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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.

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.

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.

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.

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.

Grapevine Pinot gris virus (GPGV) was firstly identified in northern Italy by deep sequencing from grapevine cv. Pinot gris, exhibiting mottling and deformation of the leaves. The objective of this study was to investigate the prevalence and genetic variability of GPGV isolates obtained from different local and imported grapevine cultivars in Turkey based on partial coat protein, movement protein and RNA-dependent RNA polymerase (RdRp) domain of the replicase (Rep) gene. Two hundred and one grapevine samples from different provinces were tested by RT-PCR assays, approximately 25% of which were found to be infected by GPGV. The PCR products were sequenced and based on the phylogenetic analysis, RdRp gene was found to be most conserved region. The phylograms of three genomic regions revealed correlation between geography and genetic structure. Furthermore, nucleotide diversity studies revealed a low divergence from the homologous sequences from GenBank and some variations within the groups were detected. The results presented in this study provide a better understanding of genetic variation and phylogenetic of GPGV isolates worldwide. © 2018, Deutsche Phytomedizinische Gesellschaft.

3rd International Symposium on Plum Pox Virus -- MAY 09-13, 2016 -- Antalya, TURKEY, WOS: 000428232700005, Plum pox virus (PPV) is the causal agent of sharka disease, which is mainly destructive on apricot (Prunus armeniaca L.), plum (Prunus domestica L.) and peach (Prunus persica L.). There are no efficient control methods except using PPV-free propagating materials and planting PPV-resistant or at least less-susceptible rootstocks. Therefore, lots of studies have been conducted in recent years on breeding of PPV-resistant plants. The objective of this study was the introduction and development of marker-assisted selection (MAS) for PPV resistance in F-1 and F-2 progenies of some Turkish apricot cultivars. Local apricot cultivars 'Adilcevaz 5', 'cologlu', 'Sekerpare' and 'cataloglul crossed with PPV-resistant 'Stark Early Orange' (SEO) were screened with molecular markers PGS1.21 and PGS2.23 co segregating with resistance to PPV in 2011. Of all combinations, seven of 20 progeny obtained from SEO x 12 of 34 progeny obtained from SEO x 'Adilcevaz 5, five of 10 progeny obtained from SEO x 'coloklu, 15 of 37 progeny obtained from SEO x `Sekerpare' and eight of 33 progeny obtained from SEO x 'cataloglu' exhibited resistant alleles., Int Soc Horticultural Sci

Symptoms resembling those associated with phytoplasma presence were observed in pomegranate (Punica granatum L.) trees in June 2012 in the Aegean Region of Turkey (Aydin province). The trees exhibiting yellowing, reduced vigour, deformations and reddening of the leaves and die-back symptoms were analysed to verify phytoplasma presence. Total nucleic acids were extracted from fresh leaf midribs and phloem tissue from young branches of ten symptomatic and five asymptomatic plants. Nested polymerase chain reaction assays using universal phytoplasma-specific 16S rRNA and tuf gene primers were performed. Amplicons were digested with Tru1I, Tsp509I and HhaI restriction enzymes, according to the primer pair employed. The phytoplasma profiles were identical to each other and to aster yellows (16SrI-B) strain when digestion was carried out on 16Sr(I)F1/R1 amplicons. However, one of the samples showed mixed profiles indicating that 16SrI-B and 16SrXII-A phytoplasmas were present when M1/M2 amplicons were digested, the reamplification of this sample with tuf cocktail primers allowed to verify the presence of a 16SrXII-A profile. One pomegranate aster yellows strain AY-PG from 16S rRNA gene and the 16SrXII-A amplicon from tuf gene designed strain STOL-PG were directly sequenced and deposited in GenBank under the Accession Numbers KJ818293 and KP161063, respectively. To our knowledge, this is the first report of 16SrI-B and 16SrXII-A phytoplasmas in pomegranate trees.

2nd International Symposium on Plum Pox Virus -- SEP 03-06, 2013 -- Olomouc, CZECH REPUBLIC, WOS: 000358036400004, Sharka disease in Turkey has firstly been reported in 1968 in Edirne (Marmara region) which is located next to the Bulgarian border. Nowadays, new PPV outbreaks have been reported in Central Anatolia (Ankara, Kayseri), Aegean (Izmir) and Mediterranean regions (Adana, Mersin, Hatay). The distribution of PPV strains was mainly related to the geographical location and the period of PPV introduction in these regions. PPV-M was mainly detected in peach, nectarine and apricot which were recently imported from abroad to the Mediterranean region. PPV-T was detected in apricot and plums in Central Anatolia and in the Aegean Regions where PPV has been endemic and existing for years. These distributions might indicate that new outbreaks may be mainly due to latently infected material that has passed through the border control. Epidemiology and rootstock susceptibility to PPV has also been recently accomplished. A breeding program has been started in 2006 and its main aim is to obtain dried apricot cultivars resistant to PPV and well adapted to Turkish conditions., Int Soc Horticultural Sci

2nd International Symposium on Plum Pox Virus -- SEP 03-06, 2013 -- Olomouc, CZECH REPUBLIC, WOS: 000358036400017, Turkey is the most important producer and exporter country of apricot, Prunus armeniaca. Production of apricots for fresh market relies on foreign cultivars grown on Mediterranean and Aegean regions while Malatya is the most important region for production of dry apricots based on local cultivars. Plum pox virus (PPV) in Turkey has been known since 1968, but it was not widespread until recent years. Malatya region has been free of sharka disease so far, but the disease has already been reported from many different provinces since 2006. Because of that, introgression of resistance to PPV in the local cultivars with good pomological characteristics became an important objective for the apricot crop. In the current breeding program, obtaining new cultivars resistant to PPV, selection of resistant seedlings by using molecular markers linked to PPV resistance was aimed at. Nineteen local apricot genitors and progenies obtained from the crosses between the PPV resistant cultivar 'Stark Early Orange' (SEO), 'Harcot' and local cultivars such as 'Hacihaliloglu', 'Kabaasi', 'Hasanbey', 'Cologlu', 'Adilcevaz5', 'Sekerpare', 'MahmudunErigi', 'Soganci' and 'Cataloglu' were screened with markers. The markers PGS1.21 and PGS2.23 co-segregating with resistance to PPV were used to screen a total of 189 apricot progenies. None of the local genitors had alleles linked to PPV resistance. Among the progenies screened, 15 seedlings from 'Sekerpare' by SEO, 12 from 'Adilcevaz5' by SEO, 7 from 'Hacihaliloglu' by SEO, 9 from 'Kabaasi' by SEO, 5 from 'Cologlu' by SEO, 9 from 'Cataloglu' by SEO, 4 from 'Hasanbey' by SEO, and 1 from 'MahmudunErigi' by SEO and none of the 'Harcot' by 'Soganci' presented resistant alleles and were selected for further studies., Int Soc Horticultural Sci

2nd International Symposium on Plum Pox Virus -- SEP 03-06, 2013 -- Olomouc, CZECH REPUBLIC, WOS: 000358036400024, Sharka disease of stone fruit trees caused by Plum pox virus (PPV) was first described in 1968 in a limited area of Turkey, but during the last decade the disease has progressively spread to a large part of the country. Although PPV-Rec and -D strains were found in Turkey, the most common PPV strains were detected as PPV-M and PPV-T. In this study, DAS-ELISA (5B-IVIA/AMR) monoclonal antibody) and Spot Real-time RT-PCR techniques have been evaluated in order to determine the best sampling time and ratio of PPV infected samples in non-infected-infected plant mixtures for detection of PPV-T and PPV-M strains. Dormant buds in winter and fresh leaves in spring from PPV-infected trees were used for testing in 2012. Six repetitions were performed by single (3 leaves or buds from infected plant) or composite plant mixture samples (3 leaves or buds from infected plant + 3 leaves from healthy plant, and the other composite samples, i.e., 3+6 to 3+27). All combinations and all repetitions of composite leaf samples of both strains were detected as positive in Spot Real-time RT-PCR. However, in DAS-ELISA, the number of PPV positive samples decreased for T and M strain in 6th composite (3 infected+12 healthy leaves) and in 9th composite (3 infected+21 healthy leaves) in spring, respectively. At least 3 repetitions in all combinations of composite samples for PPV-T and -M were found positive in dormant season by Spot Real-time RTPCR whereas it was negative only in the last composite sample (3 infected+27 healthy buds) of PPV-T by DAS-ELISA., Int Soc Horticultural Sci

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.

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.

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.