Browsing by Author "Usta, M."

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Cherry Leafroll Virus in Juglans Regia in the Lake Van Basin of Turkey
(Springer, 2008) Ozturk, M. O.; Sipahioglu, H. M.; Ocak, M.; Usta, M.
Walnut orchards of the Lake Van basin (Turkey) were surveyed from June to October 2006 to determine the incidence of viral infections. ELISA and RT-PCR were used to investigate the presence of Cherry leaf roll virus (CLRV) and Plum pox virus (PPV), testing a total of 870 samples collected from traditional seed-grown plantations. Whereas no PPNT was detected in any of the samples, CLRV was found for the first time in the surveyed locations with an average incidence of 12.9%. Two viral isolates from Edremit were mechanically transmitted to Chenopodium amaranticolor in which they caused local chlorotic spots followed by development of small leaves and apical. necrosis. A 366 bp DNA fragment was amplified by RT-PCR from the 3' non-coding region of RNA-2 of both viral isolates and sequenced. Isolate Edremit-2 was 93-98% identical to the comparable sequences of other isolates for which information is available, whereas isolate Edremit-1 had a lower sequence identity (53-46%). The size of the coat protein subunits of both viral isolates was 52.4 kDa as determined by electropboresis.
Comparison of Three Conventional Extraction Methods for the Detection of Plant Virus/Viroid Rnas From Heat Dried High-Phenolic Host Leaves
(Asian Network for Scientific Information, 2007) Sipahioglu, H.M.; Ocak, M.; Usta, M.
The presence of virus/viroid infections can go unnoticed since symptoms appear only if additional viruses are present. Detection of Plum bark necrosis stem pitting associated virus (PBNSPaV), Apricot latent virus (ApLV), Apple scar skin viroid (ASSVd), Prunus necrotic ringspot virus (PNRSV) and Potato virus Y (PVY) by reverse transcriptase polymerase chain reaction (RT-PCR) or nested-RT-PCR is possible; however, these assays could be unreliable if the tissue contains interfering compounds. This study reports on use of three extraction procedures in recently developed heat-dried virus/viroid preserved plant tissues. The methods tested were lithium chloride, silica-capture and citric buffer. The results showed that (1) the silica-capture RNA extraction method appears to be superior for total RNA extraction; (2) the increase in volume of silica improves the efficiency of RNA extraction from dried infected leaves and (3) the use of silica method minimizes the fragmentation of PCR products and improves the PCR detection of tested pathogens. The results of study indicate that the use of appropriate RNA extraction method is crucial for a successful PCR and an appreciable yield of PCR product from heat-dried infected leaves. © 2007 Asian Network for Scientific Information.
Detection and Characterization of Two Phytoplasma Lineages on Cucumber (Cucumis Sativus L.) With Same Symptomatology Based on Virtual Rflp and Nucleotide Sequence Analysis of 16s Rdna
(Centenary University, 2017) Usta, M.; Güller, A.; Sipahioğlu, H.M.
Phytoplasma-like symptoms were observed in cucumbers (Cucumis sativus L.) in Van province of Turkey. The major symptoms observed were severe dwarfing, witches’ broom, rosetting, little leaf, and sterility of plants. Genomic DNA of 8 symptomatic and non-symptomatic plant leaves was isolated for the detection of pathogenic DNA. Of the 8 cucumber leaf samples tested by nested polymerase chain reaction (Nested-PCR), the four yielded the expected 1.25-kb DNA fragments when using universal primer pairs R16mF2/R16mR1 and R16F2n/R16R2. Randomly selected two DNA bands were further cloned into a proper plasmid vector. The recombinant plasmid DNA was sequenced bidirectionally. BLAST and virtual restriction fragment length polymorphism (RFLP) analyses of the 16S rDNA sequence revealed the presence of the “Candidatus Phytoplasma solani” (similarity coefficient 1.00) (GenBank accession no: KX977570) in one of the severely symptomatic cucumber samples and the “Candidatus Phytoplasma trifolii” (similarity coefficient 0.98) (GenBank accession no.: KR080212) in the other. The isolates were designated as Van-trifolii and Van-solani isolates, respectively. No significant differences were observed between the two different phytoplasmas’ symptomatology. To the authors’ knowledge, this is the first report of ‘Ca. P. trifolii’ and ‘Ca. P. solani’ in cucumbers in Turkey. © 2017, Centenary University. All rights reserved.
Detection and Partial Characterization of Two Distinct Walnut Isolates of Cherry Leaf Roll Virus (Clrv)
(Academic Journals, 2011) Sipahioglu, H.M.; Tekin, Z.; Usta, M.
Two new cherry leaf roll virus (CLRV) isolates (Ah and Ad) were isolated and detected from traditionally-grown walnuts that showed severe apical necrosis and chlorotic spots in systemically infected Cheneopodium amaranticolor, in the eastern part of Turkey. The 404 and 405 bp long DNA fragments of the 3'-non-coding region of both isolates from PCR reactions were cloned and sequenced. A significant genetic variability (up to 14% divergence between sequences) was found within the 3' terminal region of viral genome of CLRV Turkish isolates compared with the isolates in databases. The sequence of Ad isolate was found to share 84 to 98% and the Ah isolate was found to share 85 to 97% nucleotide identity with corresponding sequences of the selected world isolates. An RNA riboprobe generated for CLRV-Ad isolate reacted also with the CLRV-Ah isolate in dot blot molecular hybridization test. Positive reactions were still visible in hybridization test when the extracts of infected fresh and dried leaf tissues of C. amaranticolor diluted 1:20. Western blot analysis revealed that the molecular mass of the coat protein of about 52 kDa for both isolates. © 2011 Academic Journals.
Detection of Arbuscular Mycorrhizal Fungi Within Colonised Roots of the Gramineae Family Members by Nested-Polymerase Chain Reaction (Pcr)
(2011) Demir, S.; Sipahioglu, H.M.; Kaya, I.; Usta, M.; Savur, O.B.
In this study, it is aimed to asses the association of arbuscular mycorrhizal (AM) fungi within colonised rhizosphere of Gramineae family members through a survey by using nested polymerase chain reaction method in Van province (Turkey). From 24 agro-ecological fields, a total of 82 samples belonging to Gramineae family were tested by molecular methods. The presence of Glomus intraradices and Glomus mosseae was ascertained in 10 plants belonging to eight different species by using fungus specific primers. Root colonisation ranged from 6 to 37% within rhizosphere of Gramineae family members and the average root colonisation by AM fungi was 22%. © 2011 Copyright Taylor and Francis Group, LLC.
Development of a Rapid Enzymatic Cdna Amplification Test for the Detection of Apple Scar Skin Viroid (Assvd) in Apple Trees From Eastern Anatolia, Turkey
(2009) Sipahioglu, H.M.; Usta, M.; Ocak, M.
A validated RT-PCR method was used to investigate the presence of Apple scar skin viroid (ASSVd) in the commercial apple orchards of eastern Anatolia. Among three modified and simplified silica-capture based extraction methods, one was used for mass extraction to ascertain the presence of ASSVd. The test was initially performed from an ASSVd-infected source and then applied to total RNA preparations from fresh leaf tissues of apple trees collected from eastern Anatolia. ASSVd was found to occur in apple trees. Among 263 apple samples, 121 were positive for ASSVd. The infected trees showed no apparent disease symptoms on the leaves other than scarring on fruit skin. Overall incidence of ASSVd was 46% in eastern Anatolia. The presence of ASSVd reported for the first time in Turkey. Among three total RNA preparation methods, Method II was determined to be the best procedure for large scale routine analysis. The improved test can be used in a certification or clean stock program to contribute to the prevention of the spread of ASSVd in the eastern Anatolia.
The Effect of Gradient Temperature Pattern on Annealing Efficiency
(int Soc Horticultural Science, 2008) Ocak, M.; Sipahioglu, H. M.; Usta, M.
The specific complementary association due to hydrogen bonding of single stranded nucleic acids is referred to as annealing. Successful primer annealing is of critical importance in a polymerase chain reaction (PCR). In this study, the effect of gradually increased and decreased annealing temperatures on PCR reactions were investigated. In the annealing tests, the isolates of Plum bark necrosis stem pitting associated virus (PBNSPaV), Apricot latent virus (ApLV), Apple scar skin viroid (ASSVd) and Potato virus Y (PVY) were used as reference isolates. The effects of annealing temperature alterations were tested by nested reverse transcriptase polymerase chain reaction (nested-RT-PCR) for PBNSPaV and RT-PCR for ApLV, ASSVd and PVY. The PCR products were analyzed and evaluated by polyacrylamide gel electrophoresis (PAGE). Gradient temperatures (2 to 4 degrees C) below and above annealing temperature resulted in increased primer annealing by both methods. Higher gradients (8-12 degrees C) never resulted in appreciable yields of a unique PCR product, as the likelihood of primer annealing was reduced. Both high and low gradients of annealing temperature gave the yield in PCR reactions, confirming flexible nature of annealing temperature.
First Report of ‘candidatus Phytoplasma Australasia’ Strain Related To Witches’-Broom of Tomato in Türkiye
(Centenary University, 2023) Usta, M.; Güller, A.; Sipahioğlu, H.M.
Phytoplasmas are dangerous bacteria severely infecting agricultural production worldwide. In the present study, the identification of phytoplasmas infecting tomato plants showing symptoms such as small leaves, flower abnormalities, stunting, witches' broom, and reddening was performed. Five plants, two symptomatic and three asymptomatic, were tested to verify phytoplasma infection. Total DNA isolated from 5 leaf samples was used as a template for PCR reactions. The phytoplasma agents were confirmed in the two symptomatic samples. BLASTn search of 16S rRNA of two sequences shared identity similarity of 99.84% with ‘Candidatus Phytoplasma australasia’. Computer-simulated virtual RFLP profiles show that the 16S rRNA sequences is identical to the reference pattern of the 16SrII-D subgroup, with a similarity coefficient of 1.00. Based on BLAST, virtual RFLP, and phylogenetic dendrogram, the identified phytoplasma strains are enclosed in the 16SrII-D subgroup. This is the first report of tomato witches' broom disease related to 16SrII-D subgroup phytoplasma strains in the Antalya province of Türkiye. © 2023, Centenary University. All rights reserved.
Molecular Identification of ‘candidatus Phytoplasma Solani’ Using Secy and Vmp1 Genes in Tomato Plants From Van Province
(Centenary University, 2021) Usta, M.; Güller, A.; Demirel, S.
Tomato (Solanum lycopersicum) is one of the most financially important vegetable crops. It is a species belonging to the Solanaceae family and is cultivated in many countries, including Turkey. The natural presence of ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’) from the Stolbur group (16SrXII) in tomato plants is extensively characterized based on the 16S rRNA gene worldwide. Tomato plants displaying abnormality and sterility of flower, purpling, and bushy appearance were observed in the Van province, Turkey. DNA extraction from tomato leaves was performed, and the extracted DNA was used to amplify 16S rRNA sequences using universal primer pairs by double PCR assays. After confirming the presence of the probable agent (‘Ca. P. solani’), nested PCR testing was performed using an appropriate primer set amplifying the SecY and Vmp1 genes of the same DNAs. Amplified PCR yields were then cloned into a pGEM T-Easy vector and sequenced by New Generation System (NGS). Sequenced 1438 bp nucleotides for Vmp1 gene (MN104838) and 905 bp nucleotides for SecY gene (MN125054) revealed 97.38% and 100% maximum nucleotide similarity with formerly published Vmp1 and SecY gene sequences of ‘Ca. P. solani’ species belonged to the Stolbur group (16Sr-XII), respectively. According to the phylogenetic tree created using our gene sequences from ‘Ca. P. solani’, Vmp1, and SecY gene sequences showed a highly phylogenetic affinity with the same sequences of the same agent from Serbia and France, respectively. To the best of our knowledge, this study is the first report to reveal the phylogenetic relationships of ‘Ca. P. solani’ in stolbur group (16Sr XII-A) using SecY and Vmp1 genes in the tomato plant in Turkey based on the non-ribosomal genes. © 2021, Centenary University. All rights reserved.
New Insights Into Tomato Spotted Wilt Orthotospovirus (Tswv) Infections in Türkiye: Molecular Detection, Phylogenetic Analysis, and in Silico Docking Study
(Academic Press, 2023) Usta, M.; Guller, A.; Demİrel, S.; Korkmaz, G.; Kurt, Z.
Seventy-eight tomato and pepper plants showing symptoms were tested for tomato spotted wilt orthotospovirus (TSWV) using specific primers targeting the full coat protein gene (CP) through RT-PCR. Plant samples were collected in Denizli region, Türkiye during September 2021. The PCR screening results revealed that 23 samples were infected with TSWV, indicating an infection rate of 29.48%. PCR products were subsequently sequenced bidirectionally, and the obtained sequences were deposited in GenBank under the accession numbers ON323583-84 for pepper, and OQ597214 for tomato. Phylogenetic analysis demonstrated a close relationship between the Turkish-Denizli TSWV isolates and previously reported isolates from tomato and pepper plants in Türkiye (KM407603), Hungary (KJ649612), and Serbia (GU369723). Moreover, in this study, three commercial chemicals and five selected phytochemicals were docked against the TSWV CP to assess their binding energies. The docking results indicate that the tested phytochemicals exhibit promising performance compared to other commercial chemicals. This study represents the first molecular and phylogenetic report on TSWV isolates in pepper and tomato plants in Denizli province, Türkiye. Furthermore, the interaction between TSWV and the selected compounds through in silico docking has been reported for the first time. © Articles by the authors; Licensee UASVM and SHST, Cluj-Napoca, Romania. The journal allows the author(s) to hold the copyright/to retain publishing rights without restriction.
Occurrence of Cherry Green Ring Mottle Virus in Turkey
(Wiley, 2008) Sipahioglu, H. M.; Usta, M.; Ocak, M.
Partial Characterisation of a Cherry Isolate of a Newly Emerging Stone Fruit Virus: Plum Bark Necrosis Stem Pitting Associated Virus
(Taylor and Francis Ltd., 2011) Sipahioglu, H.M.; Usta, M.; Oksuz, T.
Plum bark necrosis stem pitting associated virus (PBNSPaV) has occurred globally over the last two decades and is becoming one of the major agricultural issues of stone fruits. The virus was detected for the first time in plums and has been reported in the other stone fruit species. The agent, whose dissemination mode is still unknown, has been first reported in cherries in Turkey grown in Malatya province. In this study, the K1 isolate of PBNSPaV, identified in a sweet cherry plant with severe stem pitting and gumming symptoms on its trunk, was partially characterised. A fragment of Hsp70h gene located on ORF3 of viral genome has been cloned, sequenced and analysed phylogenetically (Accession number. FJ231498). The PBNSPaV-K1 isolate showed 93-96% nucleotide sequence identity to sequences of Italian and American isolates in databases. An RNA probe has been raised for fast and reliable detection of the agent by molecular hybridisation. The studies on development of genome specific primers for the detection of the isolate by one-tube RT-PCR have failed. © 2011 Taylor & Francis.
Presence of 'candidatus Phytoplasma Trifolii' in Pepper Plants Showing Yellowing and Bushy Appearance From Iǧdir Province of Turkey
(Ege Universitesi, 2022) Güller, A.; Usta, M.; Korkmaz, G.; Demirel, S.; Kurt, Z.
Objective: The objective of this study was to investigate the group/subgroup of phytoplasma agent in peppers showing phytoplasma symptoms. Material and Methods: In this study, plants collected from Iǧdir province in 2020 were analyzed using direct and nested PCR tests, and BLASTn, iphyClassifier, Mega 7, and pDRAW32 programs were used. Results: In the tests performed, approximately 1.2 kb of DNA fragments specific to phytoplasma were obtained. The 16S rRNA nucleotide sequence (1254 bp in length) (OM663745) revealed that it was showed more than 99.44% nucleotide similarity to other 'Ca. P. trifolii' members. The tentative RFLP and phylogenetic analyzes performed proved the 'Ca. P. trifolii' the infection from the Clover proliferation group (16SrVI) group and subgroup A in symptomatic pepper plants. Conclusion: The presence of 'Ca. P. trifolii' in naturally infected peppers in Iǧdir province of Turkey was detected using PCR-RFLP and cladistic analysis. © 2021 Authors.
Screening of Bean Genotypes Against Bean Common Mosaic Virus (Bcmv) by Artificial Inoculation and Molecular Confirmation
(Centenary University, 2023) Ibrahim, A.S.; Usta, M.; Şensoy, S.
Bean mosaic virus (BCMV) is a widespread plant pathogen that causes significant bean yield losses in several bean-growing regions worldwide. The use of resistant common bean varieties to BCMV is considered the most efficient and feasible approach to control its effects. Numerous genes and molecular markers associated with resistance to these pathogens have been discovered and used extensively in breeding studies around the world. Screening bean genotypes for resistance to these viruses is a critical step in developing resistant varieties. The goals of the study are to identify virus sources in the region and artificially inoculate Lake Van basin bean genotypes with BCMV. The recovered BCMV strain NL-4 was inoculated with 45 bean cultivars, most of which originated from the Lake Van basin in Turkey. Differentiation between resistant and susceptible was based on visual symptoms, and of the 45 genotypes, 29 were found to be resistant to NL-4, while 16 genotypes were susceptible (8 of them moderately susceptible and 8 of them highly susceptible). © 2023, Centenary University. All rights reserved.