Browsing by Author "Usta, Mustafa"

Now showing 1 - 20 of 55

Antiviral and Antifungal Activity of Biologically Active Recombinant Bouganin Protein From Bougainvillea Spectabilis Willd
(Ankara Univ, Fac Agr, 2018) Guller, Abdullah; Siphioglu, Hikmet Murat; Usta, Mustafa; Demirer Durak, Emre
Bouganin antiviral protein (BAP) gene. one of the ribosome inactivating proteins, isolated from Bougainvillea speclabilis Willd. was cloned, expressed and the antiviral and antifungal activities were investigated. The full-length bouganin antiviral protein gene was amplified by reverse transcription-PCR using mRNA as template extracted from mature leaves. The coding region of bouganin gene was cloned into prokaryotic expression vector pETDuet-1 after amplification with end to end gene specific primers. The recombinant plasmid was transformed into Escherichia coli cells BL21(DE3)pLysS and the expression of BAP gene was induced by isopropyl beta-D thiogalactopyranoside (IPTG). Bouganin antiviral protein having a molecular mass of 28 kDa has been isolated from transformed bacterial colonies. Antiviral activity of bouganin was assayed against Zucchini yellow mosaic virus (ZYMV) by a mechanical inoculation test. The antifungal activity of purified recombinant protein was tested against pathogenic and non-pathogenic Rhizocionia solani. Trichoderma harzianum, and Fusarium arysporurn fungi using disc diffusion method. The increased amount of antiviral protein reduced the disease severity caused by ZYMV. The bouganin antiviral protein was inhibited the growth of R. solcati by 30.7% and of T harzianum by 20% after 72 h compared to control. No growth inhibition was observed for F oayporurn. All plants including controls treated with in vitro expressed BAP protein exhibited severe growth reduction compared with negative control (not treated) plants.
Bean Common Mosaic Potyvirus (Bcmv) Characterized From Bean (Phaseolus Vulgaris L.) Crops Affected by Mosaic Disease in Denizli Province, Türkiye
(2023) Usta, Mustafa; Güller, Abdullah
Bean (Phaseolus vulgaris L.) is one of the world's oldest crops with both financial and nutritional importance. Bean common mosaic potyvirus (BCMV) is one of the prevalent viral agents that affect beans across the globe. Determining the presence of the agent in the relevant region is critical for minimizing crop losses by implementing appropriate preventive and control measures. In this study, 73 bean leaf samples were collected from bean-growing areas in the Denizli province of Türkiye in 2022. The samples were screened for the presence of viral agents using Polymerase Chain Reaction (PCR) with specific primers targeting the polypeptide gene. 26 of the bean samples were found to be positive for BCMV. The coat protein gene sequences of two randomly selected positive isolates were sequenced and deposited in the GenBank with accession numbers OQ910196 and OQ910197. The nucleotide sequences of isolates were found to have high similarity with those of isolates identified in various regions of the world. Phylogenetic analysis indicated that these isolates from Denizli, Türkiye were closely related to other Turkish isolates. However, since some Turkish isolates in the cluster associated with the Denizli isolates were identified as belonging to the US-5 or NL-6 strain, the current sequences may be related to these strains. Further research is necessary to identify the exact strain of the Denizli isolates, which could be achieved through the use of a strain differentiation set.
Bingöl İlinde Domateste Stolbur ve Yonca Proliferasyon Fitoplazma Enfeksiyonları
(2020) Güller, Abdullah; Usta, Mustafa
Bingöl ilinde yetiştiriciliği yapılan domates (Lycopersicum esculentum L.) tarlalarında fitoplazma benzeri belirtiler dikkat çekmiştir. Dikkat çeken belirtiler arasında cadı süpürgesi, rozetleme, mor renkli kıvrılmış ve gevrek yapraklar, üst dallarda küçük yapraklılık ve aşırı uzamış kaliks yer almaktadır. Simptomlu ve simptomsuz toplam 11 bitkinin yapraklarından muhtemel patojeni belirleyebilmek için genomik DNA izolasyonu yapılmıştır. Üniversal primer setleri kullanılarak gerçekleştirilen Nested-PCR testlerinde, 11 örneğin 4’ünde yaklaşık 1200 bp büyüklüğünde DNA bantları elde edilmiştir. R16F2n/R16R2 primerleri ile bant veren örneklerden rastgele ikisi seçilerek uygun bir plasmid vektörde klonlanmıştır. Saflaştırılan rekombinant plasmid DNA’sı çift yönlü olarak dizilenmiştir. 16S rDNA dizisinin moleküler analizleri, infekteli domates örneklerinde ‘Canditatus Phytoplasma solani’ (16SrXII-A grup) (benzerlik katsayısı 1.00) (Ulaşım no: MT279680) ve ‘Canditatus Phytoplasma trifolii’(16SrVI-A grup) (benzerlik katsayısı 1.00) (Ulaşım no: MT279852) doğrulamıştır. Domates örneklerindeki fitoplazma izolatları ‘Bingöl D11’ ve ‘Bingöl D90’ olarak isimlendirilmiştir. Oluşturulan filogenetik dendrogram da her iki patojenin ait olduğu yeri doğrulamıştır. Mevcut bu çalışma Bingöl ilinde domatesi doğal olarak enfekte eden “Ca. P. solani”(16SrXII-A) ve “Ca. P. trifolii”(16SrVI-A) nin ilk raporudur.
Bingöl İlinde Kabak Bitkisini (Cucurbita Pepo L.) İnfekteleyen Zucchini Yellow Mosaic Potyvirus (Zymv) Virüs İzolatının Belirlenmesi ve Moleküler Karakterizasyonu
(2019) Güller, Abdullah; Usta, Mustafa
Kabak sarı mozaik potivirüsü (ZYMV), Potyviridae ailesine ait bir virüs olup dünya çapında birçok kabakgil bitkide ciddi ekonomik kayıplara neden olmaktadır. 2018 yılı Eylül ayında, Bingöl iline bağlı kabak yetiştiriciliği yapılan alanlarda kabarıklık ve buruşukluk gibi tipik viral hastalık belirtisi gösteren bitkiler ile sağlıklı bitkilerden örnekler alınarak ZYMV enfeksiyonu bakımından Reverse Transkripsiyon Polimeraz Zircir Reaksiyonu (RT-PCR) ile testlenmiştir. RT-PCR testi pozitif reaksiyon vererek ZYMV’nin kılıf proteinine karşılık gelen yaklaşık 840 bp DNA fragmenti oluşturmuştur. Ayrıca, çoğaltılan ZYMV'nin tam kılıf protein (coat protein, CP) geni, prokaryotik klonlama vektörü olan pGEM-T Easy vektörüne klonlanmış ve genin tam nükleotid ve amino asit dizisi tespit edilmiştir. Elde edilen ZYMV-CP gen dizisinin 279 amino asiti kodladığı (yaklaşık 31.2 kDa) ve 837 nükleotidi içerdiği ortaya çıkarılmış ve bu dizi ZYMV-Bingöl izolatı olarak isimlendirilerek MK689858 erişim numarası ile gen bankasına (NCBI) kaydedilmiştir. ZYMV-Bingol CP geninin dizisi, farklı coğrafi bölgelerden elde edilen 21 izolat ile analiz edilerek filogenetik ilişkileri belirlenmiştir. Nükleotid düzeyindeki moleküler analizlere göre ZYMV CP gen dizisisi, en yüksek % 100 oranında Türkiye izolatı (JF317296) ile ve en düşük % 91.64 oranında Kore izolatı (AF062518) ile benzerlik göstermiştir. Ayrıca, filogenetik analizler, ZYMV Bingöl izolatının Türkiye- Adana izolatı (JF317296) ve Pakistan izolatı (AB127936) ile gruplandığını belirlenmiştir. Bingöl ilinde kabak bitkisini enfekte eden ZYMV’nin bulunuşu ilk kez bu çalışmayla rapor edilmiştir.
Bougainvillea Spectabilis Willd. Bitkisinin Biyolojik Olarak Aktif Rekombinant Bouganin Proteininin Antiviral Ve Antifungal Aktivitesi
(2018) Durak, Emre Demirer; Sipahioglu, Hikmet Murat; Usta, Mustafa; Güller, Abdullah
Bougainvillea spectabilis Willd. bitkisinden ribozom inaktive eden proteinlerden olan Bouganin antiviral protein (BAP) geni izole edilerek klonlanmış, ifade edilmiş, antiviral ve antifungal özellikleri araştırılmıştır. Bouganin proteinini kodlayan genin tamamı bitkinin olgun yapraklarından ekstrakte edilen mRNA’dan Reverse Transkripsiyon-PCR yöntemi ile çoğaltılmıştır. Gen, uçtan uca bölgeyi kapsayacak şekilde tasarlanan spesifik primerler yardımı ile prokaryotik ekspresyon vektörü pETDuet-1’de klonlanmıştır. BAP geninin ifadesi rekombinant plazmitin Escherichia coli (BL21(DE3)pLysS) hücrelerine transferi sonrası isopropyl β-D thiogalactopyranoside (IPTG) ile teşvik edilmiştir. Molekül ağırlığı yaklaşık 28 kDa olan BAP transforme edilmiş bakterilerden izole edilmiştir. BAP ‘ın antiviral aktivitesi Zucchini yellow mosaic virus (ZYMV) kullanılarak mekanik inokulasyon yöntemi ile araştırılmıştır. Antifungal aktivitenin belirlenmesi disk difüzyon metodu yardımıyla patojen ve patojen olmayan Rhizoctonia solani, Trichoderma harzianum ve Fusarium oxysporum fungusları ile araştırılmıştır. BAP uygulama miktarı arttıkça ZYMV’nin neden olduğu hastalık şiddetinin azaldığı tespit edilmiştir. BAP 72 saatlik kontrol uygulaması ile karşılaştırıldığında, Rhizoctonia solani’nin gelişimini % 30.7, Trichoderma harzianum’unun gelişimini ise % 20 oranında inhibe etmiştir. Fusarium oxysporum’un gelişiminde herhangi bir inhibisyon etkisi gözlenmemiştir. In vitro’da ifade edilen BAP proteininin uygulandığı tüm bitkiler (sadece BAP protieni uygulanan kontrol grubu da dahil) uygulama yapılmayan bitkilere oranla şiddetli gelişme geriliği sergilemiştir.
Cloning and Sequencing of Coat Protein Gene of Zucchini Yellow Mosaic Virus Isolated From Squash and Muskmelon in Turkey
(Tubitak Scientific & Technological Research Council Turkey, 2012) Ozer, Meryem; Sipahioglu, Hikmet Murat; Usta, Mustafa; Fidan, Hakan
The coat protein (CP) genes of the genomic RNA of 2 severe Turkish isolates of Zucchini yellow mosaic virus (ZYMV) from squash and muskmelon [ZYMV-Adana (Ad) and ZYMV-Ahlat (Ah), respectively] were cloned, and their complete nucleotide sequences and deduced amino acids were determined. The analysis revealed that both Turkish ZYMV-CP genes contained 837 nucleotides encoded for a CP of about 31.2 kDa. Phylogenetic trees based on nucleic acid sequences were constructed by the neighbor joining and unweighted pair group mean arithmetic (UPGMA) methods with 100 bootstrap replicates. A high degree of homology was detected between the 2 Turkish isolates on the nucleotide sequences (97%). The CP sequence of ZYMV-Ad and ZYMV-Ah varied among the 23 isolates with overall identity of 93%-98% and 94%-99%, respectively, at the nucleotide level. Comparison of the nucleotide sequences of 23 isolates from different geographical regions worldwide showed the ZYMV-Ad isolate clustered with isolates from Middle Eastern countries (Israel, Jordan, and Syria); ZYMV-Ah isolate was clustered with isolates from Far Eastern countries (Korea and Taiwan). The N-terminal of the Turkish ZYMV-Ah CP contained a distinctive sequence at nucleotide positions 9324-9328, which distinguished the Turkish ZYMV-Ah isolate from all previously reported ZYMV isolates. The CP cistron of the ZYMV-Ad and Ah isolates contained 279 amino acid residues. Pairwise nucleotide sequence comparison revealed sequence similarities of 58%-70% between ZYMV Turkish isolates and 22 other potyviruses. Mechanical inoculations showed that ZYMV-Ah produced faster systemic symptom induction than ZYMV-Ad on squash, suggesting that ZYMV-Ah was a more severe isolate than ZYMV-Ad (GenBank accession JF317296-JF317297).
Cloning, Bacterial Expression, Purification and the Antiviral Effect on Zucchini Yellow Mosaic Virus (zymv) of Tritin Protein Gene Which Is Inactivating Ribosome in Wheat (triticum Aestivum L.)
(2022) Demirel, Serap; Usta, Mustafa
Bitki virüsleri ekonomik olarak önemli kayıplara neden olan ve etkin mücadele yöntemleri çoğunlukla olmayan patojenlerdir. Bu tez çalışması kapsamında, buğday bitkisinden rekombinant tritin proteinin elde edilmesi ve proteinin sakız kabağı (Cucurbita pepo L.) bitkisinde Zucchini yellow mosaic virus (ZYMV)'üne karşı antiviral aktivitesi ortaya konmaya çalışılmıştır. Ekmeklik buğday çeşidi Kutluk-94'ten izole edilen total ribonükleik asit (RNA) komplementer DNA sentezinde (cDNA) kullanılmıştır. Gen spesifik primerlerle tritin geni RT-PZR ile çoğaltılarak izole edilmiştir. Tritin geni pGEM-T Easy ve pETDuet-1 vektörüne klonlanmıştır. E. coli BL21 (DE3) hücrelerinde tritin proteinin ekpresyonu sağlandıktan sonra, tritin proteini izole edilmiş ve saflaştırılmıştır. SDS-PAGE ile doğrulananan proteinin konsantrasyonu 12.56 mg/ml olarak belirlenmiştir. Sadece ZYMV uygulanan bitkilerde hastalık şiddeti %92.74 iken, 2 mg/ml tritin uygulanan bitkilerde hastalık şiddeti %46.66 olarak hesaplanmıştır. Hastalık şiddetinde azalışa neden olan tritin proteini, aynı zamanda ZYMV-CP geninin ekpresyon seviyesinde de azalışa neden olmuştur. Tritin uygulanan bitkilerde pathogenesis related gene 1 (PR1) geninin ifadesinin arttığı real time PZR ile ortaya konmuştur. Tritinin ZYMV virüsüne karşı antiviral etkisine ilaveten, morfolojik veriler farklı konsantrasyonlardaki tritin proteinin sakız kabağı bitkilerine fitotoksik etki ettiğini göstermiştir.
Coat Protein of Alfalfa Mosaic Alfamovirus (Amv) From Türkiye: Genetic Inference and in Silico Docking Analysis for Potential Antiphytoviral Purposes
(Univ Agr Sci & veterinary Med Cluj-napoca, 2024) Demirel, Serap; Guller, Abdullah; Usta, Mustafa; Kurt, Zeynelabidin; Korkmaz, Gulustan
In 2021, a study was conducted in the Denizli region of Turkiye to investigate the phylogenetic relationship and presence of alfalfa mosaic alfamovirus (AMV) infecting pepper plants exhibiting viral disease symptoms. A total of 57 samples were collected, of which twenty-four tested positive for AMV with reverse transcriptase polymerase chain reaction (RT-PCR) assays. Samples from pepper plants displaying virus symptoms gave positive bands on the agarose gel, while healthy plants yielded negative results. One of the positive samples was randomly selected, cloned and sequenced. This sequence of the Denizli AMV isolate (753 bp) was recorded in the GenBank database with accession number OQ845956. The nucleotide sequence showed a high nucleotide consensus of 97%-99% compared with the nucleotide sequences of the same variants from different origins in GenBank. According to the phylogenetic tree generated through the Neighbour Joining (NJ) method, this AMV isolate belongs to the same group as Iranian isolates from various of hosts. Furthermore, in silico docking analysis of the coat protein (CP) of the AMV isolate and promising 12 essential oil compounds was performed to enable potential antiviral drug development. Docking study showed that eucalyptol, eugenol and carvacrol can make important contributions to the advancement of drug -based strategies for the managing of plant viruses by interacting with the virus coat protein of high binding energies, -5.3, -5.2 and -5.0 kcal mol-1, respectively. Although the presence of AMV in Denizli province has been reported previously, this study reports the phylogenetic relationships and docking analysis of the new AMV isolate in pepper crops.
Comprehensive Survey of Common Potato Viruses in Eastern Anatolia Region of Turkey: New Isolates and Phylogenetic Insights
(Springer, 2025) Korkmaz, Gueluestan; Usta, Mustafa; Guller, Abdullah; Demirel, Serap
Potatoes (Solanumtuberosum L.) are a vital crop in Turkey, contributing significantly to the country's economy. However, they are susceptible to various viral infections, causing substantial yield losses if left unmanaged. This study aimed to conduct a comprehensive survey of economically significant viruses affecting potatoes in the Eastern Anatolia Region, Turkey, including the identification of new isolates and the inference of their phylogenetic relationships. A total of 1130 fresh leaf samples, including asymptomatic and symptomatic plant samples, were tested using the multiplex RT-PCR (m-RT-PCR) method to determine the prevalence of five viral pathogens. The test results revealed the presence of four viral pathogens, with an overall infection incidence of 37.08%. PVY was found in 35% of the samples, PVS in 8.5%, PVX in 1.8%, and PLRV in 1%. However, PVA was not detected. In addition, 74 of the 98 samples tested were positive for PVY + PVS, 13 for PVY + PVX, 2 for PVY + PLRV, 8 for PVY + PVS + PVX, and 1 for PVY + PVS + PLRV. From each sample that tested positive, two isolates were selected, and the complete coat protein (CP) gene regions of these isolates were cloned and sequenced. The sequences of the new potato virus isolates were deposited in the GenBank. Nucleic acid sequence comparisons revealed a consensus of over 99% among all available isolates and those from other regions of the world. However, this similarity is not specific to any particular host or region. In support of the similarities in nucleic acid, phylogenetic analyses also provided information on the evolutionary relatedness of these isolates from various origins and hosts. This finding suggests that the genetic makeup of these isolates is highly conserved across different populations and environments. This study, conducted in the Eastern Anatolia Region of Turkey, is the first comprehensive report on the presence and molecular phylogeny of four viral pathogens of economic importance in potato crops.
Concurrent Detection of Five Yellow Dwarf Viruses ( B/Cydvs) in Wheat in Mardin (Turkey) and Phylogenetic Relationship of Bydv-Pav
(Univ Agr Sci & veterinary Med Cluj-napoca, 2020) Karaozan, Abdulselam; Usta, Mustafa
Wheat (Triticum aestivum L.) is a natural host of many viruses. Yellow dwarf viruses belonging to the Luteoviridae family are important virus species that cause economic loss by restricting wheat production worldwide. Surveys were conducted in 2017 to determine Yellow dwarf viruses (BYDV-PAV, BYDV-MAV, BYDV-SGV, BYDV-RMV, and CYDV-RPV) and their infection rates in wheat production areas in Mardin province. 400 fresh leaf samples collected were tested by Multiplex reverse transcription-polymerase chain reaction (m-RT-PCR). The overall infection rate was found to be 3%. BYDV-PAV has been identified as the most widespread virus with a 2.5% presence rate. It was found out that BYDV-SGV, CYDV-RPV, and BYDVRMV infections were lower, with rates of 1.75%, 0.5% and 0.25% respectively. In the current study, double infections were detected in 8 samples. The overall infection rate of the detected viruses (BYDV-PAV, BYDVSGV, CYDV-RPV, BYDV-RMV) was found to be lower than the records reported in previous similar studies. No BYDV-MAV infection was found in any of the wheat samples tested. The cDNA of the coat protein (CP) gene of a BYDV-PAV isolate randomly selected from virus-positive samples was cloned, bidirectionally sequenced, and the phylogenetic relationship revealed. According to the phylogenetic analysis with 19 different isolates in the NCBI database of BYDV-PAV Mardin isolate, it showed the highest genetic similarity by 95.52% with the Germany isolate (KY634926) while the lowest similarity rate was 89.22% with the Germany and Pakistan isolates (KY634886 and JQ811489). The presence of BYDV-PAV, BYDV-SGV, CYDV-RPV, and BYDV-RMV were reported for the first time with this survey study conducted in Mardin.
Current Status and Molecular Phylogeny of Some Economically Important Viruses Infecting Wheat Crops of Sirnak Province, Turkey
(United Arab Emirates Univ, 2023) Kapan, Orhan; Usta, Mustafa; Guller, Abdullah
A survey of yellow dwarf viruses (YDVs)-associated diseases of wheat plants was performed for the first time in the Sirnak Province, Turkey. In plants wheat cultivated areas, symptoms such as diminished leaf size, dwarfing, yellowing, and reddening of leaves wheat plants were observed in cultivated areas. A total of 441 specimens were collected regardless of showing symptoms and assayed for Barley/Cereal Yellow Dwarf Viruses [B/CYDV] association by PCR tests using virus coat protein gene (CPG)-specific primer pairs. These assays demonstrated that 13.38% (55 samples) were positive to the occurrence of BYDV-PAV (9.73%) and CYDV-RPV (0.73%) for wheat viruses, but no BYDV-MAV,-SGV, and-RMV infections of BYDVs were found in PCR assays of collected samples. In addition, double infection of BYDV-PAV and CYDV-RPV was detected in 12 samples with a 2.91% infection rate. The CPG sequences of randomly selected two isolates were revealed using bacterial cloning and sequencing. Sequences obtained were deposited in GenBank under Accession numbers OL685734 and OL685736 for BYDV-PAV; and OL685735 and OL685737 for CYDV-RPV. BLASTn analysis of CPG sequences of four isolates shared high nucleotide homology with their intraspecies isolates, with 97.35-98.67% for two BYDV-PAV and 97.72-97.89 for two CYDV-RPV. The consensus tree constructed classified B/CYDV-associated wheat viruses with their phylogenetically closest individuals from Turkey and the world. This survey is the first report of BYDV-PAV and CYDV-RPV associated with wheat plants in Sirnak Province of Turkey.
Dedection of Viroid, Phytoplasma and Fungal Diseases of Stone Fruit Trees Grown in East Anatolia by Molecular Methods
(2005) Selçuk, Muharrem; Polat, Bülent; Usta, Mustafa; Demır, Semra; Akköprü, Ahmet; Sipahioğlu, H. Murat
Doğu Anadolu Bölgesinde yetiştiriciliği yapılan sert çekirdekli meyvelerdeki viroid, fitoplazma ve fungal hastalıkları belirlemek amacıyla 2003-2004 yıllarmm haziran-ekim aylan arasında geniş çaplı survey çalışmaları yürütülmüştür. Peach latent mosaic (PLMVd) ve Hop stunt (HSVd) viroidlerini araştırmada moleküler hibridizasyon (doku bastırma ve dot blot) testi kullanılmıştır. Testlenen 491 sert çekirdekli meyve örneğinin 16'smda viroid infeksiyonu tespit edilmiştir. Tespit edilen infeksiyonlarından 15'inin şeftalilerde PLMVd viroidine (%3) birinin ise kayısıda HSVd viroidine (%0.1) ait olduğu belirlenmiştir. Viroid hastalıklarından ileri gelen toplam infeksiyon oranı %3.2 olarak belirlenmiştir. HSVd viroidinin Doğu Anadolu bölgesindeki varlığı ilk defa bu çalışma ile tespit edilirken, bölgenin önemli kayısı yetiştiricisi illeri olan Malatya ve Elazığ'da PLMVd infeksiyonuna rastlanmamıştır. 'Candidates Phytoplasma prunorum\"un kayısılardaki varlığını tespit etmek amacıyla simptomatik örneklere gerçekleştirilen PCR testi sonucunda infekteli örneğe rastlanmamıştır. Bölgeden toplanan simptomatik sert çekirdekli meyve örneklerinden Armillaria mellea, Cytospora spp., Monilinia laxa, Stigmina carpophila, Chondrostereum purpureum, Fusarium spp., Rosellinia spp. ve Phytophthora spp fungusları tespit edilmiştir.
Detection, in Silico Analysis and Molecular Diversity of Phytoplasmas From Solanaceous Crops in Turkey
(Czech Academy Agricultural Sciences, 2022) Usta, Mustafa; Guller, Abdullah; Sipahioglu, Hikmet Murat
Phytoplasma-like symptoms of leaf yellowing and calyx malformation were observed in eggplant (Solanum melongena L.), upward leaves and fruit malformation in pepper (Capsicum annuum L.), and aerial tuber formation in potato (S. tuberosum L.) during the survey performed in the late season (August to September) of 2015 and 2016 in Van province (Turkey). A total of 100 samples were tested by nested-PCR using universal primer pairs to assess the sanitary status of the solanaceous crops and to characterise the phytoplasma isolates. Among them, seven sam-ples resulted in a 1.25 kb DNA fragment, and five (two eggplants, two peppers, and one potato) were molecularly characterised (Accession No.: KY579357, KT595210, MF564267, MF564266, and MH683601). BLAST and the virtual restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes revealed the presence of two distinct phytoplasma infections in solanaceous crops: `Candidatus Phytoplasma trifolii' a member of the clover proliferation group (16SrVI) and subgroup A and `Candidatus P. solani' a member of the stolbur group (16SrXII) and subgroup A. The virtual RFLP analysis and calculated coefficients of RFLP pattern similarities further revealed a remarkable ge-netic diversity among the `Candidatus P. solani' isolates infecting pepper (similarity coefficient of 0.90) and eggplant (similarity coefficients of 0.98 and 1.00) at the same geographical area. This is the first report of the natural occurrence of `Candidadtus P. trifolii' in potato from the Eastern Anatolia region, Turkey.
Determination of Important Potatoes Virus Diseases in the Eastern Anatolia by Multiplex Rt-Pcr and Characterization of Virus Isolates
(2022) Korkmaz, Gülüstan; Usta, Mustafa
Bu çalışmada Doğu Anadolu Bölgesi'ndeki tarlalardan toplanan 1130 adet patates bitkisi yaprak örneğinde; multipleks RT-PCR yöntemi ile önemli 5 viral etmenin yaygınlığı araştırılmıştır. Yapılan multipleks RT-PCR testi sonuçlarına göre; araştırılan viral etmenlerin Doğu Anadolu Bölgesindeki yaygınlığı sırasıyla (PVY) %35, (PVS) %8,5, (PVX) %1,8 ve (PLRV) %1 olarak teşhis edilmekle birlikte PVA varlığı tespit edilememiştir. Test edilen 1130 örnekten 419'unun araştırılan virüslerden en az bir tanesi ile bulaşık olduğu ve 74 PVY+PVS, 13 PVY+PVX, 2 PVY+PLRV, 8 PVY+PVS+PVX ve 1 PVY+PVS+PLRV olmak üzere 98'inde karışık infeksiyonlar oluştuğu tespit edilmiştir. PVY pozitif sonuç veren 396 örnekte bazı PVY ırkları multipleks RT-PCR yöntemi ile araştırılmıştır. Yapılan multipleks RT-PCR test sonuçlarına göre; örneklerin %45.2'sinde PVYN:O, %34.59'unda PVYNTN, 10.35'inde PVYNTN+PVYN:O ve %0.25'inde PVYO+PVYNTN ırklarının varlığı tespit edilmiştir. Tespit edilen virüs izolatlarının her birinden 2 adet seçilerek kılıf protein gen bölgelerinin karakterizasyonu sağlanmıştır. PLRV; Bitlis 534 (erişim no: MZ543310) ve Bitlis 581 (erişim no: MZ543311), 'PVS; Erzurum 388 (erişim no: MZ543314) ve Bitlis 694 (erişim no: MZ543315)', 'PVX; Erzurum 291 (erişim no: MZ543312) ve Erzurum 297 (erişim no: MZ543313)', 'PVY; Erzurum 391 (erişim no: OK554546) ve Bitlis 693 (erişim no: OL332045)' izolatlarının kılıf protein gen bölgelerinin tamamı klonlanmış ve genom dizilemeleri gerçekleştirilmiştir. Elde edilen kılıf protein gen dizileri dünyadaki diğer izolatlara ait gen dizileriyle karşılaştırılarak filogenetik ilişkileri ortaya konulmuştur. Tüm izolatların kendi aralarında ve dünyadaki diğer izolatlarla yapılan ikili baz çifti karşılaştırmalarında; aralarında yüksek oranda tutarlılık olduğu, aralarındaki bu benzerliğin konukçularına ya da belirli bölgelere özelleşmediği ortaya konulmuştur.
Determining Phylogenetic Relationships of Some Endemic and Rare Astragalus Taxa in the Van Lake Basin
(Kahramanmaras Sutcu Imam Univ Rektorlugu, 2023) Genli, Gulistan; Tekbudak, Ilhan Kaya; Usta, Mustafa; Mukemre, Muzaffer
Astragalus, the most diverse genus in the world, contains about 3000 different taxa. This genus has 489 taxa and 63 sections in Turkey, giving it a 51% endemism rate. 82 Astragalus taxa were collected from the Van Lake Basin in 2019 for this research. These species' morphological diagnoses revealed that four of them were rare and 17 of them were endemic. In this research, the universal primers ITS4 and ITS5 were used to amplify the ITS1 and ITS2 sections, which included the 5.8S gene of rDNA. Different programs (SnapGene, CLC DNA Workbench) were used to analyze the genome information of species in the genus Astragalus. The DNA sequences of Astragalus species have been uploaded to the GenBank database of the National Center for Biotechnology Information (NCBI) (Maryland, USA), which is open to all researchers worldwide. The length of the ITS in the study, including the 5.8S sequence, varied from 669 to 687 bp. The results showed that the phylogenetic tree combined with the most stable secondary (20) structure obtained from the universal ITS4 and ITS5 primers is an effective tool for the identification of Astragalus taxa.
Diyarbakır İlinde Yetiştirilen Hıyar Bitkilerinde Hıyar Mozayik Virüsü’nün Genetik Çeşitliliği
(2024) Güller, Abdullah; Demirel, Serap; Usta, Mustafa; Korkmaz, Gülüstan
Hıyar bitkisi (Cucumis sativus L.) dünya çapında üretilen önemli bir kültür bitkisidir. Yaygın viral hastalıklarından biri olan Hıyar mozayik virüsü (Cucumber mosaic virus, CMV) hıyar bitkisinde verim ile birlilkte kaliteyi düşürerek ekonomik kayıplara neden olmaktadır. Diyarbakır ilinde 2021 yılı Eylül ayında yapılan gözlemlerde, yapraklarda mozaik, düzensiz sarımsı lekeler ve deformite gibi virüs benzeri simptomlar gösteren hıyar bitkileri gözlenmiştir. Simptom gösteren ve göstermeyen örnekler toplanarak CMV spesifik primerler kullanılarak ters transkriptaz polimeraz zincir reaksiyonu (RT-PZR)’na tabi tutulmuş ve üretilen DNA bantları agaroz jelde görüntülenmiştir. CMV infeksiyonu 15 örnekten yedisinde belirlenmiştir. Rastgele seçilen bir örneğin bakteriyel klonlanması ve dizilenmesi sonucunda, CMV kısmi kılıf proteni geninin 593 bp uzunluğunda olduğu belirlenmiş ve NCBI veri tabanına MW962979.1 erişim numarası ile kaydedilmiştir. CMV’nin farklı izolatlarıyla gerçekleştirilen filogenetik ağaca göre, Diyarbakır CMV izolatı Subgrup IA grubunu oluşturan Avustralya, İsrail, İspanya, Macaristan, Japonya ve Kore’ye ait CMV izolatları ile kümelenmiştir. Diyarbakır bölgesinde yetiştirilen hıyar bitkilerinde CMV’nin varlığı ve grup/subgrup teşhisi ilk defa bu çalışma ile doğrulanmıştır.
Erzincan İlinde Bal Arısı (Apis Mellifera L.) Kolonilerinde Tespit Edilen Deforme Kanat Virüsünün Moleküler Karakterizasyonu
(2021) Çakar, Gözdenur; Güller, Abdullah; Kurt, Zeyneabidin; Usta, Mustafa
Iflaviridae ailesine ait olan deforme kanat virüsü (DWV), tüm dünyada bal arısı kolonilerinde ciddi maddi kayıplara neden olan yıkıcı bir virüstür. 2020'de (Kasım) Erzincan ilinden toplanan virüs şüphesi olan bal arısı örnekleri, DWV enfeksiyonuna karşı moleküler testlerle tarandı. Test edilen 12 örneğin 6'sında muhtemel virüs varlığını gösteren yaklaşık 700 bp DNA bandı elde edildi. Virüsün varlığını gösteren bantlardan rastgele seçilen iki amplifiye edilmiş DNA bandı pGEM-T Easy vektörüne klonlanmış ve elektriksel olarak kompetan E. coli bakterisine aktarılmıştır. Klonlamadan elde edilen rekombinant plazmitler, E. coli'den saflaştırılmış ve yeni nesil dizileme (NGS) ile dizilenmiştir. DWV'nin kısmi nükleotid dizilerinin 711 nükleotid içerdiği tespit edilmiş ve bu diziler MW962981 ve MW962982 erişim numarası ile NCBI GenBank'a kaydedilmiştir. Her iki izolatın en yüksek dizi benzerliği, nükleotid düzeyinde %98.03 ile %98.17 arasında değişmiştir. Ayrıca, Erzincan DWV dizilerinin filogenetik ilişkileri, GenBank'ta kayıtlı farklı ekolojik bölgelerden 15 benzersiz dizi kullanılarak ortaya çıkarılmıştır. Filogenetik ağaç, DWV Erzincan izolatlarının İngiltere (HM067438), İspanya (MK262743) ve İsveç (MN746311) izolatları ile kümelendiğini göstermiştir. Literatür bilgilerimize göre, bu çalışma ile DWV (Deforme kanat virüsü) Türkiye'nin Erzincan ili bal arılarında ilk kez rapor edilmiştir.
First Investigation of Five Tobacco Viruses Using Pcr Based Methods in Tobacco Plants Grown in Adiyaman, Turkey
(Parlar Scientific Publications (p S P), 2020) Gunay, Abidin; Usta, Mustafa
Survey studies were carried out in in 2018 in Adiyaman, Turkey, where tobacco is traditionally produced, to detect the viruses that infect tobacco including Tobacco mosaic tobamovirus (TMV), Cucumber mosaic cucumovirus (CMV), Potato potyvirus Y (PVY), Tobacco vein banding mosaic potyvirus (TVBMV), and Tobacco etch potyvirus (TEV). Accordingly, 450 fresh tobacco leaves were simultaneously analyzed by the multiplex RT-PCR method for the presence of tobacco viruses. As a result of molecular analysis, CMV (8.8%), TMV (5.5%), and TMV + CMV mixed infection (0.2%) were detected in 40 of the tobacco samples. TEV, PVY, and TVMBV viruses were not detected in any of the samples. The overall infection incidence of tobacco viruses in Adiyaman province was found to be 14.4%. Two isolates were randomly selected from each detected virus and their coat protein (CP) genes were cloned into the prokaryotic cloning vector and transformed into E. coli. Recombinant plasmids obtained from positive clones were sequenced by Next Generation Sequencing (NGS) system to reveal the viral (CP) sequence. Nucleic acid sequence similarity analyses revealed that the TMV Adiyaman isolates showed similarity in the range of 74.17-99.38% and the CMV Adiyaman isolates in the range of 75.61-99.54%. In the phylogenetic analyis using (CP) sequences, tobacco-CMV isolates were found to be in phylogenetic affinity with the Greece (AY450854) and Japan (AB448696) isolates, while both tobacco-TMV isolates showed a phylogenetic affinity with the Serbia isolate (GQ340671). According to the current literature, this is the first molecular survey study on tobacco viruses in Adiyaman and Turkey. With the present study, the molecular char acterization of the isolates selected with the presence of CMV and TMV in the tobacco plant has been re- ported for the first time.
First Report of "candidatus Phytoplasma Solani" on a New Host Marigold (Tagetes Erecta L.)
(Tubitak Scientific & Technological Research Council Turkey, 2016) Alp, Sevket; Usta, Mustafa; Sipahioglu, Hikmet Murat; Guller, Abdullah
Marigold (Tagetes erecta L.) plants, also called Mexican or Aztec marigold, with symptoms of shoot proliferation, dwarfing, and reddening were observed in ornamental gardens of Van Province (Turkey). Five plants, two of them showing reddening and three symptomless plants, were sampled at the end of September 2014. Genomic DNA isolated from symptomatic and nonsymptomatic plant leaves was used to amplify 16S rDNA fragments by nested polymerase chain reaction (PCR). Of the 5 marigold samples tested by PCR, only the two showing reddening symptoms yielded the expected 1.2-kb DNA fragments. Amplified PCR fragments were cloned into a plasmid vector and transformed into competent Escherichia coli strain JM 109. Recombinant plasmid DNA was isolated and sequenced bidirectionally. The provided sequences were 1244 bp and 1245 bp in length and were designated as isolate 1 and isolate 2, respectively. BLAST analysis of the 16S rDNA sequence and virtual restriction fragment length polymorphism (RFLP) analysis confirmed the presence of the phytoplasma "Candidatus Phytoplasma solani". The in silico virtual RFLP pattern of isolate 1, based on the 16S rDNA F2n/R2 fragment, was identical (similarity coefficient 1.00) to the reference pattern of 16Sr group XII, subgroup A (GenBank accession no.: AF248959). Isolate 1 was identified as a member of 16SrXII-A. Based on the same analyses, isolate 2 showed molecular characteristics different from reference patterns of all previously established 16Sr groups and subgroups. The most similar was the reference pattern of 16Sr group XII, subgroup A (GenBank accession no.: AF248959), with a similarity coefficient of 0.97. This is the first report of naturally occurring "Ca. P. solani" affecting T. erecta, which shows that this plant species is an alternate host of the agent.
First Report of 'candidatus Phytoplasma Trifolii' Associated With Leaf Reddening and Upright Growth in Pears (Pyrus Communis L.)
(Czech Academy Agricultural Sciences, 2021) Usta, Mustafa; Guller, Abdullah; Sipahioglu, Hikmet Murat
The natural occurrence of 'Candidatus Phytoplasma trifolii' in pear trees (Pyrus communis Linnaeus) is reported here for the first time. In 2017, a total of thirty-five pear trees, two of them exhibiting leaf rolling along the midvein, reddening, bushy appearance, and upright growth symptoms were sampled in different locations in Van province, Turkey. The total deoxyribonucleic acid was extracted from symptomatic and asymptomatic plants. The purified DNA served as a template in nested polymerase chain reaction (nested-PCR) assays, performed to amplify 16S rRNA sequences using universal primer pairs (R16mF2/R16mR1 and R16F2n/R16R2). The resulting PCR products were then cloned into a pGEM T-Easy vector and sequenced bidirectionally. The phytoplasma strain, group, and subgroup identity were determined using the in silico restriction fragment length polymorphism (RFLP) analysis of the 16S ribosomal RNA-encoding gene sequences profiling with seventeen distinct restriction enzymes. Of the thirty-five pear samples, only two yielded 1 256 bp and 1 258 bp DNA fragments and were designated as Van-Pr3 (Acc. No. MH709141) and Van-Pr4 (Acc. No. MH730561), respectively. Based on the in silico virtual RFLP pattern analysis of the 16S rRNA sequences, we confirmed the presence of 'Ca. P. trifolii' belonging to the clover proliferation group and both identified phytoplasmas were identical with the similarity coefficient of 1.00 to the reference pattern of 16Sr group VI, subgroup A (Acc. No. AY390261). Here we report that the pear tree is an alternate host of the 'Ca. P. trifolii'.