Mona GAZEL, Çiğdem ULUBAŞ SERÇE, Harun ÖZTÜRK, Kadriye ÇAĞLAYAN

Detection of ‘Candidatus Phytoplasma pyri’in different pear tissues and sampling time by PCR-RFLP analyses

Aims: In this study, the best sampling time and tissues for phytoplasma detection in twenty pear trees (cv. Deveci) infected by ‘Candidatus Phytoplasma pyri’, causal agent of pear decline disease, in Bursa province of Turkey were investigated. Methods and Results: Sampling was done throughout the year in leaf midribs, shoot and root tissues, where as the flower tissues were tested once a year in March and fruit tissues in September. All samples were analyzed by nested-PCR using P1/P7 and fU5/rU3 universal primer pairs. Nested PCR products were digested with RsaI and SspI restriction enzymes. The results revealed that the detection rate of ‘Ca. P. pyri’ in different plant tissues was greatly depending on the sample collection period. The fruit tissues, which were only sampled in September due to the ripening time of Deveci pear cultivar in Bursa, showed the highest detection rate of ‘Ca. P. pyri’ (100%) followed by flower tissues (75%). The average detection rate in root, shoot tissues and leaf midribs was found as 43.75, 39.58 and 16.25%, respectively. The present results showed that the best plant tissues for detecting ‘Ca. P. pyri’ in pear trees were fruit columella and flowers. The highest detection rate of this phytoplasma in root tissues was found from November to March, whereas it could be detected whole year around except summer months in shoot samples in Turkey. Conclusions: For 'Candidatus Phytoplasma pyri', detection, if there is no seasonal limitation for testing, the most suitable tissues are fruits and flowers. When it comes to testing throughout the year, the most suitable tissues were determined as the root, the phloem and cambium layer of the shoots and the leaves, respectively. Significance and Impact of the Study: This study on seasonal variations of ‘Candidatus Phytoplasma pyri’ in different pear tissues has been first time investigated in Turkey. This preliminary data provides important knowledge on molecular detection of Ca. P. pyri, causal agent of pear decline disease for further studies and sertification-quarantine programmes of pear trees in Turkey.

Farklı armut dokularında ve örnekleme zamanında ‘Candidatus Phytoplasma pyri’’nin PCR-RFLP analizleri ile saptanması

Amaç: Bu çalışmada, ülkemizde Bursa ilinde saptanmış olan armut yıkım fitoplazması (‘Candidatus Phytoplasma pyri’, PD) ile enfekteli 20 armut ağacı (Deveci çeşidi) seçilerek etmenin teşhis edilmesinde en uygun örnekleme zamanı ve bitki dokusunun belirlenmesi amaçlanmıştır. Yöntem ve Bulgular: Yaprak, sürgün ve kök dokularında yıl boyunca örnekleme yapılırken çiçek dokuları Mart ayı, meyve dokuları ise Eylül ayında olmak üzere yılda bir kez testlenmiştir. Tüm örnekler P1/P7 ve fU5/rU3 üniversal primer çiftleri kullanılarak nested-PCR yöntemiyle analiz edilmiştir. Nested-PCR ürünleri RsaI ve SspI restriksiyon enzimleri ile kesime tabi tutulmuştur. Elde edilen sonuçlara göre farklı bitki dokularında ‘Ca. P. pyri ‘nin saptanma oranının büyük ölçüde örnek toplama peryoduna bağlı olduğunu ortaya koymuştur. Bursa ili koşullarında Deveci armut çeşidinin olgunlaşma dönemine göre sadece Eylül ayında örneklenen meyve dokularınde yüksek oranda ‘Ca. P. pyri’ tespit edilirken (% 100), bunu çiçek dokuları (%75) izlemiştir. Kök, sürgün ve yapraklarda ortalama tespit oranı sırasıyla % 43.75, 39.58 ve 16.25 olarak bulunmuştur. Elde edilen sonuçlar armut ağaçlarında ‘Ca P. pyri’ nin saptanması için en iyi bitki dokularının meyve kolumellası ve çiçek olduğunu göstermiştir. Bu fitoplazmanın kök dokulardaki en yüksek tespit oranı Kasım-Mart ayları arasında bulunurken, ülkemizde sürgün örneklerinde yaz ayları hariç bütün yıl tespit edilebildiği belirlenmiştir. Genel Yorum: 'Candidatus Phytoplasma pyri'nin testlenmesi için mevsimsel açıdan bir sınırlama olmaması durumunda en uygun dokular meyve ve çiçekler olup yıl boyunca testleme yapılması söz konusu olduğunda ise sırasıyla en uygun dokular kök, sürgünlerin floem ve kambiyum tabakası ve yapraklar olarak belirlenmiştir. Çalışmanın Önemi ve Etkisi: Farklı armut dokularında 'Candidatus Phytoplasma pyri' varlığının mevsimsel dağılımı konusunda yapılan bu çalışma ülkemizde ilk kez yapılmıştır. Elde edilen veriler, Türkiye’de armut ağaçlarında sertifikasyon-karantina programları için ve armut yıkım fitoplazmasının etmeni Ca. P. pyri’nin moleküler tespiti konusunda önemli bilgiler sağlamıştır.

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Ahrens U, Seemüller E (1992) Detection of plant pathogenic mycoplasmalike organisms by a polymerase chain reaction that amplifies a sequence of the 16S rRNA gene. Phytopathology 82: 828-832.

Bertaccini A, Carraro L, Davies D, Laimer DC, Machado M, Martini M, Paltrinieri S, Seemüller E (2000) Micropropagation of a collection of phytoplasma strains in periwinkle and other host plants p. 101. In: 13th International Congress of IOM 14-19 July Fukuoka Japan.

Canik D, Ertunç F (2007) Ankara ve Yalova İllerindeki Armut Bahçelerinde Görülen Pear Decline Fitoplazma Enfeksiyonunun Moleküler Karakterizasyonu. II. Bitki Koruma Kongresi Isparta s.108.

Çağlayan K, Ulubaş Serçe C, Gazel M (2006) A Preliminary Account of the Presence of Pear Decline Disease (‘Candidatus Phytoplasma pyri’) in Marmara Region of Turkey. XXth International Symposium on Virus and Virus-like Diseases of Temperate Fruit Crops & XI th International Symposium of Small Fruit Virus Diseases Antalya 22-26 May 2006. p.123.

Deng S, Hiruki C (1991) Amplification of 16S rRNA genes from culturable and non culturable mollicutes. J. Microbiol. Methods 14: 53-61.

Errea P, Aguelo V, Hormaza JI (2002) Seasonal variations in detection and transmission of pear decline phytoplasma. J. Phytopathol. 150: 439-443.

Garci-Chapa M, Medina V, Viruel MA, Lavina A, Batlle A (2003) Seasonal detection of pear decline phytoplasma by nested PCR in different pear cultivars. Plant Pathol. 52: 513-520.

Gazel M, Ulubaş Serçe Ç, Çağlayan K, Öztürk H (2007) Detection of ‘Candidatus Phytoplasma pyri’ in Turkey. Bull. Insectology 60: 125-126.

Kucerova J, Kaserova R, Navratil M, Valova P (2007) Seasonal occurrence of ‘Candidatus Phytoplasma pyri’ in pear trees in the Czech Republic. Bull. Insectology 60:263-264.

Lorenz KH, Schneider B, Ahrens U, Seemuller E (1995) Detection apple proliferation and pear decline phytoplasmas by PCR amplification of ribosomal and non ribosomal DNA. Phytopathology 85: 771-776.

Nemeth M (1986) Virus mycoplasma and rickettsia diseases of fruit trees. Martinus Nijhoff Publishers Budapest Hungary.

OEPP/EPPO (2007) Pear decline phytoplasma. EPPO A2 list of pests recommended for regulation as quarantine pests. No.95 Version 2007-09.

Prince JP, Davis RE, Wolf TK, Lee IM, Mogen BD, Dally EL, Bertaccini A, Credi R, Barba M (1993) Molecular detection of diverse mycoplasmalike organisms (MLOs) associated with grapevine yellows and their classification with aster yellows X-disease and elm yellows MLOs. Phytopathology 83: 1130-1137.

Seemuller E, Lorenz KH, Lauer U (1998) Pear Decline resistance in Pyrus communis rootstocks and progenies of wild and ornamental Pyrus taxa. Acta Hortic. 472: 681-690.

Seemüller E (1989) Pear decline: In: Virus and Virus-like Diseases of Pome fruits and simulating non-infectious disorders Fridlund P.R. (ed.) Washington State University Press Pullman W.A. pp.188-201.

Seemüller E, Schaper U, Zimbelman E (1984) Seasonal variation in the colonization patterns of mycoplasmalike organisms associated with apple proliferation and pear decline. J. Plant Dis. Protect. 91: 371-382.

Seemüller E, Schneider B (2004) ‘Candidatus Phytoplasma mali’, ‘Candidatus Phytoplasma pyri’ and ‘Candidatus Phytoplasma prunorum’ the causal agents of apple proliferation pear decline and European stone fruit yellows respectively. Int. J. Syst. Evol. Micr. 54: 1217-1226.

Smart CD, Schneider B, Blomquist CL, Guerra LJ, Harrison NA, Ahrens U, Lorenz KH, Seemuller E, Kirkpatrick BC (1996) Phytoplasma-specific PCR primers based on sequences of 16S-23S rRNA spacer region. Appl. Environ. Microbiol. 62: 2988-2993.

Ulubaş Serçe C, Gazel M, Caglayan K, Baş M, Son L (2006) Phytoplasma diseases of fruit trees in germplasm and commercial orchards in Turkey. J. Plant Pathol. 88: 175-181.