PCR-RFLP analysis of Botryosphaeriaceae species isolated from the Aegean and Mediterranean vineyards

Abstract

The classical identification of Botryosphaeriaceae fungi, causing dieback and local dead arms in many plant species, is very difficult and time-consuming. Some of the molecular tools, such as PCR using species-specific primed, RFLP (Restricted Fragment Length Polymorphism) or gene sequencing are necessary for the fast identification of species. This study aims to contribute for the fast identification of some fungal species belonging Botryosphaeriaceae family reported up to now in Turkey vineyards. DNA was extracted from the 25 isolates obtained from the Aegean and Mediterranean Regions, and 4 different species (Botryosphaeria dothidea, Diplodia seriata, Lasiodiplodia theobromae, and Neofusicoccum parvum) and β-tubulin (TUB2) and ITS region (specific for Botryosphaeriaceae genus) on genomic DNA was amplified by polymerase chain reactions. This region was digested by five different restriction endonuclease enzymes (AluI, BsaHI, MboI, RsaI, TaqI) and band profiles were analyzed on an agarose gel (2%). According to the results of the analysis, the region, amplified with genus-specific primers, was not suitable for RFLP analysis. On the other hand, to discriminate these species, it was revealed that the β-tubulin gene region should be digested at least two different restriction enzymes. The enzyme BsaHI generated two different band sizes for B. dothidea and D. seriata (250 and 700 bp for the first; 400 and 600 bp for the second one) digesting β-tubulin gene of these species but this enzyme could not generate any discriminative bands for L. theobromae and N. parvum. However the enzyme TaqI could not discriminate B. dothidea and D. seriata but digest β-tubulin gene of L. theobromae and N. parvum at two points and it generated three different bands (for L. theobromae: 200-400-800 bp and for N. parvum: 200-400-650 bp).

Keywords

• grapevine,Botryosphaeria dieback,restriction enzymes,BsaHI,TaqI

Ege ve Akdeniz Bölgesi bağlarından izole edilen Botryosphaeriaceae türlerinin PCR-RFLP analizi

Öz

Birçok bitki türünde lokal kuruma ve geriye ölümlere neden olan Botryosphaeriaceae funguslarının klasik tanısı oldukça zor ve zaman alıcıdır. Türlerin hızlı tanısı için türe özgü primerlerle PCR testi, RFLP (Restricted Fragment Length Polymorphism) ya da gen sekanslama gibi bazı moleküler yöntemlerin kullanılması gerekmektedir. Bu çalışma, Türkiye bağlarında şimdiye kadar tespit edilmiş bazı Botryosphaeriaceae familyası fungus türlerinin, PCR-RFLP yöntemi ile hızlı tanısına katkı sağlamayı amaçlamaktadır. Ege ve Akdeniz Bölgesi bağlarından izole edilmiş 4 farklı türe ait (Botryosphaeria dothidea, Diplodia seriata, Lasiodiplodia theobromae ve Neofusicoccum parvum) 25 izolatttan DNA ekstraksiyonları yapılmış, genomik DNA üzerindeki β-tubulin (TUB2) ve ITS bölgesinden tasarlanmış Botryosphaeriaceae cinslerine özgü bölgeler, polimeraz zincir reaksiyonlarıyla çoğaltılmıştır. Bu bölgeler beş farklı restriksiyon endonükleaz enzimiyle (AluI, BsaHI, MboI, RsaI, TaqI) kesilmiş, agaroz jeldeki (%2) bant profilleri incelenmiştir. Analizlerin sonuçlarına göre; cinse özgü primerlerle çoğaltılan bölge RFLP analizi için uygun olmamıştır. Ancak, β-tubulin bölgesinin bu türlerin ayrımı için en az iki farklı restriksiyon enzimiyle kesilmesi gerektiği ortaya çıkmıştır. BsaHI enzimi B. dothidea ve D. seriata’nın β-tubulin genini keserek, iki ayrı büyüklükte bant profili (B. dothidea: 250 ve 700 bp ve D. seriata: 400 ve 600 bp) oluşturmuştur. Ancak bu enzim L. theobromae ve N. parvum için ayırt edici bant verememiştir. Buna karşın TaqI enzimi de B. dothidea ve D. seriata‘yı ayırt edememiş ancak L. theobromae ve N. parvum’un β-tubulin genini 2 yerden keserek her bir tür için üç ayrı bant (L. theobromae: 200-400-800 bp ve N. parvum: 200-400-650 bp) meydana getirmiştir.

Anahtar Kelimeler

• asma,Botryosphaeria geriye ölümü,kesim enzimleri,BsaHI,TaqI

Kaynakça

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