İthal ayçiçeği (Helianthus annuus L.) tohumlarında Plasmopara halstedii (Farl.)Berl. & De Toni)' nin varlığının moleküler yöntemlerle araştırılması ve mefenoxam'a karşı duyarlılığının saptanması
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Dosyalar
Tarih
2017
Yazarlar
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Yayıncı
Ege Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Özellikle, tohum kaynaklı olan ve tohumla taşınabilen etmenlerin hızlı tanılanması ve bunların kontrol altına alınması sağlıklı bir ürün elde etmenin ilk aşamasını oluşturmaktadır. Bu tez çalışmasının amacı, ithalat aşamasında analiz şartı olan tohum kaynaklı Plasmopara halstedii (Farl.) Berl. & De Toni)'nin ayçiçeği tohumundaki varlığının klasik yöntemlere göre çok daha avantajlı moleküler yöntemler kullanılarak optimizasyonunun gerçekleştirilmesi ve metalaxyl ile mefenoxam arasındaki duyarlılıklarının değerlendirilmesidir. Klasik PCR (agaroz jel elektroforez) ile ilgili hesaplamaları EPPO Standart Protokolleri Appendix 2 ve Real Time PCR (hidroliz probları) ile ilgili hesaplamalar ise EPPO Standart Protokolleri Appendix 3 referans alınarak optimize edilmiştir. Ayrıca, EPPO Standart Protokollerinde yer almayan Real time PCR (SyberGreen) çalışmasında ise, SyberGreen Mastermix kit protokolü esas alınarak optimize edilmiştir. Böylece, ülkemiz karantina laboratuarları içerisinde her üç moleküler protokülün daha uygulanabilir olması sağlanmıştır. Ayrıca, bu optimizasyon ile etmenin ayçiçeği tohumundan direkt tespitine yönelik ilk moleküler çalışma da gerçekleştirilmiştir. P. halstedii izolatlarının metalaxyl'e duyarlılıklarının azaldığı tüm dünyada bilinmektedir. Bu çalışmada da, söz konusu patojenin metalaxyl'in izomeri olan mefenoxam'a karşı da duyarlılıklarının azaldığı belirlenmiş ve aralarında çapraz dayanıklılık olduğu için metalaxyl ile aynı grupta yer almıştır.
The fact that rapid diagnosing of especially seed-borne pathogens and their control is the first step to obtain healthy crop. The aims of the study were to optimize the analyzing methods in detecting presence of Plasmopara halstedii (Farl.) Berl. & De Toni requiring analysis at the time of importation in the sunflower seed using current molecular methods that were much more advantageous than that of classical methods, and to evaluate potential variations in sensitivities of the pathogen to metalaxyl and mefenoxam. Calculations related to classical PCR (agarose gel electrophoresis) were optimized by referring to Appendix 2 of the EPPO Standard Protocols while those related to real-time PCR (the hydrolyze probes) were optimized by referring to Appendix 3 of the EPPO Standard Protocols. Moreover, SyberGreen Mastermix Kit protocol was used for optimization for the study of real-time PCR (SyberGreen) that is not present in the EPPO Standard Protocols. Thus, it was provided that all three molecular protocols were confirmed for better applicable and practical ways for the quarantine laboratories in our country. Furthermore, the first molecular studies on direct detection of the pathogen from sunflower seeds through these optimization was conducted, as well. Reduced sensitivities of P. halstedii to metalaxyl are well-known throughout the world. In this study, it was showed that the similar reduced sensitivities of the pathogen had been determined for mefenoxam that is an isomer of metalaxyl. There were found no differences statistically and both fungicides were placed in the same group because of resulting from cross-resistance between.
The fact that rapid diagnosing of especially seed-borne pathogens and their control is the first step to obtain healthy crop. The aims of the study were to optimize the analyzing methods in detecting presence of Plasmopara halstedii (Farl.) Berl. & De Toni requiring analysis at the time of importation in the sunflower seed using current molecular methods that were much more advantageous than that of classical methods, and to evaluate potential variations in sensitivities of the pathogen to metalaxyl and mefenoxam. Calculations related to classical PCR (agarose gel electrophoresis) were optimized by referring to Appendix 2 of the EPPO Standard Protocols while those related to real-time PCR (the hydrolyze probes) were optimized by referring to Appendix 3 of the EPPO Standard Protocols. Moreover, SyberGreen Mastermix Kit protocol was used for optimization for the study of real-time PCR (SyberGreen) that is not present in the EPPO Standard Protocols. Thus, it was provided that all three molecular protocols were confirmed for better applicable and practical ways for the quarantine laboratories in our country. Furthermore, the first molecular studies on direct detection of the pathogen from sunflower seeds through these optimization was conducted, as well. Reduced sensitivities of P. halstedii to metalaxyl are well-known throughout the world. In this study, it was showed that the similar reduced sensitivities of the pathogen had been determined for mefenoxam that is an isomer of metalaxyl. There were found no differences statistically and both fungicides were placed in the same group because of resulting from cross-resistance between.
Açıklama
Anahtar Kelimeler
Plasmopara Halstedii, Ayçiçeği (Helianthus Annuus), PCR, Hidroliz Probları, Optimizasyon, Metalaxyl, Mefenoxam, Duyarlılık Azalışı, Plasmopara Halstedii, Sunflower (Helianthus Annuus), PCR, Hydrolysis Probes, Optimization, Metalaxyl, Mefenoxam, Reduced Sensitivity
