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Öğe A comprehensive study on the molecular characterization of tomato spotted wilt orthotospovirus isolates and resistance genes in pepper and tomato(Tubitak Scientific & Technological Research Council Turkey, 2024) Sajid, Qurat Ul Ain; Elci, EminurTomato spotted wilt orthotospovirus (TSWV, Orthotospovirus tomatomaculae) resistance genes (Tsw, Sw5) have been identified in pepper and tomato plants. The development of resistant cultivars is one of the popular management strategies for overcoming viral infections. However, the breakdown of resistance genes has been documented from many places where resistant cultivars have been developed. This has mainly been due to the emergence of resistance-breaking (RB) lines because of the consecutive use of these cultivars. The development of these isolates may cause great losses in both tomato and pepper plants in Turkiye. To investigate the incidence of TSWV, 150 symptomatic samples were collected from widespread locations of tomato and pepper growing provinces in Turkiye. Samples were inoculated to enhance the virus titer and further confirmed by enzyme-linked immunosorbent assay (ELISA). The ELISA positive samples were confirmed by reverse transcription-polymerase chain reaction. The sequence-characterized amplified region (SCAR) and CAPS markers were used to screen resistance genes in tomato and pepper plants. Positive TSWV-isolates were partially sequenced to determine whether they had the previously reported mutations in NSs or NSm genes for RB isolates. As per the virus characterization, NSm and NSs amino acid sequences showed no previously identified mutations. Resistance gene-carrying isolates were amplified for partial genome sequences. Phylogenetic analyses revealed that both NSs and NSm were distributed in the same genetic pool. The N gene sequence comparison and phylogenetic analysis showed that the Turkish isolates were clustered in a separate clade. These findings provide insight into TSWV infections and resistance genes of pepper and tomato by phylogenetic analysis.Öğe Detection and partial characterization of grapevine leafroll-associated virus 1 in pomegranate trees in Turkey(SPRINGER, 2016) Caglayan, Kadriye; Elci, Eminur; Gazel, MonaFoliar 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.Öğe Exploring the efficacy of RNAi-mediated gene knock-down via oral delivery of dsRNA in the Colorado potato beetle (Leptinotarsa decemlineata Say)(Tubitak Scientific & Technological Research Council Turkey, 2023) Tariq, Haneef; Gokce, Ayhan; Aksoy, Emre; Elci, Eminur; Bakhsh, AllahRNA interference (RNAi) is a versatile genetic tool capable of selectively inhibiting the expression of any gene in a targeted organism. Its implementation holds great promise for safeguarding crops against insect pests and diseases. Vacuolar-ATPase represents an ideal target for RNAi-based pest management strategies since it is an enzyme essential for various physiological processes in insects. In this study, double-stranded RNA (dsRNA) was synthesized using an L4440 vector in Escherichia coli HT115 strain to silence the Vacuolar-ATPase proteolipid subunit mRNA in the Colorado potato beetle (Leptinotarsa decemlineata Say). To assess the effectiveness of RNAi, L. decemlineata larvae at different developmental stages were fed potato leaflets treated with dsRNA. The feeding bioassays using dsV-ATPase resulted in significant mortality rates, ranging from 45% to 77% across all of the instar stages of L. decemlineata. Furthermore, ingestion of dsRNAs by third-and fourth-instar larvae exerted significant effects on their body weight and foliage consumption. Notably, feeding the larvae dsV-ATPase led to a significant reduction in V-ATPase gene expression, confirming the efficacy of RNAi-mediated gene silencing in controlling L. decemlineata populations. These findings highlight the potential of RNAi-mediated gene silencing as a valuable strategy for managing L. decemlineata populations by targeting essential genes.Öğe Genomic Variability and Recombination Analysis of Grapevine leafroll-associated virus-1 Isolates from Turkey(Galenos Publ House, 2019) Elci, EminurGrapevine leafroll-associated virus-1 (GLRaV-1), one of the causal agents of Grapevine leafroll disease (GLRD), is one of the most important viral diseases of grapevine worldwide. In this study, the prevalence of GLRaV-1, genetic variation and recombination events among GLRaV-1 isolates in Turkey were investigated. Initially, 197 grapevine samples from different provinces of the country were serologically tested. Of the total samples, 109 (55.32%) were identified as GLRaV-1 infected. Subsequently, 9 samples representing different geographic distribution were selected for further sequence analysis of the heat-shock protein 70 homolog (HSP70h), open reading frame 9 (p24), coat protein (CP) and coat protein duplicate 2 (CPd2). Among the four gene regions, CPd2 was found the most divergent region while HSP70h gene exhibited the lowest genetic diversity. The phylogenetic analysis of four genomic regions including GenBank records clustered all variants in two major groups and grouped Turkish isolates mostly together. However, the isolate clusters were not correlated to their geographic origin. Furthermore, several putative recombination events were detected with trace to moderate evidence support of algorithms implemented in Recombination Detection Program (RDP). Taken together, the results provide a better understanding on genetic variation of Turkish GLRaV-1 isolates in the country and worldwide and can help to improve sanitation of propagated material programs for the grape growers.Öğe Heterodera schachtii (Nematoda: Heteroderidae) Associated with Cabbage-Cultivation Systems in Anatolia Region of Türkiye(Mdpi, 2024) Akyol, Gulsum Badel; Yuksel, Ebubekir; Elci, Eminur; Bozbuga, Refik; Dababat, Abdelfattah; Imren, Mustafa; Toktay, HalilHeterodera schachtii (Schmidt, 1871) (Nematoda: Heteroderidae) is one of the most widespread plant-parasitic nematodes (PPNs) associated with cabbages, which cause severe yield losses in cruciferous vegetables. This study aimed to improve the current understanding of the prevalence and detection of H. schachtii in the cabbage-growing areas of Ni & gbreve;de Province, T & uuml;rkiye. Field surveys were conducted between November and December 2021, and 100 soil samples were collected immediately after cabbage harvesting. Heterodera schachtii populations were identified by morphological and morphometric methods along with Internal Transcribed Spacer of the ribosomal region (ITS-rDNA) and Cytochrome Oxidase Subunit (COI-mtDNA) sequencing. The mean body length of H. schachtii was 463 +/- 7 mu m, while stylet and hyaline length ranged between 20.7-27.8 mu m and 20.1-32.1 mu m, respectively. Nearly half of the surveyed areas were infested with H. schachtii with a 41% incidence rate. However, the Merkez District had the highest proportion of infested fields with an over 51% incidence rate. The population density was determined in 41 samples with a mean of 79.5 cysts per 250 g of soil. These results will help to determine the control and management strategies of H. schachtii.Öğe Incidence and genetic diversity of grapevine leafroll-associated virus 3 (GLRaV-3) isolates in Turkey(Academic Press Ltd- Elsevier Science Ltd, 2022) Gazel, Mona; Tunc, Bahar; Elci, Eminur; Caglayan, KadriyeGrapevine leafroll-associated virus 3 (GLRaV-3) is the most important virus species within the grapevine leafroll complex. It causes significant losses to growers and vineries because of its effect on grape and wine quality. A survey was conducted in the major grape-growing provinces of Turkey in 2018 to investigate distribution and the genetic diversity of GLRaV-3 in local and foreign grapevine cultivars. The genetic diversity of GLRaV-3 isolates based on partial heat-shock protein 70 homologue (Hsp70 h), partial coat protein (CP) and partial RNA-dependent RNA polymerase (RdRp) genes were determined by RT-PCR and sequencing. The infection rate of GLRaV-3 in 141 tested grapevine samples was 13.47% by DAS-ELISA and 28.36% by RT-PCR analysis. Totally, 17 amplicons from Hsp70 h, 22 isolates from RdRp and 16 isolates from CP genes were sequenced in both directions. The sequence analysis of three genes revealed that the Turkish GLRaV-3 isolates shared 91-100% nucleotide identities with the sequences of GLRaV-3 isolated deposited in the GenBank from other parts of the world without any correlation between the distribution and geographical origin.Öğe Incidence and genetic diversity of raspberry bushy dwarf virus (RBDV) in Rubus spp. in Turkey(Wiley, 2023) Caglayan, Kadriye; Ordek, Kivilcim; Gazel, Mona; Elci, Eminur; Roumi, Vahid; Lamovsek, Janja; Plesko, Irena MavricRaspberry bushy dwarf virus (RBDV), recently renamed to Idaeovirus rubi, is one of the most common viruses infecting Rubus species worldwide but there is still a limited number of genome sequences available in the GenBank database and the majority of the sequences include partial sequences of RNA-1 and RNA-2. The distribution and incidence of RBDV in main raspberry and blackberry growing provinces in Turkey were monitored during 2015-2019 and 537 Rubus spp. samples were tested by both DAS-ELISA and RT-PCR. Among the tested samples, 36 samples tested positive for RBDV by DAS-ELISA and 67 samples by RT-PCR. There was relatively low nucleotide diversity among the Turkish isolates. Turkish isolates shared 93%-97.7%, 84.3%-98.9%, and 85%-99.2% nucleotide sequence identities with available sequences in the GenBank, in partial RNA-1, movement protein (MP) and coat protein (CP) genes, respectively. In the phylogenetic tree constructed for RNA-1, MP, and CP sequences, all Turkish raspberry isolates were clustered in a distinct clade. However, the blackberry isolates showed considerable variation in nucleotide sequences and were placed in three distinct groups. The divergent blackberry isolates showed high variability in MP (84.5%-89.3%) and CP (85.5%-89.7%) regions and were placed in a distinct group. The rest of blackberry isolates clustered together with sweet cherry RBDV isolates adjacent to the grapevine clade or together with raspberry isolates. The comparative analysis conducted on three RNA segments of RBDV highlighted the high sequence diversity of Turkish RBDV isolates. This study also emphasizes the importance of regular monitoring of RBDV infections in Turkey, with special regard to those Rubus spp. and grapevine accessions employed in conservation and selection programmes. In particular, the presence of new RBDV genetic variants and infection of Rubus species must be taken into account to choose a correct detection protocol and management strategy.Öğe Isolation and characterization of plant-pathogenic Streptomyces species associated with potato common scab disease in Turkiye(Wiley, 2024) Uysal, Nida; Bozkurt, Adem; Elci, EminurPotato common scab (PCS), a significant potato disease, negatively impacts tuber quality. The present study was conducted to isolate and characterize pathogenic Streptomyces species associated with PCS disease in Turkiye based on their morphological, physiological and molecular characteristics. Field-grown potatoes that exhibited scab lesions were collected from four provinces in 2020-2021, and 200 bacterial isolates were obtained from netted, superficial and deep-pitted common scab lesions. Pathogenicity assays, including in vitro tuber slice and in planta radish seedling bioassays, identified 150 pathogenic isolates. Morphological and physiological characterization of 92 selected isolates revealed several Streptomyces species that exhibited diverse mycelium colours, sporulation patterns and pigmentation. Molecular analysis using 16S rRNA sequencing, species-specific primers, PCR-RFLP of the 16S-23S (ITS) region with Hpy99I restriction enzyme, and multilocus sequence analysis (MLSA) based on atpD, recA, rpoB and trpB genes revealed that S. scabiei was the dominant species, followed by S. europaeiscabiei, S. caniscabiei, S. bottropensis, S. stelliscabiei and S. turgidiscabies. PCR analysis revealed the presence of the thaxtomin synthetase genes (txtAB) in all tested samples, while the necrogenic protein (nec1) and tomatinase (tomA)-encoding genes were absent from three and two isolates, respectively. Phylogenetic analysis of 32 representative isolates conducted with sequences from 16S rRNA, species-specific PCR and MLSA confirmed their morphological identification and clustered them with reference strains. This study contributes to the understanding of distribution of Streptomyces species associated with PCS, and to our knowledge, is the first molecular confirmation of S. caniscabiei and S. turgidiscabies causing potato scab in Turkiye.Öğe Knockdown of orthotospovirus-derived silencing suppressor gene by plant-mediated RNAi approach induces viral resistance in tomato(Academic Press Ltd- Elsevier Science Ltd, 2024) Sajid, Qurat ul ain; Elci, EminurRNA interference (RNAi) shows significant effectiveness in conferring resistance to viral infections in various plants. The current study was conducted to develop transgenic tomato ( Solanum lycopersicum cv. Rio Grande) lines expressing a 320 bp conserved inverted region of the integral 35S promoter region. The molecular cloning was done for the construction of expression vectors in a hairpin structure by the Tomato spotted wilt orthotospovirus (TSWV) species Orthotospovirus tomatomaculae , silencing suppressor ( NSs ) gene. It contains both sense and antisense orientation in a binary vector pFGC5941. Short hairpin RNA (shRNA) structures were employed to reduce the possibility of inducing endogenous non-specific antiviral responses, which usually target longer double -stranded RNAs (dsRNAs). To confirm the transgenic plants, molecular analysis was performed using TSWV gene -specific primers (TSWV- NSs ). Based on the EHA105 Agrobacterium strain and the pFGC5941 vector carrying NSs and nptII genes in the T -DNA region, the results validate the insertion of kanamycin resistance into the regenerated transgenic plants. The virus source harboring TSWV was used to inoculate putative transgenic plants for evaluating the confirmation of successful transformation strategy for inducing virus resistance. Quantitative real-time polymerase chain reaction (qRT-PCR) assays were performed by using qRT- NSs primer pairs to determine the relative gene expression of all constructs (TSWV- NSs both sense and antisense orientation) after inoculations. In comparison to the control groups, which contained wild type control (non-transgenic tomato plant), empty pFGC5941 vector and the pFGC5941 +sense construct, the expression of the NSs gene was noticeably reduced in the full clone (pFGC5941 +sense + antisense) construct. For the validation of RNAi effectiveness, Nicotiana tabacum plants were also inoculated with viral constructs. Specifically, they displayed severe symptoms of TSWV in plants containing the pFGC5941 +sense, pFGC5941 empty vector, and in wild -type tomato plants that are correlated with bioassay results. The lack of TSWV symptoms validated the hypothesis after careful observation of the expression of NSs genes in terms of pathogenicity. This research demonstrates the RNA interference effectiveness by targeting NSs gene as a reliable technique for achieving long lasting antiviral protection in tomatoes. The findings have practical relevance and the potential to be a tool for limiting tomato crop losses caused by TSWV worldwide.Öğe Molecular Identification of Fig Cryptic Virus and Fig Fleck-Associated Virus in Turkey(Ars Docendi, 2017) Elci, Eminur; Hancer, Tugce; Caglayan, KadriyeRecently, several new viruses infecting fig trees were identified. To assess the presence, distribution and genetic diversity of Fig cryptic virus (FCV) and Fig fleck-associated virus (FFkaV) in fig trees of Turkey, a total of 65 fig samples, which show yellowing, chlorotic, necrotic spots and vein clearing symptoms, were collected from Aegean and Mediterranean regions, which are the most important fig growing regions of Turkey, in spring 2014 and tested by molecular analysis. After cDNA synthesis, FCV and FFkaV specific primer sets of RNA dependent RNA polymerase (RdRp) regions were used for reverse transcription-polymerase chain reaction (RT-PCR) analysis and the PCR products were directly sequenced. The obtained sequences were analyzed and nucleotide sequence analysis confirmed the FCV and FFkaV infections. According to the results, some of the fig trees are infected in Turkey by FCV and FFkaV with an incidence of 20 % and 9.2 %, respectively. BLAST analysis of both FCV and FFkaV has shown high identity with Italian isolates (FCV\ref\NC015494.1 and FFkaV\ref\NC015229.1). For FFkaV, the phylogenetic analysis, constructed from partial RdRp nucleotide sequences, clustered the isolates based on their geographical origin. While the correlation between FFkaV isolates and regions was very high, no correlation between collection regions and FCV isolates was observed. It can be concluded that, fig trees from the most important fig growing regions of Turkey are infected by FCV and FFkaV and it is instrumental to overview of the viral control strategies for fig plantations in Turkey.
