GLRaV-2 Distribution and Molecular Caracterization in Croatian Autochthonous Grapevine

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Journal of Plant Diseases and Protection, 117 (5), 194200, 2010, ISSN 1861-3829. Eugen Ulmer KG, Stuttgart

J.Plant Dis.Protect. 5/2010

Distribution and partial molecular characterization ofGrapevine leafroll-associated virus 2(GLRaV-2) found in Croatian autochthonous grapevine (Vitis vinifera L.) germplasm

Verbreitung und partielle molekulare Charakterisierung des Grapevine leafroll-associated virus 2 (GLRaV-2), das inkroatischem autochthonem Keimplasma von Weinreben (Vitis vinifera L.) gefunden wurde

D. Voncina1,*, S. Simon2, E. Dermic1, B. Cvjetkovic1, I. Pejic2, E. Maletic3 & J. Karoglan Kontic31 Department of Plant Pathology, University of Zagreb Faculty of Agriculture, Zagreb, Croatia2 Department of Plant Breeding Genetics, Biometrics and Experimentation, University of Zagreb Faculty of Agriculture, Zagreb, Croatia3 Department of Viticulture and Enology, University of Zagreb Faculty of Agriculture, Zagreb, Croatia* Corresponding author, e-mail [email protected]

Received 10 May 2010; accepted 7 June 2010

Abstract

Study ofGrapevine leafroll-associated virus 2 (GLRaV-2) distri-bution was conducted on 13 Croatian autochthonous grape-

vine cultivars or cultivars which are supposed to be autochth-onous included in clonal selection: Babica, Babi , Glavinua,Grk, Ljutun, Maratina, Mladinka, Nin ua, Plavina, Plavacmali, Poip, Vlaka and Vugava. All of them are grown in thesouthern part of Croatian coastal region (Dalmatia). Sam-pling was done during autumn 2007, and only a few samplesof cultivar Babi were also collected during 2008. All sampleswere tested for the presence of GLRaV-2 using DAS-ELISAtest. Out of 1100 tested grapevine accessions, the presence ofGLRaV-2 was determined in 45 samples (4.1%). No virus wasdetected in seven grapevine varieties (Grk, Ljutun, Mladenka,Nin ua, Plavina, Poip, Vlaka). Minimal infection rate wasdetermined in the cultivar Vugava (0.8%) while the mostinfected cultivar was Babi in which out of 98 analyzed grape-vine accessions 33 (33.7%) were positive. All virus-positive

tested vines contained mixed infections of several viruses andno vine was infected by GLRaV-2 only. In 14 different, random-ly selected grapevine accessions the presence of GLRaV-2 wasalso confirmed by RT-PCR using the GLR2CP1/GLR2CP2primer pair. RT-PCR products from four different grapevine ac-cessions were sequenced. Sequences of gene segment codingfor the viral coat protein revealed between 97.9% and 99.6%similarity on the nucleotide level, with absolute similarity onamino-acid level. Sixteen grapevine accessions were selectedfor transmission to herbaceous host Nicotiana benthamiana,but GLRaV-2 was not successfully transmitted to this host. Toour knowledge this is the first report of GLRaV-2 in Croatia.

Key words: clonal selection, coat protein, DAS-ELISA,genome sequencing, RT-PCR

Zusammenfassung

Die Studie ber die Verbreitung von Grapevine leafroll-asso-ciated virus 2 (GLRaV-2), wurde an den folgenden 13 kroa-tischen autochtonen oder als autochton geltenden Rebsortendurchgefhrt: Babica, Babi , Glavinusa, Grk, Ljutun, Marati-na, Mladinka, Nin ua, Plavina, Plavac mali, Poip, Vlakaund Vugava. Alle diese Rebsorten werden im sdlichen Teilder kroatischen Kstenregion (Dalmatien) angebaut und eswerden derzeit Klone von ihnen entwickelt. Die Probennahmewurde im Herbst 2007 vorgenommen, nur einige Proben vonder Rebsorte Babi wurden erst im Laufe des Jahres 2008genommen. Alle Proben wurden mittels des DAS-ELISA-Tests

auf Anwesenheit von GLRaV-2 geprft. Von den 1100 geprftenWeinreben wurde GLRaV-2 bei 45 Proben (4,1%) festgestellt.Bei sieben Rebsorten wurde dieses Virus nicht gefunden (Grk,Ljutun, Mladenka, Nin ua, Plavina, Poip, Vlaka). Die nied-

rigste Infektionsrate wurde bei der Sorte Vugava (0,8%) fest-gestellt, whrend bei der meistinfizierten Sorte Babi von den98 analysierten Proben 33 (33,7%) positiv waren. Alle positivauf Viren getesteten Rebsorten waren mit verschiedenen Viren

mischinfiziert; keine Rebsorte war ausschlielich mit GLRaV-2infiziert. Bei den 14 verschiedenen, stichprobenweise selek-tierten Weinrebeproben wurde die Anwesenheit von GLRaV-2im RT-PCR-Verfahren mittels GLR2CP1/GLR2CP2-Primer-Paa-ren besttigt. RT-PCR-Produkte von vier verschiedenen Wein-Akzessionen wurden sequenziert. Fr das virale Hllproteinkodierende Sequenzen waren auf der Nucleotidebene zu97,799,6% und auf der Aminosureebene zu 100% iden-tisch. Sechzehn Weinrebeproben wurden fr die bertragungauf den PflanzenwirtNicotiana benthamiana selektiert, jedochkonnte GLRaV-2 nicht erfolgreich auf den genannten Wirtbertragen werden. Unseres Wissens ist dies der erste Nach-weis von GLRaV-2 in Kroatien.

Stichwrter: DAS-ELISA, Gensequenz, Hllprotein,

klonale Selektion, RT-PCR

1 Introduction

One of the economically most important and widespreadcomplex of virus diseases is Grapevine leafroll. To date nineserologically distinct viruses named Grapevine leafroll-asso-ciated virus 1 to 9 have been associated with leafroll diseasecomplex (MARTELLI et al. 2002). Two of them Grapevineleafroll-associated virus 1 (GLRaV-1) and Grapevine leafroll-associated virus 3 (GLRaV-3) are the most important onesworldwide and also present and widespread in Croatian vine-yards. GLRaV-1 has a prevalent dominance in continentalregion while GLRAV-3 is most widespread in coastal region

(KAROGLAN KONTIC et al. 2009; VONCINAet al. 2009). The thirdmajor leafroll disease associated virus is GLRaV-2, to date onlyknown member of genus Closterovirus which infects grapevine(MARTELLI et al. 2002) and is transmissible by sap inoculationto herbaceous hosts (MONETTE and GODKIN 1993; BOSCIAet al.1995; GOSZCZYNSKI et al. 1996; ABOU GHANEM-SABANADZOVIC etal. 2000). Unlike GLRaV-1 and 3 for which vectors besidehuman are known (pseudococcid mealybugs and soft scaleinsects), to date other vectors of GLRaV-2 are unknown.Infected planting material still is the main way of its dissemina-tion over medium and long distances (MARTELLI and BOUDON-PADIEU 2006). The only known way to slow its dissemination,also suggested by the EPPO standard PM 4/8(2) (2008) deal-ing with instructions for production of healthy plants forplanting, is through national systems for the production of

certified grapevine planting material. Unfortunately, mainlydue to its unknown actual distribution in vineyards and nurs-eries and its negative impact on vine performance, GLRaV-2 isnot included in grapevine certification scheme in Croatia.

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Voncina et al.: Distribution and molecular characterization of GLRaV-2 in Croatian grapevine germplasm 195


According to current Croatian legislation (Pravilnik o stavl-janju na trite materijala za vegetativno umnaanje loze,Official Gazzette of the Republic of Croatia No. 133/2006)mother plants in nurseries must be tested on four viruses:Arabis mosaic virus (ArMV), Grapevine fanleaf virus (GFLV),GLRaV-1 and GLRaV-3, rootstocks in addition also for pres-ence ofGrapevine fleck virus (GFkV).

Recently significant negative impact of GLRaV-2 on vigour,yield and fruit quality of the French cultivar Chardonnay wasreported (KOMAR et al. 2007). Beside this GLRaV-2 hasnegative impact on production of planting material throughgraft-incompatibility syndrome observed especially in case ofcertain scion-rootstock combinations (GREIF et al. 1995;MONIS and BESTWICK1997, PIROLO et al. 2006). In California avirus with genome structure closely resembling that of GL-RaV-2 was reported as a causal agent of young vines decline ofthe very popular table grapevine cultivar Redglobe grafted ondifferent rootstocks (UYEMOTO et al. 2001; ROWHANI et al.2002).

According to recent field observations, symptoms thatcould be attributed to GLRaV-2 infection (downward leaf roll-ing, premature leaf yellowing/reddening, noticeable swell-ings in graft region) were detected especially in the vineyards

planted with autochthonous cultivars which are located insouthern part of Croatian coastal region. The observed symp-toms and their frequency initiated an extensive research withthe aim to investigate the presence and the potential frequen-cy of GLRaV-2 in commercial vineyards located in this region.One of the main problems that Croatian winegrowers arefaced with is the fact that certified planting material of autoch-thonous cultivars is not available. This fact probably furtherreduces the interest of growers in rare autochthonous culti-vars. Consequently, most planting material is produced bycollecting bud-wood from vines with good agronomic traits,but unknown virus status, grown in commercial vineyards.

2 Materials and methods

2.1 Plant material

The research was conducted on 13 Croatian autochthonousgrapevine cultivars or cultivars which are supposed to beautochthonous: Babica, Babi , Glavinua, Grk, Ljutun, Ma-ratina, Mladinka, Nin ua, Plavina, Plavac mali, Poip, Vlakaand Vugava. Collecting of well wooded dormant cuttings wasperformed during October 2007 from 50 vineyards located inthe southern part of Croatian coastal region (Dalmatia). Inmost cases plants included in investigation were selected dueto their good agronomical traits (good yield, high sugar con-tent, preferable cluster shape, tolerance to grey mould Botrytis cinerea etc.). In addition, after obtaining the results ofELISA test for the cultivar Babi collected during the autumn

2007, next year during August in one vineyard with detectedoccurrence of GLRaV-2 six new samples from untested vineswere taken. Those samples were chosen according to some ofthe symptoms on already tested plants and symptoms that are,according to literature, often noticed in vines infected withGLRaV-2. The aim of this trial was to correlate expressedsymptoms with presence of GLRaV-2. In ELISA-testing falsenegative results (due to uneven distribution of virus in theplant) were tried to be avoided by taking at least three wellwooded cuttings from the different basal parts of each vineincluded in the investigation. Collected samples were labeled,sealed in plastic bags and stored at 4C until testing whichwas preformed within a period of 3 months after collecting.

2.2 Virus detection

Using ELISA-test each grapevine sample was tested for the pres-ence of nine viruses Arabis mosaic virus (ArMV), Grapevine

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fleck virus (GFkV), Grapevine fanleaf virus (GFLV), Grapevineleafroll-associated virus 1 (GLRaV-1), Grapevine leafroll-asso-ciated virus 2 (GLRaV-2), Grapevine leafroll-associated virus 3(GLRaV-3), Grapevine leafroll-associated virus 7 (GLRaV-7),Grapevine virus A (GVA) and Grapevine virus B (GVB). Detec-tion was performed using reagents produced by Agritest(Valenzano, Italy) according to manufacturers instructions (ifnot stated otherwise). Phloem scrapings obtained from col-lected dormant cuttings were used as potential source of virus.To obtain average sample and reduce possibility of false nega-tive results scraping from each cutting in sample were mixedin total amount of 0.2 g, put in mortar and by using liquidnitrogen pulverized with pestle to fine powder. The powderwas diluted with 3 ml of grapevine extraction buffer. Readingof results was done on the EL800 spectrophotometer (BioTek,Winooski, VT, USA) at a wavelength of 405 nm two hoursafter adding the enzyme substrate, p-nitrophenylphosphate(Sigma, Ronkonkoma, NY, USA).

Total number of 16 ELISA positive grapevine accessionswere selected according to their different or similar geo-graphic origin and their cuttings were self-rooted to obtainmaterial for mechanical transmission and viral detection byRT-PCR. For detection by RT-PCR total RNA was extracted

from the petioles of young leaves using RNeasy Plant minikit (Qiagen, Hilden, Germany) according to manufacturersinstructions. RT-PCR was performed using Qiagen OneStepRT-PCR kit (Qiagen) using primer pair GLR2CP1/GLR2CP2(ABOU GHANEM-SABANADZOVIC et al. 2000). cDNA synthesis andDNA amplification was conducted in 25 l reaction volume inan Eppendorf Mastercycler (Eppendorf, Hamburg, Germany):2 l of total RNA was mixed with 23 l of the reaction mixturecontaining 5 l Qiagen OneStep RT-PCR buffer, 2 l dNTPmix, 1 l of Qiagen Onestep RT-PCR enzyme mix, 5 l ofQ-solution and 0.6 M of each primer. Reverse transcriptionwas done at 47C for 50 min, initial PCR activation step at95C for 15 min followed by 35 cycles of DNA amplification:denaturation at 94C for 30 s, annealing at 52C for 30 s,primer extension at 72C for 60 s (10 min for the last cycle).

PCR products were analyzed by electrophoresis on 1.5%agarose gel in 1X TBE buffer (90 min on 70 V), stained withethidium bromide and visualized under UV transilluminator.

2.3 Sequencing and analysis of RT-PCR products

RT-PCR products obtained from four geographically distantgrapevine accessions were selected for sequencing. They werepurified using GenElute PCR clean-up kit (Sigma) accordingto manufacturer's instructions and then sequenced in bothdirections using BigDye Terminator v3.1 Cycle Sequencing Kit(Applied Biosystems, Carlsbad, CA, USA). To prepare sequenc-ing mix BigDye terminator sequencing buffer was used fordilution of BigDye Terminator v3.1 Ready Reaction Mix in

recommended ratio (4:1). Sequencing was done in 7 l reac-tion volume (2 l of cDNA, 2 l of previously preparedsequencing mix, 1 l of primer (3.2 pmol) and 2 l of ultrapure water) in an Eppendorf Mastercycler (Eppendorf) usingfollowing cycling parameters: initial denaturation at 96C for1 min followed by 35 cycles of DNA multiplication (denatur-ation at 95C for 20 s, annealing of primers at 55C for 15 sand elongation at 60C for 4 min). After sequencing reactionPCR products were purified using BigDye XTerminator purifi-cation kit (Applied BioSystems) according to manufacturer'sinstructions and sequencing was conducted using ABI 3130Genetic Analyzer (Applied Biosystems). The sequences wereedited using the BioEdit Sequence Aligment Editor ver. 7.0.9.0(HALL 1999) and with help of the ClustalW program (H IGGINSet al. 1994) aligned with each other as well as with the se-

quence of most similar completely sequenced isolate 93/955(accession number AY881628.1) obtained from the Nationalcenter for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov).


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196 Voncina et al.: Distribution and molecular characterization of GLRaV-2 in Croatian grapevine germplasm


2.4 Mechanical transmission to Nicotiana benthamiana

According to results of ELISA and RT-PCR the 16 positive, pre-viously self rooted grapevine accessions were selected formechanical transmission. During vegetation period in 2009from each potted grapevine plant petioles and main vein fromyoung leaves were used as a source of inoculum for transmis-sion to herbaceous host (Nicotiana benthamiana). The inocu-lum from each sample was prepared in three ways:

1) according to protocol described by GOSZCZYNSKI et al.(1996) with the only exception that high speed centrifugationwas done at a 25 000 rpm (59584 rotor, MSE, England) for 3 h.The pellet was dissolved in 0.7 ml of inoculation buffer (BOSCIAet al. 1993) and immediately inoculated on carborundumdusted leaves ofN. benthamiana plants (stage 46 leaves),

2) pellet was prepared as described before, covered with0.7 ml of mentioned inoculation buffer and left overnight at4C to separate the pellet from the centrifugation tube walland inoculated next day,

3) using 1 g of young leaves petioles tissue which wasgrounded in a mortar and dissolved in 3 ml of previously men-tioned inoculation buffer and inoculated on carborundumdusted leaves of test plants.

The same procedure was repeated three times during thevegetation period (in March, May and September) and inoc-ulated plants were observed for appearance of symptomsduring a 90 days period after inoculation. Plants in whichappeared any kind of symptoms (in comparison to negativecontrol) were tested for the presence of mechanical transmis-sible grapevine viruses (GLRaV-2, ArMV, GFLV, GVA, GVB) byRT-PCR using procedure as described previously. Electro-phoresis and visualization of RT-PCR products were per-formed as described before.

3 Results and Discussion

The total number of 1100 samples were collected and ana-

lyzed by ELISA-test. GLRaV-2 was not detected in seven culti-vars: Grk (analyzed 70 samples from three different vineyardson the Island Kor ula), Ljutun (85 samples, three vineyards atKatela), Mladenka (60 samples, two vineyards at Katela),Nin ua (24 samples, two vineyards at Katela), Plavina (10samples from the Island Kor ula, 10 samples from ibenik),Poip (90 samples, four vineyards on the Island Kor ula),Vlaka (49 samples, two vineyards at Katela).

In the other six varieties GLRaV-2 was detected at a mini-mum infection level of 0.8% in the cultivar Vugava, while themost frequently infected cultivar was Babi with an infectionrate of 33.7% (Table 1). Presence of GLRaV-2 was determinedin the 45 grapevine accessions (4.1%) out of mentioned 1100samples. Such infection rate is pretty high in comparison withthe sanitary status of autochthonous Italian cultivars 2.6%

(CREDI et al. 2003), grapevines in Turkey 2.41% (AKBAS et al.2007) or Egypt 1.4% (AHMED et al. 2004), but it must be stat-ed that 33 out of 45 ELISA positive samples (73.3%) belong toonly one cultivar Babi . But still the infection rate withGLRaV-2 is much lover than that determined in Palestine 8.3% (ALKOWNI et al. 1998) or Syria 9% (MSLMANIEH et al.2006) and it is similar to the infection rate determined in theneighbor country Slovenia on the autochthonous cultivarRefosk where old, non selected plants had an infection inci-dence of 3.4% (TOMAZIC et al. 2005) The investigated viticul-tural area and locations where GLRaV-2 was found accordingto results of Elisa-test are given in Fig. 2.

Almost in all infected plants symptoms were expressed asearlier leaf reddening, slightly downward leaf rolling, remark-able difference in diameter of rootstock and scion, but those

symptoms were also detected in most of the plants in whichGLRaV-2 was not detected. Unfortunately in all plants collectedduring 2007 there was no vine infected with GLRaV-2 only. Allplants (except two) were also infected with GLRaV-3 and more

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than 70% of them were infected with GVA too (Table 2).Because of the very limited number of samples analyzed, it wasimpossible to find a correlation between the observed symptomsand the virus type, but according to limited results we assumethat possible causal agent of leaf abnormalities is GLRaV-3 orsome other virus which presence was not tested. Since in plantsinfected only with GLRaV-3 and plants infected with combina-tion of GLRaV-3 + GLRaV-2 or GLRaV-3 + GVA different severityof symptoms was observed, it could be possible that GLRaV-2and GVA may have a role in intensifying the observed symptoms.Changes on wood in form of different rootstock and scion dia-meter were observed in some plants infected with GLRaV-2, butalso on plants free of this virus. Cases of vines infected withGLRaV-2 but without such changes were also not rare.

In 2008 investigation was concentrated on the most infect-ed cultivar Babi and we searched for reasons of such highinfection rate. In 2007 grapevine accessions of cultivar Babifrom Primoten vineyard 2 were selected as part of clonalselection project, but after positive ELISA-test results in 2008six new, untested plants were sampled bases on their typicalGLRaV-2 symptoms. The presence of the investigated viruswas detected in three out of six tested samples (50%) and allof them were also positive of GLRaV-3 but free of GVA. In the

mentioned vineyard changes on wood in form of swellings ofthe graft union were very common, but since scions in thisvineyard were green grafted on the rootstock Rupestris du Lotin situ, the mentioned wood changes may also have their ori-gin in the early stages of vine development when differentrootstock and scion growth occurs and can cause such swell-ings. According to literature in most cases those abnormalitiesdo not obstruct normal functioning of the whole plant (HUGLINand SCHNEIDER1998). Investigating the source of bud-wood wefound out that the vineyards 1 and 3 in Primoten wereestablished 30 years ago and some of their vines with goodagronomic traits were used as a source of bud-wood for theestablishment of the vineyard 2 in Primoten and the vineyard1 in ibenik, planted in 1998 and 2000, respectively. Even smallnumber of tested samples of the Primoten vineyards 1 and 3

revealed a very high infection rate in the original (50% and18.2% respectively) and in the newly established vineyards (inboth 33.3%) is most likely caused by using infected scions takenfrom the old vineyards. This clearly demonstrates the main wayof GLRaV-2 spreading into new vineyards and the importanceof using virus-tested certified propagation material.

Detection of GLRaV-2 from 16 ELISA positive accessions byRT-PCR using primer pair GLR2CP1/GLR2CP2 was successful

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Fig. 1: Agarose gel electrophoresis showing results ofRT-PCR detection of GLRaV-2 using primer pair GLR2CP1/GLR2CP2. Total RNA was extracted from petioles of youngleaves taken from 16 different grapevine accessions (lines 1

16).Expected size of amplified fragment was 600 bp. Line: M marker (100 bp ladder, Intron Biotechnology, Seongnam,South Korea), P positive control, N negative control (extrac-tion from healthy grapevine).


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in all samples resulting in the single band of approximately600 bp (Fig. 1). RT-PCR products from grapevine accessionsBabi 051 (BAB-051), Maratina 039 (MAR-039), Plavac mali083 (PMC-083) and Glavinusa 110 (GLA-110) were sequencedin both directions. The analyses of the molecular variability ofthe part of viral genome which codes for the part of coat protein(CP) revealed a very low level of nucleotide sequence diversity

among the isolates (between 97.9% and 99.6% sequence iden-tity). A total of 14 nucleotide differences between the mentionedfour isolates were found with PMC-083 being the most divergenthaving nine single nucleotide differences in its sequence. Chang-

ces from pyrimidine base (cytosine, guanine) to purine base(adenine, thymine) were present in 86% differences, purine topyrimidine in 7% and purine to purine in 7%. All but one basepair substitution were on the third codon position. Only on posi-tion 439 the first base in codon was different. However, differ-ences on nucleotide level did not result in changes on the aminoacid level, resulting in identical amino acid sequence in all sam-

ples. While the cause of the mentioned nucleotide differencesremain unknown, possible explanation is that they could be theresult of reverse transcriptase or Taq DNA polymerase substitu-tion errors as described in BRACHO et al. (1998).

Table 1: List of Croatian autochthonous grapevine cultivars or cultivars which are supposed to be autochthonous, locations ofsampling, number of analyzed samples and number of vines which were, according to results of ELISA-test, infected withGLRaV-2. Mentioned are only cultivars in which at least one positive sample was found.

Grapevine cultivarLocation of vineyard

No. of analyzed samples No. of infected samples Infection rate (%)

Babi 98 33 33.7

Primoten, vineyard 1 12 6 50Primoten, vineyard 2 51 17 33.3

45 (2007) + 6 (2008) 14 (2007) + 3 (2008)

Primoten, vineyard 3 11 2 18.2

ibenik, vineyard 1 24 8 33.3

Babica 90 1 1.1

Katela, vineyard 1 16 1 6.3

Katela, vineyard 2 64 0 0

Katela, vineyard 3 10 0 0

Glavinusa 16 2 12.5

Katela, vineyard 4 16 2 12.5

Maratina 88 1 1.1

Island Svetac, vineyard 1 10 0 0Island Kor ula, vineyard 1 37 1 2.7

Island Hvar, vineyard 1 13 0 0

ibenik, vineyard 1 10 0 0

Oklaj, vineyard 1 18 0 0

Plavac mali 284 7 2.5

Island Vis, vineyard 1 26 0 0


Island Svetac, vineyard 1 8 0 0

Island Kor ula, vineyard 2 3 0 0

Island Kor ula, vineyard 3 8 1 12.5

Island Kor ula, vineyard 4 18 2 11.1

Peninsula Peljeac, vineyard 1 17 0 0



Peninsula Peljeac, vineyard 4 19 1 5.3





Island Hvar, vineyard 2 20 1 5.0

Island Hvar, vineyard 3 21 2 9.5

Island Hvar, vineyard 4 13 0 0

Vugava 126 1 0.8


Island Vis, vineyard 4 17 0 0Island Vis, vineyard 5 21 0 0


Island Vis, vineyard 7 25 1 4.0

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Nucleotide similarity with completely sequenced GLRaV-2

isolate 93/955 from NCBI database was between 91.8 and92.1% while amino-acid similarity with all Croatian isolateswas 96.2%. The mentioned sequences of the Croatian isolatesare accessible in the NCBI database under the following num-

bers: BAB-051 HM185275, MAR-039 HM185276, PMC-

083 HM185277 and GLA-110 HM185278.Although transmission tryouts from grapevine toN. bentha-

miana were carried out at three times during one year usingrelatively large number of test plants (20 per grapevine acces-

Table 2: Viruses recorded by ELISA-test from cortical shavings taken from different grapevine accessions infected with GLRaV-2.Some of the samples were also tested by RT-PCR using primer pair GLR2CP1/GLR2CP2 and petioles of young leaves as a sourcefor isolation of total RNA.

Grapevineaccession

Location ArMV GFLV GFkV GLRaV-1 GLRaV-2 GLRaV-3 GLRaV-7 GVA GVB RT-PCR

Babi 009 Primoten, vineyard 2 + + + + +

Babi 010 Primoten, vineyard 2 + + +Babi 012 Primoten, vineyard 2 + + + nt

Babi 019 Primoten, vineyard 2 + + + nt


Babi 028 Primoten, vineyard 2 + + + + nt


Babi 034 Primoten, vineyard 2 + + +

Babi 037 Primoten, vineyard 2 + + nt





Babi 045 Primoten, vineyard 2 + + + ntBabi 051 Primoten, vineyard 3 + + + + +

Babi 055 Primoten, vineyard 1 + + + +


Babi 058 Primoten, vineyard 1 + + + +




Babi 068 ibenik, vineyard 1 + + + + nt


Babi 072 ibenik, vineyard 1 + + + +


Babi 077 ibenik, vineyard 1 + + + + + nt

Babi 081 ibenik, vineyard 1 + + + +

Babi 082 ibenik, vineyard 1 + + + nt

Babi 086 ibenik, vineyard 1 + + + nt

Babi 105 Primoten, vineyard 2 (2008) + + + nt

Babi 106 Primoten, vineyard 2 (2008) + + +

Babi 108 Primoten, vineyard 2 (2008) + + + nt

Babi 112 Primoten, vineyard 3 + + +

Babica 115 Katela, vineyard 1 + + + + +

Glavinusa 104 Katela, vineyard 4 + + + + +

Glavinusa 110 Katela, vineyard 4 + + + + +

Maratina 039 Island Kor ula, vineyard 1 + + + +

Plavac mali 083 Island Kor ula, vineyard 3 + + + +

Plavac mali 097 Island Kor ula, vineyard 4 + + + ntPlavac mali 103 Island Kor ula, vineyard 4 + + nt

Plavac mali 151 Peninsula Peljeac, vineyard 4 + + nt

Plavac mali 234 Island Hvar, vineyard 2 + + + +

Plavac mali 241 Island Hvar, vineyard 3 + + + + nt

Plavac mali 293 Island Hvar, vineyard 3 + + + + nt

Vugava 107 Island Vis, vineyard 7 + + + nt

Presence of virus: = negative, + = positive, nt = not tested.

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sion per trial), all tests were negative. Only in one N. bentha-miana plant infected directly with sap from grapevine acces-sion Glavinusa 110 without using ultracentrifugation symp-toms were observed similar to those reported by GLRaV-2(GOSZCZYNSKI et al. 1996; ABOU GHANEM-SABANADZOVIC et al.2000). Since this grapevine accession (according to the resultsof the ELISA-test) was apart from GLRaV-2 also infected withGVA which is also mechanically transmissible, we assume thatthose symptoms can be caused by both viruses or by oneof them. Conducting RT-PCR with primer pairs GLR2CP1/

GLR2CP2 for GLRaV-2 and H7038/C7273 for GVA (MAC-KENZIE 1997) resulted in a single band of approximate size of236 base pairs which confirmed the presence of GVA. Ob-served changes were expressed in form of vein yellowing onyoung leaves developed 30 days after inoculation and within2 days they evolved in vein necrosis. Later symptoms weredetected in form of curling and discernible reduction in size ofnewly developed leaves. These results endorse to the fact thattransmission of GLRaV-2, the only mechanically transmissiblemember of genus Closterovirus which infects grapevine, isvery difficult, demanding and depends on lots of different fac-tors and is certainly not a routine test.

Results reported by KOMAR et al. (2007) showed a signifi-cant impact of GLRaV-2 on vigour, yield, and fruit quality ofV. vinifera cv. Chardonnay and provide strong evidence for the

need to include this virus in clonal developing and certifica-tion programs. Although the presence of GLRaV-2 is wellknown in many viticultural regions and many countries world-wide, from our knowledge this is the first report of GLRaV-2 inCroatia. Importance of this discovery is even greater consider-ing increased interest among Croatian grapevine producers forestablishing new vineyards with autochthonous cultivars. Dueto the fact that GLRaV-2 is not included in Croatian grapevinecertification scheme and that in case of some autochthonousgrapevine cultivars number of potential mother plants is verynarrow (with unknown GLRaV-2 status) there is significantrisk of its spreading, while his real detrimental effect on Croat-ian autochthonous grapevine cultivars remains still unknown.On the other hand, although inclusion of GLRaV-2 in the devel-opment of clones certainly makes sense, in case of unique cul-

tivars certification system tightening could reduce availabilityof already narrow source of planting material. This can causeloss of interest for autochthonous cultivars among wine andplant material producers and also increase risk of genetic ero-

sion, so a better balanced strategy of plant health managementfor mother plants might be more appropriate.

Acknowledgements

This work was supported by the Croatian Ministry of Science,Education and Sport by the grants no. 178-1781844-2692,178-1781844-1925 and 178-1781844-2758.

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GLRaV-2 Distribution and Molecular Caracterization in Croatian Autochthonous Grapevine

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