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Öğe Burdur ilinde Plum pox virus’un tespiti ve kısmi kılıf protein geninin moleküler karakterizasyonu(Kahramanmaraş Sütçü İmam Üniversitesi Rektörlüğü, 2021) Morca, Ali Ferhan; Coşkan, Sevgi; Çelik, AliPlum pox virus (PPV), sert çekirdekli meyvelerde önemli verim kayıplarına sebep olan Şarka hastalığının etmenidir. PPV, Türkiye’nin farklı bölgelerinde sınırlı olarak tespit edilmesine rağmen, bugüne kadar Burdur iline ait herhangi bir kayıt bulunmamaktadır. Bu çalışmada, 2016-2019 yılları arasında Burdur ilinde PPV’nin varlığının belirlenmesine yönelik serolojik ve moleküler yöntemler kullanılarak yapılan araştırmanın sonuçlarına yer verilmiştir. Sürveyler süresince toplanan 47 adet sert çekirdekli meyve yaprak örneği ilk olarak Double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) ile analiz edilmiş ve 2 adet şeftali örneğinin PPV ile enfekteli olduğu belirlenmiştir. Çalışmada, PPV’nin kılıf protein bölgesi için 767 nükleotid büyüklüğünde bir bölgeyi çoğaltan bir adet dejenere primer çifti tasarlanmıştır. Yeni primerler ile gerçekleştirilen Reverse transcription-polymerase chain reaction (RT-PCR) ve sekans analizleri sonucunda elde edilen diziler GenBank’a kaydedilmiştir. Yapılan BlastN analizi neticesinde Burdur PPV izolatları en yüksek benzerlik oranını (%99.86-%98.49), PPV-M ırkı ile göstermiştir. Neighbour-joining yöntemiyle yapılan filogenetik ağaçta Burdur izolatlarının, Türkiye ve farklı ülkelere ait PPV-M izolatı ile kümelendiği belirlenmiştir. Bu çalışma ile elde edilen 2 adet PPV-M izolatı Akdeniz Bölgesi’nin PPV açısından ari alanı konumunda olan Burdur ilinde ilk kayıt niteliğindedir. Çalışma sonucunda enfekteli olduğu tespit edilen ağaçlar eradike edilerek 1 km çapında tampon bölge oluşturulmuş ve 3 yıl boyunca Prunus türlerinin yetiştiriciliğinin yasaklanmasına ve sürvey çalışmalarınnın devam edilmesine karar verilmiştir.Öğe Comprehensive surveillance and population study on plum pox virus in Ankara Province of Turkey(Springer Science and Business Media Deutschland GmbH, 2022) Coşkan, Sevgi; Morca, Ali Ferhan; Akbaş, Birol; Çelik, Ali; Santosa, Adyatma IrawanAnkara Province with its sizeable stone fruits outputs and its geographic location in the middle of Turkey might be one of the diversity centers of plum pox virus (PPV) in the country, yet the epidemiological data from there were rather limited. Multi-year extensive surveillance in all 25 districts of Ankara sampled 8131 Prunus spp. plants to be tested against PPV. DAS-ELISA detected 609 PPV positive samples (7.49%), which were all also confirmed by RT-PCR using two primer pairs to amplify P3-6K1-CI and partial NIb-CP regions. The partial genomes of 80 isolates were sequenced, then the sequences were subjected to phylogenetic and population analyses together with 170 isolates which complete genome sequences available in NCBI GenBank. The phylogenetic study showed that 73 new isolates were PPV-T, six isolates were PPV-M, and one isolate was PPV-D. The subsequent population study confirmed the interesting features of PPV with very high genetic diversities at the species level, thus it should be divided into strains that each retained much lower divergence among isolates. The obtained data also could provide new evidence to separate M, and MIs isolates into two distinct strains. Although previous studies suggested that Turkish isolates have been endemic since a long time ago in the country, the results of demographic analyses of the present study indicated that the expansions of Turkey and Ankara populations were recent, driven by relatively new mutations in their genome. © 2022, The Author(s), under exclusive licence to Deutsche Phytomedizinische Gesellschaft.Öğe Development of colorimetric and real time loop-mediated isothermal amplification (cr-LAMP) assay for rapid detection of Wheat dwarf virus (WDV)(Elseiver Science Ltd, 2021) Çelik, Ali; Morca, Ali FerhanColorimetric and real-time loop-mediated isothermal amplification (cr-LAMP) assays were developed targeting the coat protein region of genomic DNA of Wheat dwarf virus (WDV). The method was optimized relating to primer selection, determination of isothermal temperature, and determination of incubation duration. The specificity of the assay was tested using potential hosts of WDV and Maize streak virus (MSV) from Mastrevirus genus. No cross-reaction took place with MSV or with the potential hosts tested. For the naked eye observation of positive LAMP reaction, WarmStart 2X Master Mix from NEB (New England Biolabs) was used, where the change in colour from pink to yellowish points out a positive result. The amplification was monitored in real-time by smart-DART platform (Diagenetix Inc, USA) that allows connecting to a tablet or smartphone via bluetooth. The detection sensitivity of the LAMP assay was 100-fold more sensitive than conventional PCR after optimization of the cr-LAMP reaction conditions. The novel LAMP assay showed a high specificity in distinction of WDV detection from potential hosts and MSV. The cr-LAMP is considered to be fast and effective assay since it only requires very basic equipment and the results can be evaluated easily by using colorimetric and real time approaches. The simple, low-power, handheld smart-DART platform and colorimetric detection both have a major powerful advantage of the LAMP reaction in that it can be enabled in agricultural industries, where laboratory capacity is often rudimentary.Öğe First complete sequences and genetic variation of plum pox virus T strain in Prunus dulcis and Prunus cerasus(Springer Heidelberg, 2023) Akbaş, Birol; Morca, Ali Ferhan; Çoşkan, Sevgi; Santosa, Adyatma Irawan; Kılıç, Handan Çulal; Çelik, AliThe complete genome of plum pox virus strain T isolates from five different Prunus spp., including almond (P. dulcis) and sour cherry (P. ceracus) isolates, was fully sequenced using the primer pairs designed in this study. The five isolates were aligned with other 50 PPV-T isolates whose complete genome sequences were available in GenBank and then subjected to phylogenetic and diversity analyses. Recombination analysis showed no significant signal detected in the five newly sequenced isolates while confirming four recombinant isolates reported in a previous study. Nucleotide and amino acid phylogenetic trees clustered the tested isolates into three major groups: Balkan 1, 2, and 3. Strain T isolates shared high nucleotide and amino acid identities among them. Diversity analysis applied different parameters to found that the sequences of P3 and 6K1 genes were more conserved over other genes. In accordance, the highly variable P1 and CP genes were found to experience weaker purifying pressures, ? = 0.127 and 0.219, respectively, than other genes. The three neutrality tests gave negative values to all genes, suggesting that strain T populations have expanding or bottleneck selections. Genetic make-up of the only known sour cherry isolate is highly identical to isolates from other Prunus spp. Therefore, this study has updated our knowledge of T strain diversity in new hosts and provided a clear picture of genetic variation and host relationships.Öğe Global population structure of apple mosaic virus (ApMV, Genus Ilarvirus)(MDPI, 2023) Çelik, Ali; Morca, Ali Ferhan; Coşkan, Sevgi; Santosa, Adyatma IrawanThe gene sequence data for apple mosaic virus (ApMV) in NCBI GenBank were analyzed to determine the phylogeny and population structure of the virus at a global level. The phylogenies of the movement protein (MP) and coat protein (CP) genes, encoded by RNA3, were shown to be identical and consisted of three lineages but did not closely correlate with those of P1 and P2, suggesting the presence of recombinant isolates. Recombination Detection Program (RDP v.4.56) detected significant recombination signal in the P1 region of K75R1 (KY883318) and Apple (HE574162) and the P2 region of Apple (HE574163) and CITH GD (MN822138). Observation on several diversity parameters suggested that the isolates in group 3 had higher divergence among them, compared to isolates in groups 1 and 2. The neutrality tests assigned positive values to P1, indicating that only this region experiencing balanced or contracting selection. Comparisons of the three phylogroups demonstrated high Fixation index (F-ST) values and confirmed genetic separation and the lack of gene flow among them. Additionally, & PLUSMN;500 bp of partial MP + 'intergenic region' + partial CP coding regions of two Turkish isolates from apple and seven from hazelnut were sequenced and determined that their phylogenetic positions fell within group 1 and 3, respectively.Öğe Insight into Population Structure and Evolutionary Analysis of the Emerging Tomato Brown Rugose Fruit Virus(MDPI, 2022) Çelik, Ali; Coşkan, Sevgi; Morca, Ali Ferhan; Santosa, Adyatma Irawan; Koolivand, DavoudA total of 112 symptomatic tomatoes (Solanum lycopersicum L.) and 83 symptomatic pepper (Capsicum spp.) samples were collected in Ankara, Eskişehir, Bartın, and Zonguldak provinces of Turkey during 2020–2021. Six tomatoes and one pepper sample (3.6%) tested positive for tomato brown rugose fruit virus (ToBRFV, genus Tobamovirus) infection by DAS-ELISA and RT-PCR. ToBRFV-positive tomato and pepper plants were removed from greenhouses as soon as possible, and the greenhouses and tools were disinfected completely. Phylogenetic analysis on the complete CP sequences suggested the clustering of 178 GenBank isolates and 7 novel isolates into three groups. A study using DnaSP software showed very low genetic variation among current global ToBRFV isolates. All four ORFs of the virus genome were under strong negative evolutionary constraints, with a ? value range of 0.0869–0.2066. However, three neutrality tests indicated that most populations of the newly identified ToBRFV are currently expanding by assigning statistically significant negative values to them. The very low FST values (0.25 or less) obtained by all comparisons of the isolates from Europe, the Middle East, China, and America concluded that there is no clear genetic separation among currently known isolates from different geographic origins. The divergence time of ToBRFV was estimated to be in the middle of the course of the evolution of 11 tested tobamoviruses. The time to the most recent common ancestors (TMRCAs) of ToBRFV were calculated to be 0.8 and 1.87 with the genetically closest members of Tobamovirus. The results of this study could improve our understanding on the population structure of the emerging ToBRFV. © 2022 by the authors.Öğe New Detection Methods for Cryphonectria Hypovirus 1 (CHV1) through SYBR Green-Based Real-Time PCR and Loop-Mediated Isothermal Amplification (LAMP)(Mdpi, 2024) Celik, Ali; Cakar, Deniz; Dervis, Sibel; Morca, Ali Ferhan; Simsek, Secil Akilli; Romon-Ochoa, Pedro; Ozer, GokselSome mycoviruses can be considered as effective biocontrol agents, mitigating the impact of phytopathogenic fungi and consequently reducing disease outbreaks while promoting plant health. Cryphonectria parasitica, the causal agent of chestnut blight and a highly destructive pathogen, experienced a notable decrease in its virulence with the identification of cryphonectria hypovirus 1 (CHV1), a naturally occurring biocontrol agent. In this study, two innovative diagnostic protocols designed for the accurate and efficient detection of CHV1 are introduced. The ORF A and ORF B regions of CHV1 are targeted by these techniques, which employ colorimetric loop-mediated isothermal amplification (LAMP) with 2 Colorimetric LAMP Master Mix and real-time quantitative PCR (qPCR) with SYBR Green chemistry, respectively. The LAMP assay presents a discernible color transition, changing from pink to yellow after a 35 min incubation period. Comparative analysis, when assessed against two established reverse transcription-PCR (RT-PCR) techniques, reveals a significant enhancement in sensitivity for both the LAMP approach, which offers a tenfold increase, and the qPCR method, which showcases a remarkable 100-fold sensitivity improvement. Throughout the comparison phase, it was evident that the RT-PCR, LAMP, and qPCR procedures displayed superior performance compared to the Bavendamm test, relying on phenol oxidase activity, effectively distinguishing hypovirulent strains. Consequently, this study introduces two pioneer diagnostic assays for highly sensitive CHV1 detection, representing a substantial advancement in the realm of CHV1 surveillance techniques. These methodologies hold significant promise for enhancing research endeavors in the domain of the biological control of C. parasitica.Öğe A novel capillary gel electrophoresis based fragment analysis method for the rapid detection of important thrips species on alfalfa in Turkey(Plant Protection Central Research Institute, 2022) Morca, Ali Ferhan; Yücel, Cenk; Bariş, Aydemir; Atakan, Ekrem; Çelik, AliThrips cause significant yield reduction in several industrial crops. Since these pests are also included in the quarantine organisms of Turkey, the rapid detection of agents is important to prevent their spread to new areas. Mitochondrial cytochrome oxidase I (COI) barcoding gene assay; one of the molecular methods is widely used in thrips identification. However, as the COI gene has a very short fragment length, it is very difficult to distinguish fragment sizes on agarose gel after PCR. In this study, a new identification method was developed by integrating the Capillary Gel Electrophoresis (CGE) system for Thrips tabaci Lideman, Frankliniella occidentalis (Pergande) and Frankliniella intonsa (Trybom) species, using primer pairs previously used by different researchers. The assay produces strong signals obtained by minimizing the margin of error in the separation of fragment lengths close to each other, especially in the short fragment length COI gene. Therefore, by eliminating the gel electrophoresis step, reliable detections could be obtained without exposure to hazardous chemicals. The novel method shortened the detection time and minimized the process mistakes on the detection of a single thrips with a low DNA concentration. Total 83 thrips individual (52 F. intonsa, 31 F. occidentalis) were able to be detected with this capillary gel electrophoresis based fragment analysis. The novel method is evaluated as unique, specific and quick for the detection of three different thrips species. It is also thought to be able to utilize for identification of different thrips species with short fragment sizes in the foreseeable future. © 2022, Plant Protection Central Research Institute. All rights reserved.Öğe Population analysis on tomato spotted wilt virus isolates inducing various symptoms on tomato, pepper, and Chenopodium album in Turkey(Academic Press Ltd-Elsevier Science Ltd, 2022) Morca, Ali Ferhan; Çelik, Ali; Coşkan, Sevgi; Santosa, Adyatma Irawan; Akbaş, BirolMolecular identification of tomato spotted wilt virus (TSWV) has been conducted in several surveys in Turkey, but the population structure of the virus in the country remains unknown. During 2019-2020, 227 viral symptomatic tomato (Solanum lycopersicum), pepper (Capsicum annuum), and weeds leaf samples were collected from Ankara, Bartin, Eskisehir, Konya, and Zonguldak provinces. RT-PCR tests confirmed TSWV infection in 99 tomato, pepper, and Chenopodium album samples (43.6%). Phylogenetic analysis based on complete nucleotide sequences of N gene clustered the 23 newly sequenced Turkish isolates and other 281 isolates in NCBI GenBank into three major clades: p202/3WT, Tarquinia, and p105. All tested Turkish isolates were included in either p202/3WT or p105 clade. Global isolates retained high nucleotide and amino acids identity for N gene according to percentage identity analysis. N gene of TSWV was under very strong negative selection pressures, as shown by the very low omega values (<0.15) for all analyzed populations. Three neutrality tests also suggested that these populations are undergoing balancing selection by assigning negative values to most of them. The genetic differentiation and gene flow analysis among three clades demonstrated that they are genetically divergent from each other, but their medium FST values of around 0.5 showed that gene flows among clades are rare. This is the first detailed study on the genetic diversity and population structure of TSWV in Turkey.Öğe Turkish isolates of alfalfa mosaic virus belong to a distinct lineage among global population(Academic Press Ltd- Elsevier Science Ltd, 2024) Morca, Ali Ferhan; Akbas, Birol; Santosa, Adyatma Irawan; Topkaya, Serife; Celik, AliComplete RNA3 sequences of 15 alfalfa mosaic virus (AMV) isolates sampled from Afyonkarahisar, Ankara, Konya, Tokat, Kayseri, and Nevs , ehir Provinces of T & uuml;rkiye were obtained using RT-PCR with two primer pairs designed in this study. Molecular and population analyses were performed on them together with 63 global isolates which full genome sequences available in NCBI GenBank. Phylogenetic analysis based on movement protein (MP) and coat protein (CP) regions showed unrelated isolates clustering between the constructed trees, and the new Turkish isolates were dispersed in different phylogroups. Particularly, three new Turkish potato isolates: B6, K1, and K2 occupied a divergent MP lineage, together with one alfalfa isolate from China: ZM1 NX3. S , eta, and k parameters of genetic diversity confirmed that CP were more conserved than MP sequences. The 15 new Turkish isolates shared 95.4 -100% nucleotide and 94 -100% amino acid, and 92.8 -100% nucleotide and 93.3 -100% amino acid identities among themselves at MP and CP regions, respectively. All tested populations were found to be under very strict purifying constraints (dN/dS ratios of 0.061 -0.219), and had undergone recent expansion, probably due to new mutations in their genome, according to diversity and neutrality tests. Fixation index ( F ST ) values obtained by pairwise comparisons of phylogroups were all above 0.33, suggested that the isolates clustering has been done convincingly.Öğe The use of colorimetric loop-mediated isothermal amplification assay for naked-eye detection of bean common mosaic virus(Academic Press Ltd- Elsevier Science Ltd, 2023) Çelik, Ali; Morca, Ali Ferhan; Emiralioğlu, Orkun; Yeken, Mehmet Zahit; Özer, Göksel; Çiftçi, VahdettinBean common mosaic virus (BCMV), one of the most prevalent viral diseases of common beans, has been intensively studied since its initial identification. In this paper, a one-step colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method was performed by designing oligonucleotide primers to amplify the gene encoding the coat protein of BCMV. Positive LAMP reactions were observed with the naked eye using WarmStart 2 x Master Mix from NEB, changing from pink to yellowish if the test is positive. The sensitivity of this method was similar to that of conventional PCR. The method was tested on common bean and another virus, bean common mosaic necrosis virus (BCMNV), and it was shown to specifically detect BCMV and not produce false positive from other RNAs. The RT-LAMP test was quite reliable for the diagnosis of BCMV from infected samples, and the precise, sensitive. The cost analysis showed that the development of a less expensive and efficient RT-LAMP assay for BCMV detection is an important step in expanding testing capacity and improving access to accurate testing in resource-limited settings. The test can be utilized in laboratory and in the field for the diagnosis and monitoring of BCMV, and is the first reported use of RT-LAMP for BCMV detection.
