INTRODUCTION Grapevine leafroll disease (GLRD) is one of the most diffused among viral diseases for the grapevine (Vitis vinifera L.) (Almeida et al., 2013; Maare et al., 2013). To date, 11 Grapevine leafroll associated viruses (GLRaVs) have been described: GLRaV-1, -2, -3 -4, -5, -6, -7, -9, -Pr, -De, and GLRaCV, all belonging to the Closteroviridae family. Of these viruses, GLRaV-2 is the only member of the Closterovirus genus; a tentative genus called Velavirus has been created to classify GLRaV-7, while the rest of GLRaVs belong to the Ampelovirus genus. GLRaV-1 and GLRaV-3 are apparently the most common ampeloviruses in vineyards and, together with GLRaV-2, the main viruses responsible for GLRD damages. The others ampeloviruses (GLRaV-4, -5, -6, -9, -Pr, -De, and GLRaCV) belong to a common phylogenetic clade (subgroup II) in the Ampelovirus genera and may be classified as divergent variants of one virus, GLRaV-4, instead of distinct species, according to recent taxonomical classification (Martelli et al., 2012). The presence of some GLRaVs were reported from many viticultural areas in Turkey in the past. GLRaV-4 (Kaya et al., 2012) and -5 (Buzkan et al., 2010), the first GLRaV-4-like viruses, have been lately detected in some locations in Turkey. Therefore, a study was carried out to investigate the presence of GLRaV-9 in the subgroup II in Turkish autochthonous varieties and potential vector insects. MATERIALS AND METHODS A total of 116 vineyards in two viticultural areas (eastern Mediterranean and southeast Anatolia) was visited for the presence of leafroll-like symptoms and mealybug vector investigation. Grapevines were essentially self-rooted, table varieties. Plant tissues and Planococcus ficus samples were collected from July to the end of August and were stored at +4oC for PCR (mealybugs preserved in 70% ethanol solution). About 100 mg of leaves/petioles was processed for total nucleic acid (TNA) isolation with silica-capture method (Foissac et al., 2005). Five or ten insects, depending upon the size, were also used for TNA isolation (Singh et al., 1995). All TNAs from plants and insects were stored at -20oC before cDNA synthesis. Two-step reverse transcription polymerase chain reaction (RT-PCR) was performed using primers LR9-F/LR9-R (Alkowni et al., 2004). PCR amplicons were custom-sequenced directly with both primers by Medsantek (Turkey). Alignments of the obtained sequences with additional homologous sequences retrieved from GenBank after using the Blastn program (Altschul et al., 1997). A Neighbor-Joining method (Saitou and Nei, 1987) with bootstrap validation was applied to draw a phylogenetic dendrogram (from MEGA6 package). RESULTS AND DISCUSSION A total of 423 grapevine samples from 33 autocthonous varieties was tested by RT-PCR assay. GLRaV-9 was detected in 16 samples from both regions. The highest number of GLRaV-9 infected sample was obtained from southeast Anatolia region (approx. 3%). Comparative analysis of 10 Turkish (TK) and other GLRaV-4-like isolates showed close relationship. Two clustering patterns could clearly be observed in the phylogenetic tree (Fig. 1). Group one contained five TK isolates (TK 51, 53, 54, 55, 65) from three autochthonous varieties (cv. Yalova incisi, Pafu and Hönüsü) from different locations and their nucleotide identity ranged between 82% to 85% with those of GLRaV-4 strain 9 (KJ810572, AY297819). Four TK isolates grouped with the US GLRaV-4 isolate (FJ467503) (Abou-Ghanem Sabanadzovic et al., 2012) in the same cluster (Group II). The nucleotide identity of the isolate TK48 and the US GLRaV-4 isolate was 97%. The TK 78 remained out of two clusters having nucleotide identity as one of other GLRaV-4 variants. To our knowledge, This is the first report of GLRaV-9 in turkish vineyards. PCR reaction with all the P. ficus samples did not result positive for the presence of GLRaV-9. Plant samples from the same mealybug infested areas were also negative for GLRaV-9 detection.
Detection of Grapevine leafroll-associated virus 9 (GLRaV-9) in plant host and vector insect (Planococcus ficus) in Turkey
Minafra A;La Notte P;
2018
Abstract
INTRODUCTION Grapevine leafroll disease (GLRD) is one of the most diffused among viral diseases for the grapevine (Vitis vinifera L.) (Almeida et al., 2013; Maare et al., 2013). To date, 11 Grapevine leafroll associated viruses (GLRaVs) have been described: GLRaV-1, -2, -3 -4, -5, -6, -7, -9, -Pr, -De, and GLRaCV, all belonging to the Closteroviridae family. Of these viruses, GLRaV-2 is the only member of the Closterovirus genus; a tentative genus called Velavirus has been created to classify GLRaV-7, while the rest of GLRaVs belong to the Ampelovirus genus. GLRaV-1 and GLRaV-3 are apparently the most common ampeloviruses in vineyards and, together with GLRaV-2, the main viruses responsible for GLRD damages. The others ampeloviruses (GLRaV-4, -5, -6, -9, -Pr, -De, and GLRaCV) belong to a common phylogenetic clade (subgroup II) in the Ampelovirus genera and may be classified as divergent variants of one virus, GLRaV-4, instead of distinct species, according to recent taxonomical classification (Martelli et al., 2012). The presence of some GLRaVs were reported from many viticultural areas in Turkey in the past. GLRaV-4 (Kaya et al., 2012) and -5 (Buzkan et al., 2010), the first GLRaV-4-like viruses, have been lately detected in some locations in Turkey. Therefore, a study was carried out to investigate the presence of GLRaV-9 in the subgroup II in Turkish autochthonous varieties and potential vector insects. MATERIALS AND METHODS A total of 116 vineyards in two viticultural areas (eastern Mediterranean and southeast Anatolia) was visited for the presence of leafroll-like symptoms and mealybug vector investigation. Grapevines were essentially self-rooted, table varieties. Plant tissues and Planococcus ficus samples were collected from July to the end of August and were stored at +4oC for PCR (mealybugs preserved in 70% ethanol solution). About 100 mg of leaves/petioles was processed for total nucleic acid (TNA) isolation with silica-capture method (Foissac et al., 2005). Five or ten insects, depending upon the size, were also used for TNA isolation (Singh et al., 1995). All TNAs from plants and insects were stored at -20oC before cDNA synthesis. Two-step reverse transcription polymerase chain reaction (RT-PCR) was performed using primers LR9-F/LR9-R (Alkowni et al., 2004). PCR amplicons were custom-sequenced directly with both primers by Medsantek (Turkey). Alignments of the obtained sequences with additional homologous sequences retrieved from GenBank after using the Blastn program (Altschul et al., 1997). A Neighbor-Joining method (Saitou and Nei, 1987) with bootstrap validation was applied to draw a phylogenetic dendrogram (from MEGA6 package). RESULTS AND DISCUSSION A total of 423 grapevine samples from 33 autocthonous varieties was tested by RT-PCR assay. GLRaV-9 was detected in 16 samples from both regions. The highest number of GLRaV-9 infected sample was obtained from southeast Anatolia region (approx. 3%). Comparative analysis of 10 Turkish (TK) and other GLRaV-4-like isolates showed close relationship. Two clustering patterns could clearly be observed in the phylogenetic tree (Fig. 1). Group one contained five TK isolates (TK 51, 53, 54, 55, 65) from three autochthonous varieties (cv. Yalova incisi, Pafu and Hönüsü) from different locations and their nucleotide identity ranged between 82% to 85% with those of GLRaV-4 strain 9 (KJ810572, AY297819). Four TK isolates grouped with the US GLRaV-4 isolate (FJ467503) (Abou-Ghanem Sabanadzovic et al., 2012) in the same cluster (Group II). The nucleotide identity of the isolate TK48 and the US GLRaV-4 isolate was 97%. The TK 78 remained out of two clusters having nucleotide identity as one of other GLRaV-4 variants. To our knowledge, This is the first report of GLRaV-9 in turkish vineyards. PCR reaction with all the P. ficus samples did not result positive for the presence of GLRaV-9. Plant samples from the same mealybug infested areas were also negative for GLRaV-9 detection.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
