Molecular Characterization and Variation of “Bois Noir” Turkish Strains

Filiz Ertunc, Didem Canik Orel and Filiz R. Zelyut Ankara University, Faculty of Agriculture, Department of Plant Protection, 06110 Ankara, Turkey [email protected]

Abstract—Widespread “bois noir” phytoplasma (16SrXII-A) (Avramov et al. 2008; Karimi et al. 2009; Contaldo et al. 2011). infection is present in Turkish vineyards. The infection was BN is a member of “stolbur” group (16SrXII-A group) detected in wine grape varieties more than in table grapes. ‘Candidatus Phytoplasma solani’ transmitted by Hyalestes Grapevines with severe redness and inward curling of leaves were obsoletus (Cixiidae) a polyphagous vector and seldom and collected from main viticulture production areas. Collected erratically feeding on grapevine (Alma et al. 2002). The samples were subjected to nucleic acid extraction followed by 16SrXII group contains several subgroups and many of them nested PCR/RFLP analyses and, according to the results; the are defined at the ‘Canditatus Phytoplasma’ rank (Quaglino et majority were infected by “bois noir” phytoplasmas. Among the al. 2013; Bertaccini et al. 2014). collected strains 19 were sequenced on the 16S ribosomal gene and deposited in GenBank. Obtained sequences were aligned During the surveys conducted in 2009-2010 in the main using Clustal X and Bioedit and phylogenetic analyses were viticultural areas in Turkey, totally 289 plant samples bearing performed by using Mega7 program. the symptoms typical of phytoplasma-associated infections were collected. The main symptoms detected on the infected Key words–grapevine, “bois noir”, phytoplasma, Turkey. plants were severe redness and inward curling of the mature leaves which were also typical for grapevine leaf-roll virus I. INTRODUCTION diseases. The redness of the leaves was more severe on wine grape varieties comparing to table grapes. Phytoplasma Turkey is located in the subtropic climatic region in the associated infections were present in all the surveyed provinces world and Northeastern part of Anatolian peninsula between and the infection was present almost in all of the wine-grape Black sea and Caspian sea regions and it is the genetic source vineyards located in Marmara and Aegean Region of Turkey of the important grapevine varieties. Turkey is one of the (Canik et al. 2011). Phytoplasmas detected on some of the local Nations native to grapevine and is familiar with its cultivation table-grape varieties belong to 4 ribosomal groups (16SrI, for more than 6,000 years. According to the data of FAO, 16SrV, 16SrIX and 16SrXII) (Ertunc et al. 2015), but the Turkey has 540,000 ha of grapevine-cultivated area and is at the widespread infection was BN (16SrXII-A). 4th level whereas grape production is at the 6th level in the world (Soylemezoglu et al. 2015). Many of the local and international H. obsoletus is reported from the potato fields in Erzurum table grape and wine grape varieties are grown in Thrace, (Guclu and Ozbek 1988). It was very rarely present in the Central Anatolia, Mediterranean, Aegean and Eastern Anatolia vineyards inspected and also some putative vectors of BN, regions. The international varieties are Cabarnet Sauvignon, Euscelis lineolatus Brullé, Dictyophara europaea (L.) (Lessio Merlot, Syrah, Malbec, Cabarnet Franc, Pinot noir, and Alma 2008) were also present in Turkey in minor Chardonnay, Sauvignon Blanc, and some famous locals are frequency. Vectors were found less effective in dissemination Kalecik Karası, Öküzgözü, Boğazkere, Çal Karasi, Bornova of the grapevine yellows phytoplasmas in Turkey (Bayram et al. Misketi, Köhnü, Sultani seedless and Pearl of Yalova. 2014). It has been thought that the introduction of phytoplasma Grapevine is affected by several diseases, including those infected grapevines has been by infected propagation material. associated with phytoplasma presence worldwide. Plant According to the results of PCR with universal primers and pathogenic phytoplasmas are insect transmitted, wall-less, nested-PCR with group specific primers, followed by RFLP phloem-limited bacteria of the class Mollicutes with a small analyses, 53 out of 289 grapevine samples were detected as genome size, which ranges from 530-1,350 kb. In diseased infected with one of the grapevine associated phytoplasma. The plants, they reside almost exclusively in the phloem sieve tube detection rate of infected plant samples was 18.6% and 49 of the elements, to which they are introduced by phloem feeding collected samples were detected as infected with the BN hemipteran insects mainly leafhoppers (Weintraub and phytoplasma. Beandland 2006; Maixner 2011). “Bois noir” (BN) and II. MATERIAL AND METHODS “flavescence dorée” (FD) are inducing similar symptoms that Among the 49 BN strains collected in previous surveys, 19 are severe redness of red varieties and irregular yellowing of were selected according to their origin and variety, sequenced white varieties, backward curling of leaves, lack of lignification and deposited in NCBI GenBank (Table 1). Selected amplicons and shriveling of berries (Kuzmanovic et al. 2008; Martini et al. obtained with R16(I)F1/R1, M1/B6 and M1/M2 primers 1999). Both cause significant reduction in yields in many (Ertunc et al. 2015) were subjected to direct sequencing. The European countries and near Turkey in Iran, Bulgaria and Syria sequences were assembled, using Sequencher 4.1 software, compared with selected nucleotide sequences in the GenBank the sequences of Iran, Jordan, Georgia, Serbia and Italy. database, using BLAST (version BLASTN 2.2.18) (NCBI, Some of the Italian sequences of BN clustered with one Bethesda, MD, USA). Sequence alignments were performed, Canadian BN sequence (Fig. 1). It was clearly shown using Clustal X and Bio Edit (Hall 1999; Thompson et al. 1997). that ‘Ca. P. phoenicium’ was different and was in Moreover 16 BN sequences, the Turkish sequence of group completely different cluster. 16SrIX (‘Ca. P. phoenicium’) and one strain of ‘Ca. P. australiense’ (16SrXII-B) retrieved from NCBI were used in phylogenetic analysis performed using Mega 7 program (Kumar et al. 2016). Table 1. List of BN phytoplasma strain sequences

Number of Location Variety Accession numbers strains (NCBI)

Tekirdag Gamay 1 KJ810646 Pinot noir 3 KJ810644, KJ810645 KJ810647 Çanakkale Syrah 1 KP015028 Nevşehir Emir 1 KJ810642 Sauvignon 1 KJ810643 blanc Elazığ Şirfani 1 KJ810641 Unknown 5 KJ810636, KJ810637, KJ810638 810639 Kırklareli Cabernet 1 KJ810634 Sauvignon Merlot 2 KJ810635, KP010634 İzmir Alfonce 1 KJ810633 Lavallee Ankara Unknown 1 KJ810648 Trakya 1 KJ810649 ilkeren

III. RESULTS The incidence of grapevine phytoplasma infections in Turkish vineyards was quite low and the predominant phytoplasma infection was the one associated to BN presence. The main symptoms were, inward curling and severe reddening of the leaves, the phytoplasma was present mostly on wine-grape varieties of imported origin. It was detected from wine grape varieties such as Alphonse Lavallee, Chardonnay, Alicante Bouchert, Shiraz, Cabarnet Sauvignon, Sauvignon Blanc, Merlot, Malbec and Pinot Figure 1. Phylogenetic trees constructed with 19 BN sequences from this Noir and in some indigenous varieties (Fig. 1). survey and other retrieved from the GenBank using 16S rDNA region. Sequences were selected from NCBI as of the

sequences belonging to Turkey neighbor countries and IV. DISCUSSION some Europeans countries. Phylogenetic trees were constructed with 19 BN sequences from this survey using Epidemics associated with phytoplasma diseases in 16S rDNA regions of Turkish strains and others from NCBI grapevine have become the major problem in recent years database. Kırklareli Merlot sequence (GenBank accession because of the continuously spread throughout many grape number, acc. no., KP015027) and Canakkale Syrah producing regions in different European countries. sequence (acc. no. KP015028) were slightly different from Phytoplasma diseases are widespread and occur all around the the other sequences and the remaining were quite similar to world; besides Europe they are also present in Ukraine, Iran, each other except a Chilean BN strain, while some of the Syria, Lebanon, Israel, South Africa and China (Maixner Italian strains cluster together with Turkish strains. The 2011). In Turkey the attention should be focused on the presence Turkish ‘Ca. P. solani’ strains were in the same cluster with of BN especially in imported grapevine propagation materials.

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