Distribution and partial molecular characterization of ...

Journal of Plant Diseases and Protection, 117 (5), 194–200, 2010, ISSN 1861-3829. © Eugen Ulmer KG, Stuttgart

Distribution and partial molecular characterization of Grapevine 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 in kroatischem autochthonem Keimplasma von Weinreben (Vitis vinifera L.) gefunden wurde D. Voncina1,*, S. Simon2, E. Dermic1, B. Cvjetkovic1, I. Pejic2, E. Maletic3 & J. Karoglan Kontic3 Department of Plant Pathology, University of Zagreb Faculty of Agriculture, Zagreb, Croatia Department of Plant Breeding Genetics, Biometrics and Experimentation, University of Zagreb Faculty of Agriculture, Zagreb, Croatia Department of Viticulture and Enology, University of Zagreb Faculty of Agriculture, Zagreb, Croatia Corresponding author, e-mail [email protected]

1 2 3 *

Received 10 May 2010; accepted 7 June 2010

Abstract Study of Grapevine leafroll-associated virus 2 (GLRaV-2) distribution was conducted on 13 Croatian autochthonous grapevine cultivars or cultivars which are supposed to be autochthonous included in clonal selection: Babica, Babic, Glavinuša, Grk, Ljutun, Maraština, Mladinka, Nincuša, Plavina, Plavac mali, Pošip, Vlaška and Vugava. All of them are grown in the southern part of Croatian coastal region (Dalmatia). Sampling was done during autumn 2007, and only a few samples of cultivar Babic were also collected during 2008. All samples were tested for the presence of GLRaV-2 using DAS-ELISA test. Out of 1100 tested grapevine accessions, the presence of GLRaV-2 was determined in 45 samples (4.1%). No virus was detected in seven grapevine varieties (Grk, Ljutun, Mladenka, Nincuša, Plavina, Pošip, Vlaška). Minimal infection rate was determined in the cultivar Vugava (0.8%) while the most infected cultivar was Babi c in which out of 98 analyzed grapevine accessions 33 (33.7%) were positive. All virus-positive tested vines contained mixed infections of several viruses and no vine was infected by GLRaV-2 only. In 14 different, randomly selected grapevine accessions the presence of GLRaV-2 was also confirmed by RT-PCR using the GLR2CP1/GLR2CP2 primer pair. RT-PCR products from four different grapevine accessions were sequenced. Sequences of gene segment coding for the viral coat protein revealed between 97.9% and 99.6% similarity on the nucleotide level, with absolute similarity on amino-acid level. Sixteen grapevine accessions were selected for transmission to herbaceous host Nicotiana benthamiana, but GLRaV-2 was not successfully transmitted to this host. To our 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 über die Verbreitung von Grapevine leafroll-associated virus 2 (GLRaV-2), wurde an den folgenden 13 kroatischen autochtonen oder als autochton geltenden Rebsorten durchgeführt: Babica, Babic, Glavinusa, Grk, Ljutun, Maraština, Mladinka, Nincuša, Plavina, Plavac mali, Pošip, Vlaška und Vugava. Alle diese Rebsorten werden im südlichen Teil der kroatischen Küstenregion (Dalmatien) angebaut und es werden derzeit Klone von ihnen entwickelt. Die Probennahme wurde im Herbst 2007 vorgenommen, nur einige Proben von der Rebsorte Babic wurden erst im Laufe des Jahres 2008 genommen. Alle Proben wurden mittels des DAS-ELISA-Tests auf Anwesenheit von GLRaV-2 geprüft. Von den 1100 geprüften Weinreben wurde GLRaV-2 bei 45 Proben (4,1%) festgestellt. Bei sieben Rebsorten wurde dieses Virus nicht gefunden (Grk, Ljutun, Mladenka, Nincuša, Plavina, Pošip, Vlaška). Die nied-

rigste Infektionsrate wurde bei der Sorte Vugava (0,8%) festgestellt, während bei der meistinfizierten Sorte Babic von den 98 analysierten Proben 33 (33,7%) positiv waren. Alle positiv auf Viren getesteten Rebsorten waren mit verschiedenen Viren mischinfiziert; keine Rebsorte war ausschließlich mit GLRaV-2 infiziert. Bei den 14 verschiedenen, stichprobenweise selektierten Weinrebeproben wurde die Anwesenheit von GLRaV-2 im RT-PCR-Verfahren mittels GLR2CP1/GLR2CP2-Primer-Paaren bestätigt. RT-PCR-Produkte von vier verschiedenen WeinAkzessionen wurden sequenziert. Für das virale Hüllprotein kodierende Sequenzen waren auf der Nucleotidebene zu 97,7–99,6% und auf der Aminosäureebene zu 100% identisch. Sechzehn Weinrebeproben wurden für die Übertragung auf den Pflanzenwirt Nicotiana benthamiana selektiert, jedoch konnte GLRaV-2 nicht erfolgreich auf den genannten Wirt übertragen werden. Unseres Wissens ist dies der erste Nachweis von GLRaV-2 in Kroatien. Stichwörter: DAS-ELISA, Gensequenz, Hüllprotein, klonale Selektion, RT-PCR

1

Introduction

One of the economically most important and widespread complex of virus diseases is Grapevine leafroll. To date nine serologically distinct viruses named Grapevine leafroll-associated virus 1 to 9 have been associated with leafroll disease complex (MARTELLI et al. 2002). Two of them – Grapevine leafroll-associated virus 1 (GLRaV-1) and Grapevine leafrollassociated virus 3 (GLRaV-3) are the most important ones worldwide and also present and widespread in Croatian vineyards. GLRaV-1 has a prevalent dominance in continental region while GLRAV-3 is most widespread in coastal region (KAROGLAN KONTIC et al. 2009; VONCINA et al. 2009). The third major leafroll disease associated virus is GLRaV-2, to date only known member of genus Closterovirus which infects grapevine (MARTELLI et al. 2002) and is transmissible by sap inoculation to herbaceous hosts (MONETTE and GODKIN 1993; BOSCIA et al. 1995; GOSZCZYNSKI et al. 1996; ABOU GHANEM-SABANADZOVIC et al. 2000). Unlike GLRaV-1 and 3 for which vectors beside human are known (pseudococcid mealybugs and soft scale insects), to date other vectors of GLRaV-2 are unknown. Infected planting material still is the main way of its dissemination over medium and long distances (MARTELLI and BOUDONPADIEU 2006). The only known way to slow its dissemination, also suggested by the EPPO standard PM 4/8(2) (2008) dealing with instructions for production of healthy plants for planting, is through national systems for the production of certified grapevine planting material. Unfortunately, mainly due to its unknown actual distribution in vineyards and nurseries and its negative impact on vine performance, GLRaV-2 is not included in grapevine certification scheme in Croatia. J.Plant Dis.Protect. 5/2010

Voncina et al.: Distribution and molecular characterization of GLRaV-2 in Croatian grapevine germplasm According to current Croatian legislation (“Pravilnik o stavljanju na tržište materijala za vegetativno umnažanje 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 presence of Grapevine fleck virus (GFkV). Recently significant negative impact of GLRaV-2 on vigour, yield and fruit quality of the French cultivar Chardonnay was reported (KOMAR et al. 2007). Beside this GLRaV-2 has negative impact on production of planting material through graft-incompatibility syndrome observed especially in case of certain scion-rootstock combinations (GREIF et al. 1995; MONIS and BESTWICK 1997, PIROLO et al. 2006). In California a virus with genome structure closely resembling that of GLRaV-2 was reported as a causal agent of young vines decline of the very popular table grapevine cultivar Redglobe grafted on different rootstocks (UYEMOTO et al. 2001; ROWHANI et al. 2002). According to recent field observations, symptoms that could be attributed to GLRaV-2 infection (downward leaf rolling, premature leaf yellowing/reddening, noticeable swellings in graft region) were detected especially in the vineyards planted with autochthonous cultivars which are located in southern part of Croatian coastal region. The observed symptoms and their frequency initiated an extensive research with the aim to investigate the presence and the potential frequency of GLRaV-2 in commercial vineyards located in this region. One of the main problems that Croatian winegrowers are faced with is the fact that certified planting material of autochthonous cultivars is not available. This fact probably further reduces the interest of growers in rare autochthonous cultivars. Consequently, most planting material is produced by collecting 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 autochthonous grapevine cultivars or cultivars which are supposed to be autochthonous: Babica, Babic, Glavinuša, Grk, Ljutun, Maraština, Mladinka, Nincuša, Plavina, Plavac mali, Pošip, Vlaška and Vugava. Collecting of well wooded dormant cuttings was performed during October 2007 from 50 vineyards located in the southern part of Croatian coastal region (Dalmatia). In most cases plants included in investigation were selected due to their good agronomical traits (good yield, high sugar content, preferable cluster shape, tolerance to grey mould – Botrytis cinerea etc.). In addition, after obtaining the results of ELISA test for the cultivar Babic collected during the autumn 2007, next year during August in one vineyard with detected occurrence of GLRaV-2 six new samples from untested vines were taken. Those samples were chosen according to some of the symptoms on already tested plants and symptoms that are, according to literature, often noticed in vines infected with GLRaV-2. The aim of this trial was to correlate expressed symptoms with presence of GLRaV-2. In ELISA-testing false negative results (due to uneven distribution of virus in the plant) were tried to be avoided by taking at least three well wooded cuttings from the different basal parts of each vine included in the investigation. Collected samples were labeled, sealed in plastic bags and stored at 4°C until testing which was preformed within a period of 3 months after collecting.

2.2 Virus detection Using ELISA-test each grapevine sample was tested for the presence of nine viruses – Arabis mosaic virus (ArMV), Grapevine J.Plant Dis.Protect. 5/2010

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fleck virus (GFkV), Grapevine fanleaf virus (GFLV), Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine leafroll-associated 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). Detection was performed using reagents produced by Agritest (Valenzano, Italy) according to manufacturer’s instructions (if not stated otherwise). Phloem scrapings obtained from collected dormant cuttings were used as potential source of virus. To obtain average sample and reduce possibility of false negative results scraping from each cutting in sample were mixed in total amount of 0.2 g, put in mortar and by using liquid nitrogen pulverized with pestle to fine powder. The powder was diluted with 3 ml of grapevine extraction buffer. Reading of results was done on the EL800 spectrophotometer (BioTek, Winooski, VT, USA) at a wavelength of 405 nm two hours after adding the enzyme substrate, p-nitrophenylphosphate (Sigma, Ronkonkoma, NY, USA). Total number of 16 ELISA positive grapevine accessions were selected according to their different or similar geographic origin and their cuttings were self-rooted to obtain material for mechanical transmission and viral detection by RT-PCR. For detection by RT-PCR total RNA was extracted from the petioles of young leaves using RNeasy Plant mini kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. RT-PCR was performed using Qiagen OneStep RT-PCR kit (Qiagen) using primer pair GLR2CP1/GLR2CP2 (ABOU GHANEM-SABANADZOVIC et al. 2000). cDNA synthesis and DNA amplification was conducted in 25 µl reaction volume in an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany): 2 µl of total RNA was mixed with 23 µl of the reaction mixture containing 5 µl Qiagen OneStep RT-PCR buffer, 2 µl dNTP mix, 1 µl of Qiagen Onestep RT-PCR enzyme mix, 5 µl of Q-solution and 0.6 µM of each primer. Reverse transcription was done at 47°C for 50 min, initial PCR activation step at 95°C for 15 min followed by 35 cycles of DNA amplification: denaturation at 94°C for 30 s, annealing at 52°C for 30 s, primer extension at 72°C 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 with ethidium bromide and visualized under UV transilluminator.

2.3 Sequencing and analysis of RT-PCR products RT-PCR products obtained from four geographically distant grapevine accessions were selected for sequencing. They were purified using GenElute PCR clean-up kit (Sigma) according to manufacturer's instructions and then sequenced in both directions using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA, USA). To prepare sequencing mix BigDye terminator sequencing buffer was used for dilution of BigDye Terminator v3.1 Ready Reaction Mix in recommended ratio (4:1). Sequencing was done in 7 µl reaction volume (2 µl of cDNA, 2 µl of previously prepared sequencing mix, 1 µl of primer (3.2 pmol) and 2 µl of ultra pure water) in an Eppendorf Mastercycler (Eppendorf) using following cycling parameters: initial denaturation at 96°C for 1 min followed by 35 cycles of DNA multiplication (denaturation at 95°C for 20 s, annealing of primers at 55°C for 15 s and elongation at 60°C for 4 min). After sequencing reaction PCR products were purified using BigDye XTerminator purification kit (Applied BioSystems) according to manufacturer's instructions and sequencing was conducted using ABI 3130 Genetic Analyzer (Applied Biosystems). The sequences were edited using the BioEdit Sequence Aligment Editor ver. 7.0.9.0 (HALL 1999) and with help of the ClustalW program (HIGGINS et al. 1994) aligned with each other as well as with the sequence of most similar completely sequenced isolate 93/955 (accession number AY881628.1) obtained from the National center for Biotechnology Information (NCBI, www.ncbi.nlm. nih.gov).

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2.4 Mechanical transmission to Nicotiana benthamiana According to results of ELISA and RT-PCR the 16 positive, previously self rooted grapevine accessions were selected for mechanical transmission. During vegetation period in 2009 from each potted grapevine plant petioles and main vein from young leaves were used as a source of inoculum for transmission to herbaceous host (Nicotiana benthamiana). The inoculum 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 centrifugation was done at a 25 000 rpm (59584 rotor, MSE, England) for 3 h. The pellet was dissolved in 0.7 ml of inoculation buffer (BOSCIA et al. 1993) and immediately inoculated on carborundum dusted leaves of N. benthamiana plants (stage 4–6 leaves), 2) pellet was prepared as described before, covered with 0.7 ml of mentioned inoculation buffer and left overnight at 4°C to separate the pellet from the centrifugation tube wall and inoculated next day, 3) using 1 g of young leaves petioles tissue which was grounded in a mortar and dissolved in 3 ml of previously mentioned inoculation buffer and inoculated on carborundum dusted leaves of test plants. The same procedure was repeated three times during the vegetation period (in March, May and September) and inoculated plants were observed for appearance of symptoms during a 90 days period after inoculation. Plants in which appeared any kind of symptoms (in comparison to negative control) were tested for the presence of mechanical transmissible grapevine viruses (GLRaV-2, ArMV, GFLV, GVA, GVB) by RT-PCR using procedure as described previously. Electrophoresis and visualization of RT-PCR products were performed as described before.

3

Results and Discussion

The total number of 1100 samples were collected and analyzed by ELISA-test. GLRaV-2 was not detected in seven cultivars: Grk (analyzed 70 samples from three different vineyards on the Island Korcula), Ljutun (85 samples, three vineyards at Kaštela), Mladenka (60 samples, two vineyards at Kaštela), Nincuša (24 samples, two vineyards at Kaštela), Plavina (10 samples from the Island Korcula, 10 samples from Šibenik), Pošip (90 samples, four vineyards on the Island Korcula), Vlaška (49 samples, two vineyards at Kaštela). In the other six varieties GLRaV-2 was detected at a minimum infection level of 0.8% in the cultivar Vugava, while the most frequently infected cultivar was Babic with an infection rate of 33.7% (Table 1). Presence of GLRaV-2 was determined in the 45 grapevine accessions (4.1%) out of mentioned 1100 samples. Such infection rate is pretty high in comparison with the 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 stated that 33 out of 45 ELISA positive samples (73.3%) belong to only one cultivar – Babic. But still the infection rate with GLRaV-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 the neighbor country Slovenia on the autochthonous cultivar Refosk where old, non selected plants had an infection incidence of 3.4% (TOMAZIC et al. 2005) The investigated viticultural area and locations where GLRaV-2 was found according to results of Elisa-test are given in Fig. 2. Almost in all infected plants symptoms were expressed as earlier leaf reddening, slightly downward leaf rolling, remarkable difference in diameter of rootstock and scion, but those symptoms were also detected in most of the plants in which GLRaV-2 was not detected. Unfortunately in all plants collected during 2007 there was no vine infected with GLRaV-2 only. All plants (except two) were also infected with GLRaV-3 and more

than 70% of them were infected with GVA too (Table 2). Because of the very limited number of samples analyzed, it was impossible to find a correlation between the observed symptoms and the virus type, but according to limited results we assume that possible causal agent of leaf abnormalities is GLRaV-3 or some other virus which presence was not tested. Since in plants infected only with GLRaV-3 and plants infected with combination of GLRaV-3 + GLRaV-2 or GLRaV-3 + GVA different severity of symptoms was observed, it could be possible that GLRaV-2 and GVA may have a role in intensifying the observed symptoms. Changes on wood in form of different rootstock and scion diameter were observed in some plants infected with GLRaV-2, but also on plants free of this virus. Cases of vines infected with GLRaV-2 but without such changes were also not rare. In 2008 investigation was concentrated on the most infected cultivar Babic and we searched for reasons of such high infection rate. In 2007 grapevine accessions of cultivar Babic from Primošten vineyard 2 were selected as part of clonal selection project, but after positive ELISA-test results in 2008 six new, untested plants were sampled bases on their typical GLRaV-2 symptoms. The presence of the investigated virus was detected in three out of six tested samples (50%) and all of them were also positive of GLRaV-3 but free of GVA. In the mentioned vineyard changes on wood in form of swellings of the graft union were very common, but since scions in this vineyard were green grafted on the rootstock Rupestris du Lot in situ, the mentioned wood changes may also have their origin in the early stages of vine development when different rootstock and scion growth occurs and can cause such swellings. According to literature in most cases those abnormalities do not obstruct normal functioning of the whole plant (HUGLIN and SCHNEIDER 1998). Investigating the source of bud-wood we found out that the vineyards 1 and 3 in Primošten were established 30 years ago and some of their vines with good agronomic traits were used as a source of bud-wood for the establishment of the vineyard 2 in Primošten and the vineyard 1 in Šibenik, planted in 1998 and 2000, respectively. Even small number of tested samples of the Primošten vineyards 1 and 3 revealed a very high infection rate in the original (50% and 18.2% respectively) and in the newly established vineyards (in both 33.3%) is most likely caused by using infected scions taken from the old vineyards. This clearly demonstrates the main way of GLRaV-2 spreading into new vineyards and the importance of using virus-tested certified propagation material. Detection of GLRaV-2 from 16 ELISA positive accessions by RT-PCR using primer pair GLR2CP1/GLR2CP2 was successful

Fig. 1: Agarose gel electrophoresis showing results of RT-PCR detection of GLRaV-2 using primer pair GLR2CP1/ GLR2CP2. Total RNA was extracted from petioles of young leaves 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 (extraction from healthy grapevine). J.Plant Dis.Protect. 5/2010


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Table 1: List of Croatian autochthonous grapevine cultivars or cultivars which are supposed to be autochthonous, locations of sampling, number of analyzed samples and number of vines which were, according to results of ELISA-test, infected with GLRaV-2. Mentioned are only cultivars in which at least one positive sample was found. Grapevine cultivar Location of vineyard Babic Primošten, vineyard 1 Primošten, vineyard 2 Primošten, vineyard 3 Šibenik, vineyard 1 Babica Kaštela, vineyard 1 Kaštela, vineyard 2 Kaštela, vineyard 3 Glavinusa Kaštela, vineyard 4 Maraština Island Svetac, vineyard 1 Island Korc ula, vineyard 1 Island Hvar, vineyard 1 Šibenik, vineyard 1 Oklaj, vineyard 1 Plavac mali Island Vis, vineyard 1 Island Vis, vineyard 2 Island Svetac, vineyard 1 Island Korc ula, vineyard 2 Island Korc ula, vineyard 3 Island Korc ula, vineyard 4 Peninsula Pelješac, vineyard 1 Peninsula Pelješac, vineyard 2 Peninsula Pelješac, vineyard 3 Peninsula Pelješac, vineyard 4 Peninsula Pelješac, vineyard 5 Peninsula Pelješac, vineyard 6 Peninsula Pelješac, vineyard 7 Peninsula Pelješac, vineyard 8 Island Hvar, vineyard 2 Island Hvar, vineyard 3 Island Hvar, vineyard 4 Vugava Island Vis, vineyard 3 Island Vis, vineyard 4 Island Vis, vineyard 5 Island Vis, vineyard 6 Island Vis, vineyard 7

No. of analyzed samples

No. of infected samples

Infection rate (%)

98 12 51 45 (2007) + 6 (2008) 11 24 90 16 64 10 16 16 88 10 37 13 10 18 284 26 15 8 3 8 18 17 15 17 19 26 34 12 12 20 21 13 126 33 17 21 30 25

33 6 17 14 (2007) + 3 (2008) 2 8 1 1 0 0 2 2 1 0 1 0 0 0 7 0 0 0 0 1 2 0 0 0 1 0 0 0 0 1 2 0 1 0 0 0 0 1

33.7 50 33.3

in all samples resulting in the single band of approximately 600 bp (Fig. 1). RT-PCR products from grapevine accessions Babic 051 (BAB-051), Maraština 039 (MAR-039), Plavac mali 083 (PMC-083) and Glavinusa 110 (GLA-110) were sequenced in both directions. The analyses of the molecular variability of the 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 identity). A total of 14 nucleotide differences between the mentioned four isolates were found with PMC-083 being the most divergent having nine single nucleotide differences in its sequence. ChangJ.Plant Dis.Protect. 5/2010

18.2 33.3 1.1 6.3 0 0 12.5 12.5 1.1 0 2.7 0 0 0 2.5 0 0 0 0 12.5 11.1 0 0 0 5.3 0 0 0 0 5.0 9.5 0 0.8 0 0 0 0 4.0

es from pyrimidine base (cytosine, guanine) to purine base (adenine, thymine) were present in 86% differences, purine to pyrimidine in 7% and purine to purine in 7%. All but one base pair substitution were on the third codon position. Only on position 439 the first base in codon was different. However, differences on nucleotide level did not result in changes on the amino acid level, resulting in identical amino acid sequence in all samples. While the cause of the mentioned nucleotide differences remain unknown, possible explanation is that they could be the result of reverse transcriptase or Taq DNA polymerase substitution errors as described in BRACHO et al. (1998).

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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 source for isolation of total RNA. Grapevine accession

Location

Babic 009 Babic 010 Babic 012 Babic 019 Babic 025 Babic 028 Babic 033 Babic 034 Babic 037 Babic 038 Babic 039 Babic 041 Babic 044 Babic 045 Babic 051 Babic 055 Babic 057 Babic 058 Babic 060 Babic 061 Babic 062 Babic 068 Babic 070 Babic 072 Babic 074 Babic 077 Babic 081 Babic 082 Babic 086 Babic 105 Babic 106 Babic 108 Babic 112 Babica 115 Glavinusa 104 Glavinusa 110 Maraština 039 Plavac mali 083 Plavac mali 097 Plavac mali 103 Plavac mali 151 Plavac mali 234 Plavac mali 241 Plavac mali 293 Vugava 107

Primošten, vineyard 2 Primošten, vineyard 2 Primošten, vineyard 2 Primošten, vineyard 2 Primošten, vineyard 2 Primošten, vineyard 2 Primošten, vineyard 2 Primošten, vineyard 2 Primošten, vineyard 2 Primošten, vineyard 2 Primošten, vineyard 2 Primošten, vineyard 2 Primošten, vineyard 2 Primošten, vineyard 2 Primošten, vineyard 3 Primošten, vineyard 1 Primošten, vineyard 1 Primošten, vineyard 1 Primošten, vineyard 1 Primošten, vineyard 1 Primošten, vineyard 1 Šibenik, vineyard 1 Šibenik, vineyard 1 Šibenik, vineyard 1 Šibenik, vineyard 1 Šibenik, vineyard 1 Šibenik, vineyard 1 Šibenik, vineyard 1 Šibenik, vineyard 1 Primošten, vineyard 2 (2008) Primošten, vineyard 2 (2008) Primošten, vineyard 2 (2008) Primošten, vineyard 3 Kaštela, vineyard 1 Kaštela, vineyard 4 Kaštela, vineyard 4 Island Korc ula, vineyard 1 Island Korc ula, vineyard 3 Island Korc ula, vineyard 4 Island Korc ula, vineyard 4 Peninsula Pelješac, vineyard 4 Island Hvar, vineyard 2 Island Hvar, vineyard 3 Island Hvar, vineyard 3 Island Vis, vineyard 7

ArMV

GFLV

– – – – – – – – – – – – – – – – – – – – – – – – – + – – – – – – – – – – – – – – – – – – –

– – – – – – – – – – – – – – – – – – – – – – – – – + – – – – – – – – – – – + – – – – – – –

GFkV GLRaV-1 GLRaV-2 GLRaV-3 GLRaV-7 + – – – – + – – – – – – – – – – + – + – – + + – + – – – – + – + – + – – – – + – – – – – –

– – – – – – – – – – + – – – – – – – – – – – – – – – – – – – – – – + + + – + – + – – + + –

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + – + – + + + + +

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

GVA + – + + + + + – – + + + + + + + + + + + + + + + + + + + + – – – – – + + + – – – – + + + +

GVB RT-PCR – – – – – – – – – – – – – – + – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

+ + nt nt nt nt nt + nt nt nt nt nt nt + + nt + nt nt nt nt nt + nt nt + nt nt nt + nt + + + + + + nt nt nt + nt nt nt

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

Nucleotide similarity with completely sequenced GLRaV-2 isolate 93/955 from NCBI database was between 91.8 and 92.1% while amino-acid similarity with all Croatian isolates was 96.2%. The mentioned sequences of the Croatian isolates are accessible in the NCBI database under the following num-

bers: BAB-051 – HM185275, MAR-039 – HM185276, PMC083 – HM185277 and GLA-110 – HM185278. Although transmission tryouts from grapevine to N. benthamiana were carried out at three times during one year using relatively large number of test plants (20 per grapevine accesJ.Plant Dis.Protect. 5/2010


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Bjelovar

Fig. 2: Investigated viticultural area (grey) and spots where presence of GLRaV-2 was detected using ELISA-test.

sion per trial), all tests were negative. Only in one N. benthamiana plant infected directly with sap from grapevine accession Glavinusa 110 without using ultracentrifugation symptoms 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 results of the ELISA-test) was apart from GLRaV-2 also infected with GVA which is also mechanically transmissible, we assume that those symptoms can be caused by both viruses or by one of them. Conducting RT-PCR with primer pairs GLR2CP1/ GLR2CP2 for GLRaV-2 and H7038/C7273 for GVA (MACKENZIE 1997) resulted in a single band of approximate size of 236 base pairs which confirmed the presence of GVA. Observed changes were expressed in form of vein yellowing on young leaves developed 30 days after inoculation and within 2 days they evolved in vein necrosis. Later symptoms were detected in form of curling and discernible reduction in size of newly developed leaves. These results endorse to the fact that transmission of GLRaV-2, the only mechanically transmissible member of genus Closterovirus which infects grapevine, is very difficult, demanding and depends on lots of different factors and is certainly not a routine test. Results reported by KOMAR et al. (2007) showed a significant impact of GLRaV-2 on vigour, yield, and fruit quality of V. vinifera cv. Chardonnay and provide strong evidence for the need to include this virus in clonal developing and certification programs. Although the presence of GLRaV-2 is well known in many viticultural regions and many countries worldwide, from our knowledge this is the first report of GLRaV-2 in Croatia. Importance of this discovery is even greater considering increased interest among Croatian grapevine producers for establishing new vineyards with autochthonous cultivars. Due to the fact that GLRaV-2 is not included in Croatian grapevine certification scheme and that in case of some autochthonous grapevine cultivars number of potential mother plants is very narrow (with unknown GLRaV-2 status) there is significant risk of its spreading, while his real detrimental effect on Croatian autochthonous grapevine cultivars remains still unknown. On the other hand, although inclusion of GLRaV-2 in the development of clones certainly makes sense, in case of unique cultivars certification system tightening could reduce availability of already narrow source of planting material. This can cause loss of interest for autochthonous cultivars among wine and plant material producers and also increase risk of genetic eroJ.Plant Dis.Protect. 5/2010

sion, so a better balanced strategy of plant health management for 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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