Detection and Characterization of two Phytoplasma lineages on Cucumber (Cucumis sativus L.) with Same Symptomatology based on Virtual RFLP and Nucleotide Sequence Analysis of 16S rDNA

Yıl 2017, , 299 - 308, 29.09.2017

Mustafa Usta , Abdullah Güller Hikmet Murat Sipahioğlu

https://doi.org/10.29133/yyutbd.288346

Cited By: 6

Öz

Phytoplasma-like symptoms were observed in cucumbers (Cucumis sativus L.) in Van province of Turkey. The major symptoms observed were severe dwarfing, witches’ broom, rosetting, little leaf, and sterility of plants. Genomic DNA of 8 symptomatic and non-symptomatic plant leaves was isolated for the detection of pathogenic DNA. Of the 8 cucumber leaf samples tested by nested polymerase chain reaction (Nested-PCR), the four yielded the expected 1.25-kb DNA fragments when using universal primer pairs R16mF2/R16mR1 and R16F2n/R16R2. Randomly selected two DNA bands were further cloned into a proper plasmid vector. The recombinant plasmid DNA was sequenced bidirectionally. BLAST and virtual restriction fragment length polymorphism (RFLP) analyses of the 16S rDNA sequence revealed the presence of the “Candidatus Phytoplasma solani” (similarity coefficient 1.00) (GenBank accession no: KX977570) in one of the severely symptomatic cucumber samples and the “Candidatus Phytoplasma trifolii” (similarity coefficient 0.98) (GenBank accession no.: KR080212) in the other. The isolates were designated as Van-trifolii and Van-solani isolates, respectively. No significant differences were observed between the two different phytoplasmas’ symptomatology. To the authors’ knowledge, this is the first report of ‘Ca. P. trifolii’ and ‘Ca. P. solani’ in cucumbers in Turkey.

Anahtar Kelimeler

Candidatus Phytoplasma solani, Candidatus Phytoplasma trifolii, Characterization, Cucumis sativus, Detection

Hıyar (Cucumis sativus L.) Bitkisinde Aynı Simptomatolojiye Sahip İki Farklı Fitoplazma etmeninin 16S rDNA'sının RFLP ve Nükleotid Dizi Analizleri ile Teşhisi ve Karakterizasyonu

Öz

Van ilinde yetiştiriciliği yapılan hıyarlarda (Cucumis sativus L.) fitoplazma benzeri belirtiler görülmüştür. Gözlenen en önemli belirtiler arasında şiddetli cüceleşme, cadı süpürgesi görünümü, rozetleme ve küçük yapraklılık yer almaktadır. Toplanan 8 hıyar örneğine R16mF2/R16mR1 ve R16F2n/R16R2 primer çiftleri ile uygulanan Nested-PCR testi uygulanmıştır. Belirti gösteren ve göstermeyen hıyar yapraklarından izole edilen genomik DNA’ların kullanıldığı PCR reaksiyonunda fitoplazma etmenlerine ait 16S rDNA fragmentleri çoğaltılmış ve test edilen sekiz örneğin dördünde 1.25 kb uzunluğunda DNA fragmentleri elde edilmiştir. Beklenen band büyüklüğünü veren örneklerden rastgele seçilen iki örnek uygun bir plasmid vektörde klonlanmıştır. Saflaştırılan rekombinant plasmid DNA’sı çift yönlü olarak dizilenmiş ve 16S rDNA dizisinin BLAST ve RFLP analizleri yapılmıştır. Şiddetli fitoplazma belirtisi gösteren hıyar numunelerinin birinde ‘Candidatus Phytoplasma solani’ (benzerlik katsayısı 1.00) (GenBank erişim no: KX977570), diğerinde ise ‘Candidatus Phytoplasma trifolii’ (benzerlik katsayısı 0.98) (GenBank erişim no: KR080212) saptanmıştır. İzolatlardan birincisine‘Van-solani’ diğerine ise ‘Van-trifolii’ izolatı isimleri verilmiştir. İki farklı fitoplazma etmeninin oluşturduğu belirtiler arasında bir fark gözlenmemiştir. Yazarların bilgisine göre, Türkiye’de hıyarları doğal olarak enfekte eden "Ca. P. solani" ve "Ca. P. trifolii" fitoplazma etmenleri ilk defa bu çalışma ile rapor edilmiştir.

Anahtar Kelimeler

Cucumis sativus, Candidatus Phytoplasma solani, Candidatus Phytoplasma trifolii, Karakterizasyon, Survey

Kaynakça

• Ahrens U, Seemüller E (1992). Detection of DNA of plant pathogenic mycoplasma like organisms by a polymerase chain reaction that amplifies a sequence of the 16S RNA gene. Phytopathology. 82: 828-832.
• Alp Ş, Usta M, Sipahioğlu HM, Güller A (2016). First report of “Candidatus Phytoplasma solani” on a new host marigold (Tagetes erecta L.). Turk. J. Agric. For. 40: 311-318.
• Al-Saady NA, Khan AJ, Calari A, Al-Subhi AM, Bertaccini A (2008). “Candidatus Phytoplasma omanense”, associated with witches-broom of Cassia italica (Mill.) Spreng. in Oman. Int. J. Syst. Evol. Microbiol. 58: 461-466.
• Arocha Y, Lopez M, Pinol B, Fernandez M, Picornell B, Almeida R, Palenzuela I, Wilson MR, Jones P (2005). ‘Candidatus phytoplasma graminis’ and ‘Candidatus phytoplasma caricae’, two novel phytoplasmas associated with diseases of sugarcane, weeds and papaya in Cuba. Int. J. Syst. Evol. Microbiol. 55: 2451-2463.
• Başpınar H, Korkmaz S, Önelge N, Çınar A, Uygun N, Kersting U (1993). Studies on Citrus Stubborn Disease pathogen and Sesame Phyllody MLO and their assocated leafhopper vectors in sesame. J. Turk. Phytopathol. 1: 1-8.
• Bertaccini A, Duduk B, Paltrinieri S, Contaldo N (2014). Phytoplasmas and Phytoplasma Diseases: A Severe Threat to Agriculture. Am. J. Plant Sci. 5: 1763-1788.
• Choueiri E, Salar P, Jreijiri Fouad, Zammar SE, Masaad R, Abdul-Nour H, Bove´ JM, Danet JL, Foissac X (2007). Occurrence and distribution of ‘Candidatus Phytoplasma trifolii’ associated with diseases of solanaceous crops in Lebanon. Eur. J. Plant. Pathol. 118: 411-416.
• Contaldo N, Bertaccini A, Paltrinieri A, Windsor HM, Windsor GD (2012). Axenic culture of plant pathogenic phytoplasmas. Phytopathol. Mediterr. 51: 607-617.
• Çağlar BK, Elbeaino T (2013). A novel phytoplasma associated with witches’ broom disease of Ligustrum ovalifolium in Turkey. J. Plant Pathol. 137: 113-117.
• Çağlar BK, Elbeaino T, Küsek M, Pehlivan D, Fidan H, Portakaldalı M (2010). Stolbur Phytoplasma Infections in Potato and Tomato Plants from Different Locations in Turkey. J. Turk. Phytopath. 39: 1-3.
• Davis RI, Schneider B, Gibb KS (1997). Detection and differentiation of phytoplasma in Australia. Aust. J. Agric. Res. 48: 535-544.
• Duduk B, Bertaccini A (2011). Phytoplasma classification: Taxonomy based on 16S ribosomal gene, is it enough? Phytopathogenic Mollicutes. 1 (1): 3-13.
• Firrao G, Gibb K, Streten C (2005). Short taxonomic guide to the genus ‘Candidatus Phytoplasma’. J. Plant Pathol. 87: 249-263.
• Gazel M, Çağlayan K, Başpınar H, Mejia JF, Paltrinieri S., Bertaccini A, Contaldo N (2016). Detection and Identification of Phytoplasmas in Pomegranate Trees with Yellows Symptoms. J. Phytopathol. 164: 136-140.
• Griffiths HM, Sinclair WA, Smart CD, Davis RE (1999). The phytoplasma associated with ash yellows and lilac witches’- broom: ‘Candidatus Phytoplasma fraxini’. Int. J. Syst. Bacteriol. 49: 1605-1614.
• Gundersen DE, Lee IM (1996). Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer pairs. Phytopathol. Mediterr. 35: 144-151.
• Hiruki C, Wang KR (2004). Clover proliferation phytoplasma: ‘Candidatus Phytoplasma trifolii’. Int. J. Syst. Evol. Microbiol. 54: 1349-1353.
• Hogenhout SA, Oshima K, Ammar El-D, Kakizawa S, Kingdom HN, Namba S (2008). Phytoplasmas: bacteria that manipulate plants and insects. Mol. Plant Pathol. 9 (4): 403-423.
• IRPCM Phytoplasma/Spiroplasma Working Team–Phytoplasma Taxonomy Group (2004). ‘Candidatus Phytoplasma’, a taxon for the wall-less, non-helical prokaryotes that colonize plant phloem and insects. Int. J. Syst. Evol. Microbiol. 54: 1243-1255.
• Jung HY, Sawayanagi T, Kakizawa S, Nishigawa H, Miyata SI, Oshima K, Ugaki M, Lee JT, Hibi T, Namba S (2002). ‘Candidatus Phytoplasma castaneae’, a novel phytoplasma taxon associated with chestnut witches’ broom disease. Int. J. Syst. Evol. Microbiol. 52: 1543-1549.
• Jung HY, Sawayanagi T, Wongkaew P, Kakizawa S, Nishigawa H, Wei W, Oshima K, Miyata S, Ugaki M, Hibi T et al. (2003). ‘Candidatus Phytoplasma oryzae’, a novel phytoplasma taxon associated with rice yellow dwarf disease. Int. J. Syst. Evol. Microbiol. 53: 1925-1929.
• Khan AJ, Botti S, Subhi AMA, Zaidi MA, Altosaar I, Alma A, Bertaccini A (2003). Molecular Characterization of the 16S RNA gene of phytoplasma detected in two leafhopper species associated with alfalfa plants infected with witches’ broom in Oman. Phytopathol. Mediterr. 42 (3): 257-267.
• Khasa E, Gopala Taloh A, Prabha T, Madhupriyal, Rao GP (2016). Molecular characterization of phytoplasmas of ‘Clover proliferation’ group associated with three ornamental plant species in India. 3 Biotech. 6 (2): 237.
• Lee IM, Bottner KD, Secor G, Rivera-Varas V (2006). ‘Candidatus Phytoplasma americanum’, a phytoplasma associated with a potato purple top wilt disease complex. Int. J. Syst. Evol. Microbiol. 56: 1593-1597.
• Lee IM, Davis RE (1988). Detection and investigation of genetic relatedness among aster yellows and other mycoplasmalike organism by using cloned DNA and RNA probes. Mol. Plant-Microbe Interact. 1: 303-310.
• Lee IM, Gundersen-Rindal DE, Davis RE, Bartoszyk IM (1998). Revised classification scheme of phytoplasmas based on RFLP analyses of 16S rRNA and ribosomal protein gene sequences. Int. J. Syst. Bacteriol. 48: 1153-1169.
• Lee IM, Hammond RW, Davis RE, Gundersen DE (1993). Universal amplification and analysis of pathogen 16S rDNA for classification and identification of mycoplasmalike organisms. Phytopathology. 83: 834-842.
• Lee IM, Martini M, Macone C, Zhu SF (2004). Classification of phytoplasma strains in the elm yellows group (16SrV) and proposal of ‘Candidatus Phytoplasma ulmi’ for the phytoplasma associated with elm yellows. Int. J. Syst. Evol. Microbiol. 54: 337-347.
• Lou B, Bai X, Bail Y, Deng C, RoyChowdhury M, Chen C, Song Y (2013). Detection and Molecular Characterization of a 16SrII-A phytoplasma in Grapefruit (Citrus paradisi) with Huanglongbing-like Symptoms in China. J. Phytopath. 162: 387-395.
• Marcone C, Gibb KS, Streten C, Schneider B (2004a). ‘Candidatus Phytoplasma spartii’, ‘Candidatus Phytoplasma rhamni’ and ‘Candidatus Phytoplasma allocasuarinae’, respectively associated with spartium witches’-broom, buckthorn witches’-broom and allocasuarina yellows diseases. Int. J. Syst. Evol. Microbiol. 54: 1025-1029.
• Marcone C, Schneider B, Seemuller E (2004b). ‘Candidatus Phytoplasma cynodontis’, the phytoplasma associated with Bermuda grass white leaf disease. Int. J. Syst. Evol. Microbiol. 54: 1077-1082.
• Montano HG, Brioso PST, Pimentel JP, Figueiredo DV, Cunha JO (2006). Cucurbita moschata, new phytoplasma host in Brazil. J. Plant Pathol. 88 (2): 225-229.
• Montano HG, Davis RE, Dally EL, Hogenhout S, Pimentel JP, Brioso PST (2001). ‘Candidatus Phytoplasma brasiliense’, a new phytoplasma taxon associated with hibiscus witches’ broom disease. Int. J. Syst. Evol. Microbiol. 51: 1109-1118.
• Montano HG, Paulo STB, Roberta CP, Joao PP (2007). Sicana odorifera (Cucurbitaceae) a new phyto- plasma host. Bull. Insectology. 60: 287-288.
• Murray RGE, Stackebrandt E (1995). Taxonomic Note: Implementation of the Provisional Status Candidatus for Incompletely Described Procaryotes. Int. J. Syst. Bacteriol. 45 (1): 186-187.
• Murray RG, Schleifer KH (1994). Taxonomic notes: a proposal for recording the properties of putative taxa of procaryotes. Int. J. Syst. Bacteriol. 44 (1): 174-176.
• Namba S, Kato S, Iwanami S, Oyaizu H, Shiozawa H, Tsuchizaki T (1993). Detection and diffentition of plant-pathogenic mycoplasmalike organisms using Polymerase Chain Reaction. Phytopathology. 83: 786-791.
• Özdemir N, Saygılı H (2012). Batı Anadolu Bölgesi Domates Üretim Alanlarında Görülen Stolbur Hastalığının Tohumla Taşınıp Taşınmadığı İle İlgili Bir Araştırma. U. Ü. Ziraat Fakültesi Dergisi. 26 (1): 63-72.
• Özdemir N, Saygılı H, Sahin F, Karsavuran Y, Bayrak OF, Oral B (2009). Host range and genetic characterization of a phytoplasma causing tomato stolbur disease in Turkey. Proc. IInd Intl. Symposium on tomato diseases. Acta Hort. 808: 255-261.
• Sawayanagi T, Horikoshi N, Kanehira T, Shinohara M, Bertaccini A, Cousin MT, Hiruki C, Namba S (1999). ‘Candidatus Phytoplasma japonicum’, a new phytoplasma taxon associated with Japanese Hydrangea phyllody. Int. J. Syst. Bacteriol. 49: 1275-1285.
• Schneider B, Torres E, Martin MP, Schroder M, Behnke HD, Seemuller E (2005). ‘Candidatus Phytoplasma pini’, a novel taxon from Pinus silvestris and Pinus halepensis. Int. J. Syst. Evol. Microbiol. 55: 303-307.
• Seemuller E, Schneider B, Maurer R, Ahrens U, Daire X, Kison H, Lorenz KH, Firrao G, Avinent L, Sears BB (1994). Phylogenetic classification of phytopathogenic mollicutes by sequence analysis of 16S ribosomal DNA. Int. J. Syst. Bacteriol. 44 (3): 440-446.
• Seemüller E, Schneider B (2004). Taxonomic description of ‘Candidatus Phytoplasma mali’ sp. nov., ‘Candidatus Phytoplasma pyri’ sp. nov. and ‘Candidatus Phytoplasma prunorum’ sp. nov., the causal agents of apple proliferation, pear decline and European stone fruit yellows, respectively. Int. J. Syst. Evol. Microbiol. 54: 1217-1226.
• Sertkaya G, Osler R, Musetti R, Ermacora P, Martini M (2004). Detection of phytoplasmas in Rubus spp. by microscopy and molecular techniques in Turkey. Acta Hort. 656: 181-186.
• Sertkaya G, Martini M, Ermacora P, Musetti R, Osler R (2005). Detection and characterization of phytoplasmas in diseased stone fruits and pear by PCR-RFLP analysis in Turkey. Phytoparasitica. 33: 380-390.
• Sertkaya G, Martini M, Musetti R, Osler R (2007). Detection and molecular characterization of phytoplasmas infecting sesame and solanaceous crops in Turkey. Bull. Insectology. 60 (2): 141-142.
• Sertkaya G, Martini M, Osler R (2008). First report of Candidatus phytoplasma mali in Turkey. J. Plant Pathol. 90 (1): 143-149.
• Tamura K, Dudley J, Nei M, Kumar S (2007). MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24: 1596-1599.
• Tymon AM, Jones P, Harrison NA (1998). Phylogenetic relationships of coconut phytoplasmas and the development of specific oligonucleotide PCR primers. Ann. Appl. Biol. 132: 437-452.
• Ulubaş Serçe Ç, Gazel M, Çaglayan K, Baş M, Son L (2006). Phytoplasma diseases of fruit trees in germplasm and commercial orchards in Turkey. J. Plant Pathol. 88 (2): 179-185.
• Wei W, Davis RE, Lee IM, Zhao Y (2007). Computer simulated RFLP analysis of 16S rRNA genes: identification of ten new phytoplasma groups. Int. J. Syst. Evol. Microbiol. 57: 1855-1867.
• Wu Y, Hao X, Li Z, Gu P, An F, Xiang J, Wang H, Luo Z, Liu J, Xiang Y (2010). Identification of the phytoplasma associated with wheat blue dwarf disease in China. Plant Dis. 94: 977-985.
• Zhao Y, Wei W, Lee IM, Shao J, Suo X, Davis RE (2009). Construction of an interactive online phytoplasma classification tool, iPhyClassifier, and its application in analysis of the peach X-disease phytoplasma group (16SrIII). Int. J. Syst. Evol. Microbiol. 59: 2582-2593.
• Zibadoost S, Rastgou M, Asghari S, Tazehkand SA (2016). Detection and molecular identification of ‘Candidatus phytoplasma trifoli’ infecting some cultivated crops and vegetables in West Azarbaijan province. Australas. Plant Dis. Notes. 11: 3.