Sert Çekirdeklilerde Bakteriyel Kansere Neden Olan Pseudomonas syringae Pathovarlarının Klasik ve Moleküler Yöntemlerle Tanısı

Bakteriyel kanser belirtisi görülen şeftali, badem ve kiraz gibi sert çekirdekli meyve ağaçlarından izole edilmiş ve Ege Üniversitesi Ziraat Fakültesi Bitki Koruma Bölümü Bakteriyoloji laboratuar stoklarına alınmış 15 Pseudomonas syringae (Ps) pathovarlarının LOPAT (levan oluşumu, oxidase, patateste pektolitik aktivite, arginin dehidrolaz, tütünde aşırı duyarlılık reaksiyonu), GATTa (jelatinin hidrolizi, aesculinin hidrolizi, tyrosin aktivitesi, tartaric acid), karbon kaynaklarının kullanımı klasik tanı testleri ile tanısı yapılmıştır. Ayrıca patojenisite, in-vitro’da syringomycin üretimi ve buz çekirdeği oluşturma aktivitesi (INA) tanı testleri yapılmıştır. Klasik tanı testleri sonucunda kesin tanısı yapılamayan Pseudomonas syringae pathovarı izolatlarının moleküler tanısı yapılmıştır. Pseudomonas syringae pv. syringae (Pss)’nin syringomycin sentezinden sorumlu syrB geni ve Pseudomonas syringae pv. morsprunorum (Psm)’un coronatine sentezinden sorumlu cfl geni klasik PCR ile belirlenmiştir. Pss ve Psm’nin ayırt edilmesinde 4 housekeeping geni (cts, rpoD, gapA, gyrB) sekans analizleri yapılmıştır. Klasik ve moleküler tanı sonucunda 15 Ps pathovarının 9’u Pss olarak kesin tanısı yapılmıştır.

Classical and Molecular Diagnosis of Pseudomonas syringae Pathovars Causing Bacterıal Canker on Stone Fruits

15 Pseudomonas syringae (Ps) pathovars isolated from stone fruit trees such as peach, almond and cherry with bacterial cancer symptoms and kept in the Bacteriology laboratory stocks of Ege University Faculty of Agriculture, Department of Plant Protection were identified based on LOPAT (levan, oxidase, potato rot pectolytic activity, arginin dihydrolase, hypersensitive reaction on tobacco leaves), GATTa (gelatin liquefaction, aesculin hydrolysis, tyrosinase activity, utilization of tartaric acid), utilization of carbon tests classical methods. In addition, pathogenicity, syringomycin production and ice nucleation activity (INA) were used for the identification. Pseudomonas syringae pathovars were identified by PCR that cannot be diagnosed with classical tests. Molecular diagnosis of Pseudomonas syringae pv. syringae (Pss) strains were based on syringomycin synthesis (syrB) and Pseudomonas syringae pv. morsprunorum (Psm) strains were based on coronatine synthesis (cfl) were diagnosed using PCR. Also sequence analysis of 4 housekeeping genes (cts, rpoD, gapA, gyrB) were used to distinguish Pss and Psm. As a result of classical and molecular identification 9 of 15 Ps pathovars were identified as Pss.

___

  • Agrios, G.N. 2005. Plant Pathology. 5th ed. Elsevier Academic Press. United States of America, 948 pp.
  • Akbaba, M. ve Özaktan, H. 2019. Kirazda Bakteriyel Kansere Neden Olan Etmenlerin Moleküler Tanısı ve Mücadelesine Yönelik Biyolojik Yaklaşımlar. Doktora Tezi, Fen Bilimleri Enstitüsü, Ege Üniversitesi, 154s.
  • Akköprü, A. 2016. Determination of Bacterial Disease on Stone Fruits grown in Lake Van Basin, East Anatolia of Turkey Acta Horticulture ISHS II. Int. Workshop on Bacterial Disease of Stone Fruits and Nuts Acta.Hortic.1149.
  • Anonim, 2008. Tarım ve Köyişleri Bakanlığı. 2008. Zirai Mücadele Teknik Talimatları. 4. Ankara, 388s.
  • Ayers, S.H. and Johnson, W.T. 1919. A Study of the Alkali-forming Bacteria in Milk. U.S.Department of Agriculture Bull. 782:1-39.
  • Bull, C.T., Clarke, C.R., Cai, R., Vinatzer, B.A., Jardini, T.M. and Koike, S.T. 2011. Multilocus Sequence Typing of Pseudomonas syringae sensu lato Confirms Previously Described Genemospecies and Permits Rapid İdentification of P. syringae pv. coriandricola and P. syringae pv. apii Causing Bacterial Leaf Spot on Parsley. The American Phytopathological Society 11-10-0318.
  • Bultreys, A. and Gheysen, I. 1999. Biological and Molecular Detection of Toxic-Lipodepsipeptide-Producing Pseudomonas syringae Strains and PCR İdentification İn Plants. Applied and Environmental Microbiology Vol:65 (5) 1904-1909.
  • Bultreys, A. and Kaluzna, M. 2010. Bacterial Canker Caused by Pseudomonas syringae on Stone Fruit Species with Special Emphasis on the Pathovars syringae and morsprunorum race 1 and race 2. Journal of Plant Pathology 92:21-33.
  • Donmez, M.F., Karlidag, H. and Eşitken, A. 2010. Identification of Resistance to Bacterial Canker Pseudomonas syringae pv. syringae Disease on Apricot Genotypes Grown in Turkey European Journal of Plant Pathology 126 (2) 241-247.
  • FAO 2018. Agriculte and Consumer Production. Food and Agriculture Organization of the United Nations. (http://www.fao.org/faostat/). (Date accessed: June 2020).
  • Gasic, K., Prokic, A., Ivanovic, M., Kuzmanovic, N. and Obradovic, A. 2012. Differentation of Pseudomonas syringae originating from Stone Fruits. Pesticides Phytomedicine (Belgrade) 27(3) 219-229.
  • Janse, J.D. 2010. Diagnostic Methods for Phytopathıgenic Bacteria of Stone Fruit and Nuts in COST 873, Bulletin OEPP/EPPO, 40:68-85.
  • Kaluzna, M. and Sobiczewski, P. 2009. Virulence of Pseudomonas syringae Pathovars and Races Originating from Stone Fruit Trees. The Polish Phytopathological Society 54:71-79.
  • Kaluzna, M. Pulawska, J. and Sobiczewski, P. 2010. The Use of PCR Melting Profile for Typing of Pseudomonas syringae İsolates from Stone Fruit Trees. European Journal of Plant Pathology. 126:437-443.
  • Kennelly, M.M., Cazorla, F.M., Vicente, A., Ramos, C. ve Sundin, G.W. 2007. Pseudomonas syringae diseases of fruit trees progress toward understanding and control. Plant Disease, 91,1.
  • Klement, Z., Farkas, G.L. and Lourekovich, L. 1966. Hypersensitive Reaction İnduced by Phytopathogenic Bacteria in Tobacco Leaf. Phytopathology 54:474-477.
  • Kotan, R., and Şahin, F. 2002. First Record of Bacterial Canker Caused by Pseudomonas syringae pv. syringae, on Apricot Trees in Turkey. Plant Pathology, 51:798.
  • Lattore, B.A. and Jones, A.L. 1979. Pseudomonas morsprunorum, The Cause of Bacterial Canker of Sour Cherry in Michigan and Its Epiphytic Assocation with P. syringae. Phytopathology 69:335-339.
  • Lelliot, R.A. and Stead, D.E. 1987. Methods in Plant Pathology, Blackwell Scientific Publications for British Society for Plant Pathology, 2, Oxford, UK, 216p.
  • Liang, L.Z., Sobiczewski, P., Patterson, J.M. and Jones, A.L. 1994. Variation in Virulence, Plasmid Content and Genes for Coronatine Synthesis Between P. syringae pv. morsprunorum and P. s. syringae from Prunus. Plant Disease 78:389-392.
  • Mitchell, R.E. 1982. Coronatine Producing by Some Phytopathogenic Pseudomonads. Physiogical Plant Pathology 20:83-89.
  • Mitchell, R.E., Hale, C.N. and Shanks, J.C. 1983. Production of Different Pathogenic Symptoms and Different Toxins by Strains of Pseudomonas syringae pv. tomato Not Distinguishable by Gel-İmmunodiffusion Assay. Physiologicaş Plant Pathology 23:315-322.
  • Obradovic, A. 2010. Pseudomonas Pathogens of Stone Fruits and Nuts: Classical and Molecular Phytobacteriology, COST873 Stone Fruit Nut Health Trainig Course Handbook. Belgrade, 69pp.
  • Ozaktan, H., Akköprü, A., Bozkurt, A. and Erdal, M. 2008. Information of Peach Bacterial Canker in Aegean Region in Turkey, “Determination of the Incidence of the Different Pathovars of Pseudomonas syringae in Stone Fruits” COST Action 873 Bacterial Disease of Stone Fruits and Nuts.
  • Sarkar, S.F. and Guttman, D.S. 2004. Evolution of the Core Genome of Pseudomonas syringae, Highly Clonal, Endemic Plant Pathogen. Applied and Environmental Microbiology 70:1999-2012.
  • Schaad, N.W. Jones, B.J. and Chun, W. 2001. Laboratory Guide for Identification Plant Pathogenic Bacteria, APS Press, USA, 543pp.
  • Sorensen, K.N., Kim, H.K. and Takemoto, J.Y. 1998. PCR Detection of Cyclic Lipodepsinoapeptide-Producing Pseudomonas. syringae pv. syringae and similarity of Strains. Applied and Environmental Microbiology P:226-230.
  • Süle, S. and Seemüller, E. 1986. The Role of Ice Formation in the İnfection of Sour Cherry Leaves by Pseudomonas syringae pv. syringae. The American Phytopathological Society Vol.77 (2).
  • Ullrich, M., Bereswill, S., Völksch, B., Fritsche, W. and Geider, K. 1993. Molecular Characterization of Field Isolates of Pseudomonas syringae pv. glycinea Differing in Coronatine Production. J. Gen. Microbiolgy 139: 1927-1937.
  • Yamamoto, S., Kasai, H., Arnold, D.L., Jackson, R.W., Vivian, A. and Harayama, S. 2000. Phylogeny of the Genus Pseudomonas: Intragenic Structure Reconstructed from the Nucleotide Sequences of the gyrB and rpoD Genes. Microbiology 146:2385-2394.
  • Xu, G.W. and Gross, D.C. 1988. Physical and Functional Analyses of the syrA and syrB Genes Involved in Syringomycin Production by Pseudomonas syringae pv. syringae. Journal of Bacteriology 170:5680-5688.