Elimination of Grapevine rupestris stem pitting-associatedvirus (GRSPaV) from two Vitis vinifera cultivarsby in vitro chemotherapy

F. G. Skiada & V. I. Maliogka & N. I. Katis &

E. P. Eleftheriou

Accepted: 10 September 2012 /Published online: 22 September 2012# KNPV 2012

Abstract The grapevine (Vitis vinifera L.) cultivars‘Agiorgitiko’ and ‘Malagouzia’, naturally infectedwith Grapevine rupestris stem pitting-associated virus(GRSPaV), were subjected to in vitro chemotherapyusing the antiviral inosine 5′-monophosphate dehydro-genase inhibitors tiazofurin (TR), ribavirin (RBV) andmycophenolic acid (MPA). The chemotherapy lasted80 days and was carried out as two consecutive treat-ments. Severe phytotoxicity, estimated after 40 days ofculture, was observed in drug-treated explants, espe-cially when high doses of TR were used. Phytotoxicityexhibited a cultivar- and chemical compound-dependent profile. The virus eradication status of thesurvived plantlets was determined by nested RT-PCRusing total RNA templates, after 80 days of drugtreatment and one year later, after the passage of onedormancy period, in potted plants grown in a green-house. Data indicated that the highest GRSPaV elim-ination in ‘Agiorgitiko’ was obtained with 10 μg ml−1

TR, 30 μg ml−1 RBV and 20 μg ml−1 MPA. The erad-ication rates were lower in the case of ‘Malagouzia’,

where the highest ones were achieved after treatmentswith 15 μg ml−1 TR and 80 μg ml−1 MPA. This is thefirst report on GRSPaVelimination in grapevine follow-ing treatment with antiviral compounds, which couldprovide an alternative to the traditional methods of viruseradication through meristem culture and thermotherapy.

Keywords Mycophenolic acid . Nested RT-PCR .

Phytotoxicity . Ribavirin . Tiazofurin

AbbreviationsBA benzyladenineGLRaV grapevine leafroll-associated virusGRSPaV grapevine rupestris stem pitting-associated

virusGVA grapevine virus AIMPDH inosin 5′-monophosphate dehydrogenaseMPA mycophenolic acidNAA naphthaleneacetic acidRBV ribavirinRSP rupestris stem pittingTR tiazofurinWPM woody plant medium

Introduction

Several diseases caused by viruses, viroids and phyto-plasmas are known to induce severe grapevine de-cline, inferring heavy yield reduction in all the most

Eur J Plant Pathol (2013) 135:407–414DOI 10.1007/s10658-012-0097-z

F. G. Skiada : E. P. Eleftheriou (*)Department of Botany, School of Biology, AristotleUniversity of Thessaloniki,541 24 Thessaloniki, Greecee-mail: eelefth@bio.auth.gr

V. I. Maliogka :N. I. KatisPlant Pathology Laboratory, School of Agriculture,Aristotle University of Thessaloniki,541 24 Thessaloniki, Greece

important grapevine growing regions of the world(Martelli 2009). Of those, the rugose wood complexis a group of graft-transmissible and damaging grape-vine diseases worldwide, with Rupestris Stem Pitting(RSP) being the most common component of thiscomplex (Meng et al. 1999; Stewart and Nassuth2001; Nakaune et al. 2008). Although the aetiologyof these diseases is still unclear, there is evidence thatGrapevine rupestris stem pitting-associated virus(GRSPaV), a member of the genus Foveavirus withinthe family Betaflexiviridae (Martelli 2009), is closelyassociated with RSP (Zhang et al. 1998; Meng et al.1999; Nakaune et al. 2008). GRSPaV has a worldwidedistribution, and its genome has been fully character-ized (Zhang et al. 1998; Meng et al. 2005). It is nowwell known that the virus comprises a wide range ofsequence variants, which may be responsible for dif-ferent diseases that have been observed (Meng et al.2005, 2006). Thus, apart from RSP, GRSPaV is im-plicated in Syrah decline and vein necrosis of ‘Richter110’ (Bouyahia et al. 2005; Lima et al. 2006).According to the European and Mediterranean PlantProtection Organization (EPPO), the certificationscheme for grapevine should include tests for manyviruses and phytoplasmas, among them GRSPaV(Anonymous 2008). Thus, the establishment ofGRSPaV-free vineyards is an important measure forthe control of this virus and associated diseases.

The elimination of viruses is usually attempted byin vivo and in vitro thermotherapy combined withmeristem, shoot-tip or axillary bud culture and somaticembryogenesis. Although these traditional sanitationtechniques applied for several viruses produced highpercentages of healthy plants (Gambino et al. 2006,2009; Maliogka et al. 2009; Skiada et al. 2009), for therecalcitrant-to-elimination ones such as GRSPaV theyhave shown low sanitation effectiveness (Gribaudo etal. 2006; Skiada et al. 2009; and literature cited there-in). Overall, their success rates are variable due to thestrong virus-host relationship, the virus location in thehost tissue, treatment performance and experimentalconditions adopted. Moreover, the traditional eradica-tion protocols suffer some drawbacks. Thermotherapycauses thermal stress to explants, while meristem tipculture is a difficult technique and might be related topossible undesirable somaclonal variations and juve-nility in the regenerated plantlets (Gribaudo et al.2006). Shoot-tip culture following thermotherapyachieves high regeneration but low virus eradication

rates (Skiada et al. 2009), whereas somatic embryo-genesis is a difficult, time consuming and cultivardependent technique, which might also cause soma-clonal variation in plantlets (Gambino et al. 2006;Gribaudo et al. 2006). For these reasons, as pro-posed also by other researchers (Gribaudo et al.2006), alternative techniques usually adopted fromother research fields such as cryopreservation(Wang et al. 2003) and chemotherapy with anti-viral compounds, should be applied in the case ofGRSPaV-infected grapevines.

In medical research, antiviral compounds targetingdifferent steps in the replication cycle of viruses areused to eliminate them or reduce their multiplication.Some host enzymes involved in viral DNA and RNAsynthesis may also be targeted in antiviral therapy.Inosine 5′-monophosphate dehydrogenase (IMPDH)is a key enzyme that catalyzes an essential step inthe de novo biosynthesis of guanine nucleotides,namely the conversion of inosine 5′-monophosphateto xanthosine 5′-monophosphate, which is further con-verted to GMP, GDP, dGDP, GTP and dGTP (Shu andNair 2008). IMPDH can be inhibited by antiviralcompounds and is viewed as an important moleculartarget in the quest for the discovery of antiviral treat-ment (Shu and Nair 2008). Ribavirin (1-β-D-ribofur-anosyl-1,2,4-triazole-3-carboxamide) (RBV) (Sidwellet al. 1972) and tiazofurin (2-β-D-ribofuranosylthia-zole-4-carboxamide) (TR) (Srivastava et al. 1977) aresynthetic nucleotide analogues that inhibit IMPDHfunction, while mycophenolic acid (MPA) is a non-nucleotide IMPDH inhibitor (Huberman et al. 1981).The major event occurring in cells exposed to com-petitive IMPDH inhibitors such as RBV and TR oruncompetitive inhibitors such as MPA is a depletion ofthe intracellular GTP and dGTP pools required foradequate RNA and DNA synthesis (Hong andCameron 2002), which in turn results in virus elimi-nation and cytotoxicity (Shu and Nair 2008).

The application of chemotherapy to eliminate plantviruses from the vegetative propagation material has agreat potential in agriculture. Antiviral compoundsthat have been largely used against animal viruseswere then applied for the elimination of plant virusesdue to their similar action pathway. For grapevine, inparticular, chemotherapeutic protocols involving anti-viral compounds have been applied to eliminateGrapevine leafroll-associated virus-3 (GLRaV-3),Grapevine leafroll-associated virus-1 (GLRaV-1) and

408 Eur J Plant Pathol (2013) 135:407–414

Grapevine virus A (GVA) from some Italian grapevinecultivars (Panattoni et al. 2007a, b, 2011).

To our knowledge, no chemotherapeutic treatmenthas been reported to date for the elimination ofGRSPaV from infected grapevines. The objective ofthis study was therefore to eradicate GRSPaV fromtwo Greek Vitis vinifera L. cultivars by chemotherapyusing the IMPDH inhibitors RBV, TR and MPA.

Materials and methods

Plant material

In vitro established grapevine (Vitis vinifera L.) culti-vars of ‘Agiorgitiko’ and ‘Malagouzia’ from the prem-ises of the private company Vitro Hellas S.E.,Northern Greece, naturally infected with GRSPaV(Dovas and Katis 2003), were used. ‘Agiorgitiko’ isone of the most important Greek red wine cultivars,while ‘Malagouzia’ is an awarded Greek white winecultivar [bronze metal of ‘Challenge International duVin 2007’ (http://www.globalwinespirits.com), an in-ternational contest for wine held in France].

Explants of ‘Agiorgitiko’ were proliferated in aWoody Plant Medium (WPM) (Lloyd and McCown1980) supplemented with 0.3 μM benzyladenine (BA)and 0.3 μM naphthaleneacetic acid (NAA) (Skiada etal. 2009), while those of ‘Malagouzia’ were grown ina Galzy medium (Galzy et al. 1990) supplementedwith 0.5 μM BA and 0.3 μM NAA (Skiada et al.2010). The explants produced single axillary shoots,which were divided in nodal segments and trans-ferred into 500 ml glass jars containing 100 ml ofthe same proliferation medium. Stock cultureswere kept at 21±2 °C and 16 h photoperiod undercool white fluorescent light (40 μmol m−2s−1), andprovided the experimental material for the antiviralcompound applications.

Treatment with antiviral compounds

TR, RBV and MPA were used for the elimination ofGRSPaV from both cultivars. TR was a generous giftby Prof. H. N. Jayaram (Richard Roudebush VeteransAffairs Medical Center, Indianapolis, IN, USA), whileRBV and MPA were purchased from Sigma–Aldrich(St. Louis, MO, USA). TR and RBV were hydrated,while MPAwas diluted in 100 % ethanol to form stock

solutions. Immediately prior to use, they were ultra-filtrated (filters of 0.2 μm) and added to the prolifer-ation media post autoclaving. Final concentrations of1, 5, 10, 15, 20, 30, 40, 60 and 80 μg ml−1 of eachantiviral compound were added to the proliferationmedia of both cultivars, while respective media with-out antiviral drugs served as controls. RBV was nottested in the case of ‘Malagouzia’ due to the severetoxicity observed in preliminary experiments. Thirtysingle nodal explants of 1 cm in length, randomlyselected from the stock material, were used in everytreatment. Each experiment was conducted twice, thusa total of 60 explants were subjected to every drugconcentration.

The chemotherapy lasted 80 days and was com-posed of two consecutive subcultures. Drug-inducedphytotoxicity was estimated at the end of the firstsubculture (40 days) and was defined as the numberof dead explants out of the total treated ones. In eachexperiment plantlets that survived the first subculturewere transferred to fresh proliferation media supple-mented with the respective antiviral compound doseand were subjected to a second therapy cycle of40 days. Then the explants were individually num-bered and propagated in vitro for an additional 80 days,divided in two subcultures, in antiviral compound-freeproliferation media for recovering from the chemicalstress. After this period the explants were tested for thepresence of GRSPaV by nested RT-PCR. Then theplantlets were acclimatized in the greenhouse in potsand were tested again one year later, after one dor-mancy period, to confirm their sanitary status.

Virus detection by nested RT-PCR

Total RNA was extracted from 0.2 g of grapevineexplants or acclimatized plants (stems, petioles andleaves) according to the method described by Rottand Jelkmann (2001), modified by adding 6 % PVPand 0.2 M β-mercaptoethanol in the grinding buffer.GRSPaV diagnosis was done using a nested RT-PCRdeveloped for the generic detection of viti- and fovea-viruses, in which degenerate primers targeting a con-served region (198 bp) of the RNA-dependent RNApolymerase (RdRp) were used (Dovas and Katis2003). To rule out false negative results, a plant 18SrRNAwas used as an internal control for testing RNAdegradation. The amplification of a 255 bp fragmentfrom the 18S rRNA was separately performed in a

Eur J Plant Pathol (2013) 135:407–414 409

one-step RT-PCR (Du et al. 2006). In all cases 2 μl oftotal RNA were used as template. The reaction prod-ucts were analysed by electrophoresis in 1.5 % agarosegels in TAE buffer, stained with ethidium bromide,and visualized under UV light.

Results

Phytotoxicity induced by the antiviral compounds

After 40 days of the first subculture all the explantsderived from the control media survived. In those mediasupplemented with antiviral compounds, a concentra-tion and cultivar-dependent necrosis was recorded foralmost all treatments in both cultivars tested (Table 1).TR proved to be the most toxic agent for ‘Agiorgitiko’,followed by RBV, while MPA had the mildest effect; for‘Malagouzia’ the most toxic was RBV (which wasfinally not tested), followed by TR and MPA.

For ‘Agiorgitiko’ TR and RBV administrationscaused total necrosis at concentrations 20 μg ml−1

and 40 μg ml−1, respectively (Table 1). Althoughsome explants grew slightly and produced a few greenleaves in 40 and 60 μg ml−1 of RBV, their apicalmeristems died after 40 days of culture rendering theminappropriate for subculture; they were then consid-ered as dead. MPA induced a gradually increasing

toxicity up to the highest concentration applied(80 μg ml−1), where effects were severe though nottotally lethal (93.3 % mortality).

Similar reactions were observed for the ‘Malagouzia’explants, in which TR was also more toxic thanMPA (Table 1). The lethal concentration of TRwas 20 μg ml−1, while MPA toxicity increased muchslower for this cultivar, in which a considerable percent-age (50 %) of explants survived even in the highestconcentration applied (80 μgml).

GRSPaV eradication

At the end of the antiviral drug treatment and therecovery period (80+80 days), the virus eliminationefficiency was determined by nested RT-PCR using18S rRNA as a control (Fig. 1). In all cases the 255 bpfragment from the 18S rRNA was readily amplifiedeither in the presence or in the absence of GRSPaV,thus showing the efficiency of the RNA extractiontechnique. In the control treatments, no sanitationwas detected in any of the infected plantlets tested.On the other hand, in the antiviral compound-supplemented proliferation media, it was in generalobserved that, in parallel with the aforementionedincreasing phytotoxicity, the higher the antiviral com-pound concentration the higher percentage of viruseradication (Table 2).

Table 1 Phytotoxicity induced in grapevine cultivars ‘Agiorgi-tiko’ and ‘Malagouzia’ explants after 40 days of treatment withincreasing concentrations of the antiviral compounds tiazofurin(TR), ribavirin (RBV) and mycophenolic acid (MPA), expressed

as the number of dead explants out of the total number of treatedexplants. (n060 for each treatment, results are presented as %ratio±st.dev)

Antiviral compound Concentration (μgml−1)

0 1 5 10 15 20 30 40 60 80

‘Agiorgitiko’

TR 0/60 16/60 26/60 50/60 58/60 60/60 60/60 60/60 60/60 60/60

0±0 26.7±0.74 43.3±0.83 83.3±0.62 96.7±0.30 100±0 100±0 100±0 100±0 100±0

RBV 0/60 2/60 8/60 14/60 28/60 40/60 44/60 60/60 60/60 60/60

0±0 3.3±0.30 13.3±0.57 23.3±0.71 46.7±0.83 66.7±0.79 73.3±0.74 100±0 100±0 100±0

MPA 0/60 0/60 24/60 30/60 38/60 46/60 48/60 50/60 52/60 56/60

0±0 0±0 40.0±0.82 50.0±0.84 63.3±0.81 76.7±0.71 80.0±0.67 83.3±0.62 86.7±0.57 93.3±0.30

‘Malagouzia’

TR 0/60 16/60 36/60 40/60 44/60 60/60 60/60 60/60 60/60 60/60

0±0 26.7±0.74 60.0±0.82 66.7±0.79 73.3±0.74 100±0 100±0 100±0 100±0 100±0

MPA 0/60 0/60 0/60 2/60 4/60 10/60 20/60 22/60 26/60 30/60

0±0 0±0 0±0 3.33±0.30 6.7±0.41 16.7±0.62 33.3±0.79 36.7±0.80 43.3±0.83 50.0±0.84

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For ‘Agiorgitiko’, in particular, at concentrations 1, 5and 10 μg ml−1 of TR the rate of virus eradication wasincreasing rapidly, achieving a highest percentage of80 % at 10 μg ml−1 (Table 2). In 15 μg ml−1 of TR,however, no virus-free plantlets were obtained, appar-ently due to the very small number of plantlets (onlytwo) that survived the antiviral compound toxicity, asample size not representative. When this cultivar was

treated with RBV, a gradually increasing number ofvirus-free plantlets was found up to the concentrationof 30 μgml−1 (62.5 %) (Table 2). In MPA, which in-duced the mildest phytotoxicity, virus-free plantletswere produced in all concentrations used, with a highestpercentage obtained at 20 μg ml−1 (85.7 %), after whicha gradual decline was observed. Smaller percentages ofGRSPaV eradication were recorded in ‘Malagouzia’

198bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 L

255bp

a

b

Agiorgitiko MalagouziaGRSPaV-and

GRSPaV+ controls

Fig. 1 Agarose gel electrophoretic analyses of nested RT-PCRand one-tube RT-PCR products amplified with primers specificfor GRSPaV (a) and 18S rRNA (b) respectively, obtained frominfected and virus-free plant material of ‘Agiorgitiko’ and‘Malagouzia’. ‘Agiorgitiko’: Lanes 1–3: 10 μg ml−1 TR; lanes4–6: 20 μg ml−1 MPA: lanes 7, 8: 30 μg ml−1 MPA; lanes 9–11:

20 μg ml−1 RBV. ‘Malagouzia’: lanes 12, 13: 80 μg ml−1 MPA;lanes 14, 15: 15 μg ml−1 TR. Lanes 16, 17: healthy control of‘Agiorgitiko’ and ‘Malagouzia’ respectively; lanes 18–20: invitro cultured infected plant material of ‘Agiorgitiko’. Lanes21–23: in vitro cultured infected plant material of ‘Malagouzia’.L: 100 bp DNA ladder

Table 2 Elimination of GRSPaV in the grapevine cultivars‘Agiorgitiko’ and ‘Malagouzia’ explants after 80 days of in vitrotreatment with the antiviral compounds TR, RBVand MPA plus

80 days of recovering, expressed as the number of virus-freeplantlets out of the total number of plantlets that survived phyto-toxicity (cf. Table 1) (results are presented as % ratio±st.dev)

Antiviral compound Concentration (μg ml−1)

0 1 5 10 15 20 30 40 60 80

‘Agiorgitiko’

TR 0/60 6/44 14/34 8/10 0/2 0/0 0/0 0/0 0/0 0/0

0±0 13.6±0.78 41.0±1.46 80.0±4.21 0±0 na na na na na

RBV 0/60 10/58 12/52 12/46 10/32 8/20 10/16 0/0 0/0 0/0

0±0 17.2±0.65 23.0±0.81 26.0±0.96 31.2±1.47 40.0±2.51 62.5±3.12 na na na

MPA 0/60 10/60 22/36 22/30 16/22 12/14 10/12 8/10 6/8 2/4

0±0 16.6±0.62 61.1±1.37 73.3±1.49 72.7±2.07 85.7±2.59 83.3±3.24 80.0±4.21 75.0±5.78 50.0±14.43

‘Malagouzia’

TR 0/60 0/44 0/24 6/20 6/16 0/0 0/0 0/0 0/0 0/0

0±0 0±0 0±0 30.0±2.35 37.5±3.12 na na na na na

MPA 0/60 0/60 0/60 0/58 0/56 2/50 4/40 6/38 12/34 16/30

0±0 0±0 0±0 0±0 0±0 4.0±0.39 10.0±0.76 15.7±0.97 35.2±1.42 53.3±1.69

na not applicable.

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compared with ‘Agiorgitiko’, which occurred only in 10and 15 μg ml−1 of TR treatment (Table 2). At lowerconcentrations (≤5 μg ml−1) no virus-free plantlets weredetected, while in higher ones (≥20 μg ml−1) no assayscould be performed as plantlets did not survive the anti-viral compound-induced toxicity. Treatment with MPAcaused a gradually increasing rate of virus eliminationstarting from 4 % at the concentration of 20 μgml−1 andreaching 53.3 % at 80 μg ml−1 (Table 2).

The nested RT-PCR assay, applied one year later inthe acclimatized potted plants, after one dormancyperiod, confirmed their virus-free status and enhancedthe reliability of the virus detection method used.

Discussion

This study reports for the first time on the eliminationof the particularly difficult to eradicate GRSPaV fromtwo Greek grapevine cultivars, using a chemothera-peutic protocol involving the application of the anti-viral compounds TR, RBV and MPA. The sanitarystatus of the treated plants was checked with a genericnested RT-PCR, a sensitive and polyvalent assaywhich has been successfully used for the detection ofvarious GRSPaV strains (Stewart and Nassuth 2001;Dovas and Katis 2003; Meng et al. 2006).

Apparently, the most severe disadvantage of thechemotherapy protocol used was the high percentageof phytotoxicity, which displayed a chemical andcultivar-specific response, with the most toxic for‘Agiorgitiko’ being TR, followed by RBV and MPA,while for ‘Malagouzia’ the order was RBV>TR>MPA.Comparing the two cultivars, ‘Malagouzia’ appearedto be more resistant than ‘Agiorgitiko’ to TR and MPAtreatments. The different levels of resistance might beattributed to the genetic variability of Vitis viniferacultivars (Torregrosa and Bouquet 1996). Various lev-els of phytotoxicity caused by different antiviral com-pounds were also reported for the Italian grapevinevarieties ‘Sangiovese’ and ‘Sagrantino’ (Panattoni etal. 2007a, b). Similarly to our results, TR was found tobe more toxic to ‘Sangiovese’ than MPA (Panattoni etal. 2007a). However, in ‘Sangiovese’ all plants died at150 μg ml−1 TR, a much higher concentration than the20 μg ml−1 TR that was fatal for both ‘Agiorgitiko’and ‘Malagouzia’ explants. Moreover, MPA at con-centrations as high as 250 μg ml−1 caused mortality to80 % of ‘Sangiovese’ explants (Panattoni et al.

2007a), a rate obtained at 30 μg ml−1 MPA for‘Agiorgitiko’. These results reinforce the notion thatgrapevine cultivars respond differently, probably dueto their genetic variability (Torregrosa and Bouquet1996; Péros et al. 1998). Nevertheless, our results forRBV toxicity to ‘Agiorgitiko’ were similar to those ofRBVon ‘Sagrantino’ explants (Panattoni et al. 2007b),since in both cultivars the effect was lethal when drugconcentration was ≥40 μg ml−1. Moreover, our studyindicates a rather low selectivity index, i.e. the effica-cy/toxicity ratio for the TR and RBV compounds. Thisobservation is in accordance with previous reports onthe low selectivity of RBV in plants (Quecini et al.2008), which was higher when MPA was used.

As far as the GRSPaV elimination in ‘Agiorgitiko’is concerned, the highest percentages of virus-freeplantlets were obtained at 10 μg ml−1 TR (80 %),30 μg ml−1 RBV (62.5 %) and 20 μg ml−1 MPA(85.7 %) (Table 2). These results indicate a higherefficiency of chemotherapy compared to in vitro ther-motherapy combined with meristem (67.6 %) orshoot-tip (50.8 %) culture for the same cultivar andthe same virus (Skiada et al. 2009); however, thesurvival rate of plantlets was higher (52.6 %) afterthermotherapy than after antiviral compound adminis-tration. The decline of elimination rates in‘Agiorgitiko’ at high MPA concentrations could beattributed to the small rates of explant survival thatmakes the sample size not representative for the eval-uation of the eradication technique.

The elimination rates of GRSPaV varied accordingto the type of antiviral compound used and the cultivartested. Overall, higher elimination rates were achievedfor ‘Agiorgitiko’ than for ‘Malagouzia’. This could beattributed to the presence of different GRSPaV strains,composed by mixtures of genetic variants (Meng et al.2005, 2006; Nolasco et al. 2006; Nakaune et al. 2008),or even different virus titres that could affect theefficiency of elimination techniques. Another possibleexplanation of the variation observed could be thedifferences on the uptake, distribution and metabolismof the various antiviral compounds by each cultivar inaccordance with the notion of a genotype-dependenteffect on virus elimination (Torregrosa and Bouquet1996; Maliogka et al. 2009). TR, RBV and MPAmechanisms are known to be expressed by their cel-lular active metabolites; nevertheless, the actual fate ofthese and other antiviral compounds in plants has beenscarcely studied. Further experimentation is needed to

412 Eur J Plant Pathol (2013) 135:407–414

decipher the factors shaping the high variability ob-served on the efficacy of the antiviral drugs.

Novel sanitation methods have been applied toeradicate several viruses from cultivated grapevines.Cryotherapy and electrotherapy succeeded roughly60 % survival rates, of which about 40 % of GVA-free plantlets were generated (Bayati et al. 2011),while cryopreservation of in vitro-grown grapevineshoot tips resulted in high percentages of GVA elim-ination (Wang et al. 2003). Chemotherapy as a plantsanitation technique may also be considered a novelprocedure with the potential of successfully eradicat-ing severe and persistent plant viruses (Panattoni et al.2007a, b; present study). All novel techniques, how-ever, have their drawbacks and usually involve a highcost; for chemotherapy, the main disadvantage seemsto be the high phytotoxicity. Moreover, questions re-lated to the action mechanism, the theoretical accumu-lation of residues and the presumed mutagenic effectsin plants after chemotherapy need to be addressed infuture studies. As De Clercq and Field (2006)reported, with the use of correct and moderate dosesthe undesirable phytotoxic side effects can be reduced.In conclusion, chemotherapy appears to be a promis-ing technique for the effective eradication of difficultviruses such as GRSPaV from grapevines, but furtherexperimentation is needed to find more selective com-pounds and optimize conditions for higher efficacycombined with lower phytotoxicity effects.

Acknowledgments This study is part of a PhD thesis (F. G.Skiada) and was financed by a research project for the reinforce-ment of new scientists [PENED 2003, funded by 75 % of thepublic cost from the European Union, and 25 % from the GreekGovernment (Ministry of Development - General Secretariat ofResearch and Technology, GSRT), (Measure 8.3, E.P.A.N. - ThirdEuropean Community Framework)], while the infrastructure andbasic chemicals were provided by Vitro Hellas S.A. We also thankProf. H.N. Jayaram for kindly providing the tiazofurin, and thePhD student A. Katsiani for her help in some experiments.

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