Malatya, Elazığ ve Adıyaman illeri badem (Prunus dulcis Mill.) ve ceviz (Juglans regia L.) üretim alanlarında Peach latent mosaic viroid (plmvd) etmeninin araştırılması ve moleküler karakterizasyonu
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Tarih
2021
Yazarlar
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Yayıncı
Malatya Turgut Özal Üniversitesi / Lisansüstü Eğitim Enstitüsü / Bitki Koruma Ana Bilim Dalı
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Malatya, Elazığ ve Adıyaman illeri badem ve ceviz üretim alanlarında Peach latent mosaic viroid (PLMVd)'inin belirlenmesi amacıyla 2019-2020 yıllarında survey çalışmaları yürütülmüştür. Badem ve ceviz üretim alanlarından rastgele toplam 530 yaprak örneği toplanmış ve total RNA izolasyonları gerçekleştirilmiştir. PLMVd'nin toplanan örneklerdeki varlığını araştırmak için ters transkriptaz polimeraz zincir reaksiyonu (reverse transcriptase polymerase chain reaction, RT-PCR) yöntemi kullanılmıştır. RT-PCR yöntemi ile testlenen badem örneklerinin hiçbirinde PLMVd infeksiyonuna rastlanmamıştır. Testlenen ceviz örnekleri içerisinde Malatya ili Hekimhan ilçesinden alınan örneklerin tamamında (10 örnek, H izolatları) ve Elazığ ili Keban ilçesinden alınan örneklerden sadece birinde (25 örnek, K1 izolatı) PLMVd'nin varlığı tespit edilmiştir. PLMVd-K1 izolatı ve rastgele seçilen H1, H2 ve H3 izolatlarının tüm genomları, bir TA klonlama vektörü olan pGEM-T Easy vektöre aktarılarak klonlanmıştır. Saflaştırılan rekombinant plazmidler, üniversal SP6 ve T7 primerleri ile çift yönlü olarak dizilenmiş ve elde edilen PLMVd dizileri CLC Mainworkbench programında üst üste çakıştırılmış (contig) ve referans dizi baz alınarak manuel olarak düzeltilmiştir. Elde edilen konsensus dizilerine göre; PLMVd-K1 (MW294170), -H1 (MW294171), -H2 (MW294172) ve -H3 (MW294173) izolatlarının 338 bp uzunluğunda oldukları tespit edilmiştir. RNA katlanma analizlerinde, tespit edilen dört PLMVd-ceviz izolatının da referans PLMVd izolatının (M83545) oluşturduğu gibi dallanmış küre şekilinde ikincil yapı oluşturdukları görülmüştür. Cevizlerde tespit edilen PLMVd izolatlarının dünyanın farklı coğrafi bölgelerinde tespit edilen 20 farklı PLMVd izolatının nükleotid dizileri ile %96.7-99.1 arasında benzerlik gösterdiği belirlenmiştir. Malatya-Hekimhan ve Elazığ illeri ceviz üretim alanlarında PLMVd infeksiyonlarının varlığı ilk defa bu çalışma ile ortaya konmuştur.
Survey studies were conducted to investigate the presence of Peach latent mosaic viroid (PLMVd) infections in almond and walnut production areas of Malatya, Elazig and Adiyaman provinces between 2019-2020. A total of 530 leaf samples were randomly collected in almond and walnut orchards and total RNA were extracted. Reverse transcriptase polymerase chain reaction (RT-PCR) method was implemented to investigate the presence of PLMVd infections in collected samples. PLMVd was not detected in any of the almond samples tested by RT-PCR method. Among the walnut samples tested, the presence of PLMVd was identified in all of the samples taken from the Hekimhan district of Malatya province (10 samples, H isolates) and in only one sample taken from Keban district of Elazığ province (25 samples, K1 isolate). The purified recombinant plasmids were bidirectionally sequenced with universal SP6 and T7 primers, and the obtained PLMVd sequences were overlaped with the help of CLC Mainworkbench program and editid manually. According to the consensus sequences, PLMVd-K1 (MW294170), -H1 (MW294171), -H2 (MW294172) and -H3 (MW294173) isolates were found to be 338 bp in length. In RNA folding analyses, it was observed that all four detected PLMVd-walnut isolates formed a branched secondary structure, as did the reference PLMVd isolate (M83545). It was determined that PLMVd isolates detected in walnuts showed similarity between 96.7-99.1% with the nucleotide sequences of 20 different PLMVd isolates detected in different geographical regions of the world. With this study, the PLMVd was identified for the first time in the walnut production areas of Malatya-Hekimhan and Elazig provinces.
Survey studies were conducted to investigate the presence of Peach latent mosaic viroid (PLMVd) infections in almond and walnut production areas of Malatya, Elazig and Adiyaman provinces between 2019-2020. A total of 530 leaf samples were randomly collected in almond and walnut orchards and total RNA were extracted. Reverse transcriptase polymerase chain reaction (RT-PCR) method was implemented to investigate the presence of PLMVd infections in collected samples. PLMVd was not detected in any of the almond samples tested by RT-PCR method. Among the walnut samples tested, the presence of PLMVd was identified in all of the samples taken from the Hekimhan district of Malatya province (10 samples, H isolates) and in only one sample taken from Keban district of Elazığ province (25 samples, K1 isolate). The purified recombinant plasmids were bidirectionally sequenced with universal SP6 and T7 primers, and the obtained PLMVd sequences were overlaped with the help of CLC Mainworkbench program and editid manually. According to the consensus sequences, PLMVd-K1 (MW294170), -H1 (MW294171), -H2 (MW294172) and -H3 (MW294173) isolates were found to be 338 bp in length. In RNA folding analyses, it was observed that all four detected PLMVd-walnut isolates formed a branched secondary structure, as did the reference PLMVd isolate (M83545). It was determined that PLMVd isolates detected in walnuts showed similarity between 96.7-99.1% with the nucleotide sequences of 20 different PLMVd isolates detected in different geographical regions of the world. With this study, the PLMVd was identified for the first time in the walnut production areas of Malatya-Hekimhan and Elazig provinces.
Açıklama
Tez No 677460
Anahtar Kelimeler
Adıyaman, Badem, Ceviz, Elazığ, Malatya, Moleküler karakterizasyon, PLMVd, RT-PCR, Amond, Walnut, Molecular characterization
