions me ross eight russet-skinne potto ultivrs reporte s highly suseptile (HS), moertely suseptile (MS), resistnt (R), or highly resistnt (HR) to V. hlie from soils infeste t low n high pthogen levels. Brs within evlution te with the sme letter re not sttistilly ifferent se on Fisher s protete lest signifint ifferene (α=0.05) As oserve previously, vs. Dkot Trillzer n Premier Russet were the lest wilte mong the ultivrs evlute, lthough not signifintly so in every instne. Quntifition of V. hlie in Fresh Stem Tissue Signifint ifferenes mong ultivrs were oserve in oloniztion levels in stem tissue ollete 82/84 n104 DAP in 2009 n 2010 for oth the tritionl ulture plting ssys (P<0.0001; P<0.0001), s well s QPCR ssys (P<0.0001; P<0.0001) (Fig. 2). At the first smpling te, results from ulture plting ssys inite tht suseptile ontrol v. Russet Norkoth n moertely suseptile v. Russet Burnk were olonize t signifintly higher levels thn were ll other ultivrs (Fig. 2). Moertely suseptile v. Umtill Russet ws signifintly less olonize thn these two, ut more so thn the five other ultivrs. Cultivrs Alturs n Rnger Russet isplye intermeite levels of oloniztion, not signifintly ifferent from eh other. While oloniztion in Alturs ws signifintly higher thn for Dkot Trillzer, oloniztion in v. Rnger Russet ws not. Cultivrs Dkot Trillzer, Bnnok Russet n Premier Russet were lest olonize, n no signifint ifferene ws oserve mong these three. Results from QPCR ssys were similr to plting ssys; however, oloniztion of v. Russet Norkoth ws etermine to e signifintly higher thn ll other ultivrs (Fig. 2). Moertely suseptile vs. Russet Burnk n Umtill Russet were signifintly more olonize thn ll other ultivrs exept v. Russet Norkoth. Agin, vs. Dkot Trillzer, Bnnok Russet n Premier Russet were lest olonize. Signifint ifferenes were oserve etween infesttion levels with oth plting (P<0.0001) n QPCR(P=0.0012) ssys 82/ 84 DAP (t not shown). At 104 DAP, suseptile ontrol v. Russet Norkoth h signifintly higher levels of oloniztion thn ny other ultivr, when evlute with either ssy (Fig. 2 n ). Also in oth ssys, vs. Russet Burnk n Umtill Russet were olonize signifintly less thn v. Russet Norkoth, ut signifintly more thn vs. Premier Russet n Dkot Trillzer. Results from plting ssys inite tht oloniztion of v. Alturs ws not signifintly lower thn vs. Russet Burnk n Umtill Russet, ut lso ws not signifintly higher thn vs. Rnger Russet or Bnnok Russet. The QPCR ssy resulte in no signifint ifferenes mong vs. Rnger Russet, Bnnok Russet, Alturs, Dkot Trillzer n Premier Russet. However, results from oth ssys inite tht vs. Dkot Trillzer n Premier Russet re olonize t numerilly lower levels thn ll other ultivrs t this point in the growing seson. Neither plting ssys (P= ), or QPCR (P=0.0706) ssys etete signifint ifferene etween infesttion levels in these two ultivrs t 104 DAP (t not shown). Quntifition of V. hlie in Drie Stem Tissue Trens oserve from the oloniztion of stem tissue ollete t hrvest, 139 n 132 DAP in 2009 n 2010, respetively, were similr to those oserve with the oloniztion of fresh stem t (Fig. 2). Signifint ifferenes were oserve mong ultivrs using oth plting ssys (P<0.0001), s well s QPCR methos (P<0.0001). In oth ssys, the ifferene in oloniztion level ws not signifint

7 164 Am. J. Potto Res. (2013) 90: Cue root V. hlie CFU/g Russet Norkoth (VS) e Rnger Russet (R) Russet Burnk (MS/MR) Umtill Russet (MS/MR) e e 82/84 DAP 104 DAP 139/132 DAP e e Dkot Bnnok Russet Trillzer (VR) (VR) Alturs (VR) e e Premier Russet (MR) Cue root V. hlie mg/g Russet Norkoth (VS) Rnger Russet (R) Russet Burnk (MS/MR) Umtill Russet (MS/MR) Dkot Bnnok Russet Trillzer (VR) (VR) Alturs (VR) Premier Russet (MR) Fig. 2 Cue root trnsforme olony forming units of Vertiillium hlie per grm of potto stem tissue (V. hlie CFU/g) generte using the tritionl plting ssy () n mgv. hlie per grm of potto stem tissue (V. hlie mg/g) generte using the uplex QPCR ssy (). Stem tissue ws ollete 82/84, 104 n 139/132 ys fter plnting in 2009 n 2010 from soils infeste t low n high levels with V. hlie. Evlutions me ross eight russet-skinne potto ultivrs reporte s highly suseptile (HS), moertely suseptile (MS), resistnt (R), or highly resistnt (HR) to V. hlie. Brs with the sme letter re not sttistilly ifferent se on Fisher s protete lest signifint ifferene (α=0.05) mong vs. Russet Norkoth, Russet Burnk n Umtill Russet. As etermine y tritionl plting ssys, vs. Bnnok Russet n Rnger Russet lso were not signifintly less olonize thn v. Russet Norkoth (Fig. 2). Cultivrs Dkot Trillzer n Premier Russet were olonize t the lowest levels, signifintly less thn ll other ultivrs in QPCR ssys. However, no signifint ifferene ws oserve etween vs. Dkot Trillzer n Alturs in plting ssys. Here, signifint ifferenes were oserve etween low n high soil infesttion levels s etermine y oth plting (P=0.0448) n QPCR (P<0.0001) ssys (t not shown). Coloniztion of stem tissue inrese from 82/84 DAP to 104 DAP in ll ultivrs, exept Dkot Trillzer, where the erese ws very smll, n only when this ultivr ws evlute with QPCR (Fig. 2 n ). However, oloniztion levels of V. hlie s etermine y tritionl plting ssys, erese from fresh stem smples ollete 104 DAP, to stems ollete t hrvest, n rie efore plting, in five of eight russet ultivrs evlute (Fig. 2). An inrese in V.

8 Am. J. Potto Res. (2013) 90: hlie oloniztion ws oserve in vs. Bnnok Russet n Dkot Trillzer, while no hnge in oloniztion ws oserve in v. Premier Russet. Results iffere onsierly using the QPCR ssy. Coloniztion levels, expresse s mg of V. hlie DNA/g potto tissue, inrese rmtilly in ll eight ultivrs from 104 DAP to hrvest (Fig. 2). This inrese is pproximtely proportionl for ll ultivrs exept suseptile ontrol ultivr Russet Norkoth, in whih oloniztion inrese, ut to muh lesser proportion thn oserve in other ultivrs. The reltive inrese in oloniztion t hrvest ws most sustntil in vs. Dkot Trillzer n Premier Russet, whih h the lowest oloniztion levels of the ultivrs evlute t 104 DAP. Tuer Yiel n Qulity Evlutions Signifint ifferenes were oserve in totl yiel mong ultivrs (P=0.0023), however, these ifferenes were not relte to levels of reporte resistne (Tle 2). Cultivr Umtill Russet yiele signifintly higher thn vs. Russet Burnk, Dkot Trillzer, Russet Norkoth, Premier Russet, n Bnnok Russet. Cultivrs Premier Russet n Bnnok Russet yiele the lowest, ut only signifintly so when ompre to vs. Umtill Russet n Alturs. Aitionlly, no signifint ifferenes in totl yiel were oserve etween low n high infesttion levels (P=0.3335). Signifint ifferenes lso were oserve mong ultivrs for iniene of mil (P<0.0001), severe (P=0.0002) n totl (P<0.0001) vsulr isolortion (Tle 1). Unlike totl yiel, isolortion in tuer vsulr tissue followe similr trens s were oserve with oloniztion n wilt. Suseptile ontrol v. Russet Norkoth isplye signifintly higher inienes of mil isolortion thn ll other ultivrs, while v. Russet Burnk followe. Trens in levels of severe isolortion were similr to tht oserve with mil isolortion. However, v. Russet Burnk h higher iniene of severe isolortion thn v. Russet Norkoth, ut not signifintly so. Totl iniene of vsulr isolortion inite tht those ultivrs with some level of resistne h signifintly less isolortion thn oth vs. Russet Norkoth n Russet Burnk. No signifint ifferenes were oserve etween infesttion levels for iniene of mil (P=0.3012), severe (P= ) or totl (P=0.3902) tuer vsulr isolortion. Very strong n signifint reltionships existe in oloniztion etween tritionl plting n QPCR ssys t ll smpling tes s etermine y Person s orreltion (Tle 2). Aitionlly, t ll smpling tes, oloniztion s etermine y QPCR, ws losely relte to wilt on the sme te. Totl tuer vsulr isolortion lso ws losely relte to oloniztion t ll three smpling tes. However, totl yiel ws not relte to stem oloniztion s etermine y QPCR t ny te. Tle 2 Reltionship etween oloniztion of potto stems grown in fiel soils with low or high levels of Vertiillium hlie quntifie using tritionl plting ssys n the uplex VTP1 QPCR ssy, Vertillium wilt severity, totl yiel n tuer vsulr isolortion etermine y Person s orreltion oeffiient Comprison prmeters QPCR quntifition 82/84 DAP Tritionl plting quntifition 82/84 DAP r=0.92** 104 DAP 139/132 DAP 104 DAP r=0.96** 139/132 DAP r=0.85** Vertiillium wilt severity 82/84 DAP r=0.83** 104 DAP r=0.94** 139/132 DAP r=0.88** Totl yiel (mt/h) r=0.28 ns r=0.21 ns r=0.35 ns Iniene of tuer vsulr isolortion r=0.91** r=0.91** r=0.75** Stem tissue ws ollete n proesse fresh for tritionl plting n QPCR ssys t 82/84 n 104 ys fter plnting (DAP) in 2009/ Stem tissue ws ollete n proesse rie t 139/132 DAP Person orreltion oeffiients were signifint (**) or not (ns) t the α=0.05 level, n=16 Disussion Vertiillium wilt is extremely mging to potto growers, not only euse of loss of tuer yiel, size n qulity, ut lso euse of the extrorinry expense in ontrolling the isese vi the use of the soil fumignt metm soium (Rowe n Powelson 2002). In the pst, the generl lk of ultivrs with mrket eptne n resistne to Vertiillium wilt left growers with few lterntive options to mnge the isese (Powelson et l. 1993; Rowe n Powelson 2002). Reently, severl russet-skinne ultivrs were relese with purporte to Vertiillium wilt, however, for mny of these, no ttempts were me to quntify the host:pthogen intertion t the time of their relese to the inustry (Johnsen et l. 1994; Love et l. 2002, 2005, 2006; Mosley et l. 1999, 2000, 2001; Novy et l. 2002, 2003, 2006, 2008, 2010; Strk et l. 2009). Tritionl ulture plting methos re urte n relile in quntifying pthogens suh s V. hlie in potto stems (Be et l. 2008; Jnsky 2009; Jnsky n Miller 2010), however, euse they re very lor intensive n time onsuming, mny reeers o not perform these evlutions. This in turn represents n inrese in the nee for urte n rpi ssys to etet the pthogen in host

9 166 Am. J. Potto Res. (2013) 90: tissue. This stuy ws evelope, n performe, to quntify V. hlie oloniztion in eight russet-skinne potto ultivrs, n to etermine if the resistne level reporte ws truly resistne, or merely tolerne to symptom evelopment. Aitionlly, stems ollete from fiel trils were use to further vlite previously evelope uplex QPCR ssy uner fiel onitions to reple stnr plting tehniques (Pshe et l. 2012). The present reserh not only puts forth rpi n relile ssy for use in sreening ultivrs n reeing seletions for resistne to Vertiillium wilt, it preisely quntifies the egree of geneti resistne present mong the russet-skinne ultivrs evlute. This is evient not only in the levels of oloniztion present in eh ultivr, ut lso in the reltionship etween oloniztion n symptoms of Vertiillium wilt, inluing folir wilt n tuer vsulr isolortion. The iffiulty in etermining the true level of Vertiillium wilt resistne in potto ultivrs is ompoune y the miguity in the lssifition of ontrol ultivrs use for omprtive purposes (Tle 1). For instne, v. Russet Norkoth typilly is inlue s the most suseptile ontrol, ut hs een efine s oth highly (very) suseptile n suseptile (Be et l. 2007; Jnsky 2009; Lynh et l. 1997; Mosley et l. 2000). Similrly, v. Rnger Russet hs een lssifie s moertely resistnt or resistnt (Be et l. 2007, 2008; Jnsky 2009; Novy et l. 2003, 2008) While these exmples represent only slight ifferenes in lssifition, more rmti ifferenes exist for v. Russet Burnk, whih hs een lssifie s suseptile, moertely suseptile n moertely resistnt to Vertiillium wilt (Be et l. 2008; Corsini et l. 1988; Hoyos et l. 1991; Lynh et l. 1997; Mosley et l. 2000; Novy et l. 2002, 2003, 2008). The uthors elieve the ifferenes in response to V. hlie re likely ue to ifferenes in environment uner whih the stuies were onute. Temperture n moisture n hve sustntil impt on wilt evelopment, therefore, these evlutions my iffer from one growing seson to the next n from region to region (Ben-Yephet n Szmulewih 1985; Cppert et l. 1992; Nnou n Hrrison 1979; PowelsonnRowe 1993). In the trils reporte here, totl rinfll ws slightly higher, 5.1 m, in 2009 when ompre to However, euse these trils were grown uner irrigtion, this ifferene shoul not hve h mjor implitions on the results. Aitionlly, the verge mximum temperture over the ourse of the growing seson ws 22.8 n 24.4 C in 2009 n 2010, respetively. The verge minimum temperture ws 11.1 n 11.7 C, respetively. Therefore, overll environmentl onitions were very similr n most likely i not ffet the results from the first yer the fiel trils were performe to the seon. It is pprent tht situtions exist where ontrol vs. Russet Norkoth n Russet Burnk oth were lssifie s suseptile, n likewise, vs. Russet Burnk n Rnger Russet re lssifie s resistnt. These isrepnies n ompromise new ultivr evlutions n lssifitions when use s the sole sis for omprison. The present reserh inites tht signifint ifferenes exist in oloniztion mong these three ultivrs, n supports the lssifition of v. Russet Norkoth s highly (very) suseptile, v. Russet Burnk s moertely suseptile n v. Rnger Russet s resistnt. However, s emonstrte y the reserh presente here, nrrow geneti winow is represente when only these three ultivrs re use for omprison. Until now, v. Rnger Russet ws onsiere the resistnt stnr mong russetskinne ultivrs. The urrent, n other, reserh inites tht ultivrs exist with higher levels of resistne thn is present in v. Rnger Russet, inluing Dkot Trillzer n Premier Russet (Jnsky 2009). Utilizing one of these ultivrs for omprison purposes woul llow reeers to more lerly efine the lssifition of new ultivrs ompre to those urrently ville n tht one of these shoul e utilize s ontrols in Vertiillium wilt evlutions. Aitionlly, the use of v. Russet Norkoth s the suseptile ontrol shoul e reple y v. Russet Burnk. This is justifie euse ultivrs with lower levels of resistne thn v. Russet Burnk o not represent level of fiel resistne vlule to prouers. Aitionlly, v. Russet Norkoth is olonize t signifintly greter levels thn is oserve in other ultivrs, therefore, t trnsformtion is neessry in orer to meet normliztion ssumptions of the ANOVA. The use of v. Russet Burnk woul minimize the nee for suh trnsformtions. The reserh reporte here further refines the Vertiilllium wilt suseptiility lssifition from those propose in ultivr releses n other previous reserh using rpi quntifition of host oloniztion (Be et l. 2007, 2008; Frost et l. 2007; Hoyos et l. 1991; Jnsky 2009; Mosley et l. 1999; Novy et l. 2002, 2003, 2008) (Tle 1). The originl ultivr relese pulitions for Alturs, Bnnok Russet, Premier Russet n Umtill Russet were se solely on the evelopment of wilt symptoms oserve in the fiel. Cultivr Alturs originlly ws etermine to e very resistnt ompre to vs. Rnger Russet n Russet Burnk, efine s moertely resistnt n suseptile, respetively, for the evelopment of Vertiillium wilt (Novy et l. 2003). Similrly, v. Bnnok Russet lso ws efine s very resistnt, ompre to moertely suseptile v. Russet Burnk. However, se on stem oloniztion results from the urrent reserh, oth vs. Alturs n Bnnok Russet shoul e lssifie s resistnt, s oth ultivrs re olonize t rte similr to resistnt ultivr Rnger Russet (Tle 1). Cultivr Premier Russet ws rte previously s moertely resistnt, or similr to v. Rnger Russet. In this stuy, epening on the evlution te, it isplye wilt

10 Am. J. Potto Res. (2013) 90: symptoms signifintly lower thn v. Rnger Russet n sttistilly similr to tht of ultivrs rte s highly resistnt, inluing Dkot Trillzer. Therefore, the use of oloniztion ssys provie more refine nlysis of the levels of resistne n the true nture of the host:prsite intertion. Stem oloniztion y V. hlie lso ws similr to previously reporte t for the ontrol ultivrs Russet Norkoth, Rnger Russet, s well s vs. Russet Burnk n Dkot Trillzer (Be et l. 2007, 2008; Frost et l. 2007; Hoyos et l. 1991; Jnsky2009). This provies level of onfiene tht plting n QPCR tehniques employe in this reserh were relile. Suseptile ontrol ultivr Russet Norkoth onsistently h the highest levels of wilt n oloniztion. As expete, ultivrs rte s moertely suseptile, Russet Burnk n Umtill Russet, h wilt n oloniztion levels intermeite to tht of the suseptile n resistnt ontrol ultivrs. Interestingly, the four remining ultivrs, Dkot Trillzer, Bnnok Russet, Alturs n Premier Russet ll h sttistilly similr, or lower, levels of resistne to V. hlie s v. Rnger Russet. These finings re prmount to the inustry, reefining n preisely quntifying the host:pthogen intertion in urrently ville ultivrs, t time when mngement options re limite. Severl wil Solnum spp. hve een ientifie s soures of resistne to V. hlie, n in rosses me with one resistnt n one suseptile prent, this resistne ppers to e simply inherite n stle (Corsini et l. 1985, 1990; Dvis et l. 1983; Hoyos et l. 1993; Jnsky n Rouse 2000, 2003). The soures of resistne in the ultivrs evlute in this stuy re not ientifie n potentilly re not known (Mosley et l. 1999; Novy et l. 2002, 2003, 2008). However, ompring peigrees mong vs. Bnnok Russet, Premier Russet n Alturs oes revel some interesting possiilities. For exmple, Bnnok Russet n Alturs hve the sme mle prent (A ) n Premier Russet hs Bnnok Russet s grnprent, n therefore A s gret grnprent (R. Novy personl ommunition). The evelopment of this rpi, relile n urte QPCR tehnique filittes sreening the lineges of resistnt ultivrs with goo gronomi hrteristis, therefore, proviing etter unerstning of the soures of resistne. This susequently will elerte the evelopment of new Vertiillium wilt resistnt ultivrs. Attempts to evlute trils onute uner fiel onitions for the evelopment of symptoms of one isese often re onfoune y infetion of one or more non-trget pthogens. The trils performe here re no exeption to tht, however, trens in Vertiilium wilt severity mong ultivrs were onsistent ross ll four tes, even t the erliest wilt evlution te when severity ws less thn 10 %. As ws propose previously, this my llow reeers to ssess wilt symptoms erly in the growing seson n susequently hve time to ollet stems from those seletions whih show promise for resistne (Jnsky 2009; Jnsky n Rouse 2000). The three-tiere metho evelope in previous reserh ws suessful in ientifying resistne mong reeing lones using omintion of wilt symptoms, plting fresh stem sp, n plting rie stem tissue (Jnsky 2009). This pproh llows for urte sreening n ientifition of true resistne, while systemtilly eliminting lones with no resistne erly in the sreening proess, n therefore, reuing the lor require for stem oloniztion ssys. The reserh reporte here not only onfirms the use of these three prmeters for the ientifition of resistne, ut suessfully uses QPCR ssy to further reue the time n lor require to quntify stem oloniztion y V. hlie. Reports of the orreltion etween stem oloniztion n symptom expression hve een inonsistent (Dvis et l. 1983; Frost et l. 2007; Jnsky 2009; Jnsky n Rouse 2000; Lynh et l. 1997; Mohn et l. 1990). The reserh reporte here inites tht symptom expression is well orrelte with oloniztion on the sme te, n others hve me similr oservtions (Dvis et l. 1983; Mohn et l. 1990). However, some reserhers report tht no orreltion etween wilt n oloniztion exists (Jnsky 2009; Jnsky n Rouse 2000; Lynh et l. 1997). The isprity in these results inites tht tolernt ultivrs re present in some popultions evlute, n not others (Lynh et l. 1997). In the urrent stuy, wilt ws well orrelte with oloniztion, initing tht true resistne is present in those ultivrs isplying low levels of wilt symptoms n onurrent low levels of pthogen oloniztion. However, the use of visul symptoms lone nnot seprte resistne from tolerne n nnot relily istinguish ultivrs tht re resistnt or highly resistnt. Evluting the sene of wilt symptoms lone leves the possiility tht ultivrs oul e hroring high pthogen popultions. This inoulum woul e returne to the soil to infet susequent rops of potto, perhps plnte using suseptile ultivrs. Therefore, visul ssessments lone shoul not e relie upon to sreen ultivrs n germplsm for resistne to oloniztion y V. hlie. It lso hs een reporte tht stem oloniztion quntifie uring the growing seson i not orrelte well with oloniztion of rie stems t the en of the seson (Jnsky et l. 2004; Jnsky n Rouse 2003). The urrent reserh, n others, isgrees with this oservtion (Dvis et l. 1983). Stem oloniztion of fresh stems 104 DAP s etermine y QPCR methos ws well orrelte with oloniztion of rie stem tissue ollete 139/ 132 DAP (n=16; r=0.83; P<0.0001). However, results from the urrent stuy o gree with previous oservtion tht popultions reovere from fresh stems were higher thn tht reovere from rie stems, when using ulture plting tehniques (Jnsky n Rouse 2003).

11 168 Am. J. Potto Res. (2013) 90: Signifint ifferenes in totl yiel were oserve mong the ultivrs evlute, however, the tren i not follow other prmeters evlute in this stuy. This is not too surprising, s inonsistenies in yiel retions hve een oserve previously in Vertiillium wilt evlutions (Frost et l. 2007; Mohnetl.1990). Cultivrs iffer genetilly in yiel potentil, n Vertiillium wilt is ut one ftor tht n ffet tuer yiel. While tuer vsulr isolortion t only ws ollete in 1 yer of the tril, results were very ompelling. No t exist prior to this stuy on the reltionship etween wilt or stem oloniztion to the level of tuer vsulr isolortion. While this reserh shoul e onfirme in future trils, it hs the potentil to provie the inustry with vlule informtion regring tuer qulity in Vertiillium wilt resistnt ultivrs. The root lesion nemtoe P. penetrns interts synergistilly with V. hlie, resulting in lrger yiel reutions thn either pthogen lone (Mrtin et l. 1982; Rowe et l. 1985). The mehnisms of this intertion re not well unerstoo, n therefore, the effets of the presene of P. penetrns on resistne to V. hlie oserve in the ultivrs evlute in these stuies nee further exmintion. Aitionlly, previous reserh inites tht very few propgules of V. hlie re require to use high levels of isese n tht the level of inoulum n e relte to the level of stem oloniztion in v. Russet Burnk n yiel in vs. Norgol Russet n Norhip (Ben-Yephet n Szmulewih 1985; Dvis 1985; Niot n Rouse 1987; Nnou n Hrrison 1979). While two infesttion levels were evlute here, little ifferene ws oserve etween them in ny of the eight ultivrs. Therefore, further stuies re wrrnte to etermine whether or not the resistne oserve in these ultivrs will e stle uner higher inoulum pressure. The quntifition of V. hlie for the purpose of evluting resistne is n importnt tool, n hs een use extensively in potto reeing (Be et l. 2008; Corsini et l. 1985, 1990; Dvis et l. 1983; Frost et l. 2007; Hoyos et l. 1993; Jnsky 2009; Jnsky n Miller 2010; Jnsky n Rouse 2000, 2003; Jnsky et l. 2004; Mohn et l. 1990). Aitionlly, tritionl plting methos hve een utilize suessfully to evlute Vertiillium wilt ontrol mesures (Niot n Rouse 1987). However, these stuies were performe using lor-intensive plting tehniques, even fter more rpi PCR tehniques h een evelope (Atllh et l. 2007; Dn et l. 2001; Hu et l. 1993; Li et l. 1999; Mhuku et l. 1999; Mero-Blno et l. 2001; Nzr et l. 1991; Pérez-Artés et l. 2000). One stuy utilize QPCR ssy for the quntifition of V. hlie in vs. Russet Norkoth n Rnger Russet t severl points over the growing seson (Be et l. 2007). However, these methos were not use in susequent oloniztion stuies performe y similr uthors (Be et l. 2008). There my e severl resons for the lk of wiespre option of PCR ssys in V. hlie quntifition, inluing the iffiulty of suessfully inorporting of new methos into stnr protools or the reliility of the ville PCR ssys. The stuies reporte here emonstrte the use of QPCR ssy to quntify V. hlie in potto stems proue uner fiel onitions with gret ury n reliility. The use of QPCR to quntify the host:pthogen intertion will provie useful tool in more preisely efining the response of potto germplsm n ultivrs to V. hlie. Aknowlegements We re grteful to Den Peterson, Russell Benz n Roert Shermn for tehnil ssistne. We lso knowlege Curt Doetkott for ssistne with sttistil nlyses. Funing for these stuies ws provie in prt y the USDA-ARS-SCA (No ) n the Minnesot Are II n Northern Plins Potto Growers Assoitions. Referenes Atllh, Z.K., n W.R. Stevenson A methoology to etet n quntify five pthogens using potto tuer ey using reltime quntittive PCR. Phytopthology 96: Atllh, Z.K., J. Be, S.H. Jnsky, D.I. Rouse, n W.R. Stevenson Multiplex rel-time quntittive PCR to etet n quntify Vertiillium hlie oloniztion in potto lines tht iffer in response to Vertiillium wilt. Phytopthology 97: Be, J., Z.K. Atllh, S.H. Jnsky, D.I. Rouse, n W.R. Stevenson Coloniztion ynmis n sptil progression of Vertiillium hlie in iniviul stems of two potto ultivrs with iffering responses to potto erly ying. Plnt Disese 91: Be, J., D. Hltermn, n S. Jnsky Development of moleulr mrker ssoite with Vertiillium wilt resistne in iploi interspeifi potto hyris. Moleulr Breeing 22: Ben-Yephet, Y., n Y. Szmulewih Inoulum levels of Vertiillium hlie in the soils of the hot semi-ri Negev region of Isrel. Phytoprsiti 13: Bokus, W.W., n J.P. Shroyer The impt of reue tillge on soilorne plnt pthogens. Annul Review of Phytopthology 36: Cppert, M.R., M.L. Powelson, N.W. Christensen, n F.J. Crowe Influene of irrigtion on severity of potto erly ying n tuer yiel. Phytopthology 82: Corsini, D.L., J.R. Dvis, n J.J. Pvek Stility of resistne of potto to strins of Vertiillium hlie from ifferent vegettive omptiility groups. Plnt Disese 69: Corsini, D. L., Pvek, J. J., n Dvis, J. R Vertiillium wilt resistne in nonultivte tuer-ering Solnum speies. Plnt Disese 72: Corsini, D.L., J.J. Pvek, n J.R. Dvis Vertiillium wilt resistnt potto germplsm: A n A Amerin Potto Journl 67: Dn, H., S.T. Ali-Khn, n J. Ro Use of quntittive PCR ignostis to ientify tolerne n resistne to Vertiillium hlie in potto. Plnt Disese 85: Dvis, J.R Approhes to ontrol of potto erly ying use y Vertiillium hlie. Amerin Potto Journl 62: Dvis, J.R., J.J. Pvek, n D.L. Corsini A sensitive metho for quntifying Vertiillium hlie oloniztion in plnt tissue n evluting resistne mong potto genotypes. Phytopthology 73:

12 Am. J. Potto Res. (2013) 90: Dvis, J.R., O.C. Huismn, D.O. Everson, n A.T. Shneier Vertiillium wilt of potto: moel of key ftors relte to isese severity n tuer yiel in southestern Iho. Amerin Journl of Potto Reserh 78: Doinson, K.F., S.J. Grnt, n S. Kng Cloning n trgete isruption, vi Agroterium tumefiens-meite trnsformtion, of trypsin protese gene from the vsulr wilt fungus Vertiillium hlie. Current Genetis 45: Frost, K.E., D.I. Rouse, n S.H. Jnsky Consiertions for Vertiillium wilt resistne in potto. Plnt Disese 91: Hoyos, G.P., P.J. Zmino, n N.A. Anerson An ssy to quntify vsulr oloniztion of potto y Vertiillium hlie. Amerin Potto Journl 68: Hoyos, G.P., F.I. Luer, n N.A. Anerson Erly etetion of Vertiillium wilt resistne in potto reeing progrm. Amerin Potto Journl 70: Hu, X., R.N. Nzr, n J. Ro Quntifition of Vertiillium iomss in wilt isese evelopment. Physiologil n Moleulr Plnt Pthology 42: Jnsky, S.H Ientifition of Vertiillium wilt resistne in U.S. potto reeing progrms. Amerin Journl of Potto Reserh 86: Jnsky, S.H., n J.C. Miller Evlution of Vertiillium wilt resistne in Russet Norkoth n six strin seletions. Amerin Journl of Potto Reserh 87: Jnsky, S., n D.I. Rouse Ientifition of potto interspeifi hyris resistnt to Vertiillium wilt n etermintion of riteri for resistne ssessment. Potto Reserh 43: Jnsky, S.H., n D.I. Rouse Multiple isese resistne in interspeifi hyris of potto. Plnt Disese 87: Jnsky, S., D.I. Rouse, n P.J. Kuth Inheritne of resistne to Vertiillium hlie in iploi interspeifi potto hyris. Plnt Disese 88: Johnsen, R.H., B. Frnsworth, G.A. Seor, N.C. Gumest, A. Thompson-Johns, n E.T. Holm Golrush: new high qulity russet-skinne potto ultivr. Amerin Potto Journl 71: Li, K.N., D.I. Rouse, E.J. Eyestone, n T.L. Germn The genertion of speifi DNA primers using rnom mplifie polymorphi DNA n its pplition to Vertiillium hlie. Myologil Reserh 103: Love, S.L., R. Novy, D.L. Corsini, J.J. Pvek, A.R. Mosley, R.E. Thornton, S.R. Jmes, n D.C. Hne Gem Russet: long russet potto vriety with exellent fresh mrket n Frenh fry proessing qulity. Amerin Potto Journl 79: Love, S.L., R. Novy, J. Whitworth, D.L. Corsini, J.J. Pvek, A.R. Mosley, R.E. Thornton, N.R. Knowles, S.R. Jmes, n D.C. Hne Summit Russet: new russet potto vriety with goo fresh mrket n frozen proessing qulities. Amerin Journl of Potto Reserh 82: Love, S.L., R.G. Novy, J. Whitworth, D.L. Corsini, J.J. Pvek, A.R. Mosley, M.J. Pvek, N.R. Knowles, C.R. Brown, S.R. Jmes, D.C. Hne, n J.C. Miller GemStr Russet: potto vriety with high yiel, goo ulinry qulity, exellent fresh mrket pperne, n resistne to ommon s. Amerin Potto Journl 83: Lynh, D.R.K., L.M. Kwhuk, n J. Hhey Ientifition of gene onferring high levels of resistne to Vertiillium wilt in Solnum hoense. Plnt Disese 81: MRe, A., n J. Noling Overview of new EPA regultions ffeting use of metm soium n metm potssium. University of Flori Extension HS pp. pffiles/hs/hs pf. Mhuku, G.S., H.W. Pltt, n P. Mxwell Comprison of polymerse hin retion se methos with plting on mei to etet n ientify Vertiillium wilt pthogens of potto. Cnin Journl of Plnt Pthology 21: Mrtin, M.J., R.M. Rieel, n R.C. Rowe Vertiillium hlie n Prtylenhus penetrns: intertions in the erly ying omplex of potto in Ohio. Phytopthology 72: Mero-Blno, J., D. Roríguez-Juro, E. Pérez-Artés, n R.M. Jiménez-Díz Detetion of the nonefoliting pthotype of Vertiillium hlie in infete olive plnts y neste PCR. Plnt Pthology 50: Mohn, S., J. Dvis, D. Corsini, L. Sorensen, n J. Pvek Retion of potto lones n essions of Solnum spp. to Vertiillium hlie Kle. n its toxin. Potto Reserh 33: Mosley, A.R., S.R. Jmes, D.C. Hne, K.A. Rykost, C.C. Shok, B.A. Chrlton, J.J. Pvek, S.L. Love, D.L. Corsini, n R.E. Thornton Umtill Russet: full seson long russet for proessing n fresh mrket use. Amerin Journl of Potto Reserh 77: Mosley, A.R., S.R. Jmes, K.A. Rykost, D.C. Hne, C.E. Strnger, C.C. Shok, J.J. Pvek, D.L. Corsini, J.C. Miller, S.L. Love, D.G. Holm, R.E. Thornton, n R.E. Voss Century Russet: high-yieling fresh mrket ultivr with Vertiillium resistne. Amerin Potto Journl 77: Mosley, A.R., K.A. Rykost, S.R. Jmes, D.C. Hne, C.C. Shok, B.A. Chrlton, J.J. Pvek, S.L. Love, D.L. Corsini, n R.E. Thornton Klmth Russet: full seson, fresh mrket, long russet. Amerin Journl of Potto Reserh 78: Ntionl Potto Counil Potto Sttistil yerook. 77 pp. FINAL_2012Sttook_smllerfilesizeforwe.pf. Nzr, R.N., X. Hu, J. Shmit, D. Culhm, n J. Ro Potentil use of PCR-mplifie riosoml intergeni sequenes in the etetion n ifferentition of Vertiillium wilt pthogens. Physiologil n Moleulr Plnt Pthology 39: Niot, P.C., n D.I. Rouse Reltionship etween soil inoulum ensity of Vertiillium hlie n systemi oloniztion of potto stems in ommeril fiels over time. Phytopthology 77: Nnou, E.C., n M.D. Hrrison The reltionship etween Vertiillium lo-trum inoulum ensity n potto yiel. Amerin Journl of Potto Reserh 56: Novy, R.G., D.L. Corsini, S.L. Love, J.J. Pvek, A.R. Mosley, S.R. Jmes, D.C. Hne, C.C. Shok, K.A. Rykost, C.R. Brown, n R.E. Thornton Bnnok Russet: ul-purpose, russet potto ultivr with high U.S. No. 1 yiel n multiple isese resistnes. Amerin Journl of Potto Reserh 79: Novy, R.G., D.L. Corsini, S.L. Love, J.J. Pvek, A.R. Mosley, S.R. Jmes, D.C. Hne, C.C. Shok, K.A. Rykost, C.R. Brown, n R.E. Thornton Alturs: multi-purpose, russet potto ultivr with high yiel n tuer speifi grvity. Amerin Journl of Potto Reserh 80: Novy, R.G., S.L. Love, D.L. Corsini, J.J. Pvek, J.L. Whitworth, A.R. Mosley, S.R. Jmes, D.C. Hne, C.C. Shok, K.A. Rykost, C.R. Brown, R.E. Thornton, N.R. Knowles, M.J. Pvek, N. Olsen, n D.A. Inglis Defener: high-yieling, proessing potto ultivr with folir n tuer resistne to lte light. Amerin Potto Journl 83: Novy, R.G., J.L. Whitworth, J.C. Strk, S.L. Love, D.L. Corsini, J.J. Pvek, M.I. Vles, S.R. Jmes, D.C. Hne, C.C. Shok, B.A. Chrlton, C.R. Brown, N.R. Knowles, M.J. Pvek, T.L. Brnt, n N. Olsen Premier Russet: ul purpose, potto ultivr with signifint resistne to low temperture sweetening uring long-term storge. Amerin Journl of Potto Reserh 85: Novy, R.G., J.L. Whitworth, J.C. Strk, S.L. Love, D.L. Corsini, J.J. Pvek, M.L. Vles, S.R. Jmes, D.C. Hne, C.C. Shok, B.A.