Determination of Hypovirulence Strains of Cryphonectria parasitica by Using Non-Phenol-Chloroform dsRNA Analysis Method and Detection of Cryphonectria hypovirus 1 by RT-PCR

Yıl 2018, Cilt: 15 Sayı: 2, 25 - 32, 31.12.2018

Eda Mersin Serap Açıkgöz Ömer Erincik

https://doi.org/10.25308/aduziraat.415060

Cited By: 2

Öz

Chestnut blight, Cryphonectria parasitica, is a serious disease of Castanea spp. and causes bark cankers that progressively
kill branches and trunks. Hypovirulent cancers were first identified with the awareness of chestnut trees recovering in Italy in 1964,
and followed by hypovirulent strains containing the dsRNA virus used as a biological control agent. It has been determined that the
hypovirulent isolates of C. parasitica have dsRNA which obtained from chestnut areas of the Black Sea and Marmara regions of
Turkey. The dsRNA isolation and analysis method of Morris ve Dodds (1979) is usually used to determine the presence of dsRNA in
hypovirulent isolates. In this study, to determine the presence of dsRNA in C. parasitica isolates, which may be hypovirulent, the
method of Balijja and collaborators has been used (Balijja et al., 2008). This study was planned in order to investigate whether this
method is applicable for conducting research in the future due to its advantages such as shorter time requirements, less amount of
mycelium and chemicals used, and not involving phenol-chloroform compared to other isolation methods. For this purpose, the isolates
collected from healing cankers of chestnut areas in the Aegean, Black Sea and Marmara regions of Turkey, where light-white
mycelium formation was observed, were used. In this study, 30 isolates out of 68 were analyzed by Balijja et al.’s method and only 25
isolates were identified to contain dsRNA profile. dsRNA-containing six isolates were also analyzed by the method of Morris and
Dodds, and the existence of dsRNA was confirmed. It was found that 15 of 25 dsRNAs belonged to the hypovirus Cryphonectria
hypovirus 1 (CHV-1) by RT-PCR.

Anahtar Kelimeler

Cryphonectria parasitica, Chestnut bligth, dsRNA analysis, Cryphonectria hypovirus1 (CHV-1)

Cryphonectria parasitica’ nın Hipovirülent Strainlerinin Fenol ve Kloroform İçermeyen dsRNA Analiz Yöntemi ile Belirlenmesi ve Cryphonectria hypovirus 1’ in RT-PCR ile Tanılanması

Öz

Kestane Kanseri (Cryphonectria parasitica), Castanea spp., nin önemli bir hastalığı olup dal ve gövdelerde kanserlere yol
açmakta ve ağaçların zamanla ölmesine sebep olmaktadır. 1964 yılında İtalya’da iyileşmekte olan kestane ağaçlarında ilk
olarak hipovirulent kanserlerin fark edilmesini takiben biyolojik kontrol ajanı olarak kullanılan dsRNA virüsü içeren
hipovirulent strainler belirlenmiştir. Ülkemizde de Karadeniz ve Marmara Bölgeleri kestane alanlarından elde edilen C.
parasitica‘nın hipovirulent izolatlarının dsRNA içermiş oldukları belirlenmiştir. Hipovirulent izolatlardaki dsRNA’nın varlığının
belirlenmesinde genellikle Morris ve Dodds (1979)’un dsRNA izolasyon ve analiz yöntemi kullanılmaktadır. Bu çalışmada ise
hipovirulent olabileceği belirlenen C. parasitica izolatlarında dsRNA varlığı belirlemek için Balijja ve ark. (2008) tarafından
geliştirilen yöntem kullanılmıştır. Diğer izolasyon yöntemlerine kıyasla daha kısa süre gerektirmesi, kullanılan miselyum ve
kimyasal madde miktarının daha az olması ve fenol- kloroform içermemesi gibi avantajları nedeniyle gelecekte ülkemiz ve
bölgemizde bu konuda yapılacak olan araştırmalara kolaylık sağlaması amacıyla bu çalışma planlanmıştır. Bu amaçla Ege,
Karadeniz ve Marmara Bölgeleri kestane alanlarından kestane kanseri hastalığı ile bulaşık ağaçların iyileşme belirtisi gösteren
kanser yaralarının kabuk dokularından elde edilen ve açık renk-beyaz misel oluşumu gözlenen izolatlar kullanılmıştır. Çalışma
kapsamında 68 izolattan 30 tanesi dsRNA ekstraksiyonuna olanak sağlayan Balijja ve ark. (2008)’ nın yöntemi ile analiz
edilmiş ve 25 izolatın dsRNA profili içerdiği belirlenmiştir. dsRNA profili içeren altı izolat Morris ve Dodds (1979) yöntemi
ile dsRNA varlığı yönünden doğrulanmıştır. Ardından, RT-PCR yöntemi ile 15 izolattan elde edilen dsRNA’ların hipovirüs
Cryphonectria hypovirus 1 (CHV-1) olduğu belirlenmiştir. 

Anahtar Kelimeler

kestane kanseri, chestnut bligth, dsRNA analizi, Cryphonectria hypovirus1 (CHV-1)

Kaynakça

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