Study of Virulence factor of Erwinia amylovora isolates obtained from Semnan province and resistance evaluation of different pear tissues, blossom and immature fruit, in different pear cultivars to Fire blight disease

Document Type : Research paper-Persian

Authors

1 Ph.D. Graduate, Agricultural and Natural Resources Research Center of Semnan Province

2 Assistant Professor, Agricultural and Natural Resources Research Center of Semnan Province

3 Assistant Professor, Ilam University

Abstract

Background and Objectives
Fire blight, caused by Erwinia amylovora, is one of the important bacterial diseases of pome fruit trees. It causes the blight of different organs of the tree (blossoms, shoots, leaves, fruits, and limbs) and exudates' production. The main symptoms of fire blight infection are the burnt-like appearance of infected tissues, bacterial ooze released from the infected tissues, shepherd’s crook, wilting, and water-soaked appearance. Recently, it has been included in the top 10 plant pathogenic bacteria. This study aimed to collect, identify and characterize E. amylovora isolates from provinces of Semnan in Iran and determine their pathogenicity factor and the current situation of fire blight disease in this province.
Material and Methods
In the present study, 68 strains isolated from pear, quince, and apple hosts were identified in Semnan Provinces. Phenotypic, nutritional, and biochemical tests were performed on strains. The molecular identification of isolates was conducted using specific primers A and B of the plasmid pEA29. A pathogenicity test of the bacterial isolates on the immature pear fruit of the Spadona pear cultivar was performed, and the most pathogenic bacterium isolate was selected for further evaluation. Pathogenicity test was carried out on immature fruits and blooms of eight pear cultivars. Pathogenicity characteristics such as amylovoran, biofilm, and siderophore were studied in most virulence isolates.
Results and Discussion
The isolates were gram and oxidase negative, catalase-positive, and anaerobic. The isolates were able to produce a hypersensitive response in tobacco and levan production, but none of them could produce fluorescent pigment on King B medium and growth at 39 °C. The results suggested that of 68 strains collected, 52 isolates belong to Erwinia amylovora bacteria. The virulent isolate (D 43) was collected from Dibaj city. All 52 isolates under study proliferated 1000 base pairs. These tests, along with sequencing of PCR products, proved they were Erwinia amylovora species. Studying pathogenicity factors showed an almost direct relationship between all pathogenicity factors (other than biofilm production) and the severity of disease symptoms on tissues. The most resistant blooms tissues to D 43 isolate were shown to be related to Dargazi and Spadona pear cultivars; Duchess and Chini Hesar pear cultivars had the most resistant immature fruits tissues to D 43. It is therefore concluded that the susceptibility tissues to isolates of Erwinia amylovora were different in various cultivars. This point could help control fire blight disease and programs of breeding cultivars in the future.

Keywords


Afunian, M.R., Mohammadi, M., and Rahimian, H. 2000. Phenotypic characterization of Iranian strain of Erwinia amylovora the causal agent of fire blight disease pome tree. Iranian Journal Agricultural Sciences, 31(3): 463-476 (In Farsi with English summary).
Akhlaghi, M., Tarighi, S., and Taheri, P. 2018. Evaluating antibacterial effect of plant extracts against Erwinia amylovora and their role in resistance induction in pear. Biological control of pests and plant diseases, 7(2): 31-47 (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 PCR-analysis. Applied and Environmental Microbiology, 58: 3522-6.
Dye, D.W. 1968. A taxonomic study of the genus Erwinia, The amylovora group. Journal of Agricultural Science, 11: 590-607.
Ebadi, A., Erfani, J., Abdollahi, H., and Fattahi Moghaddam, J. 2014. Investigation of changes in antioxidantenzyme and total phenol level in some pear cultivars inoculated with fire blight disease. Iranian Journal of Horticultural Science, 45(2): 127-136 (In Farsi with English summary).
Expert, D., Dellagi, A., and Kachadourian, R. 2000. Iron and fire blight: role in pathogenicity of desferrioxamine E, the main siderophore of Erwinia amylovora. InVanneste, J., (Eds.), Fire blight: the disease and its causative agent Erwinia amylovora. CABI Publishing, New York, N.Y. pp. 179-195.
Fischer, T.C., Gosch, C., Mirbeth, B., Gselmann, M., Thallmair, V., and Stich, K. 2012. Potent and specific bactericidal effect of juglone (5-hydroxy-1, 4-naphthoquinone) on the fire blight pathogen Erwinia amylovora. Journal of agricultural and food chemistry, 60(49): 12074-12081.
Falkenstein, H., Bellemann, P., Walter, S., Zeller, W., and Geider, K. 1988. Identification of Erwinia amylovora, the fire blight pathogen, by colony hybridization with DNA from plasmid pEA29. Applied and Environmental Microbiology, 54: 2798-2802. 
Geier, G., and Geider, K. 1993. Characterization and influence on virulence of the levansucrase gene from the fire blight pathogen. Physiological and Molecular Plant Pathology, 42(6): 387-404.
Gross D.C., Lichens-Park, A., and Kole, C. 2015. Genomics of plant-associated bacteria. Springer press. 238 pp.
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.
Hugh, R., and Leifson, E. 1953. The taxonomic significance of fermentative versus oxidative methabolism of carbohydrates by various gram negative bacteria. Journal of Bacteriology, 66: 24-26.
Iakimova, E.T., Sobiczewski, P., Michalczuk, L., Wegrzynowicz-Lesiak, E., Mikici-nski, A., and Woltering, E.J. 2013. Morphological and biochemical characterization of Erwinia amylovora induced hypersensitive cell death in apple leaves. Plant Physiology and Biochemistry, 63: 292-305.
Khan, M.A., Zhao, Y., and Korban, S.S. 2012. Molecular Mechanisms of Pathogenesis and Resistance to the Bacterial Pathogen Erwinia amylovora, Causal Agent of Fire Blight Disease in Rosaceae. Plant Molecular Biology Reporter, 30: 247-260.
Klement, Z., Farkas, G.L., and Lovrekovich, L. 1964. Hypersensitive reaction induced by Phytopathogenic bacteria in the tobacco leaf. Phytopathology, 54: 474-477.
Koczan, J.M., Lenneman, B.R., McGrath, M.J., and Sundin, G.W. 2011. Cell surface attachment structures contribute to biofilm formation and xylem colonization by Erwinia amylovora. Applied and Environmental Microbiology, 77: 7031-7039.
Kovacs, N. 1956. Identification of Pseudomonas pyocyanea by the oxidase reaction. Nature, Lond, 178.
Lee, S.A., Ngugi, H.K., Halbrendt, N.O., Keefe, G., Lehman, B., Travis, J.W., Sinn, J.P., and McNellis, T.W. 2010. Virulence characteristics accounting for fire blight disease severity in apple trees and seedlings. Phytopathology, 100: 539-550.
Lelliot, R.A., and Stead, D.E. 1987. Methods for the Diagonosis of Bacterial Disease of Plant. Blackwell Scientific, Pub., London.
Maes, M., Orye, K., Bobev, S., Devreese, B., Van Beeumen, J., De Bruyn, A., Busson, R., Herdewijn, P., Morreel, K., and Messens, E. 2001. Influence of amylovoran production on virulence of Erwinia amylovora and a different amylovoran structure in E. amylovora isolates from Rubus. European Journal of Plant Pathology, 107: 839-844.
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).
Mohammadi, M. 2010. Enhanced colonization and pathogenicity of Erwinia amylovora strains transformed with the near-ubiquitous pEA29 plasmid on pear and apple. Plant Pathology, 59: 252-261.
Moslemkhani, K., Hemati, M., Hajnajari, H., and Aminkhaki, S. 2015. Investigating the Inheritance of Erwinia amylovora in Apple trees without symptoms of disease. Journal of Plant Protection, 29(2): 206-210 (In Farsi with English summary).
Ommati, F., and Zaker, M. 2015. Role of Infected Plant Parts in Development and Survival of Fire Blight of Pome Fruits. Applied research in plant protection, 3(2): 57-98 (In Farsi with English summary).
Parceya, M., Gaydera, S., Morley-Senklerc, V., Bakkerenc, G., Úrbez-Torresc, J.R., Ali, S., Castlee, A.J., and Svircevb, M.A. 2020. Comparative genomic analysis of Erwinia amylovora reveals novel insights in phylogenetic arrangement, plasmid diversity, and streptomycin resistance. Genomics, 112: 3762-3772.
Tavakolbakhoda, S., and Taghavi, S.M. 2010. Genotypic and phenotypic characteristics of Erwinia amylovora isolates from different hosts in Shiraz. Iranian Journal of Plant Protection Sciences, 41(1): 29-40 (In Farsi with English summary).
Schaad, N.W., Jones, J.B., and Chun, W. 2001. Laboratory guide for identification of plant pathogenic bacteria 3rd ed., The American Phytopathology Society Press, St. Paul, MN, USA. 373 pp.
Smits, T.H.,and Duffy, B. 2011. Genomics of iron acquisition in the plant pathogen Erwinia amylovora: insights in the biosynthetic pathway of the siderophore desferrioxamine E. Archives of Microbiology, 193: 693-9.
Vanneste, J.L. and Expert, D. 1990. Detection of an iron uptake system in E. amylovora. Acta Horticulturae, 273: 249-253.
Vrancken, K., Holtappels, M., Schoofs, H., Deckers, T., and Valcke, R. Pathogenicity and infection strategies of the fire blight pathogen Erwinia amylovora in Rosaceae: State of the art. Microbiology, 159: 823-832.
Yaish, M.W.F. 2006. Genetic mapping of quantitative resistance to race 5 of Pseudomonas syringae pv. phaseolicola in common bean. Euphytica, 152, 397-404.
Zakeri, Z., and Sharif Nabi, B. 1991. Pear fire blight disease in Karaj. Abstracts of the 10th Iranian Plant Protection Congress, Kerman, 157.
Zhao, Y., Wang, D., Nakka, S., Sundin, G.W., and S. S. Korban. 2009. Systems level analysis of two-component signal transduction systems in Erwinia amylovora: Role in virulence, regulation of amylovoran biosynthesis and swarming motility. BMC Genomics, 245: 1-16.