Türkiye'de önemli kiraz üretim alanlarında Prune dwarf virus (PDV)'nin tespiti ve genetik çeşitliliği
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Date
2023
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Hatay Mustafa Kemal Üniversitesi
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info:eu-repo/semantics/openAccess
Abstract
Bu çalışmada, Türkiye'nin önemli kiraz yetiştiriciliği yapılan illerinden İzmir, Konya, Kahramanmaraş, Niğde, Bursa, Manisa, Osmaniye, Adana ve Antalya'da kiraz ağaçlarını enfekte eden ve önemli viral patojenler arasında yer alan PDV'nin yaygınlığı ve izolatların genetik çeşitliliği araştırılmıştır. Yapılan sörvey çalışmasında 9 il ve 14 ilçeden virüs simptomu gösteren ve göstermeyen toplam 468 kiraz ve 4 vişne ağacından yaprak örnekleri alınmış ve bu örnekler hem DAS-ELISA hem de RT-PCR yöntemleriyle testlenmiştir. DAS-ELISA yöntemiyle testlenen toplam 472 örneğin 25 tanesinde PDV saptanırken RT-PCR analizleri ile 52 örnek pozitif bulunmuştur. Böylece testlenen örneklerde PDV'nin yaygınlık oranı DAS-ELISA yöntemiyle %5,3 olarak bulunurken bu oran RT-PCR analizlerinde %11,02 olarak tespit edilmiştir. RT-PCR analizleri sonucunda pozitif bulunan örneklerin tamamı kısmi hareketlilik protein genini (movement protein-MP) çoğaltan primerlerle testlendiği için aynı örnekler kılıf protein (CP) kısmi gen bölgesini çoğaltan primerlerle testlendiğinde 44 örnekte beklenen düzeyde amplifikasyon gözlenmiştir. Öte yandan, RNA'ya bağımlı RNA polimeraz (RdRp) kısmi gen bölgesini çoğaltan primerlerle örnekler testlendiğinde 39 örnekte beklenen düzeyde amplifikasyon elde edilmiştir. Testlenen örneklerde PDV'nin tespit edilme oranları PDV-MP spesifik primer çifti ile %11,01, PDV- CP spesifik primer çifti ile %9,32 ve PDV- RdRp spesifik primer çifti ile ise %8,26 olarak saptanmıştır. Üç farklı gen bölgesine ait kısmi gen dizileri, gen bankasında kayıtlı PDV referans izolatının yanı sıra ülkemizden ve dünyanın diğer ülkelerinden kayıtlı PDV izolatlarıyla kıyaslanarak filogenetik analizleri yapılmıştır. Yapılan BLASTn ve BLASTp analizi sonucunda, bu çalışmada elde edilen PDV kiraz izolatlarının referans PDV izolatıyla MP- nükleotid düzeyinde %88,02- 99,57 ve aminoasit düzeyinde %84,91- 99,57; CP- nükleotid düzeyinde %77,13- 100 ve aminoasit düzeyinde %54,87- 100; RdRp- nükleotid düzeyinde %74,62-99,26 amino asit düzeyinde %63,33- 100 arasında benzerlik gösterdiği tespit edilmiştir. Bu çalışmada elde edilen PDV izolatlarının MP, CP ve RdRp kısmi gen dizilerine göre çizilen filogenetik ağaçlarda iki genomik bölgede iki filogenetik grup oluşumu gözlenmiştir. Bu çalışma ile Türkiye'de kirazlarda ilk kez MP ve RdRp kısmi gen bölgelerine ait nükleotid ve aminoasit dizileri elde edilerek gen bankası kaydı oluşturulmuştur. PDV izolatlarının nükleotid ve aminoait dizileme ve filogenetik analizleri sonucu, 732 bp'lik 244 aa uzunluktaki MP, 587 bp'lik 195 aa uzunluktaki CP ve 271 bp'lik 90 aa uzunluktaki RdRp'a karşılık gelen nükleotid dizileri elde edilerek gen bankasına kayıtlı PDV izolatlarıyla filogenetik kıyaslamaları yapılmıştır.
Cherry is an important fruit crop in Turkey and all over world. In this study, the prevalence of PDV, which is one of the important viral pathogens that infects cherry trees was investigated in different provinces of Turkey where cherry production is important such as İzmir, Konya, Kahramanmaraş, Niğde, Bursa, Manisa, Osmaniye, Adana and Antalya. In this study, totally 472 plant samples, of which 468 were cherry and four were sour cherry , with and without virus symptoms were collected from 9 provinces and 14 districts. These samples were tested both by DAS-ELISA and RT-PCR assays. As a result of DAS-ELISA analysis, PDV was detected in 25 of 468 cherry samples while it was 52 by RT-PCR.. The PDV infection rate of tested cherry samples was 5.3 % by DAS-ELISA and 11.20% by RT-PCR. When all RT-PCR positive samples which were tested by primers amplifying partial movement protein gene (MP) (756 bp) were tested by primers amplifying partial CP gene (874 bp), 44 samples were found positive. On the other hand, when samples were tested with primers that amplify the RNA-dependent RNA polymerase (RdRp) partial gene (382 bp), the expected level of amplicons were observed in 39 samples. The PDV positive samples among tested plants were found in 11.01% for the PDV-MP specific primer pair, 9.32% for the PDV-CP specific primer pair, and then 8.26% for the PDV-RdRp specific primer pair, respectively. Phylogenetic analysis were performed by using partial sequences belonging to three different genes obtained in this study together with the other Turkish isolates and as well as world isolates of PDV deposited in GeneBank. As a result of the BLASTn and BLASTp analysis, the sequences of PDV reference isolate and cherry isolates obtained in this study had 88.02-99.57% similarity at MP-nucleotide level and 84.91-99.57% at amino acid level; 77.13%-100% at the CP-nucleotide level and 54.87-100% at the amino acid level; RdRp- nucleotide level 80.59% - 99.26% and amino acid level 59.09 - 100%. In the phylogenetic trees constructed according to the MP, CP and RdRp partial gene sequences of the PDV isolates obtained in this study, two phylogenetic groups were observed in two genomic regions, accordingly all the studied isolates were divided into two clusters. With this study, nucleotide and amino acid sequences belonging to MP and RdRp partial gene sequences were obtained for the first time in cherries in Turkey and they were deposited in GenBank. As a result of nucleotide and amino acid sequencing and phylogenetic analysis of PDV isolates, nucleotide sequences corresponding to MP of 732 bp of 244 aa length, CP of 587 bp to 195 aa of length and RdRp of 90 aa length of 271 bp were obtaine in this study and the sequences were compared with other world isolates of PDV deposited in GeneBank.
Cherry is an important fruit crop in Turkey and all over world. In this study, the prevalence of PDV, which is one of the important viral pathogens that infects cherry trees was investigated in different provinces of Turkey where cherry production is important such as İzmir, Konya, Kahramanmaraş, Niğde, Bursa, Manisa, Osmaniye, Adana and Antalya. In this study, totally 472 plant samples, of which 468 were cherry and four were sour cherry , with and without virus symptoms were collected from 9 provinces and 14 districts. These samples were tested both by DAS-ELISA and RT-PCR assays. As a result of DAS-ELISA analysis, PDV was detected in 25 of 468 cherry samples while it was 52 by RT-PCR.. The PDV infection rate of tested cherry samples was 5.3 % by DAS-ELISA and 11.20% by RT-PCR. When all RT-PCR positive samples which were tested by primers amplifying partial movement protein gene (MP) (756 bp) were tested by primers amplifying partial CP gene (874 bp), 44 samples were found positive. On the other hand, when samples were tested with primers that amplify the RNA-dependent RNA polymerase (RdRp) partial gene (382 bp), the expected level of amplicons were observed in 39 samples. The PDV positive samples among tested plants were found in 11.01% for the PDV-MP specific primer pair, 9.32% for the PDV-CP specific primer pair, and then 8.26% for the PDV-RdRp specific primer pair, respectively. Phylogenetic analysis were performed by using partial sequences belonging to three different genes obtained in this study together with the other Turkish isolates and as well as world isolates of PDV deposited in GeneBank. As a result of the BLASTn and BLASTp analysis, the sequences of PDV reference isolate and cherry isolates obtained in this study had 88.02-99.57% similarity at MP-nucleotide level and 84.91-99.57% at amino acid level; 77.13%-100% at the CP-nucleotide level and 54.87-100% at the amino acid level; RdRp- nucleotide level 80.59% - 99.26% and amino acid level 59.09 - 100%. In the phylogenetic trees constructed according to the MP, CP and RdRp partial gene sequences of the PDV isolates obtained in this study, two phylogenetic groups were observed in two genomic regions, accordingly all the studied isolates were divided into two clusters. With this study, nucleotide and amino acid sequences belonging to MP and RdRp partial gene sequences were obtained for the first time in cherries in Turkey and they were deposited in GenBank. As a result of nucleotide and amino acid sequencing and phylogenetic analysis of PDV isolates, nucleotide sequences corresponding to MP of 732 bp of 244 aa length, CP of 587 bp to 195 aa of length and RdRp of 90 aa length of 271 bp were obtaine in this study and the sequences were compared with other world isolates of PDV deposited in GeneBank.
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Keywords
Ziraat, Agriculture
