Host specificity, but not high-temperature tolerance, is associated with recent outbreaks of Verticillium dahliae in chrysanthemum in the Netherlands

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1 DOI /s Host specificity, ut not high-temperture tolernce, is ssocited with recent outreks of Verticillium dhlie in chrysnthemum in the Netherlnds S. K. Isphni & J. C. Goud & A. J. Termorshuizen & A. Morton & D. J. Brr Received: 7 Mrch 2007 / Accepted: 24 Jnury 2008 # KNPV 2008 Astrct Two hypotheses which might explin recent increse in the incidence of verticillium wilt of chrysnthemums in glsshouses in the Netherlnds were investigted, viz whether selection for incresed resistnce to elevted tempertures hs occurred due to frequent steming of soils in the glsshouses, or whether the strins of Verticillium dhlie occurring in chrysnthemum glsshouses re prticulrly virulent towrds this host. Following rtificil inocultion, five isoltes of V. dhlie from chrysnthemum were pthogenic on chrysnthemum ut five isoltes from potto were non-pthogenic for this host. When inoculted onto potto plnts, ll isoltes cused erly senescence with no significnt difference etween the two groups of isoltes. In mplified frgment length polymorphism nlysis, the potto isoltes formed S. K. Isphni : J. C. Goud : A. J. Termorshuizen Biologicl Frming Systems, Wgeningen University, Mrijkeweg 22, 6709 PG Wgeningen, The Netherlnds J. C. Goud Lortory of Phytopthology, P.O. Box 8025, 6700 EE Wgeningen, The Netherlnds A. J. Termorshuizen (*) Blgg, Nieuwe Knl 7F, 6709 PA Wgeningen, The Netherlnds e-mil: d.termorshuizen@lgg.nl A. Morton : D. J. Brr Wrwick HRI, University of Wrwick, Wellesourne, Wrwickshire CV35 9EF, UK cluster distinct from ll other isoltes. As group the chrysnthemum isoltes were no more diverse thn the potto isoltes ut did not form cluster distinct from 12 other isoltes tested. This suggests tht high pthogenicity to chrysnthemum hs developed on severl occsions ut tht the group of potto isoltes were possily monophyletic. Microscleroti produced in vitro from the chrysnthemum isoltes hd significntly lower verge lethl temperture tolernce thn those from the five potto isoltes suggesting tht eing le to resist the effects of soil sterilistion y stem is not fctor in wilt of chrysnthemums in the Netherlnds. Keywords Microscleroti. Host specificity. Temperture tolernce Verticillium dhlie is widespred soil-orne plnt pthogen cusing wilt in rod rnge of crops (Pegg nd Brdy 2002). In generl, hploid V. dhlie isoltes hve een viewed s not eing host-specific (Strusugh 1993), lthough specific pthotypes or host specilistion hve een reported for some crops e.g. cotton (Ashworth 1983), peppermint (Horner 1954), ell pepper nd eggplnt (Bht nd Suro 1999) nd these my e correlted with moleculr properties e.g. Jpnese pepper nd tomto pthotypes (Crder nd Brr 1994). However, isoltes from cruciferous hosts, once seen s very cler cse of host-specilistion in V. dhlie, hveeenshown to e geneticlly quite distinct from the mjority of

2 isoltes, eing mphihploid interspecific hyrids (Brr nd Clewes 2003), nd my e est thought of s distinct species (Krpp et l. 1997). In the Netherlnds, verticillium wilt is common in glsshouses, significntly ffecting rose, eggplnt nd chrysnthemum. During the lst 5 to 10 yers, necdotl reports y growers hve suggested n increse in the incidence nd severity of verticillium wilt, not only in the Netherlnds, ut lso in the UK (personl communiction, Dr C. Lne, Centrl Science Lortories, York, UK). To mnge verticillium wilt, glsshouse soils re usully stemed. This kills microscleroti, the survivl structures of V. dhlie, wherever the temperture reches C for t lest 30 min (Bollen 1985). There re two ovious resons s to why verticillium wilt might reoccur in glsshouse following steming. Firstly, chrysnthemums root deeply into the soil nd microscleroti occurring elow the treted lyer of soil my escpe the steming process. Secondly, infection cn e reintroduced, either on fresh chrysnthemum plnting mteril or from surrounding soil or crops. In the Netherlnds, V. dhlie occurs in most griculturl soils, minly ecuse most soils hve potto cropping history. During potto crop, lrge numers of V. dhlie microscleroti re formed on the senescing potto stems nd petioles (Mol et l. 1996) nd it hs een hypothesized tht potto hs een importnt in the history of most Dutch isoltes of V. dhlie, including ones isolted from other crops. It ws thought tht the esclting prolem of verticillium wilt in glsshouse-grown chrysnthemums might e ssocited with either (1) selection for incresed temperture resistnce in the microscleroti s result of mny cycles of stem tretment, or (2) enhnced pthogenicity of the V. dhlie isoltes resulting from selection imposed y the continuous cultivtion of single host. This pper reports some experiments intended to differentite these two hypotheses. Ten V. dhlie isoltes were collected from disesed chrysnthemum nd potto plnts from vrious prts of the Netherlnds (Tle 1). Potto isoltes were chosen for comprison with the chrysnthemum isoltes ecuse, s noted ove, it is thought tht pottoes hve een importnt hosts for most Dutch isoltes of V. dhlie otined from outside glsshouses. Pure cultures were prepred y incuting n infected stem for 1 dy on wet filter pper nd touching conidiophore of V. dhlie with sterile needle nd trnsferring spores to potto dextrose gr (PDA Oxoid) pltes. Chrysnthemum isoltes were single-spored shortly efore the experiments. Potto isoltes were stored s spore suspensions in glycerol t 80 C using the Micronk Bcteril nd Fungl Preservtion System (Pro-L Dignostic Inc., Ontrio, Cnd) until the strt of the experiments. Temperture sensitivity of microscleroti ws determined using mteril produced on cellophne ccording to the procedure descried y Frncl et l. (1987). Microscleroti nd mycelium were scrped off the cellophne nd wshed through 106 nd 20 μm nested sieves. Microscleroti retined on the ltter sieve were used. Approximtely 300 microscleroti per isolte were plced into microfuge tues contining 0.5 ml tp wter. Using wter ths t 25 C, 40 C, 45 C nd 50 C (ll±1 C), microscleroti smples for ll isoltes were simultneously exposed to ech temperture for 30 min. Temperture in ech th ws monitored continuously using thermocou- Tle 1 Origin of isoltes of V. dhlie from the Netherlnds used in the pthogenicity nd temperture-tolernce studies Isolte code Host Loction (town, province) Isoltion dte M Chrysnthemum Mde, Brnt April 05, 2002 S Chrysnthemum Honselersdijk, Zuid-Hollnd April 15, 2002 D Chrysnthemum Nldwijk, Zuid-Hollnd April 15, 2002 U Chrysnthemum Mslnd, Zuid-Hollnd April 15, 2002 V Chrysnthemum the Hgue, Zuid-Hollnd April 15, 2002 JKG1 Potto Wgeningen, Gelderlnd Septemer 01, 2000 JKG5 Potto Americ, Limurg Septemer 04, 2000 JKG6 Potto Heide, Limurg Septemer 04, 2000 JKG8 Potto Schore, Zeelnd Septemer 22, 2000 JKG9 Potto Achtererg, Utrecht Septemer 22, 2000

3 ples nd dt logger. After tretment, the microscleroti were spred on sterile filter pper in lminr irflow cinet nd for ech tretment 50 plced individully using fine syringe needle onto modified soil extrct gr pltes (Hrris et l. 1993) mended with 50 ppm oxytetrcyclin. Plted microscleroti were incuted t 20 C for 7 dys nd the numer germinted scored. Rooted cuttings (4 week-old) of Chrysnthemum coronrium cv. Gold-Pes were kindly provided y Fides Strthof (Msdijk, The Netherlnds). Homogeneous potto plnts (Solnum tuerosum cv. Bintje) were otined y excising eyes from potto tuers with round sclpel nd growing these for 1 week in perlite on Hoglnd s solution, followed y 2 weeks in potting compost. For ech of the ten isoltes, plug of mycelium from n ctively growing PDA-culture ws trnsferred to Czpek Dox liquid medium (Oxoid). After incuting for 1 week t 20 C with shking (100 rpm), the culture ws filtered through cheesecloth to remove the mycelium. The resulting conidil suspensions were djusted to conidi ml 1 nd for ech isolte eight chrysnthemum nd eight potto plnts were inoculted y dipping wshed roots into the suspensions for 2 min. Roots of 20 plnts of ech species were dipped into sterile wter s controls. Immeditely fter inocultion, ech plnt ws potted into compost in 10 cm plstic pot nd plced in rndomised lyout in controlled environment chmer t 20 C with 16/8 h light/drk regime. Disese severity ws scored weekly. For chrysnthemum plnts the disese scle used ws: 0, no symptoms; 1, 7 leves showing verticillium symptoms; 2, etween 7 leves nd 50% of the plnt showing symptoms; 3, mjority of the plnt showing symptoms; 4, few leves (usully t the top) without symptoms; 5, ded plnt. For potto plnts the senescence scle used ws s follows: 0, no necrosis; 1, 1 5% of plnt showing necrosis; 2, 6 25%; 3, 26 75%; 4, 76 95%; 5, %. The re-under-the-disese-progress-curve (AUDPC) over 80 dys ws clculted for every chrysnthemum plnt nd the re-under-the-senescenceprogress-curve (AUSPC) over 73 dys ws clculted for every potto plnt (Cmpell nd Mdden 1990). The presence of n effect of host on AUDPC or AUSPC ws tested y nlysis of vrince nd pirwise multiple comprisons were crried out with the Tukey test t P=0.05 using SAS version 8.0 (SAS Institute, Inc., Cry, NC, USA). Amplified frgment length polymorphism (AFLP) nlysis ws used to compre isoltes moleculrly. A commercil kit (AFLP Anlysis System II for smll genomes; Invitrogen, Pisley, UK) ws used ccording to the mnufcturer s instructions. The primers used were sed on erlier work with V. dhlie; for pre-mplifiction, the EcoRI primer hd no dditionl nucleotides while the MseI primer hd dditionl C or G ses t the 3 end. For selective mplifiction the EcoRI primer with one of three pirs of dditionl ses ws used with the MseI pre-mplifiction primers (MseI + C with EcoRI + GA, EcoRI + AT or EcoRI + GC; MseI + G with EcoRI + CC). The EcoRI primers for selective mplifiction were lelled with either Fm or Hex nd PCR products were seprted on n Applied Biosystems cpillry sequencer. Products were sized nd nlysed using stndrd softwre. For comprison, wider popultion of V. dhlie isoltes from chrysnthemums from the UK nd the Netherlnds were nlysed simultneously. These extr isoltes were collected over n extended period s prt of other studies nd re not detiled here. Prior to AFLP nlysis ll isoltes used were shown to e V. dhlie y polymerse chin rection mplifiction of the riosoml RNA internl trnscried spcer/5.8s regions, digestion of the products with restriction endonucleses nd comprison of the digestion ptterns with well-chrcterised isoltes (Collins et l. 2003). Microscleroti from ll ten isoltes were killed y het tretment t 50 C nd ll survived fter tretment t 25 C. After tretment t 40 C nd 45 C, microscleroti originting from potto plnts were significntly (P<0.05, t-test fter rcsine-root trnsformtion of proportion germinted) more het resistnt thn those from chrysnthemum plnts (Fig. 1). Chrysn- survivl % chrysnthemum potto 25 C 40 C 45 C 50 C temperture Fig. 1 Survivl of V. dhlie microscleroti produced in vitro from five isoltes of potto (open rs) nd chrysnthemum plnts (solid rs) incuted for 30 min. in wter t four tempertures. Within temperture tretments, different letters indicte results which differ significntly t p<0.05

4 themum plnts inoculted with chrysnthemum isoltes egn to show verticillium wilt symptoms from the 17th dy onwrd nd eventully showed severe wilt symptoms (Fig. 2). However, chrysnthemum plnts inoculted with potto isoltes did not show ny disese symptoms nd were not distinguishle from the control plnts t the end of the experiment. All potto plnts showed similr AUSPCs irrespective of the origin of the isolte used, nd significntly greter senescence thn showed y control plnts (Fig. 2). AFLP nlysis of the tested isoltes formed two distinct clusters with comprle levels of genetic diversity within the two (oth up to pproximtely 25%) ut with greter diversity etween ny two isoltes cross the clusters (38 53%; Tle 2). When compred with 12 other chrysnthemum isoltes collected over 25 yers (six from the Netherlnds nd six from the UK), the potto isoltes still formed discrete cluster whilst the chrysnthemum isoltes used in the other tests here did not (Fig. 3). Microscleroti formed y the potto isoltes showed significntly higher tolernce of rised tempertures thn those formed y the chrysnthemum isoltes. As the microscleroti used for the temperture sensitivity ssy were produced in vitro on cellophne nd under the sme conditions for ll isoltes, we presume this difference is geneticlly controlled. At the outset of this work, one of the possile explntions for the upsurge in verticillium wilt in glsshouse-grown chrysnthemums ws tht AUDPC/AUSPC chrysnthemum potto control isolte source Fig. 2 Are under the disese progress curve (AUDPC) for chrysnthemum plnts (solid rs) nd re under the senescence progress curve (AUSPC) for potto plnts (open rs) inoculted with chrysnthemum or potto isoltes. Within plnt species, different letters indicte results which differ significntly t P<0.05. The high AUSPC level for control potto plnts reflect the nturl senescence of the potto plnts during the course of the experiment steming of soils in glsshouses over mny consecutive yers hd selected for isoltes tolernt of higher temperture. Het dpttion of isoltes of V. dhlie hs een reported (Cstejón-Muñoz nd Bollen 1993). In the light of the dt presented here, this hs not occurred in the chrysnthemum-cultivted glsshouse soils. There is no ovious explntion s to why isoltes from potto should exhiit this higher temperture tolernce s open fields, such s re used for pottoes, re generlly exposed to lower tempertures thn glsshouse soils. It my e tht lower thn usul tolernce of elevted tempertures hs een selected for in the chrysnthemum isoltes s corollry of selection for host specificity (see elow), possily ecuse of some interction etween the two trits. However, it seems more likely tht higher thn verge temperture-tolernce is ssocited with the potto isoltes, proly s n indvertent side effect of their monophyletic origin s suggested y the moleculr nlysis. The isoltes of V. dhlie tested from potto did not cuse ny symptoms in chrysnthemum lthough they cused erly senescence on potto plnts showing tht they hd not lost their generl ility to cuse disese. However, the isoltes tested from chrysnthemum were ll highly virulent for chrysnthemum plnts. Isoltes of V. dhlie re known to e frequently unstle in culture, often losing pthogenicity nd their ility to produce microscleroti over time in n unpredictle mnner. Loss of pthogenicity in culture is usully generl in nture nd is nturl phenomenon, ut it is unlikely tht one, let lone ll of these five potto cultures, hve specificlly lost the ility to infect chrysnthemums ut retined their ility to infect pottoes whilst t the sme time none of the chrysnthemum isoltes hve lost their ility to infect this host. Moreover, these were ll recent isoltes t the time of these experiments nd hd een stored frozen t 80 C, procedure which minimizes chnges in the isoltes. These results suggest tht some degree of host specilistion of V. dhlie for chrysnthemum is ssocited with infection of this host in glsshouses in the Netherlnds. It is too erly to sy whether this is the sole cuse of the recent increse in verticillium wilt s we do not yet know how specilized the isoltes present in the crop were prior to the increse. Although undesirle for growers, development of host-specilistion of V. dhlie for chrysnthemum

5 Tle 2 Genetic diversity (%) etween five isoltes of V. dhlie from chrysnthemum nd from potto s determined y AFLP nlysis Potto isoltes Chrysnthemum isoltes M S D U V M S D U 26.2 Bnds were sized using GeneScn nd scored using Genotyper (oth Applied Biosystems) nd genetic reltionships estimted using Treecon ( with the similrity coefficient of Nei nd Li (1979) V M JKG1 JKG9 JKG8 D S JKG5 JKG6 Fig. 3 Schemtic representtion of the genetic reltionship, s determined y AFLP nlysis, for five isoltes of V. dhlie from potto (JKG1-9), the five isoltes from chrysnthemum used for pthogenicity nd temperture sensitivity testing (D, M, S, U, V) nd 12 other isoltes from chrysnthemum (six from the UK nd six from the Netherlnds, unlelled rnches). Actul reltionships for lelled isoltes re shown in Tle 2. Cldogrms were constructed from the genetic reltionships shown in Tle 2 using Treecon ( ugent.e) nd Neighour Joining U might e circumvented y crop rottion ut more informtion is required efore rtionl disese mngement plns cn e designed on this sis. For exmple, how quickly non-specilised isolte (such s one from potto) would ecome highly pthogenic to chrysnthemum plnts is not known, nor is the rte t which chrysnthemum isoltes would lose their pthogenicity during seril pssge through other hosts. Potentilly oth processes my e rpid. For exmple, Fordyce nd Green (1963) showed tht some mint isoltes initilly not pthogenic to tomto ecme so fter only one to five pssges through tomto, simultneously losing pthogenicity for mint. Also, it is known tht in some crop systems V. dhlie isoltes show reciprocl high pthogenicity, e.g. cotton nd olive isoltes from Spin Rodríguez- Jurdo et l. (1993). The host specificity encountered in this study suggests tht V. dhlie is surviving soil steming in situ in glsshouses used for chrysnthemum growing, rther thn eing introduced from unspecilized sources to ech new crop. In generl, V. dhlie, including the isoltes from chrysnthemum tested here, hs low survivl temperture (Bollen 1985) which is normlly esily reched y soil steming. However, in deep rooting crop such s chrysnthemum, some microscleroti my e produced deep in the soil where lethl tempertures re not reched with current techniques. If this is the cse, more effective tretments such s more intensive steming or introduction of root-impermele lyer t n pproprite depth in the soil will e needed.

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