Identification and genotypic characteristics of Erwinia amylovora isolates, the causal agents of fire blight on pome fruit trees in Hamadan province

Document Type : Research paper-Persian

Authors

1 Associate Professor, Lorestan University

2 Assistant Professor, Lorestan university

3 Assistant Professor, Sahid Chamran university of Ahvaz

4 M.Sc. graduate, Lorestan university

Abstract

Introduction
One of the most destructive diseases of pome fruit species which causes irreparable damage to gardening products worldwide is fire blight disease caused by Erwinia amylovora. In Iran, it was first observed in pear trees in Alborz province and then spread across fruit orchards in the country. One of the main problems in fire blight management is evaluating causal agent genotypic characteristics. Thus, the present research was conducted to characterize genotypic features of E. amylovora as the cause of fire blight disease in pome fruit species in Hamadan province, Iran.
Material and methods
In this study, the samples with symptoms of canker on shoots and blight blossoms were observed and collected from the orchards in Hamadan province located in the west of Iran. After isolation, the isolates were purified for further studies. Phenotypic, biochemical, and pathogenicity tests were performed due to standard bacterial criteria. Phenotypic and biochemical tests of strains were examined by Ntsys-pc 2.02 software. A total of 15 representative isolates were selected and analyzed due to the size of amplified DNA using primers Ea71 and genetic features of the rep-PCR test using primers ERIC and BOX. For more accuracy and higher reliability of the specific primers results, the 16S rDNA_ gene of two representative isolates was amplified and subjected to sequencing.
Results
Based on the biochemical, pathogenicity and molecular tests, a total of 34 isolates were identified as E. amylovora. Due to the numerical analysis, the data obtained by phenotypic and biochemical tests were similar at 89% level. Therefore, the isolates obtained from a specific host or region was grouped very close to each other. In the PCR tests survey, the isolates amplified 187 base pair expected fragments. The results of determining sequences indicated that both isolates were similar to E. amylovora showing 97% identity to the type of strains in the NCBI database. Due to data from the rep-PCR analysis, the isolates were divided into three groups at the similarity level of 77%.
Discussion
The results of genetic diversity using rep-PCR showed that there is no significant difference among E. amylovora bacterial isolates from different regions of Hamadan Province. Moreover, they showed high similarity to each other and placed close to each other. Our results confirm other studies regarding phenotypic characteristics of E. amylovora. Our results confirm that the isolates are homogenous in Hamadan province of Iran. To summary, these findings can be applied to breeding programs to better management of the bacterial disease.

Keywords


Abdollahi, H., and Salehi, Z. 2018. Histology of oxidative stress and generation of reactive oxygen species against progress of fire blight causal agent in pear cultivars. Seed and Plant Production Journal, 2 (33): 139-162 (In Farsi with English summary).
Aeini, M., and Taghavi, S. M. 2014. Genotypic characteristics of the causal agent of chinaberry gall. Archives of Phytopathology and Plant Protection, 47: 1466-1474.
Afunian, M.R., Mohammadi, M., and Rahimian, H. 2000. Phenotypic characterization of Iranian strains Of Erwinia amylovora, the causal agent of fire blight disease pome trees. Iranian Journal of Agriculture Science, 31(3): 464-476 (In Farsi with English summary).
Bereswill, S., Pahl, A., Bellemann, P., Zeller, W., and Geider, K. 1992. Sensitive and species-specific detection of Erwinia amylovora by polymerase chain reaction analysis. Applied and Environmental Microbiology, 58: 3522-3526.
Bonn, W.G., and Van der zwet, T. 2000. Distribution and economic importance of fire blight. In: Fire blight: the disease and its causative agent, Erwinia amylovora. J.L. Vanneste (ed.), CABI Publishing, Wallingford, Oxon, United Kingdom: 37-53.
Calzolari, A., Finelli, F., and Mazzoli, G. 1998. A severe unforeseen outbreak of fire blight in the Emilia-Romagna region. Pages 171-176. In: VIII International Workshop on Fire Blight 489.
Dagher, F., Olishevska, S., Philion, V., Zheng, J., and Déziel, E. 2020. Development of a novel biological control agent targeting the phytopathogen Erwinia amylovora. Heliyon, 6(10): 1-10.
Dye, D. 1968. A taxonomic study of the genus Erwinia. I. The'amylovora'group. New Zealand Journal of Science, 11: 590-607.
Genersch, E., and Otten, C. 2003. The use of repetitive element PCR fingerprinting (rep-PCR) for genetic subtyping of German field isolates of Paenibacillus larvae subsp. larvae. Apidologie, 34: 195-206.
Guilford, P., Taylor, R., Clark, R., Hale, C., and Forster, R. 1995. PCR-based techniques for the detection of Erwinia amylovora. Pages 53-56. In: VII International Workshop on Fire Blight 411.
Gusberti, M., Klemm, U., Meier, M. S., Maurhofer, M., and Hunger-Glaser, I. 2015. Fire blight control: the struggle goes on. A comparison of different fire blight control methods in Switzerland with respect to biosafety, efficacy and durability. International Journal of Environmental Research and Public Health, 12: 11422-11447.
Holt, J.G., Krieg, N.R., Sneath, P.H.A., Staley, J.T. and Williams, S.T. 1994. Bergey’s Manual of Determinative Bacteriology, 9th edn. Williams and Wilkins, Baltimore, Maryland, USA, 787 pp.
Holtappels, M., Noben, J.P. and Valcke, R. 2016. Virulence of Erwinia amylovora, a prevalent apple pathogen: Outer membrane proteins and type III secreted effectors increase fitness and compromise plant defenses. Proteomics, 16: 2377-2390.
Hugh, R., and Leifson, E. 1953. The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various gram negative bacteria. Journal of Bacteriology, 66: 24.
Klement, Z., Fakas, G.L., and Loverkovich, L. 1964. Hypersensitive reaction induced by pathogenic bacteria in the tobacco leaf. Phytopathology, 54: 474-477
Koczan, J.M., McGrath, M.J., Zhao, Y., and Sundin, G.W. 2009. Contribution of Erwinia amylovora exopolysaccharides amylovoran and levan to biofilm formation: implications in pathogenicity. Phytopathology, 99(11):1237-44.
Kovacs, N. 1956. Identification of Pseudomonas pyocyanea by the oxidase reaction. Nature, 178: 703.
McManus, P. S., and Jones, A. L. 1995. Genetic fingerprinting of Erwinia amylovora strains isolated from tree-fruit crops and Rubus spp. Phytopathology, 85:1547-1553.
Mirzaee, H., Taghavi, S. M., and Aeini, M. 2015. Genotypic and Phenotypic Diversity of the Causal agent of Bacterial Blight of Walnut in Fars and Lorestan Provinces. Agricultural Biotechnology, 6(1): 51-58 (In Farsi with English summary).
Mirzai, M., Aminian, H., and Roustaee, A. 2012. The study of biological control of pear fire blight caused by Erwinia amylovora by some antagonistic bacteria. Biological Control of Pests and Plant Diseases, 1: 39-47 (In Farsi with English summary).
Moarrefzadeh, N., Mohamadi, M., Sharifitehrani, A. and Zakeri, Z. 2009. Evaluation of the effectiveness of some serological and PCR-based methods for detecting bacteria in the fire blight agent of pome fruit trees. Iranian Journal of Plant Protection Sciences, 40(1): 55-64 (In Farsi with English summary).
Molaei, s., Harighi, B., and Rafipour, V. M. 2010. Investigation of phenotypice and genotypic characteristics of bud blight stains using rep-PCR technique. National genetic and biological resource congress. Tehran.
Najafipour, G., Jamali, E., and Ayazpour, K. 2015. Phenotypic and Genotypic Characterization of Erwinia amylovora in Pome Fruit Orchards in Isfahan Province. Journal of Applied Research in Plant Protection, 4(1): 177-195.
Niknejad Kazempour, M., Kamran, E., and Ali, B. 2007. Study of Fire Blight on Pear Trees in Guilan Province Caused by Erwinia amylovora. Journal of Water and Soil Science, 11 (40):257-265 (In Farsi with English summary).
Norelli, J.L., Jones, A.L., and Aldwinckle, H.S. 2003. Fire blight management in the twenty-first century: using new technologies that enhance host resistance in apple. Plant Disease, 87: 756-765.
Perombelon, M.C., and Kelman, A. 1980. Ecology of the soft rot Erwinia. Annual Review of Phytopathology, 18: 361-387.
Radunović, D., Gavrilović, V., Gašić, K., Paunović, M., Stojšin, V., and Grahovac, M. 2017. Molecular characterization of Erwinia amylovora strains originated from pome fruits and indigenous plant in montenegro. Journal of Plant Pathology, 99: 197-203.
Rico, A., Führer, M. E., Ortiz-Barredo, A., and Murillo, J. 2008. Polymerase chain reaction fingerprinting of Erwinia amylovora has a limited phylogenetic value but allows the design of highly specific molecular markers. Phytopathology, 98: 260-269.
Schaad, N.W., Jones, J.B., and Chum, W. 2001. Laboratory Guide for Identification of Plant Pathogenic Bacteria. Third eds. American Phytopathological Society, St. Paul Minnesota, USA. 373pp.
Sulsow, T.V., Schorth, M.N., and Saka, M. 1982. Application of a rapid method for gram differentiation of plant pathogenic and saprophytic bacteria without staining. Phytopathology, 72: 917–918.
Tavakol Bakhoda, Sh., and Taghavi, M. 2010. Phenotypic and genotypic features of Erwinia amylovora from different hosts in Shiraz. Iranian Plant Protection Science, 41: 29-40 (In Farsi with English summary).
Taylor, R., Guilford, P., Clark, R., Hale, C., and Forster, R. 2001. Detection of Erwinia amylovora in plant material using novel polymerase chain reaction (PCR) primers. New Zealand Journal of Crop and Horticultural Science, 29: 35-43.
Vantomme, R., Swings, J., Goor, M., Kersters, K., and De Ley, J .1982. Phytopathological, serological, biochemical and protein electrophoretic characterization of Erwinia amylovora strains isolated in Belgium. Phytopathologische Zeitschrift, 103: 349-360.
Versalovic, J., Schneider, M., de Bruijn, F. J. and Lupski, J. R. 1994. Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods of Molecular and Cellular Biology, 5: 25-40.
Yassad, C.S., Manceau, C., and Luisetti, J. 1994. Occurrence of specific reaction induced by Pseudomonas syringae pv. syringae on bean pods, lilac and pear plants. Phytopathology, 43: 528-536.
Ye, G., Hong, N., Zou, L.F. Zou H.S., Zakria, M., Wang, G.P., and Chen, G.Y. 2013. Tale-based genetic diversity of Chinese isolates of the citrus canker pathogen Xanthomonas citri subsp. citri. Plant Disease, 97: 1187–1194.