625 Pise and Gaikwad.vp Acta Bot. Croat. 72 (2), 287–294, 2013 CODEN: ABCRA 25 ISSN 0365–0588 eISSN 1847-8476 DOI: 10.2478/botcro-2013-0007 Grapevine yellows affecting the Croatian indigenous grapevine cultivar Grk MARIN JE@I]1, JASMINKA KAROGLAN KONTI]2, DARKO PREINER2, EDI MALETI]2, MIRNA ]URKOVI]-PERICA1* 1 University of Zagreb, Faculty of Science, Division of Biology, Department of Microbio- logy, Marulicev trg 9a, Zagreb, Croatia 2 University of Zagreb, Faculty of Agriculture, Department of Viticulture and Enology, Svetosimunska 25, Zagreb, Croatia Abstract – The grapevine cultivar Grk, a close relative of Crljenak ka{telanski/Zinfandel, is grown exclusively in southern Croatia. Grapevine yellows-like symptoms were ob- served on vines in the vineyards in Lumbarda (southern Croatia) and in propagated grape- vines near Zadar and Zagreb. The majority of the detected phytoplasma isolates belonged to the 16SrI group. However, RFLP pattern and R16F2n/R2 fragment sequence assigned one isolate to the 16SrIII group. Thus far, on cv. Grk, phytoplasmas belonging to three dif- ferent groups have been detected: 16SrI, 16SrIII, and 16SrXII, which was confirmed previ- ously. Aside from the 16SrI, 16SrV and 16SrXII phytoplasma groups previously found on grapevines in Croatia, the finding of 16SrIII group, which is not common on grapevines in Europe, adds to the diversity of phytoplasmas in a very small geographic region. Key words: molecular detection, phytoplasma, RFLP, Vitis vinifera cv. Grk Introduction Grk is an old Croatian grapevine cultivar grown almost exclusively on the island of Kor~ula. Its name (Grk in Croatian language means Greek) and long cultivation suggests that the cultivar was brought from the east at the time of the Greek colonization of the island (4th century BC). However, because of its close genetic relationship with Crljenak ka{telan- ski/Zinfandel (as parent and progeny, respectively), and thus its relationship with a group of Dalmatian cultivars (PEJI] et al. 2000, MALETI] et al. 2004), it is most probable that cv. Grk arose in this area. The population of cv. Grk is rather small and the most productive vine- yards are located on sandy soils near the town of Lumbarda, where its grape achieves the highest quality and gives the famous wine quality. ACTA BOT. CROAT. 72 (2), 2013 287 * Corresponding author, e-mail: mirna.curkovic-perica@biol.pmf.hr Copyright® 2013 by Acta Botanica Croatica, the Faculty of Science, University of Zagreb. All rights reserved. In recent years a decrease in the productive potential of cv. Grk vines in Lumbarda re- gion has been observed. Symptoms like leaf folding, yellowing and drying long before the fall, poor fruit set (cv. Grk has female flowers) and yield lowering indicates either a physio- logical or a phytosanitary disorder of the vines. Thus, the cv. Grk population has been screened for the economically most important vi- ruses: Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Grapevine leafroll- -associated virus 1 (GLRaV-1) and Grapevine leafroll-associated virus 3 (GLRaV-3). Us- ing ELISA, testing of viruses was carried on 70 selected vines from five vineyards more than 15 years old, out of which 37 vines were free of the tested viruses (PREINER et al. 2010). These selected mother stocks of cv. Grk, were propagated and planted in a mother vineyard in Ba{tica near Zadar in year 2008 for further propagation and clonal selection. The ob- tained results have shown that infection of the cv. Grk population with those grapevine-as- sociated viruses tested for is much lower than the average for grapevine cultivars in Dalmatia,which reaches about 90% of virus-infected vines (KAROGLAN KONTI] et al. 2009). This indicated that there had to be some other cause of the symptoms observed in Lumbarda vineyards. One of the possible causes for the decline of the Lumbarda vineyards might be phloem- -restricted prokaryotes called phytoplasmas. Phytoplasmas are classified by their 16S ribo- somal gene into less then 20 clusters (MONTANO et al. 2001, IRPCM 2004, BERTACCINI and DUDUK 2009). Infected grapevines develop very characteristic symptoms – leafyellowing and curling, aborted clusters or shriveling berries, incomplete lignification of shoots, as well as vein necrosis (CONSTABLE 2010). Diseases known as »grapevine yellows« are caused by phytoplasmas from the groups 16SrI-B (aster yellows), 16SrI-C (clover phyllody), 16Sr-II (peanut witches' broom), 16SrIII (X-disease), 16SrV-C and 16SrV-D (elm yellows) and 16SrXII-A and 16SrXII-B (stolbur) (FIRRAO et al. 2005). Studies of grapevine yellows started in Croatia in the 1950s (PANJAN 1957), but the first molecular confirmation of the causal agent of the disease – phytoplasma – came later with [ARI] et al. (1997). Since then, a few studies on phytoplasma occurrence on grapevine in Croatian vineyards have been conducted ([ERUGA et al. 2000; [ERUGA MUSI] et al. 2009, 2011a, b; [KORI] et al. 1998, 2011), but only one of them included the testing of one indige- nous cv. Grk vine from Lumbarda in Kor~ula (MIKEC et al. 2006). The aim of this research was (i) to confirm phytoplasmas as the causal agent of the grapevine yellows-like symptoms on cv. Grk vines, (ii) to determine the disease prevalence at three locations where this cultivar is planted in Croatia and (iii) to determine the diversity of the suspected pathogen (phytoplasmas). Materials and methods Samples of leaf main veins were collected in late summer of 2010 at three locations in Croatia where Grk grapevines can be found: production vineyards in Lumbarda on island Kor~ula with 10,023 plants, the National Grapevine Collection in Jazbina near Zagreb (six plants) and a mother vineyard in Ba{tica near Zadar (400 plants) (Fig. 1). The disease symp- toms were assessed on site and samples for further analysis chosen accordingly: from 11 plants with pronounced symptoms in Lumbarda and five plants with mild symptoms in Ba{tica. Only two plants showed mild symptoms in Jazbina, while the rest were asymptom- 288 ACTA BOT. CROAT. 72 (2), 2013 JE@I] M., KAROGLAN KONTI] J., PREINER D., MALETI] E., ]URKOVI]-PERICA M. atic. Therefore, in that location, the three asymptomatic plants were also included in the analysis. The procedure for total nucleic acid extraction was performed as previously described by [ERUGA et al. (2003). DNA concentration was determined using NanoDrop (Themo Fisher Scientific 2000) and adjusted to 20 ng mL–1 for the polymerase chain reaction (PCR). Direct amplification of phytoplasma 16S rDNA, was performed with a R16F1/R0 primer pair (LEE et al. 1995), and was followed by nested PCR assay, utilizing a R16F2n/R2 primer pair (GUNDERSEN and LEE 1996) with 0.5 mL from the first PCR reaction used as a template (making the template 50 x diluted in the nested PCR reaction mixture). In all experiments positive (DNA isolated from various phytoplasma strains maintained in Catharanthus roseus grown in vitro) as well as negative (water and healthy C. roseus DNA isolate) con- trols were used. Approximately 5 mL of PCR mixture were analyzed on 1% agarose gel, stained with ethidium bromide and visualized with UV. All experiments were done in tripli- cates. Restriction fragment length polymorphism (RFLP) analysis was performed on R16 F2n/R2 primer pair obtained amplicons. Besides isolates from Lumbarda, Ba{tica and Jazbina, reference strains of phytoplasmas (maintained in C. roseus plants) were included in RFLP analysis: HYDB ('Candidatus Phytoplasma asteris', 16SrI-B), KVI (Peach X disease, 16SrIII-B), FD (Flavescence dorée, 16SrV-C), GSFY/2 ('Ca. P. prunorum', 16SrX-B), SA-1 (Stolbur, 16SrXII-A). Using restriction endonucleases MseI, AluI and KpnI, digestion was performed on 200 ng of DNA according to manufacturer instructions (Fermentas, Vilnius, Lithuania) and 10 mL of reaction mixture was loaded on 5% polyacrilamide gel electropho- ACTA BOT. CROAT. 72 (2), 2013 289 PHYTOPLASMAS IN GRAPEVINE Fig. 1. Geographical locations in Croatia from which the grapevine samples were collected. resis (PAGE) vertical gels. Ethidium bromide-stained gels were photographed and restric- tion fragment patterns of phytoplasma isolates from cv. Grk were compared with patterns of standard phytoplasma strains. Amplicon (obtained with R16F2n/R2 primer pair) from sample 02L was sequenced us- ing Macrogen DNA sequencing service (Macrogen, Amsterdam, Netherlands). Results In Lumbarda on Kor~ula, 79% of 10,023 vines showed pronounced disease symptoms while 11% had mild symptoms. The rest of the vines were asymptomatic. The vineyard in Ba{tica near Zadar is much smaller, and only mild grapevine yellows symptoms were ob- served on eight out of 400 plants. In a vineyard in Jazbina near Zagreb, which serves as the national collection of grapevine cultivars, only six cv. Grk vines are grown and mild symp- toms were observed on two vines. 290 ACTA BOT. CROAT. 72 (2), 2013 JE@I] M., KAROGLAN KONTI] J., PREINER D., MALETI] E., ]URKOVI]-PERICA M. Tab. 1. Detection of phytoplasmas belonging to two different ribosomal groups in cv. 'Grk' samples collected from three locations in Croatia and expressing different symptom intensity. Pro- nounced symptoms are indicated with (++), mild symptoms with (+) and symptomless grapevines with (–) in the third column. Fourth and fifth column indicate presence or absence of specific amplicon revealed by agarose gel electrophoresis after the direct and nested PCR. Location Sample Symptoms Direct PCR R16F1/R0 Nested PCR R16F2n/R2 Phytoplasma ribosomal group Lumbarda (Kor~ula) 01L ++ – + 16SrI 02L ++ – + 16SrIII 23L ++ – + 16SrI 04L ++ – + 16SrI 05L ++ – + 16SrI 06L ++ – + 16SrI 07L ++ – + 16SrI 08L ++ – + 16SrI 09L ++ – + 16SrI 10L ++ – + 16SrI 11L ++ – + 16SrI Jazbina (Zagreb) 1J + – + 16SrI 2J + – + 16SrI 3J – – + 16SrI 4J – – + 16SrI 5J – – + 16SrI Ba{tica (Zadar) 170 + – + 16SrI 289 + – + 16SrI 290 + – + 16SrI 369 + – + 16SrI 370 + – – N/A In the direct PCR, only positive controls produced visible amplicons and therefore nested PCR was performed yielding 20 positive samples out of 21 tested. DNA fragments of expected length (1.2 kb) were obtained. In all our experiments, only one sample from Ba{tica was consistently negative, despite the mild grapevine yellows-like symptoms ob- served on that tested vine. On the other hand, phytoplasma was detected in 3 asymptomatic vines from Jazbina (Tab. 1). In RFLP analysis we digested all DNA products obtained from the first nested PCR. All RFLP patterns were consistent with 16SrI phytoplasma group, except for sample 02L (Lumbarda) which had a 16SrIII-like pattern (Fig. 2). The partial sequence obtained for that sample (JQ046414) was very similar to the Italian 16SrIII-B group phytoplasma 16SrDNA sequence available in the database (HQ589194) with only one nucleotide mismatch. There were four nucleotide mismatches between our 16SrDNA sequence and the sequence of an isolate belonging to16SrIII from the USA (AF060875) (DAVIS et al. 1998). ACTA BOT. CROAT. 72 (2), 2013 291 PHYTOPLASMAS IN GRAPEVINE Fig. 2. Restriction fragment length polymorphism patterns of phytoplasmas detected in Croatian vineyards with additional five standard strains as references. Amplicons obtained with R16F2n/R2 were digested with AluI, KpnI and MseI restriction enzymes over night and ana- lyzed on 5% polyacrylamide gels. Marker jX174 HaeIII digest. Samples: 02L and 11L (Lumbarda), 2J (Jazbina), 170 (Ba{tica); reference strains: HYDB ('Candidatus Phytoplasma asteris', 16SrI-B), KVI (Peach X disease, 16SrIII-B), FD (Flavescence dorée, 16SrV-C), GSFY/2 ('Ca. P. prunorum', 16SrX-B), SA-1 (Stolbur, 16SrXII-A). Discussion Phytoplasmas were detected in all locations included in our study, in plants with pro- nounced and also in plants without any visible disease symptoms. In samples from Lumbarda, pronounced symptoms, observed on 79% of the cv. Grk vines, were unambigu- ously connected with phytoplasma infection. Such prevalence of the disease on a high num- ber of vines might lead to wine production from this particular cultivar in southern Croatia becoming inefficient. Even more, in the tested vines sampled from two other locations, the same phytoplasma was detected even in asymptomatic or vines with only mild grapevine yellows-like symptoms. This is the first study of the prevalence of phytoplasmoses on the grapevine cv. Grk after the initial report of one vine of this cultivar sampled in Lumbarda, which was infected by phytoplasma from 16SrXII group (MIKEC et al. 2006). Infections of grapevines with phytoplasmas belonging to three groups 16SrXII, 16SrI and 16SrV were reported in Croatia previously (MIKEC et al. 2006; [ERUGA et al. 2000; [ERUGA MUSI] et al. 2009, 2011a, b; [KORI] et al. 1998, 2011). Phytoplasma belonging to group 16SrV was detected recently in two vineyards in north-western continental Croatia, near the Slovenian-Croatian border ([ERUGA MUSI] et al. 2011b); 16SrXII was found to be widely distributed, in both continen- tal and littoral Croatia while 16SrI was detected only sporadically, mostly in southern Croatia (MIKEC et al. 2006), the geographical origin of cv. Grk vines. Phytoplasmas from this group were reported previously on some other grapevine cultivars in southern littoral Croatia, namely in ^ara on island of Kor~ula and on the nearby Pelje{ac peninsula (MIKEC et al. 2006), but neither in northern Dalmatia (where our samples from Ba{tica were collected) nor in Jazbina near Zagreb. Therefore it is an interesting finding that all tested cv. Grk vines from three locations were infected by the phytoplasma belonging to the 16SrI group. Since grapevines from Lumbarda were transplanted to Ba{tica near Zadar and Jazbina near Zagreb for enological studies, there is a high possibility that grapevines were infected be- fore they were transplanted. Another possibility is a novel infection of Grk cultivar grape- vines, initiated by insect vectors in our research areas. However, in that case infections by the much more widespread 16SrXII group phytoplasma would be more probable. Yet in Jazbina in continental Croatia where infections by phytoplasmas belonging to 16SrXII were previously repeatedly confirmed on other cultivars ([ERUGA 2003, [ERUGA et al. 2000, [ERUGA MUSI] et al. 2009), on cv. Grk only phytoplasma belonging to 16SrI group was found. RFLP pattern and sequence of R16F2n/R2 obtained amplicon of one sample affiliated that phytoplasma to a distinctive 16SrIII phytoplasma group, rather rare and uncommon on grapevine in Europe. Phytoplasmas from this group were found to infect grapevines in USA, Israel and Northern Italy (MUSETTI 2008). Phytoplasma belonging to 16SrIII group was found previously in Croatia on a weed plant – Chenopodium album near Drni{ ([ERUGA 2003), but this is the first report on Vitis vinifera infected with phytoplasma belonging to this particular group. This phytoplasma was found in a single grapevine only in southern Croatia, on a geographically small and isolated area. Phytoplasmas that belong to ribosomal groups 16SrI and 16SrIII infect a broad range of plant hosts. Therefore, there is a high probability that other plant species in Croatia might be infected with these phytoplasmas as well. 292 ACTA BOT. CROAT. 72 (2), 2013 JE@I] M., KAROGLAN KONTI] J., PREINER D., MALETI] E., ]URKOVI]-PERICA M. Conclusion This study has shown an unusually high prevalence of the 16SrI group phytoplasma on cv. Grk in all three geographically distant locations where it is planted. This oddity is espe- cially prominent in plants transplanted to the continental Croatia where phytoplasmas be- longing to the 16SrXII group are well established and widespread in vineyards. We also re- port on the first occurrence of the 16SrIII group phytoplasma, rather uncommon in European grapevines. Thus far phytoplasmas belonging to three ribosomal groups (16SrI, 16SrIII and 16SrXII) were detected on cv. 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