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Aims: In this study, the best sampling time and tissues for phytoplasma detection in twenty pear trees (cv. Deveci) infected by ‘Candidatus Phytoplasma pyri’, causal agent of pear decline disease, in Bursa province of Turkey were investigated. Methods and Results: Sampling was done throughout the year in leaf midribs, shoot and root tissues, where as the flower tissues were tested once a year in March and fruit tissues in September. All samples were analyzed by nested-PCR using P1/P7 and fU5/rU3 universal primer pairs. Nested PCR products were digested with RsaI and SspI restriction enzymes. The results revealed that the detection rate of ‘Ca. P. pyri’ in different plant tissues was greatly depending on the sample collection period. The fruit tissues, which were only sampled in September due to the ripening time of Deveci pear cultivar in Bursa, showed the highest detection rate of ‘Ca. P. pyri’ (100%) followed by flower tissues (75%). The average detection rate in root, shoot tissues and leaf midribs was found as 43.75, 39.58 and 16.25%, respectively. The present results showed that the best plant tissues for detecting ‘Ca. P. pyri’ in pear trees were fruit columella and flowers. The highest detection rate of this phytoplasma in root tissues was found from November to March, whereas it could be detected whole year around except summer months in shoot samples in Turkey. Conclusions: For 'Candidatus Phytoplasma pyri', detection, if there is no seasonal limitation for testing, the most suitable tissues are fruits and flowers. When it comes to testing throughout the year, the most suitable tissues were determined as the root, the phloem and cambium layer of the shoots and the leaves, respectively. Significance and Impact of the Study: This study on seasonal variations of ‘Candidatus Phytoplasma pyri’ in different pear tissues has been first time investigated in Turkey. This preliminary data provides important knowledge on molecular detection of Ca. P. pyri, causal agent of pear decline disease for further studies and sertification-quarantine programmes of pear trees in Turkey., Amaç: Bu çalışmada, ülkemizde Bursa ilinde saptanmış olan armut yıkım fitoplazması (‘Candidatus Phytoplasma pyri’, PD) ile enfekteli 20 armut ağacı (Deveci çeşidi) seçilerek etmenin teşhis edilmesinde en uygun örnekleme zamanı ve bitki dokusunun belirlenmesi amaçlanmıştır. Yöntem ve Bulgular: Yaprak, sürgün ve kök dokularında yıl boyunca örnekleme yapılırken çiçek dokuları Mart ayı, meyve dokuları ise Eylül ayında olmak üzere yılda bir kez testlenmiştir. Tüm örnekler P1/P7 ve fU5/rU3 üniversal primer çiftleri kullanılarak nested-PCR yöntemiyle analiz edilmiştir. Nested-PCR ürünleri RsaI ve SspI restriksiyon enzimleri ile kesime tabi tutulmuştur. Elde edilen sonuçlara göre farklı bitki dokularında ‘Ca. P. pyri ‘nin saptanma oranının büyük ölçüde örnek toplama peryoduna bağlı olduğunu ortaya koymuştur. Bursa ili koşullarında Deveci armut çeşidinin olgunlaşma dönemine göre sadece Eylül ayında örneklenen meyve dokularınde yüksek oranda ‘Ca. P. pyri’ tespit edilirken (% 100), bunu çiçek dokuları (%75) izlemiştir. Kök, sürgün ve yapraklarda ortalama tespit oranı sırasıyla % 43.75, 39.58 ve 16.25 olarak bulunmuştur. Elde edilen sonuçlar armut ağaçlarında ‘Ca P. pyri’ nin saptanması için en iyi bitki dokularının meyve kolumellası ve çiçek olduğunu göstermiştir. Bu fitoplazmanın kök dokulardaki en yüksek tespit oranı Kasım-Mart ayları arasında bulunurken, ülkemizde sürgün örneklerinde yaz ayları hariç bütün yıl tespit edilebildiği belirlenmiştir. Genel Yorum: 'Candidatus Phytoplasma pyri'nin testlenmesi için mevsimsel açıdan bir sınırlama olmaması durumunda en uygun dokular meyve ve çiçekler olup yıl boyunca testleme yapılması söz konusu olduğunda ise sırasıyla en uygun dokular kök, sürgünlerin floem ve kambiyum tabakası ve yapraklar olarak belirlenmiştir. Çalışmanın Önemi ve Etkisi: Farklı armut dokularında 'Candidatus Phytoplasma pyri' varlığının mevsimsel dağılımı konusunda yapılan bu çalışma ülkemizde ilk kez yapılmıştır. Elde edilen veriler, Türkiye’de armut ağaçlarında sertifikasyon-karantina programları için ve armut yıkım fitoplazmasının etmeni Ca. P. pyri’nin moleküler tespiti konusunda önemli bilgiler sağlamıştır.

Sharka is the most damaging viral disease ofPrunusspp. and the causative agent is plum pox virus (PPV). PPV is widespread and poses a serious problem in stone fruit crops in Turkey. A unique strain of PPV has so far been identified in Turkey and called as PPV-Turkey (PPV-T). The aims of the present study were to describe the efficiency of PPV-T transmission via aphids and to identify the susceptibility of differentPrunusrootstocks against PPV-T strain under natural inoculum in Kayseri province located Central Anatolia Region of Turkey. This experiment was established in a small residential orchard where PPV-T infected apricot and plum trees were detected. Five differentPrunusrootstocks from which 200 individuals were planted in PPV-T infected orchard and experiment was designed in a randomized complete block desing with 10 replications per block. All rootstocks were regularly tested by DAS-ELISA and RT-PCR during 2015–2017. The results showed that the most susceptible rootstocks in terms of PPV-T infection were Nemaguard, Myrobalan 29B and Myrobalan 29C with the infection rate of 3.61%, 2.74% and 1.04%, respectively. On the other hand, GF677 and Garnem were never found infected with PPV-T.Aphis gossypii,A. craccivora,A. spiraecola,Brachycaudus helichrysi,Hyalopterus pruni,Myzus persicae,Macrosiphum euphorbiaewere detected as most abundant aphid species in experimental plot. In 2015, the highest percentage of viruliferous aphids was found asA. gossypii(100%), followed byB. helichrysi(77.8%),A. spiraecola(55.72%),M. persicae(46.05%) andH. pruni(30.76%) whereas it was 100% forA. gossypiiandM. euphorbiaefollowed byA. spiraecola(76.32%),M. persicae(73.1%),H. pruni(62.06%) andB. helichrysi(61.53%) in 2016 by squash real time RT-PCR. The present study showed that PPV-T was effectively transmitted by different aphid species in natural conditions in Kayseri district. It was concluded that Nemaguard and Myrobalan which were found to be the most sensitive rootstocks to PPV-T should not be preferred by growers asPrunusrootstock where PPV poses a high risk.

Son yıllarda yeni nesil sekans analiz tekniklerinin yaygın olarak kullanılması bağlarda ve diğer bitki türlerinde etiyolojisi bilinmeyen birçok virüs hastalığının etmeninin belirlenmesini sağlamıştır.. Bağlarda bu teknoloji ile saptanan yeni virüslerden en önemlileri Grapevine Pinot Gris Virus (GPGV), Grapevine Syrah Virus-1 (GSyV-1), Grapevine Red-blotch associated Virus (GRBaV) ve Grapevine Roditis Leaf Discoloration Virus (GRLDaV)‘dür. Bu çalışmanın amacı Tekirdağ ve Hatay ili bağ alanlarında GPGV, GSyV-1, GRBaV ve GRLDaV varlığının PCR ve DNA dizileme yöntemleriyle saptanması ve karakterizasyonlarının yapılmasıdır. Bu çalışma kapsamında virüs benzeri simptom gösteren omcaların yanı sıra simptomsuz omcalardan da örnekler alınmış ve toplam olarak Tekirdağ ilinden 191, Hatay ilinden ise 111 örnek toplanmıştır. Tekirdağ ilinden toplanan bağ örneklerinde %43,62 oranında GPGV, %1,04 oranında GSyV1 saptanmıştır. Hatay ilinden toplanan örneklerde ise sadece %0,9 oranında GSyV1 saptanmış ve testlenen örnekler GPGV, GRBaV, GRLDaV açısından temiz bulunmuştur. Tekirdağ örneklerinin GPGV için yapılan RT-PCR analizlerinde kısmi kılıf protein, hareketlilik, ve replikaz genlerini çoğaltan primerler kullanıldığında sırasıyla 411 bp, 302 bp ve 618 bp büyüklüğünde PCR ürünleri elde edilmiştir Bu ürünlerin doğrudan iki yönlü sekans analizi sonucunda ve her üç gen bölgesinin de nükleotid dizilimlerinin gen bankasında kayıtlı farklı GPGV izolatları ile yüksek oranda homoloji gösterdiği saptanmıştır.

Cestrum species are being used as hedge and ornamental plants in Turkey. In this study, a Cestrum elegans plant showing chlorotic spots, rings and reddening was subjected to high throughput sequencing of small RNAs, in order to clarify its etiology. Results suggested the presence of tomato chlorosis virus (ToCV), which was further confirmed by RT-PCR using two specific primers amplifying RNA-dependent RNA polymerase (RdRp) and coat protein (CP) of the virus. When 25 Cestrum samples were tested using CP primers, one symptomatic C. elegans and four symptomless C. nocturnum were infected by ToCV. The obtained sequences shared 83–99.9% nucleotide identity with ToCV isolates available in the GenBank. Phylogenetic relationship among Turkish tomato isolates of ToCV and those available in the GenBank showed that two Cestrum spp. isolates of ToCV were closely related to Turkish tomato isolates, while the other three clustered with isolates from different countries.

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Pomegranate is an important crop in the Mediterranean Basin that can be affected by a range of pathogens. With the aim to better understand the impact of viral diseases on pomegranate, two leaf samples from Turkey showing virus-like symptoms such as chlorotic spots and oak-leaf patterns were subjected to high throughput sequencing (HTS). Data analysis indicated the presence of passiflora edulis symptomless virus (PeSV: genus Roymovirus, Potyviridae family) in these two pomegranate samples, consistent with the observation by electron microscopy of flexuous filamentous viral particles 760 to 780 nm long. Further analysis of HTS reads revealed the presence of five PeSV variants in one of the samples and another single variant in the other. PeSV occurrence was also identified from publicly available SRA pomegranate RNA-Seq transcriptomic data from India and China. The genome of these PeSV-pomegranate variants share 78.0&ndash, 86.8% nucleotide identity with that of the reference isolate from passionfruit (MH379332). The presence of PeSV in pomegranate was confirmed by specific RT-PCR assays targeting either the coat protein (CP) or Nla-Pro genes in 37 cultivated and one ornamental pomegranate out of 133 samples collected from the Eastern Mediterranean region of Turkey. To our knowledge, this is the first application of HTS to assess virus occurrence in pomegranate and the first recognition of pomegranate as a new host for PeSV.

High throughput sequencing of total RNA isolated from symptomatic leaves of a sweet cherry tree (Prunus avium cv. 0900 Ziraat) from Turkey identified a new member of the genus Robigovirus designated cherry virus Turkey (CVTR). The presence of the virus was confirmed by electron microscopy and overlapping RT-PCR for sequencing its whole-genome. The virus has a ssRNA genome of 8464 nucleotides which encodes five open reading frames (ORFs) and comprises two non-coding regions, 5′ UTR and 3′ UTR of 97 and 296 nt, respectively. Compared to the five most closely related robigoviruses, RdRp, TGB1, TGB2, TGB3 and CP share amino acid identities ranging from 43–53%, 44–60%, 39–43%, 38–44% and 45–50%, respectively. Unlike the four cherry robigoviruses, CVTR lacks ORFs 2a and 5a. Its genome organization is therefore more similar to African oil palm ringspot virus (AOPRV). Using specific primers, the presence of CVTR was confirmed in 15 sweet cherries and two sour cherries out of 156 tested samples collected from three regions in Turkey. Among them, five samples were showing slight chlorotic symptoms on the leaves. It seems that CVTR infects cherry trees with or without eliciting obvious symptoms, but these data should be confirmed by bioassays in woody and possible herbaceous hosts in future studies.

The propagation materials such as rootstocks, cuttings and other types of grafting materials used as scions play a relevant role in the dissemination of phytoplasma-associated diseases. In particular in the woody plants the propagation material sanitary status plays an important role for both long-distance transmission and disease introduction in the new areas. Since the phytoplasma infection is systemic in the plants, the vegetative propagation of many horticultural crops allows their spread through cuttings, bud wood, tubers, runners and bulbs. It is, therefore, an efficient method of phytoplasma spreading and establishing infection in new plants. Although the phytoplasma spread through vegetative plant propagation occur over short distances by the use of infected propagation materials such as tubers, the worldwide movement of phytoplasmas should be mainly attributed to the man distributing infected propagation materials. The possibility for the phytoplasma vegetative propagation is present in all the shoots and roots comprizing basal shoots, stems, rhizomes, tubers, stolons, corms, buds and bulbs. Some crops like potato, sweet potato, cassava, carrot, onion, garlic, ginger, sugarcane, banana, pineapple, strawberry and many ornamentals like carnations and Chrysanthemum are only vegetatively propagated and hence they have the maximum chances of phytoplasma spread. The fruit tree propagation is usually achieved by grafting or budding of the selected variety onto a suitable rootstock, and this is the main propagation method for the stone and pome fruit trees, grapevine and other fruit trees and shrubs. Also the shoot micropropagation together with grafting, cutting, and other systems to propagate plant germplasm that avoid sexual reproduction is an efficient manner to maintain and transmit the phytoplasma diseases. The importance of phytoplasma infection spread by the vegetatively propagated plants is discussed in this chapter.

Passive “vector” plants can harbor insect viruses without supporting their replication and may have epidemiological consequences. We report complete sequence of Aphid lethal paralysis virus (ALPV, Cripavirus, Dicistroviridae) from small RNA sequencing of a new passive “vector” plant, Cestrum elegans in Turkey using de novo assembly and annotating of contigs. Its genome is 9837 nt in length which encodes for 2 ORFs (including an intergenic untranslated region (IGR) of 196 nt) flanked by a 525 nt 5′ UTR and a 596 nt 3′ UTR. Multiple alignment and phylogenetic analysis showed that the isolate shares highest identity with ALPV-An (JX480861) isolated from oleander aphid, Aphis nerii and was placed into the same clade with other isolates of passive “vector” plants comprising cucumber and maize isolates.

WOS: 000373994100018, Foliar virus-like symptoms consisting of yellowing, chlorotic spots, oak-leaf and vein clearing were observed on pomegranate cultivar Hicaz in Hatay province of Turkey in 2013. Three symptomatic out of 23 pomegranate samples reacted to Grapevine leafroll-associated virus 1 (GLRaV-1) antibodies in DAS-ELISA. In order to confirm the presence of GLRaV-1 in pomegranate, total RNA extracted from petiole samples was used in RT-PCR using specific primers designed on sequences of the heat-shock protein 70 homolog (HSP70h), coat protein (CP), coat protein duplicate 2 (CPd2) and open reading frame 9 (p24) genes of GLRaV-1. Amplicons were only obtained from symptomatic pomegranate samples for the CP, CPd2, and p24 genes but, unlike for GLRaV-1 isolates from grapevine, no amplicon was obtained for the HSP70h gene of GLRaV-1 isolates from pomegranate. The CP, CPd2 and p24 genes of GLRaV-1 from pomegranate (accession no. KP411914-KP411922) had 91-94 % nucleotide sequence identity with GLRaV-1 isolates from grapevine. Phylogenetic analyses reconstructed using the neighbor joining method showed a clustering of GLRaV-1 isolates from pomegranate and grapevine. These results suggest that pomegranate could be an alternate host for GLRaV-1.

Several viruses infecting fig trees in Turkey have been identified recently. The samples were collected from the commonest fig cultivars showing typical mosaic symptoms and from symptomless plants from different fig growing regions of Turkey. They were tested for Fig leaf mottle-associated virus 1-2 (FLMaV1-2), Fig mosaic virus (FMV), Fig latent virus-1 (FLV-1), Fig mild mottle-associated virus (FMMaV), Arkansas fig closterovirus 1-2 (AFCV1-2), Fig badnavirus-1 (FBV-1) and Fig cryptic virus (FCV) by PCR and sequence analyses. One hundred fig trees were tested, and 83% of tested samples were found to be infected by at least one virus. Complex infections were detected in most of the samples, and the most common viruses were FBV-1 and FMV with 82 and 79% infection ratios, respectively. The sequence analyses confirmed virus identity except for AFCV-1 for which no sequence data are available in GenBank. Based on phylogenetic analysis, the sequences clustered into seven groups: FLV-1, FMMaV, FBV-1, FCV, FMV, AFCV-1, FLMaV-1, as expected, and no correlation was found between Turkish isolates depending on cultivars and provinces for these viruses.

Although Plum pox virus (PPV) was first detected in Turkey 44 years ago, the virus is present in a rather limited number of trees. Our recent studies on PPV incidence showed that PPV was introduced rapidly in PPV-free regions and that there are no data available about the role of aphid species and Prunus rootstocks on these new infections. In this study the epide- miological aspect of PPV was studied in Antakya-Hatay, located in the Eastern Mediterranean region of Turkey where PPV was first detected in 2011. The susceptibility of different Prunus rootstocks to PPV was evaluated in an established experimental plot next to a PPV-infected nectarine orchard. Aphid populations were monitored in 2011 and 2012 from the last week of April to the middle of June by the sticky-plant method in both the experimental plot (EP) and the surrounding infected nectarine orchard (SNO). Regularly collected plant samples and aphids were individually tested by DASI-ELISA and squash real-time RT-PCR, respectively. The highest aphid population densities were observed at the end of May in both years. The most abundant aphid species were Aphis gossypii and A. spiraecola both in EP and SNO in both years. The percentage of PPV-viruliferous Myzus persicae, A. fabae, A. gosypii, A. spiraecola, Hyalopterus pruni, Macrosiphon euphorbiae and A. craccivora as estimated by squash real- time RT-PCR were 39.47%, 25.00%, 24.56%, 22.60%, 22.22%, 20.00% and 8.00%, respec- tively. The percentages of viruliferous aphids collected from SNO were 12.5% in A. spiraecola, 12.42% in A. gossypii and 11.11% in H. pruni. At the end of 2012, three Myrobolan 29C and two Adesoto 101 rootstocks were found infected by PPV. Molecular characterization studies showed that PPV-M was the strain present in both the originally infected nectarine plot and the Myrobolan 29C rootstocks.

During inspections in July 2016, ten sets of three stem cuttings were collected from different parts of apple trees ( Malus domestica ) cv. Red Delicious showing dappling, crinkling and irregular yellow spots on their fruits. The samples …

Fig mosaic disease (FMD) and the fig mite, Aceria ficus, are widespread in different fig growing provinces of Turkey. Fig trees (Ficus carica) cv. Bursa siyahı (D1) and an unknown seedling (D2) that showed typical FMD symptoms and was heavily infested by fig mites were used as donor plants for attempted mite transmissions to healthy fig seedlings. Transmission electron microscopy observations of donor plant samples prior to the transmission tests were performed and showed the presence of double membrane bodies (DMBs) in the palisade mesophyll cells. Electron microscopy of all experimentally inoculated fig seedlings showed the same bodies. This result reinforced the suggestion that an agent that elicits the production of DMBs in infected cells is involved in the etiology of FMD. Double-stranded (ds)RNA analyses were also performed from experimentally inoculated plants, and dsRNAs with sizes approximately 1.30 and 1.96 kb were obtained. Reverse transcription–polymerase chain reaction (RT-PCR) products of 468 and 298 bp specific to Fig mosaic virus (FMV) were amplified from both donor and experimentally inoculated plants. BLAST analyses of nucleotide sequences of these fragments showed 90% identity with FMV for the donor plant and 94 to 96% for experimentally inoculated plants. According to these results, FMV is present in both donor and experimentally inoculated plants in Turkey, and this virus is transmissible by A. ficus from fig plant to fig plant.

Sixteen Plum pox virus (PPV) isolates collected in the Ankara region of Turkey were analyzed using available serological and molecular typing assays. Surprisingly, despite the fact that all isolates except one, which was a mix infection, were typed as belonging to the PPV-M strain in four independent molecular assays, nine of them (60%) reacted with both PPV-M specific and PPV-D specific monoclonal antibodies. Partial 5' and 3' genomic sequence analysis on four isolates demonstrated that irrespective of their reactivity towards the PPV-D specific monoclonal antibody, they were all closely related to a recombinant PPV isolate from Turkey, Ab-Tk. All three isolates for which the relevant genomic sequence was obtained showed the same recombination event as Ab-Tk in the HC-Pro gene, around position 1566 of the genome. Complete genomic sequencing of Ab-Tk did not provide evidence for additional recombination events in its evolutionary history. Taken together, these results indicate that a group of closely related PPV isolates characterized by a unique recombination in the HC-Pro gene is prevalent under field conditions in the Ankara region of Turkey. Similar to the situation with the PPV-Rec strain, we propose that these isolates represent a novel strain of PPV, for which the name PPV-T (Turkey) is proposed. Given that PPV-T isolates cannot be identified by currently available typing techniques, it is possible that their presence has been overlooked in other situations. Further efforts should allow a precise description of their prevalence and of their geographical distribution in Turkey and, possibly, in other countries. (C) 2009 Elsevier B.V. All rights reserved.

Although fruit tree phytoplasmas were studied since 1999 in Turkey, there have been very limited studies and records on stone fruit tree phytoplasmas. The main symptoms reported on apricot, plum, peach and almond were chlorosis between veins, off season flowering and fruiting as a result of early bud breaking, longitudinal leaf rolling and quick die-back. More than 500 cultivated and wild Prunus plants in or nearby germplasm nurseries and commercial orchards during 2002–2009 were tested by using universal primers P1/P7 and fU5/rU3 in direct and nested PCR assays, respectively. Amplification products were digested with RsaI and SspI restriction enzymes. The average incidence of ‘Candidatus Phytoplasma prunorum’ was detected as 10.19%. The most infected stone fruit species were apricot and plum, followed by almond and peach. No phytoplasma was found in cherries and wild Prunus species.