Abstract
In 2012, a leaf spot disease on walnut seedlings was observed in Hamedan province of Iran. The spots were necrotic with yellow halos. Symptomatic samples were collected and suspected bacterial agent was isolated on nutrient agar medium. Phenotypic characteristics such as production of fluorescent pigment on KB medium, LOPAT test and utilization of various carbon sources, revealed that the strains were Pseudomonas syringae pv. syringae. All tested strains were pathogenic on peach, plum and walnut seedling. To investigate the genetic diversity among the strains, rep-PCR using BOX and ERIC primers was performed. Results showed high similarity among the strains from the same region, while variation was found among those from different areas and they were divided into two groups based on geographic regions. The phylogenetic analysis based on 16S rRNA, rpoD and gyrB sequences showed that representative isolates were closely related to P. syringae pv. syringae. To the best of our knowledge, this is the first report of the presence of P. syringae pv. syringae as a causal agent of walnut seedlings leaf spot in Iran and worldwide.
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Abbasi, V., Rahimian, H., & Tajick-Ghanbari, M. (2013). Genetic variability of Iranian strains of pseudomonas syringae pv. Syringae causing bacterial canker disease of stone fruits. European Journal of Plant Pathology, 135, 225–235.
Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215, 403–410.
Ashorpour, M., Niknejad Kazempour, M., & Ramezanie, M. (2008). Occurrence of Pseudomonas syringae pv. syringae the causal agent of bacterial canker on olives (Olea europaea) in Iran. Science Asia, 34, 323–326.
Ayers, S. H., Rupp, P., & Johnson, W. T. (1919). A study of the alkali-forming bacteria in milk. United States Department of Agriculture Bulletin, 782, 1–39.
Bahar, M., Mojtahedi, H., & Akhiani, A. (1982). Bacterial canker of apricot in Isfahan. Iranian Journal of Plant Pathology, 18, 58–68.
Banapour, A., Zakiee, Z., & Amani, G. (1990). Isolation of Pseudomonas syringae from sweet cherry in Tehran Province. Iranian Journal of Plant Pathology, 26, 67–72.
Bradbury, J. F. (1986). Guide to plant pathogenic bacteria. Kew, United Kingdom: CAB International Mycological Institute.
Bull, C. T., Clarke, C. R., Cai, R., Vinatzer, B. A., Jardini, T. M., & Koike, S. T. (2011). Multilocus sequence typing of Pseudomonas syringae sensu lato confirms previously described genomospecies and permits rapid identification of P. syringae pv. coriandricola and P. syringae pv. apii causing bacterial leaf spot on parsley. Phytopathology, 101, 847–858.
Bultreys, A., & Kaluzna, M. (2010). Bacterial cankers caused by Pseudomonas syringae on stone fruit species with special emphasis on the pathovars syringae and morsprunorum race1 and race2. Journal of Plant Pathology, 92, 21–33.
Çepni, E., & Gürel, F. (2012). Variation in extragenic repetitive DNA sequences in Pseudomonas syringae and potential use of modified REP primers in the identification of closely related isolates. Genetics and Molecular Biology, 35, 650–656.
Fahy, P. C., & Hayward, A. C. (1983). Media and methods for isolation and diagnostic tests. In P. C. Fahy & G. J. Parsley (Eds.), Plant bacteria diseases, a diagnostic guide (pp. 337–378). Australia: Academic Press.
Ferrante, P., & Scortichini, M. (2010). Molecular and phenotypic features of Pseudomonas syringae pv. actinidiae isolated during recent epidemics of bacterial canker on yellow kiwifruit (Actinidiachinensis) in Central Italy. Plant Pathology, 59, 954–962.
Gardan, L., Shafik, H., Belouin, S., Broch, R., Grimont, F., & Grimont, P. A. (1999). DNA relatedness among the pathovars of Pseudomonas syringae and description of Pseudomonas tremae sp. nov. and Pseudomonas cannabina sp. nov. (ex Sutic and Dowson 1959). International Journal of Systematic Bacteriology, 49, 469–478.
Gašić, K., Prokić, A., Ivanović, M., Kuzmanović, N., & Obradović, A. (2012). Differentiation of Pseudomonas syringae pathovars originating from stone fruits. Pesticides and Phytomedicin, 27, 219–229.
Gilbert, V., Legros, F., Maraite, H., & Bultreys, A. (2009). Genetic analyses of Pseudomonas syringae strains from Belgian fruit orchards reveal genetic variability and host preferences within pathovar syringae, and help identify both races of pathovar morsprunorum. European Journal of Plant Pathology, 124, 199–218.
Gilbert, V., Planchon, V., Legros, F., Maraite, H., & Bultreys, A. (2010). Pathogenicity and aggressiveness in populations of Pseudomonas syringae from Belgian fruit orchards European journal of plant pathology, 126, 23–277.
Gutiérrez-Barranquero, J. A., Arrebola, E., Pérez-García, A., Codina, J. C., Murillo, J., De Vicente, A., & Cazorla, F. M. (2008). Evaluation of phenotypic and genetic techniques to analyze diversity of Pseudomonas syringae pv. syringae strains isolates frommango trees. In M. B. Fatmi, A. Collmer, N. Iacobellis, J. W. Mansfield, J. Murillo, N. W. Schaad, & M. Ulrich (Eds.), Pseudomonas syringae Pathovars and related pathogens—identification, epidemiology and genomics (pp. 271–281). Netherlands: Springer.
Hajri, A., Meyera, D., Delortb, F., Guillaume’sa, J., Brina, C., & Manceaua, C. (2010). Identification of a genetic lineage within Xanthomonas arboricola pv. juglandis as the causal agent of vertical oozing canker of Persian (English) walnut in France. Journal of Plant Pathology, 59, 1014–1022.
Higgins, C. F., Ames, G. F. L., Barnes, W. M., Clement, J. M., & Hofnung, M. (1982). A novel intercistronic regulatory element of prokaryotic operons. Nature, 298, 760–762.
Hildebrand, D. C., Schroth, M. N., & Sands, D. C. (1988). Pseudomonas, In: N. W. Schaad (Ed.), Laboratory Guide for the identification of plant pathogenic bacteria (pp. 60–80). 2nd edn. St. Paul, Minnesota, USA: APS Press.
Hugh, R., & Leifson, E. (1953). The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various gram negative bacteria. Journal of Bacteriology, 66, 24–26.
Hwang, M. S. H., Morgan, R. L., Sarkar, S. F., Wang, P. W., & Guttman, D. S. (2005). Phylogenetic characterization of virulence and resistance phenotypes of Pseudomonas syringae. Applied and Environmental Microbiology, 71, 5182–5191.
Joana, G., Steven, V., & Roberts, J. (2007). Discrimination of Pseudomonas syringae isolates from sweet and wild cherry using rep-PCR. European Journal of Plant Pathology, 117, 383–392.
Kaluzna, M., Ferrante, P., Sobiczewiski, P., & Scortichini, M. (2010). Characterization and genetic diversity of Pseudomonas syringae from stone fruits and hazelnut using repetitive-PCR and MLST. Journal of Plant Pathology, 92, 781–787.
Kersters, K. W., Ludwig, M., Vancanneyt, P., De Vos, M., Gillis, D., & Schleifer, K. H. (1996). Recent changes in the classification of the Pseudomonads: an overview. Systematic and Applied Microbiology, 19, 465–477.
Kimura, M. A. (1980). Simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16, 111–120.
Lelliott, R. A., Billing, E., & Hayward, A. C. (1966). A determinative scheme for the fluorescent plant pathogenic Pseudomonads. Journal of Applied Microbiology, 29, 470–489.
Little, E. L., Bostock, R. M., & Kirkpatrick, B. C. (1998). Genetic characterization of Pseudomonas syringae pv. syringae strains from stone fruits in California. Applied and Environmental Microbiology, 64, 3818–3823.
Lonetto, M., Gribskov, M., & Gross, C. A. (1992). The sigma 70 family: sequence conservation and evolutionary relationships. Journal of Bacteriology, 174, 3843–3849.
Louws, F. J., Fulbright, D. W., Stephens, C. T., & De Bruijn, F. J. (1994). Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR. Applied and Environmental Microbiology, 60, 2286–2295.
Louws, F. J., Fulbright, D. W., Taylor, S. E., & De Bruijn, F. J. (1995). Differentiation of genomic structure by rep-PCR fingerprinting to rapidly classify Xanthomonas campestris pv. vesicatoria. Phytopathology, 85, 528–536.
Louws, F. J., Rademaker, J. L., & De Bruijn, F. J. (1999). The three Ds of PCR-based genomic analysis of phytobacteria: diversity, detection and diagnosis. Annual Review of Phytopathology, 37, 81–125.
Manceau, C., & Horvais, A. (1997). Assessment of genetic diversity among strains of Pseudomonas syringae by PCR-restriction fragment length polymorphism analysis of rRNA operons with special emphasis on P. syringae pv. tomato. Applied and Environmental Microbiology, 63, 498–505.
Marques, A. S. A., Marchaison, A., Gardan, L., & Samson, R. (2008). BOX-PCR-based identification of bacterial species belonging to Pseudomonas syringae - P. viridiflava group. Genetics and Molecular Biology, 31, 106–115.
Martín-Sanz, A., Pérez de la Vega, M., & Caminero, C. (2012). Resistance to Pseudomonas syringae in a collection of pea germplasm under field and controlled conditions. Plant Pathology, 61, 375–387.
Martín-Sanz, A., Pérez de la Vega, M., Murillo, J., & Caminero, C. (2013). Strains of Pseudomonas syringae pv. syringae from pea are phylogenetically and pathogenically diverse. Phytopathology, 103, 673–681.
Otta, J. D., & English, H. (1971). Serology and pathology of Pseudomonas syringae. Phytopathology, 61, 443–452.
Peix, A., Ramirez-Bahena, M. H., & Velazquez, E. (2009). Historical evolution and current status of the taxonomy of genus Pseudomonas. Infection, Genetics and Evolution, 9, 1132–1147.
Qing, C., Pengfei, Q., Renlin, X., Tambong, J. T., Djama, Z. R., & Wei, L. (2011). Comparison of three typing methods for evaluating the diversity of Pseudomonas fluorescens in the rhizosphere. Journal of Plant Sciences, 6, 52–65.
Rademaker, J. L. W., & de Bruijn, F. J. (1997). Characterization and classification of microbes by rep-PCR genomic fingerprinting and computer-assisted pattern analysis. In G. Caetano-Anolles & P. M. Gresshoff (Eds.), DNA markers: protocols (pp. 151–171). NY, USA, John Wiley & Sons: Application and Overviews. New York.
Renick, L. J., Cogal, A. G., & Sundin, G. W. (2008). Phenotypic and genetic analysis of epiphytic pseudomonas syringae populations from sweet cherry in Michigan. Plant Disease, 92, 372–378.
Rouhrazi, K., & Rahimian, H. (2012). Characterization of Iranian grapevine isolates of Rhizobium (Agrobacterium) spp. Journal of Plant Pathology, 94, 555–560.
Saitou, N., & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4, 406–425.
Sarkar, S. F., & Guttman, D. S. (2004). Evolution of the core genome of Pseudomonas syringae, a highly clonal, endemic plant pathogen. Applied and Environmental Microbiology, 70, 1999–2012.
Schaad, N. W., Jones, J. B., & Chun, W. (2001). Laboratory guide for the identification of plant pathogenic bacteria (3th ed.). Paul, Minnesota, USA: Phytopathological Society St.
Scortichini, M., Marchesi, U., Rossi, M. P., & Prospero, P. D. (2002). Bacteria associated with hazelnut (Corylus avellana L.) decline are of two groups: Pseudomonas avellanae and strains resembling P. syringae pv. syringae. Applied and Environmental Microbiology, 68, 476–484.
Sneath, P. H. A., & Sokal, R. P. (1973). Numerical taxonomy: the principles and practice of numerical classification. San Francisco: WH Freeman and Company.
Suslow, T. V., Schroth, M. N., & Isaka, M. (1982). Application of rapid method for gram differentiation of plant pathogenic and saprophytic bacteria without staining. Phytopathology, 72, 917–918.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28, 2731–2739.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., & Higgins, D. G. (1997). The clustal_X windows interface: flexible strategies for multiple sequence alignement aided by quality analysis tools. Nucleic Acids Research, 25, 4876–4882.
Versalovic, J., Koeuth, T., & Lupski, J. R. (1991). Distribution or repetitive DNA– sequences in eubacteria and application of fingerprinting of bacterial genomes. Nucleic Acids Research, 19, 6823–6831.
Versalovic, J., Schneider, M., de Bruijn, F. J., & Lupski, J. R. (1994). Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods in Molecular Cellular Biology, 5, 25–40.
Vicente, J. G., & Roberts, S. J. (2003). Screening Wild Cherry Micropropagated Plantlets for Resistance to Bacterial Canker. In: "Pseudomonas syringae and Related Pathogens". (Eds.): Iacobellis, N. S., et al., Biology and Genetic, Kluwer Academic Publishers, Dordrecht, The Netherlands, PP. 467–474.
Vicente, J. G., & Roberts, S. J. (2007). Discrimination of isolates of Pseudomonas syringae from sweet and wild cherry using rep-PCR. European Journal of Plant Pathology, 117, 383–392.
Watt, P. M., & Hickson, I. D. (1994). Structure and function of type II DNA topoisomerases. Biochemical Journal, 303, 681–695.
Weingart, H., & Volksch, B. (1997). Genetic fingerprinting of Pseudomonas syringae pathovars using ERIC-, REP-, and IS50-PCR. Journal of Phytopathology, 145, 339–345.
Yamamoto, S., Kasai, H., Arnold, D. L., Jackson, R. W., Vivian, A., & Harayama, S. (2000). Phylogeny of the genus Pseudomonas: intrageneric structure reconstructed from the nucleotide sequences of gyrB and rpoD genes. Microbiology, 146, 2385–2394.
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Keshtkar, A.R., Khodakaramian, G. & Rouhrazi, K. Isolation and characterization of Pseudomonas syringae pv. syringae which induce leaf spot on walnut. Eur J Plant Pathol 146, 837–846 (2016). https://doi.org/10.1007/s10658-016-0962-2
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DOI: https://doi.org/10.1007/s10658-016-0962-2